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Background Immune checkpoint inhibitors (CPIs) exhibit reduced efficacy in PD-L1 low gastroesophageal tumors. These tumors also have correspondingly lower levels of immune infiltrate relative to high PD-L1 tumors. Dendritic cells (DCs) orchestrate anti-tumor immunity and, once activated, mediate downstream T-cell and immune activation to enact tumor control. DC activation can be triggered by exposure to extracellular adenosine triphosphate (eATP) elevated by chemotherapy-driven immunogenic cell death (ICD). Inhibition of the ATPase CD39 by AB598, a therapeutic anti-CD39 antibody, increases local levels of immunostimulatory eATP and concomitant DC activation. Here we report on the development of an ATP signature and its application in assessing the effects of eATP-elevating therapies.Methods Monocyte-derived dendritic cells (moDCs) were exposed to combinations of A549 cells, docetaxel, AB598 or its isotype control, and ATP, followed by RNA-sequencing. An ATP signature was generated from genes upregulated by exogenously added ATP in the presence of AB598 and its specificity was validated in an independent in vitro DC experiment. In parallel, the relationship between PD-L1 mRNA levels and DC abundance, assessed by lineage genes, was evaluated in TCGA gastroesophageal (GEC) datasets.1 The effects of ICD chemotherapy on DC abundance and ATP signature were evaluated in data from 24 patients in the PERFECT trial,2 which evaluated neoadjuvant carboplatin, paclitaxel, and concurrent radiotherapy (CROSS) with atezolizumab in resectable esophageal cancer (ESCA).Results By comparing mRNA from moDCs cultured with exogenous ATP in the presence or absence of AB598, we derived a 23-gene ATP signature. This signature was upregulated in moDCs exposed to conditions promoting eATP release, including cocultures with A549, docetaxel, and AB598. The signature was independent of adenosine signaling when assessed in DCs treated with the adenosine receptor agonist NECA. Furthermore, genes in the ATP signature significantly overlapped with DC maturation pathways and were increased in mature vs immature DCs. Analysis of TCGA GEC patients revealed lower expression of DC lineage genes in PD-L1 low patients. 1 Expression of the DC lineage genes, CD39 mRNA, and ATP signature were increased in ESCA patients treated with the eATP-generative CROSS regimen.Conclusions While PD-L1 mRNA low GEC tumors in TCGA exhibit lower expression of DC lineage genes, ICD chemoradiotherapy increased both the levels of these genes and the ATP signature in ESCA. This suggests that PD-L1 low patients may derive benefit from inhibiting CD39 and preserving the eATP generated by ICD chemotherapies that will increase DC activation and result in an enhanced immune response.References The results shown here are in whole or part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga.Van den Ende T, de Clercq NC, van Berge Henegouwen MI, Gisbertz SS, Geijsen ED, Verhoeven RHA, Meijer SL, Schokker S, Dings MPG, Bergman JJGHM, Haj Mohammad N, Ruurda JP, van Hillegersberg R, Mook S, Nieuwdorp M, de Gruijl TD, Soeratram TTD, Ylstra B, van Grieken NCT, Bijlsma MF, Hulshof MCCM, van Laarhoven HWM. Neoadjuvant chemoradiotherapy combined with atezolizumab for resectable esophageal adenocarcinoma: a single-arm phase II feasibility trial (PERFECT). Clin Cancer Res. 2021 Jun 15;27(12):3351–3359. doi: 10.1158/1078-0432.CCR-20-4443. Epub 2021 Jan