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Intratumoral leukocyte composition of mesothelioma representing pre- and post-therapy immunotherapy, evaluated by multiplex IHC.

Purpose:Malignant pleural mesothelioma (MPM) is an aggressive cancer associated with poor prognosis. CRS-207 is a live-attenuated Listeria monocytogenes engineered to express mesothelin, a tumor-associated antigen highly expressed in MPM. CRS-207 induces antitumor immune responses and increases susceptibility of neoplastic cells to immune-mediated killing.Patients and Methods:Patients with unresectable MPM, ECOG 0 or 1, and adequate organ and pulmonary function were enrolled in this multicenter, open-label phase Ib study. They received two priming infusions of 1 × 109 CFU CRS-207, followed by pemetrexed/cisplatin chemotherapy, and CRS-207 booster infusions. Primary objectives were safety and induction of immune response. Secondary/exploratory objectives included tumor response, progression-free survival (PFS), overall survival (OS), immune subset analysis, and gene-expression profiling of tumor.Results:Of 35 evaluable patients, 89% (31/35) had disease control with one complete response (3%), 19 partial responses (54%), and 10 stable disease (29%). The estimated median duration of response was 5.0 months (95% CI, 3.9–11.5). The median PFS and OS were 7.5 (95% CI, 7.0–9.9) and 14.7 (95% CI, 11.2–21.9) months, respectively. Tumor size reduction was observed post–CRS-207 infusion prior to chemotherapy in 11 of 35 (31%) patients. No unexpected treatment-related serious adverse events or deaths were observed. IHC analysis of pre- and post–CRS-207 treatment tumor biopsies revealed possible reinvigoration and proliferation of T cells, increased infiltration of dendritic and natural killer cells, increased CD8:Treg ratio, and a shift from immunosuppressive M2-like to proinflammatory M1-like macrophages following CRS-207 administration.Conclusions:Combination of CRS-207 and chemotherapy induced significant changes in the local tumor microenvironment and objective tumor responses in a majority of treated patients.

Supplementary Data from Anti-inflammatory Triterpenoid Blocks Immune Suppressive Function of MDSCs and Improves Immune Response in Cancer

Purpose: Myeloid-derived suppressor cells (MDSC) are one of the major factors responsible for immune suppression in cancer. Therefore, it would be important to identify effective therapeutic means to modulate these cells.Experimental Design: We evaluated the effect of the synthetic triterpenoid C-28 methyl ester of 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid (CDDO-Me; bardoxolone methyl) in MC38 colon carcinoma, Lewis lung carcinoma, and EL-4 thymoma mouse tumor models, as well as blood samples from patients with renal cell cancer and soft tissue sarcoma. Samples were also analyzed from patients with pancreatic cancer treated with CDDO-Me in combination with gemcitabine.Results: CDDO-Me at concentrations of 25 to 100 nmol/L completely abrogated immune suppressive activity of MDSC in vitro. CDDO-Me reduced reactive oxygen species in MDSCs but did not affect their viability or the levels of nitric oxide and arginase. Treatment of tumor-bearing mice with CDDO-Me did not affect the proportion of MDSCs in the spleens but eliminated their suppressive activity. This effect was independent of antitumor activity. CDDO-Me treatment decreased tumor growth in mice. Experiments with severe combined immunodeficient–beige mice indicated that this effect was largely mediated by the immune system. CDDO-Me substantially enhanced the antitumor effect of a cancer vaccines. Treatment of pancreatic cancer patients with CDDO-Me did not affect the number of MDSCs in peripheral blood but significantly improved the immune response.Conclusions: CDDO-Me abrogated the immune suppressive effect of MDSCs and improved immune responses in tumor-bearing mice and cancer patients. It may represent an attractive therapeutic option by enhancing the effect of cancer immunotherapy. Clin Cancer Res; 16(6); 1812–23

Supplementary Data from Phase I Study of Taminadenant (PBF509/NIR178), an Adenosine 2A Receptor Antagonist, with or without Spartalizumab (PDR001), in Patients with Advanced Non–Small Cell Lung Cancer

Biomarkers utilized for defining immune cell lineages in IHC analysis

Supplementary Figure from Phase I Study of Taminadenant (PBF509/NIR178), an Adenosine 2A Receptor Antagonist, with or without Spartalizumab (PDR001), in Patients with Advanced Non–Small Cell Lung Cancer

