Survivors of acute COVID-19 often suffer prolonged, diffuse symptoms post-infection, referred to as "Long-COVID". A lack of Long-COVID biomarkers and pathophysiological mechanisms limits effective ...diagnosis, treatment and disease surveillance. We performed targeted proteomics and machine learning analyses to identify novel blood biomarkers of Long-COVID.
A case-control study comparing the expression of 2925 unique blood proteins in Long-COVID outpatients versus COVID-19 inpatients and healthy control subjects. Targeted proteomics was accomplished with proximity extension assays, and machine learning was used to identify the most important proteins for identifying Long-COVID patients. Organ system and cell type expression patterns were identified with Natural Language Processing (NLP) of the UniProt Knowledgebase.
Machine learning analysis identified 119 relevant proteins for differentiating Long-COVID outpatients (Bonferonni corrected P < 0.01). Protein combinations were narrowed down to two optimal models, with nine and five proteins each, and with both having excellent sensitivity and specificity for Long-COVID status (AUC = 1.00, F1 = 1.00). NLP expression analysis highlighted the diffuse organ system involvement in Long-COVID, as well as the involved cell types, including leukocytes and platelets, as key components associated with Long-COVID.
Proteomic analysis of plasma from Long-COVID patients identified 119 highly relevant proteins and two optimal models with nine and five proteins, respectively. The identified proteins reflected widespread organ and cell type expression. Optimal protein models, as well as individual proteins, hold the potential for accurate diagnosis of Long-COVID and targeted therapeutics.
Abstract
Background
Long-COVID is characterized by prolonged, diffuse symptoms months after acute COVID-19. Accurate diagnosis and targeted therapies for Long-COVID are lacking. We investigated ...vascular transformation biomarkers in Long-COVID patients.
Methods
A case–control study utilizing Long-COVID patients, one to six months (median 98.5 days) post-infection, with multiplex immunoassay measurement of sixteen blood biomarkers of vascular transformation, including ANG-1, P-SEL, MMP-1, VE-Cad, Syn-1, Endoglin, PECAM-1, VEGF-A, ICAM-1, VLA-4, E-SEL, thrombomodulin, VEGF-R2, VEGF-R3, VCAM-1 and VEGF-D.
Results
Fourteen vasculature transformation blood biomarkers were significantly elevated in Long-COVID outpatients, versus acutely ill COVID-19 inpatients and healthy controls subjects (P < 0.05). A unique two biomarker profile consisting of ANG-1/P-SEL was developed with machine learning, providing a classification accuracy for Long-COVID status of 96%. Individually, ANG-1 and P-SEL had excellent sensitivity and specificity for Long-COVID status (AUC = 1.00, P < 0.0001; validated in a secondary cohort). Specific to Long-COVID, ANG-1 levels were associated with female sex and a lack of disease interventions at follow-up (P < 0.05).
Conclusions
Long-COVID patients suffer prolonged, diffuse symptoms and poorer health. Vascular transformation blood biomarkers were significantly elevated in Long-COVID, with angiogenesis markers (ANG-1/P-SEL) providing classification accuracy of 96%. Vascular transformation blood biomarkers hold potential for diagnostics, and modulators of angiogenesis may have therapeutic efficacy.
About 50% of all primary breast cancers show a low-level expression of HER2 (HER2-low), defined as immunohistochemically 1+ or 2+ and lack of HER2 gene amplification measured by in situ ...hybridization. This low HER2 expression is a promising new target for antibody-drug conjugates (ADCs) currently under investigation. Until now, little is known about the frequency and the prognostic value of low HER2-expression in metastatic breast cancer (MBC).
The MBC-Registry of the Austrian Study Group of Medical Tumor Therapy (AGMT) is a multicenter nationwide ongoing registry for MBC patients in Austria. Unadjusted, univariate survival probabilities of progression-free survival (PFS) and overall survival (OS) were calculated by the Kaplan-Meier method and compared by the log-rank test. Multivariable adjusted hazard ratios were estimated by Cox regression models. In this analysis, only patients with known HER2 status and available survival data were included.
As of 11/15/2020, 1,973 patients were included in the AGMT-MBC-Registry. Out of 1,729 evaluable patients, 351 (20.3%) were HER2-positive, 608 (35.2%) were HER2-low and 770 (44.5%) were completely HER2-negative (HER2-0). Low HER2-expression was markedly more frequent in the hormone-receptor(HR)+ subgroup compared to the triple-negative subgroup (40% vs. 23%). In multivariable analysis, low HER2 expression did not significantly influence OS neither in the HR+ (HR 0.89; 95% CI 0.74-1.05; P = 0.171) nor in the triple-negative subgroup (HR 0.92; 95% CI 0.68-1.25; P = 0.585), when compared to completely HER2-negative disease. Similar results were observed when HER2 IHC 2+ patients were compared to IHC 1+ or 0 patients.
