anti-PD1 Checkpoint Inhibitor Research Articles

Neoadjuvant followed by adjuvant pembrolizumab in melanoma: time biases in the data analysis of the SWOG S1801 trial.

The SWOG S1801 trial investigated the role of pembrolizumab, an anti-PD1 immune checkpoint inhibitor, in the perioperative setting of stage III or IV melanoma. This phase 2 trial compared two groups: one receiving pembrolizumab both before and after surgery (neoadjuvant-adjuvant), and another receiving it only post-surgery (adjuvant-only), with event-free survival (EFS) as the primary endpoint. Neoadjuvant strategies, involving pre-surgical drug administration, potentially offer rapid tumor control and a unique opportunity to assess tumor response. However, they may expose to toxicity and delay or preclude surgery. The study met its primary endpoint, with a 72 % EFS rate in the neoadjuvant-adjuvant group, and 49 % in the adjuvant group. Here, we question the results' applicability with three potential limitations. Key concerns include an arbitrary rule in event assignment, possibly affecting the event distribution over time. Second, different rates of early censoring between groups introduce the possibility of informative censoring, which could have led to an artefactual benefit in EFS. Lastly, phase 2 trial results, by definition, carry risk of fluke results, and should be confirmed in phase 3 trial before wide adoption. Collectively, these factors must be integrated into a careful interpretation of the SWOG S1801 trial outcomes. More robust data are needed to fully appraise strengths and limitations of neoadjuvant pembrolizumab in melanoma treatment.

Abstract 2609: Vesicular LGALS3BP is a neuroblastoma biomarker and a therapeutic target for combination therapy with antibody-drug conjugate and checkpoint inhibitors

Abstract We have recently shown that targeting circulating LGALS3BP with a maytansinoid-based non-internalizing antibody-drug conjugate (ADC) resulted in tumor eradication of high-risk NMYC-amplified neuroblastoma patient-derived xenografts. LGALS3BP is a high molecular weight, hyperglycosylated and secreted protein which is enriched at the surface of cancer-derived extracellular vesicles (EVs). Here, using a neuroblastoma pseudometastatic preclinical model, we extended our previous work demonstrating that EVs-associated LGALS3BP strictly correlates with disease progression and dissemination. Moreover, to investigate whether LGALS3BP ADC, named 1959sss/DM4, possesses immune-modulating properties, we analyzed DM4 ability to induce immunogenic cell death (ICD). Interestingly, using NXS2 murine neuroblastoma cells, we found that DM4 induced a time-dependent surface expression of three different ICD marker, i.e. calreticulin, HSP70 and HSP90. As it was recently demonstrated that to limit immune surveillance NXS2 cells increase PD-L1 expression in response to IFNγ production in the tumor immune infiltrate, we evaluated potential synergism between LGALS3BP targeting ADC and an anti-PD1 immune check-point inhibitor. To this aim, we genetically manipulated NXS2 cells which resulted nearly negative for murine LGALS3BP expression. Therefore, using a lentivirus-based gene transduction system, we obtained NXS2 cells producing large amounts of vesicular human LGALS3BP. Of note, the vesicular LGALS3BP could be quantified by ELISA and imaged by confocal microscopy using 1959 antibody. Strikingly, while single agent treatment showed a limited antitumor activity in NXS2-LGALS3BP syngeneic model, the LGALS3BP ADC/anti-PD1 combination resulted in a marked inhibition of tumor growth, which was followed by a prolonged survival. In conclusion, our findings establish that vesicular LGALS3BP is a potential biomarker for liquid biopsy and reveals this protein as a suitable target for therapeutic strategy that combines 1959sss/DM4 ADC with immune checkpoint blockade for the treatment of LGALS3BP expressing neuroblastoma. Citation Format: Gianluca Sala, Emily Capone, Ilaria Cela, Giulio Lovato, Alessia Lamolinara, Manuela Iezzi, Cosmo Rossi, Vincenzo De Laurenzi, Rodolfo Ippoliti, Stefano Iacobelli. Vesicular LGALS3BP is a neuroblastoma biomarker and a therapeutic target for combination therapy with antibody-drug conjugate and checkpoint inhibitors [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 2609.

Cancer-associated Fibroblast-specific Expression of the Matricellular Protein CCN1 Coordinates Neovascularization and Stroma Deposition in Melanoma Metastasis.

