Gene Summary

Gene:RB1; retinoblastoma 1
Aliases: RB, pRb, OSRC, pp110, p105-Rb, PPP1R130
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:OMIM, VEGA, HGNC, Ensembl, GeneCard, Gene
Protein:retinoblastoma-associated protein
Source:NCBIAccessed: 17 March, 2015


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 (1990-2015)
Graph generated 17 March 2015 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 17 March, 2015 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 Publications510
Acute Lymphocytic Leukaemia (ALL)RB1 and Acute Leukaemias View Publications125
Lung CancerRB1 and Lung Cancer View Publications103
Bladder CancerRB1 and Bladder Cancer View Publications70
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 Publications69
OsteosarcomaRB1 and Osteosarcoma View Publications67
Esophageal CancerRB1 and Esophageal Cancer View Publications9
Adrenocortical CancerRB1 and Adrenocortical Carcinoma View Publications5

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)

Rodriguez-Galindo C, Orbach DB, VanderVeen D
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] Article available free on PMC after 16/04/2015 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.

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] Article available free on PMC after 16/04/2015 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] Article available free on PMC after 13/05/2015 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.

McEvoy J, Nagahawatte P, Finkelstein D, et al.
RB1 gene inactivation by chromothripsis in human retinoblastoma.
Oncotarget. 2014; 5(2):438-50 [PubMed] Article available free on PMC after 13/05/2015 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.

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] Article available free on PMC after 13/05/2015 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.

Mol BM, Massink MP, van der Hout AH, et al.
High resolution SNP array profiling identifies variability in retinoblastoma genome stability.
Genes Chromosomes Cancer. 2014; 53(1):1-14 [PubMed] Related Publications
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.

Kansara M, Leong HS, Lin DM, et al.
Immune response to RB1-regulated senescence limits radiation-induced osteosarcoma formation.
J Clin Invest. 2013; 123(12):5351-60 [PubMed] Article available free on PMC after 13/05/2015 Related Publications
Ionizing radiation (IR) and germline mutations in the retinoblastoma tumor suppressor gene (RB1) are the strongest risk factors for developing osteosarcoma. Recapitulating the human predisposition, we found that Rb1+/- mice exhibited accelerated development of IR-induced osteosarcoma, with a latency of 39 weeks. Initial exposure of osteoblasts to carcinogenic doses of IR in vitro and in vivo induced RB1-dependent senescence and the expression of a panel of proteins known as senescence-associated secretory phenotype (SASP), dominated by IL-6. RB1 expression closely correlated with that of the SASP cassette in human osteosarcomas, and low expression of both RB1 and the SASP genes was associated with poor prognosis. In vivo, IL-6 was required for IR-induced senescence, which elicited NKT cell infiltration and a host inflammatory response. Mice lacking IL-6 or NKT cells had accelerated development of IR-induced osteosarcomas. These data elucidate an important link between senescence, which is a cell-autonomous tumor suppressor response, and the activation of host-dependent cancer immunosurveillance. Our findings indicate that overcoming the immune response to senescence is a rate-limiting step in the formation of IR-induced osteosarcoma.

Price EA, Price K, Kolkiewicz K, et al.
Spectrum of RB1 mutations identified in 403 retinoblastoma patients.
J Med Genet. 2014; 51(3):208-14 [PubMed] Related Publications
BACKGROUND: Retinoblastoma (RB) is a malignant, childhood tumour of the developing retina that occurs with an estimated frequency of 1 in 20 000. Identification of oncogenic mutations in the RB1 gene aids in the clinical management of families with a heritable predisposition to RB. Here we present the spectrum of genetic and epigenetic changes identified in 194 tumours and 209 blood samples, from 403 unrelated RB patients.
METHODS: Mutation screening was carried out across all 27 RB1 exons and their associated splice sites. Small coding sequence changes were detected using fluorescent conformation analysis followed by sequencing. Large exonic deletions were detected by quantitative fluorescent PCR. Methylation specific PCR of the RB1 promoter was performed to detect epigenetic alterations. Polymorphism analysis was used to determine loss of heterozygosity in tumour samples.
RESULTS: 95% of the expected mutations were identified in the tumour samples, with 16 samples exhibiting only one mutation, while two samples had no detectable RB1 mutation. 96% of bilateral/familial RB blood samples and 9.5% of unilateral sporadic blood samples, yielded mutations. 111 were novel mutations.
CONCLUSIONS: The full range of screening techniques is required to achieve a high screening sensitivity in RB patients.

