StratoLAMP: Label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of precipitate.

Multiplex, digital nucleic acid detections have important biomedical applications, but the multiplexity of existing methods is predominantly achieved using fluorescent dyes or probes, making the detection complicated and costly. Here, we present the StratoLAMP for label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of the precipitate byproduct. The StratoLAMP designates two sets of primers with different concentrations to achieve different precipitate yields when amplifying different nucleic acid targets. In the detection, deep learning image analysis is used to stratify the precipitate within each droplet and determine the encapsulated targets for nucleic acid quantification. We investigated the effect of the amplification reagents and process on the precipitate generation and optimized the assay conditions. We then implemented a deep-learning image analysis pipeline for droplet detection, achieving an overall accuracy of 94.3%. In the application, the StratoLAMP successfully achieved the simultaneous quantification of two nucleic acid targets with high accuracy. By eliminating the need for fluorescence, StratoLAMP represents a unique concept toward label-free, multiplex nucleic acid assays and an analytical tool with great cost-effectiveness.

Benchmarking digital PCR partition classification methods with empirical and simulated duplex data.

Digital PCR (dPCR) is a highly accurate technique for the quantification of target nucleic acid(s). It has shown great potential in clinical applications, like tumor liquid biopsy and validation of biomarkers. Accurate classification of partitions based on end-point fluorescence intensities is crucial to avoid biased estimators of the concentration of the target molecules. We have evaluated many clustering methods, from general-purpose methods to specific methods for dPCR and flowcytometry, on both simulated and real-life data. Clustering method performance was evaluated by simulating various scenarios. Based on our extensive comparison of clustering methods, we describe the limits of these methods, and formulate guidelines for choosing an appropriate method. In addition, we have developed a novel method for simulating realistic dPCR data. The method is based on a mixture distribution of a Poisson point process and a skew-$t$ distribution, which enables the generation of irregularities of cluster shapes and randomness of partitions between clusters ('rain') as commonly observed in dPCR data. Users can fine-tune the model parameters and generate labeled datasets, using their own data as a template. Besides, the database of experimental dPCR data augmented with the labeled simulated data can serve as training and testing data for new clustering methods. The simulation method is available as an R Shiny app.

The Next Frontier in Noninvasive Prenatal Diagnostics: Cell-Free Fetal DNA Analysis for Monogenic Disease Assessment.

With the widespread clinical adoption of noninvasive screening for fetal chromosomal aneuploidies based on cell-free DNA analysis from maternal plasma, more researchers are turning their attention to noninvasive prenatal assessment for single-gene disorders. The development of a spectrum of approaches to analyze cell-free DNA in maternal circulation, including relative mutation dosage, relative haplotype dosage, and size-based methods, has expanded the scope of noninvasive prenatal testing to sex-linked and autosomal recessive disorders. Cell-free fetal DNA analysis for several of the more prevalent single-gene disorders has recently been introduced into clinical service. This article reviews the analytical approaches currently available and discusses the extent of the clinical implementation of noninvasive prenatal testing for single-gene disorders.

p53 as a Potential Actionable Target in Myxofibrosarcoma: A Molecular and Pathologic Review of a Single-Institute Series.

