Evolutionary conservation of VSX2 super-enhancer modules in retinal development.

Super-enhancers (SEs) are expansive regions of genomic DNA that regulate the expression of genes involved in cell identity and cell fate. We recently identified developmental stage- and cell type-specific modules within the murine Vsx2 SE. Here, we show that the human VSX2 SE modules have similar developmental stage- and cell type-specific activity in reporter gene assays. By inserting the human sequence of one VSX2 SE module into a mouse with microphthalmia, eye size was rescued. To understand the function of these SE modules during human retinal development, we deleted individual modules in human embryonic stem cells and generated retinal organoids. Deleting one module results in small organoids, recapitulating the small-eyed phenotype of mice with microphthalmia, while deletion of the other module led to disruptions in bipolar neuron development. This prototypical SE serves as a model for understanding developmental stage- and cell type-specific effects of neurogenic transcription factors with complex expression patterns. Moreover, by elucidating the gene regulatory mechanisms, we can begin to examine how dysregulation of these mechanisms contributes to phenotypic diversity and disease.

Change in stage after neoadjuvant chemoradiation is associated with survival in patients with esophageal cancer.

BACKGROUND: The aim of this study was to determine if change in stage after neoadjuvant chemoradiation (CRT) was associated with improved survival in esophageal cancer using a national database. METHODS: Using the National Cancer Database, patients with non-metastatic, resectable esophageal cancer who received neoadjuvant CRT and surgery were identified. Comparing clinical to the pathologic stage, change in stage was classified as pathologic complete response (pCR), downstaged, same-staged, or upstaged. Univariable and multivariable Cox regression models were used to identify factors associated with survival. RESULTS: A total of 7745 patients were identified. The median overall survival (OS) was 34.9 months. Median OS was 60.3 months if pCR, 39.1 months if downstaged, 28.3 months if same-staged, and 23.4 months if upstaged (p < 0.0001). On multivariable analysis, pCR was associated with improved OS compared to the other groups (downstaged: hazard ratio [HR]: 1.32 [95% confidence interval [CI]: 1.18-1.46]; same-staged: HR: 1.89 [95% CI: 1.68-2.13]; upstaged: HR: 2.54 [95% CI: 2.25-2.86]; all p < 0.0001). CONCLUSIONS: In this large database study, change in stage after neoadjuvant CRT was strongly associated with survival for patients with non-metastatic, resectable esophageal cancer. There was a significant stepwise decline in survival, in descending order of pCR, downstaged tumor, same-staged tumor, and upstaged tumor.

Orthotopic patient-derived xenografts of paediatric solid tumours.

Paediatric solid tumours arise from endodermal, ectodermal, or mesodermal lineages. Although the overall survival of children with solid tumours is 75%, that of children with recurrent disease is below 30%. To capture the complexity and diversity of paediatric solid tumours and establish new models of recurrent disease, here we develop a protocol to produce orthotopic patient-derived xenografts at diagnosis, recurrence, and autopsy. Tumour specimens were received from 168 patients, and 67 orthotopic patient-derived xenografts were established for 12 types of cancer. The origins of the patient-derived xenograft tumours were reflected in their gene-expression profiles and epigenomes. Genomic profiling of the tumours, including detailed clonal analysis, was performed to determine whether the clonal population in the xenograft recapitulated the patient's tumour. We identified several drug vulnerabilities and showed that the combination of a WEE1 inhibitor (AZD1775), irinotecan, and vincristine can lead to complete response in multiple rhabdomyosarcoma orthotopic patient-derived xenografts tumours in vivo.

Longitudinal Outcomes of Medical Student Research Mentorship: a 15-Year Analysis of the Radiation Oncology Mentorship Initiative.

