Efficacy of Lapatinib in Patients with HER2-Negative Metastatic Breast Cancer and HER2-Positive Circulating Tumor Cells-The DETECT III Clinical Trial.

BACKGROUND: The phenotypes of tumor cells change during disease progression, but invasive rebiopsies of metastatic lesions are not always feasible. Here we aimed to determine whether initially HER2-negative metastatic breast cancer (MBC) patients with HER2-positive circulating tumor cells (CTCs) benefit from a HER2-targeted therapy. METHODS: The open-label, interventional randomized phase III clinical trial (EudraCT Number 2010-024238-46, CliniclTrials.gov Identifier: NCT01619111) recruited from March 2012 until September 2019 with a follow-up duration of 19.5 months. It was a multicenter clinical trial with 94 participating German study centers. A total of 2137 patients with HER2-negative MBC were screened for HER2-positive CTCs with a final modified intention-to-treat population of 101 patients. Eligible patients were randomized to standard therapy with or without lapatinib. Primary study endpoints included CTC clearance (no CTCs at the end of treatment) and secondary endpoints were progression-free survival, overall survival (OS), and safety. RESULTS: In both treatment arms CTC clearance at first follow-up visit-although not being significantly different for both arms at any time point-was significantly associated with improved OS (42.4 vs 14.1 months; P = 0.002). Patients treated additionally with lapatinib had a significantly improved OS over patients receiving standard treatment (20.5 vs 9.1 months, P = 0.009). CONCLUSIONS: DETECT III is the first clinical study indicating that phenotyping of CTCs might have clinical utility for stratification of MBC cancer patients to HER2-targeting therapies. The OS benefit could be related to lapatinib, but further studies are required to prove this clinical observation. ClinicalTrials.gov Registration Number: NCT01619111.

Comparative analysis of EpCAM high-expressing and low-expressing circulating tumour cells with regard to their clonal relationship and clinical value.

BACKGROUND: Circulating tumour cells (CTCs) are mainly enriched based on the epithelial cell adhesion molecule (EpCAM). Although it was shown that an EpCAM low-expressing CTC fraction is not captured by such approaches, knowledge about its prognostic and predictive relevance and its relation to EpCAM-positive CTCs is lacking. METHODS: We developed an immunomagnetic assay to enrich CTCs from metastatic breast cancer patients EpCAM independently using antibodies against Trop-2 and CD-49f and characterised their EpCAM expression. DNA of single EpCAM high expressing and low expressing CTCs was analyzed regarding chromosomal aberrations and predictive mutations. Additionally, we compared CTC-enrichment on the CellSearch system using this antibody mix and the EpCAM based enrichment. RESULTS: Both antibodies acted synergistically in capturing CTCs. Patients with EpCAM high-expressing CTCs had a worse overall and progression-free survival. EpCAM high- and low-expressing CTCs presented similar chromosomal aberrations and mutations indicating a close evolutionary relationship. A sequential enrichment of CTCs from the EpCAM-depleted fraction yielded a population of CTCs not captured EpCAM dependently but harbouring predictive information. CONCLUSIONS: Our data indicate that EpCAM low-expressing CTCs could be used as a valuable tumour surrogate material-although they may be prognostically less relevant than EpCAM high-expressing CTCs-and have particular benefit if no CTCs are detected using EpCAM-dependent technologies.

Clinical relevance of circulating tumor cells in ovarian, fallopian tube and peritoneal cancer.

