Plasma Of Healthy Donors Research Articles

Prognostic significance of soluble forms of the PD-1 receptor and its ligand PD-L1 in gastric cancer

Introduction. Despite advances in the diagnosis and drug therapy of some cancers over the past decade, solid tumors, particularly gastric cancer, still have a poor prognosis and remain a leading cause of death worldwide. Therefore, the development of methods for timely diagnosis, identification of new targets for therapy and biochemical prognosis factors is an urgent problem in clinical oncology. In recent years, the focus of clinical attention has been on immune checkpoint receptors and ligands that can identify patients for immunotherapy. The clinical and prognostic significance of the levels of soluble forms of the programmed cell death receptor PD-1 and its ligand PD-L1 in the blood of patients with gastric cancer is currently not fully determined.Aim. Comparative study of the levels of sPD-1 and sPD-L1 in the blood plasma of healthy donors and patients with gastric cancer, taking into account the prevalence of the tumor process and the prognosis of overall survival.Materials and methods. The study included 100 patients with stomach cancer aged from 25 to 81 years (57 men, 43 women), who underwent examination and treatment at the N. N. Blokhin National Medical Research Center for Oncology. The concentration of sPD-L1 and sPD-1 was determined in blood plasma obtained according to standard methods before the start of specific treatment, using reagent kits for enzyme-linked immunosorbent assay “Human PD-L1 Platinum ELISA” and “Human PD-1 ELISA kit” (Affimetrix, eBioscience, USA) in accordance with the manufacturer’s instructions. The content of markers was expressed in picograms (pg) per 1 ml of blood plasma. To compare indicators and analyze their relationships, the nonparametric Mann–Whitney test was used. Overall survival analysis was performed using the Kaplan–Meier method. For all statistical tests, p values <0.05 were considered statistically significant.Results. The analysis did not reveal a connection at a threshold level of sPD-1 of 8.0 pg / ml in the blood plasma of patients with gastric cancer with overall survival rates (p = 0.59). However, an additional analysis conducted in a group of patients with gastric cancer with stages I–II demonstrated that a marker level ≥8.0 pg / ml is a favorable prognostic factor (p = 0.0039), while in advanced stages III–IV the disease it has no prognostic significance. There was no significant correlation between sPD-L1 concentrations in the blood plasma of patients with gastric cancer and overall survival rates (p = 0.35), however, the best long-term results were found at a threshold level of marker concentrations in blood plasma <35 pg / ml.Conclusion. An sPD-1 level ≥8.0 pg / ml can serve as a favorable prognostic factor in stages I–II of gastric cancer, while in advanced stages III–IV of the disease it has no prognostic significance. The prognostic role of sPD-L1 in patients with gastric cancer has not been identified. The study needs to be continued in combination with additional predictive biomarkers for gastric cancer.

TGIRT-seq of Inflammatory Breast Cancer Tumor and Blood Samples Reveals Widespread Enhanced Transcription Impacting RNA Splicing and Intronic RNAs in Plasma.

Inflammatory breast cancer (IBC) is the most aggressive and lethal breast cancer subtype but lacks unequivocal genomic differences or robust biomarkers that differentiate it from non-IBC. Here, Thermostable Group II intron Reverse Transcriptase RNA-sequencing (TGIRT-seq) revealed myriad differences in tumor samples, Peripheral Blood Mononuclear Cells (PBMCs), and plasma that distinguished IBC from non-IBC patients and healthy donors across all tested receptor-based subtypes. These included numerous differentially expressed protein-coding gene and non-coding RNAs in all three sample types, a granulocytic immune response in IBC PBMCs, and over-expression of antisense RNAs, suggesting wide-spread enhanced transcription in both IBC tumors and PBMCs. By using TGIRT-seq to quantitate Intron-exon Depth Ratios (IDRs) and mapping reads to both genome and transcriptome reference sequences, we developed methods for parallel analysis of transcriptional and post-transcriptional gene regulation. This analysis identified numerous differentially and non-differentially expressed protein-coding genes in IBC tumors and PBMCs with high IDRs, the latter reflecting rate-limiting RNA splicing that negatively impacts mRNA production. Mirroring gene expression differences in tumors and PBMCs, over-represented protein-coding gene RNAs in IBC patient plasma were largely intronic RNAs, while those in non-IBC patients and healthy donor plasma were largely mRNA fragments. Potential IBC biomarkers in plasma included T-cell receptor pre-mRNAs and intronic, LINE-1, and antisense RNAs. Our findings provide new insights into IBC and set the stage for monitoring disease progression and response to treatment by liquid biopsy. The methods developed for parallel transcriptional and post-transcriptional gene regulation analysis have potentially broad RNA-seq and clinical applications.

Beneficial effects of IVIG treatment on experimental-induced osteoporosis.

