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 mutationdependent 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 (IC 50 < 200 nmol/L in HER2 V659E vs. IC 50 > 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.

Introduction This study investigates the immune profile of the primary lung tumors and the corresponding brain metastasis from patients with NSCLC using multiplex fluorescence immunohistochemistry. Methods The study evaluated 34 patients who underwent autopsy or surgical resection for brain metastasis and autopsy, surgical resection, or core biopsy for primary lung cancer. We compared the densities of various immune cells in the primary tumors and the brain metastases by multiplex fluorescence immunohistochemical analysis. Results The density of CD4-positive (CD4 + ) T-cells, CD8-positive T-cells, and CD4 + Foxp3-positive T-cells were statistically higher in both tumor and stromal areas in primary lung cancer specimens when compared with brain metastases samples ( p < 0.0001). Only CD204-positive cells were statistically higher in the tumor areas of the brain metastases ( p = 0.0118). Tumor-infiltrating lymphocytes associated with brain metastases positively correlated with overall survival, but primary lung tumor-infiltrating lymphocytes did not. The density of CD4 + and CD4 + Foxp3-positive T-cells in brain metastases with radiation was statistically higher in the carcinoma and stromal areas compared with those without radiation ( p = 0.0343, p = 0.0173). Conclusions Our findings that CD204-positive cells were higher in brain metastases may have broader implications for treatment as these macrophages may be immunosuppressive and make the immune environment less reactive. Furthermore, the finding that the density of CD4 + T-cells was higher in cancer and stroma areas of brain metastases after radiotherapy supports the addition of immunotherapy to radiation therapy in the treatment of brain metastases in NSCLC.

Introduction: Notch receptor family dysregulation can be tumor promoting or suppressing depending on cellular context. Our studies shed light on the mechanistic differences that are responsible for NOTCH1's opposing roles in lung adenocarcinoma and lung squamous cell carcinoma. Methods:We integrated transcriptional patient-derived datasets with gene co-expression analyses to elucidate mechanisms behind NOTCH1 function in subsets of NSCLC. Differential co-expression was examined using hierarchical clustering and principal component analysis. Enrichment analysis was used to examine pathways associated with the underlying transcriptional networks. These pathways were validated in vitro and in vivo. Endogenously epitope-tagged NOTCH1 was used to identify novel interacting proteins.Results: NOTCH1 co-expressed genes in lung adenocarcinoma and squamous carcinoma were distinct and associated with either angiogenesis and immune system pathways or cell cycle control and mitosis pathways, respectively. Tissue culture and xenograft studies of lung adenocarcinoma and lung squamous models with NOTCH1 knockdown showed growth differences and opposing effects on these pathways. Differential NOTCH1 interacting proteins were identified as potential mediators of these differences.Conclusions: Recognition of the opposing role of NOTCH1 in lung cancer, downstream pathways, and interacting proteins in each context may help direct the development of rational NOTCH1 pathway-dependent targeted therapies for specific tumor subsets of NSCLC.

BACKGROUND:We have verified a mass spectrometry (MS) based targeted proteomics signature for the detection of malignant pleural mesothelioma (MPM) from the blood. METHODS:A seven-peptide biomarker MPM signature by targeted proteomics in serum was identified in a previous independent study. Here, we have verified the predictive accuracy of a reduced version of that signature, now composed of six-peptide biomarkers. We have applied liquid chromatography-selected reaction monitoring (LC-SRM), also known as multiple reaction monitoring (MRM), for the investigation of 402 serum samples from 213 MPM patients and 189 cancer-free asbestos exposed donors from the USA, Australia and Europe. RESULTS:Each of the biomarkers composing the signature was independently informative with no apparent functional or physical relation to each other. The multiplexing possibility offered by MS proteomics allowed their integration into a single signature with a higher discriminating capacity than that of the single biomarkers alone. The strategy allowed in this way to increase their potential utility for clinical decisions. The signature discriminated MPM patients and asbestos exposed donors with AUC of 0.738. For early stage MPM AUC was 0.765. This signature also was prognostic and Kaplan-Meier analysis showed a significant difference between high and low risk groups with a hazard ratio (HR) of 1.659 (95% CI [1.075, 2.562], P = 0.021).