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. Clin Cancer Res; 17(9); 2967-76. Ó2011 AACR.

Published by Elsevier B.V. All rights reserved. IntroductionGenome-wide expression profiling has demonstrated great power in deciphering molecular portraits of human breast tumors and has identified gene signatures that can be correlated to many different aspects of breast cancer such as tumor progression, prediction of outcome and sensitivity to therapy (Ayers et al., 2004;Chang et al., 2005Chang et al., , 2003Dai et al., 2005;Ma et al., 2003;Sorlie et al., 2001;van't Veer et al., 2002;Wang et al., 2004). Distinct subtypes that are associated with significant differences in overall and disease-free survival have been identified and validated in different patient cohorts (Bertucci et al., 2005;Sorlie et al., 2003;Sotiriou et al., 2003;Yu et al., 2004). Molecular profiling by microarray technologies will improve our understanding of primary tumor biology and the mechanisms behind tumorigenesis, and also provide

Subtypes DNA methylation MOMA Survival Epigenetics A B S T R A C TThe diversity of breast cancers reflects variations in underlying biology and affects the clinical implications for patients. Gene expression studies have identified five major subtypese Luminal A, Luminal B, basal-like, ErbB2þ and Normal-Like. We set out to determine the role of DNA methylation in subtypes by performing genome-wide scans of CpG methylation in breast cancer samples with known expression-based subtypes. Unsupervised hierarchical clustering using a set of most varying loci clustered the tumors into a Luminal A majority (82%) cluster, Basal-like/ErbB2þ majority (86%) cluster and a non-specific cluster with samples that were also inconclusive in their expression-based subtype correlations.Contributing methylation loci were both gene associated loci (30%) and non-gene associ-

BackgroundThe presence of disseminated tumor cells (DTCs) in bone marrow (BM) is an independent prognostic factor in early breast cancer but does not uniformly predict outcome. Tumor cells can persist in a quiescent state over time, but clinical studies of markers predicting the awakening potential of DTCs are lacking. Recently, experiments have shown that NR2F1 (COUP-TF1) plays a key role in dormancy signaling.MethodsWe analyzed the NR2F1 expression in DTCs by double immunofluorescence (DIF) staining of extra cytospins prepared from 114 BM samples from 86 selected DTC-positive breast cancer patients. Samples collected at two or more time points were available for 24 patients. Fifteen samples were also analyzed for the proliferation marker Ki67.ResultsOf the patients with detectable DTCs by DIF, 27% had ≥ 50% NR2F1high DTCs, chosen a priori as the cut-off for “dormant profile” classification. All patients with systemic relapse within 12 months after BM aspiration carried ≤ 1% NR2F1high DTCs, including patients who transitioned from having NR2F1high-expressing DTCs in previous BM samples. Of the patients with serial samples, half of those with no relapse at follow-up had ≥ 50% NR2F1high DTCs in the last BM aspiration analyzed. Among the 18 relapse-free patients at the time of the last DTC-positive BM aspiration with no subsequent BM analysis performed, distant disease-free intervals were favorable for patients carrying ≥ 50% NR2F1high DTCs compared with those with predominantly NR2F1low DTCs (p = 0.007, log-rank). No survival difference was observed by classification according to Ki67-expressing DTCs (p = 0.520).ConclusionsOur study translates findings from basic biological analysis of DTC dormancy to the clinical situation and supports further clinical studies of NR2F1 as a marker of dormancy.Electronic supplementary materialThe online version of this article (10.1186/s13058-018-1049-0) contains supplementary material, which is available to authorized users.

