Impact of age on breast cancer mortality and competing causes of death at 10 years follow-up in the adjuvant TEAM trial.

AIM: Due to increasing life expectancy, patients with breast cancer remain at risk of dying due to breast cancer over a long time. This study aims to assess the impact of age on breast cancer mortality and other cause mortality 10 years after diagnosis. METHODS: Postmenopausal patients with hormone-receptor positive breast cancer were included in the Tamoxifen and Exemestane Adjuvant Multinational (TEAM) trial between 2001 and 2006. Age at diagnosis was categorised as <65 years (n = 3369), 65-74 years (n = 1896) and ≥75 years (n = 854). Breast cancer mortality was assessed considering other cause mortality as competing event using competing risk analysis. RESULTS: After a median follow-up of 9.8 years (interquartile range 8.0-10.3), cumulative incidence of breast cancer mortality increased with increasing age (age <65 years, 11.7% [95% confidence interval {CI}: 10.2-13.2]; 65-74 years, 12.7% (11.2-14.2) and ≥75 years, 15.6% (13.1-18.0)). Univariate subdistribution hazard ratio (sHR) increased with increasing age (age: 65-74 years, sHR: 1.08, 95% CI: 0.92-1.27 and ≥75 years sHR: 1.30, 95% CI: 1.06-1.58, P = 0.013). Multivariable sHR adjusted for tumour and treatment characteristics increased with age but did not reach significance (age 65-74 years, sHR: 1.11, 95% CI: 0.94-1.31; ≥75 years, sHR: 1.18, 95% CI: 0.94-1.48, P = 0.055). CONCLUSION: Ten years after diagnosis, older age at diagnosis is associated with increasing breast cancer mortality in univariate analysis, but it did not reach significance in multivariable analysis. This is not outweighed by a substantially higher other cause mortality with older age. This underlines the need to improve the balance between undertreatment and overtreatment in older patients with breast cancer. The trial was registered in International Trial Databases (ClinicalTrials.govNCT00279448, NCT00032136, and NCT00036270; the Netherlands Trial Registry NTR267).

Dynamic prediction in breast cancer: proving feasibility in clinical practice using the TEAM trial.

BACKGROUND: Predictive models are an integral part of current clinical practice and help determine optimal treatment strategies for individual patients. A drawback is that covariates are assumed to have constant effects on overall survival (OS), when in fact, these effects may change during follow-up (FU). Furthermore, breast cancer (BC) patients may experience events that alter their prognosis from that time onwards. We investigated the 'dynamic' effects of different covariates on OS and developed a nomogram to calculate 5-year dynamic OS (DOS) probability at different prediction timepoints (tP) during FU. METHODS: Dutch and Belgian postmenopausal, endocrine-sensitive, early BC patients enrolled in the TEAM trial were included. We assessed time-varying effects of specific covariates and obtained 5-year DOS predictions using a proportional baselines landmark supermodel. Covariates included age, histological grade, hormone receptor and HER2 status, T- and N-stage, locoregional recurrence (LRR), distant recurrence, and treatment compliance. A nomogram was designed to calculate 5-year DOS based on individual characteristics. RESULTS: A total of 2602 patients were included (mean FU 6.2 years). N-stage, LRR, and HER2 status demonstrated time-varying effects on 5-year DOS. Hazard ratio (HR) functions for LRR, high-risk N-stage (N2/3), and HER2 positivity were HR = (8.427 × 0.583[Formula: see text], HR = (3.621 × 0.816[Formula: see text], and HR = (1.235 × 0.851[Formula: see text], respectively. Treatment discontinuation was associated with a higher mortality risk, but without a time-varying effect [HR 1.263 (0.867-1.841)]. All other covariates were time-constant. DISCUSSION: The current nomogram accounts for elapsed time since starting adjuvant endocrine treatment and optimizes prediction of individual 5-year DOS during FU for postmenopausal, endocrine-sensitive BC patients. The nomogram can facilitate in determining whether further therapy will benefit an individual patient, although validation in an independent dataset is still needed.

