Leukemia (2003) 17, 707715 & 2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.

00
www.nature.com/leu

HLA-DR antigen-negative acute myeloid leukemia

M Wetzler1,5, BK McElwain1, CC Stewart2, L Blumenson4, A Mortazavi1, LA Ford1, JL Slack1, M Barcos3, S Ferrone5 and MR Baer1
1 Leukemia Section, Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY, USA; 2Laboratory of Flow Cytometry, Roswell; 3Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY, USA; 4Department of Cancer Prevention, Epidemiology and Biostatistics, Roswell Park Cancer Institute, Buffalo, NY, USA; and 5Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY, USA

Human leukocyte antigen (HLA) Class II antigens are variably expressed on acute myeloid leukemia (AML) blasts. The biological and clinical signicance of HLA Class II antigen expression by AML cells is not known. Therefore, we sought to characterize cases of AML without detectable HLA-DR expression. Samples from 248 consecutive adult AML patients were immunophenotyped by multiparameter ow cytometry at diagnosis. HLA-DR antigens were not detected on AML cells from 43 patients, including 20 with acute promyelocytic leukemia (APL), and 23 with other subtypes of AML. All APL cases had t(15;17), but there were no characteristic chromosome abnormalities in non-APL cases. No direct expression of other antigens was identied in HLA-DR-negative APL and nonAPL cases. Interestingly, cells from three HLA-DR-negative non-APL patients had similar morphology to that of the hypogranular variant of APL. This morphology, however, was not present in any HLA-DR-positive AML cases. Treatment response was similar in the 23 HLA-DR-negative non-APL and the 205 HLA-DR-positive patients. Finally, relapse was infrequently associated with changes in HLA-DR antigen expression, as the HLA-DR antigen was lost at relapse in only 4% of HLA-DR-positive cases, and was gained at relapse in only 17% of HLA-DR-negative cases. We conclude that HLA-DR-negative AML includes approximately equal numbers of APL and nonAPL cases, and that the morphology of HLA-DR-negative nonAPL cases can mimic the hypogranular variant of APL. The diagnosis of APL cannot be based on morphology and lack of HLA-DR antigen expression; rather, it requires cytogenetic or molecular conrmation. Leukemia (2003) 17, 707715. doi:10.1038/sj.leu.2402865 Keywords: acute myeloid leukemia; HLA-DR; immunophenotype

HLA Class II molecules are expressed on acute myeloid leukemia (AML) blasts at diagnosis in most cases of AML, with the exception of acute promyelocytic leukemia (APL), which is characterized by lack of HLA-DR antigen expression.1417 Absence of HLA-DR antigen expression is rare in non-APL cases,18 and little information is available about the clinical signicance of lack of expression of these antigens. We sought to characterize cases of AML with subtypes other than APL in which HLA-DR antigens are not detected. Specically, we wished to determine how specic lack of HLA-DR antigen expression is in establishing the diagnosis of APL, whether it serves to identify one or more homogeneous subsets of non-APL AML, and whether it is associated with treatment response. Finally, we wanted to assess whether changes in HLA-DR antigen expression occur at relapse.

Methods

Patient samples
Bone marrow samples from 248 consecutive newly diagnosed adult AML patients referred to Roswell Park Cancer Institute (RPCI) between February 1990 and September 1998 were immunophenotyped by multiparameter ow cytometry in the Laboratory of Flow Cytometry as part of routine pretreatment studies. Relapse samples were also studied in 59 of 86 patients who relapsed. Studies were approved by the RPCI Institutional Review Board.

Introduction
Class II human leukocyte antigens (HLA) present antigenic peptides to regulatory T cells. The crucial role played by HLA Class II antigens in the generation of an immune response has stimulated interest in determining whether HLA Class II antigens expressed by tumor cells inuence the clinical course of disease. In this regard, there is conicting information about the clinical signicance of HLA Class II antigens expressed by tumor cells. For example, HLA-DR antigen loss is associated with a more aggressive course of the disease in B-cell lymphoma,13 while HLA-DR antigen expression was reported to be associated with disease progression in colon cancer.4 Furthermore, HLA-DR antigen expression was associated with improved prognosis in cervical carcinoma.57 Finally, in malignant melanoma, there is conicting information about the association of HLA Class II antigen expression with poor prognosis.813
Correspondence: Dr M Wetzler, Leukemia Section, Department of Medicine, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA; Fax: +1 716 845 2343 Supported partially by National Cancer Institute Grants CA 16056 and CA 67108.

Morphologic studies
The diagnosis of AML and morphologic categorization were according to the FrenchAmericanBritish (FAB) classication.19,20 Slides available from 22 HLA-DR-negative and 162 HLA-DR-positive non-APL cases were evaluated for the presence of morphological features resembling the hypogranular variant of APL, with varying degrees of nuclear folding, convolution or lobulation.

Cytogenetic analysis
Cytogenetic analysis was performed on pretreatment bone marrow samples from all patients. Samples were processed using short-term unstimulated cultures (2472 h). Descriptions of chromosome aberrations and clonality criteria were according to the International System for Human Cytogenetic Nomenclature.21 Patients were divided into three prognostic groups based on karyotype, as previously described.22 The prognostic groups were favorable (t(8;21), inv(16) and t(15;17)), intermediate (normal cytogenetics), and unfavorable

HLA-DR antigen-negative acute myeloid leukemia M Wetzler et al

708
(all others). Complex karyotype was dened by the presence of three or more clonal chromosomal abnormalities. Software House, Topsham, ME, USA). To identify populations of leukemia cells in each case of AML, the antibody panel was chosen that best resolved leukemia cells as a dense cluster of cells with an abnormal pattern of antigen expression or coexpression and a minimum of normal cell contamination. A new region was drawn around the abnormal cell population found in the FSC vs SSC display, creating a leukemia cell gate that was then used to analyze cells stained with all of the remaining mAb. Samples were scored as HLA-DR-negative when HLA-DR antigens were detected on o10% of cells in the leukemia cell gate. Lack of HLA-DR antigen expression on leukemia cells was conrmed by visual analysis and conrmation of HLA-DR antigen expression on lymphocytes, dened by FSC vs SSC characteristics and bright CD45 expression, in the same sample.

