Digital PCR Research Articles

Hypoxia-Induced Inflammation in In Vitro Model of Human Blood-Brain Barrier: Modulatory Effects of the Olfactory Ensheathing Cell-Conditioned Medium.

Hypoxia compromises the integrity of the blood-brain barrier (BBB) and increases its permeability, thereby inducing inflammation. Olfactory ensheathing cells (OECs) garnered considerable interest due to their neuroregenerative and anti-inflammatory properties. Here, we aimed to investigate the potential modulatory effects of OEC-conditioned medium (OEC-CM) on the response of human brain microvascular endothelial cells (HBMECs), constituting the BBB, when exposed to hypoxia. HBMECs were utilized to establish the in vitro BBB model. OECs were isolated from mouse olfactory bulbs, and OEC-CM was collected after 48h of culture. The effect of OEC-CM treatment on the HBMEC viability was evaluated under both normoxic and hypoxic conditions at 6h, 24h, and 30h. Western blot and immunostaining techniques were employed to assess NF-κB/phospho-NF-κB expression. HIF-1α, VEGF-A, and cPLA2 mRNA expression levels were quantified using digital PCR. ELISA assays were performed to measure PGE2, VEGF-A, IL-8 secretion, and cPLA2 specific activity. The in vitro formation of HBMEC capillary-like structures was examined using a three-dimensional matrix system. OEC-CM attenuated pro-inflammatory responses and mitigated the HIF-1α/VEGFA signaling pathway activation in HBMECs under hypoxic condition. Hypoxia-induced damage of the BBB can be mitigated by novel therapeutic strategies harnessing OEC potential.

Circulating tumour DNA in patients with hepatocellular carcinoma across tumour stages and treatments

ObjectiveCirculating tumour DNA (ctDNA) is a promising non-invasive biomarker in cancer. We aim to assess the dynamic of ctDNA in patients with hepatocellular carcinoma (HCC).DesignWe analysed 772 plasmas from 173...

KRAS mutations detection methodology: from RFLP to CRISPR/Cas based methods.

In personalized cancer medicine, the identification of KRAS mutations is essential for making treatment decisions and improving patient outcomes. This work presents a comprehensive review of the current approaches for detection of KRAS mutations in different cancers. We highlight the value of fast and reliable KRAS mutations discovery and the effectiveness of molecular testing for selecting individuals who might benefit from targeted therapy. We provide an overview of various methods and tools available for detecting KRAS mutations, such as digital droplet PCR, next-generation sequencing (NGS), and polymerase chain reaction (PCR). We also address the difficulties and limitations in the identification of KRAS mutations, namely tumor heterogeneity and the emergence of resistance mechanisms. This article aims to guide clinicians in KRAS mutation identification.

7826 The Identification of an Activating ESR1 Mutation in an Aggressive Prolactinoma Enabled the Use of Personalized Treatment

Abstract Disclosure: T. Paes: None. J.B. Mebarak: None. J. C. Magnotto: None. G. A. Stamatiades: None. Y. Kuang: None. C. Paweletz: None. E.R. Laws: None. R.S. Carroll: None. R. Jeselsohn: None. D. R. Mohan: None. A. Marcondes Lerario: None. W. L. Bi: None. D. A. Reardon: None. D. M. Meredith: None. U.B. Kaiser: None. A. Abreu: None. Introduction: Prolactinomas are benign tumors typically treated with dopamine agonists; few progress on medical therapy, through unclear molecular mechanisms. Although the SF3B1 mutation has been identified in one study as a driver of aggressive prolactinomas, in most cases no genetic mutations have been reported. We aimed to identify genetic alterations associated with prolactinomas using a gene panel. Method and Results: Oncopanel, a massively parallel sequencing panel, was performed to identify genomic variants and copy number variation (CNV) in a cohort of 21 patients with prolactinomas. No SF3B1 pathogenic variants were identified in this cohort. A MEN1 mutation was detected in a macroadenoma resistant to dopamine agonist. In a case of an extremely aggressive prolactinoma diagnosed in a 49-year-old woman, a somatic ESR1 [encoding estrogen receptor alpha (ERα)] mutation (ESR1Y537S) was identified in tissue from the patient’s fourth surgery. Using droplet digital PCR (ddPCR), ESR1Y537S was also detected in circulating cell-free DNA. We aimed to investigate whether ESR1Y537S may have driven initial tumor formation, or whether it was acquired as the tumor progressed. To this end, Sanger sequencing and ddPCR analyses of DNA from the first two resected tumors were performed and failed to identify ESR1Y537S. In the CNV analysis, the ESR1-mutated prolactinoma had the highest number of CNVs, compared to the rest of the cohort. Despite efforts to control tumor growth with multiple therapeutic modalities, the mutated prolactinoma had progressively enlarged post-menopausally, with invasion of surrounding structures. ESR1Y537S is a hotspot mutation in metastatic breast cancer. In breast cancer cells, ESR1Y537S activates the ERα independent of ligand binding, resulting in high cell proliferation and conferring resistance to classic hormonal treatment in ER-positive breast cancer. Elacestrant, a second-line ER degrader, increases overall free-survival in patients with resistant breast cancer and ESR1Y537S. Given the lack of response to multimodality therapy, off-label elacestrant was initiated after the third cycle of radiotherapy was completed in the patient with the aggressive prolactinoma. The combination of radiotherapy with this personalized treatment targeting ESR1 activity (elacestrant) was able to control tumor growth and significantly reduce prolactin levels. Conclusion:ESR1 regulates lactotroph differentiation and proliferation, possibly contributing to prolactinoma tumorigenesis. Our data support a role for ESR1Y537S in this prolactinoma’s unusually aggressive behavior in the postmenopausal setting. Molecular profiling revealing the mutation (ESR1Y537S) allowed the patient to receive a targeted therapy which, together with radiotherapy, resulted in a decrease in prolactin levels and a remarkable improvement in disease control. Presentation: 6/2/2024

