Mutational and expressional analysis of SMC2 gene in gastric and colorectal cancers with microsatellite instability

Structural maintenance of chromosomes 2 (SMC2) gene encodes condensin complexes that are required for proper chromosome segregation and maintenance of chromosomal stability. Although cells with defective chromosome segregation become aneuploid and are prone to harbor chromosome instability, pathologic implications of SMC2 gene alterations are largely unknown. In a public database, we found that SMC2 gene had mononucleotide repeats that could be mutated in cancers with microsatellite instability (MSI). In this study, we analyzed these repeats in 32 gastric cancers (GC) with high MSI (MSI‐H), 59 GC with low MSI (MSI‐L)/stable MSI (MSS), 43 colorectal cancers (CRC) with MSI‐H and 60 CRC with MSI‐L/MSS by single‐strand conformation polymorphism (SSCP) and DNA sequencing. We also analyzed SMC2 protein expression in GC and CRC tissues using immunohistochemistry. We found SMC2 frameshift mutations in two GC and two CRC that would result in truncation of SMC2. The mutations were detected exclusively in MSI‐H cancers, but not in MSI‐L/MSS cancers. Loss of SMC2 expression was observed in 22% of GC and 25% of CRC. Of note, all of the cancers with SMC2 frameshift mutations displayed loss of SMC2 expression. Also, both GC and CRC with MSI‐H had significantly higher incidences in SMC2 frameshift mutations and loss of SMC2 expression than those with MSI‐L/MSS. Our data indicate that SMC2 gene is altered by both frameshift mutation and loss of expression in GC and CRC with MSI‐H, and suggest that SMC2 gene alterations might be involved in pathogenesis of these cancers.     

DAB2IP with tumor-inhibiting activities exhibits frameshift mutations in gastrointestinal cancers

A scaffold protein DAB2 and its interaction partner DAB2IP have putative tumor suppressor gene (TSG) functions. Previous studies identified that both DAB2 and DAB2IP genes were inactivated by promoter hypermethylation in human cancers, but their mutational alterations in cancers remain largely unknown. The aim of our study was to find whether DAB2 and DAB2IP were mutated in gastric (GCs) and colorectal cancers (CRCs) by DNA sequencing. Both DAB2 and DAB2IP have mononucleotide repeats in their coding sequence that could be mutation targets in high microsatellite instability (MSI-H) cancers. We analyzed GC and CRC tissues and found that 8 of 34 GCs (23.5%) and 15 of 79 CRCs (20.0%) with MSI-H harbored DAB2IP frameshift mutations. DAB2 frameshift mutations were found in 2 of 79 CRCs (2.5%) with MSI-H. These mutations were not detected in microsatellite stable (MSS) cancers. We also found intratumoral heterogeneity (ITH) of DAB2IP frameshift mutations in 7 of 16 CRCs (43.8%). Loss of DAB2IP protein expression was found in approximately 20% of GCs and CRCs irrespective of MSI and DAB2IP frameshift mutation status. Our study shows that the TSG DAB2IP harbored frameshift mutations and ITH as well as expression loss. Together these tumor alterations might play a role in tumorigenesis of GC and CRC with MSI-H by down-regulating the tumor-inhibiting activities of DAB2IP.     

Accumulated frameshift mutations at coding nucleotide repeats during the progression of gastric carcinoma with microsatellite instability.

Microsatellite instability (MSI) and frameshift mutations in genes containing nucleotide repeats have been reported in a subset of gastric carcinomas, but the mutational profiles in precancerous lesions have not been characterized. To characterize the genetic events during gastric carcinogenesis, we analyzed DNA from 56 gastric adenomas and 167 gastric carcinomas for MSI using five microsatellite markers and for frameshift mutations at coding nucleotide repeats of the type II transforming growth factor beta receptor, BAX, hMSH3, hMSH6, IGF II receptor, and E2F-4 genes. On the basis of the number of markers displaying instability per tumor, the tumors were divided into three groups: those with two or more of the five markers showing instability (high MSI [MSI-H]), those with one of the five markers showing instability (low MSI [MSI-L]), and those with no instability. MSI-H was found in 8 adenomas (14%) and 19 carcinomas (11%), and MSI-L was found in 8 adenomas (14%) and 9 carcinomas (5%). These groups were tested for correlations with several clinicopathologic parameters. MSI-H gastric adenomas were related to the high histologic grade of composing dysplastic glands (p = 0.004), and MSI-H gastric carcinomas were associated with exophytic tumor growth (p = 0.005). We found 48 frameshift mutations at coding nucleotide repeats of the six genes, and all mutations except one were found in MSI-H gastric tumors. Only one of the 17 MSI-L tumors showed frameshift mutations at coding nucleotide repeats of the transforming growth factor beta receptor II gene. Compared with MSI-H gastric carcinomas, MSI-H adenomas had no mutations in the hMSH6 and the IGF II receptor genes, less frequent mutations in the transforming growth factor beta receptor II (38% versus 63%), BAX (13% versus 37%), and hMSH3 (13% versus 37%) genes, and more frequent mutations in the E2F-4 (50% versus 37%) gene. Our findings suggest that MSI and E2F-4 mutations are early genetic events and that mutations of the other five genes are accumulated during the progression of gastric carcinomas with MSI.     

