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Lung cancer is the leading cause of cancer mortality worldwide. The treatment of patients with lung cancer harboring mutant EGFR with orally administered EGFR tyrosine kinase inhibitors (TKI) has been a paradigm shift. Osimertinib and rociletinib are third-generation irreversible EGFR TKIs targeting the EGFR T790M mutation. Osimertinib is the current standard of care for patients with EGFR mutations due to increased efficacy, lower side effects, and enhanced brain penetrance. Unfortunately, all patients develop resistance. Genomic approaches have primarily been used to interrogate resistance mechanisms. Here we characterized the proteome and phosphoproteome of a series of isogenic EGFR-mutant lung adenocarcinoma cell lines that are either sensitive or resistant to these drugs, comprising the most comprehensive proteomic dataset resource to date to investigate third generation EGFR TKI resistance in lung adenocarcinoma. Unbiased global quantitative mass spectrometry uncovered alterations in signaling pathways, revealed a proteomic signature of epithelial–mesenchymal transition, and identified kinases and phosphatases with altered expression and phosphorylation in TKI-resistant cells. Decreased tyrosine phosphorylation of key sites in the phosphatase SHP2 suggests its inhibition, resulting in subsequent inhibition of RAS/MAPK and activation of PI3K/AKT pathways. Anticorrelation analyses of this phosphoproteomic dataset with published drug-induced P100 phosphoproteomic datasets from the Library of Integrated Network-Based Cellular Signatures program predicted drugs with the potential to overcome EGFR TKI resistance. The PI3K/MTOR inhibitor dactolisib in combination with osimertinib overcame resistance both in vitro and in vivo. Taken together, this study reveals global proteomic alterations upon third generation EGFR TKI resistance and highlights potential novel approaches to overcome resistance. Significance: Global quantitative proteomics reveals changes in the proteome and phosphoproteome in lung cancer cells resistant to third generation EGFR TKIs, identifying the PI3K/mTOR inhibitor dactolisib as a potential approach to overcome resistance.

Background Small cell lung cancer (SCLC) has the strongest association with smoking among lung cancers. The characteristics of never smokers with SCLC is not known. Research Question Are the clinical characteristics, prognostic factors, survival, genomic alterations, and tumor mutational burdens of SCLC in patients who have never smoked different from those who have smoked? Study Design and Methods A retrospective multicenter cohort study of patients with clinician-confirmed SCLC was performed with the use of a longitudinal and nationally representative electronic medical records database. Smoking history was assessed through technology-enabled abstraction and confirmed for never smokers via chart review. Genomic characteristics of never smoker patients with SCLC were examined with the use of a next-generation sequencing-based gene panel and whole exome sequencing. Results One hundred of 5,632 patients (1.8%) with SCLC were never smokers. Relative to smokers, never smokers were more likely to be female (66.0% vs 52.4%; P = .009) and present with extensive stage (70.0% vs 62.2%; P = .028). Never smokers had a higher proportion of patients in age groups 35 to 49 years (7.0% vs 3.0%; P = .006) and ≥80 years (17.0% vs 8.2%; P = .006). Known risk factors for lung cancer were found in Interpretation The sex- and age-specific distribution of SCLC among never smokers, along with differences that were identified by genomic analyses, suggests a distinct biology of SCLC in never smokers compared with smokers.

SUMMARY We perform an immunogenomics analysis utilizing whole-transcriptome sequencing of 657 pediatric extra-cranial solid cancer samples representing 14 diagnoses, and additionally utilize transcriptomes of 131 pediatric cancer cell lines and 147 normal tissue samples for comparison. We describe patterns of infiltrating immune cells, T cell receptor (TCR) clonal expansion, and translationally relevant immune checkpoints. We find that tumor-infiltrating lymphocytes and TCR counts vary widely across cancer types and within each diagnosis, and notably are significantly predictive of survival in osteosarcoma patients. We identify potential cancer-specific immunotherapeutic targets for adoptive cell therapies including cell-surface proteins, tumor germline antigens, and lineage-specific transcription factors. Using an orthogonal immunopeptidomics approach, we find several potential immunotherapeutic targets in osteosarcoma and Ewing sarcoma and validated PRAME as a bona fide multi-pediatric cancer target. Importantly, this work provides a critical framework for immune targeting of extracranial solid tumors using parallel immuno-transcriptomic and -peptidomic approaches., Graphical Abstract, In brief Brohl et al. perform immunogenomics analysis of a cohort of 788 pediatric extracranial solid tumors and cell lines, representing 14 diagnoses, utilizing RNA sequencing. They broadly describe prognostically relevant immunophenotypes and provide proof of concept of a transcriptomics-informed adoptive cellular therapy approach, validating PRAME as a multi-pediatric cancer immunotherapy target.

Immune checkpoint inhibitor (ICI) therapy has been a paradigm shift in the treatment of cancer. ICI therapy results in durable responses and survival benefit for a large number of tumor types. Osimertinib, a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) has shown great efficacy treating EGFR mutant lung cancers, however, all patients eventually develop resistance. ICI therapy has not benefitted EGFR mutant lung cancer. Herein, we employed stable isotope labeling by amino acids in cell culture (SILAC) quantitative mass spectrometry-based proteomics to investigate potential immune escape molecular mechanisms in osimertinib resistant EGFR mutant lung adenocarcinoma by interrogating the alterations in the human leukocyte antigen (HLA) Class I-presented immunopeptidome, Class I-interactome, and the whole cell proteome between isogenic osimertinib-sensitive and -resistant human lung adenocarcinoma cells. Our study demonstrates an overall reduction in HLA class I-presented immunopeptidome and downregulation of antigen presentation core complex (e.g., TAP1 and ERAP1/2) and immunoproteasome in osimertinib resistant lung adenocarcinoma cells. Several key components in autophagy pathway are differentially altered. S100 proteins and SLC3A2 may play critical roles in reduced antigen presentation. Our dataset also includes ~1000 novel HLA class I interaction partners and hundreds of Class I-presented immunopeptides in EGFR mutant lung adenocarcinoma. This large-scale unbiased proteomics study provides novel insights and potential mechanisms of immune evasion of EGFR mutant lung adenocarcinoma.

Simple Summary ctDNA assay is a promising non-invasive method to detect genomic alterations associated with lung cancer. In this prospective study of 25 patients with EGFR-mutant lung adenocarcinoma receiving osimertinib, ctDNA progression predated radiographic progression by 118 days in 11 of 20 patients with disease progression. Saliva-based ctDNA analysis and plasma NGS detected additional patients with ctDNA progression preceding clinical progression, suggesting the potential complementary roles of different ctDNA detection methodologies. Baseline mutant ctDNA level predicted progression-free survival while tumor volume measurements by volumetric CT did not. Serial ctDNA analysis of plasma and saliva is a clinically useful tool to monitor response and resistance to osimertinib. Abstract Background: We assessed whether serial ctDNA monitoring of plasma and saliva predicts response and resistance to osimertinib in EGFR-mutant lung adenocarcinoma. Three ctDNA technologies—blood-based droplet-digital PCR (ddPCR), next-generation sequencing (NGS), and saliva-based EFIRM liquid biopsy (eLB)—were employed to investigate their complementary roles. Methods: Plasma and saliva samples were collected from patients enrolled in a prospective clinical trial of osimertinib and local ablative therapy upon progression (NCT02759835). Plasma was analyzed by ddPCR and NGS. Saliva was analyzed by eLB. Results: A total of 25 patients were included. We analyzed 534 samples by ddPCR (n = 25), 256 samples by NGS (n = 24) and 371 samples by eLB (n = 22). Among 20 patients who progressed, ctDNA progression predated RECIST 1.1 progression by a median of 118 days (range: 61–272 days) in 11 (55%) patients. Of nine patients without ctDNA progression by ddPCR, two patients had an increase in mutant EGFR by eLB and two patients were found to have ctDNA progression by NGS. Levels of ctDNA measured by ddPCR and NGS at early time points, but not volumetric tumor burden, were associated with PFS. EGFR/ERBB2/MET/KRAS amplifications, EGFR C797S, PIK3CA E545K, PTEN V9del, and CTNNB1 S45P were key resistance mechanisms identified by NGS. Conclusion: Serial assessment of ctDNA in plasma and saliva predicts response and resistance to osimertinib, with each assay having supplementary roles.

