Multiplex Proximity Extension Assay Research Articles

Multivariate analyses of immune markers reveal increases in plasma EN-RAGE in first-episode psychosis patients

Immune cells and cytokines are largely recognized as significant factors in the pathophysiology of neuropsychiatric disorders. The possible role of other blood cells such as leukocytes in events of acute psychosis is in contrast only emerging. To study blood-born markers in acute psychosis we here evaluated plasma proteins in drug-naive first-episode psychosis (FEP) patients and healthy controls using a multiplex proximity extension assay technique. We analyzed a panel of 92 immune markers and plasma samples from 60 FEP patients and 50 controls and evaluated the changes obtained using multivariate statistical methods followed by protein pathway analyses. Data showed that 11 proteins are significantly different between FEP patients and healthy controls We observed increases in pro-inflammatory proteins such as interleukin-6, oncostatin-M, and transforming growth factor-alpha in FEP patients compared with controls. Likewise, the extracellular newly identified RAGE-binding protein (EN-RAGE) that regulates the expression of various cytokines was also elevated in the plasma of FEP patients. The results indicate that neutrophil-derived EN-RAGE could play an important role during the early phase of acute psychosis by stimulating cytokines and the immune response targeting thereby likely also the brain vasculature.

Systemic inflammatory proteins in offspring following maternal probiotic supplementation for atopic dermatitis prevention

BackgroundMaternal probiotic supplementation has a promising effect on atopic dermatitis (AD) prevention in infancy. In the randomised controlled study, Probiotics in the Prevention of Allergy among Children in Trondheim (ProPACT), maternal probiotics reduced the cumulative incidence of AD in their offspring by 40% at 2 years of age. However, our understanding on how probiotics prevented AD is still limited, and the role of inflammatory proteins in infants following maternal probiotic supplementation is unclear. We hypothesised that maternal probiotics lowered pro-inflammatory proteins and increased anti-inflammatory proteins in their 2-year-old children as a mechanism of AD prevention. We aimed to explore this hypothesis and the association between these proteins and the presence of AD, severity of AD, and the degree of preventive effect of probiotics.MethodsPlasma samples were collected from 2-year-old children (n = 202) during the ProPACT study, a randomised placebo-controlled trial of maternal probiotic supplementation. These samples were analysed for 92 inflammatory proteins using a multiplex proximity extension assay. Associations between inflammatory proteins and the presence and severity of AD, and the degree of preventive effect, was estimated individually using regression analysis and then collectively using unsupervised cluster analysis.ResultsSeveral proteins were observed to differ between the groups. The probiotic group had lower CCL11 and IL-17C, while children with AD had higher IL-17C, MCP-4, uPA, and CD6. Cytokine CCL20 and IL-18 had moderate correlation (r = 0.35 and r = 0.46) with the severity of AD. The cluster analysis revealed that children in the cluster of samples with the highest value of immune checkpoint receptors and inflammatory suppressor enzymes showed the greatest AD preventive effect from probiotics.ConclusionsThe proteins associated with both maternal probiotic supplementation and the presence and severity of AD warrant attention because of their potential biological relevance. Cluster analysis may provide a new insight when considering which subgroups benefit from probiotic supplementation. Larger studies are needed to confirm the results.Trial registration number: The study was retrospectively registered at ClinicalTrials.gov (NCT00159523) on 12nd September 2005.

SEVERE HEMATOLOGICAL TOXICITY FOLLOWING CD19 CAR‐T FOR RELAPSED/REFRACTORY LBCL IS ASSOCIATED WITH SUPPRESSIVE IMMUNE DYSREGULATION AND LIMITED CAR‐T EXPANSION

Background: Hematological toxicity represents the most frequent high-grade toxicity of CD19 CAR-T, but remains poorly understood. We recently proposed a classification system for CAR-T-related hematotoxicity, defining three unique phenotypes of neutrophil recovery: quick [“Q”] versus intermittent [“I”] versus aplastic [“A”]. Quick: sustained neutrophil recovery without a second dip below an ANC <1000/µL. Intermittent: neutrophil recovery (ANC > 1500/µL) followed by a second dip with an ANC < 1000/µL after day 21. Aplastic: continuous severe neutropenia (ANC < 500/µL) ≥14 days. Multivariable binary logistic regression was applied to analyze clinical metadata. CAR T-cell expansion dynamics were studied during the first 4 months post-CAR-T infusion. The plasma proteome was explored in 58 patients across four time points (day 0, 4, 14, 28) using a 92-protein multiplex proximity extension assay (Olink Bioscience). Progression-free (PFS) and overall survival (OS) were studied via Kaplan-Meier estimates. Results: The distribution of phenotypes was 18%, 42%, and 40% (A vs. I vs. Q). As expected, ‘aplastic’ patients displayed prolonged severe neutropenia (A vs. I vs. Q: 28 vs. 10 vs. 5 days, p < 0.001) and a higher rate of severe infections (40% vs. 22% vs. 18%). On multivariable regression of baseline risk factors (n = 344), the aplastic phenotype was independently associated with the presence of BM infiltration (p = 0.004), low ANC (p = 0.002), and increased ferritin (p = 0.039). When comparing CAR-T expansion among phenotypes, ‘intermittent’ patients displayed the greatest CAR T-cell expansion over time (Figure 1A). Conversely, ‘aplastic’ patients displayed low expansion with an unfavorable relationship between CAR-T expansion and baseline tumor burden (Figure 1B). Serum proteomics revealed higher markers of T-cell suppression, endothelial dysfunction, inflammatory cytokines, and macrophage activation in the ‘aplastic’ phenotype group (Figure 1C). Notably, both PFS and OS was poor in the ’aplastic’patients (1-yr PFS 26%, 1-yr OS 46%). On the other hand, the ‘intermittent’ phenotype, characterized by recurrent neutrophil dips, was associated with superior survival outcomes (1-yr PFS 51%, 1-yr OS 46%). Keywords: aggressive B-cell non-Hodgkin lymphoma, cellular therapies, basic and translational science – Basic and Translational Science - Other Conflicts of interests pertinent to the abstract K. Rejeski Consultant or advisory role: BMS/CELGENE Honoraria: Novartis, BMS/CELGENE Research funding: Kite/Gilead Travel grants: Kite/Gilead

Identifying Early Infections in the Setting of CRS With Routine and Exploratory Serum Proteomics and the HT10 Score Following CD19 CAR-T for Relapsed/Refractory B-NHL.

