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The organization of immune cells in human tumors is not well understood. Immunogenic tumors harbor spatially-localized multicellular ‘immunity hubs’ defined by expression of the T cell-attracting chemokinesCXCL10/CXCL11and abundant T cells. Here, we examined immunity hubs in human pre-immunotherapy lung cancer specimens, and found that they were associated with beneficial responses to PD-1-blockade. Immunity hubs were enriched for many interferon-stimulated genes, T cells in multiple differentiation states, andCXCL9/10/11+ macrophages that preferentially interact with CD8 T cells. Critically, we discovered the stem-immunity hub, a subtype of immunity hub strongly associated with favorable PD-1-blockade outcomes, distinct from mature tertiary lymphoid structures, and enriched for stem-like TCF7+PD-1+ CD8 T cells and activatedCCR7+LAMP3+ dendritic cells, as well as chemokines that organize these cells. These results elucidate the spatial organization of the human intratumoral immune response and its relevance to patient immunotherapy outcomes.

Supplemental Figure Legends

Fig. S1: Schematic of derivation 'lineages' for drug-sensitive and -resistant cell lines used in this study Fig. S2: Characterization of T790M+ cell lines Fig. S3: Characterization of A+C-resistant cell lines Fig. S4: Characterization of AZD9291-resistant cell lines

Supplementary Table 1: Tumor volume measurements during treatment Supplementary Table 2: List of mice with drug-resistant tumors Supplementary Table 3: List of mice that did not develop drug resistance. Tables listing mice used in the therapeutic studies showing tumor volumes and tumor volume changes through treatment.

Supplementary Figure 1: MRI images and tumor volume measurements. Supplementary Figure 2: Mouse body weight during drug treatment. Supplementary Figure 3: Results of molecular studies of resistant tumors.

(1) Figure S1. Sustained AKT and MAPK pathway signaling following EGFR TKI treatment in EGFR-mutant lung cancer cells. (2) Figure S2. Analysis of cells treated with TKIs after 96 hours. (3) Figure S3. HER3 reactivation after osimertinib treatment. (4) Figure S4. Combination osimertinib plus SFK inhibitor decreases SFK signaling and results in better cell growth inhibition. (5) Figure S5. Upregulation of SFKs and FAK upon EGFR inhibition in EGFR-mutant lung cancer cells. (6) Figure S6. Osimertinib compared to osimertinib plus dasatinib in an osimertinib-sensitive xenograft model. (7) Figure S7. Extracellular interactions may influence osimertinib potency. (8) Figure S8. SFK/FAK pathway inhibition most potently enhances the effects of osimertinib. (9) Figure S9. Analysis of osimertinib (AZD9291) resistant lung cancer cell lines.

Purpose: The EGFR tyrosine kinase inhibitors (TKIs), erlotinib and afatinib, have transformed the treatment of advanced EGFR-mutant lung adenocarcinoma. However, almost all patients who respond develop acquired resistance on average approximately 1 year after starting therapy. Resistance is commonly due to a secondary mutation in EGFR (EGFRT790M). We previously found that the combination of the EGFR TKI afatinib and the EGFR antibody cetuximab could overcome EGFRT790M-mediated resistance in preclinical models. This combination has shown a 29% response rate in a clinical trial in patients with acquired resistance to first-generation TKIs. An outstanding question is whether this regimen is beneficial when used as first-line therapy.Experimental Design: Using mouse models of EGFR-mutant lung cancer, we tested whether the combination of afatinib plus cetuximab delivered upfront to mice with TKI-naïve EGFRL858R-induced lung adenocarcinomas delayed tumor relapse and drug-resistance compared with single-agent TKIs.Results: Afatinib plus cetuximab markedly delayed the time to relapse and incidence of drug-resistant tumors, which occurred in only 63.6% of the mice, in contrast to erlotinib or afatinib treatment where 100% of mice developed resistance. Mechanisms of tumor escape observed in afatinib plus cetuximab resistant tumors include the EGFRT790M mutation and Kras mutations. Experiments in cell lines and xenografts confirmed that the afatinib plus cetuximab combination does not suppress the emergence of EGFRT790M.Conclusions: These results highlight the potential of afatinib plus cetuximab as an effective treatment strategy for patients with TKI-naïve EGFR-mutant lung cancer and indicate that clinical trial development in this area is warranted. Clin Cancer Res; 22(2); 426–35. ©2015 AACR.

Supplementary Figures S1-S6. Comparison of genetic alterations across multiple populations resistant to AZD9291 and other EGFR TKIs (S1); Treatment of resistant populations with AZD9291 (S2); Detection and Validation of a novel NRAS E63K mutation (S3); Lysates were prepared from parental PC9 and resistant populations analysed for levels of total and phosphorylated ERK, NRAS and KRAS by western blot (S4); The novel NRAS E63K mutation is an activating mutation that when expressed enhances resistance to cell growth inhibition by gefitinib or AZD9291 in EGFRm cell lines (S5); In vitro combination of AZD9291 with selumetinib induces more profound phenotype inhibition (S6).

Supplementary Methods and References. Description of additional methods and procedures used in the study. Also includes Supplementary References.

Legends for Supplemental Figures S1-S6

Supplementary Figure Legend. Legends for Supplementary Figures S1-S6.

Resistance to targeted EGFR inhibitors is likely to develop in EGFR-mutant lung cancers. Early identification of innate or acquired resistance mechanisms to these agents is essential to direct development of future therapies. We describe the detection of heterogeneous mechanisms of resistance within populations of EGFR-mutant cells (PC9 and/or NCI-H1975) with acquired resistance to current and newly developed EGFR tyrosine kinase inhibitors, including AZD9291. We report the detection of NRAS mutations, including a novel E63K mutation, and a gain of copy number of WT NRAS or WT KRAS in cell populations resistant to gefitinib, afatinib, WZ4002, or AZD9291. Compared with parental cells, a number of resistant cell populations were more sensitive to inhibition by the MEK inhibitor selumetinib (AZD6244; ARRY-142886) when treated in combination with the originating EGFR inhibitor. In vitro, a combination of AZD9291 with selumetinib prevented emergence of resistance in PC9 cells and delayed resistance in NCI-H1975 cells. In vivo, concomitant dosing of AZD9291 with selumetinib caused regression of AZD9291-resistant tumors in an EGFRm/T790M transgenic model. Our data support the use of a combination of AZD9291 with a MEK inhibitor to delay or prevent resistance to AZD9291 in EGFRm and/or EGFRm/T790M tumors. Furthermore, these findings suggest that NRAS modifications in tumor samples from patients who have progressed on current or EGFR inhibitors in development may support subsequent treatment with a combination of EGFR and MEK inhibition. Cancer Res; 75(12); 2489–500. ©2015 AACR.