In human patients, the expression of proangiogenic matricellular protein CCN1 in CAFs correlates positively with expression of stroma and angiogenic markers and progressive disease/resistance to checkpoint inhibitor therapy. In an animal model, loss of CCN1 from CAFs impaired metastasis of melanoma cells, neovascularization, and collagen deposition, emphasizing that CAFs coordinate cellular behavior in a tumor microenvironment and that CCN1 may be a novel target.

HLA-DR expression in melanoma: from misleading therapeutic target to potential immunotherapy biomarker.

Since the advent of anti-PD1 immune checkpoint inhibitor (ICI) immunotherapy, cutaneous melanoma has undergone a true revolution with prolonged survival, as available 5-year updates for progression-free survival and overall survival demonstrate a durable clinical benefit for melanoma patients receiving ICI. However, almost half of patients fail to respond to treatment, or relapse sooner or later after the initial response to therapy. Little is known about the reasons for these failures. The identification of biomarkers seems necessary to better understand this resistance. Among these biomarkers, HLA-DR, a component of MHC II and abnormally expressed in certain tumor types including melanoma for unknown reasons, seems to be an interesting marker. The aim of this review, prepared by an interdisciplinary group of experts, is to take stock of the current literature on the potential interest of HLA-DR expression in melanoma as a predictive biomarker of ICI outcome.

Fungal Depletion Bolsters Anti-Tumor Immune Response Elicited by Anti-PD1 Alone and in Combination with Radiation Therapy

Pembrolizumab in combination with chemotherapy has become the standard of care treatment for both metastatic and early-stage triple negative breast cancer (TNBC). Clinical trials are currently underway investigating the use of pembrolizumab with radiation in the neoadjuvant setting in early TNBC. Several groups have described a link between the microbiome and the efficacy of chemotherapy and anti-PD1 immune checkpoint inhibitors (ICIs) in preclinical models. Recent work from our lab has shown that targeting commensal fungi in the microbiome enhances the radiation induced antitumor immune response. Therefore, we hypothesized that fungal depletion might positively impact anti-PD1 therapy and combination treatment with anti-PD1 and radiation therapy (RT). This study utilized an orthotopic syngeneic breast tumor model in which the syngeneic cell line E0771 was injected into the mammary fat pad of female C57BL/6 mice. Tumor-bearing mice were then treated with and without the antifungal fluconazole, anti-PD1, and radiation (16 Gy single fraction) using the X-RAD SmART platform with CT guidance. Tumor volumes were compared using 2-way ANOVA and survival curves analyzed using log rank. In a separate set of experiments, tumor-infiltrating immune cells were isolated and analyzed by high-dimensional multiplex flow cytometry. We found that fungal depletion with fluconazole prior to treatment with anti-PD1 reduced the tumor volume and significantly improved survival in comparison to those treated with anti-PD1 alone (P = 0.0016). To identify what changes in the tumor immune microenvironment is driving this increased anti-tumor response, we performed flow cytometry on immune cells isolated from the tumors. We found that the use of fluconazole prior to anti-PD1 treatment reduced the proportion of CD11b+F4/80+ tumor-associated macrophages (TAMs) (P = 0.01) and increased tumor infiltrating cytotoxic T cell population (P = 0.04) when compared with the use of anti-PD1 alone. We also evaluated the effect of fungal depletion on combination therapy with RT and anti-PD1. Strikingly, we found that mice depleted of fungi with fluconazole prior to radiation and anti-PD1 therapy, have decreased tumor burden and significantly increased survival when compared to their fungally-intact counterparts (P = 0.043). Our data indicates that the depletion of the gut fungal populations induces an increased antitumor response following anti-PD1 alone and in combination with radiation. This increased antitumor immune response is associated with an increase in the cytotoxic CD8+ T cell compartment with concomitant decrease in immunosuppressive tumor associated macrophages.

Phase 2 trial of epidermal growth factor (EGF) vaccine CIMAvax in combination with pembrolizumab in first line and maintenance setting in advanced non–small cell lung cancer patients.

