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Purpose: Presence of disseminated tumour cells (DTCs) in the bone marrow (BM) has been described as a surrogate of residual disease in patients with early breast cancer (EBC). PADDY (Pooled Analysis of DTC Detection in Early Breast Cancer) is a large international analysis of pooled data that aimed to assess the prognostic impact of DTCs in patients with EBC., Experimental Design: Individual patient data were collected from 11 centres. Patients with EBC and available follow-up data in whom BM sampling was performed at the time of primary diagnosis before receiving any anticancer treatment were eligible. DTCs were identified by antibody staining against epithelial cytokeratins. Multivariate Cox regression was used to compare the survival of DTC-positive versus DTC-negative patients., Results: In total, 10,307 patients were included. Of these, 2814 (27.3%) were DTC-positive. DTC detection was associated with higher tumour grade, larger tumour size, nodal positivity, oestrogen receptor and progesterone receptor negativity, and HER2 positivity (all p < 0.001). Multivariate analyses showed that DTC detection was an independent prognostic marker for overall survival, disease-free survival and distant disease-free survival with hazard ratios (HR) and 95% confidence intervals (CI) of 1.23 (95% CI: 1.06-1.43, p = 0.006), 1.30 (95% CI: 1.12-1.52, p < 0.001) and 1.30 (95% CI: 1.08-1.56, p = 0.006), respectively. There was no association between locoregional relapse-free survival and DTC detection (HR 1.21; 95% CI 0.68-2.16; p = 0.512)., Conclusions: DTCs in the BM represent an independent prognostic marker in patients with EBC. The heterogeneous metastasis-initiating potential of DTCs is consistent with the concept of cancer dormancy., Competing Interests: Conflict of interest statement The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ADH, FAT and SYB report a grant from Exact Sciences. TNF received personal fees from AstraZeneca, Novartis, Roche, Pfizer, Esai, Tesaro, Teva, Celgene, Daichi Sankyo, MSD, Menarini. PAF reports grants from Cepheid, Novartis and Biontech, personal fees from Novartis, Roche, Pfizer, Celgene, Daiichi-Sankyo, TEVA, Astra Zeneca, Merck Sharp & Dohme, Myelo Therapeutics, Macrogenics, Eisai, Puma and Lilly. CAK is a member of the SAB of HiberCell, New York. All other authors declare no potential conflicts of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Introduction: From the early days of pathology back in the nineteenth century until now, there has been an ongoing search for the missing link between solid tumors such as breast cancer and distant metastases, which sometimes occur many years after removal of the primary tumor. The "seed and soil" theory hypothesizes the early dissemination of occult tumor cells into blood or bone marrow, which can persist in a dormant state for a long time and then become precursors of metastases in distant organs which offer appropriate conditions., Method: Advances in immunocytochemical methods have enabled the enrichment and visualization of those disseminated tumor cells in bone marrow (DTC-BM) or circulating tumor cells (CTC) in blood. Many studies could demonstrate prognostic significance of the detection of DTC-BM or CTC in different stages of breast cancer., Conclusion: Further characterization of those cells by immunocytochemical stainings, fluorescence in situ hybridizations, or PCR-based molecular methods will help to understand the biology of tumor cell dissemination and metastasis formation, as well as to define potential drug targets.

Objective: The aim of this study was to measure the human epidermal growth factor receptor 2 (HER2) status of disseminated tumor cells (DTCs) from bone marrow (BM) aspirates and to assess correspondence or discrepancy with the primary tumor., Methods: DTCs were isolated from the BM of 156 breast cancer patients. Cytokeratin-positive DTCs were further analyzed by the chromogenic in situ hybridization method to detect HER2 gene amplification., Results: A significant correlation (p = 0.021) was found between the HER2 status of DTCs and the primary tumors. Sixty-one (68.5%) patients had a corresponding status. However, a shift of phenotype between primary tumor and DTCs was found in the remaining patients., Conclusion: This study showed a significant grade of discordance of the HER2 status between primary tumors and DTCs in the BM of a relevant subgroup of patients. Detection of HER2 amplification on DTCs could therefore help to better stratify patients for a more tailored therapy, since they would benefit from a HER2-targeted therapy., (© 2016 S. Karger AG, Basel.)

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 × 10(6) 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., (© 2013 UICC.)

