Combined PD-L1 Research Articles

Tailored immunotherapy approach with nivolumab in advanced transitional cell carcinoma (TITAN-TCC).

446 Background: Several PD-1 immune-checkpoint inhibitors including Nivolumab (Nivo) are approved in urothelial cancer. Recently, in the front line setting, improved activity of combined PD-L1 and CTLA4 immune-checkpoint inhibition has been reported and a phase III trial with Nivolumab + Ipilimumab (Nivo+Ipi) is ongoing. Here we report a response-based tailored approach starting treatment with Nivo monotherapy using Nivo+Ipi as immunotherapeutic “boost”. Methods: Between July 2017 and April 2019 86 patients were enrolled and treated according to protocol version 3 (cohort 1). Patients started with Nivo 240 mg Q2W induction. After 4 dosings and tumor assessment at week 8 (i) responders (PR/CR) to Nivo monotherapy continued with maintenance while (ii) patients with stable (SD) or progressive disease (PD) received 2 cycles Nivo3+Ipi1 followed by another 2 cycles Nivo1+Ipi3 if not responding. Median follow-up is 8.7 months. The primary endpoint is confirmed investigator-assessed objective response rate (ORR) per RECIST1.1. Secondary endpoints include activity of Nivo monotherapy at week 8, remission rate with Nivo+Ipi “boosts”, safety, overall survival and quality of life. Results: Of the patients 42, 39 and 5 were first, second and third line, respectively. Median age was 67 years (range 45-84), 61 patients (71 %) were male and 25 female. ORR with Nivo monotherapy at first assessment (week 8) was 29 % and 23 % in first and second/third line, respectively. Of the patients 41 received Nivo+Ipi “boosts” after week 8 while 12 received later “boosts”. Best overall response (BOR) rate with Nivo induction ± Nivo+Ipi “boosts” was 48 % and 27 % in first and second/third line, respectively. In first line 7/17 (41 %) patients receiving Nivo+Ipi after week 8 had an improved response compared to 2/24 (8.3 %) in second/third line. Of the patients who continued with Nivo maintenance after week 8 and received later “boosts” 2/12 (17 %) had a PR and 2/12 (17 %) improved to SD. Treatment-related AEs will be presented. Conclusions: TITAN–TCC explores a response-driven use of Nivo+Ipi as an immunotherapeutic “boost”. In first line, this significantly improved ORR compared to the expected response rate of Nivo monotherapy, providing further evidence to the added value of Ipi in combination with Nivo. Further follow-up is ongoing to characterize duration and depth of response. Clinical trial information: NCT03219775 . Research Sponsor: Bristol-Myers Squibb[Table: see text]

A Randomized Trial of Combined PD-L1 and CTLA-4 Inhibition with Targeted Low-Dose or Hypofractionated Radiation for Patients with Metastatic Colorectal Cancer.

Prospective human data are lacking regarding safety, efficacy, and immunologic impacts of different radiation doses administered with combined PD-L1/CTLA-4 blockade. We performed a multicenter phase II study randomly assigning patients with metastatic microsatellite stable colorectal cancer to repeated low-dose fractionated radiation (LDFRT) or hypofractionated radiation (HFRT) with PD-L1/CTLA-4 inhibition. The primary endpoint was response outside the radiation field. Correlative samples were analyzed using multiplex immunofluorescence (IF), IHC, RNA/T-cell receptor (TCR) sequencing, cytometry by time-of-flight (CyTOF), and Olink. Eighteen patients were evaluable for response. Median lines of prior therapy were four (range, 1-7). Sixteen patients demonstrated toxicity potentially related to treatment (84%), and 8 patients had grade 3-4 toxicity (42%). Best response was stable disease in 1 patient with out-of-field tumor shrinkage. Median overall survival was 3.8 months (90% confidence interval, 2.3-5.7 months). Correlative IF and RNA sequencing (RNA-seq) revealed increased infiltration of CD8+ and CD8+/PD-1+/Ki-67+ T cells in the radiation field after HFRT. LDFRT increased foci of micronuclei/primary nuclear rupture in two subjects. CyTOF and RNA-seq demonstrated significant declines in multiple circulating immune populations, particularly in patients receiving HFRT. TCR sequencing revealed treatment-associated changes in T-cell repertoire in the tumor and peripheral blood. We demonstrate the feasibility and safety of adding LDFRT and HFRT to PD-L1/CTLA-4 blockade. Although the best response of stable disease does not support the use of concurrent PD-L1/CTLA-4 inhibition with HFRT or LDFRT in this population, biomarkers provide support that both LDFRT and HFRT impact the local immune microenvironment and systemic immunogenicity that can help guide future studies.

IMbrave 151: a randomized phase II trial of atezolizumab combined with bevacizumab and chemotherapy in patients with advanced biliary tract cancer.

