Kosari, Farhad

Background Immune checkpoint inhibitors (ICIs) are now a first-line treatment option for patients with pleural mesothelioma with the recent approval of ipilimumab and nivolumab. Mesothelioma has a low tumor mutation burden and no robust predictors of survival with ICI. Since ICIs enable adaptive antitumor immune responses, we investigated T-cell receptor (TCR) associations with survival in participants from two clinical trials treated with ICI. Methods We included patients with pleural mesothelioma who were treated with nivolumab (NivoMes, NCT02497508) or nivolumab and ipilimumab (INITIATE, NCT03048474) after first-line therapy. TCR sequencing was performed with the ImmunoSEQ assay in 49 and 39 pretreatment and post-treatment patient peripheral blood mononuclear cell (PBMC) samples. These data were integrated with TCR sequences found in bulk RNAseq data by TRUST4 program in 45 and 35 pretreatment and post-treatment tumor biopsy samples and TCR sequences from over 600 healthy controls. The TCR sequences were clustered into groups of shared antigen specificity using GIANA. Associations of TCR clusters with overall survival were determined by cox proportional hazard analysis. Results We identified 4.2 million and 12 thousand complementarity-determining region 3 (CDR3) sequences from PBMCs and tumors, respectively, in patients treated with ICI. These CDR3 sequences were integrated with 2.1 million publically available CDR3 sequences from healthy controls and clustered. ICI-enhanced T-cell infiltration and expanded T cell diversity in tumors. Cases with TCR clones in the top tertile in the pretreatment tissue or in circulation had significantly better survival than the bottom two tertiles (p<0.04). Furthermore, a high number of shared TCR clones between pretreatment tissue and in circulation was associated with improved survival (p=0.01). To potentially select antitumor clusters, we filtered for clusters that were (1) not found in healthy controls, (2) recurrent in multiple patients with mesothelioma, and (3) more prevalent in post-treatment than pretreatment samples. The detection of two-specific TCR clusters provided significant survival benefit compared with detection of 1 cluster (HR<0.001, p=0.026) or the detection of no TCR clusters (HR=0.10, p=0.002). These two clusters were not found in bulk tissue RNA-seq data and have not been reported in public CDR3 databases. Conclusions We identified two unique TCR clusters that were associated with survival on treatment with ICI in patients with pleural mesothelioma. These clusters may enable approaches for antigen discovery and inform future targets for design of adoptive T cell therapies.

Tiny silicon microchip–based high-throughput hanging drop arrays allow drug screening and geometric control.

A nano-resolution PDMS sensor simultaneously measures cell force and matrix stiffening dynamics in 3D.

Abstract Lung cancer is the leading cause of cancer death among both men and women in the United States. Small cell lung cancer (SCLC) results in over 30,000 deaths of Americans annually and comprises 15-17% incidences of lung cancer. SCLC is the most aggressive form of lung cancer due to its rapid doubling time and early widespread metastasis. Most SCLC patients present with advanced disease, respond to initial systemic chemotherapy, and then treatment refractory progression usually occurs within one year due to acquired drug resistance. Consequently, the median survival time of SCLC patients is only 9 to 20 months and merely 7% of SCLC patients survive beyond five years. A better molecular understanding of SCLC is necessary to develop much needed new treatment strategies. For the first time, we demonstrate that dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) and its splice variant t-DARPP stimulate tumors of neuroendocrine origin, and we show DARPP-32 proteins are aberrantly overexpressed in human SCLC. Upregulation of t-DARPP protein in a subset of SCLC patients positively correlates with overexpression of genes associated with Notch signaling, including achaete-scute homologue 1 (ASCL1). We reveal that DARPP-32 isoforms are transcriptionally activated by ASCL1 in human SCLC cells. To determine whether DARPP-32 drives SCLC growth in vivo, we utilized an orthotopic lung cancer xenograft mouse model. Mice challenged with DARPP-32-ablated DMS-53 cells show a substantial decrease in lung tumor growth relative to controls. Correspondingly, we demonstrate increased tumor growth in mice harboring lung tumors formed by administration of DMS-53 or H1048 cells overexpressing DARPP-32 and t-DARPP proteins. Mechanistically, we describe how DARPP-32 isoforms promote SCLC growth through increased proliferation, Akt/Erk-mediated survival, and anti-apoptotic signaling. Taken together, we define new regulatory mechanisms of SCLC oncogenesis that suggest DARPP-32 isoforms may represent a negative prognostic indicator for SCLC and serve as a potential target for the development of new therapies. Citation Format: Sk. Kayum Alam, Li Wang, Yanan Ren, Christina E. Hernandez, Farhad Kosari, Anja C. Roden, Rendong Yang, Luke H. Hoeppner. New molecular mechanisms of small cell lung cancer growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5956.

