Chromosomal and microsatellite instability in sporadic gastric cancer

BACKGROUND: Gastric cancer can progress through two pathways of genomic instability: chromosomal (CIN) and microsatellite instability (MSI). It is hypothesized that these two pathways are not always independent and that some tumors show overlap between these two mechanisms. METHODS: A total of 98 sporadic gastric cancers were classified based on their MSI status, using microsatellite assay with BAT26. Evidence for CIN was investigated by identifying loss of heterozygosity (LOH) events on chromosome arms, 5q, 10p, 17p, 17q, and 18q, which are regions harboring tumor suppressor genes that are significant in gastric cancer development. RESULTS: Twelve tumors (12%) showed high-frequency MSI (MSI-H). Overall, 43 of the tumors (44%) had at least one LOH event, with most frequent chromosomal losses observed on 10p and 18q (30%, respectively), followed by 5q (21%), 17p (14%), and 17q (12%). Interestingly, overlap was observed between CIN and MSI pathways. Of 43 cancers with LOH events, four (9%) were also MSI-H. It was also found that 48% of cancers without MSI-H had no LOH events identified, comprising a subgroup of tumors that were not representative of either of these two pathways of genomic instability. CONCLUSION: These results suggest that molecular mechanisms of genomic instability are not necessarily independent and may not be fully defined by either the MSI or CIN pathways in sporadic gastric cancers.     

胃癌微卫星不稳定性和抑癌基因杂合缺失

目的研究微卫星不稳和抑癌基因缺失在胃癌发生中的作用．方法采用ＰＣＲ为基础的方法,检测了５３例胃癌中６个微卫星标记突变及ＡＰＣ／ＭＣＣ和ＤＣＣ基因杂合缺失（ＬＯＨ）．结果胃癌微卫星不稳的检出率为３２１％（１７／５３）．７例（１３２％）为微卫星高频率不稳（３个以上微卫星标志）,１０例（１８９％）为微卫星低频率不稳（１或２个微卫星标记）．肠型胃癌微卫星高频率不稳的发生率（２５０％）显著高于弥漫型胃癌（３４％）（Ｐ＜００５）．高频率不稳组未发现有ＡＰＣ,ＭＣＣ和ＤＣＣ基因ＬＯＨ,微卫星高频率不稳与ＡＰＣ／ＭＣＣ和ＤＣＣ基因ＬＯＨ呈负相关．结论微卫星不稳在部分胃癌,特别是肠型胃癌早期发生中起重要作用,高频率不稳胃癌与遗传性非息肉大肠癌有共同的特点．与此相反,低频率不稳和无不稳胃癌可能通过ＬＯＨ病理途径发生     

The CpG island methylator phenotype and chromosomal instability are inversely correlated in sporadic colorectal cancer

BACKGROUND & AIMS: The CpG island methylator phenotype (CIMP) is one of the mechanisms involved in colorectal carcinogenesis (CRC). Although CIMP is probably the cause of high-frequency microsatellite instability (MSI-H) sporadic CRCs, its role in microsatellite stable (MSS) tumors is debated. The majority of MSS CRCs demonstrate chromosomal instability (CIN) with frequent loss of heterozygosity (LOH) at key tumor suppressor genes. We hypothesized that the majority of sporadic CRCs without CIN would be associated with CIMP. METHODS: We tested 126 sporadic CRCs for MSI and LOH and categorized tumors into MSI, LOH, or MSI-/LOH- subgroups. Methylation status was evaluated using 6 CIMP-related markers (MINT1, MINT2, MINT31, p16(INK4alpha), p14(ARF), and hMLH1) and 6 tumor suppressor genes (PTEN, TIMP3, RUNX3, HIC1, APC, and RARbeta2). BRAF V600E mutation analysis was performed using allele-specific polymerase chain reaction and DNA sequencing. RESULTS: We observed frequent methylation at all 12 loci in all CRCs. BRAF V600E mutations correlated with the MSI (P < .0001) and MSI-/LOH- (P = .03) subgroups. MSI and MSI-/LOH- tumors exhibited more promoter methylation than CRCs with LOH (P < .0001). We also found an inverse correlation between the frequencies of methylation and LOH (rho = -0.36; P < .0001). CONCLUSIONS: The associations between methylation frequencies at CIMP-related markers and MSI or MSI-/LOH- sporadic CRCs suggest that the majority of these tumors evolve through CIMP. These findings suggest that CIN and CIMP represent 2 independent and inversely related mechanisms of genetic and epigenetic instability in sporadic CRCs and confirm that MSI cancers arise as a consequence of CIMP.     

