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.

Focal stereotactic external beam radiotherapy as a vision-sparing method for the treatment of peripapillary and perimacular retinoblastoma: preliminary results.

AIMS: Chemotherapy with aggressive focal ablative therapy is now the mainstay of retinoblastoma therapy. Our experience presents an evolution from conventional radiotherapy by treating posterior pole tumours with focal stereotactic fractionated radiotherapy (SRT). MATERIALS AND METHODS: A retrospective chart review was conducted of five patients (six eyes) treated with SRT at the Hospital for Sick Children and Princess Margaret Hospital, Toronto, Canada, between 1999 and 2004. The prescribed dose was 40 Gy delivered in 20 fractions once daily using 6 MV photons. RESULTS: Five patients (six eyes) were treated. The median age at the time of SRT was 18 months. The median follow-up was 46.5 months as of September 2004. Four patients were treated for a posterior pole focal tumour by focal SRT, and one patient was treated for vitreous seeding with whole-eye SRT. In patients treated with focal SRT, the median doses to the tumour, optic chiasm and brainstem were 41.92, 0.25 and 0.07 Gy, respectively, and to the ipsilateral optic nerve, globe and lens were 9.98, 19.11 and 3.74 Gy, respectively. The median doses to the ipsilateral and contralateral orbital bone were 6.73 Gy (range 5.99-8.29 Gy) and 2.31 Gy (range 0.88-7.08 Gy), respectively. A complete response (residual inactive scar tissue) was seen in four of the five focal tumours treated, with one tumour responding with a partial response (suspicious residual scar tissue). No acute or late side-effects occurred in patients treated with focal SRT. Only the patient treated with whole-eye SRT developed late effects of cataract and corneal ulceration. One patient suffered recurrence within the radiation field 5 months after focal SRT. Control of this recurrence was successful using chemotherapy and focal therapy. No eye has been enucleated. CONCLUSION: Vision-sparing focal SRT for localised tumour masses in critical locations can control tumours with minimal side-effects and a minimal dose to the surrounding critical normal tissue.

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.

Spectrum of gross deletions and insertions in the RB1 gene in patients with retinoblastoma and association with phenotypic expression.

Quantitative multiplex PCR and genomic real-time PCR were used to complete an RB1 mutation analysis in 57 of 433 and 72 of 262 patients with hereditary and isolated unilateral retinoblastoma, respectively. These patients were selected because in previous analyses, which focused mainly on the identification of point mutations, no RB1 mutation was found. We identified gross deletions and insertions in peripheral blood DNA from 26 of 57 patients (46%) with hereditary retinoblastoma, and in six of 72 patients (8.3%) with isolated unilateral disease. In addition, we identified 32 somatic mutations in tumor DNA from 31 of 72 patients (43%) with isolated unilateral retinoblastoma. Together with our previous results, we found that gross RB1 alterations were present in the peripheral blood DNA from 65 of 433 (15%) and 17 of 262 (6.5%) patients with bilateral or familial and isolated unilateral retinoblastoma, respectively. Including reported gross deletions, an analysis of the frequency of breakpoints per intron length shows higher densities in introns 13, 16, 23, and 24. Genotype-phenotype analyses showed that on the whole, carriers of gross deletions develop fewer retinoblastomas compared to patients who are heterozygous for other types of RB1 null mutations. Specifically, carriers of cytogenetic and submicroscopic whole gene deletions often have unilateral tumors only. By contrast, almost all patients with gross deletions with one breakpoint in RB1 have bilateral retinoblastoma.

A retrospective review of visual outcome and complications in the treatment of retinoblastoma.

