Abstract

The expression of CD56 antigen in acute promyelocytic leukemia (APL) blasts has been associated with short remission duration and extramedullary relapse. We investigated the clinical significance of CD56 expression in a large series of patients with APL treated with all-trans retinoic acid and anthracycline-based regimens. Between 1996 and 2009, 651 APL patients with available data on CD56 expression were included in 3 subsequent trials (PETHEMA LPA96 and LPA99 and PETHEMA/HOVON LPA2005). Seventy-two patients (11%) were CD56+ (expression of CD56 in ≥ 20% leukemic promyelocytes). CD56+ APL was significantly associated with high white blood cell counts; low albumin levels; BCR3 isoform; and the coexpression of CD2, CD34, CD7, HLA-DR, CD15, and CD117 antigens. For CD56+ APL, the 5-year relapse rate was 22%, compared with a 10% relapse rate for CD56 APL (P = .006). In the multivariate analysis, CD56 expression retained the statistical significance together with the relapse-risk score. CD56+ APL also showed a greater risk of extramedullary relapse (P < .001). In summary, CD56 expression is associated with the coexpression of immaturity-associated and T-cell antigens and is an independent adverse prognostic factor for relapse in patients with APL treated with all-trans-retinoic acid plus idarubicin–derived regimens. This marker may be considered for implementing risk-adapted therapeutic strategies in APL. The LPA2005 trial is registered at http://www.clinicaltrials.gov as NCT00408278.

Introduction

Several investigators have suggested a relationship between the expression of CD56 (neural adhesion factor) antigen in the surface of leukemic blasts and both short remission duration1,2  and development of extramedullary relapse3  in patients with acute promyelocytic leukemia (APL). However, this relationship has not been yet established. In fact, only one of these studies was performed in a relatively large population of APL patients receiving a state-of-the-art treatment with all-trans retinoic acid (ATRA) and anthracycline-based chemotherapy.2  With regard to the incidence of CD56-positive (CD56+) APL and the association with other clinical and biologic variables, very little information has been published.1-5 

In this study, we set out to assess the frequency of CD56 expression, its relationship with a broad variety of clinical and hematologic features, as well as its prognostic value in a large series of patients with newly diagnosed APL who were enrolled in 3 consecutive trials of the Programa Español para el Tratamiento de Enfermedades Hematológicas (PETHEMA) and Hemato-Oncologie voor Volwassenen Nederland (HOVON) groups.

Methods

Eligibility

Patients enrolled in the consecutive multicenter PETHEMA LPA96 and LPA99 trials and PETHEMA/HOVON LPA2005 were required to have a diagnosis of de novo APL with demonstration of the t(15;17) or PML/RARA rearrangements. More details about general exclusion and inclusion criteria have been reported elsewhere.6  Of the 1208 patients included in the 3 trials (LPA96, n = 172; LPA99, n = 560; LPA2005, n = 476), 651 patients (54%) had available the percentage of APL leukemic promyelocytes expressing CD56 surface antigen and were evaluable for the present study. Informed consent was obtained from all patients. In accordance with the Declaration of Helsinki, the protocol was approved by the Research Ethics Board of each participating hospital.

Therapy of APL

Induction therapy consisted of oral ATRA and idarubicin given as an intravenous bolus on days 2, 4, 6, and 8 (ATRA plus idarubicin, ie, AIDA regimen). In the LPA99 and LPA2005 trials, patients older than 70 years of age received only the 3 first doses of idarubicin.6,7  All patients in complete remission (CR) received 3 monthly consolidation courses with anthracycline-based chemotherapy. The consolidation schedule in the 3 consecutive protocols has been previously described6,7  and is shown in the Figure 1. After completion of consolidation, patients who tested negative for PML/RARA were started on maintenance therapy, as described elsewhere,6  with intermittent ATRA and low-dose chemotherapy with 6-mercaptopurine and methotrexate for 2 years.

Figure 1

Therapeutic schedule of the PETHEMA LPA96, LPA99, and LPA2005 trials.

Figure 1

Therapeutic schedule of the PETHEMA LPA96, LPA99, and LPA2005 trials.

Multiparameter flow cytometry

Inmunophenotyping was performed on bone marrow samples collected at APL diagnosis. Leukemic cell analysis was performed at local or reference laboratories by standard inmunofluorescence methods by the use of monoclonal antibodies directed against CD2, CD7, CD9, CD11b, CD13, CD15, CD19, CD33, CD34, CD56, CD117, and HLA-DR surface antigens. The inclusion of anti-CD56 in the panel antibodies was performed at the center's discretion. Multiparametric flow analysis was performed by the use of 3 or 4 colors on a flow cytometer. Leukemic promyelocytes were gated on the basis of their unique side-scatter/CD45-positive and/or CD33hi/homogeneous immunophenotypic profile, in both hyper and hypogranulated cases; CD45hi expression with SSClo was used to further exclude T cells and other lymphocytes from the gate. In addition, CD3 and/or CD5 antigens were routinely included in the diagnostic set to rule out contamination of the gated leukemic promyelocytes by mature T cells. Following the EGIL criteria,8  a sample was defined as positive if ≥ 20% of leukemic promyelocytes expressed a specific antigen in the cell surface. The only exception was the CD34 marker, for which a cutoff level of ≥ 10% expressing cells was required in line with previous reports on APL.4,9  Expression of CD56 was systematically assessed with sensitive (eg, phycoerythrin) fluorochrome-conjugated antibody reagents.

