Prediction of metastasis of uveal melanoma: comparison of morphometric determination of nucleolar size and spectrophotometric determination of DNA.

We studied 95 cases of uveal melanoma using cytomorphometry to measure the standard deviation of nucleolar area (SD-NA) based on 200 cells per tumor and microspectrophotometry to determine the quantity of DNA in the nucleus of 100 cells per tumor. The 95 cases of uveal melanoma, in which the eye was enucleated between 1970 and 1973, were selected from the files of the REgistry of Ophthalmic Pathology. Forty-nine patients survived with a median follow-up of 15 years without evidence of metastasis at the time of the last follow-up, and 46 patients died of metastatic melanoma. Statistical analysis indicated that SD-NA was a better predictor of metastasis than DNA determination in this data set.

Uveal melanoma: mean of the longest nucleoli measured on silver-stained sections.

PURPOSE: To summarize and compare the various histologic methodologies for using nucleoli to assess the malignant potential of uveal melanoma. METHODS: This was an observational series of 100 samples of uveal melanoma in which histologic sections were studied. The cases were selected so that approximately half (n = 49) of the tumors were from patients who had died of metastatic malignant melanoma. The 51 remaining tumors were from patients who had survived at least 9 years without development of metastasis. Central sections from the uveal melanomas were stained using the colloidal silver nitrate stain for nucleolar organizing regions (AgNOR). These were compared with an adjacent hematoxylin and eosin (H&E)-stained section. A light microscope with a micrometer inset into the eyepiece (x10) was used at a final magnification of x1000 under oil immersion to measure the length of the nucleolus along the longest axis and the width perpendicular to that axis. From at least twenty cells selected from random fields throughout the tumor, the mean of the 10 longest and widest nucleoli (MLN) was calculated. Seven samples had to be discarded because the nucleoli were unmeasurable. RESULTS: T-tests and Cox proportional hazard regression analysis indicated that the MLN of nucleolar length as measured on AgNOR-stained slides was as significant as cell type but was more significant than other histopathologic prognosticating variables measured and evaluated in this study. These prognosticators included tumor size, calculated as the largest tumor dimension; MLN width; and MLN length, as measured on H&E-stained sections. CONCLUSIONS: It has previously been demonstrated that AgNOR-stained nucleoli, unlike H&E-stained nucleoli, can be captured and measured by an automated image analyzer with prognostically significant results. This new method of simple oil-immersion measurements of the longest AgNOR-stained nucleoli length in microscopic sections of uveal melanoma provides an inexpensive and highly significant method for predicting outcome in patients with uveal melanoma. Because of the high contrast with the background, the silver-stained nucleoli clearly define the nucleolar boundaries, rendering them readily discernible and allowing greater ease and speed of measurement when compared with H&E-stained nucleoli. The method of random sampling that was used was comparable with linear sampling in predicting outcome. Highly necrotic tumors, however, had to be excluded from the study because of loss of nucleolar morphology.

A comparison of prognostic covariates for uveal melanoma.

For 740 selected cases of uveal melanoma from the Armed Forces Institute of Pathology, the following features were determined: the age and sex of the patient, Callender cell type (CT), and largest tumor dimension (LTD). In addition, special morphometric devices were used to measure the standard deviation of nucleolar area (SDNA) and the mean of the largest nucleoli (MLN) from a single routine hematoxylin and eosin-stained section of each tumor. Univariate analysis using the Cox proportional-hazards model revealed that LTD, CT, SDNA, and MLN correlated equally with death from metastatic melanoma (P greater than 10(-6). Age correlated less highly (P less than 0.002), and sex had no relationship to mortality. Multivariate analysis revealed that adding LTD as a prognostic covariate to either CT, SDNA, or MLN yielded a substantial increase in prognostic value. Because MLN can be measured more easily than SDNA and is more reproducible than CT, it can be a useful cytologic index of the malignant potential of uveal melanomas.

Immunohistochemistry of infiltrating lymphocytes in uveal malignant melanoma.

