Rayan Aittaleb
Pronouns: he/him
Research Mentor(s): John Wadden
Research Mentor School/College/Department: Pediatric Hematology/Oncology / Medicine
Program: UROP
Session: Session 7 (4:40pm – 5:30pm)
Authors: Rayan Aittaleb, John Wadden
Abstract
Diffuse midline glioma(DMG) is an incurable brain tumor that primarily affects pediatric patients. A high proportion of patients with DMG also have one of three oncogenetic histone mutations (H3.3K27M/G34R and H3.1K27M). Recent work has shown that tumor progression can be tracked by monitoring the concentration of these variants found in circulating cell free tumor DNA (cf-tDNA) in plasma. Plasma has low concentrations of cf-tDNA (<1%) due to the blood brain barrier. Thus, high sensitivity assays–such as digital droplet polymerase chain reaction (ddPCR)-must be designed and validated to successfully monitor tumor progression. Assay protocols from recent work has several issues which hinder repeatability and reliability. We improve upon this work by modifying previously published ddPCR protocols to include both full pre-amplification, and metered template dilution before ddPCR, to improve experimental flexibility. We identified that dilution to 20,000 to 80,000 input copies resulted in ideal droplet occupancy over multiple replicates (n=3) and cf-tDNA concentrations (n=4). While prior work exclusively focused on H3.3K27M–due to its high occurrence rate in pediatric DMGs (~50-60%)--other hotspot mutations (H3.1K27M, H3.3G34R) also have high occurrence rates. No prior work has developed and validated ddPCR assays for these mutations. We developed multiple assays for H3.1K27M and H3.3G34R mutations and validated them using annealing temperature gradients and serial dilutions of patient samples. Our H3.1K27M assay was able to accurately reproduce tumor content in serially diluted patient samples and was selected for final evaluation. H3.3G34R assay design is ongoing. The H3.3K27M and H3.1K27M assays are currently being staged for validation in a Clinical Laboratory Improvement Amendments (CLIA) certified setting for translational use in clinic. Assay protocols from recent work had several issues which hindered repeatability and reliability. We improved upon this work by modifying previously published ddPCR protocols to include both full pre-amplification, and metered template dilution before ddPCR, to improve experimental flexibility. We identified that dilution to 20,000 to 80,000 input copies resulted in ideal droplet occupancy over multiple replicates (n=3) and cf-tDNA concentrations (n=4). While prior work exclusively focused on H3.3K27M–due to its high occurrence rate in pediatric DMGs (~50-60%)--other hotspot mutations (H3.1K27M and H3.3G34R) also have high occurrence rates. No prior work has developed and validated ddPCR assays for these mutations. We developed multiple assays for H3.1K27M and H3.3G34R mutations and validated them using annealing temperature gradients and serial dilutions of patient samples (four samples with 3 technical replicates). One of the H3.1K27M assay designs was able to accurately reproduce tumor content in serially diluted patient samples and was selected for final evaluation. Both H3.3G34R failed to generate acceptable results; H3.3G34R assay design is ongoing. The H3K27M assays are currently being staged for validation in a CLIA certified setting for translational use in clinics.
