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Prepublished online as a Blood First Edition Paper on November 7, 2002; DOI 10.1182/blood-2002-06-1714.
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Cattedra e Divisione Ematologia,
Università "Tor Vergata," Ospedale S. Eugenio, Roma, Italy.
The inability to undergo apoptosis is a crucial mechanism of
multidrug resistance in acute myeloid leukemia (AML), and the analysis
of mitochondrial apoptotic proteins may represent a significant prognostic tool to predict outcome. Bcl-2 and Bax oncoproteins were
evaluated in 255 de novo AML patients (pts) by flow cytometry using an
anti-bcl-2 monoclonal antibody (MoAb) and an anti-bax MoAb. The
results were expressed as an index (bax/bcl-2) obtained by dividing bax
mean fluorescence intensity (MFI) and bcl-2 MFI. Lower bax/bcl-2 ratio
was associated with French-American-British (FAB) M0-M1 classes
(P = .000 01) and CD34 more than 20%
(P < .000 01). There were striking inverse correlations
between CD34 or CD117 MFI and bax/bcl-2 values
(r = The primary cause of treatment failures in acute
myeloid leukemia (AML) is the emergence of multidrug
resistance.1-3 Common mechanisms of resistant disease are
defects in apoptotic pathways. Apoptosis is ultimately executed by
caspases that operate both through the receptor-mediated pathway
containing members of the tumor necrosis factor (TNF) family of death
receptors4,5 and the mitochondrial-mediated pathway
involving cytochrome c release from the mitochondria.6,7
Defects in the receptor- or mitochondrial-mediated pathway may give
important prognostic information in AML, allowing risk stratification
of the patients (pts). The mitochondrial-mediated pathway of apoptosis
is regulated by the bcl-2 family of antiapoptotic (bcl-2,
bcl-xl, mcl-1) and proapoptotic proteins (bax, bad, and
bak), and bcl-2 inhibits apoptosis by interacting and forming
inactivating heterodimers with bax/bak.8 Overexpression of
bcl-2 has been associated with drug resistance in hematologic
malignancies,9,10 whereas a high bax level was found to be
a good prognostic indicator in AML.11 It has been
suggested that the bax/bcl-2 ratio may be more important than either
promoter alone in determining apoptosis.12 However,
conventional techniques, such as immunocytochemistry and Western blot,
cannot provide reliable quantitative information and are inadequate for
calculating the bax/bcl-2 ratio.11 Flow cytometry may be
particularly well suited both for the precise, semiquantitative
measurement of bcl-2 and bax oncoprotein expression and for the
estimation of cellular heterogeneity that is frequently found in
AML.9,11 In fact, the binding of the anti-bcl-2 or bax
monoclonal antibody (MoAb) to leukemic cells is variable, which makes
it difficult to define 2 groups, one positive and one negative. On the
basis of these observations, we investigated the presence of bcl-2 and
bax proteins within a large series of 255 newly diagnosed AML cases,
using the mean fluorescence intensities rather than the percentages.
Therefore, in our study, the use of a more sensitive, quantitative flow
cytometric method allowed us to assess the strong and independent
clinical significance of bax/bcl-2 ratio.
Diagnosis of AML was made on bone marrow (BM) and peripheral
blood (PB) smears routinely stained and evaluated according to the
revised French-American-British (FAB) criteria.13 The
diagnosis of AML M0 was made following the guidelines proposed by the
same group.14
Immunophenotyping
CD34 and CD117 expressions were also evaluated in 221 pts as mean
fluorescence intensities (MFIs), calculated as the ratio of CD34 or
CD117 MoAb mean/negative control mean. Both CD34 percentages and MFI
values were determined on the blast cells identified using FSC/SSC gating.
Intracytoplasmic and nuclear stains on cytospin preparations included
terminal deoxynucleotidyl transferase; cytoplasmic CD3 (cCD3), CD22
(cCD22), and CD13 (cCD13), as well as and myeloperoxidase (MPO), as
previously described.15 Immunofluorescence was evaluated by optical examination using a Zeiss microscope equipped with an
epifluorescence set, a barrier filter set for blue fluorescence, and a
×100 phase contrast objective (Zeiss, Göttingen, Germany).
