Ras-p53 genomic cooperativity as a model to investigate mechanisms of innate immune regulation in gastrointestinal cancers

Despite increasingly thorough mechanistic understanding of the dominant genetic drivers of gastrointestinal (GI) tumorigenesis (e.g., Ras/Raf, TP53, etc.), only a small proportion of these molecular alterations are therapeutically actionable. In an attempt to address this therapeutic impasse, our group has proposed an innovative extreme outlier model to identify novel cooperative molecular vulnerabilities in high-risk GI cancers which dictate prognosis, correlate with distinct patterns of metastasis, and define therapeutic sensitivity or resistance. Our model also proposes comprehensive investigation of their downstream transcriptomic, immunomic, metabolic, or upstream epigenomic cellular consequences to reveal novel therapeutic targets in previously “undruggable” tumors with high-risk genomic features. Leveraging this methodology, our and others’ data reveal that the genomic cooperativity between Ras and p53 alterations is not only prognostically relevant in GI malignancy, but may also represent the incipient molecular events that initiate and sustain innate immunoregulatory signaling networks within the GI tumor microenvironment, driving T-cell exclusion and therapeutic resistance in these cancers. As such, deciphering the unique transcriptional programs encoded by Ras-p53 cooperativity that promote innate immune trafficking and chronic inflammatory tumor-stromal-immune crosstalk may uncover immunologic vulnerabilities that could be exploited to develop novel therapeutic strategies for these difficult-to-treat malignancies

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

Abstract 2513: MDSC-derived TNF is a novel regulator of T-cell dysfunction in pancreatic cancer

Abstract Introduction: Abundance of myeloid-derived suppressor cells (MDSC) and a dysfunctional T-cell compartment are defining hallmarks of therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC). Using congenic in vivo murine models to phenocopy extremes of T-cell enrichment or exclusion, we sought to interrogate central MDSC-mediated mechanisms that govern immune tolerance in PDAC. Methods: Orthotopically implanted T-cell-excluded (Tcelllo) vs T-cell-enriched (T-cellhi) congenic KPC tumors, and intratumoral Ly6G+F4/80- MDSCs from both clones, were subjected to RNA sequencing. Ex vivo co-cultures evaluated the effects of intratumoral MDSC on splenic T-cells. Orthotopically injected KPC-T-celllo mice were treated with etanercept vs. vehicle, and immunophenotyping via flow cytometry was performed. Results: RNA-seq of KPC T-celllo vs. T-cellhi tumors revealed enrichment of myeloid immunoregulatory pathways, and downregulation of leukocyte activation/cytotoxicity pathways. Flow cytometry revealed a dramatic increase in MDSCs infiltrating KPC-Tcelllo tumors (P<0.001). To decipher MDSC-intrinsic mechanisms associated with T-cell exclusion, RNA-seq of MDSCs infiltrating T-cellhi clones revealed relative downregulation of MAPK signaling, and cytokine profiling of MDSCs conditioned with MAPK inhibitor trametinib revealed marked reduction in TNF secretion. Confocal microscopy confirmed striking decrease in TNF in MDSCs isolated from KPC- T-cellhi vs. Tcelllo tumors. Ex vivo MDSC-T-cell co-cultures significantly attenuated T-cell proliferation and activation (via IFN-γ release) while favoring T-cell apoptosis, which could be rescued by pre-conditioning MDSCs with either etanercept (TNFR2 decoy receptor) or MAPK pathway inhibitors. Orthotopically injected KPC-T-celllo tumor-bearing mice treated with etanercept demonstrated a remodeled TME vs. vehicle-treated mice, with attenuation in MDSC trafficking, enrichment in CD4+/CD8+ T-cell infiltration, and reduction in T-cell exhaustion. Conclusion: MDSC-derived TNF regulates T-cell dysfunction in PDAC via a MAPK-dependent mechanism. Compartment-specific inhibition of TNF may be a provocative strategy to overcome immune tolerance in PDAC. Citation Format: Anna Bianchi, Iago De Castro Silva, Nilesh U. Deshpande, Siddharth Mehra, Samara Singh, Austin R. Dosch, Vanessa T. Garrido, Christine I. Rafie, Nagaraj Nagathihalli, Nipun Merchant, Jashodeep Datta. MDSC-derived TNF is a novel regulator of T-cell dysfunction in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2513

Abstract C059: Targeting tumor intrinsic p38 MAPK signaling to block IL-1α-mediated inflammatory tumor-stromal crosstalk in pancreatic cancer

