Comparison of Different Reverse Transcriptase-Polymerase Chain Reaction-Based Methods for Wastewater Surveillance of SARS-CoV-2: Exploratory Study.

BACKGROUND: Many countries have applied the wastewater surveillance of the COVID-19 pandemic to their national public health monitoring measures. The most used methods for detecting SARS-CoV-2 in wastewater are quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and reverse transcriptase-droplet digital polymerase chain reaction (RT-ddPCR). Previous comparison studies have produced conflicting results, thus more research on the subject is required. OBJECTIVE: This study aims to compare RT-qPCR and RT-ddPCR for detecting SARS-CoV-2 in wastewater. It also aimed to investigate the effect of changes in the analytical pipeline, including the RNA extraction kit, RT-PCR kit, and target gene assay, on the results. Another aim was to find a detection method for low-resource settings. METHODS: We compared 2 RT-qPCR kits, TaqMan RT-qPCR and QuantiTect RT-qPCR, and RT-ddPCR based on sensitivity, positivity rates, variability, and correlation of SARS-CoV-2 gene copy numbers in wastewater to the incidence of COVID-19. Furthermore, we compared 2 RNA extraction methods, column- and magnetic-bead-based. In addition, we assessed 2 target gene assays for RT-qPCR, N1 and N2, and 2 target gene assays for ddPCR N1 and E. Reverse transcription strand invasion-based amplification (RT-SIBA) was used to detect SARS-CoV-2 from wastewater qualitatively. RESULTS: Our results indicated that the most sensitive method to detect SARS-CoV-2 in wastewater was RT-ddPCR. It had the highest positivity rate (26/30), and its limit of detection was the lowest (0.06 gene copies/µL). However, we obtained the best correlation between COVID-19 incidence and SARS-CoV-2 gene copy number in wastewater using TaqMan RT-qPCR (correlation coefficient [CC]=0.697, P<.001). We found a significant difference in sensitivity between the TaqMan RT-qPCR kit and the QuantiTect RT-qPCR kit, the first having a significantly lower limit of detection and a higher positivity rate than the latter. Furthermore, the N1 target gene assay was the most sensitive for both RT-qPCR kits, while no significant difference was found between the gene targets using RT-ddPCR. In addition, the use of different RNA extraction kits affected the result when the TaqMan RT-qPCR kit was used. RT-SIBA was able to detect SARS-CoV-2 RNA in wastewater. CONCLUSIONS: As our study, as well as most of the previous studies, has shown RT-ddPCR to be more sensitive than RT-qPCR, its use in the wastewater surveillance of SARS-CoV-2 should be considered, especially if the amount of SARS-CoV-2 circulating in the population was low. All the analysis steps must be optimized for wastewater surveillance as our study showed that all the analysis steps including the compatibility of the RNA extraction, the RT-PCR kit, and the target gene assay influence the results. In addition, our study showed that RT-SIBA could be used to detect SARS-CoV-2 in wastewater if a qualitative result is sufficient.

Molecular findings and virological assessment of bladder papillomavirus infection in cattle.

Bovine and ovine papillomaviruses (BPVs - OaPVs) are infectious agents that have an important role in bladder carcinogenesis of cattle. In an attempt to better understand territorial prevalence of papillomavirus genotypes and gain insights into their molecular pathway(s), a virological assessment of papillomavirus infection was performed on 52 bladder tumors in cattle using droplet digital polymerase chain reaction (ddPCR), an improved version of conventional PCR. ddPCR detected and quantified BPV DNA and mRNAs in all tumor samples, showing that these viruses play a determinant role in bovine bladder carcinogenesis. OaPV DNA and mRNA were detected and quantified in 45 bladder tumors. BPV14, BPV13, BPV2, OaPV2, OaPV1, and OaPV3 were the genotypes most closely related to bladder tumors. ddPCR quantified BPV1 and OaPV4 DNA and their transcripts less frequently. Western blot analysis revealed a significant overexpression of the phosphorylated platelet derived growth factor ß receptor (PDGFßR) as well as the transcription factor E2F3, which modulate cell cycle progression in urothelial neoplasia. Furthermore, significant overexpression of calpain1, a Cys protease, was observed in bladder tumors related to BPVs alone and in BPV and OaPV coinfection. Calpain1 has been shown to play a role in producing free transcription factors of the E2F family, and molecular findings suggest that calpain family members work cooperatively to mutually regulate their protease activities in cattle bladder tumors. Altogether, these results showed territorial prevalence of BPV and OaPV genotypes and suggested that PDGFßR and the calpain system appeared to be molecular partners of both BPVs and OaPVs.

