Simple multiplex RT-PCR for identifying common fusion BCR-ABL transcript types and evaluation of molecular response of the a2b2 and a2b3 transcripts to Imatinib resistance in North Indian chronic myeloid leukemia patients.

INTRODUCTION: Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome, an abnormally shortened chromosome 22. It is the result of a reciprocal translocation of chromosomes 9 and 22, creating BCR‑ABL fusion transcripts, b3a2, b2a2, and e1a2. The aim of our study was to determine the type of BCR‑ABL fusion transcripts for molecular diagnosis and investigate the frequency of BCR‑ABL fusion transcripts in CML patients by multiplex RT‑PCR in CML. MATERIALS AND METHODS: A single reaction with multiple primers multiplex PCR was used to detect and investigate the type and frequency in 200 CML patients among which 116, 33, and 51 were in CP, AP, and BC phase, respectively. RESULTS: The study included 200 CML patients, among whom breakpoints in b3a2, b2a2 transcripts were detected in 68% and 24%, respectively, while 8% of the patients showed both b3a2/b2a2. A statistically significant difference was seen between frequency of BCR‑ABL fusion transcripts and gender (P = 0.03), molecular response (P = 0.04), and hematological response (P = 0.05). However, there was no correlation found between frequencies of BCR‑/ABL fusion transcripts and other clinicopathological parameters like age, type of therapy, thrombocytopenia, and white blood cell count. CONCLUSION: Multiplex reverse transcriptase‑polymerase chain reaction is useful and saves time in the detection of BCR‑ABL variants; the occurrence of these transcripts associated with CML can assist in prognosis and treatment of disease.

The apoptosis induction on K562 cells by the CML bcr-abl gene antisense oligonucleotides / 中国癌症杂志

Background and purpose:As the development of the completion of the human genome project (HGP), the research focus is turning to the gene function research. At present, the domestic experimental research on the apoptosis of K562 cells induced by antisense olignonucleotides is rare. This study was aimed to investigate the effect of human chronic myelogenou leukemia (CML) bcr-abl fusion gene antisense oligonucletides on autophagy and apoptosis of CMLK562 cells in vitro. Methods:By liposome as the carrier, K562 cells were transfected with the bcr-abl gene antisense olignonucleotides. Hoechst staining method was used to observe the apoptosis inducing effect of different concentrations of oligonucleotides, the expressions of LC3-Ⅱ, autophagy-related protein, were determined by the Western blot method, the cell cycles were determined by lfow cytometry (FCM), and JEM-4000EX electron microscope technology was used to detect the apoptosis morphological changes. The apoptosis was detected by DNA agarose gel electrophoresis. Results:Hoechst staining results showed that the bcr-abl gene antisense oligonucletides signiifcantly promoted the apoptosis of K562 cells in a certain concentration dependent manner. Western blot showed that the expression level of LC3-Ⅱwas obviously higher in bcr-abl gene antisense oligonucletides transfected group than the control group, showing a promoting effect on cell autophagy. FCM test results showed that bcr-abl gene antisense oligonucleotides transfected K562 cells showed obvious cell cycle arrest, visible obvious apoptosis morphology under the electron microscope, and DNA Ladder showed obvious apoptosis fragments. Conclusion:The bcr-abl gene antisense olignonucleotides can signiifcantly induce the cell apoptosis of K562. This study provides a new method for CML therapy.

Mesenchymal stem cells from patients with chronic myeloid leukemia do not express BCR-ABL and have absence of chimerism after allogeneic bone marrow transplant

Bone marrow is a heterogeneous cell population which includes hematopoietic and mesenchymal progenitor cells. Dysregulated hematopoiesis occurs in chronic myelogenous leukemia (CML), being caused at least in part by abnormalities in the hematopoietic progenitors. However, the role of mesenchymal stem cells (MSCs) in CML has not been well characterized. The objectives of the present study were to observe the biological characteristics of MSCs from CML patients and to determine if MSCs originate in part from donors in CML patients after bone marrow transplantation (BMT). We analyzed MSCs from 5 untreated patients and from 3 CML patients after sex-mismatched allogeneic BMT. Flow cytometry analysis revealed the typical MSC phenotype and in vitro assays showed ability to differentiate into adipocytes and osteoblasts. Moreover, although some RT-PCR data were contradictory, combined fluorescence in situ hybridization analysis showed that MSCs from CML patients do not express the bcr-abl gene. Regarding MSCs of donor origin, although it is possible to detect Y target sequence by nested PCR, the low frequency (0.14 and 0.34 percent) of XY cells in 2 MSC CML patients by fluorescence in situ hybridization analysis suggests the presence of contaminant hematopoietic cells and the absence of host-derived MSCs in CML patients. Therefore, we conclude that MSCs from CML patients express the typical MSC phenotype, can differentiate into osteogenic and adipogenic lineages and do not express the bcr-abl gene. MSCs cannot be found in recipients 12 to 20 months after BMT. The influence of MSCs on the dysregulation of hematopoiesis in CML patients deserves further investigation.

