Serum Samples Research Articles

Abstract B010: Systematical evaluation on alternative splicing in immune-related therapy targets reveals innate and acquired therapy resistance mechanisms in multiple myeloma

Abstract Introduction: Although multiple myeloma (MM) is considered incurable, immune-related therapies are emerging as promising approaches to prolong patient survival. Alternative splicing (AS) has been identified as a mechanism causing therapy resistance to CD19 CAR-T in B-cell malignancies, but no systematic evaluation has been made on the impact towards MM immunotherapy targets. Methods: Utilizing two datasets (MMRF N=852 and IU N=122), we selected high-confidence targets based on high RNA expression (Log2(TPM+1)>3), low toxicity (nTPM>50 in <2 normal tissues and in <2 normal blood cells) and high target potential (>3 surfaceome databases or established immune-therapy targets). Known resistance mechanisms of other therapies, such as immunomodulatory drugs (IMiDs) were also included. AS analysis was conducted for all identified targets. Expression and AS events between patient samples and 19 MM cell lines from DepMap database were also measured. Results: 102 high-confidence targets were identified in MM samples including six existing immunotherapy targets (BCMA, SLAMF7, GPRC5D, CD79A/B and CD38). From these 102 genes, 361 significant AS events were subsequently identified (|dPSI|>0.2, FDR<0.05, >10 supporting reads in >25% samples). One event of interest included an aberrant exon inclusion event in the existing target FCRL5 in ∼10% of newly diagnosed and relapse patient samples, despite being treatment naïve. A cryptic exon between exon 3 and 4 of the canonical transcript was found with higher usage in MM (PSI>0.48) compared to 8 normal bone marrow plasma cell samples (=0.13). Inclusion of this exon led to the switch from three protein-coding transcript variants (FCRL5-201, -202, -203) to a transcript encoding a truncated protein after the first Ig domain (-206). The AS event in treatment-naïve patients may lead to the selection of this variant in treated patients as a mechanism of therapy resistance. Another key event was detected in CRBN, which encodes the target of IMiDs including lenalidomide. Serial samples in one patient treated with lenalidomide identified a novel splice junction in CRBN in the later sample (dPSI=0.2). The novel splice junction led to the skipping of exon 8 encoding the LON domain, which is essential for maintaining the functional conformation of the IMiD-binding site. Lack of exon 8 will disrupt LON domain integrity, indicating a potential AS-induced resistance mechanism. Interestingly, the novel splice junction was found in 70% relapsed samples (>10 supporting reads), 60% of which were with high exon skipping level (PSI<0.5). Conclusion: We have identified potential innate and acquired AS mechanisms that affect patient response towards immune-related therapies, highlighting the importance of conducting RNA-based diagnostics for MM patients. Citation Format: Enze Liu, Travis S Johnson, Parvathi Sudha, Habib Hamidi, Faiza Zafar, Vivek S Chopra, Rafat Abonour, Brian A Walker. Systematical evaluation on alternative splicing in immune-related therapy targets reveals innate and acquired therapy resistance mechanisms in multiple myeloma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B010.

Disorders of acid-base balance promote rumen lipopolysaccharide biosynthesis in dairy cows by modulating the microbiome

IntroductionDisorders of acid-base balance in the rumen of dairy cows have a significant impact on their health and performance. However, the effect of transient differences in pH on susceptibility to subacute ruminal acidosis (SARA) and lipopolysaccharide (LPS) biosynthesis in dairy cows remains unclear.MethodsIn this study, milk, serum, and rumen fluid samples from 40 Holstein dairy cows (on d 56 postpartum) with different rumen pH (2–4 h after morning feeding) were explored to investigate the difference of susceptibility to SARA and the correlation between microbiome, LPS and inflammation. These cows were categorized into low pH (LPH, pH ≤ 6.0, n = 20) and high pH (HPH, pH ≥ 6.5, n = 20) groups.ResultsThe results showed that LPH group increased the concentrations of total volatile fatty acids, acetate, propionate, butyrate and valerate. However, milk yield and milk compositions were unaffected. Compared to the HPH group, the LPH group increased the concentrations of serum BHBA, NEFA, LPS, HIS, IL-2, IL-6, TNF-α, and MDA, and decreased the concentrations of serum IgA, IgM, IgG, SOD, T-AOC, and mTOR. In addition, the LPH group decreased the copies of Ruminococcus flavefaciens and increased the copies of Fibrobacter succinogenes. Microbial community analysis isupplendicated a significant difference in bacterial composition between the two groups. At the phylum level, Bacteroidota and Firmicutes were enriched in the LPH and HPH groups, respectively. At the genus level, the dominant bacteria in the LPH group were Prevotella. Additionally, the LPH group increased the proportions of Gram-negative phenotypes, potentially pathogenic phenotypes and LPS biosynthesis. The close correlation between two key enzymes for LPS synthesis LpxL and LpxM with rumen pH, inflammatory markers, and microorganisms indicates that low pH may increase the risk of inflammation by facilitating the lysis of Gram-negative bacteria and the release of penta-acylated LPS. Penta-acylated and hexa-acylated LPS may be mainly derived from Prevotella and Succinivibrionaceae_UCG-001, respectively.DiscussionOverall, these results support the notion that transient low pH could reflect the risk of cows suffering from SARA and associated inflammation and is strongly associated with penta-acylated LPS. Our findings provide new insights into ruminant health improvement and disease prevention strategies.

