Robert M. Conway

Vincristine- and cisplatin-induced apoptosis in human retinoblastoma. Potentiation by sodium butyrate

Chemotherapy alone has largely been unsuccessful in controlling retinoblastoma growth, and has traditionally been limited in use as an alternative to irradiation for the treatment of retinoblastoma. Recently, clinical studies combining chemotherapy with local therapies, including radiotherapy, laser therapy or cryotherapy and in some cases, cyclosporine A, have been effective in treating retinoblastoma. Differentiating agents may also be combined with chemotherapy to enhance the action of cytotoxic drugs on tumor cell growth, although this approach has not been fully investigated in retinoblastoma. In this study, we evaluated the cytotoxic response of human retinoblastoma cell lines (Y79 and WERI-Rb1) to two chemotherapy agents commonly used in treating retinoblastoma, vincristine (VCR) and cisplatin (CDDP). Retinoblastoma cells have been shown to be sensitive to the differentiating agent sodium butyrate, and cell lines were also treated with a combination of VCR or CDDP with sodium butyrate, and the effects on retinoblastoma viability assessed. Both VCR and CDDP induced dose-dependent death of Y79 and WERI-Rb1 cells, accompanied by nuclear and cytoplasmic condensation and DNA laddering, features characteristic of apoptosis. Inhibitors of macromolecular synthesis, cycloheximide and actinomycin-D, significantly reduced VCR- and CDDP-induced apoptosis, although putative endonuclease inhibitors zinc sulphate and aurintricarboxylic acid had no apparent effect. Treatment with 0.5 mM or 1 mM sodium butyrate combined with VCR or CDDP significantly increased induction of apoptosis by these agents. This augmentation of chemotherapy-induced apoptosis may have implications for retinoblastoma therapy.

Human uveal melanoma expresses NG2 immunoreactivity

Background/aims: NG2 is the rat homologue of the human melanoma proteoglycan (HMP), also known as the high molecular weight melanoma associated antigen. Most cutaneous melanomas, as well as glioblastomas, chondrosarcomas, and some leukaemias express NG2 immunoreactivity, recognised using monoclonal antibody (mAb) 9.2.27. This antibody has also been used for molecular targeting in targeted α therapy for melanoma. The purpose of this study was to evaluate the expression of NG2 immunoreactivity in human uveal melanoma and normal ocular tissue using mAb 9.2.27. Methods: Enucleated eyes from 26 patients with choroidal or ciliary body melanoma (n=26) were available as paraffin sections, and stained with haematoxylin and eosin to assess for tumour cell type and histopathology. Additional slides were investigated for NG2 immunoreactivity using mAb 9.2.27 and alkaline phosphatase anti-alkaline phosphatase (APAAP) immunostaining. Two independent observers graded immunostaining using a semiquantitative scale from 0 (negative) to 3 (strong). Results: Immunostaining for mAb 9.2.27 could not be graded in 7/26 cases with dense pigmentation of the tumour. For the remaining cases, grade 2 (moderate) or more immunostaining was seen in 18/19 tumours (95%). The retina, retinal pigment epithelium (RPE), and choroid displayed weak immunostaining (grade 0.5–1.5) in the majority of melanoma affected eyes. Normal retina and choroid (n=5) appeared negative for mAb 9.2.27. Optic nerve axon bundles in both control and melanoma affected eyes displayed moderate immunostaining. Conclusion: In the present study, the majority of human uveal melanomas expressed NG2 immunoreactivity, as detected using mAb 9.2.27. This antibody may be a suitable candidate for radioimmunotherapy to target ocular melanoma.

Expression and distribution of MMPs and TIMPs in human uveal melanoma.

Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) are involved in tumour invasion, metastasis and angiogenesis, and have been implicated as progression markers in uveal melanoma, although their topographical expression has not been fully described. In this study we compared the distribution and specificity of several classes of MMPs (MMP-1, -2, -9, -19, and MT1-MMP) and physiological MMP inhibitors (TIMP-2 and -3) in different regions of the tumour microenvironment and adjacent choroid in a series of primary uveal melanomas. Paraffin sections of untreated uveal melanomas (n=18, 3/18 spindle; 11/18 mixed, and 4/18 epithelioid) were examined for MMP-1 (collagenase 1), MMP-2 and MMP-9 (gelatinases A and B), MT1-MMP (membrane-type 1-MMP), MMP-19, TIMP-2 and TIMP-3 (tissue inhibitors of MMPs), using indirect peroxidase immunohistochemistry. The distribution and intensity of immunolabelling was graded semi-quantitatively (0-3) by 2 independent observers. Non-parametric analyses were used to test for associations between tumour cell type, and the average grade of MMP or TIMP expression. Immunostaining for MMP-1, -9, -19 and MT1-MMP was > or =Grade 2 in more than 70% of specimens, and a heterogeneous pattern of MMP-1, -9, MT1-MMP and TIMP-3 expression was observed. At the tumour-scleral interface (TSI), melanoma cells had a flattened morphology and a much reduced MMP and TIMP expression, with a high expression in tumour areas adjacent to the TSI. Tumour vasculature and stromal cells strongly expressed MMP-2. We also observed heterogeneous immunostaining of the vasculature by MMP-1, -9, MT1-MMP and TIMP-2 antibodies, and of the extravascular matrix by MMP-9 antibody. The distinct immunostaining patterns observed for MMPs and TIMPs within uveal melanoma are consistent with their involvement in tumour growth and angiogenesis. In particular, the heterogeneous expression within regions of the tumours, and the localized expression in vasculature and stromal cells emphasises the importance of the tumour microenvironment in the pathogenesis of uveal melanoma (and other tumours).