TPS2677 Background: CIMAvax-EGF is a novel growth-factor depleting immunotherapy consisting of human recombinant EGF conjugated to recombinant P64k derived from Neisseria meningitidis, that elicits an anti-EGF antibody response, resulting in reduction of circulating EGF levels. A randomized phase 3 study of CIMAvax-EGF administered as switch maintenance therapy after first-line platinum-based chemotherapy in patients with advanced NSCLC demonstrated overall survival (OS) advantage in comparison to best supportive care alone, particularly in patients with high baseline serum EGF levels. A phase 1 trial combining CIMAvax-EGF with nivolumab, an anti-PD1 immune checkpoint inhibitor, showed that the combination is safe and appears to induce higher rate of good anti-EGF antibody response within the first 8 weeks of therapy compared to historical comparison group receiving CIMAvax-EGF alone. CIMAvax-EGF is currently being investigated in combination with pembrolizumab in a multi-arm phase II trial as 1L therapy in combination with pembrolizumab for patients with EGFR/ALK wildtype NSCLC and PD-L1 > 50% (Cohort C) and as maintenance therapy after completing 1L chemoimmunotherapy for patients with PD-L1 < 50% (Cohorts D and E). Accrual to this trial is ongoing (NCT02955290). Methods: This is an open-label, non-randomized, multi-site, phase 2 basket trial. CIMAvax-EGF (2.4 mg IM every 2 weeks x 4 doses, then every 4 weeks thereafter) is being tested as first-line therapy in combination with pembrolizumab (200 mg IV every 4 weeks) in patients with advanced NSCLC with PD-L1 ≥ 50% (Cohort C), using a Simon two stage design (n up to 41). A safety lead-in consisting of the first 6 NSCLC patients was completed. There were no G3 or higher adverse events attributed to the combination within the first 4 weeks of protocol treatment. Patients with advanced squamous NSCLC (Cohort D, n=50) and non-squamous NSCLC (without EGFR/ALK/ROS-1/KRAS mutations; Cohort E, n=45) with PD-L1 expression <50% and no evidence of progression of disease by RECIST 1.1 criteria after at least 4 cycles of induction platinum-based doublet with pembrolizumab, prior to initiation of standard of care maintenance therapy will be enrolled to receive protocol treatment with pembrolizumab in combination with CIMAvax-EGF as maintenance therapy. The primary endpoint is to evaluate objective response rate (Cohort C), and 12-month PFS in Cohorts D and E. Secondary endpoints include 12-month overall survival in all cohorts. Exploratory objectives include characterizing tissue-based immune and EGFR signaling profile as well as serum EGF levels, anti-EGF antibody levels and other blood-based biomarkers in relation to clinical outcomes. Clinical trial information: NCT02955290 .

Abstract LB101: Cell-generated IL12 combined with PD-1 inhibition produces local and abscopal immune activation to eradicate metastatic melanoma and pancreatic cancer

Abstract Background: Cytokines have been FDA approved for cancer immunotherapy for treatment of metastatic melanoma and renal carcinoma for over 30 years (1, 2). To overcome stability and toxicity limitations seen with high dose cytokine immunotherapy, we developed a delivery platform, called cytokine factories, composed of genetically engineered epithelial cells encapsulated in biocompatible polymers. These clinically translatable cytokine factories are able to safely deliver high local doses of pro-inflammatory cytokines, such as interleukin-12 (IL12), and allow for controlled and predictable dosing in vivo. Results: Tumor-adjacent administration of IL12-based cytokine factories in vivo created a high local cytokine concentration (IP space) without substantial leakage into the systemic circulation. In addition, administration of cytokine factories in combination with anti-PD1 checkpoint inhibitors caused reduction of tumor burden by over 60% when delivered as a monotherapeutic to mice with metastatic melanoma (3). Further, when administered in combination with local anti-PD1 checkpoint inhibitors, these cytokine factories led to reduction of intraperitoneal tumor burden by over 80% after only 10 days of treatment. Finally, we evaluated the ability of this therapy to treat pancreatic tumors which are notoriously known to be resistant to immunotherapy. We found that the median survival for control animals was 45 days. However, 8/8 of animals treated with IL12 cytokine factories survived throughout the duration of the study (110 days), demonstrating a significant extension of overall survival time. Conclusions: Our findings demonstrate efficacy of cytokine factories as single agent and combination therapeutics in preclinical animal models and provide rationale for future clinical testing for the treatment of metastatic peritoneal cancers in humans.

