Diffuse intrinsic pontine glioma (DIPG) is a lethal childhood brain cancer and patients face a grim prognosis with few treatment options [7]. Targeted therapies based on actionable genetic mutations may offer DIPG patients novel treatment regimens [9, 10]. Although whole exome sequencing (WES) of tumor tissue can fully characterize the somatic mutational profile, it requires a surgical procedure and is relatively costly and time consuming. Consequently, less invasive and more rapid diagnostic tests are needed to detect actionable brain cancer mutations.

Brain tumors and metastases to the brain shed circulating tumor DNA (ctDNA) into the cerebrospinal fluid (CSF), which can be leveraged for the detection of tumor-associated genetic mutations from minimally invasive lumbar punctures [16]. Droplet digital PCR (ddPCR) is an ultrasensitive PCR method that can detect low copy numbers of DNA, including ctDNA, in CSF [13]. It has proven adept for the detection of ctDNA mutations in CSF from patients with primary brain tumors [3, 5, 14] and central nervous system (CNS) metastases from other cancers [3, 8, 12, 14, 15, 17]. The majority of DIPGs possess a recurrent, potentially actionable mutation to histone 3 (either H3F3A or HIST1H3B) at lysine position 27 (K27M). H3K27M detection in CSF by a combination of nested PCR and Sanger sequencing in DIPG patients [6] as well as by ddPCR in older diffuse midline glioma patients has been reported [11]. Thus far, there have been no extensive studies using ddPCR to quantify ctDNA in the CSF of