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Several risk factors have been established for colorectal cancer, yet their direct mutagenic effects in patients' tumors remain to be elucidated. Here, we leveraged whole-exome sequencing data from 900 colorectal cancer cases that had occurred in three U.S.-wide prospective studies with extensive dietary and lifestyle information. We found an alkylating signature that was previously undescribed in colorectal cancer and then showed the existence of a similar mutational process in normal colonic crypts. This alkylating signature is associated with high intakes of processed and unprocessed red meat prior to diagnosis. In addition, this signature was more abundant in the distal colorectum, predicted to target cancer driver mutations KRAS p.G12D, KRAS p.G13D, and PIK3CA p.E545K, and associated with poor survival. Together, these results link for the first time a colorectal mutational signature to a component of diet and further implicate the role of red meat in colorectal cancer initiation and progression. Significance: Colorectal cancer has several lifestyle risk factors, but the underlying mutations for most have not been observed directly in tumors. Analysis of 900 colorectal cancers with whole-exome sequencing and epidemiologic annotations revealed an alkylating mutational signature that was associated with red meat consumption and distal tumor location, as well as predicted to target KRAS p.G12D/p.G13D. This article is highlighted in the In This Issue feature, p. 2355

Purpose: Immune checkpoint inhibitors (ICI) have shown clinical benefit in many types of metastatic cancers with only a few predictive biomarkers identified so far. CDKN2A is commonly altered in human cancers, but prior studies have provided conflicting evidence regarding the association between CDKN2A genomic alterations (GA) and response to ICIs. Herein, we examined the impact of loss-of-function CDKN2A alterations on response and survival in patients treated with ICIs. Experimental Design: We studied the association between loss-of-function CDKN2A alterations and the response to ICIs in two independent cohorts of six different cancer types. Seven hundred and eighty-nine patients treated at Dana-Farber Cancer Institute (DFCI; Boston, MA) and 1,250 patients treated at Memorial Sloan Kettering Cancer Center (MSKCC; New York, NY) were included in the final analysis. Patients' tumors were sequenced using Oncopanel or MSK-IMPACT. RNA sequencing data from The Cancer Genome Atlas and IMvigor210 were used to investigate differences in the tumor microenvironment. Results: In the DFCI cohort, CDKN2A GAs were associated with poor response and survival in patients with urothelial carcinoma treated with ICIs, but not those treated with platinum-based therapy. Similarly, CDKN2A GAs were associated with worse outcomes in the MSKCC urothelial carcinoma cohort treated with ICIs. There was no association of CDKN2A status with ICI treatment outcome in five other cancers: esophagogastric, head and neck, non–small cell lung, renal cell carcinoma, and melanoma. Immuno-inflammatory pathways were significantly reduced in expression in CDKN2A-altered tumors. Conclusions: Our data show that CDKN2A GAs were associated with reduced benefit from ICI therapy in urothelial carcinoma as well as changes in the tumor–immune 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; Ptrend = 0.0004] and specifically stromal CD3+CD4+CD45RO+ cells (corresponding multivariable OR, 0.52; 95% CI, 0.32–0.85; Ptrend = 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 incidence of early-onset colorectal cancer (CRC), which occurs in individuals

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 (Ptrend 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.

Prior data have variably implicated the inactivation of the mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) complex with increased tumor sensitivity to immune checkpoint inhibitors (ICI). Herein, we examined the association between mSWI/SNF variants and clinical outcomes to ICIs. We correlated somatic loss-of-function (LOF) variants in a predefined set of mSWI/SNF genes (ARID1A, ARID1B, SMARCA4, SMARCB1, PBRM1, and ARID2) with clinical outcomes in patients with cancer treated with systemic ICIs. We identified 676 patients from Dana-Farber Cancer Institute (DFCI, Boston, MA) and 848 patients from a publicly available database from Memorial Sloan Kettering Cancer Center (MSKCC, New York, NY) who met the inclusion criteria. Multivariable analyses were conducted and adjusted for available baseline factors and tumor mutational burden. Median follow-up was 19.6 (17.6–22.0) months and 28.0 (25.0–29.0) months for the DFCI and MSKCC cohorts, respectively. Seven solid tumor subtypes were examined. In the DFCI cohort, LOF variants of mSWI/SNF did not predict improved overall survival (OS), time-to-treatment failure (TTF), or disease control rate. Only patients with renal cell carcinoma with mSWI/SNF LOF showed significantly improved OS and TTF with adjusted HRs (95% confidence interval) of 0.33 (0.16–0.7) and 0.49 (0.27–0.88), respectively, and this was mostly driven by PRBM1. In the MSKCC cohort, where only OS was captured, LOF mSWI/SNF did not correlate with improved outcomes across any tumor subtype. We did not find a consistent association between mSWI/SNF LOF variants and improved clinical outcomes to ICIs, suggesting that mSWI/SNF variants should not be considered as biomarkers of response to ICIs.

Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.

International audience; Various species of the intestinal microbiota have been associated with the development of colorectal cancer(1,2), but it has not been demonstrated that bacteria have a direct role in the occurrence of oncogenic mutations. Escherichia coli can carry the pathogenicity island pks, which encodes a set of enzymes that synthesize colibactin(3). This compound is believed to alkylate DNA on adenine residues(4,5) and induces double-strand breaks in cultured cells(3). Here we expose human intestinal organoids to genotoxic pks(+)E. coli by repeated luminal injection over five months. Whole-genome sequencing of clonal organoids before and after this exposure revealed a distinct mutational signature that was absent from organoids injected with isogenic pks-mutant bacteria. The same mutational signature was detected in a subset of 5,876 human cancer genomes from two independent cohorts, predominantly in colorectal cancer. Our study describes a distinct mutational signature in colorectal cancer and implies that the underlying mutational process results directly from past exposure to bacteria carrying the colibactin-producing pks pathogenicity island.Organoids derived from human intestinal cells that are co-cultured with bacteria carrying the genotoxic pks(+) island develop a distinct mutational signature associated with colorectal cancer.

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 (Ptrend = 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; Ptrend = 0.001, ≥15 years night shifts vs. never) but not with IRS2-negative tumors (multivariable HR = 0.90; 95% CI, 0.54–1.51; Ptrend = 0.72; Pheterogeneity for IRS2 = 0.008). Similarly, the corresponding multivariable HRs were 1.81 for IRS1-positive tumors (95% CI, 0.94–3.48; Ptrend = 0.06) and 1.13 for IRS1-negative tumors (95% CI, 0.71–1.80; Ptrend = 0.56; Pheterogeneity 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.

