Tomotsugu Ichikawa

11C-methionine (MET) and 18F-fluorothymidine (FLT) PET in patients with newly diagnosed glioma

PurposeThe purpose of this prospective study was to clarify the individual and combined role of l-methyl-11C-methionine-positron emission tomography (MET-PET) and 3′-deoxy-3′-[18F]fluorothymidine (FLT)-PET in tumor detection, noninvasive grading, and assessment of the cellular proliferation rate in newly diagnosed histologically verified gliomas of different grades.Materials and methodsForty-one patients with newly diagnosed gliomas were investigated with MET-PET before surgery. Eighteen patients were also examined with FLT-PET. MET and FLT uptakes were assessed by standardized uptake value of the tumor showing the maximum uptake (SUVmax), and the ratio to uptake in the normal brain parenchyma (T/N ratio). All tumors were graded by the WHO grading system using surgical specimens, and the proliferation activity of the tumors were determined by measuring the Ki-67 index obtained by immunohistochemical staining.ResultsOn semiquantitative analysis, MET exhibited a slightly higher sensitivity (87.8%) in tumor detection than FLT (83.3%), and both tracers were 100% sensitive for malignant gliomas. Low-grade gliomas that were false negative on MET-PET also were false negative on FLT-PET. Although the difference of MET SUVmax and T/N ratio between grades II and IV gliomas was statistically significant (P < 0.001), there was a significant overlap of MET uptake in the tumors. The difference of MET SUVmax and T/N ratio between grades II and III gliomas was not statistically significant. Low-grade gliomas with oligodendroglial components had relatively high MET uptake. The difference of FLT SUVmax and T/N ratio between grades III and IV gliomas was statistically significant (P < 0.01). Again, the difference of FLT SUVmax and T/N ratio between grades II and III gliomas was not statistically significant. Grade III gliomas with non-contrast enhancement on MR images had very low FLT uptake. In 18 patients who underwent PET examination with both tracers, a significant but relatively weak correlation was observed between the individual SUVmax of MET and FLT (r = 0.54, P < 0.05) and T/N ratio of MET and FLT (r = 0.56, P < 0.05). Total FLT uptake in the tumor had a higher correlation (r = 0.89, P < 0.001) with Ki-67 proliferation index than MET uptake (r = 0.49, P < 0.01).ConclusionsPET studies using MET and FLT are useful for tumor detection in newly diagnosed gliomas. However, there is no complimentary information in tumor detection with simultaneous measurements of MET- and FLT-PET in low grade gliomas. FLT-PET seems to be superior than MET-PET in noninvasive tumor grading and assessment of proliferation activity in gliomas of different grades.

Malignant astrocytomas with homozygous CDKN2/p16 gene deletions have higher Ki-67 proliferation indices

p16 is involved in a cell-cycle regulatory cascade that includes cyclin-dependent kinase 4 (cdk4), cyclin D1 and pRb. Alterations of each of these components have been described in primary human glioblastoma multiforme (GBM) or GBM cell lines, and alterations of the individual components of this pathway appear inversely correlated with one another. While this suggests that disruption of any individual component has similar oncogenic effects, homozygous deletions of the CDKN2/p16 gene are the most common genetic alteration. We investigated the relationship between homozygous CDKN2/ p16 deletions and cellular proliferation in 50 primary astrocytomas (2 WHO grade I pilocytic astrocytoma, 15 grade II astrocytomas, 20 grade III anaplastic astrocytomas and 13 grade IV GBMs). Using a comparative multiplex PCR assay, homozygous deletions of the CDKN2/p16 gene were detected in 5 anaplastic astrocytomas (25%) and 6 GBMs (46%), but in none of the lower-grade tumors. Ki-67 immunohistochemistry was used to assess the number of proliferating cells in the same samples used for molecular genetic analysis. In both anaplastic astrocytomas and GBMs, Ki-67 proliferation indices were significantly higher in tumors with CDKN2/p16 deletions (20%) than in those without deletions (10%; p = 0.0001). These results suggest that homozygous CDKN2/p16 deletions in high-grade astrocytomas may have a more deleterious effect on cell cycle control than the other aberrations in the p16-cdk4-cyclin D1-pRb pathway, and may provide one explanation for why homozygous CDKN2/p16 deletions are more common genetic events in high-grade astrocytomas than RB mutations or CDK4 amplification.

In vivo efficacy and toxicity of 5-fluorocytosine/cytosine deaminase gene therapy for malignant gliomas mediated by adenovirus.

