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Abstract INTRODUCTION Associations of short‐term use of menopausal hormone therapy (mHT) with Alzheimer's disease (AD) and structural magnetic resonance imaging (MRI) biomarkers were investigated 10 years after an mHT trial. METHODS Recently menopausal women with good cardiovascular health were randomized to oral conjugated equine estrogens (oCEE) or transdermal 17β‐estradiol (tE2) and micronized progesterone, or placebo for 4 years. Amyloid beta (Aβ) on positron emission tomography, hippocampal atrophy, and dorsolateral prefrontal cortex thickness on MRI were assessed 10 years after completion of the mHT trial ( n = 266). RESULTS Aβ and structural MRI biomarkers were not different in the oCEE and tE2 groups compared to placebo. Apolipoprotein E ε4 status did not modify the findings. DISCUSSION There was no evidence of adverse effects or benefits associated with 4 years of use of oral or transdermal mHT on Aβ and structural MRI biomarkers in relatively healthy women, 10 years after mHT. Findings support the long‐term safety of short‐term use of mHT on brain health. CLINICAL TRIALS REGISTRATION NCT00154180 Kronos Early Estrogen Prevention Study (KEEPS) Highlights There were no menopausal hormone therapy–related adverse effects or benefits on amyloid beta and magnetic resonance imaging biomarkers in the long term. Apolipoprotein E ε4 carrier status did not modify these findings. Findings align with neutral cognitive and cerebrovascular outcomes in this cohort.

Key Points Inclisiran for LDL cholesterol reduction was analyzed post hoc in patients with CKD. Mean percentage LDL cholesterol reduction from baseline was around 50% regardless of eGFR in phase 3 trials. Inclisiran showed sustained and effective LDL cholesterol–lowering in patients across various levels of eGFR values, without new safety findings. Background Lowering LDL cholesterol reduces the risk of atherosclerotic cardiovascular disease in patients with CKD. The efficacy and safety of inclisiran versus placebo in patients without and with CKD were investigated in a post hoc pooled analysis of three phase 3 trials (ORION-9, ORION-10, and ORION-11). Methods Patients with heterozygous familial hypercholesterolemia, atherosclerotic cardiovascular disease or its risk equivalent, and elevated LDL cholesterol were randomized 1:1 to subcutaneous inclisiran or placebo on days 1 and 90 and every 6 months thereafter for 540 days. Patients were stratified based on baseline eGFR (by CKD Epidemiology Collaboration equation): ≥90, 60 to <90, 45 to <60, and 15 to <45 ml/min per 1.73 m 2 . Coprimary end points were percentage change in LDL cholesterol at day 510 and time-adjusted percentage change after day 90 and through day 540. Safety was also evaluated. Results Of 3660 patients, 1610 (44%) had eGFR ≥90, 1608 (44%) 60 to <90, 300 (8%) 45 to <60, and 142 (4%) 15 to <45 ml/min per 1.73 m 2 . The mean (95% confidence interval) placebo-corrected percentage changes in LDL cholesterol from baseline at day 510 in patients with eGFR ≥90, 60 to <90, 45 to <60, and 15 to <45 ml/min per 1.73 m 2 were −49.9% (−53.2 to −46.6), −51.2% (−54.4 to −48.0), −54.7% (−62.5 to −47.0), and −44.7% (−57.6 to −31.8), respectively ( P < 0.001); the corresponding mean (95% confidence interval) time-adjusted placebo-corrected percentage changes in LDL cholesterol from baseline after day 90 through day 540 were −48.4% (−50.8 to −46.1), −51.8% (−54.2 to −49.4), −55.6% (−61.0 to −50.2), and −50.4% (−59.3 to −41.5; each P < 0.001). Significant decreases in total cholesterol, apolipoprotein B, non-HDL cholesterol, and lipoprotein(a) occurred in all eGFR groups. Inclisiran was well tolerated without new safety findings. Conclusions Inclisiran demonstrated sustained and effective LDL cholesterol reduction in patients with or at risk of atherosclerotic cardiovascular disease, regardless of baseline eGFR as low as 15 ml/min per 1.73 m 2 , without new safety findings. Clinical Trial registry name and registration number: ClinicalTrials.gov, ORION-9 (NCT03397121), ORION-10 (NCT03399370), and ORION-11 (NCT03400800).

