NSE/αNAE positivity in B-lineage acute lymphoblastic leukemia: revisiting a potential cytochemical diagnostic pitfall

Cytochemical staining for leukemia typing is declining in hematology laboratories, but the use of flow cytometry may not be possible in some settings. Aberrant cytochemical nonspecific esterase/α-naphthyl acetate esterase (NSE/αNAE) positive B-lymphoblasts can cause confusion with monoblasts, a potentially dangerous pitfall. This unusual cytochemical NSE/αNAE positivity had been associated with relatively poorer outcome of acute lymphoblastic leukemia (ALL) in the era prior to the advent of routine multicolor flow cytometric immunophenotyping. We reviewed morphological, cytochemical and flow-cytometric data from five cases of B-lineage ALL that showed NSE/αNAE positivity and were diagnosed definitively using multi-parametric flow cytometric immunophenotypic analysis. Diffuse or dot-like (localized) strong cytochemical NSE/αNAE activity was detected in all cases and all showed one or more features of high risk disease. The number of NSE/αNAE positive blasts in the marrow varied from 10 to 75%. The morphological differential diagnoses included T-lymphoid lineage ALL and acute monoblastic leukemia (AML-M5). Flow cytometric data revealed B-lineage antigens and the absence of monocytic or other myeloid markers resolved the diagnosis. These cases underscore the importance of immunophenotyping in all cases of suspected ALL regardless of the cytochemical findings. Although the numbers are small, the association with high risk disease observed in all five of our cases may corroborate the previously reported poor prognostic value of such aberrant cytochemical staining.     

185例儿童急性白血病流式细胞术免疫分型研究

目的：探讨儿童急性白血病流式细胞术免疫分型的意义。方法：采用流式细胞术三色荧光标记技术和CD45／SSC双参数散点图设门,检测185例儿童急性白血病的免疫表型,对抗原表达情况进行分析。结果：流式细胞术免疫分型和FAB分型的符合率为89．19％。185例儿童急性白血病中,ALL为121例,占AL的65．41％,B—ALL为113例,主要表达B系的CD19（99．12％）、CD22（98．13％）、CD79a（96．19％）、CD10（86．73％）。T—ALL占8例;主要表达CD5（100％）、CD7（100％）、cCD3（100％）、CD8（87．5％）。AML为47例,占25．41％,主要表达CD33（93．62％）、CD15（78．72％）、CD64（76．6％）、MPO（76．6％）、CD13（74．47％）。在B—ALL,AML,T—ALL中,敏感性最高的抗体分别是CD19,CD33,CD5和CD7,特异性最强的抗体分别是CD79a,MPO,cCD3。AMLL为17例,占9．19％,其中B／M为9例,T／B为5例,T／M为3例。My＋-ALL为54例,占ALL的44．63％,表达的髓系抗原为CD13、CD15、CD33、CD64。Ly＋-AML为18例,占AML的38．30％,表达的淋系抗原为CD19、CD4、CD7。系列非相关抗原CD34的表达率为67．57％,HLA—DR的表达率为85．41％,CD38的表达率为80．59％,TdT的表达率为62．59％。结论：流式细胞术免疫分型在白血病分型中起重要作用,是FAB分型的补充和修正,提高了儿童急性白血病诊断的准确率。有必要进一步加强流式细胞术免疫分型的标准化工作。     

bcr rearrangement and C-abl gene expression in Ph1-positive hybrid acute leukemia with simultaneous proliferation of lymphoid and myeloid blasts

bcr gene rearrangement and c-abl gene expression were analyzed in a patient with Philadelphia chromosome (Ph1)-positive hybrid acute leukemia with simultaneous proliferation of lymphoid and myeloid blasts. These data were compared with those from a patient with chronic myelogenous leukemia (CML) in mixed crisis. The leukemic cells of both patients showed immuno-phenotypic profiles such as non-T, non-B common ALL with some MPO-positive leukemic cells and rearranged JH genes. On analysis of molecular events associated with the Ph1 chromosome, the leukemic cells of a patient with CML in mixed crisis showed bcr rearrangement and an 8.5-kb bcr-abl chimeric mRNA, but those of a patient with Ph1-positive hybrid acute leukemia showed no 8.5-kb bcr-abl mRNA, as previously reported in a number of Ph1-positive acute lymphoblastic leukemia (ALL) cases. These results revealed that the molecular event found in Ph1-positive ALL is not only restricted to lymphoid lineage but may play an important role in the proliferation of the myeloid lineage.     

