Disseminated Tumor Cells Status Research Articles

Abstract P4-03-01: Role of disseminated tumor cell (DTC) in bone marrow (BM) detected at primary diagnosis on overall survival and central nervous system (CNS) recurrences in a cohort of 620 early breast cancer patients after 11 years follow up

Abstract Background: BM DTC detection is known to be a prognostic factor for distant metastasis and overall survival in early breast cancer. However, distant relapses may occur in patients with no DTC detected at time of primary cancer. In this study, we studied the impact of early breast cancer pathological features (including BM DTC status) on the incidence of CNS metastases. Methods: In a cohort (1998-2005) of 620 early breast cancer patients in whom BM DTC were detected using an anti-cytokeratin antibody (A45B/B3) (Bidard et al., 2008, Clinical Cancer Research, outcome updating (median follow up 11y) on overall survival (OS) and distant metastasis free survival (DMFS) was assessed by log rank test and uni- and multivariate analyses. 137 patients of this cohort have a later metastatic relapse. Chi2 test were performed to compare patient with CNS metastasis and patients with other metastases locations. CNS metastasis survivals were estimated using Kaplan-Meier method and compared by log-rank test. Results: At eleven-year median follow-up, the prognostic value of DTC detection was confirmed in the 620 patients cohort for OS (p=0.03 in multivariate analysis), but DTC were probably less involved in late metastatic events. Distant metastases were diagnosed in 137 (22%) patients. In the course of the metastatic disease, 55 of these patients (40%) have been diagnosed with CNS metastases (parenchymal=30, leptomeningeal=10, both localizations=15), as first metastatic or later event. Patients with CNS metastasis were mostly less than 50 years old (60% vs 38%, p=0.01), pN0 (31% vs 10%, p=0.002), hormonal receptor status negative (54% vs 30%, p=0.007) and HER2 status positive (50% vs 22%, p=0.01). Strikingly, although DTC detection was associated with development of distant metastasis in the whole cohort, the occurrence of CNS metastasis was found to be higher in patients who had no DTC detected at primary diagnosis (p=0.016). From CNS metastasis, median survival was 7.8 months (8.3 months for parenchymal and 2.4 months for leptomeningeal recurrences). For HER2+ patients, the median survival after diagnosis of CNS metastasis was 16.6 months (N=15) and 4.1 months for Her2- patients (N=15). Conclusions: After a long-term follow up, DTC remain an adverse prognostic factor in early breast cancer. Our study suggests that cancer cells with epithelial differentiation, as detected in the BM, are less prone to CNS dissemination. This study also confirmed previously reported determinants of CNS metastases (age, RH-, HER2+) in the pre-adjuvant trastuzumab era, but also showed that the outcome of HER2+ patients with CNS relapse was longer, probably due to HER2 targeted therapy during metastatic course. Citation Format: Delphine Loirat, François-Clément Bidard, Léonor Chaltiel, Frédérique Berger, Véronique Dieras, Yann de Rycke, Séverine Alran, Youlia Kirova, Paul Cottu, Anne Vincent- Salomon, Xavier Sastre-Garau, Jean-Yves Pierga. Role of disseminated tumor cell (DTC) in bone marrow (BM) detected at primary diagnosis on overall survival and central nervous system (CNS) recurrences in a cohort of 620 early breast cancer patients after 11 years follow up [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-03-01.

Identification of HERC5 and its potential role in NSCLC progression.

For better lung cancer diagnosis and therapy, early detection markers of tumor dissemination are urgently needed, as most lung cancers do not show symptoms until extensive metastasis formation has already taken place. Our previous studies showed that in non-small cell lung cancer (NSCLC) early tumor dissemination is associated with a loss of chromosome 4q12-q32 and the presence of disseminated tumor cells (DTC) in the bone marrow. In order to identify the potential target gene in this region, a screen for methylation-dependent expression was performed. Lung cancer cell lines showing a loss of 4q as well as a normal bronchial epithelial cell line as control were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) followed by expression profiling. Seven genes within the 4q target region, which have been associated with a positive DTC status before were found to be regulated by hypermethylation. QRT-PCR in an independent sample set identified HERC5 as a potential target gene. Quantitative methylation analysis of these lung tissue samples revealed that HERC5 promoter hypermethylation was significantly associated with positive DTC status (p = 0.020) and occurrence of brain metastases (p = 0.015). In addition, hypermethylation of the HERC5 promoter in NSCLC was identified as a predictor for poor survival for Stage I adenocarcinoma patients (p = 0.022) and also for poor overall survival in metastatic lung cancer patients (p = 0.028). In conclusion, HERC5 may function as a prognostic marker and is associated with tumor dissemination in lung cancer.

Tumor cell dissemination to the bone marrow and blood is associated with poor outcome in patients with metastatic breast cancer.

