In the present study, patients with TGF-β pathway mutation had higher incidence of MAC. This is in line with data from the TCGA where TGF-β pathway is more frequently altered in MAC histology compared to non-MAC histology (67.9% vs. 30.6%,
p < 0.001) [
18]. The TGF-β pathway is associated with cell proliferation, cell differentiation, apoptosis, and epithelial-mesenchymal transition (EMT) [
19,
20]. Although mutational inactivation of TGF- β pathway is frequently shown in colorectal cancer, the level of TGF-b production is somehow elevated in later stage tumor [
21,
22]. One of the reasons is that TGF-b level is not only affected by tumor but is also affected by stromal cells [
22,
23]. The correlation between TGF- β pathway mutation (which is usually loss-of function) and TGF-b level has not been clearly identified and maybe different among disease status. In terms of clinical relevance, there are reports showing that TGF-β pathway may have a prognostic role in CRC. CMS classifies colorectal cancer based on the gene expression profile [
2]. CMS4, which is characterized by the upregulation of genes implicated in EMT, prominent TGF-β pathway activation, stromal invasion, and angiogenesis, is associated with a worse prognosis. In addition, preclinical studies show that activation of TGF-β pathway may be linked to chemotherapy resistance in CRC cell line [
24,
25]. Recombinant TGF-β treatment caused chemotherapy resistance in CRC cell lines [
24]. However, TGF-β pathway mutation was not associated with RFS in CRC patients treated with adjuvant FOLFOX/XELOX chemotherapy, which we have previously reported [
13]. Discordance in the prognostic role of TGF-β pathway mutation among disease status could be partially explained by the fact that the response to TGF-β pathway activation is different according to cell type and status [
20,
26]. There are opposite effects of the TGF-β pathway in that it may suppress the growth of tubular adenoma but may promote the tumor growth of sessile serrated adenomas by inducing EMT and metastasis [
20,
26]. Apoptosis is the dominant feature after TGF-β pathway stimulation in classical tubular adenoma organoid culture [
26]. By contrast, inducing EMT is the main outcome after TGF-β pathway stimulation in an organoid culture model for sessile serrated adenomas [
26]. The activation of TGF-β may suppress the growth of tubular adenoma but may progress sessile serrated adenomas to CMS4 subtype cancer by inducing EMT [
26]. Moreover, evidence show that TGF-b level is also affected by stromal cells [
22]. While TGF-β pathway mutation is not associated with RFS in CRC patients treated with adjuvant FOLFOX/XELOX chemotherapy, it may be fatal in CRC patients who have recurred after curative surgery. Among the genes involved in the TGF-β pathway,
SMAD4 mutation was associated with poor SAR.
SMAD4 is a tumor suppressor gene and its mutation is involved in advanced stages, such as distant metastasis, in human colorectal carcinogenesis [
27]. In the present study, we predicted outcome of TGF-β pathway mutation using the PROVEAN, SIFT, and PolyPhen-2. Among 14 non-synonymous SNV in TGF-β pathway mutation 12(85.7%)-13(92.9%) were predicted to be damaging mutation. Other 11 mutations consisted of 5 stop gain mutation, 4 frame shift deletion mutation, and 2 non-frameshift deletion mutation which would probably lead to loss of expression. In sum, 23(92.0%)-24(96.0%) mutations were predicted to be damaging mutation in the TGF-β pathway. In addition, all SMAD4 mutations reported in the OncoKB database were loss-of-function mutation. This shows that TGF-β pathway mutation may have contributed to poor SAR due to loss of function. The correlation between TGF-β pathway mutation, TGF-b expression, and chemotherapy resistance needs to be studied in the future.
Sinicrope et al. reported that MSI-H is associated with improved SAR and that
KRAS mutation and
BRAF mutation are associated with poor SAR in stage III colon cancer patients treated with adjuvant chemotherapy [
12]. However, we could not evaluate the role of MSI-H and
BRAF mutation in SAR because there were only 3 patients with MSI-H and 5 patients with
BRAF V600E mutation. In contrast to a study by Sinicrope et al., RTK-RAS pathway mutation and
KRAS exon 2 mutation were not associated with SAR in the present study. In a study by Sinicrope et al., the negative prognostic role of
KRAS mutation was limited to patients who received adjuvant FOLFOX pus cetuximab but not in patients treated with adjuvant FOLFOX alone. In the present study, none of the patients received cetuximab in the adjuvant setting. This implicates that
KRAS mutation may not affect SAR in patients treated with adjuvant FOLFOX or XELOX alone.
The major limitation of this study was that we did not evaluate genetic characteristics other than targeted sequencing of genes associated with 5 critical pathways. Although major genes included in the TGF-β pathway were sequenced, other alterations, including copy number variation or the expression level, were not analyzed. It is also important to analyze the TGF-β pathway induced by stromal cells [
3,
23]. Therefore, comprehensive analysis of the TGF-β pathway could provide a better understanding of CRC biology in the future. Another limitation of the present study was that patients were treated differently after recurrence. However, this study was performed at a high-volume single center where the treatment plan was relatively uniform. The last limitation was the relatively small number of patients included in this cohort. However, this was the first study to evaluate the relationship between pathway mutation and SAR in CRC patients.