Use Of Immune Checkpoint Blockade Research Articles

High-Grade Pleomorphic Sarcomas Treated with Immune Checkpoint Blockade: The MD Anderson Cancer Center Experience.

Undifferentiated pleomorphic sarcomas (UPSs) are amongst the most common subtypes of soft-tissue sarcomas. Few real-world data on the use of immune checkpoint blockade (ICB) in UPS patients and other high-grade pleomorphic STS patients are available. The purpose of our study is to describe the efficacy and toxicity of ICB in patients with advanced UPSs and other high-grade pleomorphic sarcomas treated at our institution. This is a retrospective, observational study of all patients with metastatic high-grade pleomorphic sarcomas treated with FDA-approved ICB at MD Anderson Cancer Center between 1 January 2015 and 1 January 2023. Patients included in trials for which results are not yet published were excluded. Thirty-six patients with advanced/metastatic pleomorphic sarcomas were included. The median age was 52 years. A total of 26 patients (72%) had UPSs and 10 patients (28%) had other high-grade pleomorphic sarcomas. The median follow-up time was 8.8 months. The median PFS was 2.9 months. The 3-month PFS and 6-month PFS were 46% and 32%, respectively. The median OS was 12.9 months. The 12-month OS and 24-month OS were 53% and 29%, respectively. The best response, previous RT, and type of ICB treatment were significantly and independently associated with shorter PFS (p = 0.0012, p = 0.0019 and p = 0.036, respectively). No new safety signal was identified, and the toxicity was overall manageable with no toxic deaths and only four patients (11%) stopping treatment due to toxicity. Real-world retrospective data are consistent with the published literature, with a promising 6-month PFS of 32%. Partial or stable responders to ICB treatment have significantly improved PFS compared to progressors.

Abstract 7407: Melanoma clonal subline analysis reveals genetic factors driving intra-tumor heterogeneity

Abstract Melanoma is the most invasive skin cancer caused by the malignant melanocytes. The use of immune checkpoint blockade (ICB) improved the survival rate in advanced melanoma. Yet, the response rate to ICB varies across patients due to the highly heterogeneous nature of melanoma. Recent studies reported genomic and epigenetic factors contributing to the therapeutic response. Identifying these factors involved in clonal evolution in melanoma is a key to better understanding the tumor progression and divergence in the therapy response. To study melanoma heterogeneity, we generated twenty-four clonal sublines, each derived from a single cell isolated from a parental cell line derived from the M4 mouse melanoma model. DNA prepared from each subline and a spleen from a healthy mouse were subjected to long-read sequencing. Importantly, long-read sequencing allows direct detection of methylation states, small variants, and structural variants in the same run. The results thus provide excellent means to study genetic and epigenetic factors in clonal evolution. Each of these sublines was also implanted in distinct mice for survival and tumor growth analysis and further functional evaluation. We developed Severus, a structural variation (SV) caller for long reads, which works with single (germline), paired (tumor-normal), and multiple samples (e.g., multi-site or time series). Severus takes advantage of improved phasing in long-read sequencing, attributes somatic variants to germline haplotype, and builds a haplotype-specific breakpoint graph that is used to cluster multi-break rearrangements and represent the derived chromosomal structure. We benchmarked Severus using multiplatform validated COLO829 truthset and CHM1/CHM13 haploid genomes, and Severus outperformed all other methods in terms of recall and precision. The structural variation (SV) analysis using Severus with all the sublines showed enrichment of possibly clonal SVs in chr4, chr11, and mainly in chr13. Further investigation revealed SVs in chr13 were not distributed across the chromosome but clustered in certain regions. Analysis of somatic SNVs in chr13 corroborates the localized hypermutation profile, which indicates a possible kataegis. Interestingly, a loss of the chr13 copy affected by kataegis in one of the sublines coincides with a better survival rate, as compared to other sublines, in an allograft melanoma mouse model, which further supports the association between the kataegis and tumor progression. We also identified other clonal and subclonal events, including the homozygous deletion of Cdkn2a in most of the sublines, associated with their aggressiveness in vivo. Our analysis allowed us to identify clonal and subclonal genetic and epigenetic factors involved in tumor progression and evolution in a melanoma model, which can potentially translate to human disease. Citation Format: Ayse Keskus, Anton Goretsky, Yuelin Liu, Xiwen Cui, Tanveer Ahmad, Eva Perez Guijarro, Asher Bryant, Erin Malloy, Salem Malikic, Glenn Merlino, Chi-Ping Day, Cenk Sahinalp, Mikhail Kolmogorov. Melanoma clonal subline analysis reveals genetic factors driving intra-tumor heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7407.

Abstract 4049: The beta-carboline Harmine sensitives microsatellite stable colorectal cancer mouse model to anti-PD-1 through upregulating MHC-I dependent antigen presentation machinery and improving the infiltration of CD8 T cells into the tumor microenvironment

