T cell-inflamed gene expression profile is associated with favorable disease-specific survival in non-hypermutated microsatellite-stable colorectal cancer patients.

BACKGROUND: The anti-tumor immune response plays a key role in colorectal cancer (CRC) progression and survival. The T cell-inflamed gene expression profile (GEP) is a biomarker predicting response to checkpoint inhibitor immunotherapy across immunogenic cancer types, but the prognostic value in CRC is unknown. We evaluated associations with disease-specific survival, somatic mutations, and examined its differentially expressed genes and pathways among 84 sporadic CRC patients from the Seattle Colon Cancer Family Registry. METHODS: Gene expression profiling was performed using Nanostring's nCounter PanCancer IO 360 panel. Somatic mutations were identified by a targeted DNA sequencing panel. RESULTS: The T cell-inflamed GEP was positively associated with tumor mutation burden and microsatellite instability high (MSI-H). Higher T cell-inflamed GEP had favorable CRC-specific survival (hazard ratio [HR] per standard deviation unit = 0.50, p = 0.004) regardless of hypermutation or MSI status. Analysis of recurrently mutated genes having at least 10 mutation carriers, suggested that the T cell-inflamed GEP is positively associated with RYR1, and negatively associated with APC. However, these associations were attenuated after adjusting for hypermutation or MSI status. We also found that expression of genes RPL23, EPCAM, AREG and ITGA6, and the Wnt signaling pathway was negatively associated with the T cell-inflamed GEP, which might indicate immune-inhibitory mechanisms. CONCLUSIONS: Our results show that the T cell-inflamed GEP is a prognostic biomarker in non-hypermutated microsatellite-stable CRC. This also suggests that patient stratification for immunotherapy within this CRC subgroup should be explored further. Moreover, reported immune-inhibitory gene expression signals may suggest targets for therapeutic combination with immunotherapy.

Associations between molecular characteristics of colorectal serrated polyps and subsequent advanced colorectal neoplasia.

PURPOSE: BRAF mutation and DNA hypermethylation have linked sessile serrated adenomas/polyps (SSA/Ps) to serrated colorectal cancer (CRC) in cross-sectional studies, but they have not been evaluated in a longitudinal study. We aimed to evaluate the associations between molecular markers of serrated polyps and subsequent advanced colorectal neoplasia. METHODS: Study subjects included Kaiser Permanente Washington members aged 20-75 years who received an index colonoscopy between 1/1/1998 and 12/31/2007 and had hyperplastic polyps (HPs) or SSA/Ps according to study pathology review. Polyps from index colonoscopies were removed and assayed for BRAF mutation, CpG island methylator phenotype (CIMP), and MLH1 methylation. Pathology reports and biopsies from the subsequent lower gastrointestinal endoscopy through 1/1/2013 were reviewed for advanced colorectal neoplasia. We identified additional incident CRC cases through linkage to the Seattle-Puget Sound Surveillance Epidemiology and End Results registry. We used generalized estimating equations to calculate adjusted odds ratios (OR) and 95% confidence intervals (CI) for subsequent advanced colorectal neoplasia, comparing index serrated polyps with different molecular markers. RESULTS: We included 553 individuals with index serrated polyps (420 HPs and 133 SSA/Ps) and 795 subsequent endoscopies. The prevalence of BRAF-mutant, CIMP-high, and MLH1-methylated serrated polyps were 51%, 4%, and 2%, respectively. BRAF and CIMP were not associated with subsequent advanced colorectal neoplasia. MLH1-methylated SSP/As were significantly more likely to have subsequent advanced neoplasia (OR = 4.66, 95% CI 1.06-20.51). CONCLUSION: Our results suggest that BRAF-mutant and CIMP-high serrated polyps are not associated with subsequent advanced colorectal neoplasia. Among SSA/Ps, MLH1 methylation may be an important marker to identify high-risk CRC precursors.

Gene expression in breast and adipose tissue after 12 months of weight loss and vitamin D supplementation in postmenopausal women.

