leukemia 2003

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

1Leukemia 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 ParkCancer Institute, Buffalo, NY, USA

Human leukocyte antigen (HLA) Class II antigens are variablyexpressed on acute myeloid leukemia (AML) blasts. Thebiological and clinical significance of HLA Class II antigenexpression by AML cells is not known. Therefore, we sought tocharacterize cases of AML without detectable HLA-DR expres-sion. Samples from 248 consecutive adult AML patients wereimmunophenotyped by multiparameter flow cytometry atdiagnosis. HLA-DR antigens were not detected on AML cellsfrom 43 patients, including 20 with acute promyelocyticleukemia (APL), and 23 with other subtypes of AML. All APLcases had t(15;17), but there were no characteristic chromo-some abnormalities in non-APL cases. No direct expression ofother antigens was identified in HLA-DR-negative APL and non-APL cases. Interestingly, cells from three HLA-DR-negativenon-APL patients had similar morphology to that of thehypogranular variant of APL. This morphology, however, wasnot present in any HLA-DR-positive AML cases. Treatmentresponse was similar in the 23 HLA-DR-negative non-APL andthe 205 HLA-DR-positive patients. Finally, relapse was infre-quently associated with changes in HLA-DR antigen expres-sion, as the HLA-DR antigen was lost at relapse in only 4% ofHLA-DR-positive cases, and was gained at relapse in only 17%of HLA-DR-negative cases. We conclude that HLA-DR-negativeAML 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. Thediagnosis of APL cannot be based on morphology and lack ofHLA-DR antigen expression; rather, it requires cytogenetic ormolecular confirmation.Leukemia (2003) 17, 707–715. doi:10.1038/sj.leu.2402865Keywords: acute myeloid leukemia; HLA-DR; immunophenotype

Introduction

Class II human leukocyte antigens (HLA) present antigenicpeptides to regulatory T cells. The crucial role played by HLAClass II antigens in the generation of an immune response hasstimulated interest in determining whether HLA Class II antigensexpressed by tumor cells influence the clinical course of disease.In this regard, there is conflicting information about the clinicalsignificance of HLA Class II antigens expressed by tumor cells.For example, HLA-DR antigen loss is associated with a moreaggressive course of the disease in B-cell lymphoma,1–3 whileHLA-DR antigen expression was reported to be associated withdisease progression in colon cancer.4 Furthermore, HLA-DRantigen expression was associated with improved prognosis incervical carcinoma.5–7 Finally, in malignant melanoma, there isconflicting information about the association of HLA Class IIantigen expression with poor prognosis.8–13

HLA Class II molecules are expressed on acute myeloidleukemia (AML) blasts at diagnosis in most cases of AML, withthe exception of acute promyelocytic leukemia (APL), which ischaracterized by lack of HLA-DR antigen expression.14–17

Absence of HLA-DR antigen expression is rare in non-APLcases,18 and little information is available about the clinicalsignificance of lack of expression of these antigens.

We sought to characterize cases of AML with subtypes otherthan APL in which HLA-DR antigens are not detected.Specifically, we wished to determine how specific lack ofHLA-DR antigen expression is in establishing the diagnosis ofAPL, whether it serves to identify one or more homogeneoussubsets of non-APL AML, and whether it is associated withtreatment response. Finally, we wanted to assess whetherchanges in HLA-DR antigen expression occur at relapse.

Methods

Patient samples

Bone marrow samples from 248 consecutive newly diagnosedadult AML patients referred to Roswell Park Cancer Institute(RPCI) between February 1990 and September 1998 wereimmunophenotyped by multiparameter flow cytometry in theLaboratory of Flow Cytometry as part of routine pretreatmentstudies. Relapse samples were also studied in 59 of 86 patientswho relapsed. Studies were approved by the RPCI InstitutionalReview Board.

Morphologic studies

The diagnosis of AML and morphologic categorization wereaccording to the French–American–British (FAB) classifica-tion.19,20 Slides available from 22 HLA-DR-negative and 162HLA-DR-positive non-APL cases were evaluated for thepresence of morphological features resembling the hypogranularvariant of APL, with varying degrees of nuclear folding,convolution or lobulation.

Cytogenetic analysis

Cytogenetic analysis was performed on pretreatment bonemarrow samples from all patients. Samples were processedusing short-term unstimulated cultures (24–72 h). Descriptionsof chromosome aberrations and clonality criteria were accord-ing to the International System for Human CytogeneticNomenclature.21 Patients were divided into three prognosticgroups based on karyotype, as previously described.22

The prognostic groups were favorable (t(8;21), inv(16) andt(15;17)), intermediate (normal cytogenetics), and unfavorable

Correspondence: Dr M Wetzler, Leukemia Section, Department ofMedicine, Roswell Park Cancer Institute, Elm and Carlton Streets,Buffalo, NY 14263, USA; Fax: +1 716 845 2343Supported partially by National Cancer Institute Grants CA 16056 andCA 67108.