We evaluated the therapeutic efficacy and neurotoxicity of adenovirus-mediated transduction of the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) for experimental malignant brain tumors. The 5-FC sensitivity in 9 L cells infected by an adenovirus vector expressing CD (AdexCACD) was increased 1700-fold compared with control cells. Rats bearing 9 L brain tumors were treated with an intratumoral injection of AdexCACD followed by intraperitoneal administration of 5-FC. The rats demonstrated remarkable inhibition of tumor growth by magnetic resonance imaging, and 7 of 10 rats survived for >90 days. To evaluate the potential side-effects of the 5-FC/CD gene therapy, rats were treated with an intracerebral injection of AdexCACD into the right basal ganglia and with 5-FC. The magnetic resonance imaging showed a highly enhanced area on the gadollinium-enhanced T1-weighted image at 18 days postinjection. Pathologically, this corresponded to an area of necrosis with surrounding apoptotic cells. In addition, there was demyelination and gliosis with enlargement of the lateral ventricles. These results suggest that the 5-FC/CD gene therapy may provide an anticancer effect for malignant brain tumors in humans, but also show that there are neurotoxic effects on normal brain tissue.

Therapeutic effect of suicide gene-transferred mesenchymal stem cells in a rat model of glioma

We evaluated a new therapeutic strategy for malignant glioma, which combines intratumoral inoculation of mesenchymal stem cells (MSCs) expressing cytosine deaminase gene with 5-fluorocytosine (5-FC) administration. For in vitro and in vivo experiments, MSCs were transfected with adenovirus carrying either enhanced green fluorescent protein gene (AdexCAEGFP) or cytosine deaminase gene (AdexCACD), to establish MSC-expressing EGFP (MSC-EGFP) or CD (MSC-CD). Co-culture of 9L glioma cells with MSC-CD in a medium containing 5-FC resulted in a remarkable reduction in 9L cell viability. The migratory ability of MSC-EGFP toward 9L cells was demonstrated by double-chamber assay. For the in vivo study, rats harboring 9L brain tumors were inoculated with MSC-EGFP or MSC-CD. Immunohistochemistry of rat brain tumors inoculated with MSC-EGFP showed intratumoral distribution of MSC-EGFP. Survival analysis of rats bearing 9L gliomas treated with intratumoral MSC-CD and intraperitoneal 5-FC resulted in significant prolongation of survival compared with control animals. In conclusion, molecular therapy combining suicide gene therapy and MSCs as a targeting vehicle represents a potential new therapeutic approach for malignant glioma, both with respect to the antitumor potential of this system and its neuroprotective effect on normal brain tissue.

A Case of Solitary Subependymal Giant Cell Astrocytoma : Two Somatic Hits of TSC2 in the Tumor, without Evidence of Somatic Mosaicism

Subependymal giant cell astrocytoma (SEGA) is a unique brain tumor arising in tuberous sclerosis complex (TSC), an autosomal dominant inherited phacomatosis. There are several case reports of solitary SEGA without any other manifestations of TSC. Usually these cases are thought to be forme fruste of TSC due to somatic mosaicism. However, no previous reports have used molecular methodology to fully investigate mutations in TSC genes or the possibility of somatic mosaicism. Here, we report a 20-year-old woman with a brain tumor. Pathological diagnosis was consistent with SEGA, but comprehensive clinical screening found no other lesions indicative of TSC. Molecular analysis of the tumor revealed loss of heterozygosity and allelic mutation (5228G>A, R1743Q) of TSC2. To detect the small fraction of mosaic mutation in somatic cells, we developed a highly sensitive new method: triple-nested polymerase chain reaction-restriction fragment length polymorphism. The identical TSC2 missense mutation was not detected in any other tissues from the same patient, including peripheral blood, buccal mucosa, urinary sediment, nail, and hair. According to these results, this patient should be considered as having SEGA that developed from two somatic hit mutations in TSC2, rather than being a TSC2 patient with a very small fraction of somatic mosaicism.

Apoptosis induction with 5-fluorocytosine/cytosine deaminase gene therapy for human malignant glioma cells mediated by adenovirus

Previously, we evaluated the therapeutic efficacy of the adenovirus-mediated transduction of the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) for malignant gliomas. However, the molecular pathways that mediate the 5-FC/CD gene therapy-induced cell death remains to be elucidated. In this study, we examined the induction of apoptosis and the role of caspases in 5-FC/CD gene therapy using human malignant glioma cells [Gli36Δ5 (mutated p53) and U87MG (wild p53)]. The treatment with 5-FC/CD gene-therapy-induced apoptosis both in Gli36Δ5 cells and in U87MG cells according to flow cytometric analysis. Immunoblot analysis revealed that caspases 3 and 9 were processed in response to 5-FC/CD in a concentration- and time-dependent manner, but caspase 8 was not. Each caspase 3 and 9 inhibitor significantly reduced apoptosis triggered by 5-FC/CD, but the caspase 8 inhibitor did not affect apoptosis induction. 5-FC/CD significantly promoted the release of cytochorme c from mitochondria in a concentration-dependent manner. These results indicate that 5-FC/CD gene therapy induces apoptosis in human malignant glioma cells and that the apoptotic cell death is mediated by the activation of mitochondrial caspase cascades involving caspases 3 and 9. This is the first report concerning the apoptotic mechanism of 5-FC/CD gene therapy, and these findings could be used to increase the efficacy of suicide gene therapy systems for the treatment of malignant glioma.

