A71

Satellite Symposia $3

$1 THE PH~COKINETICS G. E h n i n g e r a n d U.

OF ETOPOSIDE Schuler

Etoposide, a epipodophyllotoxin derivative has shown c l i n i c a l l y b e n e f i c i a l a c t i v i t y in l y m p h o m a a n d l e u k e m i a a n d s o l i d tumors. Interaction with DNA-topois o m e r a s e II o r f o r m a t i o n of f r e e r a d i c a l s h a v e b e e n suggested to account for the cytotoxic activity. The best fit for the plasma concentration time curve is a c h i e v e d in a t w o c o m p a r t m e n t m o d e l w i t h a t e r m i n a l h a l f - l i f e of 4 to 8 hours. U r i n a r y e x c r e t i o n w a s r e p o r t e d t o b e b e t w e e n 20 t o 70%, l o w e r a m o u n t s a r e e x c r e t e d in bile. S e v e r a l m e t a b o l i t e s h a v e b e e n s e p a r a t e d a n d i d e n t i f i e d as h y d r o x y acid, t r a n s - h y d r o x y a c i d of e t o p o s i d e , as c i s - i s o m e r a n d g l u c u r o nide and sulfate conjugates. The application of h i g h - d o s e e t o p o s i d e is u s u a l l y h a m p e r e d b y t i m e - c o n suming dilution steps which yield to large volumes administered. More recent studies showed that etoposide without prior dilution might be administered v i a a c e n t r a l v e n o u s line.

Abstract not submitted

A considerable intra- and interpatient variability of t h e b i o a v a i l a b i l i t y of o r a l p r e p a r a t i o n s were noted. A r a n g e of 25% t o 75% w a s r e p o r t e d in t h e l o w e r d o s e r a n g e ; its a b s o r p t i o n d e c r e a s e s w i t h h i g h e r doses. The activity has been shown to be highly schedule d e p e n d e n t (e.g. s i n g l e a g e n t 5 - d a y - t r e a t m e n t s i g n i ficant more active than 1-day-treatment in s m a l l c e l l l u n g c a n c e r ) . In a d d i t i o n r e s p o n s e s a m o n g p a tients relapsed after standard dose have been seen w i t h 3 - w e e k - c o u r s e s of o r a l e t o p o s i d e . Address: Medizinische Universit~tsklinik, M O l l e r - S t r . i0, 7 4 0 0 T U b i n g e n , F R G

Otfried-

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$2 CLINICAL PHARMACOKINETICS OF VP-16 S P E C I A L A S P E C T S IN C H I L D R E N -

J. BOOS Universit~ts-Kinderklinik, 44 Miinster - FRG

Albert-Schweitzer-Str.

33,

The pharmacokinetics of VP-16 by different routes of application has been extensively investigated in adults and children. With continuous infusion relevant interindividual variation of pharmacokinetics in children was reported and drug targeting was proposed (Mirro et al, Haematol Blood Transf, 1989). In a current study of our own, steady state levels vary widely, too, and children with dosage by kg had lower steady state levels than those with dosage by m 2. With short term infusion the H-half life was reported to be shorter in children than in adults (D'Incalci et al, CCP 1982). In the available literature this difference is not very distinct. In a current investigation of our own, model-independent calculation resulted in t89 3,3• h (21 children). The applied dose/m 2 correlated strongly with 1.5h and 3.5h levels (r=0.9). Reduced doses for children <2 years (/kg) resulted in corresponding lower VP-16-1evels and AUC's (area under the curve). Related to dose/m 2, however, parameters of kinetics were within the expected range. Normalised parameters ( to AUC/100mg/m 2 ) showed a coefficient of variation <25%. To a certain extent pharmacokinetic variability especially in children with short term infusion may be due to the large volume of the VP-16-solution and inaccuracy of flow rate controllers resulting in inexact definition of the end of infusion,

ETOPOSIDE IN THE TREATMENT OF CHILDHOOD HODGKIN'S DISEASE (HD). THE EXPERIENCE OF THE GERMAN PEDIATRIC STUDY GROUP DAL-HD. I.SALVAGE THERAPY G. Schellong, I. H~rnig-Franz, R. P6tter Etoposide has been incorporated by the DAL-HD study-group 1986 into salvag e therapy and 1987 into front-line therapy of HD in children and adolescents. From April 1986 to July 1991, 44 pats with early progression (2) or Ist relapse (42) were enrolled in a salvage therapy study by 29 hospitals. Male/female ratio is 23/21, median age 13,5 yrs (range 6-19 yrs). Median time to failure had been 15 ms (range 1-39 ms). Front-line therapy had consisted in 29 pats of 2, 4 or 6 courses of OPA or OPA/COMP (without procarbazine; M=methotrexate) and in 15 pats of some modifications containing procarbazine or etoposide, plus involved field irradiation (IFI). Salvage therapy included the drug combinations IEP (ifosfamide 2000 mg/m 2 p.i. 24 h, day I-5; etoposide 120 mg/m 2 p.i. 60 mins, day I-5; prednisone 100 mg/m 2 p.o., day I-5), ABVD and COPP. In some pats COPP was replaced by CEP. Most of the pats received 2 courses each of the 3 combinations. Chemotherapy was followed by IFI, using 12-35 Gy. Results (Dec. 1991): 2/44 pats had progression of HD during salvage therapy and deceased later. I pat achieved PR and expired from sepsis following ABMT. 41/44 pats achieved CR, 9 of them suffered a 2nd relapse. I pat in 2nd CR developed ANLL and deceased , I pat thyroid cancer (alive). 31 pats are alive in 2nd CR after a median of 35 ms (8-66 ms). KaplanMeier estimates at 5 yrs are 0.89 (SD 0.05) for survival and 0.67 (SD 0.12) for freedom from 2nd recurrence. Side effects of IEP : Leukopenia was the most frequently observed side effect followed by alopecia, nausea/ vomiting and slight Anemia. NO severe complications arose. Conclusions: IEP/AHVD/COPP(or/CEP) chemotherapy in combination with IFI is a highly effective salvage treatment with moderate toxicity for pats with early progression or Ist relapse of HD who are pretreated with MOPP derived schedules. Adress: Univ.-Kinderklinik, Albert-Schweitzer-Str. 33, D-4400 M~nster, Germany.

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ETOPOSIDE IN THE TREATMENT OF CHILDHOOD HODGKIN'S DISEASE (HD). THE EXPERIENCE OF THE GERMAN PEDIATRIC STUDY GROUP DAL-HD. II. FRONT-LINE THERAPY I. H~rnig-Franz, G. Schellong, M. Riepenhausen

TREATMENT-RESULTS AND FUTURE STRATEGIES WITH CHOEP IN HIGH GRADE NHL. K.~H. Pfl~qert H~ Koeppler, fo r the NHL Study oroup. In two multicentre trials the CHOEP-regimen (cyclophosphamide 750mg/m 2 iv dl, doxorubicin 50 mg/m 2 iv d i, vinchristine 2mg iv d 1, etoposide 100 mg/m 2 iv d 3-5, prednisolone I00 mg po d 1-5) was evaluated in previously untreated patients with high grade NHL stage II-IV. In a first phase II trial 60 patients were included. After four courses of CHOEP an involved field irradiation with a total dose of 25 Gy was employed and followed by two additional courses of polychemotherapy. The overall response rate was 93 % and 49 patients (82 %) achieved a CR. During a median follow-up of more than 6 years 22 out of 49 CR patients relapsed. A maintained CR was seen in 55 % of all patients initially achieving CR. In a second multicentre phase III trial 175 evaluable patients with untreated hg-NHL stage II-IV were randomized to receive either four cycles of CHOEP (arm A), or four cycles of hCHOP alternating with IVEP (arm B). After 4 cycles of chemotherapy an involved field irradiation with a total dose of 35 Gy was given to all patients in CR or PR. CR was seen in 85,5 % of patients with 87 % in arm A versus 84 % in arm B. During a median follow up of 26 mo (range 860 mo) the overall survival at 36 mo is projected to be 68 % versus 62 % for arm A and B, respectively. So far, the differences in CR, survival and DFS are not statistically significant. Toxicity of all regimens was acceptable. Main side effects were mild nausea/vomiting, leukopenia and fever/infection associated with leukopenia. Therapy related deaths 3 (2 in arm A, 1 in arm B). In conclusion both treatment modalities produced high CR rates and high 3 year DFS rates. For further strategy the well tolerated C H O E P regimen will be standard therapy for normal risk patients. In high risk patients high dose chemotherapy followed by autologous BMT or hemopoietic growth factor therapy in patients older than 60 years will be evaluated.

In 1987 a pilot study was implemented to evaluate etoposide (EP) as a substitute for procarbazine (PC) in the OPPA/COPP chemotherapy applied with favorable results in the studies DAL HD-78 and HD-82. It was expected that impairment of spermatogenesis caused by PC in 30-63% of the boys could be avoided. - From Oct. 87 to Aug. 90, 46 pats with untreated HD were enrolled in this trial by 20 hospitals. Median age was 13.5 yrs (3.5-18.5). Treatment group I consisted of 26 pats with stages IA(8), IB(1) and IIA(17), group 2 of 20 pats with stages IIEA(3), IIB(9), IIIA(2) and IIIB(6). All pats received 2 courses of OEPA (VCR, PRED and ADR as in 0PPA; EP 100 or 125 mg/m 2 p.i. 60 mins, day 3-6). In group I no further chemotherapy was given, whereas 2-4 courses of COEP (CP, VCR, PRED as in COPP; EP as in group I) were added for pats in group 2. Radiotherapy was given as IFI using 25 Gy, with a boost of 5-10 Gy to fields with incomplete regression. Results (Dec. 91): I pat in group I achieved only PR and expired later after ABMT. 25/26 pats went into CR and remained there for a median of 42 ms (17-51). Kaplan-Meier estimates(KME) for survival (S) and event-free survival (EFS) are 0.96 (SD 0.04) at 4 yrs. I pat of group 2 deceased after early progression, 19/20 pats achieved CR. I pat died in CR, I developed a melanoma, 4 relapsed (alive). 13 pats are in CCR after a median of 32 ms (19-4t). KME at 3.5 yrs in group 2 are 0.79 (SD 0.15) for S, and 0.58 (SD 0.14) for EFS. Side effects: Alopecia in all pats; leueopenia, mucositis and nausea/vomiting of moderate grade. Conclusions: OEPA seems to be an effective drug combination of low toxicitiy for localized HD and should be incorporated in phase III studies, whereas the results with 0EPA/COEP in more advanced stages are less encouraging. Investigations of testicular function have to be done later. Adress: Univ.- Kinderklinik, Albert-Schweitzer-Str. 33, D-4400 M~nster

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Div. Hematol./Oncology D-3550 Marburg.

Philipps-University

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THE USE OF CHRONIC ETOPOSIDE SCHEDULES IN THE TREATMENT OF

NON-HODGKIN'S LYMPHOMA. Haiusworth J.D., Division of Oncology, Vanderbilt University. The importance of schedule in the cytotoxic eftieacy of etoposide is supported by an increasing amount of clinical data. We studied the efficacy of oral etoposlde. 50 mg/m2 daily for 21 consecutive days, repeated every 28 to 35 days, in the treatment of refractory lymphoma. Twenty-five patients were treated; all had received previous chemotherapy and were considered incurable. Fifteen patients (60%) had partial responses; 5 of these patients had received previous intravenous etoposlde by standard schedules. Median response duration was 8 months in low grade lymphoma and 3 months in intermediate or high grade lymphoma. Two patients responded to chronic oral etoposide immediately after progress'rag on intravenous etopnside containing regimens. One of these patients had a complete response to oral etoposida (better than any response with previous therapy), and is disease free more than 18 months after completion of therapy. Chronic oral etoposide provides an effective and convenient treatment option for patients with indolent lymphoma. Incorporation into combination regimens for aggressive lymphoma is currently under investigation. Recently, we have further investigated the schedule dependency of etoposide by using 25 mg/m2/day given by continuous intravenous infusion. By avoiding high peak serum levels and maintaining a serum concentration in the 1/~g/ml range, we hoped to retain efficacy and minimize or avoid myelotoxieity. Blood counts were monitored weekly; continuous infusion was continued for as long as tolerated. To data, six patients with previously treated lymphoma have received etoposide infusions ranging from 6 to 54 weeks in duration. Myelosuppression was minimal and no other toxicity except alopecia was seen. Four of 6 patients responded to treatment. These preliminary data suggest that the toxicity of etoposide can be markedly altered by this method of administration and that eytotoxicityis retained. These infusional studies are ongoing.

Abstract not submitted

of Marburg,

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MYELODYSPLASTICSYNDROMES:TREATMENTINDICATIONS AND CHOICE OF CYTOK1NES. Ganser A., Dept. of Hematology, University of Frankfurt, Theodor-Stern-Kai 7, 6000 Frankfurt/M. 70, FRG. Hematopoietic growth factors (GM-CSF, G-CSF, Interleukin-3, erythropoietin) have been used in patients with myelodysplastic syndromes to reverse cytopenia and or induce differentiation. Reversal of neutropenia can be achieved in the majority of patients treated with GM-CSF or G-CSF. Preliminary data of randomized trials indicate that the risk of infection cart be considerably reduced, while the rate of transformation to acute leukemia apparently is not incrcased in the patient population studied. Improvement of thrombocytopenia and anemia only occurs in the minority of patients after treatment with IL-3 or erythropeietin, respectively. While GM-CSF and G-CSF should be of value in the severely neutropeulr MDS patients, clinical trials still have to demonstrate a prolongation of survival after treatment with any of the hematopoietic growth factors.

THE USE OF GM-CSF AND INTERLEUKIN-3 IN THE TREATMENT OF ACUTE MYELOID LEUKEMIA (AML). Andreeff M., Tafuri A., Estey E., Drach J., Goodacre A., Zhao S., Zhang W., Sancbez-Williams G., Escudier S., Reading C., Estrov Z., Plunkett W., Mandelli F., Deisseroth A. The University of Texas M.D. Anderson Cancer Center, Houston, 'IX, USA. Cell kinetic and pharmacological studies suggest increased cytotoxic effects of irdaibitors of DNA-synthasis and topoisomerase II (Topo H) on AML cells following exposure to GM-CSF and IL-3. Recruitment of quiescent leukemic cells, increased intracellular Ara-CTP levels and Ara-CTP/dCTP ratios, increased expression of cmyc and phosphorylation of the Rb gene were found in vitro. We developed protocols for pts. with refractory/relapsed AML that combine IL-3 and Idarubicin (Ida)/Ara-C to exploit these observations clinically and to study the associated cellular, molecular and pharmacodynamic effects in vivo. Regimen A consists of IL-3 (Sandoz, 2Oug/m2 s.c.b.i.d, x 7d), followed on d 3 by Ida (12mg/m2 daily x 3) and Ara-C (1.Sg/m2 daily x4). Regimen B consists of IL-3 (25Qug/m2 s.c.q.d.xl0d) followed by Ida (8mg/m2 daily x 3) and Ara-C (Ig/m2 q 12 h x 6d). To date, 5 patients (pts.) have been entered. Two pts. expired, 3 are too early to be evaluated for response. In pt. 1 treated with reg. A, limited dilution assay ex vivo indicated Ara-C resistance on d 0 and Ara-C killing on d 3, but cell kinetics (DNA/RNA, [] 67, BUdR) and C I ~ did not change. Pt. 2 on reg A expressed IL-3 receptors, which were internalized at 24 hr. This was associated with decreased cells in C~ expressing Ki67 and PCNA. This pt. developed tumor lysis syndrome after chemotherapy. Of the 2 pts. treated with reg. B, one had tumor lysis.Another demonstrated recruitment exclusivelyof the leukemic cells as determined by fluorescence in-situ hybridization. Bone marrow and blood blasts decreased temporarily followed by an increase in maturing cells with monosomy 7, indicating induction of differentiation. We will also review the cell kinetic changes and clinical responses observed in GM-CSF/chemotherapy combinations. In summary, GM-CSF and IL-3 affect a variety of cellular and molecular pathways in AML The ongoing clinical trials will elucidate mechanisms in vivo and hopefully result in improved remission for patients with AML.

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Abstract not submitted

EFFECTS ON ARA-C CYTOTOXICITY FOR AML-COLONY F O R M I N G C E L L S BY P R I M I N G WITH rh GM-CSF AND r h I L - 3 I N V I T R O AND IN VIVO. M. Ztthlsdorf. C. Busemann. C. Ameline. S. WeiBpfennig, B. W6rmann, W. Hiddemanu. and Th. Biichner. Medizir/ische Klinik A, Wesff~ische Wilhelms-Universit~t, MUnster, FRG Cell kinetic resistance to cytosine arabinoside (am-C) might be one of the major causes of treatment failure in acute myeloid leukemia. The objective of this study was to investigate ara-C sensitivity of myeloid leukemic precursor ceils (CFU-L) after preexposure to rhGM-CSF or rhlL-3 in vitro. Also, samples of patients primed with GM-CSF in vivo in a phase II trial at this center were analyzed. Marrow cells of 14 A M L patients were obtained prior to chemotherapy, separated on Ficoll-Hypaque gradients, T-cell depleted, and incubated with 100 U/ml rhGM-CSF or rhlL-3 or medium for 48h. 7 samples were also exposed to 24h GM-CSF. Cells were then incubated with 0-100pM Ara-C for 12h, washed and plated in a CFU-L assay based on methylcellulose, 20% FBS for 14d. CFU-L colonies were scored and their morphology was verified by Pappenheim stains on cytospins. Also, 5 samples of marrow cells were investigated for CFU-GM in a similar fashion from patients achieving a complete remission (CR) after induction chemotherapy (TAD, + high-dose a r a - C and mitoxantrone). Colony numbers vs. Ara-C doses were evaluated in a median-effect plot yielding the LD50 of Ara-C for different pretreatments. The table gives the number of samples showing a significant (1.3 to 230fold) decrease in the LD50 of ara-C compared to controls without pretreatment vs. the total number of samples in the subgroup, respectively. Decrease GM-CSF primed IL-3 p r i m e d in LDS0 (ara-C) 24b 48h in vivo 48h p r i o r to t h e r a p y 4/7 10/13 3/3 6/8 at time of C R 3/5 1/1 2/2 The increase in ara-C eytotoxicity did not correlate for the two growth factors in 8 samples tested in parallel. 24h exposure to GM-CSF in vitro was less effectice than 48h. Preexposure to 24h GM-CSF in vivo yielded a measurable decrease in the LD50 of ara-C for CFU-L. Pretreatment with growth factors also enhanced ara-C toxicity for normal committed progenitors (CFU-GM) in preliminary measurements. In conclusion, priming with rhGM-CSF or rhlL-3 might overcome cell.kinetic resistance to ara-C in myeloid leukemic precursors. The effect of priming on normal stem cells must be further investigated. A phase II trial on priming with GM-CSF in A M L is ongoing and further data are accumulating.

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ABMT FOR LYMPHOID MALIGNANCIES: THE USE OF G R A N ~ MACROPHAGE COLONY STIMULATING FACTOR. Vose J.M., Bierman PJ.,

COMPARISON OF PREDNIMUSTINE/MITOXANTRONE(ProM) AND CYCLOPHOSPHAMIDEJVINCRISTINE]PREDNISOLONE (COP) IN PATIENTS WITH LOW-GRADE NON-HODGKIN LYMPHOMASFOLLOWED BYINTERFERONa MAINTENANCE. Unterhalt M., Koch P., Nahler M., Herrmann R., Hiddemann W., Dept. of Internal Medicine, University of MOnster, FRG. In 1988, Landys et al. reported on a high anti-lymphoma activity of the combination of prednimustine and mitoxantrone with a low incidence of therapy associated side effects. Based on these data a phase II study was initiated for patients with advanc~l low malignant non-Hodgkin lymphomas using prednimustine and mitoxantrone (ProM) for initial cytoreduetivn therapy and interferona as maintenance therapy. The PmM regimen in this study consisted in prednimustine 100 mg/m2/day orally days 1-5 and mitoxantrone 8 mg/rn2/day i.v. days t and 2 and was repeated every 4-6 weeks to a maximum of 6 cycles. Patients achieving a complete or partial remission (PR or CR) received 2 additional courses for consolidation followed by interferon a maintenance until progression or relapse. A total of 19 patients has been treated in this study.Thirteen of the 19 patients (68%) responded with 4 CR and 9 PR. PmM side effects consisted mainly in neutropenia requiring dose-reduction in 48% of treatment cycles. All 13 responding patients subsequently received IFN a 2 b maintenance treatment. In comparison to unmaintained first remission preceding the PmM/IFN trial a tendency towards a longer period of freedom from progression was apparent in the 13 patients receiving IFN maintenance treatment during their second PR or CR. These data provided the basis for a currently ongoing multicenter study randomly comparing initial chemotherapy by PmM vs. COP in patients with advanced CBCC and CC-non-Hodgkin's lymphomas followed by a second randomization in CR and PR patients for maintenance with IFN t~ versus observation, only. The PmM regimen is applied as indicated above. The COP regimen consists in cyclophosphamide 400 mg/m2/day Lv. days 1-5, vincristine 1,4 mg/m 2 (max. 2 rag) i.v. day 1 and prednisolone 100 mglm2/dayorally days 1-5. At the present time 203 patients have been enrolled into this ongoing study, 110 of whom are currently evaluable for response. CR was achieved in 42 cases (38%) while an additional 50 cases (45%) obtained a PR. The most important side effect for both regimens was neutropenia of less than 1000 granulocytes. This was observed for the PmM regimen after 123 of 350 treatment cycles and for the COP regimen after 101 of 280 treatment cycles. So there was no significant difference in neutropania comparing the two treatments. Further accrural of patients and longer observation times are needed to confirm these promising results and to disclose the comparative analysis of PmM vs COP and IFN maintenance versus observation, only.

Reed E.C., Armitage J.O. The University of Nebraska Medical Center, Omaha, Nebraska, USA. The use of GM-CSF following high-dose chemotherapy with autologous bone marrow transplantation for lymphoidmalignancies has now become standard practice for many transplant centers in the USA: In the original placebo controlled trial, the median days to 500 absolute granulocytes (ANC) was to day +26 in the placebo group, compared to day + 19 in the GM-CSF treated group (p<0.001). The median number of days to hospital discharge was also decreased from day +33 to day +27 in the treated GM-CSF given by 24-hour continuous infusion in this patient population. The median days to reach an ANC of 500 when GM-CSF is given by continuous infusion is day +11. The median day to hospital discharge is also reduced to day +18 in this study. However, 17/36 (47%) of the patients experienced pleural effusions when the GM-CSF was administered in this manner. This compares to a 5% pleurai effusion rate in the previous placebo arm, and a 6% pleurai effusion rate when the GM-CSF was given by a 2-hour infusion. In the continuous infusion GM-CSF patients, 5/36 (14%) had the GM-CSF stopped for symptomatic pleural effusions. Future studies may need to look at alternate dosing schedules or combination hematopdietic growth factor regimens.

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LONG-TERM RESULTS ON MITOXANTRONE TREATMENT IN PATIENTS WITH LOW-GRADE NON-HODGKIN'S LYMPHOMAS. Hansen S.W., Nissen N.L Dept. of Internal Medicine, F.A.C. Hillerod, Helsevej 2, DIC Twenty-one consecutive previouslyuntreated patients with low-grade non-Hodgkin's lymphoma were entered a phase II trial of mitoxantrone (5mg/m2 for 3 days every 3 weeks for 8 to 11 cycles) between 1985 and 1986. Seven patients had small lymphocytic lymphomas, 10 had follicular small cleaved cell lymphomas and 4 had follicular mixed small- and large-cell lymphomas.All patients obtained a response, 9 complete remission and 12 patial remission. Toxicity, both hematologic and nonhematologic; was modest. The fullow-up period is 5 to 6+ years and no long-terra slde-effects have been observed. One patient withdrew from the study, 15 patients relapsed and 5 patients remain in unmaintained complete remission. All 5 patients in complete remission had follicular small cleaved cell lymphomas, and the duration of remission is median 68+ months (range 65+ - 73+ months). Seven relapsed patients have died, 5 small lymphocyticlymphomas, 1 follicular small cleaved cell lymphoma, and 1 follicular small- and large-cell lymphoma. Follow-up studies of left ventricular ejection fraction using multiple-gated acquisition scintigraphy showed no significant decrease in cardiac function.

MITOXANTRONE, ETOPOSIDE AND CYTARABIN IN THE TREATMENT OF ACUTE MYELOCYTIC LEUKEMIA (AML) BjOrkholm M, for the Leukemia Group of Middle Sweden. Department of Medicine, Division of Hematology and Immunology, Karolinska Hospital, Stockholm, Sweden. In a phase I/II study 26 pts (median age 53 yrs;r16-77) with refractory, relapsing or untreated AML were given mitoxantrone (12mg/m2 dl-3), etoposide (100mg/m2 dl-5) and cytarabin (lg,/m2 q 12h dl-5) (MEA). Fourteen patients (54%) reached a CR; 5/10 refractory pts, 5/6 previously untreated pts and 4/10 relapsing pts. Median duration of CR was 10 months. In addition 27 elderly AML patients (median age 73 yrs;r60-84) received an attenuated MEA (all drugs for 3d and etopside 200mg/m2/d). Ten pts (37%) achieved CR; 6111 previously untreated pts with de novo AML, 2/8 refractory pts, 1 with a late and 1/3 with an early relapse and 0/4 with secondary AML. Median CR duration of untreated pts was 10 months. The median recovery time to a granulocyte count >0.5x109/1 was 23 and 25 days in the two groups, respectively. Mild to moderate stomatitis/mucositis was the most frequent nonhematological toxicity documented in the majority of patients. Encouraged by these results a 4d MEA-variant (etoposide 100mg/m2/d) was compared in a randomized phase l~/study to a combination chemotherapy schedule (POCAL-DNA) including cytarabin, thioguanine, vincristine, prednisolone and doxorubicin-calf-thymus-DNA. This protocol was previously shown to be associated with an improved survival and less cardiac abnormalities as compared to the free doxorubicin containing regimen (POCAL; Leukemia and Lymphoma 1991;3:355). In total 42 patients (median age 45 yrs;r16-60) were randomized to MEA and 44 to POCAL-DNA (median age 45 yrs;r18-59). In the interim analysis 35 (83%) and 20 (45%) pts entered CR in the two treatment arms, respectively (p<0,001). With rescue therapy the corresponding figures were 85 and 64%. OveraU survival was significantly better in MEA treated pts (p<0.005) but duration of CR did not differ between the two groups. The study was closed and preliminary data indicate that different DNA preparations may have different binding capacity, and that the DNA conjugate used in the recent study was less stable than the previous one. In conclusion, the MEA regimen has a high antileukemic activity and a moderate toxicity. A randomized study comparing MEA !-GM-CSF in AML patients >60 yrs has been initiated.

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Mitoxantrone Pharmaeokinetics E.Schleyer, A.Kamischke, F.Schneider and W.Hiddemann, Dept. Int. Med. Univ. of Mtinster, F.R.G. In spite of its broad clinical application in the treatment of malignant disorders the pharmacokinetics of Mitoxantrone are still not fully understood and warrant further investigations. In addition, limited information is available about interindividuell differences in the plasma A U C and renal elimination of Mitoxantrone and its main metabolites mono- and dicarboxyacid. In the present study the plasma concentrations of the drug and its metabolites were measured by HPLC in 18 patients with acute myeloid leukemia undergoing S-HAM therapy up to 120 hours after the end of a 10mg/m 2 30 rain Mitoxantrone infusion. Renal elimination of Mitoxantrone and its metabolites was analysed in 8 patients and was fulthermore determinated in 5 cases with chronic lymphocytic leukemia receiving 5mg/m 2 Mitoxantrone weekly in three consecutive weeks. Fitting the results of the plasma concentration measurements to a three compartment model the following phalmacokinetic parameters were obtained (median + range). Mitoxantrone: ttr2y = 75 h (42 364), A U C = 340 ngxh/ml (80 - 1030), Clearenc%,al in ~ ofdosi, = 7,5% (3,89 - 9,75). Monocarboxyacid: tlj2y = 53h (29 - 107), A U C = 62ngxlVml (29 150), Clearence~= I ~ of,~i, = 2,3% (1,59 - 4,26). Diearboxyacid: tu2y = 33h (18 - 64), A U C = 105 ngxh/ml (43 - 450), ClearenCer=~l i, ~ of do,i~ = 5,5% (4,17 - 14,25). Substantial interindividuell variability in plasma and urine kinetic parameters was observed, which may partly be due to differences in drug metabolism. Hence, patients with a high A U C of Mitoxantrone metabolites had a lower A U C of the mother drug, and vice versa. Linear regression coefficient between Mitoxantrone A U C and the A U C of metabolites was 0,6. In individual patients, however, mitoxantrone and mono- and dicarboxyacid renal elimination rates remained constant at repeated determinations at weekly intervals. These data indicate substantial differences in drug metabolism and elimination between individual patients, which might have potential impact on response and/or toxicity, especially with respect to the non-cylotoxie behavior of the Mitoxantrone metabolites. Further investigations are therefore needed to elucidate the clinical relevance of mitoxantrone pharmacokinetics.

MITOXANTRONE IN THE TREATMENT OF ACUTE MYELOGENOUS AND LYMPHOBLASTIC LEUKEMIA: THE ECOG AND UNIVERSITY OF ROCHESTER EXPERIENCE. Rowe JM, Andersen JW, Mazza J J, Hines JD, Cassileth PA, Bennett JM, Wiemik PH, Oken MM for the Eastern Cooperative Oncology Group. Hematology Unit, University (3t Rochester Medical Center, Rochester, NY, USA.

S17 IMPROVED QUALITY OF REMISSION IN ACUTE LEUKEMIA: EFFECTIVENESS OF HIGH DOSE MITOXANTRONE-ETOPOSIDEAND C"YTARMHNE. Arlin Z.A., Ahmed T., Mittelman A., Puccio C., Cook P., Chun H., Helson L. New York Medical College, Room 250, Manger Pavilion, Valhalla, New York 10595, USA. Traditional induction therapy in acute myelogeuous leukemia (AML) has either required daunorubicin (DNR) or substitutes in combination with cytarabine or in acute lymphublastie leukemia (ALL) vincristine-prednlsone with an anthracycline and/or asparaginase. Recent data has shown that one dose of high-dose mitoxantrone (M) can effectively replace DNR and can be given at more than ten times the dose intensity of DNR without penalty. In the present trial, patients received cytarabine 3 gm/rn2 over 3 hours once daily x 5 with M 80 m g / m2 x 1 and etoposida 50-150 m g / m2 on days 1, 3, and 5. Thirteen of 18 patients with AML achieved a complete remission (CR), as did 4 of 6 with relapsed ALL, 4 of 5 with blastic phase of chornic myelogenoas leukemia, and 1 of 2 with biphcnotypic leukemia. Nine of 14 patients at etoposida 100 m g / m2 or less responded, as did 3 of 3 at 125 m g / m2 and 10 of 14 at 150 m g / m2. Although stomatitis is common, the toxicity does not preclude exploration of even higher doses. It is hoped that these higher doses may be curative. If not, it is possible that post-inductlon therapy that fialed after traditional remission induction therapy may now be curative. Growth factors not used in this regimen may ultimately provide the final thrust.

102 Patients entered an ECOG trial evaluating the role ot mitoxantrone, 12 mg/m 2 for 5 days, and VP-16, 100 mg/rn 2 for 5 days, in AML patients in a) first relapse, b) second relapse, c) refractory to initial induction or reinduction therapy. The overall response rate was 37% with a CR rate of 51% in the 35 evaluable patients in first CR; 18% in the 34 evaluable refractory patients and 29% in the 24 evaluable patients in second CR. Among the patients treated in first relapse, th6 best response was seen in those who had a durable first remission (>1 6 months), who had a CR rate of 68%, compared with those who relapsed after a short first remission (<6 months) who only had a CR rate of 23%. It appears that mitoxantrone and VP-16 is an effective combination for relapsed and refractory AML - especially for first relapse patients following a durable first remission, in whom the response rate was comparable to de novo AML. An ECOG pilot study is presently evaluating the combination of mitoxantrone, 6 mg/m 2 for 6 days, cytosine arabinoside, 1 gm/m 2 for 6 days, and VP-16, 80 mg/m 2 for 6 days, for truly refractory AML patients. For patients with relapsed and refractory ALL, a pilot study was undertaken at the University of Rochester to evaluate the combination of mitoxantrone, 10 mg/m 2 for 6 days, cytosine arabinoside, 3 gm/m 2 q12h for 4 days, and VP-16, 300 mg/m 2 for 3 days. Of the 16 patients who entered the study, 4 achieved CR; 4 achieved PR, and there were 8 nonresponders. Because of the refractory nature of the majority of these patients, this response rate suggests significant activity for this combination in ALL, and this is presently being studied in a groupwide ECOG study.

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Main Sessions 21

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SOMATIC ORIGINS OF HUMAN T CELL ACUTE LEUKAEMIA T.H. Rabbitts, T, Boehm, M. Kennedy, N. Dear, V, Valge-Areher, P. Fisch, L. Foroni, P.D. Sherrington and A. Warren Abstract not submitted

The development of lymphoid turnouts is frequently associated with chromosome abnormalities which result in the activation of oncogenes. A general pattern has emerged of transcription factors activated by chromosome translocations in T.ALL Our studies are focussed on the role of two families of gene products in T-ALL The rhombodn family of putative oncogenes in T-ALL A common chromosome translocation in childhood T-ALL is t(11; 14)(p13;ql 1), and a more rare one is t(11;14)(p15;ql 1). Both of these translocations are caused by abnormal rearrangement of the TCRD/A locus at 144:111. Variant forms of the former exist (tT;1 l)(q35;p13) involving the TCRB locus at 7q35. Two related genes are found at the 1 lp junctions, and these encode cysteine-rich proteins (LIM domain proteins). The genes have been called rhombotin 1 (1 lp 15) and rhom-2 (1 lp13). A third member of the family has ben cloned and located to 12p12-13. The structure of the rhombotin proteins and their possible role in transcription will be discussed. HOXI1. a new homeobox gene involved in T-ALL The HOXll gene has been isolated from junctions of t(10;14)(q24;ql 1) and t(7;10)(q35;q24) translocations via association with TCRD (14ql 1) and TCRB (7q35). The gene encodes a protein with a homeobox domain. The gene seems to be ectopically activated by the chromosome transloeation in childhood T-ALL with such translocations. The studies at present are designed to understand the role of this DNA-binding transcription factor in leukaemia. Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England

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Abstract not submitted

Two Subtypes of Therapy-Related Leukemia J. Pedersen-Bjergsard, S.O. Larsen, P. Phillip, G. Daugaard, T.C. Sigsgaard, M. Andersson, J. Ersboll and L. Specht. Rigshospitalet, Copenhagen, Denmark. The risk of therapy-related myelodysplasia (t-MDS) and acute myeloid leukemia (t-AML) and the chromosome aberrations observed were compared for 7 well-defined cohorts of patients treated with chemotherapy and in whom s total of 61 eases of t-MDS or t-AML were observed. In patients with Hmdgkin's disease, non-Hodgkin's lymphomas and ovarian cancer treated with alkylating agents the cumulative risk increased by approximately I% per year form 2 to 8 years after start of therapy. The leukemias most often presented with t-MDS, were of FAB subtypes H1 or H2 and showed loss of whole chromosomes no 5 or no 7 various parts of their long arms. In patients with small ceil lung cancer and breast cancer treated eith a combination of drugs targeting at DNA-topoisomerase II such as etoposide, doxorubiein, 4-epi-doxorubiein or mitoxantrone and a direct-acting genotoxic drug such as eisplatin, cyclophosphamide, lomustine or prednimustine the cumulative risk of t-HDS and t-AHL increased much more steeply by 3-4% per year from only I year after start of chemotherapy. In patients with germ cell tumors treated with etoposide plus cisplatin the risk of t-NDS and t-ANL inoreased very markedly by dose. Leukemias following therapi with a combination of drugs targeting at DNA-topoisomerase II and a genotoxic drug were of two types. One aceelerated type presenting as overt t-AHL of FAB subtypes H4 or N5 with balanced chromosome translocations to bands 11q23 and 21q22, and s more delayed type identical to that observed following therapy with alkylating agents alone. Our results indicate that different steps in the multistep transformation to overt leukemia are induced or accelerated by the two types of eytostatic drug, and suggest a synergistic effect in leukemogenesis.

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INCIDENCE, BIOLOGIC FEATURES AND TREATMENT OUTCOME OF MYELOIDANTIGEN-POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA [MY + ALL). W.D. Ludwig, J. Harbott, H. Rieder, C.R. Bartram, R. Becher, B. Heinze, B. V6!kers, W. Gassmann, J, Ritter, C. Fonatsch, H. L6ffler, D, Hoelzer, H. Riehm, E. ThieL Dept. of Hemato!olly/Oncology, Klinikum Steglitz, FU Berlin, FRG. Recent studies on the incidence and prognostic impact of My + ALL reported markedly divergent results in both childhood and adult ALL, We therefore prospectively examined leukemic blasts from 733 children and 506 adults with ALL by detailed immunophenotyping, mainly focusing on the relations of myeloid-antigen (CD13/33/w65) expression to other blast-cell biologic features (karyotype, immunogenotype) and the treatment outcome of My + ALL. In 46 of 639 children (7%) and 74 of 381 (19%) adults with B-cell precursor ALL, > 20% of blast cells disclosed expression of at least one myeloid antigen. An immature (CD10-) B-cell precursor phenotype was significantly more often associated with expression of myeloid antigens (approx. 50% of patients) than a common or pre-B phenotype (5 - 20%). M y + ALL was diagnosed in 4 of 94 children (4%) and 21 of 125 adults (17%) with T-lineage ALL, and most adults with My + ALL (15/21, 71%) disclosed an immature pre-T (cyCO3/CD7 +, CD5 +/-, CD1/CD2/sCD3-) phenotype. Lymphoid lineage commitment of My + blast cells was supported in most patients by demonstration of rearranged immunoglobulin heavy-chain genes in B-cell precursor ALL or of both T-cell receptor P~and T genes in T-lineage ALL. Cytogenetic studies successfully carried out in approx. 60% of M y + ALL patients revealed a subset in both children and adults with an immature, usually CDwB5-positive, B-cell precursor phenotype (CD19 +, CD24/cylgM -/+, CD10-), rearrangements involving band 11 q23 It(4; 11 ) n = 15; t(1 ;11 ) n = 1], adverse clinical features (e.g., hyperleukocytosis) and a poor treatment outcome. The Ph chromosome was detected in only 2 patients with a M y + (CD13/33) common ALL. Other structural chromosomal abnormalities detected were t(1 ; 1ll), t(9; 12), t(1 O; 14), t(10; 11 ) (n = 4), numerical abnormalities (n = 10), and nonrandom abnormalities of 6q, 9p, and 12p (n=6). Eleven children and 7 adults with M y + ALL had normal karyotypes. The vast majority of children with ALL (My+ ALL: 98% vs. My- ALL: 99%) treated according to risk-adapted ALL-BFM 86 protocols achieved complete remission, and life-table analyses did not reveal any significant differences in event-free survival between My- (0.73) and My + (0.70) ALL patients. The treatment outcome of My + adult ALL patients entering BMFT ALL/AUL 03/87 or 04/89 protocols is now being assessed in the context of their immunophenotypic and karyotypic features.

ALL TRANS RETINOICACID (ATRA) IN ACUTE PROMYELOCYTICLEUKEMIA(APL): TARGETING DRUG FOR DIFFERENTIATION THERAPY. L. Degos* In previous reports we showed that ATRA is able to induce an in vitro and in vivo differentiation of abnormal promyelocytes into mature cells in APL. Rapid increase of leucocyte count is the major side effect, mainly occuring in newly diagnosed patients. A pilot phase was conducted in 27 newly diagnosed patients receiving 45 m g / m 2 / d a y of ATRA until CR achievement, followed by 3 courses of c h e m o t h e r a p y (DNR+ARA-C). In o r d e r to avoid side effects of hyperieucocytosis, chemotherapy was started early if leucocytes were over 5 x l 0 9 / L by day 5, 10 xl09/L by day 10 or 15 xl09/L by day 15. Twenty six achieved a CR (1 resistance), 15 with ATRA alone, 11 after addition of DNR ARA-C. An APL 91 protocol randomizing this pilot arm and chemotherapy alone in newly diagnosed APL is on going. Monitoring of patients includes in vitro culture correlated with the sensitivity to ATRA, phamarokinetics showing a lower Cp max in hyperieucocytic patients and in those with high clearance, RT-FCR for accurate diagnosis and minima! residual disease assessmenL and search for cytoplasmic retinoie acid binding protein (CRABF) possibly in relation with the resistance to ATRA therapy. We have previously reported the rearrangement of retinoic acid receptor (RAR) alpha due to the t(15;17) translocation and the presence of a hybrid mRNA made by the fusion of RARcx and PLM gene. The fused product alters gene transactivations and blocks the cell differentiation program explaining the leukemogenesis. High concentration of retinoic acid (RA) can overpass the impairment of gene transactivation and n o w w e present that RA can induce an over expression of the normal a11ele. Thus the abnormal program made b y the aberrant transcript might be overcome by RA restoring functional activity of abnormal RAR, a n d / o r high level of normal PAR. We can speculate to extend differentiation therapy to other malignancies with aberrant transcript, searching for drugs which induce an overexpression of the normal allele, or a normal activity to the aberrant product, Hematology Department - Hopital Saint Louis 75010 PARIS, FRANCE

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AUTOLOGOUS MARROW TRANSPLANTATION USING CULTURED CELLS. Dexter T.M. Cancer Research Campaign Department of Experimental Haematology, Paterson Institute for Cancer Research. Christie Hospital NHS Trust, Wiimslow Road, Withington, Manchester, UK. Haemopoiesis in rive occurs in intimate association withstromal ceils that provide the cellular microenvironments that promote self-renewal and differentiation of stem coils as well as development of committed haemopoietic progcultor cells. The precise nature of these "microenviromncnts" remains an enigma, but clearly comprise a malticomponent regulatorysystem of cell-to-cell interactions mediated by specific adhesion molecules, extracellular matrix molecules and a variety of membrance bound and soluble cytokines. When removed from the hone marrow, and cultured in the absence of added growth factors, haemopoietic cells die. Moreover, leukaemic cells from a vast majority of patients also die in the absence of added growth factors, indicating that growth of these cells is not truly independent of physiological growth regulatory factors and that other, more subtle, mechanisms are responsible for aberrant growth of these cells. Several years ago, it was shown that when long-term bone marrow cultures are established from patients with acute myeloid or lymphoid leukaemia (at presentation), in a majority of cases there was preferential expression of the (residual) normal stem cells and a "disappearance" of lcukaemia cells. The mechanism underlying this phenomenon is still unclear, but is presumably related to deficiency in the ability of the leukaemic cells to interact with the bone marrow stroma that is necessary for maintaining the normal haemopoietic cells. We have exploited this difference to "pure" marrow of leukaemic cells prior to performing autologous marrow transplantation. To date, 19 patients have been transplanted and relapsed free survival is about 80%. Our studies with cultured cells will be compared with conveational aliogeneic marrow transplantation - performed during the same period at our centre, and recent results using cultured cells from patients with CML will also be reported.

