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Prepublished online as a Blood First Edition Paper on October 24, 2002; DOI 10.1182/blood-2002-06-1801.
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Department of Oncological Sciences, University
of Torino, Torino, Italy; IRCC, Institute for Cancer
Research and Treatment, Candiolo (TO) and University Division
of Clinical Immunology and Hematology, Ospedale Mauriziano Umberto I,
Torino, Italy.
Chronic lymphocytic leukemia (CLL) has a variable clinical course.
CD38 expression and IgVH gene mutational status are
independent predictors of prognosis, but their relationships and the
CD38 cutoff level are unknown. Using cytofluorography, we analyzed CD38
in 148 patients, in 108 of whom we were able to evaluate IgVH mutations, make correlations with disease history, and
assess cumulative survival. Three different patient groups were
identified by the CD38 expression pattern: a group homogeneously
CD38 B-cell chronic lymphocytic leukemia (CLL) is the
most frequent leukemia in the Western world. It is characterized by a
variable clinical course1 with some patients having an
aggressive malignancy and others a slow, nonprogressive disease and a
virtually normal life expectancy. The possibility of establishing at
diagnosis the most likely outcome of individual patients would provide
the basis for a differential management policy. Several biologic risk factors, ranging from serum markers2 to
cytogenetics,3 have been used to assess a patient's
prognosis. Two parameters have emerged as potent predictors. One is the
mutational status of the immunoglobulin variable region
(IgVH) genes. Retrospective studies have established that
patients whose cells carry somatic mutations with less than 98%
sequence homology with the nearest germ line gene have a prognosis
significantly better than those presenting with germ line
IgVH genes.4-6 Such a difference may be
accounted for by the observation that patients with germ line IgVH genes tend to harbor high-risk genomic aberrations and
p53 dysfunctions.7-9 The other parameter is the cell's
expression of CD38, which has been shown to be an independent marker
that correlates with a shorter median survival and more need of
treatment.4,10-13
Despite a general agreement on the role of both IgVH gene
mutational status and CD38 expression in independently predicting the
prognosis, 2 major issues remain controversial. First is the CD38
cutoff level used for risk stratification. For some groups the
threshold is 30% positive cells,11,12 whereas for others it is 20%.10,13 This is not a trivial point because
numerous cases are in the "gray" zone and are not allowed a precise
prognostic categorization. Also a few patients appear to modify their
percentage of CD38+ cell expression over
time,10,12 thereby crossing the cutoff border. Second, it
has been initially suggested,4 but later challenged,12,14 that the expression of CD38 might
correlate with IgVH mutational status, meaning that
patients with 30% or more CD38+ cells are also the ones
who carry germ line IgVH genes, whereas those with less
than 30% CD38+ cells have somatically mutated
IgVH genes. Thus, it is not established which, if any,
relationship exists between the presence or absence of IgVH
mutations and the expression of CD38 nor how the 2 markers can be used
to define prognosis in individual patients. Finally, it cannot be
overlooked that the cytofluorometric evaluation of CD38 expression is
an easy and rapid technique used in most laboratories, whereas the
detection of IgVH gene mutations can be reliably used only
in a minority of sophisticated centers.