Estrogen (E2) plays a significant role in postmenopausal osteoporosis, and its deficiency is related to chronic low-grade inflammation. Intravenous immunoglobulin (IVIG) is composed of immunoglobulins derived from the plasma of healthy donors. Numerous anti-inflammatory pathways are responsible for IVIG's anti-inflammatory action The aim of this study is to investigate the effects of IVIG on experimental-induced osteoporosis. Forty adult female Wistar rats were included in the study. Thirty rats underwent bilateral dorsal ovariectomy. Rats were grouped as Group 1 (n = 10, ovariectomy and saline); Group 2 (n = 10, ovariectomy and E2); Group 3 (n = 10, ovariectomy and IVIG), and Control group (n = 10, no oophorectomy). Histopathological examination of bone tissue, and biochemical analysis for beta-catenin, plasma Tumor Necrosis Factor-α, IL-6, receptor activator of nuclear-κB ligand (RANKL), and osteoprotegerin (OPG) levels were made. The IVIG group had increased trabecular number, area, and thickness with increased bone mineral density as well as decreased trabecular separation compared with the saline group. IVIG group had lower serum RANKL and higher serum OPG levels when compared with the saline group. The bone marrow beta-catenin level was significantly higher in the control and ovariectomy + IVIG groups. IVIG has beneficial effects on experimentally induced osteoporosis with a possible action on inflammation and RANKL-β-catenin pathway.

Influence of the Immune Checkpoint Inhibitors on the Hemostatic Potential of Blood Plasma

Introduction: Immune checkpoint inhibitors (ICIs) have revolutionized classical treatment approaches of various cancer entities, but are also associated with a number of side effects. One of these may be life-threatening clotting disorders with the risk of thrombotic or hemorrhagic complications, the mechanisms of which are still poorly understood. In the present study, we analyzed the direct effects of pembrolizumab, nivolumab, and ipilimumab on platelet aggregation as well as plasma coagulation followed by fibrinolysis in an ex vivo model. Methods: Microplate spectrometry was used to analyze aggregation, coagulation, and fibrinolysis in platelet-free (PFP) and platelet-rich (PRP) healthy donor plasma samples treated with pembrolizumab, nivolumab, ipilimumab, and appropriate isotype controls. Aggregation was induced by TRAP-6. Clotting of PFP and PRP followed by lysis was initiated with a tissue factor in a mixture of phosphatidylserine:phosphatidylcholine and the addition of t-PA. Among other parameters, the area under the curve (AUC) was used to compare the effect of ICIs on aggregation, coagulation, and fibrinolysis. Results: Upon direct contact with platelets, pembrolizumab stimulated platelet aggregation in PRP, while nivolumab and ipilimumab promoted disaggregation with corresponding changes in the AUC. Pembrolizumab and nivolumab, both PD-1 receptor inhibitors, had no effect on the plasma coagulation cascade. Ipilimumab, a CTLA-4 receptor inhibitor, significantly increased the rate of PRP clotting. When clotting was followed by lysis, all ICIs were found to prolong the growth of the PRP-derived fibrin clot and delay its elimination. This was manifested by an increase in AUC relative to control PRP. Conclusion: This study characterizes the potential impact of pembrolizumab, nivolumab, and ipilimumab on hemostasis. Nivolumab and ipilimumab are able to reduce aggregation and increase the procoagulant properties of platelets, which can cause side effects associated with hemostatic imbalance leading to thrombosis or bleeding. The observed ICI-specific effects may contribute to our understanding of the mechanisms by which ICI affects platelets and suggest how, in a clinical setting, to reduce coagulation disorders during ICI treatment in the future.

Size-Exclusion Chromatography: A Path to Higher Yield and Reproducibility Compared to Sucrose Cushion Ultracentrifugation for Extracellular Vesicle Isolation in Multiple Myeloma.

Small extracellular vesicles (EVs) play a pivotal role in intercellular communication across various physiological and pathological contexts. Despite their growing significance as disease biomarkers and therapeutic targets in biomedical research, the lack of reliable isolation techniques remains challenging. This study characterizes vesicles that were isolated from conditioned culture media (CCM) sourced from three myeloma cell lines (MM.1S, ANBL-6, and ALMC-1), and from the plasma of healthy donors and multiple myeloma patients. We compared the efficacy, reproducibility, and specificity of isolating small EVs using sucrose cushion ultracentrifugation (sUC) vs. ultrafiltration combined with size-exclusion chromatography (UF-SEC). Our results demonstrate that UF-SEC emerges as a more practical, efficient, and consistent method for EV isolation, outperforming sUC in the yield of EV recovery and exhibiting lower variability. Additionally, the comparison of EV characteristics among the three myeloma cell lines revealed distinct biomarker profiles. Finally, our results suggest that HBS associated with Tween 20 improves EV recovery and preservation over PBS. Standardization of small EV isolation methods is imperative, and our comparative evaluation represents a significant step toward achieving this goal.

Balancing Donor Health and Plasma Collection: A Systematic Review of the Impact of Plasmapheresis Frequency

Most plasma used for manufacturing plasma-derived medicinal products (PDMPs) such as albumin, immunoglobulin (Ig), and clotting factors is obtained from source plasma collected via plasmapheresis, the majority of which is contributed by the United States (US). While the demand for PDMPs continues to rise, it remains unclear whether high-frequency plasmapheresis, such as the twice-weekly plasma donation allowed in the US, may have any (long-term) adverse health effects on the donor. To investigate the frequency at which plasma can be donated without harm to the donor, the current systematic review explores the impact of plasma donation frequency on cardiovascular health, protein depletion, and adverse events in healthy plasma donors. We asked the following research question: What is the impact of plasmapheresis frequency (Intervention) on the safety or health (Outcome) of healthy donors (Population)? Six databases (PubMed, Embase, Web of Science, CINAHL, the Cochrane Library, and Transfusion Evidence Library), 2 clinical trial registries (ICTRP and clinicaltrials.gov), and the PROSPERO database were searched. Four observational and 2 experimental studies were included. The results showed that very high-frequency donation (twice per week) may result in a clinically relevant decrease in ferritin and bring IgG levels below the lower threshold of 6 g/l. However, the evidence is of low to very low certainty, and solid conclusions are hindered by the healthy donor effect and methodological limitations of the included studies. To determine a safe threshold donation frequency that minimizes any possible harmful effect on the donor, more high-quality prospective cohort studies and experimental studies are needed. We should expedite such studies to support recommendations, as conclusive evidence confirming or refuting the safety of maximum allowed donation frequencies is lacking. Donor protection is essential, given that healthy donors receive no direct medical benefit from donating plasma.

