Immune microenvironmental heterogeneity according to tumor DNA methylation phenotypes in microsatellite instability-high colorectal cancers.

The detailed association between tumor DNA methylation, including CpG island methylation, and tumor immunity is poorly understood. CpG island methylator phenotype (CIMP) is observed typically in sporadic colorectal cancers (CRCs) with microsatellite instability-high (MSI-H). Here, we investigated the differential features of the tumor immune microenvironment according to CIMP status in MSI-H CRCs. CIMP-high (CIMP-H) or CIMP-low/negative (CIMP-L/0) status was determined using MethyLight assay in 133 MSI-H CRCs. All MSI-H CRCs were subjected to digital pathology-based quantification of CD3 + /CD8 + /CD4 + /FoxP3 + /CD68 + /CD204 + /CD177 + tumor-infiltrating immune cells using whole-slide immunohistochemistry. Programmed death-ligand 1 (PD-L1) immunohistochemistry was evaluated using the tumor proportion score (TPS) and combined positive score (CPS). Representative cases were analyzed using whole-exome and RNA-sequencing. In 133 MSI-H CRCs, significantly higher densities of CD8 + tumor-infiltrating lymphocytes (TILs) were observed in CIMP-H tumors compared with CIMP-L/0 tumors. PD-L1 TPS and CPS in CIMP-H tumors were higher than in CIMP-L/0 tumors. Next-generation sequencing revealed that, compared with CIMP-L/0 tumors, CIMP-H tumors had higher fractions of CD8 + T cells/cytotoxic lymphocytes, higher cytolytic activity scores, and activated immune-mediated cell killing pathways. In contrast to CIMP-L/0 tumors, most CIMP-H tumors were identified as consensus molecular subtype 1, an immunogenic transcriptomic subtype of CRC. However, there were no differences in tumor mutational burden (TMB) between CIMP-H and CIMP-L/0 tumors in MSI-H CRCs. In conclusion, CIMP-H is associated with abundant cytotoxic CD8 + TILs and PD-L1 overexpression independent of TMB in MSI-H CRCs, suggesting that CIMP-H tumors represent a typical immune-hot subtype and are optimal candidates for immunotherapy in MSI-H tumors.

Clinicopathological and molecular features of genome-stable colorectal cancers.

Colorectal cancers (CRCs) are traditionally divided into those with either chromosomal instability (CIN) or microsatellite instability (MSI). By utilizing TCGA data, the Laird team found a subset of CRCs, namely, genome-stable CRCs (GS CRCs), which lack both CIN and MSI. Although the molecular features of GS CRCs have been described in detail, the clinicopathological features are not well defined. A total of 437 CRCs were analyzed for copy number variation (CNV) statuses in eight genes (ARID1A, EGFR, FGFR1, KDM5B, MYBL2, MYC, SALL4, and SETDB1) using droplet-digital PCR. CRCs that showed CNV in ≤ one gene and no MSI were defined as GS-like CRCs. Clinicopathological and molecular features of GS-like CRCs were compared with those of CIN-like CRCs. GS-like CRCs comprised 4.6% of CRCs and showed a predilection toward the proximal colon, lower nuclear optical density, KRAS mutation, PIK3CA mutation, and aberrant expression of KRT7. Survival analysis showed no significant difference between the three subgroups. Through our study, the GS-like subtype was found to comprise a minor proportion of CRCs and have proclivity toward a proximal bowel location, hypochromatic tumor nuclei, aberrant KRT7 expression, and a high frequency of KRAS and PIK3CA mutations.

Concomitant expression patterns of CDX2 and SATB2 as prognostic factors in stage III colorectal cancers.

CDX2 and SATB2 are often used as biomarkers for identification of colorectal origin in primary or metastatic adenocarcinomas. Loss of CDX2 or SATB2 expression has been associated with poor prognosis in patients with colorectal cancer (CRC). However, little is known regarding clinicopathological features, including prognosis, of CRCs with concomitant loss of CDX2 and SATB2. A total of 431 stage III CRCs were analyzed for their expression status in CDX2 and SATB2 using tissue microarray-based immunohistochemistry and expression status was correlated with clinicopathological variables, molecular alterations, and survival. CDX2-negative (CDX2-) CRCs and SATB2-negative (SATB2-) CRCs were found in 8.1 % and 17.2 % of CRCs, respectively, whereas both CDX2-negative and SATB2-negative (CDX2-/SATB2-) CRCs comprised 3.2 % of the CRCs. On survival analysis, neither CDX2-/SATB2+ nor CDX2+/SABT2- CRCs but CDX2-/SATB2- CRCs were associated with poor prognosis. CDX2-/SATB2- CRCs showed significant associations with tumor subsite of right colon, poor differentiation, decreased expression of CK20, aberrant expression of CK7, CIMP-high, MSI-high, and BRAF mutation. In summary, our results suggest that concomitant loss of CDX2 and SATB2 is a prognostic biomarker but isolated loss of CDX2 or SATB2 is not a prognostic biomarker for stage III CRCs.

