DNA hypermethylation/boundary control loss identified in retinoblastomas associated with genetic and epigenetic inactivation of the RB1 gene promoter.

DNA hypermethylation events occur frequently in human cancers, but less is known of the mechanisms leading to their initiation. Retinoblastoma, an intraocular cancer affecting young children, involves bi-allelic inactivation of the RB1 gene (RB-/-). RB1 encodes a tumour suppressing, cell cycle regulating transcription factor (pRB) that binds and regulates the RB1 core and other E2F responsive promoters with epigenetic functions that include recruitment of histone deacetylases (HDACs). Evidence suggests that bi-allelic epigenetic inactivation/hypermethylation of the RB1 core promoter (PrE-/E-), is specific to sporadic retinoblastomas (frequency~10%), whereas heritable RB1 promoter variants (Pr-/+, frequency~1-2%) are not associated with known epigenetic phenomena. We report heritable Pr-/- retinoblastomas with the expected loss of pRB expression, in which hypermethylation consistent with distal boundary displacement (BD) relative to normal peripheral blood DNAs was detected in 4/4 cases. In contrast, proximal BD was identified in 16/16 RB-/- retinoblastomas while multiple boundaries distal of the core promoter was further identified in PrE-/E-and PrE-/E+ retinoblastomas. However, weak or no DNA hypermethylation/BD in peripheral blood DNA was detected in 8/9 Pr-/+ patients, with the exception, a carrier of a microdeletion encompassing several RB1 promoter elements. These findings suggest that loss of boundary control may be a critical step leading to epigenetic inactivation of the RB1 gene and that novel DNA methylation boundaries/profiles identified in the RB1 promoter of Pr-/- retinoblastomas, may be the result of epigenetic phenomena associated with epimutation in conjunction with loss of pRB expression/binding and/or RB1 promoter interactions with boundary control elements.

Socioeconomic and psychological impact of treatment for unilateral intraocular retinoblastoma.

PURPOSE: To identify the socioeconomic and psychosocial impacts of clinical treatment decisions for advanced unilateral intraocular retinoblastoma. DESIGN: Retrospective observational case series. SETTING: institutional study at Alexandria Main University Hospital. STUDY POPULATION: records of 66 unilateral retinoblastoma cases treated from May 2005 to May 2013 were retrospectively reviewed. Sixty cases were eligible (International Intraocular Retinoblastoma Classification [IIRC] group C, D or E). PROCEDURES: two treatment groups were compared: enucleation vs. salvage treatment. Salvage treatment eyes were further subdivided based on IIRC group. Six socioeconomic parameters (financial burden, financial impact, psychological, social, medical and tumor impacts) were scored. Parameter scores ranged from 0 to 3, for overall score range 0 (no adverse impact) to 18 (severe adverse impact). MAIN OUTCOME MEASURES: derived Socioeconomic scores were correlated with treatment and outcomes. RESULTS: The enucleation group (28 eyes) had a median overall Socioeconomic score of 4/18, significantly lower than the salvage treatment group (32 eyes), median score 11/18 (P<0.01). Socioeconomic score varied with IIRC group. Attempted eye salvage failed in 25 children, due to uncontrolled tumor (44%) and socioeconomic impact of cumulative therapies (56%). Treatment duration and Socioeconomic score were higher for the 5 children in the salvage treatment group who developed metastatic disease compared to those without metastasis (P<0.01). CONCLUSIONS: The socioeconomic and psychosocial impacts of attempted ocular salvage for unilateral intraocular retinoblastoma are severe, in comparison to primary enucleation. Primary enucleation is a good treatment for unilateral retinoblastoma.

Ultrasound biomicroscopy in the management of retinoblastoma.