12614 Induction Of Gpt2 During Metabolic Stress Is Associated With Reduced Incretin Responsiveness And Decreased β-cell Resilience To Metabolic Stress

Abstract Disclosure: S. Sen: None. A.V. Rozo: None. M. Haemmerle: None. J. Roman: None. A.V. Scota: None. J.A. Christine: None. E.M. Morrow: None. S.A. Tersey: None. C.B. Newgard: None. N.M. Doliba: None. D.A. Stoffers: None. Elevated blood glucose and lipids compromise pancreatic β-cell survival and function, but the underlying molecular mechanisms are ill-understood. We previously found that mitochondrial glutamate-pyruvate transaminase 2 (GPT2) is induced by ER and glucolipotoxic (GLT) stress. Here, we find that islets from type 2 diabetic donors exhibited markedly elevated levels of GPT2 compared to those of non-diabetic donors. GLT stress induced GPT2 in MIN6 cells, primary murine, and human non-diabetic islets. We generated β-cell specific Gpt2-deficient (Gpt2βKO) and Gpt2 floxed control (Gpt2f/f) mice to study the in vivo role of Gpt2 in β-cells. Intraperitoneal (IP) glucose tolerance (GTT) of Gpt2βKO mice was minimally affected on chow diet; therefore, we placed Gpt2f/f and Gpt2βKO mice on 45 kcal% high-fat and matched control diets. At 1 week, HFD-fed Gpt2βKO mice exhibited reduced β-cell death, measured by droplet digital PCR of circulating unmethylated preproinsulin DNA and by TUNEL analysis. Over 37 weeks, HFD-fed Gpt2βKO mice showed lower random blood glucose similar to Gpt2f/f mice under a control diet. While IPGTT was modestly improved in HFD-fed Gpt2βKO compared to HFD-fed Gpt2f/f mice, insulin secretion was not enhanced. Remarkably, oral glucose administration markedly improved glucose tolerance and enhanced insulin secretion of control- and HFD-fed Gpt2βKO mice compared to Gpt2f/f mice, while GLP-1 and GIP levels were unaltered, suggesting enhanced incretin action on the β-cell. Indeed, Gpt2βKO mice (under both control and high-fat diets) were more responsive to administration of GLP1R agonist Exendin-4 (Ex4) than Gpt2f/f mice. Isolated Gpt2βKO islets secreted more insulin when stimulated by Ex4 and GIP but not acetylcholine. Perifusion assay revealed markedly enhanced insulin secretion of Gpt2βKO islets in response to a stepwise exposure to increasing Ex4 doses, indicating enhanced incretin sensitivity. Metabolic flux analysis of Gpt2βKO and Gpt2f/f islets exposed to uniformly labeled glucose (U-13C glucose) under low (2.8mM), high (12mM) glucose, and high glucose with Ex4 conditions revealed elevated levels of key TCA cycle intermediates in Gpt2βKO islets under high glucose and high glucose+Ex4 conditions, indicating increased TCA cycle flux that likely enhances insulin secretion. Gpt2βKO mice had higher β cell mass than Gpt2f/f littermates under control diet and HFD feeding. Bulk islet RNA-seq from Gpt2f/f and Gpt2βKO mice exposed to GLT revealed upregulation of genes responsible for cell cycle and division pathways and downregulation of apoptosis and ER stress genes, consistent with enhanced survival of Gpt2βKO islets under stress conditions. In summary, β-cell GPT2 is increased during metabolic stress and T2D. Its deficiency induces a metabolic reprogramming that improves incretin sensitivity and protects pancreatic β-cells from metabolic stress. Presentation: 6/2/2024

