Combined PKC and MEK inhibition for treating metastatic uveal melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignancy and the second most common form of melanoma. UM has a strong tendency for metastatic disease, and no effective treatments have yet been identified. Activating oncogenic mutations are commonly found in GNAQ and GNA11 in UM, and inhibiting key downstream effectors of the GNAQ/11 signaling pathway represents a rational therapeutic approach for treating metastatic UM. Chen et al., doi:10.1038/onc.2013.418, now confirm activation of the MAPK and PKC pathways as a result of GNAQ and GNA11 activating mutations in melanocytes, and they demonstrate that MAPK activation occurs downstream of PKC activation. PKC inhibitors disrupt MAPK signaling and block proliferation of GNAQ/11 mutant UM cell lines and slow the in vivo growth of xenografted UM tumors without inducing their shrinkage. However, a combination of PKC and MEK inhibition led to sustained MAPK pathway inhibition and tumor regression in vivo. Hence, the authors concluded that MEK and PKC inhibition is synergistic, with superior efficacy to treatment of GNAQ/GNA11 mutant UMs with either drug alone.

p38 phosphorylates Rb on Ser567 by a novel, cell cycle-independent mechanism that triggers Rb-Hdm2 interaction and apoptosis.

The retinoblastoma protein (Rb) inhibits both cell division and apoptosis, but the mechanism by which Rb alternatively regulates these divergent outcomes remains poorly understood. Cyclin-dependent kinases (Cdks) promote cell division by phosphorylating and reversibly inactivating Rb by a hierarchical series of phosphorylation events and sequential conformational changes. The stress-regulated mitogen-activated protein kinase p38 also phosphorylates Rb, but it does so in a cell cycle-independent manner that is associated with apoptosis rather than with cell division. Here, we show that p38 phosphorylates Rb by a novel mechanism that is distinct from that of Cdks. p38 bypasses the cell cycle-associated hierarchical phosphorylation and directly phosphorylates Rb on Ser567, which is not phosphorylated during the normal cell cycle. Phosphorylation by p38, but not Cdks, triggers an interaction between Rb and the human homolog of murine double minute 2 (Hdm2), leading to degradation of Rb, release of E2F1 and cell death. These findings provide a mechanistic explanation as to how Rb regulates cell division and apoptosis through different kinases, and reveal how Hdm2 may functionally link the tumor suppressors Rb and p53.

Treatment outcomes for primary intraocular lymphoma: implications for external beam radiotherapy.

PURPOSE: Intravitreal chemotherapy for primary intraocular lymphoma (PIOL) increasingly is promoted as an alternative to radiotherapy, owing to putative high failure and complication rates of the latter modality. Our aim was to confirm whether these concerns about radiotherapy were borne out in patients treated at our institution over the last decade. DESIGN: Retrospective interventional case series. PARTICIPANTS: A total of 21 eyes of 12 patients with PIOL. METHODS: Comprehensive chart review of ophthalmologic and systemic manifestations, treatments, and outcomes. MAIN OUTCOME MEASURES: Radiation complications and local tumour control. RESULTS: Cytology-confirmed lymphoma involved one eye in three patients and both eyes in nine patients. Initial treatment included external beam radiotherapy and chemotherapy (six patients), chemotherapy alone (four patients), radiotherapy alone (one patient), and no treatment (one patient). Ocular relapses occurred in no patients receiving radiotherapy and in two patients who did not receive radiotherapy. Complications of radiotherapy included dry eye (four patients), cataract (four patients), and mild radiation retinopathy (two patients). CONCLUSIONS: Radiotherapy for PIOL is highly effective with acceptable complications. In the absence of a clear advantage to intravitreal chemotherapy, which involves repetitive injections and associated risks, radiotherapy may still be the most appropriate first-line treatment in most cases.

Association between posterior uveal melanoma and iris freckles, iris naevi, and choroidal naevi.