M O D U L A T I O N OF A R A B I N O S Y L C Y T O S I N E (ARA-C) FOR ACUTE MYELOGENOUS LEUKEMIA (AML). V. Gandhi, E. Estey, M. Keating, and W. Plunkett

THERAPY

OF

The design of a new protocol formulated to test the biochemical m o d u l a t i o n of ara-C m e t a b o l i s m in patients with r e l a p s e d AML was based on two rationales: first, the clinical response to ara-C therapy for this group of patients is strongly correlated with the area under the accumulation and retention curve (AUC) of its active m e t a b o l i t e ara-CTP in circulating blasts; second, the A U C of ara-CTP can be augmented if fludarabine is infused before ara-C. Patients (n=10) received either a 1 g/m2 or a 3 g/m2 dose of ara-C for 2 or 6 hr, followed at 20 hr by a 30 mg/m2 dose of fludsrabine for 30 min. At 24 hr, another identical dose of ara-C was infused. Comparison of ara-CTP p h a r m a c o k i n e t i c s in circulating AML cells demonstrated that the AUC of ara-CTP increased by a m e d i a n of 1.9-fold after fludarabine infusion. Plasma pharmacokinetics indicated that neither the m e d i a n ara-C concentrations nor the levels of its deamination product arabinosyluracil were significantly affected by fludarabine infusion. The m e d i a n rate of ara-CTP elimination was not significantly altered after fludarabine treatment (t89 = 2.6 hr compared with 3.2 hr), which suggests that a decrease in catabolism of ara-CTP was not responsible for the increased AUC for ara-CTP. The rate of ara-CTP accumulation by AML cells, however, was increased by a m e d i a n of 1.9-fold after fludarabine; the peak o c c u r r e d w i t h i n 4 hr after start of the infusion. Complete remissions (CR) occurred in 5/24 patients who had ist CRs of 1 yr was higher (12/17) than the rate in similar patients treated with high dose ara-C 17/16) who had comparable cytogenetics. These data demonstrate that fludarabine m o d u l a t e s ara-C m e t a b o l i s m and this r e g i m e n is effective in patients with 1st CRs >1 yr. Department of Medical Oncology, The University of Texas M.D. A n d e r s o n Cancer Center, Houston, TX 77030, U.S.A.

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MULTIPLE COURSE CHEMOTHERAPY AND GM-CSF FROM DAY MINUS ONE UNTIL NEUTROPHIL RECOVERY VERSUS CHEMOTHERAPY ALONE IN NEWLY DIAGNOSED AML Th. Biichner, W. Hiddemann, B. W6rmann, R. Rottmann, M. Ziihlsdorf, G. Maschmeyer, W. Heit, W.-D. Ludwig, E. Thiel, M.-C. Sanefland

Deisseroth A., M.D. Anderson Cancer Center, Dept. of Hematology, 1515 Holcombe Boulevard, Houston, TX 77030,.USA. Non-selective toxicity of che~aotherapy on normal tissues has limited the doses and effectiveness of treatment for leukemia and cancer. Molecular biology is being used to characterize molecular defects in the neoplastic cells which will permit more directed and specific therapy based on established modalities of treatment such as chemotherapy. Genetic modification of hematopoietie stem cells will also be used as an adjunct to established modalities of therapy. The progress in these areas will be reviewed and future directions identified.

In our 1st study starting in 1987 we could demonstrate that GM-CSF following chemotherapy in high risk AML can safely be administered and significantly reduces neutrophil recovery time and early mortality (Blood 78: 1190, 1991). The results allowed us to do the step toward standard risk AML, priming and longterm as well as multiple course administration of GM-CSF. Patients with newly diagnosed AML received GM-CSF 250 ug/m2/d starting on day minus one of chemotherapy and continuing until neutrophil recovery in each of both induction courses, the consolidation course and the two first maintenance courses. Chemotherapy in this sequence of courses was TAD, HAM, TAD, AD and AT (Sere. Hematol. 28 Suppl. 4: 76, 1991). Patients were randomized to receive the daily dose of GM-CSF either as contin i.v. infus or one or two s.c. inject or to receive chemotherapy alone. We here report on the outcome in the entire 28 GM-CSF and 15 control patients treated so far. Both groups were comparable in potential risk factors including age with equally 31% patients of 60+ years. Complete remissions were 20/28 (71%) in the GM-CSF group and 15/15 in the controls. However, relapses during the 1st 15 months occured in 4/20 GM-CSF patients but 8/15 controls (p = 0.002). During the initial days of treatment blood blasts increased by a median factor of 1.7 and up to 14.0 in 81% of GM-CSF patients and only 1.1 in 18% of controls. Neutrophil recovery time was reduced after GM-CSF (p = 0.03) in the 1st induction course and identical to the controls in the later courses. Non-hematological toxicity was acceptable. We conclude that the preliminary data allow to continue present study. Further updates will show whether GM-CSF in this setting produces higher quality complete remissions in AML. *Present address: Department of Medicine/I-Iematology, University of Miinster, Germany

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Abstract not submitted

Abstract not submitted

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CYTOGENETIC FINDINGS IN ADULT ACUTE LYMPHOBLAST1C LEUKEMIA: RESULTS OF THE GERMAN MULTICENTER ALL/ AUL STUDY C. F o n a t s c h , and H. Rieder In this study~ the high incidence of t r a n s l o c a t i o n s t(9;22) and t ( # ; l l ) in adult ALL comprising 26 % o5 the c a s e s i n v e s t i g a t e d has been c o n f i r m e d . The possible p r o g n o s t i c and biologic signif i c a n c e of t h e s e p r i m a r y and of additional s e c o n d a r y c h r o m o s o m e a b e r r a t i o n s will be discussed. The role of less f r e q u e n t l y o c c u r ring k a r y o t y p e c h a n g e s , as, e.g., n e a r - t r i p l o i d y , n e a r - t e t r a p i o i d y , p a r t i a l t r i s o m y lq, and c h r o m o s o m e 7 a b n o r m a l i t i e s in c o r r e lation to clinical f e a t u r e s and to immunological m a r k e r s will be of i n t e r e s t . As a reason for the d i f f e r e n t t h e r a p y results in childhood and in adult ALL, the varying incidences of specific c h r o m o s o m e a b n o r m a l i t i e s , a s s o c i a t e d with a bad prognosis ( P h - t r a n s l o c a t i o n ) or with a f a v o r a b l e prognosis (hyperdiploidy 50) c a n be assumed.

Abstract not submitted

Arbeitsgruppe Tumorcytogenetik, Institut f(Jr Humangenetik~ Medizinische Universit~it zu LCJbeck, R a t z e b u r g e r Allee 160, D-2#00 LLibeck FAG 34

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CANCER AND LEUKEMIA GROUP B STUDIES IN ACUTE LYMPHOBLASTIC LEUKEMIA (ALL). Larson R.A., Schiffer C.A., Bloomfield C.D. for the CALGB, Lebanon NH, 03766, USA. The CALGB has conducted a series of clinical trials in adult ALL together with centralized immunophenotyping, molecular genetics, morphology and cytogenetics studies. Protocol 8011 showed no advantage to postremission intensification with 2 courses of ara-C and daunorubicin (dnr) (J Clin Onc 1991 ;9: 2002), Protocol 8513 demonstrated equivalence between dnr and mitoxantrone for remission induction with vincristine (vcr) and prednisone (pred) (Leukemia 1991 ;5:425). In CALGB 8811 a 5-drug regimen useful in high-risk childhood ALL was tested in 214 adults. Induction consisted of cyclophosphamide (cy 1200 mg/m 2 Day 1), dnr (45 mg/m 2 Days 1,2,3), vcr (2 mg Days 1,8, 15,22), pred (60 mg/m 2 Days 1-21), and I-asparaginase (6000 U/m 2 Days 5,8,11,15, 18,22). Doses of cy (800 mg/m2), dnr (30 rag/m2), and pred (7 days) had to be reduced for patients (pts) > 60 years because of early deaths. Treatment lasts 24 mos with a modified BFM-like consolidation and maintenance and CNS prophylaxis with 2400 cGy brain RT plus ITmethotrexate. 202 patients (ages, 16-82; median, 32 yrs) were eligible and evaluable. With 18 mos median follow-up, 82% of pts have had a complete remission (CR); 72% of CRs remain disease-free. For pts < 3 0 yrs, 92% had a CR with 2% induction deaths. For pts 30-59 yrs, 85% had a CR with 6% induction deaths. Nine pts died in CR and 16 pts withdrew from protocol treatment. Estimated 2-year survival is 90% for < 30 yrs end 65% for 30-59 yrs (Iogrank, p =.05). Estimated 2-year survival is 94% for T-ALL and 62% for B-lineage ALL (p =.03). The presence of myeloid antigens has not markedly influenced CR duration or survival. Prospective molecular analyses have demonstrated BCR-ABL in 31% of patients (Proc ASCO 1991;10:222). Pts with t(9;22) had 81% CR but median duration was < 1 year. Pts with high-risk ALL (t(9;22),t(4; 11 ),t(8;14)) with HLA-matched sibling donors will now be transplanted by the CALGB in first CR. Currently, protocol 9111 tests G-CSF in a double-blind trial to decrease the induction toxicity of 8811 chemotherapy. This intensive multi-agent regimen has produced markedly improved response rates and survival over previous CALGB studies in adult ALL.

THERAPY OF ADULT ACUTE LI'MPEOBLASTIC LEUKEMIA (ALL). P R E L I M I N A R Y R E S U L T S O F T H E G I H E M A ALL/0288 STUDY. S. Amadori,L. Annino,A. Ferrari,M.L. Vegna,A. Camera,S. Ciolli~W. Deplano~F. Fabiano,F. Ferrara,S. Ladogana,G. Muti,A. Peta,A. Recohia,S. Sica,R. Stasi,A. Tabilio,G. Visani,M. Baccarani,and F. Mandelli for the GIMEMA C o o p e r a t i v e Group, Italy. The G I M E M A ALL/0288, a nationwide Italian trial for p a t i e n t s w i t h ALL a g e d 12 to 60 years (B-ALL a n d C N S + cases excluded) s t a r t e d in J a n u a r y 1988 and is still open. M a i n o b j e c t i v e s of the s t u d y w e r e to: a) e v a l u a t e the i m p a c t of a 7-day pretreatment with Prednisone on d i s e a s e outcome; bl d e t e r m i n e the r o l e of C y o l o p h o s p h a m i d e as an a d j u n c t to the 4-drug phase i induction therapy (VCR~ Pred, DAU, ASP); c) assess in a randomized m a n n e r the e f f i c a c y of an i n t e n s i v e program of consolidation therapy with 5 drug blocks administered sequentially for t h r e e cycles. CNS p r o p h y l a x i s c o n s i s t e d of p e r i o d i c i n t r a t h e c a l MTX a s s o c i a t e d w i t h the u s e of s y s t e m i c intermediate doses of the drug. 448 p a t i e n t s h a v e b e e n e n r o l l e d as of S e p t e m b e r 1991. T h e c o m p l e t e r e m i s s i o n (CR) r a t e is 84% overall, and is n e g a t i v e l y influenced by a g e greater than 50 years (63%, P< 0.001). N e i t h e r the r e s p o n s e to P r e d p r e t r e a t m e n t (defined as < 1.000 blast cells/mmc on d a y 0) nor the a d d i t i o n of Cyclo during initial induction are significantly correlated with a c h i e v e m e n t of CR. P r e s e n t l y the p r o j e c t e d e v e n t - f r e e s u r v i v a l (EFS) for all p a t i e n t s e n t e r e d in trial is 31~ (SE 4~) at 3 years. The estimated disease-free survival (DFS) for all p a t i e n t s w h o a c h i e v e d CR is 34~ (SE 5%) at 3 years, with Pred-responders doing significantly better than non-responders (3-yr DFS 40% vs 24%, P= 0.01).

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Poster Session ALL in Children 36

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RESULTS WITH RANDOMIZED BFM ADOPTED STUDIES FOR ALL THERAPY IN CHILDHOOD IN EAST GERMAN COUNTRIES F. Z i n t l , H. Malke, H. Reimann, M. Oomula, W. D O r f f e l , G. Eggers, P. Exadaktylos, E. H i l g e n f e l d , W. KoCte, I . Krause, W. Kunez~, U. H i t t l e r , D. MSbius, H. Reddem~nn, G. N e i mann, G. weiabach A t o t a l of 524 c h i l d r e n , without p r i o r treatment, were entered i n t o s m u l t i c e n t c i c c o n t r o l l e d randomized study (modif i e d BEM protocol) between September 1981 and December 1987. P a t i e n t s were divided i n t o t h r e e r i s k groups according the BFH r i s k f a c t o r score. For the duration of maintenance t h e r a py, p a t i e n t s were randomized a f t e r 78 weeks to r e c e i v e NIX and 6-MP f o r another 6 months or to r e c e i v e s l a t e i n t e n stfication.

DOSE INTENSITY AND PROGNOSIS IN ACUTE LYMPHOCYTIC LEUKEMIA (ALL) OF CHILDHOOD G.E. Janka, O. Gobrecht, and S. Gross for the COALL Study Group

STUDY V I I / 8 1

SR - 64~&

MR - 28~

HR -

9%

(1981-1987)

e ~ n ~ n NO. OF PAT.: 524 333 IOQ 146 100 45 100 PATIENTS, TOTAL COMPLETE REMISS. 322 97 139 95 42 93 97 29 48 32 22 49 RELAPSES 201 60 85 58 12 27 IN IST REMISS. LIFE-TAB. OES~SDl 0 . 6 0 [ 0 . 0 3 1 0.59[0.04• 0.37[0.081 ======================================================== 18 months maintenance therapy plus l a t e i n t e n s i f i c a t i o n i s as e f f e c t i v e as 24 months maintenance therapy. From 1988 to 1991 254 c h i l d r e n were t r e a t e d according to a new modified BFM p r o t o c o l . MTX dosage was reduced from 5 g/m z t o 1 g/m =. STUDY V I I I / 8 7 SRG - 26~ RG - 65P; EG - 9%

(1988-1991)

Intensive therapy up front has led to high cure rates in childhood ALL. Within a given protocol the individual dose intensity (DI) may vary considerab ~ ly and thus be an important prognostic factor. In a cooperative treatment study for childhood ALL 305 pts. were entered and in 217 DI was evaluated: for intensive therapy cumulative doses of drugs/time, time to complete the protocol, number of episodes with a white blood count (WBC) below I/nl, duration of leukopenia; for maintenance phase cumulative doses of 6MP a n d M T X / t i m e and mean WBC. Other prognostic factors were also analyzed. In the protocol for high-risk pts. consisting of 6 drug combinations with 12 different agents no influence of DI during intensive phase therapy could be found. The day 28 bone m a r r o w remission status was the only significant prognostic factor. In contrast in low-risk pts. treated on a less intensive short protocol a higher number of leukopenic episodes and longer exposure time to MTX were significant prognostic factors. For maintenance treatment in both risk groups neither cumulative drug doses/time nor mean WBC had an influence on prognosis. We conclude that individual DI is especially important in less intensive short protocols whereas in protocols with multiple intensive drug combinations there may be a certain margin of safety in DI without disadvantage to the pt. Although in this investigation the influence of DI on prognosis could not be shown for maintenance therapy other studies confirm the important role of this treatment phase for childhood ALL.

NO. OF PAT. : 254 n ~ n P; n PATIENTS 67 100 163 100 24 100 COMPLETE REMISS. 66 99 160 98 22 92 RELAPSES, TOTAL 1 1 17 10 5 21 I N IST RENISSIQN 63 94 132 81 11 46 LIFE-TAB. DFS(SD) 0 . 9 4 ( 0 . 0 4 ) 0.77(0.04) 0.53(0.13) SR-Standsrd, MR-Medium, HR-High r i s k , EG-Experiment. group The p r o b a b i l i t y of EFS improved to about 80 P~ a f t e r an observation time of 3 1 / 2 years. Address: U n i v . - K i n d e x ~ l i n i k , KochstraOe 2, 0 - 6900 Jena

Department of Hematology and Oncology, Children's Hospital, University of Hamburg Martinistrasse 52, 2000 Hamburg 20, FRG

37

39

T H E A L L - V I P R O T O C O L F O R N O N HIGH R I S K C H I L D H O O D A C U T E L Y M P H O B L A S T I C L E U K E M I A F R O M THE DUTCH CHILDHOOD LEUKEMIA STUDY GROUP A.J.P. Veerman, K. H~hlen, W.A. Kamps, E.F. van Leeuwen, G.A.M. de Vaan, E.R. van Wering, A. van der Does-van den Berg, G. Solbu, S. Suciu

INTENSIVE CHEMOTHERAPIE FOR ACUTE LEUKEMIAS (AL) IN CHILDHOOD WITH BFM PROTOCOLS: MOSCOW EXPERIENCES 1990-1992. FIRST SUCCESS AND FAILURES. Rumiantsev A.G., Karacbunsky A.I., Kryshanovsky O.I., Maschan A~A., Samochatova E.V., Zestkova N.M., Baidun LV. Scientific Res. Inst. of Pediatric Hematology,l Moscov, Russia. In the past thirty years the treatment results in children with acute leukemia in the GUS were dramatic: The average survival was below 10%. Treatment was not standardized at no adequate supportive care was given. High risk and relapsed patients had no chance of survival. Since 1990, the BFM protocol were used. In the past two years 95 patients with AML and ALL were treated. Twenty-three AML patients were treated according to the BFM-83-protocol. 73.5% went into complete remission, 17.4% were non-responders, 9.1% died of infectious complications. ALL patients were treated according to a modified BFM-90-protocol. 19.4% of 72 patients went into complete remission, no non-responders were observed. Four patients died during induction therapy and 4 during maintenance therapy. Three patients had bone-marrow relapse within the first 2 years. Infectious complications, e.g. hepatitis B, caused prolongation of the treatment period. Further improvements of these results necessitate better organization of hematology/oncology supportive care and prevention of hepatitis B.

In a p o p u l a t i o n - b a s e d study children with non-high risk ALL were uniformly treated according to a national protocol. Eligible were children ~0-15 yr) with ALL, excluding those with WBC 250.i0~/i, with mediastinal mass, with B-cell phenotype, or with CNS involvement. The resulting non-high risk group comprised 70% of all children with ALL. Between Dec. 1984 and July 1988 190 cases were treated with protocol ALL VI: three drug induction (VCR, dexamethasone [DXM], L-asp), three courses of 2 g/m 2 HDMTX, and maintenance during two years (alternating 6MP/MTX 5 weeks ana V C R / D X M 2 weeks). During the first year of maintenance i.t. triple chemotherapy was given every 7 weeks. In a preceding pilot study (52 pt.) prednisone (PRD) was used instead of DXM. The estimated event free survival (EFS) at 4 yrs is 83% (S.E. 3.0%) for the ALL VI study and 67% (S.E. 6.5%) for the pilot study. The proportion of BM relapses (17 out of 184 CRs) and especially the CNS relapses (2 out of 184 CRs) are quite low in ALL VI c o m p a r e d to the pilot study (7 BM and 5 CNS relapses out of 48 CRs). Therefore, DXM seems superior to PRD; with a moderately intensive protocol a large proportion (70%) of all children with ALL can be treated with very good results: 83% EFS at 4 years. Address: Dutch Childhood Leukemia Study Group, Postbox 60604, 2506LP The Hague, The Netherlands

A81 4O

42

IMPROVED PROGNOSIS IN A L L W I T H MODIFIED BFM PROTOCOLS - PRELIMINARY RESULTS FROM TURKEY L.SaNamer, L.Ulukutlu, LYd&z*

SUCCESSFUL

Regarding the considerable progress reported in the treatment of acute lymphoblastic leukemia (ALL) in contrast with the poor prognosis observed with conventional treatment in our department, a prospective clinical study was undertaken from May 1989 through June 1990 with the aim of evaluating the feasibility and success of a more intensive treatment. Twentysix newly diagnosed non-B ALL patients (17 males and 9 females), aged 4 months to 9 years, were treated with a modified ALL-BFM 81 protocol. Remission was attained in 92% of the patients with one early death and one non-response. The median follow-up period is 22 months (range 18-30+ months). The probability of survival at 2 years (Kaplan-Meier analysis) is 0.8l. There were two deaths in remission due to infectious complications and three relapses, two in the bone-marrow and one combined with central nervous system relapse. The probal~illty of event-free survlwd and fdapse-lhac interval at 2 years are 0.74 and 0.85 respectively. Since August 1990, a new study has been started with a modified ALL-BFM 90 protocol in order to improve the results especially in the high-risk patients. Twenty-two patients have entered the study up to now and remission has been achieved in 95%. There has been only one therapy-related toxic death with this aggressive treatment, and even better 10ng-term results can be expected. In conclusion, the overall efficacy of the BFM protocols and their feasibility in a clinic whose members had no experience with such an intensive therapy were confirmed. *Department of Pediatric Hematology-Oncology, Cerrahpa}a Medical School, University of Istanbul, Turkey.

( ~

~I~V(~S S~b-r~z4 I ~ I ~ S

Ws

(AT/,), I ~ ~ ~I~iES (:INS '84, '89) B. Stark, A. A b r ~ o v , D. Attias, A. ~ l i n , Y. }~3rak, Y. ~ s t ~ i n , G. (3%ividalli, I.J. (bhen, E. }~crai, Y. ~bz~Dvitz, A. PL~zani, L. J a ~ r , G. Kende, M. Mandel, Y. N e t ~ , G. Rechavi, N. l ~ u , R. Sharon, D. Sthoeger, H. ~ i , Y. Y6m_iv and R. Z a / z ~ C r a n i a l irridiation has been shown to be an effective treatment of CNS prophylaxis in c~hilci~ ALL. H c ~ r , it is associated with severe nellrc~psychologic~31 sequelae: loss in IQ score, and s e c c ~ ttunors. Ln the INS stn/dy 84, the efficacy of ~ a l t ~ t i v e a[R~r~ch to ~ a l iz~ti~ was studied in a ranclcmiz~ prospe~ive way. I/i ~ st~dard risk g r i p (Non T, WBC<50,000), patients were r~a-~ized between 18 I.T. Triple (~iIK, ARA-C, hydro~rti~Dne) injections only, versus cranial i ~ a t i o n 1800 ~ + 6 I.T. injections. From Nov. 1984 to l~c. 1988 (me~ian follow-up 5 yrs), 75 patients c~/t of 141 ~ inclt~ded in the randomization. CINS relapses occurred in 2 ~ t of 36 non-irradiated and 3 c~t of 39 i z - r ~ t ~ ~tients. similarly no differences in the Leukeaia-Free-Survival was observed in the 2 groups, 73% %~ 62% for 5 y ~ r s r ~ ti%~iy. Y_n the f o l l ~ g r~tior~l study ~ s ~ d cm AI~IK~M-86 protocol i n i t i a t ~ in Jan 1989, an e ~ e _ ~ s t ~ m r ~ risk group (Non-T, WBC <100,000) is treat~ ~rith ~Lic~nd ~ s of I.V. MX"X (20 g~n/m2) with %~P-16 (600 rag/m2) s~d 18 I.T. triple with~t cT~h~ial i r ~ a t i o n . 70 ~tients exlrolled in this group with a median f o l l ~ - u p of I .3 year. No (~qS relapses occurred so far and 5 children relapsed. The results obtained without cranial iz-zadiati~ in s t ~ and in the extended standard groups, a ~ &s g ~ %rith irr~liation snd t/~efoz~ jttstify ~x)ntinuati~ of this [motocol. *Present address : l~pt. of l ~ a t r i c H ~ t o l ~ / ( ~ c o l ( ~ , I~ilinson ~dic~l C~t~r, l~tmh Tiqva 49100, IsZael.

41

43

LONGTERM FOLLOWUP OF A PILOT STUDY OF INTERMITTENT INTENSIVE CONTINUATION THERAPY (HL-4 PROTOCOL) FOR CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA. Ueda K., Kobayashi M., Hamamoto K., Tanaka Y., Wakiguchi H.,Imai T., Department of Pediatrics,Hiroshima University School of Medicine, and Chugoku-Shikokn D i s t r i c t Leukemia Study Group, Hiroshima, Japan. The purpose of this study was to improve the relapse-free s u r v i v a l o f childhood acute lymphoblastic leukemia as close as possible up to 100%, particularly of standard risk cases. Cases were s t r a t i f i e d but not randamized. Standard risk(SR) cases were defined as total risk score less than 1 (as 2 score was given for i n i t i a l white cell count more than 25,000/zl, 1 for age less than 2 or above lO, 1 for male sex, 1 for massive splenomegaly, and 2 for delayed induction. Those with total score more than 2 were defined as high risk(HR) cases. The induction therapy for SR cases consisted of conventional 3 agents(VCR, PRD, & L-asp.). The consolidation and sanctuary phase consisted of 1 course of high dose MTX(lg) IV drip over 24 hrs. combined with intrathecal MTX and hydrocortisone, followed by cranial irradiation(18Gy). SR cases age less than 6 were not irradiated but 4 courses of high dose MTX were given. For HR cases, 2 doses of adriamycin (ADR) was added, 3 courses of high dose MTX, and a course of cyclophosphamide(CY)-VCR-PRD was given before the 24 Gy irradiation. Continuation therapy consisted of cyclic repetition of 4 intensive intermittent courses for 3 years: (~)ADR-VCRPRD-6MP,(g) VCR-MTX-EMP,@CY-VCR-PRD-6MP,(~)intermediate dose MTX(5OOmg/m2) over 5 hours. Relapse free survival of SR cases(n:50) was 73.0 %(46-124 months follow-up), and that of HR(n=39) was 64.1%(50110 mo.). There were several late relapses in SR cases but most cases with isolated CN8 relapse were salvaged and long-term survival rate of SR cases plateaued a f t e r 81 months at 86.8%. HR cases showed more early relapses(15/S9) before 50 months and only one case could be salvaged. The r e s u l t was not yet s a t i s f a ctory; The relapse-free survival rate was far below 100%, and considerable adverse late effects could be anticipated.

EFFECT OF rhG-CSF ON RELAPSED ACUTE LYMPHOBLASTIC LEUKEMIA IN CHILDREN.

I.TSUKIMOTO, A.OHARA, N.TANAKA, M. TSUCHIDA, Y.HANAWA Treatment of relapsed ALL with more aggressive therapy almost always results in serious infections. We have administered rhG-CSF to 18 children with relapsed ALL after an complete remission was achieved, and studied the effects on the neutrophil counts, the incidence of infection, the rate of accomplishment of periodic consolidation therapy, and the incidence of recurrence. All had initial bone marrow relapse and the TCCSGR-8801 protocol for relapsed ALL was employed. Block 1 induced a complete remission in all patients, and Block 2 and Block 3 were then repeatedly given as consolidation treatment.The initial course of each block of drugs served as the control,and rhG-CSF (KRN8601) 50~g/me/day was given subcutaneously in the subsequent courses from 72 hours after chemotherapy for at least i0 days. The mean nadir neutrophil counts for Block 2 and 3 during the control phase were 6. 5 and 10.4/~ l respectively, and those during the treatment phase were 4. 9 and 15.4/~1. The time required for the neutrophil counts to more than 5OO/~ l was shortened from 14.9 and 16.5 days in the control phase to 10.8 and 13 days in the treatment phase. An improvement was observed for 61% in Block 2 and 93.3% in Block 3 of ~he u'eaLmeu~ pecioa. The incidence of infection decreased from 89 and 100~ in the control phase to 50% in Block 2 and 66.7% in Block 3 of the treatment phase.Block 2 therapy was performed on schedule during both the control and treatment phase. Block 3 treatment delayed for 7.4 days during the control phase, but it could be carried out on schedule during the treatment phase. Another bone marrow relapse occurred in 2 out of 15 patients. During a total of 33 courses of treatment, only 1 patient(3.0~) developed adverse reaction (erythema at the injection seite). The adjuvant use of rhG-CSF in treatment of leukemia appeared to enable the employment of more aggressive chemotherapy, and hence could help to improve the cure rate,

Present a~dress:Ist Department of Pediatrics, Toho University School of Medicine,6-11-10mori-nishi,Otaku, Wokyo,Japan 143

A82

46

44 RometHuG-CSF

IN INDUCTION AND C O N S O L I D A T I O N

TREATMENT

OF

ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) - A PILOT STUDY. Scherrer R, Bettelheim P, Geissler K., J~iger U., Kn6bl P., Kyrle P.A., Laczika K., Mittcrbauer G., Scholtcn Ch, Schwarzinger I., Siilabcr Ch., Valent P., Watzke H., Lcclmer K. Dept. Int. Med., Div. Hematol., Univ. Vienna, Austria. Nine patiens with ALL-median age 52 years (22 to 69), 8 c-ALL (2bcr-abl positive), and 1 T-ALL were treated according to the BMF protocol (H~lzer c t a l , Blood 1984, 64:38) and in addition received r-metHuG-CSF (Neupogan" ) 200 # g/m2 i.v., in phase I from day 1 to day 21, and after day 21 until neutrophil counts were > 1000/U1 on 2 consecutive days. In phase 2, r-metHuG-CSF 200 #g/m2 s.c. was started on day 2 (after CP) and continued until neutrophiis recovered to more than 1000 on more than 2 consecutive days. These patients were compared to a historical control group treated with the same protocol. It included only patients who achieved CR within 4 weeks-median age 23 years (16 to 66), 13 c-ALL, 7 T-ALL, and 1 OALL. In phase 1 the increase of neutrophils in the G-CSF treated group was faster than in the controls; in phase 2 the decline of leucocyte counts was markedly slower than in the control group. wbc/neutrophils:

cases (n = 8)

day 1 day 8 day 15 day 22 day 29

6.173/4808 11.724/6476 4.033/3049 6.279/2571 4.006/3165

controls (n= 18) 7.759 5.067 3.610 2.445 1.843

As a result the full dose of chemotherapy could be given in 7/8 G-CSF treated patients, while in the control group only 8/18 received the protocol without dose reduction or delay. Despite the higher dose intensity G-CSF treated patients had a lower number of days with temperatures > 38~C (1.74 vs. 3.1 days). 8/9 patients achieved CR, 1 died because of fungal infection. These data indlcatc that (1) r-met HuG-CSF can bc given along with chemotherapy in induction treatment without compromising efficacy. (2) Recovery of neutrophils was faster in phase I and leucocytes were preserved in phase 2. (3) Dose intensity could be increased.

THE PROLIFEP~ATIVE ACTIVITY OF BLASTIC CELLS IN ACID PHOSPHATAS~ POSITIVE CHILDHOOD ACUTE LYMPHOBLA~TIC LEUKEMIA (ALL). J. P e r e g u d - P o g o r z e l s k i , A. K r y g l e r - - S t o J a l o w s k a , J . F y d r y k , T. U r a s l ~ s k l . I P e d i a t r i c Dept. Pomeranlan Medical Academy S z c z e c l n - P o l and. T h i s is w e l l r e c o g h i s e d o n b a s i s of clinical observation that c a s e s of acld phosphatase fa. p h . ) p o s i t i v e c h i l d h o o d A L L b e a r t h e u n f a vorable prognosis, but the biological background of this phenomenon remains unclear. The study was undertaken in order to a~l)-~e the prognostic significance of a.ph. p o s i t i tlvlty and to evaluate and compare the prollf e r a t l v e a c t l w l t y of b l a s t I c c e l l s in a. ph. C§ a n d a. p h . [ - ) c a s e s of c h i l d h o o d A L L . The studied group comprised S8 children aged "17"1 6 e rag. ( m e d i a n S e rag.) d i a g n o s e d t o h a v e A L L and treated wlth [FM 7g protocol. The followu p t i m e was w i t h i n t h e r a n g e I ~ 7 5 mo. The proliferative activity was w~asured with t h e u s e of a b s o r p t i v e c y t o m e t r y a c c o r d i n g to p r i n c i p l e s g i v e n b y D e a n et al. T h e r e w e r e 2(~ a. p h . C + ) a n d 9 8 a, p h . ( - ) c a s e s . It h a s been shown that the p-EFS and p-DFS were significantly b e t t e r f o r a. p h . ( - ) p a t i e -

nts

(

0.~3

v~ 0 . 3 1 ;

p
0.57

~

0.38;

p
45

47

CHEMOTHERAPY FOR RELAPSED CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA: RESULTS OF THE BFM STUDY GROUP

VERTEBRAL COMPRESSION FRACTURE IN CHILDREN WITH ACUTE LYMPHOBLASTIC LEUKEMIA M.Oohocka, M.Matysiak, J.Armata, G.Dobaczewski, G.Gawrofiska, D.Michalewska , D.Sofita-Jakimosyk, G.Sladkowska

Henze G., Fengler R., Hartmann R., UniverSity Children's Univerait~tsklinikum RudolfVirchow, Berlin, Germany

Hospital,

From 1983 to 1999, 401 children and adolescents with their first relapse of acute lymphoblastic leukemia (ALL) were treated in one of three subsequent relapse trials ALL-REZ 83, 95 and 87 of the BFM study group. Initial treatment consisted of alternating courses of short-term intensive polychemotherapy which was supplemented by local radiotherapy (RT) to involved extracompartments and followed by conventional maintenance. Treatment intensity and duration were stratified in three patient groups according to the presumed risk of second relapse: early bone marrow (BM) relapse (duration of 1st complete remission (CR) < 6 months after discontinuation of front-line treatment), late BM relapse, and isolated extramodullary relapse at any time. Preventive treatment to the central nervous system (CNS) consisted of intrathecal chemotherapy in studies 83 and 85. Because of e high incidence of subsequent CNS relapses cranial RT was added for all patients with systemic relapses in study 87 even if the CNS was not involved. 357 out of 401 patients (89%) attained a 2nd CR. The estimates of disease-free survival (• SD) for these patients are 0.41 (+-.03) at 3 yrs, 0.35 (+-.03) at 5 yrs, and 0.31 (+-.04) at 8 yrs. Independent risk factors were found to be (1) duration of 1st CR (unfavorable: early relapse), (2) immunology (unfavorable: T/pre-T cell), and (3) site of relapse (unfavorable: isolated BM). The prognosis of patients with isolated BM relapse was significantly improved by the introduction of preventive RT to the CNS. -Supported by the "Deutsche Krebshilfe"-

Bone pain and radiographieally detected skeletal changes are common in patients with ALL, but vertebral column is rarely involwed. Spinal osteoporosis leading to vertebral compression fractures is a very rare complication. Among 1700 children with ALL treated in seven hematological centers of Polish Leukemia-Lymphoma Study Group in years 1970-1988, twenty /1,2%/ presented vertebral compression fracture. Seventeen had back and leg pain as their initial chief complaint, twelve of them were unable to walk because of intensive pain. The duration of initial symptoms ranged from 2 to 25 weeks. Sixteen from 20 had multiple vertebral compression fracture and 4 had extensive spinal osteoporosis. Most often were thoracic /Th 7-10/ and upper lumbar /LI-3/ vertebre involved. Complete remission was induced in 18 patients. 2 had no remis sign and died in the beginning of the therapy. 12 patients are alive in first remission and 2 in second remission. They are free of leukemia for 5 months to 19 years. 13 children completely recovered from vertebral compression fracture We found that the patients with vertebral compression fracture have had usually favoravle prese~ting feature for leukemia /leuk0cytes counts median 4,6xi0 /l,no mediastinal mass or central nervous system involvement/ and that they have a relatively good treatment outcome. Among our patients 52% are estimated to be in complete remission 8 years after diagnosis of ALL. Department of Pediatric Hematolo~y/Oncolo~y Warsaw Medical School - Warsaw, Dzialdowska etr.l

A83

48

5O

EFFECT OF CHEMOTHERAPY ON BONE MARROW CFU-GM and CFU-F IN CHILDREN WITH ALL. Vladimirskaya E., Zamarayeva N, Lebedev V., Rumyantsev A. Research Institute of Pediatric Hematology, Moscow, Russia. CFM-CM (soft agar culture) and CFU-F (liquid culture by Friedenstein) were investigated in 235 children with ALL during treatment in acute phase, relapse and remission. 408 cultures were analyzed (181-granulo/macrophage, 227-fibroblastoid). It was revealed that induction therapy in the acute phase of ALL (4-6 weeks of chemotherapy) did not change the number of CFU-GM: 9.5 + 3,6/mcl; 8.3-+0.3/mcl, respectively. At the same time, bone marrow granulocytes did change significantly (from 6.9 -+1.7 x 1000/me1 to 31.8 -+9,5 x 1000 mcl), but did not reach the normal level (93.4+4.7 x 1000/mcl). The intensive chemotherapy in relapse decreased the high pretreatment level of CFU-GM from 106.2-+39.5 to 57.2+40.6 in 1 mcl of bone marrow (N = 151,9 +-23.9), but did not afflict the number of bone marrow granulocytes, that remained at normal scale. CFU-F in acute phases of ALL are very sensitive to induction chemotherapy, their level falls from 35.6 + 7.3 to 16.8 + 5.5 after 4-6 weeks of treatment (normal number of CFU-F is 85.4 -+8.1 in 1 mcl of bone marrow). It was found that chemotherapy in remission caused the decrease in CFU-GM as well as in CFU-F. But this effect became obvious only after the first year of remission, the maximum fall of CFU-GM and CFU-F was registered at the 3rd year of remission. When the treatment was stopped after 3-year therapy, levels of progenitors were capable to reparation. The effect of chemotherapy on progenitor during the first year of remission is of special interest. Iuspite of intensive chemotherapy GMand fibroblast progenitors tend to increase reaching almost normal levels at the end of the first year. So this data enables us to come to the conclusion that leukemia cells are the main target for chemotherapy during the acute phase and the first year of remission. If the remission lasts longer leukemic cells are minimized so that the residual normal hemopoietic progenitors become the target for chemotherapy, which proves the idea that antlieukemic chemotherapy must be the most intensive at the beginning but not more than for 2 years once again.

FAB C L A R I F I C A T I O N HAS NO PROGNOSTIC SIGNIFICANCE IN CHILDHOOD ACUTE LYMPHOBLAS~TIC L E U K E M I A (ALL3 T R E A T E D W I T H B F M PROTOCOL. T. U r a s i n s k i , J . P e r e g u d - P o g o r z e l s k i , B.M@~yk. I Pediatric D~pt. P o m e r a r d a n Medical A c a d e m y S z c z e c i n - Poland. S i n c e its i n t r o d u c t i o n F ~ m e r p h o l o g l c a l c l a s siflcatlon of ALL h a s b e e n c o u r t l y a c c e p t e d by hematologists. However, o p i n / o m of its p r o g n o s t i c s i g n i f i c a n c e a r e still confusing. MC-G s t a i n e d b o n e m a r r o w s m e a r s t a k e n at d i a g nosis i n 72 c h i l d r e n a g e d 1 7 - 1 0 ~ Wa.7~, w e r e reevaluated by three independent observers acc o r d i n g to FAB criteria. P a t i e n t s were stratified into standard(4t pts) and hlgh-risk (31 pts) g r o u p s and t r e a t e d w i t h B F M 7 ~ p r o tocol. The f o l l o w - u p t i m e was 1 0 - 7 3 ( m e d i a n 3~) me. R e s u l t s w e r e s t a t i s t i c a l l y e v a l u a t e d w i t h the use of l l f e - t a b l e method, I o g r a n k and chi-sqt~are tests. Triple concordance of diagnosei among observers w a s gO. ~ . T h e r e w e r e S 2 (7~. 2~) LI cases, 17 f23. O~) L~ c a s e s a n d 3 (4.2~) L 3 cases. B e c a u s e of small n u m b e r s L3 c a s e s w e r e e x c l u d e d f P o m s u b s e q u e n t analysis. T h e r e was n o s t a t i s t i c a l c o r r e l a t i o n b e t w e e n r i s k f a c t o r s a c c o r d i n g t o B F M c r l t e r l a a n d LI o r L~ m o r p h o l o g y (O. 1 ~ p ( O . 5 ) . p-EFS was not s t a t i s t i c a l l y better for L1 as c o m p a r e d t o L ~ p a t i e n t s for the w h o l e g r o u p ( 0 . 3 9 ~ O. 29; O. 1O. 5) and high-risk pts (O. 0 v~ 0.33; p>O. 5). We c o n c l u d e that FAB m o r p h o l o g y ( L t a n d L2 s u b t y p e s ) has no p r o g n o s t i c s l g n i f l e a n c e i n c h i l d r e n w i t h ALL t r e a t e d a c c o r d i n g t o B F M protocol.

49

51

CORTICOSTEROIDS CAUSE MULTIPLE CHANGES OF COAGULATION FACTORS A,H. Sutor. C.Niemever. S.Sauter. I.Witt. K.Kaufmehl. M.Brandis

P e R A S A D I A G N O S T I C T O O L IN ALL: A C O M P A R I S O N WITH CYTOGENETIC ANALYSIS IN 251 PEDIATRIC PATIENTS.

In the A L L / N H L - B F M 90 induction therapy (protocol I) for t r e a t m e n t of childhood acute lymphoblastic l e u k e m i a (ALL) p r e d n i s o n ( P R E D 60 m g / m 2 / d ) is given during induction therapy from day 1 to day 29 and then tapered, ASP (10,000 IU/mZ every 3 days) from day 12 to 30. Prior to the initiation of ASP therapy the following changes were observed a m o n g 14 children newly diagnosed with A L L between day 1 and 12: fibrinogen dropped from 319+31(mean_+SE) m g / d l to 146-+21 m g / d l . A T IH increased from 112-+7% to 130+8, protein C increased from 9 6 + 8 % to 183-+17%, plasminogen r e m a i n e d unchanged (102-+5% to 102-+5%). These changes were most likely caused by corticosteroids, although other factors, like decrease of l e u k e m i c blasts and t r e a t m e n t with vincristin and daunorubicin have to be considered. However, the same changes were observed during w e e k 21, w h e n corticosteroids ( D e x a m e t h a s o n 10 m g / m 2 / d ) were given during re-induction therapy (protocol II). F r o m these data and from our earlier results indicating that P R E D reduces capillary resistance and bleeding intensity in vivo (Europ J Paediatr 129:67-72, 1978) we conclude that corticosteroids cause multiple and severe changes of coagulation factors which may increase thrombotic a n d / o r hemorrhagic risks in patients. Universit~its-Kinderklinik, Mathildenstr. 1, D-7800 Freiburg

S.Izraeli, J.W.G. Janssen, O.A. Haas, J. H a r b o t t , F. B r o k - S i m o n i , J.U. Walther, H. K c v a r T. Henn, W.D. Ludwig, G. Rechavi, C.R. B a r t r a m , H. G a d n e r a n d T. L i o n ~. _. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C e r t a i n t y p e s of All w i t h a t ( l ; 1 9 ) ( q 2 3 ; p 1 3 ) or a t(9;22)(q34;qll) are a s s o c i a t e d w i t h p o o r p r o g n o s i s of c e r t a i n s u b t y p e s of ALL. To t e s t whether t h e e m p l o y m e n t of P e R i m p r o v e s the d e t e c t i o n r a t e of t h e s e c l i n i c a l l y r e l e v a n t genetic anomalies we have developed a multiprimer-PCR protocol which facilitates the d e t e c t i o n of e a c h of the f o u r c h i m e r i c E 2 A / P B X I a n d B C R / A B L m R N A s in a s i n g l e reaction. T h i s p r o t o c o l w a s u s e d for the e v a l u a t i o n of b o n e m a r r o w or b l o o d s a m p l e s f r o m 251 c h i l d r e n w i t h ALL in w h o m cytogenetic analyses had been performed. T w e n t y one p a t i e n t s c a r r y i n g t h e E 2 A / P B X l r e a r r a n g e m e n t and 3 w i t h the B C R / A B L t r a n s c r i p t s w e r e d e t e c t e d b y PCR. T w e l v e of these cases had escaped the detection by c o n v e n t i o n a l c y t o g e n e t i c analysis. In t w o of 12 p a t i e n t s w i t h a t y p i c a l t ( l ; 1 9 1 ( ~ 2 3 ; p 1 3 ) , n o E2A/PBXl t r a n s c r i p t s w e r e i d e n t l f l e d b y PCR, thus suggesting the presence of different molecular rearrangements. Residual leukemic c e l l s w e r e d e t e c t e d by P C R in 5 of 8 p a t i e n t s who were followed during complete clinical r e m i s s i o n . W e c o n c l u d e that the r o u t i n e u s e of PeR m a y h a v e an i m p o r t a n t impact on b o t h c l i n i c a l d i a g n o s i s and m o n i t o r i n g of m i n i m a l residual disease in patients with B-cell precursor leukemia who carry the E2A/PBXI or B C R / A B L f u s i o n genes. * Corresponding author: CCRI, Kinderspital, Kinderspitalgasse Austria.

st. Anna 6, A-1090

A84

52 THE PROGNOSTIC SIGNIFICANCE OF KARYOTYPE AT DIAGNOSIS IN CHILDHOOD ALL D.C. van der Plas* , K. H~hlen* and A~ Hagemeijer In Childhood Acute Lymphoblastic Leukemia (ALL) the leukemic cells are characterised by various karyotypic abnormalities and diverse immunologic phenotypes. Identification of prognostic factors could identify the patients that require different therapeutic approaches. In 145 children with ALL, who were diagnosed and treated in the Sophia Childrens Hospital in Rotterdam between 1980 and 1990, we investigated if karyotype at diagnosis correlated with clinical outcome. In 135 out of 145 cases cytogenetic analysis was successfully performed at diagnosis. The ploidy of the karyotype appeared to be of prognostic importance. Most of our results were in agreement with earlier reports from other investigators. New findings were: -The event free survival curves for the various karyotypes appeared to be different for clinically high risk and standard risk ALL. -An increased risk for CNS relapse was observed in childhood ALL patients with abnormalities of the short arm of chromosome 12 in combination with pre-B or common ALL phenotype. *) Present adress: Dept. of Pediatics, Div. of Hematology/Oncology, Sophia Childrens Hospital, Gordelweg 160,3038 GE Rotterdam, The Netherlands. Dept. of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands.

