Surface markers in adult acute myeloblastic leukemia: correlation of CD19+, CD34+ and CD14+/DR--phenotypes with shorter survival. Groupe d'Etude Immunologique des Leucémies (GEIL).

The immunophenotype of blast cells was investigated in a multicentric study of 154 adult acute myeloblastic leukemias (AMLs). A panel of 27 monoclonal antibodies (MoAbs) was tested in indirect immunofluorescence. Expression of CD14 (UCHM1), CD19 (SB4), CD36 (OKM5), and HLA-DR were associated with higher mean leucocyte counts. CD14 expression correlated with low hemoglobin level and the absence of CD33 (MY9) with low platelet counts. Extramedullary disease was associated with CD16 (Leu11b) and HLA-DR antigen positivity. The study of relationships between surface markers and FAB criteria confirmed the predominant expression of CD14 in the M5 sub-group (p less than 0.000001) and the association of CD19 and CD36 with monocytic M4/M5 subgroups (respectively p less than 0.00002 and p less than 0.000001). All patients received an induction therapy including an anthracycline and cytarabine. The median follow-up was 13 months. The achievement of complete remission (CR) was inversely correlated with CD34 (B13C5) and CD19 expression: CR was obtained in 31 of 59 (53%) CD34-positive AML versus 64 of 75 (85%) CD34-negative cases (p less than 0.0001) and 11 of 24 (46%) CD19-positive versus 95 of 122 (78%) CD19-negative cases (p less than 0.01). In univariate analysis, a longer survival was associated with CD33 expression and the combined phenotypes CD36+/CD19- and CD16+/CD14-. Conversely, the CD18(IOT18)+/CDw65(VIM2)- phenotype was related to shorter survival. The expression of CD19, of CD34, and of the combined phenotype CD14+/DR--correlated with shorter survival as demonstrated both in univariate and multivariate analysis (p less than 0.03 in each case in multivariate analysis).     

成人急性髓系白血病的免疫学特点及预后

目的：探讨急性髓细胞白血病（AML）的免疫表型特点。方法：使用淋系和髓系单抗,用间接免疫荧光法对70例原发性AML进行免疫表型分析。结果：所有AML患者的细胞至少被1种髓系单抗标记,各髓系抗原的表达率依次为CD33＞CD13＞CD15。所有M3患者CD9为阳性。16／70例（30％）表达CD34抗原、CD34^ AML组在年龄、外周血象及骨髓原始、幼稚细胞比例等方面与CD34^-组相比较无显著差别,但表达CD34抗原的AML常伴有HLA－DR、CD38、CD7等不成熟细胞表面标记的表达,而较成熟的髓系细胞表面标记CD15则不表达。70例AML中有16例表达淋系抗原,CD4^ 例（13．8％,M2为8．8％,M460％）;CD7^ 9例（16．9％,M1为50％,M218％,M5b16．7％）。CD4^ 的AML患者CD34为低表达,CD33表达。结论：CD9^ 、CD34^-、HLA－DR^-及CD13^ 、CD15^ 是典型M3的免疫表型特点。CD34^ AMLgn AML-M1有着密切的关系,且对化疗反应较差,证明CD34^ 的AML是一组分化程度较差的类型;提示CD7^ 和CD4^ 的AML预后差,CD7^ 的AML的一种独特类型。     

Immunological classification of acute myeloblastic leukemias: relevance to patient outcome.