IntroductionThe detection of circulating tumour cells (CTCs) in the peripheral blood and disseminated tumour cells (DTCs) in the bone marrow are promising prognostic tools for risk stratification in early breast cancer. There is, however, a need for further validation of these techniques in larger patient cohorts with adequate follow-up periods.MethodsWe assayed CTCs and DTCs at primary surgery in 733 stage I or II breast cancer patients with a median follow-up time of 7.6 years. CTCs were detected in samples of peripheral blood mononuclear cells previously stored in liquid-nitrogen using a previously-developed multi-marker quantitative PCR (QPCR)-based assay. DTCs were detected in bone marrow samples by immunocytochemical analysis using anti-cytokeratin antibodies.ResultsCTCs were detected in 7.9% of patients, while DTCs were found in 11.7%. Both CTC and DTC positivity predicted poor metastasis-free survival (MFS) and breast cancer-specific survival (BCSS); MFS hazard ratio (HR) = 2.4 (P < 0.001)/1.9 (P = 0.006), and BCSS HR = 2.5 (P < 0.001)/2.3 (P = 0.01), for CTC/DTC status, respectively). Multivariate analyses demonstrated that CTC status was an independent prognostic variable for both MFS and BCSS. CTC status also identified a subset of patients with significantly poorer outcome among low-risk node negative patients that did not receive adjuvant systemic therapy (MFS HR 2.3 (P = 0.039), BCSS HR 2.9 (P = 0.017)). Using both tests provided increased prognostic information and indicated different relevance within biologically dissimilar breast cancer subtypes.ConclusionsThese results support the use of CTC analysis in early breast cancer to generate clinically useful prognostic information.

DTC status identifies high-risk patients after FEC chemotherapy, and DTC monitoring status after secondary treatment with docetaxel correlated strongly with survival. This emphasizes the potential for DTC analysis as a surrogate marker for adjuvant treatment effect in breast cancer.

Purpose: The interaction between tumor cells, stroma, and endothelial cells is important for the dissemination of tumor cells. The aim of the present study is to examine vascularity in primary breast carcinomas and its prognostic significance and relationship with tumor cell dissemination. Experimental Design: A total of 498 invasive breast carcinomas were analyzed. Representative tumor sections were stained for CD34 and CD105, and vascularity was quantified by the Chalkley method. The relationship between Chalkley counts, vascular invasion, disseminated tumor cells (DTC) in the bone marrow, other clinicopathologic variables, and clinical outcome was evaluated. Results: High vascular grades determined by Chalkley counts were significantly associated with shorter distant disease^free survival and breast cancer^specific survival in all patients (P < 0.001, log-rank) and in node-negative patients not receiving adjuvant systemic therapy (P < 0.05). In multivariate analysis, both CD34 and CD105 Chalkley counts showed prognostic significance for distant disease^free survival (P = 0.014 and P = 0.026), whereas CD34 also showed prognostic significance for breast cancer^specific survival (P = 0.007). Vascular invasion and DTCs in the bone marrow showed independent prognostic significance. DTC did not discriminate survival for CD34 low Chalkley counts, whereas a very poor prognosis was observed for DTC-positive patients with high CD34 counts. In node-negative patients not receiving systemic chemotherapy, high CD34 and high CD105 counts in combination identified patients with unfavorable outcome, as opposed to all other CD34/CD105 combinations. Conclusions: Improved identification of risk groups could be obtained by adding CD34 and CD105 vascular analysis to DTC, vascular invasion, and other primary tumor factors. This may facilitate the selection of candidates for adjuvant systemic therapy.

The clinical relevance of isolated tumor cell (ITC: 0.2-2.0 mm) in axillary lymph nodes (ALNs) remains unknown. The aim of this study was to determine their prognostic significance. A total of 295 patients considered as pN0 after routine histological assessment, were reevaluated with ten-level cytokeratin immunohistochemistry (IHC) and two-level hematoxylin-eosin sections. Survival rates, i.e. disease-free survival (DFS), distant disease-free survival (DDFS) and breast cancer specific survival (BCSS) were compared with those of reevaluated node-negative patients. A total of 84 patients (28%) had ITC/MM identified on IHC sections. ITC had no impact on survival at a median 8.2 years of follow-up, whereas MM showed a trend toward poorer DFS (P = 0.091, log rank) and DDFS (P = 0.066) and significantly reduced BCSS (P = 0.016). In multivariate analyses, detection of MM was an independent prognostic factor for DDFS (P = 0.025) and BCSS (P = 0.01) in adjuvant un-treated patients. Micrometastases (MMs) in axillary lymph nodes have prognostic impact. This was not found for ITC. This finding supports the use of systemic adjuvant therapy in patients with MM.