Specific Antigen Research Articles

Uveal melanoma: molecular-genetic mechanisms of arising and the therapeutic approaches

Uveal melanoma (UM) is a tumor of neuroectodermal origin, which results from malignant transformation of melanocytes of the eye vasculature: iris, ciliary body and chorioidea. UM represents up to 5% of all melanoma cases, but it is extremely aggressive, since half of patients with UM develop metastases within the first 1‒2 years after the tumor appearance. Molecular mechanisms of uveal melanoma carcinogenesis are poorly understood, and have already been shown to be different from those of skin melanoma. Activating mutations in the GNAQ and GNA11 genes, encoding the large G protein subunits Gq and G11, respectively, are found in 90% of UM patients. The main signaling cascade leading to the transformation of melanocytes of the uveal tract is the signaling pathway Gaq/PKC/MAPK, and the major regulators of this cascade are targets for the development of drugs. The development of the metastatic form of UM is most often associated with mutations in the genes BAP1, EIF1AX, GNA11, GNAQ, and SF3B1. A combination of a commercial expression test panel of 15 genes and a mutation panel of 7 genes, supplemented with data on the size of the primary tumor, has been shown to be highly effective prognostic signature in prediction the risk of metastases. The risk of metastases determines the choice of therapy and patient follow-up regimen. At the same time, no systemic therapy for the treatment of metastatic UM has been developed to date; new drugs undergoing clinical trials mostly refer to either targeted therapy aimed at inhibiting the protein products of mutant genes, or immunotherapy designed to stimulate an immune response against specific antigens. In addition to these approaches, the review also considers potential therapeutic targets of epigenetic regulation of UM development.

The Immune Regulatory Functions in B Cells Are Restored by CpG to Reduce Experimental Food Allergy.

Dysfunctional immune regulation contributes to the pathogenesis of food allergy (FA). The mechanism behind regulatory B-cell dysfunction is unclear. CpG has immune regulatory functions. The purpose of this study is to use CpG to recover the immune suppressive functions of B cells in mice with FA. An FA mouse model was created using ovalbumin as the specific antigen. Flow cytometry was used to isolate B cells from the intestinal tissues. The immune regulatory functions of B cells were assessed using immunological approaches. The results showed that the FA response was linked to low IL-10 levels in gut lavage fluids of FA mice. FA mouse intestinal B cells produced lower amounts of IL-10 as compared with B cells isolated from naïve control mice. Impaired immune suppressive functions were observed in B cells isolated from the FA mouse intestine. The inducibility of the Il10 expression in naïve B cells of the intestine of FA mice was defective. The induction of Il10 expression in FA B cells could be restored by CpG through regulating the methylation status of the Cmip promoter. CpG promoted the therapeutic efficacy of allergen specific immunotherapy by restoring the induction of IL-10+ B cells in the intestine. The expression of Il10 in B cells of the FA mouse intestine was impaired. Administration of CpG could restore the expression of Il10 in B cells in the intestine and promote immunotherapy for FA.

A T cell receptor specific for an HLA-A*03:01-restricted epitope in the endogenous retrovirus ERV-K-Env exhibits limited recognition of its cognate epitope

Transposable elements (TEs) are often expressed at higher levels in tumor cells than normal cells, implicating these genomic regions as an untapped pool of tumor-associated antigens. In ovarian cancer (OC), protein from the TE ERV-K is frequently expressed by tumor cells. Here we determined whether the targeting of previously identified epitope in the envelope gene (env) of ERV-K resulted in target antigen specificity against cancer cells. We found that transducing healthy donor T cells with an ERV-K-Env-specific T cell receptor construct resulted in antigen specificity only when co-cultured with HLA-A*03:01 B lymphoblastoid cells. Furthermore, in vitro priming of several healthy donors with this epitope of ERV-K-Env did not result in target antigen specificity. These data suggest that the T cell receptor is a poor candidate for targeting this specific ERV-K-Env epitope and has limited potential as a T cell therapy for OC.

An NMR-based metabolic signature to identify clinically significant prostate cancer in patients undergoing biopsy.