We have analyzed the expression of CD38 and the occurrence of
IgVH somatic mutations in an unselected cohort of patients
and we have correlated these parameters with the patients' disease history. According to the pattern of CD38 expression we have identified 3 groups of patients. CLL cells can be homogeneously negative for CD38
(CD38 Patient population
Within this cohort, 67 men and 41 women (mean age, 70 years) were
selected on the basis of a disease history lasting more than 1 year
(median follow-up time, 90 months; range, 16-306 months) to allow the
evaluation of clinical progression (Table
1). These patients were studied in detail
by analyzing the following parameters measured at diagnosis or during
the follow-up: lymphocyte and platelet counts; lymphocyte doubling time
(LDT); hemoglobin (Hb), immunoglobulin and
In 82 of 108 cases the analysis of IgVH genes mutational status was performed on cDNA obtained from the same cell samples analyzed for CD38 expression. In selected patients further in vitro studies aimed at investigating the biologic basis of clinical differences were performed. Immunophenotypic analysis Immunophenotypic analysis was performed on fresh blood samples or on cells cryopreserved at diagnosis after having assessed that fresh and cryopreserved cells did not yield different results. In 63 cases the analysis was repeated over time either before or after any treatment. In individual cases the analysis was concomitantly performed on both PB and BM.The following antibodies were used: allophycocyanin (APC)-conjugated
anti-CD19 and phycoerythrin (PE)-labeled anti-CD23 from Caltag
Laboratories (San Francisco, CA); fluorescein isothiocyanate (FITC)-labeled anti-CD20, PE-conjugated anti-CD5, and PE-conjugated anti-CD38 from Becton Dickinson (San Jose, CA); FITC-labeled anti-CD79b from Dako (Glostrup, Denmark); and FITC-conjugated FMC-7 (Serotec, Oxford, United Kingdom). FITC-labeled anti-IgM, anti-IgG, anti-IgA heavy chain were obtained from Southern Biotechnology Associates (Birmingham, AL); FITC-conjugated F(ab)2-anti- To determine the percentage of CD38+ events, CLL cells were incubated with FITC-conjugated anti-CD5 (1:20 final dilution), PE-conjugated anti-CD38 (1:50 final dilution), and APC-conjugated anti-CD19 (1:20 final dilution). CD19+CD5+ lymphocytes were identified by properly gating on both fluorescent and light scatter parameters. Staining and acquisition conditions were standardized allowing reproducibility of the results, regardless of the actual operator. For each sample at least 10 000 events were acquired on a FACSCalibur equipped with 488 argon ion laser and 635 red diode laser (Becton Dickinson) and analyzed with the CellQuest software system (Becton Dickinson). In selected experiments CD38+ and CD38 Determination of mutational status of IgVH genes Total RNA was extracted using guanidinium thiocyanate method (TRIzol; Invitrogen Life Technologies, Paisley, United Kingdom). RNA was reverse-transcribed into cDNA using 200 U Superscript II, Rnase H Reverse Transcriptase (Invitrogen-Life Technologies
Italia, San Giuliano Milanese, Italy). After determining the
IgVH gene family used by the leukemic cells, the
VH gene sequences were determined by amplifying 2.5 µL
cDNA by polymerase chain reaction (PCR) using the appropriate sense
VH leader primer in combination with the appropriate
antisense CH primer, as described
previously.17
PCR products were sequenced directly after purification with PCR preps (Promega, Madison, WI) using an automated DNA sequencer (Applied Biosystems, Foster City, CA). Sequences were compared with those present both in the V BASE sequence directory (http://www.mrc-cpe.cam.ac.uk/imt-doc/vbase-home-page.htlm)18 and in the IMGT/V-QUEST database (http://imgt.cines.fr).19 The sequences with a germ line homology 98% or higher were considered unmutated, and those with a homology less than 98% mutated.4,5 CD40 stimulation of CLL cells Mononuclear cells from patient PB were isolated by density centrifugation over Lymphoprep (Nycomed Pharma, Oslo, Norway). Cells were washed with phosphate-buffered saline (PBS) solution and resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS; Life Technologies-Italia), 2 mM L-glutamine, and 15 µg/mL gentamicin. Cells cultured for up to 3 days at a concentration of 1 × 106 cells/mL with or without 0.1 µg/mL soluble recombinant human CD40L plus 1 µg enhancer, according to manufacturer's instructions (Alexis, San Diego, CA), were collected and phenotypically analyzed.Statistical analysis CD38 expression on CLL cells has been compared to other parametric series (lymphocyte count, Hb, LDT, 2-microglobulin) using the Spearman correlation test.
The Student t test and one-way ANOVA test were applied to
determine the statistical significance of mean value difference
among series.
Patients were grouped into 3 subsets depending on CD38 expression
(negative, bimodal, positive) and in 2 subsets depending on the
IgVH gene mutation status (unmutated, mutated). The Fisher exact test (2-tailed) and Cumulative survival analyses were obtained according to Kaplan- Meier method.
The pattern of expression of CD38 identifies 3 groups of CLL patients The range of CD38+ CLL cells in 148 patients was 0.1% to 99%. In 82 cases (Figure 1A,D) the cells were homogeneously negative with exceedingly rare and scattered positive elements (negative group; < 2%). In 26 patients most cells clustered in a homogeneously positive population (Figure 1B,E; positive group). In 40 patients a clear-cut bimodal expression of CD38 was evident (Figure 1C,F) with 2 distinct cell populations, one expressing high levels of CD38 and the other completely negative (CD38 bimodal expression group). The proportion of each population varied in individual patients (Figure 2A-E). In our cohort of CD38 bimodal patients the CD38+ subset ranged between a minimum of 8% to a maximum of 78%. It remains to be determined whether a distinct bimodal profile can also be observed in patients presenting a lower percentage of CD38+ cells (eg, < 5%).
