Detection of cytokeratin-positive cells in the bone marrow of breast cancer patients undergoing adjuvant therapy

Summary The presence of cytokeratin-positive cells in the bone marrow of breast cancer patients has been proven to be an independent prognostic factor. Their fate in primary breast cancer patients undergoing adjuvant therapy is of particular interest. We investigated the bone marrow status of 112 patients undergoing postoperative adjuvant treatment before and after therapy. A total of 373 patients with histologically confirmed primary breast cancer underwent bone marrow aspiration at the time of primary surgery. All patients were informed of their bone marrow status and offered repeat aspiration after 12 months. All patients were then treated with adjuvant chemotherapy, endocrine therapy or both based on current treatment recommendations. About 112 patients returned for a second bone marrow aspiration after a mean interval of 12 months following the initiation of adjuvant treatment. In 93 of 112 patients (83%) disseminated tumor cells had been found in the bone marrow before initiation of systemic chemo/endocrine therapy. At the time of follow-up sampling, after surgery and completion of adjuvant chemotherapy, the positivity rate dropped to 24%. Positive bone marrow status during follow-up was only associated with grading (p=0.020). Adjuvant treatment regimens are not able to completely eliminate cytokeratin-positive cells from the bone marrow. Prospective studies need to evaluate, whether these cells could become targets for additional adjuvant therapy.     

Monoclonal antibody detection of carcinoma cells in bone marrow biopsy specimens from breast cancer patients

The possibility of using immunologic methods for detecting metastatic cells in bone marrow samples from breast cancer patients was investigated. The MBr1 monoclonal antibody, which recognizes a membrane antigen on breast carcinoma cells and is unreactive on normal bone marrow cells, seemed to be an adequate reagent for this kind of approach. When human leukocyte suspensions artificially contaminated with mammary tumor cells were tested by MBr1 immunofluorescence, it was demonstrated that the added tumor cells could be specifically discriminated from normal cells and that as little as one tumor cell in 200,000 could be detected. With the same methodology we screened bone marrow biopsies from breast cancer patients with apparently uninvolved lymph nodes at the moment of surgery. Immunoreactive tumor cells were detected by the MBr1 antibody in 17% of N- patients. None of the bone marrow samples showed any evidence of tumor involvement by conventional histologic analysis.     

Monitoring of disseminated tumor cells in bone marrow in high-risk breast cancer patients treated with high-dose chemotherapy

The present study aimed to investigate the clinical relevance of disseminated tumor cells (DTC) in breast cancer patients before and after high-dose adjuvant chemotherapy with or without progenitor stem-cell support. One hundred and eighteen high-risk stage II breast cancer patients entering the Scandinavian Study Group multicenter trial were randomized to 9 cycles of tailored and dose-escalated FEC (5-fluorouracil, epirubicin, cyclophosphamide) or 3 cycles of standard FEC followed by high-dose chemotherapy. Bone marrow (BM) samples at diagnosis and 6 months after completion of chemotherapy were assessed for the presence of cytokeratin positive (CK+) cells. Before treatment, 29% of the patients were CK+ (21% in the dose-escalated group and 36% in the high-dose-group). Six months after treatment, 17% of the patients were CK+ (17 and 16% respectively). Of the 95 patients who were evaluated 6 months after treatment, 60% were consistently CK-. CK+ cells in BM was evaluated as a prognostic and predictive marker and compared to other defined prognostic factors of the primary tumor. Monitoring BM changes at the time of diagnosis and 6 months posttreatment is an independent predictive factor for breast-cancer-specific survival (BCS) (p = 0.001). Those who have consistent CK negative (-) BM findings constitute a group of patients with good prognosis. Our results suggest that changes in CK+ cells in BM before and after chemotherapy can be used clinically as a surrogate maker to predict outcome in breast cancer patients.     