Cytogenetic analysis
Bcl-2 and bax expression Bcl-2 and bax oncoproteins were evaluated by flow cytometry using the following 2 MoAbs: (1) Anti-human bcl-2 clone 124 FITC-conjugated MoAb (Dako, Glostrup, Denmark) is an immunoglobulin G1 (IgG1) that reacts specifically with bcl-2 oncoprotein associated with mitochondria, smooth endoplasmic reticulum, and perinuclear membrane18; (2) anti-bax clone Ab-2 (Oncogene, Cambridge, MA) is an IgG1 that recognizes bax, normally localized to the cytoplasm, but translocating rapidly to the mitochondria after the induction of an apoptotic signal.19 For multiparameter analysis of bcl-2 and bax combined with myeloid antigens, mononuclear cells were first incubated with CD13 and/or CD33 PE-conjugated MoAbs for 30 minutes at 4°C. Subsequently the cells, washed twice in PBS, were fixed and permeabilized in 2 steps: first in 3.5% paraphormaldeyde/PBS and then in 50% cold acetone/PBS. All samples were then incubated at 4°C for 30 minutes with either 10 µL anti-bcl-2 FITC or 20 µL unconjugated anti-bax. For bax, cells were further incubated at 4°C for 30 minutes with 100 µL FITC-conjugated F(ab)2 fragment of goat antimouse immunoglobulin (dilution 1:50; Dako, Glostrup, Denmark). Negative controls were performed by incubating cells with nonspecific isotype IgG1 antibodies. In 52 pts with AML T-residual lymphocytes identified by CD3 PE-conjugate MoAb and treated with bcl-2 and bax MoAbs were used as another negative control. KG-1 and HL-60 cell lines were used as positive controls for this study. Analysis was carried out by flow cytometry (Epics XL; Coulter). The results were obtained by gating blast cells by using SSC versus FSC and examining bcl-2 and bax expressions on CD13/CD33+ blast cells (Figure 1).
Bcl-2 and bax proteins were evaluated as relative mean fluorescence intensities (MFIs), calculated as the ratio of bcl-2 or bax MoAbs mean/negative control mean. The results were expressed as an index (bax/bcl-2), obtained by dividing MFI bax and MFI bcl-2. The threshold was set at the bax/bcl-2 median value higher than 0.3 (range, 0.01-6.0), taking into account BM immature progenitors from healthy donors as negative controls and reference cell lines (KG-1 and HL-60) as positive controls. We tried to set empirically various cutoff points for bax/bcl-2 index, and the selected threshold was sufficient to severely affect complete response and survival, identifying accurately pts at poor prognosis. Moreover, we applied a discriminant function analysis based on the squared Mahalanobis distances of each case from its group centroids (bax/bcl-2). The correct percentage (observed classification versus predicted classification) was more than 80% for this variable. Statistical analysis The relationships of bax/bcl-2 values to age or karyotype were assessed by Mann-Whitney test. Correlations between bax/bcl-2 index and FAB classification or white blood cell (WBC) counts or CD34 percentages or the complete remission (CR) rate were based on chi-square or 2-tailed Fisher exact test. Overall survival (OS), disease-free survival (DFS), and relapse-free survival curves were plotted according to the Kaplan-Meier method. Survival was measured from the date of diagnosis to the date of death or last follow-up. DFS was calculated from the date of CR until relapse. All the pts who underwent autologous or allogeneic transplantation were censored at the time of BM or peripheral blood stem cell (PBSC) infusion. For comparison of OS or DFS patterns of 2 or more groups, the log-rank test was applied. The variables for which univariate analysis had shown a significant association were studied in multivariate analysis. A logistic regression model was used to assess the independent effect of covariables, treated as dichotomous, on the CR, whereas Cox proportional hazards regression model was used to evaluate OS and DFS.
