Rhabdoid tumors are among the most aggressive malignancies seen in very young children, arising most often in the central nervous system as atypical teratoid/rhabdoid tumor, in the kidney as malignant rhabdoid tumor or rhabdoid tumor of the kidney, and at extra-renal sites. These tumors are characterized predominantly by homozygous inactivation of the SMARCB1 tumor suppressor gene, and their diagnosis and classification have long depended on comprehensive epigenomic profiling of tumor tissue.
A new study published in Acta Neuropathologica Communications reports an enzymatic approach to that profiling challenge. Researchers led by David N. Buckley, Laura A. T. Kagami and Jaclyn A. Biegel applied enzymatic conversion followed by whole genome sequencing, known as EM-Seq, to a cohort of rhabdoid tumors and to liquid biopsy samples, with the goal of unifying methylation-based classification and copy number analysis on a single platform.
The cohort comprised 24 central nervous system rhabdoid tumors, 14 renal tumors and 13 extrarenal tumors, drawn from patients with and without germline SMARCB1 predisposing mutations or copy number alterations. The researchers also analyzed five cerebrospinal fluid cell-free DNA samples from patients with atypical teratoid/rhabdoid tumor, extending the method to a minimally invasive sample type.
Previous epigenomic studies of these tumors relied on bisulfite conversion and array hybridization, and that work had already demonstrated distinct methylation subgroups, especially within atypical teratoid/rhabdoid tumor. Those subgroups have helped define the cellular origin and transcriptional programs of rhabdoid tumors arising from different anatomic sites. The new enzymatic method was tested against that established framework.
In the study cohort, global methylation patterns generated by EM-Seq confirmed the presence of the five previously described subgroups: ATRT-TYR, ATRT-SHH, ATRT-MYC, RTK-like and extra-renal-like. This agreement indicates that enzymatic conversion can reproduce the methylation-based classification that has become central to how these tumors are grouped and understood.
Beyond classification, the researchers leveraged EM-Seq to characterize genome-wide copy number alterations and copy-neutral loss of heterozygosity across the tumor genomes. The method also resolved exon-level copy number gains and losses within SMARCB1 itself, the gene whose loss defines the entity, providing a depth of resolution that integrates methylation profiling with detailed genomic analysis from the same sequencing run.
A key motivation for the work is a practical bottleneck in existing workflows. The authors note that integrating genome-wide methylation and high-resolution copy number analysis from samples with low DNA input remains challenging. That capability is expected to become essential in prospective studies with targeted treatment protocols, which increasingly use cell-free DNA from cerebrospinal fluid liquid biopsies to diagnose patients and monitor response to therapy.
The study demonstrates the feasibility of extending the EM-Seq approach to cerebrospinal fluid cell-free DNA, highlighting what the authors describe as clinical potential for minimally invasive tumor classification and molecularly informed surveillance. In principle, such surveillance could inform precision medicine for this aggressive entity, allowing molecular features of the tumor to be tracked without repeated tissue sampling.
The authors position EM-Seq as a unified platform for integrated methylation-based classification and copy number profiling of rhabdoid tumors, with applicability to both tissue and liquid biopsy samples. Because the report is being shared early as a peer-reviewed, accepted manuscript that is citable and carries a permanent DOI, the findings are available ahead of the final version of record, and the version is subject to further editorial edits.
For clinicians and researchers caring for children with rhabdoid tumors, the reported results suggest a route to obtaining methylation subgroup assignment, genome-wide copy number data and SMARCB1 exon-level detail from limited tissue, and potentially from cerebrospinal fluid, within a single sequencing workflow. The study reports feasibility and confirmation of known subgroups in the analyzed cohort; its clinical application in prospective treatment protocols remains the work the authors identify as the setting where such low-input, integrated profiling will ultimately be required.
Subject of Research: Enzymatic methylation sequencing for classification and copy number profiling of pediatric rhabdoid tumors and cerebrospinal fluid cell-free DNA
Article Title: Enzymatic based methylation profiling of tumor tissue and circulating tumor DNA from pediatric patients with rhabdoid tumors
Article References: Buckley, D. N., Kagami, L. A. T., & Biegel, J. A. (2026). Enzymatic based methylation profiling of tumor tissue and circulating tumor DNA from pediatric patients with rhabdoid tumors. Acta Neuropathologica Communications. https://doi.org/10.1186/s40478-026-02453-1
Image Credits: AI Generated
DOI: 10.1186/s40478-026-02453-1
Keywords: rhabdoid tumor, AT/RT, SMARCB1, EM-Seq, methylation profiling, copy number, cell-free DNA, cerebrospinal fluid, liquid biopsy, pediatric oncology, epigenomics, Acta Neuropathologica Communications
