Multiple Lung Cancer Research Articles

AXL Targeting Overcomes Human Lung Cancer Cell Resistance to NK- and CTL-Mediated Cytotoxicity.

Immune resistance may arise from both genetic instability and tumor heterogeneity. Microenvironmental stresses such as hypoxia and various resistance mechanisms promote carcinoma cell plasticity. AXL, a member of the TAM (Tyro3, Axl, and Mer) receptor tyrosine kinase family, is widely expressed in human cancers and increasingly recognized for its role in cell plasticity and drug resistance. To investigate mechanisms of immune resistance, we studied multiple human lung cancer clones derived from a model of hypoxia-induced tumor plasticity that exhibited mesenchymal or epithelial features. We demonstrate that AXL expression is increased in mesenchymal lung cancer clones. Expression of AXL in the cells correlated with increased cancer cell-intrinsic resistance to both natural killer (NK)- and cytotoxic T lymphocyte (CTL)-mediated killing. A small-molecule targeting AXL sensitized mesenchymal lung cancer cells to cytotoxic lymphocyte-mediated killing. Mechanistically, we showed that attenuation of AXL-dependent immune resistance involved a molecular network comprising NF-κB activation, increased ICAM1 expression, and upregulation of ULBP1 expression coupled with MAPK inhibition. Higher ICAM1 and ULBP1 tumor expression correlated with improved patient survival in two non-small cell lung cancer (NSCLC) cohorts. These results reveal an AXL-mediated immune-escape regulatory pathway, suggest AXL as a candidate biomarker for tumor resistance to NK and CTL immunity, and support AXL targeting to optimize immune response in NSCLC.

A Novel Algorithm to Differentiate Between Multiple Primary Lung Cancers and Intrapulmonary Metastasis in Multiple Lung Cancers With Multiple Pulmonary Sites of Involvement.

Differentiating between multiple primary lung cancer (MPLC) and intrapulmonary metastasis (IPM) is critical for developing a therapeutic strategy to treat multiple lung cancers with multiple pulmonary sites of involvement. We retrospectively included 252 lesions (126 pairs) from 126 patients with surgically resected multiple lung adenocarcinomas. Each pair was classified as MPLC or IPM based on histopathologic findings as the reference standard. A novel algorithm was established with four sequential decision steps based on the combination of computed tomography (CT) lesion types (step 1), CT lesion morphology (step 2), difference of maximal standardized uptake values on positron-emission tomography/CT (step 3), and presence of N2/3 lymph node metastasis or distant metastasis (step 4). The diagnostic accuracy of the algorithm was analyzed. Performances of 11 observers were assessed without and with knowledge of algorithm. Among 126 pairs, 90 (71.4%) were classified as MPLCs and 36 (28.6%) as IPMs. On applying the diagnostic algorithm, the overall accuracy for diagnosis of IPM among conclusive cases up to step 4 was 88.9%, and 65 and 44 pairs were correctly diagnosed based on step 1 and step 2, respectively. Specificity and positive predictive value for diagnosis of IPM increased significantly in all observers compared with reading rounds without the algorithm. Application of the algorithm based on comprehensive information on clinical and imaging variables can allow differentiation between MPLCs and IPMs. When both of two suspected malignant lesions appear as solid predominant lesions without spiculation or air-bronchogram on CT, IPM should be considered.

Prognostic Classification of Multiple Primary Lung Cancers Based on a Ground-Glass Opacity Component

We evaluated the prognostic impact of the presence of a ground-glass opacity (GGO) component on thin-section computed tomography for the refined clinical T classification of multiple primary lung cancers. We reviewed 272 surgically resected, clinically node-negative multiple lung cancers. Dominant tumors were classified into 2 groups based on the presence of a GGO component; that is, a GGO tumor (consolidation tumor ratio, 0 to <1.0) or pure-solid (PS) tumor (consolidation tumor ratio, 1.0). Furthermore, multifocal GGOs (MFGGOs) were defined as lesions showing a GGO component for all tumors. Their prognoses were evaluated using Cox proportional hazard model. There were 153 MFGGOs (56%) with a significantly better 5-year overall survival than non-MFGGOs (97.2% vs 68.5%, P < .001). A multivariable analysis revealed that MFGGO and absence of nodal involvement were independently significant prognosticators of better survival (P= .007 and P= .012, respectively). Furthermore, among the patients of non-MFGGO groups, multivariate analysis showed that a PS+ PS pattern and presence of nodal involvement were independently significant prognosticators of poorer survival (P= .008 and P= .001, respectively). We divided the tumors into 3 groups based on the results and focusing on the presence of a GGO; that is, MFGGO (n= 153), PS+ additional GGO (n= 81), and PS+ PS (n= 38). The 5-year overall survival was clearly split among them: MFGGO, 97.2%; PS+ additional GGO, 82.1%; and PS+ PS, 41.3% (P < .001). Our results suggest that presence of a GGO component has the ability to distinguish the survival even for multiple lung cancers. Further investigations including multicenter trials are certainly warranted to address the revision of T variable of multiple lung cancers considering a presence of GGO component.

