Caractérisation moléculaire de la résistance à l’hormonothérapie et au ciblage de la voie PI3K/mTOR dans des modèles murins de cancers du sein luminaux

Luminal breast cancer (ER+, HER2 negative) accounts for 65-75% of all breast carcinomas. Current guidelines strongly recommend endocrine treatment at both the early and advanced stages. However, more than 20% of early stage patients, and all advanced patients will eventually develop endocrine resistance.As most preclinical models (MCF7, T47D) do not recapitulate tumor biology, we have chosen to develop murine models derived from fresh tumors, hence called patient derived xenografts (PDX). We show that these models, although difficult to generate, faithfully exhibit the morphological and biological features of their parental counterpart, with high long term stability. These models have also been evaluated for their sensitivity to various endocrine treatments.In the next step, we developed from these initially endocrine sensitive models new tumors rendered resistant to endocrine therapies. We show that there is no unique biological pattern associated with endocrine resistance, although ER functional reprogramming appears to be critical. We also show that PI3K/mTOR pathway activation, may not be always related to endocrine resistance, and suggest that fulvestrant, an ER down regulator, may be highly synergistic with everolimus in specific cases.Several PI3KCA inhibitors are currently being evaluated in this setting.Les cancers du sein luminaux, exprimant le récepteur aux œstrogènes (RE) représentent 65-75% des cancers du sein soit environ 35.000 nouvelles patientes par an en France. Les référentiels thérapeutiques en vigueur recommandent une prescription systématique d’hormonothérapie au stade précoce, et quasiment constante au stade avancé. Néanmoins, il est admis que plus de 20% des patientes au stade précoce, et la quasi-totalité au stade avancé, vont échapper au traitement endocrinien, rendant impératif le développement de modèles précliniques permettant d’étudier les mécanismes d’hormonorésistance. Dans un contexte de modèles cellulaires anciens et très imparfaits (MCF7, T47D), et de quasi absence de modèles murins pertinents, nous avons choisi de développer des modèles murins dérivés de tumeurs fraîches, dits PDX (patient derived xenografts). Nous avons montré que ces modèles, difficiles à obtenir, récapitulaient avec une grande fidélité les caractéristiques morphologiques et biologiques des tumeurs d’origine. Les PDX se distinguent également par une grande stabilité de ces caractéristiques lors des passages successifs, les rendant utilisables au long cours. Nous avons également évalué les modèles obtenus pour leur profil de sensibilité à diverses modalités de traitement hormonal.Dans une seconde étape, nous avons développé des modèles résistants à partir des PDX précédemment obtenues. Quatre modèles ont pu être obtenus, qui nous ont permis d’avoir à disposition des modèles rendant compte de situations cliniques variées. Ces 4 modèles ont fait l’objet d’analyses biologiques extensives visant à identifier les caractéristiques moléculaires potentiellement associées à telle modalité de résistance : nos données suggèrent fortement qu’il y a autant de mécanismes de résistance que de situations, rendant illusoire une définition biologique unifiée de l’hormonorésistance. La reprogrammation fonctionnelle du RE semble être au centre de ces mécanismes.La voie PI3K/mTOR est une des plus fréquemment associée à l’hormonorésistance. De manière originale, nous avons mis en évidence que cette voie était activée aussi bien dans les modèles sensibles que dans les modèles résistants. La troisième étape a consisté à évaluer l’efficacité de l’everolimus, agent ciblant mTORC1. Nous avons pu montrer que l’everolimus était hautement actif dans toutes les situations considérées, sans argument pour une synergie entre everolimus et tamoxifène ou exemestane. En revanche, il existe une nette tendance à la synergie avec le fulvestrant, inhibiteur hautement spécifique du RE entraînant sa dégradation, et faisant suggérer des interactions avec la voie non génomique du RE.Nous testons actuellement des inhibiteurs spécifiques de la PI3KCA grâce à diverses collaborations industrielles qui permettront également de mener des analyses génomiques approfondies. De multiples projets académiques sont en cours

Outcome in Advanced Ovarian Cancer following an Appropriate and Comprehensive Effort at Upfront Cytoreduction: A Twenty-Year Experience in a Single Cancer Institute