PURPOSE: To investigate using immunohistochemistry the unusual finding that tumor infiltrating lymphocytes (TIL) in uveal melanomas are associated with a higher mortality rate. METHODS: We performed immunohistochemistry for B and T lymphocytes on 80 uveal malignant melanomas, which previously had been reported to contain more than 100 TIL per 20 high-powered fields. In a second study of 90 patients, we counted the number of immunohistochemically stained T lymphocytes per 20 high-powered fields in uveal melanomas from 30 patients who survived at least 15 years after enucleation, from 30 patients who died with metastasis within 2 years, and from 30 patients who died with metastasis more than 10 years after enucleation. RESULTS: T cells predominated in 73.8% of the 80 patients, and B cells were more prevalent in 13.8%. T cells were usually scattered, and B cells were usually in clumps. Post-enucleation 18-yr mortality from metastasis was 73% for patients with either T- or B-cell predominance of their TIL. The mortality rate was 32% for patients with few TIL. The patients who survived at least 15 yrs after enucleation had fewer T lymphocytes infiltrating their uveal melanomas than the two groups of patients who died with metastasis. CONCLUSIONS: The pattern of the TIL was different for T and B cells in uveal melanomas. T-lymphocytic infiltration is associated with death due to metastasis.

Inferred natural history of uveal melanoma.

We have observed that the fatality rate of patients with posterior uveal melanomas rapidly increased from a very low rate prior to enucleation to a maximum of 8% per year during the second year after enucleation. These data suggest to us that events related to enucleation have a deleterious effect on survival, and we have postulated that these events are either the iatrogenic dissemination of tumor cells or an adverse effect on the immune-defense system, or both. Others have proposed that the relationship between enucleation and increased mortality is only happenstance. They would attribute the onset of symptoms to the rapid growth of the malignant tumor and thus claim that this brings the patient to enucleation soon after the tumor becomes malignant. We have studied 2105 cases on file in the REgistry of Ophthalmic Pathology and have found that enucleation was not always performed shortly after the onset of visual disturbance. In one third of the cases, enucleation was delayed until onset oment was not obtained until the tumor measured larger than 15 mm in diameter. Using Zelen's method to infer the natural history of uveal melanoma, we found that the mean time it took for a small tumor (less than 10 mm in diameter) to become a large tumor (greater than 15 mm) was approximately 7 years. The average age of patients treated for medium-sized tumors was 5 years less than that of patients treated for larger tumors. This is interpreted as evidence of an average delay of 5 years in the treatment of large melanomas. If this delay is taken into consideration, then the patients treated with medium-sized tumors had a worse survival during the first 7 years than did patients whose treatment was delayed until the tumor became large. After the seventh year, however, the survival was better. These findings support our hypothesis that the postoperative increase in mortality during the first several years, particularly among patients with tumors of medium size, was related to enucleation.

Retinoblastoma. The relationship of proliferating cells to blood vessels.

In 150 retinoblastomas the authors found a uniform thickness of the cuff of viable retinoblastoma cells that surrounds blood vessels. The mean thickness was 98.7 microns with a standard deviation of 11.9 microns. The cross-sectional area of the cuff was negatively correlated with the mitotic activity in the cuff and positively correlated with the diameter of the central vessel. The mitotic activity in the cuff of cells was inversely related to the distance from the central blood vessel. When the cuff was divided into three concentric rings, the inner ring contained a mean of 6.2 mitotic figures, the middle ring contained a mean of 2.9 mitotic figures, and the outer ring contained a mean of 0.6 mitotic figures. This pattern of growth is similar to that observed in other rapidly growing neoplasms in humans and experimental animals. In these tumors this pattern results from reduction in oxygen tension with increased distance from the central blood vessel.

Correlation of in vitro 32P counts with histologic features of malignant melanoma of choroid and ciliary body.

The radioactive phosphorus uptake of 30 samples fron nine uveal melanomas was determined by liquid scintillation counting. These in vitro measurements correlated positively with clinical (in vivo) 32P values and with the number of mitotic figures and percentage of epithelioid cells (PEC) within the specimens. Three possible explanations were found for the failure of previous workers to demonstrate a correlation between cell type and clinical 32P. (1) Cell type, using Callender's classification, provides only a crude estimate of the PEC within a tumor; in particular, tumors of mixed cell type can vary widely in epithelioid cell content. (2) The rate of 32P absorption is not su-ficiently sensitive to distinguish spindle cell tumors from mixed cell tumors with a low PEC. (3) Clinical 32P measurements may be affected by extraneous factors that cause large deviations from the expected values.

