Adenosine A2a receptor inhibition increases the anti-tumor efficacy of anti-PD1 treatment in murine hepatobiliary cancers.

Backgrounds & Aims: The efficacy of immune checkpoint inhibitor (ICI) therapy for liver cancer remains limited. As the hypoxic liver environment regulates adenosine signaling, we tested the efficacy of adenosine A2a receptor (A2aR) inhibition in combination with ICI treatment in murine models of liver cancer. Methods: RNA expression related to the adenosine pathway was analyzed from public databases. Peripheral blood mononuclear cells of 13 patients with hepatocellular carcinoma (HCC) were examined by flow cytometry. The following murine cell lines were used: SB-1, RIL175, and Hep55.1c (liver cancer), CT26 (colon cancer), and B16-F10 (melanoma). C57BL/6 and BALB/c mice were used for orthotopic tumor models and were treated with SCH58261, an A2aR inhibitor, in combination with anti-PD1 therapy. Results: RNA expression of ADORA2A in tumor tissues derived from patients with HCC was higher than in tissues from other cancer types. A2aR+ T cells in peripheral blood from patients with HCC were highly proliferative after immunotherapy. Likewise, in an orthotopic murine model, A2aR expression on T cells increased following anti-PD1 treatment, and the expression of A2aR on T cells increased more in tumor-bearing mice compared with tumor-free mice. The combination of SCH58261 and anti-PD1 led to activation of T cells and reductions in tumor size in orthotopic liver cancer models. In contrast, SCH58261 monotherapy was ineffective in orthotopic liver cancer models and the combination was ineffective in the subcutaneous tumor models tested. CD4+ T-cell depletion attenuated the efficacy of the combination therapy. Conclusion: A2aR inhibition and anti-PD1 therapy had a synergistic anti-tumor effect in murine liver cancer models. Impact and implications: Adenosine A2a receptor (A2aR)-expressing T cells in the liver increased in tumor-bearing mice and after anti-PD1 treatment. The combination of an A2aR inhibitor and anti-PD1 treatment had potent anti-tumor effects in two murine models of orthotopic liver cancer. Adenosine A2a receptor blockade promotes immunotherapy efficacy in murine models, highlighting putative clinical benefits for advanced stage liver cancer patients.

Inhaled "Muco-Trapping" Monoclonal Antibody Effectively Treats Established Respiratory Syncytial Virus (RSV) Infections.

Respiratory syncytial virus (RSV) causes substantial morbidity and mortality in infants, the immunocompromised, and the elderly. RSV infects the airway epithelium via the apical membrane and almost exclusively sheds progeny virions back into the airway mucus (AM), making RSV difficult to target by systemically administered therapies. An inhalable "muco-trapping" variant of motavizumab (Mota-MT), a potent neutralizing mAb against RSV F is engineered. Mota-MT traps RSV in AM via polyvalent Fc-mucin bonds, reducing the fraction of fast-moving RSV particles in both fresh pediatric and adult AM by ≈20-30-fold in a Fc-glycan dependent manner, and facilitates clearance from the airways of mice within minutes. Intranasal dosing of Mota-MT eliminated viral load in cotton rats within 2 days. Daily nebulized delivery of Mota-MT to RSV-infected neonatal lambs, beginning 3 days after infection when viral load is at its maximum, led to a 10 000-fold and 100 000-fold reduction in viral load in bronchoalveolar lavage and lung tissues relative to placebo control, respectively. Mota-MT-treated lambs exhibited reduced bronchiolitis, neutrophil infiltration, and airway remodeling than lambs receiving placebo or intramuscular palivizumab. The findings underscore inhaled delivery of muco-trapping mAbs as a promising strategy for the treatment of RSV and other acute respiratory infections.

Increasing Age at the Time of Diagnosis and Evolving Phenotypes of Eosinophilic Esophagitis Over 20 Years.

