Françoise Bonnet

A refined molecular taxonomy of breast cancer

The current histoclinical breast cancer classification is simple but imprecise. Several molecular classifications of breast cancers based on expression profiling have been proposed as alternatives. However, their reliability and clinical utility have been repeatedly questioned, notably because most of them were derived from relatively small initial patient populations. We analyzed the transcriptomes of 537 breast tumors using three unsupervised classification methods. A core subset of 355 tumors was assigned to six clusters by all three methods. These six subgroups overlapped with previously defined molecular classes of breast cancer, but also showed important differences, notably the absence of an ERBB2 subgroup and the division of the large luminal ER+ group into four subgroups, two of them being highly proliferative. Of the six subgroups, four were ER+/PR+/AR+, one was ER−/PR−/AR+ and one was triple negative (AR−/ER−/PR−). ERBB2-amplified tumors were split between the ER−/PR−/AR+ subgroup and the highly proliferative ER+ LumC subgroup. Importantly, each of these six molecular subgroups showed specific copy-number alterations. Gene expression changes were correlated to specific signaling pathways. Each of these six subgroups showed very significant differences in tumor grade, metastatic sites, relapse-free survival or response to chemotherapy. All these findings were validated on large external datasets including more than 3000 tumors. Our data thus indicate that these six molecular subgroups represent well-defined clinico-biological entities of breast cancer. Their identification should facilitate the detection of novel prognostic factors or therapeutical targets in breast cancer.

High cumulative risks of cancer in patients with PTEN hamartoma tumour syndrome

Background PTEN hamartoma tumour syndrome (PHTS) encompasses several clinical syndromes with germline mutations in the PTEN tumour suppressor gene, including Cowden syndrome which is characterised by an increased risk of breast and thyroid cancers. Because PHTS is rare, data regarding cancer risks and genotype–phenotype correlations are limited. The objective of this study was to better define cancer risks in this syndrome with respect to the type and location of PTEN mutations. Methods 154 PHTS individuals with a deleterious germline PTEN mutation were recruited from the activity of the Institut Bergonié genetic laboratory. Detailed phenotypic information was obtained for 146 of them. Age and sex adjusted standardised incidence ratio (SIR) calculations, cumulative cancer risk estimations, and genotype–phenotype analyses were performed. Results Elevated SIRs were found mainly for female breast cancer (39.1, 95% CI 24.8 to 58.6), thyroid cancer in women (43.2, 95% CI 19.7 to 82.1) and in men (199.5, 95% CI 106.39 to 342.03), melanoma in women (28.3, 95% CI 7.6 to 35.4) and in men (39.4, 95% CI 10.6 to 100.9), and endometrial cancer (48.7, 95% CI 9.8 to 142.3). Cumulative cancer risks at age 70 were 85% (95% CI 70% to 95%) for any cancer, 77% (95% CI 59% to 91%) for female breast cancer, and 38% (95% CI 25% to 56%) for thyroid cancer. The risk of cancer was two times greater in women with PHTS than in men with PHTS (p<0.05). Conclusions This study shows a considerably high cumulative risk of cancer for patients with PHTS, mainly in women without clear genotype–phenotype correlation for this cancer risk. New recommendations for the management of PHTS patients are proposed.

Segmental overgrowth, lipomatosis, arteriovenous malformation and epidermal nevus (SOLAMEN) syndrome is related to mosaic PTEN nullizygosity

We describe two patients from distinct Cowden disease families with specific germline PTEN mutations whose disease differs from the usual appearance of Cowden disease. Their phenotype associates classical manifestations of Cowden disease and congenital dysmorphisms including segmental overgrowth, arteriovenous and lymphatic vascular malformations, lipomatosis and linear epidermal nevus reminiscent of the diagnosis of Proteus syndrome. We provide evidence in one of the two patients of a secondary molecular event: a loss of the PTEN wild-type allele, restricted to the atypical lesions that may explain an overgrowth of the affected tissues and the atypical phenotype. These data provide a new demonstration of the Happle hypothesis to explain some segmental exacerbation of autosomal-dominant disorders. They also show that a bi-allelic inactivation of PTEN can lead to developmental anomalies instead of malignant transformation, thus raising the question of the limitations of the tumor suppressive function in this gene. Finally, we suggest using the term ‘SOLAMEN syndrome’ (Segmental Overgrowth, Lipomatosis, Arteriovenous Malformation and Epidermal Nevus) in these peculiar situations to help the difficult distinction between the phenotype of our patients and Proteus syndrome.

