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BRCA1-associated protein 1 (BAP1) is a potent tumour suppressor gene that modulates environmental carcinogenesis. All carriers of inherited heterozygous germline BAP1-inactivating mutations (BAP1 +/-) developed one and often several BAP1 -/- malignancies in their lifetime, mostly malignant mesothelioma, uveal melanoma, and so on. Moreover, BAP1-acquired biallelic mutations are frequent in human cancers. BAP1 tumour suppressor activity has been attributed to its nuclear localization, where it helps to maintain genome integrity. The possible activity of BAP1 in the cytoplasm is unknown. Cells with reduced levels of BAP1 exhibit chromosomal abnormalities and decreased DNA repair by homologous recombination, indicating that BAP1 dosage is critical. Cells with extensive DNA damage should die and not grow into malignancies. Here we discover that BAP1 localizes at the endoplasmic reticulum. Here, it binds, deubiquitylates, and stabilizes type 3 inositol-1,4,5-trisphosphate receptor (IP3R3), modulating calcium (Ca 2+) release from the endoplasmic reticulum into the cytosol and mitochondria, promoting apoptosis. Reduced levels of BAP1 in BAP1 +/- carriers cause reduction both of IP3R3 levels and of Ca 2+ flux, preventing BAP1 +/- cells that accumulate DNA damage from executing apoptosis. A higher fraction of cells exposed to either ionizing or ultraviolet radiation, or to asbestos, survive genotoxic stress, resulting in a higher rate of cellular transformation. We propose that the high incidence of cancers in BAP1 +/- carriers results from the combined reduced nuclear and cytoplasmic activities of BAP1. Our data provide a mechanistic rationale for the powerful ability of BAP1 to regulate gene-environment interaction in human carcinogenesis.

Carriers of heterozygous germline BAP1 mutations (BAP1+/−) develop cancer. We studied plasma from 16 BAP1+/− individuals from 2 families carrying different germline BAP1 mutations and 30 BAP1 wild-type (BAP1WT) controls from these same families. Plasma samples were analyzed by liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS), ultra-performance liquid chromatography triple quadrupole mass spectrometry (UPLC-TQ-MS), and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). We found a clear separation in the metabolic profile between BAP1WT and BAP1+/− individuals. We confirmed the specificity of the data in vitro using 12 cell cultures of primary fibroblasts we derived from skin punch biopsies from 12/46 of these same individuals, 6 BAP1+/− carriers and 6 controls from both families. BAP1+/− fibroblasts displayed increased aerobic glycolysis and lactate secretion, and reduced mitochondrial respiration and ATP production compared with BAP1WT. siRNA-mediated downregulation of BAP1 in primary BAP1WT fibroblasts and in primary human mesothelial cells, led to the same reduced mitochondrial respiration and increased aerobic glycolysis as we detected in primary fibroblasts from carriers of BAP1+/− mutations. The plasma and cell culture results were highly reproducible and were specifically and only linked to BAP1 status and not to gender, age or family, or cell type, and required an intact BAP1 catalytic activity. Accordingly, we were able to build a metabolomic model capable of predicting BAP1 status with 100% accuracy using data from human plasma. Our data provide the first experimental evidence supporting the hypothesis that aerobic glycolysis, also known as the ‘Warburg effect’, does not necessarily occur as an adaptive process that is consequence of carcinogenesis, but rather that it may also predate malignancy by many years and facilitate carcinogenesis.

BRCA1-associated protein-1 (BAP1) mutations cause a new cancer syndrome, with a high rate of malignant mesothelioma (MM). Here, we tested the hypothesis that MM associated with germline BAP1 mutations has a better prognosis compared with sporadic MM. We compared survival among germline BAP1 mutation MM patients with that of all MM (N = 10 556) recorded in the United States Surveillance, Epidemiology, and End Results (SEER) data from 1973 to 2010. We identified 23 MM patients--11 alive--with germline BAP1 mutations and available data on survival. Ten patients had peritoneal MM, ten pleural MM and three MM in both locations. Thirteen patients had one or more malignancies in addition to MM. Actuarial median survival for the MM patients with germline BAP1 mutations was 5 years, as compared with1 year for the median survival in the United States SEER MM group. Five-year survival was 47%, 95% confidence interval (24-67%), as compared with 6.7% (6.2-7.3%) in the control SEER group. Analysis of the pooled cohort of germline BAP1 mutation MM showed that patients with peritoneal MM (median survival of 10 years, P = 0.0571), or with a second malignancy in addition to MM (median survival of 10 years, P = 0.0716), survived for a longer time compared with patients who only had pleural MM, or MM patients without a second malignancy, respectively. In conclusion, we found that MM patients with germline BAP1 mutations have an overall 7-fold increased long-term survival, independently of sex and age. Appropriate genetic counseling and clinical management should be considered for MM patients who are also BAP1 mutation carriers.

Mediastinal node involvement is the primary determinant for IIIA disease in NSCLC. It is locally advanced disease and curable, but remains a significant treatment challenge. There is no single treatment paradigm appropriate for all patients. The role for surgery is dictated by the ability to perform an R0 resection and by the extent of mediastinal node involvement. For most patients with N2 disease, induction therapy is recommended and survival is closely linked to the response to that treatment. Many believe that a trimodality approach using a combination of chemotherapy, radiation, and surgery provides the best hope for cure, but safety and success is highly dependent on careful patient selection and meticulous treatment delivery.

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Introduction In the Western world, malignant mesothelioma (MM) is most prevalent in the pleura of older males who have been professionally exposed to asbestos. Information about MM from rapidly industrializing countries such as China is minimal. There is concern that a proportion of MM diagnoses in China may be incorrect because most Chinese physicians do not have experience diagnosing this rare cancer. We recently reported an unusually high incidence of peritoneal MM among eastern Chinese female patients. Here, we review the accuracy of MM diagnoses in China and provide suggestions to improve the accuracy of diagnosis. Methods We reviewed 92 pathological diagnosis of MM in 2002–2015 from two reference centers in the province of Zhejiang in eastern China. We performed a large set of immunohistochemistry analyses to increase the reliability of the diagnosis. Results We confirmed the MM diagnosis in 12 of 34 of the pleural tumors (35.3%), in 38 of 56 of the peritoneal tumors (67.9%), and in two of two of the MMs of the tunica vaginalis (100%). MMs were characterized by tumor cells showing nuclear Wilms tumor 1 and calretinin staining and by strong membranous staining for cytokeratin CAM5.2. The results of staining for the epithelial markers carcinoembryonic antigen, thyroid transcription factor-1, MOC31, BerEP4, p63, p40, paired box 8, ER and PR were negative. BRCA1 associated protein 1 nuclear staining was lost in percentages similar to what has been reported for samples from Western countries. Conclusions Our findings suggest that MM—especially in its pleural localization—is often misdiagnosed in eastern China. Identifying pitfalls and possible solutions in the pathological diagnosis of MM will affect both the standard of care and research in China.

