245
-;1 ~ >
7-~7#~-~_.,y STUDIES EBRAND Changgeng
ON THE FACTOR Ruan
#~JlJ-~;I 1
MUTATIONS
AND
GENE IN CHINESE (Jiangsu Institute
POLYMORPHISMS
OF THE
VON
WlLL-
POPULATION
of H e m a t o l o g y ,
Suzhou University
Medical College,
Suzhou 215007, China) von Willebrand factor (vWF) is a highly multimerized glycoprotein that promotes platelet adhesion and aggregation ata high shear rate, while also acting a s a carrier of coagulation factor VIII. von Willebrand disease (vWD), which is caused by the qualitative and quantitative defects of ron Willebrand factor (vWF), is one of the most common inherited human bleeding disorders. In the past years, ninety one patients with vWD were consulted in our institution. Of all the vWD patients, 56 (61.5%) belonged to type 1, 26 (28.6~) type 2 and 9 (9.89~ type 3. The analysis of vWF gene was performed in some type 2 and type 3 vWD by denature gradient gel electrophoresis and sequencing. We have found six cases of point mutations of vWF gene in these patients with vWD. Among them four point mutations of vWF gene, Ala737~Glu, Gly22---~Glu, Met 37--*Val and Ser71-~stop codon, substitutions, are first reported in international database. We constructed an expression plasmid pSVA737EvWF containing full length of cDNA of vWF which included the AIa737~Glu substitution by site-direct mutagenesis. The structure of recombinant vWF within transfected COS-7 cells and the secretion of high-molecular-weight (HMW) multimers were similar to wild type vWF. HMW forms of vWF multimers were absent in plasma but present in platelets. The mutation corresponds to the group II Wpe 2A vWD characterized by normal secretion of all vWF nmltimers. Polymorphisms in the vWF gene have been found to be useful genetic markers for carrier detection and prenatal diagnosis of vWD. ATCT tandem repeated sequence and the Sma I, Hha I, Msp I and Rsa I polymorphisns in the vWF gene were analyzed by polymerase chain reaction (PCR) and restriction enzyme digestion in Chinese population. Results show that the allele frequencies of Sma I and Hha I polymorphisms are very different between Chinese and Caucasians. Two polymorphisms of vWF gene promoter and the Thr789Ala polymorphism in vWF gene ,have been reported ,are associated with arterial thrombosis.The association between the Sma I polymorphism in vWF gene and thrombotic disease was investigated by genotypic identification in two case-control studies: 107 case patients with acute ischemic stroke, 49 case patients with acute myocardial infarction (AMI), and 113 health controls age- and race matched for each case patient. The prevalence of the CC genotype in acute ischemic stroke was significantly higher than that of the normal controls (Odds ratio=3.29, 95~Cl=1.54-7.01, 0.01>p>0.001). However,the prevalence of the CC genotTpe in AMI was not significantly different from that of the normal controls. Results indicate that the Sma I polymorphism is strongly associated with increased risk of acute ischemic stroke and may represent newly identified risk factor for acute ischemic stroke in Chinese population.
7 -.:Ÿ7 ~ ,.~. t .y -> 3 > HEMATOPOIETIC STEM EASES IN TAIWAN
~.,31J-N~,~ 2
CELL TRANSPLANTATION
P o - M i n C h e n , M . D . , P h . D. ( P r e s i d e n t Medical Oncology, Department Medicine, National Yang-Ming
of t h e H e m a t o l o g y
of M e d i c i n e
OF HEMATOLOGICAL S o c i e t y of T a i w a n
Taipei Veterans
DIS-
D i v i s i o n of
G e n e r a l H o s p i t a l ; S c h o o l of
University, Taipei, Taiwan, R.O.C.)
Bone m a r r o w transplantation was first applied to patients with hematological diseases in 1983 in Taiwan. Now, with the advant of n e w biotechnology, hematopoietic s t e m cells are easily obtained from peripheral blood and cord blood to m a k e peripheral blood s t e m cell transplantation more popular. In addition, the clinical applications of hematopoietic s t e m cell transplantation ( H S C T ) b e c o m e more extensive and can now be applied to solid tumors, various i m m u n e deficiency or metabolic diseases, and even a s a part of immunotherapy. As at other sites of the world, H S C T has been so popular and m o r e t_han ten hospitals p e r f o r m e d this procedure routinely in Taiwan. W e have collected 136 patients ( F / M 64/72, child/adult 38/98) with the diagnoses as followed: A M L (n = 64), C M L (n = 38), S A A (n = 28) ancl M D S (n = 6). T h e s e patients received H S C T s b e t w e e n June 1998 and M a y 2000 in 12 hospitals in Taiwan. T h e sources of s t e m cells include B M ( n = 68), P B S C ( n = 66) and cord blood ( n = 2 ) ; and the donors include siblings ( n = 9 6 ) and matched unrelated donors ( n = 17). Disease-specific survival rates, p o s t - t r a n s p l a n t complications and mortality will be discussed. In addition, 450 cases of H S C T has been p e r f o r m e d in Taipei Veterans Generals Hospital from 1984 to 2000, and two notable characteristics were found. First, the relatively low incidence of acute g r a f f - v e r s u s - h o s t disease (aGVHD) in our institute. Secondarily, the high incidence of H B V carriers receiving H S C T . T h e relationship of H S C T and H B V infection, the patients' liver function, immunologic profile responding to H B V infection before and after H S C T , and the precore and core g e n e mutation of H B V in H B V carriers affer H S C T will be presented.
246
CURRENT STATUS AND RECENT ADVANCES OF HEMATOPOIETIC STEM CELL TRANSPLANTATION IN KOREA Dong-Jip Kim MD, PhD (Division oŸ Hematology, Department of Internal Medicine St. Mary's Hospital, Catholic Hemopoietic Stem Cell Transplantation Center The Catholic University of Korea, Seoul, Korea) In the late 1970's, we started the animal experience of allogeneic transplantation using the canine model. The first allogeneic bone marrow transplantaion was successfully perŸ in March 1983 Ÿ patient with acute lymphocytic leukemia (ALL). Affer 2 years, we periormed autologous transplants for malignant lymphoma. In October 1995, we performed successful unrelated bone marrow transplantation for chronic myelogenous leukemia (CML) patient. Cord blood transplantation was initiated in October 1996. Through August of 2000, we perŸ more than 3,000 transplants with various hematologic and solid malignancies. Among 3086 patients, 1827 recipients were allogeneic, 1239 were autologous, and 20 were cord blood transplantation. Acute myelogenous leukemia (AML) cases were 865, ALL were 357, CML were 405, aplastic anemia were 420, malignant lymphoma were 343, breast cancer were 223, myelodysplastic syndrome were 106, multiple myeloma were 87, and others were 280 cases. Use of tacrolimus (FKS06) is an approach to overcome GVHD post-transplant and probability of overall survival was higher in F K 5 0 6 + M T X group due to decrease in the incidence of severe acute GVHD even though the follow-up duration was short. Prospective randomized study comparing the effect of the two GVHD prophylaxis regimens is in progress. High dose stem cell transplantation using graft engineering was successful Ÿ poor risk aplastic anemia. The stem cell component therapy may lead to increasing survival in poor-risk SAA adult patients by reducing graff failure incidence. Recently non-myeloablative tranaplantation (NMT) has been performed to decrease transplant-related mortality and achieve a graŸ (GVL) effect. With these N M T we could achieve sustained engraftment and possible GVL effect even though we need more data to confirm this.
CELLULAR IMMUNOTHERAPY OF LEUKEMIA Dao-Pei Lu, Chunrong Tong (Peking University Institute of Hematology, Beijing 100044, PRC) Objective: Autologous cytokine induced killer (CIK) which are rich in CD3§ + T cells and have been studied for its effectiveness in 46 patients with acute leukemia since 1995 based on ex vivo and animal studies dated from 1991. Effectiveness of CIK co cultured with autologous dendritic ceUs were also evaluated in the last 15 months. Leukemia patients with concomitant HCV infection were studied at the same time. Material and Methods: For generation of CIK cells, autologous mononuclear cells (0.5~7 • 109) from peripheral blood were collected through a blood cell separator (CS-3000 Plus) for clinical use. Cells were cuItured in serum free culture medium in the presence of IFNT, IL2, mAb against CD3 and ILlc~ according to the method of Lu and Negrin. Large volume cells in a 3000 rol Baxter bag were cultivated for 10~14 days. The final yields were 5 ~ 2 9 • 109 cells. Dendritic cells (DC) isolated from patients' bone marrow were cultured in serum free culture medium in the presence of TNFc~, IL4, GM-CSF, IFNc~ Ÿ 10~14 days and then co-cultured with auto-PBMNC in the same way as CIK. A total of 46 patients were treated with CIKs (29 patients) or CIK co-cultured with DC (CIK-DC group, 17 patients). Among the former patients, 22 of 29 were in CR, 7 were in partial renfission or in retapse after either stem cell transplantation or repeated chemotherapy. Among the latter pafients, all 17 were in CR. 10 patients had leukemic markers including AML1-ETO, PML-RARc~, t(15;17), +8, 3p-, rearrangement Igtt and/or TCR8. Altogether 23 B-ALL and 23 other acute leukenfia patieuts were treated. T - A L L was not selected. 14 patients had concomitant HCV infection. Result: The probability of actuarial CCR after CIK cell therapy was above 80% (Kaplan-Maier) as shown in the following figure. t.1
~
1
0.9 0.8 0,7
"15 0.6 0.4
0.1 0
i 6
i
i
i
:
i
12
18
24
30
36
rnonthsl~~t~OKs'k~~~n
The effect CIK cell therapy was not evident in 3 hematological non-remitted patients. However, gene and cytogenetic abnormalities indicating minimal residual leukemia (MRL) disappeared in all 10 patients after only 1 course CIKs infusion. All 17 patients after CIK-DC therapy remain in CR. CIK and CIK-DC therapy lead to serum IICV-RNA negative in 13 of 14 patients with HCV inŸ and 10 remain continuous negative. CIK and CIK-DC therapy also lead to the normalization of e]evated liver enzymes and/or BIL in all 8 patients. Conelusion: (~Cellular immunotherapy with CIK cells or CIK DC is effective in acute leukemia treatment as shown by disappearance of residual leukemia markers and by the high rate of CCR. (~CIK-DC treatment was at least as eflhctive as CIK. @Most of cases with concomitant hepatitis C showed a Ÿ response.
247
728.
P-~7 r
9 3 ~
Oral Presentation
728~742
729. COMBINATION CtIEMOTHERAPY OF INTERMEDIATE-DOSE CYTARABINE, IDARUBICIN,
P L U S E T O P O S I D E AND S U B S E Q U E N T MOBIL I Z E D DONOR L E U K O C Y T E I N F U S I O N FOR R E L A P S E D ACUTE L E U K E M I A A F T E R ALL O G E N E I C BONE MARROW TRANSPLANTA-
TION
Kyoo-Hyung LEE*, Je-Hwan LEE, Shin KIM, Miee SEOL, Sang-Hee KKIM, Woo-Kun KKIM, Jung-Shin LEE. Section of Oncology-Hematology, Department of Medieine, Univer o sity oŸ Ulsan, College ol Medicine, Asan Medical Center, 388-1 Poongnap-dong, Songpa-ku, Seoul, Korea 138-736 Combination Chemotherapy of Intermediate-Dose Cytarabine, Idarubicin, plus Etop oslde and subsequent Mobinzed IDanor Leukocyte Infuslon for Relapsed Acute Le ukemle after AIIogeneic Bone Msrrow Transplsntation Kvoo-Hvunq Lee'_, Je-Hwan Lee, Shin Kim, Miee Seol, Sang-Hee Kim, Woo-Kun Kim, end Jung-~Shin Lee, Section of Oncology-Hematology, Department of Madicine, Univers ity of Ulssn. Collegs of Medicine, Asan Medical Center, 388-1 Poongnap-dong, Son9pa -ku, Seoul, Korea 138-736 The emcacy sud side effects of intemaediete-dose cytarabine, idarubicin plus etoposide and subsequent DLI were investigated in patients with acute leukemia who relapsed sft er allogeneic BMT Patients were given cytarabine continuous iv (1 g/m2/day x 5), idar ubicin ir (12 rng/m21day x 3), and etoposide Jv infusion (150 mg/m2tday x 3). Two day s later, G-CSF mobilized donor Leukocytes were infused for 2 dsys. No GVHD prophy laxis was given. Between August 1997 and February 2000, 13 patients enrolled (8 A ML and 5 ALL) AIt patients finished chemotherapy and DLI. Eleven patients (85%) a chieved CR at msdisn 27 days aRer DLI. After median follow up of 109 months 42.533.3), five of 11 pstients who uchieve CR relapsed. OveraK six of 13 patients were s urviving (618 AML and 0/5 ALL, P=O 05~)). Marrow recovery after chemotherapy and 19 LI was rapid (12 days for ANC > 5001gl). Side effects included fever with neutropenia (100%), pneumonia (46%), grade II to JV mucosifis (69%), grede fil to IV acute GVH D (45%), and extensive chronic GVHD (64%), One patient died fruto chronic GVHD. ChemotheraPY containing intermediate-dose cytarabine and DLI produced a high CR r ate in acute leukemia in relapse after allogeneic BMT. A fraction of patients ate surviv ing Iong term Side effects were substantial but manageable.
NON-MYELOABLATIVE T R A N S P L A N T A T I O N USING A NOVEL COMBINATION OF CLAFRIBINE, BUSULFAN, AND ANTI-THYMOCYTE GLOBULIN Yoshinobu KANDA, Atsushi MAKIMOTO, Takeshi SAITO, Kunihisa NAKAI, Toshihiro OHNISHI, Ryuji TANOSAKI, Kensei TOBINAI, Shin MINEISHI, Yoichi TAKAUE. National Cancer Center Hospital, Tokyo Japan We performed a phase [/II study to establish a novel nonmyeloablative regimen consisting of clad¡ busulfan, and rabbit anti-thymocyte globulin. Eligibility eriteria included patients with hematologieal disorders (age>50 or (+) organ dysfunction), and those with metastatic solid tumors which had not responded to any of available therapies. Twenty-four patients (6 AML, 6 MDS, 2 CML, 2 NHL, 1 PNH, 1 SAA and 6 solid tumors) were enrolled between July 1999 and December 2000. AII received PBSCT from ah HLA-identical or une antigen-mismatchedrelative. Regimen-related toxicifies were mild and >90% donor chime¡ was achieved before day 30 in all but 2 of evaluable patients. Transplant-relatedmortality at 100 days was 25%. Only two patients with hematological malignancies developed relapse. Three patients with renal eell carcinoma and arte with osteosareoma showed evidente of clinica~ response. In conclusion, this novel NST regimen is associated with less ¡ to eldery or patients with organ dysfunctions. Early full donor chimerism was indueed with satisfactory anti-tumor activities for both hematologiealand solid malignaneies.
730.
HIGH INCIDENCE OF CHRONIC GVHD A F T E R ALLOGENEIC PBSCT: THE NTUH EX-
PERIENCE
Jih-Luh TANG, Ming YAO, Yao-Chang CHEN. Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan Between Jan. 1996 and Jul. 2000, 89 adult patients with hematological malignancies received either allogeneic BMT (n=55) or PBSCT (n=34) fruto HLA-matched sibling donors. GVHD prophylaxis consisted of CsA and rnethotrexate. All of the 89 patients engrafted. When comparing with BM group, patients in the PB group could acquire much higher nucleated cells and CD34+ cells and had much faster recovery of the neutrophil and platelet counts. The actuarial probability of developing acute GVHD was 20 + 5% in BMT and 39 _+8% in PBSCT (p=0.05). However, the probability of chronic GVHD was significantly higher in PBSCT (70 + 10%) than in BMT (27 -+ 7%, p=0.0002). By multivariate Cox regression analysis, PBSCT and sex-mismatch (female donor/male recipient) were two independent factors for predicting developments of chronic GVHD. The majority of chronic GVHD was of de novo onset and extensive, involving mouth, liver, skin, and eye. At a median follow-up of 19 months, the actuarial probability of overall survival in BMT and PBSCT was 64 _+7% vs. 58 + 9% (p=0.14), disease-free survival 70 +_ 7% vs. 81 _+ 8% (p=0.55), and transplant-related mortality (TRM) 13 + 5% vs. 31 + 8% (p=0.02). The results suggest that allo-PBSCT was associated with high irmidence of chronic GVHD that was in favor of diseasefree survival but with higher TRM.
248
73|.
732.
H E M O P O I E T I C STEM CELL T R A N S P L A N T A TION FOR HIGH-RISK ADULT P A T I E N T S WITH S E V E R E APLASTIC ANEMIA; REDUCTION OF G R A F T F A I L U R E BY ENHANCING STEM CELL DOSE Woo Sung MIN, Chang-Ki MIN, Dong Wook KIM, Jong Wook LEE, Chi Wha HAN, Chun Choo KIM. The Catholic Hemopoietic Stem Cell Transplantation Center, College of Medicine, The Catholic University of Korea, Seoul, Korea
HEPATOCYTE GROWTH FACTOR GENE T H E R A P Y A M E R I O P A T E S ACUTE GRAFTV E R S U S - H O S T D I S E A S E AND PROMOTES DONOR CELL E N G R A F T M E N T Takanori KUROIWA, Tsuyoshi IWASAKI, Yasuro KATAOKA, Nobuo IWATA, Naoaki HASHIMOTO, Atsuhi OGATA, Teruaki HAMANO, Eizo KAKISHITA. Second Department of Internal Medicine, Hyogo College of Medicine Acute grafl-ve~sus-hostdisease (GVHD) is a major complieationof bone marrow transplantation(BMT), and is characterized by hematopoietic dysfimction, immun0sappression,and fissue injury in the skin, liver, and intestinal mucosa. Hepat0cyte growth factor (HGF), o¡ identified and clonedasa p0tem mit0genfor hepat0cytes, shows mit0genicand antiapoptotic aetivity for various epithelial eells, and plays enhaneed hematopoietic activity. In a mu¡ m0del of acute GVHD, we performed repeated transfecti0nof the humanHGF gene into skeletal musele. Gene ¨ inkibitedap0ptosisofinteslinalepithelialcellsand donorT eell inftllrationint0 the liver, thereby improvingenteropathy~ad liver injury causedby acute GVHD. It alsomarkedlysuppressedinterferon(IFN)-7 and tumor necrosis factor (TNF)-a expressi0nsin the intestineand livcx, and decreased the sea-um int0rleukin(IL)-I2. Furthermore, exlramedullary hematopoiesisby engrafleddonorceilswas inereased,and the survivalrate was improved. Theseresults suggestthat HGF gene therapymay be useful f0r con~rollingaeute GVHDaflerall0geneieBMT.
Baekground m
r
and
of
disease
ents
the
with
DeBiga
had
27.5
In
were
The
n nt
s~ival
for
infusion
ACQUIRED PROTEIN C RESISTANCE, HIGH T I S S U E FACTOR E X P R E S S I O N AND HYPERH O M O C Y S T E I N E M I A IN SYSTEMIC LUPUS ERYTHEMATOSUS Doyeun OH, Sh KIM 1, Bw NA, Sy CHUNG, Cs CHO 2, S PARK a. Department of Internal Medicine and 1Preventive Medicine, College of Medicine, Pochon CHA University, Department oI Internal Medicine, College of Medicine 2Catholic University of Korea and 3Seoul National University, KOREA Hyper-homocystalnemin, vesistancr
high fissuv factor(TF)
expression,
acquired
are highly prevalent in thromboembolic d ~
acfivated protein
C
and thromboembolism is
f~equently complicated in systemic lupus eD,thematosus(SLE ). In this study ",ve evaluated the prcvalence of APCR, high TF and homocysteinr with tho correlation of the thrombofic te~de~acy in SLE. Ninety four SLE parienta and 28 normal control we~e studied. APC ratin, TF antigens were measured using commercial kits. plasma homocysteine level was measured using HPLC. The plasma level of TF antigens in SLE pafie~ts(176.7 +ll7.Tpg/ml) were significantly higher than those in normal controls( 45.73 :i:8.2pg/ml)(p--0.0023). 2. The APC ratio m SLE patients(2.87 =t=0.80)was loweT than those in normal control(4.10 +2.10)(p=0.0093). 3. The plasma homocysteine level in SLE(12.09 •
umoFml) was not significantly differem as compared with those in normal
failuN,
1999,
of
red
40
numbers
retrospectively high-risk
eonsecutive
bloc
were
cells
enrolled,
women.
AII
pati
patienls
(72.5o/o)
interval of
the
stem
was leu
a
from
course
diagnosis
transfusions
was
cyclophosphamide,
(BM)
alone
cell
source,
respectively.
GVHD
prophylaxis
II 5
ATG,
(n=20)
method.
or T
cells
a
boost
a
SAA a
I~ge
acute
t~atment
and
the
number
with of
Boost
GVHD, 2
and
duration
of the
group.
survival
HSCT
is
duration. a
in
fallar
estimate
(range
8-67).
It
effective is of
t~atme
bi•hly graft
aehieving
Oossibl failure
engraftme
734.
"DECREASED E X P R E S S I O N OF ANTICOAGULANT P R O T E I N C IN MURINE MODELS OF INFLAMMATORY OR PROTHROMBOTIC RENAL D I S E A S E S Koji YAMAMOTO 1), Takayoshi SHIMOKAWA 1), Tetsuhito KOJIMA 2), Hidehiko SAITOIL 1)First Department of Internal Medicine, Nagoya University School of Medicine; Z)Department of Medical Technology, Nagoya University School of Health Sciences Protein C, a key anticoagulant molecule, inhibits coagulation factors Va a n d V l l h , and thedeficient state o f t h i s protein causes various thromboticcomplications. Altbeugh the liver has been the prirnary s i t e o f protein C synthesis, we detected relatively high l e v d s o f protein C tuRNA in the kidney and testis by quantitative RT-PCR assay. In situ hyb¡ and immunohistochemical analysis revealed that abundant protein C tuRNA and antigen were expressed in tubalar epithelial cells in the renal cortex, in spennatogenic cells in thetestis, andin epithelial cells in t h e e p i d d y m i s . Interestingly, protein C antigen was also Iocalized to bronchial epithelial cells in the lung, lo pyramidal neurons in the cerebrum, and to Purkinie cells in the cerebelIum. These observalions imply t h e s p e d f i c function of protein C other than being an anticoagulant in thesc organs. Therenal explession of protein C tuRNA was significanfly decreased in three different t y p e o f renal pathologies, e.g., autoimnmnelupts nephritis (MRLIpr/lpr mice), diabetic nephropathy (~o/db mice), and acutc renal injury in sepsis (encbtoxintreated mice), all of which are regarded to be prothrombolic disorder. We further e x a r a n e d (IL-1), andtransforming g r o ~ h factor-~ (TGF-~), on the protein C expression in the normal mouse becattse these cytokines were reported to h a v e a procoagulant profile in vitro andin vivo, andto play a pathological r o l e i n autoimmtmediseases, diabetes, and
thrombosis in SLE. 6. The plasma level of TI: antigens and homocysteine was correlated with each
other(r=-0.314, p=0.01). APCR, high plasma TF ievels are common in SLE but a sigmficant association was observed only betwer the prr
of APCR and thrombosis in SLE patients. This
suggests that defect of inherent anticoagulant(defensive factor) may be
more important than
increased burden of coagulation factor(offensive factor) in the thrombogenesis of SLE.
I
(p~9.042).
an
reduction
successful
patients
heart
months
B who
function.
Six
indur
40.5
lead
to
patients
marrow
CY
were
receiving
9
Kaplan-Meier
disease
is
of
The
influenced
suggest cells
BM
20xi0
GVHD
(10%)
normal
to
respectively).
chronie
Seven
(n=2),
(n=l).
platelet
0.039,
patients
recovered
BM+PBSC
treatment
and
20
pneumonia
pmlonged stem
of
and
and
(P=0.069).
adversely
results
0.5xlOg/L
(P~O.005
pneumonia
follow-up
GVHD
patients
in
alone
These
speed of engraftment. with gml~ failu~.
