Prognostic significance of serum immunoglobulin paraprotein in patients with chronic lymphocytic leukemia

Leukemia Research 35 (2011) 1060–1065

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Prognostic significance of serum immunoglobulin paraprotein in patients with chronic lymphocytic leukemia Wei Xu, Yin-Hua Wang, Lei Fan, Cheng Fang, Dan-Xia Zhu, Dong-Mei Wang, Chun Qiao, Yu-Jie Wu, Jian-Yong Li ∗ Department of Hematology, the First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, 300 Guangzhou Road, Nanjing 210029, China

a r t i c l e

i n f o

Article history: Received 16 November 2010 Received in revised form 16 November 2010 Accepted 6 December 2010 Available online 3 January 2011 Keywords: Chronic lymphocytic leukemia Immunoglobulin paraprotein Prognosis

a b s t r a c t The aim of this study was to explore the clinical and other associated laboratory features of chronic lymphocytic leukemia (CLL) patients with immunoglobulin (Ig) paraproteinemia. Serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) were performed to measure serum Ig paraprotein. The correlations between serum Ig paraprotein and other prognostic factors were analyzed. Univariate and multivariate Cox regression analyses were used to assess associations between survival time and potential risk factors. In 133 Chinese CLL patients, 27 (20.3%) patients occurred Ig paraproteinemia at diagnosis. According to the correlation analysis, advanced Binet stage (r = 0.314, P < 0.001), direct antiglobulin test (DAT)-positive (r = 0.366, P < 0.001), high level of serum ␤2-microglobulin (␤2MG) (r = 0.296, P = 0.001) and thymidine kinase (TK) 1 (r = 0.227, P = 0.037), unmutated immunoglobulin heavy chain variable gene (IGHV) status (r = 0.284, P = 0.002), ZAP-70-positive (r = 0.305, P = 0.001), CD38positive (r = 0.284, P = 0.002), and cytogenetic abnormalities of del(17p13) or del(11q22.3) (r = 0.208, P = 0.032) emerged as factors significantly related to the occurrence of Ig paraproteinemia. Survival analysis showed that the patients with Ig paraproteinemia had significantly shorter survival times than the patients without serum Ig paraprotein (P = 0.013). Binet stage (P = 0.028), high levels of lactate dehydrogenase (LDH) (P = 0.004), IgG paraproteinemia (P = 0.048), IgM paraproteinemia (P = 0.001), ZAP-70-positive (P = 0.003), DAT-positive (P = 0.013), unmutated IGHV status (P = 0.009), and del(17p13) (P = 0.001) were the adverse factors in determining overall survival (OS). Del(17p13) (P = 0.006), ZAP-70 (P = 0.030), and IgM paraproteinemia (P = 0.040) were the variables strongly associated with OS by multivariate Cox regression analysis. It was showed that serum Ig paraprotein might be applied for the assessment of prognosis in patients with CLL. © 2010 Published by Elsevier Ltd.

1. Introduction Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia in the Western world, however, infrequent in the Eastern. It shows a notable heterogeneity, with some CLL patients having an almost normal life span, others surviving only within 2 years after diagnosis despite intensive therapy. Although most patients are asymptomatic at diagnosis, some can have systemic (B type) symptoms, hepatosplenomegaly, lymphadenopathy, and cytopenias due to leukemic infiltration of bone marrow and other organs. As defined in the World Health Organization (WHO) classification, CLL is a neoplasm composed of monomorphic small, round to slightly irregular B lymphocytes that are positive for monotypic surface immunoglobulin (dim intensity), CD5, CD19, CD20 (dim intensity), and CD23 and

∗ Corresponding author. Tel.: +86 25 83781120; fax: +86 25 83781120. E-mail address: [email protected] (J.-Y. Li). 0145-2126/$ – see front matter © 2010 Published by Elsevier Ltd. doi:10.1016/j.leukres.2010.12.005

usually negative or dimly positive for CD22, CD79b, and FMC-7 [1]. Serum immunoglobulin (Ig) paraprotein can be detected in a subset of patients with CLL by serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE). The WHO classification recognizes that a “small M component” can be found in some patients with CLL, but no mention is made of the frequency of this occurrence or the range of serum paraprotein levels in patients with CLL [1]. An earlier study reported that patients with CLL with Ig paraproteinemia had an inferior survival compared with patients with CLL without serum paraprotein [2]. However, other studies have not confirmed this observation [3]. Therefore, how does the serum Ig paraprotein actually affect the CLL patients’ survival is still unknown. To better describe the prevalences of Ig paraproteinemia in patients with CLL, and the prognostic significance of a serum Ig paraprotein in CLL populations, we retrospectively analyzed 133 CLL patients followed at a single center. The main objectives of the study were to seek clinical and other associated laboratory

