Uncovering the genomic heterogeneity of multifocal breast cancer.

Multifocal breast cancer (MFBC), defined as multiple synchronous unilateral lesions of invasive breast cancer, is relatively frequent and has been associated with more aggressive features than unifocal cancer. Here, we aimed to investigate the genomic heterogeneity between MFBC lesions sharing similar histopathological parameters. Characterization of different lesions from 36 patients with ductal MFBC involved the identification of non-silent coding mutations in 360 protein-coding genes (171 tumour and 36 matched normal samples). We selected only patients with lesions presenting the same grade, ER, and HER2 status. Mutations were classified as 'oncogenic' in the case of recurrent substitutions reported in COSMIC or truncating mutations affecting tumour suppressor genes. All mutations identified in a given patient were further interrogated in all samples from that patient through deep resequencing using an orthogonal platform. Whole-genome rearrangement screen was further conducted in 8/36 patients. Twenty-four patients (67%) had substitutions/indels shared by all their lesions, of which 11 carried the same mutations in all lesions, and 13 had lesions with both common and private mutations. Three-quarters of those 24 patients shared oncogenic variants. The remaining 12 patients (33%) did not share any substitution/indels, with inter-lesion heterogeneity observed for oncogenic mutation(s) in genes such as PIK3CA, TP53, GATA3, and PTEN. Genomically heterogeneous lesions tended to be further apart in the mammary gland than homogeneous lesions. Genome-wide analyses of a limited number of patients identified a common somatic background in all studied MFBCs, including those with no mutation in common between the lesions. To conclude, as the number of molecular targeted therapies increases and trials driven by genomic screening are ongoing, our findings highlight the presence of genomic inter-lesion heterogeneity in one-third, despite similar pathological features. This implies that deeper molecular characterization of all MFBC lesions is warranted for the adequate management of those cancers.

Infrared imaging in breast cancer: automated tissue component recognition and spectral characterization of breast cancer cells as well as the tumor microenvironment.

Current evaluation of histological sections of breast cancer samples remains unsatisfactory. The search for new predictive and prognostic factors is ongoing. Infrared spectroscopy and its potential to probe tissues and cells at the molecular level without requirement for contrast agents could be an attractive tool for clinical and diagnostic analysis of breast cancer. In this study, we report the successful application of FTIR (Fourier transform infrared) imaging for breast tissue component characterization. We show that specific FTIR spectral signatures can be assigned to the major tissue components of breast tumor samples. We demonstrate that a tissue component classifier can be built based on a spectral database of well-annotated tissues and successfully validated on independent breast samples. We also demonstrate that spectral features can reveal subtle differences within a tissue component, capturing for instance lymphocytic and stromal activation. By investigating in parallel lymph nodes, tonsils and wound healing tissues, we prove the uniqueness of the signature of both lymphocytic infiltrate and tumor microenvironment in the breast disease context. Finally, we demonstrate that the biochemical information reflected in the epithelial spectra might be clinically relevant for the grading purpose, suggesting potential to improve breast cancer management in the future.

Extracellular calcium increases bisphosphonate-induced growth inhibition of breast cancer cells.

INTRODUCTION: Bisphosphonates have become standard therapy for the treatment of skeletal complications related to breast cancer. Although their therapeutic effects mainly result from an inhibition of osteoclastic bone resorption, in vitro data indicate that they also act directly on breast cancer cells, inhibiting proliferation and inducing apoptosis. METHODS: The present study examined the effects of calcium (from 0.6 to 2.0 mmol/l) on the antitumour activity of the bisphosphonate ibandronate (1 to 1,000 nmol/l) on MDA-MB-231 and MCF-7 breast cancer cells. Cell culture densities were determined using crystal violet staining assay. Apoptotic cell death was assessed by annexin V-phycoerythrin and 7-amino-actinomycin double staining. RESULTS: At low calcium concentration, 30 micromol/l ibandronate had no effect on MDA-MB-231 cells growth and only slightly inhibited MCF-7 cells growth. Higher calcium levels significantly increased growth inhibition as well as cell apoptosis induced by ibandronate. We observed similar effects with zoledronic acid. Of note, enhancement of ibandronate-induced growth inhibition was also observed in other breast cancer cell lines (T-47D, ZR-75, Hs-578T and BT-549 cells). The growth inhibitory effect of ibandronate in the presence of high concentrations of calcium was partly suppressed by the calcium chelator EGTA (ethylene glycol tetra-acetic acid). In addition, in the presence of calcium at high concentrations, cells accumulated more [14C]ibandronate than at low calcium concentrations. We obtained further evidence of enhancement of cellular ibandronate accumulation by calcium by demonstrating that high calcium levels increased the inhibition of protein prenylation induced by the bisphosphonate. CONCLUSION: Altogether, our data suggest that extracellular calcium, probably through its binding to ibandronate, markedly increased its cellular accumulation and its inhibitory activity on breast tumour cells. Thus, calcium released during the process of tumour-induced osteolysis might enhance the antitumour effects of bisphosphonates and contribute to their therapeutic efficacy.

