Colony formation in vitro by leukemic cells in acute lymphoblastic leukemia (ALL).

An assay system in vitro for the growth of malignant lymphoblastic colony-forming cells (CFC) was established. Growth of malignant myeloblastic CFC has been previously reported, but this is the first report of growth of malignant lymphoblastic CFC. Established assay systems in vitro have been very helpful in elucidating the control of growth and differentiation of both normal and malignant bone marrow cells. Lymphoblastic CFC were grown from the bone marrow aspirates of 20 children with acute lymphoblastic leukemia. Growth of these colonies was established on an agar assay system and maintained in the relative hypoxia (7% oxygen) of a Stulberg chamber. The criteria for malignancy of these colonies was based upon cellular cytochemical staining characteristics, the presence of specific cell surface markers, and the ability of these lymphoid cells to grow without the addition of a lymphoid mitogen. With this technique, specific nutritional requirements and drug sensitivities can be established in vitro, and these data may permit tailoring of individual antileukemic therapy.     

Detection of clonality by polymerase chain reaction in childhood B-lineage acute lymphoblastic leukemia

Summary DNA-based PCR with various sets of primers for TCR /, and Ig heavy chain (IgH) genes were used to study clonality in childhood B-lineage acute lymphoblastic leukemia. Amplification of the IgH CDR-III was observed in 75 of 120 analyzed cases (62.5%). From all analyzed groups, the IgH gene rearrangement was most often observed in pre-B ALL (85.7%) and was rather rare in null-ALL (34.5%). TCR delta gene rearrangement was the most common, and was observed in 77 patients (64.2%). The typical pattern of rearrangements was defined as anincomplete V2 to D3, V2 to D2, or D3 to D3 to D2 recombination product. Rearrangements of TCR gamma gene we observed in 61 cases (50.8%). TCR gamma gene rearrangements were detected predominantly in null-ALL and early B-ALL (55.2% and 60%, respectively) and were rather rare in other groups. Of all eight V segments of VI group, the most frequent gene usage concerns regions V2, V4, and V7. We have confirmed that IgH gene amplification, together with TCR gamma and delta gene amplification, provides a rapid, sensitive approach to assessing clonality in ALL almost in 100% of cases.This work was financed by KBN grants 4.0551.91.01 and 6.6346.92.03     

实时定量PCR监测造血干细胞移植后BCR/ABL融合基因的表达

目的监测慢性粒细胞白血病（CML）患者异基因造血干细胞移植（allo-HSCT）后BCR／ABL融合基因表达水平的动态变化,从而判断移植效果,指导临床治疗。方法应用Taqman实时定量RT-PCR方法,以B-actin作为内参照,检测10例初发CML患者及25例接受allo-HSCT治疗的CML患者在移植前、后不同时段外周血中BCR／ABL mRNA表达水平。结果15例接受移植的患者移植前、移植后1、2个月的NBCR／ABL（％）中位数分别为：6．57（0．14～38．83）、0．10（0～1．71）、0（0～0．52）,3个月后全部为0。移植后早期检测BCR／ABL转录本较移植前逐渐下降,移植后1、2个月NBCR／ABL（％）均较移植前下降（x^2均为13．07,P〈O．01）;移植后2个月较1个月下降（x^2=8．10,P〈0．01）。其余10例患者从移植后3～43个月不同时段起连续检测NBCR/ABL（％）也全部为0。结论实时定量RT-PCR方法检测CML患者的BCR／ABL融合基因的表达,操作迅速,灵敏度、特异性好,用于移植后微小残留病的检测对评估预后并指导临床治疗具有重要意义。     

Detection of the hybrid BCR/ABL messenger RNA in single CFU-GM colonies using the polymerase chain reaction

In order to study which hemopoietic precursor cells express the hybrid BCR/ABL fusion mRNA we have developed a technique based on the polymerase chain reaction (PCR) for the examination of single hemopoietic colonies grown on semi-solid agar. The technique was developed by examining single CFU-GM colonies grown from newly diagnosed patients with chronic myeloid leukaemia (CML). RNA was isolated from individual 14 day colonies and reverse transcribed to a complementary DNA (cDNA) copy which formed the substrate for a PCR. We have studied 3 cases of CML using this method and have found that 5 out of 5, 9 out of 10 and 20 out of 23 colonies examined were positive. Thus we describe a simple and useful technique for the study of gene expression in a limited number of hemopoietic precursor cells.     

