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Supplementary Table 3 from Expression Signatures of the Lipid-Based Akt Inhibitors Phosphatidylinositol Ether Lipid Analogues in NSCLC Cells

Supplementary Table 1, Figures 1-2 from Expression Signatures of the Lipid-Based Akt Inhibitors Phosphatidylinositol Ether Lipid Analogues in NSCLC Cells

Supplementary Table 2 from Expression Signatures of the Lipid-Based Akt Inhibitors Phosphatidylinositol Ether Lipid Analogues in NSCLC Cells

Purpose: The cross-talk between tumor cells, myeloid cells, and T cells can play a critical role in tumor pathogenesis and response to immunotherapies. Although the etiology of mesothelioma is well understood, the impact of mesothelioma tumor cells on the surrounding immune microenvironment is less well studied. In this study, the effect of the mesothelioma tumor microenvironment on circulating and infiltrating granulocytes and T cells is investigated.Experimental Design: Tumor tissues and peripheral blood from mesothelioma patients were evaluated for presence of granulocytes, which were then tested for their T-cell suppression potential. Different cocultures of granulocytes and/or mesothelioma tumor cells and/or T cells were set up to identify the mechanism of T-cell inhibition.Results: Analysis of human tumors showed that the mesothelioma microenvironment is enriched in infiltrating granulocytes, which inhibit T-cell proliferation and activation. Characterization of the whole blood at diagnosis identified similar, circulating, immunosuppressive CD11b+CD15+HLADR− granulocytes at increased frequency compared with healthy controls. Culture of healthy-donor granulocytes with human mesothelioma cells showed that GM-CSF upregulates NOX2 expression and the release of reactive oxygen species (ROS) from granulocytes, resulting in T-cell suppression. Immunohistochemistry and transcriptomic analysis revealed that a majority of mesothelioma tumors express GM-CSF and that higher GM-CSF expression correlated with clinical progression. Blockade of GM-CSF with neutralizing antibody, or ROS inhibition, restored T-cell proliferation, suggesting that targeting of GM-CSF could be of therapeutic benefit in these patients.Conclusions: Our study presents the mechanism behind the cross-talk between mesothelioma tumors and the immune microenvironment and indicates that targeting GM-CSF could be a novel treatment strategy to augment immunotherapy in patients with mesothelioma. Clin Cancer Res; 24(12); 2859–72. ©2018 AACR.

Mesothelioma granulocytes suppress T cell activation

Supplementary Figs and Tables

Mesothelioma upregulates ROS production by granulocytes

Arginase activity does not drive immunosuppressive granulocyte function

GM-CSF drives ROS release in granulocytes

Rare biallelic BLM gene mutations cause Bloom syndrome. Whether BLM heterozygous germline mutations (BLM+/−) cause human cancer remains unclear. We sequenced the germline DNA of 155 mesothelioma patients (33 familial and 122 sporadic). We found 2 deleterious germline BLM+/− mutations within 2 of 33 families with multiple cases of mesothelioma, one from Turkey (c.569_570del; p.R191Kfs*4) and one from the United States (c.968A>G; p.K323R). Some of the relatives who inherited these mutations developed mesothelioma, while none with nonmutated BLM were affected. Furthermore, among 122 patients with sporadic mesothelioma treated at the US National Cancer Institute, 5 carried pathogenic germline BLM+/− mutations. Therefore, 7 of 155 apparently unrelated mesothelioma patients carried BLM+/− mutations, significantly higher (P = 6.7E-10) than the expected frequency in a general, unrelated population from the gnomAD database, and 2 of 7 carried the same missense pathogenic mutation c.968A>G (P = 0.0017 given a 0.00039 allele frequency). Experiments in primary mesothelial cells from Blm+/− mice and in primary human mesothelial cells in which we silenced BLM revealed that reduced BLM levels promote genomic instability while protecting from cell death and promoted TNF-α release. Blm+/− mice injected intraperitoneally with asbestos had higher levels of proinflammatory M1 macrophages and of TNF-α, IL-1β, IL-3, IL-10, and IL-12 in the peritoneal lavage, findings linked to asbestos carcinogenesis. Blm+/− mice exposed to asbestos had a significantly shorter survival and higher incidence of mesothelioma compared to controls. We propose that germline BLM+/− mutations increase the susceptibility to asbestos carcinogenesis, enhancing the risk of developing mesothelioma.

Introduction Children and young adults diagnosed with malignant mesothelioma may have unique genetic characteristics. In this study, we evaluated for the presence of the anaplastic lymphoma kinase (ALK) translocations in these patients. Methods In a prospective study of mesothelioma natural history ( ClinicalTrials.gov number NCT01950572 ), we assessed for the presence of the ALK translocation in patients younger than 40 years, irrespective of the site of disease. The presence of this translocation was assessed by means of fluorescence in situ hybridization (FISH). If the patients tested positive for the ALK translocation, both immunohistochemistry and RNA sequencing were performed on the tumor specimen. Results Between September 2013 and December 2018, 373 patients were enrolled in the mesothelioma natural history study, of which 32 patients were 40 years old or younger at the time of their mesothelioma diagnosis. There were 25 patients with peritoneal mesothelioma, five with pleural mesothelioma, one with pericardial mesothelioma, and one with bicompartmental mesothelioma. Presence of an ALK translocation by FISH was seen in two of the 32 patients (6%) with mesothelioma. Both patients, a 14-year-old female and a 27-year-old male, had peritoneal mesothelioma and had no history of asbestos exposure, prior radiation therapy, or predisposing germline mutations. Neither had detectable ALK expression by immunohistochemistry. RNA sequencing revealed the presence of an STRN fusion partner in the female patient but failed to identify any fusion protein in the male patient. Conclusions Young patients with peritoneal mesothelioma should be evaluated for the presence of ALK translocations. Presence of this translocation should be assessed by FISH and these patients could potentially benefit from tyrosine kinase inhibitors targeting ALK.