Description of additional methods and procedures used in the study.

Supplementary Tables S1-S4. Generation of resistant cell populations (S1); Small molecule inhibitors (S2); IC50 (µM) values from cell growth inhibition assays comparing compound sensitivity between parental and resistant cell populations (S3); Genetic analysis of resistant cell populations (S4).

Despite the success of treating EGFR-mutant lung cancer patients with EGFR tyrosine kinase inhibitors (TKI), all patients eventually acquire resistance to these therapies. Although various resistance mechanisms have been described, there are currently no FDA-approved therapies that target alternative mechanisms to treat lung tumors with acquired resistance to first-line EGFR TKI agents. Here we found that EPHA2 is overexpressed in EGFR TKI-resistant tumor cells. Loss of EPHA2 reduced the viability of erlotinib-resistant tumor cells harboring EGFRT790M mutations in vitro and inhibited tumor growth and progression in an inducible EGFRL858R+T790M-mutant lung cancer model in vivo. Targeting EPHA2 in erlotinib-resistant cells decreased S6K1-mediated phosphorylation of cell death agonist BAD, resulting in reduced tumor cell proliferation and increased apoptosis. Furthermore, pharmacologic inhibition of EPHA2 by the small-molecule inhibitor ALW-II-41-27 decreased both survival and proliferation of erlotinib-resistant tumor cells and inhibited tumor growth in vivo. ALW-II-41-27 was also effective in decreasing viability of cells with acquired resistance to the third-generation EGFR TKI AZD9291. Collectively, these data define a role for EPHA2 in the maintenance of cell survival of TKI-resistant, EGFR-mutant lung cancer and indicate that EPHA2 may serve as a useful therapeutic target in TKI-resistant tumors. Cancer Res; 76(2); 305–18. ©2016 AACR.

Localization of EPHA2 in erlotinib resistant cells (S1); Validation of EPHA2 antibody specificity in patient tumor specimens (S2); EPHA2 kinase activity is required in maintaining cell viability of erlotinib resistant lung cancer (S3); Biochemical analysis of TKI-­�resistant tumors from EGFRL858R+T790MEphA2-­�/-­� and control mice (S4); Signaling analysis in EPHA2 knockdown cells by siRNA-­�mediated gene silencing (S5); Localization of EPHA2 upon ligand or EPHA2 inhibitor treatment (S6).

Approximately 10% of EGFR-activating mutations occur as in-frame insertion mutations in exon 20 of the EGFR kinase domain (EGFR ins20). EGFR ins20 mutations have not demonstrated the same sensitivity to early generations of EGFR tyrosine kinase inhibitors (TKI) as canonical activating EGFR mutations such as del19 and L858R. Development of effective therapies for this subset of patients has been challenging, but recent years have seen more rapid progress in these efforts. In this review, we describe the molecular and clinicopathologic features of EGFR ins20 mutations and summarize recent data on emerging therapies for patients with this subtype of EGFR-mutant non–small cell lung cancer (NSCLC).Significance:When activating mutations in EGFR were first discovered in lung cancer, the lack of sensitivity of tumors harboring EGFR ins20 mutations to early-generation EGFR TKIs resulted in this subset of EGFR-mutant tumors being initially classified as an untargetable or intrinsically resistant subpopulation. In addition, the diversity of mutations within EGFR exon 20 and resultant challenges identifying them on routine clinical genotyping tests led to underestimation of their frequency. However, recent scientific progress in targeting EGFR ins20 mutations as well as more effective identification of this clinical cohort has enhanced our ability to develop effective therapies for patients with this subtype of EGFR-mutant NSCLC.

Lineage plasticity is implicated in treatment resistance in multiple cancers. In lung adenocarcinomas (LUAD) amenable to targeted therapy, transformation to small cell lung cancer (SCLC) is a recognized resistance mechanism. Defining molecular mechanisms of neuroendocrine (NE) transformation in lung cancer has been limited by a paucity of pre/posttransformation clinical samples. Detailed genomic, epigenomic, transcriptomic, and protein characterization of combined LUAD/SCLC tumors, as well as pre/posttransformation samples, supports that NE transformation is primarily driven by transcriptional reprogramming rather than mutational events. We identify genomic contexts in which NE transformation is favored, including frequent loss of the 3p chromosome arm. We observed enhanced expression of genes involved in the PRC2 complex and PI3K/AKT and NOTCH pathways. Pharmacologic inhibition of the PI3K/AKT pathway delayed tumor growth and NE transformation in an EGFR-mutant patient-derived xenograft model. Our findings define a novel landscape of potential drivers and therapeutic vulnerabilities of NE transformation in lung cancer.The difficulty in collection of transformation samples has precluded the performance of molecular analyses, and thus little is known about the lineage plasticity mechanisms leading to LUAD-to-SCLC transformation. Here, we describe biological pathways dysregulated upon transformation and identify potential predictors and potential therapeutic vulnerabilities of NE transformation in the lung. See related commentary by Meador and Lovly, p. 2962. This article is highlighted in the In This Issue feature, p. 2945.

