The knowledge of prognostic factors such as TNM, performance status, and sex is essential for predicting patient outcome and optimal trial design and analysis. Recent advances in cytogenetics and molecular biology have yielded new prognostic factors such as DNA ploidy, oncogenes and oncogene product. New prognostic factors can predict patient outcome and should be incorporated for the multivariate analysis of prognostic factors. They can provide a guideline for selecting special patient population suitable for adjuvant chemotherapy even in the early stage of lung cancer.

Recently, remarkable progress in molecular biology has enabled isolation of genes responsible for hereditary tumors such as retinoblastoma (RB), Wilms' tumor (WT), von Recklinghausen neurofibromatosis (NF 1), and familial adenomatous polyposis (FAP). Since patients with FAP develop multiple adenomatous polyps in the colon, some of which progress to colon cancer, isolation of the FAP gene allows us a rare opportunity to study genetic events underlying the well defined morphological changes during progression of colorectal tumors. In this report, we presented an approach called "positional cloning" which has become a powerful tool for identifying genes responsible for hereditary tumors, as well as characteristics of some of such genes.

We investigated the basic technology of cell culture conditions for production of useful substances such as cytokines, and related proteins produced by Namalwa cells. Namalwa cells, human B lymphoblastoid cells, were used for large scale production of alpha-interferon. Namalwa KJM-1, a subline of Namalwa cells, adapted to serum- and albumin-free medium, can grow at a high density above 1 x 10(7) cells/ml in suspension manner by the use of a perfusion culture system, Biofermenter, containing a cone-type cell-sedimentation column as cell separator. Several kinds of cytokine cDNA can be introduced and expressed in Namalwa KJM-1 cells. Some of these were produced in large quantities by use of a gene amplification method with dhfr, even through the Namalwa KJM-1 cells contained endogenous dhfr genes. For stable production of the target protein, Namalwa KJM-1 cells are very useful host cells, because they have no effective endogenous protease activity in the conditioned medium. Using Biofermenter with micro-silicone fibers and a dialysis system, the specific productivity of the target proteins was not depressed at a high cell density.

We have established 13 esophageal cancer cell lines capable of growing in a protein-free environment. The growth of these cells was not affected by conditioned medium, but the growth of NIH3T3 cells and human fibroblasts was stimulated by conditioned medium. On the other hand, conditioned medium inhibited the growth of human endothelial cells. Amplified int-2 oncogene correlated well with the growth of cells in a protein-free environment but the number of EGF receptors and growth effect of EGF did not relate to such growth. Esophageal cancer cells grow automatically, possibly involving mesenchymal cells via the paracrine system. This results in a poor prognosis in patients.

Expression of the Cytokine genes in human astroglial cell lineage was studied. Primers for 5 different human cytokine, TNF-alpha, -beta, IFN-gamma, G-CSF and GM-CSF, were used to analyze messenger RNA transcripts in 5 cultured human astrocytoma, one neuroblastoma cell lines and fresh brain specimens by polymerase chain reaction (PCR). Three out of 5 unstimulated astrocytomas, U138, U251, U373 MG and IMR32 neuroblastoma cells expressed TNF-alpha genes. After stimulation with IL-1 beta (1000 U/ml) all these cell lines expressed TNF-alpha genes. TNF-beta genes could not be detected in these cell lines even in the presence of any cytokine stimuli. We were able to detect expression of IFN-gamma genes within 2 astrocytoma cell lines (U87MG and A172), which interestingly did not show TNF-alpha activity. Constitutive expression of mRNA transcripts of GM -CSF could be detected in all astrocytoma and two out of 5 unstimulated astrocytomas, U87MG and U138MG, expressed G-CSF genes. After stimulation with IL-1 beta, all cell lines expressed G-CSF. In addition, we also examined gene expression of these cytokines within 4 human malignant astrocytoma specimens, 2 peritumoral brain and 2 autopsied normal brains. The results show that tumor and surrounding lesions express TNF-alpha (4 of 6), TNF-beta (1/6), IFN-gamma (4/6), G-CSF (3/6) and GM-CSF (5/6) but not normal brains. One tumor specimen also expresses TNF-beta as well as TNF-alpha genes (case 2). From these results, it is suspected that astroglial cell-derived cytokines may participate in local immune reactions accompanying glioma in the brain.