W. Xu et al. / Leukemia Research 35 (2011) 1060–1065

features that might provide useful information in the management of patients with Ig paraproteinemia. 2. Patients and methods 2.1. Patients

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cells were cultured in RPMI 1640 medium supplemented with 20% fetal calf serum, 100 U/ml penicillin, and 100 ␮g/ml streptomycin, which were incubated in the presence of 200 U/ml interleukin 2 and 2 ␮M CpG-ODN DSP30 (TCGTCGCTGTC TCCGCTTCTTCTTGCC) (Invitrogen, Shanghai, China). After 72 h, colcemid (Sigma–Aldrich) was added at 0.15 ␮g/ml, and chromosome preparation, staining, and classification were performed according to standard protocols [9]. The metaphase number ≥20 was considered as successful stimulation. Patients with complex karyotype are defined by ≥3 aberrations. The karyotype reports were written using the International System for Human Cytogenetic Nomenclature [10].

Between January 2004 and October 2010, 133 consecutive patients with newly diagnosed CLL were enrolled in this study. All patients provided informed consent according to our institutional guidelines. The diagnosis of CLL was based on clinical characteristics, peripheral blood and bone marrow morphology, immunophenotype, and peripheral blood absolute lymphocyte count (ALC) ≥ 5.0 × 109 /l according to the National Cancer Institute (NCI) criteria [4]. The immunophenotype of CLL cells was co-expression of CD5, CD19 and CD23, and weak expression of surface immunoglobulin, CD20, and CD79b. The staging of CLL was performed according to the Binet staging system [5]. Data collected on the characteristics of patients at diagnosis included (either in all or in the majority of patients): age, gender, Binet stages, ␤2-microglobulin (␤2-MG), lactate dehydrogenase (LDH), direct antiglobulin test (DAT), and serum Ig paraprotein. A range of other prognostic markers was also analyzed for the majority of patients: serum thymidine kinase (TK) 1 concentration, immunoglobulin heavy chain variable gene (IGHV) mutational status, CD38 and ZAP-70 expression, and cytogenetic aberrations.

FISH analysis was performed on the sample for conventional cytogenetic studies. In order to detect prognostically relevant anomalies of chromosomal regions 6q, 11q, 13q, 14q, 17p and chromosome 12, the following fluorescent-labeled probes were used in interphase cytogenetic analyses: LSI MYB (6q23), LSI ATM (11q22.3), LSI D13S319 (13q14), LSI IGHC/IGHV (14q32), LSI p53 (17p13) and CEP12 (centromere 12) (all probes purchased from Vysis, Downers Grove, IL, USA). FISH was performed as previously described [11]. The cut-off levels for positive values (mean of normal control ± 3 SD), determined from samples of 8 cytogenetically normal persons, was 7.5% for del(6q23), and 7.7%, 10.3%, 8.9%, 5.2% and 3.0% for del(11q22.3), del(13q14), 14q32 translocation, del(17p13) and trisomy 12, respectively.

2.2. Detection of a serum Ig paraprotein

2.8. Statistical analysis

Serum Igs and light chains (␬, ␭) were measured with rate nephelometry on Beckman Array 360. SPE and IFE were run with REPII and SPIFE 2000 (Helena Laboratories, USA), respectively. M-spike was quantitated after band scanning and EZ1.51 (Helena Laboratories, USA) software analysis. The diagnosis of M protein and consensus isotype was based on IFE, SPE, Igs as well as light chain measurement. Among these methods, SPE was used for screening test, while IFE was for confirmation and isotype identification. Measurement of Igs and light chain were used for M protein quantitation and confirmation that was complementary to IFE and SPE.