IV
BM
inte~titial
median
chronic
gmup
occurred
by
to
BM
I1 t o
between
(n~2),
recovery
the
failure
followed
with only
in
theeffects o f principal cytokines, e.g., tumor necrosis factor-tl (TNF-o0, interleukin-I
and
o
consisted
65% and 6.3% respecfively. 5. A significant association was observed between the presenee of
APCR(OR:8.60, p:0.0001), but not the pre~~~nce of high pIasma TF antigen level(OR: 1.32, p::0.59)
a
BM+p
controls(11.46 • 1.08 umoFml )(p=0.0946). 4. The prevalence of high TF ant~gen level and APCR was
a
age human
received
Median with
~: p l a t e l e t s )
were
number
ma~ow
adult
Median
dono~
(CsA).
bone
as
~ceiving
Coaeluslon~. of
ste
lgraft-ver
large
the
Median
enriehment
grade
Gra~
failure
83.7%
multi-tr~sfused the
in
conditioned
neutrophil than
(35%)
GVHD
~alysis
sludy
failure
units
nine
216).
were
CD34
comparable
gml~
agd
~d a higher nt in patients
20
received
ehronie
was
multiv~iate
grat~
was
from
and
This
gmft
tmnsplant were
either
gmup, rapidly
23.1%. of
failure
ineidenee died
(n~l),
that
of 7
40
to
Twenty to
(n=20)
the
are
by
HSCT. or
December
cyclosporin 2
patients
(PBSC)
hemopoietic
(SAA)
exacerbated
methotrexate.
mo~
and
and
rer
using
BM+PBSC
while
GVHD
cells
the
graft
(15%) of
stem
12.8%
a
patients
achieved
M+PBSC had
"$80). AII
and
(52.5%)
(range,
to
befo~
than
prior
21
siblings.
(ATG)
Our depleted
(more
and
allogeneic anemia
ollen
survival
1995
p~iod
43)
months
shon-te~
incidences
22.5o/o,
e
59
10
blood and
Ionger to
globulin
were
Result*. 9/L
or
to
after
aplastir
institution.
J~uary
(HLA)-matehed
(range,
CsA
Between
our
failure
severe failure,
duration
related in
of
with gmft
dis~se
factors HSCT
16
ca~r
and
multi-transfused
was
procarbazine. PBSC
and
years
antigen
antithym~yte transplant
laterp~tatio/
733.
3
of
eripheral
prolonged
(range,
to
units
e
were
a
years
kocyte
and
main pat[ents
infection
receiving
methodL
who
nd/or
of
in
outcome
SAA
and
patients
f
The
(HSCT)
(GVHD),
tmnsfu$ion$
analyzes
nd
objectlve.
t~nsplantaion
sus-host
obesity. TNF-~ and IL-I decreased the steady slate l e v d o f protein C tuRNA in major o r g m s in a tissue-sped fic manner. 'l~e decreased expression of protein C may contribute to ah increase in the systemic and/or local procoagulan! potenlial during septic and inflammatory processes, and thus, to the progression o f histopathological changes accompaniedwit h prothrombotic state.
249
735.
U S E F U L N E S S OF LUPUS ANTICOAGULANT, ANTI-CARDIOLIPIN ANTIBODY AND ANTI-fl2GLYCOPROTEIN I ANTIBODY IN THE DIAGNOSIS OF A N T I P H O S P H O L I P I D SYNDROME
Hyun-Sook CHI, Yoon-Hee KANG. Department of Clinical Pathology, Asan Medical Center and University of Ulsan College of Medicine, Seoul, Korea INTRODUCTION : Despite improved understanding of the true nature of anti-phospholipid antibody, the clinical utility of the newer assays incl uding anti-[~2-g[ycoprotein I antibody (aGPI) is incompletely evaluated a nd the labomtory still relies predominantly on coagulation-bases assays f or lupus anticoagulant (LA) and solid phase ELISA assay for anti-cardio lipin antibody (aCL). The aim of the smdy was to compare usefulness o f LA, aCL and aGPI in the diagnosis of antiphospholipid syndrome (AP
S).
METHODS : Citrated blood was obtained from 93 patients. Presence of LA was screened by LA-sensitive reagent (PTT-LA, DIAGNOSTICA S TAGO, Fmnce) and mixing test, and confirmed by hexagonal phase phos pholipid (STACLOT| LA, DIAGNOSTICA STAGO, France). ACL-IgG/I gM and aGPI-IgG/IgM were tested semiquantitatively by ELISA kits, Va relisa (Pharmacia & Upjohn, Germany) for aCL and QUANTA LiteTM (I NOVA, USA). RESULTS : Among 93 patients, 47 patients were diagnosed as primary or secondary APS defined by Asherson et al (1989). Sensitivity of L A , aCL-IgG, aCL-IgM, aGPI-lgG and aGPI-IgM was 48.9%, 59.£ 38.3%, 19.2% and 38.3% each. Specificity of these was 30.4%, 65.2%, 39.1%, 95.7% and 78.3% each. ACL-IgG and aGPI-IgG showed higher titer in APS group, but statistically insignificant. Rank correlation between aCL -IgG and aGPI-lgG titer was 0.43. CONCLUSlON : To increase the sensitivity, all five tests should be per formed. AGPI test should not be considered a s a substitute for conventio nal LA and aCL tests. However, performance of aGPI seems to be impo rtant to improve the specificity.
737.
REAL-TIME PCR A S S A Y S FOR MONITORING OF HUMAN H E R P E S V I R U S E S REACTIVATION A F T E R STEM CELL T R A N S P L A N T A TION
Junko H. OHYASHIKI 1,2, Akitaka SUZUKI 2, Kenji ABE 1, Tomoko OJIMA 1, Norio SHIMIZU 1, Kohtaro YAMAMOTO l, Kentaro ARITAKI 3, Nahoko SHOJI 2, Yukihiko KIMURA 2, and Kazuma OHYASHIKI2 ~Department of Virology, Medical Research Insfitute, Tokyo Medical and Dental University and ZThe first Department of Internal Medicine, 3Department oŸ Pediatrics, Tokyo Medical University Viral infections ale important courses of morbidity and mortality
after stem cell transplantation. The diagnosis of human herpesviruses infection after stem cell transplantationrepresents a complex issue because the most widely used diagnostic tools, such as immunoglobulin G antibody titer determinafionand qualitative DNA PCR with blood cells, are unable to distinguish between latent and active infection. To determine whether reactivation of human herpesviruses, such as HHV-6, HCMV and EBV, play ah important role in the development of complications in patients undergoing stem cell transplantation, we developed a new, very rapid, highly sensitive, and quantitative PCR assay for the accurate measurement of viral DNA using a LightCycler. Peripheral blood cells were coUected from patients undergoing stem cell transplantation once before transplant and once per week after transplant up to 8 - 24 weeks subsequently. The real-time PCR system revealed that there was a linear correlation in the range of 10' to 106 molecules of reference. Our study shows that the rapid quantification of viral genomes by the mal-time PCR using a LightCycler may be useful not only to understand the reconstitution of immune system following martow transplantation but also to manage the care of patients.
736.
MOBILIZATION OF HEMOPOIETIC PROGENITOR CELLS INTO P E R I P H E R A L BLOOD IS A S S O C I A T E D WITH VCAM-1 CLEAVAGE IN THE BONE MARROW
Yasushi TAKAMATSU, Patfl J. SIMMONS, Jean-Pierre LEVESQUE First Department of Internal Medicine, Fukuoka University, Fukuoka, Japan and Peter MacCallum Cancer Research Institute, Melbourne, Australia Peripheral blood hemopoietic progenitor cells (HPCs) have been widely used for the source of HPC transplantafion. However, the molecular mechanisms responsible for the HPC mobilization from the bene marrow (BM) into pr blood caused by cytokines such as G-CSF remain to be determincd. Here, we report that VCAM-1 expression is strongly rr in the BM during HPC mobilization. Plasma concentrations of soluble VCAM-1 significanfly incmased du¡ HPC mobilizafion, suggesting VCAM-1 is shed in response to ah agent produced during mobilizafiom To determine which cell types produce ah activity responsible for VCAM-1 cleavage, a murme cell line FDCP-1 clone stably exptessing human VCAM-1 (FDCP-1 -huVCAM-1) was incubated with the conditioned media of normal human BM CD34- cells, BM stromal cells, bone ceUs of PB neulrophils. While media conditioned by either BM stromal ceUs of bone cells did not altex the VCAM-1 expression, media condifioned by either BM CD34- cells or PB neulrophils dmmatically reduced the expression of VCAM-1 on FDCP-I-huVCAM-I. This activity veas completely inhibited by pre-incubating these conditioned media with the serine-prolease inhibitor. Both neulrophil elastase (NE) and cathepsin G (catG), which ate serine-proteases secreted by neutrophils, cleaved rhuVCAM-1 whereas proteinase-3 did not. The concentrations ofNE and catG in the plasma and BM were significantly increased during I-IPC mobilizafiom We therefore propose that ah essenfial step contributing to the HPC mobilization is the proteolytic cleavage of VCAM-I expressed by BM stromal cells, an event triggered by the degranulation of neutmphils accumulafing in the BM in response to cytokine admiistration.
738.
THE CHANGE OF BONE MARROW CELL CONTENTS A N D T R A N S P L A N T A T I O N OUTCOME IN LENOGRASTIM ADMINISTRATION TO H E A L T H Y DONORS
Shuquan JI*, Huiren CHEN, Hengxiang WANG et al. *Department of hemato!ogy, The General Hospital of Air Force, Beijing 100036, China Twenty-four patients with myeloid leukemia underwent allo-BMT The donors of twelve patients (study goup) were given lenograstim (G-CSF) 250u~d/br seven doses prior to harvesting The marrow cell contents and outcome of subsequent en~affrnent and acute GVHD ',vas compared with twelve patients x~ithout G-CSF (control group). The BM stimulated by G-CSF yielded higher number of nucleated, CD34+, CFU-GM and CFU-MK (P<0.01). The numb~ of T lymphocyte subsets m gaft G-CSFstimulating was changed by the compaxison with control group. CD4+ cell was decreased and CD8+ was increased. The CD4+/CD8+ ratio was reduced markedly (P<0.05). The changes of CD34+, CD3+, CD4+, CD8+ percenlage and CFU-GM, CFU-MX proliferation in donor BM with and without G-CSF were analyzed. The same change charactenstic as above graft was observed. Hematopoietic reconstitution in study group after Allo-BMT were accelerated, the days of ~anuiocyte count exceeding 0.5 • I09/L and platelet count exceedmg 20 • 109/L were 16 days (rangeII-23 days) and 17 days (mnge 14-25 days) (control group 20.5 range 14-29 days and 23 range 17-32 days, P
250
739.
CHARACTERIZATION OF HUMAN LYMPHOHEMATOPOIETIC STEM/PROGENITOR CELLS DEFINED BY CD34 AND CD81 EXPRESSIONS
F e n g M A , M i k a W A D A , Shigetaka A S A N O , Tatsutoshi N A K A H A T A , Kohichiro T S U J I . Divisions of CeUular T h e r a p y and Molecular T h e r a p y , Institute of Medical Science, U n i v e r s i t y oŸ Tokyo; D e p a r t m e n t o…Pediatrics, Kyoto U n i v e r s i t y CD34§ cells represent lymphohematopoietic stem/progenitor cells, but are heterogeneous in their potentials. The sepamtion of human CD34§ cells into defined subpopulations could benefit the clinical use of fractionated stem cell transplantation. We then examined the expression of CDS1, a member of the transmembrarte 4 superfamily, on cord blood CD34+ cells. They were classified into three subpopulations based on tbeir expression levels: CD34~CDS1§ CD34~~ § and CD34+CD81 m~ cells. The lymphohematopoietic activity of each subpopulation was then examined, using suspension and clonogenic cultures for hematopoietic potential, coculture with MS-5 cells for B cell potential, NOD/SCID mouse fetal thymus organ culture for T cell potential, coculture with NOD/SCID mouse fetal liver-derived stromal cells for natural killer (NK) cell and mast cell potentials, and xenotransplantation into NOD/SCID mice for long-term repopulating ability. CD34§ * cells represented a heterogeneous population that had all the lymphohematopoietic activities, including NOD/SCID mouse-repopulating ability. CD34L~WCD81§ cells were enriched in erythroid, megakaryocytic and Ni( lineage potentials but had lost T and B potentials and long-term repopulating ability. The CD34§ ~~' fraction was depleted of most lymphohematopoietic potential except for NI< cell and mast cell potentials. Thus, along the differentiation cascade from CD34§ § lymphohematopoietic stem cells, an up-regulation of CD81 o r a down-regulation of CD34 results in a change in lymphohematopoietic properties. CD81 may serve as a marker to separate human lymphohematopoietic stem/progenitor cells.
741. C M L I N THAILAND S a e n g s u r e e J O O T A R . Division of H e m a t o l o g y , D e p a r t m e n t of Medicine, Ramathibodi Hospital, Bangkok, Thailand The prognostic importante of pretreatment clinical and laboratory features was investigated in a group of 243 patients with Philadelphia chromosome positive chronic phase chronic myeloid leukemia from 1977-1995. Chemotherapy consisted of busulfan bcfore 1993 or hydroxyurea after 1993. The overall median survival from dignosis was 28 months. The mean age of the pafients was 38 years, about lO years below that of Western populations. Univariate analysis identified 4 poor prognostic features:- thrombocytopenia, more than 5 % peripheral blasts, more than 5% erythroid precursors and less than 7 g/dl of hemoglobin. The median survival times of patients with these 4 risk factors were 5,11,11 and 12 months respectively. Multivariate analysis only identified 2 significant prognostic features:- thrombocytopenia and more than 5% peripheral blasts. Splenomegaly of more than 10 cm, basophilia and leukocytosis were associated with a shorter median survival but was not statistically significant. A risk scoring system was developed and used to classify patients into low, intermediate and high risk groups at 30.9%, 30.2% and 38.8% respectively. The median survival time according to the low, intermediate and high risk group was observed at 60, 27 and 14 months respectively. Prognostic factors for Thai patients with chronic myeloid leukemia have both similarities and differences with previously observed factors but the median patient survival time is shorter. Analysis of treatment result revealed that the overatl survival were different between groups ofpatients who received different kinds of chemotherapy (p<0.001). Patients who received interferon had longer survival (p = 0.001)
740.
THALASSEMIA IN THAILAND: SCREENING MODELS AND PRENATAL DIAGNOSIS
Kitti T O R C H A R U S , M.D. D e p a r t m e n t of Phramongkutklao Hospital, Bangkok, Thailand
Pediatrics,
Thalassemias and hemoglobinopathies ate common and heterogeneous in Thailand. About 30-40% of the population are carriers of at least one of the abnormal genes and 1.2% of the newborn or 12,000 affected cases born each year. The strategy plan for the Thailand Thalassemia Preventive Network includes University Hospitals, the Ministry of Public Health, hospitals under the Ministry of Defense, and hospitals under the Bangkok Metropolitan administration. Screening methods for the diagnosis of carriers are one tube osmotic fragility test (OFT), Dichlorophenol indophenol (DCIP) precipitation test and Mean corpuscular volume (MCV) with sensitivity and specificity of 73%, 96%; 87%, 97%; and 66%, 92% respectively. These lead to the detection of couples at risk and finally, the affected fetuses. Using advanced technology, the mutations of alpha-thalassemia ate known. For betathalassemia mutations, 25 have been detected and only 7% ate uncharacterized. Fetal tissue sampling have been well practiced. Thus, prenatal diagnosis is feasŸ in all 4 regions of the country.
742. A MULTI-CENTER
CLINICAL STUDY O N PRIMING REGIMENOF LOW DOSE CYTARABINE AND ACLARUBICIN COMBINED WITH GRANULOCYTE COLONY-STIMULATINGFACTOR FOR PATIENTS WITH REERACTORY ANDRELAPSED ACUTE MYELOGENOUS LEUKEMIA
Study Group of Priming C h e m o t h e r a p y (CAG) in China, Jun M A 1, Jianxiang W A N G z, Kugui QIU ~, Zhixiang S H E N 3, Shuyun Z H O U 4, Ru F E N G 4, Ti SHEN% IHarbin Institute of H e m a t o l o g y & Oncology, Harbin, 2Institute of Hematology, Blood Disease Hospital, C A M S , Tianjin, 3Shanghai Ruijin Hospital, Shanghai, 4Nanfang Hospital, Guangdong, 5Peking Union Medical College Hospital, CAMS, Beijing, P.R. China The treatment of patients with reffactory or relapsed acute myelogenous lettkemia (AML) still is one of knotty problems, which remains to be solved. A clinical study of priming regimen (CAG) (Cytarabine 10mg/m2 per 12 hours dl
251
743.
THE R E L A T I O N S H I P B E T W E E N A C T I V A T I O N OF NF-KB AND A P O P T O S I S O F L E U K E M I C C E L L S INDUCED BY C H E M O T H E R A P E U T I C DRUGS Xiaoping XU, Jianhui SHI, Lin LI, et al. Department of Hematology, Shanghai Changhai Hospital. Shanghai 200433. China
y~Yn~~,tz,y-L, B >
[ABSTRACT] Objective To amalyi~ethe relationship br162162activation ofNF-~ B and
apoptosis of |e.ukemiccells indur
by chemotherapeuticalDrugs and to explorethe effec!of
VCR and glucoconicoids on NF-r B activily and apoptosis induced by chemolherapeufical drugs, t91
Poster Presentation
EMSA was used to detec~ed the acfivation of NF-K B , Tunel. DNA
clectrophoresis, and other methods were adopted to observe apoptosis induced by DNR. Ara-C. Cy and VPI6 in P388 leukemic cells Resul ts The tirst one is thm the actwadon of NF- ,c B iaduceclby DI91
Ara-C. Cy and VPI6 is obviously correlated to apopIosis induced
by chemotherapeulic drugs in leukemic ce[Is. The VCR and. D X M which can suppress of
743~825
NI:- ,c B increased significamly the apoptosis mduced by chemotherapeutic dmgs The second one
is that the ability of VCR to increase apoptosis induced by chcmotherapy was related to
its ability to suppress the proliferation of leukemic cells. Conclusion The acdvalion of
NI:-te B inducedby chemotheral:muticdrugs is significantly cotietated to apoptosis induce~by chemotherapoaticdmgs in leukemicceHs. Ihe drugs which can suppress of NF- ~ B increased significanflythe apopto$isinduced by chemotherapeuticd~gs. [Key words] Chemotherapeuticdrugs; P388; NF- ~ B; Apoptosis
744. STUDY OF A P O P T O S I S INDUCED BY FRAC-
TION C (FCNNAV) Huo TAN, Yushu Af¡ Hospital,
FROM N A JA A C T R A VENOM IN H U M A N L E U K E M I A C E L L S HAO, Hongguang TING. The Second Guangzhou Medical College, Guangzhou
Objective: To study the effect and mechanism of FCNNAVinduced apoptosis in human leukemia cells. Methods: Light microscope, transmission electron microscope, DNA electrophoresis, flow cytometry and RT-PCR assay were used to observe the morphological biochemical changes and the expression levei of Bcl-2/Bax £ human leukemia cells affer their exposure to FCNNAV. Results: FCNNAV can induce HL60 cell apoptosis demonstrated by the typical morphological and biochemical changes. Flow cytometry demonstrated that J6-1, K562, HL and fresh leukemia cells could be induced to apoptosis by FCNNAV and the number of apoptofic ceIls increased with the increase of venom dosage. KT-PCR detection showed the expression level of bcl-2 gene of KL-60 cells was down regulated by FCNNAV, whereas the expression of Bax was unaffected. Conch, sion: FCNNAV could induce apoptosis in hurnan leukemia cells and this effect is related to down-regulafion of the expression level of bcl-2 gene.
745. M O N E N S I N - M E D I A T E D
GROWTH INHIBITION IN ACUTE M Y E L O G E N O U S L E U K E M I A C E L L S VIA C E L L CYCLE AND A P O P T O S I S Young Y LEE, Woo H PARK, Jae G SEOL, Eun S KIM, Byoung K KIM. Department of Internal Medicine, Han Yang University Hospital; Cancer Research Institute, Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea Monensin. Na* ionophore, regulates m a n y cellular functions including apoDtosis. However, there has been no reDort about the effect of monensin on acute myelogenous leukerrda (AML). Here, w e investigated the antiDroliferative effect of monensin on AML cells in vitro and in vivo. Monensin efficiently inhibited the proliferation of all 10 AML cell lines wit IC50 of about 0.5 uM by MTI" assay. Monensin induced G1 a n d / o r G2/M phase arrest and apoptosis in these cell lines. The levels of CDK6, cyclin Dl and cyclin A were decreased. In addition, monensin not only increased the p27, but also enhanced its binding with CDK2. Furthermore, the activities of CDK2- and CDK6-associated kinases reduced by monensin were associated with hyDophosphorylation of Rb protein. Apoptottc process was assoctated with the changes of Bax, caspase-3, caspase-8 and mitochondrial transmembrane potentiaL Monensin-treated mice had significantly smaller tumors than control mice (69% growth inhibition relative to control group; p<0.05). T u m o r from monensIn-treated mouse showed increased aI~optotic bodies and this tumor was more stained with Bax, Fas and p53 antlbodies than control tumor. In conclusion, monensln potently inhibited the Droliferation of AML cells vŸ cell cycle and apoptosis.
252
746. A N A L Y S I S OF E A R L Y STAGE APOPTOSIS
747.
Jiwei L I U , Lijun C H E N , Z h a o m i n Z H A N , Jun MA. Harbin I n s t i t u t e of H e m a t o l o g y & Oncology, H a r b i n F i r s t Hospital, Harbtil 150010, P.R. China
T i n g D U A N , Zhao W A N G , Mei XIONG. D e p a r t m e n t of Blood, Beijing F r i e n d s h i p Hospital
C E L L BY FLOWCYTOMETRY
THE E X P R E S S I O N AND R E A R R A N G E M E N T OF BCL-2 GENE IN CHRONIC LYMPHOCYTIC LEUKEMIA
Arsenic trioxide (As203) was used to reduce cell apoptosis on patients with acute promyelocytic leukemia (APL). Then the apoptosis cells were analyzed by FCM with Almexin V-FITC/PI. There were three first-visit patients. They were all male pafients. At admission to hospital, their promyelocytic cells were 42%, 51%, and 59% respectively. Results and discussion: (1) The measured results were 11.9%, 13.5%, 13.1% at 0 hour; ,nd 10.8%, 12.9%, 13% at 24 hour respectively. (2) At the early cell apoptosis, there appeared phosphatidyl se¡ (PS) translocation in the cell membrane. PS moved from internal layer of cell membrane to the out layer of cell membrane. At this time, it was very hard to analyze and determine the apoptosis cells by morphologieal observation, DNA staining and DNA electrophoresis, because cellular DNA hadn't degraded. However, Annexin V-FITC/PI could be used to analyzed and differentiate early-stage apoptosis cells and necrotic cells; (3) The expe¡ results showed that there was no significant difference br 24 hottr m~d 0 hour for apoptosis cell measurement.
In order to elucidate the role of bcl-2 gene in chronic lymphocytic leukemia (CLL), by using immunohistochemicalstaining method and polymerase chain reaction (PCR) analysis, the expression and rearrangement of bcl-2 gene in 11 CLL were detected. Over expression of bcl-2 gene was found in all cases, 1 of I 1 cases had t(14,18) (g32,g21) translocations. The data indicated hat high levels of bcl-2 gene expression occur frequently in CLL, and bcl-2 gene plays a pivotal role in pathogenesis and progression
748.
749. P L A T E L E T PRODUCTION DURING
INTERFERON GAMMA, AS WELL AS E R Y T H R O P O I E T I N , P R E V E N T S APOPTOSIS OF ERYTHROID PROGENITOR CELLS B Y R E D U C I N G COLLAPSE OF MITOCHONDRIAL TRANSMEMBRANE P O T E N T I A L VIA PI-3 KINASE PATHWAY
Kittiphong Takamitsu
PAIBOONSUKWONG, MATSUSHIMA,
N A W A T A , Koichiro M U T A
Illseung
Yasunobu
ABE,
CHOI, Hajime
(Dept. of Med. and Bioreg.
