Capillary blood self-collection for high-throughput proteomics.

In this study, we sought to compare protein concentrations obtained from a high-throughput proteomics platform (Olink) on samples collected using capillary blood self-collection (with the Tasso+ device) versus standard venipuncture (control). Blood collection was performed on 20 volunteers, including one sample obtained via venipuncture and two via capillary blood using the Tasso+ device. Tasso+ samples were stored at 2°C-8°C for 24-hs (Tasso-24) or 48-h (Tasso-48) prior to processing to simulate shipping times from a study participant's home. Proteomics were analyzed using Olink (384 Inflammatory Panel). Tasso+ blood collection was successful in 37/40 attempts. Of 230 proteins included in our analysis, Pearson correlations (r) and mean coefficient of variation (CV) between Tasso-24 or Tasso-48 versus venipuncture were variable. In the Tasso-24 analysis, 34 proteins (14.8%) had both a correlation r > 0.5 and CV < 0.20. In the Tasso-48 analysis, 68 proteins (29.6%) had a correlation r > 0.5 and CV < 0.20. Combining the Tasso-24 and Tasso-48 analyses, 26 (11.3%) proteins met these thresholds. We concluded that protein concentrations from Tasso+ samples processed 24-48 h after collection demonstrated wide technical variability and variable correlation with a venipuncture gold-standard. Use of home capillary blood self-collection for large-scale proteomics should be limited to select proteins with good agreement with venipuncture.

Identification of Circulating Plasma Proteins as a Mediator of Hypertension-Driven Cardiac Remodeling: A Mediation Mendelian Randomization Study.

BACKGROUND: This study focused on circulating plasma protein profiles to identify mediators of hypertension-driven myocardial remodeling and heart failure. METHODS: A Mendelian randomization design was used to investigate the causal impact of systolic blood pressure (SBP), diastolic blood pressure (DBP), and pulse pressure on 82 cardiac magnetic resonance traits and heart failure risk. Mediation analyses were also conducted to identify potential plasma proteins mediating these effects. RESULTS: Genetically proxied higher SBP, DBP, and pulse pressure were causally associated with increased left ventricular myocardial mass and alterations in global myocardial wall thickness at end diastole. Elevated SBP and DBP were linked to increased regional myocardial radial strain of the left ventricle (basal anterior, mid, and apical walls), while higher SBP was associated with reduced circumferential strain in specific left ventricular segments (apical, mid-anteroseptal, mid-inferoseptal, and mid-inferolateral walls). Specific plasma proteins mediated the impact of blood pressure on cardiac remodeling, with FGF5 (fibroblast growth factor 5) contributing 2.96% (P=0.024) and 4.15% (P=0.046) to the total effect of SBP and DBP on myocardial wall thickness at end diastole in the apical anterior segment and leptin explaining 15.21% (P=0.042) and 23.24% (P=0.022) of the total effect of SBP and DBP on radial strain in the mid-anteroseptal segment. Additionally, FGF5 was the only mediator, explaining 4.19% (P=0.013) and 4.54% (P=0.032) of the total effect of SBP and DBP on heart failure susceptibility. CONCLUSIONS: This mediation Mendelian randomization study provides evidence supporting specific circulating plasma proteins as mediators of hypertension-driven cardiac remodeling and heart failure.

Social Stress during the Estrus or Luteal Phase in Sheep.

In mammals, progesterone and estrogens affect the stress response. The study aimed to compare the physiological and behavioral responses to the social isolation of ewes during the estrus or luteal phase. Estrous and diestrous ewes (n = 10 and 8 respectively) were individually isolated in a novel place for 10 min. Ewes' behavior was recorded during the test. Cortisol, blood proteins and glucose concentrations, and the skin surface temperature were determined before and after the test. Cortisol increased immediately after the test ended (P = 0.02). Serum total protein (P = 0.02), globulin (P < 0.0001), and plasma glucose (P = 0.006) concentrations were greater in estrus than in the luteal phase. The abdominal skin surface temperature was greater during estrus than during the luteal phase (P = 0.02). Ewes in estrus spent more time standing up immobile than ewes in the luteal phase (P = 0.05). In conclusion, the physiological and behavioral responses changed according to the phase of the estrous cycle. These results highlight the need for future studies focusing on how reproductive status influences the stress response to different management practices in sheep.

Electrospray triple quadrupole mass spectrometry guides pathologists to suggest appropriate molecular testing in the identification of rare hemoglobin variants.