Chung J, Karkhanis V, Tae S, et al.
Protein arginine methyltransferase 5 (PRMT5) inhibition induces lymphoma cell death through reactivation of the retinoblastoma tumor suppressor pathway and polycomb repressor complex 2 (PRC2) silencing.
J Biol Chem. 2013; 288(49):35534-47 [PubMed] Article available free on PMC after 13/05/2015 Related Publications
Epigenetic regulation mediated by lysine- and arginine-specific enzymes plays an essential role in tumorigenesis, and enhanced expression of the type II protein arginine methyltransferase PRMT5 as well as the polycomb repressor complex PRC2 has been associated with increased cell proliferation and survival. Here, we show that PRMT5 is overexpressed in three different types of non-Hodgkin lymphoma cell lines and clinical samples as well as in mouse primary lymphoma cells and that it up-regulates PRC2 expression through inactivation of the retinoblastoma proteins RB1 and RBL2. Although PRMT5 epigenetically controls RBL2 expression, it indirectly promotes RB1 phosphorylation through enhanced cyclin D1 expression. Furthermore, we demonstrate that PRMT5 knockdown in non-Hodgkin lymphoma cell lines and mouse primary lymphoma cells leads to RBL2 derepression and RB1 reactivation, which in turn inhibit PRC2 expression and trigger derepression of its CASP10, DAP1, HOXA5, and HRK pro-apoptotic target genes. We also show that reduced PRMT5 expression leads to cyclin D1 transcriptional repression via loss of TP53K372 methylation, which results in decreased BCL3 expression and enhanced recruitment of NF-κB p52-HDAC1 repressor complexes to the cyclin D1 promoter. These findings indicate that PRMT5 is a master epigenetic regulator that governs expression of its own target genes and those regulated by PRC2 and that its inhibition could offer a promising therapeutic strategy for lymphoma patients.

Fu M, Landreville S, Agapova OA, et al.
Retinoblastoma protein prevents enteric nervous system defects and intestinal pseudo-obstruction.
J Clin Invest. 2013; 123(12):5152-64 [PubMed] Article available free on PMC after 13/05/2015 Related Publications
The retinoblastoma 1 (RB1) tumor suppressor is a critical regulator of cell cycle progression and development. To investigate the role of RB1 in neural crest-derived melanocytes, we bred mice with a floxed Rb1 allele with mice expressing Cre from the tyrosinase (Tyr) promoter. TyrCre+;Rb1fl/fl mice exhibited no melanocyte defects but died unexpectedly early with intestinal obstruction, striking defects in the enteric nervous system (ENS), and abnormal intestinal motility. Cre-induced DNA recombination occurred in all enteric glia and most small bowel myenteric neurons, yet phenotypic effects of Rb1 loss were cell-type specific. Enteric glia were twice as abundant in mutant mice compared with those in control animals, while myenteric neuron number was normal. Most myenteric neurons also appeared normal in size, but NO-producing myenteric neurons developed very large nuclei as a result of DNA replication without cell division (i.e., endoreplication). Parallel studies in vitro found that exogenous NO and Rb1 shRNA increased ENS precursor DNA replication and nuclear size. The large, irregularly shaped nuclei in NO-producing neurons were remarkably similar to those in progeria, an early-onset aging disorder that has been linked to RB1 dysfunction. These findings reveal a role for RB1 in the ENS.

Bond WS, Akinfenwa PY, Perlaky L, et al.
Tumorspheres but not adherent cells derived from retinoblastoma tumors are of malignant origin.
PLoS One. 2013; 8(6):e63519 [PubMed] Article available free on PMC after 13/05/2015 Related Publications
Verification that cell lines used for cancer research are derived from malignant cells in primary tumors is imperative to avoid invalidation of study results. Retinoblastoma is a childhood ocular tumor that develops from loss of functional retinoblastoma protein (pRb) as a result of genetic or epigenetic changes that affect both alleles of the RB1 gene. These patients contain unique identifiable genetic signatures specifically present in malignant cells. Primary cultures derived from retinoblastoma tumors can be established as non-adherent tumorspheres when grown in defined media or as attached monolayers when grown in serum-containing media. While the RB1 genotypes of tumorspheres match those of the primary tumor, adherent cultures have the germline RB1 genotype. Tumorspheres derived from pRb-negative tumors do not express pRb and express the neuroendocrine tumor markers synaptophysin and microtubule-associated protein 2 (MAP2). Adherent cells are synaptophysin-negative and express pRb, the epithelial cell marker cytokeratin that is expressed in the retinal pigmented epithelium and the vascular endothelial cell marker CD34. While tumorspheres are of malignant origin, our results cast doubt on the assumption that adherent tumor-derived cultures are always valid in vitro models of malignant cells and emphasize the need for validation of primary tumor cultures.

