3D Digital PCR Research Articles

Ascomycota and Basidiomycota fungal phyla as indicators of land use efficiency for soil organic carbon accrual with woody plantations

As soil fungi are major players in the carbon accumulation process, the two main fungal degraders in topsoil, Ascomycetes and Basidiomycetes, were investigated as indicators of land use effectiveness in increasing soil carbon accumulation and soil function. The study focused on the soil organic carbon content increase in a 20-year short rotation forest cycle with broadleaf woody plantations compared to a nearby arable cropping system. Total fungi, Ascomycota and Basidiomycota, were quantified in terms of DNA copy number, with specific probes using SYBR® Green I dye on the QuantStudio™ 3D digital PCR system (dPCR). Previously, next generation sequencing analysis using a general primer confirmed that Ascomycota and Basidiomycota were the most represented phyla and that fungal community composition significantly differed between treatments. A range of key soil enzyme activities for the C-cycle were also assessed. Total organic carbon content (TOC), microbial biomass in term of dsDNA and enzyme activities significantly increased in woody plantations compared to arable soil. The TOC increase differed significantly also between wood species, Salix and Robina gave the greatest increase (+30 and 20 % respectively), followed by Populus (+12), microbial biomass highly correlated with TOC showing the same trend. Total fungi, Ascomycota and Basidiomycota increased significantly in three woody plantations compared to the arable soil system. Ascomycota in the woody plantations increased two to four times (average 3144 n copies µl−1 of DNA) compared to the arable soil (1419 n copies µl−1); Basidiomycota were almost absent in arable soil (av. 94n copies µl−1) and increased five to six times in woody plantations (av. 490 n copies µl−1). Total fungi and ascomycetes correlated strongly with microbial biomass and TOC, whereas basidiomycetes did not. These findings showed that Ascomycota represent the largest portion of fungi in agricultural soils even after a 20-years short rotation forest cycle and they can be taken as indicators of carbon accumulation processes. Therefore, this study suggests that joining the Ascomycetes quantity in the topsoil to the Ascomycota:Basidiomycota ratio seems a good option when setting regional strategies for improving C accrual in farmland with the short-term afforestation.

Comparative analysis of QS3D versus droplet digital PCR for quantitative measures of EGFR T790M mutation from identical plasma

ObjectivesThe capacity of QuantStudio™ 3D (QS3D) and droplet digital PCR (dPCR) for the detection of plasma Epidermal Growth Factor Receptor (EGFR) mutations have been widely reported. Few comparative studies on the quantitative test of the identical DNA material, however, are carried out. Here we compared the performance of the two methods in detecting EGFR T790M mutation in cell-free DNA (cfDNA) from the same lung cancer patients. MethodsWe recruited 72 non-small cell lung cancer (NSCLC) patients who initially respond to tyrosine kinase inhibitor treatment but subsequently developed resistance. Two tubes of 10mL anticoagulant blood were collected and cfDNA was isolated from plasma. Identical cfDNA samples were analyzed for T790M mutation using QS3D and droplet dPCR in parallel. ResultsT790M mutation was detected in 15 and 21 cfDNA samples by QS3D and droplet digital PCR, respectively. The 6 discordant samples showed low mutation abundance (∼0.1%) and the discrepancy is caused by the stricter threshold settings for QS3D dPCR. The overall agreement between the two methods was 91.7% (66/72). The median allele frequencies for QS3D dPCR and droplet dPCR to detect T790M mutation was 2.01% and 2.62%, respectively. There was no significance in mutation abundance detected by both methods. Both methods are highly correlated with allele frequencies and copy numbers in T790M wild type and mutant, with R2 of 0.98, 0.92 and 0.95, respectively. ConclusionOur study demonstrated that QS3D dPCR are highly consistent with droplet PCR for quantitative determination of EGFR T790M mutation in plasma cfDNA.

Digital RT-PCR Chip method for detection of SARS-CoV-2 virus

The “gold standard” method for detection of SARS-CoV-2 is the real time reverse transcription-polymerase chain reaction, but due to pre-analytical and technical limitations, biological samples with low viral load are not sometimes detected. For this purpose a digital RT-PCR method on-chip was developed for detection of the SARS-CoV-2 virus, using two TaqMan™ Assays for quantification of the N Protein (Nucleocapsid) and the S Protein (Spike), and the QuantStudio™ 3D Digital PCR instrument. The method was applied to assess the nasopharyngeal swabs of asymptomatic subjects recruited in the UNICORN Study. The digital RT-PCR method is characterized by a higher sensitivity than the RT-qPCR method, even if performed with the same TaqMan™, and could be a promising tool for SARS-CoV-2 viral load quantification.

