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.

Retinoblastoma is a rare childhood cancer initiated by RB1 mutation or MYCN amplification, while additional alterations may be required for tumor development. However, the view on single nucleotide variants is very limited. To better understand oncogenesis, we determined the genomic landscape of retinoblastoma. We performed exome sequencing of 71 retinoblastomas and matched blood DNA. Next, we determined the presence of single nucleotide variants, copy number alterations and viruses. Aside from RB1, recurrent gene mutations were very rare. Only a limited fraction of tumors showed BCOR (7/71, 10%) or CREBBP alterations (3/71, 4%). No evidence was found for the presence of viruses. Instead, specific somatic copy number alterations were more common, particularly in patients diagnosed at later age. Recurrent alterations of chromosomal arms often involved less than one copy, also in highly pure tumor samples, suggesting within-tumor heterogeneity. Our results show that retinoblastoma is among the least mutated cancers and signify the extreme sensitivity of the childhood retina for RB1 loss. We hypothesize that retinoblastomas arising later in retinal development benefit more from subclonal secondary alterations and therefore, these alterations are more selected for in these tumors. Targeted therapy based on these subclonal events might be insufficient for complete tumor control.

BackgroundRetinoblastoma is a pediatric eye cancer associated with RB1 loss or MYCN amplification (RB1+/+MYCNA). There are controversies concerning the existence of molecular subtypes within RB1−/− retinoblastoma. To test whether these molecular subtypes exist, we performed molecular profiling.MethodsGenome-wide mRNA expression profiling was performed on 76 primary human retinoblastomas. Expression profiling was complemented by genome-wide DNA profiling and clinical, histopathological, and ex vivo drug sensitivity data.FindingsRNA and DNA profiling identified major variability between retinoblastomas. While gene expression differences between RB1+/+MYCNA and RB1−/− tumors seemed more dichotomous, differences within the RB1−/− tumors were gradual. Tumors with high expression of a photoreceptor gene signature were highly differentiated, smaller in volume and diagnosed at younger age compared with tumors with low photoreceptor signature expression. Tumors with lower photoreceptor expression showed increased expression of genes involved in M-phase and mRNA and ribosome synthesis and increased frequencies of somatic copy number alterations.InterpretationMolecular, clinical and histopathological differences between RB1−/− tumors are best explained by tumor progression, reflected by a gradual loss of differentiation and photoreceptor expression signature. Since copy number alterations were more frequent in tumors with less photoreceptorness, genomic alterations might be drivers of tumor progression.Research in contextRetinoblastoma is an ocular childhood cancer commonly caused by mutations in the RB1 gene. In order to determine optimal treatment, tumor subtyping is considered critically important. However, except for very rare retinoblastomas without an RB1 mutation, there are controversies as to whether subtypes of retinoblastoma do exist. Our study shows that retinoblastomas are highly diverse but rather than reflecting distinct tumor types with a different etiology, our data suggests that this diversity is a result of tumor progression driven by cumulative genetic alterations. Therefore, retinoblastomas should not be categorized in distinct subtypes, but be described according to their stage of progression.

BackgroundWhile RB1 loss initiates retinoblastoma development, additional somatic copy number alterations (SCNAs) can drive tumor progression. Although SCNAs have been identified with good concordance between studies at a cytoband resolution, accurate identification of single genes for all recurrent SCNAs is still challenging. This study presents a comprehensive meta-analysis of genome-wide SCNAs integrated with gene expression profiling data, narrowing down the list of plausible retinoblastoma driver genes.MethodsWe performed SCNA profiling of 45 primary retinoblastoma samples and eight retinoblastoma cell lines by high-resolution microarrays. We combined our data with genomic, clinical and histopathological data of ten published genome-wide SCNA studies, which strongly enhanced the power of our analyses (N = 310).ResultsComprehensive recurrence analysis of SCNAs in all studies integrated with gene expression data allowed us to reduce candidate gene lists for 1q, 2p, 6p, 7q and 13q to a limited gene set. Besides the well-established driver genes RB1 (13q-loss) and MYCN (2p-gain) we identified CRB1 and NEK7 (1q-gain), SOX4 (6p-gain) and NUP205 (7q-gain) as novel retinoblastoma driver candidates. Depending on the sample subset and algorithms used, alternative candidates were identified including MIR181 (1q-gain) and DEK (6p gain). Remarkably, our study showed that copy number gains rarely exceeded change of one copy, even in pure tumor samples with 100% homozygosity at the RB1 locus (N = 34), which is indicative for intra-tumor heterogeneity. In addition, profound between-tumor variability was observed that was associated with age at diagnosis and differentiation grades.InterpretationSince focal alterations at commonly altered chromosome regions were rare except for 2p24.3 (MYCN), further functional validation of the oncogenic potential of the described candidate genes is now required. For further investigations, our study provides a refined and revised set of candidate retinoblastoma driver genes.

