The CML-specific Philadelphia (Ph1) chromosome is relatively common cytogenetic abnormality of ALL, which has been shown 20% of adult ALL and 5% of child ALL. We analysed here the 12 patients of Ph1-positive ALL, aged 35 to 69-years old, who were experienced in our hospital for latest eight years. In comparison with Ph1-negative ALL, these 12 patients were elder and showed high peripheral and bone marrow leukemic cell counts. Of these, seven patients had 100% Ph1 abnormality in the bone marrow and another five patients showed mosaic marrow patterns of Ph1 and normal chromosomes. Remissioned eight cases had no more Ph1 abnormalities in their bone marrows. Our Ph1-positive ALL revealed B-cell lineage leukemia, since their surface phenotype were Ia+ and CD10+ and they have rearranged immunoglobulin JH genes. Four out of these nine patients had such gene rearrangement in the 5.8kb bcr (major BCR: M-BCR) as CML's patient had. Eight out of twelve Ph1-positive ALL patients (66.7%) achieved complete remission, but the prognosis was so bad since they had shorter remission duration (median 6.7 mos) and survival months (median 11.9 mos) than those of Ph1-negatives.

Clinical, immunologic, cytogenetic and molecular studies were performed on 9 patients with childhood Ph1 positive acute leukemia. FAB-L1 was found in 2 patients, L2 in 5 patients, and M1 and M2 in each patient. Six patients were older than 10 years old, and white blood cell count of 5 patients was more than 10(5)/microliters. All but one patient have died within 18 months. Immunologic analysis revealed that leukemic cells from all patients expressed lymphoid antigens CD10 and CD19, and myeloid antigen, CD13, was expressed on leukemic cells from 3 patients initially, and from 6 patients after short term in vitro culture without stimulation. bcr rearrangements were not observed in 3 patients tested. RNA analysis showed that 5 patients expressed P190bcr-abl pattern and one patient expressed P210bcr-abl pattern using polymerase chain reaction study. We conclude that Ph1 positive acute leukemia had a poor prognosis and differentiate into both myeloid and lymphoid lineages as well as chronic myelogenous leukemia (CML), and that this disease could not be possibly distinguished from CML by use of the molecular studies.

Molecular analysis and blast colony assay were performed on patients with Philadelphia chromosome-positive acute leukemia (Ph+ AL). All patients were diagnosed as ALL- L2 and 3 were My+ ALL. Nine of the 12 cases had rearranged immunoglobulin heavy chain, and 4 had rearranged TCR beta. TCR gamma rearrangement was noted in 3 patients, and TCR delta involvement was also noted in 6 patients. Clonal culture analysis revealed that leukemia cells of M-BCR rearranged Ph+ AL cases had a potential to proliferate by myelopoietic CSFs and ILs, however, M-BCR nonrearranged Ph+ AL did not. These results indicate biological and immunogenotypic heterogeneity of Ph+ AL.

We studied breakpoints within the first intron of the BCR gene in 28 Philadelphia (Ph1)-positive acute leukemias (AL) and one Ph1-positive chronic myelogenous leukemia (CML), which lacked rearrangement of major breakpoint cluster region. With a series of genomic probes from this intronic region, we have detected chromosomal breaks in 19 of 28 patients with Ph1-positive AL and one patient with CML. Breakpoints were all located within 30 kb region at the 3' portion of the intron, same as in the previously reported cases. Breakpoints in our cases were not limited within "bcr-2" or "bcr-3", proposed by Chen, et al., but, occurred within or near Alu sequences. Our findings suggest that breakpoints are not randomly distributed throughout this intron, but, there may be some specific sequences that facilitate the process of chromosomal translocation.

Cytogenetic and molecular aspects of Ph-positive leukemia are described in comparison with those of Ph-positive CML. Chromosomal characteristics of Ph+AL are; 1) mixture of a normal karyotype at diagnosis, 2) frequent combination with +Ph, +21, +6, +8, or -7, 3) recovery of a normal karyotype at remission. Additional chromosome changes at myeloid blast crisis (BC) of CML are characterized by +Ph, i(17q), +8, or +19. Meanwhile, lymphoid BC exhibits +Ph, +21, but not i(17q) or +19. There seems no cytogenetic difference between Ph+AL and lymphoid BC of CML, but i(17q) may be specific for CML BC. Eight patients with Ph+AL were studied with pulsed-field gel electrophoresis (PFGE) to examine the break site within ABL and BCR genes. One case had a M-BCR rearrangement and the remainder a rearrangement upstream of M-BCR. Minor-BCR rearrangement occurs seldom in CML but is detected in approximately a half of the reported cases of Ph+AL. ABL was rearranged within 1st or 2nd intron in all 8 cases. ABL breakpoints appear randomly distributed between exons 1b and 2 in both Ph+AL and CML.