Introduction Desmoplastic mesothelioma (DMM), a rare histological subtype of malignant pleural mesothelioma (MPM), is lethal and has very poor prognosis. Metastasis is commonly reported in DMM compared to other histological subtypes. Here we report a case of DMM with good survival (>1 year) despite lymph node metastasis. Case Report A 62-year-old female with a history of smoking and possible asbestos exposure presented with cough and wheeze in 2012. Computed tomography (CT) revealed left-sided pleural plaques and multiple ground-glass pulmonary nodules. In 2017, repeat CT showed increased diffuse left nodular pleural thickening. Positron emission tomography revealed hypermetabolic, nodular pleural thickening in the hemithorax, compatible with neoplasia. Needle biopsy in January 2018 showed pleural tissue with haphazard, nodular growth of spindled mesothelial cells, suspicious for desmoplastic mesothelioma. Definitive diagnosis was not possible due to lack of fat invasion and absence of supportive evidence by immunohistochemical stains. The patient underwent pleurectomy in February 2018. On histopathology, the majority of the tumor showed a desmoplastic pattern, composed of malignant spindled cells arranged haphazardly in a dense hyalinized stroma with chronic inflammatory infiltrate. AE1/AE3, calretinin, and D2-40 immunohistochemical stains highlighted the infiltrating neoplastic cells, which were negative for WT-1. BAP-1 was retained. The pattern of immunoreactivity supported the diagnosis of DMM. The tumor also involved visceral pleura, pulmonary parenchyma, and pericardium. It invaded into fat and displayed lymphovascular invasion. A metastatic focus of DMM was present in a lymph node. The tumor was staged as pT3N1. On recent examination, the patient only had complaints of mild breathlessness and stable hydro-pneumothorax, without any other comorbidities. Conclusion Our finding is unusual, since among the mesothelioma subtypes, DMM has the shortest survival, usually not more than 6 months. We report an extremely rare case of DMM with survival of >1 year despite invasion of lung parenchyma and lymph node metastasis.

The emergence of immune checkpoint inhibitors has changed the treatment paradigm for NSCLC. Recently, immunotherapy in combination with chemotherapy was shown to be a promising first-line treatment strategy for patients with squamous cell lung cancer, where targeted therapy has had little efficacy due to the pathological driver mutations for this disease being poorly understood. However, for both adenocarcinoma and squamous cell NSCLC, use of such immune checkpoint inhibitors has shown to improve the outcome for only a subset of patients with advanced or metastatic disease. Understanding the diversity in the tumor immune microenvironment will be critical to not only advance immune oncology research but to also determine potential clinical distinctions that dictate therapeutic response. Here, we take a multifaceted approach to compare the tumor immune microenvironments of over 50 patients diagnosed with either adenocarcinoma or squamous cell NSCLC. To this end, we performed a comprehensive multicolor flow cytometry analysis to compare tumor immune infiltrates and expression of suppressive markers to adjacent normal lung tissue as well as peripheral blood immune cells from the same patient. This analysis showed differences in the CD4+ and CD8+ T cell, CD19+ B cell, NK cell, and CD14+/CD16+ myeloid cell populations between the tumor and matched tissue and blood. These immune profiles were then compared between adenocarcinoma and squamous cell carcinoma subtypes as well as early versus late stage disease. We then integrated these findings with multiplexed immunofluorescence analysis on a panel of immune cell markers. Finally, 10x genomics single-cell RNA sequencing (scRNAseq) data were used to analyze single-cell transcriptomes from both adenocarcinoma and squamous cell lung tumors. Our result showed great heterogeneity of immune infiltrates across different patient samples, including different subtypes and different disease stages. This detailed comparative analysis will help us further understand the involvement of tumor infiltrating immune cells for different subtypes of lung cancer. Citation Format: Kayla Harmeyer, Jiehui Deng, Suhagi Shah, Ece Bagdatlioglu, Aarif Ahsan, Christopher Reid, Aatish Thennavan, Charles Perou, Kwok-Kin Wong, Harvey Pass. Profiling the tumor immune microenvironment of adenocarcinoma and squamous cell NSCLC [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 4563.

Background: Malignant mesothelioma (MM) is a rare but deadly malignancy with about 3,000 new cases being diagnosed each year in the US. Very few studies have been performed to analyze factors associated with mesothelioma survival, especially for peritoneal presentation. The overarching aim of this study is to examine survival of the cohort of patients with malignant mesothelioma enrolled in the National Mesothelioma Virtual Bank (NMVB). Methods: 888 cases of pleural and peritoneal mesothelioma cases were selected from the NMVB database, which houses data and associated biospecimens for over 1400 cases that were diagnosed from 1990 to 2017. Kaplan Meier’s method was performed for survival analysis. The association between prognostic factors and survival was estimated using Cox Hazard Regression method and using R software for analysis. Results: The median overall survival (OS) rate of all MM patients, including pleural and peritoneal mesothelioma cases is 15 months (14 months for pleural and 31 months for peritoneal). Significant prognostic factors associated with improved survival of malignant mesothelioma cases in this NMVB cohort were younger than 45, female gender, epithelioid histological subtype, stage I, peritoneal occurrence, and having combination treatment of surgical therapy with chemotherapy. Combined surgical and chemotherapy treatment was associated with improved survival of 23 months in comparison to single line therapies. Conclusions: There has not been improvement in the overall survival for patients with malignant mesothelioma over many years with current available treatment options. Our findings show that combined surgical and chemotherapy treatment in peritoneal mesothelioma is associated with improved survival compared to local therapy alone.

Nickel (Ni) compounds are widely used in industrial and commercial products including household and cooking utensils, jewelry, dental appliances and implants. Occupational exposure to nickel is associated with an increased risk for lung and nasal cancers, is the most common cause of contact dermatitis and has an extensive effect on the immune system. The purpose of this study was two-fold: (i) to evaluate immune response to the occupational exposure to nickel measured by the presence of anti-glycan antibodies (AGA) using a new biomarker-discovery platform based on printed glycan arrays (PGA), and (ii) to evaluate and compile a sequence of bioinformatics and statistical methods which are specifically relevant to PGA-derived information and to identification of putative “Ni toxicity signature”. The PGAs are similar to DNA microarrays, but contain deposits of various carbohydrates (glycans) instead of spotted DNAs. The study uses data derived from a set of 89 plasma specimens and their corresponding demographic information. The study population includes three subgroups: subjects directly exposed to Nickel that work in a refinery, subjects environmentally exposed to Nickel that live in a city where the refinery is located and subjects that live in a remote location. The paper describes the following sequence of nine data processing and analysis steps: (1) Analysis of inter-array reproducibility based on benchmark sera; (2) Analysis of intra-array reproducibility; (3) Screening of data - rejecting glycans which result in low intra-class correlation coefficient (ICC), high coefficient of variation and low fluorescent intensity; (4) Analysis of inter-slide bias and choice of data normalization technique; (5) Determination of discriminatory subsamples based on multiple bootstrap tests; (6) Determination of the optimal signature size (cardinality of selected feature set) based on multiple cross-validation tests; (7) Identification of the top discriminatory glycans and their individual performance based on nonparametric univariate feature selection; (8) Determination of multivariate performance of combined glycans; (9) Establishing the statistical significance of multivariate performance of combined glycan signature. The above analysis steps have delivered the following results: inter-array reproducibility ρ=0.920 ± 0.030; intraarray reproducibility ρ=0.929 ± 0.025; 249 out of 380 glycans passed the screening at ICC>80%, glycans in selected signature have ICC ≥ 88.7%; optimal signature size (after quantile normalization)=3; individual significance for the signature glycans p=0.00015 to 0.00164, individual AUC values 0.870 to 0.815; observed combined performance for three glycans AUC=0.966, p=0.005, CI=[0.757, 0947]; specifity=94.4%, sensitivity=88.9%; predictive (crossvalidated) AUC value 0.836.