Definitions and study end points

Remission induction response was assessed according to the revised criteria by Cheson et al10  For morphologic assessment of leukemia resistance, it was required that sufficient time had passed to allow for full terminal differentiation of the malignant promyelocytes (up to 40-50 days). Molecular remission was defined as the disappearance on an ethidium bromide gel of the PML/RARA-specific band visualized at diagnosis by use of reverse transcription polymerase chain reaction (PCR) assays with a sensitivity level of one cell in 10−4. Molecular persistence was defined as PCR positivity in 2 consecutive bone marrow samples collected at the end of consolidation therapy. Molecular relapse was defined as reported elsewhere.11  Genetic diagnosis of APL with the use of reverse transcription PCR, anti-PML staining, or cytogenetic tests was required for the diagnosis of overt hematologic relapse. Central nervous system (CNS) relapse was confirmed by lumbar puncture and cytologic examination of cerebrospinal fluid, which was performed only in patients with clinically suspected CNS relapse. Extramedullary relapse in other localization (eg, skin) was confirmed by histopathology.

Data were collected and registered prospectively. Forty patient and disease characteristics were examined to establish their relationship to CD56 expression. We analyzed the characteristics listed in Tables 1 and 2. In addition we analyzed also the following variables: total body surface; liver and spleen enlargement; coagulopathy; hemorrhagic syndrome at presentation; serum levels of lactate dehydrogenase, creatinine, uric acid, alkaline phosphatases, and total bilirubin; peripheral blood blast count and blast cell percentage; bone marrow aspirate cellularity, peroxidase reactivity, and blast cell percentage; and CD13, CD19, and CD33 surface antigen markers.

Table 1

Demographic and baseline patient characteristics according to CD56 expression

CharacteristicCD56-positive
CD56-negative
P
Median (range)No. (%)Median (range)No. (%)
Overall  72 (100)  579 (100)  
PETHEMA trial      
    LPA96  4 (6)  75 (13) .17 
    LPA99  41 (57)  289 (50)  
    LPA2005  27 (37)  215 (37)  
Age, y 41 (5-74)  40 (2-81)   
    ≤ 18  5 (7)  62 (11) .44 
    19-40  31 (43)  235 (41)  
    41-60  21 (29)  189 (33)  
    > 60  15 (21)  92 (17)  
Sex      
    Male  33 (46)  312 (54) .20 
    Female  39 (54)  267 (46)  
ECOG 1 (0-3)  1 (0-3)   
    0-1  48 (71)  431 (79) .06 
    2-3  20 (29)  106 (21)  
Fever      
    No  48 (69)  356 (62) .17 
    Yes  22 (31)  220 (38)  
WBC count, × 109/L 3.6 (0.4-162)  2.6 (0.2-460)  .03* 
    ≤ 5  39 (54)  360 (62) .19 
    5-10  10 (14)  50 (9)  
    10-50  15 (21)  132 (23)  
    > 50  8 (11)  37 (6)  
Platelet count, × 109/L 20 (2-155)  23 (1-222)   
    ≤ 40  56 (78)  444 (77) .83 
    > 40  16 (22)  135 (23)  
Relapse-risk group      
    Low  12 (17)  109 (19) .85 
    Intermediate  37 (51)  301 (52)  
    High  23 (32)  169 (29)  
Hemoglobin, g/dL 9.5 (4.6-14.3)  9.4 (2.4-15.9)   
    ≤ 10  47 (65)  349 (60) .41 
    > 10  25 (35)  230 (40)  
Fibrinogen, mg/dL 174 (40-622)  159 (0-777)   
    ≤ 170  32 (49)  301 (54) .45 
    > 170  33 (51)  255 (46)  
Albumin, g/dL 3.9 (2.3-4.9)  4.0 (1.7-6.0)  .002* 
    ≤ 3.5  19 (31)  97 (19) .04 
    > 3.5  43 (69)  406 (81)  
CharacteristicCD56-positive
CD56-negative
P
Median (range)No. (%)Median (range)No. (%)
Overall  72 (100)  579 (100)  
PETHEMA trial      
    LPA96  4 (6)  75 (13) .17 
    LPA99  41 (57)  289 (50)  
    LPA2005  27 (37)  215 (37)  
Age, y 41 (5-74)  40 (2-81)   
    ≤ 18  5 (7)  62 (11) .44 
    19-40  31 (43)  235 (41)  
    41-60  21 (29)  189 (33)  
    > 60  15 (21)  92 (17)  
Sex      
    Male  33 (46)  312 (54) .20 
    Female  39 (54)  267 (46)  
ECOG 1 (0-3)  1 (0-3)   
    0-1  48 (71)  431 (79) .06 
    2-3  20 (29)  106 (21)  
Fever      
    No  48 (69)  356 (62) .17 
    Yes  22 (31)  220 (38)  
WBC count, × 109/L 3.6 (0.4-162)  2.6 (0.2-460)  .03* 
    ≤ 5  39 (54)  360 (62) .19 
    5-10  10 (14)  50 (9)  
    10-50  15 (21)  132 (23)  
    > 50  8 (11)  37 (6)  
Platelet count, × 109/L 20 (2-155)  23 (1-222)   
    ≤ 40  56 (78)  444 (77) .83 
    > 40  16 (22)  135 (23)  
Relapse-risk group      
    Low  12 (17)  109 (19) .85 
    Intermediate  37 (51)  301 (52)  
    High  23 (32)  169 (29)  
Hemoglobin, g/dL 9.5 (4.6-14.3)  9.4 (2.4-15.9)   
    ≤ 10  47 (65)  349 (60) .41 
    > 10  25 (35)  230 (40)  
Fibrinogen, mg/dL 174 (40-622)  159 (0-777)   
    ≤ 170  32 (49)  301 (54) .45 
    > 170  33 (51)  255 (46)  
Albumin, g/dL 3.9 (2.3-4.9)  4.0 (1.7-6.0)  .002* 
    ≤ 3.5  19 (31)  97 (19) .04 
    > 3.5  43 (69)  406 (81)  

