COMPARISON OF DIFFERENT METHODS OF MOLECULAR-GENETIC ANALYSIS OF SOMATIC MUTATIONS IN K-RAS GENE IN PATIENTS WITH COLORECTAL CANCER

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Abstract


Two approaches to somatic point mutations in 12 and 13 codones of K-ras gene were analyzed: PCR/SSCP/AСRS/sequencing and allele-specific PCR in the real-life regimen (Russian set «KRAS-7M»). The comparison was carried out on 62 examples of genomic DNA extracted from frozen colon carcinomas, which underwent manual dissection. The results obtained in two attempts were consistent in 95,2% (N=59). Specificity and sensitivity of K-ras mutations detection using «KRAS-7M» set were 100 and 96,4% respectively, and 94,1 and 100% respectievly using PCR/SSCP/AСRS/ automatic sequencing. False positive results were absent when detecting with «KRAS-7M» and accounted for 2 cases (5,9%) when using PCR/SSCP/ AСRS/automatic sequencing. The only false negative response (3,6%) was obtained analyzing mutations using «KRAS-7M».


About the authors

F. A. Amosenko

Scientific centre of medical genetics, RAMS, Moscow
Blokhin Russian oncological scientific centre RAMS, Moscow

Author for correspondence.
Email: amossenko@med-gen.ru

Russian Federation кандидат биологических наук, старший научный сотрудник, ведущий научный сотрудник Медико-генетического научного центра РАМН, лаборатории ДНК-диагностики Адрес: 115478, Москва, ул. Москворечье, д. 1 Тел.: (965) 132-78-02, факс: (499) 324-07- 02

I. V. Karpov

Laboratory of genetic diagnosis «BioLink», Novosibirsk

Email: biolink@inbox.ru

Russian Federation лаборант ООО «БиоЛинк» лаборатории генодиагностики Адрес: 630117, Новосибирск, ул. Академика Тимакова, д. 2 Тел./факс: (383) 334-86-14

A. V. Polyakov

Scientific centre of medical genetics, RAMS, Moscow

Email: info@dnalab.ru

Russian Federation доктор биологических наук, профессор Медико-генетического научного центра РАМН, заведующий лабораторией ДНК-диагностики Адрес: 115478, Москва, ул. Москворечье, д. 1 Тел./факс: (495) 221-90-84

S. P. Kovalenko

Institute of molecular biology and biophysics Siberian division of RAMS, Novosibirsk

Email: sp_kovalenko@yahoo.com

Russian Federation кандидат биологических наук, заведующий лабораторией генной инженерии Института молекулярной биологии и биофизики Сибирского Отделения РАМН Адрес: 630117, Новосибирск, ул. Академика Тимакова, д. 2 Тел/факс: (383) 333-47-10

V. A. Shamanin

Laboratory of genetic diagnosis «BioLink», Novosibirsk

Email: va_shamanin@biolinklab.ru

Russian Federation кандидат биологических наук, ведущий научный сотрудник лаборатории гено- диагностики ООО «БиоЛинк» Адрес: 630117, Новосибирск, ул. Академика Тимакова, д. 2 Тел/факс: (383) 334-86-14

L. N. Lyubchenko

Blokhin Russian oncological scientific centre RAMS, Moscow

Email: clingen@mail.ru

Russian Federation доктор медицинских наук, заведующая лабораторией клинической онкогенетики Российского онкологического научного центра им. Н.Н. Блохина РАМН Адрес: 115478, Москва, Каширское шоссе, д. 24 Тел.: (916) 623-23-66, факс: (499) 324-96-29

