Whole Blood Analysis Research Articles

Low Caloric Intake Confers Cardiovascular Protection and Improves Functional Capacity Without Affecting Immunological Response in Sedentary Older Adults

Background: Aging is characterized by a decline in the cardiovascular hemodynamic response, which may be aggravated by undernutrition. However, no study has evaluated whether low caloric intake may affect cardiovascular hemodynamics and its possible relation with functional capacity and immune response in older adults. Methods: Sixty-one older adults of both genders were enrolled in this study and were classified as normocaloric (n = 18) and hypocaloric (n = 43). All volunteers were evaluated for cardiovascular hemodynamics using impedance cardiography (PhysioFlow®); functional capacity by the 1′ sit-to-stand test with SpO2 monitoring; whole-blood analysis using an automated hematocytometer (Sysmex®); and levels of IL-6, TNF-alpha, IL-10, and Klotho by ELISA. Results: The hypocaloric group presented impaired functional capacity, measured by a reduced number of sit-to-stand repetitions (p < 0.0251) and impaired delta of SpO2 (p < 0.0307). In contrast, the hypocaloric group presented an improved stroke volume (p < 0.0352), systemic vascular resistance (p < 0.0075), and systemic vascular resistance index (p < 0.0184). In addition, no changes were observed in the whole-blood analysis (p > 0.05) or for IL-6 (p > 0.05), TNF-alpha (p < 0.05), IL-10 (p < 0.05), and Klotho (p > 0.05). Conclusions: A long-term hypocaloric diet in eutrophic older adults’ resulted in an enhanced cardiovascular hemodynamic response but was associated with reduced functional capacity without changes in the immune response.

Non-classical monocyte levels correlate negatively with HIV-associated cerebral small vessel disease and cognitive performance

BackgroundDespite antiretroviral treatment (cART), aging people living with HIV (PWH) are more susceptible to neurocognitive impairment (NCI) probably due to synergistic/additive contribution of traditional cerebrovascular risk factors. Specifically, transmigration of inflammatory CD16+ monocytes through the altered blood brain barrier (BBB) may exacerbate cerebral small vessel disease (CSVD), a known cause of vascular cognitive impairment.MethodsPWH on cART (n=108) and age, sex, and Reynold’s cardiovascular risk score-matched uninfected individuals (PWoH, n=111) were enrolled. This is a longitudinal observational study but only cross-sectional data from entry visit are reported. Neuropsychological testing and brain magnetic resonance imaging (MRI) were performed. CSVD was diagnosed by Fazekas score ≥1. Flow cytometric analyses of fresh whole blood were conducted to evaluate circulating levels of monocyte subsets (classical, intermediate, and non-classical) and markers of monocyte activation (CCR2, CD40, PSGL-1, TNFR2 and tissue factor). ELISAs were used to measure sCD14, ICAM, and Osteoprotegerin. Two-way analysis of variance (ANOVA), and linear regression models were performed to study the effects of HIV status, CSVD status, and their interaction to outcome variables such as cognitive score. Two-sample t-tests and correlation analyses were performed between and within PWoH with CSVD and PWH with CSVD participants.ResultsPWH with CSVD (n=81) had significantly lower total cognitive scores, higher levels of NCMs and soluble CD14 and intracellular adhesion molecule 1 (ICAM-1) as compared to PWoH with CSVD group (n=68). sCD14 and ICAM1 were positively correlated with each other indicating that monocyte and endothelial activation are associated with each other. Cognition was negatively correlated with NCMs, especially in the PWH with CSVD group. Among other blood biomarkers measured, osteoprotegerin levels showed mild negative correlation with cognitive performance in individuals with CSVD irrespective of HIV status.ConclusionsElevated levels of NCMs may contribute to neuroinflammation, CSVD and subsequent cognitive impairment. This finding is of particular relevance in aging PWH as both HIV and aging are associated with increased levels of NCMs. NCMs may serve as a potential biomarker to address these comorbidities. Further longitudinal studies are needed to evaluate whether changes in NCM levels are associated with changes in CSVD burden and cognitive impairment.