Immunophenotyping is a major tool to assign acute leukemia blast cells to the myeloid lineage. However, because of the large heterogeneity of myeloid-related lineages, no clinically relevant immunological classification of acute myeloblastic leukemia (AML) has been devised so far. To attempt at formulating such a classification, we analyzed the pattern of expression of selected antigens, on blast cells collected at AML diagnosis. Patients were eligible if they had a first diagnosis of de novo AML and a sufficient number of blast cells for proper immunophenotyping. The relative expression of CD7, CD13, CD14, CD15, CD33, CD34, CD35, CD36, CD65, CD117, and HLA-DR were analyzed by cytometry in a test series of 176 consecutive AML cases. Statistical tools of clusterization allowed to remove antigens with overlapping distribution, leading us to propose an AML classification that was validated in a second AML cohort of 733 patients. We identified five AML subsets (MA to ME) based on the expression of seven antigens within four groups (CD13/CD33/CD117, CD7, CD35/CD36, CD15).-MA and MB-AML have exclusively myeloid features with seldom extramedullary disease and rare expression of lymphoid antigens. No cases of acute promyelocytic leukemia (APL) were observed within MB AML. MC AML have either myeloid or erythroblastic features. MD AML have more frequently high WBC counts than other subsets, which were related to the expression of CD35/CD36 and CD14 and to monoblastic differentiation. ME AML lack CD13, CD33, and CD117 but display signs of terminal myeloid differentiation. Specific independent prognostic factors were related to poor overall survival in each immunological subset: CD34+ (P<3 x 10(-4)) in MA AML, CD7+ in MB AML, non-APL cases (P<0.03) in MC AML, CD34+ (P<0.002) and CD14+ (P<0.03) in MD AML, CD14+ in ME AML (P<0.01). The inclusion of seven key markers in the immunophenotyping of AML allows a stratification into clinically relevant subsets with individual prognostic factors, which should be considered to define high-risk AML populations.     

Patterns of Membrane Antigen Expression by AML Blasts: Quantitation and Histogram Analysis

Leukaemic myeloid blasts from non-monocytic (M1-M3, n = 36) and monocytic (M4 and M5, n = 21) AML cases were examined for the expression of 12 different membrane determinants by flow cytometry. Data analyses for each antigen included the determination of (a) the mean fluorescence intensity for the whole blast cell population, (b) the relative levels of membrane fluorescence for individual events (cells), and (c) a conventional assessment of the proportion of cells staining positively (i.e. exceeding a pre-defined level of fluorescence). Three main types of staining histogram were observed and, of these, the most commonly seen (348/432 and 176/252 of non-monocytic and monocytic AML histograms respectively) was characterised by an homogenous distribution of staining intensities which did not exceed two log decades of fluorescence (S-type). The second staining pattern was characterised by a continuous spectrum of fluorescence which exceeded two log decades of fluorescence (SE-type), and the third pattern showed evidence of two leukaemic populations with different levels of fluorescent staining (BI-type). With the exception of occasional AML cases which expressed CD7 or CD19 with low staining intensity, the expression of lymphoid-associated membrane CD3, CD10, and CD22 by AML blasts was insignificant. For comparison, analysing the histogram patterns of expression for the myeloid and non-lineage associated membrane determinants revealed that CD11c, CD13, CD14, and CD38 were mainly of S- or SE-type for the non-monocytic AML variants, with a minor but significant proportion of such cases expressing CD33 (7/36), CD34 (6/36) and HLA-Dr (6/36) with a BI-type staining pattern. Similarly, histogram patterns for CD13, CD33, CD34 and CD38 expression by the monocytic AML variants were predominantly of S- or SE- type, with minor proportions of cases expressing CD11c (7/21), CD14 (10/21), and HLA-Dr with BI-type staining. Comparisons between the mean fluorescence staining intensities for the whole blast cell population and conventional positive versus negative delineations for each antigen studied further suggested that semi-quantitative measurements of fluorescent staining were more informative and potentially of greater relevance to the study and diagnostic assessment of acute myeloid leukaemia subtypes.     

AC133 expression on acute myeloid leukemia blasts: correlation to FAB and to CD34 expression and possible implications for peripheral blood progenitor cell purging in AML

AC133 is an antigen expressed on CD34+ hematopoietic progenitor cells. In acute myeloid leukemia (AML) it is expressed on leukemic blasts of most FAB subtypes. However, few data are available regarding coexpression of other surface antigens. We measured AC133 expression on AML blasts from 28 consecutive patients at initial diagnosis (n=26) or at diagnosis of first relapse (n=2) and on 26 leukapheresis products from 14 patients. In AML AC133 correlated with CD34 expression (Spearman r=0.4711, P=0.0114) and even stronger with combined CD34/CD33 expression (Spearman r=0.5083, P=0.0068). In leukapheresis products AC133 expression correlated with CD34 expression (Spearman r=0.7495, P=0.002) and the yield of the obtained amount of CD34+ cells (Spearman r=0.6484, P=0.0121). In conclusion AC133 expression is closely related to CD34 expression in AML. In leukapheresis products AC133 provides an additional marker for selection of PBPC autografts in AC133- AML.     

