RB1

Gene Summary

Gene:RB1; RB transcriptional corepressor 1
Aliases: RB, pRb, OSRC, pp110, p105-Rb, PPP1R130
Location:13q14.2
Summary:The protein encoded by this gene is a negative regulator of the cell cycle and was the first tumor suppressor gene found. The encoded protein also stabilizes constitutive heterochromatin to maintain the overall chromatin structure. The active, hypophosphorylated form of the protein binds transcription factor E2F1. Defects in this gene are a cause of childhood cancer retinoblastoma (RB), bladder cancer, and osteogenic sarcoma. [provided by RefSeq, Jul 2008]
Databases:VEGA, OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:retinoblastoma-associated protein
Source:NCBIAccessed: 11 March, 2017

Ontology:

What does this gene/protein do?
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Pathways:What pathways are this gene/protein implicaed in?
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Cancer Overview

The Retinoblastoma gene (RB1), located on chromosome 13, is a tumour suppressor gene that was discovered in genetic studies of hereditary retinoblastoma. It also has a role in other cancers including osteosarcoma. The RB1 gene is important because it helps regulates cell division, if the gene is absent then cells may proliferate in an uncontrolled way leading to tumour formation. In hereditary retinoblastoma the RB1 gene is lost and children have multiple tumours in both eyes, while in children with sporadic (non-hereditary) retinoblastoma there is usually only one tumour in one eye.

Research Indicators

Publications Per Year (1992-2017)
Graph generated 11 March 2017 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

Tag cloud generated 11 March, 2017 using data from PubMed, MeSH and CancerIndex

Specific Cancers (9)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Entity Topic PubMed Papers
RetinoblastomaRB1 mutation in Retinoblastoma
In an analysis of 192 patients with retinoblastoma with identifiable germline mutations in the RB gene (Harbour, 1998), the DNA alteration was a nonsense mutation in 83 (43%), frameshift in 67 (35%), intron mutation in 23 (12%), missense mutation in 11 (6%), in-frame deletion in 5 (3%), and promoter mutation in 3 (2%).
View Publications549
Acute Lymphocytic Leukaemia (ALL)RB1 and Acute Leukaemias View Publications125
Lung CancerRB1 and Lung Cancer View Publications103
Bladder CancerRB1 and Bladder Cancer View Publications71
Breast CancerRB1 mutations in Breast Cancer
RB1 mutations are found in some breast cancers; there is a non-random relationship between p53 mutation and loss of material from the RB1 locus.
View Publications70
OsteosarcomaRB1 and Osteosarcoma View Publications68
Esophageal CancerRB1 and Esophageal Cancer View Publications10
Adrenocortical CancerRB1 and Adrenocortical Carcinoma View Publications6

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: RB1 (cancer-related)

Mallipatna A, Marino M, Singh AD
Genetics of Retinoblastoma.
Asia Pac J Ophthalmol (Phila). 2016 Jul-Aug; 5(4):260-4 [PubMed] Related Publications
Retinoblastoma is a malignant retinal tumor that affects young children. Mutations in the RB1 gene cause retinoblastoma. Mutations in both RB1 alleles within the precursor retinal cell are essential, with one mutation that may be germline or somatic and the second one that is always somatic. Identification of the RB1 germline status of a patient allows differentiation between sporadic and heritable retinoblastoma variants. Application of this knowledge is crucial for assessing short-term (risk of additional tumors in the same eye and other eye) and long-term (risk of nonocular malignant tumors) prognosis and offering cost-effective surveillance strategies. Genetic testing and genetic counseling are therefore essential components of care for all children diagnosed with retinoblastoma. The American Joint Committee on Cancer has acknowledged the importance of detecting this heritable trait and has introduced the letter "H" to denote a heritable trait of all cancers, starting with retinoblastoma (in publication). In this article, we discuss the clinically relevant aspects of genetic testing and genetic counseling for a child with retinoblastoma.

Tang X, Liu H, Srivastava A, et al.
Transcriptome regulation and chromatin occupancy by E2F3 and MYC in mice.
Sci Data. 2016; 3:160008 [PubMed] Free Access to Full Article Related Publications
E2F3 and MYC are transcription factors that control cellular proliferation. To study their mechanism of action in the context of a regenerating tissue, we isolated both proliferating (crypts) and non-dividing (villi) cells from wild-type and Rb depleted small intestines of mice and performed ChIP-exo-seq (chromatin immunoprecipitation combined with lambda exonuclease digestion followed by high-throughput sequencing). The genome-wide chromatin occupancy of E2F3 and MYC was determined by mapping sequence reads to the genome and predicting preferred binding sites (peaks). Binding sites could be accurately identified within small regions of only 24 bp-28 bp long, highlighting the precision to which binding peaks can be identified by ChIP-exo-seq. Forty randomly selected E2F3- and MYC-specific binding sites were validated by ChIP-PCR. In addition, we also presented gene expression data sets from wild type, Rb-, E2f3- and Myc-depleted crypts and villi within this manuscript. These represent comprehensive and validated datasets that can be integrated to identify putative direct targets of E2F3 and MYC involved in the control of cellular proliferation in normal and Rb-deficient small intestines.

Yang Y, Mei Q
miRNA signature identification of retinoblastoma and the correlations between differentially expressed miRNAs during retinoblastoma progression.
Mol Vis. 2015; 21:1307-17 [PubMed] Free Access to Full Article Related Publications
PURPOSE: Retinoblastoma (RB) is a common pediatric cancer. The study aimed to uncover the mechanisms of RB progression and identify novel therapeutic biomarkers.
METHODS: The miRNA expression profile GSE7072, which includes three RB samples and three healthy retina samples, was used. After data normalization using the preprocessCore package, differentially expressed miRNAs (DE-miRs) were selected by the limma package. The targets of the DE-miRs were predicted based on two databases, followed by construction of the miRNA-target network. Pathway enrichment analysis was conducted for the targets of the DE-miRNAs using DAVID. The CTD database was used to predict RB-related genes, followed by clustering analysis using the pvclust package. The correlation network of DE-miRs was established. MiRNA expression was validated in another data set, GSE41321.
RESULTS: In total, 24 DE-miRs were identified whose targets were correlated with the cell cycle pathway. Among them, hsa-miR-373, hsa-miR-125b, and hsa-miR-181a were highlighted in the miRNA-target regulatory network; 14 DE-miRs, including hsa-miR-373, hsa-miR-125b, hsa-miR-18a, hsa-miR-25, hsa-miR-20a, and hsa-let-7 (a, b, c), were shown to distinguish RB from healthy tissue. In addition, hsa-miR-25, hsa-miR-18a, and hsa-miR-20a shared the common target BCL2L11; hsa-let-7b and hsa-miR-125b targeted the genes CDC25A, CDK6, and LIN28A. Expression of three miRNAs in GSE41321 was consistent with that in GSE7072.
CONCLUSIONS: Several critical miRNAs were identified in RB progression. Hsa-miR-373 might regulate RB invasion and metastasis, hsa-miR-181a might involve in the CDKN1B-mediated cell cycle pathway, and hsa-miR-125b and hsa-let-7b might serve as tumor suppressors by coregulating CDK6, CDC25A, and LIN28A. The miRNAs hsa-miR-25, hsa-miR-18a, and hsa-miR-20a might exert their function by coregulating BCL2L1.

Robles-Oteiza C, Taylor S, Yates T, et al.
Recombinase-based conditional and reversible gene regulation via XTR alleles.
Nat Commun. 2015; 6:8783 [PubMed] Free Access to Full Article Related Publications
Synthetic biological tools that enable precise regulation of gene function within in vivo systems have enormous potential to discern gene function in diverse physiological settings. Here we report the development and characterization of a synthetic gene switch that, when targeted in the mouse germline, enables conditional inactivation, reports gene expression and allows inducible restoration of the targeted gene. Gene inactivation and reporter expression is achieved through Cre-mediated stable inversion of an integrated gene-trap reporter, whereas inducible gene restoration is afforded by Flp-dependent deletion of the inverted gene trap. We validate our approach by targeting the p53 and Rb genes and establishing cell line and in vivo cancer model systems, to study the impact of p53 or Rb inactivation and restoration. We term this allele system XTR, to denote each of the allelic states and the associated expression patterns of the targeted gene: eXpressed (XTR), Trapped (TR) and Restored (R).

