Current status and future potential of somatic mutation testing from circulating free DNA in patients with solid tumours

The HUGO Journal - Tập 4 - Trang 11-21 - 2011
K. L. Aung1, R. E. Board1,2, G. Ellison3, E. Donald3, T. Ward1, G. Clack3, M. Ranson1,2, A. Hughes3, W. Newman4, C. Dive1,2
1Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK
2School of Cancer and Enabling Sciences, Manchester Cancer Research Centre (MCRC), University of Manchester, Manchester, UK
3AstraZeneca Pharmaceuticals, Cheshire, UK
4Genetic Medicine, St. Mary’s Hospital, School of Biomedicine, Manchester Academic Health Sciences Centre (MAHSC), University of Manchester, Manchester, UK

Tóm tắt

Genetic alterations can determine the natural history of cancer and its treatment response. With further advances in DNA sequencing technology, multiple novel genetic alterations will be discovered which could be exploited as prognostic, predictive and pharmacodynamic biomarkers in the development and use of cancer therapeutics. As such, the importance in clinical practice of efficient and robust somatic mutation testing in solid tumours cannot be overemphasized in the current era of personalized medicine. However, significant challenges remain regarding the testing of genetic biomarkers in clinical practice. Reliance on archived formalin fixed, paraffin embedded tumour, obtained from diagnostic biopsies, for testing somatic genetic alterations could restrict the scientific community in asking relevant questions about a patient’s cancer biology. Problems inherent with using formalin fixed, archival tissue are well recognized and difficult to resolve. It could be argued that to achieve rapid and efficient incorporation of genetic biomarkers into clinical practice, somatic mutation testing in cancer patients should be simpler, less invasive using a readily available clinical sample, whilst maintaining robustness and reproducibility. In this regard, use of circulating free DNA (cfDNA) from plasma or serum as an alternative and/or additional source of DNA to test cancer specific genetic alterations is an attractive proposition. In light of encouraging results from recent studies, this mini review will discuss the current role and future potential of somatic mutation testing from circulating or cell free DNA derived from the blood of patients with solid tumours.

Tài liệu tham khảo

Angenendt P, David K, Juhl H, Diehl F (2010) Detection of phosphoinositide-3-kinase, catalytic, and alpha polypeptide (PIK3CA) mutations in matched tissue and plasma samples from patients with metastatic breast cancer. J Clin Oncol (Meeting Abstracts) 28:15s (suppl 10502) Bachman KE, Argani P, Samuels Y, Silliman N, Ptak J, Szabo S, Konishi H, Karakas B, Blair BG, Lin C, Peters BA, Velculescu VE, Park BH (2004) The PIK3CA gene is mutated with high frequency in human breast cancers. Cancer Biol Ther 3(8):772–775 Board RE, Thelwell NJ, Ravetto PF, Little S, Ranson M, Dive C, Hughes A, Whitcombe D (2008) Multiplexed assays for detection of mutations in PIK3CA. Clin Chem 54(4):757–760 Board RE, Ellison G, Orr MC, Kemsley KR, McWalter G, Blockley LY, Dearden SP, Morris C, Ranson M, Cantarini MV, Dive C, Hughes A (2009) Detection of BRAF mutations in the tumour and serum of patients enrolled in the AZD6244 (ARRY-142886) advanced melanoma phase II study. Br J Cancer 101(10):1724–1730 Board RE, Wardley