Purpose: Uveal melanoma is a rare melanoma variant with no effective therapies once metastases develop. Although durable cancer regression can be achieved in metastatic cutaneous melanoma with immunotherapies that augment naturally existing antitumor T-cell responses, the role of these treatments for metastatic uveal melanoma remains unclear. We sought to define the relative immunogenicity of these two melanoma variants and determine whether endogenous antitumor immune responses exist against uveal melanoma.

Experimental Design: We surgically procured liver metastases from uveal melanoma (n = 16) and cutaneous melanoma (n = 35) patients and compared the attributes of their respective tumor cell populations and their infiltrating T cells (TIL) using clinical radiology, histopathology, immune assays, and whole-exomic sequencing.

Results: Despite having common melanocytic lineage, uveal melanoma and cutaneous melanoma metastases differed in their melanin content, tumor differentiation antigen expression, and somatic mutational profile. Immunologic analysis of TIL cultures expanded from these divergent forms of melanoma revealed cutaneous melanoma TIL were predominantly composed of CD8+ T cells, whereas uveal melanoma TIL were CD4+ dominant. Reactivity against autologous tumor was significantly greater in cutaneous melanoma TIL compared with uveal melanoma TIL. However, we identified TIL from a subset of uveal melanoma patients which had robust antitumor reactivity comparable in magnitude with cutaneous melanoma TIL. Interestingly, the absence of melanin pigmentation in the parental tumor strongly correlated with the generation of highly reactive uveal melanoma TIL.

Conclusions: The discovery of this immunogenic group of uveal melanoma metastases should prompt clinical efforts to determine whether patients who harbor these unique tumors can benefit from immunotherapies that exploit endogenous antitumor T-cell populations. Clin Cancer Res; 22(9); 2237–49. ©2015 AACR.

Translational Relevance

Although remarkable strides have been achieved in the management of metastatic cutaneous melanoma with T-cell–based immunotherapies, limited progress has been made with metastatic uveal melanoma, a rare and aggressive variant that is hypothesized to be immunotherapy-resistant. In this study, we sought to formally define the relative immunogenicity of these two melanoma variants and determine whether endogenous antitumor immune responses exist against uveal melanoma. Here, we report the novel identification of TIL from a subset of uveal melanoma metastases with robust antitumor reactivity, comparable in magnitude with that of cutaneous melanoma TIL. The discovery of this immunogenic group of uveal melanoma metastases has important clinical implications for the role of immunotherapies in the treatment of patients who harbor these unique tumors.

Uveal melanoma is a rare and aggressive variant of melanoma that has specific origin within the vascular layers of the eye, including the choroid, ciliary body, and iris (collectively known as the uvea; ref. 1). Although uveal melanoma is the most common intraocular tumor in adults, it accounts for only 3% of all melanomas (2). With an annual incidence of 5.1 per million in the United States, uveal melanoma is significantly less common than cutaneous melanomas. Interestingly, uveal melanoma and cutaneous melanoma have a shared lineage, with each arising from neural crest-derived melanocytes that are resident to their respective tissues of origin (3). Both forms of melanoma, consequently, share prominent expression of prototypic melanocytic differentiation antigens (MDA), such as MART-1, gp100, and tyrosinase (4–6). Despite these similarities, uveal melanoma can be distinguished from cutaneous melanoma by characteristic cytogenetic changes (7) and an unusual predilection to primarily metastasize to the liver (1). Further, there exists a striking dichotomy between the clinical management of patients with advanced uveal melanoma and cutaneous melanoma. Immunotherapies have become the main treatment modality for metastatic cutaneous melanoma based upon substantial evidence that tumor antigens expressed by cutaneous melanoma can be vigorously recognized by T-cell populations endogenous to the host immune system (8). By clinically augmenting these immune responses with either systemic cytokines (9), antibodies targeting T-cell checkpoint molecules (10, 11), or adoptive transfer of autologous tumor-infiltrating lymphocytes (TIL; ref. 12), significant and potentially curative cancer regression can now be achieved in advanced cutaneous melanoma patients. However, the role of these immune-based therapies for the treatment of metastatic uveal melanoma remains unclear. Patients with uveal melanoma are frequently excluded from metastatic melanoma immunotherapy clinical trials because uveal melanoma is generally thought to be an immunotherapy-resistant subtype of melanoma. It has been speculated that because the primary tumor arises in the eye, an immune-privileged site, the tumor and its metastases harbor local immunosuppressive or cellular immuno-evasive factors that render immunotherapies unsuccessful (13–16). Another theory proposes that because uveal melanoma tumors have far fewer somatic mutations compared with sun-exposed cutaneous melanoma tumors (17), there are consequentially fewer potential mutated neoepitope targets for effective antitumor immunity. The poor immunogenicity of uveal melanoma has been further suggested based upon the comparatively low response rates seen in uveal melanoma patients enrolled into small pilot trials of immune-modulating agents, such as IL2 (18) and anti–CTLA-4 antibody (19–21). Collectively, these observations have fostered the prevalent belief that uveal melanoma, in distinction to cutaneous melanoma, is a nonimmunogenic form of melanoma. However, this hypothesis has largely been based upon inference without formal comparative studies performed directly upon uveal melanoma and cutaneous melanoma metastases to accurately assess their relative immunogenicity. In this study, we aimed to address this deficiency by comparing tumor antigen expression, tumor mutational load, and endogenous antitumor immunologic reactivity found in fresh surgically resected uveal melanoma versus cutaneous melanoma metastases. By determining whether tumor-specific immune responses naturally exist against uveal melanoma metastases, we sought to provide insight into the management of this rare melanoma variant with immunotherapies that can exploit these endogenous T-cell populations.

Study population

A retrospective review of a prospectively maintained database identified 49 patients who underwent liver metastasectomy with a diagnosis of metastatic melanoma at the Surgery Branch of the NCI between 2004 and 2014. All patients signed an Institutional Review Board–approved consent for tumor tissue procurement and participation in subsequent immunotherapy protocols if the patient required further systemic therapy. Inclusion criteria included pathologically confirmed melanoma, 16 years of age or older, negative serology for HIV, Hepatitis B and C, good performance status (Eastern Cooperative Oncology Group ≤2), and life expectancy greater than 3 months. Patients were stratified into two cohorts based upon the anatomic origin of their primary melanoma. The cutaneous melanoma cohort included 35 patients; 33 of these patients had documented primary tumors arising from the cutaneous epithelium and 2 additional patients had primary tumors of unknown origin. The uveal melanoma cohort included 14 patients who had ophthalmologic documentation that their primary melanoma tumors arose specifically from the uveal tract. Patients with documented primary tumors arising from mucosal and conjunctival sites were excluded from analysis.

Tumor procurement

Patients typically underwent resection of a single metastatic liver deposit or a closely approximated cluster of tumors using standardized hepatobiliary surgical techniques. Immediately upon resection, the fresh tumor underwent pathologic assessment, dissection, and processing in the Surgery Branch Cell Production facility in conjunction with a clinical surgical pathologist and research staff. Tumor tissue was assigned a unique liver metastasis identification number (ID #) and allocated for gross and histopathologic analysis, mutational analysis, and TIL culture establishment using methods as described below. Although the main study exclusively focused upon liver metastases, a set of extrahepatic metastases from 8 additional uveal melanoma patients were incorporated into the tumor driver mutational analysis, as described below.

In-situ MRI assessment of tumor melanin content

All patients underwent preoperative MRI liver imaging as part of their radiographic tumor staging. Quantitative T1-weighted signal intensity measurements (without gadolinium enhancement) of the in-situ liver metastases and adjacent normal tissue were obtained using clinical radiology imaging software (Carestream Vue Solutions; version 11.3). Mean tumor and normal intensity were calculated by averaging three separate signal intensity measurements. Hyperintense tumors were defined as having a mean tumor/normal (T/N) intensity ratio > 1.5. Hypointense tumors had a mean T/N ratio < 0.7. Mixed intensity tumors had both hyperintense and hypointense components. The T/N signal intensity ratio for each liver metastasis was objectively calculated for each metastasis and scored as either hyperintense (2+), mixed intensity (1+), or hypointense (0), as illustrated in Supplementary Fig. S1.

