Michael H. Neale

Vascular endothelial growth factor is elevated in ocular fluids of eyes harbouring uveal melanoma: identification of a potential therapeutic window

Background: Improved local treatment of uveal melanoma makes it possible for many patients to retain the affected eye, but a proportion will develop secondary complications such as neovascularisation of the iris (NVI) and require enucleation. Although vascular endothelial growth factor A (VEGF-A) is known to correlate with NVI and can cause NVI in experimental models, this pro-angiogenic cytokine is consistently reported to be absent in uveal melanoma. Novel anti-VEGF therapies are now in clinical trial, and the authors therefore wished to determine whether VEGF-A was indeed elevated in melanoma bearing eyes. Methods: VEGF-A concentrations were measured in aqueous and vitreous from 19 and 30 enucleated eyes respectively. Results: Elevated VEGF-A concentrations (up to 21.6 ng/ml) were found in melanoma bearing eyes compared with samples from patients undergoing routine cataract extraction (all had values below 0.96 ng/ml). Immunohistochemistry showed VEGF-A protein in the iris and/or ciliary body of 54% and basic fibroblast growth factor (bFGF) in 82% of the eyes examined. VEGF was found to a limited extent and at very low levels in only 9% of these tumours. Aqueous or vitreous VEGF levels showed no apparent correlation with retinal detachment, tumour size, vascularity, or immunohistochemistry. Though limited in number, the highest VEGF levels correlated with previous radiation therapy, and with the presence neovascularisation of the iris or optic nerve head. bFGF was not significantly elevated in ocular fluids: it is known to be a pro-angiogenic agent and was detected in the majority of primary uveal melanomas. Conclusion: Based on this study, though the source of VEGF within eyes harbouring uveal melanoma is not clear, these data suggest that anti-VEGF therapy might prove useful in the management of some patients with NVI secondary to uveal melanoma.

Uveal melanomas express vascular endothelial growth factor and basic fibroblast growth factor and support endothelial cell growth

Background: Tumour microvascularity is a significant determinant of prognosis for a large number of different tumours, including uveal melanoma. The development of blood vessels within these and other tumours is partly controlled by soluble pro-angiogenic cytokines, of which basic fibroblast growth factor (bFGF) and vascular endothelial growth factor-A (VEGF) are the best described. Methods: Because VEGF has been inconsistently found within uveal melanomas and bFGF is described as an autocrine growth factor in cutaneous melanoma, the authors looked at the expression of these cytokines in uveal melanomas using immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR). The cross talk between uveal melanoma cells and endothelial cells was then assessed in an in vitro co-culture model. Results: While most tumour cells expressed bFGF at the protein level by immunohistochemistry (89%), relatively few (22%) expressed VEGF, and this was of limited extent. All 20 tumours tested by RT-PCR contained mRNA for both bFGF and VEGF. Co-culture experiments using an ATP based bioassay showed that uveal melanomas could support the growth of a rat brain endothelial cell line (GPNT) and human umbilical vein endothelial cells (HUVEC), and that this could be modulated by cytokines and anti-cytokine antibodies. Conclusion: These results suggest that angiogenesis within uveal melanoma may be the result of a complex interplay between endothelial and tumour cells, and that bFGF and VEGF could play a part.

