Iris ring melanoma: fine needle biopsy.

AIMS: To delineate the diagnostic accuracy of fine needle biopsy in iris ring melanoma and determine the tumour related mortality of this neoplasm. METHODS: A retrospective analysis of 22 patients with iris melanomas that involve the entire 360 degrees of the anterior chamber angle. RESULTS: Iris ring melanomas were correctly diagnosed in all cases. In 11 of 16 cases (69%) a fine needle biopsy performed 180 degrees away from the main mass was positive for an iris ring melanoma. The tumour related mortality in iris ring melanoma cases was four of 22 patients (18%). Actuarial survival analysis showed a 10 year mortality (Kaplan-Meier) of 15%. CONCLUSION: A fine needle aspiration biopsy can be used to diagnose an iris ring melanoma. Iris ring melanomas have significant mortality compared with focal tumours.

Inhibition of leukocyte migration by tumor-associated antigens in soluble extracts of human malignant melanoma.

Direct leukocyte migration inhibition (LMI) assays were performed to investigate whether cell-mediated immune reactions could be detected in response to tumor-associated antigens of human melanoma. The antigens were 3 M KCl-soluble extracts of different fresh melanomas, other cancers, and benign nevus tissue. A total of 48 of the 79 (61%) blood samples from melanoma patients (64 patients) reacted with extracts of melanoma tissue. Since the subjects were usually tested with two or three extracts, 57/134 (42%) tests with melanoma patients' leukocytes were inhibited by KCl extracts of melanoma tissue, whereas only 3/50 (6%) tests with leukocytes of normal donors and 4/27 (15%) with patients having other cancers gave positive results. No positive reactions were obtained when 13 melanoma patients were tested with a 3 M KCl extract of benign nevus tissue. Likewise, only 2/26 (8%) positive tests were obtained from melanoma patients tested with extracts of other cancers. Individuals in all stages of disease had similar incidences of positive reactions to the soluble melanoma extracts, except for patients with stage-1 disease who exhibited a somewhat higher incidence of reactivity. The highest incidence of reactivity was observed in patients before surgical resection of the tumor, and somewhat decreased reactivity was seen 0-14 days post surgery. The results indicate that the direct LMI assay may be used to measure cell immune reactivity against melanoma-associated antigens. Since many of the positive results were obtained with allogeneic extracts, the results also indicate that different melanomas possess common antigens.

Toxicity of a monoclonal F(ab')2:ricin A conjugate for retinoblastoma in vitro.

The toxic A chain of the poison ricin has been conjugated to the F(ab')2 fragment of a retinoblastoma-specific murine monoclonal antibody. The conjugate was toxic in vitro to Y-79 retinoblastoma cells at a concentration of approximately 5 X 10(-10) M; Y-79 retinoblastoma cells were the immunizing cells in the production of the monoclonal antibody. Against a second retinoblastoma cell line, the conjugate was toxic at a concentration of approximately 10(-9) M. Ricin A chain alone was not toxic to either of these cell lines at concentrations less than 4 X 10(-8) M. The conjugate was no more toxic than was ricin A chain against five control cell lines in vitro. The effect of the conjugate on retinoblastoma-derived cells was concentration dependent. Concentrations less than a "minimum" concentration were not cytotoxic, despite an excess of conjugate molecules over target cells. Concentrations greater than the minimum toxic concentration rapidly reached maximal toxicity, suggesting that antigenic sites were saturated. No clinical, hematologic, or renal toxicity was noted when mice were given injections of the conjugate at 1 mg/kg, or free ricin A chain at 0.375 mg/kg.

Uveal melanoma, hormonal and reproductive factors in women.

In a case-control study, we explored a potential association between uveal melanoma and reproductive factors in women. Responses from telephone interviews of 186 women diagnosed with uveal melanoma were compared with responses of 423 women without this disease. All women resided in 11 U.S. western states. We observed a decreased risk of uveal melanoma for women who had ever been pregnant [relative risk (RR) = 0.60, 95% confidence interval (CI) = 0.37 -0.95], with an increase in this protective effect with more live births after adjustment for age, menopausal status, eye color, and skin sensitivity to the sun (1-2 births, RR = 0.47,95% CI 0.29-0.78; 3-4 births, RR = 0.38, 95% CI = 0.22-0.64; 5 or more births, RR = 0.33, 95% CI = 0.15-0.71). The largest effect was observed between nulliparous and parous women. No other reproductive factors, including use of oral contraceptives or postmenopausal estrogens, were shown to be related to risk for uveal melanoma. We conclude that most reproductive factors in this population play little or no role in the etiology of uveal melanoma. The association with number of live births must be confirmed in other studies to assure that it is unrelated to confounding factors not measured in this study.