Abstract A021: Analytical platform to validate microRNA cancer biomarkers

Abstract We developed and tested a novel assay to quantify and validate microRNA (miRNA) cancer biomarkers in liquid biopsies. The assay is amplification-free, exploits a commercially available nanopore-array device for detection (MinION from Oxford Nanopore Technologies), uses total RNA isolated from biospecimen as the miRNA source, and proprietary osmylated oligonucleotides, complementary to the target miRNA, as probes. The assay exploits a bracketing experimental protocol to quantify miRNA, and yields miRNA copy numbers (copies) per 1microLiter of isolated total RNA, with an unprecedented accuracy of approximately +/- 20% across all concentrations. For development purposes and as a control/reference the combined serum of healthy men (# H6914 from Sigma-Aldrich) was used. During a period of 4 years different lots of H6914 yielded, within experimental error, comparable miRNA copies for let-7b, miR-16, miR-21, miR-15b, miR-375 and miR-141. Serum samples from healthy individuals and cancer patients were also tested. Once data were normalized to the same total RNA content, miRNA copies appeared to belong to one of two groups. The first group included miR-21, miR-375 and miR-141 copies in H6914 and in healthy samples. The second group included miR-21, miR-375 and miR-141 copies in cancer samples exhibiting an approximate 1.8-fold overexpression compared to the first group. miR-15b copies in H6914, healthy and cancer samples were all in the first group. The two groups exhibited zero data overlap which is unprecedented in the miRNA/cancer literature. Zero data overlap was the result of (i) the assay’s bracketing approach with a protocol-defined accuracy of approximately +/-20%, and (ii) the normalization of miRNA copies to the same total RNA content. Notably, these two groups included both serum and urine samples from cancer patients and healthy individuals. All subjects were deselected regarding age, sex, and ethnicity, while patients were selected for breast, prostate or pancreatic cancer, Stage I or II, pretreatment. To the best of our knowledge, this is the first-time agreement between serum and urine miRNA copies is reported and leads to the suggestion that a urine sample may replace a blood draw for the tested miRNAs. Not only clear discrimination between healthy and cancer samples was possible, but this analytical platform may be used to validate tentative cancer/disease biomarkers when miRNA overexpression between cancer/disease and healthy is equal or higher than 1.8-fold. Non-Coding RNA 2024, 10(4), 42; https://doi.org/10.3390/ncrna10040042 Citation Format: Anastassia Kanavarioti, M. Hassaan Rehman, Salma Qureshi, Aleena Rafiq, Madiha Sultan. Analytical platform to validate microRNA cancer biomarkers [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A021.

Abstract B048: Microfluidic isolation of immune cell-derived extracellular vesicles in head and neck cancer patients on immune-checkpoint blockade

Abstract Background: Immune-checkpoint blockade (ICB), with or without chemotherapy, is the standard of care for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC). However, only a minority of patients get clinical benefit from it. The combined expression of PD-L1 in a tumor section is currently the only biomarker approved for response prediction. Yet, its invasive nature and suboptimal predictive performance underscores the need for new, non- invasive surrogates of immune activity in patients undergoing ICB. Extracellular vesicles (EVs) carry key molecules critical for cell-cell communication and have the potential to be used as a non-invasive, real-time biomarker of systemic immune activity, essential for success in ICB- based therapy. Methods: We used a multichannel, microfluidic device that employs an immune- affinity approach, ‘herringbone chip’ (EVHB-Chip), to retrospectively isolate EVs from twelve HNSCC patients before and after the first cycle of ICB. One mL of plasma was used per patient per time point. For each patient, two devices were serially used, with each sample continuously following through each chip in a non-destructive manner—the first device aimed to isolate B cell EVs (antibodies against CD19 and CD20). The second device was optimized to capture T cell EVs (against CD274/PD-L1). Digital-droplet PCR was performed after EVs were isolated to detect the RNA transcripts in the EV cargo. Serial sampling was performed for six out of the twelve patients. Response assessment was conducted via RECIST1.1 criteria or clinical evaluation. Results: Most of our cohort was male 7/12 (57%) and had oral cavity 4/12 (33%) or oropharynx 4/12 (33%) tumors. Additionally, 11/12 (91%) patients received ICB monotherapy. Most of the cohort had a PD-L1 ≤ 1 (7/12; 58%), 7/12 patients experienced disease progression (PD), 4/12 experienced stable disease (SD), and one patient had a partial response (PR). Most of the RNA signal in pre-ICB samples was obtained from CD20 (12/12; 100%), followed by CD45 (10/12; 83%), CD19 (9/12; 75%), and CD274/PD-L1 (5/12; 41%). No difference was found in the RNA signal pre-ICB between responders and non-responders (p=0.1). For the patients with serial sampling available, the signal from CD274/PD-L1 increased after one infusion of ICB in 4/6 patients and remained negative in one. A trend towards a high amount of T cell-derived EVs was observed for the entire cohort (p=0.07; p=0.08) when comparing the amount of RNA via our EV marker (GAPDH, ACTB) between T and B cell devices. Patients with higher than 11.4/mL copies of CD19 (median value for the cohort) in the pre-ICB plasma sample tended to have a higher overall survival (HR 0.26, 95% CI: 0.06-1.13; p-value: 0.05) and progression-free survival (HR 0.28, 95% CI: 0.07-1.16; p-value: 0.06). Conclusion: The EVHB-Chip allowed for the detection and profiling of T and B cell-derived EVs in pre-ICB plasma samples of HNSCC patients undergoing ICB in this pilot cohort. Higher CD19 signal pre-ICB could potentially impact response and survival outcomes in ICB-based therapies. Citation Format: Andrew R. Levy, Daniel A Ruiz Torres, Uma Paithankar, Raheel Ahmad, Daniel C Rabe, Daniel L Faden, Shannon L Stott. Microfluidic isolation of immune cell-derived extracellular vesicles in head and neck cancer patients on immune-checkpoint blockade [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B048.