Malignant mesothelioma is strongly associated with asbestos exposure. Among asbestos fibers, crocidolite is considered the most and chrysotile the least oncogenic. Chrysotile accounts for more than 90% of the asbestos used worldwide, but its capacity to induce malignant mesothelioma is still debated. We found that chrysotile and crocidolite exposures have similar effects on human mesothelial cells. Morphological and molecular alterations suggestive of epithelial–mesenchymal transition, such as E-cadherin down-regulation and β-catenin phosphorylation followed by nuclear translocation, were induced by both chrysotile and crocidolite. Gene expression profiling revealed high-mobility group box-1 protein (HMGB1) as a key regulator of the transcriptional alterations induced by both types of asbestos. Crocidolite and chrysotile induced differential expression of 438 out of 28,869 genes interrogated by oligonucleotide microarrays. Out of these 438 genes, 57 were associated with inflammatory and immune response and cancer, and 14 were HMGB1 targeted genes. Crocidolite-induced gene alterations were sustained, whereas chrysotile-induced gene alterations returned to background levels within 5 weeks. Similarly, HMGB1 release in vivo progressively increased for 10 or more weeks after crocidolite exposure, but returned to background levels within 8 weeks after chrysotile exposure. Continuous administration of chrysotile was required for sustained high serum levels of HMGB1. These data support the hypothesis that differences in biopersistence influence the biological activities of these two asbestos fibers.

Introduction: Malignant pleural mesothelioma (MM) is an aggressive asbestos-associated malignancy with limited therapeutic options. This study describes the overexpression of Ephrin B2 receptor (EPHB2) in MM cell lines and tumors, and the effect of its manipulation on proliferative and invasive qualities of the disease. Methods: Using expression arrays, we investigated EPHB2 in MM tumors compared with normal mesothelium. EPHB2 and downstream target expression were evaluated using reverse-transcriptase polymerase chain reaction and immunoblotting methods. The biological significance of EPHB2 in MM was evaluated using in vitro functional assays with and without targeting by EPHB2-short hairpin RNA or blocking peptide in two mesothelioma cell lines, HP-1 and H2595. Results: EPHB2 is overexpressed in all MM cell lines, but not in benign mesothelial cells, and is significantly elevated in MM tumor tissue compared with matched normal peritoneum. Targeted knockdown of EPHB2 in HP-1 and H2595 cell lines reduced its expression and that of EPHB2 downstream targets such as matrix metalloproteinase (MMP-2) and vascular endothelial growth factor, whereas caspase 2 and caspase 8 had increased expression. Inhibition of EPHB2 resulted in a significant decrease in scratch closure (1.25-fold–1.8-fold), proliferation (1.5-fold), and invasion (1.7-fold–1.8-fold) compared with the controls. Most notably, however, EPHB2 silencing resulted in a significant increase in apoptotic proteins and activity. Conclusion: EPHB2 seems to play an important role in MM pathogenesis and these findings indicate that EPHB2 could serve as a potential novel therapeutic target for treatment of the disease.

DR. DONINGTON: Thank you for being here. The idea is to give the readers an idea of how you approach superior sulcus tumors, since you are some of the most accomplished surgeons in the country at these complex resections. So we will start with just some overview. How many of these cases do you see a year? Do you think you are seeing more or seeing less? DR. SWISHER: It’s just a hard disease to know for sure because it’s such a referral-based disease, and it goes up and down, and everybody in our group at MD Anderson Cancer Center sees these patients. I get a sense it’s going down, but I don’t know for sure. DR. BAINS: Personally, I may see a couple of them, 2 or 3 at the very least, but as a whole group, we may see 5-10 a year, but closer to 5. DR. VAILLERES: But I think it fluctuates. If you asked me that question last year, I think we did 2, and we have already done 3 this year. So I think, it comes and goes. I am not sure if the incidence is going down or not. I think it is, but I am not sure. And if it’s going down, why? Are we picking up the apical upper lobar lesions before they start invading into structures? I have no idea. DR. SWISHER: I agree, it is variable. I am also concerned because these tumors, for the most part, are tumors that should be treated at highvolume centers, and it remains to be seen how this disease is going to be affected by the changes in health care reform. It is an orphan disease since so few patients have it, but to impact it, we need to treat these patients in high-volume, multidisciplinary approaches.

Mesothelioma is diagnosed in ∼2500 patients in the United States every year, most often arising in the pleural space, but also occurring as primary peritoneal mesothelioma. The vast majority of patients with mesothelioma die of their disease within 3 years. We developed a new mouse model of mesothelioma by bladder or intraperitoneal injection of adenovirus Cre into mice with conditional alleles of each of Tp53 and Tsc1. Such mice began to develop malignant ascites about 6 months after injection, which was due to peritoneal mesothelioma, on the basis of tumor morphology and immunohistochemical staining. Mesothelioma cell lines were established, which showed loss of both Tsc1 and Tp53, with mammalian target of rapamycin complex (mTORC)1 activation. Treatment of mice with malignant ascites due to mesothelioma with rapamycin led to a marked reduction in ascites, extended survival and a 95-99% reduction in the mesothelioma tumor volume, in comparison with vehicle-treated mice. To see whether TSC1/TSC2 loss was a common genetic event in human mesothelioma, we examined nine human mesothelioma cell lines, and found that four of nine showed persistent activation of mTORC1, although none had loss of TSC1 or TSC2. A tissue microarray analysis of 198 human mesothelioma specimens showed that 33% of cases had reduced TSC2 expression and 60% showed activation of mTOR, indicating that mTOR activation is common in human mesothelioma, suggesting that it is a potential therapeutic target.

The search for biomarkers in the management of lung cancer involves the use of multiple platforms to examine changes in gene, protein, and microRNA expression. Multiple studies have been published in an attempt to describe early detection, diagnostic, prognostic, and predictive biomarkers using chiefly tissues and blood elements. Studies are characterized by a lack of commonality of specific biomarkers, and a lack of validated, clinically useful markers. The future of biomarker discovery as a means of tailoring therapy for patients with lung cancer will involve next-generation sequencing along with collaborative efforts to integrate and validate candidate markers.

INTRODUCTION: As CT screening is integrated into non-small cell lung cancer (NSCLC) care, additional parameters are needed to help distinguish cancers from benign nodules. Osteopontin (OPN), a secreted phosphoprotein, has elevated plasma levels in NSCLC. We hypothesize that changes in plasma OPN over time (i.e., OPN velocity [OPNV]) can differentiate NSCLC patients from those without cancer in a CT screening population. METHODS:A nested case-control study was conducted within a NSCLC CT screening trial. Incident cancers with serial plasma were matched to controls. OPN was measured by ELISA. Demographic, OPN, and OPNV were compared between cancers and controls using Wilcoxon Signed Rank tests. RESULTS: Ten incident cancers were identified. The pack years distributions were similar, but cancers were older (median of the paired difference: 5.35 years; p = 0.002) and their surveillance intervals were shorter (median of the paired difference: �2 months; p = 0. 03) than matched controls. Baseline OPN was similar (median of the paired difference: �5.15 ng/ml, p = 0.50), but OPNV in the cancers was significantly greater than that of matched controls, (median of the paired difference: 1.06 ng/ml/month, p = 0.01). Accuracy rate for prediction of disease status based on OPNV (adjusted for age and surveillance) was 83%. CONCLUSIONS: These are early evidence for utility of monitoring plasma OPN during CT screening to assist in identification of NSCLCs.