All statistical analyses were performed using the SPSS program for Windows (version 15.0). An effect was considered statistically significant at P < 0.05. Variables examined were age at diagnosis, gender, Binet stages, ␤2-MG, LDH, DAT, TK1, IGHV mutational status, ZAP-70 protein, CD38 expression level, cytogenetic aberrations, serum Ig paraprotein, and overall survival (OS). OS time was measured from the diagnosis date to either the last follow-up date (censored) or to the time of death by any cause. Comparisons between categoric variables were performed using the chi-square test. The Kaplan–Meier method was used to construct survival curves, and results were compared using the log-rank test. A two-variables Cox regression analysis was used to determine those factors that were independently associated with a serum Ig paraprotein result, and a multivariate Cox regression analysis was used to assess associations between survival time and potential risk factors.

2.3. Assay of serum TK1 concentration Enhanced chemiluminescent (ECL) dot blot assay was performed according to as previously described [6]. Anti-TK1 antibody was purchased from SSTK Inc., Shenzhen, China. The cut-off value of serum TK1-positive was 2 pM.

2.7. Detection of molecular cytogenetic aberrations by FISH

3. Results

2.4. Detection of ZAP-70 and CD38

3.1. Patients Flow cytometry was used to detect ZAP-70 and CD38 expression on fresh samples stained with CD5-FITC, CD19-PerCPCy5.5, ZAP-70-PE (clone 1F7.2; Caltag, Burlingame, CA, USA) and CD38-PE (clone HB-7; BD Biosciences, San Jose, CA, USA). It was performed as previously described [7]. Isotype controls were run with each sample to distinguish positive from negative cells. To detect ZAP-70, lymphocyte cells were gated further to select CD5+ CD19− cells (T cells), which were used as an internal positive control, and CD5+ CD19+ cells (CLL cells). After appropriate lymphocytes gating, cytoplasmic ZAP-70 expression was determined in CD5+ CD19+ CLL cells. CD38 expression was measured in CD5+ CD19+ CLL cells population. Data acquisition and analysis were performed using a FACSCalibur flow cytometer (BD Biosciences) and Cell Quest software (BD Biosciences). The cut-off point for ZAP-70-positive in CLL cells was >20% and CD38-positive was >30%, respectively. 2.5. Analysis of IGHV somatic mutational status To determine IGHV gene somatic mutational status in CLL cells, the cDNAs were amplified using IGHV primers, a Somatic Hypermutation Assay for Gel Detection kit including MIX I and MIX II, purchased from InVivoScribe, USA. The Hypermutation Mix I targets sequences between the leader and joining regions. Therefore the amplicon product(s) span the entire variable (V) region, which contains the FR1 (framework region 1), CDR1 (complementarity determining region 1), FR2, CDR2, and FR3 regions. The Hypermutation Mix II targets sequences were between the framework 1 (FR1) and joining (J) regions. The resulting amplicons include a portion of the FR1 region to the downstream J region. MIX I was first applied for IGHV gene amplification. The samples that failed to amplify with MIX I were amplified again with Mix II. PCR amplification performed as previously described [8]. The majority of samples were sequenced directly using an automated DNA sequencer (ABI 377; Applied Biosystems, Foster City, CA, USA). In case of the failure of direct sequencing, the sequence was determined by cloning. Nucleotide sequences were aligned and IGHV somatic mutational status was analyzed using one of the following tools: IMGT’s V-QUEST analysis tool (http://imgt.cines.fr/) and NCBI’s IgBlast tool (http://www.ncbi.nlm.nih.gov/igblast/). Two percentage deviation from a germline VH sequence was used to determine the mutational status of IGHV gene. 2.6. Conventional cytogenetics Conventional cytogenetic analyses (CCA) with R-banding technique was performed. For metaphase induction, (1–2) × 106 /ml peripheral blood mononuclear