Decoupling of Nrf2 Expression Promotes Mesenchymal State Maintenance in Non-Small Cell Lung Cancer.

Epithelial mesenchymal transition is a common mechanism leading to metastatic dissemination and cancer progression. In an effort to better understand this process we found an intersection of Nrf2/NLE2F2 (Nrf2), epithelial mesenchymal transition (EMT), and metabolic alterations using multiple in vitro and in vivo approaches. Nrf2 is a key transcription factor controlling the expression of redox regulators to establish cellular redox homeostasis. Nrf2 has been shown to exert both cancer inhibitory and stimulatory activities. Using multiple isogenic non-small cell lung cancer (NSCLC) cell lines, we observed a reduction of Nrf2 protein and activity in a prometastatic mesenchymal cell state and increased reactive oxygen species. Knockdown of Nrf2 promoted a mesenchymal phenotype and reduced glycolytic, TCA cycle and lipogenic output from both glucose and glutamine in the isogenic cell models; while overexpression of Nrf2 promoted a more epithelial phenotype and metabolic reactivation. In both Nrf2 knockout mice and in NSCLC patient samples, Nrf2low was co-correlated with markedly decreased expression of glycolytic, lipogenic, and mesenchymal RNAs. Conversely, Nrf2high was associated with partial mesenchymal epithelial transition and increased expression of metabolic RNAs. The impact of Nrf2 on epithelial and mesenchymal cancer cell states and metabolic output provide an additional context to Nrf2 function in cancer initiation and progression, with implications for therapeutic inhibition of Nrf2 in cancer treatment.

1448P - Genomic heterogeneity and clonality analysis of multiple synchronous lung cancers (MSLCs)

BackgroundIt is clinically challenging to infer the phylogenetic relationship between different tumor lesions of patient with multiple synchronous lung cancers MSLC: whether these lesions are the result of independently evolved tumor or intrapulmonary metastases. MethodsUsing Illumina X ten platform, we sequenced a total number of 49 lung adenocarcinoma samples collected from 24 patients with MSLC. All samples were analyzed for mutation spectra and phylogenetic inference. Clonality estimation was further carried out to determine the mutational similarity of different tumor lesions. ResultsAmong 24 patients, 17 of them display distinct mutational profile, suggesting these are independently evolved tumors, which is consistent with histopathological assessment. The phylogenetic analysis suggests that multiple cancerous lesions in these patients most likely evolve from different progenitor cells. On the other hand, seven patients were identified to be intrapulmonary metastasis as the mutations harbored in different lesions are clonally related. The mutation spectra of single-nucleotide variations (SNVs) were fairly consistent across different cancerous lesion within all patients. We detected genetic aberrations within genes previously reported to be recurrently altered in lung adenocarcinoma including KRAS, MET, EGFR, TP53, and BRAF. Other identified putative driver mutations are enriched in RTK-RAS signaling, TP53 signaling and cell cycle. ConclusionsOur findings show that, unlike intrapulmonary metastases, patients with MSLC harbor distinct genomic profile in different tumor lesions and we are able to distinguished MSLC from intrapulmonary metastases via clonality estimation. This study delineates the evolutionary trajectories of MSLCS and shed light on better therapeutic strategy and prognosis. Legal entity responsible for the studyThe First Affiliated Hospital of Dalian Medical University. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