BACKGROUND: The presentation of eosinophilic esophagitis (EoE) is heterogeneous, but trends over time are not known. AIM: To determine whether clinical and endoscopic phenotypes at EoE diagnosis have changed over the past 2 decades. METHODS: In this retrospective cohort study, adults and children with newly diagnosed EoE were phenotyped as follows: (1) inflammatory vs fibrostenotic vs mixed on endoscopy; (2) atopic vs non-atopic; (3) age at symptom onset; (4) age at diagnosis; (5) presence of autoimmune or connective tissue disease; and (6) responsive to steroids. The prevalence of different phenotypes was categorized by 5-year intervals. Multivariate analysis was performed to assess for changes in patient features over time. RESULTS: Of 1187 EoE patients, age at diagnosis increased over time (from 22.0 years in 2002-2006 to 31.8 years in 2017-2021; p < 0.001) as did the frequency of dysphagia (67% to 92%; p < 0.001). Endoscopic phenotypes were increasingly mixed (26% vs 68%; p < 0.001) and an increasing proportion of patients had later onset of EoE. However, there were no significant trends for concomitant autoimmune/connective tissue disease or steroid responder phenotypes. On multivariate analysis, after accounting for age, dysphagia, and food impaction, the increase in the mixed endoscopic phenotype persisted (aOR 1.51 per each 5-year interval, 95% CI 1.31-1.73). CONCLUSION: EoE phenotypes have changed over the past two decades, with increasing age at diagnosis and age at symptom onset. The mixed endoscopic phenotype also increased, even after controlling for age and symptomatology. Whether this reflects changes in provider recognition or disease pathophysiology is yet to be elucidated.

Association between time from esophageal food impaction to endoscopy and adverse events.

BACKGROUND AND AIMS: Guidelines recommend emergent or urgent EGD for esophageal food impaction (EFI), but data on how time to EGD impacts the risk of adverse events remain limited. We determined whether EFI-to-EGD time was associated with adverse events. METHODS: In this retrospective cohort study of patients with endoscopically confirmed EFI, adverse events were classified as esophageal (mucosal tear, bleeding, perforation) or extraesophageal (aspiration, respiratory compromise, hypotension, arrhythmia). Esophageal perforation and extraesophageal adverse events requiring intensive care unit admission were classified as serious adverse events. Baseline characteristics, event details, and procedural details were compared between patients with and without adverse events. Multivariable logistic regression was performed to assess for an association between EFI-to-EGD time and adverse events. RESULTS: Of 188 patients with EFI, 22 (12%) had any adverse event and 2 (1%) had a serious adverse event. Patients with adverse events were older and more likely to have an esophageal motility disorder, to tolerate secretions at presentation, and to have a higher American Society of Anesthesiologists score. EFI-to-EGD time was similar in those with and without adverse events. On multivariable analysis, EFI-to-EGD time was not associated with adverse events (odds ratio, 1.00 [95% confidence interval, .97-1.04] for 1-hour increments; odds ratio, 1.03 [95% confidence interval, .86-1.24] for 6-hour increments). Results were similar after stratifying by eosinophilic esophagitis status and after adjusting for possible confounders. CONCLUSIONS: Because the time from EFI to EGD is not associated with adverse events, emergent EGD for EFI may be unnecessary, and other considerations may determine EGD timing.

Immunosuppressive CD29+ Treg accumulation in the liver in mice on checkpoint inhibitor therapy.

