Detection and viability of tumor cells in peripheral blood stem cell collections from breast cancer patients using immunocytochemical and clonogenic assay techniques [see comments]

Although peripheral blood stem cell collections (PBSC) are thought to have less tumor involvement than bone marrow (BM), the incidence of circulating tumor cells in patients with breast cancer has not been widely investigated. We prospectively investigated the incidence and viability of tumor cell involvement in PBSC and BM collections from breast cancer patients undergoing high-dose chemotherapy/hematopoietic stem cell transplantation. Paired samples of PBSC and BM from 48 patients were analyzed using an immunocytochemical technique that detects one epithelial-derived tumor cell per 5 x 10(5) mononuclear cells. Immunostained tumor cells were detected in 9.8% (13/133) PBSC specimens from 9/48 (18.7%) patients and in 62.3% (38/61) BM specimens from 32/48 (66.7%) patients, a significantly higher rate than in PBSC (P < .005). The geometric mean concentration of tumor cells in contaminated PBSC specimens was 0.8/10(5) mononuclear cells (range 0.33 to 2.0/10(5)) compared with 22.9/10(5) mononuclear cells in BM (range 1 to 3,000/10(5), P < .0001). In culture experiments, clonogenic tumor colonies grew in 21/26 immunocytochemically positive specimens. No tumor colony growth was detected in 30/32 immunocytochemically negative specimens. Immunocytochemical detection of tumor involvement in BM and PBSC correlated significantly with in vitro clonogenic growth (P < .0001). We conclude that PBSC contain fewer tumor cells than paired BM specimens from patients with advanced breast cancer and that these tumor cells appear to be capable of clonogenic growth in vitro.     

Contamination of peripheral blood stem cell harvests by circulating neuroblastoma cells

Peripheral blood stem cells (PBSC) are being used as one alternative to autologous marrow rescue for patients with neuroblastoma and other solid malignancies. Some physicians prefer use of PBSC because less risk of tumor contamination is believed to exist. This hypothesis was evaluated by immunocytologic analysis of blood samples and concurrently drawn bone marrow (BM) samples and of PBSC harvests obtained from 31 patients with disseminated neuroblastoma. We found circulating neoplastic cells in 75% of specimens analyzed at diagnosis, in 36% during therapy, and in 14% of PBSC harvests. Tumor cells in blood obtained during therapy did not appear until 3 months after the time of diagnosis. Clearance of circulating neuroblastoma cells was documented after two courses of induction chemotherapy. Six of 13 patients with minimal or no BM disease had positive blood specimens. We conclude that substantial risk of tumor contamination of PB harvests exists and recommend that induction chemotherapy be administered before hematopoietic progenitor cells are collected from blood.     

Heterogeneous proliferative potential of occult metastatic cells in bone marrow of patients with solid epithelial tumors

Bone marrow is a major homing site for circulating epithelial tumor cells. The present study was aimed to assess the proliferative capacity of occult metastatic cells in bone marrow of patients with operable solid tumors especially with regard to their clinical outcome. We obtained bone marrow aspirates from 153 patients with carcinomas of the prostate (n = 46), breast (n = 45), colon (n = 33), and kidney (n = 29). Most of the patients (87%) had primary disease with no clinical signs of overt metastases [tumor-node-metastasis (TNM)-stage UICC (Union Internationale Contre le Cancer) I-III]. After bone marrow was cultured for 21-102 days under special cell culture conditions, viable epithelial cells were detected by cytokeratin staining in 124 patients (81%). The cultured epithelial cells harbored Ki-ras2 mutations and numerical chromosomal aberrations. The highest median number of expanded tumor cells was observed in prostate cancer (2,619 per flask). There was a significant positive correlation between the number of expanded tumor cells and the UICC-stage of the patients (P = 0.03) or the presence of overt metastases (P = 0.04). Moreover, a strong expansion of tumor cells was correlated to an increased rate of cancer-related deaths (P = 0.007) and a reduced survival of the patients (P = 0.006). In conclusion, the majority of cancer patients have viable tumor cells in their bone marrow at primary tumor diagnosis, and the proliferative potential of these cells determines the clinical outcome.     

