Gene expression prognostic of early relapse risk in low-risk B-cell acute lymphoblastic leukaemia in children.

ETV6::RUNX1 is the most common fusion gene in childhood acute lymphoblastic leukaemia (ALL) and is associated with favorable outcomes, especially in low-risk children. However, as many as 10% of children relapse within 3 years, and such early relapses have poor survival. Identifying children at risk for early relapse is an important challenge. We interrogated data from 87 children with low-risk ETV6::RUNX1-positive B-cell ALL and with available preserved bone marrow samples (discovery cohort). We profiled somatic point mutations in a panel of 559 genes and genome-wide transcriptome and single-nucleotide variants. We found high TIMD4 expression (> 85th-percentile value) at diagnosis was the most important independent prognostic factor of early relapse (hazard ratio [HR] = 5.07 [1.76, 14.62]; p = 0.03). In an independent validation cohort of low-risk ETV6::RUNX1-positive B-cell ALL (N = 68) high TIMD4 expression at diagnosis had an HR = 4.78 [1.07, 21.36] (p = 0.04) for early relapse. In another validation cohort including 78 children with low-risk ETV6::RUNX1-negative B-cell ALL, high TIMD4 expression at diagnosis had an HR = 3.93 [1.31, 11.79] (p = 0.01). Our results suggest high TIMD4 expression at diagnosis in low-risk B-cell ALL in children might be associated with high risk for early relapse.

Refining eligibility criteria of unit selection for myeloablative cord blood transplantation in acute leukemia: Real-world experience of a referral center.

The algorithm for cord blood (CB) unit selection is still somewhat ambiguous. We retrospectively analyzed 620 cases of acute leukemia between 2015 and 2020, who were treated with myeloablative single-unit umbilical CB transplantation (UCBT). We found that, when human leukocyte antigen (HLA) mismatch was ≤3/10, CD34+ cell dosage <0.83 × 105/kg-considerably lower than prevalent guidelines-was permissible without affecting survival. Moreover, synergy between donor killer-cell immunoglobulin-like receptors (KIR) haplotypes-B and donor-recipient HLA-C mismatch protected against relapse-related mortality. We submit that minimum required CD34+ cell dosage can possibly be relaxed to broaden access to UCBT, and donor KIR genotyping should be considered during unit selection.

The Composite Immune Risk Score predicts overall survival after allogeneic hematopoietic stem cell transplantation: A retrospective analysis of 1838 cases.

There has been little consensus on how to quantitatively assess immune reconstitution after hematopoietic stem cell transplantation (HSCT) as part of the standard of care. We retrospectively analyzed 11 150 post-transplant immune profiles of 1945 patients who underwent HSCT between 2012 and 2020. 1838 (94.5%) of the cases were allogeneic HSCT. Using the training set of patients (n = 729), we identified a composite immune signature (integrating neutrophil, total lymphocyte, natural killer, total T, CD4+ T, and B cell counts in the peripheral blood) during days 91-180 after allogeneic HSCT that was predictive of early mortality and moreover simplified it into a formula for a Composite Immune Risk Score. When we verified the Composite Immune Risk Score in the validation (n = 284) and test (n = 391) sets of patients, a high score value was found to be associated with hazard ratios (HR) of 3.64 (95% C.I. 1.55-8.51; p = .0014) and 2.44 (95% C.I., 1.22-4.87; p = .0087), respectively, for early mortality. In multivariate analysis, a high Composite Immune Risk Score during days 91-180 remained an independent risk factor for early mortality after allogeneic HSCT (HR, 1.80; 95% C.I., 1.28-2.55; p = .00085). In conclusion, the Composite Immune Risk Score is easy to compute and could identify the high-risk patients of allogeneic HSCT who require targeted effort for prevention and control of infection.

Surface protein profiling of prostate-derived extracellular vesicles by mass spectrometry and proximity assays.

Extracellular vesicles (EVs) are mediators of intercellular communication and a promising class of biomarkers. Surface proteins of EVs play decisive roles in establishing a connection with recipient cells, and they are putative targets for diagnostic assays. Analysis of the surface proteins can thus both illuminate the biological functions of EVs and help identify potential biomarkers. We developed a strategy combining high-resolution mass spectrometry (HRMS) and  proximity ligation assays (PLA) to first identify and then validate surface proteins discovered on EVs. We applied our workflow to investigate surface proteins of small EVs found in seminal fluid (SF-sEV). We identified 1,014 surface proteins and verified the presence of a subset of these on the surface of SF-sEVs. Our work demonstrates a general strategy for deep analysis of EVs' surface proteins across patients and pathological conditions, proceeding from unbiased screening by HRMS to ultra-sensitive targeted analyses via PLA.