OBJECTIVE: Liver metastases are often resistant to immune checkpoint inhibitor therapy (ICI) and portend a worse prognosis compared with metastases to other locations. Regulatory T cells (Tregs) are one of several immunosuppressive cells implicated in ICI resistance of liver tumours, but the role played by Tregs residing within the liver surrounding a tumour is unknown. DESIGN: Flow cytometry and single-cell RNA sequencing were used to characterise hepatic Tregs before and after ICI therapy. RESULTS: We found that the murine liver houses a Treg population that, unlike those found in other organs, is both highly proliferative and apoptotic at baseline. On administration of αPD-1, αPD-L1 or αCTLA4, the liver Treg population doubled regardless of the presence of an intrahepatic tumour. Remarkably, this change was not due to the preferential expansion of the subpopulation of Tregs that express PD-1. Instead, a subpopulation of CD29+ (Itgb1, integrin ß1) Tregs, that were highly proliferative at baseline, doubled its size in response to αPD-1. Partial and full depletion of Tregs identified CD29+ Tregs as the prominent niche-filling subpopulation in the liver, and CD29+ Tregs demonstrated enhanced suppression in vitro when derived from the liver but not the spleen. We identified IL2 as a critical modulator of both CD29+ and CD29- hepatic Tregs, but expansion of the liver Treg population with αPD-1 driven by CD29+ Tregs was in part IL2-independent. CONCLUSION: We propose that CD29+ Tregs constitute a unique subpopulation of hepatic Tregs that are primed to respond to ICI agents and mediate resistance.

Increased interleukin-6/C-reactive protein levels are associated with the upregulation of the adenosine pathway and serve as potential markers of therapeutic resistance to immune checkpoint inhibitor-based therapies in non-small cell lung cancer.

BACKGROUND: Systemic immune activation, hallmarked by C-reactive protein (CRP) and interleukin-6 (IL-6), can modulate antitumor immune responses. In this study, we evaluated the role of IL-6 and CRP in the stratification of patients with non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICIs). We also interrogated the underlying immunosuppressive mechanisms driven by the IL-6/CRP axis. METHODS: In cohort A (n=308), we estimated the association of baseline CRP with objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) in patients with NSCLC treated with ICIs alone or with chemo-immunotherapy (Chemo-ICI). Baseline tumor bulk RNA sequencing (RNA-seq) of lung adenocarcinomas (LUADs) treated with pembrolizumab (cohort B, n=59) was used to evaluate differential expression of purine metabolism, as well as correlate IL-6 expression with PFS. CODEFACS approach was applied to deconvolve cohort B to characterize the tumor microenvironment by reconstructing the cell-type-specific transcriptome from bulk expression. Using the LUAD cohort from The Cancer Genome Atlas (TCGA) we explored the correlation between IL-6 expression and adenosine gene signatures. In a third cohort (cohort C, n=18), plasma concentrations of CRP, adenosine 2a receptor (A2aR), and IL-6 were measured using ELISA. RESULTS: In cohort A, 67.2% of patients had a baseline CRP≥10 mg/L (CRP-H). Patients with CRP-H achieved shorter OS (8.6 vs 14.8 months; p=0.006), shorter PFS (3.3 vs 6.6 months; p=0.013), and lower ORR (24.7% vs 46.3%; p=0.015). After adjusting for relevant clinical variables, CRP-H was confirmed as an independent predictor of increased risk of death (HR 1.51, 95% CI: 1.09 to 2.11) and lower probability of achieving disease response (OR 0.34, 95% CI: 0.13 to 0.89). In cohort B, RNA-seq analysis demonstrated higher IL-6 expression on tumor cells of non-responders, along with a shorter PFS (p<0.05) and enrichment of the purinergic pathway. Within the TCGA LUAD cohort, tumor IL-6 expression strongly correlated with the adenosine signature (R=0.65; p<2.2e-16). Plasma analysis in cohort C demonstrated that CRP-H patients had a greater median baseline level of A2aR (6.0 ng/mL vs 1.3 ng/mL; p=0.01). CONCLUSIONS: This study demonstrates CRP as a readily available blood-based prognostic biomarker in ICI-treated NSCLC. Additionally, we elucidate a potential link of the CRP/IL-6 axis with the immunosuppressive adenosine signature pathway that could drive inferior outcomes to ICIs in NSCLC and also offer novel therapeutic avenues.