Objectives. The purpose of this retrospective evaluation of advanced-stage ovarian cancer patients was to compare outcome with published findings from other centers and to discuss future options for the management of advanced ovarian carcinoma patients. Methods. A retrospective series of 340 patients with a mean age of 58 years (range: 17–88) treated for FIGO stage III and IV ovarian cancer between January 1985 and January 2005 was reviewed. All patients had primary cytoreductive surgery, without extensive bowel, peritoneal, or systematic lymph node resection, thereby allowing initiation of chemotherapy without delay. Chemotherapy consisted of cisplatin-based chemotherapy in combination with alkylating agents before 2000, whereas carboplatin and paclitaxel regimes were generally used after 1999-2000. Overall survival and disease-free survival were analyzed by the Kaplan-Meier method and the log-rank test. Results. With a mean followup of 101 months (range: 5 to 203), 280 events (recurrence or death) were observed and 245 patients (72%) had died. The mortality and morbidity related to surgery were low. The main prognostic factor for overall survival was postoperative residual disease (P < .0002), while the main prognostic factor for disease-free survival was histological tumor type (P < .0007). Multivariate analysis identified three significant risk factors: optimal surgery (RR = 2.2 for suboptimal surgery), menopausal status (RR = 1.47 for postmenopausal women), and presence of a taxane in the chemotherapy combination (RR = 0.72). Conclusion. These results confirm that optimal surgery defined by an appropriate and comprehensive effort at upfront cytoreduction limits morbidity related to the surgical procedure and allows initiation of chemotherapy without any negative impact on survival. The impact of neoadjuvant chemotherapy to improve resectability while lowering the morbidity of the surgical procedure is discussed

Pre-operative Concomitant Radio-chemotherapy in Bulky Carcinoma of the Cervix: A Single Institution Study

Objective To evaluate the treatment results of patients (pts) with FIGO stage IB2, IIA, IIB cervical carcinoma (CC) treated with pre-operative radio-chemotherapy, followed by extended radical hysterectomy. Methods Retrospective study of 148 women treated to the Institut Curie for operable FIGO Stage IB2 to IIB, biopsy proved CC. Among them, 70 pts, median age 46 years, were treated using the same regimen associating primary radio-cisplatinum based chemotherapy, intracavitary LDR brachytherapy, followed by extended radical hysterectomy. Kaplan-Meier estimates were used to draw survival curves. Comparisons of survival distribution were assessed by the log-rank test. Results Complete histological local-regional response was obtained in 56% of the pts (n = 39). Residual macroscopic or microscopic disease in the cervix was observed in 28 pts (40%). All but one had in-situ microscopic residual CC. Lateral residual disease in the parametria was also present in 9 pts, all with residual CC. Pelvic lymph nodes were free from microscopic disease in 56 pts (80%). Eight of 55 (11%) radiological N0 patients had microscopic nodal involvement, as compared to 6/15 (40%) radiological N1 (p = 0.03). Seventeen pts (25%) had residual cervix disease but negative nodes. After median follow-up of 40 months (range, 8–141), 38/70 patients (54.1%) are still alive and free of disease, 6 (8.6%) alive with disease, and 11 (15.8%) patients were lost for follow-up but free of disease. In Conclusion The treatment of locally advanced CC needs a new multidisciplinary diagnostic and treatment approach using new therapeutic arms to improve the survival and treatment tolerance among women presenting this disease

Intra-tumor genetic heterogeneity and alternative driver genetic alterations in breast cancers with heterogeneous HER2 gene amplification

Background HER2 is overexpressed and amplified in approximately 15% of invasive breast cancers, and is the molecular target and predictive marker of response to anti-HER2 agents. In a subset of these cases, heterogeneous distribution of HER2 gene amplification can be found, which creates clinically challenging scenarios. Currently, breast cancers with HER2 amplification/overexpression in just over 10% of cancer cells are considered HER2-positive for clinical purposes; however, it is unclear as to whether the HER2-negative components of such tumors would be driven by distinct genetic alterations. Here we sought to characterize the pathologic and genetic features of the HER2-positive and HER2-negative components of breast cancers with heterogeneous HER2 gene amplification and to define the repertoire of potential driver genetic alterations in the HER2-negative components of these cases.Results We separately analyzed the HER2-negative and HER2-positive components of 12 HER2 heterogeneous breast cancers using gene copy number profiling and massively parallel sequencing, and identified potential driver genetic alterations restricted to the HER2-negative cells in each case. In vitro experiments provided functional evidence to suggest that BRF2 and DSN1 overexpression/amplification, and the HER2 I767M mutation may be alterations that compensate for the lack of HER2 amplification in the HER2-negative components of HER2 heterogeneous breast cancers.Conclusions Our results indicate that even driver genetic alterations, such as HER2 gene amplification, can be heterogeneously distributed within a cancer, and that the HER2-negative components are likely driven by genetic alterations not present in the HER2-positive components, including BRF2 and DSN1 amplification and HER2 somatic mutations

