Investigation of ENO2 as a promising novel marker for the progression of colorectal cancer with microsatellite instability-high

Immunotherapy is reportedly effective in colorectal cancers (CRCs) with high microsatellite instability (MSI-H); however, the specific cell types that respond to immune checkpoint therapy are unclear. Herein, we aimed to examine the expression of programmed cell death-ligand 1 (PD-L1) and related proteins in MSI-H and microsatellite-stable (MSS) CRCs to investigate the immune microenvironment at the tumor's invasive front. The MSI status was retrospectively assessed in 499 patients undergoing surgical resection of primary CRC; of these, 48 were classified as MSI-H. Propensity score matching was performed, and tissues from 36 and 37 patients with MSI-H and MSS CRCs, respectively, were immunohistochemically evaluated for PD-L1, PD-1, CD8 and CD68. PD-L1 expression was evaluated separately for tumor cells (PD-L1 [T]) and tumor-infiltrating myeloid cells in the stroma (PD-L1 [I]). PD-L1 (T) was positive in only 5.4% and 36.1% of MSS and MSI-H CRCs, while PD-L1 (I) was positive in 27% and 72.2% of these CRCs, respectively. The PD-L1 (T) and PD-L1 (I) expression levels in MSI-H CRCs significantly correlated with poor differentiation, lymphatic invasion and vascular invasion (p < 0.05), and with early-stage adenocarcinoma and high budding grade (p < 0.05), respectively. Significantly more PD-L1 (I), CD8-positive cells and CD68-positive macrophages were present at the invasive front than in the central tumor in MSI-H CRCs (p < 0.005). PD-L1 was expressed on both tumor cells and CD68/CD163-positive (M2) macrophages at the invasive front of MSI-H CRCs. In conclusion, PD-L1-positive tumor cells and M2-type tumor-associated macrophages may contribute to tumor invasion and immune escape at the invasive front.

High level microsatellite instability (MSI-H) occurs in about 15% of colorectal cancer (CRCs), either as sporadic cancers or in the context of hereditary non-polyposis cancer or Lynch syndrome. In MSI-H CRC, mismatch repair deficiency leads to insertion/deletion mutations at coding microsatellites and thus to the translation of frameshift peptides (FSPs). FSPs are potent inductors of T cell responses in vitro and in vivo. The present study aims at the identification of FSP-specific humoral immune responses in MSI-H CRC and Lynch syndrome. Sera from patients with history of MSI-H CRC (n = 69), healthy Lynch syndrome mutation carriers (n = 31) and healthy controls (n = 52) were analyzed for antibodies against FSPs using peptide ELISA. Reactivities were measured against FSPs derived from genes frequently mutated in MSI-H CRCs, AIM2, TGFBR2, CASP5, TAF1B, ZNF294, and MARCKS. Antibody reactivity against FSPs was significantly higher in MSI-H CRC patients than in healthy controls (P = 0.036, Mann-Whitney) and highest in patients with shortest interval between tumor resection and serum sampling. Humoral immune responses in patients were most frequently directed against FSPs derived from mutated TAF1B (11.6%, 8/69) and TGFBR2 (10.1%, 7/69). Low level FSP-specific antibodies were also detected in healthy mutation carriers. Our results show that antibody responses against FSPs are detectable in MSI-H CRC patients and healthy Lynch syndrome mutation carriers. Based on the high number of defined FSP antigens, measuring FSP-specific humoral immune responses is a highly promising tool for future diagnostic application in MSI-H cancer patients.

