Digital PCR Assay Research Articles

Antimalarial resistance risk in Mozambique detected by a novel quadruplex droplet digital PCR assay.

While the Plasmodium falciparum malaria parasite continues to cause severe disease globally, Mozambique is disproportionally represented in malaria case totals. Acquisition of copy number variations (CNVs) in the parasite genome contributes to antimalarial drug resistance through overexpression of drug targets. Of interest, piperaquine resistance is associated with plasmepsin 2 and 3 CNVs (pfpmp2 and pfpmp3, respectively), while CNVs in the multidrug efflux pump, multidrug resistance-1 (pfmdr1), increase resistance to amodiaquine and lumefantrine. These antimalarials are partner drugs in artemisinin combination therapies (ACTs) and therefore, CNV detection with accurate and efficient tools is necessary to track ACT resistance risk. Here, we evaluated ~300 clinically derived samples collected from three sites in Mozambique for resistance-associated CNVs. We developed a novel, medium-throughput, quadruplex droplet digital PCR (ddPCR) assay to simultaneously quantify the copy number of pfpmp3, pfpmp2, and pfmdr1 loci in these clinical samples. By using DNA from laboratory parasite lines, we show that this nanodroplet-based method is capable of detecting picogram levels of parasite DNA, which facilitates its application for low yield and human host-contaminated clinical surveillance samples. Following ddPCR and the application of quality control standards, we detected CNVs in 13 of 229 high-quality samples (prevalence of 5.7%). Overall, our study revealed a low number of resistance CNVs present in the parasite population across all three collection sites, including various combinations of pfmdr1, pfpmp2, and pfpmp3 CNVs. The potential for future ACT resistance across Mozambique emphasizes the need for continued molecular surveillance across the region.

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.

Overcoming Challenges in DNA Extractions from Triphala Ingredients: A Way Forward for Optimization of Conventional and Digital PCR Assays for Molecular Authentication

Overcoming Challenges in DNA Extractions from Triphala Ingredients: A Way Forward for Optimization of Conventional and Digital PCR Assays for Molecular Authentication

From genes to toxins: Profiling Prymnesium parvum during a riverine harmful algal bloom

Blooms of Prymnesium parvum, a unicellular alga globally distributed in marine and brackish environments, frequently result in massive fish kills due to the production of toxins called prymnesins by this haptophyte. In August 2022, a harmful algal bloom (HAB) of this species occurred in the lower Oder River (Poland and Germany), which caused mass mortalities of fish and other organisms. This HAB was linked to low discharge of the Oder and mining activities that caused a significant increase in salinity. In this context, we report on the molecular detection and screening of this haptophyte and its toxins in environmental samples and clonal cultures derived thereof. Both conventional PCR and droplet digital PCR assays reliably detected P. parvum in environmental samples. eDNA metabarcoding using the V4 region of the 18S rRNA gene revealed a single Prymnesium sequence variant, but failed to identify it to species level. Four clonal cultures established from environmental samples were unambiguously identified as P. parvum by molecular phylogenetics (near full-length 18S rRNA gene) and light microscopy. Phylogenetic analysis (ITS1–5.8S-ITS2 marker region) placed the cultured phylotype within a clade containing other P. parvum strains known to produce B-type prymnesins. Toxin-screening of the cultures using liquid chromatography-electrospray ionization - time of flight mass spectrometry identified B-type prymnesins, which were also detected in extracts of filter residues from water samples of the Oder collected during the HAB. Overall, our investigation provides a detailed characterization of P. parvum, including their prymnesins, during this HAB in the Oder River, contributing valuable insights into this ecological disaster. In addition, the droplet digital PCR assay established here will be useful for future monitoring of low levels of P. parvum on the Oder River or any other salt-impacted and brackish water bodies.

A multiplex digital PCR assay for detection and quantitation of porcine circovirus type 2 and type 3.

