Alterations of transforming growth factor beta receptor II, insulin growth factor receptor II genes in microsatellite unstable prostate carcinomas

DNA from 45 primary prostate tumors and corresponding normal tissues were analyzed to detect whether the alterations of transforming growth factor beta receptor II (TGFbetaRII) and insulin growth factor receptor II (IGFRII) are associated with microsatellite instability (MSI). We identified that 25 tumors were microsatellite unstable (55%). The remaining 20 tumors are found to be microsatellite stable. Loss of heterozygosity (LOH) was also tested at various loci. Results indicate that in case of TGFbetaRII, the rate of frame-shift mutation depends on the number of polyadenine [poly(A)] tracts. Twelve percent of the tumors had frame-shift alteration at BAT-RII locus which has 10 poly(A) repeats. Twenty percent of the tumors had frame-shift at BAT-25 locus which has 25 poly(A) repeats. In addition, IGFRII gene was examined for the presence of mutation in the repetitive sequences. Seven of the 25 tumors showed deletion of a G within eight poly(G) repeats. Besides these changes there were two tumors which showed a novel insertion of A within this poly(G) repeat making a change in 9 samples (R4, 36%). On the other hand, 4 tumors showed changes within the 5CT repeats. In addition, 3 tumors showed another novel insertion of C within the CT repeats.     

Microsatellite instability and frameshift mutations in genes involved in cell cycle progression or apoptosis in ovarian cancer

The loss of mismatch repair enzymes increases the mutation rate in microsatellites and coding regions of the genome and appears to be involved in drug resistance. The replication error (RER+) phenotype, associated with microsatellite instability, has been widely described for both familial and sporadic colon cancers and for gastric and endometrial tumors. For ovarian cancer, the incidence of RER+ cases among sporadic tumors is still uncertain. We analyzed epithelial ovarian tumors and ovarian carcinoma cell lines for microsatellite instability and for mutations in the coding regions of different genes, including the recently discovered human CHK-1 gene, which has an important role in controlling cell cycle progression and whose coding region contains a poly(A)9 tract. Microsatellite instability and frameshift mutations in coding regions of BAX, TGFbetaRII, IGFIIR, E2F-4, ICE, and CHK-1 genes were analyzed in ovarian cancer samples and cell lines by polymerase chain reaction (PCR). Approximately 26% of patients showed microsatellite instability in two or more loci. BAT-26 locus showed no alteration in primary tumors. We detected a BAX mutation in one tumor sample and a TGFbetaRII mutation in one cell line. Our findings confirm the presence of the RER+ phenotype in sporadic ovarian cancer. The low rate of mutation in genes previously reported to be altered in colon and gastric cancer suggests that other not yet identified genes might be altered and could play a role in tumor progression and response to treatment in RER+ ovarian tumors.     

K-ras mutation, p53 overexpression, and microsatellite instability in biliary tract cancers: a population-based study in China.

PURPOSE: The genetic alterations in biliary tract cancer and clinicopathological associations have not been studied in large population-based studies. Experimental Design: We evaluated genetic alterations such as K-ras mutation, p53 overexpression, microsatellite instability (MSI), and alterations of the polyadenine tract present in the transforming growth factor beta receptor type II (TGFbetaRII) gene in 126 biliary tract cancers: 75 gallbladder cancers, 33 bile duct cancers, and 18 ampullary cancers. These genetic alterations were compared with patient demographics and clinicopathological characteristics of the tumors. RESULTS: Mutation of the K-ras gene was present in 18 of 126 (14.3%) biliary tract cancers. K-ras mutation was present in 11 of 18 (61.1%) ampullary cancers, 5 of 33 (15.2%) bile duct cancers, and 2 of 75 (2.7%) gallbladder cancers (P = 0.000001). The mean survival of patients who had bile duct carcinomas with K-ras mutation was 3.0 +/- 2.2 months compared with 15.5 +/- 12.5 months for those without mutation (P = 0.03) but was not different for other tumor sites. p53 overexpression was present in 34 of 123 (27.6%) cancers. MSI-high (allelic shifts in 40% or more loci or alteration of the TGFbetaRII gene) was present in 4 of 126 (3.2%) biliary tract cancers without hereditary nonpolyposis colorectal cancer. MSI-high was more common in mucinous adenocarcinomas (P = 0.006) and in patients with early age of onset of cancer (P = 0.04). CONCLUSIONS: The genetic alterations in biliary tract cancers are dependent on the tumor subsite, histology, and age of onset and are associated with prognosis.     

