Diagnostic Criteria for Minimally Differentiated Acute Myeloid Leukemia (AML-M0) Evaluation and a Proposal

Hematopathology / MINIMALLY DIFFERENTIATED ACUTE MYELOID LEUKEMIA (AML-M0) Diagnostic Criteria for Minimally Differentiated Acute Myeloid Leukemia (AML-M0) Evaluation and a Proposal Zahid Kaleem, MD, and Glenda White, MT(ASCP) Key Words: Flow cytometry; Acute myeloid leukemia; AML-M0; FAB-M0; Immunohistochemistry; Immunophenotyping; Cytogenetics Abstract We studied immunophenotypic features of 30 cases of minimally differentiated acute myeloid leukemia (AML-M0) using multiparameter flow cytometry and immunohistochemistry and evaluated the immunophenotypic features of previously reported cases to facilitate correct identification of myeloid lineage. All but 1 of our 30 cases expressed CD13 and/or CD33; 2 expressed CD19; 1 expressed CD10; none expressed both CD10 and CD19. Eleven of 30 cases expressed T-cell associated antigens. All but 2 cases expressed CD34 and/or HLA-DR. Twelve of 27 cases expressed terminal deoxynucleotidyl transferase. Myeloperoxidase (MPO) expression was seen in 22 of 22 cases by immunohistochemistry and 1 of 4 by flow cytometry. None of 27 cases expressed cycd3 and cycd79a. We propose following modified criteria for AML-M0: (1) standard criteria for acute leukemia; (2) undetectable or less than 3% MPO or Sudan black B staining in blasts; (3) lack of expression of lymphoidspecific antigens, cycd3 for T lineage and cycd79 and cycd22 for B lineage; and (4) positivity for any of the myelomonocytic lineage antigens known not to be expressed on normal T or B lymphocytes or positivity for MPO as detected by ultrastructural cytochemistry, immunohistochemistry, or flow cytometry. Despite its recognition for a decade, the distinction of minimally differentiated acute myeloid leukemia (AML-M0) from acute lymphoblastic leukemia (ALL) can still be a challenge in a minority of cases for both novices and experts. It was not until 15 years after their original criteria for the classification of acute leukemia that the French-American-British (FAB) Cooperative Group recognized that many of the cases morphologically considered as examples of ALL were in fact of myeloid origin as determined by monoclonal antibodies against the myeloid-associated antigens CD13 and CD33 and by ultrastructural cytochemistry. 1,2 The subsequently proposed FAB criteria hold true for a majority of the minimally differentiated AMLs; however, a minority of cases defy correct identification. The distinction, however, is of utmost importance, as the treatment protocols, clinical behavior, and prognosis differ for the two categories. Iwate et al 3 described 1 case that initially was misclassified as AML-M0 based on original FAB criteria but later, with further laboratory investigation, was found to be a case of T-cell ALL. The bulk of errors, however, stem from misdiagnosis of AML as ALL because of the frequent expression of terminal deoxynucleotidyl transferase (TdT) and T-cell associated antigens, as well as the frequent FAB L1 or L2 morphologic features. 4 A number of publications describing AML-M0 have appeared during the past several years using FAB criteria; most of those emphasized the expression of CD13 and/or CD33 with or without myeloperoxidase (MPO), but inconsistent criteria exist as to which lymphoid markers are acceptable in the definition of AML-M0. 5-8 We sought to critically evaluate the various inclusion criteria for AML-M0, and we present our data for 30 cases to propose modifications in the original FAB criteria for AML-M0. 876 Am J Clin Pathol 2001;115:876-884 American Society of Clinical Pathologists

Hematopathology / ORIGINAL ARTICLE Materials and Methods We retrieved 30 cases from the files of Lauren V. Ackerman Laboratory of Surgical Pathology, Department of Pathology and Immunology (Washington University Medical Center, St Louis, MO) that had been examined from January 1, 1993, to December 31, 2000. Only de novo cases with bone marrow core biopsy specimen, aspirate smear, or peripheral blood smear and complete flow cytometric analysis were selected. Morphologic Examination All specimens were obtained and prepared for morphologic examination using standard techniques. The core biopsy specimens were fixed in 10% buffered formaldehyde, embedded in paraffin, and processed routinely, and the sections were stained with H&E, Leder (chloroacetate esterase), iron (Prussian blue), and reticulin stains for light microscopy. Bone marrow aspirate smears and peripheral blood specimens (when available) were air dried and stained with Wright-Giemsa technique and examined under light microscopy. Enzyme Cytochemistry For the detection of MPO, the air-dried bone marrow or peripheral blood smears were fixed for 1 minute at room temperature in 10% formalin and ethanol followed by washing with tap water for 15 to 30 seconds. The wet slides were placed for 30 seconds at room temperature in an incubator mixture containing ethyl alcohol (30%; 500 ml), benzidine dihydrochloride (1.5 g), zinc sulfate solution (5 ml), sodium acetate (5 g), hydrogen peroxide (3%; 3.5 