From www.bloodjournal.org by guest on February 6, 2015. For personal use only. Leukemia-Associated Changes Identified by Quantitative Flow Cytometry. IV. CD34 Overexpression in Acute Myelogenous Leukemia M2 With t(8;21) By Anna Porwit-MacDonald, George Janossy, Kamal Ivory, David Swirsky, Rowayda Peters, Keith Wheatley, Helen Walker, Alev Turker, Anthony H. Goldstone, and Alan Burnett During theimmunodiagnosis of517 casesof acute myelogenousleukemia(AML)entered into the Medical Research Council (MRC) AMLIO trials, we have observed the CD34 precursor cell antigen more frequently in AML of M2 morphology, especially in the 84% of cases with thet(8;21) chromosomal translocation, than in any other French-AmericanBritish classification group. CD34 expression was then quantified (using QlFl and Quantum Simply Cellular beads [Flow CytometryStandards, ResearchTriangle Park, NCI and CD34+ standard cells). When CD34 antibody-binding capacity (ABC) of normal bone marrow (BM) precursors and leukemic blasts wascompared, it was shown thatAML M2 cases with t(8;21) not only had thehighest percentages of CD34+ blasts, but in >80% of CD34+cases the individual blasts expressed higher than normal levels of CD34 antigen (>60 x I O 3 ABC per cell). In addition, in 73% of this group CD34 antigen wasoverexpressed in an asynchronous combination with cytoplasmic myeloperoxidase (MPO). Other signs of asynchrony included high CD34 expression with CD15 andl or CD56,as well as aberrant combinations of CD13 with terminal deoxynucleotidyl transferase (TdT) and CD19. These findings demonstrate that asynchrony is identifiable in virtually every case of AML with t(8;21), although it does not always involve the same antigens. M2 cases with t(8;21), mostly CD34+, had a 100% remission rate and 71% 5-year survival rate; other patients with CD34+ or CD34- AML showed 69% and 84% remission rates and 31% and 36% 5-year survival rates, respectively. Consequently, individual markers such as CD34 should be interpreted in relation to other features such as chromosomal changes. Thesesimple methods, which are well suited t o quantify the expression of ligands, are a useful contribution t o diagnosis: 60% t o 65% of M2 cases with t(8;21) are rapidly identified byCD34 overexpression alone. This aberration, together with the other signs ofasynchrony seen at presentation, can be used t o search for residual leukemia after therapy. 0 1996 b y The American Society of Hematology. T localized Sudan black B and myeloperoxidase (MPO) staining in the Golgiregion in the absence of monocytic involvement. In this early study, the nuclear immaturity amid signs of cytoplasmic maturation was also noted7-but the techniques were not yetavailable for afulldocumentation of maturation asynchrony. However, recent immunologic methods arefully suited to perform such an analysis for three main reasons: ( l ) a range of differentiationantigens,bothmembrane-associated and intracellular, are available to investigate stages of myeloid differentiation-andthesedefine theprecisesequence of development in the normalmyeloid lineage'""; (2) these markers can be studied with monoclonal antibodies (MAbs) labeled with various fluorochromes in three- and four-color combinations'","; and (3) expression of these antigens can be quantified as antibody-binding capacity (ABC) alongthe normalmaturationpathway-withthe clear intentionof comparingthesefeatures withthose seen onmalignant cells."'~'' Indeed, previous phenotypic studies on AML M2 with t(8;21) have already pointed to an aberrant CD19 expression''-Is together with interleukin-5 responsiveness'' and CD56I4.I6and CD34"-" display. To apply these recent developments, in this study we selected cases of AML M2 with t(8;21) to analyze precursor cell-associated markers such as CD34 or terminal deoxynucleotidyl transferase (TdT) in combination with differentiation antigens including MPO, CD13, and CD15. We then compared the results with those observed in other cases of AML andin fetalbone marrow (BM). Furthermore,we quantified the expression of these molecules, since it has been demonstrated that the intensity of CD34 expression in normal BM precursor cells is variable,I7 