Respective Prognostic Value of Genomic Grade and Histological Proliferation Markers in Early Stage (pN0) Breast Carcinoma

Genomic grade (GG) is a 97-gene signature which improves the accuracy and prognostic value of histological grade (HG) in invasive breast carcinoma. Since most of the genes included in the GG are involved in cell proliferation, we performed a retrospective study to compare the prognostic value of GG, Mitotic Index and Ki67 score.A series of 163 consecutive breast cancers was retained (pT1-2, pN0, pM0, 10-yr follow-up). GG was computed using MapQuant Dx(R).GG was low (GG-1) in 48%, high (GG-3) in 31% and equivocal in 21% of cases. For HG-2 tumors, 50% were classified as GG-1, 18% as GG-3 whereas 31% remained equivocal. In a subgroup of 132 ER+/HER2- tumors GG was the most significant prognostic factor in multivariate Cox regression analysis adjusted for age and tumor size (HR = 5.23, p = 0.02).In a reference comprehensive cancer center setting, compared to histological grade, GG added significant information on cell proliferation in breast cancers. In patients with HG-2 carcinoma, applying the GG to guide the treatment scheme could lead to a reduction in adjuvant therapy prescription. However, based on the results observed and considering (i) the relatively close prognostic values of GG and Ki67, (ii) the reclassification of about 30% of HG-2 tumors as Equivocal GG and (iii) the economical and technical requirements of the MapQuant micro-array GG test, the availability in the near future of a PCR-based Genomic Grade test with improved performances may lead to an introduction in clinical routine of this test for histological grade 2, ER positive, HER2 negative breast carcinoma

A genomic and transcriptomic approach for a differential diagnosis between primary and secondary ovarian carcinomas in patients with a previous history of breast cancer

<p>Abstract</p> <p>Background</p> <p>The distinction between primary and secondary ovarian tumors may be challenging for pathologists. The purpose of the present work was to develop genomic and transcriptomic tools to further refine the pathological diagnosis of ovarian tumors after a previous history of breast cancer.</p> <p>Methods</p> <p>Sixteen paired breast-ovary tumors from patients with a former diagnosis of breast cancer were collected. The genomic profiles of paired tumors were analyzed using the Affymetrix GeneChip^®Mapping 50 K Xba Array or Genome-Wide Human SNP Array 6.0 (for one pair), and the data were normalized with ITALICS (ITerative and Alternative normaLIzation and Copy number calling for affymetrix Snp arrays) algorithm or Partek Genomic Suite, respectively. The transcriptome of paired samples was analyzed using Affymetrix GeneChip^®Human Genome U133 Plus 2.0 Arrays, and the data were normalized with gc-Robust Multi-array Average (gcRMA) algorithm. A hierarchical clustering of these samples was performed, combined with a dataset of well-identified primary and secondary ovarian tumors.</p> <p>Results</p> <p>In 12 of the 16 paired tumors analyzed, the comparison of genomic profiles confirmed the pathological diagnosis of primary ovarian tumor (n = 5) or metastasis of breast cancer (n = 7). Among four cases with uncertain pathological diagnosis, genomic profiles were clearly distinct between the ovarian and breast tumors in two pairs, thus indicating primary ovarian carcinomas, and showed common patterns in the two others, indicating metastases from breast cancer. In all pairs, the result of the transcriptomic analysis was concordant with that of the genomic analysis.</p> <p>Conclusions</p> <p>In patients with ovarian carcinoma and a previous history of breast cancer, SNP array analysis can be used to distinguish primary and secondary ovarian tumors. Transcriptomic analysis may be used when primary breast tissue specimen is not available.</p

Exquisite Sensitivity of TP53 Mutant and Basal Breast Cancers to a Dose-Dense Epirubicin−Cyclophosphamide Regimen