Comparative evaluation of cell-free tumor DNA in blood and disseminated tumor cells in bone marrow of patients with primary breast cancer

Introduction  

The origin and clinical relevance of circulating cell-free tumor DNA in the blood of cancer patients is still unclear. Here we investigated whether the detection of this DNA is related to bone marrow (BM) micrometastasis and tumor recurrence in breast cancer patients.     

ERalpha-status of disseminated tumour cells in bone marrow of primary breast cancer patients

Introduction  

Isolated disseminated tumour cells (DTC) are regarded as surrogate markers for minimal residual disease in breast cancer. Characterisation of these cells could help understand the known limitations of adjuvant therapy. Of particular interest is their oestrogen-receptor (ER) status because endocrine adjuvant therapy remains a cornerstone of breast cancer treatment.     

The prognostic impact of cytokeratin-positive cells in bone marrow of patients with localized prostate cancer

Our study evaluates the prognostic significance of the cytokeratin-positive mononuclear cells (CK+ cells) in the bone marrow (BM) and peripheral blood (PB) as detected by immunocytochemistry in patients with locoregionally confined prostate cancer. BM and PB samples were obtained from 66 newly diagnosed patients with T1-4pN0M0 prostate cancer. All samples were analyzed by standardized immunocytochemical methods (anticytokeratin mononuclear antibody; AE1/AE3) applying a negative immunomagnetic cell enrichment technique. A second sampling was obtained in 60 of the 66 patients >or=2 years after definitive radiotherapy. The median follow-up after high-dose radiotherapy of the patients was 65 months. For the analysis of the postradiotherapy clinical progression-free survival (PFS) treatment, failure was defined as pelvic tumor growth or development of distant metastases. At diagnosis CK+ cells were found in BM in 14 of 66 (21%) prostate cancer patients. This was not associated with an increased risk of progression. On the other hand, the presence of CK+ cells in 12 of 60 (20%) patients at the second BM aspiration was significantly related to a shorterPFS (p = 0.02). In the multivariate analysis, the presence of CK+ cells in the posttreatment BM did not remain as an independent variable of PFS assessment if posttreatment PSA was entered into the analysis. CK+ cells in PB were found in 12% of the patients. After therapy, none of the patients had detectable CK+ cells in PB. The presence of CK+ cells in the posttreatment but not in the pretreatment BM was associated with decreased PFS in patients irradiated for pelvis-confined nonmetastatic prostate cancer. Although this association was not retained in multivariate analysis, our observations indicate that the presence of CK+ cells after local therapy define a group of patients that have a high risk of developing distant metastases.     

Sequential immunochemotherapy and edrecolomab in the adjuvant therapy of breast cancer: reduction of 17-1A-positive disseminated tumour cells.

BACKGROUND: The aim of our study was to evaluate the efficacy of the monoclonal antibody edrecolomab after chemo- and radiotherapy in the elimination of disseminated tumour cells in bone marrow in the adjuvant therapy of breast cancer. PATIENTS AND METHODS: The bone marrow of 25 patients with breast cancer was tested for the presence of disseminated tumour cells using the pancytoceratine antibody and the alkaline phosphatase-anti-alkaline-phosphatase (APAAP) technique. To characterize tumour cells simultaneously, immunofluorescent double labelling of pancytoceratine and epithelial cell adhesion molecule (antibody 17-1A) was performed on tumour cells after magneto bead enrichment. Patients positive for the 17-1A antigen in bone marrow after chemotherapy were treated with edrecolomab (500 mg Panorex) initially, then 100 mg/month over 4 months) and investigated for the presence of micrometastases 6 weeks after the last treatment. RESULTS: Of the 17 patients showing bone marrow micrometastases (BM-MM), 14 tested 17-1A positive before adjuvant chemotherapy. After chemotherapy, nine patients remained positive for the 17-1A antigen and were treated with edrecolomab. The final investigation after immunotherapy showed a complete elimination of the 17-1A-positive BM-MM in seven patients and a significant reduction of these cells in two patients. CONCLUSIONS: Sequential treatment of breast cancer with edrecolomab after adjuvant chemotherapy can reduce disseminated tumour cells in the bone marrow and eliminate 17-1A-positive micrometastases.     

