Melanoma: Folberg R

Folberg R, Salomao D, Grossniklaus HE, Proia AD, Rao NA, Cameron JD, Anonymous00355. · Department of Ophthalmology at the University of Illinois at Chicago, 60612, USA. · Hum Pathol. · Pubmed #12612878 No free full text.

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Folberg R, Kadkol SS, Frenkel S, Valyi-Nagy K, Jager MJ, Pe'er J, Maniotis AJ. · Oakland University William Beaumont School of Medicine, Rochester, Michigan 48309-4401, USA. · Invest Ophthalmol Vis Sci. · Pubmed #18689700 No free full text.

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Folberg R, Maniotis AJ. · Department of Pathology, University of Illinois Cancer Center, Chicago, Illinois 60612, USA. · APMIS. · Pubmed #15563313 No free full text.

Abstract: The term vasculogenic mimicry describes the formation of fluid-conducting channels by highly invasive and genetically dysregulated tumor cells. Two distinctive types of vasculogenic mimicry have been described. Vasculogenic mimicry of the tubular type may be confused morphologically with endothelial cell-lined blood vessels. Vasculogenic mimicry of the patterned matrix type in no way resembles blood vessels morphologically or topologically. Matrix proteins such as laminin, heparan sulfate proteoglycan, and collagens IV and VI have been identified in these patterns. The patterned matrix anastomoses with blood vessels, and systemically injected tracers co-localize to these patterns. Vasculogenic mimicry of the patterned matrix type has been identified in uveal, cutaneous and mucous membrane melanomas, inflammatory and ductal breast carcinoma, ovarian and prostatic carcinoma, and soft tissue sarcomas, including synovial sarcoma rhabdomyosarcoma, osteosarcoma, and pheochromocytoma. Because the microcirculation of many tumors may be heterogeneous -- including incorporated or co-opted vessels, angiogenic vessels, mosaic vessels, and vasculogenic mimicry of the tubular and patterned matrix types -- therapeutic regimens that target angiogenesis alone may be ineffective against highly invasive tumors that contain patterned matrices. Vasculogenic mimicry provides an opportunity to investigate the interrelationships between the genetically dysregulated invasive tumor cell, the microenvironment, and the malignant switch.

Folberg R, Hendrix MJ, Maniotis AJ. · Department of Pathology, University of Illinois at Chicago, Chicago, Illinois, USA. · Am J Pathol. · Pubmed #10666364 links to  free full text

Abstract: Tumors require a blood supply for growth and hematogenous dissemination. Much attention has been focused on the role of angiogenesis-the recruitment of new vessels into a tumor from pre-existing vessels. However, angiogenesis may not be the only mechanism by which tumors acquire a microcirculation. Highly aggressive and metastatic melanoma cells are capable of forming highly patterned vascular channels in vitro that are composed of a basement membrane that stains positive with the periodic acid-Schiff (PAS) reagent in the absence of endothelial cells and fibroblasts. These channels formed in vitro are identical morphologically to PAS-positive channels in histological preparations from highly aggressive primary uveal melanomas, in the vertical growth phase of cutaneous melanomas, and in metastatic uveal and cutaneous melanoma. The generation of microvascular channels by genetically deregulated, aggressive tumor cells was termed "vasculogenic mimicry" to emphasize their de novo generation without participation by endothelial cells and independent of angiogenesis. Techniques designed to identify the tumor microcirculation by the staining of endothelial cells may not be applicable to tumors that express vasculogenic mimicry. Although it is not known if therapeutic strategies targeting endothelial cells will be effective in tumors whose blood supply is formed by tumor cells in the absence of angiogenesis, the biomechanical and molecular events that regulate vasculogenic mimicry provide opportunities for the development of novel forms of tumor-targeted treatments. The unique patterning characteristic of vasculogenic mimicry provides an opportunity to design noninvasive imaging techniques to detect highly aggressive neoplasms and their metastases.

Mueller AJ, Freeman WR, Schaller UC, Kampik A, Folberg R. · Eye Clinic of the University, Ludwig-Maximilians-University Munich, Munich, Germany. · Ophthalmology. · Pubmed #12466160 No free full text.

