John L. Hungerford

Comparison of episcleral plaque and proton beam radiation therapy for the treatment of choroidal melanoma

PURPOSE To compare the efficacy of iodine-125 (125I) and ruthenium-106 (106Ru) episcleral plaque radiation therapy and proton beam radiation therapy (PBRT) in the treatment of choroidal melanoma. DESIGN A retrospective, nonrandomized comparative study. METHODS Charts of patients treated with 125I, 106Ru, and PBRT for choroidal melanoma between January 1988 and June 1996 at St. Bartholomews Hospital and Moorfields Eye Hospitals were reviewed. MAIN OUTCOME MEASURE Local control of choroidal melanomas after 125I, 106Ru, or PBRT. RESULTS A total of 597 patients were identified (125I = 190, 106Ru = 140, PBRT = 267). Patients treated with 106Ru had a significantly greater risk of local tumor recurrence than did patients treated with either 125I (P = 0.0133; confidence interval [CI], 1.26-7.02; risk ratio, 2.97) or PBRT (P = 0.0097; CI, 1.30-6.66; risk ratio, 2.94). A stepwise Cox proportional hazard model found maximal basal diameter to be a significant covariate (P = 0.0033). CONCLUSION Patients treated with 106Ru had a significantly greater risk of local tumor recurrence than did those patients treated with either 125I or PBRT.

The detection of melanoma cells in peripheral blood by reverse transcription-polymerase chain reaction

Both cutaneous and uveal melanoma undergo haematogenous dissemination. Detection of tyrosinase mRNA by reverse transcription-polymerase chain reaction (RT-PCR) has been described as an extremely sensitive way of detecting circulating viable melanoma cells in the peripheral venous blood, and this technique may be of value in the early detection of dissemination. Also, it has been suggested that surgical manipulation of the eye, such as occurs during enucleation, can provoke uveal melanoma dissemination. The purpose of this study was to evaluate whether tyrosinase mRNA is detectable in the peripheral blood of patients with uveal and cutaneous melanoma and in patients with uveal melanoma undergoing surgical procedures on the eye harbouring the tumour. Venous blood samples from 36 patients diagnosed as having active uveal melanoma and from six patients with advanced metastatic cutaneous melanoma were analysed. In addition, blood samples were spiked with known numbers of cells from three cell lines and four primary uveal melanoma cultures. The reported sensitivity of the technique was confirmed, with an ability to detect down to one cell per ml of blood. All 51 blood samples from the 36 patients with uveal melanoma were negative, and this included 20 perioperative blood samples. The test was also negative for the six patients with advanced cutaneous melanoma. There were two positives among 31 control samples analysed. This study demonstrates that there are far fewer circulating viable melanocytes than has been previously supposed in patients with melanoma and that the RT-PCR is of no clinical value in detecting metastatic melanoma disease. There was no evidence for surgery causing a bolus of melanoma cells to enter the peripheral circulation.

c-myc, p53, and Bcl-2 expression and clinical outcome in uveal melanoma.

AIMS Overexpression ofc-myc protein has independent prognostic significance in a variety of primary and metastatic cutaneous melanomas which suggests a possible role for this gene in melanomagenesis. We have therefore examined the importance of this oncogene in uveal melanoma and studied the coexpression of two other gene products,Bcl-2 and p53, which might contribute to its effect. METHODS The percentage of cells positive for nuclear c-mycexpression was estimated by flow cytometric analysis of nuclei extracted from paraffin blocks. The expression ofBcl-2 and p53protein was assessed by immunohistochemistry. A total of 71 tumours were studied and the results compared with survival with a mean follow up period of 6 years. RESULTS c-mycwas expressed in >50% of the cells by 70% of the tumours, and was independently associated with improved survival in a Cox multiple regression model. Although Bcl-2 was expressed by the majority of the cells in 67% tumours, it was without effect on prognosis. None of the cases studied showed convincing positivity for p53. Analysis of coexpression showed that the best survival was seen inc-myc+/Bcl-2+ tumours and the worst inc-myc−/Bcl-2−tumours. CONCLUSION The finding of improved rather than reduced survival inc-myc positive tumours is at variance with skin melanoma. There was no evidence to suggest thatc-myc was modulated by upregulation ofBcl-2 or p53inactivation/mutation. Although Bcl-2 is unlikely to have any effect on tumour growth or metastasis, it could contribute to the general lack of susceptibility to apoptosis in these tumours.