PURPOSE Given regulatory approval of immune checkpoint inhibitors in patients with mismatch repair–deficient (MMR-D) cancers agnostic to tumor type, it has become important to characterize occurrence of MMR-D and develop cost-effective screening approaches. Using a next-generation sequencing (NGS) panel (OncoPanel), we developed an algorithm to identify MMR-D frequency in tumor samples and applied it in a clinical setting with pathologist review. METHODS To predict MMR-D, we adapted methods described previously for use in NGS panels, which assess patterns of single base-pair insertion or deletion events occurring in homopolymer regions. Tumors assayed with OncoPanel between July 2013 and July 2018 were included. For tumors tested after June 2017, sequencing results were presented to pathologists in real time for clinical MMR determination, in the context of tumor mutation burden, other mutational signatures, and clinical data. RESULTS Of 20,301 tumors sequenced, 2.7% (553) were retrospectively classified as MMR-D by the algorithm. Of 4,404 samples with pathologist sign-out of MMR status, the algorithm classified 147 (3.3%) as MMR-D: in 116 cases, MMR-D was confirmed by a pathologist, five cases were overruled by the pathologist, and 26 were assessed as indeterminate. Overall, the highest frequencies of OncoPanel-inferred MMR-D were in endometrial (21%; 152/723), colorectal (9.7%; 169/1,744), and small bowel (9.3%; 9/97) cancers. When algorithm predictions were compared with historical MMR immunohistochemistry or polymerase chain reaction results in a set of 325 tumors sequenced before initiation of pathologist assessment, the overall sensitivity and specificity of the algorithm were 91.1% and 98.2%, respectively. CONCLUSION We show that targeted, tumor-only NGS can be leveraged to determine MMR signatures across tumor types, suggesting that broader biomarker screening approaches may have clinical value.

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.

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.

Despite remarkable success of immune checkpoint inhibitors, the majority of cancer patients have yet to receive durable benefits. Here, in order to investigate the metabolic alterations in response to immune checkpoint blockade, we comprehensively profile serum metabolites in advanced melanoma and renal cell carcinoma patients treated with nivolumab, an antibody against programmed cell death protein 1 (PD1). We identify serum kynurenine/tryptophan ratio increases as an adaptive resistance mechanism associated with worse overall survival. This advocates for patient stratification and metabolic monitoring in immunotherapy clinical trials including those combining PD1 blockade with indoleamine 2,3-dioxygenase/tryptophan 2,3-dioxygenase (IDO/TDO) inhibitors., Immune-checkpoint inhibition therapy has achieved success in a subset of patients. Here the authors profiled about 200 relevant metabolites in patient serum samples from three independent immunotherapy trials and found the serum kynurenine/tryptophan ratio increases to be associated with worse overall survival.

Despite considerable efforts to identify cancer metabolic alterations that might unveil druggable vulnerabilities, systematic characterizations of metabolism as it relates to functional genomic features and associated dependencies remain uncommon. To further understand the metabolic diversity of cancer, we profiled 225 metabolites in 928 cell lines from more than 20 cancer types in the Cancer Cell Line Encyclopedia (CCLE) using liquid chromatography–mass spectrometry (LC-MS). This resource enables unbiased association analysis linking the cancer metabolome to genetic alterations, epigenetic features and gene dependencies. Additionally, by screening barcoded cell lines, we demonstrated that aberrant ASNS hypermethylation sensitizes subsets of gastric and hepatic cancers to asparaginase therapy. Finally, our analysis revealed distinct synthesis and secretion patterns of kynurenine, an immune-suppressive metabolite, in model cancer cell lines. Together, these findings and related methodology provide comprehensive resources that will help clarify the landscape of cancer metabolism. Systematic metabolite profiling across cancer cell lines uncovers patterns associated with genetic and epigenetic features and reveals dysregulated metabolic states that can be exploited for anticancer therapy

Calcium intake has been associated with a lower risk of colorectal cancer. Calcium signaling may enhance T-cell proliferation and differentiation, and contribute to T-cell–mediated antitumor immunity. In this prospective cohort study, we investigated the association between calcium intake and colorectal cancer risk according to tumor immunity status to provide additional insights into the role of calcium in colorectal carcinogenesis. The densities of tumor-infiltrating T-cell subsets [CD3+, CD8+, CD45RO (PTPRC)+, or FOXP3+ cell] were assessed using IHC and computer-assisted image analysis in 736 cancer cases that developed among 136,249 individuals in two cohorts. HRs and 95% confidence intervals (CI) were calculated using Cox proportional hazards regression. Total calcium intake was associated with a multivariable HR of 0.55 (comparing ≥1,200 vs.

Background Prostaglandin-endoperoxide synthase 2 (PTGS2, cyclooxygenase-2, COX-2)-prostaglandin E2 (PGE2) pathway promotes tumour progression. Considering evidence suggesting increased PGE2 synthesis by BRAF mutation in tumour cells, we hypothesised that the association of tumour PTGS2 (COX-2) expression with colorectal cancer mortality might be stronger in BRAF-mutated tumours than in BRAF-wild-type tumours. Methods Using 1708 patients, including 1200 stage I-IV colorectal carcinoma cases in the Nurses’ Health Study (NHS) and the Health Professionals Follow-up Study (HPFS) and 508 stage III colon cancer cases in a National Cancer Institute–sponsored randomised controlled trial of adjuvant therapy ( CALGB /Alliance 89803), we evaluated tumour PTGS2 (COX-2) expression status using immunohistochemistry. We examined the prognostic association of PTGS2 (COX-2) expression in strata of BRAF mutation status by multivariable Cox proportional hazards regression models to adjust for potential confounders, including disease stage, tumour differentiation, microsatellite instability status and KRAS and PIK3CA mutations. Results In NHS and HPFS, the association of PTGS2 (COX-2) expression with colorectal cancer-specific survival differed by BRAF mutation status (Pinteraction = 0.0005); compared with PTGS2 (COX-2)-negative/low carcinomas, the multivariable-adjusted hazard ratios for PTGS2 (COX-2)-high carcinomas were 2.44 (95% confidence interval, 1.39–4.28) in BRAF-mutated cases and 0.82 (95% confidence interval, 0.65–1.04) in BRAF-wild-type cases. Differential prognostic associations of PTGS2 (COX-2) expression in strata of BRAF mutation status were similarly observed in CALGB/Alliance 89803 trial (Pinteraction = 0.03). Conclusions The association of tumour PTGS2 (COX-2) expression with colorectal cancer mortality is stronger in BRAF-mutated tumours than in BRAF-wild-type tumours, supporting interactive roles of PTGS2 (COX-2) expression and BRAF mutation statuses in prognostication of patients with colorectal cancer; ClinicalTrials.gov Identifier, NCT00003835 .

Synthetic lethality-an interaction between two genetic events through which the co-occurrence of these two genetic events leads to cell death, but each event alone does not-can be exploited for cancer therapeutics1. DNA repair processes represent attractive synthetic lethal targets, because many cancers exhibit an impairment of a DNA repair pathway, which can lead to dependence on specific repair proteins2. The success of poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors in cancers with deficiencies in homologous recombination highlights the potential of this approach3. Hypothesizing that other DNA repair defects would give rise to synthetic lethal relationships, we queried dependencies in cancers with microsatellite instability (MSI), which results from deficient DNA mismatch repair. Here we analysed data from large-scale silencing screens using CRISPR-Cas9-mediated knockout and RNA interference, and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, yet dispensable in models of cancers that are microsatellite stable. Depletion of WRN induced double-stranded DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models required the helicase activity of WRN, but not its exonuclease activity. These findings show that WRN is a synthetic lethal vulnerability and promising drug target for MSI cancers.