Patients Approval for this study was obtained from the institutional review board. Informed consent was provided according to the Declaration of Helsinki. Criteria for inclusion in this study were (1) diagnosis of AML other than acute promyelocytic leukemia (APL), because pts with APL were entered into a different protocol; and (2) eligibility for intensive chemotherapy. We studied 255 newly diagnosed AML cases, admitted at our unit, between 1995 and 2001, as shown in Table 1.
The pts had a median age of 63 years (range, 18-80 years) with a performance status of 2 or less (World Health Organization [WHO] grading system). The population studied included 116 pts aged younger than 60 and 139 pts older than 60. The first group was treated according to the European Organization of Research and Treatment of Cancer and Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto (EORTC/GIMEMA) AML-10 and AML-12 randomized trials, based, as induction scheme, on the triple association of cytarabine, etoposide, and an anthracycline (daunorubicin or idarubicin or mitoxantrone). Pts with an HLA-compatible sibling were allografted whenever eligible, whereas the others were randomly assigned to receive peripheral or BM stem cell transplantation.20,21 Pts older than 60 were entered into the EORTC/GIMEMA AML-13 randomized trial. These pts received mitoxantrone, etoposide, and cytarabine as induction therapy. On achievement of CR, pts were randomly assigned to receive either an intravenous (idarubicin, etoposide, and cytarabine) or an oral (idarubicin, etoposide, and subcutaneously administered cytarabine) consolidation program. Fifty-one patients were transplanted (40 patients with an autologous transplant and 11 patients with an allogeneic transplant) and censored at the time of BM or PBSC infusion. Characteristics of bax/bcl-2 ratio in AML The MFI of staining with anti-bcl-2 FITC MoAb (18.9 ± 17.3) and anti-bax MoAb (5.6 ± 6.7) in AMLs shifted between CD34+ cell means of normal BM (12.2 ± 11.5 and 4.4 ± 3.2, respectively) and HL-60 cell line means (24.6 ± 8.2 and 6.8 ± 3.1, respectively). Higher bcl-2 MFI median values were found within FAB M0-M1-M6 subgroups, whereas higher bax MFI median values characterized FAB M2-M4-M5 pts, as shown in Table 2.
The CD34+ small percentages (1%-5%) of immature
progenitors in 50 BM specimens from healthy donors were used as a
control group for bax/bcl-2 index: in all cases, bax/bcl-2 ratio
values, obtained by flow cytometry, were always higher than 0.3 (range,
0.5-8.5). The CD3+ T lymphocytes within 52 AML samples
showed also bax/bcl-2 ratio values higher than 0.3 (range, 0.4-6.1).
With this cutoff point, 51% (130 of 255) of the pts, showing bax/bcl-2
ratio less than 0.3, were considered as positive. No relevant
association was found between bax/bcl-2 ratio and age (Table
3). Lower than 0.3 bax/bcl-2 index was
significantly associated both with the immature FAB M0-M1 subtypes
(P = .000 01, Table 4), higher than
100 × 109/L white blood cell (WBC)
count (P = .01, Table 4) and
CD34 more than 20% (P < .000 01, Table 4). Moreover,
lower bax/bcl-2 ratio levels were significantly related to poor risk
cytogenetics (P = .0002, Table 3). There were also highly
significant inverse correlations between bax/bcl-2 ratio values and
CD34 MFI (r =
Prognostic value of bax/bcl-2 index A significant higher complete remission (CR) rate was found in pts with higher bax/bcl-2 index (79% versus 45%; P = .000 01, Table 1). Also, both a longer overall survival (OS) and disease-free survival (DFS) were observed in pts with higher bax/bcl-2 index (18% [confidence interval (CI), 10-38] versus 0% at 4 years; P = .000 01 and 26% [CI, 14-45] versus 0% at 4 years; P = .019, Figure 2A-B).However, there was no significant difference between CD34+
(> 20%) and CD34
Interestingly, lower bax/bcl-2 ratio retained its adverse prognostic
significance both within int/fav cytogenetics subgroup (n = 94 pts;
70% versus 94%, P = .002 for CR and 0% versus 26% at 2 years, P = .02 for OS, Figure
4A) and poor cytogenetics class (n = 93
pts; 11% versus 23% at 1 year, P = .01 for OS, Figure 4B, and 21% versus 70% at 6 months, P = .004 for DFS,
Figure 4C).