Immune checkpoint inhibitor and adoptive lymphocyte transfer-based therapies have shown great therapeutic potential for cancers with high tumor mutation burden (TMB). Here, we employed mass spectrometry (MS)-based proteogenomic large-scale profiling to identify potential immunogenic human leukocyte antigen (HLA) Class I-presented peptides in both melanoma, a “hot tumor” with high TMB, and EGFR mutant lung adenocarcinoma, a “cold tumor” with low TMB. We identified several classes of neopeptides, including mutated neoantigens and more than 1000 post-translationally modified peptides representing 58 different PTMs. We constructed a cancer germline (CG) antigen database with 285 antigens and identified 42 Class I-presented CG antigens. Finally, we developed a non-canonical peptide discovery pipeline to identify 44 lncRNA-derived peptides and validated Class I binding for select neopeptides. We provide direct evidence of HLA Class I presentation of a large number of neopeptides for potential vaccine or adoptive cell therapy in melanoma and mutant EGFR lung cancer.

Because tobacco is a potent carcinogen, secondary causes of lung cancer are often diminished in perceived importance. To assess the extent of inherited susceptibility to small cell lung cancer (SCLC), the most lethal type of lung cancer, we sequenced germline exomes of 87 patients (77 SCLC and 10 extrapulmonary small cell) and considered 607 genes, discovering 42 deleterious variants in 35 cancer-predisposition genes among 43.7% of patients. These findings were validated in an independent cohort of 79 patients with SCLC. Loss of heterozygosity was observed in 3 of 14 (21.4%) tumors. Identification of variants influenced medical management and family member testing in nine (10.3%) patients. Unselected patients with SCLC were more likely to carry germline RAD51 paralog D (RAD51D), checkpoint kinase 1 (CHEK1), breast cancer 2 (BRCA2), and mutY DNA glycosylase (MUTYH) pathogenic variants than healthy controls. Germline genotype was significantly associated with the likelihood of a first-degree relative with cancer or lung cancer (odds ratio: 1.82, P = 0.008; and 2.60, P = 0.028), and longer recurrence-free survival after platinum-based chemotherapy (P = 0.002), independent of known prognostic factors. Treatment of a patient with relapsed SCLC and germline pathogenic mutation of BRCA1 interacting protein C-terminal helicase 1 (BRIP1), a homologous recombination–related gene, using agents synthetically lethal with homologous recombination deficiency, resulted in a notable disease response. This work demonstrates that SCLC, currently thought to result almost exclusively from tobacco exposure, may have an inherited predisposition and lays the groundwork for targeted therapies based on the genes involved.

Mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase domain constitutively activate EGFR resulting in lung tumorigenesis. Activated EGFR modulates downstream signaling by altering phosphorylation-driven interactions that promote growth and survival. Secretory carrier membrane proteins (SCAMPs) are a family of transmembrane proteins that regulate recycling of receptor proteins, including EGFR. The potential role of SCAMPs in mutant EGFR function and tumorigenesis has not been elucidated. Using quantitative mass-spectrometry-based phosphoproteomics, we identified SCAMP3 as a target of mutant EGFRs in lung adenocarcinoma and sought to further investigate the role of SCAMP3 in the regulation of lung tumorigenesis. Here we show that activated EGFR, either directly or indirectly phosphorylates SCAMP3 at Y86 and this phosphorylation increases the interaction of SCAMP3 with both wild-type and mutant EGFRs. SCAMP3 knockdown increases lung adenocarcinoma cell survival and increases xenograft tumor growth in vivo, demonstrating a tumor suppressor role of SCAMP3 in lung tumorigenesis. The tumor suppressor function is a result of SCAMP3 promoting EGFR degradation and attenuating MAP kinase signaling pathways. SCAMP3 knockdown also increases multinucleated cells in culture, suggesting that SCAMP3 is required for efficient cytokinesis. The enhanced growth, increased colony formation, reduced EGFR degradation and multinucleation phenotype of SCAMP3-depleted cells were reversed by re-expression of wild-type SCAMP3, but not SCAMP3 Y86F, suggesting that Y86 phosphorylation is critical for SCAMP3 function. Taken together, the results of this study demonstrate that SCAMP3 functions as a novel tumor suppressor in lung cancer by modulating EGFR signaling and cytokinesis that is partly Y86 phosphorylation-dependent.

Candidate gene as well as genome-wide association studies identified several polymorphic variants to be associated with lung cancer worldwide including in India. However, contradictory results have failed to estimate the overall effect of the polymorphic variants on the disease. Textmining was conducted on PubMed following specific search strings to gather all the publications related to genetic association with lung cancer in India. Out of 211 PubMed hits only 30 studies were selected for meta-analysis following specific inclusion criteria. Heterogeneity between studies was calculated by Cochran's Q-test (P

Exportin 1 (XPO1) mediates nuclear export of many cellular factors known to play critical roles in malignant processes, and selinexor (KPT-330) is the first XPO1-selective inhibitor of nuclear export compound in advanced clinical development phase for cancer treatment. We demonstrated here that inhibition of XPO1 drives nuclear accumulation of important cargo tumor suppressor proteins, including transcription factor FOXO3a and p53 in thymic epithelial tumor (TET) cells, and induces p53-dependent and -independent antitumor activity in vitro Selinexor suppressed the growth of TET xenograft tumors in athymic nude mice via inhibition of cell proliferation and induction of apoptosis. Loss of p53 activity or amplification of XPO1 may contribute to resistance to XPO1 inhibitor in TET. Using mass spectrometry-based proteomics analysis, we identified a number of proteins whose abundances in the nucleus and cytoplasm shifted significantly following selinexor treatment in the TET cells. Furthermore, we found that XPO1 was highly expressed in aggressive histotypes and advanced stages of human TET, and high XPO1 expression was associated with poorer patient survival. These results underscore an important role of XPO1 in the pathogenesis of TET and support clinical development of the XPO1 inhibitor for the treatment of patients with this type of tumors. Cancer Res; 77(20); 5614-27. ©2017 AACR.

Background Thymic epithelial tumors are PD-L1–expressing tumors of thymic epithelial origin characterized by varying degrees of lymphocytic infiltration and a predisposition towards development of paraneoplastic autoimmunity. PD-1–targeting antibodies have been evaluated, largely in patients with thymic carcinoma. We sought to evaluate the efficacy and safety of the anti-PD-L1 antibody, avelumab (MSB0010718C), in patients with relapsed, advanced thymic epithelial tumors and conduct correlative immunological studies. Methods Seven patients with thymoma and one patient with thymic carcinoma were enrolled in a phase I, dose-escalation trial of avelumab (MSB0010718C), and treated with avelumab at doses of 10 mg/kg to 20 mg/kg every 2 weeks until disease progression or development of intolerable side effects. Tissue and blood immunological analyses were conducted. Results Two of seven (29%) patients with thymoma had a confirmed Response Evaluation Criteria in Solid Tumors–defined partial response, two (29%) had an unconfirmed partial response and three patients (two thymoma; one thymic carcinoma) had stable disease (43%). Three of four responses were observed after a single dose of avelumab. All responders developed immune-related adverse events that resolved with immunosuppressive therapy. Only one of four patients without a clinical response developed immune-related adverse events. Responders had a higher absolute lymphocyte count, lower frequencies of B cells, regulatory T cells, conventional dendritic cells, and natural killer cells prior to therapy. Conclusion These results demonstrate anti-tumor activity of PD-L1 inhibition in patients with relapsed thymoma accompanied by a high frequency of immune-related adverse events. Pre-treatment immune cell subset populations differ between responders and non-responders. Trial registration ClinicalTrials.gov - NCT01772004. Date of registration – January 21, 2013. Electronic supplementary material The online version of this article (10.1186/s40425-019-0723-9) contains supplementary material, which is available to authorized users.

BackgroundA gradual decline in renal function occurs even in healthy aging individuals. In addition to aging per se, concurrent metabolic syndrome and hypertension, which are common in the aging population, can induce mitochondrial dysfunction and inflammation, which collectively contribute to age-related kidney dysfunction and disease. Here we studied the role of the nuclear hormone receptors, the estrogen-related receptors (ERRs) in regulation of age-related mitochondrial dysfunction and inflammation. ERRs were decreased in aging human and mouse kidneys and were preserved in aging mice with lifelong caloric restriction (CR).MethodsA pan-ERR agonist was used to treat 21-month-old mice for 8-weeks. In addition, 21-month-old mice were treated with a STING inhibitor for 3 weeks.ResultsRemarkably, only an 8-week treatment with a pan-ERR agonist reversed the age-related increases in albuminuria, podocyte loss, mitochondrial dysfunction and inflammatory cytokines, including the cGAS-STING and STAT3 signaling pathways. A 3-week treatment of 21-month-old mice with a STING inhibitor reversed the increases in inflammatory cytokines and the senescence marker p21 but also unexpectedly reversed the age-related decreases in PGC-1α, ERRα, mitochondrial complexes and MCAD expression.ConclusionsOur studies identified ERRs as important modulators of age-related mitochondrial dysfunction and inflammation. These findings highlight novel druggable pathways that can be further evaluated to prevent progression of age-related kidney disease.Significance StatementThere is an increasing need for prevention and treatment strategies for age-related kidney disease. The hallmarks of aging kidneys are decreased mitochondrial function and increased inflammation. The expression of the nuclear hormone receptors estrogen-related receptors (ERRs) are decreased in aging human and mouse kidneys. This paper investigates the role of ERRs in the aging kidney. Treatment of aging mice with a pan-ERR agonist reversed the age-related increases in albuminuria and podocyte loss, mitochondrial dysfunction and inflammatory cytokines, including the cGAS-STING signaling pathways. Treatment of aging mice with a STING inhibitor decreased inflammation and increased mitochondrial gene expression. These findings identify ERRs as important modulators of age-related mitochondrial dysfunction and inflammation.

