Differential gene expression profile of retinoblastoma compared to normal retina.

PURPOSE: The retinoblastoma gene (RB1) is a tumor suppressor gene that was first discovered in a rare ocular pediatric tumor called retinoblastoma (RB). The RB1 gene is essential for normal progression through the cell cycle and exerts part of its function through the family of transcription factors (E2F) and many other intermediaries. In the absence of normal RB1, genomic instability and chromosomal aberrations accumulate, leading to tumor initiation, progression, and ultimately metastasis. The purpose of this report was to identify the molecular pathways that are deregulated in retinoblastoma. METHODS: We compared gene expression signatures of matched normal retinal tissue and retinoblastoma (RB) tumor tissue from six individuals, using microarray analysis followed by statistical and bioinformatic analyses. RESULTS: We identified 1,116 genes with increased expression and 837 with decreased expression in RB tumor tissue compared to matched normal retinal tissue. Functional categories of the cognate genes with the greatest statistical support were cell cycle (309 genes), cell death (437 genes), DNA replication, recombination and repair (270 genes), cellular growth and proliferation (464 genes), and cellular assembly and organization (110 genes). The list included differentially expressed retinal cone-cell-specific markers. These data indicated the predominance of cone cells in RB and support the idea that the latter group of cells may be the cells of origin for RB. CONCLUSIONS: The genes differentially expressed in RB as compared to normal retina belong mainly to DNA damage-response pathways, including, but not limited to, breast cancer associated genes (BRCA1, BRCA2), ataxia telangiectasia mutated gene (ATM), ataxia telangiectasia and Rad3 related gene(ATR), E2F, checkpoint kinase 1 (CHK1) genes. In addition, novel pathways, such as aryl hydrocarbon receptor (AHR) signaling, polo-like kinase and mitosis, purine metabolism pathways were involved. The molecules AHR, CHK1, and polo-like kinases are of particular interest because there are several currently available drugs that target these molecules. Further studies are needed to determine if targeting these pathways in RB will have therapeutic value. It is also important to evaluate the relative importance of these pathways in different cells that make up the normal retina

Phosphorylation of pRb: mechanism for RB pathway inactivation in MYCN-amplified retinoblastoma.

A small, but unique subgroup of retinoblastoma has been identified with no detectable mutation in the retinoblastoma gene (RB1) and with high levels of MYCN gene amplification. This manuscript investigated alternate pathways of inactivating pRb, the encoded protein in these tumors. We analyzed the mutation status of the RB1 gene and MYCN copy number in a series of 245 unilateral retinoblastomas, and the phosphorylation status of pRb in a subset of five tumors using immunohistochemistry. There were 203 tumors with two mutations in RB1 (RB1(-/-) , 83%), 29 with one (RB1(+/-) , 12%) and 13 with no detectable mutations (RB1(+/+) , 5%). Eighteen tumors carried MYCN amplification between 29 and 110 copies: 12 had two (RB1(-/-) ) or one RB1 (RB1(+/-) ) mutations, while six had no mutations (RB1(+/+) ). Immunohistochemical staining of tumor sections with antibodies against pRb and phosphorylated Rb (ppRb) displayed high levels of pRb and ppRb in both RB1(+/+) and RB1(+/-) tumors with MYCN amplification compared to no expression of these proteins in a classic RB1(-/-) , MYCN-low tumor. These results establish that high MYCN amplification can be present in retinoblastoma with or without coding sequence mutations in the RB1 gene. The functional state of pRb is inferred to be inactive due to phosphorylation of pRb in the MYCN-amplified retinoblastoma without coding sequence mutations. This makes inactivation of RB1 by gene mutation or its protein product, pRb, by protein phosphorylation, a necessary condition for initiating retinoblastoma tumorigenesis, independent of MYCN amplification