Despite clinical suspicion of prostate cancer (PCa), 20-25% of patients exhibit a tumor-negative biopsy result. To assess the serum metabolic profile of clinically significant (cs) compared to clinically insignificant (ci) PCa or benign (Be) patients. 1078 serum samples were analyzed. Nuclear magnetic resonance (NMR) spectroscopy was used to quantify 73 metabolites, Random Forest for model algorithm. We identified a 22-metabolite panel, which discriminated csPCa (ISUP 2-5, n=328) from ciPCa (ISUP 1, n=101) and benign patients (negative biopsy, n=649) with a higher performance when combined with the standard clinical parameters age, prostate specific antigen (PSA), and percentage free PSA (%fPSA) (AUC 0.84) than the clinical parameters alone (AUC 0.73). Our study further revealed significant dysregulations of the urea cycle and the choline pathway along with changes in tricarboxylic acid (TCA) cycle, cholesterol metabolism, and a significant increase of the inflammation marker GlycB in csPCa patients. In particular, ornithine and dimethylglycine were the 2 most important features to discriminate csPCa from Be+ciPCa with significantly higher ornithine and lower dimethylglycine levels in patients with csPCa (ornithine: 63.7 ± 26.5 µmol/l, dimethylglycine: 12.6 ± 6.3 µmol/l, p<0.001) compared to Be+ciPCa patients (ornithine: 50.3 ± 31.6 µmol/l, dimethylglycine: 14.9 ± 7.7 µmol/l). This study discovered a 22-metabolite panel to discriminate patients with csPCa from Be+ciPCa patients when combined with age, PSA, and %fPSA. It may therefore be used as supportive biomarker to reduce the number of unnecessary biopsies and also to identify novel therapeutic targets in the future.

T-cell engagers: model interrogation as a tool to quantify the interplay of relative affinity and target expression on trimer formation.

T-cell engagers (TCEs) represent a promising therapeutic strategy for various cancers and autoimmune disorders. These bispecific antibodies act as bridges, connecting T-cell receptors (TCRs) to target cells (either malignant or autoreactive) via interactions with specific tumour-associated antigens (TAAs) or autoantigens to form trimeric synapses, or trimers, that co-localise T-cells with target cells and stimulate their cytotoxic function. Bispecific TCEs are expected to exhibit a bell-shaped dose-response curve, with a defined optimal TCE exposure for maximizing trimer formation. The shape of the dose-response is determined by a non-trivial interplay of binding affinities, exposure and antigens expression levels. Furthermore, excessively low binding to the TCR may reduce efficacy, but mitigate risk of over-stimulating cytokine secretion or induce effector cell exhaustion. These inevitable trade-off highlights the importance of quantitatively understanding the relationship between TCE concentration, target expression, binding affinities, and trimer formation. We utilized a mechanistic target engagement model to show that, if the TCE design parameters are close to the recommended ranges found in the literature, relative affinities for TCR, TAA and target expression levels have qualitatively different, but predictable, effects on the resulting dose-response curve: higher expression levels shift the curve upwards, higher antigen affinity shifts the curve to the left, and higher TCR affinity shifts the curve upwards and to the left.

Evaluation of immune sensor responses to a viral small noncoding RNA.

Gammaherpesviruses are widespread pathogens causing persistent infections linked to the development of numerous types of lymphomas in humans. During latency, most of the viral protein-coding genes are suppressed, facilitating evasion of adaptive immune recognition of protein antigens. In contrast, many noncoding RNA (ncRNA) molecules are expressed in infected cells and can regulate key cellular pathways while simultaneously evading adaptive immune recognition. To counteract this, many cells express internal pattern recognition receptors that can intrinsically sense ongoing infections and initiate cellular defenses. Murine gammaherpesvirus 68 (MHV68) is a valuable model to study in vivo aspects of gammaherpesvirus pathogenesis. The MHV68 ncRNA TMER4 (tRNA-miRNA-encoding RNA 4) promotes lymph node egress of infected B cells: in the absence of TMER4, MHV68-infected B cells accumulate in the lymph node in a manner similar to B cells activated through specific antigen encounter. We hypothesized that TMER4 may alter intrinsic immune activation. In research described here, we aimed to explore the immunomodulatory functions of TMER4 by evaluating its impact on signaling through the critical immune sensors Toll-like receptor 4 (TLR4), TLR3, TLR7, and retinoic acid-inducible gene I (RIG-I). To accomplish this, we developed a system to test noncoding RNAs using commercially available reporter cell lines. We optimized the experimental procedure to ensure ncRNA expression and to quantify immune sensory molecule induction or inhibition by the expressed ncRNA. Expression of TMER4 RNAs from plasmid constructs did not alter TLR or RIG-I signaling. This study provides a clear experimental framework that can be applied to test other small ncRNAs for their impact on various innate immune sensor proteins.

