Noninvasive Prenatal Testing of Methylmalonic Acidemia cblC Type Using the cSMART Assay for MMACHC Gene Mutations

Noninvasive prenatal testing (NIPT) for monogenic disorders has been developed in recent years; however, there are still significant technical and analytical challenges for clinical use. The clinical feasibility of NIPT for methylmalonic acidemia cblC type (cblC type MMA) was investigated using our circulating single-molecule amplification and re-sequencing technology (cSMART). Trios molecular diagnosis was performed in 29 cblC type MMA-affected children and their parents by traditional Sanger sequencing. In the second pregnancy, invasive prenatal diagnosis (IPD) of the pathogenic MMACHC gene was used to determine fetal genotypes, and NIPT was performed using a novel MMACHC gene–specific cSMART assay. Maternal–fetal genotypes were deduced based on the mutation ratio in maternal plasma DNA. Concordance of fetal genotypes between IPD and NIPT, and the sensitivity and specificity of NIPT were determined. After removing two cases with a low P value or reads, the concordance ratio for NIPT and IPD was 100.00% (27/27), and the sensitivity and specificity were 100.00% (54.07–100.00%) and 100.00% (83.89–100.00%), respectively. This study demonstrates that NIPT using the cSMART assay for cblC type MMA was accurate in detecting fetal genotypes. cSMART has a potential clinical application as a prenatal diagnosis and screening tool for carrier and low-risk genotypes of cblC type MMA and other monogenic diseases.

Single-molecule sequencing reveals a large population of long cell-free DNA molecules in maternal plasma

In the field of circulating cell-free DNA, most of the studies have focused on short DNA molecules (e.g., <500 bp). The existence of long cell-free DNA molecules has been poorly explored. In this study, we demonstrated that single-molecule real-time sequencing allowed us to detect and analyze a substantial proportion of long DNA molecules from both fetal and maternal sources in maternal plasma. Such molecules were beyond the size detection limits of short-read sequencing technologies. The proportions of long cell-free DNA molecules in maternal plasma over 500 bp were 15.5%, 19.8%, and 32.3% for the first, second, and third trimesters, respectively. The longest fetal-derived plasma DNA molecule observed was 23,635 bp. Long plasma DNA molecules demonstrated predominance of A or G 5′ fragment ends. Pregnancies with preeclampsia demonstrated a reduction in long maternal plasma DNA molecules, reduced frequencies for selected 5′ 4-mer end motifs ending with G or A, and increased frequencies for selected motifs ending with T or C. Finally, we have developed an approach that employs the analysis of methylation patterns of the series of CpG sites on a long DNA molecule for determining its tissue origin. This approach achieved an area under the curve of 0.88 in differentiating between fetal and maternal plasma DNA molecules, enabling the determination of maternal inheritance and recombination events in the fetal genome. This work opens up potential clinical utilities of long cell-free DNA analysis in maternal plasma including noninvasive prenatal testing of monogenic diseases and detection/monitoring of pregnancy-associated disorders such as preeclampsia.

Early identification of disease progression in ALK-rearranged lung cancer using circulating tumor DNA analysis

Targeted kinase inhibitors improve the prognosis of lung cancer patients with ALK alterations (ALK+). However, due to the emergence of acquired resistance and varied clinical trajectories, early detection of disease progression is warranted to guide patient management and therapy decisions. We utilized 343 longitudinal plasma DNA samples from 43 ALK+ NSCLC patients receiving ALK-directed therapies to determine molecular progression based on matched panel-based targeted next-generation sequencing (tNGS), and shallow whole-genome sequencing (sWGS). ALK-related alterations were detected in 22 out of 43 (51%) patients. Among 343 longitudinal plasma samples analyzed, 174 (51%) were ctDNA-positive. ALK variant and fusion kinetics generally reflected the disease course. Evidence for early molecular progression was observed in 19 patients (44%). Detection of ctDNA at therapy baseline indicated shorter times to progression compared to cases without mutations at baseline. In patients who succumbed to the disease, ctDNA levels were highly elevated towards the end of life. Our results demonstrate the potential utility of these NGS assays in the clinical management of ALK+ NSCLC.

