In Vitro Detection of Occult Bone Marrow Metastases in Patients with Colorectal Cancer Hepatic Metastases Alfred M. Cohen, M.D.,* Pilar Garin-Chesa, M.D., Ph.D.,~ Morin Hanson, M.D.,* Katherine Weyhrauch, B.A.,* Nancy Kemeny, M.D.,t Yuman Fong, M.D.,* Philip Paty, M.D.,* Sydney Welt, M.D.,t Lloyd Old, M.D.t From the Departments of*Surgery and tMedicine, Memorial Sloan Kettering Cancer Center, New York, New York and the ~Oncology Research Dr. Karl Thomae GmbH, Biberach an der Riss, Germany PURPOSE: The purpose of this study was to assess the immunocytochemical status of bone marrow aspirates from patients with clinically isolated hepatic metastases to test the hypothesis that such findings would allow improved patient selection for liver-directed treatment. METHODS: All patients had biopsy-proven or presumed colorectal cancer metastatic to the liver and were scheduled for an operative procedure for hepatic resection or for hepatic artery catheter and chemotherapy pump implant. Immunocytochemical analysis of bone marrow aspirate smears was performed with a panel of monoclonal antibodies directed toward cytokeratins, Lewis Y antigen and A-33 colorectal epitopes. RESULTS: Data from 80 patients indicated that bone marrow reactivity was present in 9.5 percent of those with resectable hepatic metastases and in 34 percent of those not resected (P = 0.03). No single monoclonal antibody or combination produced better discrimination. CONCLUSIONS: Presence or absence of presumed occult colorectal cancer cells in the bone marrow of patients with isolated hepatic metastases is biologically interesting, but not useful in selecting or altering patient management. [Key words: Bone marrow; Monoclonal antibodies; Micrometastases; Colorectal hepatic metastases]
m a r r o w s , a n d this finding is an a d v e r s e p r o g n o s t i c feature. H e p a t i c r e s e c t i o n for metastatic colorectal c a n c e r results in a 30 to 40 p e r c e n t five-year survival, b u t o n l y a 20 p e r c e n t five-year d i s e a s e - f r e e survival. 16 H e p a t i c artery c h e m o t h e r a p y has b e e n f r e q u e n t l y u s e d in patients w i t h u n r e s e c t a b l e h e p a t i c metastases, b u t m a n y o f t h e s e patients fail o u t s i d e the liver. T h e p u r p o s e o f this s t u d y w a s to assess the i m m u n o c y t o c h e m i c a l status o f b o n e m a r r o w aspirates from p a tients w i t h clinically i s o l a t e d h e p a t i c m e t a s t a s e s to test the h y p o t h e s i s that s u c h findings w o u l d a l l o w imp r o v e d p a t i e n t s e l e c t i o n for l i v e r - d i r e c t e d treatment.
PATIENTS AND METHODS
Patient Eligibility All patients h a d b i o p s y - p r o v e n o r p r e s u m e d colorectal c a n c e r metastatic to the liver, a n d w e r e s c h e d u l e d for an o p e r a t i v e p r o c e d u r e either for h e p a t i c
Cohen AM, Garin-Chesa P, Hanson M, Weyhrauch K, Kemeny N, Fong Y, Paty P, Welt S, Old L. In vitro detection of occult bone marrow metastases in patients with colorectal cancer hepatic metastases. Dis Colon Rectum 1998;41:1112 - 1115.
r e s e c t i o n o r h e p a t i c artery c a t h e t e r a n d c h e m o t h e r a p y p u m p implant. Results o f chest x - r a y a n d a b d o m i n a l a n d p e l v i c c o m p u t e d t o m o g r a p h y w e r e n e g a t i v e for e x t r a h e p a t i c disease. After Institutional R e v i e w B o a r d a p p r o v a l o f this p r o t o c o l , all patients s i g n e d w r i t t e n
s s e s s m e n t o f b o n e m a r r o w aspirates for e p i t h e lial ceils using i m m u n o c y t o c h e m i c a l m a r k e r s provides presumptive evidence of tumor dissemina-
A
consents.
