anti-CTLA4 Immunotherapy Research Articles

Characterization of the inflammasome IFN-dependent in hiPSC-derived endothelial cells from ICIs-induced myocarditis patients

Immune checkpoint inhibitors (ICIs) reactivate T cells to fight tumor cells, unraveling a new approach in cancer treatment. However, cardiovascular complications induced by these ICIs, especially myocarditis, have emerged as particularly dangerous complications with a mortality rate close to 50%. A recent study on cardiac biopsies showed that the IFN-γ pathway and inflammasome regulating proteins was specifically involved in myocarditis induced by ICIs treatment; this mechanism was not found in viral myocarditis and in dilated cardiomyopathies [1] . It is therefore crucial to set up models to study these pathophysiological mechanisms so that the benefit of immunotherapies is not compromised by their adverse cardiac effects. To better understand pathophysiological mechanisms of myocarditis induced by ICIs, we characterized the response to IFN-γ of endothelial cells (hiPSC-EC) generated from cells of patients with and without ICIs-induced myocarditis. We generated human-induced pluripotent stem cell (hiPSC), differentiated in hiPSC-EC, from cells of patients followed in the MediCO Center, Marseille. We considered two groups of patients: with myocarditis (Myoc) and without myocarditis (non-Myoc) following anti-PD-1/anti-CTLA4 immunotherapy. hiPSC-EC were treated with 10 ng/mL of IFN-γ during 48 h. Endothelial cells differentiated from hiPSC clones exhibited the characteristics expected for this cell type in all clones tested, both in the Myoc and non-Myoc patient groups. Stimulation by IFN-γ activated inflammatory intracellular signaling in hiPSC-EC, in particular overexpression of MHC class I and II, and PDL-1. Moreover, changes in the expression of genes of the JAK/STAT pathway and of the inflammasome, particularly the NLRP3 inflammasome, were observed in all groups of tested cells after the IFN-γ stimulation. However, the cells from Myoc patients seemed to be more impaired than those from non-Myoc patients. In particular, hiPSC-EC proliferation was more altered in this group by IFN-γ, and the expression of genes associated with the inflammatory response was strongly increased. Further analyses are in progress to confirm these preliminary data. These preliminary results, obtained in cells reprogrammed from cells of patients with immuno-induced myocarditis, are in agreement with the data published by Finke et al. (2021) in myocardial biopsies, indicating that these hiPSCs are a good model to study the pathophysiological mechanisms associated with the cardiotoxicitiy of anticancer immunotherapy.

N6-Methyladenosine Regulator-Mediated Immue Patterns and Tumor Microenvironment Infiltration Characterization in Glioblastoma.

BackgroundEpigenetic modifications, according to emerging evidence, perform a critical role for cellular immune response and tumorigenesis. Nonetheless, the role of N6-methyladenosine modification in shaping of the glioblastoma tumor microenvironment is unknown.MethodsN6-methyladenosine(m6A) methylation patterns in GBM patients were evaluated via multiple omics analysis of 15 m6A regulators and systematically correlated with tumor immune features. For quantification of N6-methyladenosine methylation patterns of individual patients, GM-score was developed and correlated with clinical and immunological characteristics.ResultsGlioblastoma has two different m6A methylation patterns that are strongly associated with TME characteristics, tumor subtype, immunotherapy response, and patient prognosis. High-GM-score is associated with an immune tolerance phenotype dominated by the IDH1 wild molecular subtype and the Mesenchymal tissue subtype, as well as a high infiltration of immune cells and stromal cells and a poor prognosis. Furthermore, despite higher immune checkpoint expression, individuals with a high-GM-score have a poorer response to anti-CTLA4 immunotherapy regimens due to T-cells dysfunctional. Low-GM-score individuals had an immunodeficient phenotype dominated by IDH mutant molecular subtypes and Proneural tissue subtypes, with less immune cell infiltration and a better prognosis. Furthermore, patients with low-GM-scores had higher microsatellite instability (MSI) and t-cell exclusion scores, as well as a better response to anti-CTLA4 immunotherapy regimens.ConclusionThis study demonstrated that m6A modification patterns play an important role in the shaping of TME complexity and diversity. The GM-score could identify m6A modification patterns in individual patients, resulting in a more personalization and efficacious anti-tumor immunotherapy strategy.