PURPOSE: Presence of circulating tumor cells (CTCs) is associated with impaired clinical outcome in several solid cancers. Limited data are available on the significance of CTCs in gynaecological malignancies. The aims of the present study were to evaluate the dynamics of CTCs in patients with ovarian, fallopian tube and peritoneal cancer during chemotherapy and to assess their clinical relevance. METHODS: 43 patients with ovarian, fallopian tube and peritoneal cancer were included into this prospective study. Patients received chemotherapy according to national guidelines. CTC analysis was performed using the CellSearch system prior to chemotherapy, after three and six cycles. RESULTS: In 26% of the patients, ≥ 1CTC per 7.5 ml of blood was detected at baseline (17% of patients with de novo disease, compared to 35% in recurrent patients). Presence of CTCs did not correlate with other factors. After three cycles of therapy, CTC positivity rate declined to 4.8%. After six cycles, no patient showed persistent CTCs. Patients with ≥ 1 CTC at baseline had significantly shorter overall survival and progression-free survival compared to CTC-negative patients (OS: median 3.1 months vs. not reached, p = 0.006, PFS: median 3.1 vs. 23.1 months, p = 0.005). When only the subgroup with newly diagnosed cancer was considered, the association between CTC status and survival was not significant (OS: mean 17.4 vs. 29.0 months, p = 0.192, PFS: 14.3 vs. 26.9 months, p = 0.085). Presence of ≥ 1 CTC after three cycles predicted shorter OS in the entire patient cohort (p < 0.001). CONCLUSIONS: Hematogenous tumor cell dissemination is a common phenomenon in ovarian, fallopian tube and peritoneal cancer. CTC status before start of systemic therapy correlates with clinical outcome. Chemotherapy leads to a rapid decline in CTC counts; further research is needed to evaluate the clinical value of CTC monitoring after therapy.

Detection of ESR1 Mutations in Single Circulating Tumor Cells on Estrogen Deprivation Therapy but Not in Primary Tumors from Metastatic Luminal Breast Cancer Patients.

Mutations in the ligand-binding domain (LBD) of the ESR1 gene result in resistance to estrogen deprivation therapy (EDT) in breast cancer. Their detection might enable optimization of therapy strategies. However, the predictive utility of the primary tumor (PT) is limited, and obtaining serial biopsies of metastatic lesions is challenging. To underline their application as a liquid biopsy, single circulating tumor cells (CTCs) were analyzed with a next-generation sequencing approach for the ESR1 coding region. CTCs from 46 metastatic luminal breast cancer patients were enriched using CellSearch system and isolated by micromanipulation. Their genomic DNA was amplified and the ESR1 gene was sequenced. Furthermore, tissue samples from corresponding PTs and/or metastatic lesions were investigated. ESR1 mutations were detected in 12 patients-exclusively in patients treated with EDT (P = 0.048). In seven cases mutations were located in the hotspot regions in the LBD. Six novel mutations were identified. ESR1 mutations were absent in PT tissue samples and were detected only in metastases obtained after CTC characterization. Single-cell CTC analysis for ESR1 mutations could be of clinical value to identify patients who progress under EDT and therefore benefit from an early switch to an alternative endocrine therapy or other treatment regimens. Furthermore, our data indicate that mutations outside the LBD's hotspot regions might also contribute to resistance to EDT.

Determination of the androgen receptor status of circulating tumour cells in metastatic breast cancer patients.

BACKGROUND: The prognostic relevance of circulating tumour cells (CTCs) in metastatic breast cancer (MBC) patients has been confirmed by several clinical trials. However, predictive blood-based biomarkers for stratification of patients for targeted therapy are still lacking. The DETECT studies explore the utility of CTC phenotype for treatment decisions in patients with HER2 negative MBC. Associated with this concept is a plethora of translational projects aiming to identify potential predictive biomarkers. The androgen receptor (AR) is expressed in over 70% of hormone receptor-positive and up-to 45% of triple-negative tumours. Studies has indicated the promising nature of AR as a new therapy target with a clinical benefit rate for anti-AR treatment in MBC patients up to 25% The aim of this analysis was the characterization of CTCs regarding the expression of the AR using immunofluorescence. METHODS: MBC patients were screened for the HER2-status of CTCs in the DETECT studies. In a subset of CTC-positive patients (n = 67) an additional blood sample was used for immunomagnetic enrichment of CTCs using the CellSearch® Profile Kit prior to transfer of the cells onto cytospin slides. Establishment of immunofluorescence staining for the AR was performed using prostate cancer cell lines LNCaP and DU145 as positive and negative control, respectively. Staining of DAPI, pan-cytokeratin (CK) and CD45 was applied to identify nucleated epithelial cells as CTCs and to exclude leucocytes. RESULTS: Co-staining of the AR, CK and CD45 according to the above mentioned workflow has been successfully established using cell lines with known AR expression spiked into the blood samples from healthy donors. For this translational project, samples were analysed from 67 patients participating in the DETECT studies. At least one CTC was detected in 37 out of 67 patients (56%). In 16 of these 37 patients (43%) AR-positive CTCs were detected. In eight out of 25 patients (32%) with more than one CTC, AR-positive and AR-negative CTCs were observed. CONCLUSION: In 43% of the analysed CTC samples from patients with MBC the AR expression has been detected. The predictive value of AR expression in CTCs remains to be evaluated in further trials.