tion. T h e m o s t e x t e n s i v e e x p e r i e n c e w i t h this techn i q u e has b e e n in patients w i t h b r e a s t cancer, x-9 H o w e v e r , it has also b e e n a p p l i e d to e v a l u a t i o n o f p a t i e n t s with n e u r o b l a s t o m a , 1° small cell lung cancer,l~, 12 c o l o n cancer, 13 p r o s t a t e cancer, ~4 a n d m e l a n o m a . s5 In colorectal e x p e r i e n c e , n o d e - p o s i t i v e p a -
Bone Marrow Aspiration and Blood Sample
tients h a v e a h i g h e r l i k e l i h o o d of positive b o n e
Supported in part by the Ludwig Institute. Address requests for reprints to Dr. Cohen: Chief, Colorectal Service Director, Colorectal Disease Management Team, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10021.
B o n e m a r r o w aspirations from the anterior iliac crests w e r e p e r f o r m e d at the time o f the s c h e d u l e d surgical p r o c e d u r e u n d e r g e n e r a l anesthesia. Aspiration sites w e r e p r e p a r e d w i t h p o v i d o n e - i o d i n e , a n d a small incision w a s m a d e b e f o r e insertion o f the n e e dle. A single aspirate c o n s i s t e d of I to 5 ml o f h e p a rinized m a r r o w , a n d a m i n i m u m of t h r e e aspirates w e r e o b t a i n e d from e a c h patient. A m a x i m u m o f t h r e e sites w e r e used. A p p r o x i m a t e l y 20 ml o f b l o o d
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OCCULT BONE MARROW METASTASES
was sampled to test for circulating cancer cells using the technique described below.
Specimen Preparation and Preservation Up to three b o n e marrow aspirates from each patient were combined and three to five standard airdried smears were m a d e for routine cytologic evaluation. Red blood cells were separated using a FicollPaque R (Pharmacia, Uppsala, Sweden) density gradient. The resultant interface layer, containing nucleated hemopoietic cells and intact tumor cells, was centrifuged on glass slides in a cytocentrifuge. Average n u m b e r of cells per slide was 10 × 10 4. Half of the slides were fixed in cold acetone and the other half in 95 percent ethanol and were stained immediately or stored at - 7 0 ° C until use.
Immunocytochemical Staining For immunocytochemical detection of p r e s u m e d colorectal cancer cells, a panel of m o u s e monoclonal antibodies (Mabs) was used, including antibody A33, which detects a heat-stable epitope h o m o g e n e o u s l y expressed by virtually all colorectal carcinomas and normal colonic mucosa, but no other epithelial cancersX7; Mab BR55.2,18 an antibody reactive with the Lewis Y determinant, which does not react with any antigen circulating in blood, red blood cells, or other hempatopoietic elements, including b o n e marrow cells. Mabs CAM5.2, AE1, and CK18 react with different classes of cytokeratin proteins present in the cytoskeleton of all normal epithelial cells and their tumors, including colorectal carcinomas. 19' 20 The streptavidin-alkaline phosphatase method, a modification of the alkaline phosphatase procedure, 21 was used. Endogenous phosphatase activity will be inhibited b y preincubation with levamisol. Negative control experiments were performed with unrelated, isotype-matched mouse immunoglobulins. P12 was used as a positive control in all experiments. Slides were counterstained with hematoxylin and evaluated on a Nikon Optiphot microscope R(Nikon, Melville, NY). The n u m b e r of reactive cells and their morphology were evaluated on every specimen.
Control Marrows For the purpose of defining specificity, we obtained excess unstained m a r r o w slides from leukemia, melanoma, and breast cancer patients from the b o n e m a r r o w laboratory and tested them with our panel of antibodies.
