Rachel Brennan

A novel retinoblastoma therapy from genomic and epigenetic analyses

Retinoblastoma is an aggressive childhood cancer of the developing retina that is initiated by the biallelic loss of RB1. Tumours progress very quickly following RB1 inactivation but the underlying mechanism is not known. Here we show that the retinoblastoma genome is stable, but that multiple cancer pathways can be epigenetically deregulated. To identify the mutations that cooperate with RB1 loss, we performed whole-genome sequencing of retinoblastomas. The overall mutational rate was very low; RB1 was the only known cancer gene mutated. We then evaluated the role of RB1 in genome stability and considered non-genetic mechanisms of cancer pathway deregulation. For example, the proto-oncogene SYK is upregulated in retinoblastoma and is required for tumour cell survival. Targeting SYK with a small-molecule inhibitor induced retinoblastoma tumour cell death in vitro and in vivo. Thus, retinoblastomas may develop quickly as a result of the epigenetic deregulation of key cancer pathways as a direct or indirect result of RB1 loss.

Targeting the p53 Pathway in Retinoblastoma with Subconjunctival Nutlin-3a

Retinoblastoma is a rare childhood cancer of the retina that begins in utero and is diagnosed in the first years of life. The goals of retinoblastoma treatment are ocular salvage, vision preservation, and reduction of short- and long-term side effects without risking mortality because of tumor dissemination. To identify better chemotherapeutic combinations for the treatment of retinoblastoma, several groups have developed genetic mouse models and orthotopic xenograft models of human retinoblastoma for preclinical testing. Previous studies have implicated the MDMX protein in the suppression of the p53 pathway in retinoblastoma and shown that the MDM2/MDMX antagonist, Nutlin-3a, can efficiently induce p53-mediated cell death in retinoblastoma cell lines. However, Nutlin-3a cannot be administered systemically to treat retinoblastoma, because it has poor penetration across the blood-ocular barrier. Therefore, we developed an ocular formulation of Nutlin-3a, Nutlin-3a(OC), and tested the pharmacokinetics and efficacy of this new formulation in genetic and human retinoblastoma orthotopic xenograft models of retinoblastoma. Here, we show that Nutlin-3a(OC) specifically and efficiently targets the p53 pathway and that the combination of Nutlin-3a(OC) with systemic topotecan is a significantly better treatment for retinoblastoma than currently used chemotherapy in human orthotopic xenografts. Our studies provide a new standardized approach to evaluate and prioritize novel agents for incorporation into future clinical trials for retinoblastoma.

Analysis of MDM2 and MDM4 single nucleotide polymorphisms, mRNA splicing and protein expression in retinoblastoma

Retinoblastoma is a childhood cancer of the developing retina that begins in utero and is diagnosed in the first years of life. Biallelic RB1 gene inactivation is the initiating genetic lesion in retinoblastoma. The p53 gene is intact in human retinoblastoma but the pathway is believed to be suppressed by increased expression of MDM4 (MDMX) and MDM2. Here we quantify the expression of MDM4 and MDM2 mRNA and protein in human fetal retinae, primary retinoblastomas, retinoblastoma cell lines and several independent orthotopic retinoblastoma xenografts. We found that MDM4 is the major p53 antagonist expressed in retinoblastoma and in the developing human retina. We also discovered that MDM4 protein steady state levels are much higher in retinoblastoma than in human fetal retinae. This increase would not have been predicted based on the mRNA levels. We explored several possible post-transcriptional mechanisms that may contribute to the elevated levels of MDM4 protein. A proportion of MDM4 transcripts are alternatively spliced to produce protein products that are reported to be more stable and oncogenic. We also discovered that a microRNA predicted to target MDM4 (miR191) was downregulated in retinoblastoma relative to human fetal retinae and a subset of samples had somatic mutations that eliminated the miR-191 binding site in the MDM4 mRNA. Taken together, these data suggest that post-transcriptional mechanisms may contribute to stabilization of the MDM4 protein in retinoblastoma.

