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.     

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.     

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.     

Evolution of instability at coding and non-coding repeat sequences in human MSI-H colorectal cancers

A number of human genes containing coding mononucleotide repeat sequences are particularly prone to mutations in tumors with defects in mismatch repair (MMR) genes (MSI-H cancers). In a large series of MSI-H colorectal tumors, we looked for mutations in 25 coding repeats contained in eight genes already known to be mutated in these cancers or in 17 other genes with an expected role in carcinogenesis. Mutations were found in 19 of the 25 candidate genes. Using a maximum likelihood statistical method, they were separated into two different groups that differed significantly in their mutation frequencies, and were likely to represent mutations that do or do not provide selective pressures during MSI-H tumoral progression, respectively. Three new target genes were found (GRB-14, RHAMM, RAD50). Our results provide evidence that MSI-H tumoral progression involves the cumulative mutations of a large number of genes. For each MSI-H tumor we calculated indexes representing the number of mutations found in genes of these groups. We also evaluated a shortening index at both the Bat-25 and Bat-26 non-coding mononucleotide tracts that are known to be almost always unstable in MSI-H cancers. A significant correlation was observed between instability at both coding and non-coding repeats, suggesting that Bat-25 and Bat-26 could be used as simple phenotypical markers of the tumoral evolution. A preferential order of mutations was deduced. During this process, hMSH3 alterations, a target gene encoding for a MMR protein, was found to play an important role by increasing the instability phenomenon characterizing these cancers.     

Defects in mismatch repair occur after APC mutations in the pathogenesis of sporadic colorectal tumours

The roles of the intrinsic mutation rate and genomic instability in tumorigenesis are currently controversial. In most colorectal tumours, it is generally supposed that the first mutations occur at the adenomatous polyposis coli (APC) locus; APC mutations are thought to provide cells with a selective advantage but have no known effect on the mutation rate. It has also been suggested that genomic instability is the initiating event in colorectal tumorigenesis and, if this is true, mutations of DNA mismatch repair (MMR) genes (or at similar loci) are the most likely candidates. If defective MMR precedes APC mutations, the APC mutations of colon tumours with defective MMR and hence replication errors (RER+) should differ from those of RER- tumours, in at least three specific ways: (1) a higher frequency of allele loss at APC in RER- tumours; (2) more frameshift than nonsense mutations in RER+ tumours; and (3) APC mutations in simple repeat sequences [(N)_n, (N₁N₂)_n, or (N₁N₂N₃)_n] in RER+ tumours. We found no evidence that sporadic RER+ and RER- colon cancers (including cell lines) differ in any of these three ways. Although it remains possible that MMR is abnormal in tumours from HNPCC families before APC mutations occur, it is likely that in sporadic colon tumours, APC mutations, rather than genomic instability, are the initiating events in tumorigenesis. Hum Mutat 11:114–120, 1998. © 1998 Wiley-Liss, Inc.     

Defects in homologous recombination repair in mismatch-repair-deficient tumour cell lines

Loss of mismatch repair (MMR) leads to a complex mutator phenotype that appears to drive the development of a subset of colon cancers. Here we show that MMR-deficient tumour cell lines are highly sensitive to the toxic effects of thymidine relative to MMR-proficient lines. This sensitivity was not a direct consequence of MMR deficiency or alterations of DNA precursor metabolism. Instead, MMR-defective tumour cell lines are also defective in homologous recombination repair (HRR) induced by DNA double-strand breaks. Furthermore, a frameshift mutation of the human RAD51 paralog XRCC2 found in the MMR-deficient uterine tumour cell line SKUT-1 can confer thymidine sensitivity when introduced into a MMR-proficient line. Like other cells with defective XRCC2, SKUT-1 is sensitive to mitomycin C, and MMR-proficient cells expressing the mutant XRCC2 allele become more sensitive to this agent. These data suggest that the thymidine sensitivity of MMR-deficient tumour cell lines may be a consequence of defects in the HRR pathway. The increased thymidine sensitivity and the loss of an important pathway for the repair of DNA double-strand breaks create new opportunities for therapies directed specifically against this subset of tumours.     