Gene expression profiles in circulating tumor cells to predict prognosis in metastatic breast cancer patients.

BACKGROUND: A circulating tumor cell (CTC) count is an established prognostic factor in metastatic breast cancer (MBC). Besides enumeration, CTC characterization promises to improve outcome prediction and treatment guidance. Having shown the feasibility of quantifying clinically relevant mRNA transcripts in CTCs, we determined the prognostic value of CTC gene expression in MBC. PATIENTS AND METHODS: CTCs were isolated and enumerated from blood of 197 MBC patients who were about to start first-line systemic therapy. Of these, 180 were assessable for quantification of mRNA expression by RT-qPCR in relation to time-to-treatment failure (TTF). A prognostic CTC gene profile was generated by leave-one-out cross validation in a 103 patient discovery set and validated in 77 patients. Additionally, all 180 patients were randomly divided into two equal sets to discover and validate a second prognostic profile. RESULTS: CTC count predicted for TTF at baseline {≥5 versus <5 CTCs/7.5 ml blood, hazard ratio (HR) 2.92 [95% confidence interval (CI) 1.71-4.95] P < 0.0001}. A 16-gene CTC profile was generated in the first discovery set, which identified patients with death or TTF <9 months versus those with a better outcome. In multivariate analysis, the 16-gene profile was the only factor associated with TTF [HR 3.15 (95% CI 1.35-7.33) P 0.008]. Validation of this profile in the independent patient set pointed into the same direction, but was not statistically significant. A newly generated 8-gene profile showed similarly favorable test characteristics as the 16-gene profile, but did not significantly pass validation either. CONCLUSION: A 16-gene CTC profile was identified, which provided prognostic value on top of CTC count in MBC patients. However, validation of this profile in an independent cohort, nor of a second profile, reached statistical significance, underscoring the need to further fine-tune the still promising approach of CTC characterization.

Mechanisms of tumour vascularization in cutaneous malignant melanoma: clinical implications.

Malignant melanoma represents < 10% of all skin cancers but is responsible for the majority of skin-cancer-related deaths. Metastatic melanoma has historically been considered as one of the most therapeutically challenging malignancies. Fortunately, for the first time after decades of basic research and clinical investigation, new drugs have produced major clinical responses. Angiogenesis has been considered an important target for cancer treatment. Initial efforts have focused primarily on targeting endothelial and tumour-related vascular endothelial growth factor signalling. Here, we review different mechanisms of tumour vascularization described in melanoma and discuss the potential clinical implications.

Blood microvessel density, lymphatic microvessel density and lymphatic invasion in predicting melanoma metastases: systematic review and meta-analysis.

In malignant melanoma (MM) there is an urgent need to identify new markers with predictive value superior to the traditional clinical and histological parameters. Angiogenesis and lymphangiogenesis have been recognized as critical processes in tumour growth and metastasis development, and numerous studies have evaluated the significance of these parameters in predicting the prognosis in solid tumours, including MM. We set out to determine whether angiogenesis, lymphangiogenesis and lymphatic invasion (LI) are valuable prognostic markers in MM. We systematically reviewed the available literature and subsequently performed a meta-analysis on the compiled data. To be eligible for the systematic review, a study had to provide the microvessel density (MVD), the lymphatic vessel density (LVD) or information about LI, assessed by immunohistochemistry on the primary site in patients with MM. To be evaluable for the meta-analysis, a study also had to provide information on clinical outcome. We approached selected studies with the Reporting recommendations for tumour marker (REMARK) criteria, verifying whether they had followed the recommendations. In total, nine angiogenesis, seven lymphangiogenesis and 10 LI studies were included in our meta-analysis, representing 419, 474 and 802 patients, respectively. Using meta-analysis, we showed that peritumoral LVD and the presence of LI have prognostic value for patients with MM. In contrast, MVD and intratumoral LVD did not have prognostic value in these patients. LVD and LI seem to have prognostic value for patients with MM.

