Multiplex Digital PCR Research Articles

Optimization and Application of a Multiplex Digital PCR Assay for the Detection of SARS-CoV-2 Variants of Concern in Belgian Influent Wastewater

Since the beginning of the COVID-19 pandemic, the wastewater-based epidemiology (WBE) of SARS-CoV-2 has been used as a complementary indicator to follow up on the trends in the COVID-19 spread in Belgium and in many other countries. To further develop the use of WBE, a multiplex digital polymerase chain reaction (dPCR) assay was optimized, validated and applied for the measurement of emerging SARS-CoV-2 variants of concern (VOC) in influent wastewater (IWW) samples. Key mutations were targeted in the different VOC strains, including SΔ69/70 deletion, N501Y, SΔ241 and SΔ157. The presented bioanalytical method was able to distinguish between SARS-CoV-2 RNA originating from the wild-type and B.1.1.7, B.1.351 and B.1.617.2 variants. The dPCR assay proved to be sensitive enough to detect low concentrations of SARS-CoV-2 RNA in IWW since the limit of detection of the different targets ranged between 0.3 and 2.9 copies/µL. This developed WBE approach was applied to IWW samples originating from different Belgian locations and was able to monitor spatio-temporal changes in the presence of targeted VOC strains in the investigated communities. The present dPCR assay developments were realized to bring added-value to the current national WBE of COVID-19 by also having the spatio-temporal proportions of the VoC in presence in the wastewaters.

Multiplex digital PCR with digital melting curve analysis on a self-partitioning SlipChip.

Digital polymerase chain reaction (digital PCR) can provide absolute quantification of target nucleic acids with high sensitivity, excellent precision, and superior resolution. Digital PCR has broad applications in both life science research and clinical molecular diagnostics. However, limited by current fluorescence imaging methods, parallel quantification of multiple target molecules in a single digital PCR remains challenging. Here, we present a multiplex digital PCR method using digital melting curve analysis (digital MCA) with a SlipChip microfluidic system. The self-partitioning SlipChip (sp-SlipChip) can generate an array of nanoliter microdroplets with trackable physical positions using a simple loading-and-slipping operation. A fluorescence imaging adaptor and an in situ thermal cycler can be used to perform digital PCR and digital MCA on the sp-SlipChip. The unique signature melting temperature (Tm) designed for amplification products can be used as a fingerprint to further classify the positive amplification partitions into different subgroups. Amplicons with Tm differences as low as 1.5 degrees celsius were clearly separated, and multiple amplicons in the same partition could also be distinguished by digital MCA. We further demonstrated this digital MCA method with simultaneous digital quantification of five common respiratory pathogens, including Staphylococcus aureus, Acinetobacter baumannii, Streptococcus pneumoniae, Hemophilus influenzae, and Klebsiella pneumoniae. Since digital MCA only requires an intercalation dye instead of sequence-specific hydrolysis probes to perform multiplex digital PCR analysis, it can be less expensive and not limited to the number of fluorescence channels.

Generic Multiplex Digital PCR for Accurate Quantification of T Cells in Copy Number Stable and Unstable DNA Samples.

An accurate T cell quantification is prognostically and therapeutically relevant in various clinical applications, including oncology care and research. In this chapter, we describe how T cell quantifications can be obtained from bulk DNA samples with a multiplex digital PCR experiment. The experimental setup includes the concurrent quantification of three different DNA targets within one reaction: a unique T cell DNA marker, a regional corrector, and a reference DNA marker. The T cell marker is biallelically absent in T cells due to VDJ rearrangements, while the reference is diploid in all cells. The so-called regional corrector allows to correct for possible copy number alterations at the T cell marker locus in cancer cells. By mathematically integrating the measurements of all three markers, T cells can be accurately quantified in both copy number stable and unstable DNA samples.

Similar color analysis based on deep learning (SCAD) for multiplex digital PCR via a single fluorescent channel.

