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CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Cattedra e Divisione di Ematologia,
Università "Tor Vergata," Ospedale S.Eugenio, and the
Istituto di Igiene, Istituti di Ginecologia, Università Cattolica
"Sacro Cuore," Roma, Italy.
B-cell chronic lymphocytic leukemia (B-CLL) follows heterogeneous
clinical courses, and several biological parameters need to be added to
the current clinical staging systems to predict which patients will
experience an indolent or an aggressive outcome. This study
analyzed CD38 expression by flow cytometry and soluble APO1/Fas
(sAPO1/Fas), Bcl-2 (sBcl-2), and CD23 (sCD23) proteins by
immunoenzymatic methods to evaluate their effect on the clinical course
of 168 unselected B-CLL patients. Intermediate/high risk modified Rai
stages were characterized by a higher CD38+ B-cell number
(P = .0002) and higher sCD23 levels
(P < .0001). Moreover, CD38+ B-cell
percentages were significantly and directly associated both with
B-cell chronic lymphocytic leukemia (B-CLL) is the
most common leukemia in the Western world,1 characterized
by the relentless accumulation of monoclonal B cells typically positive
for CD5, CD23, and CD19 and negative for surface CD22 and
FMC7.2 These cells overexpress the Bcl-2 gene product and
are resistant to apoptosis; however, examining the relative levels of
this anti-apoptotic protein has not been particularly helpful in
predicting clinical outcome.3,4 The clinical course of
patients with B-CLL can be quite variable, with many patients surviving
for prolonged periods without any therapy, whereas others succumb
rapidly despite aggressive treatment.5 Although the 2 major staging systems have provided valuable information in addressing
this clinical heterogeneity,6,7 they have been unable to
predict an indolent or aggressive course within the intermediate risk
category. For this reason, several parameters such as lymphocyte
doubling time (LDT),8 serum levels of
Despite having several characteristics of naive B cells, such as
sequences of VH genes in germline configuration
(unmutated), B-CLL cells have been shown to have somatically mutated
immunoglobulin variable region genes in at least half of the cases,
indicating that the cell of origin has passed through the germinal
center (GC). Recent data on VH and VL
sequencing suggest that approximately 60% of B-CLL cases can be
considered post-GC memory B cells; thus B-CLL patients can be divided
into 2 subgroups according to the mutational status of the
immunoglobulin V gene.14 Along this line, Damle et
al15 demonstrated that the percentages of
CD38+ B-CLL cells in the unmutated and mutated groups were
significantly different (means, 63.9% versus 7.3%, respectively;
P = .00001). The positive predictive value of more than
30% CD38+ B cells, indicative of the unmutated genotype,
was between 95% and 100%; conversely, the predictive value of less
than 30% CD38+ B cells, indicating the mutated genotype,
was 82%. Importantly, both the unmutated and the more than 30%
CD38+ groups were characterized by a poor response to
chemotherapy and a shorter survival.15,16
In this study we investigated whether a multiparametric determination
of CD38 antigen by flow cytometry in a large cohort of unselected B-CLL
patients has a predictive value in clinical course and outcomes. If so,
then analysis of CD38 antigen might be added to current staging systems
because of its easy determination in clinical laboratories and its
strong value as a prognostic determinant of poor response to
conventional treatment.
Patients
Cellular immunophenotypic analysis
Bcl-2 enzyme-linked immunosorbent assay Peripheral blood mononuclear cells were separated by density gradient centrifugation, and CD19+ cells were highly purified by positive selection, using both CD19 Microbeads (Miltenyi Biotec, Bergish Gladbach, Germany) and the Variomacs magnetic cell separator, according to the manufacturer's instructions. This system achieved enriched CD19+ populations (92% ± 5%), as demonstrated by restaining CD19+ cells with anti-CD19-PE monoclonal antibody (Becton Dickinson Immunocytometry Systems). Cell suspension densities were adjusted to 2.5 × 106 ± 0.5 × 106 cells/mL, as recommended by the manufacturer (Endogen Human Bcl-2 ELISA; Endogen, Woburn, MA). Therefore, 250 µL of each sample was combined with 50 µL Lysis Reagent (Endogen, Woburn, MA) and then frozen at 70°C. Before running the assay, lysate samples were brought gently to room temperature and diluted 1:10 by using the
sample diluent provided. The Bcl-2 enzyme-linked immunosorbent assay
(ELISA) procedure was performed according to the manufacturer's instructions, and the results were given as units per milliliter, taking into account that sBcl-2 levels in whole blood lysates collected
from 20 apparently healthy individuals were all under 50 U/mL. The
threshold of positivity was set at the sBcl-2 median value of more than
240 U/mL.
Cytoscreen human APO-1/Fas (CD95) ELISA immunoassay This solid-phase sandwich ELISA was performed on serum samples frozen at70°C and then brought to room temperature, according to
the manufacturer's instructions (Biosource International, Camarillo, CA). The normal human APO-1/Fas values ranged from 3.3 to 7 ng/mL. Values higher than 7 ng/mL were considered positive.