ml), sodium hydroxide (1.0 N; 7.5 ml), and safranin O (1.0 g). The slides then were washed for 5 to 10 seconds in running tap water, counterstained in working Giemsa for 10 minutes followed by another wash with tap water, and air dried and mounted. For the detection of nonspecific esterase using alphanaphthyl butyrate esterase, the air-dried bone marrow or peripheral smear slides were fixed in cold formalin and acetone fixative for 30 seconds followed by one washing with tap water and a second washing with distilled water. Slides were incubated for 45 minutes at room temperature in a mixture containing M/15 phosphate buffer, ph 6.0-6.3 (18.8 ml); hexazotized pararosaniline (0.2 ml); and alpha-naphthyl butyrate esterase solution (1.0 ml). The slides were first washed with tap water and then with distilled water, counterstained with methyl green and alcian blue for 6 minutes followed by a wash with tap water, and air dried and mounted. Flow Cytometric Evaluation Bone marrow aspirates were transported immediately in sodium heparin tubes to the flow cytometry laboratory. Mononuclear WBCs were isolated and stained with various combinations of the following fluorescein isothiocyanate or phycoerythrin-labeled monoclonal antibodies against the following antigens: CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD13, CD14, CD19, CD20, CD33, CD34, CD41a, HLA-DR, and nuclear TdT. In selected cases, monoclonal antibodies against other antigens also were run with or without additional fluorochromes, including CD11c (cases 24, 26-27), CD15 (cases 24, 28-30), CD16/CD56 (cases 23, 25-30), CD61 (cases 26, 28-30), CD64 (cases 26, 28-30), CD117 (cases 26, 28-30), cytoplasmic (cy) CD3 (cases 26-30), cycd79a (cases 26-30), MPO (cases 26, 28-30), glycophorin A (cases 28-30), and T-cell receptor (TCR) alpha-beta and TCR gammadelta (case 27). For the detection of cytoplasmic (cycd79a and cycd3) and nuclear (TdT) antigens, surface staining of CD45 for gating purposes (for 4-color analysis only), as well as any for other surface antigens, was performed before processing the cells for cytoplasmic and nuclear staining. The cells then were fixed with Reagent 1 from the Beckman-Coulter- Immunotech (Miami, FL) Intraprep Permeabilization reagent kit. After 15 minutes of fixation, the cells were washed and permeabilized for 5 minutes with Reagent 2 from the kit. After a 5-minute permeabilization, appropriate antibodies were added and incubation time was adjusted for the antibody requiring the longest reaction time, 20 minutes for cytoplasmic antigens and 1 hour for nuclear antigens. The cells then were washed with Hanks balanced salt solution and fixed in the usual manner with 1% methanol-free formaldehyde before flow cytometric analysis. Two- and 4-color cytometric immunophenotyping was performed on the FACScan (2-color only; Becton Dickinson, San Jose, CA) or on the Coulter XL cytometer (2- and 4- color; Coulter, Miami, FL) by collecting 10,000 ungated list mode events, selecting an appropriate blast gate on the combination of forward and side scatter, and analyzing cells with the most appropriate blast gate. Some of the cases were gated on CD45dim vs side scatter to isolate the blast population, and 10,000 list mode events were collected on the blast gate. An antigen was considered positively expressed when at least 20% of the gated blasts expressed that antigen; however, the arbitrary cutoff limit for positive TdT expression was 10%. Analysis of CD34, CD13, CD14, CD33, CD41a, CD19, CD7, and HLA-DR was performed on all cases and for TdT in all but 3 cases. CD10 was analyzed in all except 3 cases (cases 28-30), and CD20 was analyzed in 13 cases (cases 1-12 and 20). Expression of CD1, CD2, CD4, CD5, and CD8 was analyzed in 17 cases. Immunohistochemical Analysis Immunohistochemical analysis for cycd3, cycd79a, American Society of Clinical Pathologists Am J Clin Pathol 2001;115:876-884 877

Kaleem and White / MINIMALLY DIFFERENTIATED ACUTE MYELOID LEUKEMIA (AML-M0) and MPO was performed on formalin-fixed and paraffin embedded core biopsy specimens In 22 of 27 available cases (except cases 1, 5, 9, 26, and 27). Five-micrometer-thick sections were cut and collected on lysine-coated slides and dried in a 60 C paraffin oven for 45 minutes. Sections were deparaffinized in xylene, incubated for 30 minutes in methanolic hydrogen peroxide (0.3% [vol/vol]) to quench for endogenous peroxidase and rehydrated in graded ethanol solutions followed by rinsing in distilled water and phosphate-buffered saline (ph 7.4). Heat-mediated antigen retrieval (epitope retrieval) was carried out for 12 minutes in a microwave oven in the presence of citrate buffer (ph 6.0). The sections were cooled for 20 minutes followed by rinsing in water and incubation with phosphate-buffered saline. A protein block (DAKO, Carpinteria, CA) was performed for polyclonal antibodies with a 5-minute incubation period. Primary antibodies against MPO (polyclonal 1:4,000 dilution; DAKO), CD3 (polyclonal, 1:40 dilution; DAKO), and CD79a (monoclonal, 1:100 dilution; DAKO) were applied, and the sections were incubated for 18 hours at 4 