Defects of DNA mismatch repair (MMR) cause the high level microsatellite instability (MSI-H) phenotype. MSI-H cancers may develop either sporadically or in the context of the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome that is caused by germline mutations of MMR genes. In colorectal cancer (CRC), MSI-H is characterized by a dense lymphocytic infiltration, reflecting a high immunogenicity of these cancers. As a consequence of immunoselection, MSI-H CRCs frequently display a loss of human leukocyte antigen (HLA) class I antigen presentation caused by mutations of the beta2-microglobulin (beta2m) gene. To examine the implications of beta2m mutations during MSI-H colorectal tumor development, we analyzed the prevalence of beta2m mutations in MSI-H colorectal adenomas (n=38) and carcinomas (n=104) of different stages. Mutations were observed in 6/38 (15.8%) MSI-H adenomas and 29/104 (27.9%) MSI-H CRCs. A higher frequency of beta2m mutations was observed in MSI-H CRC patients with germline mutations of MMR genes MLH1 or MSH2 (36.4%) compared with patients without germline mutations (15.4%). The high frequency of beta2m mutations in HNPCC-associated MSI-H CRCs is in line with the hypothesis that immunoselection may be particularly pronounced in HNPCC patients with inherited predisposition to develop MSI-H cancers. beta2m mutations were positively related to stage in tumors without distant metastases (UICC I-III), suggesting that loss of beta2m expression may promote local progression of colorectal MSI-H tumors. However, no beta2m mutations were observed in metastasized CRCs (UICC stage IV, p=0.04). These results suggest that functional beta2m may be necessary for distant metastasis formation in CRC patients.

Colorectal cancer (CRC) mostly develops from a variety of polyps following mainly three different molecular pathways: chromosomal instability (CIN), microsatellite instability (MSI) and CpG island methylation (CIMP). Polyps are classified histologically as conventional adenomas, hyperplastic polyps, sessile serrated adenomas/polyps (SSA/P) and traditional serrated adenomas (TSA). However, the association of these polyps with the different types of CRCs and the underlying genetic and epigenetic aberrations has yet to be resolved. In order to address this question we analyzed 140 tumors and 20 matched mucosae by array comparative genomic hybridization, by sequence analysis of the oncogenes BRAF, KRAS, PI3K3CA and by methylation arrays. MSI was tested indirectly by immunohistochemistry (IHC) and a loss of MLH1, MSH2, MSH6 or PMS2 was assigned as high microsatellite instability (MSI-H), while low microsatellite instability (MSI-L) was defined as MGMT IHC negativity only. CIN was detected in 78% of all MSI-H CRCs, most commonly as a gain of chromosome 8. Methylation data analyses allowed classification of samples into four groups and detected similar methylation profiles in SSA/P and MSI-H CRC. TSA also revealed aberrant methylation pattern, but clustered more heterogeneously and closer to microsatellite stable (MSS) CRCs. SSA/P, TSA and MSI-H CRCs had the highest degree of promotor methylation (CIMP pathway). Chromosomal instability, in contrast to the established doctrine, is a common phenomenon in MSI CRCs, yet to a lower extent and at later stages than in MSS CRCs. Methylation analyses suggest that SSA/P are precursors for MSI-H CRCs and follow the CIMP pathway.

High level microsatellite instability (MSI-H) is a hallmark of Lynch syndrome-associated colorectal cancer (CRC). MSI-H CRC express immunogenic tumour antigens as a consequence of DNA mismatch repair deficiency-induced frameshift mutations. Consequently, frameshift antigen-specific immune responses are commonly observed in patients with Lynch syndrome-associated MSI-H CRC. Dendritic cells (DC) and macrophages play a crucial role in the induction and modulation of immune responses. We here analysed DC and macrophage infiltration in MSI-H and microsatellite-stable CRC. Sixty-nine CRC (MSI-H, n=33; microsatellite-stable, n=36) were examined for the density of tumour-infiltrating DC, Foxp3-positive regulatory T cells, and CD163-positive macrophages. In MSI-H lesions, S100-positive and CD163-positive cell counts were significantly higher compared to microsatellite-stable lesions (S100: epithelium P=0.018, stroma P=0.042; CD163: epithelium P<0.001, stroma P=0.046). Additionally, numbers of CD208-positive mature DC were significantly elevated in the epithelial compartment of MSI-H CRC (P=0.027). High numbers of tumour-infiltrating Foxp3-positive T cells were detected in tumours showing a low proportion of CD208-positive, mature DC among the total number of S100-positive cells. Our study demonstrates that infiltration with DC, mature DC, and macrophages is elevated in MSI-H compared to microsatellite-stable CRC. The positive correlation of Foxp3-positive Treg cell density with a low proportion of mature DC suggests that impaired DC maturation may contribute to local immune evasion in CRC. Our results demonstrate that DC and macrophages in the tumour environment likely play an important role in the induction of antigen-specific immune responses in Lynch syndrome. Moreover, impaired DC maturation might contribute to local immune evasion in CRC.

