Jack Su

Direct Orthotopic Transplantation of Fresh Surgical Specimen Preserves CD133+ Tumor Cells in Clinically Relevant Mouse Models of Medulloblastoma and Glioma

Recent identification of cancer stem cells in medulloblastoma (MB) and high‐grade glioma has stimulated an urgent need for animal models that will not only replicate the biology of these tumors, but also preserve their cancer stem cell pool. We hypothesize that direct injection of fresh surgical specimen of MB and high‐grade glioma tissues into anatomically equivalent locations in immune‐deficient mouse brains will facilitate the formation of clinically accurate xenograft tumors by allowing brain tumor stem cells, together with their non‐stem tumor and stromal cells, to grow in a microenvironment that is the closest to human brains. Eight of the 14 MBs (57.1%) and two of the three high‐grade gliomas (66.7%) in this study developed transplantable (up to 12 passages) xenografts in mouse cerebellum and cerebrum, respectively. These xenografts are patient specific, replicating the histopathologic, immunophenotypic, invasive/metastatic, and major genetic (analyzed with 10K single nucleotide polymorphism array) abnormalities of the original tumors. The xenograft tumor cells have also been successfully cryopreserved for long‐term preservation of tumorigenicity, ensuring a sustained supply of the animal models. More importantly, the CD133+ tumor cells, ranging from 0.2%–10.4%, were preserved in all the xenograft models following repeated orthotopic subtransplantations in vivo. The isolated CD133+ tumor cells formed neurospheres and displayed multi‐lineage differentiation capabilities in vitro. In summary, our study demonstrates that direct orthotopic transplantation of fresh primary tumor cells is a powerful approach in developing novel clinical relevant animal models that can reliably preserve CD133+ tumor cell pools even during serial in vivo subtransplantations.

Genome-Wide Allelic Imbalance Analysis of Pediatric Gliomas by Single Nucleotide Polymorphic Allele Array

Using single nucleotide polymorphic (SNP) allele arrays, we analyzed 28 pediatric gliomas consisting of 14 high-grade gliomas and 14 low-grade gliomas. Most of the low-grade gliomas had no detectable loss of heterozygosity (LOH) in any of the 11,562 SNP loci; exceptions were two gangliogliomas (3q and 9p), one astrocytoma (6q), and two subependymal giant cell astrocytomas (16p and 21q). On the other hand, all high-grade gliomas had various degrees of LOH affecting 52 to 2,168 SNP loci on various chromosomes. LOH occurred most frequently in regions located at 4q (54%), 6q (46%), 9p (38%), 10q (38%), 11p (38%), 12 (38%), 13q (69%), 14q (54%), 17 (38%), 18p (46%), and 19q (38%). We also detected amplifications of epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor alpha (PDGFRalpha) in a few of the 13 cases of glioblastoma multiforme analyzed. Interestingly, the amplified EGFR and PDGFRalpha were located within regions of LOH. SNP loci with LOH and copy number changes were validated by sequencing and quantitative PCR, respectively. Our results indicate that, in some pediatric glioblastoma multiforme, one allele each of EGFR and PDGFRalpha was lost but the remaining allele was amplified. This may represent a new molecular mechanism underlying tumor progression.

HER2-Specific Chimeric Antigen Receptor–Modified Virus-Specific T Cells for Progressive Glioblastoma: A Phase 1 Dose-Escalation Trial

Importance Glioblastoma is an incurable tumor, and the therapeutic options for patients are limited. Objective To determine whether the systemic administration of HER2-specific chimeric antigen receptor (CAR)–modified virus-specific T cells (VSTs) is safe and whether these cells have antiglioblastoma activity. Design, Setting, and Participants In this open-label phase 1 dose-escalation study conducted at Baylor College of Medicine, Houston Methodist Hospital, and Texas Children’s Hospital, patients with progressive HER2-positive glioblastoma were enrolled between July 25, 2011, and April 21, 2014. The duration of follow-up was 10 weeks to 29 months (median, 8 months). Interventions Monotherapy with autologous VSTs specific for cytomegalovirus, Epstein-Barr virus, or adenovirus and genetically modified to express HER2-CARs with a CD28.&zgr;-signaling endodomain (HER2-CAR VSTs). Main Outcomes and Measures Primary end points were feasibility and safety. The key secondary end points were T-cell persistence and their antiglioblastoma activity. Results A total of 17 patients (8 females and 9 males; 10 patients ≥18 years [median age, 60 years; range, 30-69 years] and 7 patients <18 years [median age, 14 years; range, 10-17 years]) with progressive HER2-positive glioblastoma received 1 or more infusions of autologous HER2-CAR VSTs (1 × 106/m2 to 1 × 108/m2) without prior lymphodepletion. Infusions were well tolerated, with no dose-limiting toxic effects. HER2-CAR VSTs were detected in the peripheral blood for up to 12 months after the infusion by quantitative real-time polymerase chain reaction. Of 16 evaluable patients (9 adults and 7 children), 1 had a partial response for more than 9 months, 7 had stable disease for 8 weeks to 29 months, and 8 progressed after T-cell infusion. Three patients with stable disease are alive without any evidence of progression during 24 to 29 months of follow-up. For the entire study cohort, median overall survival was 11.1 months (95% CI, 4.1-27.2 months) from the first T-cell infusion and 24.5 months (95% CI, 17.2-34.6 months) from diagnosis. Conclusions and Relevance Infusion of autologous HER2-CAR VSTs is safe and can be associated with clinical benefit for patients with progressive glioblastoma. Further evaluation of HER2-CAR VSTs in a phase 2b study is warranted as a single agent or in combination with other immunomodulatory approaches for glioblastoma.

