[FISH, cytogenetic analysis].

We describe here the clinical applications of fluorescence in situ hybridization(FISH) in patients with chronic myelogenous leukemia under treatment with interferon-alpha, some cases with a false positive caused by preparation of specimen and/or probes, and the detection of chimerism after sex-mismatched bone marrow transplantation based on our clinical experience. Furthermore, we introduced our methods of performing FISH using blood smear or G banding specimens.

[A report on staff development in advanced knowledge and technology of gene diagnosis for medical technologists by the Japanese Association of Medical Technologists: the view of medical technologists].

With advancement of the molecular biology, gene diagnosis is widely utilized in clinical application of medicine. For medical technologists, it is necessary to receive continued education to practice such advanced scientific trends. The Japanese Association of Medical Technologists established a working group for a staff development program of gene diagnosis and chromosome analysis in 1996, and has continued its activities in making inquiries about the present conditions, publishing a textbook, and providing seminars. The internal laboratory utilization of gene diagnosis was 8.7%(213 of 2437 hospitals). Based on the fact that about half of the hospitals use external laboratory, staff development for the internal utilization of gene diagnosis is an urgent issue. We have been providing seminars to meet the educational needs of our members. In addition to those activities, we have continued our efforts in providing a manual with clinically useful information, standardizing methods, establishing an information network, and conducting a controlled survey. The role of the working group is now shared by the local prefecture to further increase the numbers of those with expertise in gene diagnosis. We, medical technologists, need to have a global view of professional growth, and also to cooperate with academic societies related to gene diagnosis to establish a certification system.

A case of T-lineage lymphoblastic lymphoma/leukemia with t(4;11)(q21;p15) that switched to myelomonocytic leukemia at relapse.

A 51-year-old Japanese woman, initially diagnosed with T-lineage (CD2+, CD7+, CD3-, CD4-, CD8-) lymphoblastic lymphoma with t(4;11)(q21;p15), relapsed with acute myelomonocytic leukemia with the identical chromosomal abnormality. Southern-blot analysis revealed clonal rearrangements of an immunoglobulin heavy chain gene (JH) and T-cell receptor genes (J delta 1, J gamma 1, C beta 1) at first presentation, but germ line configurations of these genes at relapse. Leukemias with t(4;11)(q21;p15) may involve a hematopoietic progenitor capable of multilineage differentiation.

Molecular features of a new human lymphoma cell line carrying both BCL2 and BCL6 gene rearrangements.

Chromosomal translocations and/or their molecular equivalents involving the BCL6 gene on 3q27 band have been suggested to be involved in the development of non-Hodgkin's lymphoma of B-cell type (B-NHL). The rearrangement of BCL6 sometimes coexists with other translocations specific to B-NHL. Here, we report a novel B-cell lymphoma cell line, YM, established from a patient with diffuse large cell lymphoma. The YM cells expressed B-cell-associated antigens in addition to mu delta/kappa monoclonal immunoglobulin. Southern blot analysis of DNA from YM cells demonstrated rearrangement of the BCL2 gene within the 5' flanking region (5'-BCL2). Polymerase chain reaction (PCR) using primer pairs for the BCL2 exons 1 and 2, and for the constant region of the immunoglobulin kappa light chain gene (IGkappa) revealed PCR products encompassing the 5'-BCL2/IGkappa fusion, indicating that the YM cells had a t(2;18)(p11;q21) translocation. The BCL6 gene was rearranged at a point within the first intron, and cloning of the rearranged BCL6 revealed unidentified sequences juxtaposed to the 5' side of the gene. The isolated clones were mapped to 16p11.2 by high resolution fluorescence in situ chromosomal hybridization. Thus, the YM cells carried a 3q27 translocation involving 16p11.2 as a partner. Chromosome painting of metaphase spreads confirmed that the YM cells had both t(2;18) and t(3;16). Northern blot analysis using a fragment immediately adjacent to the breakpoint on 16p11.2 revealed transcriptional activity within this locus. The YM cells expressed abundant transcripts with aberrant sizes from BCL2 and BCL6, indicating deregulated overexpression of the two genes resulting from the t(2;18) and t(3;16). The YM cell line will therefore be useful to study whether BCL2 and BCL6 genes collaborate in the pathogenesis of B-NHL.

