¹ Texas Children’s Cancer Center; ² Center for Cell and Gene Therapy; Departments of ³ Pediatrics, ⁴ Ophthalmology, and ⁵ Molecular and Cellular Biology, Baylor College of Medicine; ⁶ Department of Pathology, The Methodist Hospital, Houston, Texas

Requests for reprints: Richard L. Hurwitz, Texas Children’s Cancer Center, 6621 Fannin Street, M.C. 3-3320, Houston, TX 77030. Phone: 832-824-4260; Fax: 832-825-4846; E-mail: rhurwitz@txccc.org.

Retinoblastoma, the most common intraocular malignancy of childhood, metastasizes by initial invasion of the choroid and the optic nerve. There is no effective treatment for metastatic retinoblastoma, especially when the central nervous system (CNS) is involved, and prevention of this complication is a treatment priority. Seneca Valley Virus (SVV-001) is a conditionally replication-competent picornavirus that is not pathogenic to normal human cells but can kill human retinoblastoma cells in vitro with an IC₅₀ of <1 viral particle (vp) per cell. A xenograft murine model of metastatic retinoblastoma was used to examine the therapeutic potential of SVV-001. Histopathologic analysis of ocular and brain tissues after a single tail vein injection of SVV-001 (1 x 10¹³ vp/kg) showed effective treatment of choroid and ocular nerve tumor invasion (1 of 20 animals with invasive disease in the treated group versus 7 of 20 animals with invasive disease in the control group; P = 0.017) and prevention of CNS metastasis (0 of 20 animals with CNS metastatic disease in the treated group versus 4 of 20 animals with CNS disease in the control group; P = 0.036). There were no observed adverse events due to the virus in any of the treated animals. SVV-001 may be effective as a treatment of locally invasive and metastatic retinoblastoma. [Cancer Res 2007;67(22):10653–6]

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It is easy to understand that mutations resulting in defective or absent RB protein would be advantageous for cell proliferation and would initiate a cancer or contribute to increasing the malignancy of a preexisting cancer. Cryns et al. suggest in this issue of the Journal (see pages 757 to 761) that loss of RB protein is very common among parathyroid carcinomas but not parathyroid adenomas. Parathyroid adenomas frequently overexpress cyclin D1 because of somatic chromosomal rearrangements that bring the cyclin D1 gene (PRAD1) under control of the parathyroid hormone gene enhancer and thus result in high levels of expression in parathyroid tissue. Cyclin D1 can phosphorylate and inactivate RB protein. However, the association of RB mutation with parathyroid carcinoma suggests that cyclin D1 only partly inactivates RB protein, since RB gene mutation results in further growth advantage for that clone.

The tissue specificities for the initiation and progression of cancer resulting from RB gene mutations suggest that initiation and progression may be separate processes (Figure 1). Humans with one mutant germ-line RB allele are normal except for their susceptibility to cancer; their risk for the development of retinoblastoma in the first years of life is 36,000 times that of people without the mutation. Interestingly, in mice with germ-line RB mutations, it is not retinoblastoma that develops but pituitary tumors instead. Humans with germ-line RB mutations also have 2000 times the normal risk of osteosarcoma during the second decade of life and an undefined, but lower, risk of other tumors, such as melanoma and soft-tissue sarcoma. Neither parathyroid nor pituitary tumors, however, occur at an increased rate in humans with these mutations.

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William A. Zagorski¹, Erik S. Knudsen² and Michael F. Reed^1,3

¹ Division of Thoracic Surgery, Departments of Surgery and ² Cell and Cancer Biology, The Vontz Center for Molecular Studies, University of Cincinnati College of Medicine and ³ Department of Surgery, Cincinnati VA Medical Center, Cincinnati, Ohio

