Kyungja Han

Effects of multiagent chemotherapy and independent risk factors in the treatment of high-risk GTT —25 years experiences of KRI-TRD

A retrospective and comparative study of high‐risk gestational trophoblastic tumor (GTT) treated with different chemoregimen from 1971 to 1995 was performed and to find most effective chemotherapy regimen and independent risk factors.

Detection of Kinase Activity Using Versatile Fluorescence Quencher Probes

Protein phosphorylation is the most universal form of posttranslational modification of cell-signal transduction in living organisms. The human kinome comprises 518 protein kinases that control protein phosphorylation; irregular control of protein phosphorylation is a major cause of diseases such as cancer. Therefore, accurate probing of the kinase activity of a target protein is crucial for cancer diagnosis and high-throughput screening of anticancer drugs. 3] For the high-throughput analysis of kinase activity, several research groups have developed various types of protein or peptide chips using radioactive labeling with [Pg]-adenosine 5’-triphosphate (ATP) or using antibody hybridization. However, a crucial problem involved in the use of these onchip detection methods is that some kinases show decreased activities on the surfaces of chips because of reduced enzyme accessibility to the substrate. In recent years, several pharmaceutical and biotechnology companies have developed homogeneous kinase assay systems based on fluorescence polarization (FP) for developing anticancer drugs. These platforms (kinase assay systems) utilize peptide substrates with an N-terminal fluorophore and phospho-specific antibodies or phosphopeptide (or phosphoprotein)-binding nanoparticles (IMAP). However, FPbased detection has been reported to be very sensitive to fluorescence interference, and it is liable to produce false positives when used to screen a large number of compounds. Furthermore, there are no reports on the real-time monitoring of kinase activity in cell lysates through FP-based kinase detection; this is because many cellular components can bind to the fluorescent peptides and produce false positives for FP. Recently, peptideor protein-linked synthetic fluorescent probes that are sensitive to certain protein kinases have been reported by the research groups of Lawrence, Imperiali, Sames, and Hamachi. Ting, Tsien, and co-workers used fluorescent proteins to develop an in vivo probe system to detect kinase. These synthetic probes enabled real-time fluorescence monitoring of the specific activity of kinases in cellular lysates, and exhibited immense potential for use in the development of kinase activity inhibitors for certain kinases. However, it is still difficult to predict and determine the optimal sites for attaching fluorophores near the phosphorylated sites on the substrate peptides or proteins; the attachment of these fluorophores is necessary to induce significant changes in the fluorescence signal after phosphorylation of the substrate peptides or proteins by a specific kinase. Therefore, a general strategy for developing a synthetic fluorescent kinase probe is desired. We designed chemosensors Dab-DPA and PTZ-DPA (Scheme 1) to develop a simple but powerful kinase assay tool based on fluorescence intensity changes (ON/OFF). Using these chemosensors, we show for the first time the diagnosis of chronic myelogenous leukemia (CML) through real-time fluorescence monitoring of Abelson (Abl) tyrosine kinase activity and the development of a fluorescence-based homogeneous kinase assay system on a microfluidic chip. As shown in Scheme 1, Dab-DPA consists of a bis(Zndipicolylamine) complex and a dabcyl (Dab) fluorescence quencher, and PTZ-DPA consists of the dipicolylamine complex and a phenothiazine (PTZ) fluorescence quencher. Dab and PTZ quench fluorescence by F rster resonance energy transfer (FRET) and photoinduced electron transfer (PET), 11g] respectively. The bis(Zn-dipicolylamine) complex is a well-known synthetic receptor that strongly and selectively binds to phosphate in aqueous solution. DabDPA and PTZ-DPA are synthesized in a few steps (see the Supporting Information). PTZ is a good fluorescence quencher but there are very few reports on its use as such, except for the isoalloxazine ring of flavins. 11g] To prove that PTZ can be used as a general fluorescence quencher for other fluorophores, such as carboxyfluorescein (FAM) or tetramethyrhodamine (TMR), we performed electrochemical analyses of PTZ, FAM, and TMR. Figure 1a shows the cyclic voltammograms of 1 mm PTZ, TMR, and FAM; the Pt-disk working electrode is immersed in acetonitrile with 0.1m tetrabutylammonium hexafluorophosphate (TBAPF6) as supporting electrolyte. The observed waves are assigned to the oxidation of PTZ and the fluorophores (TMR and FAM). PTZ undergoes nearly Nernstian oxidation at E1/2,ox = 0.63 V with a peak separation [*] Dr. H.-W. Rhee, S. H. Lee, Dr. I.-S. Shin, S. J. Choi, Prof. Dr. T. H. Park, Prof. Dr. J.-I. Hong Department of Chemistry School of Chemical & Biological Engineering Seoul National University, Seoul 151-747 (Korea) Fax: (+ 82)2-889-1568 E-mail: thpark@snu.ac.kr jihong@snu.ac.kr

Epidemiology and time trends of gestational trophoblastic disease in Korea

Objectives: For the purpose of determining the annual incidence and time trends of gestational trophoblastic disease (GTD), the medical records from 24 university hospitals, 13 private general hospitals and the Korean Research Institute of Gestational Trophoblastic Disease (KRI‐TRD) were analyzed from 1971 to 1995.