Kim BM, Jeong CB, Lee MC, et al.
Identification of the retinoblastoma (Rb) gene and expression in response to environmental stressors in the intertidal copepod Tigriopus japonicus.
Mar Genomics. 2015; 24 Pt 3:387-96 [PubMed] Related Publications
There have been no reports thus far on the structure or molecular characterization of the retinoblastoma (Rb) gene of aquatic animals. Herein we describe the identification of the Rb gene of the intertidal copepod Tigriopus japonicus. In silico analyses revealed the conserved Rb domains of T. japonicus with those of protostomes. Phylogenetic analysis revealed that orthologs of Rb gene were evolved by an ancient split event in deuterostomes, while only a single Rb gene was conserved in protostomes except for Drosophila. The transcription of the T. japonicus Rb gene continuously increased across the molting transition from nauplius to the copepodid and adult stages, suggesting that it may play a developmental role in the molting process of T. japonicus. Information on Rb's response to environmental stressors, including toxin exposure, is lacking in copepods. To examine the transcriptional response to stressful conditions in laboratory culture conditions, copepods were exposed to UV-B radiation and different concentrations of metals, environmental toxins, and biocides. Transcription of the T. japonicus Rb gene was upregulated in response to about half of the 96 h-LD50 of UV-B radiation (12 kJ/m(2)) for 48 h, while the approximate 96 h-LD50 value (24 kJ/m(2)) of UV-B and relatively high concentrations of several toxins and biocides induced the downregulation of T. japonicus Rb mRNA expression. Taken together, our findings suggest that the T. japonicus Rb gene is sensitive to environmentally unfavorable conditions that can induce cell cycle alteration.

Murali A, Varghese BT, Kumar RR, Kannan S
Combination of genetic variants in cyclin D1 and retinoblastoma genes predict clinical outcome in oral cancer patients.
Tumour Biol. 2016; 37(3):3609-17 [PubMed] Related Publications
Oral cancer is a dreadful disease with a wide variation in geographical distribution. In order to identify some useful biomarkers for the disease prognosis, the present study assessed the influence of single nucleotide polymorphisms (SNPs) in cell cycle genes on survival in a well-annotated set of patients with oral squamous cell carcinoma (OSCC). The study examined 12 sequence variants or SNPs in selected cell cycle genes, with prognostic outcomes in 311 oral cancer patients. Our analysis showed that SNPs in cyclin D1:rs9344 and retinoblastoma:rs427686 genes showed a strong correlation with disease-free survival. In addition, the cumulative effect of these SNPs significantly and independently predicts the survival. Thus, the current study identified genotypes (SNP signature), which can be used as novel prognostic biomarkers to stratify patients based on disease-free survival and therefore maybe helpful in therapeutic decision-making.

Kim JY, Park Y
Treatment of Retinoblastoma: The Role of External Beam Radiotherapy.
Yonsei Med J. 2015; 56(6):1478-91 [PubMed] Free Access to Full Article Related Publications
The risk of radiotherapy-related secondary cancers in children with constitutional retinoblastoma 1 (RB1) mutations has led to reduced use of external beam radiotherapy (EBRT) for RB. Presently, tumor reduction with chemotherapy with or without focal surgery (chemosurgery) is most commonly undertaken; EBRT is avoided as much as possible and is considered only as the last treatment option prior to enucleation. Nevertheless, approximately 80% of patients are diagnosed at a locally advanced stage, and only 20-25% of early stage RB patients can be cured with a chemosurgery strategy. As a whole, chemotherapy fails in more than two-thirds of eyes with advanced stage disease, requiring EBRT or enucleation. Radiotherapy is still considered necessary for patients with large tumor(s) who are not candidates for chemosurgery but who have visual potential. When radiation therapy is indicated, the lowest possible radiation dose combined with systemic or local chemotherapy and focal surgery may yield the best clinical outcomes in terms of local control and treatment-related toxicity. Proton beam therapy is one EBRT method that can be used for treatment of RB and reduces the radiation dose delivered to the adjacent orbital bone while maintaining an adequate dose to the tumor. To maximize the therapeutic success of treatment of advanced RB, the possibility of integrating radiotherapy at early stages of treatment may need to be discussed by a multidisciplinary team, rather than considering EBRT as only a last treatment option.

Tasiou V, Hiber M, Steenpass L
A Mouse Model for Imprinting of the Human Retinoblastoma Gene.
PLoS One. 2015; 10(8):e0134672 [PubMed] Free Access to Full Article Related Publications
The human RB1 gene is imprinted due to integration of the PPP1R26P1 pseudogene into intron 2. PPP1R26P1 harbors the gametic differentially methylated region of the RB1 gene, CpG85, which is methylated in the female germ line. The paternally unmethylated CpG85 acts as promoter for the alternative transcript 2B of RB1, which interferes with expression of full-length RB1 in cis. In mice, PPP1R26P1 is not present in the Rb1 gene and Rb1 is not imprinted. Assuming that the mechanisms responsible for genomic imprinting are conserved, we investigated if imprinting of mouse Rb1 can be induced by transferring human PPP1R26P1 into mouse Rb1. We generated humanized Rb1_PPP1R26P1 knock-in mice that pass human PPP1R26P1 through the mouse germ line. We found that the function of unmethylated CpG85 as promoter for an alternative Rb1 transcript and as cis-repressor of the main Rb1 transcript is maintained in mouse tissues. However, CpG85 is not recognized as a gametic differentially methylated region in the mouse germ line. DNA methylation at CpG85 is acquired only in tissues of neuroectodermal origin, independent of parental transmission of PPP1R26P1. Absence of CpG85 methylation in oocytes and sperm implies a failure of imprint methylation establishment in the germ line. Our results indicate that site-specific integration of a proven human gametic differentially methylated region is not sufficient for acquisition of DNA methylation in the mouse germ line, even if promoter function of the element is maintained. This suggests a considerable dependency of DNA methylation induction on the surrounding sequence. However, our model is suited to determine the cellular function of the alternative Rb1 transcript.

Malusa F, Taranta M, Zaki N, et al.
Time-course gene profiling and networks in demethylated retinoblastoma cell line.
Oncotarget. 2015; 6(27):23688-707 [PubMed] Free Access to Full Article Related Publications
Retinoblastoma, a very aggressive cancer of the developing retina, initiatiates by the biallelic loss of RB1 gene, and progresses very quickly following RB1 inactivation. While its genome is stable, multiple pathways are deregulated, also epigenetically. After reviewing the main findings in relation with recently validated markers, we propose an integrative bioinformatics approach to include in the previous group new markers obtained from the analysis of a single cell line subject to epigenetic treatment. In particular, differentially expressed genes are identified from time course microarray experiments on the WERI-RB1 cell line treated with 5-Aza-2'-deoxycytidine (decitabine; DAC). By inducing demethylation of CpG island in promoter genes that are involved in biological processes, for instance apoptosis, we performed the following main integrative analysis steps: i) Gene expression profiling at 48h, 72h and 96h after DAC treatment; ii) Time differential gene co-expression networks and iii) Context-driven marker association (transcriptional factor regulated protein networks, master regulatory paths). The observed DAC-driven temporal profiles and regulatory connectivity patterns are obtained by the application of computational tools, with support from curated literature. It is worth emphasizing the capacity of networks to reconcile multi-type evidences, thus generating testable hypotheses made available by systems scale predictive inference power. Despite our small experimental setting, we propose through such integrations valuable impacts of epigenetic treatment in terms of gene expression measurements, and then validate evidenced apoptotic effects.

Ottaviani D, Alonso C, Szijan I
Uncommon RB1 somatic mutations in a unilateral retinoblastoma patient.
Medicina (B Aires). 2015; 75(3):137-41 [PubMed] Related Publications
Retinoblastoma (RB) is the most common primary intraocular malignancy in children. Somatic inactivation of both alleles of the RB1 tumor suppressor gene in a developing retina is a crucial event in the initiation of tumorigenesis in most cases of isolated unilateral retinoblastoma. We analyzed the DNA from tumor tissue and peripheral blood of a unilateral retinoblastoma patient to determine the RB1 mutation status and to provide an accurate genetic counseling. A comprehensive approach, based on our previous experience, was used to identify the causative RB1 mutations. Screening for RB1 mutations was performed by PCR direct sequencing, multiplex ligation-dependent probe amplification (MLPA) and Real Time-PCR analyses. Three different mutations were identified in the tumor DNA, which were absent in blood DNA. The somatic origin of these mutations was vital to rule out the heritable condition in this patient.