Flow cytometric DNA index and karyotype in childhood lymphoblastic leukemia.

Flow cytometric DNA-index (DI(FCM)) and karyotype were analysed in 82 consecutive children with acute lymphoblastic leukemia (ALL) during a 10 year period. A statistically significant correlation existed between modal chromosome number and DI(FCM) (p = 0.009). DI(FCM) could reliably identify leukemias with >51 chromosomes, whereas only three out of 12 cases with modal chromosome numbers between 47-51 were classified as aneuploid by DI(FCM). In the pseudodiploid group only one out of 20 leukemias had a DI(FCM) > 1.0. Five leukemias with a diploid karyotype showed an aneuploid DI(FCM) and in three patients the flow cytometric measurement revealed biclonality undetected by karyotyping. During treatment aneuploid clones could be detected by DI(FCM) in a substantial number of cases where the cytogenetic analysis was normal, and the opposite was also demonstrated in one case. DI(FCM) gave prognostic information, showing that cases with a DI > 1.12 (corresponding to 51 chromosomes) had a superior outcome with treatment protocols today in use.     

185例儿童急性白血病流式细胞术免疫分型研究

目的:探讨儿童急性白血病流式细胞术免疫分型的意义。方法:采用流式细胞术三色荧光标记技术和CD45/SSC双参数散点图设门,检测185例儿童急性白血病的免疫表型,对抗原表达情况进行分析。结果:流式细胞术免疫分型和FAB分型的符合率为89.19%。185例儿童急性白血病中,ALL为121例,占AL的65.41%,B-ALL为113例,主要表达B系的CD19(99.12%)、CD22(98.13%)、CD79a(96.19%)、CD10(86.73%)。T-ALL占8例;主要表达CD5(100%)、CD7(100%)、cCD3(100%)、CD8(87.5%)。AML为47例,占25.41%,主要表达CD33(93.62%)、CD15(78.72%)、CD64(76.6%)、MPO(76.6%)、CD13(74.47%)。在B-ALL,AML,T-ALL中,敏感性最高的抗体分别是CD19,CD33,CD5和CD7,特异性最强的抗体分别是CD79a,MPO,cCD3。AMLL为17例,占9.19%,其中B/M为9例,T/B为5例,T/M为3例。My十-ALL为54例,占ALL的44.63%,表达的髓系抗原为CD13、CD15、CD33、CD64。Ly+-AML为18例,占AML的38.30%,表达的淋系抗原为CD19、CD4、CD7。系列非相关抗原CD34的表达率为67.57%,HLA-DR的表达率为85.41%,CD38的表达率为80.59%,TdT的表达率为62.59%。结论:流式细胞术免疫分型在白血病分型中起重要作用,是FAB分型的补充和修正,提高了儿童急性白血病诊断的准确率有必要进一步加强流式细胞术免疫分型的标准化工作。     

Impact of prophylactic/preemptive donor lymphocyte infusion and intensified conditioning for relapsed/refractory leukemia: a real-world study

Prophylactic/preemptive donor lymphocyte infusion(p/pDLI) and intensified conditioning have shown promising results in experimental studies of refractory/relapsed acute leukemia(RRAL), but real-world data remain scarce. We conducted a multicenter, population-based analysis of 932 consecutive patients. The three-year leukemia-free survival(LFS) rates were 56% for patients receiving both p/pDLI and intensified myeloablative conditioning(MAC)(intenseMAC) and 30% for those who received neither therapy per landmark analysis. Multivariable analyses were run separately for acute myeloid leukemia(AML)and acute lymphoblastic leukemia(ALL), and p/pDLI treatment was linked to significantly higher LFS than non-DLI for both AML and ALL patients without increasing the nonrelapse mortality. IntenseMAC was associated with significantly lower relapse and higher LFS than nonintensified MAC despite higher nonrelapse mortality rates in ALL, while there was no impact of intenseMAC observed in AML. p/pDLI achieved superior outcomes in both matched-sibling donor(MSD) and haploidentical donor transplantation, while intenseMAC only influenced MSD outcomes. Data suggest that RRAL patients receiving "total therapy" by way of p/pDLI and intensified conditioning treatment have an improved chance for LFS, with p/pDLI being safer with a more extensive impact relative to intenseMAC. Patients with RRAL can tolerate both interventions and achieve a reasonable outcome.     