Background Approximately 10% of patients with SCLC develop a paraneoplastic syndrome (PNS). Neurologic PNS are thought to improve prognosis, which we hypothesized is related to increased tumor-infiltrating lymphocytes and immune recognition. Methods We queried 2,512,042 medical records from a single institution to identify patients who have SCLC with and without PNS and performed manual, retrospective chart review. We then performed multiplexed fluorescence immunohistochemistry and automated quantitative analysis (AQUA Technology) on tumors to assess CD3, CD4, and CD8 T cell infiltrates and programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) interactions. T cell infiltrates and PD-1/PD-L1 interaction scores were compared among patients with neurologic PNS, endocrinologic PNS, and a control group without PNS. Clinical outcomes were analyzed using the Kaplan-Meier method and Cox proportional hazards models. Results We evaluated 145 SCLC patients: 55 with PNS (25 neurologic and 30 endocrinologic) and 90 controls. Patients with neurologic PNS experienced improved overall survival compared to patients with endocrinologic PNS and controls (median overall survival of 24 months versus 12 months versus 13 months, respectively). Of the 145 patients, we identified tumor tissue from 34 patients that was adequate for AQUA analysis. Among 37 specimens from these 34 patients, patients with neurologic PNS had increased T cell infiltrates (p = 0.033) and PD-1/PD-L1 interaction (p = 0.014) compared to tumors from patients with endocrinologic PNS or controls. Conclusions Tumor tissue from patients with SCLC with neurologic PNS showed increased tumor-infiltrating lymphocytes and PD-1/PD-L1 interaction consistent with an inflamed tumor microenvironment.

Approximately 10% of EGFR activating mutations occur as in-frame insertion mutations in exon 20 of the EGFR kinase domain (EGFR ins20). EGFR ins20 mutations have not demonstrated the same sensitivity to early generations of EGFR TKIs as canonical activating EGFR mutations such as del19 and L858R. Development of effective therapies for this subset of patients has been challenging, but recent years have seen more rapid progress in these efforts. In this review, we describe the molecular and clinicopathologic features of EGFR ins20 mutations and summarize recent data on emerging therapies for patients with this subtype of EGFR-mutant NSCLC.

PURPOSE Plasma cell-free DNA (cfDNA) sequencing is a compelling diagnostic tool in solid tumors and has been shown to have high positive predictive value. However, limited assay sensitivity means that negative plasma genotyping, or the absence of detection of mutation of interest, still requires reflex tumor biopsy. METHODS We analyzed two independent cohorts of patients with advanced non–small-cell lung cancer (NSCLC) with known canonical driver and resistance mutations who underwent plasma cfDNA genotyping. We measured quantitative features, such as maximum allelic frequency (mAF), as clinically available measures of cfDNA tumor content, and studied their relationship with assay sensitivity. RESULTS In patients with EGFR-mutant NSCLC harboring EGFR T790M, detection of driver mutation at > 1% AF conferred a sensitivity of 97% (368/380) for detection of T790M across three cfDNA genotyping platforms. Similarly, in a second cohort of patients with EGFR or KRAS driver mutations, when the mAF of nontarget mutations was > 1%, sensitivity for driver mutation detection was 100% (43/43). Combining the two NSCLC patient cohorts, the presence of nontarget mutations at mAF > 1% predicts for high sensitivity (> 95%) for identifying the presence of the known driver mutation, whereas mAF of ≤ 1% confers sensitivity of only 26%-54% across platforms. Focusing on 21 false-negative cases where the driver mutation was not detected on plasma next-generation sequencing, other mutations (presumably clonal hematopoiesis) were detected at ≤ 1% AF in 14 (67%). CONCLUSION Plasma cfDNA genotyping is highly sensitive when adequate tumor DNA content is present. The likelihood of a false-negative cfDNA genotyping result is low in a sample with evidence of > 1% tumor content. Bioinformatic approaches are needed to further optimize the assessment of cfDNA tumor content in plasma genotyping assays.

Introduction Lung cancer is associated with severe coronavirus disease 2019 (COVID-19) infections. Symptom overlap between COVID-19 and lung cancer may complicate diagnostic evaluation. We aimed to investigate the incidence, symptoms, differential diagnosis, and outcomes of COVID-19 in patients with lung cancer. Methods To determine an at-risk population for COVID-19, we retrospectively identified patients with lung cancer receiving longitudinal care within a single institution in the 12 months (April 1, 2019 to March 31, 2020) immediately preceding the COVID-19 pandemic, including an “active therapy population” treated within the last 60 days of this period. Among patients subsequently referred for COVID-19 testing, we compared symptoms, laboratory values, radiographic findings, and outcomes of positive versus negative patients. Results Between April 1, 2019 and March 31, 2020, a total of 696 patients received longitudinal care, including 406 (58%) in the active therapy population. Among 55 patients referred for COVID-19 testing, 24 (44%) were positive for COVID-19, representing a cumulative incidence of 3.4% (longitudinal population) and 1.5% (active therapy population). Compared with patients who were COVID-19 negative, those who were COVID-19 positive were more likely to have a supplemental oxygen requirement (11% versus 54%, p = 0.005) and to have typical COVID-19 pneumonia imaging findings (5 versus 56%, p = 0.001). Otherwise, there were no marked differences in presenting symptoms. Among patients who were COVID-19 negative, alternative etiologies included treatment-related toxicity (26%), atypical pneumonia (22%), and disease progression (22%). A total of 16 patients positive for COVID-19 (67%) required hospitalization, and seven (29%) died from COVID-related complications. Conclusions COVID-19 was infrequent in this lung cancer population, but these patients experienced high rates of morbidity and mortality. Oncologists should maintain a low threshold for COVID-19 testing in patients with lung cancer presenting with acute symptoms.

While significant advancements have been made in the available therapies for metastatic non-small cell lung cancer (NSCLC), acquired resistance remains a major barrier to treatment. We have not yet achieved the ability to cure advanced NSCLC with systemic therapy, despite our growing understanding of many of the oncogenic drivers of this disease. Rather, the emergence of drug-tolerant and drug-resistant cells remains the rule, even in the face of increasingly potent targeted therapies. In this review, we provide a broad overview of the mechanisms of resistance to targeted therapy that have been demonstrated across molecular subtypes of NSCLC, highlighting the dynamic interplay between driver oncogene, bypass signaling pathways, shifting cellular phenotypes, and surrounding tumor microenvironment.

