Single Site Research Articles

Abstract PR009: Discovery of covalent NRAS inhibitors targeting the Palmitoylation site through imaging-based high-throughput screening

Abstract NRAS mutations play a crucial role as oncogenic drivers of tumor cell growth and chemo-resistance in melanoma and acute myeloid leukemia (AML). In melanoma, NRAS mutations occur in approximately 25% of cases, and are generally mutually exclusive with BRAF-mutations. NRAS mutant tumors often exhibit an aggressive phenotype, such as thicker lesions, increased mitotic activity, and higher rates of lymph node metastasis. Surgical resection of NRAS mutant melanoma has a poor prognosis with a lower median overall survival (OS) compared to non-NRAS-mutant melanoma (8.2 vs. 15.1 months for stage IV disease). Current treatment strategies for stage III/IV non-resectable metastatic NRAS mutant melanoma include immunotherapy and indirect targeting of NRAS associated signaling pathways, highlighting the urgent need to develop small molecule NRAS inhibitors. NRAS palmitoylation is essential for its oncogenic function by regulating its trafficking and anchoring to the plasma membrane. Unlike other RAS isoforms, NRAS relies on a single palmitoylation site (C181) for its plasma membrane localization and this unique dependency makes NRAS more sensitive to palmitoylation inhibitors. To identify selective NRAS inhibitors that covalently modify C181 and prevent its palmitoylation, we employed an imaging-based NRAS translocation high-throughput screen using a library of 23,000 covalent molecules. We identified a set of covalent inhibitors that selectively target NRAS translocation but do not affect HRAS localization. We further confirmed the direct binding of these compound hits to NRAS at its palmitoylation site (C181) using intact mass spectrometry and chemo-proteomic peptide mapping techniques. Moreover, we validated the functional impact of these compound hits by demonstrating their selective inhibition of cell growth in mutant NRAS-driven melanoma cell lines. In conclusion, our study presents the first direct inhibitor for NRAS, discovered through a high-throughput covalent screening approach. This finding holds promising implications for the development of novel targeted therapies for NRAS mutant tumors. Citation Format: Zhao Wang, Keith Moore, Simon Ng, Chao Wang, Ronald Tomlinson, Huay-Keng Loke, Jamie Rice, Ivan Cornella. Discovery of covalent NRAS inhibitors targeting the Palmitoylation site through imaging-based high-throughput screening [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR009.

Plasma pTau217: single vs multiple phospho‐site assays.

AbstractBackgroundBlood biomarkers have gained much attention in recent years given their increasing ability to indicate amyloid pathology and excellent performance in distinguishing diagnostic groups in the context of Alzheimer’s disease (AD). Plasma pTau217 has been considered the best candidate biomarker to serve as diagnostic and prognostic tool for clinical trials. However, biomarker assays targeting tau phosphorylation on Thr217 may differ because of their composition (e.g., targeting multiple or single phosphorylation sites) which may lead to distinct associations with pathology. Thus, we aimed to compare two plasma pTau217 immunoassays that differ in regard to their target specificity.MethodParticipants from the TRIAD cohort with available cross‐sectional plasma and imaging data, incorporating within the AD spectrum, were included in this study (Young = 25;CU‐ = 107;CU+ = 32;MCI+ = 42;AD+ = 47;MCI‐ = 19). Plasma pTau was quantified using the assays from Janssen (pTau217+; which exhibits enhanced signal with co‐phosphorylation of pTau212) and from ALZpath (single phosphorylation on pTau217), both performed on the Simoa platform. Amyloid and tau pathologies were indexed by PET using [18F]AZD4694 and [18F]MK6240, respectively. ANCOVA compared biomarker values across groups, Spearman rank tested the correlations between them. Linear regression models (LM) evaluated the association between plasma and PET biomarkers globally and at the voxel level.ResultBiomarker levels (fold mean of CU‐) were comparable across diagnostic groups, showing the expected increases in amyloid positive groups. Both plasma assays were highly correlated with amyloid (Fig.1), which was also observed on the LM adjusted by age and sex, and on the voxel‐wise analysis (Fig.2). Associations with tau PET SUVR (medial temporal) were also significant and showed similar distribution between the two pTau assays. When neocortical tau was evaluated, however, pTau217+ had a better association as well as higher correlation coefficients within amyloid+ (Fig.3) and neocortical tau+ groups than did ALZpath pTau217.ConclusionIn general, pTau217 assays with different antibody specificity showed very similar associations with PET. However, pTau217+ achieved a better association with neocortical tau as compared to ALZpath pTau217. A biomarker targeting multiple phosphorylation sites might be a better proxy of advanced tau, therefore may offer improved ability to detect and exclude participants with advanced AD pathology for clinical trials.

DNA methylation alterations in Lewy Body Dementia

AbstractBackgroundThe epigenetic mechanism of DNA methylation has been implicated in the pathophysiology of neurodegenerative disorders such as Lewy body dementia (LBD). DNA methylation profiling may help to identify novel treatment targets and early disease markers. We hypothesized that DNA methylation alterations are detectable in blood due the influence of genetic and environmental factors as well as immune system changes associated with LBD.MethodGenome‐wide DNA methylation profiling was carried out using reduced representation bisulfite sequencing in blood samples from 89 LBD patients and 67 controls. Fifty five LBD patients had amyloid PET imaging. Data processing was performed using FastQC, TrimGalore, Bismark and MethylKit. DNA methylation alterations at specific CpG sites as well as at promoter, CpG island and CpG shore regions were compared between LBD cases and controls. A log odds logistic regression was used to identify differential DNA methylation after adjusting for age, sex and smoking status. The false discovery rate (FDR) was used for correction for multiple testing. We also tested the diagnostic classification performance of the differentially methylated regions using receiver operating characteristic (ROC) curve analysis and examined the potential of DNA methylation as a predictor of amyloid PET positivity in LBD.ResultTwo single CpG sites at TNFRSF1B and EMC6 and three CpG shore regions at ABCG5, ETAA1 and TENM4 reached genome‐wide significance in the differential DNA methylation analysis comparing LBD cases and controls. The combined panel of the top three differentially methylated CpG shores reached an area under the curve (AUC) of 0.91 for the diagnosis of LBD compared to controls (sensitivity 0.89, specificity 0.85). DNA methylation at TENM4 was associated with amyloid PET positivity in LBD with an AUC of 0.84 (sensitivity 0.99, specificity 0.55).ConclusionOur data suggest that DNA methylation alterations in blood are observed in a cohort of LBD patients compared to controls. Our findings need to be replicated in larger cohorts and tested for specificity in LBD in order to explore their future potential as diagnostic markers and/or targets for novel therapeutics.

