Toshihide Tsuzuki

Analysis of prognostic and survival factors related to treatment of low-grade astrocytomas in adults.

Prognostic factors for low-grade astrocytomas have been proposed, but optimal treatment remains controversial. Eighty-eight consecutive adult patients with supratentorial low-grade astrocytomas were retrospectively reviewed to determine specific factors influencing outcome. All underwent craniotomy (43 radical resections, 45 nonradical resections). Sex, age at diagnosis, preoperative Karnofsky performance status (KPS), tumor location, estimated extent of resection, radiation, chemotherapy, histological type, p53 status, MIB-1 staining and the apoptotic index were assessed as parameters for prognostic significance. KPS (p = 0.03), tumor location (p < 0.001), extent of surgical resection (p < 0.001) and radiotherapy (p = 0.01) were significantly assoicated with longer survival rates by univariate analysis. Multivariate analysis also showed a significant correlation between radiation therapy after surgical removal and survival time (p < 0.001). p53 status was not of importance in determining the necessity for radiotherapy. Radical surgical removal is the most important factor in the management of low-grade astrocytomas. Radiation therapy appears to be effective in improving the prognosis regardless of the extent of surgical resection or the p53 status.

Comparison of ras activation in prostate carcinoma in Japanese and American men.

Comparative studies of point mutations in K‐, N‐, and H‐ras oncogenes were performed on prostate carcinoma from Japanese and American patients to clarify the racial difference.

Analyses of human gliomas by restriction landmark genomic scanning

The 16 primary gliomas were examined for changes in genomic DNA using arecently developed 2-dimensional gel electrophoresis method calledrestriction landmark genomic scanning (RLGS). This approach allows detectionof DNA amplification, deletion, methylation and potentially other geneticrearrangements represented as decreases and increases in spot/fragmentintensity on an autoradiogram. Approximately 2,000 landmark sites in tumorDNA were compared with those of DNA isolated from normal brain tissues.Seven spots showing intensified signal were consistently detected in atleast 50% of tumors, implying activation of corresponding DNAsequences, and 8 additional spots having reduced signal were observed, againin more than 50% of all tumors, suggesting inactivation by the lossof 1 allele or homozygous deletion. Decreased signal may also infer relativeCpG island methylation state. Of those spots consistently identified intumors, 2 amplified and 4 reduced spots were found to be characteristic oflow- and high-grade tumors, while the remaining 5 amplified and 4 reducedspots were associated with high-grade gliomas only, suggesting a link ofspecific mutations to degree of malignancy. A separate subset ofglioblastomas evaluated, however, showed no alterations in these ’hot spots‘which were detected in even low grade astrocytomas. The results demonstratethe genetic heterogeneity of glioblastoma and implicate the progression ofneoplasia via differing genetic pathways.

Drug resistance and apoptosis in ENU-induced rat brain tumors treated with anti-cancer drugs

To cast light on the mechanisms of drug-resistance, experimental brain tumors were immunohistochemically evaluated for expression of glutathione S-transferase (GST)-α, µ, π, p-glycoprotein and apoptosis-related factors, such as bcl-2 and p53, as well as by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) method. Rat brain tumors induced by means of prenatal exposure to ethylnitrosourea (ENU) were treated with 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) and/or vincristine. Tumors more than 2 mm in size were considered to be drug resistant. The expression of GST-µ was strongly positive in ACNU-treated brain tumors, while p-glycoprotein was overexpressed in vincristine-treated brain tumors. Neither p53 nor bcl-2 expression directly correlated with apoptosis identified by TUNEL method, but tumors lacking apoptotic cells always demonstrated the expression of either GST-µ or p-glycoprotein. These results indicate that tumors resistant to chemotherapy might not be susceptible to induction of apoptosis, and therefore that mechanisms of drug resistance are related to programmed cell death in brain tumors.

Frequent alterations of cell-cycle regulators in astrocytic tumors as detected by molecular genetic and immunohistochemical analyses

Alterations ofCDKN2A, RB, andcyclin D1 genes and expression of their products in astrocytic tumors were studied using a combination of molecular genetic and immunohistochemical assays. In addition, the association of gene status with clinical outcome was evaluated. Alterations ofCDKN2A andRB gene in 30 lesions were analyzed by single-strand conformation polymorphism of polymerase chain reaction (PCR-SSCP), direct sequencing, and Western blotting. Methylation of theCDKN2A promoter was detected by methylation-specific PCR. Immunohistochemistry was applied to determine the expression of gene products in tumors from 94 patients for whom clinical outcome was also evaluated. Analyses of theCDKN2A gene revealed 12 homozygous or hemizygous deletions, one mutation in exon 1, and three methylations in the promoter. Expression of p16 protein was not detected in 18 of 30 cases.RB mutations leading to loss of expression of the pRb were found in four (13%) cases, and six were immunohistochemically negative for this protein. Overexpression of cyclin D1 was obtained in 51 (54%) of 94 cases. Patients with pRb-negative tumors had a significantly greater risk of earlier death than those with p16 and cyclin D1 alterations. Both p16 and pRb immunohistochemistry provides useful complementary information and may provide valuable predictive information in screening. The biological consequences of deregulating individual components along cell control pathways are unequal, perhaps reflecting their hierarchical roles in the G1 checkpoint.