Sci., Grad. Sch. of Med. Sci., K y u s h u Univ.) Interferon gamma
(IFN-y) has b e e n s u g g e s t e d to delay
a p o p t o s i s of m a t u r e e r y t h r o i d p r o g e n i t o r cells. To investigate f u r t h e r t h e m e c h a n i s m b y w h i c h I F N - r affect apoptosis of t h e cells, the effects of IFN-~" on m i t o c h o n d r i a l transm e m b r a n e p o t e n t i a l ( M T P ) , r e a c t i v e o x y g e n species (ROS) p r o d u c t i o n and e x p r e s s i o n of Bcl-x w e r e d e t e r m i n e d . Addition of IFN-y, or e r y t h r o p o i e t i n ( E p o ) , induced e x p r e s s i o n of Bcl-x, t h e r e b y i n d u c e d collapse of M T P and ROS production. In t h e p r e s e n c e of PI-3 k i n a s e inhibitor, Ly294002, the effect of b o t h IFN-~" and Epo w e r e abolished. In conclusion, t h e s e r e s u l t s indicate t h a t IFN-y, as well as Epo, p l a y a significant role in p r e v e n t i n g a p o p t o s i s of erythroid p r o g e n i t o r cells b y affecting e x p r e s s i o n of Bcl-x to r e d u c e disruption of M T P , via PI-3 kinase d e p e n d e n t p a t h w a y .
of CLL.
E X VIVO
E X P A N S I O N OF HUMAN CORD BLOOD CD34 § CELLS USING THROMBOPOIETIN
K y u n g - H a R Y U 1, J u - Y o u n g S E O H 2, H e e - Y o u n g S H I N 3, Hyo-Seop A H N 3. D e p t of 1Pediatrics, 2Microbiology, College of Medicine, E w h a W o m a n s U n i v e r s i t y , Seoul, Dept of 3pediatrics, Seoul National U n i v e r s i t y College of Medicine, Seoul, K o r e a Thrombopoietin (TPO) is one of the most promising stimulants for e x v i v o expansion of hematopoietic stem cells. Previously, we have reported that "['PO induces characteristic pattern of apoptosis dufing ex v i v o expansion of human cord blood (CB) CD34§ ceUs and that the TPO-induced apoptotic fractions belong to megakaryocytic lineage. In this study, we have studied on the maturation of megakaryocytes (MKs) and platelet production in association with the TPO-induced apoptosis. CD34+ ceUs, purified from human CBs, were expanded in semm-ffee conditions stimulated with TPO. Apoptosis has been confirmed by nuclear staining with Hoechst 33258, TUNEL assay, and EM. When DNA contents were measured simultaneously with immunophenotyping, the cells with higher DNA contents (> 8N) comprised less than 5% of the CD41§ fractions until day 14, implying premature apoptosis of MKs before full polyploidization. Nevertheless, not only platelet territories but also granules and microfilaments within the produced platelets could be demonstrated by EM. Furthermore, f l o w cytometric analysis demonstrated that the platelet fraction expressed P-selectin (CD62P) and an activation morir on GPllb/IIIa recognized by PAC-1 upon stimulafion with ADP. In addifion, PAS-positive materials and non-specific esterase activities could be demonstrated. Therefore, ir is suggested that platelet producfion and accompanying processes, rather than apoptosis only, be hastened during e x v i v o expansion of CB CD34" ceUs using TPO.
253
750.
S T U D I E S ON T H E E X P R E S S I O N OF HEMATOPOIETIC G R O W T H F A C T O R S R E G U L A T E D B Y
RADIATION INDUCIBLE GENE X u e t a o PEI, Nan DU, Chengji LUO, Liang LI, Kai F E N G , Cixian BAI. Institute of Transfusion Medicine, 27 Tai-Ping Road, Beijing, 100850, China To explore the regulating effects of radiation induciblegene on the expression of hematopoietic growth factor genes.The human Flt3 ligand (FL) cDNA and EGFP cDNA were linkedtogether with IRES and then inserted into the eukaryotic expression veetor PCI-Egr. which was constructed by substituting CMV promoter in PCIneo with the Egr-1 promoter(Egr-EF).The expression of FL in HFCL/EFcells were confirmed with RI-PCR, ELISA, FACS and cell culture. The results indicated that the activityof EGFP in transfected cells increased by 3.1 fold as compared to non-transfected cells at 18h after exposure to 2.5Gy. The amounts of secreted FL in serum-free supernatants of Egr-EF increased by 605.46 _+107.21ng/ml,whichwere significantly higher than the control. FL cDNA was successfullyexpressed in the cells. Which were confirmed by ELISA and RT-PRC analysis. The results showed that at day 10 ofculture the number of CD34+cells increased by 173.09_+ 11.58 x 10:~/ml,which was significantlyhigher than that of nonradiation group. It showed that radiation can enhance the abilityof the supematants containingFL of HFCL/EFG to expand early hematopoietic progenitor cells and protected hematopoietic cells from radiation-injury effects.
752.
EXPRESSION OF EBV-BNLF-1 GENE IN MALIGNANT HEMATOLOGICAL DISEASE AND ITS CLINICAL SIGNIFICANCE X u d o n g M A 1, W e i c h e n g L I N 1, Huiping Y U 1, Wenqiao W U 1, Ruiji Z H E N 1, Lili C H E N 1, Yuanzhong C H E N L 1Zhangzhou municipal Hospital, Fujian, Zhanzhou, 363000 ZFujian Union Medical U n i v e r s i t y Hospital 350001 Objeelive: To study the expression and quantity of the EBV-BNLF-1 gene fragment in lymphoma and acute leukemia and its clinical pmgnosis m lymphoma . Method: BNLF-1 gene fragment were ampfified mad quantified by using quantitative polymerase chain reaction (P_~), and rifle VCA-IgA were detected by enzyme immunoassay. Results: [1] The positive eN~ression rate was higher in lymphoma than that i¡ acute Iymphocytic leukemia (P--0.003), in acute non-lymphocyfic leukemia (P--0.001) and in the control(P=0.001). The positive expression ro.te was higher in acute lymphocytic leukemia than that in acute non-lymphocytic leukemia (P=O.007"), m the control(P=0.001). There were no significan@ different with acute non-lymphocytic leukemia and the control (P--0.122). [2] The quantity of BNLF-1 was higher in lymphoma than that in acute leial~emia.Average quanfity of BNLF-lgene was higher in lymphoma than that in acute lymphocytic leukemia(P=0.0017), in acute non-l~~n,phocytic leukemia(P=0.0001). Average quan~.ty of BNLF-1 gene was higher m acute lymphocytic leukemia than that in acute nonlymphocytic leukemia(P=0.0104). [3] To research the relafionslaip between BNLF-1 gene and life span 50 cases of lvmphoma were followed up. COX regression analysis showed 13--0.58791 Ward X2=13.38198, OR=L800,P-=0.0003. It showed negative correlation between the quantity of BNLF-1 gene and life span. Condusions EBV-BNLF-1 may be a carcinogen. It may play an important role in carcinogenesis of lymphoma and acute lymphocyfic leukemia; It showed nega.five correlation between the quanfity and life span of these patients. The higher quantity of BNLF-1 gene, the shorter life span was. Key word: lymphoma, acute leukemia, BNLF-1, quantitafive potymerase chain reaction (PCR)
751.
ANALYSIS OF MICROSATELLITE ALTERATIONS IN HEMATOLOGICAL MALIGNANCIES T a k a f u m i O G A W A , Masayuki S U G A , Keiichi K O N D O H , Nobuko Y A M A G U C H I , Naoki K O B A Y A S H I , Masahiro O G A S A W A R A , Yoshio K I Y A M A , T o h r u N A O H A R A , Toshio HIGA, Masaharu K A S A I . D e p a r t m e n t of U n t e r n a l Medicine, Sapporo Hokuyu Hospital [Purpose] The microsatellite alterations such as microsatellite instability (MSI) and loss of heterozygosity (LOH) were reported in hereditary cancers and various types of sporadic tumors as well as in hematological malignancies. To invesfigate whether the microsatellite alterations ate involved in the malignant transformation in hematological malignancies, we examined the microsatellites at 7 loci and 4 cfitical target molecules of MSI in patients with myelodsyplastic syndrome (MDS) and malignant lymphoma (ML). [Materials and methods] Genomic DNA was extracted from bone marrow mononuclear cells of 24 MDS patients or from lymphnode biopsies of 9 ML pafients with various types of histology. DNA samples were also obtained from hair asa nomal control. DNA samples were amplified by PCR using fluorescent pfimers specific to the microsatellite loci and the target genes of MSI. The sizes of the PCR fragments were analysed using a DNA sequencer. [Results] Fourteen out of 24 (58%) patients with MDS and overt leukemia evolved from MDS had MSI. Positivity in MSI was tended to increase according to the percentage of blastic cells in blood. All the leukemia patients had MSI. In ML patients, 3 of 9 (33%) showed microsatellite alterafions. All the 3 patients with MSI showed LOH as well. On the other hand, mutafions within mononucleotide repeats located in the coding regions of BAX, TGFB type II receptor, hMSH3 and hMSH£ genes were not found in any of the pafients. [Conclusions] These findings indicated that microsatellite alterafions occur in a proportion of patients with MDS and ML. These results suggest that the genomic instability may be involved in the oncogenesis in hematological malignancies.
753. THE
EXTRACTION AND PRIMARY ANALYSIS ON N U C L E A R M A T R I X P R O T E I N S O F L E U KEMIC CELLS Juan LI, Shao-Kai LUO, W e n - D e HONG, Gou-Cai Z H A N G . T h e D e p a r t m e n t of Hematology, T h e Affiliated Hospital of Sun Yet-Sen Medical University, Guangzhou Objective: To compare nuclear matrix proteins of normal bone marrow cells with ones of leukemic cells and find special nuclear matrix proteins in leukemic cells. Methods: we extracted nuclear matrix proteins by high-salt extraction procedures, we analyzed the change o f nuclear matrix proteins by SDS-PAGE and two-dimensional (2D) electrophoresis. Results: there are obvious difference in nuclear matrix proteins of leukemic cells and normal bone marrow cells. There are many new nuclear matrix proteins in leukemic cells and difference among leukemic cells. Conclusion: There are obvious difference o f nuclear matrix proteins between norlnal bone marrow cells and the appearance of new nuclear matrix proteins in leukemic cells may related to pathogenesis o f leukemia.
254
754.
A PRELIMINARY ANALYSIS OF MUTATIONS O F Madl, Mxil AND Rox IN HEMATOLOGICAL MALIGNANCIE S Ling PAN, Hitoshi YOSHIDA, Jian Min LUO, Hirotaka M A T S U I , Yota FUJITA, Kazuyuki SHIGENO, Shinya FUJISAWA, Kensuke NAITO, Kaori SHINJO, Akihiro T A K E S H I T A , Ryuzo OHNO*, Kazunori OHNISHI Department of Medicine III, Hamamatsu University School of Medicine *Aichi Cancer Center Hospital Madl and its related members Mxil and Rox can negatively regulate Myc functions and may therefore be potential tumor suppressors. In an attempt to identify mutations in Madl, Mxil and Rox genes in human hematological malignancies, we screcned 10 hematopoietic cell lines and 26 bone marrow mononuclear celt samples from patients with acute myelogenous ieukemia (AML, 18), acute lymphocytic leukemia (ALL, 6), myelodysplastic syndrome (MI)S, 1), and multiple myeloma (MM, 1) using RT-PCR-SSCP analysis and automated sequencing. Four polymorphisms were found, two in Madl, one in Mxil, and one in Rox. Nine missense mutations were detected, two in Madl, four in Mxil and three in Rox. Among the only six pafients with acute lymphocytic leukemia, two patients had Mxil mutations and another two had Rox mutations. This is the first report to show that Madl, Mxil and Rox genes were expressed and displayed mutations in hematological malignancies. Our observafions suggest that these genes might be involved in the pathogenesis of hematological malignancies, espcciaUy in acute lymphocytic leukemia.
756. TWO NEW TCRJREC-JJ1 GENE REARRRAN-
GEMENTS IN T-ALL Yangqiu LI, Lijian YANG, Shaohua CHEN. Institute of Hematology, Medical College, Jinan University, Guangzhou 510632, P.R. China Objective To analyze the new rearrangement of TCR 6 gene in T cell-acute lymphoblasfic leukemia (T-ALL) and its distribution in peripheral blood T cells and thymocytcs from normal individuals. Methods The rearrangement segments of TCR 6 gene were arnplified in genomic DNA from peripheral blood mononuclear cells and thymocytes from 2 cases with T-ALL, 17 cases of normal individuals , respectively, by using semi-ncsted PCR or nested PCR. The clonal PCR products were farther sequenccd to define the posifion of rearrangemcnt. The expression of rearrangement gene was dctected by RT-PCR. Results Two new TCR 5 Rec-J 5 1 rearrangements could be found in 2 cases with T-ALL, and the new TCR 91 Rec-J 91 1 rearrangements could also be detected in PBMCs and thymocytes from normal individuals. RT-PCR showed that the TCR 91 Rec-J 91 1 rearrangements could be identified in 2 cases with T-ALL, but not in the healthy samples. Conelusions In the present research, two new TCR 91 Rec-J 91 1 rearrangements called TCR 91 Recl-J 91 1 and TCR 91 Rec2-J 5 1 were found. The rearrangement genes expressed in mRNA from T-ALL, but not from normal individuals, it may suggest that the rearrangement gene may relate to the initiation of T cell leukemia.
755.
ALTERED EXPRESSION ON M A F B GENE IN HUMAN MYELOMA CELLS CARRYING (14;20) CHROMOSOMAL TRANSLOCATIONS Ichiro HANAMURA 1, Shinsuke IIDA l, Yumiko AKANO 2, Hitoshi KIYOH, Tomoki NAOE 3, Shiro SHIMIZU 4, Masafumi T A N I W A K Iz, Ryuzo UEDA 1. ISecond Department of Internal Medicine, Nagoya City University Medical School, ZThird Department of Internal Medicine, Kyoto Prefectural University of Medicine; 3Department of Infectious Diseases, Nagoya University School of Medicine; 4Shimane Prefectural Central Hospital Chromosome 14q+, which represents a chromosomal rearrangement involving the immunoglobulin heavy chain gene (lgH) locus, is a genetic haUmark of human multiple myeloma (MM). Here, we report the identification of (14;20)(q32;qll) chromosomal translocations found in MM. Double color fluorescence in situ hybridization analyses pinpointed the breakpoints at the 20qll locus in two MM cell lines within a length of at most 680 kb between the KIAA0823 and MAFB gene loci. Among the transcribed sequences in the vicinity of the breakpoints, an ectopic expression of the MAFB gene, which is located at 450-680kb telomeric to one of the breakpoints and encodes a member of the MAF family basic region/leucin zipper transcription factor, was demonstrated to be associated with t(14;20). This finding, together with that of a previous study describing its transforming activity, suggests that the MAFB gene may be one of the targets deregulated by regulatory elements of the lgH asa result of t(14;20).
757Ÿ
CD7/CD34 CO-EXPRESSION AS A PROGNOSTIC FACTOR IN CHILDHOOD ACUTE MYELOID LEUKEMIA Chang Hyun YANG, Chuhl Joo LYU, Kir-Young KIM. Department of Pediatrics, Yonsei University College of Medicine, Seoul, Korea Immunophenotype is undoubtedly useful to diagnose and classify acute leukemia, but its prognostic value is still debated. Elderly patients with acute myeloid leukemia (AML) indepentently suggested a negative prognostic impact of CD7/CD34 co-expression. To evaluate the incidence of CD34 and/or CD7 expression and their prognostic implication in children, we reviewed 61 cases of de novo AML at diagnosis, together with the age, sex, hematologic manifestations, FAB subtypes, therapeutic response retrospectively. Median age was 7 yrs 8 mos. FAB subtypes were; M0-M1 5, M2 17, M3 6, M4-M5 23, M6-M7 3. We split aH patients into four phenotypic classes according to the presence of CD34, CD7 or both. Thirty-four pts were C D 7 - / C D 3 4 - , 13 C D 7 + / C D 3 4 - , 8 C D 7 - / C D 3 4 + , 6 CD7§ We classify into two groups. Positive group (27 pts) was the presence of CD7+ and/or CD34+. Negative group (34 pts) was all negative for CD7/CD34. Median age, WBC count, LDH, blast count, hepatosplenomegaly, cytogenetic abnormality were similar in both groups. Complete remission rate was 56%o and 77%o respectively (p=0.22). No difference was Iound in both CR duration and disease free survival, respectively. Our study suggests no negative prognostic impact of CD7/CD34 co-expression in childhood AML in comparison with elderly patients.
255
758.
ROLE OF MGMT REGULATION IN LEUKEMOGENE S I S
N a o y u k i M A R U O z,2), Lili L U I 3), T a k a n o r i H A T T O R I 1), S t a n t o n G E R S O N 3). 1)Pathol. of S h i g a Med. U n i r . 2)Nagitsuji H o s p i t a l 3)Hematology Oncology, C W R U Leukemia is often induced by treatment with alkylating agents, which forro O6-alkylguanine, mutagenic and lethal lesions. The repair of O6-alkylguanine is performed by action of O6-methylguanine-DNAmethyl-transferase (MGMT). To examine the relationship between MGMT levels and frequency of mutafion in EBV-transformed lymphoid cell lines (LCLs)(A,B,C cell lines) by using a shuttle rector containing gpt gene after treatment with BCNU. The MGMT levets were 16.3, 6.9 and 0 fmol/gDNA in A, B and C cells respectively. Frequency of mutation was calculated by the number of colonies on 6-thio-guanine plates. The frequency of mutation is higher in C cells than in A cells. It was shown that MGMT enhancer binding protein (MEBP), which binds to a minimal 9-roer cis element (probe) of the enhancer region, is located in the nuclei and cytoplasms of MGMT proficient cells while it is found only in the cytoplasms of MGMT deficient cells. MEBP was calculated on the basis of the fact that one mole of MEBP hybridized with one mole of DIG-labeled probe in the band shift assay using PMNC from normal human beings. The amounts of nuclear MEBP are proportional to the levels of MGMT with 0.78 (P<0.02). These findings suggest that MEBP leve] affects mutation frequency.
760. EFFECT
OF
HOMOHARRINGTONIN
ON
T E L O M E R A S E A C T I V I T Y O F K562 C E L L L I N E J. J I N l, K J Z H U 1, Y. T O N G 1, W B . Q I A N 1, R. P A R W A R E S C H z. ~ D e p a r t m e n t of H e m a t o l o g y , First Affiliated H o s p i t a l of Z h e j i a n g U n i v e r s i t y School of MedŸ cine, P.R. China, Zlnstitute of H e m a t o p a t h o l o g y , U n i v e r s i t y of Kiel, G e r m a n y The effects of Homoharringtonine (HHT), ah alkaloid extracted from the evergreen cephalotaxus harringtonia native to the southem China, on telomerase activity were examined in human erythroid leukemic (K562) cells exposed to HHT. Using Fluorescent-labeled telomeric repeat amplification protocol (TRAP) assay, we demonstrated that HHT strongly irdaibited telomerase activity in myeloid blast-like K562 eells in a dose- and time-dependent marmer. More interestingly, the inhibifion of telomerase activity in K562 cells was not due to HHT-induced cell apoptosis when concentration of HHT was 5ng/ml and 10ng/ml rather than differentiation, which was confirmed by cell morphology and surface antigen expression. But the inhibition of telomerase activity in K562 cells by large dose of HHT (more than 40ng/ml) was due to HHT-induced cell apoptosis. These findings are the first report demonstrating the inhibifion of telomerasj~ and induction of differentiation by HHT at Iower dose in leukemia cells.
759.
ANALYSIS OF GENE TRANSDUCTION SYST E M TO HUMAN LEUKEMIA AND HEMATOPOIETIC S T E M C E L L S B Y O N C O R E T R O VIRUS AND LENTIVIRUS VECTORS Y u a n s o n g B A I 1, Yasushi S O D A 1, Kiyoko I Z A W A l, E r i k a S A S A K I 1, Y u k o h N A K A Z A K I l, T s u y o s h i T A N A B E 2, A k i r a T O M O N A R I I, J u n OOI 1, F u m i t a k a N A G A M U R A 1, K a o r u U C H I M A R U 1, T o h r u I S E K I z, Hiroyuki M I Y O S H I 3, A r i n o b u T O J O 1, K e n z a b u r o T A N I 1, S h i g e t a k a A S A N O L 1Div. Mol. T h e r . , Inst. Med. Sci., U n i v . Tokyo, 2Dept. H u m . Genet., U n i v . Michigan, 3Dept. I m m u n o l . , Inst. Bas. Med. Sci. P, Univ. T s u k u b a Many gene transduetion systems have been developed in recent years, however, transduction efŸ to human blood cells are slitl low. Sinee the diffieulty of gene transduetion is ah obstacle to develop gene lherapy targetting hematologieal disorders, we developed a lentivims veetor system and eompared the transduetion effieieney of this system with tha! of oncoretrovirus system. We produeed VSV-pseudotyped HIV [HIV(VSV)] or MLV [MLV(VSV)] veetors by eotransfeetion of the eonstmet plasmids to 293T eells. GFP was used as reporter gene and the transduetion effieiency was assayed by mieroscope and flow eytometry. Gene transduetion effieieneies to primary leukemia eells by HIV(VSV) and MLV(VSV) were 65-80% and 20-30%, respectively. Continuous gene expression were found in leukemia eell lines transduced by HIV(VSV), but not by MLV(VSV). And more than 90% of human CD34+ stem eells eould be transduced by HIV(VSV) and 15% of eolonies derived from the.se eells were positive for GFP. These data suggest that this lentivirus rector system is an exeellent gene transduetion system for human blood cells, ineluding hematopoietie stem eelts, and this system is eonsidered lo become useful tool for elinieal gene lherapy.
761.
C L I N I C A L S T U D Y O N A T R A P L U S As203 I N THE TREATMENT OF REFRACTORY ACUTE PREM-YELOCYTIC LEUKEMIA (APL) J u n M A , Jiwei LIU, Z h a o m i n Z H A N . H a r b i n I n s t i t u t e of H e m a t o l o g y & Oncology, H a r b i n 150010, P.R. C h i n a From June, 1986 to June, 1999, 84 pafients with dmg-resistant mulfi-relapsed APL were treated with ATRA + AhO 3. Their age range was 16-68 years. A specific chromosome translocafion t (15;17) and PMIJRAR a posifive gene were observed in all cases. Sixty of them had relapsed for 3 times or more, and were resistant to various treatments. Therapeufic method: ATRA was given ata dose of 20mg by po twice a day, As203 at a dose of 10mg/day by IV drip. If the pafient had severe leukocytosis, ATRA should decrease to 10mg/day, and As203 to 5mg/day. Platelets should be infused when platelets were less than 20,000. Therapeufic effects and results: ~herapeulic effec~ ofATRA+As~O3in the lrea¨ ofrefractory APL CaseNo. CRtime DFS (day) CR(%) PR(%) CN(%) Death (7 years) 86 38(24-104) 56/65 9/1O 11/i3 1/13 1/13 A ~ ~ and A.s203 have been using to treat APL for nearly years. In our group, 86 APL cases retapsed twice or more were treated with ATRA + As203, and CR + PR was 66%, DSF (5 years) was 53%. Clinical observations showed that this method was one of the most effective therapies for multi-relapsed refractory APL.
256
762.
T E R M I N A L D I F F E R E N T I A T I O N OF HUMAN ACUTE PROMYELOCYTIC L E U K E M I A ( A P L ) CELLS INDUCED B Y T A N S H I N O N E II A IN P R I M A R Y CULTURE
Y o n g L I A N G , W e n X i u S O N G , e t al. D e p a r t m e n t H e m a t o l o g y , T i a n j i n M e d i c a l U n i v e r s i t y Hospital
of
The airo of this study was to investigate whether Tanshinone IIA (Tan IIA)can induce human acute promyelocytic leukemia(APL)cells to differentiate or not in primar), culture. The APL cells from 5 cases were cultured respectively with Tan I[ A at the concentration of 0.5 la g/mi for 7 days in vitro. The differentiations ofthese leukemia cells were observed cytomorphologically and examined by nitroble tetroblue tetrazolium (NBT) test. The cell DNA cycle and membrane cluster differentiation(CD) antigens (CD33, CDI16) were analyzed by flow cytometry. The results showed that 82.5% 4-4.8% of APL cells were induced into morphologically and functionally differentiated cells. The cell growth curve showed that the growth of APL cells was inhibited. The degree of differentiation and growth inhibition induced by Tan [IA was not different from that by ATRA (P)0.05). Flow cytometry analysis showed that Tan ti A arrested APL cells in Go/G~phase and inhibited cellular DNA synthesis. This study demonstrates that Tan II A can induce differentiation of APL cells in vitro, and hence it is worthy of further studies for clinical use.