Furthermore, lower bax/bcl-2 ratio was closely associated with a
shorter time to relapse (4 ± 3.6 months versus 7 ± 6.3 months; P = .03, Figure 5A).
Noteworthy, bax/bcl-2 levels accurately predicted the clinical response
and outcome of pts with normal (79 of 255) or unknown cytogenetics (68 of 255). Indeed, within this subset of 147 pts, higher bax/bcl-2 ratio
was significantly associated both with a higher CR rate (86% versus
42%; P < .000 01) and a longer OS (21% versus 0% at 4 years; P = .0016, Figure 5B).
In a logistic multivariate regression analysis of CR, bax/bcl-2 ratio
was confirmed in being a significant independent prognostic factor
(odds ratio, 3.3; CI, 1.3-8.4; P = .013). Finally, using a
multivariate Cox regression analysis both of OS and DFS, bax/bcl-2 mortality hazard ratios were 8.7 for OS (P = .003)
and 5.5 for DFS (P = .019),
respectively (Tables 5 and 6).
Lower bax/bcl-2 ratio is consistent with immaturity and a worse cytogenetic pattern in AML The biologic basis of drug resistance and relapse in AML is not well understood, and prognoses are still largely based on descriptive parameters. Several lines of evidence indicate that apoptosis plays a role in response to chemotherapy, suggesting an association between therapy-induced apoptosis and therapeutic efficacy in AML.22 Increasing evidence suggests that specific genetic alterations may abrogate apoptotic responses in myeloid tumorigenesis. High bcl-2 or low bax protein expression may contribute to leukemogenesis and drug resistance.9,23 The mechanisms linking these molecules to drug resistance have been experimentally addressed in AML by Banker et al.24 In this study, most AML samples showed less treatment-associated apoptosis, suggesting that apoptotic responses to the therapeutic agents may be attenuated. All AML samples with the highest bcl-2-positive fractions showed low apoptosis. Moreover, the relative expression of proapoptotic and antiapoptotic proteins25 has been shown to provide an indication of chemosensitivity in solid tumors.26 We hypothesized that bcl-2 measured in conjunction with bax would provide valuable information on the intrinsic chemosensitivity of AML, as reflected by the clinical response to treatment. Besides, among the polypeptides that are structurally and functionally related to bcl-2, Mcl-1 has been identified. The Mcl-1 gene encodes a 36-kD polypeptide with a carboxyl terminal domain that is 35% identical to bcl-2.27 Overexpression studies have revealed that Mcl-1 delays apoptosis induced by etoposide in myeloid leukemia cells28 and exhibits high levels at the time of leukemic relapse in AML.29 The antiapoptotic effects of bcl-2 and Mcl-1 are opposed by a number of proapoptotic bcl-2 family members, such as bax and bak. As it is well known, bcl-2 and Mcl-1 have the ability to form heterodimers with Bax, a 22-kD polypeptide, neutralizing its effect. This results in a diminished ability of the cell to undergo apoptosis.25 It has been demonstrated that, in chronic lymphocytic leukemia (CLL) cells, the conformational changes of bax and bak are among the early steps in the induction of cell death.30 Besides, after dimethyl sulfoxide (DMSO) treatment, HL-60 cells died via apoptosis, and the levels of the apoptosis effector proteins bak and bad were enhanced, whereas there was a slight down-regulation of the apoptosis suppressor protein bcl-2.31 Moreover, in myelodysplastic syndromes, a higher expression of proapoptotic proteins, such as bak, bad, and bcl-xs, was associated with a longer survival and a decreased risk of leukemic transformation.32 Our present study performed in a large series of patients identifies