Catching the Silent Culprits: TERT Promoter Mutation Screening of Minimally Invasive Follicular and Oncocytic Thyroid Carcinoma in Clinical Practice.

De-escalation of thyroid cancer treatment is crucial to prevent overtreatment of indolent disease, but it remains important to identify clinically aggressive cases. TERT promoter mutations are molecular events frequently associated with high-risk thyroid tumors with poor outcomes and may identify cases at risk of dissemination. In various international guidelines, small minimally invasive follicular thyroid carcinoma and oncocytic thyroid carcinoma (miFTC/miOTC) are classified as low-risk lesions and are not recommended adjuvant treatment. Our study aimed to explore the association between size-based risk assessment and TERT promoter mutations. Between 2019 and May 2024, 84 miFTCs/miOTCs diagnosed at our department underwent digital droplet PCR analysis targeting TERT promoter mutational hotspots C228T and C250T in clinical routine. TERT promoter mutations were found in 10 out of 84 cases (11.9%). Mutated cases were pT1 (n = 1), pT2 (n = 3), or pT3 (n = 6). Patients with mutated tumors were older compared to patients with wild-type tumors (median age of 71years vs. 57years, p = 0.041). There were no significant differences regarding patient sex, tumor size, Ki-67 labeling index, or the presence of distant metastases. Notably, 30% of mutations displayed variant allele frequencies < 10%, possibly suggesting subclonal events. To conclude, TERT promoter mutations in miFTCs and miOTCs were associated with higher patient age and were often suspected to be subclonal. However, they did not affect clinical outcomes, possibly due to short follow-up. Reflex testing for this genetic alteration in miFTCs and miOTCs could be justified regardless of tumor size, though the clinical benefit remains uncertain.

Noninvasive, microbiome-based diagnosis of inflammatory bowel disease.

Despite recent progress in our understanding of the association between the gut microbiome and inflammatory bowel disease (IBD), the role of microbiome biomarkers in IBD diagnosis remains underexplored. Here we developed a microbiome-based diagnostic test for IBD. By utilization of metagenomic data from 5,979 fecal samples with and without IBD from different geographies and ethnicities, we identified microbiota alterations in IBD and selected ten and nine bacterial species for construction of diagnostic models for ulcerative colitis and Crohn's disease, respectively. These diagnostic models achieved areas under the curve >0.90 for distinguishing IBD from controls in the discovery cohort, and maintained satisfactory performance in transethnic validation cohorts from eight populations. We further developed a multiplex droplet digital polymerase chain reaction test targeting selected IBD-associated bacterial species, and models based on this test showed numerically higher performance than fecal calprotectin in discriminating ulcerative colitis and Crohn's disease from controls. Here we discovered universal IBD-associated bacteria and show the potential applicability of a multibacteria biomarker panel as a noninvasive tool for IBD diagnosis.

The incidence of donor white blood cell survival (transfusion-associated microchimerism) in Australian pediatric patients.

Donor leucocyte survival following red blood cell (RBC) transfusion, known as transfusion-associated microchimerism (TAM), can occur in some patients. In Australia, despite the introduction of leucocyte filtration (leucodepletion) during RBC manufacture, TAM has been detected in adult trauma patients. However, the incidence of TAM in Australian pediatric patients has not been analyzed. Patients aged 0-16 years were recruited across two cohorts. Retrospective participants had RBC transfusion between January 1, 2002 and November 15, 2017 and prospective participants received RBC transfusion between December 1, 2016 and November 25, 2020. Twelve bi-allelic insertion/deletion (InDel) polymorphisms were used to detect microchimerism amplification patterns using real-time PCR (RT-PCR) and droplet digital PCR (ddPCR). Of the retrospective cohort (n = 40), six patients showed amplification of InDel sequences indicating potential microchimerism. For three patients, minor InDel sequences were detected using RT-PCR only, two patients had minor InDel amplification using ddPCR only, and one patient had minor InDel amplification that was confirmed using both techniques. Amplification of minor sequences occurred in three patients who had received a bone marrow transplant in addition to RBC transfusion. In the prospective cohort (n = 25), no InDel amplification indicating potential microchimerism was detected using RT-PCR. Cell-based therapies had been administered in three patients where microchimerism amplification patterns were detected. Three patients have microchimerism that may be attributed to RBC transfusion. In prospective patients, who received leucodepleted and gamma-irradiated RBC units, no potential microchimerism amplification were detected. ddPCR may be a suitable technique for TAM analysis but requires further evaluation.

