Healthy Donor Samples Research Articles

P0270 Standardisation of Rapid TDM Assay with WHO International Reference Material for Anti-Infliximab Antibodies

Abstract Background Point-of-care (POC) tests play a crucial role in therapeutic drug monitoring (TDM) for Inflammatory bowel disease (IBD) patients undergoing anti-TNF therapy. Infliximab (IFX), the first biologic used in IBD treatment, is a chimeric anti-TNF-alpha blocker. However, its composition can trigger an immune response in patients, leading to the production of anti-drug antibodies (ADAs). Consequently, regular monitoring for ADAs is essential during IFX therapy. Various assays are available to measure anti-IFX antibody levels, mainly based on ELISA technology. The recent introduction of an international reference material (IRM) for infliximab ADAs allows enhanced measurement traceability and enables harmonised standardisation and improved comparability across different assays. Furthermore, it is mandatory to standardise with an existing IRM for compliance with the in vitro diagnostic regulation (IVDR). This study focused on standardising the lateral flow based Quantum Blue® Anti-Infliximab assay (LF-ADIF) using the IRM and comparing its performance with the current standardisation material. Additionally, a method comparison to established ELISA assays was carried out. Methods The lateral flow assay was calibrated with both the currently used standardisation material and the new IRM. Blant-Altman analysis was performed to evaluate the comparability of both standardisation materials. To establish the cut-off, 105 healthy donor samples were measured. This cut-off will be used for discrimination of positive and negative results of the rapid test. Furthermore, a set of 30 patient samples was analysed for method comparison with established ELISA assays. Results The Blant-Altman analysis revealed that a IRM based calibration is feasible and IRM concentrations <100 IU/mL can be detected. A technical cut-off was established allowing the discrimination of positive and negative patient samples. Total agreement between the lateral flow assay and the ELISA assays was demonstrated in the method comparison. Conclusion To the best of our knowledge the here presented assay is the first IRM based and IVDR ready lateral flow assay for anti-infliximab. The total agreement of patient sample results between the Quantum Blue® Anti-Infliximab assay and commercially available ELISA assays revealed good comparability. IRM standardisation across all anti-IFX assays would further improve comparability between them.

Potential Regulation of ARID1A by miR-129-5p and miR-3613-3p and Their Prognostic Value in Gastric Cancer.

Gastric cancer (GC) remains the most common malignant tumor of the gastrointestinal tract and one of the leading causes of cancer-related deaths worldwide. Non-coding RNAs (ncRNAs), including microRNAs (miRNAs), are involved in the pathogenesis and progression of GC and, therefore, may be potential diagnostic and prognostic biomarkers. Our work was aimed at investigating the predicted regulation of ARID1A by miR-129-5p and miR-3613-3p and the clinical value of their aberrant expression in GC. The study included tumor and adjacent non-tumor tissues from 110 GC patients, 38 sectional normal gastric tissue samples, as well as 65 plasma samples of GC patients and 49 plasma samples of healthy donors. Expression levels of ARID1A and both miRNAs were quantified by reverse transcription-polymerase chain reaction (RT-PCR). We have identified significant associations of their expression with the clinical and pathological characteristics of GC patients both in tissues and plasma. To validate predicted target pairs miR-129-5p/ARID1A and miR-3613-3p/ARID1A, in vitro experiments on cancer cell lines were conducted. The obtained results suggest a complex role of ARID1A, miR-129-5p and miR-3613-3p in GC and potential regulation of ARID1A expression by both miRNAs.

Metabolic Transcriptional Activation in Ulcerative Colitis Identified Through scRNA-seq Analysis.

Ulcerative colitis is a chronic inflammatory disease affecting the colon. During chronic inflammation of epithelial cells, lipid metabolism via pro-inflammatory eicosanoids is known to modify the immune response. Starting from the Mammalian Metabolic Database, the expression of metabolic enzymes was investigated in two independent cohorts from transcriptome datasets GSE38713 and GSE11223, which analyzed ulcerative colitis tissue samples from the digestive tract. In the first cohort, 145 differentially expressed enzymes were identified as significantly regulated between ulcerative colitis tissues and normal controls. Overexpressed enzymes were selected to tune an Elastic Net model in the second cohort. Using the best parameters, the model achieved a prediction accuracy for ulcerative colitis with an area under the curve (AUC) of 0.79. Twenty-two metabolic enzymes were found to be commonly overexpressed in both independent cohorts, with decreasing Elastic Net predictive coefficients as follows: LIPG (3.98), PSAT1 (3.69), PGM3 (2.74), CD38 (2.28), BLVRA (1.99), CBR3 (1.94), NT5DC2 (1.76), PHGDH (1.71), GPX7 (1.58), CASP1 (1.56), ASRGL1 (1.4), SOD3 (1.25), CHST2 (0.965), CHST11 (0.95), KYNU (0.94), PLAG2G7 (0.92), SRM (0.87), PTGS2 (0.80), LPIN1 (0.47), ME1 (0.31), PTGDS (0.14), and ADA (0.13). Functional enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database highlighted the main implications of these enzymes in cysteine and methionine metabolism (adjusted p-value = 0.01), arachidonic acid and prostaglandin metabolism (adjusted p-value = 0.01), and carbon metabolism (adjusted p-value = 0.04). A metabolic score based on the transcriptional activation of the validated twenty-two enzymes was found to be significantly greater in Ulcerative colitis samples compared to healthy donor samples (p-value = 1.52 × 10-8). A metabolic expression score was established and reflects the implications of heterogeneous metabolic pathway deregulations in the digestive tract of patients with ulcerative colitis.