Early fever after chimeric antigen receptor T-cell (CAR-T) therapy can reflect both an infection or cytokine release syndrome (CRS). Identifying early infections in the setting of CRS and neutropenia represents an unresolved clinical challenge. In this retrospective observational analysis, early fever events (day 0-30) were characterized as infection versus CRS in 62 patients treated with standard-of-care CD19.CAR-T for relapsed/refractory B-cell non-Hodgkin lymphoma. Routine serum inflammatory markers (C-reactive protein [CRP], interleukin-6 [IL-6], procalcitonin [PCT]) were recorded daily. Exploratory plasma proteomics were performed longitudinally in 52 patients using a multiplex proximity extension assay (Olink proteomics). Compared with the CRSonly cohort, we noted increased event-day IL-6 (median 2243 versus 64 pg/mL, P = 0.03) and particularly high PCT levels (median 1.6 versus 0.3 µg/L, P < 0.0001) in the patients that developed severe infections. For PCT, an optimal discriminatory threshold of 1.5 µg/L was established (area under the receiver operating characteristic curve [AUCROC] = 0.78). Next, we incorporated day-of-fever PCT levels with the patient-individual CAR-HEMATOTOX score. In a multicenter validation cohort (n = 125), we confirmed the discriminatory capacity of this so-called HT10 score for early infections at first fever (AUCROC = 0.87, P < 0.0001, sens. 86%, spec. 86%). Additionally, Olink proteomics revealed pronounced immune dysregulation and endothelial dysfunction in patients with severe infections as evidenced by an increased ANGPT2/1 ratio and an altered CD40/CD40L-axis. In conclusion, the high discriminatory capacity of the HT10 score for infections highlights the advantage of dynamic risk assessment and supports the incorporation of PCT into routine inflammatory panels. Candidate markers from Olink proteomics may further refine risk-stratification. If validated prospectively, the score will enable risk-adapted decisions on antibiotic use.

Serum immuno-oncology markers carry independent prognostic information in patients with newly diagnosed metastatic breast cancer, from a prospective observational study

BackgroundMetastatic breast cancer (MBC) is a challenging disease, and despite new therapies, prognosis is still poor for a majority of patients. There is a clinical need for improved prognostication where immuno-oncology markers can provide important information. The aim of this study was to evaluate serum immuno-oncology markers in MBC patients and their respective relevance for prediction of survival.Patients and methodsWe investigated a broad panel of 92 immuno-oncology proteins in serum from 136 MBC patients included in a prospective observational study (NCT01322893) with long-term follow-up. Serum samples were collected before start of systemic therapy and analyzed using multiplex proximity extension assay (Olink Target 96 Immuno-Oncology panel). Multiple machine learning techniques were used to identify serum markers with highest importance for prediction of overall and progression-free survival (OS and PFS), and associations to survival were further evaluated using Cox regression analyses. False discovery rate was then used to adjust for multiple comparisons.ResultsUsing random forest and random survival forest analyses, we identified the top nine and ten variables of highest predictive importance for OS and PFS, respectively. Cox regression analyses revealed significant associations (P < 0.005) of higher serum levels of IL-8, IL-10 and CAIX with worse OS in multivariable analyses, adjusted for established clinical prognostic factors including circulating tumor cells (CTCs). Similarly, high serum levels of IL-8, IL-10, ADA and CASP8 significantly associated with worse PFS. Interestingly, high serum levels of FasL significantly associated with improved OS and PFS. In addition, CSF-1, IL-6, MUC16, TFNSFR4 and CD244 showed suggestive evidence (P < 0.05) for an association to survival in multivariable analyses. After correction for multiple comparisons, IL-8 still showed strong evidence for correlation to survival.ConclusionTo conclude, we found six serum immuno-oncology markers that were significantly associated with OS and/or PFS in MBC patients, independently of other established prognostic factors including CTCs. Furthermore, an additional five serum immuno-oncology markers provided suggestive evidence for an independent association to survival. These findings highlight the relevance of immuno-oncology serum markers in MBC patients and support their usefulness for improved prognostication.Trial registration Clinical Trials (NCT01322893), registered March 25, 2011.

Urine supernatant reveals a signature that predicts survival in clear-cell renal cell carcinoma.

