A Whole New World

We say we are in an era of precision medicine. But what can one do when the very information needed to make these informed, directed, personalized choices cannot be accessed by the clinician? Well, that is the situation we currently face as ocular oncologists for retinoblastoma (Rb).

Despite critical advances in how chemotherapy is delivered, worldwide nearly 50 percent of advanced eyes with Rb are enucleated and many more affected eyes are legally blind – even with treatment (1)(2). Why? Because there are no known molecular prognostic features that can predict the response of Rb to treatment and clinical features rely primarily on assessing the size of the tumor or presence of seeding (e.g., Group Classification); however, they still predict with only 50 percent certainty whether an advanced Group D Rb tumor will respond to intravenous chemotherapy or will require subsequent enucleation due to persistent or recurrent tumor. In 2017, for advanced eyes, we have the same predictive value as a coin flip (3).

The vast majority of Rb arises from somatic, germline or mosaic mutations in the RB1 tumor suppressor gene. And similar to other cancers – such as those found in the breast, lung and prostate – Rb DNA likely harbors specific genetic or genomic changes that will be informative regarding therapeutic response and/or prognosis. And we need this information because there is currently no targeted treatment or personalized medicine approach for Rb, despite its being one of the first cancers with a known genetic etiology for carcinogenesis. Performing genomic analyses on Rb DNA at the time of diagnosis or during treatment would allow, for the first time, clinical correlations with specific tumor mutations, genomic changes and expression profiles that were only previously available from tumor tissue from eyes that had been already enucleated – and never from those eyes that responded to therapy and were saved. This is because evaluating tumor DNA in Rb is challenging; direct biopsy of the tumors is contraindicated because of the risk of extraocular tumor spread and metastatic disease (4). As a result, the Rb field had a long-standing golden rule: the eye is inviolable during treatment, which means that tumor tissue only becomes available after enucleation.

However, the golden rule changed in 2012 as Francis Munier – an ocular oncologist in Switzerland – introduced a safety-enhanced procedure to inject melphalan into the vitreous cavity of eyes with Rb and seeding (5)(6). In this procedure, aqueous humor is withdrawn prior to the injection to lower IOP and prevent reflux of active seeds to the injection site. And it has turned out to be safe: no cases of metastatic disease have been reported with this safety-enhanced technique (7). This method of intravitreal chemotherapy as treatment for vitreous seeding in Rb has been an absolute game-changer for managing the disease, not only by providing a new, highly effective treatment strategy, but also by providing access to the aqueous humor of eyes undergoing treatment. This revolution in one aspect of Rb management has provided a critical opportunity to revolutionize another – the biopsy. We’ve managed to do just this, and have recently demonstrated that aqueous humor samples can be a “surrogate” biopsy for Rb – a liquid biopsy. In six samples obtained from three children with Rb, we identified cell-free tumor DNA through shallow whole genome sequencing using a next generation protocol, and confirmed that the chromosomal alterations in the aqueous corroborated those found in Rb tumors (8).

Our findings provide the proof of concept that, with the aqueous, we have a safe and effective way to derive genetic information from the Rb tumor without enucleation.  Finally, we can gain access to critical genomic information to help ocular oncologists decide which eyes are likely to be most responsive to therapy – and can thus be salvaged – and those which are higher risk and should undergo primary enucleation. It could also open up an entirely new research domain for Rb as well as other intraocular diseases, as the aqueous humor doesn’t only yield tumor DNA. There is also RNA, microRNA, and possibly other disease markers. In fact, there’s a whole new world to explore!