B-228 Performance Comparison of three viral RNA pathogens in MK Buffered Solution for Diagnostic Based Detection using quantitative PCR vs droplet digital PCR

Abstract Background Droplet digital PCR (ddPCR) by partitioning a single reaction into many thousands of nano-droplets allows for the absolute quantitation of a given DNA/RNA target and represents the gold standard for the identification and quantification of pathogens in clinical and environmental samples. While nucleic acid based molecular diagnostics have become fundamental for the accurate and rapid quantification of specific pathogens, these instruments are still based upon quantitative PCR (QPCR) assays. The goal of this study was to assess the compatibility of a MK Buffered solution with three different RNA viruses combined into two standard clinical negative matrices and compare performance of ddPCR and QPCR for all three pathogens. Methods Serial dilutions of heat-inactivated Sars-CoV-2 (NR-52286), Influenza A (ATCC VR-1469) and RSV-A (ATCC strain VR-26) were combined into a single tube and added to two confirmed clinically negative matrices, Nasal and Sputum (Lee Biosolutions, Maryland Heights, MO). Serial dilutions of the combined pathogens were added to sample tubes containing 500 µL of MK buffered solution yielding final concentrations of each pathogen over 4 orders of magnitude in each clinical matrix. For each sample 300 µl was processed for RNA isolation using standard RNA isolation kits (Zymo Viral RNA Kit). For ddPCR samples were quantified for all three pathogens using a BioRad® Droplet Digital PCR System (QXD200). For QPCR, samples were quantified using Quantitative real-time PCR using Promega GoTaq® Probe 1- Step RT-qPCR System. The same TAQMan® probes specific for each pathogen were used for both systems. Results Yields of all three viral RNAs were significantly lower from sputum samples as compared to nasal samples. For all three pathogens the ddPCR assay was an order of magnitude more sensitive than observed with QPCR. QPCR and ddPCR values showed a significant linear relationship for all three pathogens (Sars-CoV-2, r2 = 0.984; RSV A, r2 = 0.814; Influenza A, r2 = 0.985). Conclusions QPCR and Droplet digital PCR accurately quantified concentrations of all three RNA pathogens in both clinically negative nasal and sputum matrices across four orders of magnitude, though detected yields were significantly lower in samples spiked into the sputum matrix. For all three pathogens there was a significant linear relationship with decreasing concentration. ddPCR was an order of magnitude more sensitive than QPCR for all three pathogens.

Integrative Biomarker Panel for Improved Lung Cancer Diagnosis Using Plasma microRNAs and Sputum Bacterial DNA.

This study aimed to evaluate if integrating diverse molecular biomarkers in plasma and sputum could improve the diagnosis of lung cancer. The study analyzed miRNAs in plasma and bacterial DNA in sputum from 58 lung cancer patients and 62 cancer-free smokers using droplet digital PCR. The individual plasma miRNA and sputum bacterial biomarkers had sensitivities of 62-71% and specificities of 61-79% for diagnosing lung cancer. A panel of plasma miRNA or sputum bacterial biomarkers produced sensitivities of 79-85% and specificities of 74-82%. An integromic signature consisting of two miRNAs in plasma and three bacterial biomarkers in sputum had a higher sensitivity (87%) and specificity (89%) compared to individual biomarkers. The signature's diagnostic value was confirmed in a validation cohort of 56 lung cancer patients and 59 controls, independent of tumor stage, histological type, and demographic factors. Integrating diverse molecular biomarkers in plasma and sputum could improve the diagnosis of lung cancer.

