Bayesian risk prediction model for colorectal cancer mortality through integration of clinicopathologic and genomic data.

Routine tumor-node-metastasis (TNM) staging of colorectal cancer is imperfect in predicting survival due to tumor pathobiological heterogeneity and imprecise assessment of tumor spread. We leveraged Bayesian additive regression trees (BART), a statistical learning technique, to comprehensively analyze patient-specific tumor characteristics for the improvement of prognostic prediction. Of 75 clinicopathologic, immune, microbial, and genomic variables in 815 stage II-III patients within two U.S.-wide prospective cohort studies, the BART risk model identified seven stable survival predictors. Risk stratifications (low risk, intermediate risk, and high risk) based on model-predicted survival were statistically significant (hazard ratios 0.19-0.45, vs. higher risk; P < 0.0001) and could be externally validated using The Cancer Genome Atlas (TCGA) data (P = 0.0004). BART demonstrated model flexibility, interpretability, and comparable or superior performance to other machine-learning models. Integrated bioinformatic analyses using BART with tumor-specific factors can robustly stratify colorectal cancer patients into prognostic groups and be readily applied to clinical oncology practice.

Prognostic role of detailed colorectal location and tumor molecular features: analyses of 13,101 colorectal cancer patients including 2994 early-onset cases.

BACKGROUND: The pathogenic effect of colorectal tumor molecular features may be influenced by several factors, including those related to microbiota, inflammation, metabolism, and epigenetics, which may change along colorectal segments. We hypothesized that the prognostic association of colon cancer location might differ by tumor molecular characteristics. METHODS: Utilizing a consortium dataset of 13,101 colorectal cancer cases, including 2994 early-onset cases, we conducted survival analyses of detailed tumor location stratified by statuses of microsatellite instability (MSI), CpG island methylator phenotype (CIMP), and KRAS and BRAF oncogenic mutation. RESULTS: There was a statistically significant trend for better colon cancer-specific survival in relation to tumor location from the cecum to sigmoid colon (Ptrend = 0.002), excluding the rectum. The prognostic association of colon location differed by MSI status (Pinteraction = 0.001). Non-MSI-high tumors exhibited the cecum-to-sigmoid trend for better colon cancer-specific survival [Ptrend < 0.001; multivariable hazard ratio (HR) for the sigmoid colon (vs. cecum), 0.80; 95% confidence interval (CI) 0.70-0.92], whereas MSI-high tumors demonstrated a suggestive cecum-to-sigmoid trend for worse survival (Ptrend = 0.020; the corresponding HR, 2.13; 95% CI 1.15-3.92). The prognostic association of colon tumor location also differed by CIMP status (Pinteraction = 0.003) but not significantly by age, stage, or other features. Furthermore, MSI-high status was a favorable prognostic indicator in all stages. CONCLUSIONS: Both detailed colonic location and tumor molecular features need to be accounted for colon cancer prognostication to advance precision medicine. Our study indicates the important role of large-scale studies to robustly examine detailed colonic subsites in molecular oncology research.

Molecular Characteristics of Early-Onset Colorectal Cancer According to Detailed Anatomical Locations: Comparison With Later-Onset Cases.

INTRODUCTION: Early-onset colorectal cancer diagnosed before the age of 50 years has been increasing. Likely reflecting the pathogenic role of the intestinal microbiome, which gradually changes across the entire colorectal length, the prevalence of certain tumor molecular characteristics gradually changes along colorectal subsites. Understanding how colorectal tumor molecular features differ by age and tumor location is important in personalized patient management. METHODS: Using 14,004 cases with colorectal cancer including 3,089 early-onset cases, we examined microsatellite instability (MSI), CpG island methylator phenotype (CIMP), and KRAS and BRAF mutations in carcinomas of the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum and compared early-onset cases with later-onset cases. RESULTS: The proportions of MSI-high, CIMP-high, and BRAF -mutated early-onset tumors were lowest in the rectum (8.8%, 3.4%, and 3.5%, respectively) and highest in the ascending colon (46% MSI-high; 15% CIMP-high) or transverse colon (8.6% BRAF -mutated) (all Ptrend <0.001 across the rectum to ascending colon). Compared with later-onset tumors, early-onset tumors showed a higher prevalence of MSI-high status and a lower prevalence of CIMP-high status and BRAF mutations in most subsites. KRAS mutation prevalence was higher in the cecum compared with that in the other subsites in both early-onset and later-onset tumors ( P < 0.001). Notably, later-onset MSI-high tumors showed a continuous decrease in KRAS mutation prevalence from the rectum (36%) to ascending colon (9%; Ptrend <0.001), followed by an increase in the cecum (14%), while early-onset MSI-high cancers showed no such trend. DISCUSSION: Our findings support biogeographical and pathogenic heterogeneity of colorectal carcinomas in different colorectal subsites and age groups.