Abstract Colorectal cancer (CRC) is the most common gastrointestinal cancer and the third leading cause of cancer-related death in both sexes. Mismatch repair deficient/microsatellite instability-high (MSI) colorectal cancer patients benefit from immune checkpoint blockades (ICBs)-based immunotherapy; however, this benefit has not been translated into microsatellite stable (MSS) colorectal cancer. It has been proposed that the loss of major histocompatibility complex class I (MHC-I) in tumor cells limits the use of ICB in colorectal cancer. Harmine displays a number of biological and pharmacological properties affecting the immune response and tumor phenotype. Here, we assessed the impact of combining harmine, also referred to as ACB1801 molecule, on improving the responsiveness of ICB in the mouse MSS CRC model. Our results showed that combining ACB1801 significantly improves the therapeutic benefit of anti-PD-1 in the CT26 CRC mouse model by decreasing tumor growth and improving the survival of tumor-bearing mice. The improvement of anti-PD-1-based therapy by combining ACB-1801 was associated with a modification of the immune landscape of tumors as evidenced by an increase in the CD8+ T cells and a decrease in the Treg infiltrations into the tumor microenvironment. By profiling the cytokine/chemokine network, we showed that ACB1801 induced the expression and the release of the proinflammatory chemokine CXCL10 by CRC tumor cells in vitro, which could be responsible for driving CD8+ T cells to the tumor microenvironment. Mechanistically, we showed that ACB1801 increased the expression of MHC-I genes, including TAP1 and TAPASIN, and improved the antigen loading on MHC-I in CRC cells in vitro. Overall, our study highlights the value of combining ACB1801 and anti-PD-1 as a therapeutic opportunity to convert MSS colorectal cancer into an “immune hot” cancer, which may define the future treatment paradigm of colorectal cancer for which there is a great unmet need. Citation Format: Ruize Gao, Kris Van Moer, Coralie Pulido, Anaïs Oudin, Diane Murera, Teresa L. Ramos, Margaux Poussard, Andreas Schläpfer, Annette Ives, Christian Auclair, Bassam Janji. The beta-carboline Harmine sensitives microsatellite stable colorectal cancer mouse model to anti-PD-1 through upregulating MHC-I dependent antigen presentation machinery and improving the infiltration of CD8 T cells into the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4049.

Abstract 6391: Spatial proteomic profiling unveils pre-existing anti-tumor immunity as a hallmark of exceptional benefit from immunotherapy in NSCLC

Abstract Background: Immunotherapy is firmly established as a treatment regimen in various solid tumors due to its exceptional benefits observed in a select group of patients. Despite widespread use of immune checkpoint blockade (ICB) across diverse solid tumors, including non-small cell lung cancer (NSCLC), the quest for a clinically informative biomarker for long-term benefit remains unmet. Here we investigate the potential utility of clinicopathological and spatial proteomic profiling of tumor specimens from NSCLC patients to identify predictive biomarkers of long-term benefit to ICB. Methods: Forty-nine patients diagnosed with advanced NSCLC who received ICB at Hospital del Mar, Barcelona between 2017-2021 were included. Long-term responders (LTR, n=21) were defined as patients achieving a maintained radiologic response for more than 2 years, and short-term responders (STR, n=28) as patients presenting disease progression within the first six months of ICB initiation. Clinicopathological information, incidence of immune related adverse (irAES) and PD-L1 tumor proportion score assessed by IHC was available for all of them. DSP GeoMx was performed in subset of patients (LTR n=14 and STR n=14), to assess 49 immune biomarkers. Pancytokeratin (panCK; epithelial), CD3, CD20 and SYTO 13 (nuclear) were utilized as morphology biomarkers. Regions of interest were placed in tissue areas containing tumor and segmented in Tumor (PanCK+) and tumor microenvironment (TME) (PanCK-) that was used on downstream analysis. Digital counts were normalized using the protein expression of housekeeper proteins. Statistical analysis was performed using linear mixed models. A P value less than 0.05 was considered significant. Results: Our analysis revealed specific characteristics of LTR patients compared with STR, namely LTR population was enriched for PD-L1 positive in tumors (p=0.005) and a higher incidence of immune-related adverse events (irAEs) (p=0.001). Within the Tumor compartment, LTR patients displayed significantly higher levels of IDO1, CD8, PD-L1, CD45, HLA-DR and STING, while STR patients exhibit higher levels of B7-H3, CD56 and OX40L (p<0.05 for all proteins). Comparison analysis of the TME compartment between LTR and STR revealed augmented levels of IDO1, CD8, CD45, CD11c, CD27 and CD3 in LTR patients, and lower levels of B7H3 and OX40L compared with STR patients (p<0.05 for all proteins). Conclusions: Our comprehensive analysis of metastatic NSCLC patients treated with ICB has unveiled distinct clinicopathological and immunological features associated with long-term benefit of ICB, highlighting the presence of pre-existing antitumor immunity as a stronger predictor of long-term benefit. These findings offer insights into potential biomarkers and therapeutic strategies for enhancing ICB outcomes in metastatic NSCLC. Citation Format: Sharia Hernandez, Rafael Bach, Mario Giner, Wei Lu, Larisa Kostousov, Sean Barnes, Khaja Khan, Laura Masfarré, Xavier Villanueva, Ignacio Sanchéz, Nil Navarro, Álvaro Taus, Miguel Galindo, Max Hardy, Raúl Del Rey-Vergara, Albert Iñañez, Beatriz Sanchez-Espiridion, Laura Moliner, Sergi Clavé, Beatriz Bellosillo, Ana Rovira, Júlia Perera-Bel, Ignacio Wistuba, Edurne Arriola, Luisa M. Solis, Pedro Rocha. Spatial proteomic profiling unveils pre-existing anti-tumor immunity as a hallmark of exceptional benefit from immunotherapy in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6391.

