Abstract

The importance of coexpression of myeloid antigens in childhood acute lymphoblastic leukemia (ALL) has long been debated; results are conflicting. We studied children with ALL treated at Italian Association for Pediatric Hematology-Oncology (AIEOP) institutions over 6 years with Berlin-Frankfurt-Muenster (BFM)-based protocols and have analyzed the incidence of coexpression of six MyAg (CD11b, CD13, CD14, CD15, CD33, CD65w) to determine its prognostic impact. Criteria for MyAg coexpression (MyAg+ALL) included positivity to one or more MyAg on at least 20% of blasts and confirmation of coexpression at double-fluorescence analysis. A total of 291 of 908 cases were MyAg+ALL (32%). Incidence was similar in B-ALL and T-ALL; among common, pre-B, and pre-pre–B-ALL. CD13 and CD33 were most common. Patients with MyAg+ALL had presenting features similar to MyAgALL. They entered standard or intermediate risk protocols more frequently and had better prednisone response, but similar complete remission rates. Six-year event-free survival (EFS) was 69.0% in 291 MyAg+ALL cases and 65.3% in 617 MyAgALL cases, without significant difference. Cases expressing two or more MyAg presented similar clinical features and treatment response. MyAg+ALL had worse EFS only in infants (0% v47%) (P = .01). Therefore, in this series of homogeneously diagnosed and treated ALL, coexpression of MyAg was not associated with prognostic significance, without relevance for clinical purposes or for patient stratification, except for infants.

© 1998 by The American Society of Hematology.

RECOGNIZABLE IMMUNOLOGIC patterns usually allow to attribute childhood acute lymphoblastic leukemia (ALL) to T or B lineages.1,2 Based on experience of a large series of patients homogeneously treated, such information has been associated with some clinical and biologic features and with different prognosis in some cases.3-5 

Abnormal expression of antigens of different lineages on leukemic cells has driven much interest because it suggests aberrant hematopoietic differentiation.6 Moreover, its correlation with leukemias with peculiar clinical and biological features [eg, ALL with Ph1 chromosome or translocation t(4;11)] has long been studied as a sign of neoplastic involvement of very immature progenitors. In fact, these leukemias often present “hybrid” features as the rearrangement of different genes or the response to various growth factors. Some of them often present an aggressive clinical course, with poor response to standard therapy.7-9 The above observations have suggested we should consider the expression of myeloid antigens (MyAg) in ALL as a possible indicator of abnormal biological behavior and possibly of different prognosis.6,10 However, results have been controversial in different series and prognostic data are still awaiting a conclusive confirmation. In fact, while many adult series have shown poorer outcome for ALL with myeloid markers,10-17 this significance has not yet been firmly established for pediatric patients, about whom opinions in the literature are rather conflicting.16-25 However, differences may be due to the consistency of series, as well as to technical details about recognition of coexpression. The current widespread use of monoclonal antibodies (MoAbs) and of fluorescence activated cytometry has extended the possibility of precise immunophenotyping, thereby allowing easy confirmation of the coexpression of different markers on the surface of blasts by multiparametric analysis.

In this study, we have analyzed the pattern of expression of the most common MyAg in a large group of childhood ALL (908 cases), all homogeneously diagnosed and treated according to two cooperative studies of the Italian Association for Pediatric Hematology-Oncology (AIEOP). Three myeloid markers (CD13, CD33, and CDw65) have been analyzed as part of an obligatory panel, according to the Italian-Berlin-Frankfurt-Muenster Study Group (I-BFM-SG) recommendations.26 Three additional markers (CD11b, CD14, and CD15) have also been evaluated to extend the analysis on the most widely studied antigens in the literature. The association with the most important clinical parameters and the prognostic significance of the coexpression of MyAg are also analyzed and discussed in this report.

MATERIALS AND METHODS

Patients.

A total of 1,811 children with newly diagnosed ALL have been reported by the AIEOP institutions from January 1988 to April 1995. Of them, 1,612 (89%) were eligible for the two subsequent trials, AIEOP ALL 88 and 91, respectively. For this study, a group of 908 patients (56.3%) was analyzed, of whom the main prognostic indices and a complete immunophenotype were known. The remaining patients were not used for analysis because a full set of their records was not available.

Treatment schedule.

Patient stratification was based on tumor burden estimated as BFM Risk Factor (RF).3 They were treated using three different risk-directed regimens according to the criteria of BFM and AIEOP groups.27-29 Details of the treatment schedule have been provided elsewhere for both the AIEOP-ALL 8828 and the AIEOP-ALL 91 study.29 Treatment consisted of a classical BFM backbone3 with minor modifications. Patients in the high-risk (HR) group of the study 91 received a block-type chemotherapy, derived from the BFM experience in relapsed ALL.30 Cranial irradiation was only given to HR patients older than 1 year of age or with central nervous system (CNS) involvement. The remaining patients had extended intrathecal chemotherapy for CNS prophylaxis.

Diagnostic criteria and immunophenotype.

All cases were diagnosed as ALL according to the French-American-British (FAB) morphological and cytochemical criteria.31 Immunophenotypic studies were performed on bone marrow samples at diagnosis by indirect immunofluorescence on flow cytometry.32 Analyses were centrally performed using shipped samples at the reference Laboratory for Hemato-Oncology (Department of Pediatrics, University of Padova, Padova, Italy). All results were carefully revised and accepted by standard procedures. A base-line panel of leukocyte antigens was used in all cases, comprising B-cell antigens (CD19, CD10, CD20; CD24; surface [SIg] and cytoplasmic [CyIg] immunoglobulins), T-cell antigens (CD2, CD7, CD1a, surface and cytoplasmic CD3, CD5), myeloid antigens (CD13, CD14, CD33, CD65w, CD11b, CD15), and lineage-nonspecific markers (CD34, HLA-DR, TdT). Surface markers were considered positive when present on more than 20% of the blast cells. Antigen coexpression was always confirmed by double fluorescence tests using directly conjugated reagents (fluorescein isothiocyanate [FITC] and phycoerythrin [PE]) (Fig 1). Analyses were performed on an Epics XL (Coulter, Miami, FL) cytometer. Isotypic controls were always added. Nonspecific binding was prevented by incubation with AB human serum.