Folate receptor type beta is a neutrophilic lineage marker and is differentially expressed in myeloid leukemia

BACKGROUND: The membrane-associated folate receptor (FR) type beta is elevated in the spleen in patients with chronic myeloid leukemia (CML) and acute myeloid leukemia (AML). In this study, the authors investigated possible cell type and differentiation stage specificity of expression of FR-beta in normal and leukemic hematopoietic cells. METHODS: An affinity-purified rabbit polyclonal antibody specific for FR-beta was employed for immunostaining representative bone marrow smears and peripheral blood smears from normal individuals and from a limited number of patients with various leukemias. Multiple samples of normal bone marrow and peripheral blood were analyzed for the expression of FR-beta and selected CD antigens by two- or three-color flow cytometry. RESULTS: Of the morphologically identifiable cells, only neutrophils were positive for FR-beta. The leukemic blasts in CML patients showed expression of FR-beta with no apparent relation to the occurrence of the Philadelphia chromosome. Among acute nonlymphocytic leukemias, FR-beta was expressed in promyelocytic leukemia, in the myeloblast populations of myelomonocytic and erythroleukemias, and variably in M1/M2 AML. Neither the blasts of acute lymphocytic leukemia nor the more mature cells of chronic lymphocytic and hairy cell leukemias expressed FR-beta. The less differentiated FR-beta positive AML samples also were positive for CD34 and HLA-DR. Flow cytometric analysis of normal bone marrow and peripheral blood revealed low or insignificant coexpression of FR-beta with CD34, CD19, and CD3, whereas significant coexpression was observed with high levels of CD33, CD13, and CD11b; coexpression of FR-beta with CD14 was high in the immature bone marrow cells, comparable to that in myeloid cells, but relatively low in peripheral blood. CONCLUSIONS: The results of this study suggest a narrow expression pattern of FR-beta marking the neutrophilic lineage and the possibility of defining a subtype or subtypes of myeloid leukemia based on FR-beta expression. The identification of FR-beta positive leukemias and the absence of the receptor in normal CD34 positive cells may enable selective receptor-mediated targeting of leukemic cells.     

Haematologic and immunophenotypic profile of acute myeloid leukemia: an experience of Tata Memorial Hospital

OBJECTIVES: To study the hematologic and immunophenotypic profile of 260 cases of acute myeloid leukemia at diagnosis. MATERIAL AND METHODS: This is a retrospective analysis of 260 cases of AML diagnosed at our institution between 1998 and 2000. Diagnosis was based on peripheral blood and bone marrow examination for morphology cytochemistry and immunophenotypic studies. SPSS software package, version 10, was used for statistical analysis. RESULTS: Seventy-six percent of our cases were adults. The age of the patients ranged from one year to 78 years with a median age of 27.2 years. There were 187 males and 73 females. The commonest FAB subtype, in both children and adults, was AML-M2. The highest WBC counts were seen in AML-M1 and the lowest in AML-M3 (10-97 x 10(9)/L, mean 53.8 x 10(9)/L). The mean values and range for hemoglobin was 6.8 gm/l (1.8 gm/l to 9.2 gm/l), platelet count 63.3 x 10(9)/L (32-83 x 10(9)/L), peripheral blood blasts 41.4% (5 to 77%) and bone marrow blasts 57.6% (34-96%). Myeloperoxidase positivity was highest in the M1, M2 and M3 subtypes. CD13 and CD33 were the most useful markers in the diagnosis of AML. CD14 and CD36 were most often seen in monocytic (38%) and myelomonocytic (44%) leukemias. Lymphoid antigen expression was seen in 15% of cases. CD7 expression was the commonest (11%). CONCLUSION: AML accounted for 39.8% of all acute leukemias at this institution. The most common subtype was AML-M2. Myeloperoxidase stain was a useful tool in the diagnosis of myeloid leukemias. CD13 and CD33 were the most diagnostic myeloid markers.     

