Prognostic Factors Five Years After Enucleation for Uveal Melanoma.

Purpose: A subgroup of uveal melanoma (UM) gives rise to metastases at a late stage. Our objective was to identify patient and tumor characteristics that are associated with UM-related death in patients who survived 5 years following enucleation. Methods: A retrospective analysis was performed in 583 primary UM cases, enucleated at the Leiden University Medical Center between 1983 and 2013. Univariable and multivariable Cox regression analyses were performed in the total cohort and separately in those surviving more than 5 years (n = 297). Results: In the total cohort, the median age was 62.6 years, and the median tumor diameter was 12.0 mm. Monosomy 3 was detected in 53% of cases and gain of 8q in 47%. In the cohort surviving 5 years, the median age was 59.5 years, and the median tumor diameter was 11.0 mm. Monosomy 3 and gain of 8q were detected in 33% and 31% of cases, respectively. In the total cohort, male gender (P = 0.03), tumor diameter (P < 0.001), mitotic count (P < 0.001), extravascular matrix loops (P = 0.03), extraocular growth (P < 0.001), and gain of 8q (P < 0.001) were independently associated with UM-related death. In patients surviving 5 years after enucleation, univariable analysis revealed that age (P = 0.03), tumor diameter (P < 0.001), monosomy 3 (P = 0.04), and 8q gain (P = 0.003) were associated with subsequent UM-related death. Using a multivariable analysis, only male gender (P = 0.03) and gain of 8q (P = 0.01) remained significant. Conclusions: Predictors of UM-related death change over time. Among UM patients who survived the initial 5 years following enucleation, male gender and chromosome 8q status were the remaining factors related to UM-related death later on.

Effective cooperation of monoclonal antibody and peptide vaccine for the treatment of mouse melanoma.

mAbs binding to tumor-associated surface Ags are therapeutically applied in a range of malignancies. Therapeutic vaccination only recently met with clinical success, and the first cancer vaccine received U.S. Food and Drug Administration approval last year. To improve current protocols, we combined peptide vaccines with mAb to the tyrosinase-related protein (TRP)-1 surface Ag for the treatment of B16F10 skin melanoma. Vaccine formulations with synthetic long peptides failed to elicit strong CD8 T cell responses to self-differentiation Ags gp100 and TRP-2, whereas altered peptide sequences recruited gp100-specific CD8 T cells from the endogenous repertoire with frequencies of 40%. However, these high frequencies were reached too late; large, progressively growing melanomas had already emerged. Addition of the TRP-1-directed mAb TA99 to the treatment protocol mediated eradication of s.c. lesions. The mode of action of the Ab did not depend on complement factor C3 and did not lead to improved Ag presentation and CD8 T cell immunity; rather, it recruited FcγR-bearing innate immune cells during early tumor control, thereby creating a window of time for the generation of protective cellular immunity. These data support the concept of combination therapy, in which passive transfer of mAbs is supplemented with cancer peptide vaccines. Moreover, we advocate that tumor Ag-specific T cell immunity directed against self-proteins can be exploited from the endogenous repertoire.

Bevacizumab and intraocular tumors: an intriguing paradox.

PURPOSE: Bevacizumab, a humanized monoclonal antibody to vascular endothelial growth factor-A (VEGF-A), was originally developed as an anti-tumor treatment. In ocular oncology, it is being used to treat macular edema due to radiation retinopathy, but it may also be useful for the treatment of primary uveal melanoma (UM) or its metastases. We determined the effect of bevacizumab on the growth of B16F10 cells inside the eye and on B16F10 and UM cells cultured in vitro. METHODS: B16F10 melanoma cells were placed into the anterior chamber of the eye of C57Bl/6 mice and tumor growth was monitored after injection of different doses of bevacizumab or mock injection. In addition, the effect of bevacizumab on in vitro growth of B16F10 and human UM cells and on the expression of VEGF-A, GLUT-1, and HIF-1α was evaluated. RESULTS: Following intraocular injection of bevacizumab into murine B16 tumor-containing eyes, an acceleration of tumor growth was observed, with the occurrence of anterior chamber hemorrhages. Bevacizumab did not affect proliferation of B16F10 cells in vitro, while it inhibited UM cell proliferation. Expression analysis demonstrated that addition of bevacizumab under hypoxic conditions induced VEGF-A, GLUT-1 and HIF-1α in B16F10 cells as well as in UM cell lines and two of four primary UM tumor cultures. CONCLUSIONS: In contrast with expectations, intraocular injection of bevacizumab stimulated B16F10 melanoma growth in murine eyes. In vitro exposure of B16 and human UM cells to bevacizumab led to paradoxical VEGF-A upregulation. The use of VEGF inhibitors for treatment of macular edema (due to radiation retinopathy) after irradiation of UM should be considered carefully, because of the possible adverse effects on residual UM cells.

