Fast fluorescence in situ hybridisation for the enhanced detection of MET in non-small cell lung cancer

Autoři: David Jonathan Duncan aff001;  Michel Erminio Vandenberghe aff001;  Marietta Louise Juanita Scott aff001;  Craig Barker aff001
Působiště autorů: Precision Medicine, R&D Oncology, AstraZeneca, Cambridge, England, United Kingdom aff001
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0223926


The c-Met/hepatocyte growth factor receptor pathway is frequently dysregulated in multiple cancer types, including non-small cell lung cancer (NSCLC). MET amplification has been shown to develop as a resistance mechanism to treatment in NSCLC. The identification of increased MET copy number within tumour cells is increasingly important to stratify those tumours and patients which are susceptible to treatment targetting MET kinase inhibition. Fluorescence in situ hybridisation (FISH) has been successfully employed to identify patients with abnormal MET gene copy number with numerous probes available for use. Here we report a FISH protocol that reduces probe hybridisation time in NSCLC tissue to 1 hour and compare the results with other protocols. MET gene copy number was determined in 20 NSCLC cases using 3 FISH probes: 1. Kreatech FISH, MET (7q31) SE 7 ready to use probes, hybridised using an overnight protocol; 2. Dako MET IQFISH probe with CEP7 ready to use probe, hybridised for 2 hours; 3. Kreatech MET (7q31) SE 7 XL FISH probe, prepared in SwiftFISH buffer and hybridised for 1 hour. The MET gene copy number and MET: centromere 7 gene ratio were determined for each tissue and cases categorised as having MET high or MET low status. All three FISH probes were shown to demonstrate good agreement with each other. Overall percentage agreement between probes was ≥90%. Intraclass correlation showed good agreement (ICC ≥0.80) between all three assays for MET gene copy number and MET: centromere 7 gene ratio. These FISH protocols provide evidence that rapid laboratory developed FISH assays with short turnaround time perform consistently with standard protocols, potentially enabling faster treatment decisions.

Klíčová slova:

Biopsy – Cancer treatment – Centromeres – Fluorescent in situ hybridization – Gene amplification – Image analysis – Non-small cell lung cancer – Probe hybridization


1. Petrini I. Biology of MET: a double life between normal tissue repair and tumor progression. Ann Transl Med. 2015;3(6):82. doi: 10.3978/j.issn.2305-5839.2015.03.58 25992381

2. Schmidt L, Duh FM, Chen F, Kishida T, Glenn G, Choyke P, et al. Germline and somatic mutations in the tyrosine kinase domain of the MET proto-oncogene in papillary renal carcinomas. Nat Genet. 1997;16(1):68–73. doi: 10.1038/ng0597-68 9140397

3. Eder JP, Vande Woude GF, Boerner SA, LoRusso PM. Novel therapeutic inhibitors of the c-Met signaling pathway in cancer. Clin Cancer Res. 2009;15(7):2207–14. doi: 10.1158/1078-0432.CCR-08-1306 19318488

4. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2012;2(5):401–4. doi: 10.1158/2159-8290.CD-12-0095 22588877

5. Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal. 2013;6(269):pl1. doi: 10.1126/scisignal.2004088 23550210

6. Park S, Choi YL, Sung CO, An J, Seo J, Ahn MJ, et al. High MET copy number and MET overexpression: poor outcome in non-small cell lung cancer patients. Histol Histopathol. 2012;27(2):197–207. doi: 10.14670/HH-27.197 22207554

7. Frampton GM, Ali SM, Rosenzweig M, Chmielecki J, Lu X, Bauer TM, et al. Activation of MET via diverse exon 14 splicing alterations occurs in multiple tumor types and confers clinical sensitivity to MET inhibitors. Cancer Discov. 2015;5(8):850–9. doi: 10.1158/2159-8290.CD-15-0285 25971938

8. Bean J, Brennan C, Shih JY, Riely G, Viale A, Wang L, et al. MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc Natl Acad Sci U S A. 2007;104(52):20932–7. doi: 10.1073/pnas.0710370104 18093943

9. Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO, et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science. 2007;316(5827):1039–43. doi: 10.1126/science.1141478 17463250

10. Sequist LV, Waltman BA, Dias-Santagata D, Digumarthy S, Turke AB, Fidias P, et al. Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors. Sci Transl Med. 2011;3(75):75ra26. doi: 10.1126/scitranslmed.3002003 21430269

11. Ou SI, Agarwal N, Ali SM. High MET amplification level as a resistance mechanism to osimertinib (AZD9291) in a patient that symptomatically responded to crizotinib treatment post-osimertinib progression. Lung Cancer. 2016;98:59–61. doi: 10.1016/j.lungcan.2016.05.015 27393507