Functionalized magnetic nanoparticles remove donor-specific antibodies (DSA) from patient blood in a first ex vivo proof of principle study

The presence of donor-specific antibodies (DSA) such as antibodies directed against donor class I human leucocyte antigen (e.g., HLA-A) is a major barrier to kidney transplant success. As a proof of concept, functionalized magnetic nanoparticles have been designed to eliminate DSA from saline, blood and plasma of healthy donors and sensitized patients. Specific HLA-A1 protein was covalently bound to functionalized cobalt nanoparticles (fNP), human serum albumin (HSA) as control. fNP were added to anti-HLA class I-spiked saline, spiked volunteers’ whole blood, and to whole blood and plasma of sensitized patients ex vivo. Anti-HLA-A1 antibody levels were determined with Luminex technology. Antibodies' median fluorescent intensity (MFI) was defined as the primary outcome. Furthermore, the impact of fNP treatment on blood coagulation and cellular uptake was determined. Treatment with fNP reduced MFI by 97 ± 2% and by 94 ± 4% (p < 0.001 and p = 0.001) in spiked saline and whole blood, respectively. In six known sensitized anti-HLA-A1 positive patients, a reduction of 65 ± 26% (p = 0.002) in plasma and 65 ± 33% (p = 0.012) in whole blood was achieved. No impact on coagulation was observed. A minimal number of nanoparticles was detected in peripheral mononuclear blood cells. The study demonstrates—in a first step—the feasibility of anti-HLA antibody removal using fNP. These pilot data might pave the way for a new personalized DSA removal technology in the future.

Optimization of ultracentrifugation-based method to enhance the purity and proteomic profiling depth of plasma-derived extracellular vesicles and particles.

Circulating extracellular vesicles and particles (EVPs) are being investigated as potential biomarkers for early cancer detection, prognosis, and disease monitoring. However, the suboptimal purity of EVPs isolated from peripheral blood plasma has posed a challenge of in-depth analysis of the EVP proteome. Here, we compared the effectiveness of different methods for isolating EVPs from healthy donor plasma, including ultracentrifugation (UC)-based protocols, phosphatidylserine-Tim4 interaction-based affinity capture (referred to as "PS"), and several commercial kits. Modified UC methods with an additional UC washing or size exclusion chromatography step substantially improved EVP purity and enabled the detection of additional proteins via proteomic mass spectrometry, including many plasma membrane and cytoplasmic proteins involved in vesicular regulation pathways. This improved performance was reproduced in cancer patient plasma specimens, resulting in the identification of a greater number of differentially expressed EVP proteins, thus expanding the range of potential biomarker candidates. However, PS and other commercial kits did not outperform UC-based methods in improving plasma EVP purity. PS yielded abundant contaminating proteins and a biased enrichment for specific EVP subsets, thus unsuitable for proteomic profiling of plasma EVPs. Therefore, we have optimized UC-based protocols for circulating EVP isolation, which enable further in-depth proteomic analysis for biomarker discovery.

&lt;i&gt;LINE-1&lt;/i&gt; hypomethylation and &lt;i&gt;HIST1H4F&lt;/i&gt; hypermethylation as oncomarkers in liquid biopsy of colorectal cancer

Introduction. Local hypermethylation of gene promoters and global genome hypomethylation are well-known manifestations of aberrant methylation associated with carcinogenesis. We investigated this phenomenon as a possible diagnostic marker for liquid biopsy of colorectal cancer using the original quantitative DNA melting analysis with hybridiza-tion probes (qDMA-HP) method. Aim. To quantify the methylation of HIST1H4F promoter and LINE-1 transposon in circulating blood plasma DNA of colorectal cancer patients. Materials and methods. Bisulfite-treated DNA samples isolated from blood plasma of healthy donors and cancer patients were analyzed. HIST1H4F methylation was assessed by asymmetric polymerase chain reaction with hybridized probe and post-amplification melting of probe / amplicon hybrids. To test for repetitive and highly polymorphic LINE-1 sequences, asymmetric polymerase chain reaction with hybridized probe and SYBR Green intercalating dye was used, followed by melting of hybrids and analysis of multicomponent melt curves. Results. High diagnostic efficiency of LINE-1 and HIST1H4F methylation markers in liquid biopsy of colorectal cancer was demonstrated with the area under the ROC curve = 0.92, sensitivity – 100 %, specificity – 84 %. Cross validation supports this result. Hypermethylation of HIST1H4F and hypomethylation of LINE-1 are statistically significantly correlated (Spearman correlation coefficient r = 0.4; p = 0.01). Conclusion. The qDMA-HP is suitable for quantitative assessment of aberrant methylation of various clinically significant genes.

