bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Integrated Genomic and Transcriptomic Analysis Improves Disease Classification and Risk Stratification of MDS with Ring Sideroblasts
Authors: Todisco Gabriele , Creignou Maria , Bernard Elsa , Björklund Ann-Charlotte , Moura Pedro Luis , Tesi Bianca , Mortera-Blanco Teresa , Sander Birgitta , Jansson Monika , Walldin Gunilla , Barbosa Indira , Reinsbach Susanne E. , Hofman Isabel Juliana , Nilsson Christer , Yoshizato Tetsuichi , Dimitriou Marios , Chang David , Olafsdottir Svannildur , Venckute Larsson Sigita , Tobiasson Magnus , Malcovati Luca , Woll Petter , Jacobsen Sten Eirik W. , Papaemmanuil Elli , Hellström-Lindberg Eva
Published: October 2023

AbstractPurpose:
Ring sideroblasts (RS) define the low-risk myelodysplastic neoplasm (MDS) subgroup with RS but may also reflect erythroid dysplasia in higher risk myeloid neoplasm. The benign behavior of MDS with RS (MDSRS+) is limited to SF3B1-mutated cases without additional high-risk genetic events, but one third of MDSRS+ carry no SF3B1 mutation, suggesting that different molecular mechanisms may underlie RS formation. We integrated genomic and transcriptomic analyses to evaluate whether transcriptome profiles may improve current risk stratification.
Experimental Design:
We studied a prospective cohort of MDSRS+ patients irrespective of World Health Organization (WHO) class with regard to somatic mutations, copy-number alterations, and bone marrow CD34+ cell transcriptomes to assess whether transcriptome profiles add to prognostication and provide input on disease classification.
Results:
SF3B1, SRSF2, or TP53 multihit mutations were found in 89% of MDSRS+ cases, and each mutation category was associated with distinct clinical outcome, gene expression, and alternative splicing profiles. Unsupervised clustering analysis identified three clusters with distinct hemopoietic stem and progenitor (HSPC) composition, which only partially overlapped with mutation groups. IPSS-M and the transcriptome-defined proportion of megakaryocyte/erythroid progenitors (MEP) independently predicted survival in multivariable analysis.
Conclusions:
These results provide essential input on the molecular basis of SF3B1-unmutated MDSRS+ and propose HSPC quantification as a prognostic marker in myeloid neoplasms with RS.
Data from Specific targeting of Multiple Myeloma by Dual Split-signaling Chimeric Antigen Receptors T-cells directed against CD38 and CD138
Authors: van der Schans Jort J. , Wang Ziyu , van Arkel Jennemiek , van Schaik Thijs , Katsarou Afroditi , Ruiter Ruud , Baardemans Thomas , Yuan Huipin , de Bruijn Joost , Zweegman Sonja , van de Donk Niels W.C.J. , Groen Richard W.J. , Themeli Maria , Mutis Tuna
Published: October 2023

Abstract
Purpose: The success of BCMA-specific chimeric antigen receptor (CAR) T-cells illustrates the potential of this novel therapy for multiple myeloma (MM). Nonetheless, broadening CAR T-cell therapy beyond BCMA requires inventive strategies as there are only a few MM- or plasma cell-specific target antigens. We investigated the feasibility of achieving MM specificity by dual-split CD38/CD138 CAR targeting, whereby the stimulatory and costimulatory signals for T-cell activation are split into two separate stimulatory (sCAR) and costimulatory CARs (cCAR). Experimental Design: Using various combinations of CD38 and CD138 sCARs and cCARs with different affinities, we generated several dual-split CAR T-cells and analyzed them for MM-specific effector functions in vitro. The best-functioning CAR T-cells were tested in vivo in a murine xenograft model. Results: We found optimal designs of both CD38sCAR/CD138cCAR and CD138sCAR/CD38cCAR combinations, that effectively lysed MM cells, but spared single CD38 or CD138 positive healthy hematopoietic cells. While the CD38sCAR/CD138cCAR T-cells achieved MM-specific activity solely due to the low affinity of the CD38sCARs, the MM-specific cytotoxicity, cytokine release, and proliferation of CD138sCAR/CD38cCAR T‑cells were established through a true combinatorial stimulatory and costimulatory effect. The most optimal combination comprised a low affinity CD138sCAR combined with a high affinity CD38cCAR. These CD138sCAR/CD38cCAR T-cells also showed dual-antigen specific anti-MM effects in vivo. Importantly, they were also effective against MM cells from daratumumab pre-treated patients with decreased CD38 expression levels. Conclusions: We demonstrate the possibility to specifically target MM cells, even after CD38 targeted therapy, with carefully-designed dual-split CARs directed against CD38 and CD138.
Data from Therapeutic Vaccination against Human Papillomavirus Type 16 for the Treatment of Anal Intraepithelial Neoplasia in HIV+ Men
Authors: Gosens Karien C.M. , van der Burg Sjoerd H. , Welters Marij J.P. , Boekestijn Sanne , Loof Nikki M. , Quint Wim G.V. , van Noesel Carel J.M. , van der Wal Allard C. , Richel Olivier , Krebber Wilhelmus J.T.A. , Melief Cornelis J.M. , de Vries Henry J.C. , Prins Jan M.
Published: October 2023