Leukemia (2003) 17, 707–715& 2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00

www.nature.com/leu

(all others). Complex karyotype was defined by the presence ofthree or more clonal chromosomal abnormalities.

Reverse-transcription polymerase chain reaction(RT-PCR)

RT-PCR for PML-RARa mRNA was performed on HLA-DR-negative AML samples with morphology resembling thehypogranular variant of APL. The method was as describedbefore.23

Treatment

A total of 177 patients received high-dose cytarabine andidarubicin (HDAC/Ida) induction therapy24 and 30 receivedother induction regimens.25–27 Of the 157 patients whoachieved complete remission (CR), 126 received postremissiontherapy.

Response criteria

CR was defined by normalization of blood counts and bonemarrow morphology and disappearance of all signs of leukemia,lasting for Z4 weeks, in accordance with the recommendationsof the National Cancer Institute-Sponsored Workshop on theDefinitions of Diagnosis and Response in AML.30 Disease-freesurvival (DFS) was measured from the date of attainment of CRto the date of relapse, defined by reappearance of blasts in theblood or the finding of Z5% blasts in the bone marrow, notattributable to another cause, also in accordance with therecommendations of the National Cancer Institute-SponsoredWorkshop.28

Antibodies

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 andfrom Caltag (San Francisco, CA, USA), respectively. The panel ofmonoclonal antibodies (mAb) used to characterize AML waspreviously described.18

Multiparameter flow cytometry (MFC)

Samples were transported to the RPCI Laboratory of FlowCytometry at room temperature in tubes containing sodiumheparin and were processed as previously described.29,30 In all,228 patient samples were analyzed with three-color combina-tions of FITC, PE, and PerCP or PE/Cy5-conjugated mAb. Theremaining 20 patient samples (studied after March 1998) wereanalyzed with four-color combinations of mAb, the fourthfluorophor being allophycocyanin.31

Cell viability was determined by ethidium monoazidelabeling.32 All samples had at least 85% viable cells in thegated regions and were thus appropriate for analysis, inaccordance with the National Committee for Clinical LaboratoryStandards guidelines.33

Listmode data were acquired on either an FACScan or aFACSCalibur flow cytometer (BD Biosciences). Data wereanalyzed using WinList multiparameter analysis software (Verity

Software House, Topsham, ME, USA). To identify populations ofleukemia cells in each case of AML, the antibody panel waschosen that best resolved leukemia cells as a dense cluster ofcells with an abnormal pattern of antigen expression orcoexpression and a minimum of normal cell contamination. Anew region was drawn around the abnormal cell populationfound in the FSC vs SSC display, creating a leukemia cell gatethat was then used to analyze cells stained with all of theremaining mAb. Samples were scored as HLA-DR-negativewhen HLA-DR antigens were detected on o10% of cells in theleukemia cell gate. Lack of HLA-DR antigen expression onleukemia cells was confirmed by visual analysis and confirma-tion of HLA-DR antigen expression on lymphocytes, defined byFSC vs SSC characteristics and bright CD45 expression, in thesame sample.

Statistical analysis

All statistical calculations were performed with SAS/STATsoftware.34 The distributions of quantitative characteristics (ageand percentages of leukemic blasts expressing each antigen) indifferent patient populations were compared using the exactMann–Whitney test. Qualitative characteristics were comparedusing either the Fisher–Irwin test, the exact Mantel–Haenszeltest, or the exact Pearson w2 test. Survival curves were calculatedusing the method of Kaplan–Meier and were compared usingthe log-rank test. The method of Brookmyer–Crowley was usedto calculate confidence intervals for the median survival time.35

DFS was defined as the time from attainment of CR to relapseor last follow-up visit or death without relapse regarded as acensored event and also death without relapse regarded as acompeting risk for relapse. The crude cumulative incidence ofrelapse (or its complement, the crude survival) and correspond-ing standard errors were calculated using Equations (10.11) and(10.12), respectively.36

Results

Patient population

Ages of the 248 newly diagnosed AML patients whosepretreatment leukemia cells were immunophenotyped by MFCranged from 17 to 85 (median 65) years; 146 were male and 102were female. Data on prior history were available on 240patients: 172 patients had de novo AML, and 68 patients hadsecondary AML, defined by an antecedent hematologic disorder(48 patients), by chemotherapy/radiation therapy administeredfor a prior malignancy or other condition (18 patients), or byboth (two patients).