Autologous bone marrow transplantation in breast cancer: separation of clonogenic tumor cell colonies by gradient fractionation

A major obstacle to successful autologous bone marrow transplantation in breast cancer is infiltration of the marrow by malignant cells. We layered bone marrow samples from seven breast cancer patients on a discontinuous bovine albumin gradient, then assessed hematopoietic potential (colony-forming units-culture [CFUc]) and clonogenic tumor cells (TCFUc) by standard techniques. We found that 78% of CFUc concentrated in fraction 3 of the gradient, which contained 10% of the total nucleated cell population. TCFUc were distributed across the gradient with 14% of colonies identified in this marrow fraction. We applied these techniques to two patients with metastatic breast cancer who were treated with high-dose mitomycin-C, doxorubicin, and cyclophosphamide before receiving 1.9 x 10(9) and 1.2 x 10(9) total cells, respectively, from CFUc-rich fraction 3. We observed tumor colony growth in three patients only in separated marrow fractions, suggesting that colony growth may be a function of the cell composition after fractionation or that growth factors may be separable. Ninety percent of clonogenic breast cancer cells can be separated from hematopoietic cells by discontinuous density gradient fractionation, a technique that is applicable to the large volumes necessary in bone marrow transplantation and that may be an important initial step in marrow purging for autologous transplantation.     

A monoclonal antibody cocktail for detection of micrometastatic tumor cells in the bone marrow of breast cancer patients

We investigated whether monoclonal antibodies (MoAbs) reactive against both acidic and basic cytokeratins alone were sufficient to detect minimal numbers of contaminating epithelial tumor cells in the bone marrow of breast cancer patients. Monoclonal anti-cytokeratin antibodies (AE1 and AE3) were used to stain 14 breast carcinomas by the avidin-biotin-peroxidase technique. Nine tumors (64.3%) showed high reactivity and five (35.7%) showed low or moderate reactivity. Nine MoAbs that proved to be unreactive to light density bone marrow cells by immunoalkaline phosphatase histochemistry were screened for reactivity to breast carcinomas having only low or moderate positivity to cytokeratin antibodies. Three of nine MoAbs showed high percentages of positivity and were selected to supplement the anti-cytokeratin antibodies for immunohistochemical detection of minimal marrow disease in breast cancer patients. A MoAb cocktail was prepared, further tested for reactivity to another five breast carcinomas, and compared with cytokeratin staining alone. The cocktail labeled 100% of carcinoma cells in all the examined specimens. To determine the sensitivity of this panel for detecting minimal numbers of contaminating tumor cells in bone marrow, in vitro mixing experiments were performed. T47D breast carcinoma cells were mixed with bone marrow mononuclear cells at ratios from one tumor cell per 10 bone marrow cells up to one tumor cell per 1 x 10(6) marrow cells, and cytospin preparations were subsequently stained with the MoAb cocktail by the immunoalkaline phosphatase method. Our approach could detect one tumor cell in 1 x 10(5) hematopoietic cells.     

Dose intensive therapy with autologous blood stem cell transplantation in breast cancer

Background: Breast cancer is the commonest form of cancer in Australian women. Although approximately 50% of women with breast cancer achieve long term survival by current management methods, recurrent or metastatic disease is generally incurable. In addition, women with Stage II disease with > 10 positive axillary lymph nodes and also women with locally advanced disease (Stage III) have a poor survival even with adjuvant therapy. Aims: To assess the toxicity and efficacy of high-dose chemotherapy with autologous peripheral blood stem cell (PBSC) transplantation in women with both metastatic and poor prognosis primary breast cancer. Methods: Twenty-eight women with either metastatic (15) or poor prognosis (13) primary breast cancer were enrolled in the study between November 1988 to January 1993. PBSC were harvested using high-dose cyclophosphamide (Cy) with or without granulocyte-colony stimulating factor (G-CSF) and a myeloablative regimen of Cy, melphalan and carboplatin (CMCp) was used in the transplantation phase. Results: Optimum numbers of stem cells were harvested in 85% of patients. The use of five G/m² Cy plus G-CSF resulted in better PBSC yields and a significant reduction in haematologic morbidity when compared to mobilisation with Cy alone. Twenty-two women underwent 23 PBSC transplants (PBSCT). There have been two early deaths due to sepsis. The predominant morbidities observed following high dose chemotherapy and transplantation have been nausea, mucositis and diarrhoea. The median number of days to discharge following infusion of PBSC was 15 (range 11–21). At a median follow up time of 1.1 years (range 0 months-3.6 years), 8/22 (36%) evaluable patients remain alive and disease free while 14/22 (64%) have relapsed or progressed or died. Conclusion: High-dose chemotherapy and autologous PBSCT is a potentially highly effective treatment of women with metastatic and poor prognosis primary breast cancer. Randomised studies are required to compare this form of therapy to more standard forms of treatment in breast cancer.     

