FGA isoform as an indicator of targeted therapy for EGFR mutated lung adenocarcinoma

  • Zhi Shang
  • Xiaomin Niu
  • Kewei Zhang
  • Zhi Qiao
  • Sha Liu
  • Xiaoteng Jiang
  • Chengxi Cao
  • Shun LuEmail author
  • Hua XiaoEmail author
Original Article


Epidermal growth factor receptor (EGFR) gene is frequently mutated in non-small cell lung cancer (NSCLC), which can be targeted by EGFR tyrosine kinase inhibitors (TKIs). It is hard, however, to monitor the performance of EGFR-TKI therapy dynamically. Therefore, therapeutic indicators are urgently needed. Novel antibody microarray, containing 41,472 antibodies, was used for comprehensive analyzing of serum samples from 9 normal subjects and 9 EGFR mutated lung adenocarcinoma patients at three EGFR-TKI treatment time points, including before treatment (Baseline), partial response (PR) during treatment, and disease progression (PD) after resistance. Through microarray data analysis, five candidate antibodies were screened out for confirmation in serum samples and the verified one was utilized for candidate protein identification through immunoprecipitation-mass spectrometry strategy. A novel protein, isoform 2 of fibrinogen alpha chain (FGA2), was revealed and verified in the discovery sample set. Its performance as therapy indicator was further evaluated in another pre-validation sample set (n = 60). Our data confirmed that serum FGA2 level was correlated with EGFR-TKI response (p < 0.05). The expression and secretion of FGA2 in hepatocytes were inhibited by EGFR-TKI, partially explaining the downregulation of FGA2 in serum. Our results demonstrate that FGA2 is an indicator of targeted therapy for EGFR mutated lung adenocarcinoma.

Key messages

  • Antibody microarray was coupled with mass spectrometry for proteomics research.

  • FGA2 was discovered as an indicator of EGFR-TKI targeted therapy.

  • FGA2’s expression/secretion in hepatocytes was dramatically inhibited by EGFR-TKI.


Lung adenocarcinoma Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) Targeted therapy Isoform 2 of fibrinogen alpha chain Antibody microarray 


Author contributions

H.X. and X.M.N. contributed to the study design. Z.S., X.M.N., and K.W.Z. contributed to cell culture and sample collection. Z.S. conducted the experiments and collected the data. X.M.N. and K.W.Z. collected the clinical data. Z.S., X.M.N., K.W.Z., Z.Q., S.L., X.T.J., C.X.C., S.L., and H.X. contributed to the data analysis. The manuscript was written by Z.S., X.M.N., K.W.Z., and H.X. All the authors reviewed the manuscript. All aspects of the study were supervised by H.X. and S.L.

Funding information

This work was supported by grants from the National Natural Science Foundation of China (Nos. 21675110, 81972187, 31727801, 21305087, and 81302005), National Key Research and Development Program (Nos. 2017YFC1200204 and 2016YFC1303300), Projects of the Committee of Shanghai Science and Technology (Nos. 14DZ0501200, 15142200300, and 19ZR1449800), Clinical Research Project of Shanghai Municipal Public Health Bureau (No. 201840122), Advanced Appropriate Technology Promotion Project of Shanghai Municipal Public Health Bureau (No. 2019SY048), Research Project of Shanghai Municipal Cadre Health Bureau (No. 2017(12)), Doctoral Innovation Fund of Shanghai Jiao Tong University School of Medicine (No. BXJ201952), and Interdisciplinary Program of Shanghai Jiao Tong University (Nos. YG2014QN21, YG2015MS48, and YG2017MS80). H.X. is supported by the Recruitment Program of Global Youth Experts of China and National High-tech R&D Program of China (863 Program, No. 2014AA020545). X.M.N is supported by “Shanghai Young Physician Development Program” of Shanghai Municipal Public Health Bureau (No. 2012(105)).

Compliance with ethical standards

The current study adhered to the tenets of the Declaration of Helsinki and was performed after approval by the Institutional Review Board (IRB) of Shanghai Chest Hospital (IRB#KS1753 approved by Shanghai Chest Hospital Ethics Committee). All patients signed the IRB-approved written informed consents and were further enrolled for this study, allowing for the collection and analysis of blood samples. Serum samples from healthy control subjects were collected randomly from physical examination center of the Henan Provincial People’s Hospital (IRB#M15017 approved by Bio-X Ethics Committee of Shanghai Jiao Tong University). All control subjects provided written informed consents and had no history of any cancer.

