Disseminated Tumor Cells In Bone Marrow Research Articles

Presence of bone marrow micrometastasis is associated with different recurrence risk within molecular subtypes of breast cancer

Expression profiles of primary breast tumors were investigated in relation to disseminated tumor cells (DTCs) in bone marrow (BM) in order to increase our understanding of the dissemination process. Tumors were classified into five pre-defined molecular subtypes, and presence of DTC identified (at median 85 months follow-up) a subgroup of luminal A patients with particular poor outcome ( p = 0.008). This was not apparent for other tumor subtypes. Gene expression profiles associated with DTC and with systemic relapse for luminal A patients were identified. This study suggests that DTC in BM differentially distinguishes clinical outcome in patients with luminal A type tumors and that DTC-associated gene expression analysis may identify genes of potential importance in tumor dissemination.

Impact of disseminated tumor cells in bone marrow at diagnosis in patients with nonmetastatic prostate cancer treated by definitive radiotherapy

The purpose of this study was to explore whether detection of disseminated tumor cells (DTCs) in bone marrow (BM) of nonmetastatic prostate cancer (PC) was associated with other clinical or histopathological factors at diagnoses or clinical outcome subsequent to definitive radiotherapy (RT). We evaluated BM aspirates from 272 cT(1-4)pN(0)M(0) PC patients by immunocytochemistry employing anticytokeratin antibodies (AE1/AE3). BM-status was compared with clinical and histopathological parameters. Long-term clinical outcome was assessed in 131 of the patients who all had completed definitive RT with or without androgen deprivation (AD), initiating treatment >5 years before cut-off date June 1, 2005. They had at least 1 unfavorable prognostic feature defined as cT(3-4) or Gleason score (GS) >or= 7B or PSA >or= 10 microg/l. Overall death, cause-specific death, distant metastases (DM) as first clinical relapse, local failure as first clinical relapse and biochemical failure were defined as end-points. DTCs were detected in 18% of the patients and were associated with increasing GS (p = 0.04) and percentage of Gleason pattern 4/5 (p = 0.04). The 7-year cumulative risk of DM was 21% for BM-positive patients vs. 6% for BM-negative patients (p = 0.07). In patients receiving RT without AD (n = 75), the 7-year cumulative risk of DM for BM-positive patients was 28% vs. 9% for BM-negative patients (p = 0.03). BM-status did not have impact on other end-points. In conclusion our study shows that presence of DTCs in BM at diagnosis was associated with the histological differentiation of the primary tumor and an increased risk of developing distant metastases after RT.

Detection of cancer cells disseminated in bone marrow using real-time quantitative RT-PCR of CEA, CK19, and CK20 mRNA in patients with gastric cancer

To determine the significance of bone marrow disseminated tumor cells in gastric cancer, we investigated the mRNA expression levels of carcinoembryonic antigen (CEA), cytokeratin 19 (CK19), and cytokeratin 20 (CK20) using the real-time quantitative reverse-transcription polymerase chain reaction (RQ-PCR). Bone marrow samples were aspirated from the sternum at the time of surgery in 65 patients with resectable gastric cancer. Total RNA was extracted from bone marrow; and the expression levels of CEA, CK19, and CK20 mRNA were determined by RQ-PCR using an ABI PRISM 7000 and quantified against the GAPDH mRNA level. The detection limits of these genes were determined in the gastric cancer cell line MKN-45 and the colon cancer cell line C-1, which had been serially diluted in peripheral blood mononuclear cells (PBMCs). A rate of 1 cancer cell/million PBMCs was obtained by detecting CEA and CK19 mRNA in MKN-45 and by detecting CK20 mRNA in C-1. In the clinical samples, only 1 of the 65 gastric cancer patients (1.5%) who had stage IV disease was positive for CEA, CK19, and CK20 mRNA; none of CEA, CK19, or CK20 mRNA was positive in the remaining 64 patients. No significant correlation was observed between disseminated cancer cells in bone marrow and clinicopathological features, including simultaneous or metachronous hepatic metastasis and patient survival. The incidence of disseminated cancer cells in bone marrow in our study appears low, unlike that in previous reports. The significance of disseminated cancer cells in bone marrow may also be quite low in gastric cancer.