Serrano ML, Yunis JJ
Identification of three new mutations in the RB1 gene in patients with sporadic retinoblastoma in Colombia.
Biomedica. 2013 Jan-Mar; 33(1):53-61 [PubMed] Related Publications
INTRODUCTION: Retinoblastoma is a childhood cancer of the retina originated by altered or null retinoblastoma protein (pRb) expression. Genetic alterations in both RB1 alleles in the retinal cells are required for the development of retinoblastoma. In the sporadic form, non-hereditary RB1 gene mutations take place in a single retinoblast cell, and are therefore only present in tumor DNA (somatic mutations). Sporadic retinoblastoma is primarily unilateral, lacks family history and has no risk of transmission to descendants. Genetic tests for detection of RB1 mutation has improved the identification of carriers and facilitated accurate genetic counseling.
OBJECTIVE: To identify mutations in the RB1 gene in Colombian patients with sporadic retinoblastoma by PCR-SSCP followed by sequence.
MATERIALS AND METHODS: Four patients with sporadic retinoblastoma were analyzed by PCR-SSCP, followed by DNA sequencing to identify variations in the RB1 gene.
RESULTS: We identified five variations in RB1 gene: three new mutations (one germline and two somatic mutations), one new polymorphism and one already reported somatic mutation. Four mutations were found in three patients with unilateral retinoblastoma and one mutation was found in a patient with bilateral retinoblastoma. One of these was a germline mutation in a sporadic unilateral retinoblastoma that was not present in the parents or three siblings analyzed.
CONCLUSIONS: Our results emphasize the importance of identifying mutations for genetic counseling and clinical management of sporadic retinoblastoma patients. Description of a new RB1 gene variant is interesting since there have been a small number of polymorphisms reported for this gene.

Gonzalez-Vasconcellos I, Anastasov N, Sanli-Bonazzi B, et al.
Rb1 haploinsufficiency promotes telomere attrition and radiation-induced genomic instability.
Cancer Res. 2013; 73(14):4247-55 [PubMed] Related Publications
Germline mutations of the retinoblastoma gene (RB1) predispose to both sporadic and radiation-induced osteosarcoma, tumors characterized by high levels of genomic instability, and activation of alternative lengthening of telomeres. Mice with haploinsufficiency of the Rb1 gene in the osteoblastic lineage reiterate the radiation susceptibility to osteosarcoma seen in patients with germline RB1 mutations. We show that the susceptibility is accompanied by an increase in genomic instability, resulting from Rb1-dependent telomere erosion. Radiation exposure did not accelerate the rate of telomere loss but amplified the genomic instability resulting from the dysfunctional telomeres. These findings suggest that telomere maintenance is a noncanonical caretaker function of the retinoblastoma protein, such that its deficiency in cancer may potentiate DNA damage-induced carcinogenesis by promoting formation of chromosomal aberrations, rather than simply by affecting cell-cycle control.

Garfin PM, Min D, Bryson JL, et al.
Inactivation of the RB family prevents thymus involution and promotes thymic function by direct control of Foxn1 expression.
J Exp Med. 2013; 210(6):1087-97 [PubMed] Article available free on PMC after 13/05/2015 Related Publications
Thymic involution during aging is a major cause of decreased production of T cells and reduced immunity. Here we show that inactivation of Rb family genes in young mice prevents thymic involution and results in an enlarged thymus competent for increased production of naive T cells. This phenotype originates from the expansion of functional thymic epithelial cells (TECs). In RB family mutant TECs, increased activity of E2F transcription factors drives increased expression of Foxn1, a central regulator of the thymic epithelium. Increased Foxn1 expression is required for the thymic expansion observed in Rb family mutant mice. Thus, the RB family promotes thymic involution and controls T cell production via a bone marrow-independent mechanism, identifying a novel pathway to target to increase thymic function in patients.