We used a custom-made comparative genomic hybridization array (aCGH; average probe interval 254 bp) to screen 33 malignant mesothelioma (MM) biopsies for somatic copy number loss throughout the 3p21 region (10.7 Mb) that harbors 251 genes, including BRCA1 (breast cancer 1)-associated protein 1 (BAP1), the most commonly mutated gene in MM. We identified frequent minute biallelic deletions (

Introduction: The M component and TNM stage groupings for malignant pleural mesothelioma (MPM) have been empirical. The International Association for the Study of Lung Cancer developed a multinational database to propose evidence-based revisions for the eighth edition of the TNM classification of MPM. Methods: Data from 29 centers were submitted either electronically or by transfer of existing institutional databases. The M component as it currently stands was validated by confirming sufficient discrimination (by Kaplan-Meier analysis) with respect to overall survival (OS) between the clinical MO (cM0) and cM1 categories. Candidate stage groups were developed by using a recursive partitioning and amalgamation algorithm applied to all cM0 cases. Results: Of 3519 submitted cases, 2414 were analyzable and 84 were cM1 cases. Median OS for cM1 cases was 9.7 months versus 13.4 months (p = 0.0013) for the locally advanced (T4 or N3) cM0 cases, supporting inclusion of only cM1 in the stage IV group. Exploratory analyses suggest a possible difference in OS for single- versus multiple-site cM1 cases. A recursive partitioning and amalgamation generated survival tree on the OS outcomes restricted to cM0 cases with the newly proposed (eighth edition) T and N components indicates that optimal stage groupings for the eighth edition will be as follows: stage IA (T1N0), stage IB (T2-3N0), stage II (T1-2N1), stage IIIA (T3N1), stage IIIB (T1-3N2 or any T4), and stage IV (any M1). Conclusions: This first evidence-based revision of the TNM classification for MPM leads to substantial changes in the T and N components and the stage groupings. (C) 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Mutations in the epidermal growth factor receptor gene (EGFR) represent one of the most frequent "actionable" alterations in non-small cell lung cancer (NSCLC). Typified by high response rates to targeted therapies, EGFR tyrosine kinase inhibitors (TKIs) are now established first-line treatment options and have transformed the treatment paradigm for NSCLC. With the recent breakthrough designation and approval of the third-generation EGFR TKI osimertinib, available systemic and local treatment options have expanded, requiring new clinical algorithms that take into account individual patient molecular and clinical profiles. In this International Association for the Study of Lung Cancer commissioned consensus statement, key pathologic, diagnostic, and therapeutic considerations, such as optimal choice of EGFR TKI and management of brain metastasis, are discussed. In addition, recommendations are made for clinical guidelines and research priorities, such as the role of repeat biopsies and use of circulating free DNA for molecularstudies. With the rapid pace of progress in treating EGFR-mutant NSCLC, this statement provides a state-of-the-art review of the contemporary issues in managing this unique subgroup of patients.

Cancer Biomarkers 16 (2016) 609–617 DOI 10.3233/CBM-160602 IOS Press Serum phosphatidylethanolamine levels distinguish benign from malignant solitary pulmonary nodules and represent a potential diagnostic biomarker for lung cancer Johannes F. Fahrmann a,1 , Dmitry Grapov b,1 , Brian C. DeFelice a , Sandra Taylor c , Kyoungmi Kim c , Karen Kelly d , William R. Wikoff a , Harvey Pass e , William N. Rom f , Oliver Fiehn a,g and Suzanne Miyamoto d,∗ a University of California, Davis Genome Center, Davis, California, CA, USA CDS Creative Data Solutions, Ballwin, MO, USA c Division of Biostatistics, Department of Public Health Sciences, School of Medicine, University of California, Davis, California, CA, USA d Division of Hematology and Oncology, Department of Internal Medicine, School of Medicine, University of California , Davis Medical Center, Sacramento, CA, USA e Division of Thoracic Surgery, Department of Cardiothoracic Surgery, Langone Medical Center, New York University, New York, NY, USA f Division of Pulmonary, Critical Care, and Sleep, NYU School of Medicine, New York, NY, USA g Department of Biochemistry, Faculty of Sciences, King Abdulaziz University, Jeddah, Saudi-Arabia b Abstract. BACKGROUND: Recent computed tomography (CT) screening trials showed that it is effective for early detection of lung cancer, but were plagued by high false positive rates. Additional blood biomarker tests designed to complement CT screening and reduce false positive rates are highly desirable. OBJECTIVE: Identify blood-based metabolite biomarkers for diagnosing lung cancer. MEHTODS: Serum samples from subjects participating in a CT screening trial were analyzed using untargeted GC-TOFMS and HILIC-qTOFMS-based metabolomics. Samples were acquired prior to diagnosis (pre-diagnostic, n = 17), at-diagnosis (n = 25) and post-diagnosis (n = 19) of lung cancer and from subjects with benign nodules (n = 29). RESULTS: Univariate analysis identified 40, 102 and 30 features which were significantly different between subjects with malignant (pre-, at- and post-diagnosis) solitary pulmonary nodules (SPNs) and benign SPNs, respectively. Ten metabolites were consistently different between subjects presenting malignant (pre- and at-diagnosis) or benign SPNs. Three of these 10 metabolites were phosphatidylethanolamines (PE) suggesting alterations in lipid metabolism. Accuracies of 77%, 83% and 78% in the pre-diagnosis group and 69%, 71% and 67% in the at-diagnosis group were determined for PE(34:2), PE(36:2) and PE(38:4), respectively. CONCLUSIONS: This study demonstrates evidence of early metabolic alterations that can possibly distinguish malignant from benign SPNs. Further studies in larger pools of samples are warranted. Keywords: Lung cancer, metabolomics, phospholipids, biomarkers, solitary pulmonary nodules 1 These authors contributed equally to this work. author: Suzanne Miyamoto, Division of Hema- tology/Oncology, UC Davis Cancer Center, 4501 X Street, Room ∗ Corresponding 3016, Sacramento, CA 95817, USA. Tel.: +1 916 734 3769; Fax: +1 916 734 5415; E-mail: smiyamoto@ucdavis.edu. c 2016 – IOS Press and the authors. All rights reserved ISSN 1574-0153/16/$35.00

Background Inflammatory cytokines are at the intersection of tumor cell biology and host immune response. Peripheral cytokine expression levels may reflect the microscopic tumor milieu, and specific cytokines play an integral role in tumor initiation, growth, and metastasis. High-throughput cytokine analysis may identify panels for early-stage non-small cell lung cancer (NSCLC) diagnosis and identify individuals at high-risk for lung cancer with indeterminate lung nodules 8-20 mm in size. Methods Thirteen serum cytokines from the NYU Lung Cancer Biomarker Center cohort with early-stage NSCLC were analyzed using bead-based immunoassay technology. Results In the NYU cohort, a one unit increase in interferon-γ increased risk of lung cancer by 3% (OR = 1.03, 95% CI, 1.02-1.05) and a one unit increase in TNF-α decreased the risk of lung cancer by 53% (OR = 0.47, 95% Cl, 0.31-0.71) when both cytokines were included in a logistic regression model with adjustments for age and pack-years of smoking. The resulting AUC for the Receiver Operating Characteristic (ROC) curve was 0.88; the sensitivity and specificity at the optimal cutpoint were 78.9% and 90.3%, respectively. Conclusions Cytokines have limited value in the early diagnosis of early-stage NSCLC. Our review of the literature suggests that although inflammation is important for the development of NSCLC, that cytokines are increased in more advanced lung cancer than when the diagnosis occurs at presentation.