Limits on inferring T cell specificity from partial information

A key challenge in molecular biology is to decipher the mapping of protein sequence to function. To perform this mapping requires the identification of sequence features most informative about function. Here, we quantify the amount of information (in bits) that T cell receptor (TCR) sequence features provide about antigen specificity. We identify informative features by their degree of conservation among antigen-specific receptors relative to null expectations. We find that TCR specificity synergistically depends on the hypervariable regions of both receptor chains, with a degree of synergy that strongly depends on the ligand. Using a coincidence-based approach to measuring information enables us to directly bound the accuracy with which TCR specificity can be predicted from partial matches to reference sequences. We anticipate that our statistical framework will be of use for developing machine learning models for TCR specificity prediction and for optimizing TCRs for cell therapies. The proposed coincidence-based information measures might find further applications in bounding the performance of pairwise classifiers in other fields.

ANALYSIS OF CD10 EXPRESSION IN PROSTATE ADENOCARCINOMA AND ITS CORRELATION WITH DIFFERENT CLINICOPATHOLOGICAL PARAMETERS AT RNTMC, UDAIPUR

Objective: The study aimed to analyze the expression of CD10 in prostate adenocarcinoma to aid in early diagnosis and anti-CD10 targeted therapy. Methods: This was a cross-sectional study conducted in the Histopathology section of the Department of Pathologyat RNTMC, Udaipur, after approval from the ethical committee and institutional review board. This study was conducted on 92 patients suspected to have prostate cancer. The cases were assessed for Gleason score, Gleason Grade, and Serum Prostate Specific Antigen (PSA) levels. We performed IHC detection of CD10 in prostatic specimens and correlated the various patterns of CD10 expression concerning histopathological diagnosis. Results: In our study, we found membranous expression in low-grade carcinomas with low Gleason score and grade. The high-grade carcinomas with high Gleason score and Grade predominantly showed cytoplasmic expression. The increased CD10 cytoplasmic expression was correlated with serum PSA level. Conclusion: In our study, CD10 was found to be relevant. The low-grade carcinomas showed membranous positivity and high-grade carcinomas showed cytoplasmic expression. One hypothesis states that cytoplasmic expression is due to the localization of CD10 in the cytoplasm. Our study favors this hypothesis as there is cytoplasmic expression in high-grade tumors. In the future, this could be used as a diagnostic marker.

Polybodies: Next-generation clinical antibodies

Conventional antibodies [full-length and fragments: F(ab')2, fragment antigen-binding (Fab), single-chain variable fragment (scFv), variable heavy domain of heavy chain antibody (VHH)] are monospecific, first-generation antibodies, that have dominated the biopharmaceuticals field. However, protein engineering approaches has led to the advent of the next-generation antibodies (polybodies), which are significant improvement over the conventional antibodies. Polybodies comprise polyspecific and/or polyvalent antibodies that enable a single antibody to target multiple specific antigens simultaneously. Polybodies are superior to first-generation antibodies (more efficacious, broad-spectrum, resistance resilient, customizable, etc.) and provide a cost-effective healthcare solution. This review addresses recent developments in polybodies, highlighting their superiority over conventional antibodies and offering future perspectives to encourage the generation of innovative immunotherapies.

Improved deep learning prediction of antigen–antibody interactions

Identifying antibodies that neutralize specific antigens is crucial for developing effective immunotherapies, but this task remains challenging for many target antigens. The rise of deep learning-based computational approaches presents a promising avenue to address this challenge. Here, we assess the performance of a deep learning approach through two benchmark tests aimed at predicting antibodies for the receptor-binding domain of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Three different strategies for constructing input sequence alignments are employed for predicting structural models of antigen-antibody complexes. In our initial testing set, which comprises known experimental structures, these strategies collectively yield a significant top-ranked prediction for 61% of cases and a success rate of 47%. Notably, one strategy that utilizes the sequences of known antigen binders outperforms the other two, achieving a precision of 90% in a subsequent test set of ~1,000 antibodies, balanced between true and control antibodies for the antigen, albeit with a lower recall of 25%. Our results underscore the potential of integrating deep learning methods with single B cell sequencing techniques to enhance the prediction accuracy of antigen-antibody interactions.