Digital PCR (dPCR) has recently attracted great interest due to its high sensitivity and accuracy. However, the existing dPCR depends on multicolor fluorescent dyes and multiple fluorescent channels to achieve multiplex detection, resulting in increased detection cost and limited detection throughput. Here, we developed a deep learning-based similar color analysis method, namely SCAD, to achieve multiplex dPCR in a single fluorescent channel. As a demonstration, we designed a microwell chip-based diplex dPCR system for detecting two genes (blaNDM and blaVIM) with two kinds of green fluorescent probes, whose emission colors are difficult to discriminate by traditional fluorescence intensity-based methods. To verify the possibility of deep learning algorithms to distinguish the similar colors, we first applied t-distributed stochastic neighbor embedding (tSNE) to make a clustering map for the microwells with similar fluorescence. Then, we trained a Vision Transformer (ViT) model on 10 000 microwells with two similar colors and tested it with 262 202 microwells. Lastly, the trained model was proven to have highly accurate classification ability (>98% for both the training set and the test set) and precise quantification ability on both blaNDM and blaVIM (ratio difference <0.10). We envision that the developed SCAD method would significantly expand the detection throughput of dPCR without the need for other auxiliary equipment.

Coupling Machine Learning and High Throughput Multiplex Digital PCR Enables Accurate Detection of Carbapenem-Resistant Genes in Clinical Isolates.

Rapid and accurate identification of patients colonised with carbapenemase-producing organisms (CPOs) is essential to adopt prompt prevention measures to reduce the risk of transmission. Recent studies have demonstrated the ability to combine machine learning (ML) algorithms with real-time digital PCR (dPCR) instruments to increase classification accuracy of multiplex PCR assays when using synthetic DNA templates. We sought to determine if this novel methodology could be applied to improve identification of the five major carbapenem-resistant genes in clinical CPO-isolates, which would represent a leap forward in the use of PCR-based data-driven diagnostics for clinical applications. We collected 253 clinical isolates (including 221 CPO-positive samples) and developed a novel 5-plex PCR assay for detection of blaIMP, blaKPC, blaNDM, blaOXA-48, and blaVIM. Combining the recently reported ML method “Amplification and Melting Curve Analysis” (AMCA) with the abovementioned multiplex assay, we assessed the performance of the AMCA methodology in detecting these genes. The improved classification accuracy of AMCA relies on the usage of real-time data from a single-fluorescent channel and benefits from the kinetic/thermodynamic information encoded in the thousands of amplification events produced by high throughput real-time dPCR. The 5-plex showed a lower limit of detection of 10 DNA copies per reaction for each primer set and no cross-reactivity with other carbapenemase genes. The AMCA classifier demonstrated excellent predictive performance with 99.6% (CI 97.8–99.9%) accuracy (only one misclassified sample out of the 253, with a total of 160,041 positive amplification events), which represents a 7.9% increase (p-value <0.05) compared to conventional melting curve analysis. This work demonstrates the use of the AMCA method to increase the throughput and performance of state-of-the-art molecular diagnostic platforms, without hardware modifications and additional costs, thus potentially providing substantial clinical utility on screening patients for CPO carriage.

Accurate Quantification of T Cells in Copy Number Stable and Unstable DNA Samples Using Multiplex Digital PCR

An accurate T-cell quantification is prognostically and therapeutically relevant in various malignancies. We previously developed a digital PCR-based approach offering a precise T-cell enumeration in small amounts of DNA. However, it may be challenging to apply this method in malignant specimens, as genetic instability can disturb the underlying mathematical model. For example, approximately 24% of the tumors from The Cancer Genome Atlas pan-cancer data set carried a copy number alteration affecting the TRB gene T-cell marker, which would cause an underestimation or overestimation of the T-cell fraction. In this study, we introduce a multiplex digital PCR experimental setup to quantify T cells in copy number unstable DNA samples. By implementing a so-called regional corrector, genetic alterations involving the T-cell marker locus can be recognized and corrected for. This novel setup is evaluated mathematically in silico and validated invitro by measuring T-cell presence in various samples with a known T-cell fraction. The utility of the approach is further demonstrated in copy number altered cutaneous melanomas. Our novel multiplex setup provides a simple, but accurate, DNA-based T-cell quantification in both copy number stable and unstable specimens. This approach has potential clinical and diagnostic applications, as it does not depend on availability of T-cell epitopes, has low requirements for sample quantity and quality, and can be performed in a relatively easy experiment.

Simple, Precise, and Less Biased GMO Quantification by Multiplexed Genetic Element-Specific Digital PCR.