Complete and timely tissue genotyping is challenging, leading to significant numbers of patients with newly diagnosed metastatic non-small cell lung cancer (mNSCLC) being undergenotyped for all eight genomic biomarkers recommended by professional guidelines. We aimed to demonstrate noninferiority of comprehensive cell-free DNA (cfDNA) relative to physician discretion standard-of-care (SOC) tissue genotyping to identify guideline-recommended biomarkers in patients with mNSCLC.Prospectively enrolled patients with previously untreated mNSCLC undergoing physician discretion SOC tissue genotyping submitted a pretreatment blood sample for comprehensive cfDNA analysis (Guardant360).Among 282 patients, physician discretion SOC tissue genotyping identified a guideline-recommended biomarker in 60 patients versus 77 cfDNA identified patients (21.3% vs. 27.3%;In the largest cfDNA study in previously untreated mNSCLC, a validated comprehensive cfDNA test identifies guideline-recommended biomarkers at a rate at least as high as SOC tissue genotyping, with high tissue concordance, more rapidly and completely than tissue-based genotyping.

Introduction Patients with small cell lung cancer (SCLC) have a poor prognosis and limited treatment options. Since access to longitudinal tumor samples is very limited in patients with this disease, we chose to focus our studies on the characterization of plasma cell-free DNA (cfDNA) for rapid, noninvasive monitoring of disease burden.

Resistance to targeted EGFR inhibitors is likely to develop in EGFR-mutant lung cancers. Early identification of innate or acquired resistance mechanisms to these agents is essential to direct development of future therapies. We describe the detection of heterogeneous mechanisms of resistance within populations of EGFR-mutant cells (PC9 and/or NCI-H1975) with acquired resistance to current and newly developed EGFR tyrosine kinase inhibitors, including AZD9291. We report the detection of NRAS mutations, including a novel E63K mutation, and a gain of copy number of WT NRAS or WT KRAS in cell populations resistant to gefitinib, afatinib, WZ4002, or AZD9291. Compared with parental cells, a number of resistant cell populations were more sensitive to inhibition by the MEK inhibitor selumetinib (AZD6244; ARRY-142886) when treated in combination with the originating EGFR inhibitor. In vitro, a combination of AZD9291 with selumetinib prevented emergence of resistance in PC9 cells and delayed resistance in NCI-H1975 cells. In vivo, concomitant dosing of AZD9291 with selumetinib caused regression of AZD9291-resistant tumors in an EGFRm/T790M transgenic model. Our data support the use of a combination of AZD9291 with a MEK inhibitor to delay or prevent resistance to AZD9291 in EGFRm and/or EGFRm/T790M tumors. Furthermore, these findings suggest that NRAS modifications in tumor samples from patients who have progressed on current or EGFR inhibitors in development may support subsequent treatment with a combination of EGFR and MEK inhibition. Cancer Res; 75(12); 2489–500. ©2015 AACR.

Increased understanding of intertumoral heterogeneity at the genomic level has led to significant advancements in the treatment of solid tumors. Functional genomic alterations conferring sensitivity to targeted therapies can take many forms, and appropriate methods and tools are needed to detect these alterations. This review provides an update on genetic variability among solid tumors of similar histologic classification, using non–small cell lung cancer and melanoma as examples. We also discuss relevant technological platforms for discovery and diagnosis of clinically actionable variants and highlight the implications of specific genomic alterations for response to targeted therapy. Clin Cancer Res; 20(9); 2264–75. ©2014 AACR.

BRAF mutations occur in approximately 10% of colorectal cancers. Although RAF inhibitor monotherapy is highly effective in BRAF-mutant melanoma, response rates in BRAF-mutant colorectal cancer are poor. Recent clinical trials of combined RAF/EGFR or RAF/MEK inhibition have produced improved efficacy, but patients ultimately develop resistance. To identify molecular alterations driving clinical acquired resistance, we performed whole-exome sequencing on paired pretreatment and postprogression tumor biopsies from patients with BRAF-mutant colorectal cancer treated with RAF inhibitor combinations. We identified alterations in MAPK pathway genes in resistant tumors not present in matched pretreatment tumors, including KRAS amplification, BRAF amplification, and a MEK1 mutation. These alterations conferred resistance to RAF/EGFR or RAF/MEK combinations through sustained MAPK pathway activity, but an ERK inhibitor could suppress MAPK activity and overcome resistance. Identification of MAPK pathway reactivating alterations upon clinical acquired resistance underscores the MAPK pathway as a critical target in BRAF-mutant colorectal cancer and suggests therapeutic options to overcome resistance.RAF inhibitor combinations represent promising approaches in clinical development for BRAF-mutant colorectal cancer. Initial characterization of clinical acquired resistance mechanisms to these regimens identified several MAPK pathway alterations driving resistance by reactivating MAPK signaling, highlighting the critical dependence of BRAF-mutant colorectal cancers on MAPK signaling and offering potential strategies to overcome resistance.

Somatic mutations in kinase genes are associated with sensitivity of solid tumors to kinase inhibitors, but patients with metastatic cancer eventually develop disease progression. In EGFR mutant lung cancer, modeling of acquired resistance (AR) with drug-sensitive cell lines has identified clinically relevant EGFR tyrosine kinase inhibitor (TKI) resistance mechanisms such as the second-site mutation, EGFR T790M, amplification of the gene encoding an alternative kinase, MET, and epithelial–mesenchymal transition (EMT). The full spectrum of DNA changes associated with AR remains unknown. We used next-generation sequencing to characterize mutational changes associated with four populations of EGFR mutant drug-sensitive and five matched drug-resistant cell lines. Comparing resistant cells with parental counterparts, 18–91 coding SNVs/indels were predicted to be acquired and 1–27 were lost; few SNVs/indels were shared across resistant lines. Comparison of two related parental lines revealed no unique coding SNVs/indels, suggesting that changes in the resistant lines were due to drug selection. Surprisingly, we observed more CNV changes across all resistant lines, and the line with EMT displayed significantly higher levels of CNV changes than the other lines with AR. These results demonstrate a framework for studying the evolution of AR and provide the first genome-wide spectrum of mutations associated with the development of cellular drug resistance in an oncogene-addicted cancer. Collectively, the data suggest that CNV changes may play a larger role than previously appreciated in the acquisition of drug resistance and highlight that resistance may be heterogeneous in the context of different tumor cell backgrounds.