Abstract Introduction: Lung cancer is the leading cause of cancer death in the U.S. and in the world. In 2018, lung cancer alone resulted in 25.3% of all cancer deaths evaluated worldwide. Lung Adenocarcinoma (LUAD) is a major subtype of lung cancer and accounts for 40% of these tumors. Our group has recently discovered that about 20% of LUAD are characterized by abnormally high expression of the neuroendocrine transcription factor, ASCL1. In these tumors, ASCL1 orchestrates a network of genomic changes that create opportunities for treatment. We have shown that LUAD tumors expressing high ASCL1 have RET mRNA levels that are predictive of patient survival (p &lt; 0.004), and, these tumors were susceptible to EGFR inhibitors, with having a functional interaction between RET and EGFR. We found EGFR inhibitor, Lapatinib, to have highest efficacy, indicating it to be a primary drug treatment for ASCL1+ (A+) LUAD. However, we and others have observed Lapatinib induces resistance to EGFR therapy over time, and, Lapatinib therapy associated by-pass signaling pathways have recently gained attention. Therefore, we hypothesize that for a long lasting therapy, it is important to identify additional drugs that in combination with EGFR inhibitor Lapatinib will eliminate A+ LUAD tumors. Methodology and Results: An in vitro preliminary screen of approx. 2000 FDA approved small molecule inhibitors done with A+ and A- LUAD cell lines in collaboration at NIH revealed potential candidates for alternative pathways, 80 of which showed a differential sensitivity to A+ LUAD as single agent. We identified best 28 drugs in total: 23 selected from this pre-screen (based on their effectiveness to A+LUAD lines) and 5 additional chosen based on their biological relevance to the disease, which may act upon putative compensatory pathways in EGFR therapy resistance. Our in vitro drug screening in HCC1833 ASCL1+ and ASCL1 knockdown lines has revealed 8 out of 28 drugs to synergize as per their IC50 and fixed ratios, with Lapatinib. From these 8 leads, we have confirmed a definite synergy of Lapatinib with DDR-1 7rh, an inhibitor of ECM receptor DDR-1, using Mac-Synergy-II software. DDR-1, Discoidin domain receptor-1, is a collagen receptor highly upregulated in NSCLC, and is associated with poor prognosis in LUAD. Our microarray data on lapatinib treated tumors from mice PDX has shown ECM signatures to be among top upregulated. Additionally, TM4SF1, a signaling molecule associated with DDR-1, was found to associate with poor outcome in A+LUAD (P=0.0001). Other synergistic drugs identified include, a K+ channel opener (Retigabine), Wnt signaling and Lyn kinase inhibitors (PRI-724, and Bafetinib), Tubulin inhibitor (Docetaxel) and 3 multi-tyrosine kinase inhibitors. Interestingly, Wnt pathway was found to be elevated in Lapatinib treated ASCL1+LUAD in our microarray data. Moreover, Wnt signaling in known to crosstalk with ECM and DDR-1 in other tumors. K+ channel dysfunction is associated with neuronal/neuroendocrine disorders and Lyn kinases are the predominant SRC kinases in brain tumors, indicating their possible involvement in ASCL1+ neuroendocrine tumor pathophysiology. Conclusion: Taken together this data reveals a potential involvement of ECM (DDR-1 receptor) and Wnt signaling in effect of Lapatinib, and the identified leads as path to novel combination therapies for A+ LUAD. Citation Format: Kshama Gupta, Farhad Kosari. Identification of the synergistic drugs in combination with EGFR therapy that can target ASCL1+ Lung Adenocarcinoma (A+ LUAD) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A143. doi:10.1158/1535-7163.TARG-19-A143