To provide the consumer with choices of genetically modified organisms (GMO) or non-GMO, official food labeling systems were established in many countries. Because the threshold GMO content values were set to distinguish between "non-GMO" and "GMO" designations, GMO content quantification methods are required for ensuring the appropriateness of labeling. As the number of GMOs is continuously increasing around the world, we set out to develop a low-cost, simple and less biased analytical strategy to cover all necessary detection targets. Digital PCR methods are advantageous compared to the conventional quantitative real-time PCR methods. We developed a digital PCR-based GMO quantification method to evaluate the GMO content in maize grains. To minimize the analytical workload, we adopted multiplex digital PCR targeting the 35S promoter and the nopaline synthase terminator, which are genetic elements commonly introduced in many GMOs. Our method is significantly simpler and more precise than the conventional real-time PCR-based methods. Additionally, we found that this method enables quantification of the copy number of GMO DNA without double counting multiple elements (35S promoter and nopaline synthase terminator) tandemly placed in a recombinant DNA construct. This is the first report on the development of a genetically modified maize quantification method using a multiplexed genetic element-specific digital PCR method. The tandem effect we report here is quite useful for reducing the bias in the analytical results. Multiplexed genetic element-specific digital PCR can simplify weight-based GMO quantification and thus should prove useful in light of the continuous increase in the number of GM events.

Tunable and Contamination-Free Injection with Microfluidics by Stepinjection.

Droplet microfluidics with picoinjection provides significant advantages to multistep reactions and screenings. The T-junction design for picoinjection is convenient in adding picoliter reagents into passing droplets to initiate reactions. However, conventional picoinjectors face difficulties in eliminating cross-contamination between droplets, preventing them from widespread use in sensitive biological and molecular assays. Here, we introduce stepinjection, which uses a T-junction with a stepped channel design to elevate the diffusional buffer zone into the main channel and consequently increases the pressure difference between droplets and the inlet of the injection channel. To demonstrate the stepinjector's ability to perform contamination-sensitive enzymatic assays, we inject casein fluorescein isothiocyanate (FITC-casein) into a mixture of savinase and savinase-free (labeled with a red fluorescent dye) droplets. We observe no cross-contamination using stepinjection but find a severe cross-talk using an optimal picoinjection design. We envision that the simple, tunable, and reliable stepinjector can be easily integrated in various droplet processing devices, and facilitate various biomedical and biochemical applications including multiplex digital PCR, single-cell sequencing, and enzymatic screening.

IBCL-419: Non-Invasive Quantitative Analysis of EZH2 Mutations in Follicular Lymphoma

Context Follicular lymphoma (FL) is the most common type of indolent non-Hodgkin lymphoma, with an average overall survival rate of 15 years. The prolonged disease course raises the need for an effective disease monitoring system, which is hampered by the limited specificity and sensitivity of the most widely used, imaging-based PET-CT method. Molecular analysis of tumor-derived circulating DNA (ctDNA), frequently referred to as liquid biopsy, is a promising minimally invasive disease monitoring tool for patients suffering from malignancies. Recent studies interrogating the genomic background of FL have revealed activating mutations in the epigenetic regulator EZH2 gene in 25% of patients. Objective In our study, we tested the potential of liquid biopsy to detect and monitor EZH2 mutations in plasma samples of patients with EZH2-mutant FL treated with immunochemotherapy. Patients and Methods Eighty-five blood plasma samples were collected from 41 EZH2 mutant patients. Cell-free plasma DNA was extracted using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Germany). The EZH2 mutation status was assessed using a multiplex digital droplet PCR (ddPCR, Bio-Rad Laboratories, USA) approach, detecting seven different EZH2 hotspot mutations using only two PCR reactions. The molecular results were correlated with PET-CT images. Results Among the 41 patients, 36 responded to treatment (88%); meanwhile, 5 patients did not (12%). We detected an EZH2 mutation in 89% of the samples collected prior to treatment (17/19). Similarly, 86% of the samples (6/7) collected during therapy from patients not responding to treatment proved to be EZH2 mutated; meanwhile, only 22% of the samples were positive from responding patients (6/33). As expected, all samples collected during remission were EZH2 wild-type. EZH2 variant allele frequencies (VAF) correlated with the total metabolic tumor volume detected on the PET-CT scans. We also demonstrated spatial heterogeneity of EZH2 mutations in two cases, where different EZH2 mutations deriving from distinct anatomical sites could simultaneously be detected in the plasma. The median sensitivity of our ddPCR approach was 0.15%, with EZH2 VAFs ranging between 0.2 and 74%. Conclusions Our results demonstrate that liquid biopsy offers a sensitive, radiation-free, minimally invasive monitoring method for EZH2-mutant FL patients.

