Felix Araujo-Perez

Fusobacterium Is Associated with Colorectal Adenomas

The human gut microbiota is increasingly recognized as a player in colorectal cancer (CRC). While particular imbalances in the gut microbiota have been linked to colorectal adenomas and cancer, no specific bacterium has been identified as a risk factor. Recent studies have reported a high abundance of Fusobacterium in CRC subjects compared to normal subjects, but this observation has not been reported for adenomas, CRC precursors. We assessed the abundance of Fusobacterium species in the normal rectal mucosa of subjects with (n = 48) and without adenomas (n = 67). We also confirmed previous reports on Fusobacterium and CRC in 10 CRC tumor tissues and 9 matching normal tissues by pyrosequencing. We extracted DNA from rectal mucosal biopsies and measured bacterial levels by quantitative PCR of the 16S ribosomal RNA gene. Local cytokine gene expression was also determined in mucosal biopsies from adenoma cases and controls by quantitative PCR. The mean log abundance of Fusobacterium or cytokine gene expression between cases and controls was compared by t-test. Logistic regression was used to compare tertiles of Fusobacterium abundance. Adenoma subjects had a significantly higher abundance of Fusobacterium species compared to controls (p = 0.01). Compared to the lowest tertile, subjects with high abundance of Fusobacterium were significantly more likely to have adenomas (OR 3.66, 95% CI 1.37–9.74, p-trend 0.005). Cases but not controls had a significant positive correlation between local cytokine gene expression and Fusobacterium abundance. Among cases, the correlation for local TNF-α and Fusobacterium was r = 0.33, p = 0.06 while it was 0.44, p = 0.01 for Fusobacterium and IL-10. These results support a link between the abundance of Fusobacterium in colonic mucosa and adenomas and suggest a possible role for mucosal inflammation in this process.

The severity of nonalcoholic fatty liver disease is associated with gut dysbiosis and shift in the metabolic function of the gut microbiota

Several animal studies have emphasized the role of gut microbiota in nonalcoholic fatty liver disease (NAFLD). However, data about gut dysbiosis in human NAFLD remain scarce in the literature, especially studies including the whole spectrum of NAFLD lesions. We aimed to evaluate the association between gut dysbiosis and severe NAFLD lesions, that is, nonalcoholic steatohepatitis (NASH) and fibrosis, in a well‐characterized population of adult NAFLD. Fifty‐seven patients with biopsy‐proven NAFLD were enrolled. Taxonomic composition of gut microbiota was determined using 16S ribosomal RNA gene sequencing of stool samples. Thirty patients had F0/F1 fibrosis stage at liver biopsy (10 with NASH), and 27 patients had significant F≥2 fibrosis (25 with NASH). Bacteroides abundance was significantly increased in NASH and F≥2 patients, whereas Prevotella abundance was decreased. Ruminococcus abundance was significantly higher in F≥2 patients. By multivariate analysis, Bacteroides abundance was independently associated with NASH and Ruminococcus with F≥2 fibrosis. Stratification according to the abundance of these two bacteria generated three patient subgroups with increasing severity of NAFLD lesions. Based on imputed metagenomic profiles, Kyoto Encyclopedia of Genes and Genomes pathways significantly related to NASH and fibrosis F≥2 were mostly related to carbohydrate, lipid, and amino acid metabolism. Conclusion: NAFLD severity associates with gut dysbiosis and a shift in metabolic function of the gut microbiota. We identified Bacteroides as independently associated with NASH and Ruminococcus with significant fibrosis. Thus, gut microbiota analysis adds information to classical predictors of NAFLD severity and suggests novel metabolic targets for pre‐/probiotics therapies. (Hepatology 2016;63:764–775)

Increased rectal microbial richness is associated with the presence of colorectal adenomas in humans.

Differences in the composition of the gut microbial community have been associated with diseases such as obesity, Crohn’s disease, ulcerative colitis and colorectal cancer (CRC). We used 454 titanium pyrosequencing of the V1–V2 region of the 16S rRNA gene to characterize adherent bacterial communities in mucosal biopsy samples from 33 subjects with adenomas and 38 subjects without adenomas (controls). Biopsy samples from subjects with adenomas had greater numbers of bacteria from 87 taxa than controls; only 5 taxa were more abundant in control samples. The magnitude of the differences in the distal gut microbiota between patients with adenomas and controls was more pronounced than that of any other clinical parameters including obesity, diet or family history of CRC. This suggests that sequence analysis of the microbiota could be used to identify patients at risk for developing adenomas.

