We have experienced and treated seven patients of pre-B cell leukemia in childhood. Clinical, cytological and ultrastructural characteristics of them were studied. Most of them had higher counts of white blood cells, hepatosplenomegaly, high value of lactic dehydrogenase and various karyotype abnormalities at onset. The chromosomal translocation t (1; 19) that is supposed to be specific to pre-B cell ALL was found in four of seven of our cases. In the seven patients, survival was studied in comparison to that of 27 common ALL patients at our hospital that are common in childhood acute leukemia. Although no difference in remission duration and survival time between pre-B cell ALL patients and common ALL group, there have been seen the tendency that remission and survival were of shorter duration for patients with pre-B cell ALL.

A 7-year-old girl with an acute leukemia was reported whose blasts showed conversion from a T-lymphoid to a myeloid phenotype. At the onset of the disease, the blasts were negative for peroxidase and displayed FAB L1 morphology. Surface marker analysis revealed only CD7 antigen. Although complete remission was achieved, an extramedullary relapse was identified as having a several subcutaneous tumors 15 months later. Tumor cells showed the same marker expression as that of the blasts at the onset. After short term culture without an addition of any differentiation stimulators, the blast cells expressed CD2, CD3, CD4, CD8, and CD25 antigens. The karyotype was 46, XX, t(12; 21) (p11; q22). The intensive chemotherapy and radiation therapy were carried out, however, a hematological relapse occurred 12 months later. At this time, the blasts were strongly positive for peroxidase and expressed HLA-DR and CD33 antigens with disappearance of the CD7 antigen. Chromosome analysis revealed the additional abnormalities (del (7) (p15), -17, +der (17) t (17;?) (p13;?].

A microspectrophotometric analysis of the DNA content has been performed on 9 advanced recurrent gastric cancer patients with measurable lesions, who has either been treated by CDDP alone or with other chemotherapeutics during a three-year period since, 1984. Histograms of the DNA content were classified into four ploidy patterns. All of the 4 responder cases (CR, PR, MR) showed type, IV, although only one of 5 non-responder cases revealed the same typing. In one of the two CR cases the DNA ploidy pattern, which was examined before and after the therapy, changed from type IV to type II. Thus it appears that an analysis of the DNA content may be useful in evaluating the effectiveness of different chemotherapies.

Human germ cell tumors are an excellent model for investigating the mechanism of human early embryogenesis as well as cellular differentiation. Three human EC cell lines, NCR-G 2, 3 and 4 were newly established from testicular mixed embryonal carcinomas in vitro, G3 and G4 cells were capable of somatic cell differentiation. The G3 cells demonstrated the most noticeable antigenetical changes with the administration of retinoic acid. SSEA-1 appeared on some cells whereas expression of HLA-A, B, C as well as 2H2, 2D7 and 5D4 antigens tended to be reduced in G3 cell line. 2H2, 2D7 and 5D4 antigens which we recently produced were immature human EC specific cell surface antigens, defined by mouse monoclonal antibodies, obtained immunization with G2 cells. The production of hCG, high molecular weight cytokeratin and intercellular matrices such as type IV collagen and laminin were inducible in G3 cells. Thus, G3 cells are thought to be one of the most pluripotent human EC cells. These findings clearly indicate that the EC cell lines we established provide an opportunity to study differentiation mechanism of human germ cell tumors and also human somatic cells.

In February 1986, a 68-year-old woman was diagnosed as having acute myeloblastic leukemia (FAB-M1). At the time of diagnosis, 86.0% of the bone marrow cells were myeloblastoid, and 15% of these myeloblastoid cells were positive to myeloperoxidase. Surface marker analysis by flow cytometry disclosed granulocyte-associated antigen (MY7) and also lymphocyte-associated antigen (CALLA) on the leukemic cells. Chromosomal banding studies of bone marrow cells revealed trisomy 11 in 6 of 19 metaphases examined and normal karyotype in the others. Complete remission was attained after intensive combination chemotherapy, and has remained for 38 months. Only 19 patients with trisomy 11-associated acute nonlymphocytic leukemia (ANLL) including the present case have been reported. Morphologic analyses have revealed that the frequency of FAB-M1 is high. However, except for the present case, surface marker findings were apparent in only one M5a patient, in whom monocyte-macrophage-associated antigen was detected. Accordingly, careful surface marker studies will be needed to clarify the frequency of acute mixed lineage leukemia in such patients.

