PolyA Deletions in Hereditary Nonpolyposis Colorectal Cancer
2002; Elsevier BV; Volume: 160; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)62576-x
ISSN1525-2191
AutoresKyoung‐Mee Kim, Reijo Salovaara, Jukka‐Pekka Mecklin, Heikki Järvinen, Lauri A. Aaltonen, Darryl Shibata,
Tópico(s)Cancer Genomics and Diagnostics
ResumoMicrosatellite instability (MSI) secondary to loss of DNA mismatch repair (MMR) is present in adenomas and colorectal carcinomas from individuals with hereditary nonpolyposis colorectal cancer (HNPCC). To better characterize when MMR loss occurs during HNPCC progression, the extent of deletions in noncoding polyA sequences were compared between 6 adenomas (all ≤1.0 cm in size) and 10 cancers. Numbers of deleted bases reflect time since loss of MMR because polyA deletions are stepwise. Adenoma deletions were nearly the same (85%) as the cancers with sum total deletions at four different polyA loci of −32.7 bases in adenomas and −38.4 bases in cancers. Intervals between negative clinical examinations and tumor removal (average of 2.1 years) were known for six tumors. There were no significant differences in the extent of deletions in tumors removed under clinical surveillance (−34.8 bases) versus tumors removed without prior negative examinations (−36.5 bases). These findings illustrate that MSI is extensive in both small adenomas, and tumors which appear after negative clinical examinations, consistent with an early loss of MMR in HNPCC, even before a gatekeeper mutation. Microsatellite instability (MSI) secondary to loss of DNA mismatch repair (MMR) is present in adenomas and colorectal carcinomas from individuals with hereditary nonpolyposis colorectal cancer (HNPCC). To better characterize when MMR loss occurs during HNPCC progression, the extent of deletions in noncoding polyA sequences were compared between 6 adenomas (all ≤1.0 cm in size) and 10 cancers. Numbers of deleted bases reflect time since loss of MMR because polyA deletions are stepwise. Adenoma deletions were nearly the same (85%) as the cancers with sum total deletions at four different polyA loci of −32.7 bases in adenomas and −38.4 bases in cancers. Intervals between negative clinical examinations and tumor removal (average of 2.1 years) were known for six tumors. There were no significant differences in the extent of deletions in tumors removed under clinical surveillance (−34.8 bases) versus tumors removed without prior negative examinations (−36.5 bases). These findings illustrate that MSI is extensive in both small adenomas, and tumors which appear after negative clinical examinations, consistent with an early loss of MMR in HNPCC, even before a gatekeeper mutation. Germline mutations in mismatch repair (MMR) loci are present in individuals with HNPCC.1Kinzler KW Vogelstein B Lessons from hereditary colorectal cancer.Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4321) Google Scholar Inactivation of the normal allele, typically from loss of heterozygosity,2Hemminki A Peltomaki P Mecklin JP Jarvinen H Salovaara R Nystrom-Lahti M de la Chapelle A Aaltonen LA Loss of the wild type MLH1 gene is a feature of hereditary nonpolyposis colorectal cancer.Nat Genet. 1994; 8: 405-410Crossref PubMed Scopus (282) Google Scholar results in MMR deficiency and elevated mutation rates.1Kinzler KW Vogelstein B Lessons from hereditary colorectal cancer.Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4321) Google Scholar Mutation rates in simple repeat sequences or microsatellite (MS) loci are increased approximately 100-fold in MMR-deficient cell lines3Shibata D Peinado MA Ionov Y Malkhosyan S Perucho M Genomic instability in repeated sequences is an early somatic event in colorectal tumorigenesis that persists after transformation.Nat Genet. 