Uveal melanoma in relation to ultraviolet light exposure and host factors.

We conducted a case-control interview study among 1277 subjects (407 patients, 870 controls selected by using random digit dial) in 11 western United States to determine whether uveal melanoma and cutaneous melanoma shared common risk factors. After adjustment for other factors, the risk of uveal melanoma was increased for those with green, gray, or hazel eyes [relative risk (RR) = 2.5, P less than 0.001] or blue eyes (RR = 2.2, P less than 0.001) when compared to brown. A tendency to sunburn after 0.5 h midday summer sun exposure increased risk for uveal melanoma (burn with tanning RR = 1.5, P = 0.02; burn with little tanning RR = 1.8, P less than 0.001; burn with no tanning RR = 1.7, P = 0.002); as did exposure to UV or black lights (RR = 3.7, P = 0.003); and welding burn, sunburn of the eye, or snow blindness (RR = 7.2, P less than 0.001). An association with uveal melanoma was also noted with an increasing number of large nevi (P = 0.04 for trend), although the individual risk estimates were not remarkable. These data suggest that host factors and exposure to UV light are risk factors for uveal melanoma.

Comparative genomic hybridization in the detection of DNA copy number abnormalities in uveal melanoma.

Genomic instability appears to play an important role in the development, growth, invasiveness, and eventual metastasis of the neoplastic cell. We have used a powerful new technique, comparative genomic hybridization, to evaluate genetic alterations in 10 fresh frozen uveal melanomas. Comparative genomic hybridization utilizes dual fluorescence in situ hybridization to characterize chromosome deletions and duplications, allowing for simultaneous evaluation of the entire human genome. Several consistent chromosomal abnormalities were detected. This study confirmed previous findings obtained using standard cytogenetic techniques but demonstrated an increased incidence in abnormalities of chromosomes 3 and 8; there was loss of chromosome 3 and duplication of 8q. In addition, we identified, although less frequently, other recurrent abnormal regions including alterations on chromosomes 6p, 7q, 9p, and 13q.

Cell cycling and prognosis in uveal melanoma.

Uveal melanoma cell cycling, quantified by bromodeoxyuridine (BrdUrd)-labeling index and mitotic index, is predictive of tumor-related mortality. Serial sections from 36 formalin-fixed melanoma specimens were labeled with BrdUrd and stained with hematoxylin and eosin. All tumors were assessed for the area of highest cell cycling activity and counts for mitotic figures and BrdUrd labeling were performed in these areas in a masked manner. The BrdUrd labeling index and mitotic index were calculated and analyzed in relation to tumor-related mortality and histopathological criteria (largest tumor diameter, cell type, extra-scleral extension, ocular location). Cox multivariate analysis estimated an increased relative risk of tumor-related mortality of 2. 32 (95% confidence interval, 1.22-4.41) per doubling of BrdUrd labeling index and 2.41 (95% confidence interval, 1.29-4.49) per doubling mitotic index. Larger tumors, nonspindle cell tumors, and anterior-located tumors tended to have higher cycling rates.

Microtechniques for flow cytometric analysis of aqueous humor lymphocytes.

Flow cytometric analysis of monoclonal antibody labelled lymphocytes is an important tool in understanding the immune response. Microtechniques were developed to analyze lymphocytes in small amounts of ocular fluids. Optimal labelling volumes and concentrations for Leu 2a, Leu 3a, Leu 4 and Leu M3 antibodies were determined. The ability to label and reproducibly analyze cell samples with as few as 1000 cells is demonstrated.

Late effects of radiation on the eye and ocular adnexa.

A clinically useful classification system is suggested that can be used in prospective trials to evaluate the effects of radiation on the visual system. We review radiation-induced pathophysiological and clinical changes of the various ocular structures as well as dose-response data and management of ocular complications. The rationale for the classification scheme chosen is also discussed.

Vision following helium ion radiotherapy of uveal melanoma: a Northern California Oncology Group study.