Microsatellite instability in sporadic and inherited colon adenocarcinomas from Greek patients: correlation with several clinicopathological characteristics

BACKGROUND AND AIMS: Microsatellite instability seems to play a significant role in colorectal carcinogenesis, as it is reported to occur in HNPCC patients as well as in a proportion of sporadic cases. The aim of this study was to examine the presence of microsatellite instability in relation to other commonly observed genetic abnormalities and clinicopathological characteristics of sporadic and inherited colorectal cancers. METHODOLOGY: One hundred and three sporadic colorectal adenocarcinomas and 9 adenocarcinomas from HNPCC patients were histologically evaluated. The presence of microsatellite instability was investigated at six loci. K-ras and p53 mutations, p53 LOH, hMLH1 expression and methylation status were examined as well. Statistical analysis was performed to define possible correlations of the observed genetic alterations with the clinicopathological characteristics of the analysed tumors. RESULTS: High-grade microsatellite instability was found in 14% of sporadic adenocarcinomas and in 78% of adenocarcinomas from HNPCC patients. K-ras and p53 mutations were found in 29% and 28% of sporadic adenocarcinomas respectively and in 0% and 22% of the 9 HNPCC cases. A statistically significant correlation was noticed in sporadic tumors between the presence of MSI-H and tumor location at the proximal colon, as well as with the female gender. CONCLUSIONS: Sporadic MSI+ colon adenocarcinomas seem to represent a distinct entity with a unique profile of genetic changes, different from those observed in HNPCC or MSI negative sporadic tumors.     

Genetic aberrations common in gastric high-grade large B-cell lymphoma

Genetic aberrations associated with the development of extranodal high-grade large B-cell lymphoma originating in the stomach have not been fully identified yet. We analyzed 31 such lymphomas using 73 microsatellite markers for allelic imbalance and microsatellite instability. The highest frequency (42%) of loss of heterozygosity (LOH) was found on the long arm of chromosome 6. We identified 2 LOH hot spots on 6q21-22.1 and 6q23.3-25, flanked by markers D6S246-D6S261 and D6S310-D6S441, respectively, containing putative tumor suppressor genes (TSGs). These 6q aberrations were found to be the sole allelic imbalance in 1 patient only; they were mostly accompanied by additional abnormalities. Several known TSGs, namely, the APC, p15/p16, p53, and DCC genes, were found to suffer frequent LOH during lymphomagenesis. LOH was also detected in regions containing putative TSGs on 7q and 13q14. Frequent amplification of genomic material was found in the 2p, 3q27 at the BCL-6 gene locus, 6p, 7q, 11q23-24 at the MLL gene locus, and 18q regions. Analysis of the pattern of occurrence of these aberrations revealed an association of the amplification of the MLL gene region with LOH at the p53 locus (P =.02). Only low frequency of microsatellite instability (MSI) was detected in these lymphomas and MSI incidence increased with age (P =.01). Karyotypic instability thus plays the main role in the development of gastric high-grade large B-cell lymphoma. Common genetic aberrations responsible for lymphomagenesis are deletions of 6q, loss of p53, and amplification of the 3q27 and the MLL gene regions. (Blood. 2000;95:1180-1187)     

