The expression of IL-1 beta mRNA and IL-2 mRNA was analyzed in seven thymuses from patients with myasthenia gravis (MG) and in three normal thymuses using in situ hybridization techniques. Cells containing IL-beta mRNA were detected in all seven cases. These IL-1 beta producing cells were mainly located and clustered in the connective structures of thymic septae and peri-lobuler area like anti-keratin AE3 positive cells. The distribution of anti-CD68 positive cells was different from that of IL-1 beta producing cells in thymus of myasthenia gravis patients. IL-2 producing cells were also found in all seven cases, and their frequency (number of cells/mm2) had a tendency to correlate with the severity of the disease. On the other hand, we could not find both IL-1 beta mRNA and IL-2 mRNA in three normal thymuses. These results suggested a possible role of high IL-1 beta and IL-2 production to activate T cells in myasthenia gravis thymus. Furthermore, in situ production of IL-2 might be reflected in the severity of the disease.

Loss of heterozygosity (LOH) at the p53 and Rb genes, and its clinical correlations were examined in 58 urinary bladder carcinomas. DNA was extracted from formalin-fixed, paraffin-embedded tissues, and amplified by polymerase chain reaction (PCR). The LOH at p53 was examined by restriction fragment length polymorphism (RFLP), and the LOH at Rb by single-strand conformation polymorphism (SSCP). Among patients with urinary bladder carcinoma, 60.3% (35/58 patients) showed heterozygosity at p53 and LOH was detected in carcinoma in 60.0% (21/35), i.e., LOH in 61.9% (13/21) of superficial (< or = pT1) and in 57.1% (8/14) of muscular invasive carcinomas. Therefore, LOH at p53 was considered to occur before the beginning of muscular invasion. Except for the patients who died of other causes or were missed during the follow-up, 67.4% (29/43) showed 43.8% (7/16) died of carcinomatosis, but, only 15.4% (2/13) of the patients without p53-LOH died. The 5-year survival rate calculated by the Kaplan-Meier method was 76.9% in patients with heterozygosity at p53, and 25.1% in those with LOH at p53, being significantly lower at p < 0.05 in the former than in the latter. From an analysis of multiple bladder carcinomas, LOH at p53 occurred on the same allele in different tumors of the same bladder, suggesting the monoclonal origin of each tumor of multiple bladder carcinomas. On the other hand, 51.7% (30/58) of patients showed heterozygosity at Rb, and LOH was detected in 16.7% (5/30) of them, i.e., 6.3% (1/16) in superficial carcinoma and 28.6% (4/14) in muscular invasive carcinoma.

We compared histological effects following radiotherapy in relation to the DNA ploidy pattern and the proliferating cell nuclear antigen (PCNA) positivity rate in 37 patients with rectal cancer who underwent preoperative radiation therapy. Twelve of 23 cases in which the PCNA positivity rate before irradiation was more than 25% showed the effectiveness of radiotherapy (52.2%), against 2 of 6 cases with a rate of less than 25%. Cases in which the rate was more than 25% tended to show more effectiveness. Seventeen of 23 cases in which the PCNA positivity rate was more than 25% showed a decrease in PCNA positivity rate (73.9%). The rate in 6 cases showed no change, and no cases had an increase. In particular, in 12 cases in which the PCNA positivity rate was more than 55%, half showed effectiveness, and the PCNA rate decreased 20% on average. The PCNA positivity rate tended to decrease as a result of irradiation, and especially in diploid cases there were significant differences in the rate before and after irradiation. We suggest that cases in which the PCNA positivity rate is more than 55% with diploid DNA pattern would show most effect. The effects of irradiation could be predicted with biopsy materials and by measuring the DNA ploidy pattern and the PCNA positivity rate.

The DNA ploidy pattern and the expression of proliferating cell nuclear antigen (PCNA) were investigated in 72 gastric carcinoma patients to determine the possible correlation between proliferation kinetics and biological behavior. The diploid pattern was more often observed in cases with mucosal invasion (m cancer) than other cases (sm or advanced cancer). The PCNA labelling index in cases with mucosal invasion was significantly lower than in other cases. The sm cancer was more often observed in patients with high PCNA labelling index (> or = 30%) and aneuploid pattern, while m cancer was more common in patients with low PCNA labelling (< 20%) index and diploid pattern. As a result of this study, we conclude that proliferating kinetics of m cancer may differ from that for sm or advanced cancer. Furthermore, DNA ploidy pattern and PCNA labelling index may be useful as a parameter of depth of invasion.