Characterization of HLA-DR-negative AML at diagnosis

HLA-DR antigens were not detected on the surface of leukemicblasts from 43 patients at diagnosis. These 43 patients included20 with APL and 23 with other FAB types, including M1 (sevenpatients), M2 (10 patients), M4 (four patients), M5b (one patient)and M6 (one patient). Myeloperoxidase and/or Sudan blackpositivity was noted in over 55% of the blast cells in 21 of the 22non-APL cases. Auer rods were present in five cases, and doubleAuer rods were present in one case. Three HLA-DR-negativeFAB M2 cases showed morphological features resembling thehypogranular variant of APL, with varying degrees of nuclearfolding, convolution, or lobulation. A representative example isshown in Figure 1. The bone marrow from one of these patients

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also showed advanced reticulin and collagen myelofibrosis. Themarrow from one additional patient, with HLA-DR-negative FABM4 AML, showed a marked increase in normal promyelocytes inaddition to myeloblasts. Marrows from two additional patients,one with FAB M1 and one with FAB M2, also showed coexistentdysplastic syncitial megakaryocytic hyperplasia, suggestive ofmyeloblastic transformation from a myelodysplastic or myelo-proliferative syndrome. Finally, cells from one patient with FABM1 AML had diffuse cytoplasmic acid phosphatase staining.None of the 162 HLA-DR-positive cases with slides available forreview had APL-like morphologic features.

All of the patients with APL had t(15;17)(q22;q11-12) or oneof its variants. Of the 23 patients with HLA-DR-negative non-APL AML, 14 had a normal karyotype, four had complexcytogenetic abnormalities, one patient had t(7;11)(p10;q10), onehad 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 withany specific immunophenotypic pattern. Expression of myeloidmarkers (CD13, CD15, CD33), the stem cell antigen CD34, thestem cell and T-lymphoid marker CD7, the T-lymphoid marker

Figure 1 HLA-DR-negative AML (FAB M2) resembling APL (original magnification� 250). (a) Two myeloblasts (one contains multiple slenderAuer rods) and two abnormal promyelocytes with folded nuclei and fine-to-coarse rust-colored cytoplasmic granules. (b) Myeloblast with twodistinct Auer rods.

Table 1 Similarity in pretreatment immunophenotype of HLA-DR antigen-negative APL and non-APL cases

Immunophenotype APL (n = 20) Non-APL (N = 23) P a

CD2 Median (range)b 3.5 (0.3–70.5) 2.8 (0.6–79.0) 0.30Antigenþ casesc 5 (25%) 4 (17%) 0.71

CD7 Median (range) 10.7 (0.6–48.2) 2.8 (0.5–95.8) 0.03Antigenþ cases 11 (55%) 5 (22%) 0.03



CD15 Median (range) 8.6 (0.6–77.8) 8.8 (0.1–98.9) 0.98Antigenþ cases 8 (N¼ 19) 8 (N¼ 19) 1.0




aP-value for comparison of distributions of percent of cells positive for antigen from the exact two-sided Wilcoxon–Mann–Whitney test. P-value forcomparison of proportion of patients positive (>10% of cells positive) for antigen from the two-sided exact Fisher test.bNumbers represent percentages of gated leukemia cells that are positive for each indicated antigen.cCases were defined as positive when the indicated antigen was detected on Z10% of cells in the leukemic gate.

APL, acute promyelocytic leukemia.


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CD2, the B-lymphoid marker CD19, and the neural celladhesion molecule CD56 was compared between samples fromHLA-DR-negative APL and non-APL cases (Table 1). Although astatistically significant (Po0.05) difference was found for CD7,in larger series APL blasts rarely express this antigen37 andtherefore this difference most probably does not represent abiological phenomenon. In summary, APL cannot be distin-guished from HLA-DR-negative non-APL AML based on thepanels of antigens analyzed in this study.

Interestingly, the three patients with HLA-DR-negative non-APL (PML-RARa mRNA not detected by RT-PCR) in our serieswhose cells had morphology resembling the hypogranularvariant of APL were female, their AML cells had no chromosomalaberrations, and did not express CD2, CD7, CD19, and CD34.

Lack of correlation between HLA-DR expression andclinical characteristics in non-APL AML patients atdiagnosis

Pretreatment clinical characteristics did not differ significantlybetween HLA-DR-negative non-APL AML patients and AMLpatients whose blasts were HLA-DR-positive (Tables 2 and 3).Of note, the HLA-DR-positive group included five patients withAPL (median percentage of HLA-DR antigen expression: 45.8%,range 10.1–80.2%). The distribution of induction (P¼ 0.15) andconsolidation (P¼ 0.41 for HDAC/Ida and P¼ 0.12 for otherregimens) 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. CRrates were 73% in HLA-DR-negative patients and 61% in HLA-DR-positive patients. No HLA-DR-negative patients underwentallogeneic transplantation in first remission, as compared toseven (6%) HLA-DR-positive patients. Three (19%) HLA-DR-negative patients underwent autologous transplantation in firstremission, as compared to eight (7%) HLA-DR-positive patients.These differences in treatment between the groups are not likelyto affect outcome comparisons. The median follow-up durationwas 12.6 months (range, o1–85.4 months) for HLA-DR-negative patients and 11.2 months (range o1–99.6 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 (P¼ 0.54,

Figure 2). When death without disease was regarded as acompeting risk, the estimated DFS rates were somewhat higherin both groups of patients, while the corresponding crudesurvival curves overlapped in a similar manner. The estimatedoverall survival curves almost overlapped throughout the range0–84 months, with the estimated median survival for bothgroups 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-negativenon-APL patients have relapsed, as have 83 (40%) HLA-DR-positive AML patients.