TPS259 Background: Despite currently approved therapies, most patients with metastatic colorectal cancer (mCRC) progress through all approved treatments and eventually succumb to their disease. Thus, novel therapies for chemotherapy-refractory mCRC are desperately needed. Inhibition of the DNA unwinding enzyme TOP1 is a well-characterized and clinically successful treatment strategy for mCRC. CBX-12 is a novel peptide-drug conjugate comprised of an exatecan warhead linked to a pH-Low Insertion Peptide (pHLIP). The principle behind this targeting mechanism is based on the Warburg effect, in which tumors establish a low pH extracellular environment. The pHLIP moiety of CBX-12 preferentially forms a unidirectional, transmembrane alpha helix in a low-pH extracellular environment. Thus, CBX-12 delivers its exatecan cytotoxic warhead preferentially into tumor cells. In mouse xenograft models, CBX-12 inhibited the HCT116 CRC xenograft model more potently than an equimolar dose of free exatecan. The phase 1 study of CBX-12 reported cytopenias as the predominant AEs and confirmed responses in several solid tumors including mCRC. Methods: This is a single-arm, phase 2 trial, with a Simon 2-stage design assessing CBX-12 for the treatment of chemotherapy-refractory mCRC. The primary objective is efficacy determined by RECIST v1.1 response rate. Secondary and exploratory objectives include evaluating the PK and PD as well as the progression-free survival (PFS) on CBX-12. Key inclusion criteria include: age ≥18 years, ECOG performance status ≤1, histologically or cytologically confirmed mCRC that is MSS/pMMR based on CLIA-approved testing, and progression on 2 or more lines of chemotherapy. This design requires 17 patients for stage 1, with termination if 1 or fewer objective responses are seen out of 17. If 2 or more patients out of 17 responds, then an additional 18 patients will be enrolled for stage 2. If 5 out of the total of 35 patients (14%) respond, then the trial would be declared successful. Correlative studies will include tumor plus peripheral blood PK to evaluate effectiveness of CBX-12 tumor targeting, ctDNA, and several tumor PD markers including TOP1-DNA trapping, SLFN11 expression, DNA damage, and apoptosis. The trial opened to enrollment on 8/21/25. Clinical trial information: NCT06730100 .

Abstract Background More than 85% of Alzheimer's disease (AD) donor brains exhibit some degree of cerebral amyloid angiopathy (CAA), which is characterized predominantly by Aβ40 deposits in the brain vasculature and can cause blood brain barrier (BBB) leakage. CAA is associated with risk of infarcts, cerebral hemorrhages, and cognitive decline. Brain cell type‐specific mechanisms that influence CAA pathology, as well as related measures such as Aβ40 biochemical measures and tight junction proteins, an indicator of BBB integrity, are still elusive. Method We performed single‐nucleus RNA sequencing (snRNAseq) of temporal cortex tissue from 79 AD donors, with varying levels of CAA co‐pathology. Measurements of soluble, insoluble and membrane‐associated fractions of brain Aβ40 and tight junction protein (claudin‐5, occludin) levels were available for these donors. We correlated these measures with cell proportions. We also conducted differential gene expression and analyses for intercellular communications and regulatory networks. Result We identified 25 clusters and annotated them with major brain cell types. There were 3 oligodendrocyte, 7 excitatory and 7 inhibitory neuronal, 2 microglial, 2 astrocytic, and 1 each for endothelial, pericytic, fibroblast, and oligodendrocyte precursor cell clusters. We found that higher CAA and Aβ40 levels correlate with reduced neuronal and increased microglial, astrocytic and vascular cell proportions. Increased tight junction proteins correlate with increased inhibitory neuronal and reduced microglial, astrocytic and vascular cell proportions. Among all clusters, one microglial, both astrocytic and the pericytic clusters have greater number of genes significantly associated with CAA, Aβ40 and tight junction protein levels. Most significant genes have negative associations with CAA and membrane‐bound Aβ40 and positive with tight junction protein measures. Using these genes as input for intercellular communications analysis, we prioritized ligand‐target interactions from microglia or astrocytes to pericytes. Some of the prioritized targets are transcription factors in pericytes. Using regulon analysis, we identified genes downstream of these transcription factors that are also significantly associated with lower CAA and membrane‐bound Aβ40 and higher tight junction protein levels. Conclusion We revealed glia‐to‐pericytes intercellular interactions and pericytic gene regulatory networks that are potentially perturbed by CAA and BBB breakdown. Future directions include analyzing external snRNAseq datasets and performing functional validations.

