Browsing by Subject "clinical Chemistry"

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  • Dang, Kien (Helsingin yliopisto, 2021)
    The aim of this doctoral thesis was to utilize molecular biology techniques to detect expressed KRAS and BRAF mutations in samples at high sensitivity and specificity and evaluate the clinical role of the RNA expression of these mutations in certain MAPK/ERK-driven cancers. In the early phase of the study, we developed a novel method named extendable blocking probe reverse transcription (ExBP-RT) for detecting expressed mutations at the mRNA level. With this method we were able to detect mutations expressed in mRNA with a very high sensitivity and specificity. The ExBP-RT assay was optimized to detect expressed BRAF and KRAS mutations in a 1000-fold – 6000-fold excess of wild-type mRNA. A further improvement of the method allowed detection of expressed BRAF mutations, in thyroid cancer (TC) tissue, in a 10000-fold excess of wildtype mRNA. This novel strategy not only reveals the presence or absence of low-abundance mutations with an exceptionally high selectivity, but also provides a convenient tool for accurate determination of the mRNA levels of the mutated genes in different settings, such as quantification of allele-specific expression. We used the ExBP-RT technique to measure the mRNA levels of all seven KRAS mutations at codon 12 and 13 in primary tumour tissue samples of 571 patients with colorectal cancer (CRC). Survival data was analysed to determine the prognostic potential of this novel mRNA-based biomarker. A high level of mutated KRAS mRNA was associated with an inferior 5-year diseasespecific survival (DSS). This association was highly significant, but only in left-sided CRC (P < 0.001). Thus, the mRNA level of the mutated KRAS allele in primary tumour tissue was found to be highly prognostic in left-sided CRC, but not in right-sided disease. To study the RNA expression of BRAF mutations as a diagnostic marker in thyroid cancer (TC), we analysed 62 formalin fixed paraffin embedded (FFPE) samples from TC patients. We detected BRAF V600E mutations at the mRNA level in 56,3% (18/32) and on the DNA level in 40,6% (13/32) of thyroid cancer patients, which is in concordance with the reported prevalence of these mutations. In order to evaluate whether sensitive detection of KRAS and BRAF mutations in mRNA could serve as a biomarker for early detection of malignant transformation in patients at risk of developing colorectal cancer, we analysed esophageal atresia (EA) patient cohorts. The ExBP-RT technique was used to evaluate tissue expression of KRAS and BRAF mutations in endoscopic biopsies from 61 adults, who had been surgically treated for EA in infancy. Despite the presence of histological findings indicating an increased risk of developing cancer, we found no detectable tissue expression of KRAS or BRAF mutations in this cohort. In conclusion, we successfully established a novel technique – ExBP-RT to detect KRAS and BRAF mutations at the mRNA level with very high sensitivity and selectivity. We found a strong association between the tumour tissue expression of KRAS mutations and prognosis in left-sided, stage III CRC. We also successfully detected and quantified the level of BRAF V600E mRNA in FFPE tissue from thyroid cancer. We could not, however, detect neither KRAS nor BRAF mutations in either of the cohorts of EA patients representing potential pre-malignant conditions.