Reverse-transcription polymerase chain reaction (RT-PCR)

RT-PCR for PML-RARa mRNA was performed on HLA-DRnegative AML samples with morphology resembling the hypogranular variant of APL. The method was as described before.23

Treatment
A total of 177 patients received high-dose cytarabine and idarubicin (HDAC/Ida) induction therapy24 and 30 received other induction regimens.2527 Of the 157 patients who achieved complete remission (CR), 126 received postremission therapy.

Statistical analysis
All statistical calculations were performed with SAS/STAT software.34 The distributions of quantitative characteristics (age and percentages of leukemic blasts expressing each antigen) in different patient populations were compared using the exact MannWhitney test. Qualitative characteristics were compared using either the FisherIrwin test, the exact MantelHaenszel test, or the exact Pearson w2 test. Survival curves were calculated using the method of KaplanMeier and were compared using the log-rank test. The method of BrookmyerCrowley was used to calculate condence intervals for the median survival time.35 DFS was dened as the time from attainment of CR to relapse or last follow-up visit or death without relapse regarded as a censored event and also death without relapse regarded as a competing risk for relapse. The crude cumulative incidence of relapse (or its complement, the crude survival) and corresponding standard errors were calculated using Equations (10.11) and (10.12), respectively.36

Response criteria
CR was dened by normalization of blood counts and bone marrow morphology and disappearance of all signs of leukemia, lasting for Z4 weeks, in accordance with the recommendations of the National Cancer Institute-Sponsored Workshop on the Denitions of Diagnosis and Response in AML.30 Disease-free survival (DFS) was measured from the date of attainment of CR to the date of relapse, dened by reappearance of blasts in the blood or the nding of Z5% blasts in the bone marrow, not attributable to another cause, also in accordance with the recommendations of the National Cancer Institute-Sponsored Workshop.28

Antibodies
The peridinin chlorophyll protein (PerCP)-conjugated anti-HLADR antibody was purchased from Becton Dickinson (BD) Biosciences (San Diego, CA, USA). Fluorescein isothiocyanate (FITC)-labeled CD45 and phycoerythrin (PE)-labeled goat antimouse IgG antibodies were purchased from BD Biosciences and from Caltag (San Francisco, CA, USA), respectively. The panel of monoclonal antibodies (mAb) used to characterize AML was previously described.18

Results

Patient population
Ages of the 248 newly diagnosed AML patients whose pretreatment leukemia cells were immunophenotyped by MFC ranged from 17 to 85 (median 65) years; 146 were male and 102 were female. Data on prior history were available on 240 patients: 172 patients had de novo AML, and 68 patients had secondary AML, dened by an antecedent hematologic disorder (48 patients), by chemotherapy/radiation therapy administered for a prior malignancy or other condition (18 patients), or by both (two patients).

Multiparameter ow cytometry (MFC)

Samples were transported to the RPCI Laboratory of Flow Cytometry at room temperature in tubes containing sodium heparin and were processed as previously described.29,30 In all, 228 patient samples were analyzed with three-color combinations of FITC, PE, and PerCP or PE/Cy5-conjugated mAb. The remaining 20 patient samples (studied after March 1998) were analyzed with four-color combinations of mAb, the fourth uorophor being allophycocyanin.31 Cell viability was determined by ethidium monoazide labeling.32 All samples had at least 85% viable cells in the gated regions and were thus appropriate for analysis, in accordance with the National Committee for Clinical Laboratory Standards guidelines.33 Listmode data were acquired on either an FACScan or a FACSCalibur ow cytometer (BD Biosciences). Data were analyzed using WinList multiparameter analysis software (Verity
Leukemia

Characterization of HLA-DR-negative AML at diagnosis

HLA-DR antigens were not detected on the surface of leukemic blasts from 43 patients at diagnosis. These 43 patients included 20 with APL and 23 with other FAB types, including M1 (seven patients), M2 (10 patients), M4 (four patients), M5b (one patient) and M6 (one patient). Myeloperoxidase and/or Sudan black positivity was noted in over 55% of the blast cells in 21 of the 22 non-APL cases. Auer rods were present in ve cases, and double Auer rods were present in one case. Three HLA-DR-negative FAB M2 cases showed morphological features resembling the hypogranular variant of APL, with varying degrees of nuclear folding, convolution, or lobulation. A representative example is shown in Figure 1. The bone marrow from one of these patients