Advanced Equilibrium Studies for the Synergetic Impact of Polyaniline on the Adsorption of Rhodamine B Dye by Polyaniline/Coal Composite.

The synergetic improvement effect of the polyaniline (PANI) hybridization process on the adsorption of rhodamine B dye (RB) by PANI/coal hybrid material (PANI/C) has been evaluated using both traditional equilibrium modeling and advanced isotherm investigations. The composite was prepared by polymerizing polyaniline in the presence of coal fractions with a surface area of 27.7 m2/g. The PANI/C hybrid has an improved capacity to adsorb RB dye (423.5 mg/g) in comparison to coal particles (254.3 mg/g). The maintained increase in the elimination properties of PANI/C has been illustrated using the steric characteristics of active site density (Nm) as well as the total number of adsorbed RB on a single active site (n). However, the incorporation of PANI did not yield any substantial impact on the existing active sites' quantity, but the hybridization processes greatly influenced the selectivity and affinity of each active site, in addition to the aggregation characteristics of the dye as it interacts with the composite's surface. Whereas raw coal can only adsorb three molecules of RB, each active site throughout the PANI/C surface can adsorb approximately eight RB molecules. This is also evidence of RB dye adsorption in a vertical arrangement, which involves multimolecular processes. The Gaussian energy (4.01-5.59 kJ/mol) and adsorption energy (-4.34-4.68 kJ/mol) revealed the controllable impact of physical mechanisms. These mechanisms may include van der Waals forces, dipole-dipole interactions, and hydrogen bonds (<30 kJ/mol). The thermodynamic functions, such as enthalpy, internal energy, and entropy, that have been assessed provide evidence supporting the exothermic and spontaneous nature of the RB uptake processes by PANI/C.

TREM2‐related Polygenic Risk Score predicts defect Innate Immune Activation in Alzheimer’s Disease

AbstractBackgroundTREM2 plays a central role in immune response which, in turn, is strongly related to Alzheimer’s disease (AD) pathology. We previously found an AD‐subtype with and without innate immune activation based on CSF proteomics. Possibly, genes associated with TREM2 pathways could explain differences between these two subtypes since R47H and R62H mutations are associated with decreased microglial activation. We tested this hypothesis in a large single site cohort, and compared them on PRS including previously reported GWAS genes associated with TREM2 pathways.MethodWe included participants from the Amsterdam Dementia Cohort who had AD subtyping based on CSF proteomics available (n = 466). Based on literature, we labelled 19 of 83 known AD risk genes as belonging to TREM2 pathways and used these to construct a TREM2‐PRS. We studied associations between a standard AD‐PRS with 83 SNPs, TREM2‐PRS with 19 SNPs and non‐TREM2‐PRS with 64 SNPs between two AD‐subtypes being (1) without innate immune activation and (2) with innate immune activation, using logistic regression models (correcting for sex, age and 5 principal components).ResultThe cohort involved 146 controls (mean age 61 years, 34% female) and 195 AD cases: 97 without immune activation subtype (mean age 64 years, 52% female) and 98 with immune activation subtype (mean age 68 years, 54% female). Both subtypes showed comparable increased risk on the standard AD‐PRS compared to controls (OR 1.65, 95%CI 1.2‐2.3, p = 0.002 and OR 1.7, 95%CI 1.1‐2.5, p = 0.006) (Table 1). A higher TREM2‐PRS was associated with no immune activation subtype (OR 1.5, 95%CI 1.1‐2.1, p = 0.008). The PRS of the other 64 risk variants, which were not labelled as belonging to TREM2 pathways, was associated with immune activation subtype (OR 1.5, 95%CI 1.1‐2.1, p = 0.02).ConclusionAD individuals with a CSF proteomic‐based subtype without immune activation show more genetic variants in TREM2 pathways, whereas these SNPs are not related to the subtype with innate immune activation. These findings provide evidence for genetic differences underlying AD‐subtypes, and suggest that therapies targeting immune functioning may need to tailor therapies to AD‐subtype (i.e., additional activation vs. decreasing activation).

CO2 hydrogenation to methanol over Cu-ZnO-CeO2 catalyst: Reaction structure–activity relationship, optimizing Ce and Zn ratio, and kinetic study

The catalytic conversion of captured CO2 to MeOH is a possible solution to mitigate the rising CO2 emissions and to develop a carbon neutral society. The present study unveiled the effect of incorporating CeO2 to the precursor chemistry of Cu/ZnO catalytic system. The relation between the density of strong basic sites and H2 adsorption sites with catalytic activity is established. At 250 °C, 30 bar and 3000 mLgcat−1h−1, more than 106.7 gMeOH kgcat−1h−1 the MeOH STY is obtained for the CZCe-10 catalyst. Further, the reaction kinetics are performed for the best-screened catalyst, within the temperature 220 °C-310 °C, pressure 10 bar-30 bar, GHSV 1500–7500 mL gcat−1h−1 and H2/CO2 ratio 1:1 to 5:1 range. The literature reported single site and dual site kinetic models are applied to the experimental data, and the dual site reaction mechanism is found to be better based on model correlation’s error variances.

