TY - JOUR AU - Zaytseva, Margarita AU - Usman, Natalia AU - Salnikova, Ekaterina AU - Sanakoeva, Agunda AU - Valiakhmetova, Andge AU - Chervova, Almira AU - Papusha, Ludmila AU - Novichkova, Galina AU - Druy, Alexander PY - 2022 M3 - Original Research TI - Methodological Challenges of Digital PCR Detection of the Histone H3 K27M Somatic Variant in Cerebrospinal Fluid JO - Pathology and Oncology Research UR - https://www.por-journal.com/articles/10.3389/pore.2022.1610024 VL - 28 SN - 1532-2807 N2 - Cell-free DNA (cfDNA) in body fluids is invaluable for cancer diagnostics. Despite the impressive potential of liquid biopsies for the diagnostics of central nervous system (CNS) tumors, a number of challenges prevent introducing this approach into routine laboratory practice. In this study, we adopt a protocol for sensitive detection of the H3 K27M somatic variant in cerebrospinal fluid (CSF) by using digital polymerase chain reaction (dPCR). Optimization of the protocol was carried out stepwise, including preamplification of the H3 target region and adjustment of dPCR conditions. The optimized protocol allowed detection of the mutant allele starting from DNA quantities as low as 9 picograms. Analytical specificity was tested using a representative group of tumor tissue samples with known H3 K27M status, and no false-positive cases were detected. The protocol was applied to a series of CSF samples collected from patients with CNS tumors (n = 18) using two alternative dPCR platforms, QX200 Droplet Digital PCR system (Bio-Rad) and QIAcuity Digital PCR System (Qiagen). In three out of four CSF specimens collected from patients with H3 K27M-positive diffuse midline glioma, both platforms allowed detection of the mutant allele. The use of ventricular access for CSF collection appears preferential, as lumbar CSF samples may produce ambiguous results. All CSF samples collected from patients with H3 wild-type tumors were qualified as H3 K27M-negative. High agreement of the quantitative data obtained with the two platforms demonstrates universality of the approach. ER -