Relevance of immunophenotypes to prognostic subgroups of age,WBC, platelet count,and cytogenetics in de novo acute myeloid leukemia

Li X, Li J, Du W, Zhang J, Liu W, Chen X, Li H, Huang S, Li X. Relevance of immunophenotypes to prognostic subgroups of age, WBC, platelet count, and cytogenetics in de novo acute myeloid leukemia. APMIS 2010. Immunophenotyping is one of the independent prognostic factors in acute myeloid leukemia (AML). Relevance of immunophenotypes to prognostic subgroups of age, white blood cells (WBC), platelet count, and cytogenetics in de novo AML was comprehensively investigated in this study for the first time. Human leukocyte antigen (HLA)‐DR and CD14 expression associated with the elderly, highest WBC count, and unfavorable‐risk cytogenetics; CD4, CD7, and CD11b expression correlated with highest WBC count and unfavorable‐risk cytogenetics; CD64 expression was associated with higher WBC count while that of CD13 was associated with lower platelet count; CD22, CD34, CD123, and terminal deoxynucleotidyl transferase (TdT) expression correlated with unfavorable‐risk cytogenetics; CD5 expression was associated with normal platelet count while that of CD19 was associated with children and favorable‐risk cytogenetics; CD117 expression was associated with low WBC and lower platelet counts; myeloperoxidase (MPO) expression correlated with lower platelet count; and MPO and glycophorin A (Gly‐A) expression was associated with lower WBC count and favorable‐risk cytogenetics. The results of the relevance analysis revealed the distribution characteristics of antigen expression in different AML prognostic subgroups. The majority of antigens associated with good or poor prognostic subgroups were in accordance with the previous reports of correlation of expression of these antigens with prognosis. Antigens associated with good (or poor) prognostic subgroups were defined as good (or poor)‐risk antigens.     

Podocalyxin: a marker of blasts in acute leukemia

Podocalyxin is a CD34 family member expressed by podocytes, vascular endothelium, mesothelium, and a subset of hematopoietic progenitors. Podocalyxin expression was not observed in the hematopoietic cells of normal adult bone marrow samples. However, podocalyxin was expressed by blasts in 30 (77%) of 39 cases of acute myeloid leukemia (AML), 22 (81%) of 27 cases of acute lymphoblastic leukemia (ALL), and 13 (87%) of 15 cases of cutaneous myeloid sarcoma. No correlation with CD34 expression by immunohistochemical analysis was seen. Wilms tumor 1 (WT1) expression was detected in blasts in 17 AML cases (44%) and 21 ALL cases (78%). There was no correlation between WT1 and podocalyxin expression. We conclude that podocalyxin is expressed commonly by blasts in ALL and AML. Analysis of the expression of CD34 and podocalyxin increases sensitivity for the immunophenotypic detection of leukemic blasts compared with the analysis of CD34 alone. Therefore, podocalyxin seems to complement CD34 as a useful hematopoietic blast marker. The physiologic role of podocalyxin in leukemic blasts remains unknown.     

Expression of genes related to multiple drug resistance and apoptosis in acute leukemia: response to induction chemotherapy

Resistance to chemotherapy is a major impediment to the successful treatment of acute leukemia (AL). Expression of genes involved in drug resistance and apoptosis may be responsible for this. This study aimed to investigate the expression of drug resistance (MDR1, MRP1, LRP, BCRP, GSTP1, DHFR) and apoptotic genes (p53, BCL-2, Survivin) in adult acute leukemias and compare them with clinical and hematological findings and response to induction chemotherapy. Eighty-five patients with AL [45 with acute myeloid leukemia (AML) and 40 with acute lymphoblastic leukemia (ALL)] were used as a study group. Real-time PCR results showed that expression level of MDR1 was significantly higher in AML whereas expression of DHFR, BCRP and Survivin was significantly higher in ALL patients. In AML, significant correlation was observed between LRP and MRP1 (r(s)=0.44, p=0.016), LRP and DHFR (r(s)=0.41, p=0.02), MDR1 and BCL-2 (r(s)=0.38, p=0.03). Expression of GSTP1 and LRP correlated with high white blood count (p=0.03 and p=0.03) and BCL-2 with high peripheral blast count (p=0.009). MDR1 expression was significantly associated with the expression of immature stem cell marker CD34 (p=0.002). In ALL, significant association was found between LRP gene and female sex (p<0.0001), LRP and B-ALL patients (p=0.04) and LRP and BCR/ABL positive patients (p=0.004). High expression of MDR1 and BCL-2 in AML and MRP1 gene in ALL was associated with response to induction chemotherapy (p=0.001, p=0.02 and p=0.007 respectively). These results showed the potential clinical relevance of MDR1, MRP1 and BCL-2 in adult patients with acute leukemia in the context of induction chemotherapy.     

