Panel Of Assays Research Articles

Front-line liquid biopsy for early molecular assessment and treatment of hospitalized lung cancer patients

BackgroundMolecular characterization is pivotal for managing non-small cell lung cancer (NSCLC), although this process is often time-consuming and patients’ conditions might worsen while molecular analyses are processed.Our primary aim was to evaluate the performance of “up-front” next-generation sequencing (NGS) through liquid biopsy (LB) of hospitalized patients with newly detected lung neoplasm in parallel with conventional diagnosis. The secondary aim included longitudinal monitoring through LB of patients with oncogenic alterations at baseline. MethodsWe enrolled 47 consecutive patients immediately after hospitalization and radiological detection of symptomatic lung neoplasm. LB from peripheral blood was performed at baseline, in parallel with conventional biopsy (CB), when feasible. Additionally, LBs were repeated during treatment in patients with actionable gene alterations at baseline. Oncomine™ Lung cfTNA Research Assay panel was employed for processing plasma samples in NGS. Results47 hospitalized patients were enrolled. LB identified 28 patients with gene alterations, including mutations of EGFR (n = 7), KRAS (n = 12), ERBB2 (n = 1), TP53 (n = 2), BRAF (n = 1), one ALK rearrangement, and 4 patients with combined mutations involving EGFR, KRAS and PIK3CA.LB and CB were consistent, except for two patients. Three patients with positive LB for oncogenic drivers did not undergo CB due to contraindications.Median time to molecular results after LB was significantly lower compared to time to molecular report after CB (11 versus 22 days, p < 0.001). ConclusionsDespite limited numbers, our study supports the role of front-line LB for improving management of symptomatic patients with lung cancer, potentially leading to early targeted therapy initiation.

Semi-quantitative assessment of gastrointestinal viruses in stool samples with Seegene Allplex gastrointestinal panel assays: a solution to the interpretation problem of multiple pathogen detection?

The aim of the study was to determine and evaluate the clinical usefulness of pathogen specific semi-quantitative cut-offs in stool samples with multiple pathogen detections. The PCR (Seegene Allplex Gastrointestinal Virus Assay) data from 4527 positive samples received over 16 months were retrospectively analyzed to investigate the distribution of the Ct values of each individual viral pathogen. By using interquartile ranges for each viral pathogen, pathogen specific semi-quantitative cut-offs were determined. After a thorough analysis of the Ct values, a well-founded decision to exclude all results with a Ct value higher than 35 was made. This approach made it possible to generate a more nuanced report and to facilitate clinical interpretation in case of mixed infections by linking a lower Ct value of a pathogen to a greater likelihood of being a relevant causative pathogen. Moreover, not reporting viral pathogens with a Ct value higher than 35 led to a significant reduction (p < 0.0001) of reported mixed infections compared to oversimplified qualitative or qualitative reporting. By omitting very high Ct values and reporting semi-quantitatively, value was added to the syndromic reports, leading to an easier to read lab report, especially in mixed infections.

Longitudinal change in CSF β‐amyloid1−42 predicts PTSD symptomatology after the COVID‐19 lockdown in cognitively unimpaired older adults

AbstractBackgroundCognitively unimpaired (CU) adults with Alzheimer’s disease (AD) pathologic change may be more vulnerable to developing neuropsychiatric symptoms. We reported an association of higher β‐amyloid (Aβ) and increased anxiety/depression during the COVID‐19 lockdown in CU adults (Akinci et al., 2022). Here, we investigated whether changes in Aβ1−42 predict post‐traumatic stress disorder (PTSD) symptomatology following COVID‐19 lockdown in CU adults at risk for AD.MethodWe included 128 CU participants who underwent lumbar puncture 2.3±0.6 years before and 1.1±0.4 years after the lockdown. The outcome of interest was self‐reported PTSD symptomatology (Impact of Event Scale Revised) measured 1.7±0.1 years after the lockdown. The main predictor was annual rate of change in cerebrospinal fluid (CSF) Aβ1−42 measured with the NeuroToolKit panel of robust prototype assays (Roche Diagnostics International Ltd, Rotkreuz, Switzerland). Other predictors included sex, age, years of education, stressors during COVID‐19, and anxiety/depression (Hospital Anxiety and Depression Scale, HADS) and perceived stress (Perceived Stress Scale, PSS) measured during lockdown. We performed regression analyses to explore the association between annual rate of change in Aβ1‐42 with PTSD scores adjusting for demographic and stressor variables, and further adjusting for HADS and PSS scores. We then explored the mediating effect of HADS and PSS on the association between change in Aβ1‐42 and PTSD scores.ResultParticipant age ranged from 53−72 and 59% were women (Table 1). Independent of demographic variables and stressors experienced during COVID‐19, the rate of annual change in Aβ1‐42 predicted PTSD scores (p = .008; Table 2). This association attenuated after introducing HADS and PSS scores into the model (p = .066). We found full mediation effects of HADS and PSS scores, explaining 46% and 40% of the effect of annual change in Aβ1‐42 on PTSD scores, respectively (Figure 1).ConclusionCU older adults with longitudinal CSF Aβ1−42 increases are more vulnerable to developing PTSD symptomatology after a stressful event, such as the pandemic. Anxiety/depression is one plausible mechanism through which increases in Aβ might exacerbate PTSD symptomatology. The results suggest a window for stress regulation during stressful events to prevent further mental health worsening in people at risk for AD.

