Skip to main content
SpringerLink
Log in

Microsatellite Instability (MSI) Detection in DNA from FFPE Tissues

  • Chapter
  • First Online:
Guidelines for Molecular Analysis in Archive Tissues

  • 1925 Accesses

Abstract

Microsatellite sequences are genome-wide dispersed stretches of short tandem nucleotide repeats. Because of their repetitive nature, microsatellites are prone to undergo shortening or extension during DNA replication because of polymerase slippage or misalignment of template strands (microsatellite instability, MSI). As spontaneous mutation rate increases dramatically in the presence of a defective mismatch repair (MMR) system, MSI represents an ideal phenotypic indicator of an MMR defect. MSI occurs in approximately 15% of colorectal cancers, including those arising in the Hereditary Non-Polyposis CRC familiar syndrome (HNPCC or Lynch Syndrome). MSI tumours feature a series of molecular and clinicopathological signatures that are distinct from non-MSI ones. MSI testing therefore enables identification of patients having a unique prognosis and a different response to particular drug therapies. This chapter provides three methodological approaches for assessing MSI in FFPE samples: a basic method involving amplification of the NCI-validated microsatellite marker sequences, with PAGE run and silver stain detection of PCR products; a multiplex PCR amplification of five mononucleotide markers alternative to the NCI panel, coupled with DHPLC (Denaturating High-Performance Liquid Chromatography) analysis of PCR products; and a multiplex PCR amplification of the five mononucleotide markers coupled with capillary electrophoresis.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 129.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Notes

  1. 1.

    The solubilization of proteinase K in 50% sterile glycerol avoids the freezing effect at −20°C, thus maintaining an optimal enzymatic activity.

  2. 2.

    Clean the microtome with xylene. Cool the paraffin blocks at −20°C or on dry ice in aluminum foil to obtain thin sections.

  3. 3.

    The number may vary, according to tissue type and area.

  4. 4.

    Use some sections from a paraffin block without any tissue for the negative controls.

  5. 5.

    When working with xylene, avoid breathing fumes; it is better to perform the deparaffinization step under a chemical hood.

  6. 6.

    Discard the supernatant using a micropipette or a glass Pasteur pipette. Disposal of chemicals should be done in keeping with your laboratory safety rules.

  7. 7.

    If low amounts of DNA are obtained, digestion can be prolonged up to 72 h by adding new Proteinase K solution every 24 h.

  8. 8.

    Phenol and chloroform are toxic by inhalation; work under a chemical hood.

  9. 9.

    The concentration of dsDNA expressed in μg/μL is obtained as follows: [DNA] = A260  x dilution factor  x  50  x  10−3.

  10. 10.

    The primer sequences for D2S123 and D13S153 have been modified with respect to those previously published, in order to reduce the length of the amplified product.

  11. 11.

    It is recommended that a high-quality DNA, e.g. a DNA extracted from a cell line, be used.

  12. 12.

    6× loading buffer: 0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol in H2O.

  13. 13.

    Ethidium bromide is a potentially carcinogenic compound. Always wear gloves. Used EtBr solutions must be collected in containers for chemical waste and discharged according to the local hazardous chemical disposal procedures.

  14. 14.

    See Footnote 12.

  15. 15.

    Toxic and carcinogenic. Avoid contact with eyes and skin. Avoid breathing fumes.

  16. 16.

    The choice of amplification product volume (usually 1–5 μl) relies on band signal intensity at visual inspection of the agarose gel.

  17. 17.

    As impedance may vary according to elecrophoretic system, buffer volume, etc., voltage and run time should be adapted.

  18. 18.

    See Footnote 12.

