CD38 Expression Is an Important Prognostic Marker in Chronic Lymphocytic Leukaemia

JDu¨rig1, M Naschar1, U Schmu¨cker1, K Renzing-Ko¨hler2,THo¨lter2,AHu¨ttmann1 and U Du¨hrsen1

1Department of Haematology, Medical Faculty, University of Essen, Essen, Germany; and 2Institute of Medical Informatics, Biometry and Epidemiology, Medical Faculty, University of Essen, Essen, Germany

Employing a multicolour flow cytometry assay, 133 B-chronic CD38 is a single-chain type II transmembrane glycoprotein lymphocytic leukaemia (B-CLL) cases were analysed for sur- that is expressed by a variety of haematologic cells in an acti- face expression of CD38. Based on a cut-off value of 20%, CLL 11 vation- and differentiation-dependent manner. Its cellular (56 ؍ patients were categorised into a CD38-positive (у20%, n and separately functions include a complex ectoenzymatic activity and the (77 ؍ and a CD38-negative subgroup (<20%, n analysed for clinical and laboratory parameters. Patients in the ability to transduce signals involved in the regulation of cell CD38-positive cohort were characterised by an unfavourable proliferation and survival.12 Furthermore, it mediates a sel- clinical course with a more advanced disease stage, poor ectin-like binding to endothelial cells, thus functioning as an responsiveness to chemotherapy, short time to initiation of adhesion molecule.13 In normal human B cell development, first treatment and shorter survival. In contrast, the CD38- negative group required minimal or no treatment, remained CD38 exhibits a discontinuous expression pattern, where the treatment-free for a longer time period and had prolonged sur- molecule is detected at high levels in B cell precursors, germi- vival (P < 0.05). CD38 expression was a robust marker in the nal centre and plasma cells, while circulating peripheral majority of patients in that it was stable over time and not sig- blood and tonsillar B cells have markedly lower CD38 surface nificantly influenced by chemotherapy. In conclusion, our data expression.12 The potential role of CD38 in CLL pathophysiol- confirm recent studies suggesting a role of CD38 as a predictor ogy is presently unknown. However, it is tempting to specu- of clinical outcome in patients with B-CLL. Leukemia (2002) 16, 30–35. DOI: 10.1038/sj/leu/2402339 late that differences in CD38–ligand interaction between CLL Keywords: B-CLL; prognostic marker; CD38 clones may influence their proliferative behaviour and chance of survival.

Unlike analysis of the Ig VH gene mutation status, flow cyto- Introduction metric detection of CD38, expression can be conveniently performed in most clinical laboratories and may prove a valu- B cell chronic lymphocytic leukaemia is a heterogenous dis- able adjunct in the current staging system for predicting the ease with a highly variable clinical course. Staging systems clinical outcome in B-CLL patients. To explore this possibility devised by Rai et al1 and Binet et al2 are useful methods for further, we studied CD38 expression in 133 B-CLL patients predicting survival and treatment requirements in patients and correlated the results with clinical and laboratory with CLL. However, these staging systems are of limited prog- parameters. nostic value in early stages of the disease (Binet Aor Rai stage 0 to 2), which include most of the patients at diagnosis.3,4 Therefore, a number of studies have focused on identifying Materials and methods novel prognostic markers, which may help define patient subgroups with favourable vs poor clinical outcome in early Patients CLL.5,6 Recently, two independent studies by Damle et al7 and Hamblin et al8 have demonstrated that B-CLL may arise from Between May 1994 and December 2000, 133 consecutive either an immature pregerminal centre B cell with unmutated patients with chronic lymphocytic leukaemia from a single institution were enrolled in this study and analysed for several immunoglobulin (Ig) variable heavy chain (VH) genes or from a more mature post-germinal-centre B cell with somatically biological and clinical characteristics: age, sex, Binet stage, mutated Ig V genes. Moreover, Damle et al7 found a strong white blood cell count, haemoglobin level, platelet count, lac- H tate dehydrogenase, thymidine kinase, immunoglobulin A correlation between the Ig VH gene mutation status, CD38 surface expression of the respective B-CLL clone and clinical out- (IgA) serum-concentration, survival, treatment history and come in individual patients. B-CLL cases with mutated Ig V time from diagnosis to first treatment. In each patient morpho- H logic diagnosis of B-CLL was confirmed flow cytometrically14 genes and low numbers of CD38-positive cells exhibit a ␬ favourable clinical course, while B-CLL patients with unmu- revealing a typical CD19,20,5,23-positive Ig light chain, ( or ␭ light chain) restricted immunophenotype. Patient character- tated Ig VH genes are characterised by a poor outcome in terms of reduced survival and responsiveness to chemo- istics are shown in Table 1. The mean follow-up time was therapy. However, in a follow-up study no association 64.3 months (range 0–299). between CD38 expression and Ig VH gene mutation status was found, although the independent prognostic impact of both Ig 9 Cell surface staining and flow cytometry VH gene mutation and CD38 positivity was confirmed. By 10 contrast, Thunberg et al could not find any prognostic = significance of CD38 expression in their CLL patient cohort. Fresh heparinised peripheral blood (PB, n 91) and bone marrow (BM, n = 42) samples were prepared for flow cytometry by ammonium chloride erythrocyte lysis (Ortho-mune Lysing Correspondence: J Du¨rig, Department of Haematology, University Reagent; Ortho Diagnostic Systems, Raritan, NJ, USA). The Hospital Essen, D-45122, Essen, Germany; Fax: 0049 201 723 2304 immunophenotype was characterised using the following Received 24 April 2001; accepted 3 August 2001 panel of fluorochrome-labeled monoclonal antibodies CD38 expression in CLL JDu¨rig et al 31 Table 1 Comparison of clinical and laboratory data between CD38-positive and CD38-negative patients

