Multiplex ddPCR Research Articles

361P Development of a next-generation multiplex ddPCR assay for measurement of in-frame dystrophin mRNA in people with DMD treated with BMN 351

361P Development of a next-generation multiplex ddPCR assay for measurement of in-frame dystrophin mRNA in people with DMD treated with BMN 351

In-Depth Comparison of Adeno-Associated Virus Containing Fractions after CsCl Ultracentrifugation Gradient Separation.

Recombinant adeno-associated viruses (rAAVs) play a pivotal role in the treatment of genetic diseases. However, current production and purification processes yield AAV-based preparations that often contain unwanted empty, partially filled or damaged viral particles and impurities, including residual host cell DNA and proteins, plasmid DNA, and viral aggregates. To precisely understand the composition of AAV preparations, we systematically compared four different single-stranded AAV (ssAAV) and self-complementary (scAAV) fractions extracted from the CsCl ultracentrifugation gradient using established methods (transduction efficiency, analytical ultracentrifugation (AUC), quantitative and digital droplet PCR (qPCR and ddPCR), transmission electron microscopy (TEM) and enzyme-linked immunosorbent assay (ELISA)) alongside newer techniques (multiplex ddPCR, multi-angle light-scattering coupled to size-exclusion chromatography (SEC-MALS), multi-angle dynamic light scattering (MADLS), and high-throughput sequencing (HTS)). Suboptimal particle separation within the fractions resulted in unexpectedly similar infectivity levels. No single technique could simultaneously provide comprehensive insights in the presence of both bioactive particles and contaminants. Notably, multiplex ddPCR revealed distinct vector genome fragmentation patterns, differing between ssAAV and scAAV. This highlights the urgent need for innovative analytical and production approaches to optimize AAV vector production and enhance therapeutic outcomes.

Quadruplex Droplet Digital PCR Assay for Screening and Quantification of SARS-CoV-2.

The ongoing COVID-19 pandemic, caused by the rapid global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since early 2020, has highlighted the need for sensitive and reliable diagnostic methods. Droplet digital PCR (ddPCR) has demonstrated superior performance over the gold-standard reverse transcription PCR (RT-PCR) in detecting SARS-CoV-2. In this study, we explored the development of a multiplex ddPCR assay that enables sensitive quantification of SARS-CoV-2, which could be utilized for antiviral screening and the monitoring of COVID-19 patients. We designed a quadruplex ddPCR assay targeting four SARS-CoV-2 genes and evaluated its performance in terms of specificity, sensitivity, linearity, reproducibility, and precision using a two-color ddPCR detection system. The results showed that the quadruplex assay had comparable limits of detection and accuracy to the simplex ddPCR assays. Importantly, the quadruplex assay demonstrated significantly improved performance for samples with low viral loads and ambiguous results compared to the standard qRT-PCR approach. The developed multiplex ddPCR represents a valuable alternative and complementary tool for the diagnosis of SARS-CoV-2 and potentially other pathogens in various application scenarios beyond the current COVID-19 pandemic. The improved sensitivity and reliability of this assay could contribute to more effective disease monitoring and antiviral screening during the ongoing public health crisis.

Multi-feature next-generation liquid biopsy for diagnosis and prognosis in HPV-associated head and neck cancer.