The purpose of this study was to assess the impact of disseminated tumor cells (DTCs) on progression-free and overall survival (OS) in patients with metastatic breast cancer (MBC) and to compare it to simultaneous detection of circulating tumor cells (CTCs) from the blood in a subgroup. Disseminated tumor cells were identified in bone marrow (BM) aspirates by immunocytochemistry (pancytokeratin antibody A45-B/B3) and cytomorphology prior to the beginning of a new-line therapy. CTCs were enumerated by the CellSearch® technology. BM was obtained from 178 patients with MBC; 64/178 (36 %) patients were DTC-positive. Disseminated tumor cells occurred more frequently in patients with visceral metastases (p = 0.020) and ≥2 lines of therapy (p = 0.017). CTCs were assessed in 33 of these patients and 17/33 (52 %) patients had CTC counts ≥5 CTCs/7.5 ml blood. There was no significant association between the DTC and CTC status. Univariate analysis revealed DTC detection as a significant predictor of poor OS (p < 0.001); median OS in DTC-negative versus DTC-positive patients was 52 [95 % confidence interval (CI) 38-67] versus 28 [95 % CI 19-37] months. Moreover, as described previously, patients with ≥5 CTCs/7.5 ml blood were at an increased risk of disease progression (p = 0.026) and death (p = 0.025). Disseminated tumor cells are predictors of poor prognosis in MBC, highlighting the role of tumor cell dissemination into the BM for breast cancer progression. The absence of a significant association between concurrent DTCs and CTCs suggests they might represent different aspects of systemic BC spread.

The presence and prognostic impact of apoptotic and nonapoptotic disseminated tumor cells in the bone marrow of primary breast cancer patients after neoadjuvant chemotherapy.

IntroductionNeoadjuvant systemic therapy of primary breast cancer (PBC) patients offers the possibility to monitor treatment response. However, patients might have metastatic relapse despite achieving a pathologic complete response (pCR). This indicates that local response to therapy must not be representative for systemic treatment efficacy. Therefore, the aim of this study was to compare local response with systemic tumor cell dissemination by determining the presence of disseminated tumor cells (DTCs), including apoptotic tumor cells, in the bone marrow (BM) of PBC patients after neoadjuvant chemotherapy (NACT).MethodsDTCs were detected by immunocytochemistry (pancytokeratin antibody A45-B/B3) and cytomorphology (DTC status). The presence of apoptotic tumor cells was determined by using the M30 antibody (M30 status). This antibody detects a neo-epitope that is expressed only during early apoptosis.ResultsBM aspirates from 400 PBC patients that had completed NACT were eligible for this study. Of these, 167 (42%) patients were DTC positive (DTC status). The M30 status was investigated in 308 patients. Apoptotic (M30-positive) tumor cells were detected in 89 (29%) of these. Whereas the DTC status was not correlated (P = 0.557) to local treatment response (that is, pCR or a clinical complete/partial response), the presence of M30-positive tumor cells was significantly higher in patients that responded to therapy (P = 0.026). Additionally, DTC-positive patients were at an increased risk for disease relapse (hazard ratio, 1.87; 95% CI, 1.11 to 3.15; P = 0.019).ConclusionThe presence of DTC is independent of therapy response of the primary tumor. As patients that are DTC positive after NACT have an unfavorable outcome, they might benefit from additional systemic treatment.

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 × 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.

Pooled Analysis of the Prognostic Relevance of Disseminated Tumor Cells in the Bone Marrow of Patients With Ovarian Cancer

Detection of disseminated tumor cells (DTCs) in the bone marrow (BM) of patients with breast cancer is associated with poor outcomes. Recent studies demonstrated that DTCs may serve as a prognostic factor in ovarian cancer. The aim of this 3-center study was to evaluate the impact of BM status on survival in a large cohort of patients with ovarian cancer. Four hundred ninety-five patients with primary ovarian cancer were included in this 3-center prospective study. Bone marrow aspirates were collected intraoperatively from the iliac crest. Disseminated tumor cells were identified by antibody staining and by cytomorphology. Clinical outcome was correlated with the presence of DTCs. Disseminated tumor cells were detected in 27% of all BM aspirates. The number of cytokeratin-positive cells ranged from 1 to 42 per 2 × 10⁶ mononuclear cells. Disseminated tumor cell status did correlate with histologic subtype but not with any of the other established clinicopathologic factors. The overall survival was significantly shorter among DTC-positive patients compared to DTC-negative patients (51 months; 95% confidence interval, 37-65 months vs 33 months; 95% confidence interval, 23-43 months; P = 0.023). In the multivariate analysis, BM status, International Federation of Gynecology and Obstetrics stage, nodal status, resection status, and age were independent predictors of reduced overall survival, whereas only BM status, International Federation of Gynecology and Obstetrics stage, and resection status independently predicted progression-free survival. Tumor cell dissemination into the BM is a common phenomenon in ovarian cancer. Disseminated tumor cell detection has the potential to become an important biomarker for prognostication and disease monitoring in patients with ovarian cancer.

