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Introduction: Axi-cel utilization expanded after demonstration of superior event-free survival in patients (pts) with early relapsed or primary refractory LBCL (Locke, et al. NEJM. 2022) and was reinforced by improved overall survival (OS) among these pts (Westin, et al. NEJM. 2023). Despite increased demand, limited data exist on the impact of bridging therapies (BT) on pt outcomes. Thus, BT use between leukapheresis and lymphodepletion (LD) is largely at the discretion of treating physicians. Here, we assessed the impact of BT on effectiveness and safety outcomes after axi-cel. Methods: Pts receiving axi-cel for 3L+ LBCL in the US between 2017-2020 were prospectively collected through the CIBMTR registry in a post-authorization safety study; data up to May 2023 were included. Effectiveness outcomes were overall response rate (ORR), complete response (CR) rate, duration of response (DOR), progression-free survival (PFS), and OS. Safety outcomes were cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) per ASTCT grading, non-relapse mortality (NRM), and other key adverse events. Propensity score-based inverse probability of treatment weighting (IPTW) was used in logistic and Cox regressions to account for potential confounding in BT use. For pts who had BT, effects of BT types and responses on outcomes were evaluated using conventional logistic and Cox regressions. Results: Of 1497 pts registered, 1307 had BT data available and were included (median follow-up, 36 mo). Overall, 430 pts (33%) received BT (median time from leukapheresis to LD, 23 days [IQR, 21-28]). Systemic therapy was used in 403 pts (31%), including 305 (23%) with chemotherapy (CT)-based BT (19% treated with CT + anti-CD20 monoclonal antibody [mAb; mainly rituximab] and 4% with CT only), and 98 (7%) with non-CT systemic therapy (Table). Radiotherapy (± systemic therapy) was used in 44 pts (3%). Pts with ≥3 lines of prior therapy (odds ratio [OR], 10.67 [95% CI, 7.17−15.87]) or mild hepatic impairment prior to infusion (OR, 1.64 [1.06−2.55]) were more likely to receive BT, while those with histologic transformation (OR, 0.69 [0.51−0.92]) or longer time from diagnosis to leukapheresis were less likely to receive BT. ORRs to axi-cel were 70% and 79% (CR rates 51% and 64%) for pts with or without BT, respectively. Unadjusted DOR, PFS, and OS at 3 years were 40%, 28%, and 37%, respectively, in pts with BT vs 49%, 39%, and 49%, respectively, in those without BT. After multivariate adjustment, no statistical significance was found between BT and any effectiveness outcome. Adjusted hazard ratios (HRs) for BT were 1.13 (95% CI, 0.94-1.36) for PFS and 1.11 (95% CI, 0.92-1.34) for OS, respectively (Figure). Most safety endpoints were consistent regardless of BT. Grade ≥3 CRS and ICANS occurred in 12% and 30% of pts, respectively, who received BT vs 10% and 24% of pts who did not. Use of BT was associated with more frequent prolonged neutropenia (OR, 2.24 [1.44-3.50]) and thrombocytopenia (OR, 2.33 [1.67-3.24]), but not associated with a higher rate of infections or NRM (Table). Among pts receiving BT, 74 (17%) had a CR or partial response to BT. Of them, median PFS and median OS post infusion were 12.7 mo (3.8-not estimable [NE]) and 48.7 mo (18.6-NE), respectively. Response to BT was associated with increased CR rate (OR, 2.00 [1.07-3.75]), PFS (HR, 0.69 [0.49-0.99]), and OS (HR, 0.59 [0.40-0.88]) and decreased any-grade ICANS (OR, 0.51 [0.29-0.92]). When different BT strategies were considered, effectiveness and most safety outcomes for CT + anti-CD20 mAb vs non-CT treatment were comparable without significant difference. CT + anti-CD20 mAb was associated with improved ORR (OR, 1.90 [1.00-3.60]), PFS (HR, 0.69 [0.49-0.98]), and OS (HR, 0.66 [0.46-0.95]) but increased any-grade CRS (OR, 2.16 [1.02-4.57]) vs CT only. CT use was associated with an increased risk of prolonged thrombocytopenia (41% vs 24% for pts receiving non-CT treatment; OR, 2.37 [1.39-4.02]). No significant association was found between use of radiotherapy as BT and any outcome. Conclusion: These US real-world findings suggest that the addition of BT prior to axi-cel is associated with more frequent prolonged cytopenias but does not impact effectiveness or other critical safety outcomes. Response to BT may prognosticate more favorable outcomes after axi-cel infusion. Further study is warranted to identify the optimal bridging regimen.