Variable All patients CD38-positive patients CD38-negative P value patients

No. of patients (%) 133 (100) 56 (42.1) 77 (57.9) Median age (years) 63 62 64 0.22b Male sex (%) 93 (69.9) 44/56 (78.6) 49/77 (63.6) 0.08b Disease stage at diagnosis (%) (n = 125) Binet A 84/125 (67) 28/52 (53.8) 56/73 (75.7) 0.022b B 25/125 (20.0) 13/52 (25.0) 12/73 (16.4) C 16/125 (12.8) 11/52 (21.1) 5/73 (6.8) Disease stage during study (%) (n = 126) Binet A 57/126 (45.2) 17/52 (32.7) 40/74 (54.1) 0.053b B 24/126 (19.0) 11/52 (21.1) 13/74 (17.6) C 45/126 (35.7) 24/52 (46.1) 21/74 (28.3) White blood cell count (×10−3/␮l) (n = 127) 57.7 41.2 69.1 0.018a Haemoglobin (g/dl) (n = 131) 12.9 12.6 13.1 0.225a Platelet count (×10−3/␮l) (n = 131) 154.4 137.1 166.2 0.024a Lactate dehydrogenase (IU/l) (n = 122) 227 258 205 0.003a Ig A serum concentration (mg/dl) (n = 111) 1.9 2.1 1.7 0.638a Thymidine kinase (IU/l) (n = 27) 14.9 17.2 13.9 0.395a ␤ = a 2-microglobulin (mg/l) (n 77) 2.9 3.3 2.6 0.033 Bone marrow histology (% infiltration) (n = 85) 55.1 65.4 47.8 0.006a aThe P value is for the comparison among the CD38-positive and CD38-negative subgroups and was calculated using the Mann–Whitney U test. bThe P value is for the comparison among the CD38-positive and CD38-negative subgroups and was calculated using the chi-square test. Because of rounding, percentages do not always add to 100. employing a standard three-colour flow cytometry approach Whitney U test (for quantitative variables) and chi-square test (Figure 1):14 CD45-fluorescein isothiocyanate (FITC)/CD14- (for categorial variables). The Cox proportional model was phycoerythrin (PE)/CD20-peridinin chlorophyll (PerCP); CD4 used for multivariate analyses on overall survival. Significance (FITC)/CD8 (PE)/CD3 (PerCP); ␬ light chain (FITC)/CD19 was defined as P = 0.05, as determined by the two-tailed test. (PE)/CD5-phycoerythrin-cyanin (PeCy5); ␭ light chain (FITC)/CD19 (PE)/CD5 (PeCy5); IgM (FITC)/CD23 (PE)/CD19 (PECy5); CD10 (FITC)/CD38 (PE)/CD19 (PECy5). Antibodies Results were purchased from DAKO (Glostrup, Denmark; CD19, CD10, IgM, ␬ and ␭ light chains), Immunotech (Marseille, CD38 expression in B-CLL France; CD5) and Becton Dickinson (Heidelberg, Germany; CD38, CD4, CD8, CD3). Negative isotype-matched controls We evaluated the surface expression of CD38 in 133 cases of (Becton Dickinson) were used to define the threshold line B-CLL employing a three-colour flow cytometry approach separating surface marker positive and negative cells such that with directly conjugated monoclonal antibodies (Figure 1). As less than 1% of isotype-positive cells were present to the right per current convention, a given leukaemic population was of the line (Figure 1). ACLL population was considered CD38- considered positive for CD38 when у20% of the B-CLL cells positive when more than 20% of the gated population expressed the membrane marker.15,16 Figure 2 shows the dis- (CD19+/CD5+ cells) expressed it. To minimize potential con- tribution of CD38 in the whole patient cohort. Based on the tamination with coexisting normal B cells, only B-CLL cases 20% cut-off value, 56 patients (42%) were defined as in which Ͼ90% of CD19+ cells co-expressed CD5 were CD38-positive and 77 patients (58%) as CD38-negative, included in the study (Figure 1). In all experiments, a mini- respectively. mum of 10000 cells was analysed. The flow cytometer Twenty-seven patients (13 CD38+ and 14 CD38− patients) (FACScan; Becton Dickinson) was calibrated with CAliBRITE- were studied at two or more time-points (range 2 to 4). As 3 beads (Becton