Detection of circulating tumor cells in non-metastatic prostate cancer through integration of a microfluidic CTC enrichment system and multiparametric flow cytometry.

Prostate cancer (PCa) is the second most common cancer among men and the fifth leading cause of cancer death. Circulating tumor cell (CTC) enumeration and characterisation in PCa has been shown to provide valuable information on prognosis of disease, therapy management and detection of resistance. Here, Cellsway's microfluidic platform for high-throughput enrichment of intact CTC populations was used to isolate CTCs from the blood of 20 localised PCa patients and 10 healthy donor samples to evaluate the clinical performance of the technology. To enumerate and characterise CTCs, a multi-parameter flow cytometry analysis was performed on the enriched CTC suspension using CTC-specific biomarkers. CTCs were detected in 17 of 20 patient samples, which corresponds to 85% CTC positivity. The median CTC count per 7.5 ml blood was 2 (1-9). In 80% of patients (n = 16), the number of CTCs ranged from 1 to 5, and in 5% of patients (n = 1) the number of CTCs was above 5. No CTCs were observed in the blood samples of 10 healthy volunteers, demonstrating the high specificity and low risk of false positives of the technology.

Type 2-like polarization and elevated CXCL4 secretion of monocyte derived macrophages upon internalization of plasma-derived exosomes from head and neck cancer patients

BackgroundExosomes are closely associated with different aspects of tumor-progression in patients with head and neck squamous cell carcinoma (HNSCC), such as angiogenesis or immune regulation. As extracellular vesicles they are involved in the intercellular communication by transferring their cargo such as proteins and nucleic acids from one cell to another. However, the influence of tumor related plasma-derived exosomes on the polarization and characteristics of monocyte derived macrophages is not fully understood.MethodsExosomes were isolated from plasma samples of healthy donors (HD) and HNSCC patients and further evaluated with regard to morphology, size and protein composition via transmission electron microscopy, nanoparticle tracking, western blot analysis and cytokine assays. Differentiation and characteristics of monocyte derived macrophages upon exosome internalization were analyzed using flow cytometry and fluorescence microscopy. Macrophage cytokine secretion patterns were analyzed by human cytokine antibody arrays and ELISA measurements.ResultsOur data revealed elevated overall plasma levels of CTLA-4, PD-L1, and TIM-3 as well as elevated exosome-associated CTLA-4, PD-L2, TIM-3, and LAG-3 levels in HNSCC patients compared to HD. Furthermore, we observed a significant type 2-like polarization and elevated CXCL4 secretion of monocyte derived macrophages upon internalization of plasma-derived exosomes from HNSCC patients, which could be visualized by fluorescence microcopy of membrane stained exosomes.ConclusionsThe study provides new insights regarding exosome driven pro-tumorigenic immune regulation in the circulation of patients with head and neck cancer and could help to better understand the individual immunologic situation.

Serum levels of stearic and dihomo-γ-linolenic acids can be used to diagnose cervical cancer and cervical intraepithelial neoplasia

Despite widespread cervical cancer (CC) screening programs, low participation has led to high morbidity and mortality rates, especially in developing countries. Because early-stage CC often has no symptoms, a non-invasive and convenient diagnostic method is needed to improve disease detection. In this study, we developed a new approach for differentiating both CC and cervical intraepithelial neoplasia (CIN)2/3, a precancerous lesion, from healthy individuals by exploring CC fatty acid metabolic reprogramming. Analysis of public datasets suggested that various fatty acid metabolizing enzymes were expressed at higher levels in CC tissues than in normal tissues. Correspondingly, 11 free fatty acids (FFAs) showed significantly different serum levels in CC patient samples compared with healthy donor samples. Nine of these 11 FFAs also displayed significant alterations in CIN2/3 patients. We then generated diagnostic models using combinations of these FFAs, with the optimal model including stearic and dihomo-γ-linolenic acids. Receiver operating characteristic curve analyses suggested that this diagnostic model could detect CC and CIN2/3 more accurately than using serum squamous cell carcinoma antigen level. In addition, the diagnostic model using FFAs was able to detect patients regardless of clinical stage or histological type. Overall, the serum FFA diagnostic model developed in this study could be a powerful new tool for the non-invasive early detection of CC and CIN2/3.