Background: The presence of disseminated tumor cells (DTC) in bone marrow (BM) of breast cancer patients is associated with reduced clinical outcome. Bisphosphonate treatment was shown to eradicate DTC from BM in several studies. This controlled randomized open-label multi-center study aimed to investigate the influence of zoledronic acid (ZOL) on DTC and survival of breast cancer patients (Clinical Trial Registration Number: NCT00172068)., Methods: Patients with primary breast cancer and DTC-positive bone marrow were randomized to treatment with ZOL plus adjuvant systemic therapy (n = 40) or adjuvant systemic therapy alone (n = 46) between 03/2002 and 12/2004. DTC were identified by immunocytochemistry using the pancytokeratin antibody A45B/B3 and by cytomorphology. The change in DTC numbers at 12 months and 24 months versus baseline, as well as patient outcomes were evaluated., Results: 86 patients could be included into survival analysis (median follow-up: 88 months, range: 8-108 mths). Patients in the control group were more likely to die during follow-up than those in the ZOL-group (11% vs. 2%, p = 0.106). 15% of patients in the control group presented with relapse whereas only 8% of ZOL group patients developed metastatic or recurrent disease during follow-up (p = 0.205). At 24 months, 16% of patients from the control group were still DTC positive, whereas all patients treated with ZOL became DTC negative (p = 0.032). Patients presenting with persistent DTC 12 months after diagnosis had significantly shorter overall survival (p = 0.011)., Conclusions: Bisphosphonate therapy contributes to eradication of disseminated tumor cells. The positive influence of bisphosphonates on survival in the adjuvant setting may be due to their effects on DTC., Trial Registration: ClinicalTrials.gov Identifier: NCT00172068 [Zoledronic Acid in the Treatment of Breast Cancer With Minimal Residual Disease in the Bone Marrow (MRD-1)].

Purpose: The detection of disseminated tumor cells in the bone marrow (DTC-BM) of breast cancer patients is an independent prognostic factor. In recent years, the focus of research was on finding methods for the detection of circulating tumor cells (CTC) in peripheral blood (PB). In this study, we investigated the presence of DTC-BM and CTC by simultaneous examinations in 202 patients at different stages of the disease., Methods: Immunocytochemical examination of DTC-BM (10-20 ml of BM) with the anti-cytokeratin (CK) antibody A45B/B3 followed a standardized protocol. Analysis of PB (7.5 ml) for the presence of CTC was performed with the CellSearch Analyzer system (Veridex, Raritan, NJ, USA)., Results: Overall, DTC-BM were detected in 57/202 (28.2 %, 1->1,000 DTC) and CTC in 41/202 (20.3 %, 1-411 CTC) patients. Congruence of findings was 71.3 % (144/202, p = .002). In 147 pts with primary diagnosis, congruence of results was 69.4 % (102/147). There was no significant correlation between DTC or CTC and the established pathological factors. After a median follow-up time of 34 months (0-82), presence of CTC was borderline significant for tumor-associated death (p = .060). For 41 patients at recurrence-free follow-up, congruence of results was 75.6 % (31/41, p = .018). In this group, there was a patient with both the highest DTC (>1,000) and CTC (411) count, and she presented with distant metastases 3 months later and had died 5 months after that. Of 14 patients with metastatic disease, 9 showed both DTC and CTC (overall congruence 78.6 %, p = .176)., Conclusion: There was significant congruence between DTC and CTC, which even increased in patients at follow-up and in those with metastases. Repeated CTC examinations could be a valuable tool for monitoring patients or the effectiveness of therapies.

Purpose: Isolated tumor cells (ITC) in the bone marrow of breast cancer patients increase the risk of recurrence and decrease survival, both at primary diagnosis and during follow-up. We tested the efficacy of trastuzumab in clearing HER2/neu-positive ITC from the marrow of patients completing primary treatment., Methods: Ten recurrence-free patients with persistent HER2/neu-positive ITC after routine adjuvant treatment received trastuzumab 6 mg/kg q3w for 12 months in a non-randomized pilot phase II interventional study. Bone marrow ITC HER2/neu status was evaluated at baseline, after treatment for 3, 6 and 12 months, and yearly thereafter, in combination with clinical follow-up. Median follow-up was 23 (15-64) months after baseline bone marrow aspiration., Results: Trastuzumab for 12 months eradicated HER2/neu-positive ITC from bone marrow in all patients (P = 0.002) and significantly reduced the number of ITC-positive patients (P = 0.031). However, HER2/neu-negative ITC persisted in three patients immediately after treatment and were detected at yearly bone marrow aspiration in five patients. Two patients with ITC counts ≥5 at yearly follow-up developed metastases and one died., Conclusion: This is the first evidence that trastuzumab is effective in clearing HER2/neu-positive cells from bone marrow during recurrence-free follow-up in breast cancer patients. It also suggests, thanks to the antigen shift phenomenon, an important prognostic role for HER2/neu expression on marrow ITC as a real-time biopsy. However, treatment was mainly effective in patients with HER2/neu-positive ITC. Given the heterogeneity of minimal residual disease, these patients might benefit from a combination of targeted treatment approaches.