Purpose:The adenosine 2A receptor (A2AR) mediates the immunosuppressive effects of adenosine in the tumor microenvironment and is highly expressed in non–small cell lung cancer (NSCLC). Taminadenant (PBF509/NIR178) is an A2AR antagonist able to reactivate the antitumor immune response.Patients and Methods:In this phase I/Ib, dose-escalation/expansion study, patients with advanced/metastatic NSCLC and ≥1 prior therapy received taminadenant (80–640 mg, orally, twice a day) with or without spartalizumab (anti–programmed cell death-1, 400 mg, i.v., every 4 weeks). Primary endpoints were safety, tolerability, and feasibility of the combination.Results:During dose escalation, 25 patients each received taminadenant alone or with spartalizumab; 19 (76.0%) and 9 (36.0%) had received prior immunotherapy, respectively. Dose-limiting toxicities (all Grade 3) with taminadenant alone were alanine/aspartate aminotransferase increase and nausea [n = 1 (4.0%) each; 640 mg], and in the combination group were pneumonitis [n = 2 (8.0%); 160 and 240 mg] and fatigue and alanine/aspartate aminotransferase increase [n = 1 (4.0%) each; 320 mg]; pneumonitis cases responded to steroids rapidly and successfully. Complete and partial responses were observed in one patient each in the single-agent and combination groups; both were immunotherapy naïve. In the single-agent and combination groups, 7 and 14 patients experienced stable disease; 7 and 6 patients were immunotherapy pretreated, respectively.Conclusions:Taminadenant, with and without spartalizumab, was well tolerated in patients with advanced NSCLC. The maximum tolerated dose of taminadenant alone was 480 mg twice a day, and 240 mg twice a day plus spartalizumab. Efficacy was neither a primary or secondary endpoint; however, some clinical benefit was noted regardless of prior immunotherapy or programmed cell death ligand-1 status.

A proportion of patients with lung cancer experience long-term clinical benefit with immune checkpoint inhibitors (ICIs). However, most patients develop disease progression during treatment or after treatment discontinuation. Definitions of immune resistance are heterogeneous according to different clinical and biologic features. Primary resistance and acquired resistance, related to tumor-intrinsic and tumor-extrinsic mechanisms, are identified according to previous response patterns and timing of occurrence. The clinical resistance patterns determine differential clinical approaches. To date, several combination therapies are under development to delay or prevent the occurrence of resistance to ICIs, including the blockade of immune coinhibitory signals, the activation of those with costimulatory functions, the modulation of the tumor microenvironment, and the targeting T-cell priming. Tailoring the specific treatments with distinctive biologic resistance mechanisms would be ideal to improve the design and results of clinical trial. In this review, we reviewed the available evidence on immune resistance mechanisms, clinical definitions, and management of resistance to ICIs in lung cancer. We also reviewed data on novel strategies under investigation in this setting.

1.AbstractLung cancer is the leading cause of cancer death worldwide with 1.76 million people die as a result of the disease yearly. Analyzing high-throughput multiplexed images of tissues has recently emerged as a routine clinical procedure for early detection, clinical cancer diagnosis, treatment planning and prognosis for many cancer types including non-small cell lung cancer (NSCLC). Multiplexed images enable the precise interpretation of spatial distribution of cells and cellular states and the characterization of tumor-immune interactions in situ and at the single-cell level. Through two cornerstone image processing techniques - cell segmentation and image tiling generating quadrats - we perform an in-depth analysis of multiplexed images based on the frequency, phenotype, and spatial distribution of immune and tumor cells within the immune landscape of NSCLC. Multiplexed images were obtained from nine patients with advanced/metastatic NSCLC with progression who were treated with an oral HDAC inhibitor (vorinostat) combined with a PD-1 inhibitor (pembrolizumab). Images were collected from all patients both pre- and on-treatment (days 15-21). We profile ∼100K cells and ∼10K quadrats from tumor and adjacent tissues to characterize cellular composition and spatial neighborhoods and elucidate that distinct spatial cellular ecologies exist across progressive disease (PD) and stable disease (SD) patients where tumors of PD patients are characterized by a highly suppressed immune environment prior to treatment enabling higher chances of disease progression during treatment. These fundamentally distinct architectures across PD and SD patients enable disease progression prediction and clinical biomarker identification. Our results also provide a potential companion biomarker for PD-L1 in NSCLC. Validating the biomarker and its feasibility will require further in-depth investigation.

Cancer immunotherapy often depends on recognition of peptide epitopes by cytotoxic T lymphocytes (CTLs). The tumor microenvironment (TME) is enriched for peroxynitrite (PNT), a potent oxidant produced by infiltrating myeloid cells and some tumor cells. We demonstrate that PNT alters the profile of MHC class I bound peptides presented on tumor cells. Only CTLs specific for PNT-resistant peptides have a strong antitumor effect in vivo, whereas CTLs specific for PNT-sensitive peptides are not effective. Therapeutic targeting of PNT in mice reduces resistance of tumor cells to CTLs. Melanoma patients with low PNT activity in their tumors demonstrate a better clinical response to immunotherapy than patients with high PNT activity. Our data suggest that intratumoral PNT activity should be considered for the design of neoantigen-based therapy and also may be an important immunotherapeutic target.

There are limited real-world data about patient-reported outcomes with immunotherapies (IO) in metastatic non-small cell lung cancer (mNSCLC). We describe patient-reported distress and clinical outcomes with IO-based treatments or cytotoxic chemotherapies (Chemo).We conducted a single-institution retrospective chart review of adults with mNSCLC treated at Duke from 03/2015 to 06/2020. At each visit, patients self-reported their distress level and sources of distress using the NCCN Distress Thermometer (DT) and its 39-item Problem List. We abstracted demographic, clinical, distress, and investigator assessed-clinical response data, then analyzed these using descriptive statistics and generalized estimating equations.Data from 152 patients were analyzed in four groups: Chemo alone, IO + Chemo, single agent IO, dual agent IO. Distress was worse before treatment start in all groups, and the odds of actionable distress (DT score 4) decreased by 10 % per month. The most frequent sources of distress were physical symptoms (e.g., fatigue, pain), which remained high longitudinally. Patients receiving IO had higher clinical response rates and a lower rate of unplanned healthcare encounters compared to patients treated with Chemo alone. Only one-third of all patients were seen by palliative care.This single-center, real-world evidence study demonstrates that patients with mNSCLC experience significant distress prior to starting first-line treatment. IO treatment was associated with higher clinical benefit rates and lower healthcare utilization compared to chemotherapy. Symptom distress persists over time, highlighting potential unmet palliative and supportive care needs in mNSCLC care in the IO treatment era.