HLA-DR antigen-negative acute myeloid leukemia M Wetzler et al

709

Figure 1 HLA-DR-negative AML (FAB M2) resembling APL (original magnication 250). (a) Two myeloblasts (one contains multiple slender Auer rods) and two abnormal promyelocytes with folded nuclei and ne-to-coarse rust-colored cytoplasmic granules. (b) Myeloblast with two distinct Auer rods. Table 1 Similarity in pretreatment immunophenotype of HLA-DR antigen-negative APL and non-APL cases APL (n = 20) Median (range)b Antigen casesc Median (range) Antigen cases Median (range) Antigen cases Median (range) Antigen cases Median (range) Antigen cases Median (range) Antigen cases Median (range) Antigen cases Median (range) Antigen cases 3.5 (0.370.5) 5 (25%) 10.7 (0.648.2) 11 (55%) 3.1 (0.815.3) 2 (10%) 65.3 (1.596.4) 19 (95%) 8.6 (0.677.8) 8 (N 19) 59.9 (1.796.4) 18 (90%) 5.3 (0.490.2) 9 (45%) 2.0 (0.242.1) 2 (10%) Non-APL (N = 23) 2.8 (0.679.0) 4 (17%) 2.8 (0.595.8) 5 (22%) 1.3 (0.574.2) 1 (4%) 53.3 (3.996.6) 18 (78%) 8.8 (0.198.9) 8 (N 19) 47.5 (0.395.5) 17 (74%) 3.8 (0.492.9) 8 (35%) 1.2 (0.197.0) 7 (30%) Pa 0.30 0.71 0.03 0.03 0.02 0.59 0.29 0.19 0.98 1.0 0.26 0.25 0.49 0.55 0.89 0.14

Immunophenotype CD2 CD7 CD19 CD13 CD15 CD33 CD34 CD56

a

P-value for comparison of distributions of percent of cells positive for antigen from the exact two-sided WilcoxonMannWhitney test. P-value for comparison of proportion of patients positive (>10% of cells positive) for antigen from the two-sided exact Fisher test. b Numbers represent percentages of gated leukemia cells that are positive for each indicated antigen. c Cases were dened as positive when the indicated antigen was detected on Z10% of cells in the leukemic gate. APL, acute promyelocytic leukemia.

also showed advanced reticulin and collagen myelobrosis. The marrow from one additional patient, with HLA-DR-negative FAB M4 AML, showed a marked increase in normal promyelocytes in addition to myeloblasts. Marrows from two additional patients, one with FAB M1 and one with FAB M2, also showed coexistent dysplastic syncitial megakaryocytic hyperplasia, suggestive of myeloblastic transformation from a myelodysplastic or myeloproliferative syndrome. Finally, cells from one patient with FAB M1 AML had diffuse cytoplasmic acid phosphatase staining. None of the 162 HLA-DR-positive cases with slides available for review had APL-like morphologic features.

All of the patients with APL had t(15;17)(q22;q11-12) or one of its variants. Of the 23 patients with HLA-DR-negative nonAPL AML, 14 had a normal karyotype, four had complex cytogenetic abnormalities, one patient had t(7;11)(p10;q10), one had t(1;14)(p36.1;q31), one had der(17)t(7;17) (q11.2;p11.2), inv(8) and +11, and one had an inevaluable karyotype. Thus, there was no karyotype that was characteristic of these cases. We asked whether lack of HLA-DR expression correlated with any specic immunophenotypic pattern. Expression of myeloid markers (CD13, CD15, CD33), the stem cell antigen CD34, the stem cell and T-lymphoid marker CD7, the T-lymphoid marker
Leukemia

HLA-DR antigen-negative acute myeloid leukemia M Wetzler et al

710
Table 2 Comparison of pretreatment immunophenotypes of HLA-DR-positive and HLA-DR-negative non-APL cases HLA-DR ( ) (N = 205) Median (range)b Antigen casesc Median (range) Antigen cases Median (range) Antigen cases Median (range) Antigen cases Median (range) Antigen cases Median (range) Antigen cases Median (range) Antigen cases Median (range) Antigen cases 12.2 (0.592.0) 113 (N 201) (56%) 15.0 (0.895.3) 124 (N 201) (62%) 4.0 (075.9) 41 (N 202) (20%) 59.4 (199.3) 194 (95%) 36.9 (099.0) 139 (N 176) (79%) 31.2 (099.7) 158 (77%) 43.7 (0.399.7) 148 (N 200) (74%) 3.3 (092.7) 48 (N 203) (24%) HLA-DR () Non-APL (N = 23) 2.8 (0.679.0) 4 (17%) 2.8 (0.595.8) 5 (22%) 1.3 (0.574.2) 1 (4%) 53.3 (3.996.6) 18 (78%) 8.8 (0.198.9) 8 (N 19) (42%) 47.5 (0.395.5) 17 (74%) 3.8 (0.492.9) 8 (35%) 1.2 (0.197.0) 7 (30%) ** ** ** ** 0.0004 0.09 0.32 0.01 0.008 0.001 0.38 0.80 0.0006 ** 0.25 0.45 Pa

Immunophenotype CD2 CD7 CD19 CD13 CD15 CD33 CD34 CD56

[See Table 1.] **Po0.0001

CD2, the B-lymphoid marker CD19, and the neural cell adhesion molecule CD56 was compared between samples from HLA-DR-negative APL and non-APL cases (Table 1). Although a statistically signicant (Po0.05) difference was found for CD7, in larger series APL blasts rarely express this antigen37 and therefore this difference most probably does not represent a biological phenomenon. In summary, APL cannot be distinguished from HLA-DR-negative non-APL AML based on the panels of antigens analyzed in this study. Interestingly, the three patients with HLA-DR-negative nonAPL (PML-RARa mRNA not detected by RT-PCR) in our series whose cells had morphology resembling the hypogranular variant of APL were female, their AML cells had no chromosomal aberrations, and did not express CD2, CD7, CD19, and CD34.