Gross pathologic assessment of tumor melanin pigmentation

After surgical resection, all liver metastases underwent independent gross pathologic assessment and photo documentation by a board-certified pathologist who was blinded to the comparative analysis. Each metastasis underwent serial sectioning to assess their melanin pigmentation. Tumors were scored based on their level of pigmentation as either hyperpigmented (2+), mixed pigmented (1+), or hypopigmented (0).

Immunohistochemical staining analysis of tumor metastases

Surgically resected tumor specimens were fixed in 10% neutral-buffered formalin for up to 24 hours and routinely processed. Paraffin-embedded tissue sections of 5 mm were deparaffinized through xylene and graded series of alcohols. Immunohistochemical staining was performed following heat-induced epitope retrieval with target retrieval solution (low pH; DAKO). Slides were incubated in Tris with 3% goat serum for 15 minutes and then incubated at room temperature with primary antibody for 1 to 2 hours. Immunohistochemical staining was carried out using the Dako Autostainer or Ventana BenchMark XT Slide Stainer (for CD3 antibody) using the manufacturer-supplied reagents and standard protocols with the following primary antibodies: MART-1 (no. CMC756, 1:200; Cell Marque); HMB45 (no. 30930, 1:4; Enzo Life Sciences); Tyrosinase (no. NCL-TYROS, 1:20; Novocastra Division, Leica Microsystems); MHC Class I (HC-10, 1:1,000; provided by Dr. Soldano Ferrone); HLA-DR (TAL.1B5, 1:200; DAKO); CD20 (L26, 1:500; DAKO); CD8 (CD8/144B, 1:50; DAKO); CD4 (1F6, 1:80; Novacastra); CD3 (2GV6, prediluted; Ventana). Detection was carried out using an automated slide stainer (Autostainer; DAKO) with either horseradish peroxidase/3,3′-diaminobenzidine polymer–based detection system (Envision+; DAKO) or a red chromogen (Liquid Permanent Red Substrate-Chromogen; DAKO) for darkly pigmented tumors. The immunohistochemical staining was prospectively assessed and quantitated by two board-certified pathologists who were blinded to the comparative analysis of the study. The percentage of viable tumor cells expressing a given marker was quantified as 0% to 5%, 6% to 50%, or >50%. Staining intensity for each marker was graded on a scale of 0 (no staining), 1+, 2+, or 3+ (high intensity staining). Lymphocytic infiltrate was assessed with CD4, CD8, CD3, and CD20 staining and quantified based upon the percentage of tumor occupied by infiltrating lymphocytes as 0 (no lymphocytes detected), 1+ (<5% of tumor field), 2+ (5%–50% of tumor field), or 3+ (extensive lymphoid aggregation occupying over 50% of the tumor field).

Generation and assessment of TIL cultures

Geographically discrete 1 to 2 mm3 tumor fragments (n = 24) were freshly dissected from each tumor metastasis and placed individually in wells of a 24-well culture plate containing complete media with human AB serum and recombinant IL2 (3,000 IU/mL) as previously described (22). After approximately 2 weeks of culturing, each of the wells was assessed for successful TIL expansion based upon cell count and visual inspection. Expanded TIL cultures underwent flow cytometric phenotypic analysis after staining with anti-human CD3, CD8, and CD4 monoclonal antibodies and their respective isotype controls (BD Biosciences). Immunofluorescence, analyzed as the relative log fluorescence of live cells, was measured using a FACSCanto II flow cytometer with FACSDiva software (BD Biosciences) and FlowJo software (Tree Star, Inc.). The specific antitumor reactivity of individual TIL cultures was assessed by coculture with autologous tumor digest which had been freshly cryopreserved at the time of surgical procurement. Briefly, TIL cells (1 × 105 cells) and autologous tumor digest (1 × 105 cells) were coincubated in a 0.2-mL volume in individual wells of a 96-well plate. Supernatants were harvested from duplicate wells after 20 to 24 hours, and IFNγ secretion was measured in culture supernatants using commercially available IFNγ ELISA kits (Endogen). All data are presented as a mean of duplicate samples. Cultures with IFNγ production greater than 100 pg/mL and twice background of unstimulated TIL and autologous tumor digest alone were considered as having specific antitumor reactivity.

Whole-exomic sequencing and driver mutational analysis

Exome libraries were prepared from paired uveal melanoma metastasis and normal samples using the Agilent SureSelectXT Human All Exon V5+UTR target enrichment Kit as per the manufacturer's protocols (Agilent). The samples were pooled three samples per lane and sequenced on an Illumina HiSeq2000 sequencer with TruSeq V3 chemistry (paired-end, 101 bp read length). Basecalling was carried out using Illumina RTA 1.12.4.2 run-time analysis software, and demultiplexing was carried out using Casava 1.8.2. Each sample had >99 million pass filtered reads, with >92% bases having a base quality value >Q30 (Q30: The percentage of bases called with an inferred accuracy of 99.9% or above, a measure of basecalling quality). The percentage of unique library fragments was >90% across all samples. The capture efficiency as measured by the percentage of the reads mapping on the target regions was >60%. The mean coverage on target regions for all samples was between 60× to 90× with >82.9% of the target regions having at least 30× coverage. The quality of the raw reads was assessed using FastQC (23) and NGS QC toolkit (24). Reads were trimmed and filtered for adapters using Trimmomatic (25). Alignment was carried out to the human Hg19 reference sequence using BWA-0.7.4 (26). Alignment files were indexed, sorted, and duplicates were removed. Realignment around InDels and base-quality score recalibration was carried out as per the GATK best practices for exome-seq analysis (27). MuTect was used in the high-confidence (HC) mode for calling somatic point mutations (28). The subset of calls that passed the HC filters after the statistical analysis within MuTect was annotated using Annovar (29) to find both the location and the functional significance of the mutations. Mutations were filtered to keep only those that had Mutation_info: exonic or splicing only; Consequence: non-synonymous or stopgain_SNV. To serve as a reference group, whole-exomic sequencing (WES) data were obtained for 278 cutaneous melanoma metastases via the “Skin Cutaneous Melanoma” (SKCM) data portal of The Cancer Genome Atlas (TCGA). Somatic mutation counts for the metastatic patient samples for the SKCM cohort were extracted from the Level 2 MAF file downloaded off the GDAC Firehose resource (30). As only somatic point mutations were of interest while comparing the distribution of somatic mutations between cutaneous melanoma and uveal melanoma cohorts, InDel calls (1.2%) were removed from the TCGA-SKCM cohort. As the annotations for the point mutations in the TCGA-SKCM cohort did not have the same terminology as for the uveal melanoma cohort, the SKCM mutations were reannotated using Annovar, with the same filters applied based on the combination of “Consequence” and “Mutation_info” columns. The frequency among the cutaneous melanoma and uveal melanoma tumors for specific mutations in BRAF, GNAQ, and GNA11 was determined primarily from WES analysis. As validation in selected samples, library preparation with 10 to 20 ng genomic DNA was performed using Ion AmpliSeq Cancer Hotspot, Panel V2 and Ion AmpliSeq Library Kit 2.0, using the corresponding User Guide (Life Technologies). The amplicon panel includes 207 primer sets covering approximately 2,800 COSMIC hotspot mutations in 50 genes. Next-generation sequencing was performed in an Ion Torrent Personal genome machine, and analyzed with Torrent Suite Software (Life Technologies). Annotation and interpretation of all variants were performed in Ion Reporter that links to multiple databases, such as RefSeq, OMIM, Oncomine, COSMIC, and dbSNP. Reported mutations were confirmed by inspection of alignments using the Integrative Genomics Viewer (31).

