Flow cytometric DNA analysis was done in 76 patients of testicular germ cell tumors (GCTs) experienced in our clinic for evaluating the clinical relevance of DNA index (DI), clarifying those biological features and shedding some insights in the pathogenesis of testicular GCTs. Histological type and its incidences were seminomas in 35 adults, nonseminomatous germ cell tumors (NSGCTs) in 24 adults and prepubertal germ cell tumors (P-GCT) in 17 boys (9 in yolk sac tumors and 8 in teratomas). Totally 190 samples of paraffin embedded materials (with a mean of 2.5 samples per case) were histologically reconfirmed and employed the flow cytometry analysis with some modification of the Hedley's technique for analyzing the DNA ploidy and DIs. Coefficient of variation (CV) were acceptably ranged from 3 to 10%. In 58 evaluable cases with adult GCTs, 57 (98%) were revealed as DNA aneuploid, while DNA diploid tumor were observed in only one case with NSGCT. On the other hand, all specimens showed DNA euploid in P-GCT; DNA diploid in all teratomas and 6 yolk sac tumors, DNA tetraploid in other 3 yolk sac tumors. DNA aneuploid pattern was not apparently detected in children. Even in one adult case with pure yolk sac tumor and two adults with mature teratomas, all specimens were classified as DNA aneuploid. These results support the present hypothesis that the pathogenesis of P-GCTs is different from that of adult GCTs. DIs in adult NSGCTs (median DI = 1.56) are significantly lower than those in adult pure seminomas (median DI = 1.85) (p < 0.01). Although there was no significant correlation among the DIs in NSGCTs and the clinical staging of Japanese Urological Association, the distribution of DIs in NSGCT patients of the advanced extent were lower than that of the other extents of NSGCTs on the basis of Indiana University staging system (p < 0.05). In general, it has been postulated that the higher DI is paralleled to the more malignant nature of neoplasms. Nevertheless, this study conversely suggested that the lower DI in adult testicular GCTs is apt to related with high malignant potential determined by histological type and clinical stage. DNA heterogeneity was observed only in 4 of 23 cases with adult NSGCTs (17%), but 3 of these 4 cases (75%) with DNA heterogeneity were assigned as the advanced extent. These data suggest that the lower DI and the presence of DNA heterogeneity may have prognostic relevance for adult NSGCTs.

A case of secondary myelodysplastic syndrome (MDS) following chemotherapy for lung cancer is reported. A 78-year-old man, with a smoking history of 20 cigarettes/day for 55 years, was incidentally, diagnosed as having stage IV squamous cell carcinoma of the lung in 1987 during admission for transurethral resection of bladder cancer. He received combination chemotherapy of mitomycin C, vincristin, and cisplatin for his lung cancer between July and September 1988. His clinical course remained almost stable until October 1989, when his blood count showed severe anemia and thrombocytopenia. He was diagnosed as having secondary MDS induced by cytotoxic agents used for the treatment of lung cancer, based on the dysplastic findings of precursor cells in the bone marrow and the chromosome abnormality of 51XY, +8, +9, +21, 3p-, 5q-, +2mar. He died of infection with the progression of MDS in March 1990.

Chromosomal changes plays an important role in malignant transformation. Generally, the process of karyotype instability with grows aneuploidy occurs in tumor. But it is very difficult to obtained Flow karyotype from tumor cells. There are many technical problem in the analysis of chromosomes aberration in tumor cells. Problem is technical procedure of isolation from metaphase chromosomes. It is important to choice of swelling buffer and treatment times. Such technic affect for Flow karyotype pattern. We try to obtained Flow karyotype from chinese hamster cell and V-79 cells and we reported a recent new techniques of cell preparation and chromosomes suspension.

C-myc protein plays an important role in the regulation of cell proliferation and differentiation. In this paper, c-myc protein and DNA were doubly stained and analysed simultaneously with flow cytometry (FCM). At first, three fixatives, ethanol, methanol and paraformaldehyde, were examined using HL-60 cells, among which 50% ethanol was found to be optimal for each staining. After fixation, the cells were stained with a monoclonal antibody against c-myc protein, followed by DNA staining with PI. Simultaneous analysis demonstrated that c-myc protein was constantly expressed through the cell cycle and that the protein amount at the G2 + M phases was 1.5 times higher than that at the G1 phase. Further, the differentiation study with TPA and RA revealed that the growth, S phase and also c-myc protein expression were suppressed during the differentiation.

