Case Report: Longitudinal Evaluation and Treatment of a Melanoma-Associated Retinopathy Patient

Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with cutaneous metastatic melanoma in which patients develop vision deficits that include reduced night vision, poor contrast sensitivity, and photopsia. MAR is caused by autoantibodies targeting TRPM1, an ion channel found in melanocytes and retinal ON-bipolar cells (ON-BCs). The visual symptoms arise when TRPM1 autoantibodies enter ON-BCs and block the function of TRPM1, thus detection of TRPM1 autoantibodies in patient serum is a key criterion in diagnosing MAR. Electroretinograms are used to measure the impact of TRPM1 autoantibodies on ON-BC function and represent another important diagnostic tool for MAR. To date, MAR case reports have included one or both diagnostic components, but only for a single time point in the course of a patient’s disease. Here, we report a case of MAR supported by longitudinal analysis of serum autoantibody detection, visual function, ocular inflammation, vascular integrity, and response to slow-release intraocular corticosteroids. Integrating these data with the patient’s oncological and ophthalmological records reveals novel insights regarding MAR pathogenesis, progression, and treatment, which may inform new research and expand our collective understanding of the disease. In brief, we find TRPM1 autoantibodies can disrupt vision even when serum levels are barely detectable by western blot and immunohistochemistry; intraocular dexamethasone treatment alleviates MAR visual symptoms despite high levels of circulating TRPM1 autoantibodies, implicating antibody access to the retina as a key factor in MAR pathogenesis. Elevated inflammatory cytokine levels in the patient’s eyes may be responsible for the observed damage to the blood-retinal barrier and subsequent entry of autoantibodies into the retina.


Introduction and Case Description
Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome affecting patients with cutaneous melanoma (CM) and is characterized by impairment of rod-mediated vision and the ON retinal pathway (Elsheikh et al., 2020;Keltner et al., 2001;Lu, 2009;Milam et al., 1993).The visual symptoms of MAR include photopsia, reduced contrast sensitivity, and progressive nyctalopia, all of which are caused by autoantibodies that bind and inhibit an essential ON-bipolar cell (ON-BC) ion channel known as TRPM1 (Dhingra et al., 2011;Xiong et al., 2013).This inhibition prevents ON-BC depolarization during the light response, a phenomenon that can be identi ed as a reduction in the b-wave amplitude of the fulleld electroretinogram (ffERG) (Alexander and Fishman, 1992;Milam et al., 1993;Xiong et al., 2013).
Melanocytes, the pigment-producing cells of the skin that become cancerous in CM, are one of few cell types outside the eye that express TRPM1 (Deeds et al., 2000;Duncan et al., 2001Duncan et al., , 1998;;Fang and Setaluri, 2000).In MAR patients, B cells produce antibodies against melanocyte antigens, including TRPM1 (Dhingra et al., 2011;Kinker et al., 2021;Rodgers et al., 2022).Consequently, these circulating autoantibodies disrupt vision when they in ltrate the retina and bind TRPM1 in ON-BCs (Lei et al., 2000;Xiong et al., 2013).Serum screening for TRPM1 autoantibodies and ffERG testing serve as the most reliable combination of diagnostic tools for con rming MAR.However, melanoma treatment may in uence the presence of serum autoantibodies or the progression of a patient's visual symptoms over time, complicating diagnosis and prognosis (Casselman et al., 2023;Heng et al., 2022;Kim et al., 2019).
Several studies report ffERG recordings or serum autoantibody titers from a single timepoint in a patient's disease, yet none correlate treatment data with diagnostic testing over time to assess MAR progression and resolution.Here, we describe a case of bilateral MAR in a 79-year-old male with stage IV CM.In August 2021, the patient presented with unilateral vision impairment and a relative afferent pupillary defect (RAPD), prompting further ophthalmic testing and treatment for optic neuritis (Supplementary Table 1).The patient was receiving pembrolizumab treatment without evidence of active disease when he developed MAR visual symptoms.After observing no response to systemic corticosteroids and severe loss of inner-retinal function on ffERG without evidence of retinal degeneration on multi-modal retinal imaging (optical coherence tomography, fundus auto uorescence) the patient was suspected of having MAR.The diagnosis was supported by the patient's symptoms (nyctalopia, photopsia, blue-tinted vision, and "squiggles" in his vision) and later con rmed with a positive test for anti-ON-BC autoantibodies in the serum.Unlike previous reports, we integrate the patient's treatment history with a longitudinal evaluation of the ocular and humoral components of their disease to gain a more complete understanding of MAR pathology.