Statistical analysis

The Fisher exact test was used to determine associations between dichotomous demographic parameters, and the Wilcoxon rank-sum test was utilized for the comparison of continuous parameters such as patient age. Nonparametric comparisons between the uveal melanoma and cutaneous melanoma cohorts were performed with the Mann–Whitney test. The Student t test was used to compare the means of parametric variables. Linear regression analysis was used for correlation studies and presented as R2 values. All P values are two-tailed and have not been adjusted for multiple comparisons. In view of the exploratory analyses performed, P < 0.05 would be considered statistically significant, whereas 0.05 < P < 0.1 would be considered a trend. Excel and GraphPad Prism (v6.01) were used for analyses.

Patient demographics and procurement of liver metastases

Between 2004 and 2014, a total of 49 patients with metastatic melanoma underwent liver metastasectomy in the context of approved clinical trials in the Surgery Branch, NCI. The current study selectively analyzed liver metastases because of our previous finding that human melanoma metastases demonstrate significant heterogeneity in tumor antigen expression and lymphocytic infiltrate when stratified based upon their anatomic location in the body (6). Further, because uveal melanoma predominantly metastasizes to the liver, this homogeneous source of metastases would prevent potential site-specific bias in our comparative assessment of tumors. Patients undergoing liver metastasectomy were stratified into two cohorts, cutaneous melanoma and uveal melanoma, based upon the anatomic origin of their primary melanomas. The cutaneous melanoma cohort included 35 patients; 33 of whom had documented primary tumors arising from the cutaneous epithelium and 2 additional patients had primary tumors of unknown origin. Patients with melanoma of unknown origin were included in the cutaneous melanoma cohort based upon recent molecular genetic studies which revealed these tumors strongly resembled cutaneous melanomas (32). The uveal melanoma cohort included 14 patients who had ophthalmologic documentation that their primary melanoma arose specifically from the uveal tract. The characteristics for each of the patients who underwent liver metastasectomy are shown in Table 1, and the comparison of the cutaneous melanoma and uveal melanoma cohorts is shown in Table 2. The age (mean and range) and gender distribution of the patients in the two cohorts were similar. At the time of referral to our center, there was a greater trend for the uveal melanoma patients to have not received prior systemic therapy for their metastatic disease when compared with the cutaneous melanoma patients (uveal melanoma: 71% vs. cutaneous melanoma: 37%, P = 0.06). This finding likely reflected the growing availability of approved systemic agents and clinical trial opportunities for patients with metastatic cutaneous melanoma during the study period. Of note, 63% of the cutaneous melanoma patients had undergone prior systemic immunotherapy, whereas only 21% of uveal melanoma patients had received such treatments (cutaneous melanoma vs. uveal melanoma, P = 0.01). Although both melanoma cohorts demonstrated metastatic spread to extrahepatic sites, the uveal melanoma patients had metastases more often confined to the liver (uveal melanoma: 43% vs. cutaneous melanoma: 14%, P = 0.05), whereas the cutaneous melanoma patients demonstrated a trend toward more frequent metastases to lymph nodes and soft tissues (cutaneous melanoma: 60% vs. uveal melanoma: 29%, P = 0.06). For surgical tumor procurement, patients typically underwent resection of a single metastatic liver deposit or a closely approximated cluster of tumors. The size (mean/median) of the tumor deposits resected from the patients was not significantly different between the groups (cutaneous melanoma: 6.2/6.0 cm vs. uveal melanoma: 7.2/5.5 cm, P = 0.50). The cumulative metastatic tissue that was procured at operation was assigned a unique liver metastasis identification number (ID #) for subsequent analysis. Two uveal melanoma patients (1 and 3) developed metachronous liver metastases during the study period. These patients underwent two independent liver metastasectomy operations, and their individual tumor procurements were assigned unique liver metastasis ID #. Thus, in sum, there were 35 cutaneous melanoma liver metastases and 16 uveal melanoma liver metastases that were available for direct comparative analysis.

Table 1.

Patient demographics and procurement of liver metastases

Patient ID #AgeGenderPrimary siteSites of metastasisPrior systemic therapiesLiver metastasis ID #
Cutaneous melanoma 
35 Neck Li Immuno L-CM1 
51 Arm Li, Lu None L-CM2 
46 Scalp Li, Lu Immuno, chemo L-CM3 
62 Shoulder Li, Lu, ST None L-CM4 
44 Hip Li, Lu, kidney, LN Immuno, chemo L-CM5 
66 Back Li, LN Immune L-CM6 
61 Back Li, LN Immuno L-CM7 
44 Sacral Li, Lu, LN Immune L-CM8 
64 Back Li, ST None L-CM9 
10 37 Back Li, Lu, Bn, LN Immuno L-CM10 
11 54 Shoulder Li Immuno L-CM11 
12 42 Back Li, LN Immuno L-CM12 
13 47 Infra-auricular Li, Lu None L-CM13 
14 35 Unknown Li, Lu, LN Immuno, chemo L-CM14 
15 64 Parotid Li Immuno L-CM15 
16 62 Ear Li, Lu Immuno L-CM16 
17 50 Neck Li, Spl, Panc Immuno, chemo L-CM17 
18 62 Foot Li, Lu, Spl, LN None L-CM18 
19 45 Shoulder Li, LN None L-CM19 
20 57 Back Li Immuno L-CM20 
21 54 Back Li, ST None L-CM21 
22 50 Back Li, Lu Immuno L-CM22 
23 57 Back Li, Lu, ST None L-CM23 
24 69 Scalp Lu Immuno L-CM24 
25 48 Unknown Li, LN, ST, Bn None L-CM25 
26 55 Leg Li, Lu, LN None L-CM26 
27 65 Scalp Li, Lu, LN Immuno L-CM27 
28 53 Shoulder Li, Bn None L-CM28 
29 34 Jaw Li, Lu, LN, Bn Immuno L-CM29 
30 46 Flank Li Immuno, chemo, targeted L-CM30 
31 52 Thigh Li, LN, Adrenal Immuno L-CM31 
32 62 Back Li, LN None L-CM32 
33 56 Leg Li, LN Immuno L-CM33 
34 19 Preauricular Li, LN, Spl, Bn Immuno L-CM34 
35 27 Ear Li, Lu None L-CM35 
Uveal melanoma 
56 Uveal tract Li, Bn Immuno, TACE L-UM1a, L-UM1b 
56 Uveal tract Li TACE L-UM2 
47 Uveal tract Li, Lu, Panc, Bn None L-UM3a, L-UM3b 
68 Uveal tract Li None L-UM4 
60 Uveal tract Li, Lu, ST, LN, Per Immuno L-UM5 
55 Uveal tract Li None L-UM6 
64 Uveal tract Li None L-UM7 
56 Uveal tract Li None L-UM8 
17 Uveal tract Li, Lu, heart, LN Immuno L-UM9 
10 54 Uveal tract Li, LN None L-UM10 
11 58 Uveal tract Li, ST None L-UM11 
12 53 Uveal tract Li, Bn, Per, Om None L-UM12 
13 52 Uveal tract Li None L-UM13 
14 62 Uveal tract Li, Lu None L-UM14 
Patient ID #AgeGenderPrimary siteSites of metastasisPrior systemic therapiesLiver metastasis ID #
Cutaneous melanoma 
35 Neck Li Immuno L-CM1 
51 Arm Li, Lu None L-CM2 
46 Scalp Li, Lu Immuno, chemo L-CM3 
62 Shoulder Li, Lu, ST None L-CM4 
44 Hip Li, Lu, kidney, LN Immuno, chemo L-CM5 
66 Back Li, LN Immune L-CM6 
61 Back Li, LN Immuno L-CM7 
44 Sacral Li, Lu, LN Immune L-CM8 
64 Back Li, ST None L-CM9 
10 37 Back Li, Lu, Bn, LN Immuno L-CM10 
11 54 Shoulder Li Immuno L-CM11 
12 42 Back Li, LN Immuno L-CM12 
13 47 Infra-auricular Li, Lu None L-CM13 
14 35 Unknown Li, Lu, LN Immuno, chemo L-CM14 
15 64 Parotid Li Immuno L-CM15 
16 62 Ear Li, Lu Immuno L-CM16 
17 50 Neck Li, Spl, Panc Immuno, chemo L-CM17 
18 62 Foot Li, Lu, Spl, LN None L-CM18 
19 45 Shoulder Li, LN None L-CM19 
20 57 Back Li Immuno L-CM20 
21 54 Back Li, ST None L-CM21 
22 50 Back Li, Lu Immuno L-CM22 
23 57 Back Li, Lu, ST None L-CM23 
24 69 Scalp Lu Immuno L-CM24 
25 48 Unknown Li, LN, ST, Bn None L-CM25 
26 55 Leg Li, Lu, LN None L-CM26 
27 65 Scalp Li, Lu, LN Immuno L-CM27 
28 53 Shoulder Li, Bn None L-CM28 
29 34 Jaw Li, Lu, LN, Bn Immuno L-CM29 
30 46 Flank Li Immuno, chemo, targeted L-CM30 
31 52 Thigh Li, LN, Adrenal Immuno L-CM31 
32 62 Back Li, LN None L-CM32 
33 56 Leg Li, LN Immuno L-CM33 
34 19 Preauricular Li, LN, Spl, Bn Immuno L-CM34 
35 27 Ear Li, Lu None L-CM35 
Uveal melanoma 
56 Uveal tract Li, Bn Immuno, TACE L-UM1a, L-UM1b 
56 Uveal tract Li TACE L-UM2 
47 Uveal tract Li, Lu, Panc, Bn None L-UM3a, L-UM3b 
68 Uveal tract Li None L-UM4 
60 Uveal tract Li, Lu, ST, LN, Per Immuno L-UM5 
55 Uveal tract Li None L-UM6 
64 Uveal tract Li None L-UM7 
56 Uveal tract Li None L-UM8 
17 Uveal tract Li, Lu, heart, LN Immuno L-UM9 
10 54 Uveal tract Li, LN None L-UM10 
11 58 Uveal tract Li, ST None L-UM11 
12 53 Uveal tract Li, Bn, Per, Om None L-UM12 
13 52 Uveal tract Li None L-UM13 
14 62 Uveal tract Li, Lu None L-UM14 