HUMAN BASOPHILS EXPRESS CD22 WITHOUT EXPRESSION OF CD19

BACKGROUND Even modern automatic cell counters cannot count basophils precisely. Therefore, we need a rapid, accurate, precise, and easy method for counting basophils. METHODS Using flow cytometry, basophils (CD22+/CD19-) and B cells (CD22+/CD19+) were counted. Within a large lymphocyte light scatter gate, % basophils (G%baso) and % B cells (G%B) were determined from the total count. Another method of analysis was to make two regions (R1 for basophils and R2 for B cells) and to determine in those the % basophils (R1%baso) and % B cells (R2%B) without gating. The flow cytometric basophil counts of the blood of 21 normal controls and 43 chronic myelogenous leukemia (CML) patients were compared with manual basophil count (Ma%baso) and basophil count by Coulter electronic cell counter (Hialeah, FL) (Auto%baso). CD22+/CD19- cells were sorted by a FACSCalibur (Becton Dickinson, San Jose, CA). RESULTS The G%baso of all samples was 4.66 +/- 5.35%, and R1%baso was 4.23 +/- 4.88%, and they were well-correlated (r = 0.996, P < 0.001). The G%B of all samples was 1.55 +/- 1.68%, and R2%B was 1.59 +/- 1.67%, and they were also well-correlated (r = 0.993, P < 0.001). Their correlation was better in normal controls than in CML. G%baso was well-correlated to Ma%baso (r = 0.827) and Auto%baso (r = 0.806), and R1%baso was well-correlated to Ma%baso (r = 0.831) but showed poor correlation to Auto%baso (r = 0.734). Auto%baso revealed the poorest correlation to Ma%baso (r = 0.692). The sorted CD22+/CD19- cells were all basophils (99.48 +/- 0.30%), and they revealed CD13, CD33, and dim CD45 expression, whereas CD3, CD14, CD16, and HLA-DR were not detected on them. CONCLUSIONS We discovered a specific marker combination to identify basophils (CD22+/CD19-), and we suggest that flow cytometric analysis using these markers is an easy, reliable, and accurate method of basophil counting.

Comparison of chromosome aberrations in leiomyoma and leiomyosarcoma using FISH on archival tissues

Fluorescence in situ hybridization (FISH) with chromosome-specific probes was used to study cytogenetic changes in five cases of leiomyosarcoma (LMS) and nine cases of uterine leiomyoma (LM). Biotinylated DNA probes for the centromeric regions of chromosomes 1, 6, 8, 9, 17, and 18, painting probes for chromosomes 1 and 22, and the cosmid probe for chromosome region 21q22.3 were used on nuclei isolated from paraffin blocks. Four of five LMS cases revealed major chromosomal aberrations, while the only case with minor clonal aberrations was subsequently found not to be a typical LMS. The most common numerical aberrations found in the LMS cases were extra copies of chromosome 8 (three of five cases), loss of chromosome 1 (three of five cases), and loss of chromosome 6 (two of five cases). One of two LMS cases studied with a chromosome 1 painting probe demonstrated translocations of chromosome 1. In contrast to LMS, only five of nine uterine LM cases had abnormal clones, and these were smaller than those in LMS. Two LM cases showed 9% tetrasomy 8 with 17 or 20% monosomy 6, and three other cases had monosomy 6 clones in 18-34% of cells. These results indicate that typical LMS is characterized by multiple chromosomal aberrations affecting most of the cells, whereas borderline LMS and LM have fewer affected chromosomes and less clonal involvement.

Chromosomal numerical aberrations in gastric carcinoma: Analysis of eighteen cases using in situ hybridization

Paraffin-embedded tumor cells of 18 cases of gastric carcinoma were hybridized with digoxigenin-labeled repetitive DNA probes specific for the centromeric regions of chromosomes X, Y, 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, and 20. All cases demonstrated numerical chromosomal aberrations. The most exciting aberration, polysomy (five or more copies) of several chromosomes, was found in all cases except a case of mucinous adenocarcinoma, which showed trisomy 9 as the sole chromosomal numerical aberration. In nine cases of tubular adenocarcinoma, poorly-differentiated polysomies of several chromosomes were the consistent numerical aberration and monosomy 7, 18(2 cases each), 10, and 17(1 case each) were also found. In moderately-differentiated tubular adenocarcinoma all three cases also showed polysomies of several chromosomes. The total number of extra chromosomes (polysomy was counted as 5 copies) was higher in the intestinal type (mean 20.9) than in the diffuse type (mean 14.1). Regional lymph node metastasis, vein invasion, or perineural invasion was not related to any specific chromosomal numerical aberration in gastric cancer. Chromosomes X, 1, 2, 3, 4, 15, 17, and 20 had extra copies especially polysomy in most cases. However, chromosomes 7 and 18 revealed monosomy in many cases (31.3% and 33.3% respectively, and chromosome 9 and 11 revealed trisomy in 35.7% and 75% each. Numerically, the most conserved chromosome in gastric cancer was chromosome 12 (62.5%). By flow cytometry, two diploidy and 8 aneuploidy cases with the DNA indices from 1.30 to 1.85 were found.