Impaired nonhomologous end-joining in mismatch repair-deficient colon carcinomas

Frameshift mutations of coding mononucleotide repeat of the hRAD50 gene and formation of the mutant hMRE11 splicing variant are frequent events in tumors with mismatch repair (MMR) deficiency. Both the hRAD50 and hMRE11 proteins form a heterotrimer with the NBS1, and this heterotrimer is involved in the double strand DNA break repair by homologous recombination and nonhomologous end-joining (NHEJ). In order to clarify the role of hRAD50 and hMRE11 gene alterations in MMR-deficient tumors, we analyzed the expression of the hRAD50 and hMRE11 proteins and we evaluated NHEJ in the seven MMR-deficient and five MMR-proficient colon cancer cell lines. Frameshift mutations of the hRAD50 gene were found in five of seven MMR-deficient cell lines, and this was directly related to the decreased expression of hRAD50 mRNA and protein. The mutant hMRE11 splicing variant was found in all of the seven MMR-deficient cell lines, and this was related to the decreased hMRE11 expression in four of the seven MMR-deficient cell lines. MMR-deficient cell lines with decreased hRAD50 and hMRE11 expressions were more sensitive to gamma-irradiation, and these cell lines showed an impaired NHEJ. The impairment of NHEJ was induced after knockdown of hRAD50 and hMRE11 through small interference RNA. Our findings suggest that mutations of hRAD50 and hMRE11 genes in MMR-deficient tumors are related to the defects in NHEJ, and this may result in chromosomal changes during the progression of tumor.     

Molecular typing of colorectal cancer: applications in diagnosis and treatment

Molecular typing of colorectal cancer (CRC) is at an early stage of development with analysis of KRAS mutation status and DNA mismatch repair (MMR) deficiency representing the two major approaches currently in use for diagnosis and treatment-related purposes. RAS proteins act as molecular switches that regulate cellular processes including cell growth and survival. Activating KRAS mutations are found in 40–50% of colorectal adenomas and cancers. Detection of KRAS mutations guide the decision to use anti-EGFR antibody therapy, which is not effective in KRAS mutant cancers. MMR deficiency results in failure to repair replication-associated DNA errors, allowing persistence of mismatch mutations all over the genome, especially in regions of repetitive DNA known as microsatellites, giving rise to microsatellite instability (MSI). A high frequency of microsatellite instability (MSI-H) often with abnormal expression of MMR proteins is key to the diagnosis of Lynch Syndrome, but is also observed in ～15% of sporadic CRC.     

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.     

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.     

Spectrum of molecular alterations in colorectal, upper urinary tract, endocervical, and renal carcinomas arising in a patient with hereditary non-polyposis colorectal cancer

Hereditary nonpolyposis colon cancer (HNPCC) syndrome is the most frequent hereditary cancer syndrome predisposing to cancers of various locations, especially colon, endometrium, stomach, and upper urinary tract. Carcinomas of the kidney parenchyma are not considered as an HNPCC-related tumor. HNPCC tumors are characterized by microsatellite instability (MSI) due to a defect in mismatch repair (MMR) and carry somatic frameshift mutations in mononucleotide repeats within the coding regions of key genes. We report the first case of a papillary carcinoma of the kidney in an HNPCC patient who developed carcinomas of the upper urinary tract, endocervix, and colon. Whereas the HNPCC-related tumors demonstrated MSI phenotype, loss of MSH2 protein expression, and frameshift mutations in several of the 13 target genes analyzed, the kidney cancer displayed MSS phenotype, normal MMR protein expression, and no frameshift mutation in target genes. Our observations do not support the possibility that papillary carcinomas are part of HNPCC syndrome.     

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.     

遗传性非腺瘤病性结直肠癌结肠腺瘤病基因突变研究

目的 分析遗传性非腺瘤病性结直肠癌(hereditary non-polyposis colorectal cancers，HNPCC)结肠腺瘤病(adenomatous polyposis coli，APC)基因突变的特点及错配修复缺陷对其影响。方法 采用体外蛋白合成试验和序列分析确定19例HNPCC病例APC体细胞突变。结果 19例病例中有11例(13个突变点)发生APC突变，发生率为58％(11／19)，其中移码突变9个，无义突变4个，移码突变占多数(69％)。所有移码突变表现为1—2个碱基的缺失或插入，大多(7／9)发生在简单核苷酸重复序列，特别是单腺苷酸重复序列(A)n(5／9)。检出的由单个碱基替换而导致的无义突变都发生在CpG岛，表现为C向T的转换。结论 多于半数的HNPCC发生APC突变，其突变多发生在编码区单核苷酸重复序列(移码突变)或CpG岛(点突变)上，提示APC基因失活在HNPCC为常见的分子事件；错配修复缺陷所致的微卫星DNA不稳定性等内源性机理可能对APC突变产生影响。     

