Targeted Oncology

, Volume 12, Issue 3, pp 341–351 | Cite as

Detection of HER2 Amplification in Circulating Tumor Cells of HER2-Negative Gastric Cancer Patients

  • Yuji Mishima
  • Satoshi Matsusaka
  • Keisho Chin
  • Mariko Mikuniya
  • Sayuri Minowa
  • Tomoko Takayama
  • Harumi Shibata
  • Ryoko Kuniyoshi
  • Mariko Ogura
  • Yasuhito Terui
  • Nobuyuki Mizunuma
  • Kiyohiko Hatake
Original Research Article


A key to the successful use of targeted cancer therapy is the ability to preselect patients who are likely to benefit from the treatment according to molecular markers. Assessment for predicting therapy response is mostly done using tumor biopsies. However, these might not truly represent all of the patient’s malignant cells because of tumor heterogeneity and/or clonal evolution during disease progression. One potential strategy that can complement primary tumor biopsy is the analysis of circulating tumor cells (CTCs). In this study, we analyzed CTCs of patients with gastric cancer (GC) to find those who were likely to benefit from trastuzumab therapies. We developed an imaging-based method that enabled CTC identification simultaneously with evaluation of HER2 gene amplification (the 3D–IF-FISH method). Then we performed a study enrolling 101 GC patients in whom we analyzed CTCs by both 3D–IF-FISH and an FDA-approved CellSearch system. As compared with the CellSearch system, 3D–IF-FISH methods identified a higher number of patients whose primary tumors were HER2- but who had HER2+ CTCs, suggesting that the 3D–IF-FISH method is effective in preselecting patients for trastuzumab therapies. To demonstrate this, we performed an exploratory clinical study to evaluate the clinical benefits of trastuzumab treatment for advanced GC patients (n = 15) whose primary tumors were HER2-, but whose CTCs showed HER2 amplification. An interim evaluation after the first stage showed that these preselected patients had response rates comparable to those reported in the trastuzumab-plus-chemotherapy arm of the ToGA study. The present study offers a new, non-invasive strategy to select patients who are likely to benefit from trastuzumab-based therapies, despite their primary biopsy being HER2-negative. (UMIN ID: UMIN000008622).


Gastric Cancer Trastuzumab Overall Response Rate EpCAM Expression CellSearch System 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



Circulating tumor cells


Fluorescent in situ hybridization


Gastric cancer


Human epidermal growth factor receptor 2






Circulating tumor cells


Gastroesophageal junction


Centromere on chromosome 17


Eastern Cooperative Oncology Group


Overall response rate


Progression-free survival


Overall survival


Epithelial cell adhesion molecule


Complete response


Partial response


Stable disease


Progressive disease





We received technical support from Dr. Abe T, Ms. Asakura H, and Dr. Kojima K from Olympus corporation. We thank Ms. Suitsu C, Ms. Horikie Y, Ms. Yago M, and Ms. Kobayasi K for management of clinical samples and data collection. We also thank Dr. Dovie Wylie, of On-site English Inc. (Palo Alto, CA, USA) for English editing assistance.

Compliance with Ethical Standards


This study was supported in part by a Grant-in-Aid for Scientific Research (grant number 23501320; to YM), and a grant from the Kobayashi Institute for Innovative Cancer Chemotherapy (to SM).

Conflict of Interest Statement

The authors have no conflict of interest.

Supplementary material

11523_2017_493_MOESM1_ESM.pdf (2.2 mb)
ESM 1 (PDF 2271 kb)