Mobilization of tumor cells and hematopoietic progenitor cells into peripheral blood of patients with solid tumors [see comments]

Peripheral blood progenitor cells (PBPCs) are increasingly used for autografting after high-dose chemotherapy. One advantage of PBPCs over the use of autologous bone marrow would be a reduced risk of tumor-cell contamination. However, the actual level of tumor cells contaminating PBPC harvests is poorly investigated. It is currently not known whether mobilization of PBPCs might also result in mobilization of tumor cells. We evaluated 358 peripheral blood samples from 46 patients with stage IV or high-risk stage II/III breast cancer, small cell (SCLC) or non- small cell (NSCLC) lung cancer, as well as other advanced malignancies for the detection of epithelial tumor cells. Monoclonal antibodies against acidic and basic cytokeratin components and epithelial antigens (HEA) were used in an alkaline phosphatase-anti-alkaline phosphatase assay with a sensitivity of 1 tumor cell within 4 x 10(5) total cells. Before initiation of PBPC mobilization, circulating tumor cells were detected in 2/7 (29%) patients with stage IV breast cancer and in 2/10 (20%) patients with extensive-disease SCLC, respectively. In these patients, an even higher number of circulating tumor cells was detected after chemotherapy with VP16, ifosfamide, and cisplatin (VIP) followed by granulocyte colony-stimulating factor (G-CSF). This approach has previously been shown to be highly effective in mobilizing PBPCs. In the 42 patients without circulating tumor cells during steady state, tumor cells were mobilized in 9/42 (21%) patients after VIP+G-CSF induced recruitment of PBPCs. The overall incidence of tumor cells varied between 4 and 5,600 per 1.6 x 10(6) mononuclear cells analyzed. All stage IV breast cancer patients and 50% of SCLC patients were found to concomitantly mobilize tumor cells and PBPCs. Kinetic analyses showed two patterns of tumor cell recruitment depending on the presence or absence of bone marrow disease: (1) early after chemotherapy (between days 1 and 7) in patients without marrow infiltration, and (2) between days 9 and 16 in patients with marrow infiltration, ie, within the optimal time period for the collection of PBPCs. We show that there is a high proportion of patients with circulating tumor cells under steady-state conditions, and in addition a substantial risk of concomitant tumor cell recruitment upon mobilization of PBPCs, particularly in stage IV breast cancer patients with bone marrow infiltration. The biologic and clinical significance of this finding is unknown at present.     

Rapid and complete hematopoietic reconstitution following peripheral blood stem cell autotransplantation in a child with T cell acute lymphoblastic leukemia

Rapid and complete hematopoietic reconstitution was achieved in a child with T cell acute lymphoblastic leukemia who was autografted with peripheral blood stem cells (PBSC). A large number of PBSC was collected by two courses of 3-4 hour-lasting lymphopheresis during early remission induced by the second-line chemotherapy and then cryopreserved in liquid nitrogen. A myeloid progenitor cell dose of 203 X 10(4) CFU-GM/kg body weight was reinfused to the patient following marrow-ablative chemotherapy (MCNU 600 mg/m2, cytosine arabinoside 6 g/m2, etoposide 300 mg/m2, cyclophosphamide 160 mg/kg). Neutrophil count reached 0.5 X 10(9)/l by day + 7 and platelet count reached 20 X 10(9)/l by day + 9. Thereafter, white blood cell count continued to increase and reached a maximum of 38 X 10(9)/l on day + 14. Thus, the rapid recovery of hematopoiesis minimised marrow aplasia-related risks. This approach of stem cell rescue operation can be applied to the treatment of children with cancer, who otherwise have no hope to be cured, as an alternative to bone marrow transplantation.     

