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The brain, the testicles, and parts of the eye, including, inter alia, the vitreous, and the retina, are immune-privileged organs with relatively sealed blood-tissue barriers. These three organs may, rarely, develop aggressive primary B-cell lymphoma. Vitreoretinal lymphoma is commonly associated with primary central nervous system lymphoma (PCNSL) and with testicular lymphoma, which has a high incidence of relapse, one of the common sites of relapse is the brain. The association of testicular lymphoma with vitreoretinal lymphoma is extremely rare. In these three organs, in treating the lymphoma, the blood-tissue barrier must be overcome. We present a unique case of a patient with testicular lymphoma who 3 years later was diagnosed with monocular vitreoretinal lymphoma, and while being treated for the ocular lymphoma developed PCNSL. The lymphomas in the three organs were treated successfully: the testicular lymphoma by chemotherapy, rituximab, and radiation therapy, with intrathecal methotrexate (MTX) as a preventative measure; the vitreoretinal lymphoma by intravitreal injections of MTX alone; and the PCNSL by intravenous high-dose MTX, rituximab, dexamethasone, procarbazine, and intrathecal MTX. Ten months after completion of the last treatment, there were no signs of systemic, CNS or ocular recurrence.

A 3-year 10-month-old child initially developed locally recurrent cutaneous eruptions within the first 2 weeks after sustaining a jellyfish sting to her lower extremities. After 5 asymptomatic weeks, she developed unilateral orbital inflammation that did not respond to systemic antibiotics, antihistamines, or steroids. Imaging revealed a rapidly growing mass of the right lacrimal gland. Urgent anterior orbitotomy was performed and the lacrimal gland was biopsied. Histopathologic diagnosis revealed sclerosing dacryodenitis consistent with orbital inflammatory syndrome and/or an immune response to an antigen challenge.

PURPOSE High expression levels of insulin-like growth factor-1 (IGF-1) receptor were associated with metastatic uveal melanoma (UM). The purpose of this study was to examine the potential of serum IGF-1 in early detection of liver metastasis. METHODS IGF-1 serum levels were analyzed using enzyme-linked immunosorbent assay for 118 subjects in three different groups: 55 disease-free (DF) UM patients who did not develop metastasis within 10 years of diagnosis; 22 metastatic patients; and 41 healthy subjects. Matched pairs univariate analysis was performed for sera of 19 metastatic patients 12 and 6 months before the diagnosis of metastasis and on the day of diagnosis, both as time groups and normalized levels per patient. IGF-1 levels were compared among groups by analysis of variance and Student t-test. RESULTS Mean ± SD IGF-1 serum levels for the control, DF, and metastatic groups were 152.48 ± 49.76, 119.92 ± 60.66, and 96.99 ± 56.91 ng/mL, respectively (P < 0.001). Normalized changes in IGF-1 per metastatic patient from 6 months prior to the diagnosis of metastases compared to the day of diagnosis of metastases showed a decreasing trend. CONCLUSIONS IGF-1 levels in 10-years' disease-free UM patients were significantly lower than those in healthy subjects and were even lower in metastatic patients. IGF-1 levels decreased toward the diagnosis of metastases. Therefore, serum IGF-1 level may be used as a predictive biomarker for metastatic UM when measured repeatedly.

Purpose We examined the clinical differences in manifestation and prognosis of uveal melanoma (UM) between men and women. Methods We evaluated 723 UM patients (325 males) who were treated between 1988 and 2010 at a national referral center. Men and women were compared regarding differences in annual distribution, age at diagnosis, size and intraocular location of the tumor, symptoms leading to diagnosis, recurrence, development of metastases, and mortality. Statistical analysis included ANOVA, Pearson correlations, and competing risks for melanoma-related mortality. Results Significant gender differences were not found for annual distribution, diagnosis age, tumor size, or recurrence rate. Tumors were located more frequently posterior to the equator in men than in women. However, men were less likely than women to complain of symptoms before the diagnosis (77.10% vs. 84.65%). Men suffered more metastases. In the subgroup of patients who had metastases, the time until development of metastases was shorter in men (metastases 1 and 5 years after diagnosis of UM: 26% vs. 12.96% and 84% vs. 50%, respectively). The cumulative incidence for melanoma-related mortality was higher for men, with an almost two-fold excess of male melanoma-related mortality in the first 10 years after the diagnosis of UM. Conclusions Men have earlier and more frequent metastases in the first decade after the diagnosis of UM, a fact that may have significant implications in planning clinical trials to test adjuvant therapies to prevent metastasis.