Abstract: PURPOSE: Multiple independent laboratories have confirmed the histologic observation that some tumor microcirculation patterns (MCPs) in uveal melanomas are associated strongly with death resulting from metastatic disease. Because these patterns are imageable with confocal indocyanine green angiography (ICG), we designed a prospective study to evaluate whether these angiographically detectable MCPs predict time to tumor growth. DESIGN: Observational case series, prospective, non-randomized. PARTICIPANTS: Ninety-eight patients with unilateral, small, choroidal melanocytic tumors. METHODS: The following information and tumor characteristics were recorded for each patient: demographic parameters, best-corrected visual acuity, intraocular pressure, related visual symptoms, location and dimension of tumor, pigmentation, orange pigment, drusen, tumor-associated hemorrhage, subretinal fluid, and confocal ICG angiographically determined microcirculation patterns-silent (avascularity), normal (preexisting normal choroidal vessels within the tumor), straight vessels, parallel without and with cross-linking, arcs without and with branching, loops, and networks. MAIN OUTCOME MEASURES: Time to growth of the tumor, with growth defined as an increase in the maximal apical tumor height of 0.5 mm measured by standardized A-scan ultrasonography, photographic documentation of an increase of the largest basal diameter of at least 1.5 mm, advancement of one tumor border of at least 0.75 mm, or a combination thereof. RESULTS: Twenty-eight of the 98 tumors in this study (29%) met the predetermined criteria for tumor growth. The median time to growth was 127 days (range, 51-625 days). The following tumor characteristics were significantly associated with time to tumor growth: flashes (P = 0.0224), orange pigment (P = 0.012), subretinal fluid (P < 0.001), maximum basal tumor diameter at initial examination (P = 0.015), maximum apical tumor height (P < 0.001), parallel with cross-linking MCP (P < 0.001), arcs with branching MCP (P = 0.006), loops (P < 0.001), and networks (P < 0.001). Of these, the angiographic documentation of any of the complex MCPs (parallel with cross-linking, arcs with branching, loops, networks, or a combination thereof) showed the strongest association with the time to tumor growth in a Cox proportional hazard model. CONCLUSIONS: The characteristics of our patient cohort are comparable by clinical and echographic parameters with cohorts for predicting tumor growth, described previously in the literature. In addition, we detected a novel clinical predictor of tumor growth: the confocal ICG angiographic detection of complex MCPs.

Mueller AJ, Freeman WR, Folberg R, Schaller UC, Kampik A. · Augenklinik der Ludwig-Maximilians-Universität, Mathildenstrasse 8, 80336 München. · Ophthalmologe. · Pubmed #11917803 No free full text.

Abstract: BACKGROUND: We have previously shown that histologically described microcirculation patterns (MCP) can be visualized with indocyanine green (ICG) angiography. We have designed a prospective study to evaluate the prognostic value of these angiographically imaged MCP in small choroidal melanocytic lesions. In this report we describe the design of the study, characterize the patient collective, and present the first results. PATIENTS AND METHODS: In this prospective nonrandomized observational study, unilateral choroidal melanocytic lesions with 1.5-5.5 mm maximum apical height are observed until growth is determined according to defined criteria. Variables are demographic parameters, subjective symptoms, subretinal fluid, location and dimension of tumor, hemorrhage, color, orange pigment, and MCP determined by ICG angiography: normal, straight, parallel without crosslinking, parallel with crosslinking, arcs without branching, arcs with branching, loop, and network. RESULTS: Seventy patients (22 males, 48 females; age: 33-88 years, median: 64 years) have been included up to now: 19 tumors showed growth so far (time to growth: 51-946 days, median: 127 days). The following parameters were statistically significantly correlated with time to tumor growth: flashes (p = 0.082), orange pigment (p = 0.012), subretinal fluid (p < 0.001), maximum basal tumor diameter (p = 0.001), maximum apical tumor height (p < 0.001), parallel with crosslinking (p < 0.001), arcs with branching (p = 0.006), loop (p < 0.001), and network (p < 0.001). Of these, complex MCP (parallel with crosslinking, arcs with branching, loop and/or network) showed the strongest correlation with time to tumor growth in a Cox regression model. Based on our data, the positive predictive value of imaging complex MCP (for growth within 12 months) is 78% and the negative predictive value is 98%. CONCLUSION: Our patient collective demonstrates comparable prognostic parameters for time to growth as described in the literature. In addition, the ICG angiographic detection of complex MCP is more strongly predictive of the time to growth than other clinically determinable factors. Thus, we recommend this examination for patients with small choroidal melanocytic lesions, if the patient is to be counseled regarding the likely biologic behavior of his tumor.

Eagle RC, Grossniklaus HE, Syed N, Hogan RN, Lloyd WC, Folberg R. · Department of Pathology, Wills Eye Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA. · Arch Ophthalmol. · Pubmed #19204229 No free full text.