Thirumalairaj K, Abraham A, Devarajan B, et al.
A stepwise strategy for rapid and cost-effective RB1 screening in Indian retinoblastoma patients.
J Hum Genet. 2015; 60(9):547-52 [PubMed] Related Publications
India has the highest number of retinoblastoma (RB) patients among the developing countries owing to its increasing population. Of the patients with RB, about 40% have the heritable form of the disease, making genetic analysis of the RB1 gene an integral part of disease management. However, given the large size of the RB1 gene with its widely dispersed exons and no reported hotspots, genetic testing can be cumbersome. To overcome this problem, we have developed a rapid screening strategy by prioritizing the order of exons to be analyzed, based on the frequency of nonsense mutations, deletions and duplications reported in the RB1-Leiden Open Variation Database and published literature on Indian patients. Using this strategy for genetic analysis, mutations were identified in 76% of patients in half the actual time and one third of the cost. This reduction in time and cost will allow for better risk prediction for siblings and offspring, thereby facilitating genetic counseling for families, especially in developing countries.

Khosravi A, Shahrabi S, Shahjahani M, Saki N
The bone marrow metastasis niche in retinoblastoma.
Cell Oncol (Dordr). 2015; 38(4):253-63 [PubMed] Related Publications
BACKGROUND: Retinoblastoma (Rb) is a progressive cancer which mainly occurs in children, and which is caused by different genetic or epigenetic alterations that lead to inactivation of both alleles of the RB1 gene. Hereditary and non-hereditary forms of Rb do exist, and the hereditary form is associated with an increased risk of secondary malignancies. Metastasis to distant organs is a critical feature of many tumors, and may be caused by various molecular alterations at different stages. Recognition of these alterations and, thus, insight into the processes underlying the development of metastases may result in novel preventive as well as effective targeted treatment options. Rb is associated with metastases to various organs and tissues, including the bone marrow (BM).
METHODS: Here, we provide an overview of mutations and other molecular changes known to be involved in Rb development and metastasis to the BM. This overview is based on a literature search ranging from 1990 to 2015.
CONCLUSIONS: The various BM metastasis-related molecular changes identified to date may be instrumental for a better diagnosis, prognosis and classification of Rb patients, as well as for the development of novel comprehensive (targeted) therapies.

Butyaev A, Mavlyutov R, Blanchette M, et al.
A low-latency, big database system and browser for storage, querying and visualization of 3D genomic data.
Nucleic Acids Res. 2015; 43(16):e103 [PubMed] Free Access to Full Article Related Publications
Recent releases of genome three-dimensional (3D) structures have the potential to transform our understanding of genomes. Nonetheless, the storage technology and visualization tools need to evolve to offer to the scientific community fast and convenient access to these data. We introduce simultaneously a database system to store and query 3D genomic data (3DBG), and a 3D genome browser to visualize and explore 3D genome structures (3DGB). We benchmark 3DBG against state-of-the-art systems and demonstrate that it is faster than previous solutions, and importantly gracefully scales with the size of data. We also illustrate the usefulness of our 3D genome Web browser to explore human genome structures. The 3D genome browser is available at http://3dgb.cs.mcgill.ca/.

Norberg SM, Oros M, Manucha V, et al.
Loss of e-cadherin and retinoblastoma genes in a case of urothelial carcinoma with scrotal metastasis.
Can J Urol. 2015; 22(2):7755-7 [PubMed] Related Publications
Cutaneous metastases from urologic cancers are very uncommon, usually represent widespread metastatic disease and are associated with a very poor prognosis. They may occur in 1% of patients with urologic malignancies, most commonly from kidney, followed by bladder and prostate tumors. In this report, we describe a case of urothelial carcinoma with metastases to the scrotum treated with platinum based chemotherapy with a durable complete response lasting more than 14 months. Molecular profiling revealed deleterious mutations in e-cadherin and retinoblastoma genes, suggesting their possible role in the pathogenesis of cutaneous metastases. Further studies are needed to validate this observation.

Benavente CA, Dyer MA
Genetics and epigenetics of human retinoblastoma.
Annu Rev Pathol. 2015; 10:547-62 [PubMed] Related Publications
Retinoblastoma is a pediatric tumor of the developing retina from which the genetic basis for cancer development was first described. Inactivation of both copies of the RB1 gene is the predominant initiating genetic lesion in retinoblastoma and is rate limiting for tumorigenesis. Recent whole-genome sequencing of retinoblastoma uncovered a tumor that had no coding-region mutations or focal chromosomal lesions other than in the RB1 gene, shifting the paradigm in the field. The retinoblastoma genome can be very stable; therefore, epigenetic deregulation of tumor-promoting pathways is required for tumorigenesis. This review highlights the genetic and epigenetic changes in retinoblastoma that have been reported, with special emphasis on recent whole-genome sequencing and epigenetic analyses that have identified novel candidate genes as potential therapeutic targets.

Tsang JY, Ni YB, Ng EK, et al.
MicroRNAs are differentially deregulated in mammary malignant phyllodes tumour.
Histopathology. 2015; 67(3):294-305 [PubMed] Related Publications
AIMS: MicroRNAs (miRs) have been shown to play important roles in tumour progression. Their expression pattern can be useful for cancer classification. However, little is known about miRs in mammary phyllodes tumours (PT).
METHODS AND RESULTS: In this study, polymerase chain reaction (PCR)-based miR profiling was performed in a small PT cohort to identify deregulated miRs in malignant PT. The purported roles and targets of these miRs were further validated. Unsupervised clustering of miR expression profiling segregated PT into different grades, implicating the miR profile in PT classification. Among the deregulated miRs, miR-21, miR-335 and miR-155 were validated to be higher in malignant than in lower-grade PT in the independent cohort by quantitative PCR (qPCR) (P ≤ 0.032). Their expression correlated with some of the malignant histological features, including high stromal cellularity, nuclear pleomorphism and mitosis. Subsequent analysis of their downstream proteins, namely PTEN for miR-21/miR-155 and Rb for miR-335, also showed an independent significant negative association between miR and protein expression.
CONCLUSIONS: Differential expression of miRs in PT could be useful in diagnosis and grading of PT. Their deregulated expression, together with the altered downstream targets, implicated their active involvement in PT malignant transformation.

Musahl AS, Huang X, Rusakiewicz S, et al.
A long non-coding RNA links calreticulin-mediated immunogenic cell removal to RB1 transcription.
Oncogene. 2015; 34(39):5046-54 [PubMed] Related Publications
A subset of promoters bidirectionally expresses long non-coding RNAs (ncRNAs) of unknown function and protein-coding genes (PCGs) in parallel. Here, we define a set of 1107 highly conserved human bidirectional promoters that mediate the linked expression of long ncRNAs and PCGs. Depletion of the long ncRNA expressed from the RB1 promoter, ncRNA-RB1, reveals regulatory effects different from the RB1-controlled transcriptional program. ncRNA-RB1 positively regulates the expression of calreticulin (CALR) that in response to certain therapeutic interventions can translocate from the endoplasmic reticulum to the cell surface, hence activating anticancer immune responses. Knockdown of ncRNA-RB1 in tumor cells reduced expression of CALR, impaired the translocation of the protein to the cell surface upon treatment with anthracylines and consequently inhibited the cellular uptake by macrophages. In conclusion, co-transcription of ncRNA-RB1 and RB1 provides a positive link between the expression of the two tumor suppressors RB1 and the immune-relevant CALR protein. This regulatory interplay exemplifies disease-relevant co-regulation of two distinct gene products, in which loss of expression of one oncosuppressor protein entails the abolition of additional tumor-inhibitory mechanisms.

Rodriguez-Galindo C, Orbach DB, VanderVeen D
Retinoblastoma.
Pediatr Clin North Am. 2015; 62(1):201-23 [PubMed] Related Publications
Retinoblastoma is the most common neoplasm of the eye in childhood, and represents 3% of all childhood malignancies. Retinoblastoma is a cancer of the very young; two-thirds are diagnosed before 2 years of age and 95% before 5 years. Retinoblastoma presents in 2 distinct clinical forms: (1) a bilateral or multifocal, heritable form (25% of all cases), characterized by the presence of germline mutations of the RB1 gene; and (2) a unilateral or unifocal form (75% of all cases), 90% of which are nonhereditary. The treatment of retinoblastoma is multidisciplinary and is designed primarily to save life and preserve vision.

