Abstract Background Esophageal adenocarcinoma is a lethal disease. For locally advanced patients, neoadjuvant chemoradiotherapy followed by surgery is the standard of care. Risk stratification relies heavily on clinicopathologic features, particularly pathologic response, which is inadequate, therefore establishing the need for new and reliable biomarkers for risk stratification. Methods Thirty four patients with locally advanced esophageal adenocarcinoma were analyzed, of which 21 received a CROSS regimen with carboplatin, paclitaxel, and radiation. Capture-based targeted sequencing was performed on the paired baseline and post-treatment samples. Differentially mutated gene analysis between responders and non-responders of treatment was performed to determine predictors of response. A univariate Cox proportional hazard regression was used to examine associations between gene mutation status and overall survival. Results A 3-gene signature, based on mutations in EPHA5, BCL6, and ERBB2, was identified that robustly predicts response to the CROSS regimen. For this model, sensitivity was 84.6% and specificity was 100%. Independently, a 9 gene signature was created using APC, MAP3K6, ETS1, CSF3R, PDGFRB, GATA2, ARID1A, PML, and FGF6, which significantly stratifies patients into risk categories, prognosticating for improved relapse-free ( p = 4.73E-03) and overall survival ( p = 3.325E-06). The sensitivity for this model was 73.33% and the specificity was 94.74%. Conclusion We have identified a 3-gene signature (EPHA5, BCL6, and ERBB2) that is predictive of response to neoadjuvant chemoradiotherapy and a separate prognostic 9-gene classifier that predicts survival outcomes. These panels provide significant potential for personalized management of locally advanced esophageal cancer.
336 Background: The high incidence of esophageal adenocarcinoma (EAC) remains a major clinical challenge as majority of these cases are diagnosed at advanced stages, with poor survival outcomes. Emerging evidence indicate intriguing associations between unique microbial profiles and cancer pathogenesis. More recent data also indicates the potential clinical significance of specific microbial signatures as diagnostic and prognostic biomarkers. However, to date, no studies have systematically interrogated circulating metagenome profiling in EAC patients, particularly as non-invasive, early detection, surveillance and prognostic classifiers that may improve the current management paradigms. Methods: Metagenome sequencing was performed on 81 serum specimens collected from 51 EAC, 10 high grade dysplasia (HGD), 10 Barretts’s esophagus (BE), and 10 gastro-esophageal reflux disease (GERD) patients, respectively. Sequencing reads were classified using Bracken and MetaPhlAn3 to determine relative abundance between various classes. The Linear Discriminant Analysis effect size (LEfSe) method was performed to identify potential conserved and discrete microbial profiles between groups. Logistic regression and Kaplan-Meier analyses were used to build a model according to clinical and metagenomic classifiers to examine the diagnostic and prognostic potential of the identified metagenomic signature. Results: A significant loss of alpha and beta diversity was identified in serum specimens from EAC patients. We observed a shift in microbial taxa between each group – at the phylum, genus, and species level – with Lactobacillus sakei as the most prominent species in GERD vs. other patient groups. Interestingly, LEfSe analysis identified a complete loss of Lactobacillus ( L.Sakei and L. Curvatus), Collinsella stercoris and Bacteroides stercoris, but conversely a significant increase in Escherichia coli in patients with EAC. Finally, we developed a metagenome panel that discriminated EAC from GERD patients with an AUC value of 0.89 (95% CI, 0.78 – 0.95), and this panel in conjunction with the TNM stage was a robust predictor of overall survival (≥24 months; AUC = 0.84 [95% CI 0.66 – 0.92]) and an accompanying hazard ratio (HR) of 6.23 (95% CI, 2.65 – 14.46, P<0.001). Conclusions: This study describes unique blood-based microbial profiles in patients with GERD, BE, HGD, and EAC, that are further utilized to establish a novel circulating diagnostic and prognostic metagenomic signature in EAC.