Presence of disseminated tumor cells in bone marrow correlates with tumor stage and nodal involvement in cervical cancer patients

Detection of disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer patients is associated with poor outcome. The aim of our study was to evaluate the impact of BM status on survival in a large cohort of cervical cancer patients. Three hundred twenty‐five patients with cervical cancer were included into this prospective two‐center study (University Hospitals Tuebingen, Munich, Germany). BM was collected preoperatively. DTCs were identified by immunocytochemistry using the pancytokeratin antibody A45B/B3. DTCs were detected in 22% of all BM aspirates. The number of CK‐positive cells ranged from 1 to 93 per 2 × 10⁶ mononuclear cells. Eighteen percent of patients with T1 stage presented with DTCs in BM compared to 30% in T2 and 45% in T3/4 patients. Among nodal negative patients, 18% had tumor cells in BM compared to 32% of nodal positive patients. Positive DTC status was associated with tumor size (p = 0.007) and nodal status (p = 0.009) but not with grading (p = 0.426). DTC status did not correlate with overall or disease‐free survival. In the univariate analysis, tumor stage, nodal status, resection status and grading correlated with OS and DFS. In the multivariate analysis, only tumor stage and nodal status were independent predictors of OS and tumor stage, nodal status and grading of DFS. Tumor cell dissemination into BM is thus a common phenomenon in cervical cancer and correlates with higher tumor load but lacks prognostic relevance. Alternative detection methods may be needed to establish prognostic potential.     

Genomic analysis of single cytokeratin-positive cells from bone marrow reveals early mutational events in breast cancer

Chromosomal instability in human breast cancer is known to take place before mammary neoplasias display morphological signs of invasion. We describe here the unexpected finding of a tumor cell population with normal karyotypes isolated from bone marrow of breast cancer patients. By analyzing the same single cells for chromosomal aberrations, subchromosomal allelic losses, and gene amplifications, we confirmed their malignant origin and delineated the sequence of genomic events during breast cancer progression. On this trajectory of genomic progression, we identified a subpopulation of patients with very early HER2 amplification. Because early changes have the highest probability of being shared by genetically unstable tumor cells, the genetic characterization of disseminated tumor cells provides a novel rationale for selecting patients for targeted therapies.     

Primary systemic therapy does not eradicate disseminated tumor cells in breast cancer patients

Introduction The presence of disseminated tumor cells in the bone marrow of breast cancer patients has proven to be an independent prognostic factor. The aim of this study was to investigate the status of tumor cell dissemination after primary systemic therapy in relation to therapy response. Methods Bone marrow aspirates were obtained from 120 patients after completion of primary systemic therapy. Disseminated tumor cells were detected by immunocytochemistry using the APAAP method. Bone marrow status was correlated with clinicopathological factors as well as tumor response to primary systemic therapy. Results Sixty out of 120 patients had disseminated tumor cells in their bone marrow aspirates (50%). Response rates were 18% for pathologic complete remission, 52% for partial remission, 28% for no change and 3% for progression. Despite complete remission, 36% of these patients were bone marrow positive. In the partial remission group, the positivity rate was 48%. About 61% of patients with stable disease had disseminated tumor cells in their bone marrow. A trend to higher positivity rates was observed in the poor responder group compared to responders (61% vs. 38%, P = 0.1). Conclusion Primary systemic therapy does not completely eradicate disseminated tumor cells in the bone marrow of breast cancer patients. The biological role of persistent disseminated tumor cells needs to be further investigated to optimize current and future therapeutic strategies.     