The genetic characterization of acute promyelocytic leukemia with cryptic t(15;17) including a new recurrent additional cytogenetic abnormality i(17)(q10)

The genetic characterization of acute promyelocytic leukemia with cryptic t(15;17) including a new recurrent additional cytogenetic abnormality i(17)(q10)

AgNOR of Human Interphase Cells in Relation to Acrocentric Chromosomes

Using simultaneous detection of fluorescence in situ hybridization (FISH) to acrocentric chromosome centromeres and argyrophilic nucleolar organizer regions (AgNOR), we investigated the number of AgNOR and involvement pattern of acrocentric chromosomes in the nucleoli in various types of human interphase cells. The number of AgNOR of normal gastric mucosal epithelial cells was 2.27 +/- 1.18 and was higher than that of lymphocytes (1.08 +/- 0.28) and lower than that of gastric cancer (7.76 +/- 3.21). The number of acrocentric chromosome centromere signals of normal gastric mucosal epithelial cells was higher than that of normal leukocytes (P < 0.000), and lower than that of gastric cancer (P < 0.000). The acrocentric chromosome centromere signals in the lymphocytes and neutrophils were only half of that expected for diploid cells, perhaps related to acrocentric chromosome association. The proportion of acrocentric chromosomes attached to AgNOR in gastric cancer (0.88 +/- 0.22) was significantly higher than that of normal gastric mucosal epithelial cells (0.72 +/- 0.35, P = 0.007). In conclusion, acrocentric chromosome association appears to be present in circulating leukocytes even in interphase. The number of AgNORs and proportion of acrocentric chromosomes involved in AgNORs in human interphase cells may vary according to cell types. This could play a significant role in rDNA transcription and determination of cell phenotype, including malignant change.

In situ Hybridization Studies of Cytomegalovirus and Epstein-Barr Virus in Reactive Histiocytic Hyperplasia with Hemophagocytosis

We studied 14 adult patients presenting with fever and cytopenia of the peripheral blood and histiocytic hyperplasia with hemophagocytosis (HHH) in the bone marrow regarding an association of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) by using in situ hybridization (ISH) and also evaluated the clinical and laboratory findings according to the encountered organisms. ISH using a CMV RNA probe demonstrated infected cells in 6 out of 14 cases (43%), and ISH using an EBV EBER RNA probe demonstrated infected nuclei in 5 out of the same 14 cases (36%) of HHH. No cases showed a positive reaction with both probes. Three cases showed a negative reaction with both probes. The mean age of all patients was 29 years; and that of the CMV-positive patients was 27 years and that of the EBV-positive patients was 36 years. Organomegaly was found in 3 out of 6 CMV-positive patients (1 hepatomegaly, 1 splenomegaly, 1 hepatosplenomegaly), and 4 out of 5 EBV-positive patients (lymphadenopathy in all 4 cases, hepatosplenomegaly in 2 cases). One of the CMV-positive case had acute myeloblastic leukemia, and 2 EBV-positive cases had underlying malignancy (1 Hodgkins disease, 1 non-Hodgkins lymphoma). Seven out of the 14 HHH cases (50%) died within several months after diagnosis. Nucleic acid hybridization methods can be used for the routine examination of the association of CMV or EBV.

Acute lymphoblastic leukemia with maturation--a new entity with clinical significance.

The diagnosis of ‘ALL with maturation’ (ALLm) is proposed. One hundred and one patients with untreated ALL were entered into this study. The diagnosis of ALLm was made when more than 20% of all nucleated elements in the bone marrow showed maturation beyond prolymphocytes by light microscopic examination. The mature-appearing leukemic cells showed the same immunophenotype to remaining lymphoblasts. The number of ALLm cases was 19 (18.8%). The mean age at presentation of ALLm was 29 ± 18, older than that of 18 ± 16 of the remaining typical ALL (ALLt) (P = 0.015). Remission was induced with daunorubicin, vincristine, prednisone and L-asparaginase. Only two of 19 ALLm patients achieved CR after 4 weeks induction chemotherapy. In contrast, 57 of 82 (69.5%) ALLt patients achieved CR after the same induction chemotherapy. There was no significant difference in immunophenotype of ALLm compared with ALLt. Labeling index of DNA topoisomerase IIα (TopoLI) was studied by immunohistochemistry. Initial TopoLI of ALLm (221 ± 147) was much lower than that of ALLt (609 ± 262, P = 0.005). Furthermore, the remaining leukemic cells after chemotherapy were not labeled with anti-DNA topoisomerase IIα. The P53 protein was expressed in nine of 18 ALLm cases (50.0%) and P-glycoprotein was not expressed in ALLm cases. Twelve of 19 ALLm cases were studied for carrying bcr/abl fusion by karyotyping and/or fluorescent in situ hybridization. Only two cases revealed bcr/abl fusion. In conclusion, ALLm is a separate entity of ALL which has a very poor clinical course and is independent of other prognostic factors. The morphologically mature leukemic cells are in resting G0 phase.