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01-06-2018 | Original Article

Performance validation of an amplicon-based targeted next-generation sequencing assay and mutation profiling of 648 Chinese colorectal cancer patients

Authors: Yajian Wang, Haijing Liu, Yingyong Hou, Xiaoyan Zhou, Li Liang, Zhihong Zhang, Huaiyin Shi, Sanpeng Xu, Peizhen Hu, Zuyu Zheng, Rui Liu, Tingdong Tang, Feng Ye, Zhiyong Liang, Hong Bu

Published in: Virchows Archiv | Issue 6/2018

Abstract

Next-generation sequencing (NGS) has become a promising approach for tumor somatic mutation detection. However, stringent validation is required for its application on clinical specimens, especially for low-quality formalin-fixed paraffin-embedded (FFPE) tissues. Here, we validated the performance of an amplicon-based targeted NGS assay, OncoAim™ DNA panel, on both commercial reference FFPE samples and clinical FFPE samples of Chinese colorectal cancer (CRC) patients. Then we profiled the mutation spectrum of 648 Chinese CRC patients in a multicenter study to explore its clinical utility. This NGS assay achieved 100% test specificity and 95–100% test sensitivity for variants with mutant allele frequency (MAF) ≥ 5% when median read depth ≥ 500×. The orthogonal methods including amplification refractory mutation system (ARMS)-PCR and Sanger sequencing validated that NGS generated three false negatives (FNs) but no false positives (FPs) among 516 clinical samples for KRAS aberration detection. Genomic profiling of Chinese CRC patients with this assay revealed that 63.3% of the tumors harbored clinically actionable alterations. Besides the commonly mutated genes including TP53 (52.82%), KRAS (46.68%), APC (24.09%), PIK3CA (18.94%), SMAD4 (9.47%), BRAF (6.15%), FBXW7 (5.32%), and NRAS (4.15%), other less frequently mutated genes were also identified. Statistically significant association of specific mutated genes with certain clinicopathological features was detected, e.g., both BRAF and PIK3CA were more prevalent in right-side CRC (p < 0.001 and p = 0.002, respectively). We concluded this targeted NGS assay is qualified for clinical practice, and our findings could help the diagnosis and prognosis of Chinese CRC patients.

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Kandoth C, McLellan MD, Vandin F, Ye K, Niu B, Lu C, Xie M, Zhang Q, McMichael JF, Wyczalkowski MA, Leiserson MDM, Miller CA, Welch JS, Walter MJ, Wendl MC, Ley TJ, Wilson RK, Raphael BJ, Ding L (2013) Mutational landscape and significance across 12 major cancer types. Nature 502:333–339. https://doi.org/10.1038/nature12634 CrossRefPubMedPubMedCentral

Mendelsohn J (2013) Personalizing oncology: perspectives and prospects. J Clin Oncol 31:1904–1911. https://doi.org/10.1200/JCO.2012.45.3605 CrossRefPubMed

Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA Jr, Kinzler KW (2013) Cancer genome landscapes. Science 339:1546–1558. https://doi.org/10.1126/science.1235122 CrossRefPubMedPubMedCentral

Zhang J, Zheng J, Yang Y, Lu J, Gao J, Lu T, Sun J, Jiang H, Zhu Y, Zheng Y, Liang Z, Liu T (2015) Molecular spectrum of KRAS, NRAS, BRAF and PIK3CA mutations in Chinese colorectal cancer patients: analysis of 1,110 cases. Sci Rep 5:18678. https://doi.org/10.1038/srep18678 CrossRefPubMedPubMedCentral

De Roock W, Claes B, Bernasconi D, De Schutter J, Biesmans B, Fountzilas G, Kalogeras KT, Kotoula V, Papamichael D, Laurent-Puig P, Penault-Llorca F, Rougier P, Vincenzi B, Santini D, Tonini G, Cappuzzo F, Frattini M, Molinari F, Saletti P, De Dosso S, Martini M, Bardelli A, Siena S, Sartore-Bianchi A, Tabernero J, Macarulla T, Di Fiore F, Gangloff AO, Ciardiello F, Pfeiffer P, Qvortrup C, Hansen TP, Van Cutsem E, Piessevaux H, Lambrechts D, Delorenzi M, Tejpar S (2010) Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol 11:753–762. https://doi.org/10.1016/S1470-2045(10)70130-3 CrossRefPubMed