// Abhilash Venugopalan 1 , Min-Jung Lee 2 , Gang Niu 3 , Jose Medina-Echeverz 1 , Yusuke Tomita 2 , Martin J. Lizak 4 , Constance M. Cultraro 1 , Robert Mark Simpson 5 , Xiaoyuan Chen 3 , Jane B. Trepel 2 and Udayan Guha 1 1 Thoracic and Gastrointestinal Oncology Branch, National Cancer Institute, Bethesda, MD, USA 2 Developmental Therapeutics Branch, National Cancer Institute, Bethesda, MD, USA 3 Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, Bethesda, MD, USA 4 Mouse Imaging Facility, National Institute of Neurological Disorder and Stroke, National Institutes of Health, Bethesda, MD, USA 5 Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD, USA Correspondence to: Udayan Guha, email: // Keywords : lung cancer, EGFR, TKI, imaging, immune-infiltration Received : May 23, 2016 Accepted : July 22, 2016 Published : August 02, 2016 Abstract Lung adenocarcinoma patients harboring kinase domain mutations in Epidermal growth factor receptor (EGFR) have significant clinical benefit from EGFR-targeted tyrosine kinase inhibitors (TKIs). Although a majority of patients experience clinical symptomatic benefit immediately, an objective response can only be demonstrated after 6-8 weeks of treatment. Evaluation of patient response by imaging shows that 30-40% of patients do not respond due to intrinsic resistance to these TKIs. We investigated immediate-early effects of EGFR-TKI treatment in mutant EGFR-driven transgenic mouse models by FDG-PET and MRI and correlated the effects on the tumor and the tumor microenvironment. Within 24 hours of erlotinib treatment we saw approximately 65% tumor regression in mice with TKI-sensitive EGFR L858R lung adenocarcinoma. However, mice with EGFR L858R/T790M -driven tumors did not respond to either erlotinib or afatinib monotherapy, but did show a significant tumor response to afatinib-cetuximab combination treatment. The imaging responses correlated with the inhibition of downstream EGFR signaling, increased apoptosis, and decreased proliferation in the tumor tissues. In EGFR L858R -driven tumors, we saw a significant increase in CD45 + leukocytes, NK cells, dendritic cells, macrophages and lymphocytes, particularly CD8 + T cells. In response to erlotinib, these dendritic cells and macrophages had significantly higher MHC class II expression, indicating increased antigen-presenting capabilities. Together, results of our study provide novel insight into the immediate-early therapeutic response to EGFR TKIs in vivo .

Background KRAS mutations in NSCLC are associated with a lack of response to epidermal growth factor receptor inhibitors. Selumetinib (AZD6244; ARRY-142886) is an oral selective MEK kinase inhibitor of the Ras/Raf/MEK/ERK pathway. Patients and methods Advanced nonsmall-cell lung cancer (NSCLC) patients failing one to two prior regimens underwent KRAS profiling. KRAS wild-type patients were randomized to erlotinib (150 mg daily) or a combination of selumetinib (150 mg daily) with erlotinib (100 mg daily). KRAS mutant patients were randomized to selumetinib (75 mg b.i.d.) or the combination. The primary end points were progression-free survival (PFS) for the KRAS wild-type cohort and objective response rate (ORR) for the KRAS mutant cohort. Biomarker studies of ERK phosphorylation and immune subsets were carried out. Results From March 2010 to May 2013, 89 patients were screened; 41 KRAS mutant and 38 KRAS wild-type patients were enrolled. Median PFS in the KRAS wild-type arm was 2.4 months [95% confidence interval (CI) 1.3–3.7] for erlotinib alone and 2.1 months (95% CI 1.8–5.1) for the combination. The ORR in the KRAS mutant group was 0% (95% CI 0.0% to 33.6%) for selumetinib alone and 10% (95% CI 2.1% to 26.3%) for the combination. Combination therapy resulted in increased toxicities, requiring dose reductions (56%) and discontinuation (8%). Programmed cell death-1 expression on regulatory T cells (Tregs), Tim-3 on CD8+ T cells and Th17 levels were associated with PFS and overall survival in patients receiving selumetinib. Conclusions This study failed to show improvement in ORR or PFS with combination therapy of selumetinib and erlotinib over monotherapy in KRAS mutant and KRAS wild-type advanced NSCLC. The association of immune subsets and immune checkpoint receptor expression with selumetinib may warrant further studies.

Immune checkpoint inhibitors and adoptive lymphocyte transfer–based therapies have shown great therapeutic potential in cancers with high tumor mutational burden (TMB), such as melanoma, but not in cancers with low TMB, such as mutant epidermal growth factor receptor (EGFR)–driven lung adenocarcinoma. Precision immunotherapy is an unmet need for most cancers, particularly for cancers that respond inadequately to immune checkpoint inhibitors. Here, we employed large-scale MS-based proteogenomic profiling to identify potential immunogenic human leukocyte antigen (HLA) class I-presented peptides in melanoma and EGFR-mutant lung adenocarcinoma. Similar numbers of peptides were identified from both tumor types. Cell line and patient-specific databases (DBs) were constructed using variants identified from whole-exome sequencing. A de novo search algorithm was used to interrogate the HLA class I immunopeptidome MS data. We identified 12 variant peptides and several classes of tumor-associated antigen-derived peptides. We constructed a cancer germ line (CG) antigen DB with 285 antigens. This allowed us to identify 40 class I-presented CG antigen–derived peptides. The class I immunopeptidome comprised more than 1000 post-translationally modified (PTM) peptides representing 58 different PTMs, underscoring the critical role PTMs may play in HLA binding. Finally, leveraging de novo search algorithm and an annotated long noncoding RNA (lncRNA) DB, we developed a novel lncRNA-encoded peptide discovery pipeline to identify 44 lncRNA-derived peptides that are presented by class I. We validated tandem MS spectra of select variant, CG antigen, and lncRNA-derived peptides using synthetic peptides and performed HLA class I-binding assays to demonstrate binding to class I proteins. In summary, we provide direct evidence of HLA class I presentation of a large number of variant and tumor-associated peptides in both low and high TMB cancer. These results can potentially be useful for precision immunotherapies, such as vaccine or adoptive cell therapies in melanoma and EGFR-mutant lung cancers., Graphical Abstract, Highlights • Proteogenomics identified ∼35,000 class I-presented peptides. • CG antigen and PTM peptides identified in melanoma and lung cancer. • De novo search identified variant and lncRNA-derived peptides. • A new strategy to identify class I-presented lncRNA-derived peptides developed., In Brief Cancer immunotherapy is ineffective in low TMB EGFR-mutant lung adenocarcinoma. Qi et al. performed a comprehensive proteogenomic profiling of HLA class I-presented immunopeptides in high TMB melanoma and low TMB EGFR-mutant lung cancer. Similar numbers of immunopeptides were identified from both. Variant, CG antigen, PTM, and lncRNA-derived peptides were identified. A novel strategy to identify lncRNA-derived peptides was developed. The direct identification of class I-presented immunopeptides will potentially accelerate precision immunotherapy for low TMB tumors.