Loss of heterozygosity： An independent prognostic factor of colorectal cancer

AIM: Colorectal cancers result from the accumulation of several distinct genetic alterations. This study was to investigate the frequency and prognostic value of loss of heterozygosity (LOH) and microsatellite instability (MSI) at 14 genetic loci located near or within regions containing important genes implicated in colorectal tumorigenesis. METHODS: We studied colorectal cancers with corresponding normal mucosae in 207 patients (139 males and 68 females, mean age at the time of tumor resection 66.2±12.4 years, range 22-88 years). There were 37 right-sided colonic tumors, 85 left-sided colonic tumors and 85 rectal tumors. The distribution of tumor staging was stage Ⅰ in 25, stage Ⅱ in 73, stage Ⅲ in 68, and stage Ⅳ in 41. We analyzed the LOH and MSI of HPC1, hMSH2, hMLH1, APC, MET, P53, NH23-H1, DCC, BAT25, BAT26, D17S250, MYCL1 and D8S254 with fluorescent polymerase chain reaction and denatured gel electrophoresis. High-frequency LOH was determined to be greater than three, or more than 50% of the informative marker with LOH. High-frequency MSI (MSI-H) was determined as more than four markers with instability (>30%). Correlations of LOH and MSI with clinical outcomes and pathological features were analyzed and compared. RESULTS: The occurrence of MSI-H was 7.25%, located predominantly in the right colons (7/15) and had a higher frequency of poor differentiation (6/15) and mucin production (7/15). LOH in at least one genetic locus occurred in 78.7% of the tumors and was significantly associated with disease progression. Of the 166 potentially cured patients, 45 developed tumor recurrence within 36 mo of follow-up. Clinicopathological factors affecting 3-year disease-free survival (DFS) were TNM staging, grade of differentiation, preoperative CEA level, and high LOH status. Patients with high LOH tumors had a significantly lower DFS (50%) compared with patients with low LOH tumors (84%). Of the patients developing subsequent tumor recurrence, the number and percentage of LOH were 2.97 and 46.8% respectively, similar to the stage IV disease patients. TNM staging had the most significant impact on DFS, followed by high LOH status. CONCLUSION: Clinical manifestations of LOH and MSI are different in colorectal cancer patients. High-frequency LOH is associated with high metastatic potential of colorectal cancers.     

Microsatellite instability in patients with multiple primary cancers of the gastrointestinal tract

BACKGROUND: Little is known about the genetic alterations in multiple primary cancers of the gastrointestinal tract. Microsatellite instability (MSI) is frequently observed in hereditary non-polyposis colorectal cancer (HNPCC), and multiple primary cancers is a feature of this syndrome. AIMS: To identify MSI incidence, target gene mutation, and mismatch repair (MMR) protein status in patients with multiple primary cancers of the gastrointestinal tract. SUBJECTS: Fifty seven cancers from 22 Japanese patients with multiple primary cancers of the stomach, duodenum, colon, and rectum. METHODS: MSI was examined at 5-7 microsatellite loci. Mutation analysis for TGFbetaRII, IGFIIR, and BAX was performed in cancers with MSI. MMR protein status was examined by immunohistochemical analysis using a monoclonal antibody against hMSH2 and hMLH1. RESULTS: MSI was observed in 16 of 22 patients (73%) and in 29 of 57 lesions (51%). High frequency MSI (MSI-H) was found often in patients with multiple cancers in the same organ (p = 0.042), especially in multiple gastric cancer patients (p = 0.038). In contrast, patients with multiple cancers in different organs had a tendency to show low frequency MSI (MSI-L) or microsatellite stable (MSS) phenotype. Both target gene mutation and decreased expression of MMR protein were found only in seven lesions of three patients with MSI at more than four microsatellite loci. CONCLUSIONS: These results suggest that genetic instability may play an important role in the development of multiple gastrointestinal cancers but there may be different genetic alterations between multiple gastrointestinal cancers of the same and different organs.     

The role of clinical criteria, genetic and epigenetic alterations in Lynch-syndrome diagnosis

Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC) represents 1-3% of all diagnosed colorectal cancers (CRCs). This study aimed to evaluate the benefit of clinical criteria and several molecular assays for diagnosis of this syndrome. We examined tumors of 104 unrelated clinically characterized colorectal cancer patients for causal mismatch repair (MMR) deficiency by several methods: microsatellite instability (MSI) and loss of heterozygosity (LOH) presence, MMR protein absence, hypermethylation of MLH1 promoter and germline mutation presence. Twenty-five (24%) patients developed CRCs with a high level of MSI (MSI-H). Almost all (96%) had at least one affected relative, while this simple criterion was satisfied in only 22% (17/79) of individuals with low level MSI or stable cancers (MSI-L, MSS). Using strict Amsterdam criteria, the relative proportion of complying individuals in both sets of patients (MSI-H vs. MSI-L and MSS) decreased to 68% and 9%, respectively. The right-sided tumors were located in 54% of MSI-H persons when compared to 14% of cancers found in MSI-L or MSS patients. In 16 MSI positive patients with identified germline mutation by DNA sequencing, the gene localization of mutation could be indicated beforehand by LOH and/or immunohistochemistry (IHC) in four (25%) and 14 cases (88%), respectively. The IHC findings in MSI-H cancers with methylation in distal or both regions of MLH1 promoter have not confirmed the epigenetic silencing of the MLH1 gene. None of the patients with MSIL or MSS tumors was a carrier of the MLH1 del616 mutation, despite seven of them meeting Amsterdam criteria. The effective screening algorithm of Lynch-syndrome-suspected patients consists of evaluation of Bethesda or Revised Bethesda Guidelines fulfilling simultaneous MSI, LOH and IHC analyses before DNA sequencing. Variable methylation background in MLH1 promoter does not affect gene silencing and its role in Lynch-syndrome tumorigenesis is insignificant.     