Comparison of circulating tumor cells (CTC) and disseminated tumor cells (DTC) in patients with colorectal carcinoma for a predictive marker for tumor recurrence.

e14548 Background: Although the presence of CTC or DTC is proposed as a pridictive or a prognostic marker in the management of colorectal carcinoma patients, it is still well unknown what is different of both types of cells. The aim of this study is to compare CTC and DTC as a predictive marker for tumor recurrence by magnetic activated cell sorting system. Methods: Peripheral blood (PB) samples (n=139) and bone marrow (BM) samples (n=41) were preoperatively collected from colorectal carcinoma patients between February 2009 and March 2012. PB samples were obtainable from all patients who provided their BM samples (n=41). Enrichment of CTC was performed by direct immunomagnetic labeling of EpCAM + cells in PB. Enrichment of DTC was done by negative selection of CD45+cells. Subsequently, double immunofluorescences staining for cytokeratin and CD45 was performed to detect CTC or DTC. PB samples from 20 healthy volunteers and BM samples from 3 patients with benign disease were used as controls. Median follow-up time is 28.5 months (range 10-44). Written informed consent was obtained from all enrolled patients. Results: Preoperative positive rates of CTC and DTC were 20.9% (29/139) and 29.3% (12/41), respectively. No CTC or DTC was found in the control groups. In DTC+patients, only 4 patients were positive for CTC (33.3%), whereas 31% of DTC- patients had CTC. We found 13 patients who experienced postoperative recurrence among 139 patients, 7 patients of whom were CTC positive (53.8%). There is a significant higher incidence of recurrence in CTC+patients (risk ratio=4.4, p&lt;0.01). As for DTC, we found 5 patients with recurrence in 41 patients, 4 patients of whom were DTC positive (80%). DTC+ patients have a significant risk of recurrence (risk ratio=9.7, p=0.01). The relationship of CTC / DTC status and the recurrence rates is as follows: 50 % in CTC+/DTC+ patients (2/4); 11.1% in CTC+/DTC- patients (1/9); 25% in CTC-/DTC+ patients (2/8); 0% in CTC-/DTC-patients (0/20). Conclusions: DTC status is more predictive for postoperative recurrence than CTC status. The combination of CTC and DTC analysis might predict recurrence risk more accurately than either of the two analyses.

Persistence of disseminated tumor cells after neoadjuvant treatment for locally advanced breast cancer predicts poor survival

IntroductionPresence of disseminated tumor cells (DTCs) in bone marrow (BM) and circulating tumor cells (CTC) in peripheral blood (PB) predicts reduced survival in early breast cancer. The aim of this study was to determine the presence of and alterations in DTC- and CTC-status in locally advanced breast cancer patients undergoing neoadjuvant chemotherapy (NACT) and to evaluate their prognostic impact.MethodsBone marrow and peripheral blood were collected before NACT (BM1: n = 231/PB1: n = 219), at surgery (BM2: n = 69/PB2: n = 71), and after 12 months from start of NACT (BM3: n = 162/PB3: n = 141). Patients were included from 1997 to 2003 and followed until 2009 (or ten years follow-up). DTC- and CTC-status were determined by morphological evaluation of immunocytochemically detected cytokeratin-positive cells. The prognostic significance of DTCs/CTCs was assessed by univariate and multivariate Cox-regression analyses.ResultsBefore NACT, DTCs and CTCs were detected in 21.2% and 4.9% of the patients, respectively. At surgery, 15.9% and 1.4% had DTC- and CTC-presence, compared to 26.5% and 4.3% at 12 months from start of NACT. Of patients for whom DTC results both before NACT and at 12 months were available, concordant results were observed in 68%, and 14 out of 65 had positive DTC-status at both time points. Presence of ≥ 1 DTC 12 months from start of NACT, but not at other time points, predicted reduced disease-free survival (DFS; HR 2.3, p = 0.003), breast cancer-specific survival (BCSS; HR 3.0, p < 0.001) and overall survival (OS; HR 2.8, p < 0.001). Before NACT, presence of ≥ 3 DTCs was also associated with unfavorable outcome, and reduced BCSS was observed for CTC-positive patients (HR 2.2, p = 0.046). In multivariate analysis, DTC status (</≥ 1 DTC) at 12 months after start of NACT remained as a prognostic factor for both DFS (HR 2.2, p = 0.005), BCSS (HR 2.6, p = 0.002) and OS (HR 2.6, p = 0.002). The survival for patients with change in DTC-status was determined by the DTC-status at 12 months.ConclusionPresence of DTCs after NACT indicated high risk for relapse and death, irrespective of the DTC-status before treatment. The results supports the potential use of DTC analysis as a monitoring tool during follow up, for selection of patients to secondary treatment intervention within clinical trials.

