Recent advances in molecular biology have revealed that the alteration of multiple genes, eg., APC, K-ras, p53, DCC, are involved in multistep colorectal carcinogenesis. Some of these alterations can be used as molecular markers in genetic diagnosis. Genetic diagnoses for colorectal cancer are classified into three categories, eg., 1. identification of the career in the family of patient with hereditary disease such as FPC (Familial Polyposis Coli) or HNPCC (Hereditary Non-Polyposis Colorectal Cancer), 2. early diagnosis of colorectal cancer by identifying gene mutations in the stool, 3. assist for histopathological diagnosis, or risk assessment of the metastasis, recurrence or secondary cancer by molecular means. However, there are several problems in these genetic diagnoses. These consist of two categories, eg., 1. problems in the method of gene analyses or assay system and 2. problems in performing genetic diagnoses itself. The former includes the problem of contamination of different tissue, false positive or negative result in PCR-based analyses, heterogeneity of gene mutation in tumor tissue, and the latter includes the social, ethical or economical problems mainly related to the genetic diagnosis for hereditary colorectal cancers. In this paper, we describe the possibility of genetic diagnosis for colorectal cancers and the current problems, especially from the molecular pathological aspect, in genetic diagnosis.

Two cases of astrocytoma associated with von Recklinghausen's disease (neurofibromatosis type; NF-1) were reported. The first case wes a 60-year-old man who had been diagnosed as von Recklinghausen's disease on the basis of skin findings. Magnetic resonance imaging (MRI) showed a tumor in the left temporal lobe. Partial removal was performed with neuronavigator, and because of the existence of Rosenthal fiber the histological diagnosis was pilocytic astrocytoma. Radiation therapy was performed. The second case was a 6-year-old boy suffering from headache and left hemiparesis including his face. MRI showed a tumor with a cyst in the right thalamus and obstructive hydrocephalus. Initially CT-guided stereotactic biopsy was performed, and the histological diagnosis, on the basis of increased cellularity, pleomorphism and nuclear atypia without necrosis or vascular proliferation, was anaplastic astrocytoma. Radiation and chemo-immuno therapy were carried out after V-P shunt. It is well known that von Recklinghausen's disease (NF-1) is often associated with optic glioma (5-36%). In the literature, the glioma seldom occurs in other parts of the brain, supratentorial glioma especially is rare. Only two familial cases of supratentorial glioma associated with von Recklinghausen's disease have been reported. The prognosis of supratentorial glioma associated with NF-1 was poor in these reports. In this paper, the diagnostic and therapeutic problems are discussed.

Recent advances in molecular genetics have made a great contribution to the investigation of lymphoid malignancies. We diagnosed orbital malignant lymphomas by molecular genetic analysis. Molecular genetic analysis elucidates gene rearrangements within lymphocytes that are responsible for immunoglobulin produced by B-lymphocytes, or the expression of cell surface antigen recognition receptors in T-lymphocytes. The cells of most malignant tumors are considered to be genetically homogeneous because they are assumed to represent a single clone descended from one abnormal cell. The determination that a malignancy is of clonal origin has been used as a diagnostic aid to distinguish benign from malignant proliferations. Analysis of the immunoglobulin genes and the T-cell antigen receptor genes using DNA from biopsied specimens is very useful, as it can reveal the origin of tumor cells and clonality of tumor cell characteristics which cannot be distinguished by morphological analysis. Genetic analysis was found to be superior to immunophenotyping for identifying the monoclonality of tumor cells in instances when there were no definitive cell-lineage specific markers, or when there was a preponderance of normal counterparts with mixed appearance of neoplastic and non-neoplastic cells in malignant lymphoma. Molecular genetic analysis is used to 1) distinguish clonal from polyclonal lymphoproliferations, 2) determine the B-cell or T-cell identity of malignancies with admixed normal cells, 3) determine the genetic lineage of neoplasms lacking definitive surface antigens, and 4) determine the developmental stage of early B-cell or T-cell precursors.