Human embryonic and neuronal stem cell markers in retinoblastoma

Retinoblastoma (RB) is the most common intraocular tumor of early childhood. The early onset of RB, coupled with our previous findings of cancer stem cell characteristics in RB, led us to hypothesize that subpopulations of RB tumors harbor markers and behaviors characteristic of embryonic and neuronal origin. Our RB sources included: human pathological tissues, and the human RB cell lines Y79 and WERI-RB27. Microarray screening, single and dual-label immunocytochemistry and RT-PCR were performed to detect embryonic and neuronal stem cell markers, such as Oct3/4, Nanog, CD133, and Musashi-1. To test for functional evidence of stem cell behavior, we examined RB cells for their ability to form neurospheres and retain BrdU label as indicators of self-renewal and slow cell cycling, respectively. Microarray comparisons of human RB tumors with normal retinal tissue detected upregulation of a number of genes involved in embryonic development that were also present in Y79 cells, including Oct3/4, Nanog, Musashi-1 and Musashi-2, prominin-1 (CD133), Jagged-2, Reelin, Thy-1, nestin, Meis-1,NCAM, Patched, and Notch4. Expression of Musashi-1, Oct3/4 and Nanog was confirmed by immunostaining and RT-PCR analyses of RB tumors and RB cell lines. CD133 expression was confirmed by PCR analysis. Y79 and WERI-RB27 contained populations of Hoechst-dim/ABCG2-positive cells that co-localized with embryonic stem cell markers Oct3/4-ABCG2 and Nanog-ABCG2. Subpopulations of Y79 and WERI-RB27 cells were label-retaining (as seen by BrdU incorporation) and were able to generate neurospheres, both hallmarks of a stem cell phenotype. Small subpopulation(s) of RB cells express human embryonic and neuronal stem cell markers. There are also subpopulations that demonstrate functional behavior (label retention and self-renewal) consistent with cancer stem cells. These findings support the hypothesis that RB is a heterogeneous tumor comprised of subpopulation(s) with stem cell-like properties

Parental diet and risk of retinoblastoma resulting from new germline RB1 mutation

We conducted a case–control study of sporadic bilateral retinoblastoma, which results from a new germline RB1 mutation, to investigate the role of parents\u27 diet before their child\u27s conception. Parents of 206 cases from nine North American institutions and 269 controls participated; of these, fathers of 184 cases and 223 controls and mothers of 204 cases and 260 controls answered a food frequency questionnaire administered by phone about their diet in the year before the child\u27s conception. Cases provided DNA for RB1 mutation testing. We assessed parents\u27 diet by examining 19 food groups. Father\u27s intake of dairy products and fruit was associated with decreased risk and cured meats and sweets with increased risk. Mother\u27s intake was not associated with disease for any food group. Considering analyses adjusted for the other food groups significantly associated with disease, energy intake, and demographic characteristics as well as more fully adjusted models, the associations with father\u27s dairy products and cured meat intake were the most robust. In the fully adjusted, matched analysis, the odds ratios per daily serving were 0.70 (95% confidence interval (CI) 0.49–1.00, P = 0.047) for dairy products and 5.05 (CI 1.46–17.51, P = 0.01) for cured meat. The pattern of associations with paternal but not maternal diet is consistent with the fact that 85% of new germline RB1 mutations occur on the father\u27s allele. As few human data exist on the role of diet in any condition resulting from new germ-cell mutation, additional studies will be needed to replicate or refute our findings. Environ. Mol. Mutagen

Prognostication of uveal melanoma is simple and highly predictive using The Cancer Genome Atlas (TCGA) classification: A review.

