Kosuke Mima

Genomic Correlates of Immune-Cell Infiltrates in Colorectal Carcinoma

Summary Large-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.

Fusobacterium nucleatum in colorectal carcinoma tissue and patient prognosis

Objective Accumulating evidence links the intestinal microbiota and colorectal carcinogenesis. Fusobacterium nucleatum may promote colorectal tumour growth and inhibit T cell-mediated immune responses against colorectal tumours. Thus, we hypothesised that the amount of F. nucleatum in colorectal carcinoma might be associated with worse clinical outcome. Design We used molecular pathological epidemiology database of 1069 rectal and colon cancer cases in the Nurses’ Health Study and the Health Professionals Follow-up Study, and measured F. nucleatum DNA in carcinoma tissue. Cox proportional hazards model was used to compute hazard ratio (HR), controlling for potential confounders, including microsatellite instability (MSI, mismatch repair deficiency), CpG island methylator phenotype (CIMP), KRAS, BRAF, and PIK3CA mutations, and LINE-1 hypomethylation (low-level methylation). Results Compared with F. nucleatum-negative cases, multivariable HRs (95% CI) for colorectal cancer-specific mortality in F. nucleatum-low cases and F. nucleatum-high cases were 1.25 (0.82 to 1.92) and 1.58 (1.04 to 2.39), respectively, (p for trend=0.020). The amount of F. nucleatum was associated with MSI-high (multivariable odd ratio (OR), 5.22; 95% CI 2.86 to 9.55) independent of CIMP and BRAF mutation status, whereas CIMP and BRAF mutation were associated with F. nucleatum only in univariate analyses (p<0.001) but not in multivariate analysis that adjusted for MSI status. Conclusions The amount of F. nucleatum DNA in colorectal cancer tissue is associated with shorter survival, and may potentially serve as a prognostic biomarker. Our data may have implications in developing cancer prevention and treatment strategies through targeting GI microflora by diet, probiotics and antibiotics.

Plasma 25-hydroxyvitamin D and colorectal cancer risk according to tumour immunity status.

Objective Evidence suggests protective effects of vitamin D and antitumour immunity on colorectal cancer risk. Immune cells in tumour microenvironment can convert 25-hydroxyvitamin D [25(OH)D] to bioactive 1α,25-dihydroxyvitamin D3, which influences neoplastic and immune cells as an autocrine and paracrine factor. Thus, we hypothesised that the inverse association between vitamin D and colorectal cancer risk might be stronger for cancers with high-level immune response than those with low-level immune response. Design We designed a nested case–control study (318 rectal and colon carcinoma cases and 624 matched controls) within the Nurses’ Health Study and Health Professionals Follow-up Study using molecular pathological epidemiology database. Multivariable conditional logistic regression was used to assess the association of plasma 25(OH)D with tumour subtypes according to the degree of lymphocytic reaction, tumour-infiltrating T cells (CD3+, CD8+, CD45RO+ (PTPRC) and FOXP3+ cells), microsatellite instability or CpG island methylator phenotype. Results The association of plasma 25(OH)D with colorectal carcinoma differed by the degree of intratumoural periglandular reaction (p for heterogeneity=0.001); high 25(OH)D was associated with lower risk of tumour with high-level reaction (comparing the highest versus lowest tertile: OR 0.10; 95% CI 0.03 to 0.35; p for trend<0.001), but not risk of tumour with lower-level reaction (p for trend>0.50). A statistically non-significant difference was observed for the associations of 25(OH)D with tumour subtypes according to CD3+ T cell density (p for heterogeneity=0.03; adjusted statistical significance level of α=0.006). Conclusions High plasma 25(OH)D level is associated with lower risk of colorectal cancer with intense immune reaction, supporting a role of vitamin D in cancer immunoprevention through tumour–host interaction.

Erratum: Genomic Correlates of Immune-Cell Infiltrates in Colorectal Carcinoma (Cell Reports (2016) 15(4) (857–865) (S2211124716303643) (10.1016/j.celrep.2016.03.075))

Marios Giannakis, Xinmeng Jasmine Mu, Sachet A. Shukla, Zhi Rong Qian, Ofir Cohen, Reiko Nishihara, Samira Bahl, Yin Cao, Ali Amin-Mansour, Mai Yamauchi, Yasutaka Sukawa, Chip Stewart, Mara Rosenberg, Kosuke Mima, Kentaro Inamura, Katsuhiko Nosho, Jonathan A. Nowak, Michael S. Lawrence, Edward L. Giovannucci, Andrew T. Chan, Kimmie Ng, Jeffrey A. Meyerhardt, Eliezer M. Van Allen, Gad Getz, Stacey B. Gabriel, Eric S. Lander, Catherine J. Wu, Charles S. Fuchs, Shuji Ogino,* and Levi A. Garraway* *Correspondence: shuji_ogino@dfci.harvard.edu (S.O.), levi_garraway@dfci.harvard.edu (L.A.G.) http://dx.doi.org/10.1016/j.celrep.2016.10.009

Association of Dietary Patterns With Risk of Colorectal Cancer Subtypes Classified by Fusobacterium nucleatum in Tumor Tissue