Tafe LJ (2017) Molecular mechanisms of therapy resistance in solid tumors: chasing “moving” targets. Virchows Arch 471:155–164. https://doi.org/10.1007/s00428-017-2101-7 CrossRefPubMed

Van Cutsem E, Cervantes A, Adam R, Sobrero A, Van Krieken JH, Aderka D, Aranda Aguilar E, Bardelli A, Benson A, Bodoky G, Ciardiello F, D'Hoore A, Diaz-Rubio E, Douillard JY, Ducreux M, Falcone A, Grothey A, Gruenberger T, Haustermans K, Heinemann V, Hoff P, Kohne CH, Labianca R, Laurent-Puig P, Ma B, Maughan T, Muro K, Normanno N, Osterlund P, Oyen WJ, Papamichael D, Pentheroudakis G, Pfeiffer P, Price TJ, Punt C, Ricke J, Roth A, Salazar R, Scheithauer W, Schmoll HJ, Tabernero J, Taieb J, Tejpar S, Wasan H, Yoshino T, Zaanan A, Arnold D (2016) ESMO consensus guidelines for the management of patients with metastatic colorectal cancer. Ann Oncol 27:1386–1422. https://doi.org/10.1093/annonc/mdw235 CrossRefPubMed

(Updated November 23, 2016,Accessed January 10, 2017) National comprehensive cancer network clinical practice guidelines in oncology (NCCN Guidelines®) Colon cancer version 1.2017. nccn.org/

(Updated November 23, 2016. Accessed January 10, 2017) National comprehensive cancer network clinical practice guidelines in oncology (NCCN Guidelines®) rectal cancer version 1.2017. nccn.org/

10.

Kamps R, Brandao RD, Bosch BJ, Paulussen AD, Xanthoulea S, Blok MJ, Romano A (2017) Next-generation sequencing in oncology: genetic diagnosis, risk prediction and cancer classification. Int J Mol Sci 18. https://doi.org/10.3390/ijms18020308

11.

Hirsch B, Endris V, Lassmann S, Weichert W, Pfarr N, Schirmacher P, Kovaleva V, Werner M, Bonzheim I, Fend F, Sperveslage J, Kaulich K, Zacher A, Reifenberger G, Kohrer K, Stepanow S, Lerke S, Mayr T, Aust DE, Baretton G, Weidner S, Jung A, Kirchner T, Hansmann ML, Burbat L, von der Wall E, Dietel M, Hummel M (2018) Multicenter validation of cancer gene panel-based next-generation sequencing for translational research and molecular diagnostics. Virchows Arch. https://doi.org/10.1007/s00428-017-2288-7

12.

Kaul KL (2017) Preparing pathology for precision medicine: challenges and opportunities. Virchows Arch 471:141–146. https://doi.org/10.1007/s00428-017-2141-z CrossRefPubMed

13.

Ludyga N, Grunwald B, Azimzadeh O, Englert S, Hofler H, Tapio S, Aubele M (2012) Nucleic acids from long-term preserved FFPE tissues are suitable for downstream analyses. Virchows Arch 460:131–140. https://doi.org/10.1007/s00428-011-1184-9 CrossRefPubMed

14.

Srinivasan M, Sedmak D, Jewell S (2002) Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol 161:1961–1971. https://doi.org/10.1016/S0002-9440(10)64472-0 CrossRefPubMedPubMedCentral

15.

Do H, Dobrovic A (2015) Sequence artifacts in DNA from formalin-fixed tissues: causes and strategies for minimization. Clin Chem 61:64–71. https://doi.org/10.1373/clinchem.2014.223040 CrossRefPubMed

16.