Detection,isolation, and stimulation of quiescent primitive leukemic progenitor cells from patients with acute myeloid leukemia (AML)

Although many acute myeloid leukemia (AML) colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) directly isolated from patients are actively cycling, quiescent progenitors are present in most samples. In the current study, (3)H-thymidine ((3)H-Tdr) suicide assays demonstrated that most NOD/SCID mouse leukemia-initiating cells (NOD/SL-ICs) are quiescent in 6 of 7 AML samples. AML cells in G(0), G(1), and S/G(2)+M were isolated from 4 of these samples using Hoechst 33342/pyroninY staining and cell sorting. The progenitor content of each subpopulation was consistent with the (3)H-Tdr suicide results, with NOD/SL-ICs found almost exclusively among G(0) cells while the cycling status of AML CFCs and LTC-ICs was more heterogeneous. Interestingly, after 72 hours in serum-free culture with or without Steel factor (SF), Flt-3 ligand (FL), and interleukin-3 (IL-3), most G(0) AML cells entered active cell cycle (percentage of AML cells remaining in G(0) at 72 hours, 1.2% to 37%, and 0% to 7.6% in cultures without and with growth factors [GFs], respectively) while G(0) cells from normal lineage-depleted bone marrow remained quiescent in the absence of GF. All 4 AML samples showed evidence of autocrine production of 2 or more of SF, FL, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, 3 of 4 samples contained an internal tandem duplication of the FLT3 gene. In summary, quiescent leukemic cells, including NOD/SL-ICs, are present in most AML patients. Their spontaneous entry into active cell cycle in short-term culture might be explained by the deregulated GF signaling present in many AMLs.     

Detection of leukemic cells in the CD34(+)CD38(-) bone marrow progenitor population in children with acute lymphoblastic leukemia

Successful autologous hematopoietic stem cell (HSC) transplantation in childhood acute lymphoblastic leukemia (ALL) requires the ability to either selectively kill the leukemia cells or separate normal from leukemic HSC. Based on previous studies showing that more than 95% of childhood B-lineage ALL express CD38, this study evaluated whether normal CD34(+)CD38(-) progenitors from children with B-lineage ALL could be isolated by flow cytometry. CD34(+) cells from bone marrow samples from 10 children with B-lineage ALL were isolated at day 28 of treatment, when clinical remission had been attained. The CD34(+) progenitor cells were flow cytometrically sorted into CD34(+)CD38(+) and CD34(+)CD38(-) populations. The absolute numbers of CD34(+)CD38(-) cells that could be isolated ranged from 401 to 6245. The cells were then analyzed for the presence of clonotypic rearrangements of the T-cell receptor (TCR) Vdelta2-Ddelta3 locus. Only patients whose diagnostic marrow had an informative TCR Vdelta2-Ddelta3 rearrangement were included in this study. Detection thresholds were typically 10(-4) to 10(-5) leukemic cells in normal marrow. In 6 of 10 samples analyzed, the sorted CD34(+)CD38(-) cells had no detectable Vdelta2-Ddelta3 rearrangements. In 4 cases, the clonotypic leukemic Vdelta2-Ddelta3 rearrangement was detected in the CD34(+)CD38(-) population, indicating that the putative normal HSC population also contained leukemic cells. The data indicate that although most childhood ALL cells express CD34 and CD38, leukemic cells are also frequently present in the CD34(+)CD38(-) population. Therefore, strategies to isolate and transplant normal HSC from children with ALL will require a more stringent definition of the normal HSC than the CD34(+)CD38(-) phenotype. (Blood. 2001;97:3925-3930)     