Background: The presumptive diagnosis of hemoglobinopathies relies on routine tests such as Complete Blood Count (CBC), peripheral blood smear, Liquid Chromatography (LC), and Capillary Electrophoresis (CE), along with clinical findings. Pathologists suggest molecular sequencing of HBA and HBB genes to correlate blood picture with clinical findings in order to identify unknown rare haemoglobin (Hb) variants or variants that coelute with Hb. This paper presents a low-resolution mass spectrometry (MS)-based method for presumptive identification of variants that eluted in zone 12 of CE, followed by molecular sequencing of the HBB gene for a definitive diagnosis of hemoglobinopathies. Methods: Eight patient samples with a variant peak in zone 12 of CE (Sebia) were analyzed using MS. The mass-to-charge ratio (m/z) observed was deconvoluted to determine the mass of Hb variants. The ß variants were subsequently confirmed through molecular sequencing. Results: Based on the intact mass of the variants, there were two samples of the α variant (α + 58 Da and α + 44 Da), and six samples of the ß variant. Out of these six ß variant samples, three were the ß + 58 Da variant, and three were the ß + 30 Da variant. By correlating the intact mass information with the CE pattern and considering the ethnicity of the patients, it was presumed that the α variants were HbJ Meerut (α + 58 Da, x-axis 102) and HbJ Paris-I (α + 44 Da, x-axis 80). Molecular analysis confirmed the identity of ß variants as Hb Rambam/HbJ Cambridge, HbJ Bangkok (+58 Da), and Hb Hofu (+30 Da). Conclusion: The mass information of Hb variants obtained using Electrospray triple quadrupole MS assists pathologists in recommending the appropriate molecular sequencing for identifying unknown variants.

Multiplexed MRM-based proteomics for identification of circulating proteins as biomarkers of cardiovascular damage progression associated with diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2DM) increases the risk of coronary heart disease (CHD) by 2-4 fold, and is associated with endothelial dysfunction, dyslipidaemia, insulin resistance, and chronic hyperglycaemia. The aim of this investigation was to assess, by a multimarker mass spectrometry approach, the predictive role of circulating proteins as biomarkers of cardiovascular damage progression associated with diabetes mellitus. METHODS: The study considered 34 patients with both T2DM and CHD, 31 patients with T2DM and without CHD, and 30 patients without diabetes with a diagnosis of CHD. Plasma samples of subjects were analysed through a multiplexed targeted liquid chromatography mass spectrometry (LC-MS)-based assay, namely Multiple Reaction Monitoring (MRM), allowing the simultaneous detection of peptides derived from a protein of interest. Gene Ontology (GO) Analysis was employed to identify enriched GO terms in the biological process, molecular function, or cellular component categories. Non-parametric multivariate methods were used to classify samples from patients and evaluate the relevance of the analysed proteins' panel. RESULTS: A total of 81 proteins were successfully quantified in the human plasma samples. Gene Ontology analysis assessed terms related to blood microparticles, extracellular exosomes and collagen-containing extracellular matrix. Preliminary evaluation using analysis of variance (ANOVA) of the differences in the proteomic profile among patient groups identified 13 out of the 81 proteins as significantly different. Multivariate analysis, including cluster analysis and principal component analysis, identified relevant grouping of the 13 proteins. The first main cluster comprises apolipoprotein C-III, apolipoprotein C-II, apolipoprotein A-IV, retinol-binding protein 4, lysozyme C and cystatin-C; the second one includes, albeit with sub-grouping, alpha 2 macroglobulin, afamin, kininogen 1, vitronectin, vitamin K-dependent protein S, complement factor B and mannan-binding lectin serine protease 2. Receiver operating characteristic (ROC) curves obtained with the 13 selected proteins using a nominal logistic regression indicated a significant overall distinction (p < 0.001) among the three groups of subjects, with area under the ROC curve (AUC) ranging 0.91-0.97, and sensitivity and specificity ranging from 85 to 100%. CONCLUSIONS: Targeted mass spectrometry approach indicated 13 multiple circulating proteins as possible biomarkers of cardiovascular damage progression associated with T2DM, with excellent classification results in terms of sensitivity and specificity.

FOLFIRINOX chemotherapy modulates the peripheral immune landscape in pancreatic cancer: Implications for combination therapies and early response prediction.

