Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues

Chat-Uthai, Nunthawut; Vejvisithsakul, Pichpisith Pierre; Udommethaporn, Sutthirat; Meesiri, Puttarakun; Danthanawanit, Chetiya; Wongchai, Yannawan; Teerapakpinyo, Chinachote; Shuangshoti, Shanop; Poungvarin, Naravat

doi:10.1371/journal.pone.0198795

Cited by 7 publications

(6 citation statements)

References 61 publications

(63 reference statements)

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“…We used the reference standard DNA (Horizon Discovery, Cambridge, UK) containing 0% or 1% BRAF V600E mutant allele as negative and positive controls, respectively. This assay could detect the BRAF V600 mutation presenting in at least 1% within the background of wild-type DNA 17…”

Section: Methodsmentioning

confidence: 99%

<p>BRAF mutation in cytologically indeterminate thyroid nodules: after reclassification of a variant thyroid carcinoma</p>

Pongsapich¹,

Chongkolwatana²,

Poungvarin³

et al. 2019

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Purpose Fine-needle aspiration biopsy (FNAB) is regarded by the Bethesda system as the gold-standard investigation for stratifying the risk of malignancy of a thyroid nodule. However, some limitations affect the adequacy of the obtained materials, resulting in 30% of the cytological results remaining in the indeterminate category. We aimed to investigate the diagnostic value of the BRAF mutation in cytologically indeterminate thyroid nodules after the reclassification of a variant thyroid carcinoma. Patients and methods In this prospective diagnostic study, 76 patients with FNAB findings of atypia of undetermined significance (AUS) and suspicious for malignancy (SUS) were included. The BRAF V600 mutation from FNAB was confirmed by a PCR-based method (Sanger sequencing combined with allele-specific real-time PCR techniques) and immunohistochemistry (IHC). Pathological specimens and features, including noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), were reviewed and compared to the FNAB results. Results Using the PCR-based method, the BRAF mutation was positive in 13/76 cases (17.1%), with the diagnostic values of 16.7% sensitivity, 100% specificity, 100% positive predictive value (PPV), and 82.8% negative predictive value (NPV) in the AUS compared to 73.3% sensitivity, 100% specificity, 100% PPV, and 20% NPV in the SUS. For the IHC technique, only 20 of the 76 cytological specimens were qualified for testing. The BRAF mutation was positive in 13/20 cases, with the diagnostic values of 100% sensitivity, 63.6% specificity, 42.9% PPV, and 100% NPV in the AUS compared to 100% sensitivity and PPV in the SUS. The BRAF mutation was not found in the pathological reports for NIFTP. Conclusion The malignancy rate is high in our data, with specific and acceptable accuracy rates for the BRAF mutation from FNAB found by using the PCR-based method. NIFTP has been introduced after the pathological reclassification. Molecular diagnosis might be useful to establish the nature of the disease.

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Section: Methodsmentioning

confidence: 99%

<p>BRAF mutation in cytologically indeterminate thyroid nodules: after reclassification of a variant thyroid carcinoma</p>

Pongsapich¹,

Chongkolwatana²,

Poungvarin³

et al. 2019

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“…In our study, we used AmoyDx Mutation Detection Kit, a relatively simple real-time PCR assay, fast and less prone to external contamination [58]. It's considered to be one of the most sensitive methods available in clinical molecular laboratories [59].…”

Section: Discussionmentioning

confidence: 99%

Clinicopathological characteristics and mutational profile of KRAS and NRAS in Tunisian patients with sporadic colorectal cancer

