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.
Highlights
The presence of mutations in RAS family oncogenes is associated with a lack of response to targeted therapy: lung and colorectal carcinomas characterized by KRAS mutations and KRAS and NRAS mutations, respectively are unresponsive to treatment with anti-EGFR agents [6, 11]
In particular for 24 sites of EGFR, KRAS, NRAS, and BRAF, which are mutated at high frequency as reported by the online Catalogue Of Somatic Mutations in Cancer (COSMIC), we included both duplicate amplifications, in separate wells, and bidirectional single base pair extensions (FIG. 1)
The total 80 assays, variously combined in 8 wells depending on their extension product masses, can simultaneously detect the presence/absence of 221 mutations of KRAS, NRAS, BRAF, EGFR and PIK3CA (FIG. 2)
Summary
The introduction of personalized therapy transformed the care of selected cancer patients: detection of critical cancer gene somatic mutations in clinical tumor samples better defines patient diagnosis, prognosis and more importantly indicates highly efficient targeted therapies with both health and economic benefits [1].According to the approved antineoplastic targeted drugs, the mutational status of EGFR, KRAS, NRAS, BRAF, PIK3CA genes is routinely requested by the oncologist for the clinical management of patients with non-small cell lung carcinomas (NSCLC) [2,3,4], colorectal carcinomas (CRC) [5, 6], melanomas [7], breast carcinomas [8, 9]. Formalin induces chemical cross-links to proteins, RNA, and DNA molecules, with concomitant fragmentation of DNA [12, 13] and random nucleotide base changes, which can lead to false-positive results [14]. For these reasons, clinical mutation detection PCR-based assays should include two independent amplifications of DNA extracted from FFPE samples in order to ensure accurate results, as suggested by molecular testing guidelines [15, 16]
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