急性淋巴细胞性白血病微小残留病变筛选指标的特点分析

目的 对儿童急性淋巴细胞性白血病微小残留病变(MRD)筛选指标进行分析并评估其意义和表达特点.方法 分离35例初发B-急性淋巴细胞性白血病(ALL)患儿的单个核细胞,对符合CD38、CD45弱表达,CD58、CD21、CD22强表达,CD34和Cu同时表达,染色体相关抗原CD66c表达的,与CD10/CD34/CD19进行四色抗体组合,应用流式仪检测,如在双参数点图上所选择的四色抗体组合出现的位置明显有别于正常骨髓相应位置的,则认定该抗体组合为有效的筛选标记并进行随后的MRD监测.结果 35例患儿中31例存在至少一个MRD标记,覆盖率为88.6%;21/35例患儿(60%)存在2个或2个以上的筛选标记;TdT/CD10/CD34/CD19为最常见的四色组合.结论 TdT/CD10/CD34/CD19作为四色MRD筛选标记覆盖率高,应作为常规和首选的筛选标记;免疫表型中Pro-B缺乏有效的筛选标记,出现2个或以上的筛选标记,对提高MRD的精确度具有重要意义.     

High-throughput sequencing detects minimal residual disease in acute T lymphoblastic leukemia

High-throughput sequencing (HTS) of lymphoid receptor genes is an emerging technology that can comprehensively assess the diversity of the immune system. Here, we applied HTS to the diagnosis of T-lineage acute lymphoblastic leukemia/lymphoma. Using 43 paired patient samples, we then assessed minimal residual disease (MRD) at day 29 after treatment. The variable regions of TCRB and TCRG were sequenced using an Illumina HiSeq platform after performance of multiplexed polymerase chain reaction, which targeted all potential V-J rearrangement combinations. Pretreatment samples were used to define clonal T cell receptor (TCR) complementarity-determining region 3 (CDR3) sequences, and paired posttreatment samples were evaluated for MRD. Abnormal T lymphoblast identification by multiparametric flow cytometry was concurrently performed for comparison. We found that TCRB and TCRG HTS not only identified clonality at diagnosis in most cases (31 of 43 for TCRB and 27 of 43 for TCRG) but also detected subsequent MRD. As expected, HTS of TCRB and TCRG identified MRD that was not detected by flow cytometry in a subset of cases (25 of 35 HTS compared with 13 of 35, respectively), which highlights the potential of this technology to define lower detection thresholds for MRD that could affect clinical treatment decisions. Thus, next-generation sequencing of lymphoid receptor gene repertoire may improve clinical diagnosis and subsequent MRD monitoring of lymphoproliferative disorders.     

Copy number alterations in childhood acute lymphoblastic leukemia and their association with minimal residual disease

In vivo response to initial therapy, as assessed by determination of minimal residual disease (MRD) after 5 and 12 weeks of treatment, has evolved as a strong prognostic factor in children with acute lymphoblastic leukemia (ALL) treated according to the BFM regime. Individual treatment response may be influenced by copy number alterations (CNA) leading to altered gene expression. We aimed to evaluate CNA using high-resolution array-comparative genomic hybridization (array-CGH) in different treatment-response groups. Leukemic genomic profiles of 25 standard risk (MRD-SR) and 25 high risk (MRD-HR) patients were compared. CNAs were found in 46/50 patients (92%). The most significant difference was a gain of 1q23-qter because of an unbalanced t(1;19), found in 10/25 MRD-SR patients, but in none of the MRD-HR patients (P < 0.001). The most frequent CNAs in the MRD-HR group were deletions of genomic regions harboring the immunoglobulin genes (Ig), e.g., 2p11.2 in 60% of MRD-HR compared to 28% of MRD-SR (P = 0.045). Combining all Ig loci, significantly more MRD-HR than MRD-SR patients displayed deletions (17:8 patients, P = 0.02). Frequency of other CNAs, such as loss of 9p21 or gains of 21q, did not differ strongly between the two patient groups. This is the first study evaluating the clinical significance of CNA as detected by array-CGH in childhood ALL and the first to suggest that such analyses may provide clinically important data. This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.     