To profile the cell-free urine supernatant and plasma of a small cohort of clear-cell renal cell carcinoma (ccRCC) patients by measuring the relative concentrations of 92 proteins related to inflammation. Using The Cancer Genome Atlas (TCGA), we then performed a targeted mRNA analysis of genes encoding the above proteins and defined their effects on overall survival (OS). Samples were collected prospectively from ccRCC patients. A multiplex proximity extension assay was used to measure the concentrations of 92 inflammation-related proteins in cell-free urine supernatants and plasma. Transcriptomic and clinical information from ccRCC patients was obtained from TCGA. Unsupervised clustering and differential protein expression analyses were performed on protein concentration data. Targeted mRNA analysis on genes encoding significant differentially expressed proteins was performed using TCGA. Backward stepwise regression analyses were used to build a nomogram. The performance of the nomogram and clinical benefit was assessed by discrimination and calibration, and a decision curve analysis, respectively. Unsupervised clustering analysis revealed inflammatory signatures in the cell-free urine supernatant of ccRCC patients. Backward stepwise regressions using TCGA data identified transcriptomic risk factors and risk groups associated with OS. A nomogram to predict 2-year and 5-year OS was developed using these risk factors. The decision curve analysis showed that our model was associated with a net benefit improvement compared to the treat-all/none strategies. We defined four novel biomarkers using proteomic and transcriptomic data that distinguish severity of prognosis in ccRCC. We showed that these biomarkers can be used in a model to predict 2-year and 5-year OS in ccRCC across different tumour stages. This type of analysis, if validated in the future, provides non-invasive prognostic information that could inform either management or surveillance strategies for patients.

Systemic proinflammatory−profibrotic response in aortic stenosis patients with diabetes and its relationship with myocardial remodeling and clinical outcome

BackgroundPrevious studies have mainly focused more on how diabetes affects the valve than the myocardium in aortic stenosis (AS). In the pressure-overloaded heart, myocardial fibrosis is an important driver of the progression from compensated hypertrophy to heart failure. Using comprehensive noninvasive imaging and plasma proteomics, we investigated whether and how diabetes aggravates the remodeling of the myocardium and its relation with prognosis in AS patients.MethodsSevere AS patients were enrolled in two prospective cohorts for imaging and biomarker analysis. The imaging cohort (n = 253) underwent echocardiography and cardiac magnetic resonance, and the biomarker cohort (n = 100) blood sampling with multiplex proximity extension assay for 92 proteomic biomarkers. The composite outcome of hospitalization for heart failure admissions and death was assessed in the imaging cohort.ResultsDiabetic patients were older (70.4 ± 6.8 versus 66.7 ± 10.1 years) with more advanced ventricular diastolic dysfunction and increased replacement and diffuse interstitial fibrosis (late gadolinium enhancement % 0.3 [0.0–1.6] versus 0.0 [0.0–0.5], p = 0.009; extracellular volume fraction % 27.9 [25.7–30.1] versus 26.7 [24.9–28.5], p = 0.025) in the imaging cohort. Plasma proteomics analysis of the biomarker cohort revealed that 9 proteins (E-selectin, interleukin-1 receptor type 1, interleukin-1 receptor type 2, galectin-4, intercellular adhesion molecule 2, integrin beta-2, galectin-3, growth differentiation factor 15, and cathepsin D) were significantly elevated and that pathways related to inflammatory response and extracellular matrix components were enriched in diabetic AS patients. During follow-up (median 6.3 years), there were 53 unexpected heart failure admissions or death in the imaging cohort. Diabetes was a significant predictor of heart failure and death, independent of clinical covariates and aortic valve replacement (HR 1.88, 95% CI 1.06−3.31, p = 0.030).ConclusionsPlasma proteomic analyses indicate that diabetes potentiates the systemic proinflammatory−profibrotic milieu in AS patients. These systemic biological changes underlie the increase of myocardial fibrosis, diastolic dysfunction, and worse clinical outcomes in severe AS patients with concomitant diabetes.

Longitudinal proteomic profiling of the inflammatory response in dengue patients.

The immunopathogenesis of dengue virus (DENV) infection remains incompletely understood. To increase our understanding of inflammatory response in non-severe dengue, we assessed longitudinal changes in the inflammatory proteome in patients with an acute DENV infection. Using a multiplex proximity extension assay (PEA), we measured relative levels of 368 inflammatory markers in plasma samples from hospitalized patients with non-severe DENV infection in the acute (n = 43) and convalescence (n = 35) phase of the infection and samples of healthy controls (n = 10). We identified 203 upregulated and 39 downregulated proteins in acute versus convalescent plasma samples. The upregulated proteins had a strong representation of interferon (IFN) and IFN-inducible effector proteins, cytokines (e.g. IL-10, IL-33) and cytokine receptors, chemokines, pro-apoptotic proteins (e.g. granzymes) and endothelial markers. A number of differentially expressed proteins (DEPs) have not been reported in previous studies. Functional network analysis highlighted a central role for IFNγ, IL-10, IL-33 and chemokines. We identified different novel associations between inflammatory proteins and circulating concentrations of the endothelial glycocalyx disruption surrogate marker syndecan-1. Conclusion: This unbiased proteome analysis provides a comprehensive insight in the inflammatory response in DENV infection and its association with glycocalyx disruption.

Validation of elevated levels of interleukin-8 in the cerebrospinal fluid, and discovery of new biomarkers in patients with GBS and CIDP using a proximity extension assay.

Biomarkers for diagnosis of inflammatory neuropathies, assessment of prognosis, and treatment response are lacking. CSF and EDTA plasma from patients with Guillain-Barré syndrome (GBS), chronic inflammatory demyelinating polyneuropathy (CIDP), healthy controls (HC) and disease controls were analyzed with Olink multiplex proximity extension assay (PEA) from two independent cohorts. Levels of interleukin-8 (IL8) were further analyzed with ELISA in patients with GBS, CIDP, paraproteinemia-related demyelinating polyneuropathy (PDN), multifocal motor neuropathy (MMN), HC and disease controls. ROC analysis was performed. Outcome was measured with the GBS-disability score (GBS-ds) or Inflammatory Neuropathy Cause and Treatment (INCAT) score. In CSF, multiplex PEA analysis revealed up-regulation of IL8 in GBS compared to CIDP and HC respectively, and CIDP compared to HC. In addition, levels of IL2RA were upregulated in GBS compared to both HC and CIDP, SELE in GBS compared to HC, and ITGAM, IL6, and NRP1 in GBS compared to CIDP. In plasma, levels of MMP3, THBD and ITGAM were upregulated in CIDP compared to HC. Validation of multiplex IL8 results using ELISA, revealed increased levels of IL8 in CSF in patients with GBS and CIDP versus HC and non-inflammatory polyneuropathies (NIP) respectively, as well as in PDN versus NIP and HC. Levels of IL8 in CSF correlated with impairment in the acute phase of GBS as well as outcome at 6-months follow up. IL8 in CSF is validated as a diagnostic biomarker in GBS and CIDP, and a prognostic biomarker in GBS. Multiplex PEA hereby identifies several potential biomarkers in GBS and CIDP.