Background & objectives:The two key aspects associated with the microsatellite instability (MSI) as genetic phenomenon in colorectal cancer (CRC) are better survival prognosis, and the varying response to 5-fluorouracil (5-FU)-based chemotherapy. This study was undertaken to measure the survival of surgically treated patients with stages II and III CRC based on the MSI status, the postoperative 5-FU treatment as well as clinical and histological data.Methods:A total of 125 consecutive patients with stages II and III (American Joint Committee on Cancer, AJCC staging) primary CRCs, were followed prospectively for a median time of 31 months (January 2006 to December 2009). All patients were assessed, operated and clinically followed. Tumour samples were obtained for cytopathological verification and MSI grading.Results:Of the 125 patients, 21 (20%) had high MSI (MSI-H), and 101 patients (80%) had MSI-L or MSS (low frequency MSI or stable MSI). Patients with MSS CRC were more likely to have recurrent disease (P=0.03; OR=3.2; CI 95% 1-10.2) compared to those with MSI-H CRC. Multi- and univariate Cox regression analysis failed to show a difference between MSI-H and MSS groups with respect to disease-free, disease-specific and overall survival. However, the disease-free survival was significantly lower in patients with MSI-H CRC treated by adjuvant 5-FU therapy (P=0.03).Interpretation & conclusions:MSI-H CRCs had a lower recurrence rate, but the prognosis was worse following adjuvant 5-FU therapy.

Growth differentiation factor 15 (GDF15) is a member of the transforming growth factor beta (TGF-β) superfamily and is related to metabolism, injury, and aging. GDF15 has both tumor-promoting and tumor-suppressing effects. However, its role in colorectal cancer (CRC) with high microsatellite instability (MSI-H) must be further clarified. In our study, we found that GDF15 is generally elevated in pancarcinoma, particularly in colorectal cancer, and serves as an early indicator of the development of colorectal cancer. IHC and WB confirmed that GDF15 was elevated in MSI-H CRC clinical tissues and MSI-H CRC cell lines (HCT-116 and LoVo). Suppressing GDF15 by siRNA resulted in a substantial decrease in cell viability and proliferation. Furthermore, suppressing GDF15 can increase the sensitivity of MSI-H CRC cells to 5-fluorouracil (5-FU), which decreases cell viability and increases the apoptosis rate. In vivo experiments also demonstrated that mouse xenografts with suppressed GDF15 expression were more susceptible to 5-FU chemotherapy. We examined alterations in mitochondria via electron microscopy and changes in the mitochondrial membrane potential, ferroptosis-related signals (MDA, Fe2+), and SLC7A11/GSH/GPX4 protein pathway. Our research indicates that inhibiting GDF15 affects ferroptosis-related pathways, leading to ferroptosis and improving the MSI-H CRC response to 5-FU therapy. As a result, GDF15 could be a promising target for diagnosing and treating MSI-H CRC, potentially enhancing the overall effectiveness of therapy for patients with MSI-H CRC.

