Non-synonymous, synonymous, and non-coding nucleotide variants contribute to recurrently altered biological processes during retinoblastoma progression.

Retinoblastomas form in response to biallelic RB1 mutations or MYCN amplification and progress to more aggressive and therapy-resistant phenotypes through accumulation of secondary genomic changes. Progression-related changes include recurrent somatic copy number alterations and typically non-recurrent nucleotide variants, including synonymous and non-coding variants, whose significance has been unclear. To determine if nucleotide variants recurrently affect specific biological processes, we identified altered genes and over-represented variant gene ontologies in 168 exome or whole-genome-sequenced retinoblastomas and 12 tumor-matched cell lines. In addition to RB1 mutations, MYCN amplification, and established retinoblastoma somatic copy number alterations, the analyses revealed enrichment of variant genes related to diverse biological processes including histone monoubiquitination, mRNA processing (P) body assembly, and mitotic sister chromatid segregation and cytokinesis. Importantly, non-coding and synonymous variants increased the enrichment significance of each over-represented biological process term. To assess the effects of such mutations, we examined the consequences of a 3' UTR variant of PCGF3 (a BCOR-binding component of Polycomb repressive complex I), dual 3' UTR variants of CDC14B (a regulator of sister chromatid segregation), and a synonymous variant of DYNC1H1 (a regulator of P-body assembly). One PCGF3 and one of two CDC14B 3' UTR variants impaired gene expression whereas a base-edited DYNC1H1 synonymous variant altered protease sensitivity and stability. Retinoblastoma cell lines retained only ~50% of variants detected in tumors and enriched for new variants affecting p53 signaling. These findings reveal potentially important differences in retinoblastoma cell lines and tumors and implicate synonymous and non-coding variants, along with non-synonymous variants, in retinoblastoma oncogenesis.

Characterizing DNA methylation signatures of retinoblastoma using aqueous humor liquid biopsy.

Retinoblastoma (RB) is a cancer that forms in the developing retina of babies and toddlers. The goal of therapy is to cure the tumor, save the eye and maximize vision. However, it is difficult to predict which eyes are likely to respond to therapy. Predictive molecular biomarkers are needed to guide prognosis and optimize treatment decisions. Direct tumor biopsy is not an option for this cancer; however, the aqueous humor (AH) is an alternate source of tumor-derived cell-free DNA (cfDNA). Here we show that DNA methylation profiling of the AH is a valid method to identify the methylation status of RB tumors. We identify 294 genes directly regulated by methylation that are implicated in p53 tumor suppressor (RB1, p53, p21, and p16) and oncogenic (E2F) pathways. Finally, we use AH to characterize molecular subtypes that can potentially be used to predict the likelihood of treatment success for retinoblastoma patients.

Extracellular vesicle-associated repetitive element DNAs as candidate osteosarcoma biomarkers.

Osteosarcoma (OS) is the most common malignant bone tumor in children and young adults. Despite that high-risk factors have been identified, no test for early detection is available. This study aimed to identify circulating nucleic acid sequences associated with serum extracellular vesicle (EV) preparations at the time of OS diagnosis, as a step towards an OS early detection assay. Sequencing of small nucleic acids extracted from serum EV preparations revealed increased representation of diverse repetitive element sequences in OS patient versus control sera. Analysis of a validation cohort using qPCR of PEG-precipitated EV preparations revealed the over-representation of HSATI, HSATII, LINE1-P1, and Charlie 3 at the DNA but not RNA level, with receiver operating characteristic (ROC) area under the curve (AUC) ≥ 0.90. HSATI and HSATII DNAs co-purified with EVs prepared by precipitation and size exclusion chromatography but not by exosome immunocapture, indicative of packaging in a non-exosomal complex. The consistent over-representation of EV-associated repetitive element DNA sequences suggests their potential utility as biomarkers for OS and perhaps other cancers.

Detection of mitochondrial DNA variants at low level heteroplasmy in pediatric CNS and extra-CNS solid tumors with three different enrichment methods.

