Dysregulation of Telomere Lengths and Telomerase Activity in Myelodysplastic Syndrome

BACKGROUND: Telomere shortening is thought to be involved in the pathophysiology of myeloid malignancies, but telomere lengths (TL) during interphase and metaphase in hematopoietic malignancies have not been analyzed. We aimed to assess the TLs of interphase and metaphase cells of MDS and telomerase activity (TA) and to find out prognostic significances of TL and TA. METHODS: The prognostic significance of TA by quantitative PCR and TL by quantitative fluorescence in situ hybridization (QFISH) of interphase nuclei and metaphase chromosome arms of bone marrow cells from patients with MDS were evaluated. RESULTS: MDS patients had shorter interphase TL than normal healthy donors (P<0.001). Average interphase and metaphase TL were inversely correlated (P=0.013, p arm; P=0.029, q arm), but there was no statistically significant correlation between TA and TL (P=0.258). The progression free survival was significantly shorter in patients with high TA, but the overall survival was not different according to average TA or interphase TL groups. Multivariable Cox analysis showed that old age, higher International Prognostic Scoring System (IPSS) subtypes, transformation to AML, no history of hematopoietic stem cell transplantation and short average interphase TL (<433 TL) as independent prognostic factors for poorer survival (P=0.003, 0.001, 0.005, 0.005, and 0.013, respectively). CONCLUSIONS: The lack of correlation between age and TL, TA, and TL, and the inverse relationship between TL and TA in MDS patients reflect the dysregulation of telomere status and proliferation. As a prognostic marker for leukemia progression, TA may be considered, and since interphase TL has the advantage of automated measurement by QFISH, it may be used as a prognostic marker for survival in MDS.

The application of an in situ karyotyping technique for mesenchymal stromal cells: a validation and comparison study with classical G-banding

The cytogenetic analysis of mesenchymal stromal cells (MSCs) is essential for verifying the safety and stability of MSCs. An in situ technique, which uses cells grown on coverslips for karyotyping and minimizes cell manipulation, is the standard protocol for the chromosome analysis of amniotic fluids. Therefore, we applied the in situ karyotyping technique in MSCs and compared the quality of metaphases and karyotyping results with classical G-banding and chromosomal abnormalities with fluorescence in situ hybridization (FISH). Human adipose- and umbilical cord-derived MSC cell lines (American Type Culture Collection PCS-500-011, PCS-500-010) were used for evaluation. The quality of metaphases was assessed by analyzing the chromosome numbers in each metaphase, the overlaps of chromosomes and the mean length of chromosome 1. FISH was performed in the interphase nuclei of MSCs for 6q, 7q and 17q abnormalities and for the enumeration of chromosomes via oligo-FISH in adipose-derived MSCs. The number of chromosomes in each metaphase was more variable in classical G-banding. The overlap of chromosomes and the mean length of chromosome 1 as observed via in situ karyotyping were comparable to those of classical G-banding (P=0.218 and 0.674, respectively). Classical G-banding and in situ karyotyping by two personnel showed normal karyotypes for both cell lines in five passages. No numerical or structural chromosomal abnormalities were found by the interphase-FISH. In situ karyotyping showed equivalent karyotype results, and the quality of the metaphases was not inferior to classical G-banding. Thus, in situ karyotyping with minimized cell manipulation and the use of less cells would be useful for karyotyping MSCs.

Comparison of the Rate of Detection of Immunoglobulin Heavy Chain Gene Rearrangement by Fluoresecence In Situ Hybridization Probes in Multiple Myeloma / 대한진단검사의학회지

BACKGROUND: Immunoglobulin heavy chain (IgH) gene rearrangement, which is frequently observed in multiple myeloma, can now be detected easily by using a fluorescence in situ hybridization (FISH) method. The aim of this study was to determine the detection rate and compare the utility of the three most commonly used probes: IGH/CCND1 dual color, dual fusion probe; IGH/BCL2 dual color, dual fusion probe; and IGH dual color break apart rearrangement probe; all from Vysis Products (Downers Grove, IL, USA). METHODS: From October 1994 to July 2003, 99 patients were diagnosed as multiple myeloma at Seoul National University Hospital, Asan Medical Center and Gachon University Gil hospital. We applied the three different probes of IgH FISH on bone marrow specimens from the 99 Korean patients with multiple myeloma to detect IgH gene rearrangement. RESULTS: Forty-one (41.4%) of the 99 patients had IgH gene rearrangement. Of those 41 patients, 23 (56.1%) showed positive to all three probes, but the remaining 18 (43.9%) showed a discrepancy between the three probes: 13 (72.2%) of the 18 patients were only positive to the IGH dual color break apart rearrangement probe and the detection rate was 39.6% on the average. CONCLUSIONS: These results demonstrate that IGH dual color break apart rearrangement probe is superior to the other two probes in qualitative and quantitative ways. Thus, we recommend IGH dual color break apart rearrangement probe for the diagnosis and monitoring of multiple myeloma.

