Characterization of the CDR3 structure of the Vβ21 T cell clone in patients with P210(BCR-ABL)-positive chronic myeloid leukemia and B-cell acute lymphoblastic leukemia

The clonally expanded T cells identified in most cancer patients that respond to tumor-associated antigen such as P210(BCR-ABL) protein have definite, specific antitumor cytotoxicity. T cell receptor (TCR) Vβ CDR3 repertoire diversity was analyzed in patients with chronic myeloid leukemia (CML) and BCR-ABL(+) B-cell acute lymphoblastic leukemia (B-ALL) by GeneScan. A high frequency of oligoclonal expansion of the TCR Vβ21 subfamily was observed in the peripheral blood of CML and B-ALL patients. These clonally expanded Vβ21 T cells were correlated with the pathophysiologic process of CML. A conserved amino acid motif (SLxxV) was observed within the CDR3 region in only 3 patients with CML. Preferential usage of the Jβ segments was also observed in a minority of patients. The 3-dimensional structures of the CDR3 region containing the same motif or using the same Jβ segment displayed low similarity; on the contrary, the conformation of the CDR3 region containing no conserved motif in some T cell clones was highly similar. In conclusion, our findings indicate a high frequency of TCR Vβ21 subfamily expansion in p210(BCR-ABL)-positive CML and B-ALL patients. The characterization of the CDR3 structure was complex. Regrettably, at this time it was not possible to confirm that the Vβ21 T cell clones were derived from the stimulation of p210(BCR-ABL) protein.     

Is Wilms’ tumor gene 1 a useful biomarker for detecting minimal residual disease in chronic myeloid leukemia (BCR-ABL1-p210-positive) patients?

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder characterized by the presence of Philadelphia (Ph) chromosome. Wilms’ tumor gene 1 (WT1) plays an important role in leukemogenesis and can be useful as a molecular marker to detect minimal residual disease (MRD). The goal of this study was to evaluate WT1 expression and compare it with BCR-ABL1 expression in peripheral blood (PB) of CML patients, in order to explore the utility of WT1 as an alternative marker for MRD detection. Eighteen newly diagnosed CML patients (BCR-ABL1-p210-positive), 16 chronic CML patients with a history of imatinib therapy, and 18 normal individuals (BCR-ABL1-p210-negative) as controls were enrolled in this study. WT1 and BCR-ABL1 expression was evaluated by quantitative real-time polymerase chain reaction (Q-RT-PCR). Analysis of RT-PCR data was performed using REST Software (2009, QIAGEN, Valencia, USA). Data were analyzed by SPSS software (v.16.0) using Spearman’s rho and Kruskal-Wallis methods. WT1 expression in all PB samples of newly diagnosed CML patients was significantly higher than that of the normal individuals (P?=?0.003). WT1 expression was not different in patients on imatinib therapy compared to normal individuals (P?=?0.6), but it was significantly lower than that of the newly diagnosed CML patients (P?=?0.001). There was no significant correlation between expression of WT1 and BCR-ABL1 and hematological findings in newly diagnosed CML patients. We confirm the oncogenic role of WT1 by WT1 upregulation in CML. Due to lack of significant correlation between BCR-ABL1 and WT1 expression, using WT1 as an additional marker for CML monitoring could not be applicable.     

Follow-up of childhood chronic myelogenous leukemia with monitoring the BCR-ABL fusion gene expression in peripheral blood

Chronic myelogenous leukemia (CML) accounts for 15-20% of adult leukemias but is very rare in children (2%). Fewer than 10% of CML patients are younger than 20 years. CML is a myeloproliferative disorder characterized by the presence of the Philadelphia chromosome or the BCR-ABL fusion oncogene. The objective of this paper is to present the monitoring of imatinib therapy in two children with CML by the BCR-ABL fusion gene expression assessment from peripheral blood with quantitative real-time polymerase chain reaction (PCR) method. Patients and Methods: The 18 and six months follow-up of the patients included clinical examination, routine laboratory tests, bone marrow aspirate investigation including cytogenetic tests and the major BCR-ABL fusion gene expression measurement with qRT-PCR method from the peripheral blood. Results: Patient No. 1 diagnosed with chronic phase CML showed excellent adherence to daily 400 mg imatinib treatment and achieved complete hematologic (CHR) and cytogenetic response (CCR) by three months and major molecular response (MMR) by 12 months, with lack of side effects due to imatinib. Patient No. 2 experienced severe hematologic toxicity, which necessitated temporary withdrawal of the drug. Transient non-compliance together with imatinib dose reduction has driven to treatment failure. In this case, mutational analysis is warranted. Conclusions: BCR-ABL fusion gene expression level measurement from peripheral blood with qRT-PCR method is an excellent tool in the follow-up of CML patients.     

