Anna K. Murray

Infrequent expression of the MAGE gene family in uveal melanomas

It has previously been reported that MAGE‐1, ‐2, ‐3 and ‐4 genes are expressed in human cancers including cutaneous melanoma. MAGE‐1 and MAGE‐3 represents targets for specific immunotherapy because they encode peptide antigens which are recognised by cytotoxic T lymphocytes (CTL) when presented by HLA class I molecules, and pilot clinical trials with these peptides are currently in progress. It is likely that other members of the MAGE gene family may also encode antigens recognised by CTL. Uveal melanomas, like cutaneous melanomas, arise from melanocytes that are derived from the neural crest. To determine if uveal melanoma patients would be suitable for MAGE‐peptide immunotherapy, the expression of MAGE‐1, ‐2 ‐3 and ‐4 genes was assessed by reverse transcription followed by polymerase chain reaction (RT‐PCR) amplification and ethidium bromide staining. Expression of MAGE genes was not detected in any of 27 primary tumours. Either MAGE‐1 or MAGE‐4 was expressed in only 2 of 26 metastatic samples, but expression of MAGE‐2 or ‐3 was not detected. Our data suggest that, unlike cutaneous melanomas, uveal melanomas may not be suitable candidates for MAGE‐peptide immunotherapy.

Multiple locations on chromosome 3 are the targets of specific deletions in uveal melanoma

PurposeLoss of chromosome 3 is a frequent event in uveal melanomas, which is associated with hepatic metastases and a poor prognosis. The entire copy of chromosome 3 is usually lost (monosomy 3); however, a small subset of tumours demonstrate partial deletions of chromosome 3. Analysis of these tumours may allow the identification of tumour suppressor genes (TSGs) that are the molecular target of monosomy 3. Therefore, the purpose of this investigation was to determine the location of these partial deletions of chromosome 3 in uveal melanomas.MethodsMicrosatellite analysis and restriction fragment-length polymorphism analysis were performed on 52 primary uveal melanomas using 19 markers located on both arms of chromosome 3. Cytogenetic analysis and fluorescence in situhybridisation were performed, where possible, to confirm molecular findings.ResultsOf 52 tumours studied, five tumours (10%) demonstrated LOH at one or more informative markers, but retention of heterozygosity was observed at other loci on chromosome 3, consistent with the presence of structural abnormalities to chromosome 3. Consistent with previous findings, the pattern of LOH in these tumours indicates the presence of deletions around 3p25–26 and on 3q, and that a new target region at 3p11–14 is preferentially deleted.ConclusionsThese results indicate the presence of several tumour suppressor loci on chromosome 3 and support the notion that the high rate of monosomy 3 in uveal melanoma is driven by disruption of several TSGs located on both arms of chromosome 3.

Tumour immunology: false hopes—new horizons?

Abstract For the past 100 years, tumour immunology has witnessed peaks and troughs of interest. A recent meeting1The 5th Annual Congress of the British Society for Immunology was held at Brighton, UK, on 2–5 December 1997. 1 covered new technologies and recent developments in the field.

Reduced expression of TAP-1 and TAP-2 in posterior uveal melanoma is associated with progression to metastatic disease.

In order to determine the effects of the loss or reduced expression of molecules associated with antigen presentation (transporter associated with antigen presentation [TAP]-1, TAP-2, low molecular weight protein [LMP]-2 and LMP-7), we examined the expression of these molecules in primary uveal melanoma lesions. Paraffin-embedded sections from 29 primary uveal melanoma lesions were analysed for expression of TAP-1, TAP-2, LMP-2 and LMP-7 using specific primary antibodies followed by a three-stage immunoperoxidase technique. Microscopic examination was undertaken to determine differences in expression of these molecules on the tumour and the surrounding stroma. Overall, 72% (21 out of 29) of the tumours showed some loss or reduced expression of TAP-1, TAP-2, LMP-2 and/or LMP-7. Statistical analysis of these results showed that progression to metastatic disease was strongly associated with reduced expression of TAP-1 (P<0.05) and TAP-2 (P <0.01), taking patient age, tumour site and histology into account. We conclude that the reduced expression of molecules important in eliciting an immune response, such as TAP-1 and TAP-2, may facilitate the metastatic spread of uveal melanoma lesions and may have important implications for prospective immunotherapy.

Expression of integrins, degradative enzymes and their inhibitors in uveal melanoma: differences between in vitro and in vivo expression.