Analysis of Discrepancies Between G-banding and FISH in Hematologic Abnormalities / 대한임상병리학회지

BACKGROUND: The effective treatment of hematologic malignancies depends upon application of different therapeutic strategies by selecting patients known as the high risk group and the detection of malignant cells that can not be distinguished during following-up. We compared the results of G-banding and fluorescence in situ hybridization (FISH), which are used most frequently in detecting genetic changes, with the respect to investigating the discrepancies between these methods. METHODS: G-banding and FISH were performed on 919 consecutive specimens from 304 patients with hematologic malignancies. As for FISH, we covered most of the more frequent gene-tic changes, using 18 types of FISH probe. RESULTS: The average discrepancy between G-banding and FISH was 8.6% with a discrepancy at initial diagnosis of 6.0% and at follow-up of 11.9%, indicating greater discrepancy at follow-up after treatment. The chromosomal changes with especially large discrepancies were TEL/AML1, BCR/ABL & del(5q) (22.4%, 18.1%, and 16.2%, respectively). According to each disease, the discrepancies in acute biphenotypic leukemia (33.3%), acute lymphoblastic leukemia (14.7%), and chronic myelogenous leukemia (9.6%) were larger than average discrepancy. CONCLUSTIONS: We concluded that application of FISH is effective for detecting genetic changes in hematologic malignancies. Once genetic changes are detected, follow-up with FISH would be especially effective for making an accurate assessment of the likelihood of complete remission and recurrence.

Why Should FISH Be Done in Patients with Acute Leukemias to Detect MLL Translocation? / 대한임상병리학회지

BACKGROUND: Translocations involving the MLL gene on the long arm of chromosome 11 (11q23) are frequently observed in acute leukemia. The detection of this genetic change has a unique significance due to its implication for poor prognosis. The aim of this study was to determine the utility of fluorescence in situ hybridization (FISH) method in detecting the MLL translocation. METHODS: We applied both conventional cytogenetic analysis (CC) and MLL FISH on 289 consecutive Korean patients (children and adults) with acute leukemia and analyzed the data, placing an emphasis on the discrepancies in the results. RESULTS: Twenty-two of 289 patients (7.6%) had the 11q23/MLL translocation. In 9 cases of 22 (41%), only FISH detected the translocation. In 8 among 22 patients, a total of 19 follow-up examinations were performed, of which FISH detected a significant level of leukemia cells harboring the MLL translocation in 5 (26%) without cytogenetic evidence. Besides the MLL translocation, FISH detected submicroscopic amplification, partial deletion of the MLL gene, and trisomy 11 in 12 cases without cytogenetic evidence. CONCLUSIONS: These results demonstrate that up to 41% of the MLL translocations at initial workups and 26% during follow-up were detected by FISH without cytogenetic evidence. Thus, we recommend that MLL FISH should be performed in the diagnosis and monitoring of acute leukemia in combination with CC.

The incidence of ABL Deletion on Derivative 9 Chromosome in Chronic Myelogenous Leukemia by Interphase Fluorescence In Situ Hybridization and its Association with Progression to Blast Crisis / 대한임상병리학회지

BACKGROUND: Philadelphia(Ph) chromosome is found in about 95 percent of chronic myelogenous leukemia(CML) patients. Ph chromosome results from a reciprocal translocation between the long arms of chromosomes 9 and 22, and the fusion gene, BCR-ABL contribute to oncogenesis. Three to five years after first diagnosis, CML progresses to the blast crisis, and is accompanied by secondary cytogenetic changes in about 85% of cases. In this study, we investigated the incidence of ABL deletion of derivative 9 chromosome in CML and evaluated the association between this deletion and progression to the blast crisis by interphase fluorescence in situ hybridization(FISH). METHOD: The subjects included in this study were a consecutive series of 58 patients who were diagnosed as CML at Seoul National University Hospital between January 1997 and April 2000. On 90 archival bone marrow aspirate samples from these 58 CML patients, interphase FISH was performed with a commercially available probe. RESULTS: The ABL deletion of derivative 9 chromosome was detected in 17(29.3%) of 58 patients with CML. Eighteen of 58 patients progressed to blast crisis in this period. ABL deletion was found in 7 of 18 patients with blast crisis, and not in 11 remainders. The mean duration from the diagnosis to blast crisis was 37.1 months in 7 patients with the ABL deletion, while the mean duration was 74.2 months in 11 patients without the ABL deletion. The mean duration from the diagnosis to blast crisis in patients with ABL deletion was significantly shorter than in patients without ABL deletion(P=0.043). CONCLUSIONS: We found that 29.3% of patients with CML had the ABL deletion on derivative 9 chromosome. In these patients, the time taken for evolution to blast crisis was significantly shorter than that of the patients without ABL deletion.