HEMATOLOGICAL ONCOLOGY, VOL. 9 , 3 3 4 2 (1991)

THE VALUE OF THE IMMUNOLOGICAL SUBTYPES AND INDIVIDUAL MARKERS COMPARED TO

CLASSICAL PARAMETERS IN THE PROGNOSIS OF ACUTE LYMPHOBLASTIC LEUKEMIA

E. GOMEZ, J. F. SAN MIGUEL, M. GONZALEZ, A. ORFAO, M. c. CARIZO, J. M. MORALEDA AND A. LOPEZ BORRASCA

Servicio de Hemutologiu. Hospital Clinico, Salamanca, Spain

SUMMARY

The value of the immunophenotypical subtypes and individual markers was compared with classical parameters in the prognosis of 150 patients with acute lymphoblastic leukemia (ALL).

Regarding the immunophenotype, common-ALL had a better prognosis than T-ALL in the children’s group. However, in adults the situation was different since both null and T-ALL patients had longer survival rates than the common/pre-B group. Moreover, several individual markers add interesting prognostic infor- mation, either in ALL as a whole group or within the different immunophenotypes. Thus, the expression of CDlO and TdT had a significantly favourable influence in the outcome of the whole series of patients; within the T-ALL, those cases positive for CDlO also had a longer median survival (33 versus 17 months). In addition, in the common ALL patients group the expression of a relatively mature B marker-CDZG appeared to have a favourable prognosis (27 versus 13 months). Other non lineage specific markers, such as CD9 and CD38 did not seem to influence survival.

Regarding the more conventional parameters, our data suggest that the classical age prognostic classifi- cation in children (< 15 years) and adults can be improvcd using two cut-off points at 1 1 and 35 years. Moreover, the multivariate analysis showed that this variable, together with FAB morphology and WBC counts were the best combination of parameters for predicting survival.

The present study shows that although the immunophenotype helps us in understanding the biological heterogeneity of ALL, having also prognostic implications, there are other clinical and hematological features that yield stronger prognostic information.

KEY WORDS Immunophenotype Prognostic factors Acute lymphoblastic leukemia

INTRODUCTION

Analysis of prognostic factors in ALL has allowed the design of risk groups that facilitate the prognostic stratification of patients and the evaluation of different treatment protocols (Hoelzer er al., 1988; GTMEMA, 1989; Miller et a/., 1989; Crist et al., 1989; Wingard et a/ . , 1990; Bleyer, 1990).

Unfortunately, frequent discrepancies are observed among the prognostic factors selected in individual series. One controversial aspect is the prognostic significance of immunological pheno- types (Hoelzer et al., 1988; Crist et al., 1989; GIMEMA, 1989). This can be due to several reasons

Addressee for correspondence: E. Gbmez, Dpto. de Hernatologia, Hospital Clinico, Paseo de S. Vicente sjn 37007, Salamanca, Spain.

0278&0232/9 110 10033-10 $05.00 0 1991 by John Wiley & Sons, Ltd.

Received 5 August 1990 Revised 15 November 1990

34 E. GOMEZ ET AL.

such as: (a) The age of the patients, since prognostic factor analysis has been usually performed in series including only children or adults; series with both age groups are scanty; (b) the relationship between some prognostic factors requires the use of multivariate analyses hitherto not used on a systematic basis and (c) the treatment employed.

Moreover, it has been scarcely analysed if individual markers could add prognostic information both in ALL as a whole and within the different ALL immunological subgroups.

The aim of the present study was to explore, both with univariate and multivariate analyses, in an homogeneous series of children and adults the prognostic significance on the CR rate and survival of the different immunophenotypical subtypes and individual markers that define these phenotypes, as well as the most outstanding clinico-biological ALL characteristics. The immuno- logical markers are analysed both in the whole series and within the different immunological subgroups.

MATERIALS AND METHODS

Patients, diagnostic criteria and treatment

From 1983 to 1989 a total of 150 patients: 74 children, 76 adults, (range 2 to 79 years) diagnosed as de novo ALL according to morphological (Bennett et al., 1976) and cytochemical criteria were referred from seven hospitals to our laboratory for immunophenotyping. All patients were treated with three similar protocols including: vincristine, prednisone, L-asparaginase and daunorubicin. There were no differences either in the distribution of the clinico-biological and immunological features or in the response to treatment and survival among the three groups. The survival data were documented in all 150 patients; three of the patients refused treatment and were not evaluated in the prognostic factor analysis.