The characteristics of 133 patients with CLL are summarized in Table 1. Eighty-six patients were male and 47 were female (male:female ratio, 1.8), and the median age at diagnosis was 60 years (range, 44–84 years). 3.2. Results of serum Ig paraprotein We identified 27 patients with Ig paraproteinemia (12 patients with IgG paraprotein, 12 patients with IgM paraprotein, 3 patients with both IgG and IgM, and no patients with IgA) from a total of 133 untreated patients with CLL, for a frequency of 20.3%. There were 20 male and 7 female (male:female ratio, 2.9) with a median age of 65 years (range, 44–83 years). The clinical features of 27 CLL patients with immunoglobulin paraprotein are summarized in Table 2. The median level of serum IgG paraprotein was 28.2 g/l (range, 21.0–62.0 g/l), and IgM paraprotein was 18.1 g/l (range, 2.3–49.1 g/l). The serum monoclonal Ig light chain (␬ or ␭) was concordant with the monoclonal Ig light chain (␬ or ␭) expressed by the neoplastic cells in all patients with Ig paraproteinemia. 3.3. Correlations between serum Ig paraprotein and other prognostic factors The possibility of interaction between serum Ig paraprotein and other known prognostic factors, such as Binet stages, DAT, serum level of ␤2-MG, LDH and TK1, IGHV mutational status, ZAP-70 and CD38 expression, and cytogenetic abnormalities, was analyzed in our cohort (Table 3). Strong correlations of serum Ig paraprotein with advanced Binet stage (r = 0.314, P < 0.001), DAT-positive (r = 0.366, P < 0.001), high level of ␤2-MG (r = 0.296,

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Table 1 The characteristics of 129 patients with CLL. Characteristic Sex Male Female Age <60 years ≥60 years Binet stage A B C DAT (n = 112) Negative Positive LDH (n = 125) <250 U/l ≥250 U/l ␤2-MG (n = 123) <3.0 mg/l ≥3.0 mg/l TK1 (n = 89) <2.0 pM ≥2.0 pM IGHV (n = 124) Mutated (>2% deviation from a germline) Unmutated (≤2% deviation from a germline) ZAP-70 (n = 128) <20% ≥20% CD38 (n = 129) <30% ≥30% Cytogenetics (n = 121) Unfavorable aberration (deletion in 17p13 or 11q22.3) Favorable aberration (deletion in 13q14 as the sole abnormality) Others

Cases (%) 86 (64.7) 47 (35.3) 64 (48.1) 69 (51.9) 47 (35.3) 43 (32.3) 43 (32.3) 76 (67.9) 36 (32.1)

IgM paraprotein, 2 patients with both IgG and IgM, respectively), whereas among the 106 patients without Ig paraproteinemia, 7 (6.6%) died. Patients with Ig paraproteinemia had significantly shorter survival times than patients without serum Ig paraprotein (P = 0.013, P = 0.048 and P = 0.001, respectively) (Figs. 1–3). The estimated mean OS of patients with and without Ig paraproteinemia were 63.5 months (95% confidence interval, 53.2–73.8 months) and 77.5 months (95% confidence interval, 73.7–81.3 months), respectively; with and without IgG paraproteinemia were 62.3 months (95% confidence interval, 49.0–75.6 months) and 76.5 months (95% confidence interval, 72.6–80.4 months), respectively; with and without IgM paraproteinemia were 55.8 months (95% confidence interval, 41.1–70.4 months) and 76.8 months (95% confidence interval, 73.0–80.5 months), respectively.

89 (71.2) 36 (28.8) 57 (46.3) 66 (53.7) 70 (78.7) 19 (21.3) 74 (59.7) 50 (40.3) 92 (71.9) 36 (28.1) 80 (62.0) 49 (38.0) 33 (27.3) 17 (14.0) 71 (58.7)

P = 0.001) and TK1 (r = 0.227, P = 0.037), absence of IGHV mutations (r = 0.284, P = 0.002), ZAP-70-positive (r = 0.305, P = 0.001), CD38-positive (r = 0.284, P = 0.002), and cytogenetic abnormalities of del(17p13) or del(11q22.3) (r = 0.208, P = 0.032) were observed. However, there was no noticeable correlation between serum Ig paraprotein and serum LDH level (r = 0.095, P = 0.202). 3.4. Prognostic impact of serum Ig paraprotein on OS With a median follow-up of 36 months (range, 4–83 months), 13 patients (9.8%) died. Univariate analysis was used to assess associations between survival and potential risk factors, including serum Ig paraprotein. As expected, advanced Binet stage (P = 0.028), high levels of LDH (P = 0.004), serum Ig paraprotein (P = 0.013), IgG paraproteinemia (P = 0.048), IgM paraproteinemia (P = 0.001), ZAP-70-positive (P = 0.003), DAT-positive (P = 0.013), unmutated IGHV status (P = 0.009), and del(17p13) (P = 0.001) were the adverse factors in determining OS. The prognostic factors with statistical significance were further used in a two-variables Cox analysis, which comparing serum Ig paraprotein to advanced Binet stage, high level of LDH, ZAP-70-positive, DAT-positive, unmutated IGHV status, or del(17p13) individually to show prognostic independence. Advanced Binet stage (P = 0.016), High level of LDH (P = 0.018), ZAP-70-positive (P = 0.036), and del(17p13) (P = 0.036) were the independent prognostic factors and strongly associated with OS. The prognostic factors with statistical significance were considered in a multivariate Cox regression analysis, del(17p13) (P = 0.006), ZAP-70 (P = 0.030), and IgM paraproteinemia (P = 0.040) were the variables strongly associated with survival. Ig paraproteinemia was associated with poor outcome. Six (22.2%) patients with Ig paraproteinemia died during the observation period (2 patients with IgG paraprotein, 2 patients with