Characterization of intraocular tumors arising in transgenic mice.

PURPOSE: To characterize intraocular tumors that arise by in situ transformation in the choroid-retinal pigment epithelium (RPE) in transgenic mice bearing the SV40 oncogene under the control of the mouse tyrosinase promoter. METHODS: Tumors from TySV40 transgenic mice were characterized in vivo and in vitro by immunohistology, compound microscopy, and electron microscopy. Tumor cell lines were established and characterized for growth and metastatic potential in the eyes of nude mice. RESULTS: On light microscopy, ocular tumors were predominantly epithelioid, although occasional clusters of spindle cells were also present. Transmission electron microscopy revealed the presence of numerous basal infoldings and abundant multilaminated basement membranes on the ocular tumors. Tumors stained with antibodies to melanoma-associated antigens, gangliosides GD2 and GD3, and the SV40 T antigen. Radiolabeled transgenic tumor cells preferentially localized in the liver after intravenous injection in normal mice. Intracamerally transplanted transgenic tumors metastasized from the eyes to the livers of nude mice. CONCLUSIONS: In TySV40 transgenic mice, intraocular tumors develop that arise at the choroid-RPE interface, and they display morphologic and ultrastructural features consistent with RPE carcinomas. However, the transgenic tumors express melanoma-associated antigens and a propensity to metastasize to the liver, two features characteristic of uveal melanomas. The TySV40 transgenic murine tumors represent potentially useful tools for investigations into the biology and metastasis of intraocular neoplasms.

Primary acquired melanosis of the conjunctiva.

Forty-one cases of conjunctival primary acquired melanosis (PAM) were studied to determine the frequency of progression to malignant melanoma and to establish prognostic parameters for progression to melanoma. Two subdivisions were identified: lesions with cytologically atypical melanocytes (PAM with atypia, 28 lesions, 68.3 per cent) and those lacking cytologically atypical melanocytes (PAM without atypia, 13 lesions, 31.7 per cent). None of the lesions of PAM without atypia progressed to melanoma. Thirteen of the 28 lesions of PAM with atypia (46.4 per cent) progressed to melanoma. Progression to melanoma was more frequent in the lesions of PAM with atypia if basilar hyperplasia was not the dominant histologic pattern (90 per cent progression, P = 0.02) or if any epithelioid cells were present (75 per cent progression, P = 0.02). It was not possible to determine which lesions were atypical on the basis of clinical appearance. Lesions at risk for the development of melanoma should be totally extirpated.

Malignant melanoma of the conjunctiva.

One hundred thirty-one cases of conjunctival melanoma in which biopsies had been performed were studied to determine potential factors that might affect outcome in patients with these lesions. Two groups of lesions were identified: those associated with primary acquired melanosis (melanoma with PAM, 98 cases, 74.8 per cent) and those without primary acquired melanosis (melanoma without PAM, 33 cases, 25.2 per cent). The overall mortality rate in the 131 cases was 26 per cent (34 of 131); the mortality rate due to melanoma with PAM was 25.5 per cent (25 of 98), and that due to melanoma without PAM was 27.3 per cent (9 of 33). If PAM was associated with the lesion, the presence of atypical melanocytes within the epithelium (pagetoid invasion) was a sensitive indicator of subsequent metastasis. Tumor thickness may also be useful for predicting subsequent metastases. None of the histologic parameters studied proved useful for predicting outcome in patients who had melanomas without PAM. The presence or absence of nevi had no effect on prognosis.

Uveal melanoma: location, size, cell type, and enucleation as risk factors in metastasis.