Absence of BRAF gene mutations in uveal melanomas in contrast to cutaneous melanomas

The recent discovery of activating mutations in the BRAF gene in many cutaneous melanomas led us to screen the genomic sequence of BRAF exons 11 and 15 in a series of 48 intraocular (uveal) melanomas, together with control samples from three cutaneous melanomas and the SK-Mel-28 cell line, which has a BRAF mutation. The same mutation was detected in two-thirds of our cutaneous melanoma samples, but was not present in any uveal melanomas. This finding further underlines the distinction between uveal and cutaneous melanomas, and suggests that BRAF inhibitors are unlikely to benefit patients with uveal melanoma.

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.

Comparative Genomic Hybridization of 49 Primary Retinoblastoma Tumors Identifies Chromosomal Regions Associated With Histopathology, Progression, and Patient Outcome

Forty‐nine primary retinoblastoma (Rb) tumors were analyzed by the use of comparative genomic hybridization (CGH), and clinical/histological correlations were performed. Adverse histological factors were present in 13 patients. Chromosomal imbalance was a frequent phenomenon, seen in 96% of the tumors. Gain of 6p represented the most frequent event (69% of the tumors), whereas +1q was observed in 57%, confirming that these abnormalities are key secondary events in retinoblastoma tumor progression. Loss of 13q and 16 was significantly associated with tumors displaying adverse histo‐prognostic factors, whereas −16q was significantly associated with tumors without adverse features. In three patients who developed an extra‐ocular relapse, the tumors showed −13q and 2/3 had −5q, suggesting that these abnormalities may be associated with metastasis. Children ≥ 36 months of age at enucleation tended to have more CGH abnormalities per tumor than children < 12 months (median numbers 11 vs. 3). In addition, +1q, +13q, −16, and −16q were more frequent in children with an older age at enucleation. Identical CGH changes were found in both tumors from one patient with bilateral tumors, suggesting a common origin. It is possible that tumors displaying loss of 13q and 5q indicate those patients who may suffer an adverse outcome and who would require alternative or more intensive therapy. CGH analysis on larger cohorts and in prospective clinical trials will be invaluable in determining whether a genetic classification of retinoblastoma represents a reliable measure of prognosis.

Delay in diagnosis of retinoblastoma: risk factors and treatment outcome

BACKGROUND Delay in diagnosis of retinoblastoma causes considerable parental distress; however, the primary healthcare professional (PHP) may have difficulty detecting the most common presenting symptom—leucocoria. Alternatively, the PHP may not appreciate that retinoblastoma is the pathology underlying more common ocular symptoms in infants and young children. METHOD The parents of 100 recently diagnosed patients with retinoblastoma were interviewed to establish the extent of diagnostic delay, ascertain any associated risk factors, and to determine whether or not delay influenced treatment outcome. RESULTS Although nearly 50% of patients were referred to an ophthalmologist within 1 week of first consulting a PHP, one quarter waited more than 8 weeks. There was a significantly increased risk of diagnostic delay in younger patients, those presenting with squint rather than leucocoria, and those first presenting to a health visitor rather than to a general practitioner. The risk of local tumour invasion was significantly increased by diagnostic delay. Treatment with primary enucleation was not increased by diagnostic delay. There were no deaths during the study period. CONCLUSION Primary healthcare professionals require education about the importance of ocular symptoms, especially squint, in paediatric patients.

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

SummaryTreatment 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.

Reassessment of the PAS patterns in uveal melanoma

BACKGROUND Previous work has highlighted the prognostic importance of patterns of periodic acid Schiff (PAS) staining (the Folberg patterns) in uveal melanoma. These patterns have been ascribed to blood vessels but the patterns are different from those seen with other staining techniques for blood vessels. It has recently been shown that microvessel density is the dominant prognostic factor in uveal melanoma. This study reinvestigates the nature and significance of the PAS patterns. METHODS The PAS patterns were compared with the patterns seen with conventional connective tissue stains and with the patterns seen in sections stained for the presence of blood vessels (by immunohistochemistry for factor VIII related antigen). The PAS patterns were determined on a panel of 117 cases of uveal melanoma. The prognostic significance of each of these patterns was determined and, as more than one pattern can exist in a tumour, principal components analysis was performed to determine the number of underlying factors. RESULTS Comparison of the PAS patterns with other stains demonstrates that they are based on connective tissue including fibrovascular tissue. Five of the nine PAS patterns carried prognostic significance on univariate analysis. Principal components analysis suggested that these patterns represented three underlying factors, which were tentatively identified as representing disordered growth (factor 1), emergence of rapidly growing subclones (factor 2), and section orientation (factor 3). CONCLUSIONS The PAS patterns are based on fibrovascular tissue and can be ascribed to three underlying factors. The first two of these factors carried prognostic significance and the first (disordered growth) retained independent prognostic significance in a multivariate Cox model which included microvessel density and tumour size.

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.