Abstract
Purpose: Anal cancer is increasing in HIV-positive men who have sex with men (MSM). Treatment options for its precursor, high-grade anal intraepithelial neoplasia (HGAIN), are suboptimal. In this phase I-II dose-finding study we assessed the safety and efficacy of the human papillomavirus type 16 (HPV16) synthetic long peptide vaccine (SLP-HPV-01®) in HIV-positive MSM with HPV16-positive HGAIN. Patients and Methods: Four dosage schedules (1-5-10; 5-10-20; 10-20-40 and 40-40-40-40 mg) of SLP-HPV-01® were administered intradermally with a three-week interval in 10 patients per dose level. In each dose group, five patients also received 1 µg/kg pegylated interferon-alpha-2b subcutaneously. Primary endpoints were safety and regression of HGAIN at 3,6 and 12 months. Results: Eighty-one of 134 screened patients (60%) had HPV16-negative HGAIN lesions, leaving 53 eligible patients. Thirteen patients were excluded, leaving 40 men. The vaccine was well tolerated. One patient developed a generalized rash. The highest dosage level induced the strongest immune responses. There was no indication for stronger reactivity in the IFN-α groups. Up to 18 months of follow-up, 8/38 intention-to-treat patients had a complete clinical and histological response (CR) and one had a partial response (PR) (in total 9/38, 23.7%). At the highest dosage level the clinical response was 4/10 (40%). Stronger immune responses were detected among clinical responders. Conclusions: The highest dose level is safe, immunogenic and associated with clinical responses to HPV16-induced lesions. However, as the majority of HGAIN is caused by the other HPV types, further studies should aim at pan-HPV vaccination to prevent or treat HGAIN.
Data from HDACi-dependent Microenvironmental Normalization Overcomes Tumor Burden–induced T-cell Exhaustion
Authors: Nguyen Andrew , Brown Dominique , Krishnan Ramya , Bastin Donald , Deng Li , Chen Lan , Salem Omar , Walsh Scott R. , Bramson Jonathan L. , Wan Yonghong
Published: October 2023

AbstractPurpose:
T-cell exhaustion limits immunotherapy for the treatment of solid tumors. Although immune checkpoint blockade and adoptive T-cell therapy (ACT) can mediate tumor regression, their potency is often determined by tumor burden. Here, we identified tumor burden–related pathway changes that are conducive to T-cell exhaustion. We then determined whether microenvironmental reprogramming via epigenetic modulation could reverse T-cell exhaustion and improve immunotherapeutic responsiveness.
Experimental Design:
We developed a murine syngeneic tumor model wherein an increased burden ablated therapeutic responsiveness to ACT, which corresponded with systemic induction of T-cell exhaustion. Transcriptome analysis of these large tumors allowed us to characterize changes to immunosuppressive pathway expression during class I histone deacetylase inhibitor MS-275 treatment. We then measured the therapeutic impact of MS-275 during ACT and assessed T-cell exhaustion by transcriptome/phenotypic analysis.
Results:
ACT durably regressed small tumors but failed to control large tumors, which were associated with systemic T-cell exhaustion and ablation of T-cell responses. Large tumors were defined by an immunosuppressive pathway signature. MS-275 reversed this pathway signature and promoted durable regression of large tumors during ACT. Prototypical exhaustion marker Tim-3 was selectively upregulated in transferred T cells despite displaying a reduced exhaustion signature. Instead, we observed enhanced activation-dependent signaling correlating with enrichment of the IL2–STAT5 signaling axis. Activated CD8+ T-cell responses were predominantly skewed toward terminal effector cell–like CD44+ Tim-3hi TCF1− CD127− KLRG1+ differentiation.
Conclusions:
Tumor burden–induced pathway changes can be reversed through epigenetic reprogramming, enabling the conversion from T-cell exhaustion to effector lineage differentiation.
Data from Efficacy and Safety of Stereotactic Body Radiotherapy Combined with Camrelizumab and Apatinib in Patients with Hepatocellular Carcinoma with Portal Vein Tumor Thrombus
Authors: Hu Yue , Zhou Min , Tang Jing , Li Shuang , Liu Hongli , Hu Jianli , Ma Hong , Liu Junli , Qin Tingting , Yu Xiongjie , Chen Yongshun , Peng Jin , Zou Yanmei , Zhang Tao , Xue Jun
Published: October 2023

AbstractPurpose:
This study aimed to evaluate the efficacy and safety of camrelizumab plus apatinib with or without stereotactic body radiotherapy (SBRT) as first-line therapy for patients with hepatocellular carcinoma (HCC) with portal vein tumor thrombus (PVTT).
Patients and Methods:
This is a multicenter, open-label, noncomparative, randomized trial that recruited patients with HCC with type II/III/IV PVTT, who had not previously received systemic therapy. Patients were randomly assigned (2:1) to receive camrelizumab (200 mg, every 3 weeks) and apatinib (250 mg, every day) with or without SBRT [95% planning target volume (PTV), 36–40 Gy/6–8 Gy]. The primary endpoint was overall survival (OS), and the secondary endpoints were progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR), duration of response, time to progression, and safety.
Results:
Sixty patients were enrolled and randomly assigned to two prospective cohorts. Median OS were 12.7 months [95% confidence interval (CI), 10.2–not available (NA)] and 8.6 months (95% CI, 5.6–NA), and median PFS were 4.6 months (95% CI, 3.3–7.0) and 2.5 months (95% CI, 2.0–7.6) for the SBRT and non-SBRT cohorts, respectively. The ORR and DCR were 47.5% and 72.5% in the SBRT cohort, and 20.0% and 40.0% in the non-SBRT cohort. The most common treatment-related adverse events of any grade were hypertension (55.0%), hand-foot syndrome (51.7%), and leukopenia (50.0%). Grade ≥ 3 was reported in 13 (21.7%) patients.
Conclusions:
First-line treatment with camrelizumab–apatinib combined with or without SBRT showed clinical benefits in patients with HCC with PVTT, with an acceptable safety profile. Thus, these combination regimens may be potential options for such patients.
Data from Immune Marker Spatial Distribution and Clinical Outcome after PD-1 Blockade in Mismatch Repair–deficient, Advanced Colorectal Carcinomas
Authors: Saberzadeh-Ardestani Bahar , Graham Rondell P. , McMahon Sara , Ahanonu Eze , Shi Qian , Williams Crystal , Hubbard Antony , Zhang Wenjun , Muranyi Andrea , Yan Dongyao , Jin Zhaohui , Shanmugam Kandavel , Sinicrope Frank A.
Published: October 2023