Abbreviations: Bn, Bone; Li, Liver; LN, lymph node; Lu, lung; Om, omentum; Panc, pancreas; Per, peritoneum; Spl, spleen; ST, soft tissue.

Table 2.

Comparison of CM and UM patients who underwent liver metastasectomy

CMUMP value
Number of patients 35 14  
Age (mean) 51 54 0.157 
(Range) 19–69 17–68  
Gender, n(%) 
 F 13 (37) 7 (50) 0.52 
 M 22 (63) 7 (50)  
Prior systemic therapy, n(%) 
 Immunotherapy 22 (63) 3 (21) 0.01 
 Chemotherapy 5 (14) 0 (0) 0.30 
 None 13 (37) 10 (71) 0.06 
Extrahepatic disease, n(%) 
 Lung 17 (49) 4 (29) 0.21 
 LN/ST 21 (60) 4 (29) 0.06 
 None 5 (14) 6 (43) 0.05 
CMUMP value
Number of patients 35 14  
Age (mean) 51 54 0.157 
(Range) 19–69 17–68  
Gender, n(%) 
 F 13 (37) 7 (50) 0.52 
 M 22 (63) 7 (50)  
Prior systemic therapy, n(%) 
 Immunotherapy 22 (63) 3 (21) 0.01 
 Chemotherapy 5 (14) 0 (0) 0.30 
 None 13 (37) 10 (71) 0.06 
Extrahepatic disease, n(%) 
 Lung 17 (49) 4 (29) 0.21 
 LN/ST 21 (60) 4 (29) 0.06 
 None 5 (14) 6 (43) 0.05 

Abbreviations: CM, cutaneous melanoma; LN, lymph node; ST, soft tissue; UM, uveal melanoma.

Radiographic and pathologic comparison of the melanin content between cutaneous melanoma and uveal melanoma liver metastases

Both cutaneous melanoma and uveal melanoma primary tumors arise from transformed melanocytes. Yet, despite this common origin from melanin-producing cells, the metastases of these tumors can display significant heterogeneity in the quantitative expression of prototypic melanin-associated proteins (6). These prior findings prompted us to ask whether cutaneous melanoma and uveal melanoma metastases demonstrated fundamental differences in their melanin pigmentation. To address this question, we utilized preoperative clinical radiographic imaging and postoperative gross pathologic examination to evaluate the melanin content found in the liver metastases procured from cutaneous melanoma and uveal melanoma patients. A prior study characterizing melanoma metastases with clinical MRI imaging found the in-situ tumor signal intensity from T1-weighted sequences strongly correlated with the degree of melanin pigmentation found in those tumors after resection (33). For the current study, we utilized these defined MRI parameters to perform in-situ characterization of 30 liver metastases identified in cutaneous melanoma patients and 16 liver metastases in uveal melanoma patients. Quantitative T1-weighted signal intensity measurements (without gadolinium enhancement) of the in-situ tumor and adjacent normal tissue were obtained using clinical radiology imaging software (Carestream Vue Solutions; version 11.3). The normalized tumor signal intensity (relative to normal liver) was objectively calculated for each metastasis and scored as either hyperintense (2+), mixed intensity (1+), or hypointense (0), as illustrated in Supplementary Fig. S1. In addition, after surgical resection, metastases underwent independent gross pathologic examination, and tumor pigmentation was visually scored as either hyperpigmented (2+), mixed pigmented (1+), or hypopigmented (0). The comparison of preoperative in-situ MRI intensity scoring and postoperative pathologic pigmentation scoring for representative liver metastases is shown in Fig. 1A. Analysis of the entire population of liver metastases (n = 46) revealed a strong and direct correlation between the MRI and the pathologic scoring of pigmentation (R2 = 0.88, P < 0.0001) for the individual tumors. Next, we used these two parameters to independently compare the melanin content found in the cutaneous melanoma and uveal melanoma liver metastases. In-situ MRI tumor signal intensity was significantly different between the two melanoma cohorts (P = 0.003; Fig. 1B). Whereas, 70% of cutaneous melanoma metastases had hypointense (0) MRI signal, only 25% of uveal melanoma metastases displayed this low signal intensity. Conversely, the uveal melanoma cohort demonstrated a greater frequency of metastases with hyperintense (2+) MRI signal (uveal melanoma: 38% vs. cutaneous melanoma: 10%). When these same uveal melanoma and cutaneous melanoma metastases underwent pathologic examination after resection, we similarly found a significant difference in their gross pigmentation (P = 0.008; Fig. 1C). Cutaneous melanoma metastases were more often visually hypopigmented (0; cutaneous melanoma: 70% vs. uveal melanoma: 25%), and uveal melanoma metastases were more often hyperpigmentated (2+; uveal melanoma: 44% vs. cutaneous melanoma: 20%). Thus, we concluded that despite having common lineage from melanin-producing cells, cutaneous melanoma and uveal melanoma metastases displayed significant differences in their overall melanin content.

Figure 1.