Development of multiplex digital PCR assays for the detection of PIK3CA mutations in the plasma of metastatic breast cancer patients

With the approval of new therapies targeting the PI3K pathway, the detection of PIK3CA mutations has become a key factor in treatment management for HR+/HER2− metastatic breast cancer (MBC). We developed multiplex digital PCR (dPCR) assays to detect and quantify PIK3CA mutations. A first screening assay allows the detection of 21 mutations, with a drop-off system targeting the 542–546 hotspot mutations combined with the simultaneous detection of N345K, C420R, H1047L and H1047R mutations. In the case of a positive result, a sequential strategy based on other assays that we have developped allows for precise mutation identification. Clinical validity was determined by analyzing plasma circulating free DNA (cfDNA) from 213 HR+/HER2− MBC samples, as well as DNA extracted from 97 available matched tumors from 89 patients. Our assays have shown reliable specificity, accuracy and reproducibility, with limits of blank of three and four droplets for the screening assay. Sixty-eight patients (32%) had at least one PIK3CA mutation detectable in their plasma, and we obtained 83.1% agreement between the cfDNA analysis and the corresponding tumors. The high sensitivity and robustness of these new dPCR assays make them well-suited for rapid and cost-effective detection of PIK3CA mutations in the plasma of MBC patients.

Abstract 579: Multiplex digital PCR combined with melting curve analysis for detecting KRAS mutations towards pancreatic cancer diagnosis

Abstract Background Digital PCR (dPCR) is a promising method for liquid biopsies and is able to quantify nucleic acids more sensitively than real-time PCR. However, dPCR has a large fluctuation in the fluorescence intensity of the droplets or wells due to insufficient PCR in the small partitions, limiting the multiplexing capability of using the fluorescence intensity. In this study, we propose a measurement method that combines dPCR with melting curve analysis for highly multiplexed genotyping. Methods A sample is digitized into a silicon chip with up to ~104 wells in which asymmetric PCR was performed to obtain more single-stranded amplicons that are complimentary to molecular beacon probes. Fluorescence images were captured while controlling the temperature of the chip, and melting curve was measured for each well. Then, genotyping was performed by using the fluorescence intensity, dye color of the probe, and melting temperature (Tm). Because the Tm of the PCR products does not considerably depend on the amplification efficiency of PCR, genotyping accuracy is improved by using Tm values, enabling highly multiplexed genotyping. Results The concept was confirmed by simultaneously identifying wild-type KRAS, BRAF and, eight mutants of these genes (G12D, G12R, G12V, G13D, G12A, G12C, G12S, and V600E) through four-color melting curve analysis. G12D, G12V and G12R-mutant KRAS alleles were detected with FAM-labeled probes, G13D-mutant KRAS allele and wildtype KRAS allele were detected with HEX-labeled probes, G12S and G12C-mutant KRAS alleles were detected with Cy5-labeled probes, and V600E-mutant BRAF allele and wildtype BRAF allele were detected with CAL Fluor Red 610-labeled probes. The results showed that the peaks of the Tm distributions of the DNA groups were separated by 3.5 °C on average, while the standard deviation of the Tm distributions was 0.23 °C on average. The difference in Tm was 15 times larger than the standard deviation, enabling more accurate genotyping than when using the fluorescence intensity. Conclusion We have proposed a measurement method that combines dPCR with melting curve analysis, and successfully performed the multiplexed genotyping assay. We plan to evaluate the effect of this method on measurement using clinical specimens of pancreatic cancer patients in the future. Citation Format: Junko Tanaka, Tatsuo Nakagawa, Takeshi Ishida, Yoshio Kamura, Akiko Shiratori, Kunio Harada, Chihiro Uematsu. Multiplex digital PCR combined with melting curve analysis for detecting KRAS mutations towards pancreatic cancer diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 579.

