The pathological diagnosis of the specimens was confirmed by hematoxylin and eosin (HE) staining. To be considered as a qualified sample, the specimen needed to be ≥1 mm and the percentage of tumor cells needed to be over 20%. Subsequently, 50–200 ng of DNA extracted from the samples were broken into ~200 bp fragments and then subjected to NGS platform Illumina (San Diego, CA, USA) Nextseq 500 to >500× coverage (24 (link)).
Tumor mutation burden (TMB) was defined as somatic mutation counts in coding region per megabase of genome examined. Genes including TGFBR2, ACVR1B, ACVR2A, INHBA, SMAD2, SMAD3, SMAD4, IGF2, RUNX1, STAT3, TERT, and VEGFA have been reported to be the core members in the TGF-β pathway network and to act as cancer-related genes (21 (link)); they were thus selected to be included for analysis. A TGF-β pathway mutation was defined as the presence of at least one pathway gene with identified mutational types (missense mutation or truncating mutation), including nonsense mutation, nonstop mutation, frameshift insertion, frameshift deletion, and splice site mutation.