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Originally published as JCO Early Release 10.1200/JCO.2005.02.7227 on December 5 2005 © 2006 American Society of Clinical Oncology. Multipopulation Analysis of Polymorphisms in Five Mononucleotide Repeats Used to Determine the Microsatellite Instability Status of Human TumorsFrom the Institut National de la Santé et de la Recherche Médicale (INSERM) U434, Centre d'Etude du Polymorphisme Humain (CEPH); Service d'Anatomo-Pathologie, Hôpital Saint-Antoine, Paris, France; Dipartimento di Genetica e Microbiologica, University of Pavia, Pavia, Italy; Department of Obstetrics & Gynaecology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong; and the Institute of Genetics, Chaim Sheba Medical Center, Tel-Hashomer, Israel Address reprint requests to Richard Hamelin, INSERM U434-CEPH, 27 rue Juliette Dodu, 75010 Paris, France; e-mail: richard.hamelin{at}cephb.fr
PURPOSE: Human gastrointestinal tumors with inactivated DNA mismatch repair system (microsatellite instability [MSI] tumors) have distinct molecular and clinicopathologic profiles, and are associated with favorable prognosis. There is evidence suggesting that colorectal cancer patients with MSI tumors respond differently to adjuvant chemotherapy as compared with patients with non-MSI tumors. Finally, determination of the MSI status has clinical application for assisting in the diagnosis of suspected hereditary cases. It is thus becoming increasingly recognized that testing for MSI should be conducted systematically in all human cancers potentially of this type. We recently described a pentaplex polymerase chain reaction of five mononucleotide repeats to establish the MSI status of human tumors, and showed that this assay was 100% sensitive and specific. Moreover, these markers are quasimonomorphic in germline DNA of the white population (ie, individuals of Eurasian origin), and could be used for tumor MSI determination without the requirement for matching normal DNA in this group. PATIENTS AND METHODS: In this study, we analyzed a comparable panel of five mononucleotide markers in germline DNA from 1,206 individuals encompassing 55 different populations worldwide. RESULTS: With the exception of two Biaka Pygmies and one San individual for whom three markers showed variant alleles (three cases [0.2%]), the remaining 1,203 individuals showed no alleles of variant size (1,055 cases [87.5%]), or only one (122 cases [10.1%]) or two (26 cases [2.2%]) markers with variant alleles. All 60 MSI tumors investigated display instability in at least four of the five markers. CONCLUSION: We demonstrated that tumor MSI status can be determined using the pentaplex reaction for all human populations without the need for matching normal DNA.
Microsatellites are highly polymorphic, repetitive nucleotide sequences widely used to detect loss of heterozygosity in human cancers. The observation in a subset of tumors of new microsatellite alleles that were absent in matching normal DNA led to the discovery of the so-called microsatellite instability (MSI) phenotype1-3 in tumors that are now referred to as MSI-high (MSI-H).4 This phenotype is a marker of an underlying mismatch repair (MMR) defect that fails to recognize errors introduced during DNA replication.5-8 MSI-H is found in approximately 10% to 15% of sporadic colon, gastric and endometrial tumors and in the majority of tumors from patients with hereditary nonpolyposis colorectal cancer (HNPCC) syndrome.4 MSI-H tumors have different clinicopathologic features compared to tumors without this phenotype,9 termed microsatellite stable (MSS), and the repertoire of genetic events involved in tumoral progression of both phenotypes is different.10,11 MSI-H colonic and gastric tumors are associated with favorable prognosis.1,9,12,13 There is also evidence to suggest that colorectal cancer patients with MSI-H tumors respond differently to fluorouracil-based chemotherapy.14,15 In addition, MSI-H determination has clinical application for assisting in the diagnosis of suspected HNPCC in instances in which the current clinical criteria are not sufficiently sensitive or specific.16 Thus, it is becoming recognized that MSI-H evaluation should be performed routinely in all human tumors that potentially have this phenotype because it may provide clinically relevant information. In 1997, an international consensus meeting held in Bethesda, MD, proposed a panel of five markers for the uniform analysis of MSI.4 This included two mononucleotide (BAT-25 and BAT-26) and three dinucleotide (D5S346, D2S123, and D17S250) repeats. Tumors with instability at two or more of these markers were defined as being MSI-H, whereas those with instability at one repeat or showing no instability were defined as MSI-low and MSS tumors respectively. It is now widely accepted that only the MSI-H phenotype identifies HNPCC and sporadic tumors with MMR defects and with the distinct clinicopathologic features.17,18 The Bethesda panel of markers has been re-evaluated at a more recent international consensus meeting held in December 2002.19 It was concluded there were caveats on its use and it was recommended that dinucleotide repeats be substituted by mononucleotide repeats. We recently described a pentaplex polymerase chain reaction (PCR) assay comprising five mononucleotide repeats.20 Because of the quasimonomorphic nature of these repeats in the white population (ie, individuals of Eurasian origin), we proposed that the pentaplex assay could be used to establish the MSI status of tumors in this population without the need for matching normal DNA. To evaluate the possible application of this assay to nonwhite populations, we analyzed a similar panel of five mononucleotide repeats in a large series of normal DNA from 55 diverse worldwide populations. Various frequencies of polymorphisms in the five repeats were observed within these populations and are presented here.