Abstract: OBJECTIVES: To report an important complication of ocular evisceration therapy for blind, painful eyes that has been unreported in the literature, and to stress the need for careful preoperative evaluation to exclude occult neoplasms prior to therapy. DESIGN: Multicenter, retrospective, nonrandomized clinicopathological case series of patients found to have previously unsuspected uveal malignant melanoma during histopathological examination of blind, painful eyes treated by evisceration. RESULTS: Histopathological examination of evisceration specimens disclosed previously unsuspected uveal melanoma in 7 patients who were treated for blind, painful eyes. Inflammation caused by necrosis of the tumor and other ocular tissues led to misdiagnosis as endophthalmitis, orbital cellulitis, or idiopathic orbital inflammation in several cases. Preoperative imaging was not performed in 3 cases and failed to detect tumors in the remaining 4 cases. Failure of necrotic tumors to enhance contributed to misdiagnosis. CONCLUSIONS: The presence of a malignant intraocular neoplasm should be excluded prior to evisceration of any blind eye or blind, painful eye, particularly with opaque media. Necrosis-related inflammation can confound the clinical diagnosis of occult lesions, as can failure of necrotic tumors to enhance on imaging studies.

Kooper R, Shirk A, Lee SC, Lin A, Folberg R, Bajcsy P. · National Center for Supercomputing Applications (NCSA), University of Illinois at Urbana-Champaign (UIUC), 1205 W. Clark Street, Urbana, IL 61801, USA. · Comput Biol Med. · Pubmed #18336808 links to  free full text

Abstract: We address the problem of 3D medical volume reconstruction using web services. The use of proposed web services is motivated by the fact that the problem of 3D medical volume reconstruction requires significant computer resources and human expertise in medical and computer science areas. Web services are implemented as an additional layer to a dataflow framework called data to knowledge. In the collaboration between UIC and NCSA, pre-processed input images at NCSA are made accessible to medical collaborators for registration. Every time UIC medical collaborators inspected images and selected corresponding features for registration, the web service at NCSA is contacted and the registration processing query is executed using the image to knowledge library of registration methods. Co-registered frames are returned for verification by medical collaborators in a new window. In this paper, we present 3D volume reconstruction problem requirements and the architecture of the developed prototype system at http://isda.ncsa.uiuc.edu/MedVolume. We also explain the tradeoffs of our system design and provide experimental data to support our system implementation. The prototype system has been used for multiple 3D volume reconstructions of blood vessels and vasculogenic mimicry patterns in histological sections of uveal melanoma studied by fluorescent confocal laser scanning microscope.

Barak V, Frenkel S, Valyi-Nagy K, Leach L, Apushkin MA, Lin AY, Kalickman I, Baumann NA, Pe'er J, Maniotis AJ, Folberg R. · Immunology Laboratory for Tumor Diagnosis, Hadassah-Hebrew University Medical Center, Jerusalem, Israel. · Invest Ophthalmol Vis Sci. · Pubmed #17898257 links to  free full text

Abstract: 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.

Barak V, Frenkel S, Kalickman I, Maniotis AJ, Folberg R, Pe'er J. · Immunology Laboratory for Tumor Diagnosis, Hadassah-Hebrew University Medical Center, Jerusalem, Israel. · Anticancer Res. · Pubmed #17649791 links to  free full text

Abstract: BACKGROUND: 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. PATIENTS AND METHODS: 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. RESULTS: 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 (p < 0.005) 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%. CONCLUSION: 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.

Folberg R, Leach L, Valyi-Nagy K, Lin AY, Apushkin MA, Ai Z, Barak V, Majumdar D, Pe'er J, Maniotis AJ. · Department of Pathology, University of Illinois at Chicago, Chicago, Illinois 60612, USA. · Invest Ophthalmol Vis Sci. · Pubmed #17591861 links to  free full text

Abstract: PURPOSE: To model the behavior of uveal melanoma in the liver. METHODS: A 15-muL suspension of metastatic MUM2B or either primary OCM1 or M619 uveal melanoma cells was injected into the liver parenchyma of 105 CB17 SCID mice through a 1-cm abdominal incision. Animals were killed at 2, 4, 6, or 8 weeks after injection. Before euthanatization, 3% FITC-BSA buffer was injected into the retro-orbital plexus of one eye of three mice. Liver tissues were examined by light and fluorescence microscopy, and were stained with human anti-laminin. Vasculogenic mimicry patterns were reconstructed from serial laser scanning confocal microscopic stacks. RESULTS: OCM1a cells formed microscopic nodules in the mouse liver within 2 weeks after injection and metastasized to the lung 6 weeks later. By contrast, M619 and MUM2B cells formed expansile nodules in the liver within 2 weeks and gave rise to pulmonary metastases within 4 weeks after injection. Vasculogenic mimicry patterns, composed of human laminin and identical with those in human primary and metastatic uveal melanomas, were detected in the animal model. The detection of human rather than mouse laminin in the vasculogenic mimicry patterns in this model demonstrates that these patterns were of tumor cell origin and were not co-opted from the mouse liver microenvironment. CONCLUSIONS: There are currently no effective treatments for metastatic uveal melanoma. This direct-injection model focuses on critical interactions between the tumor cell and the liver. It provides for translationally relevant approaches to the development of new modalities to detect small tumor burdens in patients, to study the biology of clinical dormancy of metastatic disease in uveal melanoma, to design and test novel treatments to prevent the emergence of clinically manifest liver metastases after dormancy, and to treat established uveal melanoma metastases.