C. Antibody bridge assembly by the avidin-biotin-peroxidase complex (ABC) technique, using the Elite ABC kit (Vector Laboratories, Burlingame, CA), was performed the next day by 2 sequential 1-hour incubations. Chromogenic development was accomplished by immersion of the sections in 3,3'- diaminobenzidine solution (0.25 mg/ml with 0.003% hydrogen peroxide). The slides were immersed in 0.125% osmium tetroxide to enhance chromogenic precipitation, followed by light counterstaining with Harris hematoxylin. The sections were dehydrated in graded ethanol, cleared in xylene, and mounted with Cytoseal medium (Electron Microscopy Sciences, Fort Washington, PA). Cytogenetics The bone marrow and peripheral blood specimens were transported in RPMI 1640 culture medium with 15% fetal calf serum and were cultured for 24 hours at 37 C. Cells were exposed to Colcemid (0.05 µg/ml) for 2.5 hours at 4 C and harvested routinely. Routine slide preparation and Giemsa banding at 450-band resolution were performed. Results There were 13 male and 17 female patients with ages ranging from 8 to 87 years Table 1. In all cases, there were more than 30% blasts in the bone marrow. The blasts from all cases showed no or less than 3% reactivity for MPO by cytochemical analysis. No Auer rods were found in any case. None of the cases showed cytochemical reactivity for alphanaphthyl butyrate esterase. Most cases showed expression of CD34 (25/30 [83%]) and HLA-DR (25/30 [83%]) by flow cytometry (Table 1). Except for 1 case, all showed expression of one or both myeloid-associated antigens, CD13 and CD33. CD14 expression was seen in only 1 case (case 6) that also showed expression of both CD13 and CD33 and strong MPO reactivity by immunohistochemical analysis (Table 1). Only 1 of 4 cases tested (case 24) showed expression of CD15. Two cases (11 and 13) showed high-level expression of megakaryocyte-associated antigen CD41a (glycoprotein IIb) but also showed expression of CD13 and MPO by immunohistochemical analysis. Neither of the 2 cases had marrow fibrosis or other features of megakaryoblastic leukemia (AML-M7). Two (7%) of 30 cases expressed the B-cell associated antigen CD19 with concurrent expression of CD13 and CD33, and another case expressed CD10; none of the cases coexpressed CD19 and CD10. None of the cases tested expressed CD20 (by flow cytometry [13 cases]) or CD79a (by flow cytometric [5 cases] or immunohistochemical [22 cases] analysis). Expression of T-cell associated antigens was seen in 11 (37%) of 30 cases (CD7 only, 7/30 cases; CD2 only, 2/17 cases; CD2 and CD7, 1/17 cases; CD4 only, 1/17 cases). CD1, CD3, CD5, and CD8 were not expressed in any cases tested. Also, none of the 24 cases in which testing was performed expressed cycd3 by immunohistochemical (22 cases) or flow cytometric (5 cases) analysis. Twelve (44%) of 27 cases showed low level (10%-25%) or substantial (>25%) expression of TdT. This frequency is the same as the overall positivity rate in the cases reported in the literature as positive for TdT (61/140 [43.6%]) and compiled in Table 2. 2,5,7-19 In 22 cases tested, MPO as detected by immunohistochemical analysis using a polyclonal antibody was recorded as present in fewer than 1% of cells (+; 8 cases), 1% to 5% of cells (++; 8 cases), 5% to 25% of cells (+++; 4 cases), or more than 50% of cells (++++; 2 cases) Image 1. Case 26 showed no expression of CD13 and CD33 but moderate expression of CD7, TdT, and CD41a. However, almost all blasts (96%) in that case showed cytoplasmic MPO expression by flow cytometry Figure 1 without expression of cycd3, cycd79a, or CD61 (glycoprotein IIIa), confirming a myeloid origin of this acute leukemia. Furthermore, dual CD34 and MPO reactivity was present in a substantial proportion of blasts (33%) in that case. Case 27 showed expression of 3 lymphoid-associated antigens (CD2, CD7, and CD10) in addition to CD13 and CD33 but did not show cycd3, CD79a, TCR alpha-beta, and TCR gamma-delta. Except for 1 case (case 26), 3 of 4 cases (cases 28-30) in which CD117 was analyzed showed substantial CD117 expression. None of the cases in which testing was performed showed expression of CD11c, CD61, CD64, or glycophorin 878 Am J Clin Pathol 2001;115:876-884 American Society of Clinical Pathologists

Hematopathology / ORIGINAL ARTICLE Table 1 Immunophenotypic and Karyotypic Features of 30 Cases of Acute Myeloid Leukemia (AML-M0) * Case No./ Sex/ HLA- Age (y) CD34 CD13 CD33 CD14 CD41a CD19 CD7 CD2 TdT DR MPO Cytogenetics 1/M/34 84 <10 64 <10 0 0 0 ND 0 91 ND NA 2/F/61 77 25 15 <10 <10 0 47 0 0 50 + Complex 3/M/59 81 81 25 0 <10 48 0 0 17 88 + NA 4/F/8 61 0 36 14 <10 0 0 0 38 69 +++ NA 5/F/81 81 81 47 <10 <10 0 0 0 <10 87 ND NA 6/M/38 50 96 90 20 <10 0 0 0 <10 90 ++++ 45,XY, 7[10] 7/F/40 31 80 77 <10 <10 0 0 0 13 96 ++ 46,XX[23] 8/F/8 91 50 86 0 0 97 0 0 90 99 ++ 46,XX,del(22)(q11.2)[3]/46,XX[33] 9/M/87 85 0 46 0 0 0 0 0 0 14 ND 47,XY,+8; del(13)(q12q21)[12] 10/M/65 79 58 <10 0 0 0 0 0 65 72 +++ NA 11/F/70 91 20 0 <10 85 0 0 0 <10 86 ++ 46,XX,del(5)(q13q35)[3]/ 43, X, X,del(7)(q32), 21[1] 12/M/79 95 23 0 0 0 0 0 0 64 96 ++ 46,XY[25] 13/F/53 80 