Plasma proteins from several components of the immune system differentiate chronic widespread pain patients from healthy controls - an exploratory case-control study combining targeted and non-targeted protein identification.

Chronic widespread pain (CWP), including fibromyalgia (FM), is characterized by generalized musculoskeletal pain and hyperalgesia. Plasma proteins from proteomics (non-targeted) and from targeted inflammatory panels (cytokines/chemokines) differentiate CWP/FM from controls. The importance of proteins obtained from these two sources, the protein-protein association network, and the biological processes involved were investigated. Plasma proteins from women with CWP (n = 15) and CON (n = 23) were analyzed using two-dimensional gel electrophoresis analysis and a multiplex proximity extension assay for analysis of cytokines/chemokines. Associations between the proteins and group were multivarietly analyzed. The protein-protein association network and the biological processes according to the Gene Ontology were investigated. Proteins from both sources were important for group differentiation; the majority from the two-dimensional gel electrophoresis analysis. 58 proteins significantly differentiated the two groups (R2 = 0.83). A significantly enriched network was found; biological processes were acute phase response, complement activation, and innate immune response. As with other studies, this study shows that plasma proteins can differentiate CWP from healthy subjects. Focusing on cytokines/chemokines is not sufficient to grasp the peripheral biological processes that maintain CWP/FM since our results show that other components of the immune and inflammation systems are also highly significant.

Distinguishing Early Infections from CRS with Routine and Exploratory Serum Proteomics and the HT10 Score Following CD19 CAR-T for Relapsed/Refractory B-NHL

Background: Early fever after CAR-T therapy can reflect both an infection or CRS. Distinguishing both etiologies in these commonly neutropenic patients represents an unresolved clinical challenge. Accordingly, the vast majority of patients receive empiric broad-spectrum antibiotics, which can promote antibiotic resistance and negatively influence the gut microbiome, which has recently been linked to inferior CAR-T outcomes (Smith et al, Nature Medicine 2022). Here, we aimed to characterize infection- vs. CRS-specific differences in routine and exploratory serum proteomics, and to develop a clinical tool that reliably discriminates between both. Methods: Early fever events (day 0-30) were retrospectively characterized as CRS vs. infection in 62 patients treated with standard-of-care CD19 CAR-T for r/r B-NHL (23 Axi-cel, 30 Tisa-cel, 9 Brexu-cel). Severe infections were defined as requiring IV anti-infectives and/or hospitalization in the presence of microbiologic evidence or distinct clinical signs of infection; CRS was graded according to ASTCT criteria. Cumulative incidence curves were calculated as time-to-event-analysis for CRS 1°, ≥2° and severe infections (day of event = index event). Serum inflammatory markers (CRP, interleukin-6 [IL-6], procalcitonin [PCT]) were measured daily from CAR infusion until discharge and on subsequent outpatient visits. Inflammatory markers were tested using binary logistic regression for severe infection vs. CRSonlyindex events. Discriminatory thresholds were determined from receiver operating characteristic (ROC) curves by optimizing the Youden J statistic. Longitudinal plasma proteomics was performed in 52 patients across four time points (day 0, 4, 14, 28) using a 92-protein multiplex proximity extension assay (Olink Proteomics). We applied a linear mixed model (LMM) accounting for both infection vs. CRS status and time effects. Results: The cumulative 30-day incidence of CRS 1°, CRS ≥2° and severe infections was 47%, 43%, and 25%, respectively. Five patients had neither CRS nor an infection, and were subsequently excluded from further analysis. Tocilizumab was applied in the majority of CRS patients (1°: 90%, ≥2°: 100%). We did not find a significant correlation between CRS grade and severe infections (G2 = 0.13, p = n.s.). Median onset of CRS and infection was 3 and 8 days, respectively. Most events (54%) occurred in the setting of coincident severe neutropenia (ANC <500/µl). While event-day CRP was comparable between infection and CRS (median 1.8 vs. 3.4 mg/dL, n.s.), we noted increased serum IL-6 (median 2243 vs. 64 pg/mL, p=0.03) and particularly high PCT levels (median 1.6 vs. 0.3 µg/L, p<0.0001) in the infection cohort. For PCT, an optimal discriminatory threshold of 1.5 µg/L was established (AUCROC 0.78). In previous work, we demonstrated that a CAR-HEMATOTOX (HT) score ≥2 identifies patients with a high infection risk (Rejeski et al, JITC 2022). Indeed, the infection cohort displayed higher baseline HT scores (median 5 vs. 2, p=0.002). By incorporating day-of-fever PCT levels with the HT score, the discriminatory capacity for severe infection events was improved. On ROC analysis, the 'HT10 score' (addition of 10 points to the patient-specific HT score in case event-day PCT ≥1.5 µg/L) exhibited superior discrimination for severe infections (AUC 0.92, p<0.0001, sens. 80%, spec. 91%, Fig. 1A). Additionally, we identified protein-level differences between the infection (n=13) vs. CRSonly (n=39) cohorts from the Olink dataset. On day 0, we noted marked upregulation of the angiogenetic factor TNFRSF12A in the infection cohort (q=4.8E-05). Notably, we observed progressive endothelial dysfunction and immune dysregulation in the patients developing severe infections (day 28, Fig. 1B). This included an increased ANG2/1 ratio and dysregulated CD40/CD40L axis. Consistent with a response to microbial exposure, the innate immune regulators CXC3L1, CCL20, and CXCL13 were upregulated in the infection group. Conclusions: The high discriminatory capacity of the HT10 score for infections highlights the advantage of dynamic risk-assessment and emphasizes the utility of procalcitonin in routine inflammatory panels. If validated prospectively, the score will enable more patient-individual decisions on antibiotic use. Finally, the identified Olink candidate markers may further refine the discriminatory capacity of the HT10 score. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Biphasic Neutrophil Recovery after CD19 CART in R/R LBCL Is Associated with Superior PFS/OS, Robust CAR T-Cell Expansion in Relation to Baseline Tumor Volume, and a Decrease of Systemic Inflammation over Time