Characterization of immune cell populations in syngeneic murine tumor models.

Immunocompetent murine models are important tools for preclinical evaluation of immunotherapies. Here, six different immunocompetent tumor models based on four different cell lines were characterized, including metastatic lung cancer (CMT 167), triple-negative breast cancer (4T1), pancreatic cancer (KPCY), and colon cancer (MC38). The tumors were implanted subcutaneously or orthotopically before the animals were treated with anti-PD1 checkpoint inhibitor. A range of innate and adaptive immune cells were then quantified by flow cytometry of single-cell suspensions from the tumors. Furthermore, confocal laser scanning microscopy was used to quantify the density and distribution of T-cells in frozen sections. A model-dependent cellular immune landscape was observed, with variable responsiveness toward anti-PD1, ranging from the most responsive MC38 colon cancer model to the least responsive 4T1 breast cancer model. The study provides an overview of the immune landscape of these tumor models, and a foundation for further elucidation of pro-tumor and anti-tumor mechanisms behind heterogeneous responses towards immunotherapies.

Songs in the Key of Life

Songs in the Key of Life

Anti PD-1 Checkpoint Inhibitor As a First-Line Treatment for Advanced Cutaneous Squamous Cell Carcinoma.

Immune checkpoint inhibitors (ICI) have become an essential part of cancer treatment. Anti-programmed cell death receptor-1 (PD-1) is a monoclonal antibody that targets PD-1. For patients with inoperable cutaneous squamous cell carcinoma, anti-PD-1 ICI treatment has been approved as monotherapy or in adjunct with other treatment modalities. A patient primarily treated with PD-1 inhibition for local advanced moderately differentiated cutaneous squamous cell carcinoma involving the parotid and the neck is presented. Surgical therapy would be extensive including pinnectomy, radical parotidectomy, neck dissection, resection of the temporal and mastoid bones, and free flap reconstruction. Anti-PD-1 ICI was started as a first-line treatment and a complete clinical response was observed after 5 cycles of treatment. The patient is disease-free within the follow-up period of 17 months. Although a complete response to anti-PD-1 receptor antibodies was observed, off-target toxicities are a risk and not all patients will benefit from a response.

SOLTI-1904 ACROPOLI TRIAL: efficacy of spartalizumab monotherapy across tumor-types expressing high levels of PD1 mRNA.

Improved selection of cancer patients who are most likely to respond to immune checkpoint inhibitors remains an unmet clinical need. Recently, a positive correlation between levels of PD1 mRNA and clinical outcome in response to PD1 blockade across diverse tumor histologies has been confirmed in several datasets. ACROPOLI is a parallel cohort, non-randomized, phase II study that aims to evaluate the efficacy of the anti-PD1 immune checkpoint inhibitor spartalizumab as monotherapy in metastatic patients with solid tumors that express high levels of PD1 (cohort 1; n=111). An additional cohort of 30 patients with tumors expressing low levels of PD1, where PD1/PD-L1 antibodies in monotherapy are standard treatment, will also be included (cohort 2). Primary end point is overall response rate in cohort 1. Trial registration number: NCT04802876 (ClinicalTrials.gov).

Innate immune checkpoint inhibitor resistance is associated with melanoma sub-types exhibiting invasive and de-differentiated gene expression signatures.