Acute-phase innate immune responses in SIVmac239-infected Mamu-B*08+ Indian rhesus macaques may contribute to the establishment of elite control

IntroductionSpontaneous control of chronic-phase HIV/SIV viremia is often associated with the expression of specific MHC class I allotypes. HIV/SIV-specific CD8+ cytotoxic T lymphocytes (CTLs) restricted by these MHC class I allotypes appear to be critical for viremic control. Establishment of the elite controller (EC) phenotype is predictable in SIVmac239-infected Indian rhesus macaques (RMs), with approximately 50% of Mamu-B*08+ RMs and 20% of Mamu-B*17+ RMs becoming ECs. Despite extensive characterization of EC-associated CTLs in HIV/SIV-infected individuals, the precise mechanistic basis of elite control remains unknown. Because EC and non-EC viral load trajectories begin diverging by day 14 post-infection, we hypothesized that hyperacute innate immune responses may contribute to viremic control.MethodsTo gain insight into the immunological factors involved in the determination of EC status, we vaccinated 16 Mamu-B*08+ RMs with Vif and Nef to elicit EC-associated CTLs, then subjected these 16 vaccinees and an additional 16 unvaccinated Mamu-B*08+ controls to repeated intrarectal SIVmac239 challenges. We then performed whole-blood transcriptomic analysis of all 32 SIVmac239-infected Mamu-B*08+ RMs and eight SIVmac239-infected Mamu-B*08– RMs during the first 14 days of infection.ResultsVaccination did not provide protection against acquisition, but peak and setpoint viremia were significantly lower in vaccinees relative to controls. We did not identify any meaningful correlations between vaccine-induced CTL parameters and SIVmac239 acquisition rate or chronic-phase viral loads. Ultimately, 13 of 16 vaccinees (81%) and 7 of 16 controls (44%) became ECs (viremia ≤ 10,000 vRNA copies/mL plasma for ≥ 4 weeks). We identified subsets of immunomodulatory genes differentially expressed (DE) between RM groupings based on vaccination status, EC status, and MHC class I genotype. These DE genes function in multiple innate immune processes, including the complement system, cytokine/chemokine signaling, pattern recognition receptors, and interferon-mediated responses.DiscussionA striking difference in the kinetics of differential gene expression among our RM groups suggests that Mamu-B*08-associated elite control is characterized by a robust, rapid innate immune response that quickly resolves. These findings indicate that, despite the association between MHC class I genotype and elite control, innate immune factors in hyperacute SIV infection preceding CTL response development may facilitate the establishment of the EC phenotype.

Peripheral immune profiling of soft tissue sarcoma: perspectives for disease monitoring

Studying the tumor microenvironment and surrounding lymph nodes is the main focus of current immunological research on soft tissue sarcomas (STS). However, due to the restricted opportunity to examine tumor samples, alternative approaches are required to evaluate immune responses in non-surgical patients. Therefore, the purpose of this study was to evaluate the peripheral immune profile of STS patients, characterize patients accordingly and explore the impact of peripheral immunotypes on patient survival. Blood samples were collected from 55 STS patients and age-matched healthy donors (HD) controls. Deep immunophenotyping and gene expression analysis of whole blood was analyzed using multiparametric flow cytometry and real-time RT-qPCR, respectively. Using xMAP technology, proteomic analysis was also carried out on plasma samples. Unsupervised clustering analysis was used to classify patients based on their immune profiles to further analyze the impact of peripheral immunotypes on patient survival. Significant differences were found between STS patients and HD controls. It was found a contraction of B cells and CD4 T cells compartment, along with decreased expression levels of ICOSLG and CD40LG; a major contribution of suppressor factors, as increased frequency of M-MDSC and memory Tregs, increased expression levels of ARG1, and increased plasma levels of IL-10, soluble VISTA and soluble TIMD-4; and a compromised cytotoxic potential associated with NK and CD8 T cells, namely decreased frequency of CD56dim NK cells, and decreased levels of PRF1, GZMB, and KLRK1. In addition, the patients were classified into three peripheral immunotype groups: "immune-high," "immune-intermediate," and "immune-low." Furthermore, it was found a correlation between these immunotypes and patient survival. Patients classified as "immune-high" exhibited higher levels of immune-related factors linked to cytotoxic/effector activity and longer survival times, whereas patients classified as "immune-low" displayed higher levels of immune factors associated with immunosuppression and shorter survival times. In conclusion, it can be suggested that STS patients have a compromised systemic immunity, and the correlation between immunotypes and survival emphasizes the importance of studying peripheral blood samples in STS. Assessing the peripheral immune response holds promise as a useful method for monitoring and forecasting outcomes in STS.

