Circulating Cell-Free Tumor DNA-Based Liquid Biopsy of Cerebrospinal Fluid in Retinoblastoma

Introduction: Monitoring central nervous system (CNS) involvement in retinoblastoma traditionally relies on cerebrospinal fluid (CSF) cytology and MRI imaging. This study assessed the usefulness of CSF liquid biopsy as a modality for detection of early CNS dissemination and prognostication in retinoblastoma patients. Methods: Patients with stage 3 unilateral non-germline retinoblastoma were included in this study. CSF, blood, and tumor tissue (wherever available), were collected from the participants forming two distinct groups based on sample pairing: “paired CSF and tumor, and “paired CSF and blood.” CSF collection by spinal tap was done as part of the metastatic workup protocol for all extraocular retinoblastoma patients. Cell-free DNA (cfDNA) was isolated for subsequent RB1 mutation analysis by targeted next-generation sequencing (NGS). Results: CSF cytology yielded no atypical cells in all cases. Targeted NGS data of cfDNA isolated from CSF revealed RB1 mutated cfDNA in the CSF of 6 out of 11 (54.5%) patients. All 6 patients with detectable RB1 mutations in CSF developed symptoms of CNS involvement and succumbed to the disease on subsequent follow-up. Four out of 5 (80%) patients where RB1 mutated cell-free tumor DNA was not detected in CSF, are currently alive with no evidence of CNS disease at a mean follow-up of 20 months (range 10–26 months). Conclusions: CSF liquid biopsy can serve as early diagnosis modality for minimally disseminated CNS disease. Further investigations involving larger cohorts are imperative to validate these findings robustly and ascertain the clinical utility of CSF liquid biopsy in routine practice for retinoblastoma patients.