Integrating endobronchial ultrasound bronchoscopy with molecular testing of immunotherapy biomarkers in non-small cell lung cancer

Immunotherapy has transformed treatment of advanced non-small-cell lung cancer (NSCLC) patients leading to remarkable long-term survival benefit. However, only about 20% of advanced NSCLC patients typically respond to immune checkpoint inhibitors (ICIs) that target the PD-1/PD-L1 pathway. The only validated biomarker for ICI therapy is the PD-L1 immunohistochemistry (IHC) test, which is considered an imperfect assay due to several variables including availability and integrity of tumour tissue, variability in staining/scoring techniques and heterogeneity in PD-L1 protein expression within and across tumour biopsies. Herein, we discuss integrating minimally invasive EBUS bronchoscopy procedures with novel molecular approaches to improve accuracy and sensitivity of PD-L1 testing. EBUS guided bronchoscopy facilitates repeated sampling of tumour tissue to increase the probability of detecting PD-L1 positive tumours. Since intra-tumoural PD-L1 (CD274) copy number is reported to be less heterogeneous than PD-L1 protein detection, quantifying PD-L1 transcript levels may increase detection of PD-L1 positive tumours. PD-L1 transcript levels show excellent concordance with PD-L1 IHC scoring and multiplex digital droplet PCR (ddPCR) assays that quantify absolute PD-L1 transcript copy number have been developed. ddPCR can also be automated for high throughput detection of low abundant variants with excellent sensitivity and accuracy to improve the broader application of diagnostic cut-off values. Optimizing diagnostic workflows that integrate optimal EBUS bronchoscopy procedures with emerging molecular ICI biomarker assays may improve the selection criteria for ICI therapy benefit.

The Clinical Value of Measuring Circulating HPV DNA during Chemo-Radiotherapy in Squamous Cell Carcinoma of the Anus

Simple SummaryAnal cancer is treated with high dose chemoradiotherapy; but despite this, a minor patient group experience treatment failure. Anal cancer is strongly related to human papilloma virus (HPV) infection and previous smaller studies have shown that fragments of HPV DNA can be detected in blood samples from anal cancer patients. This study measured HPV DNA in 88 patients including both small- and more advanced tumors and detected six different subtypes of HPV. During treatment with chemoradiotherapy, the level of HPV DNA decreased, and three elimination patterns with clinical relevance were identified. Fast elimination correlated to a low risk of failure, slow elimination correlated to risk of failure in the pelvis, and persistent HPV DNA after treatment correlated to a high risk of later distant failure. The results add new information to the increasing interest in research of HPV DNA in HPV related cancers and holds great clinical potential.Background and purpose: Circulating tumor DNA (ctDNA) is investigated in various cancers. In squamous cell carcinoma of the anus (SCCA) infection with human papilloma virus (HPV) is found in around 90% of cases and here, plasma HPV (pHPV) can be used as ctDNA. Preliminary data have proved the ability to detect pHPV16 and -18 in SCCA. We have developed a highly sensitive method for measurement of six relevant pHPV subtypes, to investigate the elimination pattern of pHPV during chemo-radiotherapy (CRT) for SCCA and its clinical value. Material and methods: Patients treated at Aarhus University Hospital from 2016–2020 were included. P16 status in the primary biopsy was measured and 82% of patients had P16 positive tumor. Blood samples were collected prior to treatment (PT), mid treatment (MT), end of therapy (EOT), and during follow-up (FU). An in-house multiplex digital droplet PCR method measured pHPV subtypes 16, 18, 31, 33, 51, 58. Results: Samples from 88 patients were drawn PT (n = 73), MT (n = 72), EOT (n = 64) and during FU (n = 41). Plasma HPV was detectable in 52 patients and PT pHPV levels correlated to tumor stages. Three elimination patterns were observed during CRT with correlation to outcome: fast responders with no local or distant failures (0/12); slow responders with high risk of local failures (4/20), no distant failures; persistent molecular responders with high risk of distant failures (4/13), but no local failures, p < 0.01. Conclusion: During CRT, pHPV can divide patients with SCCA into three groups with significantly different risk of failure. The use of pHPV can potentially assist in clinical treatment decision.