Whole-blood RNA biomarkers for predicting survival in non-human primates following thoracic radiation

Radiation injury, either from radiotherapy or a mass-casualty event requires a health care system that can efficiently allocate resources to patients. We conducted a comprehensive transcriptome analysis of whole blood from a nonhuman primate model that received upper thoracic radiation (9.8–10.7 Gy). Blood samples were collected at multiple time points, extending up to 270 days post-irradiation with a minimum n = 6 for initial time points (Day 3-Day 40) and a total number of n = 28 primates. No males receiving the higher dose survived to Day 270. Using the Elastic Net model in R we found that pooling biomarkers from Day 3–21 increased our accuracy in discerning survival time, pleural effusion or dose compared to using biomarkers specific to a single day. For survival data, in predicting short term (less than 90 day), medium term (Day 91–269) or long-term survival (Day 270), prediction accuracy using only Day 3 data was 0.14 (95% Confidence Interval (CI) 0.1, 0.19) while pooled data for Male and Female was 0.76 (CI 0.69, 0.82). When pooled data was divided by biological sex, accuracy was 0.7 (CI 0.58, 0.8) for pooled data from Males and 0.84 (CI 0.76, 0.91) for Females. The development of RNA biomarkers as a tool to aid in clinical decision-making could significantly improve patient care in cases of radiation injury, whether from radiotherapy or mass-casualty events. Further validation and clinical translation of these findings could lead to improved patient care and management strategies in cases of radiation exposure.

A membrane-based plasma separator coupled with ratiometric fluorescent sensor for biochemical analysis in whole blood

A membrane-based plasma separator coupled with ratiometric fluorescent sensor for biochemical analysis in whole blood

Risk assessment with gene expression markers in sepsis development

Infection is a commonplace, usually self-limiting, condition but can lead to sepsis, a severe life-threatening dysregulated host response. We investigate the individual phenotypic predisposition to developing uncomplicated infection or sepsis in a large cohort of non-infected patients undergoing major elective surgery. Whole-blood RNA sequencing analysis was performed on preoperative samples from 267 patients. These patients developed postoperative infection with (n= 77) or without (n= 49) sepsis, developed non-infectious systemic inflammatory response (n= 31), or had an uncomplicated postoperative course (n= 110). Machine learning classification models built on preoperative transcriptomic signatures predict postoperative outcomes including sepsis with an area under the curve of up to 0.910 (mean 0.855) and sensitivity/specificity up to 0.767/0.804 (mean 0.746/0.769). Our models, confirmed by quantitative reverse-transcription PCR (RT-qPCR), potentially offer a risk prediction tool for the development of postoperative sepsis with implications for patient management. They identify an individual predisposition to developing sepsis that warrants further exploration to better understand the underlying pathophysiology.

Unveiling differential gene co-expression networks and its effects on levodopa-induced dyskinesia

Levodopa-induced dyskinesia (LID) refers to involuntary motor movements of chronic use of levodopa in Parkinson's disease (PD) that negatively impact the overall well-being of people with this disease. The molecular mechanisms involved in LID were investigated through whole-blood transcriptomic analysis for differential gene expression and identification of new co-expression and differential co-expression networks. We found six differentially expressed genes in patients with LID, and 13 in patients without LID. We also identified 12 co-expressed genes exclusive to LID, and six exclusive hub genes involved in 23 gene-gene interactions in patients with LID. Convergently, we identified novel genes associated with PD and LID that play roles in mitochondrial dysfunction, dysregulation of lipid metabolism, and neuroinflammation. We observed significant changes in disease progression, consistent with previous findings of maladaptive plastic changes in the basal ganglia leading to the development of LID, including a chronic pro-inflammatory state in the brain.