Objective: In CheckMate 743 (NCT02899299), nivolumab + ipilimumab significantly prolonged overall survival in patients with unresectable malignant pleural mesothelioma (MPM). We present patient-reported outcomes (PROs). Materials and Methods: Patients (N = 605) were randomized to nivolumab + ipilimumab or chemotherapy. Changes in disease-related symptom burden and health-related quality of life (HRQoL) were evaluated descriptively using the Lung Cancer Symptom Scale (LCSS)-Mesothelioma (Meso) average symptom burden index (ASBI), LCSS-Meso 3-item global index (3-IGI), 3-level EuroQol 5-dimensional (EQ-5D-3L) visual analog score (VAS), and EQ-5D-3L utility index. PROs were assessed at baseline and every 2 (nivolumab + ipilimumab) or 3 weeks (chemotherapy) through 12 weeks, every 6 weeks through 12 months, every 12 weeks thereafter, and at specified follow-ups. Mixed-effect model repeated measures (MMRM) and time to deterioration analyses were conducted. Results: Completion rates were generally > 80%. LCSS-Meso ASBI mean changes from baseline trended to improve over time with nivolumab + ipilimumab and deteriorate with chemotherapy, but did not meet clinically important difference thresholds [& PLUSMN;10 score change]. EQ-5D-3L VAS mean scores improved over time with nivolumab + ipilimumab; by week 60, patients had scores consistent with United Kingdom normal population values. MMRM analyses favored nivolumab + ipilimumab for all individual symptoms except cough. Nivolumab + ipilimumab delayed time to definitive deterioration in HRQoL (hazard ratio 0.52 [95% confidence interval 0.36-0.74]) and showed a trend in symptom delay versus chemotherapy. Conclusions: Nivolumab + ipilimumab decreased the risk of deterioration in disease-related symptoms and HRQoL versus chemotherapy and maintained QoL in patients with unresectable MPM.

Glioblastoma is the most prevalent primary brain malignancy, with a median survival of less than 21 months despite treatment with surgery, radiation, and chemotherapy. Significant intra-tumoral heterogeneity, combined with the immunosuppressive micro-environment and the limited immune population present within the privileged CNS has resulted in the failure of immunotherapy approaches. We seek to address these challenges by isolating Tumor Infiltrating Lymphocytes (TILs) which are already neoantigen specific and expanding them ex vivo before re-administering them to the patient. This allows us to greatly increase the available pool of T cells which are specific for a diversity of tumor neoantigens in the patient, while also reversing exhaustion by the addition of stimulatory co-culturing agents. We report the successful expansion of glioblastoma associated TILs from both surgically resected tumors (5 of 7 samples, 71%) and liquid aspirate from the surgical field collected in Lukens traps (4 of 6 samples, 66%). TILs were expanded with high-dose IL2 initially in the presence of tumor digest (PreREP) followed by activation and expansion with CD3/CD28 nanobeads (REP). Flow cytometry performed on PreREP TILs revealed that 50-90% of the population consisted of effector memory T cells (CCR7-CD45RA-) with 5-40% consisting of central memory T cells (CCR7+CD45RA-). The addition of stimulatory agents such as anti-4-1BB (Urelumab) during the PreREP phase increased cell yields, as well as skewing the population towards a non-exhausted CD8+ phenotype (reduced expression of PD1, LAG3, TIM3). These cells have demonstrated tumor antigen specific activation based on IFNγ and TNFα secretion following stimulation with autologous tumor digests and patient matched neurosphere cultures. TIL expansion with Urelumab doubled CD8% in the product (16% to 31%) and reduced CD4% (78% to 65%). Expanded TILs had more potent cytotoxic CD8+ T cells responses characterized by significantly increased numbers of tumor antigen specific IFNγ+ (std: 1000, Ure: 6000) and TNFα+ (std:2000, Ure:20,000))cells compared to traditionally expanded TILs. We have demonstrated that TIL isolation from resected clinical glioma is feasible and can generate large quantities of immune cells whose functional status can be altered by the addition of stimulatory agents. And most importantly these TILs are reactive and specific for tumor antigens. Following additional assessment of in vitro cytotoxicity against matched tumor neurospheres in vitro we will evaluate safety and feasibility of our novel TIL approach in patients with glioblastoma. Citation Format: Kelly M. Hotchkiss, Pamela Noldner, Kirit Singh, Anoop Patel, Peter Fecci, Scott Antonia, Beth H. Shaz, Mustafa Khasraw. Developing non-exhausted tumor infiltrating lymphocytes (TILs) as a therapy for glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3193.