Uveal melanoma (UM) liver metastases have greater melanin pigmentation when compared with cutaneous melanoma (CM) liver metastases. A, illustrative examples demonstrating the correlation between preoperative in-situ MRI intensity scoring and postoperative gross pathologic scoring of pigmentation in selected melanoma liver metastases. Arrows in the left plot indicate the tumors that underwent metastasectomy; right plot displays photos of the resected tumors. B, comparison of cutaneous melanoma and uveal melanoma liver metastases based upon MRI tumor signal intensity score and (C) gross pathologic pigmentation score. Numbers within the bubbles denote the percentage of tumors in the specified cohort with the indicated score. Bubble diameter is proportionate to these percentages. Statistical comparisons between cutaneous melanoma and uveal melanoma cohorts were performed with the Mann–Whitney test.

Figure 1.

Uveal melanoma (UM) liver metastases have greater melanin pigmentation when compared with cutaneous melanoma (CM) liver metastases. A, illustrative examples demonstrating the correlation between preoperative in-situ MRI intensity scoring and postoperative gross pathologic scoring of pigmentation in selected melanoma liver metastases. Arrows in the left plot indicate the tumors that underwent metastasectomy; right plot displays photos of the resected tumors. B, comparison of cutaneous melanoma and uveal melanoma liver metastases based upon MRI tumor signal intensity score and (C) gross pathologic pigmentation score. Numbers within the bubbles denote the percentage of tumors in the specified cohort with the indicated score. Bubble diameter is proportionate to these percentages. Statistical comparisons between cutaneous melanoma and uveal melanoma cohorts were performed with the Mann–Whitney test.

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Comparison of melanocyte differentiation antigens and MHC expression between cutaneous melanoma and uveal melanoma liver metastases

To better understand the differences observed in melanin pigmentation between the cutaneous melanoma and uveal melanoma liver metastases, we next performed immunohistochemistry (IHC) to compare the cellular expression of proteins associated with melanocyte differentiation. The tumor expression (percentage of viable cells and staining intensity) for MART-1, gp100, and tyrosinase was prospectively assessed by pathologists blinded to the comparative analysis (Fig. 2A). Among the cutaneous melanoma liver metastases, we found prominent heterogeneity in cellular MART-1 expression between individual metastases (interlesional heterogeneity) and within individual metastases (intralesional heterogeneity). In 20% of cutaneous melanoma metastases, MART-1 was either nearly absent (0%–5% of tumor cells) or expressed on a fraction of viable tumor cells (6%–50% of tumor cells). Further, only 34% of the cutaneous melanoma metastases displayed strong cellular staining (3+) for MART-1, whereas the remaining tumors had weak to intermediate staining (0 to 2+). In contrast, all of the uveal melanoma liver metastases displayed homogeneous, diffuse, and strong MART-1 staining (>50% of tumor cells with 3+ staining intensity). When the pattern of MART-1 expression was compared between the cutaneous melanoma and uveal melanoma liver metastases, we found that uveal melanoma tumors had significantly stronger MART-1 staining intensity (P < 0.0001) and a trend toward a greater percentage of MART-1–stained tumor cells (P = 0.08). Expression for gp100 was also greater in the uveal melanoma metastases compared with the cutaneous melanoma metastases based upon percentage of tumor cells stained (P = 0.05) and staining intensity (P = 0.01). Diffuse gp100 staining (>50% of tumor cells) was seen in 88% of uveal melanoma tumors versus only 58% of cutaneous melanoma tumors. Strong staining intensity (3+) for gp100 was found in 63% of uveal melanoma tumors versus only 30% of cutaneous melanoma tumors. Interestingly, tyrosinase expression (percentage of tumor cells and staining intensity) was highly variable in both cutaneous melanoma and uveal melanoma metastases and not significantly different between the melanoma cohorts (P = 0.52 and 0.37, respectively).

Figure 2.

Uveal melanoma (UM) liver metastases have greater expression of melanocyte differentiation antigens and lower MHC class II compared with cutaneous melanoma (CM) liver metastases. Comparison of cutaneous melanoma and uveal melanoma liver metastases based upon immunohistochemical staining of (A) prototypic melanocyte differentiation antigens and (B) MHC class I and II molecules. Top plots demonstrate expression based upon percentage of viable tumor cells within individual metastases that stain with the indicated marker; bottom plots demonstrate expression based upon tumor cell staining intensity. Numbers within the bubbles denote the percentage of tumors in the specified cohort with the indicated marker expression. Bubble diameter is proportionate to these percentages. Statistical comparisons between cutaneous melanoma and uveal melanoma cohorts were performed with the Mann–Whitney test.

Figure 2.

Uveal melanoma (UM) liver metastases have greater expression of melanocyte differentiation antigens and lower MHC class II compared with cutaneous melanoma (CM) liver metastases. Comparison of cutaneous melanoma and uveal melanoma liver metastases based upon immunohistochemical staining of (A) prototypic melanocyte differentiation antigens and (B) MHC class I and II molecules. Top plots demonstrate expression based upon percentage of viable tumor cells within individual metastases that stain with the indicated marker; bottom plots demonstrate expression based upon tumor cell staining intensity. Numbers within the bubbles denote the percentage of tumors in the specified cohort with the indicated marker expression. Bubble diameter is proportionate to these percentages. Statistical comparisons between cutaneous melanoma and uveal melanoma cohorts were performed with the Mann–Whitney test.

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Next, we compared the expression of MHC class I and II proteins on tumor cells in the cutaneous melanoma and uveal melanoma liver metastases (Fig. 2B). For tumor antigens to be recognized by T cells, antigens must be internally processed into peptides that are presented on the tumor cell surface by these MHC molecules. We found that MHC class I expression was equally expressed in both cohorts based upon percentage of tumor cells stained (P = 0.73) and staining intensity (P = 0.65). Diffuse MHC class I staining (>50% of tumor cells) was observed in the majority of tumors from both cohorts (cutaneous melanoma: 67% vs. uveal melanoma: 75%) with equally strong staining intensity (3+; cutaneous melanoma: 63% vs. uveal melanoma: 56%). In contrast, we found cutaneous melanoma tumors had significantly greater percentage of MHC class II–stained tumor cells (P = 0.04) and a trend toward stronger MHC class II staining intensity (P = 0.07). MHC class II expression was nearly undetectable (0%–5% of tumor cells) in 88% of uveal melanoma tumors versus only 55% of cutaneous melanoma tumors. In sum, these IHC studies demonstrated that uveal melanoma metastases had greater expression of melanocyte lineage antigens and lower expression of MHC class II molecules when compared with cutaneous melanoma metastases.

Comparison of TIL found in cutaneous melanoma and uveal melanoma liver metastases

High levels of TIL have been reported to correlate with favorable prognoses in a variety of solid organ malignancies (34–36). Specific immunologic studies of TIL expanded from cutaneous melanoma metastases have found that these infiltrating cells can often recognize antigens expressed by the tumor (37). Further, the autologous adoptive transfer of such TIL has shown durable and complete tumor regression in metastatic cutaneous melanoma (12). These findings have provided compelling evidence for the natural immunogenicity of cutaneous melanoma metastases. However, it is unclear whether uveal melanoma tumors can similarly elicit adaptive immune responses in vivo. To provide insight, we sought to compare the attributes of TIL found in uveal melanoma and cutaneous melanoma liver metastases. First, the degree of infiltrating T cells (CD3, CD4, and CD8 staining) and B cells (CD20 staining) associated with each of the metastases was prospectively assessed by pathologists blinded to the comparative analysis. From both tumor cohorts, we found significant heterogeneity in the numbers of peripheral and infiltrating T cells which ranged from no lymphocytes detected (0) to extensive lymphoid aggregation occupying over 50% of the tumor field (3+). When the cutaneous melanoma and uveal melanoma metastases were compared, we found no significant differences in the levels of peripheral and infiltrating CD3+, CD4+, or CD8+ T cells between the cohorts (Supplementary Fig. S2). Further, B cells (CD20+ cells) were undetectable in the majority of tumors and also not significantly different between the cohorts.