Accumulation of wild-type p53 in astrocytomas is associated with increased p21 expression

Abstract Approximately one quarter of human astrocytomas show immunohistochemical positivity for p53 protein but lack p53 gene mutations, which could reflect either an accumulation of wild-type p53 protein or an inadequate sensitivity of mutation detection. Since wild-type p53 up-regulates p21 expression, increased p21 expression in those astrocytomas with p53 accumulation in the absence of mutations would argue that the protein was wild type in these tumors. We therefore compared p21 expression with p53 gene and protein status in 48 primary human astrocytomas. Single-strand conformation polymorphism analysis and direct sequencing of the p53 gene showed mutations in 11 tumors (22.9%), while immunohistochemistry revealed positive staining in 19 cases (39.6%). Those tumors with p53 immunopositivity in the absence of p53 mutation had significantly increased p21 expression when compared to either mutant p53 or p53-immunonegative cases. Neither p53 nor p21 status correlated with proliferation indices, as assessed by Ki-67 immunohistochemistry. These results support the hypotheses that functionally wild-type p53 accumulates in some astrocytomas, and that alternative cell cycle checkpoints (such as the p16 pathway) may be more important than p21 in regulating proliferation in astrocytomas.

Ubiquitination-resistant p53 protein transduction therapy facilitates anti-cancer effect on the growth of human malignant glioma cells

Protein transduction therapy using poly‐arginine can deliver the bioactive p53 protein into cancer cells and inhibits the proliferation of the cells. However, one disadvantage of such therapy is the short intracellular half‐life of the delivered protein. Here, we generated mutant proteins in which multiple lysine residues in the C‐terminal were substituted by arginines. The mutant proteins were effectively delivered in glioma cells and were resistant to Mdm2‐mediated ubiquitination. Moreover, the mutant proteins displayed higher transcription regulatory activity and powerful inhibition of the proliferation of glioma cells. These results suggest that ubiquitination‐resistant p53 protein therapy may become a new effective cancer therapy.

Combined radiation and gene therapy for brain tumors with adenovirus-mediated transfer of cytosine deaminase and uracil phosphoribosyltransferase genes

Radiation therapy is an established modality for the treatment of malignant gliomas. Several reports have shown the advantage of additional radiation in combination with gene therapy. In this study, we investigated the ability of radiation therapy to enhance 5-fluorocytosine (5-FC)/cytosine deaminase (CD) plus uracil phosphoribosyltransferase (UPRT) gene therapy in malignant gliomas. In vitro study suggested evidence of a significant cytotoxic interaction between radiation therapy and 5-FC/CD+UPRT gene therapy for glioma cells. In vivo experiments demonstrated that the combination of gene therapy and radiation possessed superior antitumor effect in comparison to single therapy. However, the adverse effects of radiation therapy in combination with the gene therapy were observed with respect to normal brain. This combination therapy may be feasible for the treatment of gliomas, although the radiation dose and area should be reduced in order to prevent side effects.

Potent synergy of dual antitumor peptides for growth suppression of human glioblastoma cell lines.

Molecular targeting agents have become formidable anticancer weapons, which show much promise against the refractory tumors. Functional peptides are among the more desirable of these nanobio-tools. Intracellular delivery of multiple functional peptides forms a basis for potent, non-invasive mode of delivery, providing distinctive therapeutic advantages. Here, we examine growth suppression efficiency of human glioblastomas by dual-peptide targeting. We did simultaneous introduction of two tumor suppressor peptides (p14ARF and p16INK4a or p16INK4a and p21CIP1 functional peptides) compared with single-peptide introduction using Wr-T-mediated peptide delivery. Wr-T-mediated transport of both p14ARF and p16INK4a functional peptides (p14-1C and p16-MIS, respectively) into human glioblastoma cell line, U87ΔEGFR, reversed specific loss of p14 and p16 function, thereby drastically inhibiting tumor growth by >95% within the first 72 h, whereas the growth inhibition was ∼40% by p14 or p16 single-peptide introduction. Additionally, the combination of p16 and p21CIP1 (p21-S154A) peptides dramatically suppressed the growth of glioblastoma line Gli36ΔEGFR, which carries a missense mutation in p53, by >97% after 120 h. Significantly, our murine brain tumor model for dual-peptide delivery showed a substantial average survival enhancement (P < 0.0001) for peptide-treated mice. Wr-T-mediated dual molecular targeting using antitumor peptides is highly effective against growth of aggressive glioblastoma cells in comparison with single molecule targeting. Thus, jointly restoring multiple tumor suppressor functions by Wr-T-peptide delivery represents a powerful approach, with mechanistic implications for development of efficacious molecular targeting therapeutics against intractable human malignancies. [Mol Cancer Ther 2008;7(6):1461–71]