Matthew Genet

Infiltrating T Cells Increase IDO1 Expression in Glioblastoma and Contribute to Decreased Patient Survival

Purpose: Indoleamine 2,3 dioxygenase 1 (IDO1) mediates potent immunosuppression in multiple preclinical models of cancer. However, the basis for elevated IDO1 expression in human cancer, including the most common primary malignant brain tumor in adults, glioblastoma (GBM), is poorly understood. The major objective of this study is to address this gap in our understanding of how IDO1 expression contributes to the biology of GBM, and whether its level of expression is a determinant of GBM patient outcome. Experimental Design: Patient-resected GBM, The Cancer Genome Atlas, human T-cell:GBM cocultures, as well as nu/nu, NOD-scid, and humanized (NSG-SGM3-BLT) mice-engrafted human GBM form the basis of our investigation. Results: In situ hybridization for IDO1 revealed transcript expression throughout patient-resected GBM, whereas immunohistochemical IDO1 positivity was highly variable. Multivariate statistical analysis revealed that higher levels of IDO1 transcript predict a poor patient prognosis (P = 0.0076). GBM IDO1 mRNA levels positively correlated with increased gene expression for markers of cytolytic and regulatory T cells, in addition to decreased patient survival. Humanized mice intracranially engrafted human GBM revealed an IFNγ-associated T-cell–mediated increase of intratumoral IDO1. Conclusions: Our data demonstrate that high intratumoral IDO1 mRNA levels correlate with a poor GBM patient prognosis. It also confirms the positive correlation between increased GBM IDO1 levels and human-infiltrating T cells. Collectively, this study suggests that future efforts aimed at increasing T-cell–mediated effects against GBM should consider combinatorial approaches that coinhibit potential T-cell–mediated IDO1 enhancement during therapy. Clin Cancer Res; 23(21); 6650–60. ©2017 AACR.

Improving vaccine efficacy against malignant glioma

ABSTRACT The effective treatment of adult and pediatric malignant glioma is a significant clinical challenge. In adults, glioblastoma (GBM) accounts for the majority of malignant glioma diagnoses with a median survival of 14.6 mo. In children, malignant glioma accounts for 20% of primary CNS tumors with a median survival of less than 1 y. Here, we discuss vaccine treatment for children diagnosed with malignant glioma, through targeting EphA2, IL-13Rα2 and/or histone H3 K27M, while in adults, treatments with RINTEGA, Prophage Series G-100 and dendritic cells are explored. We conclude by proposing new strategies that are built on current vaccine technologies and improved upon with novel combinatorial approaches.

Advanced age negatively impacts survival in an experimental brain tumor model

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults, with an average age of 64 years at the time of diagnosis. To study GBM, a number of mouse brain tumor models have been utilized. In these animal models, subjects tend to range from 6 to 12 weeks of age, which is analogous to that of a human teenager. Here, we examined the impact of age on host immunity and the gene expression associated with immune evasion in immunocompetent mice engrafted with syngeneic intracranial GL261. The data indicate that, in mice with brain tumors, youth conveys an advantage to survival. While age did not affect the tumor-infiltrating T cell phenotype or quantity, we discovered that old mice express higher levels of the immunoevasion enzyme, IDO1, which was decreased by the presence of brain tumor. Interestingly, other genes associated with promoting immunosuppression including CTLA-4, PD-L1 and FoxP3, were unaffected by age. These data highlight the possibility that IDO1 contributes to faster GBM outgrowth with advanced age, providing rationale for future investigation into immunotherapeutic targeting in the future.