Diagnostic Leukapheresis Enables Reliable Transcriptomic Profiling of Single Circulating Tumor Cells to Characterize Inter-Cellular Heterogeneity in Terms of Endocrine Resistance.

Circulating tumor cells (CTCs) hold great promise with regard to prognosis, treatment optimization, and monitoring of breast cancer patients. Single CTC transcriptome profiling might help reveal valuable information concerning intra-patient heterogeneity relevant to therapeutic interventions. In this study, we combined Diagnostic Leukapheresis (DLA), which is a microfluidic enrichment using the ParsortixTM system, micromanipulation with CellCelectorTM and subsequent single cell multi-marker transcriptome profiling. First, a PCR panel consisting of 30 different endocrine resistance and phenotypic marker genes was validated for single cell profiling by using different breast cancer cell lines. Second, this panel was applied to characterize uncultured and cultured CTCs, which were enriched from a cryopreserved DLA product obtained from a patient suffering from metastatic breast cancer resistant to endocrine therapy. Gene expression profiles of both CTC populations uncovered inter CTC heterogeneity for transcripts, which are associated with response or resistance to endocrine therapy (e.g., ESR1, HER2, FGFR1). Hierarchical clustering revealed CTC subpopulations with different expressions of transcripts regarding the CTCs' differential phenotypes (EpCAM, CD44, CD24, MYC, MUC1) and of transcripts involved in endocrine signaling pathways (FOXO, PTEN). Moreover, ER-positive CTCs exhibited significant higher expression of Cyclin D1, which might be relevant for CDK4/6 inhibitor therapies. Overall, gene expression profiles of uncultured and cultured CTCs resulted in a partly combined grouping. Our findings demonstrate that multi-marker RNA profiling of enriched single uncultured CTCs and cultured CTCs form cryopreserved DLA samples may provide important insights into intra-patient heterogeneity relevant for targeted therapies and therapy resistance.

Circulating and Disseminated Tumour Cells in Breast Carcinoma: Report from the Consensus Conference on Tumour Cell Dissemination during the 38th Annual Meeting of the German Society of Senology, Berlin, 14 June 2018.

Haematogenic dissemination of tumour cells in breast carcinoma is among the most intensively researched areas in translational oncology. Large meta-analyses have shown the prognostic relevance of the disseminated tumour cells in the bone marrow and circulating tumour cells in the peripheral blood in the adjuvant as well as metastatic setting. The current status of the research was discussed in detail during the annual meeting of the German Society of Senology in Berlin. The following conference report gives an overview of the clinical study landscape and the new methodological developments for improving the detection and phenotyping of the circulating and disseminated tumour cells.

The clinical use of circulating tumor cells (CTCs) enumeration for staging of metastatic breast cancer (MBC): International expert consensus paper.