Table 2 Comparison of pretreatment immunophenotypes of HLA-DR-positive and HLA-DR-negative non-APL cases

Immunophenotype HLA-DR (þ ) (N = 205) HLA-DR (�) Non-APL (N = 23) P a

CD2 Median (range)b 12.2 (0.5–92.0) 2.8 (0.6–79.0) **Antigenþ casesc 113 (N¼201) (56%) 4 (17%) **

CD7 Median (range) 15.0 (0.8–95.3) 2.8 (0.5–95.8) **Antigenþ cases 124 (N¼201) (62%) 5 (22%) **

CD19 Median (range) 4.0 (0–75.9) 1.3 (0.5–74.2) 0.0004Antigenþ cases 41 (N¼ 202) (20%) 1 (4%) 0.09

CD13 Median (range) 59.4 (1–99.3) 53.3 (3.9–96.6) 0.32Antigenþ cases 194 (95%) 18 (78%) 0.01

CD15 Median (range) 36.9 (0–99.0) 8.8 (0.1–98.9) 0.008Antigenþ cases 139 (N¼176) (79%) 8 (N¼19) (42%) 0.001

CD33 Median (range) 31.2 (0–99.7) 47.5 (0.3–95.5) 0.38Antigenþ cases 158 (77%) 17 (74%) 0.80

CD34 Median (range) 43.7 (0.3–99.7) 3.8 (0.4–92.9) 0.0006Antigenþ cases 148 (N¼200) (74%) 8 (35%) **

CD56 Median (range) 3.3 (0–92.7) 1.2 (0.1–97.0) 0.25Antigenþ cases 48 (N¼ 203) (24%) 7 (30%) 0.45

[See Table 1.] **Po0.0001

Table 3 Similar pretreatment patient characteristics in HLA-DR-negative non-APL and HLA-DR positive cases

Characteristics HLA-DR (�)(N = 23)

HLA-DR (þ )(N = 205)

P

Age 0.16Median 70 65Range 26–84 17–85Z60 16 (70)a 125 (61)

Sex (M/F) 12/11 (52/48) 126/79 (61/39) 0.50De novo/secondary 19/4 (83/17) 145/60 (71/29) 0.33

FAB 0.39M0 0 21 (10)M1 7 (30) 27 (13)M2 10 (43) 81 (40)M3 0 5 (2)M4 4 (17) 33 (16)M5 1 (4) 14 (7)M6 1 (4) 20 (10)M7 0 1 (0.5)

Cytogenetics 0.19Favorable 0 16 (8)Intermediate 13 (59) 79 (41)Unfavorable 9 (41) 100 (51)

aNumbers in parentheses represent percentages.APL, acute promyelocytic leukemia; F, female; FAB, French–American–British classification; M, male.


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Of note, the three HLA-DR-negative non-APL patientswith the hypogranular variant morphology have remained inremission following induction and consolidation therapy, with23, 32, and 74 months follow-up.

Infrequent changes of HLA-DR antigen expression onleukemic blasts at relapse

HLA-DR antigen expression was studied at relapse in samplesfrom 59 of 93 patients who relapsed. Relapse occurred at amedian of 8.5 months (range, 3.2–55.9 months) in the patientswhose relapse samples were studied. Of six non-APL patientswhose 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 popula-tion, different from the HLA-DR-negative population that hadbeen 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 waslost 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 cellsat diagnosis, whereas a different population, which did notexpress HLA-DR antigens, was predominant at relapse.

Discussion

Analysis of a large number of AML patients showed thatapproximately 20 percent of cases of AML do not express HLA-DR antigens at diagnosis, and that cases without HLA-DRantigen expression are equally divided between APL and otherAML subtypes. Thus, whereas HLA-DR antigen expressionmakes the diagnosis of APL unlikely, absence of HLA-DRantigen expression cannot be considered to be a sufficientcriterion for establishing the diagnosis of APL. Only half of AMLcases without HLA-DR antigen expression have APL, and theother half have other AML subtypes. Further, blasts in HLA-DR-negative non-APL AML cases can have morphology resemblingthe hypogranular variant of APL. Therefore, the diagnosis of APLrequires confirmation by cytogenetic demonstration of t(15;17)

Table 4 Similar treatment response of HLA-DR-negative non-APL and HLA-DR-positive AML patients

HLA-DR (�) (N = 23) HLA-DR (+) (N = 205) P

Induction 0.15HDAC/Idaa 16 (70)b 164 (80)Otherc 7 (30) 33 (16)NT 0 8 (4)

Attainment of CR 0.90HDAC/Ida group 11 (73) 99 (61)Other group 5 (71) 18 (56)