and only CD34'"" precursors coexpress cytoplasmic MPO.I8 Such an analysis of CD34 expression, in the context of other morphologic and cytogenetic features, is of clinical relevance because several investigators have previously indicated that the expression of CD34, when investigated as an independent parameter, HE RECENT FOCUS onuniform chromosomal aberrations in acute myelogenous leukemia (AML), and the confirmation of these findings by molecular probes, has identified large groups of patients whose malignancies share common pathogenetic featuresand representwell-defined prognostic groups."3 Frequently, these cohorts also display characteristic morphologic features such as a parody of promyelocyte development in acutepromyelocyticleukemia with t( 15; 17), myelodysplasia with micromegakaryocytes and lack of Auerrods in AML with 5q abnormality, or eosinophilia in AMLwith i n ~ 1 6 . ~In - ' another form of AML, so typical that M2 with t(8;21), the morphologic changes are the aberrant morphology has guided the identification of this subgroup at the time of its cytogenetic discovery.' The main features of this disease are prominent Auer rods, heraldinga disturbed maturation of the granulocytic lineage, and typical From the Department of Clinical Immunology, Royal Free Hospital School of Medicine, London; Department of Haematology, Royal Postgraduate Medical School, London; Medical Research Council (MRC) Clinical Trial Service Unit, Radchffe Infirmary, Oxford; Department of Haematology, University College Hospital, London; and Department of Haematology, Universiiy of Wales, Cardia UK. Submitted May 22, 1995; accepted September 14, 1995. Supported by grants fromthe Leukaemia Research Fund (28/89) and the Medical Research Council of Great Britain (SPG8417830) as part of the service to the MRC United Kingdom Acute Lymphoblastoid Leukaemia and Acute Myelogenous Leukemia trials. A.P.". and G.J. have contributed equally to this paper. Address reprint requests to Prof George Janossy, DSc, Department of Clinical Immunology, Royal Free Hospital School of Medicine, London NW3 2QG, UK. The publication costsof this article were defrayedin part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1996 by The American Society of Hematology. 0006-4971/96/8702-0016$3.00/0 1162 Blood, Vol 87, No 3 (February l), 1996: pp 1162-1169 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 1163 CD34OVEREXPRESSION IN t(8;21) AML appeared to be predictive of poor clinical o u t ~ o m e . 'How~-~~ ever, this finding seems to contradict the fact that AML M2 with t(8;21) is a favorable prognostic category'*3323s24 despite CD34 positivity of the blasts."-13 In this study, a total of 517 patients entered onto Medical Research Council (MRC) AMLlO therapeutic trials were studied. We describe the overexpression of CD34 antigen in AML M2 with t(8;21) and reveal the characteristic asynchrony when CD34 is studied in combination with MPO. This asynchrony also coincides with other aberrant phenotypic features, contributing to the differential diagnosis and allowing the immunologic monitoring of patients for the presence of residual disease. PATIENTS AND METHODS Selection ofpatients. Immunodiagnostic, cytochemical, and cytogenetic results were available on 517 BM and blood samples from patients with AMLaged 17 to 55 years (mean, 47.5y). Clinical follow-up data were available from the adult AMLlO trial of the MRC of Great Britain. The hematologic diagnosis followed the guidelines of the French-American-British classification based on morphology and cytocherni~try.