Three Generations of Eminent American Chinese: Lives Intertwined With History.

This article describes the lives of 3 generations of a remarkable American Chinese family, who all excelled in their achievements. Grandfather Chan Loon Teung was the first Chinese Harvard graduate; the father, Eugene Chan, and his wife, Winifred Mao, were famous ophthalmologists, working in the United States and in China, and their daughter, Chi-Chao Chan, is a specialist in ophthalmic pathology and uveitis at the National Institutes of Health in the United States. Although the different generations all encountered dramatic situations caused by political turmoil, such as the Xinhai Revolution, World War II, and the cultural revolution in China, this did not prevent them from making major contributions to their home countries and ophthalmology. The history of this family as depicted in this article illustrates what perseverance and passion can achieve.

Changes in immunological markers and influx of macrophages following trans-scleral thermotherapy of uveal melanoma.

PURPOSE: In trans-scleral thermotherapy (TSTT), heat is applied through the sclera in order to target an intraocular uveal melanoma. Previously, it had been shown that in uveal melanoma, hyperthermia and transpupillary thermotherapy influenced expression of immunologically relevant proteins, such as S100, HLA and heat-shock proteins (HSPs). We investigated whether TSTT induced similar changes. METHODS: Experimental TSTT was applied on eleven uveal melanomas prior to enucleation. Each tumour sample was processed for histopathological examination; immunohistochemical analysis was performed to determine expression of S100, HLA, HSPs and macrophage markers. RESULTS: In TSTT-treated areas, expression of S100 and different HSPs was lost, while an upregulated expression of HSP GP96 was observed at the border of these areas. Expression levels of HLA-A and HLA-B varied between tumours and were not influenced by TSTT. The borders of the TSTT-treated areas showed high numbers of infiltrating macrophages, which were predominantly of the M2 phenotype. CONCLUSION: TSTT has an effect on immunological parameters with local loss of expression of HSPs and S100. The influx of M2 macrophages around the TSTT-treated areas indicates the presence of an innate immune reaction against the induced necrosis, suggesting that TSTT-treated tumour cells are removed by a macrophage-mediated tissue repair mechanism.

Detection of M2-macrophages in uveal melanoma and relation with survival.

PURPOSE: The presence of a high number of infiltrating macrophages in uveal melanoma is associated with a bad prognosis. However, there are several known types of macrophages, of which the M2 is considered to be proangiogenic and tumor-promoting. This study was conducted to determine whether the tumor-infiltrating macrophages in uveal melanoma are of this M2 subtype. METHODS: Macrophages were identified in sections from 43 uveal melanomas by immunofluorescence histochemistry, using monoclonal antibodies directed against CD68 and CD163. The immunopositive cell density was measured visually and with a confocal microscope and calculated per square millimeter. Results were compared with clinical and tumor characteristics. RESULTS: Infiltrating macrophages in uveal melanoma were predominantly CD68(+)CD163(+), thus of the M2 phenotype. The density of CD68(+), CD163(+), and CD68(+)CD163(+) cells was significantly increased in uveal melanomas with monosomy 3 compared with cases with disomy of chromosome 3 and was associated with ciliary body involvement. High CD68(+)CD163(+) staining was associated with an increased microvascular density. Survival was significantly better among patients with low CD68(+) and CD68(+)CD163(+) staining. CONCLUSION: The main type of macrophage present in uveal melanoma was the M2 type. Tumors with monosomy of chromosome 3 contained a higher number of M2-macrophages than tumors with disomy of chromosome 3. Infiltration of M2-type macrophages gives a worse prognosis for survival. As M2-type macrophages are proangiogenic, a high density of these cells may contribute to the previously noticed positive association between the density of CD68(+) macrophages and blood vessels.