Abstract PO4-04-08: Design and Development of the APIS ESR1 Mutations Kit to Detect Eleven Mutations Relevant to Acquired Endocrine Therapy Resistance

Abstract Introduction: Estrogen receptor 1 (ER/ESR1) mutations have arisen as key biomarkers for endocrine therapy resistance in ER positive (ER+) breast cancer (BC) patients: detecting these mutations is key to guiding researchers to better understand acquired resistance during treatment. Ongoing clinical trials, which explore the prevalence of ESR1 mutations under various treatments, commonly use next-generation sequencing (NGS)-based assays. However, the NGS panels available are costly, can have long turnaround times and require specialist equipment and software. Research use only digital PCR assays are also available but have similar drawbacks to NGS. A targeted qPCR-based assay is more time and cost effective, and does not require specialist equipment. Here, we demonstrate the APIS ESR1 Mutations Kit, a targeted, sensitive, and robust qualitative qPCR assay able to detect eleven ESR1 mutations across ESR1 exons 5 (E380Q), 7 (S463P) and 8 (P535H, L536R, L536Q, L536H, L536P, Y537C, Y537S, Y537N and D538G). The assay is designed to assess circulating-free DNA (cfDNA) samples, reducing the need for invasive procedures in clinical settings and can make use of standard cfDNA extraction kits. Therefore, the APIS ESR1 Mutations Kit provides the flexibility for researchers in any conventional molecular biology laboratory to explore ESR1 mutations with high sensitivity and specificity. Methods: The APIS ESR1 Mutations Kit provides all components and controls required to assess samples. For kit development, mutation-specific DNA fragments were spiked into wildtype background DNA (either cfDNA extracted from pooled healthy donor plasma, human genomic DNA, or DNA fragments); the Limit of Blank (LoB) and Limit of Detection (LoD) were determined using these samples. To determine linearity, a dilution series of DNA fragments ranging from 5 to 10,000 copies per reaction was assessed. All PCR runs were performed using a QuantStudio™5 Dx (ThermoFisher) instrument. Results: The performance studies showed the APIS ESR1 Mutations Kit can detect D538G at 0.4 % mutant allele frequency (MAF) and Y537S at 0.06 % MAF. All other mutations can be detected at ≤ 1.0 % MAF. By using clamp and blocking technologies, each design is mutation specific, enabling identification of specific amino acid mutations. In addition, the assays can perform in high wild-type backgrounds, enabling lower limit of blanks with ≥95% confidence and threshold cut-offs to enhance the sensitivity of the assay. The linearity of each target is within 90-110% from 50 to 10,000 copies per reaction. Conclusions: The ESR1 Mutations Kit demonstrated high sensitivity and performance as a qualitative qPCR assay to detect eleven ESR1 mutations. Table. Mutations detected with the APIS ESR1 Mutations Kit Citation Format: Anna Gasior, Colette Whitfield, Anita Campbell, Christine Hoy, Sally Holdsworth, Jesse Mukose, Ryan Nana, Sahar Shammakhi, Benjamin Scales, Joanna Gorniak. Design and Development of the APIS ESR1 Mutations Kit to Detect Eleven Mutations Relevant to Acquired Endocrine Therapy Resistance [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-04-08.

Abstract 5040: Evaluation of commercially available cell free DNA extraction kits using biosynthetic reference materials

Abstract Extraction of cell free DNA (cfDNA) from a patient’s blood sample is a crucial preanalytic step of the liquid biopsy process. Whether testing is being performed for early disease diagnosis, therapy selection, or disease and treatment surveillance, the input material must be of sufficient quantity as well as high quality to return informative data. In addition, accurate capture of the full cfDNA content from a sample is necessary to evaluate tumor growth or recurrence. Fragmentomics is an emerging field that can indicate tumor origin among other information, and again, accurate and precise cfDNA content is critical. The cfDNA content in patient plasma is highly variable, and sample mass is quite limited compared to that of resected tumors. Healthy donor plasma, though abundant, varies in cfDNA levels batch to batch and should not contain any somatic variants relevant to liquid biopsy assay development or validation. Despite the challenges associated with cfDNA isolation and ctDNA analysis there are few reference materials available to optimize laboratory process for these preanalytic challenges. Here we evaluate various commercially available extraction kits using novel Seraseq® cfDNA Extraction Reference Materials. Genomic DNA from the GM24385 cell line was blended with 4 biosynthetic DNA EGFR variants targeted at 1% allele frequency and verified using the Bio-Rad QX-200 Droplet Digital PCR (ddPCR) System. The mixed DNA was fragmented and size selected to create ctDNA-like size profiles. The ctDNA was mixed into a synthetic plasma matrix to simulate native cfDNA samples and diluted to three distinct concentrations (20, 50, and 80 ng/mL). Verification of concentration was performed using the QIAGEN QIAamp Circulating Nucleic Acid Kit for extraction. The final materials were also extracted with other kits: the Maxwell RSC ccfDNA Plasma Kit, the MagMAX Cell-Free DNA Isolation kit, the Zymo MAGicBead™ cfDNA Isolation Kit, and the Takara NucleoSnap cfDNA Kit. Yields were measured with Qubit dsDNA BR assay and EGFR variants were measured using Bio-Rad ddPCR. Other sites evaluated the materials as well, using various kits. The kits showed variable yields, but there was a clear difference between the cfDNA extracted from each reference material correlating with the intended increasing concentration. The EGFR variants were all present at ~1% regardless of the cfDNA concentration and the kit used for extraction. Seraseq ctDNA Plasma Reference Materials serve to evaluate extraction of cfDNA in liquid biopsy testing workflows without having to source donor plasma, which is limited and variable in cfDNA content. The consistency and availability of these biosynthetic materials allow for optimization without risking patient samples. Citation Format: Dana Ruminski Lowe, Sanchita Jamindar, Andrew Anfora, David Merriam, Catherine Huang, Russell Garlick, Bharathi Anekella. Evaluation of commercially available cell free DNA extraction kits using biosynthetic reference materials [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 5040.