Translation and validation of an Urdu version of the Vaccine Attitudes Examination (VAX) scale

Abstract Introduction Despite being one of the most effective methods of preventing the spread of communicable diseases1, vaccination has been subjected to criticism. Several scales have been formulated that were used to document attitudes towards vaccination. However, they either focus on parental attitudes towards children’s vaccinations or are targeted towards an individual’s attitude towards a specific vaccine. A scale that could document an individual’s attitude about vaccinations in general was developed by Martin and Petrie2. Aim The study aimed to translate and validate an Urdu version of the Vaccine Attitude Examination (VAX) scale in a selected Pakistani sample. Methods A cross sectional study was conducted for a month (Sept. 2021) in an outpatient department of a healthcare facility in Karachi, Pakistan. All adult men and women, as well as their caregivers (≥ 18 years), who visited the clinic, spoke Urdu as their first language, and were eligible for COVID-19 vaccine were invited. Convenience sampling method was selected, and sample size calculation was based on an item response ratio of 1:20. An Urdu version of the VAX scale was developed. Data were analysed using IBM SPSS version 23. The reliability was assessed using Cronbach’s alpha (α) and intraclass correlation coefficient (ICC) was calculated. The model fitness was evaluated through IBM AMOS version 25. It done carried out using confirmatory factor analysis (CFA) and reporting of fit indices, namely goodness of fit index (GFI), adjusted goodness of fit index (AGFI), Tucker Lewis index (TLI), comparative fit index (CFI), and root mean square error of approximation (RMSEA). The scale was considered as reliable if Cronbach’s (α) was &amp;gt;0.7. Model fitness was considered acceptable if most fit indices were in acceptable ranges3, i.e., GFI, TLI, CFI &amp;gt; 0.9 and RMSEA &amp;lt; 0.6, as well as p value &amp;lt; 0.05. This study was granted ethical clearance by the Institutional Review Board of Tabba Heart Institute, Karachi, Pakistan (Reference# THI/IRB/FQ/22-09-21/017). Results A total of 211 responses were analysed. The reliability of the scale, i.e., Cronbach’s alpha value was 0.821, and remained the same, i.e., &amp;gt; 0.8 during item deletion. CFA using a 4-factor model revealed the following values for fit indices; GFI = 0.944, TLI = 0.953, CFI = 0.966, and RMSEA = 0.051 (p value = 0.008). Discussion/Conclusion Although a very small scale study, the findings suggest that the Urdu version of VAX scale is reliable and a valid instrument for use in an Urdu speaking population. The scale showed good internal consistency and acceptable model fitness. The use of convenience sampling and being conducted at a single site may affect the generalisability of the results. However, this was the first time the VAX Urdu version was made available for an Urdu speaking population. It is hoped that the attitudes toward vaccines that are measured by the Urdu VAX will provide valuable information as public health officials and others work to improve vaccine uptake. Availability of the Urdu version will enable clinicians to assess the Urdu speaking persons’ attitude towards general vaccination including COVID-19 vaccination.

Synergistic Optimization of Morphology and Vacancies on Diatomic Rhodium Catalysts Dispersed on Carbon Nitride for Efficient Photocatalytic Reduction of CO2

AbstractDiatomic site catalysts (DAC) have better performance with higher metal content and more flexible active sites compared with single atomic site catalysts (SAC). Herein, the authors for the first time achieved Rh2 DAC on loose porous g‐C3N4 hollow nanospheres with N‐vacancies and applied to photocatalytic CO2 reduction reaction, overcoming the current limitations of the low electron–hole recombination rate and prolong the lifetime of the photogenerated carrier. The high specific surface area of hollow nanosphere facilitates the uniform dispersion and anchoring of Rh2 diatomic pairs, while the N‐vacancies induce a stable 3N/Rh‐Rh/1N2C coordination between the carrier and Rh2 diatomic pairs. The local charges on the support framework with N vacancies tend to be transferred to Rh2 diatomic site by 3N/Rh‐Rh/1N2C bridge, which made the charge enriched Rh2 diatomic site become the active center of reaction, enhance charge separation efficiency of Rh2/HCNS‐Nv. Compared with Rh1 SAC, further Density Functional Theory (DFT) calculation confirms that Rh2 DAC can effectively stabilize rate‐limiting intermediates CHO*, and well weaken the C─O bond strength in CH3O* species, promote the generation and separation of CH4, resulting in high CO2 reduction efficiency and CH4 electron selectivity of up to 91.65%.

Complicated target recognition by archaeal box C/D guide RNAs.

Box C/D RNAs guide the site-specific formation of 2'-O-methylated nucleotides (Nm) of RNAs in eukaryotes and archaea. Although C/D RNAs have been profiled in several archaea, their targets have not been experimentally determined. Here, we mapped Nm in rRNAs, tRNAs, and abundant small RNAs (sRNAs) and profiled C/D RNAs in the crenarchaeon Sulfolobus islandicus. The targets of C/D RNAs were assigned by analysis of base-pairing interactions, in vitro modification assays, and gene deletion experiments, revealing a complicated landscape of C/D RNA-target interactions. C/D RNAs widely use dual antisense elements to target adjacent sites in rRNAs, enhancing modification at weakly bound sites. Two consecutive sites can be guided with the same antisense element upstream of box D or D', a phenomenon known as double-specificity that is exclusive to internal box D' in eukaryotic C/D RNAs. Several C/D RNAs guide modification at a single non-canonical site. This study reveals the global landscape of RNA-guided 2'-O-methylation in an archaeon and unexpected targeting rules employed by C/D RNA.

How Donor Profile Affects the Outcome of Heart Transplant: A New Score to Predict Primary Graft Failure

Aim: This study aimed to identify demographic and clinical parameters in donors that could predict the ischaemic time tolerated by the heart transplant in order to increase the number of heart transplantation performed in our centre by expanding the area of graft recruitment. Methods: A single site retrospective study was conducted on 116 heart transplantations performed in our centre between 2013 and 2021. Multivariate logistic regression analyses were used to identify interactions between the different donor parameters. Results: Sex, weight, height, total graft ischemic time were the donor parameters independently correlated with primary graft failure (p &lt; 0.1). The logistic regression model included 11 variables, with an AUC of 0.81, this model appears to perform well in the prediction of primary graft failure in the recipient. Conclusions: A predictive score of primary graft failure was created based on identified donors parameters that may help identify grafts with a higher tolerance to ischemia.

