Background-The CpG island methylator phenotype (CIMP) characterized by widespread promoter methylation is associated with microsatellite instability (MSI) and BRAF mutation in colorectal cancer. The independent effect of CIMP, MSI and BRAF mutation on patient outcome remains uncertain.

BACKGROUND Regular use of aspirin after a diagnosis of colon cancer has been associated with a superior clinical outcome. Experimental evidence suggests that inhibition of prostaglandin-endoperoxide synthase 2 (PTGS2) (also known as cyclooxygenase-2) by aspirin down-regulates phosphatidylinositol 3-kinase (PI3K) signaling activity. We hypothesized that the effect of aspirin on survival and prognosis in patients with cancers characterized by mutated PIK3CA (the phosphatidylinositol-4,5-bisphosphonate 3-kinase, catalytic subunit alpha polypeptide gene) might differ from the effect among those with wild-type PIK3CA cancers. METHODS We obtained data on 964 patients with rectal or colon cancer from the Nurses’ Health Study and the Health Professionals Follow-up Study, including data on aspirin use after diagnosis and the presence or absence of PIK3CA mutation. We used a Cox proportional-hazards model to compute the multivariate hazard ratio for death. We examined tumor markers, including PTGS2, phosphorylated AKT, KRAS, BRAF, microsatellite instability, CpG island methylator phenotype, and methylation of long interspersed nucleotide element 1. RESULTS Among patients with mutated-PIK3CA colorectal cancers, regular use of aspirin after diagnosis was associated with superior colorectal cancer–specific survival (multivariate hazard ratio for cancer-related death, 0.18; 95% confidence interval [CI], 0.06 to 0.61; P<0.001 by the log-rank test) and overall survival (multivariate hazard ratio for death from any cause, 0.54; 95% CI, 0.31 to 0.94; P = 0.01 by the log-rank test). In contrast, among patients with wild-type PIK3CA, regular use of aspirin after diagnosis was not associated with colorectal cancer–specific survival (multivariate hazard ratio, 0.96; 95% CI, 0.69 to 1.32; P = 0.76 by the log-rank test; P = 0.009 for interaction between aspirin and PIK3CA variables) or overall survival (multivariate hazard ratio, 0.94; 95% CI, 0.75 to 1.17; P = 0.96 by the log-rank test; P = 0.07 for interaction). CONCLUSIONS Regular use of aspirin after diagnosis was associated with longer survival among patients with mutated-PIK3CA colorectal cancer, but not among patients with wild-type PIK3CA cancer. The findings from this molecular pathological epidemiology study suggest that the PIK3CA mutation in colorectal cancer may serve as a predictive molecular biomarker for adjuvant aspirin therapy. (Funded by The National Institutes of Health and others.)

Importance Evidence indicates a complex link between gut microbiome, immunity, and intestinal tumorigenesis. To target the microbiota and immunity for colorectal cancer prevention and therapy, a better understanding of the relationship between microorganisms and immune cells in the tumor microenvironment is needed. Experimental evidence suggests that Fusobacterium nucleatum may promote colonic neoplasia development by down-regulating antitumor T-cell-mediated adaptive immunity. Objective To test the hypothesis that higher amount of Fusobacterium nucleatum in colorectal carcinoma tissue is associated with lower density of T-cells in tumor tissue. Design A cross-sectional analysis was conducted on colorectal carcinoma cases in two U.S. nationwide prospective cohort studies. The amount of Fusobacterium nucleatum in colorectal carcinoma tissue was measured by quantitative polymerase chain reaction assay; we equally dichotomized positive cases (high versus low). Multivariable ordinal logistic regression analysis was conducted to assess associations of the amount of Fusobacterium nucleatum with densities (quartiles) of T-cells in tumor tissue, controlling for clinical and tumor molecular features, including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, and KRAS, BRAF, and PIK3CA mutation status. We adjusted two-sided α level to 0.013 for multiple hypothesis testing. Setting The Nurses’ Health Study and the Health Professionals Follow-up Study. Participants 598 colon and rectal carcinoma cases. Main outcomes and measures Densities of CD3+, CD8+, CD45RO (PTPRC)+, and FOXP3+ T-cells in tumor tissue, determined by tissue microarray immunohistochemistry and computer-assisted image analysis. Results Fusobacterium nucleatum was detected in colorectal carcinoma tissue in 76 (13%) of 598 cases. Compared with Fusobacterium nucleatum-negative cases, Fusobacterium nucleatum-high cases were inversely associated with the density of CD3+ T-cells (for a unit increase in quartile categories of CD3+ T-cells as an outcome: multivariable odds ratio, 0.47; 95% confidence interval, 0.26 to 0.87; Ptrend = 0.006). The amount of Fusobacterium nucleatum was not significantly associated with the density of CD8+, CD45RO+, or FOXP3+ T-cells (Ptrend > 0.013). Conclusions and relevance The amount of tissue Fusobacterium nucleatum is inversely associated with CD3+ T-cell density in colorectal carcinoma tissue. Upon validation, our human population data may provide an impetus for further investigations on potential interactive roles of Fusobacterium and host immunity in carcinogenesis.