Background: Hematological toxicity and concomitant infectious complications drive the toxicity burden of CD19 CART. We recently proposed a classification system for CART-related hematotoxicity, defining three unique phenotypes of hematopoietic recovery (Rejeski et al, Blood 2021). The underlying mechanisms of these phenotypes and their relation to survival remain poorly understood. Here, we sought to understand patterns of post-CART neutrophil recovery and their influence on clinical outcomes. Methods: In this multicenter retrospective study, we analyzed 344 pt. receiving standard-of-care axi-cel (n=208) or tisa-cel (n=136) for R/R LBCL. The phenotypes of neutrophil recovery were defined as follows: Quick: sustained neutrophil recovery without a second dip below an ANC<1000/µl.Intermittent: neutrophil recovery (ANC>1500/µl) followed by a second dip with an ANC<1000/µl after day 21.Aplastic: continuous severe neutropenia (ANC<500/µl) ≥14 days. Multivariate analysis of patient-, disease-, and therapy-related factors was performed as binary logistic regression for the aplastic vs. non-aplastic phenotypes. Variables with a p<0.2 on univariate analysis were included. Clinical outcomes were studied via Kaplan-Meier estimates. Quantification of CAR T-cells was performed by qPCR or by flow cytometry in 146 pt. according to institutional protocols. To harmonize CAR T-cell expansion across centers, patients were divided into quartiles by their respective peak expansion. Plasma proteomics were performed in 56 pts. across four time points (day 0, 4, 14, 28) using a 92-protein multiplex proximity extension assay (Olink Proteomics). Key candidate serum markers were validated in 70 pt. using the Ella automated immunoassay system (ProteinSimple). Results: Across all patients, the distribution of the 'aplastic' (A), 'intermittent' (I) and 'quick' (Q) phenotypes was 18%, 42%, and 40%, respectively. The 'aplastic' patients exhibited a longer median duration of severe neutropenia, (A vs. I vs. Q: 26 vs. 10 vs. 5 days, p<0.001), a higher rate of severe infections (A vs. I vs. Q: 41% vs. 23% vs. 18%) and a trend towards higher 1-year non-relapse mortality (A vs. I: 9% vs. 3.5%, p=0.08). On multivariate regression (n=344), the aplastic phenotype was independently associated with the presence of BM infiltration (adjusted OR=3.2, p=0.004), ANC (aOR=0.28, p=0.002), and ferritin (aOR=2.3, p=0.039). Neither high-grade CRS or ICANS nor CAR product were linked to the aplastic phenotype. In the 'intermittent' cohort, we noted a significant decrease of both serum ferritin (median 776 vs. 321 U/L) and CRP levels (median 1.6 vs. 0.2 mg/dL) between CAR infusion and day 90 follow-up. When studying CD3+ CAR T-cell dynamics over time, we found that patients with 'intermittent' recovery displayed both an earlier and more pronounced expansion peak, and longer CART persistence compared to their 'quick' counterparts. While the 'aplastic' cohort displayed comparable peak CART expansion, these patients also exhibited high baseline LDH, indicative of a higher baseline tumor burden, or 'hill to climb' (top right quadrant, Fig. 1A). Conversely, the 'quick' cohort was characterized by both low expansion and low LDH. Serum proteome analysis revealed significant disruptions of endothelial function and immune dysregulation in the 'aplastic' patients at month 1 (Fig. 1B). Importantly, we confirmed higher peak IFNγ and IL-15 levels, and an increased ratio of ANG2 to ANG1 in the 'aplastic' patients in the independent validation cohort. Notably, progression-free (PFS) and overall survival (OS) was poor in the 'aplastic' patients (1-yr PFS 26%, 1-yr OS 46%). On the other hand, 'intermittent' patients exhibited the best survival outcomes (HRPFS I vs. A: 0.45, 95% CI 0.31-0.65). Median PFS was 3 vs. 20 vs. 4 months (A vs. I vs. Q, p<0.001); median OS was 8 months vs. not-reached vs. 23 months (A vs. I vs. Q, p<0.001). Conclusion: The 'aplastic' phenotype is driven by baseline inflammation and hematopoietic function, characterized by progressive endothelial dysfunction, and associated with poor PFS/OS. In contrast, the 'intermittent' phenotype (i.e. biphasic cytopenia) was associated with high levels of CART expansion and persistence, a decrease in systemic inflammation over time, and excellent survival outcomes. Overall, these data suggest potential interactions between host hematopoiesis and CART function and efficacy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Systemic proinflammatory-profibrotic response in aortic stenosis patients with diabetes and its relationship with myocardial remodeling and clinical outcome