ES12.01 Multiple Lung Nodules

Advanced imaging techniques resulted in increased detection of multiple tumors of the lung. Distinguishing synchronous primary lung cancers from intrapulmonary metastases (separate nodules) is important because treatments are very different. In addition, patients with independent primary tumors are expected to have better prognosis. Staging of such tumors as independent primary tumors or intrapulmonary metastases is often challenging, particularly in squamous cell carcinomas. Martini and Melamed modified criteria were used as the main approach for many decades with the idea that morphology of metastases should match the primary tumor, while different morphology supports classification of tumors as unrelated separate primaries. The 8th edition of the AJCC staging of the lung cancer pretty much has replaced this classification by establishing a multidisciplinary approach to these tumors as a standard of care and by promoting the tools such as comprehensive histologic assessment, imaging studies and molecular characterization either by CGH or biomarker testing. Comprehensive histologic assessment is based on the 2015 WHO classification of lung cancers and includes determination of the main histologic tumor type, quantitative subtyping particularly of lung adenocarcinomas, and assessment of cytologic and stromal characteristics. This approach can be relatively easily applied in lung adenocarcinomas, while squamous cell carcinoma remains a great challenge. A recent study conducted by the IASLC Pathology committee showed a good agreement (κ score 0.60) among thoracic pathologists in the histologic assessment of independent primary tumors from intrapulmonary metastasis. Despite a good agreement, there were cases with split opinions supporting a need for ancillary studies. Over the past decade many studies reported different molecular approaches to analysis of multiple lung tumor nodules including DNA microsatellite analysis, CGH/aCHG and most recently next generation sequencing. The data from published reports indicate a highly variable percentage of multifocal tumors identified as clonally related (up to 70%). Discrepancy between clinical and molecular classification of originally presumed cases of multiple primary lung cancers ranged in different series from 18% to 30%. Recent recommendations for routine molecular profiling of lung adenocarcinoma resulted in a widespread use of targeted mutational profiling for oncogenic mutations (i.e. EGFR, KRAS, BRAF etc) and gene rearrangements (i.e.ALK, ROS1) which results can be used in staging of multiple lung cancers. A different mutation profile in oncogenic mutations strongly indicates two separate primary tumors. However, the presence of a common driver mutation does not necessarily indicate tumors of similar origin. Therefore, limited molecular panels may not be sufficient in some cases. The detection of shared identical breakpoints by whole genome sequencing has been recently proposed as potentially more accurate and specific for lineage determination than the analysis of driver mutations alone. Also whole exome and whole genome sequencing approaches have been reported, but these assays may be technically challenging and turnaround time may not be suitable for routine clinical use.

P2.11-26 Survival of Patients with Multiple Primaries and Single Lung Cancer - A Comparative Analysis

From the mid-1980s, lung cancer is the most commonly diagnosed malignant tumor. Longer survival is associated with an increased likelihood of new, subsequent neoplasms. The incidence of multiple primary neoplasms in the oncology population ranges from 0.73 to 11.7%. The aim of the study was to assess the survival of patients with single lung cancer in relation to patients with multiple primary neoplasms. The study was conducted based on the retrospective analysis of data collected from patients hospitalized at the Center for Pulmonary Diseases in Olsztyn (Poland). Data on multiple malignancies (140 patients) were collected from January 2013 to December 2017. Data on single primaries (601 patients) were collected from January 2016 to December 2017. Total survival was calculated beginning from the date of the first cancer diagnosis to the day of death or last observation. The statistical analysis was conducted using Statistica version 12. Based on the collected data, it was revealed that the survival of patients with a single lung cancer (0.17 years; SD 0.81) is significantly shorter than those who had multiple primaries (7.15 years; SD 7.42) (p <0.05). The average survival time of cancer patients with metachronous tumors (8,29 years; SD: 7.58) is significantly longer than patients with synchronous tumors (1.30 years; SD: 1.17) (p <0.05) and single ones (0.71 SD: 0.82) (p<0.05). It was also revealed that patients who smoked in a group of single cancer lived shorter than those with multiple malignancies (p<0.05). The average number of packyears for patients with multiple tumors was 33.95 (SD: 24.95), and with single primaries 39.90 (SD: 21.26).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Survival time for patients with single lung cancer is worse than for patients with multiple tumors, both in relation to the first and subsequent tumors. Smoking reduces survival time of patients with both multiple and single primaries.

P1.03-42 Angiotensin System Inhibitors Improve Survival in Stage IIIA NSCLC and Show Anti-Tumor Effect in Combination with Chemotherapy in a Model of NSCLC

