Follow‐up of intraocular retinoblastoma through the quantitative analysis of conserved nuclear DNA sequences in aqueous humor from patients

Abstract

Fundoscopy is the standard method for diagnosis and follow-up of intraocular retinoblastoma, but it is sometimes insufficient to discern whether tumors are inactivated following treatments. In this work, we hypothesized that the amount of conserved nuclear DNA sequences in the cell-free DNA (cfDNA) fraction of the aqueous humor (AH) might complement fundoscopy for retinoblastoma follow-up. To address our hypothesis, we developed highly sensitive droplet digital polymerase chain reaction (ddPCR) methods to quantify highly conserved DNA sequences of nucleus-encoded genes (GAPDH and B4GALNT1) and of a mitochondrial gene, MT-ATP6. We obtained AH samples during intravitreal treatments. We analyzed 42 AH samples from 25 patients with intraocular retinoblastoma and 11 AH from controls (non-cancer patients). According to clinical criteria, we grouped patients as having progression-free or progressive retinoblastoma. cfDNA concentration in the AH was similar in both retinoblastoma groups. Copy counts for nucleus-derived sequences of GAPDH and B4GALNT1 were significantly higher in the AH from patients with progressive disease, compared to the AH from progression-free patients and control non-cancer patients. The presence of mitochondrial DNA in the AH explained that both retinoblastoma groups had similar cfDNA concentration in AH. The optimal cut-off point for discriminating between progressive and progression-free retinoblastomas was 108 GAPDH copies per reaction. Among patients having serial AH samples analyzed during their intravitreal chemotherapy, GAPDH copies were high and decreased below the cut-off point in those patients responding to chemotherapy. In contrast, one non-responder patient remained with values above the cut-off during follow-up, until enucleation. We conclude that the measurement of conserved nuclear gene sequences in AH allows follow-up of intraocular retinoblastoma during intravitreal treatment. The method is applicable to all patients and could be relevant for those in which fundoscopy evaluation is inconclusive.

Aqueous VEGF-A Levels as a Liquid Biopsy Biomarker of Retinoblastoma Vitreous Seed Response to Therapy

“… 28 , 34 – 38 Importantly, these SCNAs, as well as the fraction of tumor DNA contained within the AH cfDNA, both have prognostic implications. 17 , 39 – 41 …”

Immunohistochemical expression of TFF1 is a marker of poor prognosis in retinoblastoma

“…Genomic studies to detect additional chromosomal abnormalities such as gains in 1p, 17q, and 19q, losses of 11q and mutations in BCOR and MYCN gains/amplification may provide additional information for further risk stratification. Studies of minimal residual disease or more recently, liquid biopsy may also contribute to describe a very high‐risk group warranting intensive treatment 23–26 …”

“…Studies of minimal residual disease or more recently, liquid biopsy may also contribute to describe a very high-risk group warranting intensive treatment. [23][24][25][26] Some limitations to our work need to be acknowledged. This study was done on archival specimens over a long period of time and only limited number of slides were available for some cases.…”

The Potential of Aqueous Humor Sampling in Diagnosis, Prognosis, and Treatment of Retinoblastoma

“… 39 A longitudinal study of 25 RB patients quantitatively measured conserved nucleic acid sequences of nuclear and mitochondrial genes in the AH during IViC, wherein markedly increased copy counts of nuclear genes GAPDH and B4GALNT1 (a ganglioside synthase previously identified as a putative biomarker of GD2-positive phenotype in triple negative breast cancer [TNBC] vs. non-TNBC samples 48 ) and unaltered copy counts of MT-ATP6 (mitochondrial gene) were seen in AH from RB patients with progressive disease relative to progression-free patients and controls. 49 Finally, the secretory protein associated with RB metastasis, TFF1, has been detected in the AH, where it is also correlated with increased metastasis risk. 50 Although not a nucleic acid, this novel biomarker bears mentioning given its prognostic potential.…”

“… 32 Similarly, in the genome copy study mentioned above, GAPDH copy counts decreased in patients responding to chemotherapy, while one non-responder retained high GAPDH . 49 AH sampling and AH nucleic acid evaluation might therefore be used to assess the status of the tumor during the course of RB management. Since evidence of using AH nucleic acids for treatment response is not yet so well developed, other AH biomolecules may also offer value.…”