Soluble CD23 immunoenzymometric assay The procedure was performed according to the manufacturer's instructions (Biosource Europe, Nivelles, Belgium) for the quantitative determination of heterogeneous bioactive fragments (sCD23) derived from the cleavage of the CD23 membrane molecule. As reference interval, the mean of 118 normal plasma samples was 1.3 U/mL (SD = 0.6), ranging between 0.15 U/mL and 3.3 U/mL. The threshold of positivity was set at the sCD23 value of more than 60 U/mL.Statistical methods Study statistical power.
Because we observed 4 deaths in the group with low CD38 and 14 deaths
in the group with high CD38, for an Data analysis.
All statistical analyses were performed at the end of data collection.
Associations between the CD38 percentages and some clinical variables
(modified Rai stages and response to fludarabine) were assessed by the
Mann-Whitney test and the proportional odds ordered logistic regression
model for continuous and categorical variables, respectively. To
quantify the degree of association between CD38 percentages and other
biological variables, such as sCD23, sBcl-2, sAPO1/Fas, and
Characteristics of CD38+ B cells in B-CLL CD38 antigen showed variable patterns of fluorescence intensity and percentages on CD19+CD5+ B cells (Figure 1). When the percentages of CD38+ B-CLL cells and the modified Rai stages were represented as a biparametric dot-plot (Figure 2), 2 distinct sets could be separated, empirically based on the observed distributions, 1 with more than 30% CD38+ cells (CD38+) and the other with less than 30% CD38+ cells (CD38 ). Interestingly,
more than 30% CD95 and CD10 expression was observed in 5 of 50 (10%)
and in 1 of 50 (2%) CD38+ patients, respectively,
indicating that these molecules usually found on GC B cells are
consistently low in the CD38+ B-CLL subset. Immunoglobulin
M (IgM) mean fluorescence intensity expression detected on the
CD19+ B cells was significantly and directly correlated
with the CD38 percentages (Spearman test; r = 0.20;
P = .025). The Spearman correlation between the
percentages of CD38+ cells and the serum levels of
 2-microglobulin or sCD23 was r = 0.49
(P < .0001) and r = 0.24
(P = .002), indicating a moderate and a low direct
relationship, respectively. On the contrary, no significant correlation
was found between the CD38+ percentages and the sAPO1/Fas
or sBcl-2 levels (r = 0.13; P = .089 and
r = 0.019; P = .81, respectively).
Clinical course and outcome of B-CLL cases We compared the clinical stage at the time of diagnosis by using the modified Rai system as a function of the percentages of CD38+ cells. Both higher CD38 and sCD23 levels were closely associated with the intermediate/high-modified Rai stages (P = .0002 and P < .0001, respectively; Table 1). Moreover, the presence of multiple (3 or more) intrathoracic and/or abdominal lymphadenopathies (> 3 cm in diameter) and/or splenomegaly were strongly correlated both with more than 30% CD38+ B cells and more than 7 ng/mL sAPO1/Fas levels (P < .0001 and P = .0022, respectively; Table 2). LDT less than 12 months was observed in 14 patients: 10 of 14 showed more than 30% CD38+ B cells (P = .0001; Table 2). Furthermore, CD38 was investigated as a predictor of clinical outcome. To this purpose, we used as an end point disease progression, defined as the appearance of an intermediate/high stage from a low Rai stage. The progression-free survival was 37% (confidence interval [CI], 18-65) in the CD38+ and 75% (CI, 59-84) in the CD38 cases
at 5 years from the diagnosis (P = .00006; Figure
3). Forty-eight patients started and
completed as first-line approach 6 courses of fludarabine monophosphate
at 25 mg/m2/d for 5 days, achieving a global CR rate of
35% (17 of 48). There was a significant correlation between higher
CD38 percentages and the lack of a complete response to chemotherapy
(P = .003; Table 3).
Similarly, a significantly lower CR rate was observed in patients with
higher 2-microglobulin (P = .018; Table 3), higher sCD23 (P = .001; Table 3), and higher sBcl-2
(P = .012; Table 3). A significantly shorter survival was
found in CD38+ patients as compared with CD38
patients [50% (CI, 25-71) versus 92% (CI, 78-97) at 8 years; P < .00001; Figure 4A].