Having observed that the degree of lymphocytic infiltration was similar between the cutaneous melanoma and uveal melanoma liver metastases, we next sought to assess the phenotypic and functional attributes of the TIL after ex vivo expansion. Consecutive metastatic liver tumors were procured from 8 cutaneous melanoma and 13 uveal melanoma patients during a shared time period. To account for intratumoral heterogeneity that might influence TIL growth, 24 geographically discrete tumor fragments were freshly dissected from each of the metastases and placed in culture media containing human IL2 (3,000 IU/mL). After approximately 2 weeks of culturing, we found that the percentages of tumor fragments that could successfully generate TIL were equivalent between the cutaneous melanoma and uveal melanoma tumors (95% vs. 94%, respectively). Each of these independently expanded TIL cultures were then assessed by flow cytometry to determine their percentage of CD8+ and CD4+ T cells. We observed a significant difference between the cutaneous melanoma and uveal melanoma liver metastases in the ratio of these T-cell subsets (Fig. 3A). The TIL cultures from 88% of the cutaneous melanoma metastases (7 of 8 cutaneous melanoma metastases) were composed predominantly of CD8+ T cells. In contrast, only 23% of uveal melanoma metastases (3 of 13 uveal melanoma metastases) gave rise to CD8+-enriched TIL. Cumulatively, the mean percentage of CD8+ T cells in the cutaneous melanoma–derived TIL cultures was significantly greater than in the uveal melanoma–derived TIL (cutaneous melanoma: 71% vs. uveal melanoma: 42%, P < 0.0001; Fig. 3B). Conversely, uveal melanoma–derived TIL cultures possessed a greater mean percentage of CD4+ cells when compared with the cutaneous melanoma–derived TIL cultures (uveal melanoma: 49% vs. cutaneous melanoma: 21%, P < 0.0001; Fig. 3B).

Figure 3.

Phenotypic and functional comparison of TIL cultures derived from cutaneous melanoma (CM) and uveal melanoma (UM) liver metastases. A, mean percentage of CD8+ (filled box) and CD4+ (open box) T cells found in TIL cultures derived from individual cutaneous melanoma and uveal melanoma liver metastases. T-cell subset frequency as assessed by flow cytometry and CD3 gating. Box denotes the mean of approximately 24 individual TIL cultures with error bars indicating SEM. B, cumulative comparison of cutaneous melanoma– versus uveal melanoma–derived TIL cultures based upon their percentage of CD8+ and CD4+ T cells. Bars demonstrate the mean with error bars indicating SEM. C, autologous tumor reactivity of individual TIL cultures derived from cutaneous melanoma and uveal melanoma liver metastases. Each dot represents the IFNγ production of a single TIL culture in response to overnight coculture with autologous tumor digest minus the background cytokine levels of unstimulated TIL and tumor digest alone. Bar represents the mean of approximately 24 TIL cultures. Dashed line marks 100 pg/mL of IFNγ on the y-axis. D, cumulative comparison of the antitumor reactivity of cutaneous melanoma– and uveal melanoma–derived TIL cultures based upon production of IFNγ in response to autologous tumor digest. Bars demonstrate the mean with error bars indicating SEM. Statistical comparisons between cutaneous melanoma and uveal melanoma cohorts were performed with the Student t test.

Figure 3.

Phenotypic and functional comparison of TIL cultures derived from cutaneous melanoma (CM) and uveal melanoma (UM) liver metastases. A, mean percentage of CD8+ (filled box) and CD4+ (open box) T cells found in TIL cultures derived from individual cutaneous melanoma and uveal melanoma liver metastases. T-cell subset frequency as assessed by flow cytometry and CD3 gating. Box denotes the mean of approximately 24 individual TIL cultures with error bars indicating SEM. B, cumulative comparison of cutaneous melanoma– versus uveal melanoma–derived TIL cultures based upon their percentage of CD8+ and CD4+ T cells. Bars demonstrate the mean with error bars indicating SEM. C, autologous tumor reactivity of individual TIL cultures derived from cutaneous melanoma and uveal melanoma liver metastases. Each dot represents the IFNγ production of a single TIL culture in response to overnight coculture with autologous tumor digest minus the background cytokine levels of unstimulated TIL and tumor digest alone. Bar represents the mean of approximately 24 TIL cultures. Dashed line marks 100 pg/mL of IFNγ on the y-axis. D, cumulative comparison of the antitumor reactivity of cutaneous melanoma– and uveal melanoma–derived TIL cultures based upon production of IFNγ in response to autologous tumor digest. Bars demonstrate the mean with error bars indicating SEM. Statistical comparisons between cutaneous melanoma and uveal melanoma cohorts were performed with the Student t test.

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Next, we compared the antitumor reactivity of the individual cutaneous melanoma and uveal melanoma TIL cultures by overnight coculture with tumor digests of their respective parental tumors which had been freshly cryopreserved at the time of surgical procurement. Reactive TIL cultures were defined as having tumor-induced IFNγ production ≥100 pg/mL and twice the background of unstimulated TIL and tumor digest alone. The autologous antitumor reactivity for each of the TIL cultures from their respective metastases is shown in Fig. 3C. The TIL cultures from 88% of the cutaneous melanoma metastases (7 of 8 cutaneous melanoma metastases) demonstrated mean tumor-specific IFNγ production ≥100 pg/mL. In contrast, 46% of uveal melanoma metastases (6 of 13 uveal melanoma metastases) had mean reactivity above this threshold. Cumulatively, cutaneous melanoma–derived TIL cultures produced higher mean levels of IFNγ in response to autologous tumor digest when compared with uveal melanoma–derived TIL cultures (cutaneous melanoma: 1,044 pg/mL vs. uveal melanoma: 209 pg/mL, P < 0.0001; Fig. 3D). Interestingly, however, we identified individual TIL cultures from 46% of uveal melanoma metastases (6 of 13 uveal melanoma metastases; L-UM 3b, 5, 7, 8, 12, and 14), with IFNγ production which was comparable in magnitude with the responses identified from cutaneous melanoma TIL (Fig. 3C). Thus, although specific autologous antitumor T-cell responses were more prevalent among the cutaneous melanoma liver metastases, there was a subset of uveal melanoma tumors that could also elicit strong tumor reactive T-cell responses.

Metastasis hypopigmentation identifies an immunogenic subset of uveal melanoma

Having found that a subset of uveal melanoma metastases could naturally elicit autoreactive TIL responses, we next sought to determine if there was a clinically relevant means to prospectively identify uveal melanoma patients who harbored these immunogenic tumors. Because the majority of cutaneous melanoma metastases possessed TIL with autologous tumor reactivity, we postulated that similar adaptive T-cell responses might preferentially be found in uveal melanoma metastases with attributes akin to cutaneous melanoma tumors. Preoperative MRI and gross pathologic examination had demonstrated that the majority of cutaneous melanoma liver metastases lacked melanin pigmentation (Fig. 1B). Thus, we investigated whether the in-situ melanin content of uveal melanoma metastases, as determined by preoperative MRI imaging, might correlate with the subsequent growth of autoreactive TIL populations. The liver metastases from 13 consecutive uveal melanoma patients (described in Fig. 3C) underwent stratification based upon their preoperative in-situ radiographic attributes. MRI signal intensity scores identified four metastases as hyperpigmented (2+; L-UM 1b, 4, 9, and 10), five metastases as mixed pigmented (1+; L-UM 3b, 5, 6, 11, and 13), and four metastases as hypopigmented (0; L-UM 7, 8, 12, 14). Next, the IFNγ responses from each of the TIL cultures derived from these metastases were assessed based upon the MRI characteristics of their parental tumors (Fig. 4). We found that hyperpigmented metastases (2+ MRI signal) uniformly gave rise to TIL cultures (n = 96) with low antitumor IFNγ production (mean: 35 pg/mL) that did not exceed background control levels. In contrast, the mixed pigmented metastases (1+ MRI signal) generated TIL cultures (n = 111) with significantly greater IFNγ production (mean IFNγ: 194 pg/mL; mixed vs. hyper P < 0.0001), whereas the hypopigmented metastases (0 MRI signal) were notable for producing TIL cultures (n = 87) with the highest antitumor reactivity (mean IFNγ: 419 pg/mL; hypo vs. hyper and mixed, P < 0.0001, respectively). Thus, we concluded that low to absent levels of in-situ melanin pigmentation based upon preoperative clinical MRI could identify a subset of uveal melanoma metastases capable of eliciting potent immunogenic TIL responses. In contrast, uveal melanoma metastases with high levels of melanin pigmentation identified a nonimmunogenic group of tumors.