Genetic alterations of various cancers have been clarified by recent development of molecular biology. Multiple genetic alterations occur through the development of cancer. Both activation of proto-oncogenes and inactivation of tumor suppressor genes are important for the development of cancer. Alterations of oncogenes such as K-ras, c-erbB-2/HER-2/neu and c-myc, and those of tumor suppressor genes such as p53, RB and DCC have been reported in ovarian cancer. Allelic losses of the specific chromosomes, which suggest the existence of tumor suppressor genes on those chromosomes, also have been reported in ovarian cancer. Further studies on genetic alterations of ovarian cancer will clarify the mechanisms for the development of ovarian cancer and also will develop new methods for prevention, diagnosis and treatment in clinical.

Tumor invasion and metastasis involve the interaction between tumor cells and basement membrane, which is mediated in part by laminin receptors. To search for tumor-associated-genes which can be used as new markers in colon cancers with known poor prognosis, cDNA libraries from a colon cancer cell line and colonic tissues were constructed and screened. We selected a cDNA clone which encodes 32-kD laminin-binding protein (LBP-32), and showed increased mRNA expression of LBP-32 in colon carcinoma. This mRNA expression was also correlated with clinical tumor staging. Furthermore, to investigate the role of LBP-32 in cancer invasion and metastasis, cell adhesion assays and in vitro invasion assays were performed, using anti-sense RNA of LBP-32 to block the synthesis of LBP-32. Results showed that anti-sense RNA of LBP-32 inhibits tumor cell attachment and invasiveness in vitro in transfectants of a colon cancer cell line. These data suggest that LBP-32 may play an important role in colon cancer progression, and that LBP-32 may be used as a marker of biological aggressiveness. These findings also imply that laminin receptors may provide a target for novel therapeutic strategies: modulating LBP-32 expression by anti-sense RNA or monoclonal antibodies may have clinical application in colorectal cancer therapy.

Allelic loss and mutation of the p53 gene are common events of esophageal, gastric and colorectal cancers occurring from an early stage. Moreover, p53 mutation takes place in intestinal metaplasia and adenoma of the stomach. p53 mutation spectra differ among esophageal, gastric and colorectal cancers suggesting exposure to different endogenous and exogenous mutagens. The in vivo and in vitro results indicate that the clonal expansion of p53 mutant cell may be associated with tumor progression. Although wild type p53 transfection technique may provide for cancer therapy, there is a rather serious problem about it.

I. Serial histopathologic study of esophageal squamous cell carcinoma. A review of 335 cases of squamous cell carcinoma disclosed 55 cases (16.4%) with glandular components in addition to the ordinary component of squamous cell carcinoma, suggesting that this type of esophageal tumor had originated not only from the covering squamous epithelium but from esophageal gland or ductal epithelium. Intra-epithelial carcinoma concomitant with squamous cell carcinoma was seen in 95 cases (28.4%). The incidences of coexistence in such lesion were higher in the groups of early stage esophageal cancer. These observations support the concept of field carcinogenesis of esophageal cancer. II. Histopathologic study of squamous epithelial dysplasia. Among 91 cases without preoperative treatment, there were 40 dysplastic lesions in 23 cases (25.3%). The continuity of dysplasia to the carcinoma was 48.3% and it was often encountered in severe dysplasia rather than in moderate or mild dysplasia, suggesting some relationship between the severity of dysplasia and carcinoma. III. Immunohistochemical study of EGF and c-myc. Among 27 cases, EGF was positive in 10 (37.0%). c-myc was positive in 18 (66.7%) not only cancer but normal epithelium suggesting that some change of products of oncogene occurred also in the normal epithelium of the patients of esophageal cancer.

It is important to distinguish a precancerous lesion and hepatocellular carcinoma (HCC) with diploidy or aneuploidy nuclear DNA pattern, not only in clinical cases but also in experimental carcinogenesis models. Using liver perfusion technique, we detected early HCC from persistent hyperplastic nodules (HN) which were induced in Wistar rats by intermittent 5-6 months administration of 2-acetylaminofluorene. This investigation was undertaken to assess both promotive and progressive effects of liver regeneration following partial hepatectomy (PH). Results are as follows: 1) Isolated hepatocytes of precancerous HN, which were transplanted into the spleen, didn't develop to HCC by 2 months after 70% PH of host liver. 2) Diced tissues of HCC, which were transplanted into the liver via portal vein, grew many metastasis in 10/10 by 7 weeks after PH, while 5/19 in control. 3) Nuclear DNA patterns of early HN-late HCC in rat liver were diploidy at the rate of more than 90% each. But it changed to aneuploidy, when inoculation of HCC for one month was repeated 7 times in the spleen.