Abstract Background Staphylococcus aureus (SA) infections, both methicillin-susceptible and methicillin-resistant (MSSA, MRSA), cause significant morbidity among infants in neonatal intensive care units (NICU). Colonization usually precedes infection. We studied the molecular epidemiology of SA colonization in NICU infants using whole genome sequencing (WGS) and in relation to the opening of a new NICU with single-family rooms. Methods From 2019-22, a prospective cohort of infants admitted to the Level IV NICU at Mayo Clinic, Rochester, MN was screened twice weekly for SA colonization using cultures of nares/axilla/groin swabs. Infection control procedures were consistent throughout, including use of Contact Precautions for infants colonized with MRSA but not for MSSA. The study was divided into period 1 (1/1/2019-11/30/2020) and period 2 (12/1/2020-12/31/2022) due to a move from a 34-bed unit (26 beds in four open-bay rooms and 8 beds in single-family rooms) to a new 34-bed unit (all single-family rooms). WGS was performed using Illumina MiSeq instrumentation and chemistry with Illumina Nextera XT library chemistry. Assembly and core genome multilocus sequence typing analysis were performed with Ridom SeqSphere+ software. Groups of related isolates were identified based on the number of allelic differences (related 0-8 differences, possibly related 9-29 differences, unrelated ≥ 30 differences). Groups of MRSA-related isolates were compared with reference USA genotypes. Chi square tests did not adjust for clustering. Results There were 663 admissions in period 1 and 730 in period 2. The prevalence of MSSA and MRSA colonization was unchanged in the two periods (MSSA, 18.6% vs. 16.7%, p=0.37; MRSA 1.8% vs. 3.3%, p=0.08). By WGS, most MSSA and MRSA isolates were unrelated to another isolate in both periods (Table). Groups of related MSSA and MRSA isolates differed between the two periods and the number of isolates/group was small (< 6 isolates). Groups of related MRSA isolates were associated with reference genotypes USA100 (period 1), USA1000 (periods 1 & 2), and USA300 (period 2). WGS Results for MSSA and MRSA in Periods 1 and 2 Conclusion NICU infants were usually colonized with unrelated strains of MSSA and MRSA, with only small clusters of related strains. The new NICU was not associated with a change in MSSA or MRSA colonization. Disclosures Priya Sampathkumar, MD, Merck Vaccines: Advisor/Consultant Robin Patel, MD, Abbott Laboratories: Advisor/Consultant|Adaptive Phage Therapeutics: Grant/Research Support|Adaptive Phage Therapeutics: Mayo Clinic has a royalty-bearing know-how agreement and equity in Adaptive Phage Therapeutics.|BIOFIRE: Grant/Research Support|CARB-X: Advisor/Consultant|ContraFect: Grant/Research Support|Day Zero Diagnostics: Advisor/Consultant|HealthTrackRx: Advisor/Consultant|Mammoth Biosciences: Advisor/Consultant|Netflix: Advisor/Consultant|Oxford Nanopore Technologies: Advisor/Consultant|PhAST: Advisor/Consultant|See details: Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic|See details: continued, patent on an anti-biofilm substance issued|TenNor Therapeutics Limited: Grant/Research Support|Torus Biosystems: Advisor/Consultant|Trellis Bioscience, Inc.: Advisor/Consultant W. Charles Huskins, MD, MSc, ADMA Biologics: Advisor/Consultant|Bristol Myers Squibb: Stocks/Bonds|Pfizer: Advisor/Consultant|Pfizer: Stocks/Bonds|Zimmer Biomet: Stocks/Bonds