Posterior uveal melanoma is the most common intraocular malignancy in adults. Metastasis occurs in approximately 40% of all cases and spread is primarily to the liver. Once secondary hepatic disease has developed the prognosis is poor. Metastasis involves a series of adhesion and de-adhesion events, coupled with regulated tissue degradation to facilitate tumour cell invasion and spread to both local and distant sites. These processes are assisted by the expression of integrins and degradative enzymes by both tumour and host cells. Using a series of 10 uveal melanomas, we investigated the expression of a panel of integrins, degradative enzymes and their inhibitors that have been shown to be associated with metastasis. In addition, we undertook to establish if there might be differential expression in response to growth under artificial conditions. All the tumours expressed matrix metalloproteinases (MMP)-2 and-9, tissue inhibitor of metalloproteases (TIMP)-2, urokinase plasminogen activator (u-PA), plasminogen activator inhibitor (PAI)-1 and PAI-2. Differences in the expression of the integrins α1β1, α2β1 and α6β1 were observed; in particular, these differences appeared to relate to expression as a consequence of growth in culture. In summary, uveal melanoma cells express both degradative enzymes and their respective inhibitors, which are important in metastasis. It would appear that differential expression of integrins is present, probably as a response to in vitro stimulation.

Induction of human cytotoxic T lymphocytes that preferentially recognise tumour cells bearing a conformational p53 mutant

Abstract The tumour-suppressor gene p53 is pivotal in the regulation of apoptosis, and point mutations within p53 are the commonest genetic alterations in human cancers. Cytotoxic T lymphocytes (CTL) recognise peptide-MHC complexes on the surface of tumour cells and bring about lysis. Therefore, p53-derived peptides are potential candidates for immunisation strategies designed to induce antitumour CTL in patients. Conformational changes in the p53 protein, generated as a result of point mutations, frequently expose the 240 epitope, RHSVV (amino acids 212–217), which may be processed differently from the wild-type protein resulting in an altered MHC-associated peptide repertoire recognised by tumour-specific CTL. In this study 42 peptides (37 overlapping nonameric peptides, from amino acids 193–237 and peptides 186–194, 187–197, 188–197, 263–272, 264–272, possessing binding motifs for HLA-A2) derived from the wild-type p53 protein sequence were assayed for their ability to stabilise HLA-A2 molecules in MHC class I stabilisation assays. Of the peptides tested, 24 stabilised HLA-A2 molecules with high affinity (fluorescence ratio>1.5) at 26 °C, and five (187–197, 193–200, 217–224, 263–272 and 264–272) also stabilised the complexes at 37 °C. Peptides 188–197, 196–203 and 217–225 have not previously been identified as binders of HLA-A2 molecules and, of these, peptide 217–225 stabilised HLA-A2 molecules with the highest fluorescence ratio. Peptide 217–225 was chosen to generate HLA-A2-restricted CTL in vitro; peptide 264–272 was used as a positive control. The two primary CTL thus generated (CTL-217 using peptide 217–225; and CTL-264 using peptide 264–272) were capable of specifically killing peptide-pulsed T2 or JY cells. In order to determine whether these peptides were endogenously processed and to test the hypothesis that mutants expressing different protein conformations would generate an alternative peptide repertoire at the cell surface, a panel of target cells was generated. HLA-A2+ SaOs-2 cells were transfected with p53 cDNA containing point mutations at either position 175 (R → H) or 273 (R → H) (SaOs-2/175 and SaOs-2/273). Two HLA-A2-negative cell lines, A431 and SKBr3, naturally expressing p53 mutations at positions 273 and 175 respectively, were transfected with a cDNA encoding HLA-A2. The results showed that primary CTL generated in response to both peptides were capable of killing SaOs-2/175 and SKBr3-A2 cells, which possess the same mutation, but not SaOs-2/273, A431-A2 or SKBr3 cells transfected with control vector. This suggests that these peptides are presented on the surface of SaOs-2/175 and SKBr3-A2 cells in a conformation-dependent manner and represent potentially useful target peptides for immunotherapy.

Human antigen‐presenting cell/tumour cell hybrids stimulate strong allogeneic responses and present tumour‐associated antigens to cytotoxic T cells in vitro

Most tumours do not stimulate effective antitumour immune responses in vivo. In order to enhance the immunogenicity of human tumour cells, we fused a variety of tumour cell lines with an Epstein–Barr virus transformed B‐lymphoblastoid cell line (EBV B‐LCL) in vitro, to produce stable hybrid cells. Hybrid cell lines showed a marked increase in their ability to stimulate primary allogeneic T‐cell responses in vitro, as compared with the parent tumour cells. The hybrid cells induced proliferation of naive (CD45RA+) as well as memory (CD45RO+) T lymphocytes, and both CD4+ and CD8+ subpopulations of T cells were directly stimulated. The stimulatory hybrids expressed human leucocyte antigen (HLA) class I and II, and a wide range of surface accessory molecules, including the T‐cell co‐stimulatory ligand molecules CD40, CD80 (B7.1) and CD86 (B7.2), the expression of which was required for optimal stimulation of T‐cell responses. Fusion of the EBVB‐LCL with a melanoma cell line (518.A2) yielded hybrid cells that expressed the melanoma‐associated antigens MAGE‐1 and MAGE‐3, and presented these antigens to antigen‐specific, HLA class I‐restricted cytotoxic T‐lymphocyte clones with greater efficiency than the parent melanoma cell line. These findings suggest that the generation of human antigen‐presenting cell/tumour cell hybrids offers promise as an approach to cancer immunotherapy.

