Mitsuhiro Tada

Frequent Co-Alterations of TP53, p16/CDKN2A, p14ARF, PTEN Tumor Suppressor Genes in Human Glioma Cell Lines.

In this study we established the simultaneous status of TP53, p16, p14ARF and PTEN tumor suppressor genes in 34 randomly chosen human glioma cell lines. Nine cell lines (26.4%) harbored mutations or deletions in all four tumor suppressor genes and 22 cell lines (64%) had alterations in at least three. Mutations/deletions were found at the following frequencies: TP53 (76.5%), p14ARF (64.7%), p16 (64,7%), PTEN (73.5%). Thus, there was a high incidence of alterations in the cellular pathways involving the p53 transcription factor (94.1%), the retinoblastoma protein (64.7%) and the PTEN phosphatase (73.5%) and 91% of cell lines carried mutations in two or more pathways. This provides the first clear genetic evidence that these tumor suppressors participate in biological pathways which are functioning separately/independently in glioma cells. The status of the gene alterations did not correlate with tumorigenicity in immunocompromized mice or any clinical parameters. Although the mutation rate was higher in glioma cell lines than that reported for glioma tissues, the alterations were molecularly representative of those found in adult de novo glioblastoma. This study highlights the importance of developing therapeutic approaches applicable to tumors with a broad range of genetic alterations and also provides an invaluable panel of glioma cell lines to make this possible.

Linkage of Familial Moyamoya Disease (Spontaneous Occlusion of the Circle of Willis) to Chromosome 17q25

BACKGROUND AND PURPOSE Moyamoya disease is a cerebrovascular disease of unknown cause that mainly affects Japanese children. The incidence of familial occurrence accounts for 9% of cases. The characteristic lesions of moyamoya disease are occasionally seen in neurofibromatosis type 1, of which the causative gene (NF1) has been assigned to chromosome 17q11.2. METHODS To determine whether a gene related to moyamoya disease is located on chromosome 17, we conducted microsatellite linkage analyses on 24 families containing 56 patients with moyamoya disease. Leukocyte DNA extracted from the family members was subjected to polymerase chain reaction for a total of 22 microsatellite markers on chromosome 17. The amplified polymerase chain reaction fragments were analyzed with GeneScan on an automated sequencer. RESULTS Two-point linkage analysis gave a maximum log(10) odds (LOD) score of 3.11 at the recombination fraction of 0.00 for the marker at locus D17S939. The affected pedigree member method also showed a significantly low P value (<1. 0x10(-5)) for the 5 adjacent markers at 17q25. Multipoint linkage analysis also indicated that the disease gene is contained within the 9-cM region of D17S785 to D17S836, with a maximum LOD score of 4. 58. CONCLUSIONS A gene for familial moyamoya disease is located on chromosome 17q25.

Germ cell tumours of the central nervous system: treatment consideration based on 111 cases and their long-term clinical outcomes

Germ cell tumours (GCTs) of the central nervous system (CNS) encompass various histological subtypes, and their optimal management has been the subject of debate. To indicate a better management strategy for each subtype, we analysed the records of 111 patients (median age 14 years), who underwent treatment since 1970. With a median follow-up duration of 86 months, the probability of surviving 5 years was: 96% for pure germinoma patients, 100% for mature teratoma, 67% for immature teratoma and 69% immature teratoma mixed with germinoma. The probability of cause-specific progression of germinomas producing human chorionic gonadotropin (HCG) was higher than that of non-producing germinomas (P < 0.01). GCTs that included a highly malignant component, such as embryonal carcinoma or yolk sac tumour, exhibited a poor prognosis with 38% chance of 5-year survival. Late adverse effects of therapy included stroke, secondary malignancy and cognitive, endocrinological, auditory and visual dysfunctions. Of 85 survivors with a median follow-up period of 99 months, 58 patients needed hormone replacement therapy, 26 patients showed poor performance status and, to date, only 1 patient has fathered children. Because the outcomes varied widely for each subtype, the traditional categories, that is, germinoma and non-germinomatous GCT as an extrapolation from the gonadal GCTs, are not suitable for appropriately selecting therapeutic regimen for CNS GCTs.

Roles of the functional loss of p53 and other genes in astrocytoma tumorigenesis and progression.