Molecular screening of transitional B cells as a prognostic marker of improved graft outcome and reduced rejection risk in kidney transplant.

Understanding immune cell dynamics in kidney transplantation may provide insight into the mechanisms of rejection and improve patient management. B cells have gained interest with a special relevance of the "regulatory" subsets and their graft outcome prognostic value. In this study, we aimed to prove that the direct immunophenotyping and target gene expression analysis of kidney transplant patients' fresh whole blood will help to identify graft rejection risk and assist in the monitoring of kidney transplanted patients. We employed flow cytometry and qPCR techniques to characterize B and T cell subsets within fresh whole blood samples, with particular emphasis on transitional B cells (TrB) identified as CD19+CD24hiCD38hi. TrB are a relevant population in the context of kidney transplantation and are closely associated with regulatory B cells (Bregs) in humans. Patients were monitored, tracking pertinent clinical parameters and kidney-related events, including alterations in graft function and episodes of biopsy proven rejection. Higher percentages of TrB cells at 3 months after transplantation were positively associated with better graft outcomes and lower biopsy-proven acute rejection risk. Furthermore, a novel panel of B cell regulatory associated genes was validated at 3 months post-transplantation by qPCR analysis of peripheral blood mononuclear cell (PBMC) mRNA, showing high predictive power of graft events and prognostic value. These findings suggest that monitoring TrB may provide interesting patient management information, improve transplant outcomes, and allow for personalized drug regimens to minimize clinical complications.

Inhibition of HMOX1 by MAFG potentiates the development of depression‑like behavior in mice associated with astrocyte-mediated neuroinflammation

MAF bZIP transcription factor G (MAFG)-driven astrocytes have been reported to promote inflammation in the CNS. However, its function in depression, the primary cause of disability worldwide, has not been well characterized. This study investigated the possible perturbation of heme oxygenase 1 (HMOX1, also known as HO1) by the transcription factor MAFG as an underlying mechanism of the development of depression. The GSE98793 dataset was included for gene expression analysis of whole blood from donors with major depressive disorder and controls, and the target of MAFG was predicted by multiple database mining. Mouse and cellular models of depression were established by chronic unpredictable mild stress (CUMS) and lipopolysaccharide (LPS) treatment of astrocytes, which were treated with MAFG and HMOX1 knockdown plasmids. MAFG was highly expressed in the hippocampal tissues of CUMS-challenged mice and LPS-induced astrocytes. MAFG knockdown alleviated depression-like behaviors in mice. MAFG bound to the HMOX1 promoter and repressed its transcription. Knockdown of HMOX1 exacerbated neuroinflammation in astrocytes and accelerated depression-like behavior in mice. In conclusion, MAFG knockdown attenuated CUMS-stimulated depression-like behaviors in mice by astrocyte-mediated neuroinflammation via restoration of HMOX1.

Reply to Ross, K. Comment on "Polak et al. Principles and Limitations of miRNA Purification and Analysis in Whole Blood Collected during Ablation Procedure from Patients with Atrial Fibrillation. J. Clin. Med. 2024, 13, 1898".

Thank you for your interest and insightful comments on our article [...].

Comment on Polak et al. Principles and Limitations of miRNA Purification and Analysis in Whole Blood Collected during Ablation Procedure from Patients with Atrial Fibrillation. J. Clin. Med. 2024, 13, 1898.

In the current issue of the journal, the paper by Polak et al [...].

Evaluation of the Local and Peripheral Immune Responses in Patients with Cystic Echinococcosis.