AbstractPurpose:
Targeting the programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) interaction has led to durable responses in fewer than half of patients with mismatch repair–deficient (MMR-d) advanced colorectal cancers. Immune contexture, including spatial distribution of immune cells in the tumor microenvironment (TME), may predict immunotherapy outcome.
Experimental Design:
Immune contexture and spatial distribution, including cell-to-cell distance measurements, were analyzed by multiplex immunofluorescence (mIF) in primary colorectal cancers with d-MMR (N = 33) from patients treated with anti–PD-1 antibodies. By digital image analysis, density, ratio, intensity, and spatial distribution of PD-L1, PD-1, CD8, CD3, CD68, LAG3, TGFβR2, MHC-I, CD14, B2M, and pan-cytokeratin were computed. Feature selection was performed by regularized Cox regression with LASSO, and a proportional hazards model was fitted to predict progression-free survival (PFS).
Results:
For predicting survival among patients with MMR-d advanced colorectal cancer receiving PD-1 blockade, cell-to-cell distance measurements, but not cell densities or ratios, achieved statistical significance univariately. By multivariable feature selection, only mean number of PD-1+ cells within 10 μm of a PD-L1+ cell was significantly predictive of PFS. Dichotomization of this variable revealed that those with high versus low values had significantly prolonged PFS [median not reached (>83 months) vs. 8.5 months (95% confidence interval (95% CI), 4.7–NR)] with a median PFS of 28.4 months for all patients [adjusted HR (HRadj) = 0.14; 95% CI, 0.04–0.56; P = 0.005]. Expression of PD-1 was observed on CD8+ T cells; PD-L1 on CD3+ and CD8+ T lymphocytes, macrophages (CD68+), and tumor cells.
Conclusions:
In d-MMR colorectal cancers, PD-1+ to PD-L1+ receptor to ligand proximity is a potential predictive biomarker for the effectiveness of PD-1 blockade.
Data from Molecular Results and Potential Biomarkers Identified from the Phase 3 MILO/ENGOT-ov11 Study of Binimetinib versus Physician Choice of Chemotherapy in Recurrent Low-Grade Serous Ovarian Cancer
Authors: Grisham Rachel N. , Vergote Ignace , Banerjee Susana , Drill Esther , Kalbacher Elsa , Mirza Mansoor Raza , Romero Ignacio , Vuylsteke Peter , Coleman Robert L. , Hilpert Felix , Oza Amit M. , Westermann Anneke , Oehler Martin K. , Pignata Sandro , Aghajanian Carol , Colombo Nicoletta , Cibula David , Moore Kathleen N. , del Campo Josep M. , Berger Regina , Marth Christian , Sehouli Jalid , O'Malley David M. , Churruca Cristina , Kristensen Gunnar , Clamp Andrew , Farley John , Iyer Gopa , Ray-Coquard Isabelle , Monk Bradley J.
Published: October 2023

AbstractPurpose:
We present the results of a post hoc tumor tissue analysis from the phase 3 MILO/ENGOT-ov11 study (NCT01849874).
Patients and Methods:
Mutation/copy-number analysis was performed on tissue obtained pre-randomization. The Kaplan–Meier method was used to estimate progression-free survival (PFS). Unbiased univariate analysis, Cox regression, and binary logistic regression were used to test associations between mutation status and outcomes, including PFS and binary response by local RECIST 1.1.
Results:
MILO/ENGOT-ov11 enrolled 341 patients, ranging in age from 22 to 79, from June, 2013 to April, 2016. Patients were randomized 2:1 to binimetinib or physician's choice of chemotherapy (PCC). The most commonly altered gene was KRAS (33%). In 135 patients treated with binimetinib with response rate (RR) data, other detected MAPK pathway alterations included: NRAS (n = 11, 8.1%), BRAF V600E (n = 8, 5.9%), RAF1 (n = 2, 1.5%), and NF1 (n = 7, 5.2%). In those with and without MAPK pathway alterations, the RRs with binimetinib were 41% and 13%, respectively. PFS was significantly longer in patients with, compared with those without, MAPK pathway alterations treated with binimetinib [HR, 0.5; 95% confidence interval (CI) 0.31–0.79]. There was a nonsignificant trend toward PFS improvement in PCC-treated patients with MAPK pathway alterations compared with those without (HR, 0.82; 95% CI, 0.43–1.59).
Conclusions:
Although this hypothesis-generating analysis is limited by multiple testing, higher RRs and longer PFS were seen in patients with low-grade serous ovarian cancer (LGSOC) treated with binimetinib, and to a lesser extent in those treated with PCC, who harbored MAPK pathway alterations. Somatic tumor testing should be routinely considered in patients with LGSOC and used as a future stratification factor.
Data from Axicabtagene Ciloleucel in Combination With the 4-1BB Agonist Utomilumab in Patients With Relapsed/Refractory Large B-Cell Lymphoma: Phase 1 Results From ZUMA-11
Authors: Jain Michael D. , Miklos David B. , Jacobson Caron A. , Timmerman John M. , Sun Jennifer , Nater Jenny , Fang Xiang , Patel Ankit , Davis Madison , Heeke Darren , Trinh Tan , Mattie Mike , Neumann Frank , Kim Jenny J. , To Christina , Filosto Simone , Reshef Ran
Published: October 2023