Loss of function of the p53 tumor suppressor gene due to mutation occurs early in astrocytoma tumorigenesis in about 30-40% of cases. This is believed to confer a growth advantage to the cells, allowing them to clonally expand due to loss of the p53-controlled G1 checkpoint and apoptosis. Genetic instability due to the impaired ability of p53 to mediate DNA damage repair further facilitates the acquisition of new genetic abnormalities, leading to malignant progression of an astrocytoma into anaplastic astrocytoma. This is reflected by a high rate of p53 mutation (60-70%) in anaplastic astrocytomas. The cell cycle control gets further compromised in astrocytoma by alterations in one of the G1/S transition control genes, either loss of the p16/CDKN2 or RB genes or amplification of the cyclin D gene. The final progression process leading to glioblastoma multiforme seems to need additional genetic abnormalities in the long arm of chromosome 10; one of which is deletion and/or functional loss of the PTEN/MMAC1 gene. Glioblastomas also occur as primary (de novo) lesions in patients of older age, without p53 gene loss but with amplification of the epidermal growth factor receptor (EGFR) genes. In contrast to the secondary glioblastomas that evolve from astrocytoma cells with p53 mutations in younger patients, primary glioblastomas seem to be resistant to radiation therapy and thus show a poorer prognosis. The evaluation and design of therapeutic modalities aimed at preventing malignant progression of astrocytomas and glioblastomas should now be based on stratifying patients with astrocytic tumors according to their genetic diagnosis.

Somatic Mutations of the APC Gene in Primary Breast Cancers

APC gene mutations play an important role in the initiation step of colorectal carcinogenesis in both familial adenomatous polyposis (FAP) patients and non-FAP patients. Although the APC gene is expressed in most tissues, including the lung, liver, kidney, and mammary gland, its somatic mutations have rarely been found in primary tumors affecting these organs. We have developed a sensitive yeast-based assay for screening almost the entire coding region of the APC gene. By this method, we have been able to detect somatic mutations of the APC gene in 57% of colorectal cancers and none in non-small cell lung cancers. Interestingly, the assay detected somatic APC gene mutations in 18% of breast cancers, in which APC gene mutation was previously considered rare. In the breast cancers, most of the APC mutations were distributed outside the mutation cluster region that has been advocated for colorectal cancers. We also noted a difference in the mutation pattern of the APC between colorectal and breast cancers. In colorectal cancers, all base substitutions were observed at C residues (5 of 5), whereas in breast cancers the majority of them were found at G residues (4 of 5). Furthermore, APC mutations were observed at a significantly high frequency in advanced stages of primary breast cancers (TNM classification, P < 0.05; T category, P < 0.01). Our data suggest that the etiology of the APC mutations and their biological role in carcinogenesis may differ between colorectal and breast cancers.

High frequency of p53 mutations in human oral epithelial dysplasia and primary squamous cell carcinoma detected by yeast functional assay

To determine the timing and actual incidence of p53 mutations in oral epithelial lesions, we examined 33 primary squamous cell carcinomas (SCCs), 14 dysplasias and six hyperplasias from Japanese patients by a combination of yeast functional assay and DNA sequencing. The assay detects mutations of p53 mRNA between codons 67 and 347 on the basis of the DNA-binding activity of the protein. Twenty-six SCCs (79%) and five dysplasias (36%) were positive for p53 mutation, while all six hyperplasias were negative for the mutation. Human papillomavirus type 16 E6 mRNA was detected in one of seven p53 mutation-negative SCCs by reverse transcription polymerase chain reaction (RT – PCR). We further examined p53 mutations in 17 Sri Lankan oral SCCs using the yeast functional assay and the single-strand conformation polymorphism analysis of PCR-amplified DNA fragments (PCR – SSCP) of exon 5 – 8. The mutations were confirmed by DNA sequencing and the detection sensitivity was compared between the two methods. Six samples (35%) were positive for p53 mutation in PCR – SSCP analysis, while nine samples (53%) were positive in yeast functional assay. This suggests that the incidence of p53 mutations has been considerably underestimated in the conventional SSCP analysis. The present data indicate that p53 mutations are extremely frequent in oral cancers in the Japanese, and suggest that the timing and significance of p53 mutation in oral tumor progression vary in different ethnic populations and areas.