Association between blood pressure and Alzheimer’s disease biomarkers in cognitively unimpaired adults

AbstractBackgroundHigh blood pressure is an established risk factor for dementia. However, the underlying mechanisms are not completely understood. We aimed to evaluate the association between blood pressure and Alzheimer’s disease (AD) biomarkers in cognitively unimpaired adults from the ALFA+ study.MethodWe analyzed data from 301 participants with available cross‐sectional blood pressure measures (acquired by trained staff with an oscillometric device after 5 minutes of rest) and cerebrospinal fluid (CSF) AD core biomarkers data (CSF p‐tau and t‐tau were measured with Elecsys® immunoassays and CSF Aβ42 and Aβ40 with the Roche NeuroToolKit, a panel of robust prototype assays, both from Roche Diagnostics International Ltd), and a subset of 278 participants with amyloid PET data, of whom 110 had a second follow‐up PET acquisition (3.4 years after in average). We used separate linear regression models to analyze the association of systolic blood pressure (SBP), diastolic blood pressure (DBP), and pulse pressure (PP = SBP‐DBP) with CSF and PET biomarkers at a cross‐sectional level, and their association with the annualized rate of change in amyloid PET [(follow‐up centiloids – baseline centiloids) / years between PET scan acquisition]. We adjusted all models by age, sex, APOE status, waist‐to‐hip ratio, dyslipidemia, diabetes, tobacco and alcohol consumption, physical activity, and antihypertensive treatment.ResultHigher baseline SBP (but not DBP or PP) was significantly associated with increased amyloid PET uptake over time (p = 0.043). In cross‐sectional analyses, DBP was negatively associated with Aβ42 (p = 0.013), Aβ40 (p = 0.026), and t‐tau (p = 0.045), and marginally associated with p‐tau (p = 0.069), but not with the Aβ42/40 ratio nor baseline amyloid PET uptake. Associations with Aβ42 and t‐tau were no longer significant after further adjustment by CSF Aβ40 levels. SBP and PP were not associated with CSF biomarkers or baseline amyloid PET uptake.ConclusionHigher SBP is associated with longitudinal brain amyloid accumulation, which reinforces the importance of blood pressure control in AD prevention. DBP is also associated at a cross‐sectional level with AD biomarker concentration in CSF, but the attenuation of this association after adjusting by Aβ40 suggests a non‐specific.

Targeted plasma eQTL and pQTL to identify potential novel biomarkers for Alzheimer’s Disease in African Americans

AbstractBackgroundGiven the overlap of symptoms between Alzheimer’s disease (AD) and other dementias, it is essential to develop widely accessible biomarkers that will ensure an accurate diagnosis of AD to enable its proper treatment. African Americans (AA) are twice more likely to develop AD than non‐Hispanic whites (NHW) and yet they remain underrepresented in AD research. The few studies conducted in AA suggest that there are different factors that influence AD in this population; therefore, it is possible that different biomarkers and/or therapies may be needed to effectively treat AD in AA. In this study we are testing the hypothesis that genetic variants that show association with AD‐risk in AA and with plasma levels of transcript and proteins may serve as AD biomarkers which are more accessible and less costly than cerebrospinal fluid or neuroimaging biomarkers.MethodsIn 189 AD cases and 183 cognitively unimpaired (CU) AA participants of the Florida Consortium of African American Alzheimer’s Disease Studies (FCA3DS), we performed targeted DNA sequencing of 10 AD‐associated loci. Genetic variants were tested for association with plasma transcripts and plasma total tau protein levels previously measured in this cohort using a nanoString® custom panel (Reddy J, et al. 2022) and Simoa assays (Deniz K, et al. 2021), respectively. In receiver operating characteristic (ROC) analyses, the most significant expression/protein quantitative trait loci (eQTL/pQTL) were added sequentially to a base model that included only age and sex to identify eQTL/pQTL that improved the accuracy to correctly classify AD cases vs. CU.ResultsOf the 5112 variants identified in the targeted sequencing, 71 were nominally associated (p&lt;0.05) with plasma transcript/protein levels and with AD‐risk. ROC analysis of age, sex, eQTL/pQTL in APOE, ABCA7, AKAP9, EPHA1, SORL1, and TLR4, and plasma levels of APP, ABCA7, AKAP9, CD14, CLU, and APOE‐ɛ4 dosage achieved 77.6% area under the curve (AUC) to discriminate AD vs. CU, a 19.9% improvement over a model that only included age and sex.ConclusionsPlasma transcript levels and eQTL/pQTL are promising diagnostic biomarkers for AD that may improve accessibility and reduce costs for a more accurate diagnosis of AD.

