HER2 as a target in invasive urothelial carcinoma

Joaquim Bellmunt, Lillian Werner, Aristotle Bamias, André P Fay, Rachel S Park, Markus Riester, Shamini Selvarajah, Justine A Barletta, David M Berman, Silvia de Muga, Marta Salido, Enrique Gallardo, Federico Rojo, Elizabeth A Guancial, Richard Bambury, Stephanie A Mullane, Toni K Choueiri, Massimo Loda, Edward Stack, Jonathan Rosenberg, Joaquim Bellmunt, Lillian Werner, Aristotle Bamias, André P Fay, Rachel S Park, Markus Riester, Shamini Selvarajah, Justine A Barletta, David M Berman, Silvia de Muga, Marta Salido, Enrique Gallardo, Federico Rojo, Elizabeth A Guancial, Richard Bambury, Stephanie A Mullane, Toni K Choueiri, Massimo Loda, Edward Stack, Jonathan Rosenberg

Abstract

We evaluated primary tumors from two cohorts, Spain (N = 111) and Greece (N = 102), for patients who were treated with platinum-based chemotherapy. Patients were tested for HER2 status (IHC score of 3+ or FISH ratio of ≥ 2.2) by immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), DNA copy number, mRNA expression, and mutation status in patients with metastatic urothelial carcinoma (UC), and its impact on survival. ERBB2 mutation was determined by hotspot sequencing. mRNA expression was assessed using NanoString counting. Association of overall survival (OS) and HER2 status was assessed by a Cox regression model. NIH-3T3 cells containing HER2 V777L were assessed for growth, invasion, and HER2 kinase activation. In all, 22% of Spanish and 4% of Greek cohorts had 3+ HER2 staining by IHC. FISH amplification was identified in 20% of Spanish and 4% of Greek cohorts. Kappa coefficient between FISH and IHC was 0.47. HER2 status was not associated with OS in univariate (Spanish P = 0.34; Greek P = 0.11) or multivariate (Spanish P = 0.49; Greek P = 0.12) analysis. HER2-positive tumors expressed higher levels of HER2 mRNA than HER2-negative tumors (P < 0.001). HER2 mutations (V777L and L755S) were identified in two (2%) patients. In vitro analysis of V777L results in transformation of NIH-3T3 cells, leading to increased growth, invasion on soft agar, and HER2 kinase constitutive activation. In summary, HER2 overexpression or amplification in the primary tumor did not predict OS in patients with metastatic UC. HER2 positivity rates can differ between different populations. Further trials in genomically screened patients are needed to assess HER2-targeted therapies in UC.

Trial registration: ClinicalTrials.gov NCT00949455.

Keywords: ERBB2; HER2; genomic alterations; prognosis; urothelial carcinomas.

© 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Figures

Figure 1
Figure 1
(A) HER2 expression in urothelial carcinoma. (a) HER2-positive staining scored as 3+, showing heavy membranous HER2 staining. (b) Moderate HER2 staining scored as 2+, demonstrating moderate membranous HER2 staining. (c) Weak HER2 staining, which demonstrates weak membranous staining which is scored as 1+. (d) HER2-negative urothelial cancer, showing no membranous HER2 expression, and scored as 0. (B) ERBB2 status in urothelial carcinoma. (a) Normal, nonamplified (ratio = 1), (b) polysomic, nonamplified (ratio = 1), (c) amplified (ratio = 3.5), and (d) amplified (ratio = 5). HER2, human epidermal growth factor receptor 2.
Figure 2
Figure 2
Heatmap of (A) Spanish cohort and (B) Greek cohort. Visualization of HER2 status based on different methodologies. N/A = data not available. HER2, human epidermal growth factor receptor 2.
Figure 3
Figure 3
Overall survival by HER2 status for Spanish and Greek cohorts. No difference in overall survival was observed in the two cohorts. For the Spanish cohort, the hazard ratio was 0.94 (95% CI = 0.52–1.70, P = 0.83) and for the Greek cohort, the hazard ratio was 0.2 (95% CI = 0.03–1.48, P = 0.11). Multivariable analysis incorporating known prognostic factors showed similar nonsignificant results (Tables S2 and S3). HER2, human epidermal growth factor receptor 2.
Figure 4
Figure 4
HER2 status and mRNA expression in Spanish and Greek cohorts.. The NanoString distributions of read counts HER2-positive and HER2-negative patients are visualized with box plots for both cohorts. HER2, human epidermal growth factor receptor 2.

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