Familial Cancer

, 7:319 | Cite as

Genome-wide copy neutral LOH is infrequent in familial and sporadic microsatellite unstable carcinomas

  • Marjo van Puijenbroek
  • Anneke Middeldorp
  • Carli M. J. Tops
  • Ronald van Eijk
  • Heleen M. van der Klift
  • Hans F. A. Vasen
  • Juul Th. Wijnen
  • Frederik J. Hes
  • Jan Oosting
  • Tom van Wezel
  • Hans Morreau


Mismatch repair deficiency in tumors can result from germ line mutations in one of the mismatch repair (MMR) genes (MLH1, MSH2, MSH6 and PMS2), or from sporadic promoter hypermethylation of MLH1. The role of unclassified variants (UVs) in MMR genes is subject to debate. To establish the extend of chromosomal instability and copy neutral loss of heterozygosity (cnLOH), we analyzed 41 archival microsatellite unstable carcinomas, mainly colon cancer, from 23 patients with pathogenic MMR mutations, from eight patients with UVs in one of the MMR genes and 10 cases with MLH1 promoter hypermethylation. We assessed genome wide copy number abnormalities and cnLOH using SNP arrays. SNP arrays overcome the problems of detecting LOH due to instability of polymorphic microsatellite markers. All carcinomas showed relatively few chromosomal aberrations. Also cnLOH was infrequent and in Lynch syndrome carcinomas usually confined to the locus harbouring pathogenic mutations in MLH1, MSH2 or PMS2 In the carcinomas from the MMR-UV carriers such cnLOH was less common and in the carcinomas with MLH1 promoter hypermethylation no cnLOH at MLH1 occurred. MSI-H carcinomas of most MMR-UV carriers present on average with more aberrations compared to the carcinomas from pathogenic MMR mutation carriers, suggesting that another possible pathogenic MMR mutation had not been missed. The approach we describe here shows to be an excellent way to study genome-wide cnLOH in archival mismatch repair deficient tumors.


Lynch syndrome HNPCC MSI-H Chromosomal instability Copy neutral loss of heterozygosity Mismatch repair (MMR) genes Unclassified variants MLH1 hypermethylation SNP array 



Comparative genomic hybridization


Chromosomal instability


Copy number aberrations


Copy neutral loss of heterozygosity


Colorectal cancer


Formalin-fixed paraffin-embedded


Gene call score


Gene train score




Loss of heterozygosity


Linkage panels


Mismatch repair


Microsatellite instability


Microsatellite instability


Microsatellite stable


Relative gene call score


Smallest region of overlap


Unclassified variants



We thank Illumina for providing us with part of the SNP arrays, and Ruben van ‘t Slot and Diandhra Erasmus for technical support. This study was supported by the Dutch Cancer Society, grants UL2003–2807 and UL2005–3247.