SUMMARY Clonal evolution of osimertinib-resistance mechanisms in EGFR mutant lung adenocarcinoma is poorly understood. Using multi-region whole-exome and RNA sequencing of prospectively collected pre- and post-osimertinib-resistant tumors, including at rapid autopsies, we identify a likely mechanism driving osimertinib resistance in all patients analyzed. The majority of patients acquire two or more resistance mechanisms either concurrently or in temporal sequence. Focal copy-number amplifications occur subclonally and are spatially and temporally separated from common resistance mutations such as EGFR C797S. MET amplification occurs in 66% (n = 6/9) of first-line osimertinib-treated patients, albeit spatially heterogeneous, often co-occurs with additional acquired focal copy-number amplifications and is associated with early progression. Noteworthy osimertinib-resistance mechanisms discovered include neuroendocrine differentiation without histologic transformation, PD-L1, KRAS amplification, and ESR1-AKAP12, MKRN1-BRAF fusions. The subclonal co-occurrence of acquired genomic alterations upon osimertinib resistance will likely require targeting multiple resistance mechanisms by combination therapies., In Brief Roper et al. perform multi-region whole-exome and RNA sequencing of pre- and post-resistant tumors from EGFR mutant lung cancer patients treated with osimertinib. They uncover the subclonal co-occurrence of acquired genomic alterations upon osimertinib resistance suggesting combination first-line therapies may prevent or delay key resistance mechanisms., Graphical Abstract

Lung cancer is the leading cause of cancer death in both men and women. Tumor heterogeneity is an impediment to targeted treatment of all cancers, including lung cancer. Here, we sought to characterize tumor proteome and phosphoproteome changes by longitudinal, prospective collection of tumor tissue from an exceptional responder lung adenocarcinoma patient who survived with metastatic lung adenocarcinoma for over seven years while undergoing HER2-directed therapy in combination with chemotherapy. We employed "Super-SILAC" and TMT labeling strategies to quantify the proteome and phosphoproteome of a lung metastatic site and eight distinct metastatic progressive lymph nodes collected during these seven years, including five lymph nodes procured at autopsy. We identified specific signaling networks enriched in lung compared with the lymph node metastatic sites. We correlated the changes in protein abundance with changes in copy number alteration (CNA) and transcript expression. ERBB2/HER2 protein expression was higher in lung, consistent with a higher degree of ERBB2 amplification in lung compared with the lymph node metastatic sites. To further interrogate the mass spectrometry data, a patient-specific database was built by incorporating all the somatic and germline variants identified by whole genome sequencing (WGS) of genomic DNA from the lung, one lymph node metastatic site and blood. An extensive validation pipeline was built to confirm variant peptides. We validated 360 spectra corresponding to 55 germline and 6 somatic variant peptides. Targeted MRM assays revealed two novel variant somatic peptides, CDK12-G879V and FASN-R1439Q, expressed in lung and lymph node metastatic sites, respectively. The CDK12-G879V mutation likely results in a nonfunctional CDK12 kinase and chemotherapy susceptibility in lung metastatic sites. Knockdown of CDK12 in lung adenocarcinoma cells increased chemotherapy sensitivity which was rescued by wild type, but not CDK12-G879V expression, consistent with the complete resolution of the lung metastatic sites in this patient.

SUMMARY Intratumor mutational heterogeneity has been documented in primary non-small-cell lung cancer. Here, we elucidate mechanisms of tumor evolution and heterogeneity in metastatic thoracic tumors (lung adenocarcinoma and thymic carcinoma) using whole-exome and transcriptome sequencing, SNP array for copy-number alterations (CNAs), and mass-spectrometry-based quantitative proteomics of metastases obtained by rapid autopsy. APOBEC mutagenesis, promoted by increased expression of APOBEC3 region transcripts and associated with a high-risk APOBEC3 germline variant, correlated with mutational tumor heterogeneity. TP53 mutation status was associated with APOBEC hypermutator status. Interferon pathways were enriched in tumors with high APOBEC mutagenesis and IFN-γ-induced expression of APOBEC3B in lung adenocarcinoma cells, suggesting that the immune microenvironment may promote mutational heterogeneity. CNAs occurring late in tumor evolution correlated with downstream transcriptomic and proteomic heterogeneity, although global proteomic heterogeneity was significantly greater than transcriptomic and CNA heterogeneity. These results illustrate key mechanisms underlying multi-dimensional heterogeneity in metastatic thoracic tumors., In Brief Roper et al. perform integrated exome, transcriptome, and quantitative proteomics analyses of metastases obtained through rapid autopsy of patients with thoracic malignancies. They identify APOBEC mutagenesis, driven by germline variants, mutant TP53, and the tumor microenvironment, and late copy-number alterations as key mechanisms underlying proteogenomic evolution and heterogeneity., Graphical Abstract

Elucidation of the proteogenomic evolution of metastatic tumors may offer insight into the poor prognosis of patients harboring metastatic disease. We performed whole-exome and transcriptome sequencing, copy number alterations (CNA) and mass spectrometry-based quantitative proteomics of 37 lung adenocarcinoma (LUAD) and thymic carcinoma (TC) metastases obtained by rapid autopsy and found evidence of patient-specific, multi-dimensional heterogeneity. Extreme mutational heterogeneity was evident in a subset of patients whose tumors showed increased APOBEC-signature mutations and expression of APOBEC3 region transcripts compared to patients with lesser mutational heterogeneity. TP53 mutation status was associated with APOBEC hypermutators in our cohort and in three independent LUAD datasets. In a thymic carcinoma patient, extreme heterogeneity and increased APOBEC3AB expression was associated with a high-risk germline APOBEC3AB variant allele. Patients with CNA occurring late in tumor evolution had corresponding changes in gene expression and protein abundance indicating genomic instability as a mechanism of downstream transcriptomic and proteomic heterogeneity between metastases. Across all tumors, proteomic heterogeneity was greater than copy number and transcriptomic heterogeneity. Enrichment of interferon pathways was evident both in the transcriptome and proteome of the tumors enriched for APOBEC mutagenesis despite a heterogeneous immune microenvironment across metastases suggesting a role for the immune microenvironment in the expression of APOBEC transcripts and generation of mutational heterogeneity. The evolving, heterogeneous nature of LUAD and TC, through APOBEC-mutagenesis and CNA illustrate the challenges facing treatment outcomes.

Purpose We conducted a basket clinical trial to assess the feasibility of such a design strategy and to independently evaluate the effects of multiple targeted agents against specific molecular aberrations in multiple histologic subtypes concurrently. Patients and Methods We enrolled patients with advanced non–small-cell lung cancer (NSCLC), small-cell lung cancer, and thymic malignancies who underwent genomic characterization of oncogenic drivers. Patients were enrolled onto a not-otherwise-specified arm and treated with standard-of-care therapies or one of the following five biomarker-matched treatment groups: erlotinib for EGFR mutations; selumetinib for KRAS, NRAS, HRAS, or BRAF mutations; MK2206 for PIK3CA, AKT, or PTEN mutations; lapatinib for ERBB2 mutations or amplifications; and sunitinib for KIT or PDGFRA mutations or amplification. Results Six hundred forty-seven patients were enrolled, and 88% had their tumors tested for at least one gene. EGFR mutation frequency was 22.1% in NSCLC, and erlotinib achieved a response rate of 60% (95% CI, 32.3% to 83.7%). KRAS mutation frequency was 24.9% in NSCLC, and selumetinib failed to achieve its primary end point, with a response rate of 11% (95% CI, 0% to 48%). Completion of accrual to all other arms was not feasible. In NSCLC, patients with EGFR mutations had the longest median survival (3.51 years; 95% CI, 2.89 to 5.5 years), followed by those with ALK rearrangements (2.94 years; 95% CI, 1.66 to 4.61 years), those with KRAS mutations (2.3 years; 95% CI, 2.3 to 2.17 years), those with other genetic abnormalities (2.17 years; 95% CI, 1.3 to 2.74 years), and those without an actionable mutation (1.85 years; 95% CI, 1.61 to 2.13 years). Conclusion This basket trial design was not feasible for many of the arms with rare mutations, but it allowed the study of the genetics of less common malignancies.

Proteogenomic evolution of metastatic tumors results in tumor heterogeneity that influences treatment response and overall prognosis. Here, we perform whole-exome and transcriptome sequencing, SNP array for copy number alterations (CNA) and mass spectrometry-based quantitative proteomics of lung adenocarcinoma (LUAD) and thymic carcinoma (TC) metastases obtained by rapid autopsy to elucidate mechanisms of tumor heterogeneity. APOBEC-mutagenesis driven by APOBEC3 region transcripts strongly correlated with mutational tumor heterogeneity. TP53 mutation status was associated with APOBEC hypermutator status. Interferon pathways were enriched in tumors with high APOBEC mutagenesis. Furthermore, interferon treatment of LUAD cells or immortalized lung epithelial cells in culture upregulated APOBEC3 region transcripts, suggesting a role for the immune microenvironment in the generation of mutational heterogeneity. CNA occurring late in tumor evolution correlated with downstream transcriptomic and proteomic heterogeneity between metastases. These results illustrate key mechanisms underlying multi-dimensional heterogeneity in metastatic thoracic tumors.