Searching for microsatellite mutations in coding regions in lung, breast, ovarian and colorectal cancers

RepX represents a new informatics approach to probe the UniGene database for potentially polymorphic repeat sequences in the open reading frame (ORF) of genes, 56% of which were found to be actually polymorphic. We now have performed mutational analysis of 17 such sites in genes not found to be polymorphic (<0.03 frequency) in a large panel of human cancer genomic DNAs derived from 31 lung, 21 breast, seven ovarian, 21 (13 microsatellite instability (MSI)+ and eight MSI-) colorectal cancer cell lines. In the lung, breast and ovarian tumor DNAs we found no mutations (<0.03-0.04 rate of tumor associated open reading frame mutations) in these sequences. By contrast, 18 MSI+ colorectal cancers (13 cancer cell lines and five primary tumors) with mismatch repair defects exhibited six mutations in three of the 17 genes (SREBP-2, TAN-1, GR6) (P<0.000003 compared to all other cancers tested). We conclude that coding region microsatellite alterations are rare in lung, breast, ovarian carcinomas and MSI (-) colorectal cancers, but are relatively frequent in MSI (+) colorectal cancers with mismatch repair deficits.     

Frameshift mutations in TGFbetaRII, IGFIIR, BAX, hMSH3 and hMSH6 are absent in lung cancers

A genome-wide instability at simple repeat sequences characterizes gastrointestinal and endometrial cancers of the microsatellite mutator phenotype (MMP). The genes encoding transforming growth factor-beta receptor type II (TGFbetaRII), insulin-like growth factor II receptor (IGFIIR), Bcl-2 associated X protein (BAX), hMSH3 and hMSH6 have simple repeat sequences in their coding regions. Consequently, mutations in the single repeat sequences in these genes provide one major route for carcinogenesis in these cancers. We examined 43 non-small cell lung carcinomas and 16 small cell carcinomas for frameshift mutations in simple repeat sequences of TGFbetaRII, IGFIIR, BAX, hMSH3 and hMSH6. In addition, MMP was assessed using a primer set for BAT-26. None of 59 lung cancers exhibited frameshift mutations or MMP. It is concluded that somatic frameshift mutations in these genes and MMP do not constitute important mechanisms in lung carcinogenesis. The possibility of some sort of genetic instability undetectable as a form of MMP cannot be precluded.     

Microsatellite instability and K-ras mutations in gastric adenomas, with reference to associated gastric cancers.

Gastric adenomas are often detected in the stomach resected for gastric cancer. Previous investigation have revealed that the prevalence of their malignant transformation is generally low, but the frequent coexistence with carcinoma suggests that they may share some common processes with gastric cancer in tumorigenesis. In contrast to the cumulative information about genetic alterations in gastric cancer, inquiries into the genetic changes of adenoma and coexisting carcinoma in the same individual's stomach are still few. We investigated microsatellite instability (MSI) and K-ras point mutations in codons 12 and 13 in 50 lesions of gastric adenomas in 43 cases, and 31 lesions of gastric cancers that coexisted with these adenomas. In gastric adenomas, we found seven lesions (14.0%) to have microsatellite instability (MSI) at one or more loci, and most of them (six cases) had MSI at only one locus and were not associated with alterations in presumable target molecules. MSI was detected more frequently (11/31, 35.5%) and more extensively (five lesions at multiple loci) in accompanying gastric carcinomas. The prevalence of MSI in adenomas was more frequently found in those with synchronous gastric cancer (6/37, 16.2%, vs. 1/13, 7.6%) than without, and gastric adenoma accompanied by gastric cancer with multiple MSI tended to have MSI more frequently than that accompanied by cancer without MSI (4/5, 80%, vs. 1/24, 4.2%; p = 0. 01). In at least some individuals, MSI appears to represent one step in the pathway of gastric tumorigenesis, shared by adenoma and carcinoma. We found K-ras gene alteration in 8 lesions (16.0%) out of 50 gastric flat adenomas and no difference in its prevalence between adenoma with or without cancer. Only one gastric cancer, which had adenoma without K-ras mutation, had K-ras codon 12 mutation. Adenomas with a higher grade of atypia (p < 0.05) more frequently carried K-ras point mutation, which is consistent with the situation in colorectal adenoma. We conclude that MSI, not K-ras mutation, is a shared genetic alteration in adenoma and carcinoma of the individual stomach.     