Dickinson) and FACSComp Software (Becton illustrated in Figure 3, CD38 expression in individual patients Dickinson). Data acquisition and analysis was performed was relatively stable over time and not substantially influ- using Attractors and CellQuest software (Becton Dickinson). enced by chemotherapy (five patients of the CD38− and 10 The same method of sample preparation and three-colour patients of the CD38+ subgroup received chemotherapy in the staining was used throughout the entire study period. observation period) in the majority of patients analysed. Vari- ations in CD38 staining over time were particularly high in CLL cases with CD38 levels around 50%. However, in none Statistical analysis of the cases studied did we observe a cross-over of a CD38- negative patient to the CD38-positive cohort and vice versa Survival times and censored waiting times measured from the (Figure 3). time of diagnosis were plotted by the Kaplan–Meier method In eight CLL patients, comparative analysis of CD38 in bone and compared using the log-rank test. Comparison of clinical marrow and peripheral blood samples obtained at identical and laboratory parameters between the CD38-positive and time-points yielded comparable results (Table 2) in both CD38-negative subgroups was performed using the Mann– groups, suggesting that CD38 expression levels on B-CLL cells

Leukemia CD38 expression in CLL JDu¨rig et al 32

Figure 1 Representative ﬂow cytometry dot plot analyses of CD38 expression in patients with B-CLL. Three-colour ﬂow cytometry as detailed in Materials and methods was used to analyse CD38 surface expression on lymphocyte/monocyte gated CD19+ B cells co-expressing CD5. (a) Sample of a patient positive for CD38 (20% or more). (b) Sample of a patient negative for CD38 (less than 20%). Numbers are percentages of CD19+ B CLL cells expressing CD38.

may be largely independent of the surrounding cellular The median survival of patients in the CD38+ cohort was 121 microenvironment. months, whereas the median survival for the CD38− group was not reached for the duration of follow-up (P = 0.00655). Acomparison of further clinical and laboratory parameters Correlation of CD38 expression with clinical and among the two groups is shown in Table 1. Notably, signifi- laboratory data cant differences were found for Binet stage, white blood cell count, platelet count, lactate dehydrogenase, ␤2- The treatment histories of CD38+ and CD38− patients differed microglobulin and bone marrow histology (P Ͻ 0.05). significantly (Table 3) in that the former group required more intensive chemotherapy over longer time periods than CD38− patients (P = 0.004). The higher treatment intensity in CD38+ patients correlated with a more advanced disease stage observed, both at diagnosis and study enrollment (Table 1). Univariate analysis of risk factors Furthermore, we found highly significant differences in disease progression as indicated by the treatment-free interval (Figure 4). The mean treatment-free interval (Table 1) was Univariate Cox regression analysis was used to assess associ- longer in the CD38− group (120 months) than in the CD38+ ations between survival time and potential risk factors. Binet patient cohort (32 months, P = 0.00008). Finally, we com- stage (during follow-up), haemoglobin levels, platelet counts pared overall survival among the two groups and again, and CD38 expression and IgAserum concentration were observed statistically highly significant differences (Figure 5). identified as significant factors influencing survival (Table 4).