B-231 Evaluation of the KingFisher Flex for DNA Isolation From FFPE Tissue Utilizing Autolys M Tubes and the MagMAX FFPE DNA/RNA Ultra Kit

Abstract Background Our institution is facing increasing demand for oncology testing that requires high quality DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. The current DNA extraction workflow involves a time and labor-intensive manual process that limits our ability to accommodate increasing downstream clinical testing volumes. We evaluated the KingFisher (KF) Flex utilizing Autolys M Tubes and the MagMAX FFPE DNA/RNA Ultra Kit as a possible high-throughput, automated DNA isolation workflow option that would decrease hands-on time and be conducive to volume growth without the need for additional staff. Methods DNA from 52 FFPE tissue samples (n=24 unique cases previously extracted by the current “manual method”) were extracted using Autolys M tubes, the MagMAX FFPE DNA/RNA Ultra Kit, and the KF Flex (Thermo Fisher Scientific Waltham, MA) over 4 “automated method” runs. Method modifications to the published procedure were introduced over the runs to determine any effects on DNA quality/quantity. Timings were taken during each run to compare hands-on and total run time against that of the manual method. Precision experiments were performed by extracting 4 replicates of sample on a single run (intra-assay precision) and 2 samples from 2 cases over 2 runs (inter-assay precision). Three cases were processed using varying tissue input levels to assess input-yield correlation. DNA yield was measured using HS dsDNA Qubit (Thermo Fisher Scientific, Waltham, MA). A subset of samples was tested by digital droplet PCR (ddPCR) (n=10), chromosomal microsomal microarray (CMA) (n=1), and next-generation sequencing (NGS) (n=3). Variants and QC metrics of the KF DNA were compared to those of the manual method DNA. Results The average total DNA yield was 3% lower from samples extracted following the manufacturer recommended KF procedure compared to matched samples extracted using the manual method (n=28 samples from 23 unique cases). A CV of 2.8% was observed in an intra-assay experiment (n=4 replicates from 1 case) and 3.0% in an average inter-assay experiment (n=2 replicates from 2 cases). There was a direct correlation between the tissue input amount (4 amounts of varied tissue input) and the total DNA yield (n=3 samples). We observed concordant fractional abundance values for ddPCR samples and concordant variant detection for NGS and CMA samples. We did not see a significant difference in the quality metrics tracked for any of the downstream testing assays. Conclusions This evaluation demonstrated that, as compared to our manual method, the automated method resulted in comparable DNA yields and downstream assay results. The incorporation of this automation would significantly increase our FFPE DNA extraction capacity without requiring additional staff while reducing hands-on time per run by about six hours. We would also be able to reduce ergonomic hazards related to tube cap manipulation, eliminate use of hazardous reagents, and leverage the plate format to better integrate extracted DNA into our clinical sample preparation workflows. These operational benefits highlight the value of automating high-throughput workflows.

Evaluation of Molecular Assays for Diagnosis of Amoebic Liver Abscess in India with Bayesian Latent Class Analysis.

Amoebic liver abscess (ALA) is the most common extra-intestinal complication of Entamoeba histolytica, accounting for 50,000 deaths annually, and is endemic in South Asia. Diagnosis based on microscopic examination is insensitive, and serological assays are not discerning of current infections in endemic settings with high exposure. For a rapid and confirmatory laboratory diagnosis of ALA, the performance of a polymerase chain reaction (PCR), quantitative real time PCR (qPCR), digital droplet PCR (ddPCR), and a loop-mediated isothermal amplification (LAMP) assay that detects E. histolytica DNA in liver abscess pus, and a lectin antigen detection ELISA were evaluated against clinical diagnosis (based on predefined criteria) as the gold standard. Owing to the lack of a laboratory gold standard, a Bayesian latent class analysis approach was also used to determine sensitivity and specificity of these assays. In the latent class analysis, qPCR and ddPCR showed the highest sensitivity (98% and 98.1%) and specificity (both 96.6%), and although clinical diagnosis had a comparable sensitivity to qPCR and ddPCR (95.2%), poorer specificity (64.3%) was seen. Kappa agreement analysis showed that qPCR and ddPCR had a perfect agreement of 1 followed by an agreement of 0.76 (95% CI: 0.64-0.88) with PCR. Considering the performance characteristics and relative ease of setting up qPCR as well as the wide availability of qPCR equipment needed, this would be the most optimal assay for rapid, confirmatory, molecular diagnosis of ALA in the tertiary care laboratory setting in India, whereas further optimization of LAMP or antibody-based detection is required for use at smaller or secondary hospitals.

Water and Electrolyte Content in Hypertension in the Skin (WHYSKI) in Primary Aldosteronism.