Supercharged MPNs? Automated Determination of High-Throughput Most Probable Number (htMPN) Using Chip-Based 3D Digital PCR.

Surface plating on agar and most probable number (MPN) are the standard methods for determining bacterial viability but both have limitations. Here we present a novel cell count method, high-throughput MPN (htMPN), that uses a chip-based digital PCR instrument to accelerate and to improve the quantification of viable or sublethally injured cells. This method tracks growth of up to 20,000 individual bacterial cells on a single chip. Single cells were grown in the individual wells of the chip at their optimal temperature until the cell density was high enough to detect the fluorescent signal with cell-permeant or cell-impermeant DNA-intercalating fluorescent dyes. This method based on microfluidic devices implemented in digital PCR equipment was equivalent to surface plating in determining cell counts of Escherichia coli, Salmonella enterica serovar Typhimurium, Fructilactobacillus sanfranciscensis, Pseudomonas putida, and vegetative cells but not spores of Bacillus subtilis. Viable E. coli could be enumerated within 7 h. Culture of strict aerobes was restricted to strains that are capable of nitrate respiration; organisms requiring complex media that also contain double-stranded DNA were detected after treatment of growth media with DNase before inoculation. Our approach not only monitors the frequency distribution of bacterial growth and determines cell counts with high reliability but also detected heat-injured cells of S. Typhimurium that escaped detection by the surface plating. Overall, the method accelerates detection of viable bacterial cells, facilitates automation, and offers new possibilities for the analysis of individual bacterial cells. IMPORTANCE htMPN uses chip-based fluorescence acquisition and is a simple and compact tool for automatic viable cell enumeration with applications in microbiological research. This method applies to a wide range of anaerobic or facultative anaerobic species and improves accuracy by reducing the number of pipetting steps. In addition, the method offers an additional tool for single-cell microbiology. The single cell time-to-detection times have been used as an important criterion for the physiological state of bacterial cells after sublethal stress, and htMPNs support the acquisition of such data with an unprecedented number of cells. In particular, htMPN provides an anaerobic environment and enables a long incubation time to increase the recovery rate of sublethally injured cells. Given its reproducibility and reliability, our approach can potentially be applied to quantify viable cells in samples from environmental, clinical, or food samples to reduce the risk of underestimation of the number of viable bacterial cells.

Determination of Exosome Mitochondrial DNA as a Biomarker of Renal Cancer Aggressiveness.

Simple SummaryComponents of liquid biopsy are potential non-invasive biomarkers for monitoring renal cell carcinoma (RCC) status. The aim of our study was to examine mitochondrial genes (such as HV1 and CYB) included in exosomal fractions as promising and innovative biomarkers in RCC. We found that phase C containing different types of vesicles and phase F rich in exosomes with a high mitochondrial DNA (mtDNA) content could be considered as powerful biomarkers for susceptibility to RCC. Interestingly, mtDNA was a good genetic marker when aggressiveness was evaluated.Here, the role of non-invasive biomarkers in liquid biopsy was evaluated, mainly in exosomes and mitochondrial DNA (mtDNA) as promising, novel, and stable biomarkers for renal cell carcinoma (RCC). A total of 140 fractions (named from B to F) obtained by ultracentrifugations of whole blood samples from 28 individuals (13 patients and 15 controls) were included. Nanoparticle Tracking Analysis (NTA) was conducted to characterized exosomal fraction. Subsequently, an analysis of digital PCR (dPCR) using the QuantStudio™ 3D Digital PCR platform was performed and the quantification of mtDNA copy number by QuantStudioTM 12K Flex Real-Time PCR System (qPCR) was developed. Moreover, Next Generation Sequencing (NGS) analyses were included using MiSeq system (Illumina, San Diego, CA, USA). An F fraction, which contains all exosome data and all mitochondrial markers, was identified in dPCR and qPCR with statistically significant power (adjusted p values ≤ 0.03) when comparing cases and controls. Moreover, present analysis in mtDNA showed a relevant significance in RCC aggressiveness. To sum up, this is the first time a relation between exosomal mtDNA markers and clinical management of RCC is analyzed. We suggest a promising strategy for future liquid biopsy RCC analysis, although more analysis should be performed prior to application in routine clinical practice.

Study of Early Onset Schizophrenia: Associations of GRIN2A and GRIN2B Polymorphisms.