1994; 6: 273-281Crossref PubMed Scopus (457) Google Scholar, 4Bhattacharyya NP Skandalis A Ganesh A Groden J Meuth M Mutator phenotypes in human colorectal carcinoma cell lines.Proc Natl Acad Sci USA. 1994; 91: 6319-6323Crossref PubMed Scopus (405) Google Scholar and widespread microsatellite instability (MSI) is present in hereditary nonpolyposis colorectal cancer (HNPCC) tumors.5Aaltonen LA Peltomaki P Leach FS Sistonen P Pylkkanen L Mecklin JP Jarvinen H Powell SM Jen J Hamilton SR Petersen GM Kinzler KW Vogelstein B de la Chapelle A Clues to the pathogenesis of familial colorectal cancer.Science. 1993; 260: 812-816Crossref PubMed Scopus (2608) Google Scholar Most MS loci are noncoding and selection for these mutations is unlikely. Proportions of altered MS loci have been useful for classification of tumors as either with low or high MSI.6Boland CR Thibodeau SN Hamilton SR Sidransky D Eshleman JR Burt RW Meltzer SJ Rodriguez-Bigas MA Fodde R Ranzani GN Srivastava S A National Cancer Institute Workshop on Microsatellite Instability for Cancer Detection and Familial Predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer.Cancer Res. 1998; 58: 5248-5257PubMed Google Scholar High MSI (MSI-H) is strongly associated with loss of MMR.6Boland CR Thibodeau SN Hamilton SR Sidransky D Eshleman JR Burt RW Meltzer SJ Rodriguez-Bigas MA Fodde R Ranzani GN Srivastava S A National Cancer Institute Workshop on Microsatellite Instability for Cancer Detection and Familial Predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer.Cancer Res. 1998; 58: 5248-5257PubMed Google Scholar Comparisons between tumor and normal alleles of long polyA repeats are sensitive indicators of MSI because most alterations are deletions.7Ionov Y Peinado MA Malkhosyan S Shibata D Perucho M Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis.Nature. 1993; 363: 558-561Crossref PubMed Scopus (2447) Google Scholar Deletions occur sequentially (stepwise) with loss of a single or a few bases at a time.3Shibata D Peinado MA Ionov Y Malkhosyan S Perucho M Genomic instability in repeated sequences is an early somatic event in colorectal tumorigenesis that persists after transformation.Nat Genet. 1994; 6: 273-281Crossref PubMed Scopus (457) Google Scholar, 7Ionov Y Peinado MA Malkhosyan S Shibata D Perucho M Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis.Nature. 1993; 363: 558-561Crossref PubMed Scopus (2447) Google Scholar, 8Percesepe A Pedroni M Sala E Menigatti M Borghi F Losi L Viel A Genuardi M Benatti P Roncucci L Peltomaki P Ponz de Leon M Genomic instability and target gene mutations in colon cancers with different degrees of allelic shifts.Genes Chromosomes Cancer. 2000; 27: 424-429Crossref PubMed Scopus (24) Google Scholar, 9Tran HT Keen JD Kricker M Resnick MA Gordenin DA Hypermutability of homonucleotide runs in mismatch repair and DNA polymerase proofreading yeast mutants.Mol Cell Biol. 1997; 17: 2859-2865Crossref PubMed Scopus (282) Google Scholar Therefore, polyA deletions not only indicate loss of MMR but their extent also provides information on the time since loss of MMR.10Blake C Tsao J-L Wu A Shibata D Stepwise deletions of polyA sequences in mismatch repair deficient colorectal cancers.Am J Pathol. 2001; 158: 1867-1870Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar The MSI of HNPCC adenomas suggests MMR loss occurs early in tumor progression.11Aaltonen LA Peltomaki P Mecklin JP Jarvinen H Jass JR Green JS Lynch HT Watson P Tallqvist G Juhola M Sistonen P Hamilton SR Kinzler KW Vogelstein B de la Chapelle A Replication errors in benign and malignant tumors from hereditary nonpolyposis colorectal cancer patients.Cancer Res. 1994; 54: 1645-1648PubMed Google Scholar, 12Iino H Simms L Young J Arnold J Winship IM Webb SI Furlong KL Leggett B Jass JR DNA microsatellite instability and mismatch repair protein loss in adenomas presenting in hereditary non-polyposis colorectal cancer.Gut. 2000; 47: 37-42Crossref PubMed Scopus (144) Google Scholar, 13Loeb LA A mutator phenotype in cancer.Cancer Res. 2001; 61: 3230-3239PubMed Google Scholar In theory, MMR loss could even precede a gatekeeper mutation because phenotypically normal cells may be MMR-deficient.14Parsons R Li GM Longley M Modrich P Liu B Berk T Hamilton SR Kinzler KW Vogelstein B Mismatch repair deficiency in phenotypically normal human cells.Science. 