Helen Dimaras

Characterisation of retinoblastomas without RB1 mutations: genomic, gene expression, and clinical studies.

BACKGROUND Retinoblastoma is the childhood retinal cancer that defined tumour-suppressor genes. Previous work shows that mutation of both alleles of the RB1 retinoblastoma suppressor gene initiates disease. We aimed to characterise non-familial retinoblastoma tumours with no detectable RB1 mutations. METHODS Of 1068 unilateral non-familial retinoblastoma tumours, we compared those with no evidence of RB1 mutations (RB1(+/+)) with tumours carrying a mutation in both alleles (RB1(-/-)). We analysed genomic copy number, RB1 gene expression and protein function, retinal gene expression, histological features, and clinical data. FINDINGS No RB1 mutations (RB1(+/+)) were reported in 29 (2·7%) of 1068 unilateral retinoblastoma tumours. 15 of the 29 RB1(+/+) tumours had high-level MYCN oncogene amplification (28-121 copies; RB1(+/+)MYCN(A)), whereas none of 93 RB1(-/-) primary tumours tested showed MYCN amplification (p<0·0001). RB1(+/+)MYCN(A) tumours expressed functional RB1 protein, had fewer overall genomic copy-number changes in genes characteristic of retinoblastoma than did RB1(-/-) tumours, and showed distinct aggressive histological features. MYCN amplification was the sole copy-number change in one RB1(+/+)MYCN(A) retinoblastoma. One additional MYCN(A) tumour was discovered after the initial frequencies were determined, and this is included in further analyses. Median age at diagnosis of the 17 children with RB1(+/+)MYCN(A) tumours was 4·5 months (IQR 3·5-10), compared with 24 months (15-37) for 79 children with non-familial unilateral RB1(-/-) retinoblastoma. INTERPRETATION Amplification of the MYCN oncogene might initiate retinoblastoma in the presence of non-mutated RB1 genes. These unilateral RB1(+/+)MYCN(A) retinoblastomas are characterised by distinct histological features, only a few of the genomic copy-number changes that are characteristic of retinoblastoma, and very early age of diagnosis. FUNDING National Cancer Institute-National Institutes of Health, Canadian Institutes of Health Research, German Research Foundation, Canadian Retinoblastoma Society, Hyland Foundation, Toronto Netralaya and Doctors Lions Clubs, Ontario Ministry of Health and Long Term Care, UK-Essen, and Foundations Avanti-STR and KiKa.

Pre-Enucleation Chemotherapy for Eyes Severely Affected by Retinoblastoma Masks Risk of Tumor Extension and Increases Death From Metastasis

PURPOSE Initial response of intraocular retinoblastoma to chemotherapy has encouraged primary chemotherapy instead of primary enucleation for eyes with clinical features suggesting high risk of extraocular extension or metastasis. Upfront enucleation of such high-risk eyes allows pathologic evaluation of extraocular extension, key to management with appropriate surveillance and adjuvant therapy. Does chemotherapy before enucleation mask histologic features of extraocular extension, potentially endangering the childs life by subsequent undertreatment? METHODS We performed retrospective analysis of 100 eyes with advanced retinoblastoma enucleated with, or without, primary chemotherapy, in Beijing Tongren Hospital, retrospectively, from October 31, 2008. The extent of retinoblastoma invasion into optic nerve, uvea, and anterior chamber on histopathology was staged by pTNM classification. The treatment groups were compared for pathologic stage (Cochran-Armitage trend test) and disease-specific mortality (competing risks methods). RESULTS Children who received chemotherapy before enucleation had lower pTNM stage than primarily enucleated children (P = .01). Five patients who received pre-enucleation chemotherapy died as a result of extension into brain or metastasis. No patients who had primary enucleation died. For children with group E eyes, disease-specific survival (DSS) was lower with pre-enucleation chemotherapy (n = 45) than with primary enucleation (n = 37; P = .01). Enucleation longer than 3 months after diagnosis was also associated with lower DSS (P < .001). CONCLUSION Chemotherapy before enucleation of group E eyes with advanced retinoblastoma downstaged pathologic evidence of extraocular extension, and increased the risk of metastatic death from reduced surveillance and inappropriate management of high-risk disease, if enucleation was performed longer than 3 months after diagnosis.