Indoleamine 2,3-dioxygenase 1 and overall survival of patients diagnosed with esophageal cancer

Background Indoleamine 2,3-dioxygenase 1 (IDO1) is an enzyme with immunomodulatory properties that has emerged as a potential immunotherapeutic target in human cancer. However, the role, expression pattern, and relevance of IDO1 in esophageal cancer (EC) are poorly understood. Here, we utilize gene expression analysis of the cancer genome atlas (TCGA) and immunohistochemistry (IHC) to better understand the role and prognostic significance of IDO1 in EC. Results High IDO1 mRNA levels were associated with worse overall survival (OS) in both esophageal squamous cell carcinoma (SCC) (P = 0.02) and adenocarcinoma (AC) (P = 0.036). High co-expression of IDO1 and programmed death ligand 1 (PD-L1) was associated with worse OS in SCC (P = 0.0031) and AC (P = 0.0186). IHC for IDO1 in SCC showed a significant correlation with PD-L1 (P < 0.0001) and CD3ε (P < 0.0001). Conclusions EC with high IDO1 and PD-L1 expression is significantly correlated with decreased patient survival, and may correlate with increased T-cells. These data suggest that simultaneous inhibition of IDO1 and PD-(L)1 may overcome important barriers to T-cell mediated immune rejection of EC. Materials and Methods mRNA expression data from TCGA (SCC N = 87; AC N = 97). IHC in a second cohort of EC (N = 93) were stained for IDO1, PD-L1, and CD3ε, followed by light microscopic analysis.

The Kynurenine/Tryptophan Ratio and Glioblastoma Patients Treated with Hsppc-96 Vaccine

The discovery that immunotherapy is a clinically-relevant approach for the treatment of malignant tumors is revolutionizing patient care. In adults diagnosed with glioblastoma (GBM), an aggressive and incurable primary brain tumor, autologous HSPPC-96 vaccination provides a significant increase in overall survival. However, all GBM patients eventually succumb to their disease, providing rationale for discovering new methods that proactively identify individuals that will respond, optimally. Of the immunosuppressive mediators that contribute to the inhibition of productive tumor immunity, indoleamine 2,3 dioxygenase 1 (IDO1), a rate-limiting enzyme that catabolizes tryptophan (Trp) into kynurenine (Kyn), has been demonstrated to be expressed at elevated levels in patients with malignant glioma. Recently, our group determined that a correlation exists between peripheral blood Trp and Kyn levels in GBM patients and the association with overall survival after HSPPC-96 treatment. Our findings indicate that the Kyn/Trp ratio may be a useful benchmark for identifying GBM patients with a higher likelihood to survive longer after vaccination. The relevance to future clinical trials, the limitations of brain tumor models to address these findings and the role of IDO1 versus tryptophan dioxygenase (TDO) in the maintenance of peripheral Trp and Kyn levels, is discussed.

Abstract A064: IDO1 expression stratifies glioblastoma patient survival and correlates with dominantly immunosuppressive pathways

