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Background. Liquid biopsies have been studied as an accessible method to capture spatial tumour heterogeneity and repeatedly evaluate targetable aberrations in cancer. Here, we analysed pure single and pools of circulating and disseminated tumour cells (CTC and DTC) to study genetic heterogeneity. Methods. From, three patients with metastatic breast cancer, CTC and DTC (bone marrow, pleural fluid, and cerebrospinal fluid) were CellSearch® enriched and DEPArray® sorted into 136 samples of singles and pools up to 150 cells. Sorted cells, primary tumour, circulating free (cf)DNA, and enriched CTC/DTC fractions were analysed for mutations and copy number alterations (CNA). Results. Two patients harboured a driver PIK3CA mutation in all samples. Variant allele frequencies matched the ploidy status calculated from copy number data. The majority of CNA were homogeneously present in most samples of each patient, including breast cancer subtype specific CNA. One patient with rapidly progressive disease harboured additional CNA in the CTC/DTC compartment, reflecting this aggressive course and a possible subtype switch. These clones emerged at distinct timepoints in the different compartments. Furthermore, various mutations were present in unique samples. Conclusion. Repeated tumour analysis with liquid biopsies unveils changing molecular characteristics over time, and even a difference between simultaneous primary and metastatic disease. We show that all major subclones can be captured by combined sequencing of enriched CTC and cfDNA at disease progression.

PurposeMonitoring levels of circulating tumor-derived DNA (ctDNA) represents a non-invasive snapshot of tumor burden and potentially clonal evolution. Here we describe how a novel statistical model that uses serial ctDNA measurements from shallow whole genome sequencing (sWGS) in metastatic breast cancer patients produces a rapid and inexpensive assessment that is predictive of treatment response and progression-free survival.Patients and MethodsA cohort of 188 metastatic breast cancer patients had DNA extracted from serial plasma samples (total 1098, median=4, mean=5.87). Plasma DNA was assessed using sWGS and the tumor fraction in total cell free DNA estimated using ichorCNA. This approach was compared with ctDNA targeted sequencing and serial CA 15-3 measurements. The longitudinal ichorCNA values were used to develop a Bayesian learning model to predict subsequent treatment response.ResultsWe identified a transition point of 7% estimated tumor fraction to stratify patients into different categories of progression risk using ichorCNA estimates and a time-dependent Cox model, validated across different breast cancer subtypes and treatments, outperforming the alternative methods. We then developed a Bayesian learning model to predict subsequent treatment response with a sensitivity of 0.75 and a specificity of 0.66.ConclusionIn patients with metastatic breast cancer, sWGS of ctDNA and ichorCNA provide prognostic and predictive real-time valuable information on treatment response across subtypes and therapies. A prospective large-scale clinical trial to evaluate clinical benefit of early treatment changes based on ctDNA levels is now warranted.

BACKGROUND The outcome to treatment administered to patients with metastatic castration-resistant prostate cancer (mCRPC) greatly differs between individuals, underlining the need for biomarkers guiding treatment decision making. OBJECTIVE To investigate the prognostic value of circulating tumor cell (CTC) enumeration and dynamics, in the context of second-line endocrine therapies (ie, abiraterone acetate or enzalutamide), irrespective of prior systemic therapies. DESIGN, SETTINGS, AND PARTICIPANTS In a prospective, multicentre study blood samples for CTC enumeration were collected from patients with mCRPC at baseline (n = 174). In patients who responded for minimally 10-12 weeks a follow-up sample was collected. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS For baseline analysis, patients were stratified in