Differential Gene Expression between African American and European American Colorectal Cancer Patients

The incidence and mortality of colorectal cancer (CRC) is higher in African Americans (AAs) than other ethnic groups in the U. S., but reasons for the disparities are unknown. We performed gene expression profiling of sporadic CRCs from AAs vs. European Americans (EAs) to assess the contribution to CRC disparities. We evaluated the gene expression of 43 AA and 43 EA CRC tumors matched by stage and 40 matching normal colorectal tissues using the Agilent human whole genome 4x44K cDNA arrays. Gene and pathway analyses were performed using Significance Analysis of Microarrays (SAM), Ten-fold cross validation, and Ingenuity Pathway Analysis (IPA). SAM revealed that 95 genes were differentially expressed between AA and EA patients at a false discovery rate of ≤5%. Using IPA we determined that most prominent disease and pathway associations of differentially expressed genes were related to inflammation and immune response. Ten-fold cross validation demonstrated that following 10 genes can predict ethnicity with an accuracy of 94%: CRYBB2, PSPH, ADAL, VSIG10L, C17orf81, ANKRD36B, ZNF835, ARHGAP6, TRNT1 and WDR8. Expression of these 10 genes was validated by qRT-PCR in an independent test set of 28 patients (10 AA, 18 EA). Our results are the first to implicate differential gene expression in CRC racial disparities and indicate prominent difference in CRC inflammation between AA and EA patients. Differences in susceptibility to inflammation support the existence of distinct tumor microenvironments in these two patient populations.

Differences in microbial signatures between rectal mucosal biopsies and rectal swabs

There is growing evidence the microbiota of the large bowel may influence the risk of developing colorectal cancer as well as other diseases including type-1 diabetes, inflammatory bowel diseases and irritable bowel syndrome. Current sampling methods to obtain microbial specimens, such as feces and mucosal biopsies, are inconvenient and unappealing to patients. Obtaining samples through rectal swabs could prove to be a quicker and relatively easier method, but it is unclear if swabs are an adequate substitute. We compared bacterial diversity and composition from rectal swabs and rectal mucosal biopsies in order to examine the viability of rectal swabs as an alternative to biopsies. Paired rectal swabs and mucosal biopsy samples were collected in un-prepped participants (n = 11) and microbial diversity was characterized by Terminal Restriction Fragment Length polymorphism (T-RFLP) analysis and quantitative polymerase chain reaction (qPCR) of the 16S rRNA gene. Microbial community composition from swab samples was different from rectal mucosal biopsies (p = 0.001). Overall the bacterial diversity was higher in swab samples than in biopsies as assessed by diversity indexes such as: richness (p = 0.01), evenness (p = 0.06) and Shannon’s diversity (p = 0.04). Analysis of specific bacterial groups by qPCR showed higher copy number of Lactobacillus (p < 0.0001) and Eubacteria (p = 0.0003) in swab samples compared with biopsies. Our findings suggest that rectal swabs and rectal mucosal samples provide different views of the microbiota in the large intestine.

Somatic gene mutations in African Americans may predict worse outcomes in colorectal cancer

BACKGROUND AND OBJECTIVE African Americans have worse outcomes in colorectal cancer (CRC) than Caucasians. We sought to determine if KRAS, BRAF and PIK3CA mutations might contribute to the racial differences in CRC outcome. METHODS DNA was extracted from tissue microarrays made from CRC samples from 67 African Americans and 237 Caucasians. Mutations in KRAS, BRAF, and PIK3CA were evaluated by PCR sequencing. We also examined microsatellite instability (MSI) status. Associations of mutation status with tumor stage and grade were examined using a logistic regression model. Cox proportional hazards models were used to estimate the all-cause mortality associated with mutational status, race and other clinicopathologic features. RESULTS KRAS mutations were more common in African Americans than among Caucasians (37% vs 21%, p=0.01) and were associated with advanced stage (unadjusted odds ratio (OR)=3.31, 95% confidence interval (CI) 1.03-10.61) and grade (unadjusted OR=5.60, 95% CI 1.01-31.95) among African Americans. Presence of BRAF mutations was also positively associated with advanced tumor stage (adjusted OR=3.99, 95%CI 1.43-11.12) and grade (adjusted OR=3.93, 95%CI 1.05-14.69). PIK3CA mutations showed a trend toward an association with an increased risk of death compared to absence of those mutations (adjusted for age, sex and CRC site HR=1.89, 95% CI 0.98-3.65). Among African Americans, the association was more evident (adjusted for age, sex and CRC site HR=3.92, 95% CI 1.03-14.93) and remained significant after adjustment for MSI-H status and combined education-income level, with HR of 12.22 (95%CI 1.32-121.38). CONCLUSIONS Our results suggest that African Americans may have different frequencies of somatic genetic alterations that may partially explain the worse prognosis among African Americans with CRC compared to whites.

