hMLH1 Methylation Research Articles

Expression of the hMLH1 gene is a possible predictor for the clinical response to 5-fluorouracil after a surgical resection in colorectal cancer

The loss of a DNA mismatch repair occurs in approximately 15% of sporadic colorectal cancer (CRC) and is usually caused by the lack of expression of the hMLH1 gene due to promoter methylation. Despite undergoing adjuvant 5-fluorouracil (5-FU) therapy after a curative surgical resection, some patients with advanced-stage CRC develop recurrence. In the present study, we investigated whether the hMLH1 mRNA expression or promoter methylation is a prognostic factor in CRC patients treated with adjuvant 5-FU. The hMLH1 mRNA expression levels were measured by quantitative reverse transcription PCR in cancer and normal epithelial cells that were obtained from 94 CRC patients using a laser capture microdissection. Then, the methylation status of the hMLH1 promoter in the CRC tissues was examined by methylation-specific PCR. The hMLH1 mRNA expression levels were significantly lower in the cancer cells than in the normal mucosa (p<0.01) and the hMLH1 mRNA expression levels in the cancer cells were significantly lower in the CRC tissues with methylated versus unmethylated hMLH1 (p<0.01) in the 94 patients. Among the 35 patients receiving adjuvant 5-FU, the disease-free survival rate was significantly better in the patients demonstrating a low hMLH1 mRNA expression in the cancer cells in comparison to that of the patients with a high hMLH1 mRNA expression (p<0.01). Moreover, a multivariate analysis revealed that hMLH1 mRNA expression was a significant independent prognostic factor for tumor recurrence in CRC patients treated with adjuvant 5-FU. However, hMLH1 methylation was not correlated with the survival in these 35 patients. These data suggest that the hMLH1 mRNA quantitation in colorectal cancer cells may be helpful for evaluating the prognosis of CRC patients receiving 5-FU-based adjuvant chemotherapy after a surgical resection.

Clinicopathologic and molecular features of sporadic microsatellite- and chromosomal-stable colorectal cancers

Chromosomal instability (CIN) and microsatellite instability (MSI) are two major causes of colorectal cancers. Recently, a percentage of colorectal cancers were found to be neither CIN nor MSI. This study was performed to explore whether microsatellite- and chromosomal-stable (MACS) colorectal cancers comprise a substantially distinct subtype. Sixty-nine sporadic colorectal cancers were classified into three subsets according to ploidy and microsatellite instability status: CIN+, MSI+, and MACS. Clinicopathologic, genetic, and epigenetic differences among these three groups were investigated by immunohistochemical analysis of p53, APC, hMLH1, and BAX and methylation study of pl4ARF, hMLH1, p161NK4a MGMT, and MINT1 with methylation-specific polymerase chain reaction. The 69 cases included 49 CIN+, 7 MSI+, and 13 MACS. MACS were found to differ from CIN+ and MSI+ in three aspects. The clinicopathologic features of MACS were similar to MSI+ but distinguished from CIN+. Comparatively, MACS preferred proximal location and poor differentiation (p < 0.05). An immunohistochemical study demonstrated that MACS had a lower rate of loss of hMLH1 or BAX protein than MSI+ and less loss of APC protein than CIN+. In an epigenetic aspect, both MACS and MSI+ had a high rate of CpG island methylator phenotype (46.2 and 42.9%). However, they differed in the presence of hMLH1 methylation (7.7 vs 57.1%, p < 0.05). Otherwise, compared with CIN+, MACS had a more frequent CpG island methylator phenotype and MINT1 methylation (p < 0.05) and relatively more common p161IK4a methylation with marginal significance (p= 0 .056). MACS sporadic colorectal cancers may compose a unique phenotype with distinct clinicopathologic and molecular characteristics.