Lin AY, Ai Z, Lee SC, Bajcsy P, Pe'er J, Leach L, Maniotis AJ, Folberg R. · Department of Pathology, University of Illinois at Chicago, 340 S. Wood Street, Room 110 (MC 847), Chicago, IL 60612, USA. · Appl Immunohistochem Mol Morphol. · Pubmed #17536318 links to  free full text

Abstract: We previously described techniques to generate 3-dimensional reconstructions of the tumor microcirculation using immunofluorescence histochemistry and laser scanning confocal microscopy on serial sections from archival formalin-fixed, paraffin-embedded tissues. By aligning sequential z-stacks in an immersive visualization environment (ImmersaDesk), the need to insert fiduciary markers into tissue was eliminated. In this study, we developed methods to stitch overlapping confocal z-series together to extend the surface area of interest well beyond that captured by the confocal microscope objective and developed methods to quantify the distribution of markers of interest in 3 dimensions. These techniques were applied to the problem of comparing the surface area of nonendothelial cell-lined, laminin-rich looping vasculogenic mimicry (VM) patterns that are known to transmit fluid, with the surface area of endothelial cell-lined vessels in metastatic uveal melanoma to the liver in 3 dimensions. After labeling sections with antibodies to CD34 and laminin, the surface area of VM patterns to vessels was calculated by segmenting out structures that labeled with laminin but not with CD34 from those structures labeling with CD34, or CD34 and laminin. In metastatic uveal melanoma tissues featuring colocalization of high microvascular density [66.4 microvessels adjusted for 0.313 mm2 area (range 56.7 to 72.7)] and VM patterning, the surface area of VM patterns was 11.6-fold greater (range 10.8 to 14.1) than the surface provided by CD34-positive vessels. These methods may be extended to visualize and quantify molecular markers in 3 dimensions in a variety of pathologic entities from archival paraffin-embedded tissues.

Valyi-Nagy K, Folberg R, Valyi-Nagy T, Maniotis AJ. · Department of Pathology, University of Illinois at Chicago, College of Medicine, 840 South Wood Street, Room 110, M/C 847, Chicago, IL 60612, USA. · Exp Eye Res. · Pubmed #17386925 links to  free full text

Abstract: To better understand determinants of susceptibility/resistance of uveal melanomas to herpes simplex virus type 1 (HSV-1) oncolytic therapy, uveal melanoma cell lines of low (OCM1a) and of high (M619, MUM2B) invasive potential were infected with HSV-1 either in the presence or absence of a laminin-rich extracellular matrix (Matrigel). OCM1a cultures were destroyed faster by HSV-1 than M619 and MUM2B cultures. In the presence of Matrigel, all melanoma cultures demonstrated delayed destruction by HSV-1 relative to Matrigel-free cultures. As sequestration of chromatin is a characteristic feature of highly invasive uveal melanomas that is further increased by exposure to laminin, we explored whether chromatin sequestration could be reversed by HSV-1 infection. HSV-1 infection induced a global reversal of chromatin sequestration in highly invasive uveal melanoma cells. However, this viral effect was first observed only 2h following virus infection and required novel protein synthesis from input viral DNA. These findings suggest that tumor invasiveness, the spatial relationship of tumor cells to laminin and chromatin sequestration are determinants of susceptibility/resistance of melanomas to HSV-1 oncolytic therapy. Furthermore, these findings indicate for the first time that HSV-1 infection is associated with global exposure of normally highly sequestered cellular DNA in malignant cells.

Frenkel S, Barzel I, Levy J, Lin AY, Bartsch DU, Majumdar D, Folberg R, Pe'er J. · Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel. · Eye. · Pubmed #17363922 links to  free full text

Abstract: PURPOSE: Vasculogenic mimicry patterns, formed by highly invasive melanoma cells, connect to endothelial cell-lined blood vessels and contain fluid in vitroand in vivo. This study was designed to determine if fluid leaks into vasculogenic mimicry patterns without circulation, or if fluid circulates in and clears from these patterns. METHODS: Indocyanine green (ICG) laser scanning confocal angiography (Heidelberg Retinal Angiograph (HRA); Heidelberg Engineering, Heidelberg, Germany) was performed on nine patients with posterior choroidal melanoma in an institutional setting. Blood was drawn before the ICG injection and from the contralateral arm of the ICG injection site and 1 min after the injection. Outcome measures include time to first filling of retinal vessels and vasculogenic mimicry patterns and the time at which no fluorescence could be detected by the HRA instrument. After fluorescence was no longer detected in vessels or patterns, the tubes containing the patient's blood was imaged by the Heidelberg HRA. RESULTS: Looping vasculogenic mimicry patterns were detected focally in five patients within 30 s after injection and were detectable up to 12 min post-injection. Blood drawn before ICG injection did not autofluoresce but ICG-containing blood pooled in the tube continued to fluoresce at 1-month post-injection. CONCLUSIONS: Vasculogenic mimicry patterns are not part of the endothelial cell-lined vascular system and fluid enters these patterns through leakage. The rapid infusion of ICG into these patterns after injection and the disappearance of fluorescence detectable by the Heidelberg HRA suggest that fluid circulates in these patterns and does not accumulate as a stagnant pool.