Purpose: SMAD4 has shown promise in identifying patients with colorectal cancer at high risk of recurrence or death. Experimental Design: A discovery cohort and independent validation cohort were classified by SMAD4 status. SMAD4 status and immune infiltrate measurements were tested for association with recurrence-free survival (RFS). Patient-derived xenografts from SMAD4-deficient and SMAD4-retained tumors were used to examine chemoresistance. Results: The discovery cohort consisted of 364 patients with stage I–IV colorectal cancer. Median age at diagnosis was 53 years. The cohort consisted of 61% left-sided tumors and 62% stage II/III patients. Median follow-up was 5.4 years (interquartile range, 2.3–8.2). SMAD4 loss, noted in 13% of tumors, was associated with higher tumor and nodal stage, adjuvant therapy use, fewer tumor-infiltrating lymphocytes (TIL), and lower peritumoral lymphocyte aggregate (PLA) scores (all P < 0.04). SMAD4 loss was associated with worse RFS (P = 0.02). When stratified by SMAD4 and immune infiltrate status, patients with SMAD4 loss and low TIL or PLA had worse RFS (P = 0.002 and P = 0.006, respectively). Among patients receiving 5-fluorouracil (5-FU)-based systemic chemotherapy, those with SMAD4 loss had a median RFS of 3.8 years compared with 13 years for patients with SMAD4 retained. In xenografted mice, the SMAD4-lost tumors displayed resistance to 5-FU. An independent cohort replicated our findings, in particular, the association of SMAD4 loss with decreased immune infiltrate, as well as worse disease-specific survival. Conclusions: Our data show SMAD4 loss correlates with worse clinical outcome, resistance to chemotherapy, and decreased immune infiltrate, supporting its use as a prognostic marker in patients with colorectal cancer.

Background Individuals with a family history of cancer may be at increased risk of pancreatic cancer. Ashkenazi Jewish (AJ) individuals carry increased risk for pancreatic cancer and other cancer types. Methods We examined the association between family history of cancer, AJ heritage, and incident pancreatic cancer in 49 410 male participants of the prospective Health Professionals Follow-up Study. Hazard ratios (HRs) were estimated using multivariable-adjusted Cox proportional hazards models. Results During 1.1 million person-years (1986–2016), 452 participants developed pancreatic cancer. Increased risk of pancreatic cancer was observed in individuals with a family history of pancreatic (HR, 2.79; 95% confidence interval [CI], 1.28–6.07) or breast cancer (HR, 1.40; 95% CI, 1.01–1.94). There was a trend towards higher risk of pancreatic cancer in relation to a family history of colorectal cancer (HR, 1.21; 95% CI, 0.95–1.55) or AJ heritage (HR, 1.29; 95% CI, 0.94–1.77). The risk was highly elevated among AJ men with a family history of breast or colorectal cancer (HR, 2.61 [95% CI, 1.41–4.82] and 1.92 [95% CI, 1.05–3.49], respectively). Conclusion Family history of pancreatic cancer was associated with increased risk of this malignancy. Family history of breast or colorectal cancer was associated with the increased risk among AJ men.

Evidence indicates the pathogenic role of epigenetic alterations in early-onset colorectal cancers diagnosed before age 50. However, features of colorectal cancers diagnosed at age 50–54 (hereafter referred to as “intermediate-onset”) remain less known. We hypothesized that tumor long interspersed nucleotide element-1 (LINE-1) hypomethylation might be increasingly more common with decreasing age of colorectal cancer diagnosis. In 1356 colorectal cancers, including 28 early-onset and 66 intermediate-onset cases, the tumor LINE-1 methylation level measured by bisulfite-PCR-pyrosequencing (scaled 0 to 100) showed a mean of 63.6 (standard deviation (SD) 10.1). The mean tumor LINE-1 methylation level decreased with decreasing age (mean 64.7 (SD 10.4) in age ≥70, 62.8 (SD 9.4) in age 55–69, 61.0 (SD 10.2) in age 50–54, and 58.9 (SD 12.0) in age &lt, 50, p &lt, 0.0001). In linear regression analysis, the multivariable-adjusted β coefficient (95% confidence interval (CI)) (vs. age ≥70) was −1.38 (−2.47 to −0.30) for age 55–69, −2.82 (−5.29 to −0.34) for age 50–54, and −4.54 (−8.24 to −0.85) for age &lt, 50 (Ptrend = 0.0003). Multivariable-adjusted hazard ratios (95% CI) for LINE-1 methylation levels of ≤45, 45–55, and 55–65 (vs. &gt, 65) were 2.33 (1.49–3.64), 1.39 (1.05–1.85), and 1.29 (1.02–1.63), respectively (Ptrend = 0.0005). In conclusion, tumor LINE-1 hypomethylation is increasingly more common with decreasing age of colorectal cancer diagnosis, suggesting a role of global DNA hypomethylation in colorectal cancer arising in younger adults.

BackgroundMyeloid cells represent an abundant yet heterogeneous cell population in the colorectal cancer microenvironment, and their roles remain poorly understood.MethodsWe 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.ResultsHigher intraepithelial (Ptrend=0.0002; HR for Q4 (vs Q1), 0.48, 95% CI 0.31 to 0.76) and stromal (Ptrend +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+HLA-DR+ cell versus CD14+HLA-DR− cell within a 20 µm radius, was associated with lower colorectal cancer-specific mortality (Ptrend ConclusionsMyeloid 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.

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 (

Resistance to oncogene-targeted therapies involves discrete drug-tolerant persister cells, originally discovered through in vitro assays. Whether a similar phenomenon limits efficacy of programmed cell death 1 (PD-1) blockade is poorly understood. Here, we performed dynamic single-cell RNA-Seq of murine organotypic tumor spheroids undergoing PD-1 blockade, identifying a discrete subpopulation of immunotherapy persister cells (IPCs) that resisted CD8(+) T cell–mediated killing. These cells expressed Snai1 and stem cell antigen 1 (Sca-1) and exhibited hybrid epithelial-mesenchymal features characteristic of a stem cell–like state. IPCs were expanded by IL-6 but were vulnerable to TNF-α–induced cytotoxicity, relying on baculoviral IAP repeat-containing protein 2 (Birc2) and Birc3 as survival factors. Combining PD-1 blockade with Birc2/3 antagonism in mice reduced IPCs and enhanced tumor cell killing in vivo, resulting in durable responsiveness that matched TNF cytotoxicity thresholds in vitro. Together, these data demonstrate the power of high-resolution functional ex vivo profiling to uncover fundamental mechanisms of immune escape from durable anti–PD-1 responses, while identifying IPCs as a cancer cell subpopulation targetable by specific therapeutic combinations.