荧光原位杂交技术检测慢性粒细胞白血病微小残留病

目的 研究应用荧光原位杂交技术(FISH)检测慢性粒细胞白血病微小残留病，及用FISH技术对缓解期慢性粒细胞白血病(CML)患者外周血进行检测，评价其体内微小残留病的意义。方法 应用CG和I-FI舛对30例CML(13例给予化疗、17例移植后)患者初发和／或缓解期的骨髓及外周血标本进行分析，分别检测Ph染色体和BCR/ABL融合基因的存在。结果 对13例临床给予化疗的患者初发期的外周血和骨髓进行CG分析，Ph检出率分别为15％(2／13)、100％(13／13)。同时进行I-FISH分析，均可检出BCR/ABL融合基因。对初发期骨髓的CG、I-FISH分析结果进行统计学分析，两组无显著差异；对外周血的CG、I-FISH分析结果进行统计学分析，两组有显著差异。对其缓解期骨髓CG、I-FISH分析结果进行统计学分析，两组有显著差异；对缓解期外周血CG、I-FISH结果进行统计学分析，两组有显著差异；对缓解期外周血I-FISH和骨髓I-FISH结果进行统计学分析，两组呈显著相关。对17例移植后患者CG、I-FISH结果进行统计学分析，两组有显著差异。结论 FISH技术检测慢性粒细胞白血病微小残留病敏感性大大高于常规细胞遗传学分析；采集缓解期外周血进行荧光原位杂交分析，可作为一种方便易行的手段评价微小残留病。     

Development of a new strategy for minimal residual disease monitoring in children with B-precursor acute lymphoblastic leukemia

Despite modern regimen of chemotherapy, one-third of children with acute lymphoblastic leukaemia relapse. Recent studies have shown that a high level of minimal residual disease (MRD) at the end of induction is associated with an increased risk of relapse. We have developed and validated a real-time PCR method (RQ-PCR) to quantify MRD. We monitored 57 patients using IgH and TCR (Vdelta2Ddelta3) genes rearrangements as PCR targets. RQ-PCR was performed with a primer and a TaqMan probe designed to consensus sequences in VH segments in combination with one allele specific oligonucleotide primer complementary to the junctionnal region. A sensitivity of 10(-4) was reached for 72% of the IgH alleles (n = 50) and for 54,5% of the Vdelta2Ddelta3 alleles (n = 22). We compared the results with those obtained by competitive PCR in 53 patients: no discordance between the two methods was observed. Seventeen patients were found positive (32%) and 27 negative (51%) with both techniques and 9 children (17%) were positive only with TaqMan technology. RQ-PCR is more sensitive and more specific than competitive PCR. We thus propose that RQ-PCR might be used in first intention for MRD analysis.     

Hodgkin's disease in a child with acute lymphoblastic leukemia.

Hodgkin's disease, manifested as a second malignant neoplasm in acute lymphoblastic leukemia, rarely occurs, with seventeen cases reported including this cases. We presented the clinical and pathological features of a nine-year-old male child with acute lymphoblastic leukemia in remission. He had cervical lymph node involvement 22 months after the diagnosis of leukemia as an initial presentation of Hodgkin's disease of mixed cellularity. A brief review of related literatures was also done.     

Use of fluorescence in situ hybridization to detect minimal residual disease in hematopoietic stem cell assays from peripheral blood stem cells of 2 patients with trisomy 8 acute myeloid leukemia

Fluorescence in situ hybridization (FISH) was used to analyze peripheral blood stem cells (PBSC) and stem cell assays (SCA) derived from them in 2 patients with acute myeloid leukemia (AML) with trisomy 8 as the sole chromosome abnormality prior to undergoing autologous stem cell transplantation. In both cases, the demonstration of cells containing trisomy 8 in the stem cell product led to significant changes in the patients' treatment. In the initial PBSC collections from each patient, trisomy 8 was found in aspirated granulocyte-monocyte (GM) colonies or aspirated GM clusters but not entire cell populations of SCA dish or uncultured PBSC (one patient). FISH analyses for specific cytogenetic abnormalities in hematopoietic stem cell cultures may be a more useful means of assessing the quality of the stem cell product in patients being considered for autologous stem cell transplantation.     

Detection of t(12;21) in childhood acute lymphoblastic leukemia by fluorescence in situ hybridization

Metaphase preparations from 36 patients with acute lymphoblastic leukemia (ALL) have been retrospectively screened by fluorescence in situ hybridization (FISH) to determine the incidence of translocation (12;21) and the potential usefulness of FISH as an adjunct to conventional cytogenetic analysis. With the use of specific chromosome paints, 4 of 31 patients with B-lineage childhood ALL (13%) demonstrated rearrangements of chromosomes 12 and 21, and therefore, were considered to harbor the translocation, which had not previously been detected by conventional karyotyping. However, none of these positive cases revealed the standard reciprocal t(12;21)(p12;q22) as the sole abnormality involving chromosomes 12 and 21. The study confirms the feasibility and advantages of introducing FISH screening for t(12;21) in pediatric ALL cases and demonstrates the usefulness of FISH screening as a backup to concurrent cytogenetic analysis to resolve variant translocations and aberrant results. The presence of t(12;21) has also been correlated to clinical data to assess the prognostic significance of this translocation on its own or in association with other prognostic features.     

