Vidya Mehta

Ligand-Dependent Regulation of Plasmid-Based Transgene Expression in Vivo

As gene therapy advances, the ability to regulate transgene expression will become paramount for safety and efficacy. In this study, we investigate the ability of the mifepristone-dependent GeneSwitch system to regulate the expression of trangenes delivered to mice by nonviral methods. Two plasmids, one encoding the chimeric GeneSwitch protein, the other an inducible transgene for secreted human placental alkaline phosphatase (SEAP), were delivered to the hind-limb muscles of adult mice. Modulation of the level of secretion of the transgene product into serum was achieved by intraperitoneal administration of low doses of the drug mifepristone (MFP). The EC50 for induction of transgene expression by MFP was 0.03 +/- 0.005 mg/kg. The maximal level of transgene expression after induction was equal to or higher than that displayed by a plasmid driven by the CMV enhancer/promoter. The average magnitude of induction was 14- to 19-fold. Multiple rounds of drug-dependent regulation of transgene expression in vivo were demonstrated. In BALB/c mice, the ability to regulate transgene expression persisted for approximately 3 weeks, until the appearance of neutralizing antibodies to the secreted transgene product. In immune-deficient mice, the ability to repetitively regulate transgene expression persisted for at least 5 weeks. Although the dynamic range of regulation needs improvement, the plasmid-based GeneSwitch system has features that are attractive for gene therapy applications.

Atypical teratoid/rhabdoid tumor of the pineal region in an adult: Case report

An atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant embryonal tumor most often occurring in the posterior fossa in children younger than 3 years of age. Adult cases of AT/RT are very rare, and 27 cases with a diagnosis of either AT/RT or (malignant) rhabdoid tumor have been reported to date. The authors report an adult case of an AT/RT occurring in the pineal region with molecular cytogenetic and immunohistochemical confirmation. A 33-year-old woman presented with a 2-month history of headache and blurred vision progressing to diplopia, and was admitted emergently due to deteriorating mental status. An MR image showed a heterogeneously enhancing mass involving the posterior third ventricle and pineal region with mild hydrocephalus. She underwent a subtotal resection of the tumor and was then treated with chemoradiation. Thirteen months after surgery, she was still alive with radiological evidence of recurrence/residual lesions. Histological sections showed epithelioid cellular sheets of rhabdoid tumor cells with scattered mitotic figures. Immunohistochemically, the tumor cells were diffusely and strongly positive for epithelial membrane antigen and vimentin, and showed focal expression of glial fibrillary acidic protein, pancytokeratin, and neurofilament protein. Loss of nuclear immunoreactivity for INI1 protein was observed. Fluorescence in situ hybridization analysis showed monosomy 22. Histologically, this tumor consisted exclusively of epithelioid tumor cells with rhabdoid features. The differential diagnoses include rhabdoid glioblastoma, metastatic carcinoma, and rhabdoid meningioma. Molecular testing to identify monosomy 22 or deletions of the chromosome 22q11 containing the INI1/hSNF5 gene and/or immunohistochemical staining with INI1 antibody is of great importance for the diagnosis of this tumor.

Low-level copy gain versus amplification of myc oncogenes in medulloblastoma: utility in predicting prognosis and survival : Laboratory investigation

OBJECT Medulloblastoma (MB) is a malignant embryonal tumor of the cerebellum. Amplification of c-myc or N-myc is infrequently identified and, when present, is often associated with the large cell/anaplastic (LC/A) phenotype. The frequency of low-level copy gain of myc oncogenes and its relationship to prognosis of MB has not been explored. METHODS Archival cases of MB were histologically reviewed and classified into 3 major subtypes: classic, nodular, and LC/A. Using quantitative real-time polymerase chain reaction (PCR), the authors analyzed 58 cases with a pure histological subtype for the copy number (CN) of myc (c-myc and N-myc) oncogenes. Cases with > 5-fold CN were further analyzed using the fluorescent in situ hybridization (FISH) assay. Kaplan-Meier survival analysis was performed. RESULTS A > 5-fold myc CN was noted in 5 (20.8%) of 24 LC/A, 1 (5.3%) of 19 classic, and 2 (13.3%) of 15 nodular subtypes. In a significant number of tumors (14 [56%] of 24 LC/A, 13 [68%] of 19 classic, and 10 [67%] of 15 nodular MBs) the CN was > 2-fold but < 5-fold. High-level amplification, defined as > 10-fold CN, was only seen in the LC/A subtype (5 cases), although moderate amplification (> 5-fold but < 10-fold) could be detected in other histological subtypes. Fluorescence in situ hybridization readily detected most cases corresponding to tumors with > 5-fold amplicon CN by quantitative real-time PCR, and could detect all 5 cases with > 10-fold CN by quantitative real-time PCR. The group of patients with > 5-fold myc amplicon CN showed significantly shorter survival than those with < 5-fold CN (p = 0.045), independent of histological subtype. CONCLUSIONS Since FISH could easily detect most cases in the moderate-to-high myc gene amplification (> 5-fold CN) group, the FISH assay has utility in detecting subsets of MB with poorer prognosis.