~~ Routine immunophenotyping results were obtained by immunofluorescence microscopy or flow cytometry as described later. The treatment included four courses of intensive chemotherapy consisting of daunorubicin, cytarabine, and thioguanine 3 + 10 and 3 + 8 + 5 ; methotrexate, adriamycin, cyclophosphamide, and etoposide; and MidAc (mitozantrone, daunorubicin, cytosine arabinoside).26Patients with a matched sibling were allocated to allogeneic BM transplantation (BMT). Those who lacked a donor were randomized to receive either no further treatment or an autologous BMT as the fifth course. The results of stratification according to the three choices (ie, chemotherapy and allogeneic or autologous BMT) are available from the UK Trial Office, but are omitted here since they do not influence the study conclusions. Expression of CD34 antigen was quantified in about a third of the trial cases who had CD34' leukemia. This group included 53 representative patients from various French-American-British categories of AML, 11 cases with t(8;21) and 42 without t(8;21). Finally, a selected group of 18 cases of AML M2 with t(8;21) were further investigated to analyze the various signs of phenotypic asynchrony with a panel of markers used in two- and three-color immunofluorescence (IF) tests. Controlsamples. Fetal BMwas obtained from fetal long-bone samples provided by the MRC Tissue Bank, Royal Postgraduate Medical School, London, under the ethical guidelines and permission of the Ethical Committees of both the sending and receiving institutions. The gestational ages, 8 to 18 weeks, were determined from the crowdrump length. BM cells were flushed from the bone cavity with Hanks balanced salt solution, (Gibco, Uxbridge, UK). Normal BM samples were also received from children with idiopathic thrombocytopenia for diagnosis without evidence of BM pathology. KG1 cell line was grown under standard conditions. Immunodiagnosis. Mononuclear cells were separated on FicollIsopaque density gradient 1.077 g/mL (Lymphoprep; Nycomed, Oslo, Norway). The stored cells were rapidly thawed by diluting in Hanks balanced salt solution at 20°C and washed. Cell suspensions (2.5 to 5 X lo5cells per 50 pL) were incubated for 10 to 15 minutes at20°C with MAbs conjugated with fluorescein isothiocyanate ([FITC], green, fluorescence 1: Fll), phycoerythrin ([PE], orange, F12). or Peridinin chlorophyll A (red, F13) used as premixed cocktails at pretitrated optimal conditions (Table 1). After washing in phosphate-buffered saline, the stained cells were fixed in 0.5% para- Table 1. lmmunophrnotypes of Leukemic Blasts in 18 Cases of AML With t(8;21) Case No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 CD34' TdT CD13 MP0 Cl II CD15 CD14 CD56 98 95 90 88 85 85 82 79 72 70 65 50 50 42 40 36 20 0 0 NT 60 0 0 80 65 0 0 NT 0 0 0 40 NT 92 NT 10 90 95 90 61 70 90 89 70 62 60 60 61 0 39 50 0 15 60 97 40 40 94 92 NTt 94 91 86 25 80 96 80 40 NTt 96 20 NTt 90 95 90 98 90 0 89 95 94 90 96 80 0 0 8 18 5 60 0 48 5 60 31 0 0 0 30 73 0 5 0 0 96 NT 90 93 61 NT 20 0 0 NT 43 23 0 0 NT 80 58 60 98 95 80 0 5 8 0 0 0 5 0 9 0 0 0 5 0 0 0 98 NT 0 Results are given as percentages of positive cells within the blast cell populationanalyzed by flow cytometryor the APAAP irnmunoenzyme method. Abbreviation: NT, not tested. * MAbs were directly conjugated as CD13-FITC (WM47; from Dr K. Bradstock, Westrnead Hospital, Sidney, Australia), CD15-FITC (C3D-1; Dakopatts), HLA-DR-perCP (RFDR-2; RFHSM), CD14-PE(UCHM1;from Professor P. Beverley, ICRF, London, UK),CD34-FITC(BI-C35; Seralab, Crowley, UK),CD34-PE(QBEND; Professor M.F.Greaves,LRF Labs, London, UK), CD56-PE(Leul9; Becton Dickinson, UK), anti-MP0 (Dakopatts), and H-TdT6-FITC (Supertech, Bethesda, MD) and used on two- and three-color combinations. t Strongly positive when tested by cytochemistry. formaldehyde. Isotype-matched FITC- and PE-conjugated murine Igs were used as negative controls. Five-parameter flow cytometry, ie, three-color IF with forward (FSc) and side (SSc) scatters, was performed on a FACScan with Paint-a-gatel' and Lysys-II softwares (Becton Dickinson, Oxford, UK). List mode files were also transferred to IBM-compatible computer by HP-Reader and analyzed with Winlist software (Verity Software House, Topsham, MI). The percentage of positivity for various MAbs was determined after gating blast populations by their FSclSSc features. Cases were regarded as CD34' if more than 20% of cells in the blast gate expressed this antigen. Detection of cytoplasmic and nuclear antigens. During routine immunodiagnosis, detection of cytoplasmic M P 0 and nuclear TdT was performed on cytospins using anti-MP0 MAb (Dakopatts Ltd, High Wycombe, Bucks, UK) with alkaline phosphatase anti-alkaline phosphatase (Dakopatts), and rabbit anti-TdT serum with goat anti-rabbit