B-113 Beckman Coulter DxI 9000 immunoassay analyzer assay performance meets new CLIA 2024 performance goals with highest proportion of Six Sigma performance amongst all major manufacturers

Abstract Background The Clinical Laboratory Improvement Act (CLIA) proficiency testing (PT) criteria will be updated in July 2024 with some goals tightening by up to 40%. It is assumed, but not known, that all assays and instruments will be able to achieve these goals. Using these new CLIA 2024 goals, estimates of current instrument group performance from an international proficiency testing (PT) survey have shown significant differences in quality, calling into question which assays can achieve the new CLIA goals. Currently, the highest performing diagnostic instrument achieves only 35.2% 6-Sigma, while the majority of instruments have less than 15% 6-Sigma performance indicating a higher probability of future PT failures. Methods Eighteen assays that will be governed by new CLIA 2024 goals were assessed for Sigma metric performance on the DxI 9000 immunoassay analyzer: Alpha Fetoprotein (AFP), Beta hCG, Carcinoembryonic antigen (CEA), Cortisol, Creatine Kinase MB (CK-MB), Estradiol, Ferritin, Folate, Follicle-Stimulating Hormone (FSH), Prolactin, Para-thyroid hormone (PTH), Total Prostate specific antigen (Total PSA), Testosterone, Thyroid Stimulating Hormone (TSH), Total Triiodothyronine (TT3),high sensitivity Troponin I, Total Thyroxine (TT4) and Vitamin B12. Precision and bias were determined following CLSI EP5 and EP9 guidelines, measured across 3 reagent lots. CLIA 2024 PT criteria provides the allowable total error for the standard Sigma-metric calculation: Sigma-metric = (TEa - |bias|) / SD Performance of the other major analyzers was assessed using the methodology introduced in 2006 by Westgard JO and Westgard SA. The Sigma-metric predicts future problems with PT and potential optimization of QC procedure opportunities, including fewer Westgard Rules, control levels, even reduced QC frequency and materials leading to less cost and time saved. Results Half (50%) of the assays on DxI 9000 achieves 6-Sigma, outperforming all other major manufacturers in the market who range from only 5% to 35% 6-Sigma. Further, over 78% of the DxI 9000 performance was 4Sigma and higher. Less than 8% of performance fell below 3-Sigma, which was observed at the extreme ends of the reportable range. Conclusions The DxI 9000 assay performance is well designed to meet CLIA 2024’s new PT criteria and has the highest prevalence of 6-sigma assay performance observed on market when compared to the top 5 major instrument manufacturers. Assays with Sigma metrics at 4-Sigma and higher are unlikely to face analytical PT difficulties and can be optimized for reduced Westgard Rules, control levels, and potentially even QC frequency.

B-318 Serum PSA in Extracellular Vesicles: Immunoreactivity to Commercial Kits and Elimination Kinetics

