Tumor cell contamination of peripheral blood stem cell transplants and ...

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in patients treated with high-dose chemotherapy and stem cell support.5–9 To determine ... cells in stem cell transplants and bone marrow of 21 plants (PBSCT).
Bone Marrow Transplantation, (1997) 19, 1223–1228  1997 Stockton Press All rights reserved 0268–3369/97 $12.00

Tumor cell contamination of peripheral blood stem cell transplants and bone marrow in high-risk breast cancer patients R Schulze1, M Schulze1, A Wischnik2, S Ehnle1, K Doukas3 , W Behr3, W Ehret3 and G Schlimok1 1

2 Medizinische Klinik; 2Frauenklinik; 3Institut fu¨r Laboratoriumsmedizin, Zentralklinikum, Augsburg, Germany

Summary: Twenty-one high-risk patients with primary stage II/III breast cancer were treated with high-dose chemotherapy comprising etoposide, ifosfamide, carboplatin and epirubicin (VIC-E). Tumor cells of epithelial origin were analyzed using the monoclonal antibodies CK2 (IgG1) and A45-B/B3 (IgG1) against cytokeratin (CK) components in bone marrow (BM) aspirates prior to chemotherapy, and in peripheral blood stem cell transplants (PBSCT). They were separated after the first (21/21 patients) and the second cycle (16/21 patients) of induction chemotherapy with VIP-E (etoposide, ifosfamide, cisplatin, epirubicin). Preliminary results showed CK positive tumor cells in 40% (14/35) of the analyzed transplants. In 7/12 (58.3%) patients, CK positive tumor cells were detectable in BM prior to treatment. Sixteen patients were separated after the 1st and 2nd cycle of VIP-E. PBSCT of 14/16 patients were assessable for presence of CK positive tumor cells. Our preliminary results demonstrate a lower tumor cell contamination of PBSCT separated after the 2nd cycle of induction therapy (14.3%) compared to contamination after the first induction therapy (64.3%). To date, 4/21 patients have experienced a relapse, and three of these patients had tumor cell positive transplants. Due to the small patient number only a trend towards a superior relapsefree survival in the patient group with CK negative transplants can be shown by Kaplan–Meier analysis. Keywords: tumor cells; peripheral blood stem cell transplantation; bone marrow; breast cancer

Breast cancer accounts for approximately 30% of all cancers in women. It is one of the most common malignancies and the second leading cause of death in women.1 The 10year relapse rate, increasing with the number of lymph nodes involved, is about 20% for negative lymph node stages and greater than 60% in women with one to three lymph nodes involved. Patients with stage II/III breast cancer and 10 or more involved axillary lymph nodes can currently expect only a 15–20% 10-year disease-free survival. Improvement of treatment outcome with standard-dose induction chemotherapy followed by high-dose chemoCorrespondence: Dr R Schulze, 2 Medizinische Klinik, Zentralklinikum Augsburg, Stenglinstrasse 2, D-86156 Augsburg, Germany Received 30 September 1996; accepted 18 February 1997

therapy and stem cell support appears possible. Peters et al2,3 reported a 78% overall survival (OS) in this high-risk breast cancer subgroup after high-dose chemotherapy followed by stem cell support (median follow-up 4.5 years). Gianni et al4 showed a OS of 78% after 5 years in these patients. Several authors have suggested that tumour cells present in stem cell transplants may contribute to relapse in patients treated with high-dose chemotherapy and stem cell support.5–9 To determine the significance of tumor cell contamination, we sought the presence of epithelial tumor cells in stem cell transplants and bone marrow of 21 patients with high-risk breast cancer treated by high-dose chemotherapy and stem cell support. Single tumor cells of epithelial origin disseminated to mesenchymal organs (blood, bone marrow) can be identified by using monoclonal antibodies (moAb CK2, A45-B/B3) directed against cytokeratin components, and the APAAP staining technique.10–12 In this study we aimed to analyze the influence of time of collection on tumor cell load in PBSCT and the clinical significance of these findings.