Genomic Characterization of Primary Invasive Lobular Breast Cancer.

PURPOSE: Invasive lobular breast cancer (ILBC) is the second most common histologic subtype after invasive ductal breast cancer (IDBC). Despite clinical and pathologic differences, ILBC is still treated as IDBC. We aimed to identify genomic alterations in ILBC with potential clinical implications. METHODS: From an initial 630 ILBC primary tumors, we interrogated oncogenic substitutions and insertions and deletions of 360 cancer genes and genome-wide copy number aberrations in 413 and 170 ILBC samples, respectively, and correlated those findings with clinicopathologic and outcome features. RESULTS: Besides the high mutation frequency of CDH1 in 65% of tumors, alterations in one of the three key genes of the phosphatidylinositol 3-kinase pathway, PIK3CA, PTEN, and AKT1, were present in more than one-half of the cases. HER2 and HER3 were mutated in 5.1% and 3.6% of the tumors, with most of these mutations having a proven role in activating the human epidermal growth factor receptor/ERBB pathway. Mutations in FOXA1 and ESR1 copy number gains were detected in 9% and 25% of the samples. All these alterations were more frequent in ILBC than in IDBC. The histologic diversity of ILBC was associated with specific alterations, such as enrichment for HER2 mutations in the mixed, nonclassic, and ESR1 gains in the solid subtype. Survival analyses revealed that chromosome 1q and 11p gains showed independent prognostic value in ILBC and that HER2 and AKT1 mutations were associated with increased risk of early relapse. CONCLUSION: This study demonstrates that we can now begin to individualize the treatment of ILBC, with HER2, HER3, and AKT1 mutations representing high-prevalence therapeutic targets and FOXA1 mutations and ESR1 gains deserving urgent dedicated clinical investigation, especially in the context of endocrine treatment.

Principles Governing A-to-I RNA Editing in the Breast Cancer Transcriptome.

Little is known about how RNA editing operates in cancer. Transcriptome analysis of 68 normal and cancerous breast tissues revealed that the editing enzyme ADAR acts uniformly, on the same loci, across tissues. In controlled ADAR expression experiments, the editing frequency increased at all loci with ADAR expression levels according to the logistic model. Loci-specific "editabilities," i.e., propensities to be edited by ADAR, were quantifiable by fitting the logistic function to dose-response data. The editing frequency was increased in tumor cells in comparison to normal controls. Type I interferon response and ADAR DNA copy number together explained 53% of ADAR expression variance in breast cancers. ADAR silencing using small hairpin RNA lentivirus transduction in breast cancer cell lines led to less cell proliferation and more apoptosis. A-to-I editing is a pervasive, yet reproducible, source of variation that is globally controlled by 1q amplification and inflammation, both of which are highly prevalent among human cancers.

Characterization and clinical evaluation of CD10+ stroma cells in the breast cancer microenvironment.

PURPOSE: There is growing evidence that interaction between stromal and tumor cells is pivotal in breast cancer progression and response to therapy. Based on earlier research suggesting that during breast cancer progression, striking changes occur in CD10(+) stromal cells, we aimed to better characterize this cell population and its clinical relevance. EXPERIMENTAL DESIGN: We developed a CD10(+) stroma gene expression signature (using HG U133 Plus 2.0) on the basis of the comparison of CD10 cells isolated from tumoral (n = 28) and normal (n = 3) breast tissue. We further characterized the CD10(+) cells by coculture experiments of representative breast cancer cell lines with the different CD10(+) stromal cell types (fibroblasts, myoepithelial, and mesenchymal stem cells). We then evaluated its clinical relevance in terms of in situ to invasive progression, invasive breast cancer prognosis, and prediction of efficacy of chemotherapy using publicly available data sets. RESULTS: This 12-gene CD10(+) stroma signature includes, among others, genes involved in matrix remodeling (MMP11, MMP13, and COL10A1) and genes related to osteoblast differentiation (periostin). The coculture experiments showed that all 3 CD10(+) cell types contribute to the CD10(+) stroma signature, although mesenchymal stem cells have the highest CD10(+) stroma signature score. Of interest, this signature showed an important role in differentiating in situ from invasive breast cancer, in prognosis of the HER2(+) subpopulation of breast cancer only, and potentially in nonresponse to chemotherapy for those patients. CONCLUSIONS: Our results highlight the importance of CD10(+) cells in breast cancer prognosis and efficacy of chemotherapy, particularly within the HER2(+) breast cancer disease.

Global microRNA expression profiling identifies MiR-210 associated with tumor proliferation, invasion and poor clinical outcome in breast cancer.