Multi-drug resistance (MDR1) gene expression in de novo acute leukemia cells: correlations with CD surface markers and treatment outcome.

One important mechanism of drug resistance in acute leukemia is the overexpression of the multi-drug resistance (MDR1) gene that encodes a 170-kDa membrane protein called P-glycoprotein. To estimate the incidence and role of MDR1 gene expression in patients with acute leukemia, we investigated the expression of MDR1 by using the RT-PCR method in blast cells from 40 cases of de novo acute leukemia. We found a high frequency of MDR1 gene expression: 10 out of 20 with de novo acute myeloid leukemia (AML), 8 out of 17 with de novo acute lymphoblastic leukemia (ALL), and none of the 3 with de novo acute mixed leukemia, were MDR1 mRNA-positive. No correlation between cluster designation (CD) surface markers (CD19, CD7, CD13, CD33, CD34, CD14, HLA-DR) and MDR1 gene expression in AML was found. The complete remission rate was correlated with MDR1 gene expression. Among 40 evaluable patients examined, 17% (3 of 18) with MDR1 mRNA-positive reached complete remission versus 77% (17 of 22) with MDR1 mRNA-negative (p=0.044). These results suggest that MDR1 gene expression can be used as a prognostic factor and may be helpful in determining chemotherapeutic protocol for patients with acute leukemia.     

Prognostic significance of flow cytometric immunophenotyping in acute myeloid leukemia

The prognostic significance of flow cytometric immunophenotyping (FCI) in acute myeloid leukemia (AML) has been controversial. In this study, we re-investigated the possible role of FCI in the prediction of AML relapse following standard chemotherapy. A total of 209 AML cases with follow-up information were analyzed. Among those, 78 cases were in remission (M:F=44/34; mean age of 48.9 years) and 131 had relapse (M:F=71/60; mean age of 51.3 years). The expression of CD34, HLA-DR or a combination of both was significantly different between the remission and relapse groups for all AML as well as AML without t(15;17). None of the pammyeloid markers or their combinations analyzed was found to correlate with treatment outcomes. Complex cytogenetic abnormalities were more likely associated with relapse group than with remission group, but were not statistically significant after excluding AML with t(15;17). In conclusion, FCI is useful in predicting treatment outcome and disease relapse in AML.     

CD80，CD86和ICAM-1在急性白血病细胞上的表达及意义

目的通过流式细胞术检测初诊患者急性自血病细胞上共刺激分子CD80、CD86以及黏附分子ICAM．1的表达，以了解其表达规律。方法通过流式细胞仪检测60例初治急性白血患者白血病细胞上CD80、CD86和ICAM-1的表达率；男37例，女23例，年龄2～85岁，中位年龄28岁；其中ALL20例，AML40例（M16例、M27例、M37例、M415例、M55例）。结果ALL组中的CD86的表达为（32．880±6．665）％，显著高于正常对照（P〈0．01），而CD80与正常BM对照比较无统计学意义；CD80、CD86在AML（M1、M2和M3）细胞上的表达分别为（0．766±0．187）％、（27．210±7．581）％，均显著高于相应的正常骨髓对照（P〈0．01）；在AML（M4和M5）细胞上CD80、CD86的表达与正常对照比较均无统计学意义。ICAM-1在ALL组中的表达与正常BM对照比较无统计学意义；在AML（M1、M2和M3）细胞上（63．820±7．484）％，显著高于相应的正常骨髓对照（P〈0．01）：而AML（M4和M5）细胞上表达为（50．590±7．092）％，显著低于相应的正常骨髓对照（P〈0．01）。结论CD80、CD86和ICAM．1在初治ALL和AML白血病细胞上的表达呈一定变异性，CD86在ALL上呈高表达，CD80、CD86和ICAM-1在AML（M1、M2和M3）上均呈高表达，而ICAM-1在AML（M4和M5）上均呈低表达。     

