• BMB Reports
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• v.29 no.3
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• pp.241-247
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• 1996

The Philadelphia (Ph) chromosome is the single most intensively studied chromosome alteration characterizing a human malignancy. The specific genetic alteration of chronic myelogenous leukemia (CML) is the formation of the BCR/abl fusion gene in leukemic cells. The presence of the BCR/abl gene has important diagnostic and prognostic implications in CML. The detection of BCR/abl transcripts by reverse transcriptase based polymerase chain reaction (RT-PCR) was investigated in patients with CML in whom the Ph chromosome abnormality was documented by cytogenetic analysis. In a total of 68 CML patient cases, the Ph chromosome was found in 53 cases (77.9%) by cytogenetic analysis. On the other hand, sixty two cases (91.2%) were detected to have BCR/abl gene rearrangement Of these, b3a2 was 44 cases (64.7%) and b2a2 was 17 cases (25,0%). There was one case with both b3a2 and b2a2 (1.5%). Of the fifteen cases of Ph chromosome negative by cytogenetic anlaysis, the BCR/abl gene was observed in nine cases, The results of BCR/abl fusion gene confirmed by the direct sequencing method correlated well with PCR analysis, The amplified PCR products were detected by $1{\times}10^{-5}$ dilutions. In conclusion, PCR technique is sensitive, rapid and relatively simple for a laboratory test in detecting the BCR/abl fusion gene with CML regardless of the result of cytogenetic analysis.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.22
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• pp.9961-9966
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• 2014

Background: Some reports have suggested that chronic myeloid leukemia (CML) patients have a higher prevalence of M-bcr than acute lymphoblastic leukemia (ALL) patients, which show a higher prevalence of m-bcr. However, the relationship between BCR-ABL subtypes and progression of CML and ALL remains unclear. Materials and Methods: 354 CML chronic phase (CML-CP) patients, 26 CML blastic phase (CML-BP) patients and 72 ALL patients before treatment with BCR-ABL positive were recruited for blood routine examination and bone marrow smear cytology. Some 80 CML-CP and 32 ALL patients after imatinib (IM) treatment were followed-up for BCR-ABL relative concentrations detected after treatment for 3, 6 and 9 months and 1 year. Results: Before treatment, CML-CP patients showed lower BCR-ABL relative concentrations with a higher proportion of M-bcr (42.7%) compared to CML-BP and ALL patients while ALL patients had a higher BCR-ABL relative concentration with high expression of m-bcr (51.4%). Patients with M-bcr demonstrated higher WBC counts than those with m-bcr and the mixed group and higher PLT counts were noted in the CML-CP and ALL groups. After imatinib (IM) treatment, patients with m-bcr showed higher BCR-ABL relative concentrations in both CML-CP and ALL groups. Conclusions: This study identified the BCR-ABL gene as an important factor in CML and ALL cases. The M-bcr subtype was associated more with CML while the m-bcr subtype was associated more with ALL. Patients with m-bcr seem to have a poorer response to IM in either CML or ALL patients compared to M-bcr patients.

• Korean Journal of Clinical Laboratory Science
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• v.41 no.1
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• pp.11-23
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• 2009

Chromosomal rearrangements are major pathology in hematological malignancies. The detection of minimal residual disease (MRD) for these gene rearrangements helps in monitoring treatment outcomes and predicting prognosis of patients. Recently, quantification of these gene transcripts based on real-time quantitative polymerase chain reaction (RQ-PCR) has been used as MRD detection. The purpose of this study is to ensure the usefulness of the RQ-PCR technique for detecting MRD in hamatological malignancy patients. The patients had been diagnosed to AML1-ETO positive AML, PML-RARa positive AML and BCR-ABL positive MPN at Chonnam National University Hwasun Hospital from Jan. 2006 to Aug. 2008. The fusion transcript was quntified by RQ-PCR and analyzed in comparison to conventional cytogenetics, FISH and RT-PCR. The fusion gene transcript was quantified by RQ-PCR in 57 samples from 14 patients with AML1-ETO positive AML, 79 samples from 27 patients with PML-RARa positive AML and 108 samples from 36 patients with CML. At diagnosis, the quantitative fusion transcripts for AM1-ETO, PML-RARa and BCR-ABL showed the range of 0.485552651~10.82233683 (mean 3.782217131, SD 2.998052348), 0.005300395~0.29267494 (mean 0.056901315, SD 0.080131381) and 0.1293929~12.94826849 (mean 1.701935665, SD 2.200913158). The increase of AML1-ETO fusion gene transcripts preceded morphologic relapse in two patients. Quantification of fusion gene transcripts by RQ-PCR could detected MRD in samples which were negative by in cytogenetic analysis or FISH. Our findings indicated that quantitative analysis of AML1-ETO, PML-RARa and BCR-ABL transcripts by RQ-PCR might be a useful tool for the monitoring of minimal residual disease in hematological malignancies.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.12
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• pp.4773-4780
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• 2014