Methylenetetrahydrofolate reductase C677T genotype affects promoter methylation of tumor-specific genes in sporadic colorectal cancer through an interaction with folate/vitamin B12 status

To evaluate joint effects of Methylentetra-hydrofolate reductase (MTHFR) C677T genotypes, and serum folate/vitamin B(12) concentrations on promoter methylation of tumor-associated genes among Iranian colorectal cancer patients. We examined the associations between MTHFR C677T genotype, and promoter methylation of P16, hMLH1, and hMSH2 tumor-related genes among 151 sporadic colorectal cancer patients. The promoter methylation of tumor-related genes was determined by methylation-specific PCR. Eighty six patients from whom fresh tumor samples were obtained and 81 controls were also examined for serum folate and vitamin B(12) concentrations by a commercial radioimmunoassay kit. We found 29.1% of cases had tumors with at least one methylated gene promoter. In case-case comparison, we did not find a significant association between methylation in tumors and any single genotype. However, in comparison to controls with the CC genotype, an increased risk of tumor methylation was associated with the CT genotype (OR = 2.5; 95% CI, 1.1-5.6). In case-case comparisons, folate/vitamin B(12) levels were positively associated with tumor methylation. Adjusted odds ratios for tumor methylation in cases with high (above median) versus low (below median) serum folate/vitamin B(12) levels were 4.9 (95% CI, 1.4-17.7), and 3.9 (95% CI, 1.1-13.9), respectively. The frequency of methylated tumors was significantly higher in high methyl donor than low methyl donor group, especially in those with MTHFR CT (P = 0.01), and CT/TT (P = 0.002) genotypes, but not in those with the CC genotype (P = 1.0). We conclude that high concentrations of serum folate/vitamin B(12) levels are associated with the risk of promoter methylation in tumor-specific genes, and this relationship is modified by MTHFR C677T genotypes.

A Prospective, Multicenter, Population-Based Study of BRAF Mutational Analysis for Lynch Syndrome Screening

Mismatch repair (MMR) deficiencies are the hallmark of tumors arising in Lynch syndrome, however, in approximately 15% of sporadic colorectal cancers (CRC) these deficiencies most often are associated with somatic methylation of the MMR gene MLH1. Recently, an oncogenic mutation in the BRAF gene has been involved in sporadic CRC showing MMR deficiencies as a result of MLH1 promoter methylation. The aim of this study was to evaluate the contribution of BRAF V600E mutation analysis in the identification of MSH2/MLH1 gene mutation carriers in newly diagnosed CRC patients. BRAF V600E mutation was analyzed in CRC patients with MMR deficiencies (microsatellite instability and/or lack of MLH1/MSH2 protein expression) in the EPICOLON population-based study. The effectiveness and efficiency of different strategies were evaluated with respect to the presence of MSH2/MLH1 germline mutations. MMR deficiencies were detected in 119 of the 1222 CRC patients with tumors showing either microsatellite instability (n = 111) or loss of protein expression (n = 81). BRAF mutation was detected in 22 (18.5%) of the patients. None of the patients with unambiguous germline mutation had BRAF mutation. Regardless of the strategy used to identify MSH2/MLH1 gene carriers, the introduction of BRAF analysis in these patients slightly improves their effectiveness. The introduction of BRAF mutation analysis as a step before germline genetic testing in patients with MMR deficiencies achieved a significant reduction in costs per mutation detected. Detection of BRAF V600E mutation could simplify and improve the cost effectiveness of genetic testing for hereditary nonpolyposis colorectal cancer, especially in patients whose family history is incomplete or unknown.

Integrated analysis of chromosomal, microsatellite and epigenetic instability in colorectal cancer identifies specific associations between promoter methylation of pivotal tumour suppressor and DNA repair genes and specific chromosomal alterations

