The putative tumor suppressor AIM2 is frequently affected by different genetic alterations in microsatellite unstable colon cancers

Mismatch repair (MMR) deficiency is a major mechanism of colorectal tumorigenesis that is observed in 10-15% of sporadic colorectal cancers and those associated with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. MMR deficiency leads to the accumulation of mutations mainly at short repetitive sequences termed microsatellites, constituting the high level microsatellite instability (MSI-H) phenotype. In recent years, several genes have been described that harbor microsatellites in their coding region (coding microsatellites, cMS) and are frequently affected by mutations in MMR-deficient cancers. However, evidence for a functional role of most of the known cMS-containing genes is missing, and further analyses are needed for a better understanding of MSI tumorigenesis. Here, we examined in detail alterations of the absent in melanoma 2 (AIM2) gene that shows a high frequency of cMS frameshift mutations in MSI-H colorectal, gastric, and endometrial tumors. AIM2 belongs to the HIN-200 family of interferon (IFN)-inducible proteins, its role in colon carcinogenesis, however, is unknown. Sequencing of the entire coding region of AIM2 revealed a high frequency of frameshift and missense mutations in primary MSI-H colon cancers (9/20) and cell lines (9/15). Biallelic AIM2 alterations were detected in 8 MSI-H colon tumors and cell lines. In addition, AIM2 promoter hypermethylation conferred insensitivity to IFN-gamma-induced AIM2 expression of three MSI-H colon cancer cell lines. These results demonstrate that inactivation of AIM2 by genetic and epigenetic mechanisms is frequent in MMR-deficient colorectal cancers, thus suggesting that AIM2 is a mutational target relevant for the progression of MSI-H colorectal cancers.     

Characterization of mutator pathway in younger-age-onset colorectal adenocarcinomas

The high-frequency microsatellite instability (MSI-H) phenotype, frequently identified in hereditary nonpolyposis colorectal cancer (HNPCC), also accounts for approximately 15% of sporadic colorectal cancers. Microsatellite instability (MSI) occurs from the mutational inactivation of the DNA mismatch repair genes, i.e. hMSH2 and hMLH1 in HNPCC, as well as from epigenetic inactivation of hMLH1 in sporadic colorectal tumors. The mutator pathway including microsatellite instability, hMLH1 promoter methylation, and hMSH2 and hMLH1 mutation patterns were identified in 21 sporadic colorectal adenocarcinoma patients younger than 30 yr excluding HNPCC. More than half of tumors showed MSI, with five MSI-H and six MSI-L (low-frequency microsatellite instability). Three of six MSI-H tumors showed the hMLH1 promoter methylation and did not express the hMLH1 protein. On the other hand, all MSI-L and all MSS (microsatellite stable) tumors expressed both hMSH2 and hMLH1 proteins. Two novel mutations, i.e. a missense mutation in hMLH1 and a splice-site alteration in hMSH2, were identified in two patients respectively. Although mutator pathway was implicated in younger-age-onset colorectal carcinogenesis, many tumors appeared to evolve from different genetic events other than hMSH2 and hMLH1 mutations frequently identified in HNPCC.     

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.     

Causes of microsatellite instability in colorectal tumors: implications for hereditary non-polyposis colorectal cancer screening

Microsatellite instability (MSI) analysis was performed using a "reference panel" of microsatellite markers in 345 unselected primary colorectal cancers (CRC). Thirty-five (10%) tumors were classified as high MSI (MSI-H). We identified 6 (17%) MSI-H tumors with germline mutations in mismatch repair (MMR) genes (tumors from patients with hereditary non-polyposis colorectal cancer (HNPCC) syndrome) and 29 (83%) MSI-H tumors without germline MMR mutations (sporadic MSI-H tumors). Hypermethylation of the hMLH1 promoter was found in 26/29 (90%) sporadic MSI-H tumors but only in 1/6 (17%) HNPCC tumors (P<.001). Somatic alterations were identified in both MMR genes in HNPCC tumors but mainly in the hMSH2 gene in sporadic MSI-H tumors. LOH at MMR loci was detected in 3/6 (50%) HNPCC tumors and in 4/26 (15%) informative sporadic MSI-H tumors. These results together indicate different mode of inactivation of MMR genes in sporadic MSI-H tumors versus MSI-H tumors in HNPCC patients. We therefore propose that MSI analysis of newly diagnosed primary CRC followed by methylation analysis of hMLH1 promoter in MSI-H tumors and mutational analysis of MMR genes in MSI-H tumors lacking hMLH1 promoter methylation might be an efficient molecular genetic approach for HNPCC screening.     

