Quantification of low-level human cytomegalovirus and Epstein-Barr virus DNAemia by digital PCR.

BACKGROUND: Digital PCR (dPCR) can quantify cell-free viral DNA (DNAemia), a biomarker of active viral infection. To accelerate epidemiologic investigation into low-level viral reactivation in chronic disease, we have evaluated the performance of dPCR to detect cytomegalovirus (CMV) and Epstein-Barr virus (EBV) DNAemia across platforms and blood matrices. METHODS: The droplet-based (BioRad) dPCR platform performance was compared to chip-based (BioMark), and assay validation followed dMIQE guidelines. CMV and EBV DNA reference materials were spiked into known negative plasma and serum samples. In addition, two independent cohorts of ovarian cancer patients were evaluated for viral DNAemia (n = 65 serum and 79 plasma samples). RESULTS: The limit of quantification (LOQ) was at or slightly above 100 copies/mL for both instruments: 105-135 copies/mL for droplet-based detection and 100 copies/mL for chip-based detection. DNAemia in serum had a slightly lower LOQ (105-110 copies/mL) compared to plasma (LOQ; 115-135 copies/mL). The variation (CV) coefficients for each assay and machine were less than 5 %. In patient samples, CVs ranged from 4.5 - 7.4 % and were similar for cell-free DNA derived from serum or plasma. There was good correlation between DNAemia measurements in patient samples across dPCR platforms (r > 0.90 for each assay and matrix). CONCLUSION: dPCR can quantify low-level herpes virus DNAemia with CVs below 8 %. Our results indicate that using serum-derived cell-free DNA and droplet-based dPCR is optimal for quantitating low-level viral DNAemia; however, plasma and chip-based approaches are acceptable alternatives and suitable for epidemiologic investigation.

KMT2A-D pathogenicity, prevalence, and variation according to a population database.

INTRODUCTION: The KMT2 family of genes is essential epigenetic regulators promoting gene expression. The gene family contains three subgroups, each with two paralogues: KMT2A and KMT2B; KMT2C and KMT2D; KMT2F and KMT2G. KMT2A-D are among the most frequent somatically altered genes in several different cancer types. Somatic KMT2A rearrangements are well-characterized in infant leukemia (IL), and growing evidence supports the role of additional family members (KMT2B, KMT2C, and KMT2D) in leukemogenesis. Enrichment of rare heterozygous frameshift variants in KMT2A and C has been reported in acute myeloid leukemia (AML), IL, and solid tumors. Currently, the non-synonymous variation, prevalence, and penetrance of these four genes are unknown. METHODS: This study determined the prevalence of pathogenic/likely pathogenic (P/LP) germline KMT2A-D variants in a cancer-free adult population from the Genome Aggregation Database (gnomAD). Two methods of variant interpretation were utilized: a manual genomic variant interpretation and an automated ACMG pipeline. RESULTS: The ACMG pipeline identified considerably fewer P/LP variants (n = 89) compared to the manual method (n = 660) in all 4 genes. Consequently, the total P/LP prevalence and allele frequency (AF) were higher in the manual method (1:112, AF = 4.46E-03) than in ACMG (1:832, AF = 6.01E-04). Multiple ancestry-exclusive P/LP variants were identified along with an increased frequency in males compared to females. Many of these variants identified in this population database are also associated with severe juvenile conditions. CONCLUSION: These data demonstrate that putatively functional germline variation in these developmentally important genes is more common than previously appreciated and identification in cancer-free adults may indicate incomplete penetrance for many of these variants. Future research should examine a genetic predisposing role in IL and other pediatric cancers.