Uveal melanoma (UM) is the most common primary intraocular tumor in adults and is characterized by high rates of metastatic disease. Although brachytherapy is the most common globe-sparing treatment option for small- and medium-sized tumors, the treatment is associated with severe adverse reactions and does not lead to increased survival rates as compared to enucleation. The use of irreversible electroporation (IRE) for tumor ablation has potential advantages in the treatment of tumors in complex organs such as the eye. Following previous theoretical work, herein we evaluate the use of IRE for uveal tumor ablation in human ex vivo eye model. Enucleated eyes of patients with uveal melanoma were treated with short electric pulses (50-100 μs, 1000-2000 V/cm) using a customized electrode design. Tumor bioimpedance was measured before and after treatment and was followed by histopathological evaluation. We found that IRE caused tumor ablation characterized by cell membrane disruption while sparing the non-cellular sclera. Membrane disruption and loss of cellular capacitance were also associated with significant reduction in total tumor impedance and loss of impedance frequency dependence. The effect was more pronounced near the pulsing electrodes and was dependent on time from treatment to fixation. Future studies should further evaluate the potential of IRE as an alternative method of uveal melanoma treatment. © 2013 Mandel et al.

PURPOSE. Carcinoembryonic antigen cell adhesion molecule (CEACAM)-1 is a multi-functional protein, with strong predictive value for poor prognosis when found in primary cutaneous melanoma lesions. In this study, the expression of CEACAM1 in uveal melanoma was correlated with clinicopathologic parameters. METHODS. CEACAM1 expression was immunohistochemically evaluated in 79 primary uveal melanomas and 21 liver metastases of patients who were treated at the Hadassah-Hebrew University Medical Center between the years 1986 and 2006. The findings were correlated with location, cell type, extracellular matrix patterns, tumor size, and metastatic disease. RESULTS. CEACAM1 was expressed in 45% of the primary tumors compared with 81% of the metastases (Fisher’s exact test, P 0.003). There was no significant association between CEACAM1 and location of the primary tumors. Histologically, CEACAM1 was associated with epithelioid-type tumors (69.6%), but not with spindle-type tumors (25.0%) (Cramer’s V 0.354 ;P 0.019). Also it was significantly associated with network extracellular matrix pattern (73.3%), but not with silent pattern (11.8%) (Cramer’s V 0.510 ;P 0.004). CEACAM1-positive tumors were not statistically different in size from CEACAM1-negative tumors. The higher frequency of CEACAM1 in patients who ultimately developed metastases (58.8% vs. 41.7%) was not statistically significant (likelihood ratio 2 2.069; P 0.1503). CONCLUSIONS. This report describes CEACAM1 expression in uveal melanoma. Correlation with poor prognostic factors such as epithelioid cell type and networks of extracellular matrix pattern was found, but definitive prognostic conclusions still cannot be deduced. Additional validation studies on the use of CEACAM1 expression as a prognostic marker are warranted. (Invest Ophthalmol Vis Sci. 2011;52:9368‐9372)

Iris melanomas constitute between 3% and 10% of all malignant melanomas of the uvea and, unlike posterior uveal melanoma, have a low rate of metastasis.1 The most common way of treating circumscribed iris melanoma is by partial, usually sector, iridectomy,2 with iris reconstruction where possible. When the anterior chamber angle or ciliary body is involved, iridotrabeculectomy or iridocyclectomy should be performed. Complications of iridectomy include hyphema, cataract, wound dehiscence, episcleral seeding of the tumour and vitreous loss.1 Non-resectable iris melanoma can be treated by brachytherapy using radioactive plaque3 or proton beam irradiation.4 Diffuse iris melanoma, which often involves the trabecular meshwork and causes secondary glaucoma, is usually treated by enucleation.5 We describe herein a novel technique of sector iridectomy in treating circumscribed iris melanoma. The operation is performed by pushing the tumour out of the eyeball and excising it extraocularly, without intraocular intervention, in order to prevent anterior segment complications. Six patients with growing pigmented iris lesions suspicious for iris melanoma, three of them proven histologically by punch biopsy, underwent resection using this new technique at the Hadassah-Hebrew University Medical Center between 2002 and 2007. All patients signed an informed consent for the described surgery. All …

High levels of serum VEGF have been reported in many types of cancers, especially in the metastatic stage. The aim of this study was to examine the potential of VEGF serum level as a tumor marker for metastases in uveal melanoma (UM) patients.Levels of serum VEGF were analyzed by ELISA for 23 UM patients (none of whom developed metastases within 5 years from diagnosis) at the time of diagnosis, soon after treatment, and 3 years later, and compared with serum VEGF levels of 39 metastatic patients, 58 10-year disease-free (10yDF) patients, and 23 healthy subjects. VEGF ratios were calculated per patient between diagnosis and after treatment, and between diagnosis and 3 years later. Matched pairs univariate analysis was performed for 17 metastatic patients for whom sera were available from before and after the diagnosis of metastases. Patients were followed biannually with liver ultrasonography and liver function tests for the presence of metastases.The inter-patient VEGF level range was large (e.g., the range for the metastatic patients was 46-1892 pg/ml). The mean ± SD levels for the control, 10yDF, and metastatic groups were: 329.65 ± 190.0, 407.66 ± 261.9 and 453.52 ± 270.2, respectively (p = 0.2456). The mean VEGF level ratio from after treatment to diagnosis was 1.08 (p = 0. 0024), and the ratio from 3 years after diagnosis to diagnosis was 1.53 (p = 0.0009). The mean ± SE post/premetastatic levels ratio was 1.35 ± 0.21 (p = 0.0595).Serum VEGF increased significantly after metastases developed. However, the wide inter-patient variance precludes the use of any cut-off level to determine the metastatic status of an individual patient based on a single VEGF serum level. An increase in VEGF on serial measurements may indicate the development of metastases. Further investigation is warranted to assess VEGF's value as a predictive marker for metastatic disease.