Most Early Disseminated Cancer Cells Detected in Bone Marrow of Breast Cancer Patients Have a Putative Breast Cancer Stem Cell Phenotype

The presence of disseminated tumor cells (DTC) in the bone marrow of breast cancer patients is an acknowledged independent prognostic factor. The biological metastatic potential of these cells has not yet been shown. The presence of putative breast cancer stem cells is shown both in primary tumors and distant metastases. These cells with a CD44(+)CD24(-/low) phenotype represent a minor population in primary breast cancer and are associated with self-renewal and tumorigenic potential. Recognizing the potential effect of prevalence of putative stem cells among DTC, we evaluated the bone marrow DTC. We employed the double/triple-staining immunohistochemistry protocol and modified the established bone marrow cytokeratin (CK) staining protocol by adding steps for additional antigens, CD44 and/or CD24. We evaluated 50 bone marrow specimens, previously categorized as CK(+) from early breast cancer patients. CK(+) cells were examined for CD44 and CD24 expression by light microscopy, fluorescence microscopy, and spectral imaging. We detected the putative stem cell-like phenotype in all CK(+) specimens. The mean prevalence of putative stem/progenitor cells was 72% and median prevalence was 65% (range, 33-100%) among the overall DTC per patient, compared with primary tumors where this phenotype is reported in <10% of cells. This is the first evidence of the existence of the putative stem-like phenotype within the DTC in bone marrow in early breast cancer patients. All patients had a putative stem cell phenotype among the DTC and most individual DTC showed such phenotype. Future molecular characterization of these cells is warranted.

Clinical impact of disseminated tumor cells in patients with non-metastatic prostate cancer treated by definitive radiotherapy

4575 Background: Detection of disseminated tumor cells (DTCs) in bone marrow (BM) has been proven to be an independent prognostic factor in breast cancer. To our knowledge, we here present the first larger study of radically treated prostate cancer (PC) with sufficiently long-term follow-up where the association between DTCs at diagnosis and clinical outcome has been studied. Methods: We screened 272 cT1–4pN0M0 PC patients for DTCs in BM-aspirates between 1994 and 2004. Monoclonal antibodies against cytokeratins (AE1/AE3) and standardized immunocytochemical methods were applied for detection. BM-status was compared with clinical and histopathological factors at diagnosis in all patients and with long-term clinical outcome in 131 patients. They all started treatment including definitive radiotherapy (RT) before June, 2000 and had a relatively poor prognosis defined as cT3–4 or Gleason score (GS) ≥ 7B (4 + 3) or PSA ≥ 10 μg/l. Kaplan-Meier plots were generated by BM-status with the following end-points: Overall death, cause-specific death, distant metastases (DM) as first clinical relapse, local failure as first clinical relapse and biochemical failure. Results: DTCs were detected in 18% of the patients and were significantly associated with increasing percentage Gleason grade 4/5 (p = .035, Mann-Whitney) but not with dichotomized GS, tumor-stage or PSA. In the follow-up cohort (median observation time 6.9 years), the 7-year cumulative risk of DM was 21% in BM-positive patients vs. 6% in BM-negative patients (p = .069, log rank). No association was found between DTCs and other end-points. A sub-group analysis of 73 patients with GS ≥ 7B yielded a 34% 7-year cumulative risk of DM in BM-positive patients vs. 10% in BM-negative patients (p = .039, log rank). DTCs did not predict DM in 55 patients with GS ≤ 7A (3 + 4). In a multivariate analysis, dichotomized GS (RR = 7.8 [95% confidence interval (CI) = 1.0–60.7], p = .049) and BM-status (RR = 3.0 [95% CI = .9–9.4], p = .066) had independent impact on DM. Conclusions: The presence of DTCs in BM at diagnosis of non-metastatic PC is associated with the histological differentiation of the primary tumor and seems to predict development of DM after definitive RT. No significant financial relationships to disclose.

