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.

Desmoplastic Reaction, Immune Cell Response, and Prognosis in Colorectal Cancer.

Background: The relationships between tumor stromal features (such as desmoplastic reaction, myxoid stroma, and keloid-like collagen bundles) and immune cells in the colorectal carcinoma microenvironment have not yet been fully characterized. Methods: In 908 tumors with available tissue among 4,465 incident colorectal adenocarcinoma cases in two prospective cohort studies, we examined desmoplastic reaction, myxoid stroma, and keloid-like collagen bundles. We conducted multiplex immunofluorescence for T cells [CD3, CD4, CD8, CD45RO (PTPRC), and FOXP3] and for macrophages [CD68, CD86, IRF5, MAF, and MRC1 (CD206)]. We used the inverse probability weighting method and the 4,465 incident cancer cases to adjust for selection bias. Results: Immature desmoplastic reaction was associated with lower densities of intraepithelial CD3+CD8+CD45RO+ cells [multivariable odds ratio (OR) for the highest (vs. lowest) density category, 0.43; 95% confidence interval (CI), 0.29-0.62; Ptrend <0.0001] and stromal M1-like macrophages [the corresponding OR, 0.44; 95% CI, 0.28-0.70; Ptrend = 0.0011]. Similar relations were observed for myxoid stroma [intraepithelial CD3+CD8+CD45RO+ cells (Ptrend <0.0001) and stromal M1-like macrophages (Ptrend = 0.0007)] and for keloid-like collagen bundles (Ptrend <0.0001 for intraepithelial CD3+CD8+CD45RO+ cells). In colorectal cancer-specific survival analyses, multivariable-adjusted hazard ratios (with 95% confidence intervals) were 0.32 (0.23-0.44; Ptrend <0.0001) for mature (vs. immature) desmoplastic reaction, 0.25 (0.16-0.39; Ptrend <0.0001) for absent (vs. marked) myxoid stroma, and 0.12 (0.05-0.28; Ptrend <0.0001) for absent (vs. marked) keloid-like collagen bundles. Conclusions: Immature desmoplastic reaction and myxoid stroma were associated with lower densities of tumor intraepithelial memory cytotoxic T cells and stromal M1-like macrophages, likely reflecting interactions between tumor, immune, and stromal cells in the colorectal tumor microenvironment.

Coffee Intake of Colorectal Cancer Patients and Prognosis According to Histopathologic Lymphocytic Reaction and T-Cell Infiltrates.

Given previous biologic evidence of immunomodulatory effects of coffee, we hypothesized that the association between coffee intake of colorectal cancer patients and survival differs by immune responses. Using a molecular pathologic epidemiology database of 4465 incident colorectal cancer cases, including 1262 cases with molecular data, in the Nurses' Health Study and the Health Professionals Follow-up Study, we examined the association between coffee intake of colorectal cancer patients and survival in strata of levels of histopathologic lymphocytic reaction and T-cell infiltrates in tumor tissue. We did not observe a significant association of coffee intake with colorectal cancer-specific mortality (multivariable-adjusted hazard ratio [HR] for 1-cup increase of coffee intake per day, 0.93; 95% CI, 0.84 to 1.03). Although statistical significance was not reached at the stringent level (α=.005), the association of coffee intake with colorectal cancer-specific mortality differed by Crohn disease-like lymphoid reaction (Pinteraction=.007). Coffee intake was associated with lower colorectal cancer-specific mortality in patients with high Crohn disease-like reaction (multivariable HR for 1-cup increase of coffee intake per day, 0.55; 95% CI, 0.37 to 0.81; Ptrend=.002) but not in patients with intermediate Crohn disease-like reaction (the corresponding HR, 1.02; 95% CI, 0.72 to 1.44) or negative/low Crohn disease-like reaction (the corresponding HR, 0.95; 95% CI, 0.83 to 1.07). The associations of coffee intake with colorectal cancer-specific mortality did not significantly differ by levels of other lymphocytic reaction or any T-cell subset (Pinteraction>.18). There is suggestive evidence for differential prognostic effects of coffee intake by Crohn disease-like lymphoid reaction in colorectal cancer.

Immune cell profiles in the tumor microenvironment of early-onset, intermediate-onset, and later-onset colorectal cancer.