CD117在急性白血病中的表达及临床意义

目的探讨CD117在急性白血病中的表达及其临床意义。方法应用CD45/SSC设门,直接荧光标记法,经流式细胞术对73例急性髓系白血病（AML）和47例急性淋巴细胞白血病（ALL）进行CD117的检测。结果对照组、ALL及AML3组CD117的表达阳性率差异有统计学意义（χ2=41.681,P﹤0.01）。AML组CD117的表达阳性率（58.9%）明显高于对照组（0）及ALL组（8.5%）。CD117/CD34的共表达率ALL组明显低于AML组（4.3%vs45.2%,P﹤0.05）。AML组CD117＋患者的CR率为60.5%,CD117-患者为76.7%,两者比较差异无统计学意义（P〉0.05）;而CD117＋/CD34＋患者的CR率为54.5%,明显低于CD117-/CD34-患者的CR率（88.2%,P﹤0.05）。结论 CD117可作为辅助诊断AML的髓系标志抗原,CD34＋/CD117＋可作为进一步排除ALL的指标。CD117＋/CD34＋可能是AML中一类预后不良的特殊亚型,可以作为AML预后判断的指标之一。     

Value of monoclonal anti-myeloperoxidase (MPO7) for diagnosing acute leukaemia.

The expression of myeloperoxidase (MPO) was studied in 100 cases of acute leukaemia (83 with acute myeloid leukaemia (AML) and 17 acute lymphoblastic leukaemia (ALL) by both a conventional cytochemical method and the immunocytochemical antiperoxidase (APAAP) technique using the monoclonal antibody MPO7. In each case the staining was evaluated by light microscopical examination (percentage of positive cells). Of the 83 cases of AML, 78 (93.9%) were positive for MPO7 compared with 70 (84.3%) by cytochemistry. Antibodies against the myeloid markers CD13 and CD33 were positive in 71 (85.5%) and 70 (84.3%) cases, respectively. Importantly, all cases of ALL were negative for both MPO7 and cytochemical MPO staining even when they were positive for CD13 and CD33. These results indicate that the anti-myeloperoxidase antibody MPO7 is the most sensitive and specific reagent for the diagnosis of AML and should therefore be included in routine immunophenotyping panels.     

Tim-3 is highly expressed in T cells in acute myeloid leukemia and associated with clinicopathological prognostic stratification

T cells immunoglobulin mucin 3 (Tim-3) is an important inhibitory stimulatory molecule, which has been reported to play a vital role in the tumor immune escape and be correlated with clinicopathological prognostic stratification in solid tumor. However, the related research is rare of Tim-3 in non-solid tumor, such as acute myeloid leukemia (AML). In this study, we investigated the expression characteristics of Tim-3 on the peripheral blood T cells of newly diagnosed AML patients and its clinical significance. Peripheral blood was obtained from 36 patients with newly diagnosed AML before intervention, with peripheral blood from 20 cases of healthy volunteers collected as normal control. Expression levels of Tim-3 on the peripheral blood T cells were assayed with flow cytometry. We found that Tim-3 expression on the peripheral blood CD4+ T cells and CD8+ T cells in newly diagnosed AML patients were significantly increased compared with that of normal control. CD4+ T cells/CD8+ T cell ratio (CD4/CD8) of peripheral blood in AML patients was significantly correlated with NCCN high risk group. The higher expression level of Tim-3 on CD4+ T cells in the peripheral blood of AML patients had significant correlation with FLT3-ITD mutation, the higher expression level of Tim-3 on CD8+ T cells in AML patients was significantly correlated with NCCN high risk group. To conclude, our results support the concept that Tim-3 is highly expressed on the peripheral blood T cells of AML patients, and Tim-3 expression significantly correlates with clinicopathological prognostic stratification in AMLTim-3, T cell, acute myeloid leukemia, tumor immune escape, clinicopathological prognostic stratification     

Plasma soluble interleukin-2 receptor (sIL-2R) levels in patients with acute leukemia