Backgroundβ-catenin activation plays a crucial role for tumourigenesis in the large intestine but except for Lynch syndrome (LS) associated cancers stabilizing mutations of β-catenin gene (CTNNB1) are rare in colorectal cancer (CRC). Previous animal studies provide an explanation for this observation. They showed that CTNNB1 mutations induced transformation in the colon only when CTNNB1 was homozygously mutated or when membranous β-catenin binding was hampered by E-cadherin haploinsufficiency. We were interested, if these mechanisms are also found in human CTNNB1 mutated CRCs.ResultsAmong 869 CRCs stabilizing CTNNB1 mutations were found in 27 cases. Homo- or hemizygous CTNNB1 mutations were detected in 74% of CTNNB1 mutated CRCs (13 microsatellite instabile (MSI-H), 7 microsatellite stabile (MSS)) but only in 3% (1/33) of extracolonic CTNNB1 mutated cancers. In contrast to MSS CRC, CTNNB1 mutations at codon 41 or 45 were highly selected in MSI-H CRC. Of the examined three CRC cell lines, β-catenin and E-cadherin expression was similar in cell lines without or with hetereozygous CTNNB1 mutations (DLD1 and HCT116), while a reduced E-cadherin expression combined with cytoplasmic accumulation of β-catenin was found in a cell line with homozygous CTNNB1 mutation (LS180). Reduced expression of E-cadherin in human MSI-H CRC tissue was identified in 60% of investigated cancers, but no association with the CTNNB1 mutational status was found.ConclusionsIn conclusion, this study shows that in contrast to extracolonic cancers stabilizing CTNNB1 mutations in CRC are commonly homo- or hemizygous indicating a higher threshold of β-catenin stabilization to be required for transformation in the colon as compared to extracolonic sites. Moreover, we found different mutational hotspots in CTNNB1 for MSI-H and MSS CRCs suggesting a selection of different effects on β-catenin stabilization according to the molecular pathway of tumourigenesis. Reduced E-cadherin expression in CRC may further contribute to higher levels of transcriptionally active β-catenin, but it is not directly linked to the CTNNB1 mutational status.

Colorectal cancer is a heterogeneous tumor type with regard to molecular pathogenesis and genetic instability. The majority of colorectal cancers display chromosomal instability and follow the classical adenoma-carcinoma sequence of tumor progression. A subset of about 15% of colorectal cancers, however, displays DNA mismatch repair (MMR) deficiency and the high-level microsatellite instability (MSI-H) phenotype. MSI-H colorectal cancers can occur as sporadic tumors or in the context of hereditary non-polyposis colorectal cancer (HNPCC) or Lynch syndrome. The MSI-H phenotype is a hallmark of Lynch syndrome-associated cancers, which is of diagnostic relevance for the identification of Lynch syndrome mutation carriers. MSI-H colorectal cancers are characterized by a distinct clinical behavior, which results from their particular molecular pathogenesis and gives microsatellite instability testing its clinical significance. The MSI-H phenotype shows association with proximal tumor localization, a dense local lymphocyte infiltration, and a low frequency of distant organ metastasis. Moreover, MSI-H colorectal cancers have a better prognosis than their microsatellite-stable counterparts. A distinct responsiveness of MSI-H colorectal cancer patients towards chemotherapy has been shown in several studies. The clinical characteristics of MSI-H cancers are closely linked to their molecular pathogenesis, and research on the molecular alteration characteristic of MSI-H cancers may provide the basis for novel diagnostic or therapeutic approaches.

The healthy colorectal mucosa contains many resident intraepithelial lymphocytes (IELs) consisting of partially activated yet hyporesponsive CD8(+) T cells. A predominant feature of colorectal cancers (CRCs) characterized by high levels of microsatellite instability (MSI-H) is heavy infiltration by an intraepithelial population of tumor infiltrating lymphocytes (iTILs). While it has been assumed that these iTILs originate from tumor infiltration by peripheral CD8(+) effector T cells, their origin remains unknown. In light of the phenotypic and functional differences exhibited by IELs and peripheral T cells, elucidation of the precursor population of iTILs in MSI-H CRCs could clarify the role played by these lymphocytes in tumor progression. The aim of the present study was to investigate whether MSI-H CRCs interact differently with IEL- versus peripherally-derived CD8(+) T cells. Using a Transwell assay system to mimic basolateral infiltration of tumor cells by lymphocytes, T cell migration, retention, proliferation and phenotypic alterations were investigated. Results indicate that MSI-H CRCs preferentially retain and expand IEL-derived cells to a greater degree than their microsatellite stable (MSS) counterparts. While MSI-H CRCs also retained more peripherally derived T cells, this number was considerably less than that from the IEL population. While interaction of IELs with either CRC type led to baseline lymphocyte activation, MSS CRCs induced upregulation of additional activation markers on retained IELs compared to MSI-H CRCs. These results suggest that the abundant iTILs present in MSI-H CRCs result from expansion of the preexisting mucosal IEL population and imply a limited prognostic role for iTILs in MSI-H CRC.

