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Co-amplification at Lower Denaturation-temperature PCR Combined with Unlabled-probe High-resolution Melting to Detect KRAS Codon 12 and 13 Mutations in Plasma-circulating DNA of Pancreatic Adenocarcinoma Cases

  • Wu, Jiong ;
  • Zhou, Yan ;
  • Zhang, Chun-Yan ;
  • Song, Bin-Bin ;
  • Wang, Bei-Li ;
  • Pan, Bai-Shen ;
  • Lou, Wen-Hui ;
  • Guo, Wei
  • Published : 2015.01.22

Abstract

Background: The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. Materials and Methods: We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. Results: It was found that the sensitivity of Sanger reached 0.5% with COLD-PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. Conclusions: It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.

Keywords

Plasma-circulating DNA;panreatic adenocarcinoma;KRAS gene;unlabled-probe;high-resolution melting;mutations

References

  1. Amicarelli G, Shehi E, Makrigiorgos GM, et al (2007). FLAG assay as a novel method for real-time signal generation during PCR: application to detection and genotyping of KRAS codon 12 mutations. Nucleic Acids Res, 35, 131. https://doi.org/10.1093/nar/gkm809
  2. Antonio Jimeno, Manuel Hidalgo (2006). Molecular biomarkers: their increasing role in the diagnosis, characterization, and therapy guidance in pancreatic cancer. Mol Cancer Ther, 5, 787-96. https://doi.org/10.1158/1535-7163.MCT-06-0005
  3. Arvanitakis M, Van Laethem JL, Parma J, et al (2004). Predictive factors for pancreatic cancer in patients with chronic pancreatitis in association with KRAS gene mutation. Endoscopy, 36, 535-42. https://doi.org/10.1055/s-2004-814401
  4. Boldrini L, Gisfredi S, Ursino S, et al (2007). Mutational analysis in cytological specimens of advanced lung adenocarcinoma: a sensitive method for molecular diagnosis. J Thorac Oncol, 2, 1086-90. https://doi.org/10.1097/JTO.0b013e31815ba1fa
  5. Chen CY, Shiesh SC, Wu SJ (2004). Rapid detection of KRAS mutations in bile by peptide nucleic acid-mediated PCR clamping and melting curve analysis: comparison with restriction fragment length polymorphism analysis. Clin Chem, 50, 481-9. https://doi.org/10.1373/clinchem.2003.024505
  6. Dawood S (2010). Novel biomarkers of metastatic cancer. expert review of molecular diagnostics. Expert Rev Mol Diagn. 10, 581-90. https://doi.org/10.1586/erm.10.35
  7. Deramaudt T, Rustgi AK. (2005). Mutant KRAS in the initiation of pancreatic cancer. Biochim Biophys Acta, 1756, 97-101.
  8. Di Fiore F, Blanchard F, Charbonnier F, et al (2007). Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by Cetuximab plus chemotherapy. Br J Cancer, 96, 1166-9. https://doi.org/10.1038/sj.bjc.6603685
  9. Eijk RV, Puijenbroek MV, Amiet R, et al (2010). Chhatta sensitive and specific KRAS somatic mutation analysis on whole-genome amplified DNA from archival tissues. J Molecular Diagnostics, 12, 27-34. https://doi.org/10.2353/jmoldx.2010.090028
  10. Gary Y, Timothy D, Wagner, et al (2005). Multimodality approaches for pancreatic cancer. Cancer J Clin, 55, 352-67. https://doi.org/10.3322/canjclin.55.6.352
  11. Guo W, Zhang C, Wu J, et al (2012). Unlabeled-probe high-resolution melting to detect KRAS codon 12 and 13 mutations in pancreatic adenocarcinoma tissues. Clin Chem Lab Med, 50, 1035-40.
  12. Habermann J, Bunger K, Bunger S, et al (2011). Serum biomarkers for improved diagnostic of pancreatic cancer: a current overview. J Cancer Res Clin Oncol, 137, 375-89. https://doi.org/10.1007/s00432-010-0965-x