Molecular and Pathology Features of Colorectal Tumors and Patient Outcomes Are Associated with Fusobacterium nucleatum and Its Subspecies animalis.

BACKGROUND: Fusobacterium nucleatum (F. nucleatum) activates oncogenic signaling pathways and induces inflammation to promote colorectal carcinogenesis. METHODS: We characterized F. nucleatum and its subspecies in colorectal tumors and examined associations with tumor characteristics and colorectal cancer-specific survival. We conducted deep sequencing of nusA, nusG, and bacterial 16s rRNA genes in tumors from 1,994 patients with colorectal cancer and assessed associations between F. nucleatum presence and clinical characteristics, colorectal cancer-specific mortality, and somatic mutations. RESULTS: F. nucleatum, which was present in 10.3% of tumors, was detected in a higher proportion of right-sided and advanced-stage tumors, particularly subspecies animalis. Presence of F. nucleatum was associated with higher colorectal cancer-specific mortality (HR, 1.97; P = 0.0004). This association was restricted to nonhypermutated, microsatellite-stable tumors (HR, 2.13; P = 0.0002) and those who received chemotherapy [HR, 1.92; confidence interval (CI), 1.07-3.45; P = 0.029). Only F. nucleatum subspecies animalis, the main subspecies detected (65.8%), was associated with colorectal cancer-specific mortality (HR, 2.16; P = 0.0016), subspecies vincentii and nucleatum were not (HR, 1.07; P = 0.86). Additional adjustment for tumor stage suggests that the effect of F. nucleatum on mortality is partly driven by a stage shift. Presence of F. nucleatum was associated with microsatellite instable tumors, tumors with POLE exonuclease domain mutations, and ERBB3 mutations, and suggestively associated with TP53 mutations. CONCLUSIONS: F. nucleatum, and particularly subspecies animalis, was associated with a higher colorectal cancer-specific mortality and specific somatic mutated genes. IMPACT: Our findings identify the F. nucleatum subspecies animalis as negatively impacting colorectal cancer mortality, which may occur through a stage shift and its effect on chemoresistance.

Association Between Smoking and Molecular Subtypes of Colorectal Cancer.

Background: Smoking is associated with colorectal cancer (CRC) risk. Previous studies suggested this association may be restricted to certain molecular subtypes of CRC, but large-scale comprehensive analysis is lacking. Methods: A total of 9789 CRC cases and 11 231 controls of European ancestry from 11 observational studies were included. We harmonized smoking variables across studies and derived sex study-specific quartiles of pack-years of smoking for analysis. Four somatic colorectal tumor markers were assessed individually and in combination, including BRAF mutation, KRAS mutation, CpG island methylator phenotype (CIMP), and microsatellite instability (MSI) status. A multinomial logistic regression analysis was used to assess the association between smoking and risk of CRC subtypes by molecular characteristics, adjusting for age, sex, and study. All statistical tests were 2-sided and adjusted for Bonferroni correction. Results: Heavier smoking was associated with higher risk of CRC overall and stratified by individual markers (P trend < .001). The associations differed statistically significantly between all molecular subtypes, which was the most statistically significant for CIMP and BRAF. Compared with never-smokers, smokers in the fourth quartile of pack-years had a 90% higher risk of CIMP-positive CRC (odds ratio = 1.90, 95% confidence interval = 1.60 to 2.26) but only 35% higher risk for CIMP-negative CRC (odds ratio = 1.35, 95% confidence interval = 1.22 to 1.49; P difference = 2.1 x 10-6). The association was also stronger in tumors that were CIMP positive, MSI high, or KRAS wild type when combined (P difference < .001). Conclusion: Smoking was associated with differential risk of CRC subtypes defined by molecular characteristics. Heavier smokers had particularly higher risk of CRC subtypes that were CIMP positive and MSI high in combination, suggesting that smoking may be involved in the development of colorectal tumors via the serrated pathway.

Prognostic significance of myeloid immune cells and their spatial distribution in the colorectal cancer microenvironment.