Malignant mesothelioma is an aggressive cancer with limited treatment options and poor prognosis. Better understanding of mesothelioma genomics and transcriptomics could advance novel therapies. We performed whole-exome and RNA-sequencing of germline and tumors of 122 patients with pleural, peritoneal, and tunica-vaginalis mesothelioma. We identify a 48 gene prognostic signature that is highly predictive of mesothelioma patient survival including CCNB1, whose expression is highly predictive of patient survival on its own. Using a synthetic-lethality (SL) based pipeline for analyzing the patients’ transcriptomic data, we identified SL-based signatures predictive of response to an anti-PD1 immune checkpoint inhibitor and combination therapies with pemetrexed. These SL-profiles successfully predict the overall patient-response observed across targeted, immuno- and chemotherapies in 11 independent mesothelioma clinical trials spanning 7 different treatments. These findings lay a basis for future studies aimed specifically at testing the ability of these SL profiles to serve as treatment biomarkers in mesothelioma.

Survival from malignant mesothelioma, particularly pleural mesothelioma, is very poor. For patients with breast, ovarian, or prostate cancers, overall survival is associated with increased sensitivity to platinum chemotherapy due to loss-of-function mutations in DNA repair genes. The goal of this project was to evaluate, in patients with malignant mesothelioma, the relationship between inherited loss-of-function mutations in DNA repair and other tumor suppressor genes and overall survival following platinum chemotherapy. Patients with histologically confirmed malignant mesothelioma were evaluated for inherited mutations in tumor suppressor genes. Survival was evaluated with respect to genotype and site of mesothelioma. Among 385 patients treated with platinum chemotherapy, median overall survival was significantly longer for patients with loss-of-function mutations in any of the targeted genes compared with patients with no such mutation (P = 0.0006). The effect of genotype was highly significant for patients with pleural mesothelioma (median survival 7.9 y versus 2.4 y, P = 0.0012), but not for patients with peritoneal mesothelioma (median survival 8.2 y versus 5.4 y, P = 0.47). Effect of patient genotype on overall survival, measured at 3 y, remained independently significant after adjusting for gender and age at diagnosis, two other known prognostic factors. Patients with pleural mesothelioma with inherited mutations in DNA repair and other tumor suppressor genes appear to particularly benefit from platinum chemotherapy compared with patients without inherited mutations. These patients may also benefit from other DNA repair targeted therapies such as poly-ADP ribose polymerase (PARP) inhibitors.

Introduction PARP inhibition may enhance antitumor responses in BAP1-associated mesothelioma by inducing synthetic lethality. Methods A single-center, nonrandomized, phase 2 trial was conducted, in which patients with refractory mesothelioma were given olaparib 300 mg twice daily in a 21-day cycle until disease progression or intolerable toxicity. The primary objective was to determine the objective response rate on the basis of somatic or germline mutation status of DNA repair genes. The secondary objectives were to assess safety and tolerability and to determine progression-free survival (PFS) and overall survival (OS). Whole-exome sequencing was performed on blood and tumor. Results A total of 23 previously treated patients with pleural and peritoneal mesothelioma were enrolled and treated (germline BAP1, n = 4; germline MRE11A, n = 1; somatic BAP1, n = 8 mutations). There was one (4%) partial response, 18 (78%) with stable disease at 6 weeks, and four (17%) with progressive disease. The median overall PFS and OS were 3.6 months (95% confidence interval [CI]: 2.7–4.2 mo) and 8.7 months (95% CI: 4.7 mo–not estimable), respectively. The median PFS of germline BAP1 mutants (n = 4) was 2.3 months (95% CI: 1.3–3.6 mo) versus 4.1 months (95% CI: 2.7–5.5 mo) for wild-type (n = 19; p = 0.019). The median OS was 4.6 months (95% CI: 3.1–4.9 mo) for germline BAP1 mutation versus 9.6 months (95% CI: 5.5 mo–not estimable) in no germline mutation (p = 0.0040). Olaparib was safe with no new safety concerns. Conclusions Olaparib has limited activity in previously treated mesothelioma including patients with BAP1 mutations. Germline BAP1 mutations were associated with decreased PFS and OS.

