Gene expression profiling of human adrenocortical tumors using complementary deoxyribonucleic Acid microarrays identifies several candidate genes as markers of malignancy.

International audienceThe aim of this study was to identify predictor sets of genes whose over- or underexpression in human sporadic adrenocortical tumors would help to identify malignant vs. benign tumors and to predict postsurgical metastatic recurrence. For this, we analyzed the expression of 230 candidate genes using cDNA microarrays in a series of 57 well-characterized human sporadic adrenocortical tumors (33 adenomas and 24 carcinomas). We identified two clusters of genes (the IGF-II cluster containing eight genes, including IGF-II, and the steroidogenesis cluster containing six genes encoding steroidogenic enzymes plus eight other genes) whose combined levels of expression appeared to be good predictors of malignancy. This predictive value was as strong as that of the pathological score of Weiss. The analysis of the population of carcinomas (13 tumors) for genes whose expression would be strongly different between recurring and nonrecurring tumors allowed identification of 14 genes meeting these criteria. Among these genes, there are probably new markers of tumor evolution that will deserve additional validation on a larger scale. Taken together, these results show that the parallel analysis of the expression levels of a selected group of genes on microgram quantities of tumor RNA (a quantity that can be obtained from fine needle aspirations) appears as a complementary method to histopathology for the diagnosis and prognosis of evolution of adrenocortical carcinomas

[Contribution of molecular biology to new diagnostic and/or prognostic markers characterization in cancerogenesis]

International audienceDuring malignant transformation, cells accumulate genetic and epigenetic alterations. Since ten years, the knowledge of the whole human genome, associated with the development of new molecular biology techniques allowing global analysis, encouraged the identification of these anomalies. Thus, transcriptome studies with DNA chips allowed the characterization of genes groups whose expressions vary according to the type of tumors or according to their recurrence. We analysed adrenal tumors with DNA chips and tried to characterize recurring carcinomas. On the other hand, tumor suppressor genes expression could be inhibited by epigenetic modifications like gene promoter hypermethylation. With development of sensitive methods like PCR, methylation profile could be defined. We were interested in lung and head and neck tumors and tried to evaluate if the presence of methylated gene in bronchial lavage or in saliva could be a good marker for the early detection of a primary tumor or of a recurrence

Circular RNAs and RNA Splice Variants as Biomarkers for Prognosis and Therapeutic Response in the Liquid Biopsies of Lung Cancer Patients

International audienceLung cancer, including non-small cell lung carcinoma (NSCLC), is the most frequently diagnosed cancer. It is also the leading cause of cancer-related mortality worldwide because of its late diagnosis and its resistance to therapies. Therefore, the identification of biomarkers for early diagnosis, prognosis, and monitoring of therapeutic response is urgently needed. Liquid biopsies, especially blood, are considered as promising tools to detect and quantify circulating cancer biomarkers. Cell-free circulating tumor DNA has been extensively studied. Recently, the possibility to detect and quantify RNAs in tumor biopsies, notably circulating cell-free RNAs, has gained great attention. RNA alternative splicing contributes to the proteome diversity through the biogenesis of several mRNA splice variants from the same pre-mRNA. Circular RNA (circRNA) is a new class of RNAs resulting from pre-mRNA back splicing. Owing to the development of high-throughput transcriptomic analyses, numerous RNA splice variants and, more recently, circRNAs have been identified and found to be differentially expressed in tumor patients compared to healthy controls. The contribution of some of these RNA splice variants and circRNAs to tumor progression, dissemination, or drug response has been clearly demonstrated in preclinical models. In this review, we discuss the potential of circRNAs and mRNA splice variants as candidate biomarkers for the prognosis and the therapeutic response of NSCLC in liquid biopsies

Marqueurs moléculaires et thérapies ciblées (exemple des mutations de l EGFR et de KRAS comme marqueurs prédictifs de réponse aux agents ciblant l EGFR)

