Familial Cancer

, Volume 6, Issue 3, pp 301–310 | Cite as

Incorporation of somatic BRAF mutation testing into an algorithm for the investigation of hereditary non-polyposis colorectal cancer

  • M. B. LoughreyEmail author
  • P. M. Waring
  • A. Tan
  • M. Trivett
  • S. Kovalenko
  • V. Beshay
  • M.-A. Young
  • G. McArthur
  • A. Boussioutas
  • A. Dobrovic


Patients suspected on clinical grounds to have hereditary non-polyposis colorectal cancer (HNPCC) may be offered laboratory testing in order to confirm the diagnosis and to facilitate screening of pre-symptomatic family members. Tumours from an affected family member are usually pre-screened for microsatellite instability (MSI) and/or loss of immunohistochemical expression of mismatch repair (MMR) genes prior to germline MMR gene mutation testing. The efficiency of this triage process is compromised by the more frequent occurrence of sporadic colorectal cancer (CRC) showing high levels of MSI (MSI-H) due to epigenetic loss of MLH1 expression. Somatic BRAF mutations, most frequently V600E, have been described in a significant proportion of sporadic MSI-H CRC but not in HNPCC-associated cancers. BRAF mutation testing has therefore been proposed as a means to more definitively identify and exclude sporadic MSI-H CRC cases from germline MMR gene testing. However, the clinical validity and utility of this approach have not been previously evaluated in a familial cancer clinic setting. Testing for the V600E mutation was performed on MSI-H CRC samples from 68 individuals referred for laboratory investigation of suspected HNPCC. The V600E mutation was identified in 17 of 40 (42%) tumours showing loss of MLH1 protein expression by immunohistochemistry but in none of the 28 tumours that exhibited loss of MSH2 expression (P < 0.001). The assay was negative in all patients with an identified germline MMR gene mutation. Although biased by the fact that germline testing was not pursued beyond direct sequencing in many cases lacking a high clinical index of suspicion of HNPCC, BRAF V600E detection was therefore considered to be 100% specific and 48% sensitive in detecting sporadic MSI-H CRC amongst those cases showing loss of MLH1 protein expression, in a population of patients with MSI-H CRC and clinical features suggestive of HNPCC. Accordingly, we recommend the incorporation of BRAF V600E mutation testing into the laboratory algorithm for pre-screening patients with suspected HNPCC, whose CRCs show loss of expression of MLH1. In such tumours, the presence of a BRAF V600E mutation indicates the tumour is not related to HNPCC and that germline testing of MLH1 in that individual is not warranted. We also recommend that in families where the clinical suspicion of HNPCC is high, germline testing should not be performed on an individual whose CRC harbours a somatic BRAF mutation, as this may compromise identification of the familial mutation.


Hereditary non-polyposis colorectal cancer BRAF Allele-specific PCR Microsatellite instability 



Hereditary non-polyposis colorectal cancer


Microsatellite instability


Mismatch repair


Colorectal cancer


Allele-specific polymerase chain reaction



M. B. Loughrey was supported by a Cancer Council of Victoria scholarship. We are grateful to Dr. Desiree du Sart of the Molecular Genetics Laboratory, Victorian Clinical Genetics Service for the provision of the results of MLPA analysis.