Introduction: The current T component for malignant pleural mesothelioma (MPM) has been predominantly informed by surgical data sets and consensus. The International Association for the Study of Lung Cancer undertook revision of the seventh edition of the staging system for MPM with the goal of developing recommendations for the eighth edition. Methods: Data elements including detailed T descriptors were developed by consensus. Tumor thickness at three pleural levels was also recorded. An electronic data capture system was established to facilitate data submission. Results: A total of 3519 cases were submitted to the database. Of those eligible for T-component analysis, 509 cases had only clinical staging, 836 cases had only surgical staging, and 642 cases had both available. Survival was examined for T categories according to the current seventh edition staging system. There was clear separation between all clinically staged categories except T1a versus T1b (hazard ratio = 0.99, p = 0.95) and T3 versus T4 (hazard ratio = 1.22, p = 0.09), although the numbers of T4 cases were small. Pathological staging failed to demonstrate a survival difference between adjacent categories with the exception of T3 versus T4. Performance improved with collapse of T1a and T1b into a single T1 category; no current descriptors were shifted or eliminated. Tumor thickness and nodular or rindlike morphology were significantly associated with survival. Conclusions: A recommendation to collapse both clinical and pathological T1a and T1b into a T1 classification will be made for the eighth edition staging system. Simple measurement of pleural thickness has prognostic significance and should be examined further with a view to incorporation into future staging. (C) 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

International audience; Similar to asbestos fibers, nonregulated mineral fibers can cause malignant mesothelioma (MM). Recently, increased proportions of women and young individuals with MM were identified in southern Nevada, suggesting that environmental exposure to carcinogenic fibers was causing the development of MM. Palygorskite, a fibrous silicate mineral with a history of possible carcinogenicity, is abundant in southern Nevada. In this study, our aim was to determine whether palygorskite was contributing to the development of MM in southern Nevada. While palygorskite, in vitro, displayed some cytotoxicity toward primary human mesothelial (HM) cells and reduced their viability, the effects were roughly half of those observed when using similar amounts of crocidolite asbestos. No Balb/c (0/19) or MexTAg (0/18) mice injected with palygorskite developed MM, while 3/16 Balb/c and 13/14 MexTAg mice injected with crocidolite did. Lack of MM development was associated with a decreased acute inflammatory response, as injection of palygorskite resulted in lower percentages of macrophages (p=.006) and neutrophils (p=.02) in the peritoneal cavity 3 d after exposure compared to injection of crocidolite. Additionally, compared to mice injected with crocidolite, palygorskite-injected mice had lower percentages of M2 (tumor-promoting) macrophages (p=.008) in their peritoneal cavities when exposed to fiber for several weeks. Our study indicates that palygorskite found in the environment in southern Nevada does not cause MM in mice, seemingly because palygorskite, in vivo, fails to elicit inflammation that is associated with MM development. Therefore, palygorskite is not a likely contributor to the MM cases observed in southern Nevada.

A B S T R AC T BACKGROUND New biomarkers are needed to detect pleural mesothelioma at an earlier stage and to individualize treatment strategies. We investigated whether fibulin-3 in plasma and pleural effusions could meet sensitivity and specificity criteria for a robust biomarker. METHODS We measured fibulin-3 levels in plasma (from 92 patients with mesothelioma, 136 asbestos-exposed persons without cancer, 93 patients with effusions not due to mesothelioma, and 43 healthy controls), effusions (from 74 patients with mesothelioma, 39 with benign effusions, and 54 with malignant effusions not due to mesothelioma), or both. A blinded validation was subsequently performed. Tumor tissue was examined for fibulin-3 by immunohistochemical analysis, and levels of fibulin-3 in plasma and effusions were measured with an enzyme-linked immunosorbent assay. RESULTS Plasma fibulin-3 levels did not vary according to age, sex, duration of asbestos exposure, or degree of radiographic changes and were significantly higher in patients with pleural mesothelioma (105±7 ng per milliliter in the Detroit cohort and 113±8 ng per milliliter in the New York cohort) than in asbestos-exposed persons without mesothelioma (14±1 ng per milliliter and 24±1 ng per milliliter, respectively; P

Human malignant mesothelioma is an aggressive and highly lethal cancer that is believed to be caused by chronic exposure to asbestos and erionite. Prognosis for this cancer is generally poor because of late-stage diagnosis and resistance to current conventional therapies. The damage-associated molecular pattern protein HMGB1 has been implicated previously in transformation of mesothelial cells. Here we show that HMGB1 establishes an autocrine circuit in malignant mesothelioma cells that influences their proliferation and survival. Malignant mesothelioma cells strongly expressed HMGB1 and secreted it at high levels in vitro. Accordingly, HMGB1 levels in malignant mesothelioma patient sera were higher than that found in healthy individuals. The motility, survival, and anchorage-independent growth of HMGB1-secreting malignant mesothelioma cells was inhibited in vitro by treatment with monoclonal antibodies directed against HMGB1 or against the receptor for advanced glycation end products, a putative HMGB1 receptor. HMGB1 inhibition in vivo reduced the growth of malignant mesothelioma xenografts in severe-combined immunodeficient mice and extended host survival. Taken together, our findings indicate that malignant mesothelioma cells rely on HMGB1, and they offer a preclinical proof-of-principle that antibody-mediated ablation of HMBG1 is sufficient to elicit therapeutic activity, suggesting a novel therapeutic approach for malignant mesothelioma treatment. Cancer Res; 72(13); 3290–301. ©2012 AACR.

Purpose Mesothelin is currently considered the best available serum biomarker of malignant pleural mesothelioma. To examine the diagnostic accuracy and use of serum mesothelin in early diagnosis, we performed an individual patient data (IPD) meta-analysis. Methods The literature search identified 16 diagnostic studies of serum mesothelin, measured with the Mesomark enzyme-linked immunosorbent assay. IPD of 4,491 individuals were collected, including several control groups and 1,026 patients with malignant pleural mesothelioma. Mesothelin levels were standardized for between-study differences and age, after which the diagnostic accuracy and the factors affecting it were examined with receiver operating characteristic (ROC) regression analysis. Results At a common diagnostic threshold of 2.00 nmol/L, the sensitivities and specificities of mesothelin in the different studies ranged widely from 19% to 68% and 88% to 100%, respectively. This heterogeneity can be explained by differences in study population, because type of control group, mesothelioma stage, and histologic subtype significantly affected the diagnostic accuracy. The use of mesothelin in early diagnosis was evaluated by differentiating 217 patients with stage I or II epithelioid and biphasic mesothelioma from 1,612 symptomatic or high-risk controls. The resulting area under the ROC curve was 0.77 (95% CI, 0.73 to 0.81). At 95% specificity, mesothelin displayed a sensitivity of 32% (95% CI, 26% to 40%). Conclusion In patients suspected of having mesothelioma, a positive blood test for mesothelin at a high-specificity threshold is a strong incentive to urge further diagnostic steps. However, the poor sensitivity of mesothelin clearly limits its added value to early diagnosis and emphasizes the need for further biomarker research.