Abnormalities of the transforming growth factor‐beta pathway in ocular melanoma

The majority of ocular melanomas occur in the uveal tract. Chemotherapy is generally ineffective and large tumours requiring enucleation have a greater than 50% mortality at 5 years. Monosomy for chromosome 3 is common in uveal melanoma and it is known that there is loss of responsiveness to transforming growth factor beta (TGFβ) in melanoma cell lines. Since the gene for TGFβ receptor II (TGFβR2) is located on chromosome 3p22, this study investigates the possibility that the TGFβ pathway, and TGFβR2 in particular, might be involved in the pathogenesis of this rare eye tumour. To this end, the expression of molecules in the pathway has been examined by immunocytochemistry (TGFβ, TGFβR2, SMAD2, SMAD3, SMAD4, and p27), backed up by a cell culture assay of TGFβ‐mediated growth suppression, RT‐PCR for SMAD4, and loss of heterozygosity (LOH) on 3p22. There was LOH at 3p22 in 6/19 tumours and loss of TGFβR2 expression in 10/27 tumours. Immunohistochemistry for SMADs 2, 3, and 4 showed potential loss of signal transduction in 14/27 tumours. The results indicate abnormality of the TGFβ pathway in 61% of tumours for which unequivocal results were obtained and suggest that abrogation of control of melanocyte growth by the TGFβ pathway may be important in the formation of uveal melanoma. Copyright

Relationship between expression of topoisomerase II isoforms and chemosensitivity in choroidal melanoma

Choroidal melanoma has a high mortality rate and responds poorly to existing chemotherapy, but unexpected ex vivo sensitivity of a subset of these tumours to topoisomerase II inhibitors has been noted. Since chemoresistance may be mediated by the molecular phenotype of tumours, immunohistochemistry has been used to study the expression of both isoforms of topoisomerase II (alpha and beta) in 29 choroidal melanomas for which chemosensitivity assay data for doxorubicin or mitoxantrone are also available. Of these, eight tumours were topoisomerase II beta‐positive and 11 were topoisomerase II alpha‐positive. Recent studies showing genetic abnormality (often monosomy of chromosome 3) in choroidal melanoma suggest that loss of immunostaining could be due to genomic loss rather than down‐regulation of topoisomerase II beta in these tumours. There was no convincing excess of anthracycline resistance in the topoisomerase II beta‐negative group. Addition of topoisomerase II alpha, MDR1 (11/17 positive), LRP (16/28 positive), and MRP (5/29 positive) data in multivariate analysis did not reliably predict sensitivity or resistance. Vincristine chemosensitivity showed no relation to MDR1, LRP or MRP in 18 tumours tested. While it is possible that some tumours which do express topoisomerase II beta may respond to anthracyclines, the molecular basis of resistance or sensitivity to anthracyclines or vincristine in uveal melanoma is complex and remains incompletely understood. Copyright

Culture and Characterization of Oral Mucosal Epithelial Cells on a Fibrin Gel for Ocular Surface Reconstruction

Abstract Aim of the study: To develop a clinical grade fibrin gel for the culture of oral mucosal epithelial cells (OMEC) intended for ocular surface reconstruction in the treatment of limbal stem cell deficiency (LSCD). Materials and methods: Transparent fibrin gels composed of fibrinogen and thrombin were developed for the culture of epithelial cells. Oral mucosa was harvested from the buccal region of healthy volunteers and cultured as explants on fibrin gels. Tranexamic acid (TA), a clinically approved anti-fibrinolytic agent was added to prevent the fibrin gel from digesting due to cellular activity. The gels were stained for p63α (as a marker of poorly differentiated epithelial cells), CK19, CK13 and CK3 (expressed by OMEC). Epithelial cell stratification was observed using hematoxylin–eosin staining. Results: Addition of TA prevented gels from dissolving during the culture period. OMEC proliferated on the fibrin gel and attained confluence over a 2-week period (±2 d) and exhibited a typical epithelial, cobblestone morphology. Basal OMEC exhibited positive staining for p63α while the superficial cells exhibited positive staining for CK3. The cells expressed a strong immunoreactivity for CK19 and CK13 suggesting that they retained a normal oral epithelial phenotype. Conclusion: Fibrin gels, maintained in the presence of TA, to control the rate of substrate degradation, provide a more robust yet transparent substrate for the culture and transplantation of cultured OMEC. The fibrin gels are easily standardized, the components commercially available, and produced from clinically approved materials. The resulting stratified OMEC-derived epithelium displays characteristics similar to that of a human cornea, e.g. CK3 expression. The conventional dependence on a murine feeder layer for support of epithelial cells is unnecessary with this technique and hence, provides for an attractive alternative for treatment of LSCD.