Colorectal cancer (CRC) is a complex and heterogeneous disease in which genomic instability and DNA promoter methylation play important roles. The aim of this study was to investigate the relationship between chromosomal instability (CIN), microsatellite instability (MSI) and promoter methylation of CRC-associated genes. Therefore, 71 CRCs were analysed for CIN and MSI by comparative genomic hybridization and the mononucleotide marker BAT-26, respectively. Promoter methylation of the tumour suppressor and DNA repair genes hMLH1, O(6)-MGMT, APC, p14(ARF), p16(INK4A), RASSF1A, GATA-4, GATA-5 and CHFR was analysed using methylation-specific polymerase chain reaction. These integrative analyses showed that in CIN+ CRCs, promoter methylation of GATA-4 and p16(INK4A) was inversely related to chromosomal loss at 15q11-q21 and gain at 20q13, respectively (P values: 3.8 x 10(-2) and 4.5 x 10(-2), respectively). Interestingly, promoter methylation of RASSF1A, GATA-4, GATA-5 and CHFR, as well as a high methylation index (MI), was positively related to chromosomal gain at 8q23-qter (P values: 1.5 x 10(-2), 3.8 x 10(-2), 3.9 x 10(-2), 4.9 x 10(-2) and 8.2 x 10(-3), respectively). MSI was associated with BRAF mutation, promoter methylation of hMLH1, APC and p16(INK4A) and a high MI (total number of methylated genes) (P values: 2.4 x 10(-2), 2.5 x 10(-3), 1.8 x 10(-2), 4.6 x 10(-2) and 1.0 x 10(-2), respectively). Therefore, we conclude that promoter methylation of pivotal tumour suppressor and DNA repair genes is associated with specific patterns of chromosomal changes in CRC, which are different from methylation patterns in MSI tumours.

Methylation-induced silencing of ASC/TMS1, a pro-apoptotic gene, is a late-stage event in colorectal cancer

The hypermethylation of tumor-suppressor gene promoter regions has been shown to result in the epigenetic inactivation of many genes. ASC/TMS1 is a pro-apoptotic gene that has been shown to be methylated in many different human neoplasms. The methylation status of ASC/TMS1 was analyzed in a series of colorectal cancer (CRC) cell lines, primary colorectal adenomas and cancers, and normal colorectal tissue samples using methylation-specific PCR (MSP). The gene expression of ASC/TMS1 in the CRC cell lines was analyzed using reverse-transcriptase PCR (RT-PCR). Methylation analysis showed complete methylation of ASC/TMS1 in 5 of 7 (71%) CRC cell lines. RT-PCR showed absence of mRNA expression in these same cell lines, and expression was restored after treatment with the demethylating drug 5-aza-2’-deoxyazacytidine. The two unmethylated cell lines showed ASC/TMS1 mRNA expression both before and after treatment with 5-aza-2’-deoxyazacytidine. Methylation was seen in 20 of 115 (17%) of primary colorectal cancer specimens, but no methylation was seen in 30 colorectal adenomas and 11 normal colorectal tissue samples. Methylation status of ASC/TMS1 was correlated with a series of clinicopathological variables using multivariate analysis. Methylation of ASC/TMS1 was more common in right-sided tumors (p = 0.02), concordant with hMLH1 methylation (p=0.03) and is a late stage event, occurring in 0 of 18 tubular adenomas, 0 of 12 villous adenomas, 2 of 44 (5%) Stage 1 cancers, 8 of 31 (26%) Stage 2 cancers, 8 of 21 (38%) Stage 3 cancers, and 2 of 19 (11%) Stage 4 cancers. The ASC/TMS1 gene is frequently silenced in CRC due to promoter hypermethylation. Methylation of ASC/TMS1 appears to be a late-stage event in colorectal carcinogenesis associated with invasive carcinomas but not with normal colorectal tissue or colorectal adenomas. Methylation of ASC/TMS1 may have implications for cancer prognosis.

CpG island methylation is frequently present in tubulovillous and villous adenomas and correlates with size, site, and villous component

CpG island methylator phenotype (CIMP) pathway in colorectal cancer is characterized by methylation of promoter regions of multiple putative tumor suppressor genes. Aberrant methylation also occurs in serrated and adenomatous polyps. We examined 32 tubulovillous/villous adenomas and 30 tubular adenomas for BRAF/KRAS mutations and methylation at hMLH1, p16, HIC1, RASSF2, MGMT, MINT1, and MINT31. CIMP-positive status (methylation at 3 or more loci) was observed in 44% tubulovillous/villous adenomas compared with 8 (27%) of 30 tubular adenomas (P = .08). Tubulovillous/villous adenomas showed significantly higher methylation than tubular adenomas at MGMT (87% vs 37%, P < .01) and RASSF2 (94% vs 70%, P = .02). There was no significant difference in methylation of HIC1, MINT1, MINT31, and p16. hMLH1 methylation was absent in all tubulovillous/villous adenomas and seen in only 2 (7%) tubular adenomas. CIMP-positive status correlated with large size, right-sided location, and amount of villous component in tubulovillous/villous adenomas. BRAF V600E mutation was not observed in any tubular adenoma or tubulovillous/villous adenoma. KRAS mutations were seen in 9% of tubulovillous/villous adenomas and 10% of tubular adenomas. In conclusion, CIMP-positive phenotype is common in tubulovillous/villous adenomas and increases with large size, right-sided location, and amount of villous component. Methylation of MGMT and RASSF2 increases during the progression from tubular adenoma to tubulovillous/villous adenoma. BRAF mutations are absent in tubulovillous/villous adenomas.