Abstract 6094: Detection of MRD assessment with the Personalis NeXT Personal assay using MATRIX plasma-in-plasma contrived samples

Abstract Detection of circulating tumor DNA (ctDNA) has been shown to correlate with clinical outcome of oncology patients. Molecular residual disease (MRD) profiling by ctDNA is being rapidly deployed. Current tumor-informed MRD tests assess a relatively small number of personalized variants, placing limits on their detection sensitivity and ultimately leading to false-negative results due to insufficient assay limit of detection (LOD). Here we report a performance evaluation of the Personalis NeXT Personal tumor-informed MRD assay, using a novel design for contrived samples that are highly commutable to clinical samples, enabling robust assessment of these emerging MRD assays. Novel samples were built from commercially acquired matched tumor and plasma samples, across different indications. The cancer patient plasma was diluted into a background of healthy donor plasma to create the 72 plasma-in-plasma samples in each study (total of 144). Personalis performed WGS on the matched solid tumor and normal samples from each patient. ~1800 somatic variants were selected for each patient. A personalized panel was designed and used to enrich for the selected targets in the individual plasma samples. An aggregated signal across MRD targets was evaluated to determine the presence of ctDNA in each plasma sample. MATRIX 1 panels averaged 1863 variants (range: 1818 to 1888) selected for MRD tracking. With 152 clinically relevant variants and additional assay components, an average of 2206 variants were assessed per patient. MATRIX 2 panels averaged 1836 variants (range: 1819 to 1867) selected for MRD tracking. In the MATRIX 1 study, tumor signal was detected in all analyzed samples, including 9 samples at 0.002% tumor plasma dilution, with an assay sensitivity of 100%. The MATRIX 2 study showed tumor signal detected in 14 of 16 samples at 0.001% tumor plasma dilution. The 2 false negatives were reported at the 0.001% tumor plasma dilution. Thus, the assay sensitivity was 87.5% at the lowest dilution (0.001%) and 96.8% overall. The MATRIX plasma-in-plasma approach is a robust option for assessing the analytical sensitivity of MRD assays to low levels of ctDNA. This approach addresses all parts of an MRD assay for both the plasma and tumor/normal tissues, utilizing real clinical samples while allowing direct interrogation of sensitivity. The Personalis NeXT Personal assay shows ultra-high sensitivity with reproducible data down to the 1-3 PPM range. Citation Format: Paul Labrousse, Hugh Russell, Sobia Hamid, Gabor Bartha, John Lyle, Dan Norton, Josette Northcott, Sean Michael Boyle, Richard Chen, Dan Stetson, James Hadfield. Detection of MRD assessment with the Personalis NeXT Personal assay using MATRIX plasma-in-plasma contrived samples [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 6094.

Abstract 5016: Building better contrived samples to properly evaluate Next-generation MRD and LBx assays

Abstract Circulating tumor DNA (ctDNA) is a component of cell-free DNA (cfDNA) that originates from tumor tissue in the plasma of cancer patients. Although ctDNA is a broadly applicable tumor biomarker, the absolute amount of ctDNA varies across indication and stage, with the level of ctDNA present reflective of tumor volume. As novel methods for the characterization of ctDNA develop, there is need to evaluate these assays with appropriate samples. But current commercial reference standards, generated from modified cell lines or oligonucleotides lack important features such as DNA methylation, fragmentation pattern and nucleosome positioning, whilst clinical trial samples lack the required volume to perform multiple tests and often lack proper consent. We have created a framework for generating contrived samples containing the biological features of clinical samples and the attributes of a reference standard. The plasma-in-plasma contrived samples are generated from commercially purchased and fully consented matched tumor and plasma from cancer patients with age and gender matched healthy donor plasma. Samples are characterized by an internally validated 700 gene NGS panel to identify somatic variants and copy number alterations, which establishes ‘ground truth’. A contrived plasma dilution range based on the mean variant allele frequency (VAF) of the concordant somatic variants identified is generated. Using these methods, we have been able to generate sample sets for pan-cancer and disease specific evaluations. We have tested these samples through many novel high sensitivity assays that utilize both tumor-informed and tumor agnostic methods. We have confirmed that plasma-in-plasma contrived samples are amenable to all evaluated assays to date. The FFPE tumor tissue provide results matching clinical trial samples and the extraction yields of cfDNA are within the range of plasma samples from clinical trial data. Each dilution can be generated with replicates for reproducibility metrics. Current models of the sample dilutions have been generated to increase the number of replicates at relevant levels of tumor burden (i.e., 0.01% VAF). Negative samples consisting of the background plasma were also generated for each sample used. These plasma-in-plasma contrived samples have greater commutability to clinical samples than other commercial reference standards, however there are limitations. The complexity of generating these samples restrict the number of unique patients that can be utilized. Also, due to the volume of healthy plasma used as diluent, it is difficult to generate volumes greater than 4ml. To our knowledge, this is the first report examining the generation and usage of a contrived sample set that can be applied to evaluating novel high sensitivity MRD assays with clinical sample commutability. Citation Format: Paul Labrousse, Hugh Russell, David Shera, Daniel Stetson, Barrett Nuttall, Brian Dougherty, Darren Hodgson, James Hadfield. Building better contrived samples to properly evaluate Next-generation MRD and LBx assays [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 5016.