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RESULTS Bone marrow was obtained from a total of 88 patients from July 1992 through June 1994. There were no infectious or hemorrhagic complications. Operative findings and follow-up to death or three years was obtained on all patients. Twenty control b o n e marrow specimens were all negative. Twenty five patients' b o n e marrow specimens were examined for cancer by routine methodology, and all but one were negative. All blood samples were negative for circulating tumor cells. In analyzing our data, eight patients were excluded, six with positive controls and two with inadequate data. In 42 patients with resectable hepatic metastases, only 9.5 percent were positive for ceils reacting with any of the panel of Mabs. This compares with 34 percent of patients w h o were unresectable and underwent hepatic artery p u m p implant or were treated with systemic c h e m o t h e r a p y (chi-squared; P = 0.03). Table 1 indicates reactivity of the various monoclonal antibodies w h e n positive. Of the four patients w h o underwent liver resection with a positive b o n e marrow finding, one died in the early postoperative course. The n u m b e r of remaining patients is too small to compare meaningfully the b o n e marrow positive patients to the b o n e marrow negative patients in regard to clinical outcome. Of the 22 patients w h o underwent hepatic artery chemotherapy, half were b o n e marrow positive. Kaptan-Meier survival curves for both groups were identical. However, their patterns of progression suggested a difference, but not a statistically significant one because of only 11 patients in each group. In the b o n e marrow negative group, only 27 percent failed outside the
Table 1. Reactive Tumor or Epithelial Mabs in the Presence of Negative Control Mab
Mab
Number of Reactive Patients Exclusively Total Positive
A33 AE1 CK18 CAM5 A33 and AE1 AE1 and CAM5 AE1 plus CK18 plus CAM5 CK18 plus CAM5
2 2 5 0 1 1 0 2
5 9 9 3 3 2 2 4
Mabs = monoclonal antibodies. A33 = colorectal antigen; CA5.2, AE1, CK18 = cytokeratins.
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COHEN E T A L
liver. In the marrow positive group, 73 percent failed outside (Fisher's exact probability test; P = .19). 3.
DISCUSSION In 1983, Redding et al. 4 first reported detection of presumed micrometastases in the bone marrow of patients with breast cancer using a monoclonal antib o d y to epithelial membrane antigen. Tumor cells were detected in the bone marrow of 31 of 110 patients (28 percent) studied. No tumor cells were detected in conventionally stained smears. Patients with standard indices of poor prognosis appeared more likely to have these identifiable micrometastases. Schlimok and associates zz were able to confirm the superior sensitivity of immunocytology in detecting marrow micrometastases by direct comparison with conventional cytology or histology in 212 patients with breast or colorectal carcinoma. The most relevant data to our study has been reported by Schlimok and colleagues. 23 A monoclonal antibody to a cytokeratin epitope was used to analyze bone marrow aspirates taken from the posterior iliac crest. No reactive cells were detected with this antib o d y in 102 control patients. Samples from 42 of 156 patients with colorectal cancer were positive. Reactivity by Dukes stage was as follows: A, 0 percent; B, 14 percent; C, 34 percent; D, 39 percent. Clinical follow-up of cytokeratin positive Dukes C patients revealed twice the relapse rate of negative Dukes C patients, with n o n e o f the r e c u r r e n c e s c l i n i c a l l y w i t h i n bone. In a report by O'Sullivan et al., 24 25 percent of patients with metastatic colorectal cancer were bone marrow cytokeratin cell-positive using flow cytometry. Our data indicate a statistically valid difference in resectable hepatic metastases and support the overall thrust of the above publications. No single Mab or combination produced better discrimination. However, the data indicate that such findings are not clinically useful. Bone marrow status as ascertained with this Mab panel did not provide adequate diagnostic discrimination to alter patient management.