A novel high-performance rapid screening test for the detection of total HTLV-I and HTLV-II antibodies in HTLV-I/II infected patients

Rapid diagnosis of human T-cell lymphotropic virus (HTLV) type-I and -II infections are essential for timely and cost-effective disease interventions. MP Diagnostics ASSURE HTLV-I/II Rapid Test was developed for the rapid detection of anti-HTLV-I/II antibodies in patients’ serum, plasma, and whole blood specimens. ASSURE HTLV-I/II Rapid Test employed MP Biomedicals’ proprietary HTLV-I/II Trifusion recombinant antigen conjugated with gold nanoparticles and HTLV-I / HTLV-II recombinant antigens immobilized on the nitrocellulose membrane to detect total HTLV-I and HTLV-II antibodies. The overall performance of the ASSURE HTLV-I/II Rapid Test was found to be 99.42% sensitivity (95% Confidence Interval, 98.32–99.88%) and 100% specificity (95% Confidence Interval, 99.58–100.00%) in the tested clinical samples, including a total of 518 HTLV-I/II positive specimens (396 HTLV-I infection, 97 HTLV-II infection and 25 HTLV-I/II dual infection) and 872 HTLV negative clinical specimens consisting of 691 healthy donor samples, 116 potentially cross-reactive samples, and 65 samples with interfering substances. The ASSURE HTLV-I/II Rapid Test can effectively be deployed as a screening tool in any prevalence studies, blood banks or organ transplant centres.

Broader anti-EBV TCR repertoire in multiple sclerosis: disease specificity and treatment modulation.

Epstein-Barr virus (EBV) infection has long been associated with the development of multiple sclerosis (MS). MS patients have elevated titers of EBV-specific antibodies in serum and show signs of CNS damage only after EBV infection. Regarding CD8+ T-cells, an elevated but ineffective response to EBV was suggested in MS patients, who present with a broader MHC-I-restricted EBV-specific T-cell receptor beta chain (TRB) repertoire compared to controls. It is not known whether this altered EBV response could be subject to dynamic changes, e.g., by approved MS therapies, and whether it is specific for MS. 1317 peripheral blood TRB repertoire samples of healthy donors (n=409), patients with MS (n=710) before and after treatment, patients with neuromyelitis optica spectrum disorder (n=87), myelin-oligodendrocyte-glycoprotein antibody-associated disease (n=64) and Susac's syndrome (n=47) were analyzed. Apart from MS, none of the evaluated diseases presented with a broader anti-EBV TRB repertoire. In MS patients undergoing autologous hematopoietic stem-cell transplantation, EBV reactivation coincided with elevated MHC-I-restricted EBV-specific TRB sequence matches. Therapy with ocrelizumab, teriflunomide or dimethyl fumarate reduced EBV-specific, but not CMV-specific MHC-I-restricted TRB sequence matches. Together, this data suggests that the aberrant MHC-I-restricted T-cell response directed against EBV is specific to MS with regard to NMO, MOGAD and Susac's Syndrome and that it is specifically modified by MS treatments interfering with EBV host cells or activated lymphocytes.

Development of an immunoassay for quantification of soluble human CD40L (CD154) in plasma and serum samples

When the membrane protein CD40 ligand (CD40L) on activated T cells binds the receptor CD40 on B-cells, it provides a co-stimulatory signal for B cell activation. Dysregulation of the CD40L:CD40 axis is associated with inflammatory and autoimmune diseases. The presence of soluble CD40L (sCD40L) in plasma is implicated in several diseases, from cardiovascular and autoimmune diseases to different types of cancer, and sCD40L has been suggested as a valuable marker of disease. If sCD40L is to be used as a biomarker, being able to precisely measure and quantify the levels of sCD40L in human blood samples is of utmost importance. We demonstrate the development of a sandwich-type time-resolved immunofluorometric assay for quantification of sCD40L in plasma or serum samples. For this, we generate 29 monoclonal anti-CD40L antibodies, and from these, we select the optimal combination of capture antibody and detection antibody. A number of variables were tested: the influence of the type of sample (comparing 3 different blood collection tubes for serum sampling and 4 different types of tubes for plasma sampling), the influence of freeze-thaw cycles, the influence of sampling time during night and day, and the influence of centrifugation of the samples. We found a very similar level of sCD40L in paired EDTA plasma and serum samples. Out of 100 healthy blood donor samples 61 had a level of sCD40L below the detection level of the assay, whereas the remaining 39 samples had ranging levels of sCD40L from 1.14 to 33.14 ng/mL. In summary, we present a time-resolved immunofluorometric assay based on paired monoclonal antibodies, ensuring high specificity, sensitivity, and homogeneity. The Eu3+-based assay additionally provides consistent assay readouts due to the extended decay time not seen in standard enzyme-linked immunosorbent assays. The assay paves the way for specific and consistent quantification of sCD40L in human plasma and serum samples.