Pharmacokinetic properties and gene expression of naked plasmid DNA and its cationic liposome complexes after intratumoral injection were studied. Using Walker 256 tissue-isolated tumor perfusion system, we quantified the recovery of naked plasmid DNA and cationic liposome complexes in the tumor, leakage from the tumor surface and the venous outflow after intratumoral injection. Approximately 50% of naked plasmid DNA was eliminated from the tumor at 2 hr after injection, while more than 90% of plasmid DNA was retained in the tumor when complexed with cationic liposome. However, distribution of these complexes in the tumor was restricted only in the vicinity of the injection site. Furthermore, we have examined the expression of chloramphenicol acetyltransferase (CAT) DNA constructs (naked pCMV-CAT) and its cationic liposome (DC-chol) complexes after intratumoral injection into subcutaneous rat Walker 256 carcinosarcoma. Significant gene expression was observed in both cases, but localization of gene expressing cells in the tumor tissue was limited to the vicinity of the injection site. Thus the pharmacokinetic and gene expression studies have demonstrated that in vivo gene expression in the tumor can be achieved by direct injection of naked plasmid DNA. In addition, there is a possibility that restricted localization of naked DNA and its cationic liposome complexes in tumor inhibits gene expression.

We studied the clinical significance of the extent of p53 protein expression by immunohistochemical analysis and p53 mutations by functional analysis of p53 in 42 patients with high and intermediate grade of Non-Hodgkin's Lymphoma (NHL). Neither p53 expression nor mutation correlated with short survival or resistance to chemoradiotherapy, implying that p53 mutation is not a prognostic factor in NHL.

Prevention is an important and effective measure for reducing death caused by cancer. Thus information on individual susceptibility to cancer is valuable in suggesting high-risk individuals to avoid intake of carcinogenic substances and undergo frequent physical examinations. To this end, polymorphisms found within cytochrome P450 genes implicated in metabolism of procarcinogens are expected to be good genetic targets in assessing human cancer susceptibility. The present author has shown the polymorphisms within arylhydrocarbon receptor (AHR) gene, which is responsible for inducing CYP1A1, an enzyme activating carcinogens in cigarette smoke, does not significantly associate with lung cancer susceptibility in the Japanese population, in contrast to mouse animal model, whose susceptibility is known to be governed by AhR polymorphisms. A newly identified polymorphism in CYP1A1 itself, however, may determine lung cancer susceptibility.

Ovarian cancer is the most lethal kind of gynecological malignancy. Though combination therapy has improved, the prognosis is still poor. It is a risk factor to have a member affected with breast or ovarian cancer in one's family or to have been affected with these diseases. Genetic testing will more effectively enable earlier diagnosis and cure of such diseases. Including the selected results of familial ovarian cancers, we discuss the effectiveness and difficulty of genetic testing of BRCA1 in Japan.

We examined topographic distribution of p53 mutations using in situ PCR in three primary non-small cell lung cancers, showing distinct patterns of variable p53 overexpression by immunohistochemistry (Ebina, et al:Cancer Res. 54:2496, 1994). All tumor cell nuclei, regardless of the status of p53 overexpression, demonstrated homogeneous distribution of mutant p53 with specific primers, suggesting only subgroups of the mutated cells overexpressed p53 protein. Topographic genomapping using in situ PCR is a useful method to reveal the possible correlation between cell-morphology and molecular abnormality in cancer cells.

We studied the correlation between microvessel density (MD) and gene expression of vascular endothelial growth factor (VEGF), its receptor (KDR) and basic fibroblast growth factor (bFGF) in 30 human renal cell carcinomas (RCCs). Gene expression assessed by Northern blot hybridization was quantified by a densitometer. Tissue localization of VEGF was assessed by anti VEGF-antiserum staining. MD was measured in tumor tissue sections stained for factor VIII-related antigen. VEGF gene was overexpressed in 18 (60%) of 30 RCCs. Cytoplasm of the tumor cells was stained positively for VEGF staining. Intratumoral expression of KDR gene significantly correlated with that of VEGF gene. bFGF gene expression was very weak, and no tumor overexpressed this gene. The MD in the tumors significantly correlated with the expression intensity of VEGF gene and KDR gene. These results suggest that VEGF contributes to neovascularization of RCCs through a paracrine mechanism, and could be used as a relevant indicator of angiogenesis.

