Dados do Trabalho

Título

Comparative analysis of mutational repertoire and circulating tumor DNA in metastatic right- and left-sided colorectal cancer

Introdução

Colorectal cancer (CRC) is a cancer with high mortality rate and more than 45% of CRC patients are metastatic (mCRC). Of these, little is known about the molecular mechanisms of upfront or acquired resistance to chemo and anti-EGFR therapies. Right-sided (RS) and left-sided (LS) CRC are different in carcinogenesis and a recent study demonstrated that progression also differs according to the laterality. Additionally, whether the ability of tumor cells to shed cell-free DNA (cfDNA) in the circulation differs in RS and LS CRC is not yet known.

Objetivo

Thus, this work aims to compare mutational repertoire and the ability of tumor cells to shed DNA in circulation of LS and RS colon/rectum tumors using Next Generation Sequencing (NGS)-based approaches.

Métodos

We evaluated the Genomic profiling (GP) of formalin-fixed and paraffin-embedded (FFPE) tumor tissues using Oncomine Comprehensive Assay Plus (ThermoFisher) panel. NGS was performed using Ion Proton platform (ThermoFisher). Variants were annotated and filtered using Ion Reporter (IR, ThermoFisher) and VarSeq (Golden Helix) software, with specific filters such as variant allele frequency (VAF, >1%), base coverage (≥500X) and visual inspection with Integrative Genome Viewer (IGV) tool. Tumor mutational burden (TMB, Mutations/Mb) was extract from IR and was considered TMB-high ≥10, and TMB-low <10. Somatic variants were validated through targeted-amplicon sequencing in tumor DNA to select tumor single nucleotide variants (SNVs) that were screened on cfDNA collected before any treatment at a personalized manner. Circulating tumor DNA (ctDNA) analysis was performed using Deep amplicon sequencing and positivity was considered with a base coverage ≥5,000X and VAF ≥0.5. Controls were used to classify in low and high confidence levels. Ethical approval: 2496/18.

Resultados

In this preliminary data, GP were obtained from 43 tumors, in which 93% (40/43) we detected at least one somatic SNV, with a validation rate of 94.1% (48/51). Of those, 39 cases had tumor laterality information, in which we found 122 SNVs, ranging from 1 to 7 variants per patient. Considering the LS CRC, 82 variants were detected in 24 cases, and in RS CRC, 40 were detected in 15 cases. Regarding TMB, only two mCRC presented TMB-high and both were left-sided colorectal. The most frequently mutated genes in the LS CRC group were TP53 (79.1%, 19/24), APC (58.3%, 14/24) and KRAS/NRAS/BRAF (50%, 12/24) and in the RS CRC were KRAS/NRAS/BRAF (86.6%, 13/15), APC (40%, 6/15), and TP53 (33.3%, 5/15). Regarding less frequent genes, in the LS CRC group we observed acquired mutation in FBXW7 (20.8%, 5/24), PIK3CA and SMAD4 (both 8.3%, 2/24), GATA2 and ERBB2 (both 4.1%, 1/24) and in RS CRC group in PIK3CA, NF1, EGFR, TSC2 (each one in 6.6%, 1/15). Regarding ctDNA-positive in baseline plasma samples, no significant difference was observed in LS and RS CRC (69.5% [16/23] and 78.5%, [11/14], respectively).

Conclusões

These results showed differences in the mutational repertoire between laterality in mCRC, in agreement with the literature. We observed a higher frequency in LS (79.1%) than in RS (33.3%) CRC of TP53 acquired mutations. In contrast, a higher frequency in RS (86.6%) than in LS (50%) of oncogenic mutations in KRAS/NRAS/BRAF was observed. Additional analyses are ongoing.

Palavras-chave

Metastatic colorectal cancer; Circulating tumor DNA; Right- and left-sided colorectal

Financiador do resumo

Brazilian Ministry of Health/PRONON