Table 3 Similar pretreatment patient characteristics in HLA-DRnegative non-APL and HLA-DR positive cases Characteristics HLA-DR () (N = 23) HLA-DR ( ) (N = 205) P

Age Median Range Z60 Sex (M/F) De novo/secondary FAB M0 M1 M2 M3 M4 M5 M6 M7 Cytogenetics Favorable Intermediate Unfavorable
a

0.16 70 2684 16 (70)a 12/11 (52/48) 19/4 (83/17) 0 7 (30) 10 (43) 0 4 (17) 1 (4) 1 (4) 0 0 13 (59) 9 (41) 65 1785 125 (61) 126/79 (61/39) 145/60 (71/29) 21 (10) 27 (13) 81 (40) 5 (2) 33 (16) 14 (7) 20 (10) 1 (0.5) 0.19 16 (8) 79 (41) 100 (51) 0.50 0.33 0.39

Lack of correlation between HLA-DR expression and clinical characteristics in non-APL AML patients at diagnosis
Pretreatment clinical characteristics did not differ signicantly between HLA-DR-negative non-APL AML patients and AML patients whose blasts were HLA-DR-positive (Tables 2 and 3). Of note, the HLA-DR-positive group included ve patients with APL (median percentage of HLA-DR antigen expression: 45.8%, range 10.180.2%). The distribution of induction (P 0.15) and consolidation (P 0.41 for HDAC/Ida and P 0.12 for other regimens) treatment regimens was similar in patients with HLADR-negative non-APL and HLA-DR-positive AML (Table 4), allowing comparison of outcome between the two groups. CR rates were 73% in HLA-DR-negative patients and 61% in HLADR-positive patients. No HLA-DR-negative patients underwent allogeneic transplantation in rst remission, as compared to seven (6%) HLA-DR-positive patients. Three (19%) HLA-DRnegative patients underwent autologous transplantation in rst remission, as compared to eight (7%) HLA-DR-positive patients. These differences in treatment between the groups are not likely to affect outcome comparisons. The median follow-up duration was 12.6 months (range, o185.4 months) for HLA-DRnegative patients and 11.2 months (range o199.6 months) for HLA-DR-positive patients. The estimated DFS curves, calculated with the time of death without disease censored, overlapped during the rst 12 months after induction (P 0.54,
Leukemia

Numbers in parentheses represent percentages. APL, acute promyelocytic leukemia; F, female; FAB, FrenchAmerican British classication; M, male.

Figure 2). When death without disease was regarded as a competing risk, the estimated DFS rates were somewhat higher in both groups of patients, while the corresponding crude survival curves overlapped in a similar manner. The estimated overall survival curves almost overlapped throughout the range 084 months, with the estimated median survival for both groups equal to approximately 1 year. Four (17%) HLA-DRnegative non-APL patients remain alive in CR, as do 27 (13%) HLA-DR-positive patients. In all, 10 (43%) HLA-DR-negative non-APL patients have relapsed, as have 83 (40%) HLA-DRpositive AML patients.

HLA-DR antigen-negative acute myeloid leukemia M Wetzler et al

711
Table 4 Similar treatment response of HLA-DR-negative non-APL and HLA-DR-positive AML patients HLA-DR () (N = 23) Induction HDAC/Idaa Otherc NT Attainment of CR HDAC/Ida group Other group Consolidation HDAC/Ida group VP/CYd HDAC/Ida Other group HDAC/Ida Othere AlloPBSCT in rst CR AutoPBSCT in rst CR DFS: KaplanMeier method (Censored at death without disease) Median (months) 95% condence interval for median Crude survival function: DFS in the presence of competing risk of death without disease Median (months) Overall survival Median (months) 95% condence interval of the median HLA-DR (+) (N = 205) P 0.15 16 (70)b 7 (30) 0 11 (73) 5 (71) 164 (80) 33 (16) 8 (4) 0.90 99 (61) 18 (56)

5 (45) 3 (27) 0 1 (20) 0 3 (19)

66 (67) 19 (19) 2 (11) 11 (61) 7 (6) 8 (7)

0.41

0.12

23.6 Z7.2

13.3 10.3,17.3 14.5 11.2 9.0,14.0

0.54

> 24 (55% at 24 mos) 12.6 7.3,26.2

0.83

a HDAC/Ida; high-dose cytarabine 3.0 gm/m2 every 12 h for a total of 12 doses (1.5 gm/m2 for age >50 years) and idarubicin 12 mg/m2 daily for three consecutive days. b Numbers in parentheses represent percentages. c Other; 10 were treated with and eight were treated without PSC 833 as described in Koliz et al.,25 and 15 were treated with cytarabine 100 mg/m2 continuous infusion (CIVI) over seven days and an anthracycline for three days. d VP/CY; etoposide 2.4 g/m2 by CIVI (23 patients); etoposide 3.6 g/m2 by CIVI (41 patients); and etoposide 4.2 g/m2 by CIVI (seven patients) all with cyclophosphamide 50 mg/kg for 4 days. e Other, four patients received high-dose cytarabine 3.0 gm/m2 every 12 hour for a total of six doses; four patients received cytarabine 2.0 gm/m2 every 12 h for a total of eight doses, and etoposide 160 mg/kg CIVI over four days; two patients received cytarabine 100 mg/m2 CIVI over ve days, daunorubicin 60 mg/m2 daily for two days, and etoposide 100 mg/m2 for two days; and two patients received cytarabine 100 mg/m2 CIVI over seven days and idarubicin 12 mg/m2 daily for three consecutive days. alloPBSCT, allogeneic peripheral stem cell transplantation; APL, acute promyelocytic leukemia; autoPBSCT, autologous peripheral stem cell transplantation; CR, complete remission; DFS, disease-free survival; mos, months; NT, not treated.

Of note, the three HLA-DR-negative non-APL patients with the hypogranular variant morphology have remained in remission following induction and consolidation therapy, with 23, 32, and 74 months follow-up.

present at diagnosis. In the other patient (Figure 3b), an HLADR-positive population was the major population of AML cells at diagnosis, whereas a different population, which did not express HLA-DR antigens, was predominant at relapse.