Mutant-selective EGFR tyrosine kinase inhibitors (TKI), such as osimertinib, are active agents for the treatment of EGFR-mutant lung cancer. Specifically, these agents can overcome the effects of the T790M mutation, which mediates resistance to first- and second-generation EGFR TKI, and recent clinical trials have documented their efficacy in patients with EGFR-mutant lung cancer. Despite promising results, therapeutic efficacy is limited by the development of acquired resistance. Here we report that Src family kinases (SFK) and focal adhesion kinase (FAK) sustain AKT and MAPK pathway signaling under continuous EGFR inhibition in osimertinib-sensitive cells. Inhibiting either the MAPK pathway or the AKT pathway enhanced the effects of osimertinib. Combined SFK/FAK inhibition exhibited the most potent effects on growth inhibition, induction of apoptosis, and delay of acquired resistance. SFK family member YES1 was amplified in osimertinib-resistant EGFR-mutant tumor cells, the effects of which were overcome by combined treatment with osimertinib and SFK inhibitors. In conclusion, our data suggest that the concomitant inhibition of both SFK/FAK and EGFR may be a promising therapeutic strategy for EGFR-mutant lung cancer. Cancer Res; 77(11); 2990–3000. ©2017 AACR.

Background Among patients with small cell lung cancer (SCLC), approximately 20% develop a paraneoplastic syndrome (PNS). Neurologic PNS are immune-mediated phenomena predicated on host recognition of an onconeural antigen, while endocrinologic PNS are attributed to ectopic tumor secretion of normal hormones. The presence of neurologic PNS improves prognosis and endocrinologic PNS worsens prognosis in patients with SCLC. We hypothesized that tumors from patients with neurologic PNS may have increased TILs and PD-1/PD-L1 expression compared to tumors from patients with endocrinologic PNS and this improved immune recognition accounts for prognostic differences. Methods We searched electronic medical record text stored in the Vanderbilt University Medical Center (VUMC) clinical data warehouse to identify SCLC patients with and without a PNS. We obtained clinical information through manual, retrospective chart review using an IRB-approved protocol. Overall survival (OS) and progression free survival (PFS) were compared using a log rank test. Archived formalin fixed, paraffin embedded samples were obtained from the Vanderbilt University Pathology Tissue Repository. We performed multiplexed fluorescence immunohistochemistry combined with automated quantitative analysis (AQUA® Technoloy; Navigate BioPharma Services, Inc.) to assess PD-1, PD-L1, CD4, and CD8 expression. A PD-1/PD-L1 interaction score was calculated by measuring the total area of PD-1 positive cells within the proximity of PD-L1 positive cells. This area was then divided by the total area of all non-tumor nucleated cells in the image and multiplied by a factor of 10,000. CD4, CD8, and PD-1/PD-L1 interaction scores were compared using a two sample t-test or Wilcoxon Rank Sum test. Results A total of 145 SCLC patients were identified, 55 with a PNS (25 neurologic and 30 endocrinologic) and 90 control patients. Patients with neurologic PNS exhibited significantly improved OS and PFS compared to patients with endocrinologic PNS and control patients (median OS 24mo, 95% CI 16.4mo-not reached (NR), vs 12mo, 95% CI 8.3mo-15.5mo, vs 13mo, 95% CI 12.2mo-16mo; median PFS 14mo, 95% CI 9.3mo-NR vs 6mo, 95% CI 4.6mo-9.5mo, vs 7mo, 95% CI 6.6mo-8.1mo, respectively). Tumors from patients with neurologic PNS (n=9) had statistically significantly higher PD-1/PD-L1 interaction scores (p=0.02), and increased CD4 (p=0.01) and CD8 (p=0.003) T cell infiltrates compared to tumors from patients with endocrinologic PNS (n=11). Conclusion Our study of tumor tissue from patients with SCLC and PNS demonstrated a statistically significant increase in immune modulation markers' expression in patients with neurologic PNS. Tumor immunomodulation may be the driver of the improved prognosis that has been observed in ours and other retrospective cohorts of patients with SCLC and neurologic PNS. Citation Format: Wade T. Iams, Eileen Shiuan, Catherine B. Meador, Marc Roth, Jennifer Bordeaux, Christine Vaupel, Lucy L. Wang, Joseph T. Schneider, Jeremy L. Warner, Zhiguo Zhao, Christine M. Lovly. Clinical outcomes and differential tumor immune microenvironment in patients with small cell lung cancer and paraneoplastic syndromes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1025.

e20563Background: Detecting hallmark genomic changes in peripheral blood circulating, cell-free DNA (cfDNA) in patients with small cell lung cancer (SCLC) has been increasingly validated. While the...

9071Background: Plasma cfDNA genotyping has been widely adopted for NSCLC diagnosis due to its convenience and high positive predictive value. However, limited assay sensitivity means negative plas...

Purpose: The EGFR tyrosine kinase inhibitors (TKIs), erlotinib and afatinib, have transformed the treatment of advanced EGFR-mutant lung adenocarcinoma. However, almost all patients who respond develop acquired resistance on average approximately 1 year after starting therapy. Resistance is commonly due to a secondary mutation in EGFR (EGFRT790M). We previously found that the combination of the EGFR TKI afatinib and the EGFR antibody cetuximab could overcome EGFRT790M-mediated resistance in preclinical models. This combination has shown a 29% response rate in a clinical trial in patients with acquired resistance to first-generation TKIs. An outstanding question is whether this regimen is beneficial when used as first-line therapy. Experimental Design: Using mouse models of EGFR-mutant lung cancer, we tested whether the combination of afatinib plus cetuximab delivered upfront to mice with TKI-naïve EGFRL858R-induced lung adenocarcinomas delayed tumor relapse and drug-resistance compared with single-agent TKIs. Results: Afatinib plus cetuximab markedly delayed the time to relapse and incidence of drug-resistant tumors, which occurred in only 63.6% of the mice, in contrast to erlotinib or afatinib treatment where 100% of mice developed resistance. Mechanisms of tumor escape observed in afatinib plus cetuximab resistant tumors include the EGFRT790M mutation and Kras mutations. Experiments in cell lines and xenografts confirmed that the afatinib plus cetuximab combination does not suppress the emergence of EGFRT790M. Conclusions: These results highlight the potential of afatinib plus cetuximab as an effective treatment strategy for patients with TKI-naïve EGFR-mutant lung cancer and indicate that clinical trial development in this area is warranted. Clin Cancer Res; 22(2); 426–35. ©2015 AACR.

