Justis P. Ehlers

Gene Expression Profiling in Uveal Melanoma Reveals Two Molecular Classes and Predicts Metastatic Death

Melanomas are notoriously difficult to classify because of a lack of discrete clinical and pathological stages. Here, we show that primary uveal melanomas surprisingly cluster into two distinct molecular classes based on gene expression profile. Genes that discriminate class 1 (low-grade) from class 2 (high-grade) include highly significant clusters of down-regulated genes on chromosome 3 and up-regulated genes on chromosome 8q, which is consistent with previous cytogenetic studies. A three-gene signature allows biopsy-size tumor samples to be assigned accurately to tumor classes using either array or PCR platforms. Most importantly, this molecular classification strongly predicts metastatic death and outperforms other clinical and pathological prognostic indicators. These studies offer new insights into melanoma pathogenesis, and they provide a practical foundation for effective clinical predictive testing.

Prospective trial of a blood supply-based technique of pancreaticojejunostomy: effect on anastomotic failure in the whipple procedure

BACKGROUND Anastomotic failure at the pancreaticojejunostomy after a Whipple procedure, manifested either as a pancreatic fistula or intraabdominal abscess, is still an unacceptably common postoperative complication. STUDY DESIGN A prospectively collected series of 123 patients underwent a Whipple procedure. During the pancreaticojejunostomy, the blood supply at the cut surface of the pancreas was evaluated, and if deemed inadequate, the pancreas was cut back 1.5 to 2.0 cm to improve the blood supply. The anastomosis was performed under magnification with meticulous technique. RESULTS There were 123 Whipple procedures performed. In 47 (38%), the blood supply was considered inadequate and the pancreas was cut back. Postoperatively, there were 2 pancreatic fistulas (1.6%) and 2 intraabdominal abscesses (1.6%). There was 1 (0.8%) postoperative death from aspiration pneumonia. CONCLUSIONS Pancreatic fistula, the most serious complication of the Whipple procedure, can be almost entirely eliminated by a technique that combines meticulous attention to placement and tying of sutures under magnification with optimization of blood supply to the anastomosis.

Functional gene expression analysis uncovers phenotypic switch in aggressive uveal melanomas

Microarray gene expression profiling is a powerful tool for generating molecular cancer classifications. However, elucidating biological insights from these large data sets has been challenging. Previously, we identified a gene expression-based classification of primary uveal melanomas that accurately predicts metastatic death. Class 1 tumors have a low risk and class 2 tumors a high risk for metastatic death. Here, we used genes that discriminate these tumor classes to identify biological correlates of the aggressive class 2 signature. A search for Gene Ontology categories enriched in our class-discriminating gene list revealed a global down-regulation of neural crest and melanocyte-specific genes and an up-regulation of epithelial genes in class 2 tumors. Correspondingly, class 2 tumors exhibited epithelial features, such as polygonal cell morphology, up-regulation of the epithelial adhesion molecule E-cadherin, colocalization of E-cadherin and beta-catenin to the plasma membrane, and formation of cell-cell adhesions and acinar structures. One of our top class-discriminating genes was the helix-loop-helix inhibitor ID2, which was strongly down-regulated in class 2 tumors. The class 2 phenotype could be recapitulated by eliminating Id2 in cultured class 1 human uveal melanoma cells and in a mouse ocular melanoma model. Id2 seemed to suppress the epithelial-like class 2 phenotype by inhibiting an activator of the E-cadherin promoter. Consequently, Id2 loss triggered up-regulation of E-cadherin, which in turn promoted anchorage-independent cell growth, a likely antecedent to metastasis. These findings reveal new roles for Id2 and E-cadherin in uveal melanoma progression, and they identify potential targets for therapeutic intervention.

Integration of a spectral domain optical coherence tomography system into a surgical microscope for intraoperative imaging.

PURPOSE To demonstrate an operating microscope-mounted spectral domain optical coherence tomography (MMOCT) system for human retinal and model surgery imaging. METHODS A prototype MMOCT system was developed to interface directly with an ophthalmic surgical microscope, to allow SDOCT imaging during surgical viewing. Nonoperative MMOCT imaging was performed in an Institutional Review Board-approved protocol in four healthy volunteers. The effect of surgical instrument materials on MMOCT imaging was evaluated while performing retinal surface, intraretinal, and subretinal maneuvers in cadaveric porcine eyes. The instruments included forceps, metallic and polyamide subretinal needles, and soft silicone-tipped instruments, with and without diamond dusting. RESULTS High-resolution images of the human retina were successfully obtained with the MMOCT system. The optical properties of surgical instruments affected the visualization of the instrument and the underlying retina. Metallic instruments (e.g., forceps and needles) showed high reflectivity with total shadowing below the instrument. Polyamide material had a moderate reflectivity with subtotal shadowing. Silicone instrumentation showed moderate reflectivity with minimal shadowing. Summed voxel projection MMOCT images provided clear visualization of the instruments, whereas the B-scans from the volume revealed details of the interactions between the tissues and the instrumentation (e.g., subretinal space cannulation, retinal elevation, or retinal holes). CONCLUSIONS High-quality retinal imaging is feasible with an MMOCT system. Intraoperative imaging with model eyes provides high-resolution depth information including visualization of the instrument and intraoperative tissue manipulation. This study demonstrates a key component of an interactive platform that could provide enhanced information for the vitreoretinal surgeon.