  1. 1.
    Parsons R, Li GM, Longley MJ et al (1993) Hypermutability and mismatch repair deficiency in Rer+ tumor-cells. Cell 75:1227–1236PubMedCrossRefGoogle Scholar
  2. 2.
    Lynch HT, Smyrk T (1996) Hereditary nonpolyposis colorectal cancer (Lynch syndrome)—an updated review. Cancer 78:1149–1167PubMedCrossRefGoogle Scholar
  3. 3.
    Yuen ST, Chan TL, Ho JWC et al (2002) Germline, somatic and epigenetic events underlying mismatch repair deficiency in colorectal and HNPCC-related cancers. Oncogene 21:7585–7592PubMedCrossRefGoogle Scholar
  4. 4.
    Ionov Y, Peinado MA, Malkhosyan S et al (1993) Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 363:558–561PubMedCrossRefGoogle Scholar
  5. 5.
    Thibodeau SN, Bren G, Schaid D (1993) Microsatellite instability in cancer of the proximal colon. Science 260:816–819PubMedCrossRefGoogle Scholar
  6. 6.
    Perucho M (1996) Cancer of the microsatellite mutator phenotype. Biol Chem 377:675–684PubMedGoogle Scholar
  7. 7.
    Kouri M, Laasonen A, Mecklin JP et al (1990) Diploid predominance in hereditary nonpolyposis colorectal-carcinoma evaluated by flow-cytometry. Cancer 65:1825–1829PubMedCrossRefGoogle Scholar
  8. 8.
    Kouri M (1993) Dna ploidy of colorectal-carcinoma by tumor site, gender and history of noncolorectal malignancies. Oncology 50:41–45PubMedCrossRefGoogle Scholar
  9. 9.
    Trautmann K, Terdiman JP, French AJ et al (2006) Chromosomal instability in microsatellite-unstable and stable colon cancer. Clin Cancer Res 12:6379–6385PubMedCrossRefGoogle Scholar
  10. 10.
    Kapiteijn E, Liefers GJ, Los LC et al (2001) Mechanisms of oncogenesis in colon versus rectal cancer. J Pathol 195:171–178PubMedCrossRefGoogle Scholar
  11. 11.
    Frattini M, Balestra D, Suardi S et al (2004) Different genetic features associated with colon and rectal carcinogenesis. Clin Cancer Res 10:4015–4021PubMedCrossRefGoogle Scholar
  12. 12.
    Beart RW, Melton LJ, Maruta M et al (1983) Trends in right and left-sided colon cancer. Dis Colon Rectum26:393–398PubMedCrossRefGoogle Scholar
  13. 13.
    Diep CB, Kleivi K, Ribeiro FR et al (2006) The order of genetic events associated with colorectal cancer progression inferred from meta-analysis of copy number changes. Genes Chromosomes Cancer 45:31–41PubMedCrossRefGoogle Scholar
  14. 14.
    Nakao K, Mehta KR, Moore DH et al (2004) High-resolution analysis of DNA copy number alterations in colorectal cancer by array-based comparative genomic hybridization. Carcinogenesis 25:1345–1357PubMedCrossRefGoogle Scholar
  15. 15.
    Lips EH, Dierssen JWF, van Eijk R et al (2005) Reliable high-throughput genotyping and loss-of-heterozygosity detection in formalin-fixed, paraffin-embedded tumors using single nucleotide polymorphism arrays. Cancer Res 65:10188–10191PubMedCrossRefGoogle Scholar
  16. 16.
    Sugai T, Takahashi H, Habano W et al (2003) Analysis of genetic alterations, classified according to their DNA ploidy pattern, in the progression of colorectal adenomas and early colorectal carcinomas. J Pathol 200:168–176PubMedCrossRefGoogle Scholar
  17. 17.
    Young J, Simms LA, Biden KG et al (2001) Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings: parallel pathways of tumorigenesis. Am J Pathol 159:2107–2116PubMedGoogle Scholar
  18. 18.
    Jass JR (2007) Classification of colorectal cancer based on correlation of clinical, morphological and molecular features. Histopathology 50:113–130PubMedCrossRefGoogle Scholar
  19. 19.
    de Jong AE, van Puijenbroek M, Hendriks Y et al (2004) Microsatellite instability, immunohistochemistry, and additional PMS2 staining in suspected hereditary nonpolyposis colorectal cancer. Clin Cancer Res 10:972–980PubMedCrossRefGoogle Scholar
  20. 20.
    Hendriks YM, Jagmohan-Changur S, van der Klift HM et al (2006) Heterozygous mutations in PMS2 cause hereditary nonpolyposis colorectal carcinoma (Lynch syndrome). Gastroenterology 130:312–322PubMedCrossRefGoogle Scholar
  21. 21.
    Nygren AO, Ameziane N, Duarte HM et al (2005) Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences. Nucleic Acids Res 33:e128PubMedCrossRefGoogle Scholar
  22. 22.
    Oosting J, Lips EH, van Eijk R et al (2007) High-resolution copy number analysis of paraffin-embedded archival tissue using SNP BeadArrays. Genome Res 17:368–376PubMedCrossRefGoogle Scholar
  23. 23.
    Lips EH, van Eijk R, de Graaf E et al (2008) Progression and tumor heterogeneity analysis in early rectal cancer. Clin Cancer Res 14:772–781PubMedCrossRefGoogle Scholar
  24. 24.
    de Leeuw WJ, van PM, Merx R et al (2001) Bias in detection of instability of the (C)8 mononucleotide repeat of MSH6 in tumours from HNPCC patients. Oncogene 20:6241–6244Google Scholar
  25. 25.
    Larramendy ML, El-Rifai W, Kokkola A et al (1998) Comparative genomic hybridization reveals differences in DNA copy number changes between sporadic gastric carcinomas and gastric carcinomas from patients with hereditary nonpolyposis colorectal cancer. Cancer Genet Cytogenet 106:62–65PubMedCrossRefGoogle Scholar
  26. 26.