Comprehensive Analysis of N6-methyladenosine Modification Patterns Associated With Multiomic Characteristics of Bladder Cancer.

Purpose: To comprehensively analyze N6-methyladenosine modification patterns in bladder tumors and to further systematically explore the inherent relationships between these modification patterns and multiomic tumor characteristics.Materials and Methods: A total of 901 bladder tumor samples, including 405 samples from TCGA database, 188 samples from GSE13507 and 308 samples from GSE32894, were included in this systematic analysis. The N6-methyladenosine modification patterns were identified utilizing unsupervised clustering analysis. To quantify N6-methyladenosine modification patterns, the m6Ascore of individual sample was developed using principal component analysis algorithms. Relationships among immune infiltration, tumor mutation burden, various clinical characteristics, molecular subtypes, and the m6Ascore were systematically analyzed. The guiding value of m6Ascore in immunotherapy was further validated in an external trial cohort. Genomics of Drug Sensitivity in Cancer expression references were also utilized to perform drug sensitivity analysis for patients with distinct m6A modification patterns.Results: We determined three different N6-methyladenosine modification patterns for 901 bladder tumors. The quantitative m6Ascore of individual sample derived from N6-methyladenosine modification patterns could play a significant role in predicting overall survival, immune cell infiltration, and classic oncogene mutations. A low m6Ascore combined with high tumor mutation burden indicated better survival outcomes (p < 0.001). A higher m6Ascore also indicated a higher grade, higher T and N stage, elder ages, higher death rate, and higher PD1/PDL1/CTLA4 expressions (p < 0.01). The Basal type tended to exhibit significantly higher m6Ascores than the Luminal and Neuronal subtypes. External immunotherapy cohorts demonstrated that no difference in therapeutic effects was noted between the high and low m6Ascore groups when anti-PD1 immunotherapy was exclusively administered. When anti-PD1 and anti-CTLA4 immunotherapy were simultaneously administered, the high m6Ascore group had a significantly better prognosis than the low m6Ascore group (p < 0.001). High m6A groups were potentially sensitive to various medical treatments including Bleomycin, Bortezomib, Cisplatin, Cyclopamine, Dasatinib, Docetaxe, Rapamycin, and Vinblastine in this study.Conclusions: This study systematically revealed the important roles of m6A methylation modification patterns in bladder tumors. Detailed quantification of m6A modification patterns could improve our understanding of the bladder tumor microenvironments and could provide guidance for future immunotherapy strategies.

In This Issue

In This Issue

Multi-omics analysis of m6A modification-related patterns based on m6A regulators and tumor microenvironment infiltration in lung adenocarcinoma

Epigenetic modifications, especially N6-methyladenosine (m6A) modification, play a key role in tumor microenvironment (TME) infiltration. However, the regulatory role of m6A modification in the TME of lung adenocarcinoma (LUAD) remains unclear. A total of 2506 patients with LUAD were included in the analysis and divided into different groups according to distinct m6A modification-related patterns based on 23 m6A regulators. A comprehensive analysis was performed to explore TME infiltration in different m6A modification-related patterns. Principal component analysis was performed to obtain the m6Ascore and to quantify m6A modification-related patterns in different individuals. Three distinct m6A modification-related patterns were identified by 23 m6A regulators. The pathway enrichment analysis showed that m6Acluster-A was associated with immune activation; m6Acluster-B was associated with carcinogenic activation; m6Acluster-C was prominently related to substance metabolism. M6Acluster-A was remarkably rich in TME-infiltrating immune cells and patients with this pattern showed a survival advantage. The m6Ascore could predict TME infiltration, tumor mutation burden (TMB), the effect of tumor immunotherapy, and the prognosis of patients in LUAD. High m6Ascore was characterized by increased TME infiltration, reduced TMB, and survival advantage. Patients with a high m6Ascore exhibited significantly improved clinical response to anti-cytotoxic T lymphocyte antigen-4 (anti-CTLA4) immunotherapy. This study explored the regulatory mechanisms of TME infiltration in LUAD. The comprehensive analysis of m6A modification-related patterns may contribute to the development of individualized immunotherapy and the improvement of the overall effectiveness of immunotherapy for LUAD patients.

Extracellular Matrix-Associated Pathways Promote the Progression of Gastric Cancer by Impacting the Dendritic Cell Axis.