Abstract 4102: Treatment with androgen receptor targeting vaccines provides an overall survival benefit in AR-V7 expressing tumor models

Abstract Background: Androgen receptor (AR) signaling inhibition is part of the mechanism behind many effective prostate cancer therapies, including androgen deprivation and next generation anti-androgens. However in castration resistant prostate cancer up to 50% of treatment resistance has been linked to AR amplification or expression of the constitutively active RNA splice variant, AR-V7. Although the use of immune checkpoint blockade has failed to demonstrate clinical success, immunotherapeutic interventions remain attractive as prostate cancer’s relatively slow progression provides time for anti-tumor immune responses to develop. Cancer specific vaccines have the potential to enable and enhance tumor specific immune responses by stimulating and directing T cell immunity to important oncologic targets. We hypothesized that a cancer vaccine designed to induce a T cell response against cells expressing high levels of AR would result in an anti-tumor effect and potentially prevent or delay the development of therapeutic resistance. Methods: Adenoviral vaccines against AR (Ad-AR) or AR splice variant 7 (Ad-AR-V7) were used to evaluate the potential of AR targeting vaccination in mouse models. We evaluated AR and AR variant specific adaptive responses produced by our vaccines with naïve and tumor-bearing mice by ex vivo peptide restimulation. We challenged vaccinated mice with multiple syngeneic subcutaneous tumor models that we engineered to express AR-V7, including a PTEN-/- p53-/- prostate tumor cell line. Finally we tested our vaccines in a therapeutic regimen, vaccinating mice after implanting our subcutaneous AR-V7 expressing models. Results: Across multiple strains of mice, AR or AR-V7 targeting adenoviral vaccination induced robust adaptive immune responses against the N-terminal domain of AR. Vaccination with either Ad-AR or Ad-AR-V7 resulted in significant anti-tumor responses, providing a significant overall survival benefit and complete tumor rejection by the majority of mice vaccinated against AR or AR-V7. Similarly tumor bearing mice treated with Ad-AR or Ad-AR-V7 vaccination displayed tumor growth inhibition or complete tumor regression resulting in an overall survival benefit. Additionally, we found that vaccine responses were unaffected by the use of the approved antiandrogen drug enzalutamide, suggesting that vaccinations could be deployed in tandem with standard-of-care therapies. Conclusions: Vaccination against AR and AR splice variants can induce AR specific adaptive immune responses capable of inhibiting the growth of AR-V7 expressing tumor models, illustrating the potential of AR and its splice variants as immunologic targets in prostate cancer. Citation Format: Robert D. Marek, Junping Wei, Tao Wang, Xiao-Yi Yang, Gangjun Lei, Zachary C. Hartman. Treatment with androgen receptor targeting vaccines provides an overall survival benefit in AR-V7 expressing tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4102.

Tumor immune microenvironment in odontogenic carcinomas: Evaluation of the therapeutic potential of immune checkpoint blockade.

Despite recent advances in the use of immune checkpoint blockade (ICB) across various cancer types, its efficacy in odontogenic carcinomas remains unexplored. This study aims to investigate PD-L1 expression and the tumor immune microenvironment (TIME) in odontogenic carcinomas to determine the therapeutic potential of ICB and the significance of immune markers. The expressions of PD-L1 and T cell markers (CD3, CD8, and FOXP3) were visualized by immunohistochemistry in 21 tissue samples of odontogenic carcinomas. Tumoral PD-L1 expression and the density and spatial distribution of T cell subsets were evaluated, from which TIME was determined. The associations of the variables with clinicopathological and prognostic factors were statistically analyzed. PD-L1 was positively expressed in 52.4% (11/21) of the cases studied. Among tumor types, ameloblastic carcinoma showed significantly higher PD-L1 expression (p = 0.016). TIME based on the intratumoral and stromal T cell distribution was immune-inflamed in 61.9% (13/21) and immune-excluded in 38.1% (8/21), with no immune-desert cases. PD-L1 expression was associated with the densities of all intratumoral T cell subsets (p = 0.03 for CD3, p = 0.03 for CD8, and p = 0.008 for FOXP3) but not with those of stromal T cells. High PD-L1 expression was associated with larger tumor size (p = 0.021), while the intratumoral CD8/CD3 ratio was inversely correlated with tumor size (p = 0.048). These findings indicate the involvement of adaptive immune resistance in a subset of odontogenic carcinomas and support the therapeutic potential of ICB in patients with these rare malignancies.

A dramatic response to checkpoint inhibitor in a woman with small cell carcinoma of the hypercalcemic type of the ovary

ObjectiveWe present the rare case of a 21 year old woman with small cell carcinoma of the right ovary of the hypercalcemic type with dramatic response to checkpoint inhibitor. MethodsCase report. Results and conclusionsOur patient, a 22-year old woman with small cell carcinoma of the hypercalcemic type with hepatic metastases, is currently 43 months under treatment with pembrolizumab. Last MRI revealed no viable liver metastases nor other signs of recurrence. This is the longest survival of a patient with small cell carcinoma of the ovary under therapy with checkpoint inhibitors reported in the literature so far.With this report we emphasize the importance of immunohistological testing for PD-L 1. Treating clinicians should keep off-label use of immune checkpoint blockade in mind when treating this highly aggressive tumor if all other treatment options fail.

Exploiting the Gut Microbiota to Boost Immunotherapy and Switch the Trajectory of Multiple Myeloma