'Dynamic zero-COVID' policy and viral clearance during an omicron wave in Tianjin, China: a city-wide retrospective observational study.

OBJECTIVE: To report how the Chinese mainland battled its first omicron wave, which happened in Tianjin, a metropolis with 14 million residents. We also sought to better understand how clinical features affected the timing of viral clearance. DESIGN: A retrospective study of the omicron wave in Tianjin between 8 January 2022 and 3 March 2022. SETTING: Except for the first cases on 8 January, all the omicron cases were identified through PCR mass testing in the residential communities. Residential quarantine and serial PCR mass testing were dynamically adjusted according to the trends of new cases. PARTICIPANTS: All the 417 consecutive PCR-positive cases identified through mass screening of the entire city's 14 million residents. 45.3% of the cases were male, and the median age was 37 (range 0.3-90). 389 (93%) cases had complete data for analysing the correlation between clinical features and the timing of viral clearance. MAIN OUTCOME AND MEASURE: Time to viral clearance. RESULTS: Tianjin initiated the 'dynamic zero-COVID' policy very early, that is, when daily new case number was ≈0.4 cases per 1 000 000 residents. Daily new cases dropped to <5 after 3 February, and the number of affected residential subdivisions dropped to ≤2 after 13 February. 64% (267/417) of the cases had no or mild symptoms. The median interval from hospital admission to viral clearance was 10 days (range 3-28). An exploratory analysis identified a feature cluster associated with earlier viral clearance, with HRs of 3.56 (95% CI 1.66 to 7.63) and 3.15 (95% CI 1.68 to 5.91) in the training and validation sets, respectively. CONCLUSIONS: The 'dynamic zero-COVID' policy can suppress an omicron wave within a month. It might be possible to predict in advance which cases will require shorter periods of isolation based on their clinical features.

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.

Subcutaneous adipose tissue splice quantitative trait loci reveal differences in isoform usage associated with cardiometabolic traits.

Alternate splicing events can create isoforms that alter gene function, and genetic variants associated with alternate gene isoforms may reveal molecular mechanisms of disease. We used subcutaneous adipose tissue of 426 Finnish men from the METSIM study and identified splice junction quantitative trait loci (sQTLs) for 6,077 splice junctions (FDR < 1%). In the same individuals, we detected expression QTLs (eQTLs) for 59,443 exons and 15,397 genes (FDR < 1%). We identified 595 genes with an sQTL and exon eQTL but no gene eQTL, which could indicate potential isoform differences. Of the significant sQTL signals, 2,114 (39.8%) included at least one proxy variant (linkage disequilibrium r2 > 0.8) located within an intron spanned by the splice junction. We identified 203 sQTLs that colocalized with 141 genome-wide association study (GWAS) signals for cardiometabolic traits, including 25 signals for lipid traits, 24 signals for body mass index (BMI), and 12 signals for waist-hip ratio adjusted for BMI. Among all 141 GWAS signals colocalized with an sQTL, we detected 26 that also colocalized with an exon eQTL for an exon skipped by the sQTL splice junction. At a GWAS signal for high-density lipoprotein cholesterol colocalized with an NR1H3 sQTL splice junction, we show that the alternative splice product encodes an NR1H3 transcription factor that lacks a DNA binding domain and fails to activate transcription. Together, these results detect splicing events and candidate mechanisms that may contribute to gene function at GWAS loci.

A Phenogenetic Axis that Modulates Clinical Manifestation and Predicts Treatment Outcome in Primary Myeloid Neoplasms.

