Jacob Pe'er

Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry.

Tissue sections from aggressive human intraocular (uveal) and metastatic cutaneous melanomas generally lack evidence of significant necrosis and contain patterned networks of interconnected loops of extracellular matrix. The matrix that forms these loops or networks may be solid or hollow. Red blood cells have been detected within the hollow channel components of this patterned matrix histologically, and these vascular channel networks have been detected in human tumors angiographically. Endothelial cells were not identified within these matrix-embedded channels by light microscopy, by transmission electron microscopy, or by using an immunohistochemical panel of endothelial cell markers (Factor VIII-related antigen, Ulex, CD31, CD34, and KDR[Flk-1]). Highly invasive primary and metastatic human melanoma cells formed patterned solid and hollow matrix channels (seen in tissue sections of aggressive primary and metastatic human melanomas) in three-dimensional cultures containing Matrigel or dilute Type I collagen, without endothelial cells or fibroblasts. These tumor cell-generated patterned channels conducted dye, highlighting looping patterns visualized angiographically in human tumors. Neither normal melanocytes nor poorly invasive melanoma cells generated these patterned channels in vitro under identical culture conditions, even after the addition of conditioned medium from metastatic pattern-forming melanoma cells, soluble growth factors, or regimes of hypoxia. Highly invasive and metastatic human melanoma cells, but not poorly invasive melanoma cells, contracted and remodeled floating hydrated gels, providing a biomechanical explanation for the generation of microvessels in vitro. cDNA microarray analysis of highly invasive versus poorly invasive melanoma tumor cells confirmed a genetic reversion to a pluripotent embryonic-like genotype in the highly aggressive melanoma cells. These observations strongly suggest that aggressive melanoma cells may generate vascular channels that facilitate tumor perfusion independent of tumor angiogenesis.

The Prognostic Value of Tumor Blood Vessel Morphology in Primary Uveal Melanoma

BACKGROUND It is possible to identify at least nine vascular patterns in melanomas of the ciliary body and choroid from histologic sections. An association between the presence of at least one closed vascular loop and death from metastases was shown in a matched-pair, case-control study of 40 patients whose eyes were removed for ciliary body or choroidal melanomas. METHODS Two independent observers who were masked to the follow-up of patients examined histologic preparations of 234 eyes removed for ciliary body or choroidal melanomas for the presence of each of the tumor vascular patterns. Statistical analyses included tests for interobserver reliability, Kaplan-Meier survival curves, and the fitting of Cox regression models. RESULTS The detection of each of the nine vascular patterns is highly reproducible. The Cox model indicates that the presence of vascular networks, defined as at least three back-to-back closed vascular loops, is the feature most strongly associated with death from metastatic melanoma. Other significant factors in the Cox model include (in descending order of importance) largest tumor dimension, mitoses, the parallel with cross-linking vascular pattern, age, the presence of tumor-infiltrating lymphocytes, and male gender. CONCLUSIONS The presence of vascular networks provides the most significant association with death from metastatic melanoma of all variables tested. The presence of this pattern should be recorded on pathology reports. If it becomes possible to detect this vascular pattern clinically using a noninvasive imaging technique, then ophthalmologists may be able to determine the likely biologic behavior of a melanoma before resorting to the removal of tissue.

The morphologic characteristics of tumor blood vessels as a marker of tumor progression in primary human uveal melanoma: A matched case-control study☆

Nine morphologic patterns of tumor vessels were identified in eyes removed for ciliary body or choroidal melanoma by the examination of tissue sections stained with fluorescein-conjugated Ulex europaeus I using laser scanning confocal microscopy. This technique also highlights intravascular tumor invasion. Each of these nine morphologic patterns of tumor vessels also may be demonstrated by a modification of the periodic acid-Schiff reaction, viewed with a green narrow band pass filter, but this modified histochemical technique does not accurately identify intravascular tumor invasion. Most tumors have a heterogeneous distribution of vascular patterns. Melanomas in two groups of 20 tumors each were matched by tumor size and location (one group of tumors from patients who survived at least 15 years free of metastatic melanoma after enucleation and one group of tumors from patients who died of metastatic melanoma). A matched case-control analysis indicates that the presence of at least one closed vascular loop in a uveal melanoma is the most significant vascular pattern associated with death from metastatic melanoma after enucleation. Closed loops are associated with other histologic features that are predictive of an unfavorable outcome after enucleation: epithelioid cells and mitotic figures. In this preliminary study the formation of closed vascular loops is a marker of tumor progression in ciliary body and choroidal melanomas.