Clonal proliferation of neoplastic plasma cells in the bone marrow (BM) and organ dysfunction characterizes multiple myeloma (MM), a treatable but incurable disease. Although asymptomatic smoldering MM (SMM) often precedes full blown MM, most patients affected by SMM are only offered active observation, which increases their frustration and anxiety. In transgenic Vk*MYC mice developing de novo MM, we found a direct link between gut microbiota composition and T helper 17 (Th17) lymphocytes, which migrated from the gut to the BM during asymptomatic MM (Early-MM), and favored the expansion of neoplastic plasma cells, thus fueling progression to symptomatic disease (Late-MM) [Calcinotto A. et al. Nat Commun (2018) 9(1):4832]. Similarly, in SMM patients, higher levels of BM Interleukin-17 (IL-17) predicted accelerated disease [Calcinotto A. et al. Nat Commun (2018) 9(1):4832]. Interestingly, Prevotella melaninogenica ( P.m.), a human commensal, limited MM aggressiveness in mice challenged with Vk*MYC-derived MM cells (t-Vk*MYC) by restraining Th17 cell expansion [Calcinotto et al. Nat Commun (2018) 9(1):4832]. Because the gut microbiota also contributes to the therapeutic efficacy of immune checkpoint blockade (ICB) and human neoplastic plasma cells express PD-L1, we hypothesized that modulation of the gut microbiota by P.m. could limit Th17 cell expansion in mice affected by Early-MM, thus fully exploiting the therapeutic potential of anti-PD-L1 against MM. Clinical trials combining anti-PD-1/PD-L1 antibodies, dexamethasone and immune modulatory drugs in MM were halted by US FDA because of unfavorable benefit-risk profile and life-threatening immune related adverse events (irAEs). Because IL-17 is involved in irAEs [Johnson et al. Cancer Immunol Res (2019) 7 (6): 860-865], we also hypothesized treatment with P.m. could restrain ICB-induced expansion of Th17 cells while unleashing CD8-mediated antitumor immunity. Early-MM Vk*MYC mice and t-Vk*MYC, the latter mimicking aggressive MM, were treated with P.m. and/or anti-PD-L1 and disease progression was monitored by paraprotein quantification in blood. Anemia provided clinical evidence of symptomatic disease. At sacrifice, gut, spleen and BM were analyzed by flow cytometry. In vitro assays were conducted to clarify the mechanism by which the gut microbiota influenced the induction of Th17 cells. To mimic cutaneous irAE, the skin of treated mice was exposed to imiquimod, which induces Th17-mediated inflammation [van der Fits et al. J Immunol (2009 )182(9): 5836-5845]. Body weight loss, skin inflammation, and inflammatory infiltrate into secondary lymphoid organs were taken as measure of imiquimod-induced toxicity. In Vk*MYC mice, all neoplastic plasma cells expressed PD-L1. Treatment of Early-MM Vk*MYC mice with anti-PD-L1 antibodies delayed progression to Late-MM, thus extending previous findings on the use of ICB in this model to anti-PD-L1 antibodies and Early-MM [Guillerey et al. J Clin Invest (2015 )125(5): 2077-2089]. Treatment with P.m. exerted a similar effect on disease progression, suggesting that a treatment as simple as taking a probiotic has an impact on MM evolution. When P.m. was combined with anti-PD-L1 antibodies, evolution to Late-MM was further delayed or even blocked. Single and combined treatments also reduced MM aggressiveness in t-Vk*MYC mice. Mechanistically, P.m. limited the expansion of gut-born Th17 cells and their accumulation in the BM of treated mice without dampening the antitumor cytotoxic response elicited by the ICB. Thus, only the combined treatment provided the most favorable CD8/Th17 and CD8/T regulatory cell ratios. Ex vivo, P.m.-conditioned BM DCs showed a limited propensity to produce Th17-polarizing cytokines. In vitro, stimulation of both human and mouse DCs with P.m. reduced polarization of naïve T cells toward a Th17 phenotype. Skin application of imiquimod in C57BL/6 mice induced psoriasis-like lesions and expansion of Th17 cells both in draining lymph nodes and spleen. The contextual administration of anti-PD-L1 antibodies worsened imiquimod-induced inflammation. An ongoing experiment will clarify if mice receiving P.m. and ICB experience reduced skin lesions. Taken together, our data support the development of microbiota-based strategies in combination with ICB to treat full-blown MM and to prevent progression of asymptomatic SMM to full-blown disease.

SURG-38. SAFETY AND EFFICACY OF COMBINATORIAL IMMUNE CHECKPOINT BLOCKADE AND LASER INTERSTITIAL THERMAL THERAPY

Abstract INTRODUCTION Treatment of intracranial lesions with laser interstitial thermal therapy (LITT) allows for simultaneous cytoreduction and the potential to generate an anti-tumor immune response. The safety and efficacy of combination immune checkpoint blockade (ICB) and LITT has not been determined. METHODS All patients who received LITT for neuro-oncologic indications at a single NCI-designated cancer center from 2015 – 2022 were included. Simultaneous ICB was defined as receipt of therapy within 6 weeks of LITT. Demographic, clinical, and survival data were collected. Immune-related and intracranial adverse events (iRAEs and IAEs) were graded according to Common Terminology Criteria. RESULTS Twenty-four patients received simultaneous LITT and ICB. Median age was 62 (range 40 - 78) and 12 (48%) were female. Median KPS was 80 (50 – 100). Use of ICB increased during the study period (r = 0.69). The most common pathology was non-small cell lung cancer (15 patients, 62.5%), followed by melanoma in 3 (12.5%) and 2 (8.3%) patients each with renal cell carcinoma or high-grade glioma. Twenty-three (96%) patients received single agent ICB, with pembrolizumab most common (12, 50%), followed by nivolumab (6, 25%). The median interval between LITT and ICB was 2.56 (0.85 – 4.98) weeks. The most common iRAE was hypothyroidism in 4 (16.6%) patients, with grade 3 pneumonitis in one patient. The most common IAE was seizures in 4 (16.6%) patients. Local progression was seen in 4 patients (16.6%), with a post-LITT overall survival of 20.6 (1.0 – 36.1) months for the cohort. CONCLUSIONS Combination LITT and ICB appears safe and effective, with iRAEs commensurate with trials of checkpoint inhibition. High-grade intracranial toxicity was rare. Prospective studies of combinatorial LITT + ICB are needed to establish biomarkers and optimal timing.