The accumulating data have shown that a single or certain combinations of proto-oncogenes are genetically altered and acquire oncogenic activities in certain tumor types, through a point mutation and/or overexpression. In the present study, Northern blotting analysis was applied to clinical samples of renal cell carcinomas (RCC) to elucidate expression levels of the c-myc, c-fos and Harvey ras genes in this tumor form. In 11 cases of 23 tumors examined (48%) was shown more than 3-fold expression level of the c-myc gene compared with the matching normal RNA, the c-fos gene in 6 cases (26%), and the Harvey ras gene in one case (4%) showing the concomitant overexpression of two other genes. Overexpression of both the c-myc and c-fos genes was detected in 5 cases (22%). Although the distinct correlation between overexpressions of any genes and the clinical background was not induced, it is considered that these aberrations of proto-oncogenes are involved in oncogenesis of a certain subgroup of RCC.

In vitro amplification of genomic or cDNA sequences by polymerase chain reaction (PCR) is one of the most powerful tools in recent molecular biology. More than 10(5) copies of DNA sequence ranging from 50 bp to 7 kb can be synthesized in a couple of hours. Ever since its development, PCR has attracted much attention because this strategy would allow the detection of minimal residual disease (MRD) at a very low level. The first successful application of this ultra-sensitive technique was the detection of residual tumor cells carrying a 14;18 translocation in follicular lymphoma. The abnormal transcripts caused by 9;22 translocation in chronic myelocytic leukemia (CML) was also exploited for the amplification to detect the MRD. These techniques have successfully shown the detection of one leukemic cell in 100,000 normal cells. Besides leukemic specific sequences caused by chromosome and gene translocations, unique sequences caused by rearrangements in IgH or TCR gamma, delta chain genes have been used as clonal markers for tumor cells. By targetting these sequences for PCR amplification, almost all ALL patients can be analyzed for MRD. The successive measurement of MRD might contribute to improvement of treatments for leukemic patients.

We analyzed the T-cell receptors and immunoglobulin genes in 140 cases of hematopoietic malignancies, including ALL/LBL, malignant lymphomas, AILDs, Hodgkin's disease, ATL and CLL. In many cases, the findings obtained by immunoassociated gene analysis were in parallel to the phenotypic findings. Gene analysis is useful to determine the clonality and the lineage of malignant cells. However, 10-30% cases appeared bigenotype. In some cases, oligoclonalities were recognized. The relationship to the prognosis in these cases was discussed. The development of gamma delta- and alpha beta-cells on T-cell ontogeny was elucidated. Some T-cell leukemic lines were found to be pre-alpha beta T-cells with some consensus characteristics. We also analyzed the V beta family of 15 alpha beta T-cell leukemia cell lines using six V beta cDNA probes. This approach may be useful to determine the clonality of T-cell malignancies. The method of using a PCR system on immunoassociated genes has been described. The PCR method, using the super variable region as a tumor specific fragment, is valuable in the detection of the residual cells of lymphoma and leukemia at a concentration of 1/10(5) cells.

The results of a comprehensive study on 1,000 patients who had been cytogenetically diagnosed to have leukemia in our department since 1962 are reported, and the value of cytogenetic diagnosis for leukemia emphasized. In our series, we detected patients with FAB L1 and L2 showing an abnormality rate of 60 and 66%, respectively. They included 20% with Ph1 positive ALL. In FAB L3, we found t(8;14) in 5 of the 6 patients. The FAB M1 group showed the lowest abnormality rate (50%). Forty percent of the M2 patients exhibited t(8;21), 60% of which also showed loss of sex chromosome of either X or Y. Seventy-eight percent of the M3 patients presented t(15;17) (two patients with no detectable t(15;17) showed rearrangements of retinoic acid receptor alpha gene). Inversion of chromosome 16 was found in 10% of the patients with FAB M4. Patients with M6 exhibited relatively complex chromosomes aberrations. RAEB patients showed more frequent and complex type of chromosome aberrations than PARA patients. Cytogenetic and molecular-biological analyses provided valuable information on the pathophysiology of leukemia and suggested the possible localization of novel oncogene(s).

