Metastatic ocular melanoma to the liver exhibits infiltrative and nodular growth patterns

Abstract

We examined liver specimens from 15 patients with uveal melanoma (UM) who had died of their disseminated disease. We found 2 distinct growth patterns of UM metastasis: infiltrative (n = 12) and nodular (n = 3). In the infiltrative pattern, individual UM cells with a CD133+ cancer stem cell-like phenotype were present and formed aggregates of stage I <50-μm-diameter micrometastases in the sinusoidal spaces. These micrometastases appeared to expand, destroy adjacent hepatocytes, and form stage II 51- to 500-μm-diameter and then stage III >500μm-diameter metastases, which were encapsulated by collagenized fibrous septae. In the nodular growth pattern, CD133+ melanoma cells aggregated adjacent to portal venules and subsequently appeared to grow and efface the adjacent hepatocytes to form stage II 51- to 500-μm-diameter nodules that surrounded the portal venule. These avascular nodules appeared to further expand to form stage III >500-μm-diameter nodules that exhibited vascularization with minimal fibrosis. The tumor stem cell-like phenotype seen in individual UM cells was lost as the tumors progressed. There were CD56+ natural killer cells in sinusoidal spaces and CD3+ lymphocytes in periportal areas. The nodular growth pattern showed UM cells expressing MMP9 and VEGF. UM cells in both above-described growth patterns exhibited variable BAP1 expression. We propose that changes in the liver microenvironment are related to metastatic UM growth. We hypothesize that these changes include immune regulation within the sinusoidal space for the infiltrative pattern and changes in the VEGF/PEDF ratio for the nodular pattern.

Keywords: Growth patterns; Liver metastases; Micrometastases; NK cells; Ocular melanoma; PEDF.

Figure 1

Number of metastases per area of Stage I, II and III infiltrative and nodular metastases. In the infiltrative pattern, there was a significant decrease in numbers going from StageI to Stage III metastases. In the nodular growth pattern, there was approximately one metastases per 2 cm² for all three stages. Standard deviations are shown.

Figure 2

BAP1, CD56 and CD3 expression in melanocytes, NK cells and T lymphocytes. A. There is BAP1 expression (red) in a collection of metastatic UM cells in the liver. B. Collection of metastatic UM cells which are not expressing BAP1. C. There are scattered CD56+ NK cells in the sinusoidal space. D. There are CD3+ cells in the periportal area which includes the bile duct (*) and portal venule (star). (A and B. BAP1, peroxidase anti-peroxidase with vector red chromagen, 100×. C. CD56, peroxidase anti-peroxidase, 100×. D. CD3, peroxidase anti-peroxidase, 25×)

Figure 3

Single cell and Stage I infiltrative growth pattern. A. There are metastatic UM cells present in the sinusoidal spaces of the hepatic lobule. B. These single cells include lymphocytes and cells with slightly more eosinophilic cytoplasm. C. The cells with the slightly more cytoplasm express HMB45 (arrows). D. These cell express both HMB45 and CD133 (orange). E. The individual metastatic UM cells in the sinusoidal spaces are clumping and forming small clusters. F. These clusters of melanoma cells, or micrometastases, begin to expand the sinusoidal space. G. The micrometastatic melanoma expresses HMB45. H. Dual labeling for HMB45 (green) and CD 133 (red) show that most of the larger melanoma cells in the micrometastases express only HMB45 whereas small, individual melanoma cells express both HMB45 and CD133 (orange). (A. H&E 25×, B. H&E 100×, C. HMB45, peroxidase anti-peroxidase with vector red chromagen, 100×, D. Dual labeling for HMB45 green chromagen and CD133 red chromagen, 100×, E. H&E 25×, F. H&E 100×, G. HMB45, peroxidase anti-peroxidase with vector red chromagen, 100×, GH. Dual labeling for HMB45 (green chromagen) and CD133 (red chromagen), 100×).

Figure 4

Stages II and III infiltrative growth pattern. A. The sinusoidal spaces are filled with metastatic melanoma cells and the hepatocytes have been destroyed. B. The melanoma cells express HMB45. C. Dual labeling for HMB45 (green) and CD133 (red) show that there are many melanoma cells (green) and only rare small individual dual labeled melanoma cells (orange). D. These metastatic UM cells express MMPs (red) and no VEGF (green). E. There is complete replacement of the hepatic lobule with islands of melanoma. F. The islands of melanoma express HMB45. G. Dual labeling for HMB45 (green) and CD133 (red) show a only HMB45 expression in the islands of melanoma, although there are rare, small dual labeled cells present. H. There is an absence of VEGF (green) express and MMP (red) expression by the melanoma cells. (A. H&E 100×, B. HMB45, peroxidase-anti-peroxidase with vector red chromagen, 100×, C. Dual labeling for HMB45 (green chromagen) and CD133 (red chromagen), 100×, D. Dual labeling for HMB45 (green chromagen) and MMP9 (red chromagen), 100×, E. H&E 25×, F. HMB45, peroxidase-anti-peroxidase with vector red chromagen, 25×, G. Dual labeling for HMB45 (green chromagen) and CD133 (red chromagen) 100×, H. Dual labeling for VEGF (green chromagen) and MMP (red chromagen), 100×).