At our institution, students can be mentored by radiation oncology faculty through structured research programs, such as the Medical Student Summer Research Program (MSSRP). The purpose of this study is to report the research productivity of students who engaged in radiation oncology research mentorship, whether through the MSSRP or other avenues of research mentorship. We compiled a database of abstracts and manuscripts co-authored by 58 students who conducted research with radiation oncology faculty from 2005 to 2020. The means, medians, ranges, and interquartile ranges (IQR) of co-authorships and first authorships were calculated for the overall cohort and compared for MSSRP and non-MSSRP students, who matched into radiation oncology and those who did not, and male versus female students. Among all 58 students, 106 abstracts and 70 manuscripts were identified. Of those students, 54 (93.1%) published at least one abstract or manuscript. The mean number of abstract co-authorships per student was 3.07 (median 2, range 0-25, IQR 0-4), and the mean number of manuscript co-authorships per student was 2.22 (median 1, range 0-18, IQR 1-3). There were no significant differences in research output between MSSRP and non-MSSRP students or male and female students. However, the students who matched into radiation oncology published more co-author (3.67 vs. 1.63, p = 0.01) and first-author (1.62 vs. 0.53, p = 0.006) manuscripts than those who did not. Further research is warranted to assess whether skills gained from student-directed research translate into residency and beyond.

Next-generation humanized patient-derived xenograft mouse model for pre-clinical antibody studies in neuroblastoma.

Faithful tumor mouse models are fundamental research tools to advance the field of immuno-oncology (IO). This is particularly relevant in diseases with low incidence, as in the case of pediatric malignancies, that rely on pre-clinical therapeutic development. However, conventional syngeneic and genetically engineered mouse models fail to recapitulate the tumor heterogeneity and microenvironmental complexity of human pathology that are essential determinants of cancer-directed immunity. Here, we characterize a novel mouse model that supports human natural killer (NK) cell development and engraftment of neuroblastoma orthotopic patient-derived xenograft (O-PDX) for pre-clinical antibody and cytokine testing. Using cytotoxicity assays, single-cell RNA-sequencing, and multi-color flow cytometry, we demonstrate that NK cells that develop in the humanized mice are fully licensed to execute NK cell cytotoxicity, permit human tumor engraftment, but can be therapeutically redirected to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Although these cells share phenotypic and molecular features with healthy controls, we noted that they lacked an NK cell subset, termed activated NK cells, that is characterized by differentially expressed genes that are induced by cytokine activation. Because this subset of genes is also downregulated in patients with neuroblastoma compared to healthy controls, we hypothesize that this finding could be due to tumor-mediated suppressive effects. Thus, despite its technical complexity, this humanized patient-derived xenograft mouse model could serve as a faithful system for future testing of IO applications and studies of underlying immunologic processes.

Knowledge-based planning in robotic intracranial stereotactic radiosurgery treatments.

PURPOSE: To develop a knowledge-based planning (KBP) model that predicts dosimetric indices and facilitates planning in CyberKnife intracranial stereotactic radiosurgery/radiotherapy (SRS/SRT). METHODS: Forty CyberKnife SRS/SRT plans were retrospectively used to build a linear KBP model which correlated the equivalent radius of the PTV (req_PTV ) and the equivalent radius of volume that receives a set of prescription dose (req_Vi , where Vi  = V10% , V20% … V120% ). To evaluate the model's predictability, a fourfold cross-validation was performed for dosimetric indices such as gradient measure (GM) and brain V50% . The accuracy of the prediction was quantified by the mean and the standard deviation of the difference between planned and predicted values, (i.e., ΔGM = GMpred - GMclin and fractional ΔV50%  = (V50%pred - V50%clin )/V50%clin ) and a coefficient of determination, R2 . Then, the KBP model was incorporated into the planning for another 22 clinical cases. The training plans and the KBP test plans were compared in terms of the new conformity index (nCI) as well as the planning efficiency. RESULTS: Our KBP model showed desirable predictability. For the 40 training plans, the average prediction error from cross-validation was only 0.36 ± 0.06 mm for ΔGM, and 0.12 ± 0.08 for ΔV50% . The R2 for the linear fit between req_PTV and req_vi was 0.985 ± 0.019 for isodose volumes ranging from V10% to V120% ; particularly, R2  = 0.995 for V50% and R2  = 0.997 for V100% . Compared to the training plans, our KBP test plan nCI was improved from 1.31 ± 0.15 to 1.15 ± 0.08 (P < 0.0001). The efficient automatic generation of the optimization constraints by using our model requested no or little planner's intervention. CONCLUSION: We demonstrated a linear KBP based on PTV volumes that accurately predicts CyberKnife SRS/SRT planning dosimetric indices and greatly helps achieve superior plan quality and planning efficiency.

