Molecular analysis distinguishes metastatic disease from second cancers in patients with retinoblastoma

The pediatric ocular tumor retinoblastoma readily metastasizes, but these lesions can masquerade as histologically similar pediatric small round blue cell tumors. Since 98% of retinoblastomas have RB1 mutations and a characteristic genomic copy number “signature”, genetic analysis is an appealing adjunct to histopathology to distinguish retinoblastoma metastasis from second primary cancer in retinoblastoma patients. Here, we describe such an approach in two retinoblastoma cases. In patient one, allele-specific (AS)-PCR for a somatic nonsense mutation confirmed that a temple mass was metastatic retinoblastoma. In a second patient, a rib mass shared somatic copy number gains and losses with the primary tumor. For definitive diagnosis, however, an RB1 mutation was needed, but heterozygous promoter→exon 11 deletion was the only RB1 mutation detected in the primary tumor. We used a novel application of inverse PCR to identify the deletion breakpoint. Subsequently, AS-PCR designed for the breakpoint confirmed that the rib mass was metastatic retinoblastoma. These cases demonstrate that personalized molecular testing can confirm retinoblastoma metastases and rule out a second primary cancer, thereby helping to direct the clinical management

CHD2 haploinsufficiency is associated with developmental delay, intellectual disability, epilepsy and neurobehavioural problems

BACKGROUND: The chromodomain helicase DNA binding domain (CHD) proteins modulate gene expression via their ability to remodel chromatin structure and influence histone acetylation. Recent studies have shown that CHD2 protein plays a critical role in embryonic development, tumor suppression and survival. Like other genes encoding members of the CHD family, pathogenic mutations in the CHD2 gene are expected to be implicated in human disease. In fact, there is emerging evidence suggesting that CHD2 might contribute to a broad spectrum of neurodevelopmental disorders. Despite growing evidence, a description of the full phenotypic spectrum of this condition is lacking. METHODS: We conducted a multicentre study to identify and characterise the clinical features associated with haploinsufficiency of CHD2. Patients with deletions of this gene were identified from among broadly ascertained clinical cohorts undergoing genomic microarray analysis for developmental delay, congenital anomalies and/or autism spectrum disorder. RESULTS: Detailed clinical assessments by clinical geneticists showed recurrent clinical symptoms, including developmental delay, intellectual disability, epilepsy, behavioural problems and autism-like features without characteristic facial gestalt or brain malformations observed on magnetic resonance imaging scans. Parental analysis showed that the deletions affecting CHD2 were de novo in all four patients, and analysis of high-resolution microarray data derived from 26,826 unaffected controls showed no deletions of this gene. CONCLUSIONS: The results of this study, in addition to our review of the literature, support a causative role of CHD2 haploinsufficiency in developmental delay, intellectual disability, epilepsy and behavioural problems, with phenotypic variability between individuals

Functional dissection of the Drosophila melanogaster bithoraxoid Polycomb response element

During the development of Drosophila melanogaster, the Polycomb Group (PcG) proteins act through complex, modular elements termed Polycomb response elements (PREs) to maintain the silent state of the homeotic and other loci. The bithoraxoid (bxd) 5.1 PRE of the homeotic gene Ultrabithorax (Ubx) is capable of maintaining the correct Ubx expression pattern throughout embryogenesis in a PcG-dependent manner. The bxd5.l PRE is also capable of conferring pairing-sensitive repression to the mini-white gene located in the same transposon. In order to understand how PcG proteins are recruited to PREs, a gel mobility shift assay was used to identify four fragments within the bxd5.l PRE that bind protein complexes from nuclear extracts that contain the PcG protein Polyhomeotic (PH). Chapter 2 of this thesis examines the in vivo contribution of these four PH binding sites in embryonic silencing and pairing-sensitive repression. I show, using a germline transformation assay, that deletion of each PH binding site, in the context of bxd5.\, disrupts embryonic PRE activity but not pairing-sensitive repression. Double mutant analysis of sites with related binding activities indicate that sites MHS-70 and MPA-168 constitute one functional unit of PRE activity, which is disabled by either mutation. By contrast, sites MHN-90 and S1HB-90 act synergistically to promote PRE activity. Furthermore, mutation of two d(GA)3 repeat elements within MHS-70 destabilizes PH complex formation in vitro and partially abrogates PRE activity in vivo indicating that these repeat elements are essential for PRE-mediated silencing. Chapter 3 of this thesis explores the modular structure of the core maintenance element within bxd5.l, the bxd\.5 PRE. The results indicate that the bxdl.5 PRE is a complex element built up of at least three modules, UPS, PSR and DPS, that make distinct contributions to silencing by the bxd PRE. The UPS and DPS modules directly repress the Ubx promoter in a parasegment-specific and developmental stage-specific manner. The PSR and DPS modules are capable of pairing-sensitive repression. Genetic analyses reveal that each module depends on the function of a subset of PcG and trxG genes that are required specifically for embryonic or pairing-sensitive repression or for both processes. The results clearly demonstrate that embryonic and pairing-sensitive repression are separable functions of the bxdl .5 PRE. Taken together, these studies provide insight into how the bxd PRE is built and into the nature of the functional components that read and interpret the information encoded by this complex cis-regulatory element.Science, Faculty ofZoology, Department ofGraduat