Mutations and splice variants in the estrogen receptor (ER) gene, ESR1, may yield endocrine resistance in metastatic breast cancer (MBC) patients. These putative endocrine resistance markers are likely to emerge during treatment, and therefore, its detection in liquid biopsies, such as circulating tumor cells (CTCs) and cell-free DNA (cfDNA), is of great interest. This research aimed to determine whether ESR1 mutations and splice variants occur more frequently in CTCs of MBC patients progressing on endocrine treatment. In addition, the presence of ESR1 mutations was evaluated in matched cfDNA and compared to CTCs. CellSearch-enriched CTC fractions (≥5/7.5 mL) of two MBC cohorts were evaluated, namely (a) patients starting first-line endocrine therapy (n = 43, baseline cohort) and (b) patients progressing on any line of endocrine therapy (n = 40, progressing cohort). ESR1 hotspot mutations (D538G and Y537S/N/C) were evaluated in CTC-enriched DNA using digital PCR and compared with matched cfDNA (n = 18 baseline cohort; n = 26 progressing cohort). Expression of ESR1 full-length and 4 of its splice variants ((increment)5, (increment)7, 36 kDa, and 46 kDa) was evaluated in CTC-enriched mRNA. It was observed that in the CTCs, the ESR1 mutations were not enriched in the progressing cohort (8%), when compared with the baseline cohort (5%) (P = 0.66). In the cfDNA, however, ESR1 mutations were more prevalent in the progressing cohort (42%) than in the baseline cohort (11%) (P = 0.04). Three of the same mutations were observed in both CTCs and cfDNA, 1 mutation in CTCs only, and 11 in cfDNA only. Only the (increment)5 ESR1 splice variant was CTC-specific expressed, but was not enriched in the progressing cohort. In conclusion, sensitivity for detecting ESR1 mutations in CTC-enriched fractions was lower than for cfDNA. ESR1 mutations detected in cfDNA, rarely present at the start of first-line endocrine therapy, were enriched at progression, strongly suggesting a role in conferring endocrine resistance in MBC.

Background A growing understanding of the molecular biology of cancer and the identification of specific aberrations driving cancer evolution have led to the development of various targeted agents. Tumors can exhibit significant heterogeneity and this may change over time, also as the result of selective pressure. Circulating tumor cells (CTCs), shed from multiple tumor sites, have demonstrated to represent of the overall tumor burden. We report the results of an ongoing comparative study on mutation and copy number profiles of primary and metastatic tissue, CTCs, and synchronously isolated DTCs from metastatic effusions of patients with clinically progressive MBC. Materials & Methods CTCs and DTCs were enriched from 7.5 ml blood or effusion using the CellSearch system and were further purified and sorted with the DEPArray system. For this study we isolated both 70 single and 70 pools of 10-200 CTCs or DTCs, in order have enough power to detect 5% subclones and analyse heterogeneity. Single and pooled WBCs were isolated as technical controls. DNA was isolated and amplified using the Ampli1-kit and subjected to Illumina WGS and Ion Torrent AmpliSeq panel sequencing. Fresh frozen tissue from solid metastases and the primary tumor, and bulk CTC (CellSearch Profile) were sequenced as comparators for mutation and copy number profiles. DNA of buffy coat was sequenced to enable germline variant detection. For mutational analysis, only somatic variants with good quality metrics, >20x coverage, variant allele frequencies >10%, and being non-synonymous or splice site variants, were taken into account. Results AmpliSeq panel sequencing was performed on 153 unique samples of three patients with a mean coverage depth of 1000x. In patient 1, a PIK3CA hotspot mutation was found clonally in all tumor samples at heterozygous level. Furthermore, various private mutations were found in both CTCs and DTCs, however not in WBC, including several TP53 hotspot mutations. In patient 2, another PIK3CA hotspot was present in all CTCs at heterozygous frequencies. In patient 3, an enormous heterogeneity was observed between all CTC and DTC samples. For patient 3, disease evolution was detected during multiple events of progressive disease over 2 years. At the moment, low pass WGS for CN detection for all samples is being performed and results will be present prior to the SABCS. Conclusion Based on the mutational status we conclude that both clonal mutations as well as various private variants are present in single and pools of CTCs and DTCs. In addition to the detection of targetable aberrations, the evaluation of heterogeneity is of clinical importance, as the effect of targeting subclones is currently being explored in clinical trials. Citation Format: Brouwer A, van Dam P-J, Rutten A, Prové A, Peeters M, Van Laere S, Dirix L. Evaluation of subclonality in the CTC and DTC compartment of patients with metastatic breast cancer using low pass whole genome and AmpliSeq panel sequencing [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-06-01.

