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.

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.

Cdh11 acts as a tumor suppressor in a murine retinoblastoma model by facilitating tumor cell death.

CDH11 gene copy number and expression are frequently lost in human retinoblastomas and in retinoblastomas arising in TAg-RB mice. To determine the effect of Cdh11 loss in tumorigenesis, we crossed Cdh11 null mice with TAg-RB mice. Loss of Cdh11 had no gross morphological effect on the developing retina of Cdh11 knockout mice, but led to larger retinal volumes in mice crossed with TAg-RB mice (p = 0.01). Mice null for Cdh11 presented with fewer TAg-positive cells at postnatal day 8 (PND8) (p = 0.01) and had fewer multifocal tumors at PND28 (p = 0.016), compared to mice with normal Cdh11 alleles. However, tumor growth was faster in Cdh11-null mice between PND8 and PND84 (p = 0.003). In tumors of Cdh11-null mice, cell death was decreased 5- to 10-fold (p<0.03 for all markers), while proliferation in vivo remained unaffected (p = 0.121). Activated caspase-3 was significantly decreased and beta-catenin expression increased in Cdh11 knockdown experiments in vitro. These data suggest that Cdh11 displays tumor suppressor properties in vivo and in vitro in murine retinoblastoma through promotion of cell death.

Expression analysis of 6p22 genomic gain in retinoblastoma.

To identify gene(s) targeted by 6p22 genomic gain, present in more than 50% retinoblastoma tumors, we used real-time RT-PCR to quantify the expression of seven genes in normal human retina and retinoblastoma. Six genes are located in the quantitative multiplex PCR-defined 0.6 Mb minimal region of gain at 6p22 (DEK, AOF1, TPMT, NHLRC1, KIF13A, and NUP153), and E2F3 is 2 Mb away from the minimal region of gain on 6p22. E2F3, DEK, KIF13A, and NUP153 were most frequently overexpressed in retinoblastoma with 6p genomic gain, compared with the normal adult human retina. E2F3 and DEK mRNA levels were increased in all human tumors showing 6p22 gain, as well as in mouse retinoblastoma induced by SV40 large T antigen expression in developing retina, compared with the normal controls (adult human retina and 7-day-old mouse retina, respectively). Only DEK showed statistically significant correlation of expression and genomic copy number (P = 0.019). E2F3 and DEK, but not NUP153, showed developmental regulation. E2F3 and DEK mRNA overexpression was always associated with protein overexpression, determined by immunoblotting or immunofluorescent staining of primary tumors, relative to the adjacent normal retina. E2F3 was strongly expressed in actively proliferating cells, while DEK was overexpressed in all tumor cells. Taking into account the proliferation-promoting role of E2F3, implication of E2F3 in bladder and prostate cancer, and the translocation and overexpression of DEK in leukemia, we conclude that either DEK or E2F3 (or both) are targeted by the 6p22 gain in retinoblastoma.

Distinct patterns of expression of the RB gene family in mouse and human retina.

Although RB1 function is disrupted in the majority of human cancers, an undefined cell of developing human retina is uniquely sensitive to cancer induction when the RB1 tumor suppressor gene is lost. Murine retinoblastoma is initiated only when two of the RB family of genes, RB1 and p107 or p130, are inactivated. Although whole embryonic retina shows RB family gene expression by several techniques, when E14 developing retina was depleted of the earliest differentiating cells, ganglion cells, the remaining proliferating murine embryonic retinal progenitor cells clearly did not express RB1 or p130, while the longer splice form of p107 was expressed. Each retinal cell type expressed some member of the RB family at some stage of differentiation. Rod photoreceptors stained for the RB1 protein product, pRB, and p107 in only a brief window of postnatal murine development, with no detectable staining for any of the RB family proteins in adult human and mouse rod photoreceptors. Adult mouse and human Muller glia, ganglion and rare horizontal cells, and adult human, but not adult mouse, cone photoreceptors stained for pRB. The RB gene family is dynamically and variably expressed through retinal development in specific retinal cells.