BACKGROUND: The heterogeneity of metastatic breast cancer (MBC) necessitates novel biomarkers allowing stratification of patients for treatment selection and drug development. We propose to use the prognostic utility of circulating tumor cells (CTCs) for stratification of patients with stage IV disease. METHODS: In a retrospective, pooled analysis of individual patient data from 18 cohorts, including 2436 MBC patients, a CTC threshold of 5 cells per 7.5 ml was used for stratification based on molecular subtypes, disease location, and prior treatments. Patients with ≥ 5 CTCs were classified as Stage IVaggressive, those with < 5 CTCs as Stage IVindolent. Survival was analyzed using Kaplan-Meier curves and the log rank test. RESULTS: For all patients, Stage IVindolent patients had longer median overall survival than those with Stage IVaggressive (36.3 months vs. 16.0 months, P < 0.0001) and similarly for de novo MBC patients (41.4 months Stage IVindolent vs. 18.7 months Stage IVaggressive, p < 0.0001). Moreover, patients with Stage IVindolent disease had significantly longer overall survival across all disease subtypes compared to the aggressive cohort: hormone receptor-positive (44 months vs. 17.3 months, P < 0.0001), HER2-positive (36.7 months vs. 20.4 months, P < 0.0001), and triple negative (23.8 months vs. 9.0 months, P < 0.0001). Similar results were obtained regardless of prior treatment or disease location. CONCLUSIONS: We confirm the identification of two subgroups of MBC, Stage IVindolent and Stage IVaggressive, independent of clinical and molecular variables. Thus, CTC count should be considered an important tool for staging of advanced disease and for disease stratification in prospective clinical trials.

Label-Free Enrichment and Molecular Characterization of Viable Circulating Tumor Cells from Diagnostic Leukapheresis Products.

INTRODUCTION: Circulating tumor cells (CTCs) may be used to improve cancer diagnosis, prognosis, and treatment. However, because knowledge regarding CTC biology is limited and the numbers of CTCs and CTC-positive cancer patients are low, progress in this field is slow. We addressed this limitation by combining diagnostic leukapheresis (DLA) and microfluidic enrichment to obtain large numbers of viable CTCs from metastasized breast cancer patients. METHODS: DLA was applied to 9 patients, and 7.5 mL of peripheral blood was drawn. CTCs were enriched with the Parsortix™ system. The quality of CTCs from fresh and cryopreserved DLA products was tested, and CTCs were cultured in vitro. Single uncultured and cultured CTCs were isolated by micromanipulation to determine different parameters, such as genomic aberrations and mutation profiles of selected tumor-associated genes. Expression levels of estrogen receptor and HER2/neu were monitored during in vitro culture. RESULTS: Viable CTCs from peripheral blood and fresh or frozen DLA products could be enriched. DLA increased the likelihood of successful CTC culture. Cryopreserved DLA products could be stored with minimal CTC loss and no overt reduction in the tumor cell quality and viability during an observation period of up to 3 years. The analyzed parameters did not change during in vitro culture. DLA samples with high CTC numbers and lower ratios of apoptotic CTCs were more likely to grow in culture. CONCLUSIONS: The increased CTC numbers from fresh or cryopreserved DLA products facilitate multiple functional and molecular analyses and, thus, could improve our knowledge of their biology.

Diagnostic leukapheresis for CTC analysis in breast cancer patients: CTC frequency, clinical experiences and recommendations for standardized reporting.

Diagnostic leukapheresis (DLA) is based on continuous centrifugation that collects mononuclear cells from peripheral blood with a density of 1.055-1.08 g/ml. As epithelial cells have a similar density, DLA cocollects circulating tumor cell (CTCs) along with the targeted mononuclear cells. Here, we report on our single center experience applying DLA in 40 nonmetastatic and metastatic breast cancer patients and its impact on CTC detection. We found that the use of just 5% of the DLA product (corresponding to a median peripheral blood volume of around 60 ml) in the CellSearch® assay already leads to a significant increase in CTC detection frequency and yield. The implementation of the method was unproblematic, and we did not observe any adverse events in our patient cohort. Extrapolating the CTC counts in the DLA samples to the whole DLA product indicated that enormous CTC numbers could be harvested by this approach (around 205x more CTCs than in the 7.5 ml blood sample in M1 patients). In conclusion, DLA is a clinically safe method to collect CTCs from liters of blood enabling a real liquid biopsy. Yet, further technical developments are required to process whole DLA products and exploit the full potential of this approach. As it is foreseeable that DLA will be used by several groups, and hopefully ultimately brought to the patients in a routine setting, we discuss recommendations on the minimum of required information for reporting on DLAs to allow comparison across different approaches. © 2018 International Society for Advancement of Cytometry.