Risk factors for spotted fever group rickettsioses in Kilimanjaro Region, Tanzania

Abstract Background Knowledge gaps exist on risk factors for spotted fever group rickettsioses (SFGR) in sub-Saharan Africa. We sought to identify SFGR risk factors in Kilimanjaro Region, Tanzania. Methods We recruited febrile patients presenting at two hospitals in Moshi from February 2012 through May 2014. Standardised clinical and risk factor questionnaires were administered. SFGR exposure was defined as a Rickettsia africae immunofluorescence antibody reciprocal titer ≥64, and acute SFGR as a ≥four-fold rise between paired sera. Logistitic regression was used to identify associations. Results Of 1,190 participants providing ≥1 serum sample, median (range) age was 21.8 (0.3, 100.2) years, 545 (54.3%) were female, and 650 (54.6%) had SFGR exposure. Of 731 participants with paired sera, 67 (9.2%) had acute SFGR. On multivariable analysis, odds of acute SFGR were higher in age group 0-2 years (adjusted odds ratios [aOR] for older age groups <0.36, p<0.011), rural residence (aOR 4.1, p=0.007), and areas with maximum daily temperature <26°C (aORs for higher temperature groups <0.42, p-values<0.035). Odds of SFGR exposure were higher in those working in the garden (aOR 1.8, p=0.010), and seeing a dog (aOR 1.5, p=0.010). Odds of SFGR exposure were lower in age group 0-2 years (aORs for older age groups >1.5, p<0.026), female sex (aOR 0.62, p<0.001), and being from Chaga tribe (aOR 0.68, p=0.003). Conclusion Those aged <2 years, rural residents, and persons residing in areas with cooler temperatures had increased odds of SFGR. Our results identify groups for further research on tick exposure and for targeted prevention interventions.

Impact of induction agents and maintenance immunosuppression on torque teno virus loads and year-one complications after kidney transplantation

BackgroundTorque teno virus load (TTVL) is gaining importance as a surrogate parameter to assess immunocompetence in kidney transplant recipients. Although the dynamics of TTVL have been investigated before, the impact of different induction agents and variations in immunosuppressive maintenance therapies on TTVL remain unknown.MethodsIn this retrospective study, TTVL was quantified in 537 plasma or serum samples from 134 patients transplanted between 2018 and 2021. TTVL was examined pre-transplantation and 30-, 90-, 180-, and 360-days post-transplant. To assess the influence of induction therapy on TTVL, 67 patients receiving anti-thymocyte globulin (ATG) induction were matched with 67 patients receiving an interleukin-2 receptor antagonist (IL2-RA) induction in terms of age, sex, and donor modality.ResultsFollowing transplantation, there was a steep increase in TTVL post-transplant for all patients with peak viral loads at 90 days post-transplant (median TTVL [IQR] 7.97×106, [4.50×105–1.12×108]) followed by subsequently declining viral loads. Compared to patients receiving IL2-RA as induction therapy, patients receiving ATG had significantly higher peak viral loads 3 months post-transplant (median TTVL [IQR] 2.82×107 [3.93×106–1.30×108] vs. median TTVL [IQR] 2.40×106 [5.73×104–2.60×107]; P<0.001). Throughout all post-transplant time points, patients receiving additional rituximab for induction along with higher tacrolimus target levels exhibited the highest TTVL.Patients whose TTVL 3-months post-transplant exceeded the currently proposed cutoff to predict infections within the first year post-transplant [6.2 log10] showed a trend towards a higher risk of being hospitalized with an infection in the following 9 months, albeit without being statistically significant (HR=1.642, P=0.07).ConclusionsHigher TTVL reflects the greater immunosuppressive burden in immunological high-risk patients receiving intensive immunosuppression. The choice of induction agent and intensified immunosuppressive maintenance therapy notably affects TTVL at 3 months post-transplant and beyond, necessitating careful consideration when interpreting and applying TTVL cutoffs to monitor immunocompetence post-transplant.

Assessment of Serum Adiponectin and Leptin in Chronic Preiodontitis Over Healthy Individuals

ABSTRACT Objective: The objective of the research was to ascertain whether serum levels of the biochemical markers adiponectin and leptin are related to clinical periodontal characteristics. Materials and Method: For the study, 20 people with chronic periodontitis (Group A) and 20 healthy people (Group B) were enrolled. Assessments were conducted on periodontal markers, including bleeding on probing (POP), gingival index (GI), plaque index (PI), probing pocket depth (PPD), and clinical attachment level (CAL). An enzyme-linked immunosorbent assay (ELISA) test was used to assess the levels of adiponectin and leptin in serum samples. Result: There was significant elevation in average level of leptin in chronic periodontitis in association to that in healthy group. On the other hand, there was considerable decrease in serum adiponectin in group A in association to healthy group. The ratio of leptin\adiponectin was appreciably greater amongst periodontitis group compared to healthy group. Conclusion: The current investigation leads us to the conclusion that the pathophysiology of periodontitis is significantly influenced by serum levels of adiponectin and leptin.