Background:Biliary tract cancers (BTCs) are heterogenous, highly aggressive tumors that harbor a dismal prognosis for which more effective treatments are needed. The role of cancer immunotherapy in BTC remains to be characterized. The tumor microenvironment (TME) of BTC is highly immunosuppressed and combination treatments are needed to promote effective anticancer immunity. Vascular endothelial growth factor (VEGF) drives immunosuppression in the TME by disrupting antigen presentation, limiting T-cell infiltration, or potentiating immune-suppressive cells. Many VEGF-regulated mechanisms are thought to be relevant to repressed antitumor immunity in BTC, making dual targeting of VEGF and programmed cell death protein 1 (PD-1)/PD-L1 pathways a rational approach. Gemcitabine and Cisplatin (Gem/Cis) can also modulate anticancer immunity through overlapping and complementary mechanisms to those regulated by VEGF. Anti-PD-L1/VEGF inhibition, coupled with chemotherapy, may potentiate antitumor immunity leading to enhanced clinical benefit.Methods:IMbrave 151 is a randomized, double-blind, placebo-controlled, multicenter, international phase II study to evaluate atezolizumab (a PD-L1 inhibitor) in combination with chemotherapy (gemcitabine and cisplatin) and bevacizumab (an anti-VEGF monoclonal antibody) as a first-line treatment for advanced BTC. Approximately 150 patients with previously untreated, advanced BTC will be randomized to either Arm A (atezolizumab + bevacizumab + Gem/Cis) or Arm B (atezolizumab + placebo + Gem/Cis). Randomization is stratified by the presence of metastatic disease, primary tumor location, and geographic region. The primary efficacy endpoint is investigator-assessed progression-free survival (PFS) per RECIST 1.1. Secondary endpoints include objective response rate (ORR), duration of response (DoR), disease control rate (DCR), overall survival (OS), and safety and patient reported outcomes (PROs). Tissue, blood, and stool samples will be collected at baseline and on-treatment in order to perform correlative biomarker analyses.Discussion:IMbrave 151 represents the first randomized study to evaluate combined PD-L1/VEGF blockade on a chemotherapy backbone in BTC.Trial registration:NCT identifier: NCT04677504; EUDRACT number: 2020-003759-14

PD-L1 in Combination with CD8+TIL and HIF-1α are Promising Prognosis Predictors of Head and Neck Squamous Cell Carcinoma.

ObjectiveThe aim of this study was to evaluate the prognosis effect of PD-L1 in combination with CD8+ tumor-infiltrating lymphocyte (TIL) or HIF-1α in head and neck squamous cell carcinoma (HNSCC).MethodsA total of 63 patients who underwent surgical resection were included in this study. The level of PD-L1, CD8+ TIL, and HIF-1α was determined by immunohistochemical analysis. The survival of patients was evaluated by Kaplan–Meier analysis. The prognostic power of these parameters was evaluated by C-index.ResultsWe observed that the survival of patients, who had a high level of PD-L1 in tumor cells, was significantly shorter than those who had a low level of PD-L1. However, the survival of patients who had a high level of PD-L1 in tumor microenvironment was significantly longer than patients with a low level of PD-L1 in tumor microenvironment. In addition, high level of CD8+ tumor-infiltrating lymphocyte or low level of HIF-1α level suggests a better prognosis. Moreover, we observed that PD-L1 in combination with CD8+ tumor-infiltrating lymphocyte and HIF-1α could significantly improve the prognostic effect of current TNM stage.ConclusionThe results of this study suggest that the level of PD-L1, CD8+TIL, and HIF-1α are useful prognostic biomarkers for patients with HNSCC. Incorporating these biomarkers into current TNM stage of HNSCC improve the discriminatory capability of TNM stage.

Abstract 2159: IGFBP2 regulates PD-L1 expression by activating the EGFR-STAT3 signaling pathway in malignant melanoma

Abstract Immunotherapy targeting the PD-1/PD-L1 receptor in melanoma patients has achieved great success. Although many studies have addressed the underlying mechanisms involved in the blockade of PD-1/PD-L1 and the consequent modulation of the immune system, the mechanisms of PD-L1 upregulation and reliable biomarkers to predict the efficacy of anti-PD-1/PD-L1 therapy remain unknown. The present study demonstrates the correlation between IGFBP2 and PD-L1, reveals a novel immune-associated tumour function of IGFBP2 in facilitating nuclear accumulation of EGFR and activation of the EGFR/STAT3/PD-L1 signalling pathway in melanoma cells. Our results also suggest that combined IGFBP2 and PD-L1 expression have the potential to predict the efficacy of anti-PD-1 treatment for malignant melanoma; because the combination of high IGFBP2 and PD-L1 expression characterizes melanoma patients with worse overall survival and is associated with a better immune ecosystem. These characteristics have been confirmed by both in vitro and in vivo data. So, IGFBP2 regulates PD-L1 expression by activating the EGFR-STAT3 signalling pathway and its function as a PD-L1 regulator might suggest novel therapeutic approach for melanoma. Citation Format: Jilong Yang. IGFBP2 regulates PD-L1 expression by activating the EGFR-STAT3 signaling pathway in malignant melanoma [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 2159.