BACKGROUND: Myeloid cells represent an abundant yet heterogeneous cell population in the colorectal cancer microenvironment, and their roles remain poorly understood. METHODS: We used multiplexed immunofluorescence combined with digital image analysis to identify CD14+ monocytic and CD15+ granulocytic cells and to evaluate their maturity (HLA-DR and CD33), immunosuppressive potential (ARG1) and proximity to cytokeratin (KRT)-positive tumor cells in 913 colorectal carcinomas. Using covariate data of 4465 incident colorectal cancers in two prospective cohort studies, the inverse probability weighting method was used with multivariable-adjusted Cox proportional hazards models to assess cancer-specific mortality according to ordinal quartiles (Q1-Q4) of myeloid cell densities. Immune cell-tumor cell proximity was measured with the nearest neighbor method and the G-cross function, which determines the likelihood of any tumor cell having at least one immune cell of the specified type within a certain radius. RESULTS: Higher intraepithelial (Ptrend=0.0002; HR for Q4 (vs Q1), 0.48, 95% CI 0.31 to 0.76) and stromal (Ptrend <0.0001; HR for Q4 (vs Q1), 0.42, 95% CI 0.29 to 0.63) densities of CD14+HLA-DR+ cells were associated with lower colorectal cancer-specific mortality while, conversely, higher intraepithelial densities of CD14+HLA-DR- cells were associated with higher colorectal cancer-specific mortality (Ptrend=0.0003; HR for Q4 (vs Q1), 1.78, 95% CI 1.25 to 2.55). Spatial analyses indicated that CD15+ cells were located closer to tumor cells than CD14+ cells, and CD14+HLA-DR+ cells were closer to tumor than CD14+HLA-DR- cells (p<0.0001). The G-cross proximity measurement, evaluating the difference in the likelihood of any tumor cell being colocated with at least one CD14+HLA-DR+ cell versus CD14+HLA-DR- cell within a 20 µm radius, was associated with lower colorectal cancer-specific mortality (Ptrend <0.0001; HR for Q4 (vs Q1), 0.37, 95% CI 0.24 to 0.57). CONCLUSIONS: Myeloid cell populations occur in spatially distinct distributions and exhibit divergent, subset-specific prognostic significance in colorectal cancer, with mature CD14+HLA-DR+ and immature CD14+HLA-DR- monocytic phenotypes most notably showing opposite associations. These results highlight the prognostic utility of multimarker evaluation of myeloid cell infiltrates and reveal a previously unrecognized degree of spatial organization for myeloid cells in the immune microenvironment.

Association of Screening Lower Endoscopy With Colorectal Cancer Incidence and Mortality in Adults Older Than 75 Years.

IMPORTANCE: Evidence indicates that screening for colorectal cancer (CRC) beginning at 50 years of age can detect early-stage CRC and premalignant neoplasms (eg, adenomas) and thus prevent CRC-related mortality. At present, the US Preventive Services Task Force recommends continuing CRC screening until 75 years of age and individualized decision-making for adults older than 75 years, while accounting for a patient's overall health and screening history. However, scant data exist to support these recommendations. OBJECTIVE: To examine the association of lower gastrointestinal tract screening endoscopy with the risk of CRC incidence and CRC-related mortality in older US adults. DESIGN, SETTING, AND PARTICIPANTS: This prospective cohort study of health care professionals in the US included data from the Nurses' Health Study (NHS) and Health Professionals Follow-up Study (HPFS) from January 1, 1988, through January 31, 2016, for the HPFS and June 30, 2016, for the NHS. Data were analyzed from May 8, 2019, to July 9, 2020. EXPOSURES: History of screening sigmoidoscopy or colonoscopy (routine/average risk or positive family history) to 75 years of age and after 75 years of age, assessed every 2 years. MAIN OUTCOMES AND MEASURES: Incidence of CRC and CRC-related mortality confirmed by National Death Index, medical records, and pathology reports. RESULTS: Among 56 374 participants who reached 75 years of age during follow-up (36.8% men and 63.2% women), 661 incident CRC cases and 323 CRC-related deaths were documented. Screening endoscopy after 75 years of age was associated with reduced risk of CRC incidence (multivariable hazard ratio [HR], 0.61; 95% CI, 0.51-0.74) and CRC-related mortality (HR, 0.60; 95% CI, 0.46-0.78), regardless of screening history. The HR comparing screening with nonscreening after 75 years of age was 0.67 (95% CI, 0.50-0.89) for CRC incidence and 0.58 (95% CI, 0.38-0.87) for CRC-related mortality among participants who underwent screening endoscopy before 75 years of age, and 0.51 (95% CI, 0.37-0.70) for CRC incidence and 0.63 (95% CI, 0.43-0.93) for CRC-related mortality among participants without a screening history. However, screening endoscopy after 75 years of age was not associated with risk reduction in CRC death among participants with cardiovascular disease (HR, 1.18; 95% CI, 0.59-2.35) or significant comorbidities (HR, 1.17; 95% CI, 0.57-2.43). CONCLUSIONS AND RELEVANCE: In this cohort study, endoscopy among individuals older than 75 years was associated with lower risk of CRC incidence and CRC-related mortality. These data support continuation of screening after 75 years of age among individuals without significant comorbidities.

