Establishment of a myeloid leukaemic cell line (SKNO-1) from a patient with t(8;21) who acquired monosomy 17 during disease progression.

A novel cell line SKNO-1 was established from the bone marrow cells of a 22-year-old male suffering from acute myeloblastic leukaemia (AML) M2 with t(8;21) whose disease became resistant to chemotherapy after acquisition of 17 monosomy. SKNO-1 has been maintained for more than 36 months as a granulocyte-macrophage colony-stimulating factor (GM-CSF) dependent line. Morphologically, SKNO-1 cells were myeloblasts somewhat matured. The cells grow in suspension with a doubling time of 48-72 h. The survival and growth of SKNO-1 cells was absolutely dependent on granulocyte-macrophage colony stimulating factor (GM-CSF). SKNO-1 cells possessed t(8;21) and monosomy 17 which were observed in original leukaemic cells. We confirmed that the AML1 gene, located on chromosome 21, was rearranged and the AML1-MTG8 fusion transcript was expressed in SKNO-1 cells. Over-expression and mutation of the p53 gene were also detected in SKNO-1. It is likely that alterations of AML1 or MTG8 gene and p53 gene contribute to a disease progression in this case. Since t(8;21) translocation is a common chromosome abnormality in AML, and inactivation of the p53 gene may play a crucial role in disease progression in AML, SKNO-1 would be a useful tool for analysing the molecular mechanisms in myeloid leukaemogenesis.

The SKM-1 leukemic cell line established from a patient with progression to myelomonocytic leukemia in myelodysplastic syndrome (MDS)-contribution to better understanding of MDS.

Although molecular and cytogenetic studies strongly point to the role of oncogenes, the mechanisms underlying the development of MDS and their progressive evolution to AML are still largely unknown. It has been postulated that AML has a preleukemic stage and a multi step pathogenesis, with the preleukemic stem cell able to undergo clonal evolution, with the acquisition of karyotypic abnormalities, leading to the development of acute leukemic subclones. The activations of the ras oncogenes or inactivation of the p53 anti-oncogene by point mutations have been described recently in several cases of MDS as well as AML, suggesting a critical role for these alterations in the development of these myelogenous leukemias. We reported previously establishment of a leukemic cell line, SKM-1, from the patient who initially possessed multiple point mutations of ras genes but lost these mutations during disease progression to myelomonocytic leukemia with acquisition of chromosomal abnormalities involving the p53 anti-oncogene. This process is characterized by genetic instabilities probably due to the failure of their DNA repairment leading to abnormal control of cell proliferation and differentiation. Studying this cell line, SKM-1, is a promising approach to understand the mechanisms of the initiation, disease progression, alterations of DNA repairment, and genetic instability in MDS and myelogenous malignancies.

Hemorrhagic colitis with unusual colonoscopy features, complicated with chronic graft-versus-host disease after allogeneic bone marrow transplantation.

A 32-year-old man was admitted after bone marrow transplantation because of hematochezia. He had history of chronic graft-versus-host disease (GVHD) of the skin and the liver, and cytomegaloviral pneumonia. Barium enema and colonoscopy showed multiple colon ulcers in the ascending and transverse colon. This feature is very rare in chronic GVHD and resembles the feature in autoimmune disease such as periarteritis nodosa. Thus, this ulceration is thought to be caused by vasculitis due to an autoimmune reaction in chronic GVHD.

Establishment of a leukaemic cell line from a patient with acquisition of chromosomal abnormalities during disease progression in myelodysplastic syndrome.

A cell line designated SKM-1 was newly established from leukaemic cells of a 76-year-old Japanese male patient with monoblastic leukaemia following myelodysplastic syndrome (MDS). The cells were obtained from peripheral blood of the patient when he lost multiple point mutations of ras genes with acquisition of chromosomal abnormalities during disease progression in MDS. The cells grew as a single floating cell, and have been continuously growing with the morphological characteristics of immature monoblasts by serial passages during the past 42 months with a doubling time of about 48 h. By cytochemical analysis, the cloned cells were positive for butyrate esterase, but negative for the Epstein-Barr virus associated nuclear antigen. Phenotypic analysis revealed the expression of myelomonocyte specific antigens such as CD4, CD13, CD33 and HLA-DR. Cells from the primary peripheral blood and those from 50 passages of the SKM-1 cell line both possessed no activated ras genes but showed karyotype abnormalities with 46,XY, del(9)(q13;q22), der(17) t(17;?)(p13;?). The SKM-1 cells have two mutations in p53 gene and overexpress the p53 products. This cell line may contribute to a better understanding of molecular mechanisms in the progression from MDS to myelogenous leukaemia.