Application of Second-Generation Sequencing Technology in Lower Respiratory Tract Infection.

BACKGROUND: Lower respiratory tract infection (LRTI) has long been an important threat to people's life and health, so the rapid diagnosis of LRTI is of great significance in clinical treatment. In recent years, the development of the sequencing technology provides a new direction for the rapid diagnosis of LRTI. In this review, the advantages and disadvantages of second-generation sequencing techniques represented by metagenomics next-generation sequencing (mNGS) and droplet digital polymerase chain reaction (ddPCR) in LRTI were reviewed. Furthermore, it offers insights into the future trajectory of this technology, highlighting its potential to revolutionise the field of respiratory infection diagnostics. OBJECTIVE: This review summarises developments in mechanistic research of second-generation sequencing technology their relationship with clinical practice, providing insights for future research. METHODS: Authors conducted a search on PubMed and Web of Science using the professional terms 'Lower respiratory tract infection' and 'droplet digital polymerase chain reaction' and 'metagenomics next generation sequencing'. The obtained literature was then roughly categorised based on their research content. Similar studies were grouped into the same sections, and further searches were conducted based on the keywords of each section. RESULTS: Different studies discussed the application of second-generation sequencing technology in LRTI from different angles, including the detection of pathogens of LRTI by mNGS and ddPCR, the prediction ability of drug-resistant bacteria, and comparison with traditional methods. We try to analyse the advantages and disadvantages of the second-generation sequencing technology by combing the research results of mNGS and ddPCR. In addition, the development direction of the second-generation sequencing technology is prospected.

Carrier Screening and Diagnosis for Spinal Muscular Atrophy Using Droplet Digital PCR Versus MLPA: Analytical Validation and Early Test Outcome.

Background: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular life-threatening disorder. Owing to high carrier frequency, population-wide SMA screening to quantify the copy number of SMN gene is recommended by American College of Medical Genetics and Genomics. An accurate, reliable, short runaround time and cost-effective method may be helpful in mass population screening for SMA. Methods: Multiplex ligation-dependent probe amplification (MLPA) is a gold standard to estimate the copy number variation (CNV) for SMN1 and SMN2 genes. In this study, we validated droplet digital polymerase chain reaction (ddPCR) for the determination of CNV for both SMN1 and SMN2 exon 7 for a diagnostic purpose. In total, 66 clinical samples were tested using ddPCR, and results were compared with the MLPA as a reference test. Results: For all samples, CNV for SMN1 and SMN2 exon 7 was consentaneous between ddPCR and MLPA test results (κ = 1.000, p < 0.0001). In addition, ddPCR also showed a significant acceptable degree of test repeatability, coefficient of variation < 4%. Conclusion: ddPCR is expected to be utilitarian for CNV detection for carrier screening and diagnosis of SMA. ddPCR test results for CNV detection for SMN1/SMN2 exon 7 are concordant with the gold standard. ddPCR is a more cost-effective and time-saving diagnostic test for SMA than MLPA. Furthermore, it can be used for population-wide carrier screening for SMA.

Prognostic value of poly-microorganisms detected by droplet digital PCR and pathogen load kinetics in sepsis patients: a multi-center prospective cohort study.