The retinoblastoma (RB) tumor suppressor is mutated or functionally inactivated in the majority of human malignancies, and p16^INK4a-cyclin D1-cyclin-dependent kinase 4-RB pathway aberrations are present in nearly all cases of non–small cell lung cancer (NSCLC). Here, the distinct role of RB loss in tumorigenic proliferation and sensitivity to chemotherapeutics was determined in NSCLC cells. Attenuation of RB led to a proliferative advantage in vitro and aggressive tumorigenic growth in xenograft models. Clinically, such aggressive disease is treated with genotoxic and cytotoxic chemotherapeutic agents. In vitro analysis showed that RB deficiency resulted in bypass of the checkpoint response to multiple chemotherapeutic challenges concomitant with an elevated apoptotic response. Correspondingly, RB deficiency in xenograft models led to increased chemosensitivity. However, this response was transient, and a durable response was dependent on prolonged chemotherapeutic administration. Together, these findings show that although RB deficiency enhances sensitivity to chemotherapeutic challenge, efficient and sustainable response is highly dependent on the specific therapeutic regimen, in addition to the molecular environment. [Cancer Res 2007;67(17):8264–73]

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Center for Molecular Medicine/Institute of Biotechnology, University of Texas Health Science Center at San Antonio 78245.

Models for studying the quantitative effects of RB protein in development and in tumorigenesis have been constructed. By crossing transgenic mice carrying the human Rb gene with mice heterozygous for the normal mouse Rb gene, we have generated mice that contain varying copies of the human Rb transgene and express varying amounts of human RB protein. Herein, we show that different amounts of RB protein are required to rescue two different phenotypes resulting from Rb deficiency: death during fetal development and susceptibility to cancer. Normal fetal development seems to depend on expression of critical amounts of RB protein, > or = 50% of the amount in wild-type mice. Adult mice, even if they overexpress RB protein, are prone to cancer if their cells express from only one Rb gene allele. Mice expressing small amounts of RB protein from different Rb alleles, however, are protected from cancer. Tumor suppression in cancer-prone cells, therefore, rather than depending on an absolute amount of RB protein, more directly depends on having redundancy in the Rb gene, assuring that at least one copy of a gene encoding functional RB protein is always available.

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The tumor may begin in one or both eyes. Retinoblastoma is usually confined to the eye but can spread to the brain via the optic nerve.

Retinoblastoma may be hereditary (inherited) or nonhereditary. The hereditary form may be in one or both eyes, and generally affects younger children. Retinoblastoma occurring in only one eye is often not hereditary and is more prevalent in older children. When the disease occurs in both eyes, it is always hereditary. Because of the hereditary factor, patients and their brothers and sisters should have periodic examinations, including genetic counseling, to determine their risk for developing the disease.

A statistical study by Dr Alfred G. Knudson in 1971 led to a hypothesis (later known as the Knudson hypothesis) about why some retinablastomas are hereditary and others occur by chance. This hypothesis led to the first identification of a tumor suppressor gene by a team led by Dr Thaddeus P. Dryja in 1986. Knudson won the 1998 Albert Lasker Medical Research Award for this work.

Hereditary retinoblastoma is caused by an inherited mutation in a single copy of the Rb1 gene. The remaining functional copy prevents most retinal cells from becoming cancer. However, one or more cells in the retina are likely to undergo a spontaneous loss of this functional copy, causing those cells to transform into cancer. This loss of the second copy of Rb1 is termed loss of heterozygosity, a frequent event in cancer for which retinoblastoma is the canonical example.

The patient’s choice of treatment depend on the extent of the disease within and beyond the eye. Smaller tumors can be removed with laser surgery, thermo-, or cryotherapy.

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^a Division of Thoracic Surgery, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio
^b Department of Cell Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio
^c Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

Presented at the Poster Session of the Fifty-second Annual Meeting of the Southern Thoracic Surgical Association, Orlando, FL, Nov 10–12, 2005.

BACKGROUND: Inactivation of retinoblastoma (RB) tumor suppressor function occurs frequently in lung cancer. Short-hairpin RNA can be constructed to target specific sequences and efficiently knock down protein expression. We developed a short-hairpin RNA approach to specifically target Rb in lung cancer cells to determine the influence of RB knockdown on proliferation.

METHODS: NCI-H520 human lung cancer cells (wild-type Rb) were transfected with pMSCVpuro-Rb3C, a plasmid containing a short-hairpin sequence targeted to human Rb. Transfectants harboring the construct were selected with puromycin. Loss of RB expression in selected cell populations was determined by immunoblotting. Proliferating cells were counted to establish growth rates. Retinoblastoma-proficient and RB-deficient tumor growth was monitored in nude mice.