Deans ZC, Costa JL, Cree I, Dequeker E, Edsjo A, Henderson S, Hummel M, Ligtenberg MJ, Loddo M, Machado JC, Marchetti A, Marquis K, Mason J, Normanno N, Rouleau E, Schuuring E, Snelson KM, Thunnissen E, Tops B, Williams G, van Krieken H, Hall JA, ASBL IQNP (2017) Integration of next-generation sequencing in clinical diagnostic molecular pathology laboratories for analysis of solid tumours; an expert opinion on behalf of IQN path ASBL. Virchows Arch 470:5–20. https://doi.org/10.1007/s00428-016-2025-7 CrossRefPubMed

17.

Frampton GM, Fichtenholtz A, Otto GA, Wang K, Downing SR, He J, Schnall-Levin M, White J, Sanford EM, An P, Sun J, Juhn F, Brennan K, Iwanik K, Maillet A, Buell J, White E, Zhao M, Balasubramanian S, Terzic S, Richards T, Banning V, Garcia L, Mahoney K, Zwirko Z, Donahue A, Beltran H, Mosquera JM, Rubin MA, Dogan S, Hedvat CV, Berger MF, Pusztai L, Lechner M, Boshoff C, Jarosz M, Vietz C, Parker A, Miller VA, Ross JS, Curran J, Cronin MT, Stephens PJ, Lipson D, Yelensky R (2013) Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing. Nat Biotechnol 31:1023–1031. https://doi.org/10.1038/nbt.2696 CrossRefPubMedPubMedCentral

18.

Wong SQ, Li J, Tan AY, Vedururu R, Pang JM, Do H, Ellul J, Doig K, Bell A, MacArthur GA, Fox SB, Thomas DM, Fellowes A, Parisot JP, Dobrovic A, Cohort C (2014) Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing. BMC Med Genet 7:23. https://doi.org/10.1186/1755-8794-7-23 CrossRef

19.

Ryu D, Joung JG, Kim NK, Kim KT, Park WY (2016) Deciphering intratumor heterogeneity using cancer genome analysis. Hum Genet 135:635–642. https://doi.org/10.1007/s00439-016-1670-x CrossRefPubMed

20.

Lopez JI, De Petris G (2017) Discovering intratumor heterogeneity: the next frontier for pathologists. Pathologica 109:110–113PubMed

21.

Langley RE, Rothwell PM (2013) Potential biomarker for aspirin use in colorectal cancer therapy. Nat Rev Clin Oncol 10:8–10. https://doi.org/10.1038/nrclinonc.2012.216 CrossRefPubMed

22.

Misale S, Yaeger R, Hobor S, Scala E, Janakiraman M, Liska D, Valtorta E, Schiavo R, Buscarino M, Siravegna G, Bencardino K, Cercek A, Chen CT, Veronese S, Zanon C, Sartore-Bianchi A, Gambacorta M, Gallicchio M, Vakiani E, Boscaro V, Medico E, Weiser M, Siena S, Di Nicolantonio F, Solit D, Bardelli A (2012) Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer. Nature 486:532–536. https://doi.org/10.1038/nature11156 CrossRefPubMedPubMedCentral

23.

Vagaja NN, Parry J, McCallum D, Thomas MA, Bentel JM (2015) Are all RAS mutations the same? Coexisting KRAS and NRAS mutations in a caecal adenocarcinoma and contiguous tubulovillous adenoma. J Clin Pathol 68:657–660. https://doi.org/10.1136/jclinpath-2015-202969 CrossRefPubMed

24.

Larki P, Gharib E, Yaghoob Taleghani M, Khorshidi F, Nazemalhosseini-Mojarad E, Asadzadeh Aghdaei H (2017) Coexistence of KRAS and BRAF mutations in colorectal cancer: a case report supporting the concept of tumoral heterogeneity. Cell J 19:113–117. https://doi.org/10.22074/cellj.2017.5123 PubMedPubMedCentralCrossRef

25.

Jauhri M, Bhatnagar A, Gupta S, Bp M, Minhas S, Shokeen Y, Aggarwal S (2017) Prevalence and coexistence of KRAS, BRAF, PIK3CA, NRAS, TP53, and APC mutations in Indian colorectal cancer patients: next-generation sequencing-based cohort study. Tumour Biol 39:1010428317692265. https://doi.org/10.1177/1010428317692265 CrossRefPubMed

26.