The ex vivo effect of high concentrations of doxorubicin on recurrent ovarian carcinoma

The cardiotoxicity of anthracyclines has largely prevented dose intensification, but the use of liposomal preparations (e.g. Caelyx/Doxil) allows much higher intra-tumoral concentrations to be achieved without cardiotoxicity. However, it is uncertain how much this will improve response rates over standard anthracycline therapy. The ATP-based chemosensitivity assay (ATP-TCA) has been used to develop new regimens for several tumor types, to investigate the molecular basis of chemosensitivity and shows considerable promise as a clinical method for individualizing chemotherapy. In this study, we have used the ATP-TCA to determine the concentration responsiveness of tumor-derived cells to concentrations of doxorubicin. The 22 tumor samples included were obtained from 20 heavily pretreated patients with recurrent ovarian cancer. Eight had previous anthracycline exposure, four as part of the CAP regimen. The results show more than 95% inhibition at clinically achievable concentrations in 11 of 22 tumors tested. Of the rest, seven showed a plateau effect between 80 and 95% inhibition, suggesting that there might be a subset of resistant cells present that is not inhibited by high concentrations of doxorubicin. Two tumors showed complete resistance and neither of these had previously received anthracycline therapy. As it has been suggested that gemcitabine might enhance anthracycline sensitivity in combination and we have had good results with gemcitabine modulation of alkylating agents in the assay, we have tested the combination of doxorubicin+gemcitabine under assay conditions in 11 tumors with little indication of improvement. In conclusion, doxorubicin at concentrations achievable with liposomal preparations shows strong ex vivo activity against pre-treated recurrent ovarian cancer in just over half of the cases tested.

Advanced imaging and tissue engineering of the human limbal epithelial stem cell niche.

The limbal epithelial stem cell niche provides a unique, physically protective environment in which limbal epithelial stem cells reside in close proximity with accessory cell types and their secreted factors. The use of advanced imaging techniques is described to visualize the niche in three dimensions in native human corneal tissue. In addition, a protocol is provided for the isolation and culture of three different cell types, including human limbal epithelial stem cells from the limbal niche of human donor tissue. Finally, the process of incorporating these cells within plastic compressed collagen constructs to form a tissue-engineered corneal limbus is described and how immunohistochemical techniques may be applied to characterize cell phenotype therein.

Ex vivo activity of XR5000 against solid tumors.

Topoisomerases I and II unravel DNA during transcription, DNA replication and DNA repair. Inhibitors of both enzymes are important anticancer drugs, but only now are combined inhibitors becoming available for clinical use. In this study we have used an ATP-based chemosensitivity assay to determine the activity of XR5000 and possible combinations against ovarian cancer, a tumor sensitive to current topoisomerase inhibitors, and melanoma, an insensitive tumor. A further six tumors of other types were also tested. The results from 20 ovarian cancer and 18 melanoma biopsies show remarkably little difference between the tumor types in terms of IC50, IC90 or two summary indices of chemosensitivity based on all of the concentrations tested. XR5000 on its own shows a steep concentration-response curve in most tumors, only achieving high reduction (above 95%) of ATP levels at 2440 ng/ml (6 μM). The results were often similar to the combination of etoposide and topotecan, particularly at the higher concentrations tested. The combinations with greatest activity in ovarian cancer were with paclitaxel or cisplatin, while melanoma showed greatest improvement with paclitaxel or treosulfan. The results are encouraging for the clinical introduction of this agent, and suggest that it will be effective in combination with currently available drugs for both ovarian cancer and melanoma.