Low-HER2 expression did not have any impact on prognosis of metastatic breast cancer in this real-world population.
Long-COVID occurs after SARS-CoV-2 infection and results in diverse, prolonged symptoms. The present study aimed to unveil potential mechanisms, and to inform prognosis and treatment.
Plasma proteome ...from Long-COVID outpatients was analyzed in comparison to matched acutely ill COVID-19 (mild and severe) inpatients and healthy control subjects. The expression of 3072 protein biomarkers was determined with proximity extension assays and then deconvoluted with multiple bioinformatics tools into both cell types and signaling mechanisms, as well as organ specificity.
Compared to age- and sex-matched acutely ill COVID-19 inpatients and healthy control subjects, Long-COVID outpatients showed natural killer cell redistribution with a dominant resting phenotype, as opposed to active, and neutrophils that formed extracellular traps. This potential resetting of cell phenotypes was reflected in prospective vascular events mediated by both angiopoietin-1 (ANGPT1) and vascular-endothelial growth factor-A (VEGFA). Several markers (ANGPT1, VEGFA, CCR7, CD56, citrullinated histone 3, elastase) were validated by serological methods in additional patient cohorts. Signaling of transforming growth factor-β1 with probable connections to elevated EP/p300 suggested vascular inflammation and tumor necrosis factor-α driven pathways. In addition, a vascular proliferative state associated with hypoxia inducible factor 1 pathway suggested progression from acute COVID-19 to Long-COVID. The vasculo-proliferative process predicted in Long-COVID might contribute to changes in the organ-specific proteome reflective of neurologic and cardiometabolic dysfunction.
Taken together, our findings point to a vasculo-proliferative process in Long-COVID that is likely initiated either prior hypoxia (localized or systemic) and/or stimulatory factors (i.e., cytokines, chemokines, growth factors, angiotensin, etc). Analyses of the plasma proteome, used as a surrogate for cellular signaling, unveiled potential organ-specific prognostic biomarkers and therapeutic targets.
Background Patients with kidney disease frequently experience adverse effects from medication exposure, even when drugs are cleared by nonrenal pathways. Although many studies suggest that nonrenal ...drug clearance is decreased in chronic kidney disease (CKD), there remains a paucity of in vivo studies in patients with varying degrees of decreased kidney function and those comparing the impact of dialysis modality (eg, hemodialysis HD and peritoneal dialysis PD). Study Design We performed in vivo clinical pharmacokinetic studies of midazolam, a nonrenally cleared specific probe for CYP3A4, and fexofenadine, a nonspecific probe for hepatic and intestinal transporters. Setting & Participants Healthy controls (n = 8), patients with non−dialysis-dependent (NDD)-CKD (n = 8), and patients receiving HD (n = 10) or PD (n = 8). Outcomes Exposure to midazolam and fexofenadine were quantified using area under the curve (AUC). Comprehensive pharmacokinetic parameters also were calculated for both probes. Results Midazolam AUC was significantly higher in the HD group (382.8 h·ng/mL) than in the healthy-control (63.0 h·ng/mL; P < 0.001), NDD-CKD (84.5 h·ng/mL; P = 0.002), and PD (47.4 h·ng/mL; P < 0.001) groups. Fexofenadine AUC was significantly higher in each of the NDD-CKD (2,950 h·ng/mL; P = 0.003), HD (2,327 h·ng/mL; P = 0.01), and PD (2,095 h·ng/mL; P = 0.04) groups compared with healthy controls (1,008 h·ng/mL). Limitations Small study groups had different proportions of diabetic patients, early stages of CKD not available. Conclusions Our data suggest that selection of dialysis modality is a major determinant of exposure to the CYP3A4 probe midazolam. Exposure to the intestinal and hepatic transporter probe fexofenadine is altered in patients with NDD-CKD and PD and HD patients. Thus, drug development and licensing of nonrenally cleared drugs should include evaluation in these 3 patient groups, with these results included in approved product information labeling. This reinforces the critical need for more in vivo studies of humans that evaluate the exposure to drugs cleared by these pathways.