Abstract Background It is unclear whether and how diabetes mellitus may aggravate myocardial fibrosis and remodeling in the pressure-overloaded heart. We investigated the impact of diabetes on the prognosis of aortic stenosis (AS) patients and its underlying mechanisms using comprehensive noninvasive imaging studies and plasma proteomics. Methods Severe AS patients undergoing both echocardiography and cardiovascular magnetic resonance (CMR) (n=253 of which 66 had diabetes) comprised the imaging cohort. The degree of replacement and diffuse interstitial fibrosis by late gadolinium enhancement (LGE) and extracellular volume fraction (ECV) was quantified using CMR. Plasma samples were analyzed with the multiplex proximity extension assay for 92 proteomic biomarkers in a separate biomarker cohort of severe AS patients (n=100 of which 27 had diabetes). Results In the imaging cohort, diabetic patients were older (70.4±6.8 vs. 66.7±10.1 years) and had a higher prevalence of ischemic heart disease (28.8% vs. 9.1%), with more advanced ventricular diastolic dysfunction. On CMR, diabetic patients had increased replacement and diffuse interstitial fibrosis (LGE% 0.3 [0.0–1.6] versus 0.0 [0.0–0.5], p=0.009; ECV% 27.9 [25.7–30.1] versus 26.7 [24.9–28.5], p=0.025) (Figure 1). Plasma proteomics analysis of the biomarker cohort revealed that 9 proteins (E-selectin, interleukin-1 receptor type 1, interleukin-1 receptor type 2, galectin-4, intercellular adhesion molecule 2, integrin beta-2, galectin-3, growth differentiation factor 15, and cathepsin D) are significantly elevated in diabetic AS patients (Figure 2). Pathway over-representation analyses of the plasma proteomics with Gene Ontology terms indicated that pathways related to inflammatory response and extracellular matrix components were enriched, suggesting that diabetes is associated with systemic effects that evoke proinflammatory and profibrotic response to the pressure-overloaded myocardium. During follow-up (median 6.3 years [IQR 5.2–7.2]) of the imaging cohort, 232 patients received aortic valve replacement (AVR) with 53 unexpected heart failure admissions or death. Diabetes was a significant predictor of heart failure and death, independent of clinical covariates and AVR (hazard ratio 1.88, 95% confidence interval 1.06–3.31, p=0.030). Conclusion Plasma proteomic analyses indicate that diabetes potentiates the systemic proinflammatory and profibrotic milieu in AS patients. These systemic biological changes underlie the increase of myocardial fibrosis, diastolic dysfunction, and worse clinical outcomes in severe AS patients with concomitant diabetes. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): National Research Foundation of Korea

TACE/ADAM17 substrates associate with ACS (Ep-CAM, HB-EGF) and follow-up MACE (TNFR1 and TNFR2)

Background and aimsTACE/ADAM17 is a membrane bound metalloprotease, which cleaves substrates involved in immune and inflammatory responses and plays a role in coronary artery disease (CAD). We measured TACE and its substrates in CAD patients to identify potential biomarkers within this molecular pathway with potential for acute coronary syndrome (ACS) and major adverse cardiovascular events (MACE) prediction. MethodsBlood samples were obtained from consecutive patients (n = 229) with coronary angiographic evidence of CAD admitted with ACS or electively. MACE were recorded after a median 3-year follow-up. Controls (n = 115) had a <10% CAD risk as per the HeartSCORE. TACE and TIMP3 protein and mRNA levels were measured by ELISA and RT-qPCR respectively. TACE substrates were measured using a multiplex proximity extension assay. ResultsTACE mRNA and cell protein levels (p < 0.01) and TACE substrates LDLR (p = 0.006), TRANCE (p = 0.045), LAG-3 (p < 0.001) and ACE2 (p < 0.001) plasma levels were significantly higher in CAD patients versus controls. TACE inhibitor TIMP3 mRNA levels were significantly lower in CAD patients and tended to be lower in the ACS population (p < 0.05). TACE substrates TNFR1 (OR:3.237,CI:1.514–6.923,p = 0.002), HB-EGF (OR:0.484,CI:0.288–0.813,p = 0.006) and Ep-CAM (OR:0.555,CI:0.327–0.829,p = 0.004) accurately classified ACS patients with HB-EGF and Ep-CAM levels being lower compared to electively admitted patients. TNFR1 (OR:2.317,CI:1.377–3.898,p = 0.002) and TNFR2 (OR:1.902,CI:1.072–3.373,p = 0.028) were significantly higher on admission in those patients who developed MACE within 3 years. ConclusionsWe demonstrate a possible role of TACE substrates LAG-3, HB-EGF and Ep-CAM in atherosclerotic plaque development and stability. We also underline the importance of measuring TNFR1 and TNFR2 earlier than previously appreciated for MACE prediction. We report an important role of TIMP3 in regulating TACE levels.

Quantifying the inflammatory secretome of human intermuscular adipose tissue.

Adipose tissue secretes an abundance of lipid and protein mediators, and this secretome is depot‐specific, with local and systemic effects on metabolic regulation. Intermuscular adipose tissue (IMAT) accumulates within the skeletal muscle compartment in obesity, and is associated with insulin resistance and metabolic disease. While the human IMAT secretome decreases insulin sensitivity in vitro, its composition is entirely unknown. The current study was conducted to investigate the composition of the human IMAT secretome, compared to that of the subcutaneous (SAT) and visceral adipose tissue (VAT) depots. IMAT, SAT, and VAT explants from individuals with obesity were used to generate conditioned media. Proteomics analysis of conditioned media was performed using multiplex proximity extension assays, and eicosanoid analysis using liquid chromatography–tandem mass spectrometry. Compared to SAT and/or VAT, IMAT secreted significantly more cytokines (IL2, IL5, IL10, IL13, IL27, FGF23, IFNγ and CSF1) and chemokines (MCP1, IL8, CCL11, CCL20, CCL25 and CCL27). Adipokines hepatocyte growth factor and resistin were secreted significantly more by IMAT than SAT or VAT. IMAT secreted significantly more eicosanoids (PGE2, TXB2, 5‐HETE, and 12‐HETE) compared to SAT and/or VAT. In the context of obesity, IMAT is a distinct adipose tissue with a highly immunogenic and inflammatory secretome, and given its proximity to skeletal muscle, may be critical to glucose regulation and insulin resistance.