Xu XL, Singh HP, Wang L, et al.
Rb suppresses human cone-precursor-derived retinoblastoma tumours.
Nature. 2014; 514(7522):385-8 [PubMed] Free Access to Full Article Related Publications
Retinoblastoma is a childhood retinal tumour that initiates in response to biallelic RB1 inactivation and loss of functional retinoblastoma (Rb) protein. Although Rb has diverse tumour-suppressor functions and is inactivated in many cancers, germline RB1 mutations predispose to retinoblastoma far more strongly than to other malignancies. This tropism suggests that retinal cell-type-specific circuitry sensitizes to Rb loss, yet the nature of the circuitry and the cell type in which it operates have been unclear. Here we show that post-mitotic human cone precursors are uniquely sensitive to Rb depletion. Rb knockdown induced cone precursor proliferation in prospectively isolated populations and in intact retina. Proliferation followed the induction of E2F-regulated genes, and depended on factors having strong expression in maturing cone precursors and crucial roles in retinoblastoma cell proliferation, including MYCN and MDM2. Proliferation of Rb-depleted cones and retinoblastoma cells also depended on the Rb-related protein p107, SKP2, and a p27 downregulation associated with cone precursor maturation. Moreover, Rb-depleted cone precursors formed tumours in orthotopic xenografts with histological features and protein expression typical of human retinoblastoma. These findings provide a compelling molecular rationale for a cone precursor origin of retinoblastoma. More generally, they demonstrate that cell-type-specific circuitry can collaborate with an initiating oncogenic mutation to enable tumorigenesis.

Gao R, Zhou X, Yang Y, Wang Z
Transfection of wtp53 and Rb94 genes into retinoblastomas of nude mice by ultrasound-targeted microbubble destruction.
Ultrasound Med Biol. 2014; 40(11):2662-70 [PubMed] Related Publications
Using ultrasound-targeted microbubble destruction (UTMD), we transfected both wild-type p53 (wtp53) and Rb94 genes into retinoblastomas (RBs) of nude mice to investigate the inhibitory role of these two genes in RB development. The 40 tumor-bearing mice, which had been established by sub-retinal injection of an HXO-Rb44 cell suspension, were randomly divided into five groups: blank control group (C); blank plasmid group (M); wtp53 plasmid group (p53); Rb94 plasmid group (Rb94); wtp53 + Rb94 plasmid group (p53 + Rb94). For preparation of the DNA-loaded microbubbles, a pre-determined amount of blank plasmid, pVIVO1-p53, pVIVO1-Rb94 or pVIVO1-p53-Rb94 was added and mixed with the microbubbles. Then, these DNA-loaded microbubbles were respectively transfected into the animal model by UTMD. Vascular endothelial growth factor level and microvessel density of the tumor were determined by immunohistochemical staining. Apoptosis of tissues was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Expression of wtp53 and Rb94 at both the gene and protein levels was detected by RT-PCR (reverse transcription polymerase chain reaction) and Western blot, respectively. Transfection of both genes had greater inhibitory effects on RB development and resulted in lower levels of vascular endothelial growth factor, lower microvessel density and more obvious apoptosis than single-gene transfection (p < 0.05). The results indicate that the transfection of both genes into the RB by UTMD more strongly inhibited RB growth than transfection of a single gene.

Ayari Jeridi H, Bouguila H, Ansperger-Rescher B, et al.
Genetic testing in Tunisian families with heritable retinoblastoma using a low cost approach permits accurate risk prediction in relatives and reveals incomplete penetrance in adults.
Exp Eye Res. 2014; 124:48-55 [PubMed] Related Publications
Heritable retinoblastoma is caused by oncogenic mutations in the RB1 tumor suppressor gene. Identification of these mutations in patients is important for genetic counseling and clinical management of relatives at risk. In order to lower analytical efforts, we designed a stepwise mutation detection strategy that was adapted to the spectrum of oncogenic RB1 gene mutations. We applied this strategy on 20 unrelated patients with familial and/or de novo bilateral retinoblastoma from Tunisia. In 19 (95%) patients, we detected oncogenic mutations including base substitutions, small length mutations, and large deletions. Further analyses on the origin of the mutations showed mutational mosaicism in one unilaterally affected father of a bilateral proband and incomplete penetrance in two mothers. In a large family with several retinoblastoma patients, the mutation identified in the index patient was also detected in several non-penetrant relatives. RNA analyses showed that this mutation results in an in-frame loss of exon 9. In summary, our strategy can serve as a model for RB1 mutation identification with high analytical sensitivity. Our results point out that genetic testing is needed to reveal or exclude incomplete penetrance specifically in parents of patients with sporadic disease.

Zhang T, Liao Y, Hsu FN, et al.
Hyperactivated Wnt signaling induces synthetic lethal interaction with Rb inactivation by elevating TORC1 activities.
PLoS Genet. 2014; 10(5):e1004357 [PubMed] Free Access to Full Article Related Publications
Inactivation of the Rb tumor suppressor can lead to increased cell proliferation or cell death depending on specific cellular context. Therefore, identification of the interacting pathways that modulate the effect of Rb loss will provide novel insights into the roles of Rb in cancer development and promote new therapeutic strategies. Here, we identify a novel synthetic lethal interaction between Rb inactivation and deregulated Wg/Wnt signaling through unbiased genetic screens. We show that a weak allele of axin, which deregulates Wg signaling and increases cell proliferation without obvious effects on cell fate specification, significantly alters metabolic gene expression, causes hypersensitivity to metabolic stress induced by fasting, and induces synergistic apoptosis with mutation of fly Rb ortholog, rbf. Furthermore, hyperactivation of Wg signaling by other components of the Wg pathway also induces synergistic apoptosis with rbf. We show that hyperactivated Wg signaling significantly increases TORC1 activity and induces excessive energy stress with rbf mutation. Inhibition of TORC1 activity significantly suppressed synergistic cell death induced by hyperactivated Wg signaling and rbf inactivation, which is correlated with decreased energy stress and decreased induction of apoptotic regulator expression. Finally the synthetic lethality between Rb and deregulated Wnt signaling is conserved in mammalian cells and that inactivation of Rb and APC induces synergistic cell death through a similar mechanism. These results suggest that elevated TORC1 activity and metabolic stress underpin the evolutionarily conserved synthetic lethal interaction between hyperactivated Wnt signaling and inactivated Rb tumor suppressor.

Jehanne M, Brisse H, Gauthier-Villars M, et al.
[Retinoblastoma: recent advances].
Bull Cancer. 2014; 101(4):380-7 [PubMed] Related Publications
Retinoblastoma is the most common intraocular malignancy of infancy with an incidence of 1/15,000 to 1/20,000 births. Sixty percent of retinoblastomas are unilateral, with a median age at diagnosis of two years, and in most cases are not hereditary. Retinoblastoma is bilateral in 40% of cases, with an earlier median age at diagnosis of one year. All bilateral and multifocal unilateral forms are hereditary and are part of a genetic cancer predisposition syndrome. All children with a bilateral or familial form, and 10 to 15% of children with an unilateral form, constitutionally carry an RB1 gene mutation. The two most frequent symptoms revealing retinoblastoma are leukocoria and strabismus. Diagnosis is made by fundoscopy, with ultrasound and magnetic resonance imaging (MRI) contributing both to diagnosis and assessment of the extension of the disease. Treatment of patients with retinoblastoma must take into account the various aspects of the disease (unilateral/bilateral, size, localization…), the risk to vision and the possible hereditary nature of the disease. The main prognostic aspects are still premature detection and adapted coverage by a multi-disciplinary specialized team. Enucleation is still often necessary in unilateral disease; the decision for adjuvant treatment is taken according to the histological risk factors. The most important recent therapeutic advances concern the conservative treatment which is proposed for at least one of the two eyes in most bilateral cases: laser alone or in combination with chemotherapy, cryotherapy or brachytherapy. Recently, the development of new conservative techniques of treatment, such as intra-arterial selective chemotherapy perfusion, aims at preserving visual function in these children and decreasing the number of enucleations and the need for external beam radiotherapy. The vital prognosis related to retinoblatoma is now excellent in industrialized countries, but long-term survival is still related to the development of secondary tumors, mainly secondary sarcoma. Retinoblastoma requires multi-disciplinary care as well as a long term specialized follow-up. Early counseling of patients and their family concerning the risk of transmission of the disease and the risk of development of secondary tumors is necessary.

