Hale KIVRAK, Hilal ÖZAKINCI, Duru KARASOY, Serpil DİZBAY SAK

HER2 Amplification by Next-Generation Sequencing in Lung Carcinoma: A Comparison of NGS Amplified and Non-amplified Cases by Immunohistochemistry and In Situ Hybridization

Background: Although the role of HER2 amplification and its evaluation methods are well known in breast carcinoma, methods for detection of HER2 amplification in non-small cell lung carcinoma are unclear. Next-generation sequencing is widely used in searching multiple therapeutic targets, and it is possible to evaluate copy number variation of genes by next-generation sequencing. Aims: To re-evaluate the HER2 status of non-small cell lung carcinoma cases detected as HER2 amplified and non-amplified by next-generation sequencing via the most commonly used HER2 investigation methods in routine pathology practice, namely immunohistochemistry and in situ hybridization. Study Design: Retrospective cross-sectional study. Methods: Among the 256 patients whose mutation profiles were examined by next-generation sequencing, HER2 amplified (13 cases) and non-HER2-amplified (13 cases) were determined as study and control groups, respectively, by next-generation sequencing. HER2 next-generation sequencing amplified tumors were investigated for HER2 expression and amplification using immunohistochemistry and silver in situ hybridization. Results: From a group of 256 non-small cell lung carcinoma, 33 tumors (12.8%) showed HER2 amplification with next-generation sequencing. Although we observed more frequent HER2 positivity by immunohistochemistry in next-generation sequencing-amplified cases, when compared to non-amplified cases (50% and 23% respectively), the difference was not significant (P = .221). Within the HER2 amplified group, inter-method-agreement was very good between next-generation sequencing results amplification and in situ hybridization status. Next-generation sequencing results showed a strong interclass correlation coefficient with HER2/cell (P = .009, r = 0.777) and HER2/CEP17 ratio (P = .001, r = 0.805). The median HER2/CEP17 ratio was higher in the next-generation sequencing amplified group (P = .013); however, three cases were found to be amplified by silver in situ hybridization among the next-generation sequencing non-amplified cases. EGFR and FGFR1 amplification were more frequent in HER2 next-generation sequencing amplified group than next-generation sequencing non-amplified group (P < .001). Conclusion: Until the effects of HER2 amplification on the HER2 protein are well understood and pulmonary carcinoma algorithms are defined, non-small cell lung carcinomas found to be amplified by next generation sequencing should be verified by additional methods.