ObjectiveIntegrin-linked kinase, which is relevant to neoplastic transformation, is highly expressed in malignant pleural mesothelioma. Recently, detection of integrin-linked kinase in serum of patients with ovarian cancer has been reported. This study asks whether integrin-linked kinase can also be detected in serum of patients with malignant pleural mesothelioma and whether serum level has diagnostic or prognostic relevance for that disease.MethodsA sandwich enzyme-linked immunosorbent assay was designed to detect integrin-linked kinase and applied to serum samples from 46 patients with malignant pleural mesothelioma, 98 patients with other malignant chest disease, and 23 patients with benign chest disease. Integrin-linked kinase serum concentration and clinical data were correlated statistically.ResultsMedian serum integrin-linked kinase concentration was significantly higher in malignant pleural mesothelioma (8.89 ng/mL) than in other malignant chest disease (0.66 ng/mL) or benign chest disease (0.78 ng/mL, P

Because only a small fraction of asbestos-exposed individuals develop malignant mesothelioma, and because mesothelioma clustering is observed in some families, we searched for genetic predisposing factors. We discovered germline mutations in the gene encoding BRCA1 associated protein-1 (BAP1) in two families with a high incidence of mesothelioma, and we observed somatic alterations affecting BAP1 in familial mesotheliomas, indicating biallelic inactivation. In addition to mesothelioma, some BAP1 mutation carriers developed uveal melanoma. We also found germline BAP1 mutations in 2 of 26 sporadic mesotheliomas; both individuals with mutant BAP1 were previously diagnosed with uveal melanoma. We also observed somatic truncating BAP1 mutations and aberrant BAP1 expression in sporadic mesotheliomas without germline mutations. These results identify a BAP1-related cancer syndrome that is characterized by mesothelioma and uveal melanoma. We hypothesize that other cancers may also be involved and that mesothelioma predominates upon asbestos exposure. These findings will help to identify individuals at high risk of mesothelioma who could be targeted for early intervention.

Purpose: Malignant pleural mesothelioma (MPM) is an aggressive tumor with a dismal prognosis. Unlike other malignancies, TP53 mutations are rare in MPM. Recent studies have showed that altered expression of microRNA (miRNA) is observed in human malignant tumors. In this study, we investigated the alterations of miR-34s, a direct transcriptional target of TP53, and the role of miR-34s on the pathogenesis of MPM. Experimental Design: Aberrant methylation and expression of miR-34s were examined in MPM cell lines and tumors. miR-34b/c was transfected to MPM cells to estimate the protein expression, cell proliferation, invasion, and cell cycle. Results: Aberrant methylation was present in 2 (33.3%) of 6 MPM cell lines and 13 (27.7%) of 47 tumors in miR-34a and in all 6 MPM cell lines (100%) and 40 (85.1%) of 47 tumors in miR-34b/c. Expression of miR-34a and 34b/c in all methylated cell lines was reduced and restored with 5-aza-2′-deoxycytidine treatment. Because epigenetic silencing was the major event in miR-34b/c, we investigated the functional role of miR-34b/c in MPM. miR-34b/c–transfected MPM cells with physiologic miR-34b/c expression exhibited antiproliferation with G1 cell cycle arrest and suppression of migration, invasion, and motility. The forced overexpression of miR-34b/c, but not p53, showed a significant antitumor effect with the induction of apoptosis in MPM cells. Conclusions: We show that the epigenetic silencing of miR-34b/c by methylation is a crucial alteration and plays an important role in the tumorigenesis of MPM, suggesting potential therapeutic options for MPM. Clin Cancer Res; 17(15); 4965–74. ©2011 AACR.

Malignant pleural mesothelioma (MPM) is an aggressive, asbestos-related malignancy of the thoracic pleura. Although, platinum-based agents are the first line of therapy, there is an urgent need for second-line therapies to treat the drug-resistant MPM. Cell cycle as well as apoptosis pathways are frequently altered in MPM and thus remain attractive targets for intervention strategies. Curcumin, the major component in the spice turmeric, alone or in combination with other chemotherapeutics has been under investigation for a number of cancers. In this study, we investigated the biological and molecular responses of MPM cells to curcumin treatments and the mechanisms involved. Flow-cytometric analyses coupled with western immunoblotting and gene-array analyses were conducted to determine mechanisms of curcumin-dependent growth suppression of human (H2373, H2452, H2461, and H226) and murine (AB12) MPM cells. Curcumin inhibited MPM cell growth in a dose- and time-dependent manner while pretreatment of MPM cells with curcumin enhanced cisplatin efficacy. Curcumin activated the stress-activated p38 kinase, caspases 9 and 3, caused elevated levels of proapoptotic proteins Bax, stimulated PARP cleavage, and apoptosis. In addition, curcumin treatments stimulated expression of novel transducers of cell growth suppression such as CARP-1, XAF1, and SULF1 proteins. Oral administration of curcumin inhibited growth of murine MPM cell-derived tumors in vivo in part by stimulating apoptosis. Thus, curcumin targets cell cycle and promotes apoptosis to suppress MPM growth in vitro and in vivo. Our studies provide a proof-of-principle rationale for further in-depth analysis of MPM growth suppression mechanisms and their future exploitation in effective management of resistant MPM.

The ribonuclease ranpirnase (Onconase) has been used empirically to treat malignant mesothelioma (MM) patients, and some of them had prolonged survivals. The aim of this study was to investigate the mechanisms of the therapeutic function of ranpirnase in MM cells. The effects of ranpirnase were studied in vivo and in vitro on 2 MM cell lines (epithelioid REN and sarcomatoid PPM-Mill). We found that ranpirnase was able to inhibit NF-κB nuclear translocation, evaluated by cell fractionation and immunoblotting as well as by immunofluorescence. Also, MMP9 secretion by MM cells was decreased by ranpirnase treatment, as assessed by the reduction of metalloproteinase activity, evaluated by zymography on culture-conditioned media. Ranpirnase induced apoptosis of MM cells in vitro and in vivo, causing a powerful inhibition of MM tumor growth in SCID xenografts, determined by In Vivo Imaging System (IVIS) of tumor cells engineered by lentiviral transduction of the luciferase gene. Finally, mice treated with ranpirnase showed a significantly prolonged survival. Our data provide a mechanistic rationale to explain the beneficial antitumor activity observed in some patients treated with ranpirnase and demonstrate that ranpirnase interferes with the NF-κB pathway, thus influencing MM tumor cell invasiveness and survival. It is hoped that this information will also facilitate the identification of those patients who are more likely to benefit from this drug and will also open a new frontier for the use of this drug in tumor types other than MM.