Phase 1 Study of Valproic Acid in Pediatric Patients with Refractory Solid or CNS Tumors: A Children's Oncology Group Report

Purpose: The primary purpose of this trial was to define and describe the toxicities of oral valproic acid (VPA) at doses required to maintain trough concentrations of 100 to 150 mcg/mL or 150 to 200 mcg/mL in children with refractory solid or central nervous system (CNS) tumors. Secondary objectives included assessment of free and total VPA pharmacokinetics (PKs) and histone acetylation in peripheral blood mononuclear cells (PBMC) at steady state. Patients and Methods: Oral VPA, initially administered twice daily and subsequently three times daily, was continued without interruption to maintain trough concentrations of 100 to 150 mcg/mL. First-dose and steady-state PKs were studied. Histone H3 and H4 acetylation in PBMCs was evaluated using an ELISA technique. Results: Twenty-six children, sixteen of whom were evaluable for toxicity, were enrolled. Dose-limiting somnolence and intratumoral hemorrhage were associated with VPA troughs of 100 to 150 mcg/mL. Therefore, the final cohort of six children received VPA to maintain troughs of 75 to 100 mcg/mL and did not experience any dose-limiting toxicity. First-dose and steady-state VPA PK parameters were similar to values previously reported in children with seizures. Increased PBMC histone acetylation was documented in 50% of patients studied. One confirmed partial response (glioblastoma multiforme) and one minor response (brainstem glioma) were observed. Conclusions: VPA administered three times daily to maintain trough concentrations of 75 to 100 mcg/mL was well tolerated in children with refractory solid or CNS tumors. Histone hyperacetylation in PBMCs was observed in half of the patients at steady state. Future trials combining VPA with chemotherapy and/or radiation therapy should be considered, especially for CNS tumors. Clin Cancer Res; 17(3); 589–97. ©2010 AACR.

Ototoxicity After Intensity-Modulated Radiation Therapy and Cisplatin-Based Chemotherapy in Children With Medulloblastoma

PURPOSE To report the incidence of Pediatric Oncology Group (POG) Grade 3 or 4 ototoxicity in a cohort of patients treated with craniospinal irradiation (CSI) followed by posterior fossa (PF) and/or tumor bed (TB) boost using intensity-modulated radiation therapy (IMRT). METHODS AND MATERIALS From 1998 to 2006, 44 patients with medulloblastoma were treated with CSI followed by IMRT to the PF and/or TB and cisplatin-based chemotherapy. Patients with standard-risk disease were treated with 18 to 23.4 Gy CSI followed by either a (1) PF boost to 36 Gy and TB boost to 54 to 55.8 Gy or (2) TB boost to 55.8 Gy. Patients with high-risk disease received 36 to 39.6 Gy CSI followed by a (1) PF boost to 54 to 55.8 Gy, (2) PF boost to 45 Gy and TB boost to 55.8 Gy, or (3) TB boost to 55.8 Gy. Median audiogram follow-up was 41 months (range, 11-92.4 months). RESULTS POG Grade Ototoxicity 0, 1, 2, 3. and 4 was found in 29, 32, 11, 13. and 3 ears. respectively, with POG Grade 3 or 4 accounting for 18.2% of cases. There was a statistically significant difference in mean radiation dose (D(mean)) cochlea according to degree of ototoxicity, with D(mean) cochlea increasing with severity of hearing loss (p = 0.027). CONCLUSIONS Severe ototoxicity was seen in 18.2% of ears in children treated with IMRT boost and cisplatin-based chemotherapy. Increasing dose to the cochlea was associated with increasing severity of hearing loss.