Background: Schizophrenia is a complex mental disorder with a high heritability. Dysfunction of the N-methyl-D-aspartate (NMDA)-type glutamate receptors may be involved in the pathogenesis of schizophrenia. In this study, we examined the contribution of GRIN2A and GRIN2B (Glutamate Ionotropic Receptor NMDA Type Subunit 2A/2B) polymorphisms to the clinical features of schizophrenia, such as the leading symptoms, the type of course, and the age of onset. Methods: A population of 402 Russian patients with schizophrenia from the Siberian region was investigated. Genotyping of seventeen single-nucleotide polymorphisms (SNPs) in GRIN2A and GRIN2B was performed using QuantStudio™ 3D Digital PCR System Life Technologies amplifier using TaqMan Validated SNP Genotyping Assay kits (Applied Biosystems). The results were analyzed using Chi-square and the Fisher’s exact tests. Results: We found an association of GRIN2A rs7206256 and rs11644461 and GRIN2B rs7313149 with the early onset (before the age of 18 years old) schizophrenia. We did not reveal any associations of GRIN2A and GRIN2B polymorphisms with leading (positive vs. negative) symptoms or type of course (continuous vs. episodic) of schizophrenia. Conclusions: In the study, we confirmed the involvement of the GRIN2A and GRIN2B genes in the early onset of schizophrenia in a Russian population of the Siberian region.

The Influence of Maternal Cell Contamination on Fetal Aneuploidy Detection Using Chip-Based Digital PCR Testing.

Prenatal samples obtained by amniocentesis or chorionic villus sampling are at risk of maternal cell contamination (MCC). In traditional prenatal analysis, MCC is recommended to be assayed by special tests, such as the short tandem repeat analysis and, if detected at a high level, may result in failed analysis report. The objective of this study was to test the ability of chip-based digital PCR to detect fetal aneuploidies in the presence of MCC. To determine the level of accuracy of MCC detection, an aneuploid male sample was subjected to serial dilution with an euploid female sample. DNA was extracted from prenatal samples and analyzed with QuantStudio 3D Digital PCR. Digital PCR analysis allowed the detection of trisomy 21, trisomy 18, and X monosomy accurately in samples with 90%, 85%, and 92% of MCC, respectively. Moreover, our results indicated that digital PCR was able to accurately confirm the presence of Y chromosome at up to 95% contamination. The amniotic fluid and chorionic villus sampling (CVS) received in our clinical laboratory was subjected to further analysis of MCC based on the aneuploidy assessment algorithm, resulting in the identification of 10 contaminated samples and four cases of true fetal mosaicism. We conclude that chip-based digital PCR analysis enables the detection of fetal aneuploidy with high levels of accuracy, even in cases of significant MCC. Importantly, the algorithm eliminates the need for maternal DNA and additional MCC tests, which reduces costs and simplifies the diagnostic procedure. The method is easy to set up and suitable for routine clinical practice.

Abstract 561: ALK fusions can be clearly detected in extracellular vesicles from NSCLC cell lines: A become of hope for non-invasive ALK testing

Abstract Background: Identification of ALK rearrangements in NSCLC patients has important implications as it significantly expands their therapeutic opportunities. However, the availability of tumors biopsies is sometimes limited in the clinical setting. The analysis of liquid biopsies can potentially overcome such limitation. Nevertheless, the identification of ALK rearrangements using liquid biopsies remains challenging as fusion transcripts are rapidly degraded by RNAses present in the blood. Nonetheless, encapsulated RNA inside extracellular vesicles (EVs) is protected from RNAses. The main objective of this study is to explore the potential value of exosomes for non-invasive ALK testing. Methods: H3122 and H2228 cell lines, derived from NSCLC patients with EML4-ALK variant 1 (E13;A20) and variant 3 (E6a/b;A20) respectively, were used to isolate exosomes by the sequential centrifugation of cell culture supernatants. Briefly, supernatants were centrifuged twice for 10 min at 200xg, twice for 500xg for 10 min, once for 30 min at 10,000xg and a final ultracentrifugation at 100,000×g for 2 h at 4 °C. Purified exosomes were verified in terms of morphology and size by transmission electron microscopy (TEM). In addition, specific EVs markers, namely CD63, CD81 and CD9, were measured by flow cytometry and Western-Blot. EML4-ALK variants were analyzed at the protein level by Western blot using 5A4 antibody, and RNA level by digital PCR (dPCR), using a QuantStudio® 3D Digital PCR System and specific TaqMan® assays for EML4-ALK variant 1 and 3. Samples were also submitted to NGS analysis using the Oncomine™ Pan-Cancer Cell-Free Assay kit. Results: TEM showed spherical vesicles around 100 nm in both cell lines. Flow cytometry and Western blot confirmed expression of the exosomal specific markers CD63, CD81 and CD9 tetraspanins. Western blot also showed EML4-ALK variant 1 (120 kDa) and variant 3 (87 kDa) in H3122 and H2228 purified exosomes, respectively. Isolated exosome RNA profile was composed of small RNA (< 200 nt), although 18S and 28S rRNA peaks were also present. dPCR and NGS confirmed EML4-ALK rearrangements in exosome derived from both cell lines. Conclusions: EML4-ALK fusions can be detected at the RNA levels and at the protein levels analyzing EVs derived from H3122 and H2228 cell lines. These results set the stage for the development of EV-based non-invasive ALK testing in NSCLC patients. Citation Format: Estela Sánchez-Herrero, Carmen Campos-Silva, Yaiza Cáceres Martell, Sandra Sanz-Moreno, Roberto Serna-Blasco, Alejandro Rodríguez Festa, Miguel Ángel Molina-Vila, Mariano Provencio, Mar Valés-Gómez, Atocha Romero. ALK fusions can be clearly detected in extracellular vesicles from NSCLC cell lines: A become of hope for non-invasive ALK testing [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 561.