Porcine circovirus (PCV) has become a major pathogen, causing major economic losses in the global pig industry, and PCV type 2 (PCV2) and 3 (PCV3) are distributed worldwide.We designed specific primer and probe sequences targeting PCV2 Cap and PCV3 Rap anddeveloped a multiplex crystal digital PCR (cdPCR) method after optimizing the primerconcentration, probe concentration, and annealing temperature.The multiplex cdPCR assay permits precise and differential detection of PCV2 and PCV3, with a limit of detection of 1.39 × 101 and 1.27 × 101 copies/reaction, respectively, and no cross-reaction with other porcine viruses was observed. The intra-assay and interassay coefficients of variation (CVs) were less than 8.75%, indicating good repeatability and reproducibility. To evaluate the practical value of this assay, 40 tissue samples and 70 feed samples were tested for both PCV2 and PCV3 by cdPCR and quantitative PCR (qPCR). Using multiplex cdPCR, the rates of PCV2 infection, PCV3 infection, and coinfection were 28.45%, 1.72%, and 12.93%, respectively, and using multiplex qPCR, they were 25.00%, 0.86%, and 4.31%, respectively This highly specific and sensitive multiplex cdPCR thus allows accurate simultaneous detection of PCV2 and PCV3, and it is particularly well suited for applications that require the detection of small amounts of input nucleic acid or samples with intensive processing and complex matrices.

Development and evaluation of a multi-target droplet digital PCR assay for highly sensitive and specific detection of Yersinia pestis.

Plague, caused by the bacterium Yersinia pestis, is a zoonotic disease that poses considerable threats to human health. Nucleic acid tests are crucial for plague surveillance and the rapid detection of Y. pestis. However, inhibitors in complex samples such as soil and animal tissues often hamper nucleic acid detection, leading to a reduced rate of identifying low concentrations of Y. pestis. To address this challenge, we developed a sensitive and specific droplet digital polymerase chain reaction (ddPCR) assay for detecting Y. pestis DNA from soil and animal tissue samples. Three genes (ypo2088, caf1, and pla) from Y. pestis were used to develop a multi-target ddPCR assay. The limits of detection (LoD), reproducibility, and specificity were assessed for bacterial genomic DNA samples. The ability of the assay to detect low concentrations of Y. pestis DNA from simulated soil and mouse liver tissue samples was respectively evaluated and compared with that of quantitative real-time PCR (qPCR). The results showed that the ddPCR LoDs ranged from 6.2 to 15.4 copies/reaction for the target genes, with good reproducibility and high specificity for Y. pestis. By testing 130 soil and mouse liver tissue samples spiked with Y. pestis, the ddPCR assay exhibited a better sensitivity than that of the qPCR assay used in the study, with LoDs of 102 colony forming units (CFU)/100 mg soil and 103 CFU/20 mg liver. Moreover, the assay presented good quantitative linearity (R2 = 0.99) for Y. pestis at 103-106 CFU/sample for soil and liver samples. The ddPCR assay presented good performance for detecting Y. pestis DNA from soil and mouse tissue samples, showing great potential for improving the detection rate of low concentrations of Y. pestis in plague surveillance and facilitating the early diagnosis of plague cases.

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

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

Abstract PO2-14-04: Circulating tumor DNA after neoadjuvant chemotherapy is a better prognostic test than Residual Cancer Burden in patients with triple negative breast cancer and residual tumor