Bos M, Gardizi M, Schildhaus HU, et al.
Complete metabolic response in a patient with repeatedly relapsed non-small cell lung cancer harboring ROS1 gene rearrangement after treatment with crizotinib.
Lung Cancer. 2013; 81(1):142-3 [PubMed] Related Publications
A 55-year-old Caucasian woman with lung adenocarcinoma stage IV presented with repeated relapse after treatment with cytotoxic chemotherapy (carboplatin, gemcitabine, docetaxel, pemetrexed) and targeted agents (erlotinib, cetuximab, sunitinib). Comprehensive molecular diagnostics (EGFR-, ALK-, RAS-, BRAF-, PIK3CA-, HER2- and DDR2-aberrations) were performed and failed initially to detect any driver mutation. While the patient suffered from rapid deterioration of her general condition, in particular from progressive dyspnea due to lung metastases, we implemented screening for RET- and ROS1 translocations into our molecular diagnostic program based on recent reports of these new molecular subgroups in lung adenocarcinoma. On retesting the patient's tumor sample was found to harbor a ROS1-translocation. The patient was subsequently treated with crizotinib and experienced a pronounced clinical improvement corresponding to a complete metabolic response in (18)F-FDG-PET and a good and confirmed partial response in CT (RECIST 1.1). Our case exemplifies the need for rapid implementation of newly discovered rare genetic lung cancer subtypes in routine molecular diagnostics.

Barbosa RH, Aguiar FC, Silva MF, et al.
Screening of RB1 alterations in Brazilian patients with retinoblastoma and relatives with retinoma: phenotypic and genotypic associations.
Invest Ophthalmol Vis Sci. 2013; 54(5):3184-94 [PubMed] Related Publications
PURPOSE: To identify constitutional alterations of the retinoblastoma 1 gene (RB1) in two cohorts of Brazilian patients with retinoblastoma and to analyze genotype-phenotype associations.
METHODS: Molecular screening was carried out by direct sequencing of the 27 RB1 exons and flanking regions in blood DNA of 71 patients with retinoblastoma and 4 relatives with retinoma, and with multiplex ligation-dependent probe amplification (MLPA) in 21 patients. The presumed impact of nucleotide substitutions on the structure of the retinoblastoma protein (pRB) was predicted by Polymorphism Phenotyping-2 (PolyPhen-2). Kaplan-Meier and log-rank test were used for estimating 60-month survival rates.
RESULTS: One hundred two nucleotide substitutions were detected, 92 substitutions in 59 patients with retinoblastoma and 10 substitutions in 4 individuals with retinoma. Eight substitutions were novel. The majority of substitutions were intronic (86.2%). More than one substitution was present in 37.3% of patients. Twenty-one duplications and 11 deletions were found in 12 patients; some of which with both types of alterations. Duplications/deletions were found in four patients lacking constitutional alterations when analyzed by sequencing, and in eight patients carrying one or more polymorphic intronic substitutions. The global 60-month survival rate in patients was 91.8% (Confidence Interval95% = 85.0 - 99.1). Significant, lower survival rates were found in extraocular presentation (81.0%) versus intraocular tumors (P = 0.014), first enucleation after 1 month following diagnosis (80.9%) versus earlier first enucleation (P = 0.020), and relapse (100.0%) versus absence of relapse (P = 0.0005).
CONCLUSIONS: Fifteen substitutions (4 intronic and 11 exonic) were identified as probably or likely pathogenic. Four of these 11 exonic substitutions were novel. Survival rates, however, were not affected by presence of these probably or likely pathogenic alterations, most of which not found in patients with retinoblastoma from other Latin American countries. These differences might be related to the different ethnic composition of the Latin American cohorts. Portuguese Abstract.

Ahani A, Akbari MT, Saliminejad K, et al.
Screening for large rearrangements of the RB1 gene in Iranian patients with retinoblastoma using multiplex ligation-dependent probe amplification.
Mol Vis. 2013; 19:454-62 [PubMed] Article available free on PMC after 13/05/2015 Related Publications
PURPOSE: To screen deletions/duplications of the RB1 gene in a large cohort of Iranian patients using the multiplex ligation-dependent probe amplification (MLPA) technique.
METHODS: A total of 121 patients with retinoblastoma, involving 55 unilateral and 66 bilateral or familial retinoblastomas, were included in this study. Among these patients, 121 blood and 43 tissue samples were available. DNA was extracted from the blood and tissue samples and analyzed with an RB1-specific MLPA probe set. The mutation findings were validated with SYBR Green Real-Time PCR.
RESULTS: Twenty-two mutations were found in 21 patients; of these, ten mutations were detected in patients with isolated unilateral retinoblastoma.
CONCLUSIONS: Our results suggested that MLPA is a fast, reliable, and powerful method for detecting deletions/duplications in patients with retinoblastoma.