ECOG indicates Eastern Cooperative Oncology Group; PETHEMA, Programa Español para el Tratamiento de Enfermedades Hematológicas; and WBC, white blood cell.

*

P compares continuous variables (mean WBC 17.5 vs 13.5, P = .03; and mean albumin 3.8 vs 4.0, P = .002).

Table 2

Biological features of APL according to CD56 expression

CharacteristicCD56-positive, no. (%)CD56-negative, no. (%)P
Overall 72 (100) 579 (100)  
Morphologic subtype (n = 645)    
    Hypergranular 51 (72) 461 (80) .09 
    Microgranular 20 (28) 113 (20)  
Cytogenetics (n = 476)    
    t(15;17) 37 (74) 307 (72) .77 
    t(15;17) plus other* 13 (26) 109 (28)  
FLT3-ITD mutations (n = 243)    
    Positive 10 (31) 54 (26) .49 
    Negative 22 (69) 157 (74)  
PML/RARα isoform (n = 576)    
    BCR1/BCR2 25 (38) 308 (60) < .001 
    BCR3 40 (62) 203 (40)  
CD2 (n = 538)    
    Positive 25 (46) 117 (24) < .001 
    Negative 29 (54) 367 (76)  
CD7 (n = 505)    
    Positive 9 (18) 22 (5) < .001 
    Negative 40 (82) 434 (95)  
CD9 (n = 189)    
    Positive 11 (65) 64 (37) .06 
    Negative 6 (35) 108 (63)  
CD11b (n = 464)    
    Positive 8 (17) 34 (8) .07 
    Negative 38 (83) 384 (92)  
CD15 (n = 555)    
    Positive 26 (43) 128 (26) .004 
    Negative 34 (57) 367 (74)  
CD34 (n = 607)    
    Positive 30 (48) 117 (21) < .001 
    Negative 32 (52) 428 (79)  
CD117 (n = 538)    
    Positive 50 (88) 357 (74) .02 
    Negative 7 (12) 124 (26)  
HLA-DR (n = 569)    
    Positive 10 (18) 28 (5) .001 
    Negative 46 (82) 485 (95)  
CharacteristicCD56-positive, no. (%)CD56-negative, no. (%)P
Overall 72 (100) 579 (100)  
Morphologic subtype (n = 645)    
    Hypergranular 51 (72) 461 (80) .09 
    Microgranular 20 (28) 113 (20)  
Cytogenetics (n = 476)    
    t(15;17) 37 (74) 307 (72) .77 
    t(15;17) plus other* 13 (26) 109 (28)  
FLT3-ITD mutations (n = 243)    
    Positive 10 (31) 54 (26) .49 
    Negative 22 (69) 157 (74)  
PML/RARα isoform (n = 576)    
    BCR1/BCR2 25 (38) 308 (60) < .001 
    BCR3 40 (62) 203 (40)  
CD2 (n = 538)    
    Positive 25 (46) 117 (24) < .001 
    Negative 29 (54) 367 (76)  
CD7 (n = 505)    
    Positive 9 (18) 22 (5) < .001 
    Negative 40 (82) 434 (95)  
CD9 (n = 189)    
    Positive 11 (65) 64 (37) .06 
    Negative 6 (35) 108 (63)  
CD11b (n = 464)    
    Positive 8 (17) 34 (8) .07 
    Negative 38 (83) 384 (92)  
CD15 (n = 555)    
    Positive 26 (43) 128 (26) .004 
    Negative 34 (57) 367 (74)  
CD34 (n = 607)    
    Positive 30 (48) 117 (21) < .001 
    Negative 32 (52) 428 (79)  
CD117 (n = 538)    
    Positive 50 (88) 357 (74) .02 
    Negative 7 (12) 124 (26)  
HLA-DR (n = 569)    
    Positive 10 (18) 28 (5) .001 
    Negative 46 (82) 485 (95)  
*