Abstract Background The presence of residual tumor at surgery (non-pathological complete response or non-pCR) occurs in about half of TNBCs treated with neoadjuvant chemotherapy (NAC) signals chemoresistance. Although the addition of further adjuvant chemotherapy (Capecitabine/Xeloda) significantly improves RFS in these patients, the majority of patients nevertheless have a good prognosis, and thus may not require adjuvant Capecitabine. One of the best prognostic biomarkers in these patients is Residual Cancer Burden (RCB), which relies on the pathological examination of the residual tumor bed and lymph nodes obtained at surgery. Circulating tumor DNA (ctDNA) is a plasma-based biomarker, which has been shown to accurately detect minimal residual disease in breast cancer. We have previously shown that detection of ctDNA after NAC but before surgery in patients with non-pCR signals very poor prognosis, while non-detectable ctDNA is associated with an excellent prognosis. We now compare ctDNA to RCB score in TNBC patients with non-pCR. Methods In the multicenter TRICIA study, 44 TNBC patients with non-pCR after NAC in 5 hospital centers had consented to serial plasma collection prior to and after surgery. RCB score was calculated from pathology records. Whole exome sequencing (WES) was performed on DNA extracted from primary tumor biopsies or residual tumors at surgery to identify tumor-specific mutations (5 variants/patient). Digital droplet PCR (ddPCR) assays were developed for these mutations and tests were run in an academic hospital setting. Patients with at least one detectable mutation were considered ctDNA positive for a given time point. The detection of ctDNA was correlated with relapse-free survival (RFS). Results: The median follow up was 40 months. 19 of the 44 patients relapsed within 2 years after surgery. Detection of ctDNA at the end of NAC but before surgery (T1) was correlated with very poor RFS (H.R. = 0.15, p< 0.0001 (95% CI = 0.067-0.35)). 14/15 patients (93%) without detectable ctDNA did not recur, while 76% of patients with detectable ctDNA relapsed. The RCB score distribution was as follows: 7 RCB1, 21 RCB2 and 16 RCB3. According to RCB score, 1/7 RCB1, 11/21 RCB2 and 11/16 RCB3 patients relapsed. All but one of the relapsed patients had detectable ctDNA in at least one timepoint. All 11 RCB3 patients that relapsed had detectable ctDNA while none of the 5 RCB3 patients without relapse had detectable ctDNA. The one RCB1 patient with detectable ctDNA also relapsed. In cox regression analysis, the lack of ctDNA detection was significantly associated with excellent RFS in RCB2 (HR=0.16, p=0.003, (95% CI=0.05-.53)) and RCB3 groups (HR=0.10, p=0.0005, 95% CI= 0.03-.36). Conclusion ctDNA testing using ddPCR in an academic hospital-based setting at the post-NAC time point identifies an excellent prognostic group in TNBC patients with non-pCR and can discriminate very good from very poor prognosis in both RCB2 and RCB3 score subgroups. The detection of ctDNA may be superior to RCB scores in assessing prognosis in patients with non-pCR. Citation Format: Mark Basik, Talia Roseshter, Anna Klemantovich, Luca Cavallone, Adriana Aguilar-Mahecha, Josiane Lafleur, Cathy Lan, Oluwadara Elebute, Sarah Jenna, Jean-Francois Boileau, Manuela Pelmus. Circulating tumor DNA after neoadjuvant chemotherapy is a better prognostic test than Residual Cancer Burden in patients with triple negative breast cancer and residual tumor [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 PO2-14-04.

Digital PCR as a novel way to assess chimerism of humanized immune system mice

Abstract Humanized immune system (HIS) mice contain human and murine immune cells. Analysis of chimerism in HIS mice is typically accomplished via flow cytometry, but this requires collecting a 75 µl blood sample and access to a flow cytometer. Repeated bleeding can negatively affect the health of HIS mice, limiting serial analysis. Digital PCR offers a novel method for chimerism analysis, which uses much smaller blood volumes and can be performed on clotted or frozen samples, sample types unsuitable for flow cytometry. A digital PCR assay directly comparing the presence of three human genes and one murine gene was developed. This assay was validated in the huNOG-EXL HIS mouse engrafted with CD34+ hematopoetic stem cells from 3 unique donors via blind comparison with chimerism data from flow cytometry. At 10 weeks post engraftment, the digital PCR assay showed excellent correlation for all three human genes against chimerism as measured by flow cytometry of the peripheral blood. This assay represents a new, user-friendly, rapid tool for the analysis of HIS mice and may permit serial chimerism measurement using 10 μL whole blood. This assay may be useful to investigate other mouse-human chimera models, including mice harboring functional human hepatocytes, tumors, and other cells.

Establishment of a chip digital PCR detection method for canine circovirus

Canine circovirus (CanineCV), which is a new mammalian circovirus first reported in the United States in 2012, mainly causes diarrhea and vomiting in dogs. As CanineCV evolves and new subtypes emerge, there is an urgent need for new detection technologies to improve the sensitivity and detection rates of viruses in complex scenarios. A chip digital PCR(cdPCR) assay was established for the detection of CanineCV in this study. The results showed good reproducibility, specificity and a linear relationship; the minimum detection limit of CanineCV by cdPCR was 6.62 copies/μL, which is 10 times more sensitive than quantitative real-time PCR (qPCR). The qPCR-positive detection rate was 1 %, while CanineCV cdPCR (2.1 %) exhibited a greater positive detection rate. Fifteen complete genomes were sequenced and subdivided into CanineCV-1 and CanineCV-3. In conclusion, we developed a rapid, reliable, and specific cdPCR method for screening and monitoring canine CV.

Multiplex Digital PCR-Based Development and discussion of the Detection of Genetic Association Between Staphylococcus aureus and mecA.