12. Shao D, Lin Y, Liu J, Wan L, Liu Z, Cheng S, et al. A targeted next-generation sequencing method for identifying clinically relevant mutation profiles in lung adenocarcinoma. Sci Rep. 2016;6:22338. doi: 10.1038/srep22338 26936516

13. Petersen BL, Sorensen MC, Pedersen S, Rasmussen M. Fluorescence in situ hybridization on formalin-fixed and paraffin-embedded tissue: optimizing the method. Appl Immunohistochem Mol Morphol. 2004;12(3):259–65. 15551741

14. Wynes MW, Sholl LM, Dietel M, Schuuring E, Tsao MS, Yatabe Y, et al. An international interpretation study using the ALK IHC antibody D5F3 and a sensitive detection kit demonstrates high concordance between ALK IHC and ALK FISH and between evaluators. J Thorac Oncol. 2014;9(5):631–8. doi: 10.1097/JTO.0000000000000115 24722153

15. Myers MB. Targeted therapies with companion diagnostics in the management of breast cancer: current perspectives. Pharmgenomics Pers Med. 2016;9:7–16. doi: 10.2147/PGPM.S56055 26858530

16. O'Connor C. Fluorescence In Situ Hybridization (FISH). Nature Education. 2008;1(1):171.

17. Boggs BA, Chinault AC. Analysis of DNA replication by fluorescence in situ hybridization. Methods. 1997;13(3):259–70. doi: 10.1006/meth.1997.0525 9441852

18. Zwaenepoel K, Merkle D, Cabillic F, Berg E, Belaud-Rotureau MA, Grazioli V, et al. Automation of ALK gene rearrangement testing with fluorescence in situ hybridization (FISH): a feasibility study. Exp Mol Pathol. 2015;98(1):113–8. doi: 10.1016/j.yexmp.2015.01.005 25576649

19. Viale G, Paterson J, Bloch M, Csathy G, Allen D, Dell'Orto P, et al. Assessment of HER2 amplification status in breast cancer using a new automated HER2 IQFISH pharmDx (Dako Omnis) assay. Pathol Res Pract. 2016;212(8):735–42. doi: 10.1016/j.prp.2016.06.002 27461826

20. Zanatta L, Valori L, Cappelletto E, Pozzebon ME, Pavan E, Dei Tos AP, et al. Reagent and labor cost optimization through automation of fluorescence in situ hybridization (FISH) with the VP 2000: an Italian case study. J Lab Autom. 2015;20(1):25–31. doi: 10.1177/2211068214558294 25395292

21. Chin SF, Daigo Y, Huang HE, Iyer NG, Callagy G, Kranjac T, et al. A simple and reliable pretreatment protocol facilitates fluorescent in situ hybridisation on tissue microarrays of paraffin wax embedded tumour samples. Mol Pathol. 2003;56(5):275–9. doi: 10.1136/mp.56.5.275 14514921

22. Shi SR, Cote RJ, Taylor CR. Antigen retrieval techniques: current perspectives. J Histochem Cytochem. 2001;49(8):931–7. doi: 10.1177/002215540104900801 11457921

23. Jorgensen JT, Nielsen KB, Mollerup J, Jepsen A, Go N. Detection of MET amplification in gastroesophageal tumor specimens using IQFISH. Ann Transl Med. 2017;5(23):458. doi: 10.21037/atm.2017.09.07 29285491

24. Tafe LJ, Steinmetz HB, Allen SF, Dokus BJ, Tsongalis GJ. Rapid fluorescence in situ hybridisation (FISH) for HER2 (ERBB2) assessment in breast and gastro-oesophageal cancer. J Clin Pathol. 2015;68(4):306–8. doi: 10.1136/jclinpath-2014-202787 25576545

25. Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KH, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol. 2013;31(31):3997–4013. doi: 10.1200/JCO.2013.50.9984 24101045

26. Karlsson C, Karlsson MG. Effects of long-term storage on the detection of proteins, DNA, and mRNA in tissue microarray slides. J Histochem Cytochem. 2011;59(12):1113–21. doi: 10.1369/0022155411423779 22147608

27. MET IQFISH Probe with CEP7—instructions for use. Agilent Technologies; 2015. p. 4.

28. Kreatech FISH probes MET (7q31) / SE7—instructions for use. The Netherlands: Leica Biosystems; 2015.

29. Kreatech™ FISH probes MET (7q31) / SE 7 (D7Z1)—XL for BOND—instructions for use. Rev B ed. The Netherlands: Leica Biosystems; 2016. p. 16.

30. KBI-6007 Tissue Digestion Kit I—For conventional paraffin-embedded tissues—instructions for use. D2.5 ed: Leica Biosystems; 2016. p. 2.

