RESUMO
Mutation rates determined by allele-specific PCR can be highly different in KRAS exon 2 mutant colorectal carcinoma (CRC) samples suggesting intratumoural heterogeneity. To address the effect of KRAS gene copy number on the relative mutant allele frequency the KRAS locus was individually quantified following FISH analysis in 36 cases. We observed, that mutant KRAS status was associated with an elevated KRAS locus number (2.36 ± 0.42 vs 2.63 ± 0.75; p = 0.037) reflecting an increased aneuploidy status but no true amplification of the locus. In parallel, KRAS locus copy numbers showed significant intercellular variability (1-16 copies/cell nucleus) within individual tumours also indicating to a dynamic intratumoural oscillation of the mutant allele copy number. In conclusion, aneusomy is a common feature of KRAS mutant CRC and KRAS copy number variations may have an impact on the relative mutant allele frequency detected by allele specific PCR/sequencing), potentially leading to subclonal diversity and influencing tumour behaviour.
Assuntos
Variações do Número de Cópias de DNA/genética , Dosagem de Genes/genética , Mutação/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Alelos , Neoplasias Colorretais/genética , Éxons/genética , Feminino , Frequência do Gene/genética , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Colorectal cancer (CRC) is a heterogeneous disease presenting with a wide spectrum of morphological and molecular characteristics sometimes even within the same patient. To understand the mechanisms of oscillations in the KRAS status we evaluated the collective of CRC patients tested using allele-specific PCR and Sanger-sequencing. Mutant KRAS allele was observed in 43.3% of cases. Repeated analysis of KRAS status in recurrent tumors or metastases was performed in 18/665 cases and a total of 6 cases with different KRAS status was found. In three cases the histological pattern of the tumor was identical. In one patient different histology and molecular status was seen between the primary and the recurrent tumor samples. In two further cases localization, histological type and KRAS mutational status all supported the occurrence of synchron/metachron colorectal tumors. In conclusion, both the progression of the original disease but also multiple tumor formation may contribute to mutation status differences during the course of colorectal carcinoma.
Assuntos
Adenocarcinoma/genética , Códon/genética , Neoplasias Colorretais/genética , Éxons/genética , Mutação/genética , Recidiva Local de Neoplasia/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , DNA de Neoplasias/genética , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Proteínas Proto-Oncogênicas p21(ras) , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
MicroRNAs are involved in the regulation of various pathophysiological processes such as immune regulation and cancer. Next-generation sequencing methods enable us to monitor their presence in various types of samples but we need flexible methods for validating datasets generated by high-throughput methods. Here we describe the detailed protocols to be used with our MiRNA Primer Design Tool assay design system. The presented methods allow the flexible design of the oligonucleotides needed for the RT-qPCR detection of any variant of small regulatory RNA molecules from virtually any species. This method can be used to measure miRNA levels from formalin-fixed, paraffin-embedded (FFPE) samples and various body fluids. As an example, we show the results of the hsa-miR-515-3p, hsa-miR-325, and hsa-miR-155 quantification using a specific UPL probe (Universal Probe Library) and a stem-loop RT-qPCR assay. The small nucleolar RNA RNU43 is used as endogenous control for normalization of the results. Urine from healthy pregnant women and FFPE samples from patients diagnosed with colorectal cancer and treated with antibody-based anti-EGFR monotherapy were used as samples.
