James, C. David

Object Bioluminescence imaging (BLI) offers a rapid and accurate means for longitudinal study of tumor cell growth and response to therapy in rodent models. Because this technology has only recently come into use in the field of small animal imaging, applications in this area have been limited. In the current study we have applied BLI to the analysis of clinically relevant issues involving use of the DNA methylating agent temozolomide (TMZ) in a mouse model. Methods An invasive glioblastoma multiforme xenograft was modified for BLI via transduction with a luciferase-encoding lentivirus. Supratentorial tumors were established in athymic nude mice that were subsequently assigned randomly to control and TMZ treatment groups, and the extent of intracranial tumor was monitored using BLI. Results In an experiment designed to compare the extent of antitumor effect between a single high-dose TMZ treatment and a protracted low-dose TMZ regimen, BLI revealed the protracted regimen as having superior antitumor effect, and this interpretation was consistent with results from a survival comparison between the two TMZ treatment groups. In a second experiment designed to assess the utility of BLI for testing therapies against recurrent glioblastoma multiforme, mice with intracranial tumors were retreated with TMZ at a time when BLI monitoring revealed tumor regrowth following initial TMZ treatment, and retreatment was successful in providing additional survival benefit. Conclusion The results of these experiments indicate that BLI monitoring can be used as a surrogate for predicting survival benefit from TMZ treatment, permits early determination of relative survival benefit associated with distinct TMZ therapeutic regimens, and offers a means of investigating secondary/salvage therapy efficacy following tumor regrowth from initial therapy.

Abstract Purpose: Glioblastoma multiforme is the most frequent primary brain tumor in adults and represents one of the most lethal malignancies with a median survival of 12-16 months. We have previously shown that an oncolytic measles virus derivative expressing soluble human carcinoembryonic antigen (MV-CEA) has significant antitumor activity against glioblastoma multiforme cell lines and xenografts. Radiation therapy (RT) represents one of the mainstays of glioma treatment. Here we tested the hypothesis that the combination of RT with MV-CEA would have synergistic activity against gliomas. Experimental Design: 3-(4,5-Dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and clonogenic assays were used to test cytoxicity of the combination treatment in vivo. To examine the mechanism of synergy, one-step viral growth curves, terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assays, and Western blot analyses were performed. In vivo assessment of synergistic antitumor activity was conducted in a U87 glioma model. Results: MTS and clonogenic assays showed a strong synergistic interaction between MV-CEA and RT in glioblastoma multiforme cells including both primary and established glioma lines. Furthermore, significant antitumor efficacy was observed in vivo in a subcuteneous U87 xenograph model. There was significant prolongation of survival (P = 0.001) in the combination treatment group as compared with single modality– or control-treated animals. One-step viral growth curves showed increased viral burst size by up to 2 log in MV/RT combination–treated cells, as compared with single agent MV-CEA–treated glioma cells. Changes in CEA levels and expression of viral N and H protein were also consistent with increased viral production. Furthermore, TUNEL assays and Western blot analysis showed increase in apoptosis in MV/RT combination–treated cells. The pan-caspase inhibitor Z-VAD-FMK and the caspase-8 inhibitor Z-IETD-FMK, but not the caspase-9 inhibitor Z-IEHD-FMK, protected glioma cells from MV-CEA/RT–induced cleavage of poly(ADP-ribose) polymerase (PARP), indicating that the apoptotic death in combination-treated cells is mostly mediated via the extrinsic caspase pathway. The Fas/Fas ligand interaction blocking antibody NOK-1 blocked MV/RT–induced PARP cleavage whereas the Fas agonistic antibody CH11 increased PARP cleavage in MV/RT combination–treated cells. Reverse transcription-PCR, fluorescence-activated cell sorting analysis and immunohistochemistry showed up-regulation of Fas in combination-treated tumor in vitro and in vivo cells. Conclusions: There is synergy between MV-CEA and RT in vitro and in vivo. The synergistic effect of the combination seems to be due to increase in viral burst size and increase in apoptotic cell death. This latter effect is mostly mediated via the extrinsic caspase-8 pathway, activated via increased signaling through the Fas death receptor pathway. These results could have translational implications in glioma therapy.