Materials and methods Patients For the present study 21 female patients with high-risk breast cancer and involvement of seven to nine (three patients) and 10 or more (18 patients) axillary lymph nodes were used. Informed consent was obtained from all patients. All patients had histologically proven resectable breast cancer (19 invasive ductal, two invasive lobular). After an extensive diagnostic program, patients were staged according to the UICC breast cancer classification as follows: UICC stage IIA n = 4 (19%), IIB n = 8 (38%), IIIA n = 5 (24%), IIIB n = 4 (19%). Median age of the patients at time of primary surgery was 50.8 years (34–62). No patient had received previous chemotherapy, radiotherapy or immunotherapy. Patient characteristics are shown in Table 1. Treatment Patients were treated with two cycles of induction chemotherapy with the standard-dose VIP-E regimen, which consisted of etoposide 500 mg/m2, ifosfamide 4000 mg/m2, cisplatin 50 mg/m2 and epirubicin 50 mg/m2.13 Mesna uroprotection was given according to standard protocols. For the mobilization of PBSCs G-CSF (Filgrastim, Neupogen;

Tumor cell contamination of PBSCT in breast cancer R Schulze et al

Table 1

Clinical characteristics of patients

Patient No.

Age (years)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

48 49 56 54 34 41 53 53 57 51 51 57 50 59 49 55 52 62 38 55 43

TNM classification T2 T3 T2 T1 T3 T4 T2 T2 T1 T2 T1 T1 T1 T2 T1 T3 T4 T3 T2 T4 T2

G2 G3 G2 G3 G2 G2 G2 G3 G3 G2 G2 G2 G2 G3 G3 G3 G3 G2 G3 G3 G3

N1 N1 N1 N3 N1 N1 N1 N1 N1 N1 N1 N1 N1 N1 N2 N2 N1 N1 N1 N2 N1

(20/25) M0 (17/17) M0 (10/14) M0 (in mass) M0 (12/15) M0 (15/27) M0 (7/16) M0 (15/18) M0 (10/13) M0 (9/21) M0 (13/13) M0 (12/30) M0 (26/28) M0 (18/23) M0 (14/14) M0 (15/18) M0 (8/13) M0 (16/17) M0 (19/22) M0 (18/23) M0 (11/19) M0

Breast cancer stage (UICC) II B III A II B III B III A III B II B II B II A II B II A II A II A II B III A III A III B III A II B III B II B

Amgen, Munich, Germany) 5 mg/kg was administered by subcutaneous injection, starting day 1 after chemotherapy until the time of stem cell collection. Peripheral blood stem cells (PBSC) were collected in an outpatient setting by leukapheresis on day 10 and/or 11 after each induction chemotherapy cycle. Leukaphereses were performed with a blood cell separator (AS 104, Fresenius, Oberursel, Germany). Acid citrate dextrose (ACD) anticoagulant (in a concentration ACD:0.9% sodium chloride = 1:15, Fresenius, Oberursel, Germany; 440 ml per leukapheresis) was used. 6.6 liters blood were processed per leukapheresis. Stem cells were collected with a total volume of 220 ml on each day of leukapheresis, counted, mixed with 50 ml autologous plasma, 150 ml hemofusin 10% (Pharmacia, Erlangen, Germany), 50 ml DMSO (Merck, Darmstadt, Germany) 18

CK+cells/2 × 106 analyzed cells

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16 14 12 10 8 6 4 2 0

1st transplant

2nd transplant

Figure 1 Ck positive cells in PBSCT after 1st and 2nd induction chemotherapy cycle. Number of CK positive (CK+) cells in peripheral stem cell transplants (PBSCT) of patients with transplants both after first, (m), (14 patients) and second, (M), (14 patients) induction chemotherapy cycle with VIP-E.