Minimal residual disease (MRD), i.e., isolated tumor cells (ITC) in bone marrow, may be the source of potentially fatal overt distant metastases in solid tumors even years after primary treatment. MRD can be detected by immunohistochemical methods using antibodies directed against cytokeratins, cell-surface markers, or molecular PCR-based techniques. Among solid tumors, the clinical relevance of MRD has been most extensively studied in breast cancer patients. The highest level of evidence for the prognostic impact of MRD in primary breast cancer was reached by a pooled analysis comprising more than 4,000 patients, showing poor outcome in patients with MRD at primary therapy. Yet, clinical application of MRD detection is hampered by the lack of a standardized detection assay. Moreover, clinical trial results demonstrating the benefit of a therapeutic interference derived from bone marrow status are still missing. Recent results suggest that in addition to its prognostic impact, MRD can be used for therapy monitoring or as a potential therapeutic target after phenotyping of the tumor cells. Persisting MRD after primary treatment may lead to an indication for extended adjuvant therapy. In a pooled analysis bone marrow aspirates of 726 patients from academic breast cancer units in Oslo (n=356), Munich (n=228), and Tuebingen (n=142) were analyzed during recurrence-free follow-up at a mean interval of 31.7 months after primary diagnosis of breast cancer pT1-4, pN0-3 pM0. Persistent ITC was detected in 15.4% of the patients (n=112). The Kaplan-Meier estimate for mean distant relapse-free survival estimate was 163.6 months in patients with negative and 105.2 months in patients with positive BM status. Patients without evidence of persistent ITC had a significantly longer overall survival (165.6), than patients with positive bone marrow status (103.3 months, p < .0001). Given these inspiring results on ITC in the bone marrow, several trials currently analyze the prognostic relecance of circulating tumor cells (CTC) in peripheral blood in the adjuvant setting. Persisting MRD after primary treatment may lead to an indication for extended adjuvant therapy. However, until clinical consequences of MRD detection in solid tumors and particularly in breast cancer have been validated, the detection of isolated tumor cells in bone marrow should be performed mainly in clinical trials.

Background: The prognostic significance of disseminated tumor cells from bone marrow (BM-DTCs) of breast cancer patients has been demonstrated previously. In this study, data of a standardized long term follow-up of 829 patients with examination of BM-DTCs at primary diagnosis are presented., Patients and Methods: BM aspiration and immunocytochemical examination of DTCs was performed according to a standardized protocol. Follow-up data of all patients were adjusted with the cancer registries of southern Bavaria., Results: A total of 268 patients (32%) had BM-DTCs with a median of 2 (1-1223)/2 x 10⁶ cells. Positive BM findings correlated with tumor size (p=0.032), but not with other histopathological parameters. After a median follow-up of 73 months, BM-DTCs were highly relevant for the development of distant metastases (p=0.006) and, beneath standard histological parameters, reduced overall survival (p=0.038)., Conclusion: These results confirm the prognostic relevance of the detection of BM-DTCs. Newer methods, such as detection of circulating tumor cells in blood, will have to demonstrate comparable prognostic information in the future.

Background: The prognostic significance of disseminated tumor cells (DTC) in bone marrow (BM) of breast cancer patients at the time of primary diagnosis has been confirmed by a large pooled analysis. In view of the lack of early indicators for secondary adjuvant treatment, we here evaluated whether the persistence of DTCs after adjuvant therapy increases the risk of subsequent relapse and death., Patients and Methods: Individual patient data from 676 women with primary diagnosis of early breast cancer stages I-III from 3 follow-up studies were pooled. During clinical follow-up, patients underwent BM aspiration (BMA) to determine the presence of DTC. Tumor cells were detected by the standardized immunoassays. Univariate and multivariable proportional hazards models were estimated to assess the prognostic significance of DTC for disease-free survival (DFS) and overall survival (OS)., Results: Patients were followed for a median of 89 months. BMA was performed at median 37 months after diagnosis of breast cancer. At follow-up BMA, 15.5% of patients had DTCs. The presence of DTC was an independent indicator of poor prognosis for DFS, distant DFS (DDFS), cancer-specific survival, and OS during the first 5 years following cancer diagnosis (log-rank test P < 0.001 values for all investigated endpoints)., Conclusion: Among breast cancer patients, persistent DTCs during follow-up significantly predicted the increased risk for subsequent relapse and death. Analysis of DTC might serve as a clinically useful monitoring tool and should be tested as an indicator for secondary adjuvant treatment intervention within clinical trials., (©2011 AACR.)