The plasma soluble interleukin-2 receptor (sIL-2R) level was higher in 137 patients with acute leukemia (1,489 +/- 1,798 U/ml, including 98 cases of acute myeloid leukemia (AML), 1,063 +/- 1,414 U/ml, and 39 cases of acute lymphoblastic leukemia (ALL), 2,561 +/- 2,194 U/ml), compared to 49 normal control subjects, 421 +/- 151 U/ml). The ALL patients showed elevated plasma sIL-2R levels more frequently than the AML patients (92.3% vs 44.9%). No patient with either hypoplastic AML or AML with multilineage dysplasia and only 1 of 13 patients with acute promyelocytic leukemia (APL) had an elevated plasma sIL-2R level. All the My+ ALL patients (15 cases) showed elevated plasma sIL-2R levels. Plasma sIL-2R levels were significantly lower after chemotherapy in the ALL patients, but were not significantly lower in the AML patients. IL-2R was expressed on the leukemic cells in 36 (53.7%) of 67 AML and in 9 (21.4%) of 42 ALL cases. None of the AML M3, M4, M5, M6, or M7 subgroups showed IL-2R expression. The My+ ALL patients (42.9%, 6/14) showed IL-2R expression more frequently than the other ALL subgroups (10.7%, 3/28) (p = 0.025). The plasma sIL-2R level was correlated with the proportion of leukemic cells expressing IL-2R in acute leukemia. However, there were many cases, particularly ALL cases, who had elevated plasma sIL-2R levels without IL-2R expression on their leukemic cells. These results suggest that the plasma sIL-2R level is a valuable marker for monitoring ALL after chemotherapy, particularly in My+ ALL cases, and that the T cell immune reaction to leukemia appears to be much higher in ALL patients than in AML patients.     

Utility of white blood cell suspect flags and histogram pattern in the detection of acute leukemia by Advia-60 automated hematology analyzer

Blood samples from patients with acute leukemia, when analyzed with automated hematology counters, tend to introduce inaccuracies in the automated differential count and can cause diagnostic confusion without providing definite clues to the presence of abnormal cells. We designed this study to assess the utility of white blood cell (WBC) flags and histogram pattern generated by Advia-60 automated hematology analyzer in the recognition and categorization of acute leukemia. Data printouts of 31 newly diagnosed cases of acute leukemia, 22 with acute myeloid leukemia (AML) and 9 with acute lymphoblastic leukemia (ALL) were reviewed. All cases of AML and ALL generated the WBC suspect blastflag M2 associated with two of the non blast suspectflags G1 and G2. Among the cases of AML, 95.5% of the WBC histogram patterns were definitive of the presence of abnormal cells and were indicative of the myeloid nature of cells. Only 44.4% of the histograms in the cases of ALL could be definitive of the presence of abnormal cells and 33.3% were indicative of their lymphoid nature. Significantly, 55.5% of the histograms in ALL were normal. The false positives for both AML and ALL were 10.5% when only WBC flagging was considered and were reduced to 0.05% when the flags were combined with histogram patterns for interpretation. Combined flagging and histogram recognition can be of aid in identifying cases of acute leukemia and the morphologist can then assess these samples further. This ensures that cases of acute leukemia, especially in high output laboratories, are not inadvertently missed.     

CD64在急性白血病免疫分型中的意义

目的探讨CD64在急性白血病免疫分型中的意义。方法应用直接荧光标记法,经流式细胞术对116例急性髓系白血病(AML)和70例急性淋巴细胞白血病(ALL)进行免疫表型的检测。结果 AML组CD64的表达阳性率明显高于ALL组(41.4%vs 2.9%,P<0.01)。CD64在M5和M4的表达阳性率最高,分别为77.1%和55.6%。CD64的阳性表达率在M0(0)、M1(0)、M2(7.9%)明显低于M3(47.1%)、M4与M5(P<0.01)。各亚型CD64阳性病例中,M4、M5的CD64阳性细胞表达率分别为(62.5±24.7)%、(68.7±25.9)%,均明显高于M2(28.3%±5.7%)、M3(34.3%±6.3%)(P<0.01)。CD64对诊断AML的灵敏度优于CD14,其特异度优于CD117、CD33及CD13。虽然CD64诊断M4\M5的特异度较CD14稍差(82.5%vs 100%),但是灵敏度高于CD14(69.8%vs 41.5%)。结论 CD64可作为辅助诊断AML的髓系标志抗原,有助于提高M4/M5的检出率及其与其他AML亚型的鉴别诊断。     

Clinical importance of myeloid-antigen expression in acute lymphoblastic leukemia of childhood