53 MYELOID SURFACE ANTIGEN EXPRESSION IN ADULT ACUTE LYMPHOBLASTIC LEUKEMIA G. Specchia. N. Pansini,D, Mininni,A. Mestice, G. Debellis,R, Dione, P. Ditonno, V. Liso HematologyService-Universityof Bari-ltaly The immunological characteristics of blast cells in 64 cases of Acute Lymphoblastic Leukemia (ALL) were investigated using a panel of monoclonal antibodies, (MoAb). 50 were untreated (median age 32; range 14-77) and 14 were relapsed patients (median age 27; range 15-56). The 64 ALL cases were diagnosed according to the FAB classification as L1 : 25 cases and 1.2:39 cases. Surface marker analysis was performed by the indirect immunofluorescance assay and the APAAP method. All cases reacted with 'lymphoid" monoclonal antibodies: CD19+ GDIO+: 36 cases; CD19+ CDtO-: 16 cases; CD3+ CD2+ CD7+: 7 cases; CD3+ CD2CD7+: 5 cases. Of 50 untreated patients, 13 (26%) reacted with at least one myeloid MoAb (CD13+: 13 cases; CD33+: 12 cases; CD13+ CD33+: 7 cases). The coexprassion of myeloid an,J ly,nphoid markers was confirmed by double staining. Of 14 relapsed patients, 10 (71%) reacted with at least one myeloid MoAb (CD15 in 1 case, CD13 in 6 cases and CD33 in 3 cases). In 7 (50%) relapsed patients we observed the expression of CD13 or CD33, which had been negative at the onset of the disease. 34 samples were tested for CD34 expression; out of 34 specimens 24 (70%) were CD34+; in particular, 7 of the 8 myeloid-positive cases were CD34+. No significant differences were observed between My+ and My- patients as regardsthe presenting clinical features or the complete remission rate. We confirme the relevant frequency of myeloid markers onthesurfeceof adult ALL blasts, particularly in relapsed ALL. Further studies are necessary to assess the clinical significance of myeloid antigens expression in ALL. Present address: Hematology Service, University of Bad, Policlinico, Piazza G. Cesare, 1 t - 70124 Bari, Italy.

54

~ - -Piet~s IR. , G.J. Peters2, K. H~hlen3,4, A. van der Does-van den Berg , E.R. van Wering 4, A.J.P. Veerman l, 4. Free University Hospital, Departments of PediatricsI and Oncology~, Amsterdam, Sophia's Childr.~ Hospital, subdivision Haemato/0ncology, Erasmus Uni~_rsity , Rotterdam, and Dutch Childhood Leukemia Study Group 4, The Hague, The Netherlands. Hypoxanthine-guanine phosphoribosyl-transferase (HGPRT) is responsible for the conversion of 6-mercaptopurine and 6thioguanine (6-TG) to their cytotoxic nucleotides. Decreased activity of this enzyme may cause resistance to these thiopurines in experimental leukemic syst~ns. The clinical significance of this mechanism is as yet unclear. In 83 children with untreated acute l!mi0hoblastic leukemia (ALL) we determined the prognostic value of HGPRT activity and the relation between HGPRT activity and resistance to 6-TG. HGPRT activity was determined radiochenically; In vitro resistance to 6-TG with the MTT assay. HGPRT level was signifantly lower in T-ALL ccmpared with B-lineage ALL; no differences were found between sequential differentiation stages of B-lineage ALL. HGPRT activity was inversely related to the white blood cell count !9/W~). Among patients with cALL and preB ALL with WBC < 50x10 /i, cases with a low HGPRT had a significantly poorer prognosis than those with a high HGPRT. WBC, age, Sex, organcmegaly and immunophenotype were comparable in both patient groups. No correlation was found between HGPRT activity and in vitro 6-TG resistance in cALL and preB ALL patients. T-ALL cases were not more 6-TG resistant than cALL and preB ALL cases. Cells from 6 relapsed ALL cases did not show decreased HGPRT activities. We conclude that: (a) HGPRT is lower in T- than in B-lineage ALL and is constant in sequential differentiation stages of B-lineage ALL; (b) HGPRT activity is inversely related to tt~or load; (c) low HGPRT activities are related to a poorer prognosis in precursor-B ALL but this can not be explained by thiopurine resistance because (d) there is no relation bet%t~en HGPRT activity and in vitro resistance to 6-TG. Supported by the Dutch Cancer Society (IKA 87-17 )

A85

AML in Children 55

57

EARLY RESPONSE TO TREATMENT (DAY 15 MARROW) IS AN IMPORTANI" PREDICTOR OF OUTCOME IN CHILDHOOD AML* J. Ritter, U. Creutzig, D. Grove, E. Kurzknabe, and G. Schellong for the AML-BFM-Study Group**

THERAPY OF ACUTE NYELOGENOUSLEUKEMIA IN CHILDHO00 - RESULTS OF THE NULTICENTER TRIAL AHL I I / 8 7 IN EAST G ~ ' J. Hormann, D. Fuchs, R. HSfer. A. R011er, H. Pal.me, J. Prager, W. D~rffel, R. Domula, G. Eggers, P. ExadakCylos, E. H i l g e n f e l d , T. Krauss, N. Kunect, U. ~ t t l e r , D. N6bius, H. Reddemann, V. Scharfe, E. Siegert, G. We~nman, F. Z i n t l

From December 1986 to April 1991 210 pediatric AML patients from 33 different hospitals entered the 3rd cooperative AML study BFM-87. All children and adolescents under 17 years of age Without prior malignancies or other treatment of more than 14 days were eligible. Pretherapeutical bone marrow smears from all children and day 15 bone marrow smears from 185 children after remission induction tyeatment With ARA-C, daunorubicin and etopeside (ADE) were reviewed centrally. 112 of 127 children (88%) with _<5% bone marrow blasts on day 15 achieved complete remission as compared to 39 of 58 children (67%) With >5% bone marrow blasts on day 15 (p<.05;X2-test). 84 of the 127 children with _<5% bone marrow blasts on day 15 are still in complete remission as compared to 20 of 58 children With >5% bone marrow blasts on day 15 (p<.001; X2-test). Life-table analysis revealed a probability of a 4 l/2-year eventfree interval (EFI) of 68%; SD 5% in children with -<5% bone marrow blasts on day 15 as compared to 42%; SD 9% in children With >5% bone marrow blasts on day 15 (p<.05; Iogrank test).

Si xt y one pat i ent s entered the cooperative study AHL TT/87 o f the working group " P e d i a t r i c Fmmatology and Oncology= of East Genmny. Two pat i ent s v i t h initial hyperleucecytssis died p r i o r therapy. 14 p a t i e n t s died w i t h i n t h e f i r s t 7 weeks o f therapy, and 4 p a t i e n t s did not respond t o therapy. 41 p a t i e n t s (70 Z) at t ai ned a complete remission. 17 p a t i e n t s were transplanted i n 1. CR (12 autologous BRT without purg~J~, 5 a l l o genous BraT): 13 o f them are l i v i n g and w e l l 1 t o 41 months a f t e r BKr. 10 of the 24 patients under chemothecapy relapsed, 1 p a t i e n t i s l o s t t o f o l l o w up. 13 pat i ent s are l i v i n g i n continuous complete remission. ~ne l y e table p r o b a b i l i t i e s 53 aanths a f t e r the s t a r t o f the pr ot ocol ere 0.42 f o r disease f r e e s u r v i v a l (DFS) and 0.61 f o r event f r e e i n t e r v a l (EFI). The respective r e s u l t s o f the former pr ot oco l NqL 1/82 were 0.34 f o r DFS and 0.47 f o r EFI. UniversitStskinderklinik, Kochstra6e 2, 0 - 6900 Jena

In future childhood AML trials early response to treatment may be used for stratificution of further therapy. *Supported by the Deutsche Krebshilfe **Prdsent address: Uuiversit~ts-Kinderklinik, $I~. 33, D-A400 M0nster FRG

Albert-Schweitzer-

56

58

ACUTE MEGAKARYOBLASTIC LEUKEMIA (FAB M7) IN CHILDREN: ANALYSIS OF 13 PATIENTS IN THE GERMAN COOPEl~FI'r AML-BFM-87 STUDY* J. PAtter , U. Creutzig, M. Suttorp, J. Harbott, W.D. Ludwig, H.J. Riehm and G. Schellong for the AML-BFM-Stody Group

SECOND REMISSION IN RELAPSED CHILDHOOD ACUTE MYELOGENOUS LEUKEMIA AFTER PRETREATMENT WITH THE BFM-83 PROTOCOL K. Stahnke 1, U. Creutzig 2, J. Ritter2 for the BFM study group

13 of 237 children in the German cooperative AML-BFM study -87 were classified as acute megakaryoblastic leukemia (FAB M7). There were 3 boys and I0 girls with this rare FAB subtYt~. T h e age of the 13 children ranged from 3 months to 13 2/12 years with a median of 1 5/12 years. Four of the 13 children were infants. WBC ranged from 500 to 87.000/pl with a median of 15.000/pL The diagnosis depended on the characteristic morphological and cytochemical (POX-) pattern, together with pesitivity of one or more of the following immunological markers: CDw41a, CDw42b and CD61. Cytogenetie analysis revealed the newly described traaslocation t(1;22) (p13;q13) in 3 infants. Two children died early due to infectious complications, 2 other children did not respond to the AML-BFM-87 polychemotherapy. Nine of the 13 children achieved complete remission. Seven of the responding children are still in first complete remission after a median follow-up of 2 1/2 years (range 6 months to 4 years). In 2 responding children bone marrow relapse occurred after 6 and 9 months. Acute megakaryoblastic leukemia is a rare FAB subtype in childhood AML with an incidence of 5.5% in study AML-BFM87. Therefore, final conclusions with regard to the prognostic outcome under the conditions of the AML-BFM-87 study cannot be drawn so far. *Supported by the Deutsche Krebshilfe **Present address: Universit&ts-Kinderklinik, Albert-SchweitzerStr. 33, D-4400 MOnster FRG Universit~ts-Kinderklinik Kiel, Medizinische Hochschule Hannover, FRG

Complete remissions (CR) of 80 % are achieved with the AML-BFM protocols but one third of patients relapse within the first three years. There are few reports of treatment of relapsed childhood AML, and these deal with the evaluation of new drugs for frontline therapy. We performed e retrospective analysis to investigate how patients previously treated with the BFM-83 protocol were treated after relapse end how many long term remissions were achieved. 48 patients (pts) relapsed after having achieved complete remission with the BFM-83 protocol which consists of ARA-C 100 mg/m 2 day, 1 - 8, Daunorubicin 60 mg/m 2 day 3, 4, and 5, end VP-16 150 mg/m z day 6, 7, end 8, and an 8 week consolidation therapy consisting of Prednisolone, Thioguanine, Vincristine, ADR, ARA-C, Cyclophosphamide, intrathecal ARA-C and cranial irradiation followed by maintenance therapy. Duration of first remission ranged from 1.5 months to 66.3 months. Excluding 9 pts with either isolated or combined extramedullary relapses 39 children were evaluable, 20 pts received no therapy or palliative therapy while 16 pts received chemotherapy and another 3 pts were transplanted in relapse. Although 8 different intensive chemotherapy regimens were used for reinduction, a high number (12 of 16 = 75 %) of second complete remissions was achieved. Several therapeutic options were used to maintain a second remission: regular maintenance therapy (7 pts), ellogeneous bone marrow transplantation (BMT) (2 pts), autologous BMT (3 pts). A long term survival of 0.37 was achieved on these 16 pts with follow up ranging from 33.2 to 73.0 months. We conclude that long term survival in second remission childhood AML can be achieved by various therapeutic regimens. The result is biased by selection of a group of patients with long first remission (9/16 pts with first remission > 18 months). Our analysis confirms the prognostic value of the duration of first remission. 1 Universit~its-Kinderklinik UIm, Abt.Kinderheilkunde II, Prittwitzstr. 43, D-7900 UIm, FRG 2 Universit~its-Kinderklinik MOnster, Albert-Schweitzer-Str. 33, D-4400 MOnster, FRG

A86 59

60

ONCOCYTOGENETIC INVESTIGASTIONS IN CHILDREN WITH ACUTE NON-LYMPHOCYTIC LEUKEMIA (ANLL), CHRONIC MYELOID LEUKEMIA (CML), AND MYELODYSPLASTIC SYNDROME (MDS) *

MEASLES GIANT CELL PNEUMONIA IN TWO CHILDREN WITH ACUTE MYELOID LEUKEMIA (AML) S. Sauter*, C. Niemeyer', M. Lohner', H. Ringe'*, J. RL~schoff*', and M. Brandis"

J. Ritterbach1, J. Harbott1, U. Creutzig2, J. Ritter2 and F. Lampert1 1OncocytogeneticLaboratory,Children'sUniversityHospital,6300 Giessen,FRG 2Children'sUniversityHospital,4400 MSnster,FRG

Viral infections are a serious threat to the immunocompromised child. While effective treatment is available for varicella zoster, herpes simplex and CMV, measles remains a major obstacle with a high mortality. Diagnosis is often hampered by atypical clinical features. We report on two cases of clinically unrecognized measles in children with AML and interstitial pneumonitis. Histopathological findings and antigen specific staining on lung tissue will be demonstrated. Case 1 is a 3 year old girl admitted with high fever, lymphadenopathy, hepatosplenomegaly and leukocytosis. A diagnosis of AML was made and induction therapy initiated. Throughout treatment the child remained febrile with temperatures above 39~ A rash was noted on both legs. Virus serology was nonspecific. Cough and tachypnoea developed with interstitial infiltrations on X-ray. An open lung biopsy was diagnostic. The child died due to respiratory failure despite intensive therapy. Case 2 is a 7 year old girl referred for treatment of AML in relapse. On admission conjunctivitis was noted. Antibiotic therapy had been initiated for high grade fever at an outside hospital. Reinduction chemotherapy was begun. On the third hospital day a rapidly progressing pneumonitis developed, on the sixth day a total body erythema. The child died in spite of mechanical ventilation on day 13. On autopsy giant cell pneumonia was evident. With the currently low uptake of active immunization against measles in Germany, measles and its complications remain a continuing problem in immunocompromised children.

Between January 1984 and December 1991 423 bone marrow or blood samples of 376 children with ANLL (at diagnosis, at relapse or as secondary leukemia) were studied for acquired chromosomal aberrations. Cytogenetic analysis was done prior to therapy (protocol of the AML-BFM study group). Chromosome preparation was performed by either a direct technique or after 24h and/or 48h of sample culture. Staining for GTG-banding followed routine methods. 316 (=74,7%) of the specimens could be analyzed successfully: 89 (=28,2%) of these patients showed a normal karyotype in their leukemic cells whereas the remaining 227 (=71,8%) revealed an abetrant chromosome set in their malignant cells. Numerical as well as structural anomalies could be found. Typical primary structural aberrations like t(8;21), t(15;17), inv(16) and t(9;11) were related to special FAB-subtypes. Prognostic statements were done performing life-table analysis (Kaplan-Meier). Furthermore, 60 children with the diagnosis of CML could be investigated successfully (adult type: 47; juvenile type: 13). 41 (=87%) patients with CML of the adult type showed the Philadelphia chromosome, whereas the children with CML of the juvenile type exhibited a monosomy of chromosome 7. In addition, chromosomal analysis could be performed of 32 children with MDS. 16 (=50%) patients revealed clonal changes in their bone marrow cells, the most often found aberrations were trisomy 8 and monosomy 7. .

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9) Supportedby the Kind-Philipp-Stiftungand the "Parents'InitiativeGiel3en"

Pediatric Hospital, and Institute of Pathology, University of Marburg**, and University of Freiburg', Mathildenstr. 1, D-7800 Freiburg, Germany

A87

ALL in Adults 61

63

EffiCACY OF ~N ORIBINAL CELL KINETICS BASE@CHENOTNEHAPYREGIflEHUSI~ rHU GH-CSF FOR INBUCTIONAND CONSOLIBATIOHTREATHEHTOF ALL PRELIMINARYANALYSIS. ,I.~loniecki,l.Cedrych~S.Krzemiee,E.Sudzka,S,Holo~iecka.

Analysis of BCR-ABL rearrangement by PER in adult acute lymphoblastic leukemia (ALL) : A report on 28 cases. C.Preudhomme, J.L.La'F, P.Lepelley, P.Fenaux, C.Sartiaux, M.Collyn-d'Hooghe, M . Z a n d e c k i , F.Bauters, J . P . J o u e t , J.P.Kerckaert. Ph 1 positive ALL accounts for 20 to 25% of adult ALL. However, about 40% of adult ALL have a BCR-ABL rearrangement (Lancet 1991,337, 1055-1058)., suggesting the existence of Phl negativeBCR positive ALL (as in CML). We analyzed by PCR the BCR-ABL rearrangement in 28 ALL pts (median age 36, range 15-86; immunology : 6T, 21 early B including 18 Calla positive, and 1 case not studied; karyotype : Ph 1 chromosome in 8 pts, normal in 7 pts, other abnormalities in 11 pts, failure in 2 pts). For PCR, primers for the major (Mbcr) and minor (mbcr) rearrangement were used, after reverse transcription of mRNA. Ten pts (median age 42) had a BCRABL rearrangement, 8 for mbcr and 2 for Mbcr, including the 8 pts who had a detectable Phl chromosome and 2 pts with normal karyotype. In those 2 latter pts, the number of mitoses analyzed was 7 and 18, respectively. All 9 rearranged cases studied immunofogisally were among the 18 Calla positive early B ALL..Our results confirm the high incidence of BCR positive cases in Calla positive adult ALL and their absence in T-ALL All Phl positive cases were BCR positive, with a majorityof mBCR breakpoints. Our study, which correlated cytogenetics and molecular biology, found 2/28 (7%) Ph 1 negative-BCR positive pts. The absence of Phl could have been due to technical failure (especially in the pt were only 7 mitoses were obtained) or may define a subgroup of Phl negative BCR-positive ALL, (as in CML). These results suggest that BCR-ABL rearrangement should be analyzed in all adult Ph I negative early B ALL (mainly in Calla positive cases).

8a~edon the bone marroweel} kinetics stedies suggenting that 48-% h after discontinuation of GN-CSFadministration represents a period of relative refractorinens nf neroa} progenitors to ceLl cycle specific ch~otherapy (Aglietta et al.lgg9) the following remission induction protocol has been elaborated-"Viocristin and epidnxorubicindaf~ 1,8,15,22, prednisonefor 2B day% L-asparaginaseevery 3-rd day starting from d 14 (6x), Mix i.t. d I GM~SF (Leneoma~) S oglkgld s.c, on days 2-6,9-1),lb-20~aed 23-till grannlocyte count )1.0 GII (GH-CSFis started 3& h

after eachgcr & epido~orubicioadministration and discontinued 48 h before the next ene). 8uring consolidation SH-CSFwas administeredafter two cydophessheaide § H8 Ara-Ccycles in order to induce the r~nvery of myeloid cells. The pilot groupconsisted of 10 patientsjF/H=5/S~medianage 35 (range17-49)~ FA8 LI-2t L2-S~ immoooshenetyse ) com~n-~ early -sre 8-1~ T typs-4~ W@Eloodles 3l S/I~ all eith lymph node and/or liver/spleen enlargemont~ CWS involvement-i, infoetions-4p hemorrhagicdiathesis-6. The ORwas obtained in S/9 of evaluablepts (891)~ mostly after the first cyde (S5~). The side effects were like that observed after chemotherasywithout GM-CSFend were not related to this drug. This preliiinary observationin patients with a rather onfavnrableprognosis(aBe) high proportion of l typev big tomor mass) suggests the fMlowieg aOvantageeof the pretzel under study whencomparedto enalog~usregimenswithout SN-CSF(data in bracketsh 1.a high CRrate, 2.a short time to ORequaling 3~ d in the uhele grou8 (vrs 40-57 d) and 28 die patients youngerthan 35 y (vrs 41 d), ). agrenolocytnsis persisting defied indoctio~ is ~0%of pts ~Iy (vrs 65-72%)p 4. a low infection rate during indoction -14% (vrs 48-&5%)~ 5) a close adherenceto the planned intensive consolidationmas possible, ~ randomizedstudy has beenstarted. 8egt of Hematology,Silesianlied. ~cad., 40029Katowicep8eymoata8, PL.

62 IS EARLY RESPONSE TO CHEMOTHERAPY IN ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) OF ADULTS A RELEVANT AND USEFUL PROGNOSTIC FACTOR? A STUDY FROM THE FRENCH GROUP OF ADULT ALL. C.SEBBAN, E.LEPAGE, P.DUFOUR, F.HUGUET, F.WI'IZ, A.BROUSTET, B.PIGNON, X.TROUSSARD, M.FLESCH, C.CORDONNIER, D.FIERE. In an attempt to assess prognostic significance on complete remission and survival of early response to chemotherapy, we measured percentage of residtml blasts in a bone meerow aspiration performed at Day 15 after the beginning of induction therapy in adults patients (pts) with ALL,included in two consecutive French trials(AALL85,AALL87).538 patients with a median age of 33 years received a similar induction regimen with an anthraeycline(Zorubicine 100mg/m2 D 1-3 or Cerubidine 50rag/m2 Dl-D1-3),Prednisone 60mghn2 D1-21, Vineristine 1.5rag/m2 D1,8,15,22 and Cyclophosphamide 6O0m~/m2 D1 and D8. If CR was not achieved in one co~rse, the pts received a salvage therapy with AraC lgt~n2 DI-4 and Amsacrine 120mghn2 DI-3. After obtemption of CR, pts aged 15 to 40yrs with sibling donor were ~'aasplanted, those younger than 55 years and without sibling wer~ randomized between maintenance chemotherapy or autologoas bone marrow transplantation. Complete remission is slzongly associated with the presence of <=20% blasts cells at D15 (p < 0.0001). % of residual blasts at D15 <=20% >20% CR after one course 358 23 CR after salvage therapy 14 30 Failures 30 54 Deaths 16 13 N~ patients 418 120 In a multivariate analysis, D15 blasts cells is found as the best prognostic factor for overall survival (p < 0.0001) with age younger than 35 years (p < 0.00l) and hyperlcococytosis inferior to 30g/l.(p < 0.001) but for disease-free survival (DFS), only hyperleucocytosis (p=0.005) and age (p=0.02T) were found and DI5 blasts ceils <=20% was not assneiated with best DFS (p=0.25) Moreover, we have not found statistically significant differences in term of overall survival (i)=0.8) and DFS (I~-0.7) for pts achieving CR in one course or in 2 courses. The importance of early respoase to therapy remains questionable in adults ALL and we will critically discuss methoingieal issues sterounding the usefulness of such post-therapeutic prognostic factors. Service d'H6rnntologie HOpital Edouard Herriot Lyon (FRANCE)

A88

AML in Adults 66

64

TRANSFUSIONAL SUPPORT OF AL TREATMENT: PROBLEMS A. I. Vorobiev and T. V. Golosova

SPECIAL

The achievement of CR as a prerequisite of long-term survival in AL requires complete supportive care including perfect transfusional support in addition to the antileukemic treatment. At All-Union Hematological Center in 1990/91 response data in younger patients up to 60 years were 68.2% CR, 9.2% El), and 22.6% DR. The average number of platelet transfusions from single donors was 66 during induction and 100 during induction and consolidation. Nevertheless grade III-1V hemorrhage was observed in 50% of patients indicating that platelet support was still unsufficient, though there were no deaths due to hemorrhage. In the elderly patients response data were 47.4% CR, 36.8% El), and 15.8% DR. The average number of platelet transfusions in this age group was only 33 explained by the higher early death rate. Thus, the number of platelet transfusions reflects both survival of the initial treatment and completeness of the antileukemic plus supportive treatment. Patients treated by allogeneic or autologous BMT received 40-80 and 140-160 platelet transfusions. Thus, patients completing first-line chemotherapy and BMT received 180-260 transfusions. A limiting problem of transfusional support, however, is the hepatitis virus contamination of blood products. We found an infection rate of 1% CMV, 2% hepatitis B and 3% hepatitis C in our donors. 43% of the admitted patients with leukemias had various markers of hepatitis B. From the high transfusion rate the calculated risk of contamination is as high as 100% in patients surviving long enough. An evident icterus with negative HBSAg is found in 22.5% of patients completing induction and consolidation. In transplanted patients 20% experience hepatic complications including 10% VOD, 7 % hepatitis C and 3 % SICCA probably due to previous hepatitis or virus r~activation. We conclude that an adequate curative treatment of our leukemic patients is substantially limited by hepatic complications. Only strict selection of donors may improve this situation. *Present address: All-Union Hematological Center, Moscow, USSR

AML IN THE ELDERLY (CLINICAL DATA). Parovitchnikova E.N., Savchenko V.G., Saruycheva T.G., Kulinov S.M. All-Union Hematological Center. Though the results of AML treatment have considerably improved during the last years, treatment of elderly patients is still a clinical dilemma. Two years ago, a randomized trial was started on AML treatment in the elderly. The first group of patients received 7+3 (AraC 100 mg/m 2 i.v. bid; seven days daunorubicin 45 mg/m 2 i.e. once daffy, 3 days). The second group received 3 identical cycles of AraC 50 mg/m 2 i.d. and daunorubicin 30mg/m 2. Maintenance treatment consisted of 5 days of AraC every 4 to 5 weeks and continued for 3 years. Patients who exclude from the protocol are treated with low-dose AraC (10 mg/m 2 bid.). Twenty patients between 60 and 80 years were admitted to the All-Union Hematological Center. One patient was excluded. Overall treatment results were as follows: CR 47.4%, early death 36.8%, non-responders 15.8%. Eight patients were randomized to the first protocol, 8 to the second protocol. Two patients were non-responders to low-dose AraC and were then treated with 7+3. The frequency of complications was identically in both groups. Only long term results may show a difference. Results of autoradiography studies were as follows: Patients with

IL (%) in blasts

CR DR El)

IL of eryth cells (%)

7,75 + 1,49 2,9 + 1,93

16 12

9,5 + 1,46

20,2 + 3,6

PAS+ erythroid cells (%)

+ 1,2 + 4

26,8 + 1,42 39,6 + 8,6

27,8 + 4,6

IL was significantly lower in non-responders than in responders (p=0.03). We can suppose that leukemias with low S-phase reply better to initial low-dose AraC treatment but we need more patients and time to prove it.

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RESULTS OF AML TREATMENT. Savchenko V.G., Isaev,.~V.G., Kucher R.A., Paravitchnikova E.N. ALL-Union Hematological Cent\. ter, GUS. / The results of acute leukemia treatment in the ALL Union Hematological Center are close to the world-wide standard. Patients treated between 1986 and 1989 had a CR rate of 57.7%, an early death rate of 20.3%, and non-responders 22%. The induction course in these patients was 7+3, maintenance was performed with 7+3. A new protocol was started in 1990 comparing the efficacy of the addition of VP-16 in AML M4 M5, idarubicin in M1 M2, single daunorubicin in M3, and mitoxantrone in consolidation. The longest followup is 12 months. Twenty-two patients were treated, 8 females, 14 males, the median age was 27 years. Fifteen patients achieved CR, 2 patients died early, 5 were non-responders. The increase in CR rate from 57.7% and 68.2% was mainly due to increase the early death rate. Of the 22 patients 95% had fever, 60% pneumonia, 35% septicemia, 35% hemorrhalgies, 15% hepatitis. 35% had fungal infections. In consolidation only half of the patients experienced fever, 35% pneumonia, 17% septicemia, 17% hemorrhalgies, and 7% hepatitis. Patients received a median 12-13 units of platelets, and 16 units of eryhthrocytes. If the providence of hepatitis B is 2%, of hepatitic C 3%, and CMV 1% in the donor population, we have almost 100% probability of patient infection first and second line treatment.

INTENSIVE POST-REMISSION THERAPY IN ADULT AML: COMPERATIVE STUDY OF TWO DIFFERENT STRATEGIES.- AN INTERIM ANALYSIS. U. Jehn, V. Heinemann and W. Wilmanns .

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The e f f i c a c y of postremission chemotherapy f o r 112 consesecut i v e adults 65 yr with AML of a l l FAB-subtypes was evaluated in two sequential prospective studies using an i d e n t i c a l induction regimen: DNR 45 mg/mz i . v . days I-3, VCR 1 mg/mz i . v . day 2 and araC 200 mg/m2 i . v . days I - 7 , h a l f as i . v . i n j e c t i o n qd 12 hr and h a l f as 24 hr infusion. 72% of pts reached CR (57 a f t e r I , 24 a f t e r 2 cycles), 3% a stable PR; 2 e a r l y and 12 hypoplastic deaths occurred (13%). 15/112 (13%) were r e f r a c t o r y to 2 induction cycles, h a l f of them achieved a CR a f t e r salvage with intermediate-dose (ID) araC/AMSA. The median age of a l l pts was 48 y r , 48 females and 64 males.- Pts reaching CR a f t e r 1 or 2 cycles of induction received one cycle of " e a r l y " consolidation s i m i l a r to the induction cycle, except DNR was given only day I . The f i r s t 41 pts achieving CR were randomized to e i t h e r 6 cycles of an inductiontype regimen every 6 wks (EORTC-AML 6) or to 6 cycles of an a l t e r n a t i n g - t y p e regimen consisting of high-dose (HD) araC/ AMSA or 5-AZA/AMSA (=intensive maintenance "IM"). There was no d i f f e r e n c e between both arms: med.DFS was 12.4 mo, s u r v i val 18.7 mo. Results were compared to 22 pts reaching CR in the subsequent study in which IM Was replaced by intensive consolidation ("IC") using 1 cycle of ID-araC 1 g/m2 qd 12 hr days I-6 plus AMSA 120 mg/mz days 5-7 and 1 cycle of HD-araC 3 g/m2 qd 12 hr days I-4 plus AMSA 120 mg/m2 day 5. Median DFS was 41 mo, median survival from CR has not been reached a f t e r 48 mo; both are s i g n i f i c a n t l y superior to IM.- 17 pts reaching CR refused f u r t h e r treatment with e i t h e r IM or IC or any other regimen or relapsed while waiting f o r BMT. Their median DFS was only 2.9 mo, survival 5.4 mo. Both were s i g n i f i c a n t l y i n f e r i o r to e i t h e r IM or IC. Median survival of 31 non-responders was 4.1 mo from diagnosis.- All three groups: IM, IC, refusals were comparable in age, FAB, and other c r i t e r i a . The median follow-up is 38 mo. Present address: Med.Klinik I I I , University of Munich, Marchioninistr. 15, 8000 Munich 70, FRG

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DAUNORUBICIN, ARA-C and VP-16 (DAV) FOR ADULT ACUTE MYELOBLASTIC LEUKEMIA, FOLLOWED BY A PROSPECTIVECOMPARISONOF INTENSIVEPOSTREMISSION THERAPY H. Link, G. H0bner, M. Freund, B. Schneider, H. Wandt, P. 8ch6nrock-Nabulsi, M. Gramatzki, W. Queisser, E. Fackler-Schwalbe, B. L6ffler, M. Raab, S. 0hi, U. Brack, S. Gabius, J.T. Fischer, T. Geer, G. Ehnieger from the AML-Study Group. From April 1988 to March 1991 185 patients up to the age of 50 years were enter~-~J into the trial, 141 were evaluable and analyzed as of July t 991. The induction chemotherapy consisted of ARA-C 100mg/m = civi day 1-8; DNR 60mg/m = day 3-6, VP-16 100mg/m = day 4-8 (DAV I) followed by a second cycle with ARA-C 100mg/m = civi day 1-7, DNR 45mg/m = day 3,4, VP-16 100mg day 3-7 (DAV n). If a complete remission was achieved, the first consolidation course consisted of DAV III identical to DAV I1. We then prospectively compared allogeneic bone marrow transplantation (BMT) or in a randomized fashion or by decision of the patients either high-dose Cytosine-Arabinoside/Daunorubicin (HD-ARA-C/DNR) with high dose Busulfan (Bu) and Cyclophosphamide (Cy) followed by autologous BMT. The median age was 36 years (range 16-50). 94 (68.1%) of the patients reached a complete remission. 12 patients received unpurged autologous BMT after therapy with Bu 4xl mg/kg day 14 and Cy 50mg/kg day 5-8. 39 patients were allocated for one to two courses HDARA-C/DNR: 1st course: 3g/m=i.v. over 2h every 12h day 1-6, and DNR 30 mg/m = day 7-9; 2nd course: 3g/m=i.v. over 2h every 12 h day 1-4, and DNR 30mg/m = day 5,6. 19 patients were treated with allogeneic BMT. After autologous BMT the actuarial evenffree survival (EVS) after 36 months was 15%, after HD-ARA-C/DNR 35%, after allogenaic BMT 63%. 25 patients without postremission therapy had an EVS of 6%. Concerning EVS high-dose ARA-C therapy (p=0.0t5) as wetl as atlogeneic BMT (p=0.022) were significantly more effective than Bu/Cy-therapy followed by autologous BMT, as analyzed by log-rank test. Conclusions: The remission rate of 68.1% must be further improved by intensifying the induction therapy. Hematopoietic growth factors should then be introduced to decrease the risk of severe infections due to prolonged neutropenia. In order to avoid eady relapse, the first postremission therapy should be performed as eady and intensive as possible. High-dose Busulfan/Cyclophosphamide followed by untreated autologous BMT has no benefit and should no longer be used in AML. High-dose ARA-C with Daunorubicin leads to 35% EVS after 36 months which must be further improved. After allogeneic BMT there was the highest rate of EVS (63%) probably due to the graft versus leukemia effect.

A MULTICENTER TRIAL RANDOMIZING FRACTIONATED ACLACINOMYCIN, CYTARABINE +/- ETOPOSIDE VS. "3 +7" FOR PRIMARY AML E.Koller, J.Holowiecki, H.Losoncy, R.Ihle, S.Fekete, H. Tiichler and D.Lutz for the IGCl-study group AML induction chemotherapy with high dose anthracyclines is complicated by a high rate o f toxic complications and early death in elderly patients, A pilot study with fractionated Aclacinomycin, intermediate dose Cytarabine (ID-Ara-C) and Etoposide showed a high efficacy in patients of higher age with poor prognostic indicators. In the present study 65 patients were randomized and stratified according to age < 40a > . Antkracycline dose was reduced in patients > 40 years: 32 pt~ (median age: 43 years, range: 16-69 a) ~ arm A ( ID-Ara-C 1.5 gfm~/~2h/dl-2, aclacinomycin 30(25)~/ag~am /d3-7, Cytarabine 100 m ~ m /d3-7 and Etoposide 100 mg/m /d3-7 for M4/5 subtypes) and 33 pts (mediag age 39years, range: 16-69 a).in arm B (Daunorubicin 60(45)mg/mk x 3 andcytarabine 100 mg/mL x 7). Pts achieving complete rermssion (CR) received consolidation and identicatmaifrgaaaee thera~ for both arms. An ~ evahratitm showed an overall CR-rate of 56% (18/32) in arm A and 64% (21/33) in arm B. Percentage of resistant disease was 15% vs. 24%, incidence of early death was 28 % vs. 12 %,respectively. There was no statistically significant difference on overall survival and remission duration. When patients were stratified by age ( < =40, >40 years) survival in arm B in the younger pts ( 8 vs. 17 months, p < .05) and remission duration in arm A in the elderly pts ( 7 vs. not reached, p < .05) was superior. Despite the small number of patients these results indicate that fractionated Aclacinomycin/ID-Ara-C +/- Etoposid are only effective in AML-patients of higher age. Ludwig Boltzmann-institute for Leukemia Research and Hematology and 3rd Medical Department, Hanusch Hospital, Heinrich CoUinstr.30, A - 1140 Vienna, AUSTRIA

Dept. Hematology and Oncology, Hannover Medical School, W-3000 Hannover 61, Germany; Supported by Deutsche Krebshilfe M32/90/Li1.

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MITOXANTRONE VERSUS DAUNORUBICIN CA) IN INDUCTION THERAPY OF AML W.R. Bezwoda, L. S e y m o u r

A 12-YEARS FOLLOW-UP OF 101 PATIENTS WITH ACUTE MYELOID LEUKEMIA (AML) TREATED IN TWO STUDIES M. Freund*, H. Bodenstein**, H. Kleine*, and H. Poliwoda*

+ CYTOSINE ARABINOSIDE (D+

Fifty adult patients with de novo ANLL have been entered into a study comparing mitoxantrone 12 mg/m2 x 5 days to daunorubicin 30 mg/m2 x 3 days plus cytosine arabinoside i00 mg/m2 x 7 days as induction therapy. Patients recieving mitoxantrone who failed to enter remission after one cycle were changed to the daunorubioin plus cytosine regimen while patients randomised to recieve daunorubicin plus cytosine recieved 2 cycles before evaluation. Patients who failed to respond after 2 cycles D+CA were changed to mitoxantrone. CR rate after first cycle of treatment with mitoxantrone was 18/26 (68%) and 9/24 for patient treated with D+CA. After the 2nd cycle of therapy the CR rate for patients treated with D+CA was 14/24 (58%). Another 4 patients achieved C.R. after having changed therapy to mitoxantrone. Single agent mitoxantrone treatment is effective induction treatment for ANLL with more patients achieving CR after one cycle of mitoxantrone than with the use of D+CA. An update analysis will be presented. The use of alternating regimens is being explored. Present address: Department of Medicine, Hematology/OncoIoE~y, University of the Witwatersrand, Medical School, York Road, Park Town, 2193.

Between 1978 and 1984 101 patients with AML have been treated at the Hannover Medical School in t w o studies. The first study was closed in 1982. it consisted of an induction chemotherapy with A r a - C 2 x 1 0 0 m g / m 2 IV d 1-7, and DNR 4 5 m g / m = IV d 1-3, t w o consolidation courses with Ara-C 2 x 100 mg/m 2 IV d 1-5, and DNR 45 mg/m = IV d 1-2, and a maintenance therapy with 6-TG 80 mg/m = PC d 1-4 and Ara-C 60 mg/m 2 IM d 5 given weekly for 1 1/2 years. In the second study patients have been given an induction with Ara-C 1 0 0 m g / m 2 CIVI d 1-7, DNR 4 5 m g / m 2 IV d 1-3, and 6-TG 2 x 100 mg/m 2 PC d 1-7, a first consolidation with Ara-C 2 x 100 mg/m = IV d 1-7, DNR 45 mg/m = IV d 1-2, and a second consolidation with Ara-C 100 mg/m2 CIVI d 1-7, DNR 45 mg/m 2 IV d 1-2. No maintenance was g=ven in the second study. Fifty-six patients (23 male, 33 female, mean age 47 years) have been enclosed in the first study. There were 36 AML M1 or M2, 2 M3, 13 M4, and 5 MS. Thirtyone patients achieved a CR (55.4%). Three patients suffered early death and 22 did not respond to treatment. The median duration of survival and disease-free survival was 8.7 mo • 3.0 mo resp. 15.5 mo + 3.2 rod. Five patients are alive disease-free at 10.2 to 12.4 years. Another patient is alive disease-free in a 7.0 years lasting 3rd remission after having a I s t relapse after 3.4 and a 2nd one after 1.1 years. The calculated survival and disease-free survival at 12.5 years is 12.5% and 15.4% respectively. Fourty-five patients (18 male, 27 female, mean age 50 years) have been enrolled in the second study, There were 30 AML M1 or M2, 6 M3, 7 M4, and 2 M5. Thirty patients achieved a CR (66,7%). Seven patients suffered early death and 8 did not respond. The median duration of survival and disease-free survival was 9.2 mo + 0.9 mo and 7,1 mo + 0.6 rod. Two patients are alive disease-free at 8.4 and 9.1 years. Two more had early relapses each after 3 months. Both are in a stable 2nd remission for 7.0 years. The calculated survival and disease-free survival at 9.1 years is 8.8% resp. 6.7%. The disease-free survival was inferior (p = 0.007) for patients in the second study. There was no significant difference in the overall survival. We conclude that maintenance therapy may have prolonged the remission duration after a very mild chemotherapy in the first study but has failed to improve survival. *Abteilung for H~matologie und Onl
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74

SEQUENTIAL THERAPY WITH IDARUBICIN/CYTOSINE ARABINOSID AND MITOXANTRONE/ETOPOSIDE COMBINED WITH r h G M - C S F F O R DE NOVO ACUTE MYELOGENOUS LEUKEMIA R. Haas, H. DOhncr, R.Ehrhardt, H. Goldschmidt, B. Witt, H. Huberts, M. Gocbel, F. Del Valle, A.D. Ho, and W. Hunstein

SIGNIFICANCE OF PROGNOSTIC FACTORS DURING AN I D A R U B I C I N (IDR) C O N T A I N I N G R E G I M E N IN A D U L T A M L

Since 12/90,18 patients (p) (8 female/10 male) with de novo acute myelogenous leukemia (AML) were included into the study. Their median age was 49 years (range 21 63) and the distribution accordingto FAB was: 2 M 1,7 M2, 0 M3, 6 M4, 2 M5, 0 M6,1 M7). The cytogenetic data for 8 p are as follows: 4 normal karyotype, It (8;17), 1 del (5q), 1 t (8;21) and 1-5/-7/+8. All patients started with Idambicin (12 mg/sqm/day i.v., d 1-3) and Ara-C (100 mg/sqm/d, cont. inf., d 1-7) (IDAC). Patients achieving complete or partial remission (CR/PR) received a 6 day cont. infusion of rhGM-CSF (3 ug/kg/d, Essex) starting 3 days prior to the second cycle of IDAC. For the consolidation cycle consisting ofMitoxantrone ( 10 mg/sqm/d i.v., d 1-5) and Etoposide (100 mg/sqm]d i.v., d 1-5) (NOVE) rhGM-CSF was administered withidentical dose schedule. The initialnonresponders to IDAC received NOVE without rhGM-CSF. Of the 18 p, 16 are evaluable for response so far. 10 p achieved CR (62.5%) and 5 p were refractory (31.2%) to IDAC/NOVE. Olle patient was cxcludcd after rcadfi,g t'K ~ i!h one cycle of IDAC because of severe infectious complications during the phase of neutropenia. With the exception of one patient (thrombophlebitis at the second day of administration), the rhGM- CSF administration was tolerated very well with no to xicities >WHO 2. From a baseline of 5.6/rd (median, range 2.1 - 12.4) the WB C showed an increase to 19.0/hl (median, range 10.9-45.8) after the first 3 days ofrhGM-CSF administration without the appearance ofmyeloblasts in the peripheral blood. Interestingly, platelets fell from amedian baseline of 384/nl (range 206-1010) to a median of 272/nl (range 155-803). The 10 p who initially achieved CR, are disease-free with a median follow up of 187 days (range 98-314). The response-adapted treatment with IDAC/NOVE combined with rhGM-CSF forpatients achieving PR or CR after the initial cycle is effective and very well tolerated without treatment-related mortality. To evaluate the impact of rhGM-CSF on remission duration a higher number of patients and longer follow-up are needed. DepartmentofInternalMedicineV, H0spitalstr.3, W-6900Heidelberg,FRG

Lambertenghi-beliliers G., Annaloro C., Pozzoli E., Oriani A., Mozzana R., Polli E.E. - BMT Unit, University of Milan, Italy We have evaluated the influence of traditional prognostic factors in first diagnosis adult AML patients treated with IDR during induction and post-CR and, when possible, with autologous bone marrow transplantation (ABMT) as late intensification, by means of univariate statistical analysis and discriminant analysis according to Mahalanobis. Analysis of correlations showed a satisfactory degree of relative independence among the variables. Seventy seven patients (47 male and 30 female) with a median a~le of 46 (range 15-64 years) underwent treatment, and the duration of follow up was 6-88 months. CR was achieved in 64 cases (83.1%), 55 after one induction cycle and 9 after two, with a median DFS of 26 months. Univariate analysis showed that none of the variables individually correlated with either CR or DFS. Discriminant analysis allowed a function for CR (with a predictive level of 71.4%) to be obtained which took into account age, sex, hemoglobin and platelet levels, lymphoadenopathies, spienomegaly, tonsillary infiltration and the dllrRfion of mwlndP.orassion. Median DF~ wa.~ .~ianificantlv better in patients ach~ewng GH alter only one ~naucuon cycle inan in those requiring two (27 vs 6 months), and in patients undergoing ABMT in comparison with a selected group of other patients matched for all prognostic factors except age (87.5% vs 61.7% probability of DFS after 5 years). Finally, a discriminant function with a predictive level of 74.07% was calculated, which took into account sex, hemoglobin levels, lymphoadenopathies and splenomegaly, and compared cases of relapse with those of patients in CR > 17 months (the maximum interval to relapse observed in our population). The limited relevance of traditional prognostic factors provides further evidence in favour of IDR: some of them, such as the number of leukocytes and FAB classification, were not considered in the two discriminant analyses, while others, including age, were excluded by the DFS function. This last finding also offers an interesting contribution to the discussion concerning the efficacy of A B M T in prolonging DFS, insofar as the transplanted group was not comparable with internal controls only in terms of age.