Figure 5

Single and Stage II cell nodular growth pattern. A. Rare individual mononuclear cells (arrow) with slightly more cytoplasm than lymphocytes are identified in fibrous tissue adjacent to the portal venule (*). B. A trichrome stain highlights the collagen within the portal triad which includes the portal venule (*). C. The rare mononuclear cells in this tissue express HMB45. The portal venule is present (*) and there are scattered lymphocytes also present. D. There is dual labeling for HMB45 and CD133 (orange) in rare mononuclear cells in the periportal area (arrows) and within the portal venule (*). E. A well-demarcated nodule of UM cells is present and effaces the surround hepatic lobules. A trichrome stain highlights the portal venule (arrow) within the center of the nodule. F. The melanoma cells in the nodule express HMB45 and surround the portal venule, thus effacing the surround hepatic lobules. G. Melanoma cells express HMB45 (green) and there are no cells that express CD133. H. Dual labeling of the UM cells for VEGF (green) and MMP (red) is seen. (A. H&E 100×, B. Masson trichrome 100×, C. HMB45 peroxidase anti-peroxidase vector red chromagen 100×, D. Dual labeling for HMB45 green chromagen and CD133 red chromagen, 100×, E. Masson trichrome 10×, F. HMB45 with vector red chromagen, peroxidase anti-peroxidase, 10×, G. HMB45 with green chromagen, CD133 with red chromagen, 10×, H. VEGF with green chromagen, MMP9 with red chromagen, 100×).

Figure 6

Stage III nodular growth pattern. A. A nodule of pigmented melanoma cells effaces the adjacent hepatic lobules. A trichrome stain highlights minimal fibrosis surrounding the nodule of melanoma. B. Vascular channels express CD31 in the nodule of melanoma (arrow) and immediately adjacent tissue (*). C. The melanoma expresses HMB45 (green) but not CD133 (red). D. The melanoma co-expresses VEGF (green) and MMP (red), resulting in an orange dual-labeling. (A. Masson trichrome 10×, B. CD31 peroxidase anti-peroxidase, 10×, C. HMB45 with green chromagen, CD133 with red chromagen, 10×, D. VEGF with green chromagen, MMP with red chromagen, 100×).

Figure 7

Schematic cartoon of metastatic UM to the liver. Melanoma arises in the choroid of the eye (A), extravasates into tumor blood vessels, and then travels via the systemic circulation as circulating melanoma cells with tumor stem cell-like properties (B), where it enters the liver as single cells. These single cells likely arise from hepatic arterioles and seed the sinusoidal space causing the infiltrative pattern of growth, which progresses to Stage 1 (0–50µm) micrometastases, Stage 2 intermediate (51–500µm) metastases which are bathed by serum in the sinusoidal space, and eventually Stage 3 (>501µm) metastases which replace the hepatic lobule, do not express MMP9/VEGF or display angiogenesis. Alternatively, circulating melanoma cells may enter the liver via the portal venule causing the nodular growth pattern, first as single cells, then Stage 1 (0–50µm) micrometastases that co-opt the portal venule, then Stage 2 (51–500µm) intermediate metastases that begin to efface the adjacent hepatic lobule, and finally Stage 3 (>501µm) metastases which express MMP9/VEGF, display angiogenesis and further efface the adjacent hepatic lobule.

Figure 8

Schematic cartoon of proposed mechanisms of infiltrative and nodular growth patterns of metastatic UM to the liver. UM cells with tumor stem cell-like properties extravasate from the primary ocular tumor and disseminate hematogenously to the liver. In the infiltrative growth pattern, these UM cells may metastasize to the sinusoidal spaces in the parenchyma of the hepatic lobule and grow from single melanoma cells to Stages I–III metastases. This infiltrative growth is related in part to a change in the immune microenvironment within the sinusoidal space, including downregulation of NK cells.^, Hypoxia results in MMP production in Stage II which allows for tissue dissection and creation of pseudosinusoidal spaces. Micrometastases receive their nutrition form blood in the sinusoidal space; larger metastases are bathed by blood in pseudosinusoidal spaces. In the nodular growth pattern, circulating UM cells metastasize to the periportal areas in the hepatic triad, co-opt the portal venules for nutrition, and progress from single UM cells to Stages I–III metastases. Hypoxia results in MMP production and VEGF expression. Nodular growth is related in part to MMP degradation of PEDF, increased VEGF expression by melanoma cells, an increased VEGF:PEDF ratio and angiogenesis.