Hypofractionated radiotherapy and surgery compared to standard radiotherapy in early glottic cancer.

PURPOSE: Early-stage glottic laryngeal cancer is treated with surgery or radiotherapy (RT), but limited randomized data exists to support one modality over the other. This study evaluates survival differences in early glottic cancer patients treated with either surgery or RT. MATERIALS AND METHODS: 14,498 patients with early glottic cancer diagnosed from 2004 to 2015 and treated with surgery or RT were identified in the National Cancer Database. Kaplan-Meier method was used to analyze differences in overall survival (OS) by treatment (surgery vs. RT) and radiation dose fractionation. Cox regression modeling and propensity score-matched (PSM) analysis were performed. Adjusted hazard ratios (aHR) with 95% confidence intervals (95% CI) were computed. RESULTS: Median follow-up and median OS for all patients were 49.5 and 118 months, respectively. The estimated 5-year OS for surgery and RT was 77.5% and 72.6%, respectively (P < 0.0001). On multivariate analysis, aHR (95% CI) for surgery compared to RT was 0.87 (0.81-0.94, P = 0.0004). Compared to RT regimen 63-67.5 Gray (Gy) in 28-30 fractions, worse survival was noted for RT regimen 66-70 Gy in 33-35 fractions (aHR 1.15, 95% CI 1.07-1.23, P = 0.0003). When compared with hypofractionated RT (63-67.5 Gy in 28-30 fractions), patients undergoing surgery no longer showed improved OS (aHR 0.94, 95% CI 0.86-1.02, P = 0.154). The finding was confirmed on PSM analysis (surgery aHR 0.95, 95% CI 0.87-1.05, P = 0.322). CONCLUSION: In early glottic tumors, patients treated with surgery demonstrated improved survival compared to RT, but when hypofractionation was considered, there were no significant differences in OS between patients undergoing surgery or RT.

Comparing surgical and nonsurgical larynx-preserving treatments with total laryngectomy for locally advanced laryngeal cancer.

BACKGROUND: The declining 5-year overall survival (OS) of patients with laryngeal cancer has been associated with increased nonsurgical management of stage III/IV disease. To further assess this hypothesis, the authors evaluated recent OS trends and patterns of use between larynx-preserving approaches with chemoradiation (CRT) or partial laryngectomy (PL) and total laryngectomy (TL) stratified by tumor and nodal burden. METHODS: The National Cancer Data Base was used to identify 8703 patients with stage III/IV (excluding T1 tumors) laryngeal squamous cell carcinoma treated between 2003 and 2011 with CRT or upfront PL or TL with or without adjuvant therapy. OS was analyzed using the Kaplan-Meier method and a Cox proportional hazards model. RESULTS: Among patients with non-T4, low nodal burden (T2N1 or T3N0-N1) disease, no survival differences were observed between CRT, PL, and TL. Patients who had non-T4, high nodal burden (T2-T3N2-N3) disease who underwent TL with or without adjuvant treatment had a higher risk of death compared with those who received CRT (hazard ratio, 1.25; 95% CI, 1.04-1.51; P = .016). For T4N0-N3 tumors, TL compared with CRT was associated with improved OS (hazard ratio, 0.80; 95% CI, 0.62-0.92; P = .002). No statistically significant difference in outcome was noted between CRT and PL for all stage groups. The use of CRT has declined and receipt of TL has increased since 2006 for T4 disease, whereas PL rates have remained stably low. CONCLUSIONS: No survival differences were noted between surgical and nonsurgical approaches for patients with non-T4, low nodal burden laryngeal cancer. Patients with non-T4, high nodal burden disease may benefit from definitive CRT. Total laryngectomy remains advantageous in patients with T4 disease.