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.

Background Smoking is associated with colorectal cancer (CRC) risk. Previous studies suggested this association may be restricted to certain molecular subtypes of CRC while large-scale comprehensive analysis is lacking. Methods A total of 9,789 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 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.

Background To understand fundamental mechanisms of immune escape, we leveraged our functional ex vivo platform of murine derived organotypic tumor spheroids (DOTS)1 to determine if drug-tolerant persister cells analogous to oncogene targeted therapies limit efficacy of programmed death (PD)-1 blockade, and to identify therapeutic vulnerabilities to overcome anti-PD-1 (αPD-1) resistance. Methods Murine syngeneic cancer models with well-characterized response to αPD-1 therapy were chosen: MC38 (sensitive) and CT26 (partially resistant). Bulk and single-cell (sc) RNA-sequencing (RNA-seq) were performed on αPD-1 treated DOTS. In vitro culture studies were conducted with or without cytokines (100 ng/ml) or drugs (500 nM). In vivo studies in mice bearing MC38 or CT26 tumors evaluated the combinatorial strategy with PD-1 blockade. We further evaluated our findings in scRNA-seq of an αPD-1 refractory colorectal cancer (CRC) patient tumor.2 Results Bulk RNA-seq of αPD-1 treated DOTS revealed a mesenchymal resistant phenotype with upregulated TNF-α/NFκB signaling (figure 1). scRNA-seq further identified a discrete sub-population of immunotherapy persister cells (IPCs). These cells expressed a stem-like phenotype including downregulation of E2F targets indicative of quiescence, suppression of interferon-γ response genes, induction of hybrid epithelial-to-mesenchymal state, and active IL-6 signaling (figure 1). Ly6a/stem cell antigen-1 (Sca-1) and Snai1 were found to be differentially upregulated in IPCs resistant to PD-1 blockade (not shown). Sca-1 positivity was confirmed in pre-existing tumor populations in vitro (figure 2). When enriched via sorting, these cells remained more persistently Sca-1+ at 96 hours in culture of CT26 compared to MC38 cells, related to increased autocrine IL-6 production by CT26 Sca-1+ cells. Indeed, IL-6 supplementation was capable of expanding Sca-1+ cells in culture (figure 2). Sca-1+ cells expressing ovalbumin peptide were refractory to OT-1 T cell mediated killing and failed to upregulate MHC class-1 antigen presentation (H-2Kb) in response to IL-6, in contrast to interferon-γ (not shown). Analysis of RNA-seq data further identified Birc2/3 as potential targets limiting TNF-mediated apoptosis of these cells (not shown). Notably, Birc2/3 antagonism depleted Sca-1+ IPCs in vitro and significantly potentiated the impact of PD-1 blockade in vivo in MC38, and less robustly in CT26 (figure 3). Evaluation in a microsatellite-instability high CRC patient identified a pre-existent IPC subpopulation within the αPD-1 refractory pre-treatment tumor, with high SNAI1 expression compared to CRC samples in TCGA (figure 4). Conclusions High-resolution functional ex vivo profiling identified Sca-1+/Snai1high stem-like ‘immunotherapy persister cells‘ and uncovered their anti-apoptotic dependencies targetable with Birc2/3 antagonism to augment αPD-1 efficacy. Ethics Approval This study was approved by the Dana-Farber Animal Care and Use Committee and Novartis Institutional Animal Care and Use Committee. Informed written consent to participate in Dana-Farber/Harvard Cancer Center institutional review board (IRB)-approved research protocols was obtained from the human subject. A copy of the written consent is available for review by the Editor of this journal. The study was conducted per the WMA Declaration of Helsinki and IRB-approved protocols. References Jenkins RW, Aref AR, Lizotte PH, Ivanova E, Stinson S, Zhou CW, et al. Ex Vivo Profiling of PD-1 Blockade using organotypic tumor spheroids. Cancer Discov. 2018;8(2):196–668 215. Gurjao C, Liu D, Hofree M, AlDubayan SH, Wakiro I, Su MJ, et al. intrinsic resistance to immune checkpoint blockade in a mismatch repair-deficient colorectal cancer. Cancer Immunol Res 2019;7(8):1230–6.

Purpose: Receptor tyrosine kinase fusions in colorectal cancers are rare, but potentially therapeutically relevant. We describe clinical, molecular, and pathologic attributes of RTK fusion–associated colorectal cancer. Experimental Design: We identified all cases with RTK fusions in patients with colorectal cancer seen at Dana-Farber Cancer Institute (Boston, MA) who underwent OncoPanel testing between 2013 and 2018. Clinical, histologic, and molecular features were extracted from the patient charts and molecular testing results. Results: We identified 12 driver oncogenic fusions in various RTKs. These fusions occurred exclusively in BRAF and RAS wild-type tumors and were enriched in right-sided and mismatch repair–deficient (MMR-D) colorectal cancers. All of the MMR-D colorectal cancers with RTK fusions were found in tumors with acquired MMR-D due to MLH1 promoter hypermethylation and one was associated with a sessile serrated polyp. Molecular profiles of MMR-D colorectal cancer with RTK fusions largely resembled BRAF V600E–mutated MMR-D colorectal cancer, rather than those secondary to Lynch syndrome. We describe two patients with fusion-associated microsatellite stable (MSS) colorectal cancer who derived clinical benefit from therapeutic targeting of their translocation. The first harbored an ALK-CAD fusion and received sequential crizotinib and alectinib therapy for a total of 7.5 months until developing an ALK L1196Q gatekeeper mutation. The second patient, whose tumor contained an ROS1-GOPC fusion, continues to benefit from entrectinib after 9 months of therapy. Conclusions: RTK fusions in colorectal cancer are a rare, but important disease subgroup that occurs in RAS and BRAF wild-type tumors. Despite enrichment in acquired MMR-D tumors, RTK fusions also occur in MSS colorectal cancer and provide an important therapeutic target.