Enrichment of T-lymphocytes from leukemic blood using inertial microfluidics toward improved chimeric antigen receptor-T cell manufacturing

Chimeric antigen receptor cell therapy is a successful immunotherapy for the treatment of blood cancers. However, hurdles in their manufacturing remain including efficient isolation and purification of the T-cell starting material. Herein, we describe a one-step separation based on inertial spiral microfluidics for efficient enrichment of T-cells in B-cell acute lymphoblastic leukemia (ALL) and B-cell chronic lymphocytic leukemia patient's samples. In healthy donors used to optimize the process, the lymphocyte purity was enriched from 65% (SD ± 0.2) to 91% (SD ± 0.06) and T-cell purity was enriched from 45% (SD ± 0.1) to 73% (SD ± 0.02). Leukemic samples had higher starting B-cells compared to the healthy donor samples. Efficient enrichment and recovery of lymphocytes and T-cells were achieved in ALL samples with B-cells, monocytes and leukemic blasts depleted by 80% (SD ± 0.09), 89% (SD ± 0.1) and 74% (SD ± 0.09), respectively, and a 70% (SD ± 0.1) T-cell recovery. Chronic lymphocytic leukemia samples had lower T-cell numbers, and the separation process was less efficient compared to the ALL. This study demonstrates the use of inertial microfluidics for T-cell enrichment and depletion of B-cell blasts in ALL, suggesting its potential to address a key bottleneck of the chimeric antigen receptor-T manufacturing workflow.

Lactobacillus gasseri KBL697 ameliorates psoriatic inflammation through the modulation of gut-skin axis

Abstract Psoriasis is a chronic inflammatory disease arising from aberrant interactions between various immune cells and cytokines. The current standard therapy includes topical steroids, methotrexate or anti-IL-17A/IL-23 antibodies. However, due to the limited efficacy and associated side effects, there is still an unmet medical need for the development of novel therapeutic agents for this disease. KBL697 is a strain of Lactobacillus gasseri, isolated from the vaginal samples of healthy donors. We previously reported that KBL697 ameliorated the symptoms of an inflammatory bowel disease mouse model. At the molecular level, KBL697 decreased pro-inflammatory cytokines including IFN-gamma, IL-6, and IL-17A, while an anti-inflammatory cytokine IL-10 was increased. In the current study, we tested KBL697 in pre-clinical models for psoriasis. Oral administration of KBL697 showed a significant beneficial effect in imiquimod-induced mouse models, judged by the skin inflammation, PASI score and immune profile change. Further studies revealed that IL-10 is induced by lipoteichoic acid (LTA) of KBL697, indicating the critical role of LTA/IL-10 in the anti-inflammatory function of KBL697. In the skin analysis, key cytokines in psoriasis, such as IL-17A, IL-23, and IL-36, are downregulated while genes for skin regeneration are upregulated, supporting the model for KBL697 working in the gut-skin axis. Therefore, KBL697 has great potential as a novel therapeutic agent for the treatment of psoriasis.

The Dopaminergic Cells in the Median Raphe Region Regulate Social Behavior in Male Mice.

According to previous studies, the median raphe region (MRR) is known to contribute significantly to social behavior. Besides serotonin, there have also been reports of a small population of dopaminergic neurons in this region. Dopamine is linked to reward and locomotion, but very little is known about its role in the MRR. To address that, we first confirmed the presence of dopaminergic cells in the MRR of mice (immunohistochemistry, RT-PCR), and then also in humans (RT-PCR) using healthy donor samples to prove translational relevance. Next, we used chemogenetic technology in mice containing the Cre enzyme under the promoter of the dopamine transporter. With the help of an adeno-associated virus, designer receptors exclusively activated by designer drugs (DREADDs) were expressed in the dopaminergic cells of the MRR to manipulate their activity. Four weeks later, we performed an extensive behavioral characterization 30 min after the injection of the artificial ligand (Clozapine-N-Oxide). Stimulation of the dopaminergic cells in the MRR decreased social interest without influencing aggression and with an increase in social discrimination. Additionally, inhibition of the same cells increased the friendly social behavior during social interaction test. No behavioral changes were detected in anxiety, memory or locomotion. All in all, dopaminergic cells were present in both the mouse and human samples from the MRR, and the manipulation of the dopaminergic neurons in the MRR elicited a specific social response.