Abstract Introduction: While most small cell lung cancer (SCLC) patients die within a few months, a sub-group of patients survive for many years. Factors determining long-term survivorship remain largely unknown. We present the first comprehensive comparative genomic and tumor microenvironment analyses of small cell lung cancer (SCLC) between patients with long term (LTS) and expected (EXS) survival times. Methods: We compared surgically resected tumors of 23 LTS (survival &gt; 4 years) and 18 EXS (survival ≤ 2 years). There were no differences in clinical variables including TNM staging and curative versus non-curative intend surgery between the groups. Gene expression profiling was performed by microarrays and tumor microenvironment analyses were by IHC of prominent immune related markers. Results: Immune related genes and pathways represented the majority of the differentially overexpressed genes in LTS compared to the EXS. The differences in the immunological tumor-microenvironment were confirmed by quantitative immuno-staining. Increased numbers of tumor infiltrating and associated lymphocytes were present throughout tumors of LTS. Several differentiating patterns of enhanced anti-tumor immunity were identified. While some areas of LTS tumors also harbored higher numbers of suppressive immune cells (monocytes, regulatory lymphocytes, and macrophages), ratios of these suppressive cells to CD3+ lymphocytes were generally lower in LTS tumors indicating a more tumor suppressive microenvironment. Conclusions: Our data demonstrate that long-term survivorship of SCLC patients is strongly influenced by the presence of anti-tumor immune cells in the tumor microenvironment. Characterization of the anti-tumor immune responses may identify opportunities for individualized immunotherapies for SCLC. Citation Format: Farhad Kosari, Prasuna Muppa, Simone Terra, Anurag Sharma, Aaron Mansfield, Marie Christine Aubry, Nafiseh Janaki, Aqsa Nasir, Tobias Peikert. Anti-tumor immunity is a key determinant of small cell lung cancer survivorship [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 4577.

Abstract Malignant pleural mesothelioma is a disease primarily associated with exposure to the carcinogen asbestos. Consistent with this carcinogenic exposure, cytogenetic analyses have identified multiple recurrent structural chromosomal abnormalities in this malignancy, but more recent high-throughput sequencing evaluations of point mutations suggest that there is a low mutational burden in mesothelioma. Since tumor mutational burden has been correlated with responses to treatment with immune checkpoint inhibitors such as nivolumab, it was not consistent that patients with mesothelioma and low mutation burdens would have similar response rates in clinical trials with immune checkpoint inhibitors as patients with non-small cell lung cancer which is associated with a high mutation burden. In order to reconcile these differences, and given the potential for an improved understanding of the molecular pathogenesis of mesothelioma to improve therapeutic options, we used mate-pair sequencing (MPseq) and RNA sequencing (RNAseq) to understand how structural variants affect the transcriptome. MPseq differs from standard next generation sequencing approaches by tiling the whole genome with larger fragments (2-5kb) to reliably detect structural variants such as insertions, deletions and rearrangements. Amongst 22 mesothelioma specimens there were 1535 chromosomal rearrangements (median 41, range 3-298 per specimen), that resulted in junctions or novel fusions of non-coding DNA or genes. Six-hundred thirty-seven of these rearrangements (median 22, range 5-103 range per specimen) resulted in novel fusions of genes. Many of these inter- or intra-chromosomal rearrangements were consistent with a pattern of chromoanagesis such as chromoplexy or chromothripsis. Chromosomal rearrangements detected by MPseq were used to guide analysis of RNAseq data and revealed that these chromosomal junctions resulted in the expression of 179 novel amino acid sequences (median 5, 0-51 range per specimen). To determine whether transcription of chromosomal rearrangement-related junctions have neoantigenic potential, we used in silico tools to determine whether any of the expressed junctions contained peptides that could be presented by patient-specific HLA molecules. The top candidate rearrangement-related peptides with neoantigenic potential bound patient-specific HLA molecules nearly as well or as well as a positive control in competitive binding assays. Our findings represent the discovery of potential neoantigen expression driven by structural chromosomal rearrangements. These results may have implications for the development of novel therapeutic strategies, the selection of patients to receive immunotherapy, and blood-based treatment monitoring strategies. Citation Format: Aaron S. Mansfield, Tobias Peikert, James B. Smadbeck, Julia B. Udell, Farhad Kosari, Stephen J. Murphy, Hongzheng Ren, Vishnu V. Serla, Janet L. Schaefer Klein, Giannoula Karagouga, Faye R. Harris, Carlos Sosa, Sarah H. Johnson, Wendy Nevala, Svetomir N. Markovic, Aaron O. Bungum, Eric S. Edell, Haidong Dong, John C. Cheville, Marie Christine Aubry, Jin Jen, George Vasmatzis. Rearrangement-related peptides with neoantigenic potential in malignant pleural mesothelioma [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 5726.