6064 Background: Human papillomavirus-associated head and neck squamous cell carcinoma (HPV+HNSCC) releases circulating tumor HPV DNA (ctHPVDNA) into the blood which is a highly accurate biomarker of disease status. Existing approaches based on droplet digital PCR (ddPCR) have limitations in sensitivity, genotype coverage, and do not annotate additional prognostic genomic features. In this study, we tested the diagnostic and prognostic performance of a custom HPV whole-genome next-generation sequencing (NGS) liquid biopsy, termed HPV-DeepSeek, at the time of diagnosis with HPV+HNSCC, compared to ctHPVDNA ddPCR, HPV serology, and clinical work-up. Methods: 304 participants (152 untreated HPV+HNSCC patients, 152 population-level controls) were prospectively enrolled in this single-center prospective cohort study. Plasma samples were analyzed using HPV-DeepSeek, a multiplexed serology assay, single-plex ddPCR, and a 5-genotype multiplexed ddPCR assay. We tested the hypothesis that HPV-DeepSeek would have the highest diagnostic accuracy. We secondarily examined the accuracy of HPV-DeepSeek for prognostic feature annotation including high-risk HPV16 single nucleotide polymorphisms (SNPs), integration events, and PIK3CA mutations. Results: Of 152 cases, 114 (75%) were AJCC stage 1 and 138/152 (91%) were oropharynx primary site cancers. The sensitivity (98.7%), specificity (98.7%), and diagnostic accuracy (0.974) of HPV-DeepSeek was superior (P<0.001) to singleplex ddPCR (94.2%, 98.6%, 0.928), multiplex ddPCR (90.6%, 96.3%, 0.869), HPV serology (86.4%, 96.3%, 0.827), and first attempt clinical diagnostic biopsy (78% sensitive). Utilizing HPV-DeepSeek, 8 different genotypes were detected. 4 patients were found to have multi-genotype infections. 137/152 cases were classified as HPV16, with A1 the most common sub-lineage (90/137). 21 of 137 HPV+HNSCC HPV16 cases (15%) displayed high risk SNPs. Head-to-head comparison of HPV SNP genotyping by HPV-DeepSeek, with the gold-standard tissue-based HPV whole genome sequencing assay, demonstrated 89% concordance in tissue and 85% in blood. The internal concordance rate for HPV-DeepSeek between tissue and blood was 91%. 48 of 152 cases (32%) had at least one viral integration event detected and 36 of 152 cases (24%) had a PIK3CA mutation detected by HPV-DeepSeek. Fragmentomic analysis demonstrated a mean ctHPVDNA fragment size of 148bp for ctHPVDNA versus 167bp for human cell free (cf)DNA from HPV+HNSCC patients and 168bp for control patient cfDNA. Baseline ctHPVDNA quantity was strongly correlated with viral integration status, T stage and N stage (P<0.001). Conclusions: HPV-DeepSeek has improved diagnostic accuracy relative to ddPCR, HPV serology and clinical work-up at the time of diagnosis and demonstrates detection of prognostic features including high-risk HPV16 SNPs, viral integration events, and PIK3CA mutations.

Simultaneous detection of influenza A, B and respiratory syncytial virus in wastewater samples by one-step multiplex RT-ddPCR assay

BackgroundAfter the occurrence of the COVID-19 pandemic, detection of other disseminated respiratory viruses using highly sensitive molecular methods was declared essential for monitoring the spread of health-threatening viruses in communities. The development of multiplex molecular assays are essential for the simultaneous detection of such viruses even at low concentrations. In the present study, a highly sensitive and specific multiplex one-step droplet digital PCR (RT-ddPCR) assay was developed for the simultaneous detection and absolute quantification of influenza A (IAV), influenza B (IBV), respiratory syncytial virus (RSV), and beta-2-microglobulin transcript as an endogenous internal control (IC B2M).ResultsThe assay was first evaluated for analytical sensitivity and specificity, linearity, reproducibility, and recovery rates with excellent performance characteristics and then applied to 37 wastewater samples previously evaluated with commercially available and in-house quantitative real-time reverse transcription PCR (RT-qPCR) assays. IAV was detected in 16/37 (43%), IBV in 19/37 (51%), and RSV in 10/37 (27%) of the wastewater samples. Direct comparison of the developed assay with real-time RT-qPCR assays showed statistically significant high agreement in the detection of IAV (kappa Cohen’s correlation coefficient: 0.834, p = 0.001) and RSV (kappa: 0.773, p = 0.001) viruses between the two assays, while the results for the detection of IBV (kappa: 0.355, p = 0.27) showed good agreement without statistical significance.ConclusionsOverall, the developed one-step multiplex ddPCR assay is cost-effective, highly sensitive and specific, and can simultaneously detect three common respiratory viruses in the complex matrix of wastewater samples even at low concentrations. Due to its high sensitivity and resistance to PCR inhibitors, the developed assay could be further used as an early warning system for wastewater monitoring.

Sensitive and rapid identification of pathogens by droplet digital PCR in a cohort of septic patients: a prospective diagnostic study

Background There is a critical need for a rapid and sensitive pathogen detection method for septic patients. This study aimed to investigate the diagnostic efficacy of Digital droplet polymerase chain reaction (ddPCR) in identifying pathogens among suspected septic patients. Methods We conducted a prospective pilot diagnostic study to clinically validate the multiplex ddPCR panel in diagnosing suspected septic patients. A total of 100 sepsis episodes of 89 patients were included in the study. Results In comparison to blood culture, the ddPCR panel exhibited an overall sensitivity of 75.0% and a specificity of 69.7%, ddPCR yielded an additional detection rate of 17.0% for sepsis cases overall, with a turnaround time of 2.5 h. The sensitivity of ddPCR in the empirical antibiotic treatment and the non-empirical antibiotic treatment group were 78.6% versus 80.0% (p > 0.05). Antimicrobial resistance genes were identified in a total of 13 samples. Whenever ddPCR detected the genes beta-lactamase-Klebsiella pneumoniae carbapenemase (blaKPC) or beta-lactamase-New Delhi metallo (blaNDM), these findings corresponded to the cultivation of carbapenem-resistant gram-negative bacteria. Dynamic ddPCR monitoring revealed a consistent alignment between the quantitative ddPCR results and the trends observed in C-reactive protein and procalcitonin levels. Conclusions Compared to blood culture, ddPCR exhibited higher sensitivity for pathogen diagnosis in suspected septic patients, and it provided pathogen and drug resistance information in a shorter time. The quantitative results of ddPCR generally aligned with the trends seen in C-reactive protein and procalcitonin levels, indicating that ddPCR can serve as a dynamic monitoring tool for pathogen load in septic patients.

