A 62-year old male was admitted to our hospital because of fever and dysphagia on November 14, 1987. The peripheral leukocyte count was 174,400/microliters with 93% blasts and bone marrow aspiration showed that 90.4% of nucleated cells were blasts positive for both myeloperoxidase and alpha-naphthylbutyrate esterase. Chromosome analysis revealed a karyotype of 45XY, 9q+, 16q+, -20 and 22q-. Esophageal X-ray and endoscopy showed abnormalities. Esophageal biopsy revealed squamous cell carcinoma. He was diagnosed as having Ph1 positive acute myelomonocytic leukemia (AMMoL, M4) and esophageal cancer. He was treated with BHAC-DMP and intermediate-dose ara-C therapy for leukemia and a complete remission was obtained by March 25, 1988. As treatment for esophageal cancer, radiation therapy (total 4,200 cGy) was given and followed by chemotherapy with CDDP and 5-FU. However he died on April 8, 1988. Autopsy findings showed disseminated invasion of esophageal cancer. Ph1 positive AMMoL associated with esophageal cancer is extremely rare.

Effects of pretreatment with N-methyl-N-nitrosourea (MNU) or streptozotocin (STZ) on cytotoxicity and induction of sister chromatid exchanges (SCE) in human and rodent brain tumor cells treated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or 1-(2-chloroethyl)-1-nitrosourea (CNU) were investigated. 9L-2 and SF-188 cells were more resistant to the cytotoxic effects of chloroethylnitrosourea than 9L and SF-126 cells. SF-295 cells were more resistant to the cytotoxic effects than SF-126, but more sensitive than SF-188 cells. Pretreatment of 9L-2 cells with MNU or STZ resulted in a dose-dependent increase in cytotoxicity and SCE induction by BCNU. Treatment with 1mM MNU or 1.5mM STZ completely reversed the cellular resistance of 9L-2 cells to BCNU but did not potentiate either cytotoxicity or SCE induction in 9L cells. Pretreatment of SF-188 and SF-295 cells with MNU or STZ resulted in a dose-dependent increase in cytotoxicity and SCE induction by CNU. Treatment with 500 microM MNU or 1.5mM STZ for SF-188 cells, and with 250 microM MNU or 1.5mM STZ for SF-295 cells completely reversed the cellular resistance to CNU. These results are consistent with the hypothesis that pretreatment with MNU or STZ inhibits O6-alkylguanine-DNA-alkyltransferase (O6-AT) and inhibition of the enzyme allows the formation of DNA interstrand cross-links resulting in increase in cytotoxicity and induction of SCE in resistant cells treated with chloroethylnitrosourea. In this regard, O6-AT plays an important role in determining the cytotoxicity and induction of SCE by chloroethylnitrosourea in both rodent and human brain tumor cells.

A 53-year-old woman was admitted with fever and general fatigue in December, 1988. A diagnosis of malignant histiocytosis (MH) was made based on her high level of LDH, thrombocytopenia, mild splenomegaly without systemic lymphadenopathy. There was also bone marrow infiltration large atypical cells and erythro-phagocytosis. VEPA therapy resulted in complete remission. Visual disturbance and left lagophthalmos were recognized in March 1990. These signs indicated central nervous system (CNS) relapse which disappeared after intrathecal methotrexate injection. The same symptoms and signs appeared after another, 5 months. Tumor cells were found not only in the central spinal fluid but also in bone marrow. CNS and bone marrow recurrence were treated with intrathecal methotrexate injection VEPA therapy and cranial irradiation. We diagnosed this case as MH, based on the clinical features which did not include systemic lymphadenopathy and laboratory findings although TcR-gamma rearrangement was observed in bone marrow cells. Only one case of CNS infiltration diagnosed when alive has previously been reported in Japan. We report here a very rare case in which by medical treatment CNS infiltrations was improved twice.

Human endometrial adenocarcinoma cells (Ishikawa line) constitutively express c-erbB2 coded oncoprotein p185erbB2 (p185) which is believed to be an orphan receptor for an unknown growth factor. Since we have shown that expression of p185 in primary lesions of endometrial cancer correlates well with high frequency of lymph node++ metastases and that the metastatic cells in the nodes strongly expressed p185, the role of the oncoprotein in processes of metastases was studied. Culturing the cells in the presence of 15% FCS and with monoclonal antibody to the extracellular domain of p185 (CB-1) inhibited cell growth and attenuated p185 expression on Western blotting, whereas no change occurred with the control antibody. Cells cultured without FCS achieved only approximately 1/3 growth compared to cells with FCS, and further suppression of growth was observed after adding CB-1. When cells were cultured on human term amnion, basement membrane invasion with p185 expression was observed. In nude mice, intraperitoneal seeding resulted in implant formation which was also associated with positive p185 as well as cyclin immunohistochemistry. In the two experiments, treatment of cells with CB-1 inhibited invasion or implant formation. The present study suggests that a signal through p185 receptor molecules acts as a trigger for early proliferation, and interaction with the host may enhance up-regulation of p185.