β-Glucan–conjugated anti–PD-L1 antibody enhances antitumor efficacy in preclinical mouse models

The use of immune checkpoint blockade (ICB) is a promising approach for clinical cancer treatment. However, most of cancer patients do not respond to anti–PD-1/PD-L1 antibody. In this study, we proposed a novel strategy of antibody–β-glucan conjugates (AGC) to enhance the antitumor immune response to ICB therapy. The AGC were constructed by conjugating an anti–PD-L1 antibody with a β-glucan via click chemistry. This design facilitates the delivery of β-glucan into the tumor microenvironment (TME). Furthermore, the bridging effect mediated by AGC can promote the interaction between tumor cells and dendritic cells (DCs), thereby enhancing immunotherapeutic benefits. In the MC38 tumor-bearing mouse model, AGC demonstrated powerful tumor suppression, achieving a tumor suppression rate of 86.7 %. Immunophenotyping, cytokine analysis, RNA sequencing, and FTY720-treated models were combined to elucidate the mechanism underlying AGC function. Compared with anti–PD-L1 antibody, AGC induced an earlier immune response, infiltration of DCs, and activation of preexisting T cells in the TME, with T cells predominantly proliferating locally rather than migrating from other organs. In conclusion, these data suggest that AGC could serve as a promising strategy to improve ICB therapy with prospects for clinical utilization.

GLIOMA-05 A NOVEL CONCEPT FOR TREATMENT OF GLIOBLASTOMA: VAGUS NERVE ELECTRICAL STIMULATION FOR SUPPRESSION OF CYTOKINE (IL-6) SIGNALING

Abstract Glioblastoma is an aggressive, incurable cancer. Innovative approaches are urgently needed. There is resurgent interest in harnessing the power of the immune system (O’Rourke DM, 2017; Liau LM, 2023). Glioblastoma is a key target for cytokine reprogramming of the tumor microenvironment involving macrophages, monocytes, lymphocytes, and myeloid-derived cells. Furthermore, as with other cancers, the incidence and poor prognosis is linked to the age of the patient, and chronic, low-grade inflammation, or “inflammaging” (Franceschi, 2014). There is strong clinical, genomic (Coppola, 2014), preclinical data linking the progression of glioblastoma to the cytokine, IL-6. Yi Fan and colleagues (Wang, 2018; Yang, 2021) showed that blockade of IL-6 increases survival in mouse models of glioblastoma, an effect enhanced with the use of immune checkpoint blockade (ICB). These results were validated and extended by others (Tsukamoto, 2018; Lamano,2019). Taken together, we translated the findings to a prospective, multicenter clinical trial (NRG BRAIN-010, NCT04729959; PI, Stephen Bagley) for recurrent glioblastoma, combining stereotactic radiosurgery, IL-6R blockade (tocilizumab) and ICB (atezolizumab). To date, the regimen is well-tolerated with an acceptable safety profile. Vagus nerve stimulation is FDA-approved for control of medically refractory epilepsy. It is currently being evaluated in a multicenter, pivotal trial to treat advanced rheumatoid arthritis (RESET-RA, NCT04539964). Electrical stimulation of the vagus nerve activates the inflammatory reflex (Kelly, 2022), with a reduction of inflammatory cytokines, including IL-6 (Koopman, 2016). Electrical stimulation of the vagus nerve uses the ‘inflammatory reflex’ and exploits the cholinergic effect on T-cells (Levine, 2020). Taken together, we now propose a clinical trial to assess the safety and efficacy of vagal nerve stimulation for patients with glioblastoma and are in discussion with industry (SetPoint Medical) to design a prospective, clinical trial for treatment of glioblastoma.

Identifying biomarkers and novel therapeutic targets in uveal melanoma.

Uveal melanoma (UM) is an orphan cancer despite being the most common eye tumor in adults. Patients often present to skin cancer centers for treatment of metastatic disease although there are significant genetic, biological, and clinical differences from cutaneous melanoma. The treatments most commonly used for metastatic UM are tebentafusp and combined immune checkpoint blockade, both of which yield low response rates and may be accompanied by high treatment costs and significant immune-related toxicities. Thus, it is of paramount importance to identify biomarkers and clinical profiles predictive of treatment response and to find novel therapeutic targets. The use of immune checkpoint blockade showed more favorable outcomes in patients with extrahepatic disease and normal levels of serum lactate dehydrogenase in a panel of retrospective studies, making its use more reasonable in this subgroup. To identify novel drug targets, we will analyze the expression and relevance of neural crest transcription factors in patient bio-specimens using next-generation nanopore sequencing. Computer algorithms and network-based analysis will facilitate the identification of druggable targets which will subsequently be validated in patient-derived short-term cell cultures. This approach will help to find novel and personalized treatments for UM.

Targeting immunosuppressive Ly6C+ classical monocytes reverses anti-PD-1/CTLA-4 immunotherapy resistance.

Despite significant clinical advancement with the use of immune checkpoint blockade (ICB) in non-small cell lung cancer (NSCLC) there are still a major subset of patients that develop adaptive/acquired resistance. Understanding resistance mechanisms to ICB is critical to developing new therapeutic strategies and improving patient survival. The dynamic nature of the tumor microenvironment and the mutational load driving tumor immunogenicity limit the efficacy to ICB. Recent studies indicate that myeloid cells are drivers of ICB resistance. In this study we sought to understand which immune cells were contributing to resistance and if we could modify them in a way to improve response to ICB therapy. Our results show that combination anti-PD-1/CTLA-4 produces an initial antitumor effect with evidence of an activated immune response. Upon extended treatment with anti-PD-1/CTLA-4 acquired resistance developed with an increase of the immunosuppressive populations, including T-regulatory cells, neutrophils and monocytes. Addition of anti-Ly6C blocking antibody to anti-PD-1/CTLA-4 was capable of completely reversing treatment resistance and restoring CD8 T cell activity in multiple KP lung cancer models and in the autochthonous lung cancer KrasLSL-G12D/p53fl/fl model. We found that there were higher classical Ly6C+ monocytes in anti-PD-1/CTLA-4 combination resistant tumors. B7 blockade illustrated the importance of dendritic cells for treatment efficacy of anti-Ly6C/PD-1/CTLA-4. We further determined that classical Ly6C+ monocytes in anti-PD-1/CTLA-4 resistant tumors are trafficked into the tumor via IFN-γ and the CCL2-CCR2 axis. Mechanistically we found that classical monocytes from ICB resistant tumors were unable to differentiate into antigen presenting cells and instead differentiated into immunosuppressive M2 macrophages or myeloid-derived suppressor cells (MDSC). Classical Ly6C+ monocytes from ICB resistant tumors had a decrease in both Flt3 and PU.1 expression that prevented differentiation into dendritic cells/macrophages. Therapeutically we found that addition of anti-Ly6C to the combination of anti-PD-1/CTLA-4 was capable of complete tumor eradication. Classical Ly6C+ monocytes differentiate into immunosuppressive cells, while blockade of classical monocytes drives dendritic cell differentiation/maturation to reinvigorate the anti-tumor T cell response. These findings support that immunotherapy resistance is associated with infiltrating monocytes and that controlling the differentiation process of monocytes can enhance the therapeutic potential of ICB.

