EGFR MUTATION DETECTION USING BIOLOGICAL MICROCHIPS IN NON-SMALL CELL LUNG CANCER
Keywords:
somatic mutations, lung cancer, EGFR, biochip, diagnosis
Abstract
EGFR somatic mutations predict sensitivity to targeted therapy with tyrosine kinase inhibitors, gefitinib (Iressa) and erlotinib in patients with non-small cell lung cancer. We have developed a method able to detect 13 most common EGFR mutatons including 9 deletions in exon 19 and point mutations in exons 21 (Leu858Arg) and 18 (Gly719Cys, Glyy^Ala, Gly719Ser). Wild-type seguence amplification was inhlbited using LNA oligonucleotides during polymerase chain reaction to increase test sensitivity and to detect minor tumor cell fractions in clinical specimens. Product of the LNA blocking polymerase chain reaction was hybridized with oligonucleotide probes immobilized in gel on biochip surface. The biochip was tested on 127 DNA samples from patients with non-smail cell lung cancer, mainly adenocarcinoma. Sequencing of polymerase chain reaction products was used as reference. The developed biochip test can reliably detect EGFR mutations irrespective of DNA sampling procedure even in small fractions of mutation-carrying cells.
References
1. Erlotinib in non-small cell lung cancer treatment: current status and future development / Gridelli C., Bareschino M. A., Schettino C., Rossi A., Maione Р., Ciardiello F. // Oncologist. — 2007. — Vol. 12, N 7, — P. 840—849.
2. Ciardiello F., Tortora G. EGFR Antagonists in Cancer Treatment // N. Engl. J. Med. — 2008. — Vol. 358. — Р. 1 160— 1174.
3. Clinicopathologic Signfflcance of the Mutations of the Epidermal Growth Factor Receptor Gene in Patients with Non-Sraall Cell Lung Cancer / Tomlzawa Y., lijima H., Sunaga N., Sato K., Takise A., Otani Y., Tanaka S., Suga Т., Saito R., Ishizuka Т., Dobashi K., Mlnna J. D., Nakajima Т., Mori M. // Clin. Cancer Res. — 2005. — Vol. 1 1 . — P. 68 1 6—6822.
4. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy / Paez J. G., Janne Р. Д., Lee J. C., Tracy S., Greulich H., Gabriel S., Herman Р., Kaye F. J., Llndeman N., BoggonT. J., Naoki K., Sasaki H., Fujii Y., Eck M. J., Sellers W. R., Johnson В. E., Meyerson M. // Science. — 2004. — Vol. 304. — P. 1497 — 1500.
5. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib / Lynch T. J., Bell D, W., Sordella R., Gurubhagavatula S., Oklmoto R. A., Brannigan B. W., Harris P. L., Haserlat S. M., Supko J. G., Haluska F. G., Louis D. N.,. Christiani D. C., Settleman J., Haber D. A. // N. Engl. J. Med. — 2004. — Vol. 350. — P. 2129—2139.
6. Pao W., Miller V., Zakowski M. F. EGF receptor gene mutations are common in lung cancers from «never smokers» and correlate with sensltivity of tumors to gefltlnib (Iressa) and erlotinib (Tarceva) // Proc. Natl. Acad. Scl. USA. — 2004. — Vol. 101. — P. 13 306—13 311.
7. Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways / Sordella R., Bell D. W., Haber D. A., Settleman J. // Science. — 2004. — Vol. 305. — Р. 1 163—1 167.
8. Mutations of the epiderraal growth factor receptor gene predict prolonged survival after gefitinib treatment in patients with non-smallcell lung cancer with postoperative recurrence / Mitsudomi Т., KosakaT., Endoh H., Horio Y., HidaT., MoriS., HatookaS., ShinodaM., Takahashi Т., Yatabe Y. // J. Clin. Oncol. — 2005. — Vol. 23. — P. 2513—2520.
9. Epidermal growth factor receptor gene mutations and increased сору numbers predict gefitinib sensitlvity in patients with recurrent nonsmall - cell lung cancer / Takano Т., Ohe Y., Sakamoto H., Tsuta K., Matsuno Y., Tateishi U., Yamamoto S., Nokihara H., Yamamoto N., Sekine I., Kunitoh H., Shlbata Т., SakiyamaT., YoshidaT., TamuraT. // J. Clin. Oncol. — 2005. — Vol. 23. — P. 6829—6837.
10. Prospective phase II study of gefitinib for chemotherapy-naive patients with advanced non-small-cell lung cancerwlth epidermal growth lactor receptor gene mutations / Inoue A., Suzuki Т., Fukuhara Т., Maemondo M., Kimura Y., Morikawa N., Watanabe H., Saijo Y, Nukiwa T. // J. Clin. Oncol. — 2006. — Vol. 24. — P. 3340—3346.