Lung cancer causes the highest mortality among all cancers. Patients harboring kinase domain mutations in the epidermal growth factor receptor (EGFR) respond to EGFR tyrosine kinase inhibitors (TKIs), however, acquired resistance always develops. Moreover, 30–40% of patients with EGFR mutations exhibit primary resistance. Hence, there is an unmet need for additional biomarkers of TKI sensitivity that complement EGFR mutation testing and predict treatment response. We previously identified phosphopeptides whose phosphorylation is inhibited upon treatment with EGFR TKIs, erlotinib and afatinib in TKI sensitive cells, but not in resistant cells. These phosphosites are potential biomarkers of TKI sensitivity. Here, we sought to develop modified immuno-multiple reaction monitoring (immuno-MRM)-based quantitation assays for select phosphosites including EGFR-pY1197, pY1172, pY998, AHNAK-pY160, pY715, DAPP1-pY139, CAV1-pY14, INPPL1-pY1135, NEDD9-pY164, NF1-pY2579, and STAT5A-pY694. These sites were significantly hypophosphorylated by erlotinib and a 3rd generation EGFR TKI, osimertinib, in TKI-sensitive H3255 cells, which harbor the TKI-sensitizing EGFR(L858R) mutation. However, in H1975 cells, which harbor the TKI-resistant EGFR(L858R/T790M) mutant, osimertinib, but not erlotinib, could significantly inhibit phosphorylation of EGFR-pY-1197, STAT5A-pY694 and CAV1-pY14, suggesting these sites also predict response in TKI-resistant cells. We could further validate EGFR-pY-1197 as a biomarker of TKI sensitivity by developing a calibration curve-based modified immuno-MRM assay. SIGNIFICANCE: In this report, we have shown the development and optimization of MRM assays coupled with global phosphotyrosine enrichment (modified immuno-MRM) for a list of 11 phosphotyrosine peptides. Our optimized assays identified the targets reproducibly in biological samples with good selectivity. We also developed and characterized quantitation methods to determine endogenous abundance of these targets and correlated the results of the relative quantification with amounts estimated from the calibration curves. This approach represents a way to validate and verify biomarker candidates discovered from large-scale global phospho-proteomics analysis. The application of these modified immuno-MRM assays in lung adenocarcinoma cells provides proof-of concept for the feasibility of clinical applications. These assays may be used in prospective clinical studies of EGFR TKI treatment of EGFR mutant lung cancer to correlate treatment response and other clinical endpoints.

This phase I/II study examined the safety and efficacy of Sepantronium Bromide (S), a small-molecule selective survivin suppressant, administered in combination with carboplatin (C) and paclitaxel (P).Forty-one patients were treated on study. Twenty-two patients received escalating doses of S (3.6-12 mg/m(2)) and 19 with untreated stage IV non-small-cell lung cancer (NSCLC) were treated with the maximum tolerated dose of 10 mg/m(2) in combination with standard doses of C (AUC6) and P (200 mg/m(2)) for six cycles. S was administered as a continuous intravenous infusion (CIVI) over 72 h in 21-day treatment cycles. Study end points included safety and toxic effect, response rate, progression-free and overall survival (PFS and OS), as well as exploratory pharmacodynamic correlates.Treatment with S was well tolerated, and toxic effects were mostly hematological in the phase II study. Two (11%) partial responses were observed with a median PFS of 5.7 months and median OS 16.1 months. Pharmacodynamic analysis did not demonstrate an association with response.The combination of S (10 mg/m(2)/day 72-h CIVI) administered with C and P every 3 weeks exhibited a favorable safety profile but failed to demonstrate an improvement in response rate in advanced NSCLC.NCT01100931.

The development of immune checkpoint inhibitors has altered the landscape of treatment of advanced cancers. These drugs are well tolerated and have shown clinical activity against a wide variety of solid tumors and hematological malignancies. The durability of response is particularly impressive when compared to other forms of systemic therapy. Nivolumab (Opdivo) is an IgG4 antibody that causes immune checkpoint blockade by diminishing inhibitory signaling through the programmed death receptor-1 pathway. It is approved for treatment of recurrent non-small cell lung cancer, melanoma, and renal cell carcinoma. Efforts to identify biomarkers of response to nivolumab are ongoing. Clinical trials are also being conducted to determine the benefits of combining nivolumab with other forms of treatment including chemotherapy, molecular-targeted therapy, radiation therapy, and other forms of immune therapy. This review outlines the clinical trials that have led to the emergence of nivolumab as a treatment option for patients with advanced cancers.

Because of an improved understanding of lung cancer biology and improvement in systemic treatment, an oligometastatic state in which metastatic disease is present at a limited number of anatomic sites is being increasingly recognized. An oligoprogressive state, which is a similar but distinct entity, refers to disease progression at a limited number of anatomic sites, with continued response or stable disease at other sites of disease. Such an oligoprogressive state is best described in patients with NSCLC treated with molecular targeted therapy. Possible explanations for development of the oligoprogressive state include the presence of underlying clonal heterogeneity and extrinsic selection pressure due to the use of targeted therapy. Traditionally, local ablative therapy (LAT) has been limited to symptom palliation in patients with advanced NSCLC, but the presence of oligometastatic or oligoprogressive disease provides a unique opportunity to evaluate the role of LAT such as surgery, radiation therapy, radiofrequency ablation, or cryoablation. There is increasing evidence to support the clinical benefit of LAT in patients with NSCLC with limited metastatic disease and in selected individuals in whom resistance to targeted therapies develops. In the latter instance, adequate treatment of drug-resistant clones by LAT could potentially help in avoiding switching systemic therapy prematurely. This review focuses on the biology of oligometastatic and oligoprogressive NSCLC and describes the role of LAT in the treatment of these conditions.

Background: Osimertinib is effective in EGFR-mutant NSCLC in both first- and second-line settings. However, the efficacy of osimertinib is limited by the universal development of resistance. Retrospective studies have shown a survival benefit to local ablative therapy (LAT) in those who develop oligoprogressive disease (progression at a limited number of anatomic sites) on EGFR-tyrosine kinase inhibitor (EGFR-TKI) therapy. Methods: We initiated a prospective study of LAT in patients with EGFR-mutant NSCLC and oligoprogression during treatment with osimertinib (NCT02759835). Patients with no prior EGFR-TKI therapy (cohort 1) or progression after 1st/2nd generation EGFR-TKI therapy and acquired T790M mutation (cohort 2) start osimertinib. Upon progression, eligible patients with Results: Between 04/2016 and 01/2018, 24 patients were enrolled (cohort 1: 16, cohort 2: 5, cohort 3: 3). Median age was 59.8 (range 36-77). The most common adverse events (AEs) on osimertinib treatment were rash, diarrhea, thrombocytopenia, and QTc prolongation with most of the AEs being grade 1 or 2. Among evaluable patients, confirmed objective response rates prior to LAT in cohorts 1 and 2 were 66.7 % (8/12) and 75% (3/4), respectively, with 11.2 months median PFS (95% CI: 3.6 months-undefined) in cohort 1 and 10.9 months in cohort 2 (95% CI: 6.7-12.8 months). To date, 12 patients had progressive disease, 8 of which had oligoprogression and subsequently underwent LAT (cohort 1: 3; cohort 2: 2; cohort 3: 3). Two patients were treated with combination of surgery and radiotherapy (RT), 4 patients with surgery, and 2 patients with RT. Median PFS2 was 2.3 months (95% CI: 1.0-5.9 months). Whole exome sequencing (WES) was performed on tumor tissues obtained pre-treatment and upon progression on osimertinib. MET amplification, transformation to small cell lung cancer, and EGFR C797S mutation were identified as mechanisms of resistance to osimertinib. Results of molecular analyses of tumor tissue and blood ctDNA will be presented. Conclusions: Patients with EGFR-mutant NSCLC and oligoprogression after EGFR-TKI therapy can be safely treated with LAT. In select patients, this approach could potentially maximize duration of EGFR-TKI treatment and prevent premature switching to other systemic therapies. Citation Format: Chul Kim, Nitin Roper, Chuong Hoang, Laura Wisch, Maureen Connolly, Hsien-Chao Chou, Jun Wei, Manoj Tyagi, Constance M. Cultraro, Liqiang Xi, Maryam Waris, Khoa Dang Nguyen, Eva Szabo, Emerson Padiernos, Aparna Kesarwala, Shaojian Gao, Seth M. Steinberg, Mark Raffeld, Arun Rajan, Javed Khan, Udayan Guha. Local ablative therapy for oligoprogressive, EGFR-mutant, non-small cell lung cancer (NSCLC) after treatment with osimertinib [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 CT106.