Cigarette smoking and genetic alterations in sporadic colon carcinomas

Cigarette smoking has been inconsistently associated with colon cancer risk. To evaluate the hypothesis that smoking is primarily linked to a specific colon tumor subgroup(s), we assessed associations between smoking and the occurrence of mutations in the APC, K-ras and p53 genes, p53 overexpression, and microsatellite instability (MSI) in a Dutch population-based case-control study on sporadic colon carcinomas. The study population consisted of 176 cases and 249 controls. Smoking status (never, ever), number of cigarettes smoked per day (never, <15, > or =15), total years of smoking (never, < or =30, >30), and years since first started smoking (never, < or =35, >35) were all evaluated. Cigarette smoking status was significantly differently related to p53 overexpression-positive (p53(pos)) tumors compared with p53 overexpression-negative (p53(neg)) tumors (p53(pos) versus p53(neg), OR 0.4, 95% CI 0.2-0.9), as well as to tumors with transversion mutations in APC, K-ras or p53 (transv(+)) compared with tumors without transversion mutations in one of these genes (transv(-)) (transv(+) versus transv(-), OR 2.5, 95% CI 1.0-5.9). Positive associations were observed with p53(neg) tumors and transv(+) tumors when compared with the population-based controls (ever versus transv(-), OR 1.5, 95% CI 0.9-2.8 and OR 2.2, 95% CI 0.9-5.6, respectively), inverse associations with p53(pos) tumors and transv(-) tumors (ever versus never, OR 0.5, 95% CI 0.3-1.0 and OR 0.8, 95% CI 0.5-1.3, respectively). Similar patterns of association were observed for the other smoking variables evaluated. In addition, although statistically non-significant, smoking was more notably positively associated with tumors that exhibit K-ras mutations, especially K-ras transversion mutations, than with tumors without K-ras mutations. An inverse relationship between smoking and the occurrence of APC mutations was suggested, whereas no clear associations were observed with MSI. Our data suggest that smoking-related colon cancers develop through a p53(neg) pathway and that smoking particularly results in colon carcinomas with transversion mutations.     

High EPHB2 mutation rate in gastric but not endometrial tumors with microsatellite instability

The EPH/EFN family of receptor tyrosine kinases regulates cell adhesion and migration and has an important role in controlling cell positioning in the normal intestinal epithelium. Inactivation of EPHB2 has recently been shown to accelerate tumorigenesis in the colon and rectum, and we have previously demonstrated frequent frameshift mutations (41%) in an A9 coding microsatellite repeat in exon 17 of EPHB2 in colorectal tumors with microsatellite instability (MSI). In this study, we extended these analyses to extracolonic MSI cancers, and found frameshift EPHB2 mutations in 39% (25/64) of gastric tumors and 14% (8/56) of endometrial tumors. Regression analysis of these EPHB2 mutation data on the basis of our previously proposed statistical model identified EPHB2 as a selective target of frameshift mutations in MSI gastric cancers but not in MSI endometrial carcinomas. These results suggest a functional role for EPHB2 in gastric tumor progression, and emphasize the differences between the tumorigenic processes in MSI gastrointestinal and endometrial cancer.     

High incidence of ras gene mutation in intrahepatic cholangiocarcinoma.

The DNA sequences around codons 12, 13, and 61 of the ras gene were analyzed by polymerase chain reaction and direct sequencing in 18 intrahepatic cholangiocarcinomas. The ras gene mutations were found in 9 of 18 (50%): 6 in K-ras codon 12, 1 in K-ras codon 13, 1 in K-ras codon 61, and 1 in N-ras codon 12. The incidence of mutations was higher in the hilar type of intrahepatic cholangiocarcinomas, especially when these tumors were large. The incidence and spectrum of the mutations were almost the same as those reported in colon cancers, possibly indicating similar etiologic agent(s) in the carcinogenesis of both cancers.     