Influence of platinum-based chemotherapy on disseminated tumor cells in patients with primary ovarian cancer

5067 Background: Recent studies in patients (pts) with breast cancer have demonstrated the prognostic significance of disseminated tumor cells (DTC) in the bone marrow (BM) which even can survive high-dose chemotherapy. For ovarian cancer, the impact of chemotherapy on DTC is unknown. Here we evaluated whether first-line chemotherapy with carboplatinum and paclitaxel can eliminate DTC in BM and peripheral blood (PB) of pts with primary ovarian cancer (OC, FIGO IC-IIIC). Methods: PB of 28 pts with OC and bilateral BM aspirates of 18/28 pts were assessed for DTC before and after chemotherapy using density gradient centrifugation and an immunocytochemical cytokeratin (CK) assay with the anti-CK antibody A45-B/B3. Results: We identified CK+ cells in 7/28 blood samples (25%) before therapy with a mean number of 2 cells/20 ml (range 1–3). After chemotherapy, CK+ cells were detected in 3/28 blood samples (11%) with a mean number of 9 cells/20 ml (range 1–18). In 5 out of these 7 CK+ patients prior to therapy, no CK+ cells were found after therapy, a no change and a reduction of DTC could be demonstrated in one patient each. CK+ cells were found in 10/18 BM samples (56%) before therapy with a mean number of 2 cells/9x10E6 BM cells (range 1–7) and in 8/18 BM samples (44%) after therapy with a mean number of 10 cells/9x10E6 BM cells (range 2–35). After chemotherapy, no CK+ cells were found in 6 pts, no change in DTC counts was documented in 2 pts and a significant enhancement of DTC was shown in 6 pts, including 4 pts who had no CK+ cells before chemotherapy. Conclusions: DTC are frequently present in the BM and PB of pts with OC and are not always successfully eliminated after platinum-based chemotherapy. It has to be considered, whether these pts might profit from an additive immunotherapy as already shown by the intraperitoneal application of a trifunctional antibody targeting EpCAM, CD3 and accessory cells (Proc Am Assoc Cancer Res 46: 4260, 2005). No significant financial relationships to disclose.

Comparison of HER2 status between primary tumor and disseminated tumor cells in␣primary breast cancer patients

The persistence of disseminated tumor cells (DTC) in bone marrow (BM) of breast cancer patients is associated with poor prognosis. Preliminary studies indicated that these patients might benefit from secondary adjuvant targeted therapy. HER2 protein is suggested as one of the most promising targets. The aims of this study were (1) to determine the HER2 status of DTC in BM of breast cancer patients and (2) to compare the HER2 status of DTC and corresponding primary tumors. BM aspirates from 137 primary breast cancer patients were included into the study. A double staining procedure was used for the identification of cytokeratin-positive (CK)/HER2 positive cells. HER2 status of the primary tumor was immunohistochemically assessed by the HERCEP-test. In 46 of 137 (34%) breast cancer patients CK-positive cells were detectable in BM. DTC with HER2 positivity were found in 20 (43%) of these patients. The HER2 expression on DTC was heterogeneous in 7 of 17 (41%) patients. Concordance rate of HER2 status between primary tumor and DTC was 62%. In 12 of 20 patients with HER2 negative tumors HER2 positive DTC were detected. HER2 positive DTC can be detected in patients with HER2 negative primary tumors. Therefore, the antigenic profile of DTC may be considered for treatment decision since these patients might actually benefit from trastuzumab. However, the HER2 overexpression on DTC is heterogeneous in individual patients which may reduce the efficacy of an immunotherapy based strategy directed against HER2-antigen only.