Background Elevated platelet volume is the risk factor for the development and poor overall survival of colorectal cancer (CRC) patients. Both microsatellite status and platelet glycoprotein Ibα (GPIbα) are related to platelet volume in CRC patients. This study aimed to investigate platelet GPIbα ectodomain (termed glycocalicin) levels among CRC patients and the association between the glycocalicin levels and microsatellite status in CRC. Methods The clinical and laboratory data of 430 CRC patients between January 2018 and December 2018 in Harbin Medical University Cancer Hospital were collected. The microsatellite status was determined with a polymerase chain reaction. The participants were separated into high microsatellite instability (MSI-H) and microsatellite stable (MSS) groups according to microsatellite status. The glycocalicin levels were measured with an enzyme-linked immunosorbent assay, and the cut-off point was determined with the receiver-operating characteristics curve. The clinical and pathological characteristics were collected via electronic medical records. Logistic regression was used to explore the association between glycocalicin and microsatellite status. Results Among the 430 CRC patients enrolled, 64 patients (14.9%) were identified as MSI-H and others as MSS CRC. Glycocalicin levels were significantly reduced in patients with MSI-H than those with MSS. After controlling for potential confounders, logistic regression analysis revealed that glycocalicin levels were independently associated with MSI-H CRC. Conclusions Reduced glycocalicin levels are associated with the MSI-H subtype of CRC. Further research is needed to elucidate the mechanisms of the association between glycocalicin and MSI-H in CRC patients.

Frameshift mutations of poly(adenosine diphosphate-ribose) polymerase genes in gastric and colorectal cancers with microsatellite instability

BackgroundTumor heterogeneity is contributed by tumor cells and the microenvironment. Dynamics of tumor heterogeneity during colorectal cancer (CRC) progression have not been elucidated.MethodsEight single-cell RNA sequencing (scRNA-seq) data sets of CRC were included. Milo was utilized to reveal the differential abundance of cell clusters during progression. The differentiation trajectory was imputed by using the Palantir algorithm and metabolic states were assessed by using scMetabolism. Three spatial transcription sequencing (ST-seq) data sets of CRC were used to validate cell-type abundances and colocalization. Cancer-associated regulatory hubs were defined as communication networks affecting tumor biological behaviors. Finally, quantitative reverse transcription polymerase chain reaction and immunohistochemistry staining were performed for validation.ResultsTM4SF1+, SOX4+, and MKI67+ tumor cells; CXCL12+ cancer-associated fibroblasts; CD4+ resident memory T cells; Treg; IgA+ plasma cells; and several myeloid subsets were enriched in stage IV CRC, most of which were associated with overall survival of patients. Trajectory analysis indicated that tumor cells from patients with advanced-stage CRC were less differentiated, when metabolic heterogeneity showed a highest metabolic signature in terminal states of stromal cells, T cells, and myeloid cells. Moreover, ST-seq validated cell-type abundance in a spatial context and also revealed the correlation of immune infiltration between tertiary lymphoid structures and tumors followed by validation in our cohort. Importantly, analysis of cancer-associated regulatory hubs revealed a cascade of activated pathways including leukocyte apoptotic process, MAPK pathway, myeloid leukocyte differentiation, and angiogenesis during CRC progression.ConclusionsTumor heterogeneity was dynamic during progression, with the enrichment of immunosuppressive Treg, myeloid cells, and fibrotic cells. The differential state of tumor cells was associated with cancer staging. Assessment of cancer-associated regulatory hubs suggested impaired antitumor immunity and increased metastatic ability during CRC progression.