Existence of SARS-CoV-2 RNA on ambient particulate matter samples: A nationwide study in Turkey

Coronavirus disease 2019 (COVID-19) is caused by the SARS-CoV-2 virus and has been affecting the world since the end of 2019. The disease led to significant mortality and morbidity in Turkey, since the first case was reported on March 11th, 2020. Studies suggest a positive association between air pollution and SARS-CoV-2 infection. The aim of the present study was to investigate the role of ambient particulate matters (PM), as potential carriers for SARS-CoV-2. Ambient PM samples in various size ranges were collected from 13 sites including urban and urban-background locations and hospital gardens in 10 cities across Turkey between 13th of May and 14th of June 2020 to investigate the possible presence of SARS-CoV-2 RNA on ambient PM. A total of 203 daily samples (TSP, n = 80; PM2.5, n = 33; PM2.5–10, n = 23; PM10μm, n = 19; and 6 size segregated PM, n = 48) were collected using various samplers. The N1 gene and RdRP gene expressions were analyzed for the presence of SARS-CoV-2, as suggested by the Centers for Disease Control and Prevention (CDC). According to real time (RT)-PCR and three-dimensional (3D) digital (d) PCR analysis, dual RdRP and N1 gene positivity were detected in 20 (9.8%) samples. Ambient PM-bound SARS-CoV-2 was analyzed quantitatively and the air concentrations of the virus ranged from 0.1 copies/m3 to 23 copies/m3. The highest percentages of virus detection on PM samples were from hospital gardens in Tekirdağ, Zonguldak, and Istanbul, especially in PM2.5 mode. Findings of this study have suggested that SARS-CoV-2 may be transported by ambient particles, especially at sites close to the infection hot-spots. However, whether this has an impact on the spread of the virus infection remains to be determined.

Copy Number Variations of CEP63, FOSL2 and PAQR6 Serve as Novel Signatures for the Prognosis of Bladder Cancer.

BackgroundFinding effective prognostic signatures is of great urgency due to the high risk of recurrence and progression of bladder cancer (BC). Although a lot of genetic alterations are involved in the carcinogenesis, none of them were referred in the current risk group stratifications. In this study, we aimed to find significant copy number variations (CNVs) to predict prognosis for BC patients.MethodsCNVs with high aberration frequencies in BC were explored by array-based comparative genomic hybridization in 65 tumor samples. Candidates were validated in independent groups of BC tumor samples (n=219) and urine samples (n=123). 3D digital PCR was applied for detecting accurate gene copy numbers in BC urine. In order to explore the prognostic value of candidate CNVs, all enrolled patients were followed up for the disease-free survival (DFS). Cox proportional hazards regression analysis was performed to find the independent prognostic factors for DFS.ResultsCNVs of CEP63, FOSL2 and PAQR6 with high aberration frequencies (67.7%, 56.9% and 60.0%, respectively) were found in BC tumors. Copy numbers of CEP63, FOSL2 and PAQR6 were gained in 219 tumor samples. CNVs of CEP63 and FOSL2 were correlated with advanced tumor stage and high grade. Retrospective analysis (median follow-up time: 69 months) revealed that CNVs of CEP63 and FOSL2 were independent prognostic factors for DFS of non-muscle-invasive bladder cancer (NMIBC) patients, while CNVs of FOSL2 and PAQR6 were independent prognostic factors for DFS of muscle-invasive bladder cancer (MIBC) patients. Models for predicting DFS were constructed based on CNVs of three genes. Patients with high prognostic indexes tended to have poor DFS. Prognostic index can also help to identify those with worse outcomes among high risk NMIBC patients. Copy number gains of CEP63 and FOSL2 in urine were found to be significantly correlated with poor DFS of NMIBC patients.ConclusionsCNVs of CEP63, FOSL2 and PAQR6 were capable of predicting DFS and may serve as promising signatures for prognosis of BC.

