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.

Evidence for an hMSH3 defect in familial hamartomatous polyps.

BACKGROUND: 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. METHODS: Ten hamartomatous polyposis syndrome patients were genotyped for germline mutations. Epithelial and nonepithelial polyp DNA were assayed for microsatellite instability (MSI) and PTEN frameshift mutation. DNA MMR and PTEN protein expression were assessed in all polyps by immunohistochemistry. In addition, 99 MSI-high sporadic CRCs and 50 each of hMLH1(-/-) and hMSH3(-/-) cell clones were examined for PTEN frameshifts. RESULTS: Twenty-five (58%) of 43 hamartomatous polyposis syndrome polyps demonstrated dinucleotide or greater MSI in polyp epithelium, consistent with hMSH3 deficiency. MSI domains lost hMSH3 expression, and PTEN expression was lost in polyps from germline PTEN patients; sporadic hamartomatous polyps did not show any of these findings. PTEN analysis revealed wild type exon 7 and 8 sequences suggestive of nonexistent or rare events for PTEN frameshifts; however, MSI-high sporadic CRC showed 11 (11%) of 99 frameshifts within PTEN, with 4 tumors having complete loss of PTEN expression. Subcloning hMLH1(-/-) and hMSH3(-/-) cells revealed somatic PTEN frameshifts in 4% and 12% of clones, respectively. CONCLUSIONS: Nondysplastic epithelium from hamartomatous polyposis syndrome polyps harbors hMSH3 defects, which may prime neoplastic transformation. Polyps from PTEN(+/-) patients lose PTEN expression, but loss is not a universal early feature of all hamartomatous polyposis syndrome. However, PTEN frameshifts can occur in hMSH3-deficient cells, suggesting that hMSH3 deficiency could drive hamartomatous polyposis syndrome tumorigenesis.

Activin type 2 receptor restoration in MSI-H colon cancer suppresses growth and enhances migration with activin.

BACKGROUND & AIMS: Colon cancers with high-frequency microsatellite instability (MSI-H) develop frameshift mutations in tumor suppressors as part of their pathogenesis. ACVR2 is mutated at its exon 10 polyadenine tract in >80% of MSI-H colon cancers, coinciding with loss of protein. ACVR2 transmits the growth effects of activin via phosphorylation of SMAD proteins to affect gene transcription. The functional effect of activin in colon cancers has not been studied. We developed and characterized a cell model in which we studied how activin signaling affects growth. METHODS: hMLH1 and ACVR2 mutant HCT116 cells were previously stably transferred with chromosome 2 (HCT116+chr2), restoring a single regulated copy of wild-type ACVR2 but not hMLH1. Both HCT116+chr2 and parental HCT116 cells (as well as HEC59 and ACVR2 and hMSH2 complemented HEC59+chr2 cells) were assessed for genetic complementation and biologic function. RESULTS: HCT116+chr2 cells and HEC59+chr2 cells, but not ACVR2-mutant HCT116 or HEC59 cells, acquired wild-type ACVR2 as well as expression of ACVR2 wild-type messenger RNA. Complemented ACVR2 protein complexed with ACVR1 with activin treatment, generating nuclear phosphoSMAD2 and activin-specific gene transcription. ACVR2-restored cells showed decreased growth and reduced S phase but increased cellular migration following activin treatment. ACVR2 small interfering RNA reversed these effects in complemented cells. CONCLUSIONS: ACVR2-complemented MSI-H colon cancers restore activin-SMAD signaling, decrease growth, and slow their cell cycle following ligand stimulation but show increased cellular migration. Activin is growth suppressive and enhances migration similar to transforming growth factor beta in colon cancer, indicating that abrogation of the effects of activin contribute to the pathogenesis of MSI-H colon cancers.

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 TGFbeta 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.

Use of 5-fluorouracil and survival in patients with microsatellite-unstable colorectal cancer.

BACKGROUND & AIMS: 5-Fluorouracil improves mortality in stage III colorectal cancer patients. In vitro studies suggest that microsatellite instability influences cell survival after 5-fluorouracil treatment. We investigated the survival influence of 5-fluorouracil in patients with microsatellite instability-high tumors. METHODS: We collected data and tumors on 204 consecutive stage II and III colorectal cancer patients from registries at the University of California and Veterans Administration hospitals in San Diego, California, from 1982 to 1999. Archival DNA was extracted, and microsatellite instability was assessed by National Cancer Institute-recommended markers. Cox proportional hazard modeling was used to determine survival associations for microsatellite instability and 5-fluorouracil treatment status. RESULTS: We identified 36 microsatellite instability-high (17.6%) and 168 non-microsatellite instability-high tumors (82.4%). Microsatellite instability-high tumors were significantly associated with proximal colon location, presence of mucin, and surrounding lymphoid reaction. Univariate and multivariate analyses showed no survival difference between microsatellite instability-high and non-microsatellite instability-high groups (hazard ratio, 1.04; P = 0.88). Dichotomized by use of 5-fluorouracil, there was increased risk of death in patients who received no adjuvant chemotherapy (hazard ratio, 2.02; P = 0.02). However, the benefit of 5-fluorouracil was different between microsatellite instability-high and non-microsatellite instability-high groups. Patients with non-microsatellite instability-high tumors who received 5-fluorouracil had better survival compared with patients who were not treated (P < 0.05). Conversely, patients with microsatellite instability-high tumors who were treated with 5-fluorouracil had no survival difference compared with patients without treatment (P = 0.52). CONCLUSIONS: There is improved survival in patients with non-microsatellite instability-high tumors after 5-fluorouracil-based chemotherapy that does not extend to patients with microsatellite instability-high tumors. The microsatellite instability status of a patient's colorectal cancer may indicate differences in 5-fluorouracil-based chemosensitivity; this is consistent with in vitro studies.