A 60-year-old woman was admitted to our hospital because of gastric ulcer, anemia, and leukocytosis in November 1984. Blood cell counts on admission were as follows: RBC 407 x 10(4)/microliters, Hb 9.8 g/dl, WBC 33,000/microliters (baso 8%, eo 7%, myelo 11%, meta 2%, stab 4%, seg 54%), Plt 93.7 x 10(4)/microliters. Bone marrow showed hypercellular and myeloid hyperplasia. She was diagnosed as Ph1-chromosome positive chronic myelogenous leukemia. She received natural interferon-alpha at the dosage of 600 x 10(4) IU daily for 22 days from January 14, 1985. After March 1985, she has been given intermittent administration of interferon once in 10 to 20 days, and maintained normal blood cell counts. Cytogenetic improvement was seen on 35 months after the start of IFN and complete suppression of Ph1 chromosome was observed at July 1990 (66 months after).

An autopsy case of polycythemia vera with der(15) and der(20) associated with remarkable neutrophilia was reported. A 87-year-old man was diagnosed as polycythemia vera in August 1987. The red blood cell count was 621 x 10(4)/microliters, Ht 58.5% and the white blood cell count 45,400/microliters with 92% neutrophils. The splenomegaly, increased red blood cell volume and the low erythropoietin level were present. The arterial SaO2 value was above 92%. The chromosome analysis of bone marrow cells revealed 46, XY, -15, -20, +der(15)t(15;?)(q13-15;?), +der(20)t(20;?)(q11;?). The breakpoint in No. 20 was in q11. The remarkable leukocytosis with relative and absolute neutrophilia were observed. Particularly late in the clinical course the white blood cell count was 92,900/microliters with 99% neutrophils. The Ph1 chromosome was negative and the bcr rearrangement was not detected. He died of bronchopneumonia in January 1989. At the autopsy findings neither the marrow fibrosis nor the extramedullary leukemic cell infiltration was noticed.

One of the most serious problems in cancer chemotherapy is the acquired drug resistance of tumor cells. In order to investigate the mechanism of acquired resistance to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), nine resistant cells were isolated from rat gliosarcoma 9L cells and characterized. ACNU-resistant cells were easily isolated after a single treatment even with low-dose ACNU (5 micrograms/ml). However, in spite of repeatedly high-pressure dose of ACNU, more than 30 fold increase to parental 9L cell resistance was not achieved. At this time, further increase in selection pressure resulted in cell death. The resistant ratios of these resistant cells were stable in the absence of ACNU for more than 1 year. Luria and Delbrück's fluctuation test indicated that the appearance of ACNU-resistant cells occurred spontaneously at a rate of (4.40-8.02) x 10(-7)/cell/generation. In karyotypic analysis, the mode was higher in the resistant cells than in 9L cells, but a homogeneously staining region or double minute chromosome, which was considered to be a result of gene amplification, was not observed. In growth kinetics, all the resistant cells except R1 and R12 cells had higher saturation density and plating efficacy than parental 9L cells. These findings seemed to be due to cellular modification associated with ACNU resistance.

Amplification of L-myc oncogene was noticed in a malignant meningioma originating from the right sphenoidal wing of a 54-year-old female. The patient underwent three surgical resections plus radiotherapy over a period of 11 years and then the growth rate of the tumor became much greater with a severely invasive appearance. Using Southern blot hybridization, L-myc amplification was examined on the specimen resected at the fourth operation. As a result, approximately five-fold amplification was confirmed, which has not been previously reported except for that in a small cell carcinoma of the lung. This result may suggest that L-myc amplification is responsible to some extent for the malignant transformation in this meningioma.

