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Dietary therapy successfully treats eosinophilic oesophagitis (EoE), but limited data exist on predictors of patient response.To determine response rates and to identify predictors of histologic response to elimination diets in adults with EoE METHODS: This was a retrospective, single-centre study of adults with PPI-refractory EoE undergoing dietary therapy with six food elimination diet (SFED) or extended six food elimination diet (ExSFED) in an outpatient setting from January 2012 to January 2019. Patient demographics, radiologic and endoscopic findings, endoscopic reference (EREF) scores, histology and symptoms were evaluated before and after food elimination. Histologic response was assessed via tissue obtained from endoscopically-guided biopsy or Cytosponge. Dietary therapy adherence was assessed via structured phone interview. Multivariable logistic regression analysis was performed to identify predictors of dietary response.We included 68 patients, of whom 62% had a histologic response to dietary therapy (81% to SFED, 19% to ExSFED). Median duration of follow-up was 45 months (IQR, 34-53 months). On multivariable analysis, higher pre-SFED EREF score was the only variable associated with dietary non-response (OR 0.07, 95% CI 0.49, 0.98; p = 0.04).In adults with EoE, histologic dietary non-response to SFED was associated with a higher pre-SFED EREF score, suggesting that fixed structural disease may predict dietary non-response. Our additional observations of poor correlation between symptomatic and histologic flares, and identification of ExSFED responders, suggest that histologic confirmation should be sought before committing patients to lifelong dietary changes. We also recommend the consideration of restricting legumes and corn in SFED non-responders as ExSFED detected additional dietary responders.

Supplemental Figures 1-6. Supplemental Figure 1. A typical KRAS Sanger dye termination sequencing trace for a pancreatic cancer sample. The arrow indicates the mutation site; the black peak represents a wild-type allele and the red peak represents the mutant allele. Supplemental Figure 2. (A) Copy numbers per sample of methylated KCNK12 assayed form pancreatic juice of patients with normal pancreas (NP), chronic pancreatitis (CP), high grade dysplastic main-duct intraductal pancreatic mucinous neoplasm (IPMN), and pancreatic cancer (PC) were used to calculate (B) receiver operating characteristics curves of methylated KCNK12 for the detection of PC in comparison to NP (black) and CP (grey). Supplemental Figure 3. (A) Copy numbers per sample of methylated CLEC11A assayed form pancreatic juice of patients with normal pancreas (NP), chronic pancreatitis (CP), high grade dysplastic main-duct intraductal pancreatic mucinous neoplasm (IPMN), and pancreatic cancer (PC) were used to calculate (B) receiver operating characteristics curves of methylated CLEC11A for the detection of PC in comparison to NP (black) and CP (grey). Supplemental Figure 4. (A) Copy numbers per sample of methylated NDRG4 assayed form pancreatic juice of patients with normal pancreas (NP), chronic pancreatitis (CP), high grade dysplastic main-duct intraductal pancreatic mucinous neoplasm (IPMN), and pancreatic cancer (PC) were used to calculate (B) receiver operating characteristics curves of methylated NDRG4 for the detection of PC in comparison to NP (black) and CP (grey). Supplemental Figure 5. (A) Genomic copy numbers of methylated IKZF1 in normal pancreas (NP), chronic pancreatitis (CP), high grade dysplastic main-duct intraductal pancreatic mucinous neoplasm (IPMN), and pancreatic cancer (PC) were used to calculate (B) receiver operating characteristics curves of methylated IKZF1 for the detection of PC in comparison to NP (black) and CP (grey). Supplemental Figure 6. (A) Genomic copy numbers of methylated PKRCB in normal pancreas (NP), chronic pancreatitis (CP), high grade dysplastic main-duct intraductal pancreatic mucinous neoplasm (IPMN), and pancreatic cancer (PC) were used to calculate (B) receiver operating characteristics curves of methylated PKRCB for the detection of PC in comparison to NP (black) and CP (grey).

Supplementary Figure 1. Gut microbial composition and diversity in patients with and without adenomas. A) Microbial composition was similar between groups across all taxonomic levels. The groups did not differ in terms of α-diversity: B) observed number of operational taxonomic units (OTUs) or C) the Shannon diversity index. The groups did not cluster by β-diversity: D) unweighted and E) weighted UniFrac distance metrics.