Carcinogenesis and microsatellite instability: the interrelationship between genetics and epigenetics

DNA mismatch repair (MMR) deficiency results in a strong mutator phenotype and high-frequency microsatellite instability (MSI-H), which are the hallmarks of tumors arising within Lynch syndrome. MSI-H is characterized by length alterations within simple repeated sequences, microsatellites. Lynch syndrome is primarily due to germline mutations in one of the DNA MMR genes; mainly hMLH1 or hMSH2 and less frequently hMSH6 and rarely hPMS2. Germline hemiallelic methylation of MLH1, termed epimutation, has been reported to be a new cause of Lynch syndrome. MSI-H is also observed in approximately 15% of colorectal, gastric and endometrial cancers and in lower frequencies in a minority of other tumors, where it is associated with the hypermethylation of the promoter region of hMLH1. MSI-H underlies a distinctive tumorigenic pathway because cancers with MSI-H exhibit many differences in genotype and phenotype relative to cancers without MSI-H, irrespective of their hereditary or sporadic origins. Genetic, epigenetic and transcriptomic differences exist between cancers with and those without the MSI-H. The BRAF V600E mutation is associated with sporadic MSI-H colorectal cancers (CRCs) harboring hMLH1 methylation but not Lynch syndrome-related CRCs. The differences in genotype and phenotype between cancers with and those without MSI-H are likely to be causally linked to their differences in biological and clinical features. Therefore, the diagnosis of MSI-H in cancers is thus considered to be of increasing relevance, because MSI-H is a useful screening marker for identifying patients with Lynch syndrome, a better prognostic factor and could affect the efficacy of chemotherapy. This review addresses recent advances in the field of microsatellite instability research.

Association between DNA Methylation and Shortened Survival in Patients with Advanced Colorectal Cancer Treated with 5-Fluorouracil–Based Chemotherapy

There are no good genomic markers of survival in patients with advanced colorectal cancer. The CpG island methylator phenotype (CIMP) marks a distinctive pathway in colorectal cancer. We sought to determine the prognostic significance of CIMP in advanced colorectal cancer patients treated with 5-fluorouracil (5-FU) in an Eastern Cooperative Oncology Group clinical trial. We studied 188 patients enrolled on protocol E2290, a five-arm trial comparing 5-FU, 5-FU in combination with N-phosphonoacetyl-l-aspartic acid, oral leucovorin, i.v. leucovorin, or IFNalpha-2a in patients with advanced colorectal cancer. Methylation of MINT1, MINT31, hMLH1, p14ARF, and p16INK4a in DNA extracted from formalin-fixed paraffin-embedded specimens was evaluated by combined bisulfite restriction analysis, and methylation of MINT2 was studied by methylation-specific PCR. Methylation frequencies were 21% for MINT1, 23% for MINT2, 24% for MINT31, 4% for hMLH1, 11% for p14ARF, and 17% for p16INK4a. Methylation of MINT1, MINT31, p14ARF, and p16INK4a were correlated, as expected. There was no association between methylation and clinicopathologic factors or response to therapy. Methylation of MINT1, MINT31, p14ARF, or p16INK4a was associated individually with shortened overall survival. Hazard ratios were 1.51 (P = 0.05) for MINT1, 1.70 (P = 0.006) for MINT31, 2.22 (P = 0.001) for p14ARF, and 1.51 (P = 0.05) for p16INK4a. Concurrent methylation of two or more genes of the CIMP-associated subset (MINT1, MINT31, p14ARF and p16INK4a) defined a group of cases with markedly reduced overall survival and hazard ratio was 3.22 (P < 0.0001 in multivariate analyses). CIMP is associated with poor survival in advanced colorectal cancer patients.