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.     

Somatic frameshift mutations of bone morphogenic protein receptor 2 gene in gastric and colorectal cancers with microsatellite instability

Mounting evidence exists that perturbation of bone morphogenic protein (BMP) signaling is involved in cancer development, especially in gastrointestinal cancers. However, somatic mutations of the genes encoding BMP and BMP receptors have not yet been discovered in human cancer tissues. By analyzing a public database, we found that BMP receptor 2 (BMPR2) and BMP1 genes had mononucleotide repeats in their coding sequences that could be mutation targets in cancers with microsatellite instability (MSI). In this study, we analyzed the mutation of BMPR2 and BMP1 genes in gastric (GC) and colorectal cancers (CRC) with MSI [31 GC with high MSI (MSI-H), 13 GC with low MSI (MSI-L), 38 CRC with MSI-H and 15 CRC with MSI-L] by single-strand conformation polymorphism analysis and DNA sequencing. Overall, we found seven frameshift mutations in the BMPR2 gene, but not in the BMP1 gene. The mutations were an identical deletion mutation of one base in the repeats (c.1748delA) that would result in premature stops of the amino acid synthesis (p.Asn583ThrfsX44). The BMPR2 mutations were detected in 6.5% of GC and 13.2% of CRC with MSI-H. All the cancers with the BMPR2 mutation showed loss of BMPR2 expression. Our data indicate that frameshift mutation of BMPR2 gene occurs in GC and CRC with MSI-H, and suggest that the BMPR2 mutation might contribute to cancer pathogenesis by inactivating BMPR2-mediated BMP signaling.     

Colorectal cancer with and without microsatellite instability involves different genes.

There is evidence supporting a multistep genetic model for colorectal tumorigenesis. In familial adenomatosis polyposis (FAP), the inherited defect is a mutation in the APC gene. The vast majority of all sporadic colorectal cancers also show mutations in the APC gene, and the tumorigenesis in sporadic colorectal cancer and FAP is assumed to involve the same genes. Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with germline mutations in DNA mismatch repair genes and, as a result of defective mismatch repair, microsatellite instability (MSI) is frequently seen. Tumorigenesis in HNPCC was first thought to involve mutations in the same genes as in FAP and sporadic colorectal cancer. Recently, however, an alternative pathway to development of colorectal cancer has been suggested in colorectal tumors with MSI, compared to those tumors without the MSI phenotype. We used a consecutive series of 191 sporadic colorectal cancers to find out if there were any differences between the two groups of tumors regarding the prevalence of mutations in the APC, KRAS, TP53, and TGFbetaR2 genes. As expected, 86% (19/22) of MSI-positive tumors showed a mutation in TGFbetaR2, while only one of 164 (0.6%) MSI-negative tumors did. A highly statistically significant negative association was found between MSI and alterations in APC and TP53. The MSI-positive tumors were screened for mutations in exon 3 of beta-catenin, which has been suggested to substitute for the APC mutation in the genesis of colorectal cancer, without finding mutations in any of the 22 MSI-positive tumors. The number of mutations found in KRAS was lower in MSI-positive than in MSI-negative tumors but the difference was not statistically significant. Our results strongly support the idea that carcinogenesis in MSI-positive and MSI-negative colorectal cancer develops through different pathways.     

Somatic frameshift alterations in mononucleotide repeat-containing genes in different tumor types from an HNPCC family with germline MSH2 mutation