NRP-1/CD304 expression in acute leukemia: a potential marker for minimal residual disease detection in precursor B-cell acute lymphoblastic leukemia

Neuropilin-1 (NRP-1)/CD304 is a marker for plasmacytoid dendritic cells. We determined the distribution of NRP-1/CD304 expression on normal hematopoietic cells and in 167 acute leukemias by flow cytometry. NRP-1/CD304 surface expression was frequent in precursor B-cell acute lymphoblastic leukemia (36/51 [71%]) and uncommon in acute myeloid leukemia (22.9%). In acute myeloid leukemia, expression was noted in all (4/4) acute myeloid leukemias with the M4eo subtype and in 50% of specimens (6/12) with complex cytogenetics. On hematopoietic cells, NRP-1/CD304 was expressed on normal erythroid progenitors, plasma cells, and B-cell progenitors, as well as plasmacytoid dendritic cells. Expression was not consistently detected on other hematopoietic cell types. Owing to this distribution of expression, the detection of NRP-1/CD304 alone on a hematopoietic cell cannot be used to determine plasmacytoid dendritic cell differentiation. Finally, we show that NRP-1/CD304 is overexpressed in 30% of precursor B-cell acute lymphoblastic leukemia samples compared with normal B-cell progenitors, allowing for its potential use as a marker for the detection of minimal residual disease.     

Near haploid childhood acute lymphoblastic leukemia masked by hyperdiploid line: detection by fluorescence in situ hybridization.

Near-haploid (<30 chromosomes) acute lymphoblastic leukemia (ALL) is a rare and unique subgroup of childhood common ALL associated with a very poor outcome. It may be underdiagnosed when masked by a co-existing hyperdiploid line, which has to be distinguished from the common good-prognostic hyperdiploid (>50 chromosomes) ALL. We present three children in whom, by conventional cytogenetics, near-haploid ALL was detected on relapse. Using interphase FISH probes of chromosomes X, Y, 4, 12, and 21, we were able, in two cases, to trace the hidden near-haploid lines of approximately 5% and 20% of the cells, masked by hyperdiploid cells of approximately 80% and 70%, respectively; at relapse, the proportion was reversed, with predominant near-haploid lines of over 80% and residual hyperdiploidy of less than 10%. The near-haploid lines consisted of 24 and 27 chromosomes, and always retained the second copy of chromosome 21 or its derivative, as detected in one of our patients by SKY. The hyperdiploid clones were the exact duplicates of the near-haploid ones and contained four and two copies of the chromosomes represented in two and one copies in the near-haploid stem line, respectively. Unlike the common hyperdiploid ALL, no trisomies were observed. The patients were all aged >10 years, with WBC 0.7-30 x 10(9)/L, and a common ALL phenotype. They were treated with the ALL-BFM-95 protocol, medium risk group, and responded well to 8 days of steroid therapy, but relapsed early, within 11 months, and died a few months later. Interphase FISH technique is recommended for the detection of cryptic near-haploid clones in the diagnostic survey of ALL. To assess the prognostic value of near-haploidy in the context of the ALL-BFM protocols, a larger cohort of patients is required.     

间期双色双融合荧光原位杂交技术检测成人B细胞急性淋巴细胞白血病费城染色体

目的 探讨成人B细胞急性淋巴细胞白血病(B-lineage acute lymphoblastic leukemia.BALL)中费城染色体(Philadelphia chromosome,Ph染色体)的发生率.方法 应用针对BCR-ABL的双色双融合探针和间期荧光原位杂交(dual-color dual-fusion interphase fluorescence in situ hybridization,DDFISH)技术前瞻性检测112例初诊成人B-ALL患者染色体标本中Ph染色体.并与常规细胞遗传学(conventional cytogenetics,CC)结果 进行比较.结果 成功进行CC检测的89例患者中有16例(17.98%)检出Ph染色体,而DD-FISH检测112例发现35例(31.25%)Ph染色体,阳性细胞率为18.50%～99.00%(平均66.23%).35例DD-FISH检测出Ph+的成人B-ALL患者中,10例未能成功进行CC检测,5例核型正常,16例检出Ph染色体,20例核型异常患者中13例为复杂异常.结论 Ph染色体在成人B-ALL中以DD-FISH检测发生率为31.25%;用BCR-ABL探针进行DD-FISH为检测Ph染色体提供了有效的方法 .是细胞遗传学检查的重要补充;对成人B-ALL患者应常规进行DD-FISH检测.     