Purpose The clinical significance of epidermal growth factor receptor variant III (EGFRvIII) expression in glioblastoma multiforme (GBM) and its relationship with other key molecular markers are not clear. We sought to evaluate the clinical significance of GBM subtypes as defined by EGFRvIII status. Patients and Methods The expression of EGFRvIII was assessed by immunohistochemistry in 649 patients with newly diagnosed GBM. These data were then examined in conjunction with the expression of phospho-intermediates (in a subset of these patients) of downstream AKT and Ras pathways and YKL-40 as well as with known clinical risk factors, including the Radiation Therapy Oncology Group's recursive partitioning analysis (RTOG-RPA) class. Results The RTOG-RPA class was highly predictive of survival in EGFRvIII-negative patients but much less predictive in EGFRvIII-positive patients. These findings were seen in both an initial test set (n = 268) and a larger validation set (n = 381). Similarly, activation of the AKT/MAPK pathways and YKL-40 positivity were predictive of poor outcome in EGFRvIII-negative patients but not in EGFRvIII-positive patients. Pair-wise combinations of markers identified EGFRvIII and YKL-40 as prognostically important. In particular, outcome in patients with EGFRvIII-negative/YKL-40–negative tumors was significantly better than the outcome in patients with the other three combinations of these two markers. Conclusion Established prognostic factors in GBM were not predictive of outcome in the EGFRvIII-positive subset, although this requires confirmation in independent data sets. GBMs negative for both EGFRvIII and YKL-40 show less aggressive behavior.

Abstract In the current study, we examined a panel of serially passaged glioblastoma xenografts, in the context of an intracranial tumor therapy response model, to identify associations between glioblastoma molecular characteristics and tumor sensitivity to the epidermal growth factor receptor (EGFR) kinase inhibitor erlotinib. From an initial evaluation of 11 distinct glioblastoma xenografts, two erlotinib-sensitive tumors were identified, each having amplified EGFR and expressing wild-type PTEN. One of these tumors expressed truncated EGFRvIII, whereas the other expressed full-length EGFR. Subsequent cDNA sequence analysis revealed the latter tumor as expressing an EGFR sequence variant with arginine, rather than leucine, at amino acid position 62; this was the only EGFR sequence variant identified among the 11 xenografts, other than the aforementioned vIII sequence variant. EGFR cDNAs were then examined from 12 more xenografts to determine whether additional missense sequence alterations were evident, and this analysis revealed one such case, expressing threonine, rather than alanine, at amino acid position 289 of the extracellular domain. This glioblastoma was also amplified for EGFR, but did not display significant erlotinib sensitivity, presumably due to its lacking PTEN expression. In total, our study identified two erlotinib-sensitive glioblastoma xenografts, with the common molecular characteristics shared by each being the expression of wild-type PTEN in combination with the expression of amplified and aberrant EGFR. [Mol Cancer Ther 2007;6(3):1167–74]

Abstract Among the best-characterized genetic alterations in gliomas is the amplification of the epidermal growth factor receptor (EGFR) gene, present in ∼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: HSNS (V451S, Y481N, and A527S)-CD46 blind, and HAA (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. (Cancer Res 2006; 66(24): 11840-50)

