Barry L. Burgess

Antitumor effects of the investigational selective MEK inhibitor TAK733 against cutaneous and uveal melanoma cell lines

BackgroundTAK733 is a novel allosteric, non-ATP-binding, inhibitor of the BRAF substrates MEK-1/2.MethodsThe growth inhibitory effects of TAK733 were assessed in a panel of 27 cutaneous and five uveal melanoma cell lines genotyped for driver oncogenic mutations. Flow cytometry, Western blots and metabolic tracer uptake assays were used to characterize the changes induced by exposure to TAK733.ResultsFourteen cutaneous melanoma cell lines with different driver mutations were sensitive to the antiproliferative effects of TAK733, with a higher proportion of BRAFV600E mutant cell lines being highly sensitive with IC50s below 1 nM. The five uveal melanoma cell lines had GNAQ or GNA11 mutations and were either moderately or highly sensitive to TAK733. The tested cell lines wild type for NRAS, BRAF, GNAQ and GNA11 driver mutations were moderately to highly resistant to TAK733. TAK733 led to a decrease in pERK and G1 arrest in most of these melanoma cell lines regardless of their origin, driver oncogenic mutations and in vitro sensitivity to TAK733. MEK inhibition resulted in increase in pMEK more prominently in NRASQ61L mutant and GNAQ mutant cell lines than in BRAFV600E mutant cell lines. Uptake of the metabolic tracers FDG and FLT was inhibited by TAK733 in a manner that closely paralleled the in vitro sensitivity assays.ConclusionsThe MEK inhibitor TAK733 has antitumor properties in melanoma cell lines with different oncogenic mutations and these effects could be detectable by differential metabolic tracer uptake.

Multi-Year Follow-up of Fine-Needle Aspiration Biopsy in Choroidal Melanoma

PURPOSE To report the local and systemic follow-up of patients undergoing transscleral intraoperative fine-needle aspiration biopsy (FNAB) at the time of iodine-125 plaque brachytherapy for the treatment of choroidal melanoma. DESIGN Retrospective, single-center, consecutive case cohort study. PARTICIPANTS A total of 170 consecutive patients with choroidal melanoma. METHODS All patients with choroidal melanoma treated with iodine-125 brachytherapy and intraoperative FNAB from January 2005 to January 2010 with at least 1 year of clinical follow-up were included. MAIN OUTCOME MEASURES Outcomes examined were endophthalmitis, orbital dissemination, local treatment failure, rhegmatogenous retinal detachment, monosomy 3 status, and choroidal melanoma metastasis. RESULTS A total of 170 consecutive patients with clinical diagnosis of choroidal melanoma, intraoperative FNAB, and post-brachytherapy follow-up of 1 to 6 years (mean, 2.7 ± 1.3 years) were included. For tumors with height of <3.0 mm, 3.0 to 5.0 mm, and >5.0 mm, sufficient biopsy material for fluorescence in situ hybridization (FISH) was obtained in 53%, 68%, and 91%, respectively. During the follow-up period, there was no case of postoperative endophthalmitis, orbital dissemination, or local treatment failure. Three patients developed rhegmatogenous retinal detachment. Fourteen patients developed clinical evidence of metastasis. Of the 14 patients, 8 had monosomy 3 of the primary tumor, 2 had disomy 3, 1 had trisomy 3, and 3 had insufficient material for FISH. The cumulative 5-year Kaplan-Meier metastatic rate was 13%. CONCLUSIONS Transscleral FNAB at the time of iodine-125 plaque brachytherapy was not associated with endophthalmitis, orbital dissemination, or local treatment failure in this series, and post-brachytherapy retinal detachment occurred in 3 eyes. The cumulative Kaplan-Meier 5-year metastatic rate was not statistically different from the rate of 13% reported by the Collaborative Ocular Melanoma Study for tumors of the same size treated by brachytherapy without biopsy. Rhegmatogenous retinal detachment may occur in young patients secondary to posterior vitreous detachment induced by tumor response to radiation, unrelated to FNAB.