11. Clinical course of patients with non-small cell lung cancer and epidermal growth lactor receptor exon 19 and exon 21 mutations treated with gefitinib or erlotinib / Riely G. J., Pao W,, Pham D., Rizvi N., Venkatraman E. S., Zakowski M. F., Kris M. G., Ladanyi M., Miller V, A. // Clin. Cancer Res. — 2006. — Vol. 12. — P. 839—844.
12. Exon 19 deletion mutations of epidermal growth factor receptor are associated with prolonged survival in non-small cell lung cancer patients treated with gefitinib or erlotinib / Jackman D. M, Yeap B. Y., Sequist L. V., Lindeman N., Holmes A. J. p Joshi V. A., Bell D. W., Huberman M. S., Halmos В., RabinM. S., Haber D. A., LynchT. J., Meyerson M, Johnson В. E., Janne Р. A. // Clin. Cancer Res. — 2006. — Vol. 12. — P. 3908— 3914.
13. Detection of tumor cells in blood using CD45 magnetic cell separation followed by nested mutant allele-specific amplification of p53 and K-ras genes in patients with colorectal cancer / linuma R, Okinaga K., Adachi М., Suda К., Sekine Т., Sakagawa К., Baba Y., Tamura J., Китада! Н., Ida А. // Int. J, Cancer. — 2000. — Vol. 89. — Р. 337—344.
14. А simple method of detecting K-ras point mutations in stool samples for colorectal cancer screening using one-step poiymerase chain reaction/restriction fragment length polymorphism analysis / Nishikawa Т., Maemura K., Hirata I., Matsuse R., Morikawa H., Toshina K., Murano M., Hashimoto K., Nakagawa Y., Saitoh O., Uchida K., Katsu K. // Clin. Chim. Acta. — 2002. — Vol. 318. — P. 107— 1 12.
15. Milbury С. A., Li J., Makrigiorgos G. M. PCR-based methods for the enrichment of minority alleles and mutations // Clin. Chem. — 2009. — Vol. 55. — P. 632—640.
16. Detection of tumor mutations in the presence of excess amounts of normal DNA / Sun X. r Hung K., Wu L., Sidransky D., Guo B. // Nat. Biotechnol. — 2002. —Vol. 20. — P. 186—189.
17. LNA clamped PCR: A specific method for detection of Ki-ras gene mutations in patients with sporadic colorectal carcinomas / Beranek M., Jandik Р., SachaM, Rajman M, SakraL., StumrF., SoudkovaE., Zivny Р., Havlicek K. // Klin Biochem. Metab. — 2006. — Vol. 14. — P. 217 — 220.
18. LNA (locked nucleic acids); synthesis and high-affinity nucleic acid recognition / Sanjay K., Singh S. K., Nielsen Р., Koshkin A. A, Wengel J. // Chem. Commun. — 1998. — Vol. 4. — P. 455 — 456.
19. Гагарин И. M. Молекулярные маркеры эффективности ингибиторов EGFR при немелкоклеточном раке легкого и колоректальном раке: Автореф. дис... канд. мед. наук. — М., 201 1. — 34 с.
20. Ке S-H., Madison E.L. Rapid and efficient site-directed mutagenesis by single-tube «megaprimer» PCR method // Nucleic Acids Res. — 1997. — Vol. 25, N 16. — P. 3371—3372.
21. Detectlon of rare mutant K-ras DNA in a single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe / Luo J.-D., Chan E.-C, Shih C.-L, ChenT.-L., Ying Liang Y., Hwang T.-L., Chiou C.-C. // Nucleic Acids Res. — 2006. — Vol. 34, N 2. — P. 12.
22. Hydrogel drop microchips with immobilized DNA: properties and methods for large-scale productlon / Rubina A. Y., Pan'kov S. V., Dementieva E. I., Pen'kov D. N., Butygin А. V., Vasiliskov V. A., ChudinovA. V., Mlkheikin A. L., Mikhailovich V. M., MirzabekovA. D, // Anal. Blochem. — 2004. — Vol. 325. — P. 92—106.
23. Создание биочипа для анализа полиморфизма в генах системы биотрансформации / Глотов А. С., Наседкина Т. В., Иващенко Т. Э., Юрасов Р. А., Суржиков С. А., Паньков С. В., Чудинов А. В., Баранов В. С., Заседателев А. С. // Мол. биол. — 2005. — № 39. — С. 403—412.