Purpose: The cancer genome atlas (TCGA) is a comprehensive project supported by the National Cancer Institute (NCI) in the United States to explore molecular alterations in cancer, including uveal melanoma (UM). This led to TCGA classification for UM. In this report, we review the American Joint Committee on Cancer (AJCC) classification and TCGA classification for UM from the NCI\u27s Center for Cancer Genomics (NCI CCG) (based on enucleation specimens [n = 80 eyes]) and from Wills Eye Hospital (WEH) (based on fine needle aspiration biopsy [FNAB] specimens [n = 658 eyes]). We then compare accuracy and predictability of AJCC versus (vs.) TCGA. Methods: Review of published reports on AJCC and TCGA classification for UM was performed. Outcomes based on AJCC 7th and 8th editions were assessed. For TCGA, UM was classified based on chromosomes 3 and 8 findings including disomy 3 (D3), monosomy 3 (M3), disomy 8 (D8), 8q gain (8qG), or 8q gain multiple (8qGm) and combined into four classes including Class A (D3/D8), Class B (D3/8qG), Class C (M3/8qG), and Class D (M3/8qGm). Outcomes of metastasis and death were explored and a comparison (AJCC vs. TCGA) was performed. Results: In the NCI CCG study, there were 80 eyes with UM sampled by enucleation (n = 77), resection (n = 2), or orbitotomy (n = 1) and analysis revealed four distinct genetic classes. Metastasis and death outcomes were subsequently evaluated per class in the WEH study. The WEH study reviewed 658 eyes with UM, sampled by FNAB, and found Class A (n = 342, 52%), B (n = 91, 14%), C (n = 118, 18%), and D (n = 107, 16%). Comparison by increasing class (A vs. B vs. C vs. D) revealed older mean patient age (P \u3c 0.001), worse entering visual acuity (P \u3c 0.001), greater distance from the optic disc (P \u3c 0.001), larger tumor diameter (P \u3c 0.001), and greater tumor thickness (P \u3c 0.001). Regarding outcomes, more advanced TCGA class demonstrated increased 5-year risk for metastasis (4% vs. 20% vs. 33% vs. 63%,P \u3c 0.001) with corresponding increasing hazard ratio (HR) (1.0 vs. 4.1, 10.1, 30.0,P= 0.01 for B vs. A andP \u3c 0.001 for C vs. A and D vs. A) as well as increased 5-year estimated risk for death (1% vs. 0% vs. 9% vs. 23%,P \u3c 0.001) with corresponding increasing HR (1 vs. NA vs. 3.1 vs. 13.7,P= 0.11 for C vs. A andP \u3c 0.001 for D vs. A). Comparison of AJCC to TCGA classification revealed TCGA was superior in prediction of metastasis and death from UM. Conclusion: TCGA classification for UM is simple, accurate, and highly predictive of melanoma-related metastasis and death, more so than the AJCC classification

Additional file 1: Table S1. of De novo mutational profile in RB1 clarified using a mutation rate modeling algorithm

All de novo germline variants in RB1 gene of patients with RB. “gDNA position” is the nucleotide position in the GENBANK accession number L11910 of the gene. Table S2. All ExAC variants in RB1 gene that were considered in our analysis. “gDNA position” is the nucleotide position in the GENBANK accession number L11910 of the gene. Table S3. All Nonsense variants in RB1 gene from Onadim and Houdayer groups. “gDNA position” is the nucleotide position in the GENBANK accession number L11910 of the gene. Table S4. Comparison of observed mutations and the simulated frequency of nonsense changes per exon, to find differential pathogenicity within nonsense mutations. Analysis was performed on data from Onadim and Houdayer groups. Table S5. Comparison of observed mutations and the simulated frequency of nonsense changes to find differential pathogenicity within nonsense mutations. Data shown for all amino acids and two arginine codons (99% CI) which can change to a stop codon. Analysis was performed on data from Onadim and Houdayer groups. Table S6. Polyphen predictions on the de novo germline missense mutations or some potential variants near codon 661 in RB1 gene. “Polyphen2_format” is the variant format accepted by the Polyphen2 tool. “Polyphen_prediction” is the result of Polyphen2 on the missense variant. Table S7. Comparison between observed mutations and the simulated frequency of missense changes at amino acids and codons in exon 20, to find localized pathogenicity within missense mutations. Only the significant results are reported here. Table S8. Genomic territory of RB1 gene analyzed in our study. “Position Start” is the start position of the entry as per GENBANK database. “Position End” is the end positon of the entry as per GENBANK database. “Annotation” is the description of the entry. Possible keywords are exon or donor/acceptor region in essential splice or nonessential intronic region. “Exon” corresponds to exon number of the entry. (XLSX 30 kb

Additional file 3: Figure S2. of De novo mutational profile in RB1 clarified using a mutation rate modeling algorithm

Donor splice mutations in Exons 5, 6 and 12, and their effect on codon structure. The codon structures are shown prior and after the donor splice mutation. The donor splice mutation results in exon skipping or deletion, but can also cause a frameshift mutation in certain cases. (PDF 84 kb