Different immunological groups were identified by positivity to different markers, and the criteria recommended by the I-BFM-SG Biological Cooperative Group were used for classification, thus recognizing the following categories26: pre-pre–B, common ALL, pre-B ALL, T ALL. Mature B-ALL were not eligible for these studies. We defined MyAg+ALLs as the cases coexpressing at least one MyAg among all six evaluated markers in any immunophenotypic group. Hybrid leukemias26,33 were the cases with coexpression of at least two MyAg (of CD13, CD33, CD65w) in common-ALL, pre-B-ALL, T-ALL, and of at least one MyAg in pre-pre–B-ALL.

Statistical methods.

Data were collected on protocol-specific forms. The information was stored, controlled, and analyzed by VENUS, an integrated system of software facilities manufactured by the North-East Italian Interuniversities Computing Center (CINECA, Bologna, Italy), running on an IBM mainframe. The following presenting features were evaluated34: age (cut-offs >1, 1-9, >9 years), sex, hepatomegaly, splenomegaly (below the umbilical line); CNS involvement, leukocyte count (cut-off point at 50,000/μL), hemoglobin (cut-off 8.0 g/dL), platelets (cut-off 50,000/μL), FAB morphology (L1 or L2), prednisone response (good for less than 1,000 blasts/μL after 7 days of steroid therapy)27 could be investigated only in the 816 cases (98.6%) for whom information was available. Cytogenetics and DNA index were excluded from analysis because they have become mandatory since 1995.

Difference in the distribution of single or multiple MyAg+coexpression among immunophenotypic subgroups was tested by χ2 test for heterogeneity.35 Event-free survival (EFS) was estimated by the Kaplan-Meyer method.36Time on study or time to terminal event was calculated from the day of diagnosis. Failure to achieve remission, relapse, or death from any cause were considered events, whichever occurred first. The follow-up was updated on May 31, 1996. The log-rank test37 was adopted to assess differences in univariate analysis. The Cox model was used to draw a multivariate regression.38 

RESULTS

Clinical features.

The present study included 908 patients (56.3%) for whom a complete set of clinical and immunophenotypic information was available. Their presenting features are summarized in Table1. They were compared with the remaining 704 eligible patients for whom significant differences in presenting features were not found by Fisher and χ2 tests. However, cases in study had high white blood cell (WBC) counts more often than other patients (22% v19%) P = .05.

Table 1.

Characteristics of 908 Patients From Studies AIEOP 88 and 91 and Coexpression of MyAg

Patient Characteristics  All Cases  MyAg+ALL MyAgALL  
No.  %  No.  No.  %  
Total  908  100  291  32  617  68 
Sex  
 Males  517  57  174  60  343  56 
 Females  391  43  117  40  274  44  
Age 
 <1 yr  23  3  7  2  16  2  
 1-9 yrs 726  80  238  82  488  79  
 >10 yrs  159 17  46  16  113  18  
Hepatomegaly-152 216 24  86  30  130  21  
Splenomegaly-152 253 28  81  28  172  28  
CNS disease  35  4  2  29  5  
Hemoglobin >8 g/dL  464  51  152 52  312  51  
WBC >50,000/μL  203  22  60 21  143  23  
Platelets <50,000/μL  403  44 123  42  280  45  
FAB L2  201  22  69  24 132  22  
Risk groups  
 Standard or intermediate 630  69  227  78* 403  65* 
 High 278  31  64  22  214  35  
Prednisone good response  732/816  90  242/260  93-151 490/556  88-151 
CR after induction therapy  864  95  279  96  585  95 
Patient Characteristics  All Cases  MyAg+ALL MyAgALL  
No.  %  No.  No.  %  
Total  908  100  291  32  617  68 
Sex  
 Males  517  57  174  60  343  56 
 Females  391  43  117  40  274  44  
Age 
 <1 yr  23  3  7  2  16  2  
 1-9 yrs 726  80  238  82  488  79  
 >10 yrs  159 17  46  16  113  18  
Hepatomegaly-152 216 24  86  30  130  21  
Splenomegaly-152 253 28  81  28  172  28  
CNS disease  35  4  2  29  5  
Hemoglobin >8 g/dL  464  51  152 52  312  51  
WBC >50,000/μL  203  22  60 21  143  23  
Platelets <50,000/μL  403  44 123  42  280  45  
FAB L2  201  22  69  24 132  22  
Risk groups  
 Standard or intermediate 630  69  227  78* 403  65* 
 High 278  31  64  22  214  35  
Prednisone good response  732/816  90  242/260  93-151 490/556  88-151 
CR after induction therapy  864  95  279  96  585  95 

*P = .001.

F0-151

P = .03.

F0-152

Below umbilical line.

Immunophenotype.

A total of 572 cases (63%) were diagnosed as common ALL, 185 (20.7%) were pre-B ALL, 29 (3.2%) were pre-pre–B ALL, and 122 (13.5%) were T-ALL. In 291 cases (32%), one or more myeloid antigens (MyAg+ALL) were coexpressed; most of them (n = 256) were of B lineage and 35 were of T lineage; their frequency was similar in the two groups (32.5% and 28.7%, respectively, P = .3). Frequency among the three B-lineage subtypes was also similar; in particular, the pattern of myeloid positivity in the immature pre-pre–B type (38%) was comparable to that of C-ALL (31%) and pre-B ALL (36.2%) (Table 2). T-ALL expressing MyAg were present in all stages of cell maturation; intermediate stage T-ALL was more frequently associated with expression of MyAg (19 MyAg+ of the 43 intermediate T-ALL; 7 MyAg+ of the 24 early T-ALL; 9 MyAg+ of the 55 mature T-ALL;P = .005). Ninety-four cases (10.3%) were “hybrid” according to the above-mentioned definition26; 84 were B-lineage and 10 were T-ALL, without any difference in incidence either between these two lineages (10.6% B-ALL v 8% T-ALL) or for common-ALL versus pre-B ALL.