24. Применение Ирессы (гефитиниба) в качестве терапии первой линии для лечения неоперабельных аденокарцином легкого, содержащих мутацию в гене EGFR / Моисеенко В. М., Проценко С. А., Семенов И. И., Моисеенко Ф. В., Левченко Е. В., Мацко Д. Е., Иванцов А. О., Иевлева А. Г., Митюшкина Н. В., Того А. В., Имянитов Е. Н. // Современ. онкол. — 2010. — Т. 12, № 1. — С. 60 — 66.
25. Эффективность ингибиторов рецептора эпидермального фактора роста (EGFR) у больных распространенным немелкоклеточным раком легкого / Коломейцева А. А., Гагарин И. М., Мочальникова В. В., Мазуренко Н. Н., Горбунова В. А. // Вестн. РОНЦ им. Н. Н. Блохина РАМН. — 201 1. — Т. 22, № 1. — С. 42—49.
2. Ciardiello F., Tortora G. EGFR Antagonists in Cancer Treatment // N. Engl. J. Med. — 2008. — Vol. 358. — Р. 1 160— 1174.
3. Clinicopathologic Signfflcance of the Mutations of the Epidermal Growth Factor Receptor Gene in Patients with Non-Sraall Cell Lung Cancer / Tomlzawa Y., lijima H., Sunaga N., Sato K., Takise A., Otani Y., Tanaka S., Suga Т., Saito R., Ishizuka Т., Dobashi K., Mlnna J. D., Nakajima Т., Mori M. // Clin. Cancer Res. — 2005. — Vol. 1 1 . — P. 68 1 6—6822.
4. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy / Paez J. G., Janne Р. Д., Lee J. C., Tracy S., Greulich H., Gabriel S., Herman Р., Kaye F. J., Llndeman N., BoggonT. J., Naoki K., Sasaki H., Fujii Y., Eck M. J., Sellers W. R., Johnson В. E., Meyerson M. // Science. — 2004. — Vol. 304. — P. 1497 — 1500.
5. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib / Lynch T. J., Bell D, W., Sordella R., Gurubhagavatula S., Oklmoto R. A., Brannigan B. W., Harris P. L., Haserlat S. M., Supko J. G., Haluska F. G., Louis D. N.,. Christiani D. C., Settleman J., Haber D. A. // N. Engl. J. Med. — 2004. — Vol. 350. — P. 2129—2139.
6. Pao W., Miller V., Zakowski M. F. EGF receptor gene mutations are common in lung cancers from «never smokers» and correlate with sensltivity of tumors to gefltlnib (Iressa) and erlotinib (Tarceva) // Proc. Natl. Acad. Scl. USA. — 2004. — Vol. 101. — P. 13 306—13 311.
7. Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways / Sordella R., Bell D. W., Haber D. A., Settleman J. // Science. — 2004. — Vol. 305. — Р. 1 163—1 167.
8. Mutations of the epiderraal growth factor receptor gene predict prolonged survival after gefitinib treatment in patients with non-smallcell lung cancer with postoperative recurrence / Mitsudomi Т., KosakaT., Endoh H., Horio Y., HidaT., MoriS., HatookaS., ShinodaM., Takahashi Т., Yatabe Y. // J. Clin. Oncol. — 2005. — Vol. 23. — P. 2513—2520.
9. Epidermal growth factor receptor gene mutations and increased сору numbers predict gefitinib sensitlvity in patients with recurrent nonsmall - cell lung cancer / Takano Т., Ohe Y., Sakamoto H., Tsuta K., Matsuno Y., Tateishi U., Yamamoto S., Nokihara H., Yamamoto N., Sekine I., Kunitoh H., Shlbata Т., SakiyamaT., YoshidaT., TamuraT. // J. Clin. Oncol. — 2005. — Vol. 23. — P. 6829—6837.
10. Prospective phase II study of gefitinib for chemotherapy-naive patients with advanced non-small-cell lung cancerwlth epidermal growth lactor receptor gene mutations / Inoue A., Suzuki Т., Fukuhara Т., Maemondo M., Kimura Y., Morikawa N., Watanabe H., Saijo Y, Nukiwa T. // J. Clin. Oncol. — 2006. — Vol. 24. — P. 3340—3346.
11. Clinical course of patients with non-small cell lung cancer and epidermal growth lactor receptor exon 19 and exon 21 mutations treated with gefitinib or erlotinib / Riely G. J., Pao W,, Pham D., Rizvi N., Venkatraman E. S., Zakowski M. F., Kris M. G., Ladanyi M., Miller V, A. // Clin. Cancer Res. — 2006. — Vol. 12. — P. 839—844.