Immunological phenotype

Peripheral blood and/or bone marrow mononuclear cells isolated by Ficoll-Hypaque density gradient centrifugation were analysed for sheep erythrocyte rosette formation and by direct immunofluorescence-for surface and cytoplasmatic (p) immunoglobulins-and indirect immuno- fluorescence with a TdT antiserum and a panel of MoAb. The specificities of the MoAb employed were as follows. B cell lineage: B4 (CD19), B1 (CD20); T cell lineage: 3A1 (CD7), OKTl1 (CD2), OKT3 (CD3), OKT4 (CD4), OKT8 (CD8), OKT6 (CDl); non lineage specific markers: CALLA (CDlO), Leul/Cris 1 (CDS), FMC8 (CD9), BI-3C5 (CD34), OKTlO (CD38), OKT9 (CD71), GRT2 (CD45), GRT22 (CD45R) and myeloid markers: My7 (CD13), MY9 (CD33). The reac- tivity and specificity of these MoAb in ALL have been reported previously (Sanchez-Madrid et al., 1989; San Miguel et al., 1988). One marker was considered positive when it was expressed in more than 20 per cent of the blast cells.

Most of the patients (60 per cent) showed a common phenotype (TdT+, Ia+ , CD19+, CDlOf , CD20, CIg,SIg and T-cell Ags -); 16 per cent were null (same phenotype as common, except for the negativity of both CD 10 and CD20); 16 per cent T ALL (TdT + , Ia - , T-cell Ags +) and a minority had a pre-B, 4 per cent (same phenotype as common ALL but CD20 and CIg +) or a B,4percent (TdT-, Ia+ , CD19+, CDlOf , CD20+ CIg--, SIg+)phenotype.

In children, common ALL represented 74 per cent of the whole patients with only 6 per cent and 12 per cent of null and T-ALL, respectively. However, in adults the incidence of both null and T phenotypes was higher (26 per cent and 21 per cent, respectively), whereas the percentage of common ALL cases was lower (46 per cent).

IMMUNOPHENOTYPE AND PROGNOSIS IN ALL 35

Common and pre-B phenotypes were analysed together for prognostic factors since CIg determination was not systematically performed in all CALLA + non T ALL.

Parameters evaluated In addition to immunological markers and immunophenotype, other clinical and biological data analysed included: age, sex, infection at diagnosis, bleeding, bone tenderness, general symptoms (asthenia, anorexia, weight loss), hepatomegaly, splenomegaly, lymphadenopathy, mediastinal mass, central nervous system or other infiltrations, hemoglobin, WBC counts-including the percentage and absolute number of blasts-the number of platelets, E.S.R., morphology accord- ing to FAB criteria (Bennett et al., 1976), cytochemistry (PAS and acid phosphatase stainings) serum immunoglobulins, SGOT, SGPT, alkaline phosphatase, LDH, uric acid, calcium and phosphorus levels.

Statistical methods Univariate analysis of prognostic factors regarding complete remission (CR) rate was performed with contingency tables (Mantel, 1966) that included the following statistical tests: chi-square, Yates corrected chi-square, Fisher’s exact test and the test for trend-BMDP 4F programme- (Cox, 1986). Survival curves were plotted according to the method of Kaplan and Meier (1958) and statistically compared using the MantelLCox (Mantel, 1966) and Breslow (1970) tests-BMDP 1 L programme. Subsequently, multivariate analysis was performed to examine the simultaneous effect of the different variables, both on CR using the multiple linear logistic regression model (Cox, 1970FBMDP LR programme-and on length of survival by the stepwise proportional hazards regression model (Cox, 1972FBMDP 2L programme. Variables considered for possible inclusion in the Cox regression analysis were those for which there was some indication of a significant association in the univariate analyses (p < 0.05) or for which prior studies had suggested a possible association. Multivariate analyses were performed both with the whole series of patients and excluding the L3 cases.

RESULTS

At the time of analysis, 69 (46 per cent) of the 150 patients were alive. Complete remission was achieved in 80 per cent of the cases and half of these relapsed afterwards, BM being the most frequent site of relapse (42 per cent of the patients).