Purpose Treatment of malignant pleural mesothelioma (MPM) with Ranpirnase (Onconase) results in disruption of protein translation and cell apoptosis. We hypothesize that Onconase acts via down regulation of nuclear factor kappa B (NFKβ) by specific microRNAs (miRNA) and that interference of this pathway could have implications for MPM resistance to chemotherapy. Experimental Design Three immortalized MPM cell lines (H2959, H2373, and H2591) were exposed to Onconase at 0–20 µg/mL. Cell counts were measured at 48 and 72 hours. Gene expression in miRNA-enriched RNA was validated by RT-PCR. The functional implications of miRNA expression were evaluated by transfecting miRNA mimics or inhibitors into MPM cell lines, and performing Matrigel™ invasion, cell proliferation, soft agar colony formation, and scratch closure assays. Effects on NFKβ expression and downstream targets including ABC transporters, BCL-xl, and IAP were assessed by RT-PCR and Western Blotting. Results Treatment with 20µg/mL of Onconase significantly decreased cell count and invasion. Hsa-miR-17* was significantly upregulated and hsa-miR-30c significantly down-regulated by Onconase treatment in all cell lines. Forced expression of hsa-miR-17* mimic and hsa-miR-30c inhibitor each significantly decreased functional activity of Onconase in all assays. NFKB1(p50) expression and downstream targets were also decreased with Onconase treatment as well as with forced expression miRNA mimic and inhibitors. Conclusions Onconase treatment caused a significant decrease in cell proliferation, invasion, and in expression of certain miRNAs. Recapitulation of the resultant miRNA expression pattern with hsa-miR-17* mimic and hsa-miR-30c inhibitor resulted in downregulation of NFKB1 and reduced malignant behavior in functional assays. Thus, Onconase likely exerts its anti-tumor effect through these miRNAs.

HER2 and VEGF are closely related to the progression of several tumors. The inhibitor simultaneously targeting these two proteins will effectively inhibit the progression of tumors. Here, a bispecific antibody, termed as YY0411, targeting both HER2 and VEGF as a potent anticancer therapeutic antibody is reported. YY0411 is the first bispecific antibody constructed in IgG-Decoy receptor format. It efficiently identifies and combines both HER2 and VEGF protein. YY0411 is believed to be a candidate tumor suppressor as it significantly inhibits the colony formation ability of human cancer cells (Calu-3, MDA-MB-453, and NCI-N87 cells). The phosphorylation of HER2 and VEGF downstream components are also decreased in these cells with the treatment of YY0411. Similar to other antibodies, YY0411 has the ability to promote the secretion of IFN-γ by T lymphocytes. In addition, YY0411 significantly inhibits the growth of Calu-3 cells-induced xenograft in nude mice. This work demonstrates that YY0411 may be a potential anti-lung cancer drug.

We have recently developed a novel optical technology, partial wave spectroscopic (PWS) microscopy, which is exquisitely sensitive to the nanoarchitectural manifestation of the genetic/epigenetic alterations of field carcinogenesis. Our approach was to screen for lung cancer by assessing the cheek cells based on emerging genetic/epigenetic data which suggests that the buccal epithelium is altered in lung field carcinogenesis. We performed PWS analysis from microscopically normal buccal epithelial brushings from smokers with and without lung cancer (n = 135). The PWS parameter, disorder strength of cell nanoarchitecture (Ld), was markedly (>50%) elevated in patients harboring lung cancer compared with neoplasia-free smokers. The performance characteristic was excellent with an area under the receiver operator characteristic curve of >0.80 and was equivalent for both disease stage (early versus late) and histologies (small cell versus non–small cell lung cancers). An independent data set validated the findings with only a minimal degradation of performance characteristics. Our results offer proof of concept that buccal PWS may potentially herald a minimally intrusive prescreening test that could be integral to the success of lung cancer population screening programs. Cancer Res; 70(20); 7748–54. ©2010 AACR.

The 1995 Lung Cancer Study Group consensus recommending lobectomy for stage I non-small cell lung cancer (NSCLC) has directed lung cancer resections since its publication. However, enhancements in imaging technology over the last decade have produced larger cohorts of patients presenting with localized, early-stage disease. Today, multislice computer tomography is widely available, capable of detecting NSCLC at smaller sizes, with improved spatial resolution, and is used in screening programs for high-risk individuals. Furthermore, the maturation of minimally invasive surgical resection (video-assisted thoracoscopic surgery) has reduced perioperative morbidity and mortality, improved postoperative lung function, and demonstrated equivalent oncologic effectiveness to open surgery. The mandatory use of lobectomy for patients with small stage IA NSCLC is now being challenged. Numerous single-institution trials have demonstrated that well-selected use of sublobar resection can afford comparable survival and recurrence rates to lobectomy, particularly in high-risk patients. Currently, a prospective, randomized multi-institutional phase III trial is being conducted by the Cancer and Lymphoma Group B (CALGB 140503) to determine whether patients with small (≤2 cm) peripheral NSCLC tumors can safely undergo sublobar resection while maintaining rates of survival and recurrence that are comparable to lobectomy. This review summarizes the literature from the past 15 years to assist in applying those conclusions to future research innovation.

Asbestos fibers cause chronic inflammation that may be critical to the development of malignant mesothelioma (MM). Two human MM cell lines (Hmeso, PPM Mill) were used in a SCID mouse xenograft model to assess time-dependent patterns of inflammation and tumor formation. After intraperitoneal (IP) injection of MM cells, mice were euthanized at 7, 14, and 30 days, and peritoneal lavage fluid (PLF) was examined for immune cell profiles and human and mouse cytokines. Increases in human MM-derived IL-6, IL-8, bFGF, and VEGF were observed in mice at 7 days postinjection of either MM line, and a striking neutrophilia was observed at all time points. Free-floating tumor spheroids developed in mice at 14 days, and both spheroids and adherent MM tumor masses occurred in all mice at 30 days. Results suggest that inflammation and cytokine production precede and may be critical to the development of MMs.

Malignant mesothelioma (MM) is an aggressive tumor of mesothelial tissues that has poor prognosis and limited therapeutic options. Pass et al. (N Engl J Med 2012) identified the extracellular matrix protein fibulin-3 as an accurate biomarker of MM progression, which can be detected in the tumor parenchyma and in pleural effusions. Here, we report a systematic study of the mechanisms of fibulin-3 in MM and its value as a molecular target. Fibulin-3 activated canonical NFkB signaling and regulated the expression of NFkB-downstream genes, including the proteases MMP9 and MMP13, and the proinvasive proteins TNC and MLCK. Fibulin-3 knockdown downregulated NFkB signaling, reduced MM cell viability and potentiated the cytotoxicity of cisplatin in vitro. We next developed a function-blocking antibody ("mAb428.2") against an epitope of fibulin-3 required for NFkB activation (Nandhu et al., Clin Cancer Res 2017). This antibody blocked fibulin-3 signaling in vitro and reduced the expression of this protein, as well as NFkB-regulated MMP9, in subcutaneous (sc) MM tumors implanted in nude mice. Next, we treated mice carrying sc or intrapleural models of MM with mAb428.2 or control IgG1 (8x 30 mg/kg, q24h) delivered IV. Our anti-fibulin-3 antibody caused a significant reduction of tumor burden and extension of overall survival in both models. Overall, downregulation and inhibition of fibulin-3 proved a successful approach to enhance MM therapy. Given the value of this protein as a MM biomarker, we expect that detection of fibulin-3 may serve to identify patients with the highest chance to respond to combination therapies including anti-fibulin-3 approaches, achieving maximum therapeutic against these aggressive tumors. Citation Format: Mohan Sobhana Nandhu, Chandra Goparaju, Sharon L. Longo, Harvey I. Pass, Mariano S. Viapiano. Targeting malignant mesothelioma with an antibody against the tumor extracellular matrix protein fibulin-3 [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 2769.