AM, Dixon JM, Armstrong AC, Howell S, Renshaw L, Donald E, Greystoke A, Ranson M, Hughes A, Dive C (2010) Deteection of PIK3CA mutations in circulating free DNA in patients with breast cancer. Breast Cancer Res Treat 120(2):461–467 Bokemeyer C, Bondarenko I, Makhson A, Hartmann JT, Aparicio J, de Braud F, Donea S, Ludwig H, Schuch G, Stroh C, Loos AH, Zubel A, Koralewski P (2009) Fluorouracil, leucovorin, and oxaliplatin with and without cetuximab in the first-line treatment of metastatic colorectal cancer. J Clin Oncol 27(5):663–671 Bos JL, Fearon ER, Hamilton SR, Verlaan-de Vries M, van Boom JH, van der Eb AJ, Vogelstein B (1987) Prevalence of ras gene mutations in human colorectal cancers. Nature 327(6120):293–297 Brose MS, Volpe P, Feldman M, Kumar M, Rishi I, Gerrero R, Einhorn E, Herlyn M, Minna J, Nicholson A, Roth JA, Albelda SM, Davies H, Cox C, Brignell G, Stephens P, Futreal PA, Wooster R, Stratton MR, Weber BL (2002) BRAF and RAS mutations in human lung cancer and melanoma. Cancer Res 62(23):6997–7000 Castells A, Puig P, Móra J, Boadas J, Boix L, Urgell E, Solé M, Capellà G, Lluís F, Fernández-Cruz L, Navarro S, Farré A (1999) K-ras mutations in DNA extracted from the plasma of patients with pancreatic carcinoma: diagnostic utility and prognostic significance. J Clin Oncol 17(2):578–584 Chen Z, Feng J, Buzin CH, Liu Q, Weiss L, Kernstine K, Somlo G, Sommer SS (2009) Analysis of cancer mutation signatures in blood by a novel ultra-sensitive assay: monitoring of therapy or recurrence in non-metastatic breast cancer. PLoS One 4(9):e7220 Chuang TC, Chuang AY, Poeta L, Koch WM, Califano JA, Tufano RP (2010) Detectable BRAF mutation in serum DNA samples from patients with papillary thyroid carcinomas. Head Neck 32(2):229–234 Cohen Y, Rosenbaum E, Clark DP, Zeiger MA, Umbricht CB, Tufano RP, Sidransky D, Westra WH (2004) Mutational analysis of BRAF in fine needle aspiration biopsies of the thyroid: a potential application for the preoperative assessment of thyroid nodules. Clin Cancer Res 10(8):2761–2765 Daniotti M, Vallacchi V, Rivoltini L, Patuzzo R, Santinami M, Arienti F, Cutolo G, Pierotti MA, Parmiani G, Rodolfo M (2007) Detection of mutated BRAFV600E variant in circulating DNA of stage III–IV melanoma patients. Int J Cancer 120(11):2439–2444 Di Nicolantonio F, Martini M, Molinari F, Sartore-Bianchi A, Arena S, Saletti P, De Dosso S, Mazzucchelli L, Frattini M, Siena S, Bardelli A (2008) Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer. J Clin Oncol 26(35):5705–5712 Dianxu F, Shengdao Z, Tianquan H, Yu J, Lei Ruoqing, Zurong Y, Xuezhi W (2002) A prospective study of detection of pancreatic carcinoma by combined plasma K-ras mutations and serum CA19-9 analysis. Pancreas 25(4):336–341 Diehl F, Li M, Dressman D, He Y, Shen D, Szabo S, Diaz LA Jr, Goodman SN, David KA, Juhl H, Kinzler KW, Vogelstein B (2005) Detection and quantification of mutations in the plasma of patients with colorectal tumours. Proc Natl Acad Sci U S A 102(45):16368–16373 Diehl F, Li M, He Y, Kinzler KW, Vogelstein B, Dressman D (2006) BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions. Nat Methods 3(7):551–559 Diehl F, Schmidt K, Choti MA, Romans K, Goodman S, Li M, Thornton K, Agrawal N, Sokoll L, Szabo SA, Kinzler KW, Vogelstein B, Diaz LA Jr (2008) Circulating mutant DNA to assess tumour dynamics. Nat Med 14(9):985–990 Engelman JA, Jänne