Ig labeled with rhodamine (TRITC) as a second layer (Supertech, Bethesda, MD). M P 0 and TdT were also detected with direct IF using anti-MPO-FITC (Dakopatts) and anti-TdT-FITC (HTdT-6; Supertech) after fixing and permeabilizing the cells with Permeafix (OPF; Ortho Diagnostic Systems, Inc, Raritan, NJ). The cells (2.5 to 5 X 105/50 pL) were fixed for 30 minutes at 20°C in 800 pL OPF diluted in a 1:l ratio with distilled water," and after washing in PBS, they were incubated with 5 pL anti-MPO-FITC or anti-TdT-FITC for 30 minutes at 20°C. The cells were labeled for membrane CD34-PE with QBend-PE (kindly donated by Professor M. Greaves, LRF Leukaemia Laboratories, London, UK) before fixation and then stained for intracellular antigens (see above). Cyto- From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 1164 PORWIT-MacDONALD ET AL plasmic CD22 (RFB4; Royal Free Hospital), CD79 (mb-l; kindly provided by Dr D.Y. Mason, John Radcliffe Hospital, Oxford, UK; and Dakopatts), and cytoplasmic CD3 (Leu 4-PerCP, BD) were investigated by the same methods. Quantifation of CD34 expression. FACScan was set on logarithmic scale. Instrument settings and channel compensations were controlled by calibrated fluorescence reference beads Quantum (Flow Cytometry Standard, Research Triangle Park, NC). Intensity of CD34 expression was measured as ABC per cell on standard cells with indirect IF using the QIFI kit (Biocytex, Marseille, France) as described previously.28 Boththe first layer, CD34 MAb (BI3C5; Seralab Ltd, Crowley Down, UK), and the affinity-purified second layer, goat anti-mouse Ig (GcuMIg-FITC; Southern Biotechnology Associates, Birmingham, AL), were usedat pretitrated saturated conditions. One standard was CD34+ AML blasts taken from a single patient at the start of the study (PJ-AML), frozen in small aliquots, and thawed with more than 90% viability whenever needed throughoutthe investigations. These PJ-AML blasts expressed 64 X IO’ CD34 mean ABC per cell when tested with the QIFI test. The other standard was a subclone of the KG1 cell line expressing 410 X 10’ CD34 ABC per cell, a value at the upper limit of the measured range. At the same time, QIFI tests were also performed on CD4’ normalblood lymphocytes withthe same GaMIg-HTC second layer, producing 47 to 57 x IO3 CD4 ABC.’’ These three values together represented the framework to render the quantitative IF tests comparable through the span of studies. Atthenext stage, directly conjugated CD34 MAbs, CD34-FITC (BI-3C5; Seralab) and CD34-PE (QBend, see above), were usedat saturating level in a final concentration of 5 pW100 pL on the same cellular standards (PJ-AML and KGl). The bead standards used for direct IF (Quantum Simply Cellular; QSC) from Flow Cytometry Standards (Research Triangle Park, NC)” were recalibrated to match the known values of 64 X IO3 and 410 X IO3 per cell on PJ-AML and KGl, respectively. This was a necessary step to maintain reproducibility between different directly conjugated CD34 reagents. Values for ABC were determined on Winlist with the QuickCal method following linearization of the mean fluorescence intensity.” Thus, CD34-PE provided higher M H values than CD34-FITC, but similar ABC per cell. These two reagents showed an excellent correlation on the various cases of AML (R2 = .99; not shown) and could therefore be interchangeably used in two- and three-color IF combinations. Statistics. Life-table analyses with Kaplan-Meier curves were prepared at the MRC Clinical Trials Service Unit, Cancer Studies Unit, Oxford, UK, using the Statistical Analysis System (Cary, NC) and the Mantel-Haenschel chi-square test. The nonparametric MannWhitney (Itest was also applied. RESULTS Quantitation of CD34 antigen density in normal BM. The mean percentage of CD34+ cells in the mononuclear fraction of fetal and normal BMwas6.2% (range, 1% to 20%; n = 6). There was a difference in CD34 binding capacityof small- to intermediate-size cells (mean, 26.5 X 10’ ABC per cell) versus larger cells (mean, 42 X lo’ ABC per cell), as determined by gating cells of differing sizes on the scatter plot and calculating their CD34 ABC values. This was confirmed by double staining with