To evaluate the objective proliferative activity in HCC nuclear DNA contents were measured by means of microspectrophotometry and at the same time the immunohistochemical technique using anti-PCNA antibody was employed. Surgically resected 26 HCCs were analyzed in terms of cell proliferative activity and regional heterogeneity. The analysis was performed by immunohistochemical demonstration of PCNA and pathologic histochemical study in formalin-fixed, paraffin-embedded specimens and cytophotometric measurements of nuclear DNA contents in fresh specimens. The results were as follows. 1) Nine HCCs showed regional ploidy heterogeneity. 2) PCNA labeling index and histological grade of the marginal area was much higher than that of the central area. From these results, we concluded that in the process of the HCC progression proliferative activity was decreased in the central area and was not decreased in the marginal area.

Of 33 patients surgically treated for bile duct cancer, nuclear DNA content of cancer cells was assessed by flow cytometry in order to determine whether DNA ploidy pattern was a prognostic indicator. Thirteen patients (40%) had diploid tumors and 20 patients (60%) had aneuploid tumors including 12 patients with DNA heterogeneity. No significant correlation was found between DNA ploidy pattern and clinicopathological features. Survival of patients with diploid tumor was significantly longer than those with aneuploid tumor (p < 0.01). In aneuploid cases, the prognosis of patients with DNA heterogeneity was similar to those without DNA heterogeneity. In the patients who underwent a curative resection, 5-year survival rate of diploid cases was 100%, as opposed that no patients with aneuploid tumor survived longer than 4 years. These results suggest that DNA ploidy pattern is a important prognostic determinant for bile duct cancer.

We studied DNA ploidy, point mutation of Ki-ras oncogene codon 12, and p21 expression using paraffin embedded materials from 42 cases of colorectal cancer. DNA ploidy was measured by the method of Hedley et al. flow cytometrically. Point mutation of Ki-ras oncogene was examined by the method of Bos et al. using a dot-blot screening procedure, and p21 expression was examined immunohistochemically. Incidence of aneuploidy, Ki-ras point mutation, and p21 expression was 71.4%, 26.2%, 40.5%, respectively. There was a very weak correlation between p21 expression and pathologic findings, but there was no correlation between pathologic findings and DNA ploidy, as well as Ki-ras point mutation. Patients who showed aneuploidy tended to have more point mutation of Ki-ras oncogene. There was no correlation between p21 expression and Ki-ras point mutation, as well as DNA ploidy. Although there was no correlation between Ki-ras point mutation and survival, a significant correlation between survival and DNA ploidy, as well as p21 expression was recognized. Patients who had tumors with diploidy or p21 expression tended to have better prognosis.

The ultimate goal of the human genome project to decode all the genetic information carried in the genome. Towards this goal, the physical structure of the genome, as well as the functional aspects of the genome, must be understood. We initiated a cDNA project to collect the "expression profiles" of all human genes, a database with which to describe which genes are expressed, and to what extent, in any given human cell at a particular time. Single-cycle sequencing of randomly selected members from a 3'-directed cDNA library is most appropriate for this purpose: the sequence data serve as a "gene signature" to identify the expressing gene, and the frequency of appearance of the gene signature reflects the activity of the gene. The compiled data, which usually covers some 1000 sequencing results per sample, is referred to as an "expression profile". We applied this analysis to HepG2 (a cell line derived from a hepatocellular carcinoma), liver cells and lung cells. The expression profiles shed some light upon the unique features of gene expression in the cell or tissue tested. A comparison of the expression profiles among different cells has allowed active genes to be classified as housekeepers or those with cell-specific functions. A significant fraction of the abundantly expressed genes include those that are unique to the cell. In addition, the resulting collection of thousands of gene signatures is a useful source of probes for mapping and for isolating full size cDNAs.

Mutation-specific PCR amplification, a special version of allele-specific amplification, is a method for detection of known mutation. In this method, one of the paired primers has a base at the 3' terminal that is complementary to a mutated base. With this primer, only alleles which have the same mutation can be amplified. We analyzed point mutations of N-ras protooncogene codon 61 in thyroid neoplasms using this mutation-specific PCR amplification method. This method is easy to perform, and many samples can be treated at one time. We detected 2 point mutations of N-ras codon 61 out of 7 follicular carcinomas. One was a cytosine to adenine substitution mutation at the first base, and the other was an adenine to guanine substitution mutation in the second base. These mutations were not detected in 7 follicular adenoma and 1 papillary carcinoma. The results were confirmed by both dot blot hybridization and direct sequencing. Mutation-specific amplification is a powerful method for detection of known mutations of protooncogenes or tumor suppressor genes in investigation of the cancer biology.