Monitoring twenty-six chronic myeloid leukemia patients by BCR-ABL mRNA level in bone marrow:a single hospital experience

Chronic myeloid leukemia (CML) is caused by the BCR-ABL oncogene. The Philadelphia chromosome (Ph) from a reciprocal translocation, t(9;22) (q34;q11) causes a fusion gene, BCR-ABL, that encodes a constitutively active tyrosine kinase. Treatment of CML by imatinib is effective to control the tyrosyl phosphorylation of the protein related to the cell signaling. BCR-ABL mRNA is overexpressed in the minimal residual disease (MRD), known as an early sign of relapse. Between December 2005 and June 2008, we measured BCR-ABL mRNA levels in the bone marrow (BM) from patients by quantitative real-time polymerase chain reaction (RQ-PCR) in Aomori Prefectural Central Hospital. Eighty-six samples from 26 patients were collected. Among the 26 CML patients, 11 patients (42%) were in the pretreatment group. Seven (64%) of the 11 patients achieved complete molecular response (CMR). In the post-treatment group consisting of the remaining 15 patients, 9 (60%) patients achieved CMR. The patients receiving imatinib at a dose over 300 mg per day required 13 (6-77) months [median (range)] to achieve CMR. On the other hand, the patients receiving a dose below 300 mg per day required 29.5 (11-84) months [median (range)]. When BCR-ABL mRNA was detected during the treatment course of patients with CMR, careful observation of BCR-ABL mRNA was useful for tracking the clinical course of patients. In conclusion, the BCR-ABL mRNA level was useful for monitoring the clinical course in 26 patients with CML.     

p190 BCR-ABL rearrangement in chronic myeloid leukemia and acute lymphoblastic leukemia.

A minority of chronic myeloid leukemia (CML) cases have breakpoint in the minor cluster region (m-bcr) of the BCR-ABL fusion gene. We report a patient with Ph-positive acute lymphoblastic leukemia and m-bcr breakpoint at diagnosis. The patient was treated with chemotherapy followed by an autologous peripheral blood stem cell transplantation, achieving a clinical and hematological complete remission but with persistence of the Philadelphia chromosome. One year later, she developed leukocytosis with a blood picture consistent with CML. She was treated with hydroxyurea and interferon alpha with no response. This is the second case of m-bcr CML reported presenting with features of lymphoid blast crisis or acute lymphoblastic leukemia.     

Autologous transplantation of in vivo purged PBSC in CML: comparison of FISH, cytogenetics, and PCR detection of Philadelphia chromosome in leukapheresis products

To determine the effectiveness of different methods for the detection of tumor cell contamination of collected peripheral stem cells, we performed a study on 39 chronic myelogenous leukemia (CML) patients who were consecutively treated at our department. Analyses of tumor cell contamination by fluorescence in situ hybridization (FISH), conventional cytogenetics, and polymerase chain reaction (PCR) showed marked differences in the percentage of evaluable results: Quantitative analysis of tumor cell contamination was feasible in 60 of 105 (57%) samples evaluated with the use of conventional cytogenetic analysis and in 105 of 107 (98%) samples analyzed by FISH. PCR was evaluable in all 85 samples tested (100%). Both methods were shown to be adequate overall in determining the number of BCR-ABL positive cells, although cytogenetics tended to produce slightly higher percentages. Based on these results, we conclude that FISH performed on leukapheresis products is a rapid and reliable method for assessing the quality of these products and should be used for routine evaluation of tumor cell contamination of CML stem cell products.     

实时定量RT—PCR技术检测白血病融合基因的临床应用价值

目的评估实时定量RT—PCR技术检测白血病融合基因在辅助白血病临床诊断和药物疗效判定方面的价值。方法56例慢性粒细胞白血病（CML）患者，其中男性31例，女性25例，中位年龄54（18～80）岁；9例急性早幼粒细胞白血病（APL）患者，其中男性4例，女性5例，中位年龄47（2～64）岁。另有22例非CML或APL患者（男性12例,女性10例，年龄6～55岁）。应用实时定量RT—PCR技术检测临床患者骨髓或外周血单个核细胞中白血病融合基因M—bcr／abl或PMIdRARα的表达，结合临床资料进行分析。结果①56例CML患者的M-bcr／abl融合基因检测阳性率为96．4％（54／56）；9例APL患者的PMIdRARa融合基因检测阳性率为77．8％（7／9）。②5例CML患者应用甲磺酸-伊马替尼治疗后定量检测M—bcr／abl融合基因的拷贝数较治疗前明显降低或转阴（4／5），伊马替尼治疗CML效果优于应用羟基脲和干扰素治疗的患者。结论实时定量RT—PCR技术操作简便，检测白血病融合基因的准确性高。在辅助临床诊断和白血病患者药物疗效判定方面都有较大的应用价值。     