We cultured an aspiration fluid of the sternal bone marrow of the patient having adrenal neuroblastoma and established a neuroblastoma cell line (HSNB). The HSNB line has the following biological properties. 1. They are small round in shape and proliferate in flotation while forming cell aggregate, and often they attach the bottom of plastic dish and process the nerve-like fibers. A rough-endoplasmic reticulum are poorly developed, however, a lot of free ribosomes are scattered in the cytoplasm. In the peripheral area of the cells, small spherical secretory granules (60-140 nm in diameter) are existed. One characteristic of this cell is existence of microtubules in the cell-projections. 2. They show a stable growth and the doubling time is about 50 hours. 3. Their chromosome number varied widely and the mode is 46. The double minute chromosomes were present in 50% of cells. 4. When they are transplanted in the cheek pouch of hamster, they produced the neuroblastoma. 5. They produce neuron specific enolase. 6. N-myc gene was amplified ca 250 folds.

The developmental potential of various teratocarcinomas of different origins was examined by making chimeras with mouse embryos. Of 7 teratocarcinoma lines examined, only 2 were found to contain stem cells having the ability to form live-born chimeras; one was experimentally induced OTTBALB-2 and the other was spontaneously occurring STT-3. The latter showed remarkable ability to colonize mid-gestational fetuses and adults. This result not only demonstrates that EC cells of male primordial germ cells have the ability to form viable chimeras but also suggest that this kind of tumor is a useful source of EC cells to make chimeras. The advantage of using pluripotent cell lines such as EC cells or embryonic stem (ES) cells as a vector for introducing foreign genes into mouse embryos was discussed in relation to the study on gene function and on gene regulation during development.

The effect of various anticancer drugs on alpha-fetoprotein (AFP) secretion and some other properties of human hepatoma cells was investigated in vitro with the following results. (1) There was a high correlation between AFP secretion and cell number after treatment of human hepatoma cells with anticancer drugs and the amounts of AFP secreted per 10(4) cells per 72 hours (AFP-secreting capacity) were not affected within therapeutically achievable concentrations (TAC). (2) The AFP-secreting capacity was affected with some exceptions in the cells treated with higher concentration of drugs than TAC. Furthermore, chromosomal and morphological aberrations in the similarly treated cells, were also observed, suggesting the relationship between the change of AFP-producing capacity and that of some other properties.

We previously identified the promoter region for hCG beta gene with mouse adrenal cell line Y1. However, it was not know why this cell line, which does not express hCG beta eutopically, expresses hCG beta when the gene is transfected exogenously. Therefore, to confirm the results obtained in Y1 cell experiments, it was important to establish a system in which the activity of hCG beta transcriptional regulatory elements can be tested in cells which express hCG beta eutopically. Here we report success in establishing such a system, using constructs containing hCG beta upstream elements in front of the promotorless chloramphenicol acetyl transferase (CAT) gene and also carrying the neomycin resistance gene. These constructs were transfected stably into JAr choriocarcinoma cells which express hCG beta eutopically. Then the expression of CAT in each transfected cell line was examined. The results of this experiment confirmed the presence of basic promoter elements within 78bp of the transcriptional initiation site, which we had suggested previously as a result of Y1 experiments. The results also suggest the existence of additional transcriptional regulatory elements further upstream. It was also confirmed that gene 7 is an inactive gene.

We report two leukemia patients with double minutes (DMs) chromosomes. Both patients were diagnosed as having acute myelocytic leukemia (AML) FAB M2. Cytogenetic analysis showed normal chromosome karyotype with 1-53 DMs chromosomes in the first patient, and complex chromosome aberrations including deletion of chromosome 8 at 8q24 region and 1-84 DMs chromosomes in the second patient who had a history of extensive radiotherapy for laryngeal cancer 8 years prior to the development of leukemia. Analysis of DNA from the two patients revealed that oncogene of c-myc was amplified about 5 to 10 folds in the leukemic cells. The other fourteen oncogene of c-myc was c-myb, c-abl and N-myc, showed no increases of gene content. Furthermore, a transforming gene, N-ras was detected in the first patient by in vivo selection assay method. This is the second report on AML patients with c-myc gene amplification and DMs chromosomes.