Abstract Introduction: Pancreatic cancer (PDAC) remains a major therapeutic challenge due to its innate and acquired chemoresistance. Activating KRAS mutations, a hallmark of PDAC, mediate autocrine effects and crosstalk within the tumor microenvironment (TME), by inducing cytokines and chemokines that promote a pro-inflammatory and immunosuppressive stroma. We have identified KRAS-driven interleukin-1α (IL-1α) as a critical mediator of the inflammatory response due to its pleiotropic effects on cancer-associated fibroblast (CAF) activation and immune evasion, however the mechanisms that regulate IL-1α production remain poorly understood. In our efforts to identify targetable kinase pathways downstream of KRAS that are involved in IL-1α expression, we identified p38 stress-associated MAPK α (p38α MAPK) as a key regulatory pathway involved in IL-1α production in PDAC tumor cells. Methods: KRAS-mutant tumor cells were treated with pharmacologic inhibitors to pathways downstream KRAS and IL1A levels determined in response by qPCR. Mutant KRAS G12D plasmid was overexpressed in mouse embryonic fibroblasts, and IL-1α levels were determined in response by qPCR and ELISA with and without p38 inhibition. Inhibition of phosphorylated p38 MAPK was achieved pharmacologically with ARRY-614 and genetically with an shRNA lentiviral system in human and murine PDAC cell lines. ChIP-qPCR was performed on a human PDAC cell line with and without p38 MAPK inhibition. Tumor cells pre-treated with ARRY-614 were cocultured with human pancreatic stellate cells, and inflammatory CAF genes were measured by qPCR. Ptf1aCre/+;LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice, a highly aggressive PDAC genetically engineered mouse model (GEMM), were treated daily with ARRY-614, chemotherapy, or combination therapy for downstream analysis. Results: Both pharmacologic and genetic inhibition of p38 MAPK significantly reduces IL1A transcription and protein levels in multiple human and murine PDAC tumor cell lines. Furthermore, p38 MAPK inhibition reduces binding of NF-kB to the IL1A promoter and prevents IL-1α-mediated activation of CAFs, characterized by a reduction in pro-inflammatory genes. Lastly, ARRY-614 in combination with chemotherapy significantly reduces overall tumor burden and favorably remodels the tumor microenvironment in a PDAC GEMM. Conclusions: These findings provide a compelling rationale to explore p38 MAPK inhibition in tumor cells as a novel treatment strategy to suppress IL-1α-mediated stromal activation and to combine p38 MAPK inhibition with chemotherapy to overcome therapeutic resistance through modulation of the stromal and immune microenvironment in PDAC. Citation Format: Samara P. Singh, Austin R. Dosch, Siddharth Mehra, Iago de Castro Silva, Anna Bianchi, Vanessa Tonin-Garrido, Nilesh U. Deshpande, Zhiqun Zhou, Nagaraj Nagathihalli, Jashodeep Datta, Nipun B. Merchant. Targeting tumor intrinsic p38 MAPK signaling to block IL-1α-mediated inflammatory tumor-stromal crosstalk in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C059

Abstract C021: Granulocytic MDSC-derived NLRP3 inflammasome activation is a novel regulator of inflammatory CAF skewness in pancreatic cancer