Pathogenesis of DNA repair-deficient cancers: a statistical meta-analysis of putative Real Common Target genes

DNA mismatch repair deficiency is observed in about 15% of human colorectal, gastric, and endometrial tumors and in lower frequencies in a minority of other tumors thereby causing insertion/deletion mutations at short repetitive sequences, recognized as microsatellite instability (MSI). Evolution of tumors, including those with MSI, is a continuous process of mutation and selection favoring neoplastic growth. Mutations in microsatellite-bearing genes that promote tumor cell growth in general (Real Common Target genes) are assumed to be the driving force during MSI carcinogenesis. Thus, microsatellite mutations in these genes should occur more frequently than mutations in microsatellite genes without contribution to malignancy (ByStander genes). So far, only a few Real Common Target genes have been identified by functional studies. Thus, comprehensive analysis of microsatellite mutations will provide important clues to the understanding of MSI-driven carcinogenesis. Here, we evaluated published mutation frequencies on 194 repeat tracts in 137 genes in MSI-H colorectal, endometrial, and gastric carcinomas and propose a statistical model that aims to identify Real Common Target genes. According to our model nine genes including BAX and TGFbetaRII were identified as Real Common Targets in colorectal cancer, one gene in gastric cancer, and three genes in endometrial cancer. Microsatellite mutations in five additional genes seem to be counterselected in gastrointestinal tumors. Overall, the general applicability, the capacity to unlimited data analysis, the inclusion of mutation data generated by different groups on different sets of tumors make this model a useful tool for predicting Real Common Target genes with specificity for MSI-H tumors of different organs, guiding subsequent functional studies to the most likely targets among numerous microsatellite harboring genes.     

Frequent microsatellite instability in primary esophageal carcinoma associated with extraesophageal primary carcinoma

Patients with esophageal squamous cell carcinoma (ESCC) frequently develop other primary cancers, such as gastric cancer and head and neck cancer. Details of carcinogenesis in patients with multiple primaries that include esophageal carcinoma with other primary carcinoma (ECOPC) remain uncertain. We examined microsatellite instability (MSI) status, frameshift mutation in target genes of MSI, mismatch repair protein expression and hypermethylation of the hMLH1 promoter region in ECOPC patients to better understand the underlying carcinogenic processes. High frequency MSI (MSI-H) was found in 15 (44.1%) of 34 patients with ECOPC, but in only 6 (14.3%) of 42 patients with esophageal cancer alone (p < 0.01). Frameshift mutations in TGFbetaRII, BAX, MSH3 and MSH6 genes respectively were present in 4, 1, 2 and 2 of 34 ECOPC patients. Immunohistochemical study showed that 12 (80.0%) of 15 MSI-H tumors showed loss of expression of either hMLH1 or hMSH2. In addition, 6 of 9 tumors (66.7%) that showed reduced hMLH1 expression also had hypermethylation of the hMLH1 promoter region. Our findings suggested that carcinogenesis in ECOPC was closely associated with the MSI pathway because of mismatch repair protein deficiency.     

Identification of frequently mutated genes with relevance to nonsense mediated mRNA decay in the high microsatellite instability cancers

Frameshift mutations at coding mononucleotide repeats (cMNR) are frequent in high‐microsatellite instability (MSI‐H) cancers. Frameshift mutations in cMNR result in the formation of a premature termination codon (PTC) in the transcribed mRNA, and these abnormal mRNAs are generally degraded by nonsense mediated mRNA decay (NMD). We have identified novel genes that are frequently mutated at their cMNR by blocking NMD in two MSI‐H cancer cell lines. After blocking NMD, we screened for differentially expressed genes using DNA microarrays, and then used database analysis to select 28 candidate genes containing cMNR with more than 9 nucleotide repeats. cMNR mutations have not been previously reported in MSI‐H cancers for 15 of the 28 genes. We analyzed the cMNR mutation of each of the 15 genes in 10 MSI‐H cell lines and 21 MSI‐H cancers, and found frequent mutations of 12 genes in MSI‐H cell lines and cancers, but not in microsatellite stable (MSS) cancers. Among these genes, the most frequently mutated in MSI‐H cell lines were MLL3 (70%), PHACTR4 (70%), RUFY2 (50%) and TBC1D23 (50%). MLL3, which has already been implicated in cancer, had the highest mutation frequency in MSI‐H cancers (48%). Our combined approach of NMD block, database search, and mutation analysis has identified a large number of genes mutated in their cMNR in MSI‐H cancers. The identified mutations are expected to contribute to MSI‐H tumorigenesis by causing an absence of gene expression or low gene dosage effects.     