Left‐Sided microsatellite unstable colorectal cancers show less frequent methylation of hMLH1 and CpG island methylator phenotype than right‐sided ones

We compared the methylation status of several loci between right-sided and left-sided colorectal cancers (CRCs). To the best of our knowledge, this is the first to report that left-sided CRCs show significantly less frequent methylation at several loci and CpG island methylator phenotype (CIMP) than right-sided ones. MSI CRC occurs in 10-20% of unselected series of patients with CRC. Somatic hMLH1 promoter methylation is reported to cause MSI in sporadic cases. Many researchers report that MSI CRCs are more frequently located in the right-side colon than MSS CRCs. Though the number is very small, some MSI CRCs are located in the left-side colorectum. We focused on the existence of left-sided MSI CRCs and investigated whether they arise through hMLH1 methylation as they do in right-sided ones. Thirty-eight sporadic MSI CRCs were included in the study. The methylation status of the promoter of hMLH1, p16, MINT1, 2 and 31 were examined and the proportions of methylated samples for each locus were compared. The left-sided group showed significantly less frequent methylation in hMLH1, p16, MINT1, 2 and 31. The frequency of CIMP+ samples in the left-sided group was significantly lower than the right-sided group. Left-sided MSI CRCs show significantly less frequent methylation of hMLH1. They also showed significantly less frequent occurrence of CIMP+ than right-sided ones. It is possible that left-sided MSI CRCs differ from the right-sided ones in the way of acquiring MSI.

3040 POSTER Optimal dose for an every 2 week (q2w) cetuximab (C) regimen in patients (pts) with metastatic colorectal cancer (mCRC): a phase I safety, pharmacokinetics (PK) and pharmacodynamics (PD) study of weekly (q1w) and q2w schedules

Filiform serrated adenoma (SA) is an uncommon type of polyp that shows morphological features similar to traditional serrated adenoma (TSA). Unlike TSA, filiform SA is composed predominantly of prominent, thin, elongated filiform projections lined by neoplastic epithelium with a serrated contour. However, the molecular pathogenesis underlying filiform SA is unclear and its relationship with TSA has not been explored yet. The purpose of this study was to determine the clinicopathological and molecular characteristics of filiform SA in a cohort of Korean patients.Thirteen filiform SAs were evaluated for mutations of BRAFand KRAS genes, microsatellite instability (MSI), and promoter hypermethylation of hMLH1, MGMT, p16, MINT1, MINT2, MINT31 and the APC genes. The clinicopathological and molecular results were compared to results from previously published studies of left-sided TSAs among Koreans.All but one filiform SAs were located in the left colon and showed low grade dysplasia. BRAF and KRAS mutations were observed in six (46.2%) and four (30.3%) filiform SAs, respectively. Hypermethylation of hMLH1 (using both Herman et al. and Park et al.), MGMT, p16, MINT1, MINT2, MINT31 and the APC gene was found in 30.3% and 7.7%, 38.5%, 15.4%, 53.8%, 46.2%, 38.5% and 15.4% of cases, respectively. Thirteen filiform SAs were MS stable and classified with a CpG island methylator phenotype (CIMP) of high in five, CIMP low in five and CIMP negative in three cases. Compared to TSAs in the left colon, methylation of hMLH1, APC, and MGMT was less frequent in cases of filiform SA, but the filiform SA sizes were larger.Our findings suggest that filiform SA may grow larger without acquisition of additional genetic alterations and can be categorised as a rare, less aggressive variant of TSA with unique morphology.

3038 POSTER Left-sided microsatellite unstable colorectal cancers show less frequent methylation of hMLH1 and CpG island methylator phenotype than right-sided ones

3038 POSTER Left-sided microsatellite unstable colorectal cancers show less frequent methylation of hMLH1 and CpG island methylator phenotype than right-sided ones

Methylation status of six tumor-associated genes in Japanese patients with ulcerative colitis