PD-L1 in combination with CD8+TIL and HIF-1α are promising prognosis predictors of head and neck squamous cell carcinoma.

e18545 Background: Currently, the tumor/node/metastasis (TNM) staging is the most widely used Head and neck squamous cell carcinoma (HNSCC) staging system. However, this classification only provides limit information for the prognosis of HNSCC. Recently, some studies suggest that the level of PD-L1 in tumor cells (TC-PD-L1) or in tumor microenvironment cells (TMC-PD-L1) is a promising biomarker for the prognosis of HNSCC patients. Therefore, in this study, we evaluated the prognostic value of TC-PD-L1, TMC-PD-L1, CD8+ TIL, HIF-1α. In addition, we investigated if incorporated these biomarkers into TNM stage could improve the prognostic value of TNM stage. Methods: A total of 63 patients who underwent surgical resection. The level of PD-L1, CD8+ TIL and HIF-1α was determined by immunohistochemical analysis. The survival of patients was evaluated by Kaplan-Meier analysis and Cox proportional hazards model. The prognostic power of these parameters was evaluated by C-index. Results: We observed that the survival of patients, who had high level of PD-L1 in tumor cells, was significantly shorter than those who had low level of PD-L1. However, the survival of patients who had high level of PD-L1 in tumor microenvironment was significantly longer than patients with low level of PD-L1 in tumor microenvironment. In addition, high level of CD8+ tumor-infiltrating lymphocyte or low level of HIF-1α level suggests a better prognosis. Moreover, we observed that PD-L1 in combination with CD8+tumor-infiltrating lymphocyte and HIF-1α could significantly improve the prognostic effect of current TNM stage. Conclusions: The results of this study suggest that the level of PD-L1, CD8+TIL and HIF-1α is useful prognostic biomarkers for patients with HNSCC. Incorporated these biomarkers into current TNM stage of HNSCC improve the discriminatory capability of TNM stage.

PD-L1 and neutrophil to lymphocyte ratio (NLR) as predictive panel of prognosis in bladder cancer.

e17040 Background: PD-1/PD-L1 blockade significantly improved survival for bladder cancer. PD-L1 expression might not be an ideal marker for patient selection in isolation. Several studies demonstrated that alternative markers such as NLR, a biomarker of systemic inflammation response to therapy, correlated with outcomes in a variety of cancers including bladder cancer, might be a predictor for patient selection and the response to immunotherapy. However, no reporters have been made combination of NLR and PD-L1 in predicting prognosis in bladder cancer. Methods: Suitable tissue of 100 FFPE blocks of bladder cancer patients treated in Sun Yat-Sen Memorial Hospital Sun Yat-sen University were stained with PD-L1 antibody. Clinicopathological data and pretreatment complete blood count were retrospectively collected. All patients were classified into high NLR group and low NLR group at cut-off 3.0. Kaplan-Meier and Cox proportional hazard models analyses were used to assessed the predictor of combined PD-L1/NLR for disease-free survival (DFS) and overall survival (OS). Results: Patients were mostly male (79%) with high grade () lymph node positive (21%), T3-4 (33%) . The positivity of PD-L1 expression was 22% (22/100) at cut-off 1%. Univariate analysis showed that PD-L1 expression was positively associated with larger tumor size, higher grade, positive lymph node metastases. Median DFS and OS were 35 months and 37 months, respectively. Both PD-L1 expression and NLR were associated with DFS and OS. COX analysis model showed that PD-L1 and NLR were independent prognostic factors for tumor prognosis. Of interest, when patients were divided in two groups based on PD-L1/NLR: patients with PD-L1+/high NLR as group 1 and other patients as group 2, group1 had significantly shorter DFS and OS (DFS, X2 = 4.146, P = 0.042; OS, X2 = 10.274, P = 0.001). COX proportional hazards regression models indicated that PD-L1+/high NLR was correlated with a significantly shorter DFS and OS (DFS, hazard ration [ HR] = 2.572, P = 0.05; OS, HR = 3.811, P = 0.003). Conclusions: The study indicated that combined use of PD-L-1/ NLR as predictive biomarker for survival. This feasible panel may be optional maker applied to select patients for immunotherapy.