BACKGROUND: FOLFIRINOX chemotherapy has improved outcomes for pancreatic cancer patients, but poor long-term survival outcomes and high toxicity remain challenges. This study investigates the impact of FOLFIRINOX on plasma proteins and peripheral immune cells to guide immune-based combination therapies and, ideally, to identify a potential biomarker to predict early disease progression during FOLFIRINOX. METHODS: Blood samples were collected from 86 pancreatic cancer patients before and two weeks after the first FOLFIRINOX cycle and subjected to comprehensive immune cell and proteome profiling. Principal Component Analysis and Linear Mixed Effect Regression models were used for data analysis. FOLFIRINOX efficacy was radiologically evaluated after the fourth cycle. RESULTS: One cycle of FOLFIRINOX diminished tumour-cell-related pathways and enhanced pathways related to immune activation, illustrated by an increase in pro-inflammatory IL-18, IL-15, and TNFRSF4. Similarly, FOLFIRINOX promoted the activation of CD4 + and CD8 + T cells, the proliferation of NK(T), and the activation of antigen-presenting cells. Furthermore, high pre-treatment levels of VEGFA and PRDX3 and an elevation in FCRL3 levels after one cycle predicted early progression under FOLFIRINOX. Finally, patients with progressive disease exhibited high levels of inhibitory markers on B cells and CD8 + T cells, while responding patients exhibited high levels of activation markers on CD4 + and CD8 + T cell subsets. CONCLUSION: FOLFIRINOX has immunomodulatory effects, providing a foundation for clinical trials exploring immune-based combination therapies that harness the immune system to treat pancreatic cancer. In addition, several plasma proteins hold potential as circulating predictive biomarkers for early prediction of FOLFIRINOX response in patients with pancreatic cancer.

Predicting molecular docking of per- and polyfluoroalkyl substances to blood protein using generative artificial intelligence algorithm DiffDock.

This study computationally evaluates the molecular docking affinity of various perfluoroalkyl and polyfluoroalkyl substances (PFAs) towards blood proteins using a generative machine-learning algorithm, DiffDock, specialized in protein-ligand blind-docking learning and prediction. Concerns about the chemical pathways and accumulation of PFAs in the environment and eventually in the human body has been rising due to empirical findings that levels of PFAs in human blood has been rising. DiffDock may offer a fast approach in determining the fate and potential molecular pathways of PFAs in human body.

Tuning polymer-blood and polymer-cytoplasm membrane interactions by manipulating the architecture of poly(2-oxazoline) triblock copolymers.

Bioactive moieties designed to bind to cell membrane receptors benefit from coupling with polymeric carriers that have enhanced affinity to the cell membrane. When bound to the cell surface, such carriers create a "2D solution" of a ligand with a significantly increased concentration near a membrane-bound receptor compared to a freely water-soluble ligand. Bifunctional polymeric carriers based on amphiphilic triblock copolymers were synthesized from 2-pent-4-ynyl oxazoline, 2-nonyl oxazoline and 2-ethyl oxazoline. Their self-assembly and interactions with plasma proteins and HEK 293 cells were studied in detail. The affinity of these triblock copolymers to HEK 293 cell membranes and organ tissues was tunable by the overall hydrophobicity of the polymer molecule, which is determined by the length of the hydrophobic and hydrophilic blocks. The circulation time and biodistribution of three representative triblock copolymers were monitored after intravenous administration to C57BL/6 albino mice. A prolonged circulation time was observed for polymers with longer hydrophobic blocks, despite their molecular weight being below the renal threshold.

Preclinical development and characterization of a human plasma-derived high-purity factor X concentrate for therapeutic use.

INTRODUCTION: Hereditary factor X deficiency is a rare bleeding disorder, with limited treatment options. This paper describes the approach to pre-clinical development and characterization of a high-purity plasma-derived factor X concentrate, to achieve orphan drug marketing authorization for the treatment of hereditary factor X deficiency. METHODS: A chromatographic process was developed, to purify factor X from human plasma for fractionation. The product was characterized using in vitro, in vivo and ex vivo tests for potency, purity, thrombogenicity, immunogenicity, toxicity and stability. RESULTS: The production process complied with good pharmaceutical manufacturing practice. It achieved 6000-fold purification and 100-fold concentration of the factor X protein compared to human plasma. The factor X protein was 94%-96% pure. Other residual plasma proteins were well below levels in plasma, minimizing potential interference in hemostasis after therapeutic administration. Effective virus-reduction during manufacture, and the absence of thrombogenicity, toxicity and immunogenic potential were confirmed, addressing the main safety concerns historically associated with plasma-derived therapeutics. The freeze-dried product remained stable between +2°C and +30°C for at least three years. After reconstitution with water for injections, the factor X activity was maintained for at least 48 h at +18°C to +22°C. CONCLUSION: Targeted pre-clinical development of the first highly-purified concentrate to treat hereditary factor X deficiency is described. Following international guidelines for nonclinical safety testing, particular strategies were adopted for unmodified products derived from human blood plasma. This approach may also be relevant to the development of other ultra-orphan medicinal products.