Methods
This study was performed in accordance with the Declaration of Helsinki and protection of the patient's identity.The patient was provided with written informed consent for the use of personal medical data for scienti c purposes and publication.IRB00009765.Methods details can be found in the supplementary materials.
3 Diagnostic Assessment and Results

Intraocular corticosteroids restore visual function and alleviate MAR visual symptoms
The patient presented with BCVA of 20/40 in the right eye and 20/50 in the left eye.Fundus examination was unremarkable, optical coherence tomography showed normal retinal architecture (data not shown), and wide-eld auto uorescence was normal (Figure 1A).However, wide-eld uorescence angiography (FA) showed a few focal areas of subtle vascular leakage in both eyes (Figure 1A).Wide-eld static perimetry revealed severely constricted visual elds in both eyes (Supplementary Figure 1).The ffERG showed non-recordable dim scotopic waveforms, electronegative bright scotopic waveforms, and squared a-waves of the photopic single ash and normal 30 Hz icker (Figure 2A).eye had started regressing.Indeed, the ffERG results indicated that the right eye dim scotopic b-wave amplitude had been restored to 116% of the normal range (from 6% at baseline), the electronegative character of the bright scotopic waveform had normalized, and the squared a-wave of the photopic waveform had resolved (Figure 2, for timelines see Supplementary Tables 1 and 2).The FA also showed resolution of the leakage in the right eye (Figure 1 A).In the left eye, the ffERGs resembled the baseline (Figure 2) and the FA showed recurrence of leakage in the superior arcade, con rming regression of the left eye (Figure 1A).Dexamethasone injections were repeated in both eyes in February of 2022.To potentially reduce the frequency of intraocular dexamethasone injections (every ~3 months), intravitreal uocinolone acetonide implants (0.18 mg, EyePoint Pharmaceuticals; Watertown, MA USA), which have a 2-3-year e cacy period, were injected in both eyes in April 2022.Results from the ffERG in June 2022 demonstrated restoration of the dim scotopic b-wave amplitude to 136% and 156% of normal range for the right and left eyes, respectively, and resolution of the bright scotopic electronegative waveform and photopic squared a-wave in the left eye (Figure 2, Supplementary Table 2).Moreover, wide eld static perimetry revealed signi cant expansion and improvement of visual sensitivity in both eyes (Supplemental Figure 1).However, these bene ts regressed 2-3 months later.In August 2022, an ffERG revealed that b-wave amplitudes in the right and left eye had fallen to 1% and 2% of the normal range, respectively.Unfortunately, uocinolone acetonide implant monotherapy was not effective and dexamethasone treatment was reinitiated with repeat injections for both eyes in August of 2022.Two months later in October of 2022, the b-wave in the left eye was restored to 122% of the normal range, while the right eye remained at 6% (Figure 2, Supplementary Tables 1 and 2).With continued routine dexamethasone implants every 16 weeks, there was a sustained effect of improved visual symptoms, visual elds, and normalization of the ffERG in both eyes.During the course of treatment, steroid-induced elevated intraocular pressures of 32 and 28 mmHg were observed in the right and left eye, respectively, which normalized with pressure-lowering eye drops (dorzolamide/timolol) twice per day.The patient also developed cataract in both eyes, but with cataract surgery and sustained intravitreal steroids, his BCVA stabilized to 20/30 in both eyes.