12. Exon 19 deletion mutations of epidermal growth factor receptor are associated with prolonged survival in non-small cell lung cancer patients treated with gefitinib or erlotinib / Jackman D. M, Yeap B. Y., Sequist L. V., Lindeman N., Holmes A. J. p Joshi V. A., Bell D. W., Huberman M. S., Halmos В., RabinM. S., Haber D. A., LynchT. J., Meyerson M, Johnson В. E., Janne Р. A. // Clin. Cancer Res. — 2006. — Vol. 12. — P. 3908— 3914.
13. Detection of tumor cells in blood using CD45 magnetic cell separation followed by nested mutant allele-specific amplification of p53 and K-ras genes in patients with colorectal cancer / linuma R, Okinaga K., Adachi М., Suda К., Sekine Т., Sakagawa К., Baba Y., Tamura J., Китада! Н., Ida А. // Int. J, Cancer. — 2000. — Vol. 89. — Р. 337—344.
14. А simple method of detecting K-ras point mutations in stool samples for colorectal cancer screening using one-step poiymerase chain reaction/restriction fragment length polymorphism analysis / Nishikawa Т., Maemura K., Hirata I., Matsuse R., Morikawa H., Toshina K., Murano M., Hashimoto K., Nakagawa Y., Saitoh O., Uchida K., Katsu K. // Clin. Chim. Acta. — 2002. — Vol. 318. — P. 107— 1 12.
15. Milbury С. A., Li J., Makrigiorgos G. M. PCR-based methods for the enrichment of minority alleles and mutations // Clin. Chem. — 2009. — Vol. 55. — P. 632—640.
16. Detection of tumor mutations in the presence of excess amounts of normal DNA / Sun X. r Hung K., Wu L., Sidransky D., Guo B. // Nat. Biotechnol. — 2002. —Vol. 20. — P. 186—189.
17. LNA clamped PCR: A specific method for detection of Ki-ras gene mutations in patients with sporadic colorectal carcinomas / Beranek M., Jandik Р., SachaM, Rajman M, SakraL., StumrF., SoudkovaE., Zivny Р., Havlicek K. // Klin Biochem. Metab. — 2006. — Vol. 14. — P. 217 — 220.
18. LNA (locked nucleic acids); synthesis and high-affinity nucleic acid recognition / Sanjay K., Singh S. K., Nielsen Р., Koshkin A. A, Wengel J. // Chem. Commun. — 1998. — Vol. 4. — P. 455 — 456.
19. Гагарин И. M. Молекулярные маркеры эффективности ингибиторов EGFR при немелкоклеточном раке легкого и колоректальном раке: Автореф. дис... канд. мед. наук. — М., 201 1. — 34 с.
20. Ке S-H., Madison E.L. Rapid and efficient site-directed mutagenesis by single-tube «megaprimer» PCR method // Nucleic Acids Res. — 1997. — Vol. 25, N 16. — P. 3371—3372.
21. Detectlon of rare mutant K-ras DNA in a single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe / Luo J.-D., Chan E.-C, Shih C.-L, ChenT.-L., Ying Liang Y., Hwang T.-L., Chiou C.-C. // Nucleic Acids Res. — 2006. — Vol. 34, N 2. — P. 12.
22. Hydrogel drop microchips with immobilized DNA: properties and methods for large-scale productlon / Rubina A. Y., Pan'kov S. V., Dementieva E. I., Pen'kov D. N., Butygin А. V., Vasiliskov V. A., ChudinovA. V., Mlkheikin A. L., Mikhailovich V. M., MirzabekovA. D, // Anal. Blochem. — 2004. — Vol. 325. — P. 92—106.
23. Создание биочипа для анализа полиморфизма в генах системы биотрансформации / Глотов А. С., Наседкина Т. В., Иващенко Т. Э., Юрасов Р. А., Суржиков С. А., Паньков С. В., Чудинов А. В., Баранов В. С., Заседателев А. С. // Мол. биол. — 2005. — № 39. — С. 403—412.
24. Применение Ирессы (гефитиниба) в качестве терапии первой линии для лечения неоперабельных аденокарцином легкого, содержащих мутацию в гене EGFR / Моисеенко В. М., Проценко С. А., Семенов И. И., Моисеенко Ф. В., Левченко Е. В., Мацко Д. Е., Иванцов А. О., Иевлева А. Г., Митюшкина Н. В., Того А. В., Имянитов Е. Н. // Современ. онкол. — 2010. — Т. 12, № 1. — С. 60 — 66.
25. Эффективность ингибиторов рецептора эпидермального фактора роста (EGFR) у больных распространенным немелкоклеточным раком легкого / Коломейцева А. А., Гагарин И. М., Мочальникова В. В., Мазуренко Н. Н., Горбунова В. А. // Вестн. РОНЦ им. Н. Н. Блохина РАМН. — 201 1. — Т. 22, № 1. — С. 42—49.
Published
2012-09-21
Section
Clinical studies