AIM: To investigate the association between posterior uveal melanoma and iris freckles, iris naevi, and choroidal naevi. METHODS: Cross sectional study of 65 patients with posterior uveal melanoma and 218 controls. Iris colour, iris freckles, iris naevi, and choroidal naevi were recorded for each eye of each patient. RESULTS: Iris freckles were present in 40 (61.5%) patients with melanoma and 135 (61.9%) controls (p = 0.494). Iris naevi were present in four (6.2%) patients with melanoma and nine (4.1%) controls (p = 0.955). Choroidal naevi were present in 12 (18.5%) patients with melanoma and 38 (17.4%) controls (p = 0.815). CONCLUSION: This study did not detect an association between posterior uveal melanoma and iris freckles, iris naevi, or choroidal naevi.

Association between choroidal pigmentation and posterior uveal melanoma in a white population.

BACKGROUND/AIMS: It is well known that light skin pigmentation is a risk factor for cutaneous melanoma. The aim of this study was to investigate the analogous association between choroidal pigmentation and posterior uveal melanoma. METHODS: Cross sectional study of 65 consecutive patients diagnosed with posterior uveal melanoma (melanoma group) and 218 consecutive patients referred for general retinal evaluation (control group). All patients were white. A clinical grading system for estimating choroidal pigmentation was developed and histologically validated in seven patients. RESULTS: Melanoma patients with light iris colour were significantly more likely to have darker choroidal pigmentation than controls (p = 0.005). Darker choroidal pigmentation was associated histologically with increased density of choroidal melanocytes (p = 0.005). CONCLUSIONS: Increased choroidal pigmentation, as a result of an increase in the density of pigmented choroidal melanocytes, is not protective but may actually be a risk factor for the development of posterior uveal melanoma in white patients. This finding may have implications for understanding the pathogenesis of uveal melanoma.

Molecular basis of low-penetrance retinoblastoma.

Retinoblastoma is a malignant tumor of the retina that occurs primarily in young children as a result of mutations in the retinoblastoma gene (RB), the first tumor suppressor gene to be identified. In about 35% to 40% of patients with retinoblastoma, an RB gene mutation is present in the germline, resulting in hereditary transmission of the disease. Most families with hereditary retinoblastoma demonstrate autosomal dominant inheritance with almost complete penetrance and high expressivity. However, some families display an inheritance pattern characterized by reduced penetrance and expressivity. Recent advances in our understanding of the structure and function of the retinoblastoma protein (pRB) now provide new insights into the molecular basis of this low-penetrance form of retinoblastoma. Low-penetrance retinoblastoma mutations either cause a reduction in the amount of normal pRB that is produced (class 1 mutations) or result in a partially functional mutant pRB (class 2 mutations).

The molecular biology of retinoblastoma.

Retinoblastoma, a rare pediatric eye tumor, has served as an important model for the heritable predisposition to cancer. The retinoblastoma protein, Rb, functions as a tumor suppressor by controlling progression through the cell cycle. Rb function is regulated primarily by its phosphorylation state, which is determined by the complex interaction of multiple kinases and their inhibitors that together form the 'Rb pathway'. This pathway has been found to be functionally inactivated in almost all types of cancer. Despite recent advances in our understanding of Rb function, the precise role of Rb loss in the development of retinoblastoma remains unclear. Recent work in genetically altered mice has suggested that an additional mutation in another gene is required for retinal tumor formation. An alternative model presented here is based on the noncell-autonomous functions of Rb contributing to tumorigenesis.

Deregulation of the Rb and p53 pathways in uveal melanoma.

Uveal melanoma is the most common primary eye cancer, yet its molecular pathogenesis is poorly understood. In this study, we investigated the immunohistochemical expression of proteins in the Rb and p53 tumor suppressor pathways in 33 uveal melanomas from enucleated eyes. Strong nuclear staining for Rb was present in most tumors. However, a few cases displayed weak nuclear staining and strong cytoplasmic staining (possibly indicating Rb mutation), and this aberrant staining correlated strongly with failed radiotherapy or thermotherapy before enucleation. Staining for cyclin D1 was positive in most tumors and was associated with advanced age and larger tumor size, which are both poor prognostic factors. Generally, immunostaining for p53 was weak (suggesting a lack of p53 mutations), although p53 positivity correlated strongly with staining for phosphorylated Rb, supporting the notion that inappropriate phosphorylation of Rb can induce p53. Strong immunostaining for MDM2, which can functionally block p53 activity, was observed in most tumors and correlated significantly with female sex. Strong cytoplasmic staining was observed for Bcl2, which can inhibit both p53-dependent and -independent apoptosis. We conclude that Rb and p53 are mutated infrequently in uveal melanoma, but their respective pathways may be functionally inactivated.