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GM-CSF IN A DOUBLE-BLIND RANDOMIZED, PLACEBO CONTROLLEDTRIAL IN THERAPY OF ADULT PATIENTS WITH DE-NOVO ACUTE MYELOID LEUKEMIA (AML)

MYELODu REMISSION IN DE NOVO ACUTE MYELOID LEUKAEMIA(AML); IMPLICATION FOR EARLY RELAPSE AND PUTATIVE MONOCLONAL REMISSION K. Nagal, T. Matsue, K. Kuriyama, I. Jinnai*, and M. Tomonaga

G. Hell, L. Chadid, G. Seipelt, P. Mitrou, D. Hoelzer, K. Kolbe, Ch. Huber, A. Lindemann, R. Mertelsmann, J. Frisch, U. Nicolay, W. Gaus and H. Heimpel This multicenter trial initiated in 5/90 addressed the question whether the addition of rhu GM-CSF to a standard cytotoxic regimen improves event free survival of AML patients. Furthermore we wished to reduce the incidence of infections by shorting the duration of critical neutropenia. Induction and early consolidation therapy included cytarabine (Ara-C, 100 mg/m 2, day 1 -8 civi), daunorubicin (60 mg/m =, IV, day 3 - 5,) and etoposide (100 mg/m 2, day 4 8, 2 h IV infusion) with reduced dosages in the second induction and early consolidation course. Late consolidation included one cycle with high-dose Ara-C (3g/m 2, 12 doses) and daunorubicin (30 mg/m 2, day 7 - 9) for patients aged 50 years and younger, whereas patients over 50 years received a reduced dose of Ara-C (0.6g/m =, 12 doses). Patients were randomized after the first induction course according to response to therapy and age to receive either rhu GM-CSF (E. coil, 250 pgl m2/ day, s.c.) or placebo. GMCSF/placebo therapy was started 48 hours prior to the second induction and the subsequent courses and was stopped after chemotherapy induced aplasia at absolute neutrophil count > 500//A. 35 out of 72 patients randomized so far (median age = 51 years) were evaluable for response to therapy. 24/35 patients (69%) achieved a complete remission (CR), 10 patients (28%) were treatment failures and 1 patient (3%) died during the first induction course. 90 percent of the good responders after the first induction course (< 5% bone marrow blasts) and 33% of the bad/no responders achieved a CR by the second induction course. 7/24 patients suffered a relapse of the AML after a median CR duration of 4 months. Up to now none of the patients had to be withdrawn from the protocol because of severe toxicity and no evidence was found that the relapse rate was significantly increased in the GM-CSF group. Present address: Dept. Internal Medicine III, University of UIm, 7900 UIm,

FRG.

In view of recent controversy over the b i o l o g i c a l s i g n i f i c a n c e of remi -ssion phase in AML, we investigated the incidence of myelodysplastlc f e a t u r e s in the remission marrow of AML p a t l e n t s to e l u c i d a t e whether or not they have pathophysiologlcal and prognostic implications. 46 of de nero AML cases in complete remission induced with conventional chemo - t h e r a p e u t i c protocol(behenoil ara~C or ara-C+daunorubisine+6MP+ prednl -selene) were entered into t h i s study. A s e r i e s of good q u a l i t y smears prepared in complete remission phase were observed f o r various myelodys - p l a s t i c changes according to the FAB morphological c r i t e r i a for the c l a s s i f i c a t i o n of myelodysplastic syndrome(HDS). Mieromegakaryocytes( 30.45), megakaryocytes wlth multi-separated nuclei(45.75), degranulated neutrophlls(39.15), and neutrophils with hyposegmented nuclei(34.8~) were observed with a s i g n i f i c a n t l y higher incidence compared with the i8 CR cases of common type acute lymphoblastic leukaemla. When compared with the c l i n l c a l f e a t u r e s at i n l t l a l diagnosis in AML cases, the dys -megakaryocytic changes in remission marrow were found to be s i g n i f i c a n -tly more frequent Jn patients with monocytie involvment or trillneage myelodysplasia(T-MDS AML). Disease free survival was significantly shor -ter in patients with micromegakaryocytes(p
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76 ETIOLOGIC AND CLINICAL IMPORTANCE OF MUTATIONS IN THE RAS PROTOONCOGENES IN de novo ACUTE MYELOID LEUKEMIA (AML). A.Neubauer, D.Sandler, R.Dodge, J.Taylor, D.Shore, E.BalI, R.McIntyre, C.Bloomfield, E.Liu. Abteilung H~matologie, Uni-Klinik Rudolf Virchow, Spandauer Damm 130, WI000 Berlin 19, and Lineberger Cancer Center, University of North Carolina, Chapel Hill, NC, 27599. Mutations in the ras protooncogenes are the most frequent genetic alteration observed in AML. The significance of such mutations was studied prospectively in a well characterized CALGB cohort. Using the polymerase chain reaction (PCR) and mutational specific radioactively labeled oligonuclectides, mutations were found in 18/99 AML patients: N-ras in i0 cases, K-ras in 5, and concurrent Nand K-ras in three cases. The presence of mutant ras genes was found to be associated with a low percentage of blasts in the bone marrow (p=O.016). >50% of the ras positive cases were still alive after three years, whereas the median survival of the ras negative group was 13 months (log rank p=0.07). Upon multivariate analysis, ras mutations were the most significant predictor for improved survival after age (p=0.01). This improved survival was associated with a trend towards higher CR rate in the ras positive group (83% vs 62%, p=0.1). In an overlapping group with detailed occupational data (62 patients), patients with a ras mutation were significantly more likely to have worked 5 or more years in an occupation where solvents may have been used (odds ratio [0D]=6.8, CI 1.3-36), and specifically were more likely to have breathed chemical vapors (OR=9.1, CI 1.3-63), or to have had skin contact with chemicals at work (0R=6.9, CI 1.3-37). ras mutations may define an etiologic subgroup of de novo AML and portend for a good outcome after intensive chemotherapy.

78 Inv (16) m a y be one of the only "favorable" prognostic factors in AM L : a report on 16 cases. I.Plantier-Colcher, J.Li'Laf, C.Preudhomme, N.Cambier-Lot, L.Detourmignie8, F.Bauters, P.Fenaux. C H U Lille, France. With conventional chemotherapy, about 70% of AML patients (pts) achieve complete remission (CR), but only about 30% can be cured with current post remission approaches. Prognostic factors, including age, FAB subtype, initial leukocyte count immunophenotype and karyotype have helped identify subgroups with "favorable" prognosis. However, those "favorable" subgroups generally still have a probability of long remission below 50%. Among cytogenetics findings, presence of inv(16) and its variants (del(16), t(16; 16)) has been associated with favorable prognosis, although not in all studies. Between 1985 and 1991, we found inv(16) in 15 pts and del(16) in 1 pt 3 of them had received chemotherapyand radiotherapyfor a previous neoplasm. Median age was46 (range 23-66) and M/F 1,66. Extramed ullary leukemia was present in 8 cases but no patient had initial CNS involvement. Median leukocyte count was 18.10~/L (range 2.2-176). Cytologically, all pts had M4~osino. Pts were included in 3 successive chemotherapy protocols, containing an anthracycline - AraC combination followed by moderate consolidation and prolonged maintenance (first 2 protocols) or intensive consolidation (protocol 3). All pts but 1 had CNS prophylaxis with intrathecal MTX + AraC and cranial RT. 15/16 pts achieved CR, and 1 pt died on day 2, from CNS bleeding. With a median follow up of 34 months (range 4 +-77+), 2 pts relapsed, after 26 and 40 months and 13 remained in CR (after 4* to 77+). Median actuarial disease free survival (DFS) was 78% after40 months. The relapse occurring after 26 months inyoived the CNS alone and concerned the only pt who had not received cranial irradiation. This pt has ngw been in CR2for 32 +months. The other relapse occurred in the bone m a r r o w ~ r t h e r a p y related cases, where a CR~ of 22 months was subsequently obtained (survival from diagnosis : 70 months). These results seem to confirm the "good" prognosis of AML with inv(16). By contrast, in our recently published experience in AML (Br. J. HaematoL 1989,73, 61-67) no other subgroup identified by age, leukocyte count, FAB type or eytogenetics was found to have a median actuarial DFS > 50%.

77

79

CYTOGENETIC FINDINGS DELINEATING KARYOTYPIC INSTABILITY IN ERYTHROLEUKEMIA C O R R E L A T E D WITH BAD PROGNOSIS D. Haase~ M. K r 6 g e r and C. F o n a t s c h , AG T u m o r c y t o g e n e t i k , Institut f~ir Humangenetik~ Medizinische Universit~it zu LLibeck, R a t z e b u r g e r Allee 160, D-2t400 LLibeck~ FRG. 25 p a t i e n t s with M6-ANLL a c c o r d i n g to the c r i t e r i a proposed by the F A B - g r o u p w e r e studied c y t o g e n e t i c a l l y . Alt h o u g h no e r y t h r o l e u k e m i a - s p e c i f i c a b n o r m a l i t i e s could be e l a b o r a t e d , some k a r y o t y p i c a b n o r m a l i t i e s w e r e found m o r e f r e q u e n t l y in t h e s e p a t i e n t s t h a n in a n y o t h e r FAB-subgroup. Besides an i n c r e a s e d r a t e of clonal a b e r r a t i o n s (68% vs. 52% in ANLL p a t i e n t s with all FAB-subtypes) and a high f r e q u e n c y of c o m p l e x a b n o r m a l i t i e s (65% of a b n o r m a l cases), the most striking c y t o g e n e t i c o b s e r v a t i o n in this leukemia subgroup was the k a r y o t y p i c instability which o c c u r r e d in 65% of all a b n o r m a l c a s e s (31.5% in de novo-ANLL). Non-clonal k a r y o t y p e instability o c c u r r e d as i n c r e a s e d c h r o m o somal b r e a k a g e , c h r o m o s o m a l r e a r r a n g e m e n t s and m a l d i s t r i b u tions and u n i d e n t i f i a b l e m a r k e r c h r o m o s o m e s . All of t h e s e events c o n t r i b u t e to cell to cell v a r i a t i o n s c o m p l i c a t i n g c y t o g e n e t i c analysis. H o m o g e n e o u s l y staining regions (HSR), k a r y o t y p e evolutions and polyploidizations w e r e observed as clonal p h e n o m e n a . Using c h r o m o s o m a l in situ suppression hybridization (CISS) t h e b r e a k a g e of c h r o m o s o m e s into s e v e r a l pieces and t h e i r i n t e g r a t i o n into the a b n o r m a l c h r o m o s o m e c o m p l e m e n t resulting in the g e n e r a t i o n of clonal m a r k e r c h r o m o s o m e s could be d e m o n s t r a t e d . In one c a s e the c h r o m o s o m a l instability was r e s t r i c t e d to c h r o m o s o m e band 1 2 p l l . 2 , which was the s t a r t i n g - p o i n t for the f o r m a t i o n of four d i f f e r e n t cell clones c h a r a c t e r i z e d by t r a n s l o c a t i o n s b e t w e e n 12p11.2 and different partner chromosomes. It c a n be s p e c u l a t e d t h a t the k a r y o t y p i c instability c o n t r i b u t e s to bad prognosis, b e c a u s e it m i g h t enable t h e m a l i g n a n t cell population to r e a c t rapidly to s e l e c t i v e pressures arising f r o m c h e m o t h e r a p y with the g e n e r a t i o n and expansion of r e s i s t a n t l e u k e m i c cell clones. This s'r was s u p p o r t e d by the D e u t s c h e Krebshilfe - Dr. Mildred S c h e e l - S t i f t u n g , p r o j e c t M 16/90/Fo 3.

ALL TRANSRETINOICACID (ATRA) iN NEWLY DIAGNOSED ACUTE PROMYELOCYTIC LEUKEMIA (APL) : CLINICAL EXPERIENCE OF THE FRENCH GROUP. P.Fenau:<, S. Castaiene. H. Dombret, C.Chomienne, M.Duarte, E.Archimbaud, T.Lamy, P.Tiberqhien, H.Tllly, PMaloisel, M.Cransac, A.Guerci, A.Sadoun, L.Deqos. APL group, France. ATRA is capable of inducing differentiation of abnormal promyelocytes into mature cells in APL, and may reduce the incidence of early deaths in this disease by improving DIC and avoiding a period of aplasia. In previous reports, we showed that ATRA was very effective in relapsing APL (Lancet 1990, ii, 1440), and in newly diagnosed APL in patients (pts) with contraindication to chemotherapy (Blood 1990, 76 t704). In the latter group, however, rapidly increasing leukocyte counts, often leading to fatal leukostasis, were a major side effect. Furthermore, pts rapidly relapsed ifATRAwas continued alone, and no intensive chemotherapy was administered in CR. We then conducted a pilot phase of ATRA in 27 ptswith newlydiagnosed APL and no contraindication to intensive chemotherapy. Median age was 43 (range 25-59). Pts were scheduled to receive 45 mg/ma/day of ATRA, until CR achievement. This was followed by an intensive Daunomycin (DNR) 60 mg/ma/d d~.4 + AraC 100 mg/m2/d d~_7 course. In order to avoid leukostasis due to rapidly increasing leukocyte count, however, this course was started early (ATRA being continued until CR) if leukocytes were 6 x 109/I by day 5 of ATRA therapy, or > 10 x 109/I by day 10 or 15 x 10s/ by day 15, because in our experience there was a major risk ofleukostasis above these thresholds. After the DNR - AraC course, pts received 3 consolidation courses with the same d rugs and maintenance. Twenty six pts achieved CR, after 35 to80 days, 15 of themwith ATRAalone, and 11 after the DNR AraC coursewas added to ATRAon day 2 to30, because of hyperleukocytosis (9pts) resistance toATRA (1 pt) and developement of organomegaly (1 pt). The remaining pt died from CNS bleeding, on day 4. Apart from hyperleukocytosis, side effects were mild. With a median follow up of 14 months, 2 relapses were seen after 8 and 12 months, one of them in the only pt maintained on ATRA alone. The APL91 protocol, randomizing this pilot arm and chemotherapy alone in newly diagnosed APL, has now started in France, and will start soon in other European countries. 21 pts a r e currently evaluable. In the ATRA arm, 11/12 pts achieved CR, and one pt died a few hours after treatment onset. ]n the chemotherapy arm, 8/9 pts achieved CR, and lpt had resistant leukemia (salvaged by ATRA). However one of the pts who achieved CR with the latter arm had CNS bleeding and remains severely disabled. Results will be updated, and fundamental aspects of ATRA therapy in APL will be presented at this meeting by L.Degos in another abstract.

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81

T h e r a p y related acute promyelocytic leukemia (tAPL) : a report on 11 cases, L.Detourmignies, S.Castaigne,A.M.Stoppa, I.Plantier-Colcher, A . S a d o u n , M J a n v i e r , J.L.Demory, J.L.La',', R.Berger, F B a u t e r s , P.Fenaux. C H U Lillle, St Louis Paris, St Cloud Marseille, Poitiers, France. Therapy related AML (tAML) is usually related to the use of alkylating agents~. radiotherapy, is preceded by a preleukemic phase, has chromosome 7 and/or 5 deletions and a poor prognosis. Patients with tAML and karyotype specific of de novo AML (t(8;21), inv16, t(15;17), t(9;11)), have been reported, however. In the case of t(9',11), Pedersen Bjergaard etaL showed that topoisomerase II inhibitors (especially epipodophyllotoxins) rather than alkylating agents, were causative agents. In 5 French centers, we could gather 11cases of tAPL diagnosed between 1988 and 1991. Median age was 45 (range 12-82, 9 females and 2 males). Previous neoplasm was a solid tumor in 9 pts (including 7 breast carcinomas and 2 cerebral tumors) and non Hodgkin's lymphoma in 2 pts. 1 pt had received radiotherapy (RT) alone, 1 chemotherapy (CT) alone and 9 CT + RT. Previous CT included Cyclophosphamide (Ex) in 5 pts, Adriamycin (Ad) or Mitoxantrone (MTZ) in 5 pts, 5 FU in 5 pts, vinca alkaldl'ds in 4 pts, and VP~6in only 1 pt Median interval from the onset of previous treatment and tAPL diagnosis was 24 months (range g-72). Morphologically, all lots had "classical" APL. Only 1 pt had hyperleukocytosis. DIC was present in 9 pts. A karyotype was performed in 10 pts : 9 pts had typical t(tS;17) translocation, with additional rearrangements in 3 of them (-7;16,18*;t(1 ;5)). 1 pt had normal karyotype. 4 pts were treated with conventional chemotherapy (DNR + AraC), and all 4 achieved CR, of 6, 4 +, 6 +, 10 § months duration. 7 pts received ell transretinoic acid (ATRA) and 4 achieved CRT of 2 +, 2 § 3', 36 + months duration whereas the 8 other pts did have differentiation with ATRA but died from leucostasis. Our findings suggest that tAPL is not exceptional. It largely predominates in females and in pts with previous breast carcinoma. Because the 3 drugs EX, 5FU, Ad or MTZ were combined in 4 cases, it was difficult to determine if one or several of them could be especially implicated in pathogenesis. Morphological, cytogenetic features and response to treatment of tAPL do not seem to differ from those of de novo APL (although, for treatment outcome, follow up is still limited in the present series).

MONOSYMPTOMATIC RELAPSE WITH SKIN INVOLVEMENT

OF ACUTE

MYELOID

LEUKAEMIA

Tebbe M*, Hamm H I, Bonsmann G I, Innig G 2, Kolde G I iDept Dermatol, 2Dept Internal Med, University of MUnster, MUnster, Germany In June 1991, a 26 year-old patient was diagnosed with an acute myeloid leukaemia (FAB-M5). There was no skin involvement at that time. Complete remission was achieved by induction therapy according to the standardized TAD-HAM protocol. Three months later, the patient developed disseminated oval shaped, firm, skin-coloured nodules and plaques on the trunk, scalp and arms. The lesions were arranged in l i n e s of cleavage on the trunk. Histologic and electron microscopic examination revealed dermal infiltrates of immature, atypical myelocytic cells. At this time, no leukaemic cells were found on bone marrow aspiration. The cytostatic therapy was changed to the S - H A M p r o tocol and this resulted in complete remission of the cutaneous infiltrates, on both clinical and histologic examination. This case report demonstrates the clinical features as well as the histologic, cytologic and ultrastructural characteristics of skin involvement in the course of acute myeloid leukaemia. Present address: Department of Dermatology, University of MUnster, Von-Esmarch-Str. 56, D-4400 MUnster, FRG Department of Internal Medicine, Albert-SchweitzerStr. 33, University of MUnster, D-4400 MUnster, FRG

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Bone Marrow Transplantation 82

84

ALLOGENEIC BONE MARROW TRANSPLANTATION (BMT) IN PATIENTS WITH AML IN I.CR: PROGNOSTIC FACTORS FOR SURVIVAL?

GRAFT REJECTION AFTER ALLOGENEIC BONE MARROW TRANSPLANTATION (BMT): THE IMPACT OF T CELLS, GVHR AND MARROW CELL NUMBER. Uharek L, Glass B, Gassmann W, Bolouri H. Focks B, Loeffier H, MuellerRuchholtz W. Graft rejection is a frequent complication after transplantation of T cell depleted (TCD) marrow grafts. Possible causes are: (1) lack of lymphocyteinduced hematostimulation, (2) absence of GvHR-induced immunosuppression, (3) loss of hematopoietic cells, To investigate (1), lethally irradiated (7.5 Gy) Balb/c mice received MHC-mismatched but GvH-nonreactive (BalbxC57)F1 marrrow. Rejection rates (death with neutrophils < 500/ul) after grafting of a final dose of 0.1, 1, and 10 x 106 F1 cells were 36%(n = 11), 14% (n = 14), and 0% (n = 15) for unmanipulated BM (NTCD) and 45%(n = 11), 8%(n = 12), and 0%(n = 12) after TCD with anti-Thy1. There was also no influence of TCD on chimerism: a median of 47% (NTCD) vs. 36% (TCD), 79% vs. 78%, and 83% vs. 94% of H-2b+ donor cells was detected at day 50. Thus, our data revealed a marked cell-dose effect but no engraftment-promoting activity of GvH-nonreactive T cells. To investigate (2), highly GvH-reactive C57 BM was transplanted into BalP/c mice. After injection of 0.1, 1, 10, and 40 x 106 unmanipulated cells, the incidence of rejection was 87%(n=B), 67%(n=26), 21%(n=33), and 0%(n = 17). Similar rejection rates were observed after TCD with anti-Thy-l: 87% (n = 8), 64% (n = 14), 27% (n = 33), and 0% (n = 17). Similarly, TCD with anti-CD4/CD8, or Leu-Leu-OMe successfully prevented GvHR but did not affect rejection rates and long-term chimerism. Our data demonstrate that the number of BM cells is critical for successful engraftment. Therefore, harvesting of large cell numbers and/or intensified immunosuppression is necessary when cell-consuming purging techniques are applied. Present address: Depts. of Immunol. and Int. Medicine II, Univ. of Kiel, Brunswiker Str. 4, Germany.

R. Arnold, D. Bunjes, B. Hertenstein, D. Hueske, J. Novotny, M. Stefanic, M. Theobald, M. Wiesneth, H. Heimpel Between 1980 and 1991 60 adult patients with AML in first complete remission (CR) underwent allogeneic bmt. 39 patients are alive, 21 patients are dead. Causes of death were transplant associated complications (n = 16) and relapse after brat (n = 5). Overall 8 out of 60 patients relapsed. The probability of survival is 58% and the probability of disease free survival (DFS) 52%. A statistical analysis on risk factors predictive for survival, disease free survival and transplant related mortality was performed. Neither FAB type nor leukocyte count at diagnosis, time to response to induction therapy or duration of CR before brat were significant. Regarding bmt, the kind of GvHD prophylaxis and the incidence of GvHD were tested and had no significant influence on survival or disease free survival. GvHD had a significant influence on transplant related mortality (p 0.02). In conclusion: AIIogeneic bmt for AML in 1 .CR leads to a high rate of long term survivors in complete remission (52%). But no prognostic factors for DFS were found, indicating that induction chemotherapy and conditioning therapy before brat are effective in eradicating leukaemia, when an allogeneic transplant is performed. The allogeneic effect of the graft remains to be studied. Depts. Internal Med. Ill and Transfusion Medicine, University of UIm, D-7900 UIm, FRG. supported by Deutsche Krebshilfe

83

85

COMPARISON OF HIGH-DOSE BUSULFAN (HD-BU) AND TOTAL BODY IRRADIATION ('rBI) PRIOR TO ALLOGENEIC BONE MARROW TRANSPLANTATION (BMT). Sayer H.G., Beelen D.W., Quabeck K., Mohnke M., Oidtmann M. and Schaefer U.W., Department of Bone Marrow Transplantation, University Hospital of Essen, 4300 Essen, Germany. The results of two preparative regimens prior to alidgeneic BMT for AML in 1= remission using either fractionated TBI (4 x 2.5 Gy, dose rate 3cGy/min, lung dose 8 Gy) or HD-BU (4 mg/kg body weight over 4 days PO) followed by 120 mg/kg body weight cyclophosphamide (Cy) were retrospectively compared over a 5-year period (9/86 to 9/91 ). Immunoprophylaxis of acute graftversus host disease (GvHD) with a short course of methotrexate (MTX) and cyclosporine (CSA) as welt as all other supportive measures were identical for the 41 recipients of HLA-identical sibling marrow transplants (TBI n=19 [group I], HD-BU n=22 [group liD. To estimate the influence of the allogeneic component on adverse events, 36 pts with AML in 1s' remission who had been treated with an identical HD-BU and Cy combination prior to autologous BMT during the same time period were taken as controls (group III). The median time intervall between diagnosis and BMT was 7 (3 to 21 ) months. With a median onset at day 23 (19 to 28) the cumulative incidence of developing hepatic veno occlusive disease (VOD) was 19+18% for pts of group II, while none of the pts of either group I (p<.05) or group III (p<.01) contracted VOD. Peak levels of bilirubin, alkaline phosphatase (AP) and aspartate aminotransferase (hAT) were not different between group I and group II, although peak enzyme levels were reached significantly earlier in group II. Kaplan-Meier product limit estimates of interstitial pneumonia (IP), transplant-related mortality (TRM), leukemic relapse (R), and leukemia free survival (LFS) at 5 years after BMT are given in the table:

CONTINUOUS INTRAVENOUS PENTOXIFYLLINE IN MARROW TRANSPLANT RECIPIENTS - FIRST RESULTS OF A PILOT STUDY. Beelen D.W., Sayer H.G., Herstell M., Scheulen M.E., Ouabeck K., Mohnke M., Oidtmann M., and Schaefer U.W. Dept. of Bone Marrow Transplantation, University Hospital of Essen, Germany. Recent preliminary clinical reports and experimental data suggest that pentoxitylline [(3,7-dimethyl-1-(5-oxohexyl)-xanthine] (PTX) may be useful in ameliorating or preventing some of the indispensible toxicities and adverse events associated with the marrow transplant procedure. To allow constant and high bioavailability of the compound and its major active metabolite 1-(5hydroxyhexyl)-3,7-climethylxanthine, a pilot study in which PTX is given by continuous intravenous infusion between day -10 and discharge is currently performed in allogeneic marrow transplant recipients. On each dosage level of PTX (10-15-20 mg/kg/day) five patients (pts) are enrolled in this study. A total of 14 pts (2 S, 12 o', median age 32 [20-53] yrs. HLA-identical sibling donors: 8 pts, mismatched related donors: 4 pts, matched unrelated donors: 2 pts) have entered the protocol. The major adverse effect of PTX treatment was nausea and vomiting (NAV) (grade 0 : 7 pts, grade h 4 pts, grade Ih 1 patient, grades Ill-IV: 2 pts), which necessitated dosage reduction in 1 patient treated by 20 rng/kg PTX. One patient developed myoclonia and acute obtundation during the preparative regimen, but these events were not unequivocally related to PTX administration. Comparisons of product-limit estimates of acute graft-versus-host disease (aGvHD) of grades II-IV, interstitial pneumonia (IP), and transplant-related mortality (TRM) at day 100 after transplantation between control patients matched for underlying disease, disease status, patient age, immunprophylaxis, and histocompatibility and study patients is given in the table (all comparisons not significant).

IP group I n=19

16.-1:16%

group II n=22

20•

TRM

R

LFS

24+22~

6+6%

71:1:11%

5:L5%

67+10%

j 29+20%

In conclusion, these data indicate that HD-BU and fractionated TBI lead to similar results after allegeneic BMT in pts with AML in I s' remission. The observation that hepatic VOD occured preferably in pts pretreated by HD-BU and Cy, however, suggests a more pronounced toxic effect on liver function as compared to the combination of TBI and Cy. The fact that pts in the autoIngous setting did not develop VOD may point tO a cumulative toxic effect of HD-BU and Cy and other posttransplant medications, like MTX and/or CSA.

aGvHD (%)

IP (%)

TRM (%)

controls n= 13

27=J:26

16=~?.0

23+~4

PTX n= 14

55:1.36

11 +20

11 .+.20

These preliminary results indicate that continuous infusions of PTX are tolerable up to a dosage of 15 mg/kg in marrow transplant recipients with NAV being the dose-limiting adverse effect. Pharmacokinetic studies of PTX and its major metabolite are currently performed to further define the most suitable intravenous treatment schedule.

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AUTOLOGOUS TRANSPLANTATION WITH MAFOSFAMIDE-PURGED BONE MARROW FOR ACUTE MYELOGENOUS LEUKEMIA (AML) IN FIRST AND SECOND COMPLETE REMISSION (CR) R. Ehrhardt, G. Bnschmann, S. Hohaus, E. Ogniben, B. Witt, and R. Haas

AUTOLOGOUS BMT IN PH'-POSITIVE ALL: CELL LINE BV-173 AS A

The therapeutic benefit of autologous bone marrow transplantation (ABMT) for patients (p) with AML in CR 1 and CR2 is still not clear, because prospective randomized clinicaltrials comparing autotransplantationand conventional chemotherapy are lacking. Since 9/83, we have autografted 35 p with AML in CR1 ( 18 male/17 female) and 37 p in CR2 (22 male/15 female). Their median age was: CR 137 years (range 1650) and CR2 36 years (range 17-50). The median interval between achievingthe most recent CR and autografting was: CR1 169 days (range 85-554) and CR2 108 days (range 30-633).With the exceptionof 2 p (1 CR 1/1CR2), the bone marrow was purged with mafosfamide. The pretransplantconditioningregimen consisted of total body irradiation ( 12.1 to 16.7 Gy) and cyclophosphamide (200 mg/kg bw). A total of 65 p are evaluable for leukemia-free survival (LFS), because 7 p (9.7%) died of transplantation-related complications. Of the p transplanted in CRI, 19 p (54%) have a median LFS of 39 months (range 4 - 85), whereas 13 p (37%) relapsed after a median time of 6 mo ( range 2-12) following ABMT. Interestingly,p with a short interval between CR and transplantation had a lower probability of LFS than those autografted late in CR (< 4 mo: LFS =25%, 4 to 6 mo:LFS=63.6% and > 6 too: LFS=66.7%). In the group of p autografted in CR2, l I p (29.7%) are in continuous CR after a median foUow-up of 40 me (range 1 - 80) and 22 p (59.4%) relapsed after a median time of 6 mo (range 332). In each patient group 26 p are evaluable for hematological reconstitution: CR1 (days to reach) CR2 (days to reach) WBC 1.0/nl 27(12-66) 20 (11-42) PMN O.51nl 34(13-118) 32 (14-61) PLT 20~11 77 (11-623) 38 (15-251) The relatively high LFS of patients transplanted in CR l might reflect a selection bias excluding patients with early relapse, concomitant disease or inzufficient autograft. For patients in CR2 ABMT can offer a potentiallycurative treatment which should be evaluated in controlled prospective clinical trials. Department of Internal Medicine V, Hospitalstr. 3, W-6900 Heidelberg, FRG

MODEL FOR IN VITRO PURGING.

H. Martin 1, R, Claudd1, C. Schardt 1, M. Stadler 1, J. Brficher 1, HG. Drexler 2, A. Ganser 1 and D. Hoelzer1, 1Dept. of Hematology, University Clinic of Frankfurt / Main. and 2DSM, University of Braunschweig. Philadelphia-Chromosom-positive (Ph +) ALL has a very poor prognosis (0 - < 10 % Iongterm survival) after treatment with standard chemotherapy. Autologous BMT may improve survival in patients without compatible allogeneic donor. We use incubation with Mafosfamid and/or immunomagnetic beads to purge autologous bone marrow from residual leukemia. To evaluate the effectivenas of these methods in Ph +-ALL, we used the Ph + pre-B-ALL-cell line 8V-173 as a model. BV-173 is rapidly growing in liquid cultures (doubling ;time 36-48 h). 90 -100 % of BV-173 cells express HLA-DR, CD10, CD19, and 50% CD34 -antigens. In semisolid media, about 3% of BV-173 cells form large elonogenie co~oniea. Southern blot ana!;'sis of the bcr-ab! loci rev~a!s beth M- and mbcr-abl translocatlon. After incubation for 30 rain at 37 ~ 95 % of proliferating BV-173 cells were killed at Mafo-concentrations of 10 - 30 pg/ml compared to 70-120 ,ug/ml for normal bone marrow CFU-GM. In liquid cultures however, BV-173 cells reappeared 3-8 weeks after Mafo purging at > 40 pg/ml. We conclude (1) that 14-days clonogenic assays are not sufficient to monitor Mafo-purging and (2) that long-term liquid cultures detect minimal numbers of viable BV-173 cells. BV-173 proved further useful to monitor purging with McAbs AB4 (anti-DR), anti -CD10 and -CD19 and immunomagnetic beads. In dilution experiments with normal bone marrow, BV-173 cells were detectable at concentrations of 10-4 to 10-5 using polymerase-chain-reaction (PCR) for bcr-abl. After purging marrow contaminated with 10% BV-173 cells no residual bcr-ablrearrangement was detecable by PCR. Present address: Department of Hematology, University Clinic of Frankfurt, Theodor-Stern-Kai 7, 6000 Frankfurt/M. 70

87

89

INTERLEUKIN-2 BOLUS INFUSION AS LATE C O N S O L I D A T I O N T H E R A P Y IN 2ND REMISSION OF A C U T E M Y E L O C Y T I C L E U K E M I A WITH A N D W I T H O U T A U T O L O G O U S BONE M A R R O W T R A N S P L A N T A T I O N B I O L O G I C A l EFFECTS A N D FIRST C L I N I C A L R E S U L T S Bergmann L ~ , Hell G.2 , F,l~nchel K~ , EJr~cher ~1. f - , Bunjes ~ , Lohmeyer J.'~, Gunsilius E.~, Mitrou P.S!

THE IMPACT OF NATURAL KILLER CELLS AND GVHR ON GRAFT-VERSUSLEUKEMIA (GVL) ACTIVITY. Glass B, Uharek L, Gassmann W, Bolouri H. Focks B, Loeffler H, MuellerRuchholtz W. Using two murine leukemia models, A-20 (B cell) and WEHI-3 (myelomonocytic), we have investigated GVL effects after allogeneic bone marrow transplantation. Both cell lines were of Balb/c (H-2d) origin. After injection of in-vivo passaged leukemia cells into syngeneic mice, 106 cells invariably lead to splenomegaly (> 0.2 g) and death with a median survival time (MST) of 16 (A-20) and 15 days (WEHI-3). Prior immunization with irradiated A-20 or WEHI-3 cells did not affect MST. A lethal dose of total body irradiation (7.5 Gy) followed by syngeneic BMT at day 5 after inoculation of 106 tumor cells prolonged survival significantly (A-20: MST = 27 days, WEHI3: MST = 42 days). After syngeneic BMT, relapse rates of 100% (A-20, n=23) and 39% (WEHI-3, n=18) were observed. Transplantation of 4x107 allogeneic F1 (C57xBalb/c) (H-2bxd) marrow cells revealed superior results. In case of A-20, MST increased from 27 (n=23) to 32 (n = 14) days (p < .05). The relapse rate was only marginally reduced (92 vs. 100%). This GVL-effect was absent after T-depletion of BM with a broad-range antibody (Thyl.2: MST 28 days, relapse rate 100%, n= 12). In case of WEHI-3, there was no constant antileukemic effect of unmanipulated F1(C57xBalb/c) BM in=20). In vitro experiments showed A-20 to be sensitive to NK cell mediated lysis whereas WEHI-3 was resistant to NK cells but sensitive to LAK cells. Experiments with WEHI-3bearing animals and IL-2-activated BM grafts are ongoing. Conclusions: (1) Since GvHR is excluded genetically in the FI->P system used here, our data indicate that GvH disease is no prerequisite for GvL activity. (2) Susceptibility of leukemia to in-vivo GVL effect correlates with sensibility to in-vitro NK-cell-mediated lysis. Present address: Depts. of Immunol. and Int. Medicine II, Univ. of Kiel, Brunswiker Str. 4, Germany.

In the elimination of minimal residual disease (MRD) in AML the induction of graft versus leukemia (GvL) reaction by activation of autologous cytotoxic cells may be important. Here the induction of GvL-like reaction in AML by 11-2 is an interesting approach for the prolongation of disease free survival. We conducted a phase-II trial in patients (pts.) with de hove AML in 1st relapse with an induction therapy consisting out of 2 cycles iHD-AraC (2x600 m g / m 2 d l - 4 ) and VP-16 (100 m g / m 2 dl-7). Pts. achieving, CR receive a 3rd cycle for consolidation. After 4 weeks 9 x 10 ~ lU/m 2 rll-2 (EuroCetus, Frankfurt, FRG) is applicated as l h infusion day 1-5 and 8-12. This c y c e will be repeated every 6 week. Patients undergoing a-BMT receive rll-2 starting latest 6 weeks after BMT, Up to now 9 pts. could be included into the It-2 consolidation therapy, 3 / 9 after a-BMT. The remission duration so far s 2+ 22+ mths., the mean is not yet reached. Up to now only one pt. relapsed after 11 mths. The toxicities were moderate, so that rll-2 could be applicated in selected pts. in an outpatient manner. Biological effects as rebound lymphocytosis including CD25, CD56 and DR expressing cells occurred. Additionally, we found an up-regulation of adhesion molecules. The NK- but not LAKactivity increased significantly. During 11-2 therapy secondary cytokines as TNF, IFN-y and 11-6 were induced in remarkable amounts. In conclusion, this approach with 11-2 is practicable and induces biological effects with so far promising results. 1 Divi~,ion of Haematology, J.W. Goethe University Frankf~t, FRG; ZDepartment of Haematology, University Ulm, FRG; Medical Clinic, University GieBen

A95 90

92

M i ~ PAIR i ~ I S C 0 ~ i ~ C BORE l & ~ ~ T I G N IN FIRST CI~R~R ~ I G N WITH ~u~i~z~ISSION C ~ , ~ IN r R 1 ~ r ~ GF ~aE ~xuuIES E[~4-83 ARO -aq

AUTOLOGOUS AND ALLOGENEIC BONE MARROW TRANSPLANTATION (BMT) AFTER IDENTICAL HIGH-DOSE CHEMOR A D I O T H E R A P Y 1N CI-fiLDREN W I T H R E L A P S E D A L L Sehmid H. 1, Schwerdtfeger R. I, Henze G. 1, Banmgartan E. 1, Besserer A. 2, Scbeffler A. 2, Sehmidt-Wolf 11, Sehwella N. l , Zingsem J. l , Siegert W. l , Hnhn D. 1 Freie Universi~t Berlin, UKRV/C 1 and UKS2, Federal Republic of Germany

P. h ~ , U. Creutzig, Th. K1ingebiel, W. ~oe11, Ch. SenderG~tze, W. Friedrich, U.W. Sch~fer, H. Schmid, M. Sllttorp, J. Ritter There are two approaches to prevent relapses in children with AML: pcstremiesica~ chemotherapy and allogeneic bone marrow trausplantatic~ {EMT) in I~t complete remission (CR). This analysis compares 16 children with alloge~ic BMT in I-t CR (7 and 9 children from studies -83 and -87) with children from the non-grafted groups of these studies (114 and 143 patients). To avoid bias in patient selectic~ we have chosen patients fulfilling matching criteria for blast cell reduction day 15, FAB subtypes, white b l o o d cell count, age and time to C~. In addition patients with autologous EMT and / or with early relapses (defined as remission duration shorter than the median interval between m i s s i o n and allogemeic BMT [7.3 in study -83 and 3.6 months in study 87]) in the chemotherapy group have been excluded. 12 children are in continous complete remission (CCR) after ~MT (5 of study -83, 7 of study -87), 2 patients of study -83 have died from pneumonia after EMT, 2 patients of study -87 have relapsed I year after BMT. 11 children of the matched chemotherapy group are in CCR (4 and 7 children in studies 83 and -87). The relapse rate is low (3 and 2 patients in studies -83 and -87) prrgn~hly becatt_~e early relapses {s. above) have been excl,w%e~__. The event-free interval in the two groups is similar (0.73, SD 0.12 in the transplanted group; 0.67, SD 0.12 in the chemotherapy group); relapse free interval differs slightly (0.86, SD 0.09 in the transplanted group; 0.67, SD 0.12 in the chemotherapy group, n. s.). Two childrem of the transplanted group suffer from severe s e q u e l a e (convulsive seizures / hemiparesis) in contrast to none of the chemotherapy group. According to our analysis only those children are designated for allngeneic ~MT in I-t CR who are treated according to the AML-E~Mprotocols and who are at high risk of relapse. In additic~ to these criteria the children should be treated soonest after achieving CR. Present adress: Univ. -Kinderkl inik, Str.33, D-4400 M~nster, FRS

Albert -Schweit zer-

Introduction: Children suffering from ALL with high risk factors such as relapse within 18 months of diagnosis, relapsed T- or B-ALL or multiple relapses can not be cured by conventional salvage chemotherapy. Those patients (pts.) are candidates for allogeneic (allo) BMT even if relapse occurred after 18 months provided there is an HLA-matched related donor. If no donor is available autohigous (auto) BMT can be considered as an alternative treatment. The value of autoBMT compared to alloBMT remains to be determined. In this context we report on our results of autoBMT and alloBMT in children who were uniformly treated according to BFM chemotherapy protocols prior to BMT and uniformly conditioned by fractionated total body irradiation (tTBI) and VP-16. Patients and methods: Eleven pts. (median age at diagnosis 7 years, range 3 1/12 to 12 ]1/12, ALL phenotype: 8 c-ALL, 2 pre-T-ALL, 1 T-ALL) received unmanipulated bone marrow (BM) from HLA-identical siblings. Seven pts. were in CR 2 (64%), 3 in CR 3 (27%) and 1 in CR 4 (9%). Eighteen pts. (median age at diagnosis 4 years, range 1 5/12 to 15 3/12, ALL phenntype: 12 e-ALL, 4 T-ALL, 1 pre-B-ALL, 1 B-ALL) received autohigous BM which was purged (immunobeads) in 16 eases. Remission prior to BMT was induced by chemotherapy according to BFM relapse protocols. High-dose radio-chemotherapy consisted of fI'BI (6 x 2 Gy, day -6 to -4) and VP-16 (60 mg/kg, day -3). Results: As of Dec, 1991, 2/11 allografted pts. are alive in CCR (d +290, +729), and 5/18 autografted pts. are alive in CCR (d +213, +241, +423, +969, + 1033). Transplantation related complications accounted for death in 3/11 (27%) in alloBMT and 2/18 (I1%) in autoBMT. Acute GvHD > ~ after alIoBMT occurred in 6 pts. and was fatal in one, limited chronic GvHD was observed in 2 pts. Both relapsed. In the alloBMT group probability of event-free survival (EFS) is 14% and for relapse 81% at 24 months. In the autoBMT group estimated EFS is 25% and relapse 72% at 36 months. Bone marrow was the site of relapse in all patients. Conclusions: Our results must certainly be interpreted with caution (small mtmber of pts., no prospective study, no matched pair analysis) but so far do not provide evidence of superiority of alIoBMT to autoBMT. Failure of aIIoBMT and autoBMT in our children with high risk ALL was mainly due to early relapse. The high Incidence of relapse in these groups receiving the same conditioning regimen suggests that residual malignant cells give rise to recurrent leukemia. According to our results autoBMT provides a real chance for disease-free survival in pts. who do not have an HLAidentical sibling. Regarding the outcome in both groups the value of aIIoBMT with a matched unrelated donor must be carefully weighted. Kinderklinik dar FU Berlin, Hanbnerweg 6, 1000 Berlin 19, FRG

91

93

FRAGTIONA TED TOTAL BODY IRRADIATION OR BUSULFAN IN COMBINATION WITH ETOPO$1DE AND CYCLOPHOSPHAMIDE AS CONDITIONING REGIMEN FOR BONE MARROW TRANSPLANTATION IN ACUTE LEUKEMIAS IN CHILDREN C. .P.et~.., G. M_ap~ ,H. M.ay~r,W. Emminger, W. Emminger-Schmidmaier,

BONE MARROW TRANSPLANTATION DONOR - EXPERIENCES iN CHILDREN

T h . K l i n g e b i e l 1 , R . H a n d g r e t i n g e r 1, G . E h n i n g e ~ , R . D o p f e r t, D . N i e t h a m -

~g

We have grafted 8 children (median age 9.8 years) from an unrelated donor, representing the largest series of a single center in Germany. The diagnoses were 3 ALL (CR2 T-ALL; CR3 T-ALL; CR4 c-ALL), 2 CML, 1 AML (CR2), 1 juvenile CML and 1 SAA after failure of an immuno suppressive therapy. 6 children had a conditioning regimen consisting of accelerated fractionated TBI (12 Gy, lung shielding at 10.5 Gy), VP16 4 0 m g / k g and cyclophosphamid 120 m g / m 2 in two doses. The first child grafted from an unrelated donor at all in Germany was pretreated with high dose Ara C, Cyclophosphamid and TBI. The patient with SAA was conditioned with TLI 8 Gy and cyclophosphamid 200 mg/kg. GVHD prophylaxis was done with CSA in all cases except one, plus MTX in 6 children, plus additional anti-lL2rece ptor monoclonal antibody (BB10) in 3 cases. Only two children developed an acute GVHD grade 3 or 4, and no child a chronic GVHD. 4 children relapsed, and 3 of them died of relapse. 1 patient with a long lasting C M L (10 years) died from an acute GVHD grade 4; 1 patient died 161 days after BMT from a meningitis. Disease free survival is mean 137.4 + 99.1 days (median 129.5 days), and survival is mean 242.1 _+ 200.9 days (median 154). At present 3 of the 8 children are alive, 1 with lymphomatous relapse of a T-ALL Our experiences show that matched unrelated donor transplantations are feasible in childhood without severe side effects. Results are unsatisfactory not because of the method, but because of the bad r i s k selection of the children being eligible for unrelated donor translantation in childhood. apartment of Pediatric Oncology, 2Department of Internal Medicine, Eberhard-Karls University, D-7400 T0bingen, FRG

R- _Hg_ M~_k- P.H6_c_K_a_r,_~-~aa_n_ _er_. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 conseculive patients (pts) in a single center received a intensified conditioning regimen for allogeneic (allo) (n--24) or autologous (auto) (n=14) bone marrow transplantation (8M'F).24 pts were male, 14 female, age was 3 months (rots) to 18 years (mad: 9,4 years). Median observation time (Nov 1991):19,4 rots (39-4 rots). Diagnosis before BMT ware: ALL/NHL auto- 2 pts 1st ram,4 pts early relapse (ER), 4 pts late relapse (LR),2 pts 3rd ram; ALL/NHL allo- 3 pts 1st ram. 8 pts ER, 1 pt LR, 1 pt 2nd rel, I pt 3rd ram; AML/MDS allo- 6 pts 1st ram, 1 pt 1st rel, 2 pts sac. leukemia after M. Hodgkin, 1 pt no remission; AML auto - 2 pts 1st ram. Methods: 20 lots received hyperfractionated total body irradiation (FTBI)-8xl,5 Gy day-8 to -4, 18 pts received oral Busulfan (BU) -4x3-4mg/kg on day -8 to -4,in all pts Etoposide was infused (40-60mg/kg) on day -4 and Cyclophosphamide (Cy) 2x 6o mg/kg on day -3 and -2. In allo BMT GVHD-prophylaxis consited of CyclosporinA (CyA) and MTX in pts over ten years of age and in MTX alone in children under 10 years, respectively. Autologous BM was purged with 4-Hydroxycyclophosphamide. Engrafl~nant depended on number of transplanted stem-cells, median time to achieve an ANC of >0,5x10~ was 18 days (range 11-36 days), median time to a platalet count > 25x10e/]Yvas28 days (range 17 to 96 days). Regimen related overall toxicitias were similar between TBI and BU treated pts with only skin toxicity seen significantly more frequently in the BU group and severe oropharyngeal mucositis in the TBI group. Life threatening hepatic toxicity occured in 3 pts ( 2 BU, 1 FTBI), renal failure was observed only in combination with multiorgan failure in 4 pts. Results: 24 pts are alive, (23 pts in remission, 1 pt in relapse), 14 pts died. Causes of death in the allo BMT ware: 3 toxic (VOD, multiorgan failure), 2 infection (Asperg.), 1 relapse (14 rots after BM'F'); in autoBMT: 1 inf (fungal), 1 Pneumocystic-pneumonia, 5 relapses, 1 unknown,The probability of EFS in ALL/NHL allo BMT is 86%, in ALL/NHL auto 38%, in AML 1st ram 70%, in AML 2rid or no rem 0%. Conclusion:OveraU EFS were similar among FTBI and 8U treated patients. Treatment related toxicilias were acceptable except in pts with secondary leukemias or resistant relapse. The antileucaemic efficacy seems to be higher than in standard imans used before. resent address: St. Anna Children's Hospital, Kinderspitalgasse 6, A-1090 Vienna,Austria.