Genome-Wide Analysis of Cutaneous T-Cell Lymphomas Identifies Three Clinically Relevant Classes

This study was undertaken to identify recurrent genetic alterations of the three main types of cutaneous T-cell lymphomas (CTCLs): mycosis fungoides (MF), Sézary syndrome (SS), and cutaneous anaplastic large-cell lymphoma (CALCL). Using array-based comparative genomic hybridization, the molecular cytogenetic profiles of 72 samples obtained from 58 patients with CTCL corresponding to 24 transformed MF (T-MF), 16 SS, and 18 CALCLs were determined. T-MF was characterized by gains of 1q25-31, 7p22-11.2, 7q21, 7q31, and 17q12, and losses of 9p21, 10p11.2, and 10q26. SS exhibited gains of 8q23-24.3 and 17q23-24, as well as losses of 9p21, 10p12-11.2, 10q22-24, 10q25-26, and 17p13-q11.1. Finally, CALCL exhibited 6q27 and 13q34 losses. Such imbalances were statistically associated with one CTCL subtype. Unsupervised hierarchical clustering defined three categories of clinical relevance: (1) CALCL apart from epidermotropic-CTCL, (2) an SS-only category, and (3) a mixed category with T-MF and SS cases, with both primary and secondary SS cases. In rare cases, the genetic classification did not correspond to the inclusion diagnosis, possibly reflecting the association of two diseases in the same patient or initial misdiagnosis according to follow-up. Finally, different samples in the same patient clustered together, showing reproducibility of such a classifier.

Alterations of the p53 and PIK3CA/AKT/mTOR Pathways in Angiosarcomas A Pattern Distinct From Other Sarcomas With Complex Genomics

The p53 and phosphoinositide‐3‐kinase, catalytic, alpha polypeptide/v‐akt murine thymoma viral oncogene homolog/mechanistic target of rapamycin (PIK3CA/AKT/mTOR) pathways frequently are altered in sarcoma with complex genomics, such as leiomyosarcoma (LMS) or undifferentiated pleomorphic sarcoma (UPS). The scale of genetic abnormalities in these pathways remains unknown in angiosarcoma (AS).

Molecular apocrine differentiation is a common feature of breast cancer in patients with germline PTEN mutations

IntroductionBreast carcinoma is the main malignant tumor occurring in patients with Cowden disease, a cancer-prone syndrome caused by germline mutation of the tumor suppressor gene PTEN characterized by the occurrence throughout life of hyperplastic, hamartomatous and malignant growths affecting various organs. The absence of known histological features for breast cancer arising in a PTEN-mutant background prompted us to explore them for potential new markers.MethodsWe first performed a microarray study of three tumors from patients with Cowden disease in the context of a transcriptomic study of 74 familial breast cancers. A subsequent histological and immunohistochemical study including 12 additional cases of Cowden disease breast carcinomas was performed to confirm the microarray data.ResultsUnsupervised clustering of the 74 familial tumors followed the intrinsic gene classification of breast cancer except for a group of five tumors that included the three Cowden tumors. The gene expression profile of the Cowden tumors shows considerable overlap with that of a breast cancer subgroup known as molecular apocrine breast carcinoma, which is suspected to have increased androgenic signaling and shows frequent ERBB2 amplification in sporadic tumors. The histological and immunohistochemical study showed that several cases had apocrine histological features and expressed GGT1, which is a potential new marker for apocrine breast carcinoma.ConclusionsThese data suggest that activation of the ERBB2-PI3K-AKT pathway by loss of PTEN at early stages of tumorigenesis promotes the formation of breast tumors with apocrine features.

Spectrum of CREBBP gene dosage anomalies in Rubinstein-Taybi Syndrome patients

The Rubinstein–Taybi syndrome (RTS) is a rare autosomal-dominant disease associated with 10–15% of cases with 16p13.3 microdeletions involving the CREB-binding protein gene (CREBBP). We used array-comparative genomic hybridization and Quantitative multiplex fluorescent-PCR (QMF-PCR) to search for dosage anomalies in the 16p13.3 region and the CREBBP gene. We first constructed a microarray covering 2 Mb that carries seven BAC and 34 cosmid clones, as well as 26 low-molecular-weight probes (1000–1500 bp) that are spread along the CREBBP gene. To increase further the resolution inside the CREBBP gene, we used QMF-PCR assays providing a 7 kb resolution. The deletions characterized in this work extended between as little as 3.3 kb and 6.5 Mb. Some deletions were restricted to just a few exons of CREBBP, some deleted either the 5′ or the 3′ end of the gene plus adjacent genomic segments, others deleted the whole gene away. We also identified a duplication of exon 16. We showed that CREBBP dosage anomalies constitute a common cause of RTS. CREBBP high-resolution gene dosage search is therefore highly recommended for RTS diagnosis. No correlation was found between the type of deletion and the patients’ phenotype. All patients had typical RTS, and there was no particular severity associated with certain alterations.