A growing understanding of the molecular biology of cancer and the identification of specific aberrations driving cancer evolution have led to the development of various targeted agents. Therapeutic decisions concerning these drugs are often guided by single biopsies of the primary tumor. Yet, it is well known that tumors can exhibit significant heterogeneity and change over time as a result of selective pressure. Circulating tumor cells (CTCs) are shed from various tumor sites and are thought to represent the molecular landscape of a patient's overall tumor burden. Moreover, a minimal-invasive liquid biopsy facilitates monitoring of clonal evolution during therapy pressure and disease progression in real-time. While more information becomes available regarding heterogeneity among CTCs, comparison between these studies is needed. In this review, we focus on the genomic and transcriptional heterogeneity found in the CTC compartment, and its significance for clinical decision making., SCOPUS: re.j, info:eu-repo/semantics/published

Introduction In metastatic breast cancer (MBC), discordant expression levels of the human epidermal growth factor receptor 2 (HER2) have been noted between primary tumors (PT) and matched metastatic lesions. Reassessment of HER2 status during treatment decisions in patients with advanced disease might help to optimize outcome. Circulating tumor cells (CTCs) offer the potential to provide a repeatedly accessible source of tumor cells for the real-time assessment of actual tumor characteristics. However, little is known on the concordance of HER2 expression on CTCs measured by immunofluorescence and the amplification status. Here we report on a preclinical study, using five spiked breast cancer cell lines, comparing semi-quantitative HER2 scoring on CellSearch (Riethdorf 2010) with objective DEPArray analysis, and subsequent FISH analysis on DEPArray-sorted tumor cells. At the moment these data are also being generated for 10 patient CTC samples. Expression and amplification status of CTCs will be compared with primary tumor tissue. Materials and methods MDA-MB-436, MCF-7, BT-20, KPL-4, and SKBR3 cells (increasing HER2 status) were spiked into donor blood and subjected to CellSearch enrichment. HER2/FITC intensity was scored manually on the CellSearch analyzer. All cell lines were injected into the DEPArray and exposure settings were optimized (FITC: exposure time 800 ms, gain 5%). These settings are further used for all preclinical and clinical samples. HER2 scoring was based on relative fluorescent units (rfu) of the HER2/FITC signal with background subtraction. Cells were sorted into pure batches of HER2 positive (DAPI+/CK+/HER2+/CD45-) and negative (DAPI+/CK+/HER2-/CD45-) tumor cells. Cytospins were formalin fixed and subjected to DAKO IQFISH. Results HER2 expression on CellSearch turned out to be very heterogeneous within the same cell line. DEPArray data was highly reproducible for all cell lines (p4). MDA-MB-436 and MCF-7 cells showed no gene amplification on FISH, while in KPL-4 there was a HER2/CEN17 ratio of >2. Four patient samples with HER2 positive status on CellSearch have been run on the DEPArray. For patient 1, 1005 CTC were analyzed, 32.4% were HER2+. This was 53 (69.8% HER2+), 352 (5.7% HER2+), and 622 (6.7% HER2+) for patient 2-4 respectively. These numbers are comparable with CellSearch analysis. Discussion HER2 expression analysis by immunofluorescence is comparable between CellSearch and DEPArray, however DEPArray has the advantage that it is user-independent and highly reproducible. Furthermore, CTCs can be sorted into pure batches for downstream analysis. The FISH technique on DEPArray sorted cells is now optimized and will be used to determine the correlation between the immunofluorescent HER2 scoring and the actual amplification status of the CTCs. These data will be incorporated prior to upcoming SABCS. Citation Format: Brouwer A, Verhoest F, Vermeulen P, Rutten A, Prové A, Van Laere S, Peeters M, Dirix L. Evaluation of HER2 expression and amplification on CTCs using DEPArray analysis and sorting followed by FISH [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-05-10.