The A‐splice variant of Na + ‐dependent, electrogenic bicarbonate transporter‐1 (SLC4A4, NBCe1‐A) is generally believed to be the only SLC4A4 isoform expressed in the mammalian proximal tubule, and to have critical roles in filtered bicarbonate reabsorption, proximal tubule ammonia metabolism and proximal tubule organic anion metabolism. The purpose of the current study was to examine expression of other splice variants in the mouse kidney. We used recently generated mice with TALEN‐generated deletion of A and D NBCe1 splice‐variants, but with intact expression of the B, C and E isoforms. RT‐PCR using primers specific to the B, C and E isoforms showed mRNA of non‐A,D splice variants was expressed in the kidney of mice with intact NBCe1‐A expression and with NBCe1‐A‐specific deletion. We then used an antibody that recognizes portions of NBCe1 present in all splice isoforms and examined immunolabel in the kidney of NBCe1‐A‐deletion mice. There was basolateral immunolabel in the proximal convoluted tubule in the cortex, and lesser intensity basolateral immunolabel in the proximal tubule straight tubule in both the cortex and outer medulla. Specificity of this antibody was demonstrated by identifying that mice with deletion of all splice variants had no detectable immunolabel. An antibody specific to the A‐splice variant showed basolateral immunolabel throughout the entire proximal tubule of mice with intact NBCe1‐A expression, but no detectable immunolabel in mice with NBCe1‐A‐specific deletion. Thus, an NBCe1 splice variant other than NBCe1‐A appears to be expressed in the mouse proximal tubule. In acid‐loaded NBCe1‐A‐deletion mice, immunolabel observed using the pan‐specific anti‐NBCe1 antibody showed increased immunolabel in the proximal straight tubule, particularly in the outer medulla. These studies show that splice variants of NBCe1 other than NBCe1‐A/D are present in the mouse kidney and that their expression appears to be increased with acid‐loading. We conclude that these splice variants may have important roles in proximal tubule filtered bicarbonate reabsorption, ammonia metabolism and organic anion metabolism. Support or Funding Information The studies were supported by funds from the NIH (R01–DK045788 to IDW, R01‐DK107798 to IDW and JWV, and R01‐EY017732 to MFR) and the Department of Veterans Affairs (1I01BX000818 to IDW).

PURPOSE CALGB (Alliance)/SWOG 80405 was a randomized phase III trial that in first-line patients with metastatic colorectal cancer (mCRC) treated with bevacizumab or cetuximab with chemotherapy. We aimed to discover novel mutated genes associated with prognosis and differential response to therapy with the biologics. METHODS Primary tumor DNA from 548 patients was sequenced using FoundationOne. The effect of mutated genes and mutations on overall survival (OS) was tested adjusting for microsatellite instability status, BRAF V600E, all RAS mutations, arm, sex, and age. RESULTS The median number (lower-upper quartile) of mutated genes was 5 (3-7), 5 (3-6) in microsatellite stable and 12.5 (4.5-32) in microsatellite instability-high tumors. Mutated KRAS and APC were more frequent in Black (53% and 85%) than White (27% and 65%, respectively) patients while BRAF V600E was less frequent in Black (5%) than White (14%) patients. The median OS in patients with BRAF non-V600E (2.2% of patients) was 31.9 months (95% CI, 15.1 to not applicable [NA]) similar to that of BRAF wild-type (WT) patients (31.2 months [95% CI, 29.0 to 33.9]). Mutated LRP1B (10.7% of patients) was associated with improved OS compared with WT LRP1B (hazard ratio, 0.57 [95% CI, 0.40 to 0.80]). RNF43 (5.6% of patients) interacted with treatment arms as, in the cetuximab arm, patients with mutated RNF43 had a median OS of 11.5 (95% CI, 10.8 to NA) months compared with 30.1 (95% CI, 24.9 to 35.3) months in patients with WT RNF43, whereas in the bevacizumab arm, patients with mutated RNF43 had a median OS of 25.0 (95% CI, 14.2 to NA) months compared with 31.3 (95% CI, 29.0 to 34.3) months in patients with WT RNF43. CONCLUSION These results can provide new tools to predict patient outcome and improve therapeutic decisions and trial participation in patient minorities. The molecular alterations identified in this study may direct biomarker-driven studies.