Feeding Tube Placement, Complications, and Treatment Responses in a Large Eosinophilic Esophagitis Patient Population.

OBJECTIVES: Feeding tubes can provide a temporary or long-term solution for nutritional therapy. Little is known regarding the use of feeding tubes in patients with eosinophilic esophagitis (EoE). We sought to describe the characteristics and outcomes in EoE patients requiring tube feeding. METHODS: This was a retrospective cohort study of EoE patients at a large tertiary care health system. Demographics, clinical characteristics, and endoscopic findings were extracted from medical records, and patients who had a feeding tube were identified. Patients with and without a feeding tube were compared. Details about the tube, complications, and treatment were extracted. Growth, global symptomatic, endoscopic, and histopathologic (<15 eos/hpf) responses were compared before and after the initiation of feeding tube therapy. RESULTS: We identified 39 of 1216 EoE patients who had a feeding tube (3%). Feeding tube patients were younger (mean age 6.3 years), reported more vomiting, and had a lower total endoscopic reference score than non-feeding tube patients ( P < 0.01 for all). Tubes were used for therapy for an average of 6.8 years, with most patients (95%) receiving both pharmacologic and formula treatment for EoE. An emergency department visit for a tube complication was required in 26%. Tube feeding improved body mass index z score ( P < 0.01), symptomatic response (42%), endoscopic response (53%), and histologic response (71%). CONCLUSIONS: Among EoE patients, only a small subset required a feeding tube and predominantly were young children with failure to thrive. Feeding tubes significantly improved growth and, when used in combination with other treatments, led to reduced esophageal eosinophilic inflammation.

CSF-1R+ Macrophages Control the Gut Microbiome-Enhanced Liver Invariant NKT Function through IL-18.

The gut microbiome is an important modulator of the host immune system. In this study, we found that altering the gut microbiome by oral vancomycin increases liver invariant NKT (iNKT) cell function. Enhanced iNKT cytokine production and activation marker expression were observed in vancomycin-treated mice following both Ag-specific and Ag-independent in vivo iNKT stimulations, with a more prominent effect in the liver than in the spleen. Fecal transplantation studies demonstrated that the iNKT functional regulation is mediated by altering the gut microbiome but uncoupled from the modulation of iNKT cell population size. Interestingly, when stimulated in vitro, iNKT cells from vancomycin-treated mice did not show increased activation, suggesting an indirect regulation. iNKT cells expressed high levels of IL-18 receptor, and vancomycin increased the expression of IL-18 in the liver. Blocking IL-18 by neutralizing Ab or using genetically deficient mice attenuated the enhanced iNKT activation. Liver macrophages were identified as a major source of IL-18. General macrophage depletion by clodronate abolished this iNKT activation. Using anti-CSF-1R depletion or LyzCrexCSF-1RLsL-DTR mice identified CSF-1R+ macrophages as a critical modulator of iNKT function. Vancomycin treatment had no effect on iNKT cell function in vivo in IL-18 knockout macrophage reconstituted mice. Together, our results demonstrate that the gut microbiome controls liver iNKT function via regulating CSF-1R+ macrophages to produce IL-18.

Tumor-associated macrophages trigger MAIT cell dysfunction at the HCC invasive margin.

Mucosal-associated invariant T (MAIT) cells represent an abundant innate-like T cell subtype in the human liver. MAIT cells are assigned crucial roles in regulating immunity and inflammation, yet their role in liver cancer remains elusive. Here, we present a MAIT cell-centered profiling of hepatocellular carcinoma (HCC) using scRNA-seq, flow cytometry, and co-detection by indexing (CODEX) imaging of paired patient samples. These analyses highlight the heterogeneity and dysfunctionality of MAIT cells in HCC and their defective capacity to infiltrate liver tumors. Machine-learning tools were used to dissect the spatial cellular interaction network within the MAIT cell neighborhood. Co-localization in the adjacent liver and interaction between niche-occupying CSF1R+PD-L1+ tumor-associated macrophages (TAMs) and MAIT cells was identified as a key regulatory element of MAIT cell dysfunction. Perturbation of this cell-cell interaction in ex vivo co-culture studies using patient samples and murine models reinvigorated MAIT cell cytotoxicity. These studies suggest that aPD-1/aPD-L1 therapies target MAIT cells in HCC patients.