BackgroundCD73–adenosine signaling in the tumor microenvironment is immunosuppressive and may be associated with aggressive renal cell carcinoma (RCC). We investigated the prognostic significance of CD73 protein expression in RCC leveraging nephrectomy samples. We also performed a complementary analysis using The Cancer Genome Atlas (TCGA) dataset to evaluate the correlation of CD73 (ecto-5′-nucleotidase (NT5E), CD39 (ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1)) and A2 adenosine receptor (A2AR;ADORA2A) transcript levels with markers of angiogenesis and antitumor immune response.MethodsPatients with RCC with available archived nephrectomy samples were eligible for inclusion. Tumor CD73 protein expression was assessed by immunohistochemistry and quantified using a combined score (CS: % positive cells×intensity). Samples were categorized as CD73negative(CS=0), CD73lowor CD73high(< and ≥median CS, respectively). Multivariable Cox regression analysis compared disease-free survival (DFS) and overall survival (OS) between CD73 expression groups. In the TCGA dataset, samples were categorized as low, intermediate and highNT5E,ENTPD1andADORA2Agene expression groups. Gene expression signatures for infiltrating immune cells, angiogenesis, myeloid inflammation, and effector T-cell response were compared betweenNT5E,ENTPD1andADORA2Aexpression groups.ResultsAmong the 138 patients eligible for inclusion, ‘any’ CD73 expression was observed in 30% of primary tumor samples. High CD73 expression was more frequent in patients with M1 RCC (29% vs 12% M0), grade 4 tumors (27% vs 13% grade 3 vs 15% grades 1 and 2), advanced T-stage (≥T3: 22% vs T2: 19% vs T1: 12%) and tumors with sarcomatoid histology (50% vs 12%). In the M0 cohort (n=107), patients with CD73hightumor expression had significantly worse 5-year DFS (42%) and 10-year OS (22%) compared with those in the CD73negativegroup (DFS: 75%, adjusted HR: 2.7, 95% CI 1.3 to 5.9, p=0.01; OS: 64%, adjusted HR: 2.6, 95% CI 1.2 to 5.8, p=0.02) independent of tumor stage and grade. In the TCGA analysis, highNT5Eexpression was associated with significantly worse 5-year OS (p=0.008).NT5EandENTPD1expression correlated with higher regulatory T cell (Treg) signature, whileADORA2Aexpression was associated with increased Treg and angiogenesis signatures.ConclusionsHigh CD73 expression portends significantly worse survival outcomes independent of stage and grade. Our findings provide compelling support for targeting the immunosuppressive and proangiogenic CD73–adenosine pathway in RCC.

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 (Pheterogeneity > .38). Hence, there is no sufficient evidence for differential effect of coffee intake on incidence of CRC subtypes classified by T-cell infiltrates.

Background/Objectives: 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.

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 Pinteraction = .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.

Cancer chemotherapy targeting frequent loss of heterozygosity events is an attractive concept, since tumor cells may lack enzymatic activities present in normal constitutional cells. To find exploitable targets, we map prevalent genetic polymorphisms to protein structures and identify 45 nsSNVs (non-synonymous small nucleotide variations) near the catalytic sites of 17 enzymes frequently lost in cancer. For proof of concept, we select the gastrointestinal drug metabolic enzyme NAT2 at 8p22, which is frequently lost in colorectal cancers and has a common variant with 10-fold reduced activity. Small molecule screening results in a cytotoxic kinase inhibitor that impairs growth of cells with slow NAT2 and decreases the growth of tumors with slow NAT2 by half as compared to those with wild-type NAT2. Most of the patient-derived CRC cells expressing slow NAT2 also show sensitivity to 6-(4-aminophenyl)-N-(3,4,5-trimethoxyphenyl)pyrazin-2-amine (APA) treatment. These findings indicate that the therapeutic index of anti-cancer drugs can be altered by bystander mutations affecting drug metabolic genes., Allelic losses occurring in cancer cells have been suggested as potential targets for therapy. Here, the authors show how recurring loss of heterozygosity of a drug metabolic gene in colorectal cancers can be exploited using a low molecular weight compound.

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., Large scale sequencing study is of paramount importance to unravel the heterogeneity of colorectal cancer. Here, the authors sequenced 205 cancer genes in more than 2000 tumours and identified additional mutated driver genes, determined that mutational burden and specific mutations in TP53 are associated with survival odds.

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.

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.

Background High-level plasma 25-hydroxyvitamin D [25(OH)D] has been associated with lower colorectal cancer incidence and mortality. Considering evidence indicating immunomodulatory effects of vitamin D, we hypothesised that survival benefits from high systemic vitamin D level might be stronger for colorectal carcinoma with lower immune response to tumour. Methods Using 869 colon and rectal cancer cases within the Nurses' Health Study and Health Professionals Follow-up Study, we assessed the prognostic association of postdiagnosis 25(OH)D score [derived from diet and lifestyle variables to predict plasma 25(OH)D level] in strata of levels of histopathologic lymphocytic reaction. The Cox proportional hazards regression model was adjusted for potential confounders, including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, PTGS2 (cyclooxygenase-2) expression and KRAS, BRAF and PIK3CA mutations. Results The association of postdiagnosis 25(OH)D score with colorectal cancer-specific mortality differed by levels of peritumoural lymphocytic reaction (pinteraction = 0.001). Multivariable-adjusted mortality hazard ratios for a quintile-unit increase of 25(OH)D score were 0.69 [95% confidence interval (CI), 0.54–0.89] in cases with negative/low peritumoural lymphocytic reaction, 1.08 (95% CI, 0.93–1.26) in cases with intermediate peritumoural reaction and 1.25 (95% CI, 0.75–2.09) in cases with high peritumoural reaction. The survival association of the 25(OH)D score did not significantly differ by Crohn's-like lymphoid reaction, intratumoural periglandular reaction or tumour-infiltrating lymphocytes. Conclusions The association between the 25(OH)D score and colorectal cancer survival is stronger for carcinomas with lower peritumoural lymphocytic reaction. Our results suggesting interactive effects of vitamin D and immune response may contribute to personalised dietary and lifestyle intervention strategies.

Background Evidence indicates not only carcinogenic effect of cigarette smoking but also its immunosuppressive effect. We hypothesized that the association of smoking with colorectal cancer risk might be stronger for tumors with lower anti-tumor adaptive immune response. Methods During follow-up of 134 981 participants (3 490 851 person-years) in the Nurses' Health Study and Health Professionals Follow-up Study, we documented 729 rectal and colon cancer cases with available data on T-cell densities in tumor microenvironment. Using the duplication-method Cox regression model, we examined a differential association of smoking status with risk of colorectal carcinoma subclassified by densities of CD3+ cells, CD8+ cells, CD45RO (PTPRC)+ cells, or FOXP3+ cells. All statistical tests were two-sided. Results The association of smoking status with colorectal cancer risk differed by CD3+ cell density (Pheterogeneity = .007). Compared with never smokers, multivariable-adjusted hazard ratios for CD3+ cell-low colorectal cancer were 1.38 (95% confidence interval = 1.09 to 1.75) in former smokers and 1.59 (95% confidence interval = 1.14 to 2.23) in current smokers (Ptrend = .002, across smoking status categories). In contrast, smoking status was not associated with CD3+ cell-high cancer risk (Ptrend = .52). This differential association appeared consistent in strata of microsatellite instability, CpG island methylator phenotype, or BRAF mutation status. There was no statistically significant differential association according to densities of CD8+ cells, CD45RO+ cells, or FOXP3+ cells (Pheterogeneity > .04, with adjusted α of 0.01). Conclusions Colorectal cancer risk increased by smoking was stronger for tumors with lower T-lymphocyte response, suggesting an interplay of smoking and immunity in colorectal carcinogenesis.