We developed a novel modeling paradigm to specifically recapitulate 3D tissue at the resolution of CT imaging and yet capture single cell information: the population-based model (PBM). This PBM method allowed us to seed a model with individual cells that operate at the CT scale of cubic millimeters and grow to the scale of 10 cubic centimeters or more. We used our PBM to investigate how well-known tumor traits of increased resource consumption and angiogenesis maybe differentially selected by a growing tumor in different densities of normal tissue. We found that tumor phenotypes that reduced consumption and increased angiogenesis grew the fastest in low density normal tissue, and vice versa in high density normal tissue. We ran parameter sweeps of this model using temporal patient data from the NLST dataset, which consisted of untreated tumors in the low-density lung periphery. We saw that specific movement rate and proliferation rate parameters were required to fit the growth speed of the tumors, while a low consumption and high angiogenic phenotype was required to fit the radial density distribution of the tumors. Citation Format: Rafael Ramon Bravo, Mark Robertson-Tessi, Alexander R. Anderson, Robert Gatenby, Scott Antonia, Jhanelle Gray, Amer Beg, Matthew Schabath. The impact of normal tissue density on tumor growth and evolution in a 3D whole-tumor model of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 853.

BackgroundAvailability of checkpoint inhibitors has created a paradigm shift in the management of patients with solid tumors. Despite this, most patients do not respond to immunotherapy, and there is considerable interest in developing combination therapies to improve response rates and outcomes. B7-H3 (CD276) is a member of the B7 family of cell surface molecules and provides an alternative immune checkpoint molecule to therapeutically target alone or in combination with programmed cell death-1 (PD-1)–targeted therapies. Enoblituzumab, an investigational anti-B7-H3 humanized monoclonal antibody, incorporates an immunoglobulin G1 fragment crystallizable (Fc) domain that enhances Fcγ receptor-mediated antibody-dependent cellular cytotoxicity. Coordinated engagement of innate and adaptive immunity by targeting distinct members of the B7 family (B7-H3 and PD-1) is hypothesized to provide greater antitumor activity than either agent alone.MethodsIn this phase I/II study, patients received intravenous enoblituzumab (3–15 mg/kg) weekly plus intravenous pembrolizumab (2 mg/kg) every 3 weeks during dose-escalation and cohort expansion. Expansion cohorts included non–small cell lung cancer (NSCLC; checkpoint inhibitor [CPI]–naïve and post-CPI, programmed death-ligand 1 [PD-L1] ResultsOverall, 133 patients were enrolled and received ≥1 dose of study treatment. The maximum tolerated dose of enoblituzumab with pembrolizumab at 2 mg/kg was not reached. Intravenous enoblituzumab (15 mg/kg) every 3 weeks plus pembrolizumab (2 mg/kg) every 3 weeks was recommended for phase II evaluation. Treatment-related adverse events occurred in 116 patients (87.2%) and were grade ≥3 in 28.6%. One treatment-related death occurred (pneumonitis). Objective responses occurred in 6 of 18 (33.3% [95% CI 13.3 to 59.0]) patients with CPI-naïve HNSCC and in 5 of 14 (35.7% [95% CI 12.8 to 64.9]) patients with CPI-naïve NSCLC.ConclusionsCheckpoint targeting with enoblituzumab and pembrolizumab demonstrated acceptable safety and antitumor activity in patients with CPI-naïve HNSCC and NSCLC.Trial registration numberNCT02475213.

BackgroundRecent studies show that human gut microbial composition can determine whether a patient is a responder or non-responder to immunotherapy but have not identified a common microbial signal shared by responding patients. The functional relationship between immunity, intestinal microbiota, and NSCLC response to immune checkpoint inhibitor/inhibition (ICI) in an American cohort remains unexplored.MethodsRNAlater-preserved fecal samples were collected from 65 pre-treatment (baseline) and post-treatment stage III/IV NSCLC patients undergoing ICI therapy, categorized as responders or non-responders according to RECIST criteria. Pooled and individual responder and non-responder microbiota were transplanted into a gnotobiotic mouse model of lung cancer and treated with ICIs. 16S rDNA and RNA sequencing was performed on patient fecal samples, 16S rDNA sequencing on mouse fecal samples, and flow cytometric analysis on mouse tumor tissue.ResultsResponder patients have both a different microbial community structure than non-responders (P= 0.004) and a different bacterial transcriptome (PC2 = 0.03) at baseline. Taxa significantly enriched in responders include amplicon sequence variants (ASVs) belonging to the generaRuminococcus,Akkermansia, andFaecalibacterium. Pooled and individual responder microbiota transplantation into gnotobiotic mice decreased tumor growth compared to non-responder colonized mice following ICI (P= 0.023,P= 0.019,P= 0.008, respectively). Responder tumors showed an increased anti-tumor cellular phenotype following ICI treatment. Responder mice are enriched with ASVs belonging to the generaBacteroides,Blautia,Akkermansia, andFaecalibacterium. Overlapping taxa mapping between human and mouse cohorts correlated with tumor size and weight revealed a network highlighting responder-associated ASVs belonging to the generaColidextribacter,Frisingicoccus,Marvinbryantia, andBlautiawhich have not yet been reported.ConclusionsThe role of isolate-specific function and bacterial gene expression in gut microbial-driven responsiveness to ICI has been underappreciated. This work supports further investigation using isolate-driven models to characterize the mechanisms underlying this phenomenon.