A PROSAIL model with a vegetation index lookup table optimized with in-situ statistics for rapeseed leaf area index estimation using diverse unmanned aerial vehicle sensors in the Yangtze River Basin

For winter rapeseed, timely monitoring of the leaf area index (LAI) near the winter solstice is critical for estimating its overwintering survival rate and yield. While unmanned aerial vehicle (UAV) remote sensing techniques have emerged as a promising means for crop LAI monitoring, many existing crop LAI models have only been evaluated using a single sensor system, a single cultivar, a single development stage, and/or a single site. As a result, the generalization capability of these LAI models is limited. In this study, four empirical statistical models (ESMs) and a new PROSAIL-based lookup table (LUT) method were developed to estimate the LAI of 24 winter rapeseed cultivars mainly grown in the Yangtze River Basin. The raw LUT was optimized using a vegetation index (VI) strategy after screening out inefficient parameter combinations using in-situ UAV reflectance and measured LAIs. The model transferability was evaluated across three years, six regional sites around Yangtze River Basin, different development stages, and two data sources from different sensors. An artificial neural network model in the ESMs performed best for data acquired in 2019 and 2020, but could not be used to estimate the LAI in 2018 due to the use of different sensors for data acquisition. In contrast, the optimized VI-LUT method was robust and outperformed the raw LUT for LAI retrieval with a root mean squared error of <0.7 for all data sets. These findings suggest that the optimized VI-LUT method has potential for estimating LAI at different spatial and temporal scales, especially for the situation where sensor types are difficult to be unified in multiple locations. Moreover, the genotype in these cultivars did not appear to dominate LAI generation before overwintering, but the climatic zone did. These findings have implications for the study of genotype-environment interactions in rapeseed cultivation.

Inferring multi-locus selection in admixed populations.

Admixture, the exchange of genetic information between distinct source populations, is thought to be a major source of adaptive genetic variation. Unlike mutation events, which periodically generate single alleles, admixture can introduce many selected alleles simultaneously. As such, the effects of linkage between selected alleles may be especially pronounced in admixed populations. However, existing tools for identifying selected mutations within admixed populations only account for selection at a single site, overlooking phenomena such as linkage among proximal selected alleles. Here, we develop and extensively validate a method for identifying and quantifying the individual effects of multiple linked selected sites on a chromosome in admixed populations. Our approach numerically calculates the expected local ancestry landscape in an admixed population for a given multi-locus selection model, and then maximizes the likelihood of the model. After applying this method to admixed populations of Drosophila melanogaster and Passer italiae, we found that the impacts between linked sites may be an important contributor to natural selection in admixed populations. Furthermore, for the situations we considered, the selection coefficients and number of selected sites are overestimated in analyses that do not consider the effects of linkage among selected sites. Our results imply that linkage among selected sites may be an important evolutionary force in admixed populations. This tool provides a powerful generalized method to investigate these crucial phenomena in diverse populations.

Federated Learning: Centralized and P2P for a Siamese Deep Learning Model for Diabetes Foot Ulcer Classification

It is a known fact that AI models need massive amounts of data for training. In the medical field, the data are not necessarily available at a single site but are distributed over several sites. In the field of medical data sharing, particularly among healthcare institutions, the need to maintain the confidentiality of sensitive information often restricts the comprehensive utilization of real-world data in machine learning. To address this challenge, our study experiments with an innovative approach using federated learning to enable collaborative model training without compromising data confidentiality and privacy. We present an adaptation of the federated averaging algorithm, a predominant centralized learning algorithm, to a peer-to-peer federated learning environment. This adaptation led to the development of two extended algorithms: Federated Averaging Peer-to-Peer and Federated Stochastic Gradient Descent Peer-to-Peer. These algorithms were applied to train deep neural network models for the detection and monitoring of diabetic foot ulcers, a critical health condition among diabetic patients. This study compares the performance of Federated Averaging Peer-to-Peer and Federated Stochastic Gradient Descent Peer-to-Peer with their centralized counterparts in terms of model convergence and communication costs. Additionally, we explore enhancements to these algorithms using targeted heuristics based on client identities and f1-scores for each class. The results indicate that models utilizing peer-to-peer federated averaging achieve a level of convergence that is comparable to that of models trained via conventional centralized federated learning approaches. This represents a notable progression in the field of ensuring the confidentiality and privacy of medical data for training machine learning models.

Prognostic Impact of Chromosomal Abnormalities in TP53-Mutated Acute Myeloid Leukemia and High-Risk Myelodysplastic Syndromes

