Estrogen receptor positive (ER+) breast cancers (BC) are heterogeneous with regard to their clinical behavior and response to therapies. The ER is currently the best predictor of response to the ...anti-estrogen agent tamoxifen, yet up to 30-40% of ER+BC will relapse despite tamoxifen treatment. New prognostic biomarkers and further biological understanding of tamoxifen resistance are required. We used gene expression profiling to develop an outcome-based predictor using a training set of 255 ER+ BC samples from women treated with adjuvant tamoxifen monotherapy. We used clusters of highly correlated genes to develop our predictor to facilitate both signature stability and biological interpretation. Independent validation was performed using 362 tamoxifen-treated ER+ BC samples obtained from multiple institutions and treated with tamoxifen only in the adjuvant and metastatic settings.
We developed a gene classifier consisting of 181 genes belonging to 13 biological clusters. In the independent set of adjuvantly-treated samples, it was able to define two distinct prognostic groups (HR 2.01 95%CI: 1.29-3.13; p = 0.002). Six of the 13 gene clusters represented pathways involved in cell cycle and proliferation. In 112 metastatic breast cancer patients treated with tamoxifen, one of the classifier components suggesting a cellular inflammatory mechanism was significantly predictive of response.
We have developed a gene classifier that can predict clinical outcome in tamoxifen-treated ER+ BC patients. Whilst our study emphasizes the important role of proliferation genes in prognosis, our approach proposes other genes and pathways that may elucidate further mechanisms that influence clinical outcome and prediction of response to tamoxifen.
In this study, we quantified 249 mature micro-RNA (miRNA) transcripts in estrogen receptor-positive (ER⁺) primary breast tumors of patients with lymph node-negative (LNN) disease to identify miRNAs ...associated with metastatic capability. In addition, the prognostic value of the candidate miRNAs was determined in ER⁻/LNN breast cancer. Unsupervised analysis in a prescreening set of 38 patients identified three subgroups predominantly driven by three miRNA signatures: an ER-driven luminal B-associated miRNA signature, a stromal miRNA signature, and an overexpressed miRNA cluster located on chromosome 19q23, but these intrinsic miRNA signatures were not associated with tumor aggressiveness. Supervised analysis in the initial subset and subsequent analysis in additional tumors significantly linked four miRNAs (miR-7, miR-128a, miR-210, and miR-516-3p) to ER⁺/LNN breast cancer aggressiveness (n = 147) and one miRNA (miR-210) to metastatic capability in ER⁻/LNN breast cancer (n = 114) and in the clinically important triple-negative subgroup (n = 69) (all P < 0.05). Bioinformatic analysis coupled miR-210 to hypoxia/VEGF signaling, miR-7 and miR-516-3p to cell cycle progression and chromosomal instability, and miR-128a to cytokine signaling. In conclusion, our work connects four miRNAs to breast cancer progression and to several distinct biological processes involved therein.
Clinical outcome of patients with triple-negative breast cancer (TNBC) is highly variable. This study aims to identify and validate a prognostic protein signature for TNBC patients to reduce ...unnecessary adjuvant systemic therapy.
Frozen primary tumors were collected from 126 lymph node-negative and adjuvant therapy-naive TNBC patients. These samples were used for global proteome profiling in two series: an in-house training (n = 63) and a multicenter test (n = 63) set. Patients who remained free of distant metastasis for a minimum of 5 years after surgery were defined as having good prognosis. Cox regression analysis was performed to develop a prognostic signature, which was independently validated. All statistical tests were two-sided.
An 11-protein signature was developed in the training set (median follow-up for good-prognosis patients = 117 months) and subsequently validated in the test set (median follow-up for good-prognosis patients = 108 months) showing 89.5% sensitivity (95% confidence interval CI = 69.2% to 98.1%), 70.5% specificity (95% CI = 61.7% to 74.2%), 56.7% positive predictive value (95% CI = 43.8% to 62.1%), and 93.9% negative predictive value (95% CI = 82.3% to 98.9%) for poor-prognosis patients. The predicted poor-prognosis patients had higher risk to develop distant metastasis than the predicted good-prognosis patients in univariate (hazard ratio HR = 13.15; 95% CI = 3.03 to 57.07; P = .001) and multivariable (HR = 12.45; 95% CI = 2.67 to 58.11; P = .001) analysis. Furthermore, the predicted poor-prognosis group had statistically significantly more breast cancer-specific mortality. Using our signature as guidance, more than 60% of patients would have been exempted from unnecessary adjuvant chemotherapy compared with conventional prognostic guidelines.