Uterus origin squamous cell carcinoma cell line LK-52 was established from surgical specimen of lung metastatic nodule. LK-52 produce poorly differentiated squamous carcinoma in nude mouse, and doubling time in vitro was 38 hours. Chromosome analysis show various abnormality and main mode number was 67 and 68. LK-52 shed active procoagulant substance into culture medium. The culture medium of LK-52 shortening recalcification time of normal human plasma and factor VII or factor IX deficient plasma but not factor X deficient plasma. Procoagulant activity of LK-52 product may induced with direct activation of coagulant factor X. Procoagulant activity which produced by cancer cell may play a important roll in the unbalanced haemostasis of cancer patient.

A human adenocarcinoma cell line designated as GAC-1, was established from ascites of the 56-year old male patient with rapidly progressive gastric cancer. The doubling time was about 18.5 hours in vitro, and cell cycle analysis using flow cytometry showed marked increase of S phase (46.1%). Immunohistochemical demonstration of GAC-1 cells revealed positive staining of TGF-alpha, EGF, EGF-R, FN-R, laminin and negative staining of fibronectin. Histogram of them indicated aneuploidy with modal number 57 and they formed tumors in nude mice.

The authors report a rare case of chronic myelogenous leukemia (CML) in which the Ph1 clone disappeared after remission induction of lymphoid crisis. A 58-year-old man was admitted to our hospital because of fever in July 1988. The white cell count was elevated. Bone marrow aspirate showed hypercellularity with myeloid hyperplasia. In the chromosomal analysis, Ph1 chromosomes were detected in 100% of bone marrow cells analysed. Diagnosis of CML was made and treatment was initiated with recombinant interferon-alpha 2a. Hematological remission without cytogenetic improvement was achieved. In March 1990 he developed lymphoid crisis with proliferation of CD10-positive cells. The chromosomal analysis revealed additional abnormalities including, 45, X, -Y, t(9;22) (q34;q11), +1, -8. With vincristine 0.6 mgX4, pirarubicin 15 mgX4, dexamethasone 40 mgX4 therapy complete remission was obtained. In December 1990 the Ph1 positive clone completely disappeared judging from normal karyotypes in the chromosomal analysis and the disappearance of M-bcr gene rearrangement.

The highly tumorigenic rat hepatocellular carcinoma cell line cKDH-8-cl-11 was xenogenized by transfection with an envelope (FV-env) gene derived from Friend murine leukemia virus. The transfected tumor cells, expressing the FV-env gene product on the cell surface, were injected into normal and immunosuppressed (irradiated) syngeneic rats. All the irradiated rats developed tumors at the injection site. Thirteen out of fifteen normal rats rejected the xenogenized cells and acquired tumor transplantation resistance to the parent (nontransfected) cell line. The tumor cells that grew in normal rats failed to express th FV-env gene product during growth in vivo, but resumed expression during in vitro primary culture. These results suggest that the FV-engine product, when expressed on tumor cell surfaces, displays biological characteristics which are immunologically recognized by normal rats and induces tumor rejection. Moreover the results show that the FV-env gene product is a good candidate for the xenogenization of tumor cells.

Specific DNA probes for genes encoding immunoglobulins (Ig) and the T cell receptor (TCR) are useful diagnostic tools in lymphoproliferative disorders. Gene rearrangement analysis was carried out in 2 cases of conjunctival lymphoid lesions. A 36-year-old man (case 1) had a 1-year history of left conjunctival tumor. A biopsy was performed and histopathological findings showed diffuse proliferation of small lymphocytes, but monoclonality was not revealed by immunophenotypic analysis. A right conjunctival lesion developed and five months later a biopsy of the left conjunctiva was performed again. A frozen sample was analyzed and immunoglobulin heavy chain gene rearrangement was found. A 53-year-old woman (case 2) had a 6-month history of bilateral conjunctival tumor. The first biopsy did not reveal monoclonality immunophenotypically. A second biopsy with a frozen specimen was analyzed and immunoglobulin heavy chain gene rearrangement was found. We diagnosed these two cases as B-cell lymphoma. We discuss the clinical value of gene rearrangement analysis as a diagnostic method for lymphoproliferative disorders.

Recent studies of erbB-2 expression have shown that the erbB-2 oncoprotein correlated with poor prognosis of patients with breast cancer. Surgical treatment of the bile duct carcinoma is currently unsatisfactory. To evaluate erbB-2 oncoprotein as a marker of prognosis, we analyzed 68 bile duct carcinomas immunohistologically, using monoclonal antibody against erbB-2 oncoprotein, as well as clinicopathological data and outcome. High incidence of expression of erbB-2 oncoprotein was shown in bile duct carcinoma. Positive rates of erbB-2 oncoprotein correlated with stage of bile duct carcinoma. Survival of patients with erbB-2 expression cancer was shorter than those without erbB-2 expression cancer and erbB-2 expression has a prognostic value in bile duct carcinoma.