Myxofibrosarcoma (MFS) is a common adult soft tissue sarcoma characterized by high-local recurrence rate, poorly understood molecular pathogenesis, lack of specific prognostic markers, and effective targeted therapies. To gain further insights into the disease, we analyzed a well-defined group of 133 primary MFS cases. Immunohistochemical (IHC) staining for p53, MET, RET, and RB was performed. Twenty-five cases were analyzed by targeted resequencing of known cancer driver hotspot mutations, whereas 66 and 64 MFSs were examined for the presence of genetic variants in TP53 and MET gene, respectively. All clinical, histologic, immunostaining, and genetic variables were analyzed for their impact on 5-years overall survival (OS) and 5-years event-free survival (EFS). In our series, no grade I tumors relapsed and high grade are related to a positive MET immunostaining (P = .034). Both local recurrence (P = .038) and distal metastases (P = .016) correlated to the presence of "single nucleotide variant (SNV) plus copy number variation (CNV)" in TP53. Multivariate analysis revealed that age (>60 years), metastasis at presentation, and positive IHC-p53 signal are risk factors for a poor OS (P = .003, P = .000, and P = .002), whereas age (>60 years), synchronous metastasis, and tumor size (>10 cm) predict an unfavorable 5-years EFS (P = .011, P = .000, and P = .023). Considering the smaller series (n = 66) that underwent molecular screening, the presence of "SNV+CNV" in TP53 represents a risk factor for a worse 5-years EFS (hazard ratio, 2.5; P = .017). The present series confirms that TP53 is frequently altered in MFS (86.4% of cases), appearing to play an important role in MFS tumorigenesis and being a potentially drugable target. A positive p53 immunostainings is related to a poor diagnosis, and it is the presence of a single nucleotide genetic alterations in TP53 that is essential in conferring MFS an aggressive phenotype, thus supporting the use of molecular profiling in MFS to better define the role of p53 as a prognostic factor.

Postoperative recurrence detection using individualized circulating tumor DNA in upper tract urothelial carcinoma.

Biomarkers that could detect the postoperative recurrence of upper tract urothelial carcinoma (UTUC) have not been established. In this prospective study, we aim to evaluate the utility of individualized circulating tumor DNA (ctDNA) monitoring using digital PCR (dPCR) as a tumor recurrence biomarker for UTUC in the perioperative period. Twenty-three patients who underwent radical nephroureterectomy (RNU) were included. In each patient, whole exome sequencing by next-generation sequencing and TERT promoter sequencing of tumor DNA were carried out. Case-specific gene mutations were selected from sequencing analysis to examine ctDNA by dPCR analysis. We also prospectively collected plasma and urine ctDNA from each patient. The longitudinal variant allele frequencies of ctDNA during the perioperative period were plotted. Case-specific gene mutations were detected in 22 cases (96%) from ctDNA in the preoperative samples. Frequently detected genes were TERT (39%), FGFR3 (26%), TP53 (22%), and HRAS (13%). In all cases, we obtained plasma and urine samples for 241 time points and undertook individualized ctDNA monitoring for 2 years after RNU. Ten patients with intravesical recurrence had case-specific ctDNA detected in urine at the time of recurrence. The mean lead time of urinary ctDNA in intravesical recurrence was 60 days (range, 0-202 days). Two patients with distal metastasis had case-specific ctDNA in plasma at the time of metastasis. In UTUC, tumor-specific gene mutations can be monitored postoperatively as ctDNA in plasma and urine. Individualized ctDNA might be a minimally invasive biomarker for the early detection of postoperative recurrence.

Expression analysis of NF1-mutated alleles in a rare compound heterozygous spinal NF1 patient by digital PCR.

BACKGROUD: Neurofibromatosis type 1 (NF1) is a heterogeneous neurocutaneous disorder. Spinal neurofibromatosis (SNF) is a distinct clinical entity of NF1, characterized by bilateral neurofibromas involving all spinal nerve roots. Although both forms are caused by intragenic heterozygous variants of NF1, missense variants have been associated with SNF, according to a dominant inheritance model causing haploinsufficiency. Most patients carry pathogenic variants in one of the NF1 alleles; nevertheless, patients with both NF1-mutated copies have been described. Interestingly, all NF1 variants carried by the known SNF compound heterozygotes were missense/splicing variants or in-frame insertion-deletions. AIMS: To investigate whether there is a differential expression of NF1 variant alleles in an NF1 compound heterozygous SNF patient possibly contributing to clinical phenotype. MATERIALS & METHODS: We performed an allele-specific expression study, by chip-based digital PCR, in an SNF family carrying two NF1 missense variants. We evaluated the expression levels of the two NF1-mutated alleles both carried by the compound heterozygous SNF patient and his relatives. RESULTS: Both alleles were expressed at comparable levels in the patient and hyper-expressed compared to the wild-type alleles of healthy controls. DISCUSSION: Here we provide new insights into expression studies of NF1-mutated transcripts suggesting that a novel pathogenetic mechanism, caused by gain-of-function variants, could be associated with SNF. CONCLUSIONS: Further studies should be performed in larger cohorts, opening new perspectives in the NF1 pathogenesis comprehension.

