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.

Fluorescence in situ hybridization panel for monitoring of minimal residual disease in patients with double minute chromosomes.

A double minute chromosome (dmin) is a small fragment of extrachromosomal DNA bearing amplified genes observed in malignancies. We investigated the incidence and characteristics of dmins in hematologic malignancies, and the quantitative changes during the treatment follow-up. Once a dmin was observed in conventional G-banding, it was characterized using fluorescence in situ hybridization (FISH) with the panel of MYC, NMYC, and MLL probes. Quantitative changes of malignant cells were measured using G-banding and FISH during the follow up. Dmins were observed in 1.23% of patients (6/489) at the initial diagnosis including 4 with MYC amplification, 1 with MLL and 1 with NMYC. All 6 had complex karyotypes and showed short overall survival (7.7 months). In follow-up specimens, FISH detected dmins in 11 cases out of which G-banding detected dmins in 9 cases. The number of dmins detected by FISH and G-banding did not correlate well. Amplification of NMYC in dmins is reported for the first time. A FISH panel composed of frequently amplified oncogenes (MYC, NMYC, and MLL) in dmins is useful for characterization of dmins. FISH is a sensitive method in detecting dmins and will be useful in monitoring of the minimal residual disease.

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.

RANTES polymorphisms and the risk of graft-versus-host disease in human leukocyte antigen-matched sibling allogeneic hematopoietic stem cell transplantation.

We investigated the association between RANTES (regulated upon activation, normal T cell expressed and secreted) polymorphisms and clinical outcomes in patients treated with allogeneic hematopoietic stem cell transplantation (allo-HSCT). Three RANTES gene polymorphisms, i.e., -403G/A (rs2107538), -28C/G (rs2280788) and In1.1T/C (rs2280789), were genotyped, and the effects of the genotypes and haplotypes of RANTES on clinical outcomes were analyzed. The competing risk regression analysis was used to investigate the relationship between the polymorphisms and the cumulative risk of graft-versus-host disease (GVHD). An AGC haplotype in a recessive model showed significant harmful effects on the cumulative risk of acute GVHD and relapse-free survival (adjusted hazard ratios 2.42 and 2.71, 95% confidence intervals 1.29-4.55 and 1.30-5.64; p = 0.018 and 0.024, respectively), whereas a GCT haplotype did not. RANTES polymorphisms were not significantly associated with overall survival and the risk of chronic GVHD. This study suggests that RANTES polymorphisms might be associated with the occurrence of acute GVHD rather than of chronic GVHD and also of relapse-free survival in the patients treated with allo-HSCT. Further larger prospective investigations are needed to establish the role of RANTES polymorphisms in patients treated with allo-HSCT.

Quantity of clonal cells detected by conventional cytogenetic analysis correlates with bone marrow blasts and survival in myelodysplastic syndromes.

We performed quantitative and qualitative analyses of conventional cytogenetic analysis and interphase FISH results in 87 MDS patients. The quantity of clonal cells for each chromosome of CCA did not correlate with the result of iFISH (r, range 0.0761-1.0577). The clonal cell percentage in CCA was higher in patients with >5% bone marrow blasts than those with <5% (44.7% vs. 23.1%, p=0.017). Multivariate analysis showed that a high quantity of clonal cells in CCA analysis is an independent prognostic factor for overall survival in MDS (p=0.012).

Impact of vitamin D receptor gene polymorphisms on clinical outcomes of HLA-matched sibling hematopoietic stem cell transplantation.

We hypothesized that polymorphisms of the vitamin D receptor (VDR) gene might affect clinical outcomes of allogeneic hematopoietic stem cell transplantation (HSCT). Three VDR gene polymorphisms (BsmI G>A, ApaI G>T, and TaqI T>C) were genotyped in 147 patients who underwent HLA-matched sibling allogeneic HSCT. Frequencies of infection, graft-vs.-host disease (GVHD), overall survival (OS), and disease-free survival (DFS) were compared according to genotypes and haplotypes. Infection and acute GVHD had trends to be less frequent in patients with ApaI TT genotype than non-TT genotypes (p = 0.061 and p = 0.059, respectively). For TaqI genotypes, there were no statistical differences in frequency of infection and acute GVHD (p = 0.84 and p = 0.30, respectively), but TC genotype was associated with longer OS and DFS than TT genotype (p = 0.022 and p = 0.038, respectively). In the ApaI-TaqI haplotype analysis, patients with TC haplotype had significantly longer OS and DFS than those without TC haplotype (p = 0.022 and p = 0.038, respectively). In multivariable analysis, TaqI genotype and ApaI-TaqI haplotype of recipients were independent prognostic factors for both OS and DFS. This study suggests that the genotype and haplotype of VDR in recipient might be associated with clinical outcome of sibling HLA-matched HSCT.

[Comparison of the rate of detection of immunoglobulin heavy chain gene rearrangement by fluoresecence in situ hybridization probes in multiple myeloma.].

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.

A study on 289 consecutive Korean patients with acute leukaemias revealed fluorescence in situ hybridization detects the MLL translocation without cytogenetic evidence both initially and during follow-up.

Translocations involving the MLL gene on the chromosome 11 (11q23) are frequently observed in acute leukaemia. The detection of this genetic change has a unique significance as a result of its implication of poor prognosis. To reveal the utility of fluorescence in situ hybridization (FISH) in detecting the MLL translocation, we analysed 289 consecutive Korean patients (children and adults) with acute leukaemias using both conventional cytogenetic analysis (CC) and FISH, placing an emphasis on the result discrepancies. Twenty-two of 289 patients (7.6%) had the 11q23/MLL translocation. In nine of 22 patients (41%), only FISH detected the translocation. In eight of these 22 patients, a total of 19 follow-up examinations were performed, of which FISH detected a significant level of leukaemic cells harbouring the MLL translocation in five patients (26%) without cytogenetic evidence. In addition to the MLL translocation, FISH detected submicroscopic amplification, partial deletion of the MLL gene and trisomy 11 in 12 patients without cytogenetic evidence. In summary, up to 41% of the MLL translocations at initial work-up 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 leukaemias in combination with CC.