There is increasing interest in the renin-angiotensin system and its influence on tumor microenvironment. This study investigates the anti-tumor effect of angiotensin system inhibitors (ASI) on the multi-modality treatment of stage IIIA NSCLC and examines a potential mechanism in a murine model of lung cancer. We retrospectively reviewed the medical records of 125 patients at a single institution diagnosed with clinical stage IIIA NSCLC who underwent induction chemo-radiotherapy followed by surgery between 2009-2015. Overall survival was compared in patients on or off ASI's. To assess the antitumor efficacy and mechanism of ASI’s, cell culture models of lung cancer were treated with losartan or combination losartan+cisplatin and assessed cell survival. Mechanism was investigated with markers of epithelial-mesenchymal transition or surrogates of other signaling pathways (AKT, Stat3, PD-L1). Losartan was then tested in a murine model of lung cancer. ASI was associated with improved overall survival on multi-variate analysis of patients treated with multi-modality therapy for stage IIIA NSLC (p =0.047) [Figure 1A]. Losartan(≥0.5μM) significantly inhibited proliferation and migration of multiple human lung cancer cells, (H441, H358, H1299, SW1573) and a murine lung cancer adenocarcinoma, TC-1. The combination of Losartan and cisplatin significantly improved the cytotoxic effect of cisplatin in lung cancer cells in vitro (SW1573 p =0.020, TC-1 p =0.012) and reduced the tumor volume of (SW1573-Nude mice, p =0.045 [Figure 1B]; TC-1-C57BL/6 mice, G, p =0.001) in a murine flank tumor model as compared to single drug treatment. Combination treatment also significantly inhibited epithelial to mesenchymal transition and down-regulated the expression of AKT, Stat3, and PD-L1. ASI treatment influences survival in stage IIIA NSCLC. Losartan improves the anti-tumor effect of cytotoxic chemotherapy in lung cancer invitro and significantly reduces tumor burden in a murine lung cancer model. This study suggests a role for ASI therapy in the treatment of lung cancer.

P2.16-28 Retrospective Analysis of Patients with Previous Extrathoracic Malignancies After Resection of Non-Small Cell Lung Cancer

The number of patients with previous malignancies has increased because survival time is prolonged in various malignancies. Therefore, opportunities to consider surgical indications for non-small cell lung cancer (NSCLC) patients with previous malignancies are increasing. The aim of the study is to compare the backgrounds and prognosis of NSCLC patients who had the history of previous extrathoracic malignancies with no other cancer history. Our institutional database of patients after curative resection of NSCLC from 2006 to 2013 was reviewed. Patients with synchronous or metachronous multiple lung cancer were excluded. Clinicopathological features of these patients and information about survival and recurrence were collected from the electronic medical record. Overall survival (OS) and recurrence-free probability (RFP) were estimated using the Kaplan-Meier method. In a total of 249 patients, 49 (19.7%) had previous extrathoracic malignancies. The number of previous extrathoracic malignancies was 1 in 44 patients, 2 in 3 patients and 3 in 1 patient. Sixteen patients had colorectal cancers among previous extrathoracic malignancies, 10 patients had gastric cancers and 5 had hematopoietic neoplasms. If patients with previous extrathoracic malignancies were compared to those without, the median age was 69.5 vs. 65.5 (p=0.015), male sex was 71.4% vs. 66.5% (p=0.506), average of Brinkman index was 914.2 vs. 709.7 (p=0.127) and average of serum level of carcinoembryonic antigen was 5.4 ng/mL vs. 7.5 ng/mL (p=0.506). Pathologically, stage 0 and I NSCLC account for 73.5% of patients with previous extrathoracic malignancies and 66.0% of those without it (p=0.311). The 5-year OS was 70.3% vs. 79.1% (p=0.422) and the 5-year RFP was 58.9% vs. 67.7% (p=0.223). Patients with previous extrathoracic malignancies are significantly older than those without previous extrathoracic malignancies. Patients with previous extrathoracic malignancies had relatively earlier stage NSCLC. The OS and RFP also tended to be worse in patients with previous extrathoracic malignancies. Further study is needed to consider surgical indication for NSCLC with previous extrathoracic malignancies.

EP1.17-01 Surgical Resection of Synchronous Quadruple Primary Lung Tumors Present as Three-Different Cancers and One Benign Mass

Synchronous triple lung cancer is uncommon and little is known, making it difficult to establish proper guidelines or treatment strategies. In this report, we describe a 70-year-old male with three synchronous independent and histologically different primary cancers and one benign hamartoma. Chest CT showed a 30 mm-sized lobulated lung mass in posterior basal segment of the left lower lobe (tumor 1) (Figure 1A). In addition, there were another 26 mm-sized well defined solid nodule in the right lower lobe (tumor 2) (Figure 1A). Two sub-centimeter nodules were also presented in superior segment of the right lower lobe (tumor 3) (Figure 1B) and the lingular segment of the left upper lobe (tumor 4) (Figure 1B). A transthoracic percutaneous needle aspiration biopsy performed on the 30 mm-sized nodule in the left lower lobe revealed it as squamous cell carcinoma. During the right side surgery, intraoperative frozen biopsy of tumor 2 and tumor 4 were reported as chondroid hamartoma and adenocarcinoma, respectively. As we already knew that the left side tumor was squamous cell carcinoma, we decided to continue left side surgery with mediastinal lymph node dissection. The final results of the pathological examination of tumor 1 showed squamous cell carcinoma of pT2bN1M0 (Figure 2A), stage IIB. Tumor 2 was diagnosed as chondroid hamartoma (Figure 2B). Small nodules located in the right lower lobe (tumor 3) and left upper lobe (tumor 4) were 6 mm-sized adenocarcinoma with visceral pleural invasion (pT2aNx) (Figure 2C) and 7 mm-sized adenoid cystic carcinoma (pT1aN0) (Figure 2D), respectively. There was no recurrence during the 3-year follow-up period. Appropriate preoperative staging work up including HRCT and percutaneous needle aspiration biopsy can allow timely detection of synchronous multiple lung cancer, offer proper surgical strategy, and give the possibility of implementing potentially curative treatment for patients conventionally misdiagnosed or considered as metastasized.