Two new studies demonstrate that so-called 'liquid biopsies' may reveal important genomic information needed to monitor treatment responses, forecast tumor recurrences, and provide a rationale for novel therapeutic strategies in patients with lung cancer and colon cancer.

Despite the success of treating EGFR-mutant lung cancer patients with EGFR tyrosine kinase inhibitors (TKI), all patients eventually acquire resistance to these therapies. Although various resistance mechanisms have been described, there are currently no FDA-approved therapies that target alternative mechanisms to treat lung tumors with acquired resistance to first-line EGFR TKI agents. Here we found that EPHA2 is overexpressed in EGFR TKI-resistant tumor cells. Loss of EPHA2 reduced the viability of erlotinib-resistant tumor cells harboring EGFRT790M mutations in vitro and inhibited tumor growth and progression in an inducible EGFRL858R+T790M-mutant lung cancer model in vivo. Targeting EPHA2 in erlotinib-resistant cells decreased S6K1-mediated phosphorylation of cell death agonist BAD, resulting in reduced tumor cell proliferation and increased apoptosis. Furthermore, pharmacologic inhibition of EPHA2 by the small-molecule inhibitor ALW-II-41-27 decreased both survival and proliferation of erlotinib-resistant tumor cells and inhibited tumor growth in vivo. ALW-II-41-27 was also effective in decreasing viability of cells with acquired resistance to the third-generation EGFR TKI AZD9291. Collectively, these data define a role for EPHA2 in the maintenance of cell survival of TKI-resistant, EGFR-mutant lung cancer and indicate that EPHA2 may serve as a useful therapeutic target in TKI-resistant tumors. Cancer Res; 76(2); 305–18. ©2016 AACR.

Background: Genomic alterations in the ALK tyrosine kinase, such as copy number gains, point mutations, and ALK gene fusions, have been described in a broad spectrum of malignancies. Recently a novel mechanism of ALK activation in melanoma was discovered in which ALK transcription was initiated from a de novo alternative transcription initiation (ATI) site in ALK intron 19 (ALK-ATI) [1]. In this study, we used whole transcriptome sequencing (WTS) to interrogate a cohort of clinical melanoma specimens in order to identify those that express ALK-ATI. Method: RNA-seq libraries of 33 BRAF-negative clinical metastatic melanoma specimens were prepared using the KAPA Stranded RNA-Seq Kit with RiboErase with 200ng RNA as input and sequenced on Illumina HiSeq 4000 at 2x75bp. The specimens were sequenced to an average of 69.4m total read pairs with 11.3m on-target distinct read pairs. Sequencing reads were mapped to the human genome and transcriptome, gene- and exon-level expressions were quantified. An algorithm to identify the ALK-ATI was developed based on assessing 1) transcription of intron 19 putative ATI region, 2) ALK relative RNA expression and 3) ALK 5’ to 3’ differential expression. In addition, immunohistochemistry (IHC) staining for ALK (using D5F3 antibody) was done on 10 of the 33 samples with adequate tumor material. Results: High coverage (median 30x) of intron 19 ATI region and high ALK relative expression (log ratio > 2) were observed in 4 of the 33 specimens tested, in which the ATI site is consistent with what had been reported in [1]. Among the 4 potential ALK-ATI positive cases, 3 have shown significant 3’ to 5’ overexpression of ALK. IHC staining were performed on the 4 putative ALK-ATI positive cases and 6 putative negative cases, and has confirmed ALK-ATI calls detected by WTS assay. Two of the ALK IHC positive cases harbor the highest ALK expression, and the other two ALK-ATI likely cases are ALK IHC weak positive. ALK-ATI occurred in cutaneous, mucosal, and unknown primary melanoma, and co-occurred with other driver mutations (NRAS, NF1, KIT). None of the above specimens had ALK rearrangements detected from DNAseq (by FoundationOne) or WTS. Conclusions: Our findings confirmed previously reported ALK activation through ALK-ATI in an independent melanoma cohort, and provided direct evidence that such events can be detected using whole transcriptome sequencing and align with IHC results in clinical FFPE tumor specimens. This confirmation together with future functional validation and clinical evidence may provide new therapeutic opportunities for BRAF-negative metastatic melanoma patients. 1. Wiesner, T. et al. Alternative transcription initiation leads to expression of a novel ALK isoform in cancer. Nature 526, 453-457 (2015). Citation Format: Shan Zhong, Christine Lovly, Christine Malboeuf, Kristina Brennan, Douglas Johnson, Kelsie Riemenschneider, Catherine Meador, Emily White, Geoff A. Otto, Doron Lipson, Philip Stephens, Vincent Miller, Jeffrey Ross, Jie He. Identification of ALK alternative transcription initiation in BRAF-negative metastatic melanoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2367. doi:10.1158/1538-7445.AM2017-2367