Colorectal cancer is driven by mutations that activate canonical WNT/β-catenin signaling, but inhibiting WNT has significant on-target toxicity, and there are no approved therapies targeting dominant oncogenic drivers. We recently found that activating a β-catenin–independent branch of WNT signaling that inhibits GSK3-dependent protein degradation induces asparaginase sensitivity in drug-resistant leukemias. To test predictions from our model, we turned to colorectal cancer because these cancers can have WNT-activating mutations that function either upstream (i.e., R-spondin fusions) or downstream (APC or β-catenin mutations) of GSK3, thus allowing WNT/β-catenin and WNT-induced asparaginase sensitivity to be unlinked genetically. We found that asparaginase had little efficacy in APC or β-catenin–mutant colorectal cancer, but was profoundly toxic in the setting of R-spondin fusions. Pharmacologic GSK3α inhibition was sufficient for asparaginase sensitization in APC or β-catenin–mutant colorectal cancer, but not in normal intestinal progenitors. Our findings demonstrate that WNT-induced therapeutic vulnerabilities can be exploited for colorectal cancer therapy. Significance: Solid tumors are thought to be asparaginase-resistant via de novo asparagine synthesis. In leukemia, GSK3α-dependent protein degradation, a catabolic amino acid source, mediates asparaginase resistance. We found that asparaginase is profoundly toxic to colorectal cancers with WNT-activating mutations that inhibit GSK3. Aberrant WNT activation can provide a therapeutic vulnerability in colorectal cancer. See related commentary by Davidsen and Sullivan, p. 1632. This article is highlighted in the In This Issue feature, p. 1611

While advancements in genome sequencing have identified millions of somatic mutations in cancer, their functional impact is poorly understood. We previously developed the expression-based variant impact phenotyping (eVIP) method to use gene expression data to characterize the function of gene variants. The eVIP method uses a decision tree-based algorithm to predict the functional impact of somatic variants by comparing gene expression signatures induced by introduction of wild-type versus mutant cDNAs in cell lines. The method distinguishes between variants that are gain-of-function, loss-of-function, change-of-function, or neutral. We present eVIP2, software that allows for pathway analysis (eVIP Pathways) and usage with RNA-seq data. To demonstrate the eVIP2 software and approach, we characterized two recurrent frameshift variants in RNF43, a negative regulator of Wnt signaling, frequently mutated in colorectal, gastric and endometrial cancer. RNF43 WT, RNF43 R117fs, RNF43 G659fs, or GFP control cDNA were overexpressed in HEK293T cells. Analysis with eVIP2 predicted that the frameshift at position 117 was a loss-of-function mutation, as expected. The second frameshift at position 659, was, surprisingly, predicted to be a gain-of-function mutation. Additional eVIP Pathways analysis of RNF43 G659fs predicted 10 pathways to be significantly altered, including TNF alpha via NFKB signaling, KRAS signaling, and hypoxia. To validate these predictions, we performed reporter assays and found that all eVIP2 impactful pathways tested in the assay were activated by expression of RNF43 G659fs, but not by expression of RNF43 WT, supporting that RNF43 G659fs is a gain-of-function mutation and its effect on the identified pathways. The eVIP2 method is an important step towards overcoming the current challenge of variant interpretation in the implementation of precision medicine. eVIP2 is available at https://github.com/BrooksLabUCSC/eVIP2.

SNF'ing out antitumor immunity Immune checkpoint inhibitors induce durable tumor regressions in some, but not all, cancer patients. Understanding the mechanisms that determine tumor sensitivity to these drugs could potentially expand the number of patients who benefit (see the Perspective by Ghorani and Quezada). Pan et al. discovered that tumor cells in which a specific SWI/SNF chromatin remodeling complex had been experimentally inactivated were more sensitive to T cell–mediated killing. The cells were more responsive to interferon-γ, leading to increased secretion of cytokines that promote antitumor immunity. Miao et al. examined the genomic features of tumors from patients with metastatic renal cell carcinoma who had been treated with immune checkpoint inhibitors. Tumors harboring inactivating mutations in PBRM1 , which encodes a subunit of the same SWI/SNF complex, were more likely to respond to the drugs. Science , this issue p. 770 , p. 801 ; see also p. 745

BackgroundImmune responses to cancer are highly variable, with DNA mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. Almost all tumors are infiltrated with immune cells, but the types of immune responses and their effects on tumor growth, metastasis and death, vary greatly between different cancers and individual tumors. Which of the numerous cell subsets in a tumor contribute to the response, how their interactions are regulated, and how they are spatially organized within tumors remains poorly understood.MethodsTo understand the rules governing these varied responses, we transcriptionally profiled 371,223 single cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd treatment-naive patients. We developed a systematic approach to discover cell types, their underlying gene programs, and cellular communities based on single cell RNA-seq (scRNAseq) profiles and applied it to study the distinguishing features of human MMRd and MMRp colorectal cancer. Cellular communities discovered from this analysis were spatially mapped in tissue sections using multiplex RNA in situ hybridization microscopy.ResultsTo understand the basis for differential immune responses in CRC, we first determined and compared the immune cell composition of MMRd and MMRp CRC and normal colon tissue, finding dramatic remodeling between tumor and normal tissue and between MMRd and MMRp tumors, particularly within the myeloid, T cell, and stromal compartments. Among the clusters enriched in MMRd tumors were activated CXCL13+ CD8 T cells. Importantly, gene program co-variation analysis revealed multicellular networks. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage, and an MMRd-enriched immune hub within the tumor, with activated IFNG+ and CXCL13+ T cells together with malignant and myeloid cells expressing T-cell-attracting chemokines (figure 1).ConclusionsOur study provides a rich dataset of cellular states, gene programs and their transformations in tumors across a relatively large cohort of patients with colorectal cancer. Our predictions of several multicellular hubs based on co-variation of gene expression programs, and subsequent spatial localization of two major immune-malignant hubs, organizes a large set of cell states and programs into a smaller number of coordinated networks of cells and processes. Understanding the molecular mechanisms underlying these hubs, and studying their temporal and spatial regulation upon treatment will be critical for advancing cancer therapy.Ethics ApprovalThis study was approved by the DF-HCC institutional review board (protocols 03-189 and 02-240).Abstract 641 Figure 1A coordinated network of CXCL13+ T cells with myeloid and malignant cells expressing ISGs. Image shows a portion of formalin-fixed paraffin-embedded tissue from an MMRd CRC specimen stained with multiplex RNA ISH / IF for PanCK-IF, CD3E-ISH, CXCL10/CXCL11-ISH, CXCL13-ISH, and IFNG-ISH. Note IFNG+ and CXCL13+ cells in proximity to cells expressing the chemokines CXCL10/CXCL11

Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd individuals. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across tumors from affected individuals and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T cell-attracting chemokines. By identifying interacting cellular programs, we reveal the logic underlying spatially organized immune-malignant cell networks.