Somatic mutations in the EGFR kinase domain drive lung adenocarcinoma. We have previously identified MIG6, an inhibitor of ERBB signaling and a potential tumor suppressor, as a target for phosphorylation by mutant EGFRs. Here, we demonstrate that MIG6 is a tumor suppressor for the initiation and progression of mutant EGFR–driven lung adenocarcinoma in mouse models. Mutant EGFR–induced lung tumor formation was accelerated in Mig6-deficient mice, even with Mig6 haploinsufficiency. We demonstrate that constitutive phosphorylation of MIG6 at Y394/Y395 in EGFR-mutant human lung adenocarcinoma cell lines is associated with an increased interaction of MIG6 with mutant EGFR, which may stabilize EGFR protein. MIG6 also fails to promote mutant EGFR degradation. We propose a model whereby increased tyrosine phosphorylation of MIG6 decreases its capacity to inhibit mutant EGFR. Nonetheless, the residual inhibition is sufficient for MIG6 to delay mutant EGFR–driven tumor initiation and progression in mouse models. Significance: This study demonstrates that MIG6 is a potent tumor suppressor for mutant EGFR–driven lung tumor initiation and progression in mice and provides a possible mechanism by which mutant EGFR can partially circumvent this tumor suppressor in human lung adenocarcinoma. Cancer Discov; 5(5); 534–49. ©2015 AACR. See related commentary by Izumchenko and Sidransky, p. 472. This article is highlighted in the In This Issue feature, p. 453

Background: Molecular characterization of metastatic tumors to investigate intra- and intertumor heterogeneity has proven more difficult than early stage disease as opportunities for surgical removal are limited. Rapid/warm autopsy programs that are conducted soon after death minimize tissue ischemia, thereby enabling procurement of high-quality viable tissue. To our knowledge, integrated proteo-genomics studies to characterize tumor heterogeneity of metastatic lung adenocarcinoma and thymic carcinoma are lacking. Methods: We sought to characterize and decipher mechanisms of intra- and intertumor heterogeneity of Stage IV lung adenocarcinoma and thymic carcinoma through whole exome sequencing, RNA sequencing, copy number estimation, and mass spectrometry-based proteomics of 38 tumors from five rapid/warm autopsy patients. The autopsy procedure was initiated between 2-4 hours of death. Results: One lung adenocarcinoma (RA003) and one thymic carcinoma (RA006) patient had high mutation burden and extreme mutational (SNV) heterogeneity. This was likely driven by APOBEC-mutagenesis. We constructed phylogenetic trees showing truncal, shared, and private SNVs and copy number alterations (CNAs). Interestingly, we discovered a pattern of recurrent, parallel but opposite arm level CNAs in different metastatic branches within a subset of patients (RA000, RA004, and RA006). In contrast, RA003 had only truncal CNAs and the least heterogeneity at the CNA level. Assessment of intra- and intertumor heterogeneity at the transcriptomic and proteomic levels revealed high concordance in gene expression and protein abundance within tumors from patient RA003 compared to patient RA004, potentially reflecting the downstream effect of CNA vs. APOBEC-induced mutations. Further analysis of cis vs. trans effects of CNA on gene expression and protein abundance in all the metastatic tumors revealed many trans-acting hot spots and limited cis effects, which is contrary to copy number effects on gene expression in primary lung adenocarcinoma tumors. Trans-acting hot spots differed between two major metastatic lineage of patient RA004, suggesting copy number changes may be responsible for high intermetastatic proteomic heterogeneity in this patient. Conclusions: Our results demonstrate ongoing mutational and chromosomal instability throughout the metastatic process of lung adenocarcinoma and thymic carcinoma. APOBEC mutagenesis was likely the primary driver of SNV heterogeneity. However, intertumor heterogeneity of metastatic disease at the level of the transcriptome and proteome occurred in association with CNA rather than mutational heterogeneity. APOBEC mutagenesis and CNA are complementary mechanisms of tumor evolution and heterogeneity affecting tumor metastasis. Citation Format: Nitin Roper, James Gao, Rouf Banday, Tapan Maity, Xu Zhang, Abhilash Venugopalan, Rajesh Patidar, Sivasish Sindiri, Constance Cultraro, Alexandr Goncearenco, Anna Panchenko, Romi Biswas, Jing Wang, Stephen Hewitt, David Kleiner, Bing Zhang, Javed Khan, Ludmila Prokunina-Olsson, Udayan Guha. Apobec-mutagenesis drives mutational heterogeneity while copy number alterations drive transcriptomic and proteogenomic heterogeneity in metastatic lung adenocarcinoma and thymic carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A182.

Summary Background No standard treatments are available for advanced thymic epithelial tumours after failure of platinum-based chemotherapy. We investigated the activity of sunitinib, an orally administered tyrosine kinase inhibitor. Methods Between May 15, 2012, and Oct 2, 2013, we did an open-label phase 2 trial in patients with histologically confirmed chemotherapy-refractory thymic epithelial tumours. Patients were eligible if they had disease progression after at least one previous regimen of platinum-containing chemotherapy, an Eastern Cooperative Oncology Group performance status of two or lower, measurable disease, and adequate organ function. Patients received 50 mg of sunitinib orally once a day, in 6-week cycles (ie, 4 weeks of treatment followed by 2 weeks without treatment), until tumour progression or unacceptable toxic effects arose. The primary endpoint was investigator-assessed best tumour response at any point, which we analysed separately in thymoma and thymic carcinoma cohorts. Patients who had received at least one cycle of treatment and had their disease reassessed were included in the analyses of response. The trial was registered with ClinicalTrials.gov, number NCT01621568. Findings 41 patients were enrolled, 25 with thymic carcinoma and 16 with thymoma. One patient with thymic carcinoma was deemed ineligible after enrolment and did not receive protocol treatment. Of patients who received treatment, one individual with thymic carcinoma was not assessable because she died. Median follow-up on trial was 17 months (IQR 14·0–18·4). Of 23 assessable patients with thymic carcinoma, six (26%, 90% CI 12·1–45·3, 95% CI 10·2–48·4) had partial responses, 15 (65%, 95% CI 42·7–83·6) achieved stable disease, and two (9%, 1·1–28·0) had progressive disease. Of 16 patients with thymoma, one (6%, 95% CI 0·2–30·2) had a partial response, 12 (75%, 47·6–92·7) had stable disease, and three (19%, 4·1–45·7) had progressive disease. The most common grade 3 and 4 treatment-related adverse events were lymphocytopenia (eight [20%] of 40 patients), fatigue (eight [20%]), and oral mucositis (eight [20%]). Five (13%) patients had decreases in left-ventricular ejection fraction, of which three (8%) were grade 3 events. Three (8%) patients died during treatment, including one individual who died of cardiac arrest that was possibly treatment-related. Interpretation Sunitinib is active in previously treated patients with thymic carcinoma. Further studies are needed to identify potential biomarkers of activity. Funding National Cancer Institute (Cancer Therapy Evaluation Program).

Lung cancer remains the leading cause of cancer-related deaths worldwide. Epidermal growth factor receptor(EGFR) is the primary driver oncogene in 10-30% lung adenocarcinoma. Kinase domain mutations in EGFR (L858R and E746-A750 deletion) provide sensitivity to EGFR-specific tyrosine kinase inhibitors (TKIs). Despite good tumor inhibitory effects, after approximately a year, patients acquire resistance to EGFR TKIs. We have carried out a stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative mass spectrometry (MS) on a Thermo Orbitrap Elite to identify and characterize the dynamics of tyrosine phosphorylation upon TKI treatment of human lung adenocarcinoma cell lines. The degree of inhibition of phosphorylation of several phospho-sites correlated with the extent of TKI-sensitivity. Several kinases and adaptor proteins that were significantly differentially phosphorylated include AHNAK-Y160, -Y715, STAT5A-Y694, DAPP1-Y139, ERRFI1-Y394, NEDD9-Y164, NF1-Y2579, INNPL1-Y1135, suggesting that these are a subset of potential biomarkers of TKI response. To corroborate our results, the next logical step is to carry out clinical validation of these phospho-sites in human lung adenocarcinoma patients, however no absolute quantification assay is currently available for these tyrosine phosphorylated peptides in human samples. The MS method most often used to complement shotgun methods is selected/multiple reaction monitoring (SRM/MRM) because of its high sensitivity, speed and dynamic range. We have performed SRM/MRM-MS using a nano-flow high performance liquid chromatography instrument coupled to a nano-electrospray ionization source attached to a Triple Quadrupole MS (Agilent 6495). In this method, digested proteins are chromatographically separated and ionized. The resulting precursor ions are m/z filtered in the first quadrupole(Q1) and collision induced dissociation is carried out in Q2 and the resultant product ions are m/z filtered in Q3. Each precursor-product pair is termed as a transition. For absolute quantification, heavy isotope labelled versions of the target peptides are spiked in biological samples to build a calibration curve. For the robust assay development, we have carried out the initial empirical refinement process using the heavy labelled versions of the target peptides, including selection of optimal transitions, normalizing retention times of peptides, running peptide stability assays and digestion time course experiments. We are currently validating these assays in human lung adenocarcinoma cell lines with known EGFR mutations with or without treatment with 1st and 3rd generation EGFR TKIs. The overall goal of these targeted studies is to develop absolute quantitation assays to precisely and accurately quantify these potential biomarkers in clinical samples. Citation Format: Shivangi Awasthi, Xu Zhang, Tapan Maity, Benjamin L. Oyler, David R. Goodlett, Udayan Guha. A SRM/MRM based targeted proteomics strategy for absolute quantification of potential biomarkers of TKI sensitivity in EGFR mutated lung adenocarcinoma [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 2203. doi:10.1158/1538-7445.AM2017-2203