Molecular diagnosis of diffuse glioma using a chip-based digital PCR system to analyze IDH, TERT, and H3 mutations in the cerebrospinal fluid

PurposeConventional genetic analyzers require surgically obtained tumor tissues to confirm the molecular diagnosis of diffuse glioma. Recent technical breakthroughs have enabled increased utilization of cell-free tumor DNA (ctDNA) in body fluids as a reliable resource for molecular diagnosis in various cancers. Here, we tested the application of a chip-based digital PCR system for the less invasive diagnosis (i.e., liquid biopsy) of diffuse glioma using the cerebrospinal fluid (CSF).MethodsCSF samples from 34 patients with diffuse glioma were collected from the surgical field during craniotomy. Preoperative lumbar CSF collection was also performed in 11 patients. Extracted ctDNA was used to analyze diagnostic point mutations in IDH1 R132H, TERT promoter (C228T and C250T), and H3F3A (K27M) on the QuantStudio® 3D Digital PCR System. These results were compared with their corresponding tumor DNA samples.ResultsWe detected either of the diagnostic mutations in tumor DNA samples from 28 of 34 patients. Among them, we achieved precise molecular diagnoses using intracranial CSF in 20 (71%). Univariate analyses revealed that the World Health Organization (WHO) grade (p = 0.0034), radiographic enhancement (p = 0.0006), and Mib1 index (p = 0.01) were significant predictors of precise CSF-based molecular diagnosis. We precisely diagnosed WHO grade III or IV diffuse gliomas using lumbar CSF obtained from 6 (87%) of 7 patients with tumors harboring any mutation.ConclusionWe established a novel, non-invasive molecular diagnostic method using a chip-based digital PCR system targeting ctDNA derived from CSF with high sensitivity and specificity, especially for high-grade gliomas.

Analysis of miRNA Expression Using Digital PCR and the QuantStudio™ 3D Digital PCR System.

miRNAs are short, single-stranded RNA molecules that function as posttranscriptional regulators of gene expression. miRNAs represent ideal biomarkers since they can also circulate in the bloodstream as well as in other biological fluids such as urine, saliva, and cerebrospinal fluid.miRNAs play an important role in the regulation of immune cells including cytotoxic T-lymphocytes. Circulating miRNAs can be analyzed by Real-Time PCR or microarray profiling; however data normalization represents still an unsolved problem due to the lack of widely validated house-keeping miRNAs candidates.Digital PCR (dPCR) is an end-point PCR method that is used for absolute quantification. In this chapter we will describe the applications of Digital PCR for the analysis of miRNAs that can influence immune response in serum samples and we will report a specific protocol that can be used to analyze miRNAs using the QuantStudio™ 3D Digital PCR System. The advantage of this method consists in the possibility to highlight weaker differences in miRNA circulating molecules that can be useful to monitor CTLs behavior in pathological conditions or after therapeutic intervention.

Detection of EGFR Mutations in Plasma Cell-Free Tumor DNA of TKI-Treated Advanced-NSCLC Patients by Three Methodologies: Scorpion-ARMS, PNAClamp, and Digital PCR.

Analysis of circulating cell-free tumor DNA (cftDNA) has emerged as a specific and sensitive blood-based approach to detect epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) patients. Still, there is some debate on what should be the preferential clinical method for plasma-derived cftDNA analysis. We tested 31 NSCLC patients treated with anti-EGFR tyrosine kinase inhibitors (TKIs), at baseline and serially during therapy, by comparing three methodologies in detecting EGFR mutations (L858R, exon 19 deletion, and T790M) from plasma: scorpions-amplification refractory mutation system (ARMS) methodology by using EGFR Plasma RGQ PCR Kit-QIAGEN, peptide nucleic acid (PNA) clamp and PANA RealTyper integration by using PNAClamp EGFR-PANAGENE, and digital real time PCR by using QuantStudio 3D Digital PCR System-Thermo Fisher Scientific. Specificity was 100% for all three mutations, independently from the platform used. The sensitivity for L858R (42.86%) and T790M (100%) did not change based on the method, while the sensitivity for Del 19 differed markedly (Scorpion-ARMS 45%, PNAClamp 75%, and Digital PCR 85%). The detection rate was also higher (94.23%) as measured by Digital PCR, and when we monitored the evolution of EGFR mutations over time, it evidenced the extreme inter-patient heterogeneity in terms of levels of circulating mutated copies. In our study, Digital PCR showed the best correlation with tissue biopsy and the highest sensitivity to attain the potential clinical utility of monitoring plasma levels of EGFR mutations.

