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Abstract Background Measurement of cardiac troponin (cTn) using high-sensitivity assays is recommended for the diagnosis of myocardial infarction. We determined sex-specific and uniform 99th percentile upper reference limits (URLs) using the new Elecsys® Troponin T high-sensitivity Gen 6 assay in a global, healthy reference range cohort. Methods Lithium-heparin plasma and serum samples were prospectively collected from apparently healthy individuals aged ≥20 years across 34 global sites in the United States, Europe, China, and Japan. cTnT was measured using the Troponin T high-sensitivity Gen 6 assay on the Cobas® e 801 analyzer. Exclusion criteria were defined according to the 2022 IFCC guidance. Uniform and sex-specific 99th percentile URLs and nonparametric 95% CIs were determined in plasma and serum separately and combined. Results The final study population comprised 4147 participants (52.5% female) with a median (25th–75th percentiles) age of 48.0 (33.0–59.0) years; 45.8%, 47.9%, 5.2%, and 1.1% were White, Asian, Black, and other/unknown, respectively. For sample matrices combined (n = 8294), 81.0% and 99.2% of cTnT values were above the limit of detection in females and males, respectively. Sex-specific 99th percentile URLs (95% CI) were 18 (16–23) ng/L for females and 32 (28–35) ng/L for males; the uniform 99th percentile URL was 27 (24–31) ng/L. URLs were comparable in plasma and serum samples. Conclusions This study determined sex-specific and uniform 99th percentile URLs for the Troponin T high-sensitivity Gen 6 assay that were comparable irrespective of the matrix used in a large, global, healthy reference population.

Immunotherapy has been effective in many cancer types but has failed in multiple clinical trials in prostate cancers, with the underlying mechanisms remaining largely unclear. Here, we demonstrate that androgen receptor pathway inhibitor (ARPI) plus irradiation (IR) triggered robust anticancer immunity in prostate cancers in both patients and mice. We show that androgen-activated AR suppressed innate immune signaling by inducing inhibitor of nuclear factor kappa-B kinase subunit epsilon ( IKBKE ) gene repression through HDAC2 interaction with an IKBKE enhancer RNA ( IKBKE eRNA, or IKBKE-e ). ARPI treatment caused IKBKE derepression and enhanced an IR-induced innate immune response via action of RIG-I and MDA5 dsRNA sensors. IKBKE-e ablation largely enhanced innate immunity in prostate cancer cells in culture and anticancer immunity in mice. Our results revealed AR, HDAC2, and IKBKE eRNA as critical intrinsic immune suppressors in prostate cancer cells, suggesting that rejuvenating inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKKε) signaling by targeting IKBKE-e is an actionable strategy to elicit synthetic anticancer immunity in immunologically “cold” cancers such as prostate cancer.

198 Background: The Hedgehog (Hh) signaling pathway regulates tissue patterning, growth and differentiation, including within gastrointestinal tract. Mammalian homologs include Sonic (SHH), Indian (IHH) and Desert (DHH) Hedgehog, which signal through PTCH and SMO and the GLI transcription factors. In the digestive tract, both SHH and IHH are expressed, but the functional role of Hh signaling in mCRC remains incompletely defined. Methods: We analyzed 433 mCRC patients treated with either bevacizumab (bev = 226) or cetuximab (cet = 207) plus first line chemotherapy in CALGB/SWOG 80405 (NCT00265850). RNA expression (Illumina HiSeq 2500) of 9 Hh pathway genes (SHH, IHH, DHH, PTCH1, PTCH2, SMO, GLI1, GLI2, GLI3) was evaluated. Median overall survival (mOS) and progression free survival (mPFS) in months (mo) were compared across expression tertiles (low [L], medium [M], high [H]). Likelihood ratio tests, hazard ratios and 95% confidence intervals were computed from multivariable Cox proportional hazards models, adjusting for age, sex, ethnicity, ECOG PS, tumor location, number of metastatic sites, KRAS , microsatellites instability status and treatment. Prognostic effects were modeled as marginal terms; predictive effects were modeled as multiplicative interactions. Results: Overall, IHH and GLI2 demonstrated prognostic value for OS after adjusting for multiple testing (Benjamini-Hochberg False Discovery Rate (FDR) < 0.05). High IHH expression (H group, n = 143) correlated with improved mOS (H: 36.0 mo vs M: 32.8 mo vs L: 23.5 mo; p= 0.031), whereas low GLI2 expression (L group, n = 145) was associated with longer mOS (H: 25.6 mo vs M: 33.9 mo vs L: 33.7 mo; p= 0.0016). Moreover, IHH showed predictive value for cet benefit in both OS (mOS H: 38.1 mo vs M: 34.0 mo vs L: 18.4 mo; p= 0.0032) and PFS (mPFS H: 12.6 mo vs M: 12.9 mo vs L: 8.8 mo; p= 0.0025) with significant treatment interaction for OS and PFS (p= 0.0046 and p= 0.0049, respectively; FDR< 0.05). No significant associations were found in bev treated pts. No other Hh genes showed significant prognostic or predictive effects after FDR correction. Conclusions: These results highlight a dual role of Hh pathway in mCRC. Consistent with prior preclinical evidence, reduced IHH expression appears to mark poor prognosis, likely reflecting the loss of IHH-mediated restraint on Wnt signaling, which drives proliferation and dedifferentiation. Conversely, low GLI2, a downstream transcription factor, was linked with favorable outcomes, underscoring context-specific effects of Hh signaling. The strong predictive association of IHH with cet efficacy supports its potential as a biomarker in RAS wild type mCRC. These results suggest that Hh pathway modulation, particularly IHH signaling, may influence disease biology and treatment response. Validation in independent cohorts and mechanistic studies is warranted to clarify implications.