We report the first validated proteomic signature to assess the natural course of clinical TNBC.
Most assays to detect circulating tumor cells (CTCs) rely on EpCAM expression on tumor cells. Recently, our group reported that in contrast to other molecular breast cancer subtypes, “normal-like” ...cell lines lack EpCAM expression and are thus missed when CTCs are captured with EpCAM-based technology J Natl Cancer Inst 101(1):61–66, 2009. Here, the use of CD146 is introduced to detect EpCAM-negative CTCs, thereby improving CTC detection. CD146 and EpCAM expression were assessed in our panel of 41 breast cancer cell lines. Cells from 14 cell lines, 9 of which normal-like, were spiked into healthy donor blood. Using CellSearch™ technology, 7.5 ml whole blood was enriched for CTCs by adding ferrofluids loaded with antibodies against EpCAM and/or CD146 followed by staining for Cytokeratin and DAPI. Hematopoietic cells and circulating endothelial cells (CECs) were counterstained with CD45 and CD34, respectively. A similar approach was applied for blood samples of 20 advanced breast cancer patients. Eight of 9 normal-like breast cancer cell lines lacked EpCAM expression but did express CD146. Five of these 8 could be adequately recovered by anti-CD146 ferrofluids. Of 20 advanced breast cancer patients whose CTCs were enumerated with anti-EpCAM and anti-CD146 ferrofluids, 9 had CD146+ CTCs. Cells from breast cancer cell lines that lack EpCAM expression frequently express CD146 and can be recovered by anti-CD146 ferrofluids. CD146+ CTCs are present in the peripheral blood of breast cancer patients with advanced disease. Combined use of anti-CD146 and anti-EpCAM is likely to improve CTC detection in breast cancer patients.
•Endocrine treatment resistance in estrogen receptor positive breast cancer.•Technological advancements for the discovery of biomarkers of endocrine resistance.•Opportunities and restrictions of ...biomarkers in the clinical laboratory.
Estrogen receptor (ER)-positive breast cancer represents the majority (∼70%) of all breast malignancies. In this subgroup of breast cancers, endocrine therapies are effective both in the adjuvant and recurrent settings, although resistance remains a major issue. Several high-throughput approaches have been used to elucidate mechanisms of resistance and to derive potential predictive markers or alternative therapies. In this review, we cover the state-of-the-art of endocrine-resistance biomarker discovery with regard to the latest technological developments, and discuss current opportunities and restrictions for their implementation into a clinical setting.
Molecular characterization of circulating tumor cells (CTC) holds great promise. Unfortunately, routinely isolated CTC fractions currently still contain contaminating leukocytes, which makes ...CTC-specific molecular characterization extremely challenging. In this study, we determined mRNA and microRNA (miRNA) expression of potentially CTC-specific genes that are considered to be clinically relevant in breast cancer.
CTCs were isolated with the epithelial cell adhesion molecule-based CellSearch Profile Kit. Selected genes were measured by real-time reverse transcriptase PCR in CTCs of 50 metastatic breast cancer patients collected before starting first-line systemic therapy in blood from 53 healthy blood donors (HBD) and in primary tumors of 8 of the patients. The molecular profiles were associated with CTC counts and clinical parameters and compared with the profiles generated from the corresponding primary tumors.
We identified 55 mRNAs and 10 miRNAs more abundantly expressed in samples from 32 patients with at least 5 CTCs in 7.5 mL of blood compared with samples from 9 patients without detectable CTCs and HBDs. Clustering analysis resulted in 4 different patient clusters characterized by 5 distinct gene clusters. Twice the number of patients from cluster 2 to 4 had developed both visceral and nonvisceral metastases. Comparing transcript levels in CTCs with those measured in corresponding primary tumors showed clinically relevant discrepancies in estrogen receptor and HER2 levels.