Abstract OBJECTIVE: The major drivers of therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC) are myeloid cell-derived signaling that sustains immune tolerance and interleukin-1 (IL1)-mediated inflammatory polarization of cancer-associated fibroblasts (iCAF) which promotes stromal inflammation by elaborating soluble factors (i.e., CXCL1, IL6) that further accelerate myeloid chemotaxis. We have recently shown that granulocytic myeloid-derived suppressor cell (gMDSC)-derived IL1β promotes chemoresistance in PDAC via a CAF:tumor cell IL6:STAT3 axis. Since NLRP3 inflammasome activation is the dominant source of IL1β generation in innate immune cells, we hypothesized that gMDSC-derived NLRP3 inflammasomes are a novel driver of iCAF polarization in PDAC. METHODS: We examine single-cell RNA sequencing (scRNAseq) data in human and PKT/KPC PDAC models to identify cellular source of inflammasome-derived IL1β. Gene set enrichment analysis in bulk RNAseq data and signal transduction studies examined novel pathways associated with inflammasome activation in gMDSCs. gMDSCs treated with/without novel NLRP3 inhibitor SB-NL02 were co-cultured with Il6/Acta2 dual-reporter CAF system in vitro to examine iCAF polarization. ASC oligomerization in intratumoral gMDSCs via confocal microscopy and CAF polarization via flow cytometry were evaluated in mice treated with NLRP3i SB-NL02. RESULTS: scRNAseq in human and PKT/KPC PDAC models revealed gMDSCs as the dominant source of inflammasome activation-derived IL1β. Lineage trajectory reconstruction revealed strong co-expression of Cxcr2, Nlrp3, and Il1β in gMDSC single-cell transcriptomes. Mechanistically, in vitro signal transduction and RNAseq studies revealed activation of gMDSC-intrinsic NLRP3 inflammasomes not only via canonical TLR4-MyD88 signaling but also cooperative regulation via CXCR2-p38 MAPK induction. As such, intratumoral gMDSCs retrieved from mice treated with CXCR2 inhibitor AZD5069 or those from CRISPR-edited KPC-Cxcl1KO (cognate ligand of CXCR2) tumor-bearing mice showed reduced Nlrp3 expression, inflammasome activation, and IL1β generation. To investigate the impact of gMDSC-NLRP3 on iCAF polarization, co-culture of J774M gMDSCs with Il6/Acta2 dual reporter CAFs revealed strong induction of CAF-intrinsic Il6 expression, which was mitigated by treatment of gMDSCs with NLRP3i SB-NL02 or CAFs with IL-1 receptor antagonist Anakinra. Treatment of PKT mice with NLRP3i SB-NL02 reduced primary tumor volume, as well as ASC speck oligomerization and IL1β production in intratumoral Ly6G+ gMDSCs. Moreover, we observed significant attenuation in iCAF (CD31−PDPN+Ly6C+MHCII−) skewness in PKT mice treated with NLRP3i compared with vehicle. CONCLUSIONS: gMDSC-derived NLRP3 inflammasome activation—via cooperative TLR4 and p38 signaling induction—and ensuing IL1β production is a novel regulator of iCAF polarization. Therapeutic approaches—such as gMDSC compartment-specific NLRP3 inhibition—may mitigate stromal inflammation and overcome therapeutic resistance in PDAC. Citation Format: Karthik Rajkumar, Nilesh U. Deshpande, Iago de Castro Silva, Anna Bianchi, Ifeanyichukwu Ogobuiro, Haleh Amirian, Andrews Adams, Vanessa Garrido, Siddharth Mehra, Samara Singh, Nagaraj Nagathihalli, Nipun B. Merchant, Jashodeep Datta. Granulocytic MDSC-derived NLRP3 inflammasome activation is a novel regulator of inflammatory CAF skewness in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C021

Neutrophil-Mediated Stromal-Tumor IL-6/STAT-3 Signaling Underlies the Association between Neutrophil-to-Lymphocyte Ratio Dynamics and Chemotherapy Response in Localized Pancreatic Cancer: A Hybrid Clinical-Preclinical Study

Background: Partial/complete pathologic response following neoadjuvant chemotherapy (NAC) in pancreatic cancer (PDAC) patients undergoing pancreatectomy is associated with improved survival. We sought to determine whether neutrophil-to-lymphocyte ratio (NLR) dynamics predict pathologic response following chemotherapy in PDAC, and if manipulating NLR impacts chemosensitivity in preclinical models and uncovers potential mechanistic underpinnings underlying these effects. Methods: Pathologic response in PDAC patients (n=94) undergoing NAC and pancreatectomy (7/2015-12/2019) was dichotomized as partial/complete or poor/absent. Bootstrap-validated multivariable models assessed associations between pre-chemotherapy NLR (%neutrophils÷%lymphocytes) or NLR dynamics during chemotherapy (ΔNLR=pre-surgery—pre-chemotherapy NLR) and pathologic response, disease-free survival (DFS), and overall survival (OS). To preclinically model effects of NLR attenuation on chemosensitivity, C57BL/6 mice (n=8-10/arm) were orthotopically injected with KrasG12D/+;Trp53fl/+;PdxCre(KPC) cells and 60 randomized to vehicle, NLR-attenuating anti-Ly6G, gemcitabine/paclitaxel, or gemcitabine/paclitaxel+anti-Ly6G treatments. Results: In 94 PDAC patients undergoing NAC (median:4 months), pre-chemotherapy NLR (P<0.001) and ΔNLR attenuation during NAC (P=0.002) were independently associated with partial/complete pathologic response. An NLR score=pre-chemotherapy NLR+ΔNLR correlated with DFS (P=0.006) and OS (P=0.002). Upon preclinical modeling, combining NLR-attenuating anti-Ly6G treatment with gemcitabine/paclitaxel—compared with gemcitabine/paclitaxel or anti-Ly6G alone—not only significantly reduced tumor burden and metastatic outgrowth, but also augmented tumor-infiltrating CD107a+-degranulating CD8+ T-cells (P<0.01) while dampening inflammatory cancer-associated fibroblast (CAF) polarization (P=0.006) and chemoresistant IL-6/STAT-3 signaling in vivo. Neutrophil-derived IL-1β emerged as a novel mediator of stromal inflammation, inducing inflammatory CAF polarization and CAF-tumor cell IL-6/STAT-3 signaling in ex vivo co-cultures. Conclusions: Therapeutic strategies to mitigate neutrophil-CAF-tumor cell IL-1β/IL-6/STAT-3 signaling during NAC may improve pathologic responses and/or survival in PDAC. Competing Interest Statement The authors have declared no competing interest

