Frédéric Chibon

Cancer gene expression signatures – The rise and fall?

A gene expression signature can be defined as a single or a combined gene expression alteration with validated specificity in terms of diagnosis, prognosis or prediction of therapeutic response. Since the publication of the first signature in the late 90s, high-throughput gene expression analysis has revolutionised genetics over the last 15 years. The scientific community has used this new technology to find responses to these fundamental questions; from understanding tumour biology, to prediction of progression, and treatments to which it will respond. Nevertheless, legitimate excitement about the attractiveness of molecular technologies and the promise of discovery-based research should not overlook adherence to the rules of evidence, otherwise it may result in claims that are not meaningful and lead to disappointment. This review will thus focus on the approaches developed to answer these three fundamental questions and the results evidenced both at biological and clinical level. On looking at this huge amount of data that have become increasingly minute, and at times contradictory, we discuss how gene expression signature improve our understanding of cancer biology, our ability to predict progression and response, and finally, our capacity to treat cancers more efficiently.

Prediction of HER2 gene status in Her2 2+ invasive breast cancer: a study of 108 cases comparing ASCO/CAP and FDA recommendations

Most Her2 testing guidelines recommend that all cases scoring Her2 2+ by immunohistochemistry should be analyzed by fluorescent in situ hybridization (FISH) to determine HER2 status to confirm eligibility for Trastuzumab therapy in breast cancer. The aim of our study was to determine HER2 gene and chromosome 17 (CEN17) status in a series of 108 Her2 2+ consecutive cases and study the correlation between pathological characteristics of the tumors and HER2 amplification. Invasive breast cancers were tested by FISH using the Dako HER2 FISH pharmDx® kit. The Her2 immunohistochemistry protocol was performed using the polyclonal AO485 antibody (Dako®) diluted to 1:1500. HER2 and CEN17 status were correlated to tumor SBR grade, mitotic count, estrogen receptor, progesterone receptor status and percentage of Her2 immunohistochemistry-positive cells. Following Food and Drug Administration guidelines, ie, HER2/CEN17 ratio ≥2 and an HER2 copy number >4, amplified cases were observed in 36 (33%) and 49 (45%) cases, respectively, and following American Society of Clinical Oncology/College of American Pathologists guidelines, ie, HER2/CEN17 ratio >2.2 and an HER2 copy number >6, amplified cases represented 30 and 24% of the study population, respectively. Chromosome 17 polysomy (CEN17 >2.25) was observed in 39 (36%) tumors. Significant positive correlations were found between FISH HER2 amplified cases and Her2 immunostaining >60% (P=1.1.10−5), SBR grade 3 (P=0.0001), nuclear atypia (P=0.03) and mitotic count (P=0.008). By multivariate analysis, Her2 immunostaining >60% (P<10−3) and SBR grade 3 (P<10−3) were independent factors predicting HER2 amplification status irrespective to cutoff guidelines. All SBR grade 3 cases with more than 60% Her2+ cells had an HER2/CEN17 ratio ≥2, only one had a ratio ≤2.2. In our series of consecutive Her2 2+ cases, one-third demonstrated HER2 amplification, and one-third had chromosome 17 polysomy. Pathological factors, in particular SBR grade 3 and more than 60% Her2+ cells, were significantly correlated with HER2 amplification.

Value and limitation of immunohistochemical expression of HMGA2 in mesenchymal tumors: about a series of 1052 cases