慢性髓性白血病患者β-catenin的表达及其临床意义

目的:比较β-catenin在慢性髓性白血病(CML)各期中的表达情况,并分析与bcr/abl及伊马替尼细胞遗传学疗效的关系,为探讨CML疾病进展机制及寻找新的治疗靶点提供理论依据。方法:采用RT-PCR和Western blotting方法,检测99例CML患者骨髓单个核细胞中β-catenin mRNA和蛋白质水平的表达,分析与bcr/abl的相关性;94例CML患者服用伊马替尼1年后FISH检测bcr/abl融合基因,分析β-catenin与伊马替尼细胞遗传学疗效的关系。结果:CML急变期、加速期患者β-catenin的表达明显增高(P0.01),而慢性期与正常人无明显差别(P0.05);β-catenin与bcr/abl表达水平无明显相关性(r=0.314,P0.05);未达主要细胞遗传学缓解的CML患者β-catenin明显增高(P0.01)。结论:CML疾病进展阶段β-catenin的表达明显升高,β-catenin的表达与bcr/abl无明显相关,而与伊马替尼疗效有关,β-catenin可能参与CML疾病进展机制,可作为新的治疗靶点。     

Analysis of cellular immune responses in the peripheral blood of mice using real-time RT-PCR.

Murine cancer models are commonly used in the evaluation of immunotherapeutic strategies. However, one of the major limitations in the monitoring of cellular immune responses induced by various vaccination approaches is that existing immunoassays require sacrifice of the animals for collection of the spleen or lymph nodes for analysis. We report here the development of an assay to quantitate antigen-specific T cell responses in murine blood, without euthanasia, using real-time RT-PCR for measurement of interferon-gamma mRNA levels. C57BL/6 mice were immunized with an adenoviral vector encoding the melanoma antigen gp100 (Ad2/gp100) or were left untreated. Small samples of whole blood were collected by retro-orbital puncture for analysis of T cell reactivity. The mice were then euthanized and spleen cells were isolated for comparative analyses. Blood and spleen cells were restimulated with either a peptide containing the dominant gp100 MHC Class I-restricted epitope, gp100(25-33), or a negative control peptide containing an irrelevant Class I-restricted epitope from ovalbumin. IFN-gamma mRNA was detected in gp100 peptide-pulsed whole blood as well as in spleen cells recovered from Ad2/gp100-treated mice, but not in untreated mice. In addition, there was a strong correlation in the magnitude of the gp100-specific response of spleen cells from an individual animal when measured by real-time RT-PCR with the more conventional enzyme-linked immunospot (ELISPOT) method (P<0.001). Finally, the gp100-specific immune response measured in the peripheral blood of individual animals by real-time RT-PCR or ELISPOT showed a significant correlation with the response measured in the spleen (P=0.001). We conclude that real-time RT-PCR measurement of IFN-gamma mRNA induced by antigenic stimulation is an attractive method to measure an antigen-specific cellular immune response in small samples of whole blood as it does not require euthanasia, mirrors the response observed in the spleen and correlates with the response measured using the conventional ELISPOT method.     

慢性粒细胞白血病相关的TCR Vβ T细胞的诱导及其限制性和克隆性分析

目的:了解体外诱导CML细胞相关TCRVβ亚家族T细胞克隆性增殖及其杀伤性的情况。方法:采用混合淋巴细胞/白血病细胞培养法(MLTC)体外将CML细胞、K562细胞和bcr-abl多肽与供者外周血单个核细胞混合培养和扩增,利用RT-PCR-基因扫描技术分析培养后T细胞的TCRVβ谱系的限制性利用和克隆性增殖情况,并经LDH法分析诱导增殖T细胞的杀伤性。结果:经bcr-abl多肽、CML细胞和K562细胞诱导扩增1-2周后,供者外周血T细胞表达10-13个Vβ亚家族,在Vβ16和Vβ21出现克隆性增殖T细胞,在Vβ5和Vβ13出现寡克隆生长趋势T细胞。诱导扩增后的T细胞对CML和K562细胞具有特异性杀伤作用。结论:利用CML细胞、K562细胞和bcr-abl多肽可在体外诱导出CML细胞特异性CTL,该CTL可能为优势表达的Vβ亚家族克隆性T细胞。     

Stem cell biomarkers in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a clonal multi-step myeloproliferative disease that is initially produced and ultimately sustained by a rare subpopulation of BCR-ABL+ cells with multi-lineage stem cell properties. These BCR-ABL+ CML stem cells are phenotypically similar to normal hematopoietic stem cells which are also maintained throughout the course of the disease at varying levels in different patients. Defining the unique properties of the leukemic stem cells that produce the chronic phase of CML has therefore had to rely heavily on access to samples from rare patients in which the stem cell compartment is dominated by leukemic elements. Here we review past and ongoing approaches using such samples to identify biologically and clinically relevant biomarkers of BCR-ABL+ stem cells that explain their unusual biology and that may help to design, or at least predict, improved treatment responses in CML patients. These studies are of particular interest in light of recent evidence that chronic phase CML stem cells are not only innately resistant to imatinib mesylate and other drugs that target the BCR-ABL oncoprotein, but are also genetically unstable.