Background: Background: Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) exhibit clinical heterogeneity, and genomic profiles play a crucial role in disease characteristics and survival outcomes. Elli Papaemmanuil et al. classified AML into 11 genomic subgroups (NEJM, 2016), where TP53 mutations and aneuploidy predicted dismal survival. Cases with TP53 mutations are often accompanied by complex chromosomal abnormalities (CAs), but the prognostic impact and target genes of CAs have not been fully studied. Methods: To clarify this issue, specimens of AML and MDS with excess blasts (MDS-EB) were collected from 1,001 patients enrolled at collaborating institutes of Kyoto Hematology Study Group between July 2017 and January 2023. Targeted capture sequencing with 1,216 single nucleotide polymorphism sites was used to analyze primary samples, allowing assessment of copy number alteration (CNA) profiles. The target gene panel included common mutations in myeloid neoplasms. Mutation calling was performed using the established pipeline Genomon 2. Prognostic impact of CNA, single nucleotide variants, and insertion/deletion with 10% or more frequency was evaluated, and the frequencies of CNA were calculated for the short and long arms of chromosomes 1-22 and the X chromosome. Univariate and multivariate analyses were conducted using statistical significance criteria of p &amp;lt; 0.05. Results: The average sequencing depth was 548. Among 1,001 patients, 224 (22.4%) harbored TP53 mutations. TP53-mutated cases had a median age of 70 (24-95) years old at diagnosis, with 117 (52.2%) being AML and 107 (47.8%) MDS-EB. TP53 biallelic mutation, defined by 2 or more mutations in TP53 or loss of heterogeneity of TP53 locus, was present in 200 (89.3%) of cases, more commonly associated with 3 or more CAs or CNA (complex karyotype (CK)-like) than monoallelic mutation (99% vs. 42%, p = 1.9x10 -13). The most frequent affected lesions were del(5q), del(7q), del(7p), del(12p), del(18q), gain(11q), and gain(21q) with frequencies of 76%, 59%, 34%, 33%, 28%, 27%, and 26%, respectively. In survival analysis, gain(21q), gain(19p), del(16q), del(3q), del(19p), del(13q), and del(4q), along with diagnosis of AML ( p = 2.0x10 -2), TP53 biallelic mutation ( p = 6.8x10 -3), and CK-like ( p = 3.4x10 -3), were associated with poor outcomes in univariate analysis. Multivariate analysis revealed gain(19p), del(3q), del(13q), del(4q), diagnosis of AML, and CK-like as independent poor prognostic factors. Here we have defined gain(19p), del(3q), del(13q), and del(4q) as “Risk CNA”. A significant difference in outcomes was observed between patients with and without Risk CNA (n = 110 and n = 114, respectively, Hazard ratio = 2.6, p = 6.0x10 -8): The 6-month survival rate was 70 (95% Confidence interval [60-79]) % for patients without Risk CNA and 42 [31-52] % for those with Risk CNA. Patients with two or more Risk CNAs had worse prognosis than those with one ( p = 2.7x10 -2), indicating independent or additive contributions to outcomes. Similar trends were observed when limiting the analysis to TP53-biallelic cases. Further investigation of Risk CNA target genes was performed by identifying commonly amplified/deleted lesions. Target genes of gain(19p) included DNM2, CARM1, SMARCA4, LDLR, and EPOR, while RB1 and TET2 were potential candidates of del(13q) and del(4q), respectively. Identification of candidate genes for del(3q) was challenging due to broad common deleted lesions. Conclusion: Chromosomal abnormalities stratify the prognosis of AML and MDS-EB with TP53 mutation. The number of Risk CNA also impacts survival. Investigation of target genes of Risk CNA suggests potential therapeutic targets for TP53-mutated myeloid neoplasms, warranting further study for treatment optimization.

Porous W/N/C nanoframework: A trifunctionally effective cathodic electrocatalyst for rechargeable Zn-Air/iodide hybrid battery

Different from the conventional Zinc-Air batteries which only involves oxygen reduction and evolution (ORR/OER) in the cathode, the cathode of rechargeable zinc–air/iodide hybrid batteries (ZAIHBs), with the addition of I− species, involves I− electrooxidation (IOR) during charging and oxygen/iodate reduction (ORR and IDR) during discharging. This work reports the synthesis of a hierarchically porous tungsten-based nitrogen-doped carbon framework (W/N/C) with abundant WNx active sites and numerous ultrafine WN nanoclusters and explores their application as a trifunctional IOR/ORR/IDR electrocatalyst for ZAIHBs. The formation mechanism of ZIF-8-derived W/N/C framework as well as its structural and compositional features were investigated. The catalyst demonstrates high trifunctional catalytic activity and robust durability towards IOR/ORR/IDR in alkaline condition, owing to the uniform copresence of the numerous sub-nanometer-sized WN nanoclusters and the abundant single atom WNx sites in the hierarchically structured carbon framework. When applied in homemade ZAIHBs, it revealed a high peak energy density (141.9 mW cm−2) and remarkable cycling stability (65 h), outperforming the commercial Pt/C catalysts. This work demonstrates the multi-functional electrocatalytic activity of tungsten-based non-noble metal electrocatalyst for high-performance ZAIHBs.

Loss of the OX40 Target in Lymphoma Patients after Combining an Anti-OX40 Agonist Antibody with in Situ Vaccination

INTRODUCTION:Previous clinical trials have shown that in situ vaccination using the combination of low-dose radiotherapy and intratumoral injection of CpG, a Toll-like receptor 9 (TLR9) agonist, can elicit T cell immune responses in patients with low grade lymphoma and regression at both the treated and non-treated distal tumor sites in patients with low-grade lymphoma (Brody et al., JCO 2010, Frank et al., Cancer Discovery 2018, Hammerich et al., Nat Med 2019). OX40 is a T cell activating co-receptor, belonging to the TNFR superfamily. Ligation of this receptor by an agonist anti-OX40 antibody leads to enhanced T cell activation of effector CD4 T cells while inhibiting Treg function. In preclinical models, addition of an anti-OX40 antibody to intratumoral CpG cured established tumors (Sagiv-Barfi I, et al. Sci Transl Med. 2018). Intratumoral injection of CpG up-regulated OX40 on CD4 T cells in the microenvironment enhancing the binding of agonistic anti-OX40 antibody that triggered a robust immune response. These results provided the rationale for a clinical trial of in situ vaccination (NCT03410901) using a human IgG1 agonistic anti-OX40 antibody (BMS-986178). STUDY:Fourteen patients with low-grade lymphoma received low-dose (2Gy x 2) radiotherapy to a single tumor site followed by 5 weekly intratumoral injections of 2 mg CpG-ODN (SD-101, TriSalus Life Sciences) and 3.75 mg anti-OX40 antibody into the same site. 40mg of anti-OX40 was administered intravenously starting on day 2 and continuing every 4 weeks for a total of 6 infusions. Therapy was well tolerated with no dose-limiting toxicities. Response to therapy was assessed using the revised Lugano criteria (Cheson et al., JCO 2014) based on CT scans at 12, 24, 48, 72, and 96 weeks. Fine needle aspirates (FNA) were performed at both the injected and a non-injected site outside the radiation field prior to, and at 1 and 6 weeks after initiation of treatment. Cells were analyzed by flow cytometry and droplet-based single-cell RNA sequencing to determine population dynamics, response to therapy as well as antibody binding and target occupancy by the anti-OX40 antibody. RESULTS:Of the 14 patients enrolled, 1 patient attained a partial response (PR), while 9 had stable disease (SD) and 4 had disease progression (PD) as their best overall response. Investigation into the binding of the treatment antibody and target occupancy by flow cytometry showed saturation of all the OX40 sites on CD4 T cells by week 6 at both the injected and non-injected tumors. However, further investigation also showed there was significantly less OX40 on the cell surface after treatment, suggesting a loss of the antibody target (Figure 1). This loss was reproduced by in vitro assays where activated blood CD4 T cells exhibited a loss of cell surface OX40 when cultured with BMS-986178. Additionally, Quigley et al ( Clin Cancer Res 2019) previously showed that an OX40 receptor occupancy of greater than 40% led to a loss of target in mice and in blood T cells of solid tumor patients. We further observed that the loss of cell surface OX40 occurred in all CD4 T subsets, including effector CD4 T cells and T Regulatory cells (Tregs). This result contrasted with our in vivo and in vitro findings in successful therapeutic mouse models where we did not observe this loss of cell surface OX40 on effector CD4 T cells after treatment with a mouse-specific anti-OX40 agonist antibody. C ONCLUSION: Our observations suggest that treatment with an agonistic anti-OX40 antibody induced a loss of OX40 on the cell surface of effector CD4 T cells in the tumor microenvironment. Fewer molecules of OX40 receptor may have constrained the efficacy of subsequent anti-OX40 infusions and may explain why the clinical responses observed in this study were lower than observed in our past clinical trials of in situ vaccination.