Preclinical models for neuroblastoma: establishing a baseline for treatment.

Background Preclinical models of pediatric cancers are essential for testing new chemotherapeutic combinations for clinical trials. The most widely used genetic model for preclinical testing of neuroblastoma is the TH-MYCN mouse. This neuroblastoma-prone mouse recapitulates many of the features of human neuroblastoma. Limitations of this model include the low frequency of bone marrow metastasis, the lack of information on whether the gene expression patterns in this system parallels human neuroblastomas, the relatively slow rate of tumor formation and variability in tumor penetrance on different genetic backgrounds. As an alternative, preclinical studies are frequently performed using human cell lines xenografted into immunocompromised mice, either as flank implant or orthtotopically. Drawbacks of this system include the use of cell lines that have been in culture for years, the inappropriate microenvironment of the flank or difficult, time consuming surgery for orthotopic transplants and the absence of an intact immune system. Principal Findings Here we characterize and optimize both systems to increase their utility for preclinical studies. We show that TH-MYCN mice develop tumors in the paraspinal ganglia, but not in the adrenal, with cellular and gene expression patterns similar to human NB. In addition, we present a new ultrasound guided, minimally invasive orthotopic xenograft method. This injection technique is rapid, provides accurate targeting of the injected cells and leads to efficient engraftment. We also demonstrate that tumors can be detected, monitored and quantified prior to visualization using ultrasound, MRI and bioluminescence. Finally we develop and test a “standard of care” chemotherapy regimen. This protocol, which is based on current treatments for neuroblastoma, provides a baseline for comparison of new therapeutic agents. Significance The studies suggest that use of both the TH-NMYC model of neuroblastoma and the orthotopic xenograft model provide the optimal combination for testing new chemotherapies for this devastating childhood cancer.

Topotecan and vincristine combination is effective against advanced bilateral intraocular retinoblastoma and has manageable toxicity

New, effective chemotherapeutic agents are needed for intraocular retinoblastoma.

US and MRI of pediatric ocular masses with histopathological correlation

We review our experience with unusual ocular pathologies, some mimicking retinoblastoma, that were referred to our institution during the past two decades. After presenting the imaging anatomy of the normal eye, we discuss pertinent clinical and pathological features, and illustrate the US and MRI appearance of retinoblastoma, medulloepithelioma, uveal melanoma, persistent fetal vasculature, Coats disease, corneal dermoid, retinal dysplasia and toxocara granuloma. Features useful in discriminating among these entities are emphasized.

Developmental and Adaptive Functioning in Children With Retinoblastoma: A Longitudinal Investigation

PURPOSE To determine the developmental trajectory of early cognitive and adaptive skills in young children with retinoblastoma from diagnosis to 5 years of age. PATIENTS AND METHODS Ninety-four patients with retinoblastoma treated according to an institutional protocol underwent serial assessments of cognitive and adaptive functioning at age 6 months and 1, 2, 3, and 5 years. Data were analyzed by treatment strata, with patients with 13q deletion analyzed separately. RESULTS At baseline, across all patients (except those with 13q deletion), developmental functioning was comparable with the normative mean, with mean scores for all strata within the average range. However, at age 5 years, developmental functioning was in the low average range and significantly below normative means. The trajectories of developmental functioning demonstrated significant decline over time, although this varied by treatment group/strata. Patients treated with enucleation only evidenced the greatest decline in cognitive functioning; significant change was not observed in patients treated with other modalities. Notable declines in parent-reported communication skills were observed in the majority of patients. Patients with 13q deletion evidenced delayed cognitive functioning at baseline, but minimal declines were observed through age 3 years. However, significant decreases in adaptive functioning were demonstrated over time for the 13q deletion subset. CONCLUSION The declines in functioning observed in this study were unexpected, as was the poorer performance of the enucleation-only group. This highlights the necessity of continuing to assess cognitive functioning in patients with retinoblastoma as they age. Additional research is necessary to determine the long-term trajectory of cognitive development in this population.