4. Discussion Ig paraproteinemia, or monoclonal gammopathy, is the presence of excessive amounts of a single monoclonal gammaglobulin in the blood. It denotes an underlying immunoproliferative disorder. Serum Ig paraprotein can be detected in a subset of patients with CLL by conventional methods such as SPE and can be detected in a higher number of patients by more sensitive techniques [12]. CLL is an acquired clonal disorder characterized by progressive accumulation of functionally incompetent lymphocytes. Although the median survival of CLL is around 10 years, CLL patients have a highly variable clinical course. While some patients show an indolent disease and never require treatment, others suffer from a much more aggressive course requiring intensive treatment shortly or immediately after diagnosis. For these reasons, the markers giving prognostic information for the individual patient are needed. The limitation of clinical staging systems (Rai and Binet staging systems) in CLL [5,13], which fail to identify early-stage patients most likely to progress, has lead to the search for new prognostic markers with highly predictive capabilities. In search for additional prognostic factors, several parameters have shown relevance: lymphocyte doubling time, serum LDH, ␤2-MG and TK1, ZAP-70 and CD38 expression level, cytogenetic aberrations and mutational status of IGHV. Mutational status of the IGHV gene was proved to be a strong prognostic parameter identified in CLL, even among cases with a good prognosis (Binet stage A patients) [14]. Unfortunately, the determination of the presence of somatic mutations is a difficult, expensive, and time-consuming technique which will not be readily available for routine clinical use. There is dispute about the relationship between serum Ig paraprotein and survival in CLL. Bernstein and colleagues [2], in a study of 111 patients, reported that the presence of serum monoclonal protein in patients with CLL was associated with a shorter median survival: 63 months for patients with CLL with serum Ig paraprotein compared with 103 months for patients without serum Ig paraprotein (P = 0.012). However, Yin and colleagues have not confirmed this observation (P = 0.60) [3]. The unfavorable prognostic significance of serum Ig paraprotein was observed in this study. We report an incidence of Ig paraproteinemia of 20.3% in Chinese patients with CLL. The occurrence of Ig paraproteinemia was significantly related to adverse prognostic factors. The remarkable correlation of serum Ig paraprotein with advanced Binet stage, DAT-positive, high level of ␤2-MG and TK1, absence of IGHV mutations, ZAP-70-positive and CD38-positive, cytogenetic abnormalities of del(17p13) or del(11q22.3) were observed. In this study, OS was longer in patients without Ig paraproteinemia than patients with serum Ig paraprotein. The possible explanations of discrepancy in prognostic impact of serum Ig paraprotein might be different therapeutic regimens, shorter clinical follow-up and smaller number of patients.

Table 2 The clinical characteristics of 27 CLL patients with immunoglobulin paraprotein. No

Age (yr)

Binet stage

Serum Ig paraprotein

LDH (U/l)

1

F

57

B

IgG␬

278

2 3 4

M M M

79 70 55

C A A

IgM␬ IgG␭ IgG␭

5

M

54

B

6 7

M F

72 53

A C

8 9

M M

44 68

C B

10

M

65

11 12 13

M M F

14 15

␤2-MG (mg/l)

TK1 (pM)

DAT

ZAP70 (%)

CD38 (%)

VH homology (%)

Cytogenetics

FISH

OS (months)

3.3

2.1

Positive

44.2

87.1

100

46,XX[20]