Leukemia CD38 expression in CLL JDu¨rig et al 33 Table 2 Intraindividual comparison of CD38 expression on B-CLL cells in peripheral blood (PB) and bone marrow (BM) samples obtained at simultaneous timepoints

Patient-Id (UPN) Bone marrow Peripheral blood

20 18.0 23.4 50 53.0 55.0 63 74.5 76.6 64 79.5 74.4 101 0.8 0.4 104 31.7 31.5 111 36.0 39.0 113 83.0 86.0 Mean ± s.d. 47.0 ± 30.4 48.3 ± 29.8

Table 3 Comparison of treatment histories among CD38-positive and CD38-negative patients

CD38-negative CD38-positive patients patients

Patients requiring no or 74.0% 47.2% minimal treatmenta (54/73) (25/52)

Figure 2 Distribution of CD38 expression in the B-CLL study Patients requiring 26.0% 52.8% population (n = 133). CD38 expression was analysed employing a continuous chemotherapy (19/73) (27/52) = b multicolour ﬂow cytometry assay as described in Materials and or chemotherapy with two P 0.004 methods. The dotted vertical line represents the 20% cut-off used to or more agents or separate CD38-negative from CD38-positive patients. Data are per- regimens centages of CD38+/CD19+ B-CLL cells co-expressing CD5. aMinimal treatment is deﬁned as less than 6 months of therapy in the years of follow-up.7 bThe P value was calculated using the chi-square test.

Figure 4 Probability of disease progression, as indicated by the treatment-free interval. Kaplan–Meier plot comparing time periods Figure 3 CD38 expression in B-CLL over time. Twenty-seven from diagnosis to initiation of chemotherapy in CD38-positive to patients were ﬂow-cytometrically analysed for CD38 expression at CD38-negative CLL patients. Mean time from diagnosis to ﬁrst treat- two or more time-points (range 2 to 4). Each line in the graph rep- ment (months) was 120 vs 32 months in the CD38-negative and resents an individual patient. CD38-positive group, respectively. Statistical analysis was performed using the log-rank test.

Multivariate analysis CD38, Binet stage (during follow-up), platelet count, haemo- The following patient characteristics found to impact signiﬁ- globin and IgA(Table 5). In multivariate analysis platelet cantly on survival in the univariate analysis were included in count, haemoglobin and IgA, but not CD38 and Binet stage the Cox regression model (complete case analysis, n = 102): inﬂuenced overall survival.

Leukemia CD38 expression in CLL JDu¨rig et al 34 immunoreactivity in sequential samples obtained at different time-points. This finding may be due to technical problems encountered in the gating procedure used to define CD38 expression levels, ie small ‘random’ deviations in cursor pos- itioning impact more profoundly on CLL populations with intermediate CD38 expression than on leukaemic populations with either high or low CD38 expression levels. However, due to the comparably small number of patients analysed in this subset of experiments, we cannot exclude the possibility that the observed differences indeed reflect fluctuations in CD38 expression in a minority of patients. Furthermore, intraindividual comparisons of specimens obtained simultaneously from peripheral blood and bone marrow yielded similar results. Taken together, these observations suggest that CD38 may be a robust marker that could be used reliably in most routine flow cytometry laboratories. Clearly, these data need to be confirmed in a larger patient cohort. Figure 5 Overall survival in CD38-negative and CD38-positive B- More importantly, we found that CLL cases displaying a CLL patients. Kaplan–Meier plot comparing survival based on CD38 high percentage of CD38 (у20%) are characterised by an expression. The mean survival of patients in the CD38+ cohort was − unfavourable clinical course as compared with the CD38 121 months, whereas the median survival for the CD38 group was negative patients (CD38 Ͻ20%). In particular, our data show not reached for the duration of follow-up. Statistical analysis was performed using the log-rank test. significant differences in terms of overall survival and treatment requirements between the two groups confirming the work of Damle et al7 and Hamblin and colleagues9 and Table 4 Univariate analysis of prognostic variables for survival extending it to a larger patient cohort. Furthermore, the time from diagnosis to initiation of chemotherapy, used as a surro- Variable Relative risk P valuea gate marker for progression-free survival,17 was significantly reduced in the CD38-positive, as compared to the CD38- Sex 0.89 0.84 negative patient cohort. Age 0.99 0.76 These findings contrast those of a recent report proposing CD38 1.02 0.02 that CD38 is a poor predictor of prognosis in CLL.10 This dis- White blood cell count 1.00 0.37 Platelet count 0.99 0.0013 crepancy may be at least partly explained by technical differ- Haemoglobin 0.65 0.0005 ences between the studies, ie the latter authors used a two- Binet stage at diagnosis 1.76 0.06 colour rather than a three colour flow cytometric assay and Binet stage during follow-up 2.35 0.03 thus may have included normal residual B cells in their analy- Lactate dehydrogenase 1.00 0.10 sis, also the patient number evaluated for CD38 (n = 44) was IgA serum concentration 1.49 0.003 relatively small. Bone marrow histology 1.00 0.77 The CD38 cut-off level employed for risk stratification in 10 a CLL patients remains a matter of controversy. In the initial Statistical comparisons were performed using Cox regression 7 analysis. study published by Damle et al a 30% cut-off value was chosen empirically based on a plot of CD38 expression vs Ig