Abstract B047: Monitoring treatment response and toxicity in BRAF V600-mutant metastatic melanoma with circulating cell-free DNA

Abstract Background: The DREAMseq trial (EA6134, NCT02224781) was a national multi-center randomized phase III trial coordinated by ECOG-ACRIN that found that immune checkpoint inhibitor (IO) treatment achieves improved survival outcomes compared to targeted therapy (TT) in patients with BRAF V600-mutant metastatic melanoma. Still, approximately 40% of patients do not respond to IO, and existing biomarkers fail to distinguish this subset of patients who do not benefit from IO therapy. Additionally, as many as 80% of patients may experience immune- related adverse events (irAEs), which range from mild dermatological symptoms to life- threatening myocarditis. Here, we explore the use of the methylation status of cell-free DNA (cfDNA) in serially collected blood samples to measure response and toxicity in the context of IO- and TT-treated metastatic melanoma. Methods: Serial serum samples were collected from patients with BRAF V600-mutant metastatic melanoma treated with ipilimumab/nivolumab (IO) or dabrafenib/trametinib (TT). Circulating cfDNA was isolated from serially collected serum samples, enriched for regions of interest by hybridization capture and sequenced using enzymatic methyl-seq. Cell-type deconvolution was performed to determine the abundance of cell type- specific methylation patterns of cfDNA molecules in patient serum at different time points of treatment. The BRAF V600 mutation abundance in total cfDNA was also assessed. Results: We identified melanocyte lineage-specific DNA methylation regions and demonstrate that the methylation status of these regions remains conserved in malignant melanoma. We characterized the changes in abundance of the melanocyte-lineage cfDNA over the course of treatment and show that these changes distinguish responders from non-responders to either IO or TT. Furthermore, we track the abundance of cell type-specific DNA from normal tissues to identify markers indicative of adverse effects or disease progression. Conclusions: We established melanocyte-lineage methylation markers and evaluated the use of cell-type specific DNA methylation to monitor treatment effects of immune checkpoint or BRAF/MEK inhibitors in metastatic melanoma using serially collected blood samples. Citation Format: Sidharth S Jain, A. Patrick IV McDeed, Megan E McNamara, Amber R Alley, Dori S Rosenstrauch, Harry Sun, Natalie Thompson, John M Kirkwood, Geoffrey T Gibney, Michael B Atkins, Anton Wellstein. Monitoring treatment response and toxicity in BRAF V600-mutant metastatic melanoma with circulating cell-free DNA [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B047.

Correlation Between Vitamin D Deficiency and Calcium, Phosphorus, Alkaline Phosphatase in Preterm Libyan Infants: Case-Control study

Vitamin D plays a vital role in regulating calcium and phosphorus levels and in bone mineralization. Vitamin D deficiency is a widespread issue contributing to metabolic bone disease in preterm infants. This study aimed to evaluate the biochemical parameters associated with varying levels of vitamin D deficiency. Serum samples were collected from preterm infants at the Neonatal Intensive Care Unit of Althawra Hospital Center, Al Beyda, Libya, from Feb-July 2019. The study included two groups: 62 preterm infants and a control group of 34 full-term infants. Serum levels of calcium, phosphorus, alkaline phosphatase, and 25OH-Vitamin D were measured using enzyme immunoassays and standard method. The study involved 62 preterm neonates, with a median gestational age of 32 weeks (range 28-36) and median birth weight of 1960 gm (range 900-2800 gm). The median levels recorded were calcium at 8.7 mg/dl (p=0.000), phosphorus at 4.1 mg/dl (p=0.584), ALP at 458 U/L (p=0.008), and vitamin D at 13.6 ng/ml. The prevalence of varying degrees of vitamin D deficiency was noted: very severe (9.7% < 5 ng/ml), severe (19.4% 5-10 ng/ml), and overall deficiency (45% 10-20 ng/ml), and suboptimal (25.8% 20-30 ng/ml). Comparing preterm infants to the control group revealed statistically significant differences across all parameters (Vitamin D p=0.000, Ca p=0.007, PO4 p=0.036, and ALP p=0.000). All preterm infants exhibited inadequate vitamin D levels, and there was an inverse relationship between the biochemical parameters and vitamin D deficiency. Notably, the elevated ALP level could serve as a crucial marker for diagnosing vitamin D deficiency in clinical settings.

Epidemiologic and genomic characterization of an outbreak of Rift Valley fever among humans and dairy cattle in northern Tanzania

Abstract Background A peri-urban outbreak of Rift Valley fever virus (RVFV) among dairy cattle from May through August 2018 in northern Tanzania was detected through testing samples from prospective livestock abortion surveillance. We sought to identify concurrent human infections, their phylogeny, and epidemiologic characteristics in a cohort of febrile patients enrolled from 2016-2019 at hospitals serving the epizootic area. Methods From September 2016 through May 2019, we conducted a prospective cohort study that enrolled febrile patients hospitalized at two hospitals in Moshi, Tanzania. Archived serum, plasma, or whole blood samples were retrospectively tested for RVFV by PCR. Human samples positive for RVFV were sequenced and compared to RVFV sequences obtained from cattle through a prospective livestock abortion study. Phylogenetic analysis was performed on complete RVFV genomes. Results Among 656 human participants, we detected RVFV RNA in four (0.6%), including one death with hepatic necrosis and other end-organ damage at autopsy. Humans infected with RVFV were enrolled from June through August 2018, and all resided in or near urban areas. Phylogenetic analysis of human and cattle RVFV sequences demonstrated that most clustered to lineage B, a lineage previously described in East Africa. A lineage E strain clustering with lineages in Angola was also identified in cattle. Conclusion We provide evidence that an apparently small RVFV outbreak among dairy cattle in northern Tanzania was associated with concurrent severe and fatal infections among humans. Our findings highlight the unidentified scale and diversity of inter-epizootic RVFV transmission, including near and within an urban area.