Importance Fusobacterium nucleatum appears to play a role in colorectal carcinogenesis through suppression of the hosts’ immune response to tumor. Evidence also suggests that diet influences intestinal F nucleatum. However, the role of F nucleatum in mediating the relationship between diet and the risk of colorectal cancer is unknown. Objective To test the hypothesis that the associations of prudent diets (rich in whole grains and dietary fiber) and Western diets (rich in red and processed meat, refined grains, and desserts) with colorectal cancer risk may differ according to the presence of F nucleatum in tumor tissue. Design, Setting, and Participants A prospective cohort study was conducted using data from the Nurses’ Health Study (June 1, 1980, to June 1, 2012) and the Health Professionals Follow-up Study (June 1, 1986, to June 1, 2012) on a total of 121 700 US female nurses and 51 529 US male health professionals aged 30 to 55 years and 40 to 75 years, respectively (both predominantly white individuals), at enrollment. Data analysis was performed from March 15, 2015, to August 10, 2016. Exposures Prudent and Western diets. Main Outcomes and Measures Incidence of colorectal carcinoma subclassified by F nucleatum status in tumor tissue, determined by quantitative polymerase chain reaction. Results Of the 173 229 individuals considered for the study, 137 217 were included in the analysis, 47 449 were male (34.6%), and mean (SD) baseline age for men was 54.0 (9.8) years and for women, 46.3 (7.2) years. A total of 1019 incident colon and rectal cancer cases with available F nucleatum data were documented over 26 to 32 years of follow-up, encompassing 3 643 562 person-years. The association of prudent diet with colorectal cancer significantly differed by tissue F nucleatum status (P = .01 for heterogeneity); prudent diet score was associated with a lower risk of F nucleatum–positive cancers (P = .003 for trend; multivariable hazard ratio of 0.43; 95% CI, 0.25-0.72, for the highest vs the lowest prudent score quartile) but not with F nucleatum–negative cancers (P = .47 for trend, the corresponding multivariable hazard ratio of 0.95; 95% CI, 0.77-1.17). There was no significant heterogeneity between the subgroups in relation to Western dietary pattern scores. Conclusions and Relevance Prudent diets rich in whole grains and dietary fiber are associated with a lower risk for F nucleatum–positive colorectal cancer but not F nucleatum–negative cancer, supporting a potential role for intestinal microbiota in mediating the association between diet and colorectal neoplasms.

LIN28 cooperates with WNT signaling to drive invasive intestinal and colorectal adenocarcinoma in mice and humans

Colorectal cancer (CRC) remains a major contributor to cancer-related mortality. LIN28A and LIN28B are highly related RNA-binding protein paralogs that regulate biogenesis of let-7 microRNAs and influence development, metabolism, tissue regeneration, and oncogenesis. Here we demonstrate that overexpression of either LIN28 paralog cooperates with the Wnt pathway to promote invasive intestinal adenocarcinoma in murine models. When LIN28 alone is induced genetically, half of the resulting tumors harbor Ctnnb1 (β-catenin) mutation. When overexpressed in Apc(Min/+) mice, LIN28 accelerates tumor formation and enhances proliferation and invasiveness. In conditional genetic models, enforced expression of a LIN28-resistant form of the let-7 microRNA reduces LIN28-induced tumor burden, while silencing of LIN28 expression reduces tumor volume and increases tumor differentiation, indicating that LIN28 contributes to tumor maintenance. We detected aberrant expression of LIN28A and/or LIN28B in 38% of a large series of human CRC samples (n = 595), where LIN28 expression levels were associated with invasive tumor growth. Our late-stage CRC murine models and analysis of primary human tumors demonstrate prominent roles for both LIN28 paralogs in promoting CRC growth and progression and implicate the LIN28/let-7 pathway as a therapeutic target.

Loss of CDH1 (E-cadherin) expression is associated with infiltrative tumour growth and lymph node metastasis

Background:Loss of CDH1 (E-cadherin) expression in cancer cells may promote cell migration and invasion. Therefore, we hypothesised that loss of CDH1 expression in colorectal carcinoma might be associated with aggressive features and clinical outcome.Methods:Utilising molecular pathological epidemiology database of 689 rectal and colon cancer cases in the Nurses’ Health Study and the Health Professionals Follow-up Study, we assessed tumour CDH1 expression by immunohistochemistry. Multivariate logistic regression analysis was conducted to assess association of CDH1 loss with tumour growth pattern (expansile-intermediate vs infiltrative) and lymph node metastasis and distant metastasis, controlling for potential confounders including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, and PIK3CA, BRAF and KRAS mutations. Mortality according to CDH1 status was assessed using Cox proportional hazards model.Results:Loss of tumour CDH1 expression was observed in 356 cases (52%), and associated with infiltrative tumour growth pattern (odds ratio (OR), 2.02; 95% confidence interval (CI), 1.23–3.34; P=0.006) and higher pN stage (OR, 1.73; 95% CI, 1.23–2.43; P=0.001). Tumour CDH1 expression was not significantly associated with distant metastasis or prognosis.Conclusions:Loss of CDH1 expression in colorectal cancer is associated with infiltrative tumour growth pattern and lymph node metastasis.