Supplementary Table 1. Pharmacological Inhibitors of Endocytic Pathways

Supplementary Fig. 5. Adrenomedullin increases reactive oxygen species production. Reactive oxygen/nitrogen species (ROS/RNS) were detected in INS-1 cells pre-treated with either 1 or 20 pM adrenomedullin for 24 hours. The orange filter detects superoxide and nitric oxide generation, while the green filter detects all reactive oxygen species. Positive control cells were treated with Pyocyanin (Pyo) to induce ROS production and negative control cells were treated with Pyo + N-acetyl-L-cysteine (NAC) to inhibit ROS production. Scale bar represents 10 µm.

Purpose: To determine the frequency and prognostic association of molecular markers by anatomic tumor site in patients with stage III colon carcinomas.Experimental Design: In a randomized trial of adjuvant FOLFOX ± cetuximab, BRAFV600E and KRAS (exon 2) mutations and DNA mismatch repair (MMR) proteins were analyzed in tumors (N = 3,018) in relationship to tumor location, including subsite. Cox models were used to assess clinical outcome, including overall survival (OS).Results: KRAS codon 12 mutations were most frequent at the splenic flexure and cecum; codon 13 mutations were evenly distributed. BRAF mutation frequency sharply increased from transverse colon to cecum in parallel with deficient (d) MMR. Nonmutated BRAF and KRAS tumors progressively decreased from sigmoid to transverse (all P < 0.0001). Significantly, poorer OS was found for mutant KRAS in distal [HR, 1.98; 95% confidence interval (CI), 1.49–2.63; P < 0.0001] versus proximal (1.25; 95% CI, 0.97–1.60; P = 0.079) cancers. BRAF status and outcome were not significantly associated with tumor site. Proximal versus distal dMMR tumors had significantly better outcome. An interaction test was significant for tumor site by KRAS (Padjusted = 0.043) and MMR (Padjusted = 0.010) for OS. Significant prognostic differences for biomarkers by tumor site were maintained in the FOLFOX arm. Tumor site was independently prognostic with a stepwise improvement from cecum to sigmoid (OS: Padjusted = 0.001).Conclusions: Mutation in BRAF or KRAS codon 12 was enriched in proximal cancers whereas nonmutated BRAF/KRAS was increased in distal tumors. Significant differences in outcome for KRAS mutations and dMMR were found by tumor site, indicating that their interpretation should occur in the context of tumor location. Clin Cancer Res; 21(23); 5294–304. ©2015 AACR.

Supplemental Table 2. Comparison to ICGC array data

Supplemental Table 1. Univariate Cox Proportional Hazards Regression Models for Clinical Factors and Biomarkers (TTR, DFS, and OS)

Purpose: Gastric adenocarcinoma is the third most common cause of cancer mortality worldwide. Accurate and affordable noninvasive detection methods have potential value for screening and surveillance. Herein, we identify novel methylated DNA markers (MDM) for gastric adenocarcinoma, validate their discrimination for gastric adenocarcinoma in tissues from geographically separate cohorts, explore marker acquisition through the oncogenic cascade, and describe distributions of candidate MDMs in plasma from gastric adenocarcinoma cases and normal controls.Experimental Design: Following discovery by unbiased whole-methylome sequencing, candidate MDMs were validated by blinded methylation-specific PCR in archival case–control tissues from U.S. and South Korean patients. Top MDMs were then assayed by an analytically sensitive method (quantitative real-time allele-specific target and signal amplification) in a blinded pilot study on archival plasma from gastric adenocarcinoma cases and normal controls.Results: Whole-methylome discovery yielded novel and highly discriminant candidate MDMs. In tissue, a panel of candidate MDMs detected gastric adenocarcinoma in 92% to 100% of U.S. and South Korean cohorts at 100% specificity. Levels of most MDMs increased progressively from normal mucosa through metaplasia, adenoma, and gastric adenocarcinoma with variation in points of greatest marker acquisition. In plasma, a 3-marker panel (ELMO1, ZNF569, C13orf18) detected 86% (95% CI, 71–95) of gastric adenocarcinomas at 95% specificity.Conclusions: Novel MDMs appear to accurately discriminate gastric adenocarcinoma from normal controls in both tissue and plasma. The point of aberrant methylation during oncogenesis varies by MDM, which may have relevance to marker selection in clinical applications. Further exploration of these MDMs for gastric adenocarcinoma screening and surveillance is warranted. Clin Cancer Res; 24(22); 5724–34. ©2018 AACR.