Epigenetic alterations in colorectal carcinomas and precancerous lesions

Colorectal carcinomas are the third most common malignant tumours worldwide with an incidence of 570,000 per year. According to their molecular mechanisms, sporadic colorectal carcinomas can be divided into two different phenotypes. The genetic phenotype, 50 to 70 % of all sporadic colorectal carcinomas, is characterised by a chromosomal instability (CIN) with the classical adenoma-carcinoma sequence due to alteration of the APC-betacatenin pathway with p53 mutations, SMAD alterations and LOH (loss of heterozygositiy) of 5q, 17 p 18q. On the other, the CpG island methylator phenotype (CIMP+) was described with an epigenetic inactivation of tumour suppressor genes that are typically inactivated by germline mutations in familiar cancer syndromes, e. g., Rb, VHL, hMLH1, p16 or BRCA. Colorectal carcinomas of the CIMP+ type often show a high microsatellite instability (MSI+) caused by aberrant promoter methylation of the missmatch repair gene hMLH1. Further CIMP+ are located in the proximal right-side colon and show a poor grading with mucinous or signet-cell differentiation.     

Clinicopathological significance of loss of heterozygosity and microsatellite instability in hepatocellular carcinoma in China

AIM: To determine the features of microsatellite alterations and their association with clinicopathological characteristics of hepatocellular carcinoma (HCC). METHODS: Loss of heterozygosity (LOH) and microsatellite instability (MSI) of 55 microsatellite loci were detected with PCR-based microsatellite polymorphism analyses in tumors and corresponding noncancerous liver tissues of 56 surgically resected HCCs using the MegaBACE 500 automatic DNA analysis system. RESULTS: LOH was found in 44 of 56 HCCs (78.6%) at one or several loci. Frequencies of LOH on 1p, 4q, 8p, 16q, and 17p were 69.6% (39/56), 71.4% (40/56), 66.1% (37/56), 66.1% (37/56), and 64.3% (36/56), respectively. MSI was found in 18 of 56 HCCs (32.1%) at one or several loci. Ten of fifty-six (17.9%) HCCs had MSI-H. Serum HBV infection, alpha-fetoprotein concentration, tumor size, cirrhosis, histological grade, tumor capsule, as well as tumor intrahepatic metastasis, might be correlated with LOH on certain chromosome regions. CONCLUSION: Frequent microsatellite alterations exist in HCC. LOH, which represents a tumor suppressor gene pathway, plays a more important role in hepatocarcin-ogenesis. MSI, which represents a mismatch repair gene pathway, is a rare event during liver carcinogenesis. Furthermore, LOH on certain chromosome regions may be correlated with clinicopathological characteristics in HCC.     

Telomere erosion is independent of microsatellite instability but related to loss of heterozygosity in gastric cancer

AIM: To correlate the length of the telomere to microsatellite instability (MSI) and loss of heterozygosity (LOH) of APC, MCC and DCC genes in gastric carcinomas. METHODS: Telomeric restriction fragment (TRF) length of gastric cancer was measured with Southern blot. LOH of APC, MCC and DCC genes, microsatellite instability (MSI) and frameshift mutation of hMSH6, TGF-betaRII and BAX genes were analyzed by PCR-based methods. RESULTS: Sixty-eight cases of sporadic gastric carcinoma were studied for MSI using five microsatellite markers. MSI in at least one locus was detected in 17 (25%) of 68 tumors analyzed. Frameshift mutations of hMSH6, TGF-betaRII and BAX were detected in 2,6 and 3 of gastric carcinomas respectively showing high MSI (> or = 2 loci, n = 8), but none was found in those showing low MSI (only one locus, n = 9) or MSS (tumor lacking MSI or stable, n = 51). Thirty-five cases, including all high MSI and low MSI, were studied for TRF. The mean TRF length was not correlated with clinicopathological parameters. No association was observed between TRF length and MSI or frameshift mutation. On the contrary, LOH at the DCC locus was related to telomere shortening (P<0.01). This tendency was also observed in APC and MCC genes, although there was no statistical significance. CONCLUSION: The development of gastric cancer can arise through two different genetic pathways. In high MSI gastric cancers, defective mismatch repair allows mutations to accumulate and generate the high MSI phenotype. In gastric cancers showing either low MSI or MSS, multiple deletions may represent the LOH pathway. Telomere erosion is independent of high MSI phenotype but related to the LOH pathway in gastric cancer.     