In 63 cases, blood samples were sequentially analyzed over time (12-86 months). No significant modifications of CD38 expression were observed
in 59 of 63 cases either in the absence of treatment (42 cases) or
after therapy (17 cases). In the remaining 4 cases the CD38 expression
was modified after chemotherapy. All 4 patients who had modified CD38
expression belonged to the CD38 bimodal expression group; the pattern
of modification was not clear-cut (Figure
3A-D); no one became CD38 The pattern of expression of CD38 correlates with IgVH gene mutational status The IgVH mutational status was analyzed in 82 patients, 76 surface IgM+ (sIgM+) and 6 surface IgG+ (sIgG+), and confirmed that: (1) the most frequently encountered VH genes are V3-23 (9 of 82, 11.0%), V1-69 (7 of 82, 8.5%), V3-07 (7 of 82, 8.5%), 3.30 (6 of 82, 7.3%), and V4-34 (5 of 82, 6.1%) genes5,17; (2) there is a preferential use of D3 (23 of 82, 28.0%), D2 (15 of 82, 18.3%), and D6 (13 of 82, 15.8%) genes17; and (3) the most frequently used JH segments are JH4 (45 of 82, 54.9%), JH6 (17 of 82, 20.7%) and JH3 (12 of 82, 14.6%).17Thirty-three patients (40.2%) showed no IgVH mutation.17,20 The remaining 49 (59.8%) patients showed evidence of somatic hypermutation: 41 of 49 (83.7%) cases differed 5% or more from germ line genes. All IgG cases were mutated. We evaluated the IgVH mutational status in the 3 groups of
patients defined according to the pattern of CD38 expression. Most patients (39 of 44, 88.6%) of the CD38 We then analyzed the expression of CD38 in patients categorized as
mutated or unmutated observing that 39 of 49 mutated cases (79.6%)
were CD38 These data indicate a statistically significant correlation between the
expression of CD38 and the mutational status of IgVH genes
(P < .001). Strong positive predictive values (PPVs)
predicting either the presence or the absence of somatic mutations are
found in the CD38 The 2 subsets of CD38 bimodal expression patients are differentially represented in the BM We asked which (if any) of the 2 subpopulations (CD38+ and CD38) detected in the PB of the CD38 bimodal
expression group might be prevalent in infiltrated tissues. To this
end, we compared the expression of CD38 in CLL cell samples
concomitantly obtained from both PB and BM in 14 CD38 bimodal
expression patients. Interestingly, in 10 of 14 CD38 bimodal expression
patients a significantly higher amount of CD38+
cells could be seen in the BM with an increase ranging
between 28% and 97% (Table
2).
Phenotypic characterization of leukemic cells from CD38 bimodal expression patients To evaluate whether the 2 subpopulations (CD38+ and CD38) detected in the PB of the CD38 bimodal expression
group might also differ for the expression of other markers we further
characterized by cytofluorography the surface phenotype of the 2 subpopulations in 10 bimodal expression patients. We have analyzed the
expression of: (1) CD10, CD20, CD21, CD22, CD24, CD25, CD27, CD40,
CD69, CD70, CD72, CD74, CD75, CD78, CD79b, CD80, CD81, CD86, CD95,
CD103, CD138, based on the hypothesis that these 2 subsets could differ for their differentiation status; (2) CD23, CD43, CD52, CD6, CD9, CD71,
FMC-7 based on the hypothesis that CD38 expression might correlate with
a more aggressive phenotype; and (3) CD11a, CD11c, CD18, CD31, CD44,
CD49d, CD54, CD100, CXCR3, CCR6 based on the hypothesis that the 2 subsets might have a different homing and migratory capacity.