A case of primary splenic large cell lymphoma with a t(9;14)(p13;q32).

t(9;14)(p13;q32), a subtype of 14q32 translocation, plays an essential role in the development of lymphoplasmacytoid lymphoma. t(9;14)(p13;q32) Causes juxtaposition of the PAX-5 gene on 9p13 and the IgH gene on 14q32, leading to the deregulation of the PAX-5 gene. We report a case of primary splenic lymphoma with a t(9;14). The histological diagnosis was diffuse large B-cell lymphoma without plasmacytoid differentiation. The lymphoma cells showed a complex karyotype including a t(9;14). Southern blot analysis localized the breakpoint of the PAX-5 gene within a couple of kb regions upstream of the exon 1A, although the involvement of the PAX-5 gene with the immunoglobulin heavy chain gene could not be confirmed.

Genetic analysis of 8;21 chromosomal translocation without AML1 gene involvement in MDS-AML.

We have investigated a case of acute myelocytic leukaemia derived from myelodysplastic syndrome (MDS-AML) with an 8;21 translocation. In this case the AML1/MTG8 (ETO) fusion transcript was not detected by reverse transcriptase-polymerase chain reaction (RT-PCR), and the rearrangement of the AML1 gene locus was not detected by Southern blot nor pulse field gel electrophoresis (PFGE) analyses using specific probes for the AML1 gene. Fluorescence in-situ hybridization (FISH) study using cosmid probes for 21q22 revealed that the breakpoint of 21q22 was telomeric to the AML1 gene locus and centromeric from D21S259, 351, 3421 loci. This is the first report concerning the t(8;21)(q22;q22) carrying AMLs (de novo AML, MDS-AML and therapy-related AML) to show that the breakpoint at 21q22 is located outside the AML1 gene locus. It is also noteworthy that the cell-surface antigen expression pattern of the bone marrow (BM) blasts was changed from CD7+ CD2+ CD13+ CD33+ CD19- CD11b+ CD14+ CD36+ to CD7- CD2- CD13+ CD19+ CD11b- CD14- CD33+ CD34+ CD36- CD56+ during leukaemic progression, and the pattern in leukaemic phase was similar to the characteristic phenotype of de novo AML cases with t(8;21), when the AML1/MTG8 fusion transcripts are always detected by RT-PCR.

[Significance of Aspergillus fumigatus rDNA detected by polymerase chain reaction in diagnosis of pulmonary aspergillosis].

We have studied the clinical significance of Aspergillus fumigatus rDNA detected by polymerase chain reaction (PCR) for diagnosing aspergillosis. For this purpose, a specific and sensitive PCR assay was developed to amplify the 26S rDNA/intergenic spacer region of A. fumigatus. Control experiments showed that the set of primers used was capable of amplifying A. fumigatus DNA specifically and that the DNA amount of detection limit was 1 pg. Eighteen samples from 13 patients with aspergillosis and 36 samples from 24 patients without aspergillosis were tested by means of PCR, culture, latex agglutination test for galactomannan antigen and double gel diffusion assay for precipitation of antibodies to A. fumigatus. PCR showed positive test in 8 among 13 patients with pulmonary aspergillosis. On the other hand, culture of samples detected A. fumigatus in 6 patients. Galactomannan antigen and antibodies specific to A. fumigatus were positive in 2 and 6 patients respectively. These results indicated that PCR is the most sensitive among these 4 methods. Five patients showed negative PCR test despite of having pulmonary aspergillosis. Two of these patients were proved to have aspergillosis caused by A. flavus and A. niger. On the other hand, galactomannan antigen and A. fumigatus antibody were positive in one and 2 patients, respectively. PCR was positive in 2 out of 24 patients diagnosed as not having aspergillosis: one patient had diagnosis of acute bronchitis, but she showed positive culture of A. fumigatus. The other patient had diagnosis of lobar pneumonia, because any pathogens were not detected before PCR assay. The PCR assay we developed is a useful method for diagnosing aspergillosis caused by A. fumigatus as compared with other conventional methods.