Mutational analysis of mononucleotide repeats in dual specificity tyrosine phosphatase genes in gastric and colon carcinomas with microsatellite instability

Song SY, Kang MR, Yoo NJ, Lee SH. Mutational analysis of mononucleotide repeats in dual specificity tyrosine phosphatase genes in gastric and colon carcinomas with microsatellite instability. APMIS 2010; 118: 389–93. Coordinated protein phosphorylation and dephosphorylation are crucial in the regulation of cell signaling, and disruption of the coordination is known to play important roles in cancer development. Recent reports revealed that classical protein tyrosine phosphatase (PTP)‐encoded genes are somatically mutated in human colorectal cancer. However, data on dual specificity phosphatase (DPTP) gene mutations in human cancers are lacking. By analyzing a public genomic database, we found that five DPTP genes, CDC14A, MTM1, MTMR3, SSH1, and SSH2, have mononucleotide repeats in their coding DNA sequences. To see whether these genes are mutated in cancers with microsatellite instability (MSI), we analyzed the mononucleotide repeats in 26 gastric cancers (GC) with MSI (MSI‐H), 12 GC with low MSI (MSI‐L), 45 GC with stable MSI (MSS), 33 colorectal cancers (CRC) with MSI‐H, 14 CRC with MSI‐L, and 45 CRC with MSS by single‐strand conformation polymorphism (SSCP). We found CDC14A and MTMR3 mutations in five and one cancer (s), respectively. These mutations were detected in MSI‐H cancers, but not in MSI‐L or MSS cancers. The GC and CRC with MSI‐H harbored the mutations in 15% and 6%, respectively. The CDC14A and MTMR3 mutations detected in the GC and CRC were deletion or duplication mutations of one base in the nucleotide repeats that would result in premature stops of the amino acid syntheses. Our data show that frameshift mutations of DPTP genes in MSI‐H cancers occur at moderate frequencies. The data suggested that alterations in the CDC14A and MTMR3 genes may play a role in the development of GC and CRC with MSI‐H by deregulating phosphatase functions possibly together with mutations of classical PTP genes.     

Distinct clinicopathologic and genetic profiles in sporadic gastric cancer with different mutator phenotypes

A subset of sporadic gastric cancers (GC) exhibits microsatellite instability (MSI). To define the precise role of MSI in GC, a total of 100 patients with sporadic GC were classified into three groups, i.e., high-frequency MSI (MSI-H), low-frequency MSI (MSI-L), and microsatellite stable (MSS), based on 10 microsatellite markers. Mutational analyses of TGFbetaRII, IGFIIR, BAX, MSH3, MSH6, E2F4, MSH2, MLH1, and TP53 genes, and methylation and protein expression of MLH1 and MSH2 were performed and correlated. Twenty-seven percent of GC showed MSI at least in one locus and could be further graded as MSI-H (14%) and MSI-L (13%). No clinicopathologic difference was noted between GC with MSI-L and MSS. Compared with GC with MSI-L or MSS, GC with MSI-H had a significantly higher frequency of antral location, intestinal subtype, H. pylori seropositivity, but a lower incidence of lymph node metastasis, and displayed a higher frequency of frameshift mutations of TGFbetaRII, IGFIIR, BAX, MSH3, and E2F4 genes but a lower incidence of TP53 mutations. Furthermore, hypermethylation of the MLH1 promoter was responsible for the loss of protein function in 13 of 14 MSI-H tumors. It was concluded that a specific phenotype and a distinct profile of genetic alterations exist in MSI-H GC. We speculate that epigenetic inactivation of MLH1 by methylation plays a crucial role in initiating such a pathway of carcinogenesis. In contrast, GCs with MSS and MSI-L exhibit clinicopathologic features that are distinct from MSI-H tumors and have a higher frequency of TP53 mutations, suggesting that they may evolve through an entirely different pathway.     