Is gastric cancer part of the tumour spectrum of hereditary non-polyposis colorectal cancer? A molecular genetic study

BACKGROUND: Gastric cancer is the second most common extracolonic malignancy in individuals with hereditary non-polyposis colorectal cancer (HNPCC)/Lynch syndrome. As gastric cancer is relatively common in the general population as well, it is not clear whether or not gastric cancer is a true HNPCC spectrum malignancy. AIM: To determine whether or not gastric cancer is a true HNPCC spectrum malignancy. Subjects and METHODS: The molecular and clinicopathological profiles of gastric cancers (n = 13) from HNPCC mutation carriers were evaluated and compared with the profiles of sporadic gastric cancers (n = 46) stratified by histology and microsatellite instability (MSI) status. RESULTS: This study on sporadic and HNPCC gastric cancers revealed several important universal associations. Loss of heterozygosity in the adenomatous polyposis coli (APC) region was associated with intestinal histology regardless of the MSI (p = 0.007). KRAS-mutations (p = 0.019) and frameshift mutations in repeat tracts of growth-regulatory genes (p<0.001) were associated with MSI tumours being absent in microsatellite stable (MSS) tumours. The average number of methylated tumour suppressor gene loci among the 24 genes studied (methylation index) was higher in MSI than in MSS tumours regardless of histology (p<0.001). Gastric cancers from HNPCC mutation carriers resembled sporadic intestinal MSI gastric cancers, except that MLH1 promoter methylation was absent (p<0.001) and the general methylation index was lower (p = 0.038), suggesting similar, but not identical, developmental pathways. All these lacked the mismatch repair protein corresponding to the germline mutation and displayed high MSI. CONCLUSION: The present molecular evidence, combined with the previous demonstration of an increased incidence relative to the general population, justify considering gastric cancers as true HNPCC spectrum malignancies.     

Genetic instability and aberrant DNA methylation in chronic hepatitis and cirrhosis--A comprehensive study of loss of heterozygosity and microsatellite instability at 39 loci and DNA hypermethylation on 8 CpG islands in microdissected specimens from patients with hepatocellular carcinoma

A study was conducted to examine the significance of genetic instability and aberrant DNA methylation during hepatocarcinogenesis. Genomic DNA was extracted from 196 microdissected specimens of noncancerous liver tissue that showed no marked histologic findings or findings compatible with chronic hepatitis or cirrhosis, and 80 corresponding microdissected specimens of hepatocellular carcinoma (HCC) from 40 patients. Loss of heterozygosity (LOH) and microsatellite instability (MSI) were examined by polymerase chain reaction (PCR) using 39 microsatellite markers, and DNA methylation status on 8 CpG islands was examined by bisulfite-PCR. In noncancerous liver tissues, LOH, MSI, and DNA hypermethylation were found in 15 (38%), 6 (15%), and 33 (83%) of 40 cases, respectively. The incidence of DNA hypermethylation in histologically normal liver was similar to that in chronic hepatitis and cirrhosis, although neither LOH nor MSI was found in histologically normal liver. In cancerous tissues, LOH, MSI, and DNA hypermethylation were found in 39 (98%), 8 (20%), and 40 (100%) of 40 cases, respectively. CpG islands of the p16 gene and methylated in tumor 1, 2, 12, and 31 clones were frequently methylated in cancerous tissues, although neither the thrombospondin-1 nor the human Mut L homologue (hMLH1) gene was methylated. Absence of silencing of the hMLH1 gene by DNA hypermethylation is consistent with the low incidence of MSI in HCCs. The results of this study indicate that LOH and aberrant DNA methylation contribute to hepatocarcinogenesis; DNA hypermethylation in particular, which precedes or may even cause LOH, is as an early event during hepatocarcinogenesis.     

