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Rissin et al . increase the sensitivity of sandwich ELISA by segregating beads bearing a single enzyme-labeled immunoconjugate into femtoliter-volume reaction chambers. As the small volume of each well permits detection of extremely low levels of fluorescence, protein abundance is determined by counting the number of fluorescent wells as a percentage of the number of wells containing beads. The ability to detect single protein molecules 1 , 2 in blood could accelerate the discovery and use of more sensitive diagnostic biomarkers. To detect low-abundance proteins in blood, we captured them on microscopic beads decorated with specific antibodies and then labeled the immunocomplexes (one or zero labeled target protein molecules per bead) with an enzymatic reporter capable of generating a fluorescent product. After isolating the beads in 50-fl reaction chambers designed to hold only a single bead, we used fluorescence imaging to detect single protein molecules. Our single-molecule enzyme-linked immunosorbent assay (digital ELISA) approach detected as few as ∼10–20 enzyme-labeled complexes in 100 μl of sample (∼10 −19 M) and routinely allowed detection of clinically relevant proteins in serum at concentrations (<10 −15 M) much lower than conventional ELISA 3 , 4 , 5 . Digital ELISA detected prostate-specific antigen (PSA) in sera from patients who had undergone radical prostatectomy at concentrations as low as 14 fg/ml (0.4 fM).

BackgroundSoluble PD-L1 (sPD-L1) has been associated with worse prognosis in numerous solid tumors. We determined sPD-L1 levels before and during nivolumab treatment in two prospective clinical trials of metastatic clear cell renal cell carcinoma (RCC) and melanoma patients, and investigated its relationship to clinical factors, biomarkers, and outcome.MethodsUsing a new Single Molecule Array assay, serum sPD-L1 level were determined in RCC (CheckMate 009, n=91) and melanoma (CheckMate 038-Part 1, n=78) prior to, and at two time points on treatment. Gene expression data was obtained from biopsies taken prior to, and at day 28 on treatment. Results were integrated with clinical variables, tumor PD-L1 status from immuno-histochemistry, and genomic mutation status.ResultsIn RCC patients, sPD-L1 levels were higher in patients with progressive disease as their best response. For both RCC and melanoma patients, progressive or stable disease was associated with an increase in sPD-L1 on nivolumab therapy, whereas mean sPD-L1 levels did not change or declined in patients with objective responses. By categorizing RCC patients into transcriptomic molecular subtypes, we identified a subgroup where the associations between sPD-L1 and progressive disease were particularly evident. In baseline biopsies, we identified six biological processes that were associated with sPD-L1 level in both RCC and melanoma: higher sPD-L1 is associated with lower tumor expression of the Hallmark gene sets ‘hypoxia’, ‘fatty acid metabolism’, ‘glycolysis’, ‘MTORC1 signaling’ and ‘androgen response’, and with higher expression of ‘KRAS signaling_Down’.ConclusionBaseline and on-therapy sPD-L1 levels in RCC have the potential to predict progressive disease on PD-1 inhibitor nivolumab. In a hypothesis-generating analysis of tumor gene expression, high baseline sPD-L1 is associated with a tumor metabolic state reflecting potentially targetable processes in both melanoma and RCC. In both trials, we observed associations between change in sPD-L1 on treatment and outcome metrics. sPD-L1 levels may further refine a nivolumab-refractory subtype of RCC within transcriptionally based subtypes of RCC.