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by a germline mutation in one of several DNA repair genes, which in the tumors is reflected as microsatellite instability (MSI). MSI+ tumors have been found to carry somatic frameshift mutations in mononucleotide repeats within the coding regions of several genes involved in growth control, apoptosis, and DNA repair, e.g., TGFBRII, BAX, IGFIIR, TCF4, MSH3, and MSH6. We have studied the occurrence of somatic frameshift alterations in these mononucleotide repeat-containing genes in 24 tumors (15 colorectal cancers, 1 colon adenoma, 4 endometrial cancers, 1 ovarian cancer, 1 gastric cancer, 1 urothelial cancer, and 1 duodenal cancer) from 14 individuals in an HNPCC family with germline hMSH2 mutation. Such somatic frameshift mutations occurred at a variable frequency; the long mononucleotide repeats that characterize intronic MSI markers were mutated in the majority of tumors, 13 of the tumors displayed alterations in the (A)(10) tract of TGFBII, eight tumors (all of gastrointestinal origin) had alterations in the (A)(9) repeat of TCF4, and one to five tumors had somatic frameshift alterations in the shorter mononucleotide repeats of IGFIIR, BAX, MSH3, and MSH6. Thus, longer mononucleotide repeats were more frequently affected by somatic frameshift mutations. The pattern of alterations varied between the tumors from different family members as well as between different tumors from the same individual. To what extent this variable pattern depends on the widespread mismatch repair deficiency induced by the underlying MSH2 mutation, or represents alternative ways whereby the tumors can achieve a tumorigenic phenotype, is unknown. We suggest, however, that the accumulation of somatic frameshifts, rather than the specific loci in which these occur, drives the development of the tumorigenic phenotype in HNPCC.     

Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings: parallel pathways of tumorigenesis.

High-level microsatellite instability (MSI-H) is demonstrated in 10 to 15% of sporadic colorectal cancers and in most cancers presenting in the inherited condition hereditary nonpolyposis colorectal cancer (HNPCC). Distinction between these categories of MSI-H cancer is of clinical importance and the aim of this study was to assess clinical, pathological, and molecular features that might be discriminatory. One hundred and twelve MSI-H colorectal cancers from families fulfilling the Bethesda criteria were compared with 57 sporadic MSI-H colorectal cancers. HNPCC cancers presented at a lower age (P < 0.001) with no sporadic MSI-H cancer being diagnosed before the age of 57 years. MSI was less extensive in HNPCC cancers with 72% microsatellite markers showing band shifts compared with 87% in sporadic tumors (P < 0.001). Absent immunostaining for hMSH2 was only found in HNPCC tumors. Methylation of hMLH1 was observed in 87% of sporadic cancers but also in 55% of HNPCC tumors that showed loss of expression of hMLH1 (P = 0.02). HNPCC cancers were more frequently characterized by aberrant beta-catenin immunostaining as evidenced by nuclear positivity (P < 0.001). Aberrant p53 immunostaining was infrequent in both groups. There were no differences with respect to 5q loss of heterozygosity or codon 12 K-ras mutation, which were infrequent in both groups. Sporadic MSI-H cancers were more frequently heterogeneous (P < 0.001), poorly differentiated (P = 0.02), mucinous (P = 0.02), and proximally located (P = 0.04) than HNPCC tumors. In sporadic MSI-H cancers, contiguous adenomas were likely to be serrated whereas traditional adenomas were dominant in HNPCC. Lymphocytic infiltration was more pronounced in HNPCC but the results did not reach statistical significance. Overall, HNPCC cancers were more like common colorectal cancer in terms of morphology and expression of beta-catenin whereas sporadic MSI-H cancers displayed features consistent with a different morphogenesis. No individual feature was discriminatory for all HNPCC cancers. However, a model based on four features was able to classify 94.5% of tumors as sporadic or HNPCC. The finding of multiple differences between sporadic and familial MSI-H colorectal cancer with respect to both genotype and phenotype is consistent with tumorigenesis through parallel evolutionary pathways and emphasizes the importance of studying the two groups separately.     

APC mutation and tumour budding in colorectal cancer

AIM: To determine the frequency of tumour budding and somatic APC mutation in a series of colorectal cancers stratified according to DNA microsatellite instability (MSI) status. Material/Methods: Ninety five colorectal cancers were genotyped for APC mutation in the mutation cluster region (exon 15) and scored for the presence of tumour budding at the invasive margin in haematoxylin and eosin stained sections. A subset was immunostained for beta catenin and p16. RESULTS: The frequency of both somatic APC mutation and tumour budding increased pari passu in cancers stratified as sporadic MSI high (MSI-H), hereditary non-polyposis colorectal cancer (HNPCC), MSI low (MSI-L), and microsatellite stable (MSS). Both budding and APC mutation were significantly less frequent in sporadic MSI-H cancers than in MSI-L or MSS cancers. Tumour buds were characterised by increased immunostaining for both beta catenin and p16. CONCLUSION: Tumour budding is associated with an adverse prognosis. The lack of budding in MSI-H colorectal cancer may account for the improved prognosis of this subset and may be explained by an intact WNT signalling pathway and/or inactivated p16(INK4a).     