In ammatory cytokine levels are augmented within the aqueous humor
To cause MAR symptoms, TRPM1 autoantibodies must bind and inhibit the TRPM1 ion channel in the membrane of ON-BCs (Dhingra et al., 2011;Xiong et al., 2013).Yet to reach the ON-BCs, the autoantibodies must rst breach the blood-retinal barrier (BRB), which, under healthy conditions, restricts the passage of macromolecules from the circulation into the retinal tissue (O'Leary and Campbell, 2023;

TRPM1 autoantibodies persist in patient serum throughout immunotherapy and vision treatment
To detect retinal autoantibodies circulating in the patient's blood, diluted serum from three separate blood draws was applied to cryosections of mouse retina and bound antibodies were detected with a uorescent anti-human secondary antibody.Retinal bipolar cells were labeled with all three serum draws, with much brighter labeling produced by the sera from the two later collection dates (Figure 3).This increase in signal strength may be explained by an increase in autoantibody titer, an increase in antibody a nity for retinal autoantigens, or both.When applied to retinal cryosections from TRPM1-KO mice, bipolar cell labeling with the MAR serum was dramatically reduced, indicating that anti-TRPM1 autoantibodies comprise the majority of anti-retinal autoantibodies in the patient's serum (Figure 3).Further supporting that anti-TRPM1 autoantibodies dominate the composition of retinal autoantibodies, the bipolar cell staining pattern produced by this patient's serum closely resembles that of an anti-TRPM1 mouse monoclonal antibody (Figure 3).Despite an obvious reduction in immuno uorescence intensity on retina sections from the TRPM1 KO mouse compared to the wild type, anti-retinal labeling is not entirely absent, implicating the presence of autoantibodies against additional retinal antigens in the patient's serum (Figure 3).Furthermore, the immuno uorescence on the TRPM1-KO retina sections is brighter for the later blood draws indicating an increase in titer and/or a nity of these additional antiretinal autoantibodies over time.Supplementary Figure 2 shows the ffERG recorded on the same dates as the serum draws.

Patient autoantibodies target a region of TRPM1 encoded by exons 8-10
In characterizing previous MAR patient sera, we mapped the immunoreactive epitope targeted by TRPM1 autoantibodies to a region in the N-terminal, cytoplasmic domain of TRPM1, amino acids 337-380 (encoded by exons 8-10) (Supplementary Figure 3) (Duvoisin et al., 2017).Using a slot immunoblot assay in which patient serum is reacted with puri ed TRPM1 polypeptides, we found that autoantibodies in the serum of the current patient also bind to this region of TRPM1.Sera from both the current patient and a previous MAR patient gave nearly identical results on the slot immunoblot, reacting with a TRPM1 polypeptide encoded by exons 6-10, but not with a polypeptide encoded by exons 2-7 (Figure 4A).In agreement with the retinal immuno uorescence results, sera from August 4, 2022 and January 10, 2023 reacted more strongly with the puri ed TRPM1 polypeptide than the serum from August 6, 2021 (Figure 4B).Despite the comparatively low titer/a nity of anti-TRPM1 autoantibodies in the August 6, 2021 serum, the patient began reporting vision changes as early as April 2021.Consistent with the slot immunoblot results, the patient's serum labels TRPM1 ex6-10 but not TRPM1 ex2-7 when both polypeptides are heterologously expressed by HEK293T cells (Supplementary Figure 4).