et al. 2021

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Background/aim: Colorectal cancer (CRC) is a major public health problem worldwide and in Tunisia due to its increasing incidence. KRAS and NRAS mutations have become a pivotal part of CRC diagnosis, given their association to treatment resistance with anti-epidermal growth factor receptor (EGFR) monoclonal antibodies. We aim to screen for mutations in KRAS and NRAS genes in Tunisian patients with CRC and to explore their correlations with clinicopathological features. Materials and methods: AmoyDx KRAS and NRAS mutation real-time PCR kits were used to screen for mutations in KRAS (exon 2) and NRAS (exons 2, 3 and 4) in 96 CRC tumors. Results: KRAS exon 2 mutations were found in 41.7% (40/96) of the patients. Codon 12 most abundant mutations were G12D and G12V followed by G12A, while G13D is the predominant mutation in codon 13. KRAS exon 2 mutations were associated with older patients (p = 0.029), left-sided tumors (p = 0.037) and greater differentiation (p = 0.044). The prevalence rate of NRAS mutations was 7.3%, mostly in exon 2. These mutations were associated with early stages (p = 0.039) and the absence of lymph node metastasis (p = 0.045). Conclusion: It can be inferred from this study that Tunisian CRC patients have a similar frequency of KRAS and NRAS mutations compared to those observed in other populations. Consequently, screening for KRAS and NRAS mutation is crucial to orientate the therapies and the selection of an appropriate candidate, avoiding patients' unnecessary toxicity and costs.

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“…In particular, PCR product yields have been shown to decrease by 20-fold for A:A mismatches, whereas mismatches involving T have minimal effect on PCR product yield [35]. Therefore, the design of AS-PCR assays for detection of the BRAF V600E (1799 T > A) mutation, which involves A:A or T:T mismatches, is inherently challenging, restricting assay sensitivity to about 0.1% at best [12,13,21,[36][37][38][39]. In contrast, the ExBP-RT technique used in this study discriminates between wild type and mutant alleles during a single cycle of reverse transcription, completely eliminating the problem of decay of sensitivity during subsequent qPCR amplification [16].…”

Section: Discussionmentioning

confidence: 99%

Evaluation of the expression levels of BRAFV600E mRNA in primary tumors of thyroid cancer using an ultrasensitive mutation assay

et al. 2020

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Background: The BRAF V600E gene encodes for the mutant BRAF V600E protein, which triggers downstream oncogenic signaling in thyroid cancer. Since most currently available methods have focused on detecting BRAF V600E mutations in tumor DNA, there is limited information about the level of BRAF V600E mRNA in primary tumors of thyroid cancer, and the diagnostic relevance of these RNA mutations is not known. Methods: Sixty-two patients with thyroid cancer and non-malignant thyroid disease were included in the study. Armed with an ultrasensitive technique for mRNA-based mutation analysis based on a two step RT-qPCR method, we analysed the expression levels of the mutated BRAF V600E mRNA in formalin-fixed paraffin-embedded samples of thyroid tissues. Sanger sequencing for detection of BRAF V600E DNA was performed in parallel for comparison and normalization of BRAF V600E mRNA expression levels. Results: The mRNA-based mutation detection assay enables detection of the BRAF V600E mRNA transcripts in a 10, 000-fold excess of wildtype BRAF counterparts. While BRAF V600E mutations could be detected by Sanger sequencing in 13 out of 32 malignant thyroid cancer FFPE tissue samples, the mRNA-based assay detected mutations in additionally 5 cases, improving the detection rate from 40.6 to 56.3%. Furthermore, we observed a surprisingly large, 3-log variability, in the expression level of the BRAF V600E mRNA in FFPE samples of thyroid cancer tissue. Conclusions: The expression levels of BRAF V600E mRNA was characterized in the primary tumors of thyroid cancer using an ultrasensitive mRNA-based mutation assay. Our data inspires further studies on the prognostic and diagnostic relevance of the BRAF V600E mRNA levels as a molecular biomarker for the diagnosis and monitoring of various genetic and malignant diseases.

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Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues

Cited by 7 publications

References 61 publications

<p>BRAF mutation in cytologically indeterminate thyroid nodules: after reclassification of a variant thyroid carcinoma</p>

<p>BRAF mutation in cytologically indeterminate thyroid nodules: after reclassification of a variant thyroid carcinoma</p>

Clinicopathological characteristics and mutational profile of KRAS and NRAS in Tunisian patients with sporadic colorectal cancer

Evaluation of the expression levels of BRAFV600E mRNA in primary tumors of thyroid cancer using an ultrasensitive mutation assay

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