Adult pilocytic astrocytomas: Clinical features and molecular analysis

BACKGROUND Adult pilocytic astrocytomas (PAs) are rare and have an aggressive clinical course compared with pediatric patients. Constitutive Ras/RAF/MAPK signaling appears to be an important oncogenic event in sporadic PA. We evaluated clinical data and molecular profiles of adult PAs at our institution. METHODS We identified 127 adult PAs in our institutional database. Cases with available tissue were tested for BRAF-KIAA1549 fusion/duplication (B-K fusion) by fluorescence in situ hybridization and submitted for mutation profiling using the Sequenom mutation profiling panel. Subgroup analyses were performed based on clinical and molecular data. RESULTS The majority of adult PAs are supratentorial. Twenty-two percent of cases had an initial pathologic diagnosis discordant with the diagnosis made at our institution. Recurrence was seen in 42% of cases, and 13% of patients died during follow-up. Adjuvant radiotherapy following surgical resection was associated with a statistically significant decrease in progression-free survival (P = .004). B-K fusion was identified in 20% (9 of 45) of patients but was not associated with outcome. No BRAF V600E mutations (0 of 40 tested) were found. CONCLUSION This was the largest single institution series of adult PA. A significant proportion of adult PAs follow an aggressive clinical course. Our results support a period of observation following biopsy or surgical resection. B-K fusion in adult PA does not influence outcome, and BRAF V600E mutation appears to be a very rare event. Further study of tumor biology and optimal treatment is needed, given a more aggressive clinical behavior.

Biochemical differences in Qa-2 antigens expressed by Qa-2+,6+ and Qa-2+,6- strains. Evidence for differential expression of the Q7 and Q9 genes.

Cell surface forms of Qa-6 class I molecules are biochemically indistinguishable from Qa-2 although Qa-6 maps telomeric to Qa-2 with the recombinant strain B6.K2. Analysis of appropriate F1 strains did not demonstrate the presence of a trans acting factor that could modify the Qa-2 molecule to produce the Qa-6 determinant. Also, neither a neighboring cell surface molecule nor oligosaccharides were found to block the recognition of the Qa-6 determinant in Qa-2+,6- strains. The 2-D gel profiles of neuraminidase or endoglycosidase treated anti-Qa-2 immunoprecipitates from lysates of cell surface iodinated Qa-2+,6+ strains revealed an additional basic polypeptide which was absent from that of Qa-2+,6- strains. Thus, differential sialylation/glycosylation of Qa-2 molecules masks detection of Qa-2 antigen heterogeneity when cell surface forms are analyzed. Qa-6+ phenotype associated polypeptides were also found at various stages of post-translational processing in cells metabolically labeled in the presence and absence of tunicamycin. Northern analyses using Q7 and Q9 specific oligonucleotide probes revealed appropriate sized transcripts for both genes in the Qa-2+,6+ strain B6 but only Q9 in the Qa-2+,6- strain B6.K2. These data demonstrate that there is structural heterogeneity in Qa-2 antigens expressed by Qa-2+,6+ and Qa-2+,6- strains which results from differential expression of the Q7 and Q9 genes.