Abstract PO4-15-07: ESR1 mutations emerging during neoadjuvant endocrine therapy in postmenopausal ER+/HER2- early breast cancer patients: prevalence and prognostic impact

Abstract Background Neoadjuvant endocrine therapy (NET) is currently used in several clinical trials to select patients for chemotherapy de-escalation. However, exposure to NET may induce/select endocrine-resistant tumor cell clones harboring ESR1 mutations (ESR1mut), which are well known to confer poor prognosis and ET resistance to aromatase inhibitors in the metastatic setting. In this study, we sought to determine the frequency and prognostic significance of ESR1mut after NET in postmenopausal patients with HR+/HER2- early breast cancer. Materials and methods We conducted a retrospective study in postmenopausal patients with ER+/HER2-early breast cancer diagnosed between 2000 and 2012 and treated with NET for at least three months at Institut Curie Hospitals (Paris and Saint Cloud, France). Exclusion criteria were: stage IV breast cancer, no surgery of the primary tumor, neoadjuvant radiation therapy, chemotherapy or targeted therapies. DNA from post-NET surgical samples was extracted using the phenol-chloroform DNA isolation method. ESR1mut were detected with multiplex drop-off ddPCR assay previously used in the PADA-1 trial (targeting clustered hotspot mutations in exon 8 and 5, using a BioRad QX100 system and analyzed with QuantaSoft v.1.7.4 software). Associations between ESR1mut detection and patient outcomes, distant relapse-free interval (DRFI) and overall survival (OS) were analyzed using the Kaplan-Meier method and Cox proportional hazards models. Results N=143 patients treated with NET were eligible for this study, of whom N=136 had interpretable ESR1mut detection results. Median age at NET initiation was 75.4 (IQR 66.6-81.3) years. The median duration of NET was 6.6 months (IQR 5.8 – 7.6). Most patients were treated with aromatase inhibitors (n=107, 75.9%), while 24.1% (n=34) received tamoxifen. The median time between diagnosis and surgery was 7.6 (IQR 6.4 – 8.7) months. ESR1mut were detected in post-NET surgical samples in N=7 patients (5.2%). Baseline clinicopathological characteristics were not associated with post-NET ESR1mut detection status. The median follow-up for DRFI and OS was 11.0 (IQR 7.7 – 13.8) and 18.7 (IQR 13.3 – 18.7) years, respectively. The median DRFI was 6.7 (95%CI [4.9-NR]) vs 14.0 [10.7-NR] years in ESR1mut and ESR1wt patients, respectively (HR=1.9 [0.6-6.2], p=0.29). Similarly, median OS was numerically shorter in patients with post-NET ESR1mut (6.8 [3.7-NR] vs 9.8 [8.5-NR]; HR=2.0 [0.9-4.7], p=0.09). Conclusions Post-NET tumors are enriched in ESR1mut subclones, which represent a potentially lethal threat in the context of aromatase inhibitor-based adjuvant endocrine therapy. Although the limited sample size of our study did not allow reaching statistical significance, our results suggest, for the first time, worse survival outcomes for patients with a ESR1mut detected in post-NET primary tumors. The role of next-generation oral SERDs as neoadjuvant or adjuvant endocrine therapy to prevent ESR1mut-related relapses remains to be determined. Citation Format: José Luis Sandoval, Helene Salaun, Shufang Renault, Marcela Carausu, Aurore Rampanou, Caroline Hego, Matthieu Carton, Luc Cabel, Jean-Yves Pierga, Romain Geiss, Francois-Clement Bidard, Florence Lerebours. ESR1 mutations emerging during neoadjuvant endocrine therapy in postmenopausal ER+/HER2- early breast cancer patients: prevalence and prognostic impact [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 PO4-15-07.