Tumor LINE-1 methylation level and colorectal cancer location in relation to patient survival

Colorectal tumors arise with genomic and epigenomic alterations through interactions between neoplastic cells, immune cells, and microbiota that vary along the proximal to distal axis of colorectum. Long interspersed nucleotide element-1 (LINE-1) hypomethylation in colorectal cancer has been associated with worse clinical outcome. Utilizing 1,317 colon and rectal carcinoma cases in two U.S.-nationwide prospective cohort studies, we examined patient survival according to LINE-1 methylation level stratified by tumor location. Cox proportional hazards model was used to assess a statistical interaction between LINE-1 methylation level and tumor location in colorectal cancer-specific mortality analysis, controlling for potential confounders including microsatellite instability, CpG island methylator phenotype, and KRAS, BRAF, and PIK3CA mutations. A statistically significant interaction was found between LINE-1 methylation level and tumor location in colorectal cancer-specific mortality analysis (Pinteraction = 0.011). The association of LINE-1 hypomethylation with higher colorectal cancer-specific mortality was stronger in proximal colon cancers (multivariable hazard ratio [HR], 1.66; 95% confidence interval [CI], 1.21 to 2.28) than in distal colon cancers (multivariable HR, 1.18; 95% CI, 0.81 to 1.72) or rectal cancers (multivariable HR, 0.87; 95% CI, 0.57 to 1.34). Our data suggest the interactive effect of LINE-1 methylation level and colorectal cancer location on clinical outcome.

MicroRNA let-7, T Cells, and Patient Survival in Colorectal Cancer

The population-based data presented in this study support a possible role for microRNA let-7a in the suppression of antitumor immunity in colorectal cancer patients. This may have implications for expanding the benefit of immunotherapy through targeting microRNAs. Experimental evidence suggests that the let-7 family of noncoding RNAs suppresses adaptive immune responses, contributing to immune evasion by the tumor. We hypothesized that the amount of let-7a and let-7b expression in colorectal carcinoma might be associated with limited T-lymphocyte infiltrates in the tumor microenvironment and worse clinical outcome. Utilizing the molecular pathological epidemiology resources of 795 rectal and colon cancers in two U.S.-nationwide prospective cohort studies, we measured tumor-associated let-7a and let-7b expression levels by quantitative reverse-transcription PCR, and CD3+, CD8+, CD45RO (PTPRC)+, and FOXP3+ cell densities by tumor tissue microarray immunohistochemistry and computer-assisted image analysis. Logistic regression analysis and Cox proportional hazards regression were used to assess associations of let-7a (and let-7b) expression (quartile predictor variables) with T-cell densities (binary outcome variables) and mortality, respectively, controlling for tumor molecular features, including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, and KRAS, BRAF, and PIK3CA mutations. Compared with cases in the lowest quartile of let-7a expression, those in the highest quartile were associated with lower densities of CD3+ [multivariate odds ratio (OR), 0.40; 95% confidence interval (CI), 0.23–0.67; Ptrend = 0.003] and CD45RO+ cells (multivariate OR, 0.31; 95% CI, 0.17–0.58; Ptrend = 0.0004), and higher colorectal cancer-specific mortality (multivariate hazard ratio, 1.82; 95% CI, 1.42–3.13; Ptrend = 0.001). In contrast, let-7b expression was not significantly associated with T-cell density or colorectal cancer prognosis. Our data support the role of let-7a in suppressing antitumor immunity in colorectal cancer and suggest let-7a as a potential target of immunotherapy. Cancer Immunol Res; 4(11); 927–35. ©2016 AACR.

Association Between Somatostatin Receptor Expression and Clinical Outcomes in Neuroendocrine Tumors

Objective Somatostatin receptors (SSTRs), products of gene superfamily SSTR1-5, are commonly expressed in neuroendocrine tumors (NETs). Somatostatin analogs (SSAs) bind to SSTRs and are used as therapeutic agents in patients with advanced NETs. We hypothesized that tumor SSTR expression status would be associated with clinical outcomes in NET. Methods Expression of the 5 SSTRs was evaluated by immunohistochemistry, using tissue microarrays comprising 173 primary NETs, 24 matched metastases, and 22 metastatic NETs from 195 patients. Cox proportional hazards regression analysis was used to assess the association of SSTR expression status (high vs low) with clinical outcomes, adjusting for potential confounders. Results High expression of SSTR2 was associated with longer overall survival (OS) in the cohort overall (multivariate hazard ratio, 0.42; 95% confidence interval, 0.21–0.84; P = 0.013). In a subgroup of patients with metastatic small intestine NET treated with SSAs and evaluable for progression, SSTR2 expression was associated with both longer progression-free survival (PFS) and OS. No associations with PFS or OS were observed with expression of other SSTRs. Conclusions Our study demonstrated that expression of SSTR2, but not other SSTRs, is associated with longer OS. In patients treated with SSAs, expression of SSTR2 is associated with longer PFS survival.