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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.

Immunotherapy targeting the CD274 (PD-L1)/PDCD1 (PD-1) immune checkpoint axis has emerged as a promising treatment strategy for various cancers. Experimental evidence suggests that phosphatidylinositol-4,5-bisphosphonate 3-kinase (PI3K) signaling may upregulate CD274 expression. Thus, we hypothesized that PIK3CA mutation, PTEN loss, or their combined status might be associated with CD274 overexpression in colorectal carcinoma. We assessed tumor CD274 and PTEN expression by immunohistochemistry and assessed PIK3CA mutation by pyrosequencing in 753 patients among 4,465 incident rectal and colon cancer cases that had occurred in two U.S.-wide prospective cohort studies. To adjust for potential confounders and selection bias due to tissue availability, inverse probability weighted multivariable ordinal logistic regression analyses used the 4,465 cases and tumoral data including microsatellite instability, CpG island methylator phenotype, KRAS and BRAF mutations. PIK3CA mutation and loss of PTEN expression were detected in 111 of 753 cases (15%) and 342 of 585 cases (58%), respectively. Tumor CD274 expression was negative in 306 (41%), low in 195 (26%), and high in 252 (33%) of 753 cases. PTEN loss was associated with CD274 overexpression [multivariable odds ratio (OR) 1.83; 95% confidence interval (CI), 1.22–2.75; P = .004]. PIK3CA mutation was statistically-insignificantly (P = .036 with the stringent alpha level of 0.005) associated with CD274 overexpression (multivariable OR, 1.54; 95% CI, 1.03–2.31). PIK3CA-mutated PTEN-lost tumors (n = 33) showed higher prevalence of CD274-positivity (82%) than PIK3CA-wild-type PTEN-lost tumors (n = 204; 70% CD274-positivity) and PTEN-expressed tumors (n = 147; 50% CD274-positivity) (P = .003). Our findings support the role of PI3K signaling in the CD274/PDCD1 pathway.

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 [Ptrend < 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; Ptrend = 0.002) and high GTumor:Eosinophil AUC0,20μm (Ptrend < 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 (Ptrend < 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.

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.

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.