We identified novel gene fusions in patients with lung cancer harboring the kinase domain of the NTRK1 gene that encodes the TRKA receptor. Both the MPRIP-NTRK1 and CD74-NTRK1 fusions lead to constitutive TRKA kinase activity and are oncogenic. Treatment of cells expressing NTRK1 fusions with inhibitors of TRKA kinase activity inhibited autophosphorylation of TRKA and cell growth. Three of 91 lung cancer patients (3.3%), without known oncogenic alterations, assayed by NGS or FISH demonstrated evidence of NTRK1 gene fusions.

Introduction Human epidermal growth factor receptor 2 (HER2, ERBB2) alterations have been identified as oncogenic drivers and potential therapeutic targets in lung cancers. The molecular associations of HER2 gene amplification, mutation, and HER2 protein overexpression in lung cancers have not been distinctly defined. To explore these associations, Memorial Sloan Kettering and University of Colorado combined their data on HER2 alterations in lung cancers. Methods Tumor specimens from 175 patients with lung adenocarcinomas with no prior targeted therapy were evaluated for the presence of HER2 amplification, mutation, and HER2 protein overexpression. Amplification was assessed by fluorescence in-situ hybridization (FISH) and defined as HER2/CEP17 ratio ≥2.0. Mutation was assessed by fragment analysis, mass spectrometry genotyping and Sanger sequencing. Overexpression was assessed by immunohistochemistry (IHC). The frequencies of HER2 amplification, mutation and HER2 overexpression were calculated and their overlap examined. Results HER2 amplification by FISH was detected in 5 of 175 (3%) cases. HER2 mutation was detected in 4 of 148 (3%) specimens, including 3 identical 12bp insertions (p.A775_G776insYVMA) and a 9bp insertion, all in exon 20. None of the HER2 mutant cases were amplified. HER2 overexpression (2+, 3+) on IHC was not detected in the 25 specimens available for testing and negative IHC correlated with negative results on FISH. Conclusions HER2 mutations are not associated with HER2 amplification suggesting a distinct entity and therapeutic target. “HER2-positive lung cancer” may not be an adequate term and patient cohorts for the study of HER2 targeted agents should be defined by the specific HER2 alteration present.

Targeted therapy development in head and neck squamous cell carcinoma (HNSCC) is challenging given the rarity of activating mutations. Additionally, HNSCC incidence is increasing related to human papillomavirus (HPV). We sought to develop an in vivo model derived from patients reflecting the evolving HNSCC epidemiologic landscape, and use it to identify new therapies. Primary and relapsed tumors from HNSCC patients, both HPV+ and HPV−, were implanted on mice, giving rise to 25 strains. Resulting xenografts were characterized by detecting key mutations, measuring protein expression by IHC and gene expression/pathway analysis by mRNA-sequencing. Drug efficacy studies were run with representative xenografts using the approved drug cetuximab as well as the new PI3K inhibitor PX-866. Tumors maintained their original morphology, genetic profiles and drug susceptibilities through serial passaging. The genetic makeup of these tumors was consistent with known frequencies of TP53, PI3KCA, NOTCH1 and NOTCH2 mutations. Because the EGFR inhibitor cetuximab is a standard HNSCC therapy, we tested its efficacy and observed a wide spectrum of efficacy. Cetuximab-resistant strains had higher PI3K/Akt pathway gene expression and protein activation than cetuximab-sensitive strains. The PI3K inhibitor PX-866 had anti-tumor efficacy in HNSCC models with PIK3CA alterations. Finally, PI3K inhibition was effective in two cases with NOTCH1 inactivating mutations. In summary, we have developed an HNSCC model covering its clinical spectrum whose major genetic alterations and susceptibility to anticancer agents represent contemporary HNSCC. This model enables to prospectively test therapeutic-oriented hypotheses leading to personalized medicine.

Oncogenic kinase fusions of ALK, ROS1, RET and NTRK1 act as drivers in human lung and other cancers. Residual tumor burden following treatment of ALK or ROS1+ lung cancer patients with oncogene-targeted therapy ultimately enables the emergence of drug-resistant clones, limiting the long-term effectiveness of these therapies. To determine the signaling mechanisms underlying incomplete tumor cell killing in oncogene-addicted cancer cells, we investigated the role of EGFR signaling in drug-naive cancer cells harboring these oncogene fusions. We defined three distinct roles for EGFR in the response to oncogene-specific therapies. First, EGF-mediated activation of EGFR blunted fusion kinase inhibitor binding and restored fusion kinase signaling complexes. Second, fusion kinase inhibition shifted adaptor protein binding from the fusion oncoprotein to EGFR. Third, EGFR enabled bypass signaling to critical downstream pathways such as MAPK. While evidence of EGFR-mediated bypass signaling has been reported after ALK and ROS1 blockade, our results extended this effect to RET and NTRK1 blockade and uncovered the other additional mechanisms in gene fusion-positive lung cancer cells, mouse models and human clinical specimens before onset of acquired drug resistance. Collectively, our findings show how EGFR signaling can provide a critical adaptive survival mechanism that allows cancer cells to evade oncogene-specific inhibitors, providing a rationale to co-target EGFR to reduce risks of developing drug resistance.

Introduction Low dose CT screening for lung cancer has a high false positive rate with frequent discovery of indeterminate pulmonary nodules. Noninvasive biomarkers are needed to reduce false positives and improve risk stratification. A retrospective longitudinal evaluation was performed to assess chromosomal aneusomy in sputum via fluorescence in situ hybridization (CA-FISH) in four nested case-control studies. Methods ROC analysis resulted in two grouped cohorts: High Risk (CO High Risk and CO Nodule; 68 Cases, 69 controls) and Screening (ACRIN/NLST and PLuSS; 97 Cases, 185 controls). The CA-FISH assay was a 4-target DNA panel encompassing EGFR and MYC genes, and the 5p15 and centromere 6 regions or the FGFR1 and PIK3CA genes. A 4-category scale: normal, probably normal, probably abnormal and abnormal was applied. Sensitivity, specificity, and positive and negative likelihood ratios (LR+, LR−) (with 95% CI) were estimated for each cohort. Results Sensitivity and specificity were, respectively, 0.67 (0.55, 0.78) and 0.94 (0.85, 0.98) for High Risk subjects and 0.20 (0.13, 0.30) and 0.84 (0.78, 0.89) for Screening subjects. LR+ and LR− were, respectively, 11.66 (4.44, 30.63) and 0.34 (0.24, 0.48) for High Risk; and 1.36 (0.81, 2.28) and 0.93 (0.83, 1.05) for Screening subjects. Conclusion The high positive likelihood ratio of sputum CA-FISH indicates it could be a useful adjunct to LDCT for lung cancer in high risk settings. For screening, however, its low positive likelihood ratio limits clinical utility. Prospective assessment of CA-FISH in the incidentally-identified indeterminate nodule setting is ongoing in the Colorado Pulmonary Nodule Biomarker Trial.