DDEF1 is located in an amplified region of chromosome 8q and is overexpressed in uveal melanoma.

Purpose: The molecular pathogenesis of uveal melanoma is poorly understood but is usually accompanied by amplification of chromosome 8q, suggesting the activation of one or more oncogenes. We recently identified a gene expression profile that distinguishes low-grade from high-grade melanomas. In this profile, a cluster of genes at chromosome 8q was overexpressed in high-grade tumors, providing an opportunity to search for potential oncogenes in this region. Experimental Design: Gene expression microarray analysis was done on 25 primary uveal melanomas. Microarray comparative genomic hybridization (CGH), quantitative PCR, and immunohistochemistry were done on a subset of these tumors. Cell motility was measured using a wound-healing assay. Results: In melanomas analyzed for microarray gene expression and CGH, gain of chromosome 8q correlated most strongly with expression of DDEF1, a gene located at 8q24. In contrast, the nearby MYC oncogene exhibited no significant change in expression. Confirming the microarray findings, DDEF1 mRNA levels and protein expression were significantly higher in high-grade melanomas. Furthermore, ectopic expression of DDEF1 in low-grade melanoma cells resulted in a significant increase in cell motility, a feature of high-grade metastasizing cells. Conclusions: These findings suggest that DDEF1 overexpression may be a pathogenetically relevant consequence of chromosome 8q amplification, which commonly occurs in high-grade uveal melanomas. We conclude that DDEF1 may act as an oncogene in this cancer, and it may be a useful diagnostic marker and therapeutic target.

Anatomical and visual outcomes following ocriplasmin treatment for symptomatic vitreomacular traction syndrome

Objective To evaluate the anatomical and visual outcomes of patients treated with ocriplasmin for the treatment of symptomatic vitreomacular adhesion (sVMA), including vitreomacular traction syndrome and macular holes. Design Retrospective, interventional, single centre, case series. Participants Patients with sVMA. Intervention Patients were treated with a single intravitreal injection of 0.125 mg ocriplasmin (Jetrea, Thrombogenics Inc, USA, Alcon/Novartis EU) with the reconstitution technique recommended by the manufacturer. Main outcome measures The primary study endpoint was the resolution of sVMA by spectral domain optical coherence tomography (SDOCT) at day 28. Secondary outcome measures included time to vitreous release, visual acuity (VA), changes in the optical coherence tomography (OCT) thickness and structure and macular hole closure rate. Results 17 patients were included in the study and resolution of vitreomacular adhesion (VMA) was verified by SDOCT in eight patients by day 28 (overall response rate of 47.1%, 8/17 eyes) with most patients experiencing VMA release by 7 days (41.2%, 7/17 eyes). Those who did not have VMA resolution showed no statistically significant change in VMA diameter as measured by horizontal and vertical 5-line raster scans at final follow-up (p=0.82 and p=0.75, respectively). The mean baseline Snellen VA was 20/49 and at final follow-up was 20/46 (p=0.59). The average central subfield thickness was 371 microns prior to treatment and 324 microns at final follow-up (range 191–767 microns, p=0.25). Patients meeting three of four positive predictors criteria (eg, no epiretinal membrane (ERM) at baseline, VMA diameter ≤1500 µm and phakic lens status) showed a response rate of 50.0% (seven of 14 patients); those meeting all four criteria (eg, younger than 65, no ERM at baseline, VMA diameter ≤1500 µm and phakic lens status) showed a response rate of 75.0% (three of four eyes). Transient outer segment ellipsoid zone loss was documented in seven patients and subretinal fluid presence following injection was noted in five patients. Four of the five patients with macular holes at baseline experienced resolution of their macular hole after injection. Conclusions This is the first study to quantify the extent of outer retinal changes seen in patients receiving ocriplasmin. Our initial experience with ocriplasmin shows a significant anatomical effect and is accompanied by transient changes in the outer retinal structures visualised by SDOCT.

Combination intravitreal bevacizumab/panretinal photocoagulation versus panretinal photocoagulation alone in the treatment of neovascular glaucoma.