Les cancers broncho-pulmonaires (CBNPC) et colorectaux sont les deux premières causes de mortalité par cancer dans le monde. Détectés tardivement, ils sont de très mauvais pronostic, avec une survie à 5 ans respectivement de 12 % et 5 %. Récemment, de nouvelles thérapies dites ciblées, comme par exemple les anticorps monoclonaux anti-EGFR ou les inhibiteurs de l activité tyrosine kinase (TKI) de l EGFR, ont été développées. Une faible proportion des patients porteurs d un cancer broncho-pulmonaire ou colorectal répond à ces traitements ; leur survie est augmentée et leur qualité de vie améliorée. Différentes études ont permis de préciser les facteurs prédictifs de réponse, tels que les mutations des exons 18 à 21 de l EGFR et celles de l exon 1 de KRAS. Ainsi, les anticorps monoclonaux sont indiqués dans le traitement du cancer colorectal métastatique lorsque KRAS est sauvage. Pour le traitement des CBNPC par TKIs, le statut mutationnel de l EGFR est en cours d évaluation. Au laboratoire, l analyse des mutations a dans un premier temps été réalisée par la méthode de référence, le séquençage direct, puis une technique alternative, le pyroséquençage a été développée. Cette méthode est plus sensible, plus rapide et moins coûteuse, et permet une prise en charge optimale des patients pouvant bénéficier d une thérapie ciblant l EGFR.GRENOBLE1-BU Médecine pharm. (385162101) / SudocSudocFranceF

[Aberrant methylation of tumor suppressor genes in head and neck squamous cell carcinoma: is it clinically relevant?]

International audienceDuring malignant transformation, the malignant cell accumulates epigenetic abnormalities that do not alter the DNA sequence but are transmissible during divisions and modify genes expression. The methylation of CpG islands in the tumor suppressor genes (TS genes) promoters inhibits their transcription ; it is a mecanism of gene inactivation as frequent as allelic deletions. The methylation profile (or panel of methylated genes in a tumor), similarly to allelic deletions, varies with the tumor histology. Within head and neck squamous cell carcinoma (oral cavity, larynx and oropharynx), 19 genes have been analysed, among them 5 are frequently methylated, i.e. : p16, ECAD, DAPK, MGMT et TIMP3. The method of methylation analysis, based on a bisulfite treatment followed by a PCR amplification, is sensitive and specific enough to allow the detection of abnormalities in biological fluid that drain the tumor or in circulating tumoral DNA. In the head and neck squamous cell carcinoma, correlation between the methylation profile in tumor and paired saliva is excellent ; thus methylation analysis in saliva is a very promising approach for early cancer detection in high risk patients or for the post treatment follow up and rapid diagnosis of relapse. The methylation signature might also reflect the tumor prognosis and complete the histology to define the diagnosis. Finally, DNA methylation is reversible with demethylating agents, a new avenue for cancer therapy in association with conventional chemotherapy

Pyrosequencing, a method approved to detect the two major EGFR mutations for anti EGFR therapy in NSCLC.

International audienceABSTRACT: BACKGROUND: Epidermal Growth Factor Receptor (EGFR) mutations, especially in-frame deletions in exon 19 (delta LRE) and a point mutation in exon 21 (L858R) predict gefitinib sensitivity in patients with non-small cell lung cancer. Several methods are currently described for their detection but the gold standard for tissue samples remains direct DNA sequencing, which requires samples containing at least 50% of tumor cells. METHODS: We designed a pyrosequencing assay based on nested PCR for the characterization of theses mutations on formalin-fixed and paraffin-embedded tumor tissue. RESULTS: This method is highly specific and permits precise characterization of all the exon 19 deletions. Its sensitivity is higher than that of "BigDye terminator" sequencing and enabled detection of 3 additional mutations in the 58 NSCLC tested. The concordance between the two methods was very good (97.4%). In the prospective analysis of 213 samples, 7 (3.3%) samples were not analyzed and EGFR mutations were detected in 18 (8.7%) patients. However, we observed a deficit of mutation detection when the samples were very poor in tumor cells. CONCLUSIONS: pyrosequencing is then a highly accurate method for detecting delta LRE and L858R EGFR mutations in patients with NSCLC when the samples contain at least 20% of tumor cells

Determination of dansylated polyamines in red blood cells by liquid chromatography-tandem mass spectrometry.