Detection and prognostic significance of circulating tumour cells in patients with metastatic breast cancer according to immunohistochemical subtypes.

BACKGROUND: The enumeration of circulating tumour cells (CTCs) with the EpCAM-based CellSearch system has prognostic significance in patients with metastatic breast cancer (MBC). The aim of this study was to explore potential differences in the detection and prognostic significance of CTCs in MBC according to immunohistochemical subtypes of breast cancer. METHODS: CellSearch CTC counts were obtained from 154 MBC patients before first-line systemic treatment between November 2007 and August 2012. Patients were categorised in five subgroups according to immunohistochemical surrogate definitions of intrinsic subtypes in breast cancer based on hormone receptor status, HER2/neu status and histological grade. Differences in progression-free (PFS) and overall survival (OS) were assessed relative to the cut-off value of ≥5 CTCs per 7.5 ml blood. RESULTS: No significant differences were observed in the absolute CTC counts (P=0.120) or in CTC positivity rates according to ≥1 and ≥5 CTCs per 7.5 ml blood detection thresholds (P=0.165 and P=0.651, respectively) between immunohistochemical subtypes. However, very high CTC counts, defined as ≥80 CTCs per 7.5 ml, were observed more frequently in patients with Luminal A and triple negative (TN) breast cancer (P=0.024). In the total study population, the presence of ≥5 CTCs was the single most significant prognostic factor for both PFS and OS in multivariate analysis (P<0.001). A more limited prognostic impact, not reaching statistical significance, was observed in patients with HER2-positive disease as opposed to patients with Luminal A, Luminal B-HER2-negative and TN disease. CONCLUSION: The detection of EpCAM+CTCs was not clearly associated with any of the immunohistochemical subtypes of breast cancer in patients with MBC before first-line treatment. Potentially clinically relevant differences were however observed at very high CTC counts. Furthermore, our data suggest a lower prognostic significance of CTC evaluation in HER2-positive patients with MBC.

Do type 1 receptor tyrosine kinases inform treatment choice? A prospectively planned analysis of the TEAM trial.

BACKGROUND: Epidermal growth factor receptors contribute to breast cancer relapse during endocrine therapy. Substitution of aromatase inhibitors (AIs) may improve outcomes in HER-positive cancers. METHODS: Tissue microarrays were constructed. Quantitative analysis of HER1, HER2, and HER3 was performed. Data were analysed relative to disease-free survival and treatment using outcomes at 2.75 and 6.5 years. RESULTS: Among 4541 eligible samples, 4225 (93%) had complete HER1-3 data. Overall, 5% were HER1-positive, 13% HER2-positive, and 21% HER3-positive; 32% (n=1351) overexpressed at least one HER receptor. In the HER1-3-negative subgroup, the hazard ratio (HR) for upfront exemestane vs tamoxifen at 2.75 years was 0.67 (95% confidence interval (CI), 0.52-0.87), in the HER1-3-positive subgroup, the HR was 1.15 (95% CI, 0.85-1.56). A prospectively planned treatment-by-marker analysis demonstrated a significant interaction between HER1-3 and treatment at 2.75 years (HR=0.58; 95% CI, 0.39-0.87; P=0.008), as confirmed by multivariate regression analysis adjusting for prognostic factors (HR=0.55; 95% CI, 0.36-0.85; P=0.005). This effect was time dependent. CONCLUSION: In the 2.75 years prior to switching patients initially treated with tamoxifen to exemestane, a significant treatment-by-marker effect exists between AI/tamoxifen treatment and HER1-3 expression, suggesting HER expression could be used to select appropriate endocrine treatment at diagnosis to prevent or delay early relapses.

Semiautomated isolation and molecular characterisation of single or highly purified tumour cells from CellSearch enriched blood samples using dielectrophoretic cell sorting.