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

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

Huntingtin HTT1a is generated in a CAG repeat-length-dependent manner in human tissues.

BACKGROUND: The disease-causing mutation in Huntington disease (HD) is a CAG trinucleotide expansion in the huntingtin (HTT) gene. The mutated CAG tract results in the production of a small RNA, HTT1a, coding for only exon 1 of HTT. HTT1a is generated by a block in the splicing reaction of HTT exon 1 to exon 2 followed by cleavage in intron 1 and polyadenylation. Translation of HTT1a leads to the expression of the highly toxic HTT exon 1 protein fragment. We have previously shown that the levels of HTT1a expression in mouse models of HD is dependent on the CAG repeat length. However, these data are lacking for human tissues. METHODS: To answer this question, we developed highly sensitive digital PCR assays to determine HTT1a levels in human samples. These assays allow the absolute quantification of transcript numbers and thus also facilitate the comparison of HTT1a levels between tissues, cell types and across different studies. Furthermore, we measured CAG repeat sizes for every sample used in the study. Finally, we analysed our data with ANOVA and linear modelling to determine the correlation of HTT1a expression levels with CAG repeat sizes. RESULTS: In summary, we show that HTT1a is indeed expressed in a CAG repeat-length-dependent manner in human post mortem brain tissues as well as in several peripheral cell types. In particular, PBMCs show a statistically significant positive correlation of HTT1a expression with CAG repeat length, and elevated HTT1a expression levels even in the adult-onset CAG repeat range. CONCLUSIONS: Our results show that HTT1a expression occurs throughout a wide range of tissues and likely with all CAG lengths. Our data from peripheral sample sources demonstrate that HTT1a is indeed generated throughout the body in a CAG repeat-length-dependent manner. Therefore, the levels of HTT1a might be a sensitive marker of disease state and/or progression and should be monitored over time, especially in clinical trials targeting HTT expression.

Droplet digital PCR: An effective method for monitoring and prognostic evaluation of minimal residual disease in JMML.

Juvenile myelomonocytic leukaemia (JMML) is a rare myeloproliferative neoplasm requiring haematopoietic stem cell transplantation (HSCT) for potential cure. Relapse poses a significant obstacle to JMML HSCT treatment, as the lack of effective minimal residual disease (MRD)-monitoring methods leads to delayed interventions. This retrospective study utilized the droplet digital PCR (ddPCR) technique, a highly sensitive nucleic acid detection and quantification technique, to monitor MRD in 32 JMML patients. The results demonstrated that ddPCR detected relapse manifestations earlier than traditional methods and uncovered molecular insights into JMML MRD dynamics. The findings emphasized a critical 1- to 3-month window post-HSCT for detecting molecular relapse, with 66.7% (8/12) of relapses occurring within this period. Slow MRD clearance post-HSCT was observed, as 65% (13/20) of non-relapse patients took over 6 months to achieve ddPCR-MRD negativity. Furthermore, bone marrow ddPCR-MRD levels at 1-month post-HSCT proved to be prognostically significant. Relapsed patients exhibited significantly elevated ddPCR-MRD levels at this time point (p = 0.026), with a cut-off of 0.465% effectively stratifying overall survival (p = 0.007), event-free survival (p = 0.035) and cumulative incidence of relapse (p = 0.035). In conclusion, this study underscored ddPCR's superiority in JMML MRD monitoring post-HSCT. It provided valuable insights into JMML MRD dynamics, offering guidance for the effective management of JMML.

Accuracy of quantitative viral secondary standards: a re-examination.