One hundred eighty-six uveal melanoma patients were treated with helium ion radiotherapy at Lawrence Berkeley Laboratory and followed for at least 6 months. (Follow-up times ranged from 6 to 90 months; median 26.4 months.) At last examination, 92 of 186 patients (49%) had visual acuity of 20/200 or better in the treated eye. Univariate statistical analysis revealed that post-treatment vision correlated with tumor size, distance between tumor and optic disc, distance between tumor and fovea, pretreatment visual acuity, dose delivered to the optic disc, and dose delivered to the fovea (p less than .05). Neither the maximum tumor dose nor site of tumor origin (ciliary body vs. choroid) correlated with post-treatment vision on a univariate basis. However, multivariate statistical analysis revealed that the strongest independent risk factors influencing vision outcome (p less than .05) were tumor size, pretreatment visual acuity, tumor-fovea distance, and maximum tumor dose. Neither the fovea dose nor the dose to optic disc appeared to significantly affect vision outcome when other variables were taken into account. These results suggest that post-treatment visual acuity of 20/200 or better can be achieved in one-half of uveal melanoma patients treated using helium ion irradiation. Several independent risk factors affecting vision outcome have been identified.

High intensity 125-iodine (125I) plaque treatment of uveal melanoma.

PURPOSE: Episcleral 125I plaque therapy of uveal melanoma is an important treatment modality to control tumor, salvage the globe, and potentially preserve vision. We retrospectively analyzed our experience in 239 patients to assess treatment outcome with this technique. METHODS AND MATERIALS: Between 1983 and 1990, 239 uveal melanoma patients were treated with 125I plaques at the University of California, San Francisco. High intensity 125I seeds in the range of 3-20 mCi were used to give a minimum tumor dose of 70 Gy in 4 days. Initial mean tumor size was 10.9 mm x 9.2 mm x 5.5 mm with a range in tumor diameter from 4 to 18 mm and tumor height from 1.9 to 11.1 mm. Best corrected pre-treatment visual acuity was 20/200 or better in 92% of patients. RESULTS: Local tumor control was maintained in 91.7% of patients with a mean follow-up of 35.9 months; 19 patients had local tumor progression; mean time to progression was 27.3 mo (1.8 to 60.1 mo). Actuarial local control is 82% at 5 years. Multivariate analysis demonstrates significant correlation of local failure with larger maximum tumor diameter (p = 0.0008), closer proximity to the fovea (p = 0.0001), lower radiation dose (p = 0.0437), and smaller ultrasound height (p = 0.0034). The actuarial incidence of distant metastases is 12% at 5 years with multivariate analysis showing significant correlation only with maximum tumor diameter (p = 0.0064). Visual outcome is 20/200 or better in 58% of patients. CONCLUSION: While the tumor control rates appear favorable, ocular morbidity is significant. A current randomized trial comparing 125I plaque with Helium ion therapy is in progress with specific comparison of tumor control, survival, and visual outcome.

15 years experience with helium ion radiotherapy for uveal melanoma.

PURPOSE: To review the long-term experience of helium ion therapy as a therapeutic alternative to enucleation for uveal melanoma, particularly with respect to survival, local control, and morbidity. METHODS AND MATERIALS: 347 patients with uveal melanoma were treated with helium ion RT from 1978-1992. A nonrandomized dose-searching study was undertaken, with doses progressively reduced from 80 GyE in five fractions to 48 GyE in four fractions, given in 3-15 days, mean of 7 days. RESULTS: Local control was achieved in 96% of patients, with no difference in the rate of local control being seen at 80, 70, 60, or 50 GyE in five fractions. At the lowest dose level of 48 GyE in four fractions, the local control rate fell to 87%. Fifteen of 347 patients (4%) had local regrowth in the eye requiring enucleation (12 patients), laser (1 patient) or reirradiation (2 patients). The time of appearance of local regrowth ranged from 4 months to 5 years posttreatment, with 85% occurring within 3 years. Of the 347 patients, 208 are alive as of May 1, 1997. The median follow up of all patients is 8.5 years, range 1-17 years. Kaplan-Maier (K-M) survival is 80% at 5 years, 76% at 10 years, and 72% at 15 years posttreatment. Patients with tumors not involving the ciliary body have a 15-year K-M survival of 80%. The results for patients whose tumors involved the ciliary body are poor, with a 15-year K-M survival of 43%. Seventy-five percent of patients with tumors at least 3.0 mm from the fovea and optic nerve, and initial ultrasound height less than 6.0 mm, retained vision of 20/200 or better posttreatment. Patients with tumors larger than 6 mm in thickness, or with tumors lying close to the optic nerve or fovea, have a reduced chance of retaining useful vision. The enucleation rate is 19%, 3% for local failure and 16% because of complications of the helium RT, particularly neovascular glaucoma, which occurred in 35% of patients. CONCLUSIONS: Local control and retention of the eye are excellent. Complications of therapy reduce vision and eye preservation. Twenty-four percent of patients manifested distant metastases 6 to 146 months posttreatment, mean of 43 months, median of 36 months. Late-appearing distant metastases do not appear to be caused by persistent tumor in the eye. The risk of metastases is high for patients with tumors greater than 7 mm in initial ultrasound height (37%), anterior tumors involving the ciliary body (47%), and in those with local failure (53%). Patients with tumors not involving the ciliary body and initial dimensions less than 10 mm had only an 8% chance of death from melanoma. A search for effective adjuvant therapy is needed for patients at high risk of metastases (large tumors, ciliary body involved, local regrowth in eye).