PURPOSE: To determine the role of ultrasound biomicroscopy (UBM) in the management of children affected with retinoblastoma. METHODS: A review of clinical records of children with the diagnosis of retinoblastoma at the Hospital for Sick Children from January 1995 to December 2007, for whom UBM was used to determine the extent of intraocular tumor. Clinical characteristics were compared with UBM. Pathological correlation was performed for enucleated eyes. RESULTS: In total, 101 eyes of 75 patients were included in the final analysis. Only 11 eyes were diagnosed on UBM to have extension of the tumor anterior to the ora serrata, and were enucleated. Histopathological examination confirmed the anterior extension in all the 11 eyes. In total, 50 eyes were enucleated because of various reasons, such as poor visual prognosis (12 eyes), unilateral group D or E (23 eyes), recurrences (8 eyes), and treatment failure (7 eyes). None of those patients were found to have anterior extension of the disease on histopathological examination. UBM did not yield any false negative (0/50) or any false positives (0/11). CONCLUSIONS: The UBM provided a sensitive and reproducible visualization of the anterior retina, ciliary region, and anterior segment allowing a better staging of the advanced disease process. Primary assessment of the true extent of retinoblastoma is critical for the selection of an optimal management approach.

Hereditary haemorrhagic telangiectasia: mutation detection, test sensitivity and novel mutations.

BACKGROUND: Hereditary haemorrhagic telangiectasia (HHT) is a genetic disorder present in 1 in 8000 people and associated with arteriovenous malformations. Genetic testing can identify individuals at risk of developing the disease and is a useful diagnostic tool. OBJECTIVE: To present a strategy for mutation detection in families clinically diagnosed with HHT. METHODS: An optimised strategy for detecting mutations that predispose to HHT is presented. The strategy includes quantitative multiplex polymerase chain reaction, sequence analysis, RNA analysis, validation of missense mutations by amino acid conservation analysis for the ENG (endoglin) and ACVRL1 (ALK1) genes, and analysis of an ACVRL1 protein structural model. If no causative ENG or ACVRL1 mutation is found, proband samples are referred for sequence analysis of MADH4 (associated with a combined syndrome of juvenile polyposis and HHT). RESULTS: Data obtained over the past eight years were summarised and 16 novel mutations described. Mutations were identified in 155 of 194 families with a confirmed clinical diagnosis (80% sensitivity). Of 155 mutations identified, 94 were in ENG (61%), 58 in ACVRL1 (37%), and three in MADH4 (2%). CONCLUSIONS: For most missense variants of ENG and ACVRL1 reported to date, study of amino acid conservation showed good concordance between prediction of altered protein function and disease occurrence. The 39 families (20%) yet to be resolved may carry ENG, ACVRL1, or MADH4 mutations too complex or difficult to detect, or mutations in genes yet to be identified.

Loss of p75 neurotrophin receptor expression accompanies malignant progression to human and murine retinoblastoma.

We studied the expression of pro-apoptotic neurotrophin receptor p75 (p75(NTR)) in human and murine retinoblastoma, compared to normal retina, and examined changes in p75(NTR) expression with the onset of apoptosis in the course of murine retinoblastoma progression, using immunohistochemistry and quantitative real-time RT-PCR. The murine retinoblastoma is induced by retinal specific expression of SV40 T-antigen (TAg), which blocks the function of the retinoblastoma protein (pRB) and related proteins, and is a well-studied model that closely simulates human retinoblastoma. The majority of human retinoblastoma either lacked or expressed decreased levels of p75(NTR) mRNA, compared to human retina. Moreover, p75(NTR) protein was not detected in any tumor studied, unlike normal retina. Like human retinoblastoma, advanced murine retinoblastoma did not express p75(NTR). However, before tumors emerged, small clusters of TAg-positive cells coexpressed p75(NTR) and activated caspase-3, a marker of apoptosis. Furthermore, in three rare human eyes containing retinoblastoma adjacent to regions resembling the benign retinal tumor retinoma, both normal retina and retinoma-like tissue expressed p75(NTR) protein, while the retinoblastoma did not. We suggest that p75(NTR) loss accompanies progression from retinoma to retinoblastoma.