Extensive but hemiallelic methylation of the hMLH1 promoter region in early-onset sporadic colon cancers with microsatellite instability.

BACKGROUND AND AIMS: Methylation of the hMLH1 promoter region is frequently observed in microsatellite instability (MSI)-positive sporadic colorectal carcinomas. We studied hMLH1 promoter methylation in peripheral blood lymphocytes of 87 index patients representing 29 cases of hereditary nonpolyposis colorectal cancers (HNPCCs), 28 cases of atypical HNPCCs, and 30 sporadic cases of the development of early-onset colorectal carcinomas or multiple primary cancers. METHODS: Methylation of the hMLH1 promoter region was analyzed by Na-bisulfite polymerase chain reaction/single-strand conformation polymorphism analysis or methylation-specific polymerase chain reaction. MSI, allelic status of the hMLH1 locus, and loss of hMLH1 protein expression were examined in cases for which tumor tissues were available. RESULTS: Extensive methylation of the hMLH1 promoter was detected in peripheral blood lymphocytes of 4 of 30 patients with sporadic early-onset colon cancer, among whom multiple primary cancers (1 colon and 1 endometrial cancer) developed in 2 cases. This methylation was not detected in analyses of HNPCC or atypical HNPCC groups or healthy control subjects. MSI was positive, and extensive methylation was detected in both cancers (colon and endometrial cancer) and normal tissues (colon, gastric mucosa, endometrium, and bone marrow) in all of the examined cases (3 of 3). Analysis of a polymorphic site in the hMLH1 promoter in 2 informative cases showed that methylation was hemiallelic. In 1 case, the unmethylated allele was lost in the colon cancer but not in the metachronous endometrial cancer. CONCLUSIONS: Constitutive, hemiallelic methylation of the hMLH1 promoter region was shown to be associated with carcinogenesis in sporadic, early-onset MSI-positive colon cancers.     

CpG island methylation as an early event during adenoma progression in carcinogenesis of sporadic colorectal cancer

BACKGROUND AND AIMS: CpG island methylation is present in various tumors, including colorectal carcinomas, and is thought to be an important mechanism in carcinogenesis. We evaluated the methylation status of primary colorectal tumors to determine its role in the adenoma to carcinoma sequence. METHODS: The methylation status of APC, THBS1, MGMT, hMLH1 and GSTP1, as determined by methylation specific PCR (MSP), and microsatellite instability (MSI) using three mononucleotide markers were assessed in 40 colorectal adenomas and 36 adenocarcinomas. The correlations of methylation status and MSI with the clinicopathologic parameters of the tumors were determined. RESULTS: Of the 40 adenomas, 24 (60%) were methylated at one or more loci, and 12 (30%) at two or more loci (CpG island methylation phenotype-high, CIMP-H). Of 36 carcinomas, 27 (75%) were methylated at one or more loci and 11 (30.5%) at two or more loci (CIMP-H). THBSI was the most frequently methylated locus in both adenomas (n = 19, 47.5%) and carcinomas (n = 16, 44.4%). Overall, methylation status of adenomas and carcinomas did not differ significantly (P = 0.53), nor did the methylation status of individual genes. For adenomas, size (P = 0.049) and histologic classification of the villous components (P = 0.018) were each associated with methylation status. For carcinomas, however, no clinicopathologic variable was related to methylation status. MSI was detected in three adenomas (7.5%) and five carcinomas (13.9%), and was closely correlated with hMLH1 methylation (P < 0.001). CONCLUSIONS: In colorectal tumors, CpG island methylation of tumor suppressor genes appears to be common and may be involved in the progression of adenomas.     

Refining molecular analysis in the pathways of colorectal carcinogenesis.