Abstract
Purpose: Chimeric antigen receptor (CAR) T-cell therapies have shown clinical benefit for patients with relapsed/refractory (R/R) large B-cell lymphoma (LBCL), yet approximately 60% of patients do not respond or eventually relapse. We investigated the safety and feasibility of the CD19-directed CAR T-cell therapy axicabtagene ciloleucel (axi-cel) in combination with the 4-1BB agonist antibody utomilumab as an approach to improve efficacy of CAR T-cell therapy. Methods: In Phase 1 of the single-arm ZUMA-11 trial, patients with R/R LBCL received a single axi-cel infusion (target dose: 2×106 cells/kg) plus utomilumab 10–200 mg intravenously every 4 weeks for up to 6 months in a dose-escalation design. The primary endpoint was incidence of dose-limiting toxicities (DLTs) with utomilumab. Key secondary endpoints were safety, antitumor activity, pharmacokinetics, and pharmacodynamics. Results: No DLTs were observed among patients treated with axi-cel and utomilumab (n=12). Grade ≥3 adverse events occurred in 10 patients (83%); none were Grade ≥3 cytokine release syndrome or neurologic events. The objective response rate was 75% and 7 patients (58%) had a complete response. Peak CAR T-cell levels increased in a utomilumab dose-dependent manner up to 100 mg. Patients who received utomilumab 100 mg had persistently increased CAR T cells on Days 57–168 compared with other dose levels. Utomilumab was associated with dose-dependent increases in IL-2, IFNγ, and IL-10. Conclusions: Utomilumab-mediated 4-1BB agonism combined with axi-cel therapy had a manageable safety profile. Dual 4-1BB and CD28 costimulation is a feasible therapeutic approach that may enhance CAR T-cell expansion in patients with LBCL.
Data from Safety, immunologic and clinical activity of Durvalumab in combination with Olaparib or Cediranib in advanced leiomyosarcoma: Results of the DAPPER clinical trial
Authors: Salawu Abdulazeez , Wang Ben X. , Han Ming , Geady Caryn , Heirali Alya , Berman Hal K. , Pfister Thomas D. , Hernando-Calvo Alberto , Al-Ezzi Esmail Mutahar. , Stayner Lee-Anne , Gupta Abha A. , Ayodele Olubukola , Lam Bernard , Hansen Aaron R. , Spreafico Anna , Bedard Philippe L. , Butler Marcus O. , Avery Lisa , Coburn Bryan , Haibe-Kains Benjamin , Siu Lillian L. , Abdul Razak Albiruni R.
Published: October 2023

Abstract
Purpose: Non-inflamed (cold) tumors such as leiomyosarcoma (LMS) do not benefit from immune checkpoint blockade (ICB) monotherapy. Combining ICB with angiogenesis-, or poly-ADP ribose polymerase (PARP) inhibitors may increase tumor immunogenicity by altering the immune cell composition of the tumor microenvironment (TME). The DAPPER phase II study evaluated the safety, immunologic, and clinical activity of ICB-based combinations in pre-treated LMS patients. Patients and Methods: Patients were randomized to receive durvalumab 1500mg IV q4w with either Olaparib 300mg bid PO (Arm A) or Cediranib 20mg qd PO on 5 days/week (Arm B) until unacceptable toxicity or disease progression. Paired tumor biopsies, serial radiological assessments and stool collections were performed. Primary endpoints were safety, and immune cell changes in the TME. Objective responses and survival were correlated with transcriptomic, radiomic and microbiome parameters. Results: Among 30 heavily pre-treated patients (15 on each arm), grade ≥ 3 toxicity occurred in 3 (20%) and 2 (13%) on Arms A and B, respectively. On arm A, 1 patient achieved partial response (PR) with increase in CD8 T-cells and macrophages in the TME during treatment, while 4 had stable disease ≥ 6 months (SD-6m). No patients on Arm B achieved PR or SD-6m. Transcriptome analysis showed that baseline M1-macrophage and B-cell activity were associated with overall survival. Conclusions: Durvalumab plus olaparib increased immune cell infiltration of TME with clinical benefit in some LMS patients. Baseline M1-macrophage and B-cell activity may identify LMS patients with favorable outcomes on immunotherapy and should be further evaluated.
Asymptomatic Patients With Brugada ECG Pattern: Long-Term Prognosis From a Large Prospective Study
Authors: Gaita Fiorenzo , Cerrato Natascia , Giustetto Carla , Martino Annamaria , Bergamasco Laura , Millesimo Michele , Barbonaglia Lorella , Carvalho Paula , Caponi Domenico , Saglietto Andrea , Bonacchi Giacomo , Bianchi Francesca , Silvetti Elisa , Crescenzi Cinzia , Canestrelli Stefano , De Maio Melissa , De Ferrari Gaetano Maria , Musumeci Giuseppe , Rametta Francesco , Scaglione Marco , Calò Leonardo
Published: October 2023

BACKGROUND:

Brugada syndrome poses significant challenges in terms of risk stratification and management, particularly for asymptomatic patients who comprise the majority of individuals exhibiting Brugada ECG pattern (BrECG). The aim of this study was to evaluate the long-term prognosis of a large cohort of asymptomatic patients with BrECG.

METHODS:

Asymptomatic patients with BrECG (1149) were consecutively collected from 2 Italian centers and followed-up at least annually for 2 to 22 years. For the 539 asymptomatic patients (men, 433 [80%]; mean age, 46±13 years) with spontaneous type 1 documented on baseline ECG (87%) or 12-lead 24-hour Holter monitoring (13%), an electrophysiologic study (EPS) was proposed; for the 610 patients with drug-induced–only type 1 (men, 420 [69%]; mean age, 44±14 years), multiple ECGs and 12-lead Holter were advised in order to detect the occurrence of a spontaneous type-1 BrECG. Arrhythmic events were defined as sudden death or documented ventricular fibrillation or tachycardia.

RESULTS:

Median follow-up was 6 (4–9) years. Seventeen (1.5%) arrhythmic events occurred in the overall asymptomatic population (corresponding to an event-rate of 0.2% per year), including 16 of 539 (0.4% per year) in patients with spontaneous type-1 BrECG and 1 of 610 in those with drug-induced type-1 BrECG (0.03% per year;
P
<0.001). EPS was performed in 339 (63%) patients with spontaneous type-1 BrECG. Patients with spontaneous type-1 BrECG and positive EPS had significantly higher event rates than patients with negative EPS (7 of 103 [0.7% per year] versus 4 of 236 [0.2% per year];
P
=0.025). Among 200 patients who declined EPS, 5 events (0.4% per year) occurred. There was 1 device-related death.

