Abstract
PurposeThe enucleation rate for retinoblastoma has dropped from over 95% to under 10% in the past 10 years as a result of improvements in therapy. This reduces access to tumor tissue for molecular profiling, especially in unilateral retinoblastoma, and hinders the confirmation of somatic RB1 mutations necessary for genetic counseling. Plasma cell‐free DNA (cfDNA) has provided a platform for noninvasive molecular profiling in cancer, but its applicability in low tumor burden retinoblastoma has not been shown. We analyzed cfDNA collected from 10 patients with available tumor tissue to determine whether sufficient tumorderived cfDNA is shed in plasma from retinoblastoma tumors to enable noninvasive RB1 mutation detection.MethodsTumor tissue was collected from eye enucleations in 10 patients diagnosed with advanced intra‐ocular unilateral retinoblastoma, three of which went on to develop metastatic disease. Tumor RB1 mutation status was determined using an FDA‐cleared tumor sequencing assay, MSK‐IMPACT. Plasma samples were collected before eye enucleation and analyzed with a customized panel targeting all exons of RB1.ResultsTumor‐guided genotyping detected 10 of the 13 expected somatic RB1 mutations in plasma cfDNA in 8 of 10 patients (average variant allele frequency 3.78%). Without referring to RB1 status in the tumor, de novo mutation calling identified 7 of the 13 expected RB1 mutations (in 6 of 10 patients) with high confidence.ConclusionPlasma cfDNA can detect somatic RB1 mutations in patients with unilateral retinoblastoma. Since intraocular biopsies are avoided in these patients because of concern about spreading tumor, cfDNA can potentially offer a noninvasive platform to guide clinical decisions about treatment, follow‐up schemes, and risk of metastasis.
Highlights
Retinoblastoma is the most common primary intraocular malignancy of childhood
Tumor-specific somatic RB1 mutations were identified by MSK-IMPACT from enucleation samples of all 10 patients
It was observed that plasma analysis can potentially detect somatic RB1 mutations that are present as the second hit in addition to the germline RB1 mutation in a given patient
Summary
Retinoblastoma is the most common primary intraocular malignancy of childhood. Two thirds of all cases occur in children less than 2 years old with an age-adjusted annual incidence in children aged 0-4 of 10-14 cases per 1 million.[1]. Circulating plasma cell-free DNA (cfDNA) based assays have demonstrated promise as a noninvasive tool for molecular profiling across a broad spectrum of cancer types, aiding in diagnosis,[23] detecting minimal residual disease, monitoring responses, revealing resistance mechanisms, and tracking clonal evolution during therapy.[24,25] In plasma, only a small fraction of cfDNA is tumor derived, depending on cancer type, disease stage 26,27 and tumor volume.[28] This tumor-derived cfDNA is shed into circulation from tumor cells possibly via secretion, apoptosis, and necrosis.[23] It was unclear whether RB1 mutations could be detected in cfDNA due to the low burden of disease when compared to other solid tumors In this proof of concept study, we demonstrate the feasibility of detecting somatic RB1 mutations in plasma cfDNA of patients with retinoblastoma (Figure 1). We focused on treatment-naive patients that were planned to receive primary enucleation, in order to minimize any confounding factors introduced by prior treatment
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