Original Manuscript

Leukemia (2003) 17, 707–715. doi:10.1038/sj.leu.2402865

HLA-DR antigen-negative acute myeloid leukemia

Supported partially by National Cancer Institute Grants CA 16056 and CA 67108.

M Wetzler1,5, B K McElwain1, C C Stewart2, L Blumenson4, A Mortazavi1, L A Ford1, J L Slack1, M Barcos3, S Ferrone5 and M R Baer1

  1. 1Leukemia Section, Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY, USA
  2. 2Laboratory of Flow Cytometry, Roswell
  3. 3Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY, USA
  4. 4Department of Cancer Prevention, Epidemiology and Biostatistics, Roswell Park Cancer Institute, Buffalo, NY, USA
  5. 5Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY, USA

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



Human leukocyte antigen (HLA) Class II antigens are variably expressed on acute myeloid leukemia (AML) blasts. The biological and clinical significance 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 flow 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 identified in HLA-DR-negative APL and non-APL 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 non-APL cases, and that the morphology of HLA-DR-negative non-APL 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 confirmation.


acute myeloid leukemia, HLA-DR, immunophenotype



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 influence the clinical course of disease. In this regard, there is conflicting information about the clinical significance 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,1,2,3 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.5,6,7 Finally, in malignant melanoma, there is conflicting information about the association of HLA Class II antigen expression with poor prognosis.8,9,10,11,12,13

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.14,15,16,17 Absence of HLA-DR antigen expression is rare in non-APL cases,18 and little information is available about the clinical significance 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. Specifically, we wished to determine how specific 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.



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 flow 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.

Morphologic studies

The diagnosis of AML and morphologic categorization were according to the French–American–British (FAB) classification.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 (24–72 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 (all others). Complex karyotype was defined by the presence of three or more clonal chromosomal abnormalities.

Reverse-transcription polymerase chain reaction (RT-PCR)

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


A total of 177 patients received high-dose cytarabine and idarubicin (HDAC/Ida) induction therapy24 and 30 received other induction regimens.25,26,27 Of the 157 patients who achieved complete remission (CR), 126 received postremission therapy.

Response criteria

CR was defined by normalization of blood counts and bone marrow morphology and disappearance of all signs of leukemia, lasting for greater than or equal to4 weeks, in accordance with the recommendations of the National Cancer Institute-Sponsored Workshop on the Definitions 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, defined by reappearance of blasts in the blood or the finding of greater than or equal to5% blasts in the bone marrow, not attributable to another cause, also in accordance with the recommendations of the National Cancer Institute-Sponsored Workshop.28


The peridinin chlorophyll protein (PerCP)-conjugated anti-HLA-DR antibody was purchased from Becton Dickinson (BD) Biosciences (San Diego, CA, USA). Fluorescein isothiocyanate (FITC)-labeled CD45 and phycoerythrin (PE)-labeled goat anti-mouse 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

Multiparameter flow 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 fluorophor 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 flow cytometer (BD Biosciences). Data were analyzed using WinList multiparameter analysis software (Verity 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 <10% of cells in the leukemia cell gate. Lack of HLA-DR antigen expression on leukemia cells was confirmed by visual analysis and confirmation of HLA-DR antigen expression on lymphocytes, defined by FSC vs SSC characteristics and bright CD45 expression, in the same sample.

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 Mann–Whitney test. Qualitative characteristics were compared using either the Fisher–Irwin test, the exact Mantel–Haenszel test, or the exact Pearson chi2 test. Survival curves were calculated using the method of Kaplan–Meier and were compared using the log-rank test. The method of Brookmyer–Crowley was used to calculate confidence intervals for the median survival time.35

DFS was defined 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



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, defined 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).

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 five 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 also showed advanced reticulin and collagen myelofibrosis. 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.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

HLA-DR-negative AML (FAB M2) resembling APL (original magnification times 250). (a) Two myeloblasts (one contains multiple slender Auer rods) and two abnormal promyelocytes with folded nuclei and fine-to-coarse rust-colored cytoplasmic granules. (b) Myeloblast with two distinct Auer rods.

Full figure and legend (301K)

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 non-APL 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 specific 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 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 significant (P<0.05) 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 non-APL (PML-RARalpha 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.

Lack of correlation between HLA-DR expression and clinical characteristics in non-APL AML patients at diagnosis

Pretreatment clinical characteristics did not differ significantly between HLA-DR-negative non-APL AML patients and AML patients whose blasts were HLA-DR-positive (Table 2 and Table 3). Of note, the HLA-DR-positive group included five patients with APL (median percentage of HLA-DR antigen expression: 45.8%, range 10.1–80.2%). 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 HLA-DR-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 HLA-DR-positive patients. No HLA-DR-negative patients underwent allogeneic transplantation in first remission, as compared to seven (6%) HLA-DR-positive patients. Three (19%) HLA-DR-negative patients underwent autologous transplantation in first 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, <1–85.4 months) for HLA-DR-negative patients and 11.2 months (range <1–99.6 months) for HLA-DR-positive patients. The estimated DFS curves, calculated with the time of death without disease censored, overlapped during the first 12 months after induction (P=0.54, 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 0–84 months, with the estimated median survival for both groups equal to approximately 1 year. Four (17%) HLA-DR- negative 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-DR- positive AML patients.

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

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.

Full figure and legend (66K)

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.

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, 3.2–55.9 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 HLA-DR-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 HLA-DR-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 present at diagnosis. In the other patient (Figure 3b), an HLA-DR-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.

Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Changes in HLA-DR antigen expression at relapse in AML blasts. Panel (a) demonstrates a patient whose leukemia cells were HLA-DR-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.

Full figure and legend (293K)



Analysis of a large number of AML patients showed that approximately 20 percent of cases of AML do not express HLA-DR 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 sufficient 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-DR-negative non-APL AML cases can have morphology resembling the hypogranular variant of APL. Therefore, the diagnosis of APL requires confirmation by cytogenetic demonstration of t(15;17) or one of its variants and/or molecular demonstration of PML/RARalpha or a variant gene rearrangement.

Our finding 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-DR-negative AML patients who were suggested to have APL variants based on morphology and immunophenotype, but were reclassified as FAB M2 AML after cytogenetic and molecular analyses were completed.

Distinct patterns of antigen expression on AML blasts have been associated with specific chromosomal abnormalities, including t(8;21)40,41,42,43 and inv(16).44,45,46 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.18,52,53,54 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 identified 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 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 identified by Solary et al.56 This group of patients had significantly shorter survival. However, the authors provide data neither on patient characteristics nor on treatment. Our series included only two patients with this immunophenotype (HLA-DR-, CD14+).

HLA Class II molecules play an essential role in presenting antigenic peptides to regulatory T cells.57 We did not find any difference in outcome between non-APL patients with HLA-DR-negative 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-specific 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-DR-positive 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 not represent an important mechanism of immune escape and of resistance to immunotherapy.



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