Case Report: Peripheral blood T cells and inflammatory molecules in lung cancer patients with immune checkpoint inhibitor-induced thyroid dysfunction: Case studies and literature review.

Immunotherapy has changed the paradigm of cancer treatment, yet immune checkpoint inhibitors (ICIs) such as PD-1/PD-L1 monoclonal antibodies may cause immune-related adverse events (irAEs) in some patients. In this report, two non-small cell lung cancer (NSCLC) patients treated with nivolumab presented with checkpoint inhibitor-induced thyroid dysfunction (CITD), followed by a second irAE of pneumonitis and intestinal perforation, respectively. Increases in peripheral CD8+ T cells correlated with the onset of CITD in the patients. Intriguingly, common inflammatory biomarkers, including C-reactive protein (CRP) and neutrophil/lymphocyte ratio (NLR), were not consistently increased during the onset of CITD but were substantially increased during the onset of pneumonitis and intestinal perforation irAEs. The observations suggest that unlike other irAEs such as pneumonitis, CRP levels and NLR were non-contributory in diagnosing CITD, whereas T cell expansion may be associated with immunotherapy-induced thyroiditis.

Platelets control liver tumor growth through P2Y12-dependent CD40L release in NAFLD.

Platelets, the often-overlooked component of the immune system, have been shown to promote tumor growth. Non-alcoholic fatty liver disease (NAFLD) is a common disease in the Western world and rising risk for hepatocellular carcinoma (HCC). Unexpectedly, we observed that platelets can inhibit the growth of established HCC in NAFLD mice. Through pharmacological inhibition and genetic depletion of P2Y12 as well as in vivo transfusion of wild-type (WT) or CD40L-/- platelets, we demonstrate that the anti-tumor function of platelets is mediated through P2Y12-dependent CD40L release, which leads to CD8+ T cell activation by the CD40 receptor. Unlike P2Y12 inhibition, blocking platelets with aspirin does not prevent platelet CD40L release nor accelerate HCC in NAFLD mice. Similar findings were observed in liver metastasis models. All together, our study reveals a complex role of platelets in tumor regulation. Anti-platelet treatment without inhibiting CD40L release could be considered for liver cancer patients with NAFLD.

Metformin treatment rescues CD8+ T-cell response to immune checkpoint inhibitor therapy in mice with NAFLD.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) represents the fastest growing underlying cause of hepatocellular carcinoma (HCC) and has been shown to impact immune effector cell function. The standard of care for the treatment of advanced HCC is immune checkpoint inhibitor (ICI) therapy, yet NASH may negatively affect the efficacy of ICI therapy in HCC. The immunologic mechanisms underlying the impact of NASH on ICI therapy remain unclear. METHODS: Herein, using multiple murine NASH models, we analysed the influence of NASH on the CD8+ T-cell-dependent anti-PD-1 responses against liver cancer. We characterised CD8+ T cells' transcriptomic, functional, and motility changes in mice receiving a normal diet (ND) or a NASH diet. RESULTS: NASH blunted the effect of anti-PD-1 therapy against liver cancers in multiple murine models. NASH caused a proinflammatory phenotypic change of hepatic CD8+ T cells. Transcriptomic analysis revealed changes related to NASH-dependent impairment of hepatic CD8+ T-cell metabolism. In vivo imaging analysis showed reduced motility of intratumoural CD8+ T cells. Metformin treatment rescued the efficacy of anti-PD-1 therapy against liver tumours in NASH. CONCLUSIONS: We discovered that CD8+ T-cell metabolism is critically altered in the context of NASH-related liver cancer, impacting the effectiveness of ICI therapy - a finding which has therapeutic implications in patients with NASH-related liver cancer. LAY SUMMARY: Non-alcoholic steatohepatitis represents the fastest growing cause of hepatocellular carcinoma. It is also associated with reduced efficacy of immunotherapy, which is the standard of care for advanced hepatocellular carcinoma. Herein, we show that non-alcoholic steatohepatitis is associated with impaired motility, metabolic function, and response to anti-PD-1 treatment in hepatic CD8+ T cells, which can be rescued by metformin treatment.