To examine the kinetics of the tumor marker levels: osteopontin (OPN), S-100β, melanoma inhibitory activity (MIA) and tissue polypeptide-specific antigen (TPS), and to evaluate their potential for predicting earlier liver metastasis in patients with uveal melanoma (UM).Forty-three UM patients who remained disease-free (DF) for at least 10 years, 32 patients with metastatic UM and 53 healthy controls were enrolled. Median and mean levels of the tumor markers OPN, S-100β, MIA and TPS at the time periods of 0-6, 6-12, 12-18, 18-24 and24 months prior to confirmation of metastasis by liver ultrasound, CT scan and biopsy, served in a box and whiskers analysis and were compared by Students t-test. Trends of changes in marker levels of DF and metastatic UM groups were calculated and compared by ANOVA.The lead-time for predicting metastasis was: 12-18 months both for OPN (p=0.005) and MIA (p=0.37), for S-100β 18-24 months first increase (p=0.5) followed by a second one 0-6 months (p=0.01) and for TPS 18-24 months (p=0.1). The gradient of the trendlines for the metastatic group was significantly steeper for MIA (p=0.02) and S-100β (p=0.018) than for the DF group and not statistically significant for OPN (p=0.168). For TPS, the trendline was negative. The overall increase in the levels of OPN and S-100β was significant, while for TPS and MIA, it was not.Significant increases in OPN and S-100β levels were demonstrated by a major lead time. Trendlines of the metastasis group were steeper than of the DF group predicting liver metastasis. The routine use of those markers in the follow up of UM patients, can enable earlier diagnosis of liver metastasis and effective therapeutic intervention, with an impact on survival.

To compare trends in liver function test (LFT) levels over consecutive visits before detection of liver metastasis (LM) from uveal melanoma (UM) with such trends in the serum tumor markers S-100β, melanoma inhibitory activity (MIA), osteopontin (OPN), and tissue polypeptide-specific antigen (TPS).Blood was drawn from 32 patients with metastatic UM and 43 disease-free (DF) patients semi-annually for levels of S-100β, MIA, OPN, and TPS. Abdominal ultrasonography (US) and LFTs were used to detect LM. Median LFT levels were calculated at 6-month intervals prior to the clinical detection of LM. Trends in LFT levels over consecutive visits in the groups were compared with trends in the tumor markers for these groups.Only LDH gave a statistically significant difference between the trends of the metastasis and DF groups (p=0.0041). When calculating the lead time, all of the elevations were non-significant except for gamma glutamyltransferase which showed a statistically significant elevation at time 0, the time of detection of metastasis. LDH showed a rise at 0-6 months before detection, but this was not significant. For the tumor markers, steeper trendlines were shown for the metastasis group for MIA and S-100β, and most of the markers showed a lead time of more than six months, although this was statistically significant only for OPN.Following the dynamics of tumor markers and LFTs may help to find metastatic disease in UM patients before the metastases are detectable by imaging, enabling earlier treatment.

Uveal melanoma is the most common primary intraocular tumor in adults. In the last two decades the Hadassah-Hebrew University Medical Center ocular oncology clinic has become a referral center for uveal melanoma patients.To describe the characteristics of uveal melanoma patients in Israel, their treatment modalities and outcomes during the years 1988-2007.Data were collected from the files of uveal melanoma patients in the departments of ophthalmology and oncology in our facility. Statistical analysis was performed using JMP statistical software.Data were available for 558 patients. The annual incidence of uveal melanoma in the last 5 years was 47.2 +/- 7.1 new cases per year (mean +/- standard error). There were 309 women (55.4%). The age at diagnosis was 60.8 +/- 16.5 years (range 5-95). Overall, 6.6%, 16.8% and 86.9% involved the iris, ciliary-body and choroid, respectively. Tumors were classified as small, medium and large (9.0%, 64.5% and 17.9%, respectively) according to the COMS grouping criteria. The most common primary treatment was brachytherapy (74%), followed by enucleation (17.9%). Local recurrence was noted in 11.1% of patients, while metastases developed in 13.3%. The 5, 10 and 15 year melanoma-related mortality rate was 11.4%, 17.0% and 23.3%, respectively. Of the overall study population 9.3% died of metastatic uveal melanoma.Uveal melanoma patients in Israel have tumors with characteristics similar to those in other countries. Brachytherapy is the predominant treatment, the local recurrence rate is low, and survival is comparable to that reported in the medical literature.