Melanoma is a highly aggressive skin cancer, which, although highly immunogenic, frequently escapes the body’s immune defences. Immune checkpoint inhibitors (ICI), such as anti-PD1, anti-PDL1, and anti-CTLA4 antibodies lead to reactivation of immune pathways, promoting rejection of melanoma. However, the benefits of ICI therapy remain limited to a relatively small proportion of patients who do not exhibit ICI resistance. Moreover, the precise mechanisms underlying innate and acquired ICI resistance remain unclear. Here, we have investigated differences in melanoma tissues in responder and non-responder patients to anti-PD1 therapy in terms of tumour and immune cell gene-associated signatures. We performed multi-omics investigations on melanoma tumour tissues, which were collected from patients before starting treatment with anti-PD1 immune checkpoint inhibitors. Patients were subsequently categorized into responders and non-responders to anti-PD1 therapy based on RECIST criteria. Multi-omics analyses included RNA-Seq and NanoString analysis. From RNA-Seq data we carried out HLA phenotyping as well as gene enrichment analysis, pathway enrichment analysis and immune cell deconvolution studies. Consistent with previous studies, our data showed that responders to anti-PD1 therapy had higher immune scores (median immune score for responders = 0.1335, median immune score for non-responders = 0.05426, p-value = 0.01, Mann-Whitney U two-tailed exact test) compared to the non-responders. Responder melanomas were more highly enriched with a combination of CD8+ T cells, dendritic cells (p-value = 0.03) and an M1 subtype of macrophages (p-value = 0.001). In addition, melanomas from responder patients exhibited a more differentiated gene expression pattern, with high proliferative- and low invasive-associated gene expression signatures, whereas tumours from non-responders exhibited high invasive- and frequently neural crest-like cell type gene expression signatures. Our findings suggest that non-responder melanomas to anti-PD1 therapy exhibit a de-differentiated gene expression signature, associated with poorer immune cell infiltration, which establishes a gene expression pattern characteristic of innate resistance to anti-PD1 therapy. Improved understanding of tumour-intrinsic gene expression patterns associated with response to anti-PD1 therapy will help to identify predictive biomarkers of ICI response and may help to identify new targets for anticancer treatment, especially with a capacity to function as adjuvants to improve ICI outcomes.

Molecular Markers of Response to Anti-PD1 Therapy in Advanced Hepatocellular Carcinoma

Background & AimsSingle-agent anti-PD1 checkpoint inhibitors convey outstanding clinical benefits in a small fraction (∼20%) of patients with advanced hepatocellular carcinoma (aHCC) but the molecular mechanisms determining response are unknown. To fill this gap, we herein analyze the molecular and immune traits of aHCC in patients treated with anti-PD1. MethodsOverall, 111 tumor samples from patients with aHCC were obtained from 13 centers before systemic therapies. We performed molecular analysis and immune deconvolution using whole-genome expression data (n = 83), mutational analysis (n = 72), and histologic evaluation with an endpoint of objective response. ResultsAmong 83 patients with transcriptomic data, 28 were treated in frontline, whereas 55 patients were treated after tyrosine kinase inhibitors (TKI) either in second or third line. Responders treated in frontline showed upregulated interferon-γ signaling and major histocompatibility complex II–related antigen presentation. We generated an 11-gene signature (IFNAP), capturing these molecular features, which predicts response and survival in patients treated with anti-PD1 in frontline. The signature was validated in a separate cohort of aHCC and >240 patients with other solid cancer types where it also predicted response and survival. Of note, the same signature was unable to predict response in archival tissue of patients treated with frontline TKIs, highlighting the need for fresh biopsies before immunotherapy. ConclusionInterferon signaling and major histocompatibility complex–related genes are key molecular features of HCCs responding to anti-PD1. A novel 11-gene signature predicts response in frontline aHCC, but not in patients pretreated with TKIs. These results must be confirmed in prospective studies and highlights the need for biopsies before immunotherapy to identify biomarkers of response.

The Effects of 6 Common Antidiabetic Drugs on Anti-PD1 Immune Checkpoint Inhibitor in Tumor Treatment

Diabetes and cancer are common diseases and are frequently diagnosed in the same individual. These patients need to take antidiabetic drugs while receiving antitumor drugs therapy. Recently, immunotherapy offers significant advances for cancer treatment. However, it is unclear whether antidiabetic drugs affect immunotherapy. Here, by employing syngeneic mouse colon cancer model and melanoma model, we studied the effects of 6 common antidiabetic drugs on anti-PD1 immune checkpoint inhibitor in tumor treatment, including acarbose, sitagliptin, metformin, glimepiride, pioglitazone, and insulin. We found that acarbose and sitagliptin enhanced the tumor inhibition of anti-PD1, and metformin had no effect on the tumor inhibition of anti-PD1, whereas glimepiride, pioglitazone, and insulin weakened the tumor inhibition of anti-PD1. Our study suggests that cancer patients receiving anti-PD1 antibody therapy need serious consideration when choosing antidiabetic drugs. In particular, acarbose significantly inhibited tumor growth and further enhanced the therapeutic effect of anti-PD1, which can be widely used in tumor therapy. Based on this study, further clinical trials are expected.