A Fast and Reliable Method Based on QCM-D Instrumentation for the Screening of Nanoparticle/Blood Protein Interactions.

The interactions that nanoparticles have with blood proteins are crucial for their fate in vivo. Such interactions result in the formation of the protein corona around the nanoparticles, and studying them aids in nanoparticle optimization. Quartz crystal microbalance with dissipation monitoring (QCM-D) can be used for this study. The present work proposes a QCM-D method to study the interactions on polymeric nanoparticles with three different human blood proteins (albumin, fibrinogen and γ-globulin) by monitoring the frequency shifts of sensors immobilizing the selected proteins. Bare PEGylated and surfactant-coated poly-(D,L-lactide-co-glycolide) nanoparticles are tested. The QCM-D data are validated with DLS and UV-Vis experiments in which changes in the size and optical density of nanoparticle/protein blends are monitored. We find that the bare nanoparticles have a high affinity towards fibrinogen and γ-globulin, with measured frequency shifts around -210 Hz and -50 Hz, respectively. PEGylation greatly reduces these interactions (frequency shifts around -5 Hz and -10 Hz for fibrinogen and γ-globulin, respectively), while the surfactant appears to increase them (around -240 Hz and -100 Hz and -30 Hz for albumin). The QCM-D data are confirmed by the increase in the nanoparticle size over time (up to 3300% in surfactant-coated nanoparticles), measured by DLS in protein-incubated samples, and by the trends of the optical densities, measured by UV-Vis. The results indicate that the proposed approach is valid for studying the interactions between nanoparticles and blood proteins, and the study paves the way for a more comprehensive analysis of the whole protein corona.

Vascular Effects on Cerebrovascular Permeability and Neurodegeneration.

Neurons and glial cells in the brain are protected by the blood brain barrier (BBB). The local regulation of blood flow is determined by neurons and signal conducting cells called astrocytes. Although alterations in neurons and glial cells affect the function of neurons, the majority of effects are coming from other cells and organs of the body. Although it seems obvious that effects beginning in brain vasculature would play an important role in the development of various neuroinflammatory and neurodegenerative pathologies, significant interest has only been directed to the possible mechanisms involved in the development of vascular cognitive impairment and dementia (VCID) for the last decade. Presently, the National Institute of Neurological Disorders and Stroke applies considerable attention toward research related to VCID and vascular impairments during Alzheimer's disease. Thus, any changes in cerebral vessels, such as in blood flow, thrombogenesis, permeability, or others, which affect the proper vasculo-neuronal connection and interaction and result in neuronal degeneration that leads to memory decline should be considered as a subject of investigation under the VCID category. Out of several vascular effects that can trigger neurodegeneration, changes in cerebrovascular permeability seem to result in the most devastating effects. The present review emphasizes the importance of changes in the BBB and possible mechanisms primarily involving fibrinogen in the development and/or progression of neuroinflammatory and neurodegenerative diseases resulting in memory decline.

Detection of COX-2 in liquid biopsy of patients with prostate cancer.

AIMS: To determine the profile of COX-2 gene expression in patients with prostate cancer attended at the ABC University Health Center outpatient clinic and correlate the results with patients' anatomopathological examinations. Prostate cancer is the sixth most common type of cancer worldwide and the second in Brazil. COX-2 expression is associated with an unfavourable prognosis. METHODS: 15.0 mL of peripheral blood were collected from 24 patients and 25 healthy men. RNA extraction was performed using the QIAamp RNA Blood Mini Kit. Complementary DNA synthesis was performed using SuperScript II RNAse Reverse Transcriptase. Quantitative real-time PCR was performed with specific COX-2 oligonucleotides and the endogenous GAPDH gene. RESULTS: The mean age of the patients was 69 years old. The Gleason scoring system showed 37.5% of patients with Gleason 6 (slow growth, low risk), 45.8% with Gleason 7 (intermediate risk) and 16.7% with Gleason 8 or 9 (risk of high-grade cancer). The median COX-2 expression in the study group was 0.97, while in the control group it was 0.11 (p<0.045). CONCLUSIONS: Patients with prostate cancer showed higher COX-2 expression at diagnosis compared with the control group. Since COX-2 detection associated with prostate-specific antigen dosage shows promise as a biomarker for diagnosis and prognosis in patients with prostate cancer, further research is required to confirm these findings.