80 <10 <10 68 0 0 ND 41 68 ++ 46,XX[25] 14/F/35 75 60 90 0 0 0 0 ND 0 41 + 44-45,XY, 5, 6, 7,+8, 15, 16,17,+2-3 mar[24] 15/F/36 97 98 71 0 0 0 0 ND 17 93 + 45,XX, 7[14] 16/F/62 <10 84 95 0 0 0 0 ND 0 40 + 46,XX[25] 17/F/46 <10 63 79 0 0 0 0 ND 0 0 +++ 46,XX,t(9;11)(p22;q23)[14]/46,XX[2] 18/F/57 50 50 65 0 0 0 0 0 0 <10 +++ 46,XX[20] 19/M/78 0 30 0 0 0 0 92 ND 0 95 ++ 46,X, Y,add(4)(p14),del(6)(q21q23), +9,del(16)[24] 20/M/78 83 83 58 0 15 <10 31 ND ND 90 + 46,XX[24] 21/M/35 45 42 62 0 0 0 0 ND 0 73 ++ 46,XY,t(1;14)(p35;q11.2), t(9;22)(q34;q11.2)[19] 22/F/82 72 72 <10 0 20 0 39 ND 84 95 + 46,XX,del(9)(q13q22), add(10)(q22)[3]/46,xx[17] 23/F/70 <10 0 80 0 0 0 95 0 <10 69 ++ 46,XX,der(7)t(7;8)(q32;q13)[3]/ 46,XX[17] 24/F/45 0 0 99 0 0 0 0 96 0 0 ++++ Complex 25/M/27 75 0 87 0 0 0 0 60 61 57 + 46,XY,del(7)(q22)[5]/46, del(2)(q23q35)[6] 26/M/78 33 0 0 0 37 0 33 0 22 76 ND 47,XY,+13[20] 27/M/73 98 70 55 0 0 <10 100 65 24 12 ND 46,XY[2] 28/M/75 99 93 91 0 0 0 0 ND ND 84 ND NA 29/F/84 97 0 25 0 0 0 12 ND ND 87 ND NA 30/F/79 92 88 86 0 0 0 98 ND 0 84 ND 46,X,del(X)(q22),inv(2)(p11.2q21), del(5)(q15q33)[10] MPO, myeloperoxidase; NA, not available; ND, not done; TdT, terminal deoxynucleotidyl transferase; +, present in <1% of cells; ++, 1%-5% of cells; +++, 5%-25% of cells; ++++, >50% of cells. * Immunophenotypic data are given as percentages. Case 7 expressed CD4 (77%); case 27 expressed CD10 (53%; coexpressed on CD33+ blasts). Cases 28, 29, and 30 were negative for glycophorin A but showed expression of CD117 (99%, 49%, and 80%, respectively). This case showed expression of MPO (96%) but no expression of cycd3 and cycd79a by flow cytometry. MPO also was negative by flow cytometric analysis. A. Two of 7 cases (27 and 30) expressed CD56. Seven of 23 cases had normal cytogenetics by routine Giemsa-banded karyotyping, whereas the other 16 cases showed various karyotypic abnormalities, including complex abnormalities that usually are attributed to AMLs. Discussion Until molecular therapies designed for specific genetic alterations or molecules are available as standard first-line protocols, distinction of acute leukemia as myeloid vs lymphoid will continue to dictate treatment plans and outcome. 3 The distinction, however, can be blurry in a specific category of minimally differentiated acute leukemia termed AML-M0 that not only displays minimal myeloid differentiation but also shows frequent expression of TdT and T-cell associated antigens that may lead to an erroneous diagnosis of myeloid antigen positive T-cell ALL (My+ ALL). Several cases, we believe, published in earlier reports as My+ ALL, in which patients received ALL therapy, fit best with AML-M0 and, hence, explain, at least in part, the lower complete remission rates and shorter survival than cases without expression of myeloid-associated antigens. 4,20 Rearrangement of TCR genes also can be seen in about 5% of AMLs and may not be taken as the confirmatory evidence of T-cell origin. 21 The diagnosis of AML-M0 came into existence when Lee et al 9 described a series of 10 patients among 136 American Society of Clinical Pathologists Am J Clin Pathol 2001;115:876-884 879

Kaleem and White / MINIMALLY DIFFERENTIATED ACUTE MYELOID LEUKEMIA (AML-M0) Table 2 Immunophenotype of Acute Myeloid Leukemia (AML-M0) in Published Series * Lee Matutes Campos Bennett Keenan Buccheri Sempere Yokose Venditti Segeren Creutzig Cuneo Villamor Cohen Kotylo et al et al et al et al et al et al et al et al et al et al et al et al et al et al et al Antigen (10) 9 (14) 10 (7) 11 (10) 2 (6) 12 (6) 13 (11) 14 (5) 5 (19) 15 (14) 16 (15) 17 (26) 7 (9) 18 (17) 19 (18) 8 MPO+ 8/9 8/10 NA 3/3 1/6 4/6 1/4 NA 19/19 NA NA NA 4/7 2/12 9/14 TdT+ 2/10 4/14 NA 1/4 NA NA 1/7 4/5 9/19 8/11 5/12 7/26 4/9 14/14 6/18 HLA-DR+ 8/8 NA NA NA 6/6 NA 9/11 5/5 NA 14/14 7/13 NA 9/9 16/16 16/18 CD34+ 7/8 11/12 6/7 NA 5/6 NA 3/4 NA 18/19 14/14 7/11 22/22 5/8 16/16 13/18 CD13+ 5/10 11/11 5/7 7/10 6/6 4/6 8/11 4/5 15/19 13/14 6/13 22/25 6/9 16/17 15/18 CD33+ NA 8/14 0/7 8/10 6/6 4/5 8/10 3/5 14/19 6/13 8/13 21/26 5/6 16/17 15/18 CD13 NA 0/11 2/7 0/10 0/6 1/6 NA 0/5 2/19 0/14 NA NA 0/9 0/17 0/18 CD33 CD11+ 3/8 NA NA 3/10 NA NA 4/11 NA NA NA NA 11/18 2/7 NA 8/11 CD14+ NA NA 1/7 NA NA NA 2/8 0/5 5/19 NA 0/15 NA 2/7 2/16 0/18 CD15+ 1/7 NA 5/7 1/10 NA NA NA NA 4/19 NA 4/12 8/16 NA NA 10/15 CD41+ NA NA NA 0/1 NA NA 0/7 0/3 NA NA 0/15 NA 0/9 0/16 0/16 CD1+ 1/3 0/10 NA NA NA NA NA NA NA NA NA NA NA NA NA CD2+ NA NA NA 0/10 NA NA 0/11 1/5 0/19 NA 3/12 NA 1/7 1/16 3/15 CD3+ 0/8 0/10 NA 0/10 NA NA 0/11 0/5 0/19 0/14 0/15 0/26 NA 1/16 0/16 cycd3+ NA NA NA NA NA NA NA NA 0/19 0/3 NA NA 0/9 NA NA CD4+ NA NA NA NA NA NA NA NA 0/19 NA 4/13 NA NA NA NA CD5+ NA NA NA NA NA NA NA 0/5 0/19 0/14 NA 0/26 0/8 NA NA CD7+ NA 3/10 NA 5/10 NA NA 4/8 2/5 6/19 6/14 4/13 14/26 4/9 5/16 8/18 CD8+ NA NA NA NA NA NA NA NA 0/19 NA NA 0/26 NA NA NA CD10+ 0/10 0/10 NA 0/10 NA NA 0/11 0/5 2/19 0/14 0/15 0/26 0/9 0/16 0/18 CD19+ NA 0/10 NA 0/10 NA NA 0/11 0/5 1/19 0/14 0/15 0/26 0/9 3/17 0/18 CD20+ 0/2 NA NA NA NA NA NA 0/5 0/19 NA NA NA NA 0/17 0/14 cycd22+ NA NA NA 0/10 NA NA NA NA 0/19 0/14 NA 0/26 