TRPM1 Autoantibodies in Melanoma Patients Without Self-Reported Visual Symptoms

Abstract

Purpose: Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with cutaneous malignant melanoma (CMM). Visual symptoms include night blindness, photopsia, and reduced-contrast sensitivity. An abnormal ERG b-wave and the presence of anti-bipolar cell autoantibodies, including autoantibodies reacting with the ON-bipolar cell TRPM1 channel, help to confirm the diagnosis. The goal of this study was to determine if CMM patients without visual symptoms also express anti-TRPM1 autoantibodies.

Methods: Serum samples from 15 CMM patients were tested using three assays: immunofluorescent labeling of TRPM1-transfected HEK cells, immunofluorescent labeling of retinal sections from wild-type and TRPM1 knockout mice, and immunoblot detection of a bacterially produced recombinant TRPM1 peptide.

Results: Serum specimens from 5 of the 15 CMM patients without declared visual symptoms were positive for anti-TRPM1 autoantibodies in at least one of the three assays. One of 50 control sera from patients not known to have cancer was also weakly reactive with the TRPM1 peptide.

Conclusions: Autoantibodies against TRPM1 are present in CMM patient sera without self-reported visual symptoms. Most patients had advanced (stage III and IV) disease and were undergoing aggressive treatments, including immunotherapy. It is unknown if immunotherapy affects the expression of TRPM1 autoantibodies. The presence of TRPM1 autoantibodies may predispose patients for MAR.

Figure 1

CMM patient sera react with TRPM1 in transfected cells and label retinal bipolar cells. (A) HEK293 cells transiently transfected with the N-terminal, cytoplasmic domain of human TRPM1 fused to GFP were labeled by immunofluorescence with CMM patient sera. The GFP fluorescence identifies the transfected cells, and untransfected cells serve as a control for background immunofluorescence. The left panels show immunoreactivity with CMM patient sera; the GFP images (middle) show cells transfected with TRPM1-GFP; DIC images (right) show all cells (transfected and untransfected). The scale bar represents 20 μm. (B) Serum samples CMM01 and CMM14 label retinal bipolar cells by immunofluorescence on mouse retinal cryosections. Scale bar represents 10 μm.

Figure 2

TRPM1 autoantibodies from CMM patients bind to a discrete region within the N-terminal cytoplasmic domain of TRPM1. HEK293 cell extracts with (+) and without (−) recombinant TRPM1 peptide were Western blotted with serum from a melanoma patient diagnosed with MAR, sera from five melanoma patients without a MAR diagnosis (CMM01, -03, -13, -14, -15), and sera from six patients with no known cancer (N1-N6).