UNASSIGNED: Mutations in the GCK gene cause Maturity Onset Diabetes of the Young (GCK-MODY) by impairing glucose-sensing in pancreatic beta cells. During pregnancy, managing this type of diabetes varies based on fetal genotype. Fetuses carrying a GCK mutation can derive benefit from moderate maternal hyperglycemia, stimulating insulin secretion in fetal islets, whereas this may cause macrosomia in wild-type fetuses. Modulating maternal glycemia can thus be viewed as a form of personalized prenatal therapy, highly beneficial but not justifying the risk of invasive testing. We therefore developed a monogenic non-invasive prenatal diagnostic (NIPD-M) test to reliably detect the transmission of a known maternal GCK mutation to the fetus.
UNASSIGNED: A small amount of fetal circulating cell-free DNA is present in maternal plasma but cannot be distinguished from maternal cell-free DNA. Determining transmission of a maternal mutation to the fetus thus implies sequencing adjacent polymorphisms to determine the balance of maternal haplotypes, the transmitted haplotype being over-represented in maternal plasma.
UNASSIGNED: Here we present a series of such tests in which fetal genotype was successfully determined and show that it can be used to guide therapeutic decisions during pregnancy and improve the outcome for the offspring. We discuss several potential hurdles inherent to the technique, and strategies to overcome these.
UNASSIGNED: Our NIPD-M test allows reliable determination of the presence of a maternal GCK mutation in the fetus, thereby allowing personalized in utero therapy by modulating maternal glycemia, without incurring the risk of miscarriage inherent to invasive testing.

Noninvasive prenatal diagnosis relies on the presence in maternal blood of circulating cell-free fetal DNA released by apoptotic trophoblast cells. Widely used for aneuploidy screening, it can also be applied to monogenic diseases (NIPD-M) in case of known parental mutations. Due to the confounding effect of maternal DNA, detection of maternal or biparental mutations requires relative haplotype dosage (RHDO), a method relying on the presence of SNPs that are heterozygous in one parent and homozygous in the other. Unavoidably, there is a risk of test failure by lack of such informative SNPs, an event particularly likely for consanguineous couples who often share common haplotypes in regions of identity-by-descent. Here we present a novel approach, relative genotype dosage (RGDO) that bypasses this predicament by directly assessing fetal genotype with SNPs that are heterozygous in both parents (frequent in regions of identity-by-descent). We show that RGDO is as sensitive as RHDO and that it performs well over a large range of fetal fractions and DNA amounts, thereby opening NIPD-M to most consanguineous couples. We also report examples of couples, consanguineous or not, where combining RGDO and RHDO allowed a diagnosis that would not have been possible with only one approach.

While circulating cell-free DNA (cfDNA) is becoming a powerful marker for noninvasive identification of infectious pathogens in liquid biopsy specimens, a microbial cfDNA baseline in healthy individuals is urgently needed for the proper interpretation of microbial cfDNA sequencing results in clinical metagenomics. Because noninvasive prenatal testing (NIPT) shares many similarities with the sequencing protocol of metagenomics, we utilized the standard low-pass whole-genome-sequencing-based NIPT to establish a microbial cfDNA baseline in healthy people. Sequencing data from a total of 107,763 peripheral blood samples of healthy pregnant women undergoing NIPT screening were retrospectively collected and reanalyzed for microbiome DNA screening. It was found that more than 95% of exogenous cfDNA was from bacteria, 3% from eukaryotes, and 0.4% from viruses, indicating the gut/environment origins of many microorganisms. Overall and regional abundance patterns were well illustrated, with huge regional diversity and complexity, and unique interspecies and symbiotic relationships were observed for TORCH organisms (Toxoplasma gondii, others [Treponema pallidum {causing syphilis}, hepatitis B virus {HBV}, and human parvovirus B19 {HPV-B19}], rubella virus, cytomegalovirus [CMV], and herpes simplex virus [HSV]) and another common virus, Epstein-Barr virus (EBV). To sum up, our study revealed the complexity of the baseline circulating microbial cfDNA and showed that microbial cfDNA sequencing results need to be interpreted in a more comprehensive manner. IMPORTANCE While circulating cell-free DNA (cfDNA) has been becoming a powerful marker for noninvasive identification of infectious pathogens in liquid biopsy specimens, a baseline for microbial cfDNA in healthy individuals is urgently needed for the proper interpretation of microbial cfDNA sequencing results in clinical metagenomics. Standard low-pass whole-genome-sequencing-based NIPT shares many similarities with the sequencing protocol for metagenomics and could provide a microbial cfDNA baseline in healthy people; thus, a reference cfDNA data set of the human microbiome was established with sequencing data from a total of 107,763 peripheral blood samples of healthy pregnant women undergoing NIPT screening. Our study revealed the complexity of circulating microbial cfDNA and indicated that microbial cfDNA sequencing results need to be interpreted in a more comprehensive manner, especially with regard to geographic patterns and coexistence networks.

