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Importance Tumor-infiltrating lymphocytes (TILs) are a provocative biomarker in melanoma, influencing diagnosis, prognosis, and immunotherapy outcomes; however, traditional pathologist-read TIL assessment on hematoxylin and eosin–stained slides is prone to interobserver variability, leading to inconsistent clinical decisions. Therefore, development of newer TIL scoring approaches that produce more reliable and consistent readouts is important. Objective To evaluate the analytical and clinical validity of a machine learning algorithm for TIL quantification in melanoma compared with traditional pathologist-read methods. Design, Setting, and Participants This multioperator, global, multi-institutional prognostic study compared TIL scoring reproducibility between traditional pathologist-read methods and an artificial intelligence (AI)-driven approach. The study was conducted using retrospective cohorts of patients with melanoma between January 2022 and June 2023 across 45 institutions, with tissue evaluated by participants from academic, clinical, and research institutions. Participants were selected to ensure diverse expertise and professional backgrounds. Main Outcomes and Measures Intraclass correlation coefficient (ICC) values were calculated for the manual and AI-assisted arms using log-transformed data. Kendall W values were calculated for Clark scores (brisk = 3, nonbrisk = 2, and sparse = 1). Reliabilities of ICC and W values were classified as moderate (0.40-0.60), good (0.61-0.80), or excellent (>0.80). AI TIL measurements were dichotomized using the 16.6 and median cutoffs. Univariable and multivariable Cox regression analyses assessed the prognostic value of TIL scores adjusted for clinicopathologic variables. Results There were 111 patients with melanoma in the independent testing cohort (median [range] age at diagnosis, 61.0 [25.0-87.0] years; 56 [50.5%] male) who contributed melanoma whole tissue sections. A total of 98 participants evaluated TILs on 60 hematoxylin and eosin–stained melanoma tissue sections. All 40 participants in the manual arm were pathologists, while the AI-assisted arm included 11 pathologists and 47 nonpathologists (scientists). The AI algorithm demonstrated superior reproducibility, with ICCs higher than 0.90 for all machine learning TIL variables, significantly outperforming manual assessments (ICC, 0.61 for AI-derived stromal TILs vs Kendall W , 0.44 for manual Clark TIL scoring). AI-based TIL scores showed prognostic associations with patient outcomes (n = 111) using the median cutoff approach with a hazard ratio (HR) of 0.45 (95% CI, 0.26-0.80; P = .005), and using the cutoff of 16.6, with an HR of 0.56 (95% CI, 0.32-0.98; P = .04). Conclusions and Relevance In this prognostic study of TIL quantification in melanoma, the AI algorithm demonstrated superior reproducibility and prognostic associations compared with traditional methods. Although the retrospective nature of the cohorts limits demonstration of clinical utility, the publicly available dataset and open-source AI tool offer a foundation for future validation and integration into melanoma management.

Background Topical timolol appears to provide a curative effect in managing chronic ulcers. However, the pooled evidence of its effect hasn’t been established yet. This is the first Meta-analysis to evaluate and assess the efficacy of topical timolol in accelerating wound healing in patients with chronic refractory ulcers. Methods We comprehensively searched different electronic databases; PubMed, Scopus, Web of Science, Cochrane library and ClinicalTrials.gov from their inception to November 2024. The search was conducted without imposing any restrictions on publication date or study design. The ROB-II tool was used to assess the quality of included RCTs. NIH tools were used to evaluate the quality of included cohort and single arm studies. Risk ratios (RR) and mean differences (MD), with 95% confidence intervals (CIs), were used as effect estimates. Results Six studies were included in this systematic review, comprising two single-arm studies and four double-arm studies (3 RCTs and 1 cohort study). Data from the four double-arm studies on venous ulcers were pooled for meta-analysis. The results showed that topical timolol significantly reduced ulcer area compared to control at 2 weeks (−23.63%; 95% CI: −29.26 to −18.00; p < 0.00001), 4 weeks (−41.40%; 95% CI: −49.57 to −33.23; p < 0.00001), and 12 weeks (−23.38%; 95% CI: −39.43 to −7.33; p = 0.004). Complete ulcer healing was observed at 4 weeks (RR: 12.00; 95% CI: 1.68 to 85.84; p = 0.01) and 12 weeks (RR: 3.79; 95% CI: 0.44 to 32.54; p = 0.22). Conclusion Topical timolol has the potential to promote healing in chronic unhealed ulcers. This meta-analysis, focusing on chronic venous ulcers, consistently showed favorable results across different time points. However, the small sample sizes, lack of blinding, and inclusion of only four studies limit the generalizability of the findings.