Primary aldosteronism (PA), the most common curable salt-dependent form of arterial hypertension, features renal K+ loss and enhanced Na+ reabsorption. We investigated whether the electrolyte, water, and TonEBP (tonicity-responsive enhancer binding protein)/NFAT5 (nuclear factor of activated T cells 5) content is altered in the skin of patients with PA and corrected by surgical cure. We obtained skin biopsies from 80 subjects: 49 consecutive patients with PA, optimally treated with a mineralocorticoid receptor antagonist; 6 essential hypertensives; and 25 normotensive controls. We measured Na+, K+, water content with atomic absorption spectroscopy after ashing, and NFAT5 mRNA with digital droplet polymerase chain reaction. The patients with PA were retested after adrenalectomy. We discovered a higher dry weight of the skin biopsy specimen at surgery than at follow-up (P<0.001) and a direct correlation with electrolyte and water content (all P<0.01), indicating the need for dry weight adjustment of electrolyte and water data. Surgical cure of PA markedly increased skin dry weight-adjusted K+ (from 1.14±0.1 to 2.81±0.27 µg/mg; P<0.001) and water content (from 2.92±1.4 to 3.85±0.23 mg/mg; P<0.001), but left dry weight-adjusted skin Na+ content unaffected. In patients with PA, NFAT5 mRNA was higher (P=0.031) than in normotensive controls and decreased after surgery (P=0.035). Despite mineralocorticoid receptor antagonist treatment ensuring normokalemia, the patients with PA had a skin cell K+ depletion that was corrected by adrenalectomy. The activated NFAT5/TonEBP pathway during mineralocorticoid receptor antagonist administration suggests enhanced skin Na+ lymphatic drainage and can explain the lack of overt skin Na+ accumulation in patients with PA. Its deactivation after surgical cure can account for the lack of skin Na+ decrease postadrenalectomy. URL: https://www.clinicaltrials.gov; Unique identifier: NCT06090617.

Circulating methylated HOXA9 tumor DNA as a biomarker for mortality in recurrent breast cancer: An exploratory study.

Methylated homeobox A9 (meth-HOXA9) circulating tumor DNA may be a relevant biomarker in breast cancer, although its clinical significance remains unknown. The present exploratory study aimed to investigate the association between meth-HOXA9 and mortality in patients with recurrent breast cancer. The cohort study enrolled 51 patients with breast cancer recurrence from the Department of Oncology, Vejle Hospital, University Hospital of Southern Denmark (Vejle, Denmark). Tissue samples from primary surgery and biopsies, and plasma samples obtained at the time of recurrence were analyzed for meth-HOXA9 using a methylation-specific droplet digital polymerase chain reaction. Using Cox regression, hazard ratios (HRs) for mortality with 95% confidence intervals (CIs) comparing patients with detectable and undetectable meth-HOXA9 in both tumor tissue and plasma were estimated. Among the 50 patients with data on tumor tissue meth-HOXA9, there was no association between meth-HOXA9 in the primary tumor and mortality (HR 1.09, 95% CI 0.47-2.52). A total of 34 patients had data on plasma meth-HOXA9 at the time of recurrence. Detectable plasma meth-HOXA9 was associated with higher mortality (HR 3.95, 95% CI 1.50-10.37). Among the 20 patients with data on both plasma and metastatic tissue meth-HOXA9, meth-HOXA9 was detectable in 90% of metastases and 65% of plasma samples. In conclusion, detectable plasma meth-HOXA9 was significantly associated with higher mortality in recurrent breast cancer; therefore, plasma meth-HOXA9 may prove useful as a prognostic marker in patients with breast cancer.

A-284 Development of a Low Positive Molecular Control for Mycoplasma genitalium and Trichomonas vaginalis Screening