67

184 178 199

2.8 4.0 2.5

1.0 NA 0.4

Negative NA Negative

NA 14.6 50.1

NA 0 50.8

98.5 NA 99.3

IgG␬

161

6.7

NA

Positive

66.7

60.4

99.1

192 505

2.9 5.2

NA 1.8

NA Positive

60.0 7.6

0 8.1

NA 99.9

del(13q14), del(17p13) NA Normal

36

IgM␬ IgG␬, IgM␬ IgM␭ Gig␭, IgM␭

46,XY[20] 46,XY[20] 46,XY,9q+,11,+13q[1]/46,XY[19] 46,XY,t(3;11)(q29;q13) [2]/46,XY[18] NA 46,XX[20]

del(13q14), IgH translocation, del(17p13) Normal Normal del(11q22.3)

319 177

5.0 7.7

3.6 NA

Positive Negative

29.2 85.6

22.9 0

89.8 98.4

46,XY[20] 46,XY[20]

66+ 4

C

IgG,␬

187

2.8

2.1

Negative

25.9

99.0

99.6

66 51 62

B B C

IgG,␭ IgG␭ IgM␬

350 235 776

11.6 2.7 12.1

1.6 NA 1.43

Positive Positive Negative

11.1 0 76.3

0 0 89.5

99.2 99.4 98.2

45,XY,2,add(19)(q13)[1]/48, XY,mar1,mar2[2]/ 46,XY[18] 46,XY[20] NA 46,XX[20]

del(13q14) del(13q14), IgH translocation, del(17p13) Normal

56+ 50+ 11

F M

70 71

C C

IgG␬ IgG␬

155 220

7.1 20.4

3.2 0.8

Positive Positive

0 0

54.1 9.7

100 88.4

del(11q22.3) NA del(6q23), del(13q14), IgH translocation, del(17p13) del(17p13) Normal

16 17 18

M M M

56 71 51

C C B

IgG␬ IgG␭ IgM␬

742 177 171

6.3 11.8 3.5

3.8 3.5 1.1

Positive Positive Positive

11.9 39.3 6.3

99.2 58.9 51.9

98.1 99.2 92.8

27+ 25+ 23+

19

F

62

C

IgM␬

983

5.5

0.23

Negative

0

45.0

98.6

20 21 22 23

M M F M

56 68 64 65

C C C C

IgM␭ IgM␬ IgM␬ IgM␬

595 155 276 172

6.9 9.6 5.3 3.7

3.1 1.5 1.3 0.39

Positive Negative Positive Negative

6.5 48.0 0 78.3

76.5 48.2 0 65.8

92.3 99.3 98.7 93.9

47-50,XX,1q-,+3,+3p,+3p,6q+,+7,+7,+8,9p+,90+,14,17p,+19[16][cp]/46.XX[4] NA 46,XY[20] 47,XX,+12[8]/46,XX[12] 47,XY,+12,14q+[20]

IgH translocation Normal +12, del(11q22.3), del(13q14), del(17p13) del(6q23), del(17p13)

20+ 19+ 18+ 17+

24 25

M M

66 83

B C

145 170

6.2 9.06

NA 1.3

Positive Negative

NA 7.5

2.1 91.3

92.4 85.4

46,XY[20] 46,XY[20]

26

F

61

C

IgM␬ IgG␭, IgM␭ IgG␬

123

3.55

10.1

Positive

19.1

39.6

99.5

46,XX[20]

27

M

56

A

IgM␭

826

5.29

0.61

Positive

34.0

61.0

88.6

46,XY[20]

NA Normal +12 +12, IgH translocation Normal del(6q23), del(11q22.3) del(11q22.3), del(17p13) NA

46,XX[20] 45,XY,-6[8]/46,XY,6,+mar[2]/46,XY[10] 46,XY[20] 46,XY[20] 47,XY,+12[12]/46,XY[14]

66 79+ 77+

73+ 42

57+

39+ 31+

W. Xu et al. / Leukemia Research 35 (2011) 1060–1065

Sex

22+

15+ 11+ 9+ 4+ 1063

Abbreviations: ␤2-MG, ␤2-microglobulin; DAT, direct antiglobulin test; F, female; FISH, fluorescence in situ hybridization; Ig, immunoglobulin; LDH, lactate dehydrogenase; M, male; NA, not available; OS, overall survival; TK, thymidine kinase; VH, heavy chain variable.