VH gene mutation status. Unmutated cases were found to Discussion exhibit a higher percentage of CD38 expression than mutated (post-germinal centre) B-CLL clones. However, while the This retrospective study was performed to evaluate the poten- prognostic significance of CD38 expression has been con- tial of CD38 expression as a prognostic indicator in B-CLL. In firmed in two follow-up studies9,18 and now by the data line with previous data,7 our results demonstrate that in the presented here, the association between CD38 expression and 7,9,10 majority of patients the proportion of CLL cells co-expressing Ig VH gene mutation remains conjectural. Thus, with the CD38 as determined by standard three-colour flow cytometry current state of knowledge, Ig VH gene mutation status and is relatively stable over time and does not appear to be influ- CD38 expression appear to be independent prognostic factors enced by chemotherapy. However, in a small subset of in B-CLL. The 20% CD38 cut-off value used in the present patients characterised by CD38 expression levels around report was selected by reference to previously published 50%, we observed substantial variations in CD38 surface immunophenotyping studies of haematologic malignancies

Table 5 Multivariate Cox proportional hazard model using 102 patients with B-CLL

Variable Parameter estimate Standard error Chi-square Hazard ratio P value

CD38 −0.00986 0.00949 1.0786 0.990 0.2990 Binet stage −0.35752 0.69264 0.2664 0.699 0.6057 Platelet count −0.02379 0.01076 4.8920 0.976 0.0270 Haemoglobin −0.43280 0.20906 4.2859 0.649 0.0384 IgA 0.56952 0.16191 12.3721 1.767 0.0004