Likewise a shorter survival was observed in patients with
2-microglobulin more than 2200 µg/mL
(P = .00041, Figure 4B), sCD23 more than 60 U/mL
(P = .00001, Figure 4C), sBcl-2 more than 240 U/mL
(P = .005, Figure 4D), and sAPO1/Fas more than 7 ng/mL
(P = .008, Figure 4E). These significant differences in survival were maintained when the analysis was restricted to only the
Rai intermediate risk group with regard to CD38
(P = .00015; Figure 5A),
2-microglobulin (P = .012; Figure 5B),
sCD23 (P = .00018; Figure 5C), sBcl-2
(P = .038; Figure 5D), and sAPO1/Fas (P = .034; Figure 5E). In a logistic multivariate
regression analysis of CR after fludarabine treatment, only CD38 more
than 30% (odds ratio, 7.7; CI, 1.1-56.4; P = .036) was a
significant independent prognostic factor. Finally, using a
multivariate Cox regression analysis of overall survival, we found the
mortality hazard ratios were 6.86 for CD38 more than 30%
(P = .001) and 5.39 for sCD23 more than 60 U/mL
(P = .004), respectively (Table
4). Other variables, such as modified Rai
stages, 2-microglobulin, sBcl-2, and sAPO1/Fas, were not
of prognostic significance showing colinearity with CD38 and
sCD23.
CD38+ B-CLLs are tumors of naive B cells Our present study identifies a distinct subset of B-CLL, expressing higher CD38 percentages together with higher2-microglobulin and sCD23 levels. This close
relationship between CD38 expression and 2-microglobulin
or sCD23 concentrations emphasizes that our CD38+ cases are
authentic B-CLL showing a greater disease activity.19 Damle et al15 investigated the expression of CD38 in
relation to the mutational status of the immunoglobulin variable region genes in B-CLL. They found, looking at more than 30% CD38+
expression in B-CLL, that these cases contained unmutated V genes, whereas samples expressing less than 30% CD38+ contained
all the mutated cases and only 3 unmutated cases. This finding argues
that most of our cases are tumors of memory B cells and not of naive B
cells14 because 2 of 3 of our samples express lower than
30% CD38. In addition, it has been demonstrated that B-CLLs having
mutated IgVH genes show generally a relative high frequency
of BCL-6 mutations (9 of 30; 30%), further evidence supporting their
origin from GC-related B cells.20 However, Hamblin et
al21 found no significant association between CD38 expression and mutational status. In any case, both markers (CD38 and
somatic mutations) probably reflect a common feature, such as
maturation stage or signaling dependence (eg, T dependent versus T
independent) of the leukemic B cells.16 Theoretically, a
less differentiated CD38+ lymphocyte precursor should give
rise to a subtype of CLL that has a greater malignant potential and
better probability of being resistant to chemotherapeutic-induced
apoptosis and, thus in general, a poorer prognosis.22
Conversely, Zupo et al23 have reported that
CD38+ B-CLL cells can be easily induced to undergo
apoptosis in vitro after exposure to anti-immunoglobulin antibodies,
whereas CD38 B-CLL cells are resistant to these effects.
The explanation for this apparent disagreement could be that the
quality of the antigen receptor stimulus may lead to diverse endpoints
(survival versus apoptosis). Previous studies suggest that CD38
expression identifies those B-CLL clones that are capable of
transducing signals through their antigen receptors that may increase
or decrease their chance for survival.24 It might be the
case that mature B cells can be rescued from apoptosis by CD38
triggering,25 whereas anti-CD38-mediated signaling
results in the death of immature B cells.26 In fact, Yamashita et al27 reported that a monoclonal antibody
against a murine CD38 homologue, such as CS/2, could deliver a signal to B cells for prolongation of survival and protection against apoptosis in vitro. In their study, stimulation of spleen cells with
CS/2 induced a blastoid morphology in B cells and a significant uptake
of [3H] thymidine.
Apoptotic molecules in B-CLL From the literature, it is well known that soluble Fas molecules are able to block Fas/Fas ligand interaction and thus prevent apoptosis.28,29 Moreover, in vitro, it has been demonstrated that soluble forms of Fas (sFas) can be detected in human serum and are able to inhibit apoptosis induced by either an agonistic antibody or, more importantly, by the natural Fas ligand in Fas+ cell lines.30,31 Interestingly, higher sAPO1/Fas levels were correlated with lymphadenopathy/splenomegaly (Table 2) and a shorter survival (Figures 4E and 5E) in our B-CLL patients. In these cases, a more crucial block in apoptosis could explain both the advanced disease and the poor prognosis.28 Moreover, the higher levels of sBcl-2 found in our B-CLL patients with a less favorable prognosis reinforces this hypothesis (Figures 4D and 5D). Besides, several studies show the expression of multiple apoptosis-regulating proteins in B-CLLs and suggest that the relative levels of some of these, such as Mcl-1, may provide information about in vivo responses to chemotherapy.32 Furthermore, given that many B-CLLs have been reported to be resistant to apoptosis induced by a variety of stimuli, including anti-Fas antibodies and transforming growth factor,33 it could be of interest to explore whether
cross-resistance to several apoptotic stimuli can be used to segregate
B-CLLs into apoptosis-sensitive and -resistant subgroups and further to
attempt correlating this information with clinical outcome. In any
case, the biological and clinical interrelations between these
apoptotic proteins (sAPO1/Fas and sBcl-2) and CD38 expression should be
clarified by further in vitro and in vivo studies.
