A stochastic model for dual label experiments: an analysis of the heterogeneity of S phase duration.

We develop a statistical approach for the study of S phase duration in experiments using sequential pulses of two thymidine analogues. Cell entrance into S phase is assumed to follow a possibly nonhomogeneous Poisson process, and the duration in S phase independently follows an unspecified distribution, thus allowing the possibility of variable S phase duration times for cells. Several conclusions regarding experimental design considerations are reached. The availability of three labelled cell subgroups comprising cells receiving exactly one of the two thymidine analogues or receiving both thymidine analogues at least doubles the efficiency of the mean S phase duration estimate compared to conventional estimates based on two labelled groups. Increasing the duration between the two thymidine analogue pulses can also dramatically increase the efficiency. The modelling technique was applied to fourteen uveal melanomas from patients who received in vivo injections of two thymidine analogues, bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd). Counts of labelled cells were consistent with steady-state time homogeneous entry into S phase and nonhomogeneous spatial entry into S phase.

Microwave hyperthermia for choroidal melanoma in rabbits.

Radiation has provided excellent local control rates in choroidal melanoma, but significant impairment in visual acuity has occurred in 30-60% of patients due in part to the development of radiation vasculopathy in the fovea and optic disc. Hyperthermia has been shown to have a synergistic effect when added to radiation therapy in human malignancies. The use of hyperthermia in ocular melanoma may allow a reduction in the total radiation dose necessary to achieve local control. A 2450-MHz microwave plaque applicator with integral surface cooling was used to deliver hyperthermia treatments to rabbit eyes containing choroidal melanomas. Extensive thermal mapping was done in acute eyes. In 18 survival animals, a single 23-G needle thermocouple probe with three sensors was inserted into the tumor. Target temperatures of 41.0-46.0 degrees C were maintained for 1 hour. All tumor-bearing eyes were followed for 1 month after treatment, or until tumor growth was noted, with serial ultrasound measurements and visual examinations. A 92% response rate was obtained in tumors treated at temperatures greater than 43.0 degrees C for 1 hour with no significant toxicity. Heat alone has significant tumoricidal properties in this animal model.

Alteration of human conjunctival epithelial proliferation.

We used in vitro bromodeoxyuridine to analyze the labeling indexes of conjunctival cells in several external eye diseases. Patients with superior limbic keratoconjunctivitis, squamous cell carcinoma of the conjunctiva, drug-induced pseudopemphigoid, and Stevens-Johnson syndrome had increased conjunctival DNA synthesis.

Impairment of active avoidance by the noradrenergic neurotoxin, DSP4: attenuation by post-training epinephrine.

Male Sprague-Dawley rats were treated with the selective noradrenergic neurotoxin, DSP4 prior to behavioral assessment. They were trained in an inhibitory avoidance and a one-way active avoidance task and given post-training treatment with epinephrine (EPI, 0.1 mg/kg, SC) or physiological saline. Performance on these tests was assessed at time points after treatment with DSP4 when (1) both central NE and sympathetic catecholamines were depleted and when (2) sympathetic catecholamines had returned to control levels and central NE remained depleted. Activity was also assessed at two time points after DSP4 treatment. DSP4 treatment had no effect upon inhibitory avoidance retention but impaired one-way active avoidance shuttle performance at both time points following DSP4 treatment. There was a transient depression of spontaneous activity which may indicate a deficit of behavioral initiation during the early phase after DSP4 treatment when the sympathetic catecholamine levels were depleted. This finding suggests that the peripheral sympathetic system may support some aspect of behavioral initiation. Epinephrine (0.1 mg/kg SC) administered after active avoidance training ameliorated the active avoidance retention performance deficit seen 4 days after DSP4 treatment. Post-training EPI did not significantly affect active avoidance retention performance when animals were trained and tested after peripheral sympathetic recovery (approximately 2 weeks after DSP4 treatment).(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of radiation failure after uveal melanoma brachytherapy.

We analyzed the number of cycling cells in the DNA synthesis phase and in mitosis in two uveal melanomas unsuccessfully treated with iodine 125 brachytherapy. In one patient, the tumor appeared to have been radioresistant, whereas the other patient had a clinically inapparent area of tumor that was not included in the tumoricidal radiation field. In the first patient, there was uniform tumor cell cycling, whereas the second patient had cycling cells predominantly in the area that did not receive optimal radiation. This gradient of cycling tumor cells in the second patient was observed with both standard histologic examination (mitoses per high-powered field) and bromodeoxyuridine staining (DNA synthesis). The bromodeoxyuridine technique was significantly more sensitive than standard histologic examination in the detection of cycling cells.