Methicillin-resistant Staphylococcus aureus (MRSA) is a predominant nosocomial infection-causing bacteria. The aim of this study was to develop a novel single-bacteria multiplex digital PCR assays (SMD-PCR), which is capable of simultaneously detecting and discriminating Methicillin-sensitive Staphylococcus aureus (MSSA) and MRSA. This protocol employed TaqMan probes to detect SAOUHSC_00106 and mecA genes, with the latter being linked to methicillin resistance. A total of 72 samples from various specimen types were evaluated. The accuracy rates for the sputum samples, pus samples, swab samples, ear secretion samples, and catheter samples were 94.44%, 100%, 92%, 100%, and 100%, respectively. Our results showed that the clinical practicability of SMD-PCR has applicability to the rapid detection of MRSA without DNA extraction or bacterial culture, and can be utilized for the rapid detection of Staphylococcus aureus and the timely identification of MRSA in clinical samples, thereby providing an advanced platform for the rapid diagnosis of clinical MRSA infection.

Identification and characterization of recent retrovirus in Rhinolophus ferrumequinum bats.

An investigation into retrovirus was conducted in six species of bats (Myotis aurascens, Myotis petax, Myotis macrodactylus, Miniopterus fuliginosus, Rhinolophus ferrumequinum, and Pipistrellus abramus) inhabiting South Korea. Exogenous retroviruses (XRVs) were detected in the tissue samples of R. ferrumequinum individuals by PCR assay. Proviruses were identified in all tissue samples through viral quantification using a digital PCR assay per organ (lung, intestine, heart, brain, wing, kidney, and liver), with viral loads varying greatly between each organ. In phylogenetic analysis based on the whole genome, the Korean bat retroviruses and the R. ferrumequinum retrovirus (RfRV) strain formed a new clade distinct from the Gammaretrovirus clade. The phylogenetic results determined these viruses to be RfRV-like viruses. In the Simplot comparison, Korean RfRV-like viruses exhibited relatively strong fluctuated patterns in the latter part of the envelope gene area compared to other gene areas. Several point mutations within this region (6,878-7,774 bp) of these viruses were observed compared to the RfRV sequence. One Korean RfRV-like virus (named Y4b strain) was successfully recovered in the Raw 264.7 cell line, and virus particles replicated in the cells were confirmed by transmission electron microscopy. RfRVs (or RfRV-like viruses) have been spreading since their first discovery in 2012, and the Korean RfRV-like viruses were assumed to be XRVs that evolved from RfRV.IMPORTANCER. ferrumequinum retrovirus (RfRV)-like viruses were identified in greater horseshoe bats in South Korea. These RfRV-like viruses were considered exogenous retroviruses (XRVs) that emerged from RfRV. Varying amounts of provirus detected in different organs suggest ongoing viral activity, replication, and de novo integration in certain organs. Additionally, the successful recovery of the virus in the Raw 264.7 cell line provides strong evidence supporting their status as XRVs. These viruses have now been identified in South Korea and, more recently, in Kenya since RfRV was discovered in China in 2012, indicating that RfRVs (or RfRV-like viruses) have spread worldwide.

Cell-free human papillomavirus (HPV) DNA is a sensitive biomarker for prognosis and for early detection of relapse in locally advanced cervical cancer.

Human papillomavirus (HPV) is the cause of the majority of cervical cancers and has been showed to be released as cell-free tumour DNA (ctHPV DNA) into the circulation. We here analyse if ctHPV DNA could be used as a prognostic biomarker and/or to detect relapse earlier than traditional methods in locally advanced cervical cancer (LACC). 74 patients with LACC were included, 66/74 were positive for 13 high-risk HPV-types using a bead-based assay on tumour biopsies. HPV-type-specific droplet digital PCR (ddPCR) assays were developed. Longitudinal plasma samples were then analysed for the biopsy-verified HPV-type for each patient. 418 plasma samples were analysed. Patients were followed for a median of 37 months. Results were correlated to tumour- and clinical characteristics. 92.4% of pre-treatment plasma samples were positive for ctHPV DNA. Persistent ctHPV DNA in end-of-treatment, early follow-up (1-2 months after end-of-treatment) or tumour evaluation (3-4 months after end-of-treatment) plasma was correlated with worse progression-free survival (p < 0.001) compared to if ctHPV DNA was not found. The positive predictive value of ctHPV-status at early follow-up for predicting disease progression was 87.5% and the negative predictive value was 89.3%. ctHPV DNA was found in plasma before relapse was diagnosed on radiology in all patients (n=10) who experienced relapse after complete clinical response to treatment with a median 315 days lead time. ctHPV DNA in follow-up plasma is a promising prognostic biomarker in patients with LACC, useful for analysis of response to therapy and for early detection of relapse.

