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The V600E mutation in the BRAF oncogene is associated with colorectal carcinomas, with mismatch-repair deficiency and, recently, with nonresponse to epidermal growth factor receptor inhibitor therapy. The use of reliable techniques for its detection is important. The aim of our study was to compare the performance characteristics in V600E detection of denaturing high-performance liquid chromatography (dHPLC) and high-resolution melting (HRM) with TaqMan allelic discrimination as well as direct-sequencing methods in a series of 195 colorectal paraffinembedded specimens up to the age of 15 years. The effectiveness for obtaining results on mutation status was best using TaqMan (96.9%), followed by dHPLC (93.3%), HRM (88.7%), and sequencing (88.2%). In general, TaqMan was best for analyzing older tissues, whereas sequencing was the least efficient. Heterozygotic V600E was detected in 11.6%, 9.9%, 11.6%, and 9.9% of tissues using TaqMan, dHPLC, HRM, and sequencing, respectively. Result concordances between dHPLC and TaqMan or sequencing were excellent ( ‫؍‬ 0.9411 and ‫؍‬ 0.8988, respectively); for HRM, the concordances were good ( ‫؍‬ 0.7973 and ‫؍‬ 0.7488, respectively). By using DNA dilutions from tumor tissue, a minimum of 10% of V600E harboring cancer content was required for the analysis by dHPLC and HRM. dHPLC could detect four non-V600E mutations, whereas HRM detected one. Our results indicate that dHPLC and HRM are techniques that can be reliably used for the detection of the BRAFV600E mutation in Recent advances in DNA sequencing technology have made it possible to use high-throughput sequencing on the entire human colorectal cancer (CRC) spectrum, finding 848 genes that may be somatically mutated. 1,2 The subsequent study of these mutations in CRC specimens worldwide would be facilitated by the use of less expensive and more efficient screening methods for mutation detection. Because most of these mutations are found in heterozygosis, screening techniques based on heteroduplex detection, such as denaturing high-performance liquid chromatography (dHPLC) or high-resolution melting (HRM) analysis, are strongly recommended. The V600E mutation in the BRAF oncogene has been found in CRCs with mismatch-repair gene deficiency. Familial CRCs with mismatch-repair gene deficiency do not harbor this mutation; rather, V600E is associated with the sporadic form of CRC associated with mismatch-repair gene deficiency. In addition to the diagnostic value of the BRAF mutation assessment, recent evidence 3,4 has demonstrated that stage IV CRCs with the BRAF V600E mutation do not respond to epidermal growth factor receptor inhibitor therapy, thus extending its value to predictive grounds. In previous articles, 5 V600E detection using TaqMan chemistry (Applied Biosystem, Foster City, CA) saved cost, time, and manual labor over direct sequencing, with the former showing 100% sensitivity and 100% specificity when the latter is considered as the reference method. Also, HRM has proved to be a reliable technique for V600E detection...

Background: Several clinical trials have demonstrated the efficacy and safety of osimertinib in advanced nonsmall-cell lung cancer (NSCLC). However, there is significant unexplained variability in treatment outcome.Methods: Observational prospective cohort of 22 pre-treated patients with stage IV NSCLC harboring the epidermal growth factor receptor (EGFR) p.T790M resistance mutation and who were treated with osimertinib. Three hundred and twenty-six serial plasma samples were collected and analyzed by digital PCR (dPCR) and next-generation sequencing (NGS). Results:The median progression-free survival (PFS), since the start of osimertinib, was 8.9 [interquartile range (IQR): 4.6-18.0] months. The median treatment durations of sequential gefitinib + osimertinib, afatinib + osimertinib and erlotinib + osimertinib treatments were 30.1, 24.6 and 21.1 months, respectively. The p.T790M mutation was detected in 19 (86%) pre-treatment blood samples. Undetectable levels of the original EGFR-sensitizing mutation after 3 months of treatment were associated with superior PFS (HR: 0.2, 95% CI: 0.05-0.7). Likewise, re-emergence of the original EGFR mutation, alone or together with the p.T790M mutation was significantly associated with shorter PFS (HR: 8.8, 95% CI: 1.1-70.7 and HR: 5.9, 95% CI: 1.2-27.9, respectively). Blood-based monitoring revealed three molecular patterns upon progression to osimertinib: sensitizing+/T790M+/C797S+, sensitizing+/T790M+/C797S-, and sensitizing+/T790M-/ C797S-. Median time to progression in patients showing the triplet pattern (sensitizing+/T790M+/C797S+) was 12.27 months compared with 4.87 months in patients in whom only the original EGFR sensitizing was detected, and 2.17 months in patients showing the duplet pattern (sensitizing+/T790M+). Finally, we found that mutations in exon 545 of the PIK3CA gene were the most frequent alteration detected upon disease progression in patients without acquired EGFR-resistance mutations.Conclusions: Different molecular patterns identified by plasma genotyping may be of prognostic significance, suggesting that the use of liquid biopsy is a valuable approach for tumor monitoring.

BackgroundAltered methylation patterns are driving forces in colorectal carcinogenesis. The serrated adenocarcinoma (SAC) and sporadic colorectal carcinoma showing histological and molecular features of microsatellite instability (hmMSI-H) are two endpoints of the so-called serrated pathological route sharing some characteristics but displaying a totally different immune response and clinical outcome. However, there are no studies comparing the methylome of these two subtypes of colorectal carcinomas. The methylation status of 450,000 CpG sites using the Infinium Human Methylation 450 BeadChip array was investigated in 48 colorectal specimens, including 39 SACs and 9 matched hmMSI-H.ResultsMicroarray data comparing SAC and hmMSI-H showed an enrichment in functions related to morphogenesis, neurogenesis, cytoskeleton, metabolism, vesicle transport and immune response and also significant differential methylation of 1540 genes, including CD14 and HLA-DOA which were more methylated in hmMSI-H than in SAC and subsequently validated at the CpG, mRNA and protein level using pyrosequencing, quantitative polymerase chain reaction (qPCR) and immunohistochemistry.ConclusionsThese results demonstrate particular epigenetic regulation patterns in SAC which may help to define key molecules responsible for the characteristic weak immune response of SAC and identify potential targets for treating SAC, which lacks molecular targeted therapy.Electronic supplementary materialThe online version of this article (10.1186/s13148-018-0571-3) contains supplementary material, which is available to authorized users.