Fabien Reyal

MicroRNA Sequence and Expression Analysis in Breast Tumors by Deep Sequencing

MicroRNAs (miRNA) regulate many genes critical for tumorigenesis. We profiled miRNAs from 11 normal breast tissues, 17 noninvasive, 151 invasive breast carcinomas, and 6 cell lines by in-house-developed barcoded Solexa sequencing. miRNAs were organized in genomic clusters representing promoter-controlled miRNA expression and sequence families representing seed sequence-dependent miRNA target regulation. Unsupervised clustering of samples by miRNA sequence families best reflected the clustering based on mRNA expression available for this sample set. Clustering and comparative analysis of miRNA read frequencies showed that normal breast samples were separated from most noninvasive ductal carcinoma in situ and invasive carcinomas by increased miR-21 (the most abundant miRNA in carcinomas) and multiple decreased miRNA families (including miR-98/let-7), with most miRNA changes apparent already in the noninvasive carcinomas. In addition, patients that went on to develop metastasis showed increased expression of mir-423, and triple-negative breast carcinomas were most distinct from other tumor subtypes due to upregulation of the mir~17-92 cluster. However, absolute miRNA levels between normal breast and carcinomas did not reveal any significant differences. We also discovered two polymorphic nucleotide variations among the more abundant miRNAs miR-181a (T19G) and miR-185 (T16G), but we did not identify nucleotide variations expected for classical tumor suppressor function associated with miRNAs. The differentiation of tumor subtypes and prediction of metastasis based on miRNA levels is statistically possible but is not driven by deregulation of abundant miRNAs, implicating far fewer miRNAs in tumorigenic processes than previously suggested.

Rab27a supports exosome-dependent and -independent mechanisms that modify the tumor microenvironment and can promote tumor progression

During progression from single cancer cells to a tumor mass and metastases, tumor cells send signals that can subvert their tissue microenvironment. These signals involve soluble molecules and various extracellular vesicles, including a particular type termed exosomes. The specific roles of exosomes secreted in the tumor microenvironment, however, is unclear. The small GTPases RAB27A and RAB27B regulate exocytosis of multivesicular endosomes, which lead to exosome secretion, in human HeLa cells. Here, we used mouse models to show that Rab27a blockade in mammary carcinoma cells decreased secretion of exosomes characterized by endocytic markers, but also of matrix metalloproteinase 9, which is not associated with exosomes. Rab27a blockade resulted in decreased primary tumor growth and lung dissemination of a metastatic carcinoma (4T1), but not of a nonmetastatic carcinoma (TS/A). Local growth of 4T1 tumors required mobilization of a population of neutrophil immune cells induced by Rab27a-dependent secretion of exosomes together with a specific combination of cytokines and/or metalloproteinases. Our findings offer in vivo validation of the concept that exosome secretion can exert key pathophysiologic roles during tumor formation and progression, but they also highlight the idiosyncratic character of the tumor context.

Regional copy number–independent deregulation of transcription in cancer

Genetic and epigenetic alterations have been identified that lead to transcriptional deregulation in cancers. Genetic mechanisms may affect single genes or regions containing several neighboring genes, as has been shown for DNA copy number changes. It was recently reported that epigenetic suppression of gene expression can also extend to a whole region; this is known as long-range epigenetic silencing. Various techniques are available for identifying regional genetic alterations, but no large-scale analysis has yet been carried out to obtain an overview of regional epigenetic alterations. We carried out an exhaustive search for regions susceptible to such mechanisms using a combination of transcriptome correlation map analysis and array CGH data for a series of bladder carcinomas. We validated one candidate region experimentally, demonstrating histone methylation leading to the loss of expression of neighboring genes without DNA methylation.

Oncoplastic breast surgery for cancer: analysis of 540 consecutive cases [outcomes article].