Sudden peak in tetrodotoxin in French oysters during the summer of 2021: Source investigation using microscopy, metabarcoding and droplet digital PCR

Tetrodotoxin (TTX) is a potent neurotoxin causing human intoxications from contaminated seafood worldwide and is of emerging concern in Europe. Shellfish have been shown to contain varying TTX concentrations globally, with concentrations typically higher in Pacific oysters Crassostrea gigas in Europe. Despite many decades of research, the source of TTX remains unknown, with bacterial or algal origins having been suggested. The aim of this study was to identify potential source organisms causing TTX contamination in Pacific oysters in French coastal waters, using three different techniques. Oysters were deployed in cages from April to September 2021 in an estuary where TTX was previously detected. Microscopic analyses of water samples were used to investigate potential microalgal blooms present prior or during the peak in TTX. Differences in the bacterial communities from oyster digestive glands (DG) and remaining flesh were explored using metabarcoding, and lastly, droplet digital PCR assays were developed to investigate the presence of Cephalothrix sp., one European TTX-bearing species in the DG of toxic C. gigas. Oysters analysed by liquid chromatography-tandem mass spectrometry contained quantifiable levels of TTX over a three-week period (24 June–15 July 2021), with concentrations decreasing in the DG from 424 μg/kg for the first detection to 101 μg/kg (equivalent to 74 to 17 μg/kg of total flesh), and trace levels being detected until August 13, 2021. These concentrations are the first report of the European TTX guidance levels being exceeded in French shellfish. Microscopy revealed that some microalgae bloomed during the TTX peak, (e.g., Chaetoceros spp., reaching 40,000 cells/L). Prokaryotic metabarcoding showed increases in abundance of Rubritaleaceae (genus Persicirhabdus) and Neolyngbya, before and during the TTX peak. Both phyla have previously been described as possible TTX-producers and should be investigated further. Droplet digital PCR analyses were negative for the targeted TTX-bearing genus Cephalothrix.

Abstract CT002: CTX130 allogeneic CRISPR-Cas9-engineered chimeric antigen receptor (CAR) T cells in patients with advanced clear cell renal cell carcinoma: Long-term follow-up and translational data from the phase 1 COBALT-RCC study

Abstract Background: The mainstay of therapy for clear cell renal cell carcinoma (ccRCC), the most prevalent subtype of kidney cancer, includes checkpoint inhibitors (CPIs) and tyrosine kinase inhibitors (TKIs). However, once patients progress on these drug classes, outcomes are poor. Here, we report durable response and stability observed in a Phase 1 dose-escalation study of CTX130™, a CD70-targeting allogeneic CAR T cell therapy in patients with ccRCC. Methods: In this phase I study, patients were recruited from sites in the United States, Europe and Australia. Key eligibility included advanced ccRCC with prior receipt of at least one CPI and TKI. After receipt of standard lymphodepletion with fludarabine and cyclophosphamide for 3 days (dosed at 30 mg/m2 and 500 mg/m2, respectively), patients received infusion of CTX130. Results: Among the 16 patients enrolled and treated in dose escalation, the median age was 63 (range, 53-77) and all were stage IV at the time of enrollment. Patients had received a median of 3 prior lines of therapy (range, 1-6). Doses ranging from 3 × 107 to 9 × 108 CAR T cells were administered, and no dose-limiting toxicities were encountered. Cytokine release syndrome (CRS) was observed in 8 patients (50%); all events were grade 1 or 2. Infections were observed in 4 patients (25%), all deemed to be unrelated to CTX130. No hemophagocytic lymphohistiocytosis, graft-versus host disease or treatment-related neurotoxicity was observed. As of Oct 9, 2023, 1 patient (6.3%) achieved a complete response (CR) (now extending over 36+ months) and 12 additional patients (75%) achieved stable disease (SD) (1 patient in excess of 18 months), reflecting a disease control rate (CR/partial response/SD) of 81.3%. Immunohistochemical staining of baseline and archival tissues show an initial median CD70 expression level of 100% (range, 1-100%), with no substantial antigenic loss at sequential biopsies performed at day 42. Using digital droplet PCR assays of the CAR construct, we assessed expansion. CTX130 was detected in 20 minutes after infusion, declined to a nadir 2-3 days later, followed by rapid expansion between days 7 and 15. In general, expansion increased at each dose level. Conclusions: Updated results from the COBALT-RCC study reinforce both the encouraging antitumor activity and exceptional safety profile of CTX130. The durable CR, now in excess of 3 years, reflects the first and longest seen with allogeneic CAR T cell therapy in refractory solid tumors. A study of CTX131™ (building on CTX130 with CRISPR edits to enhance potency and persistence) is ongoing. Citation Format: Sumanta Kumar Pal, Ben Tran, John B. Haanen, Michael Hurwitz, Adrian Sacher, Neeraj Agarwal, Nizar Tannir, Elizabeth Budde, Simon Harrison, Sebastian Klobuch, Sagar S. Patel, Mary-Lee Dequeant, Qiuling Ally He, Alissa Keegan, Henia Dar, Anna Ma, PK Morrow, Samer A. Srour. CTX130 allogeneic CRISPR-Cas9-engineered chimeric antigen receptor (CAR) T cells in patients with advanced clear cell renal cell carcinoma: Long-term follow-up and translational data from the phase 1 COBALT-RCC study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT002.