Monoclonal antibodies for detection of occult carcinoma cells in bone marrow of breast cancer patients

Metastases develop in 30% to 40% of patients with operable breast cancer. Investigators have reported on the detection of occult micrometastases in bone marrow using an antibody to epithelial membrane antigen (EMA) and have since reported prognostic significance for these antibody-detected cells. In this study, two anti-cytokeratin monoclonal antibodies (35 beta H11 and 34 beta E12) were used to examine bone marrow specimens from patients with breast cancer. The technique was first studied in a test system in which human or monkey bone marrow was seeded with MCF-7 cells, and was determined to be sensitive enough to detect fewer than one cancer cell in 10(4) hematopoietic cells. An immunoglucose oxidase method was used for patient specimen antibody localization and was found to be free of false-positive staining. Marrow specimens from 25 patients with breast cancer of various stages were examined. No correlation with disease stage was observed. We conclude that the technique is feasible, but prognostic import remains to be determined.     

Immunohistochemical detection of tumor cells in the bone marrow of breast cancer patients

BACKGROUND: Contamination of bone marrow and peripheral blood stem cells with tumor cells is a problem that may be encountered when autologous hematopoietic stem cell transplantation is conducted concurrently with high-dose chemotherapy. METHODS: Using monoclonal antibodies to a variety of tumors, the detection of tumor cells in the bone marrow of breast cancer patients was studied by immunohistochemistry. RESULTS: KL-1 and CAM5.2 were strongly reactive with breast cancer cells, but not with normal bone marrow cells. The reactivity of the tumor cells with EMA was not strong, and DF-3 and 115D8 yielded only slightly positive reactions. These latter antibodies also exhibited some reactivity to normal bone marrow cells. When tumor cells were admixed with normal cells, the sensitivity of CAM5.2 and EMA permitted the detection of one cell in 10(4), but with KL-1, the detection of one in 10(5) cells was possible. When immunohistochemical staining was used in testing 40 patients with advanced or recurrent breast cancer, positive reactions were obtained in four of 27 patients (14.8%) with KL-1, four of 26 (15.4%) with CAM5.2, and nine of 37 (23.7%) with KL-1 + CAM5.2, figures similar to those reported by others who studied stage IV patients. CONCLUSIONS: Immunohistochemical staining with KL-1 and CAM5.2 is therefore considered to be a useful technique for detecting contamination by tumor cells.      

A concept for the standardized detection of disseminated tumor cells in bone marrow from patients with primary breast cancer and its clinical implementation

Numerous single-institutional studies and a large pooled analysis have demonstrated that the presence of disseminated tumor cells (DTCs) in the bone marrow (BM) from patients with primary, nonmetastatic breast cancer (Stages I-III) is associated with impaired prognosis. To date, sampling of BM and assessment of DTCs is not considered a routine procedure in the clinical management of breast cancer patients; however, emerging data suggest a future role for risk stratification and monitoring of therapeutic efficacy. Because these clinical options need to be evaluated in trials to verify the principle of this concept in the clinical setting, agreement on the standardized detection of DTCs is necessary. Consequently, the German, Austrian, and Swiss Societies for Senology recently formed a panel 1) to review and discuss the existing methodologies, 2) to find a consensus for a standardized detection of DTCs, and 3) to explore the options for its clinical implementation.     

Lack of effect of adjuvant chemotherapy on the elimination of single dormant tumor cells in bone marrow of high-risk breast cancer patients.

PURPOSE: There is an urgent need for markers that can predict the efficacy of adjuvant chemotherapy in patients with solid tumors. This study was designed to evaluate whether monitoring of micrometastases in bone marrow can predict the response to systemic chemotherapy in breast cancer. PATIENTS AND METHODS: Bone marrow aspirates of 59 newly diagnosed breast cancer patients with either inflammatory (n = 23) or advanced (> four nodes involved) disease (n = 36) were examined immunocytochemically with the monoclonal anticytokeratin (CK) antibody A45-B/B3 (murine immunoglobulin G(1); Micromet, Munich, Germany) before and after chemotherapy with taxanes and anthracyclines. RESULTS: Of 59 patients, 29 (49.2%) and 26 (44.1%) presented with CK-positive tumor cells in bone marrow before and after chemotherapy, respectively. After chemotherapy, less than half of the previously CK-positive patients (14 of 29 patients; 48.3%) had a CK-negative bone marrow finding, and 11 (36. 7%) of 30 previously CK-negative patients were CK-positive. At a median follow-up of 19 months (range, 6 to 39 months), Kaplan-Meier analysis of 55 assessable patients revealed a significantly reduced overall survival (P =.011; log-rank test) if CK-positive cells were detected after chemotherapy. In multivariate analysis, the presence of CK-positive tumor cells in bone marrow after chemotherapy was an independent predictor for reduced overall survival (relative risk = 5.0; P =.016). CONCLUSION: The cytotoxic agents currently used for chemotherapy in high-risk breast cancer patients do not completely eliminate CK-positive tumor cells in bone marrow. The presence of these tumor cells after chemotherapy is associated with poor prognosis. Thus, bone marrow monitoring might help predict the response to systemic chemotherapy.     