This study aimed to investigate the prognostic value of a novel droplet digital polymerase chain reaction (DDPCR) assay in sepsis patients. In this prospective cohort study, univariable and multivariable Cox regressions were used to assess risk factors for 28-day mortality. We also monitored pathogen load together with clinical indicators in a subgroup of the cohort. A total of 107 sepsis patients with positive baseline DDPCR results were included. Detection of poly-microorganisms [adjusted hazard ratio (HR) = 3.19; 95% confidence interval (CI) = 1.34-7.62; P = 0.009], high Charlson Comorbidity Index (CCI) score (adjusted HR = 1.14; 95% CI = 1.01-1.29; P = 0.041), and Sequential Organ Failure Assessment (SOFA) score (adjusted HR = 1.18; 95% CI = 1.05-1.32; P = 0.005) at baseline were independent risk factors for 28-day mortality while initial pathogen load was not associated (adjusted HR = 1.17; 95% CI = 0.82-1.66; P = 0.385). Among 63 patients with serial DDPCR results, an increase in pathogen load at days 6-8 compared to baseline was a risk factor for 28-day mortality (P = 0.008). Also, pathogen load kinetics were significantly different between day-28 survivors and nonsurvivors (P = 0.022), with a decline overtime only in survivors and an increase from days 3 and 4 to days 6-8 in nonsurvivors. Using DDPCR technique, we found that poly-microorganisms detected and increased pathogen load a week after sepsis diagnosis were associated with poor prognosis.IMPORTANCEThis prospective study was initiated to explore the prognostic implications of a novel multiplex PCR assay in sepsis. Notably, our study was the largest cohort of sepsis with droplet digital polymerase chain reaction pathogen monitoring to date, allowing for a comprehensive evaluation of the prognostic significance of both pathogen species and load. We found that detection of poly-microorganisms was an independent risk factors for 28-day mortality. Also, pathogen load increase 1 week after sepsis diagnosis was a risk factor for 28-day mortality, and differential pathogen load kinetics were identified between day-28 survivors and nonsurvivors. Overall, this study demonstrated that pathogen species and load were highly correlated with sepsis prognosis. Patients exhibiting conditions mentioned above face a more adverse prognosis, suggesting the potential need for an escalation of antimicrobial therapy.Registered at ClinicalTrials.gov (NCT05190861).

Reliability of Droplet Digital PCR Alone and in Combination with Interleukin-6 and Procalcitonin for Prognosis of Bloodstream Infection.

Purpose: Bloodstream infection(BSI) is linked with high mortality, underscoring the significance of prompt etiological diagnosis for timely and precise treatment. This study aims to investigate the diagnostic value of droplet digital polymerase chain reaction(ddPCR) in combination with conventional inflammatory markers [interleukin-6(IL-6) and procalcitonin(PCT)] concerning disease progression and treatment prognosis in BSI patients. Furthermore, the study aims to explore a more efficient clinical application strategy. Patients and Methods: This prospective case seried study centers on 176 patients suspected of or confirmed with BSI. Blood samples were collected to extract nucleic acids for identifying pathogens (bacteria, fungi, and viruses) and determining copy loads via ddPCR. Results: The sensitivity of ddPCR was markedly higher compared to the culture method (74.71% vs 31.03%). A positive correlation existed between bacterial load and levels of inflammatory markers [IL-6 (P=0.0182), PCT (P=0.0029), and CRP (P=0.0005)]. In suspected BSI cases, the combination of ddPCR and inflammatory markers could predict sepsis risk [ROC: Area under the curve(AUC)=0.6071, P=0.0383]. Within confirmed BSI patients, the ddPCR bacterial load of those with SOFA<7 was lower than that of the SOFA≥7 (P=0.0334). ddPCR (OR: 1.789, P=0.035) monitoring combined with PCT (OR: 1.787, P=0.035) holded predictive value for SOFA progression (AUC=0.7913, P=0.0003). Similarly, BSI survivors displayed a lower burden than non-survivors (P=0.0170). Additionally, ddPCR combinated with IL-6 provided a more accurate and expedited insight into clinical outcomes prediction for BSI confirmed patients (AUC=0.7352, P=0.0030). Serial monitoring of bacterial load by ddPCR effectively mirrored the clinical course of BSI in patients. Notably, patients with positive ddPCR virus infection exhibited significantly reduced lymphocyte counts (P=0.0003). Conclusion: In a clinical context, qualitative ddPCR results and quantitative continuous monitoring can more precisely assess sepsis progression and treatment prognosis in BSI patients. Furthermore, ddPCR results offer quicker and more accurate reference points for clinical antibacterial and antiviral interventions.

Droplet digital polymerase chain reaction detection of KRAS mutations in pancreatic FNA samples: Technical and practical aspects for routine clinical implementation.