Association of Fusobacterium nucleatum with Specific T-cell Subsets in the Colorectal Carcinoma Microenvironment.

PURPOSE: While evidence indicates that Fusobacterium nucleatum (F. nucleatum) may promote colorectal carcinogenesis through its suppressive effect on T-cell-mediated antitumor immunity, the specific T-cell subsets involved remain uncertain. EXPERIMENTAL DESIGN: We measured F. nucleatum DNA within tumor tissue by quantitative PCR on 933 cases (including 128 F. nucleatum-positive cases) among 4,465 incident colorectal carcinoma cases in two prospective cohorts. Multiplex immunofluorescence combined with digital image analysis and machine learning algorithms for CD3, CD4, CD8, CD45RO (PTPRC isoform), and FOXP3 measured various T-cell subsets. We leveraged data on Bifidobacterium, microsatellite instability (MSI), tumor whole-exome sequencing, and M1/M2-type tumor-associated macrophages [TAM; by CD68, CD86, IRF5, MAF, and MRC1 (CD206) multimarker assay]. Using the 4,465 cancer cases and inverse probability weighting method to control for selection bias due to tissue availability, multivariable-adjusted logistic regression analysis assessed the association between F. nucleatum and T-cell subsets. RESULTS: The amount of F. nucleatum was inversely associated with tumor stromal CD3+ lymphocytes [multivariable OR, 0.47; 95% confidence interval (CI), 0.28-0.79, for F. nucleatum-high vs. -negative category; P trend = 0.0004] and specifically stromal CD3+CD4+CD45RO+ cells (corresponding multivariable OR, 0.52; 95% CI, 0.32-0.85; P trend = 0.003). These relationships did not substantially differ by MSI status, neoantigen load, or exome-wide tumor mutational burden. F. nucleatum was not significantly associated with tumor intraepithelial T cells or with M1 or M2 TAMs. CONCLUSIONS: The amount of tissue F. nucleatum is associated with lower density of stromal memory helper T cells. Our findings provide evidence for the interactive pathogenic roles of microbiota and specific immune cells.

The Prognostic Role of Macrophage Polarization in the Colorectal Cancer Microenvironment.

Macrophages are among the most common cells in the colorectal cancer microenvironment, but their prognostic significance is incompletely understood. Using multiplexed immunofluorescence for CD68, CD86, IRF5, MAF, MRC1 (CD206), and KRT (cytokeratins) combined with digital image analysis and machine learning, we assessed the polarization spectrum of tumor-associated macrophages in 931 colorectal carcinomas. We then applied Cox proportional hazards regression to assess prognostic survival associations of intraepithelial and stromal densities of M1-like and M2-like macrophages while controlling for potential confounders, including stage and microsatellite instability status. We found that high tumor stromal density of M2-like macrophages was associated with worse cancer-specific survival, whereas tumor stromal density of M1-like macrophages was not significantly associated with better cancer-specific survival. High M1:M2 density ratio in tumor stroma was associated with better cancer-specific survival. Overall macrophage densities in tumor intraepithelial or stromal regions were not prognostic. These findings suggested that macrophage polarization state, rather than their overall density, was associated with cancer-specific survival, with M1- and M2-like macrophage phenotypes exhibiting distinct prognostic roles. These results highlight the utility of a multimarker strategy to assess the macrophage polarization at single-cell resolution within the tumor microenvironment.

Plasma IL-6 changes correlate to PD-1 inhibitor responses in NSCLC.