Establishing a molecular toolbox of lineage-specific real-time RT-PCR assays for the characterization of foot-and-mouth disease viruses circulating in Asia

Foot-and-mouth disease (FMD) is endemic in many Asian countries, with outbreaks occurring regularly due to viruses from serotypes O, A, and Asia1 that co-circulate in the region. The ability to rapidly characterize new virus occurrences provides critical information to understand the epidemiology and risks associated with field outbreaks, and helps in the selection of appropriate vaccines to control the disease. FMD lineage-specific characterization is usually determined through sequencing; however, this capacity is not always readily available. In this study, we provide a panel of real-time RT-PCR (rRT-PCR) assays to allow differentiation of the FMD virus (FMDV) lineages known to have been co-circulating in Asia during 2020. This panel included five new rRT-PCR assays designed to detect lineages O/ME-SA/PanAsia-PanAsia-2, O/ME-SA/Ind-2001, O/SEA/Mya-98, O/CATHAY, and A/ASIA/Sea-97, along with three published rRT-PCR assays for A/ASIA/Iran-05, A/ASIA/G-VII, and Asia1 serotypes. Samples of known FMD lineage (n = 85) were tested in parallel with all eight lineage-specific assays and an established 3D pan-FMD rRT-PCR assay, and comparative limit of detection (LOD) experiments were conducted for the five newly developed assays. All samples (85/85) were assigned to the correct serotype, and the correct lineage was assigned for 70 out of 85 samples where amplification only occurred with the homologous assay. For 13 out of 85 of the samples, there was amplification in two assays; however, the correct lineage could be designated based on the strongest Ct values for 12 out of 13 samples. An incorrect lineage was assigned for 3 out of 85 samples. The amplification efficiencies for the five new rRT-PCR assays ranged between 79.7 and 100.5%, with nucleic acid dilution experiments demonstrating broadly equivalent limits of detection when compared to the 3D pan-FMD rRT-PCR assay. These new tests, together with other published lineage-specific rRT-PCR assays, constitute a panel of assays (or molecular toolbox) that can be selected for use in FMD endemic countries (individually or a subset of the assays depending on region/lineages known to be circulating) for rapid characterization of the FMDV lineages circulating in Asia at a relatively low cost. This molecular toolbox will enhance the ability of national laboratories in endemic settings to accurately characterize circulating FMDV strains and facilitate prompt implementation of control strategies, and may be particularly useful in settings where it is difficult to access sequencing capability.

Identification and Biological Evaluation of a Potent and Selective JAK1 Inhibitor for the Treatment of Pulmonary Fibrosis.

Janus kinase 1 (JAK1) plays a pivotal role in regulating inflammation and fibrosis via the JAK/STAT signaling pathway, making it a promising target for associated diseases. In this study, we explored the modification of an N-methyl 1H-pyrrolo[2,3-b]pyridine-5-carboxylate core, leading to the identification of 4-(((2S,4S)-1-(4-trifluoromethyl)-2-methylpiperidin-4-yl)amino)-N-methyl-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (36b) as a highly potent and selective JAK1 inhibitor. Compound 36b exhibited an impressive IC50 value of 0.044 nM for JAK1 and demonstrated remarkable selectivity of 382-fold, 210-fold, and 1325-fold specificity over JAK2, JAK3, and TYK2, respectively. The kinase panel assays further confirmed its specificity, and cell-based experiments established its efficacy in inhibiting JAK1-STAT phosphorylation in human L-132 or SK-MES-1 cells. Pharmacokinetic studies revealed that compound 36b boasts an oral bioavailability exceeding 36%. In a bleomycin-induced fibrosis mouse model, compound 36b significantly reduced STAT3 phosphorylation, resulting in improvement in body weight and reduced collagen deposition, all achieved without significant side effects.

Evaluation of Siltuximab and Tocilizumab Analytical Interference on Interleukin 6 Measurements

Abstract Background Interleukin 6 (IL-6) plays important roles in both innate and adaptive immunity, while dysregulated persistent production of IL-6 is involved in pathological development of various diseases associated with inflammation. Targeting the IL-6 pathway has led to many innovative treatment approaches. Siltuximab and Tocilizumab are monoclonal antibodies targeting IL-6 and IL-6 receptors respectively and have been FDA-approved for treatment of Castleman disease (Siltuximab), a rare lymphoproliferative disorder characterized by hyperproduction of IL-6 with an unknown prevalence, autoimmune diseases such as rheumatoid arthritis (Tocilizumab), and both shown potential as treatment for cytokine release syndrome in hospitalized COVID-19 patients. However, the presence of siltuximab can interfere with IL-6 assays, leading to the use of C-reactive protein (CRP) as a surrogate marker to monitor its efficacy. Aim To evaluate potential Siltuximab and Tocilizumab antibody interference on IL-6 measurements in three different IL-6 protein immunoassays. Method: Serum or EDTA plasma sample pools with different IL-6 concentrations ranging from low (2.3 – 11.1 pg/mL, around the reference interval cut-off for each assay) to high (18.5 – 1142 pg/mL), above reference interval cut-off for each assay) were utilized. Various concentrations of Siltuximab or Tocilizumab were spiked into the sample pools (at 10% of total volume) to achieve final concentrations up to 600 µg/mL and up to 300 µg/mL, respectively. Sample spiked with 10% saline were used as no-drug control. Three assays were used to measure IL-6 concentration: Beckman Access IL-6 assay on UniCel DxI 800 (Beckman Coulter), V-PLEX Plus human IL-6 assay on QuickPlex SQ120 (Meso Scale Diagnostics), and a Luminex cytokine panel assay developed in house (R&amp;D systems) with upper reference interval limits of 6.4,1.8 and 5 pg/mL respectively. Results Siltuximab interfered with the measurement of IL-6 for all three assays. A significant interference (&amp;gt; 20% reduction) was observed at even a very low siltuximab concentrations in all three assays (0.05 µg/mL). In the presence of 5 µg/mL or greater Siltuximab, a maximum reduction of 93%, 83%, and 40% was observed for Beckman Access, R&amp;D Luminex assay, and V-PLEX Plus Mesoscale assay, respectively. Tocilizumab did not interfere with the measurement of IL-6 in the three assays when serum was spiked with up to 300 µg/mL of the drug. Conclusion Siltuximab falsely reduce the measured IL-6 concentration on all three immunoassays tested in this study to differing degrees, whereas Tocilizumab does not. Consider the therapeutic range of Siltuximab (84 to 332 µg/mL) for treatment of Castleman disease, and a half-life of 20.6 days, measurement of IL-6 may not be useful in Siltuximab treated patients for an extended time-period.