Genomic alterations in human glioma cell lines detected by restriction landmark genomic scanning

Restriction landmark genomic scanning (RLGS) is a 2-dimensionalgel analysis capable of detecting amplifications, deletions andrearrangements in genomic DNA. Using RLGS, we examinedgenomic DNA from each of 6 human-derived malignantglioma cell lines and from normal brain tissuesamples. RLGS allows us to screen genomic DNAsas approximately 2,000 landmark sites in one procedurewithout any polymorphic markers. The resulting 2,000 spotsin tumor samples were compared with those innormal brain. Six spots common to 5 ofthe 6 cell lines showed intensified signal, suggestingamplification of a tumor-specific DNA fragment. In addition,25 spots common to 5 of the 6lines showed a decrease in signal intensity, converselyindicating allelic loss of homozygous deletion. These resultsimply the existence of consistent genetic alterations inhuman glioma.

Genomic alterations of human gliomas detected by restriction landmark genomic scanning

Alterations of genomic DNA in eight primary astrocytic tumors and two glioma cell lines were examined using a recently developed two-dimensional gel electrophoresis method called restriction landmark genomic scanning (RLGS). RLGS allows us to detect amplifications, deletions, and methylation in genomic DNA in one procedure without requiring any polymorphic markers. Approximately 2000 spots (landmark sites) in tumor specimens were compared with those in normal brain tissue. The 10 spots with intensified signal were reproducibly detected in at least 50% of primary tumors, implying amplification of corresponding DNA sequences. Conversely, 12 spots with reduced signal were observed in more than 50% of all tumors, suggesting inactivation by allelic loss, homozygous deletion, or CpG island methylation. These results suggest that common genetic alterations are closely correlated with the genesis or progression of human gliomas.

Progressive activity in latent prostate carcinoma defined by argyrophilic staining of the nucleolar organizer regions (AgNOR)

Argyrophilic staining of the nucleolar organizer regions (AgNOR) was studied in 30 cases of benign prostatic hyperplasias (BPH), 17 cases of latent prostate carcinomas, 50 cases of clinical carcinomas and seven cases of metastatic lesions from prostate carcinomas. The criteria for these comparisons were the number of positive‐staining dots per nucleus, the area of the dots, and a relative score determined by multiplying the number of positive‐staining dots in the nuclei by the areas of the dots. Overall, there were no significant differences in these three parameters between BPH and latent carcinomas. Among latent carcinomas, however, significantly higher AgNOR scores were observed for infiltrative lesions than for non‐infiltrative lesions. AgNOR dot number, area and score increased as tumors became less differentiated, with no significant differences detected in metastatic versus non‐metastatic carcinomas. These results suggest that some latent tumors are similar in biological behavior, such as cell proliferation, to clinical carcinoma.

Genetic variations in recurrent astrocytic tumors detected by restriction landmark genomic scanning.

The genetic mechanisms associated with recurrence of advanced astrocytic tumors are poorly understood. We therefore analyzed 24 biopsy specimens from 12 patients with a recurrent astrocytic tumor by a two-dimensional gel electrophoresis method, termed restriction landmark genomic scanning (RLGS). Four spot changes were commonly present in the primary astrocytomas, indicating that the corresponding gene alterations were early events in the development of this tumor type. Altered spots were more frequent and of different distribution in recurrent tumors than in the primary astrocytomas. In particular, specifically increased intensity for spots on chromosomes 9–12 and 18 and reduced intensity for spots on 9–12 and 18 were observed in the secondary tumors, suggesting a relation with recurrence. The same spot changes observed in primary tumors were also found in the respective secondary lesions but with strikingly different densities in some cases, suggesting increased genetic instability. The altered segments provide important candidate regions for the search for genes involved in events leading to progression and more malignant recurrent tumors.

Cerebral cortical venous reconstruction using saphenous vein graft.

Cortical venous reconstruction using saphenous vein graft was performed in a patient during a procedure for meningioma removal. A cortical vein passing by the tumour was accidentally sacrificed during the dural opening. Before tumour removal the cortical vein was reconstructed with a 2-cm length of saphenous vein graft. Intraoperative venous blood flow control was done by microvascular Doppler sonography, obtaining a venous blood flow velocity at 3 cm/s. Postoperative course of the patient was uneventful. Angiographic control at one year after operation revealed good venous graft patency. Saphenous vein graft may be an option for reconstruction of cerebral cortical veins.