Supplemental Figure 1: Quantitative allele-specific real-time target and signal amplification (QuARTS) assay.

Supplemental Table 3. Function and cancer biology associations of candidates

Supplemental Figure 2. Box plots of methylated DNA marker levels in various types of cancers were made from publically available hybridization array data from The Cancer Genome Atlas.

Supplementary Table 1. MiRKAT association P values for various adenoma characteristics in individuals with adenomas

Supplementary Figure 2. Heat map of significantly predictive taxa. Hierarchical clustering is based on the Euclidean distance of log proportion, complete linkage. The left cluster is enriched in adenoma samples (P=0.1, Fisher's exact test).

Background: Host immune response may predict the course of colorectal cancer. We examined the survival of 468 colorectal cancer patients associated with two tumor-infiltrating immune biomarkers, the number of cytotoxic T lymphocytes (CTLs), and the activated CTLs, as reflected by the number of cells expressing granzyme B (GZMB) in the prospective Iowa Women's Health Study.Methods: Using paraffin-embedded tissue samples, we constructed and immunostained tumor microarrays with CD8 (for CTL) and GZMB antibodies. We scored CTL and GZMB densities in tumor epithelial and stromal tissues and also created a composite score for each biomarker (sum of the scores across tissue compartments). Cox regression estimated the HR and 95% confidence intervals (CI) for all-cause and colorectal cancer–specific death associated with each composite score.Results: CTL and GZMB composite scores were positively correlated (r = 0.65) and each biomarker was inversely correlated with stage at diagnosis. Both composite scores were higher in proximal colon tumors and tumors characterized by MSI-high, CIMP-high, or BRAF mutation status. HRs (95% CI) were 0.53 (0.38–0.75; Ptrend = 0.0004) and 0.66 (0.51–0.86; Ptrend = 0.002) for all-cause death, respectively, and 0.30 (0.18–0.51; Ptrend < 0.0001) and 0.41 (0.27–0.63; Ptrend < 0.0001) for colorectal cancer–related death, respectively. Including CTL and GZMB scores simultaneously in the model significantly improved the predictive performance of the models for all-cause and colorectal cancer–related death.Conclusions: Higher tumor infiltration with CTL and GZMB cells is associated with improved all-cause and cancer-specific survival of colorectal cancer patients.Impact: Both the number of CTLs and GZMB appear to be useful prognostic factors in colorectal cancer, irrespective of stage. Cancer Epidemiol Biomarkers Prev; 26(4); 622–31. ©2016 AACR.

Purpose:The burden of esophageal cancer continues to rise, and noninvasive screening tools are needed. Methylated DNA markers (MDM) assayed from plasma show promise in detection of other cancers. For esophageal cancer detection, we aimed to discover and validate MDMs in tissue, and determine their feasibility when assayed from plasma.Experimental Design:Whole-methylome sequencing was performed on DNA extracted from 37 tissues (28 EC; 9 normal esophagus) and 8 buffy coat samples. Top MDMs were validated by methylation specific PCR on tissue from 76 EC (41 adeno, 35 squamous cell) and 17 normal esophagus. Quantitative allele-specific real-time target and signal amplification was used to assay MDMs in plasma from 183 patients (85 EC, 98 controls). Recursive partitioning (rPART) identified MDM combinations predictive of esophageal cancer. Validation was performed in silico by bootstrapping.Results:From discovery, 23 candidate MDMs were selected for independent tissue validation; median area under the receiver operating curve (AUC) for individual MDMs was 0.93. Among 12 MDMs advanced to plasma testing, rPART modeling selected a 5 MDM panel (FER1L4, ZNF671, ST8SIA1, TBX15, ARHGEF4) which achieved an AUC of 0.93 (95% CI, 0.89–0.96) on best-fit and 0.81 (95% CI, 0.75–0.88) on cross-validation. At 91% specificity, the panel detected 74% of esophageal cancer overall, and 43%, 64%, 77%, and 92% of stages I, II, III, and IV, respectively. Discrimination was not affected by age, sex, smoking, or body mass index.Conclusions:Novel MDMs assayed from plasma detect esophageal cancer with moderate accuracy. Further optimization and clinical testing are warranted.