Plus other additional chromosomal abnormalities.

Diagnosis and gradation of the differentiation syndrome was made according to the previously defined criteria.12  Coagulopathy was defined as a prolonged prothrombin time and/or activated partial thromboplastin time in addition to hypofibrinogenemia and/or increased levels of fibrin degradation products or D-dimers. Patients were classified as having t(15;17) with or without additional chromosomal abnormalities accordingly to previously defined criteria.13  The patient performance status at diagnosis was measured using the Eastern Cooperative Oncology Group (ECOG) scale. Risk of relapse was established at diagnosis according to a predictive model on the basis of patient leukocyte and platelet counts at diagnosis, as reported elsewhere.14 

Statistical analysis

The χ2 test, with Yates correction if necessary, was used to analyze differences in the distribution of categorical variables between patient subsets. The Student t test was used to analyze continuous variables following a normal distribution and the Mann-Whitney U test for data that failed the normality test. Unadjusted time-to-event analyses were performed by use of the Kaplan-Meier estimate15  and, for comparisons, log-rank tests.16  The probability of relapse also was estimated by the cumulative incidence method (for marginal probability).17,18  Overall survival (OS) was calculated from the date of starting induction therapy, whereas cumulative incidence of relapse (CIR) and disease-free survival (DFS) were calculated from the date of CR. In the analysis of DFS, relapse, development of secondary myelodysplastic syndrome or acute leukemia (t-MDS/t-AL), and death in CR were considered uncensored events, whichever occurred first. For cumulative incidence analysis, death in CR and development of t-MDS/t-AL were considered as a competing cause of failure. For all estimates in which the event “relapse” was considered as an end point, overt morphologic and molecular relapse, as well as molecular persistence at the end of consolidation, were each considered as uncensored events. Patient follow-up was updated on March 15, 2010. Characteristics selected for inclusion in the multivariate analysis were those for which there was some indication of a significant association in univariate analysis (P < .1) and, if available, those for which previous studies had suggested a possible relationship. Multivariate analyses were performed by use of Cox model for DFS and OS19  and Fine and Gray model for CIR.20  Missing data were substituted by the mean values from patients in whom data were available.21  All P values reported are 2-sided. Computations were performed by use of the 3D, 4F, 1L, LR, and 2L programs from the BMDP statistical library (BMDP Statistical Software), and R 2.9.2 software package for CIR and Fine and Gray model.

Results

Patient characteristics according to CD56 expression

Between December 1996 and December 2009, 651 consecutive patients in whom the results of the analysis of CD56 surface antigen expression at diagnosis were available are the subject of the study (79 patients in the LPA96, 330 in the LPA99, and 242 in the LPA2005 trial). Patients were from 85 institutions from Spain, The Netherlands, Poland, Argentina, and the Czech Republic (see supplemental Appendix, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Median follow-up of the series was 70 months (range, 3-158 months) from diagnosis.

Seventy-two of 651 patients (11%) showed expression of CD56 ranging from 20% to 100% (median, 70%). The main clinical and biologic characteristics of CD56+ APL patients are shown in Tables 1 and 2. Concerning the demographic and clinical characteristics, the white blood cell (WBC) count at baseline was greater among patients with CD56+ APL (P = .03), whereas serum albumin levels were lower (P = .002). There was a trend toward a greater proportion of patients with ECOG performance status grade 2-3 in the CD56+ group (P = .06; Table 1).

Regarding other biologic features of APL, patients with CD56+ APL presented more frequently with BCR3 isoform (P < .001), CD2+ (P < .001), CD34+ (P < .001), CD7+ (P < .001), HLA-DR+ (P = .001), CD15+ (P = .004), and CD117+ (P = .02). There was also a trend toward a greater frequency of microgranular morphology (P = .09), CD11b+ (P = .07), and CD9+ (P = .06; Table 2).

Induction results

Overall, 595 of the 651 evaluable patients achieved morphologic CR (91.4%). As shown in Table 3, 61 of 72 patients (85%) achieved CR in the CD56+ subgroup, compared with 534 of 579 patients (92%) in the CD56 group (P = .04). No significant differences were observed in the distribution of the different causes of death between the CD56+ and CD56 cohorts. The incidence and severity of differentiation syndrome were similar among patients with CD56+ and those with CD56 APL (Table 3).