Folberg R, Arbieva Z, Moses J, Hayee A, Sandal T, Kadkol S, Lin AY, Valyi-Nagy K, Setty S, Leach L, Chévez-Barrios P, Larsen P, Majumdar D, Pe'er J, Maniotis AJ. · Department of Pathology, University of Illinois at Chicago, 840 S. Wood St., 110 CSN (MC 847), Chicago, IL 60612, USA. · Am J Pathol. · Pubmed #17003493 links to  free full text

Abstract: The histological detection of laminin-rich vasculogenic mimicry patterns in human primary uveal melanomas is associated with death from metastases. We therefore hypothesized that highly invasive uveal melanoma cells forming vasculogenic mimicry patterns after exposure to a laminin-rich three-dimensional microenvironment would differentially express genes associated with invasive and metastatic behavior. However, we discovered that genes associated with differentiation (GDF15 and ATF3) and suppression of proliferation (CDKNa1/p21) were up-regulated in highly invasive uveal melanoma cells forming vasculogenic mimicry patterns, and genes associated with promotion of invasive and metastatic behavior such as CD44, CCNE2 (cyclin E2), THBS1 (thrombospondin 1), and CSPG2 (chondroitin sulfate proteoglycan; versican) were down-regulated. After forming vasculogenic mimicry patterns, uveal melanoma cells invaded only short distances, failed to replicate, and changed morphologically from the invasive epithelioid to the indolent spindle A phenotype. In human tissue samples, uveal melanoma cells within vasculogenic mimicry patterns assumed the spindle A morphology, and the expression of Ki67 was significantly reduced in adjacent melanoma cells. Thus, the generation of vasculogenic mimicry patterns is accompanied by dampening of the invasive and metastatic uveal melanoma genotype and phenotype and underscores the plasticity of these cells in response to cues from the microenvironment.

Abramson DH, Rollins IS, Lin A, Odell P, Folberg R. · · Arch Ophthalmol. · Pubmed #16832036 No free full text.

This publication has no abstract.

Kadkol SS, Lin AY, Barak V, Kalickman I, Leach L, Valyi-Nagy K, Majumdar D, Setty S, Maniotis AJ, Folberg R, Pe'er J. · Department of Pathology, University of Illinois at Chicago, 60612, USA. · Invest Ophthalmol Vis Sci. · Pubmed #16505010 links to  free full text

Abstract: PURPOSE: This was a pilot study conducted to examine the expression of osteopontin in uveal melanoma and to determine whether serum osteopontin can be used in detecting metastatic uveal melanoma. METHODS: Osteopontin mRNA was measured in three uveal melanoma cell lines of various invasive potential by real-time PCR. Tissue sections of primary and metastatic uveal melanomas were stained for osteopontin. Serum osteopontin levels were measured by ELISA assays in 15 patients with metastatic uveal melanoma and in 37 patients who were disease-free for at least 10 years after treatment of the primary tumor. Paired serum samples drawn from eight patients before and after development of metastasis were analyzed. RESULTS: By real-time PCR, highly invasive primary and metastatic uveal melanoma cells expressed 6- and 250-fold excess osteopontin mRNA, respectively, compared with poorly invasive primary uveal melanoma cells. Tissue sections of primary uveal melanomas lacking looping vasculogenic mimicry patterns either did not stain for osteopontin or exhibited weak, diffuse staining. In primary melanomas containing looping vasculogenic mimicry patterns, strong osteopontin staining was detected in the tumor periphery where patterns were located. Diffuse strong expression of osteopontin was detected in eight samples of uveal melanomas metastatic to the liver. Serum osteopontin levels were significantly higher in patients with metastatic uveal melanoma than in patients who had been disease free for at least 10 years after treatment (P = 0.0001) or in age-matched control subjects. Serum osteopontin levels were significantly higher (P = 0.008) after metastasis than before the detection of metastasis in eight patients. When a cutoff of 10 ng/mL was used, the sensitivity and specificity of serum osteopontin in detecting metastatic melanoma was 87.5%, and the area under the receiver operator characteristic curve was 96%. CONCLUSIONS: Osteopontin is expressed diffusely in tissue sections of hepatic metastases from uveal melanoma, and increased serum osteopontin levels correlate with melanoma metastasis to the liver with high specificity and sensitivity.