ConsolidationHDAC/Ida group

VP/CYd 5 (45) 66 (67) 0.41HDAC/Ida 3 (27) 19 (19)

Other groupHDAC/Ida 0 2 (11) 0.12Othere 1 (20) 11 (61)

AlloPBSCT in first CR 0 7 (6)AutoPBSCT in first CR 3 (19) 8 (7)

DFS: Kaplan–Meier method(Censored at death without disease)Median (months) 23.6 13.3 0.54

95% confidence interval for median Z7.2 10.3,17.3Crude survival function: DFS in the presence

of competing risk of death without diseaseMedian (months) > 24 (55% at 24 mos) 14.5

Overall survival Median (months) 12.6 11.2 0.8395% confidence interval of the median 7.3,26.2 9.0,14.0

aHDAC/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 forthree consecutive days.bNumbers in parentheses represent percentages.cOther; 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 withcyclophosphamide 50 mg/kg for 4 days.eOther, 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 five 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 overseven 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 celltransplantation; CR, complete remission; DFS, disease-free survival; mos, months; NT, not treated.


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or one of its variants and/or molecular demonstration of PML/RARa or a variant gene rearrangement.

Our finding that lack of HLA-DR antigen expression occurs innon-APL AML cases corroborates information in the literature.Lazarchick and Hopkins38 demonstrated absence of HLA-DRantigen expression in AML subtypes other than APL, mainly FABM2 AML. Similarly, Fenu et al39 described three HLA-DR-negative AML patients who were suggested to have APL variantsbased on morphology and immunophenotype, but werereclassified as FAB M2 AML after cytogenetic and molecularanalyses were completed.

Distinct patterns of antigen expression on AML blasts havebeen associated with specific chromosomal abnormalities,including t(8;21)40–43 and inv(16).44–46 Similarly, APL blastshave been characterized by lack of HLA-DR antigen expressionand by CD2 expression in 28% of cases.47 Further subgroupanalysis revealed that CD2 expression was associated with theBcr3 breakpoint48 or the microgranular variant of APL.49

Interestingly, the three cases with HLA-DR-negative non-APLAML whose blasts had morphology resembling hypogranularAPL did not express the CD2 antigen. Others have found APL tobe characterized by heterogeneous expression of CD13 on theleukemic blasts, with a single major blast cell populationcharacterized by CD15+ and CD34� expression.50 Of note,recent work suggests that the hypogranular morphology of APLis associated with CD34 expression.51 CD34 is expressed inapproximately two-thirds of non-APL AML cases.18,52–54 Weasked whether a unique immunophenotype could serve todistinguish between APL and HLA-DR-negative cases with otherAML subtypes. We found that these two groups could not beseparated 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 wascharacterized by lack of HLA-DR antigen expression and hadfeatures of both myeloid and natural killer (NK) cells, with CD33

and CD56 expression, but absence of CD16 expression. Theyfound this entity in 20, or 6%, of their series of 350 AMLpatients. Our series included only three patients with themyeloid/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 hadsignificantly shorter survival. However, the authors provide dataneither on patient characteristics nor on treatment. Our seriesincluded only two patients with this immunophenotype (HLA-DR�, CD14+).

HLA Class II molecules play an essential role in presentingantigenic peptides to regulatory T cells.57 We did not find anydifference in outcome between non-APL patients with HLA-DR-negative and those with HLA-DR-positive blasts. One possibleexplanation is that HLA Class II antigens play only a minimalrole in the immune response against leukemia-associatedantigens. We have previously demonstrated that HLA Class Iantigen expression is preserved on the surface of AML blasts.58

HLA Class I molecules bind antigenic peptides and present themto cytotoxic (CD8+) T cells. The recognition of these peptides bycytotoxic T cells triggers a series of events that may result in lysisof target cells. It is conceivable that the lack of HLA Class IIantigens is compensated for by cross-presentation.59 In thisphenomenon, antigen-presenting cells take up antigens shedfrom leukemic cells and present the processed antigens toantigen-specific CD4 T cells (cross-presentation), as opposed todirect presentation by leukemic blasts themselves. Alternatively,the HLA Class II-negative leukemia cells might represent cellswith limited proliferative capacity, while the small HLA-DR-positive population may actually be the stem cell populationwith unlimited proliferative capacity.

Finally, immunophenotype changes occur frequently atrelapse,18 but changes in HLA-DR antigen expression are rare.We conclude that HLA-DR antigen loss is rare in bothleukemogenesis and disease progression, and therefore should

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 withoutdisease. The bold and the broken lines represent HLA-DR-negative non-APL AML and HLA-DR-positive AML, respectively.


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

References

1 Slymen DJ, Miller TP, Lippman SM, Spier CM, Kerrigan DP, RybskiJA et al. Immunobiologic factors predictive of clinical outcome indiffuse large-cell lymphoma. J Clin Oncol 1990; 8: 986–993.