Local production of growth factors within the airway with autocrine/paracrine effects can promote features of asthma. Here, we show that glial-derived neurotrophic factor (GDNF) is a procontractile and proremodeling factor that contributes to allergen-induced airway hyperreactivity and tissue remodeling in a mouse model of asthma. Blocking GDNF signaling attenuates allergen-induced airway hyperreactivity and remodeling, suggesting a novel approach to alleviating structural and functional changes in the asthmatic airway.

Abstract Histopathology is the gold standard for distinguishing tumor recurrence from treatment-induced necrosis but differentiating pseudoprogression from true recurrence is challenging in tumors with ambiguous morphological features. Prior research reported a 36% inter-rater agreement during suspected recurrence (Holdhoff et al., 2019). While serial radiographic and laboratory assessments are common, serial histopathological assessments are not. This study evaluated agreement and factors influencing assessment of active and inactive tumors through single and multiple time-point histopathological assessments. Patients who underwent initial and subsequent reoperation were identified, and their available histology slides were examined retrospectively. Four independent neuropathologists conducted two types of reviews: single time-point reviews using most recent biopsy/surgery slides, and multiple time-point reviews comparing images from initial and repeat biopsies taken 1-3 months apart. Neuropathologists completed a questionnaire assessing various tumor characteristics. Assignments were categorized as "active tumor," "treatment effect," or "unable to classify." 324 assessments were conducted (108 single time-point, 216 multiple time-point). Intraclass correlation coefficients for single vs. multiple time-point assessments were 0.72-0.93 for active tumors and 0.57-0.86 for inactive tumors. Pathologists' assessment of % inactive tumor was influenced by mitotic activity (p= 0.0028), cellularity (p< 0.0001), hyalinized vessels (p= 0.0019), and necrosis (p= 0.0046). Evaluation of % active tumor was influenced by hyalinized vessels (p= 0.0006), mitotic activity (p< 0.0001), cellularity (p< 0.0001), and microvascular proliferation (p= 0.0093), with the latter three factors specifically affecting final sign-out pathology. Serial histopathological assessment didn’t affect determination of % active or inactive tumor. No association between % inactive tumor and fibrinoid necrosis for single time-point (p= 0.34) but a strong negative association was seen for serial assessment (p= 0.0006). Inter-rater agreement was higher for identifying active tumors than inactive tumors in both case reviews. Changes in necrosis between tissue samples in single vs. multiple timepoint assessments influenced pathologists' assessment of % inactive tumor.