764.
E X P L O R E OF P O S T R E M I S S I O N T H E R A P Y IN ACUTE MYELOID L E U L E M I A
Y i n g c h a n g MI, Y a n p i n g X U E , S h o u g e n g BIAN, Q i n g x i a n g M E N G , J i a n x i a n g W A N G . Clinical H e m a t o l o g y , I n s t i t u t e of H e m a t o l o g y & Blood D i s e a s e s Hospital, C A M S & P U M C , T i a n j i n 300020, C h i n a In acute myeloid leukemia (AML), intensive postremission treatment is needed for a long disease ffee survival (DFS). However, it is not known how lon,~ the treatment shoule last and how manv courses are necessary. "rhe obie~t of this study is to compare the results of different chemotherapy cvcles m the treatrnent of adult de novo AML. NIethods: 191 cases of unUeated de novo AML (non-M3) were Nven different induction chemctherapy, including HA, DA, mA& and HAD re~fimens (H-HomohardnNonine, D-Daunombicin, A-cvtarabine, m A Amsacrine, M-Mitoxanu:one). Doses of each dru~ were 13 40mg/m-'/d X 3d, H 2.Smg-3m~m'-/d X 7d, A 75-1COmg/m--/q12h X 7d, mA 5070mg/mŸ X 5d and NI 8mv./m:/d X 3d, respecfively. Poslxemission treatment was as follows: Pati~nts were induced by usmg HA, DA and mAA re~mens followed by two cycles of consolidafion treamaent of the same regimens. Then they received intense treatment inc!uding HA, DA, MA andmAA. For patients induced using HAD re~~mens, they received sequenfial and circulated postremission treatment ~at consisted of HA X 2, DAX2, MAX 1, m A A ~ 1. Data of clinical cbservafion were analyzed with SPSS 8.0 for Windows svstem software. Results: Tae complete remissŸ (CR) rate was 81.4%. 89.9% for che to two comes. Median disease free survival (DFS) in 140 CR patients that could be analyzed was 9.6 months. The probabiiirv of survivaI was 21.6% at 3 years, 12.9% at 5 vears. Median DFS w• 7.1 months and the probabilirv of survival was 11.4% at 3 years, 6.3% at 5 years for pafients received fess than 6 cycles of postremission themDv. For patients received 6 of more than 6 cycles of 6ostremission them~'v~ the median DFS was 35.3 months and the prcbability of survival was'43.2% at 3 years, 27.0% at 5 years. There were simaificantdifferences in the ~,/o ~oups. Conc!usions: AML paffents should receive 6 cvc!es-of i:ostremission themov at !east. 8 cyc!es seems more suitabte. It s:a22ested that treatment of A~~[Lshould maintain one year or so. ~-
763.
A P H A S E I CLINICAL TRIAL FOR RECOMB I N A N T HUMAN THROMBOPOIETIN
Y o n g q i a n g Z H A O , Jieling J I A N G , Li J I A O , et al. Division of H e m a t o l o g y , P e k i n g U n i o n Medica1 College Hospital, Beijing, C h i n a To evaluate the human tolerance for and hematopoietic response to rhTPO (Shenyang Sunshine Pharmaceutical Co., Ltd.), a phase I clinical trial was performed. In single dose tolerance investigation, 27 health volunteers were randomized to four groups and received a single subcutaneous injection of rhTPO in a dosage of 0.25 ~ g/kg, 0.5 ~ g/kg, 1 la g/kg or 2 ~ g/kg respectively. In multiple dose tolerance trial, 7 patients with NHL of acute leukemia were enrolled and received rhTPO 1.0 ~ g/kg/d for 7~14days. The body temperature increased to 37.4~ for 2 hours on the day of medication in one volunteer. One person developed mild fatigue. Slight elevation ofALT and AST occured in two persons. Single dose administration ofrhTPO was associated with 24%--52% elevation of platelet counts from baseline in a dose-related manner. No abnormalities were observed in platelet morphology and aggregations after medication. The treatment with rhTPO 1.0 q g/kg/d for 7~14 days was well tolerated by patients with hematological malignancies.
765. A P R E L I M I N A R Y RESULT OF IMVP-16 REGI-
MEN U S E D FOR R E C U R R E N T I N T E R M E D I ATE OR HIGH GRADE NON-HODGKIN LYMPHOMA
Li Z H A N G 1, Y o u j i a n H E 1, K u i f e n g L I U 2. i S u n Yat-Sen U n i v e r s i t y of M e d i c a l Sciences T u m o r Hospital, 2Guang z h o u Railway C e n t r a l Hospital, G u a n g z h o u , C h i n a 19 patients of intermediate or high grade Non-Hodgkin Lymphoma were treated with IMVP-16 regimen which was a non-crossresismnt regimen to CHOR 49 cycles of IMVP-16 (IFO 1-1.5g/M2 IV drip D1-5; VP-16 70m~M2 IV drip D1-5; MTX 40mg2M2 IV D3, D8; reFeat every 4 weeks) were totally used. Mesna and hydration were used concurrently in routine to prevent hemorrhagic cystitis. The result showed total response tate was £ with complete remission (CR) rate of 31%(6/19). The remission time was range from 3-36 weeks, median remission time was 5 weeks. The main toxicity was myelosuppression. The incidence of Neutrogena greater than ~ a d e II was 35%. One case died from infective sepsis. We concluded that IMVP-16 is one of effective reg~.mens treated with Non-Hodgk.in Lymphoma which failed to or re!apsed to standard chemotherapy. Only a few patients can get complete remission (CR) again and the remission time was also limited.
257
766.
ESHAP REGIMEN AS AN EFFECTIVE SALVAGE TREATMENT FOR REFRACTORY/ RECURRED NHL
767.
IVAM THERAPY FOR THE NHL PATIENTS REFRACTORY TO ESHAP SALVAGE REGIMEN
BS K I M , J H SEO, C W CHOI, S W S H I N , Y H K I M , J S KIM. Division of H e m a t o - O n c o l o g y , Korea U n i v e r s i t y M e d i c a l Center, Seoul, K o r e a
BS K I M , J H SEO, C W CHOI, S W S H I N , YH K I M , JS KIM. Division of H e m a t o - O n c o l o g y , K o r e a U n i v e r s i t y M e d i c a l Center, Seoul, K o r e a
Background: ESHAP regimen is known to be active for refractory/recurred NHL. This study was designed to determine the effectiveness and toxieity of this regimen in Korean patients. Method: 42 patients with refracory/relapsed NHL (32 intermediategrade and 10 high-grade) were enrolled in this study. Etoposide and solurnedrol was administered at a dose of 40 mg/m2/day as a 1 hr infusion and 500 mg/day as a 15 min infusion for 1-5 day, respectively. Ara-C 2 g/m2 was given a s a 2 lar infusion on day 5, and cisplatin was given at a dose of 25 mg/m2/day a s a continuos infusion for 1-4 day (repeated every 3 weeks). Clinical efficacy and toxicity were assessed on the basis of the WHO criteria. Results: 9 patients (21.4%) showed CR and 18(42.9%) had PR. The overall response tate was 64.3%. The medial duration of CR was 9(4-21) months. The median survival duration in the responders was 12(8-25+) months. The majority of the responders experienced relpase within 2 years. Median survival for all patients was 7 months. Major toxicity(grade 11I / IV ) was myelosuppression (leukopenia: 64.3%, thrombocytopenia; 50%). 3(7.1%) died due to sepsis associated with neutropenia. Other toxicities were alopecia(95.2%), stomatitis(85.7%), renal toxicity (19.1%), and hepatotoxicity(4.8%). Conclusion: ESHAP is an active and tolerable regimen in Korean patients with relapsed/refractory NHL, with the modest remission duration.
Background: The NHL patients refracory to salvage therapy have a poor prognosis. We designed this study to evaluate the effectiveness and toxicity of IVAM regimen in NHL refractory to ESHAP regimen. Method: 13 patients(11 :intermediate-grade, 2:high-grade) refractory to ESHAP regimen were treated with IVAM regimen(ifosfamide, 1,500 mg/m2 daily for 5 days plus mesna, 150 mg/m2 daily for 3 days, ara-C 100 mg/m2 daily for 3 days, and methotrexate 3 g/m2 on day 5, with leucovorin rescue). Clinical efficacy and toxicity were assessed on the basis of the WHO criteria. Results: 2 patients(15.4%) showed CR and 6(46.2%) had PR. The overall response rate was 61.6%. CR duration was 5 and 9 months, respectively. The median survival duration in the responders was 7(3-12+) months. The majority of the responders experienced relpase within 4 months. Median survival for all patients was 4 months. Major toxicity(grade II / IV ) was myelosuppression (leukopenia: 92.3%, thrombocytopenia; 77%). 2(15.4%) died for sepsis due to neutropenia. Other toxicities were alopecia(100%), stomatitis(92.3%), hepatotoxicity(38.5%), and renal toxicity (31%). Conclusion: IVAM regimen is an active regimen in Korean patients with refractory to ESHAP regimen with modest regimen related toxicities, but the duration of remission is brief and without significant impact on survival.
768.
CLINICAL EVALUATION OF RITUXIMAB (MABTHERA | IN THE TREATMENT OF INTERMEDIA AND HIGH GRADE NON-HODGKIN'S LYMPHOMA
J u n - M i n LI, X i a o - Y i n g Z E N G , Y u C H E N , W e n W U , Y a n g S H E N , Zhi-Ziang S H E N . D e p a r t m e n t of H e m a t o l o g y , S h a n g h a i H e m a t o l o g y I n s t i t u t e , Ruijin H o s p i t a l
Objec~ive: Rituximab (Mabthem@) is a chime¡ monoclonal antibody which tar~ets CD20. Ir has been applied to the treatment of NonHodmkin's~ Lymphema (NHL) successfi:dly, especially in low and inter¡ 2xade. We Fefformed this smdv to evaluate the clinical efficacv and ~ safetv of ¡ sin~e "drug or combined with chemo}herapy, whi91 might provide additional information for the rimximab-intergrated new regimen. Method: 19 intermediate or hig,h 2rade CD20 § B cell NHL patients (2Ce0.6-20C0.8) were eli~Ne for ritª therapy. All the patients were categorized into III to IV grade bv Ann Arbor classification. Among them, two were inidated dia~nosed, ttŸ others were multi-chemoresistant and relapsed patients. Ten ~atients received sin~e rituximab 375m~m-'/w• 4ws, and the other nine padents treated the~same dosage rim,-d¡ Ÿ combined with 1-2 courses of extra CHOP regimen. Resutt: In the nineteen e~~ible patients, 17 cases entered into efficacy evaluafion. Complete remission (CR) was acbAeved in 2 patients, partial remission (PR) in 7 and Nen-remission (NR) in 6 respectivelv. Two initial Ptis.atienrswere defined as disease stable due to the excision o~/the ori~nal ease. Severe toxic effe~ and the hematolo~cal alteNfion were not observed. The episode like asthma ememed i¡ one patient durin2 the inidal imfl~sionof ¡ Conciusion: Rituximab was effective in treatinz CD20* inte=nediate and high ~ade Non-Hodg~in's Lvmphoma without sim-dficant to.-dc effect. It be used as sal,,rage therapy in relaFsed or-chemo-resistant NHJ_ eatlents. Prot:ose: Rim:danab has aitemative mechanism which is nct crossed wkh radiochemotherapy, which m i ~ t provide additionai effect in e~drrdnatien of minimal residual disease (MRD). In this aspect, ir ceuld be inte~ared in the ccnsolidation themev of non-Hedgkin's Lymphcma to ;xnprove the _iong-~e:m survival of ~e~e patients.
769. CLINICAL STUDY ON TREATING APLASTIC ANEMIA WITH SHENGXUE MIXTURE
Y o n g m i n g Z H O U , Z h e n q i a o H U A N G , T a o H U A N G et al. Y u e y a n g Hospital of I n t e g r a t e d T r a d i t i o n a l W e s t e r n Medicine, S h a n g h a i U n i v e r s i t y of T C M ( S h a n g h a i ) 200437 C h i n a To observe the clinical effect of Shengxue Mixture(SXM) on
aplasfic anemia
and smdy the possible mechanism. 94 patients were divided hato two groups randomly: The treatment group 64 cases in Spleen-Kidney yang deficiency and in spler yin deficiency were treated with SXM-A and SXM-B respecfively,30 patients of the control group were treated with Stanozolol.. The results indiciated that basic cure was 18 cases, remission 23,markedly progress 32,not effective ll,total effective rate and cure remission tate were 86.90% and 48.81% in treatment group, Which were obviously better than that of the control group. There was significant difference br
the two
groups p<0.05) and had no obvious side-effect in ~'eatment group. While the patiants' symp~ms were alleviated ,the pe¡
blood cells increased, the ratio of T
lymphoeyte subsets tanded to balance, the level of Natural killers cells aefivity (Nka) increased, InteŸ
(IL2) reduced, and reproducfion of the bone marrow were
markedly improved in most of the patiants treated by SXM. The results suggest that SXM is an effecfive and safe drug for senile Aplastic anemia. Its meehanism might be better due to its regulating the immune flmction, facilitating the recovery of the bone marrow hematopoiesis function..
Key word: Shengxue Mixture: aplastic anemia: TCM therapy.
258
770.
T R E A T M E N T OF PLASTIC A N E M I A IN TYPE OF I N S U F F I C I E N C Y OF K I N D N E Y YANG WITH I N S T A N T P O W D E R OF BU S H E N COMPOUND
Z h o n g c h u a n QIU, Lin ZHAO, Pei C H E N , et al. D e p a r t m e n t of H e m a t o l o g y , S h a n g h a i Hospital, S h a n g h a i U n i v e r s i t y of T C M , S h a n g h a i 200021, C h i n a Objection: To explore the clinical effects with Instant Powder of Bu Shen Co (BSCIP) on aplastic anemia in type of insufficiency Kidney Yang. Method: 30 cases of the treatment one group were treated with BSCIP, 15 patients of the control two group were treated with FU FANG ZAO FAN WAN (FFZFW), 15 patients of the control third group were treated with stanazolol. Bone marrow colony-forming cell (CFC) number of the three groups of cases were measured before and after treatment. Results: The effective rate of BSCIP group (80%) was higher than that of FFIFW group (40%) and Stannazolol group (46.67%), furthermore, the recovery of bone marrow colony-forming cell (CFC) number of the responding patients was more complete in BSCIP group than in FFIFW group and Stanazolol group. Bone marrow CFC number had no sig-nificant change of all nonresponding patients in three groups. Conelusion: The res conse rate of BSCIP was higher than that of FFZFW and Stanazolol, and the recovery of bone marrow CFC number of the responding patients was more complete in BSCIP ~oup.
772.
THE STUDY OF AGGRESSIVE CHEMOTHERA P Y A N D A L T E R E D U P P E R AND LOWER H E M I BODY I R R A D I A T I O N FOR P A T I E N T S W I T H ACUTE L E U K E M I A
M i n g z h e H A N , Jialin W E I , Mei W A N G , Dchui ZOU, Donglin Y A N G , J i a n x i n C H U , Z h o n g r H A N . I n s t i t u t e of H e m a t o l o g y , C h i n e s e A c a d e m y of M e d i c a l Sciences, T i a n jin, China, 3 0 0 0 2 0 Hematopiefic stem cell transplantafion is one of most effective therapies for acute leukemia. But severe infecfion and bleeding ate often caused in the pafients with transplantation because the therapy requires strong preparative regimens to suppress hematopoisis and immunity. To decrease therapeufic related mortality, we treated pafients with acute leukemia by altered upper and lower hemi body irradiation (AHBI). From April 1999 to November 2000, we performed AI-IBI for 11 patients with acute leukemia. The Median age of them was 18 (13-22) years. Male 8 cases, female 2 cases, ALL 8 cases, AML 2 cases. All patients received high dose chemotherapy before AHBI. The interval time between chemotherapy before AHBI. The interval time between chemotherapy and upper body irradiation was 14.5 (13-22) days, interval time between upper and lower body was 20 (12-38) days. Four of 10 patients transfused platelet, 4 cases had wild infections, no pafient died with therapecutic related mortality. One patient relapsed 19 months after altered hemi body irradiadon, other 9 patients are in CCR.
771.
GIANT MEDIASTINUM L Y M P H A D E N O P A T H Y IN HODGKIN'S D I S E A S E
F e n g - J u a n LU. C h i l d r e n ' s Hospital M e d i c a l Center, F u d a n U n i v e r s i t y , S h a n g h a i , P.R. China Objective: to improve the awareness, diagnosis and treatment of giant mediastinum lymphadenopathy in Hodgldn's Disease. Method: Retrospective analysis on 29 cases of Hodgldn's Disease with giant mediastinum lymphadenopathy, In 15 of the cases, the maximal diameter of their mediastimum lymphnode mass was >1 1/3 of the inside diameter of their thoraxic cavity at 5 ~ 6 thoraxic vertebral level ( brief as giant group); the other 14 cases were attributed as non-~ant group. The therapy included MOPP,MOPP/ABVD regime, some of the patients received radiation therapy and superior venae cava syndrome(SVCS) and/or superior mediastinum syndrome (SMS) emerge,chemotherapy or dexamethasone and furoximide was given as soon as the results of pathological diagnosis were available. Results: The percentage of SVCS and/or SMS in the giant group was 33.3%, 7.1% laigher than that in the non-giant group, P<0.05. 33.3% in the giant group showed Wacheal transformaª after the compression and norte in the non-giant group, P<0.05. The mortality after relapse was 26.7% in the giant group and 7% in the non-giant group, P<0.05. Conclusion: Giant group is a progressive type Hodgkin's Disease with high risk, the extension of lesions0H and IV stage in Ann Arbor stage),the size of lymplmode lesion ( extra X in Colwolds conference), the percentage with general symptoms (Group B),the extension of lesion outside lymphnode, and the relapse rate were all higher than those in the non-giant group. Adequate dosage and tension of chemotherapy should be used and emergency treatment should be carried out when SVCS and/or SMS emerge. Key words: Hodgkin's Disease, giant lesion, mediastinttm lymphnode
773.
SECOND IMMUNOGLOBULIN D E F I C I E N C Y IN CHILDREN WITH CANCER T H E R A P Y
K u n Soo LEE, J u n g H w a LIM. D e p a r t m e n t of Pediatrics, K y u n g p o o k N a t i o n a l U n i v e r s i t y Hospital, T a e g u , K O R E A Background: Seeond immunoglobulin(Ig) deficiency is one of the late-effects of cancer therapy. Reports on impaired humoral immunity without spleneetomy were seldom. Methods: Among 85 newly diagnosed childhood acute leukemia, five cases were diagnosed as second Ig deficiency during or after chemotherapy from 1995 to 2000 in KNU Hospital. All Ig concentrations were normal at the diagnosis of leukemia. We examined CBC, T and B cell counts, immunoglobulins IgG, IgA and IgM concentrations, chest X-ray, and bacterial culture in patients with frequent severe infection. The follow-up duration was 31 (20-68) months. Results: In 5 cases, 4 were acute lymphoblastic leukemia and 1 acute nonlymphoblastic leukemia. All were male. Mean age was 5 (4-8) years. IgG, IgA and IgM were deereased in 2 cases, IgG and IgM in i, IgG or IgM alone in 1, respectively. The eoncentration of IgG were 118-367mg/dL, IgA 6.6-41.3mg/dL and IgM 8.6-43.8mg/dL. Among 41 infections, pnemonia was 15, otitis media 9, urinary tract infection 4, sepsis 4, suspicious sepsis 5, infectious diarrhea 3, meningitis 1. The time of diagnosis was 32 (20-44) months affer starting chemotherapy. Four cases were in the course of maintenanee chemotherapy and 1 at 9 months affer the completion of chemotherapy. The maintenace chemotherapeutic agents were vincristine, prednisone, 6-mercaptopurine, 6-thioguanine, methotrexate and cytosine arabinoside. Prophylactic cranial irradiation was done in 3 cases. These patients were treated with intravenous Ig and antibiofics. Two died of acute respiratory distress syndrome and 3 are alive. IgG was normalized affer 26 months in 1. Conclusion: For early diagnosis of second Ig defieiency in children with cancer chemotherapy, not only detailed history and physical examination but serial monitoring of Ig concentrations are important.
259
774.
LEG CANCER L N D U C E D BY HYDROXYUREA: 4 CASES R E P O R T Huang ZILUN. Guangzhou Provincial People's Hospital Case presentation: Case 1:36 years otd female had CNK since 1993. Hu (hydroxyurea) was used and continued. Ulcers occurred in bilateral ankles sized 1.5xl.5cm since Sept. 1999. The ulcers did not heal even after Hu was discontinued. Case 2: 64 years old female had CML since 1996. Hu was used and continued. IFN was used intermittently for 2 years. Recurrent ulcers occurred in bilateral ankles and middle toe of the right foot in Aug.1999. After skin grafting and conservative treatment, the ulcers healed and Hu continued. Case 3:36 years old female had CML since 1989. Hu was used and continued. Refractory ulcers occurred in bilateral ankles sized 6x6cm anda small ulcer in the middle toe of the left foot in 1998. Acute phase transformation of CML occurred in April 1999. Hu was discontinued. Combination chemotherapy started. GM-CSF (300ug + NS .60mi) was topically used twice a day. The ulcers healed affer 40 days treatlnent. Case 4:47 years old female had CML in Nov. 1997. Hu was used and continued. IFN was used for 6 monihs from the beginning. Ulcers occur4ed in bilateral ankles sized 3X3Cln. Hu was changed intQ myleran. GM-CSF was topically used. The ulcers healed after one-month treatment. C0nch, si0n:Hu can cause leg ulcer in CML patients. Hu stopping and GM-CSF using topically can provide cure to the ulcer
776. T H E R A P Y - R E L A T E D
CHRONIC MYELOGENOUS L E U L E M I A A F T E R R A D I O T H E R A P Y AND HIGH-DOSE CHEMOTHERAPY FOLLOWE D BY AUTOLOGOUS STEM CELL TRANSP L A N T A T I O N FOR E W I N G ' S SARCOMA Akihiko NUMATA, Yoshikiyo ITO, Daisuke MATSUOKA, Koji KATO, Kazuya SHIMODA, Yoshinobu ASANO, Hisashi GONDO, Takashi OKAMURA. First Department of Internal Medicine, Kyushu University Shcool of Medicine A 17-yearold Japanesewoman with Ewing'ssarcomawas tleatedwith convenfionalchemotherapyand localradiafiontherapy,high-dose chemotherapywith subsequentautologousperipheralbloodstem cell tmnsplantafion. She achievedcompleteremissionfor Ewing's sarcoma. After 4 years, she was diagnosedas chronicmyelogenousleukemi~ Peripheralblood stem cellsharvestedat time of pfimatytreatmentfor Ewing's sarcomadid not show the BCR/ABLfugiongene by polymerase chainreaction. Thiscase supportsthe observationthat CML may occur asa therapy-mlatedmalignancT and adds to the accumulalion,oftherapyrelated CML developingafferhigh dose chemothempyand autologous stem cell Wansplantation.
775.