FROM

AN

UNRELATED

mar 1

A96 94

96

SUCCESSFUL ADOPTIVE IMMUNOTRANSFERS IN A BOY WITH A RELAPSE OF T-ALL AFTER BONE MARROW TRANSPLANTATION FROM AN UNRELATED DONOR

FLUOROQUINOLONES FOR INFECTION PREVENTION AFTER BONE MARROW TRANSPLANTATION. W. Kern*, T. Schmeiser, B. Hertenstein, D. Bunjes, and R. Arnold.

Th.Klingebiel ~, R.Handgretinger 1, G.Ehninger2, R.Dopfer 1, A.Brand 3, G.J.A.ten Bosch 3, R.K~mpf4, D.Niethammer1

The patient fell ill with a T-ALL at an age of 6 years, and was treated according to the German ALL-trial BFM-ALL 86. After an early bone marrow relapse and a treatment with the protocol ALL-BFM REZ 89, he was grafted in CR2 with the marrow from an unrelated HLAidentical donor. He suffered from an acute GVHD-reaction of skin grade 3, but not from a chronic GVHD. 6 months after BMT the boy presented with a pleural effusion and enlarged mediastinal lymph nodes. From the effusion T-lymphoblastic cells of recipient origin could been diagnosed. Bone marrow was not involved and continued to be of donor type. The boy achieved complete but not stable remission after he had been treated with prednisolon and vinceistine and the mediastinum had been irradiated with a total dose of 24 Gy. 9 months after post-BMT relapse, following a pretreatment with c-Interferon (2 MiD U/m 2 3x/week), 5.9x108/kg nucleated cells from the donor were administered in two portions. The patient tolerated the cell infusion very well, but developed a grade 3 GVHD of skin and mucosa, which was treated by CSA and prednisolon. In spite of being in a progressive state of disease just before cell transfusion, the patient responded to the immunotransfer with a complete remission. A large pleural effusion being proved as lymphoblastic T-cells resolved without further measures as did the large lymphoma of the upper mediastinum. The boy is now in good condition without different treatment more than 1 ~,ear after a relapse of a T-cell leukemia. Department of Pediatric Oncology, 2Department of Internal Medicine and 4Department of Genetics, Eberhard-Karls University, D-7400 Ts FRG; 3Blood Bank, Nb2333 Leiden, Netherlands

95

INCREASED SERUM LEVELS OF G-CSF AFTER AUTOLOGOUS BONE MARROW TRANSPLANTATION IN PATIENTS WITH ACUTE MYELOID OR LYMPHOBLASTIC LEUKEMIA R. Haas, G. Gerieke, S. Hohaus, and W. Hunstein We evaluated the biological role of endogenously produced hemopoietic growth factors for hematological reconstitution following autologous bone marrow transplantation (ABMT) in 31 patients with acute myeloid or lymphoblastie leukemia antografted in complete remission. Serum levels for Interleukin-3 (IL-3), granuloeyte-macrophage colony-stimulating factor (OMCSF) and G-CSF were measured with commercially available enzyme-linked immunoassays. 23 patients had AML and 8 patients ALL. With the exception of one patient (AML), the bone marrow was either purged with mafosfamide (22 AML/2 ALL) or with monoclonal antibodies and immunomagnetic beads (6 ALL). The conditioning regimens used were either a combination of Cyclophosphamide and total body irradiation (29 p) or Cyclophosphamide/Busulfan (2p). For GM-CSF or IL-3 no serum levels above the normal range of healthy volunteers were found following autografting. In contrast, the G-CSF serum levels were increased during the phase of marrow aplasia with a median peak value of 2969.2 pg/ml (range 425.9-8676.0). Analyzing a total of 270 samples, a reverse correlation (R=-0.78, p <0.001 ) could be demonstratedbetween G-CSF serum levels and white blood count (WBC). An additional increase of G-CSF serum levels on days of fever (238.5oC) and/or documented infections was observed. In the first week following autografting patients without fever had a G-CSF level of 1182.7+350.3 pg/nl (mean_+SEM) versus 3013.6+546.4 pg/ml (p < 0.03) in febrile patients. The relationship between GCSF serum level and WBC supports the central role of this hemopoietic growth factor following myeloablative treatment and autotransplantation. Regulatory mechanisms ensure a higher endogenous production during periods of increased demand such as fever and/or infectious complications. Departmentof InternalMedicine,Hospitalstr.3, W-6900Heidelberg,FRG

The results of our previous study using norfloxacin (Scand. J.Infect.Dis. 20: 625) suggested that this agent may be as effective as the combination of neomycin/polymyxin or cotrimoxazole/polymyxin previously used by us in regimens for gastrointestinal ~deoontamination~ in bone marrow transplant patients. Given the higher in vitro antibacterial activity and more favourable pharmaookinetic properties of ofloxacin compared with norfloxacin, we subsequently performed an open study of ofloxacin prophylaxis in 87 adult bone marrow transplant recipients (74 allogeneic, 13 autologous). Patients were nursed in LAF or HEPA rooms with barrier nursing techniques and sterile diet, and received oral ofloxacin (200 mg q 12h) in addition to an oral antifungal agent and biweekly human intravenous immunoglobulins. The median age of the patients was 36 yrs. The median duration (from day • to granulooyte recovery (>500/pl) was 20 days (range: 9 to 83). Febrile events during this period were observed in 97% of patients, and remained unexplained (irrespective of oral mucositis) in 52 patients (60%). One patient had fever associated with pulmonary embolism. Infection was documented in 31 patients (36%): 14 had baeteremia (5 staphylococci, 4 streptococci, 2 lactobacilli, 2 Fusobacterium sp., I X.maltophilia), 13 non-bacteremic lower respiratory tract infection, and four had minor localized infections. The median duration of fever was 8 days. There was no documented fungal infection, though 13 patients (15%) received empirical antifungal therapy for unresolving fever. Of the 6 deaths occurring within one month after transplant, one was associated with bacterial infection. *Ulm University Hospital and Medical Center, Koch-Str.8, D-7900 Ulm, FRG.

Robert-

A97

Pharmacokinetics 99

97 A DOES NOT ~ VINCRISTINE, DALIkERL~ICIN

THE AWI~LEJ~MIC ~ND 6-THIOgdANINE

~mu~ IN ACUTE

R. Pieters I, D.R. Hui~mans I, A.H. Loonen I, K. H~hlen 2, A.J.P. yeerman I . Department of Pediatrics, Free University Hospital, Amsterdam, and 2Sophia's Children Hospital, Subdivision Haemato/Oncology, Erasmus University, Rotterdam, The Netherlands. It has been suggested that retinoids might potentiate the cytotoxic activity of anticancer drugs but this suggestion is based upon studies on cell lines and on animal models. No studies using cells obtained from cancer patients testing this suggestion have been reported. We studied whether isotretinoin potentiated the effect of vincristine (VCR), daunorubicin (DNR), and 6-thioguanine (6-TG) against cells obtained from 24 children with acute lyaphoblastic leukemia (ALL). The semi-automated MTT assay was used to measure leukemic cell kill (Lancet 1991; 338: 399-403). Treatment with 5 ~g/mi isotretinoin alone resulted in a leukemic cell survival (LCS) of 82% + 28%. So isotretinoin is toxic to ALL cells. Dose-response curves were obtained for VCR, ~ and 6-TG in the presence and absence of isotretinoin. Isotretinoin showed additive leukemic cell kill in combination with VCR and DNR. When corrected for cell kill by isotretinoin alone, it appeared that isotretinoin did not significantly enhance leukemic cell kill by VCR, DNR and 6TG. No differences were found between samples from patients at initial diagnosis and at relapse with respect to cell kill by isotretinoin alone and with respect to a possible synergistic effect of isotretinoin and the cytostatic drugs. It is concluded that isotretinoin has additive antileukemic effects in combination with VCR or DNR. However, isotretinoin does not potentiate the antileukenic effect of VCR, DNR and 6-TG against leukemic cells obtained from patients with ALL. Supported by the Dutch Cancer Society (IKA 87-17)

P.GLYCOPROTEIN PROFILES IN ACUTE LEUKAEMIA OF CHILDHOOD A. O'Meara*, S. Rooney*, R. Ball*, T. Tsuruo^, P. Dervan~ *Children's Research Centre, Our Lady's Hospital for Sick Children, Dublin, Ireland, "Japanese Foundation for Cancer Research, Tokyo, Japan and ~ Misericordiae Hospital, Dublin.

P-glycoprotein (P-gp), the product of the mdr-1 gene, is implicated in the development of chemoresistance of many cancers. A study was undertaken to investigate reactivity of 2 P-gp monoclonal antibodies, JSB-1 and MRK16, tecognising cytoplasmic and surface epitopes respectively on the P-gp molecule, in newly diagnosed and relapsed ALL and ANLL in childhood. JSB-1 reactivity was assessed on BM smears using an APAAP technique while MRK16 was investigated in fresh mononuclear preparations using FACScan flow cytometry. One out of 10 diagnostic ALL samples demonstrated a high degree of positivity with JSB-1 but was negative with MRK16; this patient failed to achieve remission on standard induction therapy. Three out of 6 patients in ALL relapse showed varying degrees of positivity with JSB-1, only one of whom demonstrated low grade positivity with MRK16. Among the ANLL group, Dotn MAPs snowea strong positive reactions in one patient at diagnosis who achieved remission with a cytosar, VM26 induction schedule but relapsed seven months later, and JSB was also positive in 4 out of 4 other ANLL relapses, only one of which was analysed using MRK16 and was negative. In conclusion, P-gp would appear to have a major role in the development of chemoresistance in childhood acute leukaemia. Children's Research Centre, Our Lady's Hospital for Sick Children, Crumlin, Dublin 12, Ireland

98

100

REVERSAL OF MULTI DRUG RESISTANCE BY R-VERAPAMIL.

MULTIDRUG RESISTANCE (MDR) IN MYELODYSPLASTIC SYNDROMES (MDS) AND ACUTE LEUKEMIA Runde V., Aul C., and Schneider W. Dept. Hematol., Univ. Diisseldorf, Germany

Schumacher K., Ladda E., Biihl K., Wcimer A., Eser C., Traugott U., Roller E., l..6ffier B., Eichelbaum M. Dept. of Hematology, Ontology and Immunology, and Inst. of Clinical Ph~macology, Robert-Bosch-Kraukenlaans, Stuttgart. By in vitro studies, R-Verapamil (R-vpm) has been demonstrated to be a potent modulator of multi drug resistance (mdr). On the basis of our in vitro studies we conducted a phase I clinical trial in patients with progressive malignant disease. Aims of the study were to evaluate the optimal doses of R-vpm according to plasma concentration and side effects in conjunction with several chemotherapeutic regimens. 33 patients were enroled ha the study. 13 patients with advanced breast cancer in progress were treated with doxorubicin plus Rvpm. 20 patients with different malignant diseases in progress were either continued with the prior treatment or were put on a combination of two mdrsubstances plus R-vpm. Plasma concentrations of R-vpm reached levels up to 3575 ug/ml, mean 1300 ug/ml, comparable to the effective concentrations in vitro. Arterial hypoteusion was the most frequent dose limiting side effect. Av-bloek II was registered two times, av-rhythm once and cardiomyopathie once. Av-block I was observed in 29 of 58 treatment courses. Bone marrow toxicity was significant. The clinical response rate was about 30 % in breast cancer patients, and more than 50 % in patients with refractory acute myeloid leukemia. In conelnsiun, R-vpm is an effective, non-toxic modulator of multi drug resistance. Supported by Robert-Bosch-Foundation Stuttgart and Knoll AG, Ludwlgshafen.

MDR is a well-known cellular mechanism of interference against the effects of eytotoxic drugs and is characterized by cross-resistance to different chemotherapeutic agents with no apparent structural or functional relationship. In this study we examined the presence of P-170 positive leukemic cells in bone marrow smears from 108 patients by means of an alkaline phosphatase anti-alkaline phosphatase technique (APAAP), using a monoclonal antibody (MoAb C219, CIS Isotopendiagnostic, Germany) directed against a cytoplasmic domain of P-170. The patients analysed had the following hematological malignancies: de novo AML n=41, ALL n = l I, and MDS n= 56. Several patients were investigated at different stages of their disease. Among 38 patients with newly diagnosed AML or ALL, 15 (39%) had MoAb C219-positive blast cells in the bone marrow. The respective percentages in patients with AML/ALL in first (n=16) or second relapse (n=4) were 69% and 100%. As regards the MDS group, no reactivity of medullary blasts with MoAb C219 was found among our RA and RARS patients. In contrast, blast cells were positive in 5 of 16 cases (31%) of RAEB, 3 of 10 cases (30%) of RAEB/T, 6 of 10 cases (60%) of CMML, and 13 of 21 cases (62%) investigated after evolution of MDS to AML. The proportion of positive bone marrow blast cells varied between 5% and 80%. In conclusion, patients with relapsed acute leukemia appear to be characterized by a higher frequency of MDR positive medullary blasts, as compared to patients with newly diagnosed leukemia. In the MDS group, C219 reactivity was apparently dependent on the stage of the disease. Further studies are required to evaluate the clinical significance of these in vitro findings. Volker Runde, Dept. Hematol., Univ. Dfisseldoff, Moorenstr. 5, W-4000 Dtisseldorf 1, FRG

A98

101

103

COMBINATIONS OF CYTOSTATICA WITH CALCIUM/CALMODULIN ANTAGONISTS FOR REVERSION OF MULTIDRUG RESISTANCE

CLINICAL RELEVANCE OF THE INFLUENCE OF ETOPOSIDE ON THE ARA-CTP-FORMATION IN LEUKEMIC BLAST CELLS

E. Roller, D. Bfirkle, M. Eichelbaum, B.Klumpp, J. Krause and K. Schumacher Departments of Oncology and Clinical Pharmacology, RobertBosch Hospital, Stuttgart, Germany One of the major problems in the treatment o[ leukemia is the innate or acquired resistance (MDR) due to the overexpression of the mdr-i gene which encodes the P-glycoprorein (P-170), a plasma membrane protein involved in the energy-dependent transport of antitumor drugs. Calcium /ealmodulin antagonists are able to inhibit the drug efflnx mechanism by blocking P-170. The MTT assay was used to assess sensitivity of the cells. The human lymphoblastoid cell line CCRF-CEM and the MDR subline VCRI000 which overexpresses P-170 were cultivated in CS-medium (Camon) without FCS. Resistance factors (RF) for VCRI0000 cells are: idarubicin (IDA) 10.8, doxorubicine (DX) 84.1, mitoxantrone (MIT) 73.6. Variable cytostatica/modulator combinations were added to the culture medium (B8509-035, a dihydropyridine derivate i pM or R-verapamil i0 pM). Both substances had only weak modulating activity with non-toxic concentrations of IDA, RMI 2.2/2.6 (RMI resistance modification index: IC~ of VCRI000 with cytostatica/IC~0 of VCRI000 with cytostatica and modulator). However the combination MIT and DX with B8509035 or R-verapami] reversed MDR in a dose dependant manner, even at suboptimal concentrations of cytostatic drugs (RMI MIT 15.3/12.5; RMI DX 20.9/16.]) No chemosensitizing effect was observed in the case of CCRF-CEM cells. Minor modulating activity in combination with IDA could depend on the low grade of resistance (RF 10.8) of VCRIO00 compared to CCRFCEM cells. Supported by the Robert-Bosch-Foundation, B8509-035 was provided from BYK Gulden Lomberg GmbH, Konstanz, R-verapamil from Knoll AG, Ludwigshafen

T.Menke, B.Pr~bsting, P. Schulze-Westhoff, J.Boos Universit~ts-Kinderklinik Albert-Schweiter-Str.33, 44 Miinster Etoposide is an inhibitor of the cellular nucleoside carrier. Cellular uptake by this carrier, however is precondition of Ara-C-activation to its effective metabolite Ara-C-triphosphate (Ara-CTP). Both drugs are important in anti-leukemic therapy and in many schedules they are simultaneously applied. Leukemic blasts of children with acute leukemia were exposed to Ara-C with or without Etoposide and intracellular levels of Ara-CTP were compared. Simoultaneous incubation in medium containing Etoposide (20yg/ml) and Ara-C (lyg/ml) resulted in reduced intracellular Ara-CTP levels. Freshly isolated blasts showed significantly lower levels after lh (54• of control) and after 3h (55• of control). At lower Etoposide concentrations the effect was less distinct. Considering the high plasma protein binding of Etoposide (90%) we incubated blasts of patients under the same conditions in heparinisized blood instead of medium. As well as in medium a significant reduction was measured (after lh 76• of control, after 3h 82• of control). Incubation with Etoposide (2Oyg/ml),3h) in Ara-C free medium after preincubation with Ara-C (lyg/ml,lh) increases the retention of Ara-CTP in 3 of 9 patients. Supported by the Deutsche Forschungsgemeinschaft

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104

MODULATION OF INTRACELLULAR ARA-C METABOLISM IN A M L B L A S T S BY G M - C S F P R E T R E A T M E N T I N V I T R O . Reutcr C., A. d. Landwehr U,, A. d. Landwehr U., Kiehl M., Busemann C., Zi~hlsdorf M., Schleyer E., Rolf C., W6rmann B., Biichner T. & Hiddemann W. Division of Hematology/Oncology, Department of Internal Medicine, Westf~ilische Wilhelmsuniversitfit, Albert-Schweitzer-Strage 33, D-4400 Miinstcr, Federal Republic of Germany. Cytosine-arabinoside (Ara-C) is one of the most effective single agents in the treatment of acute myeloid leukemia (AML). The cytotoxicity of Ara-C is believed to result from incorporation of its intracenular active metabolite Ara-CTP into DNA and inhibition of DNA synthesis. In vitro experiments of our group and others indicate that hematopoietic growth factors (e.g. IL-3, GM-CSF) enhance the cytotox~c effect of AraC on clenogenic leukemic cells. To elucidate this phenomenon we investigated the effect of recombinant human granulocyte-macrophage colony stimulating factor (rhGMCSF) on deoxycytidine kinasr thymidine kinase and DNA-nolymerase activity in AML blasts in vitro. Additionally, we determined 3H-TdR and 3,H-Ara-CMP incorporation into DNA, tetrazollum conversion, intracellular Ara-CTP levels and the proportion of cells in S-phase. AML blasts from bone marrow (n=27) and/or peripheral blood (n= 10) were incubated with rhGM-CSF (100 U/ml). After 48 hours AraC was added at concentrations between 0.06-100uM for another 12 hours. We observed significant enhancements of DNA polymerase, thymidine and deoxycytldine kinase activity in response to GM-CSF in vitro in the majority of the investigated cases (20/33, 8/13, 10/14). The increases in enzyme activities ranged from 10.6-965 %, 12.2-369 % and 25.6-453 %, respectively. However, the intracelhilar Ara-CTP levels were not altered by GM-CSF pretreatment. All patients investigated (4/4) showed an increase of the tetrazolium conversion in MTT-assays (20-44%). We observed a significant increase (21-187%) of the proportion of S-phase celts in 7/11 patients. In 18 of 21 cases we found significant enhancements in 3H-TdR (1.5-8.2 fold) and 3H-Ara-CMP (1..5-4.8 fold) incorporation into DNA. The enhancement of TdR and Ara-CMP uptake into DNA correlated with a rise of DNA polymerase activity in 12 of these 18 patients. 7 of 16 cases showed GM-CSF induced increases in Ara-CMP incorporation into DNA only at low extraccllular AraC concentrations (0.06-1.06 g M). In elonogenic assays we measured a considerable enhancement (2,2-229 fold) in sensitivity against Ara-C in response to GM-CSF in 10 of 13 specimens. Our results suggest that GM-CSF enhances AraC mediated cytotoxicity in AML blasts by stimulating DNA-polymerase activity and thereby increasing Ara-CMP incorporation into DNA.

RESISTANCE TO CYTOSINE ARABINOSIDE (araC). ARE MUTATIONS IN CTP SYNTHETASE (CTPs) AN IMPORTANT MECHANISM? J Whelan, 1 T Smith, 1 C Pocock,2 A Rohatiner, 1 T Lister, 1 M Meuth. 2 Imperial Cancer Research Fund 1Department of Medical Oncology, St Bartholomew's Hospital, London and 2Clare Hall Laboratories, South Mimms, Herts, UK. Resistance to the cytotoxicity of araC may result from expansion of intracellular dCTP pools. To investigate the underlying mechanism for this, chinese hamster ovary (CHO) cell lines with a distinctive phenotype of high dCTP pools and araC resistance have been studied. The biochemical basis for this elevation is a loss of normal feedback inhibition of the enzyme, CTPs by CTP. Subsequent molecular analysis has demonstrated mis-sense point mutations in three distinct areas of the CTPs gene of these CHO cells. As a 94% amino acid similarity exists between the hamster and human CTPs genes it was feasible to seek the occurrence of these mutations in patients with acute leukaemia. Samples from 36 patients (AML, 34; ALL, 2) have been examined. All had been treated with araC. DNA or RNA was prepared from peripheral blood or bone marrow samples taken at the time of failure of induction therapy (8 patients) or recurrence (28 patients). The CTPs gene sequence was amplified using the polymerase chain reaction (PCR) and further studied by direct sequence analysis of the regions implicated by the CHO model. No mutations were identified using this approach. Dilution experiments revealed that detection by sequencing of heterozygous mutations in mixed cell populations required 20% of cells to carry the mutation. Therefore more sensitive assays are being developed. Using hybridization of mutation-specific oligonucleotides against PCRamplified CTPs under conditions of very high stringency, 1 of 20 samples proved positive. Further clarification is now being sought by hybridisation of these same oligonucleotides agair;st cloned PCR fragments which allows investigation of single molecules. In conclusion, mutations in CTPS may be present in patients treated with araC but appear unlikely to prove a major mechanism of resistance. Similiar strategies should be used to study alterations in other enzymes involved in araC metabolism to determine alternative mechanisms of resistance. This will enable rational planning of therapy for AL which circumvents drug resistance.

A99 105

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INHIBITORY ACTION OF 2',2'-DIFLUORODEOXYCYTIDINE (dFdC> ON CYTIDINE 5'-TRIPHOSPHATE SYNTHETASE V. H e i n e m a n n and W. Plunkett.

CHILDHOOD ALL AND ANLL AT INITIAL D I F F E R E N C E S IN C E L L U L A R D R U G R E S I S T A N C E

dFdC shows inhibitory a c t i v i t y against h u m a n DNA polymerases, ribonucleotide reductase and dCMP deaminase. While the major a c t i o n s of dFdC are thus d i r e c t e d at the i n h i b i t i o n of DNA synthesis, RNA synthesis, m e a s u r e d by [ 3 H ] u r i d i n e i n c o r p o r a tion, was inhibited in C h i n e s e hamster o v a r y (CHO> cells incubated with dFdC. A 4-h e x p o s u r e to i00 uM dFdC r e s u l t e d in the i n t r a c e l l u l a r a c c u m u l a t i o n of 1285 uM dFdC 5 ' - t r i p h o s p h a t e (dFdCTP>. This was a c c o m p a n i e d by a specific depletion of the cellular pool of CTP, w h i l e p o o l s of ATP, UTP, and GTP did not decrease. In contrast, a CHO mutant deficient in d e o x y c y t i d i n e kinase and thus u n a b l e to a c c u m u l a t e dFdCTP maintained its CTP pools under identical c o n d i t i o n s , suggesting that the CTP pool depletion was dependent on dFdC phosphorylation. Neither I00 uM arabinosylcytosine nor 5 mM h y d r o x y u r e a a f f e c t e d CTP levels indicating that i n h i b i t i o n of DNA synthesis by analogue i n c o r p o r a t i o n or by depletion of dNTP pools were not the c a u s e s of the CTP pool perturbation. M e t a b o l i c s t u d i e s d e m o n s t r a t e d that incorporation of [ 3 H ] u r i d i n e into the UTP pool was not affected by dFdC treatment, whereas the specific a c t i v i t y of the CTP p o o l s d e c r e a s e d as a function of increasing dFdC c o n c e n t r a t i o n and time of exposure. Comparable results were obtained using 3-deazauridine, a known inhibitor of CTP synthetase. We conclude that high cellular concentrations of d F d C T P induce CTP pool d e p l e t i o n by i n h i b i t i o n of CTP synthetase. Klinikum G r o s s h a d e r n , U n i v e r s i t y of Munich, Munich FRG and The U n i v e r s i t y of T e x a s M. D. Anderson Cancer Center, Houston, Texas, USA.

DIAGNOSIS,

K a s p e r s G J L I. P i e t e r s R I, K l u m p e r E 1 , H ~ h l e n K 2,3, De W a a l FC 1,3, V a n W e r i n g ER 3, V e e r m a n A J P 1,3. IDept Pediatrics, Free University Hospital, De Boelelaan 1117, 1081 HV Amsterdam, 2Sophia's Children Hospital, Rotterdam, 3Dutch Childhood L e u k e m i a S t u d y Group, The Hague, The N e t h e r l a n d s . C h e m o t h e r a p e u t i c t r e a t m e n t is m o r e s u c c e s s f u l in c h i l d h o o d A L L t h a n in ANLL. This m i g h t be due to a r e l a t i v e drug r e s i s t a n c e of the A N L L - c e l I s . On the o t h e r h a n d the o p t i m a l t r e a t m e n t r e g i m e n m a y h a v e not yet b e e n d e s i g n e d for ANLL. W e s t u d i e d the d r u g r e s i s t a n c e of f r e s h u n t r e a t e d A L L a n d A N L L s a m p l e s u s i n g the M T T assay, w h i c h has a s u c c e s s r a t e of 85% in our l a b o r a t o r y for t h e s e samples. The r e s u l t s f r o m 66 A L L and i0 A N L L s a m p l e s w e r e analyzed. For e a c h d r u g the r e s i s t a n c e r a t i o (RR) was c a l c u l a t e d by: (median LC50 of A N L L s a m p l e s ) / (median L C 5 0 of A L L samples). LC50 is the l e t h a l c o n c e n t r a t i o n to 50% of the cells. R R ' s (p-values): p r e d n i s o l o n e >147 (.004), d e x a m e t h a s o n e >100 (.005), l-asparaginase 4.5 (.3), vincristine 1.6 (.4), 4 H O O - i f o s f a m i d e 1.6 (.07), d a u n o r u b i c i n 1.2 (.7), m a f o s f a m i d e 1.1 (.7), t e n i p o s i d e 1.0 (.6), A r a C 0.8 (.5), vindesine 0.8 (.7), 6-mercaptopurine 0.7 (.9), doxorubicin 0.6 (.16), mitoxantrone 0.4 (.15), a n d 6 - t h i o g u a n i n e (6TG) 0.5 (.004). For e a c h d r u g r a n g e s of L C 5 0 ' s of A L L and A N L L s a m p l e s o v e r l a p p e d , e x c e p t for g l u c o c o r t i c o i d s (GC). O n l y 2/10 A N L L s a m p l e s w e r e m o d e r a t e l y s e n s i t i v e to GC. 6/7 ALL cases with Ii atypical marker were r e l a t i v e l y r e s i s t a n t to GC, o n l y 1 sensitive. W e c o n c l u d e t h a t our d a t a c o r r e s p o n d to the c l i n i c a l experience. Significant differences: ANLL samples are m u c h m o r e r e s i s t a n t to GC, b u t m o r e s e n s i t i v e to 6TG t h a n A L L samples. The lack of a c t i v i t y of G C in m o s t A N L L samples, in c o n t r a s t to A L L samples, m i g h t p a r t l y e x p l a i n the w o r s e p r o g n o s i s of c h i l d h o o d ANLL. S u p p o r t e d b y the D u t c h C a n c e r Society.

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CYTOLYTIC ACTIVITY IN V I T R O O F M I T O X A N T R O N E AND A N T H R A C Y C L I N E S IN C H I L D H O O D L E U K E M I A A T D I A G N O S I S

C Y T O S T A T I C D R U G R E S I S T A N C E IN C H I L D H O O D A C U T E L Y M P H O B L A S T I C L E U K E M I A (ALL)

K a s p e r s G J L I, P i e t e r s R I, K l u m p e r E 1 , H ~ h l e n K 2,3 De W a a l FC 1,3, V a n W e r i n g ER 3, V e e r m a n A J P 1,3. iDept Pediatrics, Free University Hospital, De Boelelaan 1117, 1081 HV Amsterdam, 2Sophia's Children Hospital, Rotterdam, 3Dutch Childhood L e u k e m i a S t u d y Group, The Hague, The N e t h e r l a n d s . Mitoxantrone (Mit) has shown antileukemic a c t i v i t y s i m i l a r to t h a t of d a u n o r u b i c i n (DNR) in u n t r e a t e d a d u l t A L L a n d A N L L p a t i e n t s (pts). M i t a l s o s e e m s to h a v e a f a v o r a b l e t o x i c i t y p r o f i l e . In relapsed/refractory childhood leukemia, Mit has also shown a c t i v i t y b u t the d a t a are limited, e s p e c i a l l y for u n t r e a t e d pts. In c o n n e c t i o n w i t h a n a t i o n w i d e , p r o s p e c t i v e study, 59 u n t r e a t e d A L L a n d 10 A N L L s a m p l e s w e r e t e s t e d u s i n g the M T T a s s a y for in v i t r o r e s i s t a n c e to Mit, DNR and d o x o r u b i c i n (Dox). M e d i a n (ranges) L C 5 0 v a l u e s ( c o n c e n t r a t i o n l e t h a l to 50% of the cells), in ~g/ml:

K l u m p e r E 1 , P i e t e r s R I, K a s p e r s GJL I, v a n W e r i n g ER 4, H ~ h l e n K 2,4, H e n z e G 3 and V e e r m a n AjpI, 4. iFree U n i v Hosp, Dept. Pediatrics, P0 Box 7057, 1007 MB Amsterdam, 2Sophia's Children Hosp, Rotterdam, 3 F r e e U n i v Hosp, B e r l i n and 4 D u t c h C h i l d h o o d L e u k e m i a S t u d y Group, The Hague, NL. N e a r l y all c h i l d r e n w i t h ALL a c h i e v e a c o m p l e t e r e m i s s i o n on c o m b i n a t i o n c h e m o t h e r a p y b u t 1/3 w i l l get a relapse. D r u g r e s i s t a n c e is s u p p o s e d to p l a y a m a j o r role. B e f o r e s t u d y i n g m e c h a n i s m s of r e s i s t a n c e it is n e c e s s a r y to k n o w to w h i c h d r u g s r e s i s t a n c e o c c u r s in p a t i e n t s . W e s t u d i e d r e s i s t a n c e to 13 d r u g s in 108 c h i l d r e n w i t h u n t r e a t e d A L L and 29 w i t h r e l a p s e d ALL u s i n g the M T T assay. The r e s u l t s of this a s s a y are w e l l c o r r e l a t e d w i t h c l i n i c a l o u t c o m e in A L L (Lancet 1991; 338: 399). The LC50 ( c o n c e n t r a t i o n l e t h a l to 50% of the cells) for v i n c r i s t i n e , v i n d e s i n e , teniposide and mitoxantrone did not differ significantly between untreated and relapsed patients. Cells from relapsed patients were significantly more resistant to prednisolone (p<.0001), d e x a m e t h a s o n e (p<.0001), d a u n o r u b i c i n (p=.001), doxorubicin (p=.027), L-asparaginase (p=.001), 6-thioguanine (p<.0001), cytosine arabinoside (p=.008), 4-hydroperoxy-ifosfamide (active m e t a b o l i t e of i f o s f a m i d e ; p=.001) and m a f o s f a m i d e (active m e t a b o l i t e of c y c l o f o s f a m i d e ; p=.028) than cells from untreated patients. Cells from relapsed patients were >200-fold more resistant to prednisolone and >13-fold more r e s i s t a n t to d e x a m e t h a s o n e as c o m p a r e d to the u n t r e a t e d group. R e s i s t a n c e r a t i o s for the o t h e r drugs ranged from 2-5. No cross-resistance o c c u r r e d b e t w e e n m i t o x a n t r o n e and a n t h r a c y c l i n e s . These data are u s e f u l in d e s i g n i n g new drug r e g i m e n s in r e l a p s e d c h i l d h o o d ALL. S u p p o r t e d b y the D u t c h C a n c e r S o c i e t y (IKA 90-05)

Mit DNR Dox ALL .10 (<.001->1) .10 (<.002->2) .51 (.03-1.85) A N L L .04 (.01-.32) .12 (.05-.97) .32 (.04-.71) A L L a n d A N L L s a m p l e s d i d not s i g n i f i c a n t l y d i f f e r

in r e s i s t a n c e to t h e s e 3 drugs. T - A L L s a m p l e s w e r e s i g n i f i c a n t l y m o r e r e s i s t a n t to DNR, b u t not to M i t or Dox, t h a n B - c e l l p r e c u r s o r A L L samples. S a m p l e s f r o m pts w i t h a p r o g n o s t i c a l l y u n f a v o r a b l e age (~2 yrs, I10 yrs) w e r e s i g n i f i c a n t l y m o r e r e s i s t a n t to D N R a n d Dox as c o m p a r e d to the i n t e r m e d i a t e agegroup. For Mit these differences were not s i g n i f i c a n t . We c o n c l u d e t h a t M i t has a p r o n o u n c e d c y t o l y t i c a c t i v i t y in u n t r e a t e d c h i l d h o o d A L L a n d A N L L and s h o w s a b e t t e r a c t i v i t y in s a m p l e s f r o m pts w i t h p r o g n o s t i c a l l y u n f a v o r a b l e c h a r a c t e r i s t i c s t h a n DNR and Dox. B a s e d on c l i n i c a l d a t a a n d our in v i t r o data, Mit should be investigated in r a n d o m i z e d c l i n i c a l t r i a l s in c h i l d h o o d A L L a n d A N L L at i n i t i a l d i a g n o s i s . S u p p o r t e d b y the D u t c h C a n c e r S o c i e t y (IKA 89-06, 90-05).

RELAPSED

A IO0 109

110

COMPARISON OF VINCRISTINE PHARMACOKINETICS IN C H I L D R E N A N D ADULTS. S.S.N. de Graaf, H. Bloemhof, D.E.M.M. Vendrig*, and D.R.A. Uges.

TOPOiSOMERASE II ACTIVITY IN LEUKEMIC CELLS AND ITS RELATION TO CHEMOTHERAPY-SENSITIVITY F. Gieseler, F. Boege, B. Ruf, P. Meyer, L.A. Zwelling*

Clinical experience indicates that adults suffer more from Vincristine (VCR) neurotoxicity than children. It is unknown whether this difference is due to a higher susceptibility of adult neural tissues or to a lower drug clearance in adults, resulting in increased systemic exposure to VCR. To study this issue we developed a specific and highly sensitive HPLC-assay for the measurement of VCR in plasma and compared VCR pharmacokinetics in adults and children. Pharmacokinetic parameters of VCR were estimated in eight adults (median age 54, range 41-70 yr) and nine children (median age 3, range 1-12 yr). An open two-compartment model was used with inverse variance of the assay weighting. Results are listed below (mean values). ANOVA was used for the statistical analysis.

A large number of cytostatics inhibit the life important enzyme Topo I1. This results in DNA double strand-breakes which can be correlated to the cytostatic potency of the substance. Little is known about the physiological functions of the enzyme. We have compared in vitro experiments to the proposed way of Topo IIaction and discussed the implications for cellular sensitivity to chemotherapy. Nuclear Topo II activity derived from human promyelocytic HL-60 leukemia cells was detected by the enzyme's ability to relax, catenate or decatenate DNA, The enzyme activity is regulated by ionic strength, pH and salt composition of the reaction buffer. This is important for in vitro toxicity tests of cytostatics because these substances may alter the reaction parameters. Surprisingly, Topo II has two maxima of activity with different pH and salt requirements. The mode of enzyme action differs between the two maxima: It acts processively at pH 7.9, 60 mM KGlu and distributively at pH .8.3,120 mM KGlu.

tl/2atpha (h) tl/2beta (h) Vd ss (L'm'~) C]~tot (L.h'1.m "2)

Adults

Children

P-value

0.20 30.98 420 11.32

0.17 13.76 369 20.80

0.68 0.01 0.67 <0.01

Results indicate that VCR elimination rate is a function of age. Hence, a higher systemic exposure to VCR in adults may well be responsible for the apparent difference in VCR neurotoxicity between children and adults. *Present address: Duphar, PO Box 900, 1380DA Weesp. Departments of Pediatrics and Pharmacy, University Hospital, PO Box 30.001, 9700 RB Groningen, The Netherlands. Supported by the Groningen Foundation for Pediatric Oncology.

In vitro assays, such as DNA-relaxation, catenation or decatenation, probably do not match the in vivo situation, AIIosteric

regulations of Topo II such as pH and ionic strength influence the enzyme's DNA-binding affinity, activity and its way of action and are important factors for drug sensitivity. *MD Anderson Cancer Center, University of Texas, Houston, TX, USA Medizinische Poliklinik, University of W0rzburg, FRG. Supported by DFG SFB 172, C9 and Sander 90.038.1

A101

MDS/Secondary AML 111

113

CYTOGENETICS OF ACUTE LEUKEMIA FOLLOWING MDS D. Haase, M. KrCger and C. Fonatsch, AG Tumorcytogenetik~ lnstitut f~ir Humangenetik, Medizinische Universit~it zu L~ibeck, Ratzeburger Allee 160, D-2400 LCibeck, FRG. In a prospective study 32 patients with acute leukemias evolving from a myelodysplastic prephase were examined cytogenetically. FAB-classi:[ieation which was avaible in 19 cases of ANLL revealed an overrepresentation of M6 and an underrepresentation of M3 morphology as compared to patients with de novo-ANLL. Among the 32 patients examined, two presented with ALL and one with AUL following MDS. In comparison to de novo-ANLL patients analyzed cytogeneticalLy by our group, the patients with a preceding myelodysplasia showed an elevated aberration rate (72% vs. 52%). In 61% of all abnormal cases complex karyotypic anomalies were observed, while only 31.5% of de novo-ANLL with c y t o g e n e t i c abnormalities had complex chromosome changes. Further striking differences between de novo-ANLL and acute leukemias following MDS bec6me evident when single a f f e c t e d chromosomes are considered: Chromosomes 3, 5, 7, 11, 12, 18 and 21 were significantly more frequently as in MD5 related acute leukemias than in de novo-ANLL. On the other hand, specific anomalies like t(8;21), t(15;17) and inv(16) were not observed in these patients. The impact of sex and age on cytogenetic findings will be discussed, as well as the problem of a "bidden" mutagen exposition. Our cytogenetic findings of a high aberration rate, a high proportion of complex abnormalities and the frequent involvement of prognostically unfavourabJe chromosomes like 5 and 7 are in good accordance with the bad prognosis of this patient group observed in clinical studies. This study was supported by the Deutsche Krebshil:[e - Dr. Mildred 5cheel-StiftunR, project M 16/90/Fo 3.