Inhibition of MMP-2 and MMP-9 decreases cellular migration, and angiogenesis in in vitro models of retinoblastoma

BackgroundRetinoblastoma (Rb) is the most common primary intraocular tumor in children. Local treatment of the intraocular disease is usually effective if diagnosed early; however advanced Rb can metastasize through routes that involve invasion of the choroid, sclera and optic nerve or more broadly via the ocular vasculature. Metastatic Rb patients have very high mortality rates. While current therapy for Rb is directed toward blocking tumor cell division and tumor growth, there are no specific treatments targeted to block Rb metastasis. Two such targets are matrix metalloproteinases-2 and -9 (MMP-2, −9), which degrade extracellular matrix as a prerequisite for cellular invasion and have been shown to be involved in other types of cancer metastasis. Cancer Clinical Trials with an anti-MMP-9 therapeutic antibody were recently initiated, prompting us to investigate the role of MMP-2, −9 in Rb metastasis.MethodsWe compare MMP-2, −9 activity in two well-studied Rb cell lines: Y79, which exhibits high metastatic potential and Weri-1, which has low metastatic potential. The effects of inhibitors of MMP-2 (ARP100) and MMP-9 (AG-L-66085) on migration, angiogenesis, and production of immunomodulatory cytokines were determined in both cell lines using qPCR, and ELISA. Cellular migration and potential for invasion were evaluated by the classic wound-healing assay and a Boyden Chamber assay.ResultsOur results showed that both inhibitors had differential effects on the two cell lines, significantly reducing migration in the metastatic Y79 cell line and greatly affecting the viability of Weri-1 cells. The MMP-9 inhibitor (MMP9I) AG-L-66085, diminished the Y79 angiogenic response. In Weri-1 cells, VEGF was significantly reduced and cell viability was decreased by both MMP-2 and MMP-9 inhibitors. Furthermore, inhibition of MMP-2 significantly reduced secretion of TGF-β1 in both Rb models.ConclusionsCollectively, our data indicates MMP-2 and MMP-9 drive metastatic pathways, including migration, viability and secretion of angiogenic factors in Rb cells. These two subtypes of matrix metalloproteinases represent new potential candidates for targeted anti-metastatic therapy for Rb.

Cognitive function and social attainment in adult survivors of retinoblastoma: A report from the St. Jude Lifetime Cohort Study

Retinoblastoma has a 5‐year survival rate exceeding 95%, yet little is known about long‐term functional outcomes for these patients.

Time-Frequency Analysis of Transient-Evoked Otoacoustic Emissions in Children Exposed to Carboplatin Chemotherapy

The aims of this study were to characterize and quantify time-frequency changes in transient-evoked otoacoustic emissions (TEOAEs) recorded in children diagnosed with retinoblastoma who were receiving carboplatin chemotherapy. A signal processing technique, the wavelet transform (WT), was used to analyze TEOAE waveforms in narrow-band frequency components. Ten children (aged 3–72 months) diagnosed with unilateral or bilateral retinoblastoma were enrolled in the study. TEOAEs were acquired from the children with linear sequences of 70 dB peak equivalent SPL clicks. After WT analysis, TEOAE energy, latency and normalized energy in the narrow-band frequency components were compared before and during carboplatin chemotherapy treatment (average dose 1693 mg/m2). On a group basis, no significant differences (p > 0.05) in the TEOAE energy, latency or normalized energy before and after carboplatin treatment were observed. There were decreases in normalized energy on an individual basis in 10 out of 18 ears in the sample. Exposure to carboplatin chemotherapy did not cause significant changes in TEOAE energy, latency and normalized energy during treatment. However, long-term monitoring of hearing with measurements of TEOAEs is warranted, given the risks of delayed hearing loss in some children receiving carboplatin chemotherapy.