Mid-regional proadrenomedullin (MR-proADM), C-reactive protein (CRP) and other biomarkers in the early identification of disease progression in patients with COVID-19 in the acute NHS setting.

AIMS: There is a lack of biomarkers validated for assessing clinical deterioration in patients with COVID-19 on presentation to secondary or tertiary care. This evaluation looked at the potential clinical application of C reactive protein (CRP), procalcitonin, mid-regional proadrenomedullin (MR-proADM) and white cell count to support prediction of clinical outcomes. METHODS: 135 patients presenting to Hampshire Hospitals NHS Foundation Trust between April and June 2020 confirmed to have COVID-19 via reverse-transcription-qPCR were included. Biomarkers from within 24 hours of presentation were used to predict disease progression by Cox regression and area under the receiver operating characteristic curves. The endpoints assessed were 30-day all-cause mortality, intubation and ventilation, critical care admission and non-invasive ventilation (NIV) use. RESULTS: Elevated MR-proADM was shown to have the greatest ability to predict 30-day mortality adjusting for age, cardiovascular disease, renal disease and neurological disease. A significant association was also noted between raised MR-proADM and CRP concentrations and the requirement for critical care admission and NIV. CONCLUSIONS: The measurement of MR-proADM and CRP in patients with confirmed COVID-19 infection on admission shows significant potential to support clinicians in identifying those at increased risk of disease progression and need for higher level care, subsequently enabling prompt escalation in clinical interventions.

The usefulness of a three-protein signature blood assay (Mastocheck®) for follow-up after breast cancer surgery.

PURPOSE: Mastocheck®, a proteomic-based blood assay, has been developed for early diagnosis of breast cancer. The purpose of this study is whether Mastocheck® is useful as a postoperative follow-up. METHODS: A total of 255 patients were analyzed. The patients were classified into longitudinal monitoring and recurrence/nonrecurrence cohorts. The longitudinal monitoring cohort consisted of 111 patients. In this cohort, blood analyses were performed three times (before surgery, 8 weeks after surgery, and between 6 months and one year after surgery), and a comparative analysis of the values of Mastocheck® and individual proteins at each time point was performed. The recurrence/nonrecurrence cohort consisted of 144 patients who had been followed up for more than 1 year, and the blood marker values at the time of local recurrence were compared to those of nonrecurrence patients. RESULTS: In the longitudinal monitoring cohort analysis, in 81 of 111 patients were diagnosed with breast cancer with Mastocheck® and the sensitivity was 73.0%. Of 111 patients in the longitudinal monitoring cohort, 108 had two blood analyses (before and 8 weeks after surgery), and three serial blood analyses were performed on 53 patients. The Mastocheck® value that were in the cancer range of 73.0% (in 81 of 111 patients) of patients before surgery, was within the normal range of 68.5% (in 74 of 108 patients) at 8 weeks after surgery and 88.7% (in 47 of 53 patients) from 6 months to 1 year after surgery. The value of Mastocheck® was significantly decreased after surgery compared to before surgery (p < 0.001). In the recurrence/nonrecurrence cohort analysis, the Mastocheck® values were in the cancer range in 38 out of 63 recurrence patients and within the normal range in 66 of 81 nonrecurrence patients (sensitivity of 60.3% and specificity of 80.2%). CONCLUSIONS: Mastocheck® is expected to be used as a blood marker tool to aid in the early detection of recurrence during follow-up after breast cancer surgery.

Haptoglobin-related protein in human plasma correlates to haptoglobin concentrations and phenotypes.