Table 2.

Distribution of Immunophenotype Among 908 Cases of Childhood ALL Studied for MyAg Coexpression and Reactivity to Each MyAg

 No. of Cases MyAg+ALL  H-ALL CD15  CD65  CD33 CD13  CD14  CD11b 
No.  (%)  No.  (%)  No.  (%)  No.  (%) No.  (%)  No.  (%)  No.  (%)  No.  (%) 
Pre-pre–B ALL  29  11  (38)  8  (27.6)  (20.6)  6  (20.6)  3  (10.3)  2  (6.8)  (6.8)  3  (10.3)  
Common ALL  572  178 (31)  50  (8.7)  27  (4.7)  34  (5.9) 84  (14.6)  88  (15.3)  31  (5.4)  37  (6.4) 
Pre-B–ALL  185  67  (36.2)  26  (14)  20 (10.8)  23  (12.4)  31  (16.7)  37  (20.0)  11 (5.9)  12  (6.4)  
T-ALL  122  35  (28.6)  10 (8)  8  (6.5)  13  (10.6)  12  (9.8) 15  (12.2)  5  (4.0)  12  (9.8)  
Total  908 291  (32)  94  (10.3)  61  (6.7)  76 (8.3)  130  (14.3)  142  (15.6)  49  (5.3)  64 (7.0) 
 No. of Cases MyAg+ALL  H-ALL CD15  CD65  CD33 CD13  CD14  CD11b 
No.  (%)  No.  (%)  No.  (%)  No.  (%) No.  (%)  No.  (%)  No.  (%)  No.  (%) 
Pre-pre–B ALL  29  11  (38)  8  (27.6)  (20.6)  6  (20.6)  3  (10.3)  2  (6.8)  (6.8)  3  (10.3)  
Common ALL  572  178 (31)  50  (8.7)  27  (4.7)  34  (5.9) 84  (14.6)  88  (15.3)  31  (5.4)  37  (6.4) 
Pre-B–ALL  185  67  (36.2)  26  (14)  20 (10.8)  23  (12.4)  31  (16.7)  37  (20.0)  11 (5.9)  12  (6.4)  
T-ALL  122  35  (28.6)  10 (8)  8  (6.5)  13  (10.6)  12  (9.8) 15  (12.2)  5  (4.0)  12  (9.8)  
Total  908 291  (32)  94  (10.3)  61  (6.7)  76 (8.3)  130  (14.3)  142  (15.6)  49  (5.3)  64 (7.0) 

The distribution of positivity to each MyAg in the four different immunological subtypes is also shown in Table 2. An example of a CD13, CD33, CD15+common ALL is shown in Fig 1. Of the analyzed MyAg, CD13 and CD33 were most frequently found: 142 cases were CD13+ and 130 were CD33+. They were mostly found in B precursor ALL positive to CD10 (common and pre-B) and in T-ALL (P, not significant). Eleven pre-pre–B ALL expressed MyAg; CD15 and CD65w were the most common ones (6 positive cases each), being the association of pre-pre–B phenotype with both antigens statistically significant (P = .003 and P = .008, respectively). Seven of 23 infant patients were MyAg+ALL: all markers were equally present twice in these cases. Among 291 MyAg+ALL, 158 cases (54.2%) showed positivity to one single MyAg. CD13 and CD33 were the most frequent (53 and 35 times, respectively). However, 44 ALL were only positive either to CD11b, CD14, or CD15. In 75 cases, two MyAg were positive and in 58 cases, three or more MyAg were present. The most common association was CD13+, CD33+ (28 cases). Three cases were positive to all six MyAg: their phenotypes were the following: one pre-B, one pre-pre–B, one T-ALL. Three more cases were positive only to “minor” MyAg (CD11b, CD14, and CD15) in various combinations.

Fig. 1.

Fluorescence-activated cell sorting (FACS) multigraph images of double-fluorescence analysis in a case of MYA+ALL. The patient had a B-lineage ALL with common phenotype. Blast cells showed positivity to CD19, CD11b, CD65w, and CD15 and negativity to CD13 and CD33. Fluorochrome used are FITC (for CD19) and PE (for MyAg).

Fig. 1.

Fluorescence-activated cell sorting (FACS) multigraph images of double-fluorescence analysis in a case of MYA+ALL. The patient had a B-lineage ALL with common phenotype. Blast cells showed positivity to CD19, CD11b, CD65w, and CD15 and negativity to CD13 and CD33. Fluorochrome used are FITC (for CD19) and PE (for MyAg).

Association with presenting features.

There were no significant associations between MyAg+ALL and the clinical presenting features; in particular, the distribution of age, initial WBC counts, T or B phenotype, and CD10 reactivity was more similar than in all other ALL. However, MyAg+ALL were more frequently assigned to standard or intermediate risk protocols (78%) than MyAg cases (65%; P = .001). They were also frequently prednisone responders (93% v 88%; P = .03), although this did not result in different rate of achievement of complete remission (Table 1). When hybrid cases26 were considered separately, these patterns of results did not change.

Response to treatment.

Six-year EFS of the cohort of studied patients was 66.4% (standard deviation [SD], 1.6) with median follow-up of 48 months; this was not significantly different from EFS of the 704 nonstudied patients (71.2%; SD, 1.9) (P = .23). The only pretreatment factors found to correlate favorably with EFS, also by multivariate analysis, were: age (1 to 9 years) (P = .0001), good prednisone response (P = .0002), and lower WBC count (P = .0033).