Immunofocus-PD-L1 high throughput quantitative next generation sequencing assay.

e13521 Background: Immunohistochemical (IHC) assays are presently used as the gold standard predictive tests for immunotherapy but are compromised due to a number of potential variables. Comparative studies have demonstrated differing levels of PD-L1 staining between assays which appears independent of the antibody binding epitope. Secondly, inter-reader reliability even between expert pathologists is problematic particularly for assessment of PD-L1 positive immune cell populations. Methods: To improve predictive testing for anti PD-L1/PD1 immunotherapies we have developed and validated a Next Generation Sequencing Platform, Immunofocus, able to perform high-throughput quantitative PD-L1 gene expression levels in routine diagnostic PWET biopsies. We applied Immunofocus to a cohort of 130 NSCLCs and compared PD-L1 gene expression levels with PD-L1 IHC scores generated using the VENTANA PD-L1 (SP142) Assay. The PD-L1 IHC assessment was carried out double blinded by an independent laboratory. PD-L1 IHC scores were calculated using an algorithm combining tumour proportion score (TPS) with a PD-L1 positive immune cell (IC) score and immune cell area. Results: An exceptionally high degree of correlation was observed between the NGS PD-L1 levels with the combined PD-L1 IHC scores (P < 0.001). Therapeutic cut points for NGS PD-L1 levels were identified corresponding to PD-L1 IHC defined clinical cut points. Notably, ~20% of patients with negative PD-L1 IHC scores showed high NGS PD-L1 expression levels. We hypothesize that these cases represent false negatives and identify a cohort of patients who have shown significant response rates to anti-PD-L1/PD-directed immunotherapies. Conclusions: The Immunofocus NGS PD-L1 assay has potential to greatly improve patient selection for immunotherapy by removing the IHC assay variables and inter-reader variability which compromise current PD-L1 IHC tests while also providing standardized high throughput in the clinical setting. Immunofocus is able to integrate gene expression with somatic mutation analysis allowing capture of networks regulating the immune-checkpoint including for example adaptive and innate resistance pathways, JAK1/2 pathways, differential MHC expression, TEFF gene signature, neoantigen surrogates such as DDR defects and TMB. The integration of NGS PD-L1 expression with other putative biomarkers of response is presently ongoing to further improve prediction of response.

The path to a better biomarker: application of a risk management framework for the implementation of PD-L1 and TILs as immuno-oncology biomarkers in breast cancer clinical trials and daily practice.

Immune checkpoint inhibitor therapies targeting PD-1/PD-L1 are now the standard of care in oncology across several hematologic and solid tumor types, including triple negative breast cancer (TNBC). Patients with metastatic or locally advanced TNBC with PD-L1 expression on immune cells occupying ≥1% of tumor area demonstrated survival benefit with the addition of atezolizumab to nab-paclitaxel. However, concerns regarding variability between immunohistochemical PD-L1 assay performance and inter-reader reproducibility have been raised. High tumor-infiltrating lymphocytes (TILs) have also been associated with response to PD-1/PD-L1 inhibitors in patients with breast cancer (BC). TILs can be easily assessed on hematoxylin and eosin-stained slides and have shown reliable inter-reader reproducibility. As an established prognostic factor in early stage TNBC, TILs are soon anticipated to be reported in daily practice in many pathology laboratories worldwide. Because TILs and PD-L1 are parts of an immunological spectrum in BC, we propose the systematic implementation of combined PD-L1 and TIL analyses as a more comprehensive immuno-oncological biomarker for patient selection for PD-1/PD-L1 inhibition-based therapy in patients with BC. Although practical and regulatory considerations differ by jurisdiction, the pathology community has the responsibility to patients to implement assays that lead to optimal patient selection. We propose herewith a risk-management framework that may help mitigate the risks of suboptimal patient selection for immuno-therapeutic approaches in clinical trials and daily practice based on combined TILs/PD-L1 assessment in BC. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

IGFBP2 regulates PD-L1 expression by activating the EGFR-STAT3 signaling pathway in malignant melanoma.

Immunotherapy targeting the PD-1/PD-L1 receptor has achieved great success in melanoma patients. Although many studies have addressed the underlying mechanisms involved in the blockade of PD-1/PD-L1 and the consequent modulation of the immune system, the mechanisms of PD-L1 upregulation and reliable biomarkers to predict the efficacy of anti-PD-1/PD-L1 therapy remain unknown. The present study demonstrates the correlation between IGFBP2 and PD-L1, revealing a novel immune-associated tumor function of IGFBP2 in facilitating nuclear accumulation of EGFR and activation of the EGFR/STAT3/PD-L1 signaling pathway in melanoma cells. Our results also suggest that combined IGFBP2 and PD-L1 expression has the potential to predict the efficacy of anti-PD-1 treatment for malignant melanoma; because the combination of high IGFBP2 and PD-L1 expression characterizes melanoma patients with worse overall survival and is associated with a better immune ecosystem. These characteristics have been confirmed by both in vitro and in vivo data. Consequently, IGFBP2 regulates PD-L1 expression by activating the EGFR-STAT3 signaling pathway and its function as a PD-L1 regulator might suggest novel therapeutic approach for melanoma.