BACKGROUND: Molecular characterisation of single circulating tumour cells (CTCs) holds considerable promise for predictive biomarker assessment and to explore CTC heterogeneity. We evaluate a new method, the DEPArray system, that allows the dielectrophoretic manipulation and isolation of single and 100% purified groups of CTCs from pre-enriched blood samples and explore the feasibility of their molecular characterisation. METHODS: Samples containing known numbers of two cell populations were used to assess cell loss during sample loading. Cultured breast cancer cells were isolated from spiked blood samples using CellSearch CTC and Profile kits. Single tumour cells and groups of up to 10 tumour cells were recovered with the DEPArray system and subjected to transcriptional and mutation analysis. RESULTS: On average, 40% cell loss was observed when loading samples to the DEPArray system. Expected mutations in clinically relevant markers could be obtained for 60% of single recovered tumour cells and all groups of tumour cells. Reliable gene expression profiles were obtained from single cells and groups of up to 10 cells for 2 out of 3 spiked breast cancer cell lines. CONCLUSION: We describe a semiautomated workflow for the isolation of small groups of 1 to 10 tumour cells from whole blood samples and provide proof of principle for the feasibility of their comprehensive molecular characterisation.

Biomarker results from the AVADO phase 3 trial of first-line bevacizumab plus docetaxel for HER2-negative metastatic breast cancer.

BACKGROUND: Combining bevacizumab with first-line chemotherapy significantly improves progression-free survival (PFS) in HER2-negative metastatic breast cancer (mBC). However, identification of patients benefitting most from bevacizumab remains elusive. The AVADO trial included an extensive optional exploratory biomarker programme. METHODS: Patients with HER2-negative mBC were randomised to receive docetaxel with placebo or bevacizumab. The primary end point was PFS. Plasma samples were analysed using a multiplex ELISA. Blood mRNA expression was assessed using quantitative PCR. Tumour tissue samples were analysed by immunohistochemistry. Single-nucleotide polymorphisms (SNPs) involved in the VEGF pathway were analysed in germline DNA. RESULTS: Samples for biomarker analysis were available from 24-54% of the 736 treated patients (depending on specimen type). The most consistent potential predictive effect was observed with plasma VEGF-A and VEGFR-2; high baseline concentrations were associated with greater treatment effect. Blood mRNA analyses suggested a greater bevacizumab effect in patients with high VEGF121. No consistent predictive effect was seen for tumour neuropilin or other candidate tumour markers by immunohistochemistry, or for any of the SNPs investigated. CONCLUSION: Plasma VEGF-A and VEGFR-2 are potential predictive markers for bevacizumab efficacy, supporting findings in gastric and pancreatic cancers. Plasma VEGF-A is being evaluated prospectively in mBC in the MERiDiAN trial.

Relationship between specific adverse events and efficacy of exemestane therapy in early postmenopausal breast cancer patients.

BACKGROUND: Many adverse events (AEs) associated with aromatase inhibitors (AIs) involve symptoms related to the depletion of circulating estrogens, and may be related to efficacy. We assessed the relationship between specific AEs [hot flashes (HF) and musculoskeletal AEs (MSAE)] and survival outcomes in Dutch and Belgian patients treated with exemestane (EXE) in the Tamoxifen Exemestane Adjuvant Multinational (TEAM) trial. Additionally, the relationship between hormone receptor expression and AEs was assessed. METHODS: Efficacy end points were relapse-free survival (RFS), overall survival (OS) and breast cancer-specific mortality (BCSM), starting at 6 months after starting EXE treatment. AEs reported in the first 6 months of treatment were included. Specific AEs comprised HF and/or MSAE. Landmark analyses and Cox proportional hazards models assessed survival differences up to 5 years. RESULTS: A total of 1485 EXE patients were included. Patients with HF had a better RFS than patients without HF [multivariate hazard ratio (HR) 0.393, 95% confidence interval (CI) 0.19-0.813; P = 0.012]. The occurrence of MSAE versus no MSAE did not relate to better RFS (multivariate HR 0.677, 95% CI 0.392-1.169; P = 0.162). Trends were maintained for OS and BCSM. Quantitative hormone receptor expression was not associated with specific AEs. CONCLUSIONS: Some AEs associated with estrogen depletion are related to better outcomes and may be valuable biomarkers in AI treatment.