BACKGROUND & AIMS: In the stepwise model, specific genetic and epigenetic changes accumulate as colorectal adenomas progress to carcinomas (CRCs). CRCs also acquire global phenotypes, particularly microsatellite instability (MSI) and aneuploidy/polyploidy (chromosomal instability, CIN). Few changes specific to MSI-low or CIN+ cancers have been established. METHODS: We investigated 100 CRCs for: mutations and loss of heterozygosity (LOH) where appropriate, of APC, K-ras, BRAF, SMAD4, and p53; deletion on 5q around APC and 18q around SMAD4; total chromosomal-scale losses and gains; MSI; and CIN. RESULTS: As expected, CIN- cancers had fewer chromosomal changes overall than CIN+ lesions, but after correcting for this, 5q deletions alone predicted CIN+ status. 5q deletions were not, however, significantly associated with APC mutations, which were equally frequent in CIN+ and CIN- tumors. We therefore found no evidence to show that mutant APC promotes CIN. p53 mutations/LOH were more common in CIN+ than CIN- lesions, and all chromosomal amplifications were in CIN+ tumors. CIN- cancers could be subdivided according to the total number of chromosomal-scale changes into CIN-low and CIN-stable groups; 18q deletion was the best predictor, being present in nearly all CIN-low lesions and almost no CIN-stable tumors. MSI-low was not associated with CIN, any specific mutation, a mutational signature, or clinicopathologic characteristic. CONCLUSIONS: Overall, the components of the stepwise model (APC, K-ras, and p53 mutations, plus 18q LOH) tended to co-occur randomly. We propose an updated version of this model comprising 4 pathways of CRC pathogenesis, on the basis of 5q/18q deletions, MSI (high/low), and CIN (high/low/stable).     

Detailed deletion mapping of loss of heterozygosity on 22q13 in sporadic colorectal cancer

AIM: Both development and progression of malignancies occur as a multistep process, requiring the activation of oncogenes and the inactivation of several tumor suppressor genes. The loss of heterozygosity (LOH) of tumor suppressor genes is believed to play a key role in carcinogenesis of colorectal cancer (CRC). In this study, we analyzed the LOH of seven loci on chromosome 22q13 in an effort to identify candidate tumor suppressor genes involved in colorectal carcinogenesis. METHODS: Matched tumor and normal tissue DNA were analyzed by PCR using fluorescence-labeled polymorphic microsatellite markers in 83 CRC patients. PCR products were eletrophoresed and LOH was determined by calculating the peak height acquired through computer software. Comparisons between LOH frequency and clinicopathological features were performed by x2 test. P<0.05 was considered as statistical significance. RESULTS: The average LOH frequency of chromosome 22q13 was 28.38%. The highest LOH frequency was 64.71% on D22S1160 locus, and the lowest was 21.43% on D22S1141 locus. We detected two obvious minimal deletion regions: one between markers D22S1171 and D22S274, the other flanked by markers D22S1160 and D22S1149, each about 2.7 and 1.8 cm, respectively. None had lost in all informative loci. LOH frequency on D22S1171 is 50% on distal colon, which was higher than that on proximal one (P= 0.020); on D22S114 locus, none LOH event occurred in patients with liver metastasis, whilst 46.94% occurred in patients without liver metastasis (P= 0.008); on D22S1160 locus, LOH frequency in lymph nodes metastasis patients was 83.33%, which was much higher than 43.75% without lymph nodes metastasis ones (P= 0.016). There was no statistical significance between clinicopathological features and other loci. CONCLUSION: This study provides evidence of two minimal deletion regions, which may harbor putative tumor suppressor genes related to progression and metastasis in sporadic colorectal carcinoma on chromosome 22q13.     

Tumor suppress genes screening analysis on 4q in sporadic colorectal carcinoma

AIM： To search candidate tumor suppressor genes （TSGs） on chromosome 4q through detecting high loss of heterozygosity （LOH） regions in sporadic colorectal carcinoma in Chinese patients. METHODS： Thirteen fluorescent labeled polymorphic microsatellite markers were analyzed in 83 cases of colorectal carcinoma and matched normal tissue DNA by polymerase chain reaction （PCR）. PCR products were eletrophoresed on an ABI 377 DNA sequencer. Genescan 3.7 and Genotype 3.7 software were used for LOH scanning and analysis. Comparison between LOH frequency and clinicopathological factors were performed by χ^2 test.
RESULTS： Data were collected on all informative loci. The average LOH frequency on 4q was 28.56%, The D4S2915 locus showed highest LOH frequency （36.17%）. Two obvious deletion regions were detected： one between D4S3000 and D4S2915 locus （4q12-21.1）, another flanked by D4S407 and D4S2939 locus （4q25-31.1）. None case showed complete deletion of 4q, most cases displayed interstitial deletion pattern solely. Furthermore, compared with clinicopathological features, a significant relationship was observed between LOH frequencies on D4S3018 locus. In tumors larger than 5 cm in diameter, LOH frequency was significantly higher than tumors that were less than 5 cm （56% vs 13.79%, P = 0.01）. On D4S1534 locus, LOH was significantly associated with liver metastasis （80% vs 17.25%, P = 0.012）. No relationship was detected on other locus compared with clinicopathologial features.
CONCLUSION： By high resolution deletion mapping, two high frequency regions of LOH （4q12-21.1 and 4q25-31.1） were detected, which may contribute to locate TSGs on chromosome 4q involved in carcinogenesis and progression of sporadic colorectal carcinoma.     