Comprehensive cross-platform comparison of methodologies for noninvasive EGFR mutation testing: Results of the RING observational trial.

e21518 Background: Several platforms for non-invasive EGFR testing are currently used in the clinical setting, with sensitivities ranging from 30 to 100%. Comparison studies in prospective cohorts remain limited and reports evaluating mutant allelic fractions (MAFs) are particularly scarce. The RING observational trial (ClinicalTrials.gov identifier NCT03363139) was designed to comprehensively analyze the concordance between methodologies for EGFR mutation detection in blood. Methods: Seventy-two EGFR mutant NSCLC patients were enrolled in the trial. Plasma samples were prospectively collected at progression to first line Tyrosine Kinase Inhibitor and tested for EGFR mutations by 7 methodologies; cobas EGFR Mutation Test v2, Therascreen EGFR Plasma RGQ PCR Kit, QuantStudio 3D Digital PCR System, a 5-nuclease real-time PCR assay in presence of PNA, OncoBEAM EGFR and NGS with two different gene panels, Ion Torrent Oncomine and GeneRead QIAact Lung DNA UMI Cancer Panel. Results: The agreement between all methodologies for was almost perfect for the detection of deletions in exon 19 (K = 0.86; 95%CI: 0.76-0.96) and substantial for exon 21 point mutations (K = 0.76; 95%CI: 0.63-0.89). Regarding the p.T790M resistance mutation, concordance was lower but still substantial (K = 0.68; 95%CI: 0.57-0.79). If only NGS-based technologies were considered, the agreement was almost perfect for sensitizing mutations and substantial for the resistance mutation (K = 0.84; 95%CI: 0.68-1.00, K = 0.86; 95%CI: 0.69-1.00 and K = 0.77; 95%CI: 0.60-0.95 for exon 19, exon 21 and p.T790M, respectively). Most discordant samples between methodologies had mutant allele fractions (MAFs) ≤0.5%. Sensitizing mutations were always present at higher MAFs than concomitant p.T790M, explaining the lower concordance observed for this variant. MAFs obtained by different methodologies showed an excellent reproducibility (intraclass correlation coeficients 0.85-0.97). Similarly, Passing–Bablok regression analysis showed a high correlation between methodologies when assessing MAFs. Conclusions: Our results support the use of liquid biopsies for non-invasive EGFR testing in the clinical setting and highlight the need to systematically report MAFs.

MON-514 Detection of RAS Mutations and RET/PTC Fusions in Thyroid Cancer Using Microfluidic Digital PCR

Background:The identification of somatic mutations and gene fusions is crucial for guiding therapeutic decisions in patients with thyroid cancer. Microfluidic digital PCR is currently considered as a technique of choice for highly sensitive detection of gene mutations/fusion. We recently demonstrated that dPCR is a useful tool for detection of BRAFV600E and TERT promoter mutations in thyroid tumors.Objectives:This study aimed to determine the analytic and clinical validity of dPCR for detection of RAS mutations and RET/PTC fusions in thyroid cancer tissue.Material and Methods:Thyroid tissues from 75 patients with PTCs (58 classical PTC (CPTC) and 17 follicular variant (FVPTC) were used for DNA and RNA extraction. The rare mutation SNP genotyping assays which were multiplexed for detection of mutant and wild type NRASQ61; as well as RET/PTC1 and RET/PTC3 were synthesized by Thermo Fisher Scientific. Digital PCR was performed using a QuantStudio 3D Digital PCR platform. QuantStudio Software was used for relative and quantitative data analysis.Results:NRASQ61 was detected in 0/58 CPTC and in 6/17 (35%) FVPTC. The ratios of mutant/total varying from 11.7% to 61.5%. Among patients with FVPTC there were no significant associations between the presence of NRASQ61 and patient’s age, sex, multifocal growth, extra-thyroidal invasion and lymph node metastases. The ratios mutant/total correlated with tumor size in patients harboring NRASQ61. In 23 cases, RET/PTC1 and RET/PTC3 transcripts were examined. RET/PTC1 and RET/PTC3 transcripts were detected in 3 and 1 case, respectively. RET/PTCs were detected in CPTC, but not in FVPTC. RET/PTC positive tumors were characterized by multi-focal patterns of growth, presence of extra-thyroidal invasion, and presence of lymph node metastases (4 of 4 cases with RET/PTC). There were not RET/PTCs positive tumors harboring simultaneously anomalies in RAS oncogene.Conclusions:Microfluidic digital PCR allows specific, sensitive and rapid detection of RAS mutations and RET/PTC fusions in thyroid tissue samples. Implementation of dPCR-based assays may facilitate analysis of thyroid tumors and support research in patients with thyroid cancer.