Minimal regions of chromosomal imbalance in retinoblastoma detected by comparative genomic hybridization.

Mutation of both alleles of the retinoblastoma gene (RB1) initiate oncogenesis in developing human retina, but other common genomic alterations are present in the tumors. In order to sublocalize the altered genomic regions, 50 retinoblastoma tumors were examined by comparative genomic hybridization (CGH). The minimal regions most frequent gained were 1q31 (52%), 6p22 (44%), 2p24-p25 (30%) and 13q32-q34 (12%). The minimal region most frequently lost was 16q22 (14%). The overall total number of gains or losses evident on CGH was significantly greater in those tumors with either or both 6p or 1q gain, than in tumors with neither 6p nor 1q gain suggesting that chromosomal instability may be associated with acquisition of these changes. Genes mapping to 6p22 and 1q31 may be important in tumor development in retina subsequent to the loss of RB1 alleles.

Retinoblastoma: the disease, gene and protein provide critical leads to understand cancer.

Retinoblastoma has contributed much to the understanding of cancer. The protein product of the RB gene, pRB, is a multifaceted regulator of transcription which controls the cell cycle, differentiation and apoptosis in normal development of specific tissues. Elucidating the mechanisms in which pRB plays a critical role will enable novel therapies and strategies for prevention, not only for retinoblastoma, but for cancer in general.

Focal therapy in the management of retinoblastoma: when to start and when to stop.

PURPOSE: To describe the treatment effects and side effects of different modalities of focal treatment used for retinoblastoma. METHODS: Green (532-nm) laser, continuous wave Nd:YAG (1064-nm) laser, transpupillary or transcleral (810-nm) laser, and cryotherapy were used in the treatment of 46 eyes in 35 patients affected with retinoblastoma. The number of treatment sessions and the amount of energy applied were recorded in an attempt to determine the amount of energy required to adequately treat tumors of various sizes. In addition, we have attempted to determine when treatment becomes overtreatment and is likely to lead to complications. RESULTS: The treatment endpoint for laser in this study was calcific, gliotic, or flat scars. Small tumors (<2 mm in height, <4 DD) were successfully treated in 3 or fewer sessions of 532-nm laser. Anterior small tumors were successfully treated with transcleral 810-nm laser or cryotherapy. Medium tumors, between 2.0 and 4.0 mm in thickness, required 2 to 9 treatments to achieve a good response and often required the addition of chemotherapy to reduce the size of the tumor before or during laser treatment. Large tumors required chemotherapy combined with many laser treatments for complete control. Complications associated with excessive laser were vitreous condensation with traction, vitreous hemorrhage, retinal detachment, tumor break, cataract formation, and iris burns. CONCLUSION: Laser treatment alone for small tumors, and combined with cryotherapy and chemotherapy for larger tumors, is effective in the treatment of retinoblastoma. Complications of focal therapy can most often be avoided by using the minimal effective laser power.

[Detection of RB1 mutations by using quantitative fluorescent mutiplex PCR].

OBJECTIVE: To develop a half-automatic, simple, non-radioactive technique for rapid detection of mutation in the RB gene. METHODS: Quantitative fluorescent multiplex PCR (QFM-PCR) involves amplification of the promoter region and all 27 exons of the RB1 gene with fluorescein labeled primers in multiplex sets. Primers were divided into multiplex sets of three to seven pairs of primers, also contained were internal control primers C4. Four external controls were also tested by using nullisomic, monosomic, diploid, and trisomic for RB1. The number of copies of a fragment in a test sample was calculated by comparing fluorescence intensity of the fragment with these standards. Fragment value detection and subsequent calculations were performed automatically by Fragment Manager 2.1 Software. RESULTS: Small deletions, insertions and whole exon deletion in the RB1 gene were detected from genomic DNA. Sequencing analysis and RB tumor homozygous mutation comfirmed the defects detected by QFM-PCR. CONCLUSION: The approach is a rapid, cost-effective initial method for screening patient samples. It has been used to identify approximately 50% positives of tested samples. The mutations shown were not only small deletion and insertion, but also the single copy exons heterozygous mutation in the RB1 gene which the previously used methods were unable to detect. These features make the technique an attractive approach to clinical diagnosis of gene defects.