“Double Hit” Myeloma: Detection By FISH and Flow Cytometry in Circulating Myeloma Plasma Cells

“Double hit” myeloma is defined as the presence of 2 of the known high-risk genetic abnormalities in myeloma. This includes amp(1q), del(17p) and IGH translocations involving chromosomes 4, 16 and 20. Given the prognostic significance of these abnormalities, with poor overall survival, identification is crucial. Testing is traditionally by fluorescence in situ hybridization (FISH) on a single-site bone marrow sample at baseline or at specific disease timepoints. We explored whether amp(1q) and del(17p) “double hit” abnormalities could be detected in circulating myeloma plasma cells (CMPC). Aims: We assessed our novel imaging flow cytometry method that simultaneously integrates immunophenotyping and FISH. Called “immuno-flowFISH” this single cell method combines antigenic detection of neoplastic plasma cells, FISH for DNA loci of interest and imaging flow cytometry for automated assessment. Methods: Blood from 16 myeloma patients, at diagnosis or on therapy was studied. Following density gradient centrifugation, cells were incubated with CD38/CD138/CD319-AF647-conjugated antibodies to identify plasma cells, CD19-BV480 (to differentiate normal from neoplastic plasma cells) and CD3-BV510 (T-lymphocyte control). After fixation and cell membrane permeabilization, DNA was denatured (78 oC for 5 minutes). The cells were then incubated with FISH probes to 1q21 ( CKS1B) and 17p13 loci, and to the centromere of chromosome 17 (C17), and hybridized (24 hours for 37 oC). The nuclei were stained with SYTOX AADvanced and 50,000 to 500,000 cells acquired on the Amnis ® ImageStream ®X Mk II imaging flow cytometer (Cytek Biosciences). Digital images and quantitative data derived from computational algorithms (IDEAS software) were used to assess FISH signals overlying the counterstained nuclei. Results: A mean of 264,937 cells were acquired and 110,696 analysed. Cells with a neoplastic plasma cell phenotype (CD38/CD138/CD319-positive, CD19-negative) were present in all samples. The number of these CMPC ranged from 7 to 797, amounting to 0.011- 0.546% of all cells. FISH abnormalities were present in CMPC of 10 of the 16 cases (see Table). Isolated amp(1q) (three CKS1B FISH signals) was present in 4 cases, and isolated del(17p), (one 17p13 and two C17 FISH spots) in CMPC in 3 cases. “Double hit” abnormalities, with both amp(1q) and del(17p), were present in another 3 cases, with the following cytogenetic patterns (illustrated in the Figure): 1. amp(1q) and del(17p) in different cells (Case 8); 2. amp(1q) and del(17p) in the same cell (35.7% of CMPC), in addition to isolated amp(1q) (28.6% of CMPC) (Case 9); 3. amp(1q) and del(17p) in the same cells only (Case 10). All circulating myeloma plasma cells had diploid signals for the chromosome C17 centromeric probe and the CD3-positive T-cells diploid signals for all probes. Conclusion: Incorporating antigenic identification and FISH, with imaging flow cytometric analysis, can detect “double hit” amp(1q) and del(17p) in circulating myeloma plasma cells at high sensitivity (10 -5). This novel approach allowed sub-clonal interrogation, with amp(1q) and del(17p) “double hits” detected in the same (Cases 9, 10) and separate CMPC (Case 8). It could also identify sub-clonal acquisition of secondary del(17p) (Case 9), which may be a consequence of disease evolution or intra-tumoral clonal heterogeneity. FISH analysis of circulating plasma cells using a flow cytometric platform is achievable to low levels of disease through assessment of thousands of cells. It holds significant promise for sensitive holistic real-time blood surveillance to detect high-risk and sub-clonal “double hit” alterations at the single cell level. Blood monitoring for these genetic aberrations overcomes the limitations of single site marrow analysis and may provide insights into the disease trajectory, clonal evolution and prognosis.