DESCRIPTIVE EPIDEMIOLOGY OF MDS Aul C., Gattermann N., and Schneider W. Dept. Hematology, Heinrich Heine University, Dr_isseldorf, Germany_ Although most haematologists perceive a rising prevalence and incidence of myelodysplastic syndromes (MDS), reliable epidemiological data on these disorders are largely lacking. The bone marrow register of the University of D[isseldorf, which draws on a population of more than 1,000,(X)0 inhabitants, allowed us to assess among other epidemiologicol features the incidence of MDS, which was compared to that of AML. Among a total of 18,416 different patients registered between 1975 and 1990, 584 cases of MDS (3.2%) and 506 cases of AML (2.8%) were identified. Over the study period, the percentage of newly diagnosed MDS rose from 1.3% to 4.5%, while there was no upward trend for AML. 123 MDS patients had RA (21.I%), 142 had RARS (24.3%), 133 had RAEB (22.8%), 91 had RAEB/T (15.6%), and 95 had CMML (16.3%). The median age of MDS patients at diagnosis was 72 years (range, 16-92). An unequal sex distribution was only found in the CMML group (M/F 1.57). 31 patients (5.3%) were classified as secondary MDS because of previous treatment with cytotoxic chemotherapy and/or irradiation for a wlriety of malignancies. 12 MDS patients were identified in whom occupational exposure to organic solvents could not be ruled out. For calculating age-specific incidence rates, the analysis was confined to the town district of Diisseldorf, because exact demographical data were available for this population. In the last quinquennium of the study period, myelodysplastic syndromes were more frequent than AML in the age group 50 to 70 years (3.9 vs. 2.3/100,000/year). The incidence of MDS was more than three times that of AML in patients over 70 (22.9 vs. 6.7/100,000). Crude annual incidence (all age groups) was also higher in MDS (4. l/I 00,000) than in AML (2.4/I00,000) in recent years.We conclude that MDS are relatively common haematological neoplasias. The rising incidence in recent years is probably not due to changes in etiological factors, but may reflect increased awareness on the part of physicians and extended use of diagnostic procedures in elderly patients. Carlo Aul, Dept. Hematology, Univ. Dtisseldorf, Moorenstr. 5, W-4000 D[isseldorf 1, Gemlany

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THYMIDINE KINASE ACTIVITY IN BONE MARROW CELLS- A BASIS FOR RATIONAL TREATMENT IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES (MDS). Ntlsaler,V.,Sauer,H.,Palka-Flalscher,R., Wilmanns,W.,.Medizinischa Klinlk III Klinikum GroBhadarn, Universitltt MOnchon and Institut fiir Klinlsche H~atologie der GSF M6nchen, Germany. In e retrospective study 18 patients with refractory anemia (IRA}, 9 patients with refractory anemia with ringed sideroblasts (RARS), 29 patients with refractory anemia with excess of blasts (RAEB) and 20 petlents with refractory anemia with excess of blBsts in transformation (RAEB-TI as well as 5 patients with chronic myalomonocytic leukemia {CMML) were examined. There was e significant difference in the mean cytosolic thymidine kinase (TK) value between petlents with RARS, RAEB, RAEB-T and healthy persons; between RA and RARS; between RA and RAEB as well as between RA and RAEB-T. There were no significant difference in the biochemicel-cytoklnetic parameters thymldine incorporation {dTR), danxyurldine-incorporetion (dUR) and amounts of DNA-synthesizing S-phase-cells (%S) among RA, RARS, RAEB, RAEB-T, CMML and healthy persons. RAEB-petiants and RAEB-T-patiants with an increased TK activity showed a better response to DaunorubicinlYincristine/Adrlamycin/Ara.Ctreatment according to AML-6/AML-5-ptotocol of the EORTC (8 of 9 patients reached a complete remission [CRI) in comparing to MDS-patiants with a normal TK activity (0 of 6 patients reached CR). The response rote was very low in MDS-patients treated with low-dose Ara-C (1 of 17 MDS-patiants roached a CR} in comparison to ell MDS-patients treated with the AML-6/AML-S-protocol (8 of 15 MDS-patiants reached a CRI. Untreated MDS-patiants with an increased TK activity about 3 months at least, took e high risk of reaching a transition to acute myalogenous leukemia (AMLL This risk did not depend on the FAB-classlficaticn of MDS. It could be demonstrated, that TK activity is an independently risk factor for transition to AML in MDS-patients. This report demonstrates, that the biochemical parameter thymidine kinase is a suitable tool for a rational treatment concerning patients with MDS and useful for the early recognition of transition to AML.

MYELODYSPLASTIC SYNDROMES: RESPONSE TO AGGRESSIVE C H E M O T H E R A P Y AND P R O G N O S T I C FACTORS FOR TREATMENT OUTCOME Aul C., Runde V., Heyll A., Schneider W. Dept. Hematol., Univ. Dtisseldorf, Germany 40 patients with advanced MDS presenting with a Karnofsky score of at least 60% were treated with conventional antileukemic protocols. The median age of patients was 52 years (range, 16-68). MDS subtypes at the start of treatment were RAEB in 2 and RAEB-T in 14 cases. Twentyfour patients were treated after transformation of MDS m A M L For remission induction the following protocols were used: TAD-9 n=36, double induction TAD-9/HAM n=l, double induction TAD-9/TAD-9 n=l, HAM n=I, Ara-C/idarubicin n=l. In this series, 26 patients (65%) entered CR, and 3 patients had a PR. Early death occurred in 3 cases, and only 8 patients (20%) had refractory disease. No unusual toxicities of chemotherapy were noted. The median duratlcm of bone marrow aplasia (leukocytes < lx109/] and/or platelet count < 20x109/1) for patients achieving CR after TAD was 28 days (range, 6-39). Despite maintenance treatment, the median duration of CR was relatively short (11 months). The factors most strongly associated with successful remission induction were: 1. a comparatively low blast count (< 30%) at the start of therapy (CR rates: 81% vs. 54% ) 2. age below 50 years (78% vs. 55%) 3. presence of Auer rods in granulocyte precursors (76% vs. 57%). We conclude that intensive chemotherapy can successfully be administered to patients with MDS. Treatment is particularly successful in younger patients with Auer rod-positiv blasts and should be commenced before patients transform to overt AML. Carlo Aul, Dept. Hematol., Univ. Diisseldorf, Moorenstr. 5, 4(100 Dtisseldorf 1, Germany

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SEQUENTIAL TREATMENT WITH RECOMBINANT HUMAN GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (GM-CSF) AND HUMAN ERYTHROPOIET1N (EPO) IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES (MDS) Runde V. l, Aul C. 1, Ebert A. 2, and Schneider W. 1 1Dept. Hematol., ZDept. Nuclear Med., Univ. Diisse!dorf, Germany Preliminary studies have shown that up to 20% of MDS patients respond to recombinant human erythropoietin (EPO), by disappearance or reduction of transfusion requirements. GM-CSF, activ on early myeloid progenitors, has been shown to induce an increase in reticulocytes in at least some patients with MDS. In this study we explored the efficacy of sequentially applied GM-CSF and EPO in 6 preleukemic patients. GM-CSF was administered subcutaneously at a dosage of 150 tag/mZ/d for 10 days. EPO was given subcutaneously at a dosage of 100 UNg/d from day 11 to day 38. FABsubtypes were: RA n=2, RARS n=2, RAEB n=2. All patients were transfusion dependent (2-6 units of packed red blood cells per month) and had markedly increased endogenous serum EPO levels (range, 1593618 mU/ml ). One patient became transfusion independent with a maximal increase in hemoglobin to 9.7 g/dl. Of interest, this patient also showed an improvement in platelet counts (15 to 50 x109/1). In the remaining cases the sequential administration of GM-CSF and EPO had no beneficial effect on the hemoglobin concentration. With respect to the responder's delayed improvement in erythropoiesis (7 weeks after start of treatment) we cannot exclude, that the beneficial effect resulted from the administration of EPO alone. Side effects, observed only during application of GM-CSF, included fever in all cases and bone pain in 3 cases. Additional patients will be required to evaluate the efficacy of this treatment protocol. Volker Runde, Dept. Hematol., Univ. Dtisseldorf, Moorenstr. 5, W-4000 Dtisseldorf 1, FRG

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Leukemia Cell Biology 116

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IN SITU AMPLIFICATION OF REARRANGEDGENE SEQUENCES IN INDIVIDUAL CELLS FOR TRACING CLONAL CELLS IN SMEARS FROM BLOOD AND BONE MARROW

FLUORESCENCE IN SITU HYBRIDIZATION (FISH) ANALYSIS OF CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA WITH A PANEL OF EIGHT CHROMOSOME SPECIFIC PROBES

K. Pachmann, W. Spann, H. Zabnienska, M. Knizia and B. Emmerich

A. Berger, S. Strehl, P. Ambros, O.A. Haas and H. Gadner

The polymerase chain reaction (PCR) allows detection of minute amounts of defined DNA sequences. To date the DNA from a complete population is isolated and amplyfied. The disadvantage of this procedure is the lack of the possibility to identity the exact source of the DNA, the individual cell. This may be of importance in mixed cell populations where the gene sequence of interest is contained only in a restricted subpopulation such as in metastatic cells from a tumor in e.g. blood or bone marrow. We have overcome this disadvantage by designing a procedure which allows amplification of the DNA sequence in question directly in individual cells of conventional cell smears from bone marrow and blood. Rearranged single copy genes, such as the bcl2 rearragement can be amplified to visibility in individual lymphocytes floated from a follicular lymphhoma into the bone marrow. Ampitied material shed into the supernatant allows gel analysis showing only the expected bands. The amplified material remains highly specilically iocaiized to the cells. Thus, this method provides the possibility to trace a single altered gene in an individual cell. This method may prove useful to directly detect single leukemic cells and to enumerate the cells carrying modified gene sequences such as translocations or rearrangement even if they aare pressent only in low amounts such as aafter after therapyin e.g. leukemias. Thus malignant cells otherwise indistiguishable from normal cells can unambiguously be detected and monitored in conventionally prepared smears. Med. Klinik Innenstadt der Universit~t MOnchen H~matol. Forschungslabor Ziemssenstr.1 8000 Mnnchen

Cytogenetic analysis of childhood acute lymphoblastic leukemia (ALL) is often hampered by an insufficient quality and/or quantity of mitotic cells. We t h e r e f o r e used the f l u o r e s c e n c e in situ hybridization (FISH) technique as an alternative screening method for numerical chromosome abnormalities in interphase cells. We selected 15 cases which were previously shown to be hyperploid by DNA cytofluorometry and/or cytogenetic analysis and scanned them for the p r e s e n c e and type of numerical c h r o m o s o m e abnormalities. We used a panel of probes specific for the centromeres of chromosomes 1,8, 10, 12, 17, 18, X and Y. Eleven cases were studied with all eight probes and four cases only with six probes. Applying this method, we detected one or more additional copies of the investigated chromosomes in the blast cells of 14 patients. Comparison of the results obtained by FISH with those obtained by conventional cytogenetic analysis in 5 cases revealed a good correlation in 10 instances. Additional copies of particular chromosomes were discovered only With FISH in five instances and one clone with trisomy 12 was not detected in interphase cells. In ten cases with a hyperploid DNA-content in which no cytogenetic results were available, three copies of the a b o v e c h r o m o s o m e s were seen in 30 and four copies in two instances. Duplications of the Y and X chromsomes were seen in one and four instances from male patients, respectively. Our results therefore indicate that FISH analysis is an excellent method for studying numerical chromosomal aberrations in interphase cells of ALL CCRI, St.Anna Children's Hospital, Kinderspitalgasse 6, A-1090 Vienna, Austria

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DETECTION OF CHROMOSOMAL ABNORMALITIES ON PREVIOUSLY STAINED BLOOD AND BONE MARROW SMEARS BY FLUORESCENCE IN-SITU HYBRIDIZATION

IDENTIFICATION AND CHARACTERIZATION OF MONOCLONAL LYMPHOID POPULATIONS BY ANALYSIS OF COMPLEMENTARY RNA OF CLONESPECIFIC DNA SEQUENCES (cRNA SSCP) O.M.Koch 1, M.Volkenandt 2, E.GSker I , J.Buer 1, K.Danenberg 3, P.V.Danenberg 3, J.R. Bertino 1

Bentz M., Dbhner H., Schr6der M., Pohl S., Lichter P.* Dept. of Internal Medicine V, University of Heidelberg, Germany and *Deutsches Krebsforschungszentrum, Heidelberg, Germany With the development of fluorescence in-situ hybridization (FISH) techniques studies of chromosomal abnormalities on interphase nuclei have become possible. So far, the method has been mainly applied to slides that were prepared for routine cytogenetic analysis. By directly performing hybridization experiments on previously stained blood and bone marrow smears, investigations on archival material would become possible. We examined Wright's stained blood and bone marrow smears of three patients with AML. All three had chromosomal aneuploidies, either trisomy 8 or/and monosomy 7. For FISH the following DNA probes were used: probes recognizing highly repetitive sequences of the (~-satellite family within the centromer regions of chromosomes #7 and #8 respectively, plasmid libraries specific for #7 and #8 and a cosmid probe mapping to the c-myc protooncogene in #8q24. After destaining and methanol/acetic acid treatment, hybridization was performed as described previously (Lichter et al., Hum. Genet.1988, 80:224-234). With all DNA probes detection of the specific numerical abnormalities on blood and bone marrow smears of the AML patients was possible. In addition to the examination of cytogenetic preparations, the cell morphology was preserved and a correlation between morphological and cytogenetic features of single cells could be performed. Using appropriate probe sets, eventually the examination of cells on blood and bone marrow smears for many specific chromosomal abnormalities will become possible.

Single base pair mutations in defined genes may be detected by polymerase chain reaction (PCR) and analysis of conformational polymorphisms of complementary RNA by non denaturing polyacrylamide gel electrophoresis (cRNA SSCP, complementary RNA single strand polymorphism, (Danenberg et al. submitted). We amplified junctional regions of T-cell receptor gamma (TCR-gamma) and immunoglobulin heavy chain gene (CDR 3) rearrangements in cases of acute leukemia of both lineages by PCR. One PCR primer at the 5' end contained a T7 RNA polymerase promoter sequence. The PCR product was transcribed into cRNA and the product was then analyzed by 8% polyacrylamide gel electrophoresis and stained with ethidium bromide or visualized by autoradiography. In the presence of a monoclonal (or oligoclonal) lymphoid population either for TCR-garnma and/or for IgH gene rearrangement, multiple clonotypic cRNA bands are formed. These patterns of cRNA polymorphisms are strictly related to the primary DNA sequence of rearranging TCR or IgH genes. The cRNA SSCP methodology allows the comparison of different lymphoid clones, and may be done on specimens obtained at different time points of the course of disease. This novel method is rapid, and requires only minute amounts of DNA and may thus have advantages over the conventional Southern blot hybridization procedure. 1Memorial Sloan-KetteringCancer Center, 1275York Ave,New York, NY, 10021, USA

2DcrmatologischeUniveb3itaetsklinik,Frauenlobstrasse8-11, 8000 Muenchen2, Germany 3KennethNorris Jr. ComprehensiveCancer Center, 1305 MissionRd, Los Angeles,CA,90033, USA

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DIFFERENCES IN IMMUNOGLOBULINAND T-CELL RECEPTOR GENE REARRANGEMENTPATTERNS IN ACUTE LYMPHOBLASTIC LEUKEMIA BETWEEN DIAGNOSIS AND RELAPSE Auke Beishuizen1, Marie-Anne J. Verhoeven~, Elisabeth R. van Wering2, Karel H~thlen2'a and Jacques J.M. van Dongen T. 1. Dept of Immunology, University Hospital/Erasmus University, Rotterdam; 2. Dutch Childhood Leukemia Study Group, The Hague; 3. Dept of Pediatrics, subdivision Hematology-Oncolcgy, Sophia Children's Hospital/Erasmus University, Rotterdam, The Netherlands.

Despite the current treatment protocols 20-30% of children with acute lymphoblastic leukemia (ALL) still develop a relapse. The pathogenesis of leukemia relapse is still poorly understood. Detailed analysis of the leukemic cells at diagnosis and relapse may give insight in the process of (sub)clone selection and development of therapy resistance. We performed comparative studies on Ig and TcR gene rearrangement patterns in 20 precursor.B-ALL and 10 T-ALL at diagnosis and subsequent relapses. In 8 precursor-B-ALL multiple rearranged IgH gene bands were found at diagnosis, which were caused by b{- or oligoclonality at the Ig heavy chain (IgH) gene level. Differences in Ig and TcR gene rearrangement patterns between diagnosis and relapse were found in 55% and 30%, respectively in the 20 precursor-B-ALL (including all eight bi-/oligoclonal ALL). In the 10 T-ALL differences at the Ig and TcR gene level between diagnosis and relapse were found in 20% and 40%, respectively. In one precursor-B-ALL of the 17 ALL cases with differences in Ig and TcR gene rearrangement patterns no clonal relationship between diagnosis and relapse cell samples could be found. In the other 16 ALL cases (12 precursor-B-ALL and 4 T-ALL) clonal relationship was detected, based on the presence of at least one identical rearranged Ig and/or TcR gene band between diagnosis and relapse, proving the persistence of the original leukemic (sub)clone at relapse. No clonal evolution was found in the other 13 ALL cases. Interestingly, the duration of remission was correlated with the presence of changes at the Ig and TcR gene level, suggesting that the chance of this type of clonal evolution increases with time. Therefore, we conclude that in most ALL the changes in Ig and/or TcR gene rearrangement patterns at relapse can be regarded as a form of elonal evolution, suggesting differential sensitivity of the various (sub)clones at diagnosis for the applied cytostatic treatment.

ANALYSIS OF VNTR (VARIABLE NUMBER OF TANDEM REPEATS) REVEALS DIFFERENCES BETWEEN LEUKEMIC CELLS AND REMISSION-PHASE LEUKOCYTES IN PATIENTS WITH ACUTE LEUKEMIA G.Hiibner, K.Battmer, H.Poliwoda, and H.Link It is commonly a c c e p t e d t h a t t h e t r a n s f o r m a t i o n of cells and the development of acute leukemia is the result of an accumulation of a series of somatic changes of cellular DNA, as loss of chromosomes, deletions, other aberrations, or mutations, V N T R (Variable number of tandem repeats) analysis provides a useful approach for studying some of these changes in somatic DNA. The purpose of this study was to detect differences between the leukemic phase (at diagnosis or relapse) and remission phase DNA. We applied one simple repeat probe (GTG)~ and one minisatellite (MI3) after DNA-digestion with different restriction endonucleases (Hinf I and H A E lid and agarose gel eleetrophoresis. D N A of 20 adult patients was analysed. In 6 of ii patients with A M L and 4 of 9 patients with ALL it was possible to detect loss of bands or additional bands with at least one of ~he probes. Together the probes MI3 and (GTG)~ unveiled deviating fingerprint patterns in 50% of patients between Ieukemlc ceils and remission-phase leukecytes. In p a t i e n t s with r e l a p s e t h e VNTR p a t t e r n was i d e n t i c a l with t h a t of leukemic cells a t diagnosis. AIlogeneic bone m a r r o w t r a n s p l a n t a t i o n was performed in 6 p a t i e n t s . In each case ~he VNTR p a t t e r n of t h e donor was d i f f e r e n t from t h e r e l a p s e and t h e r e m i s s i o n - p h a s e p a t t e r n . Vie conclude from our s t u d i e s , t h a t t h e probes M13 and (GTG): are u s e f u l in d e t e c t i o n of r e s i d u a l d i s e a s e and r e l a p s e in a c u t e l e u k e m l a s a f t e r c h e m o t h e r a p y and bone marrow t r a n s p l a n t a z i o n . D e p a r t m e n t of Hematology and Oncology, H a n o v e r Medical School, P.O.Box 61 01 80, D - a 0 0 0 H a n n o v e r 61, FRG

This work was supported by the Dutch Cancer Foundation (Nederlandse Kankerbestrijding, Koningin Wilhelmina Fonds), grant IKR 89-09.

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F. Griesinger, H. Elfers, B. W~rmann, J. Ritter 2 , Th. BQchner, J. van de Loo, and W. Hiddemann. Dept. of Hematology and 2pediatrics, University of MUnster, 4400 M~nster. Progress has been made in the treatment of adult and childhood acute lymphoblastic leukemia (ALL) in the past, yet still about 25% of childhood and 40% of adult ALL relapse after successful induction therapy. Early detection of small nth~bere of residual blasts may be of value for the prediction of relapse and risk adapted post-remission therapy. We have initiated a retrospective study (mean followup 46 months after diagnosis) in children and adults with ALL in an attempt to correlate clinical outcome with the persistence of residual leukemic disease using polymerase chain reaction (pcr) for amplification of T C R 6 r e a r r a n g e ments. In non-T, non-B ALL, TCR~ frequently undergoes recombination involving V62 and D~3 with highly diversified junctional regions. Amplification of V52-(D6)-D63 rearrangements was successful in 45% (9 of 19) of childhood and 44% (4 of 9) of adult ALL. All rearrangements in adult ALL were monoallelic, while 4/9 in childhood ALL were monoallelic, 5/9 biallelic or oligoclonal. Junctional regions were found to be highly diversified by the insertion of N and P nucleotides, the involvement of more than one diversity element and extensive exonucleolytic trinuuing of coding regions, involving intronic sequences in one adult ALL. The specificity of clone specific oligonucleotides complementary to the junctional region was demonstrated, and sensitivity was determined to be below i0 -3, Results of the analysis of the remission marrows using "signature" probes for detection of minimal residual disease and the correlation with clinical outcome will be presented. In conclusion, our results of 28 childhood and adult ALL off therapy show that leukemic clone specific TCR6 rearrangements were informative in almost 50% and can be used for the detection of minimal residual disease in ALL.

TAL-1 GENE REARRANGEMENT IN ACUTE LYMPHOBLASTIC LEUKEMIA*) A.Borkhardt, R.Repp, J.Harbott, J.Ritterbach, F.Berner, C.Keller, F.Lampert Children's Univ. Hospital, Feulgenstrasse 12, 6300 Giessen, FRG Tal-1 rearrangement probably is an important genetic marker of leukemic T-cells in a significant proportion of T-cell ALL patients. By nested PCR we have analyzed bone marrow samples of 56 children with T-cell acute lymphoblastic leukemia. In 5 patients tal-1 gene rearrangements were detected which differed slightly in size as vizualized b y agarose and polyacrylamid gel electrophoresis. One patient was tested at time of diagnosis and after a relapse 11 months later. As the rearrangement was found in leucemic cells without a cytogenetically detectable deletion on chromosome 1, the site of the tal gene locus, one must assume that the alteration is beyond the resolution of karyotype analysis. Cloning in pUC 19 vector and sequencing of the fusion site confirmed sequence variation at the recombination site. By using this marker early detection of residual disease is possible in all patients with tal-1 rearrangement. Problems of quantitative PCR such as plateau effects, variable cycle efficiencies due to primer- dimer formations and variation of free cofactors and substrates can be overcome by temperature gradient gel electrophoresis (TGGE). We, therefore, have constructed a DNA probe which differed in 5 basepairs from the original sequence. After coamplification of this probe the template copy number was calculated as described by Hencc and Heibey (1990). As compared to other methods of quantitative PCR this approach offers several advantages and might be useful for monitoring minimal residual disease. Ref: Henco and Heibey (1990): Nucleic Acids Res. 18:6733-6734 ........................................ *)Supported by "Kind-Phillip-Stiftung" and the "Parents' Initative GieSen"

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MOLECULAR MONITORING OF THE PMI./RAR-a FUSION GENE IN ACUTE PROMYELOCYTIC LEUKEMIA BY POLYMERASE CHAIN REACTION. A.Biondi,A.Ramhaldi,P.P.Pandolfi,M.Alcalay,V.Rossi,G.Giudici,F.Lo Coco,D.Diverio,D.Zangrilli,F.Mandelli,F.Grignani,G.Masem,T.Barbui, P.G.Pelicci.

MUTATIONS OF THE PS3 GENE IN ACUTE LEUKEMIAS : A REPORT ON 207 CASES. C. Preudhomme, I. Quiouandon, Ph. Jonveaux, J.L. Lai', MVanrumbeke, M.H. Loucheux- Lofebvre, J.P.Kerckaert, P. Feoaux,. CHU and INSERM U 124, Lille and U 301, Paris, France. The P53 gene is a tumor suppressor gene, whose inactivation through deletions or mutations seems to play a role in neoplasia, and which has been mapped to 17p13 region. We looked for P53 gene mutations in exons 5 to 8 (where the vast majority of mutations have been found) by single strand conformation polymorphism (SSCP) analysis of DNA amplified by PCR, a sensitive method for the detection of mutations in short DNA fragments. Our preliminary findings had demonstrated the sensitivity and specificity of the technique applied to the P53 gene. 207 patients (pts) including 105 cases of AML (102 de novo, 3 therapy related) and 102 cases of ALL (9 Burkitt ALL, 51 precursor B-ALL, 42 T-ALL) were analyzed. All pts had cytogenetic examination; 13 of them (including 11 AMLand 2 ALL) had 17p monosomy, resulting from loss of a whole chromosome t 7, iso 17q formation or unbalanced translocations. SSCP analysis was normal in 197 cases, and showed an abnormal SSCP pattern in 10 cases (7 AML and 3 ALL). Sequence analysis confirmed the mutations, which involved exon 6 (1 pt), exon7 (2 pts) and exon 8 (7 pts) and consisted of nucleotide substitution (leading to an aminoacid change) in 8 cases, and deletion or insertion of 1 nucleotide (leading to a frameshift) in the 2 remaining pts. In AML, no correlation between mutations and hematological findings was found. In ALL, mutations predominated in Burkitt's type (2/9 vs 1/5 t precursor B and 0/42 T-ALL). One of the 3 therapy related cases of AML were mutated. 8 of the 21 pts (38%) with complex (ie at least 3) chromosome abnormalities had a mutation, vs 2 of the 186 other Lots. However 5/13 (40%) of the pts with 17 p monosomy (12 of whom had complex abnormalities) had a mutation vs 2/194 ptswith cytogenetically normal 17p. In addition, in 3 of the 5 mutated cases with cytogenetically normal 17p, SSCP and sequence analysis suggested loss of the normal P53 allele through cytogenetically undetectableevents. Responsetotreatment was generally poor and 9 ofthe mutated cases died within 10 months. Our findings suggest that 1) P53 gene mutations are relatively rare in acute leukemias (except perhaps in Burkitt ALL) 2) they are almost exclusively seen in pts with complex chromosome rearrangements, thus leading to question their role in leukemogenesis, in pts already having multiple genetic abnormalities. However, as they also predominate in pts with loss of the normal P53 allele, their role in the development or progression of leukemia is strongly suspected through loss of tumor suppressive activity of the normal p53 protein (as in solid tumors) 3) they are generally associated with short survival,

Acute promyelocytic leukemia (APL) is characterized by the 15;17 chromosomal tmnslocation. By cloning experiments we and others have established that the chromosome 17 breakpoints (bp) map to the RAR-0t and the 15 to the PML locus (previously designated myl). Southern blot analysis of more than 50 APLs with genomic probes derived from ehrnmosome 17 , revealed that chromosome 17 breakpoints occurred consistently within an approximately 16-Kb DNA fragment of the RAR-a intron 2. Chromosome 15 breakpoints cluster within three regions of the PML gene:intron 6 (breakpoint cluster region l or bcr 1), oxen 6 (breakpoint cluster region 2 or bcr 2) and intron 3 (breakpoint cluster region 3 or bcr 3). Two fusiune genes, PML/RAR-ct on the 15q+ derivative and RAR-tx/PML on the 17q- derivative are formed as a result of the translocation. PML/RAR-ct genes generate fusion mRNAs which encode chimeric PML/RAR-txproteins. Nucleotide sequence of PMI./RAR-tx and PML cDNAs were obtained. Primers have been identified that allow the detection of the chimeric PML/RAR-ct mRNA in all 35 APL patients analyzed by reverse PCR and nested primer approach of two rounds of amplification. The method represents the easiest and fastest way to identify the t(15; 17) even in the cases where conventional cytogenetics failed to do it. APLs that displayed similar pattern of chromosome 15 breakpoint and the same aberrant transcript showed different chimeric PML/RAR-a products , due to alternative splicings o f the PML exons, bcrl and bcr3 are the regions of PML gone most frequently involved among APL (48.5 and 34.2 of case respectively); bcr3 constitutes 62.5% of cases among M3Vas compared with 25.9% Of M3. Despite the great heterogeneity of the PML/RAR-ct fusion gone,data will be presented on the feasibility to monitor the APL clone by PCR analysis in seven APL patients who received different treatment (chemiotherapy, all-traus-retinoic acid or bone marrow transplantation ). A.Biondi,M.D. Cli.Ped.Univ,Milano H.S.Gerardo via Donizetti, 106/'20052 MONZA (MI) Italy

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SIGNIFICANCE OF LINEAGE-ASSOCIATED TRANSCRIPTS OF TDT AND ETSI FOR DETECTION OF LEUKEMIC BLASTS

WendmerC,-M., Saccbi N., BiichnorTh., HiddemannW., vmade Leo J., Wi~lllannB. Dep. of Internal MedicineA, Universityof Miinsrer,Germany; Laboratory of Molecular Ontology, National Cancer Institute, Frederick, MD, USA. Leukemic cells were examined for aberrant transcription of the differentiationassociated genes of Terminal deoxynucleotidyl Transferase (TdT) and ETS 1. TdT is predominantly detected in immature lymphoCytes in thymus and bone marrow. The ETSl gene belongs to the group of human protooncogeaes and encodes a mRNA species which is prevalently expressed in human thymocytes and T cells. A functional role during the T cell differentiation is postulated since the Ets-I protein binds to the enhancer sequence of the TCR a geae. Transcripts of TdT and ETS1 were amplified in different leukemic cell lines by the eDNA polymerase chain reaction (cDNA-PCR) using primers from different ex0ns. Conventionallyspliced TdT RNA was observed in both pre-B and pre-T cells. In contrast to RNA analyses by Northern blot minute amounts of TdT specific transcripts were amplified in mature B, T and myeloid leukemic cells as well as in non-hematopoietic cell lines and tissues, only invertebrate material proved to be TdT negative. PCR analysis of ETS 1 transcripts demonstrated an abundant expression of the gene along the lymphoid lineage with an additional minor RNA species for T cells and only minute transcripts in myeloid and non-hematopnletic cell lines in higher cycle numbers, Simultaneous amplification of RNA with specific primers both for TdT and ETS1 showed that TdT expression declined from the immature to the mature T cell lines while the transcriptional

activity of ETSI was constant along the T cell differentiation. Since the levels of TdT transcripts in mature lymphoid and myelnld malignancies as well as those of ETS! transcripts in myeloid leukemic cells were comparable to the level observed in normal tissues, an "fllegitima~" transcription was assumed.

CombinedPCR of TdT and ETS1 could be used for detectionof extramedullary residual blasts of lymphoid leukemia: after screening for leukemic blasts in

extramedullarysanctuaries and exclusion of unspecific "illegitimatetranscripts" by a low-cycle amplification, the differentiation stage of the leukemic cell could be elucidated by a high-cycle amplification dut to the qualitative and quantitative alterations of TdT and ETSI along the lymphoid differentiation pathway.

C-RAF-1 PROTO-ONCOGENE mRNA EXPRESSION IN ACUTE MYELOID LEUKEMIA BLAST CELLS C.A.Schmidt, H.Oettle, F.F.Willborn, D . H u h n , W.Siegert ........................................................................................ T h e raf-1 p r o t o - o n c o g e n e product , a c y t o p l a s m a t i c serine threonine kinase, plays a n important role in signal transduction a n d is activated in r e s p o n s e to several g r o w t h factors. Recently, activation o f raf-1 p r o t o - o n c o g e n e in m y e l o i d cell lines following h e m a t o p o i e t i c g r o w t h factor treatment w a s detected. O n c o g e n i c activation b y a m i n o terminal deletion h a s b e e n described in in vitro experiments. T o investigate alteration o f craf-1 g e n e in acute m y e l o i d l e u k e m i a ( A M L ) , blast cells s a m p l e s f r o m 74 A M L patients were investigated for D N A alteration b y S o u t h e r n Blot analysis. N o r e a r r a n g e m e n t , deletion or amplification o f the c-raf-1 g e n e could be detected b y this m e t h o d . D N A p o l y m o r p h i s m , desribed previously, w a s o b s e r v e d in 17% of A M L cases. N o r t h e r n Blot analysis w a s p e r f o r m e d in 20 o f t h e s e patients a n d revealed n o r m a l c-raf- 1 transcript size in all cases. Yet c-tar-1 m - R N A o v e r e x p r e s s i o n w a s detected in 1 o f t h e s e 20"cases. Partial s e q u e n c i n g revealed n o s e q u e n c e a b n o r m a l i t y in this c a s e s o far. W e therefore c o n c l u d e that c-raf-1 alterations, if present, are rare e v e n t s in acute m y e l o i d l e u k e m i a blast cells. Medizinisehe Kllnik und Poliklinik m.S. H~imatologie und Onkologie, UniversiEitsklinikttm Rudolf Virehow, Spandauer Datum 130, 1000 Berlin 19, FRG

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PARENTAL ORIGIN OF THE TRANSLOCATED CHROMOSOME 9 AND 22 INVOLVED IN PHILADELPHIA-POSITIVE LEUKEMIAS O.A. Haas and A. Tirita

IMMUNO-ELECTRONMICROSCOPICAL ANALYSIS OF THE CD 54 ANTIGEN (ICAM-1) EXPRESSION ON AML BLASTS AND ITS POSSIBLE ROLE IN HOMOLOGOUS AGGREGATION OF THE BLASTS J. Kranter, S. Gttz, Ch. Westphal, D. Bunjes and G. Heil

Functionally equivalent genetic material can be marked and utilized differentially depending on its maternal or paternal origin. This phenomenon is termed genomic imprinting and has been shown to play an important role in certain cancer predisposition syndromes and sporadic tumors characterized by loss of genetic material. We were interested whether such imprinting effects might also play a role in the genesis of acquired chromosome abnormalities characterizing specific haematological neoplasms. By exploiting cytogenetic polymorphisms, we have determined the parental origin of the chromosomes 9 and 22 involved in the reciprocal translocation t(9;22)(q34;q11) which is the most common translocation of Philadelphia chromosome (Ph)-positive acute and chronic leukemias. For our studies, we selected 17 consecutive Ph-positive patients (14 with CML and 3 with ALL) whose parents were available. The age of the 11 male and 6 female patients ranged from 3 to 50 years (median 11 years). Ten patients had an informative chromosome 9 heterochromatin polymorphism and the translocated chromosome 9 was of paternal origin in all patients. Furthermore, we have so far been able to determine the parental origin of the translocated chromosome 22 in three cases. In all instances it was of maternal origin.

The CD 54-antigen (ICAM-1) plays an important role in the function of the immune system and is present on a wide variety of leukocytes and stromal cells. Together with its ligand, the CD l l a antigen (LFA-1) it is involved in the homologous aggregation of phorbol ester stimulated T-lymphoeytes, B-lymphocytea and monocytes. ICAM-1 expression was also found on blasts of 30 - 40% of acute myeloid leukemias (AML), whereas its function on these cells remains unclear. To analyse antigen expression and its possible role in cell.-cell interaction monocyte and T-ceU depleted blasts of six AML cases (FAB M 4, n=4, FAB M 5, n=2) were examined by immuno-electronmieroscopical analysis of the whole cells adherent to melamine resin foils. ICAM-1 antigen expression was studied using a mouse monoclonal antibody (atui-ICAM-1, Immunotech, France) and a secondary goat-anti-mouse antibody, linked to 30 nan gold particles (GAM 30, Janssen, LSL, Belgium). For analysis of cell-ceU interaction cells were incubated as adherent monolayers for various time intervals using serum-free media either alone or with addition of the anti-ICAM-I antibody or the MY 4 antibody (CD 14). After an 18h incubation period in serum-free media cell-cell-contacts via filopodia together with cell-aggregation were found in all 6 AML-cases studied. Immunoelectron-microscopieal analysis revealed that the ICAM-1 antigen was mainly located at the ce11.-cell-contactsites. The addition of the anti-ICAM-1 antibody to the culture media significantly reduced cell-cell contacts and ceU-aggregation, while the addition of the anti-My4-antibody had no significant influence on expression of cell-cell-interactiun and aggregation. These data suggest that the CD54 antigen may play a major role in cell-cell-interaction of unstimulated myeloid blasts of FAB M4/M5 subtype. Department of Internal Medicine III, University of Ulm, 7900 Ulm, Germany

Thus, our findings provide the first evidence that genomic imprinting is also instrumental in the generation of tumor-specific chromosome rearrangements. CCRI, St.Anna Children's Hospital, Kinderspitalgasse 6, A-1090 Vienna, Austria

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REGULATION OF THE EXPRESSION OF ICAM-1 ON PRE-B CELL LINES, ~ ~ Ch.Sch~rdl~ ~, C.Schardt r H.G. Drexler ~ A. Ganser" and D. ~loelzer . Dept of Hematology, University of Frankfurt~ FRG; '~Dept of Hematology, University of Marburg,FRG; "~DSM, University of Braunschweig, FRG

UPREGULATION OF LFA-la, LFA 1B, MLA-1, AND ICAM-1 IN VlVO BY INTERLEUKIN-2 IN PATIENTS WITH AML K.Fenchel, L. Bergmann, B.Jahn, P.Mitrou

Expression of adhesion molecules is important in interactions involving cells of the immune system and can be upregulated by cytokines. To analyze the importance of cell-cellinteractions in the proliferation of acute lymphoblastic leukemic cells we have determined the baseline expression of ICAM-1 and its modulation by TPA, recombinant TNF-a and recombinant IFN-gamma in a number of human pre-B cell lines (Nalm-6, REH, MN-60, BV-173) by FACS analysis. All cell lines displayed ICAM-1 in variable degrees. In addition, immunoblots with the MoAB anti CD54 showed variably glycosylated ICAM-1 antigens resulting in different MG's of 85 KD to 92 KD. When the REH cell line was treated with IFN-gamma, TNF-G and TPA, TPA induced further maturation of REH cells along the B-lineage with monocytoid features. TPA but not IFN-gamma or TNF-a also induced adherence of REH cells to culture flasks after 12 hrs of incubation. After 12 hrs of incubation with TPA, TNF-a or IFN-gamma, upregulation of ICAM-1 expression could be determined by FACS and immunoblot analysis. Northern-blot analysis revealed de novo synthesis of ICAM-1 specific transcripts showing inducible expression for the ICAM-1 gene. IFNgamma was much more effective than TNFa in inducing ICAM-1 expression. The results of the present study indicate that variable expression of ICAM-1 is a property of pre-B cell lines and can differentially be upregulated by IFN-gamma and TNF-a. We suggest that cytokine induced expression of ICAM-1 may be important in the interaction of pre-B ALL and bone marrow stromal cells. Present address: Department of Frankfurt, Theodor-Stern-Kai 7 6000 Frankfurt

Hematology, University of

While several cytokines including IL-1, IL-4, IL-7, TNF, and IFNy are known to be able to upregulate the expression of the adhesion molecules LFA1 and ICAM-1, the influence of IL-2 on the expression of these molecules is still uncertain. In this study the effect of IL-2 on the expression of LFA-la, LFA113,VLA-1, and ICAM-1 was investigated. In 3 Patients in second remission of AML IL-2 was applicated as a late consolidation therapy in a dosage of 9 Mill. IU/m2 i.v. as a bolus infusion for about l h daily on day 1-5 and 8-12. Up to now patients received 7 courses of treatment. The expression of the adhesion molecules on peripheral blood lymphocytes was examined using flowcytometry. Soluble ICAM-1 was determined by ELISA. The cellular expression of LFA-la, LFA-11], VLA-1, and ICAM-1 showed no changes during treatment, but was upregulated 7-14 days after each therapy. This corresponded to other biologica! effects of the IL-2 therapy like rebound lymphocytosis, whicl" occurred also 7-14 days following therapy. Soluble ICAM-1 serum concentrations increased in some cases with a maximum on day 8 of treatment. Following IL-2 bolus sICAM-1 serum concentrations rose to a peak after 6-8 hours followed by a rapir decline. Immunotherapy with IL-2 induces upregulation of adhesion molecules, which may be an important factor of cytotoxicity. Div. of Hematology, Dep. of Internal Medicine, J.W.Goethe University, Frankfurt/M, FRG

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FREQUENCY OF ABERRANT SURFACE MARKER EXPRESSION IN 221 CASES OF ADULT ACUTE LEUKEMIA. AN IMMUNOLOGIC / CYTOGENETIC ANALYSIS

N~U~OPEI:~IDE u (h'~PY) I N P ~ A S AMiM~KER OF PEDIATR][C B CELL P R E C U R S O R I . ~ u ~ d g ~ . A . Bj0rk O#, Theodorsson E*, Ericsson A~, Barbany G~, Persson H~, Kogner P#*. Depts of Pediatrics# and Clinical Chemistry*, Karolinska Hospital, and Molecular Neurobiology Lab~, Karolinska Institute, S-I04 01 Stockholm, Sweden. NPY is a neurotransmitter in the central and peripheral nervous systems. Plasma NPY (P-NPY) is a marker and prognostic indicator in neuroblastoma. A series of 51 consecutive children with acute leukemia was investigated for P-NPY at diagnosis using a radioimmunoassay. Children with leukemia showed higher P-NPY than matched healthy controls ( 2 0 0 : 5 0 - 3 8 5 pmol/L, median: interquartile range, and 3 7 : 2 0 - 5 2 pmol/L resp, p<0.001). Higher P-NPY was found in children with favourable clinical risk classification (p<0.001). When compared to an age adjusted reference level, elevated P-NPY could be detected in most children with B cell precursor (BCP) ALL (31 of 40), but in no children with B- or T-cell ALL or ANLL (n=ll, p<0.00001). P-NPY correlated to CALLA positivity with highest P-NPY found in those with >60% CALLA+ lymphoblasts (p<0.001). P-NPY normalized at remission in all children. High levels of NPY mRNA were detected in leukemic bone marrow of BCP differentiation. In situ hybridization revealed NPY mRNA in CALLA+ malignant lymphoblasts. Event free survival was higher in children presenting with elevated P-NPY (79% vs 35%, 2-46 months follow up, p<0.001). We conclude that NPY is produced in leukemic BCP lymphoblasts. Furthermore, P-NPY may be a marker of BCP ALL (sensitivity 77.5%, specificity 100% in this study). P-NPY may be used as a complement to immunophenotyping using flow cytometry. A role of NPY in normal B-cell development with relation to expression of CALLA, a neutral endopeptidase, may further be suggested.