Purpose: To evaluate same-day combination intravitreal bevacizumab/panretinal photocoagulation (PRP) for the treatment of neovascular glaucoma (NVG) compared with PRP alone. Methods: This was an institutional review board-approved, retrospective, consecutive case-control study of patients receiving same-day combination bevacizumab/PRP or PRP alone as treatment of NVG from September 2004 through June 2007. Visual acuity, intraocular pressure (IOP), presence of anterior segment neovascularization, and required glaucoma interventional procedures were recorded. Results: A total of 23 patients were identified, 11 in the bevacizumab/PRP group and 12 in the PRP alone group. The bevacizumab/PRP group had a significant reduction in IOP compared with the PRP alone group (-11 vs. 0 mmHg, respectively; P = 0.03). There was a significantly higher frequency and rate of neovascular regression in the combination therapy group than in the PRP only group (11 vs 2 eyes [P < 0.001] and 12 vs 127 days [P < 0.0001], respectively). Average follow-up was 143 days for the bevacizumab/PRP group and 118 days for the PRP alone group. Conclusions: Combination treatment resulted in more rapid decrease in IOP. In addition, the combination group had increased frequency and rapidity of regression of neovascularization. This study provides a foundation for further research and suggests consideration for a possible new paradigm for the treatment of NVG.

Retinal vein occlusion: beyond the acute event.

Retinal vein occlusion is a major cause of vision loss. We provide an overview of the clinical features, pathogenesis, natural history, and management of both branch retinal vein occlusion and central retinal vein occlusion. Several recent multicenter randomized clinical trials have been completed which have changed the approach to this disorder. Management of retinal vein occlusions can be directed at the underlying etiology or the resulting sequelae. Options include surgical intervention, laser photocoagulation, intravitreal pharmacotherapy, and sustained drug delivery devices.

Integrative Genomic Analysis of Aneuploidy in Uveal Melanoma

Purpose: Aneuploidy is a hallmark of cancer and is closely linked to metastasis and poor clinical outcome. Yet, the mechanisms leading to aneuploidy and its role in tumor progression remain poorly understood. The extensive and complex karyotypic abnormalities seen in many solid tumors could hinder the identification of pathogenetically relevant chromosomal alterations. Uveal melanoma is an attractive solid tumor for studying aneuploidy because it is a relatively homogeneous cancer that is highly metastatic and has low nonspecific chromosomal instability. Experimental Design: Comparative genomic hybridization and gene expression profiling were used to analyze patterns of aneuploidy in 49 primary uveal melanomas. This analysis was supplemented by a review of cytogenetic findings in 336 published cases. Results: Three prognostically significant tumor subgroups were identified based on the status of chromosomes 3 and 6p. Discrete patterns of chromosomal alterations accumulated in these three subgroups in a nonrandom temporal sequence. Poor clinical outcome was associated with early chromosomal alterations rather than overall aneuploidy. A gene expression signature associated with aneuploidy was enriched for genes involved in cell cycle regulation, centrosome function, and DNA damage repair. One of these genes was PTEN, a tumor suppressor and genomic integrity guardian, which was down-regulated in association with increasing aneuploidy (P = 0.003). Conclusions: The relationship between aneuploidy and poor prognosis may be determined by specific, pathogenetically relevant chromosomal alterations, rather than overall aneuploidy. Such alterations can be identified using integrative genomic methods and may provide insights for novel therapeutic approaches.

Evaluation of choroidal thickness in retinitis pigmentosa using enhanced depth imaging optical coherence tomography

Objective To describe the choroidal characteristics of patients with retinitis pigmentosa (RP) using enhanced depth imaging (EDI) and spectral domain (SD) optical coherence tomography (OCT). Purpose To investigate the spectral-domain ocular coherence tomography features of the choroid in patients with RP using EDI. Methods A prospective, case–control study of 21 patients from the Cole Eye Institute with RP imaged using the Spectralis OCT and an EDI protocol. Submacular choroidal thickness measurements were obtained beneath the fovea and at 500 µm intervals for 2.5 mm nasal and temporal to the centre of the fovea. These measurements were compared to choroidal thickness measurements from 25 healthy age-matched controls with similar refractive error range and no clinical evidence of retinal or glaucomatous disease. Statistical analysis was performed to compare choroidal thickness at each location between the two groups and to correlate choroidal thickness with best-corrected visual acuity and central retinal thickness. Results Mean ages were 40.6 years for control patients and 45.1 years for RP patients (p>0.05). Mean choroidal thickness measurements were 245.6±103 µm in RP patients and 337.8.2±109 µm in controls (p<0.0001). There was no correlation between subfoveal choroidal thickness and visual acuity or retinal thickness in the RP patients when compared to the control group. Conclusions Submacular choroidal thickness, as measured by SD–OCT EDI, is significantly reduced in patients with RP, but did not correlate with visual acuity or retinal thickness in RP patients. Further research is needed to understand better the pathophysiological significance of the choroidal alterations present in RP.