Abstract 323: Optimization of a liquid biopsy assay to accommodate low cfDNA samples and increase throughput

Abstract Molecular testing that interrogates cell free DNA (cfDNA) from plasma has significant potential value across the cancer care continuum, including early cancer detection, driver mutation identification, and disease recurrence monitoring. To achieve this potential, liquid biopsy assays must be optimized to address multiple challenges. For one, cfDNA quantity in the blood is highly variable, with different amounts present depending on malignancy and stage of progression. This requires optimization to detect low concentrations of circulating tumor DNA (ctDNA) and to accommodate variable tumor fractions. In addition, minimizing assay costs will improve test accessibility and adoption. To address these issues, Genece Health is optimizing their lung cancer liquid biopsy assay to accommodate minimal cfDNA input into library preparation. Furthermore, Genece is testing algorithms that tolerate reduced read depths from low-pass whole genome sequencing (LP-WGS) while maintaining high performance. The Genece lung cancer liquid biopsy assay profiles fragment end-motif and size (FEMS) and fragment coverage signals using LP-WGS (Illumina) and proprietary machine learning algorithms. This study first tested the frequency of end motifs in pooled ctDNA after modifying library preparation (IDT) to reduce reagent volumes from 1X down to 0.5X, 0.25X, and 0.1X of manufacturer specification. A range of cfDNA input from 0.25 ng to 6 ng was tested with each library preparation volume. In addition, LP-WGS read coverage from 5X to 1X was evaluated to assess impact on end motif frequency and assay sensitivity. Performance characterization was further evaluated with a cohort of 50 clinical lung cancer and 100 healthy donor plasma samples tested at 1X and 0.25X library reagent volume. We observed an expected increase in library complexity at higher cfDNA input and library reagent volume. Assay performance was minimally impacted by variable cfDNA input and library reagent volume. However, reducing sequencing read depth is tolerated down to 1.25X depth before increasing the variability of end motif frequencies. These observations are corroborated in the 150 clinical sample cohort. This data shows that by altering standard library preparation conditions and reducing unique read depth, liquid biopsy assays can accommodate a wider range of cfDNA, while concurrently decreasing overall costs. Citation Format: Michael Salmans, Dasom Kim, Andrew Carson, Mengchi Wang, Byung In Lee, Bryan Leatham. Optimization of a liquid biopsy assay to accommodate low cfDNA samples and increase throughput [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 323.

Abstract 4788: Noninvasive longitudinal monitoring of residual disease in chemotherapy-treated colorectal cancer patients

Abstract Noninvasive monitoring of cancer shows great promise in assessing therapy response and improving patient outcomes. Recently, various groups have developed methods that detect circulating tumor DNA (ctDNA) in the plasma to measure minimal or molecular residual disease (MRD). Aberrant DNA methylation patterns are a hallmark of cancers, and robust signals can be detected by sensitive ctDNA assays. Here, we present a methylation-based approach for longitudinal monitoring of tumor burden that is tumor-naive, i.e., with no reliance on prior characterization of tumor molecular characteristics. We assess the effectiveness of our approach in a cohort of colorectal cancer (CRC) patients receiving chemotherapy with or without additional targeted agents. Longitudinal blood samples were collected from patients (n = 57) while on therapy, which averaged 4.9 months, for a total of 239 sample collection time points (median per patient = 5). Based on RECIST status, 16 patients were classified as complete responders (CR) at the end of treatment. We generated plasma cell free DNA-derived libraries for methylation sequencing targeting regions relevant to CRC. Next, we computed a disease burden score for each sample and related these scores to clinical response (CR vs. non-CR). We trained a classifier on an independent cohort of CRC and healthy donor plasma cfDNA samples, and used this classifier to check for residual disease at the end of treatment for each of our 57 patients. The classifier detected residual disease in only 3/16 CRs (19%) but 35/41 non-CRs (85%). Two of the non-CR patients were initially assessed as CRs at earlier time points. In both cases, our method successfully detected residual disease at or prior to the CR assessment and consistently showed residual disease in all follow up samples. This suggests improved sensitivity relative to RECIST and highlights the potential of using noninvasive blood tests for continuous monitoring of CRC patients receiving therapy. Citation Format: Alison D. Tang, Rebecca Gupte, Victoria Cheung, Tao Qing, Austin Cauwels, Emily Leff, Kimberly Walter, Ehsan Tabari, Alex Lovejoy, Jimmy C. Lin. Noninvasive longitudinal monitoring of residual disease in chemotherapy-treated colorectal cancer patients [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 4788.

Topographic Distribution of miRNAs (miR-30a, miR-223, miR-let-7a, miR-let-7f, miR-451, and miR-486) in the Plasma Extracellular Vesicles.