BACKGROUND. Leukemic cells in 15 to 25 percent of patients with acute lymphoblastic leukemia (ALL) express myeloid antigens as well as lymphoid antigens (the latter reflecting B-cell or T-cell lineage). The relations of myeloid-antigen expression to other features of ALL and to prognosis have been controversial. METHODS. We analyzed clinical and laboratory features present at diagnosis in 236 consecutive cases of ALL in children. Immunophenotyping, including single- and dual-fluorescence analyses, was used to classify leukemic cells as B or T lymphoblasts and also to identify myeloid-antigen expression--the simultaneous expression of lymphoid-associated antigens and at least one of three myeloid-associated antigens (CD33, CD13, and CD14) on cells classified as L1 or L2 according to the French-American-British system. RESULTS. Forty-five of 185 patients with B-lineage ALL had myeloid-antigen expression, as did 8 of 41 patients with T-lineage ALL. In 10 patients, the lineage could not be determined. Myeloid-antigen expression was associated with L2 morphology (P less than 0.05), but it did not correlate with other prognostic features recognized previously. Multivariate analysis showed that myeloid-antigen expression was an important predictor of relapse in childhood ALL and the most significant prognostic factor statistically (P less than 0.0001). A white-cell count greater than or equal to 50 x 10(9) per liter at diagnosis was also an important and highly significant prognostic feature (P less than 0.001). After 40 months, the estimated disease-free survival for patients with ALL was 84 percent for those without myeloid-antigen expression and with a low white-cell count, 57 percent for those without myeloid-antigen expression and with a high white-cell count, 47 percent for those with myeloid-antigen expression and a low white-cell count, and 26 percent for those with myeloid-antigen expression and a high white-cell count (P less than 0.00001). CONCLUSIONS. Myeloid-antigen expression is an important independent predictor of a poor response to chemotherapy in childhood ALL.     

Aberrant myeloid marker expression in precursor B-cell and T-cell leukemias

The World Health Organization (WHO) characterization of the immunophenotype of precursor B-cell acute lymphoblastic leukemia (pre-B ALL) includes the possible expression of myeloid cluster of differentiation (CD) markers CD13 and CD33. In precursor T-cell acute lymphoblastic leukemia (pre-T ALL), myeloid markers CD13 and CD33 are frequent while CD117 is rare. In the present investigation, 71 cases of confirmed pre-B ALL were evaluated for the presence of CD13 and CD33. Of the 19 (27%) cases that positively expressed myeloid markers, 10 (53%) expressed CD13, 17 (89%) expressed CD33, and 1 (5%) expressed CD117. Eight (42%) expressed both CD13 and CD33, and 1 (5%) expressed CD13, CD33, and CD117. Twenty-one cases of confirmed pre-T ALL were analyzed for myeloid markers CD13, CD33, CD117, and MPO. Of the 6 (29%) expressing myeloid markers, 4 (67%) were positive for CD13, 4 (67%) for CD33, 3 50(%) for CD117, and 1 (17%) for MPO. One (17%) was positive for both CD13 and CD117; one (17%) for CD13 and CD33; one (17%) for CD13, CD33 and CD117; and one (17%) for CD13, CD33 and MPO. These markers portend a poor prognosis compared to ALL cases without myeloid antigens, and a poor response to drug therapies targeting conventional ALL. Future studies will be directed to correlation of these markers with prognosis and therapeutic response, as well as whether drug therapies targeting myeloid antigens could be of use in treatment.     

Anti-CD10 immunoperoxidase staining of paraffin-embedded acute leukemias: comparison with flow cytometric immunophenotyping

CD10 is common in B-precursor acute lymphoblastic leukemia (ALL) but is rare in acute myeloid leukemia (AML). However, until recently, analysis for CD10 has generally required fresh or frozen tissue. 56C6 is a monoclonal antibody that is now commercially available for the detection of CD10 in routinely processed paraffin-embedded tissue. Immunoperoxidase stains for CD10 on paraffin-embedded bone marrow core biopsy specimens (B5-fixed, decalcified) and marrow aspirate clots (formalin-fixed) were compared with flow cytometric immunophenotyping for CD10 on fresh cell suspensions in 20 cases of AML and in 30 cases of ALL. CD10 detection by immunohistochemistry agreed with CD10 by flow cytometry in 98% (49 of 50) of acute leukemias. The results matched in 100% (20 of 20) of AML. Five percent (1 of 20) of AMLs expressed CD10. Two of the AMLs with monocytoid differentiation were interpreted as negative for CD10 by flow cytometry, although these had nonspecific dim immunofluorescence for multiple markers, including CD10, and these cases were negative by immunohistochemistry. CD10 detection by immunohistochemistry agreed with CD10 by flow cytometry in 97% (29 of 30) of ALL. Eighty-four percent (21 of 25) of B-precursor ALL and 40% (2/5) of T-lineage ALL expressed CD10 by immunohistochemistry. In 1 case of B-precursor ALL, CD10 was dimly positive in 24% of the blasts by flow cytometry but negative by immunohistochemistry. We conclude that immunohistochemical staining of paraffin-embedded tissue, either B5- or formalin-fixed, is an effective method for the detection of CD10 in acute leukemia. This technique is useful in distinguishing AML from ALL.     