Low-grade serous ovarian cancer (LGSOC) poses a specific clinical challenge due to advanced presentation at diagnosis and the lack of effective systemic treatments. The aim of this study was to use a precision medicine approach to identify clinically actionable mutations in a patient with recurrent LGSOC. Primary, metastatic and recurrence tissue, and blood samples were collected from a stage IV LGSOC patient. Single-gene testing for clinically actionable mutations (BRAF V600, KRAS and NRAS) and subsequent whole-exome sequencing (WES) were performed. Droplet digital PCR was used to evaluate the presence of an identified BRAF D594G mutation in the matched plasma cell-free DNA (cfDNA). No clinically actionable mutations were identified using single-gene testing. WES identified a BRAF D594G mutation in six of seven tumor samples. The patient was commenced on a MEK inhibitor, trametinib, but with minimal clinical response. A newly designed ddPCR assay detected the BRAF alteration in the matched tissues and liquid biopsy cfDNA. The identification and sensitive plasma detection of a common \"druggable\" target emphasises the impact of precision medicine on the management of rare tumors and its potential contribution to novel monitoring regimens in this field.

Donor-derived cell-free DNA (dd-cfDNA) is considered a reliable marker of organ damage with potential applications in the follow-up of transplant recipients.
In this work we present an assay based on the donor-recipient HLA-mismatch (human leukocyte antigen) at the HLA-DRB1 locus to monitor rejection by quantifying the percentage of dd-cfDNA using a droplet digital PCR (polymerase chain reaction) technique. A panel of probes targeting the HLA-DRB1 locus and covering >85% genetic variability was validated and used to assess dd-cfDNA levels in a prospective cohort of 19 adult heart transplant recipients (mean age 50.9±14.8 years). The assay was carried out on a total of 232 liquid biopsies collected at the same time as endomyocardial biopsy (EMB) during routine post-transplant follow-up.
Results show a significant increase of dd-cfDNA related to ischemia-reperfusion injury (2.22±2.09%) and to acute cellular rejection (1.71±3.10%) compared to stable conditions (0.43±1.04%, p < 0.0001). On the contrary, no increase was observed during infections or vascular complications, underlining the potential role of this biomarker for rejection monitoring. With a cut-off of 0.11%, the test showed 70.8% specificity (95% CI, 58.17% - 81.40%) and 64.2% sensitivity (95% CI, 49.80% - 76.86%) in discriminating acute rejection from no rejection.
These data demonstrate that this HLA mismatch-based droplet digital PCR method is effective for monitoring rejection in heart transplant recipients. Compared to next generation sequencing approaches, it is far more flexible, less expensive and provides faster results.

Liquid biopsies enable noninvasive therapy monitoring in patients with solid tumors. Specific serum markers such as proteins, hormones, or enzymes released from tumor cells or in response to tumor growth can be used for quantification of the tumor burden. However, only a fraction of pediatric tumors has none of these serum markers, but tumor-specific genetic alterations represent reliable alternatives. Here we describe a method for using genomic fusion sequences as liquid biopsy markers in Ewing sarcoma patients.