Bajcsy P, Lee SC, Lin A, Folberg R. · Automated Learning Group, National Center for Supercomputing Applications, University of Illinois at Urbana-Champaign, IL 61801, USA. · J Microsc. · Pubmed #16438687 links to  free full text

Abstract: The distribution of looping patterns of laminin in uveal melanomas and other tumours has been associated with adverse outcome. Moreover, these patterns are generated by highly invasive tumour cells through the process of vasculogenic mimicry and are not therefore blood vessels. Nevertheless, these extravascular matrix patterns conduct plasma. The three-dimensional (3D) configuration of these laminin-rich patterns compared with blood vessels has been the subject of speculation and intensive investigation. We have developed a method for the 3D reconstruction of volume for these extravascular matrix proteins from serial paraffin sections cut at 4 microm thicknesses and stained with a fluorescently labelled antibody to laminin (Maniotis et al., 2002). Each section was examined via confocal laser-scanning focal microscopy (CLSM) and 13 images were recorded in the Z-dimension for each slide. The input CLSM imagery is composed of a set of 3D sub-volumes (stacks of 2D images) acquired at multiple confocal depths, from a sequence of consecutive slides. Steps for automated reconstruction included (1) unsupervised methods for selecting an image frame from a sub-volume based on entropy and contrast criteria, (2) a fully automated registration technique for image alignment and (3) an improved histogram equalization method that compensates for spatially varying image intensities in CLSM imagery due to photo-bleaching. We compared image alignment accuracy of a fully automated method with registration accuracy achieved by human subjects using a manual method. Automated 3D volume reconstruction was found to provide significant improvement in accuracy, consistency of results and performance time for CLSM images acquired from serial paraffin sections.

Onken MD, Lin AY, Worley LA, Folberg R, Harbour JW. · Department of Ophthalmology & Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA. · Am J Ophthalmol. · Pubmed #16226537 No free full text.

Abstract: PURPOSE: To determine whether there is an association between gene expression profile and looping extravascular matrix patterns in primary uveal melanomas. DESIGN: Laboratory investigation. METHODS: Formalin-fixed, paraffin-embedded sections from 22 primary uveal melanomas that previously were analyzed by microarray gene expression profiling were stained for periodic acid-Schiff and scored in a masked fashion for the presence of looping extravascular matrix patterns. RESULTS: A strong association was observed between looping patterns and the unfavorable class 2 molecular prognostic signature (P < .001). CONCLUSIONS: Looping extravascular matrix patterns occur almost exclusively in class 2 uveal melanomas. This finding may provide new insights into the pathogenesis and management of uveal melanoma.

Lin AY, Maniotis AJ, Valyi-Nagy K, Majumdar D, Setty S, Kadkol S, Leach L, Pe'er J, Folberg R. · Department of Pathology, University of Illinois at Chicago, IL 60612, USA. · Arch Pathol Lab Med. · Pubmed #15974811 No free full text.

Abstract: CONTEXT: Molecular analyses indicate that periodic acid-Schiff (PAS)-positive (laminin-rich) patterns in melanomas are generated by invasive tumor cells by vasculogenic mimicry. Some observers, however, consider these patterns to be fibrovascular septa, generated by a stromal host response. OBJECTIVE: To delineate differences between vasculogenic mimicry patterns and fibrovascular septa in primary uveal melanomas. DESIGN: Frequency distributions, associations with outcome, and thicknesses of trichrome-positive and PAS-positive looping patterns were determined in 234 primary uveal melanomas. Sequential sections of 13 additional primary uveal melanomas that contained PAS-positive/trichrome-negative looping patterns were stained for type I and type IV collagens, laminin, and fibronectin. Real-time quantitative polymerase chain reaction was performed on RNA from cultured uveal melanoma cells for the expression of COL1A1, COL4A2, and fibronectin. RESULTS: Trichrome-positive loops were encountered less frequently than PAS-positive loops (10% vs 56%, respectively). Death from metastatic melanoma was strongly associated with PAS-positive (P < .001) but not with trichrome-positive (P = .57) loops. Trichrome-positive loops were significantly thicker than PAS-positive loops (P < .001). The PAS-positive patterns stained positive for laminin, type I and type IV collagens, and fibronectin. Type I collagen was detected within melanoma cells and focally within some PAS-positive patterns. Real-time quantitative polymerase chain reaction revealed 3-fold, 25-fold, and 97-fold increases, respectively, in expression of COL4A2, fibronectin, and COL1A1 by invasive pattern-forming primary melanoma cells compared with poorly invasive non-pattern-forming cells. CONCLUSIONS: Fibrovascular septa are rare and prognostically insignificant in uveal melanomas, whereas vasculogenic mimicry patterns are associated with increased mortality. Type I collagen, seen focally in some vasculogenic mimicry patterns, may be synthesized by tumor cells, independent of a host stromal response.