BACKGROUND: Pancreatic adenocarcinoma (PDAC) is associated with a 5-year survival rate of less than 6%, and current treatments have limited efficacy. The diagnosis of PDAC is mainly based on a cytologic analysis of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) samples. However, the collected specimens may prove noncontributory in a significant number of cases, delaying patient management and treatment. The combination of EUS-FNA sample examination and KRAS mutation detection can improve the sensitivity for diagnosis. In this context, the material used for molecular analysis may condition performance. METHODS: The authors prospectively compared the performance of cytologic analysis combined with a KRAS droplet digital polymerase chain reaction (ddPCR) assay for PDAC diagnosis using either conventional formalin-fixed, paraffin-embedded cytologic samples or needle-rinsing fluids. RESULTS: Molecular testing of formalin-fixed, paraffin-embedded cytologic samples was easier to set up, but the authors observed that the treatment of preanalytic samples, in particular the fixation process, drastically reduced ddPCR sensitivity, increasing the risk of false-negative results. Conversely, the analysis of dedicated, fresh needle-rinsing fluid samples appeared to be ideal for ddPCR analysis; it had greater sensitivity and was easily to implement in clinical use. In particular, fluid collection by the endoscopist, transportation to the laboratory, and subsequent freezing did not affect DNA quantity or quality. Moreover, the addition of KRAS mutation detection to cytologic examination improved diagnosis performance, regardless of the source of the sample. CONCLUSIONS: Considering all of these aspects, the authors propose the use of an integrated flowchart for the KRAS molecular testing of EUS-FNA samples in clinical routine.

Missense mutation of NRAS is associated with malignant progression in neurocutaneous melanosis.

Neurocutaneous melanosis (NCM) is a rare congenital neurocutaneous syndrome characterized by congenital melanocytic nevus of skin and abnormal proliferation of leptomeningeal melanocytes. Early acquisition of post-zygotic somatic mutations has been postulated to underlie the pathogenesis of NCM. The pathogenesis of NCM remains to be fully elucidated, and treatment options have not been established. Here, we report for the first time, multiregional genomic analyses in a 3-year-old autopsied girl with leptomeningeal melanomatosis associated with NCM, in which a ventriculo-peritoneal (VP) shunt was inserted for the treatment of hydrocephalus. The patient expired six months after the onset due to respiratory failure caused by abdominal dissemination via VP shunt. We performed multiregional exome sequencing to identify genomic differences among brain and abdominal tumors, nevus, and normal tissues. A total of 87 somatic mutations were found in 71 genes, with a significantly large number of gene mutations found in the tumor site. The genetic alterations detected in the nevus were only few and not shared with other sites. Three mutations, namely GNAQ R183Q, S1PR3 G89S and NRAS G12V, considered pathogenic, were found, although S1PR3 mutations have not been previously reported in melanocytic tumors. GNAQ and S1PR3 mutations were shared in both tumor and normal sites. Moreover, the mutant allele frequencies of the two mutations were markedly higher in tumor sites than in normal sites, with copy-neutral loss-of-heterozygosity (CN-LOH) occurring in tumor. NRAS mutation was found only in the abdominal tumor and was thought to be responsible for malignant progression in the present case. Multiregional comprehensive genetic analysis may lead to discovering novel driver mutations associated with tumorigenesis and targeted therapy.

An update of clinical value of circulating tumor DNA in esophageal cancer: a systematic review and meta-analysis.

BACKGROUND: Esophageal cancer (EC) is a deadly disease with limited therapeutic options. Although circulating tumor DNA (ctDNA) could be a promising tool in this regard, the availiable evidence is limited. We performed a systematic review and meta-analysis to summarize the clinical applicability of the next-generation sequencing (NGS) and droplet digital polymerase chain reaction (ddPCR) technology on the ctDNA detection of the EC and listed the current challenges. METHODS: We systematically searched MEDLINE (via PubMed), Embase (via OVID), ISI Web of Science database and Cochrane Library from January, 2000 to April, 2023. Progression-free survival (PFS) and overall survival (OS) were set as primary outcome endpoints. Pathologic response was evaluated by tumor regression grade (TRG), according to the eighth edition of the American Joint Committee on Cancer (AJCC). Major pathologic regression (MPR) was defined as TRG 1 and 2. The MPR was set as secondary endpoint. Hazard rate (HR) and associated 95% CI were used as the effect indicators the association between ctDNA and prognosis of EC. MPR rates were also calculated. Fixed-effect model (Inverse Variance) or random-effect model (Mantel-Haenszel method) was performed depending on the statistically heterogeneity. RESULTS: Twenty-two studies, containing 1144 patients with EC, were included in this meta-analysis. The results showed that OS (HR = 3.87; 95% CI, 2.86-5.23) and PFS (HR = 4.28; 95% CI, 3.34-5.48) were shorter in ctDNA-positive patients. In the neoadjuvant therapy, the sensitivity analysis showed the clarified HR of ctDNA-positive was 1.13(95% CI, 1.01-1.28). We also found that TP53, NOTCH1, CCND1 and CNKN2A are the most frequent mutation genes. CONCLUSIONS: Positive ctDNA is associated with poor prognosis, which demonstrated clinical value of ctDNA. Longitudinal ctDNA monitoring showed potential prognostic value in the neoadjuvant therapy. In an era of precision medicine, ctDNA could be a promising tool to individualize treatment planning and to improve outcomes in EC. PROSPERO REGISTRATION NUMBER: CRD42023412465.