Detection and quantification of clinically relevant plasmid-mediated AmpC beta-lactamase genes in aquatic systems

Abstract The extent and impact of plasmid-mediated AmpC beta-lactamase genes (pAmpCs) prevalence in aquatic environments is poorly understood. The aim of this study was to detect and quantify pAmpCs from the aquatic environment. The following pAmpCs were analysed with clinical TaqMan assays from isolated plasmids: ACC, ACT/MIR, BIL/LAT/CMY, DHA, FOX and MOX/CMY. Quantification was conducted using quantitative PCR (qPCR) and 3D chip-based digital PCR. The results of qPCR yielded 4,875.27 copies/ng DNA and dPCR, 1,640.58 copies/ng (Mann–Whitney U Test, p= 0.868). Redundancy analysis indicated that land coverage explains 90.49% (ANOVA, p= 0.601) of pAmpC variance. There was a correlation between the frequency and quantities of pAmpCs detected in each river and this could be related to anthropogenic influence. Frequencies of detection for pAmpCs were 25/36 for the Crocodile West River and 13/36 for the Marico River. Quantification resulted in higher copy numbers for the Crocodile West River and high copies in only two sites of the Marico River, thus reflecting degrees of anthropogenic influences on both rivers. The presence of these clinically relevant pAmpCs in aquatic systems are cause for concern, considering their potential impact if these genes are harboured by pathogens and become dispersed to human populations.

Salivary Amylase Gene Copy Number Is Associated with the Obesity and Inflammatory Markers in Children.

BackgroundWe have recently shown that the copy number of salivary amylase (AMY1) gene was significantly decreased, and the obesity-related salivary biomarkers resistin, MCP-1, TNF-α, IL-6, and CRP were significantly increased in overweight/obese children compared to normal weight. This study aimed to evaluate the association of AMY1 copy number variant (CNV) with obesity and inflammatory markers. Seventy-six participants aged between 6 and 10 years have participated, and the saliva samples were collected along with the anthropometric measurements.MethodsAMY1 copy number was analyzed by 3D digital PCR, and obesity-related biomarkers were performed with a Bioplex multiplex analyzer.ResultsThe mean AMY1 copy number was higher in normal weight (7.90 ± 0.38) compared to the overweight/obese group (6.20 ± 0.29). The association of AMY1 CNV with obesity and inflammatory markers showed significant negative correlation [CRP, β = −0.238 (p < 0.05); resistin, β = −0.25 (p < 0.05); MCP-1, β = −0.304 (p < 0.01)] except for complement factor D, TNF α and IL-6. The anti-inflammatory cytokine, IL-10 reported a positive correlation with AMY1 copy number with a β = 0.268 (p < 0.05). The multivariable model adjusted with age and gender depicted a similar correlation with obesity markers.ConclusionOur results report that AMY1 CNV is associated with obesity and inflammatory biomarkers in children’s saliva sample.

MiR-3940-5p/miR-8069 ratio in urine exosomes is a novel diagnostic biomarker for pancreatic ductal adenocarcinoma.

Despite the development of several therapeutic options, the prognosis of pancreatic cancer remains poor. One reason for this is the difficulty of diagnosing the disease at an early stage. For example, carbohydrate antigen (CA) 19-9, which is the most widely used biomarker for pancreatic cancer, cannot be used to detect the disease at early stages. Some studies have attempted to find novel biomarkers for pancreatic cancer. The aim of the present study was to find a novel diagnostic biomarker for pancreatic ductal adenocarcinoma (PDAC) in urine exosomes. Exosomes were isolated from urine and serum samples of patients with PDAC and control subjects, or culture media of cancer cell lines. MicroRNAs (miRNAs) were purified from exosomes. Novel biomarker candidates for PDCA were identisfied from urine exosome miRNA using expression profiling, and validated in a larger number of samples using 3D digital PCR. The results of a preliminary analysis of nine PDAC and seven control subjects revealed that the miR-3940-5p/miR-8069 ratio in urine exosomes was elevated in the patients with PDAC. Experiments using cultured cancer cell lines revealed that the elevation of the miR-3940-5p/miR-8069 ratio was specific for PDAC. Furthermore, the elevation of the miR-3940-5p/miR-8069 ratio in exosomes tended to be higher in the urine than in the serum of patients with PDAC. Validation experiments on 43 PDAC, 12 chronic pancreatitis and 25 control subjects demonstrated that the miR-3940-5p/miR-8069 ratio in urine exosomes was elevated in PDAC at a relatively early stage of the disease. When this ratio was used in combination with CA19-9 for the diagnosis of PDAC, the sensitivity and positive predictive value improved to 93.0 and 78.4%, respectively, when either of them was positive. Additionally, the positive predictive value reached 100% when both were positive. The negative predictive value also improved to 89.7% when both were negative. The miR-3940-5p/miR-8069 ratio in urine exosomes may be useful as a tool for the diagnosis of PDAC, particularly when used in combination with CA19-9.

