A more sensitive platform for the detection of low-abundance BRAFV600E mutations

Abstract

Identifying low-abundance mutations is important for the therapy and diagnose of cancer. Since the potential for tumor heterogeneity, the efficient detection of cancer-relevant mutations largely depends on the sensitivity of the methods employed. To confirm whether the mutation detection platforms affect the perceived prevalence of the BRAF(V600E) and its correlation with clinicopathologic features in papillary thyroid carcinomas (PTC), we compared Sanger Sequencing (SS), Pyrosequencing (PS), and a newly built allele-specific real-time PCR (AS-qPCR) apparatus for the detection of BRAF(V600E) in a Chinese cohort of conventional variant PTC. Accurate plasmid standards were built to assess the limit of detection of the three platforms. In this research, AS-qPCR has been found both the most sensitive and reliable at detecting mutation. The mutations detected by AS-qPCR which were not detected by SS or PS due to low abundance were confirmed by mutation enrichment platform COLD-PCR followed by SS. When analyzed by AS-qPCR, BRAF(V600E) was associated with a more aggressive phenotype. Our results indicate that the reported prevalence of the BRAF(V600E) mutations in PTC has been underestimated and more sensitive methods such as AS-qPCR should be applied in clinical settings.

Clinical impact of BRAF mutation on the diagnosis and prognosis of papillary thyroid carcinoma: a systematic review and meta‐analysis

“…), repetitive publication, unreliable reference standard and confounding result data (the data could not be extracted for statistical analysis). At last, 67 (18 diagnostic studies and 49 prognostic studies) studies were considered eligible for inclusion . The study selection process was summarized in Fig.…”

“…A total of 46 studies (9704 patients) were eligible for analysing the relationship of BRAF V600E and the gender of patients with PTC . The pooled of RR was 1·11 (95% CI: 0·98–1·25, random‐effect model, Fig.…”

“…A total of 46 studies (9704 patients) were eligible for analysing the relationship of BRAF V600E and the gender of patients with PTC [10,14,15,33,36,37,[39][40][41][42][43][46][47][48][49][50][51][52][54][55][56][57][58][59][60][61][62][63][64][65][67][68][69][70][71][72][73][74][75][76][77][78][79][80][81][82]. The pooled of RR was 1Á11 (95% CI: 0Á98-1Á25, random-effect model, Fig.…”

“…In previous studies, extrathyroidal invasion, old age, large tumour size, male gender, multifocality, LN metastasis and advanced stage are identified as the main determinants of a poor outcome in PTC patients [86]. As far as the correlation between BRAF V600E mutation and these clinicopathological parameters, opposed results are reported [39][40][41][42][43][44][45][46][47][48][49][50][51][52][53]. Variations in the criteria and protocols used for data collection, variations in the completeness of the pathological description of the tumour in the patient records, variations in the extent of the disease at the time of the initial diagnosis, and variations in the diagnostic criteria used by different pathologists may all or partially exist among different studies.…”

The use of COLD‐PCR, DHPLC and GeneScanning for the highly sensitive detection of c‐KIT somatic mutations in canine mast cell tumours

“…Hence, it is likely that those studies may have underestimated the mutation rate. In human medicine, the advent of highly sensitive techniques in recent years has led to correctly reclassifying 6–20% of cases as mutated, and to reconsidering the typology, prevalence and biological implications in many cancer types . The likelihood that previous MCT canine studies could have missed many mutations has some relevant consequences; in fact, the actual prevalence of ‘driver’ somatic mutation in canine MCTs is likely higher than that reported, and many cases should be reclassified; the prognostic and predictive value of mutational status should also be redefined; finally and more interestingly, the association with TKI treatment and mutational status may be profoundly different than that ascertained so far …”