CD47 在急性白血病干细胞中的表达及其对临床疗效及预后的影响

目的:探讨CD47在急性白血病患者骨髓白血病细胞的表达及其临床意义。方法:选择2013年5月-2015年5月在我院确诊的急性白血病患者101例作为研究对象,其中急性淋巴细胞白血病50例(ALL组),急性髓系白血病51例(AML组)。另选取同期在我院接受体检的健康志愿者39例作为对照组。采用流式细胞仪检测白血病细胞表面CD47的表达情况,并分析CD47表达与急性白血病患者临床疗效及复发情况的关系。结果:急性白血病患者白血病细胞CD47的阳性表达率明显高于健康对照组,差异具有统计学意义(P0.05);而ALL组与AML组患者白血病细胞CD47的阳性表达率比较差异无统计学意义(P0.05);CD47阴性表达的急性白血病患者CR率显著高于阳性表达者,差异具有统计学意义(P0.05);ALL组和AML组CD47阴性表达患者CR率显著高于CD47阳性表达患者,差异具有统计学意义(P0.05),但两组之间比较,差异无统计学意义(P0.05);CD47阳性表达的急性白血病患者复发率显著高于阴性表达患者,差异具有统计学意义(P0.05);ALL组和AML组CD47表达阳性患者复发率明显高于阴性患者,差异具有统计学意义(P0.05),但两组之间比较差异无统计学意义(P0.05)。结论:急性白血病患者白血病细胞表面CD47的表达异常升高,且与白血病患者的疗效和预后有关,CD47可能作为一种急性白血病的诊断及疗效和预后的辅助评估指标。     

c-erbB-2/neu protein expression, DNA ploidy and S phase in breast cancer

Abstract. DNA content and c-erbB2/neu protein (p185) expression were evaluated by flow-cytometry and ELISA, respectively, in 166 specimens of primary breast cancer. A non-diploid DNA content was found in 88 tumours (53%), with the DNA index ranging from 0.7-2.7. S phase fraction (SPF) evaluation, performed in 130 cases, showed significantly higher values in aneuploid than in diploid tumours (median values, 17.3% and 5.8%, respectively). Thirty-six tumours (21.6%) showed p185 overexpression, while 45 (27.1%) and 85 (51.3%) showed intermediate and low expression, respectively. A good correlation ( P =0.0023) was found between DNA content and p185 positivity. Tumours with high p185 values were mainly aneuploid, while tumours with intermediate or low expression had variable degrees of DNA content. Furthermore, p185 concentration was significantly higher in aneuploid than in diploid tumours ( P =0.009). The highest rate of p185 (+) cases and the highest p185 concentrations occurred in triploid (1.3相似文献   

Cytogenetic and FISH findings are complementary in childhood ALL

Primary genetic abnormalities of leukemia cells have important prognostic significance in childhood acute leukemia. In the last two years 30 newly diagnosed or recurrent childhood ALL bone marrow samples were analyzed for chromosomal abnormalities with conventional G-banding and interphase-fluorescence in situ hybridization (I-FISH) using probes to detect BCR/ABL fusions, cryptic TEL/AML1 and MLL rearrangements and p16(9p21) tumor suppressor gene deletions. G-banded karyotype analysis found clonal chromosomal aberrations in 50% of cases. With the use of complementary I-FISH techniques, ALL-specific structural and numerical changes could be identified in 70% of the patients. Nine cases (30%) had subtle chromosomal aberrations with prognostic importance that had not been detected in G-banded analysis. Conventional G-banding yielded additional information (rare and complex structural aberrations) in 19% of patients. The most common aberration (30%) was AML1 copy number increase present in G-banded hyperdiploid karyotype as a chromosome 21 tetrasomy in the majority of cases; one case displayed 5-6 copies and in another case amplification of AML1 gene on der(21) was combined with TEL/AML1 fusion of the homologue AML1 gene and deletion of the remaining TEL allele. High hiperdiploidy was detected in 6 cases, in one patient with normal G-banding karyotype. TEL/AML1 fusion signals were identified in four patients. Deletion of p16 locus was found in eight cases (23%), of which only two had cytogenetically visible rearrangements. G-banding in combination with I-FISH has produced major improvements in the sensitivity and accuracy of cytogenetic analysis of ALL patients and this method helps to achieve a more precise identification of different risk categories in order to choose the optimal treatment.     