467 PI3K Mutation in African Americans May Predict Worse Outcomes in Colorectal Cancer

Purpose: African Americans have worse outcomes in colorectal cancer (CRC) than Caucasians. Mutations in Kras, Braf and PI3K have been identified in CRC that could impact prognosis, survival or response to treatment. There is a paucity of studies looking at whether genetic alterations contribute to worse prognosis in African Americans. We sought to determine if mutations in Kras, Braf and PI3K can explain the racial differences in CRC outcome. Methods: We obtained tissue samples from 237 Caucasians and 67 African Americans from a population based study of Cancer Care Outcomes Research and Surveillance Consortium (CanCORS). CanCORS include patients who were newly diagnosed with CRC. We extracted DNA from tissue microarrays and performed polymerase chain reactions using primers for Kras codons 12 and 13, Braf exon 11, and PI3K exons 9 and 20. Somatic mutations in these genes were identified by sequencing. Mutational status, race and other clinicopathologic features were analyzed for overall survival using Cox proportional hazards model. A proportional odds model was used to examine the association between stage and grade of CRC and mutation status. Results: Frequencies of Kras mutations were higher for African Americans than Caucasians (37% vs 21%, p=0.01). We found that African Americans had significantly increased odds ratios for worse stage (OR=3.3, 95%CI 1.0-11.0) and grade (OR=5.6, 95%CI 1.0-31.0) of CRC if Krasmutations were present as compared to those without Krasmutations. African Americans with a PI3K mutation had higher hazard ratios for mortality than African Americans without a PI3K mutation in both univariate (HR=3.9, 95%CI 1.0-14.9) and multivariate (HR=11.0, 95%CI 1.4-84.8) models. Further, African Americans with PI3K mutation showed a trend toward worse overall survival when compared to Caucasians with or without PI3K mutation or African Americans without PI3K mutations (p=0.09). When adjusted for smoking, patients with Braf mutations had elevated odds of having worse stage (OR=2.7, 95%CI 1.1-6.6) or grade (OR=5.2, 95%CI 1.8-15.0) of CRC than those without mutations. Conclusions: We found that African Americans with PI3K mutations had worse overall survival and increased hazard ratios than those without PI3Kmutations. These genetic alterations may contribute to the aggressive phenotype and may partially explain the worse prognosis observed in CRC in African Americans.

Mucosal Adherent Bacteria, Inflammation and Colorectal Adenomas

G A A b st ra ct s 3 minority groups, with pancreatic cancer nationally has remained unchanged since 2002. Among all 5261 visits of minority patients to our medical center over the past 8 years, 1.3% (66) were in the Emergency Department, 11.1% (585) were in the inpatient setting, and 1.6% (84) were for surgery. These differences are statistically significant for the 3 sites of care (p<0.0001 using chi-square analysis) when compared withWhite/Non-Hispanic patients (0.2%, 7.3%, and 2.6%, respectively). CONCLUSION: A large percentage of the patients seen at our medical center for pancreatic cancer belong to a minority group but the percentage of these patients is declining, in contrast with the unchanged incidence observed nationally. Minority patients have significantly greater visits to the Emergency Department and inpatient services but significantly fewer surgical visits when compared with White/Non-Hispanic patients. This may reflect advanced stage of disease in the minority population, requiring inpatient care but precluding surgery. The causes of these trends are likely multifactorial but in need of further study in order to understand and improve the management and outcomes of minority patients with pancreas cancer.