Persistent tumor cells in bone marrow of non-metastatic breast cancer patients after primary surgery are associated with inferior outcome

BackgroundTo investigate the prognostic significance of disseminated tumor cells (DTCs) in bone marrow (BM) from non-metastatic breast cancer patients before and after surgery.MethodsPatients with non-metastatic breast cancer were consecutively recruited to this project during the years 1998–2000. Real-time RT-PCR quantification of a DTC multimarker panel consisting of cytokeratin 19, mammaglobin A and TWIST1 mRNA was performed in BM samples obtained from 154 patients three weeks (BM2) and/or six months after surgery (BM3). The results were compared to previously published data from pre-operative BM analyses for the same patients.ResultsDTCs were identified in post-operative BM samples (BM2 and/or BM3) from 23 (15%) of the 154 patients investigated. During a median follow-up of 98 months, 10 (44%) of these patients experienced systemic relapse as compared to 16 (12%) of 131 DTC-negative patients. Kaplan-Meier estimates of systemic recurrence-free- and breast-cancer specific survival demonstrated significantly shorter survival for patients with persistent DTCs in BM after surgery (p≤0.001). By multivariate Cox regression analyses, persistent DTCs after surgery was an independent predictor of both systemic recurrence-free- (HR = 5.4, p < 0.001) and breast-cancer specific survival (HR = 5.3, p < 0.001). Furthermore, the prognostic value of DTCs in BM was similar for pre- and post surgery samples. However, patients with DTCs both before and after surgery (BM1 and BM2/3) had a particularly poor prognosis (systemic recurrence-free survival: HR = 7.2, p < 0.0001 and breast-cancer specific survival: HR = 8.0, p < 0.0001).ConclusionsDetection of persistent DTCs in BM samples obtained after surgery identified non-metastatic breast cancer patients at high risk for systemic relapse, and with reduced breast-cancer specific survival. Furthermore, patients with positive DTC status both before and after surgery had a particularly poor prognosis.

Detection of disseminated tumor cells in bone marrow as an independent prognostic factor in primary ovarian cancer patients.

5042 Background: Detection of disseminated tumor cells (DTC) in the bone marrow (BM)of breast cancer patients is associated with poor outcome. Recent studies demonstratedthat DTC may serve as a prognostic factor in ovarian cancer.The aim of our study was to evaluate the impact of BM status on survival in a large cohort of ovarian cancer patients. Methods: 365 patients with primary ovarian cancer were included into this three-center prospective study. BM aspirates were collected preoperatively from iliac crest. Disseminatedtumor cells were identified by immunocytochemistry using the pancytokeratin antibodyA45B/B3 and by cytomorphology. Patient outcomes were evaluated using a multivariable Cox regression model. Results: Disseminated tumor cells were detected in 28% of all BM aspirates. The number of CK-positive cells ranged from 1 to 42 per 2x106 mononuclear cells. DTC status did not correlate with any of the established clinicopathogical factors. The overall survival was significantly shorter among DTC-positive patients compared to DTC-negative patients (51 mo, 95% CI: 35 – 67 mo versus 32 mo, 95% CI: 22 – 42 mo; p = 0.003). However, disease-free survival was not related to DTC-positivity. In the multivariable analysis, BM status, FIGO stage, nodal status, resection status and age were independent predictors of reduced overall survival. Interestingly, a subset of DTCs may have stem cell properties since a subset of these cells (128 out of 228 cases) were SOX2 positive, which is an embryonic stem cell marker. Conclusions: Tumor cell dissemination into bone marrow is a common phenomenon in ovarian cancer. DTC detection has the potential to become an important biomarker for prognostication and may be included as a therapeutic target in future concepts.

P4-06-05: Prognostic Impact of Disseminated and Circulating Tumor Cells in Patients Treated for Locally Advanced Breast Cancer.