and cryocyte frozen in bags (Hemofreeze bag; NPBI, Emmer-Compascuum, The Netherlands). Cryopreservation was performed using controlled rate freezing −1.5°C/min on an average (Kryo 10 Series II Planer Biomed; MesserGriesheim, Griesheim, Germany). Three weeks after the 2nd induction cycle, high-dose chemotherapy with the VICE regimen was given: etoposide 500 mg/m2, ifosfamide 4000 mg/m2, carboplatin 250 mg/m2 and epirubicin 50 mg/m2 day −3 until day −1.13 One PBSC transplant collected after the 2nd cycle of induction chemotherapy, and if not available, that obtained after the 1st cycle (containing 7.19 × 106 CD34+/kg on an average) was reinfused 24 h after the end of high-dose chemotherapy on day +1. The remaining PBSC transplant was kept frozen for back-up. 1 ml aliquots of each leukapheresis were used for laboratory analysis. G-CSF was administered from day +1 until the leukocyte count was >3 × 109/l. Thoracic wall irradiation (dose: 40 Gy + boost 10 Gy, delivered in standard 2 Gy fractions/day) was started, 4–6 weeks after highdose chemotherapy. Preparation and immunostaining of bone marrow specimens and stem cell collections Bone marrow samples were aspirated from both posterior iliac crests at the time of primary surgery. A mean volume of 5 ml bone marrow aspirate from both upper posterior iliac crests was taken into syringes containing 100 units heparin/ml marrow, yielding between 5 × 106 and 3 × 107 (mean 107) mononuclear cells. After dilution with 10 ml phosphate buffered saline (PBS), marrow fat was separated by centrifugation (180 g, 10 min). After density centrifugation through Ficoll–Hypaque (Seromed, Berlin, Germany; density 1.077, 900 g, 30 min) mononuclear cells were collected from the interphase. Washed twice in PBS and centrifuged (200 g, 10 min), the cells were then suspended with 1–5 ml PBS yielding a concentration of 2 × 106 cells/ml. In a cytocentrifuge (Hettich, Tuttlingen, Germany) an average number of 1 × 106 cells was centrifuged onto glass slides. After air drying for 12–24 h and acetone fixation (10 min, room temperature), two slides comprising 2 × 106 nucleated cells were routinely examined for each patient. One additional slide served as IgG isotype control. Immunostaining with the moAb (CK2 (IgG1) (Boehringer Mannheim, Tutzing, Germany) directed against the cytokeratin polypeptide 18 (CK18),14,15 and the broad-spectrum moAb A45-B/B3 (IgG1) (kindly provided by Micromet, Munich, Germany),16–18 directed against a common epitope on a variety of cytokeratin components, including CK8, 18 and 19 was used for tumor cell detection in bone marrow and in PBSC cytospin preparations.10, 19 The antibody reaction was developed with the alkaline phosphatase-antialkaline phosphatase (APAAP) technique. Stem cell collections after 1st and/or 2nd cycle of induction chemotherapy were analyzed as described for BM specimens. After density centrifugation through Ficoll– Hypaque a 1 ml aliquot of each stem cell transplant was washed twice in PBS and then suspended in 1–5 ml PBS, yielding a concentration of 2 × 106 cells/ml. An average number of 1 × 106 cells was cytocentrifuged onto glass slides followed by air drying and acetone fixation. For each

Tumor cell contamination of PBSCT in breast cancer R Schulze et al

PBSCT, two of these slides comprising 2 × 106 cells were examined after immunostaining with the moAbs CK2 and A45-B/B3. The tested sensitivity of our method was the detection of one tumor cell among 2 × 106 normal bone marrow cells or 2 × 106 cells of the PBSCT aliquots. The cytoplasm of the tumor cells stained bright orange-red. Positive smears were defined as those containing one or more tumor cells. In bone marrow samples one to nine cells were detected for most positive smears and in the PBSCT one to five cells per slide. All slides included in our study were read by two independent investigators, with an interobserver agreement of 99%. Results To date, 21 patients with high-risk breast cancer stage II/III have been treated with high-dose chemotherapy and stem cell support. Therapy can be administered safely without major side-effects. After each induction chemotherapy, peripheral blood stem cells mobilized by G-CSF were harvested, on average 8.75 × 106 CD34+/kg. The presence of cytokeratin positive cells was analyzed in the bone marrow of 14 patients prior to chemotherapy. The BM aspirates of two patients were not assessable for cytokeratin-positive tumor cells. Seven of 12 patients (58.3%) showed CK positive cells at a frequency of 5.14 CK positive cells per 2 × 106 analyzed cells (Table 2). Five of the seven patients (71.42%) with CK positive bone marrow also had CK positive stem cell transplants. The presence of CK positive cells was sought in the first (21 patients) and second (16 patients) stem cell harvest. One transplant separated after the first and one after the second cycle of induction chemotherapy were not assessable for cytokeratin positive cells. The numTable 2 Patient No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Cytokeratin positive tumor cellsa in bone marrow (BM) and peripheral stem cell transplants (PBSCT) BM prior to therapy