0/8 6/15 NA Other GlyA ccd61 CD117 CD68 CD61 CD16/56 antigens (0/3) (0/19) (3/6) (3/8) (0/16) (2/13) cy, cytoplasmic; NA, not applicable or not available. * The myeloperoxidase (MPO) in various series was detected by ultrastructural cytochemistry or monoclonal antibodies. The terminal deoxynucleotidyl transferase (TdT) was detected by immunofluorescence, immunohistochemical analysis, or flow cytometry. Numbers of cases are given in parentheses with reference information in the column headings. A commercial (Coulter, Miami, FL) preparation of My9 monoclonal antibody was used for the detection of CD33 by indirect immunofluorescence. My7 (Coulter) and My9 (Coulter) were used for the detection of CD13 and CD33, respectively, by flow cytometer (FACScan, Becton Dickinson, San Jose, CA), and a 20% cutoff was used for positive identification. One of the 2 cases negative for surface CD13 and CD33 showed cytoplasmic CD13 (50%), whereas the other case was negative for cytoplasmic CD13 as well. My7 (Coulter) and My9 (Coulter) were used for the detection of CD13 and CD33, respectively, by flow cytometer. cycd3 was not performed, and the cutoff for positivity was not given. consecutive patients with de novo acute leukemia that did not fulfill the FAB criteria for either AML or ALL. Shortly thereafter, several other reports confirmed the existence of a class of AML in which the myeloid origin could be demonstrated only by ultrastructural cytochemical analysis or the use of monoclonal antibodies for MPO. 10 Investigators included AML-M0 in their reports, and the FAB group formally recognized this subtype in 1991. 2,11 It was soon obvious that this group of AML frequently expresses TdT and lymphoid-associated antigens, particularly T-cell associated antigens, but the significance and the specificity of these markers was unknown. 22 TdT is an intranuclear DNA polymerase that catalyzes the addition of a nucleotide during rearrangements of the various regions of the TCR and immunoglobulin heavy chain genes. The frequency of TdT expression in these cases reflects only the primitive nature of these blasts and is not unusual since rearrangement of TCR-beta or TCR-gamma genes is detected in 5% to 10% of cases, and rearrangement of IgH genes is detected in approximately 15% of cases of AML. 18,21 Several lymphoid-associated antigens also are expressed frequently; however, expression of lineage-specific lymphoid antigens is not a feature of myeloid leukemia. The CD3 complex is present both in the cytoplasm and on the surface but is associated with the TCR alpha-beta or TCR gammadelta only on the surface. 23 The CD3 molecule can be identified in the cytoplasm of prothymocytes and double-negative (CD4 CD8 ) thymocytes before rearrangement of the TCRalpha chain and, hence, serves as one of the earliest T-cell markers present in all T-cell ALL. 21 None of our cases analyzed for surface (27/27) or cytoplasmic CD3 (27/27) expressed that antigen, excluding the possibility of My+ ALL. CD3 expression was reportedly positive in 1 of 16 cases tested in 1 study. 19 No additional information was provided on that case, however, to confirm the validity of true CD3 expression. We found no other case in previously published reports that showed CD3 expression and believe 880 Am J Clin Pathol 2001;115:876-884 American Society of Clinical Pathologists

Hematopathology / ORIGINAL ARTICLE Image 1 Case 10 showing expression of myeloperoxidase in approximately 10% to 15% of blasts (original magnification, 1,000). that unequivocal CD3 expression is not a feature of AML- M0. Unlike cycd3, none of the other T-cell associated markers display specificity for T cells and may be seen in myeloid leukemia. None of our 17 cases tested showed CD1 expression, and except for 1 case reported by Lee et al (patient 5 in their series), 9 none of the other studies reported CD1 expression in AML-M0. Based on our experience and the reported incidence of CD1 expression in myeloid leukemia, we believe that its true expression in AML-M0 must be a rare phenomenon, if it is unequivocally expressed at all. The expression of CD2 and CD7 in myeloid leukemia is a well-known finding, especially in AML-M0. 2,9,12,19 CD7, in fact, is normally expressed in a subset of hematopoietic stem cells, suggesting that CD7 expression in AML-M0 may represent clonal expansion of CD7+ myeloblasts, as it is the most commonly expressed T-cell associated antigen that is present in a substantial number of cases (25%-50%). 24 CD2, although less common than CD7, is the next most commonly expressed T-cell associated antigen in AML-M0 and also is not infrequently expressed in other types of AML, especially those of monocytic origin. CD4 and CD5 expression also has been reported in myeloid leukemias in general. We did not, however, observe CD5 in any of our 17 cases tested or in other reported cases of AML-M0 in the literature and believe that its expression must be uncommon in AML-M0. In contrast with T-cell associated antigens, fewer B- cell associated antigens show expression in myeloid leukemias. The most common is the pan B-cell marker CD19, which is one of the earliest B-cell antigens identified in progenitor B cells in the marrow at around the time of immunoglobulin heavy-chain gene rearrangement and is expressed throughout B-cell maturation and differentiation but is lost in most mature plasma cells. 