BACKGROUND: Blood-based biomarkers of anti-solid tumor immune checkpoint blockade (ICB) response are lacking. We hypothesized that changes in systemic cytokine levels with the initial doses of programmed cell death protein 1 (PD-1) pathway inhibitors would correlate with clinical responses. New ultrasensitive ELISA technology enables quantitation of plasma proteins in sub-picogram-per-milliliter concentrations. METHODS: We measured plasma cytokines by ultrasensitive single-molecule array assays in patients with non-small-cell lung carcinoma before and during treatment with anti-PD-1 therapy. Association with best overall response and progression-free survival (PFS) was assessed by Kruskall-Wallis test and Kaplan-Meier plots with log-rank test, respectively. RESULTS: A decrease in interleukin 6 (IL-6) levels was associated with improved PFS (n=47 patients, median PFS: 11 vs 4 months, HR 0.45 (95% CI 0.23 to 0.89), p=0.04). The extent of change in IL-6 differed between best overall response categories (p=0.01) and correlated with changes in C reactive protein levels. We also explored plasma cytokine levels in relation to immune-related adverse effects and observed some correlation. CONCLUSIONS: This study suggests the presence of a systemic, proteomic reflection of successful ICB outside the tumor microenvironment with plasma decreases in IL-6 and CRP.

Smoking Status at Diagnosis and Colorectal Cancer Prognosis According to Tumor Lymphocytic Reaction.

BACKGROUND: Smoking has been associated with worse colorectal cancer patient survival and may potentially suppress the immune response in the tumor microenvironment. We hypothesized that the prognostic association of smoking behavior at colorectal cancer diagnosis might differ by lymphocytic reaction patterns in cancer tissue. METHODS: Using 1474 colon and rectal cancer patients within 2 large prospective cohort studies (Nurses' Health Study and Health Professionals Follow-up Study), we characterized 4 patterns of histopathologic lymphocytic reaction, including tumor-infiltrating lymphocytes (TILs), intratumoral periglandular reaction, peritumoral lymphocytic reaction, and Crohn's-like lymphoid reaction. Using covariate data of 4420 incident colorectal cancer patients in total, an inverse probability weighted multivariable Cox proportional hazards regression model was conducted to adjust for selection bias due to tissue availability and potential confounders, including tumor differentiation, disease stage, microsatellite instability status, CpG island methylator phenotype, long interspersed nucleotide element-1 methylation, and KRAS, BRAF, and PIK3CA mutations. RESULTS: The prognostic association of smoking status at diagnosis differed by TIL status. Compared with never smokers, the multivariable-adjusted colorectal cancer-specific mortality hazard ratio for current smokers was 1.50 (95% confidence interval = 1.10 to 2.06) in tumors with negative or low TIL and 0.43 (95% confidence interval = 0.16 to 1.12) in tumors with intermediate or high TIL (2-sided P interaction = .009). No statistically significant interactions were observed in the other patterns of lymphocytic reaction. CONCLUSIONS: The association of smoking status at diagnosis with colorectal cancer mortality may be stronger for carcinomas with negative or low TIL, suggesting a potential interplay of smoking and lymphocytic reaction in the colorectal cancer microenvironment.

Intake of Dietary Fruit, Vegetables, and Fiber and Risk of Colorectal Cancer According to Molecular Subtypes: A Pooled Analysis of 9 Studies.

Protective associations of fruits, vegetables, and fiber intake with colorectal cancer risk have been shown in many, but not all epidemiologic studies. One possible reason for study heterogeneity is that dietary factors may have distinct effects by colorectal cancer molecular subtypes. Here, we investigate the association of fruit, vegetables, and fiber intake with four well-established colorectal cancer molecular subtypes separately and in combination. Nine observational studies including 9,592 cases with molecular subtypes for microsatellite instability (MSI), CpG island methylator phenotype (CIMP), and somatic mutations in BRAF and KRAS genes, and 7,869 controls were analyzed. Both case-only logistic regression analyses and polytomous logistic regression analyses (with one control set and multiple case groups) were used. Higher fruit intake was associated with a trend toward decreased risk of BRAF-mutated tumors [OR 4th vs. 1st quartile = 0.82 (95% confidence interval, 0.65-1.04)] but not BRAF-wildtype tumors [1.09 (0.97-1.22); P difference as shown in case-only analysis = 0.02]. This difference was observed in case-control studies and not in cohort studies. Compared with controls, higher fiber intake showed negative association with colorectal cancer risk for cases with microsatellite stable/MSI-low, CIMP-negative, BRAF-wildtype, and KRAS-wildtype tumors (P trend range from 0.03 to 3.4e-03), which is consistent with the traditional adenoma-colorectal cancer pathway. These negative associations were stronger compared with MSI-high, CIMP-positive, BRAF-mutated, or KRAS-mutated tumors, but the differences were not statistically significant. These inverse associations for fruit and fiber intake may explain, in part, inconsistent findings between fruit or fiber intake and colorectal cancer risk that have previously been reported. SIGNIFICANCE: These analyses by colorectal cancer molecular subtypes potentially explain the inconsistent findings between dietary fruit or fiber intake and overall colorectal cancer risk that have previously been reported.