Clinical Importance of Clonal Hematopoiesis in Metastatic Gastrointestinal Tract Cancers

Clonal hematopoiesis (CH) has been associated with development of atherosclerosis and leukemia and worse survival among patients with cancer; however, the association with cancer therapy efficacy, in particular immune checkpoint blockade (ICB), and toxicity has not yet been established. Given the widespread use of ICB and the critical role hematopoietic stem cell-derived lymphocytes play in mediating antitumor responses, CH may be associated with therapeutic efficacy and hematologic toxicity. To determine the association between CH and outcomes, hematologic toxicity, and therapeutic efficacy in patients with metastatic gastrointestinal tract cancers being treated with systemic therapy, both in the first-line metastatic treatment setting and in ICB. This retrospective cohort study included 633 patients with stage IV colorectal (CRC) and esophagogastric (EGC) cancer who were treated with first-line chemotherapy and/or ICB at Memorial Sloan Kettering Cancer Center. Patients underwent matched tumor and peripheral blood DNA sequencing using the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets next-generation sequencing assay between January 1, 2006, and December 31, 2020. Clonal hematopoiesis-related genetic alterations were identified by next-generation sequencing of patients' tumor and normal blood buffy coat samples, with a subset of these CH alterations annotated as likely putative drivers (CH-PD) based upon previously established criteria. Patients with CH and CH-PD in peripheral blood samples were identified, and these findings were correlated with survival outcomes (progression-free survival [PFS] and overall survival [OS]) during first-line chemotherapy and ICB, as well as baseline white blood cell levels and the need for granulocyte colony-stimulating factor (G-CSF) support. Among the 633 patients included in the study (390 men [61.6%]; median age, 58 [IQR, 48-66] years), the median age was 52 (IQR, 45-63) years in the CRC group and 61 (IQR, 53-69) years in the EGC group. In the CRC group, 161 of 301 patients (53.5%) were men, compared with 229 of 332 patients (69.0%) in the EGC group. Overall, 62 patients (9.8%) were Asian, 45 (7.1%) were Black or African American, 482 (76.1%) were White, and 44 (7.0%) were of unknown race or ethnicity. Presence of CH was identified in 115 patients with EGC (34.6%) and 83 with CRC (27.6%), with approximately half of these patients harboring CH-PD (CRC group, 44 of 83 [53.0%]; EGC group, 55 of 115 [47.8%]). Patients with EGC and CH-PD exhibited a significantly worse median OS of 16.0 (95% CI, 11.6-22.3) months compared with 21.6 (95% CI, 19.6-24.3) months for those without CH-PD (P = .01). For patients with CRC and EGC, CH and CH-PD were not associated with PFS differences in patients undergoing ICB or first-line chemotherapy. Neither CH nor CH-PD were correlated with baseline leukocyte levels or increased need for G-CSF support. These findings suggest CH and CH-PD are not directly associated with the treatment course of patients with metastatic gastrointestinal tract cancer receiving cancer-directed therapy.

Tumor CEMIP drives immune evasion of colorectal cancer via MHC-I internalization and degradation

BackgroundLoss of major histocompatibility complex class I (MHC-I) in tumor cells limits the use of immune checkpoint blockade (ICB) in colorectal cancer. Nevertheless, the regulatory mechanism of MHC-I downregulation in...

T cell exhaustion assessment algorism in tumor microenvironment predicted clinical outcomes and immunotherapy effects in glioma.

Despite the recent increase in the use of immune checkpoint blockade (ICB), no ICB medications have been approved or are undergoing large-scale clinical trials for glioma. T cells, the main mediators of adaptive immunity, are important components of the tumor immune microenvironment. Depletion of T cells in tumors plays a key role in assessing the sensitivity of patients to immunotherapy. In this study, the bioinformatics approach was applied to construct T cell depletion-related risk assessment to investigate the impact of T cell depletion on prognosis and ICB response in glioma patients. The Cancer Genome Atlas (TCGA) and GSE108474 glioma cohorts and IMvigor210 immunotherapy datasets were collected, including complete mRNA expression profiles and clinical information. We used cell lines to verify the gene expression and the R 3.6.3 tool and GraphPad for bioinformatics analysis and mapping. T cell depletion in glioma patients displayed significant heterogeneity. The T cell depletion-related prognostic model was developed based on seven prognostic genes (HSPB1, HOXD10, HOXA5, SEC61G, H19, ANXA2P2, HOXC10) in glioma. The overall survival of patients with a high TEXScore was significantly lower than that of patients with a low TEXScore. In addition, high TEXScore scores were followed by intense immune responses and a more complex tumor immune microenvironment. The "hot tumors" were predominantly enriched in the high-risk group, which patients expressed high levels of suppressive immune checkpoints, such as PD1, PD-L1, and TIM3. However, patients with a low TEXScore had a more significant clinical response to immunotherapy. In addition, HSPB1 expression was higher in the U251 cells than in the normal HEB cells. In conclusion, the TEXScore related to T cell exhaustion combined with other pathological profiles can effectively assess the clinical status of glioma patients. The TEXScore constructed in this study enables the effective assessment of the immunotherapy response of glioma patients and provides therapeutic possibilities.

