Standardization of the liquid biopsy for pediatric diffuse midline glioma using ddPCR

Abstract

Diffuse midline glioma (DMG) is a highly morbid pediatric brain tumor. Up to 80% of DMGs harbor mutations in histone H3-encoding genes, associated with poor prognosis. We previously showed the feasibility of detecting H3 mutations in circulating tumor DNA (ctDNA) in the liquid biome of children diagnosed with DMG. However, detection of low levels of ctDNA is highly dependent on platform sensitivity and sample type. To address this, we optimized ctDNA detection sensitivity and specificity across two commonly used digital droplet PCR (ddPCR) platforms (RainDance and BioRad), and validated methods for detecting H3F3A c.83A > T (H3.3K27M) mutations in DMG CSF, plasma, and primary tumor specimens across three different institutions. DNA was extracted from H3.3K27M mutant and H3 wildtype (H3WT) specimens, including H3.3K27M tumor tissue (n = 4), CSF (n = 6), plasma (n = 4), and human primary pediatric glioma cells (H3.3K27M, n = 2; H3WT, n = 1). ctDNA detection was enhanced via PCR pre-amplification and use of distinct custom primers and fluorescent LNA probes for c.83 A > T H3F3A mutation detection. Mutation allelic frequency (MAF) was determined and validated through parallel analysis of matched H3.3K27M tissue specimens (n = 3). We determined technical nuances between ddPCR instruments, and optimized sample preparation and sequencing protocols for H3.3K27M mutation detection and quantification. We observed 100% sensitivity and specificity for mutation detection in matched DMG tissue and CSF across assays, platforms and institutions. ctDNA is reliably and reproducibly detected in the liquid biome using ddPCR, representing a clinically feasible, reproducible, and minimally invasive approach for DMG diagnosis, molecular subtyping and therapeutic monitoring.

Methodological Challenges of Digital PCR Detection of the Histone H3 K27M Somatic Variant in Cerebrospinal Fluid

“…In addition to the difficulties in CSF sample collection and primary handling, their advanced molecular analysis is sophisticated. Modern strategies for analysis of somatic mutations in cfDNA are based on the use of targeted high-throughput sequencing [ 12 - 14 , 19 - 21 ] or dPCR [ 14 , 16 , 21 , 23 , 25 ]. Apart from being an established method for detecting extremely low allelic loads, dPCR provides the unique ability to quantify target DNA without external standards.…”

“…Apart from being an established method for detecting extremely low allelic loads, dPCR provides the unique ability to quantify target DNA without external standards. A number of technological platforms for dPCR are available, each of which has been comprehensively characterized on its own, whereas studies involving their comparison are scarce [ 24 , 25 ]. Here we demonstrate comparability for the highly competitive Droplet Digital™ PCR platform (Bio-Rad) and the relatively novel QIAcuity Digital PCR System (Qiagen).…”

Cell-Free Tumor DNA (cf-tDNA) Liquid Biopsy: Current Methods and Use in Brain Tumor Immunotherapy

“…By dividing a typical qPCR reaction into many isolated droplets with ~1 template copy, a precise VAF can be computed from the ratio of mutant positive to wildtype droplets, with a VAF limit of detection around 0.001% ( 43 ). ddPCR has supplanted itself as a highly-accurate technique, and is considered a gold standard approach to quantify VAFs from liquid samples ( 36 , 44 ). However, accurate and reproducible ddPCR assays require careful development and optimization of input template concentration, target-specific primers, and fluorescent probes and are restricted for use on a limited set of known hotspot mutations ( 44 ).…”

Current challenges in metastasis research and future innovation for clinical translation

“…These include mutant adenomatous polyposis coli ( APC ), epidermal growth factor receptor ( EGFR ) and Kirsten rat sarcoma virus ( KRAS ) DNA in the plasma of colorectal cancer patients as indicators of response to conventional and EGFR-targeted therapies, respectively, which are being developed for clinical use [ 34 , 35 ]. More recently, digital drop PCR pre-amplification and fluorescent probes have been developed into a promising standardized assay that detects ctDNA derived from mutant histone H3-genes in pediatric diffuse midline glioma patients, a tumor type that is generally not surgically accessible [ 36 ]. While these highly specific single-gene approaches are valuable for monitoring targeted therapy response, a panel of multiple ctDNA gene targets will be required to capture the burden of DTCs derived from diverse tumor types and highly clonal, genetically heterogeneous tumors.…”