One High Efficient Detection Panel of Multiparamer Flow Cytometry for Non-Hematopoietic Tumors

Background: In the past half century, flow cytometry has been successfully applied to detection of hematopoietic tumors. In fact, the incidence rate of non-hematopoietic tumors is higher than hematologic malignancies. However, the application of flow cytometry has been limited for a long time due to samples, lack of specific markers and other reasons. With the development of immunological technique, the detection of solid tumors by flow cytometry has attracted renewed attention. We designed a highly sensitive and specific assay panel for multiparameter flow cytometry(MFC) screening, diagnosis and follow-up of non-hematopoietic tumors. Aims:find a highly efficient panel of MFC for screening, diagnosis and follow-up of non-hematopoietic tumors. methods: From Aug. 1st, 2008 to Mar. 31st, 2022, 858 samples from patients with non-hematopoietic tumor were detected in the flow cytometry lab of Hebei Yanda Lu Daopei Hospital, of which 617 samples were BM, 3 PB, 196 cerebrospinal fluid(CSF), 9 Pleural effusion or ascites, 3 lymph node, and 30 fine needle aspiration(FNA) from various tissue. In these 850 person, 476 were males, 374 females, the median age was 4 years old (0-80 years), and the median percentage of tumor cells in the sample was 0.16% (0.01%-96.23%).After rounds of optimization, CD9 FITC/GD2 PE/CD3 percp/CD4 pecy7/CD56 APC/CD36 Apccy7/CD81 BV421/CD45 V500, cytokeratin(CK) or CD326 FITC/HLA-ABC PE/CD38 percp/CD19 pecy7/CD56 APC/CD36 Apccy7/CD7 BV421/CD45V500 were selected as the final detection panel. The analysis was performed using 3 laser 8-10 color canto flow cytometers, and diva/flowjo software. The results were compared with pathological and clinical diagnosis to evaluate the sensitivity and specificity. Results: Firstly, Sensitivity and specificity analysis. Of 858 samples, 567 were positive, of which 442 were BM, 2 PB, 84 CSF, 9 fluid, 2 lymph nodes, and 28 FNA. The coincidence rate was more than 90% with pathology, other laboratory examinations and clinical diagnosis. It covers most of tumors nominated by WHO diagnostic criteria. The coverage and specificity were more than 99%, and the sensitivity was 0.01%. Both false positive rate and false negative rate were less than 1%. Secondly, the selection and verification of immunological markers. The positive rate and specificity of each immunological markers were analyzed in 567 positive samples, as shown in Figure 1. The specificity of CK combined with GD2 in the diagnosis of non-hematopoietic tumors was almost 100%, and the positive rate of CD56 was significantly different between epithelial and non-epithelial tumors. CD3/CD19/CD4/CD38/HLA-ABC/CD36/CD7 is almost not expressed in non-hematopoietic tumors but expressed at least one by almost all hematopoietic cells, which can exclude hematological malignancies. CD9/CD81, as a sensitive but non-specific marker, can assist in judgment, and combined with CD45-/CD36-, exclude non-specific staining to prevent missed diagnosis. Thirdly, tumor sources can be roughly distinguished. PCA (Principal component analysis) can accurately classify epithelial and non-epithelial tumors by analyzing 38 cases of neuroblastoma and 5 cases of epithelial-derived tumors. Flowsom cluster analysis of some common solid tumors in children showed that the expression intensity of different tumor markers was different. As shown in Figure 2. Conclusion: Although the high heterogeneity of non-hematopoietic tumors and the lack of specific immune markers, we found one high sensitive and specific MFC panel for detection of solid tumors after rounds of trials. The panel includes some non-lineage specific markers but commonly expressed in hematologic neoplasms and rarely in non-hematopoietic tumors. Different from the precise classification of euroflow protocol, our panel focuses on the identification of tumor cells, and exclusion of hematological malignancies and non-specific staining. It is an important detection and analysis panel with high coverage, strong practicability, high sensitivity and specificity, which may reduce the rate of missed diagnosis.