Competitive PCR for quantification of minimal residual disease in acute lymphoblastic leukaemia

A very precise and reproducible polymerase chain reaction (PCR) method was developed in order to quantify minimal residual disease (MRD) in children with acute lymphoblastic leukaemia (ALL). A clone-specific competitor was constructed by introducing a restriction site in a PCR product identical to parts of the highly specific rearranged T-cell receptor delta (TCR-delta), T-cell receptor gamma (TCR-gamma), or immunoglobulin heavy chain (IgH) genes of the malignant clone. Using primers located externally to the restriction site the competitor and the DNA from the malignant clone will be amplified under identical conditions. After restriction enzyme cleavage, the PCR products originating from the competitor and the malignant clone can be distinguished by size in a gel electrophoresis step and the amount of residual disease can be determined. The method is very sensitive with a detection limit of at least one malignant cell in 10(5) normal cells. This method may be used for treatment stratification based on the early response to antileukaemic therapy.     

Detection of minimal residual T cell acute lymphoblastic leukemia by flow cytometry.

We have developed a flow cytometric assay for the determination of cellular expression of terminal deoxynucleotidyl transferase (TdT) and applied this to the detection of minimal residual T cell acute lymphoblastic leukemia (T-ALL). The flow cytometric assay for TdT demonstrated requisite specificity: TdT was localized to the nucleus, and was detected in MOLT3 T lymphoblasts, clinical T-ALL samples, and normal bone marrow B lymphoid precursors, but in neither the KG1a myeloid leukemia cell line nor normal myeloid cells. Co-expression of TdT and the pan T cell marker CD5 was used to quantify T lymphoblasts. 0.25 +/- 0.13% of normal adult bone marrow CD5+ cells were TdT+; these may represent early T lymphoid precursors. When admixed with normal bone marrow, CD5+TdT+ leukemic cells could be detected above background levels at an added concentration of 0.035% (95% confidence interval 0.028-0.43%). Long term follow-up of a large number of patients will be required to determine the clinical significance of a minimal burden of leukemic cells.     

Characterization of the translocation breakpoint sequences in Philadelphia-positive acute lymphoblastic leukemia

We have previously described a patient in whom the breakpoint occurred within the first intron of the BCR gene and have cloned the 9q+ and 22q- junctions. We have now determined the nucleotide sequence around the breakpoints on both translocation products from this patient as well as the corresponding regions from the normal chromosomes 9 and 22. We have compared the sequence with that of the breakpoint regions in the Ph1-positive leukemic patients in order to check for the presence of conserved motifs. A + T-rich sequences and ALU repeat elements are the only sequence characteristics which appear to be very common around translocation regions. The chromosome 9 ABL sequences at or adjacent to the breakpoints present in the 22q- product show homology to the consensus ALU sequence while the chromosome 22 sequences do not, suggesting a non-homologous recombination mechanism. While no sequences are deleted, there is a two-base-pair "homology" at the junction. Therefore, staggered breaks followed by ligation and repair could be part of the mechanism involved in the process of translocation in some cases of Ph1-positive ALL.     

AML1 amplification in a child with acute lymphoblastic leukemia

We report a case of de novo acute lymphoblastic leukemia with tandem amplification of the AML1 gene located in a chromosome marker that originated from chromosome 21 and a long event-free survival.     

Combined binary ratio labeling fluorescence in situ hybridization analysis of chordoma

Chordoma is a rare, low- to intermediate-grade malignant tumor involving the axial spine. Cytogenetic data on these tumors have been limited to 25 cases. The findings of clonal chromosome aberrations in five new cases are presented. One of these and two previously reported cases have been studied with multicolor combined binary ratio labeling fluorescence in situ hybridization (COBRA-FISH). The karyotypes were near-diploid, mostly with several numerical and structural aberrations. There were multiple imbalances, with loss of segments from 1p, 3p, 3q, 9p, and chromosome 10 seen in two to four of the seven cases. No clustering of breakpoints was seen and no recurrent recombination between chromosomes was detected. The findings are consistent with previous data and indicate that chordoma tumor development is associated with multiple, nonrandom losses including chromosome segments that are frequently involved in many other solid tumors.