Correction to: Imaging features of immune-mediated genitourinary disease.

The authors regret that they neglected to cite their conference report on the technical part of a 'preliminary study' presented at, and published in, the Biomedical Sciences and Engineering Conference (BSEC), 2010, May 25-26 (Fully automated segmentation and characterization of the dendritic trees of retinal horizontal neurons -DOI: 10.1109/BSEC.2010.5510843 ), as it related to the larger dataset presented as validation of the method in the Neurochemical Research article (Automated Tracing of Horizontal Neuron Processes During Retinal Development- Neurochem Res. 2011 Apr;36(4):583-93). This resulted in the lack of transparency on the re-use and duplication of introductory text, which should have been cited. No figures or tables were reproduced, but rather larger confirmatory data and different set of results were reported. Appropriate authors were cited in both papers.

Post-transcriptional gene expression control by NANOS is up-regulated and functionally important in pRb-deficient cells.

Inactivation of the retinoblastoma tumor suppressor (pRb) is a common oncogenic event that alters the expression of genes important for cell cycle progression, senescence, and apoptosis. However, in many contexts, the properties of pRb-deficient cells are similar to wild-type cells suggesting there may be processes that counterbalance the transcriptional changes associated with pRb inactivation. Therefore, we have looked for sets of evolutionary conserved, functionally related genes that are direct targets of pRb/E2F proteins. We show that the expression of NANOS, a key facilitator of the Pumilio (PUM) post-transcriptional repressor complex, is directly repressed by pRb/E2F in flies and humans. In both species, NANOS expression increases following inactivation of pRb/RBF1 and becomes important for tissue homeostasis. By analyzing datasets from normal retinal tissue and pRb-null retinoblastomas, we find a strong enrichment for putative PUM substrates among genes de-regulated in tumors. These include pro-apoptotic genes that are transcriptionally down-regulated upon pRb loss, and we characterize two such candidates, MAP2K3 and MAP3K1, as direct PUM substrates. Our data suggest that NANOS increases in importance in pRb-deficient cells and helps to maintain homeostasis by repressing the translation of transcripts containing PUM Regulatory Elements (PRE).

The role of interleukin-2, all-trans retinoic acid, and natural killer cells: surveillance mechanisms in anti-GD2 antibody therapy in neuroblastoma.

Although anti-disialoganglioside (GD2) antibodies are successfully used for neuroblastoma therapy, a third of patients with neuroblastoma experience treatment failure or serious toxicity. Various strategies have been employed in the clinic to improve antibody-dependent cell-mediated cytotoxicity (ADCC), such as the addition of interleukin (IL)-2 to enhance natural killer (NK) cell function, adoptive transfer of allogeneic NK cells to exploit immune surveillance, and retinoid-induced differentiation therapy. Nevertheless, these mechanisms are not fully understood. We developed a quantitative assay to test ADCC induced by the anti-GD2 antibody Hu14.18K322A in nine neuroblastoma cell lines and dissociated cells from orthotopic patient-derived xenografts (O-PDXs) in culture. IL-2 improved ADCC against neuroblastoma cells, and differentiation with all-trans retinoic acid stabilized GD2 expression on tumor cells and enhanced ADCC as well. Degranulation was highest in licensed NK cells that expressed CD158b (P < 0.001) and harbored a killer-cell immunoglobulin-like receptor (KIR) mismatch against the tumor-specific human leukocyte antigen (HLA; P = 0.016). In conclusion, IL-2 is an important component of immunotherapy because it can improve the cytolytic function of NK cells against neuroblastoma cells and could lower the antibody dose required for efficacy, thereby reducing toxicity. The effect of IL-2 may vary among individuals and a biomarker would be useful to predict ADCC following IL-2 activation. Sub-populations of NK cells may have different levels of activity dependent on their licensing status, KIR expression, and HLA-KIR interaction. Better understanding of HLA-KIR interactions and the molecular changes following retinoid-induced differentiation is necessary to delineate their role in ADCC.