Abstract Background Cerebral amyloid angiopathy (CAA), characterized by the accumulation of amyloid‐beta in the cerebrovasculature, affects blood vessel integrity leading to brain hemorrhages and an accelerated cognitive decline in Alzheimer's Disease (AD) patients. Our previous genome‐wide association study (GWAS) identified a LINC‐PINT splice variant associated with lower CAA levels in individuals without the APOE e4 allele and higher levels of LINC‐PINT expression in the brain of AD donors. In this study, we expand our GWAS to include additional donors with AD and those lacking significant AD neuropathology (non‐AD), with available CAA scores. Method In our prior study we assessed 853 AD donors. We expanded this by addition of genetic data from 550 AD and 502 non‐AD donors from the Mayo Clinic Brain Bank, scored for CAA. We performed QC and imputation (TOPMED) of all datasets. We conducted GWAS in AD only ( N = 1,363), non‐AD only, and all donors ( N = 1,865) by testing imputed variant dosages for association with square root transformed CAA using linear regression, adjusting for relevant covariates. To assess associations in the context of major CAA risk factors, we performed interaction analysis with APOEe4 presence and sex; and pursued stratified analyses. Result Variants at the APOE locus were identified as the most significant in our study. In addition, several other variants approached genome‐wide significance after adjusting for AD neuropathology (Braak stage and Thal phase). The LINC‐PINT splice variant remained associated with lower CAA scores in AD donors without the APOEe4 risk allele . To enhance the robustness of our findings, we are pursuing further expansion of our study cohort to include other available datasets. To explore putative functional consequences of key variants we are collecting peripheral gene expression measures in participants from Mayo Clinic with neuroimaging measures including microhemorrhages. Conclusion We expect this study will provide further insights into the genetic architecture underlying risk for CAA, both in the context of significant AD pathology, and without. Characterization of genetic variants and their functional outcomes may lead to new avenues of research aimed at identifying biomarkers and therapies to treat CAA, a common co‐pathology in those with and without AD.

Abstract Background Alzheimer's disease (AD) affects all brain cells and has complex genomic and immunological alterations. Previous research discovered missense AD risk or protective variants in microglial genes ABI3 and PLCG2, respectively. Expression levels of these genes are altered in AD and can influence microglial function. This study aims to uncover protective, and risk microglial molecular signatures associated with these variants to determine their role in microglial subtypes and states in AD by single cell expression and functional studies. Method We generated microglia‐enriched snRNAseq data from donors harboring either AD protective PLCG2 or AD risk ABI3 missense mutations, or neither mutation. After standard snRNAseq QC, we performed differential expressed gene (DEG) analysis of all microglial cells between variant carriers and non‐carriers using MAST. We defined protective signature as genes that are both down in ABI3 and up in PLCG2 mutation‐carriers. In contrast, risk signature genes are up in ABI3 and down in PLCG2 mutation‐carriers. We investigated the conservation of protective and risk signatures across multiple datasets, including scRNAseq data from iPSC‐derived microglia cells carrying PLCG2 protective variant, snRNAseq data from AD‐resilient donors, and data from AD and other diagnostic groups across multiple brain regions sourced from external datasets. Result We obtained snRNAseq profiles of 35,000 microglia from AD variant carriers. Our DEG analysis among all microglia cells provided 227 microglial protective and 293 risk signature genes defined by these variants. Using integrated analysis of multiple internal and external datasets, we further narrowed down these signatures. We determined that these high‐confidence protective signature genes are downregulated in early AD, upregulated in late AD brains and positively‐correlated with protective variant load in in vitro models. In contrast, risk signature genes are upregulated in early AD, downregulated in late AD and resilient donors. Risk signature expression is decreased with protective PLCG2 variant load, and altered with Aβ treatment in in vitro models. Conclusion Our study uncovers microglia specific protective and risk signatures associated with AD using sn/scRNAseq datasets from multiple sources and models. These findings nominate novel immune targets and pathways with implications for microglial function in health and disease, and ultimately therapeutic potential.