PA (2008) Mechanisms of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer. Clin Cancer Res 14(10):2895–2899 Fleischhacker M, Schmidt B (2007) Circulating nucleic acids (CNAs) and cancer—a survey. Biochim Biophys Acta 1775(1):181–232 Fleischhacker M, Schmidt B (2008) Cell-free DNA resuscitated for tumour testing. Nat Med 14(9):914–915 Gatalica Z, Torlakovic E (2008) Pathology of the hereditary colorectal carcinoma. Fam Cancer 7(1):15–26 Hainaut P, Hollstein M (2000) p53 and human cancer: the first ten thousand mutations. Adv Cancer Res 77:81–137 Hodgson DR, Wellings R, Orr MC, McCormack R, Malone M, Board RE, Cantarini MV (2010) Circulating tumour-derived predictive biomarkers in oncology. Drug Discov Today 15(3–4):98–101 Hosny G, Farahat N, Tayel H, Hainaut P (2008) Ser-249 TP53 and CTNNB1 mutations in circulating free DNA of Egyptian patients with hepatocellular carcinoma versus chronic liver diseases. Cancer Lett 264(2):201–208 Hsu IC, Metcalf RA, Sun T, Welsh JA, Wang NJ, Harris CC (1991) Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature 350(6317):427–428 Hussain SP, Schwank J, Staib F, Wang XW, Harris CC (2007) TP53 mutations and hepatocellular carcinoma: insights into the etiology and pathogenesis of liver cancer. Oncogene 26(15):2166–2176 Inoue A, Suzuki T, Fukuhara T, Maemondo M, Kimura Y, Morikawa N, Watanabe H, Saijo Y, Nukiwa T (2006) Prospective phase II study of gefitinib for chemotherapy-naive patients with advanced non-small-cell lung cancer with epidermal growth factor receptor gene mutations. J Clin Oncol 24(21):3340–3346 Epub 2006 Jun 19 Jackman DM, Miller VA, Cioffredi LA, Yeap BY, Jänne PA, Riely GJ, Ruiz MG, Giaccone G, Sequist LV, Johnson BE (2009) Impact of epidermal growth factor receptor and KRAS mutations on clinical outcomes in previously untreated non-small cell lung cancer patients: results of an online tumor registry of clinical trials. Clin Cancer Res 15(16):5267–5273 Kimura H, Kasahara K, Kawaishi M, Kunitoh H, Tamura T, Holloway B, Nishio K (2006) Detection of epidermal growth factor receptor mutations in serum as a predictor of the response to gefitinib in patients with non-small-cell lung cancer. Clin Cancer Res 12(13):3915–3921 Kimura H, Suminoe M, Kasahara K, Sone T, Araya T, Tamori S, Koizumi F, Nishio K, Miyamoto K, Fujimura M, Nakao S (2007) Evaluation of epidermal growth factor receptor mutation status in serum DNA as a predictor of response to gefitinib (IRESSA). Br J Cancer 97(6):778–784 Kirk GD, Lesi OA, Mendy M, Szymañska K, Whittle H, Goedert JJ, Hainaut P, Montesano R (2005) 249(ser) TP53 mutation in plasma DNA, hepatitis B viral infection, and risk of hepatocellular carcinoma. Oncogene 24(38):5858–5867 Kopnin BP (2000) Targets of oncogenes and tumor suppressors: key for understanding basic mechanisms of carcinogenesis. Biochemistry (Mosc) 65(1):2–27 Kosaka T, Yatabe Y, Endoh H, Kuwano H, Takahashi T, Mitsudomi T (2004) Mutations of the epidermal growth factor receptor gene in lung cancer: biological and clinical implications. Cancer Res 64(24):8919–8923 Kuang Y, Rogers A, Yeap BY, Wang L, Makrigiorgos M, Vetrand K, Thiede S, Distel RJ, Jänne PA (2009) Noninvasive detection of EGFR T790M in gefitinib or erlotinib resistant non-small cell lung cancer. Clin Cancer Res 15(8):2630–2636 Leary RJ, Kinde I, Diehl F, Schmidt K, Clouser C, Duncan C, Antipova A, Lee C, McKernan K, De