Hand-held high-resolution spectral domain optical coherence tomography in retinoblastoma: clinical and morphologic considerations

Purpose Hand-held spectral domain optical coherence tomography (HHSD OCT) has greatly expanded the imaging/diagnostic capacity for clinicians managing children with intraocular retinoblastoma. We present our early experience with HHSD OCT and conventional spectral domain OCT imaging in these patients. Methods In this retrospective cross-sectional observational study, infants were imaged during examination under anaesthesia with HHSD OCT in the supine position. Older cooperative retinoblastoma patients were additionally imaged with upright conventional OCT. Clinical data were derived from patient charts and from a prospectively maintained interinstitutional retinoblastoma database. Complementary imaging techniques, including RetCam™, fluorescein angiography and B-scan ultrasound, were assessed. Results Twenty-two intraocular lesions in 16 patients were imaged. HHSD OCT was used exclusively in 19 lesions, while conventional OCT was also performed in three cases. Small lesions were imaged in five cases, all of which were localised to the middle retinal layers. Clinical uses for HHSD OCT imaging identified included: diagnosis of new lesions, monitoring response to laser therapy and the identification of edge recurrences. Conclusions Although indirect ophthalmoscopy remains the gold standard for diagnosis and treatment of retinoblastoma, HHSD OCT is a valuable tool in better understanding and managing retinoblastoma.

Intra-arterial Chemotherapy for Retinoblastoma: A Systematic Review

Importance Intra-arterial chemotherapy has emerged as a treatment for intraocular retinoblastoma and has been quickly adopted by centers worldwide. Objective To conduct a systematic review and attempt a meta-analysis to summarize the reported outcomes of intra-arterial chemotherapy. Evidence Review In January 2015, we performed comprehensive searches in Medline, Embase, Cochrane, and Web of Science from inception through January 2015, including any peer-reviewed English-language publication that described outcomes related to toxicity or efficacy in at least 4 patients. Findings From a total of 208 identified publications, 28 met inclusion criteria. Twelve reports with discernable nonduplicative information were included, reporting 655 patients, 757 eyes, and 2350 catheterizations. All were single-arm case series, and 67% (8 of 12) were retrospective. Across all studies, globe salvage was achieved for 502 (66%) of all eyes. Most common reported toxicities were chorioretinal atrophy and vascular occlusions. There were at least 13 reports of children with metastases. After publication, 7 additional children had metastases. The 4 different classification systems used challenged the comparison of disease severity at presentation. Visual outcome was not addressed in most studies. Meta-analyses were not possible because no study had a comparative group. Assessment of risk of bias was not possible because no validated tool for single-arm studies was available. Conclusions and Relevance Intra-arterial chemotherapy is a promising new treatment associated with high rates of globe salvage. However, the literature is limited by the predominance of retrospective case series, absence of comparison groups, short median follow-up, heterogeneous definitions and tumor classifications, and frequent duplicate reporting. Metastases have been observed, and long-term follow-up is needed. Until the results of clinical, prospective studies are available, it is recommended that intra-arterial chemotherapy be offered selectively among other options, with fully informed discussion about all possible risks, benefits, and uncertainties.

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

We studied the expression of pro‐apoptotic neurotrophin receptor p75 (p75NTR) in human and murine retinoblastoma, compared to normal retina, and examined changes in p75NTR 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 p75NTR mRNA, compared to human retina. Moreover, p75NTR protein was not detected in any tumor studied, unlike normal retina. Like human retinoblastoma, advanced murine retinoblastoma did not express p75NTR. However, before tumors emerged, small clusters of TAg‐positive cells coexpressed p75NTR 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 p75NTR protein, while the retinoblastoma did not. We suggest that p75NTR loss accompanies progression from retinoma to retinoblastoma.