Abstract B012: M-PACT: Methylation-based predictive algorithm for CNS tumor liquid biopsies

Abstract Background: DNA methylation-based classification has been transformative in the molecular diagnostics of pediatric central nervous system (CNS) tumors. Current diagnostic pipelines integrate histopathology and molecular tools in accordance with the WHO classification of CNS tumors which rely on the availability of tissue specimens. However, some tumors are not amenable to neurosurgical resection due to their high-risk locations and tissue collection is not routinely performed at relapse. In addition, molecular biomarkers are largely lacking for longitudinal disease monitoring in pediatric neuro-oncology. Liquid biopsies (LBs) have emerged as a minimally invasive, longitudinal source of tumor-derived cell-free DNA (cfDNA) ideally suited for detecting minimal residual disease (MRD). In addition, LBs provide a sensitive means of studying tumor evolution at high temporal resolution. Success rates of LB analyses in neuro-oncology have varied drastically between studies, warranting the deployment of more robust and reproducible assays. Methods: We applied a novel cfDNA methylation-based workflow to a large cohort of cerebrospinal fluid (CSF) samples collected from pediatric brain tumor patients (n>200 patients). Enzymatic methylation sequencing (EM-seq) was adapted for CSF-derived cfDNA inputs in the picogram range, enabling the acquisition of genome-wide cfDNA methylation profiles across the cohort. Utilizing non-oncological cfDNA profiles and CNS tumor DNA methylation datasets as a reference, we developed an innovative computational pipeline that incorporates DNA methylation imputation and deep learning of a neural network. In addition, deconvolution of cfDNA profiles facilitated classification of low tumor burden samples, including those with balanced genomes that would have been missed using previous generation assays. Results: Our CSF-based classifier, M-PACT (Methylation-based Predictive Algorithm for CNS Tumors), yielded accurate tumor detection and entity prediction (AUC=0.9) in our benchmarking cohort (n>100 CSF samples). Proof-of-concept studies showed the potential of this methodology to track tumor evolution in serial CSF samples at both the genetic and epigenetic level, illuminating potential mechanisms driving treatment resistance and recurrence. Conclusions: Collectively, this study provides the framework for robust methylation- based tumor detection and classification of CSF LBs. M-PACT can be applied to aid in tumor diagnostics and to detect MRD during follow-up, warranting prospective validation of its broad applicability and clinical utility in future trials. Citation Format: Tom T. Fischer, Kyle S. Smith, Katie Han, Anna Kostecka, Hong Lin, Daniel Senfter, Tatjana Wedig, Nathalie Schwarz, Natalia Stepien, Sibylle Madlener, Christine Haberler, Esther Hulleman, Sandeep K. Dhanda, Stefan M. Pfister, Santhosh Upadhyaya, Amar Gajjar, Giles W. Robinson, Joonas Haapasalo, Hannu Haapasalo, Kristiina Nordfors, Johannes Gojo, Kendra K. Maass, Kristian W. Pajtler, Paul A. Northcott. M-PACT: Methylation-based predictive algorithm for CNS tumor liquid biopsies [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B012.

Abstract B039: Clinical liquid biopsy testing for detecting actionable genomic alterations in children and young adults with advanced solid tumors

Abstract Introduction: Given the paucity of data regarding circulating tumor DNA (ctDNA) as a modality to guide diagnosis and treatment for children with solid tumors, we conducted a prospective study to determine the frequency of diagnostic and targetable genomic variants identified by clinical ctDNA testing. Methods: GAIN is a prospective, multi-center molecular profiling study that was amended to include clinical ctDNA testing starting in January 2022. Patients were eligible if they were <30 years old with a refractory, recurrent, newly diagnosed with 2-year EFS <50%, or rare solid tumor and had measurable or evaluable disease. Paired tumor and blood samples underwent next generation sequencing (NGS) in CLIA-certified laboratories, with return of results to clinicians. Serial ctDNA samples were analyzed by F1LCDx (FoundationOne® Liquid Companion Diagnostic), an FDA-approved clinical ctDNA test that includes hybrid capture sequencing of a 300+ gene panel, comprehensive multi-omics ctDNA tumor fraction (TF) assessment, tumor mutational burden, and microsatellite instability status. We analyzed comprehensive NGS tissue-based and F1LCDx sequencing data for each participant to compare findings from each sample. The detection of alterations was stratified by ctDNA burden as assessed by ctDNA TF. We calculated the prevalence of ctDNA in our cohort, overall and by disease type. Chart review provided additional patient characteristic, diagnostic, therapeutic, and disease assessment data. The clinical impact of ctDNA results was qualitatively described. Results: The cohort included 35 patients, with a total of 70 F1LCDx samples (range 1-6 samples per patient). There were 16 unique tumor types represented, most commonly osteosarcoma (n = 8), neuroblastoma (n = 8), and Ewing sarcoma (n = 4). 77% of patients had metastatic disease at time of first ctDNA sample collection. 51% of patients had detectable ctDNA TF in at least one sample. Overall, 60% of patients had at least one actionable genomic alteration (range 0-14 variants per patient) identified in liquid biopsy samples, including 3 patients with actionable variants identified with undetectable ctDNA TF. We observed case vignettes in which ctDNA results informed clinical decisions, including disease assessment and treatment guidance. An illustrative example was the identification of ALK variants in relapse blood samples of two patients with neuroblastoma that were not identified in baseline tissue, which were targeted in both instances. Data collection and analyses as described in the methods are ongoing. Conclusion: In this prospective cohort of children and young adults with advanced solid tumors, ctDNA was detectable across a wide range of diagnoses and 60% of patients had at least one diagnostic or potentially targetable alteration identified. These data demonstrate that liquid biopsy yields actionable genomic variants, particularly when ctDNA burden is elevated. We will determine the association of these variants with those identified in tissue samples and their clinical implications in ongoing analyses. Citation Format: Holly J Roberts, Hannah Comeau, Steven G Dubois, Katerina Hoskova, Douglas I Lin, Brian D Crompton, Katherine A Janeway, David S Shulman. Clinical liquid biopsy testing for detecting actionable genomic alterations in children and young adults with advanced solid tumors [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B039.