Frameshift mutations of human gastrin receptor gene (hGARE) in gastrointestinal cancers with microsatellite instability

Gastrointestinal tumors with DNA mismatch repair (MMR) defects show microsatellite instability (MSI) and harbor frameshift mutations in coding mononucleotide repeats of cancer-related genes (targets). We assessed MSI status in 233 sporadic gastrointestinal tumors. We classified as MSI-H (high-frequency microsatellite instability) 15 (10%) of 150 colorectal cancers and 13 (16%) of 83 gastric cancers. We searched for frameshift mutations in a coding poly(T)(8) tract within the gastrin receptor gene (hGARE), which has a potential role in gastrointestinal carcinogenesis. To this purpose, we screened 43 unstable tumors (including 15 hereditary nonpolyposis colorectal cancer cases previously classified as MSI-H), 98 stable tumors, as well as 3 MMR-deficient and 4 MMR-proficient gastrointestinal cancer cell lines. We found mutations in 8 (19%) of the 43 MSI-H tumors but in none of the 98 stable cancers. hGARE mutation frequency was similar in gastric (23%) and colorectal cancers, including sporadic (13%) and hereditary (20%) cases. All mutated tumors proved to harbor frameshift mutations in other cancer-related genes that are considered as targets in MSI tumorigenesis. The MMR-deficient and gastrin-sensitive LoVo colorectal cancer cells also showed a hGARE heterozygous frameshift mutation, but expressed only the mutated allele. All detected mutations can be predicted to generate a truncated protein carrying amino acid changes. On the basis of genetic findings, we propose hGARE as a new candidate target gene in MSI tumorigenesis. Functional studies are warranted to elucidate the mechanism by which the hGARE mutation might contribute to gastrointestinal carcinogenesis.     

Laminin gene LAMB4 is somatically mutated and expressionally altered in gastric and colorectal cancers

Laminins are important in tumor invasion and metastasis as well as in maintenance of normal epithelial cell structures. However, mutation status of laminin chain‐encoding genes remains unknown in cancers. Aim of this study was to explore whether laminin chain genes are mutated and expressionally altered in gastric (GC) and colorectal cancers (CRC). In a public database, we found that laminin chain genes LAMA1, LAMA3, LAMB1 and LAMB4 had mononucleotide repeats in the coding sequences that might be mutation targets in the cancers with microsatellite instability (MSI). We analyzed the genes in 88 GC and 139 CRC [high MSI (MSI‐H) or stable MSI/low MSI (MSS/MSI‐L)] by single strand conformation polymorphism analysis and DNA sequencing. In the present study, we found LAMB4 (11.8% of GC and 7.6% of CRC with MSI‐H), LAMA3 (2.9% of GC and 2.5 of CRC with MSI‐H), LAMA1 (5.9% of GC with MSI‐H) and LAMB1 frameshift mutations (1.3% of CRC with MSI‐H). These mutations were not found in MSS/MSI‐L (0/114). We also analyzed LAMB4 expression in GC and CRC by immunohistochemistry. Loss of LAMB4 expression was identified in 17–32% of the GC and CRC. Of note, the loss expression was more common in the cancers with LAMB4 mutation or those with MSI‐H. Our data show that frameshift mutations of LAMA1, LAMA3, LAMB1 and LAMB4, and loss of LAMB4 may be features of GC and CRC with MSI‐H.     