Cystic echinococcosis (CE) cysts may persist for decades because of immune modulation mechanisms. Here, we characterize the cysts and the blood immune responses in patients with CE. We enrolled 61 patients with CE and 19 control subjects. We received tissue samples from seven patients with CE and a control subject requiring liver cystectomy. The immunohistochemistry evaluation of the immune cell subtypes and cytokines in the pericysts and surrounding liver and the antigen B (AgB)-specific response analysis of whole blood were performed. In CE, the pericyst and the surrounding liver parenchyma showed aggregates of CD3+ T lymphocytes, mainly CD4+. B lymphocyte aggregates were present in the liver tissue. Monocytes/granulocytes were rarely observed. Th2 cytokine expression was scarce, whereas IFN-γ expression was present in the CE tissues. The control subject did not show an inflammatory infiltrate. The IL-4-specific response to AgB was increased in the patients with CE compared to the control, and this result was confirmed in a larger cohort (p = 0.003), whereas the IFN-γ-response was similar between the two groups (p = 0.5570). In patients with CE, CD4+ lymphocytes infiltrate the pericyst and the surrounding liver tissue with a low IL-4/IL-13 expression level and a moderate IFN-γ expression level; moreover, an IL-4 parasite-specific response is detected in the periphery. These results support adventitia involvement in CE immunopathogenesis.

Whole-blood transcriptome analysis reveals distinct gene expression signatures in paediatric patients with sickle cell anaemia before and after exercise.

Sickle cell anaemia (SCA) patients display elevated levels of circulating pro-inflammatory cytokines and endothelial activation markers compared to healthy peers. The impact of exercise on the pro-inflammatory state in SCA remains unclear. This study aimed to characterize the whole-blood transcriptome profile in response to an acute bout of exercise in paediatric SCA patients. Twenty-three SCA participants (13 ± 3 years, 52% girls) and 17 healthy controls (14 ± 3 years, 29% girls) performed eight 2-min bouts of cycle ergometry interspersed with 1-min rest intervals. Whole-blood transcriptome profile (RNA-seq) was performed before and after exercise. At baseline, gene pathways associated with gas transport in erythrocytes were up-regulated in SCA patients compared to controls. Following exercise, gene pathways associated with innate immunity were altered in both groups. Interaction analyses revealed 160 annotated genes (101 up- and 59 down-regulated) that differentially altered by exercise in SCA patients. Moreover, genes that exhibited a blunted response to exercise in SCA patients were enriched in the IL-17 signalling pathway, suggesting an impaired innate immune response to exercise. This data will contribute to the development of evidence-based exercise prescription guidelines for this patient population.

Is alcohol and psychoactive medication use associated with excess hospital length-of-stay and admission frequency? A cross-sectional, observational study

BackgroundHospital length-of-stay and admission frequency are commonly used indicators of disease burden and health resource expenditures. However, the impact of psychoactive prescription medication use and harmful alcohol consumption on both the duration and frequency of hospital admissions is under-explored.MethodsWe conducted an analysis of data gathered from 2872 patients admitted to the Emergency Department at Lovisenberg Diaconal Hospital in Oslo, Norway. Psychoactive medicines (benzodiazepines, opioids, and z-hypnotics) were detected via liquid chromatography-mass spectrometry analysis of whole blood, while alcohol consumption was self-reported through the Alcohol Use Disorder Identification Test-4 (AUDIT-4). Using logistic regression, we examined associations with our primary outcomes, which were excess length-of-stay and admission frequency, defined as exceeding the sample median of 3.0 days and 0.2 admissions per year, respectively.ResultsCompared to the absence of psychoactive medication, and after adjusting for age, gender, malignant disease, pre-existing substance use disorder and admission due to intoxication, the detection of two or more psychoactive medicines was associated with both excess length-of-stay (odds ratio [OR], 1.60; 95% confidence interval [CI], 1.20 to 2.14) and yearly hospitalization rate (OR, 3.72; 95% CI, 2.64 to 5.23). This association persisted when increasing the definition for excess length-of-stay to 4 and 5 days and to 1.0 and 1.5 admissions per year for admission frequency. Harmful alcohol consumption (AUDIT-4 scores of 9 to 16) was not associated with excess length-of-stay, but with excess admission frequency when defined as more than 1.0 admission per year when compared to scores of 4 to 6 (OR, 2.68; 95% CI, 1.58 to 4.57).ConclusionsPsychoactive medication use is associated with both excess length-of-stay and increased antecedent admission frequency, while harmful alcohol consumption may be associated with the latter. The utility of our findings as a causal factor should be explored through intervention-based study designs.