NAFLD indirectly impairs antigen-specific CD8+ T cell immunity against liver cancer in mice.

Non-alcoholic fatty liver disease (NAFLD) has become an important etiology leading to liver cancer. NAFLD alters adaptive T cell immunity and has a profound influence on liver cancer development. However, it is unclear how NAFLD affects tumor antigen-specific T cell response. In this study, we generated a doxycycline-inducible MHC-I and -II antigen-expressing HCC cell line which allowed us to investigate tumor antigen-specific T cell response in two NAFLD mouse models. The system proved to be an effective and efficient way to study tumor antigen-specific T cells. Using this model, it was found that NAFLD impairs antigen-specific CD8+ T cell immunity against HCC. The effect was not due to reduced generation or intrinsic functional changes of tumor antigen-specific CD8+ T cells but caused by accumulated macrophages in the liver environment. The findings suggest that targeting macrophages in NAFLD-driven HCC may improve therapeutic outcomes.

Anti-PD-1 in Combination With Trametinib Suppresses Tumor Growth and Improves Survival of Intrahepatic Cholangiocarcinoma in Mice.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (iCCA) accounts for a fraction of primary liver cancers but has a 5-year survival rate of only 10%. Immune checkpoint inhibitors are effective in treating many solid cancers, but immune checkpoint inhibitor monotherapy has no clear benefit in iCCA. Mitogen-activated kinase (MEK) inhibitors, such as trametinib, have shown promising results in preclinical studies for iCCA by inhibiting cell proliferation and modifying the tumor microenvironment. This study aimed to show the potential benefit of combining trametinib with anti-programmed cell death protein 1 (PD-1) therapy in different iCCA mouse models. METHODS: Here, we assessed the in vitro cytotoxicity of trametinib in mouse (SB1 and LD-1) and human (EGI-1) cholangiocarcinoma cell lines. We examined the efficacy of single-agent trametinib, anti-PD-1, and a combination of both in subcutaneous, orthotopic, and plasmid-induced iCCA mouse models. Flow cytometry analysis was used to elucidate changes in the tumor immune microenvironment upon treatment. Whole-exome sequencing (WES) was performed on the SB1 tumor cell line to correlate this preclinical model with iCCAs in patients. RESULTS: Trametinib reduced tumor cell growth of SB1, LD-1, and EGI-1 tumor cells in vitro. Trametinib treatment led to up-regulation of major histocompatibility complex (MHC-I) and programmed cell death ligand 1 (PD-L-1) (programmed cell death ligand 1) on tumor cells in vitro. The combination of trametinib and anti-PD-1 reduced tumor burden in several iCCA tumor models and improved survival in SB1 tumor-bearing mice compared with either agent alone. Immunoprofiling of tumor-bearing mice showed an increase of hepatic effector memory CD8+ and CD4+ T cells, as well as an increased degranulation of CD8+ T cells, indicating enhanced cytotoxicity. WES and somatic mutational analysis showed no mutations of KRAS, BRAF, and ERK in SB1 tumor cells, and showed a similar genetic signature of SB1 found in a cohort of patients with iCCA. CONCLUSIONS: Altogether, our study shows that trametinib improves the immunogenicity of tumor cells by up-regulating MHC-I surface expression. The combination with anti-PD-1 results in optimal treatment efficacy for iCCA. WES of SB1 cells suggests that KRAS wild-type iCCAs also respond to this combination therapy.