Immunomagnetic purging of breast cancer from bone marrow for autologous transplantation

Intensive chemotherapy with autologous bone marrow transplantation is a promising approach for the treatment of breast cancer, provided that clonogenic tumor cells do not contaminate the patient's bone marrow. We have previously demonstrated that a combination of 4-hydroperoxycyclophosphamide (4-HC) and immunomagnetic purging (IMP) with monoclonal antibodies and microspheres could remove 4-5 logs of clonogenic breast cancer cells from a 10-fold excess of human bone marrow cells. In the present report we have evaluated an apparatus for separating tumor cells from a large volume of human marrow. This apparatus will permit preparation of large volumes of purged marrow for use in studies of intensive therapy with autologous marrow support. Bone marrow progenitor cell (CFU-GM) recovery following this IMP technique was 85% of the unpurged control, and suggests that marrow recovery following high dose systemic chemotherapy will not be adversely affected. A phase I study to evaluate marrow reconstitution following IMP is underway. Preliminary data suggest that this IMP method will not delay engraftment in breast cancer patients receiving high-dose chemotherapy and autologous bone marrow support, but further study is required.     

Characterization of possible receptor of colony-promoting activity (CPA) by comparison with that of colony-stimulating factor (CSF)

Supernatants of long-term mouse bone marrow cell cultures contain colony-promoting activity (CPA). CPA itself does not stimulate colony formation of granulocyte-macrophage progenitor cells (GM-CFC) when added to a semisolid agar culture of bone marrow cells, but augments colony formation in the presence of colony-stimulating factor (CSF). The CPA-responsive cells are postulated to be pre-GM-CFC, a cell compartment younger than GM-CFC. In absorption experiments, CSF-coated bone marrow cells failed to absorb CPA, whereas normal bone marrow cells absorbed the activity. Cholera toxin (CT) is known to inhibit GM-CFC colony formation of bone marrow cells, probably by binding to GM-CFC-receptors [8, 9]. In the present studies, preincubation of the cells with CPA, before exposure to CT, reduced the suppressive effect of CT on GM-CFC colony formation. CT also inhibited pre-GM-CFC colony formation. Such suppressive effects, however, were reduced by preincubation of the cells not only with CPA but also with CSF. These results suggest that CSF and CPA might share the same receptors. It may also suggest that CT does not bind to specific sites. A difference in the susceptibility of GM-CFC and pre-GM-CFC to suppression by CT was also observed. Incubation of the cells with a low concentration of CT resulted in the substantial decrease of the number of GM-CFC, whereas the number of pre-GM-CFC remained high. Therefore, it seems that CPA shares some but not all of the CSF receptors.     

Bone metastasis: mechanisms and therapeutic opportunities

The skeleton is one of the most common sites for metastatic cancer, and tumors arising from the breast or prostate possess an increased propensity to spread to this site. The growth of disseminated tumor cells in the skeleton requires tumor cells to inhabit the bone marrow, from which they stimulate local bone cell activity. Crosstalk between tumor cells and resident bone and bone marrow cells disrupts normal bone homeostasis, which leads to tumor growth in bone. The metastatic tumor cells have the ability to elicit responses that stimulate bone resorption, bone formation or both. The net result of these activities is profound skeletal destruction that can have dire consequences for patients. The molecular mechanisms that underlie these painful and often incurable consequences of tumor metastasis to bone are beginning to be recognized, and they represent promising new molecular targets for therapy.     

Effective in vivo purging with rituximab and autologous peripheral blood stem cell transplantation in a woman with CD5 positive primary cutaneous diffuse large B-cell lymphoma

Abstract:  Generalized subcutaneous tumors developed without any other sites of the disease in a Japanese woman. Skin biopsy revealed CD5⁺ and CD20⁺ atypical diffuse large cells infiltrating subcutaneous tissues. The diagnosis was CD5⁺ primary cutaneous diffuse large B-cell lymphoma. Tumor-specific PCR showed the existence of malignant cells in the peripheral blood and bone marrow. After three cycles of chemotherapy, she was remained in partial remission. Peripheral blood stem cells (PBSC) were harvested after the fourth cycles of chemotherapy combined with rituximab for in vivo purging. The contamination of tumor cells in PBSC was negative with PCR. She then underwent autologous peripheral blood stem cell transplantation using purged PBSC and has remained in complete remission for the past 24 month.     