Principles and Limitations of miRNA Purification and Analysis in Whole Blood Collected during Ablation Procedure from Patients with Atrial Fibrillation.

Background: MicroRNA (miRNA) have the potential to be non-invasive and attractive biomarkers for a vast number of diseases and clinical conditions; however, a reliable analysis of miRNA expression in blood samples meets a number of methodological challenges. In this report, we presented and discussed, specifically, the principles and limitations of miRNA purification and analysis in blood plasma samples collected from the left atrium during an ablation procedure on patients with atrial fibrillation (AF). Materials and Methods: Consecutive patients hospitalized in the First Department of Cardiology for pulmonary vein ablation were included in this study (11 with diagnosed paroxysmal AF, 14 with persistent AF, and 5 without AF hospitalized for left-sided WPW ablation-control group). Whole blood samples were collected from the left atrium after transseptal puncture during the ablation procedure of AF patients. Analysis of the set of miRNA molecules was performed in blood plasma samples using the MIHS-113ZF-12 kit and miScript microRNA PCR Array Human Cardiovascular Disease. Results: The miRNS concentrations were in the following ranges: paroxysmal AF: 7-23.1 ng/µL; persistent AF: 4.9-66.8 ng/µL; controls: 6.3-10.6 ng/µL. The low A260/280 ratio indicated the protein contamination and the low A260/A230 absorbance ratio suggested the contamination by hydrocarbons. Spectrophotometric measurements also indicated low concentration of nucleic acids (<10 ng/µL). Further steps of analysis revealed that the concentration of cDNA after the Real-Time PCR (using the PAXgene RNA Blood kit) reaction was higher (148.8 ng/µL vs. 68.4 ng/µL) and the obtained absorbance ratios (A260/A280 = 2.24 and A260/A230 = 2.23) indicated adequate RNA purity. Conclusions: Although developments in miRNA sequencing and isolation technology have improved, detection of plasma-based miRNA, low RNA content, and sequencing bias introduced during library preparation remain challenging in patients with AF. The measurement of the quantity and quality of the RNA obtained is crucial for the interpretation of an efficient RNA isolation.

Abstract 6562: Comprehensive liquid biopsy: Multi-analyte evaluation of cellular and plasma components of blood in prostate cancer from a single blood collection tube