Although the concept of "myeloid neoplasm continuum" has long been proposed, few comparative genomics studies directly tested this hypothesis. Here we report a multi-modal data analysis of 730 consecutive newly diagnosed patients with primary myeloid neoplasm, along with 462 lymphoid neoplasm cases serving as the outgroup. Our study identified a "Pan-Myeloid Axis" along which patients, genes, and phenotypic features were all aligned in sequential order. Utilizing relational information of gene mutations along the Pan-Myeloid Axis improved prognostic accuracy for complete remission and overall survival in adult patients of de novo acute myeloid leukemia and for complete remission in adult patients of myelodysplastic syndromes with excess blasts. We submit that better understanding of the myeloid neoplasm continuum might shed light on how treatment should be tailored to individual diseases. Significance: The current criteria for disease diagnosis treat myeloid neoplasms as a group of distinct, separate diseases. This work provides genomics evidence for a "myeloid neoplasm continuum" and suggests that boundaries between myeloid neoplastic diseases are much more blurred than previously thought.

Dynamic forecasting of severe acute graft-versus-host disease after transplantation.

Forecasting of severe acute graft-versus-host disease (aGVHD) after transplantation is a challenging 'large p, small n' problem that suffers from nonuniform data sampling. We propose a dynamic probabilistic algorithm, daGOAT, that accommodates sampling heterogeneity, integrates multidimensional clinical data and continuously updates the daily risk score for severe aGVHD onset within a two-week moving window. In the studied cohorts, the cross-validated area under the receiver operator characteristic curve (AUROC) of daGOAT rose steadily after transplantation and peaked at ≥0.78 in both the adult and pediatric cohorts, outperforming the two-biomarker MAGIC score, three-biomarker Ann Arbor score, peri-transplantation features-based models and XGBoost. Simulation experiments indicated that the daGOAT algorithm is well suited for short time-series scenarios where the underlying process for event generation is smooth, multidimensional and where there are frequent and irregular data missing. daGOAT's broader utility was demonstrated by performance testing on a remotely different task, that is, prediction of imminent human postural change based on smartphone inertial sensor time-series data.

Depression, GABA, and Age Correlate with Plasma Levels of Inflammatory Markers.

Immunomodulation is increasingly being recognised as a part of mental diseases. Here, we examined whether levels of immunological protein markers changed with depression, age, or the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). An analysis of plasma samples from patients with a major depressive episode and control blood donors (CBD) revealed the expression of 67 inflammatory markers. Thirteen of these markers displayed augmented levels in patients compared to CBD. Twenty-one markers correlated with the age of the patients, whereas 10 markers correlated with the age of CBD. Interestingly, CST5 and CDCP1 showed the strongest correlation with age in the patients and CBD, respectively. IL-18 was the only marker that correlated with the MADRS-S scores of the patients. Neuronal growth factors (NGFs) were significantly enhanced in plasma from the patients, as was the average plasma GABA concentration. GABA modulated the release of seven cytokines in anti-CD3-stimulated peripheral blood mononuclear cells (PBMCs) from the patients. The study reveals significant changes in the plasma composition of small molecules during depression and identifies potential peripheral biomarkers of the disease.

Reduced sialyl-Lewisx on salivary MUC7 from patients with burning mouth syndrome.

We analysed and compared MUC7 O-glycosylation and inflammatory biomarkers in saliva from female patients with burning mouth syndrome (BMS) and gender/age-matched controls. Oligosaccharides from salivary MUC7 from BMS and controls were released. Inflammatory mediators were measured by multiplex proximity extension assay. Presence of sialyl-Lewisx (Si-Lex) epitope on MUC7 was confirmed using Western blot. MUC7 O-glycans and measured inflammatory biomarkers were found to be similar between BMS and controls. However, oligosaccharides sialyl-Lewisx (Si-Lex) was found to be reduced in samples from BMS patients. Positive correlation (combined patients and controls) was found between levels of C-C motif chemokine 19 (CCL-19) and the amount of core-2 oligosaccharides on MUC7 as well as fractalkine (CX3CL1) and level of sialylation. Patients with BMS were shown to represent a heterogeneous group in terms of inflammatory biomarkers. This indicates that BMS patients could be further stratified on the basis of low-level inflammation. The results furthermore indicate that reduced sialylation of MUC7, particularly Si-Lex, may be an important feature in patients with BMS. However, the functional aspects and potential involvement in immune regulation of Si-Lex remains unclear. Our data suggests a chemokine driven alteration of MUC7 glycosylation.

Profiling surface proteins on individual exosomes using a proximity barcoding assay.