CLINICAL E X P E R I E N C E IN THE TREATMENT OF THE CHRONIC IDIOPATHIC THROMBOCYTOPENIA IN CHILDREN WITH ~-INTERDERON Hui-Liang XUE, Yao-Ping WANG, Jing-Yan TANG etc. Hematology Department of Shanghai Children's Medical Center 0 b j e c t s : From October 1998 t o December 1999 11 C h i l d r e n (9 boys, 2 g i r l s ) with c h r o n i c i d i o p a t h i c thrombocytopenia(CITP) were t r e a t e d w i t h o - i n t e r f e r o n . T h e i r mean age was 5 y e a r s o l d ( r a n g e : 2 - 1 0 ) . All t h e s e 11 c h i l d r e n llave b~en t r e a t e d with corticosteroid, highdose u and immunosuppressive d r u g s b u t they d i d not work. The p l a t e l e t count b e f o r e i n t e r f e r o n t r e a t m e n t was l e s s than 20 XI0g/L in 6 c a s e s : 2 0 30 XIOg/L in 3 c a s e s and 50 Xl09/L i n 2 cases. Methods: The dosage o f a - i n t e r f e r o n v a r i e d w i t h the age, 3 m i l l i o n u n i t / o n c e f o r subcutaneous i n j e c t i o m For younger c h i l d r e n , 1-2 m i l l i o n u n i t / o n c e o r 10000 u n i t / k g / o n c e subcutaneous i n j e c t i o n , once a week, l a s t e d f o r 8 weeks. D u r i a g t h e a - i n t e r f e r o n t r e a t m e n t p e r i o d , there i s no o t h e r t h e r a p y i n c l u d i n g c o r t i c o s t e r o i d or o t h e r immunosuppressive drugs. R e s u l t s : I n 4 c h i l d r e n w i t h CITP t h e p l a t e l e t count r e t u r n e d to normal with over I00 XI0g/L. The p l a t e l e t count r a i s e d t o t h e normal r a n g e w i t h a - i n t e r f e r o n treatment, s e v e r a l weeks l a t e r , i t d e c r e a s e d a g a i n , but a l i t t l e improvement in comparison w i t h a - i n t e r f e r o n t r e a t m e n t before. No e f f e c t can be seen in 3 c h i l d r e n , which m e a s the p l a t e l e t count remained the same l e v e l a s o - i n t e r f e r o n treatment before. There ~ere s i g n i f i c a n t changes i n the a n t i b o d y I e v e l compared w i t h t h a t p r i o r to t h e a - i n t e r f e r o n t r e a t m e n t . Among t h e s e 11 c M l d r e n who were good responder, The mean PAIgG l e v e l in plasma i s 1 8 7 ~ 6 . 2 2 n g / 1 0 7 p l t and 6 5 o 4 . 5 2 a g / l O T p l t r e s p e c t i v e l y b e f o r e and a f t e r a - i n t e r f e r o n t r e a t m e n t . But f o r the non-responder, the mean PAIgG l e v e l i n plasma i s 228+-8. 2 0 n g / 1 0 r p l t and 168+--8. l l / 1 0 7 p l t r e s p e c t i v e l y before and a f t e r treatment. Conclusion:We found the PAIgG l e v e l in plasma had s i g n i f i c a n t changes in t h e s e r e s p o n d e r b e f o r e and a f t e r a - i n t e r f e r o n w h i l e t h e r e was no g r e a t changes in responder. The r e a l mechanism of a - i n t e r f e r o n in i n c r e a s i n g the p l a t e l e t count s t i l l r e ~ a i n s to be f u r t h e r e l u c i d a t e d . But from the c l i n i c a l point o f view, the edvantage o f t h i s d n l g i s easy to use and has b e t t e r r e s u l t s , and was well t o l e r a t e d , So i t i s worth o f recommending to use i t f o r the p a t i e n t s with CIIP r e s i s t a n t to the g e n e r a l i z e d regimens. Key ~ords: C h i l d r e n , CITP, a - i n t e r f e r o n
777. IN A P P R O P R I A T E A N T I D I U R E T I C HORMONE
S E C R E T I O N (SIADH) FOLLOWING POST UNR E L A T E D ALLOGENEIC BMT Jie JIN, Xiujin YE, Jie ZHANG, He HUANG, Wenyuan MAI, Maofang LIN. Department of Hematology, First AŸ241 Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, P.R. China MuI~fi~al
leukoencephMopathy
irradiation(TB1)
therapy,
secretion(SIADH) sJmin~~tration secretion
has
We report
ai~r
n]]ogeneic BMT.
a case
patient
He engrai~ed
20 and 25 respectively. On days 51 aŸ disturbance generalized
suddenly
and
an
fluid pressure
urine was 672n~nolF~(normal d~y aud
careful
water
leukoencephRlopathy
rsnge
persisted
neutroph¡
an
mul~~ml HLA-identical
and platelets
presented
ds
on days
with a conscious revealed in the white
was 330 mmHg.O,serology
for CMV aud
to 124 r0mol/J~ The 24 hour Na in
130-260).He was diagnosed with SIADH.
to 42 d~ys. He received Na infusion every
restrictio~
control
with
received
TBI
of inappropriate
demyelinate
EBV were negative. His Na level reduced The episode of SIADH
(SIADH)
BMT,he
hormone
associationwith
electroencephalogram(EEG)
slow waves. A Mili revealed
matter. Cerebrospinal
in
of syndrome
hsd
of total body
antidiuretic
reported
hormone the
complication
inappropriate
been
antidiuretic
leukoencephalopathy unrelated
not
to date.
of
is a recognized but
The
SIADH
resolved
with
mult~f 91191
260
D I G I T A L F I L I N G SYSTEM F O R BLOOD CELL IMAGES D E V E L O P I N G IN TELE-HEMATOLOGY Nobuko SHIMIZU, Tomoko ARAI, Miyuki UJIIE, Midori ISHIBASHI, Takayuki MITSUHASHI, Yohko KAWAI, Kiyoaki WATANABE. Department of Laboratory Medicine, Keio University School of Medicine Tokyo, Japan 778.
Morphological tests, by evaluating pefipheral blood smears and bone marrow smears, ate essenfial for diagnosis in Hematology. Recent technical advances in Tele-medicine appreciate blood ceU images stored in a database system. We have undertaken to invesfigatethe usefulness of blood cell image ffling system, LAFIA, test data ate stored together with images a s a database. This has enabled easy referencing of blood images and bone marrow images of past episodes, and in the case of recurrences, the cytomorphology at the time of f'wst visit can be compared with recent episodes with greater efficiency than possible before. For tele-hernatology, the LAFIA server is connected to two independent networks installed in the hospital, enabling the use of blood cell image database at laboratories, conference rooms, etc., throughout the network. The image quality has been shown to be very satisfactory, and cell characteristics reproducing the microscope view almost exacfly. Thus, the use of blood cell images for research and education as well as case studies, etc., has been encouraged. In addition, because images are stored electronically,unlike specimen slides, there is no possibility of color fading or problems with storage. Telehematology and image consultation within inter-hospital networks is expected to have the most beneficial applicationsin near future, by which, opinions of experts could be more easily obtained in real-time.
KILLER INHIBITORY RECEPTOR (KIR) E X P R E S S I O N ON T CELLS IN GRANULOCYTE COLONY-STIMULATING FACTOR MOBILIZ E D P E R I P H E R A L BLOOD MONONUCLEAR CELLS Li ZHANG 1, Junji T A N A K A x, Yutaka T U T U M P , Kaoru K A H A T A z, Nobuyasu TOYOSHIMA z, Shuichi OHTA 2, Akio MORF, Masahiro ASAKA 2, Masahiro IMAMURA 1 1Hematology and Oncology, ZThird Department of Internal Medicine, Hokkaido University Graduate School of Medicine 780.
Killer cell inhibitory receptors (KIR) for HLA class I (CD94) and NKG2A play ah important role in transducing an inhibitory signal not only for NK cells but also for T cells. In this study, we have investigated that T cell expression of CD94 and NKG2A in granulocyte colonystimulating factor (G-CSF) mobilized PBMC (G-PBMC). CD94/NKG2A expression on CD3+/CD8+ T cells increased after 2 weeks allogeneic stimulation in mixed lymphocyte culture. However, CD94/NKG2A expression on CD3+ /CD8+ T cells decreased in MLC using CD14+ cells depleted G-PBMC. On the other hand, the addition of purified CD14+ cells to CD14+ cells depleted G-PBMC induced the CD94/NKG2A expression on CD3+ / CD8+ T cells as the similar level in unfractionated G-PBMC. However, the effect of purified CD14+ cells for increasing CD94/NKG2A expression on CD3+ / CD8+ T cells was inhibited by the presence of the membrane between responder cells and CD14+ cells. Proliferation of T cells in CD94 endched fraction which was obtained after 2 weeks MLC using magnetic cell sorting was suppressed up to about 90 % in MLC compared with the proliferation of T cells in CD94 depleted fraction. Therefore, CD14 cells in G-PBMC induce CD94/NKG2A expression on T cells and these cells appear to regulate alloresponse and may have some role in the regulation of immunoresponse after allo PBSCT.
779.
AUTOMATIC D E T E R M I N A T I O N OF FRAGMENTED RED CELL RATIO BY XE-2100 AND A P P L I C A T I O N FOR THE P A T I E N T S WITH MICROANGIOPATHY Katsuyasu SAIGO 1, Shunichi KUMAGAI x, Akiko KOBAYASHF, Yoshiyuki KOSAKA 2, Meiyi JIANG a, Isamu KAWASUMP, Takeshi ISHIP, Keiji FUJIMOTO 3 1Blood TransŸ Div, Kobe Univ Hosp, 2Dept of Pediat, Kobe Univ School Med, aSysmex Co. We have established a quantitative method for fragmented red cells (FRC) using en automated hematology analyzer, XE-2100 which implies flowcytometry, and studied about the usefulness of this method to follow-up the patients with hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). FRC can be detected as unusually small red cells in RET channel which distinguishes reticulocytes from mature red cells and platelets. The relationship between FRC% detected by this method and that obtained by manual counting was significant, r=0.857. In 762 healthy volunteers, the reference value was 0.03-0.56%. Five of HUS and one ofTTP patients were followed with automatic FRC%, platelet count, creatinine level, and thrombomodulin. Automatic FRC% was shown to be related to these clinical parameters, indicating the usefulness of this detection method for clinical evaluation.
HLA-DPB1 GENOTYPING BY PCR-FINGERPRINTING TECHNIQUE Ming WU, Lian-Huan LU. Department of Hematology, Shenzhen People's Hospital China 78|.
To accelerate the identificat[on of I-ILA-DPBI matched marrow donors from unrelated population, a very simple I--K,A-DPBI genotyping method called PCR-fingerprinfing (PCRF) was developed according to the theory about homoduplex and heteroduplex formation flora different PCR coding strands and non-coding ones. Unlike PCR-SSPC, strict laboratory condifion is unnecessary in the PCKF. Afier denaturing at 94"C for two minutes and cooling at 37"(2 for eight minutes, the PCR product was separated by 8% PAGE for five hours and ploymorphism band patterns would appear when the gel sta[ning was completed with either EB or silver staining procedure. To confirm its reliabi[ity, 21 individuals from n[ne family whose DPBI genotypes assigned by PCK-RFLP were verified. It was found that there were eight PCR.F patterns corresponding to the nine HLP-DPB1 genotypes from the 21 cases and the same DPB1 genotypes produced identical PCRY pattern except one pair. The factors on efficient separafion of heteroduplexes and homoduplexes were also discussed.
261
782.
ALLOGENEIC BONE MARROW T R A N S P L A N TATION U S I N G R E L A T E D DONORS STIMULATED WITH G-CSF
Hyo Seop A H N , K e o n H e e YOO, Pil Sang JANG, H y o t m g Soo CHOI, Eun Sil P A R K , H e e Young SHIN. D e p a r t m e n t of Pediatrics, Seoul National University College of Medicine, Seoul, Korea Growth factor administration to donors prior to bone marrow(BM) harvesting results in an enrichment of the grafl for myeloid precursors. Eight patients received an HLA-identical sibling, allogeneic BM transplantation using granulocyte colony-stimulating factor(G-CSF)stimulated BM. Stimulation consisted of G-CSF 150 ug/r0"/day s.c for 2 days prior to harvest. Patients were 7 acute leukemias and 1 chronic myelogenous leukemia. Conditioning regimen consisted of busulfan and cyclophosphamide. The GVHD prophylaxis consisted of cyclosporine and prednisolone. Mean number of total nucleated ceUs in the infused BM was 8.8• 10S/kg(range 4.8--11.05• The median day of ANC~ 500/uL and ~ 1,000/uL was 9 and 10, respectively. The median day of platelet count ~20• 103/uL without transfusion was 12. None of the patients developed severe GVHD. One patient relapsed 18 months afler transplantation. The remaining seven patients ate alive and in complete remission 30-610 days post-BMT. Stimulated BM may provide a valuable altemative to allageneic BM and PBSC transplantation. It may also be useful ir donor size is smalL Ideal stimulation regimens need to be investigated.
784.
R E C O N S T I T U T I O N OF T CELL R E C E P T O R REPERTOIRE DIVERSITY IN CHRONIC GRAFT-VERSUS-HOST D I S E A S E P A T I E N T S
Xin DU, Yangqou LI, Wei LIN et al. D e p e r t m e n t of H e m a t o l o g y , G u a n a g d o n g P r o v i n c e P e o p l e ' s Hospital, Guangzhou. 510080 [Abstact]
Objective The intense conditiooing used before hematological
stem cell transplantation for leukernia server two purposes: It has to eradicate residual leukemic eells and it should ablate the host hematopoiesis to prevent graft rejection and provide space for engraftment. This proeedure also eliminates the vast
majority of peripheral T cells .Because the
thymus-dependent regeneration of T cells might be severely affected with age and by conditioning, It is questionable whether a new and complete T-cell repertoire can be regenerated after transplantation. To investigate the reconstitution of T eell receptor repertoire in chronic graŸ disease (cGVHD) after allogeneic hematological stem cell transplantation. Methods
- APCR-basedmethodthatamplificationofallCDR3regionsusing
the T-cell receptor(TCR)V beta genes, was used to examine samples of peripheral blood lymphocytes
in 24 human TCR V fl subfamilies after
allogeneic stem cell transplantation. The TCR repertoire in peripheral blood mononuclear cells from 5 cases of cGVHD patients was examined after PCR amplification of 24 V fl gene subfamilies. Results
Only 2" 8V ,91 subfamily
T cens were found in samples from these patients, and there are different demonstration in different patients. We found V/3 3 T cells proliferated in 4 patients. Conclusion
9 TCR repertoire complexity was abnormal in all
patients, V fl 3 may relate to cGVHD, and the method may be demonstrated the reconstitution of T cell after transplant. Key words - TCR V q gene 9
Immunol reconstitution 9
graft-versus-host disease(cGVHD)
9
RT-PCR
chronic
783.
THE CLONAL E X P A N S I O N OF TCR Vil T CELLS IN P A T I E N T S WITH CML A F T E R D L I
Yangqiu LI, Lijian YANG, Shaohua C H E N . Institute of Hematology, Medical College, Jinan University, Guangzhou 510632, P.R. China Objective T o invesfigate the clonal expansion of TCR VI3 subfamily T cells which were considered as GVL effective cells after donor lymphocytes infª (DLI) in patients with relapse CML after allo-BMT. Methods The CDR3 of TCR VI3 24 subfamily genes were amplified in samples of peripheral blood mononuclear cells at different time points before and after DLI, which were drawn from 2 cases with relapse CML treated by allo-BMT, to observe the usage of TCR V 13repertoire, the PCR products were further labeled with fluorescent and analyzed by genescan technique for the CDR3 size, to evaluating clonality of the detectable TCR V 13 T cells. Results Only 4-11 VIB subfamily T cells could be identified in CML cases before DLI, and 12-21 VI3 subfamily T cells could be detected in samples from CML which display remission afler DLI. Genescan analysis showed that new clonal expansion TCR V 13subfamfly T cells could be found in samples after DLi. Conclusions: The skew distribution of TCR VI3 subfamily T cells could be found on patients with relapse CML after allo-BMT, and this skewing partero may stage to stage to normal pattem during the complete remission. The GVL effect may exert through some clonal expansion TCR VI~ subfamily T cells, during the treatment of iDLI in relapse CML.
785. AUTOLOGOUS BONE MARROW TRANSPLAN-
TATION FOR P H I L A D E L P H I A CHROMOSOMEPOSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA
Shuichi M I Z U T A , Akio K O H N O , Hiroshi SAO, Junki TAKAMATSU, Yoshihisa MORISHITA, Hisao S A K A M A K I , Yasuo M O R I S H I M A , Ryuzo U E D A , Hidehiko S A I T O . Nagoya B M T Group Long-term outcome after autologous bone marrow transplantation was evaluated in 13 patients with Philadelphia Chromosome (Ph)-positive acute lymphoblastic leukemia (ALL). In all patients, bone marrow was harvested after consolidation therapy a n d a cocktail of anlbCD10 monoclonal antibodies plus rabbit complement was used for ex vivo purging. The conditioning regimens consisted of cytosine arabinoside (8g/sqm) and cyclophosphamide (120mg/kg) with fractionated total body irradiation (FTBI) 12Gy in llpatients, LPAM(180mg/sqm) with FTBI in one, LPAM and BU(16mg/kg) in one patient. Eight patients died following BMT, one patient from viral infection and 7 patients from disease recurrence. The median follow-up time for all patients was 31 months. For five living patients in confinuous remission, the median follow up time is 90 months. The 5-year probability of disease free survival is 33.3 % respectively. The results of minimal residual disease study indicated that the number of residual leukemia at marrow harvest was significantly associated with subsequent relapse after BMT. These results indicate that autologous BMT is an effective therapy for Ph-positive ALL, especially for patients with relatively ]ower number of resisdual disease after consolidation therapy.
262
786.
E A R L Y CONTINUOUS I N T E N S I V E CHEMOT H E R A P Y SIGNIFICALY IMPROVED THE LONG-TERM SURVIVAL IN P A T I E N T S WITH ACUTE L E U K E M I A A F T E R AUTOLOGOUS B O N E MARROW T R A N S P L A N T A T I O N
Lugui QIU, M i n g z h e H A N , W e n w e i YAN*, Z h o n g c h a o H A N . I n s t i t u t e of H e m a t o l o g y , C A M S & P U M C , T i a n j i n 300020, C h i n a To explore more effective modality for adult acute lymphoblastic leukemia (A-ALL) and acute myeloid leukemia (AML), a protocol inctuding continuous four courses of non-cross-resistant intensiva chemotherapy followed by autologous bone marrow transptantation (ABMT) at early CR1 was designed. The intensive regimens for A-ALL included high-dose me~otrexate plus L-asparaginase, high-dose cycIophosphamide (CTX) and mitoxantrone with high-dose cymrabine (HAM). The intensive regimens for AML included HAM and MDACE (high-dose cytarabine, dexamehtasone, CTX, etoposide). Most paUents with A-ALL received total body irradiation (TBI) with high-dose melphalan (Mel) as conditioning chemotherapy before ABMT. The condifioning chemotherapy for AML included TBI+CTX and MAC 0ª Met, CTX and Ara-C). 6 patients with A-ALL and 52 patients with AML were ertrolled in this study. 30 A-ALL patients and 45 pauents with AML completed the whole protocol treatment. At a median foIlowup of 40 months (range, 1",-48), the disease-ffee survival (DFS) at 5 years after ABMT were 62.5%--12.5%and 60.5%• respecfively in patients with A-ALL and AML. The 3-5 years relapse rates in A-ALL and AML altar ABMT were 30.8% + 10.5% and 34.5% .'-4-16.5%, respecfively. Comparison with our previous results, the long-term sur~4vaI after ABMT in both of A-ALL and AML were simaificanfly improved. These results were also comparable with the results in pafients with acute leukemia unde~~,ventAllo-BivFf in our pro~am. These results indicated that continuous intensive chemotherapy in early CR could reach better "in vivo pur~Umg"and signkŸ improve the long-te:-m survival in patients with acute leukemia altar ABMT.
788.
AUTOLOGOUS P E R I P H E R A L BLOOD STEM CELL T R A N S P L A N T A T I O N FOR HIGH-RISK OR R E L A P S E D NON-HODGKIN LYMPHOMA
787.
AUTOLOGOUS P E R I P H E R A L BLOOD STEM CELL TRANSPLANTATION FOR ACUTE L E U L E M I A AND SOLID TUMOES-MULTICENTER STUDY
S h u y u n Z H O U , Y u Z H A N G . P B S C T W o r k g r o u p of Guangdong, China Baekground and Objective: Autologous pedpheral blood stem cell transplantation (APBSCT) is a thempy applied widely in the tr~tment of acute leukemia and solid tumotu's.Rer human granulocyte.-colony stimulating factor (rhG-CSF) playa very irnportant role in the currant technique of stem cell transplantation.Based on the PBSCT Workgroup of Cnmngdong,we atlcmpt to evaluate the effectivr and reliability of rhG-.CSF (lenogrostim, 250 micrograms) in APBSCs mobiHzation and hematopoiauc rceonsfitufion atter transplantation. Destgn and Methods: From October 1999 ta October 2000 tw~ty-four patients -15 with acute leukemia(AL),9 with solid tumours - were enroned in the study.The median age was 33 years(14--55). AII parienta were given intensiva chemotherapy with EA (Ara-e 2g/m2/dx3d, Vpl6 0.2---0.3g/m2/d*3d,18/24) of Cy(Cy 2-4g/m 2 ,6/24) regimans followed by rhG-CSF 250 nfierogmms/d subeutaneously to mobilize APBSCs. A rapidly rebounding white blood c•ll (WBC) count from nadir was used to p.rediet the time of peak PBSC releasr and plan leukapheresis. Leukapheresis was performed in all pafiertts by CS3000 plus blood eell separator, when the WBC count was gre..ater than 4.0 x 109/I. Condifiotª regimens were T B I + C y ( T B I 5 G y / d • Cy 6 0 m g / k g / d • 2d,7/24) or T B I + C y + V p I 6 ( V p l 6 500-600mg/ kg /d • Id ,11/24) or MACC(Mel 1 8 0 m g / r o ' I d • ld Ara-c 2 . 0 / m ' / d • Cy 6 0 m g / k g / d X2d CCNU 5 m g / k g / d X Id,5/24).thG..CSF werr given on day +3 altar stem cell infusion unfil neutrophil reeovery.Results: The nadir of WBC count was on day 4 to 15 afler eomplefing intensiva ehcmotherapy, thG..CSF to mobilize PBSCs was administered fora median of 6 days (range, 3-10 days). Twenty-two of 24 patients aehieved the target yield of greater than 2 x 106 CD34+ eells/kg of body ",veight ('BW). This was achieved in a median drena har'('ests (range, 1-2), with a median processed blood volume of 1"2 l/apheresis. A median of 3.9 x 10~ mononnclear eellx/kg BW and 5.7 x 106 CD34+ calla/kg BW for each patient were obtained. CD34+CD38- pereentage was metried in nine of 24 patients ,a medi,'m of 1.46 percent was obtained, rhG-CSF 250 mierogrnms/d subcutaneously was giran on do)' +3 afler PBSC intimida, the median times to aehieve an absoluta neutrophil count of greater than 0.5 x 109/1 wr ten days (range, 7-21 days), rapid and sustained hematopoietic engraflrnent occurred in all parienta. Conelusion: rhG-CSF 0enograstim, 250 microgram.~/d subcntaneously)can be giran in both mobilizing and post transplantation settings without significant adversa effeet.Adequate target yield ofCD34+ cells can be achieved in most eligible acute leukemia and ~lid tumours pafients ,mpid and complete hematopoieite reconstimtion oceurred in all = parienta.
789. R E L A T I O N S H I P
B E T W E E N TIME OF AUTOLOGOUS PBSCT PERFORMED AFTER COMPLETE R E M I S S I O N AND I T S P R O G N O S I S I N P A T I E N T S W I T H ACUTE MYELOID LEUKEMIA
H i r o t o K A N E K O 1, Yuri K I T A 1, Y a s u o O H K A W A R A 1, M a s a f u m i T A N I W A K I 2, Kei K A S H I M A 2, Yoshiaki SONOD A 3 1Dept. of H e m a t o l o g y , A i s e i k a i - y a m a s h i n a Hospital, 2Third D e p t . of M e d . a n d 3Dept. of H y g i e n e , Kyoto Pref. U n i v . of Med.