Abstract INTRODUCTION: Neoadjuvant systemic therapy (NST) in breast cancer patients is an established approach to reduce tumor size prior to surgery and to assess the clinical effect of therapy on the breast cancer disease. The current study was designed to identify primary tumor resistance factors to epirubicin and paclitaxel therapy in patients with locally advanced breast cancer (Chrisanthar et al PLoSOne 2008 and 2011). As a substudy, the incidence of disseminated tumor cells (DTCs) and circulating tumor cells (CTCs) before and after therapy, was investigated. The aim was to evaluate the prognostic impact of DTCs and CTCs as well as to evaluate the effect of NST on DTCs and CTCs. PATIENTS AND METHODS: Patients with locally advanced non-inflammatory breast cancer (T3-4and/or N2) were included in the study. The patients were randomly allocated to primary treatment either with epirubicin 90mg/m2 or paclitaxel 200mg/m2, with cross-over design if no response/progression, followed by mastectomy and axillary dissection. Bone marrow (BM) aspiration and peripheral blood (PB) samples were collected before NST (BM1/PB1)(n=230), at the time of surgery (BM2/PB2)(n=69; logistic reasons caused reduced sampling) and 12 months after randomization (BM3/PB3)(n=162). Detection of DTCs/CTCs was performed by standard immunocytochemical analysis of 2×106 mononuclear cells stained for cytokeratin by AE1AE3 antibodies. Patient outcomes were evaluated over a 10-year follow-up period. 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: Before NST (BM1) 21.3% were DTC positive, compared to 15.9% and 26.5% at BM2 and BM3, respectively. Of those that both had BM1 and BM3 performed, 68% concordance and 22% overlap among positive cases was observed. Presence of DTCs in BM3 predicted reduced DFS (HR 2.2; 95% CI 1.3−3.7; p=0.007) and OS (HR 3.0; 95% CI 1.8−5.2; p&amp;gt;0.001). DTC status before NST had no impact on outcome. No difference in the results was observed after exclusion of patients with limited M1 status at diagnosis (25 and 13 of those analysed for BM1 and BM3, respectively). The incidence of CTCs before NST was 4.9 % compared to 1.4% and 4.3 % at PB2 and PB3, respectively. Presence of CTC before NST was associated with reduced overall survival (HR 2.4; 95% CI 1.2−5.0; p=0.018), but CTC status was not significant for DFS or at other time points. In the multivariable analysis, DTC status at BM3 remained as a prognostic factor for both DFS (HR 2.0; 95% CI 1.1−3.6) and OS (HR 2.1; 95% CI 1.04−4.2). CONCLUSION: In patients with locally advanced breast cancer, the presence of CTCs at the time of diagnosis identified high risk patients. However, the sensitivity of the performed CTC analysis was too low for further interpretation. Presence of DTCs 12 months after neoadjuvant therapy increased the risk for relapse and death. The best clinical utility of DTC analysis appears to be as a monitoring tool during follow up, in a “window of opportunity” for selection of patients to secondary adjuvant treatment intervention within clinical trials. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-06-05.

P5-18-07: Presence of Disseminated Tumor Cells after Adjuvant Chemotherapy in Breast Cancer and Disseminated Tumor Cells Monitoring during Secondary Adjuvant Treatment.

Abstract Introduction: Detection of disseminated tumor cells (DTC) after completion of systemic adjuvant treatment is a strong predictor of early systemic relapse and death. This analysis can discover early failure of a chosen adjuvant systemic treatment. In this study, we wanted to evaluate the value of DTC detection in bone marrow (BM) as a surrogate marker for response to docetaxel rescue treatment, to predict the effect of this treatment. Further, we wanted to compare disease free survival between patients treated with docetaxel resulting in eradication of DTC after treatment and patients treated with docetaxel where DTC persists after treatment. The follow up of the study is still ongoing. Here, we present the preliminary descriptive data from the study. Materials and Methods: A total of 1128 pts with node positive or high risk node negative disease (T1c/T2GII-IIIN0) was enrolled in the period from October 2003 to May 2008. All patients had completed primary surgery and received 6 cycles of adjuvant antracycline containing chemotherapy. The first BM aspiration was performed 8–12 weeks after termination of adjuvant chemotherapy (BM1). A second BM aspiration was performed 6 months later (BM2). The processing of BM and DTC analysis (by ICC) were performed as previsously described (Wiedswang G et al, J Clin Oncol 2003). If BM2 was positive (+) for DTC, the patient was treated with docetaxel (3qw, 6 courses) Docetaxel-treated patients were reexamined at the inclusion hospital with new BM analysis at approximately 1 month (BM3) and 13 months (BM4) after the last docetaxel infusion. Results: Of 997 patients with conclusive DTC results for both BM1 and BM2, 83 patients (8.3%) were BM1 positive and 78 (7.8%) BM2 positive. Among the BM1+ patients, 15 (18.1%) were BM2+. The concordance between BM1 and BM2 were 87%. Of the patients positive at one or both time points, the concordance was 10% (15/146). The BM1 was not significantly associated with primary tumor characteristics (although borderline significance for Grade and ER status), whereas for BM2, DTC+ patients had increased frequency of node positive disease and pN2-3 stage (p=0.001, chi-square), and were positively associated with lobular carcinoma (p=0.01, chi-square). At BM1 24.7% of the DTC+ patients had &amp;gt;1 DTC, and 9.9% had ≥3 DTC. For BM2, 48.2% had &amp;gt;1 DTC, and 25.7% had ≥3 DTC. For patients with positive BM2 receiving docetaxel, the BM3 turned DTC negative in 55 of 66 evaluable cases (83.3%), and 48 of 59 were negative in BM4 (81.4%). Of 67 patients with a conclusive BM result at either BM3 or BM4, 12 were BM positive. Of 16 patients with ≥3 DTC before docetaxel treatment, only 4 patients were positive after treatment (25%). Conclusions: DTC status after adjuvant antracycline containing chemotherapy changes during the first 9 months of FU, with increased DTC positivity among patients with pN+ disease and lobular carcinomas. After docetaxel rescue treatment, the majority of patients experience disappearance of the DTCs. The clinical significance of these results awaits mature FU data, but the present results may indicate possibility for eradication of residual disease by alternative chemotherapy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-18-07.