Haptoglobin-related protein (Hpr) is a plasma protein with high sequence similarity to haptoglobin (Hp). Like Hp, Hpr also binds hemoglobin (Hb) with high affinity, but it does not bind to the Hb-Hp receptor CD163 on macrophages. The Hpr concentration is markedly lower than Hp in plasma and its regulation is not understood. In the present study, we have developed non-crossreactive antibodies to Hpr to analyze the Hpr concentration in 112 plasma samples from anonymized individuals and compared it to Hp. The results show that plasma Hpr correlated with Hp concentrations (rho = 0.46, p = .0001). Hpr accounts for on average 0.35% of the Hp/Hpr pool but up to 29% at low Hp levels. Furthermore, the Hpr concentrations were significantly lower in individuals with the Hp2-2 phenotype compared to those with the Hp2-1 or Hp1-1 phenotypes. Experimental binding analysis did not provide evidence that Hpr associates with Hp and in this way is removed via CD163. In conclusion, the Hpr concentration correlates to Hp concentrations and Hp-phenotypes by yet unknown mechanisms independent of CD163-mediated removal of Hb-Hp complexes.

Re-evalution of the measurement of haptoglobin in human plasma samples.

Haptoglobin (Hp) is an abundant plasma protein scavenging hemoglobin (Hb) via CD163 on macrophages. This process consumes Hp, which therefore negatively correlates to hemolysis. However, exact measurements of Hp plasma levels are complicated by different phenotypes (Hp1-1, Hp2-1, and Hp2-2) forming different oligomeric states with differences in immunoreactivity. In addition, humans have an immune-cross-reactive Hp-related protein. In the present study, we developed Hp-specific monoclonal antibodies for an accurate Hp analysis of the different Hp phenotypes in a panel of 112 anonymous samples from hospitalized individuals subjected to routine Hp immunoturbidimetric measurements. The data revealed immunoturbidimetry as a reliable method in most cases but also that the use of non-phenotype-specific calibrators leads to substantial bias in the measurement of the Hp-concentration, non at least in Hp1-1 individuals. Furthermore, analysis of the Hb-dependence of the CD163 interaction with Hp1-1 and Hp2-2 showed that a higher 'cost-effectiveness' in the consumption of dimeric Hp1-1 versus multimeric Hp phenotypes is a likely contribution to the observed differences in the plasma levels of the Hp phenotypes. In conclusion, the determination of Hp phenotype and the use of phenotype-specific calibrators are essential to obtain a precise estimate of the Hp level in healthy and diseased individuals.

Serum Protein Exposure Activates a Core Regulatory Program Driving Human Proximal Tubule Injury.

BACKGROUND: The kidneys efficiently filter waste products while retaining serum proteins in the circulation. However, numerous diseases compromise this barrier function, resulting in spillage of serum proteins into the urine (proteinuria). Some studies of glomerular filtration suggest that tubules may be physiologically exposed to nephrotic-range protein levels. Therefore, whether serum components can directly injure the downstream tubular portions of the kidney, which in turn can lead to inflammation and fibrosis, remains controversial. METHODS: We tested the effects of serum protein exposure in human kidney tubule microphysiologic systems and with orthogonal epigenomic approaches since animal models cannot directly assess the effect of serum components on tubules. RESULTS: Serum, but not its major protein component albumin, induced tubular injury and secretion of proinflammatory cytokines. Epigenomic comparison of serum-injured tubules and intact kidney tissue revealed canonical stress-inducible regulation of injury-induced genes. Concordant transcriptional changes in microdissected tubulointerstitium were also observed in an independent cohort of patients with proteinuric kidney disease. CONCLUSIONS: Our results demonstrate a causal role for serum proteins in tubular injury and identify regulatory mechanisms and novel pathways for intervention.

Diagnostic performance improvement with combined use of proteomics biomarker assay and breast ultrasound.