179 Background: Neuroligins are a family of postsynaptic cell adhesion molecules that play a crucial role in synapse formation and function. Recent studies suggest Neuroligin family genes (NLGN1,2,3,4X) may promote cancer cell growth and metastasis in various cancers, including colorectal cancer. Here, we investigated the association between neuroligin family gene expression and first-line treatment outcomes in mCRC. Methods: We evaluated the expression of 4 genes in the neuroligin family (NLGN1, NLGN2, NLGN3 and NLGN4X) on treatment outcomes of 433 pts with mCRC treated with either bevacizumab (Bev, n = 226) or cetuximab (Cet, n = 207) in combination with first-line chemotherapy within the CALGB/SWOG 80405 trial. mRNA was isolated from FFPE tumor samples and sequenced on the HiSeq 2500 (Illumina). Overall survival (OS) and progression-free survival (PFS) were compared between groups of pts categorized by low expression (Q1) (≤25 percentile), medium expression (Q2-3) (25th-75th percentile) and high expression (Q4)(>75th percentile) of NLGN family genes. The associations between gene expression and clinical outcomes were assessed via the multivariable Cox model and plotted with Kaplan-Meier curve. Treatment-gene interactions were evaluated using the likelihood ratio test. False discovery rate (FDR) was controlled through the Benjamini-Hochberg procedure. Results: High NLGN2 expression was associated with shorter PFS (median 10.0 mos, 95% CI (8.3, 11.3)) and OS (median 25.2mos, 95%CI (20.9, 30.0)) compared to low NLGN2 expression (PFS: median 11.2mos, 95% CI (9.8,14.3))(HR 1.41, 95% CI (1.06, 1.89)), P = 0.029). (OS: (median 31.8mos, 95%CI (26.8, 37.5)( HR 1.49, 95% CI (1.10, 2.01) P =0.0095) in multivariate analysis. Patients treated in the cetuximab arm(n=207), high HLGN2 expression significantly associated with shorter PFS (median 8.0 vs 13.4, HR 2.02, 95% CI (1.35, 3.03), P = 0.0021) and OS (median19.6 vs 36.3, HR 2.30, 95% CI (1.50, 3.53), P =8.3e-05). High NLGN3 expression was associated with shorter OS (23.9 vs 36.0 mos, P =0.0083) in cetuximab treatment arms, no significance found in PFS. We found significant treatment interaction for OS with NLGN2 and NLGN3 favoring cetuximab. In pts treated with cetuximab, High NLGN2 expression was significantly associated with shorter OS (median 29.1vs 36.3) (p=0.024) compared to pts treated with bevacizumab. For NLGN3, pts treated with cetuximab, high NLGN3 expression was significantly associated with shorter OS (median 25.0 vs 36.0) (p=0.018) compared to pts treated with bevacizumab. No significant association was found in NLGN1 and NLGN4X. Conclusions: Our results suggest NLGN2/3 gene expression may predict mCRC treatment outcomes. Potential therapeutic drug target NLGN in combination with anti-EGFR treatment in mCRC is warranted.