A phase II study of the combination of endocrine treatment and bortezomib in patients with endocrine-resistant metastatic breast cancer.

The majority of patients with hormone receptor-positive metastatic breast cancer die from disease progression despite different types of anti-hormonal treatments. Preclinical studies have indicated that resistance to anti-hormonal therapies may be the result of an activated NF-κB signalling pathway in breast cancer. Bortezomib is a proteasome inhibitor that blocks the NF-κB pathway. Recent pharmacodynamic and pharmaco-kinetic xenograft studies have shown that drug exposure may be a crucial factor for the efficacy of bortezomib in solid tumours. The aim was to investigate whether the addition of bortezomib to anti-hormonal therapy would result in regained antitumour activity in patients with progressive and measurable disease being treated with an endocrine agent. Clinical benefit was defined as patients obtaining stable disease, partial response or complete response after 2 cycles, lasting for at least another five weeks. Bortezomib was administered on days 1, 8, 15 and 22 of a 5-week regimen (1.6 mg/m2). Eight patients received an aromatase inhibitor and bortezomib, while one received tamoxifen and bortezomib. There were 3 grade 3 gastrointestinal toxicities. Median time to treatment failure was 69 days (range, 35-140). Two out of the 9 patients had stable disease for more than 10 weeks. Despite an effective target inhibition, suggested in peripheral blood mononuclear cells and available tumour samples, no objective antitumour responses were observed. Addition of a proteasome inhibitor to anti-hormonal therapy resulted in a clinical benefit rate of 22% in a limited number of patients with endocrine resistant and progressive metastatic breast cancer. The demonstrated proteasome inhibition in tumour tissue provides evidence that the lack of clinical responses is not attributed to deficient drug exposure.

Effect of trabectedin on the QT interval in patients with advanced solid tumor malignancies.

PURPOSE: The primary objective of this study was to access the potential effects of trabectedin on the QT/QTc interval in patients with locally advanced or metastatic solid tumors. METHODS: Patients (n = 75) who had received ≤3 previous lines of chemotherapy and had either relapsed or had progressive disease were enrolled. Patients were administered 3-h intravenous infusions of placebo (saline) on day 1 and trabectedin (1.3 mg/m(2)) on day 2. Time-matched serial triplicate ECG recordings and pharmacokinetic blood samples were collected over 24 h on both days. Heart rate corrected mean QT intervals and changes from predose baseline in QTc (ΔQTc) were assessed. The difference in ΔQTc between trabectedin and placebo was calculated at each time point (ΔΔQTc). RESULTS: The upper limits of the 90% confidence interval for ΔΔQTcF and ΔΔQTcB at all time points were less than the prespecified noninferiority margin of 10 ms (≤6.65 ms). No patient had a QTc > 500 ms or a time-matched increase from baseline in QTc > 60 ms at any time point. Regression analyses indicated ΔΔQTc was poorly correlated with trabectedin concentration. No adverse events suggestive of proarrhythmic potential were reported. CONCLUSION: Trabectedin did not prolong the QTc interval. Safety and pharmacokinetic profiles of trabectedin were similar to that observed in other ovarian and breast cancer studies.

VEGF-A-independent and angiogenesis-dependent tumour growth in patients with metastatic breast cancer.

BACKGROUND The mechanisms of tumour progression during anti-VEGF-A treatment are poorly understood. PATIENTS AND MATERIALS Two patients with metastatic breast cancer are described who developed new metastases while receiving anti-VEGF-A treatment. Angiogenic parameters were determined by CD34/Ki67 double staining, Chalkley counts (CC) and endothelial cell proliferation fractions (ECP). RT-PCR Taqman low-density arrays with a gene panel of 94 angiogenesis-related genes were performed on both metastases and compared to 10 unselected primary breast tumours. RESULTS Both lesions showed a high and intermediate CC of, respectively, 7.5±0.62 and 4.8±0.2. Both lesions had elevated ECP values of 14% and 8%. Low-density array screening showed that VEGFR1 mRNA was overexpressed in both samples (z-score=7.85 and 7.81) compared to control samples (out of range [min-max]). Additional analysis confirmed this finding at the protein level by immunohistochemistry. CONCLUSION These observations suggest that tumour progression under continuous anti-VEGF-A continues to be angiogenesis dependent. Further exploration is needed to identify the mechanisms of anti-VEGF-A resistance in order to design combination-targeted therapies.