Development of a multiplex droplet digital PCR method for detection and monitoring of Mycobacterium tuberculosis and drug-resistant tuberculosis

BackgroundThe prevalence of multidrug-resistant tuberculosis (MDR-TB) among Korean tuberculosis patients is about 4.1%, which is higher than the OECD average of 2.6%. Inadequate drug use and poor patient compliance increase MDR-TB prevalence through selective pressure. Therefore, prompt detection of drug resistance in tuberculosis patients at the time of diagnosis and quantitative monitoring of these resistant strains during treatment are crucial.MethodsA multiplex droplet digital PCR (ddPCR) assay was developed and assessed using DNA material of nine Mycobacterium tuberculosis strains with known mutation status that were purchased from the Korean National Tuberculosis Association. We collected a total of 18 MDR-TB residual samples referred for PCR analysis. Total DNA was extracted from the samples and subjected to the quadruplex ddPCR assay. Their results were compared to those of known resistance phenotypes.ResultsThe analytical sensitivity and specificity of the multiplex ddPCR assay for detecting INH, RIF, EMB, FQ, and SM resistance-causing mutations ranged from 71.43 to 100% and 94.12–100%, respectively. Follow-up sample results showed that the quadruplex ddPCR assay was sensitive enough to detect IS6110 and other mutations even after onset of treatment.ConclusionsWe developed a sensitive and accurate multiplex ddPCR assay that can detect the presence of tuberculosis quantitatively and resistance-conveying mutations concurrently. This tool could aid clinicians in the diagnosis and treatment monitoring of tuberculosis.

Digital PCR quantification of ultrahigh ERBB2 copy number identifies poor breast cancer survival after trastuzumab

HER2/ERBB2 evaluation is necessary for treatment decision-making in breast cancer (BC), however current methods have limitations and considerable variability exists. DNA copy number (CN) evaluation by droplet digital PCR (ddPCR) has complementary advantages for HER2/ERBB2 diagnostics. In this study, we developed a single-reaction multiplex ddPCR assay for determination of ERBB2 CN in reference to two control regions, CEP17 and a copy-number-stable region of chr. 2p13.1, validated CN estimations to clinical in situ hybridization (ISH) HER2 status, and investigated the association of ERBB2 CN with clinical outcomes. 909 primary BC tissues were evaluated and the area under the curve for concordance to HER2 status was 0.93 and 0.96 for ERBB2 CN using either CEP17 or 2p13.1 as reference, respectively. The accuracy of ddPCR ERBB2 CN was 93.7% and 94.1% in the training and validation groups, respectively. Positive and negative predictive value for the classic HER2 amplification and non-amplification groups was 97.2% and 94.8%, respectively. An identified biological “ultrahigh” ERBB2 ddPCR CN group had significantly worse survival within patients treated with adjuvant trastuzumab for both recurrence-free survival (hazard ratio, HR: 3.3; 95% CI 1.1–9.6; p = 0.031, multivariable Cox regression) and overall survival (HR: 3.6; 95% CI 1.1–12.6; p = 0.041). For validation using RNA-seq data as a surrogate, in a population-based SCAN-B cohort (NCT02306096) of 682 consecutive patients receiving adjuvant trastuzumab, the ultrahigh-ERBB2 mRNA group had significantly worse survival. Multiplex ddPCR is useful for ERBB2 CN estimation and ultrahigh ERBB2 may be a predictive factor for decreased long-term survival after trastuzumab treatment.

Application of plasma circulating KRAS mutations as a predictive biomarker for targeted treatment of pancreatic cancer.

Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations in circulating tumor deoxyribonucleic acid (ctDNA) have been reported as representative noninvasive prognostic markers for pancreatic ductal adenocarcinoma (PDAC). Here, we aimed to evaluate single KRAS mutations as prognostic and predictive biomarkers, with an emphasis on potential therapeutic approaches to PDAC. A total of 128 patients were analyzed for multiple or single KRAS mutations (G12A, G12C, G12D, G12R, G12S, G12V, and G13D) in their tumors and plasma using droplet digital polymerase chain reaction (ddPCR). Overall, KRAS mutations were detected by multiplex ddPCR in 119 (93%) of tumor DNA and 68 (53.1%) of ctDNA, with a concordance rate of 80% between plasma ctDNA and tumor DNA in the metastatic stage, which was higher than the 44% in the resectable stage. Moreover, the prognostic prediction of both overall survival (OS) and progression-free survival (PFS) was more relevant using plasma ctDNA than tumor DNA. Further, we evaluated the selective tumor-suppressive efficacy of the KRAS G12C inhibitor sotorasib in a patient-derived organoid (PDO) from a KRAS G12C-mutated patient using a patient-derived xenograft (PDX) model. Sotorasib showed selective inhibition invitro and invivo with altered tumor microenvironment, including fibroblasts and macrophages. Collectively, screening for KRAS single mutations in plasma ctDNA and the use of preclinical models of PDO and PDX with genetic mutations would impact precision medicine in the context of PDAC.