1995; 268: 738-740Crossref PubMed Scopus (297) Google Scholar It is possible to infer the past from the extent of MS mutations or drift from germline for individual MS loci. Patterns of MS mutations in CA repeat dinucleotide MS loci estimate loss of MMR occurs on average approximately 6.3 years before tumor removal (5.5 years for adenomas and 6.6 years for cancers) and can precede a gatekeeper mutation.15Tsao JL Yatabe Y Salovaara R Jarvinen HJ Mecklin JP Aaltonen LA Tavare S Shibata D Genetic reconstruction of individual colorectal tumor histories.Proc Natl Acad Sci USA. 2000; 97: 1236-1241Crossref PubMed Scopus (123) Google Scholar To further characterize the timing of MMR loss during progression, the extent of deletions at four polyA loci were compared between HNPCC adenomas and cancers. If loss of MMR occurs very early and precedes a gatekeeper mutation, then the extent of polyA deletions should be similar between adenomas and cancers, regardless of prior clinical surveillance. DNA was extracted10Blake C Tsao J-L Wu A Shibata D Stepwise deletions of polyA sequences in mismatch repair deficient colorectal cancers.Am J Pathol. 2001; 158: 1867-1870Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar from formalin-fixed, paraffin-embedded tumor sections of 6 adenomas and 10 carcinomas from 11 Finnish HNPCC patients (Table 1). Clinical information was obtained from patient charts. Surveillance intervals are defined as the time between a negative clinical examination (colonoscopy or prior surgery) and removal of a new tumor. Normal DNA was free of tumor and tumor regions were microdissected to obtain greater than 60% of tumor cells. Germline MLH1 or MSH2 mutations were confirmed by sequencing. The t-test (two-tail) was used to determine the significance between means.Table 1Patient DataPatientAge (yr)TumorSize or stageGermline mutationTotal deletionsClinical interval 146Adenoma1.0 cmMLH1−32NA46CarcinomaBMLH1−38NA 245Adenoma0.3 cmMLH1−342.8 yr 343Adenoma1.0 cmMLH1−39NA43CarcinomaCMLH1−42NA44CarcinomaBMLH1−490.5 yr 464CarcinomaBMLH1−34NA 557CarcinomaBMLH1−40NA 656Adenoma0.5 cmMSH2−292.0 yr 753Adenoma0.5 cmMSH2−282.3 yr 858Adenoma1.0 cmMLH1−343.8 yr58CarcinomaAMLH1−403.8 yr 943CarcinomaBMSH2−41NA1038CarcinomaDMLH1−26NA1157CarcinomaBMLH1−351.0 yrNA, not available. Open table in a new tab NA, not available. PolyA deletions were measured by comparing germline and tumor sizes.10Blake C Tsao J-L Wu A Shibata D Stepwise deletions of polyA sequences in mismatch repair deficient colorectal cancers.Am J Pathol. 2001; 158: 1867-1870Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar Briefly, four polyA repeat loci were examined (BAT20, BAT25, BAT26, and BAT40). Lengths of mononucleotide repeats were estimated by comparing the most intense polymerase chain reaction (PCR) product band between tumor and normal DNA after electrophoresis on 6% sequencing gels, incorporating [33P]-dCTP (NEN Research Products, Boston, MA) with 35 to 38 PCR cycles. In some cases, to better distinguish between tumor and contaminating normal alleles, tumor DNA was diluted before PCR to avoid overlapping stutter bands. Somatic deletions were present in all four polyA loci (BAT20, BAT25, BAT26, and BAT40) in the HNPCC adenomas and cancers (Figure 1 and Table 1). The sum of deletions over the four polyA loci ranged from −26 to −49 bases with overlap between adenomas and cancers (Figure 2). Adenomas had 85% of the deletions observed in the cancers (average of −32.7 versus −38.4 bases). There was a slight trend for larger deletions in larger adenomas, but among cancers, fewer deletions were present in higher stage cancers (Figure 3).Figure 2Summary of polyA deletions in adenomas and carcinomas. Average deletions for all tumors were −8.7 bases for BAT20, −6.3 bases for BAT25, −10.8 bases for BAT26, and −10.4 bases for BAT40. Average total deletions were −32.7 bases for the adenomas versus −38.4 bases for the cancers.