To investigate the roll of c-myc protooncogene in human bladder cancer, c-myc gene was transfected into T-24 human bladder cancer cells and the changes of cell characteristics were studied. C-myc gene transfection was performed, using the electroporation method described previously (J.J. Urology, 80, 1989). After electroporation, c-myc gene was transfected and neo cells were cloned in a neomycin containing medium. One typical cloned cell (myc-cl3) was obtained. And this cell clone was shown to contain more than 3 extra-copies of c-myc gene by Southern blotting analysis. Morphology and growth speed of the myc-cl3 cells were not significantly different from those of original T-24 cells. However, they easily made overlapped cell-layers in the confluent growth phase. In the soft-agarose semi-solid medium, myc-cl3 cells formed about 35 times more numerous colonies than T-24 cells. Myc-cl3 cells also formed tumors on nude-mice at a significantly higher rate than T-24 cells did. These results suggest that c-myc gene plays a key roll in clonal growth and tumor formation in human bladder cancer.

We demonstrated the clinical effectiveness of combination therapy with Busulfan and Bestatin in 13 patients with Philadelphia chromosome positive chronic myelogenous leukemia, including 9 in chronic phase and 4 in acute phase. Sequential cytogenetic studies and molecular analysis of the breakpoint cluster regions were performed every 2 months. Complete hematologic remissions were observed in all 13 patients. Five minor cytogenetic responses, 2 partial cytogenetic remissions and 2 complete cytogenetic remissions were observed. All of the 4 patients in early chronic phase achieved cytogenetic remission and in 3 of them there was a complete disappearance of rearranged restrictive fragments of the bcr gene. In contrast, 4 of the 5 patients in late chronic phase failed to achieve cytogenetic remission and only 1 achieved minor cytogenetic response, suggesting excellent effects, particularly in patients with early disease. It is clear from this pilot study that combining Bestatin with Busulfan is an effective way to reduce or eliminate Ph1 positive clone and to maintain this status.

A 75-year-old man was admitted to our hospital because of hepatosplenomegaly, generalized lymphadenopathy and lymphocytosis in February, 1989. The leukocyte counts were 93,200/microliters with 95% small lymphocytes which expressed surface membrane immunoglobulin (SmIg) M, D and kappa. Histological finding of the cervical lymph node was diffuse small cell lymphoma. A diagnosis of chronic lymphocytic leukemia (CLL) was made. He was followed up without chemotherapy. In January, 1990, he was re-admitted because of progressively enlarged lymph nodes and increased white blood cell counts, up to 183,200/microliters with 98% lymphocytes. He was treated with vincristine, cyclophosphamide, prednisolone. The leukocyte counts decreased to 5,000/microliters and lymph node swelling decreased in size. In April, 1990, generalized lymphadenopathy re-appeared. The biopsied lymph node specimen showed diffuse large cell non-Hodgkin lymphoma (NHL-DL). The lymph node cells were found to express SmIgM and kappa. The diagnosis of Richter's syndrome was made. DNA analysis using Southern blot method revealed identical immunoglobulin heavy and kappa chain gene rearrangements in the two neoplasms. These findings suggest that the CLL cells and the NHL-DL cells originate from the same clone in this case.

In order to reveal the cytological nature of polyploid cells, the cell suspension of squamous cell carcinoma was separated into low density (1.050 greater than), intermediate density (1.050 to 1.088), and high density (1.988 greater than) fractions, by density gradient centrifugation. The DNA content of the tumor cells in each fraction were measured on the smear specimens prepared by Giemsa's staining and Feulgen's stainings. As the results, it was found that the cells showing high NC ratio and having high DNA content were observed in the high density fraction. However, there was no specific relationship between the nuclear contour index and density of the tumor cell.