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.     

Development of a fluorescent multiplex assay for detection of MSI-High tumors

Determining whether a tumor exhibits microsatellite instability (MSI) is useful in identifying patients with hereditary non-polyposis colorectal cancer and sporadic gastrointestinal cancers with defective DNA mismatch repair (MMR). The assessment of MSI status aids in establishing a clinical prognosis and may be predictive of tumor response to chemotherapy. A reference panel of five markers was suggested for MSI analysis by a National Cancer Institute (NCI) workshop in 1997 that has helped to standardize testing. But this panel of markers has limitations resulting from the inclusion of dinucleotide markers, which are less sensitive and specific for detection of tumors with MMR deficiencies compared to other types of markers that are currently available. This study demonstrates that mononucleotides are the most sensitive and specific markers for detection of tumors with defects in MMR and identifies an optimal panel of markers for detection of MSI-H tumors. A set of 266 mono-, di-, tetra- and penta-nucleotide repeat microsatellite markers were used to screen for MSI in colorectal tumors. The best markers for detection of MSI-H tumors were selected for a MSI Multiplex System, which included five mononucleotide markers: BAT-25, BAT-26, NR-21, NR-24 and MONO-27. In addition, two pentanucleotide markers were added to identify sample mix-ups and/or contamination. We classified 153 colorectal tumors using the new MSI Multiplex System and compared the results to those obtained with a panel of 10 microsatellite markers combined with immunohistochemical (IHC) analysis. We observed 99% concordance between the two methods with nearly 100% accuracy in detection of MSI-H tumors. Approximately 5% of the MSI-H tumors had normal levels of four MMR proteins and as a result would have been misclassified based solely on IHC analysis, emphasizing the importance of performing MSI testing. The new MSI Multiplex System offers several distinct advantages over other methods of MSI testing in that it is both extremely sensitive and specific and amenable to high-throughput analysis. The MSI Multiplex System meets the new recommendations proposed at the recent 2002 NCI workshop on HNPCC and MSI testing and overcomes problems inherent to the original five-marker panel. The use of a single multiplex fluorescent MSI assay reduces the time and costs involved in MSI testing with increased reliability and accuracy and thus should facilitate widespread screening for microsatellite instability in tumors of patients with gastrointestinal cancers.     

RIZ, the retinoblastoma protein interacting zinc finger gene, is mutated in genetically unstable cancers of the pancreas, stomach, and colorectum

The retinoblastoma protein interacting zinc finger (RIZ) gene is a candidate tumor suppressor gene on 1p36, a region frequently rearranged in a wide variety of human tumors. As the RIZ gene harbors several microsatellites within its coding region, it is a candidate for an inactivating mutation in microsatellite instability (MSI) mediated carcinogenesis. In this study, we examined mutations of two poly adenine tracts, A(8) and A(9), within the coding region of the RIZ gene, in MSI-high (MSI-H) primary cancers occurring in the pancreas, stomach, and colorectum. Frameshift mutations were found in one (10%) of 10 pancreatic, four (36%) of 11 gastric, and two (25%) of eight colorectal cancers. These results indicate that mutations of the RIZ gene play an important role in the pathogenesis of some MSI-H cancers.     

Differential involvement of the hypermethylator phenotype in hereditary and sporadic colorectal cancers with high-frequency microsatellite instability.