Background: Adenomatous polyps are the most common precursor to colorectal cancer, the second leading cause of cancer-related death in the United States. We sought to learn more about early events of carcinogenesis by investigating shifts in the gut microbiota of patients with adenomas.Methods: We analyzed 16S rRNA gene sequences from the fecal microbiota of patients with adenomas (n = 233) and without (n = 547).Results: Multiple taxa were significantly more abundant in patients with adenomas, including Bilophila, Desulfovibrio, proinflammatory bacteria in the genus Mogibacterium, and multiple Bacteroidetes species. Patients without adenomas had greater abundances of Veillonella, Firmicutes (Order Clostridia), and Actinobacteria (family Bifidobacteriales). Our findings were consistent with previously reported shifts in the gut microbiota of colorectal cancer patients. Importantly, the altered adenoma profile is predicted to increase primary and secondary bile acid production, as well as starch, sucrose, lipid, and phenylpropanoid metabolism.Conclusions: These data hint that increased sugar, protein, and lipid metabolism along with increased bile acid production could promote a colonic environment that supports the growth of bile-tolerant microbes such as Bilophilia and Desulfovibrio. In turn, these microbes may produce genotoxic or inflammatory metabolites such as H2S and secondary bile acids, which could play a role in catalyzing adenoma development and eventually colorectal cancer.Impact: This study suggests a plausible biological mechanism to explain the links between shifts in the microbiota and colorectal cancer. This represents a first step toward resolving the complex interactions that shape the adenoma–carcinoma sequence of colorectal cancer and may facilitate personalized therapeutics focused on the microbiota. Cancer Epidemiol Biomarkers Prev; 26(1); 85–94. ©2016 AACR.

Supplementary Fig. 4. AM and PC-Exosomes modestly upregulate insulin gene expression. All studies were done using INS-1, a rat β-cell line. (A) Relative expression of insulin genes INS1 and INS2 in response to varying concentrations (1, 20, or 40 pM) of AM and AM inhibitor AM 22-52. (B) Western blot analysis of insulin protein output in response to 1, 20, or 40 pM of AM. 20 µg of protein lysate was loaded. (C) Relative expression of insulin genes INS1 and INS2 in response to the addition of 50 µg of PC-Exo. * P

Supplemental Table 1. AUC and 95% confidence intervals for the technical validation study

Supplementary Fig. 1. NanoTracker results of PC-Exosome isolated from PC patient-derived cell lines and patient plasma. (A-B) PC-Exo isolated from a PC patient-derived cell line show the majority of microparticles are of exosomal size ([A] mode, 105{plus minus}4.2 nm). In (B), microparticles are more of microvesicle size (mode, 294{plus minus}13.5 nm). Thus, depending on the cell line, a 100,000 x g isolation can yield both exosomes and microvesicles. (C-F) PC-Exo isolated from the plasma of different PC patients show that the majority of microparticles in plasma are exosomes (modes, 156{plus minus}8.6 nm; 88{plus minus}5.0 nm; 92{plus minus}3.1 nm; 100{plus minus}7.0 nm, respectively). Analysis was taken by averaging 5, 60-second movies.