Enrichment of disseminated tumor cells (DTC) in bone marrow (BM) from patients with breast cancer (BC) using a magnetic cell sorting device (MCSD)-Method and prognostic significance

9640 Background: There is increasing evidence that immunocytochemical detection (ID) of DTC in BM from patients with BC probably has prognostic significance. But the low detection rate hampers further geno- and phenotyping and the efficiency of DTC as a broadly applied prognostic tool. We studied the possibility to enrich DTC using immunomagentic beads coupled mab against human epithelial antigen (HEA) with MCSD and the prognostic significance. Methods: BM from 107 patients with stage I to IV BC were taken. After separation of mononuclear cells (MNC) (Ficoll-Hypaque density gradient), a cytospin with 1x106 MNC was made and studied for the presence of DTC using a mouse mab F(ab)2-fragment (mab A45B/B3) directed against human cytokeratines and coupled to alkaline phosphatase. The remaining portion of the MNC was incubated with mab against HEA conjugated with magnetic microbeads. Thereafter DTC were enriched with MCSD (Miltenyi-Biotec). After washing, cytospins with 1x106 MNC were made and examined for the presence of DTIC using mab A45 B/B3. Results: The detection rate and the number of DTC in enriched and not enriched samples correlates with tumor stage (Spermann-Rho 1,0;p=0,01). Using MCSD the detection rate and the number of DTC could be improved for every tumor stage. It was statistically significant for stage I and II diesease (McNemar-Test:p<0,0001). There is a trend for better PFS and OS in the group without DTC in BM (enriched and not enriched), but it was not found to be a statistically significant indipendent risk factor (Logrank-Test). Conclusions: Enrichment of DTC from BM in patients with BC using MCSD is a more sensitive method for detection and quantification of DTC than ID alone, especially in early stages of the disease. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Micromet Micromet

Disseminated tumor cells (DTC) in bone marrow (BM) and clinical outcome: Final results of pooled analysis on 10-year survival of 4,703 breast cancer patients

502 Background: Statistically powerful prognostic data on long-term outcome of DTC +ve breast cancer patients is needed to enable evidence-based therapeutic strategies designed to improve patient outcome. Methods: Individual patient data of 9 studies, involving 4,703 patients with stage I-III breast cancer, were combined to analyze long-term clinical outcome. A time-stratified Cox multivariable regression model was required because hazard ratios were not constant over the full length of the 10-year period. Results: The prevalence of DTC was 30.6%, and was significantly associated with larger tumor size, high grade, lymph-node metastasis (each P<0.001) and endocrine non-responsiveness (P=0.003). In the final multivariable model, DTC was an independent prognostic variable for early relapse and death. During the first 5 years of follow-up and thereafter, hazard ratios (HR) for OS were 1.81 (95%CI 1.51–2.16; P<0.001) and 1.58 (95% CI 1.12–2.22; P=0.009), respectively. Early relapse occurred significantly more often among DTC +ve patients (log rank, each P<0.001), with a multivariable HR of 1.85 (95%CI 1.58–2.14) for DFS and 2.03 (95%CI 1.72–2.39) for DDFS during the initial 5 years of follow-up. DTC was not an independent predictor for DFS and DDFS after 5 years. On univariate analysis, OS was significantly reduced for DTC +ve patients among subgroups on either endocrine therapy (n=1,499; incidence rate ratio [IRR] 2.49, 1.83–3.37; P<0.001) or chemotherapy alone (n=1,596; IRR 2.26, 1.80–2.85; P<0.001). Moreover, among 1,036 patients with pT1N0 disease, who received no systemic adjuvant treatment, those 229 DTC +ve patients had significantly worse OS (P=0.001), DFS (P=0.014) and DDFS (P=0.007), as compared to 807 DTC -ve patients. Conclusions: Presence of DTC is a strong prognostic factor of level I evidence for poor clinical outcome. Since for patients with DTC in BM it is reasonable to assume a differential susceptibility to current adjuvant therapy, as shown overall and in subgroups, consideration of DTC for clinical decision making might enable improved risk stratification in future clinical trials. No significant financial relationships to disclose.