P345: AN IMMUNE SCREEN IDENTIFIES 5-NONLOXYTRYPTAMINE AS A NOVEL ANTI-CANCER AGENT CAPABLE OF IMPROVING ANTI-TUMOR IMMUNITY IN COMBINATION WITH ANTI-PD1 THERAPY

Background: Exploiting the immune system, particularly CD8+ T cells to eliminate tumors has revolutionized the treatment landscape. However, despite the evident successes of CD8+ T cell-targeting immunotherapies such as anti-PD1 checkpoint inhibitors, many patients fail to respond especially those with poorly immunogenic tumors. Additional obstacles to successful immunotherapy responses include therapy-induced toxicity, lack of bio-markers of response, an immunosuppressive environment leading to T cell dysfunction and exhaustion and poor immune infiltration highlighting a need for novel combinatorial approaches to augment immunotherapeutic activity. Aims: We aimed to uncover novel therapeutic agents capable of augmenting the anti-tumoral responses of CD8+ T cells in ex vivo systems and in vivo syngenetic poorly immunogenic tumor models. Methods: To uncover novel potentiators of T cell anti-tumor immunity, we carried out an ex vivo pharmacological screen, using Lymphocytic choriomeningitis virus (LCMV)-primed splenic T cells that were combined tumor cells expressing the LCMV gp33 (B16) peptide CTL epitope and incubated with an NIH 770 compound library. Hits that increased tumor-cell killing when combined with LCMV-primed splenic T cells were further validated ex vivo. The key candidate was assessed in vivo in C57BL/6J syngeneic tumor models. Immunocompromised NSG mice as well as antibody depletion were used validate CD8+ T cell dependence. In vitro and in vivo immunomodulatory effects were deciphered using flow cytometry, immunoblot, CRISPR/Cas9 and histological analyses. Results: We identified 5-Nonyloxytryptamine (5-NL), a serotonin receptor (HTR) agonist, as increasing the ability of T cells to target tumor cells. In vitro, 5-NL induced apoptosis in melanoma and childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) at low micromolar levels. 5-NL delayed tumor growth in vivo and the phenotype was dependent on the hosts’ immune system, specifically CD8+ T cells as their depletion abrogated the phenotype (Figure 1A-B). 5-NL’s pro-immune effects were attributed to the upregulation of antigen presenting machinery in melanoma, BCP-ALL and other poorly immunogenic tumors with low MHC-I/HLAA-C expression (Figure 1C). Importantly, the upregulation of MHC-I/HLAA-C occurred without concomitant increases in PD-L1 expression and was linked to upregulation of cAMP Response Element-Binding Protein (CREB) in vitro and in vivo. As demonstrated through CRISPR/Cas9 HTR1D knockout, the immunomodulatory and pro-apoptotic effects of 5-NL occurred independently of receptor target signaling. 5-NL was successfully combined with an anti-PD1 antibody in vivo for maximal tumor growth inhibition. Figure 1: The serotonin agonist 5-Nonyloxytryptamine (5-NL) was identified as potentiating T cell mediated anti-tumor immunity. (A) C57BL/6J or (B) CD8 depleted C57BL/6J mice were subcutaneously injected with 5 x 105 gp33 expressing tumor cells. 7 days post-tumor injection, mice were randomized and treated daily 6.25 mg/kg of 5-NL or vehicle for five consecutive days and tumor volume was measured (n = 9-12). H2-Db (MHC-I, murine cell lines) and HLAA-C (human cells lines) were assessed by FACS analysis following treatment with 5-NL for 24 hours (n = 3-9). Error bars in all experiments indicate SEM; *P < 0.05 as determined by a Student´s t-test (unpaired, 2 tailed). Image:Summary/Conclusion: This study demonstrates novel therapeutic opportunities for augmenting immune responses in poorly immunogenic tumors and increasing their responsiveness to immunotherapies.

Biomarker associations of immune checkpoint inhibitor versus chemotherapy effectiveness in first-line metastatic endometrial carcinomas: A real-world study.