Abstract Background Alzheimer's disease (AD) affects all brain cells and has complex genomic and immunological alterations. Previous research discovered missense AD risk or protective variants in microglial genes ABI3 and PLCG2 , respectively. Expression levels of these genes are altered in AD and can influence microglial function. This study aims to uncover protective, and risk microglial molecular signatures associated with these variants to determine their role in microglial subtypes and states in AD by single cell expression and functional studies. Method We generated microglia‐enriched snRNAseq data from donors harboring either AD protective PLCG2 or AD risk ABI3 missense mutations, or neither mutation. After standard snRNAseq QC, we performed differential expressed gene (DEG) analysis of all microglial cells between variant carriers and non‐carriers using MAST. We defined protective signature as genes that are both down in ABI3 and up in PLCG2 mutation‐carriers. In contrast, risk signature genes are up in ABI3 and down in PLCG2 mutation‐carriers. We investigated the conservation of protective and risk signatures across multiple datasets, including scRNAseq data from iPSC‐derived microglia cells carrying PLCG2 protective variant, snRNAseq data from AD‐resilient donors, and data from AD and other diagnostic groups across multiple brain regions sourced from external datasets. Result We obtained snRNAseq profiles of 35,000 microglia from AD variant carriers. Our DEG analysis among all microglia cells provided 227 microglial protective and 293 risk signature genes defined by these variants. Using integrated analysis of multiple internal and external datasets, we further narrowed down these signatures. We determined that these high‐confidence protective signature genes are downregulated in early AD, upregulated in late AD brains and positively‐correlated with protective variant load in in vitro models. In contrast, risk signature genes are upregulated in early AD, downregulated in late AD and resilient donors. Risk signature expression is decreased with protective PLCG2 variant load, and altered with Aβ treatment in in vitro models. Conclusion Our study uncovers microglia specific protective and risk signatures associated with AD using sn/scRNAseq datasets from multiple sources and models. These findings nominate novel immune targets and pathways with implications for microglial function in health and disease, and ultimately therapeutic potential.

Abstract Background Cerebral amyloid angiopathy (CAA), characterized by the accumulation of amyloid‐beta in the cerebrovasculature, affects blood vessel integrity leading to brain hemorrhages and an accelerated cognitive decline in Alzheimer’s Disease (AD) patients. Our previous genome‐wide association study (GWAS) identified a LINC‐PINT splice variant associated with lower CAA levels in individuals without the APOE e4 allele and higher levels of LINC‐PINT expression in the brain of AD donors. In this study, we expand our GWAS to include additional donors with AD and those lacking significant AD neuropathology (non‐AD), with available CAA scores. Method In our prior study we assessed 853 AD donors. We expanded this by addition of genetic data from 550 AD and 502 non‐AD donors from the Mayo Clinic Brain Bank, scored for CAA. We performed QC and imputation (TOPMED) of all datasets. We conducted GWAS in AD only ( N = 1,363), non‐AD only, and all donors ( N = 1,865) by testing imputed variant dosages for association with square root transformed CAA using linear regression, adjusting for relevant covariates. To assess associations in the context of major CAA risk factors, we performed interaction analysis with APOEe4 presence and sex; and pursued stratified analyses. Result Variants at the APOE locus were identified as the most significant in our study. In addition, several other variants approached genome‐wide significance after adjusting for AD neuropathology (Braak stage and Thal phase). T he LINC‐PINT splice variant remained associated with lower CAA scores in AD donors without the APOEe4 risk allele. To enhance the robustness of our findings, we are pursuing further expansion of our study cohort to include other available datasets. To explore putative functional consequences of key variants we are collecting peripheral gene expression measures in participants from Mayo Clinic with neuroimaging measures including microhemorrhages. Conclusion We expect this study will provide further insights into the genetic architecture underlying risk for CAA, both in the context of significant AD pathology, and without. Characterization of genetic variants and their functional outcomes may lead to new avenues of research aimed at identifying biomarkers and therapies to treat CAA, a common co‐pathology in those with and without AD.