Both univariate and multivariate analyses suggest that the EFS of childhood ALL is not affected by MyAg coexpression. The 6-year EFS for MyAg+ALL was 69.0% (SD, 3.6). This was not significantly different from that of the 617 patients with MyAgALL who had a 6-year EFS of 65.3% (SD, 2.3) (P = .47) (Fig 2). Cases of hybrid ALL by BFM criteria did not have a poorer outcome either: their EFS (78.6%, SD, 4.4) was not different from that of nonhybrid cases (61.8%; SD, 3.1) (P = .15). The only exception was represented by the infant group: in fact, the 23 children younger than 12 months of age had a significantly worse outcome if they were MyAg+ (EFS, 0%) than if they were MyAg (EFS, 43.7%; SD, 12.4) (P = .01). In the other age groups, MyAg coexpression was not associated with any difference in EFS: 69.5% (SD, 2.5) for MyAg ALL versus 73.9% (SD, 3.7) for MyAg+ALL in children aged 1 to 9 years; 50.6% (SD, 5.3) for MyAgALL versus 54.9% (SD, 9.8) for MyAg+ALL in children older than 9 years. This pattern was not modified when hybrid cases were considered separately. Moreover, none of the six analyzed myeloid markers was independently associated with a different outcome.

Fig. 2.

Comparison of EFS in MyAg+ALL and MyAgALL. EFS in MyAg+ALL is not significantly different than in MyAgALL. Follow-up time is 72 months and median follow-up is 48 months. ev, events; MYA, My Ag.

Fig. 2.

Comparison of EFS in MyAg+ALL and MyAgALL. EFS in MyAg+ALL is not significantly different than in MyAgALL. Follow-up time is 72 months and median follow-up is 48 months. ev, events; MYA, My Ag.

MyAg coexpression was not associated with a different outcome within the different immunophenotypic subgroups. Among the 122 patients with T-lineage ALL, the 35 MyAg+ cases had a 6-year EFS of 46.7% (SD, 8.7), which was comparable to that of the 87 patients with MyAg ALL (EFS 47.1%; SD, 5.5). The 256 MyAg+ cases of B-lineage ALL had a EFS of 72.1% (SD, 3.9), which was comparable to that of the 530 patients with MyAgALL (EFS, 68.1%; SD, 2.5).

All other evaluated parameters, in particular, WBC count, organomegaly, FAB morphology, risk-directed therapy, response to steroids, CD10 expression in B-lineage ALL, B-lineage subgroups, could not demonstrate any statistically significant difference for EFS in MyAg+versus MyAgALLs.

DISCUSSION

MyAg coexpression is not a rare event in childhood ALL. In our experience, it occurs in about one third of all cases. This frequency is higher than in previous studies, which reported a range between 4% and 22%3,16-24 (Table 3). However, it is comparable to the 10% to 54% range reported for adult patients.10-15 The sensitivity of the most recent methods used in our study could possibly explain the different percentages reported by most investigators. In a recent report, Uckun et al25 found a 16.6% incidence of MyAg coexpression among 1,557 patients enrolled in the Childrens Cancer Group (CCG) protocols. Such apparently lower incidence may be explained by their choice to include only two MyAg, CD13 and CD33, in the panel of the MoAbs used for central investigation of immmunophenotype. The investigators suggest that the reported incidence of CD13 and CD33 coexpression in childhood ALL should be representative of overall MyAg expression. Although we may confirm that CD13 and CD33, alone or in combination, are the most frequently observed MyAg, in our series they might account for about one half of all cases coexpressing at least one MyAg. Actually, in our study, four more antigens were evaluated to analyze a wider panel of myeloid antigenic molecules.19,39 Using the “minor” ones (CD11b, CD14, and CD15), 47 additional cases (16%) were identified. False positive results may be easily ruled out in our setting, based on our study design: immunonophenotyping was performed centrally by flow cytometry, using highly specific MoAbs for all cases; thus even weak antigen expression might be detected. It is to be noted that limit for positivity in single fluorescence analysis was 20%, while it was 30% in the study by Uckun et al.25 Double fluorescence analysis was always performed to confirm all cases, including those that showed low percentages of positive cells as having coexpressing blasts. This should avoid the main difficulties in interpreting data on MyAg+ALL. In fact, results could be altered by using the overlapping of percentages to identify coexpression, because of the presence of residual normal myeloid cells in the samples.19,25 In our series, cases coexpressing CD13 and/or CD33 on more than 30% of blasts were 152 (16% of all cases), the same percentage found by Uckun et al.25 

Table 3.

A Comparative Review of Studies on Clinical Significance of MyAg Coexpression in ALL

Author, Year  No. of Cases  MyAg+ALL No. (%) MyAg Used  % IFI  Confirmation of Coexpression Correlation With EFS  
Pui et al, 1990  372  61 (6.4) CD11b, 13, 14 CD15, 33, 36  25  DF No (FU, 36 mo)  
Wiersma et al, 1991  236  52 (22) CD13, 33, 14  30  OVL DF  Yes (FU, 40 mo) 
Borowitz et al, 1991  1141  (7) (15)  CD13, 33 CD13, 33  30 20  30% OVL ND  No No  
Pui et al, 1991  410  25 (6.1) CD11b, 13, 14 CD15, 33, 36  20 2 MyAg+  ND  
Fink et al, 1993  206  24 (11) CD13, 33, 65, 15 14, 11b, GA, 41   OVL Yes (median FU, 44 mo)  
Ludwig et al, 1994 736  50 (7)  CD13, 33, 65  20  DF  ND  
Reiter et al, 1994  975  51 (5)  CD13, 33, 65  20  20% OVL No  
Uckun et al, 1997  1557  260 (16.6)  CD13, 33 30   No (FU, 73 mo) 
Author, Year  No. of Cases  MyAg+ALL No. (%) MyAg Used  % IFI  Confirmation of Coexpression Correlation With EFS  
Pui et al, 1990  372  61 (6.4) CD11b, 13, 14 CD15, 33, 36  25  DF No (FU, 36 mo)  
Wiersma et al, 1991  236  52 (22) CD13, 33, 14  30  OVL DF  Yes (FU, 40 mo) 
Borowitz et al, 1991  1141  (7) (15)  CD13, 33 CD13, 33  30 20  30% OVL ND  No No  
Pui et al, 1991  410  25 (6.1) CD11b, 13, 14 CD15, 33, 36  20 2 MyAg+  ND  
Fink et al, 1993  206  24 (11) CD13, 33, 65, 15 14, 11b, GA, 41   OVL Yes (median FU, 44 mo)  
Ludwig et al, 1994 736  50 (7)  CD13, 33, 65  20  DF  ND  
Reiter et al, 1994  975  51 (5)  CD13, 33, 65  20  20% OVL No  
Uckun et al, 1997  1557  260 (16.6)  CD13, 33 30   No (FU, 73 mo) 