Abstract Introduction: The data on outcomes comparing lung transplantation with ABO-identical versus ABO-compatible blood group is limited. Composite Allocation Score(CAS) was introduced on March 9th, 2023, and has considered additional points for the difficult-to-match blood group. We aimed to study the trend and outcomes of patients who underwent lung transplants with ABO identical versus compatible blood groups. Methods: We queried the United Network of Organ Sharing (UNOS) database of those who underwent lung transplantation between 10/1/1987 to 3/31/2024 and compared the outcomes between those who had ABO-identical vs ABO-compatible blood groups. Combined organ transplants and recipients age &lt;18 were excluded. Results: A total of 50,406 patients were included in our study, out of which 4,111 (8.15%) were ABO-compatible and 47,347 (91.85%) were ABO-identical. Among the recipient demographics, the ABO-compatible group compared to the ABO-identical group showed a lesser lung allocation score (40 ± 16 vs 42 ± 16), but higher A-blood group (59% vs 38%), obstructive lung disease (38% vs 33%), CAS (49 ± 13 vs 47 ± 10), mechanical ventilator support pre-transplant (6.2% vs 5.2%), need for ex-vivo lung perfusion (9.6% vs 5.8%) and listing for single lung transplant (39% vs 34%), all p&lt;0.05. The donors for the ABO-compatible group had a closer distance to the transplant center (205 ± 285 vs 226 ± 265 nautical miles), lesser white race (61% vs 64%), and gender mismatch (30% vs 32%) but higher male gender (64% vs 61%), O-blood group (81% vs 49%) and ethnicity mismatch (45% vs 41%), all p&lt;0.05. The univariate analysis showed a higher 30-day (7.2% vs 5.8%), 1-year (22% vs 19%), and 5-year (54% vs 50%) mortality among patients with ABO-compatible compared to ABO-identical recipients, all p&lt;0.001. However, the adjusted odds ratio of 30-day (0.76, 95% CI 0.36-1.47, p=0.4), 1-year (1.07, 95% CI 0.69-1.66, p=0.7) and 5-year (0.69, 95% CI 0.46-1.05, p=0.081) mortality were not statistically significant with ABO-identical when compared to ABO-compatible group on adjusting to the covariates in the multivariate analysis. The trend of ABO-compatible lung transplantation has been increasing with the introduction of CAS (figure 1). Conclusion: Patients who underwent lung transplantation with the ABO-compatible blood group donor organs had similar outcomes compared to the ABO-identical blood group. The findings of our study reinforce the importance of increasing the use of ABO-compatible organs to enhance the lung transplantation rate, aligning with the observed trend following the introduction of CAS.

ABSTRACT The rare human neuroendocrine tumors pheochromocytoma and paraganglioma (PPGL) can result from loss of mitochondrial succinate dehydrogenase. The resulting succinate accumulation is tumorigenic in certain neuroendocrine cells. Here, we explore two theoretical approaches to mitigate tumorigenic succinate accumulation in a cell culture model of PPGL. We first study a gene replacement strategy using transposition technology, and conclude that many of the changes in mitochondrial morphology, oxidative cell metabolism and succinate accumulation can be reversed by this process. We then investigate whether riboflavin supplementation has the potential to rescue succinate dehydrogenase activity in the intact SDHA catalytic subunit to suppress succinate accumulation even in the absence of SDHB. We show that this latter strategy is not successful.

