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We present a cohort of 41 patients with osimertinib resistance biopsies, including 2 with an acquired CCDC6–RET fusion. Although RET fusions have been identified in resistant EGFR-mutant non–small cell lung cancer (NSCLC), their role in acquired resistance to EGFR inhibitors is not well described. To assess the biological implications of RET fusions in an EGFR-mutant cancer, we expressed CCDC6–RET in PC9 (EGFR del19) and MGH134 (EGFR L858R/T790M) cells and found that CCDC6–RET was sufficient to confer resistance to EGFR tyrosine kinase inhibitors (TKI). The selective RET inhibitors BLU-667 and cabozantinib resensitized CCDC6–RET-expressing cells to EGFR inhibition. Finally, we treated 2 patients with EGFR-mutant NSCLC and RET-mediated resistance with osimertinib and BLU-667. The combination was well tolerated and led to rapid radiographic response in both patients. This study provides proof of concept that RET fusions can mediate acquired resistance to EGFR TKIs and that combined EGFR and RET inhibition with osimertinib/BLU-667 may be a well-tolerated and effective treatment strategy for such patients. Significance: The role of RET fusions in resistant EGFR-mutant cancers is unknown. We report that RET fusions mediate resistance to EGFR inhibitors and demonstrate that this bypass track can be effectively targeted with a selective RET inhibitor (BLU-667) in the clinic. This article is highlighted in the In This Issue feature, p. 1494

Atlantic killifish ( Fundulus heteroclitus) inhabiting the Atlantic Wood Industries region of the Elizabeth River, Virginia, have passed polycyclic aromatic hydrocarbon (PAH) resistance to their offspring as evidenced by early life stage testing of developmental toxicity after exposure to specific PAHs. Our study focused on environmentally relevant PAH mixtures in the form of Elizabeth River sediment extract (ERSE). Juvenile (5 month) F1 progeny of pollution-adapted Atlantic Wood (AW) parents and of reference site (King’s Creek [KC]) parents were exposed as embryos to ERSE. Liver alterations, including nonneoplastic lesions and microvesicular vacuolation, were observed in both populations. ERSE-exposed KC fish developed significantly more alterations than unexposed KC fish. Interestingly, unexposed AW killifish developed significantly more alterations than unexposed KC individuals, suggesting that AW juveniles are not fully protected from liver disease; rapid growth of juvenile fish may also be an accelerating factor for tumorigenesis. Because recent reports show hepatic tumor formation in adult AW fish, the differing responses from the 2 populations provided a way to determine whether embryo toxicity protection extends to juveniles. Future investigations will analyze older life stages of killifish to determine differences in responses related to chronic disease.

The cornerstone of treatment for advanced ALK-positive lung cancer is sequential therapy with increasingly potent and selective ALK inhibitors. The third-generation ALK inhibitor lorlatinib has demonstrated clinical activity in patients who failed previous ALK inhibitors. To define the spectrum of ALK mutations that confer lorlatinib resistance, we performed accelerated mutagenesis screening of Ba/F3 cells expressing EML4–ALK. Under comparable conditions, N-ethyl-N-nitrosourea (ENU) mutagenesis generated numerous crizotinib-resistant but no lorlatinib-resistant clones harboring single ALK mutations. In similar screens with EML4–ALK containing single ALK resistance mutations, numerous lorlatinib-resistant clones emerged harboring compound ALK mutations. To determine the clinical relevance of these mutations, we analyzed repeat biopsies from lorlatinib-resistant patients. Seven of 20 samples (35%) harbored compound ALK mutations, including two identified in the ENU screen. Whole-exome sequencing in three cases confirmed the stepwise accumulation of ALK mutations during sequential treatment. These results suggest that sequential ALK inhibitors can foster the emergence of compound ALK mutations, identification of which is critical to informing drug design and developing effective therapeutic strategies. Significance: Treatment with sequential first-, second-, and third-generation ALK inhibitors can select for compound ALK mutations that confer high-level resistance to ALK-targeted therapies. A more efficacious long-term strategy may be up-front treatment with a third-generation ALK inhibitor to prevent the emergence of on-target resistance. Cancer Discov; 8(6); 714–29. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 663

Summary Personalized cancer therapy is based on a patient’s tumor lineage, histopathology, expression analyses, and/or tumor DNA or RNA analysis. Here, we aim to develop an in vitro functional assay of a patient’s living cancer cells that could complement these approaches. We present methods for developing cell cultures from tumor biopsies and identify the types of samples and culture conditions associated with higher efficiency of model establishment. Toward the application of patient-derived cell cultures for personalized care, we established an immunofluorescence-based functional assay that quantifies cancer cell responses to targeted therapy in mixed cell cultures. Assaying patient-derived lung cancer cultures with this method showed promise in modeling patient response for diagnostic use. This platform should allow for the development of co-clinical trial studies to prospectively test the value of drug profiling on tumor-biopsy-derived cultures to direct patient care., Graphical Abstract, Highlights • Successful culture of cancer cells from tumor biopsies • An immunofluorescence-based assay to quantify drug sensitivity in mixed cell cultures • NSCLC patients’ biopsy culture sensitivities reflect clinical response, Kodack et al. report on the development of cancer models from tumor biopsies and technologies toward a functional approach for personalized medicine. They describe the ability to reliably test drug response in patient-derived samples of mixed cell populations. In doing so, they show that patient biopsy cultures may predict patient clinical responses.