Brg1 coordinates multiple processes during retinogenesis and is a tumor suppressor in retinoblastoma.

Retinal development requires precise temporal and spatial coordination of cell cycle exit, cell fate specification, cell migration and differentiation. When this process is disrupted, retinoblastoma, a developmental tumor of the retina, can form. Epigenetic modulators are central to precisely coordinating developmental events, and many epigenetic processes have been implicated in cancer. Studying epigenetic mechanisms in development is challenging because they often regulate multiple cellular processes; therefore, elucidating the primary molecular mechanisms involved can be difficult. Here we explore the role of Brg1 (Smarca4) in retinal development and retinoblastoma in mice using molecular and cellular approaches. Brg1 was found to regulate retinal size by controlling cell cycle length, cell cycle exit and cell survival during development. Brg1 was not required for cell fate specification but was required for photoreceptor differentiation and cell adhesion/polarity programs that contribute to proper retinal lamination during development. The combination of defective cell differentiation and lamination led to retinal degeneration in Brg1-deficient retinae. Despite the hypocellularity, premature cell cycle exit, increased cell death and extended cell cycle length, retinal progenitor cells persisted in Brg1-deficient retinae, making them more susceptible to retinoblastoma. ChIP-Seq analysis suggests that Brg1 might regulate gene expression through multiple mechanisms.

CONSERTING: integrating copy-number analysis with structural-variation detection.

We developed Copy Number Segmentation by Regression Tree in Next Generation Sequencing (CONSERTING), an algorithm for detecting somatic copy-number alteration (CNA) using whole-genome sequencing (WGS) data. CONSERTING performs iterative analysis of segmentation on the basis of changes in read depth and the detection of localized structural variations, with high accuracy and sensitivity. Analysis of 43 cancer genomes from both pediatric and adult patients revealed novel oncogenic CNAs, complex rearrangements and subclonal CNAs missed by alternative approaches.

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.

The Childhood Solid Tumor Network: A new resource for the developmental biology and oncology research communities.

Significant advances have been made over the past 25 years in our understanding of the most common adult solid tumors such as breast, colon, lung and prostate cancer. Much less is known about childhood solid tumors because they are rare and because they originate in developing organs during fetal development, childhood and adolescence. It can be very difficult to study the cellular origins of pediatric solid tumors in developing organs characterized by rapid proliferative expansion, growth factor signaling, developmental angiogenesis, programmed cell death, tissue reorganization and cell migration. Not only has the etiology of pediatric cancer remained elusive because of their developmental origins, but it also makes it more difficult to treat. Molecular targeted therapeutics that alter developmental pathway signaling may have devastating effects on normal organ development. Therefore, basic research focused on the mechanisms of development provides an essential foundation for pediatric solid tumor translational research. In this article, we describe new resources available for the developmental biology and oncology research communities. In a companion paper, we present the detailed characterization of an orthotopic xenograft of a pediatric solid tumor derived from sympathoadrenal lineage during development.

Development and characterization of a human orthotopic neuroblastoma xenograft.