Plasma protein biomarker profiling reveals major differences between acute leukaemia, lymphoma patients and controls

Aiming to accommodate the unmet need for easily accessible biomarkers with a focus on biological differences between haematological diseases, the diagnostic value of plasma proteins in acute leukaemias and lymphomas was investigated. A multiplex proximity extension assay (PEA) was used to analyze 183 proteins in diagnostic plasma samples from 251 acute leukaemia and lymphoma patients and compared with samples from 60 healthy controls. Multivariate modelling using partial least square discriminant analysis revealed highly significant differences between distinct disease subgroups and controls. The model allowed explicit distinction between leukaemia and lymphoma, with few patients misclassified. Acute leukaemia samples had higher levels of proteins associated with haemostasis, inflammation, cell differentiation and cell-matrix integration, whereas lymphoma samples demonstrated higher levels of proteins known to be associated with tumour microenvironment and lymphoma dissemination. PEA technology can be used to screen for large number of plasma protein biomarkers in low µL sample volumes, enabling the distinction between controls, acute leukaemias and lymphomas. Plasma protein profiling could help gain insights into the pathophysiology of acute leukaemia and lymphoma and the technique may be a valuable tool in the diagnosis of these diseases.

P1458: PATTERNS OF LONG-TERM HEMATOPOIETIC RECOVERY IMPACT SURVIVAL OUTCOMES AFTER CD19-DIRECTED CAR T-CELL THERAPY FOR R/R LBCL

Background: Real-world evidence has underlined the role of hematological toxicity and infections in driving the toxicity burden of CD19 CAR-T. We have recently developed a classification system for post-CAR-T hematotoxicity, identifying three unique phenotypes of neutrophil recovery (Rejeski et al Blood 2021). However, their prognostic impact remains poorly understood. Aims: We sought to understand potential pathomechanisms underlying hematopoietic recovery patterns, and characterize their influence on toxicity and clinical outcomes. Methods: In this multicenter retrospective observational study, we analyzed 316 pts. receiving Axicabtagene ciloleucel (n=194) or Tisagenlecleucel (n=122) for R/R LBCL in a real-world setting. The phenotypes of neutrophil recovery were defined as follows: 1) Quick Recovery: sustained neutrophil recovery without a second dip below an ANC<1000/µl. 2) Intermittent Recovery: neutrophil recovery (ANC>1500/µl) followed by a second dip with an ANC<1000/µl after day 21. 3) Aplastic: continuous severe neutropenia (ANC<500/µl) ≥14 days. Multivariate analysis was performed as stepwise binary logistic regression for the aplastic vs. non-aplastic phenotypes. Variables with a p<0.2 on univariate analysis were included. Clinical outcomes were studied via Kaplan-Meier estimates. Serum cytokines were analyzed using the Ella automated immunoassay system (ProteinSimple) in 70 pt. from Moffitt Cancer Center. The plasma proteome was characterized in a further 38 pts. from the LMU Munich across four time points (day 0, 4, 14, 28) using a 92-protein multiplex proximity extension assay (Olink Bioscience). Results: The overall distribution of the ‘quick’ (Q), ‘intermittent’ (I) and ‘aplastic’ (A) phenotypes was 40%, 41%, and 19%, respectively. Prior to lymphodepletion, ‘aplastic’ patients displayed increased systemic inflammation (median ferritin 1168 ng/ml), impaired hematopoietic function (median platelet count 101 G/l) and high tumor burden (median LDH 344 U/l). ‘Aplastic’ patients displayed a longer median duration of severe neutropenia, (A vs. I vs. Q: 28 vs. 10 vs. 5 days, p<0.001), higher rate of severe infections (A vs. I vs. Q: 40% vs. 22% vs. 18%), and a trend towards higher 1-year non-relapse mortality (A vs. I: 10% vs. 4%, p=0.09). Progression-free (PFS) and overall survival (OS) was poor in the ‘aplastic’ patients (Fig. 1, 1-yr PFS 24%, 1-yr OS 48%). On the other hand, ‘intermittent’ patients exhibited the best survival outcomes (HR for PFS, I vs. A: 0.46, 95% CI 0.32-0.69). Median PFS was 3 vs. 4 vs. 20 months (A vs. Q v. I, p<0.001); median OS was 8 vs. 23 months vs. not reached (A vs. Q v. I, p=0.0025). On multivariate regression (n=316), the aplastic phenotype was primarily driven by baseline ECOG (OR=1.6, p=0.05), ferritin (OR=2.5, p=0.01), ANC (OR=0.3, p=0.01), and hemoglobin (OR=0.8, p=0.04). Of note, high-grade CRS/ICANS and CAR product were not associated with the aplastic phenotype. Initial analyses of inflammatory patterns revealed higher peak IL-15 levels and an increased ratio of ANG2 to ANG1 in ‘aplastic’ patients in both cohorts, with ANG-1 levels decreasing over time. Image:Summary/Conclusion: Hematopoietic recovery patterns impact disease outcomes after CD19 CAR-T. Surprisingly, a phenotype associated with recurrent neutrophil dips (i.e. prolonged cytopenia) was associated with improved survival outcomes. The poor-risk ‘aplastic’ phenotype was driven by baseline inflammation and hematopoietic function, and characterized by progressive endothelial dysfunction. Further mechanistic exploration is warranted and ongoing.