Chromatin remodeling and Rb activity.

Progression of cells through the cell cycle is central to normal cell proliferation, and checkpoints that regulate this cycle are targets of tumorigenic mutations. One of these checkpoints is the Rb family of proteins that seems to regulate exit of cells from both G(1) and S phase of the cell cycle. Recent studies have linked the function of the Rb family to chromatin remodeling enzymes.

Inactivation of retinoblastoma protein in uveal melanoma by phosphorylation of sites in the COOH-terminal region.

Uveal melanoma is the most common malignancy of the eye, but little is known about its underlying genetic defects. Melanomas of uveal origin, unlike those of the skin, are rarely familial and have not been linked consistently to mutations in tumor suppressor genes. Here, we investigated the Rb pathway in uveal melanoma. Most tumors displayed strong immunostaining for Rb and p16, suggesting that they were not mutationally inactivated. However, Rb was frequently phosphorylated at serine-807 and serine-811, and cyclin D1 was expressed in many of the tumors. Mutation of these serine residues prevented cyclin D-dependent phosphorylation from inactivating Rb in cultured cells. We conclude that Rb is frequently inactivated in uveal melanoma by phosphorylation of residues in the COOH-terminal region that regulate its activity, and one mechanism for this phosphorylation is overexpression of cyclin D.

Exit from G1 and S phase of the cell cycle is regulated by repressor complexes containing HDAC-Rb-hSWI/SNF and Rb-hSWI/SNF.

We present evidence that Rb forms a repressor containing histone deacetylase (HDAC) and the hSWI/SNF nucleosome remodeling complex, which inhibits transcription of genes for cyclins E and A and arrests cells in the G1 phase of the cell cycle. Phosphorylation of Rb by cyclin D/cdk4 disrupts association with HDAC, relieving repression of the cyclin E gene and G1 arrest. However, the Rb-hSWI/SNF complex persists and is sufficient to maintain repression of the cyclin A and cdc2 genes, inhibiting exit from S phase. HDAC-Rb-hSWI/SNF and Rb-hSWI/SNF then appear to maintain the order of cyclin E and A expression during the cell cycle, which in turn regulates exit from G1 and from S phase, respectively.

Rb function in cell-cycle regulation and apoptosis.

Loss of cell-cycle control is a hallmark of neoplastic cells. One regulator of the critical G1 to S-phase transition in the cell cycle is the retinoblastoma tumour suppressor protein Rb, which interacts with the E2F family of cell-cycle transcription factors to repress gene transcription required for this transition. Through its interaction with E2F, Rb also regulates genes that control apoptosis. Here we review the roles of Rb in regulating the cell cycle and apoptosis and discuss recent results linking these Rb functions to chromatin-remodelling enzymes.

Tumor suppressor genes in ophthalmology.

Tumor suppressor genes have a diversity of functions, but they have in common the property of inhibiting neoplastic transformation. When they become inactivated, a constraint is removed that allows cells to grow inappropriately. Mutations in these genes are now thought to be the initiating events in most cancers. The first tumor suppressor gene was discovered through its role in retinoblastoma, and many other tumor suppressor genes also have important ophthalmic manifestations. The first group of tumor suppressor genes to be discussed are those involved in retinoblastoma and uveal melanoma. These are among the most frequently mutated genes in human cancer and are key regulators of growth and homeostasis. The second group of genes is associated with specific hereditary tumor syndromes with ophthalmic manifestations. These genes function in a variety of molecular pathways and are associated with neoplastic and non-neoplastic abnormalities in restricted tissue distributions. Research on tumor suppressor genes continues to shed light on the molecular pathophysiology of ophthalmic tumors and will increasingly yield diagnostic and therapeutic applications.