基于粒子群优化算法的白血病基因表达样本分类研究

目的基于分子生物学的微阵列基因表达数据和智能优化算法对白血病肿瘤样本进行分类研究。方法给出基于粒子群优化(PSO)算法用于分类模型的训练和测试,选取含7129个基因的72个白血病基因表达样本,从中选取包含50、100和200个特征基因的3组数据,在不同基因数条件下分别执行10次分类测试。建立基于K-均值算法的分类模型,在同等条件下验证PSO算法分类性能。使用准确率、精确率、召回率、F1值等机器学习指标及Boxplot和Heatmap图谱用于分析对比。结果PSO算法用于分类测试的数据分别含20例急性淋巴细胞白血病(ALL)和14例急性髓细胞白血病(AML)样本。10次分类结果的平均分类准确率均在90%左右;PSO算法的分类准确率并不稳定,10次分类测试中,准确率的平均值和最优值间存在明显差异;ALL亚型的召回率明显高于AML亚型,均接近100%,但AML亚型的精确率明显高于ALL亚型,均接近100%,F1值可比性不大。K-均值算法与PSO算法类似,分类性能随着基因数的增加而降低;K-均值算法在200基因数条件下分类结果较差,分类稳定性和准确率均出现大幅下降,且低于同等条件下PSO算法分类结果;100个基因数条件下,ALL亚型召回率为100%,高于AML亚型;AML亚型精确率为100%,高于ALL亚型;200个基因数条件下,平均值中ALL亚型召回率和F1值高于AML亚型,AML亚型精确率高于ALL亚型,其最优值的统计学指标差异不大。相同白血病肿瘤样本的不同特征基因数条件下,PSO算法可获得较高准确率的分类结果,但分类稳定性不足,整体上优于K-均值算法。结论PSO算法能够应用于白血病基因表达样本的分类研究。     

Aberrant phenotypes in childhood and adult acute leukemia and its association with adverse prognostic factors and clinical outcome

Occurrence of aberrant phenotypes in childhood and adult acute leukemia (AL) differs considerably in independent studies and their association with prognostic factors is still controversial. In the present study, 214 patients with AL (106 children and 108 adults) were evaluated for the aberrant expression of CD33 in ALL (B cell and T cell) and CD3, CD5, CD7, and CD19 in AML. In B-ALL, aberrant expression of CD33 was found in 39 and 23% cases of adult and children, respectively. In T-ALL, CD33 was seen in 33% cases of adults while in children CD33 was not observed. In AML, aberrant expression of CD19 was expressed in 52 and 32% while CD7 was expressed in 14 and 15% cases of childhood and adult AML, respectively. Among FAB subtypes, aberrant expression of CD19 and CD7 was more commonly seen in M5 subtype. One adult patient (AML-M5) showed expression of CD3, CD5, and CD19. In summary, aberrant phenotype was commonly seen in adults than childhood B-ALL while in AML, aberrant phenotype was more common in children than adults. CD19 was most commonly expressed antigen followed by CD7 in both childhood and adult AML. Interestingly, aberrant phenotype was not found in childhood T-ALL; however, it was seen in 33% cases of adults. We did not find any association of aberrant phenotype with adverse prognosis factors, CD34 marker, and clinical outcome except the absence of auer rod which was found to be significantly associated with aberrant phenotype of childhood AML (P = 0.01).     