CONCLUSIONS:

The entire population of asymptomatic patients with BrECG exhibits a relatively low event rate per year, which is important in view of the long life expectancy of these young patients. The presence of spontaneous type-1 BrECG associated with positive EPS identifies a subgroup at higher risk. Asymptomatic patients with drug-induced–only BrECG have a minimal arrhythmic risk, but ongoing follow-up with 12-lead Holter monitoring is recommended to detect the appearance of spontaneous type-1 BrECG pattern.
Geographical Requirements for the Applicability of the Results of the RACECAT Study to Other Stroke Networks
Authors: Schuler Florian A. F. , Ribó Marc , Dequatre‐Ponchelle Nelly , Rémi Jan , Dobrocky Tomas , Goeldlin Martina B. , Gralla Jan , Kaesmacher Johannes , Meinel Thomas R. , Mordasini Pasquale , Seiffge David J. , Fischer Urs , Arnold Marcel , Kägi Georg , Jung Simon
Published: October 2023

Background

The RACECAT (Transfer to the Closest Local Stroke Center vs Direct Transfer to Endovascular Stroke Center of Acute Stroke Patients With Suspected Large Vessel Occlusion in the Catalan Territory) trial was the first randomized trial addressing the prehospital triage of acute stroke patients based on the distribution of thrombolysis centers and intervention centers in Catalonia, Spain. The study compared the drip‐and‐ship with the mothership paradigm in regions where a local thrombolysis center can be reached faster than the nearest intervention center (equipoise region). The present study aims to determine the population‐based applicability of the results of the RACECAT study to 4 stroke networks with a different degree of clustering of the intervention centers (clustered, dispersed).

Methods and Results

Stroke networks were compared with regard to transport time saved for thrombolysis (under the drip‐and‐ship approach) and transport time saved for endovascular therapy (under the mothership approach). Population‐based transport times were modeled with a local instance of an openrouteservice server using open data from OpenStreetMap.The fraction of the population in the equipoise region differed substantially between clustered networks (Catalonia, 63.4%; France North, 87.7%) and dispersed networks (Southwest Bavaria, 40.1%; Switzerland, 40.0%). Transport time savings for thrombolysis under the drip‐and‐ship approach were more marked in clustered networks (Catalonia, 29 minutes; France North, 27 minutes) than in dispersed networks (Southwest Bavaria and Switzerland, both 18 minutes).

Conclusions

Infrastructure differences between stroke networks may hamper the applicability of the results of the RACECAT study to other stroke networks with a different distribution of intervention centers. Stroke networks should assess the population densities and hospital type/distribution in the temporal domain before applying prehospital triage algorithms to their specific setting.
Abstract P1001: Comprehensive “Tool Box” For Delineating Adrenergic Receptor Function
Authors: Elmquist Joel , Caron Alexander , Wyler Steven , Lee Syann , Choi Yun-Hee , Lord Caleb , Liu Chen , Fujikawa Teppei
Published: October 2023

Sympathetic regulation of the cardiovascular system is required to maintain cardiac output and vascular tone in response to varied physiological stressors. This includes the release norepinepherine and epinephrine. Nor/epinephrine acts on nine, evolutionarily conserved, adrenergic, G-protein coupled receptors. Three alpha 1 (
Adra1
), 3 alpha 2 (
Adra2
), and 3 beta (
Adrb
) receptors. Understanding the distribution and cell-specific function of each of these receptors may lead to new targeted therapies for cardiovascular disease. To delineate the relative roles of each of the 9 adrenergic receptors, we are taking a genetic approach to study these receptors in mice; specifically, by using the Cre-loxP system to target the varied receptors in a tissue-specific manner. Unfortunately, there is a dearth of such tools to study these receptors. We therefore are in the process of generating three sets of tools for each of the 9 adrenergic receptors using the CRISPR-Cas9 method. 1. Adrenergic receptor floxed mice. 2. Adrenergic receptor reactivation (transcriptional blocking) mice and 3. Adrenergic receptor Cre mice. Thus far, we have generated
Adra1a
,
Adra1b
,
Adra1d
,
Adra2a
,
Adrb2
, and
Adrb3
floxed mice by inserting loxP sites flanking at least one exon near the 5’ end of the gene. The floxed mice were crossed to a germline-Cre (CMV-Cre), generating mice with whole-body loss of function alleles. Complementing the conditional loss of function alleles, we are also generating reactivation alleles by inserting a loxP-flanked transcriptional blocking cassette (TB) in each of the 9 endogenous adrenergic receptor genes. These TB alleles will allow us to reactivate endogenous expression of each adrenergic receptor in a Cre-dependent manner in an otherwise global knockout background. This model will allow us to identify tissues which are
sufficient
for the function of each adrenergic receptor. Finally, the ires-Cre models will allow for targeting of each adrenergic receptor-expressing cells with genetic reporters, and physiological studies using chemo and/or optogenetics. These new tools will assist in identifying the site of action and physiological function of various receptor subtypes and provide mechanistic insights into adrenergic receptor signaling.
Abstract P1031: An Optogenetic Approach To Study Atrial-fibrillation-associated Remodelling In Atrial Engineered Human Myocardium
Authors: Popp Fiona , Rubio Tony , Seibertz Fitzwilliam , Liutkute Aiste , Bruegmann Tobias , Zimmermann Wolfram H , Shi Runzhu , Diaz Nuria , Voigt Niels
Published: October 2023

Background:
Atrial fibrillation (AF) is the most common clinically reported arrhythmia. It is associated with electrophysiological and structural alterations that promote the maintenance of the disease. The limited longevity of human cardiac samples has historically restricted mechanistic investigation into these remodeling processes of human atrial cardiomyocytes and has hampered antiarrhythmic-therapy development.