La Vega FM, Kinzler KW, Vogelstein B, Diaz LA Jr, Velculescu VE (2010) Development of personalized tumour biomarkers using massively parallel sequencing. Sci Transl Med 2(20):20ra14 Lee JW, Soung YH, Kim SY, Lee HW, Park WS, Nam SW, Kim SH, Lee JY, Yoo NJ, Lee SH (2005) PIK3CA gene is frequently mutated in breast carcinomas and hepatocellular carcinomas. Oncogene 24(8):1477–1480 Leon SA, Shapiro B, Sklaroff DM, Yaros MJ (1977) Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res 37(3):646–650 Levine DA, Bogomolniy F, Yee CJ, Lash A, Barakat RR, Borgen PI, Boyd J (2005) Frequent mutation of the PIK3CA gene in ovarian and breast cancers. Clin Cancer Res 11(8):2875–2878 Li M, Diehl F, Dressman D, Vogelstein B, Kinzler KW (2006) BEAMing up for detection and quantification of rare sequence variants. Nat Methods 3(2):95–97 Maher CA, Kumar-Sinha C, Cao X, Kalyana-Sundaram S, Han B, Jing X, Sam L, Barrette T, Palanisamy N, Chinnaiyan AM (2009) Transcriptome sequencing to detect gene fusions in cancer. Nature 458(7234):97–101 Maheswaran S, Sequist LV, Nagrath S, Ulkus L, Brannigan B, Collura CV, Inserra E, Diederichs S, Iafrate AJ, Bell DW, Digumarthy S, Muzikansky A, Irimia D, Settleman J, Tompkins RG, Lynch TJ, Toner M, Haber DA (2008) Detection of mutations in EGFR in circulating lung-cancer cells. N Engl J Med 359(4):366–377 Mandel P, Metais P (1948) Les Acides Nucleiques Du Plasma Sanguin Chez L’Homme. P. C R Acad Sci Paris 142:241–243 Mok TS, Wu YL, Thongprasert S, Yang CH, Chu DT, Saijo N, Sunpaweravong P, Han B, Margono B, Ichinose Y, Nishiwaki Y, Ohe Y, Yang JJ, Chewaskulyong B, Jiang H, Duffield EL, Watkins CL, Armour AA, Fukuoka M (2009) Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med 361(10):947–957 Olsen CC, Schefter TE, Chen H, Kane M, Leong S, McCarter MD, Chen Y, Mack P, Eckhardt SG, Stiegmann G, Raben D (2009) Results of a phase I trial of 12 patients with locally advanced pancreatic carcinoma combining gefitinib, paclitaxel, and 3-dimensional conformal radiation: report of toxicity and evaluation of circulating K-ras as a potential biomarker of response to therapy. Am J Clin Oncol 32(2):115–121 Perrone F, Lampis A, Orsenigo M, Di Bartolomeo M, Gevorgyan A, Losa M, Frattini M, Riva C, Andreola S, Bajetta E, Bertario L, Leo E, Pierotti MA, Pilotti S (2009) PI3KCA/PTEN deregulation contributes to impaired responses to cetuximab in metastatic colorectal cancer patients. Ann Oncol 20(1):84–90 Epub 2008 Jul 31 Pinzani P, Salvianti F, Cascella R, Massi D, De Giorgi V, Pazzagli M, Orlando C (2010) Allele specific Taqman-based real-time PCR assay to quantify circulating BRAFV600E mutated DNA in plasma of melanoma patients. Clin Chim Acta 411(17–18):1319–1324 Pleasance ED, Cheetham RK, Stephens PJ, McBride DJ, Humphray SJ, Greenman CD, Varela I, Lin ML, Ordóñez GR, Bignell GR, Ye K, Alipaz J, Bauer MJ, Beare D, Butler A, Carter RJ, Chen L, Cox AJ, Edkins S, Kokko-Gonzales PI, Gormley NA, Grocock RJ, Haudenschild CD, Hims MM, James T, Jia M, Kingsbury Z, Leroy C, Marshall J, Menzies A, Mudie LJ, Ning Z, Royce T, Schulz-Trieglaff OB, Spiridou A, Stebbings LA, Szajkowski L, Teague J, Williamson D, Chin L, Ross MT, Campbell PJ, Bentley DR, Futreal PA, Stratton MR (2009) Complex landscapes of somatic rearrangement in human breast cancer genomes. Nature 462(7276):1005–1010 Plesec TP, Hunt JL (2009) KRAS mutation testing in colorectal