AC133 antigen as a prognostic factor in acute leukemia

AC133 is a novel 5-transmembrane antigen present on a CD34((bright)) subset of human hematopoietic stem cells (HSCs) and it is also expressed on the subset of CD34 positive (CD34(+)) leukemias. But the clinical significance of AC133 expression on leukemic blasts is not yet known. We investigated the expression of AC133 antigen on blast cells of acute leukemia. Forty-one cases of acute leukemia were examined for expression of AC133, CD34, and other antigens using multicolor flow-cytometry. Samples were considered positive if at least 20% of the cells specifically stained with monoclonal antibodies (MoAbs) revealed a higher fluorescence intensity compared to cells of corresponding negative control samples (=20% cut-off level). 14/36 (38.9%) acute myelogenous leukemia (AML) samples and 6/20 (30%) acute lymphoblastic leukemia (ALL) samples were positive for AC133, the difference was not significant. All AC133 positive (AC133(+)) leukemias expressed CD34, whereas 13 of 33 CD34(+) leukemias were negative for AC133, and AC133(+)/CD34(-) leukemia was not found. Expression rates of CD31, CD62L, CD62E, CD105 and CD144 were significantly higher in AC133(+) leukemia compared to those of AC133(-) leukemia (P=0.045, P<0.001, P<0.001, P<0.001, P=0.003, respectively), but bcl-2, CXCR-1, CXCR4, VLA-4, CD106 expression rates were not significantly different between AC133(+) and AC133(-) leukemias. None of the clinical prognostic markers such as age, hemogram, lactate dehydrogenase, and chromosomal aberration were significantly different between AC133(+) and AC133(-) leukemias. CR rates of AC133(+) AML and AC133(-) AML were not significantly different, although there was a trend toward higher CR rates in AC133(-) AML (18/22[81.8%] AC133(-) AML versus 9/14[64.3%] AC133(+) AML), but the 1-year relapse rate of AC133(+) AML was significantly higher than that of AC133(-) AML (8/9 (88.9%) versus 7/19 (36.8%), P=0.016). Median disease-free survival (DFS) times of AC133(+) and AC133(-) AML were significantly different (11 and 18 months, respectively, P=0.006), although overall survival (OS) times were not significantly different (AC133(+) 15 months versus AC133(-) 20 months, respectively, P=0.06). Similar results regarding clinical outcomes were found when AC133(+)/CD34(+) and AC133(-)/CD34(+) were analyzed separately, but the difference did not attain statistical significance. In ALL, 9/11 (81.8%) AC133(-) and 2/4 (50%) AC133(+) cases achieved CR, but the difference was not significant. Four of 11 AC133(-) ALL (36.4%) and 2 of 3 AC133(+) ALL (66.7%) relapsed within 1 year. In survival analysis, median DFS time and OS time of the AC133(+) group were 7 and 18 months, respectively, and these were not significantly different from those of the AC133(-) group (median DFS 15, OS 22 months, respectively). Our results demonstrate that AC133 expression in AML blasts is associated with poor clinical outcomes in terms of higher early relapse and shorter disease-free survival, suggesting that the AC133 antigen might provide the prognostic stratification of acute leukemia. However, to verify the effect of AC133 expression on the therapeutic outcomes of adult acute leukemia, further study including more cases is needed.     

CD117在急性白血病中的表达及其临床意义

 目的 探讨细胞表面抗原CD117在各型急性白血病（AL）中的表达规律及其临床意义。方法 采用流式细胞术（FCM）检测139例AL患者骨髓或外周血中CD117的表达情况,并进行分析和比较。结果 急性淋巴细胞白血病（ALL）患者CD117阳性细胞表达水平与正常对照组相比差异无统计学意义（P＞0.05）;急性髓细胞白血病（AML）患者CD117阳性患者比例及阳性细胞表达水平较正常对照组和ALL组均升高,差异有统计学意义（均P＜0.05）。在AML各亚型中,CD117阳性患者比例及阳性细胞表达水平以M0／M1最高,M5b最低;在5种髓系膜抗原标志中,虽然CD117检测AML的灵敏度低于CD13和CD33,但特异性都较高;CD117与CD34及HLA-DR在AML中的表达呈显著正相关,但在M3中,CD117表达明显高于CD34和HLA-DR。结论 CD117可作为AL患者MIC诊断分型的重要髓系免疫表型指征,在AML的诊断分型以及AML和ALL的鉴别诊断中具有重要意义。     

