Here we provide the first strategy to use a competitive Extendable Blocking Probe (ExBP) for allele-specific priming with superior selectivity at the stage of reverse transcription. In order to analyze highly similar RNA variants, a reverse-transcriptase primer whose sequence matches a specific variant selectively primes only that variant, whereas mismatch priming to the alternative variant is suppressed by virtue of hybridization and subsequent extension of the perfectly matched ExBP on that alternative variant template to form a cDNA–RNA hybrid. This hybrid will render the alternative RNA template unavailable for mismatch priming initiated by the specific primer in a hot-start protocol of reverse transcription when the temperature decreases to a level where such mismatch priming could occur. The ExBP-based reverse transcription assay detected BRAF and KRAS mutations in at least 1000-fold excess of wild-type RNA and detection was linear over a 4-log dynamic range. This novel strategy not only reveals the presence or absence of rare mutations with an exceptionally high selectivity, but also provides a convenient tool for accurate determination of RNA variants in different settings, such as quantification of allele-specific expression.

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.

The mitogen‐activated protein kinase (MAPK) pathway plays a central role in colorectal cancers (CRC). In particular, BRAF V600E‐mutant tumors, which represent around 10% of CRCs, are refractory to current therapies. Overexpression and secretion of serine peptidase inhibitor Kazal type 1 (SPINK1) are observed in around 50% of CRCs, and its serum level can be used as a biomarker for poor prognosis. Utilizing a recently developed extendable blocking probe assay, we analyzed the BRAF mutation status in a CRC patient cohort (N = 571) using tissue‐derived RNA as the starting material. From the same RNA samples, we measured the relative SPINK1 expression levels using a quantitative real‐time PCR method. Expression of mutant BRAF V600E correlated with poor prognosis, as did low expression of SPINK1 mRNA. Further, BRAF V600E correlated negatively with SPINK1 levels. In order to investigate the effect of MAPK pathway‐targeted therapies on SPINK1 secretion, we conducted in vitro studies using both wild‐type and V600E CRC cell lines. BRAF inhibitor vemurafenib, and subsequent MAPK pathway inhibitors trametinib and SCH772984, significantly increased SPINK1 secretion in V600E CRC cell lines Colo205 and HT‐29 with a concomitant decrease in trypsin‐1 and ‐2 secretion. Notably, no SPINK1 increase or trypsin‐1 decrease was observed in BRAF wild‐type CRC cell line Caco‐2 in response to MAPK pathway inhibitors. In further mechanistic studies, we observed that only trametinib was able to diminish completely both MEK and ERK phosphorylation in the V600E CRC cells. Furthermore, the key regulator of integrated stress response, activating transcription factor 4 (ATF‐4), was downregulated both at mRNA and at protein level in response to trametinib treatment. In conclusion, these data suggest that sustained inhibition of not only MAPK pathway activation, but also ATF‐4 and trypsin, might be beneficial in the therapy of BRAF V600E‐mutant CRC and that SPINK1 levels may serve as an indicator of therapy response.

Despite the presence of histological findings indicating long-standing gastroesophageal reflux in 25%, as well as symptomatic gastroesophageal reflux in more than 40%, there was no detectable tissue expression of KRAS or BRAF mutations in this cohort of patients.