Infiltration of CD163-positive macrophages in glioma tissues after treatment with anti-PD-L1 antibody and role of PI3Kγ inhibitor as a combination therapy with anti-PD-L1 antibody in in vivo model using temozolomide-resistant murine glioma-initiating cells

Although chemoimmunotherapy often lengthens glioblastoma (GBM) survival, early relapses remain problematic as immunosuppressive M2 macrophages (Mϕ) that function via inhibitory cytokine and PD-L1 production cause immunotherapy resistance. Here, we detail anti-PD-L1 antibody effects on the tumor microenvironment, including Mϕ infiltration, using a temozolomide (TMZ)-treated glioma model. In addition, we tested combinations of anti-PD-L1 antibody and the M2Mϕ inhibitor IPI-549 on tumor growth. We simulated late TMZ treatment or relapse stage, persistent GBM cells by generating TMZ-resistant TS (TMZRTS) cells. M2Mϕ-associated cytokine production and PD-L1 expression in these cells were investigated. TMZRTS cells were then subcutaneously implanted into C57BL/6 mice to determine the effectiveness of an anti-PD-L1 antibody and/or IPI-549 treatment on infiltration of CD163-positive Mϕ, usually considered as an M2Mϕ marker into tumor tissues. CD163 expression in samples from human GBM patients were also evaluated. CD163-positive Mϕ heavily infiltrated TMZRS tumor tissues after in vivo anti-PD-L1 antibody treatment. Tumor growth was strongly inhibited by anti-PD-L1 antibody and IPI-549 combination therapy. Anti-PD-L1 antibody treatment significantly reduced infiltration of CD163-positive Mϕ into tumors, while combined PD-L1 antibody and IPI-549 therapy remarkably inhibited tumor growth. These therapies may be useful for recurrent or chronic GBM after TMZ treatment, but clinical safety and effectiveness studies are needed.

Abstract B58: A phase I/II study of durvalumab and stereotactic radiotherapy in locally advanced pancreatic cancer

Abstract Purpose/Objective: Blockade of the PD-1/PD-L1 pathway with radiotherapy (RT) can enhance both local and systemic control in varying tumor types. Yet, no significant responses have been seen in pancreatic cancer (PC). We have previously shown that RT induces PD-1 and PD-L1 in PC following upregulation of interferon-gamma. This mechanism of adaptive immune resistance by PC may serve as an ideal therapeutic target for combined RT and PD-L1 blockade. We report interim results of an ongoing phase 1/2 clinical trial in locally advanced (LA) and borderline resectable (BR) PC (NCT03245541). Materials/Methods: Patients with LA or BR PC treated with standard-of-care gemcitabine and nab-paclitaxel for 3 to 6 cycles were enrolled. Treatment consisted of durvalumab (750 mg Q14 days) on D1. Stereotactic ablative radiotherapy (SABR; 6.6 Gy/fraction) was delivered every other day x 5 fractions beginning D8. Durvalumab continued as maintenance Q14 days until resection or progression. The run-in phase I utilized a standard 3+3 design prior to phase 2 expansion. Dose-limiting toxicities (DLTs), adverse events (AEs), and serious adverse events (SAEs) were assessed during the first 10 weeks of study treatment. CT scans were obtained every 2 months for response assessment. Endoscopic research biopsies were obtained pre- and 6-8 weeks post-SABR, and weekly blood samples were obtained on D1, weekly for 10 weeks, and then every 2 months until resection or progression to assess for immune correlates of response. Results: From 8/2017-5/2019, 18 of 30 planned patients were enrolled of whom 13 (72%) were LA and 5 (28%) were BR. Median age was 70 and 44% were female. No DLTs were identified in the phase 1 run in, and enrollment in the phase 2 study continues. Median follow-up from diagnosis is 14 months (range 6-26 months). Median cycles of chemotherapy received prior to enrollment were 4. Grade 3 toxicities have been identified in 2 patients (nausea, anorexia). No patients experienced dose-limiting toxicities. Median PFS was 14 months with median OS not reached. 9 of 18 patients have undergone resection and all were margin negative. BOR: PR (54%); CR (6%), SD (34%), PD (6%). Conclusion: To our knowledge, this is the first report of a PD-L1 inhibitor-RT combination in LA PC. The regimen was safe, well tolerated and appears to be clinically active with high rates of margin-negative resection. Citation Format: Richard Tuli, Nicholas Nissen, Simon Lo, Mourad Tighiouart, Veronica Placencio, Andrew Hendifar. A phase I/II study of durvalumab and stereotactic radiotherapy in locally advanced pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B58.

Isolated autoimmune adrenocorticotropic hormone deficiency: From a rare disease to the dominant cause of adrenal insufficiency related to check point inhibitors.