CD10 MAb: CD34+/ CD10+ cells (B-cell precursors) displayed lower CD34 ABC (mean, 29.5 X lo’ ABC per cell) than CD34+/CD10- cells with larger size that include early myeloid precursors (mean, 44 X lo3 ABC per cell). Very few, if any, cells could be observed with more than 60 X 10’ CD34 ABC per cell; this value can be considered the upper limit of CD34 expression on precursor cells in fetal and normal BM. Double staining for CD34 and MP0 in normal and fetal BM showed that among CD34+ cells, the MPO- majority population (R2 gate in Fig lb) expressed higher levels of CD34 (median, 25 X lo3 CD34 ABC per cell; Fig IC) than the rare MPO+ forms (R3 gate in Fig lb; median, 16 X lo’ CD34 ABC per cell; Fig Id). Incidence of CD34 positivity in AML. When grouped by French-American-British criteria, among 5 17 cases of AML tested, 103 (19.9%) were MOM1, 152 (29.4%) M2, and 65 (12.6%) M3, and 174 (33.8%) showed signs of monocytic differentiation, including 121 cases of M4and 53 of M5 (Fig 2), with 21 cases (4.1%) in other minor categories (not shown). Itwas found that CD34 positivity, identified by more than 20% CD34’ cells within the blast population, was less frequent in AML M3 and M5 (18% and 28% of cases, respectively) than in the other AML subtypes (39% to 42% of cases); nevertheless, these differences were not statistically significant (Fig 2). The incidence of CD34 expression was checked among M2 cases with t(8;21). In this group, higher CD34 expression was seen than in the other cases with M2 morphology without t(8;21) ( P < .001). In 32 of 38 t(8;21) cases tested (84.2%) more than 20% leukemic blasts were CD34+, with a mean of 68.5% (range, 20%to 98%). Nevertheless, six cases of M2 with t(8;21) representing 15.8% of t(8;21) cases were CD34-. Quantitation of CD34 antigen expression in AML. Since both microscopic and flow-cytometricobservations indicated that in a number of AMLM2 cases CD34 staining was particularly strong, the CD34 ABC was quantified in about one third of all CD34’ leukemias entered into the trial. These were morphologically characterized as seven MO, 18M1, eight M2 without t(8;21), l1 M2 with t(8;21), and nine with monocytic differentiation (M4/5). CD34 ABC was significantly higher in AML with t(8; 2 l) than in any other group of AML (Fig 3); six of 11 cases displayed values more than twofold higher (300 to >400 X lo3 ABC per cell) than the highest CD34 ABC (144 X lo3 ABC per cell) found in other cases of AML without t(8;21). In nine of 11 cases (82%), CD34 ABC was higher than the upper limit of the CD34 range for normal BM (60 X 10’ ABC per cell). CD34 ABC was also higher than the mean CD34 expressionseen in normalBM (30 X loq ABC) insevenof 18 (38%) M1, three of eight (37.5%) M2 without t(8;21), and three of nine (33%) AML. Nevertheless, only seven of 42 cases (16.7%) of AML without t(8;21) had higher than the maximal levels of CD34 expression seen in normal fetal BM (Fig 3). Aberrant features of myeloblasts in AML M2 with t(8;21). A total of 18 cases were investigated in further detail, all of which showed t(8;21)-associated cytologic and cytochemical features’such as M2 morphology displaying varying degrees of granulocytic maturation, with Auer rods seen in all cases. There was no evidence of any monocytic component on either morphology or esterase staining or any demonstrable erythroid or megakaryocytic abnormalities. The blasts also displayed the typical nuclear indentation with a prominent Golgi region. Granulocyte maturation was abnor- From www.bloodjournal.org by guest on February 6, 2015. For personal use only. CD34 OVEREXPRESSION IN t(8;21) AML 1165 mean ABC value (x103 CD34 ABClcell): Fig 1. Quantitative assessment ofCD34 expression in normal infant BM la t o d)and in AML M2 with t(8;21) (e t o h).Values in the gateR2 in normal BM (b) correspond to the mean values of 25 x l o 3ABC per cell and includebothintermediateand large cells. Only some of these cells are MPO-positive (16 x lo3 CD34 ABC per cell). As a marked contrast, MPO-positive cells (gated in R51 in AML with t(8;21) were as high as 165 x lo3 CD34 ABC per cell, above the normal range. R5 in f can be used as a live gate t o search for residual aberrant