Expression of cancer-testis antigens (MAGE-A1, MAGE-A3/6, MAGE-A4, MAGE-C1 and NY-ESO-1) in primary human uveal and conjunctival melanoma

Aim Metastatic disease in ocular melanoma remains untreatable, is associated with late detection and is resistant to conventional systemic therapies. Many tumours including cutaneous melanoma express specific cancer-testis (CT) antigens and vaccines targeting these antigens can induce T-cell-mediated and humoural immune responses. The authors examined primary uveal and conjunctival melanomas for expression of CT antigens to assess their potential as targets for ocular melanoma immunotherapy. Methods Paraffin-embedded uveal (n=32) and conjunctival (n=15) melanomas were assessed by immunohistochemistry for melanocyte differentiation antigens (gp100, Melan-A/MART-1 and tyrosinase), and CT antigens (MAGE-A1, MAGE-A3/6, MAGE-A4, MAGE-C1 and NY-ESO-1). Results Melanoma differentiation antigens, gp100, Melan-A/MART1 and tyrosinase, were expressed in >75% of tumour cells in all uveal and conjunctival melanomas tested. Expression of all five CT antigens tested was low in uveal melanomas, and when present, stained <25% of the tumour cells. MAGE-A1, MAGE-A4 and NY-ESO-1 were expressed in <10% of tumour cells in conjunctival melanomas, while MAGE-C1 and MAGE-A3/6 were expressed in ∼20% and ∼35% of tumour cells in this malignancy, respectively, with variable expression levels. Conclusions Uveal and conjunctival melanomas consistently expressed high levels of the differentiation antigens (gp100, Melan-A/MART1 and tyrosinase). However, compared with other tumours, including cutaneous melanoma, only low levels of CT antigens were found in ocular melanomas. These observations suggest that immunotherapy directly targeting the CT antigens studied may not be effective for ocular melanoma.

Beta-lapachone inhibits proliferation and induces apoptosis in retinoblastoma cell lines.

AimsTo investigate the cytotoxicity of beta-lapachone, a potent agent that may selectively target tumour cells, in retinoblastoma (RB) cell lines.MethodsGrowth inhibitory effects of beta-lapachone were evaluated in Y79, WERI-RB1, and RBM human retinoblastoma cell lines. Pro-apoptotic effects of beta-lapachone were evaluated in Y79 cells by detection of caspase 3/7 activity, by enzyme-linked immunosorbent assay for nucleosome fragments, and by cellular morphological analysis.ResultsBeta-lapachone induced significant dose-dependent growth inhibitory effects in all three retinoblastoma cell lines. The 50% growth inhibitory concentration (IC50) of this agent was 1.9 μM in Y79 cells, 1.3 μM in WERI-RB1 cells, and 0.9 μM in RBM cells. Beta-lapachone also induced proapoptotic effects in RB cells. Treatment of Y79 cells with 1.9 μM beta-lapachone (IC50) resulted in a peak, fourfold induction of caspase 3/7 activity at 72 h post-treatment; a peak, 5.6-fold increase in nucleosome fragments at 96 h post-treatment; and a peak, 1.7-fold increase in the frequency of apoptotic cells at 48 h post-treatment, relative to vehicle-treated controls.ConclusionBeta-lapachone induced potent cytotoxic effects in RB cell lines at low micromolar concentrations, suggesting this agent could be useful in the clinical management of RB.

Tapioca melanoma of the iris mimicking a vascular tumour: a clinicopathological correlation

Although vascular iris processes are usually benign, they may be a clinical manifestation of iris melanoma, and this should be considered in the differential diagnosis. The case is reported of a 20‐year‐old man with a highly vascularized tapioca melanoma of the left iris without involvement of the anterior chamber angle or ciliary body, but with observed growth tendency. Sector iridectomy was performed 2 months after first examination. In addition, two separate floating tumour nodules were observed in the anterior chamber angle at 6 o’clock and were removed by aspiration. After 1 year follow up, no recurrence of melanoma cells was observed. In tapioca iris melanomas, diagnostic differentiation from congenital, inflammatory, vascular and neoplastic iris processes is required. Careful biomicroscopic examination of the iris in patients with vascular iris tumours may help the ophthalmologist to identify malignant iris melanomas (tapioca type) and plan appropriate surgical treatment.

Human retinoblastoma: in vitro differentiation and immunoglobulin superfamily antigen modulation by retinoic acid.

Abstract Suspension and attachment cultures of Y79 human retinoblastoma cells were treated with all-trans retinoic acid (RA) for up to 10 days to assess its effect on growth and cell-surface expression of immunoglobulin superfamily antigens MHC class I and class II, ICAM-1, NCAM and Thy1. RA up to 10 μM induced growth inhibition, and marked morphological differentiation with extension of prominent processes resembling neurites was seen in attachment cultures. However, above 10 μM RA produced extensive cell death. We also observed increased cell-surface expression of MHC class I, ICAM-1, NCAM and Thy1 on Y79 cells treated with 10 μM over 10 days; constitutive MHC class II expression was not apparent, nor did RA treatment appear to induce Y79 cells to express MHC class immunoreactivity. The up-modulation of cell-adhesion molecules (NCAM, ICAM-1 and Thy1) and immune recognition molecules (NCAM, ICAM-1 and MHC class I), associated with reduced growth and tumour cell differentiation, suggests that RA may have a potential role in regulating the growth and development of retinoblastoma tumours.