Abstract Background Prostate Specific Antigen (PSA) also circulates in blood bound to extracellular vesicles (EVs), and observed higher ratio of PSA in EVs respect total serum PSA when serum concentrations are &amp;lt;4 μg/L, and can represent up to 59% of total circulating PSA. Moreover, this molecular form of PSA (ev-PSA) is recognized and can be quantified with commercial methodologies designed for serum quantification. We aimed to characterize ev-PSA studying its immunoreactivity to different commercial assays and analyzing its presence in international standards used for assays standardization. Serum half-life and elimination kinetics of ev-PSA were also studied. Methods Serum EVs from 32 prostate cancer (PCa) patients were isolated by ultracentrifugation (100,000xg, 90 minutes). PSA was assessed in serum (srm-), serum supernatant free of EVs (sn-) and EVs (ev-) with total PSA (T-PSA) commercial immunoassays (Elecsys® from Roche Diagnostics and Immulite® 2000 from Siemens Healthineers), and Immulite/Elecsys ratio was calculated in all samples. The analysis of the differences between methods was performed with Bland-Altman test, representing the percentage of the difference respect the mean. WHO 96/670 PSA Standard was ultracentrifuged and analyzed by Nanoparticle Tracking Analysis (NTA) in a NanoSight LM20.For half-life estimation, serum samples from 10 localized PCa patients were collected basal and 2 days after radical prostatectomy, and EVs were isolated by Size Exclusion Chromatography using Exo-spinTM kit (Cell Guidance System). The Research Ethic Committee approved the study. Results Significant differences were observed between Elecsys and Immulite immunoassays in ev-T-PSA (p&amp;lt;0.001) concentrations. ev-T-PSA was quantified in all samples with Elecsys (median: 0.134 μg/L; Q1-Q3: 0.033-0.391 μg/L), but only in 22 patients (68.75%) with Immulite (median: 0.206 μg/L; Q1-Q3: 0.074-0.340 μg/L). In those 22 quantifiable patients, the median Immulite/Elecsys ratio was similar (p=0.057) between sn-T-PSA and srm-T-PSA (0.96 and 0.92, respectively), but much lower for ev-T-PSA (0.77; p&amp;lt;0.001). Bland-Altman graphical analysis showed that Elecsys overestimates the concentrations of T-PSA in all samples compared to Immulite, being this overestimation more marked in the case of PSA contained in EVs, and amplified at lower PSA concentrations. Besides, NTA analysis of WHO 96/670 PSA Standard showed that it does not contain EVs, and therefore, ev-PSA. The percentage of remnant PSA calculated in samples obtained post-surgery respect to baseline concentration in prostatectomized patients, was significantly higher for ev-T-PSA compared to soluble srm-T-PSA (p=0.014) and srm-F-PSA (p=0.002). The elimination half-life of ev-T-PSA was 3.99 days, much higher than those estimated for serum T-PSA and F-PSA, that were 1.76 days and 0.64 days, respectively. Conclusions The quantification of ev-PSA is affected by the different immunoreactivity of the diverse commercial assays. Thus, Elecsys T-PSA immunoassay shows higher immunoreactivity compared to Immulite technique, being this different reactivity more marked at lower PSA concentrations. ev-PSA is not considered in the International WHO 96/670 PSA Standard used for methods standardization. PSA bound to EVs remains longer in circulation than soluble serum PSA.

B-348 Performance of the Atellica IM Analyzer’s HighSensitivity PSA Assay as an Aid for Monitoring Prostate Cancer Recurrence

Abstract Background Prostate specific antigen (PSA) is a suitable marker for monitoring men with prostate cancer (PC), and for detecting recurrence after therapy in conjunction with other diagnostic measures. Studies were performed to evaluate performance of the Atellica® IM High-Sensitivity PSA assay (hsPSA)* versus the Atellica® IM PSA assay in patients undergoing management (monitoring) of PC. Methods A total of 868 serum samples from 170 subjects with PC (92 [54%] subjects had a radical prostatectomy (RP) before enrollment) were collected at 7 sites across the United States. Subjects had baseline and &amp;gt;2 follow-up visits during treatment (average 5.106 visits, SD=1.414). Samples were tested using the Atellica IM hsPSA assay and the commercially available Atellica IM PSA assay (comparative method). For RP and non-RP combined, 644 paired samples for each assay were tested. Subjects were designated “No Progression” (responding, stable, and no evidence of disease subjects) or “Progression” at each visit based on clinical classification. Results In this study the Atellica IM hsPSA assay demonstrated equivalent performance as commercial Atellica IM PSA in predicting disease status (table). The PPA between the assays was 95.5% and the NPA was 98.9%. Concordance to clinician classification was similar for both assays (∼80% PPA and 90% NPA). Conclusions Preliminary results demonstrated equivalent performance of the Atellica IM hsPSA assay as the commercially available Atellica IM PSA assay in terms of predicting disease status. This hsPSA assay can detect very low levels of PSA, which is particularly important for the monitoring of RP patients. *CAUTION: Investigational Device. Not available for sale. Future availability cannot be guaranteed. The results are preliminary and were achieved in unique setting with no expected timeline for completion.

B-346 Diagnostic Performance of the Atellica IM Free PSA Assay in Men Undergoing Prostate Cancer Screening

