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Tumor stroma and tumor-infiltrating lymphocytes (TILs) are major constituents of the tumor microenvironment, although they have different effects on the prognosis of patients with colorectal cancer (CRC). Combinatory statuses of tumor-stromal percentage (TSP) and TILs are expected to provide more powerful prognostic information but have never been studied in CRCs.Stage III CRCs from patients (n = 487) treated with adjuvant chemotherapy were assessed for their TSP and CD3-TIL or CD8-TIL densities using computer-aided methodology. With cut-off values set at median values for intraepithelial TIL (iTIL) and stromal TIL (sTIL) densities, CRCs were sorted into low and high iTIL or sTIL groups. CRCs were classified into five quintile (Q1-Q5) groups according to their TSP and divided into high TSP (Q5) and low TSP (Q1-4) groups.The combination of CD8 iTIL density and TSP was found to be an independent prognostic parameter in multivariate survival analysis in terms of cancer-specific survival and recurrence-free survival. CRCs with low CD8 iTIL density and high TSP showed the worst survival. The combinatory status showed more prognostic power than CD8 iTIL density or TSP alone. Multivariate survival analysis in an independent cohort of stage III CRC validated the prognostic power of the combinatory statuses.The findings suggest that the combinatory status might serve as a prognostic parameter in stage III CRCs. Further research in a large-scale cohort of patients with stage III CRC is needed to validate the prognostic power of the combinatory status.

Several studies have reported that the prognostic effect of KRAS mutations on colorectal cancers (CRCs) varies depending on the type of mutation. Considering the effect of KRAS mutations on tumor microenvironment, we analyzed the prognostic significance of KRAS mutation types after adjusting for the tumor-infiltrating lymphocytes (TIL) and tumor-stromal percentage (TSP) statuses. In two independent cohorts, KRAS mutations were analyzed by Sanger sequencing and/or next-generation sequencing. TIL density and the TSP were quantified from whole-slide immunohistochemical images. KRAS-mutant CRCs were divided into three subgroups (G12D/V, other codon 12 mutations and codon 13 mutations) to examine their differential effect on TIL density, the TSP and recurrence-free survival (RFS). Among the KRAS mutations, only the G12D/V subgroups showed significantly less TIL infiltration than the wild-type CRCs. According to survival analysis, G12D/V mutations were associated with short RFS; codon 13 mutations showed discordant trends in the two cohorts, and other codon 12 mutations showed no significant association. Multivariate analysis further supported the prognostic value of G12D/V mutations. This result is not only consistent with a recent study suggesting the immunosuppressive effect of mutant KRAS but also provides insight into the type-specific prognostic effect of KRAS mutations.

Aims The precise profile of aberrant expression of thyroid transcription factor-1 (TTF-1) according to antibody clones in colorectal carcinomas (CRCs) has been controversial. Moreover, the detailed clinicopathological and molecular features of CRCs with TTF-1 expression have rarely been investigated. Here, we evaluated the TTF-1 expression status in a large series of CRC cases using three different antibody clones. Methods and Results Immunohistochemistry for TTF-1 using clones 8G7G3/1, SPT24, and SP141 was performed on tumour tissues of 1,319 primary CRCs and 98 corresponding metastatic lesions. Among the 1,319 CRCs, TTF-1 expression was detected in 68 cases using both clones SPT24 and SP141. TTF-1 expression was not detected in any of the cases when clone 8G7G3/1 was used. The 68 CRCs with TTF-1 expression detected using both the SPT24 and SP141 clones were considered TTF-1-positive in this study. TTF-1 positivity was significantly associated with distal tumour location, non-mucinous histology, intact CDX2 expression, and low frequency of KRAS mutations in CRCs. Nearly all TTF-1-positive CRCs showed microsatellite-stable and CpG island methylator phenotype-negative statuses. TTF-1 positivity was also found in all metastatic lesions of the five TTF-1-positive primary CRCs. TTF-1 negativity was maintained in all metastatic lesions of the 93 TTF-1-negative primary CRCs. Conclusions Our study confirmed that the frequency and characteristics of aberrant TTF-1 expression in CRCs vary depending on the antibody clone. Aberrant TTF-1 expression detected by clone SPT24 or SP141 may be encountered preferentially in distally located, conventional pathway-type CRCs. This article is protected by copyright. All rights reserved.

In the present study, we developed a computational method and panel markers to assess microsatellite instability (MSI) using a targeted next-generation sequencing (NGS) platform and compared the performance of our computational method, mSILICO, with that of mSINGS to detect MSI in CRCs. We evaluated 13 CRC cell lines, 84 fresh and 119 formalin-fixed CRC tissues (including 61 MSI-high CRCs and 155 microsatellite-stable CRCs) and tested the classification performance of the two methods on 23, 230, and 3,154 microsatellite markers. For the fresh tissue and cell line samples, mSILICO showed a sensitivity of 100% and a specificity of 100%, regardless of the number of panel markers, whereas for the formalin-fixed tissue samples, mSILICO exhibited a sensitivity of up to 100% and a specificity of up to 100% with three differently sized panels ranging from 23 to 3154. These results were similar to those of mSINGS. With the application of mSILICO, the small panel of 23 markers had a sensitivity of ≥95% and a specificity of 100% in cell lines/fresh tissues and formalin-fixed tissues of CRC. In conclusion, we developed a new computational method and microsatellite marker panels for the determination of MSI that does not require paired normal tissues. A small panel could be integrated into the targeted NGS panel for the concurrent analysis of single nucleotide variations and MSI.