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

109_2019_1848_MOESM1_ESM.docx (5.2 mb)
ESM 1 (DOCX 5.21 mb)
109_2019_1848_MOESM2_ESM.xlsx (479 kb)
ESM 2 (XLSX 479 kb)


  1. 1.
    Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A (2018) Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 68:394–424CrossRefGoogle Scholar
  2. 2.
    Chen Z, Fillmore CM, Hammerman PS, Kim CF, Wong KK (2014) Non-small-cell lung cancers: a heterogeneous set of diseases. Nat Rev Cancer 14:535–546CrossRefGoogle Scholar
  3. 3.
    Pao W, Chmielecki J (2010) Rational, biologically based treatment of EGFR-mutant non-small-cell lung cancer. Nat Rev Cancer 10:760–774CrossRefGoogle Scholar
  4. 4.
    Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, Louis DN, Christiani DC, Settleman J, Haber DA (2004) Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350:2129–2139CrossRefGoogle Scholar
  5. 5.
    Wu YL, Zhong WZ, Li LY, Zhang XT, Zhang L, Zhou CC, Liu W, Jiang B, Mu XL, Lin JY et al (2007) Epidermal growth factor receptor mutations and their correlation with gefitinib therapy in patients with non-small cell lung cancer: a meta-analysis based on updated individual patient data from six medical centers in mainland China. J Thorac Oncol 2:430–439CrossRefGoogle Scholar
  6. 6.
    Dearden S, Stevens J, Wu YL, Blowers D (2013) Mutation incidence and coincidence in non small-cell lung cancer: meta-analyses by ethnicity and histology (mutMap). Ann Oncol 24:2371–2376CrossRefGoogle Scholar
  7. 7.
    Khozin S, Blumenthal GM, Jiang X, He K, Boyd K, Murgo A, Justice R, Keegan P, Pazdur R (2014) U.S. Food and Drug Administration approval summary: erlotinib for the first-line treatment of metastatic non-small cell lung cancer with epidermal growth factor receptor exon 19 deletions or exon 21 (L858R) substitution mutations. Oncologist 19:774–779CrossRefGoogle Scholar
  8. 8.
    Kazandjian D, Blumenthal GM, Yuan W, He K, Keegan P, Pazdur R (2016) FDA approval of gefitinib for the treatment of patients with metastatic EGFR mutation-positive non-small cell lung cancer. Clin Cancer Res 22:1307–1312CrossRefGoogle Scholar
  9. 9.
    Sequist LV, Yang JC, Yamamoto N, O’Byrne K, Hirsh V, Mok T, Geater SL, Orlov S, Tsai CM, Boyer M et al (2013) Phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations. J Clin Oncol 31:3327–3334CrossRefGoogle Scholar
  10. 10.
    Wu YL, Cheng Y, Zhou X, Lee KH, Nakagawa K, Niho S, Tsuji F, Linke R, Rosell R, Corral J, Migliorino MR, Pluzanski A, Sbar EI, Wang T, White JL, Nadanaciva S, Sandin R, Mok TS (2017) Dacomitinib versus gefitinib as first-line treatment for patients with EGFR-mutation-positive non-small-cell lung cancer (ARCHER 1050): a randomised, open-label, phase 3 trial. Lancet Oncol 18:1454–1466CrossRefGoogle Scholar
  11. 11.
    Greig SL (2016) Osimertinib: first global approval. Drugs 76:263–273CrossRefGoogle Scholar
  12. 12.
    Jackman D, Pao W, Riely GJ, Engelman JA, Kris MG, Janne PA, Lynch T, Johnson BE, Miller VA (2010) Clinical definition of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer. J Clin Oncol 28:357–360CrossRefGoogle Scholar
  13. 13.
    Maemondo M, Inoue A, Kobayashi K, Sugawara S, Oizumi S, Isobe H, Gemma A, Harada M, Yoshizawa H, Kinoshita I, Fujita Y, Okinaga S, Hirano H, Yoshimori K, Harada T, Ogura T, Ando M, Miyazawa H, Tanaka T, Saijo Y, Hagiwara K, Morita S, Nukiwa T, North-East Japan Study Group (2010) Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med 362:2380–2388CrossRefGoogle Scholar