A 39-year-old woman was admitted to our hospital complaining of hemosputum and right neck swelling. Pharyngography, neck CT scan and laryngoscopy revealed moderately differentiated squamous cell carcinoma of the right pyriform sinus. After series of examinations, it was found that she had pancytopenia, hypoplastic bone marrow, hyperpigmentation of the skin, cardiac anomaly, small stature, hypogonadism, chromosomal aberrations and consanguinity in her parents. These findings suggested that she was the congenital aplastic anemia, that is Fanconi's anemia, variant form. Although pepleomycin and corticosteroids were administrated for the treatment of squamous cell carcinoma and aplastic anemia, she died of cardiovascular shock due to massive hemorrhage. Flow cytometric analysis of squamous cell carcinoma showed an unusual aneuploidy DNA histogram. This is the first report on Fanconi's anemia with squamous cell carcinoma in Japan. It is said that chromosomal aberrations and impairment of DNA cross-links repair may play an important role developing of malignancy in Fanconi's anemia.

We report a patient with acute nonlymphocytic leukemia (FAB classification: M 2) with trisomy 4, which is the first case in our country. A 42-year-old man was admitted to our hospital because of fever and general fatigue in May, 1988. His WBC count was 8,100/microliters with 90% of leukemic cells and bone marrow smear showed 76.1% leukemic cells. The chromosomal analysis of marrow cells by G-banding revealed 47, XY, +4. In spite of administration of chemotherapy complete remission was not obtained, and he died of septic shock and severe liver damage 4 months after making the diagnosis. Chromosomal abnormality of trisomy 4 has been reported to be associated with predominantly either M 4 or M 2, and to be less than 0.1% of incidence in ANLL, according to the Second MIC Cooperative Study Group. It is suggested that trisomy 4 may be caused by exposure to some environmental factors such as toxic substances, since this chromosomal abnormality has been reported in the last 10 to 20 years.

Ras genes (H-,K-,N-ras) are converted to active oncogenes by point mutations occurring in either codon 12, 13 or 61. We analyzed 19 pancreatic tumors (formalin fixed paraffin embedded tissue) of these codons by a method to directly sequence nucleotides around codons 12/13 and 61 of the three ras genes, using polymerase chain reaction and direct sequencing method. Of 19 pancreatic tumors, all 17 duct cell carcinomas involving 2 mucous producing pancreatic cancers had point mutations of the K-ras codon 12, but 2 islet cell tumors had ano point mutation around codons 12, 13, 61 of the three ras genes. Extremely high incidence of ras gene mutation may be relevant to certain pathogenesis of pancreatic cancers.

Recent advances of cytogenetics in human hematological malignancies and solid tumors were reviewed. In leukemia and lymphoma, many non-random chromosome aberrations have been found in the last decade. Further specific chromosome aberrations, which existed usually in less than five percent of acute leukemia cases, were recently found, including t (1 ; 3) in MDS or AML M4, +der (1) t (1 ; 7) in MDS, t (1 ; 11) in AML M4 or M5 and +4 in AML M2 or M4. Recurrent chromosome deletions of 17p-, 9q- and 2p- were also found as secondary aberrations in association with tumor development. Accumulation of the data from variant translocation for the 9 ; 22, 8 ; 21 and 15 ; 17 gave us important informations of critical sites of the chromosome in leukemia development. A new trial for the simultaneous analysis of morphology, immunologic phenotype and karyotype on the same metaphase clearly demonstrated stem cell origin of leukemia in some cases, specializing the affected cell lineage. Progress in non-radioactive in situ hybridization techniques now allows approaches to the recognition of particular chromosome abnormality in metaphase and also in interphase cell by means of specific repetitive probes for each chromosome. Though a hypothesis that fragile sites may act as factors predisposing to chromosomal rearrangements have attracted attention in past few years, recent results appear to be conflicting without any direct proof. Cytogenetic studies in solid tumor have been remarkably progressed with advances of methodology. Recurrent chromosome aberrations in solid tumor were found, such as t (X ; 18) in synovial sarcoma, t (12 ; 16) in liposarcoma, and i (12p) in seminoma. Studies on the correlation between specific chromosome changes and histologic subtypes resulted in an useful orientation to the diagnosis and the therapy. Advance in cytogenetics may serve as new concepts for patho-physiology of malignant tumors and contribute to further understandings of molecular genetics in human solid tumors.