cancer. Adv Anat Pathol 16(4):196–203 Ramirez JL, Sarries C, de Castro PL, Roig B, Queralt C, Escuin D, de Aguirre I, Sanchez JM, Manzano JL, Margelí M, Sanchez JJ, Astudillo J, Taron M, Rosell R (2003) Methylation patterns and K-ras mutations in tumour and paired serum of resected non-small-cell lung cancer patients. Cancer Lett 193(2):207–216 Riely GJ, Politi KA, Miller VA, Pao W (2006) Update on epidermal growth factor receptor mutations in non-small cell lung cancer. Clin Cancer Res 12(24):7232–7241 Rosell R, Moran T, Queralt C, Porta R, Cardenal F, Camps C, Majem M, Lopez-Vivanco G, Isla D, Provencio M, Insa A, Massuti B, Gonzalez-Larriba JL, Paz-Ares L, Bover I, Garcia-Campelo R, Moreno MA, Catot S, Rolfo C, Reguart N, Palmero R, Sánchez JM, Bastus R, Mayo C, Bertran-Alamillo J, Molina MA, Sanchez JJ, Taron M, Spanish Lung Cancer Group (2009) Screening for epidermal growth factor receptor mutations in lung cancer. N Engl J Med 361(10):958–967 Rumore PM, Steinman CR (1990) Endogenous circulating DNA in systemic lupus erythematosus. Occurrence as multimeric complexes bound to histone. J Clin Invest 86(1):69–74 Ryan BM, Lefort F, McManus R, Daly J, Keeling PW, Weir DG, Kelleher D (2003) A prospective study of circulating mutant KRAS2 in the serum of patients with colorectal neoplasia: strong prognostic indicator in postoperative follow up. Gut 52(1):101–108 Sartore-Bianchi A, Martini M, Molinari F, Veronese S, Nichelatti M, Artale S, Di Nicolantonio F, Saletti P, De Dosso S, Mazzucchelli L, Frattini M, Siena S, Bardelli A (2009) PIK3CA mutations in colorectal cancer are associated with clinical resistance to EGFR-targeted monoclonal antibodies. Cancer Res 69(5):1851–1857 Sequist LV, Martins RG, Spigel D, Grunberg SM, Spira A, Jänne PA, Joshi VA, McCollum D, Evans TL, Muzikansky A, Kuhlmann GL, Han M, Goldberg JS, Settleman J, Iafrate AJ, Engelman JA, Haber DA, Johnson BE, Lynch TJ (2008) First-line gefitinib in patients with advanced non-small-cell lung cancer harboring somatic EGFR mutations. J Clin Oncol 26(15):2442–2449 Sequist LV, Engelman JA, Lynch TJ (2009) Toward noninvasive genomic screening of lung cancer patients. J Clin Oncol 27(16):2589–2591 Shah SP, Morin RD, Khattra J, Prentice L, Pugh T, Burleigh A, Delaney A, Gelmon K, Guliany R, Senz J, Steidl C, Holt RA, Jones S, Sun M, Leung G, Moore R, Severson T, Taylor GA, Teschendorff AE, Tse K, Turashvili G, Varhol R, Warren RL, Watson P, Zhao Y, Caldas C, Huntsman D, Hirst M, Marra MA, Aparicio S (2009) Mutational evolution in a lobular breast tumour profiled at single nucleotide resolution. Nature 461(7265):809–813 Shinozaki M, O’Day SJ, Kitago M, Amersi F, Kuo C, Kim J, Wang HJ, Hoon DS (2007) Utility of circulating B-RAF DNA mutation in serum for monitoring melanoma patients receiving biochemotherapy. Clin Cancer Res 13(7):2068–2074 Sluiter MD, van Rensburg EJ (2011) Large genomic rearrangements of the BRCA1 and BRCA2 genes: review of the literature and report of a novel BRCA1 mutation. Breast Cancer Res Treat. 