Synchronous multiple lung cancers presenting as multifocal pure ground glass nodules: are whole-body positron emission tomography/computed tomography and brain enhanced magnetic resonance imaging necessary?

Multifocal ground glass nodules (GGNs) represent a special radiological pattern indicative of synchronous multiple lung cancers (SMLCs), especially adenocarcinoma. However, the necessity of performing whole-body positron emission tomography/computed tomography (PET-CT) scanning and brain enhanced magnetic resonance imaging (MRI) as a staging workup for multifocal pure GGN (pGGN) patients remains unclear. The purpose of this study was to determine the utility of these two imaging scans for patients with multifocal pGGNs. This retrospective study was reviewed and approved by the ethics committee of the Cancer Hospital of the Chinese Academy of Medical Sciences. The study cohort was retrospectively selected from patients with multifocal pGGNs who underwent whole-body PET-CT examinations and/or brain enhanced MRIs between January 2010 and February 2019 at our institution. The additional value of the two exams for detecting nodal and distant metastases was evaluated. In total, 73 patients (male-to-female ratio, 20:53; median age, 57 years) with multifocal pGGNs who underwent whole-body PET-CT (55 patients) and/or brain enhanced MRI (25 patients) were enrolled. No clearly metastatic lesions were detected. Among the enrolled patients, 53 (128 pGGNs) underwent complete surgical resection. All pGGNs were adenocarcinomas and/or preneoplasias, and no lymph node metastases were found on final pathology. Whole-body PET-CT and brain enhanced MRI added no definite benefit compared with chest CT alone before surgery. Whole-body PET-CT scans and brain enhanced MRIs are not necessary for patients with multifocal pGGNs.

THE RESULTS OF SURGICAL TREATMENT OF PATIENTS WITH MULTIPLE PRIMARY LUNG CANCERS ACCORDING TO ORENBURG REGIONAL ONCOLOGICAL CLINIC DATA

Aim. To assess the immediate and long-term results of treating patients with bilateral lung cancer (LC). Patients and methods. Radically operated 5 patients with primary multiple synchronous LC (PMSLC) and 5 patients with primary multiple metachronous LC (PMMLC) were included in the study. Clinical and morphological data, results of instrumental studies, immediate and long-term treatment results were analyzed. Statistical analysis was performed using the Statistica6 software. Results. Tumors corresponding to T1‑T2 prevailed in both groups. At the same time, both in the PMSLC and PMMLC group, only in one case both tumors corresponded to T1–2N0M0, in the others — one or both tumors were with metastases in lymph nodes of the root or mediastinum. In early postoperative period, the number of surgical complications was 10%, somatic — 20%. All complications were successfully treated conservatively. Postoperative mortality was not observed. In PMMLC and PMSLC cases, one-year relapse-free survival (RFS) was 100% and 80%; two-year — 80% and 60%; three-year — 60% and 60%; four-year — 60% and 20%, respectively. The five-year RFS in patients with PMMPC was 40%. One patient with PMSLC was alive without relapse of disease for 4.5 years. Accordingly, one-year overall survival (OS) was 100% and 100%; two-year — 80% and 80%; three-year — 80% and 60%; four-year — 80% and 40%. Five- and six-year OS in patients with PMMLC were 60% and 20%, respectively. Conclusion. The absence of postoperative mortality and low number of postoperative complications indicate the safety of bilateral operations in patients with bilateral LC. Low rates of patients’ 5‑year survival are connected with problems of primary diagnostics and low efficiency of existing algorithms of regular medical checkup of patients with this pathology. To improve long-term results of LC treatment, it is necessary to more actively introduce the modern programs of dynamic observation and complex examination of patients, undergone surgery for LC, using the annual chest CT and fibrobronchoscopy.