Validation of a real-time PCR panel for detection and quantification of nine pathogens commonly associated with canine infectious respiratory disease

Canine infectious respiratory disease (CIRD) is a complicated respiratory syndrome in dogs [1–3]. A panel PCR was developed [4] to detect nine pathogens commonly associated with CIRD: Mycoplasma cynos, Mycoplasma canis, Bordetella bronchiseptica; canine adenovirus type 2, canine herpesvirus 1, canine parainfluenza virus, canine distemper virus, canine influenza virus and canine respiratory coronavirus [5–16]. To evaluate diagnostic performance of the assay, 740 nasal swab and lung tissue samples were collected and tested with the new assay, and compared to an older version of the assay detecting the same pathogens except that it does not differentiate the two Mycoplasma species. Results indicated that the new assay had the same level of specificity, but with higher diagnostic sensitivity and had identified additional samples with potential co-infections. To confirm the new assay is detecting the correct pathogens, samples with discrepant results between the two assays were sequence-confirmed. Spiking a high concertation target to samples carrying lower concentrations of other targets was carried out and the results demonstrated that there was no apparent interference among targets in the same PCR reaction. Another spike-in experiment was used to determine detection sensitivity between nasal swab and lung tissue samples, and similar results were obtained.•A nine-pathogen CIRD PCR panel assay had identified 139 positives from 740 clinical samples with 60 co-infections;•High-concentration target does not have apparent effect on detecting low-concentration targets;•Detection sensitivity were similar between nasal swab and lung tissue samples.

BIOM-26. RAPID IDENTIFICATION OF IDH1-R132 VARIANTS IN GLIOMA WITH LOCKED NUCLEIC ACID LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LNA-LAMP)

Abstract Point mutations that impact the catalytic site of isocitrate dehydrogenase 1 (IDH1) are associated with an extended overall survival of approximately 3.8 years for glioma patients compared to 1 year for those with wild-type IDH1. The difference in survival benefit is associated with the maximal surgical resection of IDH1 mutant tumors, making intraoperative knowledge of the mutational status valuable. Unfortunately, current methods used to determine IDH1-R132 mutational status are incompatible with obtaining real-time results. We have developed a panel of assays that use LNA-LAMP to identify commonly encountered IDH1-R132 variants (R132H, R132C, R132G, R132S, R132L) within 40 minutes. These assays require little preanalytical processing, no complex equipment, and yield results which are easily interpreted by the naked eye. Tumor samples are initially subjected to a 5-minute alkaline digest, then added directly to LNA-LAMP reactions containing primers unique to each IDH1-R132 variant. Discrimination between the wild-type and mutant allele is achieved with the integration of locked nucleic acids within primers. A reaction with an IDH1-R132R specific primer serves as the sample positive control, as all tumor lysates are expected to contain wild-type IDH1 DNA. Lysates are incubated for 35 minutes in a hot water bath, followed by visual interpretation using a pH-based colorimetric indicator, where samples positive for the mutation appear a vibrant yellow while those lacking the mutation remain pink. This method has been rigorously tested with patient-derived tumor lysates, yielding reproducible and accurate results, and has an analytical sensitivity of about 20% mutant DNA in a background of wild-type DNA. This sensitivity is comparable to that of Sanger Sequencing while simultaneously offering a more cost-effective and efficient workflow. LNA-LAMP is amenable for use in the intraoperative window to allow patients with an IDH1-R132 mutation the benefit of a maximal surgical resection.

Multiplex screening for biomarkers associated with subsequent heart failure hospitalisation in patients with atrial fibrillation: insights from the ARISTOTLE trial

Abstract Background Atrial fibrillation (AF) is associated with heart failure (HF) with a complex cause and effect relationship. We performed multiplex screening of plasma proteins in order to identify biomarkers and pathways associated with subsequent HF hospitalisation in patients with AF. Methods Using a case-cohort design of patients with AF from the ARISTOTLE trial, the study cohort consisted of 596 cases of HF hospitalisations during follow-up and 4029 randomly selected controls without HF hospitalization. The median follow-up time was 1.9 years. Plasma obtained at randomisation was analysed with conventional immunoassays and three OLINK proximity extension assay panels; CVDII, CVDIII and the inflammation panel. Biomarker associations with subsequent HF hospitalisation was evaluated using Random Survival Forest (RF), Boruta and Cox-regression analyses adjusted for baseline characteristics, renal function, and N-terminal pro-B-type natriuretic peptide (NT-proBNP) as well as cardiac troponin-T (cTnT-hs). Results The biomarkers most associated with HF hospitalisation, using all three statistical methods (Table), were (hazard ratio for inter-quartile comparison [95% CI]), NT-proBNP (2.07 [1.76 - 2.4]), cTnT-hs (1.35 [1.15 - 1.58]), fibroblast growth factor 23 (FGF-23; 1.26 [1.15 - 1.39]), spondin-1 (SPON-1; 1.21 [1.10, 1.33]), TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2; 1.19 [1.07 - 1.32]), insulin Growth Factor Binding Protein 7 (IGFBP7; 1.25 [1.13 - 1.39]), urokinase receptor (uPAR) 1.4 [1.19 - 1.66]), vascular Endothelial Growth Factor D (VEGF-D; 1.28 [1.08 - 1.53]), pro-adrenomedullin (ADM; 1.20 [1.03 - 1.40]), and C-C motif chemokine 3 (CCL3; 1.15 [1.06 - 1.24]). Conclusions Out of 269 evaluated biomarkers, this comprehensive screening study identified biomarkers representing cardiorenal dysfunction (NT-proBNP, cTnT-hs, FGF-23), cell adhesion (SPON-1), apoptosis (TRAILR2), growth factors (IGFBP7, VEGF-D), fibrinolysis (uPAR), vasodilation (ADM) and inflammation (CCL3) as being the most strongly associated with subsequent HF hospitalisation in patients with AF. These findings highlight the most important pathophysiological pathways involved in new or worsening HF in patients with AF.Biomarkers associated with HF hospitalis