Filtration based assessment of CTCs and CellSearch® based assessment are both powerful predictors of prognosis for metastatic breast cancer patients.

BACKGROUND: The assessment of circulating tumor cells (CTCs) has been shown to enable monitoring of treatment response and early detection of metastatic breast cancer (MBC) recurrence. The aim of this study was to compare a well-established CTC detection method based on immunomagnetic isolation with a new, filtration-based platform. METHODS: In this prospective study, two 7.5 ml blood draws were obtained from 60 MBC patients and CTC enumeration was assessed using both the CellSearch® and the newly developed filtration-based platform. We analyzed the correlation of CTC-positivity between both methods and their ability to predict prognosis. Overall survival (OS) was calculated and Kaplan-Meier curves were estimated with thresholds of ≥1 and ≥5 detected CTCs. RESULTS: The CTC positivity rate of the CellSearch® system was 56.7% and of the filtration-based platform 66.7%. There was a high correlation of CTC enumeration obtained with both methods. The OS for patients without detected CTCs, regardless of the method used, was significantly higher compared to patients with one or more CTCs (p < 0.001). The median OS of patients with no CTCs vs. ≥ 1 CTC assessed by CellSearch® was 1.83 years (95% CI: 1.63-2.02) vs. 0.74 years (95% CI: 0.51-1.52). If CTCs were detected by the filtration-based method the median OS times were 1.88 years (95% CI: 1.74-2.03) vs. 0.59 years (95% CI: 0.38-0.80). CONCLUSIONS: The newly established EpCAM independently filtration-based system is a suitable method to determine CTC counts for MBC patients. Our study confirms CTCs as being strong predictors of prognosis in our population of MBC patients.

Discordance in Human Epidermal Growth Factor Receptor 2 (HER2) Phenotype Between Primary Tumor and Circulating Tumor Cells in Women With HER2-Negative Metastatic Breast Cancer.

PURPOSE: Discordance in human epidermal growth factor receptor 2 (HER2) status between primary tumor and metastases might have important implications for treatment response and therapy decisions. Here, we evaluate both the frequency of circulating tumor cells (CTCs) and the factors predicting HER2 discordance between primary tumor and CTCs as a potential surrogate for tumor biology and tumor heterogeneity in patients with metastatic breast cancer. PATIENTS AND METHODS: The number of CTCs in 7.5 mL of peripheral blood and HER2 status were evaluated in 1,123 women with HER2-negative metastatic breast cancer. HER2 discordance was defined as the presence of at least one CTC with a strong immunocytochemical HER2 staining intensity. Factors predicting discordance in HER2 phenotype were assessed using multivariable logistic regression. RESULTS: Overall, 711 (63.3%) of 1,123 screened patients were positive for CTCs (≥ one CTC). Discordance in HER2 phenotype between primary tumor and CTCs was observed in 134 patients (18.8%) and was significantly associated with histologic type (lobular v ductal; odds ratio [OR], 2.67; 95% CI, 1.63 to 4.39; P < .001), hormone receptor status (positive v negative; OR, 2.84; 95% CI, 1.15 to 7.02; P = .024), and CTC number (≥ five v one to four; OR, 7.64; 95% CI, 3.97 to 14.72; P < .001). CONCLUSION: HER2 discordance between primary tumor and CTCs was observed in 18.8% of patients and was associated with histologic type, hormone receptor status of the primary tumor, and CTC number. The clinical utility of CTCs as liquid biopsy to assess tumor heterogeneity of metastatic disease and guide treatment decisions must be evaluated in prospective randomized trials.