125(2):325–349 Stratton MR, Campbell PJ, Futreal PA (2009) The cancer genome. Nature 458(7239):719–724 Stroun M, Anker P, Lyautey J, Lederrey C, Maurice PA (1987) Isolation and characterization of DNA from the plasma of cancer patients. Eur J Cancer Clin Oncol 23(6):707–712 Stroun M, Anker P, Maurice P, Lyautey J, Lederrey C, Beljanski M (1989) Neoplastic characteristics of the DNA found in the plasma of cancer patients. Oncology 46(5):318–322 Stroun M, Maurice P, Vasioukhin V, Lyautey J, Lederrey C, Lefort F, Rossier A, Chen XQ, Anker P (2000) The origin and mechanism of circulating DNA. Ann NY Acad Sci 906:161–168 Stroun M, Lyautey J, Lederrey C, Olson-Sand A, Anker P (2001) About the possible origin and mechanism of circulating DNA apoptosis and active DNA release. Clin Chim Acta 313(1–2):139–142 Szymańska K, Lesi OA, Kirk GD, Sam O, Taniere P, Scoazec JY, Mendy M, Friesen MD, Whittle H, Montesano R, Hainaut P (2004) Ser-249TP53 mutation in tumour and plasma DNA of hepatocellular carcinoma patients from a high incidence area in the Gambia, West Africa. Int J Cancer 110(3):374–379 Tsao MS, Sakurada A, Cutz JC, Zhu CQ, Kamel-Reid S, Squire J, Lorimer I, Zhang T, Liu N, Daneshmand M, Marrano P, da Cunha Santos G, Lagarde A, Richardson F, Seymour L, Whitehead M, Ding K, Pater J, Shepherd FA (2005) Erlotinib in lung cancer—molecular and clinical predictors of outcome. N Engl J Med 353(2):133–144 Tsiatis AC, Norris-Kirby A, Rich RG, Hafez MJ, Gocke CD, Eshleman JR, Murphy KM (2010) Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications. J Mol Diagn 12(4):425–432 Van Cutsem E, Köhne CH, Hitre E, Zaluski J, Chang Chien CR, Makhson A, D’Haens G, Pintér T, Lim R, Bodoky G, Roh JK, Folprecht G, Ruff P, Stroh C, Tejpar S, Schlichting M, Nippgen J, Rougier P (2009) Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. N Engl J Med 360(14):1408–1417 van der Vaart M, Semenov DV, Kuligina EV, Richter VA, Pretorius PJ (2009) Characterisation of circulating DNA by parallel tagged sequencing on the 454 platform. Clin Chim Acta 409(1–2):21–27 Wang JY, Hsieh JS, Chang MY, Huang TJ, Chen FM, Cheng TL, Alexandersen K, Huang YS, Tzou WS, Lin SR (2004) Molecular detection of APC, K-ras, and p53 mutations in the serum of colorectal cancer patients as circulating biomarkers. World J Surg 28(7):721–726 Wang S, An T, Wang J, Zhao J, Wang Z, Zhuo M, Bai H, Yang L, Zhang Y, Wang X, Duan J, Wang Y, Guo Q, Wu M (2010) Potential clinical significance of a plasma-based KRAS mutation analysis in patients with advanced non-small cell lung cancer. Clin Cancer Res 16(4):1324–1330 Xing M, Westra WH, Tufano RP, Cohen Y, Rosenbaum E, Rhoden KJ, Carson KA, Vasko V, Larin A, Tallini G, Tolaney S, Holt EH, Hui P, Umbricht CB, Basaria S, Ewertz M, Tufaro AP, Califano JA, Ringel MD, Zeiger MA, Sidransky D, Ladenson PW (2005) BRAF mutation predicts a poorer clinical prognosis for papillary thyroid cancer. J Clin Endocrinol Metab 90(12):6373–6379 Yancovitz M, Yoon J, Mikhail M, Gai W, Shapiro RL, Berman RS, Pavlick AC, Chapman PB, Osman I, Polsky D (2007) Detection of mutant BRAF alleles in the plasma of patients with metastatic melanoma. J Mol Diagn 9(2):178–183 Yang CH, Yu CJ, Shih JY, Chang YC, Hu FC, Tsai MC, Chen KY, Lin ZZ, Huang CJ, Shun CT, Huang CL, Bean J, Cheng AL, Pao W, Yang PC (2008) Specific EGFR mutations predict treatment outcome of stage IIIB/IV patients with chemotherapy-naive non-small-cell lung cancer receiving first-line gefitinib monotherapy. J Clin Oncol 26(16):2745–2753 Yung TK, Chan KC, Mok TS, Tong J, To KF, Lo YM (2009) Single-molecule detection of epidermal growth factor receptor mutations in plasma by microfluidics digital PCR in non-small cell lung cancer patients. Clin Cancer Res 15(6):2076–2084