DNA Samples The Human Genome Diversity panel is a collection of immortalized lymphoblastoid cell lines (LCL) developed by the Human Genome Diversity Project/Centre d'Etude du Polymorphisme Humain (HGDP/CEPH).21,22 It contains 1,064 LCL from different individuals belonging to 52 populations scattered throughout the world (Fig 1). In addition, germline DNA was genotyped from 68 Hong Kong Chinese women with an endometrial carcinoma, and from 50 non-Ashkenazi and 50 Ashkenazi Jewish Israeli women with breast cancer.
MSI-H tumors were selected from a larger series of primary colorectal tumors obtained from patients undergoing surgery at Saint-Antoine Hospital (Paris, France). DNA was extracted from frozen tissues using standard methods.
Pentaplex PCR
Reading the Individual PCR Profiles All PCR amplification profiles were scored individually, and the size of both alleles was determined for each mononucleotide repeat, and for each individual. As expected for such long repeats, amplification products obtained are not a single unique peak, but rather are seen as several peaks differing by 1 bp due to stuttering by Taq polymerase. Several comments should be made regarding our reading procedure. First, sizes were deduced with Genescan software (Genotyper 2.1; Applera France, Courtaboeuf, France) but can be considered only as approximations and not real values. This is indicated by the fact that the software provides decimal values, although the bp distribution of a PCR fragment is by definition an integer. We systematically rounded the experimental values up or down to the closest integer. We noticed that sizes deduced from analysis with an ABI PRISM 310 Genetic Analyzer were 2 bp shorter than those obtained with an ABI PRISM 3100 Genetic Analyzer. This fact—a result of the use of a different performance-optimized polymer, and possibly other technical factors as well—needs to be considered when using different apparatus for the same analysis. In our case, we applied a correction coefficient to the few ABI PRISM 310 results because most of our analyses were performed with a 3100. To measure the size of both alleles in each individual and to harmonize our readings, we took advantage of the few DNA samples containing a variant allele with a clearly distinct size (Fig 2A). In these particular cases, there is a single allele in the normal size range indicating the characteristic profile of a homozygous sample for this marker (Fig 2B) and, by inference, the profiles of heterozygous samples in which both alleles have 1- or 2-bp differences in size (Fig 2C).
Reading the Multiethnic Germline DNA Profiles Of the 1,064 individuals from the HGDP/CEPH diversity panel, amplification was successful for the five markers in 1,042 samples. It was successful for all 68 germline DNA samples from the Hong Kong Chinese and for 96 of the 100 Jewish samples. Altogether, the five markers were successfully amplified in a total of 1,206 individuals representing 55 worldwide populations. The data analysis pertains to these samples and thus concerns 2,412 alleles for each marker. Table 2 indicates the sizes of the alleles for each marker, and their number and percentage in the worldwide population. It should be noted these values obtained experimentally are slightly different from the sizes deduced from sequence databases (Table 1).