Abstract α-PD-1 checkpoint blockade is a promising immunotherapeutic approach for many solid tumors; however, this strategy is minimally effective for GBM patients as a monotherapy. One potential reason why α-PD-1 checkpoint blockade is ineffective is the tumor microenvironment and systemic derangements of the immune system which are prevalent in GBM patients. We have successfully modeled peripheral immune derangements in experimental murine GBM models. These include low peripheral blood CD4 T cell counts, reduced MHC class II expression on monocytes, and atrophy of primary immune organs in animals harboring gliomas. We therefore hypothesized that extended half-life IL-2, a potent cytokine which promotes the proliferation, differentiation, and killing activity of T cells, could overcome immune derangements and could potentially synergize with α-PD-1 checkpoint blockade therapy. In cohorts of C57Bl/6 mice, we supplemented α-PD-1 checkpoint blockade with multiple bio-engineered extended half-life IL-2 molecules to treat GL261 and CT2A orthotopic glioma models which are normally resistant to monotherapy. We observe a statistically significant increase in survival in GL261-bearing animals receiving the combination of extended half-life IL-2 reagents and αPD-1 therapy relative to control-treated mice. Some animals receiving this combination therapy had a reduction in tumor burden to levels below detection. These long-term survivors underwent a rechallenge experiment with a second inoculation of GL261-Luc and effectively cleared tumor without further therapeutic intervention. This intervention causes a significant shift in the immune profile, including the formation of mature T cell responses, as well as restoring the diminished peripheral immune cell counts in GL261-bearing animals. Finally, combining extended half-life IL-2 with α-PD-1 checkpoint blockade is effective independently of the CD8 T cell response. Together, these data suggest that combination immunotherapies employing extended half-life IL-2 fusion proteins are translational to the clinic and do not require the identification of tumor specific antigens for efficacy

Abstract Glioblastoma (GBM) is aggressive form of brain cancer that is associated with severe peripheral immunosuppression. This immunosuppression is a critical barrier to patient survival and the success of immunotherapies. Immunocompetent mice harboring intracranial gliomas present with the hallmark features of peripheral immunosuppression observed in GBM patients, including T cell lymphopenia. Using parabiosis, we determined that T cell lymphopenia can be transferred from glioma-bearing to healthy mice via circulation This led to the identification of a high molecular weight immunosuppressive factor in sera of glioma-bearing mice that inhibits T cell proliferation ex vivo. Proteomics analysis identified 119 proteins uniquely upregulated in the sera of glioma-bearing mice compared to controls. These proteins are associated with pathways involved in DNA response elements, cell death, and DNA-histone complexes which imply DNA release. Additional analysis revealed serum isolated from glioma-bearing mice harbor significantly higher concentrations of double stranded cell free DNA (cfDNA) compared to controls. Reducing DNA content using DNase partially reverses the proliferation defects observed in T cells when exposed to serum of glioma-bearing mice. This is while, addition of cfDNA purified from serum of glioma-bearing mice, but not genomic DNA, potently inhibited T cell function. These results were AIM2 independent implicating a novel pathway of cfDNA sensing in T cells. We contend that cfDNA induces immunosuppressive effects in experimental GBM. Devising strategies to reduce circulating cfDNA is therefore a therapeutic approach that could improve outcomes in GBM patients.

Introduction: Genome-wide association studies (GWAS) have discovered >300 associations for CAD. Few loci are functionally characterized and may represent new mechanisms of disease. Hypothesis: Can an unbiased transcriptional screen for the >2000 candidate GWAS genes in endothelial cells (ECs) identify causal genes and disease-relevant pathways at CAD GWAS loci. Methods: We applied CRISPRi-Perturb-seq to knock down expression of genes within 500 Kb of coronary artery disease GWAS loci (2,300 genes in total) and measure their transcriptomic effects using single-cell RNA-seq. Validation of candidate causal genes was conducted in primary human ECs and zebrafish. Results: We identified 50 programs of co-expressed genes, which represent core cellular pathways (i.e. ribosome biogenesis) and 13 EC-specific pathways such as flow response and angiogenesis. The EC-specific programs show the greatest contribution to CAD heritability. 5 EC-specific programs have the greatest number of GWAS candidate genes. We find that 43 of 306 CAD GWAS signals converge onto these 5 gene programs, and all are regulated by genes in the cerebral cavernous malformations (CCM) pathway—which coordinates KLF2/4 transcriptional responses in endothelial cells, but has not been linked to CAD risk. The strongest regulator of these programs is TLNRD1 , which we show is a new CAD gene and novel regulator of the CCM pathway. TLNRD1 loss-of-function alters actin organization and barrier function in endothelial cells in vitro , and heart development in zebrafish in vivo . Conclusions: High-throughput functional analysis of 2,300 genes proximal to CAD GWAS loci prioritized pathways—such as angiogenesis and EC migration—that are regulated by multiple risk SNPs. Our study identifies new genes that likely influence risk for CAD, identifies convergence of CAD genes into certain pathways in endothelial cells, and prioritizes study of genes in the CCM-signaling pathway for contribution to CAD risk.