Cory Allen

Analysis of PTCH/SMO/SHH pathway genes in medulloblastoma

Inactivation of the PTCH tumor suppressor gene occurs in a subset of sporadic medulloblastomas, suggesting that alterations in the PTCH pathway may be important in the development of this tumor. In order to address the frequency of genetic alterations affecting genes in this pathway, we used a combination of loss of heterozygosity (LOH) analysis, single‐stranded conformational polymorphism (SSCP) analysis, and direct sequencing of DNA samples from sporadic primitive neuroectodermal tumors (PNETs). To identify alterations in the PTCH gene, we performed LOH analysis on 37 tumor DNA samples. Of those with matched constitutional DNA samples, one demonstrated LOH. Of those without matched constitutional DNA, six were homozygous with all markers. All exons of the PTCH gene were sequenced in these seven tumors, and three mutations were found. To identify alterations in the SHH and SMO genes, we analyzed all exons of both genes in 24 tumors with SSCP and sequenced any exons that showed aberrant band patterns. No mutations were found in either SHH or SMO in any tumor. We also identified the following genes as candidate tumor suppressors based on their roles in controlling hh/ptc signaling in Drosophila: EN‐1 and EN‐2, deletion of which results in a lack of cerebellar development in mice; SMAD family members 1–7, and protein kinase A subunits RIα, RIβ, RIIβ, Cα, and Cβ. Each of these genes was investigated in a panel of 24 matched constitutional and tumor DNA samples. Our search revealed no mutations in any of these genes. Thus, PTCH is the only gene in this complex pathway that is mutated with notable frequency in PNET. Genes Chromosomes Cancer 27:44–51, 2000.

Evidence That Haploinsufficiency of Ptch Leads to Medulloblastoma in Mice

The PTCH gene encodes a putative tumor suppressor protein; germline alterations in PTCH have been found in patients with the nevoid basal cell carcinoma syndrome (NBCCS). Medulloblastoma, a brain tumor, develops in about 3% of NBCCS patients, and mutations in PTCH have also been described in a subset of sporadic medulloblastomas. The search for the causes of medulloblastoma has been hindered by the lack of an appropriate model system for this tumor type. Recently, a transgenic mouse hemizygous for the Ptch gene was generated by homologous recombination. Medulloblastomas were found in about 19% of these mice within the first 25 weeks after birth. The status of the wild‐type PTCH allele in these tumors has not been investigated. For clearer definition of the role of PTCH as a tumor suppressor in medulloblastoma, 13 cerebellar tumors from transgenic Ptch+/− mice were examined for alterations in the remaining Ptch allele. A single mutation was found in one tumor, a C‐to‐A substitution changing a tyrosine to a stop codon; all other tumors exhibited a wild‐type sequence. Two tumors with normal Ptch cDNA were examined by in situ hybridization. Ptch cDNA was found in tumor cells but not in associated tumor stroma. We also examined the mRNA expression levels for the remaining Ptch allele, as well as for Gli1, a gene known to be transcriptionally activated by Ptch inactivation. Blot analysis of RNA from the 13 tumors shows that Ptch mRNA of appropriate size is expressed in all tumors at varying levels. Expression of Gli1 was increased in tumors compared to normal cerebellum. These results suggest that deletion of one copy of Ptch may be sufficient to promote medulloblastoma development in mice. Genes Chromosomes Cancer 28:77–81, 2000.

Retargeted Oncolytic Measles Strains Entering via the EGFRvIII Receptor Maintain Significant Antitumor Activity against Gliomas with Increased Tumor Specificity

Among the best-characterized genetic alterations in gliomas is the amplification of the epidermal growth factor receptor (EGFR) gene, present in approximately 40% of glioblastoma multiforme, and frequently associated with the EGFRvIII gene rearrangement. We have previously shown that attenuated vaccine strains of measles virus have potent antitumor activity against gliomas, and identified H protein mutations, which ablate recognition of the natural measles virus receptors CD46 and SLAM. Retargeted recombinant viruses were generated from the measles Edmonston-NSe vaccine strain displaying a single-chain antibody against EGFRvIII at the COOH terminus of H and containing the marker green fluorescent protein (GFP) gene in position 1. Two different H mutants were employed: H(SNS) (V451S, Y481N, and A527S)-CD46 blind, and H(AA) (Y481A and R533A)-CD46 and SLAM blind. MV-GFP virus was used as a positive control. Both EGFRvIII-retargeted viruses had significant antitumor activity against EGFRvIII-expressing glioblastoma multiforme but no cytopathic effect against normal cells. In an orthotopic model of EGFRvIII-expressing GBM39 xenografts, there was comparable therapeutic efficacy between retargeted strains and unmodified MV-GFP and statistically significant prolongation of survival in treated animals compared with the control group (P = 0.001). Formation of syncytia was observed in tumors treated with retargeted viruses, with a surrounding infiltrate consisting of macrophages and natural killer cells. In summary, EGFRvIII-retargeted oncolytic measles virus strains have comparable therapeutic efficacy with the unmodified MV-GFP strain against EGFRvIII-expressing glioma lines and xenografts with improved therapeutic index, a finding with potential translational implications in glioma virotherapy.