Abstract 4319: Clinical utility of multiplex digital PCR to detect androgen receptor amplification in cell-free DNA for prognosis of metastatic castration-resistant prostate cancer

Abstract Background: Androgen receptor (AR) amplification is a common genomic event in advanced prostate cancer, in particular, the metastatic castration-resistant prostate cancer (mCRPC) and is associated with poor treatment response and short overall survival (OS). Recent studies also show more frequent amplification at AR enhancer region in the mCRPC patients. The aim of this study is to evaluate clinical utility of multiplex digital PCR assay to detect amplification at AR locus in cell-free DNA (cfDNA) for prognostication of mCRPC. Methods: Plasma samples were collected from 108 patients with mCRPC before stage-specific treatment. The target regions were selected based on our previous sequencing data showing frequent amplification in mCRPC patients at AR loci, including AR enhancer, AR (AR exon 1 and exon 8) and OPHN1 (downstream of AR). Multiplex digital PCR assays to detect four targets were developed by adjusting the concentrations of primers and probes. The Kaplan-Meier (KM) estimate was used for OS analysis. Results: We first examined existing databases to determine relative stable genomic regions in prostate cancer. This analysis showed most stable regions in three gene loci including C2orf16 at 2p23.2, FAM111B at 11q12.1, and GRIA3 at Xq25, which were then used as reference controls for copy number normalization. We then designed two multiplex digital PCR assays to quantify copy number of selected targets. To separate two different targets with same fluorescence, we adjusted ratio of FAM-labeled probes with 1: 0.4 and HEX-labeled probes with 1:0.4. The ratio of FAM to HEX probes was 1:1.5 in final assays. The combination of four different targets in one reaction gave 9 possible clusters in the 2D amplitude space. We defined the amplification as the normalized ratio ≥ 1.8 between target and reference loci. By applying this cutoff in 108 mCRPC samples, we detected the amplification of AR-enhancer, AR and OPHN1 in 38, 34, and 25 samples, respectively. KM analysis showed the significant association of amplification in AR-enhancer (p=0.0053, HR=2.16[1.41-6.53]), AR (p=0.04, HR=1.74[1.04-4.03]) and OPHN1 (p=0.031, HR=1.91[1.07-5.53]) with OS. This result was consistent with whole genome sequencing data in the same group of patients. Conclusions: We established a highly sensitive multiplex digital PCR assay to detect copy number changes of AR, AR enhancer and OPHN1 in plasma cfDNA and demonstrated its potential clinical use as non-invasive testing tool for mCRPC prognosis. The easy-to-use and low-cost multiplex digital PCR assay covers multiple gene loci, which can aid in improving the sensitivity of detecting amplification at AR locus in the highly heterogeneous mCRPC patients. Citation Format: Meijun Du, Chiang-Ching Huang, Winston Tan, Manish Kohli, Liang Wang. Clinical utility of multiplex digital PCR to detect androgen receptor amplification in cell-free DNA for prognosis of metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4319.

Abstract 166: Absolute quantification of DNA methylation markers using methylation-sensitive restriction enzymes and multiplex digital PCR

Abstract Aberrant DNA methylation plays important roles in the initiation and progression of cancer. Therefore, DNA methylation markers have important diagnostic and prognostic potential. However, accurate quantification of these markers remains challenging in clinical samples of limited quality or quantity. We introduce a novel, digital approach for the absolute quantification of the amount, density and allele-specificity of DNA methylation. We designed an experimental setup combining efficient methylation-sensitive restriction enzymes with quantitative digital PCR. This allowed to measure a targeted density of DNA methylation independent of bisulphite conversion. In-silico simulations were performed to demonstrate the mathematical validity of the setup. A practical validation was carried out by quantifying RASSF1A promotor methylation in a variety of healthy and malignant reference samples and in an innovative calibration curve. Furthermore, we extended our setup to a multiplex digital PCR experiment. Hereby, methylation markers may be analyzed together with physically linked genetic markers to determine the allele-specificity of the methylation. Overall, our results show an accurate quantification of DNA methylation markers with digital PCR, independent of bisulphite conversion. Effective measurements with high precision could be obtained, even in minute amounts of DNA. Moreover, as the context-density and allele-specificity of methylation can also be determined, mechanisms in cancer biology can now be quantitatively assessed in a digital experiment. Citation Format: Rogier J. Nell, Pieter A. Van der Velden. Absolute quantification of DNA methylation markers using methylation-sensitive restriction enzymes and multiplex digital PCR [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 166.