Little MP, Schaeffer ML, Reulen RC, et al.
Breast cancer risk after radiotherapy for heritable and non-heritable retinoblastoma: a US-UK study.
Br J Cancer. 2014; 110(10):2623-32 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Retinoblastoma is a rare childhood eye cancer caused by germline or somatic mutations in the RB1 gene. Previous studies observed elevated breast cancer risk among retinoblastoma survivors. However, there has been no research on breast cancer risk in relation to radiation (primarily scatter radiation from the primary treatment) and genetic susceptibility of retinoblastoma survivors.
METHODS: Two groups of retinoblastoma survivors from the US and UK were selected, and breast cancer risk analysed using a case-control methodology, nesting within the respective cohorts, matching on heritability (that is to say, having bilateral retinoblastoma or being unilateral cases with at least one relative with retinoblastoma), and using exact statistical methods. There were a total of 31 cases and 77 controls.
RESULTS: Overall there was no significant variation of breast cancer risk with dose (P>0.5). However, there was a pronounced and significant (P=0.047) increase in the risk of breast cancer with increasing radiation dose for non-heritable retinoblastoma patients and a slight and borderline significant (P=0.072) decrease in risk of breast cancer with increasing radiation dose for heritable retinoblastoma patients, implying significant (P=0.024) heterogeneity in radiation risk between the heritable and non-heritable retinoblastoma groups; this was unaffected by the blindness status. There was no significant effect of any type of alkylating-agent chemotherapy on breast cancer risk (P>0.5).
CONCLUSIONS: There is significant radiation-related risk of breast cancer for non-heritable retinoblastoma survivors but no excess risk for heritable retinoblastoma survivors, and no significant risk overall. However, these results are based on very small numbers of cases; therefore, they must be interpreted with caution.

Riba A, Bosia C, El Baroudi M, et al.
A combination of transcriptional and microRNA regulation improves the stability of the relative concentrations of target genes.
PLoS Comput Biol. 2014; 10(2):e1003490 [PubMed] Free Access to Full Article Related Publications
It is well known that, under suitable conditions, microRNAs are able to fine tune the relative concentration of their targets to any desired value. We show that this function is particularly effective when one of the targets is a Transcription Factor (TF) which regulates the other targets. This combination defines a new class of feed-forward loops (FFLs) in which the microRNA plays the role of master regulator. Using both deterministic and stochastic equations, we show that these FFLs are indeed able not only to fine-tune the TF/target ratio to any desired value as a function of the miRNA concentration but also, thanks to the peculiar topology of the circuit, to ensure the stability of this ratio against stochastic fluctuations. These two effects are due to the interplay between the direct transcriptional regulation and the indirect TF/Target interaction due to competition of TF and target for miRNA binding (the so called "sponge effect"). We then perform a genome wide search of these FFLs in the human regulatory network and show that they are characterized by a very peculiar enrichment pattern. In particular, they are strongly enriched in all the situations in which the TF and its target have to be precisely kept at the same concentration notwithstanding the environmental noise. As an example we discuss the FFL involving E2F1 as Transcription Factor, RB1 as target and miR-17 family as master regulator. These FFLs ensure a tight control of the E2F/RB ratio which in turns ensures the stability of the transition from the G0/G1 to the S phase in quiescent cells.

McEvoy J, Nagahawatte P, Finkelstein D, et al.
RB1 gene inactivation by chromothripsis in human retinoblastoma.
Oncotarget. 2014; 5(2):438-50 [PubMed] Free Access to Full Article Related Publications
Retinoblastoma is a rare childhood cancer of the developing retina. Most retinoblastomas initiate with biallelic inactivation of the RB1 gene through diverse mechanisms including point mutations, nucleotide insertions, deletions, loss of heterozygosity and promoter hypermethylation. Recently, a novel mechanism of retinoblastoma initiation was proposed. Gallie and colleagues discovered that a small proportion of retinoblastomas lack RB1 mutations and had MYCN amplification [1]. In this study, we identified recurrent chromosomal, regional and focal genomic lesions in 94 primary retinoblastomas with their matched normal DNA using SNP 6.0 chips. We also analyzed the RB1 gene mutations and compared the mechanism of RB1 inactivation to the recurrent copy number variations in the retinoblastoma genome. In addition to the previously described focal amplification of MYCN and deletions in RB1 and BCOR, we also identified recurrent focal amplification of OTX2, a transcription factor required for retinal photoreceptor development. We identified 10 retinoblastomas in our cohort that lacked RB1 point mutations or indels. We performed whole genome sequencing on those 10 tumors and their corresponding germline DNA. In one of the tumors, the RB1 gene was unaltered, the MYCN gene was amplified and RB1 protein was expressed in the nuclei of the tumor cells. In addition, several tumors had complex patterns of structural variations and we identified 3 tumors with chromothripsis at the RB1 locus. This is the first report of chromothripsis as a mechanism for RB1 gene inactivation in cancer.

Jones K, Minassian BA
Genetic testing in infantile spasms identifies a chromosome 13q deletion and retinoblastoma.
Pediatr Neurol. 2014; 50(5):522-4 [PubMed] Related Publications
BACKGROUND: Infantile spasms is an epileptic encephalopathy and the common final manifestation of numerous disparate insults to the developing brain during infancy. The varied etiologies may be structural, metabolic, genetic, or unknown. Etiological diagnosis is important as it may lead to specific therapy, which may affect developmental outcome.
PATIENT: We report a case of infantile spasms of unknown etiology with dysmorphic features, in which genetic copy number variation microarray testing was included in the investigation of the cause of the disease.
RESULTS: A large deletion of chromosome 13 was identified in the region 13q13 to 13q21.3 encompassing the retinoblastoma gene (13q14.2). Urgent ophthalmological evaluation revealed an asymptomatic retinoblastoma of the left eye, leading to early treatment.
CONCLUSION: This is the first case report of infantile spasms specifically associated with a chromosome 13q deletion. Chromosomal region 13q13 to 13q21.3 may contain one or more genes whose hemizygous loss leads to infantile spasms. Copy number variation testing for cryptogenic infantile spasms led to the discovery of a mutation responsible for retinoblastoma, enabling early diagnosis and treatment of a potentially life-threatening cancer. High-sensitivity molecular diagnosis improves health care and substantially reduces expenses. This shift in diagnostic evaluation is broadly relevant to health care.

Pinto-Leite R, Carreira I, Melo J, et al.
Genomic characterization of three urinary bladder cancer cell lines: understanding genomic types of urinary bladder cancer.
Tumour Biol. 2014; 35(5):4599-617 [PubMed] Related Publications
Several genomic regions are frequently altered and associated with the type, stage and progression of urinary bladder cancer (UBC). We present the characterization of 5637, T24 and HT1376 UBC cell lines by karyotyping, fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH) and multiplex ligation-dependent probe amplification (MLPA) analysis. Some cytogenetic anomalies present in UBC were found in the three cell lines, such as chromosome 20 aneuploidy and the loss of 9p21. Some gene loci losses (e.g. CDKN2A) and gains (e.g. HRAS, BCL2L1 and PTPN1) were coincident across all cell lines. Although some significant heterogeneity and complexity were detected between them, their genomic profiles exhibited a similar pattern to UBC. We suggest that 5637 and HT1376 represent the E2F3/RB1 pathway due to amplification of 6p22.3, concomitant with loss of one copy of RB1 and mutation of the remaining copy. The HT1376 presented a 10q deletion involving PTEN region and no alteration of PIK3CA region which, in combination with the inactivation of TP53, bears more invasive and metastatic properties than 5637. The T24 belongs to the alternative pathway of FGFR3/CCND1 by presenting mutated HRAS and over-represented CCND1. These cell lines cover the more frequent subtypes of UBC and are reliable models that can be used, as a group, in preclinical studies.

Cimino PJ, Robirds DH, Tripp SR, et al.
Retinoblastoma gene mutations detected by whole exome sequencing of Merkel cell carcinoma.
Mod Pathol. 2014; 27(8):1073-87 [PubMed] Related Publications
Merkel cell carcinoma is a highly aggressive cutaneous neuroendocrine tumor that has been associated with Merkel cell polyomavirus in up to 80% of cases. Merkel cell polyomavirus is believed to influence pathogenesis, at least in part, through expression of the large T antigen, which includes a retinoblastoma protein-binding domain. However, there appears to be significant clinical and morphological overlap between polyomavirus-positive and polyomavirus-negative Merkel cell carcinoma cases. Although much of the recent focus of Merkel cell carcinoma pathogenesis has been on polyomavirus, the pathogenesis of polyomavirus-negative cases is still poorly understood. We hypothesized that there are underlying human somatic mutations that unify Merkel cell carcinoma pathogenesis across polyomavirus status, and to investigate we performed whole exome sequencing on five polyomavirus-positive cases and three polyomavirus-negative cases. We found that there were no significant differences in the overall number of single-nucleotide variations, copy number variations, insertion/deletions, and chromosomal rearrangements when comparing polyomavirus-positive to polyomavirus-negative cases. However, we did find that the retinoblastoma pathway genes harbored a high number of mutations in Merkel cell carcinoma. Furthermore, the retinoblastoma gene (RB1) was found to have nonsense truncating protein mutations in all three polyomavirus-negative cases; no such mutations were found in the polyomavirus-positive cases. In all eight cases, the retinoblastoma pathway dysregulation was confirmed by immunohistochemistry. Although polyomavirus-positive Merkel cell carcinoma is believed to undergo retinoblastoma dysregulation through viral large T antigen expression, our findings demonstrate that somatic mutations in polyomavirus-negative Merkel cell carcinoma lead to retinoblastoma dysregulation through an alternative pathway. This novel finding suggests that the retinoblastoma pathway dysregulation leads to an overlapping Merkel cell carcinoma phenotype and that oncogenesis occurs through either a polyomavirus-dependent (viral large T antigen expression) or polyomavirus-independent (host somatic mutation) mechanism.

