Ryan T. Doctolero

Diet, Lifestyle, and Genomic Instability in the North Carolina Colon Cancer Study

Objective: Microsatellite instability (MSI) is one form of genomic instability that occurs in 10% to 20% of sporadic colon tumors and almost all hereditary nonpolyposis colon cancers. However, little is known about how environmental factors (e.g., diet) may influence MSI in sporadic colon cancer. Methods: We used data from a population-based case-control study in North Carolina (486 colon cancer cases and 1,048 controls) to examine associations of diet (total energy, macronutrients, micronutrients, and food groups) with MSI. In-person interviews elicited information on potential colon cancer risk factors, and a previously validated food frequency questionnaire adapted to include regional foods was used to assess diet over the year before diagnosis or interview date. MSI was classified as MSI-high (MSI-H) and MSI-low or microsatellite stable (MSI-L/MSS). Multivariate logistic regression models estimated energy-adjusted and non-energy-adjusted odds ratios (OR). Results: Ten percent of the cases (n = 49) had MSI-H tumors (29% African American). The strongest associations between diet and MSI were observed in case-control comparisons: there was a robust inverse association between MSI-H status and β-carotene [OR, 0.4; 95% confidence interval (95% CI), 0.2-0.9] and positive associations with energy-adjusted refined carbohydrates (OR, 2.2; 95% CI, 0.9-5.4) and non-energy-adjusted read meat intake (OR, 2.0; 95% CI, 0.9-4.2). Compared with controls, MSI-L/MSS tumors were statistically significantly associated with energy-adjusted vitamin C, vitamin E, calcium, dietary fiber, and dark green vegetables and positively associated with total energy intake (all Ps for trend < 0.05). In case-case comparisons, no dietary factors were significantly differently related to MSI-H compared with MSI-L/MSS tumors. Conclusion: Refined carbohydrate and red meat consumption may promote development of MSI-H tumors, whereas β-carotene may be associated with lower risk.

Influence of race on microsatellite instability and CD8+ T cell infiltration in colon cancer

African American patients with colorectal cancer show higher mortality than their Caucasian counterparts. Biology might play a partial role, and prior studies suggest a higher prevalence for microsatellite instability (MSI) among cancers from African Americans, albeit patients with MSI cancers have improved survival over patients with non-MSI cancers, counter to the outcome observed for African American patients. CD8+ T cell infiltration of colon cancer is postively correlated with MSI tumors, and is also related to improved outcome. Here, we utilized a 503-person, population-based colon cancer cohort comprising 45% African Americans to determine, under blinded conditions from all epidemiological data, the prevalence of MSI and associated CD8+ T cell infiltration within the cancers. Among Caucasian cancers, 14% were MSI, whereas African American cancers demonstrated 7% MSI (P = 0.009). Clinically, MSI cancers between races were similar; among microsatellite stable cancers, African American patients were younger, female, and with proximal cancers. CD8+ T cells were higher in MSI cancers (88.0 vs 30.4/hpf, P<0.0001), but was not different between races. Utilizing this population-based cohort, African American cancers show half the MSI prevalence of Caucasians without change in CD8+ T cell infiltration which may contribute towards their higher mortality from colon cancer.

Influence of target gene mutations on survival, stage and histology in sporadic microsatellite unstable colon cancers