Abstract 1980: Multiplex digital PCR combined with melting curve analysis for detecting KRAS and BRAF mutations

Abstract Background Digital PCR (dPCR) is a promising method for liquid biopsies and is able to quantify nucleic acids more sensitively than real-time PCR. However, dPCR has a large fluctuation in the fluorescence intensity of the droplets or wells due to insufficient PCR in the small partitions, limiting the multiplexing capability of using the fluorescence intensity. In this study, we propose a new measurement method that combines dPCR with melting curve analysis for highly multiplexed genotyping. Methods A sample is digitized into a silicon chip with up to ~104 wells in which asymmetric PCR was performed to obtain more single-stranded amplicons that are complimentary to molecular beacon probes. Fluorescence images were captured while controlling the temperature of the chip, and melting curve was measured for each well. Then, genotyping was performed by using the fluorescence intensity, dye color of the probe, and melting temperature (Tm). Because the Tm of the PCR products does not considerably depend on the amplification efficiency of PCR, genotyping accuracy is improved by using Tm values, enabling highly multiplexed genotyping. Results The concept was confirmed by measuring four common KRAS mutations (G12D, G12V, G12R, and G13D) and one common BRAF mutation (V600E). G12D, G12V and G12R-mutant KRAS alleles were detected with FAM-labeled probes, G13D-mutant KRAS allele and wildtype KRAS allele were detected with HEX-labeled probes, and V600E-mutant BRAF allele and wildtype BRAF allele were detected with CAL Fluor Red 610-labeled probes. The results showed that the peaks of the Tm distributions of the DNA groups were separated by more than 2°C. By combining the fluorescence intensities and the Tm values, the wildtype and mutant groups were clearly identified and able to be quantified. Conclusion We have proposed a new measurement method that combines dPCR with melting curve analysis, and successfully performed the multiplexed genotyping assay. To our knowledge, this is the first demonstration of genotyping of seven DNA groups with a single mutation of cancer-related genes by combining dPCR with melting curve analysis. Citation Format: Junko Tanaka, Tatsuo Nakagawa, Akiko Shiratori, Kunio Harada, Chihiro Uematsu, Erina Takai, Shinichi Yachida. Multiplex digital PCR combined with melting curve analysis for detecting KRAS and BRAF mutations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1980.

Abstract 4113: Discriminatory 11-plex assay designed to monitor estrogen receptor mutations using a novel, melt-analysis capable digital PCR platform

Abstract In a large fraction of ER+/HER2- metastatic breast cancer (MBC) patients, treatment with aromatase inhibitors fails due to emerging resistance. A key mechanism of this resistance is associated with a limited set of missense mutations in the gene encoding for the estrogen receptor (ESR1). These mutations are primarily found in the ligand binding domain of the protein. They result in a constitutively active estrogen receptor, are infrequently found in primary tumors (&amp;lt;5%) and instead likely coincide with the emergence of an endocrine resistant phenotype. The occurrence of ESR1 mutations following treatment has varied from study to study depending on patient selection and method of detection, but prevalence may be as high as 50%. Because these patients require second line therapies as soon as possible, ESR1 mutations are an area of intense focus in research and clinical trials for ER+/HER2- breast cancer. Studies utilizing droplet digital PCR (ddPCR) assays have correlated an increase of ESR1 mutations in patient liquid biopsy samples with therapeutic resistance approximately 8 months prior to conventional methods, however the number of individual mutations evaluated in these studies was limited due to an inability to accurately and sensitively identify more than one individual mutated SNP allele within a single reaction. We have developed a multiplex digital PCR assay that utilizes melt-based probes to detect and discriminate between 10 unique, clinically relevant ESR1 mutations in circulating tumor DNA (ctDNA) samples while simultaneously quantifying total cell-free DNA (cfDNA). Designed for use with an innovative, array-based dPCR system, the assay requires a hands-on time of only 10-15 minutes and a total run time of 2.5 hours. This assay will allow breast cancer researchers to further refine our understanding of the role ESR1 mutations play in personalized treatment strategies for MBC. Citation Format: Jennifer N. Bernier, Johanna E. Takach, Tiffany Wang, Steve Gallagher, Robert Lin, Andrew Zayac, Paul Hung, Matthew Lesho, Douglas Whitman. Discriminatory 11-plex assay designed to monitor estrogen receptor mutations using a novel, melt-analysis capable digital PCR platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4113.