Significance We found that the probability of carrying heterozygous germline BLM mutations is significantly higher among mesothelioma patients than in the general population. In vitro and in vivo experiments suggest that heterozygous BLM mutations increase susceptibility to mesothelioma. BLM-mutation carriers are at higher risk of developing mesothelioma, and their risk increases upon asbestos exposure. Therefore, BLM mutation carriers should benefit from prevention and screening for early detection. We suggest that genetic testing to identify carriers of BLM heterozygous mutations and genetic counseling would help identify individuals at higher risk of mesothelioma and provide mutation carriers the option to implement simple preventive measures to reduce exposure to asbestos and other carcinogenic fibers to decrease their risk of developing mesothelioma., Rare biallelic BLM gene mutations cause Bloom syndrome. Whether BLM heterozygous germline mutations (BLM+/−) cause human cancer remains unclear. We sequenced the germline DNA of 155 mesothelioma patients (33 familial and 122 sporadic). We found 2 deleterious germline BLM+/− mutations within 2 of 33 families with multiple cases of mesothelioma, one from Turkey (c.569_570del; p.R191Kfs*4) and one from the United States (c.968A>G; p.K323R). Some of the relatives who inherited these mutations developed mesothelioma, while none with nonmutated BLM were affected. Furthermore, among 122 patients with sporadic mesothelioma treated at the US National Cancer Institute, 5 carried pathogenic germline BLM+/− mutations. Therefore, 7 of 155 apparently unrelated mesothelioma patients carried BLM+/− mutations, significantly higher (P = 6.7E-10) than the expected frequency in a general, unrelated population from the gnomAD database, and 2 of 7 carried the same missense pathogenic mutation c.968A>G (P = 0.0017 given a 0.00039 allele frequency). Experiments in primary mesothelial cells from Blm+/− mice and in primary human mesothelial cells in which we silenced BLM revealed that reduced BLM levels promote genomic instability while protecting from cell death and promoted TNF-α release. Blm+/− mice injected intraperitoneally with asbestos had higher levels of proinflammatory M1 macrophages and of TNF-α, IL-1β, IL-3, IL-10, and IL-12 in the peritoneal lavage, findings linked to asbestos carcinogenesis. Blm+/− mice exposed to asbestos had a significantly shorter survival and higher incidence of mesothelioma compared to controls. We propose that germline BLM+/− mutations increase the susceptibility to asbestos carcinogenesis, enhancing the risk of developing mesothelioma.

Purpose: The cross-talk between tumor cells, myeloid cells, and T cells can play a critical role in tumor pathogenesis and response to immunotherapies. Although the etiology of mesothelioma is well understood, the impact of mesothelioma tumor cells on the surrounding immune microenvironment is less well studied. In this study, the effect of the mesothelioma tumor microenvironment on circulating and infiltrating granulocytes and T cells is investigated. Experimental Design: Tumor tissues and peripheral blood from mesothelioma patients were evaluated for presence of granulocytes, which were then tested for their T-cell suppression potential. Different cocultures of granulocytes and/or mesothelioma tumor cells and/or T cells were set up to identify the mechanism of T-cell inhibition. Results: Analysis of human tumors showed that the mesothelioma microenvironment is enriched in infiltrating granulocytes, which inhibit T-cell proliferation and activation. Characterization of the whole blood at diagnosis identified similar, circulating, immunosuppressive CD11b+CD15+HLADR− granulocytes at increased frequency compared with healthy controls. Culture of healthy-donor granulocytes with human mesothelioma cells showed that GM-CSF upregulates NOX2 expression and the release of reactive oxygen species (ROS) from granulocytes, resulting in T-cell suppression. Immunohistochemistry and transcriptomic analysis revealed that a majority of mesothelioma tumors express GM-CSF and that higher GM-CSF expression correlated with clinical progression. Blockade of GM-CSF with neutralizing antibody, or ROS inhibition, restored T-cell proliferation, suggesting that targeting of GM-CSF could be of therapeutic benefit in these patients. Conclusions: Our study presents the mechanism behind the cross-talk between mesothelioma tumors and the immune microenvironment and indicates that targeting GM-CSF could be a novel treatment strategy to augment immunotherapy in patients with mesothelioma. Clin Cancer Res; 24(12); 2859–72. ©2018 AACR.

Introduction BRCA1-associated protein-1 (BAP1), a nuclear deubiquitinase thought to be involved in DNA double-strand break repair, is frequently mutated in mesothelioma. Because poly(adenosine diphosphate-ribose) polymerase inhibitors (PARPIs) induce synthetic lethality in BRCA1/2 mutant cancers, we evaluated whether BAP1 inactivating mutations confer sensitivity to PARPIs in mesothelioma and if combination therapy with temozolomide (TMZ) would be beneficial. Methods A total of 10 patient-derived mesothelioma cell lines were generated and characterized for BAP1 mutation status, protein expression, nuclear localization, and sensitivity to the PARPIs, olaparib, and talazoparib, alone or in combination with TMZ. BAP1 deubiquitinase (DUB) activity was evaluated by ubiquitin with 7-amido-4-methylcoumarin assay. BAP1 knockout mesothelioma cell lines were generated by CRISPR-Cas9. Because Schlafen 11 (SLFN11) and O6-methylguanine-DNA methyltransferase also drive response to TMZ and PARPIs, we tested their expression and relationship with drug response. Results BAP1 mutations or copy-number alterations, or both were present in all 10 cell lines. Nonetheless, four cell lines exhibited intact DUB activity and two had nuclear BAP1 localization. Half maximal-inhibitory concentrations of olaparib and talazoparib ranged from 4.8 μM to greater than 50 μM and 0.039 μM to greater than 5 μM, respectively, classifying them into sensitive (two) or resistant (seven) cells, independent of their BAP1 status. Cell lines with BAP1 knockout resulted in the loss of BAP1 DUB activity but did not increase sensitivity to talazoparib. Response to PARPI tended to be associated with high SLFN11 expression, and combination with temozolomide increased sensitivity of cells with low or no MGMT expression. Conclusions BAP1 status does not determine sensitivity to PARPIs in patient-derived mesothelioma cell lines. Combination of PARPI with TMZ may be beneficial for patients whose tumors have high SLFN11 and low or no MGMT expression.