BACKGROUND: Despite heightened interest in early-onset colorectal cancer (CRC) diagnosed before age 50, little is known on immune cell profiles of early-onset CRC. It also remains to be studied whether CRCs diagnosed at or shortly after age 50 are similar to early-onset CRC. We therefore hypothesized that immune cell infiltrates in CRC tissue might show differential heterogeneity patterns between three age groups (< 50 "early onset," 50-54 "intermediate onset," ≥ 55 "later onset"). METHODS: We examined 1,518 incident CRC cases with available tissue data, including 35 early-onset and 73 intermediate-onset cases. To identify immune cells in tumor intraepithelial and stromal areas, we developed three multiplexed immunofluorescence assays combined with digital image analyses and machine learning algorithms, with the following markers: (1) CD3, CD4, CD8, CD45RO (PTPRC), and FOXP3 for T cells; (2) CD68, CD86, IRF5, MAF, and MRC1 (CD206) for macrophages; and (3) ARG1, CD14, CD15, CD33, and HLA-DR for myeloid cells. RESULTS: Although no comparisons between age groups showed statistically significant differences at the stringent two-sided α level of 0.005, compared to later-onset CRC, early-onset CRC tended to show lower levels of tumor-infiltrating lymphocytes (P = 0.013), intratumoral periglandular reaction (P = 0.025), and peritumoral lymphocytic reaction (P = 0.044). Compared to later-onset CRC, intermediate-onset CRC tended to show lower densities of overall macrophages (P = 0.050), M1-like macrophages (P = 0.062), CD14+HLA-DR+ cells (P = 0.015), and CD3+CD4+FOXP3+ cells (P = 0.039). CONCLUSIONS: This hypothesis-generating study suggests possible differences in histopathologic lymphocytic reaction patterns, macrophages, and regulatory T cells in the tumor microenvironment by age at diagnosis.

Smoking and Incidence of Colorectal Cancer Subclassified by Tumor-Associated Macrophage Infiltrates.

BACKGROUND: Biological evidence indicates that smoking can influence macrophage functions and polarization, thereby promoting tumor evolution. We hypothesized that the association of smoking with colorectal cancer incidence might differ by macrophage infiltrates. METHODS: Using the Nurses' Health Study and the Health Professionals Follow-up Study, we examined the association of smoking with incidence of colorectal cancer subclassified by macrophage counts. Multiplexed immunofluorescence (for CD68, CD86, IRF5, MAF, and MRC1 [CD206]) combined with digital image analysis and machine learning was used to identify overall, M1-polarized, and M2-polarized macrophages in tumor. We used inverse-probability-weighted multivariable Cox proportional hazards regression models to control for potential confounders and selection bias because of tissue data availability. All statistical tests were 2-sided. RESULTS: During follow-up of 131 144 participants (3 648 370 person-years), we documented 3092 incident colorectal cancer cases, including 871 cases with available macrophage data. The association of pack-years smoked with colorectal cancer incidence differed by stromal macrophage densities (Pheterogeneity = .003). Compared with never smoking, multivariable-adjusted hazard ratios (95% confidence interval) for tumors with low macrophage densities were 1.32 (0.97 to 1.79) for 1-19 pack-years, 1.31 (0.92 to 1.85) for 20-39 pack-years, and 1.74 (1.26 to 2.41) for 40 or more pack-years (Ptrend = .004). In contrast, pack-years smoked was not statistically significantly associated with the incidence of tumors having intermediate or high macrophage densities (Ptrend > .009, with an α level of .005). No statistically significant differential association was found for colorectal cancer subclassified by M1-like or M2-like macrophages. CONCLUSIONS: The association of smoking with colorectal cancer incidence is stronger for tumors with lower stromal macrophage counts. Our findings suggest an interplay of smoking and macrophages in colorectal carcinogenesis.

Spatial Organization and Prognostic Significance of NK and NKT-like Cells via Multimarker Analysis of the Colorectal Cancer Microenvironment.