In this series of 3,432 cases of malignant melanoma of the choroid and ciliary body, mortality from metastasis 15 years after enucleation was 46 per cent. This mortality was at least ten times greater than has been observed with tumors of the iris, probably owing to the greater size and more malignant cytology of choroidal and ciliary body tumors. In 56 per cent of the 3,432 cases, the melanomas were composed of a mixture of spindle and epithelioid cells. The 15-year mortality of patients with melanomas of mixed cell type was three times that of patients with tumors of pure spindle cell type. In 30 per cent of the cases in this series, the melanomas of the choroid and ciliary body were larger than 15 mm in diameter. Size was highly correlated with mortality. The distribution of deaths following enucleation in the 3,432 cases was a log-normal function of time from enucleation. This indicated that metastasis occurred in these fatal cases close to the time of enucleation. The authors were also able to infer that many years were usually required for these uveal melanomas to grow from small (7 to 10 mm in diameter) to large (greater than 15 mm in diameter). These observations are consistent with the hypothesis that dissemination of tumor cells at the time of enucleation has been a major cause of metastasis with small and medium-sized uveal melanomas.

Computerized histologic assessment of malignant potential: a method for determining the prognosis of uveal melanomas.

The authors evaluated 50 cases of primary melanoma of the choroid and ciliary body. In each case, 100 cells were randomly selected from a single histologic slide, and on each cell computer-assisted measurements were made of 18 nuclear and nucleolar features. Means and standard deviations were calculated for each of these features in each tumor. Thirteen calculated variables (six means and seven standard deviations) were found to correlate significantly with patient mortality following enucleation. Standard deviations of statistically significant nuclear and nucleolar features demonstrated significantly greater correlation with mortality than the means of these features, thus confirming the great value of nuclear pleomorphism for predicting the malignant potential of uveal melanomas. Furthermore, when the standard deviation of the nucleolar circumference, a feature highly correlated with survival (P less than 0.00001), was combined with the measurement of the largest dimension of the tumor, linear discriminant analysis correctly predicted the clinical course of 88 per cent of cases.

Sebaceous carcinomas of the ocular adnexa: A clinicopathologic study of 104 cases, with five-year follow-up data.

One hundred four cases of sebaceous carcinoma that arose from ocular adnexa, with at least five years' follow-up information following diagnosis, were studied to extend the authors' previous observations on various prognostic factors in these tumors. Twenty-three patients died from metastatic disease. Although sebaceous carcinomas elsewhere in the skin are rare, this study establishes that these neoplasms occur much more frequently in the ocular adnexa and have significant morphologic features that identify the more highly lethal carcinomas. The various clinicopathologic features that indicated a bad prognosis were vascular, lymphatic, and orbital invasion; involvement of both upper and lower eyelids; poor differentiation; multicentric origin; duration of symptoms greater than six months; tumor diameter exceeding 10 mm; a highly infiltrative pattern; and pagetoid invasion of the overlying epithelia of the eyelids. In many cases pagetoid change appeared to originate in the underlying sebaceous glands and from there extended to invade the overlying epithelia.

Quantitative analysis of the Callender classification of uveal melanoma cells.

Six experienced ophthalmic pathologists independently classified 90 uveal melanoma cells according to the Callender classification. They frequently disagreed among themselves, but at least four of the six agreed on the classification of 77 cells. In an effort to delineate those cytologic features that are most useful in the application of Callender classification, 12 factors were evaluated for each of these cells. Multivariate analysis (MVA) proved that six factors (chromatin clumping, minimum nuclear diameter, nucleolar diameter, chromatin margination, maximum nuclear diameter, and abundant cytoplasm) were useful for determining the cell type selected by the majority of pathologists, although some cells could not be sharply classified. It was concluded that uveal melanoma cells occupy a continuous cytologic spectrum, from benign-appearing spindle A cells to very malignant-appearing epithelioid cells.

Malignant melanoma of the choroid and ciliary body in black patients.

In a retrospective study of choroidal and ciliary body malignant melanomas in black patients, we found that the death rates due to metastatic melanoma after enucleation were equivalent for 39 blacks and 3,339 whites. When compared with a control group of whites, it was found that blacks were more likely to have secondary glaucoma and inflammation prior to enucleation. Of the histopathologic features analyzed, the presence of necrosis or mixed or epithelioid type cells within the tumor was associated with increased mortality in blacks. The histopathologic features of malignant melanomas in blacks and whites were similar, except that tumor necrosis and heavy pigmentation were found more frequently in tumors of blacks.

Phakomatous choristoma of eyelid: electron microscopical confirmation of lenticular derivation.