Abstract C033: KRAS-TP53 cooperativity regulates Cxcl1 to sustain tumor-permissive circuitry via granulocyte-derived CXCR2-TNF signaling in pancreatic cancer

Abstract Objective: We have recently shown that KRAS-TP53 genomic co-alteration is associated with innate immune-enriched and T-cell-excluded tumor microenvironments (TME), chemotherapy resistance, and poor survival in pancreatic ductal adenocarcinoma (PDAC) patients. We sought to define the multi-cellular crosstalk that underlies these effects by dissecting how cancer cell-autonomous transcriptional programs orchestrate tolerogenic circuitries to mediate chemoresistance in KRAS-TP53 cooperative PDAC. Methods: Spatial neighborhood analysis via Imaging Mass Cytometry (IMC) was performed in patient-derived PDAC sections. Immune profiling and bulk RNA-seq in whole tumors, as well as bulk-RNAseq in intratumoral F4/80-Ly6Ghi neutrophilic(PMN)-MDSCs in orthotopic KPC tumors with/without CRISPR/Cas9 editing of Cxcl1 was performed. Effect of TNFR2 inhibition via etanercept on ex vivo co-cultures of intratumoral PMN-MDSC with KPC tumor cells/CAFs and T-cells, as well as in orthotopic KPC models in vivo with/without gemcitabine+paclitaxel chemotherapy was performed. Results: Interrogation of cancer cell transcriptomes and IMC architecture in human tumors reveals disproportionate enrichment of Cxcl1 in KRAS-TP53 co-altered PDAC. IMC-enabled spatial neighborhood analysis in KRAS-TP53 co-altered human PDAC TMEs demonstrates strong spatial contiguity between PanCK+CXCL1+ tumor islands and cognate CD15+CXCR2+ PMN-MDSCs, with exclusion of CD8+ T-cells from tumor cell:PMN-MDSC communities. In murine orthotopic models that phenocopy T-cell excluded human PDAC, genetic silencing of tumor cell-intrinsic Cxcl1 overcomes CD8+ T-cell exclusion and controls tumor growth in a CD8+ T-cell dependent manner in vivo. Transcriptomes from KPC-Cxcl1KO tumors not only reveal enrichment in pathways encoding for T-cell effector activity but also attenuation in pathways related to innate immune function. These immune potentiating effects upon Cxcl1 silencing are driven in large part by reprogramming of trafficking dynamics and immunosuppressive potential in intratumoral CXCR2+ PMN-MDSCs. To identify neutrophil-intrinsic mechanisms that govern remodeling of the TME following Cxcl1 silencing, transcriptomes in intratumoral KPC-Cxcl1KO PMN-MDSCs reveal strong downregulation of MAPK and TNF pathways, with signaling studies implicating a novel Cxcr2-Ikk-Map3k8-Tnf axis. We uncover novel effects of neutrophil-derived TNF in promoting tumor cell-Cxcl1 production, inflammatory CAF polarization, and T-cell dysfunction in ex vivo co-cultures, predominantly via a membraneTNF-TNFR2 dependent mechanism. Systemic TNFR2 inhibition via etanercept not only augments T-cell activation, but also mitigates tumor-wide Cxcl1 production, stromal inflammation, and CAF:tumor cell IL6-STAT3 signaling to improve sensitivity to gemcitabine+paclitaxel chemotherapy in vivo. Conclusion: These data uncover novel tumor-permissive/chemoresistant circuitries in which cancer cell-intrinsic Cxcl1 sustains innate immunoregulatory and tolerogenic signaling via neutrophil-derived TNF in the PDAC TME. Citation Format: Anna Bianchi, Iago De Castro Silva, Nilesh U. Deshpande, Siddharth Mehra, Vanessa T. Garrido, Samara Singh, Christine I. Rafie, Zhou Zhiqun, Ifeanyichukwu C. Ogobuiro, Austin R. Dosch, Nagaraj Nagathihalli, Nipun B. Merchant, Jashodeep Datta. KRAS-TP53 cooperativity regulates Cxcl1 to sustain tumor-permissive circuitry via granulocyte-derived CXCR2-TNF signaling in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C033