Exosomes have been implicated in numerous biological processes, and they may serve as important disease markers. Surface proteins on exosomes carry information about their tissues of origin. Because of the heterogeneity of exosomes it is desirable to investigate them individually, but this has so far remained impractical. Here, we demonstrate a proximity-dependent barcoding assay to profile surface proteins of individual exosomes using antibody-DNA conjugates and next-generation sequencing. We first validate the method using artificial streptavidin-oligonucleotide complexes, followed by analysis of the variable composition of surface proteins on individual exosomes, derived from human body fluids or cell culture media. Exosomes from different sources are characterized by the presence of specific combinations of surface proteins and their abundance, allowing exosomes to be separately quantified in mixed samples to serve as markers for tissue-specific engagement in disease.

A targeted proteomics approach reveals a serum protein signature as diagnostic biomarker for resectable gastric cancer.

BACKGROUND: Gastric cancer (GC) is the third leading cause of cancer death. Early detection is a key factor to reduce its mortality. METHODS: We retrospectively collected pre- and postoperative serum samples as well as tumour tissues and adjacent normal tissues from 100 GC patients. Serum samples from non-cancerous patients were served as controls (n = 50). A high-throughput protein detection technology, multiplex proximity extension assays (PEA), was applied to measure levels of over 300 proteins. Alteration of each protein was analysed by univariate analysis. Elastic-net logistic regression was performed to select serum proteins into the diagnostic model. FINDINGS: We identified 19 serum proteins (CEACAM5, CA9, MSLN, CCL20, SCF, TGF-alpha, MMP-1, MMP-10, IGF-1, CDCP1, PPIA, DDAH-1, HMOX-1, FLI1, IL-7, ZBTB-17, APBB1IP, KAZALD-1, and ADAMTS-15) that together distinguish GC cases from controls with a diagnostic sensitivity of 93%, specificity of 100%, and area under receiver operating characteristic curve (AUC) of 0·99 (95% CI: 0·98-1). Moreover, the 19-serum protein signature provided an increased diagnostic capacity in patients at TNM I-II stage (sensitivity 89%, specificity 100%, AUC 0·99) and in patients with high microsatellite instability (MSI) (91%, 98%, and 0·99) compared to individual proteins. These promising results will inspire a large-scale independent cohort study to be pursued for validating the proposed protein signature. INTERPRETATION: Based on targeted proteomics and elastic-net logistic regression, we identified a 19-serum protein signature which could contribute to clinical GC diagnosis, especially for patients at early stage and those with high MSI. FUND: This study was supported by a European H2020-Marie Sklodowska-Curie Innovative Training Networks grant (316,929, GastricGlycoExplorer). Funder had no influence on trial design, data evaluation, and interpretation.

Monitoring of Protein Biomarkers of Inflammation in Human Traumatic Brain Injury Using Microdialysis and Proximity Extension Assay Technology in Neurointensive Care.

Traumatic brain injury (TBI) is followed by secondary injury mechanisms strongly involving neuroinflammation. To monitor the complex inflammatory cascade in human TBI, we used cerebral microdialysis (MD) and multiplex proximity extension assay (PEA) technology and simultaneously measured levels of 92 protein biomarkers of inflammation in MD samples every three hours for five days in 10 patients with severe TBI under neurointensive care. One µL MD samples were incubated with paired oligonucleotide-conjugated antibodies binding to each protein, allowing quantification by real-time quantitative polymerase chain reaction. Sixty-nine proteins were suitable for statistical analysis. We found five different patterns with either early (<48 h; e.g., CCL20, IL6, LIF, CCL3), mid (48-96 h; e.g., CCL19, CXCL5, CXCL10, MMP1), late (>96 h; e.g., CD40, MCP2, MCP3), biphasic peaks (e.g., CXCL1, CXCL5, IL8) or stable (e.g., CCL4, DNER, VEGFA)/low trends. High protein levels were observed for e.g., CXCL1, CXCL10, MCP1, MCP2, IL8, while e.g., CCL28 and MCP4 were detected at low levels. Several proteins (CCL8, -19, -20, -23, CXCL1, -5, -6, -9, -11, CST5, DNER, Flt3L, and SIRT2) have not been studied previously in human TBI. Cross-correlation analysis revealed that LIF and CXCL5 may play a central role in the inflammatory cascade. This study provides a unique data set with individual temporal trends for potential inflammatory biomarkers in patients with TBI. We conclude that the combination of MD and PEA is a powerful tool to map the complex inflammatory cascade in the injured human brain. The technique offers new possibilities of protein profiling of complex secondary injury pathways.

