Rapid targeted somatic mutation analysis of solid tumors in routine clinical diagnostics

Abstract

Tumor genotyping is an essential step in routine clinical practice and pathology laboratories face a major challenge in being able to provide rapid, sensitive and updated molecular tests.We developed a novel mass spectrometry multiplexed genotyping platform named PentaPanel to concurrently assess single nucleotide polymorphisms in 56 hotspots of the 5 most clinically relevant cancer genes, KRAS, NRAS, BRAF, EGFR and PIK3CA for a total of 221 detectable mutations. To both evaluate and validate the PentaPanel performance,we investigated 1025 tumor specimens of 6 different cancer types (carcinomas of colon, lung, breast, pancreas, and biliary tract, and melanomas), systematically addressing sensitivity, specificity, and reproducibility of our platform. Sanger sequencing was also performed for all the study samples.Our data showed that PentaPanel is a high throughput and robust tool, allowing genotyping for targeted therapy selection of 10 patients in the same run, with a practical turnaround time of 2 working days. Importantly, it was successfully used to interrogate different DNAs isolated from routinely processed specimens (formalin-fixed paraffin embedded, frozen, and cytological samples), covering all the requirements of clinical tests.In conclusion, the PentaPanel platform can provide an immediate, accurate and cost effective multiplex approach for clinically relevant gene mutation analysis in many solid tumors and its utility across many diseases can be particularly relevant in multiple clinical trials, including the new basket trial approach, aiming to identify appropriate targeted drug combination strategies.

Molecular profiling of lung cancer specimens and liquid biopsies using MALDI-TOF mass spectrometry

“…We observed a relationship between the frequency of a mutated allele in the sample and the amount of DNA required for detection. This is partially in contrast to the findings of Magliacane et al (2015), who reported that mutations could be detected even using very small amounts of DNA (1 ng per reaction) [28]. In particular, we observed that, in the presence of highly represented mutations (about 20%), only a very small amount of DNA (about 5 ng) was sufficient for the detection.…”

A Novel Functional Splice Variant of AKT3 Defined by Analysis of Alternative Splice Expression in HPV-Positive Oropharyngeal Cancers

“…(33) New mutational analyses have led to understanding cancer mutations that occur across organ systems to allow for the design of specific basket trials, where therapies can be rationally targeted towards specific mutations. (34) Alternative splicing is another mechanism by which alternative isoforms may have significant impact on gene expression and function, independent of genetic mutations. (8) In this study, a novel algorithm is utilized to systematically identify alternative splicing events unique to tumors to show that alternative splicing can represent an important functional mechanism of oncogenesis in HPV-related oropharyngeal cancer that has not been previously appreciated.…”

Identification of skin-related lncRNAs as potential biomarkers that are involved in Wnt pathways in keloids

“…Therefore, we assume that it might be a LINC00312-dependent network which provided subtle yet decisive regulation of cell proliferation and transformation in keloids, which might also be found in other highly recurrent benign tumours. The third most up-expressed lncRNA was HOXA11-AS, which was also documented to increase the progression of glioma [27, 28] and human uterine cervix carcinoma [29, 30]. Also, the functional variant of HOXA11-AS has been revealed to inhibit the oncogenic phenotype of epithelial ovarian cancer [30].…”

“…The third most up-expressed lncRNA was HOXA11-AS, which was also documented to increase the progression of glioma [27, 28] and human uterine cervix carcinoma [29, 30]. Also, the functional variant of HOXA11-AS has been revealed to inhibit the oncogenic phenotype of epithelial ovarian cancer [30]. To the best of our knowledge, keloid is the only benign tumour in which HOXA11-AS has been documented aberrantly changed.…”