Modeling mechanisms of in vivo variability in methotrexate accumulation and folate pathway inhibition in acute lymphoblastic leukemia cells

Methotrexate (MTX) is widely used for the treatment of childhood acute lymphoblastic leukemia (ALL). The accumulation of MTX and its active metabolites, methotrexate polyglutamates (MTXPG), in ALL cells is an important determinant of its antileukemic effects. We studied 194 of 356 patients enrolled on St. Jude Total XV protocol for newly diagnosed ALL with the goal of characterizing the intracellular pharmacokinetics of MTXPG in leukemia cells; relating these pharmacokinetics to ALL lineage, ploidy and molecular subtype; and using a folate pathway model to simulate optimal treatment strategies. Serial MTX concentrations were measured in plasma and intracellular MTXPG concentrations were measured in circulating leukemia cells. A pharmacokinetic model was developed which accounted for the plasma disposition of MTX along with the transport and metabolism of MTXPG. In addition, a folate pathway model was adapted to simulate the effects of treatment strategies on the inhibition of de novo purine synthesis (DNPS). The intracellular MTXPG pharmacokinetic model parameters differed significantly by lineage, ploidy, and molecular subtypes of ALL. Folylpolyglutamate synthetase (FPGS) activity was higher in B vs T lineage ALL (p<0.005), MTX influx and FPGS activity were higher in hyperdiploid vs non-hyperdiploid ALL (p<0.03), MTX influx and FPGS activity were lower in the t(12;21) (ETV6-RUNX1) subtype (p<0.05), and the ratio of FPGS to γ-glutamyl hydrolase (GGH) activity was lower in the t(1;19) (TCF3-PBX1) subtype (p<0.03) than other genetic subtypes. In addition, the folate pathway model showed differential inhibition of DNPS relative to MTXPG accumulation, MTX dose, and schedule. This study has provided new insights into the intracellular disposition of MTX in leukemia cells and how it affects treatment efficacy.     

In vitro investigation of the apoptotic effect of heparin on lymphoblasts by using flow cytometric DNA analysis and fluorometric caspase-3 and -8 activities

The apoptotic effect of heparin on the lymphoblasts obtained from 12 newly diagnosed children with acute lymphoblastic leukemia (ALL) was investigated in vitro. The lymphoblasts were incubated with 0, 10, and 20 U/mL heparin concentrations at 0, 1, and 2 h. The percentages of the apoptotic lymphoblasts were calculated by flow cytometry (FCM), and activities of caspase-3 and -8 were simultaneously measured by fluorometric protease activity method. The apoptotic effect of heparin on the lymphoblasts was determined in 10 and 20 U/mL heparin concentrations (p < 0.005 and p < 0.001, respectively) while no apoptosis was detected in 0 U/mL heparin concentration at 0, 1, and 2 h. The apoptotic percentages of the lymphoblasts were higher at the first hour than those at 0 and 2 h in 10 and 20 U/mL heparin levels (p < 0.001). The highest apoptosis was found at first hour in 20 U/mL heparin concentration. Increased concentrations of heparin had an increasing effect on the percentages of the apoptotic lymphoblasts. Significantly higher caspase-3 and -8 activities were determined in 10 and 20 U/mL heparin concentrations than those in 0 U/mL heparin concentration at 0, 1, and 2 h (p < 0.001). There were no significant differences between the caspase-3 and -8 activities in 10 and 20 U/mL heparin concentrations at 1 and 2 h (p > 0.05), while statistically significant differences were simultaneously detected in the apoptotic rates of the lymphoblasts (p < 0.001). This may be due to that the study included the limited patients, or measurement of the caspase activities is a more sensitive method than the FCM analysis for determination of apoptosis because the activation time of the caspases takes a long time period. It was concluded that the apoptotic effect of heparin in vitro on lymphoblasts developed due to the extrinsic pathway of apoptosis via the caspase-3 and -8 activations in newly diagnosed ALL patients.     