Infrequent changes of HLA-DR antigen expression on leukemic blasts at relapse

HLA-DR antigen expression was studied at relapse in samples from 59 of 93 patients who relapsed. Relapse occurred at a median of 8.5 months (range, 3.255.9 months) in the patients whose relapse samples were studied. Of six non-APL patients whose cells did not express HLA-DR antigens at diagnosis, one (17%) had HLA-DR-positive disease at relapse, and the HLADR-positive cells present at relapse represented a new population, different from the HLA-DR-negative population that had been present at diagnosis (Figure 3a). Of 53 patients with HLADR-positive disease at diagnosis, samples from two (4%) demonstrated a loss of HLA-DR antigen expression at relapse. In one of these two patients, HLA-DR antigen expression was lost at relapse on an HLA-DR-positive population that had been

Discussion
Analysis of a large number of AML patients showed that approximately 20 percent of cases of AML do not express HLADR antigens at diagnosis, and that cases without HLA-DR antigen expression are equally divided between APL and other AML subtypes. Thus, whereas HLA-DR antigen expression makes the diagnosis of APL unlikely, absence of HLA-DR antigen expression cannot be considered to be a sufcient criterion for establishing the diagnosis of APL. Only half of AML cases without HLA-DR antigen expression have APL, and the other half have other AML subtypes. Further, blasts in HLA-DRnegative non-APL AML cases can have morphology resembling the hypogranular variant of APL. Therefore, the diagnosis of APL requires conrmation by cytogenetic demonstration of t(15;17)
Leukemia

HLA-DR antigen-negative acute myeloid leukemia M Wetzler et al

712

Figure 2 Similar DFS of HLA-DR-negative non-APL and HLA-DR-positive AML patients. Time to relapse was censored at date of death without disease. The bold and the broken lines represent HLA-DR-negative non-APL AML and HLA-DR-positive AML, respectively.

or one of its variants and/or molecular demonstration of PML/ RARa or a variant gene rearrangement. Our nding that lack of HLA-DR antigen expression occurs in non-APL AML cases corroborates information in the literature. Lazarchick and Hopkins38 demonstrated absence of HLA-DR antigen expression in AML subtypes other than APL, mainly FAB M2 AML. Similarly, Fenu et al39 described three HLA-DRnegative AML patients who were suggested to have APL variants based on morphology and immunophenotype, but were reclassied as FAB M2 AML after cytogenetic and molecular analyses were completed. Distinct patterns of antigen expression on AML blasts have been associated with specic chromosomal abnormalities, including t(8;21)4043 and inv(16).4446 Similarly, APL blasts have been characterized by lack of HLA-DR antigen expression and by CD2 expression in 28% of cases.47 Further subgroup analysis revealed that CD2 expression was associated with the Bcr3 breakpoint48 or the microgranular variant of APL.49 Interestingly, the three cases with HLA-DR-negative non-APL AML whose blasts had morphology resembling hypogranular APL did not express the CD2 antigen. Others have found APL to be characterized by heterogeneous expression of CD13 on the leukemic blasts, with a single major blast cell population characterized by CD15+ and CD34 expression.50 Of note, recent work suggests that the hypogranular morphology of APL is associated with CD34 expression.51 CD34 is expressed in approximately two-thirds of non-APL AML cases.18,5254 We asked whether a unique immunophenotype could serve to distinguish between APL and HLA-DR-negative cases with other AML subtypes. We found that these two groups could not be separated based on the panels of antigens analyzed in this study. Our data differ from those of Scott et al55 and Solary et al.56 Scott et al55 identied a unique subset of AML that was characterized by lack of HLA-DR antigen expression and had features of both myeloid and natural killer (NK) cells, with CD33
Leukemia

and CD56 expression, but absence of CD16 expression. They found this entity in 20, or 6%, of their series of 350 AML patients. Our series included only three patients with the myeloid/NK immunophenotype (HLA-DR, CD33+, CD56+, CD16). A different HLA-DR-negative subset, expressing CD14, was identied by Solary et al.56 This group of patients had signicantly shorter survival. However, the authors provide data neither on patient characteristics nor on treatment. Our series included only two patients with this immunophenotype (HLADR, CD14+). HLA Class II molecules play an essential role in presenting antigenic peptides to regulatory T cells.57 We did not nd any difference in outcome between non-APL patients with HLA-DRnegative and those with HLA-DR-positive blasts. One possible explanation is that HLA Class II antigens play only a minimal role in the immune response against leukemia-associated antigens. We have previously demonstrated that HLA Class I antigen expression is preserved on the surface of AML blasts.58 HLA Class I molecules bind antigenic peptides and present them to cytotoxic (CD8+) T cells. The recognition of these peptides by cytotoxic T cells triggers a series of events that may result in lysis of target cells. It is conceivable that the lack of HLA Class II antigens is compensated for by cross-presentation.59 In this phenomenon, antigen-presenting cells take up antigens shed from leukemic cells and present the processed antigens to antigen-specic CD4 T cells (cross-presentation), as opposed to direct presentation by leukemic blasts themselves. Alternatively, the HLA Class II-negative leukemia cells might represent cells with limited proliferative capacity, while the small HLA-DRpositive population may actually be the stem cell population with unlimited proliferative capacity. Finally, immunophenotype changes occur frequently at relapse,18 but changes in HLA-DR antigen expression are rare. We conclude that HLA-DR antigen loss is rare in both leukemogenesis and disease progression, and therefore should