Purpose : The objective of this study was to evaluate the prognostic significance of the immunosuppressive molecule indoleamine 2, 3-dioxygenase ( IDO1 ) mRNA expression in patients with low-grade glioma (LGG) and glioblastoma (GBM). Experimental Design : Hi-RNA-seq. Illumina data mined from the cancer genome atlas (TCGA) reflecting 475 LGG (WHO grade II and III) and 172 GBM patients were utilized to characterize the pattern of IDO1 mRNA expression. Kaplan-Meier (KM) survival analysis and multivariate Cox proportional hazards regression were conducted to evaluate the significance of IDO1 expression as a stratification marker in glioma patients. mRNA expression of proinflammatory and immunosuppressive factor genes were also investigated for their correlation with IDO1 mRNA expression. Ivy GAP analysis was further utilized to localize IDO1 mRNA within different GBM anatomic niches. Results: Expression profiling of the TCGA revealed a distinct pattern of progressively increasing IDO1 mRNA levels correlating with decreased survival both in LGG and GBM patients. In grade III LGG and GBM, IDO1 mRNA expression was significantly decreased when mutant isocitrate dehydrogenase 1 and 2 (m IDH1/2 ) was coincidently expressed when compared to samples with wild-type IDH1/2 expression (mean difference = 1.51, P P IDO1 mRNA expression levels to be an independent prognostic factor for survival among LGG ( P = 0.0115; HR = 1.68) and GBM ( P = 0.0076; HR = 1.82) patients. Pearson9s correlation analysis identified significant associations among GBM between IDO 1 and the genes encoding immunosuppressive factors including PD-L1 ( r = 0.2993, P PD-L2 ( r = 0.4871, P PD-1 ( r = 0.3416, P CTLA-4 ( r = 0.3534, P STAT3 ( r = 0.2366, P = 0.0018), CD39 ( r = 0.2691, P = 0.0004), BTLA ( r = 0.2981, P Lag3 ( r = 0.2567, P = 0.0007), FoxP3 ( r = 0.1865, P FGL2 ( r = 0.4267, P IDO1 mRNA levels were higher in the cellular (1.398 ± 0.1257, mean ± SEM) and necrotic (1.543 ± 0.1489) GBM zones, when compared to infiltrating (0.9303 ± 0.0786) and leading margins (0.9064 ± 0.1224). Additionally, for the IDO1 paralog genes IDO2 and TDO2 , which encodes indoleamine 2, 3-dioxygenase 2 and tryptophan dioxygenase respectively, no overall differential expression pattern were observed between different pathological grades nor a significant stratification in LGG and GBM patient survival. Conclusions : Our data show for the first time that IDO1 mRNA levels can be used as an independent prognostic variable for patients with LGG and/or GBM. Given that ongoing IDO1-targeted immunotherapy clinical trials aim to inhibit enzymatic activity in malignant glioma, our data suggest that targeted subject enrollment may result in a better clinical response to therapy. Citation Format: Lijie Zhai, Matthew Genet, Erik Ladomersky, Kristen Lauing, Meijing Wu, David Binder, Leo Kim, Jeremy Rich, Craig Horbinski, C. David James, Jeffrey A. Sosman, Orin Bloch, Derek A. Wainwright. IDO1 expression stratifies glioblastoma patient survival and correlates with dominantly immunosuppressive pathways [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A064.

Abstract B018: A novel IDO1 inhibitor combined with targeted immunotherapy durably increases survival in a mouse model of glioblastoma

Glioblastoma (GBM) is the most common primary brain tumor, accounting for 54% of malignant glioma diagnoses. Over the course of the past 30 years, a diagnosis of GBM has remained fatal even after maximum surgical resection, radiotherapy (RT), and chemotherapy, with a median overall survival of 14.6 months. The immunosuppressive microenvironment of GBM is a major contributor to the poor patient outcome. Expression of IDO1, as well as the accumulation of tumor-infiltrating regulatory T cells contribute to the avoidance of immune surveillance. Although current immunotherapies have had some success in extending patient survival, combinatorial treatment approaches addressing both tumor growth and the potent immunosuppression may prove to be more effective. This work aimed to determine the efficacy of a novel, pharmaceutical-grade, blood brain barrier-penetrating small molecule IDO1 inhibitor, BGB-5777, in combination with PD-1 blockade and/or whole brain radiation in an immunocompetent mouse GBM model. METHODS/RESULTS: All mice were intracranially-engrafted 2×105 GL261 (syngeneic to B6 background) cells to recapitulate brain tumors. At 14 days post-intracranial injection (dp-ic.), mice were treated with IgG alone as a control (n = 7), 2Gy RT for 5 days (n = 8), 500 ug (loading dose), followed by three 200 ug maintenance doses given every 3 days, of PD-1 mAb (J43) (n = 8), or 100mg/kg BGB-5777 for 4 weeks (n = 10), with a median overall survival (OS) of 25, 25, 32, and 26.5 days, respectively. Mice treated with dual therapies including RT and PD-1 mAb (n = 10), RT and BGB-5777 (n = 8), or PD-1 mAb and BGB-5777 (n = 9), had a median OS of 30, 39, and 32 days, respectively. All mice treated with mono- or dual-therapy succumbed to tumor burden. In contrast, mice treated with concurrent RT, PD-1 mAb and BGB-5777 (n = 9) showed a significant increase in median OS to 53 days (P Citation Format: Erik Ladomersky, Lijie Zhai, Galina Gritsina, Kristen L. Lauing, Matthew Genet, C. David James, Derek A. Wainwright. A novel IDO1 inhibitor combined with targeted immunotherapy durably increases survival in a mouse model of glioblastoma [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B018.