Cancer in relatives of retinoblastoma patients.

Medical histories with emphasis on malignant disease were obtained on 1,269 first and second degree relatives of 93 probands with retinoblastoma and on 671 first and second degree relatives of 50 age-matched control children. The number of nonocular malignancies expected to occur in first and second degree relatives of the probands were calculated using the observed number of neoplasms in control families as the standard. Using a Poisson distribution to evaluate the findings, a statistically significant excess of cancer was found in relatives of the probands with retinoblastoma. Gastrointestinal malignancies were particularly common in this population.

Flow cytometry of uveal melanomas.

Tumor cell kinetic parameters were determined for 36 uveal melanomas retained in fixed tissue sections using flow cytometric techniques and computerized morphometry. By flow cytometry the majority of cells comprising the 36 tumors were in the G0/G1 phase (55.7%). The DNA index was 1.40 +/- 0.57 units. Using Spearman's rank order correlation test, the correlation between DNA index and the inverse standard deviation of nucleolar area was found to be statistically significant. The potential usefulness of flow cytometry in predicting tumors of high malignant potential is discussed.

Lymphocyte subpopulations before therapy in patients with uveal malignant melanoma.

T-cell and B-cell lymphocyte subpopulations, monocytes, granulocytes, and immunoglobulin receptors were measured with monoclonal antibodies and flow cytometric techniques in the peripheral blood of 266 patients with posterior uveal melanoma before therapy. Statistically significant differences were found in T-helper/inducer (OKT4), T-suppressor/cytotoxic (OKT8), and B-lymphocyte populations between patients with uveal melanoma and age-matched controls.

Metastatic uveal melanoma: an ocular melanoma associated antigen in the serum of patients with metastatic disease.

Two monoclonal antibodies, MAb8-1H and ME491, which bind to different determinants of the same highly glycosylated melanoma-associated antigen, were used to determine melanoma-associated antigen levels in serum samples from patients treated for primary choroidal or ciliary body melanoma and who subsequently developed systemic metastasis. An immunoassay was developed in which ME491 was absorbed to polystyrene beads in order to bind the melanoma-associated antigen present in serum. 125I-MAb8-1H was used to detect the bound antigen. This double-determinant immunoassay is both sensitive and reproducible. Supernatant fluids of tissue cultured melanoma cell lines served as positive standards for the calculation of melanoma-associated antigen units. The mean serum levels of melanoma-associated antigen were 7.7 units for patients with benign ocular conditions, 9.51 units for patients with choroidal melanoma without documented metastatic disease, and 48.3 units for patients with choroidal melanoma and documented systemic metastasis. The clinical implications of using this test as a screening method for the detection of metastatic disease is discussed.

Antigenic modulation in retinoblastoma: a flow cytometric study.

Flow cytometry (FCM) was used to investigate antigenic expression and modulation during the cell cycle of Y-79 and WERI-Rb1 tissue cultured retinoblastoma cell lines using a polyclonal anti-Y-79 antibody and fluorescein conjugated lectins. Several Y-79 resting cell populations were identified by FCM analysis of antibody binding, while only a single population with uniform antigen expression was found to exist in the synthetic and mitotic phases. WERI-Rb1 cells bound antibody approximately equally in each phase of the cell cycle. Multiple cell populations with different lectin binding affinities were seen in the resting phase with FITC-concanavalin A, FITC-ricinus communis-60 and FITC-ricinus communis-120 (FITC-RCA-120). During the S-phase of the cell cycle, a higher percentage of cells bound FITC-RCA-120 and FITC wheat germ agglutinin. The relationship between antigenic expression during the cell cycle and treatment considerations in retinoblastoma is discussed.

Lymphocyte subpopulations in Graves' ophthalmopathy.