HLA-DR antigen-negative acute myeloid leukemia M Wetzler et al

713

a
10
4

DIAGNOSIS
120 0 10 20 30 40 50 60 70 80 90 100

C D 4 5
10 20 30 40 50 60 70 80 90100 120 g

S S C

10

FSC

HLA-DR
10 4

10 g

10

10

1 10

10

FL1-Height 2 3 10 10

10

10

4 10 i s o t y p e

control FL3-Height 10 i s o t y p e

0 10 20 30 40 50 60 70 80 90 100

120

RELAPSE

C D 4 5
10 20 30 40 50 60 70 80 90100 120 g

S S C

10 1

10

10

10

10

1 10

FL1-Height 2 3 10 10

10 2

10 3

FSC

g HLA-DR

control

0 10 20 30 40 50 60 70 80 90100 110 120

10 4

DIAGNOSIS i s o t y p e
10
1

C D 4 5

S S C

10 20 30 40 50 60 70 80 90100110 120 g

10 1

10 2

10 3

FSC

10

10

10

g HLA-DR 10 4

control

0 10 20 30 40 50 60 70 80 90100 110 120

RELAPSE i s o t y p e
10
1

C D 4 5
10 20 30 40 50 60 70 80 90100110 120 g

S S C

10 1

10 2

10 3

FSC

10

10

10

g HLA-DR

control

Figure 3 Changes in HLA-DR antigen expression at relapse in AML blasts. Panel (a) demonstrates a patient whose leukemia cells were HLADR-negative at diagnosis (shown in the ellipse), whereas an HLA-DR-positive population (shown in the square) became more prominent at relapse. Panel (b) demonstrates a patient whose leukemia cells were predominantly HLA-DR-positive at diagnosis but in whom an HLA-DR-negative population (shown in the square) was more prominent at relapse. Control represents isotype control antibodies.

not represent an important mechanism of immune escape and of resistance to immunotherapy.

References
1 Slymen DJ, Miller TP, Lippman SM, Spier CM, Kerrigan DP, Rybski JA et al. Immunobiologic factors predictive of clinical outcome in diffuse large-cell lymphoma. J Clin Oncol 1990; 8: 986993.

2 Pilkington G, Juneja S, Tan L, Matthews J, Quirk J, Lee G et al. Correlation of immunological surface antigens with survival in diffuse large cell lymphoma. Hematol Oncol 1993; 11: 195205. 3 Riemersma SA, Jordanova ES, Schop RF, Philippo K, Looijenga LH, Schuuring E et al. Extensive genetic alterations of the HLA region, including homozygous deletions of HLA class II genes in B-cell lymphomas arising in immune-privileged sites. Blood 2000; 96: 35693577.
Leukemia