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.

Abstract LB106: Ultra-short ctDNA: A new paradigm for ctDNA liquid biopsy

Abstract Mononucleosomal ctDNA (mnctDNA, 157-bp, double-stranded) spearheads liquid biopsy (LB) for cancer detection, treatment and clinical care. The bottleneck of mnctDNA LB are low copy number of ctDNA and limit of detection of platform technologies. Recent discovery of ultra-short ctDNA (usctDNA, 52-54 nt, single-stranded) that harbors somatic mutations prompted us to explore the quantification and stoichiometry of usctDNA to mnctDNA in plasma and saliva of NSCLC patients. Paired plasma and saliva from 20 advanced NSCLC patients with tissue genotyped positive for L858R were processed for cfDNA extraction and quantification with Qubit and Tape Station. Digital PCR assays targeting L858R somatic mutation to generate amplicons of 57-bp (usctDNA) and 100-bp (mnctDNA) were performed by UCLA and independently by NIST. Results in Table 1 revealed that three findings of usctDNA that will have potential to impact ctDNA liquid biopsy. First, the somatic mutation (L858R) is present in usctDNA. Second, The stoichiometric ratio of usctDNA to mnctDNA is similar in plasma and saliva at 1.6X. For the wild-type counterpart, the ratio is 1.1X for plasma and 0.6X for saliva. Third, the copy number of usctDNA and mnctDNA in saliva to plasma is 7.8X and 7.7X. Conclusion: Detection of tumor-associated/specific EGFR L858R somatic mutations in uscfDNA is translationally impactful, adding 1.6X of the ctDNA targets for molecular detection, effectively cumulating 2.6X of total ctDNA targets, permitting earlier cancer detection, treatment monitoring and therapy prognostication. The finding that saliva harbors 7X of the us- and mnctDNA further raises the prospect of advancing saliva ctDNA liquid biopsy for NSCLC detection as it may effectively allow 15.5X (7.8X+7.7X) ability to detect malignancy-associated ctDNA. Table 1. Quantification of usctDNA and mnctDNA in Plasma and Saliva Mut usctDNA (L858R) WT cfDNA usctDNA mnctDNA U/M uscfDNA mncfDNA U/M Plasma 1555 ± 326 964 ± 209 1.6X 15201 ± 1509 13261 ± 1424 Saliva 12158 ± 1731 7442 ± 1020 1.6X 340896 ± 27170 601733 ± 71573 1.1X Saliva/Plasma (fold X) 7.8 7.7 1.6X 22.4 45.3 0.6X Citation Format: Feng Li, Fang Wei, Hua-Jun He, Chien-Chung Lin, Szu-Chun Yang, Allen Wei-Lun Huang, Cheuk Ying Tang, Yong Kim, David T. Wong. Ultra-short ctDNA: A new paradigm for ctDNA liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB106.