BackgroundGastric cancer (GC) is the third most frequent malignant tumour in the Chinese population, let alone the whole world. Recently, most prognostic models have only focused on the levels of several genes, miRNAs, lncRNAs, gene mutations, or DNA methylation; however, the activation status of biological pathways is more stable and can reflect the comprehensive inner conditions of tumours.MethodsWe collected samples from the Cancer Genome Atlas Stomach Adenocarcinoma (TCGA-STAD) cohort and GSE62254 cohort, with a total of 594 patients. We employed GSEA to first compare the diverse activated signalling pathways between dead GC patients and living patients. The least absolute shrinkage and selection operator (LASSO) regression analysis was subsequently performed by the “glmnet” package to generate a prognostic signature.ResultsWe extracted a total of 218 genes from the KEGG Focal Adhesion and KEGG ECM Receptor Interaction pathways, which showed significant activation in dead GC patients in two enrolled cohorts, for subsequent LASSO analysis. In the TCGA-STAD cohort, patients in the high-risk group faced a significantly poorer prognosis than those in the low-risk group (P < 0.001, HR: 4.62, 95% CI: 3.447–6.183), with an AUC of 0.694. In the GSE62254 cohort, the HR value was 4.94 (95% CI: 3.413–7.165), and the AUC value was as high as 0.834. A high-risk score and poor prognosis correlated with infiltrated dendritic cells, and the receptor of IFN-α was also positively linked with the risk score, as well as poor prognosis. GC patients with high-risk scores were more likely to respond to CTLA4 treatment but not PD1 treatment.ConclusionTaken together, we established and verified an extracellular matrix prognostic model of gastric cancer patients. The model can be used to evaluate the risk of death of GC patients, as well as the response to anti-CTLA4 immunotherapy.

Early B-cell factors involve in the tumorigenesis and predict the overall survival of gastric cancer.

Gastric cancer (GC) is a heavy health burden around the world, which is the fifth most frequent tumor and leads to the third most common cancer-related deaths. It is urgent to identify prognostic markers as the guideline for personalized treatment and follow-up. We accessed the prognostic value of Early B-cell factors (EBFs) in GC. A total of 415 GC tissues and 34 normal tissues from The Cancer Genome Atlas Stomach Adenocarcinoma (TCGA-STAD) cohort, 616 external patients from GSE15459, GSE22377, GSE51105, GSE62245 were enrolled for analysis. Univariate and multivariate Cox regression analyses were employed to evaluate the sole and integrative prognostic value of EBFs, respectively. Genetic alterations, DNA methylation of EBFs were also evaluated, as well as the involved signaling pathways. We revealed that increased EBFs associated with the poor prognosis of GC patients, the prognostic model was established in TCGA-STAD cohort, and validated in Gene Expression Omnibus (GEO) cohorts, with effectiveness in both HER2 positive and negative patients. DNA methylation was involved in the impact on prognosis. Cell cycle, immune-associated, and MAPK pathways were influenced by EBFs. Anti-CTLA4 immunotherapy is more suitable for EBFs determining high-risk groups, but not anti-PD-1/PD-L1 therapy. 5-Fluorouracil, methotrexate, vorinostat are suitable to inhibit the function of EBFs. Our new findings provide novel insight into the prediction of prognosis and clinical treatment of GC patients based on EBFs.

First line immunotherapy extends brain metastasis free survival, improves overall survival, and reduces the incidence of brain metastasis in patients with advanced melanoma.