LMB-100 is an immunotoxin targeting the cell surface protein mesothelin, which is highly expressed in many cancers including mesothelioma. Having observed that patients receiving pembrolizumab off protocol after LMB-100 treatment had increased tumor responses; we characterized these responses and developed animal models to study whether LMB-100 made tumors more responsive to antibodies blocking programmed cell death protein 1 (PD-1). The overall objective tumor response in the 10 patients who received PD-1 inhibitor (pembrolizumab, 9; nivolumab, 1) after progression on LMB-100 was 40%, and the median overall survival was 11.9 months. Of the seven evaluable patients, four had objective tumor responses, including one complete response and three partial responses, and the overall survival for these patients was 39.0+, 27.7, 32.6+, and 13.8 months. When stratified with regard to programmed death ligand 1 (PD-L1) expression, four of five patients with tumor PD-L1 expression had objective tumor response. Patients with positive tumor PD-L1 expression also had increased progression-free survival (11.3 versus 2.1 months, P = 0.0018) compared with those lacking PD-L1 expression. There was no statistically significant difference in overall survival (27.7 versus 6.8 months, P = 0.1). LMB-100 caused a systemic inflammatory response and recruitment of CD8(+) T cells in patients’ tumors. The enhanced antitumor effects with LMB-100 plus anti–PD-1 antibody were also observed in a human peripheral blood mononuclear cell–engrafted mesothelioma mouse model and a human mesothelin–expressing syngeneic lung adenocarcinoma mouse model. LMB-100 plus pembrolizumab is now being evaluated in a prospective clinical trial for patients with mesothelioma.

Background Malignant mesothelioma (MM) is an aggressive cancer with limited treatment options and poor prognosis. Malignant pleural mesothelioma comprises 80% of the cases and has worse outcome than malignant peritoneal mesothelioma. An in-depth knowledge of genetic, transcriptomic and immunogenic events involved in MM is critical for successful development of prognostics and therapeutic modalities. Methods We performed whole-exome sequencing of germline and tumors of 122 patients with pleural (n=59), peritoneal (n=61) and tunica vaginalis (n=2) mesothelioma, and RNA-sequencing of 100 tumors to identify pathogenic variants, somatic mutational signatures, and prognostic gene expression signatures, predictive of patient survival and tumor response to therapies. We validated our findings using the TCGA and Bueno et al. mesothelioma datasets. Results The important findings from this study include: a) Key somatic mutational signatures are associated with DNA repair pathways and BRCA1 associated protein-1 (BAP1) is the most commonly mutated gene (~13% with germline mutation). b) We identified a set of 48 genes, a “mesothelioma prognostic signature”, whose high expression level is associated with poor survival (Cox regression, FDR < 0.1). These genes are enriched for genes related to cell cycle and DNA repair. This signature is highly predictive of patient survival in two other independent, pleural mesothelioma cohorts: TCGA (Hazard ratio (HR) = 2.6, P = 6.94e-10) and Bueno et al. mesothelioma dataset (HR = 1.49, P = 4.34e-07), after controlling for age and gender. c) Among the 48 genes, the expression of CCNB1 is highly predictive of patient survival suggesting its important role in MM, possibly via its involvement in the CDK1-CCNB1-CCNF complex (HR = 2.54, P = 1.89e-08 for TCGA; HR = 1.40, P = 1.65e-05 for Bueno et al. dataset). d) Using a synthetic lethality (SL) based precision-oncology computational framework for analyzing the patients' transcriptomic data, we were able to accurately predict response to an anti-PD1 immune checkpoint inhibitor and combination therapies with pemetrexed (chemotherapy) in mesothelioma patients. The SL profiles successfully predicted the overall patient-response observed across targeted, immuno- and chemotherapies in 11 independent mesothelioma clinical trials (Spearman's ρ = 0.64, P = 0.0348). This is the first analysis shown to successfully predict overall patient-response for various treatments within a cancer type. Conclusions By analyzing the tumor genomic and transcriptomics data of a large cohort of MM patients, we identify gene expression prognostic markers predictive of patient survival and response to therapy, both as independent signatures and via their SL interactions. These findings lay a basis for the future development of personalized therapy approaches for mesothelioma patients. Citation Format: Nishanth Ulhas Nair, Qun Jiang, Jun S. Wei, Vikram A. Misra, Betsy Morrow, Leandro C. Hermida, Joo Sang Lee, Idrees Mian, Jingli Zhang, Manjistha Sengupta, Javed Khan, Eytan Ruppin, Raffit Hassan. Genomic and transcriptomic profiling of malignant mesothelioma patients identifies gene signatures predictive of survival and response to immuno and chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 667.