Immune checkpoint inhibitors have introduced a new and heterogeneous class of immune-related adverse effects, with the endocrine system being a predominant target for autoimmunity. Autoimmune hypothalamic-pituitary-adrenal axis (HPA) diseases induced by checkpoint inhibitors are being increasingly recognized. We aimed to characterize the spectrum of checkpoint associated hypothalamic-pituitary-adrenal axis endocrinopathies. A retrospective cohort study of a tertiary cancer center. Patients were characterized for HPA axis abnormalities based on clinical and pituitary axes evaluation. The risk for developing HPA endocrinopathies was compared by log- rank test, by the time since checkpoint inhibitors initiation. Additionally, the risk for developing HPA endocrinopathies after adjusting for covariates was assessed using multivariable logistic regression analysis. Among 1615 patients, fourteen (0.87%) patients developed isolated adrecocorticotrophic hormone deficiency (IAD), six (0.37%) - hypophysitis and no case of adrenalitis was identified. IAD presented with mild and non-specific symptoms, mainly asthenia. In multivariable analysis, exposure to both PD-1/PD-L1 and Ipilimumab and female gender were associated with an increased odds ratio (OR) for developing IAD (6.98 [95% CI 2.38-20.47, p<.001] and 3.67 [95% CI 1.13-11.84, p=.03]), respectively. IAD, a rare disease before the immunotherapy era, has become a predominant checkpoint related HPA axis autoimmune injury. Despite its life threatening potential, IAD may be missed due to its subtle presentation. Patients exposed to Ipilimumab and PD-1/PD-L1 in combination or sequentially and women have an increased risk for developing IAD.

Association of combined PD-L1 expression and tumour-infiltrating lymphocyte features with survival and treatment outcomes in patients with metastatic melanoma.

Recent advances obtained with immune checkpoint inhibitors (ICIs) targeting the programmed cell death-1 (PD-1) protein have significantly improved the outcome of patients with metastatic melanoma. The PD-L1 expression in tumour cells as detected by immunohistochemistry is a predictive biomarker in some solid tumours, but appears insufficient as prognostic or predictive factor of response to ICIs in metastatic melanomas. We investigated whether the presence and the features of pretreatment CD8+ tumour-infiltrating T lymphocytes (TILs) could be a complementary prognostic or predictive biomarker in patients with metastatic melanoma. In this retrospective study, we evaluated the association of PD-L1 expression ≥5% of tumour cells combined with TIL features (CD8, CD28, Ki67) with the overall survival (OS) among 51 patients treated with ICIs and 54 patients treated with other treatment options (non-ICIs). PD-L1 positivity was observed in 33% and 39% of primary melanomas and matched metastases, respectively, with, however, poor concordance between the primary and the matched metastatic site (κ=0.283). No significant association was noted between PD-L1 expression and CD8+ TIL profile analysed as single markers and OS or response to immunotherapy. Instead, their combined analysis in primary melanoma samples showed that the PD-L1-/CD8+ status was significantly associated with prolonged OS in the whole population (P=0.04) and in the subgroup treated with non-ICIs (P=0.009). Conversely, the PD-L1+/CD8+ status was a good prognostic factor in patients treated with ICIs (P=0.022), whereas was significantly associated with poor prognosis in patients treated with non-ICIs (P=0.014). While the expression of CD28 was not related to outcome, the Ki67 expression was significantly associated with poor OS in the subgroup CD8+ TIL+/PD-L1- (P=0.02). The pretreatment combination of PD-L1 expression with the level of CD8+ TILs could better assess OS and predict therapeutic response of patients with metastatic melanoma treated by either immunotherapy or other treatment regimens.

P2.04-44 Combined Immune Checkpoint Blockade in Mesothelioma: In Vivo Investigation of in Vitro Data

Malignant pleural mesothelioma (MPM) is an aggressive cancer that is causally associated with asbestos exposure. Due to its aggressive nature and despite the effectiveness of conventional anti-cancer treatment, the prognosis of patients diagnosed with MPM remains dismal, highlighting the urgent need for new therapeutic strategies. Our group and others have recently demonstrated PD-1 and PD-L1 expression in MPM patients, providing rationale to evaluate their suitability as immunotherapeutic targets in MPM. Tree human cell lines representative for the epithelioid and sarcomatoid subtypes of MPM were placed in allogeneic co-cultures with healthy donor peripheral blood mononuclear cells. The co-cultures were treated with the following immune checkpoint blocking antibodies: anti PD-1 (Nivolumab®, BMS) or anti PD-L1 (Durvalumab®, AstraZeneca) in combination with anti TIM-3 or anti LAG-3. Supernatant was collected and enzyme-linked immunosorbent assays and multiplex electrochemo-luminescence were used to look at the secretion of 7 cytokines, being IFNγ, IL-2/5/6/10, IL-1band TNF-α, as well as the enzyme granzyme B. Significant differences were found for the secretion of IFNγ, granzyme B, IL-2, IL-5 and IL-10. Though the differences were not always significant for the 3 MPM cell lines, the same trends were observed among them. Interestingly, highest concentrations of the aforementioned cytokines were all noticed for monotherapy treatment with anti PD-1, anti PD-L1 or or their combination with anti TIM-3. In vivo investigation of PD-1, PD-L1 and TIM-3 blockade, alone or in combination is required for validation of our in vitro results and is currently ongoing. Our data show that treatment with anti PD-1, anti PD-L1 or their respective combination with anti TIM-3 resulted in the highest secretion of cytokines and granzyme B, suggesting that these treatments stimulate the antitumor response the most. Results of our in vivo validation are awaited in order to confirm our in vitro findings.