Only series analyzed by flow cytometry have been shown.

Abbreviations: % IFI, percentage of cells positive by single fluorescence analysis; DF, double fluorescence staining; OVL, overlapping of percentages of positive cells; FU, follow-up; mo, months; ND, not done; GA, glycophorin A.

The frequency of myeloid coexpression was comparable in T- and in B-ALL, as well as in all subgroups of B-lineage ALL. In particular, CD10, B-lineage ALL (pre-pre–B ALL) had the same incidence of MyAg coexpression than other more mature types, while Ludwig et al16 suggested a particularly high frequency (50%). Moreover, CD13 and CD33 were not typical of this group, while the most frequent MyAg was CD15, in agreement with previous reports.22,40 The presence of CD15 in infant ALL has already been found in a larger group of our patients,32even if it could not be confirmed in this limited subset of infants. The correlation of MyAg coexpression, the peculiar chromosomal translocation t(4;11), and poor prognosis in infant and pre-pre–B ALL has often been suggested.41 T-ALL of intermediate maturational stage showed a particularly high number of MyAg+ cases. This difference with previous observations19,22 may be due to the small numbers of T-ALL MyAg+ cases analyzed therein.

Coexpression of MyAg+ALL was not associated with any of the presenting features, in particular previous reports34prompted us to explore any association with those presenting features known to bear an adverse prognostic value, such as infant or older age and higher leukocyte count. Interestingly, poor steroid response on day 7, indeed a powerful independent adverse prognostic factor in the BFM-chemotherapy setting,3,42 was not associated with MyAg coexpression. On the contrary, MyAg+ cases were more frequently good steroid responders. Accordingly, they were more frequently eligible for the non-high (standard and intermediate) risk groups. This also agrees with a recent report from our group, describing a higher incidence of MyAg coexpression in the ALL subset identified by the prospective molecular screening of the t(12; 21) translocation,43 which is associated with favorable prognosis.44,45 Thus, our data confirm that MyAg coexpression is usually associated with a constellation of favorable presenting features. For this reason, the MyAg+ cases have a nonrandom distribution toward the lower risk groups. Finally, myeloid antigen coexpression is not associated with a different outcome in childhood ALL. Because only infant patients with MyAg+ALL have shown a worse prognosis, their presence could affect statistical analysis in small series. This conclusion on our large series of unselected, newly diagnosed childhood ALL cases is in agreement with the recent report from the CCG25 for patients who are treated with a BFM-based chemotherapy.

In conclusion, our large, prospective study of coexpression of six different MyAg in childhood ALL confirms it is not associated either with immunophenotype, response to therapy, or long-term outcome and thus has no prognostic value, as far as aggressive treatment is used.46,47 Therefore, it should not be used for any therapeutic choice, including patient stratification or indication for bone marrow transplantation, with the exception of infant leukemia. Nevertheless, continuous use of a wide panel, including many different MyAg (up to six in our experience), is justified by the possibility of identifying cases of undifferentiated leukemia and FAB type Mo AML48 and also to define in the majority of cases a leukemia-associated immunophenotype for individual patients.48-51 This information, jointly with molecular studies, might be highly valuable when drawing prospective studies of minimal residual disease aimed to tailor treatment.

We also suggest that the term “hybrid” leukemia for MyAg+ALL is not supported by any clinical evidence of heterogeneity. On the contrary, it could induce incorrect impressions of “myeloid” or “atypical” characteristics and might be misleading.

ACKNOWLEDGMENT

The authors thank all investigators from AIEOP Institutions: L. Felici (Cl. Pediatrica, Ancona); N. Santoro (Cl. Pediatrica I, Bari); T. Santostasi (Cl. Pediatrica II, Bari); P. Cornelli (O. Riuniti, Bergamo); A. Pession (Cl. Pediatrica III, Bologna); A. Arrighini (Cl. Pediatrica, Brescia); G. M. Fiori (Div. Oncoematologia Pediatrica, Cagliari); R. Galanello (Cl. Pediatrica II; Cagliari); A. Sciotto (Div. Oncoematologia Pediatrica, Catania); S. Magro (Div. Ematologia, Catanzaro); A. Lippi (O. Meyer, Firenze); M. Cominetti (O. Galliera, Genova); C. Rosanda (O. Gaslini, Genova); F. Massolo, (Cl. Pediatrica II, Modena); A. Murano (Cl. Pediatrica I, Napoli); M.F. Pinta (O. Pausillipon, Napoli); S. Auricchio, (Cl. Pediatrica II, Napoli); A. Correra (O. SS. Annunziata, Napoli); L. Zanesco (Cl. Pediatrica II, Padova), G. Fugardi (Cl. Pediatrica I, Palermo); G. Izzi (Div. Ematologia Pediatrica, Parma); M. Aricò (Cl. Pediatrica, Pavia); A. Amici (Cl. Pediatrica, Perugia); R. Di Lorenzo (Div. Ematologia, Pescara); C. Favre (Cl. Pediatrica III, Pisa); F. Nobile (Div. Ematologia, Reggio Calabria); A.M. Testi (Cattedra di Ematologia, La Sapienza, Roma); G. De Rossi (O. Bambino Gesù, Roma); B. Werner (Cl. Pediatrica, La Sapienza, Roma); D. Gallisai (Cl. Pediatrica, Sassari); C. D'Ambrosio (Cl. Pediatrica, Siena); E. Barisone (Cl. Pediatrica, Torino); P. Tamaro (Cl. Pediatrica, Trieste); L. Nespoli (Cl. Pediatrica, Varese); G. Marradi (Cl. Pediatrica, Verona). We also wish to thank Dr Lucia Masiero for statistical assistance and Dr M. Spinelli, G. Giacometti and B. Michielotto for technical work.