163 Background: Being physically inactive is an independent predictor of poor outcomes in patients with stage III colon cancer. Whether physical activity can mitigate the oncologic risk of postoperative MRD is unknown. Methods: We concluded a post hoc analysis of a phase III trial evaluating celecoxib vs. placebo and 3 vs. 6 months of adjuvant chemotherapy in patients with resected stage III colon cancer. Self-reported physical activity was assessed in standardized measures of metabolic equivalent hours per week (MET-h/wk) at 2 timepoints: 1) midway through and 2) 6 months after completing adjuvant chemotherapy, modeled as a time-weighted average. MRD was determined via a clinically validated, tumor-informed circulating tumor DNA (ctDNA) assay (Signatera, Natera, Inc.). Disease-free survival (DFS) and overall survival (OS) were estimated using flexible parametric survival models. Results: Among 763 patients (median follow-up 6.0 years), 24% were highly active (≥18 MET-h/wk) and 32% were inactive (<3 MET-h/wk); 18% were ctDNA positive. Adjusting for covariates, DFS improved in patients who were ctDNA negative and moderately active (Ref: inactive; 3-17.9 MET-h/wk; HR=0.62, 95% CI [0.40-0.97]) or highly active (HR = 0.53 [0.30-0.94]). No benefit was seen among patients who were ctDNA positive (Ref: inactive; moderately active: HR = 1.20 [0.75-1.92]; highly active: HR=0.83 [0.45-1.54]). The interaction was not significant (Pint=0.269). Physical activity did not impact OS regardless of ctDNA status. The interaction of physical activity and adjuvant celecoxib use is shown in Table. Conclusions: Among patients who were ctDNA negative, physical activity was associated with improved DFS, and may demonstrate increased survival benefit alongside adjuvant celecoxib. The additive effects of both interventions were inconclusive among those who were ctDNA positive. Support: U10CA180821, U10CA180882, U24CA196171; https://acknowledgments.alliancefound.org; Natera, Pfizer; NCT01150045. Disease-free survival by physical activity level and celecoxib use, stratified by ctDNA status. 3-yr event # at risk Adjusted HR (95% CI) P-value Pint ctDNA Negative 0.258 Inactive + placebo 23 182 Ref Active + placebo 17 132 0.74 (0.43-1.28) 0.283 Inactive + celecoxib 23 167 0.92 (0.57-1.48) 0.720 Active + celecoxib 10 146 0.40 (0.22-0.79) 0.007 ctDNA Positive 0.221 Inactive + placebo 26 33 Ref Active + placebo 15 20 0.96 (0.50-1.84) 0.903 Inactive + celecoxib 34 54 0.62 (0.37-1.05) 0.076 Active + celecoxib 14 29 0.34 (0.18-0.64) 0.001 ctDNA, circulating tumor DNA; HR, hazard ratio; CI, confidence interval. Active = ≥9 MET-h/week, inactive = <9 MET-h/week. Celecoxib use defined by assignment in CALGB/SWOG 80702. Models adjusted by T and N stage, age, sex, race, assigned cycles of fluorouracil, leucovorin, and oxaliplatin, KRAS and BRAF status, BMI, and Western diet.

Key Points α Klotho as an antiaging protein can extend the lifespan of healthy animals; serum α Klotho was lower in patients with autosomal dominant polycystic kidney disease. Transgenic α Klotho and recombinant α Klotho delayed cyst growth in Pkd1 mutant mouse kidneys. α Klotho exerted its proapoptotic and anti-inflammatory function through interacting with and destabilizing cellular inhibitor of apoptosis protein 1. Background α -Klotho ( α Klotho) is expressed mainly in the kidney, which is encoded by the KL gene, existing in a membrane-bound form and a soluble circulating form. Soluble serum α Klotho was lower in patients with autosomal dominant polycystic kidney disease (ADPKD). However, the role of α Klotho in ADPKD remains unknown. Methods To investigate the roles of α Klotho in ADPKD, we generated transgenic α Klotho and Pkd1 mutant Pkd1-HOMO-KL Tg mice to evaluate cyst growth and investigated underlying mechanisms with single-cell RNA sequencing analysis. Results We showed that α Klotho was lower in diverse kidney cell types in Pkd1 mutant kidneys, which was restored by transgenic α Klotho. We identified differential expression genes and pathways mediated by transgenic α Klotho at single-cell resolution and determined the cytokines and their receptors mediated cell-to-cell communication networks in Pkd1 mutant and Pkd1-HOMO-KL Tg kidneys by CellChat analysis. We further showed that α Klotho exerted a proapoptotic and anti-inflammatory function through interacting with and destabilizing cellular inhibitor of apoptosis protein 1, which disturbed TNF and its receptor mediated prosurvival complex, leading to ( 1 ) the formation of predeath complex to increase cystic cell death and ( 2 ) the decrease of the activation of NF-κB, the expression of cytokines, and the recruitment of macrophages, resulting in a delay of cyst growth. Treatment with recombinant α Klotho also delayed cyst growth in Pkd1 mutant kidneys. Conclusions This study defined an unknown role and mechanism of α Klotho in the regulation of a TNF-dependent cystic cell apoptosis and inflammation in Pkd1 mutant kidneys.