A novel method to assess copy number variations in melanocytic neoplasms: Droplet digital PCR for precise quantitation of MYC and MYB genes.

INTRODUCTION: While most melanocytic neoplasms can be classified as either benign or malignant by histopathology alone, ancillary molecular diagnostic tests can be necessary to establish the correct diagnosis in challenging cases. Currently, the detection of copy number variations (CNVs) by fluorescence in situ hybridization and chromosomal microarray (CMA) are the most popular methods, but remain expensive and inaccessible. We aim to develop a relatively inexpensive, fast, and accessible molecular assay to detect CNVs relevant to melanoma using droplet digital polymerase chain reaction (ddPCR) technology. METHODS: In this proof-of-concept study, we evaluated CNVs in MYC and MYB genes from 73 cases of benign nevi, borderline melanocytic lesions, and primary and metastatic melanoma at our institution from 2015 to 2022. A multiplexed ddPCR assay and CMA were performed on each sample, and the results were compared. RESULTS: Concordance analysis of ddPCR with CMA for quantification of MYC and MYB CNVs revealed a sensitivity and specificity of 89% and 86% for MYC and 83% and 74% for MYB, respectively. CONCLUSION: We demonstrate the first use of a multiplexed ddPCR assay to identify CNVs in melanocytic neoplasms. With further improvement and validation, ddPCR may represent a low-cost and rapid tool to aid in the diagnosis of histopathologically ambiguous melanocytic tumors.

Accurate Detection of Multiple Tumor Mutations in Formalin-Fixed Paraffin-Embedded Tissues by Coupling Sequence Artifacts Elimination and Mutation Enrichment With MeltArray.

Formalin-fixed paraffin-embedded (FFPE) tissues are the primary source of DNA for companion diagnostics (CDx) of cancers. Degradation of FFPE tissue DNA and inherent tumor heterogeneity constitute serious challenges in current CDx assays. To address these limitations, we introduced sequence artifact elimination and mutation enrichment to MeltArray, a highly multiplexed PCR approach, to establish an integrated protocol that provides accuracy, ease of use, and rapidness. Using PIK3CA mutations as a model, we established a MeltArray protocol that could eliminate sequence artifacts completely and enrich mutations from 23.5- to 59.4-fold via a single-reaction pretreatment step comprising uracil-DNA-glycosylase excision and PCR clamping. The entire protocol could identify 13 PIK3CA hotspot mutations of 0.05% to 0.5% mutant allele fractions within 5 hours. Evaluation of 106 breast cancer and 40 matched normal FFPE tissue samples showed that all 47 PIK3CA mutant samples were from the cancer tissue, and no false-positive results were detected in the normal samples. Further evaluation of 105 colorectal and 40 matched normal FFPE tissue samples revealed that 11 PIK3CA mutants were solely from the cancer sample. The detection results of our protocol were consistent with those of the droplet digital PCR assays that underwent sequence artifact elimination. Of the 60 colorectal samples with next-generation sequencing results, the MeltArray protocol detected 2 additional mutant samples with low mutant allele fractions. We conclude that the new protocol provides an improved alternative to current CDx assays for detecting tumor mutations in FFPE tissue DNA.

Effect of BCR::ABL1 transcript type and droplet digital polymerase chain reaction on successful treatment-free remission in chronic myeloid leukemia patients who discontinued tyrosine kinase inhibitor.