Maniotis AJ, Valyi-Nagy K, Karavitis J, Moses J, Boddipali V, Wang Y, Nuñez R, Setty S, Arbieva Z, Bissell MJ, Folberg R. · Department of Pathology, University of Illinois at Chicago, 1819 W. Polk Street, 446 CMW (MC 847), Chicago, IL 60612, USA. · Am J Pathol. · Pubmed #15793298 links to  free full text

Abstract: Given that expression of many genes changes when cells become malignant or are placed in different microenvironments, we asked whether these changes were accompanied by global reorganization of chromatin. We reasoned that sequestration or exposure of chromatin-sensitive sites to restriction enzymes could be used to detect this reorganization. We found that AluI-sensitive sites of nonmalignant cells were relatively more exposed compared to their malignant counterparts in cultured cells and human tumor samples. Changes in exposure and sequestration of AluI-sensitive sites in normal fibroblasts versus fibrosarcoma or those transfected with oncogenes, nonmalignant breast cells versus carcinomas and poorly metastatic versus highly invasive melanoma were shown to be independent of the cell cycle and may be influenced by proteins rich in disulfide bonds. Remarkably, regardless of degree of malignancy, AluI-sensitive sites became profoundly sequestered when cells were incubated with laminin, Matrigel, or a circular RGD peptide (RGD-C), but became exposed when cells were placed on collagen I or in serum-containing medium. Disruption of the actin cytoskeleton led to exposure, whereas disruption of microtubules or intermediate filaments exerted a sequestering effect. Thus, AluI-sensitive sites are more sequestered with increasing malignant behavior, but the sequestration and exposure of these sites is exquisitely sensitive to information conferred to the cell by molecules and biomechanical forces that regulate cellular and tissue architecture.

Buerk BM, Pulido JS, Chiong I, Folberg R, Edward DP, Duffy MT, Thulborn KR. · Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, USA. · Trans Am Ophthalmol Soc. · Pubmed #15747759 links to  free full text

Abstract: PURPOSE: Because signal-to-noise performance improves with increased magnetic field strength, the quality of magnetic resonance images is greater at 3.0 tesla (T) than at 1.5 T. Because of the longer T1 values at higher field strength, intravenously administered magnetic resonance contrast agents provide improved T1 enhancement at 3.0 T. We have used these factors to obtain high-quality contrast-enhanced imaging of small intraocular lesions using a standard head radiofrequency volume coil. Specifically, we have examined lesion size and magnitude of maximum contrast enhancement in a series of intraocular melanomas before and during therapy. METHODS: Eighteen patients with intraocular masses were examined by 3.0 T magnetic resonance imaging (MRI) including intravenous contrast enhancement. Precontrast images were acquired through the orbits followed by sequential postcontrast images at 1-minute intervals for 5 minutes. The magnitude of contrast enhancement of the lesion, extraocular muscles, and brain parenchyma was measured as a percentage increase in magnetic resonance signal over the preenhancement signal intensity. RESULTS: Lesions demonstrated different levels of enhancement ranging up to 130%. Three patterns of enhancement--0% to 20%, 20% to 50%, and >50%-were identified. Brain parenchyma, benign lesions, and responsive tumors following brachytherapy with 125I demonstrated enhancement of less than 20%. Four choroidal melanomas showed intermediate (20% to 50%) levels of enhancement. Four malignant lesions (three melanomas, one metastatic tumor), as well as the extraocular muscles, showed strong, rapid enhancement (>50%). Four patients who had MRI studies before and following plaque brachytherapy ultimately demonstrated a decline in the contrast enhancement following treatment. CONCLUSIONS: Contrast enhancement of intraocular lesions measured by 3.0 T MRI demonstrates different patterns of enhancement that may be useful for indicating the degree of malignancy and in monitoring response to therapy.

Coleman DJ, Silverman RH, Rondeau MJ, Boldt HC, Lloyd HO, Lizzi FL, Weingeist TA, Chen X, Vangveeravong S, Folberg R. · Department of Ophthalmology, Weill Medical College of Cornell University, A-856, 1300 York Avenue, New York, NY 10021, USA. · Ophthalmology. · Pubmed #15019336 No free full text.