25 Two (7%) of 30 cases in our series and 3 (1.9%) of 154 cases reported in the literature expressed CD19. In contrast, expression of CD10 is less common in AML-M0 than CD19, and we are unaware of a single case that coexpressed CD10 and CD19 in a bona fide AML-M0. Similar to the T-cell specific antigen, cycd3, we did not identify expression of the B-cell specific antigen, cycd79a, in 26 cases tested. CD79a, in a heterodimer with CD79b, forms the components of B-cell receptor signal transduction and as such is restricted to B-lineage cells; non- B cells are not known to express CD79a or CD79b. 26 None of the myelomonocytic antigens commonly in diagnostic use is lineage restricted; preferential expression is seen either in myeloid or monocytic cells. Thus, expression of CD11, CD13, CD14, CD15, CD33, CD64, and CD68 can be seen in cells of myeloid and monocyte lineages and, hence, in leukemic blasts differentiating toward or originating from these lineages. 27 Expression of CD11 and CD68 also can be seen in lymphocytes and, thus, is nonspecific for myelomonocytic lineage. 27 In contrast with myelomonocytic antigens, expression of MPO is considered specific for cells of myeloid lineage; its expression in leukemic cells excludes a lymphoid origin. 28 Lack of MPO expression, however, does not exclude a myeloid origin, as a minority of bona fide cases of AML-M0 do not show detectable MPO. This may reflect either inability to detect MPO or a true absence of MPO in very early myeloid stem cells. Monocytes and macrophages may express MPO to a lesser degree (unpublished observation), as we have seen a few cases otherwise typical of monocytic cells but that expressed MPO by cytochemical analysis. Whether detectable MPO is present in all cells of monocyte or macrophage origin or in a few is unknown, but the important message is the lack of MPO in lymphoid cells. It is of much interest, in this context, that a few cases in literature reported the expression of MPO messenger RNA (mrna) in precursor B-cell ALL and T-cell ALL, as well as precursor protein in lymphoid cell lines. 29-32 Whether this disqualifies MPO specificity for myeloid cells is far from established, since other reports did not confirm this finding. 33 Furthermore, inappropriate activation of certain genes in only some malignant but not in normal cells may result in a positive signal. The lack of detectable MPO protein by cytochemical analysis or antibodies in MPO-mRNA positive cases of ALL suggests loss of transcribed mrna and, hence, functional protein. It is widely accepted, however, that expression of mature MPO protein as detected by light-microscopic cytochemical or ultrastructural cytochemical analysis or American Society of Clinical Pathologists Am J Clin Pathol 2001;115:876-884 881

Kaleem and White / MINIMALLY DIFFERENTIATED ACUTE MYELOID LEUKEMIA (AML-M0) CD34-PE A 1,000 100 10 1.1 C 1,000 CD1-PE 100 10 1.1 38% 1 2 D 3 4.1 1 10 100 1,000 CD13-FITC 1 2 D 3 4 33%.1 1 10 100 1,000 CD7-FITC antibodies is not a feature of any lymphoblastic leukemia, thus testifying to the specificity of MPO for myeloid lineage, at least for diagnostic purposes. 21 Both CD34 and HLA-DR are present in most cases, and the odds that either will be present are more than 90%. In other words, lack of expression of both CD34 and HLA-DR is uncharacteristic for this type of acute leukemia, whereas HLA-DR is expressed less commonly in T-cell ALL and may help in the differential diagnosis. Based on our 30 cases and approximately 200 reported cases, we propose the following modified criteria for AML- M0: 1. Standard criteria for the diagnosis of acute leukemia (more than 20% blasts in the bone marrow or peripheral blood [proposed World Health Organization classification]) 2. Undetectable or less than 3% positivity for MPO or Sudan black B by light-microscopic enzyme cytochemical analysis and no other definitive evidence of lymphoid differentiation 3. Lack of expression of lymphoid-specific antigens: cycd3 for T cells and cycd79 and cycd22 for B cells 1,000 CD34-PE CD34-PE B 100 10 1.1 D 1,000 100 10 1.1 1 2 C 33% 3 4 63%.1 1 10 100 1,000 MPO-FITC 1 2 E 3 4 6%.1 1 10 100 1,000 cycd3-fitc Figure 1 Case 26 showed no expression of CD13 on CD34 positive cells (A), but cells expressed myeloperoxidase (MPO) (B). A large proportion of blasts also expressed CD7 (C), but no cycd3+ cells were identified (D). cy, cytoplasmic; FITC, fluorescein isothiocyanate; PE, phycoerythrin. 4. Positivity for any one of the myelomonocytic lineage antigens known not to be expressed on normal B- or T-lymphoid cells (such as CD13, CD14, CD15, CD33, or CD64) or positivity for MPO as detected by ultrastructural cytochemical analysis, immunohistochemical analysis, or flow cytometric analysis. The most important criterion is not the obligate detection of CD13, CD33, or MPO, but the lack of T- or B- cell specific antigens because most undifferentiated leukemias are treated with protocols designed for AMLs. In fact, a minor proportion of AML-M0 cases are negative for both CD13 and CD33, as seen in case 26 in our series and in rare reported cases. 