Engineered Measles Virus as a Novel Oncolytic Therapy Against Prostate Cancer

No curative therapy is currently available for locally advanced or metastatic prostate cancer. Oncolytic viruses represent a novel class of therapeutic agents that demonstrates no cross‐resistance with existing approaches and can therefore be combined with conventional treatment modalities. Measles virus strains deriving from the Edmonston (MV‐Edm) vaccine strain have shown considerable oncolytic activity against a variety of solid tumers and hematologic malignancies. In this study, we investigated the antitumor potential of recombinant MV‐Edm derivatives as novel oncolytic agents against prostate cancer.

Noninvasive Imaging and Radiovirotherapy of Prostate Cancer Using an Oncolytic Measles Virus Expressing the Sodium Iodide Symporter

Prostate cancer cells overexpress the measles virus (MV) receptor CD46. Herein, we evaluated the antitumor activity of an oncolytic derivative of the MV Edmonston (MV-Edm) vaccine strain engineered to express the human sodium iodide symporter (NIS; MV-NIS virus). MV-NIS showed significant cytopathic effect (CPE) against prostate cancer cell lines in vitro. Infected cells effectively concentrated radioiodide isotopes as measured in vitro by Iodide-125 ((125)I) uptake assays. Virus localization and spread in vivo could be effectively followed by imaging of (123)I uptake. In vivo administration of MV-NIS either locally or systemically (total dose of 9 x 10(6) TCID(50)) resulted in significant tumor regression (P < 0.05) and prolongation of survival (P < 0.01). Administration of (131)I further enhanced the antitumor effect of MV-NIS virotherapy (P < 0.05). In conclusion, MV-NIS is an oncolytic vector with significant antitumor activity against prostate cancer, which can be further enhanced by (131)I administration. The NIS transgene allows viral localization and monitoring by noninvasive imaging which can facilitate dose optimization in a clinical setting.

Oncolytic measles virus strains in the treatment of gliomas.

Background: Recurrent gliomas have a dismal outcome despite use of multimodality treatment including surgery, radiation therapy and chemotherapy. Objective: In this article the authors discuss potential applications of oncolytic measles virus strains as novel antitumor agents in the treatment of gliomas. Methods: Important aspects of measles virus development as an anticancer therapeutic agent including engineering, retargeting and combination studies with other therapeutic modalities are discussed. The translational process that led to the first clinical trial of an engineered measles virus derivative in patients with recurrent glioblastoma multiforme is also described. Results/conclusions: Oncolytic measles virus strains hold promise as novel antitumor agents in the treatment of gliomas.

Interleukin-13 displaying retargeted oncolytic measles virus strains have significant activity against gliomas with improved specificity.

The majority of glioblastoma multiforme (GBM) tumors (80%) overexpress interleukin-13 receptor α2 (IL-13Rα2), but there is no expression of IL-13Rα2 in normal brain. Vaccine strains of measles virus have significant antitumor activity against gliomas. We tested the hypothesis that measles virus entry could be retargeted via the IL-13Rα2. MV-GFP-HAA-IL-13 was generated from the Edmonston-NSe vaccine strain, by displaying human IL-13 at the C-terminus of the H protein, and introducing CD46 and signaling lymphocyte activation molecule (SLAM)-ablating mutations in H. The IL-13 retargeted virus showed significant cytopathic effect (CPE) against IL-13Rα2 overexpressing glioma lines, and lack of CPE/viral replication in normal human astrocytes and normal human fibroblasts not expressing IL-13Rα2. In vivo treatment of orthotopically implanted GBM12 xenografts demonstrated significant prolongation of survival in mice treated with the retargeted strain (P < 0.0001), and comparable activity between the IL-13R retargeted strain and MV-GFP (P = 0.6377). In contrast to MV-GFP-treated mice, administration of the retargeted strain in the central nervous system of measles replication-permissive Ifnarko CD46 Ge mice resulted in lack of neurotoxicity. Strains of measles virus retargeted against the glioma-specific IL-13Rα2 receptor have comparable therapeutic efficacy, and improved specificity as compared with the unmodified measles virus strain MV-GFP in vitro and in vivo.The majority of glioblastoma multiforme (GBM) tumors (80%) overexpress interleukin-13 receptor alpha2 (IL-13Ralpha2), but there is no expression of IL-13Ralpha2 in normal brain. Vaccine strains of measles virus have significant antitumor activity against gliomas. We tested the hypothesis that measles virus entry could be retargeted via the IL-13Ralpha2. MV-GFP-H(AA)-IL-13 was generated from the Edmonston-NSe vaccine strain, by displaying human IL-13 at the C-terminus of the H protein, and introducing CD46 and signaling lymphocyte activation molecule (SLAM)-ablating mutations in H. The IL-13 retargeted virus showed significant cytopathic effect (CPE) against IL-13Ralpha2 overexpressing glioma lines, and lack of CPE/viral replication in normal human astrocytes and normal human fibroblasts not expressing IL-13Ralpha2. In vivo treatment of orthotopically implanted GBM12 xenografts demonstrated significant prolongation of survival in mice treated with the retargeted strain (P < 0.0001), and comparable activity between the IL-13R retargeted strain and MV-GFP (P = 0.6377). In contrast to MV-GFP-treated mice, administration of the retargeted strain in the central nervous system of measles replication-permissive Ifnar(ko) CD46 Ge mice resulted in lack of neurotoxicity. Strains of measles virus retargeted against the glioma-specific IL-13Ralpha2 receptor have comparable therapeutic efficacy, and improved specificity as compared with the unmodified measles virus strain MV-GFP in vitro and in vivo.