Abstract 7657: Detection of KMT2A-PTDs and KMT2A fusions using next generation sequencing

Abstract Introduction: KMT2A (MLL) rearrangements (fusions) is a therapeutic biomarker for Menin inhibitors. KMT2A-PTDs (partial tandem duplications) are considered a prognostic biomarker in myeloid malignancies. KMT2A fusions and PTDs are traditionally detected by RT-qPCR (quantitative real-time PCR). This study investigates KMT2A PTDs levels in healthy donors and myeloid malignancy samples to establish a threshold to report samples with high PTDs, using next generation sequencing (NGS) with OncomineTM Myeloid Assay GX v2. We also report KMT2A fusions in myeloid malignancies. Methods: We sequenced 8483 research samples with known myeloid malignancies at Sonora Quest LaboratoriesTM and at Thermo Fisher ScientificTM. We acquired 20 healthy donor whole blood samples (total 127 replicates) from StanfordTM Blood Center and Discovery Life SciencesTM and sequenced them at 3 different sites of Thermo Fisher ScientificTM. Samples were sequenced with the Ion TorrentTM GenexusTM 6.6 or Ion GeneStudioTM S5 System. They were profiled for 6 different KMT2A-PTD variants and 199 KMT2A fusion isoforms. Results: The mean read length of this data set is 90 - 120 bp and the mean mapped fusion reads is 20,000 - 30,000. KMT2A-PTDs were detected in both healthy donors and myeloid samples. Healthy donor PTD read counts were consistently <2000 and averaged 1/3 of myeloid sample PTD read counts. About 33% of myeloid samples had higher PTD read counts than any healthy donor sample. BLAT (BLAST-Like Alignment Tool) analysis confirmed specific exon matching on the KMT2A gene in both cohorts. Among the 8483 myeloid samples, 162 samples contained a total of 5 unique KMT2A PTDs, and 105 samples contained a total of 30 unique KMT2A fusion isoforms with KMT2A-MLLT1 and KMT2A-MLLT3 being the most prevalent KMT2A fusion gene pairs. Conclusions: We characterize the KMT2A fusions present in myeloid malignant samples. We also describe the abundance of KMT2A PTDs in both healthy donor and myeloid samples, with myeloid cases showing significantly higher PTD read counts. KMT2A PTD read count >2000 is present only in malignant samples but not in healthy donors. This intriguing finding opens opportunities for prospective studies to monitor individuals with elevated PTD levels for myeloid malignancy development and retrospective studies to explore whether healthy donors identified with this alteration years ago after blood donation were subsequently recorded in the national health system with myeloid malignancies. (For research use only. Not for use in diagnostic procedures. © 2023 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Stanford is a trademark of the Board of Trustees of the Leland Stanford Junior University. Discovery Life Sciences is a trademark of Discovery Life Sciences. Sonora Quest Laboratories is a trademark of Sonora Quest Laboratories.) Citation Format: Jiajie Huang, Haigang Gu, Janet Orton, Marina Sedova, Amir Marcovitz, Jennifer Burke, Sarah Brozio, Paul Williams, Scott Myrand, Nate Olowo, Adam Broomer, Brendan Deal, Collyn Seeger, Seth Sadis, Sophie Rozenzhak, Fiona Hyland, Guang Liu. Detection of KMT2A-PTDs and KMT2A fusions using next generation sequencing [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 7657.

Abstract 3669: Clinical application of urinary DNA methylation biomarkers for identifying patients with non-muscle invasive bladder cancer