Background: To investigate the effects of small interference RNA (siRNA) targeting BCR/ABL mRNA on proliferation and apoptosis in the K562 human chronic myeloid leukemia (CML) cell line and to provide a theoretical rationale and experimental evidence for its potential clinical application for anti-CML treatment. Materials and Methods: The gene sequence for BCR/ABL mRNA was found from the GeneBank. The target gene site on the BCR/ABL mRNA were selected according to Max-Planck-Institute (MPI) and rational siRNA design rules, the secondary structure of the candidate targeted mRNA was predicted, the relevant thermodynamic parameters were analyzed, and the targeted gene sequences were compared with BLAST to eliminate any sequences with significant homology. Inhibition of proliferation was evaluated by MTT assay and colony-formation inhibiting test. Apoptosis was determined by flow cytometry (FCM) and the morphology of apoptotic cells was identified by Giemsa-Wright staining. Western blotting was used to analyze the expression of BCR/ABL fusion protein in K562 cells after siRNA treatment. Results: The mRNA local secondary structure calculated by RNA structure software, and the optimal design of specific siRNA were contributed by bioinformatics rules. Five sequences of BCR/ABL siRNAs were designed and synthesized in vitro. Three sequences, siRNA1384, siRNA1276 and siRNA1786, which showed the most effective inhibition of K562 cell growth, were identified among the five candidate siRNAs, with a cell proliferative inhibitory rate nearly 50% after exposure to 12.5nmol/L~50nmol/L siRNA1384 for 24,48 and 72 hours. The 50% inhibitory concentrations ($IC_{50}$) of siRNA1384, siRNA1276 and siRNA1786 for 24hours were 46.6 nmol/L, 59.3 nmol/L and 62.6 nmol/L, respectively, and 65.668 nmol/L, 76.6 nmol/L, 74.4 nmol/L for 72 hours. The colony-formation inhibiting test also indicated that, compared with control, cell growth of siRNA treated group was inhibited. FCM results showed that the rate of cell apoptosis increased 24 hours after transfecting siRNA. The results of annexinV/PI staining indicated that the rate of apoptosis imcreased (1.53%, 15.3%, 64.5%, 57.5% and 21.5%) following treamtne with siRNAs (siRNA34, siRNA372, siRNA1384, siRNA1276 and siRNA1786). Morphological analysis showed td typical morphologic changes of apoptosis such as shrunken, fragmentation nucleus as well as "apoptotic bodies" after K562 cell exposure to siRNA. Western blot analysis showed that BCR/ABL protein was reduced sharply after a single dose of 50nmol/L siRNA transfection. Conclusions: Proliferation of K562 cells was remarkbly inhibited by siRNAs (siRNA1384, siRNA1276 and siRNA1786) in a concentration-dependent manner in vitro, with effective induction of apoptosis at a concentration of 50 nmol/L. One anti-leukemia mechanism in K562 cells appeared that BCR/ABL targeted protein was highly down-regulated. The siRNAs (siRNA1384, siRNA1276 and siRNA1786) may prove valuable in the treatment of CML.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.11
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• pp.5469-5475
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• 2012

Background and Objectives: Acute lymphoblastic leukemia (ALL) is a complex genetic disease involving many fusion oncogenes (FO) having prognostic significance. The frequency of various FO can vary in different ethnic groups, with important implications for prognosis, drug selection and treatment outcome. Method: We studied fusion oncogenes in 101 pediatric ALL patients using interphase FISH and RT-PCR, and their associations with clinical features and treatment outcome. Results: Five most common fusion genes i.e. BCR-ABL t (22; 9), TCF3-PBX1 (t 1; 19), ETV6-RUNX1 (t 12; 21), MLL-AF4 (t 4; 11) and SIL-TAL1 (del 1p32) were found in 89/101 (88.1%) patients. Frequency of BCR-ABL was 44.5% (45/101). BCR-ABL positive patients had a significantly lower survival ($43.7{\pm}4.24$ weeks) and higher white cell count as compared to others, except patients with MLL-AF4. The highest relapse-free survival was documented with ETV6-RUNX1 (14.2 months) followed closely by those cases in which no gene was detected (13.100). RFS with BCR-ABL, MLL-AF4, TCF3-PBX1 and SIL-TAL1 was less than 10 months (8.0, 3.6, 5.5 and 8.1 months, respectively). Conclusions: This is the first study from Pakistan correlating molecular markers with disease biology and treatment outcome in pediatric ALL. It revealed the highest reported frequency of BCR-ABL FO in pediatric ALL, associated with poor overall survival. Our data indicate an immediate need for incorporation of tyrosine kinase inhibitors in the treatment of BCR-ABL+ pediatric ALL in this population and the development of facilities for stem cell transplantation.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.9
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• pp.3793-3797
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• 2015