Supplementary Figure Legends

Purpose: Pancreatic cancer frequently causes diabetes. We recently proposed adrenomedullin as a candidate mediator of pancreatic β-cell dysfunction in pancreatic cancer. How pancreatic cancer–derived adrenomedullin reaches β cells remote from the cancer to induce β-cell dysfunction is unknown. We tested a novel hypothesis that pancreatic cancer sheds adrenomedullin-containing exosomes into circulation, which are transported to β cells and impair insulin secretion.Experimental Methods: We characterized exosomes from conditioned media of pancreatic cancer cell lines (n = 5) and portal/peripheral venous blood of patients with pancreatic cancer (n = 20). Western blot analysis showed the presence of adrenomedullin in pancreatic cancer-exosomes. We determined the effect of adrenomedullin-containing pancreatic cancer exosomes on insulin secretion from INS-1 β cells and human islets, and demonstrated the mechanism of exosome internalization into β cells. We studied the interaction between β-cell adrenomedullin receptors and adrenomedullin present in pancreatic cancer-exosomes. In addition, the effect of adrenomedullin on endoplasmic reticulum (ER) stress response genes and reactive oxygen/nitrogen species generation in β cells was shown.Results: Exosomes were found to be the predominant extracellular vesicles secreted by pancreatic cancer into culture media and patient plasma. Pancreatic cancer-exosomes contained adrenomedullin and CA19-9, readily entered β cells through caveolin-mediated endocytosis or macropinocytosis, and inhibited insulin secretion. Adrenomedullin in pancreatic cancer exosomes interacted with its receptor on β cells. Adrenomedullin receptor blockade abrogated the inhibitory effect of exosomes on insulin secretion. β cells exposed to adrenomedullin or pancreatic cancer exosomes showed upregulation of ER stress genes and increased reactive oxygen/nitrogen species.Conclusions: Pancreatic cancer causes paraneoplastic β-cell dysfunction by shedding adrenomedullin+/CA19-9+ exosomes into circulation that inhibit insulin secretion, likely through adrenomedullin-induced ER stress and failure of the unfolded protein response. Clin Cancer Res; 21(7); 1722–33. ©2014 AACR.See related commentary by Korc, p. 1508

Supplemental Table 1: Discrimination for gastric adenocarcinoma (GAC) from normal gastric mucosa by the top 22 methylated DNA marker candidates in Discovery and Technical Validation Sets. Supplemental Table 2: Function of Genes Containing Differentially Methylated Regions Identified as Targets for Candidate Markers Supplemental Table 3: Covariate Effects on Tissue Levels of Methylated DNA Marker Candidates (Data shown represent p-values).

Supplementary Fig. 2. PC-derived exosomes from additional PC cell lines and PC patient plasma can decrease insulin secretion. (A-B) PC-Exo isolated from 2 PC patient-derived cell lines decreased insulin secretion in INS-1 cells. (C-D) PC-Exo isolated from 2 different PC patient blood plasma samples can decrease insulin secretion at varying levels. * P

Purpose: Discriminant markers for pancreatic cancer detection are needed. We sought to identify and validate methylated DNA markers for pancreatic cancer using next-generation sequencing unbiased by known targets.Experimental Design: At a referral center, we conducted four sequential case–control studies: discovery, technical validation, biologic validation, and clinical piloting. Candidate markers were identified using variance-inflated logistic regression on reduced-representation bisulfite DNA sequencing results from matched pancreatic cancers, benign pancreas, and normal colon tissues. Markers were validated technically on replicate discovery study DNA and biologically on independent, matched, blinded tissues by methylation-specific PCR. Clinical testing of six methylation candidates and mutant KRAS was performed on secretin-stimulated pancreatic juice samples from 61 patients with pancreatic cancer, 22 with chronic pancreatitis, and 19 with normal pancreas on endoscopic ultrasound. Areas under receiver-operating characteristics curves (AUC) for markers were calculated.Results: Sequencing identified >500 differentially hyper-methylated regions. On independent tissues, AUC on 19 selected markers ranged between 0.73 and 0.97. Pancreatic juice AUC values for CD1D, KCNK12, CLEC11A, NDRG4, IKZF1, PKRCB, and KRAS were 0.92*, 0.88, 0.85, 0.85, 0.84, 0.83, and 0.75, respectively, for pancreatic cancer compared with normal pancreas and 0.92*, 0.73, 0.76, 0.85*, 0.73, 0.77, and 0.62 for pancreatic cancer compared with chronic pancreatitis (*, P = 0.001 vs. KRAS).Conclusions: We identified and validated novel DNA methylation markers strongly associated with pancreatic cancer. On pilot testing in pancreatic juice, best markers (especially CD1D) highly discriminated pancreatic cases from controls. Clin Cancer Res; 21(19); 4473–81. ©2015 AACR.