Landscape of somatic single nucleotide variants and indels in colorectal cancer and impact on survival.

Colorectal cancer (CRC) is a biologically heterogeneous disease. To characterize its mutational profile, we conduct targeted sequencing of 205 genes for 2,105 CRC cases with survival data. Our data shows several findings in addition to enhancing the existing knowledge of CRC. We identify PRKCI, SPZ1, MUTYH, MAP2K4, FETUB, and TGFBR2 as additional genes significantly mutated in CRC. We find that among hypermutated tumors, an increased mutation burden is associated with improved CRC-specific survival (HR = 0.42, 95% CI: 0.21-0.82). Mutations in TP53 are associated with poorer CRC-specific survival, which is most pronounced in cases carrying TP53 mutations with predicted 0% transcriptional activity (HR = 1.53, 95% CI: 1.21-1.94). Furthermore, we observe differences in mutational frequency of several genes and pathways by tumor location, stage, and sex. Overall, this large study provides deep insights into somatic mutations in CRC, and their potential relationships with survival and tumor features.

Prognostic Significance of Immune Cell Populations Identified by Machine Learning in Colorectal Cancer Using Routine Hematoxylin and Eosin-Stained Sections.

PURPOSE: Although high T-cell density is a well-established favorable prognostic factor in colorectal cancer, the prognostic significance of tumor-associated plasma cells, neutrophils, and eosinophils is less well-defined. EXPERIMENTAL DESIGN: We computationally processed digital images of hematoxylin and eosin (H&E)-stained sections to identify lymphocytes, plasma cells, neutrophils, and eosinophils in tumor intraepithelial and stromal areas of 934 colorectal cancers in two prospective cohort studies. Multivariable Cox proportional hazards regression was used to compute mortality HR according to cell density quartiles. The spatial patterns of immune cell infiltration were studied using the GTumor:Immune cell function, which estimates the likelihood of any tumor cell in a sample having at least one neighboring immune cell of the specified type within a certain radius. Validation studies were performed on an independent cohort of 570 colorectal cancers. RESULTS: Immune cell densities measured by the automated classifier demonstrated high correlation with densities both from manual counts and those obtained from an independently trained automated classifier (Spearman's ρ 0.71-0.96). High densities of stromal lymphocytes and eosinophils were associated with better cancer-specific survival [P trend < 0.001; multivariable HR (4th vs 1st quartile of eosinophils), 0.49; 95% confidence interval, 0.34-0.71]. High GTumor:Lymphocyte area under the curve (AUC0,20µm; P trend = 0.002) and high GTumor:Eosinophil AUC0,20µm (P trend < 0.001) also showed associations with better cancer-specific survival. High stromal eosinophil density was also associated with better cancer-specific survival in the validation cohort (P trend < 0.001). CONCLUSIONS: These findings highlight the potential for machine learning assessment of H&E-stained sections to provide robust, quantitative tumor-immune biomarkers for precision medicine.

Metabolic Profiling of Formalin-Fixed Paraffin-Embedded Tissues Discriminates Normal Colon from Colorectal Cancer.

Accumulating evidence suggests that metabolic reprogramming has a critical role in carcinogenesis and tumor progression. The usefulness of formalin-fixed paraffin-embedded (FFPE) tissue material for metabolomics analysis as compared with fresh frozen tissue material remains unclear. LC/MS-MS-based metabolomics analysis was performed on 11 pairs of matched tumor and normal tissues in both FFPE and fresh frozen tissue materials from patients with colorectal carcinoma. Permutation t test was applied to identify metabolites with differential abundance between tumor and normal tissues. A total of 200 metabolites were detected in the FFPE samples and 536 in the fresh frozen samples. The preservation of metabolites in FFPE samples was diverse according to classes and chemical characteristics, ranging from 78% (energy) to 0% (peptides). Compared with the normal tissues, 34 (17%) and 174 (32%) metabolites were either accumulated or depleted in the tumor tissues derived from FFPE and fresh frozen samples, respectively. Among them, 15 metabolites were common in both FFPE and fresh frozen samples. Notably, branched chain amino acids were highly accumulated in tumor tissues. Using KEGG pathway analyses, glyoxylate and dicarboxylate metabolism, arginine and proline, glycerophospholipid, and glycine, serine, and threonine metabolism pathways distinguishing tumor from normal tissues were found in both FFPE and fresh frozen samples. This study demonstrates that informative data of metabolic profiles can be retrieved from FFPE tissue materials. IMPLICATIONS: Our findings suggest potential value of metabolic profiling using FFPE tumor tissues and may help to shape future translational studies through developing treatment strategies targeting metabolites.