  1. 1.
    Cuello M, Ettenberg SA, Clark AS, Keane MM, Posner RH, Nau MM, et al. Down-regulation of the erb B-2 receptor by trastuzumab (herceptin) enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in breast and ovarian cancer cell lines that overexpress erb B-2. Cancer Res. 2001;61(12):4892–900.PubMedGoogle Scholar
  2. 2.
    Bang YJ, Van Cutsem E, Feyereislova A, Chung HC, Shen L, Sawaki A, et al. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. Lancet. 2010;376(9742):687–97.CrossRefPubMedGoogle Scholar
  3. 3.
    Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol. 2007;25(1):118–45.Google Scholar
  4. 4.
    Santinelli A, Pisa E, Stramazzotti D, Fabris G. HER-2 status discrepancy between primary breast cancer and metastatic sites. Impact on target therapy. Int J Cancer. 2008;122(5):999–1004.Google Scholar
  5. 5.
    Simmons C, Miller N, Geddie W, Gianfelice D, Oldfield M, Dranitsaris G, et al. Does confirmatory tumor biopsy alter the management of breast cancer patients with distant metastases? Ann Oncol. 2009;20(9):1499–504.Google Scholar
  6. 6.
    Fehm T, Muller V, Aktas B, Janni W, Schneeweiss A, Stickeler E, et al. HER2 status of circulating tumor cells in patients with metastatic breast cancer: a prospective, multicenter trial. Breast Cancer Res Treat. 2010;124(2):403–12.CrossRefPubMedGoogle Scholar
  7. 7.
    Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Matera J, Miller MC, et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med. 2004;351(8):781–91.CrossRefPubMedGoogle Scholar
  8. 8.
    Nagrath S, Sequist LV, Maheswaran S, Bell DW, Irimia D, Ulkus L, et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature. 2007;450(7173):1235–9.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Schneck H, Gierke B, Uppenkamp F, Behrens B, Niederacher D, Stoecklein NH, et al. EpCAM-independent enrichment of circulating tumor cells in metastatic breast cancer. PLoS One. 2015;10(12):e0144535.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Riethdorf S, Fritsche H, Muller V, Rau T, Schindlbeck C, Rack B, et al. Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the cell search system. Clin Cancer Res. 2007;13(3):920–8.Google Scholar
  11. 11.
    Newton SS, Dow A, Terwilliger R, Duman R. A simplified method for combined immunohistochemistry and in-situ hybridization in fresh-frozen, cryocut mouse brain sections. Brain Res Brain Res Protoc. 2002;9(3):214–9.Google Scholar
  12. 12.
    Kurokawa Y, Sugimoto N, Miwa H, Tsuda M, Nishina S, Okuda H, et al. Phase II study of trastuzumab in combination with S-1 plus cisplatin in HER2-positive gastric cancer (HERBIS-1). Br J Cancer. 2014;110(5):1163–8.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Kataoka H, Mori Y, Shimura T, Nishie H, Natsume M, Mochizuki H, et al. A phase II prospective study of the trastuzumab combined with 5-weekly S-1 and CDDP therapy for HER2-positive advanced gastric cancer. Cancer Chemother Pharmacol. 2016;77(5):957–62.CrossRefPubMedGoogle Scholar
  14. 14.
    Gong J, Liu T, Fan Q, Bai L, Bi F, Qin S, et al. Optimal regimen of trastuzumab in combination with oxaliplatin/capecitabine in first-line treatment of HER2-positive advanced gastric cancer (CGOG 1001): a multicenter, phase II trial. BMC Cancer. 2016;16:68.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Ryu MH, Yoo C, Kim JG, Ryoo BY, Park YS, Park SR, et al. Multicenter phase II study of trastuzumab in combination with capecitabine and oxaliplatin for advanced gastric cancer. Eur J Cancer. 2015;51(4):482–8.CrossRefPubMedGoogle Scholar
  16. 16.
