Keeping up with Precision Medicine

Advances in common cancers – breast, lung, prostate, colorectal and more – are in the news every day. But while those fields are blazing new trails in cancer treatment, ocular oncology is falling behind. For the most part, the treatment of ocular and orbital (abbreviated as “ocular”) tumors have not yet benefited from many of the emerging and established technologies in oncology and other disciplines to personalize medicine – and there has been no exploitation of cancer-causing genetic and epigenetic changes for diagnostics and treatment. For instance, some types of breast cancers can be treated by epidermal growth factor receptor (EGFR) inhibitors, and patients with certain lung cancers can receive anaplastic lymphoma kinase (ALK) inhibitors. These therapies are based on knowing the gene mutations in oncogenes and tumor suppressors, or copy number alterations that drive the growth of the cancer. Epigenetics – modifications to DNA or histones that aberrantly switch tumor-promoting or cancer-preventing genes on or off, without changing the genetic sequence – is an emerging field that has deepened our understanding of how tumors form. No epigenetic-based therapies currently exist for ocular cancers because epigenetic regulation of those cancers remains poorly understood. We’re working to help change that. Here’s our story so far...

Expanding the horizon with epigenetics

When my colleagues in ophthalmology and I first started paying attention to epigenetics in 2008, the focus was on histone methylation – the addition of methyl groups to histone tails – and how that affects gene expression. Though a “hot” area, no one had really examined it in the developing mammalian retina. So we decided to investigate, and published a paper showing how the histone and methyl marks change during retinal development in the mouse (1). We discovered some interesting trends: a lot of these histone marks were upregulated in the inner retina, and some of the enzymes involved were developmentally regulated. One of these, the histone methyl-transferase EZH2, was expressed only during the growth phase of the retina, and then switched off when the retina was formed. Interestingly EZH2 was emerging as a major target for cancer therapies because it appears to be enriched in cells that grow quickly, such as fetal cells as well as cancer cells.

From studying the developing human retinae, we found that EZH2 was highly expressed in fetal retinae, but not postnatally (2). Because embryonic proteins can be expressed in cancer cells (3), we studied retinoblastoma samples, and found that EZH2 was highly expressed in the tumor cells from this childhood cancer. We also found it to be a good biomarker; immunohistochemistry on these tumor samples for EZH2 was almost a black and white indication of where tumor cells are… and where they are not. Histopathologic detection of EZH2 expression allowed identification of single tumor cells that were invading into the optic nerve or adjacent tissues. Because the decision to use systemic chemotherapy is linked to whether retinoblastoma cells have spread to the optic nerve – and beyond – staining these specific markers could help provide better indications of whether further treatment beyond surgery is actually warranted.

EZH2 was already under scrutiny by several companies, and small molecule inhibitors against it were in trials for other tumor types, such as lymphoma. We tested some of those inhibitors and found that they selectively killed the retinoblastoma cells, sparing the normal retinal cells. This was our entry point and, since then, we’ve published papers showing high levels of EZH2 in different tumors including vitreoretinal and orbital lymphomas, medulloepithelioma, and basal cell skin cancer, which can occur on the eyelid and the orbit, where it is difficult to treat (4)(5)(6). Right now, we are looking at some of the pathways downstream of EZH2, including DNA methylation.

A precise approach

An important part of our studies is to help patients with ocular cancers gain access to more targeted treatments. A close collaborator, Scott Tomlins, helped develop the panel for the National Cancer Institute MATCH trial (see “The NCI-MATCH Trial”). Together, we’ve been using the same technology to study eye and orbit tumors. But because ocular cancers are quite rare – there are only about 5,000 cases per year in the United States (7) – acquiring tissue for study can be challenging. I use the analogy that studying cancer and human tissues is like studying freshwater; just like most freshwater is “locked up” in ice at the poles, most human tissues are embedded in wax and archived in hospitals and medical centers. But this fixation process and the wax can make downstream research applications more difficult, especially when trying to study the epigenetics. It’s why we’ve had to take a unique approach.

Using a scalpel, we “shave” off sections of samples to collect DNA, which we then put on a next-generation sequencing (NGS) panel to find mutations, gene alterations or copy number changes in those tumors. The panel is enriched for gene targets for which drugs have already been approved by the FDA (or in trial) for other cancer indications. So far, we’ve discovered many actionable alterations in genes, including MYD88, ARID1A, EZH2, PTEN, TP53, HRAS and NRAS (2)(4)(5)(6)(8). We’ve also found that eye lymphomas have a certain “flavor” and abundance of mutations that are not present elsewhere; MYD88 was commonly mutated in orbital marginal zone lymphomas, but uncommonly in marginal zone lymphomas elsewhere in the body (6). As there is already a drug in trial against the mutation we identified, we hope that our orbital lymphoma patients might be attractive candidates.