It has been recognized that patients with longstanding ulcerative colitis (UC) have an increased risk of colorectal cancer. The major carcinogenic molecular pathways that lead to sporadic colorectal cancer, namely chromosomal instability, microsatellite instability, and hypermethylation, are also believed to correlate with the developing of colitis associated colorectal cancer (CAC). We detected the methylation status of promoter regions of estrogen receptor gene (ER), TP53, p14, p16, p21 and hMLH1 in rectal inflammatory mucosa of patients with UC, and investigated the association between DNA methylation rate and the clinical manifestations. Enrolled in the study were 36 Japanese patients with UC, including 20 cases of male and 16 cases of female. The average age was 40.4±9.6 years old, and the clinical duration was 9.1±4.8 years. A total of 51 specimens, including 38 biopsies at inflammatory mucosa from the rectum and 13 at normal mucosa from the terminal ileum were obtained during colonoscopic examinations. DNA extraction and methylation specific polymerase chain reaction were performed to detect the methylation in promoter CpG islands of the above six genes. The results showed that methylation rate of ER promoter in the rectal inflammatory mucosa was significant higher than that in the controls (76.3% vs 46.2%, p=0.0251). The methylation rate of ER was also remarkably higher in the longstanding patients (90.9% in those longer than 7 years, 57.1% in the shorter, p=0.0175). Methylation rates in promoters of p14 and p16 in inflammatory mucosa were higher than those in the controls, respectively, but the differences were not of statistic significance. Meanwhile, methylation in TP53 promoter was found in only one case and p21 and hMLH1 methylation were negative in all cases. In addition, significant association was found between p14 methylation and patient's age, however, no significant association was found in methylation rates of ER, p14 and p16 to gender, inflammatory degree or extension of colitis. These results suggests that methylation in promoter regions of ER, p14 and pl6 may play a role in the development of mucosal inflammation in chronic UC, and methylation analysis of ER in rectal mucosa may be useful on the identification of UC patients at high risk of carcinogenesis. In addition, our experiments also prove that inflamed rectal mucosa, unlike normal colorectal mucosa, demonstrates significant abnormalities in DNA methylation even before any histological evidence of neoplastic transformation.

Low Frequency of Promoter Methylation ofO6-Methylguanine DNA Methyltransferase and hMLH1 in Ulcerative Colitis–Associated Tumors

To cast light on the contribution of methylation to genesis of ulcerative colitis (UC)-associated tumors, promoter methylation and expression of O6-methylguanine DNA methyltransferase (MGMT), hMLH1, p16INK4, and E-cadherin were examined in 14 low-grade dysplasias (LGDs), 15 high-grade dysplasias (HGDs), and 14 adenocarcinomas associated with UC and, for comparison, in 30 sporadic adenomas with LGD, 30 adenomas with HGD, and 60 adenocarcinomas, using methylation-specific polymerase chain reaction and immunohistochemical analysis. The frequency of MGMT and hMLH1 methylation in UC-associated tumors was low, with a significant difference between HGD and sporadic adenomas with HGD of the left hemicolon. The methylation frequency of p16INK4 in UC-associated tumors was also relatively low compared with sporadic colonic tumors. For E-cadherin, methylation was limited in both types of tumor. Decrease of expression of MGMT, hMLH1, and p16INK4 was significantly correlated with methylation. Thus, compared with the sporadic type, contribution of methylation to UC-associated tumorigenesis seems to be low.

Low Frequency of Promoter Methylation of O 6 -Methylguanine DNA Methyltransferase and hMLH1 in Ulcerative Colitis-Associated Tumors: Comparison With Sporadic Colonic Tumors

To cast light on the contribution of methylation to genesis of ulcerative colitis (UC)-associated tumors, promoter methylation and expression of <italic>O6</italic>-methylguanine DNA methyltransferase (MGMT), hMLH1, p16INK4, and E-cadherin were examined in 14 low-grade dysplasias (LGDs), 15 high-grade dysplasias (HGDs), and 14 adenocarcinomas associated with UC and, for comparison, in 30 sporadic adenomas with LGD, 30 adenomas with HGD, and 60 adenocarcinomas, using methylation-specific polymerase chain reaction and immunohistochemical analysis. The frequency of MGMT and hMLH1 methylation in UC-associated tumors was low, with a significant difference between HGD and sporadic adenomas with HGD of the left hemicolon. The methylation frequency of p16INK4 in UC-associated tumors was also relatively low compared with sporadic colonic tumors. For E-cadherin, methylation was limited in both types of tumor. Decrease of expression of MGMT, hMLH1, and p16INK4 was significantly correlated with methylation. Thus, compared with the sporadic type, contribution of methylation to UC-associated tumorigenesis seems to be low.