We assessed the percentages of T-lymphocyte subsets, B lymphocytes, monocytes, and granulocytes in the mononuclear cell preparations of euthyroid patients with minimal and severe Graves' ophthalmopathy. Patients with active Werner class 4-5 and class 6 ophthalmopathy had statistically significant elevations of suppressor/cytotoxic T8+ lymphocytes. During successful corticosteroid therapy, the number of T8+ lymphocytes returned to the normal range. Mirroring these results, the T4/T8 ratio was initially depressed in patients with class 4-5 ophthalmopathy and increased during therapy. Our previous observations noted a decreased number of rosette-forming T lymphocytes in patients with severe ophthalmopathy. In this study, however, there were normal percentages of T3+ and T11+ lymphocytes, suggesting that the T lymphocytes are present in peripheral blood but they cannot form rosettes. There was a slight reduction of the percentage of the T11+ (erythrocyte receptor) lymphocytes in the patients with class 4-5 ophthalmopathy; however, it was not statistically significant. No significant differences were evident in any of the other T-lymphocyte subsets, B lymphocytes, monocytes, or granulocytes studied. We conclude that Graves' ophthalmopathy is characterized by a surface membrane defect associated with increased percentages of suppressor/cytotoxic T8+ lymphocytes. Successful corticosteroid therapy reverses these findings.

Retinal S-antigen and retinoblastoma: a monoclonal antibody and flow cytometric study.

Retinal S-antigen was demonstrated in the WERI-Rb1 and to a lesser extent the Y-79 tissue cultured retinoblastoma cell lines as well as an ethanol-fixed, paraffin-embedded retinoblastoma by an indirect immunoperoxidase technique using monoclonal antibody MAbA9-C6, and by flow cytometric analysis (FCM) using MAbA9-C6 and MAbA1-G5. In fixed tissue sections, S-antigen immunoreactivity was restricted and localized to small numbers of retinoblastoma cells, including Flexner-Wintersteiner rosettes. By FCM, MAbA9-C6 bound to 15.4% of WERI-Rb1 cells and to 10.99.7% of Y-79 cells whereas MAbA1-G5 bound to 14.66% of WERI-Rb1 and to 4.23% of Y-79 cells respectively. Cell cycle analysis showed that S-antigen was predominately expressed in the resting (G0/G1) phase. The usefulness of MAbA9-C6 in studying the embryological development of the retina and as a marker protein for studying antigenic expression and modulation in retinoblastomas is discussed.

Flow cytometry of leukocytes after intravenous fluorescein angiography.

Blood specimens were obtained for T lymphocyte analysis from patients undergoing intravenous fluorescein angiography (IVFA). Mononuclear cells were prepared by differential density centrifugation and analyzed by flow cytometry (FCM). Prior to IVFA, there was very little autofluorescence in the mononuclear cell population. After IVFA, there was an increase in the fluorescence of the mononuclear cells. Though barely visible by fluorescence microscopy, the fluorescence was uniformly distributed throughout the cytoplasm. By FCM, fluorescein was detected in 27% of the lymphocytes, 61% of the monocytes, and 75% of the granulocytes. Care must be taken when interpreting flow-cytometric lymphocyte subset data because of the increased nonspecific fluorescence of mononuclear cells in patients who have undergone IVFA.

Thymus-derived lymphocyte enumeration in patients with uveal malignant melanoma.

Thymus-derived lymphocytes (T lymphocytes) were enumerated in patients with uveal malignant melanoma. Two T-lymphocyte subpopulations were determined, the active rosette forming cells (A-RFC) and the total rosette forming cells (T-RFC). Subjects were divided into the following groups: (a) pretreatment patients, (b) patients treated by enucleation, (c) patients treated by photocoagulation, (d) patients treated by cobalt plaque radiotherapy, (e) patients treated by enucleation who developed clinically detectable metastasis, and (f) normal controls. There were no differences in the numbers of A-RFC or T-RFC in the control population, pretreatment patients, and those treated in the different ways. Statistically significant depressions of A-RFC and T-RFC levels were seen in patients with metastatic lesions, suggesting that they had an impairment of immunocompetence, as measured by T-lymphocyte rosette formation.

Schirmer's test. A closer look.

In 50 normal subjects, results of kinetics studies of Schirmer's test demonstrated nonlinear wetting with an initial rapid phase of wetting followed by a progressive reduction in rate. This initial rapid phase of wetting indicated a reflex secretion of tears. In the majority of subjects, topical anesthesia with 0.5% proparacaine hydrochloride dampened the initial reflex secretion but could not completely suppress it. The data indicated that Schirmer's test with anesthesia was not capable of measuring a basic tear secretion independent of reflex components.