Background and Objectives: Recently the highest level of evidence indicated the prognostic value of isolated tumor cells (ITC) in bone marrow of patients with breast cancer, both at primary diagnosis and during recurrence-free follow-up. The aim of the present study was to investigate the therapeutic efficacy of zoledronate in reducing persistence of ITC in the bone marrow of patients with breast cancer after they had completed primary therapy., Patients and Methods: In a non-randomized phase II pilot study, 4 mg of zoledronate were administered once every four weeks for six months, after a initial loading dose of 8 mg, to 31 patients with persisting ITC in bone marrow. All patients had completed surgery and adjuvant chemotherapy, if indicated, at least 6 months previously. The bone marrow was re-examined after 7.9 months (std 0,89). ITC were detected by immunocytochemical staining, using the monoclonal pan-cytokeratin antibody A45-B/B3 and the APAAP technique. Patients were followed-up prospectively for a median of 39 months after the first aspiration., Results: ITC were detected in all 31 patients at the time of first bone marrow aspiration, but 27 of them (87 %) were free of ITC 6 months after the end of zoledronate therapy. The reduction in cell numbers between first and second aspiration were statistically significance (P < 0,0001). Ten of 12 patients without detection of ITC in bone marrow after treatment and who had undergone additional aspirations, still had no ITC a median time of 19 months (range 4.7 - 38.7 months) after the end of treatment. Zoledronate treatment was well tolerated, bone pain having been the most common side effect in 45 % of patients (n = 14)., Conclusion: These results indicate a potential antineoplastic effect of zoledronate, a cell-cycle independent drug, on persisting ITC in a dormant state. Our data provide the basis for investigating the efficacy of zoledronate on ITC in treating primary breast cancer in prospectively randomized trials. ITC in bone marrow represents a useful marker in selecting patients at risk for recurrence and for monitoring therapeutic efficacy.

Purpose: The presence of disseminated tumor cells in the bone marrow (DTC-BM) of breast cancer patients has shown independent prognostic impact. Immunomagnetic enrichment of such cells is an approach to increase the number of detected cells with limited sample volume, especially for circulating tumor cells (CTCs) in blood. The Thomsen-Friedenreich (TF) antigen (CD 176) is a specific oncofetal carbohydrate epitope (Galbeta1-3GalNAcalpha-O) expressed on the surface of various carcinomas. Own studies demonstrated a nearly complete TF expression on DTC-BM, indicating its suitability as marker for immunomagnetic enrichment., Methods: BM samples of 65 and peripheral blood samples of 11 breast cancer patients were examined immunocytochemically by staining with the anti-Cytokeratin antibody A45-B/B3 before and after immunomagnetic enrichment. Enrichment was done by incubation with the primary antibody TF 2 (IgM), followed by secondary magnetically labelled rat-anti mouse IgM. Cytospin slides were screened manually by bright-field microscopy., Results: 15/65 pts (23%) showed DTC-BM in primary screening with a median of 2/2 mio cells (range 1-10). By enrichment, a median of 23.3 mio cells (0.8-218) could be analysed, increasing positivity to 72% (47/65 pts) with a med. of 4 DTCs (1-105, P < .0001). Blood from 1/11 pts before and 5/11 (45%) after enrichment showed CTCs (med. 2, 1-20), at a med. of 12.4 mio (2.6-38.5) cells analysed. Comparing BM and blood of the same patients after enrichment, 5 were positive in both compartments, 4 showed DTC-BM without presence of CTCs., Conclusion: The positive immunomagnetic enrichment technique with TF-antibodies enables to analyse larger sample volumes and increase tumor cell detection rate. This could allow monitoring and characterisation of CTCs as targets for therapies.