Microsatellite instability in the development of DNA mismatch repair deficient tumors

Microsatellites are highly abundant short repetitive sequences found in the genomes across different species. They have gained increasing interest in recent years because length alterations in several coding as well as non-coding microsatellites are associated with a variety of different disorders. Particularly, microsatellite mutations play an important role in tumorigenesis of DNA mismatch repair deficient tumors that account for up to a 15% of colorectal, endometrial, and various other cancers. The systematic analysis of the distribution and function of affected microsatellite sequences has facilitated to unravel important steps in the selection processes that drive tumorigenesis. Here, we review the role of microsatellite mutations in the development of cancers with DNA mismatch repair deficiency, outlining biostatistical approaches for the identification of MSI target genes with relevance to MSI associated carcinogenesis. Knowledge about the biological impact of microsatellite mutations in these genes will potentially help to develop modified clinical concepts for diagnosis, prevention, and treatment of microsatellite unstable human cancers.     

Detection of K-ras mutations in pancreatic and hepatic neoplasms by non-isotopic mismatched polymerase chain reaction.

Mutations within codon 12 leading to activation of Kirsten-ras (K-ras) genes occur in a wide variety of human tumors, but have been reported most frequently in pancreatic carcinomas. We studied twenty-four paraffin-embedded pancreatic and hepatic tumors and two colon carcinoma cell lines with a rapid and simple approach that exploits allele-specific amplification of genomic DNA in a polymerase chain reaction (PCR). We extend the utility of this technique, which is dependent on an exact match at the 3' nucleotide between synthetic oligonucleotides and template DNA, to analyse paraffin-embedded tumor samples for the presence of point mutations at the first and second base of codon 12 of the K-ras gene. The PCR mismatch amplification technique demonstrated a 66% incidence of K-ras mutations at codon 12 in the group of pancreatic neoplasms as a whole. The percentage of mutations varied only slightly in the pancreatic cancer subcategories: 75% in ampullary, 66% in bile duct and 57% in the ductal adenocarcinomas. One islet cell carcinoma and normal tissues adjacent to the tumors revealed wild-type alleles only. One hepatoblastoma and one of six hepatocellular carcinomas also had codon 12 mutations. The PCR mismatch is a sensitive and rapid method that may be useful in screening neoplasms for K-ras point mutation and can be applied to archival material. This application allows a retrospective analyses of a wide range of pathological specimens to determine the role of K-ras mutations in human tumorigenesis.     

Synchronous multiple primary gastrointestinal cancer exhibits frequent microsatellite instability

Colorectal (CRC) and gastric cancers (GC), the most common gastrointestinal malignancies, have been known to develop occasionally in a same patient. Previous studies have focused on the etiology of patients with multiple primary gastric and colorectal cancer (MPGCC); however, the carcinogenic process of MPGCC remains unclear. In this study, we have examined the genetic alterations in MPGCC in order to clarify the carcinogenic pathway. Twenty patients with sporadic MPGCC were examined for microsatellite instability (MSI) and frameshift mutations of target genes such as TGFbetaRII, BAX and IGFIIR. In 10 (50%) of 20 patients with MPGCC, MSI was present at least at 1 lesion of GC or CRC. Four (50%) of 8 cases with synchronous MPGCC displayed MSI in both GC and CRC, while only 1 (8%) of 12 cases of metachronous MPGCC exhibited MSI in both organs. Carcinogenic process of MPGCC was fairly associated with the MSI pathway, particularly in cases of synchronous MPGCC. MSI was found in 5 (25%) of 20 GCs and in 10 (50%) of 20 CRCs. MSI was involved more closely in CRC than in GC among MPGCC. Although most frameshift mutations at target genes were found in the MSI-positive MPGCC, infrequent mutations were observed in the genes. Frameshift mutation was found in only 1 of 5 cases of MSI-positive GC at TGFbetaRII. Only 2 of 10 cases of CRC with MSI showed mutation at TGFbetaRII, and 1 case also showed mutation at BAX and IGFIIR. Our findings suggest that TGFbetaRII, BAX and IGFIIR are not the main target genes for carcinogenesis in MSI-positive MPGCC.     

Microsatellite instability is uncommon in breast cancer.