Macrophages in uveal melanoma and in experimental ocular tumor models: Friends or foes?

Macrophages belong to the innate immune system and as such constitute one of the first barriers against infection. They play an important role in wound healing, in inflammation and in angiogenesis, but are also essential in the first stage of a "danger response". After scavenging debris, they can digest cellular proteins into smaller pieces, and protein-derived peptides can subsequently be presented to the immune system. Depending on the activation state of the macrophage, this antigen presentation may trigger a full-blown active immune response, or may suppress a potential immune reaction. Macrophages constitute a heterogeneous cell population described by many names, with varying phenotypic characteristics, depending on their tissue location and state of activation. They play important roles in different ocular tissues, including the cornea and the choroid, and have been found to be involved in anti-tumor immune responses in mouse ocular tumor models. One would thus expect macrophages to belong to the "good guys" that help to protect our body against dangers such as cancer. In human uveal melanoma however, a high density of macrophages is associated with a poor prognosis for the patient. Macrophages play a role in promoting angiogenesis, and thus may stimulate tumor growth; in addition, macrophages have also been found to suppress anti-melanoma immune responses. These functions may shift during aging. Taken together, these new observations extend our understanding of the diverse functions of macrophages and show us their different faces, making them either "friends or foes" in human uveal melanoma. A better understanding of these multifaceted cells will help in developing new treatments to prevent the growth of metastases in uveal melanoma patients.

Peptide vaccination after T-cell transfer causes massive clonal expansion, tumor eradication, and manageable cytokine storm.

Adoptive T-cell transfer (ACT) is successfully applied as a cancer treatment that is based on the activation and effector functions of tumor-specific T cells. Here, we present results from a mouse model in which ACT is combined with a long peptide-based vaccine comprising gp100 T-cell epitopes. Transferred CD8(+) T cells expanded up to 1,000-fold after peptide vaccination, leading to a 3-fold increase in white blood cell count and a very high frequency in the generation of antigen-specific memory T cells, the generation of which tended to correlate with effective antitumor responses. An enormous pool of effector T cells spread widely to different tissues, including the skin and the immune-privileged eye, where they mediate tumor eradication. Importantly, these striking T-cell dynamics occurred in immunocompetent mice without prior hematologic conditioning. Continued activation of the specific T-cell pool by vaccination led to strong T-cell-mediated cytokine storm and lethality due to multi-organ failure. However, this immunopathology could be prevented by controlling the rapid biodistribution of the peptide or by using a weakly agonistic peptide. Together, these results identify a peptide vaccination strategy that can potently accentuate effective ACT in non-lymphodepleted hosts.

In aged mice, outgrowth of intraocular melanoma depends on proangiogenic M2-type macrophages.

Macrophages are part of the tumor microenvironment and have been associated with poor prognosis in uveal melanoma. We determined the presence of macrophages and their differentiation status in a murine intraocular melanoma model. Inoculation of B16F10 cells into the anterior chamber of the eye resulted in rapid tumor outgrowth. Strikingly, in aged mice, tumor progression depended on the presence of macrophages, as local depletion of these cells prevented tumor outgrowth, indicating that macrophages in old mice had a strong tumor-promoting role. Immunohistochemistry and gene expression analysis revealed that macrophages carried M2-type characteristics, as shown by CD163 and peroxisome proliferator-activated receptor gamma expression, and that multiple angiogenic genes were heavily overrepresented in tumors of old mice. The M2-type macrophages were also shown to have immunosuppressive features. We conclude that tumor-associated macrophages are directly involved in tumor outgrowth of intraocular melanoma and that macrophages in aged mice have a predisposition for an M2-type profile.

Inflammatory cytokines in eyes with uveal melanoma and relation with macrophage infiltration.

