Detection of minimal residual disease in chronic myeloid leukemia patients after bone marrow transplantation by polymerase chain reaction

We used a modification of the polymerase chain reaction (PCR) to amplify the specific bcr-abl mRNA from 14 patients with chronic myeloid leukemia (CML) who had previously received non T cell depleted allogenic bone marrow transplantation (BMT). Two types of reactions were used: a single step amplification with 5' and 3' primers, and a double step PCR in which products of the first amplification were reamplified using nested primers. The latter procedure was highly sensitive and capable of detecting one abnormal cell in 10(7) cells. At the time of PCR analysis, all 14 patients were in hematological remission, and 13 were in cytogenetic remission. PCR analysis revealed rearranged bcr-abl mRNA in five patients. The interval from transplant in those five patients ranged from 3 to 63 months. Two of the five positive patients were reexamined after 3 months and were found negative by double step PCR. Our findings suggest that after non-T cell depleted BMT the eradication of the leukemic clone probably occurs in some patients. Other patients, however, proved to have a small number of abnormal cells even at long intervals after BMT, although these cells could only be detected transiently in some patients. The significance of these abnormal cells with respect to the risk of leukemic relapse remains to be determined.     

依据免疫球蛋白基因重排构建的实时定量聚合酶链反应体系在B淋巴细胞白血病微小残留病监测中的应用

目的 依据B淋巴细胞白血病存在的特异免疫球蛋白(Ig)轻、重链基因重排构建白血病细胞的特异定量检测方法,并将其用于B淋巴细胞白血病微小残留病的监测.方法 利用欧洲BIOMED-2体系和方法分析15例B淋巴细胞白血病患者的Ig轻、重链基因的重排序列,并据此为患者分别设计白血病细胞特异性引物,建立个体化实时定量聚合酶链反应(qPCR)体系,用于监测患者治疗后白血病微小残留病.结果 15例患者中,14例检测出个体及白血病细胞特异的Ig轻、重链基因重排序列,其中10例患者构建的Ig-qPCR反应体系具有高度特异性,检测敏感度达到10-5水平,符合国际标准,满足临床需要.该方法与其他临床常用检测白血病微小残留病的方法结果相似.结论 依据Ig基因重排设计的白血病负荷检测方法对大多数B淋巴细胞白血病患者敏感、特异、准确,能为治疗的选择提供可靠依据.     

Monitoring of residual disease in chronic myelogenous leukemia by quantitative polymerase chain reaction.

The polymerase chain reaction (PCR) has become a standard method for highly sensitive detection of the bcr/abl rearrangement in patients with chronic myelogenous leukemia (CML) or acute lymphoblastic leukemia (ALL). The exquisite sensitivity of the PCR facilitates the detection of residual leukemic cells after chemotherapy or after bone marrow transplantation. However, the detection of minimal residual disease does not yield any information on the malignant potential of the bcr/abl-rearranged cells. Qualitative PCR is therefore of limited prognostic value in the monitoring of residual leukemia. We have adapted the PCR for quantitative evaluation of cells carrying the bcr/abl rearrangement and by means of two exemplary cases of CML patients after bone marrow transplantation and under treatment with alpha-interferon, respectively, we show that this new technique is suitable for the long term follow-up of the activity of the residual bcr/abl rearranged clone. Longitudinal monitoring of residual disease by the technique presented provides a novel tool for detection of incipient relapse at a very early stage.     

Detection of minimal residual disease in acute myeloid leukemia.

The distinction of clonogenic leukemic cells (CFU-L) and normal myeloid progenitors (GM-CFU) is a problem because both types of cells respond to the same growth factors and their clones resemble each other morphologically in culture. We investigated by means of an indirect enzyme-immunoassay the expression of "early" and "late" differentiation markers on bone marrow cell suspensions, as well as on agar clones in 18 cases of newly diagnosed acute myeloid leukemia (AML) as compared with 13 normal controls. Uncultured AML cells carried only low amounts of "late" myeloid differentiation antigen (CD15) but expressed nearly normal levels when cultured in agar with colony-stimulating factor (CSF). In contrast to normal bone marrow, AML cells were strongly reactive with "early" differentiation markers (CD10, CD20, CD34) and remained so during culture. Normal and leukemic agar clones could be specifically distinguished by CD20- and CD34 antibodies. By means of a double marker technique, it could be shown that "late" myeloid differentiation markers (CD15) and "early" markers (CD10, CD20, CD34) were coexpressed on the same cells only in AML but not in normal bone marrow. Leukemic clones were identified by phenotyping of agar clones in 17 of 19 cases investigated during complete clinical remission (CR) of the disease. A formal proof of the leukemic origin of CD20/CD34 positive clones grown in CR was made possible in four cases either by Southern blot analysis or by a cytogenetic marker. These results demonstrate that AML cells can partially differentiate in vitro in the presence of CSF. A distinction of AML from normal clones, however, is possible by their reactivity with "early" differentiation markers, because this is maintained under the differentiating influence of CSF. The technique described here identifies residual leukemic clones in the majority of AML in CR, which persist at a constant rate and increase 6 months before cytological relapse.     