myeloblasts in patients during therapy. W W l0 90 110 Forward Scatter 3 MPO-FITC mal in all cases, with asynchronously poor nuclear maturation and cytoplasmic hypogranularity from the myelocyte stage onward. Sudan black B staining revealed a typical localized positivity in the Golgi region within the blasts, and a small proportion of blasts were chloroacetate esterasepositive. The eosinophils, where present, usually showed variable granule size and amphophilic staining. When studied by flow cytometry (Table 1) the blast cells had a similar pattern of FSc/SSc, with most cells locating within the myeloid blast region (54% to 91%; mean, 72.5%) and spreading toward the differentiating myeloid cell region (Fig le). Four markers showed a relative uniformity of this patient group. The high proportions of CD34' cells were accompanied by similarly high percentages of blasts positive for class 11, CD13, and MP0 in all except one or two unusual cases with each single marker studied (Table l ) . CD14 nega- IO' + 10' IO' CDM-PE ?, CD34-PE + tivity, confirming the lack of monocytic differentiation, was also uniform, whereas the expression of CD15 was heterogeneously positive in 39%of cases when tested witha VIMDSlike antibody reacting withthe nonsialated determinant of the Lewis' antigen.' Among I8 cases of AML with t(8;2 I), 13 were studied for CD56 expression. Six of these (46%) had more than 40% CD56' myeloblasts coexpressing CD34-a leukemia-associated combination.'" A sign of asynchronous development was the expression of nuclear TdT in myeloblasts that coexpressed membrane CD13 or cytoplasmic MP0 in six of 14 cases tested(42%)-and again, these features are leukemia-associated.3' Nevertheless, the most frequent aberration in AML M2 with t(8;21) was the overexpression of CD34 antigen, which was formally quantified and found to be above the levels in 39 Mofhll MO 0 20 40 60 80 100 Fig 2. Incidence of CD34* leukemias among cases of AML. In this study of 494 patients, cells with >5 x lo3 CD34 ABC per cell were regarded as positive, and cases with >20% CD34' cells within the blast gate werescored as positive. The numbers shownare percentage positive cases within each French-American-British category. MI - t(8;21) no t(8;Zll M2 M4 MS Fig 3. Quantitation of ABC per cell in 53 casesof CD34' leukemia. Mean ABC values in blast cell populations are shown followingquantitation withCD34-FITC calibrated on the standard AML and KG1 cell line. The highest expression is observed in M2 with t(8;211. In six cases, values are >250 x lo3 ABC per cell. From www.bloodjournal.org by guest on February 6, 2015. For personal use only. PORWIT-MacDONALD ET AL 1166 were still all in remission, and 32 CD34' patients hadan excellent 62% 5-year survival rate (Fig 5); nevertheless, this CD34- versus CD34' comparison, due to the small sample size of CD34- patients with t(8;21), is uninterpretable. When the non-t(8;21) group was analyzed separately, CD34- patients had a significantly higher CR rate (84%) and marginally better survival (36%) than the CD34' group (69% CR and 3 1 % survival). However, the difference between CD34and CD34' subgroups is relatively small compared with the greater impact of cytogenetics. CD34 over-expression CD34 MP0 CD34 CD66 TdT MP0 or CD13 t J cumulative of these DISCUSSION D 20 40 60 80 100 % of cases tested Fig 4. Aberrant and asynchronous staining combinations in AML M2 with t(8;211. The relative frequency of CD34 overexpression, asynchronous combination with M P 0 and CD56, and aberrant combination of TdT staining with M P 0 andlor CD13 are shown. The cumulative data reveal that aberrant or asynchronous features are seen in every case of AML with t(8;211, but these alterations are not fully uniform (see Table 11. The AML M2-associated t(8;21)(q22;q22.3) results in fusion of the AMLl gene on chromosome 21 with a gene on chromosome 8 referred to as ET0.".33 These molecular findings already provide insights into the altered function of the AMLIETO protein in the processes normally regulated by AMLl gene during myeloid differentiation.3 In this report, we characterize the complexity of these changes when measured in these malignant AML blasts