Electronic DNA Analysis of CSF Cell-free Tumor DNA to Quantify Multi-gene Molecular Response in Pediatric High-grade Glioma.

PURPOSE: Pediatric high-grade glioma (pHGG) diagnosis portends poor prognosis and therapeutic monitoring remains difficult. Tumors release cell-free tumor DNA (cf-tDNA) into cerebrospinal fluid (CSF), allowing for potential detection of tumor-associated mutations by CSF sampling. We hypothesized that direct, electronic analysis of cf-tDNA with a handheld platform (Oxford Nanopore MinION) could quantify patient-specific CSF cf-tDNA variant allele fraction (VAF) with improved speed and limit of detection compared with established methods. EXPERIMENTAL DESIGN: We performed ultra-short fragment (100-200 bp) PCR amplification of cf-tDNA for clinically actionable alterations in CSF and tumor samples from patients with pHGG (n = 12) alongside nontumor CSF (n = 6). PCR products underwent rapid amplicon-based sequencing by Oxford Nanopore Technology (Nanopore) with quantification of VAF. Additional comparison to next-generation sequencing (NGS) and droplet digital PCR (ddPCR) was performed. RESULTS: Nanopore demonstrated 85% sensitivity and 100% specificity in CSF samples (n = 127 replicates) with 0.1 femtomole DNA limit of detection and 12-hour results, all of which compared favorably with NGS. Multiplexed analysis provided concurrent analysis of H3.3A (H3F3A) and H3C2 (HIST1H3B) mutations in a nonbiopsied patient and results were confirmed by ddPCR. Serial CSF cf-tDNA sequencing by Nanopore demonstrated correlation of radiological response on a clinical trial, with one patient showing dramatic multi-gene molecular response that predicted long-term clinical response. CONCLUSIONS: Nanopore sequencing of ultra-short pHGG CSF cf-tDNA fragments is feasible, efficient, and sensitive with low-input samples thus overcoming many of the barriers restricting wider use of CSF cf-tDNA diagnosis and monitoring in this patient population.

CERNA SEARCH METHOD IDENTIFIED A MET-ACTIVATED SUBGROUP AMONG EGFR DNA AMPLIFIED LUNG ADENOCARCINOMA PATIENTS.

Given the diverse molecular pathways involved in tumorigenesis, identifying subgroups among cancer patients is crucial in precision medicine. While most targeted therapies rely on DNA mutation status in tumors, responses to such therapies vary due to the many molecular processes involved in propagating DNA changes to proteins (which constitute the usual drug targets). Though RNA expressions have been extensively used to categorize tumors, identifying clinically important subgroups remains challenging given the difficulty of discerning subgroups within all possible RNA-RNA networks. It is thus essential to incorporate multiple types of data. Recently, RNA was found to regulate other RNA through a common microRNA (miR). These regulating and regulated RNAs are referred to as competing endogenous RNAs (ceRNAs). However, global correlations between mRNA and miR expressions across all samples have not reliably yielded ceRNAs. In this study, we developed a ceRNA-based method to identify subgroups of cancer patients combining DNA copy number variation, mRNA expression, and microRNA (miR) expression data with biological knowledge. Clinical data is used to validate identified subgroups and ceRNAs. Since ceRNAs are causal, ceRNA-based subgroups may present clinical relevance. Using lung adenocarcinoma data from The Cancer Genome Atlas (TCGA) as an example, we focused on EGFR amplification status, since a targeted therapy for EGFR exists. We hypothesized that global correlations between mRNA and miR expressions across all patients would not reveal important subgroups and that clustering of potential ceRNAs might define molecular pathway-relevant subgroups. Using experimentally validated miR-target pairs, we identified EGFR and MET as potential ceRNAs for miR-133b in lung adenocarcinoma. The EGFR-MET up and miR-133b down subgroup showed a higher death rate than the EGFR-MET down and miR-133b up subgroup. Although transactivation between MET and EGFR has been identified previously, our result is the first to propose ceRNA as one of its underlying mechanisms. Furthermore, since MET amplification was seen in the case of resistance to EGFR-targeted therapy, the EGFR-MET up and miR-133b down subgroup may fall into the drug non-response group and thus preclude EGFR target therapy.