Abstract Background Mycoplasma genitalium (MG) is a sexually transmitted gram -negative bacteria that infects the urinary and genital tracts. Trichomonas vaginalis (TV) is a protozoan parasite and is the most common curable sexually transmitted infection (STI) worldwide. TV/ MG infections often go unrecognized because infected individuals are either asymptomatic or have symptoms similar to other infections of the urogenital tract (cervicitis in females and urethritis in males). Left untreated, TV/MG can lead to severe health complications including pelvic inflammatory disease, adverse pregnancy outcomes such as preterm delivery and miscarriage, increased risk of HIV acquisition, sexually acquired reactive arthritis, and infertility. The rates of STIs are increasing and differentiating among different STIs is critical to ensure patients receive effective treatment. Amplified nucleic acid based tests (NAATs) have emerged as the gold standard for diagnosis of TV/MG infections due to their high sensitivity and high throughput using a variety of clinical samples including male and female urine. This study describes work to develop a low positive quality control material in human urine for TV/MG amplified nucleic acids tests. Methods A method was developed to eliminate residual enzymatic activity from raw, pooled human urine from both genders to create a homogeneous diluent that supports analyte stability. The urine diluent was tested for viral pathogens (HIV-1, HCV, HBV) and ensure urine proteins and chemistry analytes were within acceptable limits. TaqMan® assays for TV (targeting RNA polymerase Rpb1 gene) and MG (targeting SMC Chromosome segregation gene) were developed and qualified for digital PCR quantitation of these organisms using the BioRad QX200 system. Cultured Trichomonas vaginalis strain C-1:NIH and Mycoplasma genitalium strain G37 were spiked into the urine diluent along with 0.09% sodium azide as a preservative. The concentration of TV and MG was guided by digital PCR quantitation throughout formulation and the final product was tested on Abbott Alinity m STI Panel, Hologic Aptima® Mycoplasma genitalium Assay, and Hologic Aptima® Trichomonas vaginalis Assay. Results Three independent lots were produced that were each detected for both TV and MG on the Abbott Alinity m STI panel. The concentration of the controls targeted a cycle number (CN) of ∼27 for TV and ∼32 for MG and were within acceptable limits. The lots were also positive for TV and MG when tested on the Hologic Aptima TV and MG assays. Accelerated stability studies showed that the product is stable throughout 2 years of shelf life when stored frozen at -20 °C, but is also stable for up to 30 days at 2-8 °C refrigerated storage. Conclusions LGC Clinical Diagnostics is developing a stable, whole organism molecular control material for use with in vitro diagnostic test methods that detect TV and MG. The product will fulfill an unmet need for molecular quality controls for STI testing that are formulated in a true patient-like matrix like urine. This study helps ensure the molecular control will be a low positive on leading diagnostic assays, be consistent batch to batch, and have the needed stability to meet the needs of clinical testing labs.

Protocol for detection of pathogenic enteric RNA viruses by regular monitoring of environmental samples from wastewater treatment plants using droplet digital PCR

BackgroundThe present comprehensive protocol is focused on the detection of pathogenic enteric RNA viruses, explicitly focusing on norovirus genogroup Ⅱ (GⅡ), astrovirus, rotavirus, Aichi virus, sapovirus, hepatitis A and E viruses in wastewater treatment plants through droplet digital PCR (ddPCR). Enteric viruses are of significant public health concern, as they are the leading cause of diseases like gastroenteritis. Regular monitoring of environmental samples, particularly from wastewater treatment plants, is crucial for early detection and control of these viruses. This research aims to improve the understanding of the prevalence and dynamics of enteric viruses in urban India and will serve as a model for similar studies in other regions. Our protocol objective is to establish a novel ddPCR-based methodology for the detection and molecular characterization of enteric viruses present in wastewater samples sourced from Bhopal, India. Our assay is capable of accurately quantifying virus concentrations without standard curves, minimizing extensive optimization, and enhancing sensitivity and precision, especially for low-abundance targets. MethodsThe study involves fortnightly collecting and analyzing samples from nine wastewater treatment plants over two years, ensuring comprehensive coverage and consistent data. Our study innovatively applies ddPCR to simultaneously detect and quantify enteric viruses in wastewater, a more advanced technique. Additionally, we will employ next-generation sequencing for detailed viral genome identification in samples tested positive for pathogenic viruses. ConclusionThis study will aid in understanding these viruses’ genetic diversity and mutation rates, which is crucial for developing tailored intervention strategies. The findings will be instrumental in shaping public health responses and improving epidemiological surveillance, especially in localities heaving sewage networks.

Using qPCR and ddPCR to study biodistribution of cell therapy products: a multi-site evaluation