Regarding to the pancreatic cancer, outcomes of the patients surgically treated have been poor. By using polymerase chain reaction (PCR), paraffin-embedded specimens of the pancreatic carcinoma were confined point mutation in Kirsten (K)-ras codon 12. Then, incidence and type of point mutation of this oncogene and correlative studies with stage, T or N factor of pancreatic cancer were analysed. Extremely high incidence of K-ras gene mutation was shown in present report. The highest mode of point mutation of K-ras oncogene was GGT to GAT coded aspartic acid. Cases without point mutation in K-ras codon 12 were significantly frequent in papillary adenocarcinoma than in tubular type. There were not correlative result among mutation types, stage and T factor of pancreatic cancer. Most patients with pancreatic cancer who survived more than 2 years have not shown mutation to aspartic acid. Four cases including two cases of mucin producing pancreatic cancer did not have point mutation in K-ras codon 12. Pathogenesis of mucin producing cancer can be distinguished from typical pancreatic cancer by detection of point mutation in K-ras codon 12 using PCR.

This report reviewed recent remarkable progresses on the cytomolecular mechanisms in colorectal carcinogenesis. Colorectal carcinoma is a good model for the study of multi-step progression, because we can obtain adenomatous polyps which are considered as a precancerous form. Furthermore, a familial syndrome, which is characterized by numerous adenomas of the colon, is available for linkage analysis. Recently, the p53 and DCC genes have been identified as candidate tumor suppressor genes on chromosome 17p and 18q respectively. In this paper, we present the multiple genetic alterations in colorectal carcinoma, including activation of K-ras gene and inactivation of tumor suppressor gene such as p53 and DCC genes as well as loss of heterozygosity and approach to the gene responsible for adenomatous polyposis coli by reverse genetics.

We found M-proteins with two peaks by agarose electrophoresis in the serum of a myeloma patient. The M-proteins were identified as both IgG 1-kappa type, and classified as IgG-F (fast mobility) and IgG-S (slow mobility). 1) The possibility that the two M-proteins were derived from the post translational differences of sugar moieties of the same IgG molecule was unlikely, because no migration changes were observed in IgG-F and IgG-S after the treatment with 4 different sugar enzymes. 2) Fab fractions of IgG-F and IgG-S were analyzed. After papain or pepsin digestion, western blotting with anti-Fab antiserum revealed that the Fab fraction of IgG-F and IgG-S had identical mobility by agarose electrophoresis. However the Fc fractions of IgG-F and IgG-S analyzed by the same procedures with anti-Fe antiserum, were different. 3) Anti-idiotype antiserum prepared in rabbits against IgG-S, or -F, and absorbed by normal IgG and normal human serum showed a fused precipitin line with IgG-F and IgG-S. These findings suggest that two M-proteins with both IgG 1 and kappa type, have the same VH and VL regions but have different constant regions of heavy chain. Since one copy of IgG 1 constant gene is found in each human haploid gene. It is speculated that the switching of the rearranged VDJ gene to constant region gene occurred not only between cis chromosome but also between trans chromosome.

A 43 year-old man admitted to our hospital because of fever and splenomegaly. Laboratory findings were as follows: Hb 9.5 g/dl, Plts 4.9 X 10(4)/microliter, LDH 2,348 IU/l. Bone marrow findings showed tumor cell 47% with or without phagocytosis. The tumor cells were stained positive lysozyme and alpha 1 antitrypsin. Cytogenetic study was 47, XY, -7, -8, +9, -11, -12, -19, -21, 3q+, 6p+, +6 markers. This case was diagnosed as malignant histiocytosis. Complete remission was achieved with CHOP-E chemotherapy. Remission has been maintained with repeated this therapy. Etoposide deserves a good evaluation in the treatment of malignant histiocytosis. Some cases of malignant histiocytosis with a t(2; 5) (p23; q35) translocation were often reported in Europe and America, while there was no specific chromosomal abnormalities with malignant histiocytosis in Japan.

We established new cell line designed TMCC-2.U, which suggested transformation to undifferentiated carcinoma, derived from endometrial clear cell carcinoma cell line (TMCC-2). The monolayer culture cell showed a pavement arrangement and spindle like shape. A rough-endoplasmic reticulum, mitochondria etc. are well developed. But cytoplasmic endocrine granulosa were so poorly, it suggests functional developments are poor. The TMCC-2.U cells were transplanted to nude mice which showed no typical pattern suggested undifferentiated carcinoma. Their chromosome number varied and the mode is 78. Marker chromosome were found frequency. Growth pattern and production of tumor marker are clearly differentiate from TMCC-2. As mensioned above, TMCC-2.U cell line will be very valuable in basic research on mechanism of transformation and effects of patient's serum on hystogenesity.