Vendrell JA, Grand D, Rouquette I, Costes V, Icher S, Selves J, Larrieux M, Barbe A, Brousset P, Solassol J (2017) High-throughput detection of clinically targetable alterations using next-generation sequencing. Oncotarget 8:40345–40358. https://doi.org/10.18632/oncotarget.15875 CrossRefPubMedPubMedCentral

27.

Eifert C, Pantazi A, Sun R, Xu J, Cingolani P, Heyer J, Russell M, Lvova M, Ring J, Tse JY, Lyle S, Protopopov A (2017) Clinical application of a cancer genomic profiling assay to guide precision medicine decisions. Per Med 14:309–325. https://doi.org/10.2217/pme-2017-0011 CrossRefPubMedPubMedCentral

28.

Liang J, She Y, Zhu J, Wei L, Zhang L, Gao L, Wang Y, Xing J, Guo Y, Meng X, Li P (2016) Development and validation of an ultra-high sensitive next-generation sequencing assay for molecular diagnosis of clinical oncology. Int J Oncol 49:2088–2104. https://doi.org/10.3892/ijo.2016.3707 CrossRefPubMed

29.

Fontanges Q, De Mendonca R, Salmon I, Le Mercier M, D'Haene N (2016) Clinical application of targeted next generation sequencing for colorectal cancers. Int J Mol Sci 17. https://doi.org/10.3390/ijms17122117

30.

Zhang L, Chen L, Sah S, Latham GJ, Patel R, Song Q, Koeppen H, Tam R, Schleifman E, Mashhedi H, Chalasani S, Fu L, Sumiyoshi T, Raja R, Forrest W, Hampton GM, Lackner MR, Hegde P, Jia S (2014) Profiling cancer gene mutations in clinical formalin-fixed, paraffin-embedded colorectal tumor specimens using targeted next-generation sequencing. Oncologist 19:336–343. https://doi.org/10.1634/theoncologist.2013-0180 CrossRefPubMedPubMedCentral

31.

Singh RR, Patel KP, Routbort MJ, Reddy NG, Barkoh BA, Handal B, Kanagal-Shamanna R, Greaves WO, Medeiros LJ, Aldape KD, Luthra R (2013) Clinical validation of a next-generation sequencing screen for mutational hotspots in 46 cancer-related genes. J Mol Diagn 15:607–622. https://doi.org/10.1016/j.jmoldx.2013.05.003 CrossRefPubMed

32.

Robbe P, Popitsch N, Knight SJL, Antoniou P, Becq J, He M, Kanapin A, Samsonova A, Vavoulis DV, Ross MT, Kingsbury Z, Cabes M, Ramos SDC, Page S, Dreau H, Ridout K, Jones LJ, Tuff-Lacey A, Henderson S, Mason J, Buffa FM, Verrill C, Maldonado-Perez D, Roxanis I, Collantes E, Browning L, Dhar S, Damato S, Davies S, Caulfield M, Bentley DR, Taylor JC, Turnbull C, Schuh A (2018) Clinical whole-genome sequencing from routine formalin-fixed, paraffin-embedded specimens: pilot study for the 100,000 Genomes Project. Genet Med. https://doi.org/10.1038/gim.2017.241

33.

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 12:425–432. https://doi.org/10.2353/jmoldx.2010.090188 CrossRefPubMedPubMedCentral

34.

Ogasawara N, Bando H, Kawamoto Y, Yoshino T, Tsuchihara K, Ohtsu A, Esumi H (2011) Feasibility and robustness of amplification refractory mutation system (ARMS)-based KRAS testing using clinically available formalin-fixed, paraffin-embedded samples of colorectal cancers. Jpn J Clin Oncol 41:52–56. https://doi.org/10.1093/jjco/hyq151 CrossRefPubMed

35.

Huang T, Zhuge J, Zhang WW (2013) Sensitive detection of BRAF V600E mutation by amplification refractory mutation system (ARMS)-PCR. Biomark Res 1:3. https://doi.org/10.1186/2050-7771-1-3 CrossRefPubMedPubMedCentral

36.