Reactions to and Desire for Prognostic Testing in Choroidal Melanoma Patients

To determine if choroidal melanoma patients want cytogenetic prognostic information. Ninety-nine choroidal melanoma patients completed a questionnaire regarding their opinions about receiving prognostic information. The perceived usefulness of prognostic information was evaluated in patients who had undergone cytogenetic testing. Depressive symptoms, quality of life, and interest in supportive counseling during test receipt were assessed. Ninety-seven percent of respondents reported that they would have wanted prognostic information at the time of their treatment and 98% of respondents reported that supportive counseling should be offered when prognostic information is given. Patients who had received a more favorable prognostic result were more likely to endorse the usefulness of cytogenetic testing than were patients who had received a less favorable prognostic result. Psychological status did not vary significantly as a function of cytogenetic test result. Prognostic information was important to patients with choroidal melanoma, even in the absence of prophylactic measures which might improve prognosis.

Genomic Identification of Significant Targets in Ciliochoroidal Melanoma

PURPOSE To identify genomic targets for ciliochoroidal melanoma diagnosis, prognosis, and therapy. METHODS Fifty-eight ciliochoroidal melanomas were analyzed by high-resolution, genome-wide, single nucleotide polymorphism (SNP) mapping arrays. The 58 SNP arrays were compared to 48 HapMap normals representing both sexes and assessed with a systematic statistical method, Genomic Identification of Significant Targets in Cancer (GISTIC), to identify significant ciliochoroidal chromosomal abnormalities including chromosome-arm-sized as well as focal events of amplification and deletion. The 58 SNP arrays were also analyzed to assess copy number. RESULTS The 58 ciliochoroidal melanomas analyzed by GISTIC showed large regions of chromosome amplification on 6p and 8q in addition to focal amplification peaks on 1q31.3, 4p16.2, 9p23, and 9q33.1. The melanomas also showed large regions of deletion on 1p and all of 3, 6q, 8p, and 16q, as well as focal deletion peaks on 2p12, 2q14.3, 4q26, 5q21.1, 7q21.11, 8p21.3, 9p21.1, 13q21.31, 13q31.3, and 16q23.3. For each large region and focal peak, the statistical significance was computed, and known genes were specified. CONCLUSIONS High-resolution analysis of ciliochoroidal melanoma cytogenetic aberration patterns supports the utility of systematic characterization of the cancer genome by corroborating known melanoma-related genomic aberrations and identifying additional melanoma-related genomic abnormalities that can be used to identify potential targets for diagnosis, prognosis and therapy.

Three-dimensional organization of nascent rod outer segment disk membranes

Significance A vertebrate photoreceptor cell depends on the elaboration of its cilium to generate its light-sensitive organelle, the outer segment (OS), which is made up of a stack of membrane disks, containing the visual receptor, opsin. How this elaboration occurs has been the subject of recent controversy. Here we used electron microscope tomography to obtain a 3D analysis of the membrane organization at the base of the OS, where new membrane disks are continually made to replace the older ones. We show that the nascent disk membrane is continuous with the ciliary plasma membrane, and appears to form by a complex reshaping of this membrane, involving an invagination, followed by outward growth, and, finally, the completion of a disk rim. The vertebrate photoreceptor cell contains an elaborate cilium that includes a stack of phototransductive membrane disks. The disk membranes are continually renewed, but how new disks are formed remains poorly understood. Here we used electron microscope tomography to obtain 3D visualization of the nascent disks of rod photoreceptors in three mammalian species, to gain insight into the process of disk morphogenesis. We observed that nascent disks are invariably continuous with the ciliary plasma membrane, although, owing to partial enclosure, they can appear to be internal in 2D profiles. Tomographic analyses of the basal-most region of the outer segment show changes in shape of the ciliary plasma membrane indicating an invagination, which is likely a first step in disk formation. The invagination flattens to create the proximal surface of an evaginating lamella, as well as membrane protrusions that extend between adjacent lamellae, thereby initiating a disk rim. Immediately distal to this initiation site, lamellae of increasing diameter are evident, indicating growth outward from the cilium. In agreement with a previous model, our data indicate that mature disks are formed once lamellae reach full diameter, and the growth of a rim encloses the space between adjacent surfaces of two lamellae. This study provides 3D data of nascent and mature rod photoreceptor disk membranes at unprecedented z-axis depth and resolution, and provides a basis for addressing fundamental questions, ranging from protein sorting in the photoreceptor cilium to photoreceptor electrophysiology.