Upregulated expression of vascular endothelial growth factor in proliferative diabetic retinopathy.

AIMS/BACKGROUND: Vascular endothelial growth factor (VEGF) is a hypoxia induced angiogenic factor. Recent studies have shown that high levels of VEGF accumulate in the vitreous of patients with proliferative diabetic retinopathy (PDR). The purpose of the present study was to identify the retinal cells that upregulate VEGF expression in human PDR patients representing progressive stages of retina deterioration. METHODS: Thirteen formalin fixed and paraffin embedded enucleated eyes with PDR were used (eyes were enucleated because of being blind and painful as a result of neovascular glaucoma). Thin retina sections were hybridised in situ with a VEGF specific probe, to identify cells producing VEGF mRNA. RESULTS: All eyes with PDR showed upregulated expression of VEGF mRNA, specifically in the cells of the neurosensory retina. VEGF expression was upregulated in all three nuclear layers--namely, the ganglion cell layer, the inner nuclear layer, and the outer nuclear layer. However, in each patient, VEGF producing cells were mostly distributed in a different layer, or even confined to a specific region in that layer. For example, expression by the outer nuclear layer was mostly detected in detached (presumably hypoxic) regions of the retina. CONCLUSIONS: Progression of PDR is distinguished by a sustained, upregulated expression of VEGF by the neurosensory retina. Cells in all retina layers can potentially contribute to augmented VEGF production. The restricted population of VEGF producing cells in each case is likely to represent cells residing in ischaemic regions of the retina. Thus, VEGF may function as a linking factor between retinal ischaemia and PDR associated neovascularisation.

Molecular determinants of human uveal melanoma invasion and metastasis.

The molecular analysis of cancer has benefited tremendously from the sequencing of the human genome integrated with the science of bioinformatics. Microarray analysis technology has the potential to classify tumors based on the differential expression of genes. In the current study, a collaborative, multidisciplinary approach was utilized to study the molecular determinants of human uveal melanoma invasion and metastasis. Uveal melanoma is considered the most common primary intraocular cancer in adults, resulting in the death of approximately 50% of patients affected. Unfortunately, at the time of diagnosis, many patients already harbor microscopic metastases, thus underscoring a critical need to identify prognostic markers indicative of metastatic potential. The investigative strategy consisted of isolating highly invasive vs. poorly invasive uveal melanoma cells from a heterogeneous tumor derived from cells that had metastasized from the eye to the liver. The heterogeneous tissue explant MUM-2 led to the derivation of two clonal cell lines: MUM-2B and MUM-2C. Further morphological and functional analyses revealed that the MUM-2B cells were epithelioid, interconverted (expressing mesenchymal and epithelial phenotypes) highly invasive, and demonstrated vasculogenic mimicry. The MUM-2C cells were spindle-like, expressed only a vimentin mesenchymal phenotype, poorly invasive, and were incapable of vasculogenic mimicry. The molecular analysis of the MUM-2B vs. the MUM-2C clones resulted in the differential expression of 210 known genes. Overall, the molecular signature of the MUM-2B cells resembled that of multiple phenotypes – similar to a pluripotent, embryonic-like genotype. Validation of select genes that were upregulated and down-regulated was conducted by semiquantitative RT-PCR measurement. This study provides a molecular profile that will hopefully lead to the development of new molecular targets for therapeutic intervention and possible diagnostic markers to predict the clinical outcome of patients with uveal melanoma.

Cyclosporine Eyedrops for the Treatment of Severe Vernal Keratoconjunctivitis

Twelve children with severe chronic vernal keratoconjunctivitis participated in a pilot study aimed at evaluating the effect of 2% cyclosporine eyedrops on the clinical course and symptoms of this disease. Eleven of the 12 children showed improvement after the first week of treatment. Nine patients demonstrated persistent improvement at the completion of the treatment schedule after six weeks. However, although seven of the children were symptom-free while being treated, only three were free of disease two months after cessation of the cyclosporine eyedrops. The other nine showed rapid recurrence of their symptoms.