  1. 1.
    de la Chapelle A (2004) Genetic predisposition to colorectal cancer. Nat Rev Cancer 4(10):769–780PubMedCrossRefGoogle Scholar
  2. 2.
    Peltomaki P, Vasen HF (1997) Mutations predisposing to hereditary nonpolyposis colorectal cancer: database and results of a collaborative study. The International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer. Gastroenterology 113(4):1146–1158PubMedCrossRefGoogle Scholar
  3. 3.
    Marra G, Boland C (1995) Hereditary nonpolyposis colorectal cancer: the syndrome, the genes, and historical perspectives. J Natl Cancer Inst 87(15):1114–1125PubMedCrossRefGoogle Scholar
  4. 4.
    Wu Y, Berends MJ, Sijmons RH, Mensink RG, Verlind E, Kooi KA et al (2001) A role for MLH3 in hereditary nonpolyposis colorectal cancer. Nat Genet 29(2):137–138PubMedCrossRefGoogle Scholar
  5. 5.
    Lothe RA, Peltomaki P, Meling GI, Aaltonen LA, Nystrom-Lahti M, Pylkkanen L et al (1993) Genomic instability in colorectal cancer: relationship to clinicopathological variables and family history. Cancer Res 53(24):5849–5852PubMedGoogle Scholar
  6. 6.
    Thibodeau SN, Bren G, Schaid D (1993) Microsatellite instability in cancer of the proximal colon. Science 260(5109):816–819PubMedCrossRefGoogle Scholar
  7. 7.
    Boland CR, Thibodeau SN, Hamilton SR, Sidransky D, Eshleman JR, Burt RW et al (1998) 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 58(22):5248–5257PubMedGoogle Scholar
  8. 8.
    Burgart LJ (2005) Testing for defective DNA mismatch repair in colorectal carcinoma: a practical guide. Arch Pathol Lab Med 129(11):1385–1389PubMedGoogle Scholar
  9. 9.
    Kane MF, Loda M, Gaida GM, Lipman J, Mishra R, Goldman H et al (1997) Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res 57(5):808–811PubMedGoogle Scholar
  10. 10.
    Jass JR (2003) Hyperplastic-like polyps as precursors of microsatellite-unstable colorectal cancer. Am J Clin Pathol 119(6):773–775PubMedCrossRefGoogle Scholar
  11. 11.
    Hawkins NJ, Ward RL (2001) Sporadic colorectal cancers with microsatellite instability and their possible origin in hyperplastic polyps and serrated adenomas. J Natl Cancer Inst 93(17):1307–1313PubMedCrossRefGoogle Scholar
  12. 12.
    Hawkins NJ, Bariol C, Ward RL (2002) The serrated neoplasia pathway. Pathology 34(6):548–555PubMedGoogle Scholar
  13. 13.
    Jass JR, Young J, Leggett BA (2000) Hyperplastic polyps and DNA microsatellite unstable cancers of the colorectum. Histopathology 37(4):295–301PubMedCrossRefGoogle Scholar
  14. 14.
    Vasen HF, Watson P, Mecklin JP, Lynch HT (1999) New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC. Gastroenterology 116(6):1453–1456PubMedCrossRefGoogle Scholar
  15. 15.
    Jass JR (2004) HNPCC and sporadic MSI-H colorectal cancer: a review of the morphological similarities and differences. Fam Cancer 3(2):93–100PubMedCrossRefGoogle Scholar
  16. 16.
    Stormorken AT, Bowitz-Lothe IM, Noren T, Kure E, Aase S, Wijnen J et al (2005) Immunohistochemistry identifies carriers of mismatch repair gene defects causing hereditary nonpolyposis colorectal cancer. J Clin Oncol 23(21):4705–4712PubMedCrossRefGoogle Scholar
  17. 17.
    Umar A, Boland CR, Terdiman JP, Syngal S, de la Chapelle A, Ruschoff J et al (2004) Revised Bethesda guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability. J Natl Cancer Inst 96(4):261–268PubMedCrossRefGoogle Scholar
  18. 18.
    Jass JR, Walsh MD, Barker M, Simms LA, Young J, Leggett BA (2002) Distinction between familial and sporadic forms of colorectal cancer showing DNA microsatellite instability. Eur J Cancer 38(7):858–866PubMedCrossRefGoogle Scholar
  19. 19.
    Shia J, Ellis NA, Paty PB, Nash GM, Qin J, Offit K et al. (2003) Value of histopathology in predicting microsatellite instability in hereditary nonpolyposis colorectal cancer and sporadic colorectal cancer. Am J Surg Pathol 27(11):1407–1417PubMedCrossRefGoogle Scholar
  20. 20.
    Young J, Simms LA, Biden KG, Wynter C, Whitehall V, Karamatic R 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(6):2107–2116PubMedGoogle Scholar
  21. 21.
    Bouzourene H, Taminelli L, Chaubert P, Monnerat C, Seelentag W, Sandmeier D et al. (2006) A cost-effective algorithm for hereditary nonpolyposis colorectal cancer detection. Am J Clin Pathol 125(6):823–831PubMedCrossRefGoogle Scholar
  22. 22.
    Esteller M, Fraga MF, Guo M, Garcia-Foncillas J, Hedenfalk I, Godwin AK et al. (2001) DNA methylation patterns in hereditary human cancers mimic sporadic tumorigenesis. Hum Mol Genet 10(26):3001–3007PubMedCrossRefGoogle Scholar
  23. 23.