PURPOSE: Aberrant microRNA (miRNA) expression is associated with cancer and has potential diagnostic and prognostic value in various malignancies. In this study, we investigated miRNA profiling as a complementary tool to improve our understanding of breast cancer (BC) biology and to assess whether miRNA expression could predict clinical outcome of BC patients. EXPERIMENTAL DESIGN: Global miRNA expression profiling using microarray technology was conducted in 56 systemically untreated BC patients who had corresponding mRNA expression profiles available. Results were further confirmed using qRT-PCR in an independent dataset of 89 ER-positive BC patients homogeneously treated with tamoxifen only. MiR-210 functional analyses were performed in MCF7 and MDA-MB-231 BC cell lines using lentiviral transduction. RESULTS: Estrogen receptor (ER) status, tumor grade and our previously developed gene expression grade index (GGI) were associated with distinct miRNA profiles. Several miRNAs were found to be clinically relevant, including miR-210, its expression being associated with tumor proliferation and differentiation. Furthermore, miR-210 was associated with poor clinical outcome in ER-positive, tamoxifen-treated BC patients. Interestingly, the prognostic performance of miR-210 was similar to several reported multi-gene signatures, highlighting its important role in BC differentiation and tumor progression. Functional analyses in BC cell lines revealed that miR-210 is involved in cell proliferation, migration and invasion. CONCLUSIONS: This integrated analysis combining miRNA and mRNA expression demonstrates that miRNA expression provides additional biological information beyond mRNA expression. Expression of miR-210 is linked to tumor proliferation and appears to be a strong potential biomarker of clinical outcome in BC.

HER2-positive circulating tumor cells in breast cancer.

PURPOSE: Circulating Tumor Cells (CTCs) detection and phenotyping are currently evaluated in Breast Cancer (BC). Tumor cell dissemination has been suggested to occur early in BC progression. To interrogate dissemination in BC, we studied CTCs and HER2 expression on CTCs across the spectrum of BC staging. METHODS: Spiking experiments with 6 BC cell lines were performed and blood samples from healthy women and women with BC were analyzed for HER2-positive CTCs using the CellSearch®. RESULTS: Based on BC cell lines experiments, HER2-positive CTCs were defined as CTCs with HER2 immunofluorescence intensity that was at least 2.5 times higher than the background. No HER2-positive CTC was detected in 42 women without BC (95% confidence interval (CI) 0-8.4%) whereas 4.1% (95%CI 1.4-11.4%) of 73 patients with ductal/lobular carcinoma in situ (DCIS/LCIS) had 1 HER2-positive CTC/22.5 mL, 7.9%, (95%CI 4.1-14.9%) of 101 women with non metastatic (M0) BC had ≥1 HER2-positive CTC/22.5 mL (median 1 cell, range 1-3 cells) and 35.9% (95%CI 22.7-51.9%) of 39 patients with metastatic BC had ≥1 HER2-positive CTC/7.5 mL (median 1.5 cells, range 1-42 cells). In CTC-positive women with DCIS/LCIS or M0 BC, HER2-positive CTCs were more commonly detected in HER2-positive (5 of 5 women) than HER2-negative BC (5 of 12 women) (p = 0.03). CONCLUSION: HER2-positive CTCs were detected in DCIS/LCIS or M0 BC irrespective of the primary tumor HER2 status. Nevertheless, their presence was more common in women with HER2-positive disease. Monitoring of HER2 expression on CTCs might be useful in trials with anti-HER2 therapies.

Cytostatic and apoptotic effects of bisphosphonates on prostate cancer cells.

BACKGROUND: Bisphosphonates are potent inhibitors of bone resorption frequently used for breast cancer and myeloma-induced bone disease. Zoledronic acid has been recently shown to also reduce skeletal morbidity from prostate cancer. METHODS: We have investigated the biological effects of bisphosphonates on PC-3 cell survival (MTT assay and DNA content). We compared four bisphosphonates at doses ranging from 10(-6) to 10(-4) M: clodronate, pamidronate, ibandronate and zoledronic acid. We analyzed cell cycle phases and assessed apoptotic effects of bisphosphonates by three different methods. RESULTS: Clodronate exhibited only a slight inhibitory effect on cell growth. In contrast, aminobisphosphonates markedly decreased cell growth in a time- and dose-dependent manner exerting cytostatic and apoptotic effects. The largest effects were observed after six days of exposure to 10(-4) M bisphosphonates. Cytostatic effects were observed with all three aminobisphosphonates whereas apoptotic effects were especially evident after zoledronic acid incubation. CONCLUSIONS: Aminobisphosphonates, especially zoledronic acid, markedly inhibited PC-3 cancer cell growth, through a variable combination of cytostatic and apoptotic effects. This activity could potentially contribute to the beneficial effects of bisphosphonates in prostate cancer patients with bone metastases.