A 16-year-old boy was operated upon for synovial sarcoma of the right thigh and underwent chemotherapy consisted of adriamycin (320 mg), cisplatin (780 mg), etoposide (4,200 mg) and ifosfamide (30,000 mg). He developed secondary leukemia 18 months after the chemotherapy. Acute lymphoblastic leukemia (L3) was initially diagnosed because of poor staining of alpha-naphtyl butylate esterase and induction chemotherapy with the LVP regimen (L-asparaginase 5,000 U/m2 day 8-21, vincristine 1.5 mg/ m2 day 1, 6, 11, 16, 21, 26, prednisolone 40 mg/m2 day 1-28) was performed. After the therapy was initiated, the leukemia was finally diagnosed as acute momocytic leukemia (M5a) because of the following data; blasts were positive for CD33 and HLA-DR and negative for CD10, CD19 and CD20; serum lysozyme was 104.0 micrograms/ml; re-evaluation revealed that blasts were strongly positive for alpha naphtyl butyrate esterase in a small part of the slides; 95% of the bone marrow cells showed t (9; 11) chromosomal aberration; gene rearrangement was positive for MLL and negative for JH, JK and TCR C beta 1. Nevertheless, complete remission was obtained after 1 course of LVP therapy. He received bone marrow transplantation from an unrelated volunteer donor after 3 courses of consolidation therapy. He has remained in complete remission for 16 months.

A 27-year-old female was diagnosed as having atypical aplastic anemia in 1979 because of hypercellular bone marrow with abnormal erythroblasts and megakaryocytes. Afterward the diagnosis was corrected to myelodysplastic syndrome (RA) due to the reevaluation of the bone marrow smears. In March, 1995, thirst and polyurea occurred. In April, 1995, bone marrow aspiration biopsy showed the proliferation of atypical blasts (28%), and two months later, the number of the blasts increased (30%) and leukemic progression was noticed. Only 0.5 percent of the blasts showed weak peroxidase activity, and most of the blasts had CD13, CD33 and several adhesion molecules as CD11a, CD11b, CD44, CD54 and CD56. Karyotype of the bone marrow cells was 45, XX, -7. Her polyurea was caused by central diabetes insipidus. She was also complicated by pleuritis, colon ulcer, sinusitis and hypothalamic dysfunction. The etiology of these signs was due to the leukemic cell infiltration. She died despite of receiving multi-drug chemotherapy.

While not all circulating tumor cells survive, one could postulate that patients with circulating tumor cells might be candidates for adjuvant chemotherapy because of the risk of relapse. Recently, reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of circulating tumor cells has been suggested as a potential technique for staging of cancer. In malignant melanoma, the detection of circulating melanoma cells by tyrosinase RT-PCR correlated with the clinical stage of patients and was an independent prognostic factor for recurrence. A strong association was found between the detection of neuroblastoma cells in circulation by tyrosine hydroxylase RT-PCR with bone marrow micrometastasis. This method may be of use to identify early signs of relapse in disease-free patients. In prostatic cancer, the frequency of positivity for detection of circulating tumor cells in peripheral blood by PSA RT-PCR increased with tumor stages but a significant proportion of patients with metastatic disease were negative. The prognostic significance of the detection of tumor cell in blood in other epithelial tumors is not established and will require further long-term follow-up study.