References

  1. Marshall C.J. Small GTPases and cell cycle regulation. Biochem. Soc. Trans. 1999; 27 (4): 363−370.
  2. Chang F., Lee J.T., Navolanic P.M. et al. Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy. Leukemia. 2003; 17: 590−603.
  3. McCubrey J.A., Steelman L.S., Chappell W.H. et al. Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochim. Biophys. Acta. 2007; 1773: 1263−1284.
  4. Bos J.L. К-ras oncogenes in human cancer: a review. Cancer Res. 1989; 49: 4682−4689.
  5. Minamoto T., Mai M., Ronai Z. K-ras mutation: early detection in molecular diagnosis and risk assessment of colorectal, pancreas, and lung cancers ― a review. Cancer Detect. Prev. 2000; 24: 1−12.
  6. Parkin D.M., Bray F., Ferlay J. et al. Global cancer statistics. 2002; CA. Cancer J. Clin. 2005; 55 (2): 74−108.
  7. Brink M., de Goeij A.F., Weijenberg M.P. et al. K-ras oncogene mutations in sporadic colorectal cancer in the Netherlands cohort study. Carcinogenesis. 2003; 24 (4): 703−710.
  8. Samowitz W.S., Curtin K., Schaffer D. et al. Relationship of Ki-ras mutations in colon cancers to tumor location, stage, and survival: a population-based study. Cancer Epidemial. Biomarkers Prev. 2000; 9 (11): 1193−1197.
  9. Slattery M.L., Curtin K., Anderson K. et al. Associations between dietary intake and Ki-ras mutations in colon tumors: a population-based study. Cancer Res. 2000; 60 (24): 6935−6941.
  10. Amosenko F.A., Korchagina E.L., Matveeva T.I. et al. Mutation analysis of K-ras protooncogene in colorectal adenocarcinomas and polyps in Russian patients. Russian J. Genetics. 2010; 46 (5): 617−624.
  11. Vogelstein B., Fearon E.R., Stanley S.R. et al. Genetic alterations during colorectal tumor development. N. Engl. J. Med. 1988; 319: 525−532.
  12. Lowy D.R., Willumsen B.M. Function and regulation of ras. Annu. Rev. Biochem. 1993; 62: 851−891.
  13. Al-Mulla F., Milner-White E.J., Going J.J. et al. Structural differences between valine-12 and aspartate-12 Ras proteins may modify carcinoma aggression. J. Pathol. 1999; 187: 433−438.
  14. Tuveson D.A., Shaw A.T., Willis N.A. et al. Endogenous oncogenic K-ras (G12D) stimulates proliferation and widespread neoplastic and developmental defects. Cancer Cell. 2004; 5: 375−387.
  15. Smakman N., Borel Rinkes I. H., Voest E. E. et al. Control of colorectal metastasis formation by K-ras. Biochim. Biophys. Acta. 2005; 1756: 103−114.
  16. Castagnola P., Giaretti W. Mutant KRAS, chromosomal instability and prognosis in colorectal cancer. Biochim. Biophys. Acta. 2005; 1756: 115−125.
  17. Pajkos G., Kiss I., Sandor J. et al. The prognostic value of the presence of mutations at the codons 12, 13, 61 of Ki-ras oncogene in colorectal cancer. Anticancer Res. 2000; 20: 1695−1701.
  18. Colin A., Smith G., Carey F. A. et al. The prognostic significance of K-ras, p53, and APC mutations in colorectal carcinoma. Gut. 2005; 54 (9): 1283−1286.
  19. Lievre A., Bachet J.B., Boige V. et al. KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J. Clin. Oncol. 2008; 26: 374−379.
  20. Amado R.G., Wolf M., Peeters M. et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J. Clin. Oncol. 2008; 26: 1626−1634.
  21. Taniguchi K., Okami J., Kodama K. et al. Intratumor heterogeneity of epidermal growth factor receptor mutations in lung cancer and its correlation to the response to gefitinib. Cancer Sci. 2008; 99: 929−935.
  22. Fox J.C., England J., White P. et al. The detection of K-ras mutations in colorectal cancer using the amplification ― refractory mutation system. Br. J. Cancer. 1998; 77: 1267−1274.
  23. Poehlmann A., Kuester D., Meyer F. et al. K-ras mutation detection in colorectal cancer using the pyrosequencing technique. Pathol. Res. Pract. 2007; 203 (7): 489−497.
  24. Taback B., Bilchik A.J., Saha S. et al. Peptide nucleic acid clamp PCR: a novel K-ras mutation detection assay for colorectal cancer micrometastases in lymph nodes. Int. J. Cancer. 2004; 111 (3): 409−414.
  25. Lleonart M.E., Ramon Y., Cajal S. et al. Sensitive and specific detection of K-ras mutations in colon tumors by short oligonucleotide mass analysis. Nucl. Acids Res. 2004; 32: 53.
  26. Bjorheim J., Lystad S., Lindblom A. et al. Mutation analyses of KRAS exon 1 comparing three different techniques: temporal temperature gradient electrophoresis, constant denaturant capillary electrophoresis and allele specific polymerase reaction. Mutat. Res. 1998; 403: 103−112.
  27. Do H., Krypuy M., Mitchell P. et al. High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies. BMC Cancer. 2008; 8: 142.
  28. Li J., Wang L., Mamon H. et al. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat. Med. 2008; 14 (5): 579−584.
  29. Ogino S., Kawasaki T., Brahmandam M. et al. Sensitive sequencing method for KRAS mutation detection by pyrosequencing. J. Mol. Diagn. 2005; 7 (3): 413−421.
  30. Ronaghi M., Uhlen M., Nyren P. A sequencing method based on real-time pyrophosphate. Science. 1998; 281 (5375): 363−365.
  31. Clayton S.J., Scott F.M., Walker J. et al. K-ras point mutation detection in lung cancer: comparison of two approaches to somatic mutation detection using ARMS allele-specific amplification. Clin. Chem. 2000; 46 (12): 1929−1938.
  32. Levi S., Urbano-Ispizua A., Gill R. et al. Multiple K-ras codon 12 mutations in cholangiocarcinomas demonstrated with a sensitive polymerase chain reaction technique. Cancer Res. 1991; 51: 3497−3502.
  33. Suzuki Y., Orita M., Shiraishi M. et al. Detection of ras gene mutations in human lung cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene. 1990; 5: 1037−1043.
  34. Jean G.W., Shah S.R. Epidermal growth factor receptor monoclonal antibodies for the treatment of metastatic colorectal cancer. Pharmacotherapy. 2008; 28 (6): 742−754.
  35. Peeters M., Balfour J., Arnold D. Review article: panitumumab ― a fully human anti-EGFR monoclonal antibody for treatment of metastatic colorectal cancer. Aliment. Pharmacol. Ther. 2008; 28 (3): 269−281.
  36. Iqbal S., Lenz H.J. Integration of novel agents in the treatment of colorectal cancer. Cancer Chemother. Pharmacol. 2004; 54 (Suppl. 1): 32−39.
  37. de Castro-Carpeno J., Belda-Iniesta C., Casado Saenz E. et al. EGFR and colon cancer: a clinical view. Clin. Transl. Oncol. 2008; 10 (1): 6−13.
  38. Porebska I., Harlozinska A., Bojarowski T. Expression of the tyrosine kinase activity growth factor receptors (EGFR, ERB B2, ERB B3) in colorectal adenocarcinomas and adenomas. Tumour Biol. 2000; 21 (2): 105−115.
  39. Tigue C., Fitzner K., Alkhatib M. et al. The value of innovation: the economics of targeted drugs for cancer. Targeted Oncol. 2007; 2 (2): 113−119.
  40. Monzon F.A., Ogino S., Hammond E.H. et al. The role of KRAS mutation testing in the management of patients with metastatic colorectal cancer. Arch. Pathol. Lab. Med. 2009; 133: 1600−1606.

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