Circulating tumor cells in breast cancer--current status and perspectives.

The phenomenon of tumor cell dissemination through the blood stream has been known since the 19th century. Circulating tumor cells (CTCs) may be detected in peripheral blood of patients with breast cancer and may serve as a surrogate marker for minimal residual disease. Prognostic relevance of CTCs has already been demonstrated in early and metastatic breast cancer and commercially available detection systems are currently employed in various clinical trials. Since peripheral blood is an easily accessible compartment, serial reevaluation of CTCs is possible and may contribute to better therapy monitoring. Another potential of CTCs lies in the characterization of tumor cells. Expression profiles may differ between CTCs and primary tumor, which may result in different responses to treatment. Assessment of molecular features of CTCs may be an important step for the optimization of adjuvant and metastatic systemic therapy.

Therapeutic intervention based on circulating tumor cell phenotype in metastatic breast cancer: concept of the DETECT study program.

PURPOSE: The aim of the ongoing DETECT study program is to evaluate therapeutic intervention based on phenotypes of circulating tumor cells (CTC) in patients with metastatic breast cancer (MBC). Currently (as of July 2015) more than half of the projected about 2000 patients with MBC have already been screened for CTC. METHODS: Women with HER2-negative primary tumor and presence of CTC are recruited into different DETECT trials according to the HER2-phenotype of CTC. Patients with HER2-positive CTC are randomized to treatment with physicians' choice therapy (standard chemo- or endocrine therapy) with or without additional HER2-targeted therapy with lapatinib in the DETECT III trial. In DETECT IVa, postmenopausal patients with hormone-receptor positive primary cancer and HER2-negative CTC receive everolimus and standard endocrine therapy. For women with HER2-negative CTC and triple negative MBC or hormone-receptor positive tumor and indication for chemotherapy, a treatment with eribulin is offered (DETECT IVb). The clinical efficacy is investigated by CTC-Clearance and progression-free survival (PFS). The DETECT V/CHEVENDO trial extends the DETECT study program for women with HER2-positive and hormone-receptor positive MBC. The primary objective of this trial is to compare safety and quality of life (QoL) as assessed by the occurrence of adverse events in patients treated with dual (trastuzumab plus pertuzumab) HER2-targeted therapy plus either endocrine or chemotherapy. The translational research projects of the DETECT study program focus on further molecular characterization of CTC and evaluation of markers for their suitability to predict treatment response and to facilitate the development of more personalized treatment options.

Molecular profiling of single circulating tumor cells with diagnostic intention.

Several hundred clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non-random whole genome amplification method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 CTC-positive breast cancer patients. We defined a genome integrity index (GII) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of > 90% for successful molecular analysis of high-quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered pre-existing cells resistant to ERBB2-targeted therapies suggesting ongoing microevolution at late-stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance.

Genomic high-resolution profiling of single CKpos/CD45neg flow-sorting purified circulating tumor cells from patients with metastatic breast cancer.

BACKGROUND: Circulating tumor cells (CTCs) are promising surrogate markers for systemic disease, and their molecular characterization might be relevant to guide more individualized cancer therapies. To enable fast and efficient purification of individual CTCs, we developed a work flow from CellSearch(TM) cartridges enabling high-resolution genomic profiling on the single-cell level. METHODS: Single CTCs were sorted from 40 CellSearch samples from patients with metastatic breast cancer using a MoFlo XDP cell sorter. Genomes of sorted single cells were amplified using an adapter-linker PCR. Amplification products were analyzed by array-based comparative genomic hybridization, a gene-specific quantitative PCR (qPCR) assay for cyclin D1 (CCND1) locus amplification, and genomic sequencing to screen for mutations in exons 1, 9, and 20 of the phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) gene and exons 5, 7, and 8 of the tumor protein p53 (TP53) gene. RESULTS: One common flow-sorting protocol was appropriate for 90% of the analyzed CellSearch cartridges, and the detected CTC numbers correlated positively with those originally detected with the CellSearch system (R(2) = 0.9257). Whole genome amplification was successful in 72.9% of the sorted single CTCs. Over 95% of the cells displayed chromosomal aberrations typical for metastatic breast cancers, and amplifications at the CCND1 locus were validated by qPCR. Aberrant CTCs from 2 patients harbored mutations in exon 20 of the PIK3CA gene. CONCLUSIONS: This work flow enabled effective CTC isolation and provided insights into genomic alterations of CTCs in metastatic breast cancer. This approach might facilitate further molecular characterization of rare CTCs to increase understanding of their biology and as a basis for their molecular screening in the clinical setting.