Purpose: The Thomsen-Friedenreich antigen (TF, CD176) is a specific oncofetal carbohydrate epitope (Gal beta1-3GalNAc alpha-O-Ser/Thr) expressed on the surface of various carcinomas. It mediates endothelium adhesion and formation of metastases. As it also causes immune response, its prognostic impact is indeterminate. The presence of disseminated tumor cells in the bone marrow of breast cancer patients (DTC-BM) indicates worse prognosis. We examined the expression of TF in primary breast cancer tissue of 265 patients with known BM status at the time of first diagnosis., Methods: BM aspiration, cytospin preparation and immunocytochemical staining with the anti-Cytokeratin antibody A45 B/B3 was done following a standardised protocol. TF expression was examined immunohistochemically on Tissue Micro Arrays (TMA) with the anti-TF antibody A78-G/A7. Evaluation was done using the immunoreactive score (IRS)., Results: Median IRS for TF expression was 2 (0-12). 68 of 265 patients (25.7%) showed DTC-BM with a median of 2/2 x 10(6) cells (1-1500). There was no correlation between TF expression and DTC-BM. After a median follow up of 60.1 months (7-119), the detection of DTC-BM showed prognostic significance for overall survival (OS, p = 0.034), whereas TF positivity (IRS > 2) indicated prolonged disease-free (p = 0.01), distant disease-free (p = 0.005), and overall survival (p = 0.005)., Discussion: Patients with TF-positive tumors had a significantly better prognosis. Dissemination routes, TF-mediated metastasis formation, and the immunogeneity of TF might determine the prognostic impact of TF expression in different tumor entities. Further characterisation of primary tumors and DTC-BM could help to improve the biological understanding of metastases and develop targeted therapies.

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.

Purpose: The presence of isolated tumor cells in the bone marrow (ITC-BM) is an independent prognostic factor in all stages of breast cancer. Both the expression/amplification of human epithelial growth factor receptor 2 (HER2) and Topoisomerase IIalpha (TOP IIa), a key enzyme of DNA replication and main target of anthracyclins, in breast cancer tissue seem to have predictive value regarding the effectiveness of systemic therapies., Methods: To investigate the correlation between these factors and their influence on clinical outcome, tumor tissue of 54 patients who were screened for ITC-BM before and after anthracyclin-based chemotherapy (abCTX) was examined for HER2 and TOP IIa by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH)., Results: By IHC, 31% of the tumors showed positive for HER2 (2+/3+), 14.6% were amplified in FISH. TOP IIa expression (>50%) was found in 13/53 patients (25%), FISH was positive in 5/47 cases (11%). TOP IIa amplification was not seen in cases without HER2 amplification, five of the seven HER2 amplified cases also were amplified for TOP IIa (71% co-amplification). Forty-three patients had adjuvant, seven neo-adjuvant, four palliative abCTX. ITC-BM were present in 24% of patients before and 31% after CTX. Patients with HER2 (IHC, P = 0.29) and TOP IIa (FISH, P = 0.16) positive tumors tended to stay or become negative in BM status after abCTX and vice versa. After a median follow-up of 44 months (6-127), none of the factors reached significance for overall survival. Yet, patients with HER2 (P = 0.16) and TOP IIa (P = 0.09) positive tumors showed a trend towards prolonged disease-free survival. Remarkably, none of the TOP IIa FISH-positive patients developed distant metastases (P = 0.099) or died (P = 0.19) after CTX so far., Conclusions: HER2- and TOP IIa positivity seem to improve the effect of abCTX. The combination of the prognostic value of ITC-BM and the predictive capacity of HER2 and TOP IIa could help to stratify patients for certain therapies. The direct examination of those factors on ITC-BM is the focus of ongoing studies.

Minimal residual disease (MRD), or isolated tumor cells (ITCs) in bone marrow, may be the source of potentially fatal overt distant metastases in solid tumors even years after primary treatment. MRD can be detected by immunohistochemical methods using antibodies directed against cytokeratins or cell-surface markers or molecular, polymerase chain reaction-based techniques. Among solid tumors, the clinical relevance of MRD has been most extensively studied in breast cancer patients. Recently, the highest level of evidence for the prognostic impact of MRD in primary breast cancer was reached by a pooled analysis comprising more than 4,000 patients, showing poor outcome in patients with MRD at primary therapy. Yet the clinical application of MRD detection is hampered by the lack of a standardized detection assay. Moreover, clinical trial results demonstrating the benefit of a therapeutic intervention determined by bone marrow status are still absent. Recent results suggest that, in addition to its prognostic impact, MRD can be used for therapy monitoring or as a potential therapeutic target after phenotyping of the tumor cells. Persistent MRD after primary treatment may lead to an indication for extended adjuvant therapy. However, until clinically relevant data regarding successful therapy of MRD are available, treatment interventions on the basis of MRD should only be performed within clinical trials.