Background: Colorectal cancer (CRC) is the second most common cancer in China. Autophagy plays an important role in the initiation and development of CRC. Here, we assessed the prognostic value and potential functions of autophagy-related genes (ARGs) using integrated analysis using single-cell RNA sequencing (scRNA-seq) data from the Gene Expression Omnibus (GEO) and RNA sequencing (RNA-seq) data from The Cancer Genome Atlas (TCGA). Methods: We analyzed GEO-scRNA-seq data from GEO using various single-cell technologies, including cell clustering, and identification of differentially expressed genes (DEGs) in different cell types. Additionally, we performed gene set variation analysis (GSVA). The differentially expressed ARGs among different cell types and those between CRC and normal tissues were identified using TCGA-RNA-seq data, and the hub ARGs were screened. Finally, a prognostic model based on the hub ARGs was constructed and validated, and patients with CRC in TCGA datasets were divided into high- and low-risk groups based on their risk-score, and immune cells infiltration and drug sensitivity analyses between the two groups were performed. Results: We obtained single-cell expression profiles of 16,270 cells, and clustered them into seven types of cells. GSVA revealed that the DEGs among the seven types of cells were enriched in many signaling pathways associated with cancer development. We screened 55 differentially expressed ARGs, and identified 11 hub ARGs. Our prognostic model revealed that the 11 hub ARGs including CTSB, ITGA6, and S100A8, had a good predictive ability. Moreover, the immune cell infiltrations in CRC tissues were different between the two groups, and the hub ARGs were significantly correlated with the enrichment of immune cell infiltration. The drug sensitivity analysis revealed that the patients in the two risk groups had difference in their response to anti-cancer drugs. Conclusion: We developed a novel prognostic 11-hub ARG risk model, and these hubs may act as potential therapeutic targets for CRC.

In sporadic colorectal tumours the BRAFV600E is associated with microsatellite instability (MSI-H) and inversely associated to KRAS mutations. Tumours from hereditary non-polyposis colorectal cancer (HNPCC) patients carrying germline mutations in hMSH2 or hMLH1 do not show BRAFV600E, however no consistent data exist regarding KRAS mutation frequency and spectrum in HNPCC tumours. We investigated KRAS in 158 HNPCC tumours from patients with germline hMLH1, hMSH2 or hMSH6 mutations, 166 MSI-H and 688 microsatellite stable (MSS) sporadic carcinomas. All tumours were characterized for MSI and 81 of 166 sporadic MSI-H colorectal cancer (CRCs) were analysed for hMLH1 promoter hypermethylation. KRAS mutations were observed in 40% of HNPCC tumours, and the mutation frequency varied upon the mismatch repair gene affected: 48% (29/61) in hMSH2, 32% (29/91) in hMLH1 and 83% (5/6) in hMSH6 (P = 0.01). KRAS mutation frequency was different between HNPCC, MSS and MSI-H CRCs (P = 0.002), and MSI-H with hMLH1 hypermethylation (P = 0.005). Furthermore, HNPCC CRCs had more G13D mutations than MSS (P < 0.0001), MSI-H (P = 0.02) or MSI-H tumours with hMLH1 hypermethylation (P = 0.03). HNPCC colorectal and sporadic MSI-H tumours without hMLH1 hypermethylation shared similar KRAS mutation frequency, in particular G13D. In conclusion, we show that depending on the genetic/epigenetic mechanism leading to MSI-H, the outcome in terms of oncogenic activation may be different, reinforcing the idea that HNPCC, sporadic MSI-H (depending on the hMLH1 status) and MSS CRCs, may target distinct kinases within the RAS/RAF/MAPK pathway.