Chen Z, Moran K, Richards-Yutz J, et al.
Enhanced sensitivity for detection of low-level germline mosaic RB1 mutations in sporadic retinoblastoma cases using deep semiconductor sequencing.
Hum Mutat. 2014; 35(3):384-91 [PubMed] Free Access to Full Article Related Publications
Sporadic retinoblastoma (RB) is caused by de novo mutations in the RB1 gene. Often, these mutations are present as mosaic mutations that cannot be detected by Sanger sequencing. Next-generation deep sequencing allows unambiguous detection of the mosaic mutations in lymphocyte DNA. Deep sequencing of the RB1 gene on lymphocyte DNA from 20 bilateral and 70 unilateral RB cases was performed, where Sanger sequencing excluded the presence of mutations. The individual exons of the RB1 gene from each sample were amplified, pooled, ligated to barcoded adapters, and sequenced using semiconductor sequencing on an Ion Torrent Personal Genome Machine. Six low-level mosaic mutations were identified in bilateral RB and four in unilateral RB cases. The incidence of low-level mosaic mutation was estimated to be 30% and 6%, respectively, in sporadic bilateral and unilateral RB cases, previously classified as mutation negative. The frequency of point mutations detectable in lymphocyte DNA increased from 96% to 97% for bilateral RB and from 13% to 18% for unilateral RB. The use of deep sequencing technology increased the sensitivity of the detection of low-level germline mosaic mutations in the RB1 gene. This finding has significant implications for improved clinical diagnosis, genetic counseling, surveillance, and management of RB.

Further References

Harbour JW
Overview of RB gene mutations in patients with retinoblastoma. Implications for clinical genetic screening.
Ophthalmology. 1998; 105(8):1442-7 [PubMed] Related Publications
OBJECTIVE: This study aimed to determine the distribution of germline mutations in the retinoblastoma (RB) gene in patients with retinoblastoma to design more effective genetic testing.
DESIGN: A meta-analysis.
PARTICIPANTS: 192 cases identified from literature.
METHODS: All identifiable reported cases of bilateral retinoblastoma, which included DNA sequence analysis of the RB gene, were reviewed.
MAIN OUTCOME MEASURE: Type of genetic mutation.
RESULTS: Among 192 patients with retinoblastoma with identifiable germline mutations in the RB gene, the DNA alteration was a nonsense mutation in 83 (43%), frameshift in 67 (35%), intron mutation in 23 (12%), missense mutation in 11 (6%), in-frame deletion in 5 (3%), and promoter mutation in 3 (2%). Mutations were distributed throughout 24 of the 27 exons of the RB gene with no single mutational "hotspot." Exons 8, 17, 18, and 23 were involved most often, and 189 (98%) of the mutations were predicted to affect the RB large pocket domain.
CONCLUSIONS: A single genetic test is unlikely to detect all germline RB gene mutations in patients with retinoblastoma because of the variety of types and locations of mutations that occur. However, a series of complementary tests may be able to rapidly detect mutations based on the observation that most mutations alter the protein size and disrupt the large pocket domain.

Toguchida J, Ishizaki K, Sasaki MS, et al.
Chromosomal reorganization for the expression of recessive mutation of retinoblastoma susceptibility gene in the development of osteosarcoma.
Cancer Res. 1988; 48(14):3939-43 [PubMed] Related Publications
Recent evidence indicates that the mutation of retinoblastoma susceptibility (RB) gene is also involved in the development of osteosarcoma. We studied 30 cases of osteosarcoma for the structural anomalies of the RB gene by Southern hybridization analysis with cDNA probes of the RB gene. Thirteen cases (43%) showed structural anomalies of the RB gene. They included the total or partial deletion, or rearrangement of the RB gene; seven with homozygous deletions and six with hemizygous deletions or rearrangements. By the use of restriction fragment length polymorphism fragments as chromosome markers, those seven tumors having homozygous deletions and four of six tumors having hemizygous anomalies showed the loss of heterozygosity at other loci on chromosome 13. Among those tumors with no apparent structural changes of the RB gene, seven cases showed the loss of heterozygosity on chromosome 13, and altogether the loss of heterozygosity by either homozygosity or hemizygosity was found in 18 (64%) of 28 informative cases. The loss of heterozygosity was also found for nine of 10 other chromosomes, of which chromosome 17 showed the highest frequency (77%). The tumors with loss of chromosome 13 alleles also showed additional losses of alleles on other chromosomes, while tumors retaining heterozygosity of chromosome 13 also retained heterozygosity at the informative loci on other chromosomes. Southern hybridization and karyotype analysis in some selected cases suggest that the concerted loss of heterozygosity at multiple loci may be a consequence of the polyploidization-segregation process.

Miller CW, Aslo A, Won A, et al.
Alterations of the p53, Rb and MDM2 genes in osteosarcoma.
J Cancer Res Clin Oncol. 1996; 122(9):559-65 [PubMed] Related Publications
Molecular defects affecting tumor-suppressor genes are an important step in the genesis of sarcomas. For example, inheritance of a defective Rb or p53 gene predisposes the carrier to develop osteosarcoma, among other malignancies. In this study, we have assessed the occurrence of p53, Rb and MDM2 alterations in the same samples of osteosarcomas, along with representative samples of various other sarcomas. Point mutations of the p53 gene were found in 13 of 42 osteosarcomas and 1 of 8 leiomyosarcomas, and gross rearrangement of the p53 gene was demonstrated in 5 of 37 osteosarcomas. The retinoblastoma susceptibility gene (Rb) was either rearranged or deleted in 7 of 37 osteosarcomas, 1 of 7 soft-tissue sarcomas and 1 of 4 Ewing sarcomas. Remarkably, 5 of the osteosarcomas having Rb alterations also had p53 mutations. Amplification and overexpression of the MDM2 oncogene may lead to increased MDM2-p53 binding resulting in inactivation of p53 function. A two- to threefold increase in the copy number of MDM2 was detected in 7 of 37 samples, 5 of which were osteosarcomas. Amplification of the MDM2 gene occurred independently of p53 mutation; one sample having threefold amplification of MDM2 also had a p53 mutation. In summary, 34 alterations of the p53, Rb and MDM2 genes were found in 26 of 42 (62%) osteosarcomas.

Kelley MJ, Nakagawa K, Steinberg SM, et al.
Differential inactivation of CDKN2 and Rb protein in non-small-cell and small-cell lung cancer cell lines.
J Natl Cancer Inst. 1995; 87(10):756-61 [PubMed] Related Publications
BACKGROUND: The CDKN2 gene encodes the human cyclin-dependent kinase 4 inhibitor. This inhibitor protein is believed to be a tumor suppressor that plays an essential role in cell cycle regulation. One half of all cancer cell lines and one fourth of lung cancer cell lines examined to date contain homozygous deletions (i.e., both alleles lost) of CDKN2. However, the relative frequency of homozygous CDKN2 deletions in non-small-cell lung cancers (NSCLC) and in small-cell lung cancers (SCLC) has not been determined. Inactivation or loss of another tumor suppressor encoded by the retinoblastoma gene (the Rb protein) is more common in SCLC than in NSCLC.
PURPOSE: We measured the frequency of homozygous CDKN2 deletions in 77 NSCLC and in 93 SCLC tumor cell lines. In addition, possible associations were explored between CDKN2 gene loss, the presence or absence of Rb protein, and the clinical status of lung cancer patients.
METHODS: DNA was isolated from each tumor cell line and from the primary tumor and normal tissue of one NSCLC patient. Sequences corresponding to exons 1 and 2 of the CDKN2 gene were amplified by use of the polymerase chain reaction, and the resulting amplification products were analyzed by agarose gel electrophoresis and DNA blotting. Genomic DNA blotting was also used to evaluate CDKN2 gene deletions. The frequency of homozygous CDKN2 loss and the presence or absence of functional Rb protein (reported previously) in the cell lines were compared.
RESULTS: Homozygous deletion of CDKN2 was detected in 18 (23%) of 77 cell lines established from patients with NSCLC, compared with one (1%) of 93 cell lines established from patients with SCLC (P < .001). No CDKN2 gene loss was observed in the normal tissue of an NSCLC patient whose tumor cell line showed homozygous deletion of the gene; however, the primary tumor from this patient had evidence of CDKN2 loss. Homozygous CDKN2 deletion was detected in 13 (28%) of 46 tumor cell lines from patients with stage III or stage IV NSCLC, compared with zero of 10 tumor cell lines from patients with stage I or stage II NSCLC. Coincident loss of CDKN2 genes and functional Rb protein was rarely observed (in two of 135 cell lines).
CONCLUSION: The frequency of homozygous CDKN2 gene deletion in NSCLC cell lines is greater than that observed for any other known, or candidate, tumor suppressor gene.
IMPLICATION: Further study of the role of CDKN2 gene alteration in the pathogenesis of NSCLC is needed.