Phorbol ester stimulates the activity of a protein tyrosine phosphatase containing SH2 domains (PTP1C) in HL-60 leukemia cells by increasing gene expression.

The affinity-purified antibody to a protein tyrosine phosphatase (PTP) containing two src homology 2 domains (PTP1C) was generated. The antibody recognized two types of PTP1C (PTP1C-alpha and -beta) of which the molecular sizes were 66 (alpha) and 62 kDa (beta), respectively, and these two types were expressed differentially in various cell types. The immune complex phosphatase assay using the antibody demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) and a vitamin D metabolite increased the PTP activity of immunoprecipitated PTP1C to 230 and 150% of control, respectively. By contrast, neither dimethyl sulfoxide nor retinoic acid significantly affected the PTP activity of PTP1C in HL-60 cells. The time course increment by TPA of PTP1C activity was closely correlated with that of the acquisition by HL-60 cells of a macrophage-like phenotype. In addition, TPA increased the amount of PTP1C detected by immunoblotting and immunoprecipitation and raised the level of expression of PTP1C mRNA in HL-60 cells. The increase of PTP1C mRNA induced by TPA treatment was inhibited by cycloheximide, suggesting that new protein synthesis is required for the increase by TPA of PTP1C mRNA expression. Furthermore, TPA increased the rate of transcription of the PTP1C gene without affecting the stability of PTP1C mRNA. These results suggest that (i) two subtypes of PTP1C may exist and function in various cell types, and (ii) TPA stimulates the PTP activity of PTP1C by increasing the transcription rate of PTP1C gene expression. The possible role of PTP1C in the macrophage differentiation will be also discussed.

Mesenteric fibromatosis presenting as fever of unknown origin.

We report a case of fibromatosis, soft tissue tumors that are benign histologically, but exhibit behavior intermediate between benign and malignant diseases. Mesenteric fibromatosis grows vigorously without a general inflammatory reaction, and its symptoms are the result of obstruction and/or compression of the intestine. However, in our case, the general inflammatory reactions of fever and C-reactive protein (CRP) elevation was present at an early stage. Because this inflammatory reaction disappeared after surgical resection, it may have been induced by some inflammatory factors produced in the tumor, such as those produced in inflammatory fibrous histiocytoma. We found that computed tomography was useful in the detection of the cause of fever of unknown origin, and suggest that it should be recommended in cases of long-lasting unexplainable fever.

Successful treatment of angioimmunoblastic lymphadenopathy with dysproteinemia with cyclosporin A.

Angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is a lymphoma-like disease. Although the histologic appearance of this disease is benign, it is clinically malignant, with chemotherapy being ineffective and the prognosis being poor. In this article, the authors report the results of treating two patients with AILD with cyclosporin A. One patient had cervical lymphadenopathy and fever, and AILD was refractory to both chemotherapy and alpha-interferon. However, treatment with cyclosporin A achieved complete remission. The other patient had generalized lymphadenopathy, and cyclosporin A was used as the treatment of first choice. Complete remission also was achieved in this patient, who still is in remission. These two cases demonstrate that cyclosporin A can be effective for treating AILD.

[Monitoring of cyclosporin A concentration in whole blood with fluorescence polarization immunoassay after bone marrow transplantation].

Cyclosporin A, a strong immunosuppressive agent, has been used to prevent rejection or graft-versus-host disease (GVHD) after organ transplantations including bone marrow transplantations. Monitoring of cyclosporin A concentration in whole blood is necessary for its high frequency of side effects. The authors to measured its concentration by means of radioimmunoassay (RIA) as well as fluorescence polarization Immunoassay (FPIA) a cases after bone marrow transplantation. There was a good correlation between these two methods. FPIA is less specific for cyclosporin A than RIA, but it is an easier method. It is important to measure the concentration of cyclosporin with FPIA because there are metabolites of cyclosporin A which cannot be measured with RIA. Thus FPIA can be applied to the routine monitoring of cyclosporin.