with quantitative immunologic techniques. In all of these cases, irregular control of cellular differentiation can be demonstrated with simple combination staining for precursor cell features such as CD34 or TdT versus signs of myeloid differentiation such as MPO, CD1 3, and CD15 display. Furthermore, many of these alterations appear to be characteristic for this particular disorder. Further extended use of the quantitative techniques used in this study on a larger cohort of patients willbe required to see whether such changes occur at all in AML without t(8;21). The most obvious of these changes is the overexpression of CD34 antigen in MPO-positive malignant myeloblasts. Nevertheless, these changes are not fully uniform (Fig 4), and16% of AML with t(8;21) are CD34-. Thus, the complexity of the changes argues for a disturbed regulatory control rather than for mere involvement of genes that directly regulate these particular proteins. One of the main features of our study has been the quantitation of CD34 antigen on normaland leukemic cells in combination staining with other MAbs, eg, for cytoplasmic MPO. This necessitated the use of CD34-FITC and CD34- CD34' cells seen in normal and fetal BM in nine of I 1 cases (81%; Fig 3). Furthermore, in eight of these samples (73%), CD34 showed an asynchronous combination with MP0 above the levels seen in normal BM'8 (Fig l). A distinctive live gating (R5 in Figs I f and h) clearly identified large proportions of malignant blasts, indicating which parameters should primarily be used when remission samples are analyzed from the same patient. The high levels of CD34 expression in combination with another marker of granulocytic differentiation, CD15, were also recorded in seven of 18 cases analyzed (39%), although the double-stained blasts were relatively few in two cases (Table l ) . Finally, strong CD19 expression byCD34' myeloblasts was detected in three cases, whereas in other samples the CD19' peak, when studied with the RFB9 MAb used, showed weak positivity but could notbe fully separated from negative control; the CD19 ABC per cell waslow (not shown). Cytoplasmic CD22 and CD79a, bona fide Blineage-specific antigens, were negative in all cases, including those that were TdT-positive. Taken together, these obTable 2. Clinical Outcome in 517 Patients Entered Into the MRC servations demonstrate that aberrant andor asynchronous AMLlO Trial-Effects of t(8;21) and CD34 Expression combinations are identifiable in virtually every case of AML t(8:21)-* t(8;21)" M2 with t(8;21), although these changes are not fully uniform (Fig 4) and in 15% to 20% of AML M2 with t(8;21) CD34CD34' CD34CD34' do not express strong CD34 positivity (Figs 2 and 3). 143 336 patients No. of 6 32 Correlation with clinical outcome. Clinical outcome for CR rate (%l 69t 84t 100 100 38 patients with t(8;21) and 479 patients with other forms Reason for failure (%l of AML is shown in Table 2 and Fig 5 . All patients with Induction death 9 15 0 0 t(8;21) obtained complete remissions ([CRs] 100%); in this Resistant disease 7 16 0 0 group, significantly fewer relapses and deaths were seen 36* 31$ l005 625 Survival (%) from entry at 5 years (71% 5-year survival) than among patients who hadno P < ,0005 for both CR rate (x2 test) and survival (log-rank test1 t(8;21) (34% survival, P < .0005;Table 2). Taken overall, differences between t(8;211-positive and -negative patients. the CR rate was significantly better in CD34- patients than t P = .0003 by x* test. in CD34' patients (84% v 75%, P = .008), but there was P = .05 by log-rank test. no survival difference (38% v 37% at 5 years). When the 5 P is not interpretabledue to the small number of CD34- patients t(8;21) group was studied separately, six CD34- patients in the t(8;211 group. * From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 1167 CD34 OVEREXPRESSION IN t(8;21) AML Fig 5. Kaplan-Meier survivalcurvesfor patients from the time of entry onto the AMLlO trial according to the presence or absence of tl8;21) and CD34 antigen on blasts at presentation.The criterion for CD34 positivity was ~ 2 0 % CD34' cells among blasts. tl8;21)-, CD34- (-, n = 336); t18;21)-, CD34+ n = 