BACKGROUND: In breast cancers, only a minority of patients fully benefit from the different chemotherapy regimens currently in use. Identification of markers that could predict the response to a particular regimen would thus be critically important for patient care. In cell lines or animal models, tumor protein p53 (TP53) plays a critical role in modulating the response to genotoxic drugs. TP53 is activated in response to DNA damage and triggers either apoptosis or cell-cycle arrest, which have opposite effects on cell fate. Yet, studies linking TP53 status and chemotherapy response have so far failed to unambiguously establish this paradigm in patients. Breast cancers with a TP53 mutation were repeatedly shown to have a poor outcome, but whether this reflects poor response to treatment or greater intrinsic aggressiveness of the tumor is unknown. METHODS AND FINDINGS: In this study we analyzed 80 noninflammatory breast cancers treated by frontline (neoadjuvant) chemotherapy. Tumor diagnoses were performed on pretreatment biopsies, and the patients then received six cycles of a dose-dense regimen of 75 mg/m(2) epirubicin and 1,200 mg/m(2) cyclophosphamide, given every 14 days. After completion of chemotherapy, all patients underwent mastectomies, thus allowing for a reliable assessment of chemotherapy response. The pretreatment biopsy samples were used to determine the TP53 status through a highly efficient yeast functional assay and to perform RNA profiling. All 15 complete responses occurred among the 28 TP53-mutant tumors. Furthermore, among the TP53-mutant tumors, nine out of ten of the highly aggressive basal subtypes (defined by basal cytokeratin [KRT] immunohistochemical staining) experienced complete pathological responses, and only TP53 status and basal subtype were independent predictors of a complete response. Expression analysis identified many mutant TP53-associated genes, including CDC20, TTK, CDKN2A, and the stem cell gene PROM1, but failed to identify a transcriptional profile associated with complete responses among TP53 mutant tumors. In patients with unresponsive tumors, mutant TP53 status predicted significantly shorter overall survival. The 15 patients with responsive TP53-mutant tumors, however, had a favorable outcome, suggesting that this chemotherapy regimen can overcome the poor prognosis generally associated with mutant TP53 status. CONCLUSIONS: This study demonstrates that, in noninflammatory breast cancers, TP53 status is a key predictive factor for response to this dose-dense epirubicin–cyclophosphamide regimen and further suggests that the basal subtype is exquisitely sensitive to this association. Given the well-established predictive value of complete responses for long-term survival and the poor prognosis of basal and TP53-mutant tumors treated with other regimens, this chemotherapy could be particularly suited for breast cancer patients with a mutant TP53, particularly those with basal features

Genetic Heterogeneity in Therapy-Naïve Synchronous Primary Breast Cancers and Their Metastases

Purpose:; Paired primary breast cancers and metachronous metastases after adjuvant treatment are reported to differ in their clonal composition and genetic alterations, but it is unclear whether these differences stem from the selective pressures of the metastatic process, the systemic therapies, or both. We sought to define the repertoire of genetic alterations in breast cancer patients with; de novo; metastatic disease who had not received local or systemic therapy.; Experimental Design:; Up to two anatomically distinct core biopsies of primary breast cancers and synchronous distant metastases from nine patients who presented with metastatic disease were subjected to high-depth whole-exome sequencing. Mutations, copy number alterations and their cancer cell fractions, and mutation signatures were defined using state-of-the-art bioinformatics methods. All mutations identified were validated with orthogonal methods.; Results:; Genomic differences were observed between primary and metastatic deposits, with a median of 60% (range 6%-95%) of shared somatic mutations. Although mutations in known driver genes including; TP53, PIK3CA; , and; GATA3; were preferentially clonal in both sites, primary breast cancers and their synchronous metastases displayed spatial intratumor heterogeneity. Likely pathogenic mutations affecting epithelial-to-mesenchymal transition-related genes, including; SMAD4, TCF7L2; , and; TCF4; (; ITF2; ), were found to be restricted to or enriched in the metastatic lesions. Mutational signatures of trunk mutations differed from those of mutations enriched in the primary tumor or the metastasis in six cases.; Conclusions:; Synchronous primary breast cancers and metastases differ in their repertoire of somatic genetic alterations even in the absence of systemic therapy. Mutational signature shifts might contribute to spatial intratumor genetic heterogeneity