To systematically define molecular features in human tumor cells that determine their degree of sensitivity to human allogeneic natural killer (NK) cells, we quantified the NK cell responsiveness of hundreds of molecularly annotated ‘DNA-barcoded’ solid tumor cell lines in multiplexed format and applied genome-scale CRISPR-based gene-editing screens in several solid tumor cell lines, to functionally interrogate which genes in tumor cells regulate the response to NK cells. In these orthogonal studies, NK cell–sensitive tumor cells tend to exhibit ‘mesenchymal-like’ transcriptional programs; high transcriptional signature for chromatin remodeling complexes; high levels of B7-H6 (NCR3LG1); and low levels of HLA-E/antigen presentation genes. Importantly, transcriptional signatures of NK cell–sensitive tumor cells correlate with immune checkpoint inhibitor (ICI) resistance in clinical samples. This study provides a comprehensive map of mechanisms regulating tumor cell responses to NK cells, with implications for future biomarker-driven applications of NK cell immunotherapies. The use of natural killer (NK) cells in immunotherapy as an alternative to allogeneic T cells is gaining ground. Here, two genome-scale high-throughput platforms are used to identify genes that modulate the sensitivity of multiple solid tumor cell lines to NK-mediated killing.

While advancements in genome sequencing have identified millions of somatic mutations in cancer, their functional impact is poorly understood. We previously developed the expression-based variant impact phenotyping (eVIP) method to use gene expression data to characterize the function of gene variants. The eVIP method uses a decision tree-based algorithm to predict the functional impact of somatic variants by comparing gene expression signatures induced by introduction of wild-type (WT) versus mutant cDNAs in cell lines. The method distinguishes between variants that are gain-of-function, loss-of-function, change-of-function, or neutral. We present eVIP2, software that allows for pathway analysis (eVIP Pathways) and usage with RNA-seq data. To demonstrate the eVIP2 software and approach, we characterized two recurrent frameshift variants in RNF43, a negative regulator of Wnt signaling, frequently mutated in colorectal, gastric, and endometrial cancer. RNF43 WT, RNF43 R117fs, RNF43 G659fs, or GFP control cDNA were overexpressed in HEK293T cells. Analysis with eVIP2 predicted that the frameshift at position 117 was a loss-of-function mutation, as expected. The second frameshift at position 659 has been previously described as a passenger mutation that maintains the RNF43 WT function as a negative regulator of Wnt. Surprisingly, eVIP2 predicted G659fs to be a change-of-function mutation. Additional eVIP Pathways analysis of RNF43 G659fs predicted 10 pathways to be significantly altered, including TNF-α via NFκB signaling, KRAS signaling, and hypoxia, highlighting the benefit of a more comprehensive approach when determining the impact of gene variant function. To validate these predictions, we performed reporter assays and found that each pathway activated by expression of RNF43 G659fs, but not expression of RNF43 WT, was identified as impacted by eVIP2, supporting that RNF43 G659fs is a change-of-function mutation and its effect on the identified pathways. Pathway activation was further validated by Western blot analysis. Lastly, we show primary colon adenocarcinoma patient samples with R117fs and G659fs variants have transcriptional profiles similar to BRAF missense mutations with activated RAS/MAPK signaling, consistent with KRAS signaling pathways being GOF in both variants. The eVIP2 method is an important step towards overcoming the current challenge of variant interpretation in the implementation of precision medicine. eVIP2 is available at https://github.com/BrooksLabUCSC/eVIP2., Author summary Cancer is often caused by a DNA change, called a mutation, that creates an atypical protein variant. Many cancer-associated mutations remain uninvestigated because experimental validation of their effect on gene function is costly, time-consuming, and requires prior knowledge of a gene’s function. We present our computational tool, eVIP2, which advances our previous approach called expression-based variant impact phenotyping (eVIP) and can be used with RNA sequencing data. This method uses gene expression data to characterize a gene variant’s function, requiring no prior knowledge of the wild-type gene’s function. With eVIP2, we can predict if a mutation causes a gain, loss, or change in function, or if it is neutral. Here, we found two recurrent frameshift mutations in RNF43 that have different effects on gene function where one mutation causes multiple cancer pathways to be activated. This study highlights the usefulness of using the eVIP2 tool for profiling a mutation’s impact at the pathway level.

Background: Biological evidence indicates that smoking influences immune responses through interacting with various immune cell populations. We therefore hypothesized that the association of smoking with colorectal cancer (CRC) incidence differs by T-cell, tumor-associated macrophage (TAM), and myeloid cell densities. Methods: We developed multiplexed immunofluorescence assays to identify immune cells, including T-cell subsets (CD3, CD4, CD8, CD45RO, and FOXP3), TAMs (overall, M1-like, M2-like), and myeloid cell subsets (CD14, CD15, HLA-DR, ARG) in tumor intraepithelial and stromal areas. Utilizing the Nurses' Health Study and the Health Professionals Follow-up Study, we examined the association of smoking with incidence of CRC subtypes classified by each immune cell subset. We applied inverse probability weighted Cox proportional hazards regression models to control for selection bias and potential confounders. Results: During follow-up of 131,144 participants (3,648,371 person-years), we documented 3,203 incident CRC cases including 871 CRC cases with available data on immune cell densities in tumor tissue. The association of pack-years smoked with colorectal cancer incidence was stronger for tumors with lower CD3+CD8+ T-cell densities (Pheterogeneity=0.074) and lower overall TAM densities (Pheterogeneity=0.004) in tumor stromal areas. Further, the association of pack-years smoked with colorectal cancer incidence was stronger for tumors with higher CD14+HLA-DR− immature monocytic myeloid cell densities (Pheterogeneity=0.001) in tumor intraepithelial areas. Conclusions: The association of smoking with CRC incidence differed by stromal CD3+CD8+ T-cell densities, stromal TAM densities, and intraepithelial CD14+HLA-DR− myeloid cell densities. Our findings suggest a complex interplay of T-cells, TAMs, and myeloid cells in smoking-related colorectal carcinogenesis. Cumulative Pack-years Smoked and Colorectal Cancer Incidence, Overall and by Immune Cell DensityCumulative Pack-years Smoked01-1920-39≥40P for trendP for heterogeneityAll colorectal cancer (N=871)361209149152--Multivariable HR (95% CI)1 (referent)1.06 (0.93-1.22)1.08 (0.92-1.25)1.25 (1.07-1.46)0.007-Stromal CD3+CD8+ cell density------Low (N=310)124685761--Multivariable HR (95% CI)1 (referent)1.07 (0.79-1.45)1.25 (0.91-1.73)1.46 (1.07-2.00)0.0120.074Intermediate (N=280)111764449--Multivariable HR (95% CI)1 (referent)1.24 (0.92-1.66)1.04 (0.73-1.47)1.38 (0.98-1.94)0.15-High (N=281)126664643--Multivariable HR (95% CI)1 (referent)0.91 (0.68-1.23)0.89 (0.64-1.26)0.98 (0.70-1.39)0.85-Stromal Macrophage density------Low (N=288)106715061--Multivariable HR (95% CI)1 (referent)1.31 (0.97-1.78)1.30 (0.92-1.84)1.70 (1.23-2.35)0.0030.004Intermediate (N=294)125605356--Multivariable HR (95% CI)1 (referent)0.82 (0.60-1.13)1.03 (0.75-1.43)1.36 (0.99-1.86)0.045-High (N=289)130784635--Multivariable HR (95% CI)1 (referent)1.12 (0.84-1.48)0.95 (0.67-1.33)0.80 (0.55-1.17)0.20-Intraepithelial CD14+HLA-DR− cell density-----Low (N=290)130763945--Multivariable HR (95% CI)1 (referent)1.08 (0.82-1.44)0.77 (0.54-1.11)0.99 (0.70-1.40)0.510.001Intermediate (N=285)120724350--Multivariable HR (95% CI)1 (referent)1.04 (0.77-1.40)0.89 (0.63-1.27)1.26 (0.90-1.77)0.36-High (N=279)98596656--Multivariable HR (95% CI)1 (referent)1.18 (0.85-1.62)1.85 (1.34-2.55)1.75 (1.26-2.44) Citation Format: Tomotaka Ugai, Juha P. Väyrynen, Koichiro Haruki, Naohiko Akimoto, Mai Chan Lau, Rong Zhong, Sara A. Väyrynen, Melissa Zhao, Andressa Dias Costa, Jennifer Borowsky, Jennifer L. Guerriero, Charles S. Fuchs, Xuehong Zhang, Molin Wang, Marios Giannakis, Jeffrey A. Meyerhardt, Jonathan A. Nowak, Shuji Ogino. Smoking and colorectal cancer Iincidence by tumor microenvironment in cancer tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2733.