  13. Huang C, Wang WM, Gong JP, et al (2013). Oncogenesis and the clinical significance of K-ras in pancreatic adenocarcinoma. Asian Pac J Cancer Prev, 14, 2699-701. https://doi.org/10.7314/APJCP.2013.14.5.2699
  14. Karnoub AE, Weinberg RA (2008). RAS oncogenes: split personalities. Nat Rev Mol Cell Biol, 9, 517-31. https://doi.org/10.1038/nrm2438
  15. Krypuy M, Newnham GM , Thomas DM, et al (2006). High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer. BMC Cancer, 6, 295-306. https://doi.org/10.1186/1471-2407-6-295
  16. Laurent-Puig P, Lecomte T, Berger A, et al (2002). Detection of free-circulating tumor-associated DNA in plasma of colorectal cancer patients and its association with prognosis. Intl J Cancer, 100, 542-8. https://doi.org/10.1002/ijc.10526
  17. Liew M, Pryor R, Palais R, et al (2004). Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem, 50, 1156-64. https://doi.org/10.1373/clinchem.2004.032136
  18. Li J, Wang L, Mamon H, et al (2008). Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nature Med, 14, 579-84. https://doi.org/10.1038/nm1708
  19. Lipsky RH, Mazzanti CM, Rudolph JG, et al (2001). DNA melting analysis for detection of single nucleotide polymorphisms. Clin Chem, 47, 635-44.
  20. Lokshin A, Brand E, Nolen RE, et al (2011). Serum biomarker panels for the detection of pancreatic cancer. Clin Cancer Res, 17, 805-16. https://doi.org/10.1158/1078-0432.CCR-10-0248
  21. Maheswaran S, Sequist LV, Nagrath S, et al (2008). Detection of mutations in EGFR in circulating lung-cancer cells. N Engl J Med, 359, 366-77. https://doi.org/10.1056/NEJMoa0800668
  22. Malfertheiner P, Fry L, Monkemuller CK, et al (2008). Molecular markers of pancreatic cancer: development and clinical relevance. Langenbecks Arch Surg, 393, 883-90. https://doi.org/10.1007/s00423-007-0276-0
  23. Massarelli E, Varella-Garcia M, Tang X, et al (2007). KRAS mutation is an important predictor of resistance to therapy with epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer. Clin Cancer Res, 13, 2890-6. https://doi.org/10.1158/1078-0432.CCR-06-3043
  24. Ogino S, Kawasaki T, Brahmandam M, et al (2005). Sensitive sequencing method for KRAS mutation detection by pyrosequencing. J Mol Diagn, 7, 413-21. https://doi.org/10.1016/S1525-1578(10)60571-5
  25. Paez JG, Janne PA, Lee JC, et al (2004). EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science, 304, 1497-500. https://doi.org/10.1126/science.1099314
  26. Pitman MB, Lewandrowski K, Shen J, et al (2010). Fernandez-del Castillo, C. Pancreatic cysts preoperative diagnosis and clinical management. Cancer Cytopathology, 118, 1-13. https://doi.org/10.1002/cncy.20059
  27. Robert M, Beazley, lsidore Cohn, Jr (1981). Pancreatic cancer. CA Cancer J Clin, 31, 346-58. https://doi.org/10.3322/canjclin.31.6.346
  28. Maheswaran S, Sequist LV, Nagrath S, et al (2008). Detection of mutations in EGFR in circulating lung-cancer cells. N Engl J Med, 359, 366-77. https://doi.org/10.1056/NEJMoa0800668
  29. Vossen RH, Aten E, Roos A, et al (2009). High-resolution melting analysis (HRMA)-more than just sequence variant screening. Human Mutation, 30, 860-6. https://doi.org/10.1002/humu.21019
  30. Wittwer CT, Reed GH, Gundry CN, et al (2003). High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem, 49, 853-60. https://doi.org/10.1373/49.6.853
  31. Wu CC, Hsu HY, Liu HP, et al (2008). Reversed mutation rates of KRAS and EGFR genes in adenocarcinoma of the lung in Taiwan and their implications. Cancer, 113, 3199-208. https://doi.org/10.1002/cncr.23925

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Acknowledgement

Supported by : Ministry of Education