Comprehensive analysis of PTEN status in breast carcinomas

PTEN plays a well‐established role in the negative regulation of the PI3K pathway, which is frequently activated in several cancer types, including breast cancer. A nuclear function in the maintenance of chromosomal stability has been proposed for PTEN but is yet to be clearly defined. In order to improve understanding of the role of PTEN in mammary tumorigenesis in terms of a possible gene dosage effect, its PI3K pathway function and its association with p53, we undertook comprehensive analysis of PTEN status in 135 sporadic invasive ductal carcinomas. Four PTEN status groups were defined; complete loss (19/135, 14%), reduced copy number (19/135, 14%), normal (86/135, 64%) and complex (11/135, 8%). Whereas the PTEN complete loss status was significantly associated with estrogen receptor (ER) negativity (p=0.006) and in particular the basal‐like phenotype (p<0.0001), a reduced PTEN copy number was not associated with hormone receptor status or a particular breast cancer subtype. Overall, PI3K pathway alteration was suggested to be involved in 59% (79/134) of tumors as assessed by human epidermal growth factor receptor 2 overexpression, PIK3CA mutation or a complete loss of PTEN. A complex PTEN status was identified in a tumor subgroup which displayed a specific, complex DNA profile at the PTEN locus with a strikingly similar highly rearranged pan‐genomic profile. All of these tumors had relapsed and were associated with a poorer prognosis in the context of node negative disease (p=1.4 × 10−13) thus may represent a tumor subgroup with a common molecular alteration which could be targeted to improve clinical outcome.

Deletion of Chromosomes 13q and 14q Is a Common Feature of Tumors with BRCA2 Mutations

Introduction Germline BRCA1 or BRCA2 mutations account for 20–30% of familial clustering of breast cancer. The main indication for BRCA2 screening is currently the family history but the yield of mutations identified in patients selected this way is low. Methods To develop more efficient approaches to screening we have compared the gene expression and genomic profiles of BRCA2-mutant breast tumors with those of breast tumors lacking BRCA1 or BRCA2 mutations. Results We identified a group of 66 genes showing differential expression in our training set of 7 BRCA2-mutant tumors and in an independent validation set of 19 BRCA2-mutant tumors. The differentially expressed genes include a prominent cluster of genes from chromosomes 13 and 14 whose expression is reduced. Gene set enrichment analysis confirmed that genes in specific bands on 13q and 14q showed significantly reduced expression, suggesting that the affected bands may be preferentially deleted in BRCA2-mutant tumors. Genomic profiling showed that the BRCA2-mutant tumors indeed harbor deletions on chromosomes 13q and 14q. To exploit this information we have created a simple fluorescence in situ hybridization (FISH) test and shown that it detects tumors with deletions on chromosomes 13q and 14q. Conclusion Together with previous reports, this establishes that deletions on chromosomes 13q and 14q are a hallmark of BRCA2-mutant tumors. We propose that FISH to detect these deletions would be an efficient and cost-effective first screening step to identify potential BRCA2-mutation carriers among breast cancer patients without a family history of breast cancer.

An array CGH based genomic instability index (G2I) is predictive of clinical outcome in breast cancer and reveals a subset of tumors without lymph node involvement but with poor prognosis.

BackgroundDespite entering complete remission after primary treatment, a substantial proportion of patients with early stage breast cancer will develop metastases. Prediction of such an outcome remains challenging despite the clinical use of several prognostic parameters. Several reports indicate that genomic instability, as reflected in specific chromosomal aneuploidies and variations in DNA content, influences clinical outcome but no precise definition of this parameter has yet been clearly established.MethodsTo explore the prognostic value of genomic alterations present in primary tumors, we performed a comparative genomic hybridization study on BAC arrays with a panel of breast carcinomas from 45 patients with metastatic relapse and 95 others, matched for age and axillary node involvement, without any recurrence after at least 11 years of follow-up. Array-CGH data was used to establish a two-parameter index representative of the global level of aneusomy by chromosomal arm, and of the number of breakpoints throughout the genome.ResultsApplication of appropriate thresholds allowed us to distinguish three classes of tumors highly associated with metastatic relapse. This index used with the same thresholds on a published set of tumors confirms its prognostic significance with a hazard ratio of 3.24 [95CI: 1.76-5.96] p = 6.7x10-5 for the bad prognostic group with respect to the intermediate group. The high prognostic value of this genomic index is related to its ability to individualize a specific group of breast cancers, mainly luminal type and axillary node negative, showing very high genetic instability and poor outcome. Indirect transcriptomic validation was obtained on independent data sets.ConclusionAccurate evaluation of genetic instability in breast cancers by a genomic instability index (G2I) helps individualizing specific tumors with previously unexpected very poor prognosis.