Nuclear localization conferred by the pocket domain of the retinoblastoma gene product.

The tumor suppressor Rb is a nuclear phosphoprotein that controls cell growth and differentiation by modulating the activity of certain transcription factors. Transport of Rb to the nucleus is affected by both a bipartite nuclear localization signal (NLS) in the C-terminus of the protein and a central domain, termed A/B or pocket, through which Rb interacts with transcription factors and viral oncoproteins. Mutations in either the A or B subdomains of the pocket render a NLS-deficient Rb completely cytoplasmic. Fusing the A/B domain of Rb to the Escherichia coli beta-galactosidase, to create betagal-A/B, confers nuclear localization upon this bacterial protein. Moreover, co-expression with the adenovirus oncoprotein, E1A, further augments nuclear localization of betagal-A/B. These findings provide direct evidence that the pocket domain of Rb is not only required but also sufficient to induce nuclear transport by a 'piggyback' mechanism. Thus, nuclear localization of Rb is dictated by two independent and autonomous domains: (i) the bipartite NLS and (ii) the pocket domain. We suggest that via these domains, Rb chaperons and co-compartmentalizes with its associated factors and preempts their activity prior to nuclear transport.

Egocentric localization: visually directed alignment to projected head landmarks in binocular and monocular observers.

PURPOSE: To determine the accuracy and precision with which humans align moving, non-moving, point-like and linear stimuli to three head landmarks projected to a frontal plane. Monocularly enucleated and binocularly normal subjects, performing monocularly, were tested. METHODS: Experimental tasks consisted of aligning the stimulus, a luminescent target, using a remote steering wheel control, to three projected landmarks; the nose, and the outside edge of each ear. Experiments were performed with a fixed [static] linear stimulus and a small dynamic stimulus, and then with a fixed stimulus at both a very close and an intermediate distance. RESULTS: The data from the monocular enucleated observers showed better accuracy in some conditions but their variable errors were larger than those of the controls. CONCLUSIONS: We propose that, for the alignments of the enucleated monocular subjects, a) the increase in accuracy is consistent with a shift of the egocenter in the direction of the remaining eye, and b) the decrease in precision may be due to the fact that the egocenter does not correspond to the mid-sagittal or median plane. This disparity would require adjustments to be learned in order to perform tasks involving visually directed alignments to the self.

Cumulative effect of phosphorylation of pRB on regulation of E2F activity.

The product of the retinoblastoma susceptibility gene, pRB, is a nuclear phosphoprotein that controls cell growth by binding to and suppressing the activities of transcription factors such as the E2F family. Transactivation activity is inhibited when E2F is bound to hypophosphorylated pRB and released when pRB is phosphorylated by cyclin-dependent kinases (CDKs). To determine which of 16 potential CDK phosphorylation sites regulated the pRB-E2F interaction, mutant pRB proteins produced by site-directed mutagenesis were tested for the ability to suppress E2F-mediated transcription in a reporter chloramphenicol acetyltransferase assay. Surprisingly, no one CDK site regulated the interaction of pRB with E2F when E2F was bound to DNA. Instead, disruption of transcriptional repression resulted from accumulation of phosphate groups on the RB molecule.

Developmental basis of retinal-specific induction of cancer by RB mutation.