Regulation of expression of TL genes of the mouse Mhc

The expression of thymus leukemia (TL) antigens and genes in thymocytes and activated T cells was examined by immunoprecipitation, flow cytometric, northern, and nuclear run-off transcription analyses. Cell surface forms of TL were detectable by immunoprecipitation on activated peripheral T cells from Tla haplotypes except Tlab, in agreement with expression observed on thymocytes. Approximately 40%–50% of concanavalin A (Con A) or anti-CD3-activated T cells were TL+, with expression detected on both the CD4 and CD8 subsets by dual-color analysis. Activated T cells expressed detectable levels of TL mRNA 48 h after stimulation, but no TL transcripts were detectable in unstimulated splenocytes. However, TL mRNA expression in mature activated T cells did not precisely mimic thymocyte expression: the level of expression was considerably lower in activated T cells, and in most haplotypes the transcripts produced in activated T cells appeared to represent a subset of the transcripts produced in thymocytes. By run-off transcription assays in isolated nuclei, TL gene expression was detected in activated but not resting T cells indicating that lack of expression of TL in resting T cells is not due to message instability. These data demonstrate that TL genes are inducible and transcriptionally regulated.

Xenotransplantation of pediatric low grade gliomas confirms the enrichment of BRAF V600E mutation and preservation of CDKN2A deletion in a novel orthotopic xenograft mouse model of progressive pleomorphic xanthoastrocytoma

To identify cellular and molecular changes that driver pediatric low grade glioma (PLGG) progression, we analyzed putative cancer stem cells (CSCs) and evaluated key biological changes in a novel and progressive patient-derived orthotopic xenograft (PDOX) mouse model. Flow cytometric analysis of 22 PLGGs detected CD133+ (<1.5%) and CD15+ (20.7 ± 28.9%) cells, and direct intra-cranial implantation of 25 PLGGs led to the development of 1 PDOX model from a grade II pleomorphic xanthoastrocytoma (PXA). While CSC levels did not correlate with patient tumor progression, neurosphere formation and in vivo tumorigenicity, the PDOX model, IC-3635PXA, reproduced key histological features of the original tumor. Similar to the patient tumor that progressed and recurred, IC-3635PXA also progressed during serial in vivo subtransplantations (4 passages), exhibiting increased tumor take rate, elevated proliferation, loss of mature glial marker (GFAP), accumulation of GFAP−/Vimentin+ cells, enhanced local invasion, distant perivascular migration, and prominent reactive gliosis in normal mouse brains. Molecularly, xenograft cells with homozygous deletion of CDKN2A shifted from disomy chromosome 9 to trisomy chromosome 9; and BRAF V600E mutation allele frequency increased (from 28% in patient tumor to 67% in passage III xenografts). In vitro drug screening identified 2/7 BRAF V600E inhibitors and 2/9 BRAF inhibitors that suppressed cell proliferation. In summary, we showed that PLGG tumorigenicity was low despite the presence of putative CSCs, and our data supported GFAP−/Vimentin+ cells, CDKN2A homozygous deletion in trisomy chromosome 9 cells, and BRAF V600E mutation as candidate drivers of tumor progression in the PXA xenografts.

FOXG1 expression shows correlation with neuronal differentiation in cerebellar development, aggressive phenotype in medulloblastomas, and survival in a xenograft model of medulloblastoma

FOXG1 is a transcription factor that interacts with multiple signaling pathways and modulates neuronal differentiation in the telencephalon. Dysregulation of FOXG1 expression has been previously reported in medulloblastoma. In this study, we demonstrate a regional specific expression of FOXG1 and its colocalization with Nestin expression in the premigratory mitotically active (outer) layer of the external granular layer of the cerebellum. An inverse expression of the granular precursor cell markers, Math1 and Musashi1, in the inner nonmitotic migratory layer of the external granular layer and in the internal granular layer was observed. Furthermore, modulation of FOXG1 in the medulloblastoma cell line, DAOY, was associated with the induction of neuronal differentiation markers and significant changes in multiple signaling pathways regulating cell proliferation, differentiation, survival, and apoptosis. Additionally, we observed enhanced survival in intracerebellar mice xenografts injected with DAOY cells bearing shFOXG1 constructs versus shLuciferase construct. Overall, these findings suggest that down-modulation of FOXG1 is a prerequisite for the onset of neuronal differentiation during cerebellar development and that a decrease of FOXG1 in medulloblastoma cells offers a survival advantage in mice. We propose that the disruption of signaling pathways that promote mature neuronal differentiation by overexpressed FOXG1 is a contributing event in the neoplastic transformation of cerebellar stem cells.