31. Slide Processing For Paraffin Embedded Tissue Samples. Empire genomics. p. 1.

32. Varella-Garcia M, Diebold J, Eberhard DA, Geenen K, Hirschmann A, Kockx M, et al. EGFR fluorescence in situ hybridisation assay: guidelines for application to non-small-cell lung cancer. J Clin Pathol. 2009;62(11):970–7. doi: 10.1136/jcp.2009.066548 19861557

33. Ahn M, Han J, Sequist L, Cho BC, Lee JS, Kim S, et al. OA 09.03 TATTON Ph Ib Expansion Cohort: Osimertinib plus Savolitinib for Pts with EGFR-Mutant MET-Amplified NSCLC after Progression on Prior EGFR-TKI. Journal of Thoracic Oncology. 2017;12(11):S1768.

34. Giavarina D. Understanding Bland Altman analysis. Biochem Med (Zagreb). 2015;25(2):141–51. doi: 10.11613/BM.2015.015 26110027

35. Koo TK, Li MY. A Guideline of Selecting and Reporting Intraclass Correlation Coefficients for Reliability Research. J Chiropr Med. 2016;15(2):155–63. doi: 10.1016/j.jcm.2016.02.012 27330520

36. Matthiesen SH, Hansen CM. Fast and non-toxic in situ hybridization without blocking of repetitive sequences. PLoS One. 2012;7(7):e40675. doi: 10.1371/journal.pone.0040675 22911704

37. Press MF, Ellis CE, Gagnon RC, Grob TJ, Buyse M, Villalobos I, et al. HER2 Status in Advanced or Metastatic Gastric, Esophageal, or Gastroesophageal Adenocarcinoma for Entry to the TRIO-013/LOGiC Trial of Lapatinib. Mol Cancer Ther. 2017;16(1):228–38. doi: 10.1158/1535-7163.MCT-15-0887 27811012

38. Starczynski J, Atkey N, Connelly Y, O'Grady T, Campbell FM, di Palma S, et al. HER2 gene amplification in breast cancer: a rogues' gallery of challenging diagnostic cases: UKNEQAS interpretation guidelines and research recommendations. Am J Clin Pathol. 2012;137(4):595–605. doi: 10.1309/AJCPATBZ2JFN1QQC 22431536

39. Singh K, Tantravahi U, Lomme MM, Pasquariello T, Steinhoff M, Sung CJ. Updated 2013 College of American Pathologists/American Society of Clinical Oncology (CAP/ASCO) guideline recommendations for human epidermal growth factor receptor 2 (HER2) fluorescent in situ hybridization (FISH) testing increase HER2 positive and HER2 equivocal breast cancer cases; retrospective study of HER2 FISH results of 836 invasive breast cancers. Breast Cancer Res Treat. 2016;157(3):405–11. doi: 10.1007/s10549-016-3824-x 27180259

40. Tsao MS, F.R. H, IASLC Y. Y. Atlas of ALK Testing in Lung Cancer. 2013.

41. van der Logt EM, Kuperus DA, van Setten JW, van den Heuvel MC, Boers JE, Schuuring E, et al. Fully automated fluorescent in situ hybridization (FISH) staining and digital analysis of HER2 in breast cancer: a validation study. PLoS One. 2015;10(4):e0123201. doi: 10.1371/journal.pone.0123201 25844540

42. Virzi AR, Gentile A, Benvenuti S, Comoglio PM. Reviving oncogenic addiction to MET bypassed by BRAF (G469A) mutation. Proc Natl Acad Sci U S A. 2018;115(40):10058–63. doi: 10.1073/pnas.1721147115 30224486

43. Savic S, Bubendorf L. Common Fluorescence In Situ Hybridization Applications in Cytology. Arch Pathol Lab Med. 2016;140(12):1323–30. doi: 10.5858/arpa.2016-0202-RA 27479335

44. Young R, Pailler E, Billiot F, Drusch F, Barthelemy A, Oulhen M, et al. Circulating tumor cells in lung cancer. Acta Cytol. 2012;56(6):655–60. doi: 10.1159/000345182 23207444

45. Pailler E, Oulhen M, Borget I, Remon J, Ross K, Auger N, et al. Circulating Tumor Cells with Aberrant ALK Copy Number Predict Progression-Free Survival during Crizotinib Treatment in ALK-Rearranged Non-Small Cell Lung Cancer Patients. Cancer Res. 2017;77(9):2222–30. doi: 10.1158/0008-5472.CAN-16-3072 28461563

46. Zhang T, Boominathan R, Foulk B, Rao C, Kemeny G, Strickler JH, et al. Development of a Novel c-MET-Based CTC Detection Platform. Mol Cancer Res. 2016;14(6):539–47. doi: 10.1158/1541-7786.MCR-16-0011 26951228

Článek vyšel v časopise


2019 Číslo 10