High-frequency microsatellite instability (MSI-H) due to defective DNA mismatch repair occurs in the majority of hereditary nonpolyposis colorectal cancers (HNPCCs) and in a subset of sporadic malignant tumors. Clinicopathologic and genotypic features of MSI-H colorectal tumors in HNPCC patients and those in sporadic cases are very similar but not identical. Correlation between the MSI phenotype and aberrant DNA methylation has been highlighted recently. A strong association between MSI and CpG island methylation has been well characterized in sporadic colorectal cancers with MSI-H but not in those of hereditary origin. To address the issue, we analyzed hereditary and sporadic colorectal cancers for aberrant DNA methylation of target genes using methylation-specific polymerase chain reaction. DNA methylation of the MLH1, CDKN2A, MGMT, THBS1, RARB, APC, and p14ARF genes was found in 0%, 23%, 10%, 3%, 73%, 53%, and 33% of 30 MSI-H cancers in HNPCC patients and in 80%, 55%, 23%, 23%, 58%, 35%, and 50% of 40 sporadic colorectal cancers with MSI-H, respectively. Cases showing methylation at three or more loci of six genes other than MLH1 were defined as CpG island methylator phenotype-positive (CIMP +), and 23% of HNPCC tumors and 53% of sporadic cancers with MSI-H were CIMP+ (P = 0.018). Differences in the extent of CpG island methylation, coupled with the differential involvement of several genes by methylation, in HNPCC tumors and sporadic MSI-H colorectal cancers may be associated with diverging developmental pathways in hereditary and sporadic cancers despite similar MSI-H phenotypes.     

Lack of microsatellite instability in neoplasms of ampulla of Vater

To clarify the genetic background of ampullary neoplasm, we investigated the occurrence of microsatellite instability (MSI) in 64 samples of neoplasm of the ampulla of Vater. Eight out of 22 adenomas (34.6%), nine out of 32 carcinomas (28.1%) and one metastatic lesion (10.0%) showed MSI in 1-3 of the nine dinucleotide markers; those cases are categorized into microsatellite instability-low (MSI-L). The remaining samples were stable with respect to all of the tested markers. None of the samples showed a frameshift mutation in the poly A-tract of BAT-26 or transforming growth factor-beta type II receptor, which are frequently mutated in gastric or colorectal cancers showing microsatellite instability. To confirm our finding, we stained 93 ampullary neoplasms with antibodies against the mismatch repair proteins: hMLH1 and hMSH2. All tumors were found to express mismatch repair proteins. In contrast to gastric or colorectal cancers, MSI does not play an important role in the carcinogenesis of ampullary carcinoma.     

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.     

Distinct PTEN mutational spectra in hereditary non-polyposis colon cancer syndrome-related endometrial carcinomas compared to sporadic microsatellite unstable tumors

Germline PTEN mutations cause Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome (BRR), two hamartoma-tumor syndromes with an increased risk of breast, thyroid and endometrial cancers. Somatic genetic and epigenetic inactivation of PTEN is involved in as high as 93% of sporadic endometrial carcinomas (EC), irrespective of microsatellite status, and can occur in the earliest precancers. EC is the most frequent extra-colonic cancer in patients with hereditary non-polyposis colon cancer syndrome (HNPCC), characterized by germline mutations in the mismatch repair (MMR) genes and by microsatellite instability (MSI) in component tumors. To determine whether PTEN is involved in the pathogenesis of EC arising in HNPCC cases, and whether PTEN inactivation precedes MMR deficiency, we obtained 41 ECs from 29 MLH1 or MSH2 mutation positive HNPCC families and subjected them to PTEN expression and mutation analysis. Immunohistochemical analysis revealed 68% (28/41) of the HNPCC-related ECs with absent or weak PTEN expression. The remaining 27% (11/41) of tumors had normal expression and 5% (2/41) with mixed populations showing weak/absent as well as normal expression. Mutation analysis of 20 aberrant PTEN-expressing tumors revealed that 17 (85%) harbored 18 somatic PTEN mutations. All mutations were frameshift, 10 (56%) of which involved the 6(A) tracts in exon 7 or 8. These results suggest that PTEN plays a significant pathogenic role in both HNPCC and sporadic endometrial carcinogenesis, unlike the scenarios for colorectal cancer. Furthermore, we have shown that somatic PTEN mutation, especially frameshift, is a consequence of profound MMR deficiency in HNPCC-related ECs. In contrast, among 60 previously reported MSI+ sporadic ECs with 70 somatic mutations in PTEN, 39 (56%) were frameshift, of which only eight (21%) were affecting the 6(A) tracts in exon 7 or 8 (P = 0.01), suggesting that PTEN mutations may precede MMR deficiency.     

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.     