GDF-15 adds to GRACE 2.0 for long-term risk prediction in ACS but echocardiographic parameters do not: A SWEDEHEART prognostication study

Abstract Background The risk stratification score GRACE 2.0 has lately been validated to predict all-cause death up to three years after acute coronary syndrome (ACS). GRACE 2.0 does not take important prognostic biomarkers and imaging measurements that may improve long-term risk prediction in to account. This study set out to evaluate the clinical value of such parameters analyzed on top of GRACE 2.0. Methods The study was performed in a subset of 773 patients from the TOTAL-AMI cohort with ACS subjects recruited from three Swedish hospitals between 2008 and 2014. Biomarkers were analyzed with a Proximity Extension Assay panel and the echocardiographic parameters, Left Ventricular Ejection Fraction (LVEF) and Global Longitudinal Strain (GLS), were assessed at a core lab. LVEF, GLS and two high yield biomarkers (NTproBNP and GDF-15) were analyzed with Cox proportional hazards regression by stepwise addition of each variable to a baseline model with GRACE 2.0. The C-index was assessed for each model. All models were adjusted for treatment with betablockers and/or RAAS-inhibitors. Outcome was all-cause death at three years. Results Forty-one patients had died at three years follow-up. The mean population age was 64 years and 77% were men. Median LVEF was 55 % (inter quartile range: 47-60). LVEF, GLS and GDF-15 all emerged as independent predictors of three-year death in the multivariable models with GRACE 2.0 and medical treatment. Only GDF-15 rendered substantial improvement in the C-index on top of GRACE 2.0 (0.755 to 0.810). The echocardiographic measurements did not improve prognostication on top of GRACE 2.0 and GDF-15. Conclusions GRACE 2.0 emerged as a strong long-term risk-predictor irrespective of the biomarkers and imaging measurements. Although LVEF, GLS and GDF-15 also emerged as independent predictors of three-year death only GDF-15 improved the C-index to an extent that may be considered clinically meaningful.

Abstract 11983: Biomarker Profile of Patients With Atrial Fibrillation and Heart Failure With Preserved Ejection Fraction or Reduced Ejection Fraction: Insights From the ARISTOTLE Trial

Introduction: Heart failure with preserved ejection fraction (HFpEF) is a poorly understood entity with phenotypic differences to heart failure with reduced ejection fraction (HFrEF). To shed light on the potential pathophysiological difference between HFrEF and HFpEF, multiplex screening of plasma proteins was performed. Methods: From the ARISTOTLE trial with atrial fibrillation (AF) patients, 562 patients with HFrEF and 562 with HFpEF at randomisation were included. Heart failure status at baseline was defined according to systolic left ventricular function; for HFrEF ≤40% and for HFpEF &gt;40%. Plasma samples were analysed with conventional immunoassays and OLINK proximity extension assay panels; CVDII, CVDIII and the inflammation panel. Associations between biomarkers and HF subtype was evaluated with Wilcoxon–Mann–Whitney test with Bonferroni-Holm adjustment for multiplicity. Results: Out of 269 biomarkers, 53 biomarkers differed between the HF subtypes. After adjustment for multiplicity, nine biomarkers had the strongest associations with difference between HFrEF and HFpEF (Figure). In HFrEF, the proteins levels of N-terminal pro-B-type natriuretic peptide (NT-proBNP), brain natriuretic peptide (BNP), cardiac troponin-T (cTnT-hs), renin, growth differentiation factor-15 (GDF-15), angiotensin-converting enzyme 2 (ACE-2) and interleukin-6 (IL-6) were higher than in HFpEF, whereas levels of stem cell factor (SCF) and leptin were higher (Figure) in HFpEF. Conclusions: HFrEF was more strongly associated with elevated markers of cardiorenal dysfunction, the renin-angiotensin-aldosterone system and oxidative stress/inflammation while HFpEF was more strongly associated with higher levels of adipose tissue metabolism and vasculogenesis and tissue repair proteins. These findings add valuable knowledge to the understanding of the underlying differing biological mechanisms in the HF subtypes HFrEF and HFpEF in an AF population.

A Novel and Potent EZH1/2 Dual Inhibitor, HM97662 Demonstrated a Wide Spectrum of Therapeutic Potential for Hematological Malignancies