Background: Droplet digital polymerase chain reaction (ddPCR) is an exact method of measurement. Objectives: We conducted this study to identify the prognostic factors for successful treatment-free remission in patients with chronic-phase chronic myeloid leukemia who discontinued tyrosine kinase inhibitors (TKIs). We also aimed to validate ddPCR for predicting molecular relapse. Design: This is a prospective, multicenter study. Methods: We enrolled patients treated with TKIs for at least 3 years with a confirmed sustained deep molecular response (DMR) for at least 1 year. TKI was re-administered in patients who experienced the loss of major molecular response (MMR). Results: A total of 66 patients from five institutions in South Korea were enrolled. During a median follow-up period of 16.5 months, 29/66 (43.9%) patients experienced molecular relapse; the probability of molecular relapse-free survival (RFS) at 6 or 12 months after TKI discontinuation was 65.6% or 57.8%, respectively, with most molecular relapses occurring within the first 7 months. All patients who lost MMR were re-treated with TKI, and all re-achieved MMR at a median of 2.8 months. E14a2 transcript type (p = 0.005) and longer DMR duration (⩾48 months) prior to TKI discontinuation (p = 0.002) were associated with prolonged molecular RFS and with sustained DMR. Patients with both e13a2 transcript type and detectable BCR::ABL1 (⩾MR5.0) by ddPCR at the time of TKI discontinuation showed shorter duration of molecular RFS (p = 0.015). Conclusion: Our data suggest that transcript type and BCR::ABL1 transcript levels on ddPCR should be taken into consideration when deciding whether to discontinue TKI therapy.

Usefulness of bronchial washing fluid for detection of EGFR mutations in non-small cell lung cancer.

INTRODUCTION: The implementation of bronchial washing fluid (BWF) as a diagnostic specimen may complement the low diagnostic yields of plasma in detecting EGFR mutation (mEGFR) in non-small cell lung cancer. However, the diagnostic value of BWF in detecting mEGFR has yet to be clarified. MATERIALS AND METHODS: From March 2021 to August 2022, patients with histologically confirmed NSCLC with matched tumor tissue, BWF, and/or plasma samples were enrolled. Patients were classified into either initial diagnosis or rebiopsy groups. Diagnostic yields of mEGFR in BWF and plasma were evaluated using droplet digital polymerase chain reaction and compared to mEGFR in tumor tissue as standard. RESULTS: The study included 123 patients (74.1 %) in the initial diagnosis and 43 patients (25.9 %) in the rebiopsy group. BWF showed higher sensitivity, specificity, and concordance rates than plasma in both the initial diagnosis (57.4 %, 96.4 %, and 74.0 % vs. 16.4 %, 96.2 %, and 53.1 %) and the rebiopsy group (87.9 %, 60.0 %, and 81.4 % vs. 25.0 %, 75.0 %, and 41.7 %). In the initial diagnosis group, mEGFR was detected in the BWF of 13 out of 16 patients, even in the absence of tumor cells in the tissue biopsy. In these cases, EGFR test results obtained from BWF showed concordance with EGFR test results from the tumor tissue obtained through repeated biopsy or surgery later. In the rebiopsy group, T790M was detected in 16 patients (37.2 %) by tissue biopsy. The combined use of tissue biopsy and BWF increased detection, confirming T790M in 22 patients (51.2 %). DISCUSSION: The detection of mEGFR using BWF shows higher diagnostic yields than plasma for both initial diagnosis and rebiopsy. T790M was detected earlier in BWF than in tissue rebiopsy in some cases, providing patients with an early opportunity to access third-generation EGFR-TKIs. The complementary use of BWF with tumor tissue may improve precision in EGFR-mutated NSCLC treatment strategies.

PIK3CA mutations in cutaneous squamous cell carcinoma.

Oncogenic PIK3CA mutation activates phosphoinositide 3-kinase (PI3K) enzyme, and PI3K-AKT signaling activation induces several growth-regulatory transcription factors. PIK3CA mutations have attracted attention as biomarker in clinical trials of various inhibitors including PI3K inhibitors. About 80% of PIK3CA mutations in human cancers are observed in 'hot spot' regions: exon 9 (E542K and E545K) and exon 20 (H1047R). There were few reports about clinical significance of PIK3CA mutations in cutaneous cell carcinoma (cSCC). Thus, we investigate the prevalence of three PIK3CA hot spot mutations in 143 cases with cSCC and evaluate the correlation between the presence of these mutations and clinical characteristics by using ddPCR. The frequency of each E542K, E545K and H1047R PIK3CA mutations was 1.4% (2/143), 2.8% (4/143), and 0.7% (1/143) respectively. No significant correlation was found between PIK3CA mutations and clinical characteristics. Although additional basic researches and clinical trials are necessary, various inhibitors may be effective therapeutics for PIK3CA mutation-positive cSCC. Our study revealed the prevalence of PIK3CA mutations in cSCC.

First application of a droplet digital PCR to detect Toxoplasma gondii in mussels.

Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is one of the main food-, water- and soil-borne zoonotic disease worldwide. Over the past 20 years many papers were published on the transmission of T. gondii by marine animals, including mollusks, which can concentrate the oocysts and release them. Sporulated oocysts may remain viable and infective for 18 months in seawater. Therefore, raw or undercooked bivalve mollusks pose a risk to humans. This study aimed to apply and validate for the first time a very sensitive digital droplet polymerase chain reaction (ddPCR) protocol to detect and quantify T. gondii DNA in mussels. Four concentration levels: 8000 genomic copies (gc)/µL, 800 gc/µL, 80 gc/µL, 8 gc/µL of a T. gondii reference DNA were tested. DNA was extracted from 80 pools of mussels (Mytilus galloprovincialis). Forty pools were contaminated with T. gondii reference DNA and used as positive controls, while 40 pools were used as negative controls. DdPCR reaction was prepared using a protocol, previously developed by the authors, for detection of T. gondii in meat. Amplification was obtained up 8 gc/µL. All infected replicates resulted positive, as well as no droplets were detected in negative controls. The droplets produced in the reaction ranged from 8,828 to 14,075 (average 12,627 droplets). The sensitivity and specificity of ddPCR were 100% (95%CI = 94.3-99.9). In addition, 100 pools of mussels collected in the Gulf of Naples were used to validate the protocol. Of these 16% were positive (95% CI = 9.7-25.0) for T. gondii. Samples were also tested by real-time PCR and no positive samples were found. Data obtained from ddPCR showed good identification of negative and positive samples with higher specificity and efficiency than real-time PCR. This tool could be very useful for a rapid sensitive detection of low DNA concentrations of T. gondii in mussels, reducing the risk of toxoplasmosis in humans.

Non-invasive prenatal screening & diagnosis of ß-thalassaemia in an affected foetus.

Background & objectives: Non-invasive prenatal testing (NIPT) of maternally inherited alleles of ß-thalassaemia (MIB) remains to be a challenge. Furthermore, current techniques are not available for use as routine tests. NIPT for ß-thalassaemia disease was developed by using a specific droplet digital polymerase chain reaction (ddPCR) assay to analyze the cell-free foetal DNA (cffDNA) derived from maternal plasma. Methods: Pregnant women and their spouses who are at risk of bearing an offspring with ß-thalassaemia disease from common MIB mutations (CD 41/42-TCTT, CD17A>T, IVS1-1G>T and CD26G>A) were enrolled. The ddPCR assay sets were constructed for each of the four mutations. All cell-free DNA samples were first screened for the paternally inherited ß-thalassaemia (PIB) mutation. The PIB-negative samples were considered as non-disease and were not further analyzed. For PIB-positive samples, DNA fragments of 50-300 base pairs in size were isolated and purified, and further analyzed for MIB mutation. The allelic ratio between the mutant and the wild-type was used to determine the presence of MIB in cffDNA. All cases underwent a prenatal diagnosis by amniocentesis for a definite diagnosis. Results: Forty two couples at risk were enrolled. Twenty two samples were positive for PIBs. Among these 22 samples, there were 10 cases with allelic ratio >1.0 (MIB positive). All foetuses with over-represented mutant alleles were further diagnosed with ß-thalassaemia disease; eight with compound heterozygous and two with homozygous mutations. The 20 PIB-negative and 12 MIB-negative foetuses were non-affected. Interpretation & Conclusions: The results of this study suggest that NIPT utilizing the ddPCR assay can be effectively used for the screening and diagnosis of foetal ß-thalassaemia in at risk pregnancies.

Allogeneic haematopoietic stem cell transplantation for myelofibrosis: prognostic indicators and the role of JAK2V617F measurable-residual disease monitoring by droplet-digital polymerase chain reaction.