Ocular Response of Choroidal Melanoma With Monosomy 3 Versus Disomy 3 After Iodine-125 Brachytherapy

PURPOSE To report the ocular response of choroidal melanoma with monosomy 3 vs. disomy 3 after (125)I brachytherapy. METHODS AND MATERIALS We evaluated patients with ciliochoroidal melanoma managed with fine needle aspiration biopsy immediately before plaque application for (125)I brachytherapy between January 1, 2005 and December 31, 2008. Patients with (1) cytopathologic diagnosis of melanoma, (2) melanoma chromosome 3 status identified by fluorescence in situ hybridization, and (3) 6 or more months of follow-up after brachytherapy were sorted by monosomy 3 vs. disomy 3 and compared by Kruskal-Wallis test. RESULTS Among 40 ciliochoroidal melanomas (40 patients), 15 had monosomy 3 and 25 had disomy 3. Monosomy 3 melanomas had a median greatest basal diameter of 12.00 mm and a median tumor thickness of 6.69 mm before brachytherapy; at a median of 1.75 years after brachytherapy, median thickness was 3.10 mm. Median percentage decrease in tumor thickness was 48.3%. Disomy 3 melanomas had a median greatest basal diameter of 10.00 mm and median tumor thickness of 3.19 mm before brachytherapy; at a median of 2.00 years after brachytherapy, median tumor thickness was 2.37 mm. The median percentage decrease in tumor thickness was 22.7%. Monosomy 3 melanomas were statistically greater in size than disomy 3 melanomas (p < 0.001) and showed a greater decrease in tumor thickness after brachytherapy (p = 0.006). CONCLUSION In this study, ciliochoroidal melanomas with monosomy 3 were significantly greater in size than disomy 3 melanoma and showed a significantly greater decrease in thickness at a median of 1.75 years after brachytherapy. The greater decrease in monosomy 3 melanoma thickness after brachytherapy is consistent with other malignancies in which more aggressive pathology has been shown to be associated with a greater initial response to radiotherapy.

Identification of Candidate Tumor Oncogenes by Integrative Molecular Analysis of Choroidal Melanoma Fine-Needle Aspiration Biopsy Specimens

OBJECTIVE To report integrative molecular analysis of choroidal melanoma fine-needle aspiration biopsy specimens to identify candidate tumor oncogenes. METHODS Thirty-one choroidal melanoma fine-needle aspiration biopsy specimens were analyzed using cytopathologic diagnosis of melanoma, fluorescence in situ hybridization for chromosome 3, cytogenetic characterization (GeneChip Human 250K NSPI Mapping Arrays; Affymetrix, Santa Clara, California), and gene expression profiles (GeneChip Human Genome U133 Plus 2.0 Arrays, Affymetrix). These analyses were performed by clustering of cytogenetic aberrations, sorting by chromosome 3 loss and chromosome 6p gain, and comparing gene expression profiles in chromosome 3 loss- and chromosome 6p-gain tumors to identify genes with differential expression based on cytogenetic characteristics. RESULTS Of 31 choroidal melanoma biopsy specimens included in this study, 19 tumors had chromosome 3 loss, and 12 tumors without chromosome 3 loss had chromosome 6p gain. Comparative RNA analysis for these 2 groups revealed 49 genes with greater than 4-fold higher expression and 31 genes with greater than 4-fold lower expression in chromosome 3-loss tumors relative to chromosome 6p-gain tumors. CONCLUSIONS Molecular analysis of choroidal melanoma fine-needle aspiration biopsy specimens demonstrated 2 cytogenetically distinct groups characterized by chromosome 3 loss or chromosome 6p gain. In chromosome 3-loss melanomas relative to chromosome 6p-gain melanomas, integrative RNA analysis revealed genes with higher expression and lower expression and identified several genes that have not been reported in previous studies. CLINICAL RELEVANCE Genes differentially expressed between chromosome 3-loss and chromosome 6p-gain melanomas may provide new knowledge about the biologic nature of choroidal melanoma and may contribute to the development of targeted therapies.