The mitochondrial genome is small, 16.5 kb, and yet complex to study due to an abundance of mitochondria in any given cell or tissue. Mitochondrial DNA (mtDNA) mutations have been previously described in cancer, many of which were detected at low heteroplasmy. In this study we enriched the mitochondrial genome in primary pediatric tumors for detection of mtDNA variants. We completed mtDNA enrichment using REPLI-g, Agilent SureSelect, and long-range polymerase chain reaction (LRPCR) followed by next generation sequencing (NGS) on Illumina platforms. Primary tumor and germline genomic DNA from a variety of pediatric central nervous system (CNS) and extra-CNS solid tumors were analyzed by the three different methods. Although all three methods performed equally well for detecting variants at high heteroplasmy or homoplasmy, only LRPCR and SureSelect-based enrichment methods provided consistent results for variants that were present at less than five percent heteroplasmy. We then applied both LRPCR and SureSelect to three successive samples from a patient with multiply-recurrent gliofibroma and detected a low-level novel mutation as well as a change in heteroplasmy levels of a synonymous variant that was correlated with progression of disease. IMPLICATION: This study demonstrates that LRPCR and SureSelect enrichment, but not REPLI-g, followed by NGS are accurate methods for studying the mtDNA variations at low heteroplasmy, which may be applied to studying mtDNA mutations in cancer.

Developmental stage-specific proliferation and retinoblastoma genesis in RB-deficient human but not mouse cone precursors.

Most retinoblastomas initiate in response to the inactivation of the RB1 gene and loss of functional RB protein. The tumors may form with few additional genomic changes and develop after a premalignant retinoma phase. Despite this seemingly straightforward etiology, mouse models have not recapitulated the genetic, cellular, and stage-specific features of human retinoblastoma genesis. For example, whereas human retinoblastomas appear to derive from cone photoreceptor precursors, current mouse models develop tumors that derive from other retinal cell types. To investigate the basis of the human cone-specific oncogenesis, we compared developmental stage-specific cone precursor responses to RB loss in human and murine retina cultures and in cone-specific Rb1-knockout mice. We report that RB-depleted maturing (ARR3+) but not immature (ARR3-) human cone precursors enter the cell cycle, proliferate, and form retinoblastoma-like lesions with Flexner-Wintersteiner rosettes, then form low or nonproliferative premalignant retinoma-like lesions with fleurettes and p16INK4A and p130 expression, and finally form highly proliferative retinoblastoma-like masses. In contrast, in murine retina, only RB-depleted immature (Arr3-) cone precursors entered the cell cycle, and they failed to progress from S to M phase. Moreover, whereas intrinsically highly expressed MDM2 and MYCN contribute to RB-depleted maturing (ARR3+) human cone precursor proliferation, ectopic MDM2 and Mycn promoted only immature (Arr3-) murine cone precursor cell-cycle entry. These findings demonstrate that developmental stage-specific as well as species- and cell type-specific features sensitize to RB1 inactivation and reveal the human cone precursors' capacity to model retinoblastoma initiation, proliferation, premalignant arrest, and tumor growth.

Genomic cfDNA Analysis of Aqueous Humor in Retinoblastoma Predicts Eye Salvage: The Surrogate Tumor Biopsy for Retinoblastoma.

Tumor-derived cell-free DNA (cfDNA) has biomarker potential; therefore, this study aimed to identify cfDNA in the aqueous humor (AH) of retinoblastoma eyes and correlate somatic chromosomal copy-number alterations (SCNA) with clinical outcomes, specifically eye salvage. AH was extracted via paracentesis during intravitreal injection of chemotherapy or enucleation. Shallow whole-genome sequencing was performed using isolated cfDNA to assess for highly recurrent SCNAs in retinoblastoma including gain of 1q, 2p, 6p, loss of 13q, 16q, and focal MYCN amplification. Sixty-three clinical specimens of AH from 29 eyes of 26 patients were evaluated; 13 eyes were enucleated and 16 were salvaged (e.g., saved). The presence of detectable SCNAs was 92% in enucleated eyes versus 38% in salvaged eyes (P = 0.006). Gain of chromosome 6p was the most common SCNA found in 77% of enucleated eyes, compared with 25% of salvaged eyes (P = 0.0092), and associated with a 10-fold increased odds of enucleation (OR, 10; 95% CI, 1.8-55.6). The median amplitude of 6p gain was 1.47 in enucleated versus 1.07 in salvaged eyes (P = 0.001). The presence of AH SCNAs was correlated retrospectively with eye salvage. The probability of ocular salvage was higher in eyes without detectable SCNAs in the AH (P = 0.0028), specifically 6p gain. This is the first study to correlate clinical outcomes with SCNAs in the AH from retinoblastoma eyes, as such these findings indicate that 6p gain in the aqueous humor is a potential prognostic biomarker for poor clinical response to therapy.Implications: The correlation of clinical outcomes and SCNAs in the AH identified in the current study requires prospective studies to validate these finding before SCNAs, like 6p gain, can be used to predict clinical outcomes at diagnosis. Mol Cancer Res; 16(11); 1701-12. ©2018 AACR.