5596 Background: Limited evidence exists comparing outcomes of single-agent anti-PD1 immune checkpoint inhibitors (ICPI) vs. chemotherapy in the 1st line among metastatic endometrial carcinoma (mEC) patients. dMMR, MSI status, and Tumor Mutational Burden (TMB) have been characterized as predictive biomarkers of ICPI response in many solid tumors, including mEC. We sought to evaluate ICPI vs. chemotherapy effectiveness in 1st line mEC, stratified by TMB ≥ 10 mut/MB and MSI as assessed by next-generation sequencing. Methods: Following a prespecified analysis plan, this study used the nationwide (̃280 US cancer clinics) de-identified, EHR-derived, Flatiron Health-Foundation Medicine mEC clinico-genomic database (FH-FMI CGDB), with tissue CGP between 1/2011 - 6/2021. Cohort inclusion criteria: recurrent mEC patients with stage I-III at original diagnosis, 1st line ICPI or platinum chemotherapy treatment with TMB and MSI assessments available. Adjusted hazard ratios (aHR) from multivariable Cox proportional hazard models were utilized for time to next treatment (TTNT) and overall survival (OS) comparisons between 1L chemotherapy and 1L ICPI from start of 1L treatment stratified by TMB (≥ 10 vs. &lt; 10) and adjusted for ECOG, BMI, and stage at diagnosis. Results: Among the 1st line cohort, patients received either Chemotherapy (n = 139, 87%) or first-line ICPI (n = 20, 13%). While the overlap between the TMB ≥ 10 (n = 46) population and MSI-H (n = 39) populations was high, with 38/46 (83%) of TMB ≥ 10 classified as MSI-H and 38/39 (97%) of MSI-H with a TMB ≥ 10, there were also 8 (17%) TMB ≥ 10 patients who were MSS. TMB ≥ 10 was associated with more favorable TTNT (aHR: 0.11 [0.03 - 0.44]) and OS (aHR: 0.1 [0.01 - 0.82]) on single-agent ICPIs vs. chemotherapy; however, TMB &lt; 10 patients exhibited no observed differences in TTNT (aHR: 1.66 [0.79 - 3.48]) or OS (aHR: 1.79 [0.66 - 4.8]) between treatments. MSI-H pts were associated with more favorable TTNT (aHR: 0.1 [0.02 - 0.39]) and OS (aHR: 0.1 [0.01 - 0.86]) on single-agent ICPIs over chemotherapy; however, MSS pts did not observe differences in TTNT (aHR: 1.5 [0.65 - 3.5]) or OS (aHR: 2.14 [0.63 - 7.26]) between ICPI vs. chemotherapy. Conclusions: Recurrent 1st line mEC patients with TMB ≥ 10 and/or MSI-H exhibit more favorable outcomes on single-agent ICPI than standard chemotherapy in real-world settings. These findings warrant prospective randomized validation. Future studies should assess the utility of a combined biomarker approach across TMB and MSI status to identify the broadest group of patients who might benefit from ICPI.

Evaluation of checkpoint inhibitor therapies using a mixed lymphocyte reaction (MLR) assay

Abstract Checkpoint inhibitors block the ‘off’ signals that are engaged when T cells interact with antigen-presenting cells, such as dendritic cells (DCs). This increases T cell activation and enhances the anti-tumor response. Here we demonstrate the use of advanced flow cytometry to evaluate these immunotherapies in vitro by co-culturing immune cells from two donors in an MLR assay. Assays were set up as either one-way or two-way MLR co-cultures. One-way assays combined isolated DCs from one donor with CD4+ T cells from another. Two-way assays mixed PBMCs from two donors. Cells and checkpoint inhibitors were plated in 96 or 384-well formats for 3–6 days. Samples taken during the time course were analyzed for cell subsets and cytokine concentrations using the iQue® Advanced Flow Cytometry Platform. Expression of activation marker CD25 on T cells was 3-fold greater after 6 days in co-culture with DCs (62 ± 2 %) compared to T cells in monoculture (22 ± 9 %). The addition of an anti-PD1 checkpoint inhibitor induced a further, concentration dependent increase in CD25 expression. This was accompanied by increased production of cytokines indicative of activation and inflammation, such as TNFα and CCL2. Throughout the study, there were large differences in sensitivity to drug response between donor pairs. For example, IFNγ release increased 13.5-fold upon addition of anti-CTLA4 (17 ng/mL) with one donor pair, compared to only 1.3-fold with another. This highlights the need to characterize drug activity using multiple immune cell donor pairs. These data exemplify the use of advanced flow cytometry to generate pharmacological data for checkpoint inhibitor activity on T cells in MLR, with the potential to profile libraries of novel therapeutics in minimal time.