It has been reported that the break point of 8q24 in t (8; 14) (q24; q32) is located far up-stream from c-myc gene locus in endemic EBV (Epstein-Barr virus)-positive BL, while the break-point is located close to the 1st intron of c-myc gene in sporadic EBV-negative BL. Considering that no statistical analysis is available regarding BL in Japan, the break-point of chromosome No.8 was investigated in 13 BL/L3 cell lines (having t(8; 14)) and 4 fresh samples derived from Japanese patients, including 3 EBV-positive BL cell lines, by using long-distance PCR. In this PCR, one primer was set in the 2nd intron of the c-myc gene, and the other primer in Ig constant region gene, mu, gamma, alpha and epsilon. This long distance PCR can cover up to 30 kb. Thus, this PCR does'nt generate product, if the 8q24 break-point is located far up-stream (more than 50 kb) fom c-myc gene. In 2 of the 3 t (8; 14) EBV-positive BL lines, no product was generated in two lines(N831 and Middle 91), while a product was synthesized in one line(Akata), indicating that the 8q24 break-point is near the c-myc gene in Akata. In all the other BL/L3 lines, a product was synthesized. A larger number of BL cases are necessary to investigate in order to know which 8q24 break-point pattern is exhibited by EBV-positive BL in Japan, while this method is suitable for testing a large number of case materials.

The authors reviewed the clinical, phenotypic, genotypic, and Epstein-Barr virus (EBV)-findings of 18 patients with nasal T-cell lymphoma(NTL). The clinical features were characterized as prolonged fever, widespread dissemination into distant sites, and poor prognosis with median survival of only 6 months. EBV-encoded small nuclear early region(EBER) transcripts were identified in 16 of 18 patients. Monoclonal EBV genome, EBV-encoded nuclear antigen(EBNA)-1, and latent membrane protein(LMP)-1 were also detected in all EBER-positive cases tested. All EBV-positive NTL showed coexpression of natural killer(NK) cell phenotype CD56 and CD2. Of 9 EBV-positive NTL, seven cases expressed T-cell receptor (TCR)-delta chain with rearranged beta-, gamma- and/or delta- genes. These data suggest that some cases of EBV-positive NTL may be derived from the lineage of NK-like T-cells or gamma delta T-cells, and that EBV may play a role in the lymphomagenesis.

Epstein-Barr virus (EBV), a ubiquitous human herpes virus, was recently identified in 2-16% of gastric carcinomas. EBV-encoded small RNA was found in nearly all of the carcinoma cells even at the intramucosal stage. EBV in EBV associated gastric carcinoma (EBVaGC) is monoclonal based on Southern blot hybridization using probes adjacent to the unique terminal repeat of EBV-DNA. Furthermore, the genetic pathway of this carcinogenesis is different of EBVaGC: deletion of 5q and/or 17p and microsatellite instability are extremely rare in EBVaGC, in contrast to their high frequency in EBV-negative carcinomas. We also examined the relationship between the expression of CD44 variants and EBVaGC, and found the expression of CD44 variants was significantly correlated with EBV-etiology.

Many antineoplastic drugs and cytotoxic irradiation induce apoptosis in cancer cells. ICE and ICE-like proteases play important roles in drug-induced apoptosis of cancer cells. We evaluated the cellular factors affecting susceptibility to apoptosis using gene-transfected cells. Introduction of bcl 2 gene into human small cell lung cancer cells conferred resistance to mitomycin C and irinotecan. DNA fragmentation was reduced in these cells. These results indicate apoptosis is one of the mechanisms of cell death caused by some antineoplastic drugs. Investigations are ongoing to elucidate the contribution of the Bcl 2 family proteins to antineoplastic drug induced apoptosis. Wild type p53-transfected cancer cells were sensitive to anticancer drugs. On the other hand, p53-depleted cells were reported to be more sensitive to taxanes than p53-proficient cells. Introduction of Rb gene and p16-gene enhanced cytotoxicity of taxanes and topoisomerase I inhibitors, respectively. In clinical studies, patients of non small cell lung cancer with high expression of Bcl-2 were reported to show longer survival than patients with lower expression. However, this result may be confusing because Bcl-2 reduced the efficacy of antineoplastic drugs. Further evaluation is required to determine the cellular proteins serving as markers for treatment efficacy or prognosis.