Purpose: To infer the prognostic value of simultaneous androgen receptor (AR) and TP53 profiling in liquid biopsies from patients with metastatic castration-resistant prostate cancer (mCRPC) starting a new line of AR signaling inhibitors (ARSi). Experimental Design: Between March 2014 and April 2017, we recruited patients with mCRPC (n = 168) prior to ARSi in a cohort study encompassing 10 European centers. Blood samples were collected for comprehensive profiling of CellSearch-enriched circulating tumor cells (CTC) and circulating tumor DNA (ctDNA). Targeted CTC RNA sequencing (RNA-seq) allowed the detection of eight AR splice variants (ARV). Low-pass whole-genome and targeted gene-body sequencing of AR and TP53 was applied to identify amplifications, loss of heterozygosity, mutations, and structural rearrangements in ctDNA. Clinical or radiologic progression-free survival (PFS) was estimated by Kaplan–Meier analysis, and independent associations were determined using multivariable Cox regression models. Results: Overall, no single AR perturbation remained associated with adverse prognosis after multivariable analysis. Instead, tumor burden estimates (CTC counts, ctDNA fraction, and visceral metastases) were significantly associated with PFS. TP53 inactivation harbored independent prognostic value [HR 1.88; 95% confidence interval (CI), 1.18–3.00; P = 0.008], and outperformed ARV expression and detection of genomic AR alterations. Using Cox coefficient analysis of clinical parameters and TP53 status, we identified three prognostic groups with differing PFS estimates (median, 14.7 vs. 7.51 vs. 2.62 months; P < 0.0001), which was validated in an independent mCRPC cohort (n = 202) starting first-line ARSi (median, 14.3 vs. 6.39 vs. 2.23 months; P < 0.0001). Conclusions: In an all-comer cohort, tumor burden estimates and TP53 outperform any AR perturbation to infer prognosis. See related commentary by Rebello et al., p. 1699

The efficiency of the adaptive immune system is dependent on the diversity of T- and B-cell receptors, which is created by random rearrangement of receptor gene segments. AmpliCot is an experimental technique that allows the measurement of the diversity of the T- and B-cell repertoire. This procedure has the advantage over other cloning and sequencing techniques of being time- and expense-effective. In previous studies, receptor diversity, measured with AmpliCot, has been inferred assuming a second-order kinetics model. The latter implies that the relation between diversity and concentration × time (Cot) values is linear. We show that a more detailed model, involving heteroduplex and transient-duplex formation, leads to significantly better fits of experimental data and to nonlinear diversity-Cot relations. We propose an alternative fitting procedure, which is straightforward to apply and which gives an improved description of the relationship between Cot values and diversity.

We present a single-cell application to determine PIK3CA mutations in CTCs, which uncovered the degree of intra-patient heterogeneity in patients with metastatic hormone receptor-positive breast cancer (HR+ MBC) and high CTC count (>10 CTCs/7.5mL). Using CellSearch and DEPArray we isolated circulating tumor cells (CTCs) and white blood cells (WBCs) from peripheral blood and sequenced PIK3CA exons 9 and 20 by targeted amplicon sequencing. Comparative analysis between the primary tumor (PT, n=27 patients), circulating cell-free DNA (cfDNA, n=31 patients), single (n=146 CTCs) and pools (n=70 CTC suspensions, ranging 5-120 cells/suspension) of CTCs from 26 patients and metastases/DTCs (n=11 patients) was performed. Mutations were frequent in PT (15/27 (55.5%)) and showed slight and substantial agreement with cfDNA (n=21; kappa=0.14) and CTCs (n=22; kappa=0.6733), respectively. A wild-type genotype in WBCs indicates a high specificity. Inter-compartmental concordance was observed in 13/18 (72.2%) patients and temporal heterogeneity in 4/18 patients (22.2%). CTC analysis reveals both mutational homo- and heterogeneity with cases showing the presence of different mutant and wild-type CTC subpopulations. Additionally, unique double-mutated CTCs were detected in 8/26 patients (30.7%). The developed liquid biopsy provides an insight into inter and intra-patient PIK3CA mutational heterogeneity in patients with HR+ MBC, paving the way towards the application of a more personalized medicine.

BACKGROUND: Preclinical and clinical studies have reported that human epidermal growth factor receptor 2 (HER2) overexpression yields resistance to endocrine therapies. Here the prevalence and prognostic impact of HER2-positive circulating tumor cells (CTCs) were investigated retrospectively in metastatic breast cancer (MBC) patients with a HER2-negative primary tumor receiving endocrine therapy. Additionally, the prevalence and prognostic significance of HER2-positive CTCs were explored in a chemotherapy cohort, as well as the prognostic impact of the estrogen receptor (ER) CTC status in both cohorts. METHODS: Included were MBC patients with a HER2-negative primary tumor, with ≥1 detectable CTC, starting a new line of treatment. CTCs were enumerated using the CellSearch system, characterized for HER2 with the CellSearch anti-HER2 phenotyping reagent, and characterized for ER mRNA expression. Primary end point was progression-free rate after 6 months (PFR6months) of endocrine treatment in HER2-positive versus HER2-negative CTC patients. RESULTS: HER2-positive CTCs were present in 29% of all patients. In the endocrine cohort (n=72), the PFR6months was 53% for HER2-positive versus 68% for HER2-negative CTC patients (P=.23). In the chemotherapy cohort (n=82), no prognostic value of HER2-positive CTCs on PFR6months was observed either. Discordances in ER status between the primary tumor and CTCs occurred in 25% of all patients but had no prognostic value in exploratory survival analyses. CONCLUSION: Discordances regarding HER2 status and ER status between CTCs and the primary tumor occurred frequently but had no prognostic impact in our MBC patient cohorts.