Clinical application of non-invasive prenatal diagnosis of phenylketonuria based on haplotypes via paired-end molecular tags and weighting algorithm

Background Phenylketonuria (PKU) is a metabolic disease that can cause severe and irreversible brain damage without treatment. Methods Here we developed a non-invasive prenatal diagnosis (NIPD) technique based on haplotypes via paired-end molecular tags and weighting algorithm and applied it to the NIPD of PKU to evaluate its accuracy and feasibility in the early pregnancy. A custom-designed hybridization probes containing regions in phenylalanine hydroxylase (PAH) gene and its 1 Mb flanking region were used for target sequencing on genomic and maternal plasma DNA (7–13 weeks of gestation) to construct the parental haplotypes and the proband’s haplotype. Fetal haplotype was then inferred combined with the parental haplotypes and the proband’s haplotype. The presence of haplotypes linked to both the maternal and paternal mutant alleles indicated affected fetuses. The fetal genotypes were further validated by invasive prenatal diagnosis in a blinded fashion. Results This technique has been successfully applied in twenty-one cases. Six fetuses were diagnosed as patients carrying both of the mutated haplotypes inherited from their parents. Eleven fetuses were carriers of one heterozygous PAH variants, six of which were paternal and five of which were maternal. Four fetuses were absence of pathogenic alleles. All results were consistent with the prenatal diagnosis through amniotic fluid. Conclusions The results showed that our new technique applied to the genotyping of fetuses with high risk for PKU achieves an accurate detection at an early stage of pregnancy with low fetal fraction in cell free DNA.

Clinical Utility of Circulating Tumor DNA in Advanced Rare Cancers

PurposePatients with advanced/relapsed rare cancers have few treatment options. Analysis of circulating tumor DNA in plasma may identify actionable genomic biomarkers using a non-invasive approach.Patients and MethodsRare cancer patients underwent prospective plasma-based NGS testing. Tissue NGS to test concordance was also conducted. Plasma DNA alterations were assessed for incidence, functional impact, therapeutic implications, correlation to survival, and comparison with tissue NGS.ResultsNinety-eight patients were analyzed. Diseases included soft-tissue sarcoma, ovarian carcinoma, and others. Mean turn-around-time for results was 9.5 days. Seventy-six patients had detectable gene alterations in plasma, with a median of 2.8 alterations/patient. Sixty patients had a likely pathogenic alteration. Five received matched-therapy based on plasma NGS results. Two developed known resistance mutations while on targeted therapy. Patients with an alteration having VAF ≥5% had a significantly shorter survival compared to those of lower VAF. Tissue NGS results from eleven of 22 patients showed complete or partial concordance with plasma NGS.ConclusionPlasma NGS testing is less invasive and capable of identifying alterations in advanced rare cancers in a clinically meaningful timeframe. It should be further studied as a prospective enrollment assay in interventional studies for patients with rare advanced stage cancers.Clinical Registration[https://www.umin.ac.jp/ctr/index-j.htm], identifier UMIN000034394.

Association of plasma DNA integrity and long fragment ALU247 in the diagnosis of epithelial ovarian cancer

DNA Integrity index (DNA Int) and cell-free DNA (cf-DNA) represent promising biomarkers for epithelial ovarian cancer (EOC) detection. Tumor necrosis produces DNA fractions of different sizes, which contrasts apoptosis in normal tissue that releases smaller and more regular DNA fragments. Using ALU gene primers in quantitative PCR, the amplified cf-DNA is supposed to be either short fragments of 115 bp (ALU 115) or long fragments of 247 bp (ALU 247). ALU levels and DNA Int were determined in the plasma of 30 EOC patients, 30 benign cysts, and 15 healthy individuals. The mean values of DNA Int, ALU115, and ALU247 were elevated in malignant patients (0.51±0.09, 3.93 ng/ul ±1.93, 2.35 ng/ul ±1.1) respectively in comparison to healthy females (0.37±0.05; p < 0.001, 2.56 ng/ul ±0.9; p=0.027, 1.26±0.44; p< 0.01). A significant increase was shown in the mean values of DNA Int and ALU247 of EOC patients compared to those with benign cysts (0.4±0.06, p <0.001; 1.69±0.66, p =0.008) respectively. The area under the curve (AUC) for EOC versus healthy females achieved 0.913 (DNA Int), 0.696 (ALU115), and 0.809 (ALU247) with sensitivities and specificities were (86.7% and 93.3%) for DNA Int, (63.3% and 86.7%) for ALU115 and (76.7% and 86.7%) for ALU247 respectively. Furthermore, comparing patients with EOC versus those with benign cysts gave AUC of 0.834 (DNA Int), 0.564 (ALU115), and 0.681 (ALU247) with sensitivities and specificities were (80% and 80%) for DNA Int, (63.3% and 60%) for ALU115 and (60% and 80%) for ALU247 respectively. Higher DNA Int and plasma ALU247 could help in the assessment of EOC, and their measurements seem to have clinical value in diagnosis.