Interlaboratory agreement of viral load assays depends on the accuracy and uniformity of quantitative calibrators. Previous work demonstrated poor agreement of secondary cytomegalovirus (CMV) standards with nominal values. This study re-evaluated this issue among commercially produced secondary standards for both BK virus (BKV) and CMV, using digital polymerase chain reaction (dPCR) to compare the materials from three different manufacturers. Overall, standards showed an improved agreement compared to prior work, against nominal values in both log10 copies/mL and log10 international unit (IU)/mL, with bias from manufacturer-assigned nominal values of 0.0-0.9 log10 units (either copies or IU)/mL. Standards normalized to IU and those values assigned by dPCR rather than by real-time PCR (qPCR) showed better agreement with nominal values. The latter reinforces prior conclusions regarding the utility of using such methods for quantitative value assignment in reference materials. Quantitative standards have improved over the last several years, and the remaining bias from nominal values might be further reduced by universal implementation of dPCR methods for value assignment, normalized to IU. IMPORTANCE: Interlaboratory agreement of viral load assays depends on accuracy and uniformity of quantitative calibrators. Previous work, published in JCM several years ago, demonstrated poor agreement of secondary cytomegalovirus (CMV) standards with nominal values. This study re-evaluated this issue among commercially produced secondary standards for both BK virus (BKV) and CMV, using digital polymerase chain reaction (dPCR) to compare the materials from three different manufacturers. Overall, standards showed an improved agreement compared to prior work, against nominal values, indicating a substantial improvement in the production of accurate secondary viral standards, while supporting the need for further work in this area and for the broad adaption of international unit (IU) as a reporting standard for quantitative viral load results.

Characterization of integrated Marek's disease virus genomes supports a model of integration by homology-directed recombination and telomere-loop-driven excision.

IMPORTANCE: Marek's disease virus (MDV) is a ubiquitous chicken pathogen that inflicts a large economic burden on the poultry industry, despite worldwide vaccination programs. MDV is only partially controlled by available vaccines, and the virus retains the ability to replicate and spread between vaccinated birds. Following an initial infection, MDV enters a latent state and integrates into host telomeres and this may be a prerequisite for malignant transformation, which is usually fatal. To understand the mechanism that underlies the dynamic relationship between integrated-latent and reactivated MDV, we have characterized integrated MDV (iMDV) genomes and their associated telomeres. This revealed a single orientation among iMDV genomes and the loss of some terminal sequences that is consistent with integration by homology-directed recombination and excision via a telomere-loop-mediated process.

Combining germline, tissue and liquid biopsy analysis by comprehensive genomic profiling to improve the yield of actionable variants in a real-world cancer cohort.

BACKGROUND: Comprehensive next-generation sequencing is widely used for precision oncology and precision prevention approaches. We aimed to determine the yield of actionable gene variants, the capacity to uncover hereditary predisposition and liquid biopsy appropriateness instead of, or in addition to, tumor tissue analysis, in a real-world cohort of cancer patients, who may benefit the most from comprehensive genomic profiling. METHODS: Seventy-eight matched germline/tumor tissue/liquid biopsy DNA and RNA samples were profiled using the Hereditary Cancer Panel (germline) and the TruSight Oncology 500 panel (tumor tissue/cfDNA) from 23 patients consecutively enrolled at our center according to at least one of the following criteria: no available therapeutic options; long responding patients potentially fit for other therapies; rare tumor; suspected hereditary cancer; primary cancer with high metastatic potential; tumor of unknown primary origin. Variants were annotated for OncoKB and AMP/ASCO/CAP classification. RESULTS: The overall yield of actionable somatic and germline variants was 57% (13/23 patients), and 43.5%, excluding variants previously identified by somatic or germline routine testing. The accuracy of tumor/cfDNA germline-focused analysis was demonstrated by overlapping results of germline testing. Five germline variants in BRCA1, VHL, CHEK1, ATM genes would have been missed without extended genomic profiling. A previously undetected BRAF p.V600E mutation was emblematic of the clinical utility of this approach in a patient with a liver undifferentiated embryonal sarcoma responsive to BRAF/MEK inhibition. CONCLUSIONS: Our study confirms the clinical relevance of performing extended parallel tumor DNA and cfDNA testing to broaden therapeutic options, to longitudinally monitor cfDNA during patient treatment, and to uncover possible hereditary predisposition following tumor sequencing in patient care.

Circulating Tumor DNA in Patients with Desmoid Fibromatosis during Active Surveillance.