Although tumor-infiltrating T cells hold a beneficial prognostic role in colorectal cancer, other lymphocytic populations are less characterized. We developed a multiplexed immunofluorescence assay coupled with digital image analysis and machine learning to identify natural killer (NK) cells (NCAM1+CD3-), natural killer T-like (NKT-like) cells (NCAM1+CD3+), and T cells (NCAM1-CD3+) within the PTPRC+ (CD45+) cell population and to measure their granzyme B (GZMB; cytotoxicity marker) and FCGR3A (CD16a; NK-cell maturity marker) expression. We evaluated immune cell densities and spatial configuration in 907 incident colorectal carcinoma cases within two prospective cohort studies. We found that T cells were approximately 100 times more abundant than NK and NKT-like cells. Overall, NK cells showed high GZMB expression and were located closer to tumor cells than T and NKT-like cells. In T and NKT-like cells, GZMB expression was enriched in cells in closer proximity to tumor cells. Higher densities of both T and NKT-like cells associated with longer cancer-specific survival, independent of potential confounders (P trend < 0.0007). Higher stromal GZMB+ and FCGR3A+ NK-cell densities associated with longer cancer-specific survival (P trend < 0.003). For T and NKT-like cells, greater proximity to tumor cells associated with longer cancer-specific survival (P trend < 0.0001). These findings indicate that cytotoxic NCAM1+CD3-GZMB+ NK cells and NCAM1+CD3+ NKT-like cells are relatively rare lymphocytic populations within the colorectal cancer microenvironment and show distinct spatial configuration and associations with patient outcome. The results highlight the utility of a quantitative multimarker assay for in situ, single-cell immune biomarker evaluation and underscore the importance of spatial context for tumor microenvironment characterization.

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 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.

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.

Tumour budding, poorly differentiated clusters, and T-cell response in colorectal cancer.

BACKGROUND: Tumour budding and poorly differentiated clusters (PDC) represent forms of tumour invasion. We hypothesised that T-cell densities (reflecting adaptive anti-tumour immunity) might be inversely associated with tumour budding and PDC in colorectal carcinoma. METHODS: Utilising 915 colon and rectal carcinomas in two U.S.-wide prospective cohort studies, and multiplex immunofluorescence combined with machine learning algorithms, we assessed CD3, CD4, CD8, CD45RO (PTPRC), and FOXP3 co-expression patterns in lymphocytes. Tumour budding and PDC at invasive fronts were quantified by digital pathology and image analysis using the International tumour Budding Consensus Conference criteria. Using covariate data of 4,420 incident colorectal cancer cases, inverse probability weighting (IPW) was integrated with multivariable logistic regression analysis that assessed the association of T-cell subset densities with tumour budding and PDC while adjusting for selection bias due to tissue availability and potential confounders, including microsatellite instability status. FINDINGS: Tumour budding counts were inversely associated with density of CD3+CD8+ [lowest vs. highest: multivariable odds ratio (OR), 0.50; 95% confidence interval (CI), 0.35-0.70; Ptrend < 0.001] and CD3+CD8+CD45RO+ cells (lowest vs. highest: multivariable OR, 0.44; 95% CI, 0.31-0.63; Ptrend < 0.001) in tumour epithelial region. Tumour budding levels were associated with higher colorectal cancer-specific mortality (multivariable hazard ratio, 2.13; 95% CI, 1.57-2.89; Ptrend < 0.001) in Cox regression analysis. There were no significant associations of PDC with T-cell subsets. INTERPRETATION: Tumour epithelial naïve and memory cytotoxic T cell densities are inversely associated with tumour budding at invasive fronts, suggesting that cytotoxic anti-tumour immunity suppresses tumour microinvasion.

Immune checkpoint inhibitor-associated celiac disease.