We applied restriction fragment length polymorphism (RFLP) analysis to 24 cases of renal cell carcinomas (RCC), 18 cases of prostate adenocarcinomas (PC), and 11 cases of transitional cell carcinomas (TCC) in the renal pelvis to study the oncogene amplification and inactivation of tumor suppressor genes. All of the cases showed no amplification nor gross rearrangements of the Harvey ras, c-myc, c-fos, c-myb, EGFR and PDGFR. In contrast, RFLP analyses demonstrated allelic losses interpreted as inactivational events of TSGs among the tumor forms studied. RCC had allelic losses on the short arm of chromosome 3 (3p) (68%), the long arm of chromosome 18 (18q) (33%), Y chromosome (29%), and 17p (27%) at high frequencies. PC showed frequent allelic losses on 16q (67%), 8p (50%), 18q (43%), 10p (40%), and 10q (38%). TCC had allelic losses on 17p (73%), 11p (64%), and 9p (40%). It was likely that the cases with the more malignant grade tumor had the more allelic losses.

In 43 cases of adenoid cystic carcinomas of the salivary glands (ACC), expressions of the oncogene products such as epidermal growth factor receptor (EGF-R), erbB-2 product, c-myc product and N-myc product were investigated immunohistochemically. First, we confirmed the specificity of the antibodies used with the 13 cell lines. Of the anti-EGF-R antibodies, clone 29. 1. 1 reacted only with A431 but not with the other cell lines overexpressing EGF-R, so that it was most likely to cross-react with the blood type A antigen. Also, the anti-N-myc product antibody revealed the presence of a certain cross-reacting antigen in Lu65. Overexpression of EGF-R was observed in only one case. Nine cases (20.9%) showing tubular and cribriform patterns overexpressed the erbB-2 product, but the signals were mainly localized in the cytoplasm as a granular appearance. Eighteen cases (41.9%) with slight cellular atypia showed an overexpression of the c-myc product. The immunolocalization of the c-myc product was at the nuclei in most cases, or both the nuclei and the cytoplasm in some cases. None of the ACC expressed the N-myc product. It is speculated that the multiple oncogene expressions might be partly related to the acquirement of the differentiated or malignant phenotype in the ACC.

In this study, immunocytochemical and biochemical detection of c-myc protein in gynecological cultured cancer cells were performed together with gene expression of the cells. OMC-1 (cervical squamous carcinoma cell line), OMC-2 (endometrial adenocarcinoma cell line), OMC-3 (ovarian mucinous adenocarcinoma cell line) and OMC-4 (cervical adenocarcinoma cell line) were used. Immunocytochemically, c-myc protein was detected in both nuclei and cytoplasms of cultured cells when they were fixed in 95% ethanol, 10% formalin and 4% paraformaldehyde (PFA) including 10mM NaCl. However, it was detected in the nuclei of almost all of the cells in nuclei of the cells when they were fixed in 4% PFA including 1,000mM NaCl. Western blotting against a nuclear fraction of the cells demonstrated 66Kd protein in OMC-1 and 62Kd protein in OMC-2,3,4, respectively. They were completely absorbed by c-myc synthetic peptide. However, there was no reaction against the cytoplasmic fraction of the cells. Slot blot hybridization against the DNA of the cells demonstrated 15 times and 5 times c-myc gene amplification in OMC-2 and OMC-4, respectively. These results suggest that OMC-1,2,3,4 can be used as positive controls for the immunocyto- and histochemical detection of c-myc protein in clinical materials. However, it must be noted that the redistribution of c-myc nuclear protein into the cytoplasm may occur in the fixation process.

We report a case involving mixed hematopoietic chimerism after an allogeneic bone marrow transplantation (BMT) from a sex mismatched donor. A 31 year-old-man who was diagnosed as having chronic myelogenous leukemia in the accelerated phase received an allogenic BMT from his HLA-identical sister in March, 1989. To determine the mixed chimerism we used the Y-chromosome specific repeated sequence of DNA using a specific probe (PHY 10). The donor's DNA 3.5 kb band appeared in 1-10% of male DNA by Southern blot hybridization in the peripheral blood 21 days after BMT. The Y-chromosome DNA band decreased day by day, and disappeared 110 days after BMT. The Y-chromosome DNA band could be detected, even though few metaphases were obtained immediately after BMT. Thus this method is very sensitive for determining which cells contain the Y-chromosome, and is therefore useful for detecting mixed chimerism after sex-mismatched BMT. Using this method the clinical significance of mixed chimerism can be assessed.