Cdk phosphorylation triggers sequential intramolecular interactions that progressively block Rb functions as cells move through G1.

We present evidence that phosphorylation of the C-terminal region of Rb by Cdk4/6 initiates successive intramolecular interactions between the C-terminal region and the central pocket. The initial interaction displaces histone deacetylase from the pocket, blocking active transcriptional repression by Rb. This facilitates a second interaction that leads to phosphorylation of the pocket by Cdk2 and disruption of pocket structure. These intramolecular interactions provide a molecular basis for sequential phosphorylation of Rb by Cdk4/6 and Cdk2. Cdk4/6 is activated early in G1, blocking active repression by Rb. However, it is not until near the end of G1, when cyclin E is expressed and Cdk2 is activated, that Rb is prevented from binding and inactivating E2F.

Overview of RB gene mutations in patients with retinoblastoma. Implications for clinical genetic screening.

OBJECTIVE: This study aimed to determine the distribution of germline mutations in the retinoblastoma (RB) gene in patients with retinoblastoma to design more effective genetic testing. DESIGN: A meta-analysis. PARTICIPANTS: 192 cases identified from literature. METHODS: All identifiable reported cases of bilateral retinoblastoma, which included DNA sequence analysis of the RB gene, were reviewed. MAIN OUTCOME MEASURE: Type of genetic mutation. RESULTS: Among 192 patients with retinoblastoma with identifiable germline mutations in the RB gene, the DNA alteration was a nonsense mutation in 83 (43%), frameshift in 67 (35%), intron mutation in 23 (12%), missense mutation in 11 (6%), in-frame deletion in 5 (3%), and promoter mutation in 3 (2%). Mutations were distributed throughout 24 of the 27 exons of the RB gene with no single mutational "hotspot." Exons 8, 17, 18, and 23 were involved most often, and 189 (98%) of the mutations were predicted to affect the RB large pocket domain. CONCLUSIONS: A single genetic test is unlikely to detect all germline RB gene mutations in patients with retinoblastoma because of the variety of types and locations of mutations that occur. However, a series of complementary tests may be able to rapidly detect mutations based on the observation that most mutations alter the protein size and disrupt the large pocket domain.

Metastatic risk for distinct patterns of postirradiation local recurrence of posterior uveal melanoma.

OBJECTIVE: The purpose of the study is to compare the prognostic significance of horizontal/marginal versus vertical/diffuse patterns of postirradiation local recurrence of posterior uveal melanoma. DESIGN: The study design was a nonrandomized, retrospective clinical study. Semiparametric and nonparametric statistical techniques were used. PARTICIPANTS: Seven hundred sixty-six posterior uveal melanoma patients were studied. INTERVENTION: Either iodine-125 plaque or helium ion radiation therapy was performed. MAIN OUTCOME MEASURES: Local tumor recurrence and systemic metastasis were measured. RESULTS: Local tumor recurrence was detected in 66 (8.6%) of 766 irradiated tumors. The 5-year actuarial rate of local recurrence was 10%. The recurrence pattem was horizontal/marginal in 27 patients (41%) and vertical/diffuse in 39 patients (59%). Systemic metastasis was detected in 5 patients (19%) with horizontal/marginal recurrence and in 19 patients (49%) with vertical/diffuse recurrence. After known metastatic risk factors were controlled, the relative risk for metastasis was 2.2 for horizontal/marginal recurrence and 5.1 for vertical/diffuse recurrence (P = 0.05). The actuarial rate of systemic metastasis was 2.9% per year for all patients, 6.3% per year for patients with horizontal/marginal recurrence, and 15.5% per year for patients with vertical/diffuse recurrence. CONCLUSIONS: Postirradiation local recurrence of posterior uveal melanoma is a risk factor for systemic metastasis. Vertical/diffuse recurrences may be associated more strongly with metastatic disease than horizontal/marginal recurrences.