Lack of Serological and Molecular Evidence of Duck Tembusu Virus Infection in Ducks from South Korea

The duck Tembusu virus (DTMUV), an emerging flavivirus, has led to severe neurological disorders and substantial economic losses in the duck industry throughout Asia. Considering South Korea’s increasing duck production and its strategic location along the East Asian–Australasian Flyway, this study aimed to assess the presence of DTMUV in South Korea to evaluate potential risks to the poultry industry. We performed a comprehensive serological survey of 1796 serum samples from broiler and breeder ducks collected between 2011 and 2023, alongside molecular detection tests on 51 duck flocks exhibiting suspected clinical signs of DTMUV infection. The absence of serological and molecular evidence for DTMUV or other flavivirus infections suggests that these viruses have not yet affected South Korean duck populations. These findings underscore the critical need for ongoing surveillance, given the virus’s potential to disrupt agriculture and pose public health risks. The study also emphasizes the importance of maintaining stringent biosecurity measures and conducting further research to monitor and prevent DTMUV transmission, particularly due to the possible role of migratory birds and other vectors in spreading zoonotic diseases.

Abstract A051: Identifying patterns of cell-free DNA associated with elevated risk of diffuse large B-cell lymphoma in adult dogs

Abstract Background: Nonrandom patterns of cell-free DNA (cfDNA) are closely associated with malignancy. We hypothesize that they are also found in the systemic microenvironment that gives rise to cancer, and that such signatures can inform a risk stratification test. To achieve this, we have initiated the Lymphoma Risk Assessment (LyRA) study, using machine learning on a training set of dogs with known health status and a test set of otherwise healthy adult pet dogs to identify cfDNA signatures that are associated with elevated risk of developing diffuse large B- cell lymphoma (DLBCL). We will validate the predictive accuracy of these signatures, including the persistence of the risk environment and the interval between risk detection and development of clinical disease. Since cfDNA is the primary input of the LyRA test, we first performed a pilot study to assess how sample characteristics affect cfDNA quality and subsequent low-pass whole genome sequencing (WGS) data. Methods: Blood samples were collected from pet dogs with the owner’s informed consent. Paired samples were collected into potassium (K3)EDTA tubes and additive-free red top tubes (n = 5 dogs) or K3EDTA tubes and Cell-free DNA BCT® Streck Tubes (n = 6 dogs). Additional serum samples (n=3) were generously provided by the Morris Animal Foundation Golden Retriever Lifetime Study. cfDNA was isolated using the QIAamp® Circulating Nucleic Acid kit. Total cfDNA concentration was measured by PicoGreen™ and fragment distribution by Agilent High Sensitivity D500 ScreenTape® Assay, with comparisons made by starting material (plasma vs. serum) and collection tubes. Results: Serum had both higher total concentrations of cfDNA (PicoGreen™) and calibrated concentrations of long fragment cfDNA (515-658 bp Agilent traces) compared to plasma. In addition, total cfDNA concentrations increased with malignancy, following the pattern: no cancer < cancer < hematological cancer. Median fragment lengths of Agilent peaks also increased with malignancy for short (125-181 bp) and medium (340-425 bp) fragments, in both serum and plasma, but decreased for long fragments. In these pilot data the trends seen were consistent with our hypothesis and with prior studies, although due to sample size, no statistical testing was performed. Conclusions: The pilot study demonstrated that the effects of sample parameters on cfDNA quality are consistent with existing literature, validating our methods and supporting the rationale for the LyRA study. These features may prove valuable when integrated with genomic data to identify high-risk microenvironments. Recent research has shown that a majority of cfDNA in tumor patients originates from non-malignant cells and current methods often overlook potentially important patterns in this so-called 'discard cfDNA.' By focusing on these overlooked patterns, with an emphasis on risk stratification, the LyRA test could shift clinical management to a prevention-focused approach, serving as a model of testing and interception for DLBCL and other cancers in dogs, and potentially in humans. Citation Format: Lauren E Burt, Amy E Treeful, Mitzi Lewellen, Kimberly Demos-Davies, Andrea Chehadeh, Sara Pracht, Ashley J Schulte, Caitlin Feiock, Julia Medland, Davis Seelig, Kelly M Makielski, Ali Khammanivong, Antonella Borgatti, Michael S Henson, Michael Linden, Daniel Vallera, Gary R Cutter, Aaron L Sarver, Jaime F Modiano. Identifying patterns of cell-free DNA associated with elevated risk of diffuse large B-cell lymphoma in adult dogs [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A051.