Leukemia CD38 expression in CLL JDu¨rig et al 35 and the recent proposals of the EGIL group.15,16 To better 6 Binet JL. Prognostic factors in chronic lymphocytic leukaemia. compare our data with those of previous studies7,9,18 survival Haematologica 1999; 84: 96–97. analyses were recalculated using the 30% cut-off value 7 Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL, Buch- 7,9,18 binder A, Budman D, Dittmar K, Kolitz J, Lichtman SM, Schulman employed by other authors. Interestingly, the resulting P, Vinciguerra VP, Rai KR, Ferrarini M, Chiorazzi N. Ig V gene survival curves did not differ significantly from those pre- mutation status and CD38 expression as novel prognostic indi- viously obtained with the 20% cut-off value (data not shown). cators in chronic lymphocytic leukemia. Blood 1999; 94: 1840– Furthermore, the median survival of our CD38-positive group 1847. (121 months) was very similar to that reported by Damle et 8 Hamblin T, Davis Z, Gardiner A, Oscier DG, Stevenson FK. al7 (120 months) and Hamblin and colleagues9 (105 months). Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999; 94: 1848– In conclusion, our data confirm previous studies showing 1854. that CD38 expression is a novel prognostic marker in B-CLL. 9 Hamblin T, Orchard JA, Gardiner A, Oscier DG, Davis Z, Steven- It will be important to determine whether this parameter in son FK. Immunoglobulin V genes and CD38 expression in CLL. conjunction with other established prognostic factors can Blood 2000; 95: 2455–2457. improve risk stratification in the routine diagnostic work-up of 10 Thunberg U, Johnson A, Roos G, Thorn I, Tobin G, Sallstrom J, CLL patients. Sundstrom C, Rosenquist R. CD38 expression is a poor predictor for VH gene mutational status and prognosis in chronic lymphocytic leukemia. Blood 2001; 97: 1892–1894. 11 Deaglio S, Mehta K, Malavasi F. Human CD38: a (r)evolutionary story of enzymes and receptors. Leuk Res 2001; 25: 1–12. Acknowledgements 12 Kumagai M, Coustan-Smith E, Murray DJ, Silvennoinen O, Murti KG, Evans WE, Malavasi F, Campana D. Ligation of CD38 sup- presses human B lymphopoiesis. J Exp Med 1995; 181: 1101– This work represents a part of MNs MD thesis. We are 1110. indebted to numerous colleagues for generously contributing 13 Deaglio S, Mallone R, Baj G, Arnulfo A, Surico N, Dianzani U, information on the clinical course and treatment histories of Mehta K, Malavasi F. CD38/CD31, a receptor/ligand system ruling the study patients. adhesion and signaling in human leukocytes. Chem Immunol 2000; 75: 99–120. 14 Ho¨ffkes HG, Schmidtke G, Uppenkamp M, Schmu¨cker U. Multi- parametric immunophenotyping of B cells in peripheral blood of healthy adults by flow cytometry. Clin Diagn Lab Immunol 1996; References 3: 30–36. 15 Bene MC, Bernier M, Casasnovas RO, Castoldi G, Knapp W, 1 Rai KR, Sawitsky A, Cronkite EP, Chanana AD, Levy RN, Pas- Lanza F, Ludwig WD, Matutes E, Orfao A, Sperling C, van’t Veer ternack BS. Clinical staging of chronic lymphocytic leukemia. MB. The reliability and specificity of c-kit for the diagnosis of Blood 1975; 46: 219–234. acute myeloid leukemias and undifferentiated leukemias. The Eur- 2 Binet JL, Auquier A, Dighiero G, Chastang C, Piguet H, Goasguen opean Group for the Immunological Classification of Leukemias J, Vaugier G, Potron G, Colona P, Oberling F, Thomas M, Tchernia (EGIL). Blood 1998; 92: 596–599. G, Jacquillat C, Boivin P, Lesty C, Duault MT, Monconduit M, 16 Bene MC, Castoldi G, Knapp W, Ludwig WD, Matutes E, Orfao Belabbes S, Gremy F. Anew prognostic classification of chronic A, van’t Veer MB. Proposals for the immunological classification lymphocytic leukemia derived from a multivariate survival analy- of acute leukemias. European Group for the Immunological sis. Cancer 1981; 48: 198–206. Characterization of Leukemias (EGIL). Leukemia 1995; 9: 1783– 3 Rai KR. Chronic lymphocytic leukaemia. Current strategy and new 1786. perspectives of treatment. Haematologica 1999; 84: 94–95. 17 Do¨hner H, Stilgenbauer S, Benner A, Leupolt E, Krober A, Bul- 4 Rai KR, Peterson BL, Appelbaum FR, Kolitz J, Elias L, Shepherd L, linger L, Do¨hner K, Bentz M, Lichter P. Genomic aberrations and Hines J, Threatte GA, Larson RA, Cheson BD, Schiffer CA. Fludara- survival in chronic lymphocytic leukemia. New Engl J Med 2001; bine compared with chlorambucil as primary therapy for chronic 343: 1910–1916. lymphocytic leukemia. New Engl J Med 2000; 343: 1750–1757. 18 Matrai Z, Lin K, Dennis M, Sherrington P, Zuzel M, Pettitt AR, 5 Zwiebel JA, Cheson BD. Chronic lymphocytic leukemia: staging Cawley JC. CD38 expression and Ig VH gene mutation in B cell and prognostic factors. Semin Oncol 1998; 25: 42–59. chronic lymphocytic leukemia. Blood 2001; 97: 1902–1903.

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