Otterson GA, Kratzke RA, Coxon A, et al.
Absence of p16INK4 protein is restricted to the subset of lung cancer lines that retains wildtype RB.
Oncogene. 1994; 9(11):3375-8 [PubMed] Related Publications
Cell cycle dependent phosphorylation of the RB tumor suppressor protein is mediated by a family of G1 cyclin dependent kinases (cdks) and cyclins including the activated cdk4:cyclin D complex. The identification of a cdk4 inhibitor, p16INK4, as a target for mutations in cultured tumor lines and primary tumors suggested that RB activity may be affected in these cells. We have examined 88 lung cancer lines for p16INK4 protein expression and have observed a striking inverse correlation between the presence of p16INK4 and wildtype RB. We demonstrated that only 6/55 (11%) of small cell lung cancer (SCLC) samples had absent p16INK4 protein, and all 6 belonged to the rare subset of SCLC with wildtype RB expression. Conversely of 48 SCLC samples with absent or mutant RB, all showed detectable levels of p16INK4 protein. In contrast, we observed that 23/33 (70%) of non-SCLC samples had loss of p16INK4. Twenty-two of 26 non-SCLC lines with wildtype RB had absent p16INK4 while 6 of 7 non-SCLC lines with absent or mutant RB had detectable p16INK4. The inverse correlation of RB and p16INK4 expression and the absence of p16INK4 inactivation in RB (-/-) SCLC lines (0/48) confirms a common p16INK4/RB growth suppressor pathway in human cancers and provides evidence that p16INK4, and not an adjacent gene on chromosome 9p, is a specific target for mutational events.

Harbour JW, Lai SL, Whang-Peng J, et al.
Abnormalities in structure and expression of the human retinoblastoma gene in SCLC.
Science. 1988; 241(4863):353-7 [PubMed] Related Publications
Small cell lung cancer (SCLC) has been associated with loss of heterozygosity at several distinct genetic loci including chromosomes 3p, 13q, and 17p. To determine whether the retinoblastoma gene (Rb) localized at 13q14, might be the target of recessive mutations in lung cancer, eight primary SCLC tumors and 50 cell lines representing all major histologic types of lung cancer were examined with the Rb complementary DNA probe. Structural abnormalities within the Rb gene were observed in 1/8 (13%) primary SCLC tumors, 4/22 (18%) SCLC lines, and 1/4 (25%) pulmonary carcinoid lines (comparable to the 20 to 40% observed in retinoblastoma), but were not detected in other major types of lung cancer. Rb messenger RNA expression was absent in 60% of the SCLC lines and 75% of pulmonary carcinoid lines, including all samples with DNA abnormalities. In contrast, Rb transcripts were found in 90% of non-SCLC lung cancer lines and in normal human lung. The finding of abnormalities of the Rb gene in SCLC and pulmonary carcinoids (both neuroendocrine tumors) suggests that this gene may be involved in the pathogenesis of a common adult malignancy.

Ceccarelli C, Santini D, Chieco P, et al.
Retinoblastoma (RB1) gene product expression in breast carcinoma. Correlation with Ki-67 growth fraction and biopathological profile.
J Clin Pathol. 1998; 51(11):818-24 [PubMed] Free Access to Full Article Related Publications
AIMS: To investigate the expression of retinoblastoma protein (pRb) in invasive breast tumours and compare its expression with the major biopathological prognostic indicators to identify more aggressive subgroups.
MATERIAL: Archival paraffin embedded tissues from 153 consecutive primary breast carcinomas.
METHODS: pRb, Ki-67, and oestrogen receptor/progesterone receptor proteins were identified by immunohistochemistry and score values were recorded by image cytometric analysis; p53 and EGFr expression was also evaluated.
RESULTS: pRb scores correlated strongly with proliferation activity as determined by Ki-67 staining. Positive relations were also observed between pRb scores, tumour size, nuclear and histological grade, and oestrogen receptor/progesterone receptor content, while abnormal p53 accumulation was not associated with pRb expression. Among the high proliferating carcinomas it was possible to identify 13 cases with loss of pRb expression.
CONCLUSIONS: pRb expression paralleled proliferative activity in the majority of breast carcinomas examined, suggesting that in these cases the protein behaves normally in regulating the cell cycle. Conversely in cases with loss of pRb immunostaining, the combined expression of specific highly aggressive factors (EGFr and p53 expression, oestrogen receptor/progesterone receptor negative status, and high K67) seems to characterise a more aggressive phenotype showing growth advantage and cellular "progression" rather than significant nodal involvement.

Eyfjörd JE, Thorlacius S
Genetic changes in breast carcinomas in an Icelandic population.
Pharmacogenetics. 1992; 2(6):309-16 [PubMed] Related Publications
We have examined breast tumour samples from 109 unselected breast cancer patients for genetic changes on chromosomes 13 and 17. We have looked for allelic losses, firstly, at the retinoblastoma locus, RB1, on chromosome 13q, and secondly, on both arms of chromosome 17. We have also studied the same samples for amplification of the erbB2 oncogene. We searched for mutations in four well conserved areas of the p53 gene using constant denaturant gradient electrophoresis (CDGE). Allelic loss or rearrangement was detected in a large proportion of the tumours, affecting 37-51% of cases with different probes. The areas most frequently affected were 17p13.1 and 17p13.3. Point mutations and small deletions in the p53 gene on 17p13.1 were detected in 16% of the tumours. The data on genetic changes were then analyzed for three different correlations: 1) co-operation between different lesions, 2) association with family history of breast cancer, 3) correlation with clinical factors and prognosis. There was association between losses at the retinoblastoma locus and losses on 17p and 17q. We also found an association between p53 mutations and amplification of the erbB2 oncogene. Relatives of patients having deletions at the retinoblastoma locus and/or sites on chromosome 17 in the tumours have a significantly increased relative risk of developing breast cancer. No such correlation is found for p53 mutations or erbB2 amplification. No p53 germline mutations were detected. P53 mutations do, however, appear to be a strong indication of poor prognosis in this population.

Sauerbrey A, Stammler G, Zintl F, Volm M
Expression of the retinoblastoma tumor suppressor gene (RB-1) in acute leukemia.
Leuk Lymphoma. 1998; 28(3-4):275-83 [PubMed] Related Publications
In this report we review current studies concerning the RB-1 gene expression in acute leukemias. The RB-1 gene was analyzed in several studies by protein-, RNA and DNA-techniques in acute lymphoblastic leukemia (ALL) as well as in acute myelogenous leukemia (AML). The frequency of RB-1 inactivation in ALL-patients ranged between 30% and 64% in several studies. Structural abnormalities of the RB-1 gene were reported in 18% of ALL-patients and in 27% of Philadelphia chromosome-positive ALL, respectively. The proportion of AML-patients with absent RB-1 protein expression ranged between 19% and 55%. Structural RB-1-abnormalities in AML were predominantly reported in leukemias with monocytic differentiation. Furthermore, the prognostic value of an abnormal RB-1 gene expression was also estimated in some studies. In childhood ALL RB-1 inactivation was reported to have prognostic significance while in contrast, in another study on adults no prognostic value of RB-1 was found. In 4 out of 5 documented studies AML-patients with RB-1 inactivation generally had a poorer prognosis. In conclusion, RB-1 inactivation is frequently observed in acute leukemia. The prognostic value of low RB-1 expression is controversial but the majority of published studies found low RB-1 expression to be a negative prognostic predictor, in acute leukemia.