Abstract Background Liquid biopsy samples offer a non-invasive method for investigating drug targets and biomarkers in solid tumors. In this study, we describe a comprehensive liquid biopsy analysis of whole blood to identify circulating tumor cells (CTCs), large extracellular vesicles and CTC fragments, and CTC-white blood cell hybrids; quantify protein biomarker expression on CTCs; perform targeted mutation profiling on CTCs and cell-free DNA (cfDNA) from plasma; and analyze protein expression on white blood cells (WBCs) from a single 7.5 mL blood sample. This array of tests was applied to blood samples from patients with Stage IV prostate cancer to demonstrate clinical feasibility. Methods A novel dual biomarker CTC assay was developed to measure protein expression of PSMA and ARv7 on CTCs from stage IV prostate cancer patients. Nucleated cells from clinical samples were processed to microscope slides using AccuCyte®, an unbiased, density-based separation method that also allows for plasma collection for downstream analysis. Slides were fixed and stained with antibodies to cytokeratin, EpCAM, and CD45 to detect CTCs and exclude WBCs, and with PSMA and ARv7 to evaluate protein biomarker expression on identified CTCs. The slides were imaged with the CyteFinder® automated immunofluorescence scanning microscope that identifies CTCs for confirmation by trained reviewers and quantitatively measures protein mean fluorescence intensity (MFI) on any cells detected on the slides (CTCs, CTC-WBC hybrids, large extracellular vesicles and CTC fragments and WBCs). Individual CTCs and control pools of WBCs from clinical samples with at least 4 CTCs were picked using CytePicker®, a needle-based system used to isolate single cells. cfDNA was isolated from plasma removed during the AccuCyte® process using a QIAamp MinElute ccfDNA kit. Picked CTCs, WBC pools, and isolated ctDNA were sequenced using the OncoZoom Cancer HotSpot panel from Paragon Genomics. Results Nine progressing Stage IV prostate cancer patient blood samples were stained with a novel PSMA/ARv7 dual biomarker assay. CTCs were identified in all clinical samples (median 4, range 1 - 384). PSMA positivity in clinical samples ranged from 0% to 75%, and ARv7 positivity ranged from 33% to 100%, and. Additional analysis using QuPath was performed to determine PSMA/ARv7 protein expression on WBCs and CTC fragments. ctDNA analysis from 5 individual samples was compared to sequencing results from individual CTCs from the same samples. Conclusions Using a single 7.5 mL blood liquid biopsy, we can measure protein expression on enumerated CTCs and quantify these same protein biomarkers on WBCs and CTC fragments/oncosomes, determine the genetic makeup of the tumor through the sequencing of cfDNA, and monitor genetic mutations in intact CTCs in circulation. Citation Format: Jon Ladd, Erin Bayer, Brock Bartels, Arista Tischner, Rachel Ponting, Eric Kaldjian, Arturo Ramirez. Comprehensive liquid biopsy: Multi-analyte evaluation of cellular and plasma components of blood in prostate cancer from a single blood collection tube [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6562.

Abstract 6038: Copper-67 based targeted radiotherapy primes immunologically cold PDAC for immunotherapy

Abstract Background and Purpose: Immune checkpoint therapy (ICT) has revolutionized cancer treatment however efficacy remains dismal for some cancers, including pancreatic cancers. Strategies to enhance immune responses include combining ICT with other existing cancer therapies. Targeted radiotherapy (TRT) uses a radiolabeled cancer-targeting vector, allowing for systematic delivery of specific radiation to all tumor sites while minimizing radiation exposure to healthy tissues. We propose Targeted Copper Theranostics (TCTs) as a TRT, utilizing copper-67 to irradiate and prime immunologically “cold” pancreatic adenocarcinoma (PDAC) and evaluate the efficacy of copper-67-based TRT-ICT combination therapy. Methods: We evaluated two tumor-targeting vectors conjugated to a sarcophagine macrobicyclic chelator (SAR), an antibody—5B1, targeting the PDAC-specific biomarker carbohydrate antigen-19-9 (CA19-9), and a peptide RGD, targeting the αvβ3 integrin receptor, which is highly expressed in some PDAC tumors. We administrated ~1.1 MBq of [64Cu]Cu-SAR-bisRGD (~0.1 μg) or [64Cu]Cu-SAR-5B1 (~10 μg) to FC1245 Ft2Ab PDAC (a murine PDAC cell line expressing CA19-9) tumor-bearing C57BL/6 mice and performed biodistribution studies at 1 and 4 h for [64Cu]Cu-SAR-bisRGD-injected mice and at 24 and 48 h for [64Cu]Cu-5B1-injected mice. [67Cu]Cu-SAR-bisRGD- and [67Cu]Cu-SAR-5B1-based dose escalation studies and the combination of a copper-67-based TRT and ICT were performed to evaluate therapeutic efficacy of copper-67-based TRT and potential synergistic effects of combination therapy. Results: Tumor uptake of [64Cu]Cu-SAR-bisRGD is ~5 %ID/g at 4h-post injection, and tumor uptake of [64Cu]Cu-SAR-5B1 is ~55 %ID/g 24h-post injection. In dose escalation studies, administration of ~11, 22, and 37 MBq of [67Cu]Cu-SAR-bisRGD (~1 μg) or administration of ~4, 11, 18 MBq of [67Cu]Cu-5B1(~150 μg) improved overall survival to 25 or 32 days respectively, compared to the control cohort (14 days). Based on weekly whole-blood analyses, no significant radiotoxicity was observed in mice receiving various doses of TRTs. The combination of 11 MBq of either [67Cu]Cu-SAR-bisRGD or [67Cu]Cu-SAR-5B1 in combination with either antiPD-L1/PD1 or antiCTLA4, improved overall survival by ~7 days compared to ICT-treated control groups. Conclusion: Biodistribution studies demonstrated relatively high tumor specific uptake for [64Cu]Cu-SAR-bisRGD and [64Cu]Cu-SAR-5B1. Dose escalation study results suggest copper-67-based TRT could effectively inhibit tumor growth with minimal radiotoxicity. The combination of TRT with ICTs improved overall survival compared to single-ICT-treated control groups. Collectively, our results strongly support the hypothesis that TRT with [67Cu]Cu-SAR-bisRGD or [67Cu]Cu-SAR-5B1 could prime immunologically “cold” pancreatic adenocarcinoma (PDAC) to be responsive to ICTs. Citation Format: Yi Rao, Tara Viray, Kurt Gehlsen, Matt Harris, Lachlan McInnes, Olufolake Majekodunmi, Jason Lewis. Copper-67 based targeted radiotherapy primes immunologically cold PDAC for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6038.