Purpose:The adenosine 2A receptor (A2AR) mediates the immunosuppressive effects of adenosine in the tumor microenvironment and is highly expressed in non–small cell lung cancer (NSCLC). Taminadenant (PBF509/NIR178) is an A2AR antagonist able to reactivate the antitumor immune response.Patients and Methods:In this phase I/Ib, dose-escalation/expansion study, patients with advanced/metastatic NSCLC and ≥1 prior therapy received taminadenant (80–640 mg, orally, twice a day) with or without spartalizumab (anti–programmed cell death-1, 400 mg, i.v., every 4 weeks). Primary endpoints were safety, tolerability, and feasibility of the combination.Results:During dose escalation, 25 patients each received taminadenant alone or with spartalizumab; 19 (76.0%) and 9 (36.0%) had received prior immunotherapy, respectively. Dose-limiting toxicities (all Grade 3) with taminadenant alone were alanine/aspartate aminotransferase increase and nausea [n = 1 (4.0%) each; 640 mg], and in the combination group were pneumonitis [n = 2 (8.0%); 160 and 240 mg] and fatigue and alanine/aspartate aminotransferase increase [n = 1 (4.0%) each; 320 mg]; pneumonitis cases responded to steroids rapidly and successfully. Complete and partial responses were observed in one patient each in the single-agent and combination groups; both were immunotherapy naïve. In the single-agent and combination groups, 7 and 14 patients experienced stable disease; 7 and 6 patients were immunotherapy pretreated, respectively.Conclusions:Taminadenant, with and without spartalizumab, was well tolerated in patients with advanced NSCLC. The maximum tolerated dose of taminadenant alone was 480 mg twice a day, and 240 mg twice a day plus spartalizumab. Efficacy was neither a primary or secondary endpoint; however, some clinical benefit was noted regardless of prior immunotherapy or programmed cell death ligand-1 status.

Clinical studies have shown that pre-existing tumor-reactive T cells are critically important for effective cancer immunotherapy. Methods to augment the number and functionality of tumor-reactive T cells are therefore vital. Given that cytotoxic CD8 T cell priming requires conventional dendritic cells (cDC), we hypothesized that strategies to enhance cDC functionality in tumors would augment anti-tumor CD8 T cell responses. CD40 and type 1 IFN signaling pathways each promote the cross-presentation function of cDCs to activate CD8 T cells. However, it is unclear whether these pathways are functionally independent and whether their combined activation in tumor cDC leads to higher levels of T cell priming. Using adenovirus vectors, we tested the impact of expression of IFNβ and a chimeric membrane-stable CD40 ligand (MEM40) in syngeneic mouse tumor models. Compared to intratumoral administration of each individual transgene-encoding adenovirus, combined MEM40 + IFNβ expression resulted in the best tumor growth control, highest levels of tumor infiltrating lymphocytes (TILs), and systemic CD8 T cell responses. MEM40 and IFNβ were independently capable of enhancing T cell responses as we found IFNβ in CD40-/- mice and MEM40 in type 1 IFN receptor IFNAR1-/- mice induced a strong T cell response. These results were consistent with a cDC-mediated priming mechanism. Studies with cDC1 deficient BATF3-/- mice revealed a crucial role of this cDC subset in MEM40 + IFNβ induced T cell responses. IFNβ alone and MEM40 + IFNβ triggered increase in lymph node (LN) homing receptor CCR7 expression in cDC1 concurrent with a reduction in cDC1 in tumors and gain in tumor draining LNs. Simultaneously, combined MEM40 + IFNβ combination led to the highest levels of expression of CD80 and CD86 co-stimulatory molecules in both tumor and LN cDC1. Single cell (sc)-RNA sequencing of tumor DCs further showed that MEM40 + IFNβ led to the highest proportion of a recently defined mature/regulatory cDC subset characterized by high expression of CCR7, CD40 and IL12b. Applying these results towards an effective therapeutic modality, we developed a dual-transgene-armed MEM40 + IFNβ expressing conditionally replicative adenovirus (MEM-288) that simultaneously confers tumor-specific oncolysis and promotes tumor antigen release. MEM-288 drives MEM40 + IFNβ expression in freshly resected human lung tumors and generates potent anti-tumor responses as a monotherapy and in combination with immune checkpoint inhibitors. Furthermore, MEM-288 induced enhancement of tumor-reactive TILs and provides a rationale for studies to determine whether MEM-288 pre-treatment generates a superior TIL product for more effective adoptive T cell therapy. Citation Format: Hong Zheng, Scott Antonia, Mark J. Cantwell, Xiaoqing Yu, Amer A. Beg. Oncolytic adenovirus encoding transgenes for IFN-beta and recombinant CD40 ligand promotes conventional dendritic cell activation and trafficking with enhanced tumor infiltrating lymphocytes for effective cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3542.