Mutation and methylation of hMLH1 in gastric carcinomas with microsatellite instability

AIM: To appraise the correlation of mutation and methylation of hMSH1 with microsatellite instability (MSI) in gastric cancers.METHODS: Mutation of hMLH1 was detected by Twodimensional electrophoresis (Two-D) and DNA sequencing;Methylation of hMLH1 promoter was measured with methylation-specific PCR; MSI was analyzed by PCR-based methods.RESULTS: Sixty-eight cases of sporadic gastric carcinoma were studied for mutation and methylation of hMLH1 promoter and MSI. Three mutations were found, two of them were caused by a single bp substitution and one was caused by a 2 bp substitution, which displayed similar Two-D band pattern.Methylation of hMLH1 promoter was detected in 11(16.2%)gastric cancer. By using five MSI markers, MSI in at least one locus was detected in 17/68(25%) of the tumors analyzed.Three hMLH1 mutations were all detected in MSI-H (≥2 loci,n=8), but no mutation was found in MSI-L (only one locus,n=9) or MSS (tumor lacking MSI or stable, n=51). Methylation frequency of hMLH1 in MSI-H (87.5%, 7/8) was significantly higher than that in MSI-L (11.1%, 1/9) or MSS (5.9%, 3/51)(P&lt;0.01-0.001), but no difference was found between MSI-L and MSS (P&gt;0.05).CONCLUSION: Both mutation and methylation of hMLH1 are involved in the MSI pathway but not related to the LOH pathway in gastric carcinogenesis.     

Molecular characteristics of serrated adenomas of the colorectum

BACKGROUND: Serrated adenomas (SAs) of the colorectum combine architectural features of hyperplastic polyps and cytological features of classical adenomas. Molecular studies comparing SAs and classical adenomas suggest that each may be a distinct entity; in particular, it has been proposed that microsatellite instability (MSI) distinguishes SAs from classical adenomas and that SAs and the colorectal cancers arising from them develop along a pathway driven by low level microsatellite instability (MSI-L). AIMS: To define the molecular characteristics of SAs of the colorectum. MATERIALS AND METHODS: We analysed 39 SAs from 27 patients, including eight SAs from patients with familial adenomatous polyposis (FAP). We screened these polyps for selected molecular changes, including loss of heterozygosity (LOH) close to APC (5q21) and CRAC1 (15q13-q22), MSI, and mutations of K-ras, APC, p53, and beta-catenin. Expression patterns of beta-catenin, p53, MLH1, MSH2, E-cadherin, and O(6)-methylguanine DNA methyltransferase (MGMT) were assessed by immunohistochemistry. Comparative genomic hybridisation was performed on several polyps. RESULTS: MSI was rare (<5% cases) and there was no loss of expression of mismatch repair proteins. Wnt pathway abnormalities (APC mutation/LOH, beta-catenin mutation/nuclear expression) occurred in 11 SAs, including 6/31 (19%) non-FAP tumours. CRAC1 LOH occurred in 23% of tumours. K-ras mutations and p53 mutations/overexpression were found in 15% and 8% of SAs, respectively. Loss of MGMT expression occurred in 18% of polyps and showed a borderline association with K-ras mutations. Aberrant E-cadherin expression was found in seven polyps. Comparative genomic hybridisation detected no gains or deletions of chromosomal material. CONCLUSIONS: The serrated pathway of colorectal tumorigenesis appears to be heterogeneous. In common with classical adenomas, some SAs develop along pathways involving changes in APC/beta-catenin. SAs rarely show MSI or any evidence of chromosomal-scale genetic instability. K-ras mutations may however be less common in SAs than in classical adenomas. Some SAs may harbour changes in the CRAC1 gene. Changes in known genes do not account for the growth of the majority of SAs.