Preferentially Expressed Antigen in Melanoma Immunohistochemistry Labeling in Uveal Melanomas

Abstract

Introduction: Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, and despite treatment of the primary tumor, approximately 15%-50% of patients will develop metastatic disease. Based on gene expression profiling (GEPs), UM can be categorized as Class 1A (low metastatic risk), Class 1B (intermediate metastatic risk), or Class 2 (high metastatic risk). PReferentially expressed Antigen in MElanoma (PRAME) status is an independent prognostic UM biomarker and a potential target for immunotherapy in metastatic UM. PRAME expression status can be detected in tumors using reverse-transcription polymerase chain reaction (RT-PCR). More recently, immunohistochemistry (IHC) has been developed to detect PRAME protein expression. Here, we employed both techniques to evaluate PRAME expression in 18 UM enucleations.

Methods: Tumor material from the 18 UM patients who underwent enucleation was collected by fine-needle aspiration before or during enucleation and sent for GEP and PRAME analysis by RT-PCR. Histologic sections from these patients were stained with an anti-PRAME monoclonal antibody. We collected patient demographics and tumor characteristics and included this with our analysis of GEP class, PRAME status by RT-PCR, and PRAME status by IHC. PRAME IHC and RT-PCR results were compared.

Results: Twelve males (12/18) and 6 females (6/18) with an average age of 60.6 years underwent enucleation for UM. TNM staging of the UM diagnosed Stage I in 2 patients (2/18), Stage II in 7 patients (7/18), Stage III in 8 patients (8/18), and Stage IV in 1 (1/18). GEP was Class 1A in 6 tumors (6/18), Class 1B in 6 tumors (6/18), and Class 2 in 6 tumors (6/18). PRAME IHC showed diffusely positive labeling of all UM cells in 2/18 enucleations; negative IHC labeling of UM cells in 9/18 enucleations; and IHC labeling of subsets of UM cells in 7/18 enucleations. Eleven of the 17 UMs tested for PRAME by both RT-PCR and IHC had consistent PRAME results. In the remaining 6/17 cases tested by both modalities, PRAME results were discordant between RT-PCR and IHC.

Conclusions: We find that PRAME IHC distinguishes PRAME-positive and PRAME-negative UM tumor cells. Interestingly, IHC reveals focal PRAME expression in subsets of tumor cells consistent with tumor heterogeneity. PRAME RT-PCR and IHC provide concordant results in most of our cases. We suggest that discordance in PRAME results could arise from spatial or temporal variation in PRAME expression between tumor cells. Further studies are required to determine the prognostic implications of PRAME IHC in UM.

Keywords: Immunohistochemistry; Metastasis; Preferentially expressed antigen in melanoma; Prognosis; Reverse-transcription polymerase chain reaction; Uveal melanoma.

Fig. 1

UM enucleation (case 1) with diffusely positive PRAME IHC expression. a Cross-section of enucleation at the pupil-optic nerve shows choroidal melanoma tumor toward the superior-posterior pole (H&E stain). b High-power image of the malignant melanoma cells in the tumor (H&E stain). c MART-1 immunolabeling (red cytosolic staining) highlights melanoma cells. d PRAME immunolabeling (brown nuclear staining) with diffuse immunolabeling of tumor cells. Nuclei in b–d are counterstained with hematoxylin (blue). Scale bars, 2 mm (a) and 20 μm (b–d).

Fig. 2

UM with negative PRAME IHC expression (case 17). a Cross-section of enucleation shows choroidal melanoma tumor toward the posterior pole (H&E stain). b High-power image of the malignant melanoma cells in the tumor (H&E stain). c MART-1 immunolabeling (red cytosolic staining) highlights melanoma cells. d PRAME immunolabeling (brown nuclear staining) is negative. Nuclei in b–d have been counterstained with hematoxylin (blue). Scale bars, 2 mm (a) and 20 μm (b–d).

Fig. 3

UM with focally positive PRAME expression (case 16). a Cross-section of enucleation shows choroidal melanoma tumor toward the inferior pole (H&E stain). b Low-power image of the malignant melanoma cells in the tumor (H&E stain). c MART-1 immunolabeling (red cytosolic staining) highlights the tumor cells. d–f Focally positive PRAME immunolabeling with intermixed PRAME-positive (brown nuclear staining) and PRAME-negative tumor cells (no brown nuclear staining) present in the same enucleation. Nuclei in (b–f) have been counterstained with hematoxylin (blue). Scale bars, 2 mm (a), 50 μm (b–d), and 10 μm (e, f).

Fig. 4

UM with focally positive PRAME expression (case 14). a Cross-section of enucleation shows choroidal melanoma tumor toward the inferior pole (H&E stain). b Low-power image of the malignant melanoma cells in the tumor (H&E stain). c MART-1 immunolabeling (red cytosolic staining) highlights the tumor cells. d–f Focally positive PRAME immunolabeling (brown nuclear staining) with intermixed PRAME-positive and PRAME-negative tumor cells in the same enucleation. Nuclei in (b–f) have been counterstained with hematoxylin (blue). Scale bars, 2 mm (a), 50 μm (b–d), and 10 μm (e, f).