Protein measurements in venous plasma, earlobe capillary plasma and in plasma stored on filter paper.

In this study, levels of inflammatory protein biomarkers in venous plasma, plasma derived from capillary blood from the earlobe, and capillary plasma stored as dried plasma spots (DPS) were compared. Samples from 12 male individuals were assessed with a panel of 92 inflammation-related proteins using multiplex proximity extension assay. Correlations between sample types varied greatly between analytes. A high correlation of ρ > 0.8 was observed between capillary plasma and DPS for 32 analytes. At this level of correlation, 13 analytes correlated between venous and capillary plasma and 5 analytes in the comparison of venous blood with DPS.

Protein Profiling in Serum and Cerebrospinal Fluid Following Complex Surgery on the Thoracic Aorta Identifies Biological Markers of Neurologic Injury.

Surgery on the arch or descending aorta is associated with significant risk of neurological complications. As a consequence of intubation and sedation, early neurologic injury may remain unnoticed. Biomarkers to aid in the initial diagnostics could prove of great value as immediate intervention is critical. Twenty-three patients operated in the thoracic aorta with significant risk of perioperative neurological injury were included. Cerebrospinal fluid (CSF) and serum were obtained preoperatively and in the first and second postoperative days and assessed with a panel of 92 neurological-related proteins. Three patients suffered spinal cord injury (SCI), eight delirium, and nine hallucinations. There were markers in both serum and CSF that differed between the affected and non-affected patients (SCI; IL6, GFAP, CSPG4, delirium; TR4, EZH2, hallucinations; NF1). The study identifies markers in serum and CSF that reflect the occurrence of neurologic insults following aortic surgery, which may aid in the care of these patients.

Ibrutinib induces rapid down-regulation of inflammatory markers and altered transcription of chronic lymphocytic leukaemia-related genes in blood and lymph nodes.

In chronic lymphocytic leukaemia (CLL) patients, treatment with the Bruton tyrosine kinase inhibitor ibrutinib induces a rapid shift of tumour cells from lymph nodes (LN) to peripheral blood (PB). Here, we characterized in depth the dynamics of ibrutinib-induced inflammatory, transcriptional and cellular changes in different compartments immediately after treatment initiation in seven relapsed/refractory CLL patients. Serial PB and LN samples were taken before start and during the first 29 days of treatment. Changes in plasma inflammation-related biomarkers, CLL cell RNA expression, B-cell activation and migration markers expression, and PB mononuclear cell populations were assessed. A significant reduction of 10 plasma inflammation markers, the majority of which were chemokines and not CLL-derived, was observed within hours, and was paralleled by very early increase of CD19+ circulating cells. At the RNA level, significant and continuous changes in transcription factors and signalling molecules linked to B-cell receptor signalling and CLL biology was observed in both PB and LN CLL cells already after 2 days of treatment. In conclusion, ibrutinib seems to instantly shut off an ongoing inflammatory response and interfere with diverse sensitive pathways in the LN.

GABA Regulates Release of Inflammatory Cytokines From Peripheral Blood Mononuclear Cells and CD4+ T Cells and Is Immunosuppressive in Type 1 Diabetes.

The neurotransmitter γ-aminobutyric acid (GABA) is an extracellular signaling molecule in the brain and in pancreatic islets. Here, we demonstrate that GABA regulates cytokine secretion from human peripheral blood mononuclear cells (PBMCs) and CD4+ T cells. In anti-CD3 stimulated PBMCs, GABA (100nM) inhibited release of 47 cytokines in cells from patients with type 1 diabetes (T1D), but only 16 cytokines in cells from nondiabetic (ND) individuals. CD4+ T cells from ND individuals were grouped into responder or non-responder T cells according to effects of GABA (100nM, 500nM) on the cell proliferation. In the responder T cells, GABA decreased proliferation, and inhibited secretion of 37 cytokines in a concentration-dependent manner. In the non-responder T cells, GABA modulated release of 8 cytokines. GABA concentrations in plasma from T1D patients and ND individuals were correlated with 10 cytokines where 7 were increased in plasma of T1D patients. GABA inhibited secretion of 5 of these cytokines from both T1D PBMCs and ND responder T cells. The results identify GABA as a potent regulator of both Th1- and Th2-type cytokine secretion from human PBMCs and CD4+ T cells where GABA generally decreases the secretion.