    Wong DD, Kumarasinghe MP, Platten MA, de Boer WB. Concordance of HER2 expression in paired primary and metastatic sites of gastric and gastro-oesophageal junction cancers. Pathology. 2015;47(7):641–6.CrossRefPubMedGoogle Scholar
  17. 17.
    Park SR, Park YS, Ryu MH, Ryoo BY, Woo CG, Jung HY, et al. Extra-gain of HER2-positive cases through HER2 reassessment in primary and metastatic sites in advanced gastric cancer with initially HER2-negative primary tumours: results of GASTric cancer HER2 reassessment study 1 (GASTHER1). Eur J Cancer. 2016;53:42–50.CrossRefPubMedGoogle Scholar
  18. 18.
    Paoletti C, Muniz MC, Thomas DG, Griffith KA, Kidwell KM, Tokudome N, et al. Development of circulating tumor cell-endocrine therapy index in patients with hormone receptor-positive breast cancer. Clin Cancer Res. 2015;21(11):2487–98.Google Scholar
  19. 19.
    Zhang S, Li L, Wang T, Bian L, Hu H, Xu C, et al. Real-time HER2 status detected on circulating tumor cells predicts different outcomes of anti-HER2 therapy in histologically HER2-positive metastatic breast cancer patients. BMC Cancer. 2016;16:526.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Aktas B, Kasimir-Bauer S, Muller V, Janni W, Fehm T, Wallwiener D, et al. Comparison of the HER2, estrogen and progesterone receptor expression profile of primary tumor, metastases and circulating tumor cells in metastatic breast cancer patients. BMC Cancer. 2016;16:522.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Beije N, Onstenk W, Kraan J, Sieuwerts AM, Hamberg P, Dirix LY, et al. Prognostic impact of HER2 and ER status of circulating tumor cells in metastatic breast cancer patients with a HER2-negative primary tumor. Neoplasia. 2016;18(11):647–53.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Agelaki S, Dragolia M, Markonanolaki H, Alkahtani S, Stournaras C, Georgoulias V, et al. Phenotypic characterization of circulating tumor cells in triple negative breast cancer patients. Oncotarget. 2017;8(3):5309–22.PubMedGoogle Scholar
  23. 23.
    Fehm T, Becker S, Duerr-Stoerzer S, Sotlar K, Mueller V, Wallwiener D, et al. Determination of HER2 status using both serum HER2 levels and circulating tumor cells in patients with recurrent breast cancer whose primary tumor was HER2 negative or of unknown HER2 status. Breast Cancer Res. 2007;9(5):R74.Google Scholar
  24. 24.
    Pestrin M, Bessi S, Galardi F, Truglia M, Biggeri A, Biagioni C, et al. Correlation of HER2 status between primary tumors and corresponding circulating tumor cells in advanced breast cancer patients. Breast Cancer Res Treat. 2009;118(3):523–30.CrossRefPubMedGoogle Scholar
  25. 25.
    Tewes M, Aktas B, Welt A, Mueller S, Hauch S, Kimmig R, et al. Molecular profiling and predictive value of circulating tumor cells in patients with metastatic breast cancer: an option for monitoring response to breast cancer related therapies. Breast Cancer Res Treat. 2009;115(3):581–90.CrossRefPubMedGoogle Scholar
  26. 26.
    Flores LM, Kindelberger DW, Ligon AH, Capelletti M, Fiorentino M, Loda M, et al. Improving the yield of circulating tumour cells facilitates molecular characterisation and recognition of discordant HER2 amplification in breast cancer. Br J Cancer. 2010;102(10):1495–502.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Mayer JA, Pham T, Wong KL, Scoggin J, Sales EV, Clarin T, et al. FISH-based determination of HER2 status in circulating tumor cells isolated with the microfluidic CEE platform. Cancer Genetics. 2011;204(11):589–95.CrossRefPubMedGoogle Scholar
  28. 28.
    Hayashi N, Nakamura S, Tokuda Y, Shimoda Y, Yagata H, Yoshida A, et al. Prognostic value of HER2-positive circulating tumor cells in patients with metastatic breast cancer. Int J Clin Oncol. 2012;17(2):96–104.CrossRefPubMedGoogle Scholar