Circulating tumour cells in the central and the peripheral venous compartment in patients with metastatic breast cancer.

BACKGROUND: The enumeration of circulating tumour cells (CTC) has prognostic significance in patients with metastatic breast cancer (MBC) and monitoring of CTC levels over time has considerable potential to guide treatment decisions. However, little is known on CTC kinetics in the human bloodstream. METHODS: In this study, we compared the number of CTC in both 7.5 ml central venous blood (CVB) and 7.5 ml peripheral venous blood (PVB) from 30 patients with MBC starting with a new line of chemotherapy. RESULTS: The number of CTC was found to be significantly higher in CVB (median: 43.5; range: 0-4036) than in PVB (median: 33; range: 0-4013) (P=0.001). When analysing samples pairwise, CTC counts were found to be significantly higher in CVB than in PVB in 12 out of 26 patients with detectable CTC. In contrast, only 2 out of 26 patients had higher CTC counts in PVB as compared with CVB, whereas in 12 remaining patients no significant difference was seen. The pattern of CTC distribution was independent of the sites of metastatic involvement. CONCLUSION: A substantial difference in the number of CTC was observed between CVB and PVB of patients with MBC. Registration of the site of blood collection is warranted in studies evaluating the role of CTC assessment in these patients.

International expert panel on inflammatory breast cancer: consensus statement for standardized diagnosis and treatment.

BACKGROUND: Inflammatory breast cancer (IBC) represents the most aggressive presentation of breast cancer. Women diagnosed with IBC typically have a poorer prognosis compared with those diagnosed with non-IBC tumors. Recommendations and guidelines published to date on the diagnosis, management, and follow-up of women with breast cancer have focused primarily on non-IBC tumors. Establishing a minimum standard for clinical diagnosis and treatment of IBC is needed. METHODS: Recognizing IBC to be a distinct entity, a group of international experts met in December 2008 at the First International Conference on Inflammatory Breast Cancer to develop guidelines for the management of IBC. RESULTS: The panel of leading IBC experts formed a consensus on the minimum requirements to accurately diagnose IBC, supported by pathological confirmation. In addition, the panel emphasized a multimodality approach of systemic chemotherapy, surgery, and radiation therapy. CONCLUSIONS: The goal of these guidelines, based on an expert consensus after careful review of published data, is to help the clinical diagnosis of this rare disease and to standardize management of IBC among treating physicians in both the academic and community settings.

Integrated miRNA and mRNA expression profiling of the inflammatory breast cancer subtype.

BACKGROUND: MicroRNAs (miRNAs) are key regulators of gene expression. In this study, we explored whether altered miRNA expression has a prominent role in defining the inflammatory breast cancer (IBC) phenotype. METHODS: We used quantitative PCR technology to evaluate the expression of 384 miRNAs in 20 IBC and 50 non-IBC samples. To gain understanding on the biological functions deregulated by aberrant miRNA expression, we looked for direct miRNA targets by performing pair-wise correlation coefficient analysis on expression levels of 10 962 messenger RNAs (mRNAs) and by comparing these results with predicted miRNA targets from TargetScan5.1. RESULTS: We identified 13 miRNAs for which expression levels were able to correctly predict the nature of the sample analysed (IBC vs non-IBC). For these miRNAs, we detected a total of 17,295 correlated miRNA-mRNA pairs, of which 7012 and 10 283 pairs showed negative and positive correlations, respectively. For four miRNAs (miR-29a, miR-30b, miR-342-3p and miR-520a-5p), correlated genes were concordant with predicted targets. A gene set enrichment analysis on these genes demonstrated significant enrichment in biological processes related to cell proliferation and signal transduction. CONCLUSIONS: This study represents, to the best of our knowledge, the first integrated analysis of miRNA and mRNA expression in IBC. We identified a set of 13 miRNAs of which expression differed between IBC and non-IBC, making these miRNAs candidate markers for the IBC subtype.