Valproic Acid Prolongs Survival Time of Severe Combined Immunodeficient Mice Bearing Intracerebellar Orthotopic Medulloblastoma Xenografts

Purpose: To develop novel orthotopic xenograft models of medulloblastoma in severe combined immunodeficient mice and to evaluate the in vivo antitumor efficacy of valproic acid. Experimental Design: Orthotopic xenografts were developed by injecting 103 to 105 tumor cells from four medulloblastoma cell lines (D283-MED, DAOY, MHH-MED-1, and MEB-MED-8A) into the right cerebellum of severe combined immunodeficient mice. Animals were then examined for reproducibility of tumorigenicity, cell number-survival time relationship, and histopathologic features. Tumor growth was monitored in vivo by serially sectioning the xenograft brains at 2, 4, 6, and 8 weeks postinjection. Valproic acid treatment, administered at 600 μg/h for 2 weeks via s.c. osmotic minipumps, was initiated 2 weeks after injection of 105 medulloblastoma cells, and treated and untreated animals were monitored for differences in survival. Changes in histone acetylation, proliferation, apoptosis, differentiation, and angiogenesis in xenografts were also evaluated. Results: Tumorigenicity was maintained at 100% in D283-MED, DAOY, and MHH-MED-1 cell lines. These cerebellar xenografts displayed histologic features and immunohistochemical profiles (microtubule-associated protein 2, glial fibrillary acidic protein, and vimentin) similar to human medulloblastomas. Animal survival time was inversely correlated with injected tumor cell number. Treatment with valproic acid prolonged survival time in two (D283-MED and MHH-MED-1) of the three models and was associated with induction of histone hyperacetylation, inhibition of proliferation and angiogenesis, and enhancement of apoptosis and differentiation. Conclusion: We have developed intracerebellar orthotopic models that closely recapitulated the biological features of human medulloblastomas and characterized their in vivo growth characteristics. Valproic acid treatment of these xenografts showed potent in vivo anti-medulloblastoma activity. These xenograft models should facilitate the understanding of medulloblastoma pathogenesis and future preclinical evaluation of new therapies against medulloblastoma.

A clinically relevant orthotopic xenograft model of ependymoma that maintains the genomic signature of the primary tumor and preserves cancer stem cells in vivo

Limited availability of in vitro and in vivo model systems has hampered efforts to understand tumor biology and test novel therapies for ependymoma, the third most common malignant brain tumor that occurs in children. To develop clinically relevant animal models of ependymoma, we directly injected a fresh surgical specimen from a 9-year-old patient into the right cerebrum of RAG2/severe complex immune deficiency (SCID) mice. All five mice receiving the initial transplantation of the patient tumor developed intracerebral xenografts, which have since been serially subtransplanted in vivo in mouse brains for 4 generations and can be cryopreserved for long-term maintenance of tumorigenicity. The xenograft tumors shared nearly identical histopathological features with the original tumors, harbored 8 structural chromosomal abnormalities as detected with spectral karyotyping, maintained gene expression profiles resembling that of the original patient tumor with the preservation of multiple key genetic abnormalities commonly found in human ependymomas, and contained a small population (<2.2%) of CD133(+) stem cells that can form neurospheres and display multipotent capabilities in vitro. The permanent cell line (BXD-1425EPN), which was derived from a passage II xenograft tumor and has been passaged in vitro more than 70 times, expressed similar differentiation markers of the xenograft tumors, maintained identical chromosomal abnormalities, and formed tumors in the brains of SCID mice. In conclusion, direct injection of primary ependymoma tumor cells played an important role in the generation of a clinically relevant mouse model IC-1425EPN and a novel cell line, BXD-1425EPN. This cell line and model will facilitate the biological studies and preclinical drug screenings for pediatric ependymomas.

Imaging Changes in Pediatric Intracranial Ependymoma Patients Treated With Proton Beam Radiation Therapy Compared to Intensity Modulated Radiation Therapy