Disseminated tumor cells in pancreatic cancer—an independent prognosticator of disease progression and survival

Pancreatic cancer is one of the most devastating cancers with a 6-month median survival and a 5-year survival rate of 3-5%. Still important aspects of its aggressive biology remain elusive and advanced therapeutic regimens have not been substantially successful. We investigated the prognostic role of disseminated tumor cells (DTC) in bone marrow, a reservoir for early DTC potentially contributing to metastatic progression, of pancreatic cancer patients. After exclusion of patients with different postsurgery diagnosis or missing DTC status (n = 40) a total of 175 patients remained for final analyses. One-hundred and nineteen patients were male and 96 female with a median age of 67 years, 96 patients underwent complete resection. Bone marrow aspirates taken at primary surgery were analyzed for DTC by an immunocytochemical cytokeratin assay and correlated to survival data. Overall 13.7% of patient samples (24/175) harbored DTC in their bone marrow. Histopathological parameters did not correlate significantly. Univariate survival analysis revealed a borderline significant correlation between DTC and decreased progression-free survival (p = 0.069), and was significant for overall survival (p = 0.036). Regarding patients with resected tumors, the respective p-values were 0.058 for progression-free and 0.016 for overall survival. Importantly, the prognostic influence was independent from other risk factors as shown by multivariate analyses for progression-free (p = 0.030, HR: 2.057; CI (95%): 1.073-3.943) and overall survival (p = 0.006, HR: 2.283; CI (95%): 1.260-4.135). The presence of DTC in bone marrow is a strong and independent prognostic factor of survival in patients with pancreatic cancer. Thus, bone-targeting may be a new future therapeutic option for DTC-positive patients.

Bone marrow versus sentinel lymph node involvement in breast cancer: a comparison of early hematogenous and early lymphatic tumor spread

The early spread of tumor cells in primary breast cancer patients may occur either through lymphatic or hematogenous dissemination. Lymph node (LN) status and presence of disseminated tumor cells (DTC) in bone marrow (BM) are independent predictors of poor outcome. It is unknown which factors determine one or the other route of tumor cell spread and whether lymphatic and hematogenous tumor cell dissemination are two independent processes. This study is aimed to compare the DTC status in clinically node-negative (N0) breast cancer patients with their sentinel LN status and to investigate predictors of BM and LN involvement. The DTC status of 1,345 clinically N0 breast cancer patients who underwent SLN biopsy during initial surgery was investigated. BM and LN status were compared and predictors of hematogenous and lymphatic tumor cell spread were investigated. DTCs were present in the BM of 181 (13%) patients. LN involvement was found in 348 (26%) patients. There was no correlation between LN and BM status: 137 of 997 nodal-negative patients (14%) had BM involvement and 44 of 348 nodal-positive patients (13%) were positive for DTCs (P = 0.649). The presence of DTCs was not influenced by tumorbiological factors. Conversely, a high correlation between nodal status and tumor size, histology, ER-status and lymph vessel invasion was found. Hematogenous and lymphatic tumor spread seem to be because of independent pathways of cancer progression.

A QPCR-Based Circulating Tumor Cell Detection Platform Predicts Survival in 776 Early Stage Breast Cancer Patients.