Rapid screening and sensitive detection of NPM1 (nucleophosmin) exon 12 mutations in acute myeloid leukaemia

Nucleophosmin mutations of exon 12 (NPM1 mutations) represent the most frequent molecular aberration that can be found in patients with acute myeloid leukaemia (AML) and can be detected in about 35% of AML patients. NPM1 mutations are characterised by four basepair insertions within the region corresponding to the C-terminus of the protein leading to its translocation out of the nucleus. Until now, more than 40 different subsets of mutations have been identified and about 90% of NPM1 mutations are represented by subtype A and B (78% versus 12%, respectively). So far, standard screening of NPM1 mutations using conventional polymerase chain reaction (PCR) followed by capillary electrophoresis is rather time consuming. We established a new method for rapid screening of NPM1 mutations using the fluorescence resonance energy transfer (FRET) principle. Furthermore, based on individual NPM1 mutations type A and B, we designed mutation specific primers to perform a highly sensitive PCR assay that can be applied for the detection of minimal residual disease (MRD). In summary, we demonstrate new methodological approaches for rapid screening of NPM1 mutations as well as for MRD analyses based on the most frequent NPM1 mutations.     

Detection of minimal residual disease in acute myeloid leukemia

Acute myeloid leukemia (AML) is a curable malignancy, but its cure rate remains disappointingly low. Accurate quantitation of residual AML cells would allow individualization of therapy and thereby increase the likelihood of cure for each patient. Techniques that are being studied for residual disease detection include molecular assays for abnormalities that are present in subsets of AML patients, and multiparameter flow cytometry, which has broader applicability. Although significant advances have been made in the development of assays for monitoring residual disease, and particularly in techniques for molecular quantitation, additional refinement and validation are needed before these assays can be applied routinely in clinical management.     

Monitoring of minimal residual disease in acute myeloid leukemia

Two highly sensitive methods, multiparameter flow cytometry (MFC) and real-time quantitative PCR (RQ-PCR), are increasingly used to monitor minimal residual disease (MRD) and to guide risk-adapted management in acute myeloid leukemia (AML). An independent prognostic impact has been demonstrated for MRD levels obtained by both methods. MFC has been found particularly useful for assessment of early clearance of malignant cells and after consolidation therapy. At the latter checkpoint, MRD levels quantified by RQ-PCR in AML with fusion genes also have the strongest prognostic power. In addition, highly predictive initial expression levels have been identified by RQ-PCR. Both methods are capable of early detection of relapse. Through the use of all available markers including NPM1 mutations and FLT3 mutations in addition to fusion genes, RQ-PCR-based MRD assessment is possible in more than half of patients, whereas MFC is applicable to most AML cases. With a sensitivity of 10(-4) (PML-RARA) to 10(-7) (patient-specific primers, FLT3 and NPM1 mutations), RQ-PCR is more sensitive in most cases. Large clinical trials will determine the exact role and place of immunologic and RQ-PCR-based monitoring of MRD in the therapy of patients with AML.     

Minimal residual disease in hairy cell leukemia patients assessed by clone-specific polymerase chain reaction.

Cladribine induces long-term complete remission in hairy cell leukemia (HCL) patients but does not clear minimal residual disease (MRD) according to high-sensitivity PCR assays. To quantify MRD in patients after anti-CD22 recombinant immunotoxin BL22 and other agents, we used a relative quantitative PCR (RQ-PCR) assay using a primer and probe, both patient specific for the immunoglobulin heavy chain rearrangement. Using this method, we were able to detect one Bonna 12 HCL cell in either 10(6) Jurkat cells or in 10(6) normal mononuclear cells. We studied 84 samples from 10 patients, taken before or after treatment with BL22 and other agents. Patient-specific RQ-PCR was much more sensitive than flow cytometry, which in turn was (as recently reported) more sensitive than PCR using consensus primers. RQ-PCR was positive in 62 of 62 (100%) flow-positive samples in 10 patients and in 20 of 22 (91%) flow-negative samples in six patients. The relative level of MRD as quantified by RQ-PCR correlated with disease status and remission. Thus, patient-specific RQ-PCR is the most sensitive test for MRD in HCL patients and could be used to determine maximal response in patients obtaining multiple cycles of nonmyelotoxic biological treatment for this disease.     

Molecular monitoring of minimal residual disease in acute leukemia

Treatment of patients with acute leukemia is based on antineoplastic drug therapy (mainly chemotherapy) and/or immunotherapy, such as allogeneic stem cell transplantation, both associated with the risk of severe toxicity, including treatment-related mortality. Therefore, the extent of therapy should ideally be adapted to the patient’s individual relapse risk. The latter can be estimated taking into account leukemia subtype as well as conventional and molecular cytogenetics, as determined at the time of diagnosis. Furthermore, particularly in acute lymphoblastic leukemia (ALL), early and subsequent assessment of treatment response is routinely incorporated into the global risk stratification. Multiparameter flow cytometry and molecular methods allow for the detection of minimal residual disease that remains obscure to conventional cytology. While molecular monitoring of the treatment efficacy has entered clinical routine in chronic myelogenous leukemia, acute promyelocytic leukemia and ALL, this concept is still evolving in acute myeloid leukemia. This short review is aimed to give an overview of current methods as well as established and candidate indications of molecular disease monitoring in patients with acute leukemia.     