Niclosamide, an antihelmintic drug, enhances efficacy of PD-1/PD-L1 immune checkpoint blockade in non-small cell lung cancer

BackgroundPD-1/PD-L1 blockade has received approval for clinical application due to its encouraging benefit with improving prognosis in selected populations. Unfortunately, the response to immunotherapy for many patients remains unsatisfactory. It remains a great challenge to generate potential combinations that will outperform single agents alone with regard to anti-tumor activity.MethodsUsing NSCLC cell lines and mouse models, we explored the effects of combined niclosamide and PD-L1 blockade on tumor growth and T cell function. Furthermore, we investigated the relationship between PD-L1 and p-STAT3 expression in tumor samples from patients with NSCLC using IHC, as well as their relationship to patient survival.ResultsIn vitro, niclosamide, an antihelmintic drug, enhanced the cancer cell lysis mediated by T cells in the presence of PD-L1 blockade. Accordingly, mice treated with niclosamide and PD-L1 antibody showed significant delay in tumor growth and increased survival which were associated with the increase of tumor infiltrating T cells and granzyme B release. Importantly, we found niclosamide could decrease the expression of PD-L1 in both a concentration- and time-dependent manner in NSCLC cells, which was linked to the blockage of p-STAT3 binding to the promoter of PD-L1.ConclusionsAn enhancement of PD-L1 antibody by niclosamide was observed in inhibition of NSCLC growth in vitro and in vivo, which was involved in blockage of p-STAT3 binding to promoter of PD-L1 and finally downregulation of PD-L1 expression. These encourage the combination therapy of niclosamide and PD-1/PD-L1 blockade to be further studied in clinic.

The Obesity Paradox in Cancer, Tumor Immunology, and Immunotherapy: Potential Therapeutic Implications in Triple Negative Breast Cancer.

Cancer immunotherapy has been heralded as a breakthrough cancer treatment demonstrating tremendous success in improving tumor responses and survival of patients with hematological cancers and solid tumors. This novel promising treatment approach has in particular triggered optimism for triple negative breast cancer (TNBC) treatment, a subtype of breast cancer with distinct clinical features and poor clinical outcome. In early 2019, the FDA granted the first approval of immune checkpoint therapy, targeting PD-L1 (Atezolizumab) in combination with chemotherapy for the treatment of patients with locally advanced or metastatic PD-L1 positive TNBC. The efficacy of immuno-based interventions varies across cancer types and patient cohorts, which is attributed to a variety of lifestyle, clinical, and pathological factors. For instance, obesity has emerged as a risk factor for a dampened anti-tumor immune response and increased risk of immunotherapy-induced immune-related adverse events (irAEs) but has also been linked to improved outcomes with checkpoint blockade. Given the breadth of the rising global obesity epidemic, it is imperative to gain insight into the immunomodulatory effects of obesity in the peripheral circulation and within the tumor microenvironment. In this review, we resolve the impact of obesity on breast tumorigenesis and progression on the one hand, and on the immune contexture on the other hand. Finally, we speculate on the potential implications of obesity on immunotherapy response in breast cancer. This review clearly highlights the need for in vivo obese cancer models and representative clinical cohorts for evaluation of immunotherapy efficacy.

Combination PD-1 and PD-L1 Blockade Promotes Durable Neoantigen-Specific T Cell-Mediated Immunity in Pancreatic Ductal Adenocarcinoma

SUMMARYPancreatic ductal adenocarcinoma (PDA) is a lethal cancer resistant to immunotherapy. We create a PDA mouse model and show that neoantigen expression is required for intratumoral T cell accumulation and response to immune checkpoint blockade. By generating a peptide:MHC tetramer, we identify that PDA induces rapid intratumoral, and progressive systemic, tumor-specific T cell exhaustion. Monotherapy PD-1 or PD-L1 blockade enhances systemic T cell expansion and induces objective responses that require systemic T cells. However, tumor escape variants defective in IFNγ-inducible Tap1 and MHC class I cell surface expression ultimately emerge. Combination PD-1 + PD-L1 blockade synergizes therapeutically by increasing intratumoral KLRG1+Lag3−TNFα+ tumor-specific T cells and generating memory T cells capable of expanding to spontaneous tumor recurrence, thereby prolonging animal survival. Our studies support that PD-1 and PD-L1 are relevant immune checkpoints in PDA and identify a combination for clinical testing in those patients with neoantigen-specific T cells.