Objective:
We aim to assess if classical AF-associated remodeling events can be elicited in atrial engineered human myocardium (EHM) constructed from atrial subtype-specific induced pluripotent stem cell derived cardiomyocytes (iPSC-CM).

Methods:
To allow for long-term optical tachypacing at 3 Hz (TP), EHM were generated from cells expressing the light-gated ion-channel f-Chrimson (CRISPR-Cas9 technique). 24-hour or 7 day TP was applied to EHMs. Action potentials (AP) were acquired using the sharp microelectrode impaling technique.

Results:
24-hour TP of atrial EHMs resulted in action potential shortening (APD
90
: 215.7±4.9 vs. 243.7±7.3,
P
<0.01) compared to EHMs paced at 1 Hz. In addition, TP attenuated the response to the M-receptor agonist carbachol. Resting membrane potential was unaffected. In order to more closely simulate persistent AF (clinically diagnosed after 7 consecutive days of AF events), 7 day TP was applied to the EHMs. Similar to 24 hour TP, this produced AP shortening and a blunted response to M-receptor agonists. Interestingly, this also led to hyperpolarization of the resting membrane potential (RMP: -75.1±0.8 vs. -72.5±0.8,
P
<0.05) compared to EHMs paced at 1 Hz.

Conclusion:
Classical characteristics of AF-associated remodeling were elicited through TP of EHM. This unique optogenetic technique represents the first
in vitro
model to investigate the time-dependence of cellular AF-associated remodeling in the long-term. This offers immense potential for drug development studies and personalized approaches for antiarrhythmic therapy.
Abstract P1045: Replacement Of Wearable-device-measured Sedentary Time By Physical Activity, Genetic Susceptibility, And Risk Of Coronary Heart Disease
Authors: Kim Youngwon , Jang Haeyoon , Wang Mengyao , Strain Tessa , Sharp Stephen J , Shi Qiaoxin , Au Yeung Shiu Lun , Luo Shan , Griffin Simon , Wareham Nicholas J , Brage Soren
Published: October 2023

Backgrounds:
Excess sedentary time (ST) is recognized as an important modifiable risk factor for coronary heart disease (CHD). However, whether the associations of genetic susceptibility with CHD incidence can be modified by replacing wearable-device-measured ST with physical activity (PA) is unknown. This study aims to examine the combined associations of genetic susceptibility to CHD, and wearable-device-measured ST replaced by moderate-to-vigorous PA (MVPA) or light PA in relation to incident CHD.

Methods and Findings:
This study included 75,545 white British (57% female) with valid wrist-worn accelerometer data and without prevalent CHD/stroke from UK Biobank. Genetic susceptibility to CHD was quantified through weighted polygenic risk scores for CHD based on 300 single-nucleotide polymorphisms. Wrist-worn accelerometer data were used to derive ST, light PA and MVPA. The hazard ratio of CHD was 1.36 (95% CI: 1.03-1.80) for every-30-minute/day-increase in ST relative to other movement behaviors, after adjusting for genetic risk and confounders. Reallocation of any amount of ST (as little as 1 minute/day) into the same MVPA time (but not light PA) was associated with lower CHD hazards overall and within strata of genetic risk: no evidence of interaction between genetic risk and ST (p-value: 0.739). However, differences in absolute risk of CHD for ST replaced by MVPA were greater at high genetic risk
versus
low genetic risk. Limitations of our study include no causal evidence derived, limited generalizability to individuals of other ethnic groups or with sub-clinical symptoms, and potential residual confounding.

Conclusions:
Replacing any amount of ST with an equal amount of MVPA time is associated with a lower relative risk of developing CHD at all levels of genetic susceptibility to CHD. Potential reductions in absolute risk of CHD through replacement of ST by MVPA in equivalent amounts may be greater in individuals at high genetic risk of CHD.
Abstract P1068: Yap Induces A Neonatal Like Pro-renewal Niche In The Adult Heart
Authors: Li Rich Gang , Li Xiao , Grisanti-Canozo Francisco , Morikawa Yuka , Meng Fansen , Kim Jong H , Xie Bing , Klysik Elzbieta , Liu Shijie , Samee Md Abul Hassan , Martin James F
Published: October 2023

The heart is a poorly renewable organ, meaning that tissue architecture and the cardiac microenvironment are irreversibly disrupted after injury. This presents a major challenge in treatment of ischemic heart disease, the leading cause of death worldwide. To date, most studies have focused on cardiomyocytes (CMs). However, very few studies have examined CM-extrinsic mechanisms, such as innate immunity, that complement CM-intrinsic mechanisms in heart renewal. Inactivation of the Hippo signaling pathway can rebuild the post ischemic microenvironment and improve cardiac function. We investigated spatially resolved cellular relationships of neonatal and adult renewal-competent hearts to gain insight into inefficient mammalian heart renewal. Spatial transcriptomics and single-cell sequencing of adult control hearts and hearts expressing YAP5SA, an active version of the Hippo signaling pathway effector YAP, which models heart renewal, revealed a conserved, renewal-competent CM population in control hearts and amplified in YAP5SA hearts. This CM population was also found in the wildtype, renewal competent neonatal heart after myocardial infarction, as well as the adult human heart. In YAP5SA hearts and neonatal hearts post myocardial infarction, resident macrophages and cardiac fibroblasts expressing complement pathway genes colocalized with these CMs, creating a pro-renewal microenvironment. Complement loss-of-function in fibroblasts or macrophages suppressed cardiomyocyte proliferation in both neonatal injured hearts and adult YAP5SA hearts, indicating that the complement system plays a direct role in heart renewal.
Abstract P1094: Mesenchymal Stem Cells Educate Lung Megakaryocytes To Favor Cardiac Repair Post Myocardial Infarction
Authors: Hu Shiqi , Cheng Ke
Published: October 2023