Experiences and Outcomes of Fertility Testing in Male Adolescents and Young Adults with Sickle Cell Disease

Introduction: Most adolescents and young adults (AYAs) with sickle cell disease (SCD) desire future biological parenthood. Recent research shows abnormal semen parameters in men with SCD, yet few studies have been conducted to elucidate fertility related impacts of SCD and its treatments among male AYAs with SCD. AYAs face many barriers to receiving care that could also impact their ability to complete fertility testing (i.e., via semen analysis). Thus, the aims of this study were to: 1) assess the feasibility of obtaining semen samples at a reproductive diagnostic center from male AYAs with SCD, 2) describe AYAs' and caregivers' experiences with testing, and 3) examine semen parameters among AYAs who completed testing. Methods Male AYAs with SCD (14-22 years, any genotype) and their caregivers were recruited to a single site study from November 2022-April 2023; the protocol involved completion of a novel web-based reproductive health program (i.e., FUTURES) describing potential fertility-related aspects of SCD and its treatments. AYA participants ≥18 years of age and AYA-caregiver dyads (if the AYA was &amp;lt;18 years old) were subsequently informed of the option for the AYA to obtain a semen analysis at an off-site reproductive diagnostic center (with costs of testing and transportation covered). Up to three attempts were made to contact AYA participants about their interest in testing. If interested, an order was sent to the testing center who then coordinated testing with the AYA. Research staff made up to three attempts to follow up with AYAs who reported interest but did complete testing. AYAs (and caregivers, if &amp;lt;18) who completed testing were notified of their results by a hematologist and invited to complete a facilitators interview. AYAs (and caregivers, if &amp;lt;18) who did not complete testing were invited to complete a barriers interview. Research staff conducted, transcribed, and analyzed interview transcripts for themes that demonstrated strong interrater reliability (κ=.94). Testing was considered feasible if ≥25% of AYAs completed testing. Demographic and treatment characteristics were abstracted from the medical record. Results Thirty-three AYAs completed FUTURES; five (15%) completed testing (Table 1) and a facilitators interview. Ten AYAs completed a barriers interview. Of the 18 enrolled caregivers, one completed a facilitators interview and seven completed a barriers interview. Facilitators included: 1) interest in learning about reproductive health [“ I told him ... sometimes people don't know if it is too late {to have a biological child} and if we checked we could have done something about it.” - Caregiver], 2) assistance coordinating testing appointments [“ Helping to coordinate {made testing easier} because I kept on missing the appointments.” -AYA] 3) alleviating costs [“ It {the funding} helped a lot because they {research team} had to pay and we didn't have to worry about it.” - Caregiver]. Barriers included: 1) discomfort [“ I don't think anything could have helped with the decision... he was just a little uncomfortable.” -Caregiver], 2) time [“ It is something I'm interested in, but not at the moment. I'm catching up on a lot of things.” -AYA], 3) transportation [e.g., “ I don't have a car. I didn't have a way to get there.” -AYA], and 4) forgetting the appointment [“ We just forgot about the appointment.” -Caregiver]. Conclusions Despite providing reproductive health education, compensation, and access to a local testing center, obtaining semen samples at an offsite center from male AYAs with SCD was not feasible. Abnormal semen parameters were noted across genotypes. While azoospermia and cryptozoospermia were noted in those on prolonged hydroxyurea treatment, oligospermia was also found in an individual with less severe disease and no hydroxyurea exposure. These findings underscore the importance of larger, longitudinal studies to examine semen parameters in male AYAs with SCD, ideally starting in mid-puberty. Further, qualitative findings highlight the need for: 1) developmentally and culturally appropriate fertility education, 2) innovative methods to collect semen samples (e.g., mail-in testing), and 3) advocacy to increase access to affordable reproductive healthcare. This is crucial to elucidate the fertility impacts of SCD and hydroxyurea and because the curative options that can also affect fertility are expanding for this underserved minority population.

Novel Multidimensional Pain Assessment Tool Is a Feasible, Valid, and Enjoyable Approach to Communication of Pain Symptoms in Pediatric Sickle Cell Disease

Background: Pain is the most common symptom of sickle cell disease (SCD) and is especially difficult to assess in the pediatric population. Children have varied developmental stages and cognitive abilities making the communication of this subjective experience hard to operationalize. Both the assessment of pain and validation of these assessments is challenging. No pediatric pain assessment tool has been deemed valid and reliable across all ages and types of pain. Commonly used pediatric tools include the Wong-Baker Faces scale, the numeric rating scale, and the visual analog scale (VAS), which combines the visuals of faces and numeric rating. These are unidimensional - only assessing the severity of pain, and in the case of faces, can cause confusion between pain severity and patient affect. In children with SCD, accurate pain assessment is crucial to current and lifelong management. “Painimation” is a technology-based pain assessment tool that incorporates conceptual animations, rather than numbers or words, to characterize pain. These animations are intended to help clinicians better understand pain from the patient's perspective. They are dynamic and transcend language. For example, animations are designed to communicate dull pulsating pain, sharp electric pain, or anything in between. “Painimation” does not rely on verbal communication skills, making it ideal for use in the pediatric population. “Painimation” has been validated in adults with SCD and is currently in implementation trials. Our study aims to determine the feasibility and validity of “Painimation” in pediatric SCD. Methods: This is a single site non-randomized cross-sectional feasibility and validity study. Participants were 10 - 21 years old with SCD who presented to clinic in baseline health. Participants completed the Painimation application, which consists of a numeric pain rating scale (1-10), a front and back 2-dimensional body image that can be shaded to indicate areas affected by pain, and eight abstract animations intended to represent different pain qualities (tingling, shooting, stabbing, throbbing, pounding, cramping, electrifying, and burning). The patient chose up to three animations and the intensity of each chosen animation was adjusted using a sliding bar without numeric labels. The animations were presented to the patient without labeling the intended quality. The patients then completed a survey consisting of questions regarding the usability of Painimation, clinical questions regarding their disease, Lansky Play-Performance Scale, and validated patient reported outcomes questions (PedsQL, Ped-PRO-CTCAE, PROMIS). Results: We enrolled 30 participants between April 1 and July 31, 2023, with recruitment ongoing. Five records were incomplete and were not analyzed at this time. The mean ratings when asked whether 1) Painimation was easy to use, 2) the patient enjoyed using Painimation, and 3) the patient would use Painimation to communicate pain with their provider were respectively 3.28±.74 (SD), 3.08±.95, and 3.12±1.17 (scale 0-4), indicating a generally favorable result for feasibility. The median VAS score was 5.10 (IQR = 0.25, 8.25). Pain was most reported in the back lower chest (60%), front stomach (48%), and front chest (44%). The stabbing animation was the most chosen at 56% followed by the cramping animation at 20%. Electrifying and Cramping animations had the highest agreement with their intended McGill descriptors at 40%. VAS scores and Painimation severity scores demonstrated excellent positive correlation (Pearson r=.74). Conclusion: Painimation is a feasible pain assessment tool in pediatric SCD with high user satisfaction. Severity scores on Painimation correlate strongly with VAS scores. The pain characteristic conveyed by each animation adds unique dimensions to this measure and will be further analyzed. Painimation is a novel pediatric pain assessment that holds the promise of greatly improving the communication of pain and transforming care in Pediatric SCD.