黑色素瘤特异性抗原基因定量检测在急性髓系白血病及微小残留病监测中的应用

目的 探讨黑色素瘤特异性抗原(PRAME)mRNA在初诊急性髓系白血病(AML)患者中的表达,评估其在微小残留病(MRD)监测中的作用.方法 收集63例初诊AML患者骨髓标本,并在28个月内追踪了11例患者的34份样本,用Taqman探针的荧光定量PCR技术检测PRAME mRNA的表达.结果 初诊患者中PRAME基因表达阳性率52.4 ％(33/63),不同类型的AML中PRAME基因表达以M3最高;治疗缓解后PRAME基因转阴,形态学复发前数月可检测到PRAME mRNA的升高.结论 PRAMEmRNA在AML中高表达,可作为一个广谱的白血病标志物,用于MRD的监测.     

PCR结合限制性内切酶、异源双链形成分析检测白血病微小残留病的研究

目的:探讨急性淋巴细胞白血病(ALL)微小残留病的检测方法,并进一步研究了该方法对完善微小残留病检测的价值和临床意义.方法:采用PCR结合限制性内切酶酶切、异源双链形成分析的方法,分析了30例急性淋巴细胞白血病患者发病及病程中的TCR_γ、IgH基因重排.结果:30例ALL患者, 16例呈TCR_γ,重排,6例呈IgH重排,可作为微小残留病的标志.10例进行了酶谱分析和异源双链形成实验的患者,每例均有不同的V区参与重排,6例形成互不相同的异源双链.10例重排中5例有新的N区插入,形成了新的酶切位点与异源双链,亦即产生新的基因标志.结论:PCR结合限制性内切酶酶谱和异源双链形成的方法,能进一步区别不同的克隆,识别微小残留病.     

Tandem application of flow cytometry and polymerase chain reaction for comprehensive detection of minimal residual disease in childhood acute lymphoblastic leukemia.

Children with acute lymphoblastic leukemia (ALL) with > or = 0.01% leukemic cells in the bone marrow after remission induction are at a greater risk of relapse. The most promising methods of detecting minimal residual disease (MRD) are flow cytometric identification of leukemia-associated immunophenotypes and polymerase chain reaction (PCR) amplification of antigen-receptor genes. However, neither assay can be applied to all patients. Moreover, both assays carry the risk of false-negative findings due to clonal evolution. The simultaneous use of both assays might resolve these problems, but the correlation between the methods is unknown. We studied serial dilutions of normal and leukemic cells by flow cytometry and PCR amplification of IgH genes and found the two methods highly sensitive (one leukemic cell among 10(4) or more normal cells), accurate (r2 was 0.999 for flow cytometry and 0.960 for PCR by regression analysis) and concordant (r2 = 0.962). We then examined 62 bone marrow samples collected from children with ALL in clinical remission. In 12 samples, both techniques detected MRD levels > or = 1 in 10(4). The percentages of leukemic cells measured by the two methods correlated well (r2 = 0.978). Of the remaining 50 samples, 48 had MRD levels < 1 in 10(4). In only two samples results were discordant: 2 in 10(4) and 5 in 10(4) leukemic cells by PCR but < 1 in 10(4) by flow cytometry. We conclude that immunologic and molecular techniques can be used in tandem for universal monitoring of MRD in childhood ALL.     

Comparative analysis of flow cytometry and polymerase chain reaction for the detection of minimal residual disease in childhood acute lymphoblastic leukemia.

Minimal residual disease (MRD) is an independent prognostic factor in childhood acute lymphoblastic leukemia (ALL). The most widely applied MRD assays in ALL are flow cytometric identification of leukemia immunophenotypes and polymerase chain reaction (PCR) amplification of antigen-receptor genes. We measured MRD by both assays in 227 patients with childhood B-lineage ALL. Of 1375 samples (736 bone marrow and 639 peripheral blood) examined, MRD was <0.01% in 1200, and > or =0.01% in 129 by both assays; MRD levels measured by the two methods correlated well. Of the remaining 46 samples, 28 had MRD > or =0.01% by flow cytometry but <0.01% by PCR. However, PCR (which had a consistent sensitivity of 0.001%) detected leukemic gene rearrangements in 26 of these 28 samples. Conversely, in 18 samples, MRD was > or =0.01% by PCR but <0.01% by flow cytometry. In nine of these samples, flow cytometry had a sensitivity of 0.001%, and detected aberrant immunophenotypes in eight samples. Therefore, the two most widely used methods for MRD detection in ALL yield concordant results in the vast majority of cases, although the estimated levels of MRD may vary in some. The use of the two methods in tandem ensures MRD monitoring in all patients.