Gamma-glutamyl transpeptidase in the prognosis of patients with uveal malignant melanoma.

A study of 116 patients with histologically proven uveal malignant melanoma suggested that hepatic metastases were present at the same time that the ocular tumor was diagnosed in 15 patients (13%) by serum gamma-glutamyl transpeptidase assay. The average pretherapy gamma-glutamyl transpeptidase levels were statistically higher (P less than .01) in the 28 patients who developed metastases compared with those in 88 patients who have remained clinically free of metastases for a follow-up period of at least two years. The predictive value test indicated that pretherapy gamma-glutamyl transpeptidase levels greater than or equal to 22 IU/ml were increased in patients developing metastasis. With this upper-normal limit, 15 (54%) of the 28 patients who later developed clinical evidence of metastasis had increased pretherapy levels. In comparison, only 12 (14%) of the 88 patients who remained tumor-free for at least two years had increased pretherapy levels. Although not all patients who developed metastases had increased pretherapy levels, most developed increases at some time during the course of their disease. Because the gamma-glutamyl transpeptidase assay can detect hepatic metastases before other liver function assays, it should be included as a routine liver function study for patients with uveal malignant melanoma.

Presence of circulating immune complexes in patients with peripheral corneal disease.

Seventeen patients with peripheral corneal thinning and ulcers (four with Mooren's corneal ulcer, five with Terrien's marginal degeneration, and eight with collagen vascular disease) were tested for immune complexes in their serum. Circulating immune complexes, measured by Raji cell and C1q binding assays, were compared with levels in serum samples from normal controls and seven patients with staphylococcal marginal corneal ulcers. Comparison with normal controls showed significantly higher levels of circulating immune complexes in patients with collagen disease by the C1q binding assay and in patients with Mooren's ulcer by the Raji cell assay. Circulating immune complexes may play a role in the pathogenesis of Mooren's ulcer and marginal ulceration in the presence of collagen vascular disease. Their presence, however, may represent an epiphenomenon nonspecifically associated with corneal ulceration.

Association of HLA antigen BW35 with severe Graves' ophthalmopathy.

Human leukocyte antigens (HLA) in patients with Graves' disease were investigated according to the ophthalmic classification system of the American Thyroid Association. All prior HLA studies of Graves' disease have disregarded the patients' specific ophthalmic manifestations. Examination of 18 A and 34 B loci antigens disclosed an increased frequency (corrected P = 0.002, relative risk = 13.1) of HLA-BW35 in patients improving with oral corticosteroids who have severe extraocular muscle and orbital inflammation (class 4-5) when compared to a geographically and racially matched control population. No statistically significant associations were found when patients without ophthalmic manifestations or with other categories of Graves' ophthalmopathy were compared to controls. The association of severe Graves' ophthalmopathy with HLA-BW35 may provide immunologic evidence to explain both the unpredictable association of the orbital and thyroid disturbances of Graves' disease as well as the unpredictable response of the orbital inflammation to oral corticosteroids.

In vitro characteristics of uveal malignant melanomas.

During a 17-month period, 33 histologically proved posterior uveal melanomas were prepared for in vitro cultivation in our laboratory. Nine of these remained in culture from five to 12 passages. Four tumors histologically classified as heavily pigmented spindle B cell type remained as pigmented spindle cell tumors in primary culture. During subcultivation one culture lost pigmentation and exhibited mixed cells. Three other tumors, classified histologically as lightly pigmented spindle B cell type, appeared as amelanotic mixed cells in primary culture and grew as epithelioid cells during subcultivation. Two tumors classified as mixed cell type grew as spindle cells in primary culture. One of these last cultures reverted to mixed cell type on subcultivation. One cell culture has shown pigmentary and morphologic changes with variations in carbon dioxide level and media composition.

Antibody to Toxocara canis in the aqueous humor.