In thyroid there is a wide variety of tumors varying from slowly growing differentiated adenocarcinomas to rapidly proliferating undifferentiated carcinomas. There has been some histopathological evidence that the undifferentiated thyroid carcinomas are derived from differentiated carcinomas. Moreover, it is suspected that some genetic events might be associated with such changes. To identify these genetic events that must be involved in thyroid tumor progression, we initially investigated p53 gene alterations in 10 papillary adenocarcinomas, 4 follicular adenocarcinomas, and 8 undifferentiated carcinomas. Base substitutional mutations in exons 5 to 8 and loss of heterozygosity (LOH) of the p53 gene were not detected in papillary or follicular adenocarcinomas. However, 7 of 8 undifferentiated carcinomasa were carrying base substitutional mutations, and LOH was detected in 3 of 5 informative cases. These results strongly suggest that the mutated p53 gene plays a crucial role in de-differentiation during the progression of thyroid tumors.

We examined serum levels of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) in 32 patients with renal cell carcinoma. Serum IL-1 beta was elevated in only one patient (3.1%), IL-6 in 17 (53.1%) and TNF-alpha in 6 (18.8%). Positive rate of serum IL-6 increased with progression of the stage (stage I and II: stage III and IV = 14.3%: 88.9%, p < 0.0001) and was higher for rapid growth type (slow and intermediate growth type: rapid growth type = 36.4%: 90.0%, p < 0.005). However, increased serum levels of IL-1 beta and TNF-alpha were not correlated with stage, grade, or growth type. Gene expressions of IL-1 beta, IL-6, IL-6 receptor and TNF-alpha in the tissues resected from 15 patients with renal cell carcinoma and 3 cell lines established from renal cancers were evaluated by Northern blot analysis. Gene expression of IL-1 beta and TNF-alpha were not detected in any of the tissues or cell cultures, but highly enhanced expressions of IL-6 were revealed in two tumor specimens (13.3%, 1 rapid and 1 slow growth type) and 2 of 3 cell lines. Expressions of IL-6 receptor in tissues and cells were weak. These results suggest that IL-6 plays an important role in the progression of renal cell carcinoma, and that measurement of IL-6 in patients with renal cell carcinoma may be useful clinically.

We describe a characteristic case of chromosomal abnormalities in a 47-year-old man with chronic myelogenous leukemia (CML). He had been diagnosed with the chronic phase of CML on May 1990, but he refused treatment. He was readmitted in October 1991 because of peripheral lymphadenopathy. Hematologic studies showed that he remained in the chronic phase of CML. There were no additional chromosomal findings in the bone marrow, but cervical lymph node biopsy revealed complete replacement with blastoid cells. The blasts were positive for CD13 and CD33. DNA analysis for the breakpoint cluster region gene showed rearrangement pattern, but immunoglobulin heavy chain joining region genes and T-cell antigen receptor gene rearrangement were not observed. The karyotype of lymph node cells showed various additional chromosomal abnormalities. Therefore, we concluded that the patient had an extramedullary blastic crisis in his peripheral lymph nodes. Abnormal clones were composed of one main clone and three subclones, with the latter occupying more than 10% of the volume each other. Considering the types of the additional chromosomal abnormalities, all four types of clones were thought to have evolved through independent pathways.

Various kinds of nonrandom chromosomal aberrations have been reported in hematopoietic malignancies. Since the 1980s, many translocation-associated oncogenes and several suppressor oncogenes have been identified and applied for the clinical diagnosis of these malignancies. The former is of major, clinical importance for specific diagnosis made on the basis of molecular detection of the chromosomal translocation, the deregulated expression, and the chimeric mRNA of those genes. Both BCL-1 and BCL-2 genes, associated with mantle zone lymphoma and follicular lymphoma, respectively, belong to the representative deregulated oncogenes by juxtaposition with an immunoglobulin gene enhancer as well as an MYC gene in Burkitt's lymphoma. On the other hand, the MLL gene, associated with infant leukemia, acute monocytic leukemia and secondary leukemia, produces chimeric mRNAs between LTG4, 9, and 19 genes as well as the BCR-ABL chimeric gene in chronic myelogenous leukemia. The detection of minimal residual disease (MRD) by either polymerase chain reaction (PCR) or reverse transcriptase (RT)-PCR is becoming an essential test during the course of treatment containing bone marrow transplantation, because positive results of the MRD are closely related to poor prognosis and would have great influence on the choice of treatment plans.