REFERENCES 1. Ellis G, Ferguson M, Yamanaka E, Livingston RB, Gown AM. Monoclonal antibodies for detection of occult carcinoma cells in bone marrow of breast cancer patients. Cancer 1989;63:2509-14. 2. Deamaley DP, Sloane JP, Ormerod MG, et al. Increased detection of mammary carcinoma cells in marrow
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
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smears using antisera to epithelial membrane antigens. Br J Cancer 1981;44:85-90. Ceci G, Franciosi V, Nizzoli R, et al. The value of bone marrow biopsy in breast cancer at time of diagnosis. A prospective study. Cancer 1988;61:96-8. Redding WH, Coombes RC, Monaghan P, et al. Detection of micrometastases in patients with primary breast cancer. Lancet 1983;2:1271-4. Cote RJ, Rosen PP, Hakes TB, et al. Monoclonal antibodies detect occult breast carcinoma metastases in the bone marrow of patients with early stage disease. AmJ Surg Pathol 1988;12:333-40. Coombes RC, Berger U, Mansi J, et al. Prognostic significance of micrometastases in bone marrow in patients with primary breast cancer. Natl Cancer Inst Monogr 1986;1:51-3. Porto G, Menard S, Tagliabue E, et al. Monoclonal antibody detection of carcinoma cells in bone marrow biopsy specimens from breast cancer patients. Cancer 1988;61:2407-11. Singletary SE, Lille L, Tucker SL, Spitzer G. Detection of micrometastatic tumor cells in bone marrow of breast carcinoma patients. J Surg Oncot 1991;47:32-6. Berger U, Bettelheim R, Mansi JL, Easton D, Coombes RC, Neville AM. The relationship between micrometastases in the bone marrow, histopathologic features of the primary tumor in breast cancer, and prognosis. Am J Clin Pathol 1988;90:1-6. Moss TJ, Reynolds CP, Sather HN, Romansky SG, Hammond GD, Seeger RC. Prognostic value of immunocytologic detection of bone marrow metastases in neuroblastoma. N Engl J Med 1991;324:219-26. Hay FG, Ford A, Leonard RC. Clinical applications of immunocytochemistry in the monitoring of the bone marrow in small cell lung cancer. Int J Cancer Suppl 1988;2:8-10. Canon JL, Humblet Y, Lebacq-Verheyden AM, et al. Utilization of three monoctonal antibodies to monitor bone marrow involvement by small cell lung cancer. Proc Am Assoc Cancer Res 1987;28:216. Schneider BM, Schlimok G, Riethmuller G, Witte J. Bone marrow micrometastases in colon cancer. Fortschr Med 1989;107:59-63. Mansi JL, Berger U, Wilson P, Shearer R, Coombes RC. Detection of tumor ceils in bone marrow of patients with prostatic cancer by immunocytochemical techniques. J Urol 1988;139:545-8. Dantas ME, Brown JP, Thomas MR, Robinson WA, Glode LM. Detection of melanoma cells in bone marrow using monoclonal antibodies. A comparison of flourescence activated cell sorting (FACS) and conventional immunoflourescence (IF). Cancer 1983;52:949-53. Fong Y, Cohen AM, Fortner JG, et al. Liver resection for colorectal metastases. J Clin Oncol 1997;15:938-46.
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17. Welt S, Divgi CR, Real FX, Yeh SD, et al. Quantitative analysis of antibody localization in human metastatic colon cancer: a phase I study of monoclonal antibody A33. J Clin Oncol 1990;8:1894-906. 18. Blaszczyk-Thurin M, Thurin J, Hindsgaul O, Karlsson KA, Steplewski Z, Koprowski H. Y and blood group B type 2 glycolipid antigens accumulate in human gastric carcinoma cell lines as detected by monoclonal antibody. Isolation and characterization by mass spectrometry and NMR. J Biol Chem 1987;262:372-9. 19. Garin-Chesa P, Rettig WJ, Melamed MR. Expression of cytokeratins in normal and neoplastic colonic epithelial cells: implications for cellular differentiation and carcinogenesis. Am J Surg Pathol 1986;10:829-35. 20. Moll R, Franke WW, Schiller DL, Geiger B, Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors, and cultured cells. Cell 1982;31:11-24.
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21. Cordell JL, Fauni B, Erber WN, et aL Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal antialkaline phosphatase (APAAP complexes). J Histochem Cytochem 1984;32:219-29. 22. Schlimok G, Funke I, Holzmann B, et al. Micrometastatic cancer cells in bone marrow: in vitro detection with anticytokeratin and in vivo labeling with anti-17-1A monoclonal antibodies. Proc Nail Acad Sci U S A 1987;84:8672--6. 23. Schlimok G, Funke I, Bock B, Schwerberer B, Witte J, Riethmuller G. Epithelial tumor cells in bone marrow of patients with colorectal cancer: immunocytochemical detection, phenotypic characterization, and prognostic significance. J Clin Oncol 1990;8:831-7. 24. O'Sullivan GC, Collins JK, O'Brien FO, et al. Micrometastases in bone marrow of patients undergoing "curative" surgery for gastrointestinal cancer. Gastroenterology 1995;109:1535--40.