A Case of Multiple Lung Cancer Harboring Different Mutations in EML4-ALK and EGFR

A Case of Multiple Lung Cancer Harboring Different Mutations in EML4-ALK and EGFR

Abstract 2731: A novel PDE10/beta-catenin inhibitor, MCI-048, suppresses lung tumorigenesis to block metastasis

Abstract We previously reported that phosphodiesterase 10A (PDE10) is overexpressed during early stages of lung cancer and is essential for lung tumor cell growth. Here we characterize a novel PDE10 inhibitor, MCI-048, that was identified by screening a library of indene compounds. MCI-048 potently inhibited the growth and induced apoptosis of multiple human lung cancer cell lines expressing PDE10, while normal human airway epithelial cells lacking PDE10 expression were appreciably less sensitive. The mechanism of action of MCI-048 involves PDE10 inhibition, cGMP elevation, PKG activation, and phosphorylation of β-catenin at key residues that induce ubiquitination and proteosomal degradation of the oncogenic pool of β-catenin in cytoplasm to suppress the translocation of active β-catenin to the nucleus and Lef/Tcf-mediated transcription of genes encoding for proteins such as c-myc, cyclin D, and survivin, which are essential for tumor cell proliferation and survival. Pharmacokinetic and tissue distribution studies revealed a unique feature of MCI-048 to accumulate at high concentrations in lungs relative to plasma and other tissues. To assess the potential of MCI-048 for the treatment of lung cancer and blocking metastasis, we tested the drug in two mouse models of lung cancer involving either orthotopic implantation of KRAS mutant A549 lung tumor cells or the chemical carcinogen, urethane. Oral administration of MCI-048 significantly inhibited tumor growth and extended survival in the orthotopic model using two different protocols to either treat the primary tumor or metastasis. Similarly, MCI-048 significantly reduced tumor burden as measured by both surface counting and histopathological analysis in the urethane-induced model of lung tumorigenesis. Biochemical analysis showed that MCI-048 reduced levels of urethane-induced active Ras-GTP and PDE10 levels, as well as other oncogenic markers, including pEGFR and c-Myc. Both mouse models revealed that MCI-048 was well tolerated with no discernable toxicity, thus supporting preclinical development for the treatment of lung cancer. Funding provided by NCI grants R21CA182941, R01CA131378, R01CA148817, R01CA197147, and R01CA155638. Citation Format: Bing Zhu, Veronica Ramirez-Alcantara, Antonio Ward, Kristy Berry, Adam B. Keeton, Michael R. Boyd, Yulia Maxuitenko, Xi Chen, Gary A. Piazza. A novel PDE10/beta-catenin inhibitor, MCI-048, suppresses lung tumorigenesis to block metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2731.

Abstract 4342: Direct genome editing of patient-derived xenografts for rapid in vivo functional genomics

Abstract Patient-derived xenografts (PDXs) are high fidelity in vivo tumor models that have become valuable tools for cancer research, particularly in the study of emerging cancer therapeutics. However, PDXs are rarely used in mechanistic and functional genomic studies, due in part to the technical barriers imposed by continuous passaging in vivo. We sought to develop a CRISPR-Cas9 platform that would leverage the robust nature of PDXs and enable in vivo functional genomic studies in these cancer models. To this end, we created pSpCTRE-CD4, an all-in-one doxycycline-inducible Cas9 lentiviral vector, which features a novel truncated CD4 selection marker that enables selection of transduced PDX cells by flow cytometry, bypassing the need to culture these cells ex vivo. The inducible TRE3GS promoter controlling Cas9 expression is in the reverse orientation, which reduces potential leaky expression of Cas9 and also places this promoter in close proximity to the constitutive EFS promoter controlling CD4 and rtTA expression. Interestingly, this promoter orientation causes a dramatic ~100-fold increase in CD4 expression over basal levels in the presence of doxycycline and thus provides an indicator for cells that induce Cas9. This property of pSpCTRE-CD4 allows for the study of heterogeneous tumor cell populations without the need to select for single cell clones, which is a common practice in inducible cell line systems. In vitro functional studies of pSpCTRE-CD4 showed that this vector is tightly regulated by doxycycline. We transduced 27 lung cancer PDXs with pSpCTRE-CD4 lentivirus and observed transduction efficiencies ranging from 0-30%. Of these, we successfully enriched 8 Cas9 PDX models derived from multiple lung cancer subtypes. We functionally validated these Cas9 PDXs by introducing an sgRNA targeting the essential gene RPA1 in an in vivo clonal competition assay. For each of the Cas9 PDXs tested, we observed a significant decrease in the sgRPA1:sgNon-targeting ratio for two independent RPA1 sgRNAs in mice treated with doxycycline, indicating a loss of fitness in these PDXs when RPA1 is depleted. Similarly, in JHU-LX55a-Cas9, a KRAS mutant lung adenocarcinoma PDX, we observed a significant decrease in the fitness of cells with an sgRNA targeting KRAS. We are using this system to investigate genetic dependencies within these PDXs and have designed a small sgRNA library targeting the druggable genome to perform CRISPR screens in these models. Additionally, we have designed a recombinant AAV vector that expresses a single sgRNA as well as template to introduce point mutations via homology-directed repair and we are using this system to interrogate variants of unknown significance identified in large-scale tumor sequencing studies. The CRISPR-Cas9 platform we have developed will greatly expand the use of PDXs and enable functional genomic studies to be performed in this robust in vivo model system. Citation Format: Christopher H. Hulton, Emily A. Costa, Charles M. Rudin, John T. Poirier. Direct genome editing of patient-derived xenografts for rapid in vivo functional genomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4342.

