Betsy Morrow

Molecular Profiling and Targeted Therapy for Advanced Thoracic Malignancies: A Biomarker-Derived, Multiarm, Multihistology Phase II Basket Trial

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.

Malignant Mesothelioma Effusions Are Infiltrated by CD3+ T Cells Highly Expressing PD-L1 and the PD-L1+ Tumor Cells within These Effusions Are Susceptible to ADCC by the Anti–PD-L1 Antibody Avelumab

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‐&ggr;–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.

Mutations of epigenetic regulatory genes are common in thymic carcinomas

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.

Expression signatures of the lipid-based Akt inhibitors phosphatidylinositol ether lipid analogues in NSCLC cells

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.

Tumor-Derived GM-CSF Promotes Granulocyte Immunosuppression in Mesothelioma Patients

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.

Elevated Serum Megakaryocyte Potentiating Factor as a Predictor of Poor Survival in Patients with Mesothelioma and Primary Lung Cancer

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.

Abstract 3247: 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

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.

Abstract 3103: 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

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.

Abstract 624: Synergistic effects of combining the Akt inhibitor triciribine with erlotinib in EGFR TKI-resistant NSCLC

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC 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.