Neuroblastoma is a pediatric cancer of the developing sympathoadrenal lineage. The tumors are known to develop from the adrenal gland or paraspinal ganglia and have molecular and cellular features of sympathetic neurons such as dense core vesicles and catecholamine production. Here we present the detailed molecular, cellular, genetic and epigenetic characterization of an orthotopic xenograft derived from a high-risk stage 4 neuroblastoma patient. Overall, the xenografted tumor retained the high risk features of the primary tumor and showed aggressive growth and metastasis in the mouse. Also, the genome was preserved with no additional copy number variations, structural variations or aneuploidy. There were 13 missense mutations identified in the xenograft that were not present in the patient's primary tumor and there were no new nonsense mutations. None of the missense mutations acquired in the xenograft were in known cancer genes. We also demonstrate the feasibility of using the orthotopic neuroblastoma xenograft to test standard of care chemotherapy and molecular targeted therapeutics. Finally, we optimized a new approach to produce primary cultures of the neuroblastoma xenografts for high-throughput drug screening which can be used to test new combinations of therapeutic agents for neuroblastoma.

Expression of podocalyxin separates the hematopoietic and vascular potentials of mouse embryonic stem cell-derived mesoderm.

In the mouse embryo and differentiating embryonic stem cells, the hematopoietic, endothelial, and cardiomyocyte lineages are derived from Flk1+ mesodermal progenitors. Here, we report that surface expression of Podocalyxin (Podxl), a member of the CD34 family of sialomucins, can be used to subdivide the Flk1+ cells in differentiating embryoid bodies at day 4.75 into populations that develop into distinct mesodermal lineages. Definitive hematopoietic potential was restricted to the Flk1+Podxl+ population, while the Flk1-negative Podxl+ population displayed only primitive erythroid potential. The Flk1+Podxl-negative population contained endothelial cells and cardiomyocyte potential. Podxl expression distinguishes Flk1+ mesoderm populations in mouse embryos at days 7.5, 8.5, and 9.5 and is a marker of progenitor stage primitive erythroblasts. These findings identify Podxl as a useful tool for separating distinct mesodermal lineages.

Pharmacokinetics and efficacy of the spleen tyrosine kinase inhibitor r406 after ocular delivery for retinoblastoma.

PURPOSE: Retinoblastoma is a childhood cancer of the retina. Clinical trials have shown that local delivery of broad spectrum chemotherapeutic agents is efficacious. Recent studies characterizing the genomic and epigenomic landscape of retinoblastoma identified spleen tyrosine kinase (SYK) as a promising candidate for targeted therapy. The purpose of this study was to conduct preclinical testing of the SYK antagonist R406 to evaluate it as a candidate for retinoblastoma treatment. METHODS: The efficacy of the SYK antagonist R406 delivered locally in a human orthotopic xenograft mouse model of retinoblastoma was tested. Intraocular exposure of R406 was determined for various routes and formulations. RESULTS: There was no evidence of efficacy for subconjunctival. R406. Maximal vitreal concentration was 10-fold lower than the minimal concentration required to kill retinoblastoma cells in vitro. Dosage of R406 subconjunctivally from emulsion or suspension formulations, direct intravitreal injection of the soluble prodrug of R406 (R788), and repeated topical administration of R406 all increased vitreal exposure, but failed to reach the exposure required for retinoblastoma cell death in culture. CONCLUSION: Taken together, these data suggest that R406 is not a viable clinical candidate for the treatment of retinoblastoma. This study highlights the importance of pharmacokinetic testing of molecular targeted retinoblastoma therapeutics.

The search for the retinoblastoma cell of origin.

The cellular effects of the genetic defects associated with tumorigenesis are context dependent. To better understand the reasons that different cell types require distinct combinations of mutations to form tumours, it is essential to identify and characterize a tumour's 'cell of origin'. Retinoblastoma, a rare childhood cancer of the retina that is caused by RB inactivation, is a good model in which to search for a tumour cell of origin, because retinal development is well understood and the initiating genetic lesion is well characterized. Identifying the cell of origin for this tumour would advance our understanding of how cellular context affects the requirement of specific mutations for cancer initiation and progression.