There are many articles on the quantitative analysis of miRNAs contained in a population of EVs of different sizes under various physiological and pathological conditions. For such analysis, it is important to correctly quantify the miRNA contents of EVs. It should be considered that quantification is skewed depending on the isolation protocol, and different miRNAs are degraded by nucleases with different efficiencies. In addition, it is important to consider the contribution of miRNAs coprecipitating with the EVs population, because the amount of miRNAs in the EVs population under study is skewed without appropriate enzymatic treatment. By studying a population of EVs from the blood plasma of healthy donors, we found that the absolute amount of miRNA inside the vesicles is commensurate with the amount of the same type of miRNA adhered to the outside of the EVs. The inside/outside ratio ranged from 1.02 to 2.64 for different investigated miRNAs. According to our results, we propose the hypothesis that high occupancy of miRNAs on the outer surface of EVs influence on the transporting RNA repertoire no less than the inner cargo received from the host cell.

Development and application of an ultra-sensitive ctDNA mutation profiling assay in monitoring therapy response and drug resistance.

264 Background: Next-generation sequencing (NGS) assays offer efficient high-throughput profiling methods for analyzing circulating tumor DNA (ctDNA) in blood or other liquid biopsy specimens. The ctDNA mutant allele fractions of patients after treatment is often very low and below the detection limit of regular cfDNA assays. Here we report the development and clinical application of a CLIA-certified ultra-sensitive NGS cfDNA assay in detecting the low-frequency ctDNA mutations and monitoring therapy response and drug resistance in patients with metastatic gastroesophageal adenocarcinoma under combined blockade treatments. Methods: PredicineCARE ULTRA cfDNA assay with 100,000X sequencing depth was validated under CLIA settings. Limit of blank (LoB) and limit of detection (LoD) were established by profiling 38 healthy donor plasma samples, 23 contrived human cfDNAs samples from patients with various cancers, and 30 replicates of well-established ctDNA reference standards. In silico down-sampling of FASTQ files was carried out to test the sensitivity of this assay with a lower coverage (50,000X). This assay was then adopted for longitudinal sequencing of 57 serial plasmas from 6 patients with HER2+ gastroesophageal adenocarcinomas (GEC) who underwent combined treatments (capecitabine, oxaliplatin, bevacizumab and trastuzumab) at different time points (baseline, response, and progression). Results: PredicineCARE ULTRA cfDNA assay exhibited exceptional sensitivity in calling single-nucleotide variants and indels, with a 98.0% detection rate at a LoD of 0.075% mutant allele frequency (MAF) for 60 ng cfDNA. This is significantly lower than the LoD (0.25% MAF) of a standard PredicineCARE cfDNA assay. The ultra-sensitive assay also displayed decent analytical specificity (&gt;99.99%) and precision (intra-assay 100%; inter-assay 99.41%). In 6 GEC patients’ longitudinal samples, PredicineCARE ULTRA cfDNA assay detected 36 additional somatic mutations with the MAFs spanning in a range from 0.024% to 0.1% beyond 124 baseline mutations identified using standard PredicineCARE. In two representative cases presented here, all baseline mutations (genes TP53 and PIK3CA etc.) were consistently reported in the follow-up time points with a noticeable MAF reduction (lowest MAFs from 0.046% to 0.024%) during response. Subsequent elevations were observed in more genes, including low-frequency mutations (KRAS and ERBB2) not detected at baseline or only detected in PredicineCARE ULTRA. Conclusions: This study demonstrated the promise and clinical utility of the ultra-sensitive cfDNA molecular profiling at 100,000X sequencing depth in treatment selection, therapy monitoring, and drug resistance studies.

Tandem SERS and MS/MS Profiling of Plasma Extracellular Vesicles for Early Ovarian Cancer Biomarker Discovery.

Extracellular vesicles (EVs) are vectors of biomolecular cargo that play essential roles in intercellular communication across a range of cells. Protein, lipid, and nucleic acid cargo harbored within EVs may serve as biomarkers at all stages of disease; however, the choice of methodology may challenge the specificity and reproducibility of discovery. To address these challenges, the integration of rigorous EV purification methods, cutting-edge spectroscopic technologies, and data analysis are critical to uncover diagnostic signatures of disease. Herein, we demonstrate an EV isolation and analysis pipeline using surface-enhanced Raman spectroscopy (SERS) and mass spectrometry (MS) techniques on plasma samples obtained from umbilical cord blood, healthy donor (HD) plasma, and plasma from women with early stage high-grade serous carcinoma (HGSC). Plasma EVs were purified by size exclusion chromatography and analyzed by surface-enhanced Raman spectroscopy (SERS), mass spectrometry (MS), and atomic force microscopy. After determining the fraction of highest EV purity, SERS and MS were used to characterize EVs from HDs, pooled donors with noncancerous gynecological ailments (n = 6), and donors with early stage [FIGO (I/II)] with HGSC. SERS spectra were subjected to different machine learning algorithms such as PCA, logistic regression, support vector machine, naïve Bayes, random forest, neural network, and k nearest neighbors to differentiate healthy, benign, and HGSC EVs. Collectively, we demonstrate a reproducible workflow with the potential to serve as a diagnostic platform for HGSC.