PURPOSE The clinical significance of magnetic resonance imaging (MRI) changes after radiation therapy (RT) in children with ependymoma is not well defined. We compared imaging changes following proton beam radiation therapy (PBRT) to those after photon-based intensity modulated RT (IMRT). METHODS AND MATERIALS Seventy-two patients with nonmetastatic intracranial ependymoma who received postoperative RT (37 PBRT, 35 IMRT) were analyzed retrospectively. MRI images were reviewed by 2 neuroradiologists. RESULTS Sixteen PBRT patients (43%) developed postradiation MRI changes at 3.8 months (median) with resolution by 6.1 months. Six IMRT patients (17%) developed changes at 5.3 months (median) with 8.3 months to resolution. Mean age at radiation was 4.4 and 6.9 years for PBRT and IMRT, respectively (P = .06). Age at diagnosis (>3 years) and time of radiation (≥3 years) was associated with fewer imaging changes on univariate analysis (odds ratio [OR]: 0.35, P = .048; OR: 0.36, P = .05). PBRT (compared to IMRT) was associated with more frequent imaging changes, both on univariate (OR: 3.68, P = .019) and multivariate (OR: 3.89, P = .024) analyses. Seven (3 IMRT, 4 PBRT) of 22 patients with changes had symptoms requiring intervention. Most patients were treated with steroids; some PBRT patients also received bevacizumab and hyperbaric oxygen therapy. None of the IMRT patients had lasting deficits, but 2 patients died from recurrent disease. Three PBRT patients had persistent neurological deficits, and 1 child died secondarily to complications from radiation necrosis. CONCLUSIONS Postradiation MRI changes are more common with PBRT and in patients less than 3 years of age at diagnosis and treatment. It is difficult to predict causes for development of imaging changes that progress to clinical significance. These changes are usually self-limiting, but some require medical intervention, especially those involving the brainstem.

A single intravenous injection of oncolytic picornavirus SVV-001 eliminates medulloblastomas in primary tumor-based orthotopic xenograft mouse models

Difficulties of drug delivery across the blood-brain barrier (BBB) and failure to eliminate cancer stem cells (CSCs) are believed to be the major causes of tumor recurrences in children with medulloblastoma (MB). Seneca Valley virus-001 (SVV-001) is a naturally occurring oncolytic picornavirus that can be systemically administered. Here, we report its antitumor activities against MB cells in a panel of 10 primary tumor-based orthotopic xenograft mouse models. We found that SVV-001 killed the primary cultured xenograft cells, infected and replicated in tumor cells expressing CSC surface marker CD133, and eliminated tumor cells capable of forming neurospheres in vitro in 5 of the 10 xenograft models. We confirmed that SVV-001 could pass through BBB in vivo. A single i.v. injection of SVV-001 in 2 anaplastic MB models led to widespread infection of the preformed intracerebellar (ICb) xenografts, resulting in significant increase in survival (2.2-5.9-fold) in both models and complete elimination of ICb xenografts in 8 of the 10 long-term survivors. Mechanistically, we showed that the intracellular replication of SVV-001 is mediated through a subverted autophagy that is different from the bona fide autophagic process induced by rapamycin. Our data suggest that SVV-001 is well suited for MB treatment. This work expands the current views in the oncolytic therapy field regarding the utility of oncolytic viruses in simultaneous targeting of stem and nonstem tumor cells.

A phase I trial of veliparib (ABT-888) and temozolomide in children with recurrent CNS tumors: a pediatric brain tumor consortium report.

BACKGROUND A phase I trial of veliparib (ABT-888), an oral poly(ADP-ribose) polymerase (PARP) inhibitor, and temozolomide (TMZ) was conducted in children with recurrent brain tumors to (i) estimate the maximum tolerated doses (MTDs) or recommended phase II doses (RP2Ds) of veliparib and TMZ; (ii) describe the toxicities of this regimen; and (iii) evaluate the plasma pharmacokinetic parameters and extent of PARP inhibition in peripheral blood mononuclear cells (PBMCs) following veliparib. METHODS TMZ was given once daily and veliparib twice daily for 5 days every 28 days. Veliparib concentrations and poly(ADP-ribose) (PAR) levels in PBMCs were measured on days 1 and 4. Analysis of pharmacokinetic and PBMC PAR levels were performed twice during study conduct to rationally guide dose modifications and to determine biologically optimal MTD/RP2D. RESULTS Twenty-nine evaluable patients were enrolled. Myelosuppression (grade 4 neutropenia and thrombocytopenia) were dose limiting. The RP2Ds are veliparib 25 mg/m(2) b.i.d. and TMZ 135 mg/m(2)/d. Only 2 out of 12 patients treated at RP2Ds experienced dose-limiting toxicities. Although no objective response was observed, 4 patients had stable disease >6 months in duration, including 1 with glioblastoma multiforme and 1 with ependymoma. At the RP2D of veliparib, pediatric pharmacokinetic parameters were similar to those in adults. CONCLUSIONS Veliparib and TMZ at the RP2D were well tolerated in children with recurrent brain tumors. A phase I/II trial to evaluate the tolerability and efficacy of veliparib, TMZ, and radiation in children with newly diagnosed brainstem gliomas is in progress.