2 Pilkington G, Juneja S, Tan L, Matthews J, Quirk J, Lee Get al. Correlation of immunological surface antigens with survivalin diffuse large cell lymphoma. Hematol Oncol 1993; 11:195–205.

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-celllymphomas arising in immune-privileged sites. Blood 2000; 96:3569–3577.

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Figure 3 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-negativepopulation (shown in the square) was more prominent at relapse. Control represents isotype control antibodies.


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4 Norazmi M, Hohmann AW, Skinner JM, Bradley J. Expression ofMHC class I and class II antigens in colonic carcinomas. Pathology1989; 21: 248–253.

5 Hilders CG, Houbiers JG, van Ravenswaay Claasen HH, Veldhui-zen RW, Fleuren GJ. Association between HLA-expression andinfiltration of immune cells in cervical carcinoma. Lab Invest 1993;69: 651–659.

6 Coleman N, Stanley MA. Analysis of HLA-DR expressionon keratinocytes in cervical neoplasia. Int J Cancer 1994; 56:314–319.

7 Cromme FV, van Bommel PF, Walboomers JM, Gallee MP, SternPL, Kenemans P et al. Differences in MHC and TAP-1 expression incervical cancer lymph node metastases as compared with theprimary tumours. Br J Cancer 1994; 69: 1176–1181.

8 Lopez-Nevot MA, Garcia E, Romero C, Oliva MR, Serrano S,Garrido F. Phenotypic and genetic analysis of HLA class I andHLA-DR antigen expression on human melanomas. Exp ClinImmunogenet 1988; 5: 203–212.

9 Ruiter DJ, Mattijssen V, Broecker EB, Ferrone S. MHC antigens inhuman melanomas. Semin Cancer Biol 1991; 2: 35–45.

10 Colloby PS, West KP, Fletcher A. Is poor prognosis really related toHLA-DR expression by malignant melanoma cells? Histopathology1992; 20: 411–416.

11 Moretti S, Pinzi C, Berti E, Spallanzani A, Chiarugi A, Boddi V et al.In situ expression of transforming growth factor beta is associatedwith melanoma progression and correlates with Ki67, HLA-DR andbeta 3 integrin expression. Melanoma Res 1997; 7: 313–321.

12 Konstadoulakis MM, Vezeridis M, Hatziyianni E, Karakousis CP,Cole B, Bland KI et al. Molecular oncogene markers and theirsignificance in cutaneous malignant melanoma. Ann Surg Oncol1998; 5: 253–260.

13 Ostmeier H, Fuchs B, Otto F, Mawick R, Lippold A, Krieg V et al.Can immunohistochemical markers and mitotic rate improveprognostic precision in patients with primary melanoma? Cancer1999; 85: 2391–2399.

14 Hanson CA, Gajl-Peczalska KJ, Parkin JL, Brunning RD. Immuno-phenotyping of acute myeloid leukemia using monoclonalantibodies and the alkaline phosphatase–antialkaline phosphatasetechnique. Blood 1987; 70: 83–89.

15 Scott CS, Patel D, Drexler HG, Master PS, Limbert HJ, Roberts BE.Immunophenotypic and enzymatic studies do not support theconcept of mixed monocytic–granulocytic differentiation in acutepromyelocytic leukaemia (M3): a study of 44 cases. Br J Haematol1989; 71: 505–509.

16 De Rossi G, Avvisati G, Coluzzi S, Fenu S, LoCoco F, Lopez Met al. Immunological definition of acute promyelocytic leukemia(FAB M3): a study of 39 cases. Eur J Haematol 1990; 45: 168–171.

17 Stone RM, Mayer RJ. The unique aspects of acute promyelocyticleukemia. J Clin Oncol 1990; 8: 1913–1921.

18 Baer MR, Stewart CC, Dodge RK, Leget G, Sule N, Mrozek K et al.High frequency of immunophenotype changes in acute myeloidleukemia at relapse: implications for residual disease detection(Cancer and Leukemia Group B Study 8361). Blood 2001; 97:3574–3580.

19 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA,Gralnick HR et al. Proposed revised criteria for the classification ofacute myeloid leukemia A report of the French–American–BritishCooperative Group. Ann Intern Med 1985; 103: 620–625.

20 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA,Gralnick HR et al. Proposal for the recognition of minimallydifferentiated acute myeloid leukaemia (AML-MO). Br J Haematol1991; 78: 325–329.

21 ISCN. In: Mitelman F (ed). An International System for HumanCytogenetic 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 forthrombopoietin, in acute myeloid leukemia blasts identifies agroup of patients with poor response to intensive chemotherapy. JClin Oncol 1997; 15: 2262–2268.

23 Slack JL, Bi W, Livak KJ, Beaubier N, Yu M, Clark M et al. Pre-clinical validation of a novel, highly sensitive assay to detect PML-RARalpha mRNA using real-time reverse-transcription polymerasechain reaction. J Mol Diagn 2001; 3: 141–149.