Abstract: PURPOSE: Primary malignant melanoma of the choroid and ciliary body has traditionally been treated without histologic staging, using purely clinical indicators. The presence of extravascular matrix patterns (EMP) in histologic sections of uveal melanoma has been shown to be an independent indicator of metastatic risk. These patterns are of a dimension and physical composition that are likely to be detected with ultrasound backscatter analysis. Our aim was to determine whether ultrasound parameter imaging could detect the presence of EMP at a diagnostically significant level for treatment staging and for planning investigational studies of therapeutic modalities. DESIGN: Prospective, masked ultrasound-pathologic correlative study. PARTICIPANTS: One hundred seventeen patients diagnosed with previously untreated choroidal melanoma were scanned within 2 weeks before enucleation. METHODS: Tumors were evaluated histologically and divided into high-risk and low-risk groups on the basis of the presence of 2% or more histologic cross-sectional area composed of EMP patterns. Digital ultrasound data were processed to generate parameter images representing the size and concentration of ultrasound scatterers. Histologic and ultrasound images and data were correlated, and linear and nonlinear statistical methods were used to create multivariate models for noninvasive differentiation of high-risk and low-risk tumors. MAIN OUTCOME MEASURES: Presence or absence of high-risk EMP and associated ultrasound parameter classification models. RESULTS: Of the 117 tumors, 69 were classified as low risk, and 48 were classified as high-risk with histologic analysis. A classification that used ultrasound parameter image features with linear discriminant analysis could correctly identify 79.5% of cases retrospectively and 75.2% of cases by use of cross-validation, an estimate of prospective classification ability. By use of a more powerful classification technique (support vector machine), 93.1% of cases were correctly classified retrospectively. With a cross-validation procedure, 80.10% of cases were correctly classified. CONCLUSIONS: Ultrasound can be used noninvasively to classify tumors into high-risk and low-risk groups by detecting the presence of EMP patterns. By the use of previous studies that compared the histologic presence of EMP patterns with patient survival, estimates of hazard rates associated with ultrasound risk groups can be made. The noninvasive ultrasound classification is potentially useful as a prognostic variable and as a tool for stratification of patient populations for tumor treatment evaluation.

Silverman RH, Folberg R, Rondeau MJ, Boldt HC, Lloyd HO, Chen X, Lizzi FL, Weingeist TA, Coleman DJ. · Department of Ophthalmology, Weill Medical College of Cornell University, New York, NY 10021, USA. · Ultrasound Med Biol. · Pubmed #12878240 No free full text.

Abstract: Specific extracellular matrix patterns in uveal melanoma are associated with metastatic risk. The laminin-rich composition and dimensions (on the order of a wavelength or less) of these structures suggest that acoustic backscatter might be affected by their presence. In this study, 10-MHz radiofrequency (RF) ultrasound (US) data were acquired before surgical removal of 117 eyes with uveal malignant melanoma. Histologic sections were evaluated for the presence of matrix patterns and acoustic backscatter was characterized using calibrated spectrum analysis. Statistical correlations between acoustic and histologic patterns were determined and linear discriminant analysis (LDA) and radial basis networks (RBN) were used to develop classification models for histologically based risk groups. Statistically significant correlations were found between acoustic parameters and the presence of histologic matrix-rich patterns. Retrospective classification accuracies of 74.4% and 78.6% were obtained with LDA and RBN, respectively. Leave-one-out analyses indicated estimated predictive accuracies of 71.8% and 75.0% for LDA and RBN, respectively.

Chen X, Ai Z, Rasmussen M, Bajcsy P, Auvil L, Welge M, Leach L, Vangveeravong S, Maniotis AJ, Folberg R. · Department of Pathology, University of Illinois at Chicago, Chicago, Illinois 60612, USA. · Invest Ophthalmol Vis Sci. · Pubmed #12824220 links to  free full text

Abstract: PURPOSE: Looping patterns rich in laminin are present in tissue samples of primary aggressive human uveal melanomas and their metastases. Because these extravascular patterns connect to blood vessels and transmit fluid in vitro and in vivo, the three-dimensional configuration of these patterns has been the subject of considerable speculation. In the current study, methods were devised to describe the three-dimensional configuration of looping extravascular matrix patterns in archival human uveal melanoma tissue. METHODS: Twenty-five serial 4-microm-thick sections from primary uveal melanoma tissue were labeled with fluorescence-tagged laminin and examined by confocal microscopy to generate a Z-series within each 4-microm-thick section. The z-series from each section was stacked using an immersive three-dimensional environment (ImmersaDesk; Fakespace, Kitchener, Ontario, Canada) to allow for precise alignment and compensation for distortion artifact. RESULTS: Extravascular matrix patterns that appeared to form loops in two dimensions were shown to represent thin wrappings around branching and twisting cylindrical groupings of melanoma cells. Blood vessels joined with some of these laminin-positive cylindrical wrappings. CONCLUSIONS: In this preliminary study, periodic acid-Schiff (PAS)-positive laminin-rich looping patterns in two-dimensional tissue sections appear to outline cylindrical branching packets of melanoma cells rather than spheroidal nests. The conduction of fluid through this extravascular system may provide a novel delivery system for contrast and diagnostic agents.