[General properties and kinds of antibodies for enzyme immunoassay--characteristics and advantages of polyclonal and monoclonal antibodies].

General properties and advantages between monoclonal and polyclonal antibodies are described with regard to their specificity, cross reaction, practical consideration, affinity and isotype. Although monoclonal antibodies are superior in specificity, they have less affinity than those in polyclonal antibodies. Since monoclonal antibodies usually react with a single epitope, they sometimes show a particular characteristic to an antigen. It should be emphasized that investigation of required properties is important to obtain the best combination of antibodies for immunoassay purposes.

Acute lymphoblastic leukemia associated with a t(1;19)(q23;p13) in an adult.

A cytogenetic study of a 38-year-old patient with acute lymphoblastic leukemia (ALL) revealed a t(1;19)(q23;p13), which is a characteristic translocation of childhood ALL. The leukemic cells were positive for the CD10, CD19, HLA-DR, TdT and cytoplasmic mu-chain. Both of the immunoglobulin heavy chain gene loci were rearranged and the RNA-based polymerase chain reaction demonstrated the E2A/PBX1 fusion transcript which is the result of the t(1;19). This finding suggests that the t(1;19) is implicated not only in childhood ALL but also in adult ALL patients.

Characterization of a novel T-cell lymphoma cell line established from a patient with systemic lupus erythematosus-associated lymphoma.

A malignant lymphoma developed in a 46-year-old male patient who had had systemic lupus erythematosus (SLE) for 18 years. The lymphoma was at disease stage IV at initial examination, and the patient died shortly thereafter. The lymphoma cells were cultured in vitro, and a continuous cell line, named SMZ-1, was established. The SMZ-1 cells, as well as the parental lymphoma cells, were of helper/inducer T-cell immunophenotype; they were positive for CD2, CD3, and CD4 antigens, and negative for CD8. Expression of CD5 and CD7 antigens was observed in a small percentage of the cells. The activation markers identified by antibodies against CD25, CD71, and HLA-DR antigens were positive. Cytogenetic analysis revealed that the SMZ-1 cells had a characteristic translocation between chromosomes 6 and 14 [t(6;14)(p21.1;q24)]. Southern blot analysis of DNA extracted from the cells demonstrated clonal rearrangement of the T cell receptor beta-chain gene. Integration of the human T-cell lymphotrophic virus type I (HTLV-I) genome was negative. The SMZ-1 cell lines should thus provide a useful model for characterization of peripheral T-cell lymphomas.

A novel B cell line established from Ki-1-positive diffuse large cell lymphoma.

A novel cell line, designated KIS-1, was established from a patient with Ki-1-positive diffuse large cell lymphoma. Multiple phenotypic analysis of the KIS-1 cells was carried out with a total of 22 monoclonal antibodies defining hematopoietic cell subsets and lineages. The KIS-1 cells were positive for Ki-1, B4, HLA-DR, and 2D1 (common leucocyte) antigens, but were negative for the antigens reportedly specific for T cells, natural killer cells, granulocytes, monocytes, interdigitating reticulum cells and dendritic reticulum cells. The genomic analysis of the KIS-1 cells showed not only the rearrangement of JH and J kappa genes but also the probable rearrangement of C lambda genes. Moreover, the cells produced immunoglobulin lambda chains. Thus, KIS-1 was considered to be of B-cell lineage. The lymphoma-cell derivation of KIS-1 was based on the following facts. The cytochemical, immunologic, cytogenetic properties and the results of the molecular genomic analysis in the KIS-1 cells were essentially the same as those of the original tumor cells, and the KIS-1 cells were negative for Epstein-Barr virus-associated nuclear antigen. KIS-1 is the only known B-cell line derived from Ki-1-positive diffuse large cell lymphoma, and should be useful for defining the biological implications of Ki-1 antigen.