Introduction: In metastatic breast cancer (MBC), discordant expression levels of the human epidermal growth factor receptor 2 (HER2) have been noted between primary tumours (PT) and matched metastatic lesions (Meta). Therefore reassessment of this predictive marker at time of metastatic disease might help to optimize treatment. Circulating tumour cells (CTCs) offer the potential to provide a repeatedly accessible source of tumour cells for the real-time assessment of actual tumour characteristics. Here we report on a retrospective study analysing over two and a half thousand CTCs in order to evaluate the inter-observer variability when using the semi-quantitative scale (0-3+) described by Riethdorf in 2010. Furthermore we designed a digital scoring system using 13 parameters selected by ImageJ. HER2 status in CTCs was compared to PT and/or Meta of patients with MBC. Materials and methods: 65 patients starting first or second line systemic therapy for MBC and harbouring more than 5 CTC/7.5 mL blood were selected. HER2 status of 2721 CTCs was determined by immunofluorescence using the CellSearch system. HER2 status of the solid lesions was determined by IHC or FISH. Inter-observer variability was calculated using the Kendalls tau tests. 284 CTCs were analysed with the digital scoring system using ImageJ 'plot profile' and 'analyse particles'. Selected parameters comprise cell size, mean and maximum intensity of the cell and its surrounding, and both ratio's and differences of the aforementioned. Dissimilarity matrix was calculated using Pearson Correlation-Distance. Results: Of 2721 CTCs, 1485 cells (55%) were scored 0+ and 2263 cells (83%) were found to be HER2- (0+ or 1+) by both observers. 458 cells (17%) were scored HER2+ (2+ or 3+) by at least one of the observers, however only 175 (6%) by both observers. Inter-observer variability was 0.703, but when omitting the usually undebatable 0+ cells, this variability showed to be 0.278. HER2 scoring of CTC by two observers. HER2- (0+/1+)HER2+ (2+/3+)HER2- (0+/1+)2263 (83%)131 (4.8%)HER2+ (2+/3+)152 (5.6%)175 (6.4%) 24 of 65 patients had at least 80% 0+ CTCs (≥96% HER2- cells). Of these patients, 5 were HER2+ based on their PT. Oppositely, 10 and 20 patients harboured at least 40% and 10% HER2+ CTCs respectively. From these 20 patients only 10 were diagnosed with HER2+ disease on PT or Meta and 1 was shifted form HER2- PT to HER2+ Meta. The digital scoring system was able to identify four groups with different HER2 expression levels. When comparing the identified clusters with the manually scored cells the two moderately related clusters showed to contain almost only 2+ and 3+ CTCs. A very isolated cluster contained almost solely 0+ CTCs. Discussion: The manual scoring system showed to be feasible, however we noticed that there are some discrepancies regarding the scoring of 1+ to 3+ cells. The digital scoring is able to predict the outcome and can by itself cluster CTCs into 4 groups. It strongly distinguishes between the HER2+ and HER2- cells. HER2+ status can change during disease progression, both with gain and loss of HER2 positivity. This can be monitored using CTCs. Citation Format: Brouwer A, van de Wiel M, Peeters B, van Dam P-J, Vermeulen P, Peeters M, Van Laere S, Peeters D, Dirix L. Manual and digital detection of HER2 status of 2721 circulating tumour cells in patients with metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-04-01.