Nutrient deprivation and hypoxia alter T cell immune checkpoint expression: potential impact for immunotherapy

AimUse of immune checkpoint blockade to enhance T cell-mediated immunity within the hostile tumour microenvironment (TME) is an attractive approach in oesophageal adenocarcinoma (OAC). This study explored the effects of the hostile TME, including nutrient deprivation and hypoxia, on immune checkpoint (IC) expression and T cell phenotypes, and the potential use of nivolumab to enhance T cell function under such conditions.Methods and ResultsICs were upregulated on stromal immune cells within the tumour including PD-L2, CTLA-4 and TIGIT. OAC patient-derived PBMCs co-cultured with OE33 OAC cells upregulated LAG-3 and downregulated the co-stimulatory marker CD27 on T cells, highlighting the direct immunosuppressive effects of tumour cells on T cells. Hypoxia and nutrient deprivation altered the secretome of OAC patient-derived PBMCs, which induced upregulation of PD-L1 and PD-L2 on OE33 OAC cells thus enhancing an immune-resistant phenotype. Importantly, culturing OAC patient-derived PBMCs under dual hypoxia and glucose deprivation, reflective of the conditions within the hostile TME, upregulated an array of ICs on the surface of T cells including PD-1, CTLA-4, A2aR, PD-L1 and PD-L2 and decreased expression of IFN-γ by T cells. Addition of nivolumab under these hostile conditions decreased the production of pro-tumorigenic cytokine IL-10.ConclusionCollectively, these findings highlight the immunosuppressive crosstalk between tumour cells and T cells within the OAC TME. The ability of nivolumab to suppress pro-tumorigenic T cell phenotypes within the hostile TME supports a rationale for the use of immune checkpoint blockade to promote anti-tumour immunity in OAC.Graphical abstractStudy schematic: (A) IC expression profiles were assessed on CD45+ cells in peripheral whole blood and infiltrating tumour tissue from OAC patients in the treatment-naïve setting. (B) PBMCs were isolated from OAC patients and expanded ex vivo for 5 days using anti-CD3/28 + IL-2 T cell activation protocol and then co-cultured for 48 h with OE33 cells. T cell phenotypes were then assessed by flow cytometry. (C) PBMCs were isolated from OAC patients and expanded ex vivo for 5 days using anti-CD3/28 + IL-2 T cell activation protocol and then further cultured under conditions of nutrient deprivation or hypoxia for 48 h and T cell phenotypes were then assessed by flow cytometry. Key findings: (A) TIGIT, CTLA-4 and PD-L2 were upregulated on CD45+ immune cells and CTLA-4 expression on CD45+ cells correlated with a subsequent decreased response to neoadjuvant regimen. (B) Following a 48 h co-culture with OE33 cells, T cells upregulated LAG-3 and decreased CD27 co-stimulatory marker. (C) Nutrient deprivation and hypoxia upregulated a range of ICs on T cells and decreased IFN-γ production by T cells. Nivolumab decreased IL-10 production by T cells under nutrient deprivation-hypoxic conditions.

CtDNA in treatment response monitoring in patients with relapsed gynecologic malignancies.

e17501 Background: The role of immunotherapy in gynecologic malignancies continues to evolve. Use of immune checkpoint blockade (ICB) agents have consistently demonstrated less than a 15% response rate in ovarian, cervical, and subsets of endometrial cancer. Given the limited number of responders, a novel approach to treatment monitoring is needed to better sequence therapies. Circulating tumor DNA (ctDNA) surveillance in patients treated with ICB is one such approach that has been shown to predict clinical benefit. We sought to evaluate this approach in patients with recurrent gynecologic malignancies managed with ICB therapy. Methods: In this retrospective analysis of real-world data, plasma samples (n = 208) from 28 patients with recurrent/metastatic ovarian (n = 8), endometrial (n = 14), and cervical (n = 6) cancers were identified as having received ICB therapy. A personalized and tumor-informed multiplex PCR assay (Signatera™ bespoke mPCR NGS assay) was used for the detection of ctDNA in plasma samples. Serial time points were collected to monitor ctDNA levels in response to immunotherapy. Results: Response assessment results and longitudinal plasma samples were available for 19 patients. Pre-ICB plasma samples were available for 11 patients. Of these ctDNA was detected in 73% (8/11) of patients. All patients with progressive disease (10/10) had rising ctDNA levels that preceded radiological findings by a median of 2.8 months. One patient with rising ctDNA concentration presented with stable disease by imaging at the last follow-up. The remaining 8 patients demonstrated response to therapy that coincided with a declining or negative ctDNA level; 5 with partial response had a decline in ctDNA level prior to imaging (n = 3) or lack of ctDNA detection (n = 2), and 3 with complete response remained ctDNA negative, prior to imaging. Patients that exhibited a rise in ctDNA concentration (n = 11) (consecutive time points) while undergoing ICB therapy were associated with resistance to treatment (HR = 4.58 95%CI: 1.048-20.03, p = 0.04). TMB and MSI status (binary) were not predictive of response in univariate (p = 0.8, p = 0.9) analyses. Conclusions: ctDNA monitoring during the course of immunotherapy for relapsed gynecologic malignancies allows for accurate determination of therapeutic response and early prediction of disease progression. This enables timely assessment with imaging, and change in treatment plan. Our data suggest that patients with gynecologic malignancies may benefit from personalized, tumor-informed ctDNA testing to guide treatment decisions. Prospective studies are needed to establish the clinical utility of ctDNA monitoring in patients with gynecologic malignancies undergoing systemic therapies.