Watanabe M, Sowa Y, Yogosawa M, Sakai T
Novel MEK inhibitor trametinib and other retinoblastoma gene (RB)-reactivating agents enhance efficacy of 5-fluorouracil on human colon cancer cells.
Cancer Sci. 2013; 104(6):687-93 [PubMed] Related Publications
Chemotherapy for colorectal cancer has become more complicated and diversified with the appearance of molecular-targeting agents. 5-Fluorouracil (5-FU) has been a mainstay of chemotherapy for colorectal cancer, but it is still unknown whether the combining of 5-FU with novel molecular-targeting agents is effective. Thymidylate synthase (TS) is a direct target of 5-FU, and the low TS level has been generally supposed to sensitize 5-FU's efficacy. We therefore hypothesized that RB-reactivating agents could enhance the efficacy of 5-FU, because the RB-reactivating agents could suppress the function of transcription factor E2F of TS gene promoter. We used three RB-reactivating agents, trametinib/GSK1120212 (MEK inhibitor), fenofibrate (PPARα agonist), and LY294002 (PI3K inhibitor), with 5-FU against human colon cancer HT-29 and HCT15 cells. Trametinib induced p15 and p27 expression and reduced cyclin D1 levels in HT-29 cells. Fenofibrate also dephosphorlated ERK1/2 and reduced cyclin D1 levels in HT-29 cells. LY294002 induced p27 expression in HCT15 cells. All three agents caused dephosphorylation of RB protein and G1-phase arrest with a reduction of TS expression. As a consequence, the combination of 5-FU with each of the agents resulted in a significant decrease of colony numbers in HT-29 or HCT15 cells. These results suggest "RB-reactivation therapy" using molecular-targeting agents to be a new strategy for 5-FU-based chemotherapy. In particular, we strongly expect trametinib, which was discovered in Japan and was recently submitted to FDA for approval, to be used together with established regimens for colorectal cancer.

Youn JI, Kumar V, Collazo M, et al.
Epigenetic silencing of retinoblastoma gene regulates pathologic differentiation of myeloid cells in cancer.
Nat Immunol. 2013; 14(3):211-20 [PubMed] Article available free on PMC after 13/05/2015 Related Publications
Two major populations of myeloid-derived suppressor cells (MDSCs), monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs) regulate immune responses in cancer and other pathologic conditions. Under physiologic conditions, Ly6C(hi)Ly6G(-) inflammatory monocytes, which are the normal counterpart of M-MDSCs, differentiate into macrophages and dendritic cells. PMN-MDSCs are the predominant group of MDSCs that accumulates in cancer. Here we show that a large proportion of M-MDSCs in tumor-bearing mice acquired phenotypic, morphological and functional features of PMN-MDSCs. Acquisition of this phenotype, but not the functional attributes of PMN-MDSCs, was mediated by transcriptional silencing of the retinoblastoma gene through epigenetic modifications mediated by histone deacetylase 2 (HDAC-2). These data demonstrate a new regulatory mechanism of myeloid cells in cancer.