Stem cell therapy in myocardial infarction (MI) have mixed clinical results, and the mechanism of its potential benefits is uncertain. Intravenously administrated mesenchymal stem cells (MSCs) are trapped in the lung capillary bed and hardly reach to the heart. Despite their extremely low retention in the heart, cardiac repair benefits are still seen in multiple clinical trials. There is a large knowledge gap exists regarding the interconnection and bidirectional pathways between heart and lung. It has been established that lung is a reservoir of megakaryocytes (MKs), which produce platelets. We found that MSCs trapped in the lung capillary altered the transcription profiles of lung MKs and circulating platelets. Single cell analysis revealed that MSCs reduced the activation of megakaryocyte progenitor cells (MKp) and promoted MK maturation in the lung. Additionally, MSC treatment reduced the activation of immune cells and the ratio of proinflammatory monocytes in the lung and macrophage infiltration in the infarcted area. CellChat analysis demonstrated that reduced communication between lymphocytes and MKp after MSC treatment. Lastly, we demonstrated that cell-cell contact was needed for this education process, and MSC secretome didn’t promote the megakaryopoiesis process
in vitro
and
in vivo
. Intravenously injection of MSCs showed a better therapeutic effect compared to MSC secretome in MI mice. Collectively, this study unevils the role of pulmonary MK in lung-heart crosstalk and the interaction of MSCs trapped in the lung tissue with pulmonary MK during MI.

Scheme 1. Schematic illustration of heart-lung crosstalk and the role of lung MK during MI.
Abstract P1100: The PD1/PD-L1 Pathway Provides An Immunosuppressive Environment Necessary For Neonatal Heart Regeneration
Authors: Vargas Aguilar Stephanie , Cui Miao , Liu Ning , Bassel-Duby Rhonda S , Olson Eric N
Published: October 2023

The neonatal mouse heart regenerates after injury without scarring or impairment in heart function, but this ability is lost within the first week of life. This regenerative time window coincides with important changes in immune cell composition, maturation, and function. To understand how these changes support regeneration over scarring in the neonatal heart, we compared the immune response in regenerative and non-regenerative mouse hearts following myocardial infarction (MI). The immune response in regenerative hearts is characterized by a low degree of inflammation that is rapidly resolved, whereas non-regenerative hearts exhibit an exacerbated and prolonged immune response that involves strong T cell activation. Accordingly, we discovered that the PD1/PD-L1 pathway, an immune-modulatory pathway that acts mainly through the suppression of T cell activation, is highly activated in regenerative hearts but rapidly silenced during the postnatal period. Deletion of the PD1 receptor or inactivation of its main ligand, PD-L1, prevented regeneration of neonatal hearts after MI. The immune response in neonatal hearts from PD1 knockout mice was characterized by increased monocyte infiltration, a pro-inflammatory gene signature and exacerbated T cell activation. Our findings reveal a previously unrecognized role of the PD1/PD-L1 pathway in heart regeneration through modulation of the immune response and provide new insights into the importance and characteristics of a regeneration-permissive environment.
Abstract P1125: Development Of A Multicellular Sinoatrial Node Organoid For Modeling Robust Pacemaking And Conduction
Authors: Son Young Hoon , Sheng Christina , Cho Hee Cheol , Park Sung-Jin
Published: October 2023

The prevalence and healthcare burden of sinoatrial (SA) node dysfunction have been continuously increasing due to the growth in the elderly population, emphasizing the need for more detailed studies of SA node functions to enable effective therapy for treating and preventing SA node dysfunction. However, current SA node or pacemaker models are limited to isolated single cell-type cells or cell clusters, leaving a gap in the ability to model autonomous cardiac contraction. In contrast, the SA node is a natural organoid with elaborate insulated architecture and heterogeneous cellular composition. Furthermore, current single cell-type pacemakers have worsened heart rhythm stability during one-month in vivo integration, limiting their application as clinically viable biological pacemakers that can generate robust pacemaking and conduction. To address the current limitations of SA node models, we developed a three-dimensional multicellular SA node organoid by reproducing the human SA node's multicellular tissue structure and fail-safe mechanisms. First, inspired by the SA node's partial electrical insulation, we structurally isolated a group of cardiomyocytes with a single exit pathway that allowed for electrical connection to neighboring cardiomyocytes. We found that the geometrically isolated cardiomyocytes successfully activated neighboring quiescent cardiomyocytes without any pacemaker-related gene expression, and they maintained the rhythm for three months. Second, the overexpression of HCN4, a key pacemaker channel, in these isolated cardiomyocytes further improved heart rhythm variability down to the millisecond level, suggesting the interplay between pacemaker gene expression and nodal architecture for long-term, high-fidelity pacing. Finally, we successfully fabricated and integrated the SA node organoid into our 3D pacemaker organoid-myocardium platform, and the integrated organoid dominated rhythm control in 3D cardiac muscle constructs for 30 days. These studies will help define if tissue-level architecture and multicellular compositions mediate SA node's robust pacemaking and conduction and may reveal a high-fidelity tissue-level biological pacemaker as a novel therapeutic strategy for SA node dysfunctions.
Abstract P1126: A Direct Comparison Of Cardiomyocyte Maturation In Vivo And In Vitro At Single Cell Resolution
Authors: Chen Elaine Z , Kannan Suraj , Zhu Renjun , Guo Jason , Kwon Chulan
Published: October 2023