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1. WilliamD, Travis EB, Burke AP, Marx A, Andrew GN. In: Bosman ESJFT, Lakhani SR, Ohgaki H, eds. WHO Classification of Tumours of the Lung, Pleura, Tyhmus and Heart. Tumours of the lung. 4th ed. Lyon, France: International Agency for Research on Cancer (IARC); 2015.
2. Cancer Genome Atlas Research Network. Comprehensive molecular profiling of lung adenocarcinoma. Nature. 2014;511:543-550. [CrossRef]
3. Rakha EA, Ellis IO, Horii R, et al. Invasive breast carcinoma: general overview. In: WHO Classification of Tumours Editorial Board, eds. Breast Tumours, WHO Classification of Tumours. 5th ed. Lyon, France: International Agency for Research on Cancer (IARC); 2019:82-101.
4. Li BT, Ross DS, Aisner DL, et al. HER2 amplification and HER2 mutation are distinct molecular targets in lung cancers. J Thorac Oncol. 2016;11:414-419. [CrossRef]
5. Heinmöller P, Gross C, Beyser K, et al. HER2 status in non-small cell lung cancer: results from patient screening for enrollment to a phase II study of Herceptin. Clin Cancer Res. 2003;9:5238-5243. [CrossRef]
6. Hotta K, Aoe K, Kozuki T, et al. A phase II study of trastuzumab emtansine in HER2-positive non-small cell lung cancer. J Thorac Oncol. 2018;13:273-279. [CrossRef]
7. Suzuki M, Shiraishi K, Yoshida A, et al. HER2 gene mutations in non-small cell lung carcinomas: concurrence with Her2 gene amplification and Her2 protein expression and phosphorylation. Lung Cancer. 2015;87:14-22. [CrossRef]
8. Mazières J, Peters S, Lepage B, et al. Lung cancer that harbors an HER2 mutation: epidemiologic characteristics and therapeutic perspectives. J Clin Oncol. 2013;31:1997-2003. [CrossRef]
9. Visscher DW, Yadrandji S, Tabaczka P, Kraut M, Sarkar FH. Clinicopathologic analysis of k-ras, p53, and ERBB-2 gene alterations in pulmonary adenocarcinoma. Diagn Mol Pathol. 1997;6:64-69. [CrossRef]
10. Wolff AC, Hammond MEH, Allison KH, et al. Human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline focused update. J Clin Oncol. 2018;36:2105-2122. [CrossRef]
11. Lokuhetty D, White V, Watanabe R, Cree I. WHO Classification of Breast Tumours. Geneva: WHO; 2019.
12. Abrahao-Machado LF, Scapulatempo-Neto C. HER2 testing in gastric cancer: an update. World J Gastroenterol. 2016;22:4619-4625. [CrossRef]
13. Takezawa K, Pirazzoli V, Arcila ME, et al. 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. 2012;2:922-933. [CrossRef]
14. Yu HA, Arcila ME, Rekhtman N, et al. Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in 155 patients with EGFR-mutant lung cancers. Clin Cancer Res. 2013;19:2240-2247. [CrossRef]
15. Mar N, Vredenburgh JJ, Wasser JS. Targeting HER2 in the treatment of non-small cell lung cancer. Lung Cancer. 2015;87:220-225. [CrossRef]
16. Langer CJ, Stephenson P, Thor A, Vangel M, Johnson DH, Eastern Cooperative Oncology Group Study 2598. Trastuzumab in the treatment of advanced non-small cell lung cancer: is there a role? Focus on Eastern Cooperative Oncology Group study 2598. J Clin Oncol. 2004;22:1180-1187. [CrossRef]
17. Clamon G, Herndon J, Kern J, et al. Lack of trastuzumab activity in nonsmall cell lung carcinoma with overexpression of erb-B2: 39810: a phase II trial of cancer and leukemia group B. Cancer. 2005;103:1670-1675. [CrossRef]
18. Gatzemeier U, Groth G, Butts C, et al. Randomized phase II trial of gemcitabine cisplatin with or without trastuzumab in HER2-positive non-small-cell lung cancer. Ann Oncol. 2004;15:19-27. [CrossRef]
19. Liu L, Shao X, Gao W, et al. The role of human epidermal growth factor receptor 2 as a prognostic factor in lung cancer: a meta-analysis of published data. J Thorac Oncol. 2010;5:1922-1932. [CrossRef]
20. Lee K, Jung HA, Sun JM, et al. Clinical characteristics and outcomes of non-small cell lung cancer patients with HER2 alterations in Korea. Cancer Res Treat. 2020;52:292- 300. [CrossRef]
21. cBioPortal. cBioPortal for cancer genomics [internet]. Available at: https://www. cbioportal.org/.
22. Eijkelenboom A, Tops BBJ, van den Berg A, et al. Recommendations for the clinical interpretation and reporting of copy number gains using gene panel NGS analysis in routine diagnostics. Virchows Arch. 2019;474:673-680. [CrossRef]
23. Yao R, Yu T, Qing Y, Wang J, Shen Y. Evaluation of copy number variant detection from panel-based next-generation sequencing data. Mol Genet Genom Med. 2019;7:e00513. [CrossRef]
24. Yoshizawa A, Sumiyoshi S, Sonobe M, et al. HER2 status in lung adenocarcinoma: a comparison of immunohistochemistry, fluorescence in situ hybridization (FISH), dual-ISH, and gene mutations. Lung Cancer. 2014;85:373-378. [CrossRef]
25. Kim EK, Kim KA, Lee CY, Shim HS. The frequency and clinical impact of HER2 alterations in lung adenocarcinoma. PLoS One. 2017;12:e0171280. [CrossRef]
26. Tan D, Deeb G, Wang J, et al. HER-2/neu protein expression and gene alteration in stage I-IIIA non-small-cell lung cancer: a study of 140 cases using a combination of high throughput tissue microarray, immunohistochemistry, and fluorescent in situ hybridization. Diagn Mol Pathol. 2003;12:201-211. [CrossRef]
27. Cox G, Vyberg M, Melgaard B, Askaa J, Oster A, O’Byrne KJ. Herceptest: HER2 expression and gene amplification in non-small cell lung cancer. Int J Cancer. 2001;92:480-483. [CrossRef]
28. Kuyama S, Hotta K, Tabata M, et al. Impact of HER2 gene and protein status on the treatment outcome of cisplatin-based chemoradiotherapy for locally advanced non small cell lung cancer. J Thorac Oncol. 2008;3:477-482. [CrossRef]
29. Nakamura H, Saji H, Ogata A, et al. Correlation between encoded protein overexpression and copy number of the HER2 gene with survival in non-small cell lung cancer. Int J Cancer. 2003;103:61-66. [CrossRef]
30. Rüschoff J, Dietel M, Baretton G, et al. HER2 diagnostics in gastric cancer-guideline validation and development of standardized immunohistochemical testing. Virchows Arch. 2010;457:299-307. [CrossRef]
31. Osako T, Miyahara M, Uchino S, Inomata M, Kitano S, Kobayashi M. Immunohistochemical study of c-erbB-2 protein in colorectal cancer and the correlation with patient survival. Oncology. 1998;55:548-555. [CrossRef]
32. Demirbaş S, Sücüllü I, Yildirim S, Celenk T. Influence of the c-erb B-2, nm23, bcl-2 and p53 protein markers on colorectal cancer. Turk J Gastroenterol. 2006;17:13-19.
33. Varella-Garcia M, Mitsudomi T, Yatabe Y, et al. EGFR and HER2 genomic gain in recurrent non-small cell lung cancer after surgery: impact on outcome to treatment with gefitinib and association with EGFR and KRAS mutations in a Japanese cohort. J Thorac Oncol. 2009;4:318-325. [CrossRef]
34. Cappuzzo F, Varella-Garcia M, Shigematsu H, et al. Increased HER2 gene copy number is associated with response to gefitinib therapy in epidermal growth factor receptor-positive non-small-cell lung cancer patients. J Clin Oncol. 2005;23:5007- 5018. [CrossRef]
35. Hirashima N, Takahashi W, Yoshii S, Yamane T, Ooi A. Protein overexpression and gene amplification of c-erb B-2 in pulmonary carcinomas: a comparative immunohistochemical and fluorescence in situ hybridization study. Mod Pathol. 2001;14:556-562. [CrossRef]
36. Hirsch FR, Varella-Garcia M, Franklin WA, et al. Evaluation of HER-2/neu gene amplification and protein expression in non-small cell lung carcinomas. Br J Cancer. 2002;86:1449-1456. [CrossRef]