Aims: To evaluate the posthepatectomy survival of uveal melanoma patients with liver metastases. Methods: Data were collected from the files in the Departments of Ophthalmology, General Surgery and Oncology, for uveal melanoma patients who were seen in the Ocular Oncology Clinic at the Hadassah Medical Center from 1988 to 2007. The main outcome was posthepatectomy survival. Statistical analysis was performed using JMP statistical software. Results: Of the 558 patients, 74 (13%) developed metastases after a median of 35.0 months from the initial diagnosis. Thirty-five patients underwent hepatectomy. These patients had similar clinical characteristics as those who did not undergo hepatectomy. The median survival time from the detection of metastasis was 3.7-fold higher in the operated patients in comparison with the non-operated patients. Posthepatectomy survival of patients who were found in surgery to have 1–5 metastatic nodules was 3.1 times longer than those with six or more lesions. The hepatectomies of 13 patients resulted in complete resection of the hepatic metastases with clean histological margins (R0). These patients survived 1.9 times longer than those with residual disease (R1/R2). Conclusion: It is possible to extend significantly the life expectancy of uveal melanoma patients who develop isolated hepatic metastases by complete resection of the lesions.

PURPOSE. Many malignancies are known to be associated with abnormal activation of the PI3K-AKT pathway. Recently, a somatic mutation in the AKT1 gene (E17K) was identified in a small proportion of human tumors. This mutation activated AKT1 by means of abnormal membrane recruitment and stimulated downstream signaling. This study was designed to analyze AKT1 mutations in retinoblastoma and gain insights into the role PI3K-AKT pathway plays in the development of this tumor. METHODS. Twenty-four samples of retinoblastoma from children were analyzed for mutations in the AKT1, PTEN and K-RAS genes, using a chip-based matrix-assisted laser desorption-time-of-flight (NLkLDI-TOF) mass spectrometer. Mutations in the PIK3CA gene were analyzed in 16 retinoblastoma samples using direct sequencing. RESULTS. These results show that the mutation E17KlAKT1 was not detected in the 24 samples of retinoblastoma analyzed. K-RAS mutations were identified in two samples. There were no mutations in any of the other genes analyzed by a mass array system. On direct sequencing of 16 samples for the PIK3CA gene, one sample showed gain of function mutation in exon 9. In another sample, a genetic polymorphism of unknown significance (rs17849079) was detected in exon 20. CONCLUSIONS. Although the PI3K-AKT pathway is known to be dysregulated in retinoblastoma, the low frequency of oncogenic mutations in the AKT1, PIK3CA, and PTEN genes, suggests a different activating mechanism.

Ocular adnexal lymphoma is a hematopoietic tumor that arises in the conjunctiva, orbit, eyelid, lacrimal gland, or lacrimal sac. The treatment options in children have not been addressed in the literature. The authors describe a 13-year-old child with ocular adnexal lymphoma and discuss the treatment options.

Authentication of cell lines in biomedical research has been elevated to a very high priority. From a review of the literature, Lacroix1 reviewed the issue of cross-contamination of cell lines including the well known contamination of cell lines with HeLa cells,2 and the mis-identification of the ECV304 cell line as “immortalized endothelial cells” when these cells in fact originated from T24 bladder carcinoma cells.3 Lacroix 1 estimated that between 18 and 36% of cell lines have been misclassified. One survey at a large research institution suggested that fewer than 50% of researchers authenticate their cell lines.4 Nardone5 proposed recently that identification of cell lines be required of investigators before grants are awarded, and the National Institutes of Health subsequently called for researchers to authenticate cell lines as a prerequisite for grant funding.6

Aim: To determine the incidence and characteristics of maculopathy associated with blood–brain barrier disruption (BBBD) which is used in treating primary central nervous system lymphoma (PCNSL). Methods: Files of all 56 patients with PCNSL, with or without intraocular lymphoma (IOL), treated at Hadassah University Hospital during the years 1997–2007 were reviewed. Data on 46 patients for whom we had documentation of ocular examination were studied. Those who were alive at the time of the data collection were invited for further evaluation of the presence or absence of maculopathy. The patients were divided into four groups according to treatment protocol. Group 1: systemic intravenous chemotherapy; Group 2: systemic intravenous chemotherapy and intravitreal methotrexate (MTX); Group 3: systemic intra-arterial (IA) chemotherapy and BBBD; Group 4: systemic IA chemotherapy, BBBD, and intravitreal MTX. Results: Of the 23 patients of Groups 1 and 2 who were not treated by BBBD, none developed maculopathy. Of those 23 patients who were treated by BBBD, 12 of 17 (70.5%) in Group 3 and three of six (50%) in Group 4, for a total of 15 of 23 (65.2%) developed maculopathy. The maculopathy did not significantly affect visual acuity in any of them. Conclusions: BBBD may cause maculopathy in almost two-thirds of patients treated for PCNSL, without affecting significantly the visual acuity. Intravitreal injection of MTX, according to the protocol which we apply, is not associated with maculopathy.

