Somatic mutational spectrum analysis in a prospective series of 104 gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors distinguished by driver mutations in proto-oncogenes KIT or PDGFRA in 85-90% of cases. These mutations have been linked to the response to imatinib, a multikinase inhibitor, and have independent prognostic impact. Here, we describe the prospective study of the molecular characteristics of 104 GISTs from French adult patients analyzed routinely through the National Hospital Program of Molecular Cancer Diagnosis. All patients with GISTs diagnosed at the University Hospital of Besançon between August 2005 and October 2014 were prospectively included in the present study. KIT, PDGFRA and KRAS-codons 12 and 13 as well as BRAF codon 600 mutations were analyzed by Sanger sequencing or SNaPshot. KIT and PDGFRA mutations were detected in 71.2 and 19.2% of the cases, respectively. A total of 43 different mutations were detected of which 13 had never been described. As expected, KIT exon 9 and PDGFRA exon 18 mutations were associated with small bowel and gastric localizations respectively. No mutation was found in KRAS and BRAF. Molecular studies are critical to improve the management of GISTs. Our study enhances the current knowledge by describing 13 new mutations in KIT. A common molecular pattern in all KIT exon 11 substitutions is also described for the first time in this study but its significance remains unknown since genetic and environmental risk factors favoring the development of GISTs such as DNA repair defects and exposure to carcinogens are not currently known.

[Analysis of MGMT methylation with the therascreen(®) MGMT Pyro(®) Kit (Qiagen). A method verification]. / Étude du statut de méthylation de MGMT par pyroséquençage avec la trousse therascreen(®) MGMT Pyro(®) (Qiagen). Vérification de la méthode.

Promoter methylation of the MGMT gene, encoding the enzyme O6-methylguanine-ubiquitous methyltransferase, is a theranostic good prognosis marker of glioblastomas treated with alkylating chemotherapy (temozolomide, Temodal(®)). Among the methylation analysis techniques, pyrosequencing is a reproducible and sensitive quantitative method. As part of the accreditation of the hospital platform of molecular genetics of cancer, Besançon, our objective was to verify the performance of the pyrosequencing commercial kit therascreen(®) MGMT Pyro(®) (Qiagen) in terms of repeatability, reproducibility, limit of blank (LOB), limit of detection (LOD), linearity and contamination by the guide SH GTA 04 delivered by the Cofrac. The repeatability tests show an average methylation of 3.22% [standard deviation (SD) = 0.41, coefficient of variation (CV) = 12.75%] for the unmethylated control and 70.16% (SD = 2.20, CV = 3.14%) for the methylated control. Reproducibility demontrates an average methylation of 1.39% (SD = 0.25, CV = 18.25%) for the unmethylated control and of 94.03% (SD = 2.56, CV = 2.73%) for the methylated control. The percentages of LOB and LOD are respectively 3.43% and 6.22% methylation. The regression coefficient of 0,983 confirms the linearity of the assay from 0% to 100% methylation. No contamination has been observed. Over 40% of glioblastomas studied in 2013 in our laboratory have shown a methylated MGMT gene. Our results confirms that the theraScreen(®) MGMT Pyro(®) kit (Qiagen) is performant in compliance with the quality requirements of the NF EN ISO 15189 for the routine analysis of methylation status of MGMT in glioblastomas.

A multiplex SNaPshot assay as a rapid method for detecting KRAS and BRAF mutations in advanced colorectal cancers.

The analysis of KRAS mutations has become a prerequisite for anti-epidermal growth factor receptor therapy in patients with metastatic colorectal cancers. KRAS mutations are associated with resistance to treatment by monoclonal antibodies such as cetuximab and panitumumab and thus are correlated with a shorter progression-free survival. BRAF mutations also may play a role in treatment decisions. The widespread use of these targeted therapies has generated the need to develop cost-effective methods for routine KRAS and BRAF analysis. The aim of this study was to compare a multiplex SNaPshot assay with DNA sequencing and high-resolution melting analysis for identifying KRAS codons 12 and 13 and BRAF codon 600 mutations. Thus 110 routinely formalin-fixed and paraffin-embedded tissue blocks were tested by each method. The SNaPshot analysis detected KRAS and BRAF codon 600 mutations in, respectively, 34.5% (n = 38) and 10% (n = 11) of these tissue blocks. These results were confirmed by direct DNA sequencing and by high-resolution melting analysis. The costs and time constraints of each detection method were compared at the same time. In conclusion, our newly designed multiplex SNaPshot assay is a fast, inexpensive, sensitive, and robust technique for molecular diagnostic practices and patient selection.

Molecular and ultrastructural characterization of two ascomycetes found on sunken wood off Vanuatu islands in the deep Pacific ocean.

A new genus of a deep-sea ascomycete with one new species, Alisea longicolla, is described based on analyses of 18S and 28S rDNA sequences and morphological characters. A. longicolla was found together with Oceanitis scuticella, on small twigs and sugar cane debris trawled from the bottom of the Pacific Ocean off Vanuatu Islands. Molecular and morphological characters indicate that both fungi are members of Halosphaeriaceae. Within this family, O. scuticella is phylogenetically related to Ascosalsum and shares similar ascospore morphology and appendage ontogeny. The genus Ascosalsum is considered congeneric with Oceanitis and Ascosalsum cincinnatulum, Ascosalsum unicaudatum and Ascosalsum viscidulum are transferred to Oceanitis, an earlier generic name.

Analysis of the guinea-pig estrogen-regulated gec1/GABARAPL1 gene promoter and identification of a functional ERE in the first exon.

The gec1/GABARAPL1 (GABA(A)-receptor-associated protein like-1) gene has been identified as an early estrogen-regulated gene in guinea-pig cultured endometrial glandular epithelial cells (GEC). Guinea-pig and human gec1/GABARAPL1 proteins share 87% identity with GABARAP, which acts as a protein linker between microtubules and the GABA(A) receptor. To investigate the molecular mechanisms regulating gec1/GABARAPL1 gene expression, the 1.5-kbp region upstream of the translation initiation codon of the guinea-pig gec1/GABARAPL1 gene was cloned. A 300-bp fragment encompassing a pyrimidine-rich initiator element (INR) and the transcription start site (+1) was sufficient to initiate transcription. Transfection and gel shift experiments showed that a sequence located at +36/+50 in the first exon permitted induction of expression of this gene by estradiol acting via ERalpha. This sequence (GGGTCAACGTGACGT) differs only by one base pair from the consensus estrogen response element ERE (GGGTCAACGTGACCT). It can be concluded that the ERE located in the first exon encoding the 5'-untranslated region is sufficient for E2 activation of gec1/GABARAPL1 transcription.

Expression of gec1/GABARAPL1 versus GABARAP mRNAs in human: predominance of gec1/GABARAPL1 in the central nervous system.

GABARAP and gec1/GABARAPL1 genes encode very similar proteins belonging to a new microtubule-associated protein (MAP) family. These proteins could participate in a complex clustering, targeting and/or degrading the GABA(A) receptors on post-synaptic membrane of neurons. Using specific cDNA probes, we investigated the differential expression of both genes in 76 human tissues. Against all odds, gec1/GABARAPL1 was more expressed than GABARAP in the central nervous system (CNS), while GABARAP was more expressed in endocrine glands.