Abstract The phosphatidylinositol 3-kinases (PI3K) are a family of enzymes that relay important cellular growth control signals. Recently, a large-scale mutational analysis of eight PI3K and eight PI3K-like genes revealed somatic mutations in PIK3CA, which encodes the p110α catalytic subunit of class IA PI3K, in several types of cancer, including glioblastoma multiforme. In that report, 4 of 15 (27%) glioblastomas contained potentially oncogenic PIK3CA mutations. Subsequent studies, however, showed a significantly lower mutation rate ranging from 0% to 7%. Given this disparity and to address the relation of patient age to mutation frequency, we examined 10 exons of PIK3CA in 73 glioblastoma samples by PCR amplification followed by direct DNA sequencing. Overall, PIK3CA mutations were found in 11 (15%) samples, including several novel mutations. PIK3CA mutations were distributed in all sample types, with 18%, 9%, and 13% of primary tumors, xenografts, and cell lines containing mutations, respectively. Of the primary tumors, PIK3CA mutations were identified in 21% and 17% of pediatric and adult samples, respectively. No evidence of PIK3CA gene amplification was detected by quantitative real-time PCR in any of the samples. This study confirms that PIK3CA mutations occur in a significant number of human glioblastomas, further indicating that therapeutic targeting of this pathway in glioblastomas is of value. Moreover, this is the first study showing PIK3CA mutations in pediatric glioblastomas, thus providing a molecular target in this important pediatric malignancy. (Mol Cancer Res 2006;4(10):709–14)

Abstract The biological and clinical implications of p16 gene methylation in multiple myeloma (MM) are still unclear despite previous studies. In this comprehensive study, using methylation-specific PCR (MS-PCR), we show that p16 methylation is relatively common and occurs in monoclonal gammopathy of undetermined significance (MGUS; n = 17), smoldering multiple myeloma (SMM; n = 40), and MM (n = 522) at a prevalence of 24%, 28%, and 34%, respectively. However, p16 methylation does not appear to affect gene expression level. In a large cohort of patients with long-term follow-up information (n = 439), there was no difference in overall survival between patients with or without p16 methylation. We also found no association between p16 methylation and the main cytogenetic categories, although it was more common among patients with 17p13.1 deletions (p53 locus), a genetic progression event in MM. In addition, p16 methylation has no apparent effect on the cycle because there was also no difference in the plasma cell labeling index (a direct measurement of proliferation) between patients with and without p16 methylation. Our results question a major role for p16 methylation in the oncogenesis of the PC neoplasm, and we now believe p16 methylation may be a marker for overall epigenetic changes associated with disease progression, with no obvious direct biological or clinical consequences.

Abstract Purpose: The influence of epidermal growth factor receptor (EGFR) amplification on glioblastoma patient prognosis following definitive radiotherapy has been extensively investigated in clinical studies, and yet the relationship between EGFR status and radiation response remains unclear. The intent of the current study was to address this relationship using several EGFR-amplified glioblastoma xenografts in an orthotopic athymic mouse model. Experimental Design: We examined the effect of radiation on the survival of nude mice with intracranial xenografts derived from 13 distinct patient tumors, 7 of which have amplified EGFR. Mice with established intracranial tumors were randomized to sham treatment or 12-Gy radiation in six fractions delivered over 12 days. Results: For six of the xenografts, radiation of mice with intracranial tumor significantly extended survival, and four of these xenografts had EGFR amplification. For seven other xenografts, radiation treatment did not significantly extend survival, and three of these, including GBM12, had EGFR amplification. Similar to EGFR, the tumor genetic status of p53 or PTEN did not show preferential association with radiation-sensitive or radiation-resistant xenografts whereas hyperphosphorylation of Akt on Ser473 was associated with increased radioresistance. To specifically investigate whether inhibition of EGFR kinase activity influences radiation response, we examined combined radiation and EGFR inhibitor treatment in mice with intracranial GBM12. The combination of oral erlotinib administered concurrently with radiation resulted only in additive survival benefit relative to either agent alone. Conclusions: Our results indicate that EGFR amplification, as a biomarker, is not singularly predictive of glioblastoma response to radiation therapy, nor does the inhibition of EGFR enhance the intrinsic radiation responsiveness of glioblastoma tumors. However, efficacious EGFR inhibitor and radiation monotherapy regimens can be used in combination to achieve additive antitumor effect against a subset of glioblastoma.