Histologic features distinguish microsatellite-high from microsatellite-low and microsatellite-stable colorectal carcinomas, but do not differentiate germline mutations from methylation of the MLH1 promoter

The detection of microsatellite-unstable (microsatellite instability [MSI]) colorectal carcinomas (CRCs) has prognostic value and can help screen for Lynch syndrome. We determined which histologic features are associated with MSI status and presence of germline mutation and/or methylation of MLH1 promoter. Patients diagnosed with CRC were offered participation in the Columbus-area hereditary nonpolyposis colorectal cancer syndrome study regardless of age or family history. Tumors were evaluated for MSI using a modified Bethesda panel of microsatellite markers. Methylation status of the MLH1 promoter was evaluated by methylation-specific polymerase chain reaction and bisulfite PCR followed by restriction digestion of tumor DNA. All patients with microsatellite-unstable tumors underwent mutation analysis of the MLH1, MSH2, and MSH6 genes by full sequencing of genomic DNA and by multiplex ligation probe assay of MLH1 and MSH2. Histologic end points were tumor type, grade, percentage of mucin, border, and lymphoid host response. Of the 482 CRCs, 87 were MSI with 69 MSI high (MSI-H), 18 MSI low (MSI-L), and 395 microsatellite stable (MSS). Of 87 MSI tumors, 12 had germline mutations and 34 had methylation of the MLH1 promoter. Younger age, but not histologic features, was significantly associated with a germline mutation. Percentage of mucin, histologic type, grade, and lymphoid host response differed significantly between MSI-H when compared with MSI-L or MSS. No difference was found between MSI-L versus MSS. Histologic features are associated with MSI-H CRC and are helpful to differentiate MSI-H from MSI-L and MSS. These features are not useful to distinguish MSI-L from MSS carcinomas, and those with a deleterious germline hereditary nonpolyposis colorectal cancer syndrome mutation from those with methylation of the MLH1 promoter region.     

RAS突变和微卫星不稳定与Ⅲ期结直肠癌患者临床病理特征及预后的相关性分析

目的:检测Ⅲ期结直肠癌患者中RAS和BRAF基因突变以及微卫星不稳定(microsatellite instability,MSI)状态,并分析其临床病理关系及预后。方法:收集2010~2015年广东省人民医院共281例经病理学证实的Ⅲ期结直肠癌组织标本,采用PCR-Sanger测序法和免疫组织化学法对石蜡切片进行分析,检测RAS/BRAF基因突变和MSI状态,并探讨其与结直肠癌临床病理特征和预后的关系。结果:281例患者中,RAS/BRAF突变率为48.4%(136/281),其中KRAS突变率最高(116/281,41.3%)。RAS/BRAF基因突变与癌胚抗原水平密切相关(P0.05)。免疫组织化学法检测到高度MSI(MSI-H)患者18例(6.4%),MSI-H状态在淋巴结转移N2b期患者中更为常见(P0.05)。BRAF基因在MSI-H肿瘤中的突变率较高(P0.01)。RAS/BRAF野生型或者MSI-H患者的总生存期和无进展生存期均明显高于突变型或低度MSI/微卫星稳定患者。结论:RAS/BRAF突变和MSI检测有助于结直肠癌生物学行为分析和患者预后判断。     

Frameshift mutations of Wnt pathway genes AXIN2 and TCF7L2 in gastric carcinomas with high microsatellite instability

Frameshift mutations of genes with mononucleotide repeats are features of colorectal and gastric cancers with microsatellite instability (MSI). Deregulation of Wnt pathway is involved in the mechanisms of cancer development, and mutations of the Wnt-pathway genes have frequently been detected in cancers, indicating somatic mutations are important deregulation mechanisms of the Wnt signaling in cancer development. Both AXIN2 and TCF7L2 genes in the Wnt pathway possess mononucleotide repeats in their coding sequences and are considered as candidate tumor suppressor genes. The aim of this study was to see whether AXIN2 and TCF7L2 are altered by frameshift mutations in gastric carcinomas with MSI. For this, we analyzed human AXIN2 exon 8 and TCF7L2 exon 14 in 32 gastric carcinomas with high MSI, 13 gastric carcinomas with low MSI, and 47 gastric carcinomas without MSI by a single-strand conformation polymorphism analysis. Overall, we detected 9 AXIN2 and 6 TCF7L2 frameshift mutations in the mononucleotide repeats in the cancers with MSH-H, and all of them were found in MSH-H cancers (AXIN2, 28.1%; TCF7L2, 18.8%). Of the 32 high MSI cancers, 13 cancers (40.6%) harbored at least one of AXIN2 and TCF7L2 mutation, whereas 19 cancers (59.4%) harbored neither. The present data indicate that frameshift mutations in both AXIN2 and TCF7L2 genes are common in gastric carcinomas with high MSI and suggest that these mutations may contribute to development of gastric cancers with high MSI by deregulating the Wnt signaling in the affected cancer cells.     