International audienceThe concentration of polyamines in red blood cells (RBCs) is considered to be an index of cell proliferation. This index has been demonstrated to be of clinical importance for the follow-up and treatment of some cancer patients. The concentration of polyamines in RBCs is usually determined by high-performance liquid chromatography (HPLC) with fluorescence detection. In the current work, we present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of putrescine, spermidine, and spermine, the three major polyamines in RBCs. The polyamines were dansylated and analyzed by an LC gradient of 20-min duration on a C18 column on-line with a tandem mass spectrometer. An internal standard (1,8-diaminooctane) was used for quantification. This method exhibited excellent linearity for the three polyamines with regression coefficients higher than 0.99. The limits of detection for putrescine, spermidine, and spermine were 0.10, 0.75, and 0.50 pmol/ml, respectively. The intrarun precision values for putrescine, spermidine, and spermine all were better than 10%, and the interrun precision values were 13%, 9%, and 20%, respectively. The LC-MS/MS method is sufficiently simple and reliable enough to replace the currently used HPLC method with fluorescence detection in which putrescine is not always detectable

[Study of aberrant methylation of TSG in saliva in case of upper-aerodigestive-tract cancer.]

International audienceCURRENT SITUATION: Early detection of the relapse in case of tumor located in the upper aerodigestive tract (UADT) is an important point for improving prognosis. Clinical control has not been efficient and, until today, no biological biomarker has been validated for surveillance of patients. In a preliminary study, we have shown the benefit of the methylation status of six tumor suppressor genes (TSG): TIMP3, ECAD, p16, MGMT, DAPK, and RASSF1A in saliva for early diagnosis of tumor relapse. The main objective of this second study is to confirm the initial results. MATERIAL AND METHODS: This is a bicenter, prospective, diagnostic, single-blind study. The study started in December 2007, running for one year. The main inclusion criterion is a patient with squamous-cell carcinoma of the oral cavity or the oropharynx, stages I to II, without previous treatment for this location. Eighty patients will be included. The data analysis will use a multivariate Cox model. EXPECTED RESULTS: If our preliminary results are confirmed, identification of methylations in saliva would be able to constitute the first usuable biomarker, for the follow-up of patients with UADT cancer

Tumor-specific methylation in saliva: a promising biomarker for early detection of head and neck cancer recurrence.

PURPOSE: Our goal was to define tumor and saliva gene methylation profile of head and neck squamous cell carcinoma and to evaluate its prognostic significance and its biomarker potential for early detection of relapse. EXPERIMENTAL DESIGN: We prospectively analyzed 11 genes by methylation-specific PCR on primary tumors, histologically normal adjacent mucosa, and saliva from 90 French patients at diagnosis and during follow-up as well as on 30 saliva specimens from control-matched patients with nonmalignant head and neck pathology. Five additional genes were analyzed on 50 tumors of the series. RESULTS: Methylation of TIMP3, ECAD, p16, MGMT, DAPK, and RASSF1 was the most frequently observed in tumors and paired saliva samples were analyzed at diagnosis, with an excellent agreement between both samples. At least one of these six genes was methylated in >75% of the samples without additional positive samples when other genes were analyzed. Methylation profile was similar in newly diagnosed and second primary cancers. Aberrant methylation was not associated with a worse prognosis. Ninety percent of normal adjacent mucosa and all control saliva samples were negative. Twenty-two patients were followed after treatment; abnormal methylation was detectable in the saliva of five patients few months before clinical and 2-deoxy-2[(18)F]fluoro-d-glucose-positron emission tomography signs of relapse, allowing curable surgery. Saliva samples were negative for the 17 other patients: 16 were in remission and only 1 relapsed. CONCLUSIONS: Gene methylation in saliva is a promising biomarker for the follow-up and early detection of still curable relapses of head and neck squamous cell carcinoma patients

Recherche d'une signature proteomique du cancer du poumon.

International audienceCe travail est consacré à la recherche d'une signature protéomique du cancer du poumon. A partir de spectres acquis avec un spectromêtre de masse SELDI, nous recherchons un ensemble de protéines permettant de discriminer les patients atteints d'un cancer du poumon des sujets sains. Le problème est difficle en particulier parce qu'il y a plus de protéines que de patients et parce qu'il y a une grande variabilité dans les spectres d'une même population. Nous cherchons aussi à nous affranchir des effets de l'âge et du sexe. Nous avons choisi d'utiliser Random Forest. Cette méthode de classification naturellement multivariée, permettant d'identifier les variables discriminantes et étant capable de travailler avec plus de variables que d'échantillons nous semble bien adaptée au problème posé. Nous avons ainsi trouvé un ensemble de protéines discriminantes dont certaines ont pu être identifiées