Circulating cell-free DNA (cfDNA) consists of small fragments of DNA that circulate freely in the bloodstream. In cancer patients, a fraction of cfDNA is derived from tumour cells, therefore containing the same genetic and epigenetic alterations, and is termed circulating cell-free tumour DNA. The potential use of cfDNA, the so-called \'liquid biopsy\', as a non-invasive cancer biomarker has recently received a lot of attention. The present review will focus on studies concerning the potential clinical applications of cfDNA in ovarian cancer patients.

UNASSIGNED: Gastrointestinal stromal tumors (GISTs) are characterized by oncogenic KIT mutations that cluster in two exon 11 hotspots. The aim of this study was to develop a single, sensitive, quantitative digital droplet PCR (ddPCR) assay for the detection of common exon 11 mutations in both GIST tumor tissue and in circulating tumor DNA (ctDNA) isolated from GIST patients\' plasma.
UNASSIGNED: A ddPCR assay was designed using two probes that cover both hotspots. Available archival FFPE tumor tissue from 27 consecutive patients with known KIT exon 11 mutations and 9 randomly selected patients without exon 11 mutations were tested. Plasma samples were prospectively collected in a multicenter bio-databank from December 2014. ctDNA was analyzed of 22 patients with an exon 11 mutation and a baseline plasma sample.
UNASSIGNED: The ddPCR assay detected the exon 11 mutation in 21 of 22 tumors with exon 11 mutations covered by the assay. Mutations in ctDNA were detected at baseline in 13 of 14 metastasized patients, but in only 1 of 8 patients with localized disease. In serial plasma samples from 11 patients with metastasized GIST, a decrease in mutant droplets was detected during treatment. According to RECIST 1.1, 10 patients had radiological treatment response and one patient stable disease.
UNASSIGNED: A single ddPCR assay for the detection of multiple exon 11 mutations in ctDNA is a feasible, promising tool for monitoring treatment response in patients with metastasized GIST and should be further evaluated in a larger cohort.

This editorial article summarizes the achievements and current challenges for the Journal of Circulating Biomarkers (JCB) regarding a more strategic approach to branding and attracting a high quality variety of articles. More emphasis is placed on fostering engagement with academic and industry sources operating at the cutting-edge of translational technologies applied to the field of circulating biomarkers (interface between extracellular vesicles including exosomes and microvesicles, circulating tumour cells, cell-free circulating DNA and circulating protein markers) and with those in the investment arena seeking and providing private funding for this area of research.

While various clinical applications especially in oncology are now in progress such as diagnosis, prognosis, therapy monitoring, or patient follow-up, the determination of structural characteristics of cell-free circulating DNA (cirDNA) are still being researched. Nevertheless, some specific structures have been identified and cirDNA has been shown to be composed of many \"kinds.\" This structural description goes hand-in-hand with the mechanisms of its origins such as apoptosis, necrosis, active release, phagocytosis, and exocytose. There are multiple structural forms of cirDNA depending upon the mechanism of release: particulate structures (exosomes, microparticles, apoptotic bodies) or macromolecular structures (nucleosomes, virtosomes/proteolipidonucleic acid complexes, DNA traps, links with serum proteins or to the cell-free membrane parts). In addition, cirDNA concerns both nuclear and/or mitochondrial DNA with both species exhibiting different structural characteristics that potentially reveal different forms of biological stability or diagnostic significance. This review focuses on the origins, structures and functional aspects that are paradoxically less well described in the literature while numerous reviews are directed to the clinical application of cirDNA. Differentiation of the various structures and better knowledge of the fate of cirDNA would considerably expand the diagnostic power of cirDNA analysis especially with regard to the patient follow-up enlarging the scope of personalized medicine. A better understanding of the subsequent fate of cirDNA would also help in deciphering its functional aspects such as their capacity for either genometastasis or their pro-inflammatory and immunological effects.