Anterior segment sparing to reduce charged particle radiotherapy complications in uveal melanoma.

PURPOSE: The purpose of this investigation is to delineate the risk factors in the development of neovascular glaucoma (NVG) after helium-ion irradiation of uveal melanoma patients and to propose treatment technique that may reduce this risk. METHODS AND MATERIALS: 347 uveal melanoma patients were treated with helium-ions using a single-port treatment technique. Using univariate and multivariate statistics, the NVG complication rate was analyzed according to the percent of anterior chamber in the radiation field, tumor size, tumor location, sex, age, dose, and other risk factors. Several University of California San Francisco-Lawrence Berkeley National Laboratory (LBNL) patients in each size category (medium, large, and extralarge) were retrospectively replanned using two ports instead of a single port. By using appropriate polar and azimuthal gaze angles or by treating patients with two ports, the maximum dose to the anterior segment of the eye can often be reduced. Although a larger volume of anterior chamber may receive a lower dose by using two ports than a single port treatment. We hypothesize that this could reduce the level of complications that result from the irradiation of the anterior chamber of the eye. Dose-volume histograms were calculated for the lens, and compared for the single and two-port techniques. RESULTS: NVG developed in 121 (35%) patients. The risk of NVG peaked between 1 and 2.5 years posttreatment. By univariate and multivariate analysis, the percent of lens in the field was strongly correlated with the development of NVG. Other contributing factors were tumor height, history of diabetes, and vitreous hemorrhage. Dose-volume histogram analysis of single-port vs. two-port techniques demonstrate that for some patients in the medium and large category tumor groups, a significant decrease in dose to the structures in the anterior segment of the eye could have been achieved with the use of two ports. CONCLUSION: The development of NVG after helium-ion irradiation is correlated to the amount of lens, anterior chamber in the treatment field, tumor height, proximity to the fovea, history of diabetes, and the development of vitreous hemorrhage. Although the influence of the higher LET deposition of helium-ions is unclear, this study suggests that by reducing the dose to the anterior segment of the eye may reduce the NVG complications. Based on this retrospective analysis of LBNL patients, we have implemented techniques to reduce the amount of the anterior segment receiving a high dose in our new series of patients treated with protons using the cyclotron at the UC Davis Crocker Nuclear Laboratory (CNL).

Precision, high dose radiotherapy: helium ion treatment of uveal melanoma.

We report on 75 patients with uveal melanoma who were treated by placing the Bragg peak of a helium ion beam over the tumor volume. The technique localizes the high dose region very tightly around the tumor volume. This allows critical structures, such as the optic disc and the macula, to be excluded from the high dose region as long as they are 3 to 4 mm away from the edge of the tumor. Careful attention to tumor localization, treatment planning, patient immobilization and treatment verification is required. With a mean follow-up of 22 months (3 to 60 months) we have had only five patients with a local recurrence, all of whom were salvaged with another treatment. Pretreatment visual acuity has generally been preserved as long as the tumor edge is at least 4 mm away from the macula and optic disc. The only serious complication to date has been an 18% incidence of neovascular glaucoma in the patients treated at our highest dose level. Clinical results and details of the technique are presented to illustrate potential clinical precision in administering high dose radiotherapy with charged particles such as helium ions or protons.

Inhibition of leukocyte migration with melanoma-associated antigens in choroidal tumors.

Patients with choroidal melanomas and simulating choroidal tumors were tested in a masked manner in a leukocyte-migration inhibition (LMI) assay with a 3M KCl antigen derived from a metastatic choroidal melanoma. Good discrimination petween patient groups was observed with this antigen. The sensitivity and specificity of this assay are discussed.