The high mobility group A (HMGA2) gene encodes a protein that alters chromatin structure and regulates the transcription of many genes; it is implicated in both benign and malignant neoplasias, but its rearrangements are a feature of development of several mesenchymal tumors. Given its implication in these tumors and particularly adipocytic tumors, and the availability of antibodies usable on paraffin-embedded tissues, we evaluated the immunohistochemical expression of this gene in a series of 1052 mesenchymal tumors. The objective was to define the value and limitations of HMGA2 immunohistochemical expression for histotyping, and compare with molecular data reported in the literature. We thus analyzed 880 cases on tissue microarray and 182 cases on whole sections (211 adipocytic tumors, 628 sarcomas, 213 benign mesenchymal tumors, and 10 normal adipose tissues). A nuclear immunostaining was detected in 86% of conventional and intramuscular lipomas, in 86% of well-differentiated liposarcomas and in 67% of dedifferentiated liposarcomas, as opposed to 16% of other benign adipose tumors and to 15% of non-well-differentiated liposarcoma/dedifferentiated liposarcoma sarcomas. Among benign mesenchymal tumors and lesions, it was detected in 90% of nodular fasciitis and in 88% of benign fibrous histiocytomas with respective specificities of 85 and 100%, and in 90% of aggressive angiomyxoma, contrary to other vulvovaginal tumor types, which expressed HMGA2 only rarely. The normal adipose tissue was always negative for HMGA2. Although not specific, immunohistochemical detection of the HMGA2 protein is helpful for the distinction of normal adipose tissue from well-differentiated lesions, particularly on biopsy or on re-excision. It is less sensitive than MDM2/CDK4 for dedifferentiated liposarcomas diagnosis, but it appears more specific to distinguish dedifferentiated liposarcomas from other poorly differentiated sarcomas. Finally, and may be more importantly, HMGA2 is useful for the diagnosis of benign fibrous histiocytoma, nodular fasciitis and vulvovaginal benign mesenchymal tumors.

Nuclear-Receptor-Mediated Telomere Insertion Leads to Genome Instability in ALT Cancers

The breakage-fusion-bridge cycle is a classical mechanism of telomere-driven genome instability in which dysfunctional telomeres are fused to other chromosomal extremities, creating dicentric chromosomes that eventually break at mitosis. Here, we uncover a distinct pathway of telomere-driven genome instability, specifically occurring in cells that maintain telomeres with the alternative lengthening of telomeres mechanism. We show that, in these cells, telomeric DNA is added to multiple discrete sites throughout the genome, corresponding to regions regulated by NR2C/F transcription factors. These proteins drive local telomere DNA addition by recruiting telomeric chromatin. This mechanism, which we name targeted telomere insertion (TTI), generates potential common fragile sites that destabilize the genome. We propose that TTI driven by NR2C/F proteins contributes to the formation of complex karyotypes in ALT tumors.

Monosomy 7 and absence of 12q amplification in two cases of spindle cell liposarcomas.

Spindle cell liposarcoma (SCL) is a rare malignant adipose tissue tumor presently regarded as a variant of well-differentiated liposarcoma (WDLPS). While WDLPS is cytogenetically characterized by the presence of supernumerary ring or giant chromosomes containing MDM2 amplification, data concerning the genomic alterations of SCL are scarce. Here, we describe the molecular cytogenetic characterization of two SCL cases. In these two cases, we did not identify supernumerary ring or giant chromosomes containing 12q amplification or any other chromosome 12 rearrangement. Instead, we observed a partial or complete monosomy 7 as the sole anomaly or among a few additional simple numeric and structural abnormalities. Monosomy 7 is not usual in adipose tissue tumors. It has been described in myelodysplastic syndromes and acute myeloid or lymphoblastic leukemias, as well as in several benign or malignant solid tumors. Our data suggest that the loss of material from chromosome 7 might play a crucial role in the pathogenesis of some SCL probably through the inactivation of tumor suppressor genes located on chromosome 7. On the basis of cytogenetic and molecular findings, some SCL may constitute an independent entity rather than being regarded as variants of WDLPS.