Prognostic relevance of induced and spontaneous apoptosis of disseminated tumor cells in primary breast cancer patients.

BACKGROUND: An imbalance between cell proliferation and programmed cell death can result in tumor growth. Although most systemic cytotoxic agents induce apoptosis in tumor cells, a high apoptotic rate in primary breast cancer correlates with poor prognosis. The aim of this study was to investigate the incidence and the prognostic significance of apoptotic disseminated tumor cells (DTC) in the bone marrow (BM) of breast cancer patients who either underwent primary surgery or primary systemic chemotherapy (PST). METHODS: A total of 383 primary breast cancer patients with viable DTC in the BM were included into this study. Eighty-five patients were initially treated with primary systemic chemotherapy whereas 298 patients underwent surgery first. Detection of apoptotic DTC were performed by immunocytochemistry using the M30 antibody which detects a neo-epitope expressed after caspase cleavage of cytokeratin 18 during early apoptosis. The median follow up was 44 months (range 10-88 months). RESULTS: Eighty-two of 298 (27%) primary operated patients and 41 of 85 (48%) patients treated with primary systemic systemic therapy had additional apoptotic DTC (M30 positive). In the neoadjuvant group M30-positive patients were less likely to suffer relapse than those without apoptotic DTC (7% vs. 23% of the events, p=0.049). In contrast, the detection of apoptotic DTC in patients treated by primary surgery was significantly associated with poor overall survival (5% vs. 12% of the events, p=0.008). CONCLUSIONS: Apoptotic DTC can be detected in breast cancer patients before and after systemic treatment. The presence of apoptotic DTC in patients with PST may be induced by the cytotoxic agents. Thus, both spontaneous and chemotherapy-induced apoptosis may have different prognostic significance.

Expression of stem cell and epithelial-mesenchymal transition markers in circulating tumor cells of breast cancer patients.

Evaluation and characterization of circulating tumor cells (CTCs) have become a major focus of translational cancer research. Presence of CTCs predicts worse clinical outcome in early and metastatic breast cancer. Whether all cells from the primary tumor have potential to disseminate and form subsequent metastasis remains unclear. As part of the metastatic cascade, tumor cells lose their cell-to-cell adhesion and undergo epithelial-mesenchymal transition (EMT) in order to enter blood circulation. During EMT epithelial antigens are downregulated; thus, such tumor cells might elude classical epithelial marker-based detection. Several researchers postulated that some CTCs express stem cell-like phenotype; this might lead to chemoresistance and enhanced metastatic potential of such cells. In the present review, we discuss current data on EMT and stem cell markers in CTCs of breast cancer and their clinical significance.

Clinical validity of circulating tumour cells in patients with metastatic breast cancer: a pooled analysis of individual patient data.