We’re basically looking for the “low-hanging fruit” – tumors that haven’t yet been sequenced. And with orbit and eye tumors being so much rarer compared with other cancers, we have had to dig deep into our archives or collaborate with other centers to find tumor samples for study; in some cases we are analyzing samples that are 30 years old! But science and sequencing technologies have moved so fast that, for relatively little money, we can use small amounts (as little as 5 ng of tumor DNA) of these archived samples – including those that we thought were “locked up in ice” – and still retrieve usable information. Our initial goal is for patients with eye cancer to have the same options and treatments as patients who have other cancers through basket trials (clinical trials that target cancers based only on whether they contain specific genetic alterations, rather than the part of the body they come from). We hope to exploit drugs in our armamentarium that may be more specific than chemotherapy because they target genetic alterations present in the tumor, but not in normal tissue. In the future, we want to help other ophthalmologists, ocular oncologists and pathologists who see patients diagnosed with eye cancer by using these precision medicine strategies to identify diagnostic or druggable targets in their patients’ ocular cancers – and thus which clinical trials a patient may be eligible for. And if the drug receives approval, we’ll have made an important link in bridging a drug from other types of cancer or diseases to the field of eye cancer, and helped our patients access more targeted therapies.

The NCI-MATCH Trial

The NCI-MATCH Trial (NCT02465060) is an ongoing precision medicine cancer treatment trial (11). In this study, biopsies from patients are analyzed using an NGS-based panel to identify actionable genomic alterations for precision medicine-based treatment strategies. Patients with advanced solid tumors, lymphomas, or myeloma who have progressed on standard treatment for their cancer, or patients with a rare cancer for which there is no standard treatment, are eligible for MATCH. Currently, there are 19 treatment arms open to patients, including genetic changes in EGFR, ALK and mTOR (11). In the NCI-MATCH “basket trial,” patients can be enrolled in a trial no matter what kind of cancer they have, provided they have the same mutation that the trial drug is targeting. The trial utilizes the Oncomine Comprehensive Panel (OCP) to detect genomic variants in patient samples (12). The OCP was developed from analysis of over 700,000 tumor samples, and combination of genomic alterations with available therapeutics and ongoing clinical trials.

A call for collaboration

We’re also hoping to improve diagnosis. Vitreoretinal lymphoma is rare – there are only around 400 cases per year in the US (9). Because of its link to CNS (brain) lymphoma, it’s a deadly disease, with only a quarter of patients surviving more than five years after diagnosis (10). It represents a crucial unmet need, not only because there is no standardized treatment – but also because it is very difficult to diagnose. In small-volume vitreous samples taken from four patients with confirmed or suspected vitreoretinal lymphoma, we identified alterations in MYD88, CDKN2A and PTEN, showing that it is feasible to perform targeted NGS on intraocular liquid biopsies (as small as 500 µl or 5 ng of DNA) and identify the presence of tumor-causing mutations or copy number alterations (8) (see Figure 1).

This approach could potentially enhance vitreoretinal lymphoma diagnosis and influence patient care. For instance, prior vitreous biopsies from one of the study cases had all been read as cytologically negative, with neither eye showing the presence of cancer cells. Two years after initial consultation, the patient presented with vision loss and right hemianopia; he had a brain lymphoma mass that was pressing on his visual centers. When we analyzed his original vitreous samples which had been stored in the freezer when collected two years prior to the brain lymphoma, we found tumor DNA confirming what later showed up in the brain: diffuse large B cell lymphoma. Vitreoretinal lymphoma can be difficult to diagnose through standard cytology approaches, but because our test is so sensitive, as little as 5 or 6 ng of DNA is needed – which can come from just a few tumor cells in the eye. In the near future, we’d like to develop a diagnostic test that can be used by anybody. We already have the basis for the test, but because our research was only based on four samples – about one percent of all cases in the US – we want to collaborate with others; we need more samples to show that diagnosis using this precision medicine approach could be accurate and beneficial.

Making strides forwards

The key take home message is this: the technology we need is there, but to make more progress in the field, different teams need to talk. Just as ophthalmology has led the way in stem cells and gene therapy, we can learn from other fields, like pathology, that are leading in precision medicine, and then apply it to ocular oncology. We can use today’s technologies to make powerful advances to improve both diagnosis and treatment for our patients. Because ocular tumors are so rare, collaboration is really the key – it helps us bring the power of NGS, “big data” bioinformatics, precision medicine and epigenetic technologies to bear on this intractable problem.

Rajesh C. Rao is vitreoretinal surgeon, clinician-scientist, Assistant Professor of Ophthalmology and Visual Sciences at the Kellogg Eye Center, and Assistant Professor of Pathology, at the University of Michigan, USA. Rao is also the Leslie H. and Abigail S. Wexner Emerging Scholar at the A. Alfred Taubman Medical Research Institute, University of Michigan, USA.