BACKGROUND: Sporadic desmoid fibromatosis (DF) is a rare locally aggressive tumor characterized by mutation in exon 3 of CTNNB1 (T41A, S45F, and S45P). Standard of care is active surveillance (AS), but 30% require treatment. DF clinical course is unpredictable and identification of prognostic markers is needed to tailor strategy. In this prospective study, we investigated the consistency between mutation detected in tumor biopsies with that detected in plasma by digital droplet PCR (ddPCR) and the association between circulating tumor DNA (ctDNA) abundancy with clinical outcome. PATIENTS AND METHODS: A total of 56 patients and 10 healthy donors were included. CTNNB1 mutation status of DF biopsies was determined by Sanger and in case of WT CTNNB1 with NGS. In matched plasma samples at enrollment and during AS at specific timepoints, we evaluated cfDNA quantity and ctDNA. RESULTS: ctDNA levels were measured in 46 patients with CTNNB1 mutation. Detection rate for T41A, S45F and S45P was 68%, 42% and 100%, respectively. S45P variant has been detected in all patients with S45P mutation. Longitudinal assessment of ctDNA during AS in nine patients (four with regression and five with progression as first event according to RECIST) showed a concordance between the event and ctDNA level change in six out of nine patients tested (4/5 with progression and 2/4 with regression). CONCLUSIONS: Results of ctDNA analysis support its potential clinical implementation as diagnostic tool in specific clinical scenarios where biopsy can be challenging. A prospective clinical trial needs to be performed to evaluate the potential role of ctDNA as predictive biomarker.

Mitochondrial DNA in bronchoalveolar lavage fluid is associated with the prognosis of idiopathic pulmonary fibrosis: a single cohort study.

BACKGROUND: Extracellular mitochondrial DNA (mtDNA) is released from damaged cells and increases in the serum and bronchoalveolar lavage fluid (BALF) of idiopathic pulmonary fibrosis (IPF) patients. While increased levels of serum mtDNA have been reported to be linked to disease progression and the future development of acute exacerbation (AE) of IPF (AE-IPF), the clinical significance of mtDNA in BALF (BALF-mtDNA) remains unclear. We investigated the relationships between BALF-mtDNA levels and other clinical variables and prognosis in IPF. METHODS: Extracellular mtDNA levels in BALF samples collected from IPF patients were determined using droplet-digital PCR. Levels of extracellular nucleolar DNA in BALF (BALF-nucDNA) were also determined as a marker for simple cell collapse. Patient characteristics and survival information were retrospectively reviewed. RESULTS: mtDNA levels in serum and BALF did not correlate with each other. In 27 patients with paired BALF samples obtained in a stable state and at the time of AE diagnosis, BALF-mtDNA levels were significantly increased at the time of AE. Elevated BALF-mtDNA levels were associated with inflammation or disordered pulmonary function in a stable state (n = 90), while being associated with age and BALF-neutrophils at the time of AE (n = 38). BALF-mtDNA ≥ 4234.3 copies/µL in a stable state (median survival time (MST): 42.4 vs. 79.6 months, p < 0.001) and ≥ 11,194.3 copies/µL at the time of AE (MST: 2.6 vs. 20.0 months, p = 0.03) were associated with shorter survival after BALF collection, even after adjusting for other known prognostic factors. On the other hand, BALF-nucDNA showed different trends in correlation with other clinical variables and did not show any significant association with survival time. CONCLUSIONS: Elevated BALF-mtDNA was associated with a poor prognosis in both IPF and AE-IPF. Of note, at the time of AE, it sharply distinguished survivors from non-survivors. Given the trends shown by analyses for BALF-nucDNA, the elevation of BALF-mtDNA might not simply reflect the impact of cell collapse. Further studies are required to explore the underlying mechanisms and clinical applications of BALF-mtDNA in IPF.

Droplet digital polymerase chain reaction-based quantitation of therapeutic lentiviral vector copies in transduced hematopoietic stem cells.