9023 Background: Intratumor heterogeneity has been characterized among multiple cancer types. In lung adenocarcinoma, APOBEC-mutagenesis has been shown to be a source of heterogeneity. However, these data are largely limited to early stage primary tumors. There is limited information about the role of APOBEC-mutagenesis and somatic variants, copy number changes, transcript and protein expression in influencing tumor heterogeneity in metastatic lung adenocarcinoma and other thoracic tumors. Methods: We applied whole exome sequencing, RNA-seq, OncoScan CNV and mass spectrometry-based proteomic analyses on 46 tumor regions from metastatic sites including lung, liver and kidney, obtained by rapid/warm autopsy from 4 patients (pts) with stage IV lung adenocarcinoma, 1 pt each with pleural mesothelioma and thymic carcinoma. The autopsy procedure was initiated between 2-4 hours of death. Results: All tumors displayed organ-specific, branched evolution that was consistent across exome, transcriptome and proteomic analyses. The degree of heterogeneity at the genomic and proteomic level was patient-specific. There was extensive heterogeneity within the tumors of one of four patients with lung adenocarcinoma and in the thymic carcinoma patient (both non-smokers) with multiple driver mutations and copy number changes occurring in only some of the tumors suggesting ongoing late tumor evolution. Further examination of the heterogenous thymic and lung adenocarcinoma tumors showed strong enrichment with the APOBEC-mutagenesis pattern and high associated levels of APOBEC3B mRNA. Conclusions: Metastatic lung adenocarcinoma, thymic carcinoma and mesothelioma evolve through a branched, organ-specific process with marked differences in the acquisition of significant driver mutations and copy number changes. APOBEC3B is a potential driver of heterogeneity in pts with advanced, heterogeneous metastatic lung adenocarcinoma and thymic carcinoma and needs to be evaluated further.

e20545 Background: EGFR tyrosine kinase inhibitors (EGFR-TKIs) improve progression-free survival (PFS) in patients with EGFR-mutant NSCLC, but disease progression limits efficacy. Retrospective studies show a survival benefit to LAT in patients with oligoprogressive disease (progression at a limited number of anatomic sites). Methods: This is a prospective study of LAT in patients with oligoprogressive EGFR-mutant NSCLC. Patients with no prior EGFR-TKI therapy (cohort 1) or progression after 1st/2ndgeneration EGFR-TKIs with acquired T790M mutation (cohort 2) receive osimertinib. Upon progression, eligible patients with < = 5 progressing sites undergo LAT and resume osimertinib until disease progression. Patients previously treated with osimertinib qualifying for LAT upon disease progression are also eligible for treatment (cohort 3). Primary endpoint: evaluation of safety and efficacy of reinitiation of osimertinib after LAT (assessed by PFS). Additional goals are assessment of mechanisms of resistance to osimertinib by multi-omics analyses of tumor, blood, and saliva. Results: Between 04/2016 and 01/2017, 15 patients were enrolled (cohort 1: 9, cohort 2: 3, cohort 3: 3). Median age was 57 (range 36-71). Treatment was well tolerated. The most common adverse events (AEs) were rash, diarrhea, thrombocytopenia, and alanine transaminase elevation. Grade 3/4 AEs were observed in 4 (27%) patients. Among evaluable patients, objective response rates prior to LAT in cohorts 1 and 2 were 71% (5/7) and 100% (2/2), respectively, with 6.8 months median PFS (95% CI: 3.4 months-undefined) in cohort 1 and no progressions in cohort 2. To date, 5 patients (33%; cohort 1: 2; cohort 3: 3) had LAT. Two patients with 3 progressing sites underwent a combination of surgery and radiation. Three patients with 1 progressing site underwent surgery alone. Post-LAT PFS and results of molecular analyses will be presented. Conclusions: Patients with EGFR-mutant NSCLC and oligoprogression after EGFR-TKI therapy can be safely treated with LAT. In selected patients, this approach could potentially maximize duration of EGFR-TKI treatment and prevent premature switching to other systemic therapies. Clinical trial information: NCT02759835.

We used next-generation sequencing to identify somatic alterations in multiple metastatic sites from an “exceptional responder” lung adenocarcinoma patient during his 7-yr course of ERBB2-directed therapies. The degree of heterogeneity was unprecedented, with ∼1% similarity between somatic alterations of the lung and lymph nodes. One novel translocation, PLAG1-ACTA2, present in both sites, up-regulated ACTA2 expression. ERBB2, the predominant driver oncogene, was amplified in both sites, more pronounced in the lung, and harbored an L869R mutation in the lymph node. Functional studies showed increased proliferation, migration, metastasis, and resistance to ERBB2-directed therapy because of L869R mutation and increased migration because of ACTA2 overexpression. Within the lung, a nonfunctional CDK12, due to a novel G879V mutation, correlated with down-regulation of DNA damage response genes, causing genomic instability, and sensitivity to chemotherapy. We propose a model whereby a subclone metastasized early from the primary site and evolved independently in lymph nodes.

Background: The mutually exclusive pattern of the major driver oncogenes in lung cancer suggests that other mutually exclusive oncogenes exist. We conducted a systematic search for tyrosine kinase fusions by screening all tyrosine kinases for aberrantly high RNA expression levels of the 3′ kinase domain (KD) exons relative to more 5′ exons. Methods: We studied 69 patients (including five never smokers and 64 current or former smokers) with lung adenocarcinoma negative for all major mutations in KRAS, EGFR, BRAF, MEK1, HER2, and for ALK fusions (termed “pan-negative”). A NanoString-based assay was designed to query the transcripts of 90 tyrosine kinases at two points: 5′ to the KD and within the KD or 3′ to it. Tumor RNAs were hybridized to the NanoString probes and analyzed for outlier 3′ to 5′ expression ratios. Presumed novel fusion events were studied by rapid amplification of cDNA ends (RACE) and confirmatory reverse transcriptase PCR (RT-PCR) and FISH. Results: We identified one case each of aberrant 3′ to 5′ ratios in ROS1 and RET. RACE isolated a GOPC-ROS1 (FIG-ROS1) fusion in the former and a KIF5B-RET fusion in the latter, both confirmed by RT-PCR. The RET rearrangement was also confirmed by FISH. The KIF5B-RET patient was one of only five never smokers in this cohort. Conclusion: The KIF5B-RET fusion defines an additional subset of lung cancer with a potentially targetable driver oncogene enriched in never smokers with “pan-negative” lung adenocarcinomas. We also report in lung cancer the GOPC-ROS1 fusion originally discovered and characterized in a glioma cell line. Clin Cancer Res; 18(24); 6599–608. ©2012 AACR.

Recent large-scale cancer genome sequencing studies have uncovered extensive diverse mutational landscapes in lung cancer patients. Furthermore, tumor heterogeneity has been widely recognized and it has significant clinical implications in selection of targeted treatment strategies as well as treatment response. Using whole genome sequencing, we demonstrate an unprecedented genomic heterogeneity between sequentially acquired lung and lymph node metastatic sites from an African American never-smoker lung adenocarcinoma patient who has survived with metastatic disease for over seven years while being treated with single or combination HER2-directed therapies. We determined that less than 1% of somatic variants were common between the two tumor sites. Copy number variations were more intense in the lung tumor than in the metastatic lymph node. We identified several novel somatic mutations in key cancer genes in both sites. Interestingly, one novel translocation, PLAG1-ACTA2 was highly expressed in both the lung and lymph node metastases resulting in overexpression of ACTA2, which has been suggested to increase the metastatic potential in lung adenocarcinoma. Using ultra deep targeted re-sequencing, we validated all non-synonymous variants, and approximately 80% of those identified in the metastatic lymph node were also present in a second lymph node biopsied two years after the first one. Although this degree of tumor heterogeneity was surprising, somatic variants affected key hallmarks of tumorigenesis in both sites. These findings suggest a model of early metastatic spread and parallel clonal evolution in disparate metastatic sites. Citation Format: Shaojian Gao, Constance Cultraro, Romi BIswas, Corey A. Carter, Tapan K. Maity, Anish Thomas, Arun Rajan, Paul Meltzer, David Schrump, Giuseppe Giaccone, Javed Khan, Udayan Guha. Whole genome sequencing of sequentially acquired lung and lymph node metastatic sites from a never smoker lung adenocarcinoma patient revealed extensive genomic heterogeneity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4759. doi:10.1158/1538-7445.AM2015-4759

7525 Background: There are no standard treatments for patients with advanced TETs after failure of platinum-based chemotherapy. In thymic carcinoma, KIT over-expression and mutations are found in a...