Rationale: Examination of multiplexed images of tissues has recently emerged as a routine clinical procedure for cancer diagnosis and prognosis. The simultaneous detection of numerous biomarkers enables the interpretation of cellular states and the characterization of tumor-immune interactions in situ and at the single-cell level. However, image processing and the subsequent interpretive and predictive tools for multiplexed image data remain limited. Methods: We developed a computational multiplexed-image analysis pipeline using cell-segmentation and quadrat-based approaches to analyze the spatial and temporal features of multiplexed non-small cell lung cancer (NSCLC) images, and predict disease progression and identify clinical biomarkers. Images were obtained from nine patients with advanced/metastatic NSCLC who were treated with the oral HDAC inhibitor vorinostat combined with the PD-1 inhibitor pembrolizumab. Images were collected from all patients both pre- and on-treatment (during the third week). Results: Both cell-segmentation and quadrat-based approaches confirm that different spatial neighborhoods exist that distinguish progressors (PD) from non-progressors (SD): PD patients have distinct ecologies with higher colocalization of PanCK+PD-1+FoxP3 indicating an immunosuppressive environment, whereas SD patients have a higher colocalization of PanCK+PD-L1 along with T cells suggesting immunoactive tumor regions. These can be considered as potential biomarker candidates for predicting tumor progression. Further, from the single-cell analysis, we note there is a higher abundance of immune cells across the tumor border in PD patients than SD patients. Using the quadrat approach for species distribution modeling, we were able to predict treatment response with 91.4 percent accuracy given each patient’s spatial distribution of cell types from pre-treatment images. Further, we can generate risk maps for each image to identify tumor areas indicating higher probabilities of progression during treatment. Conclusions: We leveraged both single-cell and quadrat-resolution analysis of multiplexed imaging data and identified fundamentally distinct spatial ecologies between PD and SD patients. The ecology in PD patients appears to be primed for immune resistance even before treatment. This ecological diversity between SD and PD patients acts as a biomarker that enables accurate disease progression prediction. Citation Format: Sandhya Prabhakaran, Chandler Gatenbee, Mark Robertson-Tessi, Amer A. Beg, Jhanelle Gray, Scott Antonia, Robert A. Gatenby, Alexander R. Anderson. Distinct tumor-immune ecologies in NSCLC patients predict progression and define a clinical biomarker of therapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5037.

Small cell lung cancer (SCLC) is an aggressive malignancy and carries a poor prognosis with limited effective treatments in the advanced setting. SCLC is characterized by a high tumor mutation burden and alterations in Notch signaling and DNA damage repair pathways, providing rationale for the use of immunotherapy and targeted therapies. Immunotherapies have led to the most significant advances in treating SCLC in decades, and several promising targeted approaches have emerged from the increased understanding of the biology of SCLC. However, responses to these novel approaches are far from universal, and efforts to refine these therapies are ongoing.

Purpose of study: Non-small cell lung cancer (NSCLC) is the most common and fatal of cancers. In this work, we examine multiplexed images of NSCLC tumors to investigate both the spatial and spatiotemporal eco-evolutionary interactions between the tumor and its microenvironment, to better understand NSCLC tumor progression and therapy response. Methods: We developed a scalable, computational image analysis pipeline using cell-segmentation and quadrat-based approaches to analyze the spatial and temporal features of high-dimensional multiplexed NSCLC images. Multiplexed images enable the spatial readouts of numerous biomarkers per tissue sample and allow the interpretation of cellular states and the characterization of tumor-immune interactions across tissue ensembles. We also implement statistical approaches for ecological niche modelling combined with machine learning and deep learning models to predict disease progression and identify clinical imaging biomarkers. Data: Images were obtained from two 9-patient cohorts having advanced/metastatic NSCLC who were treated with the oral HDAC inhibitor vorinostat combined with the PD-1 inhibitor pembrolizumab. The first cohort had 4 progressors (PD) and 5 with stable disease (SD). The second cohort had 3 patients each in the PD, SD and partial response (PR) categories. Images were collected from all patients both pre- and on-treatment (during the third week). Results: Using our computational framework based on cell segments and quadrats, we confirm that different spatial neighborhoods exist that distinguish PD from SD, and that these spatial ecologies aid disease progression: PD patients have distinct ecologies with higher colocalization of PanCK+PD-1+FoxP3 indicating an immunosuppressive environment, whereas SD patients have a higher colocalization of PanCK+PD-L1 along with T cells, suggesting immunoactive tumor regions. These can be considered as potential biomarker candidates for predicting tumor progression. In an additional experiment where we include PR samples in our analyses, these distinct spatial neighborhoods are reinforced amongst PD, SD and PR patient groups corroborating the existence of spatiotemporal patterns. Further, we were able to predict treatment response with >91% accuracy given each patient’s spatial distribution of cell types from pre-treatment images. Using these predictions, we can generate risk maps at the patient level to identify areas of the tumor that are indicators of a higher probability of progression during treatment. Conclusions: We leveraged both single-cell and quadrat-resolution analysis of multiplexed imaging data and identified fundamentally distinct spatial ecologies between PD and SD patients. The ecology in PD patients appears to be primed for immune resistance even before treatment. This ecological diversity between SD and PD patients acts as a biomarker that enables accurate disease progression prediction. Our disease progression predictions can be used in conjunction with standard PD-L1 status to further strengthen personalized treatment strategies. Citation Format: Sandhya Prabhakaran, Chandler Gatenbee, Mark Robertson-Tessi, Amer A. Beg, Jhanelle Gray, Scott Antonia, Robert A. Gatenby, Alexander R.A. Anderson. Distinct spatiotemporal tumor-immune ecologies enable disease prediction in NSCLC patients [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B020.