Technical Performance Evaluation of Olink Proximity Extension Assay for Blood-Based Biomarker Discovery in Longitudinal Studies of Alzheimer's Disease.

The core Alzheimer's disease (AD) cerebrospinal fluid (CSF) biomarkers; amyloid-β (Aß), total tau (t-tau), and phosphorylated tau (p-tau181), are strong indicators of the presence of AD pathology, but do not correlate well with disease progression, and can be difficult to implement in longitudinal studies where repeat biofluid sampling is required. As a result, blood-based biomarkers are increasingly being sought as alternatives. In this study, we aimed to evaluate a promising blood biomarker discovery technology, Olink Proximity Extension Assays for technical reproducibility characteristics in order to highlight the advantages and disadvantages of using this technology in biomarker discovery in AD. We evaluated the performance of five Olink Proteomic multiplex proximity extension assays (PEA) in plasma samples. Three technical control samples included on each plate allowed calculation of technical variability. Biotemporal stability was measured in three sequential annual samples from 54 individuals with and without AD. Coefficients of variation (CVs), analysis of variance (ANOVA), and variance component analyses were used to quantify technical and individual variation over time. We show that overall, Olink assays are technically robust, with the largest experimental variation stemming from biological differences between individuals for most analytes. As a powerful illustration of one of the potential pitfalls of using a multi-plexed technology for discovery, we performed power calculations using the baseline samples to demonstrate the size of study required to overcome the need for multiple test correction with this technology. We show that the power of moderate effect size proteins was strongly reduced, and as a result investigators should strongly consider pooling resources to perform larger studies using this multiplexed technique where possible.

Temporal patterns of inflammation-related proteins measured in the cerebrospinal fluid of patients with aneurysmal subarachnoid hemorrhage using multiplex Proximity Extension Assay technology

BackgroundThe complexity of the inflammatory response post subarachnoid hemorrhage (SAH) may require temporal analysis of multiple protein biomarkers simultaneously to be more accurately described.MethodsVentricular cerebrospinal fluid was collected at days 1, 4 and 10 after SAH in 29 patients. Levels of 92 inflammation-related proteins were simultaneously measured using Target 96 Inflammation ® assay (Olink Proteomics, Uppsala, Sweden) based on Proximity Extension Assay (PEA) technology. Twenty-eight proteins were excluded from further analysis due to lack of >50% of measurable values. Temporal patterns of the remaining 64 proteins were analyzed. Repeated measures ANOVA and its nonparametric equivalent Friedman’s ANOVA were used for comparisons of means between time points.ResultsFour different patterns (Groups A-D) were visually observed with an early peak and gradually decreasing trend (11 proteins), a middle peak (10 proteins), a late peak after a gradually increasing trend (30 proteins) and no specific pattern (13 proteins). Statistically significant early peaks defined as Day 1 > Day 4 values were noticed in 4 proteins; no significant decreasing trends defined as Day 1 > Day 4 > Day 10 values were observed. Two proteins showed significant middle peaks (i.e. Day 1 < Day 4 > Day 10 values). Statistically significant late peaks (i.e. Day 4 < Day 10 values) and increasing trends (i.e. Day 1 < Day 4 < Day 10 values) were observed in 14 and 10 proteins, respectively. Four of Group D proteins showed biphasic peaks and the rest showed stable levels during the observation period.ConclusionThe comprehensive data set provided in this explorative study may act as an illustration of an inflammatory profile of the acute phase of SAH showing groups of potential protein biomarkers with similar temporal patterns of activation, thus facilitating further research on their role in the pathophysiology of the disease.

Periodontal Disease Augments Cardiovascular Disease Risk Biomarkers in Rheumatoid Arthritis.

Objectives: Periodontal disease (PD) and rheumatoid arthritis (RA) are known chronic conditions with sustained inflammation leading to osteolysis. Cardiovascular diseases (CVD) are frequent comorbidities that may arise from sustained inflammation associated with both PD and RA. In order to determine CVD risk, alterations at the molecular level need to be identified. The objective of this study, therefore, was to assess the relationship of CVD associated biomarkers in RA patients and how it is influenced by PD. Methods: The study consisted of patient (26 RA with PD, 21 RA without PD, 51 patients with PD only) and systemically and periodontally healthy control (n = 20) groups. Periodontal parameters bleeding on probing, probing pocket depth, and marginal bone loss were determined to characterize the patient groups. Proteomic analysis of 92 CVD-related protein biomarkers was performed using a multiplex proximity extension assay. Biomarkers were clustered using the search tool for retrieval of interacting genes (STRING) to determine protein–protein interaction (PPI) networks. Results: RA patients with PD had higher detection levels for 47% of the measured markers (ANGPT1, BOC, CCL17, CCL3, CD4, CD84, CTRC, FGF-21, FGF-23, GLO1, HAOX1, HB-EGF, hOSCAR, HSP 27, IL16, IL-17D, IL18, IL-27, IL6, LEP, LPL, MERTK, MMP12, MMP7, NEMO, PAPPA, PAR-1, PARP-1, PD-L2, PGF, PIgR, PRELP, RAGE, SCF, SLAMF7, SRC, THBS2, THPO, TNFRSF13B, TRAIL-R2, VEGFD, VSIG2, and XCL1) as compared to RA without PD. Furthermore, a strong biological network was identified amongst these proteins (clustering coefficient = 0.52, PPI enrichment p-value < 0.0001). Coefficients for protein clusters involved in CVD (0.59), metabolic (0.53), and skeletal (0.51) diseases were strongest in the PD group. Conclusion: Periodontal disease augments CVD-related biomarkers in RA through shared pathological clusters, concurrently enhancing metabolic and skeletal disease protein interactions, independent of autoimmune status.