Activation of receptor‐interacting protein kinase 1 (RIPK1), a broadly expressed serine/threonine protein kinase, by pro‐inflammatory cytokines and pathogens can result in apoptosis, necroptosis, or inflammation. RIPK1 inhibition has been shown to reduce inflammation and cell damage in preclinical studies and may have therapeutic potential for degenerative and inflammatory diseases. SIR2446 is a potent and selective novel small molecule RIPK1 kinase inhibitor. This phase I, randomized, double‐blind, placebo‐controlled study in Australia (ACTRN12621001621808) evaluated the safety (primary objective), pharmacokinetics, and pharmacodynamics of single (3–600 mg) and multiple (5–400 mg for 10 days) ascending oral doses of SIR2446M (SIR2446 magnesium salt form) in healthy adults from Nov 24, 2021, until May 01, 2023. All treatment‐emergent adverse events (TEAEs) were mild/moderate. The most reported TEAEs were vascular access site pain, headache, and rash morbilliform. SIR2446M plasma half‐lives ranged from 11 to 19 h and there were no major deviations from dose proportionality for maximum concentration and area under the curve across doses. Renal excretion of unchanged SIR2446 was minimal. No marked accumulation was observed (mean accumulation ratio, 1.2–1.6) after multiple daily doses. A high‐fat meal mildly reduced the exposure but was not considered clinically significant. SIR2446M had a rapid and sustained inhibitory effect on the activity of RIPK1, with an overall 90% target engagement at repeated doses ranging from 30 to 400 mg in peripheral blood mononuclear cells ex vivo stimulated to undergo necroptosis. The favorable safety, pharmacokinetic, and pharmacodynamic profile of SIR2446M in healthy participants supports its further clinical development in patients with degenerative and inflammatory diseases.

Receptor‐interacting serine/threonine kinase 1 (RIPK1) regulates inflammatory signaling and induces apoptosis and necroptosis. Pharmacological inhibition of RIPK1 kinase activity has demonstrated efficacy in animal models of neurodegenerative, autoimmune and inflammatory diseases. SIR9900 is a potent and selective novel small molecule RIPK1 inhibitor. This first‐in‐human, phase I, randomized, double‐blind, placebo‐controlled study evaluated the safety, pharmacokinetics, and pharmacodynamics of single (3–200 mg) and multiple (3–60 mg daily for 10 days) ascending oral doses of SIR9900 in healthy adult (18–64 years, n = 80) and elderly participants (≥ 65 years, multiple doses 30 mg, n = 8). The study included a food effect component. Overall, SIR9900 was safe and well tolerated with no concerning dose‐dependent trends in safety observed. SIR9900 was rapidly absorbed with a plasma maximum concentration time (Tmax) of 3.0–4.0 h and plasma half‐life (t1/2) of 31.92–37.75 h following single doses. Similar Tmax and t1/2 results were observed following multiple doses. Systemic exposure to SIR9900 increased in a dose‐proportional manner and was similar between adult and elderly participants. No appreciable food effect was observed. The cerebrospinal fluid to unbound plasma ratio was 1.15. A robust pharmacodynamic effect was demonstrated with approximately 90% peripheral target engagement at 3 h post‐dose, and sustained RIPK1 inhibition over the 10‐day treatment period. The promising safety, pharmacokinetic, and pharmacodynamic profile of SIR9900 with central nervous system penetrating potential in healthy adult and elderly participants supports its further clinical development in patients with inflammatory and degenerative diseases, particularly in the central nervous system.

Backgroud: The tumor immune microenvironment influences the efficiency of concurrent chemoradiotherapy (CCRT) in high-grade glioma (HGG). This study investigated peripheral blood T lymphocyte subsets as clinical indicators of therapeutic response and prognosis in pediatric high-grade glioma (pHGG). Methods: This retrospective study included 77 patients with postoperative pHGG who were treated concurrently with temozolomide and external beam radiotherapy between January 1, 2012, and December 31, 2018. The median follow-up was 26 (range: 5-106) months. Peripheral venous blood samples were collected before and after CCRT. The proportions of peripheral blood T lymphocytes and their association with treatment outcome and survival were determined. Results: Sixty-four (83.1%) patients achieved complete remission, partial remission, and stable disease, and 13 (16.9%) patients had progressive disease. Higher CD3+ T cell, CD4+ T cell, and CD8+ CD28+ T cell ratios were predictive of better response, while a higher CD8+ CD28− T cell ratio was predictive of poorer response. Binary logistic regression analysis showed that the CD8+ CD28+ T cell ratio was a significant independent predictor of CCRT response (odds ratio [OR] = 53.521, 95% confidence interval [CI] = 4.294-667.119, P = .002). Univariate and multivariate analysis of prognostic factors associated with survival showed that the CD8+ CD28+ T lymphocyte ratio was a significant independent predictor of progression-free survival (hazard ratio [HR] = 1.80, 95% CI = 1.06-3.08, P = .03), but none of the subsets were significantly associated with overall survival. Conclusion: Peripheral blood T lymphocytes have potential as predictors of CCRT response and prognosis in pHGG.