Abstract Background: Esophageal adenocarcinoma (EAC) is a leading cause of cancer-related mortality. Sitravatinib is a small molecule spectrum-selective tyrosine kinase inhibitor (TKI) that targets TAM receptors (AXL, MERTK and TYRO3), VEGF, PDGF, and c-Kit. Previously, dual inhibition of TAM and VEGF receptors has shown to successfully modulate the tumor immune microenvironment towards a less immunosuppressive state, by depletion of MDSCs and repolarization of TAMs towards a proinflammatory M1 phenotype. In clinical trials, TKIs have shown to improve patient outcomes, alone or in combination with immune checkpoint inhibitors, in multiple solid tumors. In the present study, the antitumor activity of sitravatinib alone and in combination with PD-1 blockade was investigated in a de novo EAC rat model. Methods: Ninety-six rats underwent an end-to-side esophagojejunostomy to induce gastroesophageal reflux, resulting in EAC carcinogenesis. At 32 weeks post-operatively, tumor bearing animals were randomized to a dose of 10mg/kg of sitravatanib (S) or vehicle control (VC), for a total of three 14 day cycles. A PD-1 inhibitor, AUNP-12 (IO), was administered at a dose of 3mg/kg or placebo, on day 12 of each cycle. At 38 weeks post-operatively animals were euthanized. Safety and efficacy was evaluated by on-treatment mortality, MRI, immunofluorescence and qRT PCR. Results: The S+/-IO groups demonstrated a higher mortality when compared to the control groups (p<0.001). Pre- to post-treatment, mean MRI tumor volume decreased by 32% and 73% in the S-IO and S+IO and increased by 90% and 160% in the VC+IO and VC-IO, respectively (p<0.001). Enhanced CD3+CD8+ T-cell densities were observed in the treatment groups when compared to the control groups (p<0.001). Additionally, a higher CD86/CD206 ratio was observed in the S-IO and S+IO groups than in the control groups (p<0.001), indicating a repolarization from M2 to M1 macrophage phenotype. Gene expression analysis of post treatment samples demonstrated upregulation of pro-inflammatory cytokines TNF-α, IFN-γ, IL-1β, IL-6, IL-12 and downregulation of anti-inflammatory cytokines including TGF-β, IL-4, IL-10, and IL-13 in the treated versus control animals (p<0.05). Pre- to post-treatment qRT-PCR levels demonstrated significant inhibition of pathway genes, AXL, AKT, MERTK and PI3K, in the treated animals (p=<0.001). Moreover, pre-treatment AXL levels were significantly higher in major responders (>80% tumor reduction n=8) compared to the non-responders (<25% tumor reduction/progression n=6), in the merged S +/- IO group (p=0.009). Lastly, increased apoptosis and decreased proliferation were confirmed through Cas-3 and Ki-67, respectively, in both treatment groups (p<0.001). Conclusion: This study establishes a promising combination strategy using the multi-gene TKI sitravatinib to overcome PD-1/PD-L1 resistance and potentiate immune checkpoint inhibition in an EAC model. Citation Format: Ryan W. Sweeney, John T. Fitzpatrick, Ashten N. Omstead, Ping Zheng, Anastasia Gorbunova, Patrick L. Wagner, Juliann E. Kosovec, Blair A. Jobe, Ronan J. Kelly, Ali H. Zaidi. Sitravatanib enhances immune checkpoint blockade in a de novo esophageal adenocarcinoma model [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B60.
333 Background: Esophageal adenocarcinoma (EAC) is a leading cause of cancer deaths. Pexidartinib, is a multi-gene tyrosine kinase inhibitor, targeting FLT3, KIT and CSF1R. In particular, pexidartinib by targeting CSF-1R down modulates macrophage mediated pro-survival tumor signaling. Recently, CSF-1R inhibitors have successfully shown to enhance antitumor activity of PD-1/PD-L1 inhibitors by suppressing tumor immune evasion, in multiple solid tumors. Methods: Seventy-two rats underwent an end-to-side esophagojejunostomy to induce gastroesophageal reflux, resulting in EAC carcinogenesis. At 32 weeks postoperatively, tumor bearing animals were randomized to a dose of 30mg/kg of pexidartinib or vehicle control, 5 days on and 2 days off (14 day cycle), for a total of 3 cycles. A PD-1 inhibitor, AUNP-12, at a dose of 3mg/kg or placebo, was administered on day 12 of each cycle. At 38 weeks postoperatively animals were euthanized. Safety and efficacy was evaluated by objective health assessments, pre and posttreatment MRI, RT-PCR, immunofluorescent labeling and a serum cytokine assay. Results: The pexidartinib +/- PD-1 inhibitor (Px+P and Px-P) groups demonstrated no significant difference in daily weight change (p = 0.98) or in mortality (p = 0.59) when compared to vehicle control (V+P and V-P) groups. On post-mortem serum analysis AST (p = 0.01) and ALT (p = 0.001) were elevated, while albumin (p = 0.57) and BUN/creatinine ratio (p = 0.09) were reported within normal ranges, when comparing treatment to placebo groups. Pre to posttreatment, mean MRI tumor volume decreased by 37% and 54% in the Px-P and Px+P animals and increased by 147% and 88% in the V+P and V-P animals, respectively (p = < 0.0001). Overall response rate was 69% and 87% in Px-P and Px+P animals compared to 21% and 13% in V-P and V+P animals, respectively (p = < 0.0001). Downstream gene expression demonstrated upregulation of TNF-α (p = 0.003), IFN-γ (p = 0.002) and IL-6 (p = 0.0001) and downregulation of IL-13 (p = 0.051), IL-10 (p = 0.051), TGF-β (p = 0.024) and Arg-1 (p = 0.0003), in treatment vs. placebo animals. Increased apoptosis (Cas-3 p = 0.0005) and reduced proliferation (Ki-67 p = < 0.0001) were reported in the treatment animals. Higher CD3 + CD8 + T cell densities and CD86/CD206 macrophage ratio was reported in treatment groups compared to placebo (p = < 0.0001). Serum cytokine levels significantly displayed upregulation of TNF-α and IFN-γ and downregulation of IL-4 and IL-10 in the treatment animals compared to the placebo. Conclusions: Overall, this study established a promising combinatorial strategy using a CSF-1R inhibitor to overcome resistance to PD-1/PD-L1 axis blockade in an esophageal cancer model, providing the rationale for future clinical strategies.