PURPOSE: The presence of an inflammatory phenotype, characterized by an increased expression of HLA antigens and an immunologic infiltrate, carries a bad prognosis in uveal melanoma. This study was conducted to determine whether the aqueous humor (AqH) from eyes with uveal melanoma contains inflammatory cytokines and whether their presence is associated with inflammation. METHODS: Immediately after enucleation, AqH was obtained from 37 eyes containing uveal melanoma. Samples were stored at -80°C until use. Fifteen different cytokines were measured with a multiplex bead array. Intratumoral macrophages were analyzed by immunohistochemistry and immunofluorescence staining. The presence of specific cytokines was compared with histopathologic, genetic, and clinical tumor characteristics, as well as patient survival. RESULTS: Several cytokines showed significantly higher expression in the AqH of uveal melanoma-containing eyes than in the AqH of eyes undergoing cataract surgery. MCP-3 was associated with the presence of CD68(+) macrophages. Correlations were found between some cytokine levels and a few known prognostic factors of uveal melanoma, but cytokine levels were not of predictive value for survival. CONCLUSIONS: Uveal melanoma-containing eyes often carry increased levels of inflammation-related cytokines in their AqH. However, the presence of most specific cytokines was not related to the presence of macrophages, clinical or histopathologic parameters, or prognosis.

Regulation of VEGF-A in uveal melanoma.

PURPOSE: Blood vessels are important constituents of intraocular uveal melanoma (UM), but whether angiogenesis is regulated by environmental factors such as ischemia or by genetic mechanisms is not known. This study was undertaken to examine the regulation of the proangiogenic factor vascular endothelial growth factor (VEGF-A). METHODS: Cell lines and primary tumors were tested for expression of VEGF-A, under normoxic and hypoxic conditions, using quantitative PCR, ELISA, WST-1 viability, and in-cell Western experiments. VEGF-A serum levels were determined by ELISA. RESULTS: Hypoxia induced expression of HIF-1alpha and VEGF-A in UM cell lines and primary tumor cultures, but it did not influence proliferation. VEGF-A expression in primary tumors was variable, demonstrating no correlation with specific histologic markers or prognosis. However, VEGF-A levels were significantly raised in UM patients with metastases compared with those without metastases (P < 0.001). CONCLUSIONS: VEGF-A expression by UM cells is mainly controlled by hypoxia and involves the HIF-1alpha pathway, thus indicating an important role for the tumor cell environment. Metastases led to increased serum VEGF-A levels, indicating that VEGF-A may be involved in the growth of metastases.

Impact of tumor-associated macrophages in LH(BETA)T(AG) mice on retinal tumor progression: relation to macrophage subtype.

PURPOSE: To determine the distribution of tumor-associated macrophages (TAMs) during retinoblastoma tumor development, examine the contribution of bone marrow-derived TAMs in retinoblastoma tumors, and evaluate the supportive role of TAMs in tumor growth in a transgenic retinoblastoma mouse model. METHODS: The time course of macrophage infiltration in transgenic retinoblastoma tumors was assessed by immunohistochemistry at different time points in tumorigenesis. The origin of TAMs in transgenic retinoblastoma tumors was determined by transplanting 10(7) bone marrow cells from green fluorescent protein (GFP)-positive 16-week-old mice into age-matched, irradiated LH(BETA)T(AG) mice via tail vein injections. Macrophage depletion was performed by subconjunctival (SC) delivery of liposomal clodronate. RESULTS: The density of TAMs increased from 4 to 12 weeks of age in mice with small to medium tumors (P = 0.037) and remained stable in the later stages of disease (i.e., 16 weeks old with large tumors; P = 0.20). In 16-week-old mice, 38% (2.5 +/- 3.2 cells per 400x high-power field) of TAMs were GFP-positive, bone marrow-derived macrophages. Total TAM depletion was associated with a significant decrease in the expression levels of MMP-9 (P = 0.014) and mature vessels (P < 0.001) and a nonsignificant decrease in the density of neovessels (P = 0.94). The density of M2-polarized TAMs did not change significantly after TAM depletion (P = 0.68). After M1-polarized TAM depletion, the tumor burden increased (P = 0.056). CONCLUSIONS: This work extends understanding of the complex role that macrophages play in retinoblastoma. Macrophage modulation in the tumor microenvironment is a critical factor in retinoblastoma tumor progression.

Intravascular presence of tumor cells as prognostic parameter in uveal melanoma: a 35-year survey.