An integrated analysis of lymphocytic reaction, tumour molecular characteristics and patient survival in colorectal cancer.

BACKGROUND: Histological lymphocytic reaction is regarded as an independent prognostic marker in colorectal cancer. Considering the lack of adequate statistical power, adjustment for selection bias and comprehensive tumour molecular data in most previous studies, we investigated the strengths of the prognostic associations of lymphocytic reaction in colorectal carcinoma by utilising an integrative database of two prospective cohort studies. METHODS: We examined Crohn's-like reaction, intratumoural periglandular reaction, peritumoural reaction and tumour-infiltrating lymphocytes in 1465 colorectal carcinoma cases. Using covariate data of 4420 colorectal cancer cases in total, inverse probability-weighted Cox proportional hazard regression model was used to control for selection bias (due to tissue availability) and potential confounders, including stage, MSI status, LINE-1 methylation, PTGS2 and CTNNB1 expression, KRAS, BRAF and PIK3CA mutations, and tumour neoantigen load. RESULTS: Higher levels of each lymphocytic reaction component were associated with better colorectal cancer-specific survival (Ptrend < 0.002). Compared with cases with negative/low intratumoural periglandular reaction, multivariable-adjusted HRs were 0.55 (95% CI, 0.42-0.71) in cases with intermediate reaction and 0.20 (95% CI, 0.12-0.35) in cases with high reaction. These relationships were consistent in strata of MSI status or neoantigen loads (Pinteraction > 0.2). CONCLUSIONS: The four lymphocytic reaction components are prognostic biomarkers in colorectal carcinoma.

Night-Shift Work Duration and Risk of Colorectal Cancer According to IRS1 and IRS2 Expression.

BACKGROUND: We hypothesized that the risk of colorectal cancer in night-shift workers might be different according to insulin receptor substrate status. METHODS: Among 77,470 eligible women having night work assessed in the Nurses' Health Study, we documented a total of 1,397 colorectal cancer cases, of which 304 or 308 had available data on IRS1 and IRS2, respectively. We used duplication-method Cox proportional hazards regression analysis for competing risks to calculate HRs and 95% confidence intervals (CI) for each colorectal cancer subtype. We measured tumor IRS1 or IRS2 expression by immunohistochemistry (IHC). RESULTS: Compared with women who never worked night shifts, those working ≥15 years night shifts had a marginal trend of increased overall risk of colorectal cancer (P trend = 0.06; multivariable HR = 1.20; 95% CI, 0.99-1.45). Longer duration of night-shift work was associated with a higher risk of IRS2-positive tumors (multivariable HR = 2.69; 95% CI, 1.48-4.89; P trend = 0.001, ≥15 years night shifts vs. never) but not with IRS2-negative tumors (multivariable HR = 0.90; 95% CI, 0.54-1.51; P trend = 0.72; P heterogeneity for IRS2 = 0.008). Similarly, the corresponding multivariable HRs were 1.81 for IRS1-positive tumors (95% CI, 0.94-3.48; P trend = 0.06) and 1.13 for IRS1-negative tumors (95% CI, 0.71-1.80; P trend = 0.56; P heterogeneity for IRS1 = 0.02). CONCLUSIONS: Our molecular pathologic epidemiology data suggest a potential role of IRS in mediating carcinogenesis induced by night-shift work. IMPACT: Although these findings need validation, rotating night shift might increase colorectal cancer risk in women with abnormal insulin receptor pathways.

Dietary Intake of Branched-Chain Amino Acids and Risk of Colorectal Cancer.