High‐frequency microsatellite unstable (MSI‐H) colon tumors develop as a consequence of mutations at repetitive sequences in target genes. TGFBR2 and ACVR2, encoding TGFβ superfamily receptors, and the proapoptotic gene BAX are frequent targets for frameshift mutation. We analyzed the effect of these mutations on survival and histology in 2 separate cohorts. Forty‐eight MSI‐H Dukes B2 colon tumors from a cohort of 172 patients had mutations in TGFBR2, BAX and ACVR2 correlated with patient survival. Further, 54 population‐based MSI‐H colon cancers of all stages from a cohort of 503 patients had mutations correlated with tumor stage, grade and size. Of 44 amplifiable MSI‐H Dukes B2 tumors, 70% harbored TGFBR2, 63% BAX and only 4.5% ACVR2 mutations. While mutation alone did not influence survival, concomitant mutation of TGFBR2 and BAX was associated with an improved prognosis in Dukes B2 patients (p = 0.05). ACVR2 mutations were more frequent in the second, population‐based cohort (stage II: 32.5%, p < 0.05). While no target gene mutation correlated with stage in this cohort, poor histological grade and large tumor volume were associated with mutant ACVR2, but not TGFBR2 or BAX mutations, and likely accounts for the lower prevalence of ACVR2 mutations in the first, well‐differentiated Dukes B2 cohort. Because target gene mutations did not correlate with stage, they likely occur early in the pathogenesis of MSI‐H cancers. Mutations in TGFBR2 and BAX may improve survival in MSI‐H Dukes B2 patients, and mutations of ACVR2 may augment histological changes consistent with poor tumor grade that is characteristic of MSI‐H colon cancers, and increase tumor size.

Evidence for an hMSH3 defect in familial hamartomatous polyps

Patients with hamartomatous polyposis syndromes have increased risk for colorectal cancer (CRC). Although progression of polyps to carcinoma is observed, pathogenic mechanisms remain unknown. The authors examined whether familial hamartomatous polyps harbor defects in DNA mismatch repair (MMR), and assayed for somatic mutation of PTEN, a gene inactivated in the germline of some hamartomatous polyposis syndrome patients.

Nuclear accumulation of β-catenin occurs commonly in the epithelial cells of juvenile polyps

In the two conditions juvenile polyps (JPs) and juvenile polyposis coli (JPC), colonic polyps may have overlapping histologic and phenotypic appearance, but JPC confers a significant risk for colon adenocarcinoma. Although not thought to contain adenomatous polyposis coli (APC) mutations, the status of β-catenin and full-length APC protein expression in JPs is not known. We evaluated β-catenin and full-length APC protein expression in JPs from children with JPs and JPC. Cases were identified through endoscopic procedure records. Immunohistochemistry was performed for β-catenin and full-length APC protein. Loss of heterozygosity at the APC gene locus on chromosome 5 was assessed using two APC-linked microsatellite markers. Polyp and normal colonic tissue were analyzed from 36 children with JPs and 9 with JPC. Both APC and β-catenin immunoreactivity were present in epithelial cells from all samples but in different patterns. In all normal colon and polyp samples, APC expression was cytoplasmic with maximal immunoreactivity in the goblet cells. In contrast, β-catenin immunoreactivity in epithelial cells was limited to the plasma membrane in normal colon but was both cytoplasmic and nuclear in all 45 JPs. No evidence of APC gene loss of heterozygosity was found. In polyps from children with JPs and JPC, nuclear β-catenin accumulation is a consistent feature, and it is not due to APC gene mutation or loss of full-length APC protein expression. Thus, β-catenin accumulation may be intrinsic to the formation of juvenile-type polyps through an as-yet-undefined mechanism.

Erratum: Influence of race on microsatellite instability and CD8+ T cell infiltration in colon cancer (PLoS ONE (2014) 9, 6 (e100461) DOI: 10.1371/journal.pone.0100461)

1. Carethers JM, Murali B, Yang B, Doctolero RT, Tajima A, et al. (2014) Influence of Race on Microsatellite Instability and CD8 T Cell Infiltration in Colon Cancer. PLoS ONE 9(6): e100461. doi:10.1371/journal.pone.0100461 Figure 1. CD8 T cell infiltration in colon cancers. (A) Immunohistochemistry of CD8 T cells within malignant epitheilial glands of a colon cancer. Note the presence of intraepithelial CD8 T cells (inset). (B) CD8 T cell counts between MSI and MSS cancers