Abstract 1200: AXL targeting enhances lymphocyte-mediated cytotoxicity of lung cancer cells

Abstract As immunotherapies are now used to treat a large proportion of NSCLC patients, defining mechanisms of immune resistance is critical. Immune resistance may arise from both genetic instability and tumor heterogeneity driven by microenvironmental stresses such as hypoxia that promotes carcinoma cell plasticity as well as extrinsic or intrinsic mechanisms of immune resistance. AXL, a member of the TAM receptor tyrosine kinase family is widely expressed human cancers and increasingly recognized for its role in cell plasticity and drug resistance. In this study, we used a model of hypoxia-induced tumor plasticity to generate multiple lung cancer clones with mesenchymal and epithelial features to address mechanisms of immune resistance. We demonstrate that AXL expression is dramatically increased in mesenchymal lung cancer clones. Moreover, expression of AXL in the cells was correlated with an increased cancer cell intrinsic resistance to both NK and CTL-mediated killing, Notably, small molecule AXL targeting potently sensitized mesenchymal lung cancer cells to cytotoxic lymphocyte-mediated killing. Mechanistically, we showed that attenuation of AXL-dependent immune resistance to immune cells involved a novel molecular network comprising NF-κB activation, increased ICAM1 expression, and upregulation of ULBP1 expression coupled with MAPK inhibition. Congruently, higher ICAM1 and ULBP1 tumor expression, correlated with improved patient survival in two NSCLC cohorts. These results reveal a novel AXL-mediated immune escape regulatory pathway, suggest AXL as a novel candidate biomarker for tumor resistance to NK and CTL immunity, and support AXL targeting to optimize immune response in NSCLC. Citation Format: Sallem Chouaib, Stephane Terry, Stephanie Buart, Agnete Engelsen, Gro Gausdal, James Lorens, Jean Paul Thiery, Fathia Mami-Chouaib. AXL targeting enhances lymphocyte-mediated cytotoxicity of lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1200.

Electronic Health Record-Based Screening for Major Cancers: A 9-Year Experience in Minhang District of Shanghai, China.

Background: An electronic health record (e-HR) system has been developed in Minhang District of Shanghai, China, since 2005, making it convenient for local health institutions to provide integrative and comprehensive health care and management for major diseases.Methods: In 2008, an e-HR-based cancer prevention program was initiated to screen multiple cancers, including colorectal, gastric, liver, lung, cervical, and breast cancers, and provide subsequent health education and health management to cancer patients and high-risk individuals. This study was designed in prospective analysis, based on the constructive analysis of key information, observation of cancer screening and healthcare processes and organizations, and stages of cancers detected by the e-HR-based programs.Results: From 2008 to 2016, health education was conducted for over 5 million attendances, and more than 3 million screening tests were performed for eligible residents over 40 years old. A total of 2,948 cancer cases were detected, accounting for 13.3% of all newly diagnosed cancers in the district during the 9-year period. Thirty point seven percent detected cancer cases were at the early stage, significantly higher than the 22.9% in cases identified by e-HR-based follow-up and 13.8% in cases diagnosed due to signs or symptoms. More than 136,000 residents were identified as individuals at high risk of cancer and subject to sustainable clinical follow-up and health management.Conclusions: The successful application of e-HR system in cancer prevention and control in Minhang district of Shanghai, China, implies that the system may act as an extendable and sustainable infrastructure for comprehensive health care and services for a broad spectrum of diseases and health events.