Prognostic significance of STING expression in solid tumor: a systematic review and meta-analysis.

Objective: Stimulator of interferon genes (STING) is a key regulator in initiating innate immune response from sensing cytosolic DNA. Recent studies have revealed that the cGAS-STING signaling pathway has a crucial role in tumor development and progression across cancer types. Herein, we conducted a meta-analysis to explore the relationship between the immunoexpression of STING and the survival outcome of patients in various solid tumors. Studies relevant to the subject were searched from PubMed, Embase, and Web of Science. Results: Eleven studies including 2,345 patients were eligible for the analysis. STING expression in tumor cells was related to improved disease-free survival/recurrence-free survival (DFS/RFS) (HR = 0.656, 95% CI = 0.455-0.946, p = 0.024) but not with overall survival (OS) (HR = 0.779, 95% CI = 0.534-1.136, p = 0.194). STING expression in stromal cells, however, did not show significant correlation with DFS/RFS and OS (HR = 0.979, 95% CI = 0.565-1.697, p-value = 0.940 and HR = 1.295, 95% CI = 0.845-1.985, p = 0.235, respectively). In a subgroup analysis, STING expression in tumor cells was associated with better DFS (HR = 0.622, 95% CI = 0.428-0.903, p = 0.012). In tumor cells, favorable DFS/RFS were also related to studies from univariate analysis and the gastrointestinal system (HR = 0.667, 95% CI = 0.482-0.923, p = 0.015 and HR = 0.566, 95% CI = 0.330-0.971, p = 0.039). Conclusions: STING expression in tumor cells is associated with favorable outcome in solid tumors. Systematic review registration: https://www.crd.york.ac.uk/prospero/, registration number: CRD42023427027.

Prognostic and clinicopathological significance of Fusobacterium nucleatum in colorectal cancer: a systemic review and meta-analysis.

BACKGROUND: Fusobacterium nucleatum has been identified to promote tumor progression in colorectal cancer (CRC). However, association between F. nucleatum and prognostic or clinicopathological features has been diverse among studies, which could be affected by type of biospecimen (formalin-fixed paraffin-embedded or fresh frozen [FF]). METHODS: Articles were systemically reviewed for studies that included the correlation between F. nucleatum and prognosis or clinicopathological features in CRC. RESULTS: Ten articles, eight studies with survival-related features involving 3,199 patients and nine studies with clinical features involving 2,655 patients, were eligible for the meta-analysis. Overall survival, disease-free survival, and cancer-specific survival were all associated with worse prognosis in F. nucleatum-high patients (p<.05). In subgroup analysis, only studies with FF tissues retained prognostic significance with F. nucleatum. In meta-analysis of clinicopathological variables, F. nucleatum level was associated with location within colon, pT category, MLH1 hypermethylation, microsatellite instability status, and BRAF mutation regardless of type of biospecimen. However, lymph node metastasis and KRAS mutation was only associated with F. nucleatum level in FF-based studies. CONCLUSIONS: In conclusion, type of biospecimen could affect the role of F. nucleatum as a biomarker associated with clinicopathological features and prognosis.

Identification of tissue of origin in cancer of unknown primary using a targeted bisulfite sequencing panel.

Aim: To construct a targeted bisulfite sequencing panel predicting origin of cancer of unknown primary. Methods: A bisulfite sequencing panel targeting 2793 tissue-specific markers was performed in 100 clinical samples. Results: The authors' prediction model showed 0.85 accuracy for the 'first-ranked' tissue type and 0.93 accuracy for the 'second-ranked' tissue type using 2793 tissue-specific markers and 0.84 accuracy for the 'first-ranked' tissue type and 0.92 accuracy for the 'second-ranked' tissue type when the number of tissue-specific markers was reduced to 514. Conclusion: Targeted bisulfite sequencing is a useful method for predicting the tissue of origin in patients with cancer of unknown primary.

When patients with cancer present with tumors that have migrated from elsewhere in the body, it is difficult for clinicians to identify where the cancer originated. DNA methylation profiling is a promising test to help identify where the cancer originated because it reflects cell of origin and is compatible with formalin-fixed, paraffin-embedded tissues. Because next-generation sequencing has already been implemented in clinical laboratories, the authors developed a targeted bisulfite sequencing panel that could predict the tissue of origin using genomic DNA extracted from formalin-fixed, paraffin-embedded tissues. The authors found that a hybrid capture-based targeted bisulfite sequencing panel is a useful method for predicting the tissue of origin in patients with cancer of unknown primary origin in clinical practice.

Combinatory statuses of tumor stromal percentage and tumor infiltrating lymphocytes as prognostic factors in stage III colorectal cancers.

BACKGROUND AND AIM: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Methylation statuses of NCOR2, PARK2, and ZSCAN12 signify densities of tumor-infiltrating lymphocytes in gastric carcinoma.