Chromatin remodeling is a crucial process for transcriptional regulation, of which dysregulation is often observed in various human cancers. The enhancer of zeste homolog 2 (EZH2) and its homolog EZH1 are catalytic components of polycomb repressive complex 2 (PRC2), which tri-methylate histone H3 at lysine 27 (H3K27me3) to repress transcription of their target genes. Hyperinduction of H3K27me3 mediated by EZH2 overexpression or EZH2 gain-of-function (GOF) mutation (e.g. Y641, A677, and A687) has been associated with the progression of hematological malignancies including B-cell lymphoma, T-cell lymphoma, and multiple myeloma. Hence, it has been suggested that suppressing PRC2 activity by EZH2 inhibition is a potential therapeutic target for hematological malignancies. Although the methyltransferase activity of PRC2 is mainly contributed by EZH2, EZH1 also conducts a compensatory role to maintain tri-methylation of H3K27. Moreover, EZH1 directly binds to chromatin and modulates its condensation. Therefore, dual inhibition of EZH1 and EZH2 can give better effect than EZH2 inhibition alone in blocking PRC2 function as an anti-cancer therapy. Herein, we presented a novel EZH1/2 dual inhibitor, HM97662, which simultaneously inhibited the methyltransferase activity of wild-type EZH1 as well as wild-type and GOF mutant EZH2 at nanomolar concentrations. SPR analysis of HM97662 on trimeric complexes (EZH1/EZH2, SUZ12, and EED) demonstrated that HM97662 binds both EZH1- and EZH2-PRC2 complexes. HM97662 potently repressed tri-methylation of H3K27 in KARPAS 422 DLBCL, HH PTCL, and MM.1S MM cell lines. Furthermore, HM97662 showed broader and stronger activities than EZH2 selective inhibitors in long-term proliferation assay against a variety of hematological cancer cell lines. HM97662 induced the differentiation of DLBCL to plasma cells with the increment of cell lineage specific markers (e.g. PRDM1 and CD38), and caused cell cycle G0/G1 arrest and apoptosis in KARPAS 422 DLBCL cells harboring EZH2 Y641N GOF mutation. Additionally, HM97662 increased mRNA expression of several target genes inducing cell cycle arrest and apoptosis in gene panel assay performed in HH PTCL cell line. Two cell cycle repressors, CDKN2A (p16) and CDKN1C (p57), and a pro-apoptotic marker, BTG-2, were significantly increased by HM97662. We further conducted a chromatin accessibility assay and identified that the chromatin structures of that genes were loosened and highly transcribed after the treatment of HM97662. As a result, HM97662 showed potent antitumor activity in various hematological cell lines xenograft mouse models including B-cell lymphoma, T-cell lymphoma, and multiple myeloma (e.g. KARPAS 422, HuT-102, and MM.1S). Once daily oral dosing of HM97662 greatly inhibited tumor growth in a dose-dependent manner without significant clinical signs compared to the known EZH2 selective or EZH1/2 dual inhibitor. In conclusion, the present preclinical studies demonstrated that HM97662, an EZH1/2 dual inhibitor, had a promising and wide spectrum of therapeutic potential for hematological malignancies. It is urgent to assess the effectiveness of HM97662 in further clinical trials.

Structure-based design, synthesis and biological evaluation of N-substituted 6H-thiochromeno[2,3–c]quinolin-12(12H)-one as potential breast cancer drugs

Tetraheterocyclic compounds, derived from natural sources and contemporary pharmaceuticals, have shown promise as multitarget therapeutic agents. However, their mechanisms of action remain partially understood. In this study, we synthesized a series of 6H-thiochromeno[2,3-c]quinolin-12(12H)-one derivative, totaling 26 compounds, and assessed their potential for therapeutic application. We evaluated their effects on cell proliferation and conducted NCI-60 cell panel assays. MTT assays revealed that select compounds exhibited notable antiproliferative activity against two breast cancer cell lines (MCF-7 and MDA-MB-468). Notably, compounds 17 and 18 displayed the highest cytotoxicity against these cell lines. Furthermore, one-dose assays of the NCI-60 human tumor cell line screening program identified compounds 6, 7, 16, 18, 20, 24, 25, and 30 for further investigation.Subsequent five-dose cytotoxicity studies focused on compounds 18 and 20, which met the threshold inhibition criteria across a panel of cell lines. Our study highlights the effectiveness of compounds 18 and 20 in targeting breast cancer cell lines. Molecular docking simulations revealed that these compounds bind to the active sites of topoisomerase I (TOPO I). Our findings suggest that these novel compounds are promising anticancer agents, particularly against breast cancer, and are worthy of consideration as lead pharmacological candidates.

Stability Assessment of Fresh or Cryopreserved Whole Blood and Bone Marrow Samples from AML and B-ALL Patients to Monitor CD123 Receptor and Immune Cells Subsets