Regulatory requirements in the good manufacturing practice production of an epithelial cell graft for ocular surface reconstruction

In the past decade, stem cell therapy has been increasingly employed for the treatment of various diseases. Subsequently, there has been a great interest in the manufacture of stem cells under good manufacturing practice, which is required by law for their use in humans. The cells for sight Stem Cell Therapy Research Unit, based at UCL Institute of Ophthalmology, delivers somatic cell-based and tissue-engineered therapies to patients suffering from blinding eye diseases at Moorfields Eye Hospital (London, UK). The following article is based on our experience in the conception, design, construction, validation and manufacturing within a good manufacturing practice manufacturing facility based in the UK. As such the regulations can be extrapolated to the 28 members stated within the EU. However, the principles may have a broad relevance outside the EU.

Treosulfan and gemcitabine

Contrary to the authors’ assertion, the paper by Terheyden et al. confirms the results of earlier in vitro studies, which suggested that low doses of treosulfan will be ineffective. The title is therefore misleading and the paper contains many other inaccuracies. Our in vitro papers (Myatt et al. 1997; Neale et al. 1999) showed enhancement of the cytotoxicity of treosulfan by gemcitabine in uveal melanoma cells obtained from the primary tumour. We used an arbitrary threshold to compare the sensitivity of the agents tested, equivalent to 50% inhibition of the ATP content of the cells across the range of concentrations tested (Neale et al. 1999). Our data showed that DTIC was inactive, in keeping with clinical results, and that treosulfan alone showed the best activity of any of the alkylating agents tested (6% with >50% inhibition) (Neale et al. 1999). This was modulated by gemcitabine, a cytidine analogue we postulated might inhibit repair of treosulfan-induced DNA damage, with 70% of tumours then showing >50% inhibition in the assay. It should be noted that the logarithmic kill hypothesis requires around 99% inhibition to produce a clinical response in six cycles. We were extremely careful to emphasise that these comparative figures would therefore not be expected to translate directly into clinical practice. Our papers simply suggested that treosulfan + gemcitabine might be more active in patients than other drugs and combinations tested, and stated that clinical trials were necessary. With colleagues from Cologne, we designed a regimen based on the concentrations tested in vitro and the presumed mechanism of action. We have found this to be active in uveal melanoma, skin melanoma, and breast and ovarian carcinoma (Kurbacher et al. 2003; Sharma et al. 2003). We have suggested a mechanism of action of the combination similar to that for cisplatin + gemcitabine, which has been more extensively studied (Peters et al. 1996). Treosulfan causes DNA damage proportional to its concentration—more drug produces more damage. The cell will growth arrest via a p53-dependent mechanism (Neale 2001), and may die by apoptosis (Hartley et al. 1999). The effect of treosulfan can be modulated by the addition of gemcitabine, which has been shown to inhibit the function of DNA polymerases, preventing cells from repairing the damage caused by treosulfan. It is also possible that there is greater incorporation of gemcitabine into DNA due to increased DNA repair. Certainly, giving gemcitabine before treosulfan results in loss of inhibition. Our data suggest that it is important to give the highest possible dose of treosulfan: the gemcitabine is acting as a modulating agent and reduction of the dose seems to be possible if it proves necessary to reduce haematological toxicity (C.M. Kurbacher, unpublished results). Gemcitabine is actively transported into the cell and this process is probably saturated at this level in most tumours: giving more may be possible, but is not necessary. The optimal regimen is therefore likely to be treosulfan 5–7 g/m, as recommended by the manufacturers for single agent use, combined with gemcitabine 1,000 mg/m, both on day 1, three weekly. Most groups basing their work on our data have used this regimen, usually with treosulfan 5 g/m in the palliative setting. This contrasts with the regimen used by Terheyden et al., which was not designed by us. We would expect a lower dose of treosulfan to be less effective, and the authors have simply confirmed this. In contrast to its title, the study of Terheyden et al. does not represent a clinical evaluation of in vitro chemosensitivity testing at all, since the investigators neither used an individualized strategy with chemoI. A. Cree (&) Translational Oncology Research Centre, Queen Alexandra Hospital, Portsmouth, PO6 3LY, UK E-mail: ian.cree@porthosp.nhs.uk Fax: +44-2392-286379