INTRODUCTION: Osimertinib is a third-generation, mutant-selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) recently approved for the treatment of T790M-positive EGFR-mutant lung cancer. The mutant-selective nature of osimertinib improves its tolerability by limiting its inhibition of wild-type EGFR compared to first- and second-generation EGFR inhibitors, making it a good candidate for rationally-designed combination therapies. One such combination, osimertinib plus the MEK inhibitor selumetinib (AZD6244; ARRY-142886), is currently being studied as part of the phase 1b TATTON trial (NCT02143466). Previous work by our group demonstrated that selumetinib may delay or reverse resistance to osimertinib in some cases; however, we anticipate that resistance to this combination will ultimately develop in patients. Here, we investigate acquired resistance to osimertinib and selumetinib combination therapy in vitro and in vivo. DESIGN: In cells sensitive to osimertinib plus selumetinib (hereafter referred to as combination-sensitive cells), ERK phosphorylation was monitored via Western blot over the course of a 7-day treatment with 200 nM osimertinib plus 1 µM selumetinib. To detect RAS-GTP, an active RAS precipitation was performed in combination-sensitive and combination-resistant cell lines following treatment with osimertinib. In combination-resistant cells, pharmacologic inhibitors of various MAPK pathway components were used to assess whether they could restore potency. Western blot analysis was used to confirm on-target effects. Lastly, an analysis of differences in gene expression between four isogenic sets of combination-sensitive and combination-resistant cell lines is currently ongoing. RESULTS: In cell lines that are sensitive to growth inhibition by the combination of osimertinib plus selumetinib, we observed complete re-activation of the MAPK pathway after 5 days of continuous exposure to both inhibitors. Additionally, RAS activity was elevated in combination-resistant cell lines, and these cells remained sensitive to the ERK inhibitor SCH772984. Likewise, the addition of a pan-RAF inhibitor restored the growth inhibitory effects of osimertinib plus selumetinib in combination-resistant cells, suggesting that incomplete inhibition of MAPK is responsible for resistance in these cells. Furthermore, an alternative MEK inhibitor, trametinib, was efficacious in combination with osimertinib in cell lines that were resistant to osimertinib plus selumetinib. CONCLUSION: These data identify a potential mechanism of resistance to a combination therapy that is currently being tested in the clinic. Specifically, these data demonstrate that re-activation of the MAPK pathway is a mechanism of resistance to osimertinib plus selumetinib and that this pathway is still targetable in combination-resistant cells. Citation Format: David Westover, Catherine B. Meador, Eiki Ichihara, Hayden F. Byrd, Cath Eberlein, Yingjun Yan, Darren A. Cross, Christine M. Lovly. Sustained MAPK activation as a mechanism of resistance to osimertinib plus selumetinib in models of EGFR-mutant cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3160. doi:10.1158/1538-7445.AM2017-3160

Introduction: Small cell lung cancer (SCLC) is a highly lethal neuroendocrine malignancy that accounts for approximately 10-15% of all lung cancers and is responsible for approximately 30,000 deaths annually in the United States and 200,000 deaths worldwide every year1. There is an urgent need to develop novel treatment strategies for patients with this disease. We sought to improve the quality of patient care by establishing a liquid biopsy assay for rapid, noninvasive monitoring of disease burden. Design: The SCLC assay relies on targeted next-generation DNA sequencing of cell-free DNA (cfDNA) collected from patient plasma. The assay targets a panel of 14 genes that are frequently mutated in SCLC2. We examined a total of 141 plasma samples from a cohort of 27 patients. 11 patients had limited stage SCLC and 16 patients had extensive stage SCLC. The analyzed plasma samples were collected during the course of patient treatment and included time points before and after chemotherapy or immunotherapy. Results: We detected somatic, disease-associated mutations in the cfDNA of 78% of patient samples (21/27). The allele frequency of cfDNA ranged from ≤0.5% to ≥85%. The most commonly mutated genes were TP53 and RB1, which were found in 17/27 and 10/27 samples, respectively. We also detected single nucleotide variants in PIK3CA (3/27) and PTEN (1/27) as well as copy number variants in MYC and MYCL1 (2/27). The observed mutant allele frequencies in longitudinal samples tracked closely with treatment responses. Strikingly, we found instances where the assay detected the reappearance of tumor-associated markers several weeks before clinical evidence of relapse was detected. Conclusions: cfDNA sequencing allows for improved monitoring of disease burden, depth of responses to treatment, and timely warning of disease relapse in patients with SCLC. References: 1. Society, A.C., Cancer Facts and Figures 2015. American Cancer Society, Atlanta, Ga, 2015. 2. George, J. et al., Comprehensive genomic profiles of small cell lung cancer, Nature 524:47 Citation Format: Christine M. Lovly, Karinna Almodovar, Wade T. Iams, Catherine B. Meador, Sally York, Leora Horn, Christopher K. Raymond, Jennifer Hernandez, Lee P. Lim. Longitudinal monitoring of cell-free DNA in patients with small cell lung cancer reveals dynamic insights into treatment efficacy and disease relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4949. doi:10.1158/1538-7445.AM2017-4949

8571 Background: Advances in the treatment of small cell lung cancer (SCLC), an aggressive disease with poor prognosis, will require the development of diagnostic methods during treatment that are more sensitive and descriptive than are currently available. The emergence of “liquid biopsy” technologies coupled with comprehensive genomic information about common mutations in SCLC prompted us to implement a blood-based assay for simultaneous sequencing of circulating, cell-free tumor DNA (cfDNA) and determination of the peripheral a and b T cell receptor repertoire in patients with SCLC. Methods: Our SCLC assay uses targeted hybrid capture and next generation sequencing of cfDNA extracted from patient plasma to detect somatic mutations in 14 frequently mutated genes in SCLC. We also sequenced rearranged T cell receptor α and β genes, each with 5000 unique α and β CDR3 open reading frames per microgram of gDNA. Over 26 months we followed 27 patients with SCLC (16 with extensive stage and 11 with limited stage disease) and examined cfDNA from 141 plasma samples and T cell repertoire from 41 samples. Results: We detected somatic, disease-associated mutations in 85% of patient samples (23/27) with allele frequencies of cfDNA ranging from ≤0.5% to ≥85%. The most commonly mutated genes were TP53 (17/27 patients) and RB1 (10/27 patients) . We detected 87 unique genomic alterations in 12 different genes (in addition to TP53 and RB1 these included PTEN, NOTCH1-4, MYC, MYCL1, PIK3CA, KIT, and BRAF). The observed mutant allele frequencies in longitudinal samples tracked with treatment response, including cases in which cfDNA allele frequencies increased before clinical evidence of relapse. Longitudinal monitoring of T cell repertoire demonstrated both variability between patients and sequential changes during therapy, including one case of decreased T cell numbers accompanying disease relapse. Conclusions: As the field of immuno-oncology matures, we anticipate that coupled determination of T cell repertoire together with cfDNA monitoring will merge into a clinically useful “molecular image” of each patient’s disease status and real-time host immune response.