Somatic mutations in mismatch repair genes in sporadic gastric carcinomas are not a cause but a consequence of the mutator phenotype

In hereditary nonpolyposis colorectal cancer (HNPCC), patients' mismatch repair (MMR) gene mutations cause MMR deficiency, leading to microsatellite instability (MSI-H). MSI-H is also found in a substantial fraction of sporadic gastric carcinomas (SGC), mainly due to MLH1 promoter hypermethylation, although somatic mutations in MMR genes have been described. We aimed to investigate which MMR defects are present in SGC. Twenty-nine MSI-H SGC investigated previously for MLH1 promoter hypermethylation were screened for somatic mutations in MLH1, MSH2, MSH6, MLH3, and MBD4 by denaturing gradient gel electrophoresis and sequencing. Five truncating mutations (three in MSH6, one in MLH3, and one in MBD4) and one missense mutation (MLH1) were identified. Of these, three truncating mutations were in MSI-H cases that lack MLH1 hypermethylation. As all truncating mutations were found in the coding poly-A tracts, it seems likely that they result from the MSI phenotype rather than cause it. In summary, somatic mutations in MMR genes are rare in SGC and do not explain the development of these tumors reflecting, rather than causing, the mutator phenotype. Other MMR genes are probably involved in MSI-H gastric cancer without MLH1 hypermethylation.     

Endometrial and colorectal tumors from patients with hereditary nonpolyposis colon cancer display different patterns of microsatellite instability

The colorectum and uterine endometrium are the two most commonly affected organs in hereditary nonpolyposis colon cancer (HNPCC), but the genetic basis of organ selection is poorly understood. As tumorigenesis in HNPCC is driven by deficient DNA mismatch repair (MMR), we compared its typical consequence, instability at microsatellite sequences, in colorectal and endometrial cancers from patients with identical predisposing mutations in the MMR genes MLH1 or MSH2. Analysis of non-coding (BAT25, BAT26, and BAT40) and coding mononucleotide repeats (MSH6, MSH3, MLH3, BAX, IGF2R, TGF beta RII, and PTEN), as well as MLH1- and MSH2-linked dinucleotide repeats (D3S1611 and CA7) revealed significant differences, both quantitative and qualitative, between the two tumor types. Whereas colorectal cancers displayed a predominant pattern consisting of instability at the BAT loci (in 89% of tumors), TGF beta RII (73%), dinucleotide repeats (70%), MSH3 (43%), and BAX (30%), no such single pattern was discernible in endometrial cancers. Instead, the pattern was more heterogeneous and involved a lower proportion of unstable markers per tumor (mean 0.27 for endometrial cancers versus 0.45 for colorectal cancers, P < 0.001) and shorter allelic shifts for BAT markers (average 5.1 bp for unstable endometrial cancers versus 9.3 bp for colorectal cancers, P < 0.001). Among the individual putative "target" loci, PTEN instability was associated with endometrial cancers and TGF beta RII instability with colon cancers. The different instability profiles in endometrial and colorectal cancers despite identical genetic predisposition underlines organ-specific differences that may be important determinants of the HNPCC tumor spectrum.     

Mutations in the ataxia telangiectasia and rad3-related-checkpoint kinase 1 DNA damage response axis in colon cancers

In response to certain types of DNA damage, ataxia telangiectasia and rad3-related (ATR) phosphorylates checkpoint kinase 1 (CHEK1) resulting in cell cycle arrest and subsequent DNA repair. ATR and CHEK1 contain mononucleotide microsatellite repeat regions, which are mutational targets in tumors with defective mismatch repair (MMR). This study examined the frequency of such mutations in colon cancers and their impact on biologic behavior. Screening for ATR mutations in 48 tumors was performed using denaturing high-performance liquid chromatography (DHPLC) and confirmed with sequencing analysis. The CHEK1 exon 7 A(9) region was sequenced in 20 of the 27 (74%) tumors with high frequency of microsatellite instability (MSI-H). Univariate and multivariate analyses were used to examine associations with clinical outcomes. Frequent mutations in MSI-H colon cancers were identified within the ATR (37%)/CHEK1(5%) damage response pathway. Stage and MSI status both independently predicted overall survival (OS) and disease-free survival (DFS). ATR status was not associated with stage, but was associated with a trend toward improved DFS: 0/9 cancers recurred in MSI-H cases harboring ATR mutations vs. 4/18 recurrences in MSI-H cases without ATR mutations. This suggests that ATR mutations may affect clinical behavior and response to therapy in MSI-H colon cancers.     

