Serial assessment of measurable residual disease in medulloblastoma liquid biopsies

Abstract

Nearly one-third of children with medulloblastoma, a malignant embryonal tumor of the cerebellum, succumb to their disease. Conventional response monitoring by imaging and cerebrospinal fluid (CSF) cytology remains challenging, and a marker for measurable residual disease (MRD) is lacking. Here, we show the clinical utility of CSF-derived cell-free DNA (cfDNA) as a biomarker of MRD in serial samples collected from children with medulloblastoma (123 patients, 476 samples) enrolled on a prospective trial. Using low-coverage whole-genome sequencing, tumor-associated copy-number variations in CSF-derived cfDNA are investigated as an MRD surrogate. MRD is detected at baseline in 85% and 54% of patients with metastatic and localized disease, respectively. The number of MRD-positive patients declines with therapy, yet those with persistent MRD have significantly higher risk of progression. Importantly, MRD detection precedes radiographic progression in half who relapse. Our findings advocate for the prospective assessment of CSF-derived liquid biopsies in future trials for medulloblastoma.

Keywords: biomarkers; cell-free DNA; cerebrospinal fluid; childhood cancer; liquid biopsy; measurable residual disease; medulloblastoma; microscopic residual disease; minimal residual disease; relapse disease.

Figure 1.

Detection of MRD from serial CSF-derived cfDNA samples in children with medulloblastoma. (A) Study cohort composition with patients stratified by disease risk and sample availability, see also Table S2. (B) Outline of experimental workflow, comprising prospective, serial CSF banking, cfDNA extraction and quantification, low-coverage whole-genome sequencing, CNV calling and clinical correlation. See also Figure S1. cfDNA; cell-free DNA; CSF, cerebrospinal fluid; CNV, copy-number variation; MRD, minimal residual disease; WGS, whole-genome sequencing.

Figure 2.

Tumor-associated CNVs are detectable in CSF-derived cfDNA. Exemplary data comparing CNV profiles of primary tumor and longitudinal cfDNA samples in three patients: sj099 (disease relapse after initial response), sj048 (persistent refractory disease) and sj056 (complete response).

Figure 3.

Patient-matched comparison of CNVs from baseline cfDNA and primary tumors. (A) Genome-wide CNV profiles arranged by molecular subgroup and sample origin (low-coverage whole-genome sequencing of cfDNA vs. methylation profiling of primary tumors). (B) Heatmap and (C) histogram summarization of pairwise Pearson correlation between CNVs from baseline cfDNA and primary tumors.

Figure 4.

MRD detectability in serial CSF mirrors disease course. (A) MRD re-emergence 3-months before radiographic diagnosis of a solitary metastatic relapse in a patient with Group 3-MB (sj026). (B-C) MRD detectability and corresponding MRI and CSF cytology findings in patients with (B) relapsed or refractory disease, or (C) without progression. Evaluation time-points are only shown where data from both MRD and conventional assessments are available. (D) Number of patients with MRD detectability summarized according to protocol time-points and disease status. m, month; NED, no evidence of disease; RT, radiotherapy; tx, treatment.

Figure 5.

MRD detectability stratifies patients by risk of progression. (A-D) Progression-free survival (PFS) for patients from the longitudinal cohort stratified by MRD at baseline, post-radiotherapy (RT), mid-chemotherapy, and end-of-therapy. (E-H) PFS for patients from the longitudinal cohort stratified by MRD and clinical risk group at the respective time-points. See also Figures S3 and S4. AR, average-risk; CI, confidence interval; HR, high-risk; neg, negative; pos, positive.

Figure 6.

Serial cfDNA profiles reveal tumor evolution at progression. (A) Heatmap depicting evolution of genome-wide CNV profiles in paired early and late cfDNA samples in patients with progression. Patients are grouped by conserved (sj024-sj078) or divergent (sj026-sj099) CNV profiles. (B) Loss of chromosome 10q acquired in cfDNA samples from 6 patients at relapse. (C) Comparison of cfDNA profiles at diagnosis and progression in patient sj097 illustrating relapse-specific focal amplicons on chromosomes 6 and 18 that target PIM1 and SMAD2, respectively.

Figure 7.

Illustrative vignettes highlighting the utility of serial cfDNA profiling in children with medulloblastoma. (A) CNV profiles of bulk tumor and CSF-derived cfDNA at diagnosis/baseline and metastatic relapse in a patient with Group 4-MB (sj024). (B) Patient with SHH-MB and rapidly progressive right temporal lesion histologically diagnosed as high-grade neuroepithelial tumor with EGFR and PDGFRA amplification (sj028). cfDNA analysis at baseline retrospectively indicated presence of PDGFRA and EGFR amplification, suggesting concurrent medulloblastoma and high-grade glioma (HGG).