Challenging the global retinoblastoma survival disparity through a collaborative research effort

Imagine two children with unilateral retinoblastoma. One lives in North America, while the other lives in sub-Saharan Africa. In North America, a mother notices a funny white-eye reflex in photographs of her child. It is consistently present in various photos taken over the last month. Curious, she surfs the internet and discovers that a white-eye reflex could be a sign of cancer. Mother and child visit their family doctor, who confirms the mothers worst fear: it is retinoblastoma. Two days later, the affected eye is removed at the local childrens hospital. The next week, histopathology results confirm no risk of tumour spread, while a few weeks later, genetic testing of the tumour and blood indicates the RB1 mutation was sporadic. The child is fitted with a prosthetic eye and goes on to lead a normal, productive life. In Africa, a mother notices a white reflection in her childs eye, most prominent at dusk. Since it does not cause her child pain or discomfort, she ignores it at first, distracted as she continues to care and provide for all of her children. Months later, the white reflection is still prominent. She takes the child to the local dispensary, where the healthcare worker reassures her that there is nothing wrong. When the eye appears to worsen, she consults a traditional healer. He prescribes a herbal medication for the child, and instructs her to administer it twice daily. The eye shows no improvement, and it eventually begins to protrude from the socket. The mother takes the baby to the city hospital, struggling to gather the funds needed for transportation to the city. The hospital doctors tell her the eye must come out; it is cancer of the eye. When the eye is removed, mother …

The p75NTR neurotrophin receptor is a tumor suppressor in human and murine retinoblastoma development

The transition from the benign retinal tumor retinoma to its malignant counterpart retinoblastoma is accompanied by the loss of expression of the p75NTR neurotrophin receptor. This change in expression is mimicked in the TAg‐RB murine model of retinoblastoma, where early tumors retain expression of p75NTR and advanced tumors lack it. We sought to determine the functional effect on tumor development of absence of p75NTR from the onset of TAg‐RB tumor initiation. TAg‐RB mice were crossed with either p75NTR exon 3 (E3KO) or exon 4 knockout (E4KO) mice to produce TAg‐RB offspring that lacked one or both normal p75NTR alleles. The average tumor area per eye as a percentage of retinal area was measured. TAg‐RB/E3KO (TAg‐RBE3KO) and heterozygous mice showed no significant difference in tumor area compared to the TAg‐RB control mice at any time point studied. However, TAg‐RB/E4KO (TAg‐RBE4KO) and heterozygous mice displayed a significantly larger tumor area than the TAg‐RB control mice. Furthermore, adenoviral‐mediated expression of p75NTR in a p75NTR‐deficient human retinoblastoma cell line resulted in increased apoptosis. Our results confirm that p75NTR suppresses progression of both human and TAg‐RB murine retinoblastoma, and holds promise as a target for future therapy of the disease.

The TAg-RB Murine Retinoblastoma Cell of Origin Has Immunohistochemical Features of Differentiated Muller Glia with Progenitor Properties