Many of the genetic alterations related to carcinogenesis and progression such as gene amplification, deletion, mutation and overexpression can be analyzed on paraffin-embedded clinical materials. Genetic abnormalities of tumor suppressor gene such as p53 and APC (adenomatous polyposis coli) are good markers for differential diagnosis of gastrointestinal cancers. Gene amplification and overexpression of oncogenes and growth factors/receptors such as c-met, K-sam, c-erbB2, EGF and EGF receptor are biological marker of biological malignancy. Molecular diagnosis has been done in Hiroshima Medical Association Laboratory to make an objective diagnosis for border line lesions and to obtain information on the biological behavior of gastrointestinal cancers based on genetic alterations. Molecular analysis is a powerful tool to complement histological diagnosis of gastrointestinal lesions.

For lung cancer diagnosis, the diagnostic significance of the ras, myc, erbB2 oncogenes, and the p53 anti-oncogene was reviewed. Point mutation of the ras oncogene, amplification of the myc oncogene, and overexpression of the erbB2 oncogene are associated with poor prognosis of lung carcinomas. Mutation of p53 anti-oncogene is a common event of lung carcinoma and the differences in its mutation pattern can be used for the molecular diagnosis of multicentric lung carcinomas.

To study the alteration of nuclear DNA content of cancer cells after peplomycin (PEP) treatment, DNA cytofluorometry was performed in combination with 3H-thymidine (3H-TdR) autoradiography using cultured A431 cells. The cells in the logarithmic growth were treated with PEP (1.25 micrograms/ml) for 24 hr, during the first 4 hr of which they were pulse-labeled with 3H-TdR (2.4 x 10(4) Bq/ml). After washing with PBS, the cells were then cultured without both PEP and 3H-TdR, fixed at different times and stained with propidium iodide (PI) for the auto-stage cytofluorometry, which enabled DNA content analysis for labeled and unlabeled cells by repeated scanning of the same cell population. The nuclear DNA content histograms demonstrated that A431 cells were mostly arrested in G2 phase of 4C stem line by treatment with PEP for 24 hr. This G2 block lasted up to 8 hr after removal of the drug, and thereafter, marked polyploidization associated with DNA synthesis occurred, showing almost no mitotic figures, while only a few cells returned to G1 phase via M phase. During the period of 72-120 hr, however, the fractions of advanced polyploid cells (DNA content > or = 8C) gradually decreased and the DNA content distribution pattern became eventually similar to the original one as seen before PEP treatment. From these results we hypothesized as follows: 1) At S-G2 boundary, there is some control mechanism that checks whether the cells, after S phase, can enter the M phase or not. 2) The cells, which are not permitted to enter mitosis by the control mechanism, show marked polyploidization. 3) Only the cells that enter into mitosis can live and proliferate, though the advanced polyploid cells die shortly. 4) This control mechanism might be related to the precision of DNA repair that is checked at the G2-M checkpoint.

Multiple endocrine neoplasia (MEN)2a is known to be genetically linked to a locus on chromosome 10. The application of polymorphic DNA probes (RFLP) for the region made presymptomatic diagnosis possible. The validity and significance of genetic diagnosis has been proved in Caucasian MEN2a families in terms of early diagnosis and cure. In addition, non-carriers of the gene can be discriminated with a high degree of certainty and can be relieved from years of conventional screening program. An example of genetic diagnosis in a Japanese MEN2a family is shown. DNA analysis was done employing MEN203 as a probe. The usefulness of genetic diagnosis was confirmed in Japanese as well as Caucasians. Clinical consideration in carrying DNA analysis and the future of genetic diagnosis is also discussed.

Multiple endocrine neoplasia (MEN) type 2A (MEN 2A) and type 2B (MEN 2B) are dominantly inherited with a predisposition to endocrine tumors. The responsible genes for MEN 2A and 2B have recently been localized to chromosome 10q 11.2 by genetic and physical mapping. The DNA segment encompasses the RET proto-oncogene. This is a receptor tyrosine kinase gene, which is expressed in medullary thyroid carcinoma and pheochromocytoma. Point mutations in the cysteine-rich domain of the RET were demonstrated in patients with MEN 2A. The cosegregation of these mutations and disease in MEN 2A families indicates that they possess a predisposition endocrine organs to develop into tumors. Biological assessment of the mutant forms in cell culture and transgenic mouse lines should provide further insight as to the role of the RET in the tumor development.