Increased pre-therapeutic serum vascular endothelial growth factor in patients with early clinical relapse of osteosarcoma

To investigate the clinical significance of circulating angiogenic factors, especially in association with early relapse of osteosarcoma, we quantified pre-therapeutic levels of vascular endothelial growth factor, basic fibroblast growth factor and placenta growth factor in the sera of 16 patients with osteosarcoma using an enzyme-linked immunosorbent assay. After a 1-year follow-up, the serum level of angiogenic factors was analysed with respect to microvessel density of the biopsy specimen and clinical disease relapse. The serum vascular endothelial growth factor levels were positively correlated with the microvessel density with statistical significance (P=0.004; Spearman rank correlation) and also significantly higher in seven patients who developed pulmonary metastasis than the remaining nine patients without detectable disease relapse (P=0.0009; The Mann–Whitney U-test). In contrast, the serum levels of basic fibroblast growth factor or placenta growth factor failed to show significant correlation with the microvessel density or relapse of the disease. Although there was no significant correlation between serum vascular endothelial growth factor levels and the tumour volume, the serum vascular endothelial growth factor levels were significantly higher in patients with a vascular endothelial growth factor-positive tumour than those with a vascular endothelial growth factor-negative tumour. These findings suggest that the pre-therapeutic serum vascular endothelial growth factor level reflects the angiogenic property of primary tumour and may have a predictive value on early disease relapse of osteosarcoma.

Evaluation of application techniques of fibrin sealant to prevent cerebrospinal fluid leakage: a new device for the application of aerosolized fibrin glue.

OBJECTIVE This report evaluates the sealing effects of fibrin sealant applied on the dura mater using different techniques. METHODS Three application methods were studied: a sequential layer method, a simultaneous method using a cannula, and a spray method using a newly developed spray device. The sealing effects of these methods were compared using in vitro histological analysis and a pressure resistance test. The clinical efficacy of the fibrin sealant to prevent water leakage through the dura mater was retrospectively analyzed in a total of 509 patients. The process of absorption of a clinically applied fibrin clot in vivo was examined using surgical specimens. RESULTS The fibrin plate made using the spray method withstood a hydrostatic pressure greater than 200 cm H2O. A scanning electron microscopic study of the fibrin clots showed that the sequential and simultaneous methods produced a fibrin fiber network; in contrast, our spray method formed a dense fibrin tissue in which the fibrin molecules fused together forming stratified laminae. Of the 295 supratentorial craniotomies during which spraying was used, postsurgical cerebrospinal fluid leakage occurred in 9 cases (3.1%), whereas of the 214 craniotomies during which spraying was not used, cerebrospinal fluid leakage occurred in 19 cases (8.9%). Histological examinations of 10 surgical specimens obtained during second craniotomies revealed that the spray-made fibrin clots had been gradually replaced by mature granulation composed of collagenous connective tissue. CONCLUSION The optimal technique for applying fibrin sealant is the spray method that aerosolizes fibrin glue and produces a tough fibrin plate.

Cells with TP53 mutations in low grade astrocytic tumors evolve clonally to malignancy and are an unfavorable prognostic factor.

It is important to understand how low grade tumors recur and progress to malignant lesions since this dramatically shortens patient survival. Here, we evaluated the concept that malignant progression and poor prognosis of low grade astrocytic tumors are TP53 dependent through clonal expansion of mutated cells. TP53 status was established in primary and recurrent tumors from 36 patients with WHO grade II astrocytic tumors and two tumor types were found. Tumors from 14 patients (39%; type 1) had TP53 mutated cells, and 92% of these recurred with 57% progressing to malignancy. The evolution of TP53 mutated cells before and after progression was examined using a clonal analysis procedure in yeast. Malignant progression was accompanied by an increased percentage of mutant TP53 (red) yeast colonies resulting from monoclonal expansion of cells with mutated TP53. The presence of TP53 mutations in WHO grade II astrocytic tumors was associated with malignant progression (P=0.034, χ2 test) and shorter progression-free survival (PFS; 47.6±9.6 months for TP53-mutated tumors vs 67.8±8.2 months for TP53-wild type tumors, P<0.05, log-rank test). Tumors from 22 patients (61%; type 2) were without TP53 mutations, and 64% of these recurred without a change in TP53 status, although 41% progressed to malignancy. This suggests that TP53 mutation is not an initiating or progression event in the majority of low grade astrocytic tumors. Our study also indicates that irradiation for WHO grade II astrocytic tumors might be associated with poor outcome (P<0.0001) and this was independent of TP53 status. These findings have important implications in the clinical management of patients with low grade astocytoma and provide new support to the clonal evolution model for tumor progression.

Familial occurrence of Moyamoya disease

The authors report four cases of familial occurrence of moyamoya disease. Although the pathogenesis of moyamoya disease is not clear, there is extensive evidence that this disease has a tendency to show multifactorial inheritance. Therefore, a screening test for those at high risk, i.e., who have a moyamoya patient among their blood relatives, is clinically important. Magnetic resonance angiography (MRA) successfully revealed abnormal findings specific to moyamoya disease in members of the four probands families. MRA is a powerful and noninvasive way of detecting individuals at high risk of developing moymoya disease.