Abstract The detection of circulating tumor cells (CTCs) in the blood of cancer patients is a promising tool for risk stratification, treatment tailoring, and monitoring of the disease state. We previously developed a QPCR-based platform for the detection of CTCs in peripheral blood, which proved to be prognostic in a cohort of metastatic breast cancer patients.In the current study, we aimed to build on this work by determining the prognostic value of our CTC assay in an independent, prospectively collected, retrospective patient series of 776 non-metastatic, stage I-III breast cancer patients (median follow-up 7.3 years). Assay positivity was observed in 83 patients (10.7%), while maintaining 96% specificity in a group of 25 healthy female controls (however a second blood sample from the healthy control that gave a positive result was found to be negative). CTC positivity as determined by our assay corresponded to a significantly poorer relapse-free survival (RFS) (HR = 1.9, 95% CI = 1.3-2.9, p = 0.02) and overall survival (HR = 1.8, 95% CI = 1.1-2.8, p = 0.02) in this group. This platform demonstrated similar prognostic power to the pan-anticytokeratin (AE1/AE3) immunohistochemistry-based detection of disseminated tumor cells (DTCs) in the bone marrow of these patients (RFS HR = 1.9, 95% CI = 1.3-2.7, p = 0.02). Importantly, multivariate analyses including tumor size, histological grade, lymph node status, and hormone receptor status, both CTC and DTC status remained significant, independent predictors of RFS (CTC status HR = 1.6, 95% CI = 1.0-2.6, p = 0.037; DTC status HR = 1.6; 95% CI = 1.1-2.5, p = 0.023). However, CTC status was prognostic in both lymph node negative patients (HR = 2.0, p = 0.04) and lymph node positive patients (HR = 1.7, p = 0.043), whereas DTC status was only prognostic in lymph node positive patients (HR = 2.0, p = 0.002).This CTC detection platform represents an objective assay with both high specificity and prognostic power, and could be an effective clinical tool for outcome prediction in breast cancer patients.This work was supported by the Sixth Framework Program of the European Commission as part of the international DISMAL collaboration for research into disseminated epithelial malignancies. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3002.

Prognostic implications of isolated tumour cells and micrometastasis in lymph nodes in non-adjuvant treated breast cancer patients.

Abstract Abstract #205 Introduction : The status of axillary lymph nodes (ALNs) is the most powerful predictor of survival and has clinical implications for adjuvant treatment of breast cancer patients. Occult metastases in ALNs of patients initially staged as negative have been detected up to 40%, using the various methods with serial sectioning and/or immunohistochemical (IHC) staining. However, there is no consensus concerning the prognostic significance for occult metastases. The aim of this study was to estimate the prognostic value of isolated tumor cells (ITC, ≤0.2 mm) and micrometastases (MM, 0.2-2.0 mm) in invasive breast cancer.&amp;#x2028; Materials and Methods: The ALNs of 295 patients with axillary node-negative invasive breast cancer treated from 1995 to 1998 was evaluated with serial sectioning and cytokeratin IHC (a single section from each of 10 levels) and hematoxylin-eosin (one section from 3rd and 10th level). Disseminated tumor cell status in bone marrow was also known. Of 295 patients, 232 were offered no systemic adjuvant therapy. Patients were categorized by largest cluster size (≤0,2mm, 0.2-2.0mm), and then their survival rates, i.e. disease-free survival (DFS), distant disease-free survival (DDFS) and breast cancer specific survival (BCSS) were compared with those of node-negative patients. We also investigated the correlation between occult metastases in ALNs and bone marrow (BM).&amp;#x2028; Results: The median patient age was 59 years and the median tumour size was 1.5 cm. At a median observation time of 98 months (9-127 months), 33 patients had developed relapse,2 local and 31 systemic. 17 patients had died of breast cancer. Occult metastases were found in 92 of 295 patients (31%); ITC – 58% (53/92), MM -34% (31/92) and macrometastases (&amp;gt;2.0mm)- 8% (8/92). The cases with macrometastases were excluded from survival analysis. The results presented in the table below demonstrate significant prognostic impact of MM on BCSS, borderline significance on DFS and DDFS. This was not shown for ITC. No significant correlation was found between occult metastases in ALN and BM in both total patients (Χ2-test, p=0.680).&amp;#x2028; &amp;#x2028; Conclusions: Our observation of a prognostic impact of MM in non-adjuvant treated patients indicates that adjuvant therapy might be warranted in this group. Lack of a similar finding in patients with ITC questions the need of adjuvant therapy in this group. The lack of correlation between occult metastases in ALN and BM indicates different “homing” mechanisms for the two compartments. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 205.

Disseminated tumor cells in prostate cancer patients after radical prostatectomy and without evidence of disease predicts biochemical recurrence.