Supported by AIRC (Associazione Italiana per la Ricerca sul Cancro); by AIL (Associazione Italiana Leucemie), Progetto “30 Ore per la Vita”.

Presented in part at the XXIX Congress of SIOP, held in Instanbul, September 23-27, 1997.

Address reprint requests to Maria Caterina Putti, MD, Dipartimento di Pediatria, Via Giustiniani, 3, 35128 Padova, Italy.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.

REFERENCES

REFERENCES
1
Pui
C-H
Behm
FG
Crist
WM
Clinical and biologic relevance of immunologic marker studies in childhood acute lymphoblastic leukemia.
Blood
82
1993
343
2
Pui
C-H
Crist
WM
Biology and treatment of acute lymphoblastic leukemia.
J Pediatr
124
1994
491
3
Reiter
A
Schrappe
M
Ludwig
W-D
Hiddeman
W
Sauter
S
Henze
G
Zimmermann
M
Lampert
F
Havers
W
Niethammer
D
Odenwald
E
Ritter
J
Mann
G
Welte
K
Gadner
H
Riehm
H
Chemotherapy in 988 unselected childhood acute lymphoblastic leukemia patients. Results and conclusions of the multicenter trial ALL-BFM86.
Blood
84
1994
3122
4
Schorin
MA
Blattner
S
Gelber
RD
Tarbell
NJ
Donnelly
M
Dalton
V
Cohen
HJ
Sallan
SE
Treatment of childhood acute lymphoblastic leukemia: Results of Dana-Farber Cancer Institute — Children Hospital Acute Lymphoblastic Leukemia Consortium Protocol 85-01.
J Clin Oncol
12
1994
740
5
Raimondi
SC
Behm
FG
Roberson
PK
Williams
DL
Pui
C-H
Crist
WM
Look
AT
Rivera
GK
Cytogenetics of pre-B acute lymphoblastic leukemia with emphasis on prognostic implications of the t(1;19).
J Clin Oncol
8
1990
1380
6
Greaves
MF
Chan
LC
Furley
AJW
Watt
SM
Molgaard
HV
Lineage promiscuity in hemopoietic differentiation and leukemia.
Blood
67
1986
1
7
Pui
C-H
Frankel
LS
Carroll
AJ
Raimondi
SC
Shuster
JJ
Head
DR
Crist
WM
Land
VJ
Pullen
DJ
Steuber
CP
Behm
FG
Borowitz
MJ
Clinical characteristics and treatment outcome of childhood acute lymphoblastic leukemia with the t(4;11)(q21;q23): A collaborative study of 40 cases.
Blood
77
1991
440
8
Pui
C-H
Crist
WM
Look
AT
Biology and clinical significance of cytogenetic abnormalities in childhood acute lymphoblastic leukemia.
Blood
76
1990
1449
9
Crist
WM
Carrol
A
Shuster
J
Jackson
J
Head
D
Borowitz
M
Behm
F
Link
M
Steuber
P
Ragab
A
Hirt
A
Brock
B
Land
V
Pullen
J
Philadelphia chromosome positive childhood acute lymphoblastic leukemia: Clinical and cytogenetic characteristics and treatment outcome. A Pediatric Oncology Group study.
Blood
76
1990
489
10
Drexler
HG
Thiel
E
Ludwig
W-D
Review of the incidence and clinical importance of myeloid antigen-positive acute lymphoblastic leukemia.
Leukemia
5
1991
637
11
Sobol
RE
Mick
R
Royston
I
Davey
FR
Ellison
RR
Newman
R
Cuttner
J
Griffin
JD
Collins
H
Nelson
DA
Bloomfield
CD
Clinical importance of myeloid antigen expression in adult acute lymphoblastic leukemia.
N Engl J Med
316
1987
1111
12
Childs
CC
Hirsch-Ginsberg
C
Walters
RS
Andersson
BS
Reuben
J
Trujillo
JM
Cork
A
Stass
SA
Freireich
EJ
Zipf
TF
Myeloid surface antigen positive acute lymphoblastic leukemia: Immunophenotypic, ultrastructural, cytogenetic and molecular characteristics.
Leukemia
3
1989
777
13
Guyotat
D
Campos
L
Shi
Z-H
Charrin
C
Treille
D
Magaud
JP
Fiere
D
Myeloid surface antigen expression in adult acute lymphoblastic leukemia.
Leukemia
4
1990
664
14
Lauria
F
Raspadori
D
Martinelli
G
Rondelli
D
Ventura
MA
Farabegoli
F
Tosi
P
Testoni
N
Visani
G
Zaccaria
A
Increased expression of myeloid antigen markers in adult acute lymphoblastic leukemia patients: Diagnostic and prognostic implications.
Br J Haematol
87
1994
286
15
Boucheix
C
David
B
Sebban
C
Racadot
E
Bené
MC
Bernard AM Campos
L
Jouault
H
Sigaux
F
Lepage
E
Hervé
P
Fière
D
Immunophenotype of adult acute lymphoblastic leukemia, clinical parameters and outcome: An analysis of a prospective trial including 562 tested patients (LALA87).
Blood
84
1994
1603
16
Ludwig
W-D
Reiter
A
Loeffler
H
Goekbuget
Hoelzer
D
Riehm
H
Thiel
E
Immunophenotypic features of childhood and adult acute lymphoblastic leukemia: Experience of the German multicenter trials ALL-BFM and GMALL.
Leuk Lymphoma
13
1994
71
17
Urbano-Ispizua
A
Matutes
E
Vilamor
N
Ribera
JM
Feliu
E
Montserrat