成人Ph+急性淋巴细胞白血病不同BCR-ABL转录本特征及预后比较

目的 比较成人Ph+急性淋巴细胞白血病(ALL)不同融合基因转录本临床特征的差异,并探讨其与预后的关系.方法 回顾性分析1996年1月至2007年12月我院诊断为Ph+ALL患者(年龄≥15岁)的资料,比较两个BCR-ABL转录本组间的临床特征,探讨两组患者预后的差异.结果 (1)106例患者中位年龄34岁,中位白细胞数28.5×109/L.p190组67例,p210组35例,p210组较p190组中位年龄偏大(40岁、31岁,P=0.002),初诊血小板数高(49.5×109/L、31.5×109/L,P=0.012),中、重度脾大的发生率高(48.6%、25.4%,P=0.019).(2)两组患者完全缓解(CR)率分别为92.2%(59/64)和93.9%(31/33).(3)p190组中位总生存(OS)和中位无复发生存(RFS)分别为13个月和10个月;p210组中位OS和RFS分别为15个月和10个月.两组之间比较差异无统计学意义(P>0.05).结论 成人Ph+ALL中BCR-ABL以p190表达为主;p210患者较p190具有年龄偏大,初诊血小板数偏高,中、重度脾大发生率高的特点;两组在CR率、RFS和OS上无明显差异.     

Detection and quantitation of neoplastic cells in acute lymphoblastic leukaemia, by use of the polymerase chain reaction

We report a simple and robust method for sensitive quantitation of leukaemic cells in acute lymphocytic leukaemia. Chain determining region 3 (CDR3) of the immunoglobulin heavy chain gene is a precise genetic marker for a patient's leukaemic clone. Quantitation of the leukaemic lymphocytes was achieved by use of the polymerase chain reaction to detect CDR3 at limiting dilution of DNA samples. Five patients were studied and high levels (1 in 1 to 1 in 10) of leukaemic cells were detected at diagnosis or relapse. Leukaemic cells were detected in remission marrows from three patients, at levels of 1 in 1000 to 1 in 100,000. All five patients showed a 1000 to 100,000-fold reduction in the levels of leukaemic cells after induction therapy. This technique should prove useful for monitoring therapy and may help predict outcome.     

Association between HLA-DQB1 gene and patients with acute lymphoblastic leukemia (ALL)

Acute lymphoblastic leukemia (ALL) affects both children and adults. Survival in ALL has improved in recent decades due to recognition of its biological heterogeneity. Although children have higher remission and cure rates than adults, both populations have benefited from these improvements. Our aim in this study is to determine the association between HLA-DQB1 genes with childhood and adult ALL patients. To define this association, we compared HLA-DQB1 allele frequencies and allele carrier frequencies in a cohort of 135 adults and children with ALL with 150 controls, using polymerase chain reaction with sequence-specific primers. Allele carrier frequencies in childhood ALL show a deficiency in DQ2 (*0201) (P 0.049 and RR 0.75), but an increase in DQ5 (*0501-*0504) and DQ7 (*0301, *0304) compared to the control group (P 0.001 RR 1.89, P 0.003 RR 1.48, respectively). Allele carrier frequencies in adult ALL indicated an increase in DQ5 (*0501-*0504) (P0.045 RR 2.28). Allelic frequencies in childhood ALL revealed the same increase in DQ5 and DQ7, and a decrease in DQ2. In adult ALL it shows a decrease in DQ7. Therefore, our results in adult ALL were similar to childhood ALL addressing DQ5 allele carriers, which showed an increase in both age groups. We suggest that DQ5 could be more strongly considered as an ALL susceptibility allele, and that this allele may underlie a pathogenic phenotype with a major role in the immunologic process involved in both adults and children with ALL.     

Heterogeneity of genomic fusion of BCR and ABL in Philadelphia chromosome-positive acute lymphoblastic leukemia.