Self-Association of Von Willebrand Factor (VWF) into Prothrombotic Fibers Is Most Potently Enhanced By the Highly Atherogenic Small, Dense Fraction of Low-Density Lipoprotein (sd-LDL)

We recently demonstrated that plasma lipoproteins play a major role in regulating the function of the hemostatic system by either inhibiting or promoting the self-association of von Willebrand factor (VWF) into large fibers and bundles that are hyperadhesive for platelets. Such fibers can form on the surface of activated endothelium such as during thrombotic microangiopathies. We showed that high-density lipoprotein (HDL) attenuates VWF self-association and reduces the formation of VWF-platelet thrombi, both in vitro and in mice (PMID:26552698). By contrast, low-density lipoprotein (LDL) enhances VWF self-association and promotes the formation of VWF-platelet thrombi ( Blood, in press). Consequently, wild-type mice with elevated plasma LDL display intense and prolonged VWF-platelet thrombosis in stimulated mesenteric microvessels, exceeding even the thrombosis observed in ADAMTS13-deficient mice with normal LDL levels. All the lipoproteins of low density carry apolipoprotein (apo) B100 and comprise fractions of very low density (VLDL), intermediate density (IDL), and low density (LDL). The LDL fraction comprises two subfractions: small-dense (sd) LDL and large-buoyant (lb) LDL. Epidemiologic studies suggest that sd-LDL is the most atherogenic subfraction (PMID:33586462). Another atherogenic lipoprotein also carries apoB-100, Lp(a), in which the apoB-100 is complexed with apo(a). Here, we sought to determine the effect of LDL subfraction(s) on VWF self-association. We isolated LDL, sd-LDL and lb-LDL from healthy donor plasma by sequential KBr density centrifugation. Lp(a) was purified from patients with high circulating Lp(a) (&amp;gt;50 mg/dL) as described by Mueller et al. (PMID:35650291). We first tested these lipoproteins in microfluidic devices adapted from the design of Herbig and Diamond (PMID:26178390). In these devices, high shear stress and flow acceleration are generated around a 30-um-wide pillar within a 60-um-wide flow channel and induce the attachment and self-association of VWF into fibers on and around the pillar. We quantified VWF fiber accumulation from differential interference contrast (DIC) images. Of all the lipoprotein fractions, sd-LDL was the most potent in accelerating VWF strand accumulation (see figure). The effect of lipoproteins in this device correlated with their impact on microvascular thrombosis in vivo. We infused apoB100-containing lipoproteins into wild-type mice (3-4 weeks old) before inducing VWF-platelet thrombi deposition in the mesenteric microvasculature with ionophore. All the apoB100 lipoproteins enhanced microvascular thrombosis [we did not test Lp(a), which had no effect in the microfluidic channel], but sd-LDL was by far the most potent; the treated mice had twice the number of large thrombi (&amp;gt;30-um diameter) as the mice treated with an equivalent quantity of lb-LDL. The effect also persisted for much longer before returning to baseline with the sd-LDL. We have explored possible reasons why sd-LDL is so much more potent. Compared to the other apoB-containing lipoproteins, sd-LDL is smaller and denser, with a greater surface curvature. Whether this fact changes accessibility to sites on apoB100 is not known. We also compared the lipidomes and proteomes of sd-LDL and lb-LDL for clues to the difference. Compared to lb-LDL, sd-LDL contained a much higher content of cholesterol and cholesterol esters per particle. The proteomes of the two particles were also different, with several proteins being more abundant in lb-LDL, in particular apoC-III, apoE, apoA-IV, and apoC-II. Only apoF was significantly more abundant in sd-LDL. We are currently testing to determine if any of these difference accounts for the differences between the two LDL subfractions in promoting VWF self-association. In summary, we show that the lipoprotein associated with the greatest atherosclerotic burden and most cardiovascular disease is also the one with the most potent effect in promoting a prothrombotic state by enhancing VWF self-association. These findings have important implications for the development of the atherosclerotic lesion, the formation of the thrombus after the lesion ruptures, and for the microvascular dysfunction often observed after revascularization.

Identifying a core protein signature of small extracellular vesicles derived from B-cell precursor acute lymphoblastic leukaemia.

Acute paediatric leukaemia is diagnosed and monitored via bone marrow aspirate assessment of blasts as a measure of minimal residual disease. Liquid biopsies in the form of blood samples could greatly reduce the need for invasive bone marrow aspirations, but there are currently no blood markers that match the sensitivity of bone marrow diagnostics. Circulating extracellular vesicles (EVs) represent candidate biomarkers that may reflect the blast burden in bone marrow, and several studies have reported on the utility of EVs as biomarkers for adult haematological malignancies. Increased levels of EVs have been reported for several haematological malignancies, and we similarly report here elevated EV concentrations in plasma from paediatric BCP-ALL patients. Plasma EVs are very heterogeneous in terms of their cellular origin, thus identifying a cancer selective EV-marker is challenging. Here, we undertook a reductionistic approach to identify protein markers selectively associated to plasma EVs derived from BCP-ALL patients. The EV proteome of primary BCP-ALL cell-derived EVs were compared against EVs from healthy donor B cells and the BCP-ALL cell line REH, and further against EVs isolated from plasma of healthy paediatric donors and paediatric BCP-ALL patients. With this approach, we identified a signature of 6 proteins (CD317, CD38, IGF2BP1, PCNA, CSDE1, and GPR116) that were specifically identified in BCP-ALL derived EVs only and not in healthy control EVs, and that could be exploited as diagnostic biomarkers.