Intravitreal methotrexate for treating vitreoretinal lymphoma: 10 years of experience

Aim: To describe our experience in treating vitreoretinal involvement of primary central nervous system lymphoma, by intravitreal injections of methotrexate (MTX). Methods: Patients with suspected intraocular lymphoma underwent a diagnostic vitrectomy. Samples were sent for cytology, genetic evaluation and for interleukin level measurements. Treatment protocol included injection of 400 μg/0.1 ml MTX intravitreally twice weekly for 4 weeks, once weekly for 8 weeks, and then once monthly for 9 months, for a total of 25 injections. Data were collected from the patients’ records and included, inter alia, response to intravitreal MTX measured by time to disappearance of vitreal cells and retinal infiltrates, changes in visual acuity, and clinical recurrence rate. Results: In the past 10 years we have treated 44 eyes of 26 patients; seven patients had monocular involvement, and 19 binocular. Six patients were initially diagnosed as having a non-responsive uveitis, and 16 with either CNS or systemic lymphoma with later involvement of the eye. Four patients had systemic lymphoma; one of them was found to have CNS lymphoma after the ocular involvement. Three patients had T cell lymphoma, and the rest had B cell lymphoma. Clinical remission was reached after 6.4 (3.4) (2–16) injections of MTX (mean (SD) (range)), with 95% of the eyes needing 13 injections or less to be cleared of malignant cells. None of the patients had an intraocular recurrence. Among the side effects, the most common was corneal epitheliopathy, which usually appeared after the third injection and began to subside when the intervals between injections increased. Conclusions: Vitreoretinal involvement of lymphoma can be controlled effectively and without serious adverse reactions by intravitreal MTX injections. The treatment protocol described herein has resulted in no intraocular recurrence so far and has had bearable side effects. The accumulating clinical results bring us to propose the consideration of this protocol as a good first-line treatment option for intraocular lymphoma.

Relation between the Microcirculation Architecture and the Aggressive Behavior of Ciliary Body Melanomas

PURPOSE To study the relation between vascular patterns and the biologically aggressive behavior of ciliary body melanomas. METHODS The authors compared the frequency distribution of vascular patterns by location for 234 uveal melanomas (54 tumors involving the ciliary body, and 180 without ciliary body involvement). Stepwise Cox regressions (for the endpoint of time-to-death due to melanoma), performed separately for melanomas with and without ciliary body involvement, included the following variables: size, vascular patterns, cell type, mean of the largest nucleoli, mitoses, tumor infiltrating lymphocytes, age, and sex. A separate Cox regression procedure included the variable of tumor location. Kaplan-Meier survival curves were generated for time to melanoma death with ciliary body involvement and melanomas without ciliary body involvement for tumors containing or lacking vascular networks. RESULTS These vascular patterns appear more often in the ciliary body than in the choroid: parallel vessels (P = 0.022), arcs (P = 0.003), and parallel with cross-linking, arcs with branching, and loops and networks (all P = 0.0001). Stepwise regression for tumors confined to the choroid indicated that the presence of networks was the most significant variable (P = 0.0001); stepwise regression for tumors with ciliary body involvement suggested that only one variable, networks, was significant (P = 0.0066). Kaplan-Meier survival estimates indicated that the survival of patients with tumors containing networks in the ciliary body was comparable to those containing networks in the choroid. CONCLUSION Regardless of location, ciliary body or choroid, the presence of vascular networks shortens survival. The tumor location does not enter a stepwise Cox regression model when vascular patterns are included as variables. Therefore, the aggressive behavior of ciliary body melanomas appears to be related to the tendency for vascular networks to develop in this location.

Long-term survival of uveal melanoma patients after surgery for liver metastases

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.

The microcirculation of choroidal and ciliary body melanomas

The microcirculation of ciliary body and choroidal melanomas is remodelled into patterns. The presence of microvascular networks, composed of back-to-back loops that encircle microdomains of tumour, and parallel vessels with cross-linking, are associated with death from metastatic melanoma. The formation of these complex vascular patterns may result from reciprocal interactions between the tumour cell and the extracellular matrix, and pattern formation may reflect an invasive tumour cell phenotype. Ciliary body and choroidal melanomas are among the few forms of cancer treated before a pathologist assigns a grade to indicate whether tumour is likely to follow a benign or aggressive course. There is evidence to suggest that prognostically significant microcirculatory patterns may be detectable by non-invasive imaging techniques that may provide a substitute for biopsy to guide the clinical management of patients with these sight- and life-threatening tumours.