408 Background: Esophageal adenocarcinoma (EAC) is a deadly disease with poor prognosis due to limited treatment options. STING is a transmembrane protein that activates the transcription of type I IFN genes, resulting in the stimulation of APCs and enhanced CD8+ T-cell infiltration. Recently, STING agonists have demonstrated durable anticancer activity in solid tumors when used alone and in combination with either chemotherapy, radiation or immunotherapy. In this study we evaluated the efficacy and immunomodulatory effects of STING agonist +/- radiation in an established EAC model. Methods: Esophagojejunostomy was performed on rats to induce reflux leading to the development EAC. At 32 weeks post operatively, rats received either STING (ADU-S100) or placebo (PBS), +/- 16Gy radiation. Drug efficacy was evaluated by pre- and post- treatment MRI, serial biopsies, histology and RT-PCR. Additionally, immunofluorescence was performed using CD8 and PD-L1 antibodies. Results: A comparison of MRIs in the study groups between 32 and 40 weeks demonstrated a mean increase in percentage tumor volume of 76.7 % and 152.4% in the P and P+R arms and a decrease of 30.1 % and 50.8% in the S and S+R arms, respectively (ANOVA test p < 0.0001) Overall, the S+R group demonstrated the best results with maximum mean volume reduction with all cases responding. Downstream gene expression, pre, on, and post- treatment demonstrated significant upregulation of IFNβ, TNFα, IL-6 and CCL-2 in the treatment groups compared with placebo. On and post treatment, radiation alone, ADU-S100 alone and ADU-S100 + radiation groups demonstrated enhanced PD-LI expression, induced by higher densities of IFNγ producing CD8+ T-cells (p < 0.01). Conclusions: ADU-S100 +/- radiation exhibits potent anti-tumor efficacy and a promising immunomodulatory profile in a de novo EAC model providing the rationale for clinical testing, likely concurrently in combination with immune checkpoint inhibitors.
Alcohol provokes different changes in velocity parameters of cardiac electrical activity in patients with alcohol heart disease. These changes enhance electrical heterogenity, which may result in significant arrhythmogenic sequallae. Acute alcohol withdrawal is associated with cardiac arrhythmias, ventricular arrythmias may lead to sudden cardiac death. Purpose: To evaluate diagnostic potential of the first ECG derivative in assessing velocity parameters of cardiac eletrical activity in patients with alcohol heart disease in acute alcohol withdrawal. Methods: A cross-sectional study of men (n=114), alcohol addicted, aged 35-55 years, with acute alcohol withdrawal syndrome. Patients underwent clinical examination, ECG, first ECG derivative. Holter monitoring was performed in 35 patients. Key parameter of first ECG derivative - ventricular activation rate (VAR) was used in analysis. VAR is quantative parameter of QRS detection, algorithm suggested by professor Volkova E.G. (1976). Statistical analysis was performed using t-test, Pearson's correlation and linear regression analyses. Results: Examination demonstrated VAR 38,8±2,2 sec-1 (mean±SD). Holter monitoring revealed atrial fibrillation in 5 (14%), atrial ectopics in 8 (23%), high grade ventricular arrhythmias (grade 3 and above) in 19 patients (54%). Data did not reveal correlation between atrial arrythmias and VAR. Data demonstrated negative correlation between VAR and ventricular arrhythmias, detected with Holter monitoring (r=-0,887, p=0,0001). Linear regression analyses demonstrated significant relationship between VAR and ventricular arrhythmias (adjusted R square=0,78, β=-0,887, p=0,0001). Conclusions: Acute alcohol withdrawal in men with alcohol heart disease is associated with different heart rhythm disorders. Patients with lower ventricular activation rate exhibit different ventricular arrhythmias, including life-threatening. First ECG derivative method allows us to predict ventricular arrhythmias in alcoholics. Assessment of first ECG derivative may be useful in complex evaluation of patients with alcohol heart disease.