From results shown in Table 2, the different alleles were classified as variant (light gray shading) or within the quasimonomorphic variation range (QMVR) of each marker (dark gray shading). NR-24 is remarkably stable with the size of quasimonomorphic alleles ranging from 124 to 128 bp. Only two alleles of 114 and 130 bp were clearly different from the QMVR, and were detected once each. For NR-21, BAT-25, and BAT-26, there is a clear bimodal or trimodal distribution distinguishing the QMVR from variant alleles. For NR-21, the QMVR is 105 to 110 bp. We considered every allele equal to or below 104 bp as a variant, with 103 bp as the main variant allelic size. Since 111-bp alleles were all found in the same population (Biaka Pygmies), we also considered them as variant alleles although of a larger size than the QMVR of NR-21. All other variants from any marker present a smaller allelic size than corresponding QMVR. For BAT-25, the QMVR was considered 146 to 151 bp with variant alleles below or equal to 145 bp, comprising two distinct groups around 136 and 144 bp. The QMVR of BAT-26 is 176 to 182 bp and two groups of allelic variants are clearly distinct around 167 and 171 bp. The allelic distribution for NR-27 is not bimodal, but because 85-bp alleles were over-represented in the African populations, we considered the QMVR for NR-27 as 86 to 89 bp, and every allele equal to or below 85 bp as a variant. In the complete series with amplification of all five markers, the percentage of variant alleles were for the different markers: 2.6% (62 of 2,412) for NR-27, 2.6% (62 of 2,412) for NR-21, 0.1% (two of 2,412) for NR-24, 1.7% (40 of 2,412) for BAT-25, and 1.2% (30 of 2,412) for BAT-26 (Fig 3A). Figure 4A shows the percentage of each allelic size, variant or within the QMVR, for each marker in the complete worldwide series. It appears that all markers conserve their quasimonomorphic nature and that variant alleles are barely visible due to their low incidence.
For each marker, we have indicated the populations showing variant alleles in a decreasing order (Table 3). Besides variant alleles, the quasimonomorphic nature of the more common alleles for each marker is conserved in all analyzed worldwide populations. We then grouped the 55 populations within seven major geographic regions and indicated for each marker the total percentage of variant alleles (Fig 5, left panels). Subsaharan Africans show variant alleles above or approaching 10% for NR-27, BAT-25, and BAT-26, whereas Oceanians show variant above 10% for NR-21. None of the other geographic regions reached a frequency of 10% variant alleles for any of the markers.
We also calculated a polymorphic index for each population, indicating the mean value of genetic variation for the five markers. When classified in decreasing order, the top six populations are from Subsaharan Africa, and four of these show a polymorphic index above 10% (Table 4). Among the major geographic regions, only Africa approaches a polymorphic index of 10% (Fig 5, left panels).
We then looked at the number of variant alleles for each individual. This is indicated in Figure 5 (right panels) as the number of individuals in the major geographic regions with 0, 1, or more allelic variants. In the Middle East/North Africa, Central/South Asia, East Asia, America, and Europe regions, more than 95% of individuals show variant alleles in none of the five markers, whereas the remaining individuals present variant alleles in only one of the markers. In Oceania, 69% of the 39 individuals analyzed show no polymorphism. The remaining 31% show variant alleles on only one marker, NR-21. We analyzed 125 individuals from Subsaharan Africa. Forty-two (33.6%) of these have a single variant allele and 24 (19.2%) have two. Three individuals (two Biaka Pygmies and one San) were polymorphic on three markers. The remaining 56 African individuals (44.8%) showed no polymorphism. When the same analysis was performed on the complete worldwide series (Fig 3B), 1,055 individuals (87.5%) showed no polymorphism for any of the markers, whereas 122 (10.1%) and 26 (2.2%) showed variant alleles at one or two markers respectively. The two Pygmies and one San with three variant markers represent only 0.2% of the complete multiethnic series.