Tirier C. 1, B e c h e r R. 2, Heir W. 1 We report about our experience in phenotyping 221 cases of de novo acute leukemia in adults between 1985 and 1991 being treated in our department. Morphologically / immunologically 167 were AML and 54 ALL. Aberrant surface marker expression was found in 28 cases (12.7%). In AML 14 out of 164 (8.4%) were aberrant, in ALL 14 out of 54 cases (26%). In these, following surface marker patterns were found: In AML 11/14 cases were CD7 positiv, two of them additionally CD5, another two CD10.3/14 were CD19, one additionally CD10 positiv. Cytogenetic analysis revealed the Philadelphia chromosome in only 1/14 cases, coexpressing CD10 and CD19. Both AML solely aberrant for CD19 showed involvement of chromosome 8 [ t(8;19) and t(8;21) ]. CD7 coexpression showed no distinct chromosomal aberration. 2/14 AML had a normal karyotype. In ALL 9/14 were common-ALL, 3/14 pre-pre-B, 2/14 T-lineage cornmired. C-ALL were CD13 + (3), CD33 + (4), CD15 + (3), CD14 + (2), CDw65 + (1), CD2+(2), CD5 + (2). 3 c-ALL had more than one aberrant marker, 2 of them were My + and T+.The pre-pre B ALL were CD15 + (2) or CD33 + (1), T-ALL CD13 § (1) and CDw65 + (t). Cytogenetic analysis showed the Philadelphia chromosome in 3/14 ALL, all were c-ALL. T (4;11) was found in 2/14, both were CD15 + pre-pre-B ALL. 5/14 had a normal karyotype, 3/14 were hyperdiploid. 1. Dept. of Internal Mad., Hematology/Oncology,Evang. Hospital, Pattbergstr.l-3, 4300 Essen-Werden, 2. Dept. of Internal Med./CancerResearch, CytogeneticLab., Univ. Clinics, 4300 Essen, Germany

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133 O-ACETYL-DISIALOGANGLIOSIDE T-ALL

GD3 IS COEXPRESSED ON

U. Wargalla, G. Mfinstermann, T. Niemeyer, J.Ritter The finding that GD3 is expressed at the cell surface in a large population of T-All and is detectable with a well described anti-GD3 monoclonal antibody, which mediates leucaemic cell-lysis in vitro suggesting a possible clinical utility for anti-GD3 antibodies in the immunotherapy of T-All in childhood (Merrit,W.D et al). Therefore we determined whether GD3 positive T-All cells do also coexpress a melanoma associated Oacetyl-GD3 epitope. Using two monoclonal antibodies U-2AI0 (anti-O-acetyl-GD3) and 6H4 (anti-GD3) in an indirect immunofluorescenceassay, we examined several blood smears from T-All patients for their disialoganglioside expression. However, mAb U-2AI0 shows a strong reactivity only with single T-cell lymphoblasts whereas mAb 6H4 reacts with a wide range of T-cell lymphoblasts. Department of Paediatric Haematology and Oncology University of MUnster, Germany Albert Schweitzer Strasse 33 4400 HGnster, FRG

CLINICAL APPLICATIONS OF STROMAL CELL STUDY IN ACUTE LEUKEMIA. Zaritskey A.Yu., Strijak O.V., Zubarovskaya L.S., Efimov K.V., Afanasiev B.V. Bone Marrow Transplantation Centre, City Hospital N 31, pr. Dynamo 3, 197042 St. Petersburg. Alterations of stromal cells seem to be crucial in the genesis of haematological malignancies and bone marrow graft take. The aim of this study is to reveal clinical significance of stromal cell abnormalities. Routine (CFUf, long-term bone marrow culture) and new (culture of hematoms and CFUf in semisolid plasma dot system, culture of fetal bone and bone marrow core) methods. The results are the following: Normal trephine core, fetal bone, fibroblasts and stromal cells in different manner stimulated proliferation of GM-precursors. When engrafted in rats fetal bone facilitated posteytostatie granulocyterecovery. CFUf assay appeared to be prognostie in AL, especially in AML. Trephine cores, libroblasts of AL patients stimulated GM-colony formation in different manner. Stromal cell and fibroblast ability to stimulate normal haemapoietic cell 3H-thymidine uptake varied in different patients and in different stages of the disease. The ability to stimulate 3-thymidine uptake and to differentiate AML blast cells correlates with remission induction rate. Plasma clot study of herontons permits to evaluate the association of normal or leukemic cells with stromal cells. Preliminary data show association of blast and stromal cells in untreated patients (microenvironment dependency).

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Expression of Granulocyte Macrophage Colony-stimulatingFactor Receptor on Blasts of Acute myelogenous Leukemia (AML) M.Bommer, H.Serve, E.Gunsilius and G.Hell In several institutions clinical trials are under way to examine a possible beneficial role of granalocyte macrophage colony stimulating factor (GM-CSF) in acute myelogenous leukemia (AML). Because no extensive data on GM-CSF receptor expression on lenkemio blasts of a larger patient population and no data on the regulation of GM-CSF receptors on AML - blasts were available, we examined the expression of GM-CSF receptors on separated leukemic blasts from 58 untreated AML patients. Previously eryopreserved peripheral blood samples from patients with a differential blood count of > 80% leukemic blasts were thawed, Ficolla separated and depleted of adherent cells to a purity of leukemic blasts of > 97 %. They were subsequentlyincubated for lh with 500000 cpm radioiodinated GM-CSF with and without the addition of a 500-fold excess of unlabeled GM-CSF. Radioiodination of recombinant human GM-CSF (kindly provided by Dr, Martin Opper Behringwerke, Marburg, FRO was performed by the JodegenR method and yielded high specific radienetivity (60-110 ~Ci/~g) with good conservation of biological activity (85 % as ~ r e d in colony assays of normal human bone marrow) and maximal binding capacity of > 7 0 % after iodination. 20 out of 58 patients samples showed significant specificbinding of ~z~I,.GM-CSF. Seatchard analysisof these cases revealed between 200 and 4000 binding sitesper cellwith a K D of about I nM. On 5 of 20 cases evidence for a very low number of an additionalhigh affinityreceptorwas found (KD ~ 1O - I00 pM; 20 - 500 binding sitesper cell).Studieson the effectof in-vitropreexposure to O M - C S F on the expression of G M - C S F receptorsare under way. Preliminary resultssuggest thatin contrast to peripheral blood granulocytes AML blasts do not show significant down regulation of GM.-CSF receptors after 3hr exposure to rhGM-CSF (10 ng/ml). In conclusion, considerable heterogenity in the expression of GM-CSF receptors on the blasts exists. Additionalexperiments are required to correlate in vitro proliferative and self renewal response of AML blasts to GM-CSF with the data presented here.

EFFECTS OF TNF(z ON AML-CELLS DETERMINED BY FLOW CYTOMETRIC TECHNIQUE BASED ON BROMODESOXYURIDINE INCORPORATION H.D. Kleine, U. Wagner, E. Lux, G. D0ring, H. Poliwoda and M. Frsund TNFa exerts cytotoxic and antiproliferative effects on neoplastic cells. It has been used as therapeutic agent for solid turnouts and hematological malignancies. We report on the ex vivo determination of the effect of rhuoTNFa on bone marrow aspirates of fourteen patients by a Bromocleoxyuridina/propidiumiodide-method. Cell samples were drawn after 0.5, 2, 4, 6, 8, 10, 22 and 25 hours from short term suspension bone marrow cultures from patients with AML. Flow cytometric celt-cycle analysis is performed after double DNA staining with propidiumlodide and anti-BrdU-antibodles. By this method the effect of mu-TRFa on cell-prolifemi.ioncan be evakrated after 35 hours only. In about two third of the bona marrow aspirates of AML an inhibiting effect on rhu-TNFa can be demonstrated developing to its full extent after 10 hours. Department of Hematology and Oncology, Hannover Medical School, Konstsnty-Gutsohow-StraSe8, W-3000 Hannover 61, FRG

University of ULM Dept.Innere Medizin 3, Robert-Koeh-Str.8. 7900 Ulna

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GROWTH FACTORS INCREASE THE NUMBER OF CYCLING LEUKEMIC CLONOGENIC CELLS AND ENHANCE ARA-C TOXICITY. N. Van Der Lely, T. De Witte, R. Raymakers, J. Wessels, P. Brons

INTERLEUKIN-7: A GROWTH FACTOR FOR TWO NEW LEUKEMIA CELL LINES WITH A T ( 4 ; l l ) CHROMOSOMAL REARRANGEMENT. Greil j . t P e l t n e r M.1, G r a m a t z k i M.2, Burger R. 2, T r a u t m a n n U2, S t e h r K. 1, Beck J J, ~Kinderklinik, 2Medizinische Klinik III, a l n s t i t u t fiir H u m a n g e n e t i k , U n i v e r s i t y of E r l a n g e n , FRG. Since 1987 t h r e e p e d i a t r i c p a t i e n t s with a b i p h e n o t y p i c a c u t e l e u k e m i a and a t ( 4 ; l l ) t r a n s l o c a t i o n in t h e i r leukemic cells had b e e n t r e a t e d a t our i n s t i t u t i o n . From two of t h o s e t h r e e p a t i e n t s we were able to e s t a b l i s h new l e u k e m i a cell lines. The f i r s t cell line, d e s i g n a t e d SEM, had been e s t a b l i s h e d from t h e leukemic cells of a f i v e y e a r old girl with r e l a p s e of ALL and t h e second cell line, d e s i g n a t e d L L M , from a f e m a l e i n f a n t with ALL a t diagnosis. A c y t o g e n e t i c a n a l y s i s r e v e a l e d in both cell lines a clonal t ( 4 ; l l ) chromosomal r e a r r a n g e m e n t . Both cell lines showed b e s i d e s t h e e x p r e s s i o n of B - c e l l a n t i g e n s CD19, CD22 and m a t u r e B - c e l l m a r k e r CDw75 t h e myeloid a n t i g e n s CD13, CD]5 and CDw65. The e s t a b l i s h e d SEM cells were able to grow in s e r u m f r e e medium w i t h o u t t h e addition of c y t o k i n e s . H o w e v e r addition of 500 U/ml recombinant human i n t e r l e u k i n - 7 (rhIL-7), a growth factor for i m m a t u r e B - c e l l s and T - c e l l s , e n h a n c e d SEM cell growth t h r e e f o l d compared to medium controls. In c o n t r a s t to SEM cells, g r o w t h of LLM cells was d e p e n d e n t on t h e p r e s e n c e of r h i L - 7 . LLM cells c u l t u r e d in s e r u m free medium in t h e a b s e n c e of r h I L - 7 died w i t h i n s e v e r a l d a y s . T h u s we conclude t h a t in v i t r o t h e g r o w t h of leukemic cells from two out of t h r e e patients with t ( 4 ; l l ) chromosomal r e a r r a n g e m e n t was promoted by r h l L - 7 .

The antileukemic drug cytosine-arabinoside (Ara-C) is preferentially cytotoxic to proliferating cells. Therefore, resting leukemic colony-forming cells (CFU-L) may eScape chemotherapy. Previous in vitro experiments showed that growth factors like interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) could increase the number of CFU-L and enhance Ara-C toxicity. This suggested that growth factors could increase the number of clonogenic cells in S-phase. In the present stud:K we investigated the influence of growth factors on the number of cycling CFU-L and c0rrelated this with Ara-C toxicity. Blasts of both spontaneous (n=2) and non-spontaneous (n--5) growing leukemias were cultured in suspension in the absence and presence of stimulatory growth factors for a period of 5 days. During the last 3 days the cells were exposed to various concentrations of Ara-C. Iododeoxyuridine (IdUrd), which is incorporated into the DNA during Sphase, was added during the whole Ara-C exposure period. Subsequently, the cells were washed and the number of surviving CFU-L was determined by agar assay. After 7 days aggregates were counted and the agar cultures were lifted onto glass slides and dried by centrifugation. By immunological detection with peroxidase, individual colonies were analyzed for IdUrd positive and negative cells. In the absence of CSFs, only 3-23 % of the CFU-L from non-spontaneous growing leukemias were IdUrd positive. The IdUrd positive aggregates are postulated to originate from leukemic colony-forming cells that incorporated IdUrd into the DNA during the 3 day Ara-C exposure. This means that only a small minority of leukemic clonogenic ceils have been in S-phase during that period. The Ara-C concentration resulting in 50 % reduction of CFU-L (IDS0) varied from > 105 - 4 x 10"7 M. In the presence of stimulating CSFs, the number of ldUrd positive aggregates increased to 65-89 %. The IDS0 values were much lower, 10,7 to 5 x 10-9 M Ara-C. When spontaneously growing leukem~as were tested, 93-99% of the CFU-L were IdUrd positive regardless whether cultured in the absence or presence of growth factors. The ID50 values varied from 2 x l0 "8 1 x 10-9 M Ara-C. Our data show that stimulation with IL-3, GM-CSF and/or GCSF results in an increased number of proliferating leukemic colony-forming cells and an enhanced Ara-C toxicity. Some leukemias have a spontaneous high proportion of CFU-L in S-phase and are highly Ara-C sensitive. Div. of Hematology, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.

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LYMPHOCYTESUBSETS AND I L - I PRODUCTIONIN CHILDREN WITH. ACUTE LYMPHOBLASTICLEUKEMIA (ALL) DURING INTENSIVE CHEMO-ANDRADIOTHERAPY. A.Chybicka, J. Bogus~awska-Oaworska,W.Jaworski Department of Children Hematology and Oncology,

IN VITRO ISOLATION OF HUMAN CYTOTOXIC T CELL CLONES SPECIFIC FOR AUTOLOGOUS LEUKEMIC BLASTS D.Montagna, R.Maccario.

M.Arico,

E,Montini, F.De Benedetti, A.Martini,

Department of Pediatrics, University of Pavia, IRCCS Policlinico S.Matteo, Pavia, Italy. The role of cytotoxic T lymphocytes in anti-tumor surveWance has been investigated in patients with solid tumors, while a few data are available in patients with lymphoprolipherative disorders. The aim of our study was to find suitable condtions for in vitro activation and cloning of cytotoxic T cells specific for autologous leukemic blasts. Specific were defined those human cytotoxic T cell clones (TCC) able to kill autologous leukemic blasts but not autologous cells from bone marrow during remission in a 5-hr cytotoxic assay. We investigated one child with acute myeloid leukemia (AML) during remission. 352 TCC were obtained from culture of peripheral blood lymphocytes, by using different experimental approaches; cut ,~f ,,,e ,,~,_ &,,,c,,, ,~ ~,. . . . . . . b~ ~,~o, .~ or autologous leukemic blasts. The specific clones were not able to kill autologous T and B lymphoblastoid cell lines nor K562 eryhthroleukemic cell line. 9 of the 10 TCC were alpha/beta+, CD3+, CD8+, CD4-, CD56+; one was gamma/delta+, CD3+,CD8,CD4-, CD56-. These results suggest the presence of in vitro-inducible cytotoxic T cells specifically directed against autologous leukemic blasts, in PBL obtained from a child with AML during remission. The role of these cells in the in vice anti-tumor immune surveillance remains to be established.

Medical Academy, Wroc~aw, Poland Department of Children Surgery, Medical Academy, WrocZaw, Poland In atempt to find an immune defect in children with ALL and to examine the influence of chemo-and radiotherapy on the immune system of the patients we studied the lymphocyte subsets by fluorescence method using a panel of monoclonal antibodies: CD4, CDB, C03, COl6 and IL-I production by mononuclears in 70 children with ALL. Twenty five healthy children served as the control group, The IL-I production was measured by method based on the inhibition of CBA mouse thymocytes autologous rosette formation (acc.Zimecki and Wieczorek). In ALL children the circulating lymphocytes 8 and Tsubsets (CD4,CDB,CDI6) were significantly lower than taht obtained in control group of healthy children (p.O,05). The IL-l production before any therapy was started showed variability from 0-64 units. During the whole period of cytostatic therapy the striking decrease of IL-I production was observed. One year after the end of the therapy IL-I growed up, but never reached values of the control group (median 18 units v 28 units). Based on these observations and on the known multiple activities on hematopoiesis IL-1 appears to be a suitable candidate for experimental stimulation of hematopoiesis "in vivo".

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T gamma/delta lymphocyte subpopulations before and after intensive chemotherapy in patients with Acute myeloid leukemia (AML) H.S.P. Garritsen, J. BQcher, M. Kuhlmann, B.G. de Grooth, J. Greve, W. Hiddemann and B. W6rmann Dept. Int. Med. A, University of Mfinster, MQnster, Germany Dept. Applied Physics, Univ. of Twente, Enschede, the Netherlands

SELECTIVE T LYMPHOCYTE TARGETING AGAINST AUTOLOGOus NON-LYMPHOCYTIC LEUKEMIA AND NONMALIGNANT C E L L S - MOLECULAR BASIS AND IMPLICATIONS FOR IMMUNOTHERAPY M. Notter, H. Dfirkop, W.-D: Ludwig, E. TaM

T gamma delta (T g/d) cells are T lymphocytes expressing a gamma and or delta chain instead of the alpha/beta chain expressed by the majority of T cells. Recent data provide evidence for an immune modulatory role of T gamma delta cells, but the underlying mechanisms have not been evaluated so far. With the availability of different monoclonal antibodies against subpopulations of gamma/delta T cells it is possible to identify subpopulations. In the peripheral blood of healthy individuals the T g/d subsets coexpressing the Vgammag-Vdelta2 gene product form the majority of T g/d cellls. The study described here, using double and triple color immunofluorescence flow cytometric techniques, compared the relative and absolute numbers of different T g/d subsets in the peripheral blood of 10 AML patients before and after intensive chemotherapy. Substantial differences in the percentages and absolute numbers of the different T g/d subsets were observed. The subset distribution in the majority of patients (n=8) before chemotherapy resembled the pattern of T g/d subsets of normal healthy individuals. After intensive chemotherapy the distribution patterns of different subsets changed in a number of patients. In 3 patients a more prominent population of T g/d cells using Vdeltal gene products was observed. From this study we conclude that the immunophenotype of circulating Tg/d lymphocytes can alter during treatment of AML.

Development of rational immunotherapeutic strategies requires the detailed analysis of tumor-host interaction on a molecular and cellular level. Interleukin-2 01-2) activated T lymphocytes of 15 non-selected patients with acute leukemia (12 AML, 3 ALL) were screened for cytotoxic activity against autologous tumor cells and autologous non-malignant Epstein-Barrvirus transformed B (EBV-B) lymphoblasts by standard Chromium release assay. In all AML patients studied, killing of AML but not of EBV-B cells was enhaced 2 to 9,2 fold, when the screening was performed in the presence of the anti-CD3 monoclonal antibody (MAB) OKT3. T callmediated lysis of COS cells transfected with CD64 and CD32 was enhanced 3,2- and 1,g-fold, respectively, in the presence of OKT3. In cold target inhibition studies, CD64 + COS cells completely blocked OKT3-targeted T cell killing of autologous AML blasts, and CD32 + COS cells had a small inhibitory effect, providing definite proof that the major target molecule for OKT3-eoated T cells on AML blasts is CD64. Expression of CD64 on leukemic cells could bc up-regulated by Interferon (IFN)-r (n=4) and IFNc~ (n=l) resulting in T cell targeting being further improved. T cells preincubated with OKT3 and IgG2a (control) were co-cultured with autologous peripheral blood mononuclear cells and the leukemic cell line U937 in a colony formation assay. U937 colonies were completely eliminated by OKT3-coated T cells. The total number of normal hemopoetic progenitor colonies derived from peripheral blood was not affected by targeted T cells, but a shift from CFU-GEMM and CFU-GM to CFU-E and BFU-E occured. Human non-malignant endothelial cells cutured in the presence and absence of IFN-r lacked expression of both CD64 and CD32. Consequently, they were not lysed by T lymphoeytes targeted with OKT3. These in vitro data provide a rationale for the combined clinical use of 11-2, Interferuns, and anti-CD3 MAB in the therapy of AML in stages of minimal residual disease. Dept. of Hematology, Dept. of Pathology, Free University, Klinikum Steglitz, Hindenburgdamm 30, 1000 Berlin 45, FRG.

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INHALATION OF A M P H O T E R I C I N B AS P R O P H Y L A C T I C ANTIF U N G A L T R E A T M E N T DURING I N T E N S I V E L E U K E M I A T H E R A P Y V. Heinemann, P. Scholz, and U. Jehn

O N C E D A I L Y D O S I N G OF N E T I L M I C I N IN T H E E M P I R I C A L T R E A T M E N T OF F E V E R IN THE N E U T R O P E N I C PATIENT. E. Rozdzinski, W. Kern*, A. Reichle, T. Moritz, T. S c h m e i s e r , a n d E. Kurrle.

Pulmonary i n f e c t i o n s by Aspergillus and Candida species have remained a major cause of m o r t a l i t y in leukemia patients. S t u d i e s p e r f o r m e d in a n i m a l s indicate a pulmonary accumulation of inhaled amphotericin B (Antimicrob Agents Chemother 34: 2 9 - 3 2 , 1 9 9 0 ) . F o l l o w i n g t h i s r a t i o n a l e we performed a p r o s p e c t i v e study to evaluate prophylactic inhalation o f a m p h o t e r i c i n B d u r i n g 44 c o u r s e s o f intensive a n t i l e u k e m i c chemotherapy. Median duration of fever > 38 ~ was 5 . 5 days. Systemic antibiotic t r e a t m e n t was performed i n 35 p t s f o r a median o f 18 days; a d d i t i o n a l systemic a n t i f u n g a l treatment w i t h a m p h o t e r i c i n S i . v . was a p p l i e d i n

6 treatment courses for a m e d i a n of 13.5 days. Amphotericin B was inhaled as aerosol at a d o s e of 3 x I0 m g / d a y for a m e d i a n of 27 days. Inhalation was performed from start of chemotherapy to recovery of g r a n u l o c y t e s in the p e r i p h e r a l blood. Inhalation of a m p h o t e r i c i n S was well tolerated, and significant toxic side effects were not observed. HPLC analysis performed during I0 treatment c o u r s e s did not reveal amphotericin B in p t - s e r u m (limit of detection = 0.i ug/ml) indicating that s y s t e m i c e x p o s u r e to a m p h o t e r c i n B did not occur. Weekly analysis of microbial colonization of nose, throught, and sputum resulted in the d e t e c t i o n o f Candida s p e c i e s a t a

rate of O, 16, and 27Z r e s p e c t i v e l y . Aspergillus species were not observed. None of the e v a l u a t e d patients s u f f e r e d from invasive fungal infection of the lungs, and overall m o r t a l i t y from fungal i n f e c t i o n s d u r i n g g r a n u l o z y t o p e n i a was zero. Klinikum Orosshadern, University of M e d i z i n i s c h e Klinik~ Munich, G e r m a n y

Munich,

III

A prospective randomized trial was performed to comp a r e t h e e f f i c a c y a n d s a f e t y of n e t i l m i c i n (6 m g / k g ) g i v e n o n c e p e r d a y (OD) w i t h t h e c o n v e n t i o n a l t h r i c e d a i l y d i v i d e d d o s i n g r e g i m e n (TD) for i n i t i a l e m p i rical combination treatment with B-lactam antibiot i c s of f e b r i l e n e u t r o p e n i o p a t i e n t s . Of t h e 116 ass e s s a b l e p a t i e n t s , 25% h a d b a c t e r e m i a (most of t h e m g r a m - p o s i t i v e b a c t e r e m i a ) , 3 5 % h a d n o n b a c t e r e m i c doc u m e n t e d infection, a n d 40% h a d s u s p e c t e d i n f e c t i o n . Peak serum concentrations of n e t i l m i c i n in t h e OD g r o u p w e r e h i g h e r (median: 18.3 v e r s u s 5.9 mg/l), and t r o u g h s e r u m l e v e l s l o w e r (median: 0.2 versus 0.9 mg/l) t h a n in t h e TD group. No d i f f e r e n c e s w e r e s e e n in r e s p o n s e r a t e s a n d t o x i c i t y . The overall r e s p o n s e r a t e w a s 72% in t h e O D g r o u p c o m p a r e d w i t h 69% in the TD group. C o m p a r a b l e r a t e s in b o t h g r o u p s w e r e a l s o s e e n in t e r m s of r e s p o n s e to t h e i n i t i a l u n m o d i f i e d (i.e. no c h a n g e or a d d i t i o n of B - l a c t a m s , or a d d i t i o n of g l y c o p e p t i d e a n t i b i o t i c s ) treatment (57% v e r s u s 51%), a n d in t h e n u m b e r of d e a t h s d u e to i n f e c t i o n (one p a t i e n t in e a c h group). M u l t i v a r i a t e statistics using the Cox proportional hazards model i d e n t i f i e d t h e t y p e of infection, and the increase in neutrophils but not the allocated treatment group, age, u n d e r l y i n g disease, or i n i t i a l n e u t r o p h i l c o u n t s as a s i g n i f i c a n t p r o g n o s t i c f a c t o r for the response to t r e a t m e n t . An increase in s e r u m creatinine concentration >50% a b o v e baseline was s e e n in t w o (OD) a n d t h r e e p a t i e n t s (TD), r e s p e c tively. In the neutropenic patient, once daily d o s i n g of n e t i l m i c i n a p p e a r s to b e as s a f e a n d as e f f e c t i v e as t h r i c e d a i l y dosing. *Ulm University Hospital and Medical K o c h - S t r . 8 , D - 7 9 0 0 Ulm, FRG.

Center,

Robert-

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F R E Q U E N C Y OF B A C T E R I A L AND F U N G A L I N F E C T I O N S IN AC U T E L E U K E M I A (AL) A C C TO THE FAB CLASSFICATION. J.Nowicka

INTERVENTIONAL TREATMENT OF UNEXPLAINED FEVER (FUO) AND DOCUMENTED INFECTIONS IN NEUTROPENIC PATIENTS WITH HEMATOLOGIC MALIGNANCIES: FLUCONAZOLE V E R S U S A M P H O B / 5 - F C G . S i l l i n g - E n g e l h a r d t , W . F e g e l e r , N.Roos, C . S c h w a r z , M. Essink, W. H i d d e m a n n , T h . B ~ c h n e r

T h e i n c i d e n c e of i n f e c t i o u s e p i s o d e s h a s been studied in 140 p a t i e n t s [pts] wi!h AL ~MO~ i i . ! ~ - - 2 ~ ! . M2-25.MS-3.M4-22,M5-8,M6-6.LI-20,L2-22,L3-!) during the c o u r s e of the d i s e a s e on the b a s i s of clinical, laboratory, bacteriological and fungal findings. The seven studied groups (M0,MI,M2+M3,M4+M5.M6,LI, L2+L3) s h o w e d no d i f f e r e n c e s in the n u m b e r of infections w h e n a n a l y z e d w i t h the T - S t u d e n t and C h i 2 tests. Poisson's m o d e l w a s a p p l i e d to study the frequency of infections. This statistical method t a k e s into a c c o u n t not o n l y the i n c i d e n c e of infections but a l s o s u r v i v a l time. Six pts w i t h s u r v i val time longer than 60 m o n t h s w e r e e x c l u d e d from the study. Frequency coefficient L (infect i o n s / m o n t h ) w a s e s t i m a t e d i n d i v i d u a l l y for e a c h of the r e m a i n i n g 134 pts. F o r b a c t e r i a l and funga] infections this coefficient was 0.3815 whereas for bacterial i n f e c t i o n s L = 0 . 2 8 5 3 and for fungal L = 0,0962. In the g r o u p of A M L pts these c o e f f i c i e n t s w e r e equal to 0.5306, 0.3995 and 0.1311 r e s p e c t l v e ly, and f o r the g r o u p of L L A pts 6) 2106, 0.1542 and 0.0561 resp. The likelihood-ratio method revealed statistically significantly higher frequency of bacterial and fungdl i n f e c t i o n s in pts with AML c o m p a r e d to pts w i t h ALL. The highest frequency was f o u n d in M5 pts (L = 0 . 8 8 0 9 for b a c t e r i a ] and funga] infections, L = 0 . 5 4 7 6 for b a c t e r i a l infections and L = 0 . 3 3 3 3 for funga] infections) and in M4 pts (L = 0.6108, L = 0 . 4 7 3 0 and L = 0 1377 resp.) T h e p e r f o r m e d a n a l y s i s i n d i c a t e s that b a c t e rial and funsal i n f e c t i o n s i n f l u e n c e the survival t i m e in M5 and M 4 in a g r e a t e r d e g r e e than in o t h e r forms of acute l e u k e m i a s and thus m a y be an important p r o g n o s t i c factor. Dept

of H e m a t o l o g y , M e d i c a l

Academy,PL50367

Wroclaw.

In o r d e r to e v a l u a t e the therapeutic e f f e c t of fluconazole (F) in c o m p a r i s o n to A m p h o t e r i c i n B/ 5-Flucytosine (A) 69 patients at first fever received a combination of a n t i b i o t i c s i.v. and because of non-response after 4 days another combination of a n t i b i o t i c s plus randomly either fluconazole 5.7mg/kg/day i.v. or A m p h o B 0 . 7 5 m g / k g / d a y p l u s 5 - F C 3 7 . S m g / k g 4 t i m e s a day. In c a s e of n o n - r e s p o n s e F w a s r e p l a c e d a f t e r i w e e k b y A. U n d e r l y i n g d i s e a s e s w e r e AML, All, M D S or M P S a n d lymphomas. O n l y p t s w e r e i n c l u d e d w h e n t h e n u m b e r of n e u t r o p h i l s w a s l e s s t h a n 1000/cmm. U n t i l n o w 38 p t s (F) a n d 31 p t s (A) are e v a l u a b l e , 30(F) a n d 26(A) s u f f e r e d f r o m FUO, 8(F) a n d 4(A) f r o m d o c u m e n t e d i n f e c t i o n s . R e s p o n s e of f e v e r w a s achieved b y 16/30(F) and 20/26(A), respectively 4/8(F) and 3/5(A). This difference was not significant 5/6 Non-responder (F) responded when Ampho B/5-FC was given. Radiologic signs i m p r o v e d in 6/12(F) a n d 7/13(A). A s p e r g i l l o s i s w a s e v i d e n t in 5(F) a n d 4(A). In t h i s h i g h - r i s k g r o u p 16 pat. died, 7(F) a n d 9(A), 6 of e a c h g r o u p f r o m infections and 4(F) and 5(A) from mycosis, We conclude t h a t in o p p o r t u n i s t i c infections of n e u t r o p e n i c p t s a f t e r c h e m o t h e r a p y o~ h e m a t o l o g i c malignancies the well tolerable fluconazole should b e a d m i n i s t e r e d e a r l y a n d e m p i r i c a l l y a n d in c a s e of r e f r a c t o r i n e s s s h o u l d be r e p l a c e d a f t e r i w e e k b y A m p h o B/5-FC. Department University

of M e d i c i n e of M U n s t e r ,

(Hematology/Oncology) Germany

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INVASIVE ASPERGILLOSIS DURING CHEMOTHERAPY CHILDREN Th. B6melburg*, N. Roos, W. Fegeler, J. Ritter

IN

Invasive aspergillosis is known to be a serious infection in immunocompromised adults, often diagnosed postmortem, only. It has also become a serious complication during the treatment of oncological disease in childhood due to intensification of chemotherapy a n d longer periods of bone marrow depression. We present 9 children (5 boys, 4 girls, age 4-14 years) all with acute leukemia, who developed invasive aspergillosis within a 5-year-period (1986-1991). Diagnosis was proven by histological examination and/or cultures. All but 2 children were treated with systemic antifungal therapy, i.e. amphotericin B and fluorocytosine. In the 2 children without therapy, aspergillosis was diagnosed postmortem after a short fulminant course of fungal infection. The outcome was fatal in 6 patients and related to fungal infection in 5 of them. Two children with pseudomycetoma who had pulmonary lobe resection during chemotherapy are in continuous remission of their leukemia since 2 and 3 years. The third patient has a non-progressive aspergillosis with multiple pulmonary pseudomycetomas and is awaiting bone marrow transplantation. The relevance of clinical course as well as a variety of pertinent diagnostic tools is illustrated by the case histories presented here. Univ.-Kinderklinik, 4400 MUnster

Albert-Schweitzer-Str.

33,

EFFICACY OF LIPOSOMAL AMPHOTERICIN B (AMBISOME) IN NEUTROPENIC PATIENTS WITH FUNGAL INFECTIONS G.Silling-Engelhardt, M.Z~hlsdorf, E.Aguion-FreireInnig, F.Griesinger, T.B~chner, J.van de Loo Deep or disseminated fungal infections are major causes of morbidity and mortality in neutropenic pts. Amphotericin B (Amp B) is the drug of choice but administration is limited because of nephrotoxicity and side effects as fever, chills and hypotonia. In order to reduce toxicity and improve efficacy Amp B was incorporated into liposomes. We used the Amp B preparation Ambisome (Vestar Inc.) in i0 neutropenic pts with ii episodes. Neutrophils were less than 1000/cmm, 8 pts suffered from Aml, 2 from NHL. All pts were resistent to antibiotic therapy and pretreated with conventional Amp B and 5-FC. Change to Ambisome was due to toxicity in 6 and /or progression of pulmonary infiltrates in 5 and/or persistent fever in 4 pts. Ambisome was given in a dose of 3 mg/kg/day. The median duration was 27 days (4-40 days). 7/10 pts survived, response to Ambisome was achieved in 8/11 episodes, 7/9 with pulmonary infiltrates and 5/6 of those with radiological signs of pulmonary aspergillosis improved. Only one patient improved with recovery of neutrophils, all other pts were still neutropenic. 3 pts died: 1 pulmonary bleeding because of aspergilloma, 1 aspergilloma of pranasal sinus and pseudomonas infection, 1 fungal pneumonia. Although higher doses of Amp B were given it was well tolerated and nephrotoxicity was reduced. We conclude that in neutropenic pts Ambisome provides a well tolerated antifungal drug and suggests to be highly effective even in pulmonary aspergillosis. Department of Int. Medicine (Hematology/Oncology) University of MQnster, Germany

149

151

LIPOSOMAL AMPHOTERICIN B (AMBISOME R) IN PATIENTS WITH SEVERE NEUTROPENIA AND ANTIBIOTIC RESISTANT FEVER U. Hahn, A. Dcboben, G. Egerer, H. Goldsehmidt, R. Haas, W. Hunstein

Differential cell count and lymphocyte subsets of bronchoalveolar lavage in patients with severe granulocytopenia M. yon Eiff, O. Schlingheider, F. Schulze, N. Roos, J. van de Loo

Systemic mycoses are frequent complications during chemotherapy induced neutropenia. The administrationof Amphotericin B (AmphoB) is often limited by adverse reactions such as chills, fever and impairment ofrenai function. AmbisomeR is a new formulation of AmphoB, the substance being packed in so-called liposomes, i.e. bilayer lipid vesicles. We evaluated whether treatment with AmbisomeR offers adequateorlmproved effector at leastless sideeffects. Patienteharacteristies;wetreated 15 patients (pts), twelve underwent autologousbone marrow transplantation or blood stem cell transplantation (ABMT, ABSCT), whereas 3 pts. had intensive chemotherapy for hematologicalmalignancies. All pts were severely neutropenic (>0.5/nl) and had fever (>38,5oc) resistant to broad spectrum antibiotic therapy (median 6 days, range 3-10 days). Conventional AmpboB was given (0,75 mg/kg B W) for a median time of 6 days (range 4-18 days) before AmbisomeR was started. Allpts had either severe side effects or deteriorated clinically on conventional AmpboB. 9 pts had pulmonary infiltrates(4 proven pulmonary candidosis, 1 proven candida and Aspergilius pneumonia and 4 with only suspected pulmonary mycosis). 6 pts. had FUO or only suspected mycosis. Treatment: AmbisomeR was given in a dose of 3 mg/kg body weight. The median duration of treatment was 21 days (range 8-45 days).Daily therapy cost amounted to DM 1700,--/pt. Results: 12/15 pts survived. 8/15 pts and 7/9 of those with pulmonary infiltrates improved although still neutropenic. 3/15 pts died (1 candida pneumonia, 1 candida/aspergilluspneumonia, 1 pt with disseminatedencephalitis of unknown origin.) On conventional AmphoB 13 pts suffered from severe side effects. On AmbisomeR - despite the 4- fold higher dose - no patient had severe adverse reactions and only 3 pts had very mild reactions (shivering). In 4 pts pathological values of S-Creatinin and S-Potassium even norm aiized under treatment with AmbisomeR. Conclusion: AmbisomeR provides an effective therapy in systemic mycosis with very low toxicity. On the other hand the very high market prize prevents - at this time - its wide spread routine use. Department oflntemal Medicine, Universityof Heidelberg, Hospitalstr. 3, Germany

Because of early empirical antibiotic and/or antimycotic treatment bronchoalveolar lavage often can not clarify the etiology of pneumonia in patients with acute leukemia and severe granulocytopenia. Looking for characteristic BAL cell profile for infective pulmonary disease we determined BAL cell populations in 90 immunocompromisedpatients with fever and pulmonary infiltrates and 12 controls (c), respectively. In Patients with acute leukemia and severe peripheral granulocytopenia (A,n=32 had a lymphocytic alveolitis, whereas patients with other underlying diseases and normal peripheral cell count B,n=58 had increased p e r c e n t a g e s o f l ~ p h o c y t e s and polymorphic neutrophils in bronchoalveolar lavage: alveolar macophages A 68• t B 48• C 85• lymphocytes 25• 28• 9• polymorphonuclear cells 4+9; 20• 3 • 2. Patients with and without peripheral granulocytopeniahad similar findings in B A L 1 y m p h o cyte subsets, but CD 4- and CD 8-positive lymphocytes differed from controls: CD 4 34• 35• 45• CD 8 49• 49• 35• CD 4/ CD 8 0,8• 0,8• 1,3• CD 19 16• 14• 7• CD 57 7• 5• 3• A predominance of polymorphic neutrophils (17 • 22) was seen in specimen from patients with bacterial infections, whereas in specimen from patients with pneumocystis infections a predominance of lymphocytes (38• was seen. The lowest CD 4/ CD 8 ratio in BAL was measured in patients with pneumocystis pneumonia (CD 4/ CD 8 fungi 0,9• bacteria 0,7• pneumocystis 0,2• Pneumonias with m o r e t h a n one infectious agents showed a lower ratio than patiens with only one strain of bacteria (0,6• vs. 0,8•

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THERAPEUTIC USE OF G-CSF IN FEBRILE NEUTROPENIA IN ACUTE MYELOBLASTIC LEUKEMIA AND LYMPHOID NEOPLASIAS H. Link ~, A. Prahst t, G. HiJbner~, D. Richard2, A. Yver2, H.Wandt 3 Patients with chemotherapy-induced neutropenia < 1000/1~1have a high risk of febrile infections. If the neutrophil count is not rising at the onset of fever, despite adequate antibiotic therapy, the risk of death is 8% in fever of unknown origin, 20.4% in documented infections and 26.8 % in patients with pneumonia, as described by the Study of the Paul Ehrlich Geseilschaft (PEG) for Chemotherapy in Germany in 1573 neutropenic episodes. This risk further increases with the age of the patient. The aim of this phase I/II trial was to the determine the optimal dose of G-CSF (Chugai-Rh6ne-Poulenc) in order to maximally accelerate the regeneration of granulopoiesis, when used therapeutically in fever with neutrophils less than 500/Id. All patients were treated with standard antibiotic combination therapy. Six patients were treated and evaluable with G-CSF at each dosage with 2, 5 and 10 Ixg/kg and 7 patients with 20 I~g per kg. Glycosylated G-CSF was genetically engineered and derived from CHO-cells given as 30min i.v. infusion. Only patients without leukemic cells in the bone marrow smear were entered into the trial. Their average age was 49 years, 13 were male and, 12 female. 19 patients had acute myeloblastic leukemia, 3 acute lymphoblasic leukemia, 1 high grade lymphoma and 1 multiple myeloma. No side effects attributable to G-CSF were observed. There was no progression of leukemia during G-CSF therapy. The detailed results will be presented at the meeting.

EMEkGING BACTERIAL PATHOGENS IN PATIENTS WITH ACUTE LEUKEMIA: VIRIDANS ~ T R E P T O C O C C I AND N Q N - F E R M E N T A T I V E GRAM-NEGATIVE BACILLI. W. Kern*, E. Rozdzinski, T. Schmeiser, B. Hertenstein, R. Arnold, and E. Kurrle.

Dept. Hematology and Oncology, Hannover Medical School~, Narnberg3, Germany; Chugai-Rh6ne-Poulenc, Paris2, France.

Several recent reports indicate that the spectrum of bacterial pathogens causing major infections in patients with acute leukemia may have substantially changed over the past years. This report reviews the epidemiology and clinical significance of viridans group streptococci (VS) and n o n - f e r m e n t a t i v e gramnegative bacilli (NF, other than P.aeruginosa) causing bloodstream infections in acute leukemia patients at our center. Both infections showed a steady increase during the years 1983 to 1991 and are now (besides coagulase-negative staphylococci) the most frequent blood culture isolates in patients developing fever after chemotherapy at this center. Analysis of 55 cases of VS (single organism) bacteremia revealed frequent association w i t h the presence of oral mucositis, the development of lower respiratory tract symptoms, need for mechanical ventilation, and prolonged fever, but persistent bacteremia was never documented in such cases. The death rate was 16%. NF bacteremia, in contrast, frequently appeared associated with c a t h e t e r - r e l a t e d infection and short-duration fever. U n l i k e VS bacteremia, NF bacteremia was relatively common in periods unrelated to profound neutropenia, and had a low mortality (<5%). The most frequent organisms isolated from NF bacteremia included X.maltophilia, Acinetobacter spp., and v a r i o u s non-aeruginosa Pseudomonas spp. We conclude that the significance of ~gram-positive~ and ~gram-negative~ bacteremia may have profoundly changed with the emergence of these new bacterial pathogens in the neutropenic host. *Ulm University Hospital and Medical Center, RobertKoch-Str.8, D-7900 Ulm, FRG.

153 CYTOMEGALOVIRUS CMV INFECTION IN CHILDREN WITH BLOOD MALIGNANCIES AND APLASTIC ANEMIAS A.Skrobowska-Wo2niak.,R.Rokicka-Milewska., W.Paciorkiewicz., M.Jastrz~bska. CMV is the cause of the most freguent viral infecttion in patients with hematological malignao~ies and chronic blood diseases. The study included 68 children treated for acute leukemias and bone marrow aplasia. Cytyomegalovirus antigen was detected by immunofluorescence in urinary sediment cells and in cell cultures after their inoculation with CMV and the activity of antibodies to CMV was determined. Presence of one or more markers of CMV infection was demonstrated in 31 children, that is 45.5%. Active CMV infection on the basis of coexistence of clinical symptoms like fever,pneumonia,hepatitis, rash with markers of CMV was found in 11,7% of children. Active CMV infection caused deterioration of the general condition of the patients and was the cause of temporary discon• of intensive chemotherapy for from 2 weeks to 2 months. In cases of active CMV infection regression of clinical symptoms and elimination of virus antigen from urine was achieved by i.v. administration of Cytotect / Biotest/ in a dose of 2-4 ml/kg b.w. every day or every other day during 2-5 weeks. Cytotect was highly effective in the treatment of children with leukemias and aplastic anemias in whom active CMV infection was diagnosed. Address: Department of Pediatric Hematology/Oncology of Medical Academy, Marsza~kowska 24, 00-576 Warsaw, Poland.