HER2 expression in disseminated tumor cells (DTC) and corresponding primary tumors of breast cancer patients

9580 Background: The presence of disseminated tumor cells (DTC) in bone marrow (BM) of breast cancer patients is associated with poor prognosis. Several studies have indicated that these patients may benefit from secondary adjuvant immunotherapy. HER2 protein is suggested as one of the promising targets. The phenotype of the primary tumor is the current gold standard to make antibody based treatment decisions. However, the antigenic profile of primary tumors and DTC may be different. The aims of this study were (1) to determine the HER2 status of DTC in BM of breast cancer patients (2) and to compare the HER2 status of DTC and corresponding primary tumors. Method: Cytospins from bone marrow aspirates of 124 primary breast cancer patients (stage I-III) were analyzed for this study. A double labeling procedure was used for the identification of cytokeratin-positive (CK+)/HER2 positive cells. Slides were incubated with the pan-anti-cytokeratin antibody C11 conjugated to FITC and the polyclonal rabbit antibody CB11 against the HER2 protein. CB11 was detected by a secondary antibody labeled with Texas Red. HER2 status of the primary tumor was immunohistochemically assessed by the HERCEP-test (Dako Co., CA). Results: Disseminated tumor cells (DTC) were detected in the bone marrow of 47 patients. The number of DTC ranged from 1 to 70 per slide. 24 of these patients had HER2 positive DTC. HER2 positivity (score: 2+/3+) could be demonstrated in 12 of 46 primary tumors. Concordance rate between primary tumor and DTC was 63%. In three patients with HER2 positive tumors HER2 negative DTC were detected. In contrast, 14 patients with HER2 negative tumors (score: 0/1+) had HER2 positive DTC. The HER2 expression in DTC was heterogeneous in 8 of 20 patients with more than one CK-positive cell. Conclusions: (1) HER2 positive DTC can be detected in patients with HER2 negative primary tumors. Therefore, the antigenic profile of DTC should be included in the treatment decision for antibody based strategies. (2) The DTC are heterogeneous with regard to HER2 positivity. The effect of heterogeneity on the patient’s response is yet to be determined. No significant financial relationships to disclose.

Human antibody SC-1 reduces disseminated tumor cells in nude mice with human gastric cancer

Advanced gastric cancer is a systemic disease that requires adjuvant therapy targeted at eliminating disseminated tumor cells (DTCs). We investigated whether the apoptosis-inducing human monoclonal IgM antibody SC-1 was able to reduce the number of disseminated gastric cancer cells in blood and bone marrow. Human gastric tumor specimens with positive expression of the SC-1 receptor were transplanted in nude mice with metastasizing gastric cancer. After tumor growth (4-6 weeks) animals were randomly allocated to intraperitoneal 100 microg SC-1 (n=23) or 100 microg human IgM (n=23). One week later, animals were sacrificed and blood and bone marrow specimens were obtained. A nested RT-PCR for cytokeratin 20 (CK-20) from blood and bone marrow of mice was performed for detection of disseminated tumor cells. Animals receiving SC-1 had significantly fewer DTCs than did control animals (p=0.0011). None of the SC-1 mice had DTCs simultaneously in both blood and bone marrow versus four of the control animals (p=0.0363). The reduction of DTCs in SC-1 animals was due to reduction in bone marrow (p=0.032 compared to controls), but not in blood (p=0.1158). Treatment with SC-1 significantly reduced the number of DTCs in bone marrow in this animal model.