BackgroundRecent advances in targeted therapy and immunotherapy have improved the prognosis of melanoma patients but brain metastasis remains a major challenge. Currently, it is unclear how existing therapies can be best used to prevent or treat brain metastasis in melanoma patients.AimsWe aimed to assess brain metastasis free survival (BMFS), overall survival (OS), incidence of brain metastases, and sequencing strategies of immunotherapy and targeted therapy in patients with BRAF‐mutated advanced melanoma.Methods and resultsWe retrospectively analyzed 683 patients with BRAF‐mutated advanced melanoma treated with first line (1L) immunotherapy (N = 266) or targeted therapy (N = 417). The primary outcome was BMFS. Secondary outcomes included OS of all patients and incidence of brain metastases in patients without documented brain metastases prior to 1L therapy. The median BMFS was 13.7 months [95% confidence interval (CI): 12.4–16.0] among all patients. The median BMFS for patients receiving 1L immunotherapy was 41.9 months [95% CI: 22.8–not reached (NR)] and targeted therapy was 11.0 months (95% CI: 8.8–12.5). Median OS results were qualitatively similar to BMFS results. The cumulative incidence of brain metastases for patients receiving 1L targeted therapy was higher than for patients receiving 1L immunotherapy (P < .001). Patients receiving 1L anti‐CTLA4 plus anti‐PD1 combination immunotherapy only or followed by second line (2L) targeted therapy had better BMFS (HR 0.40, 95% CI: 0.24–0.67, P = .001), improved OS (HR 0.49, 95% CI: 0.30–0.81, P = .005), and reduced incidence of brain metastases (HR 0.47, 95% CI: 0.24–0.67, P = .047) than patients receiving 1L combination BRAF and MEK targeted therapy followed by 2L immunotherapy.ConclusionPatients with advanced BRAF mutant melanoma treated with 1L immunotherapy have significantly longer BMFS and OS, and reduced incidence of brain metastases, compared with those treated with 1L targeted therapy. Further studies evaluating the ability of immunotherapy and targeted therapy to improve OS and prevent brain metastases are warranted

A Novel Mice Model for Studying the Efficacy and IRAEs of Anti-CTLA4 Targeted Immunotherapy.

BackgroundPatient-derived orthotopic xenograft (PDOX) is a popular animal model for translational cancer research. Immunotherapy is a promising therapy against glioblastoma (GBM). However, the PDOX model is limited to evaluating immune-related events. Our study aims to establish GBM humanized PDOX (HPDOX) mice models to study the mechanism of anti-CTLA4 immunotherapy and immune-related adverse events (IRAEs).MethodsHPDOX models were established by culturing GBM tissues and intracranially implanting them in NSG mice. Meanwhile, peripheral blood mononuclear cells (PBMCs) were separated from peripheral blood and of GBM patients and administrated in corresponding mice. The population of CD45+, CD3+, CD4+, CD8+, and regulatory T (Treg) cells was estimated in the peripheral blood or tumor.ResultsT cells derived from GBM patients were detected in HPDOX mice models. The application of anti-CTLA4 antibodies (ipilimumab and tremelimumab) significantly inhibited the growth of GBM xenografts in mice. Moreover, residual patient T cells were detected in the tumor microenvironment and peripheral blood of HPDOX mice and were significantly elevated by ipilimumab and tremelimumab. Additionally, Treg cells were decreased in mice with IRAEs. Lastly, the proportion of CD4+/CD8+ T cells dramatically increased after the administration of ipilimumab. And the degree of IRAEs may be related to CD56+ expression in HPDOX.ConclusionsOur study established HPDOX mice models for investigating the mechanism and IRAEs of immunotherapies in GBM, which would offer a promising platform for evaluating the efficacy and IRAEs of novel therapies and exploring personalized therapeutic strategies.

Early memory differentiation and cell death resistance in T cells predicts melanoma response to sequential anti-CTLA4 and anti-PD1 immunotherapy.

Immune checkpoint blockers (ICBs)-based immunotherapy has revolutionised oncology. However, the benefits of ICBs are limited to only a subset of patients. Herein, the biomarkers-driven application of ICBs promises to increase their efficacy. Such biomarkers include lymphocytic IFNγ-signalling and/or cytolytic activity (granzymes and perforin-1) footprints, whose levels in pre-treatment tumours can predict favourable patient survival following ICB-treatment. However, it is not clear whether such biomarkers have the same value in predicting survival of patients receiving first-line anti-CTLA4 ICB-therapy, and subsequently anti-PD1 ICB-therapy (i.e., sequential ICB-immunotherapy regimen). To address this, we applied highly integrated systems/computational immunology approaches to existing melanoma bulk-tumour transcriptomic and single-cell (sc)RNAseq data originating from immuno-oncology clinical studies applying ICB-treatment. Interestingly, we observed that CD8+/CD4+T cell-associated IFNγ-signalling or cytolytic activity signatures fail to predict tumour response in patients treated with anti-CTLA4 ICB-therapy as a first-line and anti-PD1 ICB-therapy in the second-line setting. On the contrary, signatures associated with early memory CD8+/CD4+T cells (integrating TCF1-driven stem-like transcriptional programme), capable of resisting cell death/apoptosis, better predicted objective response rates to ICB-immunotherapy, and favourable survival in the setting of sequential ICB-immunotherapy. These observations suggest that sequencing of ICB-therapy might have a specific impact on the T cell-repertoire and may influence the predictive value of tumoural immune biomarkers.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Genomic analysis uncovers prognostic and immunogenic characteristics of ferroptosis for clear cell renal cell carcinoma