[Percutaneous transhepatic intraportal administration of amphotericin B to a patient with multiple liver abscesses due to Candida albicans: with monitoring by fungal index as a parameter of Candida volume].

A 59-year-old female with acute promyelocytic leukemia in remission was admitted to our center because of an episode of incidental high fever with general fatigue. She was found to have hepatomegaly. Abdominal CT revealed multiple liver abscesses and a positive culture was obtained for candida albicans from an aspirated abscess. She was treated with percutaneous transhepatic intraportal administration of amphotericin B in addition to oral and intravenous administration. We confirmed the remission of these abscesses by means of the fungal index which is the difference between the values of the limulus test and endotoxin specific test. The fungal index appears to be useful for early diagnosis and treatment of fungal infection.

Multiple point mutation of N-ras and K-ras oncogenes in myelodysplastic syndrome and acute myelogenous leukemia.

We analyzed activating mutations of N-ras and K-ras by the polymerase chain reaction and oligonucleotide hybridization in hematological disorders. Activating mutations of these codons were detected in 4 of 20 cases of myelodysplastic syndrome (MDS) and 15 of 77 cases of acute myelogenous leukemia (AML). Our of 19 cases of MDS and AML who carried active mutations, 7 cases were found to have two or more distinct mutations in activating codons of N-ras and K-ras. Ras mutation was found preferentially in progressive disease such as refractory anemia with excess of blasts (RAEB) of RAEB in transformation (RAEB-t). A relatively high incidence of ras mutation was found in M5 AML (40%). No ras mutations were found in other hematological disorders, such as acute lymphoblastic leukemia and chronic myelogenous-leukemia. The most frequent amino acid substitution was that of an aspartate for glycine at codon 12 of N-ras resulting from G to A mutation (11/35). The survival of AML patients who carried ras mutations showed no significant differences from those without ras mutations calculated by Kaplan-Meier. Seven cases of MDS and 7 cases of AML patients could be investigated at various points during their clinical course. Among these 14 cases, we found 2 interesting cases of MDS. The first case lost multiple clones carrying ras mutations during disease progression, the second case acquired mutation of the ras gene during disease progression. These results suggested that multiple point mutations of ras genes may not be initiating events but may contribute to a clonal evolution of MDS and AML.

RAS gene mutations in multiple myeloma and related monoclonal gammopathies.

The presence of RAS gene mutations in precancerous lesions suggests that they participate in the early stages of neoplastic development. Furthermore, neoplastic progression from monoclonal gammopathy of undetermined significance (MGUS) to overt multiple myeloma (MM) have been frequently observed. These observations prompted us to study the pathogenetic role of RAS genes in MM and related monoclonal gammopathies. DNA from 18 patients with monoclonal gamma-globulinemia including 12 MM were investigated for the presence of N- and K-RAS gene mutations by polymerase chain reaction (PCR)/oligonucleotide hybridization. Mutations involving codons 12, 13 or 61 of N-RAS gene were identified in 3 of the 12 MM patients, 1 of the solitary plasmacytoma patients and none of the 3 of the MGUS patients. In the case of plasmacytoma, RAS mutations were detected in his bone marrow specimens.

[Isolated gingival relapse during hematological remission in a patient with erythroleukemia].

A 54-year-old man was admitted to Kitano Hospital because of nuchal pain in November, 1984. The bone marrow was hypercellular with 18% of myeloblasts and 45% of erythroblasts many of which were megaloblastoid and positive for PAS-staining. BH-AC . DMP therapy induced a complete remission which was maintained for more than 3 years until April, 1988, when gingival swelling occurred. A biopsy of the gingiva revealed a diffuse infiltration of myeloblasts together with a few erythroblasts. Local irradiation resulted in he disappearance of the swelling. However, the microscopic infiltration of the leukemic cells in the gingiva still remained in December, 1988 when there was no apparent gingival swelling and the patient was in a second hematological remission. Bone marrow relapse occurred in September, 1988 and twice thereafter. Complete remissions were again achieved for the first two relapses and the leukemic infiltration of the gingiva disappeared after another irradiation. The third relapse was refractory to an intensive chemotherapy and the gingival swelling also recurred in July, 1989. The patient died of pneumonia in October, 1989. The gingiva may be more common site of extramedullary relapse. Careful observation of this organ would be necessary throughout the course of leukemias.