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) offer vast potential for clinical applications including cell therapy, drug screening, and disease modeling. However,
in vitro
-derived PSC-CMs incompletely mature with fetal-like phenotype, which drastically limits their clinical applications. Currently, it remains unknown why PSC-CMs fail to fully mature in culture. We sought to understand CM maturation process by generating high-quality single-cell RNA-sequencing (scRNA-seq) reference of
in vivo
mouse CMs sampled from embryonic stage to adult stage. Subsequently, we generated isogenic embryonic stem cells and established an
in vitro
scRNA-seq reference of differentiated CMs across a range of differentiation timepoints. By directly comparing the
in vivo
and the
in vitro
maturation trajectories, we identified a group of dysregulated transcription factors (TFs), NFE2L2, NRF1, MEF2A, JUN, YY1, ESRRA, SRF, SOX9, PPARA, which may be important to activate the CM maturation program. Therefore, we increased their levels in PSC-CMs and subsequently examined their transcriptome changes. Our initial analysis showed that higher levels of the TFs increase the expression of electron transport chain protein genes and decrease the expression of glycolysis protein genes, suggesting the initiation of metabolic maturation. Our study presents a first direct comparison of CM maturation
in vivo
and
in vitro
at single-cell resolution, which illuminates new research directions for understanding PSC-CM maturation.
Abstract P1129: Directing Revascularization In Vivo With Local Controlled Release Of An Optimized Angiogenic Growth Factor Cocktail
Authors: Minor Alicia , Roser Stephanie , Kant Rajeev , Zahiri Keyana , Polucha Collin , Coulombe Kareen L
Published: October 2023

Reduced vascular perfusion due to aging, ischemia, or injury impedes healing and tissue function in conditions like skin ulcers and myocardial infarction, motivating development of therapies to restore vasculature locally in target tissues. We hypothesize that specific combinations of signaling molecules stimulate angiogenesis and vascular remodeling in the local tissue environment to increase perfused vessel density. Using a vascular tissue engineering approach with a fractional factorial design of experiments (DOE), we evaluated
in vivo
angiogenesis of combinations of ten growth factors, cytokines and/or enzymes including VEGF, bFGF, Shh, PDGF, IGF-1, EGF, TGF-β1, Ang-1, MCP-1 and MMP-9 in a subcutaneous gel plug assay in the WT immune competent rat. Our initial assessment with Matrigel identified 5 factors that maximized CD31+ vessel lumen density and aSMA+ encoated vessels at 7 days, warranting further evaluation. We developed a fully defined biomaterial system with covalently bound heparin to alginate mixed with collagen type 1 as an injectable gel. This defined biomaterial prolonged protein release up to 14 days
in vitro
versus Matrigel and unmodified alginate (2-4 days). The defined biomaterial was used for a second round DOE to deliver high (2 μg) and low (0.5 μg) doses of VEGF, bFGF, Shh, PDGF, and/or IGF-1. The defined biomaterial alone enhanced average lumen diameter 15-fold and endothelial cell infiltration 12-fold versus Matrigel. Perfused lumen density increased 16-fold with high Shh; aSMA-positive lumen wall area increased 4-fold with high VEGF, PDGF, and IGF-1; perfused vessel size increased 3-fold with high VEGF, bFGF, and Shh; and endothelial cell recruitment increased 2-fold with high IGF-1 relative to unloaded controls after 7 days
in vivo.
Empirical testing of 2- to 5-factor combinations showed that cocktail potency required initiation of angiogenic sprouting by VEGF or bFGF and vascularization responses were strongest in the presence of Shh and IGF-1. In conclusion, we identified sets of highly potent cocktails and leveraged controlled release from a defined biomaterial as a tailored revascularization therapy for local angiogenesis, vascular remodeling, and tissue regeneration.
Abstract P1134: RNA Elongation Mediates Rapid Induction Of Gene Expression During Endothelial-to-mesenchymal Transition
Authors: Kokot Karoline Elizabeth , Hoba Julian , Erbe Stephan , Andritschke Michael , Sheikh Bilal N , Laufs Ulrich , Boeckel Jes-Niels
Published: October 2023

Endothelial-to-mesenchymal transition (EndMT) is triggered by stress stimuli and characterized by the conversion of endothelial cells (EC) into mesenchymal cells. Stress stimuli such as hypoxia or inflammation depend on rapid gene expression mediated by RNA elongation. RNA elongation is controlled by the super elongation complex (SEC) through transient pausing of RNA Polymerase II (RNAPII). We aimed to analyze the regulatory role of RNA elongation on rapid gene expression during the onset of EndMT. In an in vitro EndMT model, SEC scaffolds AFF1 and AFF4 were upregulated compared to controls. SEC inhibition using chemical compounds reduced the protein levels of AFF1 and AFF4. EndMT was reduced by SEC inhibition, whereas overexpression of AFF4 increased EndMT as measured by mesenchymal marker gene expression. Rapid gene expression depends on the release of paused RNAPII at promoter sites. ChIP-sequencing revealed a reduction of RNAPII occupancy at promoter sites in EndMT compared to controls, suggesting the release of RNAPII during EndMT. Transcriptome-wide RNA elongation rates were increased as early as 10 min after induction of EndMT, as determined by analysis of nascent 4sU-labeled RNA. SEC inhibition reduced global RNA elongation rates, suggesting a regulatory contribution of RNA elongation in EndMT. Among the genes that were immediately upregulated after the induction of EndMT was the SPARC mRNA. RNA initiation and elongation of SPARC mRNA were increased 10 min after EndMT induction, which was reduced by SEC inhibition. At 72 h after EndMT induction, inhibition of SEC decreased SPARC mRNA, protein concentration and SPARC delivery to the supernatant. Silencing of SPARC reduced the angiogenic potential of EC and enhanced EndMT induction, which was attenuated by treatment with recombinant SPARC. Treatment with recombinant SPARC also improved endothelial impedance under EndMT conditions. Spatial transcriptomics revealed increased SPARC expression in brachiocephalic artery plaques from LDLR-/- mice. In conclusion, we observed increased RNA elongation rates associated with reduced RNAPII pausing during EndMT that mediate rapid gene expression of target genes such as SPARC. Our results suggest that RNA elongation plays a regulatory role in EndMT.