In this study, the characteristic patterns of ferroptosis in clear cell renal cell carcinoma (ccRCC) were systematically investigated with the interactions between ferroptosis and the tumor microenvironment (TME). On the mRNA expression profiles of 57 ferroptosis-related genes (FRGs), three ferroptosis patterns were constructed, with distinct prognosis and immune cell infiltrations (especially T cells and dendritic cells). The high ferroptosis scores were characterized by poorer prognosis, increased T cell infiltration, higher immune and stromal scores, elevated tumor mutation burden, and enhanced response to anti-CTLA4 immunotherapy. Meanwhile, the low ferroptosis scores were distinctly associated with enhanced tumor purity and amino acid and fatty acid metabolism pathways. Following validation, the ferroptosis score was an independent and effective prognostic factor. Collectively, ferroptosis could be involved in the diverse and complex TME. Evaluation of the ferroptosis patterns may heighten the comprehension about immune infiltrations in the TME, assisting oncologists to generate individualized immunotherapeutic strategies.

Energy status dictates PD-L1 protein abundance and anti-tumor immunity to enable checkpoint blockade

Aberrant energy status contributes to multiple metabolic diseases, including obesity, diabetes, and cancer, but the underlying mechanism remains elusive. Here, we report that ketogenic-diet-induced changes in energy status enhance the efficacy of anti-CTLA-4 immunotherapy by decreasing PD-L1 protein levels and increasing expression of type-I interferon (IFN) and antigen presentation genes. Mechanistically, energy deprivation activates AMP-activated protein kinase (AMPK), which in turn, phosphorylates PD-L1 on Ser283, thereby disrupting its interaction with CMTM4 and subsequently triggering PD-L1 degradation. In addition, AMPK phosphorylates EZH2, which disrupts PRC2 function, leading to enhanced IFNs and antigen presentation gene expression. Through these mechanisms, AMPK agonists or ketogenic diets enhance the efficacy of anti-CTLA-4 immunotherapy and improve the overall survival rate in syngeneic mouse tumor models. Our findings reveal a pivotal role for AMPK in regulating the immune response to immune-checkpoint blockade and advocate for combining ketogenic diets or AMPK agonists with anti-CTLA4 immunotherapy to combat cancer.

ARIH1 signaling promotes anti-tumor immunity by targeting PD-L1 for proteasomal degradation

Cancer expression of PD-L1 suppresses anti-tumor immunity. PD-L1 has emerged as a remarkable therapeutic target. However, the regulation of PD-L1 degradation is not understood. Here, we identify several compounds as inducers of PD-L1 degradation using a high-throughput drug screen. We find EGFR inhibitors promote PD-L1 ubiquitination and proteasomal degradation following GSK3α-mediated phosphorylation of Ser279/Ser283. We identify ARIH1 as the E3 ubiquitin ligase responsible for targeting PD-L1 to degradation. Overexpression of ARIH1 suppresses tumor growth and promotes cytotoxic T cell activation in wild-type, but not in immunocompromised mice, highlighting the role of ARIH1 in anti-tumor immunity. Moreover, combining EGFR inhibitor ES-072 with anti-CTLA4 immunotherapy results in an additive effect on both tumor growth and cytotoxic T cell activation. Our results delineate a mechanism of PD-L1 degradation and cancer escape from immunity via EGFR-GSK3α-ARIH1 signaling and suggest GSK3α and ARIH1 might be potential drug targets to boost anti-tumor immunity and enhance immunotherapies.