Changes in the concentration of EGFR-mutated plasma DNA in the first hours of anti-EGFR therapy allow the prediction of tumor response in patients with EGFR-driven lung cancer

This study aimed to analyze changes in the plasma concentration of EGFR-mutated DNA occurring immediately after the start of therapy with EGFR tyrosine kinase inhibitors (TKIs). The study included 30 patients with EGFR mutation-driven non-small cell lung cancer (NSCLC). Serial plasma samples were collected before intake of the first tablet and at 0.5, 1, 2, 3, 6, 12, 24, 36 and 48 hours after the start of the therapy. EGFR-mutated plasma DNA (EGFR+ ctDNA) was detectable at diagnosis in 25 out of 30 study participants. There were different patterns of changes of the amount of circulating tumor DNA, i.e., the consistent decline of ctDNA content, or continuing increase of the number of circulating EGFR mutant copies, or alternating spikes and drops in the ctDNA concentration. Correlation with the disease outcome was observed only for the measurement performed at 48 hours. Twelve (50%) out of 24 informative patients showed >25% reduction of the ctDNA content at 48 h time point; all these patients demonstrated disease control after 4 and 8-12 weeks of therapy. The remaining 12 individuals showed either stable content of circulating EGFR+ DNA (n = 5) or the elevation of ctDNA concentration (n = 7). 10 of 12 patients with elevated or stable ctDNA level achieved an objective response at 4 weeks, but only 5 of 10 evaluable patients still demonstrated disease control at 8-12 weeks (p = 0.014, when compared to the group with ctDNA decrease). The decline of the amount of circulating EGFR mutant copies also correlated with longer progression-free survival (PFS; 14.7 months vs. 8.5 months, p = 0.013). Conclusion: Monitoring of plasma EGFR-M+ concentration within the first hours of the TKI therapy may be used as an immediate predictor of tumor response to the treatment.

Concentration of Circulating Cell-Free DNA in the Peripheral Blood Plasma of Patients with Acute Endogenous and Exogenous Etiology Psychoses

Introduction: cell-free plasma DNA (cfDNA) is used as a marker refl ecting the level of apoptosis in the human body under stress. Acute psychosis caused by endogenous (schizophrenia) and exogenous (alcohol intoxication) factors in the patient’s body is associated with oxidative stress. Presumably, cfDNA concentration in the blood plasma of patients with acute psychoses of endogenous and exogenous etiology is increased. The purpose of the study: comparative analysis of the cfDNA concentration in the blood plasma of treated and untreated patients with paranoid schizophrenia during the disease exacerbation, patients with alcoholic psychosis and healthy volunteers. Patients and methods: the concentration of cfDNA was determined in the blood plasma samples of 476 people: control group (n = 95); patients with schizophrenia in the acute stage of the disease (n = 334); patients with alcoholic psychosis (n = 47). Results: the concentrations of cfDNA in the plasma of patients with schizophrenia (median 931 ng/ml) is 2.2 times higher than in the control group (median 428 ng/ml) and 1.8 times higher than in the patients with alcoholic psychosis (504 ng/ml). For the patients with schizophrenia with high PANSS, we found the highest values of the cfDNA concentration in the blood plasma during psychosis, which indicates a more pronounced systemic process, which is accompanied by the cell death level increase. Conclusions: the concentration of cfDNA in the blood plasma could be used as a biochemical marker that refl ects the severity of the schizophrenia patient’ state upon admission to the hospital.

Noninvasive prenatal diagnosis of monogenic disorders based on direct haplotype phasing through targeted linked-read sequencing

Background Though massively parallel sequencing has been widely applied to noninvasive prenatal screen for common trisomy, the clinical use of massively parallel sequencing to noninvasive prenatal diagnose monogenic disorders is limited. This study was to develop a method for directly determining paternal haplotypes for noninvasive prenatal diagnosis of monogenic disorders without requiring proband’s samples. Methods The study recruited 40 families at high risk for autosomal recessive diseases. The targeted linked-read sequencing was performed on high molecular weight (HMW) DNA of parents using customized probes designed to capture targeted genes and single-nucleotide polymorphisms (SNPs) distributed within 1Mb flanking region of targeted genes. Plasma DNA from pregnant mothers also underwent targeted sequencing using the same probes to determine fetal haplotypes according to parental haplotypes. The results were further confirmed by invasive prenatal diagnosis. Results Seventy-eight parental haplotypes of targeted gene were successfully determined by targeted linked-read sequencing. The predicted fetal inheritance of variant was correctly deduced in 38 families in which the variants had been confirmed by invasive prenatal diagnosis. Two families were determined to be no-call. Conclusions Targeted linked-read sequencing method demonstrated to be an effective means to phase personal haplotype for noninvasive prenatal diagnosis of monogenic disorders.