Ottaviani D, Parma D, Giliberto F, et al.
Spectrum of RB1 mutations in argentine patients: 20-years experience in the molecular diagnosis of retinoblastoma.
Ophthalmic Genet. 2013; 34(4):189-98 [PubMed] Related Publications
BACKGROUND: Retinoblastoma is a hereditary cancer of childhood caused by mutations in the RB1 tumor suppressor gene. An early diagnosis is critical for survival and eye preservation, thus identification of RB1 mutations is important for unequivocal diagnosis of hereditary retinoblastoma and risk assessment in relatives.
METHODS: We studied 144 families for 20 years, performing methodological changes to improve detection of mutation. Segregation analysis of polymorphisms, MLPA, FISH and cytogenetic assays were used for detection of "at risk haplotypes" and large deletions. Small mutations were identified by heteroduplex/DNA sequencing.
RESULTS: At risk haplotypes were identified in 11 familial and 26 sporadic cases, being useful for detection of asymptomatic carriers, risk exclusion from relatives and uncovering RB1 recombinations. Ten large deletions (eight whole gene deletions) were identified in six bilateral/familial and four unilateral retinoblastoma cases. Small mutations were identified in 29 cases (four unilateral retinoblastoma patients), being the majority nonsense/frameshift mutations. Genotype-phenotype correlations confirm that the retinoblastoma presentation is related to the type of mutation, but some exceptions may occur and it is crucial to be considered for genetic counseling. Three families included second cousins with retinoblastoma carrying different haplotypes, which suggest independent mutation events.
CONCLUSION: This study enabled us to obtain information about molecular and genetic features of patients with retinoblastoma in Argentina and correlate them to their phenotype.

Sheck LH, Ng YS, Watson M, Vincent AL
Clinical findings and molecular diagnosis of retinoblastoma in older children.
Ophthalmic Genet. 2013; 34(4):238-42 [PubMed] Related Publications
BACKGROUND: Retinoblastoma (RB) is the most common primary childhood intraocular malignancy and usually presents before the age of 4 years. RB in late childhood is rare and may pose a diagnostic challenge to clinicians.
MATERIALS AND METHODS: Patients over the age of 4 years with RB were identified retrospectively. Clinical data, histological findings, and molecular genetic diagnoses were obtained.
RESULTS: Two cases of late onset RB were identified. Case 1 was a 10-year-old boy who presented with floaters, and was found to have a unilateral exudative retinal detachment and RB on clinical examination. Genetic testing showed a novel homozygous mutation in exon 20 of the RB1 gene in the tumor sample, c.2027_2034dup, resulting in p.Ile679X. No mutation was found in the DNA obtained from the peripheral blood sample. Case 2 was a 6-year-old boy who presented with loss of vision and pain in the left eye. RB was diagnosed on clinical examination with exudative retinal detachment. Genetic testing showed no mutation in the RB1 gene, but complete methylation of the RB1 promoter region.
CONCLUSIONS: RB can rarely present in late childhood. Clinicians should consider RB as a diagnosis when faced with a patient with unexplained exudative retinal detachment.

Temming P, Lohmann D, Bornfeld N, et al.
Current concepts for diagnosis and treatment of retinoblastoma in Germany: aiming for safe tumor control and vision preservation.
Klin Padiatr. 2012; 224(6):339-47 [PubMed] Related Publications
Retinoblastoma affects approximately 40 children in Germany per year. Most children are diagnosed early with localized intraocular disease, and the overall survival rate exceeds 95%. However, the prognosis of metastasized retinoblastoma remains poor. In 40% of the patients, retinoblastoma occurs bilaterally and, especially for these children, the salvage of the eye and visual function is of major importance. The variety of conservative treatment options for localized retinoblastoma includes laser coagulation, thermotherapy, cryotherapy, brachytherapy and chemotherapy. While systemic chemotherapy has nearly completely replaced external beam radiotherapy in the primary treatment of intraocular retinoblastoma, intra-arterial, intravitreal and periocular application of chemotherapy was also shown to be effective in treating intraocular retinoblastoma in case series. Genetic testing is an integral part of the routine diagnostics of all patients. Available tumor material should be analyzed to detect mutational mosaicism, that affects >10% of children with unilateral retinoblastoma. Genetic testing also identifies children with heritable (50% of patients) retinoblastoma. These children have a genetic predisposition for second malignancies. For this reason, late effects are an increasing concern and the care of patients with retinoblastoma requires a multidisciplinary approach to tailor therapy and long-term follow-up. Multicenter clinical trials are being developed to evaluate evidence-based treatment concepts for localized and metastasized retinoblastoma to improve survival rates and quality of life of children with retinoblastoma.