Role of the BRAF Mutations in the Microsatellite Instability Genetic Pathway in Sporadic Colorectal Cancer

Between 10 and 15% of all cases of colorectal cancer are the result of microsatellite instability (MSI) in the genetic pathway due to an alteration in the DNA repair genes. Tumors with high MSI are characterized by a better prognosis. The BRAF oncogene has been linked to the MSI pathway in tumorogenesis. The objective of this study was to determine whether alterations in BRAF are related to MSI and whether they can result in differences in survival rates. The study cohort comprised 351 patients diagnosed with sporadic colorectal cancer. MSI was determined in accordance with the National Cancer Institute's (NCI) recommendations by means of PCR and sequence analyses. Mutational analysis of the BRAF gene was performed by real-time PCR and subsequent sequencing of the altered samples. The methylation pattern of the hMLH1 gene was determined using methylation-specific PCR analyses of bisulfite-treated DNA and the results confirmed by sequencing. Of the patients tested, 6.9% showed high MSI and 3.7% showed a BRAF gene mutation. hMLH1 methylation was observed in 67.2% of the patients with MSI and/or the BRAF alteration. The BRAF mutation was related to the MSI genetic pathway (P < 0.0001) and with hMLH1 methylation. In the analysis of overall survival only MSI had an independent prognostic value for the risk of death. Patients with the BRAF mutation showed a higher risk of death, although this association was found not to be statistically significant. There is a subgroup of carcinomas which develop via the MSI pathway that carry an alteration of the BRAF gene. This alteration confers a poorer outcome on these patients within the total group of patients with MSI who have a better prognosis. This hypothesis should be further investigated in a larger study population due to the low incidence of BRAF mutations in colorectal cancer.

Methylation of Serum DNA Is an Independent Prognostic Marker in Colorectal Cancer

Aberrant CpG island hypermethylation is a feature of a subgroup of colorectal cancers, which can be detected in the serum of affected patients. This study was designed to identify methylation targets with prognostic significance in the serum of patients with colorectal cancer. In a gene evaluation set consisting of sera from 24 patients with local colorectal cancers, 14 with metastasized disease, and 20 healthy controls, the genes HPP1/TPEF, HLTF, and hMLH1 were identified as potential serum DNA methylation markers. These genes were further analyzed in a test set of sera of 104 patients with colorectal cancer. Methylation of HLTF, HPP1/TPEF, and hMLH1 was found to be significantly correlated with tumor size, and methylation of HLTF and HPP1/TPEF was significantly associated with metastatic disease and tumor stage. Moreover, methylation of HPP1/TPEF was also associated with serum carcinoembryonic antigen. The prognostic relevance of methylation of these genes was tested in pretherapeutic sera of 77 patients with known follow-up. Patients with methylation of HPP1/TPEF or HLTF were found to have unfavorable prognosis (P = 0.001 and 0.008). In contrast, serum methylation of hMLH1 was not associated with a higher risk of death. Multivariate analysis showed methylated HPP1 and/or HLTF serum DNA to be independently associated with poor outcome and a relative risk of death of 3.4 (95% confidence interval, 1.4-8.1; P = 0.007). These data show that the methylation status of specific genes in the serum of patients with colorectal cancer has the potential to become a pretherapeutic predictor of outcome.

Colorectal serrated adenocarcinoma

Colorectal cancer (CRC) ranks among the three most common cancers in terms of both cancer incidence and cancer-related deaths in most Western countries. Serrated adenocarcinoma is a recently described, distinct variant of CRC, accounting for about 7.5% of all CRCs and up to 17.5% of most proximal CRCs. It has been postulated that about 10-15% of sporadic CRCs would have their origin in serrated polyps that harbour a significant malignant potential. These lesions include hyperplastic-type aberrant crypt foci, hyperplastic polyps, sessile serrated adenomas, admixed polyps and serrated adenomas, and constitute the so-called 'serrated pathway', which is distinct from both the conventional adenoma-carcinoma pathway and the mutator pathway of hereditary non-polyposis CRC and is characterized by early involvement of oncogenic BRAF mutations, excess CpG island methylation (CIM) and subsequent low- or high-level DNA microsatellite instability (MSI). Methylation of hMLH1 is likely to explain the increased frequency of high-level MSI (16%) and methylation of MGMT is postulated to explain the low-level MSI (29%) in serrated adenocarcinomas. Reproducible histopathological criteria for serrated adenocarcinoma have recently been established and they have been qualified by DNA expression analysis for 7928 genes, showing clustering of serrated adenocarcinomas into a molecular entity apart from conventional adenocarcinoma, and representing with distinct down-regulation of EPHB2, PTCH and up-regulation of HIF1alpha.