BACKGROUND AIMS: Gene therapy using lentiviral vectors (LVs) that harbor a functional ß-globin gene provides a curative treatment for hemoglobinopathies including beta-thalassemia and sickle cell disease. Accurate quantification of the vector copy number (VCN) and/or the proportion of transduced cells is critical to evaluate the efficacy of transduction and stability of the transgene during treatment. Moreover, commonly used techniques for LV quantification, including real-time quantitative polymerase chain reaction (PCR) or fluorescence-activated cell sorting, require either a standard curve or expression of a reporter protein for the detection of transduced cells. In the present study, we describe a digital droplet PCR (ddPCR) technique to measure the lentiviral VCN in transduced hematopoietic stem and progenitor cells (HSPCs). METHODS: After HSPCs were transduced with an LV encoding the therapeutic ß-globin (ßA-T87Q) gene, the integrated lentiviral sequence in the host genome was amplified with primers that targeted a sequence within the vector and the human RPP30 gene. The dynamic range of ddPCR was between 5 × 10-3 ng and 5 × 10-6 ng of target copy per reaction. RESULTS: We found that the ddPCR-based approach was able to estimate VCN with high sensitivity and a low standard deviation. Furthermore, ddPCR-mediated quantitation of lentiviral copy numbers in differentiated erythroblasts correlated with the level of ßA-T87Q protein detected by reverse-phase high-performance liquid chromatography. CONCLUSIONS: Taken together, the ddPCR technique has the potential to precisely detect LV copy numbers in the host genome, which can be used for VCN estimation, calculation of infectious titer and multiplicity of infection for HSPC transduction in a clinical setting.

Analysis of three characterization assays reveals ddPCR of LIN28A as the most sensitive for the detection of residual pluripotent stem cells in cellular therapy products.

BACKGROUND AIMS: With the continuous development and advancement of human pluripotent stem cell (PSC)-derived cell therapies, an ever-increasing number of clinical indications can benefit from their application. Due to the capacity for PSCs to form teratomas, safety testing is required to ensure the absence of residual PSCs in a cell product. To mitigate these limitations, in vitro analytical methods can be utilized as quality control after the production of a PSC-derived cell product. Sensitivity of these analytic methods is critical in accurately quantifying residual PSC in the final cell product. In this study, we compared the sensitivity of three in vitro assays: qPCR, ddPCR and RT-LAMP. METHODS: The spike-in samples were produced from three independent experiments, each spiked with different PSC lines (PSC1, NH50191, and WA09 referred to as H9) into a background of primary fibroblasts (Hs68). These samples were then subjected to qPCR, ddPCR and RT-LAMP to determine their detection limit in measuring a commonly used PSC marker, LIN28A. RESULTS: The results indicated that the three analytic methods all exhibited consistent results across different cell-line spiked samples, with ddPCR demonstrating the highest sensitivity of the three methods. The LIN28A ddPCR assay could confidently detect 10 residual PSCs in a million fibroblasts. DISCUSSION: In our hand, ddPCR LIN28A assay demonstrated the highest sensitivity for detection of residual PSCs compared to the other two assays. Correlating such in vitro safety results with corresponding in vivo studies demonstrating the tumorigenicity profile of PSC-derived cell therapy could accelerate the safe clinical translation of cell therapy.

Validation of one-step reverse transcription digital PCR assays for Norovirus GI.

Regular monitoring of Norovirus presence in environmental and food samples is crucial due to its high transmission rates and outbreak potential. For detecting Norovirus GI, reverse transcription qPCR method is commonly used, but its sensitivity can be affected by assay performance. This study shows significantly reduced assay performance in digital PCR or qPCR when using primers targeting Norovirus GI genome 5291-5319 (NC_001959), located on the hairpin of the predicted RNA structure. It is highly recommended to avoid this region in commercial kit development or diagnosis to minimizing potential risk of false negatives.

Revealing parental mosaicism: the hidden answer to the recurrence of apparent de novo variants.