a distinct AML subset (130 pts), expressing lower than 0.3 bax/bcl-2 ratio, characterized by immaturity (higher CD34 and CD117 levels) and by a poor karyotype. The use of a quantitative flow cytometric method based both on bcl-2 and bax mean fluorescence intensities and their ratio allowed us to precisely define its biologic and clinical potentiality. In fact, the quantitation of fluorescence intensity seems technically adequate and useful for a more correct evaluation of clinical and prognostic correlations, as previously demonstrated.33 Moreover, Ong et al11 observed that immunocytochemical techniques were both inadequate for calculating the bax/bcl-2 ratio and unable to find any significant correlation between this ratio and outcome parameters. All our AML cases were found to be bcl-2 positive, probably because of a more sensitive technical approach by direct immunofluorescence, as observed also by other researchers.10 Concerning the FAB classification, our data demonstrated significantly higher bax/bcl-2 ratio values in monocytic AMLs (M4 and M5), because of a lower bcl-2 MFI. This finding is in contrast to the report of Campos et al9 who found a significantly higher percentage of bcl-2-positive cells in these FAB subtypes. However, our results confirm other observations of higher bcl-2 expression in more immature cells34 and in immature AMLs.10,35 The prognostic value of bax/bcl-2 ratio may be also due to a correlation of bcl-2 with maturation. In fact, this ratio was found to be low in our CD34+ AML cases (P < .000 01), as observed by others.35,36 Besides, CD34 MFI values were significantly correlated with bax/bcl-2 ratio (r = .40,
P < .000 001). Moreover, a significant correlation was
found between higher bcl-2 expression (MFI > 14) and coexpression of
the CD34 antigen, further suggesting that the higher bcl-2 expression
is more evident in less differentiated AMLs.10 These findings could contribute to the prognostic relevance of bax/bcl-2 ratio, because CD34+ AMLs are considered to have an
unfavorable outcome.35,37 On the contrary, despite the
strict correlation between CD34 expression and bax/bcl-2 ratio, in our
experience CD34 alone did not show any clinical significance with
regard to CR, OS, and DFS in univariate analysis. In addition, the
strong inverse correlation between bax/bcl-2 ratio and MFI CD117 values
(P = .000 002) in our cases confirms the link between
this index and maturation pathways, because CD117 is expressed by
myeloid precursor cells38 and its activation prevents
apoptosis.39 Besides, a lower bax/bcl-2 ratio was able to
identify clearly pts at unfavorable prognosis within the
CD34+ subset (Figure 3), demonstrating that its strong
independent prognostic value is bound not only to immaturity but also
probably to a disregulation of the apoptotic pathways. Furthermore,
bax/bcl-2 ratio was found to be expressed at lower levels in our
poor-risk cytogenetics AML subset (P = .0002), as
corroborated also by other studies.35,40 From literature,
it is well known that chromosomal translocations associated with
specific subtypes of acute leukemia result in the rearrangement of a
variety of transcription factor genes. These resulting hybrid genes
encode chimeric or aberrantly expressed proteins that may contribute to
malignant transformation via the suppression of apoptosis. Banker et
al24 observed that some karyotypic abnormalities, such as
t(15;17), del(7), and t(9;11), exhibited lower basal apoptosis in AML.
On the contrary, other researchers10,11 showed that the
bcl-2 expression (bcl-2 mRNA, bcl-2/bax ratio, or bcl-2 MFI) did not
correlate to prognostic groups defined by karyotype.