Purging breast cancer cells in preparation for autologous bone marrow transplantation

Mixtures of the T-47D human breast cancer cell line and normal human bone marrow cells were used for studying a new approach for purging epithelial tumor cells for autologous bone marrow transplantation (BMT) in breast cancer. Breast cancer cell line T-47D cells were shown to bind soybean agglutinin (SBA) in a specific fashion that could be blocked by D-galactose. Tumor cells were effectively purged by both SBA agglutination and depletion of cells bound to magnetic beads (0.7-5.0 micron) covalently linked to SBA. A depletion of 3-4 orders of magnitude of tumor cells was consistently accomplished by combining one step of agglutination followed by one cycle of SBA-magnetic bead depletion. Neither procedure affects stem cell recovery. We suggest that effective purging of breast cancer cells can be accomplished using SBA for autologous BMT in patients with advanced breast cancer.     

47例骨髓转移癌的临床及血液学特点

目的 研究骨髓转移癌的临床及血液学特点。方法 骨髓活检塑料包埋切片，H-Gmiesa-E,Gomori,PAS,Alcin blue染色。结果 47例患者中24例找到原发灶，其中胃癌15例，前列腺癌4例，肺癌2例，乳腺癌，结肠癌，肝癌各1例，34例做骨髓活检者骨髓均找到转移癌细胞，而其中10例骨髓象未能发现转移癌细胞，临床表现以贫血最常见（91.5%)，其次是血小板减少（64.7%)，骨痛（61.7%)和发热（48.9%)。结论 对于不明显原因的贫血和骨痛患者，骨髓活检对转移癌具有重要诊断价值。     

Detection of disseminated tumor cells in patients with cervical cancer

PURPOSE: Detection of disseminated tumor cells in a cohort of patients presenting the entire spectrum of invasive cervical cancer. METHODS: Disseminated tumor cells were detected in blood samples taken at different times during surgery or in bone marrow aspirates by a HPV type-specific nested PCR enzyme immunoassay (n-PCR-EIA). A group of 24 patients with HPV-positive cervical cancers representing early and late stages were evaluated, and 15 patients with breast cancer and without HPV-related genital disease served as controls. RESULTS: Disseminated tumor cells were detected in blood samples and/or bone marrow aspirates of 6 of 24 patients. A significant association was found between detectable disseminated tumor cells and recurrent disease ( P=0.013) and between disseminated tumor cells and survival of the patients ( P=0.0054). There was also a clear association between the presence of disseminated tumor cells and tumor size and/or positive lymph node status which, however, was not statistically significant. There was no evidence of increased shedding of tumor cells during surgery. CONCLUSION: Detection of disseminated tumor cells in blood or bone marrow may prove to be of prognostic value, particularly for early-stage cervical cancers.     

Faster engraftment of neutrophils and platelets with peripheral blood stem cells from unrelated donors: a comparison with marrow transplantation

Engraftment was achieved in 43/45 (95%) recipients of peripheral blood stem cells (PBSC) from HLA-compatible unrelated donors (n = 45), compared to all 45 patients in matched controls receiving bone marrow and 14/18 (78%) recipients of CD34-selected PBSC (P < 0.01). The time to reach ANC >0.5 x 10(9)/l was a median of 16 days in the PBSC and CD34 groups, compared to 20 days in the bone marrow controls (P < 0.001 vs PBSC). The time to reach platelets >50 x 10(9)/l was a median of 23 days in the PBSC group and 24 days in the CD34 group, which was significantly faster than 29 days in the bone marrow controls (P < 0.01). Acute GVHD grades II-IV developed in 30% in the PBSC group, 20% in the recipients of bone marrow and 18% in the CD34 group. The corresponding figures for chronic GVHD were 59%, 85% and 0% (P < 0.01) in the three groups, respectively. The probability of non-relapse death was 27% in the recipients of PBSC, 21% in the bone marrow controls and 60% in the CD34 group (NS). The 2-year leukaemia-free survival was 46% in the PBSC group, 41% in the bone marrow group and 25% in the CD34 group (NS).     

