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Cell-Free DNA Tumor Fraction in the Aqueous Humor Is Associated With Therapeutic Response in Retinoblastoma Patients

ABSTRACT: Purpose The aqueous humor (AH) liquid biopsy enables in vivo evaluation of tumor-derived cell-free DNA (cfDNA) from retinoblastoma (RB) eyes. Herein, we test our hypothesis that longitudinal dynamics of AH cfDNA—including tumor fraction (TFx) and somatic copy number alteration (SCNA) amplitude—correspond to therapeutic response. Methods Eyes with ?3 AH extractions during intravitreal chemotherapy (IVM) or at secondary enucleation between 2015 to 2019 were included. AH cfDNA was sequenced to assess RB SCNA amplitude; ichorCNA software was used to estimate TFx. Eyes without SCNAs or with TFx < 0.10 across all samples were excluded. Therapeutic responses for each eye were determined from clinical records. Statistical analyses included Mann-Whitney U and Pearson correlation tests. Results Twenty eyes of 20 patients underwent ?3 AH extractions; 6 eyes lacked SCNAs or had TFx < 0.10 throughout sampling and were excluded. Clinical progression was associated with significantly higher SCNA amplitudes and TFx values than regression (P ? 0.04). Relative increases in TFx (?TFx 1.86 ± 2.22) were associated with disease progression, whereas relative decreases in TFx (?TFx 0.53 ± 0.36) were associated with disease regression (P < 0.00001). A ?15% increase in TFx relative to baseline during treatment was associated with an over 90-fold increased likelihood of clinical progression (odds ratio = 90.67, 95% confidence interval = 8.30–990.16, P = 0.0002). TFx and SCNA amplitude were significantly positively correlated throughout sampling (P ? 0.002). Conclusions Longitudinal changes in AH-derived cfDNA TFx and SCNA amplitude are concordant with clinical responses of intraocular RB during active therapy. Translational Relevance Longitudinal evaluation of AH cfDNA may provide an objective, quantitative way to monitor therapeutic response and disease burden in RB patients.

SUBMITTER: Polski A

PROVIDER: S-EPMC7533735 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Project description:ImportanceRetinoblastoma (Rb) is one of the first tumors to have a known genetic etiology. However, because biopsy of this tumor is contraindicated, it has not been possible to define the effects of secondary genetic changes on the disease course.ObjectiveTo investigate whether the aqueous humor (AH) of Rb eyes has sufficient tumor-derived DNA to perform genetic analysis of the tumor, including DNA copy number alterations.Design, setting, and participantsThis investigation was a case series study at a tertiary care hospital (Children's Hospital Los Angeles) with a large Rb treatment center. Cell-free DNA (cfDNA) was isolated from 6 AH samples from 3 children with Rb, including 2 after primary enucleation and 1 undergoing multiple intravitreous injections of melphalan for vitreous seeding. Samples were taken between December 2014 and September 2015.Main outcomes and measuresMeasurable levels of nucleic acids in the AH and identification of tumor-derived DNA copy number variation in the AH. The AH was analyzed for DNA, RNA, and micro-RNA using Qubit high-sensitivity kits. Cell-free DNA was isolated from the AH, and sequencing library protocols were optimized. Shallow whole-genome sequencing was performed on an Illumina platform, followed by genome-wide chromosomal copy number variation profiling to assess the presence of tumor DNA fractions in the AH cfDNA of the 3 patients. One child's cfDNA from the AH and tumor DNA were subjected to Sanger sequencing to isolate the RB1 mutation.ResultsSix AH samples were obtained from 3 Rb eyes in 3 children (2 male and 1 female; diagnosed at ages 7, 20, and 28 months). A corroborative pattern between the chromosomal copy number variation profiles of the AH cfDNA and tumor-derived DNA from the enucleated samples was identified. In addition, a nonsense RB1 mutation (Lys→STOP) from 1 child was also identified from the AH samples obtained during intravitreous injection of melphalan, which matched the tumor sample postsecondary enucleation. Sanger sequencing of the AH cfDNA and tumor DNA with polymerase chain reaction primers targeting RB1 gene c.1075A demonstrated this same RB1 mutation.Conclusions and relevanceIn this study evaluating nucleic acids in the AH from Rb eyes undergoing salvage therapy with intravitreous injection of melphalan, the results suggest that the AH can serve as a surrogate tumor biopsy when Rb tumor tissue is not available. This novel method will allow for analyses of tumor-derived DNA in Rb eyes undergoing salvage therapy that have not been enucleated.

Project description:PurposeRetinoblastoma (Rb) is the most common primary intraocular cancer in children. Unlike with most solid tumors, direct biopsy is contraindicated due to risk of tumor dissemination. However, recent therapeutic techniques have allowed for the safe extraction of aqueous humor (AH) from eyes undergoing therapy, providing the unique opportunity to use AH as a liquid biopsy for Rb. Although the extraction of AH in Rb eyes undergoing therapy is new, the consideration of whether there are tumor biomarkers in the AH is not. The current manuscript is a systematic review of all studies that have examined biomarkers in the AH of Rb eyes. The authors hypothesized that AH sampling and analysis of tumor biomarkers may have new clinical relevance for the diagnosis, prognosis, and/or management of Rb.MethodsA comprehensive database search (PubMed, Web of Science, Embase, and Cochrane Databases) was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement to identify articles on AH markers in Rb eyes. Inclusion criteria included English language articles with original reports on AH markers in the eyes of patients with confirmed Rb. Data on marker type, number of eyes, marker means and ranges, and when available, control values and clinicopathological correlations were collected. Articles were stratified based on marker type, and assessed quantitatively and qualitatively.ResultsAn initial database search produced 325 articles, and an additional 11 articles were identified through searching citations. After removing duplicates and applying the eligibility criteria, we selected 27 articles to be included in the current review. A total of 463 eyes with histologically confirmed Rb were included in this review. The various markers and their values, with comparison to controls and clinicopathological correlations, are discussed.ConclusionsAH sampling and tumor biomarker analysis in eyes without undergoing enucleation have the potential to revolutionize the management of Rb.Translational relevanceAlthough previous studies evaluated markers in the AH only after enucleation and not at diagnosis or during therapy, the clinical relevance of these markers was limited. However, recent changes in the management of Rb have allowed for safe sampling of the aqueous during therapy and, thus, correlation of tumor biomarkers with disease course. Thus, the authors felt it important to revisit previous research to evaluate whether these markers may now be applicable for the diagnosis, prognosis, or management of Rb.

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Cell-Free DNA Tumor Fraction in the Aqueous Humor Is Associated With Therapeutic Response in Retinoblastoma Patients

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