PBSCT after 1st ind cycle

7 6 Not done 0 Not done Not done Not done 1 1 Not done 0 0 9 9 Not assessable Not done 0 Not assessable 3 0 Not done

0 3 3 0 1 0 4 0 1 0 3 2 5 1 1 2 0 0 1 Not assessable 0

CK positive cells per 2 × 106 cells analyzed. ind cycle = cycle of induction chemotherapy.

a

ber of CK positive cells in each transplant is shown in Table 2. Altogether 14 of 35 transplants analyzed (40%) showed CK positive cells as a sign of tumor cell contamination. In the 14 patients with stem cell separates collected after both the 1st and 2nd induction chemotherapy cycle, tumor cells were found on an average of 2.25 CK positive cells per 2 × 106 cells analyzed after the 1st and on an average of 1.5 CK positive cells per 2 × 106 transplant cells analyzed after the 2nd cycle. Extent of tumor cell positivity of the transplants obtained after the 1st cycle of induction chemotherapy yields 64.3% (9/14 patients), but after the 2nd cycle only 14.3% (2/14 patients). The cumulative number of CK positive cells of the patients with transplants separated after the 1st and 2nd induction cycle is presented in Figure 1. The lower number of CK positive cells in the second stem cell separate is apparent. Stratifying the positive transplants according to tumor stage, revealed no correlation. We carried out follow-up analysis of all patients. The median follow-up is 13.57 months (4–27). To date 17 patients are in complete remission, four patients experienced a relapse, two patients died due to tumor relapse. Three of the four patients with a relapse demonstrated CK positive cells in the stem cell transplants. Altogether, 25% of the patients with tumor cell positive transplants (3/12) had a relapse, whereas recurrence of disease was seen in only 11% of the patients with CK negative transplants (1/9). Thus, a trend towards a superior relapse-free survival is present in the patient group with CK negative transplants. Kaplan–Meier analysis (Figure 2) yields a slightly better relapse-free survival for the patients with CK negative transplants. However, these results are not statistically significant due to the low patient number.

PBSCT after 2nd ind cycle Not Not Not Not

done done done done 0 0 2 0 0 0 Not done 0 0 0 1 0 0 0 0 0 Not assessable

Relapse Yes/No no no no yes no no yes no no no no no no no yes yes no no no no no

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1.0 0.9 Probability of RFS

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1

P = 0.5

0 0

10

20

30

Months

Figure 2 Relapse-free survival (RFS) of patients with CK positive (ªª) (12 patients) and CK negative (JJ) (nine patients) peripheral stem cell transplants (PBSCT); median follow-up 13.6 (4–27) months.

Discussion Autologous peripheral blood stem cell transplantation permits an alternative to bone marrow transplantation for patients treated with high-dose chemotherapy in solid tumors and hematological malignancies. Main advantages of this method are the ready availability of peripheral blood stem cells and their more rapid hematopoietic engraftment. After chemotherapy and use of growth factors, peripheral blood stem cells can be processed and harvested in large amounts. Chemotherapy followed by growth factor administration may mobilize tumor cells into peripheral blood.5 Notably, residual tumor cells can be detected in bone marrow in about 30% of patients with curatively resected solid tumors. 20 In breast cancer patients, minimal residual disease in bone marrow is an independent prognostic factor for disease-free and overall survival.21–25 Thus, a possible cause of tumor relapse in patients treated with high-dose chemotherapy and stem cell rescue could be the reinfusion of these contaminating tumor cells.5,21,26–28 The reinfusion of malignant cells by autologous bone marrow transplantation has been shown to be a prognostic factor for relapse in B cell lymphoma by Gribben et al29 and in acute myeloid leukemia by Brenner et al.30 Minimal residual disease in breast cancer patients can be detected by immunocytochemistry. 31 The sensitivity of immunocytochemical (ICC) methods for tumor cell detection is limited by the number of cells examined. The 95% confidence limit of sensitivity is about one tumor cell in 106 nucleated cells for 3 × 106 cells examined.32 In immunocytochemical analyses, the specificity of moAbs against cytokeratin (CK), the cytoskeleton of epithelial cells, has been shown to be superior when compared with that of moAbs directed against cell surface antigens such as the epithelial membrane antigen (EMA), the human epithelial antigen (HEA) 125 or the tumor associated glycoprotein (TAG) 72, which may crossreact with hematopoietic cells.10,19 Nevertheless, in bone marrow samples of occasional patients (,5%) without known epithelial malignancies, CK positive cells have been found in very small numbers. The same mechanisms which underlie false-positive isotype controls (,5%), for instance non-specific (Fc-receptor)-binding of moAb could be