Axillary surgery in patients with breast cancer has been a history of de-escalation; however, surgery for clinically node-positive breast cancer remained at the dogmatic level of axillary lymph node ...dissection (ALND). In these patients, currently the only way to avoid ALND is neoadjuvant systemic treatment (NST) with nodal pathologic complete response (pCR) as diagnosed by selective lymph node removal. However, pCR rates are highly dependent on tumor biology, with luminal tumors being most present yet showing the lowest pCR rates. Therefore, the TAXIS trial is investigating whether in clinically node-positive patients, either with residual disease after NST or in the upfront surgical setting, ALND can be safely omitted. All patients undergo tailored axillary surgery (TAS), which includes removal of the biopsied and clipped node, the sentinel lymph nodes as well as all palpably suspicious nodes, turning a clinically positive axilla into a clinically negative. Feasibility of TAS was recently confirmed in the first pre-specified TAXIS substudy. TAS is followed by axillary radiotherapy to treat any remaining nodal disease. Disease-free survival is the primary endpoint of this non-inferiority trial, and morbidity as well as quality of life are the main secondary endpoints, with ALND being known for having a relevant negative impact on both. Currently, 663 of 1500 patients were randomized; accrual completion is projected for 2025. The TAXIS trial stands out in including clinically node-positive patients in both the neoadjuvant and upfront surgery setting, thereby investigating surgical de-escalation at the far-end of the risk spectrum of patients with breast cancer.
•Axillary lymph node dissection is currently indicated in most patients with clinically node-positive disease.•The TAXIS trial investigates the de-escalated concept of tailored axillary surgery.•Tailored axillary surgery reduces the tumor load in the axilla to the point where radiation should be able to control it.•TAXIS accrual completion is projected for 2025 and primary endpoint analysis for 2029.
The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, are important drugs used in the treatment and prevention of cardiovascular disease. Although statins are well tolerated, ...many patients develop myopathy manifesting as muscle aches and pain. Rhabdomyolysis is a rare but severe toxicity of statins. Interindividual differences in the activities of hepatic membrane drug transporters and metabolic enzymes are known to influence statin plasma pharmacokinetics and risk for myopathy. Interestingly, little is known regarding the molecular determinants of statin distribution into skeletal muscle and its relevance to toxicity.
We sought to identify statin transporters in human skeletal muscle and determine their impact on statin toxicity in vitro.
We demonstrate that the uptake transporter OATP2B1 (human organic anion transporting polypeptide 2B1) and the efflux transporters, multidrug resistance-associated protein (MRP)1, MRP4, and MRP5 are expressed on the sarcolemmal membrane of human skeletal muscle fibers and that atorvastatin and rosuvastatin are substrates of these transporters when assessed using a heterologous expression system. In an in vitro model of differentiated, primary human skeletal muscle myoblast cells, we demonstrate basal membrane expression and drug efflux activity of MRP1, which contributes to reducing intracellular statin accumulation. Furthermore, we show that expression of human OATP2B1 in human skeletal muscle myoblast cells by adenoviral vectors increases intracellular accumulation and toxicity of statins and such effects were abrogated when cells overexpressed MRP1.
These results identify key membrane transporters as modulators of skeletal muscle statin exposure and toxicity.
Organic anion transporting polypeptide 2B1 (OATP2B1) is a widely expressed membrane transporter with diverse substrate specificity. In vitro and clinical studies suggest a role for intestinal OATP2B1 ...in the oral absorption of medications. Moreover, OATP2B1 is highly expressed in hepatocytes where it is thought to promote liver drug clearance. However, until now, a shortcoming of studies implicating OATP2B1 in drug disposition has been a lack of in vivo models. Here, we report the development of a knockout (KO) mouse model with targeted, global disruption of the
gene to examine the disposition of two confirmed mOATP2B1 substrates, namely, fexofenadine and rosuvastatin. The plasma pharmacokinetics of intravenously administered fexofenadine was not different between KO and wild-type (WT) mice. However, after oral fexofenadine administration, KO mice had 70% and 41% lower maximal plasma concentration (
) and area under the plasma concentration-time curve (AUC
) than WT mice, respectively. In WT mice, coadministration of fexofenadine with grapefruit juice (GFJ) or apple juice (AJ) was associated with reduced
by 80% and 88%, respectively, while the AUC
values were lower by 35% and 70%, respectively. In KO mice, AJ coadministration reduced oral fexofenadine
and AUC
values by 67% and 59%, respectively, while GFJ had no effects. Intravenous and oral rosuvastatin pharmacokinetics were similar among WT and KO mice. We conclude that intestinal OATP2B1 is a determinant of oral fexofenadine absorption, as well as a target for fruit juice interactions. OATP2B1 does not significantly influence rosuvastatin disposition in mice. SIGNIFICANCE STATEMENT: A novel mouse model with targeted disruption of the
gene revealed that OATP2B1 is a determinant of oral absorption but not systemic disposition of fexofenadine, as well as a target of fruit juice interactions. Rosuvastatin oral and intravenous pharmacokinetics were not dependent on OATP2B1. These findings support the utility of the
KO mouse model for defining mechanisms of drug disposition at the intersection of in vitro and clinical pharmacology.
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