Yoshiaki M O R I Y A M A , Hiroyuki S A I T O , F u m i n o r i S A T O , S a t o r u Y A M A S I T A . Division of H e m a t o l o g y , T s u b a m e Rosai Hospital for L a b o u r W e l f a r e C o r p o r a t i o n
High dose chemotherapy has recently been safely available with autologous peripheral blood stem cell tmnsplantation (APBSCT).We analyzed treatment outeome of patients underwent APBSCT for non-Hodgkin lymphoma (NHL) classified as high-risk group according to the Internalional Prognostie Index or relapsed. 16 patients were eligible. Age mnged between 22 and 69 (median 55). They consist of 13 with inilially Ireated NHL and 3 relapsed. Histological glade was aggressive in 12 and indolent in 4. 19 transplants were carried out sinee 3 reeeived the second APBSCT for relapse after the first one..6 cycles of CHOP (ME CP for relapse), high dose etoposide for PBSC mobilization, and myeloablative therapy followed by APBSCT were performed. 1.05-21.15x lOS/kg of CFU-GM were transplanted in each patient and hematological reeonstitution was rapidly acquired. Complete remission (CR) was aehieved in 10 of 13 (77%) parienta with inilial presentation and 2 of 6 (33%) in relapse. In CR patients, median disease free survival is 32 months and relapse rete is 8%. All 3 with the seeond APBSCT died of progressive disease or sepsis. We suggest that APBSCT is useful for patients with high-¡ or relapsed NHL,while the thempeulie slrategy for those with relapse after APBSCT remains to be investigated.
The optimal strategy for autologous peripheral blood stem cell transplantation (PBSCT) for patients with acute myeloid leukemia (AML) in first remission (CR)is not well defined. In addition, less is known about the best time of PBSCT to be performed afler CR was achieved in AML. We studied the effect of the time of PBSCT performed after CR on the prognosis of Patients with AML. Out of 22 patients with AML who obtained CR and then their peripheral blood stem cells (CD34 + cells) were harvested under G-CSF stimulation afler 2 courses of consolidation chemotherapy (in vivo purging) , 16 enrolled in PBSCT and 6 in allogeneic PBSCT or BMT. A total of 11 patients (7:CR, 2:2nd CR, and 2:relapse) were treated. Three patients died of relapse before PBSCT, and 2 were droped-out. The median age was 57 years. AII 11 patients received PBSCT according to "AII Japan-PBSCT protocal" : G-CSF primed BUS / VP16 / HDAC regimen. Kaplan-Meier analysis showed that patients auto-transplanted within 10 months from an initial diagnosis can live significantly longer than those over 10 months (P ~ 0.05) . Survival for patients who developed relapse before PBSCT was limited, even though PBSCT was carried out after 2nd CR. There results suggest that timing of PBSCT performed after CR in AML may be important fora long-term engraftment, and that PBSCT should be considered soon after 2 courses of consolidation chemotherapy.
263
790.
DIFFERENTIATION OF DENDRITIC CELLS FROM CD34 + CELLS OF HUMAN CORD BLOOD, BONE MARROW AND CORD BLOOD
Hyo-Seop AHN 1, Sang Hyeok KOH 1, Hee-Young SHIN 1, Kyung-Ha RYU 2, Ju-Young SEOH a. Dept of 1Pediatrics, Seoul National University College of Medicine, Dept of ZPediatrics, 3Microbiology, College of Medicine, Ewha Womans University, Seoul, Korea Dendritic cells (DC) are potent antigen-presenting cells with capacity to stimulate a primary T lymphocyte immune response and therefore of interest for potential immunotherapeutic applications. But the clinical use ofDCs was timited because the biologic characte¡
of DCs were not well known and generation and
isolation of these cells were so diffieult. CD34+ eells were separated from cord blood, mobilized peripherat blood and bone marrow mononuclear eells using MACS and treated with rhGM-CSF, rhlL-4, TNF-6, SCF and flt3 ligand for 14 days. At day 7, non-adherent cells were transferred to the other well and cultured separately. Afler 14 days culture, celts were treated with FITC labeled mouse anti human CDI4, CD83, HLA-DR mAbs and PE labeled anti CDla, CD58, CD86 mAbs and surfaee phenotype was analyzed using FACS. These cells also were evaluated
morphologically with
phase
contrast microscope
and
electronmieroscope. Multiple nonadherent cellular aggregate was visible under the inverted microscope from 10 day of culture The phase contrast microscope showed that cellular aggregates release typical dendritic eells. The percentage of CDS3§
and
CDIa'CD86+CD58+celIs was
increased
according to culture duration, and from adherent cells than nonadherent cetls In conclusion, the functional DCs were differentiated successfully from CD34+cells of cord blood, peripheral blood, and bone marrow, but we couldn't find the differences among these groups.
792. ALLOGENEIC AND AUTOLOGOUS HEMOPOI-
ETIC STEM CELL TRANSPLANTATION FOR REFRACTORY/RECURRED NHL
BS KIM, JH SEO, CW CHOI, SW SHIN, YH KIM, JS KIM. Division of Hemato-Oncology, Korea University Medical Center, Seoul, Korea P u r p o s e : In this study, w e r e p o r t our e x p e r i e n c e s o f a u t o l o g o u s of a l l o g e n e i c s t e m cell t r a n s p l a n t a t i o n ( S C T ) for r e f r a c t o r y / r e l a p s e d N H L patients. M e t h o d : 8 p a t i e n t s r e c e i v e d a u t o l o g o u s S C T and 3 patients r e c e i v e d a l l o g e n e i c SCT. AII o f t h e p a t i e n t s w e r e treated w i t h E S H A P s a l v a g e r e g i m e n . In the p a t i e n t s u n d e r g o n e a u t o l o g o u s SCT, 5 p a t i e n t s w e r e c h e m o - s e n s i t i v e a n d others w e r e c h e m o - r e s i s t a n t . 3 patients undergone allogeneic SCT were chemo-resistant. The c o n d i t i o n i n g r e g i m e n w a s B E A M c h e m o t h e r a p y c o n s i s t e d with c a r m u s t i n e 300 m g / m 2 ( d a y -6), e t o p o s i d e 2 0 0 m g / m 2 ( d a y -5 to -2), a r a - C 2 0 0 m g / m 2 ( t w i c e daily, d a y -5 to -2) and m e l p h a l a n 140 m g / m 2 ( d a y -1). Clinical e f f i c a c y a n d toxicity w e r e a s s e s s e d on the b a s i s o f the W H O c r i t e ¡ Results: All o f the c h e m o - r e s i s t a n t patients w e r e died d u e to disease p r o g r e s s i o n w i t h the m e d i a n d u r a t i o n o f 4 5 ( 1 8 - 1 7 5 ) d a y s a f t e r SCT. A m o n g the 5 c h e m o - s e n s i t i v e patients, 2 e x p e r i e n c e d disease r e l a p s e after 4 a n d 7 m o n t h s o f SCT. T h r e e is in C R status w i t h the m e d i a n duration o f 2 5 ( 1 8 - 3 0 ) m o n t h s . M a j o r n o n - h e m a t o l o g i c toxicities(grade m / i V ) were mucositis (100%), diarrhea(81.8%), and h e p a t o t o x i c i t y ( 3 6 . 3 % ) . R e g i m e n related d e a t h w a s not observed. C o n c l u s i o n : T h e s e d a t a s h o w s that the r e s p o n s i v e n e s s o f s a l v a g e t h e r a p y is the m a i n d e t e r m i n e n t for the success o f S C T using B E A M r e g i m e n in the r e l a p s e d N H L patients, r e g a r d l e s s o f the m o d a l i t i e s o f SCT.
791.
IV BUSULFAN AND CYCLOPHOSPHAMIDE (BuCy) CONDITIONING FOR ALLOGENEIC STEM CELL TRANSPLANTATION (SCT) IN YOUNG CHILDREN WITH ACUTE LEUKEMIA
Jong-Jin SEO, Yoon-Jeong KIM, Hyung-Nam MOON, Thad T. GHIM. Division of Hematology/Oncology/BMT, Department of Pediatrics, Asan Medical Center & Ulsan University School of Medicine Oral Bu and Cy is a well established pretmnsplant conditioning regimen, but the use of oral Bu is limited by the diffieulty of oral administration i n young children and by the liver toxicity following unpredietable intestina I drug absorption. Several studies on IV Bu (Busulfex TM) showed more rel iable pharmaeokinetie parameters than that seen with oral Bu in children. We experienced IV BuCy as eonditioning regimen for SCT in 2 tases of acute pediatric leukemia, and report the tolerability, toxieity and engrattme nt data. Case 1 was diagnosed as mixed lineage infant leukemia with t(4: ll)(q21;q23) at 2 months of age, and case 2, AML(M7) at 28 months of age. Both cases received SCT using IV BuCy as conditioning regimen at second remission. Case 1 received full-matched unrelated SCT at 7 months of age, and case 2, full-matched sibling peripheral SCT at 46 months of age. IV Bu was given at 0.8 mg/kg every 6 hr x 16, Cy at 60 mg/kg daily x 2. Both of them received short-term MTX and Cyclosporine A as GVHD prophylaxis, and G-CSF starting at day +5. The total nueleated cell doses/kg were 8.4 xl0 s and 16.6x10 s, and CD34+ cell doses/kg were 12.2x106 and 15.5x10 £ respeetively. Engraftment to >500 neutrophil/p.L was at day +13 and +16, and >20,000 platelet/gL was at day +30 and +39 respectively. Early treat ment-related toxieitias were acceptable; grade 11 mucositis and hepatotoxicit y in both cases, mild VOD in case t whicb resolved, and there was no CNS toxicity. The follow-up periods are 8 and 9 months posttransplant re spectively. The chemerism status documented by STR-PCR reveals complet e chimerism for case 1 and mixed chimerism for case 2 until now. IV Bu was well tolerated, and could avoid the difficulty of oral administration i n younger children, with moderate toxicity. Ir should be considered as repl acement for oral busulfan in conditioning regimen prior to SCT in younge r children with leukemia.
793.
SUCCESSFUL TREATMENT OF A CHILD WITH RELAPSED AML WITH HIGH-DOSE CHEMOTHETAPY & ALLO-BMT Xiaotian XIE. Department of PediatrŸ Tongi Hospital, P.R. China We report the result of allogeneie bone marrow transplantation (alIo-BMT) for a ehild with relapsed acute myeloid leukemia (AML). A boy (8 years old ) with AML (M4) was administed to our hospital for relapsed afler got CRI for 8 months. Afler a reioduelion protocol (DAE) with DNR, Ara-C and VPI6, the hoy acbieved CR2, and foIIowed by two eourses of high-dose Ara-C based protoeols (DNR + HDARAC) as consolidate therapy, then alIo---BMT was given. Bone marrow stem cells were from his younger brother (4 years old). The HLA-matehed sibling donor rer
G-CSF
(Lenograstim, Chugai Phannaceutieal Co., Ltd., Japan) 5ug/kg/day for 5 days as for bone marrow mobilization. 1.5 X q
ofMNC and 3.45 X 106/kg of CD34+ eells
were •olleeted fiero bone mm'row. The eonditioning protoeols consisted of CTX 60mg/kg for 2 days, Ara-C 1000mg/m2 for t day (+dl) and VP16 600mg/mz for I day (+ d2) plus 800 eGY (600 eGY for lunes shielding) total body irradiation (TBI). Lenograstim 10ug/kg/day was used for promoting graI~. Cyclosporin A (CSA) and MTX were regularly used for GVHD prophylaxis. The patient's peripberal WBC reached to 0.Sx109/L at the da)' 16 (+dl6) afler BMT and the bone marrow was recovered at the day 40. lOGVHD was found at day 25 witb skin rash and disappeared by CSA therapy. Now the boy has reaehed CCR for more than 3 years after alIo-BMT. Conelasions: AIIo-BMT is very effective for ehild relapsed leukemia. Lenograstim (CHYGAI, JAPAN) is ver), useful for bone marrow mobilization and grafl promotion.
264
794.
ALLOGENEIC TRANSPLANTATION OFPURIFIED HAPLOIDENTICAL STANDARD DOSAGE CD34+ PERIPHERAL BLOOD STEM CELLS WITH SUITABLET CELL REINFUSION TO TREAT PH +ALL H u i L I A N G l, Y u n W A N G z, R U O B I N G G U O 1, Z h i y i n g C H E N G 1, H u i l i a n g X U E z, L o n g j u n G U z 1Dept. of H e m a . , X i n h u a H o s p . Affiliated to S h a n g h a i S e c o n d M e d i c a l U n i v . , 2Dept. o f H e m a , S h a n g h a i C h i l d r e n ' s M e d i c a l C e n t e r S h a n ghai, C h i n a Objeefive: We uscd standard dosage of CD34+ ceUs with suitable T cells reinfusion method to avoid severe GVHD and HVG for one case of HLA l~t:› PBSCT from the mother to the son (ALL Ph+) .Pafients and : Recipient: male, 9 years vld, ALL-L2,CR2 Chromosome study: 47xy, +1, Ph+, t(9;22). PCR analysis: bcr/abl (+). The second remission lamed 6 months up to transplantation. DOnor: mother of the recipient, 30 years old. Type of HLA for the donor and rec~ent: one site of HLA-B mismarched, other fivesites matched. Erythrocyte blood type: identical for the recipient and the donor. Mobilized with G-(~F for 5 days. Two leukapheresis p._~_uctswere selected to CD34+ cells using only one CD34 anfibo~, kit(Cl]niMACS). The pu¡ sample, 5xlO"/kg CD34+ cetls and 5.3 xlO'/kg CD3+ cells, were inf~sed to tlae.p~ent immediately ; The next day,Sml tmmanipulatextPBSC wea'e infused wmca contained lxl0~/kg CD34+ cells and lxlO'/kg CD3+ cells. The p~~.tt received 6x 106 C-~D34+cetls/kg and t.05x 107 C~3+ cell/kg in total , clifi~omj~regimen: . B ~ a n 4.mg/k~,/d for 4 days; CTX 50mg/kg/d for 2 E s . uvl-tt~ prophylaxis: ATG (rabbit, Germauy) 10mg/d, on days -7, -6 and -5 _ ore transplantafion; CyclosL~rinA from the day -1^on. MTX was not used. Remlts: ~NBC > 1.0• IO~/Laad ANC > 0.5 X IO~/Lwere on day +12. Hb > 70g/L, on day +16. The platelet > 20X 109/L on day +13 and> 50X 109/L on day +35. Bonr marrow examhaationon day +15,+30, +60, +90, +140 and +180 showed consistent remission. Grade II super-acu~ GVHD took place on day +9 ,Treated with methyl-prednisolone i .O.mg/J.x~..d, the diarrhea stopped on the third day of treamaent and the skin rash diminished. On day +55, the skin rash completely disappeatcd. DNA analysis of STR site: converted to the donor type on aay +30, and still maintained on day +180. Chromosome examination: converted from 46xy to chimera 46xy/46xx on day 30 and to 46xx on day +180. ~ showed that Ph+ t (9;.22) and Ix:r/abl fusion gene disappeared. Conclusion: standa(d, dosage of CD34+ cell plus suilable amount of CD3+ T cells .tya~lantation. can conquer the di~culty of I-ILA partial ~ a t c h e d , espedally m~ naplomentacaltransohntation.
796.
WHO-QOL IN PATIENTS WITH HEMATOLOGICAL MALIGNANCIES: BEFORE AND AFTER ALLOGENEIC BONE MARROW TRANSPLANTATION Akira HIRAOKA, Hideki MITUI, Takahiro KARASUNO, Toyoko OKABE, Ayako SOH, Emiko WAKA, Eturo MIYAOKA, Miyako TAZAKI. Osaka Medical Center Ÿ Cancer and Cardiovascular Diseases, Osaka. Science University of Tokyo, Tokyo, Japan
The purpose of this study was to measure the impact of allogeneic BMT on QOL in patients with hematological malignancies who received a transplant at our institution from April 1997 to November 1999. Using the Japanese version of WHOQOL-BREF, their QOL were measured for three times; before (n=48/49 consecutive patients;a), three months after (b), and within 1-2 years afler the transplant (n=40/40 relapse-free survivors;c), and compared with QOL obtained from 50 healthy donors (d) who donated their marrows during the same period. QOL scores of (b) were statistically lower in Overall, as well as in each of physical, social, and environmental domains than those of (c) and (d) by Tukey HSD test. However, there were no significant differences between (c) and (d). These results indicate that the impact of the transplant was big enough to drop patients* QOL, but within 1-2 years affer BMT the), could recover at the same level as healthy polulation. As WHOQOL was developed for the use of intemational comparison, we would like to conduct a cooperative study with other Asian countries in near future.
795.
ALLOGENEIC PERIPHERAL BLOOD STEM CELL TRANSPLANTATION FOR A PATIENT WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA Liangling HU, Hu CHEN, Min JIANG. The Hemotopoietic S t e m Cell T r a n s p l a n t a t i o n C e n t e r , Bei T a i p i n g R o a d H o s p o t a l , B e i j i n g 100039, C H I N A To observe the efficacy of allogeneic peripherat blood stem cell transplanting (PBSCT) in the treatment of paroxysmal nocturnal hemoglobinuria (PNH), HLA compatible donor peripheral blood mobilized by G-CSF wasinfused after conditioning regiment with CTX and ATG.The infused mononuclear cells (MNC) and CD34 + cells were 5.98 X q and CD34+5.11 • 106/kg, respectively. Hematopoietic reconstruction occurred on day 13, Engraftment was Completely documented on day 30 by DNA fingerprinting. PNH colons disappeared, The patient has been disease-free survival for 30 months post-transplant without acute and chronic graft-versus-host disease (GVHD). This is the first repon oftreatment ofPNH with PBSCT in China, indicating that allogeneic PBSCT is the effective method oftreatment of PNH.
797.
INCREASED CONCENTRATIONS OF SERUM HEPATOCYTE GROWTH FACTOR IN ACUTE GRAFT-VERSUS-HOST DISEASE Takahiro OKAMOTO, Hiroyuki TAKATSUKA, Yoshihiro FUJIMORI, Hiroshi WADA, Tsuyoshi IWASAKI, EIZO K a k i s h i t a . S e c o n d d e p a r t m e n t of I n t e r n a l M e d i c i n e , H y o g o College of M e d i c i n e Hepatocyte growth factor (HGF) is known to have mitogenic, motogenic and morphogenic effects on various cells and also has antiapototic activity. HGF was reported to be effective in preventing acute graft-versus-host disease (GVHD) in a mufine model. We examined semm HGF concen¨ in 38 patients receiving aUogeneic bone marrow la'ansplants, and investigated the relationship o f serum HGF concentrations to severity o f acute GVHD. More HGF was present in sera from patients with than without acute GVHD. Serum HGF correlated significanfly with grade o f acute GVHD. Futhermore, serum HGF COiTelatedwith semm concentrafions o f C-reactive protein, T glutamyltranspeptidase (GTP), and asparate aminotransferase (AST). Serum concen~'ations o f HGF in transplanted patients without GVHD were consistently low, while those in patients with acute GVHD increased with exacerbation. We concluded that HGF was produced during induction o f the GVH reaction, and would tend to protect tissue from injury caused by inflammatory cytokines.
265
798.
D E T E C T I O N OF WT1 GENE E X P R E S S I O N AS A P O S S I B L E M A R K E R FOR MINIMAL R E S I D UAL D I S E A S E (MRD) IN CHILDHOOD LEUKEMIA Hyoung Soo CHOI l, Sang Hyeok KOH 2, Hee Young SHIN 2, Hyo Seop AHN 2. Dept. of Pediatrics, 1Kangwon National Unir. College of Medicine, Chunchon, ZSeoul National Univ. College of Medicine, Seoul, Korea T h i s study ~
l:~forrlaed to determine the ex~ession levd of the WTI
gene by a s e m i ~ t a t i v e metl~ mad to invmfigatethe signifir~m~of the W'F1 gene in childhood lethkemia. Monom~ear calla were isolated fixan bene marrow ast• (BIv0 and/cr immpher~ blood cells
gene. Tbese results show that WTI gev,e exlm~,~n is frequentiy noted in ehildhood hmke~aiaand might be a tme~ and sensitivepanleukemic for MRD monitoring, to evaluate remission status and predict early relapse in childhood leukm~a ata molecular levd.
800.
TWO-HUNDRED CASES OF A L L O G E N E I C H E M A T O P O I E T I C STEM CELL T R A N S P L A N TATION IN C H I L D R E N : SINGLE C E N T E R STUDY Hack-Ki KIM, Bin CHO, Nak-Gyun CHUNG, Soh-Yeon KIM, Dong-Wook KIM, Chun-Choo KIM. Catholic Hematopoietic Stem Cell Transplantation Center, The Catholic University of Korea, Seoul, Korea We a n a l ~ d 200 csses of allogeneic hematopoietie stem eell tmnsplantatioc in childmn. Two hundred children underwent allogeneie hamatopoietic stem eell transplantation between Nov. 1983 and Sep. 2000 in Catholic Hematopoietie Stem CeI] Transplantafion Cantar of Korea. Out of 146 tases of HLA-matched sibling transplantation, 50 childrr wr trarmplant~i for ~vem aplastie anemia (SAA), 33 for acute lymphoblestic leukemia (AL[.), 45 for aeute myelogenous leukemia (AML), 11 for chronic myelogenous leukemiatmyelodysplastic syndrome (CML/MDS) and 7 for nonmalignant r a ~ disr162 Therr were 78 males and 68 females with a median age of 10 years and median follow-up of 47 months. The 5y ~ r estimated event-frr survival (EFS) of SAA was 96 %. The 5-year estimatr EFS of ALL in CRI (18 cases), CR2 (14 caos) and refractory state (1 coses) were 83 %, 7 1 % and 0 %, mspeetively. The 5-ye~r estimated EFS of AML in CR1 (40 coses) and CR2 (5 casas) were 74 % and 60 %, respeetively. The 5-year estimated EFS of CML/MDS was 73 %. The 5-year estimated EFS of nonmalignant rara disea~ was 86 %. Twenty-six children underwent unrelated bone mmrrow transplantafion (UBMT), 15 underwent eord blood tmmplantation,(CBT) and 13 underwent haploidentical familial transplantntion (HVr). There were 35 males and 19 f~males with a median age of 6.5 years mad median follow-up of 18.5 months. The astimated 2-yem" EI~ of UBMT, CBT and HFT were 56 %, 58 % aed 34 %, rt~pectively. ~ data ~ w s that allol~neic kematopoietic stem cell trnnsplantation is a eumfive ~ for ehildmn with hematopoietic stem eell disordevs and we wish to share these resulta.
799.
UNRELATED DONOR BONE MARROW TRANSPLANTATION (UDBMT) IN 102 P A T I E N T S WITH H E M A T O L O F I C A L MALIGNANCIES: RESULTS FROM A SINGLE INSTITUTION Hiroshi SAO ~, Hiroatsu IIDA 1, Satoko MAEDA 1, Tatsuya ADACHP, Yasuo MORISHIMA z. 1Department of Hematology, Meitetsu Hospital. 2Department of Hematology and Chemotherapy, Aichi Cancer Center, Nagoya We performed a retrospeetive review of 102 patients with hematological malignancies at our institutinn, who underwent a UD BMT from HLA-A,B and DR serologically matched donor. The diagnoses were AML (N=26), ALL (N=28), CML (N=31), MDS (N=ll), ML (N=5) and MM (N=I). The median age wos 29 (range 10-54). 9, 8 and 23 patients were mismatched at HLA-A, B, and DRBI by molecular typing, respectively. GVHD prophylaxis consisted ofCSP + MTX in 33 patients, FK-506 + MTX in 57 patients and T-cell depletion + CSP + MTX in 12 patients, respeetively. 2 patients failed to obtain engraflment, both were transplamed from lILA mismatched donors. More severe acute GVHD were secn in patients transplanted ffom HLA mismatched donors of in patients who were treated with CSP + MTX for GVHD prophylaxis. The probability of disease-ffee survival for standard risk (AML, ALL /CR1 and CML/CP) and high risk patients was 65% and 30%, respectively. 8 of 10 patients in whom grada I]I-IV sr acute GVHD developed died. The main causes of death were severe GVHD and TMA in standard risk patients and RRT, lP and relapse of leukemia in high risk patients We coneluded that better control of acute GVHD and other early stage complications such as TMA, RRT and lP ara clearly important to improving the outcome ofUD BMT.
801.