11,13,15 Any myelomonocytic-lineage antigen not known to be expressed on normal B- or T- lymphoid cells can serve the same purpose as CD13 or CD33. The original FAB criteria for AML-M0 require the expression of either CD13 or CD33 to determine the myeloid lineage; this might misclassify rare cases of M0 without CD13 and CD33 as ALL if MPO also is not detected. In disagreement with Stasi and Amadori, 34 we do not suggest the use of reverse transcriptase polymerase chain reaction for MPO mrna detection in the light of its positivity in a few well-documented cases of both precursor B-cell ALL and T-cell ALL. An argument can be made for the biphenotypic nature of those reported cases. As a corollary to our definition of AML-M0 and to explicitly define the boundaries of M0, it is appropriate that we also define acute undifferentiated leukemia and acute biphenotypic leukemia in this context. Acute undifferentiated leukemia should satisfy the following criteria: 1. No definitive evidence of lymphoid differentiation (no expression of lymphoid specific antigens, cycd3 for T cells and cycd79 and cycd22 for B cells) 2. No definitive evidence of myelomonocytic differentiation by cytochemical analysis, ultrastructural analysis, and immunophenotyping (no expression of any myelomonocytic lineage specific antigens and no expression of MPO by light-microscopic cytochemical or ultrastructural cytochemical analysis and antibodies against MPO) 3. No convincing evidence of erythroid and megakaryocytic lineage differentiation 4. Expression of the following antigens in any combination may be seen: CD34, HLA-DR, TdT, and CD7 Acute biphenotypic leukemia should satisfy the following criteria: 1. Definitive cytochemical, ultrastructural, or immunophenotypic evidence of dual differentiation (expression of 2 lineage-specific antigens: cycd3 and cycd79/ cycd22 [B and T lineage] or cycd3 and MPO [T 882 Am J Clin Pathol 2001;115:876-884 American Society of Clinical Pathologists

Hematopathology / ORIGINAL ARTICLE and myelomonocytic lineage] or cycd79/cycd22 and MPO [B and myelomonocytic lineage]) 2. Expression of any number of additional differentiation markers Cases that do not fit the aforementioned criteria may remain and may fall into the categories of My+ ALL or lymphoid antigen positive AML. From a practical viewpoint, acute undifferentiated and acute biphenotypic leukemia are treated with protocols designed for AML, and as such, extensive undue laboratory testing for strict differentiation of an acute leukemia as AML-M0, undifferentiated or biphenotypic, is unnecessary as long as ALL is excluded. However, such distinction helps us understand the biology of hematopoietic stem cell differentiation and may prove valuable for future studies. For all practical purposes, expression of CD19 is seen universally in precursor B-cell ALL, and, therefore, a diagnosis of precursor B-cell ALL in the absence of CD19 must be made with great caution. CD7 serves the same purpose as CD19 for precursor T-cell ALL. We believe that implementation of the proposed criteria would exclude a false diagnosis of ALL in all cases and provide latitude for the positive identification of myeloid differentiation in cases of AML-M0. From the Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO. Address reprint requests to Dr Kaleem: Washington University School of Medicine, Dept of Pathology and Immunology, Division of Surgical Pathology, Box 8118, 660 S Euclid Ave, St Louis, MO 63110. References 1. Bennett JM, Catovsky D, Daniel MT, et al, for the French- American-British (FAB) Cooperative Group. Proposals for the classification of the acute leukemias. Br J Haematol. 1976;33:451-458. 2. Bennett JM, Catovsky D, Daniel MT, et al. Proposals for the recognition of minimally differentiated acute myeloid leukemia (AML-M0). Br J Haematol. 1991;78:325-329. 3. Iwate H, Kami M, Kishi Y, et al. Limitations of the diagnostic criteria for minimally differentiated acute myeloid leukemia (AML-M0). Leukemia. 2000;14:2013-2014. 4. Childs C, Hirsch-Ginsberg C, Walters RS, et al. Myeloid surface antigen positive acute lymphoblastic leukemia (My+ ALL): immunophenotypic, ultrastructural, cytogenetic and molecular characteristics. Leukemia. 1989;3:777-783. 5. Yokose N, Ogata K, Ito T, et al. Chemotherapy for minimally differentiated acute myeloid leukemia: a report of five cases and review of the literature. Ann Hematol. 1993;66:67-70. 6. Stasi R, Del Poeta G, Venditti A, et al. Analysis of treatment failure in patients with minimally differentiated acute myelogenous leukemia (AML-M0). Blood. 1994;83:1619-1625. 7. Cuneo A, Ferrant A, Michauz JL, et al. Cytogenetic profile of minimally differentiated (FAB-M0) acute myeloid leukemia: correlation with clinicopathologic findings. Blood. 1995;85:3688-3694. 