Abstract Introduction Differences in DNA methylation along cell-free DNA (cfDNA) fragments are known to exist between cancer patients and healthy individuals. Profiling these aberrant methylation patterns in urinary cfDNA (ucfNDA) holds great promise for the non-invasive detection and monitoring of bladder tumors, including non-muscle invasive bladder cancer (NMIBC). Here, we analyzed fragment-level DNA methylation patterns in urine samples obtained prior to repeat transurethral resection of a bladder tumor (reTURBT) from a cohort of NMIBC patients. The results were compared to traditional clinical disease assessment and mutation-based tumor fractions. We found a strong concordance between DNA methylation abnormality and clinical diagnosis, as well as tumor fractions, highlighting the potential of epigenetic alterations as a non-invasive indicator of bladder cancer status. Methods Patients with high-risk NMIBC (n = 30) were prospectively enrolled prior to reTURBT. The PredicineEPIC™ genome-wide methylation analysis and PredicineBEACON™ MRD profiling was performed on pre-repeat TURBT urine samples. Abnormally methylated genomic fragments were detected against a background model built from a panel of plasma samples from healthy donors. Results DNA methylation abnormality was significantly higher (p = 4.8 × 10−6) in samples classified as NIMBC positive by a clinical pathologist (n = 19) compared to negative samples (n = 11), as well as compared to hold-out healthy donor samples (n=24; p = 7.0 × 10−9). Classifying the samples based on a DNA methylation score cut-off derived from healthy donor samples, 24/30 samples showed concordant disease status. Furthermore, DNA methylation abnormality scores correlated positively with mutation-based tumor fractions (r = 0.62), with higher grade samples showing a stronger correlation (r = 0.81; n = 13) than lower grade samples (r = 0.54; n = 17). Conclusion We demonstrate the utility of methylation patterns extracted ucfDNA samples obtained prior to reTURBT to detect and monitor NMIBC. Urinary DNA methylation patterns were highly concordant with clinical pathology status and as well as tumor fractions determined from mutation analysis. Citation Format: Giancarlo Bonora, Ziqi Zhu, Billie Gould, Binggang Xiang, Kemin Zhou, Shidong Jia, Roger Li, Pan Du. Clinical application of urinary DNA methylation biomarkers for identifying patients with non-muscle invasive bladder cancer [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 3669.

Abstract 5023: Analytical validation of the Oncomine™ Dx Express Test using liquid biopsy samples as an IUO assay in a CAP/CLIA lab

Abstract Introduction: The Oncomine™ Dx Express Test (ODxET) is a next-generation sequencing (NGS) test. It can be used as an investigational use only (IUO**) test to detect somatic DNA and RNA alterations in human cell-free total nucleic acid (cfTNA) isolated from plasma samples. This study outlines the analytical validation of the ODxET for relevant single nucleotide variants (SNV) and insertion/deletions (INDEL) in liquid biopsy specimens. The Ion Torrent Genexus™ platform has a short turnaround time with minimal operator input. This study was performed in a College of American Pathologists (CAP™) accredited and Clinical Laboratory Improvement Act (CLIA) licensed laboratory. Methods: Forty-seven unique samples (39 clinical, 3 derived, 2 reference and 3 commercially available DNA controls) were analyzed. All experiments began with sample extraction, except the lower input limit of detection (LLOD). We evaluated limit of detection, variant calling accuracy, and precision. Two control NIST™ samples were sequenced with 3 technical replicates each for INDEL and SNVs analytical accuracy. The LOD was evaluated at both the target input of 2ng/µl (40ng), and the minimum input,0.33ng/µl (6.6ng) with allele frequency (AF) levels at 1%, 0.5%, and 0.25%. The 39 clinical plasma samples were used for variant calling accuracy. Inter-assay reproducibility was assessed by sequencing DNA controls across four runs by different operators over multiple days/systems. Intra-assay repeatability was assessed by sequencing 4 samples in replicate on the same run by one operator on the same day/system. Results: The analytical accuracy and NPV was 100% for SNV/INDEL variants. The 40ng input LOD sensitivity was 96% for SNVs at 0.5% AF and 100% for INDELs at 0.25% Minimum AF. The lower input LOD sensitivity was 89% for SNVs and 100% for INDEL at 1% AF. Variant calling accuracy on samples with pre-characterized data was 100% for the 13 positive samples. Specificity was >99% for all samples including both healthy donor and positive samples. Repeatability was 100% for INDELs and >96% for SNVs, reproducibility was 100% for both variant classes. Conclusion: This validation study to estimate analytical accuracy, limit of detection, variant calling accuracy and precision has demonstrated that the ODxET assay is highly robust in detecting SNV and INDEL mutations in plasma samples and is suitable for use in a CAP/CLIA laboratory as an IUO assay.**=In Vitro Diagnostic use only. Not available in all regions including North America. Citation Format: Juan C. Espinoza, Rajendra Ramsamooj, Akemi Yuki, John Williamson, Diarra Hassell, Madhu Jasti, Suzanne Salazar, David Ginzinger. Analytical validation of the Oncomine™ Dx Express Test using liquid biopsy samples as an IUO assay in a CAP/CLIA lab [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 5023.