Background: Chronic myeloid leukemia (CML) is a myeloproliferative disorder of pluripotent stem cells, caused by reciprocal translocation between the long arms of chromosomes 9 and 22, t(9;22)(q34;q11), known as the Philadelphia chromosome. Materials and Methods: A total of 51 CML patients were recruited in this study. Complete blood counts of all CML patients were performed to find out their total leukocytes, hemoglobin and platelets. FISH was performed for the detection of BCR-ABL fusion and cryptogenic tests using bone marrow samples were performed for the conformation of Ph (9;22)(q34;q11) and variant translocation mechanisms. Results: In cytogenetic analysis we observed that out of 51 CML patients 40 (88.9%) were Ph positive and 4 (8.88%) had Ph negative chromosomes. Mean values of WBC 134.5 $10^3/{\mu}l$, hemoglobin 10.44 mg/dl, and platelets 288.6 $10^3/{\mu}l$ were observed in this study. Conclusions: In this study, Ph positive translocation between chromosome (9:22)(q34;q11) were observed in 40 (88.9%) CML patients.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.12
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• pp.7501-7508
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• 2013

Background: Chronic myeloid leukemia (CML) is a myeloproliferative disorder of hematopoietic stem cell scarrying the Philadelphia (Ph) chromosome and an oncogenic BCR-ABL1 fusion gene. The tyrosine kinase inhibitor (TKI) of BCR-ABL1 kinase is a treatment of choice for control of CML. Objective: Recent studies have demonstrated that miRNAs within exosomes from cancer cells play crucial roles in initiation and progression. This study was performed to assess miRNAs within exosomes of K562 cells. Methods: miRNA microarray analysis of K562 cells and K562 cell-derived exosomes was conducted with the 6th generation miRCURYTM LNA Array (v.16.0). Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were also carried out. GO terms and signaling pathways were categorized into 66 classes (including homophilic cell adhesion, negative regulation of apoptotic process, cell adhesion) and 26 signaling pathways (such as Wnt). Results: In exosomes, 49 miRNAs were up regulated as compared to K562 cells, and two of them were further confirmed by quantitative real-time PCR. There are differentially expressed miRNAs between K562 cell derived-exosomes and K562 cells. Conclusion: Selectively expressed miRNAs in exosomes may promote the development of CML via effects on interactions (e.g. adhesion) of CML cells with their microenvironment.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.7
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• pp.3349-3355
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• 2012

Background and objectives: Chromosomal abnormalities play an important role in genesis of acute lymphoblastic leukemia (ALL) and have prognostic implications. Five major risk stratifying fusion genes in ALL are BCR-ABL, MLL-AF4, ETV6-RUNX11, E2A-PBX1 and SIL-TAL1. This work aimed to detect common chromosomal translocations and associated fusion oncogenes in adult ALL patients and study their relationship with clinical features and treatment outcome. Methods: We studied fusion oncogenes in 104 adult ALL patients using RT-PCR and interphase-FISH at diagnosis and their association with clinical characteristics and treatment outcome. Results: Five most common fusion genes i.e. BCR-ABL (t 9; 22), TCF3-PBX1 (t 1; 19), ETV6-RUNX1 (t 12; 21), MLL-AF4 (t 4; 11) and SIL-TAL1 (Del 1p32) were found in 82/104 (79%) patients. TCF3-PBX1 fusion gene was associated with lymphadenopathy, SIL-TAL1 positive patients had frequent organomegaly and usually presented with a platelets count of less than $50{\times}10^9/l$. Survival of patients with fusion gene ETV6-RUNX1 was better when compared to patients harboring other genes. MLL-AF4 and BCR-ABL positivity characterized a subset of adult ALL patients with aggressive clinical behaviour and a poor outcome. Conclusions: This is the first study from Pakistan which investigated the frequency of5 fusion oncogenes in adult ALL patients, and their association with clinical features, treatment response and outcome. Frequencies of some of the oncogenes were different from those reported elsewhere and they appear to be associated with distinct clinical characteristics and treatment outcome. This information will help in the prognostic stratification and risk adapted management of adult ALL patients.