References

  1. Dutrillaux B (1995) Pathways of chromosome alteration in human epithelial cancers. Adv Cancer Res 67:59–82

    Article  PubMed  CAS  Google Scholar 

  2. Lengauer C, Kinzler KW, Vogelstein B (1997) Genetic instability in colorectal cancers. Nature 386(6625):623–627

    Article  PubMed  CAS  Google Scholar 

  3. Sweezy MA, Fishel R (1994) Multiple pathways leading to genomic instability and tumorigenesis. Ann NY Acad Sci 726:165–177

    Article  PubMed  CAS  Google Scholar 

  4. Fearon ER, Vogelstein B (1990) A genetic model for colorectal tumorigenesis. Cell 61(5):759–767

    Article  PubMed  CAS  Google Scholar 

  5. Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M et al (1988) Genetic alterations during colorectal-tumor development. N Engl J Med 319(9):525–532

    Article  PubMed  CAS  Google Scholar 

  6. Aaltonen LA, Salovaara R, Kristo P, Canzian F, Hemminki A, Peltomaki P et al (1998) Incidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screening for the disease. N Engl J Med 338(21): 1481–1487

    Article  PubMed  CAS  Google Scholar 

  7. Greenson JK, Bonner JD, Ben-Yzhak O, Cohen HI, Miselevich I, Resnick MB et al (2003) Phenotype of microsatellite unstable colorectal carcinomas: Well-differentiated and focally mucinous tumors and the absence of dirty necrosis correlate with microsatellite instability. Am J Surg Pathol 27(5):563–570

    Article  PubMed  Google Scholar 

  8. Gryfe R, Kim H, Hsieh ET, Aronson MD, Holowaty EJ, Bull SB et al (2000) Tumor microsatellite instability and clinical outcome in young patients with colorectal cancer. N Engl J Med 342(2):69–77

    Article  PubMed  CAS  Google Scholar 

  9. Gryfe R, Swallow C, Bapat B, Redston M, Gallinger S, Couture J (1997) Molecular biology of colorectal cancer. Curr Probl Cancer 21(5):233–300

    Article  PubMed  CAS  Google Scholar 

  10. Thibodeau SN, French AJ, Roche PC, Cunningham JM, Tester DJ, Lindor NM et al (1996) Altered expression of hMSH2 and hMLH1 in tumors with microsatellite instability and genetic alterations in mismatch repair genes. Cancer Res 56(21):4836–4840

    PubMed  CAS  Google Scholar 

  11. 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):5849–5852

    PubMed  CAS  Google Scholar 

  12. Thibodeau SN, Bren G, Schaid D (1993) Micro­satellite instability in cancer of the proximal colon. Science 260(5109):816–819

    Article  PubMed  CAS  Google Scholar 

  13. Anthoney DA, McIlwrath AJ, Gallagher WM, Edlin AR, Brown R (1996) Microsatellite instability, apoptosis, and loss of p53 function in drug-resistant tumor cells. Cancer Res 56(6):1374–1381

    PubMed  CAS  Google Scholar 

  14. Elsaleh H, Joseph D, Grieu F, Zeps N, Spry N, Iacopetta B (2000) Association of tumour site and sex with survival benefit from adjuvant chemotherapy in colorectal cancer. Lancet 355(9217):1745–1750

    Article  PubMed  CAS  Google Scholar 

  15. Fink D, Aebi S, Howell SB (1998) The role of DNA mismatch repair in drug resistance. Clin Cancer Res 4(1):1–6

    PubMed  CAS  Google Scholar 

  16. Hendriks YM, de Jong AE, Morreau H, Tops CM, Vasen HF, Wijnen JT et al (2006) Diagnostic approach and management of Lynch syndrome (hereditary nonpolyposis colorectal carcinoma): a guide for clinicians. CA Cancer J Clin 56(4):213–225

    Article  PubMed  Google Scholar 

  17. 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):5248–5257

    PubMed  CAS  Google Scholar 

  18. Bocker T, Diermann J, Friedl W, Gebert J, Holinski-Feder E, Karner-Hanusch J et al (1997) Microsatellite instability analysis: a multicenter study for reliability and quality control. Cancer Res 57(21):4739–4743

    PubMed  CAS  Google Scholar 

  19. Dietmaier W, Wallinger S, Bocker T, Kullmann F, Fishel R, Ruschoff J (1997) Diagnostic microsatellite instability: definition and correlation with mismatch repair protein expression. Cancer Res 57(21):4749–4756

    PubMed  CAS  Google Scholar 

  20. 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):261–268

    Article  PubMed  CAS  Google Scholar 

  21. Suraweera N, Duval A, Reperant M, Vaury C, Furlan D, Leroy K et al (2002) Evaluation of tumor microsatellite instability using five quasimonomorphic mononucleotide repeats and pentaplex PCR. Gastroenterology 123(6):1804–1811