9054 Background: BRCA1 associated protein 1 ( BAP1), a nuclear deubiquitinase involved in DNA double-strand break repair is frequently mutated in MM. Because poly(ADP-ribose) polymerase inhibitors (PARPIs) induce synthetic lethality in BRCA1/2 mutant cancers, we sought to evaluate efficacy of olaparib in patients with MM and correlate it with pathogenic germline and somatic mutations in DNA repair genes. Methods: Phase II single-center study (NCT03531840) enrolled patients with advanced pleural or peritoneal mesothelioma who had progressed on prior therapies, age >18 years, ECOG performance status

Background There is an urgent need for a companion assay to work with mesothelin-targeted therapeutic agents and for noninvasive and accurate prognostication of malignant mesothelioma (MM) patients. We report the development and validation of a blood-based assay for megakaryocyte potentiating factor (MPF) and the evaluation of its effectiveness for prognosis in MM and lung cancer patients. Methods Using electrochemiluminescence technology, we developed a sensitive MPF assay and performed both analytical and clinical validations. Further, the effectiveness of the MPF assay in predicting prognosis was evaluated for 95 MM and 272 lung cancer patients. Results We performed comprehensive analytical and clinical validation, including precision and accuracy, interference, preanalytical variables, sensitivity, and specificity for mesothelioma. In MM patients, increased serum MPF is a predictor of poor survival with a hazard ratio (HR) = 2.46 (log-rank P = 0.003; n = 95). In refractory MM patients, increased MPF is a strong predictor of poor outcome with an HR = 6.12 (log-rank P = 0.0007; n = 57). In a lung cancer patient cohort, increased MPF is a predictor of poor survival, with an HR = 1.57 (log-rank P = 0.003; n = 272). Conclusions The MPF assay has robust technical characteristics, with strong analytic and clinical validation. Clinical studies indicate that increased serum MPF is a predictor of poor survival for MM patients, throughout the course of the disease. Increased MPF is also associated with poor overall survival for patients with newly diagnosed lung cancer.

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

Nelfinavir is an HIV protease inhibitor being repurposed as an anti-cancer agent in preclinical models and in small oncology trials, yet the MTD of nelfinavir has not been determined. Therefore, we conducted a Phase Ia study to establish the maximum tolerated dose (MTD) and dose limiting toxicities (DLT) of nelfinavir in subjects with advanced solid tumors. Adults with refractory cancers were given oral nelfinavir twice daily with pharmacokinetic and pharmacodynamic analyses. Twenty-eight subjects were enrolled. Nelfinavir was generally well tolerated. Common adverse events included diarrhea, anemia, and lymphopenia, which were mostly mild. The DLT was rapid-onset neutropenia that was reversible. The MTD was established at 3125 mg twice daily. In an expansion cohort at the MTD, one of 11 (9%) evaluable subjects had a confirmed partial response. This, plus two minor responses, occurred in subjects with neuroendocrine tumors of the midgut or pancreatic origin. Thirty-six percent of subjects had stable disease for more than 6 months. In peripheral blood mononuclear cells, Nelfinavir inhibited AKT and induced markers of ER stress. In summary, nelfinavir is well tolerated in cancer patients at doses 2.5 times the FDA-approved dose for HIV management and showed preliminary activity in tumors of neuroendocrine origin.

Lessons Learned This is the first human interventional study in patients with Cowden syndrome that is driven by inactivation of germline PTEN gene. Single-agent sirolimus, a mTOR inhibitor, suppressed mTOR signaling in surrogate human tissues without significant toxicity. Background Cowden syndrome is characterized by inactivating germline PTEN mutations, which can lead to activation of the PI3K-Akt-mTOR pathway. Methods Adult subjects with germline PTEN mutation who met international diagnostic criteria for Cowden syndrome and who had Eastern Cooperative Oncology Group (ECOG) performance status 0–2 and adequate organ function were enrolled. Subjects were treated with a 56-day course of daily oral sirolimus. In addition to symptom assessment and physical examination, dermatologic, endoscopic, neurologic (cerebellar), and radiographic assessments were conducted. Inhibition of the mTOR pathway in benign skin and gastrointestinal (GI) lesion was assessed by immunohistochemistry. Results A total of 18 patients and 16 families were enrolled. PTEN mutations were located at exons 1–8. Regression of skin and GI lesions was observed by dermoscopy or endoscopy. Neurological evaluation showed improvement in cerebellar function score at 1 month. Immunohistochemistry (IHC) analysis in skin and GI benign lesions showed a decrease in the ratio of phosphorylated (p)S6 to total S6 in response to sirolimus. Ratios of pS6K to total S6 at days 14 and 56 were significantly lower than at baseline (p = .0026, p = .00391, respectively). A 56-day course of sirolimus was well tolerated. Conclusion A 56-day course of sirolimus was well tolerated in subjects with Cowden syndrome and was associated with some evidence of improvement in symptoms, skin and GI lesions, cerebellar function, and decreased mTOR signaling.

Introduction The functional aspects of programmed death 1 (PD-1) and PD ligand 1 (PD-L1) immune checkpoints in malignant mesothelioma have not been studied. Methods Tumor samples from 65 patients with mesothelioma were evaluated for PD-L1 expression by immunohistochemistry, and its prognostic significance was examined. Malignant effusions from patients with pleural and peritoneal mesothelioma were evaluated for PD-1–positive and PD-L1–positive infiltrating lymphocytes and their role in inducing PD-L1 expression in tumor cells. Antibody-dependent cellular cytotoxicity (ADCC) of avelumab, a fully humanized immunoglobulin G1 anti PD-L1 antibody against primary mesothelioma cell lines, was evaluated in presence of autologous and allogeneic natural killer cells. Results Of 65 pleural and peritoneal mesothelioma tumors examined, 41 (63%) were PD-L1–positive, which was associated with slightly inferior overall survival compared to patients with PD-L1–negative tumors (median 23.0 versus 33.3 months, p = 0.35). The frequency of PD-L1 expression was similar in patients with pleural and peritoneal mesothelioma, with 62% and 64% of samples testing positive, respectively. In nine mesothelioma effusion samples evaluated, the fraction of cells expressing PD-L1 ranged from 12% to 83%. In seven patients with paired malignant effusion and peripheral blood mononuclear cell (PBMC) samples, PD-L1 expression was significantly higher on CD3-positive T cells present in malignant effusions as compared with PBMCs ( p = 0.016). In addition, the numbers of CD14-positive PD-1–positive cells were increased in malignant effusions compared with PBMCs ( p = 0.031). The lymphocytes present in malignant effusions recognized autologous tumor cells and induced interferon-γ–mediated PD-L1 expression on the tumor cell surface. Of the three primary mesothelioma cell lines tested, two were susceptible to avelumab-mediated ADCC in the presence of autologous natural killer cells. Conclusions Most pleural as well as peritoneal mesotheliomas express PD-L1. Malignant effusions in this disease are characterized by the presence of tumor cells and CD3-positive T cells that highly express PD-L1. In addition, mesothelioma tumor cells are susceptible to ADCC by the anti–PD-L1 antibody avelumab.

