Burgart, Lawrence J.

Abstract Purpose: Colon tumors with defective DNA mismatch repair (dMMR) have a well-characterized phenotype and accounts for ∼15% to 20% of sporadic colon cancer as well as those colon cancer patients with Lynch syndrome. Although the presence of dMMR seems to be a favorable prognostic marker, data suggest that these patients do not respond as well to adjuvant chemotherapy. Experimental Design: In this study, we examined the prognostic significance of tumor MMR deficiency and the presence of a specific mutation in BRAF (V600E) in a group of patients (n = 533) who participated in a randomized prospective clinical trial through the North Central Cancer Treatment Group. Results: Tumors with dMMR were found to be associated with higher tumor grade (P = 0.001), proximal location (P < 0.0001), and improved overall and disease-free survival (P = 0.05 and 0.04, respectively). Among all cases examined, evaluation of the BRAF V600E mutation status revealed no statistically significant differences in either disease-free or overall survival. Patients were then grouped into four categories for further analysis: dMMR/BRAF(−), dMMR/BRAF(+), pMMR/BRAF(−), and pMMR/BRAF(+). The dMMR/BRAF(−) group had a significantly improved overall survival (5-year overall survival of 100% versus 73%, P = 0.002) compared with all others. The remaining three groups had very similar survival outcomes. An additional cohort of tumors previously classified as having dMMR were also tested for the BRAF V600E alteration. Results remained significant (P = 0.006) when the two groups were combined for analysis. Conclusions: Overall, these data suggest that the underlying molecular etiology of those tumors having dMMR may influence the disease outcome in these patients.

Abstract The frequency of colorectal cancer (CRC) among the Alaska Native people is the highest of any ethnic group in the United States. In this study, CRC from 329 Alaska Native people (165 Eskimo, 111 Indians, and 53 Aleut) were evaluated for evidence of defective DNA mismatch repair (MMR) by testing tumors for altered protein expression (hMLH1, hMSH2, and hMSH6) and for the presence of microsatellite instability. Of the 329 samples tested, 46 (14%) showed both microsatellite instability and altered protein expression; 42 (91%) with a loss of hMLH1, 3 (7%) hMSH2, and 1 (2%) hMLH1/hMSH6. Tumors with loss of hMLH1 were further evaluated for hMLH1 promoter hypermethylation and for the presence of the BRAF-V600E mutation. Among cases tested, all 19 (100%) tumors among the Eskimo patients showed hMLH1 promoter hypermethylation, whereas this was the case for only 3 of the 7 (42%) tumors among the Aleut (P = 0.002) and 5 of the 10 (50%) tumors from the Indians (P = 0.002). The majority of hypermethylated cases (23 of 27) tested positive for the V600E alteration. Of the nine tumors from the Aleut and Indian patients that did not exhibit hMLH1 hypermethylation, eight tested negative for V600E. Overall, the frequency of defective MMR among the three groups was not statistically different (P = 0.75). However, the data suggest that the pathogenesis of CRC may differ between the three groups. The CRC with defective MMR among the Eskimo sample fit the typical profile for hMLH1-related cancer associated with sporadic CRC, whereas the pattern in the Aleut and Indian suggests the possibility that germ line hMLH1 mutations may be present. (Cancer Epidemiol Biomarkers Prev 2007;16(11):2344–50)