Multiplex immunofluorescence and immunohistochemistry (mIF/IHC) are increasingly employed antibody-based technologies that use tissue sparingly and facilitate the detection of co-localized or neighboring biomarkers. Specifically, these platforms enable spatial analyses of the tumor microenvironment as well as extended applications, for example, describing normal tissue anatomy, autoimmunity, infectious diseases, etc. mIF/IHC has greatly enhanced biomarker discovery efforts, and a growing number of studies suggest superiority to traditional IHC. Standardization of staining approaches, reporting of image analysis strategies and resultant data is critical for facilitating cross-study comparisons, validation, deployment, and generalization of findings. To address this challenge, The Society for Immunotherapy of Cancer (SITC) previously published two articles providing best practice guidelines for mIF/IHC staining, image analysis, and data sharing. Here, SITC convened stakeholders to develop the third article in the series, a consensus checklist for scientific reporting of mIF/IHC data to support and complement the best practice guidelines. The checklist includes critical components of mIF/IHC applications to be defined within publications such as detailed descriptions of analytical validation; image acquisition, selection, and registration methods; and cell clustering and spatial analysis strategies, amongst others. Such information will help with data reproducibility and comparison across studies towards future drug and assay development.

Background: Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare, disabling, genetic disorder characterized by progressive heterotopic ossification (HO). FOP care pathways are complex, and there are many underrepresented patients globally. Objectives: This scoping review aimed to identify global FOP case reports and investigate real-world management practices. Evidence sources, eligibility criteria, and charting methods: International/local databases and congress proceedings were searched. Publications reporting patient-level data for ⩾1 patient with an FOP primary diagnosis, published January 2000–January 2024, were eligible. Publication details, patient demographics, disease presentation, and care pathway were extracted. Data were charted through categorization of themes and frequency analyses. Results: Of 6064 publications screened, 369 were eligible, reporting on 541 patients with FOP. Most publications (82.4% (304/369)) reported an individual case; the remaining reported 2–25 cases. Cases were from 57 countries, most commonly in the Asia/Pacific region (47.0% (254/541)). Most patients had reportedly seen a single specialist (64.8% (59/91)), and care from multidisciplinary teams was reported for only 5.5% (5/91). HO (84.7% (458/541)) and great toe malformations (66.7% (361/541)) were the most commonly reported signs, though mobility limitations (46.2% (96/208)) and growths/lesions (38.5% (80/208)) were most common before diagnosis. The predominant drug classes reported for FOP treatment were corticosteroids (52.6% (102/194)) and nonsteroidal anti-inflammatory drugs (39.7% (77/194)). Surgeries were reported in 35.1% (190/541) of patients. Where specified, the most common surgery types were malformation correction/bone resection (43.6% (78/179)) and biopsies (34.1% (61/179)). Conclusion: This scoping review synthesized valuable real-world insights from case reports of FOP, potentially supporting outreach initiatives in underserved regions and facilitating shared learnings on diagnosis and treatment. Although the inherent selection bias of case studies toward atypical cases limits definitive conclusions, some global trends in the clinical management practices were revealed.