DNA damaging agents such as nitrosoureas are widely used for the treatment of malignant gliomas. Therefore, quantitative measurement of DNA damages induced by antineoplastic drugs is useful to judge the efficacy of the drug and understand the pharmacological action of the drug. We have utilized in situ nick translation method to demonstrate "nicks" in DNA of glioma cells treated by various antineoplastic agents. Exponentially growing rat 9 L glioma cells (4 x 10(4] were seeded in the chamber slide. After fourty eight hours, the medium was changed to that containing various concentration of the drug (ACNU, cis-DDP, BLM, ADM and VP-16) and the cell was treated for 1 hour. Then, the cell was fixed for 10 minutes in methanol-acetic acid (v/v 3:1). Following fixation, the cell was incubated in the nick translation mixture containing E. coli DNA polymerase I, 3H-TTP, and 4 dNTP's (ATP, GTP, CTP, CTP and TTP) for 10 minutes at room temperature. The slide was dipped in the autoradiographic emulsion, exposed for 4 days at 4 degrees C, and then developed, the number of the silver grains over nuclei was counted under the microscope. For comparison of the effect of the drug to glioma cells, IC50 (inhibitory concentration of the drug for 50% cell kill) of each drug was determined by treating the cell for 48 hours at the various concentration of the drug. Small number of the silver grains was noted in cells with no treatment. Over IC50 as the concentration of the drug increased, the number of the nick increased in cells treated with bleomycin or adriamycin which are known to produce single strand breaks in DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Twenty-five cases of gastrointestinal leiomyosarcomas were subjected to clinico-pathological studies in an attempt to correlate the prognosis with tumor size, mitosis, cellularity and DNA ploidy pattern. Leiomyosarcomas greater than 5cm in diameter had poorer prognosis. Those with mitotic index larger than 3.0/mm2, cellularity larger than 3.0/0.0004mm2, DNA aneuploidy had poorer prognosis. By multiple regression analysis, mitotic index was useful for the prediction of tumor recurrence in earlier postoperative period, but cellularity, tumor size were useful for the prediction of recurrence in later postoperative period. As for the type of tumor recurrence, hematogenous metastasis was observed in 7 cases, peritoneal dissemination in 2 and local recurrence in 4. Four cases with local recurrence had all hematogenous metastasis. Two cases of gastric leiomyosarcomas developing local recurrence were greater than 10cm in diameter and gastric local resection was done for them. One case was diagnosed benign leiomyoma in the rectum histologically, but after trans-anal tumor resection local recurrence and metastasis to the lung occurred. We must pay attention to the surgical margin and the surface of tumor dissection during of the tumor, especially in larger tumors. Aggressive surgical resection is efficient for the treatment of recurrent tumors.

Flow cytometric cell cycle analysis using a monoclonal antibody to Bromodeoxyuridine was performed on 117 gastric cancers. Dissociated cells were stained with indirect immunostaining for BrdU (FITC-BrdU) and propidium iodide. Bivariate BrdU/DNA distribution were obtained using EPICS-C flow cytometry. Tumor ploidy was classified as follows, D1: diploidy, D2: diploidy + aneuploidy, A1: single aneuploidy, A2: multiple aneuploidies. The ploidies of noncancerous gastric mucosa were all diploidy and that of S-phase fraction(SPF) were ranged from 0.0% to 1.2%. In 117 gastric cancers, aneuploidy was observed in 80 cases, D2: 38, A1: 15, A2: 27. SPF was higher in aneuploidy (14.5 +/- 5.1%) than diploidy (6.1 +/- 5.1%). Significant differences were observed between that of D1 (6.1 +/- 2.5) and D2 (13.6 +/- 6.2), A1 (12.5 +/- 4.5), A2 (16.0 +/- 3.2), and A1 and A2 (p less than 0.01). The patients with aneuploid tumors had poor prognosis than diploid tumors (p less than 0.05). In concerned with DNA ploidy pattern, the patients with A2 had most poor prognosis than the other (p less than 0.05). Furthermore, the patients with SPF over 10% had poor prognosis than that of SPF below 10%. These results indicated that DNA ploidy pattern and SPF may possibly be useful prognostic markers for gastric cancers.