Significance of mutations in TGFBR2 and BAX in neoplastic progression and patient outcome in sporadic colorectal tumors with high-frequency microsatellite instability

The mutator pathway implied in the development of colorectal cancer is characterized by microsatellite instability (MSI), which is determined by alterations of mismatch repair (MMR) genes. Defects in MMR genes affect repetitive DNA tracts interspersed mostly between coding sequences, and therefore it cannot be expected that they play a role during tumor progression. Genes containing repetitive sequences within their coding regions could be targets for MSI tumorigenesis, but this does not necessarily imply a causal role for the affected gene, because most are probably passenger mutations. We analyzed MSI and TGFBR2 and BAX frameshift mutations to further clarify the relationships between inactivation of the two genes and genomic instability in sporadic colorectal cancer (CRC), and to address how mutations in these genes influence the development of tumors and, eventually, patient outcome. One hundred and fifty-five patients with sporadic CRC were classified according to their MSI status. Frameshift mutations in the two genes were recurrent in high-frequency MSI (MSI-H) tumors, but these tended to be more common in poorly differentiated tumors. A high rate of mutations of TGFBR2 was found in tumors at Dukes' B stage, showing a greater extent of vascular invasion. Finally, in MSI-H tumors, mutations of either gene were associated with a significant decrease in survival. Our results contribute to the understanding of how the TGFBR2 and BAX gene mutations contribute to tumor progression in the mutator phenotype pathway for MSI colorectal cancers.     

RIS1, a gene with trinucleotide repeats, is a target in the mutator pathway of colorectal carcinogenesis

Microsatellite instability (MSI) due to mismatch repair system (MMR) alterations characterizes the mutator pathway implied in colorectal cancer development. In the present study, we have analyzed the gene RIS1 (Ras-induced senescence 1) in relation to loss of heterozygosity (LOH) and its frameshift mutations for an imperfect trinucleotide repeat (GCN) located at the 3'-OH end. Additionally, we have compared the status of RIS1 with a number of genetic and clinicopathological variables. RIS1 did not display LOH in any informative tumor of our series, but exhibited frameshift mutations in a high percentage (43.8%) of high-frequency MSI tumors (MSI-H), and its alteration was correlated with mutations in two target genes: BAX and TGFBR2. Moreover, mutations in RIS1 in MSI-H tumors correlated with the epigenetic silencing of MLH1 (P = 0.04). Finally, RIS1 seemed to be functionally involved in tumor development, as low-frequency MSI tumors (MSI-L) with RIS1 mutated usually were associated with a worse prognosis: 83% of them developed metastasis, and no patient with MSI-L tumor and RIS1 mutated (35.3% of MSI-L) survived >25 months after surgery (log rank P < 0.001). All these results indicate, according to the Bethesda criteria, that RIS1 is a target gene in the mutator pathway.     

Frameshift mutations of autophagy‐related genes ATG2B,ATG5, ATG9B and ATG12 in gastric and colorectal cancers with microsatellite instability

Mounting evidence indicates that alterations of autophagy processes are directly involved in the development of many human diseases, including cancers. Autophagy‐related gene (ATG) products are main players in the autophagy process. In humans there are 16 known ATG genes, of which four (ATG2B, ATG5, ATG9B and ATG12) have mononucleotide repeats with seven or more nucleotides. Frameshift mutations of genes with mononucleotide repeats are features of cancers with microsatellite instability (MSI). It is not known whether ATG genes with mononucleotide repeats are altered by frameshift mutations in gastric and colorectal carcinomas with MSI. For this, we analysed the mononecleotide repeats in ATG2B, ATG5, ATG9B and ATG12 in 32 gastric carcinomas with high MSI (MSI‐H), 13 gastric carcinomas with low MSI (MSI‐L), 43 colorectal carcinomas with MSI‐H and 15 colorectal carcinomas with MSI‐L by a single‐strand conformation polymorphism (SSCP) analysis. We found ATG2B, ATG5, ATG9B and ATG12 mutations in 10, 2, 13 and 0 cancers, respectively. The mutations were detected in MSI‐H cancers but not in MSI‐L cancers. Gastric and colorectal cancers with MSI‐H harboured one or more ATG mutations in 28.1% and 27.9%, respectively. Our data indicate that frameshift mutations in ATG genes with mononucleotide repeats are common in gastric and colorectal carcinomas with MSI‐H, and suggest that these mutations may contribute to cancer development by deregulating the autophagy process. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Inactivating mutations of class II transactivator (CIITA) gene in gastric and colorectal cancers