Advances in D-dimer testing: progress in harmonization of clinical assays and innovative detection methods.

The D-dimer is a sensitive indicator of coagulation and fibrinolysis activation, especially valuable as a biomarker of intravascular thrombosis. Measurement of plasma D-dimer levels plays a crucial role in the diagnosis and monitoring of conditions such as deep vein thrombosis, pulmonary embolism, and disseminated intravascular coagulation. A variety of immunoassays, including enzyme-linked immunosorbent assays, latex-enhanced immunoturbidimetric assays, whole-blood aggregation analysis, and immunochromatography assays, are widely used in clinical settings to determine D-dimer levels. However, the results obtained from different D-dimer assays vary significantly. These assays exhibit intra-method coefficients of variation ranging from 6.4% to 17.7%, and the measurement discrepancies among different assays can be as high as 20-fold. The accuracy and reliability of D-dimer testing cannot be guaranteed due to the lack of an internationally endorsed reference measurement system (including reference materials and reference measurement procedures), which may lead to misdiagnosis and underdiagnosis, limiting its full clinical application. In this review, we present an in-depth analysis of clinical D-dimer testing, summarizing the existing challenges, the current state of metrology, and progress towards harmonization. We also review the latest advancements in D-dimer detection techniques, which include mass spectrometry and electrochemical and optical immunoassays. By comparing the basic principles, the definition of the measurand, and analytical performance of these methods, we provide an outlook on the potential improvements in D-dimer clinical testing.

VEXAS syndrome is characterized by inflammasome activation and monocyte dysregulation

Acquired mutations in the UBA1 gene were recently identified in patients with severe adult-onset auto-inflammatory syndrome called VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic). However, the precise physiological and clinical impact of these mutations remains poorly defined. Here we study a unique prospective cohort of VEXAS patients. We show that monocytes from VEXAS are quantitatively and qualitatively impaired and display features of exhaustion with aberrant expression of chemokine receptors. In peripheral blood from VEXAS patients, we identify an increase in circulating levels of many proinflammatory cytokines, including IL-1β and IL-18 which reflect inflammasome activation and markers of myeloid cells dysregulation. Gene expression analysis of whole blood confirms these findings and also reveals a significant enrichment of TNF-α and NFκB signaling pathways that can mediate cell death and inflammation. This study suggests that the control of the nflammasome activation and inflammatory cell death could be therapeutic targets in VEXAS syndrome.