A 31 year-old male who was treated with radiation under the diagnosis of malignant lymphoma was admitted to our hospital because of systemic erythema and tumor of bilateral upper arms in October, 1987. Leucocyte count of peripheral blood showed 4,400/microliters with 36% leukemic cells and bone marrow was hypercellular with 85.6% leukemic cells. Leukemic cells were negative for peroxidase reaction and lineage specific monoclonal antibodies such as CD3, CD4, CD8, CD10, CD19 and CD20. T cell receptor (TCR) delta gene was rearranged but TCR beta, TCR gamma and immunoglobulin (Ig) genes were in germline configuration. He was treated with combination regimen of doxorubicin, vindesine, prednisolone and L-asparaginase, and complete remission was obtained. These observations suggest that TCR delta gene rearrangement is useful for determination of clonality in cases without rearrangements of the other TCR and Ig genes.

The karyotype of 124 children with acute lymphoblastic leukemia (ALL), who were diagnosed on the basis of the FAB classification, were studied according to numerical, structural abnormalities and the presence or absence of translocations. Clonal chromosome abnormalities were found in 85 (68.5%) of the 124 patients. Firstly, chromosome abnormalities were classified according to modal number. Twenty-four patients with ALL had hyperdiploidy (51-66 chromosomes). They had favorable prognostic factors, including low leukocyte count, age between 2 and 10 years and a low level of serum LDH. They had the most favorable outcome. Thirty-nine patients with normal karyotype had a relatively favorable outcome. In contrast 8 patients with near hyperdiploidy (47, 48 chromosomes), 47 with pseudodiploidy, and 4 with hypodiploidy (45 chromosomes) had a poor outcome. Secondly, chromosome abnormalities were also classified according to specific structural abnormalities. The 1; 19 translocation, which is associated with the pre-B phenotype, was found in 13 patients. Their outcome was better than has hitherto been reported. The abnormality of the short arm of chromosome 12 was found in 11 patients, who had a good outcome. The chromosome abnormality of the breakpoint in the T-cell receptor gene locus, containing bands 14q11 and 7q35, was found in 7 patients. Four patients had mediastinal tumors and the T phenotype. Their outcome was intermediate. The 14q32 translocation was found in 8 patients. The 8; 14 translocation was closely associated with L3 (FAB) and the B phenotype, but another 14q32 translocation was not. Their outcome was poor. The 11q23 translocation was found in 4 Patients, who had null cell blasts. Three of the 4 had unfavorable prognostic factors, including both a high leukocyte count and age under one year. They had a poor outcome. The partial deletion of the long arm of chromosome 6 was found in 4 patients. The structural abnormality of the short arm of chromosome 9 was found in 4 patients with absence of T cell phenotype. Two patients had a mass. Prognosis of the patients with 6q- and 9p abnormalities was unclear because of the small number of cases. Thirdly, chromosome abnormalities were also classified according to the presence or absence of translocations. The chromosomal translocations, which have an adverse effect in ALL (p = 0.004), were ones of the strongest predictor of treatment outcome. This study demonstrated that chromosome modal number, specific structural abnormalities and the presence or absence of translocations were significantly correlated with clinical features and survival rates of Japanese childhood ALL.

A 26-year-old male was admitted to our hospital because of fever and leukocytosis. On admission, a white blood cell count was 28,300/microliters with 46.5% blast cells and 16.0% atypical monocytoid cells, a hemoglobin level 13.7 g/dl, and a platelet count 15.0 X 10(4)/microliters. Bone marrow contained 58.8% of peroxidase-negative blast cells. He was diagnosed as acute lymphoblastic leukemia (ALL L2) according to the FAB classification. Chromosome analysis revealed the marrow cells to contain 45, XY, -7, t(9; 22) (q34; q11). On surface marker analysis, the leukemic cells were positive for both lymphoid (CD10) and myeloid markers (CD13). Two color flow-cytometric analysis showed two distinct populations with CD10 and CD1 3, respectively. Rearrangements of both immunoglobulin heavy chain and T cell receptor beta-chain were observed. The "breakpoint cluster region" on chromosome 22 was not rearranged. On the basis of these findings, we thought this case being acute mixed leukemia. He was refractory to AdVP therapy and BHAC-DMP therapy. He is now under treatment with A-Triple-V therapy.