Our study shows that molecular profiling of low numbers of CTCs in a high background of leukocytes is feasible and shows promise for further studies on the clinical relevance of molecular characterization of CTCs.
Splicing factors (SFs) act in dynamic macromolecular complexes to modulate RNA processing. To understand the complex role of SFs in cancer progression, we performed a systemic analysis of the ...co-regulation of SFs using primary tumor RNA sequencing data. Co-regulated SFs were associated with aggressive breast cancer phenotypes and enhanced metastasis formation, resulting in the classification of Enhancer- (21 genes) and Suppressor-SFs (64 genes). High Enhancer-SF levels were related to distinct splicing patterns and expression of known oncogenic pathways such as respiratory electron transport, DNA damage and cell cycle regulation. Importantly, largely identical SF co-regulation was observed in almost all major cancer types, including lung, pancreas and prostate cancer. In conclusion, we identified cancer-associated co-regulated expression of SFs that are associated with aggressive phenotypes. This study increases the global understanding of the role of the spliceosome in cancer progression and also contributes to the development of strategies to cure cancer patients.
Lysosome-associated membrane protein 3 (LAMP3) is a member of the LAMP-family of proteins, which are involved in the process of autophagy. Autophagy is induced by tamoxifen in breast cancer cells and ...may contribute to tamoxifen resistance. In this study, the significance of LAMP3 for tamoxifen resistance in breast cancer was examined. The methods employed included use of clonogenic assays to assess the survival of MCF7 breast cancer cells with LAMP3 knockdown after tamoxifen treatment and of quantitative real-time PCR of LAMP3 to evaluate its predictive value for first-line tamoxifen treatment in patients with advanced breast cancer. Results show that tamoxifen treatment of MCF7 cells induced LAMP3 mRNA expression. LAMP3 knockdown in these cells increased tamoxifen sensitivity. Evaluation of expression of the autophagy markers, LC3B and p62, after LAMP3 knockdown showed increased expression levels, indicating that cells with LAMP3 knockdown have a suppressed ability to complete the autophagic process. In addition, knockdown of autophagy-associated genes resulted in sensitization to tamoxifen. Next, tamoxifen-resistant MCF7 cells were cultured. These cells had a sevenfold higher LAMP3 mRNA expression, showed elevated basal autophagy levels, and could be significantly resensitized to tamoxifen by LAMP3 knockdown. In patients treated with first-line tamoxifen for advanced disease (n=304), high LAMP3 mRNA expression was associated with shorter progression-free survival (P=0.003) and shorter post-relapse overall survival (P=0.040), also in multivariate analysis. Together, these results indicate that LAMP3 contributes to tamoxifen resistance in breast cancer. Tamoxifen-resistant cells are resensitized to tamoxifen by the knockdown of LAMP3. Therefore, LAMP3 may be clinically relevant to countering tamoxifen resistance in breast cancer patients.
The molecular basis for breast cancer metastasis to the brain is largely unknown. Brain relapse typically occurs years after the removal of a breast tumour, suggesting that disseminated cancer cells ...must acquire specialized functions to take over this organ. Here we show that breast cancer metastasis to the brain involves mediators of extravasation through non-fenestrated capillaries, complemented by specific enhancers of blood-brain barrier crossing and brain colonization. We isolated cells that preferentially infiltrate the brain from patients with advanced disease. Gene expression analysis of these cells and of clinical samples, coupled with functional analysis, identified the cyclooxygenase COX2 (also known as PTGS2), the epidermal growth factor receptor (EGFR) ligand HBEGF, and the 2,6-sialyltransferase ST6GALNAC5 as mediators of cancer cell passage through the blood-brain barrier. EGFR ligands and COX2 were previously linked to breast cancer infiltration of the lungs, but not the bones or liver, suggesting a sharing of these mediators in cerebral and pulmonary metastases. In contrast, ST6GALNAC5 specifically mediates brain metastasis. Normally restricted to the brain, the expression of ST6GALNAC5 in breast cancer cells enhances their adhesion to brain endothelial cells and their passage through the blood-brain barrier. This co-option of a brain sialyltransferase highlights the role of cell-surface glycosylation in organ-specific metastatic interactions.
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