Clinical significance of bax/bcl-2 ratio From a clinical point of view, higher bax/bcl-2 ratio was significantly correlated with a higher CR rate (Table 1), longer time to relapse (Figure 5A), and a longer OS and DFS (Figure 2A-B). Accordingly, Campos et al9 demonstrated that AML pts with high levels of bcl-2 expression by immunofluorescent staining had a CR rate of 29% versus an 85% CR rate in pts with low levels. Likewise, high levels of bcl-2 translated into decreased OS. Bcl-2 remained an important predictor of survival in multivariate analysis over other known prognostic markers, including age and WBC count of more than 30. Besides, increased levels of bax expression by immunocytochemical analysis correlated with improved rates of OS in a study of 56 pts with de novo AML.11 On the contrary, a study of 165 pts with newly diagnosed AML41 reported that levels of bax expression by immunoblotting did not correlate with response to induction chemotherapy or survival. However, in the same study, high ratios of bcl-2 to bax protein conferred a poor prognosis with decreased rates of CR and OS. Moreover, there was a significant correlation between bcl-2-to-bax expression ratio and clinical response (P < .005) in 14 AML pts examined by using an immunophosphatase staining method.12 Along these lines, it has been demonstrated that the ratio of bcl-2 to bax correlates inversely with the sensitivity of B-cell chronic lymphocytic leukemia (B-CLL) cells to cytotoxic drugs in vitro.42 Therefore, the prognostic discrimination offered by the ratio of the proteins is greater than the bcl-2 levels alone. Also in our hands, the use of a more sensitive, quantitative method, eg, quantitative flow cytometry, allowed us to realize the clinical prognostic potential value of this ratio, as demonstrated by our clinical results. Moreover, lower bax/bcl-2 ratio retained its unfavorable prognostic value both within int/fav and poor cytogenetic classes with regard to CR, OS, and DFS (Figure 4). In myelodysplastic syndromes (MDSs) and AML secondary to MDS, bax/bcl-2 ratio was inversely correlated with both the International Prognostic Scoring System (IPI) score and cytogenetics: the highest levels were observed in pts with low IPI score and/or good risk cytogenetics.43 Andreeff et al44 reported an unexpectedly poor survival rate associated with low bcl-2 in a group of AML pts with unfavorable cytogenetics. These preliminary data were confirmed by Kornblau et al41 who demonstrated that AML pts with a lower bcl-2/bax ratio had an inferior response rate to induction therapy (35% versus 78%, P = .0006) and an inferior survival outcome (median survival, 11 versus 53 weeks, P = .000 02). On the contrary, we found that a low bax/bcl-2 ratio predicted a poor outcome also within the unfavorable cytogenetic class. The explanation for this discrepancy may be in the particular karyotypic abnormalities, such as del(7), complex karyotypes, and 11q23 translocations, notoriously inhibiting apoptosis24,45 and very frequently found in our poor-risk cytogenetics subgroup. Furthermore, we found that bax/bcl-2 ratio maintained its strong prognostic significance with regard to CR (P < .000 01) and OS (P = .0016) also within AML pts with normal or unknown cytogenetics. These data suggested that cytogenetics and bax/bcl-2 ratio were independent predictors of response and survival. In multivariate analysis a low bax/bcl-2 ratio confirmed its worse significant effect on the outcomes.In conclusion, this biologic ratio is feasible and reliable through flow cytometric methods46 and is a potentially useful prognostic indicator for tailoring the intensity of treatment. Its striking prognostic effect confirms the key role of mitochondrial apoptotic proteins, such as bcl-2 and bax, both in chemosensitivity and in future strategies for overcoming multidrug resistance in AML.
Submitted June 12, 2002; accepted October 31, 2002.
Prepublished online as Blood First Edition Paper, November 7, 2002; DOI 10.1182/blood-2002- 06-1714.
Supported in part by MURST, Programmi di ricerca di interesse nazionale 2000.
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 U.S.C. section 1734.
Presented in part at the 42nd annual meeting of the American Society of Hematology, San Francisco, CA, December 1-5, 2000.46 Reprints: Giovanni Del Poeta, Cattedra e Divisione Ematologia, Università "Tor Vergata," Ospedale S. Eugenio, P. le Umanesimo, 10, 00144 Roma, Italy; e-mail: g.delpoeta{at}tin.it.
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Top
Abstract
Introduction
(< 20%) pts with regard to CR
(P = .06), OS (P = .4), and DFS (P = .8). However, lower bax/bcl-2 ratio was capable of
clearly identifying pts at poorer prognosis within the
CD34+ subgroup (n = 150 pts; 42% versus 81%,
P = .000 03 for CR; 3% versus 21% at 3 years,
P = .000 01 for OS, Figure
45 yrs old in first CR: results of the EORTC-GIMEMA AML-10 trial [abstract].
Blood.
2001;98(suppl 1):2010a.
modulate the prognostic impact of Bcl-2 expression in acute myelogenous leukemia.
Clin Cancer Res.
2000;6:1401-1409