Genomic index predicts clinical outcome of intermediate-risk gastrointestinal stromal tumours, providing a new inclusion criterion for imatinib adjuvant therapy

PURPOSEnImatinib mesylate is the front-line targeted therapy for gastrointestinal stromal tumours (GISTs). Patients eligibility to adjuvant imatinib after primary tumour resection is currently based on histological and clinical risk assessment. While therapeutic options are clear for the very-low, low and high-risk subpopulations, no standard is actually available for the tumours classified as intermediate. Since we recently validated genomic index (GI), a measure of the level of genomic alterations, as a strong predictor of clinical outcome in GIST, we asked whether it could also represent a novel prognostic factor for the intermediate subgroup.nnnEXPERIMENTAL DESIGNn82 intermediate risk patients were selected based on the Armed Forces Institute of Pathology (AFIP) classification for genomic profiling.nnnRESULTSnData revealed that even if studied samples generally harboured a combination of the typical genetic aberrations found in GIST, i.e. 1p, 14q 22q deletions and frequently lost CDKN2A locus on chromosome 9, they profoundly differed from each other on the total number of genomic changes and GI value. Kaplan-Meier analyses of metastatic-free survival unveiled that stratification of the tumours by the GI value at a cutoff of 10 separated the good from the poor prognosis patients, proven that metastatic-risk in GIST intermediate patients is strongly associated with high GI values and genome complexity.nnnCONCLUSIONnGI is validated here as a robust marker to predict intermediate-GIST clinical outcome. Applicable in numerous Pathology Laboratories already using array comparative genomic hybridisation (CGH) with formalin-fixed paraffin-embedded (FFPE) samples, this assay presently stands as an efficient tool for the clinical management of intermediate GIST-patients.

Robust gene expression signature is not merely a significant P value

Since the seminal publication in 1999, the role of gene expression signatures in oncology has grown, particularly for predicting outcome or response to chemotherapy. Nevertheless, legitimate excitement about the attractiveness of molecular technologies and the promise of discovery-based research should not overlook adherence to the rules of evidence, otherwise it may result in claims that are not meaningful and lead to disappointment. The very first of these rules has to be the significance of the findings, both at the statistical and biological levels. Few studies based on the outcomeassociation argument report negative controls to check whether their signature of interest is actually more strongly related to outcomes than signatures with no oncological rationale. Faced with this fact, Venet et al. recently reported a very interesting study aiming to test the null hypothesis assuming a background of no associations with outcomes. They compared 47 published breast cancer outcome signatures to signatures of identical size constructed from random genes and showed that 60% of the published signatures were not significantly better outcome predictors than random signatures. Here we report exactly the same approach regarding the CINSARC signature recently published and validated

Biologie moléculaire en pathologie des tissus mous : utile ou nécessaire ?

Sarcomas are a heterogeneous group of tumors. Their diagnosis is based on morphology and immunohistochemical profile, with categories of tumors according to the type of tissue that they resemble. Nevertheless, for several tumors, cellular origin is unknown. Molecular analysis performed in recent years allowed, combining histophenotype and genomics, better classifying such sarcomas, individualizing new entities and grouping some tumors. Simple and recurrent genetic alterations, such as translocation, mutation, amplification, can be identified in one of two sarcomas and appear as new diagnostic markers. Their identification in specialized laboratories in molecular pathology of sarcomas is often useful and sometimes necessary for a good diagnosis, leading to a heavy and multidisciplinary multi-step treatment.

Automate à inclusion rapide : l’expérience bordelaise

Resume Le conditionnement actuel des prelevements tissulaires en pathologie, base sur la fixation formolee et l’inclusion par un automate sous vide, n’est plus adapte aux normes sanitaires, a la necessite de preserver les acides nucleiques et a la reduction des delais de rendu des diagnostics. Nous testons depuis 3 ans une nouvelle methode de conditionnement tissulaire basee sur une fixation alcoolique dans le Molecular Fixative ® (MF) et l’automate Xpress ® a inclusion rapide de Sakura™. En utilisant ce systeme, il est possible de traiter les fragments tissulaires en une a deux heures, a condition d’adapter leur prise en charge macroscopique. Le rendu morphologique varie peu de celui des tissus fixes au formol et se traduit par une retraction cellulaire et tissulaire variable selon le tissu et son epaisseur. Des ajustements protocolaires permettent les analyses immunohistochimiques et FISH de la meme facon qu’avec les fixateurs classiques. Les differentes techniques montrent l’integrite de l’ADN et de l’ARN des tissus fixes dans le MF et conditionnes dans l’Xpress ® .