The detection of disseminated tumor cells in the bone marrow (DTC-BM) of breast cancer patients has proved prognostic significance in all stages of the disease. Further characterisation of those cells could help to improve the biological understanding of metastases, develop targeted therapies and define surface markers for enrichment techniques. The Thomsen-Friedenreich (TF) antigen has been shown to be a tumor specific antigen in breast cancer. The aim of this study was to investigate the expression of TF on DTC-BM in 25 patients. Bone marrow samples were first double-stained by a Cy3 conjugated cytokeratin (CK) antibody (ab) A45 B/B3 (IgG) and anti-TF ab Nemod 2 (IgM), followed by Cy2 conjugated goat anti-mouse IgM ab. For further characterisation samples were also double-stained with anti-TF ab Nemod 2 (IgM), followed by Cy2 conjugated goat anti-mouse IgM ab, and anti MUC1 ab A76-A/C7 IgG, followed by Cy3 conjugated goat anti-mouse IgG. CK positive DTC-BM showed co-expression of TF antigen in 22/23 patients (96%) and 61 of 62 detected cells (98%). Mononuclear BM cells without CK expression were also negative for TF. All of the TF positive cells showed strong MUC1 expression. This is the first study showing co-expression of CK and TF as markers of DTC-BM. Double staining experiments of TF and MUC1 expression showed that MUC1 is the carrier protein of TF in these cells. As TF is a specific marker of DTC-BM, it could be used as a target for antibody based therapy and immunomagnetic enrichment techniques for the isolation of DTC-BM.

Background: The prognostic significance of isolated tumor cells (ITCs) in bone marrow (BM) from patients with breast carcinoma at the time of their primary diagnosis recently was been confirmed by a large pooled analysis. If the persistence of ITCs after adjuvant therapy confers a similar risk for recurrence, then it would be an indication to consider secondary adjuvant therapy., Methods: The authors analyzed BM aspirates from 228 patients during recurrence-free follow-up at a median interval +/- standard deviation (SD) of 21.3 +/- 29.1 months after a primary diagnosis of breast carcinoma (pathologic T1 [pT1]-pT2, pN0-pN3, pM0). Carcinoma cells were detected using a standardized immunoassay with monoclonal antibody A45-B/B3 directed against cytokeratin (CK). Patients were followed for a median +/- SD of 49.8 +/- 32.1 months after their primary diagnosis., Results: Persistent ITCs in BM were detected in 12.7% of patients (n=29 patients). Positive BM status was more frequent (15.7%) within the first 21 months after primary diagnosis than after a follow-up > 21 months (9.7%). The Kaplan-Meier estimate for mean recurrence-free survival was 149.7 months (95% confidence interval [95% CI], 139.6-159.8 months) in patients with negative BM status and 86.5 months (95% CI, 65.7-107.4 months; P=0.0003) in patients with positive BM status at the time patients underwent follow-up BM aspiration. Patients who were without evidence of persistent ITCs had a significantly longer overall survival (162.1 months; 95% CI, 152.1-172.0 months) compared with patients who had positive BM status (overall survival, 98.7 months; 95% CI, 79.7-117.9 months; P=0.0008). In multivariate Cox regression analysis that included BM status, tumor size, lymph node status, and histopathologic grade, evidence of ITCs was an independent significant predictor for reduced disease-free survival (relative risk [RR], 4.57; P <0.0001) and overall survival (RR, 5.57; P=0.002). Persistent ITCs had the greatest prognostic relevance when they were detected between 25 months and 42 months after primary diagnosis (RR, 7.68)., Conclusions: Evidence of persistent ITCs in BM from patients with breast carcinoma indicated an increased risk for subsequent recurrence. Prospective trials should investigate the benefit of secondary adjuvant treatment on the basis of BM marrow status., (2005 American Cancer Society.)

Blood-borne distant metastasis is the leading cause of cancer-related death in breast cancer. The onset of this fundamental process can now be assessed in cancer patients by using ultrasensitive immunocytochemical and molecular assays able to detect even single metastatic cells. To date, clinical studies with large study populations, validated immunoassays, and adequate follow-up time provide evidence for the independent prognostic value of the presence of disseminated tumor cells in the bone marrow. At present, bone marrow evaluation may therefore be considered a diagnostic tool for improved risk assessment with regard to distant relapse and death, especially among potentially curable breast cancer patients. Except for promising pilot studies, no data are available to support the immediate use of bone marrow evaluation for treatment monitoring in order to enable the prediction of response to adjuvant therapy.