Non-invasive Molecular Detection of Minimal Residual Disease in Papillary Thyroid Cancer Patients.

Background: Papillary thyroid cancer (PTC) is the most common type of thyroid malignancy. Serum thyroglobulin (Tg) levels are used to monitor PTC treatment response and recurrences however, in about 25% of the cases the sensitivity of this method is compromised due to either the presence of neutralizing anti-Tg antibodies (TgAb) or the absence of Tg in less differentiated tumors. Up to 80% of PTC tumors harbor the c.1799T>A hotspot mutation in the BRAF gene (BRAFV600E). Here, we assessed the potential use of plasma cell-free BRAFV600E mutant tumor DNA (ctDNA) levels in determining the minimal residual tumor status of PTC patients.Methods: Patients were classified as either having persistent disease (PD) or no evidence of disease (NED) based on clinicopathological assessments. Tumor BRAFV600E status was determined by both direct sequencing and digital PCR. Plasma total cell-free BRAFV600 wild type DNA (cfDNA) and ctDNA fractions circulating in the plasma of PTC patients were determined by an emulsion based-digital PCR and total ctDNA was quantified by 3D digital PCR. The total ctDNA levels (copies/ml) were then compared to patients' clinicopathological features.Results: About 74% (28/38) of tumors harbored the BRAFV600E mutation. Percent plasma ctDNA fractions for PD patients with BRAFV600E tumors ranged from 0 to 2.07%, whereas absolute plasma ctDNA copies ranged from 0 to 62 copies. The ctDNA levels accurately detected tumor burden of PTC patients whose tumors harbored BRAFV600E; median plasma ctDNA copy numbers were significantly higher (Wilcoxon test, p = 0.03) in patients with metastasis (MET) (20 copies/ml) compared to patients with non-metastatic (non-MET) tumors (1 copy/ml). The plasma ctDNA levels (copies/ml) accurately determined the disease status of PTC patients with sensitivity of 86% and specificity of 90% as compared to 78% sensitivity and 65% specificity determined by serum Tg levels (ng/ml) with areas under the curves (AUC) of 0.88 and 0.71, respectively. Intriguingly, plasma total cfDNA levels were significantly higher in patients with no evidence of residual disease (NED) compared to persistent disease (PD) patients.Conclusions: Our study supports the clinical applicability of plasma ctDNA as biomarker to determine the residual tumor status and tumor burden of PTC patients.

Chip-Based Digital PCR Approach Provides A Sensitive and Cost-Effective Single-Day Screening Tool for Common Fetal Aneuploidies-A Proof of Concept Study.

In the prenatal period, the copy number aberrations of chromosomes 13, 18, 21, X and Y account for over 80% of the clinically significant chromosome abnormalities. Classical cytogenetic analysis is the gold standard in invasive prenatal diagnostics but the long test waiting time affects its clinical utility. Several molecular rapid tests have been developed and employed in clinical practice, however all have substantial drawbacks. The aim of the study was to design and evaluate an optimized tool for rapid molecular detection of fetal aneuploidies. We established a novel single-day method using a chip-based platform, the QuantStudio 3D Digital PCR system. In order to assess the clinical usefulness of our screening test, we analyzed 133 prenatal samples. The difference in distributions of euploid and aneuploid samples identified the ploidy of each of the target chromosomes with high precision. The distribution of the chromosome ratio for euploid and aneuploid samples showed a statistically significant result (p = 0.003 for trisomy 13, p = 0.001 for trisomies 18 and 21, Mann–Whitney U test). Our results suggest that this novel chip-based approach provides a tool for rapid, technically simple, cost-effective screening for common fetal aneuploidies.