Activation of the serine/threonine kinase Akt contributes to the formation, maintenance, and therapeutic resistance of cancer, which is driving development of compounds that inhibit Akt. Phosphatidylinositol ether lipid analogues (PIA) are analogues of the products of phosphoinositide-3-kinase (PI3K) that inhibit Akt activation, translocation, and the proliferation of a broad spectrum of cancer cell types. To gain insight into the mechanism of PIAs, time-dependent transcriptional profiling of five active PIAs and the PI3K inhibitor LY294002 (LY) was conducted in non–small cell lung carcinoma cells using high-density oligonucleotide arrays. Gene ontology analysis revealed that genes involved in apoptosis, wounding response, and angiogenesis were upregulated by PIAs, whereas genes involved in DNA replication, repair, and mitosis were suppressed. Genes that exhibited early differential expression were partitioned into three groups; those induced by PIAs only (DUSP1, KLF6, CENTD2, BHLHB2, and PREX1), those commonly induced by PIAs and LY (TRIB1, KLF2, RHOB, and CDKN1A), and those commonly suppressed by PIAs and LY (IGFBP3, PCNA, PRIM1, MCM3, and HSPA1B). Increased expression of the tumor suppressors RHOB (RhoB), KLF6 (COPEB), and CDKN1A (p21Cip1/Waf1) was validated as an Akt-independent effect that contributed to PIA-induced cytotoxicity. Despite some overlap with LY, active PIAs have a distinct expression signature that contributes to their enhanced cytotoxicity. Mol Cancer Ther; 10(7); 1137–48. ©2011 AACR.

8504Background: Identifying the profile of DNA repair genes predisposing to MM will enable treatment options for patients (pts) and risk assessment for their families. Methods: In this prospective study of the natural history of MM (NCT01950572) we enrolled 239 consecutive pts independent of site of disease, family history of cancer, age at diagnosis, ethnicity, or asbestos exposure. Germline DNA was sequenced for all 239 pts, identifying mutations of all classes in 73 DNA repair genes. Tumor DNA from 12 pts with germline BAP1 mutations was evaluated by whole exome sequencing. Results: Of the 239 pts, 29 (12%) carried a pathogenic germline mutation in a DNA repair gene: BAP1 (N = 17 pts), CHEK2 (N = 5), PALB2 (N = 2), and BRCA2, MLH1, POT1, TP53, and MRE11A (N = 1 each). Pts with mutations were more likely to be female (P = 0.02) and to have been diagnosed with another cancer (P = 0.009). Pts with germline mutations were more likely to have a 1o relative with a diagnosis of MM (P < 0.0001), melanoma (P = ...

Genetic alterations and etiology of thymic epithelial tumors (TETs) are largely unknown, hampering the development of effective targeted therapies for patients with TETs. Here TETs of advanced-stage patients enrolled in a clinical trial of molecularly-guided targeted therapies were employed for targeted sequencing of 197 cancer-associated genes. Comparative sequence analysis of 78 TET/blood paired samples obtained from 47 thymic carcinoma (TC) and 31 thymoma patients revealed a total of 86 somatic non-synonymous sequence variations across 39 different genes in 33 (42%) TETs. TCs (62%; 29/47) showed higher incidence of somatic non-synonymous mutations than thymomas (13%; 4/31; p < 0.0001). TP53 was the most frequently mutated gene in TETs (n = 13; 17%), especially in TCs (26%), and was associated with a poorer overall survival (p < 0.0001). Genes in histone modification [BAP1 (n = 6; 13%), SETD2 (n = 5; 11%), ASXL1 (n = 2; 4%)], chromatin remodeling [SMARCA4 (n = 2; 4%)], and DNA methylation [DNMT3A (n = 3; 7%), TET2 (n = 2; 4%), WT1 (n = 2; 4%)] pathways were recurrently mutated in TCs, but not in thymomas. Our results suggest a potential disruption of epigenetic homeostasis in TCs, and a substantial difference in genetic makeup between TCs and thymomas. Further investigation is warranted into the roles of epigenetic dysregulation in TC development and its potential for targeted therapy.

