Minimally invasive classification of pediatric solid tumors using reduced representation bisulfite sequencing of cell-free DNA: a proof-of-principle study

Abstract

In the clinical management of pediatric solid tumors, histological examination of tumor tissue obtained by a biopsy remains the gold standard to establish a conclusive pathological diagnosis. The DNA methylation pattern of a tumor is known to correlate with the histopathological diagnosis across cancer types and is showing promise in the diagnostic workup of tumor samples. This methylation pattern can be detected in the cell-free DNA.Here, we provide proof-of-concept of histopathologic classification of pediatric tumors using cell-free reduced representation bisulfite sequencing (cf-RRBS) from retrospectively collected plasma and cerebrospinal fluid samples. We determined the correct tumor type in 49 out of 60 (81.6%) samples starting from minute amounts (less than 10 ng) of cell-free DNA. We demonstrate that the majority of misclassifications were associated with sample quality and not with the extent of disease. Our approach has the potential to help tackle some of the remaining diagnostic challenges in pediatric oncology in a cost-effective and minimally invasive manner. Translational relevanceObtaining a correct diagnosis in pediatric oncology can be challenging in some tumor types, especially in renal tumors or central nervous system tumors. Furthermore, the diagnostic odyssey can result in anxiety and discomfort for these children. By applying a novel technique, reduced representation bisulfite sequencing on cell-free DNA (cf-RRBS), we show the feasibility of obtaining the histopathological diagnosis with a minimally invasive test on either plasma or cerebrospinal fluid. Furthermore, we were able to derive the copy number profile or tumor subtype from the same assay. Given that primary tumor material might be difficult to obtain, in particular in critically ill children or depending on the tumor location, and might be limited in terms of quantity or quality, our assay could become complementary to the classical tissue biopsy in difficult cases.

Genome-wide study of the effect of blood collection tubes on the cell-free DNA methylome

“…In order to evaluate changes in cell contributions to cfDNA content, we performed non-negative least squares (NNLS) on the cfDNA samples in combination with a previously published cfDNA reference atlas (generated on Illumina arrays) 3 . In order to compare cf-RRBS with Illumina HM450K or EPIC, we grouped individual CpGs into regions as described before 2 . We observed similar proportions of erythrocyte progenitors as described by Moss et al (35.80 % [28.70-41.80]).…”

“…The analysis of cell-free tumor-derived DNA (ctDNA) isolated from liquid biopsies, including blood plasma, has emerged as a complementary assay for tumor tissue genomic profiling 1,2 . The methylation pattern of cell-free DNA is currently under investigation as a biomarker for the non-invasive diagnosis of both benign 3 and malignant 4 conditions.…”