PURPOSE: Invasion of tumor cells into blood vessels is essential for metastasis of uveal melanoma. The occurrence of ingrowth of tumor cells in blood vessels in uveal melanoma was analyzed, and this parameter was compared with the survival of the patients. METHODS: Between 1972 and 2007, 643 eyes primarily enucleated for uveal melanoma were evaluated histopathologically. Survival data were obtained from charts and from the Integral Cancer Center patient registry. RESULTS: No vascular ingrowth of tumor cells occurred in 59% of the eyes, whereas 18% had tumor cell ingrowth in vessels inside the tumor, 10% in vessels outside the tumor, and 8% in vessels inside as well as outside the tumor. The presence of any intravascular ingrowth of tumor cells correlated significantly with the diameter (P < 0.01) and prominence of the tumor (P < 0.01), as well as with non-spindle-cell type (P = 0.03) and intrascleral ingrowth (P < 0.01), and was associated with a worse survival. When extravascular matrix patterns were not included in the multivariate analysis, intravascular ingrowth came out as an independent prognostic factor, but this was not the case when extravascular matrix patterns were included in the multivariate model. CONCLUSIONS: Intravascular ingrowth of tumor cells in uveal melanoma occurs frequently in combination with well-known histopathologic factors such as large tumor size, epithelioid cell type, and intrascleral ingrowth.

Evidence for natural killer cell-mediated protection from metastasis formation in uveal melanoma patients.

PURPOSE: In uveal melanoma, low human leukocyte antigen (HLA) class I expression on primary tumors is associated with a decreased risk of metastasis. Consequently, it has been suggested that natural killer (NK) cells, which detect decreased expression of HLA class I, are involved in the immune control of metastases. In this study, three novel lines of evidence were identified that support a role for NK cells. METHODS: Uveal melanoma cell lines were used to determine the expression of NK cell receptor ligands (MICA, MICB, ULBP1-3, CD112, CD155, and HLA class I) and to examine sensitivity to lysis by human NK cell lines. Because interactions between polymorphic killer immunoglobulin receptors (KIRs) and HLA regulate NK cell function, KIR and HLA genotyping was performed on 154 patients with uveal melanoma and 222 healthy control subjects. RESULTS: First, all 11 uveal melanoma cell lines tested expressed ligands for activating as well as inhibitory NK cell receptors. Second, such cell lines were lysed efficiently by human NK cells in vitro. Finally, the HLA-C genotype was related to the risk of metastasis-related death in patients with uveal melanoma: The patients carrying HLA-C alleles encoding ligands for KIR2DL1 and KIR2DL2/3 (HLA-C group 1/group 2 heterozygous patients), both inhibitory NK receptors, had a longer metastasis-free survival than did those carrying HLA-C ligands for either KIR2DL1 (HLA-C group 2 homozygotes) or KIR2DL2/3 (HLA-C group 1 homozygotes). CONCLUSIONS: Together, the data support a role for NK cells in the prevention of uveal melanoma metastases.

Monosomy of chromosome 3 and an inflammatory phenotype occur together in uveal melanoma.

PURPOSE: In uveal melanoma, different predictors of poor prognosis have been identified, including monosomy of chromosome 3, HLA expression, and the presence of infiltrating leukocytes and macrophages. Each of these parameters can be used to differentiate prognostically the favorable tumors from the unfavorable ones, and thus the hypothesis for the present study was that they are related, and that monosomy of chromosome 3 occurs in the same tumors as the unfavorable inflammatory phenotype. METHODS: Tumor tissue was obtained from 50 cases of uveal melanoma treated between 1999 and 2004. After enucleation, nuclei were isolated from paraffin-embedded tissue for fluorescence in situ hybridization, to determine the chromosome 3 copy number. Each tumor-containing globe was further processed for conventional histopathologic examination and for immunohistochemical analysis with HLA class I and II-specific antibodies and with macrophage marker CD68. RESULTS: Of 50 uveal melanomas, 62% (31/50) were categorized as having monosomy of chromosome 3. Monosomy 3 was associated with the presence of epithelioid cells, an increased density of tumor-infiltrating macrophages, and a higher HLA class I and II expression. Survival analyses showed a correlation between monosomy 3 and decreased survival and identified monosomy 3, ciliary body involvement, and largest basal tumor diameter as the best prognostic markers. CONCLUSIONS: Monosomy 3 in uveal melanoma is associated with the presence of an inflammatory phenotype, consisting of a high HLA class I and II expression as well as an increased number of tumor-infiltrating macrophages. In a multivariate Cox regression analysis, the presence of monosomy 3 was one of the best prognostic markers of metastatic disease and survival, although the follow-up time was short.