Development of a low-cost and accurate carrier screening method for spinal muscular atrophy in developing countries

Heterozygous carriers of the survival of motor neuron 1 (SMN1) gene deletion in parents account for approximately 95% of neonatal spinal muscular atrophy cases. Given the severity of the disease, professional organizations have recommended periconceptional spinal muscular atrophy carrier screening to all couples, regardless of race or ethnicity. However, the prevalence of screening activities in mainland China remains suboptimal, mainly attributed to the limitations of the existing carrier screening methods. Herein, we aimed to develop a low-cost, accessible, and accurate carrier screening method based on duplex droplet digital PCR (ddPCR), to cover a wider population in developing countries, including China. The receiver operating characteristic curve was used to determine the cut-off value of SMN1 copy numbers. Performance validation was conducted for linearity, precision, and accuracy. In total, 482 cases were considered to validate the concordance between the developed ddPCR assay and multiplex ligation-dependent probe amplification. Linear correlations were excellent between the expected concentration of the reference gene and the observed values (R2 > 0.99). Both the intra- and inter-assay precision of our ddPCR assays were less than 6.0%. The multiplex ligation-dependent probe amplification and ddPCR results were consistent in 480 of the 482 cases (99.6%). Two cases with multiplex ligation-dependent probe amplification, suggestive of two copies of SMN1 exon 7, were classified into three copies by ddPCR analysis. The overall correct classification of the samples included in our ddPCR assay was 100%. This study demonstrates that an appropriate cut-off value is an important prerequisite for establishing a semi-quantitative method to determine the SMN1 copy numbers. Compared to conventional methods, our ddPCR assay is low-cost, highly accurate, and has full potential for application in population spinal muscular atrophy carriers screening.

Clinically accessible amplitude-based multiplex ddPCR assay for tryptase genotyping

Hereditary α tryptasemia (HαT) is an autosomal dominant trait characterized by increased TPSAB1 copy number (CN) encoding α-tryptase. The determination of HαT is being discussed as an important biomarker to be included in risk assessment models and future diagnostic algorithms for patients with mastocytosis and anaphylaxis. Due to the complex genetic structure at the human tryptase locus, genetic testing for tryptase gene composition is presently notably limited and infrequently pursued. This study aimed to develop, optimise and validate a multiplex droplet digital PCR (ddPCR) assay that can reliably quantify α- and β-tryptase encoding sequences in a single reaction. To optimise the ddPCR conditions and establish an amplitude-based multiplex ddPCR assay, additional primers and probes, a thermal gradient with varying annealing temperatures, different primers/probe concentrations, and various initial DNA quantities were tested. Results obtained from all 114 samples analysed using multiplex ddPCR were identical to those obtained through the use of original duplex assays. Utilizing this multiplex ddPCR assay, in contrast to conducting distinct duplex ddPCRs, presents noteworthy benefits for tryptase genotyping. These advantages encompass a substantial threefold decrease in material costs and considerable time savings. Consequently, this approach exhibits high suitability and particularly captures interest for routine clinical implementation.

Clinical evaluation of a multiplex droplet digital PCR for pathogen detection in critically ill COVID-19 patients with bloodstream infections