Orlandi EM, Bernasconi P, Pascutto C, et al.
Chronic lymphocytic leukemia with del13q14 as the sole abnormality: dynamic prognostic estimate by interphase-FISH.
Hematol Oncol. 2013; 31(3):136-42 [PubMed] Related Publications
This study analyzed 140 patients with isolated del13q14 on interphase FISH (I-FISH), to identify subsets with a different progression risk and to assess the acquisition of additional chromosomal abnormalities (clonal evolution) in treatment-naïve del13q14 patients. A monoallelic deletion (del13qx1) was detected in 123 cases (88%), a biallelic deletion (del13qx2) in eight and a mosaic of monoallelic and biallelic deletions (del13qx1/del13qx2) in nine. In 33% of cases, deletion encompassed the Rb1 locus The median percentage of abnormal nuclei was 50% (15%-96%), and it was higher in patients with a biallelic/mosaic pattern in comparison with patients with monoallelic deletion. Sixty two patients (44%) have been treated; 5-year treatment free survival rate was 56% and the median treatment free survival was 65 months. The baseline percentage of deleted nuclei, as a continuous variable, was related to progression (HR: 1.02; p = 0.001). According to deletion burden, three groups were identified: 64 cases (46%) had <50% deleted nuclei, 47 (33%) had 50-69% deleted nuclei, and 29 (21%) had ≥70% deleted nuclei. The 5-year untreated rate was 70.5% , 52.6% and 28.7% (p < 0.0001), respectively. In multivariate analysis using IGHV mutational status, presence of a nullisomic clone, CD38 expression and percentage of deleted nuclei as covariates, only IGHV mutational status and the percentage of deleted nuclei were independent risk factors for treatment. In 103 patients serially monitored by I-FISH before starting any treatment, we observed a significant increase in the proportion of del13q14 cells, and this increase affected the risk of subsequent treatment requirement (HR 2.54, p = 0.001). The appearance of a new clone was detected in 16 patients (15.5%) and chromosome 13 was involved in 14 of them. I-FISH monitoring proves worthwhile for a dynamic risk stratification and for planning clinical surveillance.

Song H, Yao E, Lin C, et al.
Functional characterization of pulmonary neuroendocrine cells in lung development, injury, and tumorigenesis.
Proc Natl Acad Sci U S A. 2012; 109(43):17531-6 [PubMed] Article available free on PMC after 13/05/2015 Related Publications
Pulmonary neuroendocrine cells (PNECs) are proposed to be the first specialized cell type to appear in the lung, but their ontogeny remains obscure. Although studies of PNECs have suggested their involvement in a number of lung functions, neither their in vivo significance nor the molecular mechanisms underlying them have been elucidated. Importantly, PNECs have long been speculated to constitute the cells of origin of human small-cell lung cancer (SCLC) and recent mouse models support this hypothesis. However, a genetic system that permits tracing the early events of PNEC transformation has not been available. To address these key issues, we developed a genetic tool in mice by introducing a fusion protein of Cre recombinase and estrogen receptor (CreER) into the calcitonin gene-related peptide (CGRP) locus that encodes a major peptide in PNECs. The CGRP(CreER) mouse line has enabled us to manipulate gene activity in PNECs. Lineage tracing using this tool revealed the plasticity of PNECs. PNECs can be colabeled with alveolar cells during lung development, and following lung injury, PNECs can contribute to Clara cells and ciliated cells. Contrary to the current model, we observed that elimination of PNECs has no apparent consequence on Clara cell recovery. We also created mouse models of SCLC in which CGRP(CreER) was used to ablate multiple tumor suppressors in PNECs that were simultaneously labeled for following their fate. Our findings suggest that SCLC can originate from differentiated PNECs. Together, these studies provide unique insight into PNEC lineage and function and establish the foundation of investigating how PNECs contribute to lung homeostasis, injury/repair, and tumorigenesis.

Kuehl WM, Bergsagel PL
Molecular pathogenesis of multiple myeloma and its premalignant precursor.
J Clin Invest. 2012; 122(10):3456-63 [PubMed] Article available free on PMC after 13/05/2015 Related Publications
Multiple myeloma is a monoclonal tumor of plasma cells, and its development is preceded by a premalignant tumor with which it shares genetic abnormalities, including universal dysregulation of the cyclin D/retinoblastoma (cyclin D/RB) pathway. A complex interaction with the BM microenvironment, characterized by activation of osteoclasts and suppression of osteoblasts, leads to lytic bone disease. Intratumor genetic heterogeneity, which occurs in addition to intertumor heterogeneity, contributes to the rapid emergence of drug resistance in high-risk disease. Despite recent therapeutic advances, which have doubled the median survival time, myeloma continues to be a mostly incurable disease. Here we review the current understanding of myeloma pathogenesis and insight into new therapeutic strategies provided by animal models and genetic screens.

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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