    Gaasenbeek M, Howarth K, Rowan AJ et al (2006) Combined array-comparative genomic hybridization and single-nucleotide polymorphism-loss of heterozygosity analysis reveals complex changes and multiple forms of chromosomal instability in colorectal cancers. Cancer Res 66:3471–3479PubMedCrossRefGoogle Scholar
  27. 27.
    Hemminki A, Peltomaki P, Mecklin JP et al (1994) Loss of the wild-type Mlh1 gene is a feature of hereditary nonpolyposis colorectal-cancer. Nat Genet 8:405–410PubMedCrossRefGoogle Scholar
  28. 28.
    Lu SL, Akiyama Y, Nagasaki H et al (1996) Loss or somatic mutations of hMSH2 occur in hereditary nonpolyposis colorectal cancers with hMSH2 germline mutations. Jpn J Cancer Res 87:279–287PubMedGoogle Scholar
  29. 29.
    Tannergard P, Liu T, Weger A et al (1997) Tumorigenesis in colorectal tumors from patients with hereditary non-polyposis colorectal cancer. Hum Genet 101:51–55PubMedCrossRefGoogle Scholar
  30. 30.
    Kuismanen SA, Holmberg MT, Salovaara R et al (2000) Genetic and epigenetic modification of MLH1 accounts for a major share of microsatellite-unstable colorectal cancers. Am J Pathol 156:1773–1779PubMedGoogle Scholar
  31. 31.
    Potocnik U, Glavac D, Golouh R et al (2001) Causes of microsatellite instability in colorectal tumors: implications for hereditary non-polyposis colorectal cancer screening. Cancer Genet Cytogenet 126:85–96PubMedCrossRefGoogle Scholar
  32. 32.
    de Abajo AS, de la Hoya M, van Puijenbroek M et al (2006) Dual role of LOH at MMR loci in hereditary non-polyposis colorectal cancer? Oncogene 25:2124–2130CrossRefGoogle Scholar
  33. 33.
    Tuupanen S, Karhu A, Jarvinen H et al (2007) No evidence for dual role of loss of heterozygosity in hereditary non-polyposis colorectal cancer. Oncogene 26:2513–2517PubMedCrossRefGoogle Scholar
  34. 34.
    Ollikainen M, Hannelius U, Lindgren CM et al (2007) Mechanisms of inactivation of MLH1 in hereditary nonpolyposis colorectal carcinoma: a novel approach. Oncogene 26:4541–4549PubMedCrossRefGoogle Scholar
  35. 35.
    Konishi M, KikuchiYanoshita R, Tanaka K et al (1996) Molecular nature of colon tumors in hereditary nonpolyposis colon cancer, familial polyposis, and sporadic colon cancer. Gastroenterology 111:307–317PubMedCrossRefGoogle Scholar
  36. 36.
    Horii A, Han HJ, Sasaki S et al (1994) Cloning, characterization and chromosomal assignment of the human genes homologous to yeast Pms1, a member of mismatch repair genes. Biochem Biophys Res Commun 204:1257–1264PubMedCrossRefGoogle Scholar
  37. 37.
    Nicolaides NC, Carter KC, Shell BK et al (1995) Genomic organization of the human Pms2 gene family. Genomics 30:195–206PubMedCrossRefGoogle Scholar
  38. 38.
    De Vos M, Hayward BE, Picton S et al (2004) Novel PMS2 pseudogenes can conceal recessive mutations causing a distinctive childhood cancer syndrome. Am J Hum Genet 74:954–964PubMedCrossRefGoogle Scholar
  39. 39.
    Hayward BE, De Vos M, Valleley EMA et al (2007) Extensive gene conversion at the PMS2 DNA mismatch repair locus. Hum Mutat 28:424–430PubMedCrossRefGoogle Scholar
  40. 40.
    Li LS, Kim NG, Kim SH et al (2003) Chromosomal imbalances in the colorectal carcinomas with microsatellite instability. Am J Pathol 163:1429–1436PubMedGoogle Scholar
  41. 41.
    Camps J, Armengol G, del Rey J et al (2006) Genome-wide differences between microsatellite stable and unstable colorectal tumors. Carcinogenesis 27:419–428PubMedCrossRefGoogle Scholar
  42. 42.
    Douglas EJ, Fiegler H, Rowan A et al (2004) Array comparative genomic hybridization analysis of colorectal cancer cell lines and primary carcinomas. Cancer Res 64:4817–4825PubMedCrossRefGoogle Scholar
  43. 43.
    Haiman CA, Le ML, Yamamato J et al (2007) A common genetic risk factor for colorectal and prostate cancer. Nat Genet 39:954–956PubMedCrossRefGoogle Scholar
  44. 44.
    Poynter JN, Figueiredo JC, Conti DV et al (2007) Variants on 9p24 and 8q24 are associated with risk of colorectal cancer: results from the colon cancer family registry. Cancer Res 67:11128–11132PubMedCrossRefGoogle Scholar
  45. 45.
    Tomlinson I, Webb E, Carvajal-Carmona L et al (2007) A genome-wide association scan of tag SNPs identifies a susceptibility variant for colorectal cancer at 8q24.21. Nat Genet 39:984–988PubMedCrossRefGoogle Scholar
  46. 46.
    Zanke BW, Greenwood CM, Rangrej J et al (2007) Genome-wide association scan identifies a colorectal cancer susceptibility locus on chromosome 8q24. Nat Genet 39:989–994PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Marjo van Puijenbroek
    • 1
  • Anneke Middeldorp
    • 1
  • Carli M. J. Tops
    • 2
  • Ronald van Eijk
    • 1
  • Heleen M. van der Klift
    • 2
  • Hans F. A. Vasen
    • 3
  • Juul Th. Wijnen
    • 2
  • Frederik J. Hes
    • 2
  • Jan Oosting
    • 1
  • Tom van Wezel
    • 1
  • Hans Morreau
    • 1
  1. 1.Department of PathologyLeiden University Medical CenterLeidenThe Netherlands
  2. 2.Center Human and Clinical GeneticsLeiden University Medical CenterLeidenThe Netherlands
  3. 3.The Netherlands Foundation for the Detection of Hereditary TumorsLeidenThe Netherlands

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