HLA-DR antigen-negative acute myeloid leukemia M Wetzler et al

714
4 Norazmi M, Hohmann AW, Skinner JM, Bradley J. Expression of MHC class I and class II antigens in colonic carcinomas. Pathology 1989; 21: 248253. 5 Hilders CG, Houbiers JG, van Ravenswaay Claasen HH, Veldhuizen RW, Fleuren GJ. Association between HLA-expression and inltration of immune cells in cervical carcinoma. Lab Invest 1993; 69: 651659. 6 Coleman N, Stanley MA. Analysis of HLA-DR expression on keratinocytes in cervical neoplasia. Int J Cancer 1994; 56: 314319. 7 Cromme FV, van Bommel PF, Walboomers JM, Gallee MP, Stern PL, Kenemans P et al. Differences in MHC and TAP-1 expression in cervical cancer lymph node metastases as compared with the primary tumours. Br J Cancer 1994; 69: 11761181. 8 Lopez-Nevot MA, Garcia E, Romero C, Oliva MR, Serrano S, Garrido F. Phenotypic and genetic analysis of HLA class I and HLA-DR antigen expression on human melanomas. Exp Clin Immunogenet 1988; 5: 203212. 9 Ruiter DJ, Mattijssen V, Broecker EB, Ferrone S. MHC antigens in human melanomas. Semin Cancer Biol 1991; 2: 3545. 10 Colloby PS, West KP, Fletcher A. Is poor prognosis really related to HLA-DR expression by malignant melanoma cells? Histopathology 1992; 20: 411416. 11 Moretti S, Pinzi C, Berti E, Spallanzani A, Chiarugi A, Boddi V et al. In situ expression of transforming growth factor beta is associated with melanoma progression and correlates with Ki67, HLA-DR and beta 3 integrin expression. Melanoma Res 1997; 7: 313321. 12 Konstadoulakis MM, Vezeridis M, Hatziyianni E, Karakousis CP, Cole B, Bland KI et al. Molecular oncogene markers and their signicance in cutaneous malignant melanoma. Ann Surg Oncol 1998; 5: 253260. 13 Ostmeier H, Fuchs B, Otto F, Mawick R, Lippold A, Krieg V et al. Can immunohistochemical markers and mitotic rate improve prognostic precision in patients with primary melanoma? Cancer 1999; 85: 23912399. 14 Hanson CA, Gajl-Peczalska KJ, Parkin JL, Brunning RD. Immunophenotyping of acute myeloid leukemia using monoclonal antibodies and the alkaline phosphataseantialkaline phosphatase technique. Blood 1987; 70: 8389. 15 Scott CS, Patel D, Drexler HG, Master PS, Limbert HJ, Roberts BE. Immunophenotypic and enzymatic studies do not support the concept of mixed monocyticgranulocytic differentiation in acute promyelocytic leukaemia (M3): a study of 44 cases. Br J Haematol 1989; 71: 505509. 16 De Rossi G, Avvisati G, Coluzzi S, Fenu S, LoCoco F, Lopez M et al. Immunological denition of acute promyelocytic leukemia (FAB M3): a study of 39 cases. Eur J Haematol 1990; 45: 168171. 17 Stone RM, Mayer RJ. The unique aspects of acute promyelocytic leukemia. J Clin Oncol 1990; 8: 19131921. 18 Baer MR, Stewart CC, Dodge RK, Leget G, Sule N, Mrozek K et al. High frequency of immunophenotype changes in acute myeloid leukemia at relapse: implications for residual disease detection (Cancer and Leukemia Group B Study 8361). Blood 2001; 97: 35743580. 19 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR et al. Proposed revised criteria for the classication of acute myeloid leukemia A report of the FrenchAmericanBritish Cooperative Group. Ann Intern Med 1985; 103: 620625. 20 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR et al. Proposal for the recognition of minimally differentiated acute myeloid leukaemia (AML-MO). Br J Haematol 1991; 78: 325329. 21 ISCN. In: Mitelman F (ed). An International System for Human Cytogenetic Nomenclature. Basel: S. Karger, 1995. 22 Wetzler M, Baer MR, Bernstein SH, Blumenson L, Stewart C, Barcos M et al. Expression of c-mpl mRNA, the receptor for thrombopoietin, in acute myeloid leukemia blasts identies a group of patients with poor response to intensive chemotherapy. J Clin Oncol 1997; 15: 22622268. 23 Slack JL, Bi W, Livak KJ, Beaubier N, Yu M, Clark M et al. Preclinical validation of a novel, highly sensitive assay to detect PMLRARalpha mRNA using real-time reverse-transcription polymerase chain reaction. J Mol Diagn 2001; 3: 141149. 24 Baer MR, Pixley LA, Ford LA, Donohue K, OLoughlin KL, Minderman H et al. High-dose cytarabine and idarubicin induction produces a high complete remission rate in previously untreated de novo acute myeloid leukemia patients. Blood 2000; 96(Suppl 1): 322a (Abstract). Kolitz JE, George SL, Hurd D, Hoke E, Dodge RK, Velez-Garcia E et al. Parallel phase I trials of multidrug resistance modulation with PSC-833 in untreated patients with acute myeloid leukemia o60 years old: preliminary results of CALGB 9621. Blood 1999; 94(Suppl 1): 384a (Abstract). Kolitz JE, George SL, Hurd D, Hoke E, Dodge RK, Caligiuri MA et al. Cytogenetic risk-adapted intensication followed by immunotherapy with recombinant interleukin-2 (rIL-2) in patients (PTS) o60 years old with acute myeloid leukemia (AML) in rst complete remission (CR): preliminary results of CALGB 9621. Blood 1999; 94(Suppl 1): 579a (Abstract). Baer MR, George SL, Dodge RK, OLoughlin KL, Minderman H, Caligiuri MA et al. Phase 3 study of the multidrug resistance modulator PSC-833 in previously untreated patients 60 years of age and older with acute myeloid leukemia: Cancer and Leukemia Group B Study 9720. Blood 2002; 100: 12241232. Cheson BD, Cassileth PA, Head DR, Schiffer CA, Bennett JM, Bloomeld CD et al. Report of the National Cancer Institutesponsored workshop on denitions of diagnosis and response in acute myeloid leukemia. J Clin Oncol 1990; 8: 813819. Stewart CC, Stewart SJ. Cell preparation for the identication of leukocytes. In: Darzynkiewicz Z, Crissman H, Robinson JP (eds). Methods of Cell Biology, Vol. 64. New York: Academic Press, Inc, 2001, pp 218270. Stewart CC, Stewart SJ. Multiparameter data acquisition and analysis of leukocytes by ow cytometry. In: Darzynkiewicz Z, Crissman H, Robinson JP (eds). Methods of Cell Biology, Vol. 64. New York: Academic Press, Inc, 2001, pp 289312. Baer MR, Stewart CC, Lawrence D, Arthur DC, Mrozek K, Strout MP et al. Acute myeloid leukemia with 11q23 translocations: myelomonocytic immunophenotype by multiparameter ow cytometry. Leukemia 1998; 12: 317325. Riedy MC, Muirhead KA, Jensen CP, Stewart CC. The use of a photolabeling technique to identify nonviable cells in xed homologous or heterologous cell populations. Cytometry 1991; 12: 133139. Clinical Applications of Flow Cytometry: Immunophenotyping of Leukemic Cells; Proposed Guidelines. Document H43-P, National Committee for Clinical Laboratory Standards, Villanova, PA, 1993. The SAS System. Release 8.2. SAS Institute Inc.: Cary, NC, USA, 2000. Lee ET. Statistical Methods for Survival Data Analysis, 2nd edn. New York: John Wiley & Sons, 1995. Marubini E, Valsecchi MG, Emmerson M. Analyzing Survival Data From Clinical Trials and Observational Studies. New York: John Wiley & Sons, 1995. Paietta E, Andersen J, Gallagher R, Bennett J, Yunis J, Cassileth P et al. The immunophenotype of acute promyelocytic leukemia (APL): an ECOG study. Leukemia 1994; 8: 11081112. Lazarchick J, Hopkins M. HLA-Dr negative acute non-lymphocytic leukemia. Ann Clin Lab Sci 1998; 28: 150152. Fenu S, Carmini D, Mancini F, Guglielmi C, Alimena G, Riccioni R et al. Acute myeloid leukemias M2 potentially misdiagnosed as M3 variant FrenchAmericanBritain (FAB) subtype: a transitional form? Leuk Lymphoma 1995; 18(Suppl 1): 4955. Hurwitz CA, Raimondi SC, Head D, Krance R, Mirro Jr J, Kalwinsky DK et al. Distinctive immunophenotypic features of t(8;21)(q22;q22) acute myeloblastic leukemia in children. Blood 1992; 80: 31823188. Kita K, Nakase K, Miwa H, Masuya M, Nishii K, Morita N et al. Phenotypical characteristics of acute myelocytic leukemia associated with the t(8;21)(q22;q22) chromosomal abnormality: frequent expression of immature B-cell antigen CD19 together with stem cell antigen CD34. Blood 1992; 80: 470477. Adriaansen HJ, Jacobs BC, Kappers-Klunne MC, Hahlen K, Hooijkaas H, van Dongen JJ. Detection of residual disease in AML patients by use of double immunological marker analysis for terminal deoxynucleotidyl transferase and myeloid markers. Leukemia 1993; 7: 472481. Baer MR, Stewart CC, Lawrence D, Arthur DC, Byrd JC, Davey FR et al. Expression of the neural cell adhesion molecule CD56 is associated with short remission duration and survival in acute