Abscisic acid regulates dormancy of prostate cancer disseminated tumor cells in the bone marrow

Prostate cancer (PCa) commonly metastasizes to the bone where the cells frequently undergo dormancy. The escape of disseminated tumor cells from cellular dormancy is a major cause of recurrence in marrow. Abscisic acid (ABA), a phytohormone, is known to regulate dormancy of plant seeds and to regulate other stress responses in plants. Recently, ABA was found to be synthesized by mammals cells and has been linked to human disease. Yet the role of ABA in regulating tumor dormancy or reactivation is unknown. We found that ABA is produced by human marrow cells, and exogenous ABA inhibits PCa cell proliferation while increasing the expression of p27, p21, and p16 and decreasing the expression of the proliferation marker, Ki67. Further, ABA significantly increased the percentage of PCa cells in the G₀ phase of the cell cycle as well as the duration the cells were arrested in G₀. We found that ABA regulates an increase of PPARγ receptor expression and suppressed phosphorylation of mTOR/p70S6K signaling and resulting in the induction of the cellular dormancy. We then confirmed that ABA regulates G₀ cell cycle arrest through PPARγ receptor signaling in vitro and under co-culture conditions with osteoblasts. Finally, we demonstrate that ABA regulates PCa dormancy in vivo following intratibial injection in an animal model. Together these data suggest that the ABA and PPARγ signaling pathways contribute to the establishment of PCa cellular dormancy in the bone marrow microenvironment. These findings may suggest critical pathways for targeting metastatic disease.     

Detection of tumor cells in bone marrow of patients with primary breast cancer: a prognostic factor for distant metastasis.

PURPOSE: At the time of primary surgery, approximately 90% of all patients with breast cancer are free of metastases, but in the next 5 years almost 50% of them will relapse. We evaluated the significance of the presence of tumor cells in bone marrow of patients with primary breast cancer to investigate their predictive value for relapse. PATIENTS AND METHODS: Two hundred sixty patients with primary breast cancer were examined for tumor cells in bone marrow aspirates taken from six sites of the skeleton. After density centrifugation, cells in interphase were smeared and stained. For the immunocytologic reaction, we used a new monoclonal antibody (2E11) that was reactive with the core protein of the tumor-associated glycoprotein TAG12. TAG12 is secreted by nearly all human breast carcinomas. RESULTS: A significant correlation was found between tumor-cell detection and tumor stage (P < .0001), nodal status (P < .0001), and tumor grading (P = .002). A good relation to progesterone receptor (PR; P = .008) was found, but there was no correlation to estrogen receptor (ER) and menopausal status. Follow-up examinations showed distant metastases in 26 of 211 patients (15%). Twenty-two relapses occurred among the 81 patients with 2E11-positive cells in bone marrow, but only four occurred among the 130 patients without tumor-cell detection. CONCLUSIONS: This study suggests that tumor-cell detection in bone marrow of patients with primary breast carcinoma is a good predictor for all distant relapses (P < .0005, Cox multiple regression analysis) and provides additional information in regard to other prognostic factors. The highest predicting value for distant metastasis results from the combination of nodal status, negative PR, and tumor-cell presence in bone marrow.