Abstract Hereditary tumor predisposition syndromes pose a challenge for early detection and timely treatment of tumors. In von Hippel–Lindau disease, desirable personalized surveillance programs are lacking due to insufficient data on genotype-specific risk profiles of individual mutations. To describe neoplastic risk profiles for carriers of pathogenic and likely pathogenic VHL germline mutations, our observational study recruited 1,350 participants from 40 centers worldwide. 432 different VHL germline mutations were observed, with p.Asn78Ser, p.Arg161Ter, p.Arg161Gln, p.Arg167Gln, p.Arg167Trp and p.Tyr98His being the six most frequent, occurring in a total of 493 carriers (36.5%) and in ≥30 patients each. Age-related penetrance risks for retinal hemangioblastoma, central nervous system hemangioblastoma, renal cell carcinoma, pancreatic neuroendocrine tumors and pheochromocytoma/paraganglioma in carriers of the most frequent VHL mutations were assessed. In addition, the number of organs affected, the frequency of surgery and the outcome are reported. Pairwise comparisons of the age-dependent tumor penetrance of these six mutations showed that 47 out of 90 pairs were significantly different. The most significant associations were found in p.Tyr98His (n = 19), followed by p.Arg161Ter (n = 10). All pairwise comparisons of mutations affecting different codons showed at least one significant (P &lt; 0.05) difference, except for p.Asn78Ser vs p.Arg161Ter. Thus, tumor risk varied by VHL mutation type and location, but did not differ between the truncating mutation p.Arg161Ter and the missense mutation p.Asn78Ser. Our study demonstrates the importance of mutation-specific phenotype prediction. With appropriate validation, the data have important implications for risk assessment and decision making in tumor prevention for carriers of the respective VHL mutations.

ABSTRACT Cellular senescence is an irreversible form of cell-cycle arrest caused by excessive stress or damage. While various biomarkers of cellular senescence have been proposed, there are currently no universal, stand-alone indicators of this condition. The field largely relies on the combined detection of multiple biomarkers to differentiate senescent cells from non-senescent cells. Here we introduce a new approach: unbiased cell culture selections to identify senescent cell-specific folded DNA aptamers from vast libraries of trillions of random 80-mer DNAs. Senescent mouse adult fibroblasts and their non-senescent counterparts were employed for selection. We demonstrate aptamer specificity for senescent mouse cells in culture, identify a form of fibronectin as the molecular target of two selected aptamers, show increased aptamer staining in naturally aged mouse tissues, and demonstrate decreased aptamer staining when p16 expressing cells are removed in a transgenic INK-ATTAC mouse model. This work demonstrates the value of unbiased cell-based selections to identify new senescence-specific DNA reagents.

Abstract Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor of adults. Current therapeutic options yield dismal prognoses that have remained essentially unchanged over nearly two decades. Diffuse growth patterns, high intratumoral heterogeneity, and variable blood–brain barrier integrity limit treatment efficacy, creating challenges that rational small molecule design has not overcome. Antibody–drug conjugates have shown some promise, leading us to hypothesize that smaller folded DNA aptamers, developed in vivo via principles of natural selection, might eventually have advantages for drug delivery. Here, we document the first in vivo DNA aptamer selection involving an orthotopic patient-derived xenograft GBM mouse model to identify tumor-homing DNA aptamers. We demonstrate the preferential accumulation of these aptamers in the tumor relative to other tissues 4 h after intraperitoneal injection. The aptamers can be detected by quantitative polymerase chain reaction, fluorescent tumor staining, and stain GBM tumor section from untreated mice and the GBM tumor cells in culture. Two of three candidates are selective for the target cell line in vitro and do not bind other human tumor cells. In vivo selection of tumor-specific DNA aptamers demonstrates a novel approach for diagnostics or toxin delivery that might allow for the development of individualized therapies.