Introduction: RNF43 is a transmembrane E3 ubiquitin ligase and WNT signaling suppressor that is commonly mutated in colorectal cancer (CRC). A C-terminal RNF43 hotspot mutation, RNF43_G659fs, occurs in approximately 36% (55/151) of microsatellite-instability (MSI)-high CRCs, but its underlying mechanism and function remain poorly understood. This study investigated the functional role of RNF43_G659fs in order to evaluate potential novel therapeutic approaches for tumors harboring this mutation. Methods: Isogenic RNF43117mut and RNF43659mut cell line models were generated using the CRISPR/Cas9 system to evaluate CRC tumorigenesis and WNT dependency. RNF43659mut was screened with a novel high-throughput drug repurposing library that employed a set of 5363 small molecules to identify compounds capable of selectively inhibiting RNF43659mut cell growth. Small molecules that selectively killed the RNF43659mut cells were validated in organoid models. Proteomic analysis, RNA-Seq and gene set enrichment analysis (GSEA) were performed to characterize mechanistic interactions and related signaling pathways of RNF43659mut in CRC. Results: Unlike N-terminal RNF43 frameshift mutations, we observed that RNF43659mut conferred a growth advantage over RNF43WT cells independent of WNT signaling. Furthermore, RNF43659mut and RNF43WT exhibited differential drug responses in the high-throughput drug repurposing screen which revealed that RNF43659mut cells were vulnerable to PI3K/AKT/mTOR inhibitors, including BYL-719 (Alpelisib). Enhanced AKT and mTOR activation was observed in RNF43659mut cell and attenuated by BYL-719 treatment in a dose-dependent manner. These results were subsequently validated in patient-derived organoid models. Furthermore, immunoprecipitation and proteomic analysis revealed interactions between RNF43_G659fs and p85, a negative regulator of PI3K. We also demonstrated that the RNF43_G659fs mutant activated PI3K/AKT/mTOR signaling through binding and degradation of p85. Consistent with the role of PI3K in immunomodulation, our RNA-Seq results showed that the RNF43_G659fs mutation was positively related to NF-kB activation (Normalized Enrichment Score=1.842, p Conclusion: This study confirms that RNF43659mut is an essential driver mutation in CRC and provides evidence that patients harboring RNF43_G659fs-mutant tumors may respond favorably to PI3K inhibition. Citation Format: Lishan Fang, Dane Ford-Roshon, Max Russo, Casey O'Brien, Carino Gurjao, Maximilien Grandclaudon, Steven M. Corsello, Srivatsan Raghavan, Namrata Udeshi, James Berstler, Ewa Sicinska, Kimmie Ng, Marios Giannakis. RNF43 G659fs is an oncogenic mutation in colorectal cancer and sensitizes tumor cells to PI3K/mTOR inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 960.

Background: The release of ATP from dying cancer cells in response to platinum-based chemotherapy increases immunosuppressive extracellular adenosine, which can bind and activate the A2a and A2b receptors on immune cells, diminishing the effectiveness of the anti-tumor immune response. Concomitant dual adenosine receptor blockade may therefore enhance the therapeutic efficacy of some chemotherapeutic agents. Etrumadenant (etruma, AB928) is the first clinical-stage small-molecule, selective dual antagonist of A2aR and A2bR for which combination data with mFOLFOX-6 in mCRC were previously presented1,2. Here we present updated results for safety in all patients (pts) and efficacy in 3L+ pts. Methods: ARC-3 (NCT03720678) was a phase 1/1b, multicenter, open-label study to evaluate etruma + mFOLFOX-6 in pts with mCRC. Eligible pts had histologically confirmed mCRC with ≥1 measurable lesion. Pts received 75 mg (phase 1 only) or 150 mg etruma orally once daily (QD) + mFOLFOX-6. The primary objective was to assess the safety of the combination; secondary objectives included evaluation of clinical activity. Results: As of 20Nov2020, 44 pts were enrolled and received etruma (75mg: n=4; 150mg: n=40) + mFOLFOX-6. Etruma-related AEs were reported in 34 pts and were mostly Grade 1 or 2; those reported in >30% of pts were fatigue and nausea. One pt had an etruma-related Grade 3 acute kidney injury and there were no Grade 4-5 etruma-related SAEs. There were 22 efficacy-evaluable 3L+ subjects, all of which had received prior FOLFOX and/or FOLFIRI. In this subgroup, the ORR was 9.1% and the median PFS and OS were 3.9 and 15.7 mos respectively. Conclusions: Updated results confirm etruma + mFOLFOX-6 was well tolerated in pts with mCRC without significant additive toxicity and was associated with disease control in heavily pretreated pts. Based on these encouraging data, a platform study (ARC-9) has been initiated to further explore this and other combinations in mCRC pts. References 1. Cecchini M, et.al. AACR Annual Meeting 2020, Abstract# 99532. Udyavar A, et.al. SITC Annual Meeting 2020, Abstract# 338 Citation Format: Michael Cecchini, Cheng Quah, Shiyao Liu, Rachel Woloski, Akshata Udyavar, Marios Giannakis. ARC-3: Updated results of etrumadenant (AB928) + modified FOLFOX-6 (mFOLFOX-6) in metastatic colorectal cancer (mCRC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT129.