Abstract 2302: Innovative blood-based cell-free RNA whole transcriptome sequencing for comprehensive RNA profiling in patient plasma samples and its clinical applications

Abstract Background: Plasma cell-free RNA (cfRNA) holds promise for non-invasive cancer biomarker discovery, particularly in diseases like prostate cancer where specific biomarkers (AR splicing variants and TMPRSS2:ERG fusion) are detectable at the RNA level. Despite its potential, the technical challenges posed by blood cell-originated cfRNA abundance necessitates the development of robust and reliable assays for non-invasive cancer diagnostics. This study establishes and demonstrates the clinical utility of an innovative protocol for isolating and identifying tumor-originated cfRNA in plasma samples. Methods: A novel methodology was developed for the simultaneous extraction of cfDNA and cfRNA from a single blood sample tube, enabling whole transcriptome sequencing (WTS) analysis with minimal input as low as 1ng. A proprietary method was developed to selectively deplete blood cell-originated transcripts in the library. An enrichment-based WTS assay was conducted, and a customized RNASeq analysis pipeline was employed to enable expression profiling and cancer biomarker testing from the cfRNA samples. Results: The developed extraction method yielded substantial amounts of cfDNA and cfRNA from a 4 mL blood sample. Efficient depletion of blood cell-originated transcripts, achieving over 99% removal, enhanced the detection of a broader range of gene expressions in plasma. Implementation of the cfRNA WTS assay across healthy donor and cancer samples demonstrated consistency with documented biomarker status. The assay revealed significant differential clustering between cancer samples and healthy donors, with characteristic cancer genes contributing to the distinction. Reproducibility and analytical sensitivity of the developed cfRNA WTS assay were successfully validated. Conclusions: This study introduces Predicine's proprietary cfRNA WTS assay, enabling concurrent expression profiling and RNA biomarker detection in plasma samples. Overcoming technical challenges associated with blood cell-originated cfRNA, this assay represents a significant advancement in liquid biopsy-based cancer diagnostics. Citation Format: Fang Liu, Yong Huang, Min Wang, Xiangying Sun, Binggang Xiang, Shidong Jia, Pan Du, Shujun Luo. Innovative blood-based cell-free RNA whole transcriptome sequencing for comprehensive RNA profiling in patient plasma samples and its clinical applications [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 2302.

Abstract 4878: Enhancing non-invasive detection of urothelial carcinoma through combined cytology and methylation profiling of urinary cell-free DNA

Abstract Detection of urothelial carcinoma (UC) faces a significant challenge in sensitivity due to the limitations of existing methodologies such as cytology. Identifying tumors through epigenetics pattern presents a promising advancement in diagnostic precision. This study aims to explore a non-invasive and more sensitive strategy by analyzing urinary cell-free DNA (ucfDNA) methylation profiles in conjunction with traditional urine cytology. The cohort for this study were collected from 20 patients with UC and 17 patients with benign or non-tumor lesions which histopathologic and cytopathologic results were reviewed by pathologist. Additional 16 cancer samples and 40 healthy donor samples were collected from independent research centers. We employed PredicineEPIC, a comprehensive whole-genome methylation assay, to investigate the DNA methylation characteristics in urine samples. The dataset was splited into 1:2 proportions for modeling and validation. The methylation signals were fed to three machine learning algorithms for determining the most effective classification model. Differentiating cancer and benign samples through urinary cytology has high specificity (1.0) but low sensitivity (0.3). Then we examined differentially methylated regions and implemented four most abnormally methylated regions as classify features. We found that the Gradient Boosting Machine (GBM) model achieved optimal performance in the validation set, with an AUC of 0.93, sensitivity of 0.81 (21/26), and specificity of 1.0 (36/36). The combination of methylation- and urine cytology-based models raised 2% prediction accuracy without reducing precision, suggesting that this assay could be applied to augment the cytology testing for UC detection. This study proposes a new and promising approach, leveraging cytology combined with epigenetic profiles of urinary cell-free DNA, for the precise diagnosis of urothelial carcinoma. Citation Format: Huan Zhao, Hang Dong, Haoran Tang, Zhihui Zhang, Na Wei, Jingjing Xu, Pan Du, Shidong Jia, Ting Xiao, Huiqin Guo. Enhancing non-invasive detection of urothelial carcinoma through combined cytology and methylation profiling of urinary cell-free DNA [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 4878.

High CD62L expression predicts the generation of chimeric antigen receptor T cells with potent effector functions.