  29. 29.
    Frithiof H, Aaltonen K, Ryden L. A FISH-based method for assessment of HER-2 amplification status in breast cancer circulating tumor cells following cell search isolation. Onco Targets and Therapy. 2016;9:7095–103.CrossRefGoogle Scholar
  30. 30.
    Konigsberg R, Obermayr E, Bises G, Pfeiler G, Gneist M, Wrba F, et al. Detection of EpCAM positive and negative circulating tumor cells in metastatic breast cancer patients. Acta Oncol. 2011;50(5):700–10.CrossRefPubMedGoogle Scholar
  31. 31.
    Deng G, Herrler M, Burgess D, Manna E, Krag D, Burke JF. Enrichment with anti-cytokeratin alone or combined with anti-EpCAM antibodies significantly increases the sensitivity for circulating tumor cell detection in metastatic breast cancer patients. Breast Cancer Res. 2008;10(4):R69.Google Scholar
  32. 32.
    de Wit S, van Dalum G, Lenferink AT, Tibbe AG, Hiltermann TJ, Groen HJ, et al. The detection of EpCAM(+) and EpCAM(−) circulating tumor cells. Sci Rep. 2015;5:12270.Google Scholar
  33. 33.
    Dent BM, Ogle LF, O’Donnell RL, Hayes N, Malik U, Curtin NJ, et al. High-resolution imaging for the detection and characterisation of circulating tumour cells from patients with oesophageal, hepatocellular, thyroid and ovarian cancers. Int J Cancer. 2016;138(1):206–16.Google Scholar
  34. 34.
    Lapin M, Tjensvoll K, Oltedal S, Buhl T, Gilje B, Smaaland R, et al. MINDEC-an enhanced negative depletion strategy for circulating tumour cell enrichment. Sci Rep. 2016;6:28929.Google Scholar
  35. 35.
    Rüschoff J, Hanna W, Bilous M, Hofmann M, Osamura RY, Penault-Llorca F, et al. HER2 testing in gastric cancer: a practical approach. Mod Pathol. 2012;25(5):637–50.Google Scholar
  36. 36.
    Stahl P, Seeschaaf C, Lebok P, Kutup A, Bockhorn M, Izbicki JR, et al. Heterogeneity of amplification of HER2, EGFR, CCND1 and MYC in gastric cancer. BMC Gastroenterol. 2015;15:7.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Kim MA, Lee HJ, Yang HK, Bang YJ, Kim WH. Heterogeneous amplification of ERBB2 in primary lesions is responsible for the discordant ERBB2 status of primary and metastatic lesions in gastric carcinoma. Histopathology. 2011;59(5):822–31.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Yang J, Luo H, Li Y, Li J, Cai Z, Su X, et al. Intratumoral heterogeneity determines discordant results of diagnostic tests for human epidermal growth factor receptor (HER) 2 in gastric cancer specimens. Cell Biochem Biophys. 2012;62(1):221–8.CrossRefPubMedGoogle Scholar
  39. 39.
    Amato M, Perrone G, Righi D, Pellegrini C, Rabitti C, Di Matteo F, et al. HER2 status in gastric cancer: comparison between primary and distant metastatic disease. Pathol Oncol Res: POR. 2017;23(1):55–61.CrossRefPubMedGoogle Scholar
  40. 40.
    Jordan NV, Bardia A, Wittner BS, Benes C, Ligorio M, Zheng Y, et al. HER2 expression identifies dynamic functional states within circulating breast cancer cells. Nature. 2016;537(7618):102–6.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2017

Authors and Affiliations

  • Yuji Mishima
    • 1
  • Satoshi Matsusaka
    • 2
  • Keisho Chin
    • 2
  • Mariko Mikuniya
    • 1
  • Sayuri Minowa
    • 1
  • Tomoko Takayama
    • 1
  • Harumi Shibata
    • 1
  • Ryoko Kuniyoshi
    • 1
  • Mariko Ogura
    • 2
  • Yasuhito Terui
    • 1
    • 2
  • Nobuyuki Mizunuma
    • 2
  • Kiyohiko Hatake
    • 1
    • 2
  1. 1.Clinical Chemotherapy, Cancer Chemotherapy CenterJapanese Foundation for Cancer ResearchTokyoJapan
  2. 2.Cancer Institute Hospital of the Japanese Foundation for Cancer ResearchTokyoJapan

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