Figure 4.

Metastasis hypopigmentation identifies an immunogenic subset of uveal melanoma. Association between the preoperative melanin content of uveal melanoma (UM) metastases and the autologous antitumor reactivity of their derived TIL cultures. Uveal melanoma TIL cultures (n ∼24) were established from individual liver metastases from 13 uveal melanoma patients. The cultures were stratified into cohorts based upon the melanin pigmentation status of their parental tumors as assessed by preoperative in-situ MRI signal intensity scoring. Each dot represents the IFNγ production of a single uveal melanoma TIL culture in response to overnight coculture with autologous tumor digest minus the background cytokine levels of unstimulated TIL and tumor digest alone. Statistical comparisons between the stratified pigmentation cohorts were performed with the Student t test.

Figure 4.

Metastasis hypopigmentation identifies an immunogenic subset of uveal melanoma. Association between the preoperative melanin content of uveal melanoma (UM) metastases and the autologous antitumor reactivity of their derived TIL cultures. Uveal melanoma TIL cultures (n ∼24) were established from individual liver metastases from 13 uveal melanoma patients. The cultures were stratified into cohorts based upon the melanin pigmentation status of their parental tumors as assessed by preoperative in-situ MRI signal intensity scoring. Each dot represents the IFNγ production of a single uveal melanoma TIL culture in response to overnight coculture with autologous tumor digest minus the background cytokine levels of unstimulated TIL and tumor digest alone. Statistical comparisons between the stratified pigmentation cohorts were performed with the Student t test.

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Comparison of tumor mutational profile between cutaneous melanoma and uveal melanoma metastases

Although normal differentiation antigens are common targets for endogenous T cells in melanoma patients, recent studies have demonstrated that unique somatic mutations expressed by tumors can also elicit autologous T-cell responses (37–39). Further, comparative WES has revealed sun-exposed cutaneous melanoma tumors to have the highest number of somatic mutations among common malignancies (40). These observations have fostered the theory that the unique responsiveness of metastatic cutaneous melanoma to a variety of immunotherapy approaches is a direct consequence of endogenous immune responses against neoepitopes encoded by these large numbers of mutations. Thus, we next sought to determine if the identified subset of immunogenic uveal melanoma metastases also harbored a greater mutational load that might explain their enhanced T-cell recognition. Previously, it has been reported that sun-shielded melanomas, including uveal melanoma, have far fewer nonsynonymous mutations when directly compared with sun-exposed cutaneous melanoma tumors (17). However, these analyses were based upon a limited number of uveal melanoma samples which included a mixture of primary and metastatic tumors. Thus, we first sought to better determine the frequency and characteristics of the nonsynonymous mutations occurring in cutaneous melanoma and uveal melanoma metastases. To provide adequate sample numbers for this analysis, we obtained WES data for 278 cutaneous melanoma metastases via TCGA data portal and compared these against data from 14 uveal melanoma metastases from our cohort. Of note, because the TCGA database does not denote the anatomic site of the metastases, the cutaneous melanoma data represent metastases from a variety of sites. Protein-altering somatic point mutations for each tumor were determined using a common analytical workflow based upon comparison with matched germline DNA. We found that cutaneous melanoma metastases had a broad range in mutation number (range: 6–31,250) when compared with uveal melanoma metastases (range: 15–168). Further, as a group, cutaneous melanoma metastases had significantly more somatic mutations when compared with uveal melanoma metastases (median counts; cutaneous melanoma: 282 vs. uveal melanoma: 73, P < 0.0001; Fig. 5A).

Figure 5.

Tumor mutational profiles of cutaneous melanoma (CM) and uveal melanoma (UM) metastases. A, comparison of the number of nonsynonymous mutations identified in cutaneous melanoma and uveal melanoma metastases. Box extends from 25th to 75th percentiles, line through the box indicates median, and bars extend from the smallest to largest values. Statistical comparison between cutaneous melanoma and uveal melanoma cohorts was performed with the Student t test. B, frequency of BRAF, GNAQ, and GNA11 mutations identified in cutaneous melanoma and uveal melanoma metastases. Statistical comparison of oncogene frequency between cutaneous melanoma and uveal melanoma was performed with the Fisher exact test. C, correlation between the number of nonsynonymous mutations identified in uveal melanoma metastases and the autologous antitumor reactivity of their derived TIL cultures. Data represent uveal melanoma TIL cultures (n ∼24) established from metastases from 12 uveal melanoma patients. Each dot plots the mutation frequency of the parental tumor (x-axis) versus the IFNγ production of the derived uveal melanoma TIL cultures in response to overnight coculture with autologous tumor digest minus the background cytokine levels of unstimulated TIL and tumor digest alone (y-axis). Linear regression analysis was used to derive R2 values.

Figure 5.

Tumor mutational profiles of cutaneous melanoma (CM) and uveal melanoma (UM) metastases. A, comparison of the number of nonsynonymous mutations identified in cutaneous melanoma and uveal melanoma metastases. Box extends from 25th to 75th percentiles, line through the box indicates median, and bars extend from the smallest to largest values. Statistical comparison between cutaneous melanoma and uveal melanoma cohorts was performed with the Student t test. B, frequency of BRAF, GNAQ, and GNA11 mutations identified in cutaneous melanoma and uveal melanoma metastases. Statistical comparison of oncogene frequency between cutaneous melanoma and uveal melanoma was performed with the Fisher exact test. C, correlation between the number of nonsynonymous mutations identified in uveal melanoma metastases and the autologous antitumor reactivity of their derived TIL cultures. Data represent uveal melanoma TIL cultures (n ∼24) established from metastases from 12 uveal melanoma patients. Each dot plots the mutation frequency of the parental tumor (x-axis) versus the IFNγ production of the derived uveal melanoma TIL cultures in response to overnight coculture with autologous tumor digest minus the background cytokine levels of unstimulated TIL and tumor digest alone (y-axis). Linear regression analysis was used to derive R2 values.

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Next, we compared the tumor cohorts for the frequency of prototypic melanoma associated oncogenic driver mutations, including, BRAF, GNAQ, and GNA11 (Fig. 5B). We found BRAF mutations in 53% of the cutaneous melanoma metastases (n = 278). However, BRAF was not mutated in any of the uveal melanoma tumors (n = 22; BRAF mutation frequency; cutaneous melanoma vs. uveal melanoma metastases, P < 0.0001). In contrast, activating mutations in either of the homologous genes, GNAQ or GNA11, were identified in 91% of the uveal melanoma metastases, but in only 5% of the cutaneous melanoma metastases (GNAQ/GNA11 mutation frequency; cutaneous melanoma vs. uveal melanoma metastases, P < 0.0001).

Finally, we investigated in 12 uveal melanoma patients whether the mutational frequency identified in their metastases correlated with the autologous antitumor reactivity of their respectively derived TIL cultures (n ∼24 cultures/tumor). When the tumor-induced IFNγ production from each of the TIL cultures was assessed against the number of nonsynonymous mutations identified in their respective parental tumors, we found no correlation between the parameters (Fig. 5C).

