Custom multi­tumor next­generation sequencing panel for routine molecular diagnosis of solid tumors: Validation and results from three­year clinical use.

Molecular testing is extremely important in cancer care, starting as early as at diagnosis. In order to address the challenge of providing reliable results within the timeframe adapted to patient management and suitable to guide clinical decisions, a capture­based next­generation sequencing (NGS) panel focusing on ten genes known to harbor genetic variations which may be targeted by approved drugs in patients with cancer was designed and validated. Very favorable analytical performances were obtained for both solid and liquid biopsies. For solid biopsies, a low read depth (80X per nucleotide) led to the genotype detection accuracy of 100%. The read of raw data for liquid biopsies resulted in the 91.19% result concordance between paired solid and liquid samples. The present method met all the requirements for the ISO15189 certification. During our three­year experience of routinely using this panel, almost 2,300 samples from lung and colorectal cancers, melanomas and gastrointestinal stromal tumors have been analyzed. It was found that our panel detected slightly more gain­of­function variants than described in the literature. Surprisingly, loss­of­function variants were also detected in certain of the analyzed genes. Finally, liquid biopsy data revealed statistically different mutated allele frequencies between tumor types, but also between mutated genes and variants themselves. In conclusion, the use of our capture­based NGS panel is perfectly adapted to perform relevant molecular diagnosis in a time frame compatible with patient care.

[Validation steps for the detection of L858R mutation in EGFR gene by real time quantitative PCR]. / Validation de méthode de la recherche de la mutation L858R par PCR en temps réel utilisant les sondes fluorescentes Taqman® dans les cancers bronchiques non à petites cellules.

Since January 16(th) 2010, the French legislation requires that the medical laboratories must be accredited according to ISO 15189 standards. This concerned all the biological medical technics, including molecular biology technics. In this work, we described the validation steps by real time quantitative PCR of L858R mutation in EGFR gene, frequently detected in non-small lung cancers (NSCLC). Epidermal growth factor - tyrosine kinase inhibitors (EGFR-TKIs) are authorized in Europe for the treatment of metastatic NSCLC after failure of, at least one, prior chemotherapy. Thus, in view of accreditation of this analysis, we have used the recommendation of the COFRAC (Comité français d'accréditation) and INCa (Institut national du cancer). Several parameters have been tested, such as the primers, the limit of detection, and the sensitivity and specificity of the method. In addition, a risk study has been evaluated. Although long and fastidious, the method of validation is required to perform analysis in optimal conditions to guaranty optimal results for the patients.

Survivin-3B potentiates immune escape in cancer but also inhibits the toxicity of cancer chemotherapy.

Dysregulation in patterns of alternative RNA splicing in cancer cells is emerging as a significant factor in cancer pathophysiology. In this study, we investigated the little known alternative splice isoform survivin-3B (S-3B) that is overexpressed in a tumor-specific manner. Ectopic overexpression of S-3B drove tumorigenesis by facilitating immune escape in a manner associated with resistance to immune cell toxicity. This resistance was mediated by interaction of S-3B with procaspase-8, inhibiting death-inducing signaling complex formation in response to Fas/Fas ligand interaction. We found that S-3B overexpression also mediated resistance to cancer chemotherapy, in this case through interactions with procaspase-6. S-3B binding to procaspase-6 inhibited its activation despite mitochondrial depolarization and caspase-3 activation. When combined with chemotherapy, S-3B targeting in vivo elicited a nearly eradication of tumors. Mechanistic investigations identified a previously unrecognized 7-amino acid region as responsible for the procancerous properties of survivin proteins. Taken together, our results defined S-3B as an important functional actor in tumor formation and treatment resistance.