Efficient generation of chimeric antigen receptor (CAR) T cells is highly influenced by the quality of apheresed T cells. Healthy donor-derived T cells usually proliferate better than patients-derived T cells and are precious resources to generate off-the-shelf CAR-T cells. However, relatively little is known about the determinants that affect the efficient generation of CAR-T cells from healthy donor-derived peripheral blood mononuclear cells (PBMC) compared with those from the patients' own PBMC. We here examined the efficiency of CAR-T cell generation from multiple healthy donor samples and analyzed its association with the phenotypic features of the starting peripheral blood T cells. We found that CD62L expression levels within CD8+ T cells were significantly correlated with CAR-T cell expansion. Moreover, high CD62L expression within naïve T cells was associated with the efficient expansion of T cells with a stem cell-like memory phenotype, an indicator of high-quality infusion products. Intriguingly, genetic disruption of CD62L significantly impaired CAR-T cell proliferation and cytokine production upon antigen stimulation. Conversely, ectopic expression of a shedding-resistant CD62L mutant augmented CAR-T cell effector functions compared to unmodified CAR-T cells, resulting in improved antitumor activity in vivo. Collectively, we identified surface expression of CD62L as a concise indicator of potent T cell proliferation. CD62L expression is also associated with functional properties of CAR-T cells. These findings are potentially applicable to selecting optimal donors to massively generate CAR-T cell products.

Microplastics in human urine: Characterisation using μFTIR and sampling challenges using healthy donors and endometriosis participants

Microplastics (MPs) are found in all environments, within the human food chain, and have been recently detected in several human tissues. The objective herein was to undertake an analysis of MP contamination in human urine samples, from healthy individuals and participants with endometriosis, with respect to their presence, levels, and the characteristics of any particles identified. A total of 38 human urine samples and 15 procedural blanks were analysed. MPs were characterised using μFTIR spectroscopy (size limitation of 5 μm) and SEM-EDX. In total, 123 MP particles consisting of 22 MP polymer types were identified within 17/29 of the healthy donor (10 mL) urine samples, compared with 232 MP particles of differing 16 MP polymer types in 12/19 urine samples from participants with endometriosis. Healthy donors presented an unadjusted average of 2589 ± 2931 MP/L and participants with endometriosis presented 4724 ± 9710 MP/L. Polyethylene (PE)(27%), polystyrene (PS)(16%), resin and polypropylene (PP)(both 12%) polymer types were most abundant in healthy donor samples, compared with polytetrafluoroethylene (PTFE) (59%), and PE (16%) in samples from endometriosis participants. The MP levels within healthy and endometriosis participant samples were not significantly different. However, the predominant polymer types varied, and the MPs from the metal catheter-derived endometriosis participant samples and healthy donors were significantly smaller than those observed in the procedural blanks. The procedural blank samples comprised 62 MP particles of 10 MP polymer types, mainly PP (27%), PE (21%), and PS (15%) with a mean ± SD of 17 ± 18, highlighting the unavoidable contamination inherent in measurement of MPs from donors. This is the first evidence of MP contamination in human urine with polymer characterisation and accounting for procedural blanks. These results support the phenomenon of transport of MPs within humans, specifically to the bladder, and their characterisation of types, shapes and size ranges identified therein.

Size-exclusion chromatography combined with DIA-MS enables deep proteome profiling of extracellular vesicles from melanoma plasma and serum

Extracellular vesicles (EVs) are important players in melanoma progression, but their use as clinical biomarkers has been limited by the difficulty of profiling blood-derived EV proteins with high depth of coverage, the requirement for large input amounts, and complex protocols. Here, we provide a streamlined and reproducible experimental workflow to identify plasma- and serum- derived EV proteins of healthy donors and melanoma patients using minimal amounts of sample input. SEC–DIA-MS couples size-exclusion chromatography to EV concentration and deep-proteomic profiling using data-independent acquisition. From as little as 200 µL of plasma per patient in a cohort of three healthy donors and six melanoma patients, we identified and quantified 2896 EV-associated proteins, achieving a 3.5-fold increase in depth compared to previously published melanoma studies. To compare the EV-proteome to unenriched blood, we employed an automated workflow to deplete the 14 most abundant proteins from plasma and serum and thereby approximately doubled protein group identifications versus native blood. The EV proteome diverged from corresponding unenriched plasma and serum, and unlike the latter, separated healthy donor and melanoma patient samples. Furthermore, known melanoma markers, such as MCAM, TNC, and TGFBI, were upregulated in melanoma EVs but not in depleted melanoma plasma, highlighting the specific information contained in EVs. Overall, EVs were significantly enriched in intact membrane proteins and proteins related to SNARE protein interactions and T-cell biology. Taken together, we demonstrated the increased sensitivity of an EV-based proteomic workflow that can be easily applied to larger melanoma cohorts and other indications.