Introduction: Compounds targeting CD123 are currently under clinical development for the treatment of acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (MDS). As part of clinical trials, CD123 expression and immune cell subsets need be monitored by quantitative flow cytometry (qFC) in bone marrow (BM) aspirates and whole blood (WB) samples before and on-treatment. One critical parameter for analysis is the poor stability of the fresh biological BM or WB samples. No general rule can predict the stability of the expression of a given receptor on diseased or healthy cells thus the pre-analytical stability of each biomarker should be evaluated to design the sample logistics. The pre analytical stability of clinical samples is the main constraint for quantitative flow cytometry (qFC) in multicenter and worldwide clinical studies. The transportation of the samples is one of the limiting factor. The classical freezing method of peripherical whole blood (WB) or bone marrow (BM) isolated by Ficoll requires high skilled operators, is time consuming and may be difficult to set up at sampling site in hospitals. One option to make the clinical samples suitable for subsequent high dimensional analysis by qFC is to use a simple freezing method at -80°C. Our cryopreservation method could be a new option for easier preparation of clinical samples prior to centralized samples analysis. This study aims to evaluate the pre-analytical fresh or cryopreserved stability of each pharmacodynamic monitoring endpoint over time: CD123 expression and receptor density, T-cell subsets and activation profile. Methods: FreshBM and WB were collected in EDTA tubes from 5 AML/MDS patients at hospitals, in accordance with local regulations. The samples were stored at room temperature (RT) for 4, 24, 48 or 72 h before immunostaining with the standard operation procedures and analysis by qFC or mixed with the cryopreserved media (BioCytex) within 24 h post-collection before freezing at -80°C for months. After storage, the cryopreserved samples were thawed and immunostained before analysis by qFC. Using a qFC assay (Multiplex FC panel assay developed by BioCytex, Marseille, France), CD123 receptor density/cell was collected. T-cell and blast cell immunophenotypes were also assessed on fresh samples for time point 4, 24, 48, or 72 h post-collection but also for cryopreserved WB sample within the 24hs post collection. The assay profiled blasts, monocytes, plasmacytoid dendritic cells, basophils and T-cells. Results: CD123 density in blast and normal cells was stable at RT from 4 h up to 48 h after collection. The profiles of blast subsets were very similar in BM and WB samples from a particular patient, whether a single blast cell subset (CD45lowCD11b-CD34+CD38med) or several blast cell subsets (expressing CD34 and CD38 at different levels) were identified. Both T-cell distribution and activation markers were stable up to 48 h for BM and WB. A similar pattern of T-cell subsets was also observed in the BM and WB paired samples from the same donor. In addition, CD123 receptor density, immunophenotyping and percentage of blast subsets and T cells from fresh WB samples was comparable to paired cryopreserved samples with a new method and for at least 1 month. Conclusions: Based on these observations, we conclude that CD123 density, blast cells and T-cell subsets in WB and BM samples from AML patients were stable for 48 h in our experimental conditions. As a consequence, fresh WB and BM samples can be processed and analyzed by qFC within 48 h of sample collection. Furthermore, the new cryopreservation method showed sample stabilization for blasts and T cells immunophenotyping. This data opens new perspectives in clinical studies for AML This new strategy would also help to standardize preparation of clinical samples and to centralize sample analysis.

Differential patterns of lysosomal dysfunction are seen in the clinicopathological forms of primary progressive aphasia.

Increasing evidence implicates endo-lysosomal dysfunction in frontotemporal dementia (FTD). 18 proteins were quantified using a mass spectrometry assay panel in the cerebrospinal fluid of 36 people with the language variant of FTD, primary progressive aphasia (PPA) (including 13 with non-fluent variant (nfvPPA), 11 with semantic variant (svPPA), and 12 with logopenic variant (lvPPA)) and 19 healthy controls. The concentrations of the cathepsins (B, D, F, L1, and Z) as well as AP-2 complex subunit beta, ganglioside GM2 activator, beta-hexosaminidase subunit beta, tissue alpha L-fucosidase, and ubiquitin were decreased in nfvPPA compared with controls. In contrast, the concentrations of amyloid beta A4 protein, cathepsin Z, and dipeptidyl peptidase 2 were decreased in svPPA compared with controls. No proteins were abnormal in lvPPA. These results indicate a differential alteration of lysosomal proteins in the PPA variants, suggesting those with non-Alzheimer's pathologies are more likely to show abnormal lysosomal function.

Biological age estimation using circulating blood biomarkers

Biological age captures physiological deterioration better than chronological age and is amenable to interventions. Blood-based biomarkers have been identified as suitable candidates for biological age estimation. This study aims to improve biological age estimation using machine learning models and a feature-set of 60 circulating biomarkers available from the UK Biobank (n = 306,116). We implement an Elastic-Net derived Cox model with 25 selected biomarkers to predict mortality risk (C-Index = 0.778; 95% CI [0.767–0.788]), which outperforms the well-known blood-biomarker based PhenoAge model (C-Index = 0.750; 95% CI [0.739–0.761]), providing a C-Index lift of 0.028 representing an 11% relative increase in predictive value. Importantly, we then show that using common clinical assay panels, with few biomarkers, alongside imputation and the model derived on the full set of biomarkers, does not substantially degrade predictive accuracy from the theoretical maximum achievable for the available biomarkers. Biological age is estimated as the equivalent age within the same-sex population which corresponds to an individual’s mortality risk. Values ranged between 20-years younger and 20-years older than individuals’ chronological age, exposing the magnitude of ageing signals contained in blood markers. Thus, we demonstrate a practical and cost-efficient method of estimating an improved measure of Biological Age, available to the general population.

Detection and genotyping of Dientamoeba fragilis from symptomatic patients: New insights from Italy into a little-known gastrointestinal protozoan

Dientamoeba fragilis (D. fragilis) represents a common protozoan in both high and low income countries. Despite this, epidemiological data on dientamoebiasis are still limited, and it is possible that the actual prevalence rates of D. fragilis have been underestimated due to the challenges in its detection and identification. In the present study, symptomatic patients from Rome (Central Italy) were surveyed for two years to determine D. fragilis percentage of infection and genotypes. Stool samples collection was performed over 864 patients, DNA extracted, and RT-PCR performed by the SeeGene Allplex™ Gastrointestinal Parasite Panel Assays. Seventy-nine resulted positive for D. fragilis (9.1%). Co-infections were detected in 22 isolates: 21 displayed Blastocystis sp. + D. fragilis (27.8%). Based on the sequence of a central fragment of the SSU rRNA gene, only genotype 1 was identified. These findings are among the few available data regarding genetic diversity of D. fragilis in Italy. Large-scale human and animal research are required to enhance our knowledge of prevalence, host range, genetic variability and zoonotic transmission of this little-known intestinal protozoan.