BACKGROUND: We aimed to assess the clinical validity of circulating tumour cell (CTC) quantification for prognostication of patients with metastatic breast cancer by undertaking a pooled analysis of individual patient data. METHODS: We contacted 51 European centres and asked them to provide reported and unreported anonymised data for individual patients with metastatic breast cancer who participated in studies between January, 2003, and July, 2012. Eligible studies had participants starting a new line of therapy, data for progression-free survival or overall survival, or both, and CTC quantification by the CellSearch method at baseline (before start of new treatment). We used Cox regression models, stratified by study, to establish the association between CTC count and progression-free survival and overall survival. We used the landmark method to assess the prognostic value of CTC and serum marker changes during treatment. We assessed the added value of CTCs or serum markers to prognostic clinicopathological models in a resampling procedure using likelihood ratio (LR) χ(2) statistics. FINDINGS: 17 centres provided data for 1944 eligible patients from 20 studies. 911 patients (46·9%) had a CTC count of 5 per 7·5 mL or higher at baseline, which was associated with decreased progression-free survival (hazard ratio [HR] 1·92, 95% CI 1·73-2·14, p<0·0001) and overall survival (HR 2·78, 95% CI 2·42-3·19, p<0·0001) compared with patients with a CTC count of less than 5 per 7·5 mL at baseline. Increased CTC counts 3-5 weeks after start of treatment, adjusted for CTC count at baseline, were associated with shortened progression-free survival (HR 1·85, 95% CI 1·48-2·32, p<0·0001) and overall survival (HR 2·26, 95% CI 1·68-3·03) as were increased CTC counts after 6-8 weeks (progression-free survival HR 2·20, 95% CI 1·66-2·90, p<0·0001; overall survival HR 2·91, 95% CI 2·01-4·23, p<0·0001). Survival prediction was significantly improved by addition of baseline CTC count to the clinicopathological models (progression-free survival LR 38·4, 95% CI 21·9-60·3, p<0·0001; overall survival LR 64·9, 95% CI 41·3-93·4, p<0·0001). This model was further improved by addition of CTC change at 3-5 weeks (progression-free survival LR 8·2, 95% CI 0·78-20·4, p=0·004; overall survival LR 11·5, 95% CI 2·6-25·1, p=0·0007) and at 6-8 weeks (progression-free survival LR 15·3, 95% CI 5·2-28·3; overall survival LR 14·6, 95% CI 4·0-30·6; both p<0·0001). Carcinoembryonic antigen and cancer antigen 15-3 concentrations at baseline and during therapy did not add significant information to the best baseline model. INTERPRETATION: These data confirm the independent prognostic effect of CTC count on progression-free survival and overall survival. CTC count also improves the prognostication of metastatic breast cancer when added to full clinicopathological predictive models, whereas serum tumour markers do not. FUNDING: Janssen Diagnostics, the Nuovo-Soldati foundation for cancer research.

Analysing the mutational status of PIK3CA in circulating tumor cells from metastatic breast cancer patients.

The frequently altered phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway is involved in the regulation of cellular processes required for breast carcinogenesis. The aim of the project was to develop a method to identify hotspot mutations in the PIK3CA gene in circulating tumor cells (CTCs) of metastatic breast cancer (metBC) patients. From 44 enrolled CTC-positive metBC patients a total number of 57 peripheral blood samples were analysed by CellSearch(®). Genomic DNA of enriched CTCs was isolated, amplified and analyzed for PIK3CA mutations in exons 9 and 20 which lead to E542K, E545K or H1047R amino acid changes and result in increased PI3K activity. The mutations were detected by using SNaPshot-methodology comprising PCR amplification and single nucleotide primer extension. SNaPshot analysis was established using genomic DNA from different breast cancer cell lines and then successfully transferred to investigate blood samples and single cells. Overall, twelve hotspot mutations in either exon 9/E545K (6/12, 50%) or exon 20/H1047R (6/12, 50%) could be determined within 9 out of 57 (15.8%) blood samples from 7 out of 44 (15.9%) patients; CTC counts ranged from 1 to 9748. PIK3CA variants E542K, E545G and E545A were not detected. Analysing the PIK3CA genotype of CTCs has clinical relevance with respect to drug resistance, e.g. against HER2-targeted therapy. The herein described approach including SNaPshot technology provides a simple method to characterize hotspot mutations within CTCs enriched from peripheral blood and can be easily adopted for analysing further therapeutically relevant SNPs.