We have shown that in up to half of the patients with metastatic breast cancer (MBC), higher numbers of circulating tumour cells (CTCs) are present in the central venous blood (CVB) compared to the peripheral venous blood (PVB), suggesting that the lungs might retain a substantial number of CTCs. Here we report the presence of tumour cell emboli (TCE) in the microvasculature of the lungs in three out of eight patients with MBC and one patient with metastatic cervical carcinoma who had markedly elevated numbers of CTCs in the blood. All these patients suffered from symptomatic dyspnoea not easily attributable to other causes. No TCE were observed in five patients with MBC and elevated CTC counts and three patients with MBC who had low CTC counts (

Tuberculosis (TB) of the breast is a rare entity, and can be confused with many other breast disorders, like mamma carcinoma or inflammatory breast cancer. When finding granulomatous mastitis (GM) on histology, it is important to make a differential diagnosis and seek actively for clues on the presence of tuberculosis, sarcoidosis, Wegener's granulomatosis, or idiopathic granulomatous mastitis, since treatment strategies differ and maltreatment has major implications on morbidity and mortality. An extensive clinical evaluation, laboratory work up, and imaging will lead in most cases to the right diagnosis. Anti-tuberculous therapy is the core treatment for breast TB, and surgery is indicated for extensive or persistent residual disease. Here we present a case of tuberculous mastitis and a review of literature on GM.

Introduction: Circulating tumour cells (CTC) found in the blood of patients with cancer offer the potential to provide a repeatedly accessible source of tumour cells for the real-time assessment of tumour characteristics in patients with metastatic breast cancer (MBC). Questions remain to what extent CTC are truly representative of the actually present tumour mass in a patient at a specific moment in time and the molecular heterogeneity within the CTC population is only now being explored. Here, we report on the first results of an ongoing comparative study of mutation profiles of CTC and synchronously isolated disseminated tumour cells (DTC) from metastatic effusions or biopsies of solid metastases of patients with clinically progressive MBC. Materials and methods: For this project CTC are isolated from 7.5 ml blood samples of patients with MBC using the CellSearch system. CellSearch enriched CTC samples are subsequently further purified and sorted into several batches of 1-125 CTC per patient using the DEPArray system. DNA is isolated and amplified using the Ampli1 whole genome amplification (WGA) kit and subjected to whole exome paired-end sequencing (WES). DTC from metastatic effusions, fresh frozen tissue from solid metastases or the primary tumour, or - in patients with extremely high CTC counts (>10.000/7.5 ml) - pooled CTC from the CellSearch Profile sample, are sequenced as a comparator for mutation profiles. DNA from the buffy coat of white blood cells are sequenced to enable somatic mutation analysis. Results: Eight samples of 1-125 CTC and a CellSearch Profile sample of one patient with MBC who had ca. 30.000 CTC/7.5 ml of blood (patient 1) and 4 CTC samples of 5-10 CTC, 2 temporally matched DTC samples of 10 and 20 DTC from a pleural effusion and a fresh frozen tissue sample of the primary tumour of a second patient (patient 2) have been sequenced so far. Average base coverages were 13.6x (patient 1) and 11.8x (patient 2) for CTC/DTC samples and 175x and 120x for the CellSearch profile sample (patient 1) and the primary tumour sample (patient 2) respectively. Between 29.64% and 53.57% of the exomes of amplification products of CTC/DTC DNA were uncovered, probably due to technical limitations of the WGA procedure. Overall, if adequately covered, good concordances were observed for variants identified with MuTect in 28 frequently mutated genes in breast cancer between samples of amplification products of 1-125 CTC and the CellSearch Profile sample of patient 1. In patient 2, the same H1047R PIK3CA mutation was identified in the primary tumour and all CTC and DTC samples. In-depth analyses of the full exome data are being conducted. Discussion: Our data provide insight into clinically relevant questions to what extent CTC reflect mutational profiles in temporally matched metastatic tumour cells, and – by analysing multiple CTC samples of the same patient – genetic heterogeneity between CTC in patients with MBC. Sample accrual and analysis is being expanded and updated results will be presented at the conference. Citation Format: Dieter JE Peeters, Ken Op De Beeck, Anja Brouwer, Geert Vandeweyer, Patrick Pauwels, Marc Peeters, Peter B Vermeulen, Peter A van Dam, Steven J Van Laere, Guy Van Camp, Luc Y Dirix. Whole exome sequencing of circulating and disseminated tumour cells in patients with metastatic breast cancer [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 P4-01-14.