Epithelial membrane antigen (EMA) or MUC1 expression in monocytes and monoblasts

AIMS: Epithelial membrane antigen (EMA) or MUC1 belongs to a heterogeneous group of heavily glycosylated proteins and is expressed in most normal and epithelial neoplastic cells. EMA is also expressed in plasma cells, anaplastic large cell lymphoma (Ki-1 antigen), malignant histiocytosis and erythroleukaemia. In 1996, Cheong et al. (Hematology 1996; 1: 223) demonstrated the positive expression of EMA in monoblasts. Since there were very few useful markers for differentiating subtypes of acute myeloid leukaemia with a monocytic component from the those without, a study was conducted to evaluate the prevalence of EMA expression and its relationship with known markers for monocytic-macrophage lineage (CD11c, CD14 and intracellular CD68) in monocytes and monoblasts. METHODS: EMA detection was performed by flow cytometry in monocytes and monoblasts. EMA expression was compared with other known markers of monocytic-macrophage lineage (CD11c, CD14 and intracellular CD68). Samples of purified monocytes were obtained from 20 healthy volunteers. Twenty-two cases of monocytic AML (M4 and M5) were studied and controls were selected from 20 cases of acute lymphoblastic leukaemia (ALL) and 18 cases of non-monocytic AML (M0, M1, M2, M3, and M7). RESULTS: EMA was shown to be expressed strongly on the surface of all purified monocytes. EMA expression was observed on blast cells in 18/22 (81.8%) cases of AML M4 and M5, but not in that of non-monocytic AML or ALL. In this study EMA monoclonal antibody has demonstrated a strong association (P<0.001) with all the other known markers of monocytic-macrophage lineage in acute leukaemia subtypes. EMA had also shown 100% specificity and 81.8% sensitivity in the diagnosis of AML M4 and M5. CONCLUSIONS: The monoclonal antibody EMA (clone E29) is a useful marker in the classification of acute myeloid leukaemia and can be used as a supplementary analysis for the diagnosis of acute leukemia with monocytic involvement.     

Syndecan-1/CD138 expression in normal myeloid,acute lymphoblastic and myeloblastic leukemia cells

Stabilization of cell surface antigens and preservation of ultrastructural integrity are important aspects of immunoelectron microscopical studies. In the present study, 4 anti-syndecan-1/CD138 (B-B2, B-B4, MI15, 1D4) monoclonal antibodies (mAbs) were applied in combination with periodatelysine-paraformaldehyde (PLP) fixation and indirect pre-embedding peroxidase electron microscopical immunocytochemistry to analyse the localization and function of these molecules in normal myeloid cells, acute lymphoblastic leukemia (ALL) cells and acute myeloblastic leukemia (AML) cells. One case of normal human bone marrow, 3 cases of untreated AML and 2 cases of untreated ALL were studied. Samples were immediately fixed for 4 h in freshly-prepared PLP fixative in 0.037 mol/L phosphate buffer, pH 7.4, containing 10 mmol/L sodium metaperiodate, 75 mmol/L lysine, and 2% paraformaldehyde. Expression of syndecan-1 was found at the plasma membrane of all cell types. Staining intensity at the membrane of AML cells was stronger than that on the membrane of normal myeloid and ALL cells. We conclude that anti-syndecan-1/CD138 mAbs in combination with the method described here are a suitable tool for detection of cell surface syndecan molecules in cells originating from progenitor cells that can differentiate in both myeloid and lymphoid cells.     

Combination assay of IAP and ADA in hematologic malignancies

We measured the levels of adenosine deaminase (ADA) and immunosuppressive acid protein (IAP) in 10 patients with acute myeloid leukemia (AML), 5 with acute lymphoblastic leukemia (ALL), 8 with chronic myeloid leukemia (CML), 7 with myelodysplastic syndrome (MDS), 5 with malignant lymphoma (ML), 3 with multiple myeloma (MM) and one with adult T cell leukemia. On admission, the level of IAP was abnormally high in all cases of AML and ALL 50% of CML cases, 71.4% of MDS cases, 60% of ML cases and none of MM cases. ADA was elevated in all cases of ALL, 77.8% of AML and CML cases, 57.1% of MDS cases, 60% of ML cases and 33.3% of MM cases. In 7 patients with AML, the level of IAP returned to normal when they achieved complete remission. On the other hand, the level of ADA had already returned to normal even during induction therapy. ADA showed a positive correlation with the absolute number of peripheral blasts and lactic dehydrogenase both in AML and ALL. These results suggest that ADA indicates the activity of leukemia and IAP indicates the immunocompetence of the host.