Characterization of the fatty acid metabolism in colorectal cancer to guide clinical therapy

Colorectal cancer (CRC) is one of the most common malignant tumors, with the second-highest mortality of all 36 cancers worldwide. The roles of fatty acid metabolism in CRC were investigated to explore potential therapeutic strategies. The data files were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Univariate and least absolute shrinkage and selection operator (LASSO) Cox regression analyses were used to construct a prognostic risk score model with fatty acid metabolism-related genes for predicting prognosis in CRC. Patients with a high-risk score had a poorer prognosis in TCGA cohort than those with a low-risk score and were confirmed in the GEO cohort. Further analysis using the “pRRophetic” R package revealed that low-risk patients were more sensitive to 5-fluorouracil. A comprehensive evaluation of the association between prognostic risk score model and tumor microenvironment (TME) characteristics showed that high-risk patients were suitable for activating a type I/II interferon (IFN) response and inflammation-promoting function. Tumor Immune Dysfunction and Exclusion (TIDE) and SubMap algorithm results also demonstrated that high-risk patients are more suitable for anti-CTLA4 immunotherapy. Therefore, the evaluation of the fatty acid metabolism pattern promotes our comprehension of TME infiltration characteristics, thus guiding effective immunotherapy regimens.

Enhancing the Generation of Eomeshi CD8+ T Cells Augments the Efficacy of OX40- and CTLA-4-Targeted Immunotherapy.

CTLA-4 blockade in combination with an agonist OX40-specific monoclonal antibody synergizes to augment antitumor immunity through enhanced T-cell effector function, leading to increased survival in preclinical cancer models. We have shown previously that anti-OX40/anti-CTLA-4 combination therapy synergistically enhances the expression of Eomesodermin (Eomes) in CD8+ T cells. Eomes is a critical transcription factor for the differentiation and memory function of CD8+ T cells. We hypothesized that EomeshiCD8+ T cells were necessary for anti-OX40/anti-CTLA-4 immunotherapy efficacy and that further enhancement of this population would improve tumor-free survival. Indeed, CD8+ T cell-specific deletion of Eomes abrogated the efficacy of anti-OX40/anti-CTLA-4 therapy. We also found that anti-OX40/anti-CTLA-4-induced EomeshiCD8+ T cells expressed lower levels of checkpoint receptors (PD1, Tim-3, and Lag-3) and higher levels of effector cytokines (IFNγ and TNFα) than their Eomeslo counterparts. Eomes expression is negatively regulated in T cells through interleukin-2-inducible T-cell kinase (ITK) signaling. We investigated the impact of modulating ITK signaling with ibrutinib, an FDA-approved tyrosine kinase inhibitor, and found that anti-OX40/anti-CTLA-4/ibrutinib therapy further enhanced CD8+ T cell-specific Eomes expression, leading to enhanced tumor regression and improved survival, both of which were associated with increased T-cell effector function across multiple tumor models. Taken together, these data demonstrate the potential of anti-OX40/anti-CTLA-4/ibrutinib as a triple therapy to improve the efficacy of immunotherapy.

Abstract PO064: Baseline steroids impair immunotherapy efficacy in a mouse model of melanoma

Abstract While checkpoint inhibition has changed the landscape of treatment for multiple tumor types, many questions remain, particularly about the use of concurrent immunosuppressive agents such as steroids. Many patients starting immunotherapy are on baseline steroids, such as patients with autoimmune conditions or patients with brain metastases. In vitro, steroids are well-known to disrupt dendritic cell priming, activation, and maturation, steps that are key for appropriate mobilization of CD8+ cytotoxic T cells that are critical for antitumor immunity, but few studies directly study the interaction of baseline steroids with immunotherapy. In this work, we studied this interaction using the YUMMER model (Yale University Mouse Melanoma Exposed to Radiation) 1.7 model, a syngeneic cell line that originates from BrafV600E; Pten-/-; and Cdkn2a-/- genetically engineered mouse melanomas. YUMMER1.7 (YR1.7) has been irradiated to increase immune system recognition via induction of neoantigens and thus exhibits reproducible responses to anti-CTLA4 and anti-PD1 immunotherapy. In this study, 6-8 week old C57Bl6/j mice were injected subcutaneously with 500K YR1.7 cells and treated with dual biweekly injections of anti-CTLA4 and anti-PD1 therapy starting on day 7 (dCPI, n = 10), dCPI + baseline steroids administered on the day prior to tumor implantation and every other day for 10 days thereafter (n = 10), baseline steroids alone (n = 10), or nothing (n = 10). We found that survival of mice receiving baseline steroids with dCPI was significantly reduced (p = 0.0355) compared to mice receiving only dCPI. Tumor volumes on day 18 were significantly larger (p = 0.0052) in the group both dCPI and baseline steroids compared to the group receiving only dCPI. In mice receiving dCPI with steroids started on day 7, as opposed to day -1, this reduction in effect was not seen. Serum cytokine analysis showed that on day 12, mice receiving baseline steroids had significantly lower concentrations of many crucial antitumor cytokines, most notably IFN-gamma, MIG, IP-10, RANTES, TARC, IL-12, IL-7, IL-15, MIP-1a, and 6CKine. Weights of tumor-draining lymph nodes were significantly lower from mice receiving baseline steroids and dCPI (p = 0.0411), suggesting that steroids blunted the proliferation of antitumor immune cells in the LN. Finally, IHC staining demonstrated that CD8 cells were absent in the tumor microenvironment of mice receiving baseline steroids. Together, these data suggest that baseline steroids may be deleterious to immunotherapy-induced antitumor immunity, likely through impairing T-cell activation and migration, possibly through interfering with DC priming. Clinically, this raises the question of whether patients on baseline steroids on the cusp of initiating immunotherapy should be switched to a more narrow immunosuppressive regimen that achieves disease control without compromising the potentially helpful effect of immunotherapy. Citation Format: Michelle Ferreira, Drew Daniels, Marcus Bosenberg. Baseline steroids impair immunotherapy efficacy in a mouse model of melanoma [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO064.