Purpose: Vitreoretinal involvement occurs in approximately one-quarter of patients with primary CNS lymphoma (PCNSL), and in some patients with systemic lymphoma who may later develop CNS lymphoma. The traditional method for treating vitreoretinal lymphoma, which is the common type of intraocular lymphoma, is by external beam radiation, or intravenous or intrathecal chemotherapy; however, recurrences are common. We describe our ten years of experience in treating vitreoretinal involvement of PCNSL by intravitreal injections of methotrexate (MTX). Methods: Biopsy-proven vitreoretinal lymphoma has been treated by intravitreal injections of 400 μg/ml methotrexate. According to our protocol, injections are administered twice weekly for 4 weeks, once weekly for 8 weeks, and then once monthly for 9 months for a total of 25 injections. Results: Forty-four eyes of 26 patients have been treated; 7 patients had monocular involvement and 19 binocular. Twenty-three patients had B-cell lymphoma and 3 patients had T-cell lymphoma. In all patients remission of the intraocular lymphoma was achieved after 6.1 + 3.5 injections (range 2 – 16 injections). None of the patients had an intraocular recurrence. Among the side effects, the most common were conjunctival hyperemia and corneal epitheliopathy, which usually subsided when the interval between the injections increased. Conclusions: In our experience, treatment by intravitreal injections of methotrexate provides complete remission of vitreoretinal lymphoma with, so far, no recurrences and with bearable side effects, and can be considered as alternative treatment to radiotherapy and systemic or intrathecal chemotherapy.

PURPOSE. Aberrant promoter hypermethylation of CpG islands is thought to play an important role in the inactivation of tumor-suppressor genes (TSGs) in cancer. Studies of cutaneous melanoma have reported a high methylation rate for MGMT, DAPK, RAR-b2, and RASSF1A. In colon cancer, SOCS-1, IGF-2, RUNX3, NEUROG1, and CACNA1G are commonly inactivated. The concomitant methylation of at least three of these genes may represent a distinct trait, the CpG island methylator phenotype (CIMP). The purpose of the present study was to investigate the role of epigenetic inactivation of multiple genes in uveal melanoma. METHODS. Twenty samples of uveal melanoma were analyzed for the methylation status of nine candidate cancer-related genes: MGMT, DAPK, RAR-b2, RASSF1A, SOCS-1, IGF-2, RUNX3, NEUROG1, and CACNA1G, using real-time quantitative methylation-specific polymerase chain reaction after sodium bisulfite modification. RESULTS. Methylation rates of the genes commonly inactivated in cutaneous melanoma were 70% for RASSF1A, 5% for MGMT and DAPK, and 0 for RAR-b2. The rates for the CIMP-related genes were 25% for RUNX3, 5% for NEUROG1 and CACNA1G, and 0 for SOCS-1 and IGF-2. None of the samples was CIMP- positive. CONCLUSIONS. In this study uveal melanoma was negative for CIMP, with hypermethylation of RASSF1A. The negative CIMP phenotype and frequent RASSF1A methylation in uveal melanoma is in accord with its known lack of BRAF mutations. Given that mutations in genes of the RAS pathway are rarely observed in uveal melanoma, epigenetic inactivation of RASSF1A may be an alternative mechanism of tumorigenesis. The low frequency of promoter methylation of TSGs commonly inactivated in cutaneous melanoma further stratifies the different tumorigenesis pathway in cutaneous and uveal melanoma.

Epigenetic silencing of tumor suppressor genes by methylation of discrete regions of the CpG islands is a major mechanism underlying tumorigenesis. Methylation of at least three of five specific genes may represent a distinct trait, termed the CpG island methylator phenotype (CIMP). Positive CIMP is associated with BRAF mutations. The present study sought to investigate the presence of BRAF mutations in human retinoblastoma and the role of epigenetic silencing of multiple tumor suppression genes in a search for methylation phenotype. Twenty-five archival retinoblastoma samples were analyzed for BRAF mutations with polymerase chain reaction, Mutector assay, and direct sequencing. Nineteen samples were also analyzed for the promoter methylation status of eight candidate cancer-related genes using real-time quantitative methylation-specific polymerase chain reaction after sodium bisulfite modification. The CIMP status was determined. No BRAF mutations were found. The frequencies of cancer-related gene methylation were as follows: 89% for RASSF1A, 52% for NEUROG1, 5% for DAP-kinase, RUNX3 and CACNA1G, and 0 for RAR-beta2, SOCS-1 and IGF-2. The lack of BRAF mutations in retinoblastoma is in accord with the negative CIMP status and the high hypermethylation rate for RASSF1A. The high methylation status for NEUROG1 may point to an alternative pathway in the development and progression of retinoblastoma, but further studies are needed.