Examination of mutation signature and mutation spectrum as predictors of response to immune checkpoint blockade therapy.

2537 Background: In recent years, there has been promising progress in the use of immune checkpoint blockade (ICB) as a treatment for various cancer types such as lung, kidney, bladder, skin, colon, and breast cancer. In order for this modality to have increased success, more precise selection tools are needed to predict which patients will benefit from treatment. Tumor Mutational burden (TMB) has been implicated as a biomarker for ICB response due to the increased tumor immunogenicity present in high TMB samples. However, we hypothesize that the quality of mutations may also have an impact in determining immune response, rather than just the quantity of mutations alone. Methods: A retrospective analysis of 2041 patients across multiple solid tumor types was conducted using an open-access, open-source cancer genomics database (Gao, Sci Signal, 2013), with the goal of assessing the influence of mutational signature on patient response to ICB (including CTLA-4, PD-1, and PDL-1). Patient demographics, treatment variables/outcomes, and tumor variables, including mutation spectrum, and mutation count, were evaluated. A paired two sample test for means was used to analyze data with p < 0.05 considered statistically significant. Response outcomes were determined per RECIST v 1.1. Results: Our data demonstrate that mutational spectrum profiles show distinct patterns when considering response to ICB, independent of TMB. Patients with breast invasive ductal carcinoma who responded to ICB had less C > A mutations ( p = 0.020) and more C > T mutations ( p = 0.017) compared to non-responders. Lung adenocarcinoma responders had less C > T mutations ( p = 0.003) and more C > A mutations ( p = 0.0003) compared to non-responders. Furthermore, both head and neck squamous cell carcinoma and glioblastoma multiforme responders had more T > A mutations ( p = 0.047 and p = 0.011, respectively) than non-responders. However, in other cancer types studied (urothelial, colon, renal, lung squamous, and melanoma), there were no obvious trends to distinguish responders from non-responders. Preferentially mutated gene targets were also considered for all 10 cancer types (including CARD11, SMARCA4, GLI1, PIK3R1, PTPRT, among others), many of which were also tied to the unique mutational signatures observed and which may be separately identified as novel ICB therapy targets. Conclusions: We found that specific mutation type may impact response to ICB in at least 4 cancer types, including breast, lung adenocarcinoma, head and neck squamous cell carcinoma and glioblastoma multiforme. Of note, this trend was not found in other cancer types which are typically found to have higher TMB. Moving away from solely considering the magnitude of mutations in tumor samples and towards identifying specific mutational signatures may aid in providing necessary specificity for selecting patients for ICB cancer therapy.

Abstract P2-08-15: Clinical, pathologic, and molecular associations of tumor mutational burden in metastatic breast cancer

Abstract Background: Tumor mutational burden (TMB) is a biomarker approved to predict response to immune checkpoint blockade (ICB) in solid tumors irrespective of their tissue of origin. However, there are limited data in patients with breast cancer and high TMB to support the use of ICB. The goal of this analysis is to describe clinical, pathological and molecular associations with TMB within a cohort of patients with metastatic breast cancer. Methods: We included patients enrolled onto an ongoing prospective study titled Individualized Molecular Analyses Guide Efforts (IMAGE)-II. Patients eligible for IMAGE-II have metastatic breast cancer of any subtype that had progressed on at least one standard-of-care therapy. Genetic profiling of tumor tissue was performed at the discretion of the treating team using one of several commercially available next generation sequencing platforms. For purposes of this analysis, only patients who underwent tissue-based Foundation Medicine analysis are included as TMB assessments are different across different platforms. Data are summarized by descriptive statistics. Linear and logistic regression analyses are conducted to evaluate the association between TMB and other clinical, pathological and molecular factors. We will present data on associations with specific mutations (i.e. ESR1, ERBB2, DNA repair, Pi3K signaling, TP53) and ctDNA TMB at a later time. Results:Of 117 patients in the IMAGE-II database, median age was 57 (range 23-86), 65% were White, 29% Black, and 6% Other. TMB data were available on 62 patients. Of those with both TMB and subtype information, 35 (70%) had ER+HER2- tumors, and 15 (30%) had ER-HER2- tumors. Median TMB was 4 mutations/megabase and ranged from 0 to 27. We did not observe significant differences in TMB in patients with ER+HER2- and those with ER-HER2- tumors (median TMB of 4 [0-27] and 5 [1-25], respectively), nor between White versus Black patients (median TMB of 4 [0-27] and 5 [0-12], respectively). However, we did observe that age was positively associated with higher TMB (p-value = 0.02). Additionally, we observed that the time between metastatic diagnosis and TMB measurement was positively associated with TMB (p-value < 0.01); this significant association was also observed in ER+HER2- patients (p-value < 0.01) but not in ER-HER2- patients. Median time to obtaining TMB since metastatic diagnosis was 1.1 (range -0.8 - 12.8) years. More lines of chemotherapy prior to TMB assessment was not observed to be associated with higher TMB. Conclusions: We observed that TMB was higher in patients who have had a longer disease course. Further research is required to understand changes in TMB over time, and how TMB is correlated with. other genomic and tumor microenvironment characteristics. A deeper understanding of TMB may help refine it as a predictive biomarker for ICB. Citation Format: Mohamed A Mohamed, Chenghuang Wang, Morgan Buckley, Jennifer Lehman, Jenna Canzoniero, Christopher D Gocke, Raquel Nunes, Ben Ho Park, Karen L Smith, Jessica Tao, Hanna Tukachinsky, Mary Wilkinson, Antonio C Wolff, Vered Stearns, Cesar A Santa-Maria. Clinical, pathologic, and molecular associations of tumor mutational burden in metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-08-15.