The frequency of the type of mutations in the RB1 gene in our unbiased national cohort is the same as the mutation spectrum described worldwide. Furthermore, our RB1 mutation detection regimen achieves a high scanning sensitivity.

Both hereditary and nonhereditary retinoblastoma (Rb) are commonly initiated by loss of both copies of the retinoblastoma tumor suppressor gene (RB1), while additional genomic changes are required for tumor initiation and progression. Our aim was to determine whether there is genomic heterogeneity between different clinical Rb subtypes. Therefore, 21 Rb tumors from 11 hereditary patients and 10 nonhereditary Rb patients were analyzed using high-resolution single nucleotide polymorphism (SNP) arrays and gene losses and gains were validated with Multiplex Ligation-dependent Probe Amplification. In these tumors only a few focal aberrations were detected. The most frequent was a focal gain on chromosome 2p24.3, the minimal region of gain encompassing the oncogene MYCN. The genes BAZ1A, OTX2, FUT8, and AKT1 were detected in four focal regions on chromosome 14 in one nonhereditary Rb. There was a large difference in number of copy number aberrations between tumors. A subset of nonhereditary Rbs turned out to be the most genomic unstable, while especially very young patients with hereditary Rb display stable genomes. Established Rb copy number aberrations, including gain of chromosome arm 1q and loss of chromosome arm 16q, turned out to be preferentially associated with the nonhereditary Rbs with later age of diagnosis. In contrast, copy number neutral loss of heterozygosity was detected mainly on chromosome 13, where RB1 resides, irrespective of hereditary status or age. Focal amplifications and deletions and copy number neutral loss of heterozygosity besides chromosome 13 appear to be rare events in retinoblastoma.

IntroductionStress has been shown to be a tumor promoting factor. Both clinical and laboratory studies have shown that chronic stress is associated with tumor growth in several types of cancer. Corticotropin Releasing Factor (CRF) is the major hypothalamic mediator of stress, but is also expressed in peripheral tissues. Earlier studies have shown that peripheral CRF affects breast cancer cell proliferation and motility. The aim of the present study was to assess the significance of peripheral CRF on tumor growth as a mediator of the response to stress in vivo.MethodsFor this purpose we used the 4T1 breast cancer cell line in cell culture and in vivo. Cells were treated with CRF in culture and gene specific arrays were performed to identify genes directly affected by CRF and involved in breast cancer cell growth. To assess the impact of peripheral CRF as a stress mediator in tumor growth, Balb/c mice were orthotopically injected with 4T1 cells in the mammary fat pad to induce breast tumors. Mice were subjected to repetitive immobilization stress as a model of chronic stress. To inhibit the action of CRF, the CRF antagonist antalarmin was injected intraperitoneally. Breast tissue samples were histologically analyzed and assessed for neoangiogenesis.ResultsArray analysis revealed among other genes that CRF induced the expression of SMAD2 and β-catenin, genes involved in breast cancer cell proliferation and cytoskeletal changes associated with metastasis. Cell transfection and luciferase assays confirmed the role of CRF in WNT- β-catenin signaling. CRF induced 4T1 cell proliferation and augmented the TGF-β action on proliferation confirming its impact on TGFβ/SMAD2 signaling. In addition, CRF promoted actin reorganization and cell migration, suggesting a direct tumor-promoting action. Chronic stress augmented tumor growth in 4T1 breast tumor bearing mice and peripheral administration of the CRF antagonist antalarmin suppressed this effect. Moreover, antalarmin suppressed neoangiogenesis in 4T1 tumors in vivo.ConclusionThis is the first report demonstrating that peripheral CRF, at least in part, mediates the tumor-promoting effects of stress and implicates CRF in SMAD2 and β-catenin expression.

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