Using Genomics to Differentiate Multiple Primaries From Metastatic Lung Cancer

IntroductionGenomic technologies present a promising mechanism of resolving the clinical dilemma of distinguishing independent primary tumors from intrapulmonary metastases in NSCLC. We evaluated the utility of discordant mapping somatic junctions from chromosomal rearrangements in diagnosing metastatic disease compared to the current standard histologic review. Material and MethodsMate-pair sequencing was performed on DNA extracted from 76 distinct tumors from 37 cases of multiple lung cancers. Discordant mapping junctions and chromosomal copy levels were assessed for each tumor. Blood-derived DNA was available on 22 of these cases for germline assessments. A lung cancer next-generation sequencing panel was additionally performed on tumor pairs from 17 patients. ResultsWhereas mate-pair sequencing was able to classify lineage in all tumor pairs, histologic review appeared to misclassify lineage in 9 of 33 (27%) same-histology tumor pair comparisons. Based on disagreement between the reviewing pathologists, histopathologic lineage was classified as indeterminate in seven cases. In two cases where pathologists agreed on a metastatic call, no shared junctions were found suggesting independent primaries. Although germline junctions passing algorithmic filters were common, on average less than three were present and all had predictable structures of small focal rearrangements or transposons. Evaluation of shared chromosomal copy changes and driver mutations through a lung cancer next-generation sequencing panel, while informative, were nondefinitive in calling lineage in all cases. ConclusionsThe highly unique nature and prevalence of chromosomal rearrangement in lung cancers provide a useful and definitive technique for calling lineage in multifocal lung cancer.

In silico prediction of housekeeping long intergenic non-coding RNAs reveals HKlincR1 as an essential player in lung cancer cell survival

Prioritising long intergenic noncoding RNAs (lincRNAs) for functional characterisation is a significant challenge. Here we applied computational approaches to discover lincRNAs expected to play a critical housekeeping (HK) role within the cell. Using the Illumina Human BodyMap RNA sequencing dataset as a starting point, we first identified lincRNAs ubiquitously expressed across a panel of human tissues. This list was then further refined by reference to conservation score, secondary structure and promoter DNA methylation status. Finally, we used tumour expression and copy number data to identify lincRNAs rarely downregulated or deleted in multiple tumour types. The resulting list of candidate essential lincRNAs was then subjected to co-expression analyses using independent data from ENCODE and The Cancer Genome Atlas (TCGA). This identified a substantial subset with a predicted role in DNA replication and cell cycle regulation. One of these, HKlincR1, was selected for further characterisation. Depletion of HKlincR1 affected cell growth in multiple lung cancer cell lines, and led to disruption of genes involved in cell growth and viability. In addition, HKlincR1 expression was correlated with overall survival in lung adenocarcinoma patients. Our in silico studies therefore reveal a set of housekeeping noncoding RNAs of interest both in terms of their role in normal homeostasis, and their relevance in tumour growth and maintenance.

Clinicopathologic Characteristics and Prognosis of Patients With Non–Small Cell Lung Cancer Who Undergo Pulmonary Segmentectomy

This study retrospectively investigated the clinical characteristics and prognosis of the non–small cell lung cancer (NSCLC) patients who underwent segmentectomy as either intentional or passive limited surgery. The subjects included in this study comprised 72 patients who underwent segmentectomy at our hospital. There were 23 patients who underwent intentional segmentectomy. The histologic type was adenocarcinoma in all of these patients. Regarding the tumor size, 9 tumors were 1 cm or less in diameter, 13 were 1.1 to 2 cm, and 1 was 2.5 cm. All patients who underwent intentional segmentectomy were alive without any recurrence as of the last follow-up examination. A passive segmentectomy was performed for 49 patients. The reasons for passive segmentectomy were due to the presence of metachronous multiple lung cancer in 14 patients, synchronous multiple lung cancer in 9 patients, age above 80 years in 12 patients, reduced pulmonary function in 8 patients, and moderate to severe cardiovascular disease in 4 patients; another 2 patients were receiving treatment for another primary cancer. The 5-year survival rate of the patients who underwent passive segmentectomy was 69.8%. There was no postoperative mortality in either of the groups. This study showed that all of the patients who underwent intentional segmentectomy were alive without any recurrence, thus suggesting that the procedure is associated with sufficient oncologic efficacy, along with preservation of the lung function. The passive segmentectomy was a feasible procedure and was an alternative procedure with an acceptable overall survival rate in high-risk patients with underlying medical comorbidities.