reponsible for this phenomenon. Crossreactivity of CK moAbs with hematopoietic cells was excluded in previous double-marker analyses, demonstrating exclusive positivity of CK components and a simultaneous lack of CD45 and vimentin.10,11,19 On the other hand, CK positive cells showed tumor-associated characteristics, such as downregulation of MHC class I antigens and overexpression of the erbB2 oncogene.10,33 Furthermore, combination of ICC and fluorescence in situ hybridization (FISH) demonstrated amplification of the erb-B2 gene in disseminated tumor cells in bone marrow samples of breast cancer patients.34 Theoretically, polymerase chain reaction analysis (PCR) may overshadow the well-demonstrated specificity and sensitivity of immunocytochemical assays for detection of tumor cells in hematopoietic organs, such as BM and peripheral blood. 35,36 Reverse transcriptase polymerase chain reaction (RT-PCR) based on cytokeratin 19 showed an approximately 1 log greater sensitivity compared to the ICC method, but increased sensitivity may decrease specificity.32 The specificity of RT-PCR may not be absolute but may rather reflect quantitative differences in the level of expression of malignant cells and the surrounding autochthonous cells.37 Thus, amplification of pseudogenes19 or illegitimate transcription of CK19 by mononuclear cells decrease the specificity of RT-PCR.38 Similarly, genes encoding for other tumor-associated molecules may not be expressed uniquely in tumor cells but may also exert some mRNA expression in certain normal tissues.39–43 The rate of false-positive findings can be lowered by decreasing the number of PCR cycles and by using fewer cells, but this modification automatically limits the sensitivity of the method.32 Zippelius et al40,41 revealed that the RT-PCR assay in BM specimens of patients with prostate or colorectal cancer was less sensitive than the standard immunocytochemical cytokeratin assay. 10 At the present time, ICC is the best method for detecting isolated disseminated tumor cells in hematopoietic tissues, and PCR methods are best for demonstrating negativity for micrometastasis.32 We attempted to detect tumor cell contamination immunocytochemically in peripheral blood stem cell harvests of breast cancer patients by using moAbs directed against CK components. Peripheral blood stem cells were harvested after the 1st and the 2nd induction cycles with VIP-E chemotherapy followed each time by stimulation with GCSF. The higher tumor cell contamination of the stem cell harvests obtained after the 1st cycle of induction chemotherapy compared to the 2nd indicates that chemotherapy is capable of reducing minimal residual tumor cell load (in vivo purging). The number of CK positive cells was higher in bone marrow compared to PBSC harvests. These data confirm the results of other authors.7,44,45 The prognostic meaning of epithelial tumor cells in peripheral blood stem cell transplants (PBSCT) is, in contrast to bone marrow, as yet unknown.21 Ross et al44,45 revealed that tumor cells in both marrow and PBSC collections appear to be capable of clonogenic growth in vitro, thus possibly contributing to relapse. Ybanez et al46 found no correlation of tumor contamination in PBSC collections in advanced stage IV breast cancer patients with time to progression, sites of relapse, or overall survival. Contrary to these data, Peters et al3 revealed that tumor involvement of the autologous marrow