Aim: AZD9291 is an oral, irreversible EGFR-TKI effective against EGFR-TKI-sensitising (EGFRm+) and resistance T790M mutations. It is efficacious in EGFRm+ advanced NSCLC patients with acquired resistance to EGFR-TKIs. We report on the evaluation of AZD9291 in the first-line setting. Methods: In vitro and in vivo xenograft studies were performed utilising PC-9 (EGFR exon 19 del) and H3255 (EGFR L858R) cells. The efficacy of AZD9291 was compared with known EGFR inhibitors: gefitinib, afatinib and WZ4002. Transgenic mice with lung tumours driven by EGFR L858R or exon 19 del were also dosed with AZD9291. In the clinic, AZD9291 was given to patients with advanced stage NSCLC with a documented EGFR-TKI-sensitising mutation as first-line therapy (treatment-naive), in an ongoing Phase I trial (NCT01802632). Results: In vitro, compared to afatinib at 0.8nM, resistance to AZD9291 at 10nM was delayed by an average of 43 days in PC9 cells. Notably, 73% (8/11) of cell populations exposed to gefitinib or afatinib developed T790M mutation compared to none (0/14) with AZD9291. In PC9 and H3255 xenograft studies, AZD9291 demonstrated strong efficacy, evidenced by profound and sustained tumour reduction. Robust tumour shrinkage was also noted in exon 19 del and L858R in vivo transgenic models. These lines of evidence prompted the clinical investigation of AZD9291 in treatment-naive patients with advanced NSCLC. As of 2 April 2014, 22 patients ( 7 males; median age 61 yrs; WHO PS 0/1, 14/6; Asian ethnicity 18; EGFR-TKI-sensitising mutation exon 19del 6, L858R 8, other 1, unknown 7) were treated with AZD9291 at 80 mg/day. Safety and efficacy data from all evaluable patients will be presented. Conclusions: AZD9291 is a novel, mutation-specific EGFR inhibitor which demonstrates good activity in pre-clinical EGFR mutant xenograft models. Early clinical data with AZD9291 in treatment-naive patients with advanced NSCLC supports further evaluation in first-line therapy of EGFRm+ NSCLC. Disclosure: S.S. Ramalingam: Advisory boards: AstraZeneca, Boehringer Ingelheim, Genentech; N. Nogami: Speaker: AstraZeneca (not related to AZD9291); J.C. Yang: Advisory boards: Boehringer Ingelheim, Eli Lilly, Pfizer, Novartis, Roche/Genetech, Astrazeneca, Merck, Bayer, Clovis Oncology. Corporate-sponsored research: Boehringer Ingelheim; C. Eberlein, S. Ashton, M. Mellor, D. Cross, P. Ballard, G. Hughes, P. Frewer, S. Ghiorghiu and M. Cantarini: Employment and stock ownership: AstraZeneca; W. Pao: Rights to EGFR T790M testing licensed on WP's behalf by MSKCC to MolecularMD. Research funding and travel and consulting funds: AstraZeneca; P.A. Janne: Consultant or advisory role: AstraZeneca, Boehringer Ingelheim, Clovis Oncology, Pfizer, Merrimack Pharmaceuticals, Chugai, Immunogen. Stock ownership: Gatekeeper. Other: Lab Corp. All other authors have declared no conflicts of interest.

Aim: First-generation EGFR-TKIs, such as gefitinib, are active in first-line, EGFR-TKI-sensitising-mutant (EGFRm+), advanced NSCLC, but the duration of clinical benefit is limited by acquired tumour resistance. A common resistance mechanism is gain of an additional mutation, T790M, and about 5–15% of patients also develop MET amplification, with or without T790M. The recently developed novel third-generation EGFR-TKI, AZD9291 (a selective EGFR-TKI of EGFRm+ and T790M mutations), overcomes T790M-mediated resistance. Combining EGFR-TKIs with selective molecularly targeted agents has the potential to delay the emergence of EGFR-TKI resistance across lines of therapy. Finally, immunotherapeutics, such as checkpoint inhibitors, have the potential to target cancers orthogonally; thus studies to optimise their use with EGFR-TKIs would be of high value to patients. Methods: In vitro and in vivo preclinical models representing EGFRm+ and T790M NSCLC were used to investigate the potential of combining AZD9291 or gefitinib with immunotherapeutics, and selective small molecule kinase inhibitors, namely selumetinib (AZD6244, ARRY-142886; MEK1/2 inhibitor) and AZD6094 (MET inhibitor). Results: Preclinical models harbouring MET overexpression demonstrated AZD6094 plus EGFR-TKI was well tolerated and effective in reversing MET-driven resistance. In models of acquired resistance mediated by increased MEK dependency, resistance could be delayed in vitro and in vivo by addition of selumetinib. Finally, a genetically engineered mouse model of mutant EGFR NSCLC was used to assess the impact of AZD9291 treatment on T-cell infiltration; results will be presented. Conclusions: These preclinical studies provide a strong rationale for the clinical evaluation of AZD9291 and gefitinib in combination with molecularly targeted agents, such as AZD6094 and selumetinib, to delay and/or overcome acquired resistance to single-agent EGFR-TKI therapy. Rational combinations with immunotherapeutics are also worth further investigation. Disclosure: D. Cross, C. Eberlein, S. Ashton, M. Mellor, P. Smith, M. Frigault and P.J. Jewsbury: Employment and stock ownership: AstraZeneca; C. D'Cruz: Employment: AstraZeneca; R. Stewart: Employment: MedImmune. Stock ownership: AstraZeneca; A. Schuller: Employment: AstraZeneca; W. Pao: Rights to EGFR T790M testing licensed on WP's behalf by MSKCC to MolecularMD. Research funding and travel and consulting funds: AstraZeneca. All other authors have declared no conflicts of interest.