Endoscopic Ultrasound–Guided Fine Needle Aspirate Microsatellite Loss Analysis and Pancreatic Endocrine Tumor Outcome

The clinical course of pancreatic endocrine tumor (PET) varies depending on tumor aggressiveness, disease extent, and possibly accumulated molecular alterations. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) results, although accurate in diagnosing PET, correlate poorly with PET outcome. The role of detecting key molecular abnormalities in predicting PET behavior and clinical outcome from EUS-FNA material remains unknown. Patients with confirmed PET who underwent EUS-FNA during a 32-month period were included. Patient demographics and clinical data were recorded and follow-up information was obtained by contacting their physician to evaluate disease progression. Representative tumor cells were microdissected from the FNA material. DNA was harvested and amplified, targeting a panel of 17 polymorphic microsatellite markers on chromosomes 1p, 3p, 5q, 9p, 10q, 11q, 17p, 17q, 21q, and 22q. The polymerase chain reaction products were subjected to fluorescent capillary gel electrophoresis to detect microsatellite loss. The fractional allelic loss (FAL) was calculated. Twenty-five patients were studied. Thirteen were classified histologically as benign PET limited to the pancreas and 12 as malignant PET (invasive or metastatic). The mean FAL in the benign and malignant PET was 0.03 and 0.37 (P<.0001), respectively. In addition, the mean FAL was significantly greater in those with disease progression as compared with patients with stable disease (0.45 vs 0.09 respectively, P<.0001). Microsatellite loss analysis of EUS-FNA material from PET can be performed reliably and an FAL value of more than 0.2 is associated with disease progression. This technique may have value in the preoperative assessment and risk stratification of patients with PET.

Relationship between point gene mutation, chromosomal abnormality, and tumour suppressor gene methylation status in colorectal adenomas

Epigenetic mechanisms in carcinogenesis may have a significant role in the development of colorectal cancer. To investigate this phenomenon in early-stage disease, promoter methylation status in the tumour suppressor genes APC, MGMT, hMLH1, P14/P14ARF, and CDKN2A/P16 was investigated in 78 colorectal adenomas. These had previously been characterized for mutations of APC, KRAS, and TP53 genes and for chromosomal abnormality by comparative genomic hybridization (CGH). APC hypermethylation was seen in 52 tumours (66.7%). APC showed either methylation or mutation in 66 lesions (84.6%), but these events were not statistically associated. MGMT methylation was detected in 39 cases (50%). Adenomas with this abnormality showed a significantly lower number of chromosomal changes by CGH (p < 0.02), confirming that DNA repair defect of this type is associated with a lower level of chromosomal instability. An hMLH1 methylation defect was seen in only one adenoma (1.3%), from a patient who had a synchronous cancer showing the same defect. Methylation of P14 (P14ARF) was seen in 31 adenomas (39.7%) and CDKN2A (P16) abnormality in 25 (32.1%). DNA methylation at two or more loci was seen in 46 tumours (59%), while 11 lesions (14.1%) showed no evidence of hypermethylation at any of the loci studied. Methylation at any or all of MGMT, P14 or P16 was significantly associated with APC methylation (p = 0.01). Those neoplasms with more than two methylated genes showed significantly fewer chromosomal abnormalities than adenomas with one or no methylated loci (p < 0.001). There was no association between specific individual chromosomal abnormalities, APC, KRAS or TP53 mutations and any pattern of methylation abnormality. We conclude that methylation abnormality is very common in pre-invasive colorectal neoplasia, and that high level methylation is associated with low level chromosomal instability.