The role of myeloid derived suppressor cells (MDSCs) in mediating tumor immunosuppression in patients with malignant mesothelioma has not been well characterized. The goal of our study was to analyze for the presence of both monocytic (Mo) and granulocytic (Gr) MDSCs in blood of mesothelioma patients, and characterize their mechanism of suppression of T cells responses. We evaluated the peripheral blood of patients (n = 23) for the presence of Gr-MDSC (CD11b+CD15+CD14-HLADR-) and Mo-MDSC (CD11b+CD14+HLADR-). Gr-MDSC (62.3±13.9% vs. 47.8±17.8%; p = 0.005) as well as monocytic MDSC (0.2±0.4% vs. 0.1±0.3%; p = 0.01) were significantly elevated in peripheral blood of mesothelioma patients as compared to healthy donors (n = 21). The frequency of Gr-MDSC was higher than Mo-MDSC. Thus, Gr-MDSCs appeared to be the main immunosuppressive population and were investigated further.Gr-MDSC from mesothelioma patients were found to inhibit the proliferation of both autologous CD4 and CD8 T cells (mean inhibition of proliferation of 61.9% for CD4 and 75.5% for CD8 at 1:2 T: Gr-MDSC ratio) and was accompanied by a decrease in IFN-γ released by T cells in the co-culture supernatants (92.4 pg/mL at 1:2 ratio). To determine the mechanism by which Gr-MDSC inhibit T cells, we tested the levels of reactive oxygen species (ROS), and nitrite oxide species and arginase activity in Gr-MDSC sorted from peripheral blood. Both arginase (0.3±0.3 vs. 0.2±0.3 μM) and nitrite levels (1.8±2.5 vs. 0 μM) in Gr-MDSC were below detection limit in both patients and donors and were marginally affected by the addition of their respective inhibitors (nor-NOHA and L-NMMA). However, the levels of ROS in Gr-MDSCs derived from mesothelioma patients were on average 3 folds higher than the Gr-myeloid cells from healthy donors (average MFI ∼15000 vs. ∼5000). In summary our results show that the major immunosuppressive cell population in mesothelioma patients are Gr-MDSCs and they suppress T cell proliferation and activation through release of reactive oxygen species. Citation Format: Swati Khanna, Francis Mussai, Anish Thomas, Gary Middleton, Constance Yuan, Betsy Morrow, Jingli Zhang, Ira Pastan, Maryalice Stetler-Stevenson, Raffit Hassan. Granulocytic MDSCs (CD11b+CD15+CD14-HLADR-) in peripheral blood of mesothelioma patients are elevated and suppress T cell proliferation and function via reactive oxygen species. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3247.

Mesothelin is a tumor differentiation antigen that is highly expressed in many cancers including malignant mesothelioma. RG7787 is a second-generation anti-mesothelin immunotoxin that is designed to be less immunogenic in patients and has reduced non-specific toxicity in pre-clinical studies. In this study, we evaluated the anti-tumor efficacy of RG7787 alone and in combination with nab-paclitaxel using primary mesothelioma cell lines obtained from patients with pleural or peritoneal mesothelioma. Four early passage malignant mesothelioma cell lines: NCI-Meso16, NCI-Meso19, NCI-Meso21 and NCI-Meso29 were evaluated for mesothelin expression and have 18×103- 56×103 mesothelin sites/cell. All four cell lines were sensitive to RG7787 with IC50s of 0.3 to 10 ng/ml. Three of the cell lines: NCI-Meso 16, NCI-Meso 21 and NCI-Meso 29 were also sensitive to nab-paclitaxel with IC50s of 4, 40 and 2 ng/ml respectively while, NCI-Meso 19 was resistant with IC50 >100 ng/ml. Synergistic in vitro cell cytotoxicity was observed with both NCI-Meso16 and NCI-Meso21 cells, when treated with the combination of RG7787 and nab-paclitaxel. To evaluate the efficacy of RG7787 with nab-paclitaxel in vivo, athymic nude mice were inoculated subcutaneously with 5×106 NCI-Meso 16 cells. When the tumors reached 100 mm3 in size, mice (n = 7 in each group) were treated with 2 cycles (separated by 3 days) of either RG7787 2.5 mg/kg every other day x 3 doses, nab-paclitaxel 100 mg/kg on day 1, a combination of RG7787 and nab-paclitaxel at the same dose and schedule as for single agent therapy, or received no treatment (control). Although treatment with RG7787 and nab-paclitaxel alone resulted in tumor shrinkage compared to control mice, none of the mice had complete tumor regressions. However, in mice treated with RG7787 plus nab-paclitaxel all mice had complete tumor regressions by day 64 that persisted in 7/7 mice when the experiment was terminated 100 days after starting treatment. In the slow growing NCI-Meso 21 in-vivo model, we also observed similar synergistic anti-tumor effects with the combination of RG7787 and nab-paclitaxel. Taken together, our findings show that RG7787 in combination with nab-paclitaxel has significant anti-tumor activity against primary mesothelioma cell lines both in vitro and in vivo, suggesting that this combination treatment could be useful to treat patients with mesothelioma. Citation Format: Jingli Zhang, Qun Jiang, Christine Alewine, Swati Khanna, Betsy Morrow, Anish Thomas, Ira Pastan, Raffit Hassan. The reduced immunogenicity anti-mesothelin immunotoxin RG7787 in combination with nab-paclitaxel has significant anti-tumor efficacy against primary mesothelioma cell lines and patient derived mesothelioma tumor xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3103.