Background: Synchronous plastic and oncological surgery is undertaken to improve the security of excision margins and yield high-quality aesthetic outcomes when conventional breast-conserving therapy either anticipates poor results or is not possible. Methods: A total of 540 consecutive patients underwent primary oncoplastic breast surgery for cancer with high tumor-to-breast volume ratios and locations precluding a good aesthetic result with simple tumor excision. A variety of techniques were employed at the Institut Curie between 1986 and 2007, and aesthetic outcomes were assessed on a five-point scale from 1 (excellent) to 5 (poor). Results: The median age was 52 years (range, 28 to 90 years), and median follow-up was 49 months (6 to 262 months). Median tumor size was 29.1 mm (range, 4 to 100 mm), with most patients (72.3 percent) having a brassiere cup size of B or C. Close or involved margins occurred in 18.9 percent, with mastectomy being necessary in 9.4 percent. A satisfactory aesthetic outcome (ratings of 1 to 3) at 5 years was obtained in 90.3 percent. Five-year overall and distant disease-free survival rates were 92.9 and 87.9 percent, respectively, with local recurrence in 6.8 percent. Conclusions: With local recurrence and survival rates similar to those for breast-conserving therapy, this series confirms the safety of oncoplastic breast surgery for tumors both high in volume and difficult in location. Highly satisfactory cosmetic outcomes extend the indications for conservative surgery, further reduce the mastectomy rate, and limit adverse aesthetic sequelae.

A comprehensive analysis of prognostic signatures reveals the high predictive capacity of the Proliferation, Immune response and RNA splicing modules in breast cancer

IntroductionSeveral gene expression signatures have been proposed and demonstrated to be predictive of outcome in breast cancer. In the present article we address the following issues: Do these signatures perform similarly? Are there (common) molecular processes reported by these signatures? Can better prognostic predictors be constructed based on these identified molecular processes?MethodsWe performed a comprehensive analysis of the performance of nine gene expression signatures on seven different breast cancer datasets. To better characterize the functional processes associated with these signatures, we enlarged each signature by including all probes with a significant correlation to at least one of the genes in the original signature. The enrichment of functional groups was assessed using four ontology databases.ResultsThe classification performance of the nine gene expression signatures is very similar in terms of assigning a sample to either a poor outcome group or a good outcome group. Nevertheless the concordance in classification at the sample level is low, with only 50% of the breast cancer samples classified in the same outcome group by all classifiers. The predictive accuracy decreases with the number of poor outcome assignments given to a sample. The best classification performance was obtained for the group of patients with only good outcome assignments. Enrichment analysis of the enlarged signatures revealed 11 functional modules with prognostic ability. The combination of the RNA-splicing and immune modules resulted in a classifier with high prognostic performance on an independent validation set.ConclusionsThe study revealed that the nine signatures perform similarly but exhibit a large degree of discordance in prognostic group assignment. Functional analyses indicate that proliferation is a common cellular process, but that other functional categories are also enriched and show independent prognostic ability. We provide new evidence of the potentially promising prognostic impact of immunity and RNA-splicing processes in breast cancer.

Integrated Genomic and Transcriptomic Analysis of Ductal Carcinoma In situ of the Breast

Purpose: To gain insight into genomic and trancriptomic subtypes of ductal carcinomas in situ of the breast (DCIS). Experimental Design: We did a combined phenotypic and genomic analysis of a series of 57 DCIS integrated with gene expression profile analysis for 26 of the 57 cases. Results: Thirty-two DCIS exhibited a luminal phenotype; 21 were ERBB2 positive, and 4 were ERBB2/estrogen receptor (ER) negative with 1 harboring a bona fide basal-like phenotype. Based on a CGH analysis, genomic types were identified in this series of DCIS with the 1q gain/16q loss combination observed in 3 luminal DCIS, the mixed amplifier pattern including all ERBB2, 12 luminal and 2 ERBB2-/ER- DCIS, and the complex copy number alteration profile encompassing 14 luminal and 1 ERBB2-/ER- DCIS. Eight cases (8 of 57; 14%) presented a TP53 mutation, all being amplifiers. Unsupervised analysis of gene expression profiles of 26 of the 57 DCIS showed that luminal and ERBB2-amplified, ER-negative cases clustered separately. We further investigated the effect of high and low copy number changes on gene expression. Strikingly, amplicons but also low copy number changes especially on 1q, 8q, and 16q in DCIS regulated the expression of a subset of genes in a very similar way to that recently described in invasive ductal carcinomas. Conclusions: These combined approaches show that the molecular heterogeneity of breast ductal carcinomas exists already in in situ lesions and further indicate that DCIS and invasive ductal carcinomas share genomic alterations with a similar effect on gene expression profile.

