Potential of Aqueous Humor as a Surrogate Tumor Biopsy for Retinoblastoma

Abstract

Importance: Retinoblastoma (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.

Objective: To 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 participants: This 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 measures: Measurable 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.

Results: Six 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 relevance: In 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.

Figure 1.. Case 1: Chromosomal Copy Number Variation Profile Demonstrating the Chromosomal Changes (Both Gains and Losses) That Vary From a Diploid Cell With a Full Complement of Chromosomes

This profile is from a patient after primary enucleation showing overlapping gains and losses in the aqueous humor (AH) and tumor demonstrating that the cell-free DNA in the AH is derived from the tumor. A, Chromosomal copy number variation profile from cell-free DNA in the AH (gain of 1q, 6p, and 17q22-17q25 and loss of 17p13.3-p13.1). B, Chromosomal copy number variation profile from tumor (gain of 1q, 6p, and 17q22-17q25 and loss of 17p13.3-p13.1). C, Overlay of the chromosomal copy number variation profiles (AH is blue, and tumor is red).

Figure 2.. Case 2: Chromosomal Copy Number Variation Profile Taken After Primary Enucleation in a Patient With a 13q− Deletion Indicating Predisposition to the Development of Retinoblastoma

A and B, Chromosomal analysis demonstrated a heterogeneous sample with clonal differences in the chromosomal copy number variations between the cell-free DNA in the aqueous humor (AH) (A) and tumor (B). C, Genomic DNA from a buccal swab demonstrates the same 13q deletion (13q13.3-q21.32). D, The overlay of the chromosomal copy number variation profiles is shown (AH is blue, tumor is red, and buccal swab is green).

Figure 3.. Case 3: Chromosomal Copy Number Variation Profile Showing Similar Chromosomal Gains and Losses Over Time in a Patient Treated With Multiple Intravitreous Injections of Melphalan With Concurrent Aqueous Humor (AH) Extraction During Each Injection

A-D, Chromosomal copy number variation profile from cell-free DNA in the AH from sequential samples (AH 1 through AH 4). E, Chromosomal copy number variation profile from tumor after subsequent (secondary) enucleation showing that the same chromosomal changes seen initially in the AH were also found in the tumor. F, Overlay of the AH and tumor chromosomal copy number variation profiles (AH is blue, and tumor is red).

Figure 4.. Single Aqueous Humor (AH) Chromosomal Copy Number Variation Compared With Summative Tumor Chromosomal Copy Number Variation by Kooi et al

A and B, Chromosomal copy number variation from the AH cell-free DNA in all 3 patients (A) compared with a summation of the chromosomal copy number variation profiles from 71 retinoblastoma tumor samples in the study by Kooi et al (reproduced with permission) (B).