The presence of occult micrometastases in bone marrow (BM) of patients with early breast cancer increases the risk of relapse. Detection of circulation tumor cells in peripheral blood (PB) may also influence the patient's prognosis. Few data are available on the correlation between tumor cell dissemination in BM and PB in solid epithelial tumors. Twenty-milliliter blood samples were collected from PB of 42 patients with advanced breast cancer and centrifuged using the density gradient OncoQuick (OncoQuick Greiner BioOne, Frickenhausen, Germany). The BM aspirates available from 11 of the 42 patients were centrifuged using density centrifugation Ficoll. Tumor cell detection was performed by microscopy after cytospin preparation and immunocytochemical staining with the monoclonal antibody A45-B/B3. Cytokeratin-positive cells were detected in 23 patients (55%) in the PB and in three patients (27%) in the BM. A cohort with bone lesions as the only metastatic side showed a correlation as follows: 7 of the 11 patients (64%) had negative findings in BM and PB, whereas cytokeratin-positive cells in PB were present in 3 of these 11 patients (27%). The presence of visceral metastases was associated with the detection of cytokeratin-positive cells in the PB in 20 of the 31 patients (65%) in this subgroup. The density gradient OncoQuick in combination with immunocytochemical staining allows the detection of cytokeratin-positive cells in PB of patients with advanced breast cancer. The immunocytochemical detection of cytokeratin-positive cells in PB seems to be associated with the site of metastatic manifestation.

Purpose: Based on conventional tumor staging, primary ovarian cancer is viewed as an intraperitoneal disease that rarely spreads to extraperitoneal organs. However, autopsy studies reveal a much higher rate of occult metastasis, indicating that extraperitoneal spread occurs with much greater frequency than previously appreciated. Consequently, we investigated the incidence of early hematogenous dissemination and its association with distant disease-free and overall survival., Patients and Methods: Bone marrow aspirates from 108 patients newly diagnosed with International Federation of Gynecology and Obstetrics stage I to III ovarian cancer were prospectively analyzed with the novel anti-cytokeratin (CK) antibody A45-B/B3. We investigated the frequency of CK-positive tumor cells in bone marrow and their effect on prognosis in relation to established risk factors for tumor progression., Results: Tumor cells in bone marrow were detected in 32 (30%) of 108 patients. A CK-positive finding was unrelated to established risk parameters, except for poor nuclear grading of the primary tumor. At a median follow-up of 45 months (range, 12 to 77 months), the presence of occult metastatic cells in bone marrow was associated with the occurrence of clinically overt, extraperitoneal (predominantly extraskeletal) distant metastasis (relative risk [RR], 16.5; 95% confidence interval [CI], 6.2 to 56.9; P < .0001) and death from cancer-related causes (RR, 2.3; 95% CI, 1.2 to 4.3; P = .01). Multivariate analysis identified a positive bone marrow finding as an independent prognostic factor of reduced distant disease-free survival for all patients (RR, 13.8; 95% CI, 5.4 to 52.9; P < .0001) and for the 64 stage R0-1 patients (RR, 7.3; 95% CI, 1.5 to 56.8; P = .0021)., Conclusion: Our data signal that hematogenous dissemination of tumor cells occurs early and throughout all stages of ovarian cancer. The clinical significance of our findings is supported by the unfavorable prognosis in association with the presence of occult metastatic cells, especially in those patients who received an effective locoregional therapy.

Introduction and Objective: A clinically important myelosuppression due to adjuvant chemotherapy is seen more frequently as dosage is intensified and new drugs are used. The assessment of the cytopenia expected is frequently hampered by a lack of directly comparable data. The aim of this study was to compare - in our own patient population - the chemotherapy-associated myelosuppression of four chemotherapeutic regimens used in gynecological oncology., Methods: 79 patients with primary breast cancer and 26 patients with epithelial ovarian carcinoma underwent cytostatic treatment, and the associated myelosuppression was evaluated by the determination of cytopenia and the need for supportive therapy. The chemotherapy regimens investigated were CMF (cyclophosphamide 600 mg/m(2), methotrexate 40 mg/m(2), 5-fluorouracil 600 mg/m(2), 6xq3w), EC/CMF (epirubicin 90 mg/m(2), cyclophosphamide 600 mg/m(2), 4xq3w, followed by CMF, 3xq3w), DE (docetaxel 75 mg/m(2), epirubicin 90 mg/m(2), 6xq3w) and CC (cyclophosphamide 600 mg/m(2), carboplatin AUC 6, 6xq3w)., Results: The EC/CMF and DE regimens were used significantly more frequently for more advanced tumor stages, but there were no differences concerning tumor-dependent prechemotherapeutic myelosuppression. Hemopoiesis was most impaired in the CC group with a mean drop of serum hemoglobin of 1.5 g/dl to the end of the cytostatic treatment; correspondingly, most transfusions of concentrated erythrocytes were needed in this group. The strongest suppression of leukopoiesis was found in the DE group, with a mean drop in leukocyte counts of 6.2 x 10(3)/microliter per cycle; in this group, a mean of 7.6 ready-made syringes with 263 microgram Lenogastrim was used to stimulate leukopoiesis. The severest drop in the mean thrombocyte count, i.e. 171.7 x 10(3)/microliter, was found in the CC group., Conclusions: The CC regimen impairs thrombo- and erythropoiesis most, whereas the DE regimen causes marked leukopenia. The regimen with the smallest myelosuppression was CMF. No severe cytopenia-associated complications were detected in any of the cases investigated.