Abstract C040: CREB-LIF axis drives immune suppression by promoting macrophage polarization in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) remains an aggressive malignancy, and its unique genetic makeup and immunosuppressive tumor microenvironment (TME) produce a lack of response to current therapies. Previously, we have identified Cyclic AMP Response Element Binding protein 1 (CREB) as an oncogenic transcriptional factor that promotes disease aggressiveness, poor survival, and immune suppression. Based on these, we sought to determine the role of tumor intrinsic CREB in promoting immunosuppressive TME in PDAC. Methods: We have generated a genetically engineered mouse model (GEMM) of pancreas-specific CREB deletion (CREBfl/fl) in LSL-KrasG12D/+; Trp53 R172H/+; Pdx1Cre/+ (KPC) mice that phenocopy human PDAC disease. CRISPR/CAS9-based genomic editing was utilized to ablate CREB (CREBKO) in KPC tumor cells. RNA-sequencing analysis was performed in KPC CREB wild type (CREBWT) vs. CREBKO tumor cells to identify CREB-mediated transcriptomic changes. Chromatin immunoprecipitation (ChIP-qPCR) analysis was performed in KPC tumor cells. Syngeneic orthotopic tumor implantation of these cells was performed in the pancreata of mice. Immunophenotyping was accomplished to assess changes in the immune subsets with CREB deletion invivo. Additionally, these tissues were also processed for single-cell RNA (scRNA) transcriptomics analysis to evaluate changes on different cellular constituents. Results: Pancreas-specific CREB deletion in the KPC GEMM led to a significant reduction in the primary tumor burden, liver metastases, and improved overall survival compared to wild-type KPC. In assessing the immune repercussions of CREB deletion in pancreatic tumors, we observed a decreased infiltration of tumor-promoting CD11b+ F4/80+ CD206+ [M2-like tumor-associated macrophages (TAMs)] and a concomitant increase in the antigen-presenting M1-like macrophages (F4/80+MHC-IIhighCD86high). Additionally, scRNA sequencing analysis within the macrophage compartment in CREBKO tumors revealed significant enrichment of M1 hallmark signaling pathways. Also, CREB ablation in these tumors further facilitated increased infiltration of activated effector memory CD8+ T cells and resulted in enhanced adaptive immune response within the PDAC TME. Mechanistically, RNA transcriptomic-based analysis of CREBKO tumor cells revealed, Leukemia inhibitory factor (LIF) as one of the downstream targets of CREB. ChIP qPCR analysis after CREB1 pulldown confirmed its occupancy on LIF promoter regulatory region. Incubation of macrophages with CREBWT conditioned media in the presence of LIF neutralizing antibody or blocking its receptor expression using EC359 pushed these macrophages towards an M1-like phenotype, confirming its role as a mediator of tumor cell macrophage crosstalk. Conclusion: These findings broaden our understanding of the tumor cell-intrinsic role of CREB and provide new insights into its molecular underpinnings in fostering immunosuppressive profile by promoting skewness of TAMs towards M2 phenotype in PDAC. Citation Format: Siddharth Mehra, Vanessa T. Garrido, Samara Singh, Iago De Castro Silva, Anna Bianchi, Luis A. Nivelo, Nilesh U. Deshpande, Austin R. Dosch, Zhou Zhiqun, Supriya Srinivasan, Christine I. Rafie, Ifeanyichukwu C. Ogobuiro, Xi Chen, Alejandro Villarino, Jashodeep Datta, Nipun B. Merchant, Nagaraj Nagathihalli. CREB-LIF axis drives immune suppression by promoting macrophage polarization in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C040