Abstract Background Measurement of free prostate specific antigen (fPSA) in serum is used to aid in distinguishing prostate cancer (PC) from benign prostate conditions in men 50 years or older with hsPSA of 4.0-10.0 ng/mL (µg/L) and digital rectal exam (DRE) nonsuspicious for PC. Studies were performed to evaluate clinical performance for the Atellica® IM fPSAII assay* [fPSAII/hsPSAx100 ratio]. Methods Serum samples were prospectively collected from 1250 patients referred to an urologist for PC evaluation at 26 sites throughout the United States. At diagnostic cutoffs for %fPSAII spanning the spectrum of %fPSAII values, the percentage of PCs detected (sensitivity) and the corresponding percentage of biopsies avoided in men without PC (specificity) were tabulated. At a pre-specified sensitivity near 90%, %fPSAII specificity (relative to biopsy result) was compared to hsPSA specificity. The mean PC probabilities on biopsy and nonparametric 95% CI (2.5th and 97.5th percentiles) were calculated. The difference between these proportions was evaluated to estimate the improvement in specificity of %fPSAII relative to hsPSA alone. Difference in specificity was significant if the zero value was not bounded by 2.5th and 97.5th percentile values of the improvement estimate with a minimum of 5% improvement in specificity. Results Sensitivity and specificity results are presented in the table. The cutoffs provided a range of sensitivity estimates from 86%-100%. At 22% cutoff, sensitivity (90.3%) plus specificity (21.4%) is greater than 110.0%. Significant improvement in %fPSAII specificity over hsPSA alone was 8.9% with 95% CI of 6.8%-11.5%. Conclusions The clinical performance of the Atellica IM Analyzer fPSAII assay was acceptable and demonstrated an improvement in detection of PC than using hsPSA alone in this study. *CAUTION: Investigational Device. Not available for sale. Future availability cannot be guaranteed. The results are preliminary and were achieved in unique setting with no expected timeline for completion.

B-121 Optimizing Test Ordering: Leveraging Heuristics in Naming Conventions - A PSA Case Study

Abstract Background A common screening test for prostate cancer is the Prostate-Specific Antigen (PSA) test, which may also be referred to as total PSA or total PSA plus free PSA, among other variations. Looking at the test menus of two major reference laboratories, it's apparent that PSA can be ordered under thirteen different test names. From an end-user perspective, the question arises: which of these thirteen tests should be ordered, and why are there so many options? This plethora of choices can lead to confusion and uncertainty for clinicians and patients alike. According to various guidelines, ordering Free PSA is only warranted when PSA values exceed 4 ng/ml. In such cases, the ratio of free PSA to total PSA can aid in further clinical decision-making and patient workup. At Zuckerberg San Francisco General Hospital (ZSFGH), we offer two tests for PSA. The first is named “Prostate Specific Antigen” is total PSA done in house, while the second is labeled “Prostate Spec. Ag. Total and Free.” which is sent out to a reference lab. Upon reviewing our send-out orders, we observed a significant increase (376%) from 2022 to 2023 in the use of the send out order. Methods The ordering data for our providers was collected from February 2022 to January 2024 for both in-house and send-out orders. The data was analyzed using descriptive statistics in Microsoft Excel. Results In our population, we observed an abnormal concentration of send-outs originating from a select group of physicians. Additional we also identified a group of providers who only selected the send out test order. Analysis of in-house assay results for PSA revealed that only 15% (1292 out of 8136) exceeded the cutoff of 4 ng/ml, necessitating further testing to inform clinical decision-making. Yet our send out volume of 4157 test is in discordance with how many PSA would need further evaluation. Based on a cost analysis reducing the send out volume by 80% could result in $53,210 savings. Therefore, to reduce the send out amount and to keep the naming convention similar we decided to implement a new test name that would reflex the PSA to Free PSA if the PSA is above 4 ng/ml. We decided on the name “Prostate Specific Antigen total with reflex to Free Prostate Specific Antigen”. Conclusions Our investigation into the variability in PSA test ordering revealed a significant disparity in the number of send-out orders, particularly originating from a select group of physicians. However, the disproportionately high volume of send-out tests suggested an inefficiency in the current testing approach. To address this issue and align with clinical guidelines, we decided to process a request to our medical executive committee for approval for this reflex test in order to meet reimbursement compliance with the Office of the Inspector General. This change aims to streamline the process, reduce unnecessary send-out tests, and ultimately improve the efficiency and accuracy of prostate cancer screening at our institution.