Identification of peptide epitopes of MAGE-1, -2, -3 that demonstrate HLA-A3-specific binding

Abstract The MAGE gene family of tumour antigens are expressed in a wide variety of human cancers. We have identified 43 nonamer peptide sequences, from MAGE-1, -2 and -3 proteins that contain binding motifs for HLA-A3 MHC class I molecules. The T2 cell line, transfected with the cDNA for the HLA-A3 gene, was used in a MHC class I stabilisation assay performed at 37°C and 26°C. At 37°C, 2 peptides were identified that stabilised HLA-A3 with high affinity (fluorescence ratio, FR >1.5), 4 peptides with low affinity (FR 1.11 – 1.49) and 31 peptides that did not stabilise this HLA haplotype (FR <1.1). At 26°C, 12 peptides were identified that stabilised HLA-A3 with high affinity, 8 peptides with low affinity and 17 peptides that did not stabilise this HLA haplotype. Two peptides stabilised HLA-A3 at both temperatures. Small changes in one to three amino acids at positions distinct from the anchor residues altered peptide affinity. Data were compared to a similar study in which a peptide competition assay was used to investigate MAGE-1 peptide binding to several HLA haplotypes. This study demonstrates that anchor residues do not accurately predict peptide binding to specific HLA haplotypes, changes in one to three amino acids at positions distinct from anchor residues influence peptide binding and alternative methods of determining peptide binding yield different results. We are currently investigating the ability of these peptides to induce antitumour cytotoxic T lymphocyte activity as they may be of potential therapeutic value.

Apoptotic cell death in conjunction with CD80 costimulation confers uveal melanoma cells with the ability to induce immune responses

Uveal melanoma is a rare malignancy with a poor prognosis despite current therapeutic intervention. The current investigation focuses on the immunogenicity of uveal melanoma cells genetically modified with recombinant adenovirus encoding CD80 (AdCD80) in contrast to their parental counterpart. We demonstrate that costimulation provided by uveal melanoma cells improved immune responses in vitro as determined by mixed lymphocyte tumour cell cultures and cytotoxic T‐cell assays using lymphocytes from healthy donors and uveal melanoma patients. Flow cytometry revealed T‐cell stimulation by activated CD4+ and CD8+ T cells. Additionally, autologous lymphocytes proliferated in response to CD80‐expressing primary uveal melanomas, indicating that this patient group is suitable for immunotherapy. Moreover, this study utilized AdCD80 modified and parental apoptotic tumour cells, loaded onto immature dendritic cells, as a source of tumour antigen. The ability of live or apoptotic tumour cells to stimulate lymphocyte proliferation and activation was determined. Apoptotic uveal melanoma cells expressing CD80 were efficient at inducing an immune response and served as a potent immunogen. The use of apoptotic uveal melanoma cells in combination with expression of costimulatory molecules could prove a novel adjuvant therapy for the treatment of this disease.

Recognition of Melanoma-Associated Peptides by Peripheral Blood Mononuclear Cells of Ocular Melanoma Patients

Melanoma of the eye is the most common intraocular malignancy in adults with approximately 50% of patients subsequently developing metastases, usually of the liver. In the United Kingdom there are 300–400 new cases of ocular/uveal melanoma each year. Tumour-associated antigens have been described for ocular melanoma and there is evidence to suggest that choroidal melanoma cells can elicit an immune response to tumour antigens [1]. It has already been shown that although uveal melanoma cells do not express the MAGE genes, they do express high levels of the melanocyte lineage specific genes, tyrosinase, gp100 and melan-A/MART-1 [2] (Table 1). Three nonamer melanoma-associated peptides, restricted by HLA-A0201, were selected from the genes tyrosinase, gp100 and melan-A/MART-1, for the study, and coded, T9368, G9280 and M927 respectively [4-6] (Table 2). These peptides were considered most suitable for the study since, HLA-A0201 is the most frequently expressed Class I allele (49% of Caucasians), and they have been shown to have both the ability to bind to HLA-A0201, and are capable of eliciting a CTL response, restricted by this haplotype, in cutaneous melanoma (Table 2).