Bone marrow—disseminated tumor cells in patients with carcinoma of the esophagus or cardia

Background. The long-term prognosis after surgical therapy for esophageal carcinoma depends on tumor stage and completeness of resection. Similarly to other epithelial tumors, the presence of micro deposits of neoplastic cells in the bone marrow may indicate residual disease and the potential for recurrence. This study assesses the prevalence of bone marrow-disseminated tumor cells in patients undergoing surgical resection for esophageal carcinoma. In addition, we investigated the agreement between immunohistochemical and molecular techniques for the detection of micrometastases in a subgroup of patients. Methods. Between January 1998 and November 1999, forty-eight patients with adenocarcinoma of the esophagogastric junction (n = 29) or squamous cell carcinoma of the thoracic esophagus (n = 19) and no evidence of overt metastatic disease entered the study. An immunohistochemical assay (capable of detecting 1 carcinoma cell in 7 × 10 5 bone marrow cells) was used to test bone marrow obtained by flushing a resected rib or by needle aspiration either of the iliac crest or of a rib. A polymerase chain reaction (PCR) molecular technique was also used to identify bone marrow and peripheral blood epithelial cells. Results. Cytokeratin-positive cells were found in 79.1% of the bone marrow samples obtained from the rib, and in only 8% of the needle aspirates either from the iliac crest or from a contiguous rib: This difference is probably explained by the improved removal of metastatic cells with the flushing of the rib. Comparable results were obtained at a qualitative level by the PCR technique on bone marrow. In addition, PCR-positive results were found in 3 of 18 peripheral blood samples. There was no association with tumor type, neoadjuvant therapy, or lymph node status. Patients with a pT3 or pT4 tumor showed, at a borderline statistical level, a higher proportion ofcytokeratin-positive cells in the flushed rib. Conclusions. Bone marrow-disseminated tumor cells are present in the resected rib of a high proportion of patients undergoing esophagectomy for carcinoma, and immunohistochemistry seems to be the method of choice for their quantitative assessment. However, the prognostic and therapeutic implications of this finding need further investigation. (Surgery 2001;129:15-22.)

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<h3>Objectives:</h3> Disseminated tumor cells (DTCs) in the bone marrow (BM) were shown to be of prognostic significance in gynecological cancers. Bone marrow aspiration is less accepted by patients compared with blood drawing. In this pilot study, we applied the AdnaTest BreastCancer based on immunomagnetic enrichment, targeting common antigens on epithelial gynecological cancers, followed by multiplex reverse transcriptase-polymerase chain reaction for selection and detection of circulating tumor cells (CTCs) in the blood of 122 ovarian cancer patients at primary diagnosis and/or after platinum-based chemotherapy. Results were compared with detection of DTC in BM. <h3>Methods:</h3> Ten-milliliter blood was obtained before surgery (n = 86) and/or after chemotherapy (n = 70) and analyzed for CTC with the AdnaTest BreastCancer for the detection of EpCAM-, MUC-1-, and HER-2-transcripts. CA 125 was assessed in an additional single-plex reverse transcriptase-polymerase chain reaction. Bone marrow aspirates were analyzed in duplicate by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. <h3>Results:</h3> Before surgery, CTCs were detected in 19% of patients, expressing EpCAM (31%), MUC-1 (50%), HER-2 (31%), and CA 125 (50%), respectively. After chemotherapy, the overall detection rate for CTC was 27%, thereof EpCAM (68%), MUC-1 (47%), HER2 (21%), and CA 125 (37%). The overall detection rate for DTC in the BM was 35% before surgery and 31% after therapy. A comparison between DTC and CTC resulted in a concordance rate of 59% before surgery and 56% after chemotherapy. CTC positivity significantly correlated with shorter overall survival before surgery (<i>P</i> = 0.0054) and after chemotherapy (<i>P</i> = 0.047). <h3>Conclusions:</h3> This methodological approach might help to identify molecular targets for specific biological therapies. Blood analysis could give additional information complimentary to that obtained by DTC.