Overcoming bioprocess bottlenecks in the large-scale expansion of high-quality hiPSC aggregates in vertical-wheel stirred suspension bioreactors

Abstract Background Human induced pluripotent stem cells (hiPSCs) hold enormous promise in accelerating breakthroughs in understanding human development, drug screening, disease modeling, and cell and gene therapies. Their potential, however, has been bottlenecked in a mostly laboratory setting due to bioprocess challenges in the scale-up of large quantities of high-quality cells for clinical and manufacturing purposes. While several studies have investigated the production of hiPSCs in bioreactors, the use of conventional horizontal-impeller, paddle, and rocking-wave mixing mechanisms have demonstrated unfavorable hydrodynamic environments for hiPSC growth and quality maintenance. This study focused on using computational fluid dynamics (CFD) modeling to aid in characterizing and optimizing the use of vertical-wheel bioreactors for hiPSC production. Methods The vertical-wheel bioreactor was modeled with CFD simulation software Fluent at agitation rates between 20 and 100 rpm. These models produced fluid flow patterns that mapped out a hydrodynamic environment to guide in the development of hiPSC inoculation and in-vessel aggregate dissociation protocols. The effect of single-cell inoculation on aggregate formation and growth was tested at select CFD-modeled agitation rates and feeding regimes in the vertical-wheel bioreactor. An in-vessel dissociation protocol was developed through the testing of various proteolytic enzymes and agitation exposure times. Results CFD modeling demonstrated the unique flow pattern and homogeneous distribution of hydrodynamic forces produced in the vertical-wheel bioreactor, making it the opportune environment for systematic bioprocess optimization of hiPSC expansion. We developed a scalable, single-cell inoculation protocol for the culture of hiPSCs as aggregates in vertical-wheel bioreactors, achieving over 30-fold expansion in 6 days without sacrificing cell quality. We have also provided the first published protocol for in-vessel hiPSC aggregate dissociation, permitting the entire bioreactor volume to be harvested into single cells for serial passaging into larger scale reactors. Importantly, the cells harvested and re-inoculated into scaled-up vertical-wheel bioreactors not only maintained consistent growth kinetics, they maintained a normal karyotype and pluripotent characterization and function. Conclusions Taken together, these protocols provide a feasible solution for the culture of high-quality hiPSCs at a clinical and manufacturing scale by overcoming some of the major documented bioprocess bottlenecks

Modeling the functional heterogeneity of leukemia stem cells: role of STAT5 in leukemia stem cell self-renewal

Although the cancer stem cell (CSC) concept implies that CSCs are rare, recent reports suggest that CSCs may be frequent in some cancers. We hypothesized that the proportion of leukemia stem cells would vary as a function of the number of dysregulated pathways. Constitutive expression of MN1 served as a 1-oncogene model, and coexpression of MN1 and a HOX gene served as a 2-oncogene model. Leukemia-initiating cell (LIC) number and in vitro expansion potential of LICs were functionally assessed by limiting dilution analyses. LIC expansion potential was 132-fold increased in the 2-compared with the 1-oncogene model, although phenotypically, both leukemias were similar. The 2-oncogene model was characterized by granulocyte-macrophage colony-stimulating factor (GM-CSF) hypersensitivity and activated STAT/ERK signaling. GM-CSF hypersensitivity of the 2-oncogene model (MN1/HOXA9) was lost in Stat5b(-/-) cells, and the LIC expansion potential was reduced by 86- and 28-fold in Stat5b(-/-) and Stat1(-/-) cells, respectively. Interestingly, in 201 acute myeloid leukemia (AML) patients, coexpression of MN1 and HOXA9 was restricted to patients with the poorest prognosis and was associated with highly active STAT signaling. Our data demonstrate the functional heterogeneity of LICs and show that STAT signaling is critical for leukemia stem cell self-renewal in MN1- and HOXA9-expressing leukemias. (Blood. 2009; 114: 3983-3993