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

2532 Background: CS is characterized by germline PTEN mutations. Because tumors from CS patients show increased activation of the PI3K/Akt/mTOR pathway, mTOR inhibitor such as S might have activity in such patients. Methods: Eligibility: subjects with germline PTEN mutation who meet international diagnostic criteria for CS, age 18, ECOG PS 0-2, and adequate organ function. Subjects were treated with a 56-day course of daily oral S (2 mg). Objective: Inhibition of the mTOR pathway in benign skin/GI lesion, as assessed by IHC (P-AKT, Total S6, P-S6, P-4E-BP1, score 0-4), changes in benign or malignant tumor by CT/MRI/PET, digital dermoscopy/endoscopy, and changes in cerebellar testing by modified SARA (Neurology 2006). Protocol was amended to allow up to 20 subjects. Results: A total of 18 pts/16 families were enrolled. Median age 42 (range 19-69). Male/Female: 9/9. Involvement in skin, thyroid, GI polyps, breast, CNS, and all of the five organs was observed in 18, 15, 13, 8, 18, 5 subjects, respectively. 7 had h/o malignancies: 3 renal cell, 4 breast, 3 thyroid, 4 others. 3 cerebellar gangliocytomas and 2 others were measurable by CT or MRI. PTEN mutations: 6 families in Exon 1-2, 1 in Exon 4, 9 in Exon 5-8. All but one (D24H) were truncating mutations. 11 of 16 pts who completed a 56-day course reported subjective improvement in energy, mood, focus or skin lesion. Pts with Ex6-8 mutation (n=4) had a median SUV decrease of 29.4%. Regression of skin and GI lesions was observed by dermoscopy or endoscopy. Cerebellar evaluation showed a significant improvement in a total SARA score at 1 month (n=9, p=0.034). 3 pts were treated for only 28 days due to voluntary withdrawal. IHC analysis in skin and GI benign lesions showed a decrease in average P-S6K, P-S6, Total S6, P-S6/Total S6 in response to S. P-S6K/Total S6 ratios at d14 and d56 were significantly lower than at baseline (p=0.0026, 0.0391, respectively). The most common AEs (all grades >25%) were LFTs/Hb (39%), fatigue/hypercholesterolemia (28%). Grade 3 AEs: 1 pt hypophosphatemia/lymphopenia. Conclusions: A 56-day course of S was well tolerated in subjects with CS and was associated with improvement in symptoms, skin/GI lesions, cerebellar function and decreased mTOR signaling. Clinical trial information: NCT00971789.

Increasing number of oncogenic mutations important to the clinical management of advanced thoracic malignancies is being identified. To translate this into routine clinical testing environment, we developed and validated a targeted exome sequencing method using next generation sequencing (NGS) technology that is designed to detect both single nucleotide variants and short insertions or deletions (indels) by employing a custom designed target selection system. In each individual assay, 197 cancer related genes, including 12 clinically relevant genes (AKT1, AKT2, BRAF, EGFR, ERBB2, HRAS, KIT, KRAS, MEK1, NRAS, PIK3CA, and PTEN) are simultaneously sequenced from 100-500ng of total DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. To date, we have performed this assay on over 250 FFPE tissues from Non-Small-Cell Lung Cancers (NSCLC), Small-Cell Lung Cancers (SCLC), and thymic malignancies. Mutations in all 12 clinically relevant genes including EGFR L858R, KRAS codon 12 missense mutations as well as EGFR exon 19 deletions, EGFR exon 20 insertions, and ERBB2 exon 20 insertions have been identified and validated with alternate technology in approximately 30% of all samples tested. Compared with conventional capillary sequencing, which typically requires in excess of 25% mutant DNA, this assay can detect mutations with 5% - 10% mutant allele frequency. This is a robust, reliable and cost effective assay that not only is suitable for prospective molecular profiling of clinically relevant targets, but also provides a powerful tool for retrospective analysis of existing FFPE tissues from clinical trials. Citation Format: Christopher C. Lau, Keith Killian, Yuelin J. Zhu, Yonghong Wang, Marbin Pineda, Anish Thomas, Yisong Wang, Betsy Morrow, Liqiang Xi, Mark Raffeld, Giuseppe Giaccone, Paul S. Meltzer. A targeted exome sequencing platform for routine clinical molecular profiling of advanced thoracic malignancies relevant to targeted therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1999. doi:10.1158/1538-7445.AM2013-1999

EGFR tyrosine kinase inhibitors (TKIs) are clinically useful agents for the treatment of NSCLC, yet resistance ultimately develops. In an effort to develop strategies to combat resistance and prolong patient benefit, EGFR TKIs were combined with the Akt inhibitor triciribine in a panel of EGFR TKI-resistant NSCLC cell lines. The combination synergistically reduced proliferation in cells that expressed a variety of resistance mechanisms, including intrinsic EGFR T790M mutations, K-Ras mutations, and Met amplification. The combination of erlotinib and triciribine significantly increased apoptosis in H1975 and H1650 cells. It also decreased the phosphorylation of Akt and PRAS40, C-Raf, MEK, and ERK to a greater extent than either drug alone. Unbiased analysis using reverse phase lysate arrays confirmed decreased activation of PI3K/Akt pathway components as well as ERK. The effect of the combination appeared to depend on downregulation of Akt, since a constitutively active form of Akt abrogated the decreased proliferation and increase in cell death caused by the combination. In vivo, the combination inhibited the growth of H1975 xenografts to a greater extent than either drug alone. When tested in an invariably progressive, transgenic model of EGFR-driven lung tumorigenesis (EGFR L858R/T790M), the combination did not cause regression of tumors but stabilized disease in approximately 80% of mice. We conclude the combination of triciribine with EGFR TKIs is promising and should be explored clinically, and that combining inhibitors of the PI3K/Akt pathway with EGFR TKIs may be a useful strategy to overcome EGFR TKI resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 624.