PURPOSE Human retinoblastoma arises from an undefined developing retinal cell after inactivation of RB1. This is emulated in a murine retinoblastoma model by inactivation of pRB by retinal-specific expression of simian virus 40 large T-antigen (TAg-RB). Some mutational events after RB1 loss in humans are recapitulated at the expression level in TAg-RB, supporting preclinical evidence that this model is useful for comparative studies between mouse and human. Here, the characteristics of the TAg-RB cell of origin are defined. METHODS TAg-RB mice were killed at ages from embryonic day (E)18 to postnatal day (P)35. Tumors were analyzed by immunostaining, DNA copy number PCR, or real-time quantitative RT-PCR for TAg protein, retinal cell type markers, and retinoblastoma-relevant genes. RESULTS TAg expression began at P8 in a row of inner nuclear layer cells that increased in number through P21 to P28, when clusters reminiscent of small tumors emerged from cells that escaped a wave of apoptosis. Early TAg-expressing cells coexpressed the developmental marker Chx10 and glial markers CRALBP, clusterin, and carbonic anhydrase II (Car2), but not TuJ1, an early neuronal marker. Emerging tumors retained expression of only Chx10 and carbonic anhydrase II. As with human retinoblastoma, TAg-RB tumors showed decreased Cdh11 DNA copy number and gain of Kif14 and Mycn. It was confirmed that TAg-RB tumors lose expression of tumor suppressor cadherin-11 and overexpress oncogenes Kif14, Dek, and E2f3. CONCLUSIONS TAg-RB tumors displayed molecular similarity to human retinoblastoma and origin in a cell with features of differentiated Müller glia with progenitor properties.

Retinoblastoma CSF Metastasis Cured By Multimodality Chemotherapy Without Radiation

Objective: Cerebrospinal fluid (CSF) metastasis is the most difficult type of retinoblastoma metastasis to cure, even with bone marrow transplant. Most metastatic retinoblastoma cells express P-glycoprotein causing multidrug resistance (MDR). P-glycoprotein-rich blood vessels form blood-brain and blood-eye barriers, inhibit drug entry into central nervous system (CNS) and eyes. High-dose craniospinal radiation is too morbid for treatment of young children. To cure CSF metastasis without radiation, we designed an intensive multimodality chemotherapy regimen. Method: After left eye enucleation, a 4-month-old boy with bilateral International Intraocular Retinoblastoma Classification Group E eyes and CSF metastasis was treated with 7-cycle high-dose carboplatin and etoposide, standard-dose vincristine, and high-dose/short-infusion cyclosporine to inhibit P-glycoprotein. Intraventricular drugs, non-substrate of P-glycoprotein (cytarabine), or less susceptible to MDR (topotecan), contributed to treatment of the metastasis. On achieving complete response, he was consolidated with supralethal-dosage carboplatin, etoposide, and cyclophosphamide, and his bone marrow rescued with autologous cord blood stem cells. Results: Following 1-cycle systemic chemotherapy and 2-dose intraventricular chemotherapy, the CSF metastasis cleared. The right eye tumor regressed completely. The patient remains in remission 8.3 years after diagnosis and 7.8 years post-transplant. Conclusion: Intensive multimodality chemotherapy can cure CSF metastasis in retinoblastoma without incurring extreme morbidity from craniospinal radiation.

The incidence and distribution of retinoblastoma in Kenya

We estimated the Kenyan retinoblastoma incidence, and distribution by age, sex, family history, ethnicity and province. A nationwide chart-review identified retinoblastoma cases between 1 January 2006 and 31 December 2007 (supplementary methods). We observed 206 cases in 46 Kenyan healthcare facilities, of which 58 (28%) were lost after referral, and 148 (72%) were traceable (figure 1). We excluded 16 cases, comprising 3 missing files and 13 with histology inconsistent with retinoblastoma (figure 2), leaving 132 cases. The concordance of 91% of available histological reports with the initial clinical diagnosis indicates that initial clinical diagnosis of retinoblastoma is performed adequately. Figure 1 Retinoblastoma cases observed in 2006 and 2007 (n=206). From the total number of cases clinically diagnosed as having retinoblastoma (Rb), seen over the 2-year period in 46 Kenyan healthcare facilities, we excluded cases lost after referral, with missing files or later showing histology inconsistent with Rb. This group of confirmed Rb cases was further narrowed down to the traceable population diagnosed during 2007. We added the estimated confirmed Rb cases from the untraceable study population to estimate the total number of newly diagnosed Rb cases in 2007. Figure 2 Histology of excluded cases (n=13). Histopathology reports showed 13 cases clinically reported …