Table 3

Treatment results according to CD56 expression

CharacteristicCD56-positive
CD56-negative
P
No. of patients%No. of patients%
Overall 72 100 579* 100  
Induction outcome      
    Complete remission 61 85 534 92 .04 
    Causes of induction death      
        Hemorrhage 6.9 26 4.5 .53 
        Infection 4.2 11 1.9 .41 
        Differentiation syndrome 1.4 0.9 .99 
        Thrombosis/infarction 2.8 0.4 .09 
Differentiation syndrome      
    Severe 11 16 69 12 .69 
    Moderate 10 14 80 14  
    Absent 49 70 417 74  
Postremission outcomes at 5 y      
    CIR  22  10 .006 
    CIR (extramedullary)  7.0  0.7 < .001 
    Disease-free survival  73  85 .03 
    Overall survival  78  84 .09 
CharacteristicCD56-positive
CD56-negative
P
No. of patients%No. of patients%
Overall 72 100 579* 100  
Induction outcome      
    Complete remission 61 85 534 92 .04 
    Causes of induction death      
        Hemorrhage 6.9 26 4.5 .53 
        Infection 4.2 11 1.9 .41 
        Differentiation syndrome 1.4 0.9 .99 
        Thrombosis/infarction 2.8 0.4 .09 
Differentiation syndrome      
    Severe 11 16 69 12 .69 
    Moderate 10 14 80 14  
    Absent 49 70 417 74  
Postremission outcomes at 5 y      
    CIR  22  10 .006 
    CIR (extramedullary)  7.0  0.7 < .001 
    Disease-free survival  73  85 .03 
    Overall survival  78  84 .09 

CIR indicates cumulative incidence of relapse.

*

One patient among the CD56 cohort was considered as resistant.

A total of 636 patients were evaluable for differentiation syndrome.

After multivariate analysis, the regression model for induction death selected the following adverse factors: abnormal creatinine level (P < .0001), WBC count greater than 10 × 109/L (P < .0001), age older than 60 years (P < .0001), male sex (P = .0004), ECOG more than 1 (P = .009), and CD56 positivity (P = .02).

Postremission outcomes

Sixty relapses occurred among the 595 patients who had achieved CR (4 molecular persistence, 15 molecular relapses, and 41 clinical relapses), 12 among 61 CD56+ patients and 48 among 534 CD56 patients. Seven relapses involved extramedullary sites (6 in CNS and 1 in skin), of which 4 occurred among CD56+ patients. In addition, 13 patients died in CR (1 in the CD56+ group), and 10 patients developed t-MDS/t-AL (2 in the CD56+ group).

Relapse rate.

The 5-year CIR rate in the CD56+ cohort was 22%, whereas for CD56 APL it was 10% (P = .006; Table 3 and Figure 2A). For patients in the LPA96 trial, the 5-year CIR rates for CD56+ and CD56 patients were 50% and 10%, whereas in the LPA99 trial they were 18% and 11%, and in the LPA2005 they were 25% and 7%, respectively (P = .003, P = .29, and P = .01, respectively). According to relapse risk groups, the 5-year CIR rates for CD56+ and CD56 patients were 21% and 5% (P = .07) in the low-risk group, 17% and 6% (P = .02) in the intermediate group, and 35% and 22% (P = .24) in high-risk patients (Figures 2B-D). In the multivariate analysis, CD56 expression retained the independent predictive value along with the WBC counts (Table 4). The 5-year cumulative incidence of extramedullary relapse was significantly greater in CD56+ patients compared with those CD56 (7.0% vs 0.7%, P < .001; Table 3 and Figure 3).

Figure 2

Cumulative incidence of relapse in APL patients according to CD56 expression. (A) Overall series; (B) low-risk patients; (C) intermediate-risk patients; and (D) high-risk patients.

Figure 2

Cumulative incidence of relapse in APL patients according to CD56 expression. (A) Overall series; (B) low-risk patients; (C) intermediate-risk patients; and (D) high-risk patients.

Table 4

Statistically significant variables in univariate and multivariate analysis for relapse

CovariateUnfavorable categoryUnivariate analysis PMultivariate analysis
HR (95% CI)P
WBC count > 10 × 109/L < .001 3.9 (1.2-12.9) .03 
Relapse risk category High > intermediate > low < .001   
PML/RARA isoform BCR3 .002   
Morphologic subtype Microgranular .001   
CD56 Positive .01 2.3 (1.2-4.6) .01 
CD2 Positive .04   
CD34 Positive .002   
CovariateUnfavorable categoryUnivariate analysis PMultivariate analysis
HR (95% CI)P
WBC count > 10 × 109/L < .001 3.9 (1.2-12.9) .03 
Relapse risk category High > intermediate > low < .001   
PML/RARA isoform BCR3 .002   
Morphologic subtype Microgranular .001   
CD56 Positive .01 2.3 (1.2-4.6) .01 
CD2 Positive .04   
CD34 Positive .002   

CI indicates confidence interval; HR, hazard ratio; and WBC, white blood cell.