Branched-chain amino acids (BCAA) are essential amino acids, and emerging evidence suggests that BCAAs may mediate pathways related to cancer progression, possibly due to their involvement in insulin metabolism. We investigated the association between dietary intake of BCAAs with colorectal cancer risk in three prospective cohorts: the Nurses' Health Study I [(NHS), number of participants (n) at baseline = 77,017], NHS II (n = 92,984), and the Health Professionals Follow-up Study [(HPFS) n = 47,255]. Validated food frequency questionnaires were administered every 4 years and follow-up questionnaires on lifestyle biennially. Hazard ratios (HR) and 95% confidence intervals (CI) were calculated using Cox proportional hazards regression models. Pooled HRs were obtained using random effect models. After up to 28 years of follow-up, 1,660 cases were observed in NHS, 306 in NHS II, and 1,343 in HPFS. In multivariable adjusted models, we observed a weak inverse association between BCAA intake and colorectal cancer [highest vs. lowest quintile, pooled HR including all three cohorts (95% CI): 0.89 (0.80-1.00), P trend = 0.06, HR per standard deviation (SD) increment 0.95 (0.92-0.99)]. However, after including dairy calcium to the models, BCAA intake was no longer associated with risk of colorectal cancer [HR 0.96 (0.85-1.08), P trend = 0.50, HR per SD increment 0.97 (0.93-1.01)]. We did not find evidence that higher dietary BCAA intake is associated with higher risk of colorectal cancer. As this is the first prospective study to examine the association between BCAA intake and colorectal cancer, our findings warrant investigation in other cohorts.

Association of autophagy status with amount of Fusobacterium nucleatum in colorectal cancer.

Fusobacterium nucleatum (F. nucleatum), which has been associated with colorectal carcinogenesis, can impair anti-tumour immunity, and actively invade colon epithelial cells. Considering the critical role of autophagy in host defence against microorganisms, we hypothesised that autophagic activity of tumour cells might influence the amount of F. nucleatum in colorectal cancer tissue. Using 724 rectal and colon cancer cases within the Nurses' Health Study and the Health Professionals Follow-up Study, we evaluated autophagic activity of tumour cells by immunohistochemical analyses of BECN1 (beclin 1), MAP1LC3 (LC3), and SQSTM1 (p62) expression. We measured the amount of F. nucleatum DNA in tumour tissue by quantitative polymerase chain reaction (PCR). We conducted multivariable ordinal logistic regression analyses to examine the association of tumour BECN1, MAP1LC3, and SQSTM1 expression with the amount of F. nucleatum, adjusting for potential confounders, including microsatellite instability status; CpG island methylator phenotype; long-interspersed nucleotide element-1 methylation; and KRAS, BRAF, and PIK3CA mutations. Compared with BECN1-low cases, BECN1-intermediate and BECN1-high cases were associated with lower amounts of F. nucleatum with odds ratios (for a unit increase in three ordinal categories of the amount of F. nucleatum) of 0.54 (95% confidence interval, 0.29-0.99) and 0.31 (95% confidence interval, 0.16-0.60), respectively (Ptrend < 0.001 across ordinal BECN1 categories). Tumour MAP1LC3 and SQSTM1 levels were not significantly associated with the amount of F. nucleatum (Ptrend > 0.06). Tumour BECN1, MAP1LC3, and SQSTM1 levels were not significantly associated with patient survival (Ptrend > 0.10). In conclusion, tumour BECN1 expression is inversely associated with the amount of F. nucleatum in colorectal cancer tissue, suggesting a possible role of autophagy in the elimination of invasive microorganisms. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Myc targeted CDK18 promotes ATR and homologous recombination to mediate PARP inhibitor resistance in glioblastoma.

PARP inhibitors (PARPis) have clinical efficacy in BRCA-deficient cancers, but not BRCA-intact tumors, including glioblastoma (GBM). We show that MYC or MYCN amplification in patient-derived glioblastoma stem-like cells (GSCs) generates sensitivity to PARPi via Myc-mediated transcriptional repression of CDK18, while most tumors without amplification are not sensitive. In response to PARPi, CDK18 facilitates ATR activation by interacting with ATR and regulating ATR-Rad9/ATR-ETAA1 interactions; thereby promoting homologous recombination (HR) and PARPi resistance. CDK18 knockdown or ATR inhibition in GSCs suppressed HR and conferred PARPi sensitivity, with ATR inhibitors synergizing with PARPis or sensitizing GSCs. ATR inhibitor VE822 combined with PARPi extended survival of mice bearing GSC-derived orthotopic tumors, irrespective of PARPi-sensitivity. These studies identify a role of CDK18 in ATR-regulated HR. We propose that combined blockade of ATR and PARP is an effective strategy for GBM, even for low-Myc GSCs that do not respond to PARPi alone, and potentially other PARPi-refractory tumors.