Most markers were homogeneously either positive or negative on the
leukemic clones as a whole; that is, no differential expression could
be detected in the 2 cell subsets. Few markers (eg, CD11c, CD49d, CD69,
CD78, CCR6, CXCR3) had a more dispersed expression with a mixture of
positive and negative cells but the pattern of expression was
superimposable in both CD38+ and CD38 Functional characterization of leukemic cells from CD38 bimodal expression patients Next we investigated whether the presence of 2 subsets in the PB of CD38 bimodal expression patients might be associated with a different response to microenvironmental stimuli. We have previously described that cells obtained from PB of unselected CLL patients stimulated through CD40 can have a different outcome.21 Leukemic cells from the majority of the patients (66%) respond to such stimulation by prolonging their survival in vitro and up-regulating CD80 and CD95 on the surface (CD40L responders), whereas the remaining (33%) are not modified by this stimulus (CD40L nonresponders). Therefore, we cultured CLL cells from PB of 46 CLL patients (31 CD38, 5 CD38+, and 10 CD38
bimodal expression) in the presence or the absence of soluble human
CD40 ligand (sCD40L) and analyzed cell survival and CD80/CD95
expression after up to 3 days of culture. Twenty-three of the 31 CD38 as well as all 5 of the CD38+ patients
were CD40L responders. Among the 10 CD38 bimodal expression patients, 3 were CD40L nonresponders and the remaining 7 had a homogeneous
up-regulation of both CD80 and CD95 on the surface. In these cases,
after up to 3 days of culture, both CD38+ and
CD38 compartments prolonged their survival, and the
relative ratios of the 2 subsets were unmodified.
The phenotypic and functional homogeneity shown by both subsets of CLL cells in CD38 bimodal expression patients is also matched by the molecular evidence that in 25 CD38 bimodal expression patients specifically analyzed the cells belonging to the 2 CD38 subsets carry the same IgVH rearrangement. Repetitive reverse transcription-PCR amplification produced only one discrete band from each sample and the direct sequencing of this band, performed in different occasions from both 5' and 3' ends, always produced the same nucleotide sequence. Concordance of CD38 pattern of expression, IgVH mutation status, and traditional prognostic factors The increasing value of CD38 significantly correlated with low platelet counts (r = 0.278; P = .004), low Hb levels
(r = 0.360; P < .001), and high
2-microglobulin serum levels (r = 0.372;
P = .001).
CD38+ and CD38 bimodal expression patients had
significantly higher Likewise, patients belonging to the unmutated group had significantly
higher
The pattern of CD38 expression predicts CLL patients at risk of disease progression At diagnosis the majority of patients (78 of 108, 72.2%) were in the low-risk group16: specifically, 53 of 62 (85.5%) of CD38 patients, 18 of 30 (60.0%) of CD38
bimodal expression group, and 7 of 16 (43.8%) of the CD38+
group (Table 1).
When disease progression criteria were correlated with CD38 expression
(Figure 4A) the vast majority of
CD38
The relationship of CLL cell IgVH mutation status with
disease progression was statistically significant
(P < .001), though at a lower degree than CD38 expression
alone (Figure 5; Table 2; PPV = 77.6%
for mutated and stable disease; PPV = 69.7% for unmutated and
progressive disease). The concomitant analysis of both somatic
mutations and CD38 expression improved the capacity in predicting
disease progression only in the presence of the association of
unmutated Ig and CD38+ or bimodal expression cells
(PPV = 90.0% and 81.3%, respectively).
Only 16 of 62 (25.8%) CD38 Among the 20 patients who presented autoimmune hemolytic anemia7 or thrombocytopenia,13 5 were CD38+ and 9 were CD38 bimodal expression, indicating that patients presenting with even a small population of CD38+ cells are more prone to develop autoimmune manifestations (P = .03; Table 1). The analysis of cumulative survival curves (Figure
6A) indicates that the median survival
for the bimodal expression group, regardless of the proportion of
CD38+ cells, is 156 months and overlaps the median survival
of the CD38+ group (183 months). As shown in Figure 6B, the
median survival of CD38 bimodal expression patients did not vary
significantly when the patients were analyzed according to the 30%
positivity cutoff. The mean survival of the CD38
CLL is a frequently diagnosed blood malignancy whose management still defies clear-cut practice guidelines. One major reason for this failure is the clinical heterogeneity of the disease. Predicting prognosis would allow patients optimal counseling and more adequate treatment planning. Starting from the classical staging systems,22,23 many efforts have been devoted to the identification of prognostic parameters that might help dissect the clinical heterogeneity and could indicate at diagnosis which natural history can be reasonably expected in individual patients. The rationale of focusing on CLL cell expression of CD38 and IgVH mutational status is that the clinical relevance of these 2 markers is matched by their biologic significance. In vitro studies have shown that the CLL cell expression of CD38 defines 2 groups of patients strikingly differently in their