Purpose Nivolumab, a fully human immunoglobulin G4 programmed death-1 immune checkpoint inhibitor antibody, has demonstrated improved survival in previously treated patients with advanced non–small-cell lung cancer (NSCLC). CheckMate 012, a phase I, multicohort study, was conducted to explore the safety and efficacy of nivolumab as monotherapy or combined with current standard therapies in first-line advanced NSCLC. Here, we report results for nivolumab plus platinum-based doublet chemotherapy (PT-DC). Patients and Methods Patients (N = 56) received nivolumab (intravenously) plus PT-DC concurrently every 3 weeks for four cycles followed by nivolumab alone until progression or unacceptable toxicity. Regimens were nivolumab 10 mg/kg plus gemcitabine-cisplatin (squamous) or pemetrexed-cisplatin (nonsquamous) or nivolumab 5 or 10 mg/kg plus paclitaxel-carboplatin (all histologies). The primary objective was to assess safety and tolerability. Secondary objectives included objective response rate and 24-week progression-free survival rate (per Response Evaluation Criteria in Solid Tumors version 1.1); exploratory objectives included overall survival (OS) and response by tumor programmed death ligand-1 expression. Results No dose-limiting toxicities occurred during the first 6 weeks of treatment. Forty-five percent of patients (25 of 56 patients) reported grade 3 or 4 treatment-related adverse events (AEs); 7% of patients (n = 4) had pneumonitis. Twenty-one percent of patients (n = 12) discontinued all study therapy as a result of treatment-related AEs. Objective response rates for nivolumab 10 mg/kg plus gemcitabine-cisplatin, nivolumab 10 mg/kg plus pemetrexed-cisplatin, nivolumab 10 mg/kg plus paclitaxel-carboplatin, and nivolumab 5 mg/kg plus paclitaxel-carboplatin were 33%, 47%, 47%, and 43%, respectively; 24-week progression-free survival rates were 51%, 71%, 38%, and 51%, respectively; 2-year OS rates were 25%, 33%, 27%, and 62%, respectively. Responses were achieved regardless of tumor programmed death ligand-1 expression. Conclusion The safety profile of nivolumab plus PT-DC was consistent with that expected for individual agents; however, treatment discontinuation related to AEs was greater with the combination. Encouraging activity was observed, especially for the nivolumab 5 mg/kg plus paclitaxel-carboplatin group, with a 2-year OS rate of 62%.

PURPOSE: Malignant pleural mesothelioma (MPM) is an aggressive cancer associated with poor prognosis. CRS-207 is a live-attenuated Listeria monocytogenes engineered to express mesothelin, a tumor-associated antigen highly expressed in MPM. CRS-207 induces antitumor immune responses and increases susceptibility of neoplastic cells to immune-mediated killing. PATIENTS AND METHODS: Patients with unresectable MPM, ECOG 0 or 1, and adequate organ and pulmonary function were enrolled in this multicenter, open-label phase Ib study. They received two priming infusions of 1 × 10(9) CFU CRS-207, followed by pemetrexed/cisplatin chemotherapy, and CRS-207 booster infusions. Primary objectives were safety and induction of immune response. Secondary/exploratory objectives included tumor response, progression-free survival (PFS), overall survival (OS), immune subset analysis, and gene-expression profiling of tumor. RESULTS: Of 35 evaluable patients, 89% (31/35) had disease control with one complete response (3%), 19 partial responses (54%), and 10 stable disease (29%). The estimated median duration of response was 5.0 months (95% CI, 3.9–11.5). The median PFS and OS were 7.5 (95% CI, 7.0–9.9) and 14.7 (95% CI, 11.2–21.9) months, respectively. Tumor size reduction was observed post–CRS-207 infusion prior to chemotherapy in 11 of 35 (31%) patients. No unexpected treatment-related serious adverse events or deaths were observed. IHC analysis of pre-and post–CRS-207 treatment tumor biopsies revealed possible reinvigoration and proliferation of T cells, increased infiltration of dendritic and natural killer cells, increased CD8:T(reg) ratio, and a shift from immunosuppressive M2-like to proinflammatory M1-like macrophages following CRS-207 administration. CONCLUSIONS: Combination of CRS-207 and chemotherapy induced significant changes in the local tumor microenvironment and objective tumor responses in a majority of treated patients.

Purpose Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) blockade with tremelimumab (CP-675,206), a fully human anti-CTLA4 monoclonal antibody, was tolerated and demonstrated antitumor activity in a single dose, dose-escalation phase I trial in patients with solid tumors. This phase I/II trial was conducted to examine safety of multiple doses of tremelimumab, to further assess efficacy, and to identify an appropriate dosing regimen for further development. Patients and Methods Twenty-eight patients with metastatic melanoma received monthly intravenous infusions of tremelimumab at 3, 6, or 10 mg/kg for up to 1 year to determine recommended monthly phase II dose. During phase II, 89 patients received tremelimumab 10 mg/kg once every month or 15 mg/kg every 3 months. Results No dose-limiting toxicity was observed in phase I once every month dosing. In phase II, 8 (10%) of 84 response-assessable patients attained objective antitumor responses; best overall objective response was one complete response and three partial responses in each dosing regimen. Most responses were durable (range, 3 to 30+ months). Most frequent treatment-related adverse events (AEs) were diarrhea, rash, and pruritus. Frequency of grade 3/4 AEs was 13% in the 15 mg/kg every 3 months arm and 27% in the 10 mg/kg once every month. Serious AEs were also less frequent in the 15 mg/kg once every 3 months cohort (9% v 23% in 10 mg/kg arm). Conclusion Multiple infusions of tremelimumab were generally tolerable and demonstrated single-agent antitumor activity. Both phase II regimens generated durable tumor responses. Based on its more favorable safety profile, 15 mg/kg every 3 months was selected for further clinical testing.