Rabbit model of ocular indirect photodynamic therapy using a retinoblastoma xenograft.

PURPOSE: The goal of this project was to demonstrate the feasibility of coupling the indirect ophthalmoscope laser delivery system with the 690 nm wavelength diode laser used to perform photodynamic therapy (PDT) in the treatment of retinoblastoma. METHODS: For phase 1, a total of six pigmented rabbits were treated with the indirect laser delivery system. The laser source was provided by the Lumenis Opal 690 nm laser unit, delivered through a 810 nm Indirect ophthalmoscope headpiece and a hand-held 28-diopter indirect lens (1.0 mm spot size). Four rabbits received intravenous verteporfin at doses of 0.43 or 0.86 mg/kg, and two rabbits did not receive verteporfin (controls). A second phase of the study involved eight rabbits using a retinoblastoma xenograft to determine the effect of indirect PDT on subretinal tumors. RESULTS: For phase 1, a total of 20 laser treatments were performed in the right eyes of six rabbits. Laser power levels ranged between 40 and 150 mW/cm2 and treatment duration ranged between 1 and 3 min. In the four rabbits that received verteporfin, focal retinal scars were noted at 40 mW/cm2 and higher power levels. In the two control rabbits that did not receive verteporfin, thermal burns were confirmed at 75 mW/cm2 and higher power levels. Histopathology showed focal retino-choroidal scars at the site of PDT treatment, without evidence of generalized ocular damage. Using the retinoblastoma xenograft, the indirect PDT system was shown to cause areas of tumor necrosis on histopathology. CONCLUSIONS: The results of this pre-clinical study suggest verteporfin may be activated in the rabbit retina with the indirect delivery system and the 690 nm laser unit (i.e., Indirect PDT). Using verteporfin, treatment effects were observed at 40-50 mW/cm2 in the rabbit retina, while photocoagulation was achieved at 75 mW/cm2 and higher power levels. Fundoscopic and histopathologic examination of treated areas showed circumscribed areas of retinal damage and a lack of generalized ocular toxicity, suggesting that this modality may represent a safe and localized method for treating intraocular retinoblastoma.

Potential of Aqueous Humor as a Surrogate Tumor Biopsy for Retinoblastoma.

Importance: Retinoblastoma (Rb) is one of the first tumors to have a known genetic etiology. However, because biopsy of this tumor is contraindicated, it has not been possible to define the effects of secondary genetic changes on the disease course. Objective: To investigate whether the aqueous humor (AH) of Rb eyes has sufficient tumor-derived DNA to perform genetic analysis of the tumor, including DNA copy number alterations. Design, Setting, and Participants: This investigation was a case series study at a tertiary care hospital (Children's Hospital Los Angeles) with a large Rb treatment center. Cell-free DNA (cfDNA) was isolated from 6 AH samples from 3 children with Rb, including 2 after primary enucleation and 1 undergoing multiple intravitreous injections of melphalan for vitreous seeding. Samples were taken between December 2014 and September 2015. Main Outcomes and Measures: Measurable levels of nucleic acids in the AH and identification of tumor-derived DNA copy number variation in the AH. The AH was analyzed for DNA, RNA, and micro-RNA using Qubit high-sensitivity kits. Cell-free DNA was isolated from the AH, and sequencing library protocols were optimized. Shallow whole-genome sequencing was performed on an Illumina platform, followed by genome-wide chromosomal copy number variation profiling to assess the presence of tumor DNA fractions in the AH cfDNA of the 3 patients. One child's cfDNA from the AH and tumor DNA were subjected to Sanger sequencing to isolate the RB1 mutation. Results: Six AH samples were obtained from 3 Rb eyes in 3 children (2 male and 1 female; diagnosed at ages 7, 20, and 28 months). A corroborative pattern between the chromosomal copy number variation profiles of the AH cfDNA and tumor-derived DNA from the enucleated samples was identified. In addition, a nonsense RB1 mutation (Lys→STOP) from 1 child was also identified from the AH samples obtained during intravitreous injection of melphalan, which matched the tumor sample postsecondary enucleation. Sanger sequencing of the AH cfDNA and tumor DNA with polymerase chain reaction primers targeting RB1 gene c.1075A demonstrated this same RB1 mutation. Conclusions and Relevance: In this study evaluating nucleic acids in the AH from Rb eyes undergoing salvage therapy with intravitreous injection of melphalan, the results suggest that the AH can serve as a surrogate tumor biopsy when Rb tumor tissue is not available. This novel method will allow for analyses of tumor-derived DNA in Rb eyes undergoing salvage therapy that have not been enucleated.