Synergies Radiotherapy-Immunotherapy in Head and Neck Cancers. A New Concept for Radiotherapy Target Volumes-"Immunological Dose Painting".

The combination of immune checkpoint inhibitors and definitive radiotherapy is investigated for the multimodal treatment of cisplatin non-eligible locally advanced head and neck cancers (HNC). In the case of recurrent and metastatic HNC, immunotherapy has shown benefit over the EXTREME protocol, being already considered the standard treatment. One of the biggest challenges of multimodal treatment is to establish the optimal therapy sequence so that the synergistic effect is maximal. Thus, superior results were obtained for the administration of anti-CTLA4 immunotherapy followed by hypofractionated radiotherapy, but the anti-PD-L1 therapy demonstrates the maximum potential of radio-sensitization of the tumor in case of concurrent administration. The synergistic effect of radiotherapy–immunotherapy (RT–IT) has been demonstrated in clinical practice, with an overall response rate of about 18% for HNC. Given the demonstrated potential of radiotherapy to activate the immune system through already known mechanisms, it is necessary to identify biomarkers that direct the “nonresponders” of immunotherapy towards a synergistic RT–IT stimulation strategy. Stimulation of the immune system by irradiation can convert “nonresponder” to “responder”. With the development of modern techniques, re-irradiation is becoming an increasingly common option for patients who have previously been treated with higher doses of radiation. In this context, radiotherapy in combination with immunotherapy, both in the advanced local stage and in recurrent/metastatic of HNC radiotherapy, could evolve from the “first level” of knowledge (i.e., ballistic precision, dose conformity and homogeneity) to “level two” of “biological dose painting” (in which the concept of tumor heterogeneity and radio-resistance supports the need for doses escalation based on biological criteria), and finally to the “third level“ ofthe new concept of “immunological dose painting”. The peculiarity of this concept is that the radiotherapy target volumes and tumoricidal dose can be completely reevaluated, taking into account the immune-modulatory effect of irradiation. In this case, the tumor target volume can include even the tumor microenvironment or a partial volume of the primary tumor or metastasis, not all the gross and microscopic disease. Tumoricidal biologically equivalent dose (BED) may be completely different from the currently estimated values, radiotherapy treating the tumor in this case indirectly by boosting the immune response. Thus, the clinical target volume (CTV) can be replaced with a new immunological-clinical target volume (ICTV) for patients who benefit from the RT–IT association (Image 1).