Bevacizumab in the treatment of patients with advanced breast cancer: where have we landed?

Vast preclinical and clinical evidence has made angiogenesis one of the hallmarks of cancer. In many human tumours, vascular endothelial growth factor (VEGF) has been identified as the crucial mediator of this process. Initial studies suggested that angiogenesis, and VEGF in particular, could be inhibited without the risk of major side effects. After the pivotal data in first-line studies in patients with colorectal cancer, numerous clinical trials have been undertaken in patients with breast cancer. This review attempts to update these investigations and define the role of anti-VEGF antibody treatment in advanced breast cancer.

Circulating tumour cell detection: a direct comparison between the CellSearch System, the AdnaTest and CK-19/mammaglobin RT-PCR in patients with metastatic breast cancer.

BACKGROUND: The detection, enumeration and isolation of circulating tumour cells (CTCs) have considerable potential to influence the clinical management of patients with breast cancer. There is, however, substantial variability in the rates of positive samples using existing detection techniques. The lack of standardisation of technology hampers the implementation of CTC measurement in clinical routine practice. METHODS: This study was designed to directly compare three techniques for detecting CTCs in blood samples taken from 76 patients with metastatic breast cancer (MBC) and from 20 healthy controls: the CellSearch CTC System, the AdnaTest Breast Cancer Select/Detect and a previously developed real-time qRT-PCR assay for the detection of CK-19 and mammaglobin transcripts. RESULTS: As a result, 36% of patients with MBC were positive by the CellSearch System, 22% by the AdnaTest, 26% using RT-PCR for CK-19 and 54% using RT-PCR for mammaglobin. Samples were significantly more likely to be positive for at least one mRNA marker using RT-PCR than using the CellSearch System (P=0.001) or the AdnaTest (P<0.001). CONCLUSION: We observed a substantial variation in the detection rates of CTCs in blood from breast cancer patients using three different techniques. A higher rate of positive samples was observed using a combined qRT-PCR approach for CK-19 and mammaglobin, which suggests that this is currently the most sensitive technique for detecting CTCs.

The presence of circulating total DNA and methylated genes is associated with circulating tumour cells in blood from breast cancer patients.

Circulating tumour cells (CTC) and tumour-related methylated DNA in blood have been separately assessed for their utility as a marker for subclinical metastasis in breast cancer. However, no studies have looked into the relation between the both molecular markers in this type of cancer. In this study, we investigated the correlations between total/methylated DNA and CTC in the blood from metastatic breast cancer patients. We simultaneously obtained whole blood, plasma and serum samples from 80 patients and 20 controls. The CellSearch System was used to enumerate CTC in blood samples. Plasma total DNA levels were determined by a QPCR method. Sera were analysed by methylation-specific QPCR for three markers: adenomatous polyposis coli (APC), ras association domain family protein 1A (RASSF1A) and oestrogen receptor 1 (ESR1). Total DNA levels in patients were significantly increased when compared with controls (P<0.001) and correlated with the number of CTC (r=0.418, P<0.001). Hypermethylation of one or more genes was detected in 42 (53%) serum samples from breast cancer patients and in three (16%) serum samples from controls (P=0.003). APC was hypermethylated in 29%, RASSF1A in 35% and ESR1 in 20% of breast cancer cases. Detection of a methylated gene in serum was associated with the detection of CTC in blood (P=0.03). The detection of large amounts of circulating total/methylated DNA correlated with the presence of CTC in the blood from patients with breast cancer. This can be interpreted in two ways: (a) CTC are a potential source of circulating tumour-specific DNA; (b) high numbers of CTC and circulating methylated DNA are both a phenotypic feature of more aggressive tumour biology.