Amyloid β (Aβ) peptides are proteolytic products from amyloid precursor protein (APP) and are thought to play a role in Alzheimer disease (AD) pathogenesis. While much is known about molecular mechanisms underlying cerebral Aβ accumulation in familial AD, less is known about the cause(s) of brain amyloidosis in sporadic disease. Animal and postmortem studies suggest that Aβ secretion can be up-regulated in response to hypoxia. We employed a new technology (Single Molecule Arrays, SiMoA) capable of ultrasensitive protein measurements and developed a novel assay to look for changes in serum Aβ42 concentration in 25 resuscitated patients with severe hypoxia due to cardiac arrest. After a lag period of 10 or more hours, very clear serum Aβ42 elevations were observed in all patients. Elevations ranged from approximately 80% to over 70-fold, with most elevations in the range of 3-10-fold (average approximately 7-fold). The magnitude of the increase correlated with clinical outcome. These data provide the first direct evidence in living humans that ischemia acutely increases Aβ levels in blood. The results point to the possibility that hypoxia may play a role in the amyloidogenic process of AD.

Measurement of prostate-specific antigen (PSA) in prostate cancer patients following radical prostatectomy (RP) has been hindered by the limit of quantification of available assays. Because radical prostatectomy removes the tissue responsible for PSA production, postsurgical PSA is typically undetectable with current assay methods. Evidence suggests, however, that more sensitive determination of PSA status following RP could improve assessment of patient prognosis and response to treatment and better target secondary therapy for those who may benefit most. We developed an investigational digital immunoassay with a limit of quantification 2 logs lower than current ultrasensitive third-generation PSA assays. We developed reagents for a bead-based ELISA for use with high-density arrays of femtoliter-volume wells. Anti-PSA capture beads with immunocomplexes and associated enzyme labels were singulated within the wells of the arrays and interrogated for the presence of enzymatic product. We characterized analytical performance, compared its accuracy with a commercially available test, and analyzed longitudinal serum samples from a pilot study of 33 RP patients. The assay exhibited a functional sensitivity (20% interassay CV) <0.05 pg/mL, total imprecision <10% from 1 to 50 pg/mL, and excellent agreement with the comparator method. All RP samples were well within the assay measurement capability. PSA concentrations following surgery were found to be predictive of prostate cancer recurrence risk over 5 years. The robust 2-log improvement in limit of quantification relative to current ultrasensitive assays and the validated analytical performance of the assay allow for accurate assessment of PSA status after RP.

Glial fibrillary acidic protein (GFAP), microtubule-associated protein tau, and amyloid β peptide (Aβ42) have been proposed as diagnostic and prognostic biomarkers in traumatic brain injury (TBI). Single molecule array (Simoa) is a novel technology that employs highly sensitive immunoassays for accurate measurements of candidate biomarkers found at low concentration in biological fluids. Our objective was to trace the trajectory of tau, GFAP, and Aβ42 levels in plasma from the acute through subacute stages after TBI, compared with controls. Samples from 34 TBI subjects enrolled in the Citicoline Brain Injury Treatment Trial (COBRIT) were studied. Injury severity was assessed by Glasgow Coma Scale (GCS) and admission CT. Glasgow Outcome Scale Extended (GOSE) was assessed 6 months after injury. Plasma was collected within 24 h (Day 0), and 30 and 90 days after the TBI. Plasma collected from 69 healthy volunteers was used for comparison. At every time point, increases were noted in plasma GFAP (p < 0.0001 for all comparisons), tau (p < 0.0001, p < 0.0001, and p = 0.0044, at Days 0, 30, and 90, respectively), and Aβ42 (p < 0.001, p < 0.0001, and p = 0.0203, respectively) in TBI cases compared with controls. The levels were maximal at Day 0 for GFAP and tau and at Day 30 for Aβ42. Area under curve (AUC) analyses for Day 0 GFAP and tau were excellent for discrimination of complicated mild TBI (cmTBI) from controls (0.936 and 0.901, correspondingly). Discriminant component analysis (DCA) for all three biomarkers at Days 0 and 30 differentiated controls from cmTBI (91.1% and 89.7% correctly classified, at each time point). Duration of post-traumatic amnesia (PTA) correlated weakly with tau levels at 30 days (Spearman's r = 0.40; 95% CI 0.0003–0.60, p = 0.044). The Marshall CT Grade on admission correlated weakly with Day 30 tau levels (Spearman's r = 0.41; 95% CI 0.04–0.68, p = 0.027). Day 30 Aβ42 correlated with GOSE (standardized β −0.486, p = 0.042). GFAP, tau and Aβ42 were increased up to 90 days after TBI compared with controls. Total tau levels correlated with clinical and radiological variables of TBI severity. Plasma Aβ42 correlated with clinical outcome. Combination of all three biomarkers at Days 0 and 30 can be used to differentiate controls from cmTBI populations, and may be useful as biomarkers of TBI in both acute and subacute phases.