Seroprevalence and associated risk factors of peste des petits ruminants in sheep and goats in three districts of the Central Oromia Region, Ethiopia

BackgroundPeste des petits ruminants (PPR) is a viral disease that affects domestic and wild small ruminants and camels in Africa, the Middle East, and Asia. Following the successful eradication of rinderpest, the World Organization for Animal Health (WOAH) and the Food and Agriculture Organization (FAO) have undertaken to eradicate PPR by 2030. Regular surveillance and monitoring of the disease in various regions of Ethiopia are crucial to achieving this goal. This study aimed to estimate the prevalence of PPR, assess community awareness of PPR infection, and examine the associated risk factors of the disease in selected districts of the Central Oromia Region, Ethiopia.MethodThe study collected 384 serum samples from 73 flocks containing 217 sheep and 167 goats using a multi-stage sampling technique. A competitive enzyme-linked immunosorbent assay (c-ELISA) was used to detect antibodies against the PPR virus. Additionally, a pre-tested questionnaire was used to gather information on community awareness and potential risk factors for PPRV infection in the study area.ResultsThe study found that the overall prevalence of PPR in flocks was 71.2% [95% confidence interval (CI): 59.4%−81.2%]. The prevalence of PPR at the animal level was 50% (95% CI: 44.9%−55.1%), with sheep having a prevalence of 54.4% (95% CI: 47.0%−60.6%) and goats having a prevalence of 44.3% (95% CI: 36.6%−52.2%). The study also found that districts, flock size, and agroecology were independent predictors of PPRV seropositivity in sheep, whereas districts, origin, and mixed species were independent predictors of PPRV seropositivity in goats.ConclusionThe study revealed a high prevalence of PPR in sheep and goats in the study area. To prevent the spread of the disease, the study suggests quarantining animals before introducing them to districts, regular PPR vaccination, and isolation and molecular characterization of the PPR virus circulating in the study area.

Abstract 4146303: A Single-Dose of a Novel CasX-Editor Lowers APOC3 Levels In Vivo

Background: Apolipoprotein C-III (APOC3) is a key regulator of lipid metabolism that inhibits the catabolism and clearance of triglyceride (TG)-rich lipoproteins in the blood. Loss-of-function APOC3 variants are associated with significantly lower TG levels and a reduced risk of heart disease, underscoring the potential of APOC3 inactivation as a therapeutic strategy for patients with familial chylomicronemia syndrome (FCS) and severe hypertriglyceridemia (SHTG). Here, we developed STX-1400 lead molecules as a novel investigational gene editing treatment using a highly engineered CRISPR-CasX mRNA and a guide RNA (gRNA) encapsulated into lipid nanoparticles (LNPs) to edit the APOC3 gene and knock out its hepatic expression. Methods: STX-1400 lead molecules were evaluated for editing potency and resulting functional effects on APOC3 reduction in vitro in primary human hepatocytes (PHHs) and primary cynomolgus hepatocytes (PCHs). In vivo studies were conducted in PXB mice, a humanized liver mouse model. Liver biopsies and serum samples were collected at two weeks post-dose. Human APOC3 mRNA and APOC3 protein levels were measured in liver biopsies and serum samples, respectively. Lipid profile evaluation, which included measuring TGs and total cholesterol (TC) levels, was also performed. Results: Treatment of PHHs with LNPs containing STX-1400 lead molecules showed a dose-dependent increase in editing activity; up to 90% editing at the APOC3 locus was associated with >70% reduction in secreted APOC3 protein levels. Potency was also observed in PCHs, with up to 70% editing. A single dose of LNPs encapsulating STX-1400 lead molecules delivered into PXB humanized mice resulted in >60% editing at the APOC3 locus in the liver, which was associated with >90% reduction of serum hAPOC3 protein levels, accompanied by reduction in TG and TC levels. Conclusions: These data demonstrate that the STX-1400 lead molecules efficiently knock down hepatic expression of APOC3 in preclinical models. This work suggests that the CRISPR-CasX-based editor approach could be developed as a single dose therapy for the treatment of hypertriglyceridemia.

Effect of Different Rates of Rambutan Peel Extract on Visfatin and Cardiac Troponin I Response in Type 2 Diabetic Rats Induced with Streptozotocin

Background: The aim of this study was to investigate the effects of STZ (Streptozotocin) and Rambutan peel extract (RPE) on serum visfatin and cardiac response in experimental Type 2 diabetic rats. Methods: In this study, 64 adult male Wistar albino rats, aged between 8-10 weeks, were used. 8 groups were randomly selected, each containing 8 rats as Control (C), R100 (RPE 100 mg/kg group given with oral gavage route of administration (OGRA), R200 (RPE 200 mg/kg group given with OGRA), R300 (RPE 300 mg/kg group given with OGRA); Diabetes Control (DC): STZ 50 mg/kg i.p administered group, DR100 (D+100 mg/kg JPE), DR200 (D+200 mg/kg JPE), DR300 (D+300 mg/kg JPE). The study lasted a total of 31 days, including adaptation (7 days), induction of diabetes (3 days), and trial period (21 days). Blood samples were taken from the tail vein (vena caudalis) of all subjects on days 0 and 21 of the study. Visfatin and cTnI levels in the serum samples were measured and evaluated by ELISA method. Result: While the increase in mean serum visfatin and cTnI levels in diabetic groups was seen mostly in DC groups, the most significant decrease in mean serum visfatin level due to the addition of RPE was determined in DR100 groups (p less than 0.01).As a result, it was concluded that the administration of 100 mg/kg RPE in rats had no adverse effects. It was concluded that RPE, which has a cardioprotective effect by regulating the visfatin response that plays a role in the development of diabetes, may be useful in supporting the impaired cardiovascular system function that occurs in diabetes.