Dilution effect of nontumor cells on flow cytometric DNA S-phase fraction in breast cancer fine needle aspirates

OBJECTIVE: To analyze the proportion of nontumor cells in fine needle aspirates of breast carcinoma and its influence on flow cytometric S-phase fraction (SPF) estimation. STUDY DESIGN: We analyzed the proportion of nontumor cells in fine needle aspiration biopsy smears, performed flow cytometric analysis of DNA ploidy and SPF on freshly aspirated tumor material and analyzed histograms manually and automatically using Multi-Cycle AV software (Phoenix Flow Systems, San Diego, California, U.S.A.) for cell cycle analysis. We corrected SPF of diploid tumors for the dilution effect using an individually established percentage of nontumor cells (individual correction) and the mean proportion of nontumor cells in diploid tumors (factor correction). RESULTS: The proportion of nontumor cells ranged from 0.5% to 76.6% (mean, 12.6; SD, 15.7) in 55 diploid tumors and from 0.5% to 53% (mean, 8.6; SD, 8.9) in 84 aneuploid tumors (p=0.178). In 14 of 139 (10%) samples, the proportion of nontumor cells exceeded 20%. The mean SPF values of diploid tumors without correction were 4.9% (manually) and 6.5% (automatically) and of aneuploid tumors, 9.5% and 11.0%, respectively. In univariate Cox survival analysis, noncorrected SPF was a significant prognostic factor in overall survival (p < 0.001). Neither individual nor factor correction of SPF significantly changed its prognostic value. CONCLUSION: Fine needle aspirates contain low proportions of nontumor cells, having an insignificant dilution effect on SPF estimation. Most probably, SPF could be reliably estimated usingfreshly aspirated tumor material without any correction or adjustment.     

Fluorocytometric study of proliferation antigens, nuclear proteins and ploidy in acute leukosis]

P53 protein, Ki67 proliferative associated antigen and DNA content have been studied by flow cytometry in the blood blastic cells from 41 patients with acute leukemia. The results were compared with the F.A.B. classification. Cells were permeabilised and fixed by PLP solution before using the FITC conjugated Ki67 MoAb and the p53 MoAb (clone 1801). Propidium iodide and RNAase has been used in order to determine ploidy. Ki67 and p53 protein were found to be expressed at higher level in leukemia cells than in normal bone marrow cells; however there was no correlation between Ki67 and p53 expression and F.A.B. subtype. In acute leukemia patients the range of positivity of Ki67 was 1.1-52.1% while it ranged from 1.8% to 80.1% for the p53 protein. On the basis of these findings we conclude that the flow cytometry evaluation of Ki67 and p53 represents a useful tool for the study of the biologic characteristics of the leukemic cells in patients with acute leukemia.     

Cytometric evaluation of transferrin receptor 1 (CD71) in childhood acute lymphoblastic leukemia

Transferrin receptor 1 (CD71) is a transmembrane glycoprotein responsible for cellular iron uptake. Higher expression of CD71 has been identified as a negative prognostic marker for numerous solid tumor types and for some lymphomas. The aim of this study was to evaluate CD71 expression on acute lymphoblastic leukemia (ALL) cells and to follow its possible clinical correlations. Sixty one patients, aged 1-17 years and diagnosed with ALL, were enrolled in the study. CD71 expression was analyzed on the bone marrow blastic cells by flow cytometry. CD71 expression on the leukemic blasts was diversified; in most patients, all blastic cells showed expression of CD71, but levels of expression varied. CD71 expression was statistically higher on T-lineage leukemias. Within the B lineage ALL, a significant difference in CD71 expression existed between precursor B ALL and mature B-ALL, which showed higher CD71 expression. CD71 expression positively correlated with Hgb concentration at diagnosis. Initial risk group assessment and therapy response were not correlated with CD71 expression, although disease free and overall survival times tended to be shorter in patients with B-lineage leukemias with initial high CD71 expression.     