Implementation of amyloid biomarkers in clinical practice would be accelerated if such biomarkers could be measured in blood. We analyzed plasma levels of Aβ42 and Aβ40 in a cohort of 719 individuals (the Swedish BioFINDER study), including patients with subjective cognitive decline (SCD), mild cognitive impairment (MCI), Alzheimer’s disease (AD) dementia and cognitively healthy elderly, using a ultrasensitive immunoassay (Simoa platform). There were weak positive correlations between plasma and cerebrospinal fluid (CSF) levels for both Aβ42 and Aβ40, and negative correlations between plasma Aβ42 and neocortical amyloid deposition (measured with PET). Plasma levels of Aβ42 and Aβ40 were reduced in AD dementia compared with all other diagnostic groups. However, during the preclinical or prodromal AD stages (i.e. in amyloid positive controls, SCD and MCI) plasma concentration of Aβ42 was just moderately decreased whereas Aβ40 levels were unchanged. Higher plasma (but not CSF) levels of Aβ were associated with white matter lesions, cerebral microbleeds, hypertension, diabetes and ischemic heart disease. In summary, plasma Aβ is overtly decreased during the dementia stage of AD indicating that prominent changes in Aβ metabolism occur later in the periphery compared to the brain. Further, increased levels of Aβ in plasma are associated with vascular disease.

Objective The delivery quality assurance (DQA) of craniospinal irradiation (CSI) due to the target length results in no ideal verification devices. Delivery Analysis (DA) could calculate the dose distribution based on the measured multi leaf open time in helical tomotherapy (HT). This study aimed to evaluate the efficacy of DA for DQA of CSI in HT. Material and Methods 32 CSI plans were classified into two groups based on γ analysis of the PTV‐cranial and PTV‐spine plans using a 2D ionization chamber matrix (MatriXX). Plans with γ passing rates ≥ 95% at 3%/2 mm were classified as the passed group, while those < 95% were classified as the failed group. Receiver operating characteristic (ROC) curves identified optimal passing rate threshold for DA in HT. Logistic regression analyzed risk factors for DQA failure, and failed plans were reoptimized according to the adjusted parameter. Results For PTV‐cranial plans, 30 passed and two failed; for PTV‐spine plans, 21 passed and 11 failed. ROC analysis revealed areas under the curve of 0.858 (PTV‐cranial, threshold: 89.0%) and 0.714 (PTV‐spine, threshold: 86.0%). Logistic regression identified planned modulation factor (MF‐plan; p = 0.046; p = 0.023) and actual modulation factor (MF‐actual; p = 0.027; p = 0.008) as independent risk factors for DQA failure in both MatriXX and DA. Additionally, beam on time (p = 0.043), gantry period (p = 0.007) and maximum leaf open time (p = 0.007) were identified as independent risk factors for DA. Reoptimization of failed plans with MF‐plan = 2.6 significantly improved passing rates in DA (73.70% ± 13.30% vs. 88.20% ± 12.30%; p = 0.010) and MatriXX (91.20% ± 2.60% vs. 96.10% ± 1.40%; p < 0.001). Conclusion Delivery Analysis could be a feasible tool for DQA of CSI in HT. Increasing the MF‐plan is recommended to enhance the passing rate.