139例急性髓系白血病免疫分型特点分析

目的探讨急性髓性白血病（AML）的免疫分型特点及意义。方法采用单克隆抗体和流式细胞仪检测AML的免疫表型。结果（1）139例AML病例中各种抗原的阳性表达率依次为为MPO（92.1%）,CD33（92.1%）,CD13(89.2%),其中53例AML伴淋巴系抗原表达,分别为CD19(20.9%),CD7(16.2%),CD2(7.2%),CD10(0.72%)。(2)CD14在M4、M5型AML中高表达。(3)干祖细胞分化抗原表达率依次为CD117（83.8%）〉HLA DR(80.3%)>CD34(67.6%),CD34阳性的完全缓解率（CR）分别明显低于CD34阴性组（P=0.034）。（4）CD7阳性患者CR明显低于其抗原表达阴性者（P=0.041）。结论白血病免疫分型能确诊某些特殊类型的白血病,对免疫分型的研究将有助于指导临床诊断、治疗及判断预后。     

Diagnostic value of CD117 in differential diagnosis of acute leukemias

C-kit receptor (CD117) and its ligand, stem cell factor, play a key role in normal hematopoiesis. It has been demonstrated that its expression extremely increases in leukemias with myeloid commitment. We analyzed findings on CD117 expression together with other myeloid related markers in 203 de novo acute leukemias, referred to Iranian immunophenotyping centers: Iranian Blood Transfusion Organization (IBTO) and Baghiatallah Hospital (BH). All cases were characterized based on the French American British cooperative group (FAB) and European Group for Immunological Classification of Leukemias (EGIL). The cases comprised of 111 acute myeloblastic leukemia (AML), 86 acute lymphoblastic leukemia (ALL), and 6 acute undifferentiated leukemia (AUL). CD117 was positive in 75 % of AML and 50 % of AUL, whereas none of the ALL cases was positive for this marker. Although CD117 was positive in 100 % of M5a cases, no M5b positive was found (p?=?0.036). The calculated specificity for myeloid involvement was 100 % for CD117 and CD33, and 98 % for CD13 and CD15 (p?<?0.001). The calculated sensitivity for myeloid involvement was 83, 76, 64, and 41 % for CD13, CD117, CD33, and CD15, respectively (p?<?0.001). We concluded that CD117 expression is a specific and rather sensitive marker for differential diagnosis between AML and ALL, and except for M5 subtypes, it fails to determine FAB subtypes; lack of expression in M5 can identify M5b. Therefore, it should be included in the routine primary panel for diagnosis of acute leukemias.     

Differentiation of leukemic cells induced by short-course high-dose methylprednisolone in children with different subtypes of acute myeloblastic leukemia.

Differentiation of myeloid leukemic cells to mature granulocytes by high-dose methylprednisolone (HDMP, 20-30 mg/kg/day) with a favorable antileukemic effect has previously been demonstrated in children with acute promyelocytic leukemia and acute myeloblastic leukemia (AML) M4. In the present study, three children with other morphological subtypes of AML (two AML M1, one AML M2) were given methylprednisolone (30 mg/kg/day) orally in a single dose. After a short-course (3 or 7 days) of HDMP treatment alone, a striking decrease in blast cells associated with an increase in maturing and abnormally nucleated polymorphonuclear-like cells some containing Auer rods were detected in all patients in peripheral blood or bone marrow smears. During HDMP treatment, in parallel to morphological improvements, marked increases in the percentage of cells expressing granulocytic antigen (CD15) were observed. The increase of CD15 expression on myeloid cells, together with the steady expression of CD34 and CD117 antigens in Casel(AML M1) , is suggestive of aberrant CD34 + CD117 + CD15 + cells, which may indicate the leukemic origin of the maturing myeloid cells. These results suggest that HDMP treatment may induce differentiation of myeloid leukemic cells in some children with different morphological subtypes of AML, and that the differentiation-inducing effect of HDMP should be explored in other malignant diseases.     