BACKGROUND: Rare cases of immune checkpoint inhibitor (ICI)-associated celiac disease (ICI-CeD) have been reported, suggesting that disruption of tolerance mechanisms by ICIs can unmask celiac disease (CeD). This study aims to characterize the clinicopathological and immunophenotypic features of ICI-CeD in comparison to ICI-associated duodenitis (ICI-Duo) and usual CeD. METHODS: A medical and pathological records search between 2015 and 2019 identified eight cases of ICI-CeD, confirmed by tTG-IgA. Nine cases of ICI-Duo, 28 cases of moderate CeD, as well as 5 normal controls were used as comparison groups. Clinical information was collected from the electronic medical records. Immunohistochemistry for CD3, CD8, T-cell receptor gamma/delta (γδ), programmed death ligand 1 (PD-L1), and programmed death 1 (PD-1) were performed, with quantification of intraepithelial lymphocyte (IEL) subsets in three well-oriented villi. CD68, PD-L1, and PD-1 were assessed as a percentage of lamina propria surface area infiltrated by positive cells. Statistical significance was calculated by the Student's t-test and Fisher's exact test. RESULTS: The eight patients with ICI-CeD (F:M=1:3) and nine patients with ICI-Duo (F:M=5:4) presented similarly with diarrhea (13/17) and abdominal pain (11/17) after a median of 1.6 months on ICI therapy. In patients with ICI-CeD, tTG-IgA ranged from 104 to >300 IU/mL. Histological findings in ICI-CeD and ICI-Duo were similar and included expansion of the lamina propria, active neutrophilic duodenitis, variably increased IELs, and villous blunting. Immunohistochemistry showed that the average number of IELs per 100 enterocytes is comparable between ICI-CeD and ICI-Duo, with increased CD3+ CD8+ T cells compared with normal duodenum but decreased γδ T cells compared with CeD. Average PD-L1 percentage was 9% in ICI-CeD and 18% in ICI-Duo, in comparison to <1% in CeD and normal duodenum; average PD-1 percentage was very low to absent in all cases (<3%). On follow-up, five patients with ICI-CeD improved on a gluten-free diet (GFD) as the sole therapeutic intervention (with down-trending tTG-IgA) while the other three required immunosuppression. All patients who developed ICI-Duo received immunosuppression with variable improvement in symptoms. CONCLUSIONS: ICI-CeD resembles ICI-Duo clinically and histologically but shares the serological features and response to gluten withdrawal with classic CeD. Immunophenotyping of IELs in ICI-CeD and ICI-Duo also shows similar CD3, CD8, γδ T cell subsets, and PD-L1 populations, all of which differed quantitatively from usual CeD. We conclude that ICI-CeD is biologically similar to ICI-Duo and is likely a variant of ICI-Duo, but treatment strategies differ, with ICI-CeD often improving with GFD alone, whereas ICI-Duo requires systemic immunosuppression.

PET Imaging Quantifying 68Ga-PSMA-11 Uptake in Metastatic Colorectal Cancer.

At diagnosis, 22% of colorectal cancer (CRC) patients have metastases, and 50% later develop metastasis. Peptide receptor radionuclide therapy (PRRT), such as 177Lu-PSMA-617, is used to treat metastatic prostate cancer. 177Lu-PSMA-617 targets prostate-specific membrane antigen (PSMA), a cell-surface protein enriched in prostate cancer and the neovasculature of other solid tumors, including CRC. We performed 68Ga-PSMA-11 PET/CT imaging of 10 patients with metastatic CRC to assess metastasis avidity. Eight patients had lesions lacking avidity, and 2 had solitary metastases exhibiting very low avidity. Despite expression of PSMA in CRC neovasculature, none of the patients exhibited tumor avidity sufficient to be considered for 177Lu-PSMA-617 PRRT.

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.

Alterations in signaling pathways that accompany spontaneous transition to malignancy in a mouse model of BRAF mutant microsatellite stable colorectal cancer.

The serrated neoplasia pathway gives rise to a distinct subgroup of colorectal cancers distinguished by the presence of mutant BRAFV600E and the CpG Island Methylator Phenotype (CIMP). BRAF mutant CRC are commonly associated with microsatellite instability, which have an excellent clinical outcome. However, a proportion of BRAF mutant CRC retain microsatellite stability and have a dismal prognosis. The molecular drivers responsible for the development of this cancer subgroup are unknown. To address this, we established a murine model of BRAFV600E mutant microsatellite stable CRC and comprehensively investigated the exome and transcriptome to identify molecular alterations in signaling pathways that drive malignancy. Exome sequencing of murine serrated lesions (mSL) and carcinomas identified frequent hot spot mutations within the gene encoding ß-catenin (Ctnnb1). Immunohistochemical staining of ß-catenin indicated that these mutations led to an increase in the presence of aberrant nuclear ß-catenin that resulted in gene expression changes in targets of ß-catenin transcription. Gene expression profiling identified a significant enrichment for transforming growth factor-ß (TGF-ß) signaling that was present in mSL and carcinomas. Early activation of TGF-ß suggests that this pathway may be an early cue directing mSL to microsatellite stable carcinoma. These findings in the mouse model support the importance of alterations in WNT and TGF-ß signaling during the transition of human sessile serrated lesions to malignancy.

Coffee Intake and Colorectal Cancer Incidence According to T-Cell Response.