Prognostic factors of CR Table 1 shows only those factors that significantly influenced the achievement of CR in the univariate analysis.

Immunophenotype was a significant prognostic factor for CR, with a progressive decrease in the proportion of CR rates from common ALL (89 per cent) to null ALL (70 per cent), T (65 per cent) and B ALL (28 per cent), p = 0.003. After excluding the seven cases with B ALL, the differences were also significant ( P = O . O l ) , as well as between common and null ALL (p=O.O3). When immunological markers were analysed individually, only the expression of CDI 0 and the presence of TdT activity had a favourable impact on CR (p < 0-05). The absence of CD7 and CD38 and the positivity for CD9 also favoured CR but the differences did not reach a significant level (p > 0.1).

The multivariate analysis showed that the factors with significant independent predictive information were increasing age, bleeding tendency, high WBC counts and morphology (other than Ll), which adversely influenced CR in the whole series (Table 2): The next characteristic selected in the Cox model was the common/pre-B phenotype but this variable did not significantly

36 E. GOMEZ ET AL.

Table 1. Prognostic factors of CR. Univariate analysis

Ncases CR(%) P

Age (a) 2-1 5 years > I5 years

(b) 2-1 1 years 11-14 years 15-35 years > 35 years

(c) < 11 years 11-35 years > 35 years

Infection Yes No

Bleeding Yes No

WBC (lO’/l) < 100 > 100

Platelets (109/1) t 4 0 > 40

FAB L1 L2 L3

Immunophenotype Null Common/pre-B T B

CDlO+

TdT + CD7 + CD38 + CD9 +

-

-

-

-

-

69 60 53 16 39 21 53 55 21 40 60 57 51

108 21 46 71 64 44

7 20 89 23 7

81 28

108 7

16 47 19 32 52 11

88.4 68.3 92.5 75.0 71.8 61.9 93.1 72.7 61.9 65.0 83-3 66.7 90.2 82.4 61.9 67.4 83.1 85.9 68.2 28.5 70.0 88.8 65.2 28.6 87.7 67.9 85.1 28.6 75.0 89.4 57.9 78.1 82.7 63.6

0.006

0.02 *

0.003 t

0.04

0.004

0.04

0.05

0.004 1

0.004 9:

0.02

0.009

>0-1

>0.1

>0.1

*Test for trend: p=0402. ?Test for trend: p=0.001. $L1 versus L2: p=0.003. §Upon excludingB ALL:p=0.01.

improve the predictive model. When L3 cases were excluded, then morphology no longer appeared in the model.

Prognostic factors of survival Table 3 shows the parameters at diagnosis with a significant predictive value on survival. Among the clinical parameters, an inverse relationship was observed between age and length of survival. Interestingly, patients from 11 to 14 years had very similar survival to those from 15 to 35 years.

When the immunological phenotypes were compared in relation to survival in the whole series, there were no statistically significant differences, except for the poor prognosis of B ALL.

IMMUNOPHENOTYPE A N D PROGNOSIS IN ALL 37

Table 2. Prognostic factors of CR. Multivariate analysis

Variable P

Age* 0.002 Bleeding tendency? 0.001 WBC counts* 0.033 L1 t 0.050 Common/pre-B phenotype 0.086

*Continuous values. ?Yes or no. Hazard ratio: p/l-p=exp[1.9-0.05(age) + 1,8(bleed- ing) -0+)07(WSC) - 1 . I ( L 1 )]

Nevertheless, in children common ALL was associated with a longer survival than T ALL (Figure l), while in adults (Figure 2) the situation was the opposite. In both age groups null ALL pheno- type had the longest survival, but the differences did not reach statistical significance. TdT was the only individual marker that showed prognostic value (Table 3). The expression of CDlO and the absence of CD38 and CD71 were related to a longer survival, although the differences did not reach significance. We also investigated whether or not the expression of individual markers might differenciate prognostic subtypes within each immunophenotypical subgroup. Although the differences did not reach statistical significance, in common ALL (Table 4) it was observed that the positivity for CD20 ( > 20 per cent) and a low expression of CDlO ( < 50 per cent) were associated with longer survivals. In T ALL the presence of the CDlO antigen was also related with a better prognosis (33 months ofmedian in CDIO+T ALL versus 17 months in CD10-T ALL) although the differences did not achieve statistical significance.