Background: Using cytokeratin (CK) as a histogenetic marker of epithelial tumor cells in the bone marrow of patients with primary breast carcinoma, a subgroup of patients with decreased survival can be identified. This study was designed to evaluate the frequency and prognostic relevance of such cells in patients with recurrent breast carcinoma., Methods: Bone marrow aspirates from 65 patients were analyzed immunocytochemically for the presence of CK positive cells. A quantitative immunoassay with monoclonal anti-CK antibody A45-B/B3 was used and 2 x 10(6) bone marrow cells per patient were evaluated. For prognostic evaluation the authors calculated a cutoff value of micrometastatic tumor cells by analogy to classification and regression tree (CART) analysis. Patients were monitored prospectively for a median of 37 months (range, 11-63 months)., Results: Bone marrow micrometastases were present in 5 of 32 patients (16%) with locoregional recurrence and in 24 of 33 patients (73%) with distant recurrence. The bone marrow status yielded no prognostic indication for patients with locoregional recurrence. In contrast, a cutoff value of 2.5 tumor cells per 1 million bone marrow cells analyzed (2.5 x 10(-6) tumor cells) correlated with a significantly different prognosis for women with distant disease. Patients with metastatic disease and a micrometastatic tumor load of > 2.5 x 10(-6) tumor cells survived for a mean of 6 months (95% confidence interval [95% CI], 2.0-9.1) compared with 17 months (95% CI, 11.6-22.0) for patients with < or = 2.5 x 10(-6) tumor cells (P < 0.0001). Multivariate analysis, allowing for hormone receptor status, disease free interval prior to recurrence, manifestation site of metastases, age, and micrometastases in bone marrow, revealed that bone marrow involvement was an independent risk factor, with a hazard ratio of 7.4 (95% CI, 1.6-13.3) for disease-related death., Conclusions: An increased number of micrometastases identified in the bone marrow of patients with metastatic breast carcinoma represents an independent prognostic factor that may influence future therapeutic strategies for patients with metastatic breast carcinoma.

Background: Cytokeratins are specific markers of epithelial cancer cells in bone marrow. We assessed the influence of cytokeratin-positive micrometastases in the bone marrow on the prognosis of women with breast cancer., Methods: We obtained bone marrow aspirates from both upper iliac crests of 552 patients with stage I, II, or III breast cancer who underwent complete resection of the tumor and 191 patients with nonmalignant disease. The specimens were stained with the monoclonal antibody A45-B/B3, which binds to an antigen on cytokeratins. The median follow-up was 38 months (range, 10 to 70). The primary end point was survival., Results: Cytokeratin-positive cells were detected in the bone marrow specimens of 2 of the 191 control patients with nonmalignant conditions (1 percent) and 199 of the 552 patients with breast cancer (36 percent). The presence of occult metastatic cells in bone marrow was unrelated to the presence or absence of lymph-node metastasis (P=0.13). After four years of follow-up, the presence of micrometastases in bone marrow was associated with the occurrence of clinically overt distant metastasis and death from cancer-related causes (P<0.001), but not with locoregional relapse (P=0.77). Of 199 patients with occult metastatic cells, 49 died of cancer, whereas of 353 patients without such cells, 22 died of cancer-related causes (P<0.001). Among the 301 women without lymph-node metastases, 14 of the 100 with bone marrow micrometastases died of cancer-related causes, as did 2 of the 201 without bone marrow micrometastases (P<0.001). The presence of occult metastatic cells in bone marrow, as compared with their absence, was an independent prognostic indicator of the risk of death from cancer (relative risk, 4.17; 95 percent confidence interval, 2.51 to 6.94; P<0.001), after adjustment for the use of systemic adjuvant chemotherapy., Conclusions: The presence of occult cytokeratin-positive metastatic cells in bone marrow increases the risk of relapse in patients with stage I, II, or III breast cancer.

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.