The abundance of tumour-infiltrating T-cells has been associated with microsatellite instability (MSI) and a favorable prognosis in colorectal cancer. Because molecular alterations in colon cancer including MSI, the CpG island methylator phenotype (CIMP), BRAF mutation and global DNA hypomethylation have been associated with clinical outcome, potential confounding by these molecular features needs to be controlled when assessing the prognostic significance of tumour-infiltrating T-cells. We utilized a database of clinically and molecularly-annotated colon and rectal carcinoma cases (N=768; stage I-IV) in two prospective cohort studies (the Nurses' Health Study and the Health Professionals Follow-up Study). Using tissue microarray and automated Ariol image analysis system, we quantified densities of CD3 + , CD8 + , CD45RO + (PTPRC) and FOXP3 + -cells within neoplastic epithelial areas. We used Cox proportional hazard models to compute mortality hazard ratio, adjusting for clinical and molecular features including KRAS, BRAF, and PIK3CA mutations, MSI, CIMP and LINE-1 hypomethylation. The densities of CD8 + , CD45RO + and FOXP3 + -cells were significantly associated with patient survival in univariate analyses (P trend <0.007). In the multivariate model, tumour-infiltrating CD45RO + -cell density, but not that of CD3 + , CD8 + or FOXP3 + -cell, was significantly associated with survival (p=0.0032). In multivariate linear regression analysis, MSI-high (p<0.0001) and high-level tumour LINE-1 methylation (p=0.0013) were independently associated with higher CD45RO + -cell NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript density. Nonetheless, the survival benefit associated with CD45RO + -cells was independent of MSI and LINE-1 status. In conclusion, tumour-infiltrating CD45RO + -cell density is a prognostic biomarker associated with longer survival of colorectal cancer patients, independent of clinical, pathological and molecular features. In addition, MSI-high and tumour LINE-1 methylation level are independent predictors of CD45RO + -cell density. Our data offer a possible mechanism by which MSI confers an improved clinical outcome, and support efforts to augment host immune response in the tumour microenvironment as a strategy of targeted immunotherapy.

Table S3 has been updated. The updated version includes all somatic mutations used in our analyses.

Purpose: Host immune response to tumor may be an important prognostic factor for colon cancer patients. However, little is known on prognostic significance of histopathologic lymphoid reaction to tumor, independent of the number of lymph nodes examined and tumoral molecular alterations, including microsatellite instability (MSI) and the CpG island methylator phenotype (CIMP), both of which are associated with lymphocytic reaction and clinical outcome. Experimental Design: Using 843 colorectal cancer patients in two independent prospective cohorts, we examined patient prognosis in relation to four components of lymphocytic reaction (i.e., Crohn's-like reaction, peritumoral reaction, intratumoral periglandular reaction, and tumor-infiltrating lymphocytes) and overall lymphocytic score (0-12). CIMP was determined using eight markers including CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1. Cox proportional hazard models computed hazard ratio for mortality, adjusted for covariates including tumor stage, body mass index, lymph node count, KRAS, BRAF, p53, cyclooxygenase-2 (PTGS2), MSI, CIMP, and LINE-1 methylation. Results: Increasing overall lymphocytic reaction score including tumor-infiltrating lymphocytes was associated with a significant improvement in colorectal cancerspecific and overall survival (log-rank P < 0.003). These findings remained significant (adjusted hazard ratio estimates, 0.49-0.71; P trend < 0.009) in multivariate models that adjusted for covariates, including body mass index, MSI, CIMP, LINE-1 hypomethylation, and cyclooxygenase-2. The beneficial effect of tumoral lymphocytic reaction was consistent across strata of clinical, pathologic, and molecular characteristics. Conclusions: Lymphocytic reactions to tumor were associated with improved prognosis among colorectal cancer patients, independent of lymph node count and other clinical, pathologic, and molecular characteristics. (Clin Cancer Res 2009;15(20): 6412-20)

SummaryLarge-scale genomic characterization of tumors from prospective cohort studies may yield new insights into cancer pathogenesis. We performed whole-exome sequencing of 619 incident colorectal cancers (CRCs) and integrated the results with tumor immunity, pathology, and survival data. We identified recurrently mutated genes in CRC, such as BCL9L, RBM10, CTCF, and KLF5, that were not previously appreciated in this disease. Furthermore, we investigated the genomic correlates of immune-cell infiltration and found that higher neoantigen load was positively associated with overall lymphocytic infiltration, tumor-infiltrating lymphocytes (TILs), memory T cells, and CRC-specific survival. The association with TILs was evident even within microsatellite-stable tumors. We also found positive selection of mutations in HLA genes and other components of the antigen-processing machinery in TIL-rich tumors. These results may inform immunotherapeutic approaches in CRC. More generally, this study demonstrates a framework for future integrative molecular epidemiology research in colorectal and other malignancies.

To understand the genetic drivers of immune recognition and evasion in colorectal cancer, we analyzed 1,211 colorectal cancer primary tumor samples, including 179 classified as microsatellite instability-high (MSI-high). This set includes The Cancer Genome Atlas colorectal cancer cohort of 592 samples, completed and analyzed here. MSI-high, a hypermutated, immunogenic subtype of colorectal cancer, had a high rate of significantly mutated genes in important immune-modulating pathways and in the antigen presentation machinery, including biallelic losses of and genes due to copy-number alterations and copy-neutral loss of heterozygosity. WNT/β-catenin signaling genes were significantly mutated in all colorectal cancer subtypes, and activated WNT/β-catenin signaling was correlated with the absence of T-cell infiltration. This large-scale genomic analysis of colorectal cancer demonstrates that MSI-high cases frequently undergo an immunoediting process that provides them with genetic events allowing immune escape despite high mutational load and frequent lymphocytic infiltration and, furthermore, that colorectal cancer tumors have genetic and methylation events associated with activated WNT signaling and T-cell exclusion. This multi-omic analysis of 1,211 colorectal cancer primary tumors reveals that it should be possible to better monitor resistance in the 15% of cases that respond to immune blockade therapy and also to use WNT signaling inhibitors to reverse immune exclusion in the 85% of cases that currently do not. .