24 Baer MR, Pixley LA, Ford LA, Donohue K, O’Loughlin KL,Minderman H et al. High-dose cytarabine and idarubicin

induction produces a high complete remission rate in previouslyuntreated de novo acute myeloid leukemia patients. Blood 2000;96(Suppl 1): 322a (Abstract).

25 Kolitz JE, George SL, Hurd D, Hoke E, Dodge RK, Velez-Garcia Eet al. Parallel phase I trials of multidrug resistance modulation withPSC-833 in untreated patients with acute myeloid leukemia o60years old: preliminary results of CALGB 9621. Blood 1999;94(Suppl 1): 384a (Abstract).

26 Kolitz JE, George SL, Hurd D, Hoke E, Dodge RK, Caligiuri MAet al. Cytogenetic risk-adapted intensification followed by im-munotherapy with recombinant interleukin-2 (rIL-2) in patients(PTS) o60 years old with acute myeloid leukemia (AML) in firstcomplete remission (CR): preliminary results of CALGB 9621.Blood 1999; 94(Suppl 1): 579a (Abstract).

27 Baer MR, George SL, Dodge RK, O’Loughlin KL, Minderman H,Caligiuri MA et al. Phase 3 study of the multidrug resistancemodulator PSC-833 in previously untreated patients 60 years ofage and older with acute myeloid leukemia: Cancer and LeukemiaGroup B Study 9720. Blood 2002; 100: 1224–1232.

28 Cheson BD, Cassileth PA, Head DR, Schiffer CA, Bennett JM,Bloomfield CD et al. Report of the National Cancer Institute-sponsored workshop on definitions of diagnosis and response inacute myeloid leukemia. J Clin Oncol 1990; 8: 813–819.

29 Stewart CC, Stewart SJ. Cell preparation for the identification ofleukocytes. In: Darzynkiewicz Z, Crissman H, Robinson JP (eds).Methods of Cell Biology, Vol. 64. New York: Academic Press, Inc,2001, pp 218–270.

30 Stewart CC, Stewart SJ. Multiparameter data acquisition andanalysis of leukocytes by flow cytometry. In: Darzynkiewicz Z,Crissman H, Robinson JP (eds). Methods of Cell Biology, Vol. 64.New York: Academic Press, Inc, 2001, pp 289–312.

31 Baer MR, Stewart CC, Lawrence D, Arthur DC, Mrozek K, StroutMP et al. Acute myeloid leukemia with 11q23 translocations:myelomonocytic immunophenotype by multiparameter flowcytometry. Leukemia 1998; 12: 317–325.

32 Riedy MC, Muirhead KA, Jensen CP, Stewart CC. The use of aphotolabeling technique to identify nonviable cells in fixedhomologous or heterologous cell populations. Cytometry 1991;12: 133–139.

33 Clinical Applications of Flow Cytometry: Immunophenotyping ofLeukemic Cells; Proposed Guidelines. Document H43-P, NationalCommittee for Clinical Laboratory Standards, Villanova, PA, 1993.

34 The SAS System. Release 8.2. SAS Institute Inc.: Cary, NC, USA,2000.

35 Lee ET. Statistical Methods for Survival Data Analysis, 2nd edn.New York: John Wiley & Sons, 1995.

36 Marubini E, Valsecchi MG, Emmerson M. Analyzing Survival DataFrom Clinical Trials and Observational Studies. New York: JohnWiley & Sons, 1995.

37 Paietta E, Andersen J, Gallagher R, Bennett J, Yunis J, Cassileth Pet al. The immunophenotype of acute promyelocytic leukemia(APL): an ECOG study. Leukemia 1994; 8: 1108–1112.

38 Lazarchick J, Hopkins M. HLA-Dr negative acute non-lymphocyticleukemia. Ann Clin Lab Sci 1998; 28: 150–152.

39 Fenu S, Carmini D, Mancini F, Guglielmi C, Alimena G, Riccioni Ret al. Acute myeloid leukemias M2 potentially misdiagnosed as M3variant French–American–Britain (FAB) subtype: a transitionalform? Leuk Lymphoma 1995; 18(Suppl 1): 49–55.

40 Hurwitz CA, Raimondi SC, Head D, Krance R, Mirro Jr J,Kalwinsky DK et al. Distinctive immunophenotypic features oft(8;21)(q22;q22) acute myeloblastic leukemia in children. Blood1992; 80: 3182–3188.

41 Kita K, Nakase K, Miwa H, Masuya M, Nishii K, Morita N et al.Phenotypical characteristics of acute myelocytic leukemia asso-ciated with the t(8;21)(q22;q22) chromosomal abnormality:frequent expression of immature B-cell antigen CD19 togetherwith stem cell antigen CD34. Blood 1992; 80: 470–477.