Chen Q, Lu P, Jones AV, Cross NC, Silver RT, Wang YL (2007) Amplification refractory mutation system, a highly sensitive and simple polymerase chain reaction assay, for the detection of JAK2 V617F mutation in chronic myeloproliferative disorders. J Mol Diagn 9:272–276. https://doi.org/10.2353/jmoldx.2007.060133 CrossRefPubMedPubMedCentral

37.

Shen Y, Wang J, Han X, Yang H, Wang S, Lin D, Shi Y (2013) Effectors of epidermal growth factor receptor pathway: the genetic profiling of KRAS, BRAF, PIK3CA, NRAS mutations in colorectal cancer characteristics and personalized medicine. PLoS One 8:e81628. https://doi.org/10.1371/journal.pone.0081628 CrossRefPubMedPubMedCentral

38.

Lan YT, Jen-Kou L, Lin CH, Yang SH, Lin CC, Wang HS, Chen WS, Lin TC, Jiang JK, Chang SC (2015) Mutations in the RAS and PI3K pathways are associated with metastatic location in colorectal cancers. J Surg Oncol 111:905–910. https://doi.org/10.1002/jso.23895 CrossRefPubMed

39.

Ye JX, Liu Y, Qin Y, Zhong HH, Yi WN, Shi XY (2015) KRAS and BRAF gene mutations and DNA mismatch repair status in Chinese colorectal carcinoma patients. World J Gastroenterol 21:1595–1605. https://doi.org/10.3748/wjg.v21.i5.1595 CrossRefPubMedPubMedCentral

40.

Ling YYJ, Qiu T et al (2012) Detection of KRAS, BRAF, PIK3CA and EGFR gene mutations in colorectal carcinoma. Zhonghua Bing Li Xue Za Zhi 41:4

41.

Al-Shamsi HO, Jones J, Fahmawi Y, Dahbour I, Tabash A, Abdel-Wahab R, Abousamra AO, Shaw KR, Xiao L, Hassan MM, Kipp BR, Kopetz S, Soliman AS, McWilliams RR, Wolff RA (2016) Molecular spectrum of KRAS, NRAS, BRAF, PIK3CA, TP53, and APC somatic gene mutations in Arab patients with colorectal cancer: determination of frequency and distribution pattern. J Gastrointest Oncol 7:882–902. https://doi.org/10.21037/jgo.2016.11.02 CrossRefPubMedPubMedCentral

42.

Korphaisarn K, Morris VK, Overman MJ, Fogelman DR, Kee BK, Raghav KPS, Manuel S, Shureiqi I, Wolff RA, Eng C, Menter D, Hamilton SR, Kopetz S, Dasari A (2017) FBXW7 missense mutation: a novel negative prognostic factor in metastatic colorectal adenocarcinoma. Oncotarget 8:39268–39279. https://doi.org/10.18632/oncotarget.16848 PubMedPubMedCentralCrossRef

43.

Marmol I, Sanchez-de-Diego C, Pradilla Dieste A, Cerrada E, Rodriguez Yoldi MJ (2017) Colorectal carcinoma: a general overview and future perspectives in colorectal cancer. Int J Mol Sci 18. https://doi.org/10.3390/ijms18010197

Title: Performance validation of an amplicon-based targeted next-generation sequencing assay and mutation profiling of 648 Chinese colorectal cancer patients
Authors: Yajian Wang
Haijing Liu
Yingyong Hou
Xiaoyan Zhou
Li Liang
Zhihong Zhang
Huaiyin Shi
Sanpeng Xu
Peizhen Hu
Zuyu Zheng
Rui Liu
Tingdong Tang
Feng Ye
Zhiyong Liang
Hong Bu
Publication date: 01-06-2018
Publisher: Springer Berlin Heidelberg
Published in: Virchows Archiv / Issue 6/2018
Print ISSN: 0945-6317
Electronic ISSN: 1432-2307
DOI: https://doi.org/10.1007/s00428-018-2359-4

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Performance validation of an amplicon-based targeted next-generation sequencing assay and mutation profiling of 648 Chinese colorectal cancer patients

Abstract

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Abstract

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