Molecular profiling of patient-derived breast cancer xenografts

IntroductionIdentification of new therapeutic agents for breast cancer (BC) requires preclinical models that reproduce the molecular characteristics of their respective clinical tumors. In this work, we analyzed the genomic and gene expression profiles of human BC xenografts and the corresponding patient tumors.MethodsEighteen BC xenografts were obtained by grafting tumor fragments from patients into Swiss nude mice. Molecular characterization of patient tumors and xenografts was performed by DNA copy number analysis and gene expression analysis using Affymetrix Microarrays.ResultsComparison analysis showed that 14/18 pairs of tumors shared more than 56% of copy number alterations (CNA). Unsupervised hierarchical clustering analysis showed that 16/18 pairs segregated together, confirming the similarity between tumor pairs. Analysis of recurrent CNA changes between patient tumors and xenografts showed losses in 176 chromosomal regions and gains in 202 chromosomal regions. Gene expression profile analysis showed that less than 5% of genes had recurrent variations between patient tumors and their respective xenografts; these genes largely corresponded to human stromal compartment genes. Finally, analysis of different passages of the same tumor showed that sequential mouse-to-mouse tumor grafts did not affect genomic rearrangements or gene expression profiles, suggesting genetic stability of these models over time.ConclusionsThis panel of human BC xenografts maintains the overall genomic and gene expression profile of the corresponding patient tumors and remains stable throughout sequential in vivo generations. The observed genomic profile and gene expression differences appear to be due to the loss of human stromal genes. These xenografts, therefore, represent a validated model for preclinical investigation of new therapeutic agents.

EGFR as a potential therapeutic target for a subset of muscle-invasive bladder cancers presenting a basal-like phenotype

A subtype of aggressive human muscle-invasive bladder cancer expresses basal epithelial markers and is sensitive to EGFR inhibition in preclinical models. Bladder Cancer’s Basal Instincts Like most cancers, bladder tumors are much easier to treat when they do not invade deep into the tissue, accounting for the poor outcomes of patients with muscle-invasive bladder cancer. By performing genetic analysis on a large number of these tumors, Rebouissou et al. identified a specific subgroup of muscle-invasive bladder cancers expressing basal markers. Although these are aggressive tumors, the authors showed that they have a weak spot and are unusually dependent on the activity of a signaling pathway called epidermal growth factor receptor (EGFR). As a result, these tumors are sensitive to treatment with drugs that inhibit the EGFR pathway, which the authors confirmed in preclinical models. Muscle-invasive bladder carcinoma (MIBC) constitutes a heterogeneous group of tumors with a poor outcome. Molecular stratification of MIBC may identify clinically relevant tumor subgroups and help to provide effective targeted therapies. From seven series of large-scale transcriptomic data (383 tumors), we identified an MIBC subgroup accounting for 23.5% of MIBC, associated with shorter survival and displaying a basal-like phenotype, as shown by the expression of epithelial basal cell markers. Basal-like tumors presented an activation of the epidermal growth factor receptor (EGFR) pathway linked to frequent EGFR gains and activation of an EGFR autocrine loop. We used a 40-gene expression classifier derived from human tumors to identify human bladder cancer cell lines and a chemically induced mouse model of bladder cancer corresponding to human basal-like bladder cancer. We showed, in both models, that tumor cells were sensitive to anti-EGFR therapy. Our findings provide preclinical proof of concept that anti-EGFR therapy can be used to target a subset of particularly aggressive MIBC tumors expressing basal cell markers and provide diagnostic tools for identifying these tumors.