B-347 Analytical Performance Evaluation of the High Sensitivity PSA Assay and the Free PSAII Assay on the Atellica IM Analyzer

Abstract Background Measurement of serum prostate specific antigen (PSA) levels may be used in prostate cancer (PC) screening, but elevated PSA levels also are observed in patients with other etiologies such as benign prostatic hypertrophy. Studies suggest that measuring PSA with or before digital rectal examination (DRE) provides a better method of detecting PC than DRE alone. The free prostate specific antigen (fPSA) assay is an aid for distinguishing PC from benign prostate conditions in men 50 years or older with PSA values 4.0-10.0 ng/mL (µg/L) and DRE findings nonsuspicious for PC. The goal of this study was to evaluate the analytical performance of the high sensitivity PSA (hsPSA)* and free PSA (fPSAII)* assays on the Atellica® IM Analyzer. Methods Detection capability studies were performed per CLSI EP17-A2. Precision testing was performed per CLSI EP05-A3 with native human serum and QC samples, 3 reagent lots, 1 analyzer, 2 runs/day, 2 replicates/run for 20 days. For Atellica® hsPSA and fPSAII assays a multi-site reproducibility study was conducted per CLSI EP-05 A3 using native and contrived serum samples, 3 reagent lots, and 3 different sites and analyzers; samples were assayed in triplicate for five days, two runs/day ∼2 hours apart. Linearity testing was performed per CLSI EP06-A on a dilution series made from a high PSA sample (native human male serum pool) and a low sample (native human female serum), 4 replicates, 3 reagent lots, and 1 analyzer. Specimen equivalency was determined by Weighted Deming regression model per CLSI EP09c-ed3. Results Results are presented in the table. Conclusions The Atellica IM hsPSA assay and fPSAII assays detection capability, precision, linearity, and specimen equivalency were acceptable. *CAUTION: Investigational Device. Not available for sale. Future availability cannot be guaranteed. The results are preliminary and were achieved in unique setting with no expected timeline for completion.

Antibodies to specific domains of Plasmodium falciparum erythrocyte membrane protein-1 and its relationship with protection from severe malarial anemia: A prospective study among Ghanaian children.

Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) is important in malaria pathogenicity as it mediates Pf-infected erythrocytes cytoadherence to host endothelial microvasculature receptors. Naturally acquired antibodies against specific PfEMP-1 antigens may be beneficial in clinical malaria protection. This study determined antibodies to DBLα2, CIDRα1, DBLβ12, and DBLγ6 domains of PfEMP-1 in children with P. falciparum malaria in Tamale, Ghana. Sixty P. falciparum-infected children, and 30 controls, aged 1-12 years were recruited for this case-control study from April to July 2023 in Northern Ghana. Participants with uncomplicated malaria had asexual P. falciparum in peripheral blood and Hb ≥ 5.0 g/dL, and severe malaria was diagnosed when participants had Hb < 5.0 g/dL in addition to asexual P. falciparum in peripheral blood. Blood cell indices were measured using hematology analyzer, and IgG antibodies to DBLα2, CIDRα1, DBLβ12, and DBLγ6 domains of PfEMP-1 and pro-inflammatory cytokines were detected using enzyme-linked immunosorbent assay. Data were analyzed using SPSS version 26.0. The prevalence of PfEMP-1 IgG antibodies among P. falciparum-infected children and the uninfected group was 65.0% and 6.7%, respectively. PfEMP-1 IgG antibodies were present in 83.3% of uncomplicated malaria cases, and 46.7% in severe malaria subjects. Plasma levels of PfEMP-1 IgG antibodies were elevated in participants with uncomplicated malaria compared to those with severe malaria (p < 0.001). Hemoglobin, RBC, HCT, and platelet were significantly lower among P. falciparum-infected children without PfEMP-1 IgG antibodies than among those with the antibodies. Prevalence of anemia among children with PfEMP-1 IgG antibodies and those without the antibodies were 74.4% and 100%, respectively. The high prevalence of PfEMP-1 IgG antibodies to DBLα2, CIDRα1, DBLβ12, and DBLγ6 domains observed in participants with uncomplicated malaria, and the relationship between PfEMP-1 IgG antibodies and blood cell parameters could indicate that the antibodies may be related to effective erythropoietic response in P. falciparum malaria. Immune antibodies against DBLα2, CIDRα1, DBLβ12, and DBLγ6 domains of PfEMP-1 may suppress the deteriorating effects of PfEMP-1 antigens and provide immune protection against severe malarial anemia in children.

Next-generation cell-penetrating antibodies for tumor targeting and RAD51 inhibition.

