FLASH is an essential component of Cajal bodies.

Cajal bodies are small nuclear organelles with a number of nuclear functions. Here we show that FLICE-associated huge protein (FLASH), originally described as a component of the apoptosis signaling pathway, is mainly localized in Cajal bodies and is essential for their structure. Reduction in FLASH expression by short hairpin RNA results in disruption of the normal architecture of the Cajal body and relocalization of its components. Because the function of FLASH in the apoptosis receptor signaling pathway has been strongly questioned, we have now identified a clear function for this protein.

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.

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.

Comparison of the ex vivo chemosensitivity of uveal and cutaneous melanoma.

Cutaneous and uveal melanoma both have a poor prognosis and chemotherapy is usually unsuccessful. We have previously reported the activity of a number of cytotoxic agents against metastatic cutaneous and primary choroidal uveal melanoma using an ex vivo adenosine triphosphate (ATP)-based chemosensitivity assay (ATP-TCA). In this study we compare the results obtained with the two types of melanoma. Cutaneous melanoma deposits in skin and lymph nodes (n = 58) and choroidal melanomas (n = 77) were tested using the ATP-TCA. Analysis of the data based on an arbitrary threshold for sensitivity shows that both types of melanoma exhibit heterogeneity of sensitivity to all the agents and combinations tested. With all the single agents except gemcitabine, cutaneous melanomas showed greater sensitivity in the assay, though this did not achieve statistical significance. This was also true with the drug combinations, with the exception of treosulfan + gemcitabine, which had similar activity in each type of melanoma. Of all the single agents tested, doxorubicin (47% of specimens classed as sensitive), vinorelbine (43%), treosulfan (41%) and paclitaxel (33%) showed the greatest activity with cutaneous melanoma. In the uveal melanoma samples, mitoxantrone (33%), gemcitabine (22%) and treosulfan (21%) showed the greatest activity. In contrast to the cutaneous melanomas, 13% of the uveal melanomas were sensitive to paclitaxel, 4% were sensitive to doxorubicin and 11% were found to be sensitive to vinorelbine. Both tumour types showed greater sensitivity to combinations of cytotoxic agents. The combination of treosulfan + gemcitabine was universally effective, with 72% of cutaneous melanomas and 80% of uveal melanomas exhibiting activity at the level selected to indicate sensitivity in the assay, though this will not necessarily indicate a similar level of clinical sensitivity.

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 (TGFbeta) in melanoma cell lines. Since the gene for TGFbeta receptor II (TGFbetaR2) is located on chromosome 3p22, this study investigates the possibility that the TGFbeta pathway, and TGFbetaR2 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 (TGFbeta, TGFbetaR2, SMAD2, SMAD3, SMAD4, and p27), backed up by a cell culture assay of TGFbeta-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 TGFbetaR2 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 TGFbeta pathway in 61% of tumours for which unequivocal results were obtained and suggest that abrogation of control of melanocyte growth by the TGFbeta pathway may be important in the formation of uveal melanoma.

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 microM). 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.

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.

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 pretreated recurrent ovarian cancer in just over half of the cases tested.

Heterogeneity of chemosensitivity of metastatic cutaneous melanoma.

Advanced melanoma has a poor prognosis and chemotherapy provides little benefit for most patients. This may be related to heterogeneity of chemosensitivity as well as frequent constitutive resistance to individual cytotoxic drugs. We have therefore examined the heterogeneity of chemosensitivity in metastatic cutaneous melanoma specimens using an ex vivo ATP-based chemosensitivity assay (ATP-TCA). Melanoma deposits (n=55) in skin or lymph node were tested using the ATP-TCA, performed in three separate laboratories. Analysis of the data collected (based on an arbitrary sensitivity index < 300) shows considerable heterogeneity of chemosensitivity. The most active single cytotoxic agents in the assay were identified as cisplatin, treosulfan, paclitaxel, vinblastine, gemcitabine and mitoxantrone. There was also a limited direct inhibition of melanoma cell growth by interferon-alpha2b, although this agent is known to have a number of indirect biological antitumor effects. Exposure of tumor cells to combinations of drugs at the concentrations tested as single agents showed the most active combinations to be treosulfan+gemcitabine, cisplatin+paclitaxel and vinblastine+paclitaxel. There was considerable heterogeneity of chemosensitivity: some tumors responded well to one agent or combination, while others showed no response to this and instead responded to one of the alternatives tested. Occasional highly resistant tumors showed no response to any of the single agents or combinations tested. The degree of heterogeneity observed suggests that the ATP-TCA could be used to select patients who might benefit from specific chemotherapeutic agents alone or in combination. This provides the rationale for future randomized controlled trials of ATP-TCA-directed chemotherapy versus physician's choice to determine whether assay-directed chemotherapy can improve patient response and survival.

Combination chemotherapy for choroidal melanoma: ex vivo sensitivity to treosulfan with gemcitabine or cytosine arabinoside.

Treatment of choroidal melanoma by chemotherapy is usually unsuccessful, with response rates of less than 1% reported for dacarbazine (DTIC)-containing regimens which show 20% or more response rates in skin melanoma. Recently, we reported the activity of several cytotoxic agents against primary choroidal melanoma in an ATP-based tumour chemosensitivity assay (ATP-TCA). In this study, we have used the same method to examine the sensitivity of choroidal melanoma to combinations suggested by our earlier study. Tumour material from 36 enucleated eyes was tested against a battery of single agents and combinations which showed some activity in the previous study. The combination of treosulfan with gemcitabine or cytosine arabinoside showed consistent activity in 70% and 86% of cases, respectively. Paclitaxel was also active, particularly in combination with treosulfan (47%) or mitoxantrone (33%). Addition of paclitaxel to the combination of treosulfan + cytosine analogue added little increased sensitivity. For treosulfan + cytosine arabinoside, further sequence and timing experiments showed that simultaneous administration gave the greatest suppression, with minor loss of inhibition if the cytosine analogue was given 24 h after the treosulfan. Administration of cytosine analogue 24 h before treosulfan produced considerably less inhibition at any concentration. While we have so far been unable to study metastatic tumour from choroidal melanoma patients, the combination of treosulfan with gemcitabine or cytosine arabinoside shows activity ex vivo against primary tumour tissue. Clinical trials are in progress.