Intraocular granuloma: a Schistosoma mansoni model of ocular inflammation.

An experimental uveitis model was developed in New Zealand rabbits by an intraocular injection of Schistosoma mansoni eggs. An inflammatory response was clinically apparent after 5 days and histologically was characterized by an eosinophilic infiltrate into the vitreous and choroid. The chorioretinitis that developed resulted in the disruption of the photoreceptor layer. After 30 days, eggs were enveloped by a granulomatous host response similar to that observed in animals infected systemically with Schistosoma mansoni. Reduction (immunomodulation) of granuloma size and cellularity compared with controls was observed in paraffin sections of eyes challenged (100 eggs) 4 weeks after a priming injection (500 eggs) in the contralateral eye. The granulomatous response was not evident when heat-killed eggs were injected intraocularly. Extracts made from viable eggs also induced an intense vitreous infiltrate 12 hr after injection. Serum collected from rabbits injected with 500 or more eggs showed antibody (7s) reactivity for 125I-labeled bovine S antigen, as demonstrated by immunoprecipitation with Staphylococcus aureus (Pansorbin). This model is useful for analyzing immunologic parameters involved in ocular granulomatous and parasitic diseases, humoral and cellular responses mediating autosensitization to retinal or other ocular antigens, and possible for screening chemotherapeutic agents for immunomodulation of potentially injurious host inflammatory responses.

Thymus-derived lymphocytes in the Vogt-Koyanagi-Harada syndrome.

Previous investigations have suggested that immunologic factors may play a role in the Vogt-Koyanagi-Harada syndrome, a disease involving the eye, central nervous system, and dermis. Among patients with this disease we have observed a decrease in their peripheral blood lymphocytes, as measured by two types of rosette assays. These results strengthen the concept that an altered immunologic status may be important in the pathophysiology of this disease.

Fc and C3b receptors on cultured retinoblastoma cells.

Fc and C3b receptors were identified on cultured retinoblastoma cells. Labeled receptor protein bound to affinity gels prepared with IgG, Aggregated IgG, and Fc but not to control gels prepared from Fab'2 or Sepharose-4B alone. Eluted Fc receptors was partially characterized by polyacrylamide gel electrophoresis. Molecular weight of the isolated receptor or its subunit was approximately 4.3 X 10(4) daltons. Cultured retinoblastoma cells were found to rosette with human indicator erythrocytes specific for C3b and Fc receptors. This study indicates that considerable reactivity between retinoblastoma patients' sera and cultured tumor cells may be mediated by these receptors as well as by reactivity toward retinoblastoma antigens.

Binding of retinoblastoma and normal sera to retinoblastoma-derived cultured cells.

We have observed increased binding of retinoblastoma patients' sera to a retinoblastoma-derived cultured cell line (Y-79). This reactivity was mediated by the serum IgG fraction and was directed toward different tumor or target cell (Y-79, Molt, Raji, and fibroblasts) cultured in media containing fetal calf serum. Normal pooled serum IgG fractions did not demonstrate any similar binding. When target cells were cultured in media containing human serum instead of fetal calf serum, a considerable reduction in retinoblastoma sera binding activity was observed. Reactivity against target retinoblastoma cells could be reduced but not entirely eliminated by quantitative absorption with nonretinoblastoma (Molt) cells grown in media with fetal calf serum. Retinoblastoma and normal sera binding to autologous fibroblasts, nonautologous fibroblasts, and cultured melanoma cells was also minimal. These findings suggest that residual binding activity in the sera tested may be directed against retinoblastoma tumor antigens. The fetal calf serum component responsible for reactivity with certain retinoblastoma sera was shown by immunoprecipitation, competitive inhibition, and gel electrophoresis to be bovine serum albumin.

Aqueous immune complexes in immunogenic uveitis.

A primary immunogenic uveitis was produced in rabbits by the intravitreal injection of bovine plasma albumin or ovalbumin. A secondary or recurrent uveitis was induced by intravenous injection of the specific soluble antigen several months after the cessation of primary inflammation. Aqueous antigen-antibody complexes were studied at times of maximal clinical response in both primary and recurrent forms during resolution of primary immunogenic uveitis, and in a nonspecific protein extravasation induced by aqueous paracentesis. Immune complexes could be demonstrated only during times of clinically evident inflammation in immunogenic uveitis. The results indicate the importance of antigen-antibody complex formation in the pathogenesis of this form of experimental ocular inflammatory disease.