143); t(8;21)+, CD" I - . - . - , n = 6); and t(8;21)+, CD34+ I - . -. , n = 32). ( 9 . ., -. - PE reagents. If a single reagent is used in an investigation, the molecules equivalent to soluble fluorochromes (MESF units) are adequate to obtain comparative data.30 Inour case, it was more appropriate to introduce standard CD34' cells that expressed known values for ABC per cell established by the QIFI test in indirect IF28,29,34 to secure the reproducibility of the performance of directly conjugated CD34 used in diagnostic staining combinations (see the methods). In this way, quantitative IF becomes a practical possibility. The same methods have already been applied to document the overexpression of CD10 antigen on 44% of cases with acute lymphoblastic leukemia.34 Expression of CD34 in AML has previously been associated with the poorly differentiated French-American-British subgroups MO and Ml,35.36and several studies have suggested that CD34 positivity may be related to a poor clinical response in terms of CR and/or s u r v i ~ a l . ' ~In. ~this ~ -context, ~~ the observations of Lanza et al,37 who also find that the "bright" CD34 expression is associated with poor prognosis, are particularly interesting. This is because cytogenetic analysis reveals that their AML M2 group lacks cases with t(8;21) but primarily shows -5 and 5q- involvement, a known poor prognostic sign.38Thus, our investigations, based on large numbers of patients who were uniformly treated in the AMLlO multicenter may resolve the apparent discrepancies seen in the literature that appear to be due to different proportions of patients with various chromosomal abnormalities in the individual studies. On one hand, when we excluded t(8;21) patients from the evaluation, CD34+ AML cases were less likely to obtain CRand showed slightly inferior 5-year survival as compared with the CD34- group (Fig 5). These findings are reminiscent of the poor prognostic significance of CD34 expression, save the t(8;21) group. On the other hand, we confirm the survival advantage of patients with t(8;21) despite the obviously bright CD34 expression on most of these cases.1.23In conclusion, these findings indicate that individual markers such as CD34 positivity should not be taken out of the context of the overall clinical and cytogenetic presentation, but should be used for patient assessment as part of the diagnosis. Our studies also confirm other observations that emphasize the relevance of chromosomal changes in interpreting the prognostic significance of lymphoid marker expression such as CD19 and CD211.39, as well as CD7,40in the various forms of AML. A clear example of the diagnostic advance inherent in our investigation is the use of quantitative IF for rapid diagnosis, as well as remission assessment, in t(8;21) AML. Sensitive polymerase chain reaction techniques have uniformly revealed the persistence of AML1-ET0 fusion gene in patients during remission, sometimes as long as 52 months after therapy.3,41-44 This is a clear step toward a better understanding of a multistep process in malignancy, but it questions the clinical utility of these molecular probes during the remission phase. Residual-disease assessment with immunologic methods in these diseases should now include analysis of CD341 CD56,L4,16CD13/TdT?' CD19/CD13,'1,'2"4.39and CD34/ MPOLs(and this study), together with a quantitative analysis of CD34 expression as delineated in this report. It will be interesting to observe whether patients with identifiable molecular disease retain or lose signs of asynchrony in myeloid development while remaining in an apparent clinical remission. ACKNOWLEDGMENT A.P.M. was on leave from the Department of Pathology, Karolinska Hospital, Stockholm, Sweden. 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CD34 overexpression in acute myelogenous leukemia M2 with t(8;21) A Porwit-MacDonald, G Janossy, K Ivory, D Swirsky, R Peters, K Wheatley, H Walker, A Turker, AH Goldstone and A Burnett Updated information and services can be found at: http://www.bloodjournal.org/content/87/3/1162.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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