Evaluating the impact of hydroxychloroquine on mouse lymphocyte proliferation and cytokine production in vivo and in vitro.

Immunomodulatory drugs can alter lymphocyte function. Hydroxychloroquine (HCQ) is prescribed for many autoimmune diseases and is under investigation as an anti-tumor autophagy inhibitor. Here, we describe a protocol to evaluate the influence of HCQ on lymphocyte function by measuring the in vitro and ex vivo proliferation and cytokine production. The protocol can provide insights into potential immunomodulatory effects of HCQ and can be used for assessing other medications' effects on lymphocyte functions. For complete details on the use and execution of this protocol, please refer to Wabitsch et al. (2021).

Hydroxychloroquine can impair tumor response to anti-PD1 in subcutaneous mouse models.

Hydroxychloroquine (HCQ) is a well-known anti-inflammatory drug but is also known as an anti-inflammatory drug. Here, we evaluate the influence of HCQ treatment on the effect of anti-PD1 tumor immunotherapy. Anti-PD1 therapy-sensitive tumor lines MC38, CT26, and RIL-175 were used to investigate the impact of HCQ on anti-PD1 therapy efficacy. In vitro assays demonstrated that HCQ directly inhibited tumor cell growth in all the tested tumor cell lines. HCQ treatment impaired both antigen-specific and nonspecific T-cell production of TNFα and IFNγ in vitro and in vivo. Importantly, in all the three tumor models, HCQ treatment significantly impaired the response to anti-PD1 treatment, accompanying diminished in vivo T-cell activation and reduced tumor-infiltrating, antigen-specific CD8+ T cells. This study shows that HCQ treatment can result in immunotherapy failure due to its immunosuppressive effects that offset both increased MHC-I expression by tumor cell and direct cytotoxicity.

Characterization of Immune Checkpoint Inhibitor-Related Cardiotoxicity in Lung Cancer Patients From a Rural Setting.

BACKGROUND: Immune checkpoint inhibitor (ICI)-related cardiotoxicity (iRC) is uncommon but can be fatal. There have been few reports of iRC from a rural cancer population and few data for iRC and inflammatory biomarkers. OBJECTIVES: The purpose of this study was to characterize major adverse cardiac events (MACE) in ICI-treated lung cancer patients based in a rural setting and to assess the utility of C-reactive protein (CRP) and neutrophil-lymphocyte ratio (NLR) in the diagnosis of iRC. METHODS: Patients with lung cancer treated with ICIs at Vidant Medical Center/East Carolina University (VMC/ECU) between 2015 and 2018 were retrospectively identified. MACE included myocarditis, non-ST-segment elevated myocardial infarction (NSTEMI), supraventricular tachycardia (SVT), and pericardial disorders. Medical history, laboratory values, pre-ICI electrocardiography (ECG), and echocardiography results were compared in patients with and without MACE. RESULTS: Among 196 ICI-treated patients, 23 patients (11%) developed MACE at a median of 46 days from the first ICI infusion (interquartile range [IQR]: 17 to 83 days). Patients who developed MACE experienced myocarditis (n = 9), NSTEMI (n = 3), SVT (n = 7), and pericardial disorders (n = 4). Ejection fraction was not significantly different at the time of MACE compared to that at baseline (p = 0.495). Compared to baseline values, NLR (10.9 ± 8.3 vs. 20.7 ± 4.2, respectively; p = 0.032) and CRP (42.1 ± 10.1 mg/l vs. 109.9 ± 15.6 mg/l, respectively; p = 0.010) were significantly elevated at the time of MACE. CONCLUSIONS: NLR and CRP were significantly elevated at the time of MACE compared to baseline values in ICI-treated patients. Larger datasets are needed to validate these findings and identify predictors of MACE that can be used in the diagnosis and management of ICI-related iRC.