We examined the effects of cadmium on the mRNA levels of several genes in cultured retinoblastoma (Y79) cells. After Y79 cells were treated with 15 microM CdCl2, RNA was extracted at a given time. The levels of retinoblastoma gene (Rb) mRNA decreased after cadmium treatment, although it was unlikely that the Rb gene product is functional in this cell line. The N-myc gene (oncogene) is constitutively expressed in untreated Y79 cells but its mRNA levels also decreased following cadmium treatment. On the other hand, the mRNA levels of both heat-shock protein (hsp 70) and metallothionein gene, both having physiological protective effects, increased under these conditions. These results indicate that Y79 cells have physiological protective responses to such a heavy metal as cadmium and that both Rb and N-myc gene expressions are down-modulated in the presence of cadmium.

PRAD1 (parathyroid adenoma 1) gene at chromosome 11q13 has been cloned from parathyroid adenomas as a putative oncogene, activated by translocation with the parathyroid hormone gene. 4.5 kb and 1.7 kb mRNA are transcribed and both have the same open reading frame of 885 bp encoding 34 kd protein of a cyclin gene family, cyclin D1. Recently, overexpression of PRAD1 gene has been reported to be correlated closely with the rearrangement of bcl-1 locus, particularly in centrocytic lymphoma. In our study, overexpression of PRAD1 gene was shown in five B cell lines with t(11;14)(q13;q32) including one centrocytic lymphoma line and 4 myeloma lines, when compared with other hematopoietic cell lines without translocation. One of the cell lines, SP-49, demonstrated a truncated mRNA of 3.4 kb, in addition to 1.7 kb of normal size. Southern blot analysis demonstrated a rearrangement with PRAD1 cDNA probe, suggesting that the gene is altered in this particular cell line. By cloning analysis, we confirmed that 1.8 kb deletion in 3' region of PRAD1 gene eliminating the destabilizing signal of PRAD1 mRNA, gave rise to the aberrant mRNA of 3.4 kb. These findings suggest that PRAD1 gene is most likely the candidate oncogene for bcl-1 activated by t(11; 14)(q13;q32) translocation. The gene alteration found in one cell line, SP-49, might also play an important role for deregulation of the gene.

The deleted in colorectal carcinomas (DCC) gene, located in human chromosome band 18q21, was identified as a potential tumor suppressor gene by Fearon et al. in 1990. The DCC gene encodes a protein which is highly similar to neural cell adhesion molecules and other related cell surface glycoproteins. In colorectal carcinoma, the expression of the DCC gene is reduced or absent in 88% of cases. We examined the expression of the DCC gene using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Its expression was reduced or absent in some leukemias. Our findings suggest the possibility that this gene may play a role in leukemogenesis.

The p53 gene encodes a nuclear phosphoprotein and is now considered as a tumor suppressor gene. Mutations of the p53 gene have frequently been observed in several types of solid tumors and are believed to be implicated in the development of these tumors. Recent studies have shown that the p53 gene is altered in chronic myelogenous leukemia (CML) in blast crisis. In CML, alterations of the p53 gene may play an important role in the development of blast crisis. More recently, p53 mutations have been reported in other types of hematologic neoplasms, such as acute leukemia, adult T-cell leukemia, and malignant lymphoma. These observations suggest that inactivation of the p53 gene is involved in the tumorigenesis of various types of hematologic neoplasms.

The retinoblastoma susceptibility gene (RB) is expressed in all lineages of normal hematopoietic cells and plays an important role in controlling cell cycle progression at G1/S. Abnormalities of the RB gene may, therefore, predispose to the development of hematologic malignancies. DNA rearrangement was reported to be present in 1.5-12.1% of cases with primary leukemias and the absence of the RB gene product was also observed in 6.3-23.2%. The abnormalities were frequently observed in blastic crisis of CML, especially of the megakaryoblastic phenotype, AML with monocytic differentiation and Ph1-positive leukemias. These results indicate that abnormalities of RB are relatively common in hematologic malignancies and loss of RB function may contribute to the altered growth of these cells.

Specific chromosomal translocations are one of the cytogenetic characteristics of hematopoietic malignancies. The application of molecular biological techniques has led to important insight into the molecular consequences, accompanied by the chromosomal translocations. Specific oncogenes and related genes activated by the translocations have been identified at the translocation breakpoints. Translocated oncogenes are deregulated by their juxtaposition to enhancers of Ig or TCR genes, resulting in their overexpressions in some lymphoid tumors. Alternatively, they are activated by gene fusion, resulting in a chimeric oncoprotein, as seen in Ph1-positive leukemia. The molecular consequences by chromosomal translocation in hematopoietic malignancies is focused here.