BackgroundNosocomial bloodstream infections (nBSI) have emerged as a clinical concern for physicians treating COVID-19 patients. In this study, we aimed to evaluate the effectiveness of a multiplex ddPCR in detecting bacterial pathogens in the blood of COVID-19 critically ill patients.MethodsThis prospective diagnostic study included RT-PCR-confirmed COVID-19 patients admitted to our hospital from December 2022 to February 2023. A multiplex ddPCR assay was used to detect common bacterial pathogens and AMR genes in blood samples of the patients, along with antimicrobial susceptibility testing (AST). The diagnostic performance of the ddPCR assay was evaluated by comparing the results with those obtained through blood culture and clinical diagnosis. Additionally, the ability of ddPCR in detecting bacterial resistance was compared with the AST results.ResultsOf the 200 blood samples collected from 184 patients, 45 (22.5%) were positive using blood culture, while 113 (56.5%) were positive for bacterial targets using the ddPCR assay. The ddPCR assay outperformed blood culture in pathogen detection rate, mixed infection detection rate, and fungal detection rate. Acinetobacter baumannii and Klebsiella pneumoniae were the most commonly detected pathogens in COVID-19 critically ill patients, followed by Enterococcus and Streptococcus. Compared to blood culture, ddPCR achieved a sensitivity of 75.5%, specificity of 51.0%, PPV of 30.9%, and NPV of 87.8%, respectively. However, there were significant differences in sensitivity among different bacterial species, where Gram-negative bacteria have the highest sensitivity of 90.3%. When evaluated on the ground of clinical diagnosis, the sensitivity, specificity, PPV and NPV of ddPCR were 78.1%, 90.5%, 94.7%, and 65.5%, respectively. In addition, the ddPCR assay detected 23 cases of blaKPC, which shown a better consistent with clinical test results than other detected AMR genes. Compared to blaKPC, there were few other AMR genes detected, indicating that the application of other AMR gene detection in the COVID-19 critically ill patients was limited.ConclusionThe multiplex ddPCR assay had a significantly higher pathogen detection positivity than the blood culture, which could be an effective diagnostic tool for BSIs in COVID-19 patients and to improve patient outcomes and reduce the burden of sepsis on the healthcare system, though there is room for optimization of the panels used.- Adjusting the targets to include E. faecalis and E. faecium as well as Candida albicans and Candida glabrata could improve the ddPCR' s effectiveness. However, further research is needed to explore the potential of ddPCR in predicting bacterial resistance through AMR gene detection.

A duplex droplet digital PCR assay for absolute quantification and characterization of long self-amplifying RNA

Self-amplifying messenger ribonucleic acid (saRNA) provides extended expression of genes of interest by encoding an alphavirus-derived RNA replicase and thus is 2–3 times larger than conventional messenger RNA. However, quality assessment of long RNA transcripts is challenging using standard techniques. Here, we utilized a multiplex droplet digital polymerase chain reaction (ddPCR) assay to assess the quality of saRNA produced from an in vitro transcription reaction and the replication kinetics in human cell lines. Using the one-step reverse transcription ddPCR, we show that an in vitro transcription generates 50–60% full-length saRNA transcripts. However, we note that the two-step reverse transcription ddPCR assay results in a 20% decrease from results obtained using the one-step and confirmed using capillary gel electrophoresis. Additionally, we provided three formulas that differ in the level of stringency and assumptions made to calculate the fraction of intact saRNA. Using ddPCR, we also showed that subgenomic transcripts of saRNA were 19-to-108-fold higher than genomic transcripts at different hours post-transfection of mammalian cells in copies. Therefore, we demonstrate that multiplex ddPCR is well suited for quality assessment of long RNA and replication kinetics of saRNA based on absolute quantification.

Establishment and application of multiplex droplet digital polymerase chain reaction assay for bovine enterovirus, bovine coronavirus, and bovine rotavirus.

Bovine enterovirus (BEV), bovine coronavirus (BCoV), and bovine rotavirus (BRV) are still the major worldwide concerns in the health care of cattle, causing serious economic losses in the livestock industry. It is urgent to establish specific and sensitive methods to detect viruses for the early control of diseases. Droplet digital PCR (ddPCR) has been proposed to effectively detect viral particles, and it does not involve Ct values or standard curves. In this study, we designed specific primers and probes, based on conserved regions of viral genomes, to optimize protocols for a dual ddPCR assay for detecting BCoV and BRV and a multiplex ddPCR assay for BEV, BCoV, and BRV. Sensitivity assays revealed that the lower limit of detection for qPCR was 1,000 copies/μL and for ddPCR for BEV, BCoV, and BRV, 2.7 copies/μL, 1 copy/μL and 2.4 copies/μL, respectively. Studying 82 samples collected from diarrheal calves on a farm, our dual ddPCR method detected BCoV, BRV, and co-infection at rates of 18.29%, 14.63%, and 6.1%, respectively. In contrast, conventional qPCR methods detected BCoV, BRV, and co-infection at rates of 10.98%, 12.2%, and 3.66%, respectively. On the other hand, studying 68 samples from another farm, qPCR detected BCoV, BRV, BEV, and co-infection of BCoV and BEV at rates of 14.49%, 1.45%, 5.80%, and 1.45%, respectively. Our multiplex ddPCR method detected BCoV, BRV, BEV, co-infection of BCoV and BEV, and co-infection of BRV and BEV. at rates of 14.49%, 2.9%, 8.7%, 2.9%, and 1.45%, respectively. Studying 93 samples from another farm, qPCR detected BCoV, BRV, BEV, and co-infection of BCoV and BEV was detected at rates of 5.38%, 1.08%, 18.28%, and 1.08%, respectively. Co-infection of BCoV, BRV, BEV, BCoV, and BEV, and co-infection of BRV and BEV, were detected by multiplex ddPCR methods at rates of 5.38%, 2.15%, 20.45%, 1.08%, and 1.08%, respectively. These results indicated that our optimized dual and multiplex ddPCR methods were more effective than conventional qPCR assays to detect these viral infections.