Understanding why children with RB mutations specifically develop retinoblastoma will contribute to the understanding of the fundamental principles of cancer. Only a subset of developing retinal cells are at risk for developing cancer when RB is mutant because rod photoreceptor and bipolar cells never normally express RB. Retinoblastomas are observed to arise commonly in the inner nuclear layer, where they can show features attributed to outer nuclear layer cells (photoreceptors). The best-studied function of RB is control of the cell cycle, and the usual tissue consequence of loss of RB is apoptosis. Perhaps the specificity of RB mutation for retinal cancer resides in the dependency of this tissue on programmed cell death to achieve a precise architecture of individual types of interconnecting neurons. The additional mutations that are present in all retinoblastoma, such as the i(6p) marker chromosome, may interrupt signals that normally would induce apoptosis when RB is absent. A combination of loss of cell cycle control and loss of signals that delete extra cells would result in retinoblastoma.

Frequency of somatic and germ-line mosaicism in retinoblastoma: implications for genetic counseling.

Although mosaicism can have important implications for genetic counseling of families with hereditary disorders, information regarding the incidence of mosaicism is available for only a few genetic diseases. Here we describe an evaluation of 156 families with retinoblastoma; the initial oncogenic mutation in the retinoblastoma gene had been identified in these families. In 15 ( approximately 10%) families, we were able to document mosaicism for the initial mutation in the retinoblastoma gene, either in the proband or in one of the proband's parents. The true incidence of mosaicism in this group of 156 families is probably higher than our findings indicate; in some additional families beyond the 15 we identified, mosaicism was likely but could not be proven, because somatic or germ-line DNA from key family members was unavailable. Germ-line DNA from two mosaic fathers was analyzed: in one of these, the mutation was detected in both sperm and leukocyte DNA; in the other, the mutation was detected only in sperm DNA. Our data suggest that mosaicism is more common than is generally appreciated, especially in disorders such as retinoblastoma, in which a high proportion of cases represent new mutations. The possibility of mosaicism should always be considered during the genetic counseling of newly identified families with retinoblastoma. As demonstrated here, genetic tests of germ-line DNA can provide valuable information that is not available through analysis of somatic (leukocyte) DNA.

Visual prognosis of Coats' disease.

PURPOSE: Our purpose was to determine the visual prognosis of retinal telangiectasia (Coats' disease). METHODS: We performed a retrospective review of 35 patients with Coats' disease seen at the Hospital for Sick Children, Toronto, Canada between 1987 and 1996. Ten patients were excluded because of incomplete records. Treatment modalities consisted of no treatment, cryotherapy with and without 532 nm laser through the indirect ophthalmoscope, and enucleation. Visual outcome was determined where possible. RESULTS: Median follow-up was 4.5 years. Deterioration in visual acuity was associated with the presence of greater than 5 clock hours of involved retina and retinal detachment at diagnosis. Final visual acuity did not correlate with age of onset of disease. No eye treated with cryotherapy progressed to retinal detachment. CONCLUSIONS: Aggressive treatment of Coats' disease with cryotherapy with or without 532 nm laser, before retinal detachment, is likely to stabilize vision and decrease the risk of future total retinal detachment.

Deletion of RB exons 24 and 25 causes low-penetrance retinoblastoma.

