Fragmentation patterns and personalized sequencing of cell‐free DNA in urine and plasma of glioma patients

Abstract

Glioma‐derived cell‐free DNA (cfDNA) is challenging to detect using liquid biopsy because quantities in body fluids are low. We determined the glioma‐derived DNA fraction in cerebrospinal fluid (CSF), plasma, and urine samples from patients using sequencing of personalized capture panels guided by analysis of matched tumor biopsies. By sequencing cfDNA across thousands of mutations, identified individually in each patient’s tumor, we detected tumor‐derived DNA in the majority of CSF (7/8), plasma (10/12), and urine samples (10/16), with a median tumor fraction of 6.4 × 10−3, 3.1 × 10−5, and 4.7 × 10−5, respectively. We identified a shift in the size distribution of tumor‐derived cfDNA fragments in these body fluids. We further analyzed cfDNA fragment sizes using whole‐genome sequencing, in urine samples from 35 glioma patients, 27 individuals with non‐malignant brain disorders, and 26 healthy individuals. cfDNA in urine of glioma patients was significantly more fragmented compared to urine from patients with non‐malignant brain disorders (P = 1.7 × 10−2) and healthy individuals (P = 5.2 × 10−9). Machine learning models integrating fragment length could differentiate urine samples from glioma patients (AUC = 0.80–0.91) suggesting possibilities for truly non‐invasive cancer detection.

Transcending Blood—Opportunities for Alternate Liquid Biopsies in Oncology

“…There is some debate as to whether similar conclusions based on size analysis can be drawn regarding non-blood cfDNA. cfDNA with a similar size profile to blood has been reported in various fluids [ 22 , 32 , 79 , 80 ], and tumour enrichment in short cfDNA fragments has been demonstrated in CSF and urine [ 81 , 82 ]. However, distinct size profiles have also been observed.…”

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

“…As glioma cells proliferate and die via apoptosis, necrosis, or immune response, tumor DNA is immediately shed into the surrounding interstitial fluid and CSF. During apoptosis, tumor chromosomal DNA is fragmented via endonucleases around nucleosome boundaries (~140bp-180bp) resulting in a characteristic pattern of fragmentation ( 24 ). cf-tDNA fragments spread throughout the central nervous system before eventually permeating the blood-brain barrier ( 25 ).…”

Recent Advances in Liquid Biopsy of Brain Cancers

“…Very recently, with the detection of somatic copy number alterations and the analysis of DNA fragmentation patterns, scientists can identify glioma patients using untargeted and low-coverage whole-genome sequencing technique, which makes diagnosis and subtyping cheaper and more time-saving (Mouliere et al, 2018). Notably, a recent study had integrated multiple cfDNA fragmentation patterns to build a machine learning classifier on urine samples, which shows promising accuracy in differentiating glioma patients from negative controls (Mouliere et al, 2021). Besides early diagnosis and subtyping, liquid biopsy assays have also been developed for disease progression monitoring and therapeutic methods assessments of brain cancer patients.…”

“…Among patients with brainstem gliomas, 97.3% of cases with detectable alterations in the primary tumors are identified in CSF and the detection rates of mutated fragments are much higher in CSF (100%) than in plasma (38%; Pan et al, 2019). Other groups also show that ctDNA is more abundant and contains more comprehensive information in CSF than that of plasma in patients with GBM, medulloblastoma, and metastatic brain cancers (De Mattos-Arruda et al, 2015;Escudero et al, 2020;Mouliere et al, 2021). The advantage of CSF may be of particular value in developing liquid biopsy assays for prognosis monitoring and cancer relapse prediction.…”