Enhanced myeloid specificity of CD117 compared with CD13 and CD33

The c-kit proto-oncogene encodes a 145 kd tyrosine kinase transmembrane receptor, which plays a key role in haemopoiesis. The c-kit has been classified as CD117 and is especially useful in the differential diagnosis of acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL). We analysed 104 consecutive cases (55 AML, 23 B-cell lineage ALL, three T-cell ALL, 11 blast crisis of chronic myeloproliferative disorders and 12 cases of myelodysplastic syndromes with more than 10% of blasts) referred to our Hospital for immunophenotypic diagnosis and compared the expression pattern of CD13, CD33 and CD117 using the same fluorochrome (phycoerythrin-PE). The recommendations of the EGIL group were followed in order to establish lineage involvement of the blastic population. The threshold used to assign positivity for CD117 was 10%. Bcr/abl, TEL/AML-1 and MLL rearrangements were assessed by molecular methods. CD117 expression was detected in 91% of AML and MDS. All the negative cases corresponded to acute monocytic leukemias. The calculated specificity for myeloid involvement was 0.86 for CD117, 0.36 for CD13 and 0.44 for CD33 (P < 0.005). CD117 was also positive in four cases of ALL. None of these cases showed bcr/abl or MLL rearrangements. In the light of these findings, CD117 expression should yield a higher score, at least one point, in the system currently applied for the diagnosis of biphenotypic acute leukemias (BAL) as its myeloid specificity is greater than that of CD13 and CD33. Moreover, its absence in AML could identify two subgroups of M5b cases. The coexpression of CD117 with cytoplasmic CD79a is often associated with CD7 reactivity, suggesting a stem cell disorder. CD117 should be included on a routine basis for the immunophenotypic diagnosis of acute leukemias.     

The expression pattern of CD56 (N-CAM) in human bone marrow biopsies infiltrated by acute leukemia

In hematological neoplasms CD56 (N-CAM) is expressed by T/natural killer (NK) cell lymphoma, by most neoplastic plasma cells in multiple myeloma and also in a subset of acute myelogenous leukemias (AML). In the latter, it is an indicator of poor clinical outcome. Most of the data on CD56 expression in acute leukemia have been obtained by flow cytometric analysis. Up to now, no systematic analysis of the expression pattern of CD56 in formalin fixed paraffin embedded bone marrow biopsies of acute leukemias has been performed. We immunohistochemically studied the expression of CD56 in a series of 141 bone marrow biopsies fixed in Sublimat Mercury II Chloride (SUSA) including 100 cases of AML FAB M0-M7, 11 cases of AML not further specified, 3 cases of biphenotypical leukemia, 20 cases of acute lymphoblastic leukemia (ALL) and 7 cases of reactive bone marrow biopsies. Overall, 14 of 134 (10%) leukemia cases were positive for CD56. Detail analysis revealed positivity in 5/13 cases of AML M5 (38%), 3/9 AML M1 (33%), 1/8 AML M0 (13%), 1/11 AML not specified (9%), 2/31 AML M2 (7%) and 2/26 AML M4 (8%). All cases of ALL and biphenotypic leukemias were CD56 negative. The CD56 expression in AML M5 was statistically significant (p=0.003). On paraffin embedded bone marrow biopsies CD56 expression occurs in de novo AML with an overall frequency of 13%. It is significantly correlated with AML M5, which is positive in 38% of the cases. Cases of ALL are consistently CD56 negative.     

Clinical Significance of Co-expression of Aberrant Antigens in Acute Leukemia: A Retrospective Cohort Study in Makah Al Mukaramah,Saudi Arabia