Oncolytic measles virus strains have significant antitumor activity against glioma stem cells

Glioblastoma (GBM) is the most common primary brain tumor in adults and has a dismal prognosis despite multimodality treatment. Given the resistance of glioma stem cells (GSC) to chemotherapy and radiation therapy, their eradication could prevent tumor recurrence. We sought to evaluate the antitumor activity of measles virus (MV) derivatives against GSC. We generated neurosphere cultures from patient-derived primary tumor GBM xenografts, and we characterized them for the GSC markers CD133, SOX2, Nestin, ATF5 and OLIG2. Using the MV-strains MV-GFP, MV-CEA and MV-NIS we demonstrated infection, viral replication and significant cytopathic effect in vitro against GSC lines. In tumorigenicity experiments, GBM44 GSC were infected with MV in vitro and subsequently implanted into the right caudate nucleus of nude mice: significant prolongation of survival in mice implanted with infected GSC was observed, compared with mock-infected controls (P=0.0483). In therapy experiments in GBM6 and GBM12 GSC xenograft models, there was significant prolongation of survival in MV-GFP-treated animals compared with inactivated virus-treated controls (GBM6 P=0.0021, GBM12 P=0.0416). Abundant syncytia and viral replication was demonstrated in tumors of MV-treated mice. Measles virus derivatives have significant antitumor activity against glioma-derived stem cells in vitro and in vivo.

Expression of immunomodulatory neutrophil-activating protein of Helicobacter pylori enhances the antitumor activity of oncolytic measles virus

Helicobacter pylori neutrophil-activating protein (NAP) is a major virulence factor and powerful inducer of inflammatory reaction and Th1-polarized immune response. Here, we evaluated the therapeutic efficacy of measles virus (MV) strains engineered to express secretory NAP forms against metastatic breast cancer. Recombinant viruses encoding secretory NAP forms (MV-lambda-NAP and MV-s-NAP) efficiently infect and destroy breast cancer cells by cell-to-cell viral spread and large syncytia formation independently of hormone receptor status. Intrapleural administration of MV-s-NAP doubled the median survival in a pleural effusion xenograft model: 65 days as compared to 29 days in the control group (P < 0.0001). This therapeutic effect correlated with a brisk Th1 type cytokine response in vivo. Secretory NAP was expressed at high levels by infected tumor cells and increased tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-12/23 cytokine concentrations were detected in the pleural effusion. In an aggressive model of lung metastatic breast cancer, MV-lambda-NAP and MV-s-NAP also significantly improved survival of the treated animals (P < 0.05) as compared to the control MV strain. These data suggest that potent immunomodulators of bacterial origin, such as H. pylori NAP, can enhance the antitumor effect of oncolytic viruses and support the feasibility and potential of a combined viroimmunotherapy approach.

Effective Radiovirotherapy for Malignant Gliomas by Using Oncolytic Measles Virus Strains Encoding the Sodium Iodide Symporter (MV-NIS)

Engineered measles virus (MV) strains deriving from the vaccine lineage represent a promising oncolytic platform and are currently being tested in phase I trials. In this study, we have demonstrated that MV strains genetically engineered to express the human sodium iodide symporter (NIS) have significant antitumor activity against glioma lines and orthotopic xenografts; this compares favorably with the MV strain expressing the human carcinoembryonic antigen, which is currently in clinical testing. Expression of NIS protein in infected cells results in effective concentration of radioactive iodine, which allows for in vivo monitoring of localization of MV-NIS infection by measuring uptake of (123)I or (99m)Tc. In addition, radiovirotherapy with MV-NIS followed by (131)I administration resulted in significant increase of MV-NIS antitumor activity as compared with virus alone in both subcutaneous (p=0.0003) and orthotopic (p=0.004) glioblastoma models. In conclusion, MV-NIS-based radiovirotherapy has significant antitumor activity against glioblastoma multiforme and represents a promising candidate for clinical translation.