RESULTS: Transfection with pMSCVpuro-Rb3C dramatically diminished RB expression and led to aberrant expression of RB-regulated genes. Cells harboring pMSCVpuro-Rb3C grew at an increased rate compared with control cells: 480.6 ± 37.7 versus 159.4 ± 36.2 (relative cell count at 12 days). Tumor growth in nude mice also increased with RB knockdown compared with control mice: 135.2 ± 73.6 mm³ versus 40.0 ± 17.0 mm³ (tumor volume at 10 days).

CONCLUSIONS: Inhibition of RB expression is efficiently achieved in lung cancer cells with short-hairpin RNA. Genetic targets of RB are deregulated with RB knockdown. Retinoblastoma depletion increases growth in vitro and in murine xenografts. These studies indicate that even in the context of an established tumor cell line, RB limits tumorigenic proliferation. Additionally, this model will serve as an ideal system to evaluate the role of RB activity on therapeutic response.

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Divisions of Cancer Informatics and Cellular and Molecular Biology, Ontario Cancer Institute/Princess Margaret Hospital, University Health Network, Toronto, Ontario, Canada; Departments of ² Medical Biophysics, ³ Molecular and Medical Genetics, and ⁴ Ophthalmology, University of Toronto, Toronto, Ontario, Canada; ⁵ Retinoblastoma Solutions, Toronto, Ontario, Canada; and ⁶ Department of Ophthalmology, West China Hospital, Faculty of Medicine, Sichuan University, Chengdu, People’s Republic of China

Retinoblastoma is initiated by loss of both RB1 alleles. Previous studies have shown that retinoblastoma tumors also show further genomic gains and losses. We now define a 2.62 Mbp minimal region of genomic loss of chromosome 16q22, which is likely to contain tumor suppressor gene(s), in 76 retinoblastoma tumors, using loss of heterozygosity (30 of 76 tumors) and quantitative multiplex PCR (71 of 76 tumors). The sequence-tagged site WI-5835 within intron 2 of the cadherin-11 (CDH11) gene showed the highest frequency of loss (54%, 22 of 41 samples tested). A second hotspot for loss (39%, 9 of 23 samples tested) was detected within intron 2 of the cadherin-13 (CDH13) gene. Furthermore, deletion of the exons of CDH11 and/or WI-5835 was shown by quantitative multiplex PCR in 17 of 30 (57%) of previously untested tumors. Immunoblot analyses revealed that 91% (20 of 22) retinoblastoma exhibited either a complete loss or a decrease of the intact form of CDH11 and 8 of 13 showed a prevalent band suggestive of the variant form. Copy number of WI-5835 for these samples correlated with CDH11 protein expression. CDH11 staining was evident in the inner nuclear layer in early mouse retinal development and in small transgenic murine SV40 large T antigen–induced retinoblastoma tumors, but advanced tumors frequently showed loss of CDH11 expression by reverse transcription-PCR, suggestive of a role for CDH11 in tumor progression or metastasis. CDH13 protein and mRNA were consistently expressed in all human and murine retinoblastoma compared with normal adult human retina. Our analyses implicate CDH11, but not CDH13, as a potential tumor suppressor gene in retinoblastoma.

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Authors’ Affiliations: Departments of ¹ Developmental Neurobiology and ² Pharmaceutical Sciences, St. Jude Children’s Research Hospital, Memphis, Tennessee and ³ University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania

Requests for reprints: Michael A. Dyer, Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Mail Stop 323, 332 North Lauderdale, Memphis, TN 38105-2794. Phone: 901-495-2257; Fax: 901-495-3143; E-mail: michael.dyer@stjude.org