Background: Aberrant phenotypes in acute leukemia have variable frequency and their prognostic andpredictive relevance is controversial, despite several reports of clinical significance. Aims: To determinethe prevalence of aberrant antigen expression in acute leukemia, assess clinical relevance and demonstrateimmunophenotype-karyotype correlations. Materials and Methods: A total of 73 (40 AML and 33 ALL) newlydiagnosed acute leukemia cases presenting to KAMC, Kingdom of Saudi Arabia, were included. Diagnosis wasbased on WHO criteria and FAB classification. Immunophenotyping by flow cytometry, conventional karyotypingand fluorescence in situ hybridization for gene rearrangements were performed. Results: Aberrant antigens weredetected in 27/40 (67.5%) of AML and in 14/33 (42.4%) in ALL cases. There were statistically significant higherTLC in Ly+ AML than in Ly-AML (p=0.05) and significant higher blast count in ALL with aberrant antigensat presentation and day 14 (p=0.005, 0.046). There was no significant relation to clinical response, relapse freesurvival (RFS) or overall survival (p>0.05), but AML cases expressing ≥2 Ly antigens showed a lower medianRFS than those expressing a single Ly antigen. In AML, CD 56 was expressed in 11/40. CD7 was expressed in7/40, having a significant relation with an unfavorable cytogenetic pattern (p=0.046). CD4 was expressed in 5/40.CD19 was detected in 4/40 AML associated with M2 and t (8; 21). In ALL cases, CD33 was expressed in 7/33and CD13 in 5/33. Regarding T Ag in B-ALL CD2 was expressed in 2 cases and CD56 in 3 cases. Conclusions:Aberrant antigen expression may be associated with adverse clinical data at presentation. AML cases expressing≥2 Ly antigens may have shorter median RFS. No specific cytogenetic pattern is associated with aberrant antigenexpression but individual antigens may be related to particular cytogenetic patterns. Immunophenotype-karyotypecorrelations need larger studies for confirmation.     

Geographic heterogeneity of cellular characteristics of acute myeloid leukemia: a comparative study of Australian and Japanese adult cases.

We assessed a large number of adults (368 from Australia and 494 from Japan) with de novo acute myeloid leukemia (AML) to define the biological differences between the two populations. In this study, AML was classified using the French-American-British (FAB) criteria into seven groups (M1-M7). M2 was more common in Japan than in Australia, whereas M4 occurred more frequently in Australia than in Japan. Other FAB subtypes were evenly distributed. Cytogenetically, Japanese M2 displayed a higher frequency of t(8;21) than Australian (33.1% vs 15.3%, P < 0.05). The t(15;17), inv/del(16), 11q23 aberrations and 5/7/8 abnormalities were seen at similar frequencies. Immunophenotypically, Japanese M4/M5 more frequently displayed CD13 and CD14 than Australian, whereas the stem cell markers, CD34 and HLA-DR were observed at a relatively higher rate in Australian M3 than in Japanese M3. The B cell antigen, CD19 was more frequently seen in Japanese M2 than in Australian M2, but found more often in Australian M5 than in Japanese M5. In both populations, a close relationship was observed between the expression of CD19 and t(8;21). These findings suggest different biological characteristics of AML between the two populations, the main differences being generated by a higher frequency of t(8;21) chromosomal abnormality in Japanese AML. Leukemia(2000) 14, 163-168.     

Acute myelogenous leukemia with t(8;21)--identification of a specific immunophenotype

Association between certain surface markers and acute myelogenous leukemia (AML) with t(8;21) has been described. The specificity and the predictive values of these markers have never been assessed. In this study, we aimed, to explore whether a specific pattern could predict for this translocation. Of 405 consecutive AML, 18 (4.4%) had the t(8;21). Patients with this cytogenetic abnormality showed higher frequency of CD34 (P = 0.003), HLA-DR (P = 0.03), Tdt (P = 0.02), CD19 (P < 0.0001), and CD56 (P < 0.0001) and lower CD33 (P = 0.0001). Taken singly, the sensitivity of these markers for AML with t(8;21) ranged between 39 and 100% with CD34+ having the highest and CD33- having the lowest and the positive predictive values (PPV) ranged between 5 and 21% with CD19+ having the highest and HLA-DR+ having the lowest. When combinations of different markers were analyzed by multivariate analysis, the pattern CD34+/HLA-DR+/MPO+ was found to have the highest sensitivity (100%) with a PPV of 14% and the pattern CD34+/CD19+/CD56+ had the highest PPV (100%) with a sensitivity of 67%. We conclude that AML with t(8;21) is better identified by a combination of markers than by a single antigen pattern, the absence of CD34+, HLA-DR+ or MPO+ would preclude and the expression of the pattern CD34+/CD19+/CD56+ is highly predictive and could serve as a screening criteria for the t(8;21).     

