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... Show morePURPOSE. 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. Show less
Ly, L.V.; Sluijter, M.; Burg, S.H. van der; Jager, M.J.; Hall, T. van 2013
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... Show morePURPOSE 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. Show less
Lange, J. de; Ly, L.V.; Lodder, K.; Verlaan-de Vries, M.; Teunisse, A.F.A.S.; Jager, M.J.; Jochemsen, A.G. 2012
The prognosis of patients with uveal melanoma is poor. Because of the limited efficacy of current treatments, new therapeutic strategies need to be developed. Because p53 mutations are uncommon in... Show moreThe prognosis of patients with uveal melanoma is poor. Because of the limited efficacy of current treatments, new therapeutic strategies need to be developed. Because p53 mutations are uncommon in uveal melanoma, reactivation of p53 may be used to achieve tumor regression. We investigated the use of combination therapies for intraocular melanoma, based on the p53 activators Nutlin-3 and reactivation of p53 and induction of tumor cell apoptosis (RITA) and the topoisomerase I inhibitor Topotecan. Nutlin-3 treatment induced p53-dependent growth inhibition in human uveal melanoma cell lines. The sensitivity to Nutlin-3 of the investigated cell lines did not correlate with basal Hdm2 or Hdmx levels. Nutlin-3 synergized with RITA and Topotecan to induce apoptosis in uveal melanoma cell lines and short-term cultures. Drug synergy correlated with enhanced induction of p53-Ser46 phosphorylation, which was attenuated by ATM inhibition. Nutlin-3 and Topotecan also significantly delayed tumor growth in vivo in a murine B16F10 model for ocular melanoma. Combination treatment appeared to inhibit tumor growth slightly more efficient than either drug alone. Nutlin-3, RITA and Topotecan lead to comparable p53 activation and growth inhibition under normoxia and hypoxia. Treatment with Nutlin-3 or RITA had no effect on HIF-1α induction by hypoxia, whereas the combination of these two drugs did inhibit hypoxia-induced HIF-1α. Also Topotecan, alone or in combination with Nutlin-3, reduced HIF-1α protein levels, suggesting that a certain level of DNA damage response is required for p53-mediated downregulation of HIF-1α. In conclusion, combination treatments based on small-molecule-induced p53 activation may have clinical potential for uveal melanoma.Oncogene advance online publication, 18 July 2011;doi:10.1038/onc.2011.309. Show less
This thesis describes the role of the immune system as an important phenomenon in the most frequently occurring form of eye cancer in adults, namely in uveal melanoma. We show that the immune... Show moreThis thesis describes the role of the immune system as an important phenomenon in the most frequently occurring form of eye cancer in adults, namely in uveal melanoma. We show that the immune system can be the cause of the tumor, and also plays a role in the development of a tumor, and may be an entry for therapy. In the first chapters of this thesis, we describe the phenomenon of "an inflammatory phenotype" in uveal melanoma. It appears that when this type of cancer shows more inflammation, the survival of patients decreases. A possible explanation for this observation is that one of the key players among the immune cells, the macrophage, plays an essential role in intra-ocular tumor growth. We demonstrate this in patient material, but also in experimental studies; we are able to inhibit tumor growth when we modulate the presence of macrophages. In this thesis, we also show that the immune system can be used effectively to eradicate eye melanomas in experimental models. T cell vaccination in combination with monoclonal antibodies gave promising results for treating eye cancer. Further research has to be performed to translate this into the clinic. Show less
Jager, M.J.; Ly, L.V.; Filali, M. el; Madigan, M.C. 2011
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... Show morePURPOSE. 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. CONCLUSIONS. 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. (Invest Ophthalmol Vis Sci. 2011;52:643-650) DOI:10.1167/iovs.10-5979 Show less
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,... Show morePurpose: 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. Show less
Ly, L.V.; Bronkhorst, I.H.G.; Beelen, E. van; Vrolijk, J.; Taylor, A.W.; Versluis, M.; ... ; Jager, M.J. 2010
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... Show morePURPOSE. 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 degrees 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. (Invest Ophthalmol Vis Sci. 2010; 51: 5445-5451) DOI: 10.1167/iovs.10-5526 Show less
Ly, L.V.; Sluijter, M.; Versluis, M.; Luyten, G.P.M.; Burg, S.H. van der; Melief, C.J.M.; ... ; Hall, T. van 2010
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... Show moreAdoptive 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. Cancer Res; 70(21); 8339-46. (C) 2010 AACR. Show less
Ly, L.V.; Baghat, A.; Versluis, M.; Jordanova, E.S.; Luyten, G.P.M.; Rooijen, N. van; ... ; Jager, M.J. 2010
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... Show moreMacrophages 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. The Journal of Immunology, 2010, 185: 3481-3488. Show less
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,... Show morePURPOSE. 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 LHBETA 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 400 x 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. (Invest Ophthalmol Vis Sci. 2010; 51: 2671-2677) DOI: 10.1167/iovs.09-4255 Show less
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... Show morePURPOSE. 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. (Invest Ophthalmol Vis Sci. 2010; 51: 658-665) DOI: 10.1167/iovs.09-3824 Show less
Filali, M. el; Missotten, G.S.O.A.; Maat, W.; Ly, L.V.; Luyten, G.P.M.; Velden, P.A. van der; Jager, M.J. 2010