The aim of this study was to look at the visual outcome and treatment complications of children diagnosed with Retinoblastoma during the years 1985-2003 inclusive. A retrospective review of all patients records was performed. Patient characteristics, treatment methods and complications were recorded. Twenty eight children presented to Temple street Hospital between 1985-2003. Six of these infants had bilateral tumours. The mean age at presentation was 23.7 months. Sixty-nine percent presented with Leucocoria, of these 33% also had a squint. The mean duration of symptoms was only known in 58% and this figure was approximately 19.8 months. Enucleation was performed in 24 eyes of 24 patients. Three patients required adjuvant chemotherapy post enucleation. Two eyes was treated with external beam radiation and one eye with plaque radiotherapy. One eye (second eye) was treated with systemic chemotherapy and radiation. Five eyes of three patients were treated with systemic chemotherapy followed by adjuvant Argon laser, cryotherapy and diode laser to each eye.The complications of each treatment group was recorded. The visual outcome in the salvaged eyes was favourable. There were no deaths recorded. Though chemotherapy with adjuvant local treatments provide adequate treatment for early tumours, enucleation still plays a major role in the treatment of Retinoblastoma. The total eye salvage rate in this study was 29% with an enucleation rate of 90% in unilateral cases and 33% in bilateral cases. Sixty-six percent of bilateral eyes affected were salvaged. Seventy-one percent of tumours were diagnosed after a parent noticed a gross abnormality of the eye. This highlights the possible need for screening for retinoblastoma in the infant population.

Visual results in children treated for macular retinoblastoma.

PURPOSE: To evaluate the results of patching treatment in children with macular retinoblastoma in one eye. METHODS: Fifteen children affected by macular retinoblastoma received instructions for patching treatment for amblyopia. Data were collected on age at diagnosis of the tumor, presence of unilateral or bilateral disease, area of posterior pole involvement by the scar of the regressed tumor and its relationship to the fovea; and the onset, duration, and compliance of patching. The visual acuities recorded were expressed in logMAR (logarithm minimum angle of resolution) equivalents. RESULTS: Twelve children (80%) had bilateral retinoblastoma with the macular involved in one eye and three children had unilateral macular tumors. The median age at which patching was initiated was 15 months (range 4-36). Compliance to patching was good in 80% of children, with a median duration of 4 h (range 0.5-8) per day, 7 days per week, with total occlusion of the better eye. The median percentage of posterior pole involvement was 34% (range 11-100%). Eighty percent of children had some improvement in their visual acuity, and of the children in whom final logMAR acuity was recorded, 73% had an acuity of 1.0 logMAR or better and 53% an acuity of 0.5 logMAR or better after patching. There was no evidence of association between age of patient, sex, duration of patching, or percentage of posterior pole involvement and the improvement in visual acuity. CONCLUSIONS: In spite of the macular involvement of eyes with retinoblastoma, some visual recovery was achieved in 80% of children. Hence a trial of patching therapy is recommended for all children with involvement of the macula by 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.

Cost comparison of predictive genetic testing versus conventional clinical screening for familial adenomatous polyposis.

BACKGROUND: Mutations of the APC gene cause familial adenomatous polyposis (FAP), a hereditary colorectal cancer predisposition syndrome. AIMS: To conduct a cost comparison analysis of predictive genetic testing versus conventional clinical screening for individuals at risk of inheriting FAP, using the perspective of a third party payer. METHODS: All direct health care costs for both screening strategies were measured according to time and motion, and the expected costs evaluated using a decision analysis model. RESULTS: The baseline analysis predicted that screening a prototype FAP family would cost $4975/ pound3109 by molecular testing and $8031/ pound5019 by clinical screening strategy, when family members were monitored with the same frequency of clinical surveillance (every two to three years). Sensitivity analyses revealed that the genetic testing approach is cost saving for key variables including the kindred size, the age of screening onset, and the cost of mutation identification in a proband. However, if the APC mutation carriers were monitored at an increased (annual) frequency, the cost of the genetic screening strategy increased to $7483/ pound4677 and was especially sensitive to variability in age of onset of screening, family size, and cost of genetic testing of at risk relatives. CONCLUSIONS: In FAP kindreds, a predictive genetic testing strategy costs less than conventional clinical screening, provided that the frequency of surveillance is identical using either strategy. An additional significant benefit is the elimination of unnecessary colonic examinations for those family members found to be non-carriers.

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.