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Shreya Sirivolu, Liya Xu, Peter Kuhn, James Hicks, Jesse L Berry; Chromosome 6p amplification detected in blood cell-free DNA in advanced retinoblastoma. Invest. Ophthalmol. Vis. Sci. 2022;63(7):2332 – A0001.
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Both aqueous humor (AH) and blood-based liquid biopsies can carry tumor-derived cell-free DNA (cfDNA) in retinoblastoma (RB) patients. Detection of somatic copy number alterations (SCNAs) requires approximately 5% tumor fraction, as such have only previously been detected in AH, which is an enriched source of tumor DNA. We analyzed AH and blood cfDNA taken at the time of primary enucleation to compare SCNA profiles and somatic RB1 mutation detection between both biospecimens.
One patient diagnosed with advanced unilateral RB (Group D/AJCC Stage cT2B) was included. After isolating cfDNA from AH and blood, constructed whole genome libraries were sequenced on an Illumina platform to assess genome-wide SCNAs, which were considered positive at 20% deflection from the baseline. Correlation between SCNA profiles was calculated using Pearson correlation coefficient. The libraries were also used to identify somatic RB1 pathogenic variants using a comprehensive cancer panel that includes all RB1 exons.
In the peripheral blood, a germline RB1 mutation was not detected, however a heterozygous mutation (c.3920T>A) in the APC gene was reported from clinical testing. Genomic analysis of the tumor revealed two somatic RB1 mutations that were not present in germline, c.1589_1590del with a variant allele frequency (VAF) of 84.0% and c.2330dupC with VAF of 40.0%. These mutations were similarly detected in the AH, with VAFs of 78.9% and 45.27% respectively, but were not detected in the blood. Genomic analysis of both tumor and AH demonstrated highly recurrent RB SCNAs such as gains on 1q, 2p, and 6p, in addition to other alterations. The AH SCNA profile was highly concordant with the tumor profile (r=0.987; p<0.0001). 6p gain was detected in the blood and comparison of the 6p amplitude between AH and blood suggests approximately 12% tumor fraction in the blood.
To our knowledge, this is the first time an SCNA has been detected in the blood of an RB patient, suggesting a high enough tumor fraction. No metastasis has been identified, however monitoring is ongoing. The results illustrate the benefits and limitations of both AH and blood-based liquid biopsies. While AH is composed of more highly enriched eye-specific tumor information, only blood can provide information regarding systemic disease and metastasis.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
SCNA profiles comparing (A) AH and tumor and (B) blood and tumor.
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