We established a new cell line (FU-UrC-1) derived from a human primary ureteral carcinoma xenografted in a nude mouse. This cell line exhibited epithelial characteristics and formed clusters in monolayer cultures. The cells were subcultured in vitro for more than 20 passages and had a doubling time of 53 hours. The modal number of chromosomes was 66. The cell line, which was xenografted again to nude mice, produced tumors essentially identical to the original tumor. Furthermore, the cultured cells expressed carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) that were secreted in the culture media. This cell line appears to provide a useful system for studying ureteral carcinoma in vivo and in vitro.

There are two main mechanisms of origin for complete hydatidiform mole; a) fertilization of an empty egg by a haploid sperm followed by duplication, and b) fertilization of such an egg by two haploid spermatozoa. It is widely accepted that, of all forms of pregnancy that had to choriocarcinoma, the risk associated with moles is by far the highest. Homozygous expression of a recessive mutation of moles has been assumed to associate with this propensity to malignancies. Alterations of several tumor suppressor genes together with activation of oncogenes are assumed to be necessary for choriocarcinogenesis. Genetic characteristics shown in the moles suggest that the former alterations are especially important to explore the multistep conversions to malignancies. Thus, individual chromosomes derived from normal cells were introduced into choriocarcinoma cells via microcell fusion. Evaluation of tumorigenicity in microcell hybrids suggested that chromosome #7 carried a putative tumor suppressor gene for choriocarcinoma. The gene imprinting or recessive mutation may be responsible for the inactivation of such a gene, being able to correspond with the high propensity to malignancy of moles.

NIH3T3 cells transfected with c-H-ras and/or c-myc genes were examined for differences in drug sensitivity. The two transfectants used were NIH3T3-nm-1 (nm-1), pT22-3-nm-2. They were transfected with c-myc, c-myc plus activated c-H-ras, respectively. The relative resistances (IC50 values of transfectants/those of NIH3T3 cells) to cisplatin, adriamycin, 4-hydroperoxycyclophosphamide, melphalan, and CPT-11 were 2.1, 1.6, 4.7, 4.9, 1.6, respectively for nm-1 and 1.6, 2.2, 3.3, 9.1 and 2.2, respectively for nm-2. These results strongly suggest that the expression of the c-myc gene plays a role for the acquisition of drug resistance. The c-myc gene is believed to provide us an important clue for determining the mechanism of drug resistance.

Inactivation of two tumor suppressor genes, RB and p53, is associated with tumor formation. To elucidate the molecular basis of the tumorigenesis of human osteosarcoma, structural and expressional alterations of these two genes were examined in five human osteosarcoma cell lines, two of which were from Japanese patients. In addition, I analyzed two adenovirus E1A-binding proteins, p107 and p300, putative "tumor suppressor gene products", which share similar properties with the RB protein in binding to the E1A oncoprotein. Detailed analyses of DNA, mRNA, and protein showed that (1) 3 lines including both Japanese lines lost the expression of the RB protein due to either the absence or the alteration of mRNA caused by DNA rearrangement, (2) abnormality of p53 gene was detected in all cell lines : 4 lines lost p53 expression due to either gene loss or the absence of mRNA, and one line expressed an abnormal form of the protein without detectable DNA and mRNA alterations and (3) no significant alteration of p107 or p300 was detected in all cell lines. These results further confirm that inactivating mutations of p53 and RB genes are deeply involved in the carcinogenesis of human osteosarcoma and suggest that p107 and p300 may not play a role in the tumorigenesis.

A human gastric cancer cell line, designated GCIY, was established from ascites of a patient with scirrhus type gastric cancer. Doubling time of this cell line was 55 hours. The karyotype indicated that these cells were human cells and the chromosome number varied widely, the mode being 57. GCIY cells were of moderate size and showed monolayer arrangement. They had a large nucleus and a clear nucleolus. Papanicolaou staining showed that they had the characteristics of adenomatous epithelia. They could be subcutaneously transplanted into nude mice, and they histologically resembled the original tumor. They were immunohistochemically recognized by anti-CA19-9 antibody and weakly by anti-CA125, CEA and alpha FP antibodies. Cultured medium also contained CA19-9, CA125, CEA and alpha FP.