Philadelphia chromosome-positive acute lymphoblastic leukemia occurs in two molecular forms, those with and those without rearrangement of the breakpoint cluster region on chromosome 22. The molecular abnormality in the former group is similar to that found in chronic myelogenous leukemia. To characterize the abnormality in the breakpoint cluster region-unrearranged form, we have mapped a 9;22 translocation from the Philadelphia chromosome-positive acute lymphoblastic leukemia cell line SUP-B13 by using pulsed-field gel electrophoresis and have cloned the DNA at the translocation junctions. We demonstrate a BCR-ABL fusion gene on the Philadelphia chromosome. The breakpoint on chromosome 9 is within ABL between exons Ia and II, and the breakpoint on chromosome 22 is approximately equal to 50 kilobases upstream of a breakpoint cluster region in an intron of the BCR gene. This upstream BCR breakpoint leads to inclusion of fewer BCR sequences in the fusion gene, compared with the BCR-ABL fusion gene of chronic myelogenous leukemia. Consequently, the associated mRNA and protein are smaller. The exons from ABL are the same. Analysis of leukemic cells from four other patients with breakpoint cluster region-unrearranged Philadelphia chromosome-positive acute lymphoblastic leukemia revealed a rearrangement on chromosome 22 close to the breakpoint in SUP-B13 in only one patient. These data indicate that breakpoints do not cluster tightly in this region but are scattered, possibly in a large intron. Given the large size of BCR and the heterogeneity in breakpoint location, detection of BCR rearrangement by standard Southern blot analysis is difficult. Pulsed-field gel electrophoresis should allow detection at the DNA level in every patient and thus will permit clinical correlation of the breakpoint location with prognosis.     

Detection of minimal residual disease in T-cell acute lymphoblastic leukemia using polymerase chain reaction predicts impending relapse.

After achieving remission, approximately one-third of patients with T-cell acute lymphoblastic leukemia (T-ALL) relapse due to the resurgence of residual leukemic cells that cannot be detected in remission by morphologic methods. Thus, the early detection of residual disease is highly desirable to monitor the efficacy of therapy, or to institute an alternative mode of therapy. Toward this aim, we have examined the applicability of polymerase chain reaction (PCR) amplification in the detection of minimal residual disease (MRD) in bone marrow samples from patients with T-ALL in morphologic remission. Two different approaches were taken to identify leukemic clone-specific sequences that could be used as targets for PCR amplification. The first technique used T-cell receptor-delta (TCR-delta) gene rearrangements that were sequenced directly after PCR amplification of leukemic DNA. This method was successful in generating clone-specific probes for 76% of T-ALL patients screened. An alternative method was used to clone and sequence a TCR-beta chain gene from leukemic cells to generate a specific probe. The PCR assays that we used were specific for each patient's leukemic clone, and were capable of routinely detecting one leukemic cell in 10(4) normal cells. Using these sensitive PCR-based assays, we found no evidence for persistence of the leukemic clone in any of the bone marrow samples from four T-ALL patients who are in long-term (3.9 + to 8.1 + years) remission. In contrast, we detected residual disease in clinical remission samples from two patients who subsequently relapsed. In one patient, where we had appropriate samples, we observed a dramatic expansion of the leukemic clone 3 months before clinical relapse. These results suggest that PCR-based assays for detection of MRD in T-ALL patients have great potential in predicting impending relapse, and in determining the efficacy of the anti-leukemic therapy. These methods may also allow the identification of long-term survivors.     

T-cell acute lymphoblastic leukemia with severe leukopenia: evidence for suppression of myeloid progenitor cells by leukemic blasts

A patient with T-cell acute lymphoblastic leukemia presented with leukopenia due to neutropenia, no circulating blasts and normal hemoglobin level. Marrow leukemic T lymphoblasts inhibited in vitro normal CFU-GM colony growth and released an activity that stimulated normal BFU-E growth. This patient demonstrates that immature T cells may modify hematopoietic stem cell growth both in vitro and in vivo.