Chemotherapy combined with laser therapy and cryotherapy has improved the ocular salvage rate for children with bilateral retinoblastoma. However, children with late-stage disease often experience recurrence shortly after treatment. To improve the vision salvage rate in advanced bilateral retinoblastoma, we have developed and characterized two new rodent models of retinoblastoma for screening chemotherapeutic drug combinations. The first model is an orthotopic xenograft model in which green fluorescent protein– or luciferase-labeled human retinoblastoma cells are injected into the eyes of newborn rats. The second model uses a replication-incompetent retrovirus (LIA-E^E1A) encoding the E1A oncogene. Clonal, focal tumors arise from mouse retinal progenitor cells when LIA-E^E1A is injected into the eyes of newborn p53^–/– mice. Using these two models combined with pharmacokinetic studies and cell culture experiments, we have tested the efficacy of topotecan combined with carboplatin and of topotecan combined with vincristine for the treatment of retinoblastoma. The combination of topotecan and carboplatin most effectively halted retinoblastoma progression in our rodent models and was superior to the current triple drug therapy using vincristine, carboplatin, and etoposide. Vincristine had the lowest LC₅₀ in culture but did not reduce tumor growth in our preclinical retinoblastoma models. Taken together, these data suggest that topotecan may be a suitable replacement for etoposide in combination chemotherapy for the treatment of retinoblastoma.

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Alfred G. Knudson, Herbert W. Hethcote, and Barry W. Brown

The incidences of some childhood cancers have been shown to fit a two-mutation hypothesis for cancer initiation. According to this hypothesis, the first mutation can be either germinal or somatic while the second is always somatic. A probabilistic model involving the mean number of tumors per genetically susceptible individual is developed as a function of age and is compared with age incidence data for retinoblastoma. The change in the mean number of tumors with time is interpreted in terms of the growth of retinal cells. In patients who are not genetically susceptible, the times of occurrence of the first and second somatic mutations can be inferred from a comparison of familial and non-familial unilateral case incidences. The total incidences of hereditary and nonhereditary forms of retinoblastoma are related to germinal and somatic mutation rates. The even distribution of certain childhood cancers throughout the world suggests that their incidences are determined by spontaneous

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Ruth A. Kleinerman, Margaret A. Tucker, Robert E. Tarone, David H. Abramson, Johanna M. Seddon, Marilyn Stovall, Frederick P. Li, Joseph F. Fraumeni, Jr

From the Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Department of Health and Human Services; International Epidemiology Institute, Rockville, MD; Ophthalmic Oncology Service, Memorial Sloan- Kettering Cancer Center, New York, NY; Massachusetts Eye and Ear Infirmary; Dana-Farber Cancer Institute, Boston, MA; and Department of Radiation Physics, The University of Texas M.D. Anderson Cancer Center, Houston, TX

PURPOSE: Many children diagnosed with retinoblastoma (Rb) survive into adulthood and are prone to subsequent cancers, particularly hereditary patients, who have germline Rb-1 mutations. We have extended the follow-up of a large cohort of Rb patients for 7 more years to provide new information on the risk of additional cancers after radiotherapy in long-term survivors.

PATIENTS AND METHODS: We analyzed the risk of new cancers through 2000 in 1,601 Rb survivors, diagnosed from 1914 to 1984, at two US medical centers. The standardized incidence ratio (SIR) was calculated as the ratio of the observed number of cancers after hereditary and nonhereditary Rb to the expected number from the Connecticut Tumor Registry. The cumulative incidence of a new cancer after hereditary and nonhereditary Rb and radiotherapy was calculated with adjustment for competing risk of death.

RESULTS: Subsequent cancer risk in 963 hereditary patients (SIR, 19; 95% CI, 16 to 21) exceeded the risk in 638 nonhereditary Rb patients (SIR, 1.2; 95% CI, 0.7 to 2.0). Radiation further increased the risk of another cancer in hereditary patients by 3.1-fold (95% CI, 2.0 to 5.3). Hereditary patients continued to be at significantly increased risk for sarcomas, melanoma, and cancers of the brain and nasal cavities. The cumulative incidence for developing a new cancer at 50 years after diagnosis of Rb was 36% (95% CI, 31% to 41%) for hereditary and 5.7% (95% CI, 2.4% to 11%) for nonhereditary patients.

CONCLUSION: Hereditary Rb predisposes to a variety of new cancers over time, with radiotherapy further enhancing the risk of tumors arising in the radiation field.

Supported by N02-CP-81121 (National Cancer Institute) to Westat Inc (for field work), Rockville, MD.

Authors’ disclosures of potential conflicts of interest are found at the end of this article.

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