Abstract Purpose: Most colorectal cancers that have high levels of microsatellite instability (MSI-H) show loss of immunohistochemical expression of proteins that participate in the DNA mismatch repair process, most often involving MLH1 and MSH2. Less commonly, a third DNA mismatch repair protein, MSH6, may also be lost as the primary event. Rarely, tumors with MSI-H show normal expression of these three proteins. The genetic deficiency leading to the MSI-H phenotype in such cases is unknown. PMS2 is another member of the DNA mismatch repair complex. Its expression is generally lost in tumors with MLH1 loss of expression. Rarely, there is selective loss of PMS2 expression. We sought to describe the frequency and clinical correlates of selective loss of expression of PMS2 with the MSI-H tumor phenotype. Experimental Design: Two thousand seven hundred nineteen colorectal cancers from both clinic- and research-based ascertainment were studied. Tumor MSI testing and immunohistochemistry for MLH1, MSH2, MSH6, and PMS2 were conducted. Medical records were abstracted for age at diagnosis, gender, colorectal cancer site, and family history. Results: Five hundred thirty-five of the 2,719 tumors were MSI-H. Of these, 93% showed loss of expression of MLH1, MSH2, and/or MSH6. Thirty-eight showed normal expression for these proteins. PMS2 immunohistochemical staining was successful in 32 of 38 of these tumors. Of the 32, 23 showed selective loss of expression of PMS2. This was associated with young age of diagnosis and right-sided location but not with a striking family history of cancer. Conclusions: Overall, 97% of the MSI-H tumors showed loss of expression for one or more of these four mismatch repair proteins. Selective loss of expression of PMS2 was present in 72% of cases in which colorectal cancers had an MSI-H phenotype but no alteration of expression of MLH1, MSH2, and MSH6. The underlying mechanism involved cannot be determined from this study but could involve point mutations in other DNA mismatch repair genes with retention of immunohistochemical expression, somatic inactivation of PMS2, or germ line mutation of PMS2.

Abstract Celiac disease is associated with an increased risk of small bowel adenocarcinoma. The aims of this study were to investigate the molecular basis, assess outcomes, and identify clinicopathologic characteristics of small bowel adenocarcinoma in celiac disease. Retrospective case control cohort study of all celiac disease patients treated at our institution for small bowel adenocarcinoma and matched control patients with sporadic small bowel adenocarcinoma from July 1960 to November 2002. Mismatch repair (MMR) status was accessed by testing tissue for microsatellite instability (MSI) and for hMLH1 and hMSH2 protein expression. Over a 40-year time period, 18 patients with small bowel adenocarcinoma and celiac disease were treated at the Mayo Clinic. One celiac disease patient was excluded. High-frequency MSI (MSI-H) was identified in 8 of 11 (73%) and 2 of 22 (9%) available small bowel adenocarcinoma specimens in the celiac disease and control groups, respectively. In the celiac disease group, MSI-H was associated with loss of hMLH1 and hMSH2 in 6 and 1 specimens, respectively. Loss of hMLH1 occurred in both control tumors. Stage was associated with celiac disease status (P = 0.018), and 78% of controls were stage III or IV compared with 47% of celiac disease patients. Overall, survival was better (P = 0.025) in the celiac disease group compared with stage-matched controls. Celiac disease patients with small bowel adenocarcinoma had a high incidence defective MMR (73%) compared with controls and had better survival compared with stage-matched controls. In addition, celiac disease patients presented more frequently with early-stage small bowel adenocarcinoma. The better survival and earlier presentation of small bowel adenocarcinoma in celiac disease appears to be biologically associated with defective MMR.

Abstract The MutLα heterodimer formed by mismatch repair (MMR) proteins MLH1 and PMS2 is a major component of the MMR complex, yet mutations in the PMS2 gene are rare in the etiology of hereditary nonpolyposis colorectal cancer. Evidence from five published cases suggested that contrary to the Knudson principle, PMS2 mutations cause hereditary nonpolyposis colorectal cancer or Turcot syndrome only when they are biallelic in the germline or abnormally expressed. As candidates for PMS2 mutations, we selected seven patients whose colon tumors stained negative for PMS2 and positive for MLH1 by immunohistochemistry. After conversion to haploidy, truncating germline mutations of PMS2 were found in two patients (2192delTAACT and deletion of exon 8). These mutations abrogated PMS2 protein in germline cells by Western analysis. In two additional patients, PMS2 protein from one allele also was abrogated. Novel or previously described missense variants of PMS2 were detected, but their pathogenicity is undetermined. We detected and characterized a new transcript, PMS2CL, showing 98% sequence identity with exons 9 and 11–15 of PMS2 and emanating from a locus close to PMS2 in chromosome 7p. Its predicted protein product was not detected. Thus, in addition to several previously described PMS2-related genes resembling the 5′ end of PMS2, at least one related gene resembles the 3′ end of PMS2. In conclusion, both detectable and presently undefined germline mutations are deleterious and produce susceptibility to cancer by the two-hit mechanism. Paralogous genes interfere with mutation detection, resulting in underdiagnosis of PMS2 mutations. Mutation detection in PMS2 requires haploid DNA.