AbstractCopy number variation (CNV) is a common form of structural variation detected in human genomes, occurring as both constitutional and somatic events. Cytogenetic techniques like chromosomal microarray (CMA) are widely used in analyzing CNVs. However, CMA techniques cannot resolve the full nature of these structural variations (i.e. the orientation and location of associated breakpoint junctions) and must be combined with other cytogenetic techniques, such as karyotyping or FISH, to do so. This makes the development of a next‐generation sequencing (NGS) approach capable of resolving both CNVs and breakpoint junctions desirable. Mate‐pair sequencing (MPseq) is a NGS technology designed to find large structural rearrangements across the entire genome. Here we present an algorithm capable of performing copy number analysis from mate‐pair sequencing data. The algorithm uses a step‐wise procedure involving normalization, segmentation, and classification of the sequencing data. The segmentation technique combines both read depth and discordant mate‐pair reads to increase the sensitivity and resolution of CNV calls. The method is particularly suited to MPseq, which is designed to detect breakpoint junctions at high resolution. This allows for the classification step to accurately calculate copy number levels at the relatively low read depth of MPseq. Here we compare results for a series of hematological cancer samples that were tested with CMA and MPseq. We demonstrate comparable sensitivity to the state‐of‐the‐art CMA technology, with the benefit of improved breakpoint resolution. The algorithm provides a powerful analytical tool for the analysis of MPseq results in cancer.

Abstract Purpose: Pulmonary carcinoid tumors account for up to 5% of all lung malignancies in adults, comprise 30% of all carcinoid malignancies, and are defined histologically as typical carcinoid (TC) and atypical carcinoid (AC) tumors. The role of specific genomic alterations in the pathogenesis of pulmonary carcinoid tumors remains poorly understood. We sought to identify genomic alterations and pathways that are deregulated in these tumors to find novel therapeutic targets for pulmonary carcinoid tumors. Experimental Design: We performed integrated genomic analysis of carcinoid tumors comprising whole genome and exome sequencing, mRNA expression profiling and SNP genotyping of specimens from normal lung, TC and AC, and small cell lung carcinoma (SCLC) to fully represent the lung neuroendocrine tumor spectrum. Results: Analysis of sequencing data found recurrent mutations in cancer genes including ATP1A2, CNNM1, MACF1, RAB38, NF1, RAD51C, TAF1L, EPHB2, POLR3B, and AGFG1. The mutated genes are involved in biological processes including cellular metabolism, cell division cycle, cell death, apoptosis, and immune regulation. The top most significantly mutated genes were TMEM41B, DEFB127, WDYHV1, and TBPL1. Pathway analysis of significantly mutated and cancer driver genes implicated MAPK/ERK and amyloid beta precursor protein (APP) pathways whereas analysis of CNV and gene expression data suggested deregulation of the NF-κB and MAPK/ERK pathways. The mutation signature was predominantly C&gt;T and T&gt;C transitions with a minor contribution of T&gt;G transversions. Conclusions: This study identified mutated genes affecting cancer relevant pathways and biological processes that could provide opportunities for developing targeted therapies for pulmonary carcinoid tumors. Clin Cancer Res; 24(7); 1691–704. ©2018 AACR.

Abstract TMPRSS2-ERG gene fusions occur in over 50% of prostate cancers, but their impact on clinical outcomes is not well understood. Retention of interstitial genes between TMPRSS2 and ERG has been reported to influence tumor progression in an animal model. In this study, we analyzed the status of TMPRSS2-ERG fusion genes and interstitial genes in tumors from a large cohort of men treated surgically for prostate cancer, associating alterations with biochemical progression. Through whole-genome mate pair sequencing, we mapped and classified rearrangements driving ETS family gene fusions in 133 cases of very low-, low-, intermediate-, and high-risk prostate cancer from radical prostatectomy specimens. TMPRSS2-ERG gene fusions were observed in 44% of cases, and over 90% of these fusions occurred in ERG exons 3 or 4. ERG fusions retaining interstitial sequences occurred more frequently in very low-risk tumors. These tumors also frequently displayed ERG gene fusions involving alternative 5′-partners to TMPRSS2, specifically SLC45A3 and NDRG1 and other ETS family genes, which retained interstitial TMPRSS2/ERG sequences. Lastly, tumors displaying TMPRSS2-ERG fusions that retained interstitial genes were less likely to be associated with biochemical recurrence (P = 0.028). Our results point to more favorable clinical outcomes in patients with ETS family fusion-positive prostate cancers, which retain potential tumor-suppressor genes in the interstitial regions between TMPRSS2 and ERG. Identifying these patients at biopsy might improve patient management, particularly with regard to active surveillance. Cancer Res; 77(22); 6157–67. ©2017 AACR.