    Article  PubMed  CAS  Google Scholar 

  22. Perucho M, (1999) Correspondence re: C.R. Boland et al, 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 58:5248–5257, 1998. Cancer Res 59(1):249–256

    Google Scholar 

  23. Bacher JW, Flanagan LA, Smalley RL, Nassif NA, Burgart LJ, Halberg RB et al (2004) Development of a fluorescent multiplex assay for detection of MSI-High tumors. Dis Markers 20(4–5):237–250

    PubMed  Google Scholar 

  24. Zhou XP, Hoang JM, Cottu P, Thomas G, Hamelin R (1997) Allelic profiles of mononucleotide repeat microsatellites in control individuals and in colorectal tumors with and without replication errors. Oncogene 15(14):1713–1718

    Article  PubMed  CAS  Google Scholar 

  25. Berginc G, Glavac D (2009) Rapid and accurate approach for screening of microsatellite unstable tumours using quasimonomorphic mononucleotide repeats and denaturating high performance liquid chromatography (DHPLC). Dis Markers 26(1):19–26

    PubMed  CAS  Google Scholar 

  26. Buhard O, Suraweera N, Lectard A, Duval A, Hamelin R (2004) Quasimonomorphic mononucleotide repeats for high-level microsatellite instability analysis. Dis Markers 20(4–5):251–257

    PubMed  Google Scholar 

  27. Goel A, Nagasaka T, Hamelin R, Boland CR (2010) An optimized pentaplex PCR for detecting DNA mismatch repair-deficient colorectal cancers. PLoS ONE 5(2):e9393

    Article  PubMed  Google Scholar 

  28. Nardon E, Glavač D, Benhattar J, Groenen P, Höfler G, Höfler H et al (2010) A multicenter study to validate the reproducibility of MSI testing with a panel of five quasimonomorphic mononucleotide repeats. Diagn Mol Pathol 19(4):236–242

    Article  PubMed  CAS  Google Scholar 

  29. Shibata DK, Arnheim N, Martin WJ (1988) Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction. J Exp Med 167(1):225–230

    Article  PubMed  CAS  Google Scholar 

  30. Muller A, Giuffre G, Edmonston TB, Mathiak M, Roggendorf B, Heinmoller E et al (2004) Challenges and pitfalls in HNPCC screening by microsatellite analysis and immunohistochemistry. J Mol Diagn 6(4):308–315

    Article  PubMed  Google Scholar 

  31. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT et al (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239(4839):487–491

    Article  PubMed  CAS  Google Scholar 

  32. Sieben NL, ter Haar NT, Cornelisse CJ, Fleuren GJ, Cleton-Jansen AM (2000) PCR artifacts in LOH and MSI analysis of microdissected tumor cells. Hum Pathol 31(11):1414–1419

    Article  PubMed  CAS  Google Scholar 

  33. Bovo D, Rugge M, Shiao YH (1999) Origin of spurious multiple bands in the amplification of microsatellite sequences. Mol Pathol 52(1):50–51

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Molecular Genetics, Institute of Pathology, University of Ljubljana, Ljubljana, Slovenia

    Damjan Glavač

  2. Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy

    Ermanno Nardon

Authors
  1. Damjan Glavač
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ermanno Nardon
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. c/o Ospedale di Cattinara, University of Trieste, Strada di Fiume 447, Trieste, 34149, Italy

    Giorgio Stanta

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer-Verlag Berlin Heidelberg

About this chapter

Cite this chapter

Glavač, D., Nardon, E. (2011). Microsatellite Instability (MSI) Detection in DNA from FFPE Tissues. In: Stanta, G. (eds) Guidelines for Molecular Analysis in Archive Tissues. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-17890-0_28

Download citation

  • DOI: https://doi.org/10.1007/978-3-642-17890-0_28

  • Published:

  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-642-17889-4

  • Online ISBN: 978-3-642-17890-0

  • eBook Packages: MedicineMedicine (R0)

Publish with us

Policies and ethics

Search

Navigation