Abstract Background Stigma associated with tuberculosis (TB) and TB diagnosis is a major social factor in delay of diagnosis and non-adherence to TB treatment among Asian American and Pacific Islander (AAPI) populations. Main sources of stigma include misconceptions regarding TB transmission and lack of knowledge about symptoms or treatment.Figure 1.Respondent demographicsBreakdown of respondent demographics by age, gender, and AAPI subgroup.Figure 2.Demographic breakdown of TB awarenessTB awareness by AAPI subgroup, age, and gender. Methods A cross-sectional survey of 666 AAPI individuals analyzed TB symptom recognition, transmission misconceptions, and awareness in two AAPI community centers. Data were segmented by AAPI subgroup, metropolitan area (Seattle/Tacoma vs. Las Vegas), age, and gender.Figure 3.TB transmission beliefs.Figure 4.TB treatment, curability, and latent TB infection awareness. Results Overall, 76.6% of respondents reported TB awareness, varying by AAPI subgroup. Awareness was highest among Korean (95%) and Filipino (91%) participants, and lowest among Vietnamese (52%) and Cambodian (50%) respondents. Regionally, TB awareness was similar between Tacoma (76%) and Las Vegas (79%). Age and gender significantly impacted awareness. Respondents under 18 had the lowest TB awareness (59%), while those aged 18-34 had the highest (84%). Females reported higher TB awareness (79%) and symptom recognition (61%) than males (74% and 46%, respectively). Symptom knowledge varied. 55% were aware of TB symptoms, but only 16% were able to recognize five common TB symptoms. Misconceptions about transmission also persisted broadly. Out of the 71% of respondents who were aware that TB is transmissible, many incorrectly believed TB spread via shaking hands (35%), sharing food/drinks (50%), or touching items in public areas (37%). The 45-64 and 65+ age groups were most prone to misconceptions about transmission. While 72% of respondents were aware that TB could be treated, only 57% knew TB could be cured. 30% of respondents were aware of latent TB infection. Conclusion Significant disparities in TB awareness exist in AAPI communities. Females demonstrated greater TB awareness perhaps due to cultural differences (“caretaker role”), while TB transmission misconceptions were prevalent among older individuals. Limited knowledge on LTBI and TB treatment represent significant barriers to effective TB control. Tailored educational efforts will be required to improve TB awareness and reduce misconceptions within AAPI communities. Disclosures All Authors: No reported disclosures

Abstract Background Previous studies demonstrate that certain asymptomatic MAPT variant carriers (Asymp‐ MAPT ) exhibit altered baseline gray matter volume (GMV) and functional connectivity (FC) profiles. It remains unclear whether these imaging measures may herald risk for earlier conversion to the symptomatic phase. To address this, we stratified Asymp‐ MAPT based on baseline GMV and longitudinal FC changes to assess whether any Asymp‐ MAPT subgroups showed signs of longitudinal neurodegeneration or cognitive decline. Method We studied 25 Asymp‐ MAPT recruited from the UCSF Memory and Aging Center and ALLFTD Consortia. Using a median split, we divided Asymp‐ MAPT into groups with smaller (LowGMV; n = 12) and larger baseline GMV (HighGMV; n = 13) within regions atrophied in symptomatic MAPT carriers ( n = 20). Next, we stratified Asymp‐ MAPT based on longitudinal FC changes using k‐means clustering on rates of change in FC for four intrinsic connectivity networks associated with common MAPT ‐related syndromes: salience (SN), corticobasal syndrome (CBS), progressive supranuclear palsy (PSP), and default mode (DMN) networks. We compared Asymp‐ MAPT subgroups from each stratification on baseline and longitudinal changes in demographics, plasma neurofilament light (NfL) concentration, GMV within 246 Brainnetome regions, and neuropsychological test scores. Result Stratification of Asymp‐ MAPT based on baseline GMV showed that LowGMV had higher education levels compared to HighGMV, with no other demographic differences. Though LowGMV and HighGMV did not differ in baseline NfL, LowGMV showed greater longitudinal NfL increases. These groups did not differ in baseline or longitudinal GMV changes. LowGMV had lower baseline attention and working memory scores, and greater longitudinal decline in set‐shifting and visuospatial performance. Stratification based on FC changes yielded one group (DecFC; n = 17) exhibiting longitudinal decreases in the SN, PSP, and DMN and another (IncFC; n = 8) showing increases in each network. DecFC and IncFC did not differ in demographics, baseline or longitudinal NfL, or baseline GMV. Longitudinally, DecFC showed greater decline in posterior superior temporal and inferior parietal cortices. DecFC showed lower baseline attention and semantic fluency scores, and greater longitudinal decline in set‐shifting and visuospatial memory performance. Conclusion Lower baseline GMV within regions atrophied during the symptomatic phase and longitudinal FC declines within MAPT ‐relevant networks may signal earlier symptomatic conversion in Asymp‐ MAPT .