Reading the MSI-H DNA Profiles
We analyzed the allelic sizes of a panel of five mononucleotide markers in a series of germline DNA from 1,206 individuals belonging to 55 different worldwide populations. The frequency of polymorphism for each marker is highly variable from one population to another. The aim of this study is not a population genetic analysis, but some observations can be highlighted. With the exception of NR-24, an African population shows the highest polymorphic rate for each marker: Biaka Pygmies for NR-27 and BAT-26 (34.7% and 15.3%, respectively), Bantus from South Africa for NR-21 (18.8%), and the San people for BAT-25 (57.1%). For NR-27, BAT-25, and BAT-26, the 11 populations showing polymorphic frequencies above 10% are all from Subsaharan Africa. The NR-21 variant alleles are more widespread: Among the six populations showing a polymorphic frequency above 10%, one is from South Africa (Bantus), two from Oceania (Melanesian and Papuan), one from Central/South Asia (Makrani from Pakistan) and two Chinese minorities from East Asia (Dai and Tujia). In this work, seven populations were from Africa. Bantus from South Africa show variant frequencies above 10% for three of the markers (NR-21, BAT-26, and BAT-25). Variant alleles frequencies above 10% were also present for BAT-25 and NR-27 in the San and Mbuti Pygmies, for NR-27 and BAT-26 in Biaka Pygmies and Yorubas, and for BAT-25 in only Bantus from Kenya. The only Subsaharan African population that did not show a high polymorphic frequency for any of the markers was Mandenkas from Senegal. Taking all markers together, six of the seven Subsaharan African populations (excluding again Mandenkas) comprised the worldwide populations showing the highest polymorphic index (Table 4). Polymorphisms in BAT-26 and BAT-25 were reported previously for African Americans and for a small population of Nigerians.25,26 We confirm here a relatively high incidence of polymorphism for other mononucleotide repeats in the African populations. Apart from the African ethnic groups, a relatively high polymorphic index for any of the markers was rare and isolated. NR-21 was polymorphic in more than 10% of at least one population from four of the seven major geographic regions. For Papuans and Melanesians from Oceania, 17.6% and 13.6% of the alleles for NR-21 have a variant size, and as a consequence, 31% of the 39 individuals analyzed in this series are monomorphic at four markers, but polymorphic at NR-21 (no homozygous variant alleles in this series). For BAT-26 or BAT-25, the best known markers for MSI-H evaluation,27,28 the only non-African population approaching a 10% variant frequency are the Pimas from Mexico (8% for BAT-26). The white population is considered as originating from Eurasia (Europe, Middle East/North Africa and South-Central Asia). Of the 22 different white populations analyzed in this study, only the French Basques (8.3% for NR-27), and Makrani from Pakistan (14% for NR-21), approached or reached 10% polymorphic index for any of the markers. When considering the entire worldwide series, all markers were monomorphic in the great majority of the cases (1,055 individuals [87.5%]). Variant alleles for one or two markers were observed in 122 and 26 individuals (10.1% and 2.2%, respectively), respectively. There were only three individuals (0.2%) showing allelic variants at three markers. Alternatively, four or five of these markers were unstable in nine (15%) and 51 (85%), respectively, of the 60 MSI-H colorectal tumors studied here. The exact ethnic origin of the cancer patients is unknown, but they most likely all originate from Europe or from North Africa, with possibly some individuals from Africa and/or the French Antilles. We have thus not analyzed the five markers of the pentaplex assay in MSI-H tumors from a worldwide population, but there is no reason to believe that mechanisms of microsatellite instability differ from one world population to another. A simple, sensitive, and specific method to determine the MSI status of human tumors that are potentially of this type is becoming increasingly important for clinicians due to the genetic, prognostic and diagnostic implications of this phenotype. NR-24 was monomorphic in all but two individuals of the worldwide series of normal DNA analyzed here. Its analysis would not be sufficient, however, to establish the MSI status of tumors, since it showed instability in only 90% of the MSI-H tumors analyzed in this study. When using the pentaplex of mononucleotide repeats, we have demonstrated a clear difference between the number of polymorphisms in germline worldwide DNA (zero to three) and their instability in gastrointestinal MSI-H tumors (four or five). The three individuals who showed variant alleles on three of five markers were two Biaka Pygmies and one San individual. In our earlier article describing the pentaplex assay,20 we reported an MSI-H gastric tumor sample showing instability at only three markers. This sample would be the only one for which MSI determination without matching normal DNA would be problematic using the pentaplex assay. However, the patient was white, in which populations germline DNA is never polymorphic at two markers. We therefore conclude that a gastrointestinal tumor is MSI-H if instability is detected in at least three of the five markers of the pentaplex assay and this conclusion can be reached without the need to analyze matching normal DNA. This is true for any population worldwide, with the exception of Biaka Pygmies and the San, for which a minimum of four unstable markers is required to establish MSI-H.
The authors indicated no potential conflicts of interest.
We thank Barry Iacopetta, PhD, for critical reading of the manuscript. DNAs from the Human Genome Diversity Project were obtained thanks to Howard Cann, MD, and Claudia de Toma at the Centre d'Etude du Polymorphisme Humain (Paris, France).
Supported in part by Association de la Recherche Contre le Cancer, and an Interface grant from INSERM/Assistance Publique-Hôpitaux de Paris. Exchange agreements were obtained from INSERM–Consiglio Nazionale delle Ricerche and Procore-France/Hong-Kong Joint Research Scheme grants for Italy and Hong-Kong, respectively. Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
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