Detection and quantification of integrated vector copy number by multiplex droplet digital PCR in dual-transduced CAR T cells

The success of chimeric antigen receptor (CAR) Tcell therapies in refractory hematologic malignancies has prompted investigation of their efficacy in solid tumors. AUTO6NG is a dual-transduced GD2-targeting CAR that encodes distinct modules designed to enhance Tcell activity in relapsed/refractory neuroblastoma. The ability to detect and precisely quantify vector copy number (VCN) for each integrated vector is essential for assessing the effect of each module on Tcell tumor infiltration, persistence, and clinical activity. Droplet digital PCR (ddPCR) enables accurate, sensitive, and absolute quantification of specific nucleic acid sequences. Compared to standard detection of two targets, multiplex ddPCR assays allow simultaneous detection of up to four targets by selective modulation of signal amplitude while retaining the ability to quantify the target. We have developed a multiplex assay based on the two-channel system for simultaneous detection and quantification of three targets in AUTO6NG CAR Tcells. The assay was highly specific, sensitive, accurate, and reproducible across time and samples. No differences were observed in measuring VCN between standard duplex and multiplex assays. Our results demonstrate that ddPCR is an accurate and cost-effective method for simultaneous detection of multiple targets in genomic DNA derived from engineered CAR Tcells.

Whole-genome informed circulating tumor DNA analysis by multiplex digital PCR for disease monitoring in B-cell lymphomas: a proof-of-concept study.

Analyzing liquid biopsies for tumor-specific aberrations can facilitate detection of measurable residual disease (MRD) during treatment and at follow-up. In this study, we assessed the clinical potential of using whole-genome sequencing (WGS) of lymphomas at diagnosis to identify patient-specific structural (SVs) and single nucleotide variants (SNVs) to enable longitudinal, multi-targeted droplet digital PCR analysis (ddPCR) of cell-free DNA (cfDNA). In 9 patients with B-cell lymphoma (diffuse large B-cell lymphoma and follicular lymphoma), comprehensive genomic profiling at diagnosis was performed by 30X WGS of paired tumor and normal specimens. Patient-specific multiplex ddPCR (m-ddPCR) assays were designed for simultaneous detection of multiple SNVs, indels and/or SVs, with a detection sensitivity of 0.0025% for SV assays and 0.02% for SNVs/indel assays. M-ddPCR was applied to analyze cfDNA isolated from serially collected plasma at clinically critical timepoints during primary and/or relapse treatment and at follow-up. A total of 164 SNVs/indels were identified by WGS including 30 variants known to be functionally relevant in lymphoma pathogenesis. The most frequently mutated genes included KMT2D, PIM1, SOCS1 and BCL2. WGS analysis further identified recurrent SVs including t(14;18)(q32;q21) (IGH::BCL2), and t(6;14)(p25;q32) (IGH::IRF4). Plasma analysis at diagnosis showed positive circulating tumor DNA (ctDNA) levels in 88% of patients and the ctDNA burden correlated with baseline clinical parameters (LDH and sedimentation rate, p-value <0.01). While clearance of ctDNA levels after primary treatment cycle 1 was observed in 3/6 patients, all patients analyzed at final evaluation of primary treatment showed negative ctDNA, hence correlating with PET-CT imaging. One patient with positive ctDNA at interim also displayed detectable ctDNA (average variant allele frequency (VAF) 6.9%) in the follow-up plasma sample collected 2 years after final evaluation of primary treatment and 25 weeks before clinical manifestation of relapse. In summary, we demonstrate that multi-targeted cfDNA analysis, using a combination of SNVs/indels and SVs candidates identified by WGS analysis, provides a sensitive tool for MRD monitoring and can detect lymphoma relapse earlier than clinical manifestation.