Abstract LB099: Identifying actionable genomic alterations in non-small cell lung cancer through the analysis of extracellular vesicles in plasma

Abstract Lung cancer is the second most common cancer worldwide, accounting for 2.2 million new cases and 1.8 million deaths in 2020. Personalized targeted therapy based on actionable molecular markers has completely transformed the therapeutic landscape in NSCLC, which has improved considerably the overall survival. However, ~50% of NSCLC patients with tissue biopsy inaccessible or inadequate, limiting biomarker testing and access to targeted therapies. The availability of a wide range of highly effective targeted therapy options for NSCLC justifies the significance of developing non-invasive and cost-effective detection assays for guiding targeted therapy selection in NSCLC.Extracellular vesicles (EVs) are lipid-bilayer-enclosed vesicles of sub-micrometer size that are secreted by virtually all cell types. They contain hundreds of different proteins, thousands of intact RNA species, and double-stranded DNA (dsDNA) fragments that sample the entire human genome. Recent findings further corroborate the potential role of EVs in early cancer screening, cancer diagnosis, treatment selection and monitoring. In this study, we used lipid nanoprobe (LNP) for EV isolation from plasma and droplet digital PCR assay for detecting actionable genomic alternation to aid targeted therapy selection in NSCLC. Specifically, we extracted EV associated DNA/RNA from plasma first, and then we used the individual ddPCR assays for detecting KRAS mutations, EGFR mutations, and ALK fusions from isolated EVs associated nucleic acids, respectively. We identified KRAS mutations G12C/G12S/G13C/G12A/Q61H, EGFR L858R mutation and exon 19 deletion, and ALK-EML4 gene fusion variant 3 from 12 NSCLC patients that matched the mutation status obtained from patient tissue specimens. Only in 1 out 13 patients, EV mutation status did not match the mutation status from the tissue specimen. This data indicated that clinical actionable gene mutations are present in EVs associated nucleic acids isolated from plasma samples of NSCLC patients. We anticipate that this novel non-invasive EVs-based actionable genomic alterations detection test would efficiently assist physicians/oncologists in selecting optimal targeted therapies for cancer treatment, as recommended by National Comprehensive Cancer Network (NCCN) guidelines for NSCLC. Citation Format: Hongzhang He, Jadranka Milosevic, Siyang Zheng. Identifying actionable genomic alterations in non-small cell lung cancer through the analysis of extracellular vesicles in plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB099.

Laboratory-developed Droplet Digital PCR Assay for Quantification of the JAK2 V617F Mutation.

Precise quantification of the JAK2 V617F mutation using highly sensitive assays is crucial for diagnosis, treatment process monitoring, and prognostic prediction in myeloproliferative neoplasms' (MPNs) patients. Digital droplet polymerase chain reaction (ddPCR) enables precise quantification of low-level mutations amidst a high percentage of wild type alleles without the need for external calibrators or endogenous controls. The objective of this study was to optimize a ddPCR assay for detecting the JAK2 V617F mutation and establish it as a laboratory-developed ddPCR assay in our center. The optimization process involved fine-tuning five key parameters: primer/probe sequences and concentrations, annealing temperature, template amount, and PCR cycles. Our ddPCR assay demonstrated exceptional sensitivity, and the limit of quantification (LoQ) was 0.01% variant allele frequency with a coefficient of variation of approximately 76%. A comparative analysis with quantitative PCR on 39 samples showed excellent consistency (r = 0.988). In summary, through rigorous optimization process and comprehensive analytic performance validation, we have established a highly sensitive and discriminative laboratory-developed ddPCR platform for JAK2 V617F detection. This optimized assay holds promise for early detection of minimal residual disease, personalized risk stratification, and potentially more effective treatment strategies in MPN patients and non-MPN populations.

Optimizing Droplet Digital PCR Assay for Precise Assessment of MEIS1 Gene Promoter Methylation

Abstract DNA methylation is characterized as a gene regulatory mechanism that involves the methylation of the 5-carbon (C5) position of cytosine, resulting in the formation of 5-methylcytosine. The analysis of aberrantly methylated cytosine-phosphate-guanine (CpG) dinucleotides, primarily in the promoter regions of tumor suppressor genes, can serve as promising prognostic and predictive markers of cancer development. Meis homeobox 1 (MEIS1) gene, crucial for cell growth and differentiation, exhibits dysregulation linked to various cancer types, acting as both a positive and negative regulator. The selection of an appropriate method for the evaluation of gene promoter methylation status is important for clinical implementation without biases regarding false positive and false negative outcomes. The study focuses on the optimization of a novel droplet digital PCR (ddPCR) assay for identifying the methylation status of MEIS1. Compared to traditional methods, ddPCR offers an increased sensitivity and specificity, presenting a promising tool for precise DNA methylation assessment with potential implications for cancer diagnostics and prognostics.