Expression of MHC class II, which is important against cancer immunity, depends on the transcactivator CIITA. These data suggest a possibility that CIITA gene might be inactivated in cancers. In this study, we studied inactivating mutation status of CIITA gene in gastric (GC) and colorectal (CRC) cancers by analyzing the C7 repeat in the CIITA (exon 11) gene. We found frameshift mutations in 3 GCs and 6 CRCs in cancers with high microsatellite instability (MSI-H) (9/113), but not in those with microsatellite-stable (MSS) (0/90) (P = 0.004). They were all deletion or duplication of one base in the C7 repeat that would result in truncation of amino acid synthesis. Immune therapy is now a major option in cancer therapy and our results on the genetic alterations of MHC II-related CTIITA in MSH-H GC and CRC might provide useful information for the treatment of MSI-H cancers.     

MUC gene abnormalities in sporadic and hereditary mucinous colon cancers with microsatellite instability

Aim of this study was verifying whether mucin producing colon cancers (CRCs) could develop through a molecular pathway involving microsatellite instability (MSI) and MUC gene alterations. Out of 49 CRCs expressing variable amounts of mucin, 22 (44.9%) were MSI-H and 5 (10.2%) were MSI-L. MUC genes were analyzed by Southern blotting and extra bands were evident in the Variable Number Tandem Repetition (VNTR) regions of MUC2 (5 cases) and MUC5AC (2 cases), but not MUC1 and MUC4 genes. Since the somatic VNTR abnormalities were detected in 6 MSI-H and in 1 MSI-L tumors, they seem to be peculiar of mismatch repair defective CRCs. Our finding suggests that alteration and/or loss of structurally normal MUC genes may be an important step in the neoplastic molecular pathway of a subset of CRCs and that mutations involving VNTR repetitive sequences may exist in MSI tumors as a direct and/or indirect consequence of an inefficient MMR system.     

Colorectal carcinogenesis: MSI-H versus MSI-L

Microsatellite instability (MSI) is a well-recognized phenomenon that is classically a feature of tumors in the hereditary non-polyposis colorectal syndrome. Ten to 15% of sporadic colorectal cancers, however, will have MSI. Microsatellite unstable tumors can be divided into two distinct MSI phenotypes: MSI-high (MSI-H) and MSI-low (MSI-L). MSI sporadic colorectal cancers with a high level of MSI (MSI-H) form a well defined group with distinct clinicopathologic features characterized by an overall better long-term prognosis. These sporadic MSI-H colorectal tumors most often arise from the epigenetic silencing of the mismatch repair gene MLH1. In contrast, MSI-L colorectal tumors have not been shown to differ in their clinicopathologic features or in most molecular features from microsatellite stable (MSS) tumors. Unlike MSI-H tumors, MSI-L tumors appear to arise through the chromosomal instability carcinogenesis pathway, similar to MSS tumors. Some groups have reported more frequent mutations in K-ras and in the methylation of methylguanine transferase in MSI-L tumors, but others have questioned these findings. Therefore, although the use of the MSI-L category is widespread, there continues to be some debate as to whether a discrete MSI-L group truly exists. Rather, it has been suggested that MSI-L tumors differ quantitatively from MSS tumors but do not differ qualitatively. Future studies will need to evaluate the specific mutations in non-MSI-H tumors in an attempt to sub-classify MSI-L tumors with regard to MSS tumors so that subtle differences between these two sub-groups can be identified.     

Classification of colorectal cancer based on correlation of clinical, morphological and molecular features