In some tumors, defects in mismatch repair enzymes lead to errors in the replication of simple nucleotide repeat segments. This condition is commonly known as microsatellite instability (MSI) because of the frequent mutations of microsatellite sequences. Although the MSI phenotype is well recognized in some colon, gastric, pancreatic, and endometrial cancers, reports of MSI in breast cancer are inconsistent. We report here our experience with >10,000 amplifications of simple nucleotide repeats in noncoding genomic regions using DNA from 267 cases of breast cancer, including cases that represent all major histological types of breast cancer. We rarely (10 reactions) found unexpected bands in amplifications of tumor DNA that were not present in amplifications of normal DNA. Moreover, repeats of these reactions did not confirm microsatellite instability in a single case. We also evaluated the simple nucleotide repeats in the transforming growth factor type II receptor, insulin-like growth factor type II receptor, BAX, and E2F-4 genes, which are frequently mutated in tumors with microsatellite instability. No mutations of these genes were found in any of the 30 breast cancer cell lines and 61 primary breast cancer samples examined. These results indicate that mismatch repair errors characteristic of the MSI phenotype are uncommon in human breast cancer.     

Frequent alterations of the beta-catenin and TCF-4 genes, but not of the APC gene, in colon cancers with high-frequency microsatellite instability

High-frequency microsatellite instability (MSI-H) due to defective DNA mismatch repair (MMR) is a characteristic of the majority of tumors from kindreds with hereditary nonpolyposis colorectal cancer (HNPCC) and a subset of sporadic cancers. To better understand the molecular characteristics of colon cancers with MSI-H, we analyzed these cancers for alterations of genes, such as APC, beta-catenin, and TCF-4 genes, involved in the Wnt signaling pathway. Following the National Cancer Institute (NCI) criteria, 385 unselected colon cancers were classified as follows: 50 (13%) MSI-H tumors, 36 (9%) low-frequency MSI (MSI-L) tumors, and 299 (78%) microsatellite stable (MSS) tumors. The frequency of APC mutations was significantly lower in MSI-H tumors (9 out of 50) than in MSI-L (12 out of 20) and MSS (66 out of 100) tumors (P = 0.0005 and P < 0.0001, respectively). In contrast, the frequency of exon 3 mutations in the beta-catenin gene was higher in MSI-H tumors (10 out of 50) than in MSI-L tumors (0 out of 30; P = 0.0110) and MSS tumors (3 out of 100; P = 0.0010). Frameshift mutations in a (A)9 tract of the TCF-4 gene were detected in 44% (22 out of 50) of MSI-H tumors, but not in any of the 20 MSI-L tumors or 40 MSS tumors. In total, 78% of MSI-H tumors and 84% of the remaining tumors had at least one alteration in APC, beta-catenin, or the TCF-4 genes. Although further analysis is needed to functionally characterize the consequences of each of these alterations on beta-catenin/TCF target gene expression, our results suggest that the activation of the Wnt signaling pathway plays a pivotal role in colon tumorigenesis, irrespective of MSI status.     

Microsatellite mutations of transforming growth factor-beta receptor type II and caspase-5 occur in human precursor T-cell lymphoblastic lymphomas/leukemias in vivo but are not associated with hMSH2 or hMLH1 promoter methylation

In solid cancers, defective DNA mismatch repair (MMR) is most commonly caused by hMSH2 or hMLH1 mutations, or epigenetic silencing of hMLH1 by promoter hypermethylation, and results in the acquisition of characteristic frameshift microsatellite mutations of mononucleotide repeats located within the coding regions of defined target genes. We previously identified hMSH2 mutations in T-cell lymphoblastic lymphoma (T-LBL) patient tumor samples and others have reported coding region microsatellite mutations in T-cell acute lymphoblastic leukemia (T-ALL) cell lines. Thus, while MMR gene mutations are known to occur in some human T-lymphoblastic tumors in vivo, it is still unknown if the coding region microsatellite mutations detected in human cell lines also occur in vivo or if hMLH1 or hMSH2 promoter hypermethylation contributes to defective MMR in these tumors. We analyzed the TGFbetaRII (A)10 and caspase-5 (A)10 coding region repeats in 16 human T-LBL/ALL patient tumor samples and identified six with microsatellite mutations in one or both repeats. There was no evidence of hMSH2 or hMLH1 promoter methylation as assessed by standard methylation specific PCR or by a novel temporal temperature gradient electrophoresis (TTGE) method that analyzed 25 and 30 CpG sites in the hMLH1 and hMSH2 promoters, respectively. Our results indicate that coding region microsatellite mutations characteristic of defective MMR occur in some human T-LBL/ALL in vivo but not as a consequence of hMLH1 or hMSH2 promoter hypermethylation. Furthermore, the identification of TGFbetaRII and caspase-5 coding region mutations in vivo implicates these genes in the pathogenesis of human T-LBL/ALL.