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Extensive total polyA deletions regardless of adenoma size or cancer stage.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Some of the HNPCC patients were under surveillance with six known intervals (average of 2.1 years) between tumor removal and a prior negative clinical examination (Table 1). There was a slight trend for fewer deletions in tumors with longer surveillance intervals (Figure 4). Surveillance did not significantly reduce mutations in the polyA sequences because tumors removed after negative clinical examinations had average deletions of −34.8 bases versus −36.5 bases (P = 0.66) for tumors removed without prior surveillance. Loss or inactivation of the functional MMR allele in HNPCC marks a “second-hit” and starts the accumulation of mutations in MS loci. Frame-shift mutations characteristic of MMR deficiencies are found in a number of tumor suppressor loci such as TGFBRII and BAX in HNPCC tumors,16Markowitz S Wang J Myeroff L Parsons R Sun L Lutterbaugh J Fan RS Zborowska E Kinzler KW Vogelstein B Brattain M Willson JKV Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability.Science. 1995; 268: 1336-1338Crossref PubMed Scopus (2158) Google Scholar, 17Rampino N Yamamoto H Ionov Y Li Y Sawai H Reed JC Perucho M Somatic frame-shift mutations in the BAX gene in colon cancers of the microsatellite mutator phenotype.Science. 1997; 275: 967-969Crossref PubMed Scopus (1238) Google Scholar suggesting loss of MMR precedes these mutations. However, designating exactly when MMR loss occurs during progression is difficult because “time” is typically based on morphological and frequency criteria. Mutations present in both adenomas and cancers are “early” mutations whereas “late” mutations are found more frequently in cancers.18Fearon ER Vogelstein B A genetic model for colorectal tumorigenesis.Cell. 1990; 61: 759-767Abstract Full Text PDF PubMed Scopus (10351) Google Scholar The extensive MSI observed in HNPCC adenomas and carcinomas suggest loss of MMR occurs early in progression.3Shibata D Peinado MA Ionov Y Malkhosyan S Perucho M Genomic instability in repeated sequences is an early somatic event in colorectal tumorigenesis that persists after transformation.Nat Genet. 1994; 6: 273-281Crossref PubMed Scopus (457) Google Scholar, 11Aaltonen LA Peltomaki P Mecklin JP Jarvinen H Jass JR Green JS Lynch HT Watson P Tallqvist G Juhola M Sistonen P Hamilton SR Kinzler KW Vogelstein B de la Chapelle A Replication errors in benign and malignant tumors from hereditary nonpolyposis colorectal cancer patients.Cancer Res. 1994; 54: 1645-1648PubMed Google Scholar, 12Iino H Simms L Young J Arnold J Winship IM Webb SI Furlong KL Leggett B Jass JR DNA microsatellite instability and mismatch repair protein loss in adenomas presenting in hereditary non-polyposis colorectal cancer.Gut. 2000; 47: 37-42Crossref PubMed Scopus (144) Google Scholar, 13Loeb LA A mutator phenotype in cancer.Cancer Res. 2001; 61: 3230-3239PubMed Google Scholar Determining when a mutation occurs may be confounded by the requirement that only mutations accompanied by a gatekeeper mutation are detectable. A gatekeeper mutation1Kinzler KW Vogelstein B Lessons from hereditary colorectal cancer.Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4321) Google Scholar is defined here as the first mutation that allows for visible clonal expansion. Progenitors with mutations other than gatekeeper mutations are “invisible”. Such scenarios are illustrated in Figure 5 using an “imaginary” observer with the omniscient ability to see a MMR “clock” which may start whether or not it is accompanied by a tumor. Imaginary and real observers will agree on late MMR loss because tumors with and without MMR deficiency can be sampled. In contrast, only the imaginary observer can see the early MMR loss and accumulation of MS mutations that precede a gatekeeper mutation because a real observer will see nothing. However, a real observer may be just as knowledgeable because the clock can be examined when the tumor is removed. Assuming an ability to recognize and interpret this clock, a real observer can infer when it started. PolyA deletions were similar between HNPCC adenomas and cancers, and between tumors regardless of clinical surveillance. The scenario most consistent with these observations is MMR loss that greatly precedes a gatekeeper mutation. With this scenario (Figure 5), adenomas have as nearly as many polyA deletions as cancers because most MS mutations occur before a gatekeeper mutation. Tumors arising with or without prior negative clinical examinations would have similar numbers of deletions because most deletions accumulate in occult progenitors. With early MMR loss, the final extent of MSI in HNPCC adenomas or cancers would not be surprising for an imaginary omnipresent observer (Figure 5), but a real observer might conclude that MMR loss is associated with catastrophic MSI because intermediate MSI states are seldom observed.19Thibodeau SN Bren G Schaid D Microsatellite instability in cancer of the proximal colon.Science. 1993; 260: 816-819Crossref PubMed Scopus (2843) Google Scholar, 20Thibodeau SN French AJ Cunningham JM Tester D Burgart LJ Roche PC McDonnell SK Schaid DJ Vockley CW Michels VV Farr Jr, GH O'Connell MJ Microsatellite instability in colorectal cancer: different mutator phenotypes and the principal involvement of hMLH1.Cancer Res. 1998; 58: 1713-1718PubMed Google Scholar A real observer may also question whether polyA deletions reflect times since loss of MMR because proliferation kinetics vary during progression. However, a key feature of very early MMR loss is that phenotypic differences between adenomas or cancers would have relatively minor effects on the polyA clock because most of their histories are written in normal colon. Most MS mutations accumulate unobserved in phenotypically normal progenitors that are likely to have similar proliferation rates. In addition, polyA repeat sequences exhibit molecular clock properties in many different MMR deficient cells including normal murine intestines and human cancer cell lines.3Shibata D Peinado MA Ionov Y Malkhosyan S Perucho M Genomic instability in repeated sequences is an early somatic event in colorectal tumorigenesis that persists after transformation.Nat Genet. 1994; 6: 273-281Crossref PubMed Scopus (457) Google Scholar, 7Ionov Y Peinado MA Malkhosyan S Shibata D Perucho M Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis.Nature. 1993; 363: 558-561Crossref PubMed Scopus (2447) Google Scholar, 8Percesepe A Pedroni M Sala E Menigatti M Borghi F Losi L Viel A Genuardi M Benatti P Roncucci L Peltomaki P Ponz de Leon M Genomic instability and target gene mutations in colon cancers with different degrees of allelic shifts.Genes Chromosomes Cancer. 2000; 27: 424-429Crossref PubMed Scopus (24) Google Scholar, 9Tran HT Keen JD Kricker M Resnick MA Gordenin DA Hypermutability of homonucleotide runs in mismatch repair and DNA polymerase proofreading yeast mutants.Mol Cell Biol. 1997; 17: 2859-2865Crossref PubMed Scopus (282) Google Scholar, 10Blake C Tsao J-L Wu A Shibata D Stepwise deletions of polyA sequences in mismatch repair deficient colorectal cancers.Am J Pathol. 2001; 158: 1867-1870Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar Total deletions are not precise time measures because mutations are stochastic10Blake C Tsao J-L Wu A Shibata D Stepwise deletions of polyA sequences in mismatch repair deficient colorectal cancers.Am J Pathol. 