  14. 14.
    Takezawa K, Pirazzoli V, Arcila ME, Nebhan CA, Song X, de Stanchina E, Ohashi K, Janjigian YY, Spitzler PJ, Melnick MA, Riely GJ, Kris MG, Miller VA, Ladanyi M, Politi K, Pao W (2012) HER2 amplification: a potential mechanism of acquired resistance to EGFR inhibition in EGFR-mutant lung cancers that lack the second-site EGFRT790M mutation. Cancer Discov 2:922–933CrossRefGoogle Scholar
  15. 15.
    Remaud A, Cornu C, Guevel A (2009) Agonist muscle activity and antagonist muscle co-activity levels during standardized isotonic and isokinetic knee extensions. J Electromyogr Kinesiol 19:449–458CrossRefGoogle Scholar
  16. 16.
    Ohashi K, Sequist LV, Arcila ME, Moran T, Chmielecki J, Lin YL, Pan Y, Wang L, de Stanchina E, Shien K, Aoe K, Toyooka S, Kiura K, Fernandez-Cuesta L, Fidias P, Yang JC, Miller VA, Riely GJ, Kris MG, Engelman JA, Vnencak-Jones CL, Dias-Santagata D, Ladanyi M, Pao W (2012) Lung cancers with acquired resistance to EGFR inhibitors occasionally harbor BRAF gene mutations but lack mutations in KRAS, NRAS, or MEK1. Proc Natl Acad Sci U S A 109:E2127–E2133CrossRefGoogle Scholar
  17. 17.
    Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, Kris MG, Varmus H (2005) Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 2:e73. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Hanash SM, Pitteri SJ, Faca VM (2008) Mining the plasma proteome for cancer biomarkers. Nature 452:571–579CrossRefGoogle Scholar
  19. 19.
    Xiao H, Zhang L, Zhou H, Lee JM, Garon EB, Wong DT (2012) Proteomic analysis of human saliva from lung cancer patients using two-dimensional difference gel electrophoresis and mass spectrometry. Mol Cell Proteomics 11(M111):012112. CrossRefPubMedGoogle Scholar
  20. 20.
    Michot A, Alet JM, Pelissier P, Grolleau-Raoux JL, Bodin F, Chaput B (2016) Morbidity in combined-procedure associating abdominoplasty and breast surgery: a systematic review. Ann Chir Plast Esthet 61:e9–e19CrossRefGoogle Scholar
  21. 21.
    MacBeath G (2002) Protein microarrays and proteomics. Nat Genet 32:S526–S532CrossRefGoogle Scholar
  22. 22.
    Sutandy FX, Qian J, Chen CS, Zhu H (2013) Overview of protein microarrays. Curr Protocols Protein Sci Chapter 27: Unit 27.21. DOI Google Scholar
  23. 23.
    Hu CJ, Pan JB, Song G, Wen XT, Wu ZY, Chen S, Mo WX, Zhang FC, Qian J, Zhu H, Li YZ (2017) Identification of novel biomarkers for Behcet disease diagnosis using human proteome microarray approach. Mol Cell Proteomics 16:147–156CrossRefGoogle Scholar
  24. 24.
    Yang L, Wang J, Li J, Zhang H, Guo S, Yan M, Zhu Z, Lan B, Ding Y, Xu M, Li W, Gu X, Qi C, Zhu H, Shao Z, Liu B, Tao SC (2016) Identification of serum biomarkers for gastric cancer diagnosis using a human proteome microarray. Mol Cell Proteomics 15:614–623CrossRefGoogle Scholar
  25. 25.
    Schroder C, Srinivasan H, Sill M, Linseisen J, Fellenberg K, Becker N, Nieters A, Hoheisel JD (2013) Plasma protein analysis of patients with different B-cell lymphomas using high-content antibody microarrays. Proteomics Clin Appl 7:802–812CrossRefGoogle Scholar
  26. 26.
    Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, Dancey J, Arbuck S, Gwyther S, Mooney M, Rubinstein L, Shankar L, Dodd L, Kaplan R, Lacombe D, Verweij J (2009) New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 45:228–247CrossRefGoogle Scholar
  27. 27.
    Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, Speed TP (2002) Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30:e15CrossRefGoogle Scholar
  28. 28.
    Sheng L, Luo M, Sun X, Lin N, Mao W, Su D (2013) Serum fibrinogen is an independent prognostic factor in operable nonsmall cell lung cancer. Int J Cancer 133:2720–2725CrossRefGoogle Scholar
  29. 29.
    Zhang L, Xiao H, Zhou H, Santiago S, Lee JM, Garon EB, Yang J, Brinkmann O, Yan X, Akin D, Chia D, Elashoff D, Park NH, Wong DTW (2012) Development of transcriptomic biomarker signature in human saliva to detect lung cancer. Cell Mol Life Sci 69:3341–3350CrossRefGoogle Scholar
  30. 30.
    Sun ZQ, Han XN, Wang HJ, Tang Y, Zhao ZL, Qu YL, Xu RW, Liu YY, Yu XB (2014) Prognostic significance of preoperative fibrinogen in patients with colon cancer. World J Gastroenterol 20:8583–8591CrossRefGoogle Scholar
  31. 31.
    Zhu W, Liu M, Wang GC, Peng B, Yan Y, Che JP, Ma QW, Yao XD, Zheng JH (2014) Fibrinogen alpha chain precursor and apolipoprotein A-I in urine as biomarkers for noninvasive diagnosis of calcium oxalate nephrolithiasis: a proteomics study. Biomed Res Int 2014:415651PubMedPubMedCentralGoogle Scholar
  32. 32.
    Kinoshita A, Onoda H, Imai N, Iwaku A, Oishi M, Tanaka K, Fushiya N, Koike K, Nishino H, Matsushima M, Tajiri H (2013) Elevated plasma fibrinogen levels are associated with a poor prognosis in patients with hepatocellular carcinoma. Oncology 85:269–277CrossRefGoogle Scholar
  33. 33.
    Arigami T, Uenosono Y, Matsushita D, Yanagita S, Uchikado Y, Kita Y, Mori S, Kijima Y, Okumura H, Maemura K, Ishigami S, Natsugoe S (2016) Combined fibrinogen concentration and neutrophil-lymphocyte ratio as a prognostic marker of gastric cancer. Oncol Lett 11:1537–1544CrossRefGoogle Scholar
  34. 34.
    Kobayashi S, Boggon TJ, Dayaram T, Janne PA, Kocher O, Meyerson M, Johnson BE, Eck MJ, Tenen DG, Halmos B (2005) EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 352:786–792CrossRefGoogle Scholar
  35. 35.
    Sahni A, Simpson-Haidaris PJ, Sahni SK, Vaday GG, Francis CW (2008) Fibrinogen synthesized by cancer cells augments the proliferative effect of fibroblast growth factor-2 (FGF-2). J Thromb Haemost 6:176–183CrossRefGoogle Scholar
  36. 36.
    Ernst E (1993) The role of fibrinogen as a cardiovascular risk factor. Atherosclerosis 100:1–12CrossRefGoogle Scholar
  37. 37.
    Fuller GM, Zhang Z (2001) Transcriptional control mechanism of fibrinogen gene expression. Ann N Y Acad Sci 936:469–479CrossRefGoogle Scholar
  38. 38.
    Li L, Han R, Xiao H, Lin C, Wang Y, Liu H, Li K, Chen H, Sun F, Yang Z, Jiang J, He Y (2014) Metformin sensitizes EGFR-TKI-resistant human lung cancer cells in vitro and in vivo through inhibition of IL-6 signaling and EMT reversal. Clin Cancer Res 20:2714–2726CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, School of Life Sciences and BiotechnologyShanghai Jiao Tong UniversityShanghaiChina
  2. 2.Department of Shanghai Lung Cancer Center, Shanghai Chest HospitalShanghai Jiao Tong UniversityShanghaiChina
  3. 3.Department of Vascular and Endovascular Surgery, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou UniversityPeople’s Hospital of Henan UniversityZhengzhouChina
  4. 4.Department of Instrument Science and Engineering, School of Electronic Information and Electrical EngineeringShanghai Jiao Tong UniversityShanghaiChina

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