The aim of this study is to establish heat shock protein 110 (HSP110) as a prognostic biomarker of colorectal carcinomas (CRCs) with microsatellite instability-high (MSI-H) by considering the intratumoral heterogeneity of HSP110 expression. We performed whole-section immunohistochemistry (IHC) for wild-type HSP110 (HSP110wt) in 164 MSI-H CRCs. The intensity of the HSP110wt expression in tumor cells was semiquantitatively scored (0/1/2/3), and the HSP110wt expression status of each tumor was classified as low or high using the following four scoring criteria: H-score, dominant intensity score, lowest intensity score, and highest intensity score. Among the four criteria, only the dominant intensity score-based dichotomous classification of HSP110wt expression was significantly associated with a difference in disease-free survival (log-rank p = 0.035) in 164 MSI-H CRCs. The HSP110wt-low MSI-H CRCs were significantly correlated with larger deletions in the HSP110 T17 mononucleotide repeat (≥4 bp; p

Acetyl-CoA synthetase-2 is an emerging key enzyme for cancer metabolism, which supplies acetyl-CoA for tumor cells by capturing acetate as a carbon source under stressed conditions. However, implications of acetyl-CoA synthetase-2 in colorectal carcinoma may differ from other malignancies, because normal colonocytes use short-chain fatty acids as an energy source, which are supplied by fermentation of the intestinal flora. Here we analyzed acetyl-CoA synthetase-2 mRNA expression by reverse-transcription quantitative PCR in paired normal mucosa and tumor tissues of 12 colorectal carcinomas, and subsequently evaluated acetyl-CoA synthetase-2 protein expression by immunohistochemistry in 157 premalignant colorectal lesions, including 60 conventional adenomas and 97 serrated polyps, 1,106 surgically resected primary colorectal carcinomas, and 23 metastatic colorectal carcinomas in the liver. In reverse-transcription quantitative PCR analysis, acetyl-CoA synthetase-2 mRNA expression was significantly decreased in tumor tissues compared with corresponding normal mucosa tissues. In acetyl-CoA synthetase-2 immunohistochemistry analysis, all 157 colorectal polyps showed moderate-to-strong expression of acetyl-CoA synthetase-2. However, cytoplasmic acetyl-CoA synthetase-2 expression was downregulated (acetyl-CoA synthetase-2 low expression) in 771 (69.7%) of 1,106 colorectal carcinomas and 21 (91.3%) of 23 metastatic lesions. The colorectal carcinomas with acetyl-CoA synthetase-2-low expression were significantly associated with advanced TNM stage, poor differentiation, and frequent tumor budding. Regarding the molecular aspect, acetyl-CoA synthetase-2-low expression exhibited a tendency of frequent KRT7 expression and decreased KRT20 and CDX2 expression. In survival analysis, acetyl-CoA synthetase-2-low expression was an independent prognostic factor for poor 5-year progression-free survival (hazard ratio, 1.39; 95% confidence interval, 1.08-1.79; P=0.01). In conclusion, these findings suggest that downregulation of acetyl-CoA synthetase-2 expression is a metabolic hallmark of tumor progression and aggressive behavior in colorectal carcinoma.

Background: The aim of this study was to reveal the clinicopathological and molecular characteristics of microsatellite instability-high (MSI-H) colorectal cancers (CRCs) showing programmed death ligand-1 (PD-L1) positivity, which are good candidates for anti-PD-1/PD-L1 immunotherapy. Methods: The PD-L1 expression status of 208 MSI-H CRCs was retrospectively analysed using immunohistochemistry. PD-L1 positivity in tumour cells (PD-L1+(T)) and PD-L1 positivity in immune cells (PD-L1+(I)) were separately evaluated. Results: Programmed death ligand-1 positivity in tumour cells and PD-L1+(I) were observed in 26 (12.5%) and 62 (29.8%) MSI-H CRCs, respectively, and occasionally overlapped (n=12; 5.8%). Programmed death ligand-1 positivity tumours in MSI-H CRCs were significantly associated with older age, female sex, non-mucinous-type poor differentiation, infiltrating growth, tumour budding, advanced stage, CpG island methylator phenotype-high, MLH1 promoter methylation, and BRAF V600E mutations. However, PD-L1+(I) MSI-H CRCs were characterised by high-density tumour-infiltrating immune cells, including T cells and macrophages, and intense peritumoural lymphoid reactions. In patients with stage IV MSI-H CRCs who had undergone metastatectomy (n=4), the PD-L1 status of primary tumours was maintained in corresponding distant metastatic lesions. Conclusions: In MSI-H CRCs, PD-L1+(T) and PD-L1+(I) are linked to a sporadic hypermethylated subtype and an immune cell-rich subtype, respectively. Potential differential therapeutic implications of PD-L1+(T) and PD-L1+(I) in CRCs should be further investigated.