Distinct patterns of KRAS mutations in colorectal carcinomas according to germline mismatch repair defects and hMLH1 methylation status

In sporadic colorectal tumours the BRAFV600E is associated with microsatellite instability (MSI-H) and inversely associated to KRAS mutations. Tumours from hereditary non-polyposis colorectal cancer (HNPCC) patients carrying germline mutations in hMSH2 or hMLH1 do not show BRAFV600E, however no consistent data exist regarding KRAS mutation frequency and spectrum in HNPCC tumours. We investigated KRAS in 158 HNPCC tumours from patients with germline hMLH1, hMSH2 or hMSH6 mutations, 166 MSI-H and 688 microsatellite stable (MSS) sporadic carcinomas. All tumours were characterized for MSI and 81 of 166 sporadic MSI-H colorectal cancer (CRCs) were analysed for hMLH1 promoter hypermethylation. KRAS mutations were observed in 40% of HNPCC tumours, and the mutation frequency varied upon the mismatch repair gene affected: 48% (29/61) in hMSH2, 32% (29/91) in hMLH1 and 83% (5/6) in hMSH6 (P = 0.01). KRAS mutation frequency was different between HNPCC, MSS and MSI-H CRCs (P = 0.002), and MSI-H with hMLH1 hypermethylation (P = 0.005). Furthermore, HNPCC CRCs had more G13D mutations than MSS (P < 0.0001), MSI-H (P = 0.02) or MSI-H tumours with hMLH1 hypermethylation (P = 0.03). HNPCC colorectal and sporadic MSI-H tumours without hMLH1 hypermethylation shared similar KRAS mutation frequency, in particular G13D. In conclusion, we show that depending on the genetic/epigenetic mechanism leading to MSI-H, the outcome in terms of oncogenic activation may be different, reinforcing the idea that HNPCC, sporadic MSI-H (depending on the hMLH1 status) and MSS CRCs, may target distinct kinases within the RAS/RAF/MAPK pathway.     

PTEN mutational spectra,expression levels,and subcellular localization in microsatellite stable and unstable colorectal cancers

PTEN on 10q23.3 encodes a dual-specificity phosphatase that negatively regulates the phosphoinositol-3-kinase/Akt pathway and mediates cell-cycle arrest and apoptosis. Germline PTEN mutations cause Cowden syndrome and a range of several different hamartoma-tumor syndromes. Hereditary nonpolyposis colon cancer (HNPCC) syndrome is characterized by germline mutations in the mismatch repair (MMR) genes and by microsatellite instability (MSI) in component tumors. Although both colorectal carcinoma and endometrial carcinoma are the most frequent component cancers in HNPCC, only endometrial cancer has been shown to be a minor component of Cowden syndrome. We have demonstrated that somatic inactivation of PTEN is involved in both sporadic endometrial cancers and HNPCC-related endometrial cancers but with different mutational spectra and different relationships to MSI. In the current study, we sought to determine the relationship of PTEN mutation, 10q23 loss of heterozygosity, PTEN expression, and MSI status in colorectal cancers (CRCs). Among 11 HNPCC CRCs, 32 MSI+ sporadic cancers, and 39 MSI- tumors, loss of heterozygosity at 10q23.3 was found in 0%, 8%, and 19%, respectively. Somatic mutations were found in 18% (2 of 11) of the HNPCC CRCs and 13% (4 of 32) of the MSI+ sporadic tumors, but not in MSI- cancers (P = 0.015). All somatic mutations occurred in the two 6(A) coding mononucleotide tracts in PTEN, suggestive of the etiological role of the deficient MMR. Immunohistochemical analysis revealed 31% (14 of 45) of the HNPCC CRCs and 41% (9 of 22) of the MSI+ sporadic tumors with absent or depressed PTEN expression. Approximately 17% (4 of 23) of the MSI- CRCs had decreased PTEN expression, and no MSI- tumor had complete loss of PTEN expression. Among the five HNPCC or MSI+ sporadic CRCs carrying frameshift somatic mutations with immunohistochemistry data, three had lost all PTEN expression, one showed weak PTEN expression levels, and one had mixed tumor cell populations with weak and moderate expression levels. These results suggest that PTEN frameshift mutations in HNPCC and sporadic MSI+ tumors are a consequence of mismatch repair deficiency. Further, hemizygous deletions in MSI- CRCs lead to loss or reduction of PTEN protein levels and contribute to tumor progression. Finally, our data also suggest that epigenetic inactivation of PTEN, including differential subcellular compartmentalization, occurs in CRCs.