Figure 3

Cumulative incidence of extramedullary relapse in APL patients according to CD56 expression.

Figure 3

Cumulative incidence of extramedullary relapse in APL patients according to CD56 expression.

DFS and OS.

The 5-year DFS rates were 73% in the CD56+ cohort and 85% in the CD56 cohort (P = .03). The probability of remaining alive after 5 years was 78% in the CD56+ group and 84% in the CD56 group (P = .09; Table 3).

Discussion

This study shows a prevalence of CD56+ APL in 11% of newly diagnosed patients with APL. The expression of CD56 antigen was correlated with the BCR3 isoform and the coexpression of other surface antigens, such as CD2, CD34, HLA-DR, and CD7. The results presented here confirm that the expression of CD56 antigen is an independent risk factor for predicting relapse in patients with APL treated with ATRA and anthracycline-based regimens, along with the APL relapse-risk score, which is a composite of WBC and platelet counts.14  In addition, CD56+ APL had a significantly higher risk of extramedullary relapse.

The present study analyzes the clinical significance of CD56 expression in a significantly larger series of APL patients compared with previous studies, in which only the Gruppo Italiano Malattie Ematologiche dell'Adulto (GIMEMA) study included a sizable number of patients (100 patients). As in previous studies, we used the cut-off level of 20% of leukemic promyelocytes expressing CD56 antigen to define CD56+ APL.1-3  The frequency of 11% of CD56+ APL here reported is not dissimilar to the12% to 15% values found in other studies.2-4  It should be noted that in the current multicenter study, as in previous studies that analyzed the prognostic impact of CD56 in APL,1,2  immunophenotypic analyses were not performed centrally, preventing a systematic standardization of flow cytometry. A further limitation of our study is the possible selection bias because not all centers performed cytometric analysis that included anti-CD56 in the diagnostic panel. This limitation leads to a considerable reduction in the sample size to almost half compared with the total series.

Concerning the biologic features of CD56+ APL, our study shows a significant correlation with the BCR3 isoform1,5  and CD34 coexpression,3  as has been previously observed. Furthermore, we found an association between CD56+ APL and expression of additional immaturity-associated markers, such as CD117 and HLA-DR antigens, as well as natural killer (NK) and T-cell antigens, such as CD2 and CD7, which have not been previously reported. The higher frequency of coexpression of stem-cell and NK-cell antigens in CD56+ APL may suggest that in some of these cases the APL might have arisen in progenitors that have not undergone lineage restriction.4,22  Interestingly, we found a trend indicating an association with the M3 variant morphology, which has been previously related to CD2 and CD344,5,9  but never CD56 expression. The relationship between CD56 expression and WBC counts confirmed in our series had been suggested in previous studies, although without statistical significance, probably because of the small sample size in those studies.1,2  However, we were unable to confirm the relationship between fibrinogen levels and CD56 expression suggested by others.1 

The higher induction mortality rate in CD56+ patients observed in our series treated with the AIDA regimen confirms a similar observation previously reported,1  although many of these patients did not receive a state-of-the-art treatment. Probably, the higher induction mortality rate in CD56+ APL was attributable to its association with other recognized adverse factors for induction response23 ; however, multivariate analysis showed that this immunophenotypic feature had an independent prognostic value.

As suggested in previous studies,1-3  we demonstrated that CD56 expression has an impact on the relapse rate and also confirmed its independent prognostic value as in the GIMEMA study.2  Interestingly, the CD56 expression was able to distinguish a subset of patients with a greater risk of relapse in the intermediate-risk category, whereas its usefulness in low-risk patients needs to be confirmed in larger series. Moreover, the prognostic value of CD56 in high-risk APL seems insignificant. Of note, the expression of CD56 was associated with a higher relapse rate in all 3 PETHEMA trials, although the differences were significant in the LPA96 and LPA2005 trials, but not in the LPA99.