Men with apparently localized prostate cancer often relapse years after radical prostatectomy. We sought to determine if epithelial-like cells identified from bone marrow in patients after radical prostatectomy, commonly called disseminated tumor cells (DTC), were associated with biochemical recurrence. We obtained bone marrow aspirates from 569 men prior to radical prostatectomy and from 34 healthy men with prostate-specific antigens <2.5 ng/mL to establish a comparison group. Additionally, an analytic cohort consisting of 98 patients with no evidence of disease (NED) after radical prostatectomy was established to evaluate the relationship between DTC and biochemical recurrence. Epithelial cells in the bone marrow were detected by magnetic bead enrichment with antibodies to CD45 and CD61 (negative selection) followed by antibodies to human epithelial antigen (positive selection) and confirmation with FITC-labeled anti-BerEP4 antibody. DTC were present in 72% (408 of 569) of patients prior to radical prostatectomy. There was no correlation with pathologic stage, Gleason grade, or preoperative prostate-specific antigens. Three of 34 controls (8.8%) had DTC present. In patients with NED after radical prostatectomy, DTC were present in 56 of 98 (57%). DTC were detected in 12 of 14 (86%) NED patients after radical prostatectomy who subsequently suffered biochemical recurrence. The presence of DTC in NED patients was an independent predictor of recurrence (hazard ratio 6.9; 95% confidence interval, 1.03-45.9). Approximately 70% of men undergoing radical prostatectomy had DTC detected in their bone marrow prior to surgery, suggesting that these cells escape early in the disease. Although preoperative DTC status does not correlate with pathologic risk factors, persistence of DTC after radical prostatectomy in NED patients was an independent predictor of recurrence.

Bone marrow disseminated tumor cells, loco-regional and adjuvant treatment are prognostic factors for loco-regional relapses in early breast cancers.

Abstract Abstract #307 Purpose: To study in early breast cancer (BC) patients (pts) if bone marrow (BM) disseminated tumor cells (DTC) are associated with a specific loco-regional dissemination of cancer cells, according to the radiotherapy fields and to the pattern of loco-regional relapses.&amp;#x2028; Patient and methods: BM aspirates were prospectively screened for cytokeratin positive (CK+) cells at primary treatment in stage I-III BC pts. Local recurrence (LR) was defined as recurrence of the breast or chest wall; Regional recurrence (RR) was defined as lymph node recurrence after loco-regional treatment. Irradiation fields, BM DTC status, clinical and pathological variables of the pts were correlated with loco-regional relapse-free survival (LRRFS).&amp;#x2028; Results: BM DTC were detected in 94 out of 621 pts (15%) and were not associated with the axillary lymph node status and other prognostic factors. After a median follow-up of 56 months, 18 pts (2,9%) experienced loco-regional relapses: 15 LR occurred in the RT fields whereas 2 LR and 1 RR were out. 8 of the 18 relapsing pts (44%) were DTC positive (8 LR in the RT fields). BM DTC detection was the only prognostic factor for LRRFS (p=0.0005, OR=5.2 [2.0-13.1]) in multivariate analysis. In BM DTC positive patients, longer LRRFS were observed in those who were given adjuvant hormonal therapy (p=0.03) and RT on supra-clavicular (SCN) and internal mammary (IMN) nodes (p=0.055) (multivariate analysis).&amp;#x2028; Conclusions: BM DTC strongly influence loco-regional relapse in early BC patients. Our data support the hypothesis of a different lymphatic spread of BM DTC positive BC and of a possible re-seeding of BM DTC to the breast. Survival analyses suggest that BM DTC positive patients take advantage of adjuvant hormone therapy but also of adjuvant SCN and IMN irradiation. This observation could be one explanation of the influence of systemic treatment on locoregional relapse and of radiotherapy on distant metastatic dissemination. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 307.

Presence of apoptotic and nonapoptotic disseminated tumor cells reflects the response to neoadjuvant systemic therapy in breast cancer

IntroductionNeoadjuvant systemic therapy (NST) is an established strategy to reduce tumor size in breast cancer patients prior to breast-conserving therapy. The effect of NST on tumor cell dissemination in these patients is not known. The aim of this study was to investigate the incidence of disseminated tumor cells (DTC), including apoptotic DTC, in breast cancer patients after NST, and to investigate the correlation of DTC status with therapy response.MethodsBone marrow aspiration was performed in 157 patients after NST. DTC were detected by immunocytochemistry using the A45–B/B3 anticytokeratin antibody. To detect apoptotic DTC the antibody M30 (Roche Diagnostics, Germany) was used, which detects a neo-epitope expressed only after caspase cleavage of cytokeratin 18 during early apoptosis.ResultsThe incidence of DTC in breast cancer patients was 53% after completion of NST. Tumor dissemination was observed more frequently in patients with no change/progressive disease (69%) than in patients with partial remission or complete remission of the primary tumor (46%) (P < 0.05). Ten out of 24 patients with complete remission, however, were still bone marrow positive. Apoptotic DTC were present in 36 of 157 (23%) breast cancer patients. Apoptotic cells only were detected in 14% of the patients with partial remission or complete remission, but were detected in just 5% of the patients with stable disease. Apoptotic DTC were detectable in none of the patients with tumor progression.ConclusionThe pathological therapy response in breast cancer patients is reflected by the presence of apoptotic DTC. Patients with complete remission, however, may still have nonapoptotic DTC. These patients may also benefit from secondary adjuvant therapy.