Relapse after allogeneic haematopoietic stem cell transplantation (HSCT) is one of the key determinants of outcome in myelofibrosis (MF) and remains an important unmet need. In this retrospective single-centre study, we evaluated 35 consecutive patients with MF receiving allogeneic HSCT. At 30 days post-HSCT, full donor chimerism was achieved in 31 patients (88.6%). The median time to neutrophil engraftment was 16.8 (10-42) days and the median time to platelet engraftment was 26 (12-245) days. Four patients (11.4%) experienced primary graft failure. With a median duration of follow-up of 33 (1-223) months, with the 5-year overall survival (OS) and progression-free survival (PFS) were 51.6% and 46.3%, respectively. Relapse after HSCT (P < 0.001), leucocyte count ≥ 18 × 109/L at HSCT (P = 0.003) and accelerated/blast phase disease at HSCT (P < 0.001) were significantly associated with worse OS. Age at HSCT ≥ 54 years (P = 0.01), mutated ETV6 (P = 0.03), leucocyte count ≥ 18 × 109/L (P = 0.02), accelerated/blast phase MF (P = 0.001), and grade 2-3 bone marrow reticulin fibrosis at 12 months post-HSCT (P = 0.002) were significantly associated with worse PFS. JAK2V617F MRD ≥ 0.047 [sensitivity 85.7%; positive predictive value (PPV) 100%; AUC 0.984; P = 0.001] at 6 months and JAK2V617F MRD ≥ 0.009 (sensitivity 100%; PPV 100%; AUC 1.0; P = 0.001) at 12 months were highly predictive of post-HSCT relapse. Inferior OS and PFS were significantly associated with detectable JAK2V617F MRD at 12 months (P = 0.003 and P = 0.0001, respectively).

Plasma exosomal hsa_circ_0079439 as a novel biomarker for early detection of gastric cancer.

BACKGROUND: Due to the poor prognosis of gastric cancer (GC), early detection methods are urgently needed. Plasma exosomal circular RNAs (circRNAs) have been suggested as novel biomarkers for GC. AIM: To identify a novel biomarker for early detection of GC. METHODS: Healthy donors (HDs) and GC patients diagnosed by pathology were recruited. Nine GC patients and three HDs were selected for exosomal whole-transcriptome RNA sequencing. The expression profiles of circRNAs were analyzed by bioinformatics methods and validated by droplet digital polymerase chain reaction. The expression levels and area under receiver operating characteristic curve values of plasma exosomal circRNAs and standard serum biomarkers were used to compare their diagnostic efficiency. RESULTS: There were 303 participants, including 240 GC patients and 63 HDs, involved in the study. The expression levels of exosomal hsa_circ_0079439 were significantly higher in GC patients than in HDs (P < 0.0001). However, the levels of standard serum biomarkers were similar between the two groups. The area under the curve value of exosomal hsa_circ_0079439 was higher than those of standard biomarkers, including carcinoembryonic antigen, carbohydrate antigen (CA)19-9, CA72-4, alpha-fetoprotein, and CA125 (0.8595 vs 0.5862, 0.5660, 0.5360, 0.5082, and 0.5018, respectively). The expression levels of exosomal hsa_circ_0079439 were significantly decreased after treatment (P < 0.05). Moreover, the expression levels of exosomal hsa_circ_0079439 were obviously higher in early GC (EGC) patients than in HDs (P < 0.0001). CONCLUSION: Our results suggest that plasma exosomal hsa_circ_0079439 is upregulated in GC patients. Moreover, the levels of exosomal hsa_circ_0079439 could distinguish EGC and advanced GC patients from HDs. Therefore, plasma exosomal hsa_circ_0079439 might be a potential biomarker for the diagnosis of GC during both the early and late stages.

Development and validation of a model gene therapy biodistribution assay for AVGN7 using digital droplet polymerase chain reaction.

Biodistribution assays are integral to gene therapy commercialization and have traditionally used real-time qPCR. Droplet digital PCR (ddPCR), however, has distinct advantages including higher sensitivity and absolute quantification but is underused because of lacking regulatory guidance and meaningful examples in the literature. We report a fit-for-purpose model process to validate a good laboratory practice (GLP)-compliant ddPCR assay for AVGN7, a Smad7 gene therapeutic for muscle wasting. Duplexed primer/probe sets for Smad7 and mouse TATA-box binding protein were optimized using gBlock DNA over a dynamic range of 10-80,000 copies/reaction in 250 ng mouse gDNA. Linearized plasmid and mouse gDNA were used for validation, which determined precision, accuracy, ruggedness/robustness, selectivity, recovery, specificity, dilution linearity, and stability. Inter-run precision and accuracy met previously established criteria with bias between -5% and 15%, coefficient of variation (CV) less than 19%, and total error within 8%-35%. The limit of detection was 2.5 copies/reaction, linearity was confirmed at 40-80,000 copies/reaction, specificity was demonstrated by single droplet populations and assay stability was demonstrated for benchtop, refrigerated storage, and repeated freeze-thaw cycles. The procedural road map provided exceeds recently established standards. It is also relevant to many IND-enabling processes, as validated ddPCR assays can be used in biodistribution studies and with vector titering and manufacturing quality control.