Identification of Recurrent Activating HER2 Mutations in Primary Canine Pulmonary Adenocarcinoma

Abstract

Purpose: Naturally occurring primary canine lung cancers share clinicopathologic features with human lung cancers in never-smokers, but the genetic underpinnings of canine lung cancer are unknown. We have charted the genomic landscape of canine lung cancer and performed functional characterization of novel, recurrent HER2 (ERBB2) mutations occurring in canine pulmonary adenocarcinoma (cPAC).

Experimental design: We performed multiplatform genomic sequencing of 88 primary canine lung tumors or cell lines. Additionally, in cPAC cell lines, we performed functional characterization of HER2 signaling and evaluated mutation-dependent HER2 inhibitor drug dose-response.

Results: We discovered somatic, coding HER2 point mutations in 38% of cPACs (28/74), but none in adenosquamous (cPASC, 0/11) or squamous cell (cPSCC, 0/3) carcinomas. The majority (93%) of HER2 mutations were hotspot V659E transmembrane domain (TMD) mutations comparable to activating mutations at this same site in human cancer. Other HER2 mutations were located in the extracellular domain and TMD. HER2 ^V659E was detected in the plasma of 33% (2/6) of dogs with localized HER2 ^V659E tumors. HER2 ^V659E cPAC cell lines displayed constitutive phosphorylation of AKT and significantly higher sensitivity to the HER2 inhibitors lapatinib and neratinib relative to HER2-wild-type cell lines (IC₅₀ < 200 nmol/L in HER2 ^V659E vs. IC₅₀ > 2,500 nmol/L in HER2 ^WT).

Conclusions: This study creates a foundation for molecular understanding of and drug development for canine lung cancer. These data also establish molecular contexts for comparative studies in dogs and humans of low mutation burden, never-smoker lung cancer, and mutant HER2 function and inhibition.

Figure 1.. The Genomic Landscape of Primary Canine Lung Cancers.

(A) Recurrent likely pathogenic somatic mutations in cancer genes identified in primary canine lung cancers through multi-platform sequencing. SNVs were determined from combined tumor/normal exome and/or amplicon sequencing across 88 total tumors and cell lines. (B) CNVs were determined from tumor/normal exome data in five cPAC cases. (C) Somatic mutation burden (SNVs, CNVs, and SVs) identified by exome sequencing of five tumor/normal cPAC cases. (D) Distribution of somatic HER2 mutations within the HER2 protein identified in primary canine lung cancers. The length of the lollipops is proportional to the number of mutations found. (E) Detection of HER2 hotspot mutations in plasma from 11 canine primary lung cancer cases.

Figure 2.. HER2^V659E constitutively activates downstream HER2 signaling and is associated with response to HER2 inhibitors in primary canine pulmonary adenocarcinoma (cPAC) cell lines.

(A) HER2 signaling activation in canine lung cancer cell lines. Phospho-AKT and AKT levels were assessed by Western blot under serum starvation in the presence and absence of EGFR activation by hNRG in HER2^V659E and HER2^WT cPAC cell lines. (B) Neratinib drug-dose-response studies in primary lung cancer cell lines. Five canine cell lines (three HER2^WT and two HER2^V659E) and two human lung cancer positive controls with known HER2 activating mutations (BT474 – HER2-amplified, and H1781 – HER2^G776V) and HER2 inhibitor responses were treated with 14 neratinib doses ranging from 100 μM to 5.5×10⁻² nM for 72 hours with CellTiterGlo viability endpoints. Survival is shown relative to DMSO vehicle control.