M2 macrophage infiltration into tumor islets leads to poor prognosis in non-small-cell lung cancer.

Background: Lung cancer is the leading cause of cancer-related death worldwide. Although the macrophages can affect the development of tumor, the contribution of macrophages to the prognosis of non-small-cell lung cancer (NSCLC) is still controversial. Moreover, anti-PD-1 therapy can redirect macrophages from an M2 to an M1 phenotype, suggesting that tumor PD-L1 may affect the prognostic role of macrophages. Therefore, in this study, we aimed to display a macrophage landscape to clarify the function of macrophages, considering the localization and polarization of the macrophages, and evaluate the effect of M2 macrophages and tumor PD-L1 in combination on the prognosis of NSCLC.Methods: We performed multiplex quantitative immunofluorescence staining of pan-cytokeratin (CK), CD68, CD163, PD-L1, and DAPI on one tissue specimen simultaneously from 137 NSCLC patients.Results: M2 macrophages, involved marginM2 (M2 macrophages in tumor stroma), and centralM2 (M2 macrophages infiltrating into tumor islets) increased as the tumor stage increased. More macrophages were found in lung squamous cell carcinoma (LUSC) patients, patients with wild-type EGFR, and smokers than in patients with lung adenocarcinoma (LUAD), patients with EGFR mutations, and non-smokers. Infiltration of centralM2 was an independent prognostic factor of poor overall survival (OS) and disease-free survival (DFS) for NSCLC patients (P<0.05), which was superior to total macrophages and total M2 macrophages. Moreover, patients with centralM2lessPD-L1− tumors showed the best OS and DFS, while the patients with centralM2morePD-L1+ tumors showed the worst OS and DFS, and the two groups with centralM2lessPD-L1+ and centralM2morePD-L1− were in the middle (P=0.002, 0.034, respectively).Conclusion: Tumor islet-infiltrating M2 macrophages influence the prognosis of NSCLC patients. The analysis of M2 macrophages and tumor PD-L1 in combination may enhance the accuracy of prognostic prediction. This study provides a new understanding of macrophages in the development of NSCLC through the analysis of macrophage landscape.

Tumor mutational burden (TMB) and PD-L1 expression as predictors of response to immunotherapy (IO) in NSCLC.

2630 Background: PD-L1 expression and TMB, as a proxy for neoantigen burden, have been correlated with response to IO in advanced NSCLC (aNSCLC) clinical trials, but their combined utility is unclear. We assessed TMB and PD-L1 as predictors of response in aNSCLC patients (pts) after IO monotherapy in a real-world setting. Methods: Pts had a diagnosis of aNSCLC, comprehensive genomic profiling of 186-315 genes/1.1 megabase (Mb), PD-L1 testing of pre-IO specimens, and were treated in the Flatiron Health network (1/2011 - 6/2018). Clinical characteristics and real-world tumor response (rwTR) were obtained via technology-enabled abstraction of clinician notes and radiology/pathology reports, and linked to genomic data in the Flatiron Health-Foundation Medicine Clinico-Genomic Database. A general additive model examined the predictive value of TMB (as continuous measure) and PD-L1 level on rwTR. A reduced PD-L1-only model was compared to the full model using Akaike Information Criterion (AIC). rwTR predictions at representative TMB and PD-L1 levels were calculated. Results: Of 426 pts, PD-L1 expression was high (≥50%) in 140, low (1-49%) in 123, and negative (&lt;1%) in 163. Median TMB was 9.6 mut/Mb (IQR 4.4 - 14.8) overall, 11.3 in responders and 8.7 in non-responders. TMB did not correlate with PD-L1 level (Kruskal-Wallis p=0.29). The TMB + PD-L1 model had superior prediction of rwTR than the PD-L1 model, as assessed by lower AIC score. In the combined model, higher TMB and PD-L1 levels were each associated with higher rwTR likelihood (Table). Predicted rwTR probability, % (95% CI), by TMB and PD-L1 in line 1. Conclusions: TMB and PD-L1 expression are independent markers that, when combined, have increased predictive power for response to IO. High TMB + low/neg PD-L1 behaved similarly to low TMB + high PD-L1, and high TMB + high PD-L1 predicted the highest rwTR. Investigation of these biomarkers as complementary predictors of progression and overall survival is ongoing.[Table: see text]