E
Granena
A
Vives-Corrons
JL
Rozman
C
Clinical significance of the presence of myeloid associated antigens in acute lymphoblastic leukemia.
Br J Haematol
75
1990
202
18
Pui
C-H
Raimondi
SC
Head
DR
Schell
MJ
Rivera
GK
Mirro
J
Crist
WM
Behm
FG
Characterization of childhood acute leukemia with multiple myeloid and lymphoid markers at diagnosis and at relapse.
Blood
78
1991
1327
19
Wiersma
SD
Ortega
J
Sobel
E
Wienberg
KI
Clinical importance of myeloid antigen expression in acute lymphoblastic leukemia of childhood.
N Engl J Med
324
1991
800
20
(correspondence)
Borowitz
MJ
Shuster
JJ
Land
VJ
Steuber
CP
Vitti
TJ
Myeloid antigen coexpression in childhood acute lymphoblastic leukemia.
N Engl J Med
325
1991
1379
21
Pui C-H, Schell MJ, Raimondi SC, Head DR, Rivera GK, Crist WM, Behm FG: Myeloid antigen expression in childhood acute lymphoblastic leukemia N Engl J Med 325:1378, 1991 (correspondence)
22
Cantù-Rajnoldi
A
Putti
MC
Granchi
D
Foà
R
Shirò
R
Castagni
M
Valeggio
C
Jankovich
M
Miniero
R
Paolucci
P
Basso
G
Co-expression of myeloid antigens in childhood acute lymphoblastic leukemia: Relationship with the stage of differentiation and clinical significance.
Br J Hematol
79
1991
40
23
Kurec
AS
Belair
P
Stefanu
C
Barret
DM
Dubowy
RL
Davey
FR
Significance of aberrant immunophenotypes in childhood acute lymphoid leukemia.
Cancer
67
1991
3081
24
Fink
M-M
Koeller
U
Mayer
H
Haas
OA
Gruemayer-Panzer
R
Urban
C
Dengg
K
Mutz
I
Tuechler
H
Gattere-Menz
I
Knapp
W
Gadner
H
Prognostic significance of myeloid associated antigen expression on blast cells in children with acute lymphoblastic leukemia.
Med Pediatr Oncol
21
1993
340
25
Uckun
FM
Sather
HN
Gaynon
PS
Arthur
DC
Trigg
ME
Tubergen
DG
Nachman
J
Steinherz
PG
Sensel
MG
Reaman
GH
Clinical features and treatment outcome of children with myeloid antigen positive acute lymphoblastic leukemia: A report from the Children's Cancer Group.
Blood
90
1997
28
26
Van der Does-van der Berg
A
Bartram
C
Basso
G
Benoit
YC
Biondi
A
Debatin
KM
Haas
O
Harbott
J
Kamps
WA
Koeller
U
Lampert
F
Ludwig
W-D
Niemayer
C
van Wering
ER
Minimal requirements for diagnosis, classification and evaluation of the treatment of childhood acute lymphoblastic leukemia (ALL) in the “BFM” Family Cooperative Group.
Med Pediatr Oncol
20
1992
497
27
Riehm
H
Reiter
A
Schrappe
M
Berthold
F
Dopfer
R
Gerein
V
Ludwig
R
Ritter
J
Stollmann
B
Henze
G
Corticosteroid-dependent reduction of leukocyte count in blood as a prognostic factor in acute lymphoblastic leukemia in childhood (therapy study ALL-BFM 83).
Klin Padiatr
199
1987
151
28
Conter
V
Arico'
M
Valsecchi
MG
Rizzari
C
Testi
AM
Messina
C
Mori
PG
Miniero
R
Colella
R
Basso
G
Rondelli
R
Pession
A
Masera
G
Extended intrathecal methotrexate may replace cranial irradiation for prevention of CNS relapse in children with intermediate-risk lymphoblastic leukemia treated with Berlin-Frankfurt-Munster based invasive chemotherapy. The Associazione Italiana di Ematologia e Oncologia Pediatrica.
J Clin Oncol
13
1995
2497
29
Conter
V
Schrappe
M
Arico'
M
Reiter
A
Rizzari
C
Doerdelmann
M
Valsecchi
MG
Zimmermann
M
Ludwig
D-W
Basso
G
Masera
G
Riehm
H
Role of cranial radiotherapy for childhood T-cell acute lymphoblastic leukemia with high WBC count and good response to prednisone.
J Clin Oncol
15
1997
2786
30
Schrappe
M
Reiter
A
Sauter
S
Ludwig
WD
Harbott
J
Bender-Gotze
C
Dorffel
Dopfer
R
Frey
E
Concept and interim results of the ALL-BFM 90 therapy study in treatment of acute lymphoblastic leukemia in children and adolescents: The significance of initial therapy response in blood and bone marrow.
Klin Padiatr
206
1994
208
31
Bennet
JM
Catovsky
D
Daniel
MT
Flandrin
G
Galton
DAG
Gralnik
HR
Sultan
C
The FAB Cooperative Group. The morphological classification of acute lymphoblastic leukemia: Concordance among observers and clinical correlation.
Br J Haematol
47
1981
553
32
Basso
G
Putti
MC
Cantù-Rajnoldi
A
Saitta
M
Santostasi
T
Santoro
N
Lippi
A
Comelli
A
Felici
L
Favre
C
Russo Mancuso
G
Guglielmi
C
Paolucci
P
Biondi
A
Rondelli
R
Pession
A