AbstractMost esophageal adenocarcinomas arise within Barrett's esophagus but the cause of this increasingly prevalent condition remains unknown. Early detection improves survival and discriminant screening markers for Barrett's esophagus and cancer are needed. This study was designed to explore the natural history of eyes absent 4 (EYA4) gene methylation in the neoplastic progression of Barrett's esophagus and to evaluate methylated EYA4 as a candidate marker. Aberrant promoter methylation of EYA4 was studied by methylation-specific PCR using bisulfite-treated DNA from esophageal adenocarcinomas, Barrett's esophagus, and normal epithelia, and then confirmed by sequencing. Eight cancer cell lines were treated with the demethylation agent 5-aza-2′-deoxycytidine, and EYA4 mRNA expression with and without treatment was quantified by real-time reverse-transcription PCR. EYA4 hypermethylation was detected in 83% (33 of 40) of esophageal adenocarcinomas and 77% (27 of 35) of Barrett's tissues, but only in 3% (2 of 58) of normal esophageal and gastric mucosa samples (P < 0.001). The unmethylated cancer cell lines had much higher EYA4 mRNA expression than the methylated cancer cell lines. Demethylation caused by 5-aza-2′-deoxycytidine increased the mRNA expression level by a median of 3.2-fold in methylated cells, but its effect on unmethylated cells was negligible. Results indicate that aberrant promoter methylation of EYA4 is very common during tumorigenesis in Barrett's esophagus, occurs in early metaplasia, seems to be an important mechanism of down-regulating EYA4 expression, and represents an intriguing candidate marker for Barrett's metaplasia and esophageal cancer.

Purpose Molecular studies of colon cancer have provided insights into pathogenesis, yet it is unclear how important these markers are in predicting prognosis. This study investigated the prognostic significance of TUNEL, bcl-2, p53, proliferation marker Ki-67 and DNA mismatch repair (MMR) status in patients with Dukes' stage B2 and C colorectal adenocarcinomas. Patients and Methods Tumor tissue from 366 patients (75% Dukes' C, 25% Dukes' B2) from four randomized North Central Cancer Treatment Group phase III surgical adjuvant trials were used. Eighty-one percent of patients received adjuvant treatment, which was primarily fluorouracil (FU) based (90%). Tumor location was predominantly (87%) the colon. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), Ki-67, p53, bcl-2, and MMR were assayed using immunohistochemistry. Stage, grade, MMR, Ki-67, and previously determined flow cytometry markers (ploidy and S phase) were explored for associations with each other and with overall survival (OS) and disease-free survival (DFS). Results Univariately, stage B2, low grade, diploid, Ki-67 more than 27%, normal p53, and FU-based adjuvant treatment were significantly associated with improved OS and DFS (P < .05). After adjusting for stage, grade, and ploidy in multivariate analysis, Ki-67 remained significantly related to both OS and DFS (P < .01). Active FU-based adjuvant treatment was significant only for OS in this multivariate model. Neither bcl-2 nor TUNEL were significant. Conclusion This retrospective study indicates that Ki-67 and ploidy may have stronger prognostic impact on OS and DFS than other parameters investigated after adjusting for stage and tumor grade. Prospective studies to elucidate the mechanism and prognostic significance of these findings are necessary.