The reason why an elevated CD56 expression leads to a greater risk of relapse remains uncertain but at least 2 hypotheses can be proposed. First, as we have alluded to previously, CD56+ APL may emerge from a more immature undifferentiated and pluripotent leukemic stem cell that is less sensitive to the combination of ATRA and anthracyclines. CD56 expression has been associated with the expression of the multidrug resistant marker P-glycoprotein in patients with acute myeloid leukemia with t(8;21).24  Unfortunately, in the current study the expression of the multidrug resistance markers has not been assessed. Second, the implication of CD56 expression in the development of extramedullary relapses found in our study has already been hypothesized in a previous study by Ito et al.3  In this study, 3 of the 4 CD56+ APL patients developed extramedullary relapse, compared with none of the 24 CD56 patients. Of note in this respect, the expression of CD56 has been associated with extramedullary involvement in nonpromyelocytic acute myeloid leukemia.25-29 

In addition to confirm the prognostic value of CD56 expression, this study also provides new insights into the clinical features of CD56+ APL. CD56 expression is associated with some clinical and biologic features, such as increased WBC counts, BCR3 isoform, and coexpression of immaturity and NK-cell antigen markers. It should be noted that, despite this association with WBC counts, the regression model for relapse risk selected both as independent adverse factors, WBC count greater than 10 × 109/L and CD56 positivity. If the independent prognostic value of CD56 expression in APL cells is confirmed, it should be considered for designing future risk-adapted strategies.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Acknowledgments

We thank Alberto Orfao for the critical review of the section addressing the immunophenotypic analyses and Leticia Llopis for data collection and management.

This study was supported in part by the Fundación para la Investigación Hospital Universitario La Fe-Ayudas Bancaja (grant 2006/0137) and Red Temática de Investigación Cooperativa en Cáncer (RD06/0020/0031).

A complete list of participating institutions and clinicians appears in the online supplemental Appendix.

Authorship

Contribution: P.M. and M.A.S. conceived the study and analyzed and interpreted the data; P.M., B.L., and M.A.S. wrote the paper; P.M. performed the statistical analyses; and C.R., E.V., S.B., J.G., M.G., A.H., J.E., J.B., J.D.G., C.R., M.T., V.R., J.B., F.M., G.M., J.d.l.S., I.P., M.P.-E., I.K., J.M.R., and L.E. included data of patients treated in their institutions, reviewed the manuscript, and contributed to the final draft.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Miguel A. Sanz, Hospital Universitario La Fe, Avenida Campanar 21, Valencia 46009, Spain; e-mail: msanz@uv.es.

References

1
Murray
 
CK
Estey
 
E
Paietta
 
E
, et al. 
CD56 expression in acute promyelocytic leukemia: a possible indicator of poor treatment outcome?
J Clin Oncol
1999
, vol. 
17
 
1
(pg. 
293
-
297
)
2
Ferrara
 
F
Morabito
 
F
Martino
 
B
, et al. 
CD56 expression is an indicator of poor clinical outcome in patients with acute promyelocytic leukemia treated with simultaneous all-trans-retinoic acid and chemotherapy.
J Clin Oncol
2000
, vol. 
18
 
6
(pg. 
1295
-
1300
)
3
Ito
 
S
Ishida
 
Y
Oyake
 
T
Satoh
 
M
, et al. 
Clinical and biological significance of CD56 antigen expression in acute promyelocytic leukemia.
Leuk Lymphoma
2004
, vol. 
45
 
9
(pg. 
1783
-
1789
)
4
Albano
 
F
Mestice
 
A
Pannunzio
 
A
, et al. 
The biological characteristics of CD34+ CD2+ adult acute promyelocytic leukemia and the CD34 CD2 hypergranular (M3) and microgranular (M3v) phenotypes.
Haematologica
2006
, vol. 
91
 
3
(pg. 
311
-
316
)
5
Paietta
 
E
Goloubeva
 
O
Neuberg
 
D
, et al. 
A surrogate marker profile for PML/RAR alpha expressing acute promyelocytic leukemia and the association of immunophenotypic markers with morphologic and molecular subtypes.
Cytometry B Clin Cytom
2004
, vol. 
59
 
1
(pg. 
1
-
9
)
6
Sanz
 
MA
Martin
 
G
Gonzalez
 
M
, et al. 
Risk-adapted treatment of acute promyelocytic leukemia with all-trans-retinoic acid and anthracycline monochemotherapy: a multicenter study by the PETHEMA group.
Blood
2004
, vol. 
103
 
4
(pg. 
1237
-
1243
)
7
Sanz
 
MA
Montesinos
 
P
Rayon
 
C
, et al. 
Risk-adapted treatment of acute promyelocytic leukemia based on all-trans retinoic acid and anthracycline with addition of cytarabine in consolidation therapy for high-risk patients: further improvements in treatment outcome.
Blood
2010
, vol. 
115
 
25
(pg. 
5137
-
5146
)
8
Bene
 
MC
Castoldi
 
G
Knapp
 
W
, et al. 
Proposals for the immunological classification of acute leukemias. European Group for the Immunological Characterization of Leukemias (EGIL).
Leukemia
1995
, vol. 
9
 
10
(pg. 
1783
-
1786
)
9
Guglielmi
 
C
Martelli
 
MP
Diverio
 
D
, 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
, vol. 
102
 
4
(pg. 
1035
-
1041
)
10
Cheson
 
BD
Bennett
 
JM
Kopecky
 
KJ
, et al. 
Revised recommendations of the International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia.
J Clin Oncol
2003
, vol. 
21
 