We hypothesized that the associations between coffee intake and colorectal cancer (CRC) incidence might differ by immune cell densities in CRC tissue. Using the Nurses' Health Study and the Health Professionals Follow-up Study, we examined the association of coffee intake with incidence of CRC classified by intraepithelial or stromal T-cell subset densities by multiplex immunofluorescence assay for CD3, CD4, CD8, CD45RO (PTPRC), and FOXP3. We applied an inverse probability-weighted Cox proportional hazardsregression model to control for selection bias and potential confounders. During follow-up of 133 924 participants (3 585 019 person-years), we documented 3161 incident CRC cases, including 908 CRC cases with available data on T-cell densities in tumor tissue. The association between coffee intake and CRC was not statistically significantly different by intraepithelial or stroma T-cell subset (P heterogeneity > .38). Hence, there is no sufficient evidence for differential effect of coffee intake on incidence of CRC subtypes classified by T-cell infiltrates.

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.

Clinicopathological features of pyloric gland adenomas of the duodenum: a multicentre study of 57 cases.

AIMS: To determine the clinicopathological features of pyloric gland adenomas (PGA) that arise in the duodenum. METHODS AND RESULTS: Fifty-seven cases of duodenal PGA were identified and analysed from 56 patients. Clinicopathological and immunohistochemical analyses were performed. PGA tend to occur in older individuals (median age = 73.5), with a slight female predominance (25 males, 31 females). PGA arise more commonly in the proximal duodenum (68.75% in D1, 25% in D2 and 6.25% in D3) and usually present as mucosal nodules (98.2%) or plaques (1.8%), with a mean size of 14.8 mm. There is associated gastric heterotopia in 22.8% of cases. PGA showing features of high-grade dysplasia were significantly larger in size than PGA, showing only low-grade dysplasia (23.1 versus 8.7 mm; P = 0.0001) and more likely to show a tubulovillous rather than a pure tubular architecture (P = 0.025). In our series, 10 of 56 patients had intramucosal or invasive carcinoma associated with the duodenal PGA (17.9%). Three of these carcinomas showed lymph node metastasis. Following definitive treatment, local recurrence occurred in only three patients. CONCLUSIONS: Duodenal PGA tend to occur in the proximal duodenum of older individuals. Larger size and tubulovillous architecture correlates with high-grade dysplasia and associated adenocarcinoma. The low recurrence rate of these lesions would suggest that endoscopic management is appropriate, provided that the lesion can be completely resected.

Spectrum of gastrointestinal tract pathology in a multicenter cohort of 43 Cowden syndrome patients.

Most patients with Cowden syndrome have lesions in the gastrointestinal tract, characterized by multiple polyps of various histologic types in the large bowel, polyps in the upper gastrointestinal tract, and esophageal glycogenic acanthosis. However, pathologists are often unaware of the distinctive polyposis phenotype of Cowden syndrome. In this multicenter study, we report the spectrum of gastrointestinal manifestations in a series of 43 Cowden syndrome patients who had at least one endoscopy. The median age at the first endoscopy was 46 years and 58% were women. In 24 of 29 (83%) tested patients, a pathogenic germline mutation in PTEN was identified. The histology from 199 endoscopy procedures (67 upper gastrointestinal endoscopy and 132 colonoscopies) was reviewed. Hamartomatous polyps of the large bowel were the most common lesions, present in 85% of patients. Hamartomatous polyps showed varied histology, including lymphoid aggregates in 55% of patients, a lipomatous component in 52%, a ganglioneuromatous component in 52%, and a fibrous-rich component in 14%. Polyps with at least two different stromal components were found in 55% of patients. Inflammatory polyps were present in 21% of patients. Conventional adenomas and serrated polyps were identified in 48% and 62% of patients, respectively. In the upper gastrointestinal tract, the most common lesions were esophageal glycogenic acanthosis (37%), gastric hamartomatous polyps (47%), and duodenal hamartomatous polyps (20%). All patients with glycogenic acanthosis who had a colonoscopy had hamartomatous polyps of the large bowel. In five patients, the diagnosis of Cowden syndrome was established after the pathology report raised suspicion for the diagnosis. Pathologists who are aware of the characteristic admixture of lesions in Cowden syndrome can play an essential role in recommending referral to genetic counseling and gene testing. Early diagnosis of Cowden syndrome is important, as these patients and their relatives are at increased risk for developing multiple cancers.