A deletion in the tumor-suppressor gene, RB, discovered by quantitative multiplex PCR, shows low penetrance (LP), since only 39% of eyes at risk in this family develop retinoblastoma. The 4-kb deletion spanning exons 24 and 25 (delta24-25) is the largest ever observed in an LP retinoblastoma family. Unlike the usual RB mutations, which cause retinoblastoma in 95% of at-risk eyes and yield no detectable protein, the delta24-25 allele transcribed a message splicing exon 23 to exon 26, resulting in a detectable protein (pRBdelta24-25) that lacks 58 amino acids from the C-terminal domain, proving that this domain is essential for suppression of retinoblastoma. Two functions were partially impaired by delta24-25-nuclear localization and repression of E2F-consistent with the idea that LP mutations generate "weak alleles" by reducing but not eliminating essential activities. However, delta24-25 ablated interaction of pRB with MDM2. Since a homozygous LP allele is considered nontumorigenic, the pRB/MDM2 interaction may be semi- or nonessential for suppressing retinoblastoma. Alternatively, some homozygous LP alleles may not cause tumorigenesis because an additional event is required (the "three-hit hypothesis"), or the resulting imbalance in pRB function may cause apoptosis (the "death allele hypothesis"). pRBdelta24-25 was also completely defective in suppressing growth of Saos-2 osteosarcoma cells. Targeting pRBdelta24-25 to the nucleus did not improve Saos-2 growth suppression, suggesting that C-terminal domain functions other than nuclear localization are essential for blocking proliferation in these cells. Since delta24-25 behaves like a null allele in these cells but like an LP allele in the retina, pRB may use different mechanisms to control growth in different cell types.

Expression patterns of the E2F family of transcription factors during mouse nervous system development.

The E2F family of transcription factors consists of two subgroups termed E2F and DP. E2F is required for cell proliferation, and is necessary for fruit fly development. E2F activity is a target for regulation by the retinoblastoma gene family, which includes pRB, p107 and p130. Mutant RB-/-, RB-/-:p107-/- and p107-/-:p130-/- mice develop abnormally, probably as a result of dysregulation in the activity of E2F, indicating the importance of E2F in mammalian development. To investigate the role of E2F in murine development, we have examined the patterns of expression of E2F-1 through E2F-5, and DP-1 in the developing nervous system by in situ hybridization. E2F-1, E2F-2 and E2F-5 are first detected in the 9.5 days post-coitus (dpc) forebrain. Expression of these E2F forms extends caudally thereafter and includes the developing brain and the upper half of the 10.5 dpc spinal cord. By 11.5 dpc, these E2F factors are expressed throughout the central nervous system. In 12.5 dpc embryos, E2F-1, E2F-2 and E2F-5 are highly expressed in proliferating, undifferentiated neuronal precursors. As neurons differentiate and migrate to the outer marginal zones in the nervous system, expression of these E2F members is extinguished. In the developing retina, another neuronal tissue, E2F-1 expression is also confined to the proliferating, undifferentiated retinoblastic layer. In contrast, E2F-3 expression is up-regulated as retinoblasts differentiate into the ganglion cell layer. In non-neuronal tissues, high E2F-4 transcript levels are present in regions corresponding to proliferative chondrocytes, whereas E2F-2 and E2F-4 transcripts are very abundant in the thymic cortex, which contains immature thymocytes. We conclude that individual E2F forms are differentially regulated during the development of distinct tissues, and especially during neuronal development.

Multidrug resistance protein (MRP) expression in retinoblastoma correlates with the rare failure of chemotherapy despite cyclosporine for reversal of P-glycoprotein.

Failure of chemotherapy associated with expression of the multidrug resistance protein p170 frequently occurs in retinoblastoma (RB). Despite using cyclosporine, which inhibits p170 and improves our chemotherapy results, rare failures occur. In nonmetastatic primarily enucleated RBs, we show expression of p170 in 3 of 18 samples and expression of multidrug resistance protein (MRP), the second protein associated with resistance to chemotherapy, in 1 of 18 samples. All three RBs that failed chemotherapy without cyclosporine expressed MRP with p170. All three RBs that were enucleated immediately when chemotherapy failed despite the addition of cyclosporine expressed only MRP. One RB enucleated 2 years after failing chemotherapy with cyclosporine, despite radiation and salvage chemotherapy, expressed both p170 and MRP. Two metastatic RBs that expressed both p170 and MRP at diagnosis and at recurrence failed chemotherapy without cyclosporine, whereas one metastatic RB that expressed neither protein was cured by chemotherapy without cyclosporine. MRP may result in failure of chemotherapy despite the elimination of p170-expressing clones by cyclosporine.