This article presents the sixth recorded example of phakomatous choristoma of the eyelid. The tumor occurred in a 4-month-old boy and was located in the lower lid near the inner canthus. The findings from light microscopy included epithelial, tubular structures with associated patches of thick, irregular basement membrane. The results of electron microscopy (ie, epithelial cells with a scarcity of organelles, homogeneous type of basement membrane, cytoplasmic microfilaments, segments of fascia occludens, and degenerated cells with peculiar interdigitations of their plasmalemmas) are all consistent with lenticular derivation, as originally proposed by Zimmerman.

Uveal melanoma in children and adolescents.

Five-year survival data were available in 78 cases originally classified as uveal melanomas in patients less than 20 years old. Forty-two were malignant melanomas arising from the choroid and/or ciliary body; 13 patients from this group died of metastatic disease. Factors that significantly correlated with fatality were a red, painful eye, extraocular extension at enucleation, basal tumor diameter greater than 10 mm, increased mitotic activity, and tumor necrosis. There were 36 iridic tumors; nine were reclassified as nevi. Of the 27 patients with iridic melanomas, only four died of metastases. The predominant factors relating to fatal outcome were glaucoma, extension of tumor into the ciliary body, diffuse growth, deep angle invasion, scleral invasion by tumor cells, and increased mitotic activity. Except for their relative rarity, uveal melanomas in children and adolescents did not differ significantly from their counter-parts in adults.

Is there a blood-brain barrier at the optic nerve head?

The retina and optic nerve have been demonstrated to possess a blood-brain barrier that prevents the passive passage of protein and certain dyes from the blood vessels into the extracellular space. Our observations suggest that an exception is present at the normal optic disc. Using horseradish peroxidase as a tracer for electron microscopy and the normal rhesus monkey as the experimental animal, we have demonstrated that in certain regions of the optic nerve head, horseradish peroxidase from the blood stream reaches the axons of the optic nerve through the border tissue of Elschnig from the adjacent choridal tissues. A barrier formed by a series of cell junctions between glial cells at the edge of the optic disc prevented spread of the tracer from the optic disc into the subretinal space.

Herpes simplex virus in primary graft failure.

OBJECTIVE: To determine the frequency of herpes simplex virus type 1 (HSV-1) in primary corneal graft failures. METHODS: The clinical data submitted about all cases of corneal graft failure on file at the Armed Forces Institute of Pathology, Washington, DC, from the last 25 years (1970-1995) were evaluated. Cases that met the definition of primary graft failure were examined microscopically and analyzed using polymerase chain reaction (PCR) for the DNA of HSV-1. RESULTS: Three (2.8%) of the 106 cases of graft failure were primary graft failures. The DNA from 2 of the 3 corneal buttons was amplifiable by PCR analysis and results of the PCR analysis and Southern blotting were positive for HSV-1. None of the results of the PCR analysis and Southern blotting of the corneal buttons from the 3 graft failures occurring later than 30 days were positive for HSV-1. Results of the PCR analysis and Southern blotting indicated that 2 of 3 corneal buttons in the control group of clinically suspected herpetic keratitis were positive for HSV-1. The cornea from the first case of primary graft failure showed acute inflammation with stromal necrosis. The cornea in the second case had loss of endothelium without inflammation. CONCLUSIONS: The finding of DNA from HSV-1 in corneal buttons from 2 cases of primary graft failure supports similar observations by Cleator et al and suggests that HSV-1 may be pathogenic in some cases of primary graft failure. A larger study is needed to determine if HSV-1 is a causative factor in primary graft failure.

Silica granuloma of eyelid and ocular adnexa.

Seven cases of subcutaneous epithelioid granulomas caused by silica and involving the eyelids and periorbital region are described. Histologically, all cases disclosed epithelioid tubercles and varying numbers of foreign-body-type giant cells, areas of scarring, and birefringent crystals. Light microscopy with polarized readily facilitated visualization of the crystals. Scanning electron microscopy and energy dispersive x-ray analysis clearly established that the crystals were composed of silica in both its pure and combined forms. This study emphasizes the need for examination of all epithelioid granulomas with polarized light. If crystalline foreign bodies are identified, further special techniques should be used for chemical characterization of the crystals.