2001; 158: 1867-1870Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar and confidence intervals (not calculated here) would be expected to be large. The polyA repeat tumor clock analysis also concurs with a quantitative analysis of CA-dinucleotide repeats,15Tsao JL Yatabe Y Salovaara R Jarvinen HJ Mecklin JP Aaltonen LA Tavare S Shibata D Genetic reconstruction of individual colorectal tumor histories.Proc Natl Acad Sci USA. 2000; 97: 1236-1241Crossref PubMed Scopus (123) Google Scholar with similar and small differences between adenomas and cancers, with or without surveillance (Table 2). Although the current polyA sequence analysis is not formally quantitative, both CA- and polyA-repeat mutation patterns support the conclusion that loss of MMR often precedes a gatekeeper mutation. The mutation spectrum of APC is also consistent with MMR loss preceding APC mutation in most MSI-H cancers.21Huang J Papadopoulos N McKinley AJ Farrington SM Curtis LJ Wyllie AH Zheng S Willson JK Markowitz SD Morin P Kinzler KW Vogelstein B Dunlop MG APC mutations in colorectal tumors with mismatch repair deficiency.Proc Natl Acad Sci USA. 1996; 93: 9049-9054Crossref PubMed Scopus (298) Google Scholar APC mutations likely lag BAT polyA repeat deletions because APC coding regions lack long repeats (≤A8) and mutation rates are markedly lower for shorter polyA repeats.9Tran HT Keen JD Kricker M Resnick MA Gordenin DA Hypermutability of homonucleotide runs in mismatch repair and DNA polymerase proofreading yeast mutants.Mol Cell Biol. 1997; 17: 2859-2865Crossref PubMed Scopus (282) Google ScholarTable 2Total PolyA Deletions versus CA-Repeat Clock DataSpecimenPolyA deletionsCA-repeat tumor clock: intervals since MMR loss*From Tsao et al.15Adenoma−32.7 bases2000 divisionsCancer−38.4 bases2400 divisionsRatio0.850.83Interval tumors−34.8 bases6.0 yearsNon-interval tumors−36.5 bases6.2 yearsRatio0.950.97* From Tsao et al.15Tsao JL Yatabe Y Salovaara R Jarvinen HJ Mecklin JP Aaltonen LA Tavare S Shibata D Genetic reconstruction of individual colorectal tumor histories.Proc Natl Acad Sci USA. 2000; 97: 1236-1241Crossref PubMed Scopus (123) Google Scholar Open table in a new tab The start of progression has been usually defined as a visible change in phenotype.22Nowell PC The clonal evolution of tumor cell populations.Science. 1976; 194: 23-28Crossref PubMed Scopus (5019) Google Scholar However, the start of genetic progression (the accumulation of mutations) does not need to coincide with visible HNPCC tumor progression because MMR deficient tissues may accumulate MS mutations and remain phenotypically normal.14Parsons R Li GM Longley M Modrich P Liu B Berk T Hamilton SR Kinzler KW Vogelstein B Mismatch repair deficiency in phenotypically normal human cells.Science. 1995; 268: 738-740Crossref PubMed Scopus (297) Google Scholar, 23Vilkki S Tsao JL Loukola A Poyhonen M Vierimaa O Herva R Aaltonen LA Shibata D Extensive somatic microsatellite mutations in normal human tissue.Cancer Res. 2001; 61: 4541-4544PubMed Google Scholar Many or most mutations may potentially accumulate before the onset of visible neoplasia. Loss of MMR may not even mark the start of genetic progression because some mutations found in MSI-H tumors lack features characteristic of MMR loss.21Huang J Papadopoulos N McKinley AJ Farrington SM Curtis LJ Wyllie AH Zheng S Willson JK Markowitz SD Morin P Kinzler KW Vogelstein B Dunlop MG APC mutations in colorectal tumors with mismatch repair deficiency.Proc Natl Acad Sci USA. 1996; 93: 9049-9054Crossref PubMed Scopus (298) Google Scholar Further systematic studies of tumor mutations may help better characterize the frequencies and lengths of occult genetic progression periods. We thank Sylvia I. Lambrechts and Sarka Cernosek for technical assistance.
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