Expression of HOX genes, HOX cofactors, and MLL in phenotypically and functionally defined subpopulations of leukemic and normal human hematopoietic cells.

To explore the possibility that deregulated HOX gene expression might commonly occur during leukemic hematopoiesis, we compared the relative levels of expression of these and related genes in phenotypically and functionally defined subpopulations of AML blasts and normal hematopoietic cells. Initially, a semi-quantitative RT-PCR technique was used to amplify total cDNA from total leukemic blast cell populations from 20 AML patients and light density cells from four normal bone marrows. Expression of HOX genes (A9, A10, B3 and B4), MEIS1 and MLL was easily detected in the majority of AML samples with the exception of two samples from patients with AML subtype M3 (which expressed only MLL). Low levels of HOXA9 and A10 but not B3 or B4 were seen in normal marrow while MLL was easily detected. PBX1a was difficult to detect in any AML sample but was seen in three of four normal marrows. Cells from nine AML patients and five normal bone marrows were FACS-sorted into CD34+CD38-, CD34+CD38+ and CD34-subpopulations, analyzed for their functional properties in long-term culture (LTC) and colony assays, and for gene expression using RT-PCR. 93 +/- 14% of AML LTC-initiating cells, 92 +/- 14% AML colony-forming cells, and >99% of normal LTC-IC and CFC were CD34+. The relative level of expression of the four HOX genes in amplified cDNA from CD34- as compared to CD34+CD38- normal cells was reduced >10-fold. However, in AML samples this down-regulation in HOX expression in CD34- as compared to CD34+CD38- cells was not seen (P < 0.05 for comparison between AML and normal). A similar difference between normal and AML subpopulations was seen when the relative levels of expression of MEIS1, and to a lesser extent MLL, were compared in CD34+ and CD34- cells (P < 0.05). In contrast, while some evidence of down-regulation of PBX1a was found in comparing CD34- to CD34+ normal cells it was difficult to detect expression of this gene in any subpopulation from most AML samples. Thus, the down-regulation of HOX, MEIS1 and to some extent MLL which occurs with normal hematopoietic differentiation is not seen in AML cells with similar functional and phenotypic properties.     

The immunophenotype of minimally differentiated acute myeloid leukemia (AML-M0): reduced immunogenicity and high frequency of CD34+/CD38- leukemic progenitors.

Minimally differentiated acute myeloid leukemia (AML-M0) is a rare FAB subtype (2-3% of AMLs) of poor prognosis. The aim of our study was to characterize AML-M0 expression and regulation of adhesion/costimulatory molecule involved in immune recognition, to test blast in vitro immunogenicity, and to determine the percentage of leukemia progenitor cells. Here, we demonstrate that alloimmune recognition of AML-M0 in primary mixed lymphocyte reaction, as evaluated by IL-2 secretion of responding T cells, is reduced in comparison with more differentiated subtypes (128 +/- 95 pg/ml vs304 +/- 159 pg/ml, P < 0.05). These data are in line with low blast cell expression of major histocompatibility complex (MHC) class II DR molecules, and of the CD28 ligand B7-2, which plays an important role in AML immune recognition. Adhesion/costimulatory molecules were up-regulated by leukemic cell stimulation via CD40, and, although less efficiently, by gamma-IFN; both stimuli improved blast cell immunogenicity. We also demonstrate that AML-M0 have a very high percentage (40% +/- 30) of CD34+/CD38- leukemic clonogenic precursors in comparison with more differentiated AMLs (2.5% +/- 2) or non-leukemic CD34+hematopoietic precursors (1.8% +/- 0.8). Since the presence of a leukemic cell population at an early differentiation stage has been identified as a poor prognostic factor, we conclude that the high frequency of CD34+/CD38- blasts in AML-M0 may converge with already identified poor prognosis factors such as chemotherapy resistance and cytogenetic abnormalities. The clinical implications of AML-M0 impaired in vitroimmunogenicity and a high percentage of CD34+/CD38- blasts will require comparative analysis of additional patients. The increased immunogenicity of blast cells after CD40 triggering provide interesting clues for AML-M0 immunotherapy, that have to be confirmed with an in vivo leukemia model in mice.