Aim: To obtain iris biopsy samples of sufficient quality and quantity for histopathological analysis using a novel punch biopsy technique. Methods: Two patients underwent iris tumour biopsy at an ocular oncology service. A trabeculectomy punch (Kelly Descemet’s membrane punch) with a 1.0 mm diameter head and a 0.75 mm deep bite was inserted through a clear cornea perforated by a SatinSlit 3.2 mm angled slit knife into a viscoelastic-filled anterior chamber. The Kelly punch was placed over the lesion and pressed down before the punch was made. After obtaining the sample, the Kelly punch was removed from the eye and then opened over a dry cellulose sponge. Tissue samples were placed in 4% formalin and processed routinely for standard staining with H&E, periodic acid Schiff and immunostains. Results: In both patients, by using the punch biopsy technique with the Kelly punch, we were able to obtain a 0.8×0.6 mm piece of tissue, large enough for any histological analysis. H&E staining showed spindle cell melanoma. Tissue sections, stained positive with MART-1 (melanoma antigen recognised by T cells) and negative with cytokeratin, established the diagnosis of melanoma of the iris in each of these patients. Conclusions: Iris biopsy with the punch technique yields a tissue biopsy specimen, as opposed to cytology samples obtained by fine needle aspiration biopsy. This technique is quick, simple to perform and requires non-expensive and easily available equipment. The tissue obtained is of sufficient quality and quantity to enable routine and special stainings.

Osteopontin (OPN) is overexpressed in metastatic uveal melanoma (UM). S-100beta and melanoma-inhibitory activity (MIA) serum levels are elevated in metastatic cutaneous melanoma. The ability of OPN, S-100beta and MIA serum levels to be used as non-invasive markers for detecting metastatic UM was tested.OPN, S-100beta and MIA levels were measured by ELISA assays in 18 patients with metastatic UM and in 38 patients who were disease-free (DF) for at least 10 years after treatment of the primary tumor. Paired serum samples from 8 patients before and after development of metastasis were analyzed. Forty-four healthy controls (C) were compared to the other two groups.Serum OPN, MIA, and S-100beta levels were significantly higher in patients with metastatic UM as compared to patients who were DF for at least 10 years after treatment (p = 0.0001) or with age-matched controls. Serum OPN, MIA and S-100beta levels were significantly higher (p0.005) after metastasis formation than before the clinical detection of metastasis in the 8 patients. Receiver operator characteristic analysis was performed for metastatic patients vs. DF and vs. C, and the area under the curve was calculated for each marker and for the combination of the 3 markers, which was 91%.Elevated serum OPN, MIA and S-100beta levels correlate with metastatic UM to the liver. When used in combination, these markers provide a highly sensitive and specific method to detect hepatic metastases and therefore provide for earlier therapeutic intervention that can prolong survival.

BACKGROUND AND OBJECTIVE The feasibility and reproducibility of scanning laser polarimetry performed through dilated pupils rather than through non-dilated pupils was tested. PATIENTS AND METHODS One eye each of 36 subjects (12 normal, 12 suspected glaucoma, and 12 glaucoma) was scanned using a single GDx unit with variable corneal compensator (GDx-VCC; Laser Diagnostic Technologies, Inc., San Diego, CA). Two scans prior to and two scans after dilation were performed on each study eye, resetting the cornea compensation prior to each scan. The dilated eye was viewed off-center, such that the whitish focusing patch was projected on the 9-o’clock peripheral iris. After adequate anteroposterior focus, the pupil was centered and a scan was acquired. Each of 5 GDx parameters was evaluated comparing the pre-dilation and post-dilation scans. RESULTS No statistically significant difference was found between pre-dilation and post-dilation measurements. There was a high pre-dilation to post-dilation correlation of 98%, 98%, 98%, 93%, and 95% for nerve fiber indicator, TSNIT average, TSNIT standard deviation, superior average, and inferior average, respectively. Less than 5% of the measurement variability was attributed to changes in pupil size (R2 ranging from 0.024 to 0.047). Stratifying the data by diagnostic groups yielded similar results. CONCLUSIONS Pharmacologic mydriasis was not found to influence the retinal nerve fiber layer measurements acquired using the GDx-VCC. Results were comparable to scans achieved in the same eyes prior to dilation. [Ophthalmic Surg Lasers Imaging 2006;37:212-216.] AUTHORS From the Department of Ophthalmology, Hebrew University-Hadassah Medical Center, Jerusalem, Israel. Accepted for publication October 30, 2005. Dr. Blumenthal has received research support from Laser Diagnostic Technologies, Inc., San Diego, CA. Address reprint requests to Eytan Z. Blumenthal, MD, Department of Ophthalmology, Hadassah University Hospital, P. O. Box 12000, Jerusalem 91120, Israel.

Four masked examiners studied the usefulness of the glass-rod for estimating intraocular pressure (IOP) in a living, anesthetized rabbit eye manometrically set to 135 different random values. With training, the average error in IOP estimation improved from 7.42 +/- 5.56 mmHg to 5.13 +/- 4.48 mmHg. Gentle palpation of the corneal apex with a glass-rod provides tactile and visual clues that may assist in estimating the IOP.