Supplementary Figure 1. Receiver operator curve (ROC) plots in the logistic regression model (Model 2) for all-cause (B) and CRC-death (A) within 5 years after diagnosis (adjusted for age of diagnosis, BMI, smoking status, stage and grade at diagnosis). AUC=area under the ROC curve. Supplementary Table 1. Characteristics of women diagnosed with CRC across tumor composite scores for cytotoxic T lymphocytes (CTL) and granzymeB (GZMB)a. Supplementary Table 2. Tumor stromal and epithelial cytotoxic T lymphocytes (CTL) scoresa and risk of all-cause and CRC-specific death in the CRC patients, IWHS, 1986-2011. Supplementary Table 3. Tumor stromal and epithelial granzyme B (GZMB) scores and risk of all-cause and CRC-specific death the CRC patients, IWHS, 1986-2011. Supplementary Table 4. Tumor composite scores for cytotoxic T lymphocytes (CTL) and granzyme B (GZMB) for all-cause and CRC-specific death in the CRC patients followed for 5 years, IWHS, 1986-2011.

Supplementary Fig. 3. Chlorpromazine and Nocodazole do not inhibit exosome internalization. INS-1 cells were treated with varying concentrations of chlorpromazine (A) or Nocodazole (C) for 30 minutes and 10 minutes, respectively. PKH67-dyed PANC-1 exosomes were then added to the cells for 5 hours to allow internalization. Scale bars represent 10 µm. (B) and (D) show the quantification of internalized green fluorescence per cell for chlorpromazine (B) and Nocodazole (D) treated cells.

Supplementary Table 1 from Expression and Regulatory Role of GAIP-Interacting Protein GIPC in Pancreatic Adenocarcinoma

Supplementary Figure 2 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Supplementary Figure 1 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Supplementary Figure 5 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Regulator of G-protein signaling–GAIP-interacting protein COOH terminus (GIPC) is involved in protein trafficking, endocytosis, and receptor clustering and is associated with insulin-like growth factor I receptor (IGF-IR), a receptor important for proliferation and anchorage-independent growth. Here, we described GIPC expression in different human pancreatic adenocarcinoma (PCA) cell lines and we examined the role of GIPC in the regulation of IGF-IR protein levels in PCA. Interestingly, inhibition of GIPC expression by RNA interference led to reduced IGF-IR protein levels and a subsequent decrease in proliferation of PCA cells. We also determined that the PDZ domain of GIPC is essential for the post-translational regulation and the binding of IGF-IR. The importance of GIPC in pancreatic cancer development and progression is supported by tissue microarray data of 300 pancreatic cancer specimens where GIPC is highly expressed in PCA. Taken together, our data suggest that GIPC is a central molecule for the stability of IGF-IR and could be a target for future therapy. (Cancer Res 2006; 66(21): 10264-8)

Supplementary Figure 3 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Supplementary Tables 1-2 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Supplementary Figure 6 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Supplementary Figure Legends 1-6 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Supplementary Figure 4 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

BACKGROUND/AIMS: Although T-cell intrinsic expression of G9a has been associated with murine intestinal inflammation, mechanistic insight into the role of this methyltransferase in human T-cell differentiation is ill defined, and manipulation of G9a function for therapeutic use against inflammatory disorders is unexplored. METHODS: Human naive T cells were isolated from peripheral blood and differentiated in vitro in the presence of a G9a inhibitor (UNC0642) before being characterized via the transcriptome (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin by sequencing), protein expression (cytometry by time of flight, flow cytometry), metabolism (mitochondrial stress test, ultrahigh performance liquid chromatography–tandem mas spectroscopy) and function (T-cell suppression assay). The in vivo role of G9a was assessed using 3 murine models. RESULTS: We discovered that pharmacologic inhibition of G9a enzymatic function in human CD4 T cells led to spontaneous generation of FOXP3(+) T cells (G9a-inibitors–T regulatory cells [Tregs]) in vitro that faithfully reproduce human Tregs, functionally and phenotypically. Mechanistically, G9a inhibition altered the transcriptional regulation of genes involved in lipid biosynthesis in T cells, resulting in increased intracellular cholesterol. Metabolomic profiling of G9a-inibitors–Tregs confirmed elevated lipid pathways that support Treg development through oxidative phosphorylation and enhanced lipid membrane composition. Pharmacologic G9a inhibition promoted Treg expansion in vivo upon antigen (gliadin) stimulation and ameliorated acute trinitrobenzene sulfonic acid–induced colitis secondary to tissue-specific Treg development. Finally, Tregs lacking G9a expression (G9a-knockout Tregs) remain functional chronically and can rescue T-cell transfer–induced colitis. CONCLUSION: G9a inhibition promotes cholesterol metabolism in T cells, favoring a metabolic profile that facilitates Treg development in vitro and in vivo. Our data support the potential use of G9a inhibitors in the treatment of immune-mediated conditions including inflammatory bowel disease.