8. Kotylo PK, Seo I-S, Smith FO, et al. Flow cytometric immunophenotypic characterization of pediatric and adult minimally differentiated acute myeloid leukemia (AML-M0). Am J Clin Pathol. 2000;113:193-200. 9. Lee EJ, Pollak A, Leavitt RD, et al. Minimally differentiated acute non-lymphocytic leukemia: a distinct entity. Blood. 1987;70:1400-1406. 10. Matutes E, DeOliveira MP, Foroni L, et al. The role of ultrastructural cytochemistry and monoclonal antibodies in clarifying the nature of undifferentiated cells in acute leukemia. Br J Haematol. 1988;69:205-211. 11. Campos L, Guyotat D, Archimbaud E, et al. Surface marker expression in adult acute myeloid leukemia: correlations with initial characteristics, morphology and response to therapy. Br J Haematol. 1989;72:161-166. 12. Keenan FM, Barnett D, Reilly JT. Clinicopathologic features of minimally differentiated acute myeloid leukemia (AML- M0). Br J Haematol. 1992;81:458-459. 13. Buccheri V, Shetty V, Yoshida N, et al. The role of an antimyeloperoxidase antibody in the diagnosis and classification of acute leukemia: a comparison with light and electron microscopy cytochemistry. Br J Haematol. 1992;80:62-68. 14. Sempere A, Jarque L, Guinot M, et al. Acute myeloblastic leukemia with minimal myeloid differentiation (FAB AML- M0): a study of eleven cases. Leuk Lymphoma. 1993;12:103-106. 15. Venditti A, Del Poeta G, Stasi R, et al. Minimally differentiated acute myeloid leukemia (AML-M0): cytochemical, immunophenotypic and cytogenetic analysis of 19 cases. Br J Haematol. 1994;88:784-793. 16. Segeren CM, de Jong-Gerrits GC, van t Veer MB, et al. AML-M0: clinical entity or waste basket for immature blastic leukemias? a description of 14 patients. Ann Hematol. 1995;70:297-300. 17. Creutzig U, Harbott J, Sperling C, et al. Clinical significance of surface antigen expression in children with acute myeloid leukemia: results of study AML-BFM-87. Blood. 1995; 86:3097-3108. 18. Villamor N, Zarco M-A, Rozman M, et al. Acute myeloblastic leukemia with minimal myeloid differentiation: phenotypical and ultrastructural characteristics. Leukemia. 1998;12:1071-1075. 19. Cohen PL, Hoyer JD, Kurtin PJ, et al. Acute myeloid leukemia with minimal differentiation: a multiple parameter study. Am J Clin Pathol. 1998;109:32-38. 20. Wiersma S, Ortega J, Sobel E, et al. Clinical significance of myeloid-associated antigen expression in acute lymphoblastic leukemia of childhood. N Engl J Med. 1991;324:800-808. 21. Van Dongen JJM, Adriaansen HJ. Immunobiology of leukemia. In: Henderson ES, Lister TA, Greaves MF, eds. Leukemia. 2nd ed. Philadelphia, PA: Saunders; 1996:83-130. 22. Parreira A, Pombo de Oliveira MS, Matutes E, et al. Terminal deoxynucleotidyl transferase positive acute myeloid leukaemia: an association with immature myeloblastic leukaemia. Br J Haematol. 1988;69:219-224. 23. Saito T, Yamazaki T. CD3 workshop panel report. In: Kishimoto T, Kikutani H, von dem Borne AEG, et al, eds. Leukocyte Typing VI: White Cell Differentiation Antigens. New York, NY: Garland Publishing; 1998:44-48. American Society of Clinical Pathologists Am J Clin Pathol 2001;115:876-884 883

Kaleem and White / MINIMALLY DIFFERENTIATED ACUTE MYELOID LEUKEMIA (AML-M0) 24. Bowen MA. CD7 workshop panel report. In: Kishimoto T, Kikutani H, von dem Borne AEG, et al, eds. Leukocyte Typing VI: White Cell Differentiation Antigens. New York, NY: Garland Publishing; 1998:62-63. 25. Sato S, Tedder TF. CD19 workshop panel report. In: Kishimoto T, Kikutani H, von dem Borne AEG, et al, eds. Leukocyte Typing VI: White Cell Differentiation Antigens. New York, NY: Garland Publishing; 1998:133-134. 26. Nakamura T. CD79 workshop panel report. In: Kishimoto T, Kikutani H, von dem Borne AEG, et al, eds. Leukocyte Typing VI: White Cell Differentiation Antigens. New York, NY: Garland Publishing; 1998:180-182. 27. Shaw S. CD Guide. In: Kishimoto T, Kikutani H, von dem Borne AEG, et al, eds. Leukocyte Typing VI: White Cell Differentiation Antigens. New York, NY: Garland Publishing; 1998:1109-1220. 28. Goasguen JE, Bennett JM, Henderson ES. Biologic diagnosis of leukemia. In: Henderson ES, Lister TA, Greaves MF, eds. Leukemia. 2nd ed. Philadelphia, PA: Saunders; 1996:8-33. 29. Ferrari S, Mariano MT, Tagliafico E, et al. Myeloperoxidase gene expression in blast cells with a lymphoid phenotype in cases of acute lymphoblastic leukemia. Blood. 1998;72:873-876. 30. Zhou M, Findley HW, Zaki SR, et al. Expression of myeloperoxidase mrna by leukemia cells from childhood acute lymphoblastic leukemia. Leukemia. 1993;7:1180-1183. 31. Serrano J, Roman J, Sanchez J, et al. Myeloperoxidase gene expression in acute lymphoblastic leukaemia. Br J Haematol. 1997;97:841-843. 32. Crisan D, Topalovski M, O Malley B. Myeloperoxidase mrna analysis in acute lymphoblastic leukemia. Diagn Mol Pathol. 1996;5:236-248. 33. Imamura N. Sensitive detection technique of myeloperoxidase precursor protein by flow cytometry with monoclonal antibodies. Am J Hematol. 1998;58:241-243. 34. Stasi R, Amadori S. AML-M0: a review of laboratory features and proposal of new diagnostic criteria. Blood Cells Mol Dis. 1999;25:120-129. 884 Am J Clin Pathol 2001;115:876-884 American Society of Clinical Pathologists