Clinical course of CD38+ B-CLLs From a clinical point of view, higher CD38 percentages and higher sCD23 levels were significantly correlated with more advanced Rai stages (Table 1). Moreover, our CD38+ patients most often presented with large intrathoracic/abdominal lymphadenopathies, splenomegaly (P < .0001), and a LDT less than 12 months (P = .0001), showing an active and aggressive disease. Complete response to fludarabine as initial therapy34 was significantly correlated with CD38 percentages (P = .003) and with sCD23 (P = .001) and sBcl-2 (P = .012) levels, demonstrating that these biological factors might be used to predict the chemosensitivity of B-CLL patients. In any case, only CD38 more than 30% resulted in being an independent prognostic factor with regard to CR achievement in multivariate analysis (P = .036). Moreover, CD38+ patients progressed more rapidly to advanced Rai stages (P = .00006) and showed a very significant shorter survival (P < .00001). Accordingly, both Damle et al15 and Hamblin et al21 demonstrated that the CD38+ patients responded poorly to continuous multiregimen chemotherapy and had a significantly poorer prognosis. Besides, in a series of 131 patients with B-CLL, the same investigators demonstrated that the B-CLL cases with unmutated VH genes had a distinctly more malignant disease and a much shorter survival than those with somatic mutations.15,35 Finally we performed these same biological determinations (CD38,2-microglobulin, sCD23, sBcl-2, and sAPO1/Fas) among
those patients who presented initially in the Rai intermediate stage.
It is often difficult for clinicians to choose which patients in this
group have to be treated because they can show either an indolent
course or a rapid downhill outcome despite aggressive treatment. In
univariate analysis, CD38 positivity as well as the other biological
variables was able to segregate those Rai intermediate risk patients
who experienced a shorter survival. The prognostic effect of CD38
expression on clinical outcome of B-CLL patients was also corroborated
by the results of the multivariate analysis. Therefore, our study
proposes CD38 expression as a novel and significant prognostic
indicator that delineates similar overlapping groups of B-CLL patients.
In addition, this parameter can be determined easily and rapidly by
flow cytometry in most hematologic laboratories, and, consequently, it
may be a very useful addition to the current staging systems. Actually, this simple test may enable physicians to accurately predict a favorable or an unfavorable clinical course. Last, our experience suggests that CD38+ B-CLL patients are more likely than
CD38 patients to have a progressive and an unfavorable
disease, and, therefore, they should be considered as candidates for
experimental and more tailored therapeutic approaches.
We thank Dr Andrea Stoler for revising the style and the presentation of this paper and the members of our Department of Hematology clinical staff for their support to our B-CLL clinical research program.
Submitted June 21, 2000; accepted June 10, 2001.
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.
Reprints: Giovanni Del Poeta, Cattedra e Divisione di Ematologia, Università "Tor Vergata," Via Fiume Giallo, 430 MA, 00144 Roma, Italy; e-mail: g.delpoeta{at}tin.it.
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© 2001 by The American Society of Hematology.
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  L. Kujawski, P. Ouillette, H. Erba, C. Saddler, A. Jakubowiak, M. Kaminski, K. Shedden, and S. N. Malek Genomic complexity identifies patients with aggressive chronic lymphocytic leukemia Blood, September 1, 2008; 112(5): 1993 - 2003. [Abstract] [Full Text] [PDF]
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L. Z. Rassenti, S. Jain, M. J. Keating, W. G. Wierda, M. R. Grever, J. C. Byrd, N. E. Kay, J. R. Brown, J. G. Gribben, D. S. Neuberg, et al. Relative value of ZAP-70, CD38, and immunoglobulin mutation status in predicting aggressive disease in chronic lymphocytic leukemia Blood, September 1, 2008; 112(5): 1923 - 1930. [Abstract] [Full Text] [PDF] |
Top
Abstract
Introduction
level (2-sided) = 0.05, our
study identified the following mortality hazard ratios for the overall
survival at 5 years: 3 with a power 1-
the best single prognostic parameter in chronic lymphocytic leukemia: a multivariate survival analysis of 329 cases.
Blood.
1984;64:642-648