42 Adriaansen HJ, Jacobs BC, Kappers-Klunne MC, Hahlen K,Hooijkaas H, van Dongen JJ. Detection of residual disease inAML patients by use of double immunological marker analysis forterminal deoxynucleotidyl transferase and myeloid markers.Leukemia 1993; 7: 472–481.

43 Baer MR, Stewart CC, Lawrence D, Arthur DC, Byrd JC, Davey FRet al. Expression of the neural cell adhesion molecule CD56 isassociated with short remission duration and survival in acute


714

Leukemia

myeloid leukemia with t(8;21)(q22;q22). Blood 1997; 90: 1643–1648.

44 Larson RA, Williams SF, Le Beau MM, Bitter MA, Vardiman JW,Rowley JD. Acute myelomonocytic leukemia with abnormaleosinophils and inv(16) or t(16;16) has a favorable prognosis.Blood 1986; 68: 1242–1249.

45 Haferlach T, Gassmann W, Loffler H, Jurgensen C, Noak J, LudwigWD et al., for the AML Cooperative Group. Clinical aspects ofacute myeloid leukemias of the FAB types M3 and M4Eo. The AMLCooperative Group. Ann Hematol 1993; 66: 165–170.

46 Paietta E, Wiernik PH, Andersen J, Bennett J, Yunis J. Acutemyeloid leukemia M4 with inv(16) (p13q22) exhibits a specificimmunophenotype with CD2 expression. Blood 1993; 82: 2595.

47 Guglielmi C, Martelli MP, Diverio D, Fenu S, Vegna ML, Cantu-Rajnoldi A et al. Immunophenotype of adult and childhood acutepromyelocytic leukaemia: correlation with morphology, type ofPML gene breakpoint and clinical outcome. A cooperative Italianstudy on 196 cases. Br J Haematol 1998; 102: 1035–1041.

48 Claxton DF, Reading CL, Nagarajan L, Tsujimoto Y, Andersson BS,Estey E et al. Correlation of CD2 expression with PML genebreakpoints in patients with acute promyelocytic leukemia. Blood1992; 80: 582–586.

49 Rovelli A, Biondi A, Cantu Rajnoldi A, Conter V, Giudici G,Jankovic M et al. Microgranular variant of acute promyelocyticleukemia in children. J Clin Oncol 1992; 10: 1413–1418.

50 Orfao A, Chillon MC, Bortoluci AM, Lopez-Berges MC, Garcia-Sanz R, Gonzalez M et al. The flow cytometric pattern of CD34,CD15 and CD13 expression in acute myeloblastic leukemia ishighly characteristic of the presence of PML-RARalpha generearrangements. Haematologica 1999; 84: 405–412.

51 Foley R, Soamboonsrup P, Carter RF, Benger A, Meyer R, Walker Iet al. CD34-positive acute promyelocytic leukemia is associated

with leukocytosis, microgranular/hypogranular morphology, ex-pression of CD2 and bcr3 isoform. Am J Hematol 2001; 67: 34–41.

52 Tucker J, Dorey E, Gregory WM, Simpson AP, Amess JA, Lister TAet al. Immunophenotype of blast cells in acute myeloid leukemiamay be a useful predictive factor for outcome. Hematol Oncol1990; 8: 47–58.

53 Terstappen LW, Safford M, Konemann S, Loken MR, Zurlutter K,Buchner T et al. Flow cytometric characterization of acute myeloidleukemia. Part II. Phenotypic heterogeneity at diagnosis. Leukemia1992; 6: 70–80.

54 Reading CL, Estey EH, Huh YO, Claxton DF, Sanchez G,Terstappen LW et al. Expression of unusual immunophenotypecombinations in acute myelogenous leukemia. Blood 1993; 81:3083–3090.

55 Scott AA, Head DR, Kopecky KJ, Appelbaum FR, Theil KS, GreverMR et al. HLA-DR�, CD33+, CD56+, CD16� myeloid/naturalkiller cell acute leukemia: a previously unrecognized form of acuteleukemia potentially misdiagnosed as French–American–Britishacute myeloid leukemia-M3. Blood 1994; 84: 244–255.

56 Solary E, Casasnovas RO, Campos L, Bene MC, Faure G, MaingonP et al. Surface markers in adult acute myeloblastic leukemia:correlation of CD19+, CD34+ and CD14+/DR-phenotypes withshorter survival. Groupe d’Etude Immunologique des Leucemies(GEIL). Leukemia 1992; 6: 393–399.

57 Sartoris S, Accolla RS. Transcriptional regulation of MHC class IIgenes. Int J Clin Lab Res 1995; 25: 71–78.

58 Wetzler M, Baer MR, Stewart SJ, Donahue K, Ford L, Stewart CCet al. HLA class I antigen cell surface expression is preserved onacute myeloid leukemia blasts at diagnosis and at relapse.Leukemia 2001; 15: 128–133.

59 Heath WR, Carbone FR. Cross-presentation in viral immunity andself-tolerance. Nat Rev Immunol 2001; 1: 126–134.


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