Kornblau SM, Andreeff M, Hu SX, et al.
Low and maximally phosphorylated levels of the retinoblastoma protein confer poor prognosis in newly diagnosed acute myelogenous leukemia: a prospective study.
Clin Cancer Res. 1998; 4(8):1955-63 [PubMed] Related Publications
A prior retrospective study suggested that the level of retinoblastoma protein (RB) expression was prognostic for survival in acute myelogenous leukemia (AML). Individuals with no/low RB protein expression were considered to have loss of RB function, and those with maximally phosphorylated (maxphos) RB were also felt to have nonfunctional RB. To confirm this, we prospectively investigated whether the level of RB expression was prognostic in AML in a larger cohort of patients. RB level was measured by Western blot and immunohistochemical analysis on peripheral blood samples from 210 newly diagnosed AML patients. Patients were divided into three groups based on the level of RB protein expression (i.e., no or low, elevated, and maxphos) or into two groups on the basis of presumed RB function, altered function (AF-RB, low and maxphos RB), versus normal function (NF-RB, elevated RB). By combined results of Western blot and immunohistochemical analysis, 20%, 65%, and 15% of patients had low, elevated, and maxphos RB, respectively. Most patients with acute promyelocytic leukemia (APL) with a French-American-British classification of M3 were in the low RB group, likely reflecting a lower proliferative rate of promyelocytes. Analysis was performed with and without these APL patients. The median survival was significantly shorter for both patients with low RB expression (48 weeks, P = 0.05, including APL patients; 34 weeks, corrected P = 0.008, with APL patients excluded) and maxphos RB expression (51 weeks, P = 0.007) compared to those with elevated RB expression (122 weeks including and 98 weeks excluding APL patients). Differences were greatest among patients with nonfavorable prognosis cytogenetics (median survival, 34 weeks versus 85 weeks; corrected P = 0.001 for AF-RB versus NF-RB). Remission duration was also significantly shorter for non-APL patients with AF-RB versus NF-RB (median survival, 36 weeks versus not reached; corrected P = 0.02). In multivariate analyses, including cytogenetics, performance status, age, antecedent hematological disorder, and RB status, with and without APL patients included, no/low and maxphos-RB protein expression were independent predictors for poorer survival. This prospective study confirms that the level of expression of RB is a strong prognostic factor in AML, with an inferior survival experience being associated with no/low RB and maxphos RB expression. Therefore, therapeutic decisions based on the level of RB expression may be indicated, and protocols to incorporate this are currently under development.

Ahuja HG, Jat PS, Foti A, et al.
Abnormalities of the retinoblastoma gene in the pathogenesis of acute leukemia.
Blood. 1991; 78(12):3259-68 [PubMed] Related Publications
The retinoblastoma-susceptibility (Rb) gene is an antioncogene that is frequently altered in retinoblastomas, sarcomas, and some epithelial tumors. We examined the structure of the Rb gene by Southern blotting in 215 cases of leukemias and lymphomas of diverse phenotype and in 15 leukemic cell lines. In selected cases Rb protein expression was examined with specific monoclonal antibodies. Structural abnormalities of the Rb gene with absent protein expression were frequent in all types of human acute leukemia, but were particularly common (27% incidence) in M4 and M5 myeloid leukemia with monocytic differentiation and in Philadelphia chromosome (Ph1)-positive leukemia of lymphoid phenotype (11% to 29% incidence). Changes in Rb were observed early in the transition to acute leukemia in cases of myelodysplastic syndrome and in the accelerated phase of chronic myelocytic leukemia in transition to blast crisis. In one case, molecular changes in Rb could be correlated with leukemia remission and relapse. We conclude that the Rb antioncogene is commonly involved in the evolution of human acute leukemias, particularly in those of a monocytic phenotype and in lymphoid leukemia in which there is an antecedent alteration of the Ph1 chromosome.

Cote RJ, Dunn MD, Chatterjee SJ, et al.
Elevated and absent pRb expression is associated with bladder cancer progression and has cooperative effects with p53.
Cancer Res. 1998; 58(6):1090-4 [PubMed] Related Publications
Rb protein (pRb) expression was evaluated in 185 cases of transitional cell carcinoma of the bladder from patients that underwent radical cystectomy. Tumors were stratified into three categories based on the percentage of nuclei expressing pRb: (a) 0, 0% of tumor cells showing nuclear reactivity; (b) 1+, 1-50% of tumor cells showing nuclear reactivity; and (c) 2+, >50% of tumor cells showing nuclear reactivity. Cases with undetectable (pRb 0) and high (pRb 2+) pRb reactivity had identical rates of recurrence. These cases had significantly higher recurrence (P = 0.0001) and lower survival rates (P = 0.0002) compared to cases with moderate (pRb 1+) pRb reactivity, indicating that high levels of pRb expression may reflect a dysfunctional (altered) Rb pathway. The tumors were also examined for alterations in p53 expression; patients with tumors altered in both p53 and pRb had significantly increased rates of recurrence (P < 0.0001) and survival (P < 0.0001) compared to patients with no alterations in either p53 or pRb; patients with alterations in only one of these proteins had intermediate rates of recurrence and survival. These results suggest that: (a) bladder cancers with high pRb expression do not show the tumor suppressor effects of the protein; and (b) alteration in both p53 and pRb may act in cooperative or synergistic ways to promote tumor progression.

Grossman HB, Liebert M, Antelo M, et al.
p53 and RB expression predict progression in T1 bladder cancer.
Clin Cancer Res. 1998; 4(4):829-34 [PubMed] Related Publications
The optimal clinical management of minimally invasive (stage T1) bladder cancer is controversial. T1 bladder cancers share characteristics of both noninvasive (Ta) papillary cancer and high stage, muscle-invasive bladder cancers. Patients with T1 bladder cancer have a higher risk of cancer progression and death than do patients with Ta bladder cancer. However, this risk is much lower than that of patients with high-stage bladder cancers. Methods of identifying T1 bladder cancer patients at greatest risk for progression may significantly improve clinical management. We retrospectively evaluated two tumor suppressor genes, p53 and RB, as potential prognostic markers for progression in a cohort of 45 patients with pT1 bladder cancer. Median follow-up for these individuals was greater than 3.5 years. Of this group, 58% had altered p53 expression based on positive p53 immunostaining. Three patterns for RB nuclear protein staining were observed: absent, heterogeneous (normal), and strongly homogeneous. Progression-free survival was similar for patients with loss of RB protein expression and those with apparent overexpression of RB protein. Therefore, both staining patterns were considered abnormal. Patients with normal expression of both proteins (i.e., p53 negative and RB heterogeneously positive) had an excellent outcome, with no patient showing disease progression, whereas patients with abnormal expression of either or both proteins had a significant increase in progression (P = 0.04 and P = 0.005, respectively). These data support the stratification of T1 bladder cancer patients based on p53 and RB nuclear protein status and suggest that patients with normal protein expression for both genes can be managed conservatively, whereas patients with alterations in one and particularly both genes require more aggressive treatment.

Niehans GA, Kratzke RA, Froberg MK, et al.
G1 checkpoint protein and p53 abnormalities occur in most invasive transitional cell carcinomas of the urinary bladder.
Br J Cancer. 1999; 80(8):1175-84 [PubMed] Free Access to Full Article Related Publications
The G1 cell cycle checkpoint regulates entry into S phase for normal cells. Components of the G1 checkpoint, including retinoblastoma (Rb) protein, cyclin D1 and p16INK4a, are commonly altered in human malignancies, abrogating cell cycle control. Using immunohistochemistry, we examined 79 invasive transitional cell carcinomas of the urinary bladder treated by cystectomy, for loss of Rb or p16INK4a protein and for cyclin D1 overexpression. As p53 is also involved in cell cycle control, its expression was studied as well. Rb protein loss occurred in 23/79 cases (29%); it was inversely correlated with loss of p16INK4a, which occurred in 15/79 cases (19%). One biphenotypic case, with Rb+p16- and Rb-p16+ areas, was identified as well. Cyclin D1 was overexpressed in 21/79 carcinomas (27%), all of which retained Rb protein. Fifty of 79 tumours (63%) showed aberrant accumulation of p53 protein; p53 staining did not correlate with Rb, p16INK4a, or cyclin D1 status. Overall, 70% of bladder carcinomas showed abnormalities in one or more of the intrinsic proteins of the G1 checkpoint (Rb, p16INK4a and cyclin D1). Only 15% of all bladder carcinomas (12/79) showed a normal phenotype for all four proteins. In a multivariate survival analysis, cyclin D1 overexpression was linked to less aggressive disease and relatively favourable outcome. In our series, Rb, p16INK4a and p53 status did not reach statistical significance as prognostic factors. In conclusion, G1 restriction point defects can be identified in the majority of bladder carcinomas. Our findings support the hypothesis that cyclin D1 and p16INK4a can cooperate to dysregulate the cell cycle, but that loss of Rb protein abolishes the G1 checkpoint completely, removing any selective advantage for cells that alter additional cell cycle proteins.

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