The last 30 years have provided substantial evidence that the human immune system can naturally generate potent immunologic responses against tumor antigens expressed by metastatic cutaneous melanoma (8). Cancer regression can now be achieved in patients with metastatic cutaneous melanoma with mechanistically diverse forms of immunotherapy that augment naturally existing tumor-specific T-cell responses (9–12). However, the role of these immune-based therapies for the treatment of metastatic uveal melanoma patients remains unclear. Patients with uveal melanoma are frequently excluded from metastatic melanoma immunotherapy clinical trials because uveal melanoma is generally thought to be a nonimmunogenic form of melanoma (13–16). However, there have not been formal comparative studies performed directly upon uveal melanoma and cutaneous melanoma metastases to accurately assess their relative immunogenicity. In this study, we compared the tumor antigen expression, tumor mutational load, and endogenous antitumor immunologic reactivity found in fresh surgically resected uveal melanoma and cutaneous melanoma metastases. By defining the tumor-specific immune responses that are naturally found in these metastases, we sought to provide insight into the role for immune-based therapies for the management of uveal melanoma patients. We previously reported that melanoma metastases demonstrate significant heterogeneity in tumor antigen expression and lymphocytic infiltrate based upon their anatomic location in the body (6). Thus, to avoid potential site-specific bias in the current study, we focused our comparative analysis selectively upon liver metastases resected from uveal melanoma and cutaneous melanoma patients. Our findings revealed that despite having common melanocytic lineage, uveal melanoma and cutaneous melanoma liver metastases were highly dichotomous in their melanin content, tumor differentiation antigen expression, and somatic mutational profile. The majority of cutaneous melanoma liver metastases lacked gross melanin pigmentation, whereas uveal melanoma liver metastases were more commonly hyperpigmented in appearance. In support of this observation, immunohistochemical profiling revealed that cutaneous melanoma metastases had lower cellular expression of proteins associated with melanocyte differentiation, including MART-1 and gp100. Further, we found significant differences in the overall somatic mutational profile between cutaneous melanoma and uveal melanoma liver metastases. Comparative whole-exomic sequencing revealed that cutaneous melanoma metastases had significantly greater mutational burden compared with uveal melanoma metastases with the melanoma variants also possessing quite different oncogenic driver mutations of the MAPK pathway. Similar to previous reports (41–43), nearly all of the uveal melanoma metastases had GNAQ and GNA11 mutations, whereas cutaneous melanoma metastases commonly had BRAF mutations. Collectively, these comparative studies demonstrate cutaneous melanoma metastases to be far more dedifferentiated from their melanocytic origin when compared with uveal melanoma metastases in terms of their mutational profile, tumor antigen expression, and gross melanin pigmentation.

When endogenous immune responses in these highly divergent forms of melanoma were characterized, we further identified marked differences in the phenotype and antitumor reactivity of their respective infiltrating lymphocytes. Cutaneous melanoma TIL were predominantly composed of CD8+ T cells, whereas uveal melanoma TIL were CD4+ dominant. Reactivity against autologous tumor was significantly greater in cutaneous melanoma TIL compared with uveal melanoma TIL. However, we identified TIL from a subset of uveal melanoma patients which had robust antitumor reactivity that was comparable in magnitude with that of cutaneous melanoma TIL. The identification of this immunogenic group of uveal melanoma metastasis has not been previously reported and thus has fostered our interest in determining the specific antigenic targets that are recognized by these uveal melanoma–derived TIL. Interestingly, the level of in-situ melanin pigmentation found in parental tumors strongly correlated with the generation of tumor-reactive uveal melanoma TIL. Hyperpigmented uveal melanoma metastases generated TIL cultures with poor tumor reactivity, whereas the metastases that lacked pigmentation produced the most reactive TIL cultures. Although the precise mechanism underlying the relationship between tumor pigmentation and TIL reactivity is not completely understood, the loss of pigment proteins by metastatic tumor cells is thought to be driven by the stochastic genetic instability of tumor cells combined with the nonstochastic selective pressures of the host immune system (immunoediting; refs. 44–48). Animal models have demonstrated the development of vitiligo and the loss of tumor pigment proteins through the action of antigen-specific CD8+ T cells (49). Further, it is well known that human cutaneous melanoma TIL frequently possess T cells specific for melanocyte pigment proteins, such as MART-1 and gp100 (50). Thus, in the current study, we hypothesize that the loss of tumor pigmentation in this subset of uveal melanoma metastases could signify the presence of a vigorous immune response targeting pigment antigens. However, beyond the targeting of these normal differentiation antigens, recent analyses have found that unique somatic mutations expressed by tumors can generate neoepitopes that also elicit robust autologous T-cell responses (37–39). Although our study found no correlation between the mutational frequencies identified in uveal melanoma metastases and the autologous antitumor reactivity of their respectively derived TIL cultures, there may still be individual mutations that are recognized. Thus, we have begun studies to assess the tumor-reactive uveal melanoma TIL for recognition of both nonmutated and mutated antigen targets.

Although not completely validated as a clinical biomarker, the MRI assessment of melanin content in uveal melanoma metastases was found in this study to accurately identify tumors that can elicit a strong endogenous immune response. We are, thus, interested in determining whether immune-based therapies may be more effective in the subset of uveal melanoma patients who harbor these unique immunogenic tumors. To help address these questions, we are conducting the first-in-human adoptive T-cell transfer trial dedicated to patients with metastatic uveal melanoma (NCT01814046). In this phase II study, patients with metastatic uveal melanoma undergo surgical metastasectomy to procure tumor tissue for TIL generation. The expanded lymphocytes are then adoptively transferred back into the host in conjunction with a nonmyeloablative lymphodepleting regimen. The primary endpoint of this study is to define the objective response rate of TIL immunotherapy in patients with metastatic uveal melanoma. The results of this trial should provide valuable insight into the role of immune-based therapies for the treatment of metastatic uveal melanoma.

No potential conflicts of interest were disclosed.

Conception and design: L.D. Rothermel, A.C. Sabesan, D.J. Stephens, U.S. Kammula, T.H. Pham

Development of methodology: L.D. Rothermel, A.C. Sabesan, D.J. Stephens, S.S. Chandran, B.C. Paria, A.K. Srivastava, U.S. Kammula, L. Xi, T.H. Pham

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L.D. Rothermel, A.C. Sabesan, D.J. Stephens, S.S. Chandran, B.C. Paria, A.K. Srivastava, J.R. Wunderlich, C.-C.R. Lee, M. Raffeld, U.S. Kammula, L. Xi

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L.D. Rothermel, A.C. Sabesan, D.J. Stephens, S.S. Chandran, B.C. Paria, A.K. Srivastava, M. Raffeld, P. Jailwala, M. Kasoji, U.S. Kammula, L. Xi, T.H. Pham

Writing, review, and/or revision of the manuscript: L.D. Rothermel, A.C. Sabesan, D.J. Stephens, S.S. Chandran, B.C. Paria, A.K. Srivastava, C.-C.R. Lee, M. Raffeld, U.S. Kammula, L. Xi

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L.D. Rothermel, A.C. Sabesan, D.J. Stephens, R. Somerville, P. Jailwala, U.S. Kammula, T.H. Pham

Study supervision: U.S. Kammula

The authors thank the Surgery Branch cell production facility and the immunotherapy clinical and support staff for their contributions. They also thank Li Jia for assistance in bioinformatics analysis. The Prospective Procurement of Solid Tumor Tissue to Identify Novel Therapeutic study was supported by the Intramural Research Program of the NCI, NIH, Department of Health and Human Services. Whole-exome raw data were uploaded to the NIH database for Genotypes and Phenotypes (dbGaP) under accession number phs001003.v1.p1.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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