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Main Sessions 155

157

COMPARATIVE EVALUATIONOF INTENSIVE POST-REMISSION THERAPY WITH DIFFERENT DOSE SCHEDULES OF ARA-C IN ADULTS WITH ACUTE MYELOID LEUKEMIA (AML): INITIAL RESULTS OF A CALGB PHASE III STUDY. Mayer RJ., Davis R.B., Schiffer C.A., Berg D.T., Powcll B.L, Schulman P., Frel HI E. for the Cancer and Leukemia Group B (CALGB). While complete remission (CR) may be achieved in 65 % of previously untreated adults having de novo AML, such CR's lack durability when convontional postremission is administered. Uncontrolled trials have suggested that intensive postremission therapy may prolong these CR's. To evaluate this concept, 1085 adults (median age: 52 years; range: 16-86 years) received induction therapy with ara-C and dannorubicin (DNR); 695 patients (pts) achieved CR. 59% of these patients were randomized to receive 4 courses of single agent ara-C in one of 3 dose schedules. 3 gm/m2 in 3 hr infusion q 12 hr x 2 qod x 3 (HiDAc) (187 pts.); 400 mg/m2/d x 5 days continuous infusion (CI) (206 pts.); or 100 mg/m2/d x 5 days CI (203 pts.). All pts then received 4 coarses of monthly maintenance treatment with ara-C (100 mg/n~ subcut q 12 hr x 10) and DNR (45 mg/m2 x 1). Hematologic toxicity was related to the ara-C dose during intensification with hospitalizations due to fever and ncutropenia required in 70 % of HiDAc courses, 59% of 400 mg/m2 courses, and 16 % of 100 mg/m2 courses. CNS toxicity occurred in 22/187 (12%) of the HiDAC cohort. Remission deaths occurred in 4.8 %, 6.3 %, and 1.0 % of the HiDAc, 400 mg/m2 cohorts respectively. The median follow-up time is now 33 months. Prognosis is statistically related to age and treatment schedule. Projected probabilities (PP) for continuous CR (CCR) after 3 years are; 38%, 36%, and 21% for pts. < age 40, age 40-60, and > age 60 respectively; and 42%, 35%, and 22% for pts. who received HiDAc, 400 mg/m2 and 100 mg/m2 respectively. In pts. < age 40, the HiDAc and 400 mg/m2 cohorts had equally superior outcoumes (PP for CCR after 3 years: 46%) compared to the 100 mg/m2 group (PP for CCR after 3 years: 24 %). In pts. ages 40-60, HiDAc treatment was more effective than 400 mg/m2 or 100 mg/m~ therapy (PP for CCR after 3 years: 48% vs. 33% vs. 25% respcctivcly). In pts. > age 60, prognosis was equally poor among the 3 groups (PP for CCR after 3 years: 18-25%). These data support the concept of a dose,response effect in AML pts. < age 60 and presently favor the HiDAc schedule. Further follow-up will be needed before final conclusions can be drawn.

MORPHOLOGICAL, CYTOCHEM!CAL AND CLINICAL ASPECTS OF ACUTE MYELOGENOUS LEUKEMIAS OF THE FAB-TYPES M3 AND M4-eo H. L6ffler*, W. Gassmann, Ch. JSrgensen, J. Noak, W.-D. Ludwig, M.R. Nowrousian, H. Eimermacher, H.J. Weh, D. Braumann, E. Lengfelder, G. Maschmeyer, B. Schlegelberger, R. Becher, C. Aul, D. Haase, Ch. Fonatsch, P. Koch, A. Heinecke, Th. BOchner .

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Until 30th of September 1991, marrow samples of 1167 patients with AML were prospectively examined by the central morphological, cytochemical and cytogenetical review institutions of the German AML study group. Distribution of th e FAB-subtypes was as follows: M1 21%, M2 31%, M3 3,3%, M3v 1,1%, M4 20%, M4eo 5,7%, MSa 8,1%, M5b 4,7%~ M6 3,8%, M7 0,7%. Morphological, cytochemical, cytogenetical and clinical data on the 39 cases of M3, 14 cases of M3v and 68 cases of M4eo will be presented at the meeting. Special attention will be paid to the prognostic significance of these three subtypes. In M3, a preliminary analysis revealed complete remission and remission duration rates of 64,1% and 61,8%/4 years, respectively, and 68,8% and 30,2%/6 years, respectively, in the whole patient population. For M4eo this analysis is ongoing and will be presented at the meeting. Address: Second Department of Medicine, Christian-AlbrechtsUniversit~t, Chemnitzstr. 33, D 2300 Kiel, Germany

156

158

LONGTERM RESULTS IN ADULT AML: CONVENTIONALINDUCTION WITH MAINTENANCE VERSUS DOUBLE INDUCTION WITH MAINTENANCE VERSUS ALLOGBNEIC OR AUTOLOGOUSBMT. DATA FROM AMLCG. Th. BQchner, W. Hiddemenn, B. W6rmann, H. Lbfl]er, G. Maschmeyer, W.-D. Ludwig, C. Aul, E. Lengfelder, U. W. Schaefer, N. Schmitz, A. Heine~ke

R A N D O M I Z E D S T U D Y OF I N D I V I D U A L I Z E D I N D U C T I O N T H E R A P Y WITH OR W I T H O U T VCR, A N D OF M A I N T E N A N C E T H E R A P Y TO 4 OR 12 C O U R S E S IN A D U L T AML : JALSG-AML87. R. Ohno and Japan Adult Leukemia Study Group

In order to further improve long-term results in AML we combined a prolonged postremission chemotherapywith a very early intensificationby double induction. The results of this strategy were compared with those of Conventionalinduction used in the previous study and prospectively compared with those of BMT on the basis of comparablepts. 60 pts with newly diagnosed AML entered the study. Median age was 49 yrs ranging from 16 to 80 yrs with 205 pts 60 yrs of age and over. Pts were treated for induction by TAD (J. Clin. Oncol. 3:1583, 1985). All pts under 60 yrs of age received a second induction course starting on day 21 even in aplasia with no b.m. blasts. 2nd induction was randomly either TAD or HAM (Blood 69:744, 1987). Pts of 60+ yrs received another TAD induction only in case of persistent b.m. blasts. In this age group pts were randomized to receive daonorubicin in TAD either at full (60 mg/mVd x 3) or half dose. TAD consolidation end monthly maintenance by 5 day an-C together with daunorobicin or thioguanine or cyclophosphamlderotatingly was given uniformly to all responders in both age groups. In the entire group of pts CR rate was 62% and 5 yrs CCR 34%. In the younger age group CR was 71% and CCR 37%. Randomization resulted in 69% CR and 35% CCR after TAD-TAD and 73% CR and 38% CCR after TAD-HAM (n.s,). In the older age group CR was 46% and CCR 26 % and acx~rding to randomization50 % CR and 31% CCR after full dose and 42% CR and 7.0% CCR after half dose dauno (n.s.). For comparison conventional induction in the previous study produced 59% CR (n.s.) and 25% CCR (n.s.) in the entire pts, in the younger pts 65% CR end 24% CCR (p =0.07) and in the older pts 41% CR and 31% CCR (n.s.). 38 pts received allogeneic and 20 pts autologous BMT in Ist CR. They were prospectively compared to chemotherapypts of same age and CR duration at time of BMT by nmtched pair analysis. Continuous relapse free survival after nllo BMT is 66 % after 3 1/2 yrs vs 46 % (n.s.) in the controls. Correspondingdata after auto BMT are 38% vs 33% (n.s.). We conclude that age adapted very early intensification seems to add to the cure rate produced by prolonged maintenance and improved longtenn CCR to as much as 37 % in younger pts and 34 % in pts of all ages. Allo BMT shows a trend to superior iongterm-rosultswhile the cure rate after autoBMT resembles that of best chemotherapywhen comparing comparable pts. *Present address: Department of Medicine/Hematology, University of Mfinster, Gern~my

We asked 2 questions in this study. First is the additional effect of VCR in induction therapy, and the second is the duration of maintenance therapy. Adult AML was treated by a r e s p o n s e - o r i e n t e d individualized therapy with behenoyl Ara-C 200 mg/sq.m, daily + 6MP 70 mg/sq.m, daily with 300 mg allopurinol + prednisolone 40 mg/sq.m on day 1-4 + DNA 40 mg/sq.m, on day 1-3 and optionally on day 7-8 and day 11-12 (for M3 DNR 50 mg/sq.m.+BHAC+PSL daily) (BHAC-DMP) until bone marrow became severely hypoplastic with < 5% of blasts. Patients were randomized to BHAC-DMP or BHACDMP + VCR 0.35 mg/sq.m, on day 1-4. After obtaining CR, 3 courses of intensive consolidation therapy were given with I.T. MTX+ Ara-C. Maintenance therapy was randomized to 4 or 12 courses given every 6 weeks. Pts. of age >_60 received about 2/3 reduced doses. From July 1987 to Aug. 1989, 265 consecutive adult AML were registered and 258 were evaluable. Age ranged from 15 to 79 (reed., 48). Out of 258, 78% achieved CR (80% in 209 pts. of age < 60 and 65% in 49 of age _> 60). Addition of VCR reduced the high CR rate of BHACDMP significantly (84% to 70%, p=0.007). At the median follow-up of 37 mo., overall survival is 38%, and event-free survival 20%. In CR cases, continuing CR rate is 37%, and disease-free survival (DFS) 35%. Patients received 12 courses of maintenance/intensification showed higher DFS (48% vs 33%, P=0.0555). Multivariate analyses revealed that age (<60), P.S. (0-2) and VCR (-) are significant factors for the achievement of CR, and no. of course of induction to reach CR (one), FAB (M3 or M5) and age (<50) were for the longer DFS. This study confirmed the high CR rates of the individualized response-oriented therapy, and showed a beneficial effect of fairly intensive maintenance/intensification therapy in adult AMU Presentaddress:Departmentof Medicine,Nagoya UniversitySchool of Medicine The Branch Hospital, 1-1-20Daiko-Minami,Higashiku,Nagoya461,Japan

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161

THEHAPY OF ACUTEITELOID LEUKEMIAIN ELDERLYPATIENTS. PRELIIdlNARYRESULTS OF A 61MEIIASTUDY GROUP TRIAL P. ]landellifor The ItalianCooperativeOroup 61](EllA

ADULT ACU-IE MYELOGENOUS LEUKEMIA: RECENT STUDIES FROM THE EASTERN COOPERATIVEONCOLOGY GROUP (ECOG). Rowe JM, Andersen JW, Cassileth PA, Oken MM, Wiernik PH for the Eastern Cooperative Oncology Group. Hematology Unit, University of Rochester Medical Center, Rochester, NY USA.

In december 1980the OIIIEMAgroup started a randomizedclinical trial for the treatmentof AIILpts aged ~60 years designed to: i) evaluate the efficacy and toxicity of a regimencombiningintermediate-doseAra-C (IDAC) and Uitoxantrone(MTX) for remission induction;2l determinenhether a consolidation phasewith a drug regimen other than that used for inductionwould improvedisease free survival; 3) assess the efficacyof recombinantalfa-2 b IFN (raIFN) in maintainingthe remission status. Complete remission(CR) was induced with a singlecourse of IDAC (Igls2 continuousinfusionover 6 hrs daily for 6 days) and MXT (Hmg/n2daily for 6 days), Responderswere randomizedto receivea consolidation courseeither with the same regimen (art A) or nith a combinationof ONE 130mglm2i.v. bolus days I-2l, Ara-C (lOOmglm2continuousinfusiondays I-5) and VP 16 (60mglm2 i.v. days I-5) (Arm B). Patients still in CH after consolidation were randomized either to receive maintenancetreatmentwith ralFN s.c. 2 millionsU/n2 far 5 days every week for 0 months (Arm l} or to stop therapy (Arm 2). 204 patients,median age 64,8 years (range 61-77,9), are evaluablefor remissioninduction.1051204151.5~)achievedCR (median time to CR was 29 days), 371204 (18.I~,)patientswere resistantand 62 (30.4~.) died during induction.83/105responders here randomized:43 to Arm A and 40 to Arm H; subsequently30 patientshere randomizedto receive INF maintenancetherapyand 34 to stop therapy.Overall49 patientsrelapsed:10 after consolidationand 39 after the second randomization.The preliminarydata suggest that the combinationof IDAC+I(X'f is an effectiveregimenfor inducingCR in elderlypts with AIL and that the whale treatmentprogram is yell tolerated. Ematology-DepartmentHuman Riopathology

University"La Sapienza" Via Beneventon.6 - 00101Rosa-Italy

160

The majority of adults with de nova AML can now be expected to enter complete remission. Major emphasis at present is directed at improving post-remission therapy. Recent ECOG studies have shown that escalating the intensity of therapy after complete remission prolongs the disease-free survival (DFS). Preliminary analysis from the most recently completed study, EST 3483, reveals a trend towards improved DFS when a single course of high dose consolidation therapy is compared with prolonged maintenance therapy and similarly when allogeneic bone marrow transplantation was compared with intensive consolidation therapy. Similarly, preliminary analysis from a recently completed ECOG pilot study employing autologous bone marrow transplantation for post-remission therapy looks very promising with a DFS of 50% at 3 years. A major prospective randomized intergroup. study, EST 3489, is now underway comparing both autologous and allogeneic bone marrow transplantation with consolidation therapy for patients <55 years of age. Another major study is underway assessing prospectively, in a randomized placebo controlled fashion' the role of GM-CSF in reducing the period of neutropenia in elderly patients (55-70) with de nova AML. Studies in relapsed and refractory patients have evaluated the combinations of cytosine arabinoside with amsacrine as well as mitoxantrone with etoposide. A' post-remission randomization,following high dose cytosine arabinoside and amsacrine induction, compared low dose cytosine arabinoside, 10 mg sc bid for 21 days every 8 weeks, with no additional treatment. It appears, for the first time in a large prospective randomized study, that there may indeed be a clear benefit for maintenance therapy in prolonging the CR duration for relapsed and refractory patients when compared with no further therapy (8 months vs. 3 months).

162

RECRUITMENT OF LEUKEMIA TO CYTOTOXICITY WITH BZOMODULATION COMBINED WITH TIMED SEQUENTIAL THERAPY (TST). P.J.Burke, K.A.Wendel, P.D.Nicholls, K.Cowan, L.Morrall, E.M.Bonnem, and K.L.HalI.

Abstract not submitted Pharmacologic amounts of granulocytic-macrophage colony stimulating factor(hGM-CSF) given throughout therapy were added to a sequence of drugs timed to coincide with tumor growth recruited by host stimulatory factor (HSA).To define an effect i v e d o s e o f G M - C S F , 20 p a t i e n t s with relapsed AML received 1 course of induction therapy and 5 received 1 c o u r s e in r e m i s s i o ~ with GM-CSF at either i(4), 5(15) or i0(i) ug/kgm/d given by continuous infusion beginning 3 d prior to TST with AC-D-VPI6 and continuing through dl3. Measurements of changes in bone marrow morphology, growth by flow cytometry, and serum activities by DNA synthesis assays made during the initial period defined a doubling of noLmal and leukemic cells, serum stimulation and leukemic c e l l "S" p h a s e . P r i o r t o V P I 6 o n d l l , the serum stimulating activitiy doubled, reflecting the additive effect of GM-CSF and HSA, as predicted in in-vitro pretreatment assays. Similar results were obtained with assays using remission bone marrow. Toxicities included pericarditis (l,dl3) and DIC (l,d3). There was no evidence of prolongation of bone marrow aplasia or uncontrolled recruitment of leukemia. The dose of 5 ug/mg/d f o r 13 d a y s s e e m s r e a s o n a b l e and effective. The Johns Hopkins Oncology Center, Wolfe Street, Baltimore, MD 21205

600 North

163

Abstract not submitted

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166

Autologous Bone Marrow Transplantation in Acute Leukemia with Edelfosine Purged Marrow. W. R. Vogler and W. E. Berdel. Emory University, Atlanta, GA and University of Berlin, Berlin, Germany. Edelfosine is one of several ether ph0spholipid analogs of lysophosphatidylcholine which is directly cytocidal to neoplastic cells. Although the mechanism of action is not clearly delineated, it does have a major effect on cell membranes. The unique feature is the selective cytotoxicity to leukemic cells and the relative sparing of normal marrow progenitor cells. Thus, edelfosine would appear to be an ideal candidate for purging remission marrows in acute leukemia prior to autologous bone marrow transplantation (ABMT). We have conducted a phase I trial to determine the optimal purging dose of edelfosine. Twenty-five patients had marrows purged at a dose of 50/zg/ml for 4 hours and 10 were transplanted. Forty patients had marrow purged at 75/zg/ml and 18 were transplanted. One patient undergoing a second transplant had marrow purged at 100/~g/ml, but failed to recover blood counts after ABMT. Edelfosine had little effect on progenitor cell assays. There were 10 ALL (15 in second and third CR) and 18 AML (4 in 1st CR, i1 in 2nd CR and 3 in early relapse) patients transplanted. The ablative program consisted of high dose cytarabine and fractionated total body irradiation (TBI) (15 AML and 3 ALL), TB! and etoposide (8 ALL) cyclophosphamide and TBI (1 AML and 1 ALL). At the 50 /zg/ml edelfosine dose, the mean time to granulocyte recovery to 500 #1 was 29.6 + 2.8 days and to platelet recovery to 25,000//~1 was 49.6 + 8 days. At the 75/~g/ml dose, the time to granulocyte recovery to 500//zl was 35.8 _ 4.39 days and the time to platelet recovery was 38.4 __+4.8 days. Currently, 6 of 10 patients with ALL are alive and 3 are in remission (121, 715 and 942 days). The median survival of the ALL patients is 248 days. In AML, 10 patients are alive and 7 are in remission from 55 to 1385 days (median 301 days). The median survival of all AML patients is 153 days. These results in this high risk population indicate that 75 #g/ml is a safe dose to test in a phase II trial.

CURRENT RESULTS OF ST. JUDE STUDIES FOR CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) C.-H. Pui*, G.K. Rivera, M.L. Hancock, W.E. Evans, J.T. Sandlund, F.G. Behm, S.C. Raimondi, R.C. Ribeiro, W.L. Furman, H. Mahmoud, M.V. Relling, and W.M. Crist St. Jude Study XI (1984-1988) featured seven-drug intensive induction and consolidation therapy. Patients achieving complete remission were randomized on the basis of risk group assignment to conventional continuation treatment or to four pairs of drugs rotated weekly or every 6 weeks. The overall 5-year event-free survival (EFS) rate (+SE) for the 358 patients enrolled is 72% + 4%; all 262 patients remaining in remission are off therapy for 9 months or more. While the outcome within risk groups is not significantly affected by the treatment schedule, the 84 higher-risk patients who received epipodophyUotoxins in weekly doses have a significantly higher 6-year cumulative risk of secondary acute myeloid leukemia than the other 148 higher-risk patients who received the agents every 2 weeks (12.3%; 95% CI = 5.7% to 25.4% vs. 1.6%; 0.4% to 6.1%; p<0.01). The 5-year EFS of various high-risk subgroups have apparently improved: 66% + 8% in adolescents >10 years old, 80% __+21% in the t(1;19) ALL, 67% + 16% in blacks, 51% + 11% in T-cell ALL, and 36% + 21% in Ph § ALL. Cases with myeloid-associated antigen expression fare as well as those without expression of the antigens (p=0.58). Independent adverse prognostic features were abnormal karyotype and lack of CD10 expression among T-cell ALL, and high leukocyte counts and infant age in B-precursor-cell ALL. Based on the excellent results of Study XI and low-risk of myeloid leukemia in one of the treatment arms, we have initiated a new study which will be described. *Present Address: St. Jude Children's Research Hospital 332 N. Lauderdale, P.O. Box 318, Memphis, TN 38101-0318

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167

PROSPECTIVE STUDY ON THE CLINICAL RELEVANCE OF RESIDUAL DISEASE IN AML PATIENTS IN COMPLETE REMISSION. B. W~rmann, M. Safford, S. KGnemann, K. Zurlutter, K, Piechotka, K. Schreiber, M. R. Lokan,Th. BOchner,W. Hiddemann and L.

CNS PROPHYLAXIS WITH HIGH DOSE METHOTREXATE (HDMTX). A PHARMACOKINETIC AND CLINICAL STUDY. R E S U L T S O F P R O T O C O L F R A L L E 89.

W. M. M. Terstappan 80 % of patients with newly diagnosed acute myeloid leukemia (AML} reach a complete remission (CR) through intensive chemotherapy, but the majority of them will relapse within 2 years. Recently developed cell biological techniques can detect leukemic cells in CR with higher sensitivity than light microscopy. The critical level of residual leukemic cells and the clinical impact of these results have not been determined. A prospective study was performed in 45 patients with newly diagnosed AML in CR. Treatment was standardized and consisted of induction, consolidation and maintanance chemotherapy. The leukemic cells had been identified at diagnosis based on the aberrant expression of cell surface antigens using multiparameter flow cytometry. Bone marrow aspirates were analyzed at achievement of hematological complete remission and 4 - 6 months in continuous CR (CCR). Residual cells with the leukemic phenotype were detected in 30 / 45 patients (67 %) at achievement of CR with a median percentage of leukemic cells of 3 % (range 0.3 - 55 %). The projected rate for continuous CR (CCR) was 63 % for patients without detectable leukemic cells compared to 17 % for patients with residual leukemic cells (p = 0.0036). 25 patients were reanalyzed 4 - 6 months in CCR, and residual cells with the leukemic phenotype were detected in 13 patients. All patients have relapsed within 22 months compared to 3 of 12 patients without detectable disease (p = 0.0004). We conclude, that two third of AML patients had evidence of residual leukemic cells at achievement of hematologic remission. The frequency of cells with the leukemic immunophenotype was relatively high. Patients with evidence of residual disease in QR had a high dsk of relapse. Background.

60 -

Department 0t Internal Medicine A, University of M0nster, Albert Schweitzer - Str. 33, 4400 MOnster, Germany.

G. Schaison*, M.F. Auclerc, I. Madeleine, A. Baruchel, T. Leblanc, Y. Perel, G. Couillault, G. Comu, C. Sehmitt and SHIP. Late sequelae of CNS irradiation included growth and endocrine disorders, neuropsychological and intellectual impairment. Since 10 y it has been shown that HDMTX can replace irradiation for CNS prophylaxis with far less side effects. In protocol Fralle 89 all patients (pts) (except those with initial CNS involvement) were treated with HDM-IX. HDMTX was given as a 24 h continuous infusion with 1 / 3 injecte~ in 1 h. Folinic acid rescue was started at H. 28 with 15 mg/m~every 6 h x 12. Since Feb. 1989, 509 pts were included and 499 (98 %) achieved CR after a five drug induction. Mean follow up is 14 m (3-27). In standard risk (SR) pts were randomized in two arms. Arm A (125 pts) pts received 4 courses MTX 3 g and 10 single drug intrathecal (IT) chemotherapy with MTX : in arm B (124 pts) MTX 8 g and 5 ITMTX. Mean plasma level at H 24 was 26.5/~M in arm A and 101 in arm B whereas CSF drug concentration was respectively 1.09 p,M (0.10-7.60) and &.35 (0.32-37,50). Drug CNS concentration greater or equal to !0" M was achieved in 37 % of pts in arm A and 93 % in arm B. There were 1 CNS relapse in arm A (0.8 %) and 12 in arm B (10 %) (p = 0.007). In high risk (HR) group after the same induction pts received 4 courses MTX 8 g and 9 triple drug IT followed by a consolidation with Ara-C and VP16. Among 129 pts, 6 developed CNS relapses (4.6 %). In the mediastin group (MM) 80 pts received MTX 8 g and 12 triple IT and a meningeal relapse occured in 9 (11.3 %). In very HR group most of 41 pts were transplanted and CNS relapses were rare. We conclude : .) high drug CSF concentration after MTX 8 g and a reduced number of ITMTX are inadequate for CNS prophylaxis (SR) 2) increasing the level of systemic MTX don't allow to reduce the number of IT 3) MTX 8 g with numerous triple IT chemoprophylaxis combined with aggressive systemic therapy can be substituted for cranial irradiation without loss of efficacy (HR) 4) the same regimen is clearly inadequate in patients with MM and it appears that skull irradiation is still necessary for these pts. * Saint-Louis Hospital, 75010 Paris, France. 168

Abstract not submitted

169 Abstract not submitted

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CLINICAL RELEVANCE OF CHROMOSOMAL ABERRATIONS IN CHILDHOOD ALL CYTOGENETIC DATA OF THE GERMAN THERAPY STUDIES *) J.Harl0ott 1), J.Ritterbach1), W.-D.Ludwig2), C.R.Bartram3), A.Borkhardt 1), A.Reiter4) G.Janka-Schaubs),and F.Lamperti) 0Children's Univ. Hospital, Feulgenstrasse12, 6300 Giessen, FRG; ~)Dept. of Hematology/Ontology Klinikum Steglitz, 1000 Berlin, FRG; 3)Dept. of Moleculargenetics, Univ. Children's Hospital, 7900 UIm, FRG; 4)Dept. of Pediatrics, Hannover Medical School, 3000 Hannover, FRG; S)Dept. HematoIogy/Oncology,Children'sUniv. Hospital, 2000Hamburg,FRG From January 1984 to December 1991 1906 bone marrow or blood samples of children with ALL were sent to our laboratory by mail from more than 70 hospitals. 1089 of these samples (57%) could be analysed successfully, All patients were treated uniformly in the West-German ALL-BFM or CoALL trials. An abnormal leukemia karyotype was found in 695 (64%) patients and in nearly three quarters non-random chromosome aberrations appeared (n=493). Most frequently only one typical abnormality was found in the immunophenotypic subgroUps, e.g. t(8;14) in BALL, t(4;11) in pre-pre-B-ALL, or 14q11-aberrations in T-ALL. In children with common ALL, however, three of the non-random aberrations were detected: 250 children had a hyperdiploid karyotype with more than 50 chromosomes with a modal number of 51-63. In pseudodiploid metaphases the t(9;22) (Philadelphiachromosome) was very frequent (n=34), and also the t(1;19) appeared very often (n=16). The latter aberration, which is described to be typical for pre-B-ALL, was found in 8 children with this immnunophenotype. Wether the non-random or random chromosomal aberrations found in ALL represent an independent factor for a higher or lower risk of relapse within the therapy trials remains to be seen.

CRANIAL IRR~IATION IS ESSENTIAL IN THE TREATMENT OF CHILDHOOD AML: RESULTS OF STUDY AML-BFM-87.* Ursula Creutzig, J. Ritter, and G. Schellong for the AML-BFMStudy Group** Study AML-BFM-87 attempted to determine whether cranial irradiation could be replaced by t w o blocks of intensification with HD-ARA-C]VP-16 after consolidation and whether prognosis could be improved by this intensification.H Since December 1986 210 children with AML have been enrolled in study BFM-87. 163 patients (78%) achieved CR. Results after a median follow up of 2.3 years for the total group are: eventfree survival (EFS) 45%, SD 4%, event-free interval (EFI) 58%, SD 5%. In the first 84 CR patients cranial irradiation with 18 Gy was randomized in 31 pts., selected or not in 24 pts., and determined in 29 children because of initial C N S involvement and/or leukocyte counts >70.000//tl. Since 1989 cranial irradiation has been abandoned except in children with initial CNS involvement because interim analysis gave no evidence of a positive outcome of randomization. However, latest results are inferior in non-irradiated patients, relapse-free interval 47%, SD 8% vs. 72%, SD 6%, p<.04, and are not due to a higher incidence of isolated or combined CNS relapses but to bone marrow relapses. The increase in relapse rate is seen especially in nonirradiated patients of a low risk group as defined according to study AML-BFM-83 (BLOOD 75, 1932, 1990). The high risk group, however, shows slightly better results in children having received irradiation: 64%, SD 8%, compared to 44%, SD 6% (n.s.) in the previous study. We conclude, cranial irradiation is essential in childhood AML and cannot be replaced by intensification with HD-ARA-C, and fm-thermore, patients of the high risk group may profit from intensification. *Supported by the Deutsche Krebshilfe **Present address: Universit~ts-Kinderklinik, Str. 33, D-4400 Mfinster F R G

.................................................. * ) Supported by the "Kind-Philipp-S~ng" and the "Parents' Initiative GieBen"

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Albert-Schweitzer-

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MULTIDRUG RESISTANCE (MDR) REVERSAL UTILIZING MITOXANTRONE, ETOPOSIDE AND CONTINUOUS INFUSION CYCLOSPORINE (MEC) IN PEDIATRIC ANLL. Dahl G., Grier H., Sikic B., Weinstein H, Arceei R. Stanford University, Pale Alto, CA and Dana-Farber Cancer Institute, Boston, MA, USA. A majority of ANLL cells are known to express high levels of mdrl gene encoding the P-glycoprotein (P-gp). This may explain, in part, the frequently seen clinical resistance to certain drugs, e.g. mitoxantrone, etoposide. Cyclosporine (C) is a potent MDR reversal agent and in vitro reverses MDR at serum level >2 micromolar. Administration of C during chemotherapy with agents of MDR phenotype may reverse this resistance by inhibiting the P-gp efflux pump. In a pilot study, six children with highly refractory ANLL were treated with a 3-day IV MEC course consisting of: Mitoxantrone (M) 100 mg/qm/day x 3 + etoposide (E) 150 mg/qm/day x 3 + cyclosporine (C) 6mg/kg over 2 hours, then 18 mg/kg/day continuously for 60 hours (total 62 hours). C dose was adjusted at hours 14, 26 and 38 to maintain a 2 to 4 micromolar (2400-4800 ng/ml) serum level. All patients required inmreases in C dose to achieve the desired serum C levels. All patients developed elevated bilirubin levels which returned to normal after C was stopped. Marked mucositis and myelotoxicity was noted in all patients treated. One patient developed flushing and rash with C infusion but treatment with benadryl allowed for therapy to continue. This pilot study shows that C levels >2 micromolar can be maintained in children without undue toxicity. Profound oncolytic responses were seen in all patients and 2 achieved complete remission. As toxicities were tolerable in these heavily pretreated patients, a group-wide phase II study is planned.

Prevention of b a c t e r i a l infections in profound granulocytopenia: experience J u l e s Bordet

patients of the

with Institut

P. Van der Auwera, Institut Jules Bordet, Brussels, Belgium. The c h o i c e o f a n t i b i o t i c r e g i m e n u s e d f o r d i g e s t i v e t r a c t decontamination has been a matter of controversy. Cotrimoxazole was found succesfull in many centers in double-blind placebo controlled study but not in our center due to high resistance in entcrobaeteriaeeac. Fluoroquinolones have been tested although no double-blind placebo controlled study has been done yet. Wc have run t h r e e open trials (cnoxacin, ciprofloxacin, pefloxacin) s u g g e s t i n g that infection with G r a m negative bacilli have been almost e r a d i c a t e d , a l t h o u g h replaced by G r a m - p o s i t i v e c o c c i (Coagulasc negative staphylococci, CNS; & streptococci). All patients having received oral quinolones are colonized in the feces by quinolonc-resistant CNS. The number of patients requiring empiric antibiotics for fever has not declined with prophylaxis nOr the delay to the first fever. Quinolonc resistance did not emerge in G r a m negative bacilli. A collaborative study with oral penicillin (with pefloxacin, against a placebo) suggested that less strop. bacteremia occurred, probably those originated in the oral cavity. A small open study with a macrolidc (spiramycin) failed to show any convincing efficacy. Resistance to penicillin and macrolidcs in oral strep, is increasing and might impair this approach. To control the gut reservoir of CNS we performed two pilot studies in healthy volunteers with oral vancomycin and teicoplanin. In both studies , highly glycopeptide-resistant strains of Ent. faceium emerged in the feces ( V a n A phenotype). These strains were still h i g h l y susceptible to ramoplanin , a new lipopeptide developed for topical treatment. Although few of these strains have been reported to cause infection, extreme caution should be taken. Some reports have suggested that bactercmia due to oral streptococci might be prevented by the use of acyclovir. This was probably due to the role of herpes simplex reactivation in the p a t h o g e n e s i s of severe mueositis and streptococcal bacteremia. Future directions are : ramoplanin, new quinoloncs active against resistant staphylococci, new macrolidcs active against resistant streptococci, & ramoplanin.

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PREVENTION AND TREATMENT OF INFECTIONS: NEW DRUGS FOR THE PREVENTION AND TREATMENT OF Pneumoeystis carinii PNEUMONIA W.T. Hughes

INTERVENTIONAL ANTIMICROBIAL STRATEGY FOR THE MANAGEMENT OF INFECTIONS IN NEUTROPENIC PATIENTS WITH HEMATOLOGIC MALIGNANCIES-RESULTS OF A MULTICENTER COMPARATIVE ANALYSIS. Hiddemann, W., Link H., Maschmeyer G., Meyer P., Helmerking M, Adam D. for the Paul Ehrlich Society for Chemotherapy Study Group, Dept. Int. Medicine, University of Miiuster, MOnster, FRG. The present study investigated a three step interventional antimicrobiai strategy in patients of fever of unknown origin (FUO) and clinically and/ur microbiologieaUy documented infectious. During phase 1 patients with FUO were randomly assigned to penicillin plus aminogiycoside (PEN, AG), penicillin plus ccphalosporin (PEN, CEPH) or AG plus CEPH. Non-responders were randomized in phase II between PEN/CEPH/AG versus PEN/CEPH/vancomycin. Failures on phase II or recurrent. fever after initial response to phase I entered a randomized comparison of rifampicin, amphotericin B and 5-fluorocytosine supplemented with either PEN/CEPH or imipenem. In patients with documented infections empiric therapy was adapted for microbiologic findings and results of in-vitro sensitivity testing as well as for infection sites. 1770 patients at ages 15-84 (median 48) entered the trial and 1573 cases are currently evaluable. 800 patients (50.9%) had FUO while 773 cases (49.1%) had documented infections. In FUO no differences emerged from the randomized comparison at the different intervention steps with complete responses in 68.4% of cases during phase I, 50.3% during phase II, and 72.7% at phase III. The cumulative response rate was 91.3%. Among the clinically documented infectious lung infections comprised 24.4% of cases at initial presentation with a significant increase to 53.3% after the first week of treatment. Response to therapy was significantly inferior as compared to other clinically documented infections (61.3% versus 82.1% - p < 0.001). In patients with mierobiologicany verified infectious gram-positive bacteria were the predominant microorganisms at first presentation with 51.5% as compared to 39.7% gram-negative organisms and 8.2% fungal infectious. A significant increase of fungal infections was observed after the first week of treatment comprising 51.9% of cases. These data indicate a high cumulative response rate of 91.3% to the applied interventional antimicrobial strategy in FUO and 75.4% in documented infections. After the first week of therapy fungai infections are encountered in more than half of cases thus necessitating the early incorporation of systemic antifungal treatment. FUrther improvements are also needed in the therapy of lung infections.

Pneumocvstis carinii pneumonitis (PCP) is a life-threatening infection in immunosuppressed patients with leukemia. The incidence of PCP is related to the type and intensity of chemotherapy. In high risk groups the pneumonitis can be prevented with the administration of trimethoprim-sulfamethoxazole (TMP-SMZ). Two drugs, TMP-SMZ and pentamidine isethionate, are effective in the treatment of 75-80% of cases of PCP. However, each of these drugs has unacceptable adverse effects. In experimental animal models and in clinical trials we have discovered other drugs with potent anti-P, earinii activity. These include diaminodiphenylsulfone (dapsone) alone and in combination with TMP; erythromycin-sulfisoxazole, clarithromycin-sulfamethoxazole, azithromycin-sulfamethoxazole and a new hydroxynaphthoquinone designated 566C80. The most promising of these compounds is 566C80. 566C80 has no discernible effect on hematopoiesis, can be taken orally and phase I studies in infants, children and adults fail to identify any significant toxic effects. Preliminary studies show therapeutic efficacy in PCP in adults. It also has activity against Toxoplasma gondii and Plasmodium sp. Further studies are warranted to evaluate 566C80 as a prophylactic agent for high risk cancer and bone marrow transplant patients. Present address: Department of Infectious Diseases, St. Jude Children's Research Hospital, 332 North Lauderdale, Mempis, TN 38105 USA.

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TREATMENTOF FUNGALINFECTIONS.BodeyG.P. Dept. of Medical Specialties, Section of Infectious Diseases, The University of Texas M.D. Anderson Cancer Center, 1515HolcombeBlvd., Houston,TX 77030, USA. Fungal infectionshave emerged as a commoncause of fatal infectionin leukemic patients (pts.) Especially disturbing is the fact that many of these infections are occurring during initial remissioninduction chemotherapy.The major predisposing factors are prolonged neutropenia, disruption of the mucocutaneousbarrier, and administration of corticosteroids and broad-spectrum antibiotics. Whereas Candida albicans had been the predominantpathogen, Candida trouicalis has become a more frequent cause of infection at some institutions.At others, aspergillosisis more of a problem than candidiasis. Other pathogenshave emerged, including Triehosooron spp. and Furasium spp. The diagnosis of ftmgal infections is often difficult because the only signs may be persistent fever and progressivedebilitation in apt. receiving antibacterial agents. Culturesof blood and sputumare often of little value and tissue biopsy is often contraindicated. Serological tests appear to be promisingbut still unproven as diagnostic tools and are not routinely available. Therapy of fungal infections has been unsatisfactory,since the most critical factor in response is recovery of the patient's neutrophil count. In the past, amphoteriein B has been the mainstayof therapy, but a few organismshave becomeresistant and its toxicities are troublesome. Lipid preparationsof amphotericinB havebeen developed that greatly improve its therapeutic indexand enhance its efficacy.Flucytosine and ketoconazole play a limited role in leukemicpts. Fluconazoleand itraconazole are new agentswith potentially important applications in these pts. Growth factors may serve as useful adjuvants to antifungal therapy, but experience in this setting is limited.

INHALATION OF A M P H O T E R I C I N B AS P R O P H Y L A C T I C ANTIFUNGAL TREATMENT DUPING INTENSIVE LEUKEMIA THERAPY V. H e i n e m a n n , P. S c h o l z , and U. J e h n Pulmonary infections by Aspergillus and Candida species have remained a major cause of mortality in leukemia patients. Studies performed in a n i m a l s indicate a pulmonary accumulation of inhaled amphotericin B (Antimicrob Agents Chemother 3A: 29-32,1990). Following this rationale we p e r f o r m e d a prospective study to evaluate prophylactic inhalation of a m p h o t e r i c i n B d u r i n g 4 4 c o u r s e s of intensive antileukemic chemotherapy. Median duration of f e v e r > 38 ~ was 5.5 days. Systemic antibiotic treatment was performed in 3 5 p t s f o r a median o f 18 d a y s ; additional systemic antifungal treatment with amphutericin B i.v. w a s a p p l i e d in 6 treatment courses for a median of 13.5 days. Amphotericin B w a s i n h a l e d as a e r o s o l at a d o s e o f 3 x I0 m g / d a y for a m e d i a n o f 2 7 d a y s . Inhalation was performed from start of chemotherapy to recovery of g r a n u l o c y t e s in t h e p e r i p h e r a l blood. Inhalation of a m p h o t e r i c i n B was well t o l e r a t e d , and significant toxic side effects were not observed. HPLC analysis performed during I0 treatment c o u r s e s did not reveal amphotericin B in pt-serum (limit of detection 0.I ug/ml) indicating that systemic exposure to a m p h o t e r c i n B did not occur. Weekly analysis of microbial colonization of nose, throught, and sputum resulted in t h e detection o f C a n d i d a s p e c i e s at a rate of O, 16, and 2 7 % r e s p e c t i v e l y . Aspergillus speEies were not observed. N o n e of the e v a l u a t e d patients suffered from invasive fungal infection of the lungs, and overall mortality from fungal infections during granulozytopenia w a s zero. Klinikum Grosshadern, University of Medizinische Klinik, Munich, Germany

182 CALGB STUDIF~ WITH HEMATOPOIETIC GROWTH FACTORS IN PATIENTS WITH AML C.A. Schiffer*, R.M. Stone, B.A. Peterson, J. Moore, for the CALGB .......................................................................................... In an attempt to shorten the duration of neutropenia and attenuate infectious complications in elderly pts with AML, the Cancer and Leukemia Group B (CALGB) is conducting a study in which pts z 60 years are randomized to receive either placebo or 5 #g/kg GM-CSF IV over 6 hours beginning the day after completion of standard induction therapy with daunorubicin and ARA-C. GM-CSF is continued until the granulocyte count is > 1000//zl, or the development of refractory leukemia or unacceptable toxicity attributed to the infusion. 73/124 (59%) pts (median age 68 yrs) evaluable to date have achieved CR (68/73 with 1 course of induction therapy) on this ongoing, double blinded study. In AML pts < 60 yrs in first CR, GCSF is being evaluated as a means of shortening the long periods-of aplasia noted in an earlier cohort of pts after the administration of mitoxantrone/diaziquone as intensive post remission consolidation. This study will provide information about the effect of growth factors after the use of agents with cytotoxicity against "stem cells". In pts with AML in first relapse or CML in myeloid blast crisis, the CALGB is evaluating whether "recruitment" of blasts into cell cycle by growth factor enhances the effect of cytotoxic therapy. Patients are randomized to receive GM-CSF (3 Fg/kg/day by CI for a maximum of 5 days before the chemotherapy) or to no pretreatment, followed by ARA-C, 2 grn/n~ q 12 h x 12 doses. GM-CSF is continued for 24 hrs after the ARA-C. It. is hoped that this attempt to circumvent "cytokinetic resistance" will have eventual application to the therapy of more resistant subtypes of AML. *Univ of Maryland Cancer Ctr, 22 S. Greene St., Baltimore, MD 21201

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RISK FACTORS OF ACUTE GRAFT-VERSUS-HOST DISEASE (GvHD) AFTER HLA-IDENTICAL MARROW TRANSPLANTATION WITH SPECIAL REFERENCE TO INTESTINAL BACTERIAL DECONTAMINATION. Schaefer U.W., Beelen D.W., Haralambie E., Brandt H., Quabeck K., MOiler K.D.. Sayer H.G. Depts. of Bone Marrow Transplantation and Microbiology, University Hospital of Essen, Germany. A retrospective study COvering a 13-yrs-time period of factors influencing the occurense of grades II-IV acute GvHD after allogeneic HLA-identical sibling marrow transplantation was performed. A total of 194 predominantly adult patients (pts) (97 e', 97 =, median age 28 [7*49] yrs, acute leukemias 109 pts, chronic myeloid leukemia [CML] 58 pts, other diseases 27 pts) were enrolled in the analysis. Covariates included were the underlying disease, categorized patient/donor age, patient/donor sex combination, type of immunprophylactic regimen and protective environment, and the quality of intestinal bacterial decontamination. Forty-five patients (23%) developed acute GvHD and univariate analysis identified four features, which significantly increased the risk for this reaction: CML as the underlying disease compared to all other disease categories (p<.0001), female marrow donors for male recipients compared to other sex combinations (p<.005), methotrexate as the sole immunprophylactic compound compared to ciclosporin containing regimens (p<.05), and ineffective compared to successful growth suppression of intestinal anaerobic bacteria (p<.02). Proportional hazards regression analysis confirmed these features as independent predictors for acute GvHD with relative risk estimates of 3.2 for the underlying disease (p<.0001), of 2.5 for the donor/recipient gender COmbination (p<.008), of 2.5 for the immunprophylactic regimen (p<.03), and of 3.1 for anaerobic decontamination (p<.007), respectively. Best subset selection modeling identified the quality of anaerobic decontamination as the second most important predictor for acute GvHD when all four significant features were included. Estimates of acute GvHD by increasing numbers of risk factors stratified by the quality of anaerobic decontamination showed the strongest influence of anaerobic decontamination in patients already burdened by more than one risk factor (p<.02). In Conclusion this study indicates that growth suppression of intestinal anaerobic bacteria after clinical sibling marrow transplantation may independently modulate the occurrence of grades II to IV acute GvHD, which is in concordance with previous results from animal transplantation models.

WHAT IS THE BEST THERAPY OF ACUTE MYELOGENOUS LEUKEMIA. Gale R.P., Butturini A. UCLA, Los Angeles, USA. Three therapies are widely-used to treat acute myelogenons leukemia (AML): allogenic bone marrow transplants, autotransplants and chemotherapy. Which therapy or combination of therapies is best is controversial. Analyzing and comparing results of these therapies is complex and controversial. Comparisons of published data are flawed by biased reporting and subject selection. Randomized trials are also reported but are limited by small numbers of subjects and the question of whether their condnsions apply to most persons with A/vIL. Hypotheses being tested in randomized trials are often misstated or misunderstood. Furthermore, in many trials a substantial proportion of subjects do not receive their intended treatment. Observational data bases have also been used to compare results of different AML therapies. Although this is probably the most effective analytic approach, substantial differences in outcome-related variable between treatment groups make complex statistical adjustment necessary. However, validity of these adjustments is controversial. Limitations of these diverse approaches to comparing results of different therapies of AML suggests that a predse answer to the question of how best to treat AML is unlikely to be forthcoming. This uncertainty is further confounded by the considerable subjectivity that influences how physicians choose between alternative therapies. The net results of these considerations is a consensus approach to treating AML is unlikely to develop, probably because results of diverse treatments are not very different.

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