PURPOSE: To investigate the combined use of blood-based 3-protein signature and breast ultrasound (US) for validating US-detected lesions. METHODS: From July 2011 to April 2020, women who underwent whole-breast US within at least 6 months from sampling period were retrospectively included. Blood-based 3-protein signature (Mastocheck®) value and US findings were evaluated. Following outcome measures were compared between US alone and the combination of Mastocheck® value with US: sensitivity, specificity, positive predictive value (PPV), negative predictive value, area under the receiver operating characteristic curve (AUC), and biopsy rate. RESULTS: Among the 237 women included, 59 (24.9%) were healthy individuals and 178 (75.1%) cancer patients. Mean size of cancers was 1.2 ± 0.8 cm. Median value of Mastocheck® was significantly different between nonmalignant (- 0.24, interquartile range [IQR] - 0.48, - 0.03) and malignant lesions (0.55, IQR - 0.03, 1.42) (P < .001). Utilizing Mastocheck® value with US increased the AUC from 0.67 (95% confidence interval [CI] 0.61, 0.73) to 0.81 (95% CI 0.75, 0.88; P < .001), and specificity from 35.6 (95% CI 23.4, 47.8) to 64.4% (95% CI 52.2, 76.6; P < .001) without loss in sensitivity. PPV was increased from 82.2 (95% CI 77.1, 87.3) to 89.3% (95% CI 85.0, 93.6; P < .001), and biopsy rate was significantly decreased from 79.3 (188/237) to 72.1% (171/237) (P < .001). Consistent improvements in specificity, PPV, and AUC were observed in asymptomatic women, in women with dense breast, and in those with normal/benign mammographic findings. CONCLUSION: Mastocheck® is an effective tool that can be used with US to improve diagnostic specificity and reduce false-positive findings and unnecessary biopsies.

CXCR4/ACKR3/CXCL12 axis in the lymphatic metastasis of vulvar squamous cell carcinoma.

AIMS: Vulvar squamous cell carcinoma (VSCC) spreads early and mainly locally via direct expansion into adjacent structures, followed by lymphatic metastasis to the regional lymph nodes (LNs). In the lymphatic metastasis, cancer cells bearing CXCR4 and ACKR3 (CXCR7) receptors are recruited to the LNs that produce the CXCL12 ligand. Our study aimed to assess the role of the CXCR4/ACKR3/CXCL12 axis in VSCC progression. METHODS: Tumour and LN tissue samples were obtained from 46 patients with VSCC and 51 patients with premalignant vulvar lesions. We assessed CXCR4, ACKR3 and CXCL12 by immunohistochemistry (IHC) in the tissue samples. Additionally, CXCL12 levels were determined by ELISA in the sera of 23 patients with premalignant lesions, 37 with VSCC and 16 healthy volunteers. RESULTS: CXCR4 and ACKR3 proteins were virtually absent in vulvar precancers, while in VSCC samples the IHC staining was strong. In the LNs of patients with VSCC, 98% of metastatic cells expressed CXCR4 and 85% expressed ACKR3. Neither CXCR4 nor ACKR3 presence was correlated with tumour human papilloma virus status. Few CXCL12-positive cells were found in the analysed tissue samples, but serum CXCL12 levels were significantly increased in both patients with premalignant vulvar lesions and with VSCC compared with healthy volunteers. CONCLUSIONS: It appears that during progression and lymphatic spread of VSCC, the CXCR4/ACKR3/CXCL12 axis is activated. Moreover, our data suggest that CXCR4 antagonists merit further attention as a possible therapeutic option in patients with VSCC.

Targeted proteomics for evaluating risk of venous thrombosis following traumatic lower-leg injury or knee arthroscopy.

INTRODUCTION: Patients with lower-leg cast immobilization and patients undergoing knee arthroscopy have an increased risk of venous thrombosis (VT). Guidelines are ambiguous about thromboprophylaxis use, and individual risk factors for developing VT are often ignored. To assist in VT risk stratification and guide thromboprophylaxis use, various prediction models have been developed. These models depend largely on clinical factors and provide reasonably good C-statistics of around 70%. We explored using protein levels in blood plasma measured by multiplexed quantitative targeted proteomics to predict VT. Our aim was to assess whether a VT risk prediction model based on absolute plasma protein quantification is possible. METHODS: We used internal standards to quantify proteins in less than 10 µl plasma. We measured 270 proteins in samples from patients scheduled for knee arthroscopy or with lower-leg cast immobilization. The two prospective POT-(K)CAST trails allow complementary views of VT signature in blood, namely pre and post trauma, respectively. From approximately 3000 patients, 31 patients developed VT who were included and matched with double the number of controls. RESULTS: Top discriminating proteins between cases and controls included APOC3, APOC4, APOC2, ATRN, F13B, and F2 in knee arthroscopy patients and APOE, SERPINF2, B2M, F13B, AFM, and C1QC in patients with lower-leg cast. A logistic regression model with cross-validation resulted in C-statistics of 88.1% (95% CI: 85.7-90.6%) and 79.6% (95% CI: 77.2-82.0%) for knee arthroscopy and cast immobilization groups respectively. CONCLUSIONS: Promising C-statistics merit further exploration of the value of proteomic tests for predicting VT risk upon additional validation.