Cell-Free RNA from Plasma in Patients with Neuroblastoma: Exploring the Technical and Clinical Potential

Neuroblastoma affects mostly young children, bearing a high morbidity and mortality. Liquid biopsies, e.g., molecular analysis of circulating tumor-derived nucleic acids in blood, offer a minimally invasive diagnostic modality. Cell-free RNA (cfRNA) is released by all cells, especially cancer. It circulates in blood packed in extracellular vesicles (EV) or attached to proteins. We studied the feasibility of analyzing cfRNA and EV, isolated by size exclusion chromatography (SEC), from platelet-poor plasma from healthy controls (n = 40) and neuroblastoma patients with localized (n = 10) and metastatic disease (n = 30). The mRNA content was determined using several multiplex droplet digital PCR (ddPCR) assays for a neuroblastoma-specific gene panel (PHOX2B, TH, CHRNA3) and a cell cycle regulation panel (E2F1, CDC6, ATAD2, H2AFZ, MCM2, DHFR). We applied corrections for the presence of platelets. We demonstrated that neuroblastoma-specific markers were present in plasma from 14/30 patients with metastatic disease and not in healthy controls and patients with localized disease. Most cell cycle markers had a higher expression in patients. The mRNA markers were mostly present in the EV-enriched SEC fractions. In conclusion, cfRNA can be isolated from plasma and EV and analyzed using multiplex ddPCR. cfRNA is an interesting novel liquid biopsy-based target to explore further.

Non-invasive prenatal testing for fetal Ss, Kidd, and CTL2 blood group prediction by multiplex digital droplet PCR.

Some blood groups, such as S and s blood groups in the MNS blood group system, and Kidd and CTL2 blood group systems, can cause severe fetal and newborn alloimmune disorders. Non-invasive prenatal testing (NIPT) to predict fetal blood groups and knowledge of local blood group gene frequency are both important for pregnancy management decisions. Droplet digital PCR (ddPCR) has high specificity and sensitivity in detecting fetal single nucleotide variation. The objective is to predict fetal Ss, Kidd, and CTL2 blood groups using multiplex ddPCR. The gene frequencies of three blood groups were detected by ddPCR in northwest China. This is a prospective study. Cell-free fetal DNA isolated from 26 healthy single pregnant women at different gestational stages was tested with QX200 Droplet Digital PCR. Results were compared with fetal genotypes. DNA samples purified from 20 blood pools containing a total of 1000 donors in northwest China were subjected to ddPCR to detect the gene frequency of three blood groups. Ss, Kidd, and CTL2 blood groups of 26 pregnant fetuses were accurately detected by multiplex ddPCR. The multiplex ddPCR results were consistent with the Sanger sequencing results of 26 fetal blood samples after birth. The gene frequencies of the three blood groups detected by ddPCR were 9.30% for S, 90.70% for s, 48.43% for Jka, 51.57% for Jkb, 66.57% for HNA-3A, and 33.43% for HNA-3B. It is reliable to predict fetal Ss, Kidd, and CTL2 blood groups by multiplex ddPCR. Meanwhile, we designed a simple and efficient method for inferring the gene frequency of three blood groups based on ddPCR.

Peripheral blood marker of residual acute leukemia after hematopoietic cell transplantation using multi-plex digital droplet PCR.

BackgroundRelapse remains the primary cause of death after hematopoietic cell transplantation (HCT) for acute leukemia. The ability to identify minimal/measurable residual disease (MRD) via the blood could identify patients earlier when immunologic interventions may be more successful. We evaluated a new test that could quantify blood tumor mRNA as leukemia MRD surveillance using droplet digital PCR (ddPCR).MethodsThe multiplex ddPCR assay was developed using tumor cell lines positive for the tumor associated antigens (TAA: WT1, PRAME, BIRC5), with homeostatic ABL1. On IRB-approved protocols, RNA was isolated from mononuclear cells from acute leukemia patients after HCT (n = 31 subjects; n = 91 specimens) and healthy donors (n = 20). ddPCR simultaneously quantitated mRNA expression of WT1, PRAME, BIRC5, and ABL1 and the TAA/ABL1 blood ratio was measured in patients with and without active leukemia after HCT.ResultsTumor cell lines confirmed quantitation of TAAs. In patients with active acute leukemia after HCT (MRD+ or relapse; n=19), the blood levels of WT1/ABL1, PRAME/ABL1, and BIRC5/ABL1 exceeded healthy donors (p<0.0001, p=0.0286, and p=0.0064 respectively). Active disease status was associated with TAA positivity (1+ TAA vs 0 TAA) with an odds ratio=10.67, (p=0.0070, 95% confidence interval 1.91 – 59.62). The area under the curve is 0.7544. Changes in ddPCR correlated with disease response captured on standard of care tests, accurately denoting positive or negative disease burden in 15/16 (95%). Of patients with MRD+ or relapsed leukemia after HCT, 84% were positive for at least one TAA/ABL1 in the peripheral blood. In summary, we have developed a new method for blood MRD monitoring of leukemia after HCT and present preliminary data that the TAA/ABL1 ratio may may serve as a novel surrogate biomarker for relapse of acute leukemia after HCT.