Discussion
Melanoma-associated retinopathy is a rare paraneoplastic syndrome that affects individuals with cutaneous melanoma, causing visual impairment due to a humoral immune response against ON-BC neurons in the retina.A de nitive MAR diagnosis requires assessment of visual dysfunction and autoimmune activity.The bright scotopic ffERG b-wave amplitude provides a useful assessment of ON-BC function and serves as a proxy for the degree of visual impairment.To complement the ffERG, immuno uorescent detection of anti-TRPM1 autoantibodies in the serum indicates the presence and intensity of the immune response against ON-BCs.Together, these tests can be used to monitor disease progression and response to ocular or systemic treatment, such as intraocular corticosteroids or cancer immunotherapy.Here we present the rst longitudinal case report of a MAR patient that integrates ffERG recordings, serum autoantibody measurements, aqueous humor cytokine pro ling, and the patient's treatment record.The synthesis and integration of these data shed new light on poorly understood aspects of MAR pathogenesis which may prove useful to the greater community of autoimmune retinopathy researchers and clinicians.
First, although the patient developed symptoms in April 2021 (Supplementary Table 1), we note that ON-BC autoantibodies were barely detectable by immunohistochemistry or immunoblot in the rst serum draw from August 6, 2021 (Figure 3, Figure 4A).Consistent with the patient's symptoms, the ffERG recorded on August 4, 2021 revealed an undetectable dim scotopic waveform and electronegative bright scotopic waveform in both eyes, indicating severe bipolar cell dysfunction (see Supplementary Figure 2).Together, these ndings indicate that even low levels of circulating ON-BC autoantibodies can profoundly impact vision.Next, between August 6, 2021 and August 4, 2022, there was a substantial increase in detectable TRPM1 autoantibodies in the patient's serum (Figure 3, Figure 4), which preceded a melanoma relapse (leptomeningeal disease) in September 2022.This spike in retinal autoantibodies occurred in the absence of cancer immunotherapy, though other reports suggest immunotherapy potentiates autoimmune retinopathy (Chauhan et al., 2022;Chen et al., 2022;Heng et al., 2022).Our patient underwent ~17 months of nivolumab and pembrolizumab therapy before reporting visual symptoms (Supplementary Table 1), whereas other studies report the onset of visual dysfunction after 3-6 weeks of immunotherapy.Therefore, any direct effect of the immunotherapy on visual function appears unlikely in this case.However, in one previous study, a patient who developed MAR visual symptoms after 18 months of pembrolizumab reported greatly improved vision 5 days after stopping treatment.Our patient reported no such improvement to vision after stopping pembrolizumab in August 2021; however, visual function improved after dexamethasone treatment two months later.One possible explanation for the autoantibody surge is that the melanoma relapse reinvigorated the immune response against the cancer, leading to increased production of autoantibodies.A progressive increase in the immuno uorescent labeling intensity on the TRPM1 KO retina sections across serum draws demonstrates the development of additional anti-retinal autoantibodies, consistent with a reinvigorated immune response (Figure 3).Increased autoantibody levels in the serum may therefore serve as useful predictors for the progression of cancer prior to PET or CT imaging but are not necessarily linked to cancer immunotherapy.
In agreement with previous case reports on MAR, our patient responded positively to intraocular dexamethasone implants, which restored the normal ffERG and correlated with signi cant improvement in visual elds (Figure 2, Supplementary Figure 1) (Chaves et al., 2022;Patel et al., 2018;Xie et al., 2019).While ffERG b-wave amplitudes appeared maximally restored during June through August 2022 (Figure 2), high levels of TRPM1 autoantibodies remained detectable in the patient's serum during this same period (Figure 3, Figure 4), con rming that circulating TRPM1 autoantibodies alone are not su cient to cause visual impairment (Duvoisin et al., 2019).Instead, these data suggest additional pathological factors may be required for autoantibodies to inhibit retinal function.Given that the blood-retinal barrier (BRB) typically prevents antibodies and other macromolecules from accessing the retina, anti-retinal autoantibodies could circulate through the blood yet be restricted from ON-BCs, in which case no visual impairment would occur (Duvoisin et al., 2019;Platt et al., 2017).This concept is supported by a previous study demonstrating the presence of TRPM1 autoantibodies in patients without self-reported visual symptoms (Duvoisin et al., 2019).Anti-retinal autoantibodies must rst circumvent this barrier before reaching the retina, suggesting that BRB damage or malfunction may be a prerequisite for the development of MAR symptoms.
As in ammation is a known modulator of BRB integrity (Byrne et al., 2021;Capaldo et al., 2014;Ferreira et al., 2023;Frank and Lisanti, 2008;Ogura et al., 2017), we sought to examine the in ammatory state of the eye to assess the potential for BRB damage in this MAR patient.Our cytokine array detected a heightened in ammatory state in the patient's aqueous humor on all three collection dates when compared to a non-MAR control cohort (Figure 1B).Furthermore, several cytokines linked to BRB or blood-brain barrier (BBB) damage appeared elevated in this patient and their persistence could potentially have reduced the integrity of the BRB and enabled autoantibody access to the retina (Platt et al., 2017).Coinciding with the heightened risk of BRB damage caused by in ammation, uorescein angiograms taken on September 1, 2021 revealed multiple sites of vascular leakage to indicate the loss of BRB integrity (Supplementary Figure 2).In response to treatment with intraocular dexamethasone, these leakage sites resolved and the patient's vision improved, supporting the hypothesis that in ammation damages the BRB, allowing for the passage of autoantibodies and the onset of MAR symptoms.The reappearance of vascular leakage on uorescein angiograms February 23, 2022 may   Anti-retinal autoantibodies in the sera were then visualized by incubating the tissue with an Alexa Fluor 488-conjugated anti-human secondary antibody (green).The anti-TRPM1 monoclonal antibody was visualized using an Alexa Fluor 488-conjugated anti-mouse secondary antibody (green).In all three serum draws, autoantibodies were seen to label bipolar cells.However, the intensity of anti-bipolar cell staining was noticeably higher on sections labeled with the sera from 08/04/22 and 01/10/23.The ON-BC labeling visible in the WT retina is signi cantly reduced in the TRPM1 KO retina, indicating the Figure 4