Excision repair cross complementing 1 (ERCC1) and ribonucleotide reductase M1 (RRM1) are molecular determinants that predict sensitivity or resistance to platinum agents and gemcitabine, respectively. Tailored therapy using these molecular determinants suggested patient benefit in a previously reported phase 2 trial. Here, we report an individual patient analysis of prospectively accrued patients who were treated with the "personalized therapy" approach versus other "standard," noncustomized approaches.Patients who had nonsmall cell lung cancer (NSCLC) with extranodal metastatic disease and an Eastern Cooperative Oncology Group performance status of 0/1 were accrued to 4 phase 2 clinical trials conducted at the H. Lee Moffitt Cancer Center: Trial A (first-line carboplatin/gemcitabine followed by docetaxel), Trial B (docetaxel and gefitinib in patients aged ≥70 years), Trial C (combination therapy with carboplatin/paclitaxel/atrasentan), and Trial D (personalized therapy based on ERCC1 and RRM1 expression). Patients with low RRM1/low ERCC1 expression received gemcitabine/carboplatin, patients with low RRM1/high ERCC1 expression received gemcitabine/docetaxel, patients with high RRM1/low ERCC1 expression received docetaxel/carboplatin, and patients with high RRM1/high ERCC1 expression received vinorelbine/docetaxel. Patients who were treated on Trials A, B, and C were pooled together and analyzed as the "standard therapy" group. Patients accrued to Trial D were called the "personalized therapy" group. Individual patient data were updated as of February 8, 2011. Overall survival (OS) and progression-free survival (PFS) were estimated using the Kaplan-Meier method.There were statistically significant improvements between the personalized therapy group versus the standard therapy group in response (44% vs 22%; P = .002), OS (median: 13.3 months vs 8.9 months; P = .016), and PFS (median: 7.0 months vs 4.3 months; P = .03).The results from individual patient analyses suggest that ERCC1 and RRM1/tailored selection of first-line therapy improved survival over standard treatment-selection approaches.

Purpose To determine the maximum-tolerated dose (MTD) and assess the safety, pharmacokinetics, and preliminary evidence of antitumor activity of YM155, a small-molecule inhibitor of survivin. Patients and Methods Patients with advanced solid malignancies or lymphoma were treated with escalating doses of YM155 administered by 168-hour continuous intravenous infusion (CIVI). Plasma and urine samples were assayed to determine pharmacokinetic parameters and excretion. Results Forty-one patients received 127 cycles of YM155 at doses ranging from 1.8 to 6.0 mg/m2/d by 168-hour CIVI every 3 weeks. Overall, the most common grade 1 to 2 toxicities were stomatitis, pyrexia, and nausea, whereas grade 3 and 4 toxicities were rare. Reversible elevation in serum creatinine in two patients, with one developing acute tubular necrosis, was dose-limiting at 6.0 mg/m2. The MTD was 4.8 mg/m2. At the MTD, the mean steady-state concentration, clearance, volume of distribution at steady-state, and terminal elimination half-life were 7.7 ng/mL, 47.7 L/h, 1,763 L, and 26 hours, respectively. One complete and two partial responses lasting 8, 24+ and 48+ months occurred in three patients with non-Hodgkin's lymphoma, two patients with hormone- and docetaxel-refractory prostate cancer had prostate-specific antigen responses, and one patient with non–small-cell lung cancer had a minor response. Conclusion YM155 can be administered safely at 4.8 mg/m2/d 168 hours CIVI every 3 weeks. The absence of severe toxicities, attainment of plasma concentrations active in preclinical models, and compelling antitumor activity warrant further disease-directed studies of this agent alone and in combination with chemotherapy in a broad array of tumors.

We studied the fate of myeloid-derived suppressor cells (MDSC) in cancer. Unexpectedly, MDSC had lower viability and a shorter half-life than their control counterparts neutrophils and monocytes. This effect was due to increased apoptosis mediated by the changes in TRAIL receptors (TRAIL-R) expression. Targeting TRAIL-R did not affect myeloid cells from naïve mice, but dramatically reduced the presence of MDSC and improved the immune responses in tumor-bearing mice. Pro-inflammatory cytokines did not affect TRAIL-R expression. However, induction of ER stress recapitulated changes in TRAIL-R expression observed in tumor-bearing hosts. ER stress response was detected in MDSC isolated from cancer patients and tumor-bearing mice, but not in control neutrophils or monocytes. Block of ER stress abrogated changes in TRAIL-R. Thus, MDSC pathophysiology is linked to ER stress, which shortens their lifespan in the periphery and promotes their expansion in bone marrow. In conclusion, TRAIL-R can be considered for selective targeting of MDSC.