24
(pg. 
4642
-
4649
)
11
Bolufer
 
P
Barragan
 
E
Sanz
 
MA
, et al. 
Preliminary experience in external quality control of RT-PCR PML-RAR alpha detection in promyelocytic leukemia.
Leukemia
1998
, vol. 
12
 
12
(pg. 
2024
-
2028
)
12
Montesinos
 
P
Bergua
 
JM
Vellenga
 
E
, et al. 
Differentiation syndrome in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline chemotherapy: characteristics, outcome, and prognostic factors.
Blood
2009
, vol. 
113
 
4
(pg. 
775
-
783
)
13
Cervera
 
J
Montesinos
 
P
Hernández-Rivas
 
JM
, et al. 
Additional chromosome abnormalities in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and chemotherapy.
Haematologica
2010
, vol. 
95
 
3
(pg. 
424
-
431
)
14
Sanz
 
MA
Lo Coco
 
F
Martin
 
G
, et al. 
Definition of relapse risk and role of nonanthracycline drugs for consolidation in patients with acute promyelocytic leukemia: a joint study of the PETHEMA and GIMEMA cooperative groups.
Blood
2000
, vol. 
96
 
4
(pg. 
1247
-
1253
)
15
Kaplan
 
EL
Meier
 
P
Nonparametric estimations from incomplete observations.
J Am Stat Assoc
1958
, vol. 
53
 (pg. 
457
-
481
)
16
Mantel
 
N
Evaluation of survival data and two new rank order statistics arising in its consideration.
Cancer Chemother Rep
1966
, vol. 
50
 
3
(pg. 
163
-
170
)
17
Gray
 
RJ
A class of K-sample test for comparing the cumulative incidence of a competing risk.
Ann Stat
1988
, vol. 
16
 
3
(pg. 
1141
-
1154
)
18
Pepe
 
MS
Mori
 
M
Kaplan-Meier, marginal or conditional probability curves in summarizing competing risk failure time data?
Stat Med
1993
, vol. 
12
 
8
(pg. 
737
-
751
)
19
Cox
 
DR
Regression models and life tables (with discussion).
J R Stat Soc B
1972
, vol. 
34
 (pg. 
187
-
220
)
20
Fine
 
J
Gray
 
R
A proportional hazards model for the subdistribution of a competing risk.
J Am Stat Assoc
1999
, vol. 
94
 (pg. 
496
-
509
)
21
Lee
 
ET
Statistical Methods for Survival Data Analysis
1980
Vermont, CA
Wadsworth Inc
22
Chapiro
 
E
Delabesse
 
E
Asnafi
 
V
, et al. 
Expression of T-lineage-affiliated transcripts and TCR rearrangements in acute promyelocytic leukemia: implications for the cellular target of t(15;17).
Blood
2006
, vol. 
108
 
10
(pg. 
3484
-
3493
)
23
de la Serna
 
J
Montesinos
 
P
Vellenga
 
E
, et al. 
Causes and prognostic factors of remission induction failure in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and idarubicin.
Blood
2008
, vol. 
111
 
7
(pg. 
3395
-
3402
)
24
Raspadori
 
D
Damiani
 
D
Lenoci
 
M
, et al. 
CD56 antigenic expression in acute myeloid leukemia identifies patients with poor clinical prognosis.
Leukemia
2001
, vol. 
15
 
8
(pg. 
1161
-
1164
)
25
Xu
 
N
Liu
 
XL
DU
 
QF
, et al. 
CD56 and CD11b antigen expressions in patients with acute monocytic leukemia and the clinical implications.
Nan Fang Yi Ke Da Xue Xue Bao
2009
, vol. 
29
 
8
(pg. 
1605
-
1608
)
26
Ando
 
T
Mitani
 
N
Matsui
 
K
, et al. 
Recurrent extramedullary relapse of acute myelogenous leukemia after allogeneic hematopoietic stem cell transplantation in a patient with the chromosomal abnormality t(8;21) and CD56-positivity.
Int J Hematol
2009
, vol. 
90
 
3
(pg. 
374
-
377
)
27
Chang
 
H
Brandwein
 
J
Yi
 
QL
, et al. 
Extramedullary infiltrates of AML are associated with CD56 expression, 11q23 abnormalities and inferior clinical outcome.
Leuk Res
2004
, vol. 
28
 
10
(pg. 
1007
-
1011
)
28
Baer
 
MR
Stewart
 
CC
Lawrence
 
D
, et al. 
Expression of the neural cell adhesion molecule CD56 is associated with short remission duration and survival in acute myeloid leukemia with t(8;21)(q22;q22).
Blood
1997
, vol. 
90
 
4
(pg. 
1643
-
1648
)
29
Tallman
 
MS
Extramedullary acute myeloid leukemia infiltrates.
Leuk Res
2004
, vol. 
28
 
10
(pg. 
1005
-
1006
)