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Table 3 Correlations between Ig paraprotein and other prognostic factors. Factors

Sex Male Female Age <60 years ≥60 years Binet stage A B C DAT (n = 112) Negative Positive LDH (n = 125) <250 U/l ≥250 U/l ␤2-MG (n = 123) <3 mg/l ≥3 mg/l TK1 (n = 89) <2.0 pM ≥2.0 pM IGHV (n = 119) Mutated (>2% deviation from a germline) Unmutated (≤2% deviation from a germline) ZAP-70 (n = 128) <20% ≥20% CD38 (n = 129) <30% ≥30% Cytogenetics (n = 121) Unfavorable aberration (deletion in 17p13 or 11q22.3) Favorable aberration (deletion in 13q14 as the sole abnormality) Others

With Ig paraprotein (%)

Without Ig paraprotein (%)

20 (23.3) 7 (14.9)

66 (78.7) 40 (85.1)

10 (15.6) 17 (24.6)

54 (84.4) 52 (75.4)

3 (6.4) 8 (18.6) 16 (37.2)

44 (93.6) 35 (81.4) 27 (62.8)

9 (11.8) 16 (44.4)

67 (88.2) 20 (55.6)

17 (19.1) 10 (27.8)

72 (80.9) 27 (72.2)

5 (8.8) 22 (33.3)

52 (91.2) 44 (66.7)

13(18.6) 8 (42.1)

57 (81.4) 11 (57.9)

8 (10.8)

66 (89.2)

17 (34.0)

33 (66.0)

11 (12.0) 14 (38.9)

81 (88.0) 22 (61.1)

9 (11.3) 17(24.7)

71 (88.8) 32 (65.3)

11 (33.3)

22 (66.7)

1 (5.9)

16 (94.1)

11 (15.5)

60 (84.5)

r-Value

Pvalue

−0.099

0.179

0.112

0.141

0.314

<0.001

0.366

<0.001 Fig. 2. Overall survival curve of patients with and without IgG paraproteinemia.

0.095

0.202

0.296

0.001

0.227

0.037

0.284

0.002

0.305

0.001

0.284

0.002

0.208

0.032

Fig. 3. Overall survival curve of patients with and without IgM paraproteinemia.

retained their capability of isotype switching, a process independent of immunoglobulin heavy chain gene mutation [16]. As an alternative, these additional M components may represent biclonal or triclonal lymphoproliferative disorders as described in the literature [17] or the development of a subclone within the original tumor cells due to clonal evolution. In summary, patients with CLL with serum Ig paraprotein represent a heterogeneous group with inferior clinical outcome. Serum Ig paraprotein might be applied for the assessment of prognosis in patients with CLL. Conflict of interest The authors declare no conflict of interest. Acknowledgments

Fig. 1. Overall survival curve of patients with and without serum Ig paraprotein.

In the present study, the surface Ig light chain expressed by the CLL cells was identical to that of the serum Ig paraprotein in all patients with Ig paraproteinemia, suggesting that the serum paraprotein might be the secreted product of the CLL cells. Furthermore, we noted a monoclonal serum IgG paraprotein coexisting with IgM paraprotein in 3 patients, with an identical Ig light chain. The presence of both IgM and IgG monoclonal proteins suggests that neoplastic transformation may have occurred at the time of isotype switching from IgM to IgG [15] or that the tumor cells

This study was supported by National Natural Science Foundation of China (30871104, 30971295, 30971296), National Science & Technology Pillar Program (2008BAI61B01), Jiangsu Province’s Outstanding Medical Academic Leader Program (LJ200623), Jiangsu Province’s Medical Elite Program (RC2007042), Natural Science Foundation of Jiangsu Province (BK2010584), “Qing Lan” project of Jiangsu Province, and “Liu Da Ren Cai Gao Feng” of Jiangsu Province. References [1] Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, et al. WHO classification of tumours of haematopoietic and lymphoid tissues. 4th ed. Lyon: IARC Press; 2008. [2] Bernstein ZP, Fitzpatrick JE, O’Donnell A, Han T, Foon KA, Bhargava A. Clinical significance of monoclonal proteins in chronic lymphocytic leukemia. Leukemia 1992;6:1243–5.

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