25

26

27

28

29

30

31

32

33 34 35 36 37 38 39

40

41

42

43

Leukemia

HLA-DR antigen-negative acute myeloid leukemia M Wetzler et al

715
44 myeloid leukemia with t(8;21)(q22;q22). Blood 1997; 90: 1643 1648. Larson RA, Williams SF, Le Beau MM, Bitter MA, Vardiman JW, Rowley JD. Acute myelomonocytic leukemia with abnormal eosinophils and inv(16) or t(16;16) has a favorable prognosis. Blood 1986; 68: 12421249. Haferlach T, Gassmann W, Lofer H, Jurgensen C, Noak J, Ludwig WD et al., for the AML Cooperative Group. Clinical aspects of acute myeloid leukemias of the FAB types M3 and M4Eo. The AML Cooperative Group. Ann Hematol 1993; 66: 165170. Paietta E, Wiernik PH, Andersen J, Bennett J, Yunis J. Acute myeloid leukemia M4 with inv(16) (p13q22) exhibits a specic immunophenotype with CD2 expression. Blood 1993; 82: 2595. Guglielmi C, Martelli MP, Diverio D, Fenu S, Vegna ML, CantuRajnoldi A et al. Immunophenotype of adult and childhood acute promyelocytic leukaemia: correlation with morphology, type of PML gene breakpoint and clinical outcome. A cooperative Italian study on 196 cases. Br J Haematol 1998; 102: 10351041. Claxton DF, Reading CL, Nagarajan L, Tsujimoto Y, Andersson BS, Estey E et al. Correlation of CD2 expression with PML gene breakpoints in patients with acute promyelocytic leukemia. Blood 1992; 80: 582586. Rovelli A, Biondi A, Cantu Rajnoldi A, Conter V, Giudici G, Jankovic M et al. Microgranular variant of acute promyelocytic leukemia in children. J Clin Oncol 1992; 10: 14131418. Orfao A, Chillon MC, Bortoluci AM, Lopez-Berges MC, GarciaSanz R, Gonzalez M et al. The ow cytometric pattern of CD34, CD15 and CD13 expression in acute myeloblastic leukemia is highly characteristic of the presence of PML-RARalpha gene rearrangements. Haematologica 1999; 84: 405412. Foley R, Soamboonsrup P, Carter RF, Benger A, Meyer R, Walker I et al. CD34-positive acute promyelocytic leukemia is associated 52 with leukocytosis, microgranular/hypogranular morphology, expression of CD2 and bcr3 isoform. Am J Hematol 2001; 67: 3441. Tucker J, Dorey E, Gregory WM, Simpson AP, Amess JA, Lister TA et al. Immunophenotype of blast cells in acute myeloid leukemia may be a useful predictive factor for outcome. Hematol Oncol 1990; 8: 4758. Terstappen LW, Safford M, Konemann S, Loken MR, Zurlutter K, Buchner T et al. Flow cytometric characterization of acute myeloid leukemia. Part II. Phenotypic heterogeneity at diagnosis. Leukemia 1992; 6: 7080. Reading CL, Estey EH, Huh YO, Claxton DF, Sanchez G, Terstappen LW et al. Expression of unusual immunophenotype combinations in acute myelogenous leukemia. Blood 1993; 81: 30833090. Scott AA, Head DR, Kopecky KJ, Appelbaum FR, Theil KS, Grever MR et al. HLA-DR, CD33+, CD56+, CD16 myeloid/natural killer cell acute leukemia: a previously unrecognized form of acute leukemia potentially misdiagnosed as FrenchAmericanBritish acute myeloid leukemia-M3. Blood 1994; 84: 244255. Solary E, Casasnovas RO, Campos L, Bene MC, Faure G, Maingon P et al. Surface markers in adult acute myeloblastic leukemia: correlation of CD19+, CD34+ and CD14+/DR-phenotypes with shorter survival. Groupe dEtude Immunologique des Leucemies (GEIL). Leukemia 1992; 6: 393399. Sartoris S, Accolla RS. Transcriptional regulation of MHC class II genes. Int J Clin Lab Res 1995; 25: 7178. Wetzler M, Baer MR, Stewart SJ, Donahue K, Ford L, Stewart CC et al. HLA class I antigen cell surface expression is preserved on acute myeloid leukemia blasts at diagnosis and at relapse. Leukemia 2001; 15: 128133. Heath WR, Carbone FR. Cross-presentation in viral immunity and self-tolerance. Nat Rev Immunol 2001; 1: 126134.

45

53

46 47

54

55

48

56

49 50

57 58

51

59

Leukemia