A Phase 2 Trial of SCRI-CAR19 Demonstrates Favorable Leukemia-Free Survival (LFS), with a Pilot Study of CD19-Expressing T-Cell Antigen Presenting Cells (CD19t T-APCs) Demonstrating Safety and Tolerability

The value of CD19 directed CAR T cell therapy for relapsed, refractory B-ALL has been well established. Iterative and complementary clinical trials have identified subgroups with improved outcomes, including those with low disease burden at the time of treatment as well as those with prolonged CAR T cell in vivo persistence. PLAT-02 was a phase 1/2 study of SCRI-CAR19, a 2 nd generation murine CAR with 41BB co-stimulation. Phase 1 previously reported 1 year LFS, event-free (EFS) and overall survival (OS) of 55%, 51.2%, and 69.5%. Subjects with low CD19 antigen burden in the bone marrow (&amp;lt;15%) prior to lymphodepletion (LD) and those with a rapid contraction of CAR T cells between Day 10 and 14 (ratio ≥1.5) were at higher risk of early loss of persistence. Phase 2 of PLAT-02 was a multisite trial along with a single site pilot study, PLAT-03, to explore administration of exogenous CD19 antigen designed to boost CAR T cell engraftment in subjects at high risk of early loss of persistence. PLAT-02 (NCT02028455) enrolled subjects 1-26 years of age with relapsed, refractory CD19+ B-ALL. The primary efficacy cohort was defined as those who received fludarabine/cyclophosphamide (flu/cy) LD and were infused with CAR19. PLAT-03 (NCT03186118) was a pilot study of CD19t T-APCs (manufactured T cells with CD19 tag) administered at doses of 10x10 6/kg or flat dosing of 5x10 8 for subjects &amp;gt;25kg every 4 weeks up to 6 total doses. Cohort A included subjects with low CD19 antigen burden, and cohort B included those with rapid early contraction of CAR19. 88 subjects with B-ALL were enrolled on PLAT-02 with plans for treatment at the recommended phase 2 dose (RP2D) of 1x10 6 CAR+ T cells/kg. Eighty-six percent had at least one prior relapse and 42% had undergone prior hematopoietic cell transplant (HCT). The feasibility of manufacturing was 98%. 71 subjects were in the primary efficacy cohort with an 89% minimal residual disease negative complete remission (MRDneg CR) rate. EFS and OS probability estimates with 95% CI at 1 year following CAR19 were 0.65 (0.53, 0.75) and 0.76 (0.64, 0.84). LFS at 1 year from entering MRDneg CR was 0.71 (0.58, 0.81). Cytokine release syndrome (CRS) was grade (Gr) 1 (53%), Gr 2 (26%), Gr 3 (10%), and Gr 4 (3%). Neurotoxicity occurred in 53%: Gr 1 (24%), Gr 2 (13%), Gr 3 (15%), and Gr 5 (1%). 25 subjects were treated on PLAT-03, 10 as cohort A (10 received CAR19 on PLAT-03, 9 received T-APCs), and 11 as cohort B (5 received CAR19 on PLAT-02, 6 received CAR19 on PLAT-03, and 9 total received T-APCs). One was cohort C (retreatment) and 3 were cohort D (no T-APCs). A total of 72 infusions of T-APCs were administered, with no occurrence of CRS or neurotoxicity. Two subjects had grade 3 toxicities related to T-APCs, 1 febrile neutropenia and 1 infusion related reaction. EFS and OS probability estimates at 1 year following CAR19 were 0.67 (0.43, 0.83) and 0.95 (0.71, 0.99). Overall LFS at 1 year was 0.63 (0.38, 0.80); by cohort: cohort A, 0.44 (0.13, 0.72) and cohort B, 0.80 (0.41, 0.95). Median duration of persistence, as determined by cumulative incidence using B cell aplasia (BCA) as a surrogate, among all subjects who received RP2D of CAR19 with flu/cy LD across PLAT-02 and PLAT-03, engrafted and achieved MRDneg CR, was 12.9 months (m). For subjects who did not receive T-APCs, the median persistence was 12.1m. Impact of antigen burden prior to LD was again observed; those with &amp;gt;15% CD19 antigen burden had persistence of &amp;gt;12.1m vs. 3.9m in the low antigen burden group. For subjects treated with T-APCs, the median persistence was 16m; 9.9m in cohort A and &amp;gt;16m in cohort B. The improvement in LFS after SCRI-CAR19 in phase 2 compared to phase 1 is likely multi-factorial, with contributing factors including earlier treatment, uniform use of LD, enhanced persistence, and better allocation of patients who benefit from consolidative HCT. The use of T-APCs was well tolerated without recurrent CAR T cell mediated toxicity. Although not directly comparable, there did appear to be enhancement of CAR T cell persistence with the use of T-APCs in populations predicted to have inferior persistence, including those with low antigen burden. There was not a clear improvement of LFS in those who received T-APCs. However, subjects were not randomized to receive T-APCs and those enrolled on PLAT-03 were more heavily treated and more likely to have received a prior HCT. Further exploration of T-APCs is warranted to define and evaluate their impact on persistence and durable responses.