Epidermal Growth Factor Receptor (EGFR) mutant lung adenocarcinomas are sensitive to the tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib. EGFR is one member of a family of four receptor tyrosine kinases (EGFR, ERBB2, ERBB3 and ERBB4) that can homo- and/or hetero-dimerize leading to activation of the RAS-MAPK and phosphoinositide 3-kinase (PI3K) pathways. In EGFR mutant lung cancer cell lines, ERBB3 has been implicated in activation of the PI3K pathway. However, the requirement for ERBB3 in the initiation, progression and therapeutic response of EGFR mutant tumors remains undetermined. Using genetically engineered mouse models we investigated the contribution of Erbb3 to the development of EGFR-induced lung adenocarcinomas and their response to TKIs. We found that upon conditional deletion of Erbb3 in the lungs of mice that express mutant EGFR tumorigenesis and erlotinib sensitivity were unaffected. In the absence of Erbb3, tumors retained activation of the Pi3k pathway via the adapter proteins Gab1 and Gab2. Acute loss of ERBB3 in human lung adenocarcinoma cell lines with sensitizing EGFR mutations had a modest effect on phosphorylation of AKT and cell viability that varied among cell lines. These data demonstrate that activation of the Pi3k pathway in EGFR mutant tumors is independent of Erbb3 and has implications for the potential of ERBB3 as a therapeutic target in lung cancer. Knowledge of the role EGFR heterodimerization partners play in mutant EGFR-driven lung cancer can help guide the selection of which EGFR family members to target in the treatment of this disease. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C113. Citation Format: Xiaoling Song, Pang-Dian Fan, Udayan Guha, David Threadgill, Harold Varmus, Katerina Politi. ERBB3 independent activation of the PI3K pathway in EGFR mutant lung adenocarcinomas. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C113.

7513 Background: CUSTOM is the first completed prospective clinical trial using molecular selection for treatment assignments into multiple targeted therapy arms and in multiple cancer histological subtypes concurrently. Methods: All patients with advanced NSCLC, SCLC or TM were eligible to participate in the study. Oncogenic mutations, amplifications or translocations in 12 genes detected in CLIA-certified laboratories were used to assign patients to 1 of 5 biomarker/treatment groups per histological subtype: EGFR mutations/erlotinib; KRAS, NRAS, HRAS or BRAF mutations/AZD6244; PIK3CA, AKT or PTEN mutations/MK2206; ERBB2 mutations or amplifications/lapatinib; and KIT or PDGFRA mutations/sunitinib; or to standard-of-care therapy. For each arm, the study was conducted as an optimal two-stage phase II trial in favor of a response rate of 40% or more. Results: 668 patients were enrolled at two academic institutions. The most frequent genetic alterations in NSCLC were KRAS and EGFR mutations (25.2 and 19.7% respectively), ALK rearrangements 7.8%, HER2 amplifications 2.7% and mutations in PIK3CA 2.5%, BRAF 1.9%, HRAS 1.5%, ERBB2 1.7%, AKT1 0.4%, and NRAS 0.7%. PTEN mutation analysis was only feasible in 13 patients with NSCLC of which 3 were positive (23%). The most frequent genetic alterations in SCLC were mutations in PIK3CA 6.5%, ERBB2 amplifications 5.3% and mutations in HRAS 3.4%, AKT1 2.2%, BRAF 2% and KRAS 2%. The most frequent genetic alterations in TMs were HER2 amplifications 7.7% and mutations in HRAS 4.7%, PIK3CA 1.4% and EGFR 1.4%. Only 6.2% (n=42) of patients met criteria for enrollment into the treatment arms of the study. Efficacy analyses including response rates will be presented. Conclusions: CUSTOM is the first completed prospective clinical trial demonstrating the feasibility of conducting efficacy analyses of multiple biomarker-matched therapies in multiple cancer histological subtypes concurrently. CUSTOM is also the largest prospective molecular profiling study of patients with SCLC and TMs. Clinical trial information: NCT01306045.

8026 Background: KRAS mutations are present in 20% of NSCLC and are associated with primary resistance to erlotinib (E). KRAS mutations result in constitutive activation of the Ras/Raf/MEK/ERK pathway. Selumetinib (S) (AZD6244, ARRY-142886) is a selective and uncompetitive inhibitor of MEK kinase. Preclinical studies demonstrated increased activity in NSCLC cell lines using S+E regardless of KRAS status. Methods: Advanced NSCLC patients with progressive disease after platinum based chemotherapy +/- one other treatment were stratified by KRAS status, which was centrally assessed using macrodissection and pyrosequencing for all mutations involving codons 12, 13, and 61. Patients were randomized to receive either the standard of care E or a combination of S+E in KRAS wild type (wt) and S alone or S+E in patients with KRAS mutations. Single agent E and S were administered orally at 150 mg daily and 75 mg twice daily respectively. Combination dosing were S 150 mg every morning and E 100 mg every evening. The primary endpoint for the KRAS wt group was PFS and for the KRAS mutant group objective response rate. Results: From March 2010 to January 2013, 79 patients screened; 78 enrolled: KRAS mutant, 39; KRAS wt, 40; M/F (39:39); median age: 64 years (33-84); median WHO PS 1(0-2); 66 former and 13 never smokers; 67 adenocarcinoma, 9 squamous cell. Three patients died of complications prior to first evaluation (1 coronary disease, 1 pulmonary fibrosis, and 1 disease progression) of which none were related to S. Dose reductions occurred in 5% E, 40% S, and 56% E+S. Most grade 3/4 AEs occurred in combination therapy; diarrhea (23%), fatigue (23%) lymphopenia (13%), myositis (10%), dyspnea (10%), rash (7%). Discontinuation due to AEs was 8% all occurring in S+E cohorts. Conclusions: This study failed to show improvement of combination therapy over single agent in KRAS wt and mutant patients. Toxicity was increased in the combination arms. Interestingly, the KRAS mutant cohort had longer PFS than KRAS wt patients, although not statistically significant (p=0.11). Clinical trial information: NCT01229150. [Table: see text]

Lung cancer is the leading cause of cancer related mortality in the United States and is the most common cancer worldwide. Somatic mutations in the kinase domain of EGFR such as in-frame deletion of LREA amino acids in exon 19 and L858R point mutation in exon 21 largely account for 15-30% of lung adenocarcinoma. Although lung cancer patients carrying either of these mutations respond dramatically to EGFR-directed tyrosine kinase inhibitors (TKIs) such as erlotinib and gefitinib, acquired resistance to these drugs has been the main obstacle in treating these patients for the long term. Secondary mutation at the gatekeeper residue of the kinase domain (T790M) of EGFR accounts for drug resistance in about 50% of patients. We have used doxycycline inducible mutant EGFR transgenic mice to model human lung adenocarcinoma that is sensitive and resistant to TKIs. Our overall goal is to examine the treatment response to EGFR-directed TKIs in lung adenocarcinoma harboring various EGFR mutants. Our approach is aimed to identify imaging surrogates and proteomics profile of treatment response in mouse models of mutant EGFR-driven lung tumorigenesis. In this study, we have used transgenic mice that express exon 19 deletion mutant, L858R mutant, and L858R/T790M double mutant in type II pneumocytes upon doxycycline induction. Tumor burden in these mice was monitored by serial MRI imaging. Each group of mice with significant tumor burden was treated with erlotinib, a reversible EGFR inhibitor, BIBW-2992, an irreversible EGFR/ERBB2 dual inhibitor, or a combination of BIBW-2992 and cetuximab, an antibody directed against EGFR followed by 2[18F]Fluoro-2-deoxy-d-glucose (FDG)-PET imaging to assess any tumor regression. We observed a significant reduction of 18FDG uptake by tumors after the treatment with erlotinib in L858R mutant mice. However, in L858R/T790M mutant mice, no significant reduction of 18FDG uptake was observed upon treatment with erlotinib and BIBW-2992 had only mild effect. We are currently designing rational combination treatments based on the response in 18FDG-PET imaging. Finally, we are performing global phosphoproteomic analysis using quantitative mass spectrometry and immunohistochemistry with phospho-specific antibodies on tumor tissue before and after various treatments and correlating with the response in PET imaging. Results from these experiments will help us identify potential biomarkers (imaging and proteomic) of treatment response in lung adenocarcinoma driven by both TKI-sensitizing and resistant mutants of EGFR. Citation Format: Abhilash Venugopalan, Niu Gang, Jinxia Guo, Tapan Maity, Xu Zhang, Constance Cultraro, Xiaoyuan Chen, Udayan Guha. In vivo imaging and quantitative proteomics to study responsiveness to EGFR targeted therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 725. doi:10.1158/1538-7445.AM2013-725