Monitoring of bcr-abl Fusion Transcript Levels by Quantitative Real-Time Polymerase Chain Reaction in Chronic Myeloid Leukemia after Bone Marrow Transplantation / 대한진단검사의학회지

BACKGROUND: The bcr-abl gene rearrangement is a very important genetic marker in at least 95% of cases of chronic myeloid leukemia (CML). The technique that allows the detection of bcr-abl gene rearrangement seem to be the testing for the diagnoses and the detection of minimal residual disease (MRD) and may help in monitoring patients. Recent introduction of real-time quantitative PCR should eventually allow actual quantification of the target gene during amplification. In the present study, we investigated bcr-abl chimeric mRNA quantification in CML after bone marrow transplantation (BMT) by real-time RT-PCR and its clinical applicability to the decision of therapeutic intervention. METHODS: The subjects were a consecutive series of 9 CML patients with serial quantification of bcr-abl chimeric mRNA by real-time RT-PCR in peripheral blood or bone marrow aspirates. The sensitivity of real-time RT-PCR was compared to conventional RT-PCR and then the correlation with FISH results was analyzed. RESULTS: Seventeen bone marrow samples and 40 peripheral bloods were obtained from patients. The sensitivity of real-time RT-PCR was 10(-6) and positive signals were detected in negative cases by conventional RT-PCR. A significant correlation between the bcr-abl/G6PDH ratio by real-time RT-PCR and the proportion of positive cells for bcr-abl gene rearrangement by FISH was obtained (r=0.64). In the serial blood and bone marrow specimens, we found a progressive decrease in the bcr-abl/G6PDH ratio in all the patients. CONCLUSIONS: Our findings indicated that quantitative analysis of bcr-abl chimeric mRNA by real-time RT-PCR might be a useful tool for the monitoring of minimal residual disease in bcr-abl positive leukemic patients.

A Case of Chronic Myelogenous Leukemia Manifested bcr-abl Gene Rearrangement in Pleural Effusion / 대한임상병리학회지

Chronic myelogenous leukemia (CML) has been reported to show extra-medullary involvement in 10% of cases and affect mostly lymph nodes and spleen. Extensive pleura involvement of leukemic cells is very rare and no cases of CML infiltrating pleura have been reported in Koreans. Authors recently experienced a case of CML presenting pleural effusion with malignant leukemic cells. A 76-year-old woman was admitted at Inha University Hospital because of dyspnea and rectal bleeding 3 months ago. The patient had a past history of treatment for massive pleural effusion in local clinic 4 month ago. In biochemical test, LD (lactate dehydrogenase) was 620 IU/dL, and serum uric acid was 8.1 ug/dL. Peripheral blood smear showed 3% of blasts and 65% of basophils and bone marrow biopsy revealed marked increase of cellularity. Total cell number of pleural fluid was 14,100/uL and cytological examination of the pleural fluid revealed cells with the morphological features of myeloblasts and occasional megakaryocytes. The bcr-abl gene rearrangement was detected in cells isolated from pleural fluid and bone marrow aspirates. The patient refused treatment with chemotherapeutic for CML and was discharged.

Expression changes of SHIP1 and the possible mechanism in the chronic mylogenous leukemia / 解放军医学杂志

Objective To explore the expression changes in SHIP1 gene in patients with chronic granulocytic leukemia (CGL), healthy volunteers and the K562 cells (one of human CGL cell lines), and to explore the effects of BCR/ABL expression in K562 cells on the SHIP1 gene by blocking the BCR/ABL expression in K562 cells with a specific siRNA. Methods The expression levels of SHIP1 mRNA in leukocytes of the patients with CGL, healthy controls and K562 cells were assessed with RQ-PCR. In addition, K562 cells were divided into three groups: control group (untreated), non-specific interference group (treated with non-specific siRNA), and specific interference group (treated with specific siRNA to block BCR/ABL gene). RQ-PCR and Western blotting were employed to examine the expression level of mRNA and protein (P210 and P145) of BCR/ABL and SHIP1 genes. Results The mRNA expression of SHIP1 in CGL patients and K562 cells was significantly lower than that of the healthy controls. The expression of BCR/ABL gene was down-regulated, while SHIP1 gene up-regulated, in the specific interference group. No statistical significant difference was found between the non-specific interference group and the control group. Conclusions The mRNA expression of SHIP1 is lower in patients with CGL than in the healthy control. The specific siRNA can block the expression of BCR-ABL and further suppress the expression of SHIP1.