in vitro response to anti-Ig antibodies.24,25 In clinical studies CD38 has proved to be an independent and reliable prognostic marker.10-13 The IgVH mutational status is used to classify the cellular origin of blood malignancies26 because IgVH somatic mutations appear to occur only in B cells that have been somehow exposed to antigenic stimulation. In CLL it has been shown4,5 and confirmed6 that the presence of IgVH somatic mutations carries a favorable prognosis, whereas its absence is an unfavorable marker. The use of these parameters in clinical practice would be extremely useful provided that 2 major discrepancies in the literature are clarified. The first concerns the relationship between immunoglobulin somatic mutations and CD38 expression that some authors have observed,4 but others12,27 have questioned. The second even more important problem is the numerical definition of which cutoff value of CD38+ cells should be used to have the most useful clinical information. Different authors have used 30%, 20%, or even 7% of CD38+ cells as the cutoff point that provides the best separation for prognostic subgroups.4,7,10-13 A nonsecondary corollary of the whole issue is that cytofluorographic determination of CD38 is an easily manageable test, whereas the definition of IgVH somatic mutations requires a definitely less common expertise. We have shifted the perspective under which the cytofluorometric
analysis of CD38 expression in CLL has to be assessed. In this work we
demonstrate that the important clinical discriminant is not a CD38
numerical value, but rather it is its pattern of expression that
correlates with IgVH gene mutational status and allows us
to predict which patients are at risk for disease progression. We
started from the observation that CLL patients can be divided into 3 different subsets: a CD38 Using this approach it has become evident that the CD38 The relevance of our observation is 2-fold. First, it indicates that a reproducible, well-disseminated technique can be used to identify patients who, irrespective of the clinical stage at presentation, will have progressive disease. This approach may be used to design new trials aimed at differentially managing patients whose outcome can be reasonably foreseen. It is irrelevant whether the CD38+ population is present in a high or in a low proportion (Figures 4B and 6B). What really matters is the presence of a CD38+ population whose existence proves to be a simple and effective prognostic parameter that can be also used to reconcile the literature discrepancies. The second point concerns the biologic background of these clinical observations. It becomes obvious to ask whether the 2 populations detected in the PB of the bimodal expression group may have distinctive features. We here show that the 2 populations carry the same IgVH rearrangement and do not differ in terms of expression of differentiation status, aggressive phenotype, homing, and migratory capacity markers. Likewise no differences have been observed in terms of response to microenvironmental stimuli represented by the in vitro response to CD40L. Of interest, the CD38+ subset of bimodal patients appears to be increased in infiltrated BM as compared to PB, suggesting that the BM environment, which is known to be a privileged site of relapse in CLL patients, more easily hosts cells that are associated with an adverse outcome. The quest for the biologic bases of the observed clinical differences will need painstaking experiments of cell culture and molecular investigation of extensively purified populations.
Submitted June 19, 2002; accepted September 29, 2002.
Prepublished online as Blood First Edition Paper, October 24, 2002; DOI 10.1182/blood-2002-06-1801.
Supported by AIRC (Associazione Italiana per la Ricerca sul Cancro), Milano, by Ministero dell'Università e Ricerca Scientifica (MIUR), Roma, and by MDACC Laboratory Study Agreement LS01-039 (Houston, TX). G.G. and C.S. are recipients of a fellowship from Comitato Gigi Ghirotti (Torino).
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: Federico Caligaris-Cappio, Ospedale Mauriziano Umberto I, Largo Turati 62, 10128 Torino, Italy; e-mail: fcaligaris{at}mauriziano.it.
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Top
Abstract
Introduction
and PE-anti-
light chain were purchased from Dako.
2 test were performed to
determine the difference between the observed proportions of events
among patients' subsets (nonparametric analysis). The events examined
were the stage of disease, presence or absence of disease progression,
history of treatment, and history of hematologic autoimmune disease.
). In contrast, most
of CD38+ patients had a progressive disease (
) as did a
large proportion of patients belonging to the bimodal expression group.
The differences among the 3 groups are highly significant
(P < .001). (B) The strong tendency to develop
progressive disease was not related to the percentage of
CD38+ cells as shown by the analysis of CD38+
bimodal expression patients divided according to the 30% positivity
cutoff (< 30% and 
30%).
indicates a patient with stable
disease; 
, one with progressive disease). For clarity, 2 horizontal lines encompass the CLL patients with a CD38 bimodal
expression profile and a vertical line divides mutated (< 98%) from
unmutated (