Abstract The A1 domain in Von Willebrand Factor (VWF) initiates coagulation through binding to platelet glycoprotein GPIbα receptors. Von Willebrand Disease (VWD)-Mutations in A1 that either impair (type 2M) or enhance (type 2B) platelet adhesion to VWF can locally destabilize and even misfold the domain. We leveraged misfolding in the gain-of-function type 2B VWD phenotype as a target, distinct from the normal conformation. Two nuclease-resistant 2′-fluoropyrimidine RNA aptamers were selected to discriminate normal A1 domains from a type 2B V1314D A1 variant in a glycosylated A1A2A3 tri-domain VWF-fragment. Two aptamers, W9 and V1, were isolated that selectively recognize, bind, and inhibit the A1–GPIbα interaction with WT A1A2A3 and V1314D A1A2A3, respectively. These aptamers were tested against their respective recombinant targets, plasma VWF, VWF concentrates, and patient plasma with the heterozygous type 2B VWD R1306W variant using clinical assays, surface plasmon resonance and inhibition assays of platelet adhesion to recombinant A1 and A1A2A3 domains under shear stress. The specificity of W9 and V1 aptamers confirms that pathological conformations of VWD Type 2B proteins are different from normal VWF. The availability of aptamers that distinguish normal plasma-derived VWF from VWD suggests potential applicability in clinical diagnosis of severe gain-of-function phenotypes.

Abstract Disclosure: L. Rahimi: None. F. Al-Khazal: None. N. Becker: None. L. Maher, III: None. Carriers of loss-of-function alleles in genes encoding the four succinate dehydrogenase (SDH) subunits exhibit a heightened risk for pheochromocytoma/paraganglioma (PPGL), a rare neuroendocrine tumor of chromaffin cells. Tumorigenesis begins with the somatic loss of the remaining functional gene copy, resulting in a cell devoid of functional SDH. Compared to wild-type immortalized chromaffin cells (WT imCCs), SDHB-KO immortalized chromaffin cells (SDHB-KO imCCs) are larger, grow more slowly, display deformed mitochondria, show strikingly heightened succinate accumulation, and altered metabolism. Among several mechanisms proposed to link SDH deficiency with PPGL tumorigenesis, the accumulation of succinate as an oncometabolite is a leading hypothesis. We describe two attempts to suppress succinate accumulation in SDHB-KO imCCs. First, riboflavin has previously been shown to enhance residual SDHA flavinylation and enzymatic function, possibly reducing succinate levels by supporting conversion of accumulated succinate to fumarate in the absence of SDHB. We tested this by treating WT and SDHB-KO imCCs with 5 µM riboflavin for 2 weeks. Metabolomic analysis did not show decreased succinate, suggesting that enzyme activity of any remaining SDHA subunit was not rescued by increased flavinylation. Second, we envisioned a gene therapy approach to replace defective SDHB with a rescuing transgene. SDHB-KO imCCs received DNA encoding SDHB or SDHC cDNAs via transposon technology, confirmed by PCR and analyzed by western blot analyses. These studies showed that whereas WT imCCs successfully expressed processed transgene products in mitochondria, SDHB-KO imCCs could not process or transport rescuing SDH subunits into mitochondria. This shows that transposed SDHB-KO imCCs cannot properly express a rescuing SDHB subunit and do not revert to a wild-type phenotype. It is likely that the severely damaged mitochondria of SDHB-KO imCCs are incompetent to process and import rescuing SDHB subunits, so unprocessed protein accumulates without effect. This defect may lie in the mitochondrial targeting peptide recognition system because the targeting peptide of rescuing SDHB protein is not removed in SDHB-KO imCCs as judged by western blotting. In summary, we show the failure of two conceptually simple tactics to suppress succinate accumulation in SDHB-KO imCCs with highly defective mitochondria: rescue of SDHA by supporting flavinylation, or rescue of SDHB with a replacement gene copy. Further research may allow screening for small molecules that suppress succinate accumulation by other mechanisms. It remains to be determined if blocking succinate accumulation would be therapeutic in SDH-loss PPGL tumors. Presentation: 6/1/2024