Adipose tissue is involved in the etiology of postmenopausal breast cancer, possibly through increased sex steroid hormone production, inflammation, and altered adipokines. Vitamin D may affect these pathways but its effect on gene expression in different tissues has not been examined. Within a double-blind, 12-month placebo-controlled randomized trial, we compared 2000 IU/day oral vitamin D3 supplementation (N = 39) vs. placebo (N = 40) on the expression of 5 genes in breast and adipose tissue in overweight/obese postmenopausal women (50-75 years). All participants had serum 25-hydroxyvitamin D (25(OH)D) levels ≥ 10-<32 ng/mL ("insufficient") and concurrently completed a behavioral weight loss program. Random periareolar fine needle aspiration (RPFNA) and abdominal subcutaneous adipose tissue biopsies were performed at baseline and 12 months. Changes in expression of aromatase (CYP19A1), peroxisome proliferator-activated receptor gamma (PPARG), adiponectin (ADIPOQ), monocyte-chemoattractant protein 1 (MCP-1), and vitamin D receptor (VDR) were analyzed by qRT-PCR. Compared to placebo, 2000 IU vitamin D did not show significant effects on gene expression in breast or adipose tissue. Replete women (i.e., 25(OH)D ≥ 32 ng/mL; N = 17) showed a small decrease in MCP-1 expression compared to an increase among women who remained 'insufficient' despite supplementation (N = 12) (Replete:-1.6% vs. Non-replete: 61.2%, p = 0.015) in breast, but not adipose tissue. No statistically significant differences in gene expression were detected according to degree of weight loss. Vitamin D repletion during weight loss may have different effects on gene expression in breast and adipose tissue. Further research on the localized effects of vitamin D is needed to determine its effect on breast cancer risk.

Tissue-specific patterns of gene expression in the epithelium and stroma of normal colon in healthy individuals in an aspirin intervention trial.

Regular aspirin use reduces colon adenoma and carcinoma incidence. UDP-glucuronosyltransferases (UGT) are involved in aspirin metabolism and clearance, and variant alleles in UGT1A6 have been shown to alter salicylic acid metabolism and risk of colon neoplasia. In a randomized, cross-over, placebo-controlled trial of 44 healthy men and women, homozygous for UGT1A6*1 or UGT1A6*2, we explored differences between global epithelial and stromal expression, using Affymetrix U133 + 2.0 microarrays and tested effects of 60-day aspirin supplementation (325 mg/d) on epithelial and stromal gene expression and colon prostaglandin E2 (PGE2) levels. We conducted a comprehensive study of differential gene expression between normal human colonic epithelium and stroma from healthy individuals. Although no statistically significant differences in gene expression were observed in response to aspirin or UGT1A6 genotype, we have identified the genes uniquely and reproducibly expressed in each tissue type and have analyzed the biologic processes they represent. Here we describe in detail how the data, deposited in the Gene Expression Omnibus (GEO) - accession number GSE71571 - was generated including the basic analysis as contained in the manuscript published in BMC Medical Genetics with the PMID 25927723 (Thomas et al., 2015 [9]).

Tissue-specific patterns of gene expression in the epithelium and stroma of normal colon in healthy individuals in an aspirin intervention trial.

BACKGROUND: Regular aspirin use reduces colon adenoma and carcinoma incidence. UDP-glucuronosyltransferases (UGT) are involved in aspirin metabolism and clearance, and variant alleles in UGT1A6 have been shown to alter salicylic acid metabolism and risk of colon neoplasia. METHODS: In a randomized, cross-over, placebo-controlled trial of 44 healthy men and women, homozygous for UGT1A6*1 or UGT1A6*2, we explored differences between global epithelial and stromal expression, using Affymetrix U133 + 2.0 microarrays and tested effects of 60-day aspirin supplementation (325 mg/d) on epithelial and stromal gene expression and colon prostaglandin E2 (PGE2) levels. RESULTS: No statistically significant differences in gene expression were observed in response to aspirin or UGT1A6 genotype, but tissue PGE2 levels were lower with aspirin compared to placebo (p <0.001). Transcripts differentially expressed between epithelium and stroma (N = 4916, P <0.01, false discovery rate <0.001), included a high proportion of genes involved in cell signaling, cellular movement, and cancer. Genes preferentially expressed in epithelium were involved in drug and xenobiotic metabolism, fatty acid and lipid metabolism, apoptosis signaling, and ion transport. Genes preferentially expressed in stroma included those involved in inflammation, cellular adhesion, and extracellular matrix production. Wnt-Tcf4 pathway genes were expressed in both epithelium and stroma but differed by subcellular location. CONCLUSIONS: These results suggest that, in healthy individuals, subtle effects of aspirin on gene expression in normal colon tissue are likely overwhelmed by inter-individual variability in microarray analyses. Differential expression of critical genes between colonic epithelium and stroma suggest that these tissue types need to be considered separately.