Abstract D075: Investigating the immuno-biology underlying differential response to immunotherapy in White and non-White patients with metastatic acral melanoma

Abstract Background: Acral melanoma (ACML), the rarest form of melanoma (MEL) represents 4-6% of all MEL cases however it is the most common subtype darker pigmented individuals accounting for up to 70% of all cases. Checkpoint inhibitors (CPI) have emerged as an efficacious treatment option. In cutaneous melanoma (CtMEL), tumor mutational burden (TMB) correlates with response rate. Despite low TMB in AMEL, responses to CPI parallel those in CtMEL. Given the differences in ethnicity among ACML patients compared CtMEL patients we aimed to study: (i) the correlation between response and TMB, (ii) the relationship between ethnicity and response and (iii) the underlying immune-biology accounting for differential responses. Method: All advanced and metastatic ACML patients (pts) treated with anti-CTLA4 (ipilimumab) or anti-PD1 (pembrolizumab or nivolumab) immunotherapy between March 2011 and January 2019 at MD Anderson Cancer Center, Texas, were included in this retrospective analysis. Clinical response, progression free survival and overall survival (PFS and OS) and their correlation to ethnicity and TMB were evaluated. Objective response was measured using RECIST 1.1 and analyzed using logistic regression. PFS and OS were assessed using the Kaplan Meir method and log-rank test. TMB was predicted using validated algorithm based on a defined gene mutation set obtained using next generation sequencing. Results: 44 pts with Stages III-IV ACML (IIIA: 2%, IIIB 9%, IIIC 27%, IV-M1A: 16%, IV-M1B: 25%, IV-M1C: 20%, IV-M1D2%) were included in the analysis. Median age was 63 years old (39-88) and 60% were men. Of 44 patients 12 (27%) self-identified as Hispanic, 2 as (5%) Asian and 30 (68%) as White. The overall response rate was nearly 5 times times higher in Hispanic compared to White pts (OR 4.60, p-value 0.04). The median TMB in Hispanic patients was 16 mutations/mb compared to 7 mutations/mb in White pts. The median PFS and OS for White pts were 7.2 months and 34.3 months respectively while for Hispanic pts the median PFS and OS were 6.7 (log-rank p=0.69) and 26.1 months (log-rank p=0.38) respectively. In addition to this data deep immune analysis of tumor tissue including nanostring, gene expression and TCR sequencing will be assessed and reported. Conclusions: The data from this small retrospective study suggests that White pts with ACML had low response rates to CPI presumably due to low TMB while interestingly, Hispanic pts, despite relatively low TMB have high response rates paralleling those seen in the overall CtMEL population. Despite higher response rates in Hispanic patients, there was no significant difference in OS. This data together with further immune analysis provides the rationale to design prospective studies to investigate how tumor micro-environment varies with ethnicity. Additionally we will investigate the social and biological determinants that limit survival in Hispanic pts despite higher response rates. Citation Format: Kaysia Ludford, Chantal Saberian, Sara K Nabhan, Stephen Gruschkus, Chantale Bernatchez, Natalie Jackson, Cara L Haymaker, Patrick Hwu, Adi Diab. Investigating the immuno-biology underlying differential response to immunotherapy in White and non-White patients with metastatic acral melanoma [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr D075.

Prognostic significance of tumor poliovirus receptor and CTLA4 expression in patients with surgically resected non-small-cell lung cancer.

Poliovirus receptor (PVR) is a tumor promoter and a regulatory checkpoint that enhances immunosuppression. We investigated PVR expression by applying immunohistochemistry (IHC) staining. A positive association existed between PVR expression and cytotoxic T lymphocyte-associated antigen 4 (CTLA4) expression in patients with surgically resected non-small-cell lung cancer (NSCLC). PVR expression is a prognosis predictor of lung adenocarcinoma. To investigate the prognostic significance of PVR expression and CTLA4 expression for surgically resected NSCLC. The medical records of 108 Chinese patients with primary NSCLC who underwent surgery were retrospectively reviewed. The expression of PVR and CTLA4 were measured through IHC. Clinical characteristics, the association between PVR and CTLA4, and the prognostic significance of PVR were analyzed. A significant positive association was observed between PVR and CTLA4 expression in NSCLC (P = 0.016). PVR had a high positive rate among females, nonsmokers, and patients with adenocarcinoma and advanced lung cancer. The overall survival (OS) of patients with negative PVR expression was significantly longer than that of patients with positive PVR expression (P = 0.049), especially among females (P = 0.03) and nonsmokers (P = 0.025). Multivariate analysis results showed that advanced tumor stage and PVR expression were independent prognosis predictors of poor OS. PVR can potentially serve as a prognostic predictor and biomarker for NSCLC and cancer anti-CTLA4 immunotherapy response.