The aqueous humor and serum samples of patients with presumed ocular toxocariasis were examined by an enzyme-linked immunosorbent assay (ELISA) for antibody to the embryonated egg antigen. Using a single dilution (1:8), higher amounts of antibody have been found in the aqueous humor than in the serum samples. Antibody was not found in the serum or aqueous humor of patients with retinoblastoma, Coats' disease, uveal malignant melanoma, or central retinal artery obstruction. While two patients with presumed ocular toxocariasis had normal serum levels of antibody, the aqueous humor ELISA titers were greatly elevated, supporting the clinical diagnosis and suggesting localized antibody production.

The clinical immunology of Graves' ophthalmopathy.

Graves' ophthalmopathy is frequently a diagnostic and therapeutic dilemma for practicing ophthalmologists. We have reviewed the clinical immunology of this disorder with regard to clinical manifestations, humoral immunity, and cellular immunity. Special emphasis with applicable clinical and laboratory data is given to the hypothesis that Graves' thyrotoxicosis and ophthalmopathy may be frequently associated diseases rather than disorders caused by a common pathogenetic mechanism. Dysthyroidism may be the substrate for the development of the ophthalmopathy, but the two disorders probably are caused by different immunopathogenetic mechanisms.

Familial pars planitis.

Pars planitis has rarely been noted to occur in more than one member of a family. We report the occurrence of this disorder in more than one member of two unrelated families. The affected members of one of these families included a 37-year-old woman and three of her six children. The affected members of the other family were two teenaged brothers. No specific cause for the familial clustering of pars planitis has been identified in the affected members of these families.

Graves' ophthalmopathy--immunologic parameters related to corticosteroid therapy.

Patients with severe Graves' ophthalmopathy may or may not improve with systemic corticosteroids. In an attempt to find immunologic distinctions to correspond with this clinical phenomenon, we have evaluated various parameters in corticosteroid-responsive and corticosteroid-resistant individuals. Percentages and absolute numbers of thymus-derived active and total rosette-forming cells (A-RFC and T-RFC) underwent statistically significant (p < 0.001) increases during successful prednisone therapy in 17 patients . B lymphocytes and complement receptor mononuclear cells did not significantly change with steroid therapy. In the second group, five patients therapeutically resistant to corticosteroids presented with elevated A-RFC and normal T-RFC. When treated with oral prednisone, these patients' A-RFC decreased (p < 0.001), and the T-RFC were unchanged. Corticosteroids increased the lymphoblast transformation response to phytohemagglutinin (PHA) of a steroid-responsive patient, whereas steroids decreased the PHA lymphoblast transformation response of a corticosteroid-resistant patient. A disease activity index to correlate the clinical and immunologic data has been devised. The findings may allow the clinician to predict which patients with Graves' ophthalmopathy are likely to benefit corticosteroid therapy and which patients may be managed better by other methods.

Intracranial malignancy in patients with bilateral retinoblastoma.

Tumors from two patients with bilateral retinoblastoma and intracranial malignancies were studied from a clinical, histopathological, and an immunohistochemical standpoint. An antiserum derived against tissue cultured Y-79 retinoblastoma cells was utilized in order to examine the surface and cytoplasmic antigens of the two brain tumors, histopathologically diagnosed as an embryonal cell carcinoma (case 1), a pinealoblastoma (case 2), and one ocular retinoblastoma. The two brain tumors reacted with the antiserum to different degrees. In comparison, the ocular tumor reacted strongly. None of the tissues examined were found to contain retinal S- or P-antigen. The concept of retinoblastomas arising de novofrom the pineal gland and the association of intracranial malignancy occurring in retinoblastoma is stressed. The antigenic and immunologic relationships between normal retina and pineal gland and retinoblastoma, neuroblastoma, and pinealoblastomas are discussed.

Immunodiagnosis of late, recurrent retinoblastoma.

The clinical, pathological, and immunohistochemical features of a patient with bilateral retinoblastoma who developed a nasopharyngeal mass seven years following irradiation treatment of the left eye is described. Diagnosis of the new tumor was difficult by routine light microscopy. Immunohistochemical staining of the original globes, the nasopharyngeal mass, and a cervical lymph node biopsy specimen with an antiserum prepared against tissue cultured retinoblastoma cells suggested the diagnosis of metastatic retinoblastoma rather than a "radiation-induced" sarcoma.