Table 5 shows the characteristics defining the regression equation obtained with the best combi- nation of variables and their prognostic weight. The hazard ratio or relative risk of death was greater when morphology was not L1, and even higher when it was L3 (or B phenotype) and with increasing age and WBC count. Finally, two groups were separated according to their relative risk, using the median hazard ratio as the cut-off point. The median survival for the good prognosis group of patients had not been reached versus the 13 months of the other group, p = 0.000 I .

As in the analysis of CR, the stepwise proportional regression model was repeated after exclud- ing L3 cases and showed that the variables selected were essentially the same. Moreover, in both cases the following variables to appear with favourable prognosis were female sex and null phenotype, although these parameters did not significantly improve the quality of the predictive model.

DISCUSSION

Prognostic factor analysis in ALL has been the subject of multiple studies in the literature. How- ever, there still are some controversial areas, such as the prognostic influence of the immunopheno- type. One of the more sustained concepts is the good prognosis of common ALL as compared to the other groups. Upon analysing this, it should be noticed that these data have been mainly obtained from childhood ALL series (Greaves et al., 1981; Miller et af., 1983; Crist et af., 1985; Pinkel, 1985; Ortega et al., 1987). In our study, common ALL had a better prognosis than T ALL in the children’s group. However, in adults the situation was different since both null and T ALL had longer survival rates than the common/pre-B group. These results together with those reported by

38 E. ~ M E Z ET AL.

Table 3. Prognostic factors of survival. Univariate analysis

N cases Survival (median months) P

Age (a) 2-1 5 years > 15 years

(b) 2-1 1 years 11-14 years 15-35 years > 35 years

( c ) 2-1 1 years 11-35 years > 35 years

Sex Males Females

Infection Ye5 N o

Bone tend. Yes No

WBC (a) < 20 > 20

(b) <20 ( 1 OY' I 1 )

2&100 > 100

Hypogamma *Yes

FAB L1 L2 L3

(mg/d 1 ) No

Phenotype Null Common/pre-B T B

TdT + C D l O t

CD38 + CD71+

-

-

-

-

74 73 63 16 43 25 63 59 25 95 52 44 62 43 61 12 66

72 44 22 14 48 70 48

7 23 92 25 6

140 7

85 32 22 32 6

34

28-2 9.8

36.1 17.8 22.5 7.1

36. I 18.6 7. I

16-8 26. I 12.8 29-7 12-4 25- 1 31.6 13.9

31.6 15.5 9.5

14.9 30.9 33.8 11.5 2-5

32.0 23.5 15.7 3.0

24.8 4.5

25.5 19.3 10.8 27.8 5.3

28.3

< 0.003

0~0001

0~0000

< 0.07

< 0.03

< 0.2 (Breslow < 0.06)

< 0.03

< 0.05

< 0.05

0.0002

0.0002

< 0.005

< 0.6

< 0.2

< 0.2

*IgG < 500 and/or IgA < 40 and/or IgM < 80 in children or < 15 in adults. Cases: common-ALL: 7; B-ALL: 4and T-ALL: 2 cases. TLI versus L2: p<0.002. $Upon excluding B-ALL: p <0.5.

Marcus et al. (1986), Sobol et al. (1 987) Hoelzer and Gale (1987) and Hoelzer et al. ( 1 988) suggest that the expression of the CALLA antigen does not have the favourable prognostic influence previously described, at least in adult patients. The poor outcome of adults with common ALL could be due to other associated adverse prognostic factors in this group, such as a greater incidence of chromosome abnormalities, i.e. the Ph' chromosome or the coexpression of myeloid

100

eo

60

40

20

0

-?---I- L-. I -

IMMUNOPHENOTYPE AND PROGNOSIS IN ALL

IllDIloLocIcAL P l m m r P I s IY CIIxLDm

I... ........................... -\ -y.:. ...................................................................... ... Conman ( 20 months ) : ____ -

0 3 6 9 12 15 18 21 24 21 SO 33 36 39 42 45 48 5 1 54 5 1 60 survival ( months )

Figure 1 . Survival expressed in months of survival from diagnosis. In brackets: median survival. Common (n = 64), T (n = 10) and null ( n = 5 )

7

1W

80

60

40

20

0

"1 I- -L-----,

- L - - - - - - - - - - _.