We discovered that heterozygous germline BAP1 mutations (BAP1+/-) cause a novel cancer syndrome that we named “The BAP1 cancer syndrome” characterized by development of malignant mesothelioma (MM), uveal and cutaneous melanoma, and other cancers. We demonstrated how germline BAP1 mutations are transmitted over the centuries across generations and found that 100% of individuals who inherited germline BAP1 mutations have developed one or more malignancies in their lifetime. Very recently we demonstrated that the potent BAP1 tumor-suppressor activity is linked to its dual function in the nucleus, where it regulates DNA repair, and in the cytoplasm, where it regulates apoptosis by deubiquitylating and stabilizing the IP3R3 endoplasmic reticulum (ER) Ca2+ channel. In parallel we discovered that cells carrying heterozygous BAP1 mutations derive energy largely via aerobic glycolysis, Warburg effect. Now we have found the mechanisms regulated by BAP1 that are responsible for the changes in metabolism. We discovered that reduced BAP1 levels lead to reduced release of Ca2+ from the ER into the cytoplasm and reduced intra-mitochondrial Ca2+ levels. Reduced mitochondrial Ca2+ levels directly impair aerobic respiration and cells switch to glycolysis even in the presence of oxygen. We demonstrate that aerobic glycolysis can be induced in apparently any human cell type by reducing BAP1 levels using siRNAs or other strategies, and that in these cells glycolysis can be reverted to aerobic respiration upon re-establishing normal Ca2+ levels. In conclusion, our findings indicate how BAP1 deficiency regulates DNA repair and apoptosis and how low BAP1 levels induce the Warburg effect, an effect seen in all “normal” cell types from individuals born with inherited germline heterozygous BAP1 mutations. In these individuals aerobic glycolysis predates by many years malignant transformation. Citation Format: Michele Carbone, Angela Bononi, Carlotta Giorgi, Simone Patergnani, Harvey I. Pass, Wei Jia, Paolo Pinton, Haining Yang. BAP1 modulates gene-environment interaction in carcinogenesis [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 5519.

Treating KRAS mutant lung adenocarcinoma (LUAD) remains a major challenge in cancer treatment given the difficulties associated with directly inhibiting the KRAS oncoprotein1. One approach to addressing this challenge is to define frequently co-occurring mutations with KRAS, which themselves may lead to therapeutic vulnerabilities in tumors. Approximately 20% of KRAS mutant LUAD tumors carry loss-of-function (LOF) mutations in Kelch-like ECH-associated protein 1 (KEAP1)2-4, a negative regulator of nuclear factor erythroid 2-like 2 (NFE2L2; hereafter NRF2), which is the master transcriptional regulator of the endogenous antioxidant response5-10. The high frequency of mutations in KEAP1, which would be predicted to result in activation of the NRF2 pathway, suggests an important role for the oxidative stress response in lung tumorigenesis. To test this directly, we used a CRISPR/Cas9-based approach in a mouse model of Kras-driven LUAD to examine the effects of loss of KEAP1 function in lung cancer progression. We show that loss of KEAP1 hyper-activates Nrf2 and promotes Kras-driven LUAD. Combining CRISPR/Cas9-based genetic screening and metabolomic analyses, we also show that KEAP1/NRF2 mutant cancers are dependent on increased glutaminolysis, and this property can be therapeutically exploited through the pharmacologic inhibition of glutaminase. Finally, we provide a rationale for sub-stratification of human lung cancer patients with KRAS-KEAP1 or -NRF2 mutant tumors as likely to respond to glutaminase inhibition. Citation Format: Rodrigo Romero, Volkan I. Sayin, Davidson M. Shawn, Matthew Bauer, Simranjit X. Singh, Sarah LeBoeuf, Triantafyllia R. Karakousi, Donald C. Ellis, Arjun Bhutkar, Francisco Sanchez-Rivera, Lakshmipriya Subbaraj, Britney Martinez, Roderick T. Bronson, Justin R. Prigge, Edward E. Schmidt, Craig J. Thomas, Angela Davies, Igor Dolgalev, Adriana Heguy, Viola Allaj, John T. Piorier, Andre L. Moreira, Charles M. Rudin, Harvey I. Pass, Matthew G. Vander Heiden, Tyler Jacks, Thales Papagiannakopoulos. Loss of Keap1 promotes KRAS-driven lung cancer and results in genotype-specific vulnerabilities [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A43.

PurposePlasma osteopontin (OPN) levels in advanced non–small-cell lung cancer (NSCLC) correlate with therapeutic response and survival, but the utility of plasma OPN for diagnosis and monitoring of early-stage NSCLC has not been investigated. We hypothesize that plasma OPN levels are elevated in early-stage NSCLC and decrease with resection.Patients and MethodsPresurgery plasma OPN levels (in ng/mL) were measured by enzyme-linked immunosorbent assay (ELISA) in a discovery set of 60 patients with early-stage NSCLC and were compared with data from 56 cancer-free smokers. Presurgery OPN was validated in an independent cohort of 96 patients with resectable NSCLC. The presurgery levels in the latter cohort were compared with matched postsurgery levels. Perioperative OPN levels were correlated with demographics, tumor characteristics, and perioperative events. OPN was monitored during follow-up.ResultsDiscovery set presurgery NSCLC OPN (271 ± 31 ng/mL) was higher than smokers (40 ± 2 ng/mL; P = .001). Presurgery OPN was similar in the NSCLC validation cohort (324 ng/mL ± 20 ng/mL; P = .134). Postsurgery OPN (256 ng/mL ± 21 ng/mL) measured at mean of 9.8 weeks (range, 2 to 46 weeks) was lower than presurgery OPN (P = .005). Time from surgery significantly impacted postsurgery OPN: OPN ≤ 6 weeks postsurgery (303 n/mL ± 26 ng/mL) was higher than OPN greater than 6 weeks postsurgery (177 ng/mL ± 29 ng/mL; P = .003). Multivariate analysis noted correlations between albumin and creatinine to presurgery OPN and use of thoracotomy to postsurgery OPN. Recurrence rate was 5% at 29 weeks mean follow-up. OPN at recurrence was elevated from postsurgery nadir.ConclusionPlasma OPN levels are elevated in early-stage NSCLC. They are reduced after resection and appear to increase with recurrence. Plasma OPN may have utility as a biomarker in early-stage NSCLC.

DNA copy number analysis was performed, using single-nucleotide polymorphism mapping arrays, to fine map genomic imbalances in human malignant mesothelioma (MM) cell lines derived from primary tumors. Chromosomal losses accounted for the majority of genomic imbalances. All 22 cell lines examined showed homozygous deletions of 9p21.3, centering at the CDKN2A/ARF and CDKN2B loci. Other commonly underrepresented segments included 1p36, 1p22, 3p21-22, 4q13, 4q34, 11q23, 13q12-13, 14q32, 15q15, 18q12, and 22q12, each observed in 55-90% of cell lines. Focal deletions of 11q23 encompassed the transcriptional repressor gene promyelocytic leukemia zinc finger (PLZF), which was validated by analysis of genomic DNA using real-time polymerase chain reaction (PCR). Semi-quantitative RT-PCR and immunoblot analysis revealed that PLZF is greatly downregulated in MM cell lines compared with non-malignant mesothelial cells. Ectopic expression of PLZF in PLZF-deficient MM cells resulted in decreased cell viability, reduced colony formation, as well as increased apoptosis, the latter based on results of various cell death assays and the observation of increased cleavage of caspase 3, PARP, and Mcl-1. These data indicate that deletions of PLZF are a common occurrence in MM and that downregulation of PLZF may contribute to MM pathogenesis by promoting cell survival.