T R E A T M E N T OF V E R Y HIGH-RISK LYMPHOID MALIGNANCIES W I T H ALLOGENEIC BONE MARROW T R A N S P L A N T A T I O N FROM U N R E L A T E D DENOR Takashi ASHIDA, Yoshiyasu SUMIMOTO, Kazunobu KAWANISHI, Junichi MIYATAKE, Mitsuhiro MATSUDA, Yoichi TATSUMI, Yasuhiro MAEDA, Akihisa KANAMARU Third Department of Internal Medicine, Kinki University School of Medicine Philadelphia chromosome (Ph) -posotive acute lymphoblastic leukemia (ALL) and adull T cell leukemia (ATL) are hardly treated with conventional chemotherapy to achieve a tong-term remission. We report three cases with such high-risk lymphoid malignancy treated with allogeneic bone marrow transplantafion (BMT) from HLA-matched unrelated donors. Preparative regimen consisted of busulfan + cyclophosphamide (CY) + total body irradiafion (TBI) for Ph+-ALL, and CY + TBI for ATL. Short-term methotrexate + cyclospofinA were usedas GVHD prophylaxis. A 32-year old female anda 44-year old male with Ph+-ALL in non-CR state were successfully treated with BMT. A 50-year old male with ATL who could not obtaln CR was also treated with BMT. We will discuss the efficacy of BMT with those preparative regimen and GVHD prophylaxis for very high-risk lymphoid malignancies.
266
802.
UMBILICAL CORD BLOOD TRANSPLANTATION FROM UNRELATED DONORS (UCBT); A REPORT FROM KINKI CORD BLOOD BANK
S h t m r o K A I , Hiroshi H A R A , Keisei K A W A . Kinki Cord Blood B a n k We p r e s e n t clinical results of UCBT reported to Kinki C o r d Blood Bank from 17 transplant centers. T h i r t y - f i v e patients with non-malignancies(9) and h e m a t o l o g i c malignancies(26;8 were transplanted in CRI of CR2 and the others in a d v a n c e d stage) received 37times of UCBTs. The m e d i a n age and body weight at transplant were 5years (range;0.3-2l) and 18kg(6-65), respectively. Six pts had p r e v i o u s l y received ah autologous or allogeneic blood or m a r r o w transplants. The d e g r e e of HLA m a t c h i n g was 4/6 in 14 pts,5/6 in 20 pts and 6/6 in 3 pts. The m e d i a n infused cell dose was 3.8 x 10E7/kg(range;l.54-14.2).Results. Neutrophil r e c o v e r y at day60 was 82% and p l a t e l e t recovery at day 100 was 58%. Two pts died early(dayl3 and 16) and 7 did not engraft. The incidence of A G V H D (>=grade II)was 37% and that of >=grade III was 7%. Chronic G V H D was observed in 5 out of 24 evaluable p t s . T w o y e a r EFS was 53% in n o n - m a l i g n a n c i e s and 26% in m a l i g n a n c i e s ( 1 4 % for pts t r a n s p l a n t e d in advanced ssage vs 49% for those in CRI/CR2; p < 0 . 0 5 ) . I n conclusion, despite a high level of HLA disparity, ah incidence of severe AGVHD and CGVHD was low, and these results suggest that UCBT is ah optional source of s t e m cells for patients lacking a m a t c h e d donor, e s p e c i a l l y in CRI or CR2.
804.
CORD
BLOOD
TRANSPLANTATION
803.
COLLECTION, PROCESSING AND TESTING
OF PLACENTAL CORD BLOOD UNITS IN B E I J I N G C O R D B L O O D BANK K. Y. LIU, W. C. DONG, Y. L. W A N G , Y. J. J I A N G , D. LI, D. P. LU. P e k i n g U n i v e r s i t y P e o p l e ' s H o s p i t a l a n d I n s t i t u t e of H e m a t o l o g y , Beijing 100044, China
Placental cord blood (PCB) has been shown to successfully reconstitute haematopoiesis following allogeneic transplantation for a variety of diseases in children and more recently in an increasingly number of adult patients. Banks of CPB cryopreserved after HLAtesting are required in order to provide various HLA-types for unrelated transplantation. Between July 1997 and July 2000, 4008 placental cord blood (PCB) units were stored in Beijing Cord Blood Bank. All of PCB units had been HLA-typed with molecular (SSP/SSO) and serological method. PCB units were collected by trained midwives while the placentas remained in utero. The mean volume of collected ffom 4008 PCB units was 934-22 ml (mean_.+ SD; range45-198ml). The mean total nucleated ceU counts~per unit was 1 1 . 2 _ 5.32 (rang4.46-45.1). Collected PCB units were temporarity stored ant room temperature and processed with in 18 hours after collection. Depletion of red blood cells was performed using hydroxythyl starch. Afler depletion of red blood cells, the mean nucleated cell count was 9.374- 4.59(rang-3.12-30.5) with mean nucleated cell recovery of 79%. PCB units were cryopreserved with DMSO and hydroxythyl starch. 40 PCB units were thawed for testing, typan blue viability of nucleated cell was 88.2%. The recoveries of CFU-GM and CD34 § cells (ProCOUNT) were 97.6%, 86.4% respectively. A search of PCB units in 109 registry of patients showed that HLA matched or mismatched at 1, 2 HLA loci were 7.33%, 35.78%, 57.21%, respecfively.
FROM
U N R E L A T E D B O N E MARROW TRANSPLAN(UBMT) FOR EBV-ASSOCIATED T/NK-LYMPHOPROLIFE RATIVE DISEASES (T/NK-LPD) Takayuki OKAMURA, Masahiro YASUI, Tomoko K I S H I M O T O , M a s a m i I N O U E , Naoki S A K A T A , Keiko YAGI, Keisei K A W A . D e p a r t m e n t of P e d i a t r i c s , Osaka Medical C e n t e r a n d R e s e a r c h I n s t i t u t e for M a t e r n a l a n d Child H e a l t h
This report describes the results obtained from 40 pediatric patients with leukemia and E)S who recieved cord blood transplantation(CBT) from ltlA-mismatched unrelated donors of [anagawa Cord blood Bank(KCBB).In particular, the lILA disparity and disease-related factors that have affected event-free survival, the incidence of acute GVI~I)have been analysed. Patients: This retrospective study included 21 children with Al,L, 15 with hJIL and one each with CML,Ill)S and J]iliL. Tbose subjected to CBT during the f i r s t or second complete remission(CR) and IIDS without blasts were assigned to the standard risk(SR) group(n=16). Patients in third or subsequent remission, relapse or partial remission with refractory leukemia were considered to be in high-risk(tlR) group(n=ll). J~IL and the 2nd CR a f t e r relapse(n=8),or BII failure a f t e r BIT(n=3) were included in the }IR group. Results:Kaplan-meier estimates for neutropbil and platelet recovery were 83. 7-+12.25 at day 60 and 55. 4-+16.65 at day 10O respectively. The incidence of gardes ll-ff acute GV~I)vas 58. 5-+16.85. Three-year event-free survival rate vas 49. 2-+16.65. From multivariate analysis, the most important factor influencing EFS vas disease status at CBT: standard risk p a t i e n t s h a d a 3-year EFS of 74. 0-+21.65, compared vith 29. 6-+20.6~ for those with high risk disease(P=0. 013, Ih~ 4. 746, 95~CI1.382-16. 293). Conclusion:These data confir~ that t]LA-mis~atched unrelated CBT is a feasible procedure to cure a significant proportion of children vith leukemia, especially if conducted in a favorable phase of the disease.
EBV-associated T/NK-LPD consists of several disorders such as chronic active EBV infection (CAEBV), EBV-AHS, hydroa vacciniforme (HV), and NK lymphoma/leukemia. It is known to be refractory and fatal disease in many instantes. Thus lar, we have treated four such patients with unrelated BMT.
H L A - M I S M A T C H E D U N R E L A T E D DONORS IN C H I L D R E N W I T H H E M A T O L O G I C A L MALIGNANCIES: A REPORT FROM K A N A G A W A CORD BLOOD BANK IN J A P A N H. N I S H I H I R A , K. O H N U M A , K. I S O Y A M A , K. I K U T A . Dept. of P e d i a t r , S h o w a U n i v e r s i t y F u j i g a o k a Hospital. Div of Clin I m m u n o l , I n s t of M e d i c a l Sci U n i v of Tokyo. Yokoh a m a City U n i v School of M e d .
805.
TATION
Age/Sex
diagnosis/ Clinical
M.I. 18/F EBV-AHS (CR at BMT)
course preeonditionin TB[+VP+CY outcome
alive(16m+) eGVHD
K.N. Y.I. 15/M 7/F CAEBV, Vasculardisease, CAEBV, NK-LPD, NK-LPD NK lymphoma TBI+VP+LPAM TBI+VP+CY dead(14d) infection, renal alive(7m+) failure
S.M. 17/M CAEBV,HV, NK-LPD T8I+VP+CY alive(7m+)
Three out of 4 patients are in CR and EBV-genome was not detected by PCR methods after BMT. We conclude that stem cell transplantation including UBMT should be considered in this disorder.
267
806.
D E T E R M I N A T I O N OF CORD BLOOD HEMATOPOIETIC S T E M C E L L
Lijun CHEN, Jiwei LIU, ChunŸ JIANG, Zhaomin ZHAN, J u n M A . H a r b i n I n s t i t u t e oŸ H e m a t o l o g y & O n c o l o g y , H a r bin First Hospital, Harbtil 150010, P.R. China
OF PBSC COLLECTION I N THE RYUKYU ISLANDS. - - A BLOOD BANK'S IMPLICATION I N S T E M C E L L T R A N S P L A N T - -
807.
SUMMARY
Tsuyoshi MIYAKUNI, Tomio UEZU, Sumiko TAIRA The Japanese Red Cross Okinawa Blood Center, OKINAWA
The numbers of mononuclear cell (MNC), hematopoieticprogenitor cell,
BackgroundzThe
and CD34+ cells were measurcd. Method: the cord blood was collectcd from 20 normal newborn fetuscswith 20 minutcs, and prcserved at room tclnperature.After M N C separation by 60% B K L gravity centrifugafion, thcy wcrc culturcd and measured for CFU-GM, BFU-E, and CFU-Meg
Center
Red Cross(JRC)
Japanese
embarked on the PBSC collection
on Sep. 1994 as well We are
as therapeutic apheresis
responsible
proces sing,
not
only
collected
blood.
method was used for LTC-IC. Results: M N C numbcr was 1.2l + 1.57 (0.0l-lS)• CFU-GM, 1210+2260 (70-8600)/mi; BFU-E, 768+
Method~z
Peripheral
were
1363 (36-4150)/nfl;CFU-Meg, 64+413 (0-1014)/ml; CD34+ cell, 1.4+ 4.2%o; LTC-IC numbcr, 410+21/mi. Discussion:Hematopoietic stem cclls
Spectra, and CS-3000. Those non-program freezing. Resultsz
werc nch in cord blood,but werc not enough for CBSCT. The combination of EPO, IL-6, G M - C S F and T P O were used for LTC-IC. The
Age-'0.5-68
(Mz43,
A 35-year-old malo with ehronic myelogcnous leukemia m monocytic blast crisis sucr rcceived a pcriphetal blood stvm cell transplantation (PBSCT). The donor was his 65-yr mother who was identical with ou~ patient at the HLA-A,B and HLA-DRBI,DRB3/B4 1or On MLR testing, the donor's cells showed no response, but the paaent's cclls showed a low response to the dmaor's cells (relative rcspomc ~ 0.22). Ba.~linc cytogcnetlcs revealed oomplex aberrant karyotype inoluding t(9;22) m 100% metaphases. Bone marrow analysis shows madr hypereeUularity and blast crisis phase of CML. Conditioning rcgimens included CTX and TBI. GVHD prophylax~ was CyA and short r MTX PBSC were coUected using G-CSF mobilL~ationin the dose of 10mcg/kg and the infi.tsext CD34+ dose was 2.03 • 106/kg The patient showed rapid hemopoietic engraftment and attended a complete ~ytogenetie response. He only developed mild graft-versus-~Ÿ disease; ( 1 grade GVHD) which were successfully treated with prednisone I m~rkg per day. The pafient is currenfly 12 months post-transplant with hematologlcal and cytogenefic remission and has a normal daily social life [ Key words l penpheral blood stem ceU transplantation, Leukerma, myeloid, chronic blast crisis
ailogeneic
Solid
tumor
of
activities.
(PMCs)
harvest
solution
used
(mean 2.4
28 cases.
In
Diseases
and now widely
Ryukyu.
In addition,
allo-PBSCT
covered
by national
medica1
Because
of the
frequent to often
by COBE
alIo-PBSCT,
of the pts were
implemented
was albo insurance
request operate
as an even
in
ad~nitted
to be
on April
2000.
of stem cell even
times).
malignancy
PBSCT was considered
technique
have
our
Hematological
44 times.
Disr
1980s.
but for
were frozen at -80~ by As for auto-PBSCT, 69 pts
efficient
our ataffs
ALLOGENEIC PERIPHERAL BLOOD STEM CELL TRANSPLANTATION FOR CHRONIC MYELOGENOUS LEUKEMIA IN BLASTIC PHASE USING A PHENOTYPICALLY IDENTICAL FONOR Weihun FU, Hian HOU, Dongxing WANG, Zhenggang YUAN, Qiusheng CHEN. Department of H e m a t o l o g y , ChangZheng Hospital, ShangHai, P.R. China
1 ).
after
166 times
27).
were harvested
H.M. (except
808.
cells
just
Blood
distribution
mononuclear
(median
41 cases,
18 donors
and here
were harvested y-o
in early collection
We sua~arized
for CD34
F~26)
(H.M.)
hematopoietic stem cellswere much increased.
for
cryopreservation
colony. And CD34+ cells were mcasured by flowcytometry. Dexter
measured
Okinawa
program in Okinawa
collection,
on Sunday.
809.
ALLOGENEIC PERIPHERAL STEM CELL TRANSPLANTATION FOR PURE RED CELL APLASIA--A CASE REPORT Sheng-Fung LIN, Rs-Bin TZNG, Yu-Chieh SU, Ta-Chih LIU, Tyen-Po CHEN. Division of Hematology-Oncology, Department of I n t e r n a l M e d i c i n e , K a o h s i u n g M e d i c a l University, Kaohsiung, Taiwan PRCA is a rare hematologieal disease charar by marrow erythroid aplasia. Many tases of aequired PRCA may respond to convantional immunosuppressive therapies, but drug dependent ate noted in majority of cases. To date, stem r transplantation has been used for congenital PRCA (Diamond-Blackfan anemia), but not r for aequired PRCA. Herein we successfully treated a case o f idiopathic PRCA with allogeneie peripheral stem eell transplantation and reported it. A 37-year-old malr suffered from progressive dyspnea for 2-3 momhs. Then, he was admitted due to one episode o f syncope. At that time CBC showed Hb:4.9gm/dl,WBC:4330/ul,Pl:293000/ul with normal differentiation of white cells. Bone marrow aspiration and biopsy revealed marked hypoplasia (10-20% of cellularity) and no megakaryocytes was found through the whole examinational fir Other laboratory data inr chest X-ray showed nothing particular, so acquired idiopathir PRCA was diagnosed. Prednisolone was prescribed with good response but anemia recurred when prednisolone was tapered even combination with other immunosuppressive drug. PB stem cell transplant was pr 21 months later with one HLA-identieal older sister. Conditioning was as for sr aplastic anemia with TBI 300 rad and cyelophosphamide 4 x 50 mg/kg. GVHD prophylaxis was Cyclosporine and MTX. ANC was > 500/ul on D8. Complete donor typ• engraftm•nt at D32. CBC showed all within normal range after 6 months post PBSCT. From this case, stem cell transplantation should be r162 for patients with idiopathic acquired PRCA when an HLA-identical donor was available.
268
810.
N O N - M Y E L O A B L A T I V E STEM CELL TRANSP L A N T A T I O N WITH F L U D A R A B I N E B A S E D P R E P A R A T O R Y R E G I M E N IN ADVANCED
HEMATOLOGIC MALIGNANCIES Kouzou MASUDA, Katsuji SHINAGAWA, Kazuma IKEDA, Kinuyo KANEDA, Takayuki FUJIWARA, Hiromichi DANSAKO, Kensuke KOJIMA, Fumihiko ISHIMARU, Mine HARADA. Department of Internal Medicine II, Okayama University, Okayama, Japan Introduction We performed phase I / [ I s t u d y of non-myeloablative stem cell transplantation(NMSCT) for advanced hematologic maligmmcies to evaluate the feasibility and graft versus leukemia(GVL) effect. P a t i e n t s and M#tho• 7 pts (3 pts with refractory NHL:~ttuse large, follicular, mantle cell, 2 pts with reŸ AML:M4&M5, ref ALL:Phl and RAEB) were treated by NMSCT between Sep.2000 ~ad Jan.2001. The preparatory regimen consisted of CY 30mg/kg ftom day - 7 to - 6 and fludarabine 25mg/m 2 from day - 5 to - I ( F C regimen) was applied for 2 ref NHL, ref AML(M4) and RAEB. Along with FC regimen, BUS 4mg/kg f o r 2 days were added in reŸALL and rituximab in reŸNHL(m~atle cell). FLAGIda(fludarabine, ara-C, Idarubicin, G-CSF) and A T G was applied for reŸAML(MS). GVHD prophylaxis were CSP and sMTX. 5 pts received G-CSF mobilized PBSC al]ografts from HLA-identical siblings and 2 pts from 1 or 2 locus mismŸ related donors. 3 pts received donor lymphocyte infusion(DLl) aŸ NMSCT.
R#~ults Transplanted CD34+ cells was 3.5(1.8-5.1)* 106/kg and hematotogic recovery was ANC>500//LI in day13(ll-18) and PLT>20,000/q in day17(8-24). Engrattment was confirmed by VNTR analysis in borla lymphocytes(CD3+) and other nuclear oell fractions . 2 pts tL*'veloped Grademacute-GVHD. Pts with M4 and A L L achieved hematologic remission, NHL(mant]e cell) and RAEB were in stable disease, NHl.(~ffuse large) and M5 were in progressive £ and remaining NHL(follicular) died of acute GVHD. Conr Our preliminary data suggests:(1)NMSCT with Fiudarabine based preparatoty regimen was feasible and initial engraitment was successful. (2)To evaluate GVL effects for advanced disease, further investigation was needed using carefully defined inclusion criteria.
812.
CYTOGENETIC O B S E R V A T I O N ON THE INFUS I O N OF DONOR P E R I P H E R A L BLOOD M O N O N U C L E A R CELL TO TREAT R E L A P S E
A F T E R TRANSPLANTATION FOR CML Xiaoli LIU, Shuyun ZHOU, Lanlin SONG. Department 0f Hemat010gy, Nanfang Hospital, The First Military Medical University To i n ~ the ~I1~~/ of Inlma0n of aonor im~hanl W0od
mononuclmr ceU (MNC) to treat relapse altar ellogemeic bone marrow tflmt~lafltatJofl (BMT) for chronie myeloid leukernis (CML). Two pe'dente~ CML who aro male and donors me female rdal~mr slter BMT and received the Infu=ion of donor MNC wem eemmlnsd by three Idnds of Cytogen~lo me(hod: i n t e r l ~ e Fk~H, chromosome analysls of pedpheral btoo4 (BP) wltll PIdA and bone rnsrrow ~ (BM) wlthout PHA. One paUent after non-myeloablatJve allogmleic hem=topoletic atem r tmnslfl=nt=tion. Condi'dorting reghllen were Fludarablne (50mg.r (Smgkg'~.d'~x4), CTX(60mg-kg'~.d'lx2). At day +30 Cytogenetic show that 60% of i ~ nucleus wem X'Y and 40% were XX by FISIl;, but karyotypes were 46, XX [8] by bandlng cf BP with PHA (T cell). And 5% of I ~ wem 46, XY by bandlng of BM. At day +66 the patlent relal~md. Metaphase cytogenatics conflrmed pedpheral biood Ind bone marrow cell were 46, X~, t (9; 22) =11.At d,,y *80,§ the I~ti~t were tmated by Infu=donof dono~ perlpheml blood MNC (0.5x 10=.kg'~,lOx107.kg-~).Following rnanlfested GVHD and plncytopenia, the patient re-enter mmi=r,ion at de'/+210. Parieras bone marrow (~tls dlsl~ay 100% of chromosomes XX by FISH. The enothe~ re]epsed after BMT with oondiUoning mgimen "rBI+CYX (TBI 10GY, CTX 60rng,l~'l.r 2).Kan/otype of BM cells result46. ~ [4)'46, XY, t (S;22X2] at day +40, 46, X Y, t(9;22)[f10] at da]/+lg0. The patient were treated by infusion of donor MNC(1.0X 107.1~t,2.0 x 107.kg4) the P~ient obtalned major oq rmnisdon by FiSil and lasted for 10 months with GVHD. For patients with CML w1~orelapse a#er allogenetc BMT, donor MNC Infuslons can induce s pot=nt gm/t-versur,-leukernia reactJon and rtHmtabhh ~~nplete cytogefl#dc =nd molecular rernissions in the majodty of pariente It b posslble to manlpulate the donor MNC product in en attemld to enhance the effl~cy of GVL Induction, whlle llrnlting toxidty from marrow aplasia, GVi..ID, and other complication. Cytogenetic eocarnlnation induding: interphase FISH, banding analysis of pedphm'al blood and bone marmw cells provide a useful tool to understand the detalled mechanisms of GVL and GVHD.
811.
MINI-ALLOGRAFT FOR REFRACTORY OR POOR-RISK NON-HODGKIN'S LYMPHOMA USING Flu/Mel REGIMEN
Toshio KITAWAKI, Masakatsu HISHIZAWA, Momoko NISHIKORI, Norimitsu KADOWAKI, Tatsuo ICHINOHE, Takayuki ISHIKAWA, Hitoshi OHNO, Takashi UCHIYAMA Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University We have conducted allogeneic peripheral blood stern cell mmsplantation with non-myeloablative conditioning regimen for two patients with non-Hodgldn's lymphoma. Both cases were either refractory or poor-risk. One case was a 63-year-old ma.le with refractory lgG-~-type lymphoplasmacytoid lymphoma with abundant M-protein in serum. He had never achieved CR with several previous conventional chemotherapies. He was not eligible for eonventional myeloablative transplantation because of older age. The other case was a 38-year-old female with stage l NK/T cell lymphoma involving nasopharyngeal region, indueed to CR with local radiotherapy. She was considered to have poor prognosis with higher risk of relapse, and allogeneic transplantafion was supposed to reduce the incidence of relapse with the GVL effect. Conditioning regimen consisted of fludarabine 25mg/m2/day for 5 days and melphalan 70mg/m2/day for 2 days. FK506 and short-term MTX was administered for prophylaxis of acute GVHD. Peripheral blood stem cell gratis were harvested by G-CSF administration from HLA-matched sibling donors. Successful engraflment was achieved in all patients with 100% donor-type hematopoiesis by day 30, proved by microsatellite chimerism assay. The regimen was well-tolerated and no grade III-IV n0n-hemat01ogical t0xicity 0ccurred. In this series 0fpatients, no grade III-IV acute GVHD 0oeurred. In one patient with lymph0plasmaeyt0id lymph0ma, remarkable GVL effeet was 0bserved. C0nr Mini-all0gra~ with fludarabine and melphalan c0nditioning regimen is effeetive and sale f0r p00r-risk 0r rcfractory n0n-H0dgkin's lymph0ma.
813.
EFFECT OF INDUCED GVHD IN L E U K E M I A R E L A P S I N G P A T I E N T S A F T E R ALLO GENEIC BONE MARROW TRANSPLANTATION: SINGLE-CENTER E X P E R I E N C E OF 33 ADULT PATIENTS
Chang-Ki MIN, Ki-Seong EOM, Dong Wook KIM, Jong Wook LEE, Woo Sung MIN, Chun Choo KIM. The Catholic Hemopoietic Stem Cell Transplantation Center, College ol Medicine, The Catholic University of Korea, Seoul, Korea In a retrospr h~t
$ingle
diseose
antation (15
acute
immune
hat consequently SC)
(n=lS),
A)
by
(n=3),
patients
and
had
speetively. Ilowed
CsA
aeutr
showed
was
r achieving
Overall 33.9%
Eight
CI:
12 due
20-52~
who
leukemia.
Twelve
(57.6%) and
resultr had
both
4
patients
in a higher acute
We eonelude
that
GVHD aeute
died.