The immunophenotype in infant acute lymphoblastic leukemia: Correlation with clinical outcome. An Italian multicenter study (AIEOP).
Br J Haematol
81
1981
184
33
Gale
RP
Ben Bassat
I
Hybrid acute leukemia.
Br J Haematol
65
1987
261
34
Smith
M
Arthur
D
Camitta
B
Crist
W
Gaynon
P
Gelber
R
Heerema
N
Korn
EL
Link
M
Sharon
M
Pui
C-H
Pullen
J
Reaman
G
Sallan
SE
Sather
H
Shuster
R
Simon
R
Trigg
M
Tuberegn
D
Uckun
F
Ungerleider
R
Uniform approach to risk classification and treatment assignment for children with acute lymphoblastic leukemia.
J Clin Oncol
14
1996
18
35
Pearson ES, Hartley HO: Biometrika Tables for Statisticians (vol 1, ed 3). New York, NY, Cambridge, 1966
36
Kaplan
EL
Meier
P
Nonparametric estimation from incomplete observations.
J Am Stat Assoc
53
1958
457
37
Mantel
N
Haenszel
W
Statistical aspects of the analysis of data from retrospective studies of disease.
J Natl Cancer Inst
22
1959
719
38
Cox
DR
Regression models and life-tables.
J R Stat Soc B
34
1972
187
39
Basso
G
Rondelli
R
Putti
MC
Cantu'Rajnoldi
A
Granchi
D
Cocito
MG
Saitta
M
Santostasi
T
Guglielmi
C
Lippi
A
Santoro
N
Pession
A
Arico'
M
Biondi
A
Incidence and prognostic significance of immunophenotypic subgroups in childhood acute lymphoblastic leukemia: The experience of the AIEOP Cooperative study.
Recent Results Cancer Res
131
1993
298
40
Lenkei
R
Bjork
O
Oest
A
Biberfield
P
Expression of CD15 as a predictor of relapse in children wih acute lymphoblastic leukemia of the pre-B type.
Leuk Res
15
1991
189
41
Ludwig
WD
Bartram
CR
Harbott
J
Koeller
U
Haas
OA
Hansen-Hagge
T
Heil
G
Seibt-Jung
H
Teichmann
JV
Ritter
J
Knapp
W
Gadner
H
Theil
E
Riehm
H
Phenotypic and genotypic heterogeneity in infant acute leukemia I. Acute lymphoblastic leukemia.
Leukemia
3
1989
431
42
Cimino
G
Lo Coco
F
Biondi
A
Elia
L
Luciano
A
Croce
CM
Masera
G
Mandelli
F
Canaani
E
ALL-1 gene at chromosome 11q23 is consistently altered in acute lymphoblastic leukemia of early infancy.
Blood
82
1993
544
43
Arico'
M
Basso
G
Mandelli
F
Rizzari
C
Colella
R
Barisone
E
Zanesco
L
Rondelli
R
Pession
A
Masera
G
Good steroid response in vivo predicts a favourable outcome in children with T-cell acute lymphoblastic leukemia. The Associazione Italiana Ematologia Oncologia Pediatrica.
Cancer
75
1995
1684
44
Borkhardt
A
Cazzaniga
G
Viehmann
S
Valsecchi
MG
Ludwig
W-D
Mangioni
S
Burci
L
Schrappe
M
Rierhm
H
Lampert
F
Basso
G
Masera
G
Harbott
J
Biondi
A
Incidence and clinical relevance of TEL/AML1 fusion genes in children with acute lymphoblastic leukemia enrolled in the German and Italian multicenter therapy trials.
Blood
90
1997
571
45
McLean
TW
Ringold
S
Neuberg
D
Stegmaier
K
Tantravahi
R
Ritz
J
Koeller
HP
Takeuchi
S
Janssen
JWG
Seriu
T
Bartram
CR
Sallan
SE
Gillland
DG
Golub
TR
TEL/AML1 dimerizes and is associated with a favorable outcome in childhood acute lymphoblastic leukemia.
Blood
88
1996
4252
46
Schurtleff
SA
Buijs
A
Behm
FG
Rubnitz
JE
Raimondi
SC
Hancock
ML
Chan
GC
Pui
C-H
Grosveld
G
Downing
JR
TEL/AML1 fusion resulting from criptic t(12;21) is the most common genetic lesion in pediatric ALL and defines a subgroup of patients with an excellent prognosis.
Leukemia
9
1995
1985
47
Pinkel
D
Selecting treatment for children with acute lymphoblastic leukemia.
J Clin Oncol
14
1996
4
48
Campana
D
Hansen-Hagge
TE
Matutes
E
Coustan-Smith
E
Yokota
S
Shetty
V
Bartram
C
Janossy
G
Phenotypic, genotypic, cytochemical and ultrastructural characterization of acute undifferentiated leukemia.
Leukemia
4
1990
620
49
Campana
D
Freitas
RO
Coustan-Smith
E
Detection of residual leukemia with immunologic methods: Technical developments and clinical implications.
Leuk Lymphoma
13
1994
31
50
Orfao
A
Ciudad
J
Lopez-Berges
MC
Vidriales
B
Caballero
MD
Valverde
B
Gonzales
M
San Miguel
JF
Acute lymphoblastic leukemia: Detection of minimal residual disease at flow cytometry.
Leuk Lymphoma
13
1994
87
51
Van Dongen
JJM
Szczepansky
T
de Bruijn
MAC
van den Beemd
MWM
de Bruin-Versteeg
S
Wijkhuijs
JM
Tibbe
GJM
van Gastel-Mol
EJ
Groeneveld
K
Hooijkaas
H
Detection of minimal residual disease in acute leukemia patients.
Cytokines Mol Ther
2
1996
121