Abstract Background Mitochondrial dysfunction in Alzheimer's Disease (AD) is characterized by impaired energy production, oxidative stress, and disrupted calcium homeostasis, which collectively contribute to neuronal damage, synaptic dysfunction, and the progression of AD pathology. A significant knowledge gap in studying mitochondrial dysfunction in Alzheimer's Disease (AD) lies in understanding the precise molecular mechanisms linking mitochondrial metabolic alterations to neuronal damage, particularly how sex‐specific and cell‐type‐specific mitochondrial processes contribute to the progression of AD pathology. Method This study integrates multiple datasets, post‐mortem brain RNAseq from ROSMAP, Mayo Clinic brain bank and Mount Sinai Brain Bank cohort data available via AD Knowledge Portal hosted by Sage Bionetworks), Human Protein Atlas (HPA), MitoCarta3, genome‐scale human metabolic reconstruction, mitochondriome analysis, and brain & blood metabolomics data (data generated by the Alzheimer's Disease Metabolomics Consortium (ADMC)) to identify mitochondrial genes associated with disrupted metabolism in AD. Additionally, biodomain analysis was used to identify key processes associated with pathology. Result A total of 62 common genes were identified across these approaches, highlighting their potential significance in mitochondrial processes. We also developed the framework to perform metabolite‐gene correlation analyses revealing key mitochondrial‐metabolic interactions (eg. homocysteine and MTHFR gene; acetyl‐CoA and PDHA1 gene). Integrated in silico metabolic analysis identified reaction fluxes, uncovering sex‐specific differences in mitochondrial transport and tricarboxylic acid (TCA) cycle subsystems, with notable alterations in females. Biodomain enrichment analysis focused on mitochondrial processes, identifying disruptions in pathways critical for energy production and redox homeostasis. Additionally, we analyzed datasets related to metformin and complex I inhibitors, linking mitochondrial complex I modulation with metabolic alterations in AD. This analysis identified druggable targets and potential therapeutic candidates for mitochondrial metabolic pathways. Conclusion Our findings integrate multiple data sources to provide a systems‐level understanding of mitochondrial metabolism in AD. Sex‐specific differences associated with mitochondrial dysfunction in AD were identified. We identify important mitochondrial transport proteins and metabolites that could potentially be used as druggable targets in AD.

Abstract Hereditary hemorrhagic telangiectasia (HHT) causes severe recurrent epistaxis, chronic gastrointestinal bleeding, solid organ arteriovenous malformations, and significant anemia. Although it is the second most common inherited bleeding disorder worldwide, no approved therapies exist. The multicenter, US randomized, controlled PATH-HHT trial demonstrated efficacy of short-term pomalidomide treatment of epistaxis in HHT. However, questions regarding long-term safety, effectiveness, and utility for HHT-associated gastrointestinal bleeding remain. The PATH-HHT ATLAS (after trial longitudinal assessment study) was a multicenter US longitudinal observational study evaluating patients enrolled in PATH-HHT who continued pomalidomide after PATH-HHT through a poststudy drug access program, with ongoing close monitoring per PATH-HHT protocol and as mandated by the US Food and Drug Administration. Sixty-two patients treated with pomalidomide for HHT for up to 4.4 years were included. Significant, durable improvements in mean epistaxis severity score (5.55 points [baseline] to 2.80 points [month 12]; P&lt; .0001) and mean hematologic support score (9.11 red cell unit equivalents [RUEs] during 6 months pretreatment to 5.73 RUEs [months 7-12]; P = .0056) were observed. Pomalidomide was less effective for gastrointestinal bleeding than for epistaxis, particularly in patients with high red blood cell transfusion requirements. Thirty-one patients (50%) underwent dose reduction from 4 mg daily, primarily due to neutropenia, but most maintained effectiveness at 2 or 3 mg daily. Over 105.2 patient-years of pomalidomide treatment, 0 patients developed peripheral neuropathy, 1 developed venous thromboembolism, and 5 developed serious infections. In conclusion, pomalidomide was effective for HHT-associated epistaxis over extended durations, albeit with nontrivial potential toxicities, and may be considered for certain patients with HHT. This trial was registered at www.clinicaltrials.gov as NCT07018401.