The efficacy of high pressure liquid chromatography (HPLC) in detecting congenital glycosylation disorders (CDG)

Abstract Objectives Congenital disorders of glycosylation (CDG) are a family of rare inherited metabolic disorders. This study aimed to examine the carbohydrate-deficient transferrin (CDT) screening results of 1,328 patients with suspected CDG by using transferrin- high pressure liquid chromatography (Tf- HPLC) method and to evaluate the performance of the method as a reference diagnostic tool. Methods Relative CDT levels (CDT concentrations expressed as percent of total transferrin) were determined in serum samples by HPLC. The method sensitivity, specificity and positive predictive value (PPV) were further calculated. Results Abnormal transferrin isoform profile consistent with CDG Type-I and CDG Type-II were determined in 50 cases; in 44 cases asiolo-Tf (7.63 ± 5.44 %) and disialo-Tf (36.29 ± 9.04 %), in six cases monosialo-Tf (3.95 ± 0.95 %) and trisialo-Tf (25.05 ± 4.46 %) were determined and decreased tetrasialo-Tf (49.75 ± 11.59 %) was identified in all cases. Two cases having abnormal CDT pattern were molecularly diagnosed with hereditary fructose intolerance and galactosemia and 11 cases diagnosed with CDG based on clinical and molecular analysis showed a normal pattern. The sensitivity, specificity and positive predictive values of Tf-HPLC method were 81.96 %, 99% and 96 %, respectively. Conclusions Tf-HPLC is a useful, highly sensitive, cost-advantageous and reliable method for the detection and preliminary diagnosis of CDG for laboratories working with large sample series.

Biomarkers of response to ocrelizumab in relapsing–remitting multiple sclerosis

ObjectiveTo ascertain the changes of serum neurofilament light chain (sNfL) and glial fibrillary acidic protein (sGFAP) values in relapsing–remitting multiple sclerosis (RRMS) patients treated with ocrelizumab and their association with treatment response.MethodsMulticenter prospective study including 115 RRMS patients initiating ocrelizumab treatment between February 2020 and March 2022 followed during a year. Serum samples were collected at baseline and every 3 months to measure sNfL and sGFAP levels using single-molecule array (SIMOA) technology. Based on age and body mass index, sNfL values were standardized using z-score. NEDA (non-evidence of disease activity)-3 status was defined for patients free of disease activity after a year of follow-up. Inflammation (INFL) was considered when new relapses occurred during follow-up or new MRI lesions were found at 1-year exploration. PIRA (progression independent of relapse activity) was defined as disability progression occurring in the absence of relapses or new MRI activity.ResultsAfter a year on ocrelizumab, 85 patients (73.9%) achieved NEDA-3. Thirty patients did not achieve NEDA: 20 (17.4%) because of INFL and 10 (8.7%) because of PIRA. Of INFL patients, 6 (30.0%) had relapses, and 17 (85.0%) had at least one new MRI lesion at the 12-month examination. At baseline, INFL patients had higher sNfL (p = 0.0003) and sGFAP (p = 0.03) than the NEDA-3 group. PIRA patients mostly exhibited low sNfL and heterogeneous sGFAP levels. After a year, NEDA-3 and INFL patients showed similar decreases in sNfL (p < 0.0001) and sGFAP (p < 0.0001 for NEDA-3 and p = 0.001 for INFL ones). However, the decrease occurred earlier in NEDA-3 patients. Accordingly, sNfL > 1.5 z-score 3 months after ocrelizumab initiation indicated a higher risk of inflammation (OR = 13.6; p < 0.0001). Decrease in sGFAP values occurred later in both groups, with significant reductions observed at 12 months for INFL and 6 and 12 months for NEDA-3. No significant changes in sNfL or sGFAP were observed in PIRA patients.ConclusionOcrelizumab induced normalization of sNfL and sGFAP in the majority of NEDA-3 and inflammatory patients but did not cause changes in the PIRA group. Our data suggest that normalization of sNfL and sGFAP is associated with the lack of inflammatory-associated disease progression but it may not affect non-inflammatory PIRA.

Effect of SARS-CoV-2 Infection on Selected Parameters of the Apelinergic System in Repeat Blood Donors

Background: SARS-CoV-2 enters cells primarily by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, thereby blocking its physiological functions, affecting the apelinergic system, and inhibiting the cleavage of its peptides. The appropriate concentration of peptides in the apelinergic system influences the maintenance of homeostasis and protects against cardiovascular diseases. In our research, we determined the level of selected parameters of the apelinergic system—apelin (AP), elabela (ELA), and the apelin receptor (APJ)—in repeat blood donors. Methods: We analyzed 120 serum samples obtained from 30 repeat donors (study group) within four time periods after a SARS-CoV-2 infection: <60 days, 61–90 days, 91–120 days, and >120 days. We compared the results from the study groups with those of the control group, which consisted of 30 serum samples collected from donors donating blood in the years 2018–2019. Results: We observed that the AP, ELA, and APJ concentrations in the control group are higher than in any period in the study group. In the study group, the concentrations of AP and ELA increased in subsequent study periods. AP and ELA concentrations were lower shortly after SARS-CoV-2 transfection and then slowly increased in subsequent periods. APJ concentrations, on the other hand, were lowest at 61–90 days after the infection, but the decrease, relative to their level in healthy subjects, was significant in every period studied. Conclusions: The results suggest that infection with SARS-CoV-2 causes changes in the parameters of the apelinergic system, both after a short period of time has passed since the onset of the SARS-CoV-2 infection, and even up to 4 months after the infection.