PURPOSE The recommended duration of adjuvant fluoropyrimidine and oxaliplatin chemotherapy for patients with stage III colon cancer is based on tumor classification into clinically low-risk (T1-3 N1) and high-risk (T4 or N2) groups. We determined whether Immunoscore can enhance prognostication within these risk groups. MATERIALS AND METHODS Patients with stage III colon carcinomas (N = 600) were randomly selected from the infusional fluorouracil, leucovorin, and oxaliplatin arm of adjuvant trial NCCTG N0147 (Alliance for Clinical Trials in Oncology). Tumors were evaluated for Immunoscore that quantifies CD3+ and CD8+ T-cell densities in the tumor center and invasive margin by digital image analysis. Disease-free survival (DFS) by Immunoscore was analyzed using a multivariable Cox regression model in each risk group with adjustment for covariates including KRAS, BRAF V600E, and mismatch repair status. RESULTS Of 559 cancers with Immunoscore data, 299 (53.5%) were classified as clinically low-risk (T1-3 N1) and 260 (46.5%) as clinically high-risk (T4 and/or N2). Among patients with low-risk tumors, those with Immunoscore-Low versus Immunoscore-High tumors had significantly worse 5-year DFS rates (77.5% v 91.8%; hazard ratio, 1.70; 95% CI, 1.03 to 2.79; P = .037). Among patients with high-risk tumors, those with Immunoscore-Low versus Immunoscore-High tumors also had significantly worse DFS (55.3% v 70.3%; hazard ratio, 1.65; 95% CI, 1.11 to 2.47; P = .013). Tumors that were low-risk/Immunoscore-Low had similar outcomes as did tumors that were high-risk/Immunoscore-High ( P = .174). Prognostication was significantly improved in multivariable models where Immunoscore was added to clinical risk parameters and limited biomarkers (likelihood ratio test P = .0003). CONCLUSION Immunoscore can refine patient prognosis beyond clinical risk group classification, suggesting its potential utility for adjuvant decision making.

3525 Background: Tumor-infiltrating lymphocytes (TILs) are significantly associated with clinical outcomes in patients with colon cancer. However, the potential for a differential effect of TILs on prognosis based on primary tumor sidedness has not been studied. We determined the interaction between TILs and tumor sidedness in relationship to disease-free survival (DFS). Methods: We analyzed data on TIL densities by primary tumor sidedness in 1532 stage III colon carcinomas from participants in a phase III trial of FOLFOX-based adjuvant chemotherapy (NCCTG N0147). TIL densities were dichotomized as low or high (≤3 /HPF) based on an optimized cutoff previously identified for DFS in this cohort. Right-sided tumors were defined as proximal to the splenic flexure. Analysis of TILs and sidedness with DFS were examined using Kaplan-Meier methodology and multivariable Cox regression. Results: Overall, tumors with high vs low TILs had the best DFS [HRadj: 0.58 (95%CI: 0.45-0.74); Padj interaction (adj) = 0.045). Among right-sided tumors, high vs low TILs were significantly associated with improved DFS ( Padj adj =0.173)[Table]. Similar results for TILs and DFS by sidedness were found for pMMR cancers. We then analyzed our data in low risk (T1-3, N1) and high risk (T4 and/or N2) tumors. Among low risk tumors, high vs low TILs was significantly associated with improved 5-yr DFS only in right-sided tumors ( Padj =0.006) [Table]. Among high risk tumors, high vs low TILs were significantly associated with better DFS in both right-sided ( Padjadj =0.024) tumors. Conclusions: Overall, tumors with high TIL densities had significantly better DFS in right-sided but not left-sided cancers. Among low risk patients, the association of high TILs with better DFS was limited to right-sided tumors. These findings suggest that TILs should be interpreted by sidedness for prognostication. ClinicalTrials.gov Identifier: NCT00079274 . [Table: see text]