Multiplex Digital PCR to Detect Amplifications of Specific Androgen Receptor Loci in Cell-Free DNA for Prognosis of Metastatic Castration-Resistant Prostate Cancer.

Amplification of androgen receptor (AR) is a common genomic event in metastatic castration-resistant prostate cancer (mCRPC). To evaluate the prognostic value of the amplifications of specific loci in the AR gene in cell-free DNA, we developed a multiplex digital PCR (dPCR) assay that targeted AR enhancer (AR-En), AR exon 1 (AR-E1), AR exon 8 (AR-E8) and OPHN1 (downstream of AR). We selected three relatively stable genes, C2orf16, FAM111B, and GRIA3, as reference controls for copy number normalization. One hundred and eight mCRPC patients were recruited to test the association of specific AR loci amplification with clinical outcome. Using a normalized ratio ≥ 1.92 as cutoff, amplification of AR-En, AR-E1, AR-E8 and OPHN1 was observed in 28, 25, 24 and 19 of 108 mCRPC patients, respectively. Among the 41 patients with AR region amplification, 9 (21.9%) showed amplification at all four selected regions and 15 (36.6%) showed amplification at AR-En, AR-E1, and AR-E8. Six (14.6%) patients showed independent AR-En amplification, while the remaining 3 (7.3%) demonstrated AR-E8 amplification only. Kaplan–Meier analysis showed overall survival’s association with the amplification of AR-En (p = 0.02, HR = 1.68 (1.07–2.65)), AR-E8 (p = 0.02, HR = 1.78 (1.08–2.92)) and AR-En-E8 (the combination of AR-En and AR-E8 (p = 0.009, HR = 1.77 (1.15–2.73)). Multivariate models that included AR-En-E8 amplification and clinical factors significantly improved prognostic performance (p = 0.0001). With further validation, the multiplex dPCR assay may assist in prognostication of mCRPC patients.

Intact proviral DNA assay analysis of large cohorts of people with HIV provides a benchmark for the frequency and composition of persistent proviral DNA

A scalable approach for quantifying intact HIV-1 proviruses is critical for basic research and clinical trials directed at HIV-1 cure. The intact proviral DNA assay (IPDA) is a novel approach to characterizing the HIV-1 reservoir, focusing on the genetic integrity of individual proviruses independent of transcriptional status. It uses multiplex digital droplet PCR to distinguish and separately quantify intact proviruses, defined by a lack of overt fatal defects such as large deletions and APOBEC3G-mediated hypermutation, from the majority of proviruses that have such defects. This distinction is important because only intact proviruses cause viral rebound on ART interruption. To evaluate IPDA performance and provide benchmark data to support its implementation, we analyzed peripheral blood samples from 400 HIV-1+ adults on ART from several diverse cohorts, representing a robust sample of treated HIV-1 infection in the United States. We provide direct quantitative evidence that defective proviruses greatly outnumber intact proviruses (by >12.5 fold). However, intact proviruses are present at substantially higher frequencies (median, 54/106 CD4+ T cells) than proviruses detected by the quantitative viral outgrowth assay, which requires induction and in vitro growth (∼1/106 CD4+ T cells). IPDA amplicon signal issues resulting from sequence polymorphisms were observed in only 6.3% of individuals and were readily apparent and easily distinguished from low proviral frequency, an advantage of the IPDA over standard PCR assays which generate false-negative results in such situations. The large IPDA dataset provided here gives the clearest quantitative picture to date of HIV-1 proviral persistence on ART.

58P Detection of PIK3CA mutations in plasma ctDNA by Crystal Digital PCR for the selection of alpelisib treatment in routine clinical practice in advanced breast cancer patients

Alpelisib (a PI3Kα-specific inhibitor) has demonstrated clinical benefit in combination with fulvestrant and is approved for PIK3CA-mutated, ER +,HER- metastatic breast cancer (MBC) patients who relapsed after aromatase inhibitor (Andre F et al, NEJM, 2019). The PI3CA status may be determined either in tumour biopsy and/or cell-free DNA (cfDNA) (Andre F et al, NEJM, 2019). In our center we use a multiplex digital droplet PCR assay (ddPCR) for the detection of circulating PIK3CA mutations.