Philadelphia chromosome-positive acute lymphoblastic leukemia- current concepts and future perspectives

Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL) is diagnosed rarely in children, but constitutes the most frequent cytogenetic abnormality in adults with ALL. In contrast to chronic myeloid leukemia (CML), patients with Ph-positive ALL usually demonstrate expression of a truncated version of the BCR-ABL protein called p190bcr-abl. Irrespective of age and breakpoint location, Ph-positive ALL carries a poor prognosis. Although remission rates are identical to those of Ph-negative ALL, relapse is almost universal and long-term survival remains rare. Given the poor outcome with current chemotherapy consolidation programs, stem cell transplantation is usually recommended for these patients in first remission or as soon as feasible. Even with transplantation the impact on outcome is limited and new therapeutic concepts are urgently needed. One of the most promising developments in recent years has been the introduction of the tyrosine kinase inhibitors such as STI571. An overview of current treatment modalities in Ph-positive ALL will be provided and the rationale for new therapies will be discussed.     

Promoter methylation of p16 and EDNRB gene in leukemia patients in Taiwan

Both epigenetic and genetic alternations are involved in cancer formation. In this study, we have identified the methylation frequency of p16 and endothelin receptor type B (EDNRB) of 26 leukemia patients and 8 randomly selected normal blood donors in Taiwan. Promoter methylation of p16 was detected in 85% of acute lymphocytic leukemia (ALL), 83% in acute myeloid leukemia (AML) whereas no methylation was detected in chronic myeloid leukemia (CML) in blast crisis. Hypermethylation of EDNRB was observed in 92% of ALL, 75% AML and 100% in CML in blast crisis. No aberrant methylation of p16 and EDNRB was found in 8 normal blood donors. Taken together, aberrant methylation of p16 and EDNRB was highly prevalent in leukemia patients in Taiwan.     

Expression of CD13/aminopeptidase N in precursor B-cell leukemia: role in growth regulation of B cells

Expression of cell surface CD13 in acute B-cell leukemia (ALL-B) is often viewed, as an aberrant expression of a myeloid lineage marker. Here, we attempted to study the stage specific expression of CD13 on ALL-B blasts and understand its role in leukemogenesis as pertaining to stage of B-cell ontogeny. A total of 355 cases of different hematological malignancies were diagnosed by immunophenotyping. Among 68 cases of early B-cell ALL, 22 cases with distinct immunophenotype was identified as immature B-cell ALL. Blasts from these ALL-B patients demonstrated prominent expression of CD10, CD19, CD22, but neither cytoplasmic nor surface IgM receptors. This strongly indicates leukemogenesis at an early stage of B-cell development. We also identified, the existence of a subpopulation of cells with remarkably similar phenotype in non-leukemic marrow from healthy subjects (expressing CD10, CD19, CD22, CD24, Tdt together with the co-expression of CD13). This sub-population of B cells concomitantly expressing CD13 appeared to be a highly proliferating group. By blocking their cell surface CD13 in leukemic blasts with monoclonal antibody we were able to inhibit their proliferation. We hypothesized that neoplastic transformation at this stage may be facilitated by CD13. CD13 may thus be an important target for novel molecular therapy of early stage acute B-cell leukemia.     

Prognostic significance of proliferative activity, DNA-ploidy, p53 and Ki-ras point mutations in colorectal liver metastases

Paired colorectal liver metastases (CLM) and normal tissue samples from a consecutive series of 36 patients were studied prospectively. MIB-1 expression was studied by immunohistochemistry on paraffin-embedded sections. DNA ploidy and S-phase fraction (SPF) measurements were performed by flow cytometry on frozen tissues. Mutations within the p53 (exons 5-8) and c- Ki-ras (codons 12 and 13) genes were detected by PCR single-strand conformation polymorphism analysis followed by sequencing. A high correlation was observed between the MIB-1 LI and SPF value (rho=0.81; P <0.01). Moreover, p53 gene mutations were associated with either high MIB-1 LI and high SPF. In univariate analysis, SPF and MIB-1 levels were related to risk of death. The association between overall survival and DNA-ploidy or p53 mutations did not reach statistical significance, but a slightly better survival was observed for patients either with DNA-diploid tumours or without mutations ( P =0.05 and P =0.06, respectively). SPF was shown by multivariate Cox model analysis to be an independent prognostic variable and thus it might be a useful prognostic factor in patients with CLM.