    Deng G, Bell I, Crawley S, Gum J, Terdiman JP, Allen BA et al. (2004) BRAF mutation is frequently present in sporadic colorectal cancer with methylated hMLH1, but not in hereditary nonpolyposis colorectal cancer. Clin Cancer Res 10(1 Pt 1):191–195PubMedCrossRefGoogle Scholar
  24. 24.
    Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S (2002) Mutations of the BRAF gene in human cancer. Nature 417(6892):949–954PubMedCrossRefGoogle Scholar
  25. 25.
    Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B, Velculescu VE (2002) Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature 418(6901):934PubMedCrossRefGoogle Scholar
  26. 26.
    Kambara T, Simms LA, Whitehall VL, Spring KJ, Wynter CV, Walsh MD et al (2004) BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum. Gut 53(8):1137–1144PubMedCrossRefGoogle Scholar
  27. 27.
    Wang L, Cunningham JM, Winters JL, Guenther JC, French AJ, Boardman LA et al (2003) BRAF mutations in colon cancer are not likely attributable to defective DNA mismatch repair. Cancer Res 63(17):5209–5212PubMedGoogle Scholar
  28. 28.
    McGivern A, Wynter CV, Whitehall VL, Kambara T, Spring KJ, Walsh MD et al (2004) Promoter hypermethylation frequency and BRAF mutations distinguish hereditary non-polyposis colon cancer from sporadic MSI-H colon cancer. Fam Cancer 3(2):101–107PubMedCrossRefGoogle Scholar
  29. 29.
    Domingo E, Laiho P, Ollikainen M, Pinto M, Wang L, French AJ et al (2004) BRAF screening as a low-cost effective strategy for simplifying HNPCC genetic testing. J Med Genet 41(9):664–668PubMedCrossRefGoogle Scholar
  30. 30.
    Miyaki M, Iijima T, Yamaguchi T, Kadofuku T, Funata N, Mori T (2004) Both BRAF and KRAS mutations are rare in colorectal carcinomas from patients with hereditary nonpolyposis colorectal cancer. Cancer Lett 211(1):105–109PubMedCrossRefGoogle Scholar
  31. 31.
    Domingo E, Niessen RC, Oliveira C, Alhopuro P, Moutinho C, Espin E et al (2005) BRAF-V600E is not involved in the colorectal tumorigenesis of HNPCC in patients with functional MLH1 and MSH2 genes. Oncogene 24(24):3995–3999PubMedCrossRefGoogle Scholar
  32. 32.
    Halvarsson B, Lindblom A, Rambech E, Lagerstedt K, Nilbert M (2004) Microsatellite instability analysis and/or immunostaining for the diagnosis of hereditary nonpolyposis colorectal cancer? Virchows Arch 444(2):135–141PubMedCrossRefGoogle Scholar
  33. 33.
    Jover R, Paya A, Alenda C, Poveda MJ, Peiro G, Aranda FI et al (2004) Defective mismatch-repair colorectal cancer: clinicopathologic characteristics and usefulness of immunohistochemical analysis for diagnosis. Am J Clin Pathol 122(3):389–394PubMedCrossRefGoogle Scholar
  34. 34.
    Lindor NM, Burgart LJ, Leontovich O, Goldberg RM, Cunningham JM, Sargent DJ et al (2002) Immunohistochemistry versus microsatellite instability testing in phenotyping colorectal tumors. J Clin Oncol 20(4):1043–1048PubMedCrossRefGoogle Scholar
  35. 35.
    Southey MC, Jenkins MA, Mead L, Whitty J, Trivett M, Tesoriero AA et al (2005) Use of molecular tumor characteristics to prioritize mismatch repair gene testing in early-onset colorectal cancer. J Clin Oncol 23(27):6524–6532PubMedCrossRefGoogle Scholar
  36. 36. International Society for Gastrointestinal Hereditary Tumours databaseGoogle Scholar
  37. 37.
    Gille JJ, Hogervorst FB, Pals G, Wijnen JT, van Schooten RJ, Dommering CJ et al (2002) Genomic deletions of MSH2 and MLH1 in colorectal cancer families detected by a novel mutation detection approach. Br J Cancer 87(8):892–897PubMedCrossRefGoogle Scholar
  38. 38.
    Pollock PM, Harper UL, Hansen KS, Yudt LM, Stark M, Robbins CM et al (2003) High frequency of BRAF mutations in nevi. Nat Genet 33:19–20PubMedCrossRefGoogle Scholar
  39. 39.
    Young J, Barker MA, Simms LA, Walsh MD, Biden KG, Buchanan D et al (2005) Evidence for BRAF mutation and variable levels of microsatellite instability in a syndrome of familial colorectal cancer. Clin Gastroenterol Hepatol 3(3):254–263PubMedCrossRefGoogle Scholar
  40. 40.
    Young J, Jass JR (2006) The case for a genetic predisposition to serrated neoplasia in the colorectum: hypothesis and review of the literature. Cancer Epidemiol Biomarkers Prev 15(10):1778–17784PubMedCrossRefGoogle Scholar
  41. 41.
    Hamilton SR, Aaltonen LAe (2000) World Health Organisation classification of tumours. Pathology and genetics of tumours of the gastrointestinal tract. IARC press, LyonGoogle Scholar
  42. 42.
    Rodriguez-Bigas MA, Boland CR, Hamilton SR, Henson DE, Jass JR, Khan PM et al (1997) A National Cancer Institute Workshop on Hereditary Nonpolyposis colorectal cancer syndrome: meeting highlights and Bethesda guidelines. J Natl Cancer Inst 89(23):1758–1762PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science + Business Media B.V. 2007