Ki-67 and bromodeoxyuridine labeling of human choroidal melanoma cells.

Understanding tumor growth patterns has implications for prognosis as well as for response and susceptibility to treatment. The antibody Ki-67 was used as a marker of cycling cells and bromodeoxyuridine (BrdUrd) was used as a marker of proliferating cells to characterize the cycling and proliferative rates of cells from human choroidal melanoma. The BrdUrd labeling indices varied from 0-1.1% and the Ki-67 labeling indices ranged from 0-3.0.3%. Linear regression modeling showed good correlation defined by the equation: Ki-67 index = 0.237 + 1.63 x BrdUrd labeling index with r = 0.919. Correlations between these indices and clinical and histologic parameters were not significant.

Infiltrating lymphocytes and antigen expression in uveal melanoma.

Monoclonal antibodies and flow cytometry were used to study lymphocyte, monocyte and tumor cell antigen expression in uveal melanoma. Forty-one melanomas were studied after various forms of treatment. An antimelanoma antibody, 13A3E, stained 81.7% of the cells. Tumors treated with helium ions or 125I plaques had 13A3E staining reduced to 62.5%, p = 0.011. HLA-A,B,C stained 85.4%, and HLA-DR stained 7.0% of the cells. T cells (CD3 positive) comprised 4.5% (range 0.1-29.2%) of total cell population. Natural killer (NK) cells, B cells and macrophages were present in small numbers (mean less than 2.5% in each case). Tumors with numerous T cells (greater than 5%), were used for T cell subtype studies. The mean helper/inducer (CD4) to cytotoxic/suppressor (CD8) ratio was 1.00 (range 0.11-3.15). CD8 decreased, and CD4 increased with age in unirradiated tumors (p less than 0.02).

Retinoblastoma cell cycling.

Techniques for the measurement of bromodeoxyuridine (BrdUrd) positive cells generally include either microscopic evaluation of paraffin embedded sections or measurements on cell suspensions using a fluorescent activated cell sorter. The accuracy of these measurements and their correlations can be affected by a number of technical and intrinsic tumor factors. Extrinsic parameters including degree of necrosis and tumor growth fraction are less easily analyzed in BrdUrd stained material. Retinoblastoma tumor cell cycling was prospectively studied in 11 children using in vivo and one child using in vitro BrdUrd. BrdUrd measurements were made by staining cell suspensions or sections of paraffin embedded tumor and analyzing by microscopy. Approximately 14% of viable cells were in the synthesis-phase of the cell cycle. The correlation between BrdUrd in cell suspensions and BrdUrd in paraffin embedded sections did not reach significance (r = 0.48). DNA analysis of these tumors was also performed using flow cytometry. Nine tumors were found to have a normal diploid DNA content, one had a G1 peak below the diploid control, two had a G1 peak above the diploid control, and one had two G1 peaks (a diploid and a hyperdiploid peak). There was no correlation between abnormal DNA content and the percent of cells in synthesis.

[Cytokines in uveal melanoma. Original title: uveal melanoma cytokines]. / Zytokine in Uveamelanomen. Originaltitel: Uveal Melanoma Cytokines.

Cytokines may be important in both the pathophysiology an ocular morbidity associated with treated and untreated uveal melanomas. In this study we used message amplification phenotyping to identify cytokines present in uveal melanoma samples. A number of cytokines were noted that have been associated with tumor progression, neovascularization, and inflammation in other malignancies. Interferongamma was commonly found in spindle cell tumors. Only one of 16 tumors had detectable levels of TNF-alpha and this occurred in one of two patients who developed metastatic disease.

Cytomorphometry of uveal melanoma. Comparison of fine needle aspiration biopsy samples with histologic sections.

A number of approaches are being investigated to increase the prognostic accuracy for uveal melanoma patients; the standard deviation of nucleolar area measurements and the DNA content appear to correlate better with survival than do classic histologic parameters. The utility of performing cytomorphometric measurements on fine needle aspiration (FNA) biopsy samples was prospectively analyzed for 24 eyes containing uveal melanomas that were examined with both 25-gauge FNA biopsy and standard histologic techniques. "Masked" analysis of the cellular composition of the 24 cases showed the presence or absence of epithelioid cells to be accurately predicted on the FNA samples in all cases. Image analysis cytomorphometric measurements of nucleolar area showed marked variability (with r less than 0.4) when FNA and histologic samples from the same case were compared. The relationship between these measurements was affected by cell type, sampling, specimen processing and investigator experience.