Integrin αvβ5 is not required for the phagocytosis of photoreceptor outer segments by cultured retinal pigment epithelial cells

Abstract The phagocytosis of photoreceptor outer segments (OS) by the retinal pigment epithelium (RPE) is a receptor mediated process. A key component of this process is the receptor tyrosine kinase, Mer. RPE cells from the RCS rat, which lacks a functional mer gene, and do not express Mer protein, are able to bind OS, but are unable to ingest them, suggesting that both a binding receptor and an ingestion receptor (Mer) are required for phagocytosis to occur. These rats become blind shortly after birth. To date the binding receptor has not been identified. Recent studies, using an SV40 transformed rat RPE cell line, RPE-J, or cultured human RPE cells, have suggested that the receptor for OS binding is the integrin αvβ5. However, the results presented here clearly show that this integrin plays at most a minor role in the phagocytosis of OS by primary cultures of rat RPE cells. OS phagocytosis by normal RPE cells is not affected by a function-blocking antibody to αvβ5 integrin, nor by the integrin-specific blocking peptide GRGDSP. Additionally, RPE-J cells do not express the Mer receptor protein, which has been shown to be obligatory for OS phagocytosis, or RPE65, a specific marker for RPE cells. We suggest that the RPE-J cell line is not a valid model with which to study the complex process of OS phagocytosis.

Association of positive dual-modality positron emission tomography/computed tomography imaging of primary choroidal melanoma with chromosome 3 loss and tumor size.

Purpose: To evaluate the positive dual-modality positron emission tomography/computed tomography (PET/CT) of choroidal melanoma with chromosome 3 loss and tumor size. Methods: Thirty-seven consecutive patients with choroidal melanoma with known chromosome 3 status who underwent whole-body PET/CT imaging were retrospectively reviewed. Cytology and chromosome 3 loss were identified by fine-needle aspiration biopsy. Fluorescent in situ hybridization and whole genome microarray by single-nucleotide polymorphism were used to evaluate the chromosome 3 status. Metabolic activity of primary choroidal melanoma by PET/CT imaging was evaluated. Results: Thirteen of 37 (35%) primary choroidal melanomas had loss of chromosome 3; 7 of the 13 (54%) melanomas were positive for metabolic activity identified by PET/CT imaging. All 24 of 37 melanomas without chromosome 3 loss were inactive for metabolic activity. There was a statistically significant association between positive metabolic activity and chromosome 3 loss (P = 0.00017 Fisher exact test); positive PET/CT imaging was 54% sensitive and 100% specific for loss of chromosome 3. Seven of 37 (19%) choroidal melanomas with positive metabolic activity by PET/CT were statistically significantly larger in size than the 30 metabolically inactive melanomas (P < 0.001, Kruskal-Wallis test). Conclusion: Positive metabolic activity of choroidal melanoma identified by PET/CT imaging was statistically significantly associated with chromosome 3 loss and larger tumor size.

Further Studies on the Phagocytosis of Photoreceptor Outer Segments by Rat Retinal Pigment Epithelial Cells

The shedding of photoreceptor outer segments (OS) and their phagocytosis by the adjacent retinal pigment epithelium (RPE) is a fundamental process which occurs in all vertebrate species studied to date. Numerous studies have showed that OS shedding and phagocytosis is light entrained and follows a circadian rhythm (1, 2). When this process goes awry, such as in the RCS rat (3–5) and possibly in the vitiligo mouse (6, 7) degeneration of the OS soon follows. There is an accumulating body of evidence (8) that the phagocytosis of OS is a receptor mediated process, whereby a specific receptor on the RPE (the phagocytosis receptor) recognizes a ligand on the surface of the shed OS. After binding to the receptor, the shed OS is internalized by the RPE cell, where it undergoes digestion by lysosomal enzymes.