Figures Figure 1
Figures

Figure 3 TRPM1
Figure 3 majority of ON-BC labeling in the WT retina is caused by autoantibodies against TRPM1.Residual bipolar cell staining in the TRPM1 KO retina is likely caused by additional anti-retinal autoantibodies against other antigens.The similarity in the staining pattern between the anti-TRPM1 monoclonal antibody and the MAR serum on the WT retina is further evidence that the retinal autoantibodies in the MAR patient serum primarily target TRPM1.Scale bars = 10µ; ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer.
stages of treatment, the concentrations of forty in ammatory cytokines were examined in aqueous humor samples using a cytokine array (Figure1B).Fifteen cytokines, which were undetectable in a non-MAR control cohort, appear elevated in the MAR patient, several of which have been implicated in regulating BRB integrity, including IL-1β, IL-17A, TNFα , and VEGF-A (Figure1B) (Byrne et al., 2021; Capaldo et al., 2014; Capaldo and Nusrat, 2009; Ferreira et al., 2023; Frank and Lisanti, 2008).In accordance with the heightened in ammatory state, evidence of vascular leakage indicative of a compromised BRB was visible in the patient's uorescein angiograms on September 1, 2021 (Figure1A).Over two months later in November 2021, the vascular leakage appeared resolved, following the initiation of dexamethasone treatment on October 6, 2021.ByFebruary 23, 2022, leakage reappeared at one site in the left eye within the superior arcade, which correlated with regression of symptoms and abnormal ffERG in the left eye.
Yang et al., 2011).Currently, it is not understood how TRPM1 autoantibodies cross the BRB to reach the retina.Several studies report that in ammation in or near the eye can negatively impact the integrity of the BRB(Bora etal., 2023; Byrne et al., 2021; Capaldo et al., 2014; Capaldo and Nusrat, 2009; Ferreira et al., 2023; Ogura et al., 2017; Platt et al., 2017), which may present an avenue for circulating proteins, such as autoantibodies, to access this restricted space.To assess the in ammatory status of the patient's eyes at several