Mosaicism refers to the presence of two or more populations of genetically distinct cells within an individual, all of which originate from a single zygote. Previous literature estimated the percentage of parental mosaicism ranged from 0.33 to 25.9%. In this study, parents whose children had previously been diagnosed with developmental disorders with an apparent de novo variant were recruited. Peripheral blood, buccal and semen samples were collected from these parents if available for the detection of potential parental mosaicism using droplet digital PCR, complemented with the method of blocker displacement amplification. Among the 20 families being analyzed, we report four families with parental mosaicism (4/20, 20%). Two families have maternal gonosomal mosaicism (EYA1 and EBF3) and one family has paternal gonadal mosaicism (CHD7) with a pathogenic/ likely pathogenic variant. One family has a paternal gonosomal mosaicism with a variant of uncertain significance (FLNC) with high clinical relevance. The detectable variant allele frequency in our cohort ranged from 8.7-35.9%, limit of detection 0.08-0.16% based on our in-house EBF3 assay. Detecting parental mosaicism not only informs family with a more accurate recurrence risk, but also facilitates medical teams to create appropriate plans for pregnancy and delivery, offering the most suitable care.

Usefulness of KIT mutant transcript levels for monitoring measurable residual disease in t (8;21) acute myeloid leukemia.

In addition to RUNX1::RUNX1T1 transcript levels, measurable residual disease monitoring using KIT mutant (KITmut ) DNA level is reportedly predictive of relapse in t (8; 21) acute myeloid leukemia (AML). However, the usefulness of KITmut transcript levels remains unknown. A total of 202 bone marrow samples collected at diagnosis and during treatment from 52 t (8; 21) AML patients with KITmut (D816V/H/Y or N822K) were tested for KITmut transcript levels using digital polymerase chain reaction. The individual optimal cutoff values of KITmut were identified by performing receiver operating characteristics curve analysis for relapse at each of the following time points: at diagnosis, after achieving complete remission (CR), and after Course 1 and 2 consolidations. The cutoff values were used to divide the patients into the KITmut -high (KIT_H) group and the KITmut -low (KIT_L) group. The KIT_H patients showed significantly lower relapse-free survival (RFS) and overall survival (OS) rates than the KIT_L patients after Course 1 consolidation (p = 0.0040 and 0.021, respectively) and Course 2 consolidation (p = 0.018 and 0.011, respectively) but not at diagnosis and CR. The <3-log reduction in the RUNX1::RUNX1T1 transcript levels after Course 2 consolidation was an independent adverse prognostic factor for RFS and OS. After Course 2 consolidation, the KIT_H patients with >3-log reduction in the RUNX1::RUNX1T1 transcript levels (11/45; 24.4%) had similar RFS as that of patients with <3-log reduction in the RUNX1::RUNX1T1 transcript levels. The combination of KITmut and RUNX1::RUNX1T1 transcript levels after Course 2 consolidation may improve risk stratification in t (8; 21) AML patient with KIT mutation.

Detection and quantification of JAK2V617F copy number by droplet digital PCR versus real-time PCR.

Myeloproliferative neoplasm (MPN) usually has an adverse prognosis, progressing to acute leukemia or splanchnic vein thromboses (SVTs). Therefore, early diagnosis and intervention are significantly important. Clinically, the burden of JAK2V617F is a vital diagnostic basis, which can be detected during the early stage of MPN. Thus, an accurate and rapid detective technique is urgently required. In recent years, real-time quantitative PCR (qPCR) has primarily been applied to detect the copies of JAK2V617F, whereas droplet digital PCR (ddPCR), a novel and promising detective tool, can conduct precise and repeatable quantification of nucleic acid copies without relying on the standard curve. In our study, both qPCR and ddPCR are used to evaluate the mutation burden of JAK2V617F in a series of gradient diluted standards and clinical JAK2V617F-positive MPN patients' bone marrow samples collected, while using next-generation sequencing technology (NGS) as a contrast. With the help of statistical methods, our study concluded that ddPCR had a better performance in accuracy, sensitivity, and stability, especially in a low burden. Regarding the accuracy, ddPCR showed a better linearity (Pearson R2 = 0.9926; P < 0.0001) than qPCR (Pearson R2 = 0.9772; P < 0.0001). What is more, ddPCR showed lower intra-assay and inter-assay CVs and the limit of detection (LOD) for the series of diluted standards than qPCR, demonstrating better stability and lower LOD. In a nutshell, ddPCR is a more promising technique for the detection and quantification of JAK2V617F.