Tumor-immune microenvironment revealed by Imaging Mass Cytometry in a metastatic sarcomatoid urothelial carcinoma with a prolonged response to pembrolizumab.

Sarcomatoid urothelial carcinoma (SUC) is a rare subtype of urothelial carcinoma (UC) that typically presents at an advanced stage compared to more common variants of UC. Locally advanced and metastatic UC have a poor long-term survival following progression on first-line platinum-based chemotherapy. Antibodies directed against the programmed cell death 1 protein (PD-1) or its ligand (PD-L1) are now approved to be used in these scenarios. The need for reliable biomarkers for treatment stratification is still under research. Here, we present a novel case report of the first Imaging Mass Cytometry (IMC) analysis done in SUC to investigate the immune cell repertoire and PD-L1 expression in a patient who presented with metastatic SUC and experienced a prolonged response to the anti-PD1 immune checkpoint inhibitor pembrolizumab after progression on first-line chemotherapy. This case report provides an important platform for translating these findings to a larger cohort of UC and UC variants.

Performance status (PS) as a predictor of poor response to immune checkpoint inhibitors (ICI) in recurrent/metastatic head and neck cancer (RMHNSCC) patients.

Anti-PD1 checkpoint inhibitors (ICI) represent an established standard-of-care for patients with recurrent/metastatic head and neck squamous cell carcinoma (RMHNSCC). Landmark studies excluded patients with ECOG performance status (PS) ≥2; the benefit of ICI in this population is therefore unknown. We retrospectively reviewed RMHNSCC patients who received 1+ dose of ICI at our institution between 2013 and 2019. Demographic and clinical data were obtained; the latter included objective response (ORR), toxicity, and any unplanned hospitalization (UH). Associations were explored using uni- and multivariate analysis. Overall survival (OS) was estimated using a Cox proportional hazards model; ORR, toxicity, and UH were evaluated with logistic regression. Of the 152 patients, 29 (19%) had an ECOG PS ≥2. Sixty-six (44%) experienced toxicity; 54 (36%) had a UH. A multivariate model for OS containing PS, smoking status, and HPV status demonstrated a strong association between ECOG ≥2 and shorter OS (p< 0.001; HR=3.30, CI=2.01-5.41). An association between OS and former (vs. never) smoking was also seen (p< 0.001; HR=2.17, CI=1.41-3.35); current smoking did not reach statistical significance. On univariate analysis, poor PS was associated with inferior ORR (p= 0.03; OR=0.25, CI=0.06-0.77) and increased UH (p= 0.04; OR=2.43, CI=1.05-5.71). There was no significant association between toxicity and any patient characteristic. We observed inferior OS, ORR, and rates of UH among ICI-treated RMHNSCC patients with ECOG 2/3. Our findings help frame discussion of therapeutic options in this poor-risk population.

Cell death-induced immunogenicity enhances chemoimmunotherapeutic response by converting immune-excluded into T-cell inflamed bladder tumors

Chemoimmunotherapy has recently failed to demonstrate significant clinical benefit in advanced bladder cancer patients; and the mechanism(s) underlying such suboptimal response remain elusive. To date, most studies have focused on tumor-intrinsic properties that render them “immune-excluded”. Here, we explore an alternative, drug-induced mechanism that impedes therapeutic response via disrupting the onset of immunogenic cell death. Using two immune-excluded syngeneic mouse models of muscle-invasive bladder cancer (MIBC), we show that platinum-based chemotherapy diminishes CD8+ T cell tumor infiltration and constraines their antitumoral activity, despite expression of activation markers IFNγ and granzyme B. Mechanistically, chemotherapy induces the release of prostaglandin E2 (PGE2) from dying cancer cells, which is an inhibitory damage-associated molecular pattern (iDAMP) that hinderes dendritic cell maturation. Upon pharmaceutical blockade of PGE2 release, CD8+ T cells become tumoricidal and display an intraepithelial-infiltrating (or inflamed) pattern. This “iDAMP blockade” approach synergizes with chemotherapy and sensitizes bladder tumors towards anti-PD1 immune checkpoint inhibitor therapy. These findings provide a compelling rationale to evaluate this drug combination in future clinical trials.