Individual cell types of human tissues have their own CpG site methylation profiles, which might be utilized for the development of methylation markers to denote tumor-infiltrating lymphocytes (TILs). We aimed to develop DNA methylation markers that recapitulate the densities of TILs in gastric carcinoma (GC). Through genome-wide methylation profiling, NCOR2, PARK2, and ZSCAN12 were found to be highly methylated in CD3-positive and CD8-positive cells and rarely methylated in tumor cells. Scores of the three methylation markers were analyzed for their relationship with the overall survival and recurrence-free survival of patients with advanced GC (n = 471). The scores of three methylation markers were closely associated with densities of CD3-positive or CD8-positive cells at the tumor center or invasive front of GCs and found to be a significant prognostic factor in univariate analysis of overall survival and recurrence-free survival. In multivariate analysis, the highest score showed hazard ratios of 0.513 (CI 0.306-0.857) and 0.434 (CI 0.261-0.720) for overall survival and recurrence-free survival, respectively. The findings suggest that methylation markers signifying TILs might be utilized for the recapitulation of TIL density in GCs and serve as biomarkers for predicting prognosis in patients with GC.

Comprehensive clinicopathologic, molecular, and immunologic characterization of colorectal carcinomas with loss of three intestinal markers, CDX2, SATB2, and KRT20.

Caudal-type homeobox 2 (CDX2), special AT-rich sequence-binding protein 2 (SATB2), and keratin 20 (KRT20) are frequently used as intestinal epithelium-specific markers in immunohistochemical studies. However, subsets of colorectal carcinomas (CRCs) show loss of these markers. We analyzed The Cancer Genome Atlas data to explore molecular correlates of CDX2, SATB2, and KRT20 genes in 390 CRCs. The decreased mRNA expression of each of the three genes commonly correlated with microsatellite instability-high (MSI-H), CpG island methylator phenotype-high (CIMP-H), BRAF/RNF43 mutations, consensus molecular subtype 1, and high tumor mutational burden. The downregulation of CDX2 or SATB2 was dependent on both MSI-H and CIMP-H, whereas that of KRT20 was more dependent on MSI-H than on CIMP-H. Next, we evaluated the immunohistochemical expression of CDX2, SATB2, and KRT20 in 436 primary CRCs. In contrast to RNA-level expression, decreased expression of CDX2 and SATB2 was more dependent on CIMP-H than on MSI-H. However, consistent with RNA-level expression, decreased expression of KRT20 was more dependent on MSI-H than on CIMP-H. CIMP-H and lymphatic invasion were consistently associated with both CDX2 loss and SATB2 loss in CRCs, regardless of MSI status. In microsatellite stable CRCs, CDX2 loss correlated with BRAF mutation, whereas SATB2 loss was associated with KRAS mutations and decreased T-cell infiltration. Cases with concurrent loss of all three markers were found exclusively in MLH1-methylated MSI-H/CIMP-H CRCs. In conclusion, MSI-H and/or CIMP-H are major common correlates of decreased CDX2/SATB2/KRT20 expression in CRCs, but the specific features associated with the loss of each marker are different in CRCs.

Genomic and transcriptomic characterization of heterogeneous immune subgroups of microsatellite instability-high colorectal cancers.

BACKGROUND: Colorectal cancers (CRCs) with microsatellite instability-high (MSI-H) are hypermutated tumors and are generally regarded as immunogenic. However, their heterogeneous immune responses and underlying molecular characteristics remain largely unexplained. METHODS: We conducted a retrospective analysis of 73 primary MSI-H CRC tissues to characterize heterogeneous immune subgroups. Based on combined tumor-infiltrating lymphocyte (TIL) immunoscore and tertiary lymphoid structure (TLS) activity, MSI-H CRCs were classified into immune-high, immune-intermediate, and immune-low subgroups. Of these, the immune-high and immune-low subgroups were further analyzed using whole-exome and transcriptome sequencing. RESULTS: We found considerable variations in immune parameters between MSI-H CRCs, and immune subgrouping of MSI-H CRCs was performed accordingly. The TIL densities and TLS activities of immune-low MSI-H CRCs were comparable to those of an immune-low or immune-intermediate subgroup of microsatellite-stable CRCs. There were remarkable differences between immune-high and immune-low MSI-H CRCs, including their pathological features (medullary vs mucinous), genomic alterations (tyrosine kinase fusions vs KRAS mutations), and activated signaling pathways (immune-related vs Wnt and Notch signaling), whereas no significant differences were found in tumor mutational burden (TMB) and neoantigen load. The immune-low MSI-H CRCs were subdivided by the consensus molecular subtype (CMS1 vs CMS3) with different gene expression signatures (mesenchymal/stem-like vs epithelial/goblet-like), suggesting distinct immune evasion mechanisms. Angiogenesis and CD200 were identified as potential therapeutic targets in immune-low CMS1 and CMS3 MSI-H CRCs, respectively. CONCLUSIONS: MSI-H CRCs are immunologically heterogeneous, regardless of TMB. The unusual immune-low MSI-H CRCs are characterized by mucinous histology, KRAS mutations, and Wnt/Notch activation, and can be further divided into distinct gene expression subtypes, including CMS4-like CMS1 and CMS3. Our data provide novel insights into precise immunotherapeutic strategies for subtypes of MSI-H tumors.