Potential contribution of cytochrome P450 2B6 to hepatic 4-hydroxycyclophosphamide formation in vitro and in vivo.

Results from retrospective studies on the relationship between cytochrome P450 (P450) 2B6 (CYP2B6) genotype and cyclophosphamide (CY) efficacy and toxicity in adult cancer patients have been conflicting. We evaluated this relationship in children, who have faster CY clearance and receive different CY-based regimens than adults. These factors may influence the P450s metabolizing CY to 4-hydroxycyclophosphamide (4HCY), the principal precursor to CY's cytotoxic metabolite. Therefore, we sought to characterize the in vitro and in vivo roles of hepatic CYP2B6 and its main allelic variants in 4HCY formation. CYP2B6 is the major isozyme responsible for 4HCY formation in recombinant P450 Supersomes. In human liver microsomes (HLM), 4HCY formation correlated with known phenotypic markers of CYP2B6 activity, specifically formation of (S)-2-ethyl-1,5-dimethyl-3,3-diphenyl pyrrolidine and hydroxybupropion. However, in HLM, CYP3A4/5 also contributes to 4HCY formation at the CY concentrations similar to plasma concentrations achieved in children (0.1 mM). 4HCY formation was not associated with CYP2B6 genotype at low (0.1 mM) or high (1 mM) CY concentrations potentially because CYP3A4/5 and other isozymes also form 4HCY. To remove this confounder, 4HCY formation was evaluated in recombinant CYP2B6 enzymes, which demonstrated that 4HCY formation was lower for CYP2B6.4 and CYP2B6.5 compared with CYP2B6.1. In vivo, CYP2B6 genotype was not directly related to CY clearance or ratio of 4HCY/CY areas under the curve in 51 children receiving CY-based regimens. Concomitant chemotherapy agents did not influence 4HCY formation in vitro. We conclude that CYP2B6 genotype is not consistently related to 4HCY formation in vitro or in vivo.

Determinants of aspirin metabolism in healthy men and women: effects of dietary inducers of UDP-glucuronosyltransferases.

BACKGROUND/AIMS: Interindividual variation in aspirin (ASA) metabolism is attributed to concomitant use of drugs or alcohol, urine pH, ethnicity, sex, and genetic variants in UDP-glucuronosyltransferases (UGT). Little is known about the effects of diet. METHODS: We evaluated cross-sectionally whether urinary excretion of ASA and its metabolites [salicylic acid (SA), salicyluric acid (SUA) phenolic glucuronide (SUAPG), salicylic acid acyl glucuronide (SAAG) and salicylic acid phenolic glucuronide (SAPG)] differed by UGT1A6 genotype and dietary factors shown to induce UGT. Following oral treatment with 650 mg ASA, urine was collected over 8 h in 264 men and 264 women (21-45 years old). RESULTS: There were statistically significant differences in metabolites excreted between sexes and ethnicities. Men excreted more SUA; women more ASA (p = 0.03), SA, SAAG and SAPG (p ≤ 0.001 for all). Compared to Caucasians, Asians excreted more ASA, SA and SAAG, and less SUA and SUAPG (p ≤ 0.03 for all); African-Americans excreted more SAAG and SAPG and less SUA (p ≤ 0.04). There was no effect of UGT1A6 genotypes. Increased ASA and decreased SUAPG excretion was observed with increased servings of vegetables (p = 0.008), specifically crucifers (p = 0.05). CONCLUSION: Diet may influence the pharmacokinetics of ASA, but effects may be through modulation of glycine conjugation rather than glucuronidation.

Associations between polymorphisms in glucuronidation and sulfation enzymes and sex steroid concentrations in premenopausal women in the United States.

Glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGT) and sulfation, catalyzed by sulfotransferases (SULT), are pathways through which sex steroids are metabolized to less active compounds. These enzymes are highly polymorphic and genetic variants frequently result in higher or lower activity. The phenotypic effects of these polymorphisms on circulating sex steroids in premenopausal women have not yet been investigated. One hundred and seventy women aged 40-45 years had a blood sample drawn during the follicular phase of the menstrual cycle for sex steroid measures and to obtain genomic DNA. Urine was collected for 2-hydroxy (OH) estrone (E(1)) and 16α-OH E(1) measures. Generalized linear regression models were used to assess associations between sex steroids and polymorphisms in the UGT1A and UGT2B families, SULT1A1, and SULT1E1. Women with the UGT1A1(TA7/TA7) genotype had 25% lower mean estradiol (E(2)) concentrations compared to the wildtype (TA6/TA6) (p=0.02). Similar associations were observed between SULT1A1(R213/H213) and E(1) (13% lower mean E(1) concentration vs. wildtype; p-value=0.02) and UGT2B4(E458/E458) and dehydroepiandrosterone (DHEA) (20% lower mean DHEA vs. wildtype; p-value=0.03). The SULT1E1(A/C) and the UGT1A1(TA7)-UGT1A3(R11) haplotypes were associated with reduced estrogen concentrations. Further study of UGT and SULT polymorphisms and circulating sex steroid measures in larger populations of premenopausal women is warranted.

Associations between polymorphisms in glucuronidation and sulfation enzymes and mammographic breast density in premenopausal women in the United States.

OBJECTIVE: Sex hormones are metabolized to less active compounds via (a) glucuronidation catalyzed by UDP-glucuronosyltransferases (UGT) and (b) sulfation catalyzed by sulfotransferases (SULT). Functional UGT and SULT polymorphisms can affect clearance of sex hormones, thereby influencing exposure in hormone-sensitive tissues, such as the breast. We assessed relationships between functional polymorphisms in the UGT and SULT genes and breast density in premenopausal women. METHODS: One hundred seventy-five women ages 40 to 45 years, who had a screening mammogram taken within the previous year, provided a genomic DNA sample. Mammograms were digitized to obtain breast density measures. Using generalized linear regression, we assessed associations between percent breast density and polymorphisms in the UGT1A and UGT2B families, SULT1A1, and SULT1E1. RESULTS: Women with the SULT1A1(H213/H213) genotype had 16% lower percent breast density compared with women with the SULT1A1(R213/R213) genotype after controlling for ethnicity (P = 0.001). Breast density was 5% lower among women carrying at least one copy of the UGT1A1(TA7)-UGT1A3(R11)-UGT1A3(A47) haplotype compared with the UGT1A1(TA6)-UGT1A3(W11R)-UGT1A3(V47A) haplotype (P = 0.07). No associations were observed between polymorphisms in the UGT2B family or SULT1E1 and breast density. CONCLUSION: Polymorphisms in SULT1A1 and the UGT1A locus may influence percent breast density in premenopausal women.

Genetic variability, haplotypes, and htSNPs for exons 1 at the human UGT1A locus.

UDP-Glucuronosyltransferases (UGTs) are a superfamily of enzymes responsible for glucuronidation of xenobiotics and endobiotics. Genetic polymorphisms have been identified in the promoter and exonic regions of several UGT genes. The UGT1As on chromosome 2q37 have unique exons 1 but share the remainder of their coding sequence. We screened exon 1 of each of the nine functional UGT1As in Asians (n=46) and Caucasians (n=92) with the aim of determining linkage disequilibrium (LD) and haplotypes across the entire locus in both populations. For polymorphisms in UGT 1A3, 1A4, 1A5, 1A7, and 1A8, we observed significant differences in the allele frequency between the two populations. The haplotype block structure across the UGT1A locus was constructed using all 83 polymorphisms and showed four and five haplotype blocks in Caucasians and Asians, respectively. There was long-distance LD between UGT pairs: 1A8 and 1A10; 1A1 and 1A3; 1A1 and 1A6; 1A6 and 1A7; and 1A7 and 1A9. Whereas both ethnic groups shared some haplotype-tagging SNPs (htSNPs), Caucasians and Asians also had unique htSNPs. This was partly due to the fact that rare variants (<5% allele frequency) were included in our analyses. Haplotypes with frequencies >5% represented only 60% of Caucasian and 65% of Asian UGT1A haplotypes. Differences in haplotype distribution patterns suggest individual and ethnic differences in glucuronidation capacity.