RESUMO
The standard for detecting chimeric genes of neurotrophic receptor tyrosine kinases (NTRK) is next generation sequencing (NGS). However, this analysis is expensive and takes several days. As a rapid screening method for the detection of NTRK3-dependent papillary thyroid cancer, an analysis of the expression imbalance between 5' and 3' NTRK3 mRNA fragments was used (5'/3' RT-PCR). The reference method for detection of NTRK3 rearrangements was fluorescent in situ hybridization (FISH), and the most frequent rearrangements in papillary thyroid cancer were tested using reverse transcription PCR (RT-PCR). Using 5'/3' RT-PCR, 18 samples of papillary thyroid cancer carrying chimeric transcripts of NTRK3 mRNA were detected. The sensitivity of the developed technique was 88.9% and specificity was 99.3%. Thus, a fast and cost-effective method of screening samples of papillary thyroid cancer in paraffin blocks is proposed with acceptable sensitivity and specificity.
Assuntos
Neoplasias da Glândula Tireoide , Humanos , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Hibridização in Situ Fluorescente , RNA Mensageiro/genética , Receptores Proteína Tirosina Quinases/genéticaRESUMO
Solid tumors resulting from oncogenic stimulation of neurotrophin receptors (TRK) by chimeric proteins are a group of rare tumors of various localization that respond to therapy with targeted drugs entrectinib and larotrectinib. The standard method for detecting chimeric TRK genes in tumor samples today is considered to be next generation sequencing with the determination of the prime structure of the chimeric transcripts. We hypothesized that expression of the chimeric tyrosine kinase proteins in tumors can determine the specific transcriptomic profile of tumor cells. We detected differentially expressed genes allowing distinguishing between TRK-dependent tumors papillary thyroid cancer (TC) from other molecular variants of tumors of this type. Using PCR with reverse transcription (RT-PCR), we identified 7 samples of papillary TC carrying a EVT6-NTRK3 rearrangement (7/215, 3.26%). Using machine learning and the data extracted from TCGA, we developed of a recognition function for predicting the presence of rearrangement in NTRK genes based on the expression of 10 key genes: AUTS2, DTNA, ERBB4, HDAC1, IGF1, KDR, NTRK1, PASK, PPP2R5B, and PRSS1. The recognition function was used to analyze the expression data of the above genes in 7 TRK-dependent and 10 TRK-independent thyroid tumors obtained by RT-PCR. On the test samples from TCGA, the sensitivity was 72.7%, the specificity - 99.6%. On our independent validation samples tested by RT-PCR, sensitivity was 100%, specificity - 70%. We proposed an mRNA profile of ten genes that can classify TC in relation to the presence of driver NTRK-chimeric TRK genes with acceptable sensitivity and specificity.
Assuntos
Proteínas Proto-Oncogênicas c-ets , Receptor trkC , Receptores de Fator de Crescimento Neural , Proteínas Repressoras , Neoplasias da Glândula Tireoide , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptor trkC/genética , Receptor trkC/metabolismo , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
The authors modified the method for silver staining of argyrophilic nucleolar organizer regions (AgNORs). This modification eliminates defects of an original technique. The method accelerates the staining and prevents fallout of deposits. The modification is recommended for wide application.