Sevcn
By
~ grade GVHD
foll
died
by acute
di
we~
Kaplan-Meier remain
(median
patients
(21.
patients
responses
2 ALL
months
lo
hema
Seven
Two the
re
acute
complete
their
(Cs
of 33
( n = l I, s i
or CML.
using
5 patients
t t
9 patients,
ehronic
of GVHD.
multivariate
probability
(78.9%)
including
3-93
(PBL)
GVHD;
novo
including
(n=7)
toxicities;
infeetions.
six
and
and
cells (PB
of cyclosporin
I 1, 3 a n d
at 3 years
patients
~mission
stem
chronic
immunotherapy
estimated
to t~atment-related
by infections,
oecurrenee
patients
ive without
of patients,
blood
leukemias
unde~
lmmunotherapies
lymphocytes
de
a remission
of the oecurrence
of starting
or eytogenetie
reaction,
Twenty
and
mye[oid
(OVL)
eessation
IV in 3,
for acute
15 ehronic
center,
developed (n=4)
obtained
in spite
(95%
Nineteen and
followed
GVHD
survival
patients
transpl
leukemia
BMT
blood
(n=2).
graft-vcr•us-
marrow with
allogeneic
peripheral
abrupt
IFN-a
respeetively
2 months
by
peripheral
chronic
patients
(n=5)
rr
e progression ronic
within
hematological
y GVHD
(72.7~ remission,
at our
1. II. II1. a n d
limited
and
6) after
OVHD
(n=3),
with
g~de
(n=3),
no remi$sion
GVHD
evaluable.
method,
6).
Twenty-four
patienrs
mplete
(n=2),
eombined
bonr
grat~-versus-leukemia
alone
induced
patients
leukemia,
followed
PBL
of
allogr
33 adult
(median
chronic
tmnsplant
ovr
ehronic
of cytogenetic
ed of aeute not
to induce
Eightr162 (64.3*/o) of 28 evaluab]r
x extensive). 2%)
GVHD;
after
2000.
lymphocytic
of: chemothr
withdrawal
the outcome
patients May
months
and/or
(IFN)-C~ (n~5),
by extensive
tologieal
1-36
acute
non-myeloablative
interferon
cxamined
and
3 acutr
manipulations
eomprised
wr
relapsing 1992
or relapsing
developed
o elicit OVHD owed
study March
leukemia,
persisting
various
r
in leukemia
Betwer
myelogr
leukemia) ent
(GVHD)
(BMT).
due
in co 12) aftr
to discas
GVHD,
3 b
Cox
analysis,
only
of disease-free
survival
(/~0.02
1 and
chronic
is a s s o e i a t e d
(3VHD
with
were
considerable
eh
all al tox
269
814.
RELATIONSHIP BETWEEN INVERSIONS OF FACTOR VlII GENE AND FACTOR VIII INHIBITOR DEVELOPMENT IN THE JAPANESE PATIENTS WITH SEVERE HEMOPHILIA A Kazuyoshi F U K U D A , Hiroyuki N A K A , Shogo M O R I T I K A , M a s a r u S H I B A T A , Ichiro T A N A K A , Midori S H I M A , A k i r a Y O S H I O K A . D e p a r t m e n t of Pediatrics, N a r a Medical University In about 40% of severe hemophilia A patients, inversion exists due to recombination between int22h-1(intron 22 hemologous region 1) in intron 22 of the FVIII gene and 2 other extragenic copies (int22h-2 or 22h-3) Iocated 500kb upstream from int2•h-1. We discuss that relationship between inversions of FVlII gene and inhibitor development in the Japanese patients with severe hemophilia A. We tested blood samples from total100 patients with severe hemophilla A whose FVIII activity and antigen were all less than 1%. Twenty patients out of them (20%) developed inhibitor. Genomic DNA of each patient was digested with Bcl I followed by Southern blot analysis using p482.6 (0.9kb EcoRI/Sac I fragment ) asa probe. We found inversions in 42 (42%)of the 100 patients.36 and 6 patients showed distal inversion pattern (type I) and proximal inversion pattern (type II) respectively. The incidence of inhibitor in the patients with inversion was 16.7%(7/42). AII cases with inhibitor were type I. In the patients without inversions, the incidence of inhibitor was 22.4% (13/58) The frequency of inversion in the Japanese hemophiliacs is equal to that in the USA and European coutries. There was no relationship between the incidence of inhibitor and the presence of inversions in severe hemophilia A.
816.
ANALYSES OF GENETIC ABNORMALITIES IN T Y P E I CD36 D E F I C I E N C Y I N J A P A N H. K A S H I W A G I , Y. T O M I Y A M A , T. KIYOI, S. T A D O K O R O , S. H O N D A , S. K O S U G I , H. K A T O , Y. K U R A T A , Y. M A T S U Z A W A . Internal M e d i c i n e and Molecular Science, G r a d u a t e School of Medicine, Osaka University To elucidate genetic abnormalities in type I CD36 defieiency, we analyzed 28 Japanese subjects, whose platelets and monocytes/maerophages lack CD36 on their sufface. We have already reported three mutalions that ale responsible for CD36 deficiency; a subslitulion of T for C at nucleotide (nt) 478 of CD36 cDNA in codon 90, a dinucleolide (AC) delelion at nt 539 in codon 110, anda single nucleotide (A) insertion at nt 1159 in codon 317. In this study we identified two novel mutations that ale responsible for CD36 deficiency. One was a delelion/insertion mutation; three base pairs (bps), GAG, were deleted at nucleofide (nt) 839-841 and 5 bps, AAAAC, were inserted at the same position (S39GAG'*AAAAC). The mutafion was located in exon 7 of the CD36 gene. S39GAG--*AAAACleads to frameshift and appeatance of a premature stop codon, sagGAG--" AAAAC was also accompanied with marked reduction in the amount of CD36 mRNA. The other was a 12 bps delefion at nt 1438-1449 (1438A-1449Tdd), and we deteeted two types of CD36 tuRNA by RTPCR; one had only 143SA-1449T,ld and the other had 143SA-1449T,td anda delelion of nt 959-1028, corresponding to exon 9. Anelysis of the probund's CD36 gene revealed that 143gA-1449Tddwas located in exon 13 and there was no mutafion in the exon/inlron boundaries of exon 9. ~43SA-~449Tdd leads to inframe 4 amino acids delelion, and exon 9 skipping leads to frameshift a_ad appearanee of premature stop codon. Expression assay revealed that each of 1438A-1449Tdd and exon 9 skipping directly caused impairment of surface expression of CD36. Survey of the 5 known mutations in 28 type I CD36 deficient subjects demonstrated that more than 50% of their alleles hada substitution of T for C at nt 478 and about 90% of them had one of these mutation.
815.
ROLE OF Fe RECEPTOR y-CHAIN IN PLATELET GLYCOPROTEIN Ib-MEDEATED SIGNALING W u YI, Yutaka Y A T O M I , Yukio OZAKI. D e p a r t m e n t of Clinical and Laboratory Medicine, Yamanashi Medical University, Yamanashi The glycoprot•in Ib/IX (GPIb/IX) compl•x on the platelet membrane plays an important tole in thrombus formation, especially under the condition where high shear stress or collagen/von Willebrand factor (vWF) interacfion occurs. The binding between vWF and GPIb stimulates tyrosine kinases and subsequent tyrosine phosphorylation events in platelets, which eomprise the important signal transduction pathway. We have found that the combination of vWF and botrocetin, by interacting with GPIb, induced tyrosine phosphorylation of Fe receptor y-chain (FcR 7-chain), Syk, linker for activation of T cells (LAT), and phospholipase C ,q (PLCy2), which are the signaling molecules involved in GPVI-, a collagen receptor, mediated platelet activation. In spite of tyrosine phosphorylation of PLC,q however, there was no intracellular calcium release and inositol 1,4,5-trisphosphate production, which is distinct from collagen stimulation. FcR ,/-chain was found to be constitutively associated with GPIb. Src and Lyn formed a complex with FcR 7chain and Syk upon GPIb stimulation. Aggregation and tyrosine phosphorylation of Syk, LAT and PLC72 in response to vWF plus botrocetin were significantly suppressed in FcR ?-chain deficient platelets. These results suggest that FcR ,/-chain and its downstream signals are required for the full activation of platelets mediated by vWF-GPIb interaction.
8|7.
SYNDECAN-4 DEFICIENCY INCREASES SUSCEPTIBILITIES TO T H R O M B O S I S I N T H E FETAL VESSELS OF THE PLACENTA, KAPPA-CARRAGEENAN-INDUCED RENAL DAMAGE AND LIPOPOLYSACCHARIDE-INDUCED LETHALITY Kazuhiro ISHIGURO, Tetsuhito KOJIMA, Kazuo K U S U G A M I , Kenji K A D O M A T S U , Takashi M U R A M A T S U , Hidehiko S A I T O . First D e p a r t m e n t of Internal Medicine, D e p a r t m e n t of Medical T e c h n o l o g y and Department of Biochemistry, Nagoya U n i v e r s i t y School of Medicine and School of Health Sciences Syndecan-4 is a type I heparan sulfate proteoglycan, belonging t o the syndecan family. Syndecan-4 is expressed in many tissues and binds to various factors such as midldne, basic fibroblast growth factor and tissue factor pathway inhibitor. Thus, syndecan-4 is supposed to be involved in diverse biological functions. To investigate its biological roles, we generated syndecan-4-deficient mice by gene targeting. Until now, we have demonstrated that syndecan-4 deficiency impairs focal adhesion formation of fibroblasts only under restricted conditions and fetal vessels in placental labyrinth presumably through thrombosis and that syndecan-4 deficiency increases susceptibility to kappacarrageenan, which induces degeneration of renal inner medulla and obstructive nephropathy. Furthermore, we have demonstrated that syndecan-4-deficient mice are more susceptible to lipopolysaccharide, which induces endotoxin shock and disseminated intravascular coagulation, than wild-type mice. It is possible that in the human syndecan-4 also play critical roles under certain conditions such as procoagulant stress, and endotoxemia, and the syndecan-4-deficient mouse is a good model to study the mode of action of this molecule.
270
LOW-DOSE LIPO PGE1 (Eglandin | IS MORE EFFECTIVE THAN ORAL ANTIOXIDANTS FOR THE PREVENTION OF HEPATIC VENOOCCLUSSIVE DISEASE AFTER HEMATOPOIETIC STEM CELL TRANSPLANTATION IN CHILDREN WITH HEMATOLOGIC MALIGNANCIES Bin CHO, N a k - G y u n C H U N G , S o h - Y e o n KIM, D o n g - W o o k KIM, Chun-Choo KIM, Hack-Ki KIM. Catholic H e m a t o p o i e t i c S t e m Cell T r a n s p l a n t a t i o n Center, T h e Catholic U n i v e r s i t y of Korea, Seoul, K o r e a
RECOMBINANT HUMAN SOLUBLE THROMBOMODULIN (ART-123); NOVEL ANTICOAGULANT SPECIFICALLY SUPPRESSING CLOT EXTENSION M i t s u n o b u M O H R I . I n s t i t u t e for Life S c i e n c e R e s e a r c h , A s a h i Kasei C o r p o r a t i o n
Lipo PGEI (EglandinTM, WeIFide Korea Co., Ltd, Korea) was used to prevent hepatie veno-occlusive disease (VOD) afler hematopoietic stem cell transplantation (HSCT) in children with hr malignanciesin our center from Nov. 1999 to Jun. 2000. We compared the effect of Lipo PGEv for the prevention of VOD with that of vitamin F_Jglutathione(antioxidant group, from Apr. 1998 to Jun 1999). Patients were prepared with va¡ vonditioning regimens according to the disease states and types of transplant. For the
thrombin generation in human plasma by activafing protein C a n d by subsequent
818.
prevention of VOD, twentypatientsreceivedLipo PGEt from one day prior to the start of eonditioning to day 30 aRer stem cell transplantation in continuous intravenous infusion ata dosr of 1 pg/kg/day(0.042 iag/kg/hr).Forty-onepaticnts received vitamin E with of without glutatione. Patients received 400 IU (25
kg) oral vitamin E once daily. Oral glutathionewas given 100 mg/day (<25 kg) or 150 mg/day(>25 kg), divided in two of three doses. Twentyfour out of 41 patients Rceived vitamia E alone sinee one day before the conditioning umil day 7 (n-9) of until day 14 (n-17). Remaining 17 out of 41
paUents received vitamin E and glutathione until day 28. According to the McDonald cfil~ria, 4 ola of 43 patients (9 %) werc diagnosed as V O D in antioxidant group, and no cese o f V O D oucnrred in Lipo PGE~ group. Two casca
of dr
VOD (day 34 and day 35) octurred ,in antioxidant group. In Lipo
819.
ART-123 is a soluble form of recombinant human thrombomodulin comprising all extracellular domalns of thrombomodulin. Bound to thrombin, ART-123 inhibits its procoagulant aefivity and promotes aefivafion of protein C. The antieoagulant effects of ART-123 were examined in vitro and in vivo experiments. In vitro, ART-123 attenuated inaefivafion of factor Va and prothrombinase. ART-I23 and acfivated protein C suppressed the level of thrombin without affecting start of thrombin burst, while thrombin inhibitor and low molecular weight heparin delayed the thrombin burst with little affecting thrombin level. ART-123 aŸ
inifiafion of clot formation. A higher concenlrafion of ART-123 was needed to affect clotfing time and platelet aggregation need higher concenlrafion than thrombin generation. These data suggest that ART-123 is a potent anticoagulant with less affecting haemostatsis.
antieoagulaats. The plasma half-life of ART-123 is long, especially on subcutaneous injection. ART-123 may be a promising anticoagulant for the Ireatment for the prophylaxis of thrombotic disorder.
820.
821.
DIAGNOSIS AND THERAPY OF ACQUIRED VON WILLEBRAND SYNDROME H i r o s h i M O H R I . D e p a r t m e n t of I n t e r n a l Medicine, Ise-Keio Hospital, Keio U n i v e r s i t y P u r p o s e ; We evahate the laboratory features, possible pathogenctic mechanisms, and therapeutic strategies for AvWS. M e t h o d s ; A retrospecª analysis of patients with AvWS according to review of the fiterature (1968-2000). R e s u l t s ; AvWS was associated with lympho-proliferative or mydoproliferative disorders, cardiovascular diseases, neoplasia, immunologic disorders and other diseases. AvWS is characterized mairdy by mucosal bleeding, vWF:RCo of vWF:CBA ate usually abnormal, while FVIII:C can be sometimes normal. Multimeric analysis showed selective loss of large multimers or normal pattern with decrease of vWF:Ag. Anti-FVIII/vWF activities, most of which recognized A l domaln of vWF, have been found in patients with immunological abnormalities. A close relaUonship was found between the inhibitor and bleeding tendency. Bleeding episodes were managed malnly by treatment of undeflying diseases and/or DDAVP. In some patients, FVIII/vWF concentrates and plasmapheresis might be effective. Intravenous immunoglobulin therapy (0.3g/kg, 3 days) was effective to correcta hemostatic defect in patients with inhibitors. C o n c l u s i o n ; AvWS is more frequent than previously reported and should be suspected with appropriate tests in patients with underlying diseases. The patients with inhibitors ate likely to have bleeding problems and might be resistant to DDAVP therapy. Intravenous immunoglobulin infusions should be reserved to correct vWF levels in these patients.
Intravenous adminis~ation ofART-123 is effective in animal
models of DIC, AV-shunt and others with a wider safety margin than conventional
PGEI group onc ~ of dr V O D on post-U'an.splant day 58 occurrcd. There was no toxicity attfibute to antioxidants or Lipo PGE~, We concluded that Lipo
PGEI is more effective than oral antioxidants (vitamin F_Jglutathion) for the prevention of VOD aRer hematopoietic stern cell transplantation in ehildren with hematoSogic malignancies.
the extension of the clot by
inhibiting further thrombin generation on elots, while other anticoagulants inhibited the
CLINICAL SIGNIFICANCE OF HEMOSTATIC MOLECULAR MARKERS IN ACUTE LEUKEMIA X-Y ZENG, F W U , X - F W A N G , X-Y Z H E N B QI, J C H E N G , Y S H E N , H-L W A N G , Z-X S H E N . S h a n g h a i Institute of H e m a t o l o g y , Ruijin Hospital, S h a n g h a i Second Medical U n i v e r s i t y , S h a n g h a i , China Abstrat In order to study the changes of hemostatic molecular markers in acute leukemia for elucidate its clinical significance, a se¡ of hemostatic molecular markers were measured among acute leukemia , which includingTF, TFPI, TAT, PAP, u-PA. u-PARo The plasma level ofTF, TAT, u-PA and u-PAR was elevated significantly at diagnosis in AL, while TFPI, PAP only in AML. Afler treatment, TF and TAT remained high level in AML. u-PA u-PAR were still abnormal except in patients who obtained complete remission .PAP, u-PA was remarkable elevated in patient with severe hemorrhage. Results indicated that their existed hemostatic abnormal as well as hypertibrinolysis ,which can be varied with different type and can be ameliorated with clinical improvement .The measure of TF. TAT, PAP may provide useful information for the diagnosis of DIC; u-PA, u-PAR may be considered asa useful marker for prognosis. Patient with sever hemorrhage should be treated with antifibrinolysis drugs. Great attention should be paid to prevention hypercoagulability in patient with AML afier chemotherapy treatment. Key words acute leukemia; hemostatsis and coagulation; Fibrinolysis
271
822.
VITAMIN K DEFICIENCY ASSOCIATED WlTH INTRACRANIAL BLEEDING DUE TO CYROME GALOVIRUS HEPATITIS IN A BREAST-FED INFANT DESPITE THE ORAL PROPHYLACTIC ADMINISTRATION Kouichi Y O S H I D A , Seiki K A M I S U E , Ichiro T A N A K A , Midori S H I M A , Yukihiro T A K A H A S H I , Akira Y O S H I 0 KA. D e p a r t m e n t of Pediatrics, Nara Medical U n i v e r s i t y Wr report h e r e a case of intracranial bleeding in a 54-day-old breast-fed baby with vitamin K defieieney, in spite of the prophylactic administrafion of three oral doses of vitamin K2. He was anemie and exhibited hyperbilirubinemia and elevation of serum transaroin**es and total bile aeid. Level of IgM anfibody to cytomegalovirus(CMV) was inereased. CMV anfigen (C7-HRP) was detected in bis peripheral leukocytes and CMV DNA was isolated from bis urine and the breast milk of his mother. These results suggested that his eholestatie liver dise,ase resulted from CMV infection, and interfered with the gasu'ointestinal absorpfion of the fat-soluble vitamin K. The breast milk of bis mother also eontained lower levels of vitamin K. The prophylaetie administration of oral vitamin K ~ recommended in Japan did not prevent hemorrhagie eomplieations in this case.The findings demonstrated that it may be important to use alternative protocol to alleviate vitamin K deficiency in neonates with eholestatic liver disease.
824.
E V A L U A T I O N O F G L O B A L T E S T F O R T H E DIAGNOSIS OF DIC A y a k o N O D A , Hideo W A D A , T s u t o m u NOBORI. D e p t of Clin Lab, Mie U n i v e r s i t y School of Medicine, T s u Evaluation of the diagnostic criteria of disseminated intravascular coagulation (DIC) established by the Japanese Ministry of Health and Welfare (JMHW) was carried out in 375 patients with hematopoietic tumors and in 555 with non-hematopoietic tumors. Bleeding symptoms were observed in more than 60 % and symptoms of organ failure were frequently observed in the patients with non-hematopoietic tumor. Comparison between patients with DIC and non-DIC, changes o f prothrombin time (PT) ratio and fibrinogen were significant, but those of fibrinogen and fibrin degradation products (FDP) were not. The frequency of DIC score of more than 1 point was highest for FDP but lowest for fibrinogen.
The frequency of DIC score of 0
point in non-DIC was highest for fibrinogen but lowest for FDP. ROC analysis showed that the specificity of DIC dŸ
was
high for FDP and PT ratio, but low for platelet counts and fibrinogen.
These findings suggest that the cut-off value of FDP
should be elevated, that of PT ratio reduced and that fibrinogen omitted in the diagnostic criteria of DIC.
823.
PROGRESSING STROKE AND ANT-PHOSPHOLIPID SYNDROME OR LOWERED PROTEIN S ACTIVITY H. Y A K A H A S H I , T. U R A N O * , Y. T A K A D A * , A. T A K A DA*. Dept. of Neurosurgery, Fujinomiya City General Hosp. & Dept. of Physiology*, H a m a m a t s u U n i r . Sch. of Med. "Progressing Stroke" is defined as small (lacunar) infarction showing progressive neurological deficits. To analyze the mechanism of the progression, we evaluated coagulation and fibrinolytic parameters in the patienIs witb Progressing Stroke, and found that these patients were in a hypercoagulable state and more frequently associated witb antiphospholipid syndrome or lowered Protein-S activity. Methods: We recruited 130 patients with first-ever cerebral infarction. Fifty-five were thrombotic infarction (TH) and 75 were lacunar infarction. Fourteen out of 75 patients revealed progressive deterioration (Progressing Stroke; PROG), and the other 61 patients were ordinary lacunars (LAC). Results: Eight of 14 PROG patients were diagnosed as antiphospholipid syndrome (APS) and 7 patients were accompanied with Iowered protein-S activity (low-PS). The incidence of these two abnormalities were significantly higher than those of other groups; APS; 4/55 (TH) and 2/61 (LAC, p<0.000l), and low-PS; 2/55 (TH) and 0/61 (LAC, p<0.0001). Thrombin-ATIl/ complex (p<0.0001) and soluble fibrin monomer (P<0.005) as well as plasma fibrinogen (p<0.05) in the PROG patients were significantly higher than those in TH or I_AC patients. No significant difference was observed among three groups in tPA and PAI-I level. Conelusions: The etiology of the progressing stroke is quite different from either tbrombotic or lacunar infarction. Hypercoagulable state due to either antiphospholipid syndrome or lowered protein-S activity seems to be involved in the pathogenesis of "progressing stroke".
825.
F R E S H F R O Z E N P L A S M A I N CHILDHOOD IDIOPATHIC THROMBOCYTOPENIC PURPURA A r u n e e J E T S R I S U P A R B , Patcharee K O M W I L A I S A K , Surapon W I A N G N O N , Charoon J E T S R I S U P A R B . Department of Pediatrics, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand Back~round: Idiopathic thrombocytopeaic purpura (ITP) is a disease in which platelets are destroyed mostly by immtmologieal processes. The most se¡ eomplicalion is intracranial hemorrhage, especially when the platelel count is less than 20,000/ mm 3. The treatmems of choiee are intravenous immunoglobulin (IVIG) and/or corticosteroid, however, IVIG is very expensive and most of our patients eannot afford to take ir. Some patients are refraetory to cortieosteroid treatment and some have eomplieations to the drug. Objeetive: To find ah alternative treatment for severe thrombocytopenia in ehildhood ITP for those who cannot afford IVIG and/or those who are refraetory to eorficosteroid treatment, and/or those who experience complieations to steroid treatment. Setting: Department of Pediatries, Faeulty of Medicine, Khon Kaen University. Design: Deseriptive, retrospective and prospective study. Patients and Methodology: Twenty four episodes of severe tttromboeytopenia (with platelet eounts _<20,000/ mm 3) were recorded in ten patients. The slides of blood smear were examined to eonfirm the low platelet cotmts. They were transfused with 10 to 20 mlJkg/day of fresh frozen plasma for three consecutive days. Furosemide, 1 mg/kg/dose, was pushed intravenously before every transfusion. Measurements: AŸ the third day of transfusions, the platelet counts were evaluated using the same automated blood cell count. BIeeding, blood pressure and complieations of transfusions were also rer A 'good' result was a platelet cotmt > 20,000/mm 3 and no bleeding. Results: Good results were aehieved ia eight of nine (88.88%) transfusion-episodes when preeeded by 10 mL of FFP/kg/day and in eleven of fifleen (73.33%) transfusion-episodes when preceded by 20 roL of FFP/kg/day. In the tnansfused group given with 10 mL of FFP/kg/day, six were in a single patient who suffered ffom hypertension and convulsinns as a result of steroid complications; eaeh transfusion had good results and normal blood pressure. AII patients had normal blood pressure during the three days of transfusions, and one had an allergic rash in response to the ehlorpheniramine. Suggestions: Trensfusions given with 10 to 20 roL of FFP/kg/day for three eonsecutive days may be ah altemative treatment for severe thrombocytopenia in ehildhood ITP. More eases and more informafion are needed to confirm this protoeol.