Authors and Affiliations

  • M. B. Loughrey
    • 1
    • 2
    Email author
  • P. M. Waring
    • 3
    • 4
  • A. Tan
    • 1
  • M. Trivett
    • 3
  • S. Kovalenko
    • 3
  • V. Beshay
    • 3
  • M.-A. Young
    • 5
  • G. McArthur
    • 6
  • A. Boussioutas
    • 7
    • 8
  • A. Dobrovic
    • 1
    • 9
  1. 1.Molecular Pathology Research LaboratoryPeter MacCallum Cancer CentreMelbourneAustralia
  2. 2.Department of PathologyRoyal Group of HospitalsBelfastN. Ireland, UK
  3. 3.Department of PathologyPeter MacCallum Cancer CentreMelbourneAustralia
  4. 4.Genentech Inc.South San FranciscoUSA
  5. 5.Familial Cancer CentrePeter MacCallum Cancer CentreMelbourneAustralia
  6. 6.Division of Haematology and Medical OncologyPeter MacCallum Cancer CentreMelbourneAustralia
  7. 7.Trescowthick Research LaboratoryPeter MacCallum Cancer CentreMelbourneAustralia
  8. 8.Department of MedicineUniversity of MelbourneMelbourneAustralia
  9. 9.Department of PathologyUniversity of MelbourneMelbourneAustralia

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