The role of nipple-sparing mastectomy in breast cancer: a comprehensive review of the literature.

Background: The role of nipple-sparing mastectomy for breast cancer is controversial, as there is concern regarding its oncologic safety and complication rate. The authors reviewed the literature to quantify the incidence of occult nipple malignancy in breast cancer, identify the factors influencing occult nipple malignancy, quantify locoregional recurrence rates, and quantify nipple-sparing mastectomy complication rates. Methods: A search of the literature was performed using PubMed. Key words used were “mastectomy,” “nipple involvement,” “nipple-sparing mastectomy,” “skin-sparing mastectomy,” “occult nipple malignancy,” “occult nipple disease,” and “breast cancer recurrence.” Articles were analyzed regarding incidence of occult nipple malignancy, potential factors influencing the incidence of occult malignancy, and recurrence/complications following nipple-sparing mastectomy. The incidence of occult nipple disease was compared between groups using chi-square or Fisher’s exact tests for categorical variables and t tests for continuous variables. Values of p < 0.05 were considered significant. Results: The overall rate of occult nipple malignancy was 11.5 percent. Primary tumor characteristics influencing occult nipple malignancy were tumor-nipple distance less than 2 cm, grade, lymph node metastasis, lymphovascular invasion, human epidermal growth factor receptor-2–positive, estrogen receptor/progesterone receptor–negative, tumor size greater than 5 cm, retroareolar/central location, and multicentric tumors. The overall nipple recurrence rate considered significant was 0.9 percent, and the skin flap recurrence rate was 4.2 percent. Full- and partial-thickness nipple necrosis rates were 2.9 and 6.3 percent, respectively. Conclusions: Nipple-sparing mastectomy for primary breast cancer is appropriate in carefully selected patients. All patients should have retroareolar sampling. There is strong evidence to suggest that suitable cases are well circumscribed single or multifocal lesions that have a tumor-to-nipple distance greater than 2 cm. Tumors should be grade 1 to 2 and not have lymphovascular invasion, axillary node metastasis, or human epidermal growth factor receptor-2 positivity.

Identification of a pharmacologically tractable Fra-1/ADORA2B axis promoting breast cancer metastasis

Metastasis confronts clinicians with two major challenges: estimating the patients risk of metastasis and identifying therapeutic targets. Because they are key signal integrators connecting cellular processes to clinical outcome, we aimed to identify transcriptional nodes regulating cancer cell metastasis. Using rodent xenograft models that we previously developed, we identified the transcription factor Fos-related antigen-1 (Fra-1) as a key coordinator of metastasis. Because Fra-1 often is overexpressed in human metastatic breast cancers and has been shown to control their invasive potential in vitro, we aimed to assess the implication and prognostic significance of the Fra-1–dependent genetic program in breast cancer metastasis and to identify potential Fra-1–dependent therapeutic targets. In several in vivo assays in mice, we demonstrate that stable RNAi depletion of Fra-1 from human breast cancer cells strongly suppresses their ability to metastasize. These results support a clinically important role for Fra-1 and the genetic program it controls. We show that a Fra-1–dependent gene-expression signature accurately predicts recurrence of breast cancer. Furthermore, a synthetic lethal drug screen revealed that antagonists of the adenosine receptor A2B (ADORA2B) are preferentially toxic to breast tumor cells expressing Fra-1. Both RNAi silencing and pharmacologic blockade of ADORA2B inhibited filopodia formation and invasive activity of breast cancer cells and correspondingly reduced tumor outgrowth in the lungs. These data show that Fra-1 activity is causally involved in and is a prognostic indicator of breast cancer metastasis. They suggest that Fra-1 activity predicts responsiveness to inhibition of pharmacologically tractable targets, such as ADORA2B, which may be used for clinical interference of metastatic breast cancer.