Monoclonal antibody therapies for cancer have demonstrated extraordinary clinical success in recent years. However, these strategies are thus far mostly limited to specific cell surface antigens, even though many disease targets are found intracellularly. Here we report studies on the humanization of a full-length, nucleic acid binding, monoclonal lupus-derived autoantibody, 3E10, which exhibits a novel mechanism of cell penetration and tumor specific targeting. Comparing humanized variants of 3E10, we demonstrate that cell uptake depends on the nucleoside transporter ENT2, and that faster cell uptake and superior in vivo tumor targeting are associated with higher affinity nucleic acid binding. We show that one human variant retains the ability of the parental 3E10 to bind RAD51, serving as a synthetically lethal inhibitor of homology-directed repair in vitro. These results provide the basis for the rational design of a novel antibody platform for therapeutic tumor targeting with high specificity following systemic administration.

Detection of Apical Cancer with Novel Imaging Modalities to Predict Apical Margin Positivity in Robotic Assisted Radical Prostatectomy

ObjectivesTo evaluate margin positivity at apex utilizing preoperative magnetic resonance imaging [MRI], micro-ultrasound [MUS], prostate specific membrane antigen positron emission tomography PSMA PET] scan, biopsy location and intraoperative timing of deep venous complex [DVC] ligation during robot assisted radical prostatectomy [RARP]. MethodsInstitution review board approved retrospective study underwent RARP between November 2022 to March 2024. All patients underwent preoperative MRI, MUS and PSMA PET scan. Patients underwent RARP using either standard DVC [done prior apical dissection] ligation or delayed DVC [after prostate removal] technique. All patients underwent intra operative frozen section analysis by an experienced genitourinary pathologist. Descriptive statistics were performed. Data analyzed using R software version 4.3.3. ResultsTotal 619 prostate cancer patients underwent RARP. Of these, 365 men underwent RARP using delayed DVC ligation technique and 254 men using standard DVC ligation technique. There was no statically significant difference in 2 groups on demographic parameters, MRI, MUS and PSMA-PET scan features. Sensitivity of MRI, MUS, PSMA-PET and prostate biopsy for detection of apical positive margin were 66%, 81%, 81% and 73% respectively. Specificity of MRI, MUS, PSMA-PET and prostate biopsy for detection of apical positive margin were 45%, 14%, 16% and 30% respectively. When all modalities are used accumulatively, apical cancer was missed only in 1% of cases. ConclusionsWith proper preoperative understanding of apical lesion location, timing of DVC ligation [standard vs delayed] doesn't impact apical positive surgical margins. Combination of MRI, MUS, PSMA-PET and prostate biopsy reduce apical positive surgical margin rates significantly.

Investigating the pattern of prostate specific antigen screening among E-cigarette smokers within the behavioral risk factor surveillance system

Introduction and objectivesE-cigarettes use has recently increased, even among older individuals quitting smoking. Though past studies suggest tobacco smokers may avoid cancer screening, the relationship between e-cigarette uses and preventive health behaviors like prostate specific antigen screening is uncertain. We assessed the relationship between self-reported e-cigarette smoking and prostate specific antigen screening utilization among US adults with a history of e-cigarette use. Materials and methodsWe included men aged 50–69 years, who provided responses regarding PSA screening receipt and smoking status, from Behavioral Risk Factor Surveillance System 2020 and 2022 surveys. Primary outcome was PSA screening receipt. Multivariable regression model was performed to investigate the association between smoking status (never-smokers, current or former e-cigarette smokers, current or former tobacco smokers) and PSA screening. ResultsWe included a weighted population of 8.1 million men aged 50–69. 2.3 million (28.3%) received PSA screening. 3.9 million (48.2%) were never-smokers. 1.3 million (16.6%) were from e-cigarettes smokers group, and 2.9 million (35.2%) were from tobacco smokers group. E-cigarettes smokers were less likely to receive PSA screening within last 2 years (0.76 [0.66–0.88]) than never-smokers. No significant difference in PSA screening was detected between never-smokers and tobacco smokers (0.91 [0.82–1.02]). E-cigarette smokers were less likely to receive PSA screening within the selected time frame (0.84 [0.72–0.97]) than tobacco smokers. When examining potential mediation by primary care visits, e-cigarette smokers were less likely to have had a check-up visit in past 2 years than tobacco smokers (0.77 [0.65–0.92]). ConclusionsE-cigarette smokers were less likely to undergo PSA screening than never-smokers and tobacco smokers, possibly due to reduced use of primary care services.