Backgroud aimsRegenerative therapies employing cell therapy products (CTPs) have attracted considerable attention. Biodistribution (BD) evaluation of CTPs is mainly performed to clarify the cell survival time, engraftment, and distribution site. This evaluation is crucial for predicting the efficacy and safety profiles of clinical studies based on non-clinical BD study outcomes. However, no internationally unified method has been established for assessing cell BD after administration. Here, we aimed to standardize the BD assay method used for CTPs, conducting the following evaluations using the same protocol across multiple study facilities: (1) in vitro validation of quantitative polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR) analyses using the primate-specific Alu gene, and (2) in vivo BD studies after the intravenous administration of human mesenchymal stem cells (hMSCs) to immunodeficient mice, commonly used in non-clinical tumorigenicity studies. MethodsQuality control samples were prepared and analyzed by adding a fixed number of human-derived cells to several mouse tissues. The respective quantitative performances of the qPCR and ddPCR methods were compared for accuracy and precision. hMSCs were intravenously administered to immunodeficient mice, and tissues were collected at 1, 4, and 24 h after administration. ResultsBoth methods demonstrated an accuracy (relative error) generally within ±50% and a precision (coefficient of variation) generally less than 50%. While differences in calibration curve ranges were observed between qPCR and ddPCR, no significant differences in quantification were found among the assay facilities. The BD of hMSCs in mice was evaluated at seven facilities (qPCR at three facilities; ddPCR at four facilities), revealing similar tissue distribution profiles in all facilities, with the lungs showing the highest cell distribution among the tissues tested. ConclusionsQuantitative evaluation of qPCR and ddPCR using Alu sequences was conducted, demonstrating that the test method can be adapted for BD evaluation.

Exposure dose, light distribution and wavelength affect the fate of introduced bacterial biological control agents in the phyllosphere of greenhouse grown tomato

Societal Impact StatementThe use of chemical plant protection products must be reduced to promote sustainability in food production. One possible alternative is biological control agents (BCAs), but their efficacy under commercial conditions does not always reach the standard of chemical control agents. Previously, light has been found to induce mechanisms in bacterial BCAs that can affect their distribution and establishment. This could promote BCA efficacy. We looked into how monochromatic and polychromatic (which is what growers use) light treatments affected the occurrence of three BCAs post‐application. By combining two non‐chemical methods: a biological (BCA) and a cultural (light) control method, this offers a new integrated pest control strategy.Summary The dynamics and functionality of beneficial and non‐beneficial, non‐phototrophic bacteria can be influenced by light quality. We investigated if light could aid the survival of three bacterial biological control agents (BCAs; Bacillus amyloliquefaciens DSM7, Pseudomonas chlororaphis 50083 and Streptomyces griseoviridis CBS904.68) in the canopy of greenhouse‐grown tomatoes at four light treatments. Tomato plants were exposed to 50 μmol m−2 s−1 of either polychromatic light (white) or monochromatic light (blue: 420 nm, green: 530 nm and red: 660 nm) using DYNA LED lamps for a total of 48 h post foliar application of the BCAs. Leaves were harvested from two levels in the canopy at the top and middle of each plant at 0, 2, 4, 8, 12, 24 and 48 h post inoculation. The occurrences of the BCAs were quantified by plate count and droplet digital PCR (ddPCR). S. griseoviridis persisted under most treatments, whereas P. chlororaphis and B. amyloliquefaciens preferred the polychromatic and green light treatments as depicted by the viable count analyses. Significant differences between the DNA and cDNA concentrations were only noted for P. chlororaphis, with prominent wavelength effects. Light exposure dose, placement in the canopy and wavelength were found to be decisive factors for BCA re‐isolation, indicating that they have different optimal light environments.

453P Characterisation of cell culture models of myotonic dystrophy type I for drug screening by in-cell western and digital droplet PCR

453P Characterisation of cell culture models of myotonic dystrophy type I for drug screening by in-cell western and digital droplet PCR

LAMP-based electrochemical platform for monitoring HPV genome integration at the mRNA level associated with higher risk of cervical cancer progression.

Human papillomaviruses (HPVs) represent a diverse group of double-stranded DNA viruses associated with various types of cancers, notably cervical cancer. High-risk types of HPVs exhibit their oncogenic potential through the integration of their DNA into the host genome. This integration event contributes significantly to genomic instability and the progression of malignancy. However, traditional detection methods, such as immunohistochemistry or PCR-based assays, face inherent challenges, and thus alternative tools are being developed to fasten and simplify the analysis. Our study introduces an innovative biosensing platform that combines loop-mediated amplification with electrochemical (EC) analysis for the specific detection of HPV16 integration. By targeting key elements like the E7 mRNA, a central player in HPV integration, and the E2 viral gene transcript lost upon integration, we show clear distinction between episomal and integrated forms of HPV16. Our EC data confirmed higher E7 expression in HPV16-positive cell lines having integrated forms of viral genome, while E2 expression was diminished in cells with fully integrated genomes. Moreover, we revealed distinct expression patterns in cervical tissue of patients, correlating well with digital droplet PCR, qRT-PCR, or immunohistochemical staining. Our platform thus offers insights into HPV integration in clinical samples and facilitates further advancements in cervical cancer research and diagnostics.