1

........... :

Null ( 25 months )

T ( 17 months )

...............

.- Common( 9 months I ----

0 7 14 21 28 35 42 49 56 63 7 0 7 7 84

Figure 2. Survival expressed in months of survival from diagnosis. In brackets: median survival. Null (n = 23), common (n =42) and T (n = 18)

Survival f month*)

39

40 E. GOMEZ ET AL.

Table 4. Survival of immunological subgroups in common ALL

N CR Survival cases (%) P (months of median) P

CD20 >20% < 20%

CDlO >50% < 50%

Ia >50% < 50%

TdT >50% < 50%

38 29 66 18 65 15 64 6

86 N.S.* 88 85 N.S.

100 91 N.S. 89 89 N.S.

I00

27 N.S. 13 24 N.S.

N . R . t 25 N.S.

N.R. 25 N.S. 28

*N.S. = Non significant. 7N.R. =Not reached

Table 5 . Prognostic factors of survival. Multivariate analysis

Variable P

L1* L3* (B phenotype) Age t WBCt

Sex Null phenotype

0.0000 0.003 1 0.0 175 0.03 I9

0.2171 0.3972

*Yes or no. fContinuous values. Hazard ratio: ht/hOt=exp[O.S(Ll)- 1.3(L3)+ 0,01(age) + OW3(WBC)]

antigens (Sobol et al., 1987). The opposite is true for patients with T ALL, since several more recent studies (Sobol et al., 1989; Hoelzer et al., 1988; GIMEMA, 1989) indicate a favourable evolution for adult patients with this phenotype.

One aspect that has not received much attention in the literature is the prognostic value of the expression of individual markers in ALL cases and whether these markers can separate within each immunophenotypical subgroup, subsets of patients with a different prognosis. Our results show that some markers may add prognostic information. In the whole series the expression of CDlO and TdT had a favourable influence on CR and on survival, respectively (p = 0.02, p < 0.005). In T ALL, as in Dowell’s et al. (1987) series it was outstanding that CDIO-T ALL had a shorter survival than CDlO+T ALL, although this was not statistically significant. Within common ALL patients, slightly longer survivals were achieved in the more mature subgroup (CD20 + ), as well as in those cases with low CDlO expression (< 50 per cent of+cells).

In relation to the more conventional disease characteristics, it is classically accepted the age limit for separating children from adults to be 15 years (Clarkson et al., 1985; Gingrich et al., 1985; Crist et a!., 1985; Sanchez-Fayos et al., 1986; Sobol et al., 1987). Since prognostic factor analyses are

IMMUNOPHENOTYPE AND PROGNOSIS IN ALL 41

usually performed separately in children and in adults, other age limits have not been generally explored. The present results show that stratification according to age could be improved by classifying the patients in three prognostic groups: a good prognosis group (patients between 2 and 1 1 years), an intermediate group (from 11 to 35 years) and the group of worst prognosis (patients older than 35 years). This finding would have been overlooked if prognostic factors had been analysed separately in children and in adults. In agreement with this observation, Baccarani et ul. (1982) found no differences in the CR rate between adolescents and young adults (below 30 years).

Regarding the two major morphological subtypes (L1 and L2) most series, like the present one, show a better prognosis for L1 cases according to both CR (Miller et al., 1983; Marcus et al., 1986) and survival (Baccarani et al., 1982; Miller et al., 1983). However, a controversial point is whether this favourable influence has an independent value with respect to the cell morphology, or whether it is associated with other characteristics present in the L1 patient group, such as its lower age. For this, it is essential to perform multivariate analyses. In our series, both the FAB morphology (L1 and L2 subtypes) and age appeared as independent prognostic factors on survival, suggesting that the morphology of blast cells provides additional information in predicting survival in ALL patients.

The present findings suggest that simple clinical and hematological variables such as age, WBC counts and the FAB morphology are probably more important for prognosis than the immuno- logical phenotype although it is unquestionable that the latter provides valuable information concerning the clinical and biological heterogeneity of ALL.

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