335 Background: There is a critical need to develop effective therapeutic approaches for patients with esophageal cancer, a leading cause of cancer mortality. Recently, immune checkpoint blockade (ICB) has demonstrated modest survival benefit in gastroesophageal cancer patients. Epigenetic therapies, including DNA methyltransferases (DNMT) and histone deacetylases (HDACs), have shown to enhance tumor immune recognition by directly upregulating antigen presentation and by activation of type-1 interferon response triggered by cytoplasmic double-stranded RNA, a phenomenon known as viral mimicry. In other cancer models, epigenetic therapy has shown early promise by enhancing the effectiveness of ICB via addictive antiproliferative effects, enhanced immune activation and reversal of T cell exhaustion towards a memory-effector T-cell phenotypes. Methods: Esophagojejunostomy was performed to induce gastroduodenojejunal reflux in rats, leading to development of EAC. At 32 weeks postop, animals were randomized into eight treatment and control groups: placebo ± radiation, epigenetic treatment ± radiation, immunotherapy treatment ± radiation, and concurrent epigenetic and immunotherapy ± radiation. Animals underwent three 14 day cycles of epigenetic treatment or placebo. Each cycle consisted of one week of DNMT treatment or placebo at a dose of 0.5mg/kg, followed by one week of HDAC treatment or placebo at a dose of 2mg/kg. Animals received one 3mg/kg dose of PD1 inhibitor or placebo on day 12 of each cycle. Safety and efficacy were evaluated via objective health assessments, change in tumor volume, immunofluorescent labeling and RT-PCR. Results: Seventy of 74 randomized animals reached study end-point with no significant differences in mortality observed across groups (p = 0.556). There were no significant differences in observed health scores or weights between all groups (p = 0.431 and p = 0.882, respectively). Pre to posttreatment, mean MRI tumor volume increased by 263.1% and 90.9% in the placebo and the placebo plus radiation groups, respectively. The largest tumor reduction was observed in the concurrent treatment groups, 84.5% and 61.8% with and without radiation, respectively. Additionally, independent epigenetic and immunotherapy treatments with or without radiation also exhibited decrease in tumor volume, but to a lesser extent than the concurrent regimens (p = 0.005). Immunofluorescent labeling demonstrated a significant increase in CD3+ CD8+ T cells in all treatment groups compared to placebo (p = < 0.001). Gene expression displayed upregulation of CD206 (p = 0.002), TNFα (p = < 0.001), and Arg1 (p = < 0.001) in treatment arms compared to placebo. Conclusions: This study establishes addictive antitumor efficacy and enhanced immune sensitization for a concurrently sequenced treatment, combining epigenetic therapy and radiation with immunotherapy, to treat EAC.
'%3e%3cg id='b'%3e%3cpath id='a' stroke='%23000' stroke-width='2' d='M-6-25H6m-12 3H6m-12 3H6'/%3e%3cuse xlink:href='%23a' y='44'/%3e%3c/g%3e%3cpath stroke='%23fff' d='M0 17v10'/%3e%3ccircle r='12' fill='%23cd2e3a'/%3e%3cpath fill='%230047a0' d='M0-12A6 6 0 0 0 0 0a6 6 0 0 1 0 12 12 12 0 0 1 0-24z'/%3e%3c/g%3e%3cg transform='rotate(-123.69)'%3e%3cuse xlink:href='%23b'/%3e%3cpath stroke='%23fff' d='M0-23.5v3M0 17v3.5m0 3v3'/%3e%3c/g%3e%3c/svg%3e)
'/%3e%3c/defs%3e%3cpath fill='%23012169' d='M0 0h10080v5040H0z'/%3e%3cpath stroke='%23fff' stroke-width='.6' d='m0 0 6 3m0-3L0 3' clip-path='url(%23b)' transform='scale(840)'/%3e%3cpath stroke='%23e4002b' stroke-width='.4' d='m0 0 6 3m0-3L0 3' clip-path='url(%23c)' transform='scale(840)'/%3e%3cpath stroke='%23fff' stroke-width='840' d='M2520 0v2520M0 1260h5040'/%3e%3cpath stroke='%23e4002b' stroke-width='504' d='M2520 0v2520M0 1260h5040'/%3e%3cg fill='%23fff'%3e%3cuse xlink:href='%23d' x='2520' y='3780'/%3e%3cuse xlink:href='%23a' x='7560' y='4200'/%3e%3cuse xlink:href='%23a' x='6300' y='2205'/%3e%3cuse xlink:href='%23a' x='7560' y='840'/%3e%3cuse xlink:href='%23a' x='8680' y='1869'/%3e%3cuse xlink:href='%23e' x='8064' y='2730'/%3e%3c/g%3e%3c/svg%3e)