Compound heterozygosity for two MSH6 mutations in a patient with early onset colorectal cancer, vitiligo and systemic lupus erythematosus

Lynch syndrome (hereditary non-polyposis colorectal cancer, HNPCC) is an autosomal dominant condition caused by heterozygous germline mutations in the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, or PMS2. Rare cases have been reported of an inherited bi-allelic deficiency of MMR genes, associated with multiple café-au-lait spots, early onset CNS tumors, hematological malignancies, and early onset gastrointestinal neoplasia. We report on a patient with vitiligo in segments of the integument who developed systemic lupus erythematosus (SLE) at the age of 16, and four synchronous colorectal cancers at age 17 years. Examination of the colorectal cancer tissue showed high microsatellite instability (MSI-H) and an exclusive loss of expression of the MSH6 protein. Immunohistochemical analysis of normal colon tissue also showed loss of MSH6, pointing to a bi-allelic MSH6 mutation. Sequencing of the MSH6 gene showed the two germline mutations; c.1806_1809delAAAG;p.Glu604LeufsX5 and c.3226C > T;p.Arg1076Cys. We confirmed that the two mutations are on two different alleles by allele-specific PCR. To our knowledge, neither parent is clinically affected. They did not wish to be tested for the mutations identified in their daughter. These data suggest that bi-allelic mutations of one of the MMR genes should be considered in patients who develop early-onset multiple HNPCC-associated tumors and autoimmune disorders, even in absence of either hematological malignancies or brain tumors.     

Involvement of PTEN mutations in the genetic pathways of colorectal cancerogenesis

So far, somatic mutations of the PTEN gene have been found in several different neoplasms but not in colorectal tumours. As exons 7 and 8 of the PTEN coding sequence contain an (A)(6)repeat and mononucleotide repeat sequences are targets for mutations in tumours with microsatellite instability (MI), we screened a panel of sporadic colorectal tumours exhibiting MI to test whether PTEN gene repeats are frequently mutated in MI(+)colorectal cancers. Of 32 cases studied, seven mutations were found in six (18.75%) patients, as a PTEN biallelic frameshift mutation was observed in one case, with consequent loss of function of the gene. Loss of heterozygosity, evaluated in the remaining five cases using the microsatellite marker D10S541, was detected in two of three informative samples. To further address the role of the PTEN gene in MI(+)colorectal cancer, in the six patients with mutated PTEN, we analysed the mononucleotide repeats of six other genes: BAX, hMSH3, hMSH6, TGFbRII, IGFIIR and APC. In two of these six patients, mutations of the TGFbRII gene only were present, indicating that PTEN may have a role in the mutator pathway of colorectal tumorigenesis. Overall, these results indicate that PTEN mutations are selected for during tumorigenesis in MI(+)colorectal tumours. The mutation of both PTEN alleles and evidence that the PTEN protein is expressed in normal colon suggest that loss of function of this gene could play a direct role in tumorigenesis.     

Colorectal pretumor progression before and after loss of DNA mismatch repair

A pretumor progression model predicts many oncogenic cancer mutations may first accumulate in normal appearing colon. Although direct observations of early pretumor mutations are impractical, it may be possible to retrospectively reconstruct tumor histories from contemporary cancer mutations. To infer when and in what order mutations occur during occult pretumor progression, we examined 14 cancers from individuals with heterozygous germline mutations in DNA mismatch repair (MMR) genes or hereditary nonpolyposis colorectal cancer (HNPCC). Somatic inactivation of the normal allele occurs sometime during a lifetime and results in loss of MMR, elevated mutation rates, and subsequent widespread somatic microsatellite mutations in HNPCC cancers. Patient ages at MMR loss can be estimated because intervals between MMR loss and cancer removal can be inferred from numbers of microsatellite tumor mutations. The relative order of MMR loss during pretumor progression may also be inferred from its collective ages of occurrence. Somatic MMR loss preceded cancer removal by an average of 6.1 years, occurred relatively late in life (average of 41.6 versus 47.7 years at cancer removal), and was a surprisingly late (fifth or sixth) step. Calculations indicate five or six oncogenic mutations could accumulate with relatively normal replication fidelity in normal appearing colon. HNPCC pretumor progression essentially begins from birth and ends with MMR loss, implying elevated mutation rates and tumorigenesis may be unnecessary for most progression.