PURPOSE: Accurate assessment of the retinal nerve fibre layer (RNFL) is central to the diagnosis and follow-up of glaucoma. The in vivo measurement of RNFL thickness by a variety of digital imaging technologies is becoming an important measure for early detection, as well as for follow-up, of glaucomatous damage. However, when drawing clinical inference concerning the state of the RNFL, it is important to have valid reference data on RNFL thickness in both healthy and diseased eyes. In this review, we summarize the knowledge currently available about RNFL thickness in human and primate eyes. METHODS: A review of the literature on histological analysis of RNFL thickness in the context of glaucomatous damage. CONCLUSIONS: Six studies have so far analysed RNFL thickness. Despite the diverse study methodology taken, a consistent feature of all the data is that the superior and inferior quadrants of the peripapillary retina are thicker than the nasal and temporal quadrants; that the RNFL thickness rapidly diminishes with increasing distance from the disc margin; and that apparently at different locations the ratio of axons to supportive tissue varies significantly. We conclude that limited data are available to describe the normal variation in RNFL thickness in the normal human eye. Further studies may help better characterize the RNFL thickness in health and disease and to facilitate the correlation with clinical methods for nerve fibre layer assessment.

Purpose: To evaluate the presence, duration and magnitude of a learning effect in serial visual field (VF) testing, using the commercially available frequency doubling technology (FDT) instrument. Patients and Methods: 21 healthy adults with no prior VF experience underwent 6 serial VF tests, using the full-threshold C-20 program of the Zeiss-Humphrey FDT analyzer, on one randomly chosen eye. Tests were spaced at least two days apart. Results: The average mean sensitivity was 32.37 ± 2.6 dB; the average mean deviation (MD) was 1.22 ± 1.8 dB. The MD at the first examination (0.28 ± 2.1 dB) was significantly poorer than at any of the other testing sessions (p

We thank Dr Chia Seen Nee for the interest in our research.1 As Dr Chia Seen Nee have described,2 the cases which are presented in our manuscript demonstrate retinal detachment that …

Microphthalmia (mi) is a transcription factor that plays a major role in the regulation of growth and function in mast cells and melanocytes. Association of mi with other proteins is a critical step in the regulation of mi-mediated transcriptional activation. We found protein kinase C-interacting protein 1 (PKCI) specifically associated with mi in yeast two-hybrid screening. Immunoprecipitation of mi from quiescent rat basophilic leukemic cells or mouse melanocytes resulted in the specific co-immunoprecipitation of PKCI. This association was significantly reduced on engagement of the surface FcepsilonRI of mast cells or engagement of the Kit receptor on melanocytes. Hence, cell activation caused disengagement of mi from PKCI. Microphthalmia was previously shown to activate the mouse mast cell protease 6 (mMCP-6) promoter. Cotransfection of mi with PKCI in NIH 3T3 fibroblasts containing an mMCP-6 promoter-luciferase reporter demonstrated an up to 94% inhibition of mi-mediated transcriptional activation. PKCI by itself, although localized in the cytosol and nucleus of the cells, has no known physiological function and did not demonstrate transcriptional activity. Its ability to suppres mi transcriptional activity in the transient transfected fibroblast system suggests that it can function in vivo as a negative regulator of mi-induced transcriptional activation.

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy accounting for 80% of leukemias. The involvement of the central nervous system (CNS) by ALL is a major clinical problem and occurs in about 50% of the children without adequate treatment. The introduction of CNS-directed therapy consisting of intrathecal and high dose systemic chemotherapy and, occasionally, cranial irradiation, reduced relapse rate to less than 5% and has become a prerequisite for treating children with ALL. However, substantial neurotoxicity associated with this therapy is a major concern. Moreover the CNS is involved in up to a third from all relapses. To date very little is known about the pathogenesis of CNS leukemia. Our research was promoted by the previous observation that high mRNA expression of interleukin 15 (IL15) in leukemic blasts is associated with increased risk for CNS involvement (Cario et al JCO 2007;25:4813-20). As IL15 is a strong stimulant of Natural Killer (NK) cells, we hypothesized that the increased expression of IL15 may activate NK cells which, in turn, will control residual ALL cells in the peripheral blood but not in the relatively protected central nervous system. To investigate this hypothesis, we utilized two mouse models, a S49-derived T lymphoblastic leukemia syngeneic model and a novel human xenograft ALL model in immune-deficient mice. We found that constitutive expression of IL15 in mouse T lymphoblastic leukemia cells transplanted in neonatal Balb/c mice markedly slowed the development of systemic disease and caused CNS leukemia characterized by pronounced clinical CNS symptoms and subarachnoid infiltration of leukemia cells. This phenotype was accompanied by increase in activated natural killer (NK) cells (from 0.16% to 18.6% P Taken together we show here, for the first time, a crucial role for NK cells in the control of CNS leukemia. We suggest that the association between IL15 expression in ALL blasts and isolated CNS relapse might be explained by activation of NK cells leading to increased surveillance of residual leukemia in the bone marrow but not in the CNS which serve as a sanctuary site for tumor growth. Disclosures: No relevant conflicts of interest to declare.