Tumor cell contamination of PBSCT in breast cancer R Schulze et al

graft (30/83 patients, ie 36%) was significantly associated with a shorter disease-free and overall survival in stage II and III patients. Further, the authors found that relapse-free and overall survival were a function of the number of tumor cells detected in bone marrow, with an increasing number of cells being associated with shorter survival.45 However, in this study only two of 57 patients (4%) showed tumor cell contamination of PBSCT, and hence a similar analysis could not be done. Thus, at the present time, there is no published study in stage II/III breast cancer patients, treated with adjuvant high-dose chemotherapy discussing the clinical significance of tumor cell contamination in PBSCT. Our preliminary results describing the extent of tumor cell contamination in PBSCT in stage II and III breast cancer patients show a trend to a higher relapse rate in patients with CK positive transplants. Further clinical trials are urgently warranted to address whether CK positive cells in PBSCT only reflect a high tumor burden or whether these cells constitute a new independent risk factor in breast cancer. References 1 Henderson BE, Ross RK, Pike MC. Toward the primary prevention of cancer. Science 1991; 254: 1131–1137. 2 Peters WP, Ross M, Vredenburgh JJ et al. High-dose chemotherapy and autologous bone marrow support as consolidation after standard-dose adjuvant therapy for high-risk primary breast cancer. J Clin Oncol 1993; 11: 1132–1143. 3 Peters WP, Berry D, Vredenburgh JJ et al. Five-year followup of high-dose combinating alkylating agents with ABMT as consolidation after standard-dose CAF for primary breast cancer involving >10 axillary lymph nodes (DUKE/CALGB 8782). Proc Am Soc Clin Oncol 1995; 14: A933 (Abstr.). 4 Gianni AM, Siena S, Bregni M et al. 5-year results of highdose sequential (HDS) adjuvant chemotherapy in breast cancer with >10 positive nodes. Proc Am Soc Clin Oncol 1995; 14: A61 (Abstr.). 5 Brugger W, Bross K, Glatt M et al. Mobilization of tumor cells and hematopoietic progenitor cells into peripheral blood of patients with solid tumors. Blood 1994; 83: 636–640. 6 Moss TJ, Cairo MS, Bostrom B et al. Using bone marrow (BM) immunocytochemical (ICC) analysis to determine optimal timing of peripheral blood stem cell (PBSC) harvest for patients with breast cancer and neuroblastoma. Blood 1994; 84 (Suppl. 1): 1401 (Abstr.). 7 Sharp JC, Kessinger A, Mann S et al. Detection and clinical significance of minimal tumor cell contamination of peripheral blood stem cell harvests. Int J Cell Clon 1992; 10 (Suppl. 1): 92–94. 8 Shpall EJ, Jones RB. Release of tumor cells from bone marrow. Blood 1994; 83: 623–625. 9 Weaver CH, Hazelton B, Ross AA, Schwartzberg L. The incidence of tumor cell contamination in peripheral blood progenitor cell (PBPC) collections in patients with breast cancer. Blood 1994; 84 (Suppl. 1): 1389a (Abstr.). 10 Pantel K, Schlimok G, Angstwurm M et al. Methodological analysis of immunocytochemical screening for disseminated epithelial tumor cells in bone marrow. J Hematother 1994; 3: 165–173. 11 Schlimok G, Funke I, Holzmann B et al. Micrometastatic cancer cells in bone marrow: in vitro detection with anti-cytokeratin and in vivo labelling with anti-17-1A monoclonal antibodies. Proc Natl Acad Sci USA 1987; 84: 8672–8676.

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40 Zippelius A, Honold G, Ku¨fer P et al. Potential and limitations of RT-PCR for detection of isolated carcinoma cells in bone marrow. Exp Hematol 1995; 23: 763 (Abstr.). 41 Zippelius A, Kufer P, Honold G et al. Limitations of reverse transcriptase-polymerase chain reaction analyses for detection of micrometastatic epithelial cancer cells in bone marrow. Blood 1996; 88 (Suppl. 1): 2431 (Abstr.). 42 Corradini P, Cinque F, Astolfi M et al. Detection of minimal residual disease in patients with breast cancer: a novel nestedPCR assay using maspin gene. Blood 1996; 88 (Suppl. 1): 972 (Abstr.). 43 Jung K, Henke W, Lein M et al. Polymerase chain reaction in the detection of micrometastases and circulating tumor cells. Cancer 1996; 78: 2445–2447. 44 Ross AA, Cooper BW, Warner NE et al. Detection and viability of tumor cells in peripheral blood stem cell collections from breast cancer patients using immunocytochemical and clonogenic assay techniques. Blood 1993; 82: 2605–2610. 45 Ross AA, Layton TJ, Ostrander AB et al. Comparative analysis of breast cancer contamination in mobilized and nonmobilized hematopoietic grafts. J Hematother 1996; 5: 549–552. 46 Ybanez J, Lazarus HM, Ross AA et al. Impact of occult tumor reinfusion on outcome after high-dose chemotherapy in patients with advanced breast cancer. Blood 1995; 86: 386a (Abstr.).