Circulating autologous stem cells collected in very early remission from acute non-lymphoblastic leukaemia produce prompt but incomplete haemopoietic reconstitution after high dose melphalan or supralethal chemoradiotherapy

Haemopoietic reconstitution (HR) using autologous peripheral blood stem cells (PBSC) was attempted after intensive chemotherapy or chemoradiotherapy in two patients with relapsed acute non-lymphoblastic leukaemia (ANLL). The PBSC were collected by leukapheresis very early in first remission and cryopreserved in liquid nitrogen. Both patients demonstrated early evidence of trilineage engraftment. The first patient received melphalan 200 mg/m2 followed by rescue with 1.3 X 10(8) mononuclear cells/kg body weight containing 29 X 10(4) granulocyte-macrophage progenitor cells (CFU-GM)/kg, and HR was evident by Day 14. The second patient was treated with supralethal chemoradiotherapy followed by rescue with 3.0 X 10(8) mononuclear cells/kg containing 23 X 10(4) CFU-GM/kg. He demonstrated early engraftment with near normal peripheral blood counts by Day 16. There was a subsequent fall in both bone marrow cellularity and peripheral blood counts to a level of low but persistent activity. There was a further phase of haematological recovery from 8 weeks following transplantation with an increase in peripheral blood counts and bone marrow cellularity until final relapse at 13 weeks. This study demonstrates that circulating stem cells have haemopoietic reconstitutive capacity, previously only shown with buffy coat cells from chronic granulocytic leukaemia. The minimum number of PBSC required for satisfactory engraftment remains unknown, although it seems probable that the ratio of pluripotent stem cells to committed progenitor cells is lower in very early remission peripheral blood than in either allogeneic normal bone marrow or autologous bone marrow collected later in stable remission. The question of leukaemic contamination of the PBSC remains to be answered.     

Autotransplants in men with breast cancer. ABMTR Breast Cancer Working Committee. Autologous Blood and Marrow Transplant Registry.

The purpose of this study was to determine the outcome of high-dose therapy with autologous hematopoietic stem cell support (autotransplants) in men with breast cancer. We studied 13 men receiving autotransplants for breast cancer and reported to the Autologous Blood and Marrow Transplant Registry (ABMTR) by 10 centers. Six men had stage 2 breast cancer, four had stage 3, and three had metastatic breast cancer. Of twelve tumors tested, all were estrogen receptor positive. Median age at transplant was 50 years. The most common conditioning regimen was cyclophosphamide, thiotepa and carboplatin (n = 5); the remaining eight men received other alkylator-based regimens. Three men received bone marrow, eight received blood stem cells, and two received both for hematopoietic support. All patients had hematopoietic recovery. There were no unexpected regimen-related toxicities. Of 10 men receiving autotransplants as adjuvant therapy, three relapsed 3, 5 and 50 months post-transplant and died 16, 19 and 67 months post-transplant. Seven of 10 are disease-free with median follow-up of 23 months (range 6-50 months). Of three men treated for metastatic breast cancer, one had progressive disease and two recurrent disease at 6, 7 and 16 months post-transplant. In conclusion, results of autotransplants for male breast cancer appear similar to those reported for women receiving autotransplants for breast cancer.     

Addition of peripheral blood stem cells collected without mobilization techniques to transplanted autologous bone marrow did not hasten marrow recovery following myeloablative therapy.

A randomized prospective trial was conducted to determine if the addition of cryopreserved autologous peripheral blood stem cells (PBSC) collected without mobilization techniques to autologous cryopreserved bone marrow for patients receiving an autologous bone marrow transplant (ABMT) affected the time to marrow function recovery. Thirty-five evaluable patients with various malignancies were studied. Sixteen received PBSC + ABMT and 19 received ABMT alone. The PBSC were collected with 4 h leukapheresis procedures on 3 consecutive days. No manipulations to increase the number of circulating stem cells were used during the collections. The median time to recover 0.5 x 10(9)/l circulating granulocytes was 20 days after transplantation in the ABMT group and 27 days in the PBSC + ABMT group (p = 0.12). The median time to recover 20 x 10(9)/l platelets was 22 days after transplantation in the ABMT group and more than 27 days in the PBSC + ABMT group (p = 0.29). The day of discharge from the hospital was earlier for the ABMT group (median 29 days) than the PBSC + ABMT group (median 35 days, p = 0.03). We did not find that the addition of non-mobilized PBSC to infused autologous marrow accelerates marrow recovery.