PURPOSE RASSF1A inactivation in uveal melanoma (UM) is common and methylation-induced. We investigated the effect of RASSF1A re-expression on the UM phenotype in vivo and in vitro. METHODS The phenotypic effect of methylation-induced inactivation of RASSF1A in UM was explored using a stable RASSF1A-expressing UM-15 clone. RASSF1A expression was assessed using QRT-PCR. Proliferation was evaluated in vitro using MTT assays. Additionally, athymic NOD/SCID mice were injected subcutaneously or intraocularly with RASSF1A-expressing and -non-expressing UM-15 clones, and euthanized when tumors reached a volume of 1500 mm(3), or at 56 or 46 days, respectively. Tumor tissues, eyes, and livers were analyzed histologically. RESULTS In vitro analysis confirmed the lack of RASSF1A expression and full methylation of the RASSF1A promoter region in the UM-15 cell line, which was reversible following treatment with 5-Aza-2-deoxycytidine. Cells expressing exogenous RASSF1A showed slower proliferation than controls and regained sensitivity to cisplatin. Compared to mice injected with control cells, mice treated with UM-15 cells expressing exogenous RASSF1A did not acquire intraocular tumors, and their subcutaneous tumors were relatively delayed and small. Neither group had liver metastases. CONCLUSIONS UM cells reduced tumorigenicity in the presence of activated RASSF1A. RASSF1A apparently has an important role in the development of UM, and its reactivation might be applied in the development of new treatments.

PURPOSE: To develop a method to screen for serum biomarkers of early hepatic metastasis from uveal melanoma. METHODS: Cytokeratin 18 (TPS) was identified from gene expression profiles as protein generated by highly invasive uveal melanoma cells. Sera were collected from two groups of 15 SCID mice 2 weeks after injection of either tissue culture medium or MUM2B human metastatic uveal melanoma cells into the mouse liver. Serum TPS levels were assayed in 53 healthy human controls, 64 uveal melanoma patients who were disease free for at least 10 years, and 37 patients with metastatic uveal melanoma. RESULTS: After 2 weeks, small hepatic nodules (0.1-2.8 mm; mean, 0.80 mm) developed in 11 of 15 mice injected with MUM2B cells. Serum TPS levels in media-injected mice (84.7 U/L) were substantially lower than levels in MUM2B-injected mice (601 mug/L). TPS levels were significantly higher (P < 0.0001) in patients with metastatic uveal melanoma (139.63 +/- 22.20) than in healthy controls (54.23 +/- 0.01) or in patients free of disease (69.29 +/- 9.76). Significant differences were found between TPS levels before and after the development of hepatic metastases (P < 0.01), and serum TPS levels became elevated in four patients at least 6 months before the detection of hepatic metastases by abdominal ultrasonography. CONCLUSIONS: The direct-injection model of uveal melanoma in the mouse liver may be used to screen for potential serum biomarkers of metastatic uveal melanoma.

Purpose To quantify the magnitude of test-retest variability (TRV) for normal subjects in serial visual fields (VF) using the frequency doubling technology (FDT) instrument. Methods Twenty-one healthy adults, aged 23 to 60 years, underwent four serial FDT VF tests, using the full-threshold C-20 program of the Zeiss-Humphrey FDT analyzer, on one randomly chosen eye. The VF tests were spaced 2 to 4 days apart. All subjects performed two preliminary FDT tests in order to minimize any learning effect. Test-retest variability was calculated as the standard deviation of each location's sensitivity value across the four VF tests. Results Mean TRV (±SD) for the entire field was 2.44±1.32 dB. Mean TRV (±SD) for the superior, inferior, nasal, and temporal hemifields were 2.48±1.3, 2.40±1.4, 2.40±1.3, and 2.48±1.3 dB, respectively. Mean TRV (±SD) for the foveal location, the 4 central, and the 12 peripheral locations were 2.49±1.4, 2.16±1.2, and 2.54±1.4 dB, respectively. Conclusions TRV was found to be rather uniform across the visual field of the commercially available FDT device, with only a mild, clinically insignificant, effect of both eccentricity and age on TRV. Variability in the FDT VF, for normal subjects, was found to be more uniform than that of both standard and short wavelength automated perimetry. In addition, a strong inverse correlation was found, in normal subjects, between the mean sensitivity and TRV. (Eur J Ophthalmol 2007; 17: 203–7)

Multiple transcription factors are activated in the cytoplasm and translocated to the nucleus where they exert positive or negative control over cellular genes. Such subcellular traffic of transcription factors usually requires the presence of a positively charged nuclear localization sequence (NLS). Upstream stimulating factor 2 (USF2) is one of the few transcription factors that contain two potential domains for nuclear localization. In addition to the conventional basic NLS, USF2 contains a highly conserved USF-specific region that is involved in its nuclear translocation. In the present work, the induction of translocation of USF2 into the mast cell nucleus was observed and found to be dependent on activation of the cells either by IL-3 or IgE-Ag. It was also observed that the prevention of the translocation of USF2 to the nucleus, using a peptide derived from the specific USF-NLS region, significantly inhibited their IL-3-mediated survival. Thus, our findings show a direct connection between mast cell surface receptor-mediated USF2 nuclear translocation and cell viability.