Over the last 20 years it has become clear that colorectal cancer (CRC) evolves through multiple pathways. These pathways may be defined on the basis of two molecular features: (i) DNA microsatellite instability (MSI) status stratified as MSI-high (MSI-H), MSI-low (MSI-L) and MS stable (MSS), and (ii) CpG island methylator phenotype (CIMP) stratified as CIMP-high, CIMP-low and CIMP-negative (CIMP-neg). In this review the morphological correlates of five molecular subtypes are outlined: Type 1 (CIMP-high/MSI-H/BRAF mutation), Type 2 (CIMP-high/MSI-L or MSS/BRAF mutation), Type 3 (CIMP-low/MSS or MSI-L/KRAS mutation), Type 4 (CIMP-neg/MSS) and Type 5 or Lynch syndrome (CIMP-neg/MSI-H). The molecular pathways are determined at an early evolutionary stage and are fully established within precancerous lesions. Serrated polyps are the precursors of Types 1 and 2 CRC, whereas Types 4 and 5 evolve through the adenoma-carcinoma sequence. Type 3 CRC may arise within either type of polyp. Types 1 and 4 are conceived as having few, if any, molecular overlaps with each other, whereas Types 2, 3 and 5 combine the molecular features of Types 1 and 4 in different ways. This approach to the classification of CRC should accelerate understanding of causation and will impact on clinical management in the areas of both prevention and treatment.     

MSI-L gastric carcinomas share the hMLH1 methylation status of MSI-H carcinomas but not their clinicopathological profile

Sporadic gastric carcinomas (SGC) with microsatellite instability (MSI) exhibit mutations in target genes and display a particular clinicopathological profile. In SGC the MSI phenotype has been associated with hMLH1 promoter hypermethylation. Fifty-seven SGC, classified as high-frequency MSI (MSI-H), low-frequency MSI (MSI-L), and microsatellite stable (MSS), were analyzed for hMLH1 promoter methylation status and clinicopathological features. hMLH1 mutations and hMLH1 expression, as well as target gene mutations, were also evaluated. Our aims were to characterize the molecular and clinicopathological features of SGC, with and without hMLH1 promoter hypermethylation, and to compare the molecular and clinicopathological features of MSI-L, MSI-H, and MSS tumors in an attempt to clarify the place of MSI-L tumors in the mismatch repair (MMR) pathway. Hypermethylation of hMLH1 promoter occurred in 27 of 57 SGC (47.3%) and was significantly associated with MSI status, target gene mutations, and expansive pattern of growth of the tumors. Seventy-five percent of the MSI-H and 50% of MSI-L carcinomas showed hypermethylation (Met+) of hMLH1 in contrast to 0% in MSS carcinomas. No hMLH1 expression was observed in MSI-L/Met+ and MSI-H/Met+ cases. MSS and MSI-L tumors share the same clinicopathological profile regardless of the methylation status of the latter and are distinct from MSI-H tumors. We conclude that mutations in target genes, more than hypermethylation or absence of expression of hMLH1, are the link between MSI status and most of the clinicopathological features of SGC.     

Somatic mutations and losses of expression of microRNA regulation‐related genes AGO2 and TNRC6A in gastric and colorectal cancers

Mounting evidence indicates that deregulation of microRNAs (miRNAs) are involved in development of many human diseases, including cancers. Regulation of miRNA is a complicated process and some components in the regulation are known to be altered in human cancers. Among the miRNA regulation‐related genes, we found that AGO1, AGO2, TNRC6A, TNRC6C, TARBP2 and EXPORTIN5 genes have mononucleotide repeats in their coding sequences. To see whether these genes are mutated in cancers with microsatellite instability (MSI), we analysed the mononucleotide repeats in 27 gastric cancers (GCs) with high MSI (MSI‐H), 18 GC with low MSI (MSI‐L), 45 GC with stable MSI (MSS), 41 colorectal cancers (CRCs) with MSI‐H, 14 CRCs with MSI‐L and 45 CRCs with stable MSI (MSS) by single‐strand conformation polymorphism (SSCP) analysis and DNA sequencing. We found AGO2, TNRC6A, TARBP2, TNRC6C and EXPORTIN5 mutations in 10, six, one, one and one cancer(s), respectively. They were detected in MSI‐H but not in MSI‐L or MSS cancers. The GCs and CRCs with MSI‐H harboured one or more mutations of the genes in 22% and 27%, respectively. We also analysed Ago2 and TNRC6A protein expressions in GCs and CRCs with MSI‐H. In cancers with MSI‐H, loss of Ago2 expression was observed in 40% of GCs and 35% of CRCs, while loss of TNRC6A was observed in 52% of the GCs and 54% of the CRCs. Our data indicate that frameshift mutations in AGO2 and TNRC6A and their losses of expression are common in GCs and CRCs with MSI‐H, and suggest that these alterations may contribute to the cancer development by deregulating miRNA regulation. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.