Abstract
Several retrospective studies have shown that a high level of microsatellite instability (MSI‐H) is an important prognostic factor of a more favorable outcome in stage II and III colorectal cancer (CRC) patients. In this study, three commonly used polymerase chain reaction (PCR)‐based MSI analysis techniques were compared (polyacrylamide gel electrophoresis followed by silver‐staining [SSPAGE], fluorescence capillary electrophoresis [FCE], and denaturing high‐performance liquid chromatography [DHPLC]) on a limited group of CRC patients, to identify the most optimal detection technique. Pathology blocks of 26 CRC patients were subjected to microdissection and the Bethesda reference panel was used for MSI analysis. Considering the samples analyzed by both SSPAGE and FCE, 8.7% were MSI‐H, 8.7% were MSI‐L, and 82.6% were MSS using SSPAGE. FCE resulted in 16% MSI‐H, 4% MSI‐L, and 80% MSS. Due to difficulties in analyzing the dinucleotide markers on DHPLC, we only analyzed the mononucleotide markers with this technique. The results were 100% concordant to those obtained by FCE. SSPAGE is time consuming, subjective, and less user‐friendly and interpretable. DHPLC was not feasible due to interpretation difficulties for the dinucleotide markers. We recommend the use of FCE to analyze MSI status. This technique is sensitive, reproducible, user‐friendly and leads to easy interpretation and high‐throughput. J. Clin. Lab. Anal. 20:52–61, 2006. © 2006 Wiley‐Liss, Inc.
Keywords: colorectal cancer, MSI, PAGE, fluorescence capillary electrophoresis, DHPLC
| Abbreviations | |
|---|---|
| LOH | loss of heterozygosity |
| MMR | mismatch repair |
| MSI | microsatellite instability |
| SSPAGE | silver‐staining polyacrylamide gel electrophoresis |
| DHPLC | denaturing high‐performance liquid chromatography |
| TEAA | triethylammonium acetate |
| H&E | hematoxylin and eosin |
| MSI‐H | microsatellite instability–high grade |
| MSI‐L | microsatellite instability–low grade |
| MSS | microsatellite stable. |
REFERENCES
- 1. Grady WM, Rajput A, Lutterbaugh JD, Markowitz SD. Detection of aberrantly methylated hMLH1 promoter DNA in the serum of patients with microsatellite unstable colon cancer. Cancer Res 2001;61:900–902. [PubMed] [Google Scholar]
- 2. Edmonston TB, Cuesta KH, Burkholder S, et al. Colorectal carcinomas with high microsatellite instability: defining a distinct immunologic and molecular entity with respect to prognostic markers. Hum Pathol 2000;31:1506–1514. [DOI] [PubMed] [Google Scholar]
- 3. Nash GM, Gimbel M, Shia J, et al. Automated, multiplex assay for high‐frequency microsatellite instability in colorectal cancer. J Clin Oncol 2003;21:3105–3112. [DOI] [PubMed] [Google Scholar]
- 4. Atkin NB. Microsatellite instability. Cytogenet Cell Genet 2001;92:177–181. [DOI] [PubMed] [Google Scholar]
- 5. Gryfe R, Kim H, Hsieh ET, et al. Tumor microsatellite instability and clinical outcome in young patients with colorectal cancer. N Engl J Med 2000;342:69—77. [DOI] [PubMed] [Google Scholar]
- 6. Samowitz WS et al. Microsatellite instability in sporadic colon cancer is associated with an improved prognosis at the population level. Cancer Epidemiol Biomarkers Prev 2001;10:917—923. [PubMed] [Google Scholar]
- 7. Boland CR, Thibodeau SN, Hamilton SR, 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 1998;58:5248—5257. [PubMed] [Google Scholar]
- 8. Schlegel J, Vogt T, Munkel K, Ruschoff J. DNA fingerprinting of mammalian cell lines using nonradioactive arbitrarily primed PCR (AP‐PCR). Biotechniques 1996;20:178–180. [DOI] [PubMed] [Google Scholar]
- 9. Carethers JM, Smith EJ, Behling CA, et al. Use of 5‐fluorouracil and survival in patients with microsatellite‐unstable colorectal cancer. Gastroenterology 2004;126:394–401. [DOI] [PubMed] [Google Scholar]
- 10. Lindforss U, Papadogiannakis N, Zetterquist H, Lindberg G, Olivecrona H. Distribution of genetic variants in preneoplastic areas of colorectal tumours. European J Surgical Oncol 2003;29:491–496. [DOI] [PubMed] [Google Scholar]
- 11. Kim IJ, Shin Y, Kang HC, et al. Robust microsatellite instability (MSI) analysis by denaturing high‐performance liquid chromatography (DHPLC). J Hum Genet 2003;48:525–530. [DOI] [PubMed] [Google Scholar]
- 12. Dietmaier W, Wallinger S, Bocker T, Kullmann F, Fishel R, Ruschoff J. Diagnostic microsatellite instability: definition and correlation with mismatch repair protein expression. Cancer Res. 1997;57:4749–4756. [PubMed] [Google Scholar]
- 13. Thibodeau SN, French AJ, Cunningham JM, et al.. Microsatellite instability in colorectal cancer: different mutator phenotypes and the principal involvement of hMLH1. Cancer Res 1998;58:1713–1718. [PubMed] [Google Scholar]
- 14. Cawkwell L, Li D, Lewis FA, Martin I, Dixon MF, Quirke P. Microsatellite instability in colorectal cancer: improved assessment using fluorescent polymerase chain reaction. Gastroenterology 1995;109:465–471. [DOI] [PubMed] [Google Scholar]
- 15. Loukola A, Eklin K, Laiho P, et al. Microsatellite marker analysis in screening for hereditary nonpolyposis colorectal cancer (HNPCC). Cancer Res 2001;61:4545–4549. [PubMed] [Google Scholar]
- 16. Pedroni M, Tamassia MG, Percesepe A, et al. Microsatellite instability in multiple colorectal tumors. Int J Cancer 1999;81:1–5. [DOI] [PubMed] [Google Scholar]
- 17. Ward R, Meagher A, Tomlinson I, et al. Microsatellite instability and the clinicopathological features of sporadic colorectal cancer. Gut 2001;48:821–829. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Smyrk TC, Watson P, Kaul K, Lynch HT. Tumor‐infiltrating lymphocytes are a marker for microsatellite instability in colorectal carcinoma. Cancer 2001;91:2417–2422. [PubMed] [Google Scholar]
- 19. Prall F, Duhrkop T, Weirich V, et al. Prognostic role of CD8+tumor‐infiltrating lymphocytes in stage III colorectal cancer with and without microsatellite instability. Human Path 2004;35:808–816. [DOI] [PubMed] [Google Scholar]
- 20. Guda K, Upender MB, Belinsky G, et al. Carcinogen‐induced colon tumors in mice are chromosomally stable and are characterized by low‐level microsatellite instability. Oncogene 23:3813–3821. [DOI] [PubMed] [Google Scholar]
- 21. Halford S, Sasieni P, Rowan A, et al. Low‐level microsatellite instability occurs in most colorectal cancers and is a nonrandomly distributed quantitative trait. Cancer Res 2002;62:53–57. [PubMed] [Google Scholar]
- 22. Pan K, Liu W, Lu YY, et al. High throughput detection of microsatellite instability by denaturing high‐performance liquid chromatography. Hum Mutat 2003;22:388–394. [DOI] [PubMed] [Google Scholar]