The activation of protooncogenes (ras, fms and myc genes) by point mutations in hematological malignancies are described in this review. Ras mutations are found in a variety of human malignancies at codon 12, 13, and 61. Generally, N-ras mutations are frequent in hematological malignancies. Fms mutation at codon 301 and 969, which are seen in 10 to 20% cases of AML and MDS, increase tyrosine kinase activity of the fms products. Ras and fms mutations are postulated to influence leukemogenesis at rather early stages. Burkitt lymphomas are characterized by specific chromosomal translocations between c-myc gene and one of the immunoglobulin genes. Furthermore, mutations in the 3' border of the exon 1 of c-myc are frequent, and may play an additional role in pathogenesis of Burkitt lymphoma.

Tumor-specific chromosomal translocations in lymphoid malignancy frequently involve the fusion of immunoglobulin (Ig) or T-cell receptor (TCR) gene loci with oncogenes, thereby activating the latter to contribute to the malignant phenotype. The preponderance of chromosomal translocations involving Ig or TCR genes suggests that the enzymatic machinery, the lymphocyte recombinase, is used in the formation of abnormal chromosomes. However, the role of ectopic recombination signals for recombinase in chromosomal translocations has been controversial. In this paper, molecular studies of genes adjacent to the breakpoints of these chromosomal translocation are reviewed, and the mechanism of chromosomal translocation and clinical characteristics of the tumor with specific chromosomal translocation are discussed.

T-cell receptor genes encode proteins related to their specific recognition structures. As is true for immunoglobulins, T-cell receptor genes consist of separate germ line segments that encode the variable and constant regions of the chain. The variable region genes are assembled from two or three separate germ line segments that somatically rearrange during differentiation. The diversity in the variable region of T-cell receptors results from germ line diversity of V, (D), and J sequences, combinatorial joining of the segments, junctional diversity leading to codon changes at the end of gene segments, N-region diversity arising from addition of template independent nucleotides at junction. Each T-cell clone has its own unique structure of the variable region, and in T-cell malignancies, this variable region is retained after malignant transformation. This enables the nucleotide sequences of the variable region genes to be used as a molecular marker for the malignant clone. By amplification of these sequences of variable genes, it is now possible to study the pathological features of the leukemic clones in vivo, as well as to follow the patients for minimal residual disease.

More detailed identification and understanding of the heterogeneity of leukemias using a broad panel of markers seems to be essential for the successful design of more sophisticated and effective treatments. Based on the FAB system, immunological phenotypes using a panel of monoclonal antibodies, and rearrangements of immunoglobulin and T-cell receptor genes, acute leukemia can be divided into six subtypes such as B-lineage, T-lineage, AML, NK-lineage, AUL and mixed lineage leukemia. The definition of B-lineage and T-lineage cells, a new classification for mixed lineage leukemia, incidence of dual rearrangements and their clinical significance are discussed.

Molecular genetical analysis using Ig and TCR genes has been applied for hematological malignancies. In acute leukemias, this method is used to clarify the cellular lineage, and in malignant lymphomas to detect the monoclonal component. In this article, we describe the mechanism of immune gene rearrangement and the present status and future directions of immunogenetic analysis of leukemias and lymphomas.

In situ hybridization provides a direct method to localize DNA sequences on metaphase chromosomes. Initially, nucleic acid probes were labeled isotopically and detected by autoradiography. Recently, fluorescent in situ hybridization (FISH) have been developed. FISH has resolved several problems, such as radiolysis of probes, long exposure times, high background noise, and unclear localization of silver-grains visualized by autoradiography. In addition, FISH allows us to study numerical and structural chromosome aberrations using chromosome-specific DNA probes, not only in metaphase chromosome, but also in interphase nuclei. Therefore, FISH can be applied to a broad spectrum of biological and clinical problems in hematological malignancies.

Clonal chromosome abnormalities are found in most patients with non-Hodgkin's lymphoma. The role of the chromosome abnormalities in predicting the prognosis of lymphoma patients has not been fully clarified, because of different histological classifications being used in different areas and the complexity of the chromosome abnormalities often found in lymphoma. Recent studies have shown distinct correlation of rather rare abnormalities with specific histologic and immunologic phenotypes and prognoses. Many chromosome abnormalities seem to specifically correlate with these parameters of non-Hodgkin's lymphomas, as seen in leukemias. The chromosome data also seem to support the histological observations that some lymphomas may show uneven geographical distributions.

The chromosomal classification system of childhood acute leukemia according to ploidy or structural chromosomal changes of leukemia cells is clinically useful for the prediction of the treatment outcome. While patients with hyperdiploid (greater than 50 chromosomes) ALL enjoy the best prognosis, those with hypodiploid or pseudodiploid ALL are expected to have unfavorable clinical outcome. Among various translocations or deletions in ALL, t(4;11), t(9;22) and t(8;14) predict the worst clinical outcome. AML patients with t(8;21) or inv (16) have better 50% survival than those with other chromosome changes or normal diploidy. Chromosome analysis of leukemia cells should be included in the diagnostic workup of childhood acute leukemia.