Leukemia & Lymphoma, 2014; Early Online: 1–4 © 2014 Informa UK, Ltd. ISSN: 1042-8194 print / 1029-2403 online DOI: 10.3109/10428194.2014.926345

LETTER TO THE EDITOR

High levels of CD160 expression up-regulated counts of chronic lymphocytic leukemia cells and were associated with other clinical parameters in Chinese patients with chronic lymphocytic leukemia Zheng-Hao Zhang1, Shu Chen2, Shuang Chen1, Yang Liu1 & Jian-Hua Qu1 1Department of Hematology and 2Department of Pathology, First Affiliated Hospital of Xinjiang Medical University,

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Urumqi, Xinjiang, China

of CLL cells, and it may also be associated with the progression and prognosis of the disease. In this study, therefore, we measured the CD160 expression of CLL, and correlated the positive expression rate of CD160 with CLL cells, immune phenotypes, clinical parameters and other well-established prognostic indicators such as clinical stage, IgVH mutational status, ZAP-70, CD38 and chromosomal abnormalities. The study population consisted of 57 consecutive patients diagnosed with CLL between January 2008 and June 2013. All patients signed written informed consents and the University and Institutional Review Boards gave their approval for this research. According to the National Cancer Institute (NCI) criteria, the diagnosis of CLL requires persistent B lymphocytosis of more than 5.0 ⫻ 109/L, and the typical CLL phenotype includes the coexpression of cell surface markers CD5, CD19 and CD23, weak surface immunoglobulin and negative/weak FMC7. All patients were either untreated or at least 6 months from the last treatment. Biological and clinical data were collected during the first hospitalization: age, gender, white blood cell (WBC) count, lymphocyte (LY) count, Binet stage, IgVH, cytogenetics, β2-microglobulin, lymphadenopathy and splenomegaly. Cell surface antigens were analyzed by four-color immunofluorescence. Alexa Fluorâ647–CD160 (clone BY55), fluorescein isothiocyanate (FITC)–CD19 (clone 4G7), allophycocyanin (APC)–CD5 (clone L17F12), phycoerythrin (PE)–CD23 (clone M-L233), FITC–FMC7 (clone FMC7), PE–CD20 (clone 2H7), APC–CD38 (clone HB7), PE–CD200 (clone MRC OX-104), peridinin chlorophyll protein complex (PerCP)–CD45 (clone 2D1) and PE–ZAP-70 (clone 1 E7.2) were purchased from BD Biosciences. Three to five millilters of fresh peripheral blood or bone marrow samples for each patient were collected at CLL diagnosis. Samples were prepared according to the manufacturer’s instructions. All antibodies were conjugated directly and approved for diagnostic use. Isotype controls were run with each sample to distinguish positive from negative cells. The CD160 ⫹

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal B cells expressing CD19, CD5 and CD23 together with low surface expression of B-cell receptors and related molecules such as FMC7 [1]. CLL cells grow out of control and accumulate in the bone marrow and blood, where they crowd out healthy blood cells. CLL is the most frequent type of adult leukemia in the Western world, accounting for nearly 30% of all reported leukemias, but its prevalence in Asia is relatively low [2]. CLL is a disease of adults, and the median age of newly diagnosed patients with CLL is 65 years [3]. It is a heterogeneous disease with a highly variable clinical course and prognosis. Clinical staging of CLL and determination of the extent of the disease are conducted with the Rai staging system or Binet classification [4]. The clinical course and survival of patients with B-CLL are quite variable, and the two major clinical staging systems are unable to discriminate prospectively between an indolent or aggressive course within the low and intermediate risk categories. For this reason, some biological parameters have been used for the CLL staging systems to differentiate subsets of prognostics. Recently, several new prognostic markers have been reported for the prediction of disease progression in CLL, such as β2-microglobulin, immunoglobulin variable heavy chain (IgVH) gene mutational status and the expression of ZAP-70 and CD38 [5,6]. CD160 is a 27 kDa glycoprotein that is known as an essential natural killer (NK) cell activating receptor [7]. Its expression is associated tightly with peripheral blood NK cells and CD8 ⫹ T lymphocytes with cytolytic effector activity [8]. Recently, some reports have shown that CD160 is expressed by CLL cells, but not in normal B lymphocytes [9]. It has also been demonstrated that CLL cells can be regulated by the CD160 pathway for in vitro proliferation and activation. Further, activated CD160 protects CLL cells from rapid spontaneous apoptosis in vitro via the phosphatidylinositol 3-kinase (PI3K)/AKT pathway [10]. Thus, we hypothesized that expression of CD160 may be associated with high levels

Correspondence: Jian-Hua Qu, Department of Hematology, First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang, 830054, China. E-mail: [email protected] Received 15 October 2013; revised 21 April 2014; accepted 12 May 2014.

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Table I. Correlations between expression of CD160 cells and clinical and biological parameters in CLL.

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Factor Gender Male Female Age ⬍ 70 ⱖ 70 Lymphadenopathy No palpable Palpable Splenomegaly No palpable Palpable WBC ⬍ 35 ⫻ 109/L ⱖ 35 ⫻ 109/L Binet stage A B C β2-Microglobulin (n ⫽ 51) ⬍ 3.0 mg/L ⱖ 3.0 mg/L IgVH (n ⫽ 44) Mutated Unmutated ZAP-70 (n ⫽ 44) ⱕ 20% ⬎ 20% Cytogenetics (n ⫽ 50) Normal or sole 13q del Trisomy 12 11q del or 17p del

n

Mean ⫾ SD (%)

Range (%)

43 14

30.4 ⫾ 23.1 23.1 ⫾ 17.9

0.4–88.5 1.9–61.2

26 31

28.1 ⫾ 23.3 29.0 ⫾ 21.1

0.4–88.5 1.9–81.5

13 44

18.4 ⫾ 16.2 31.6 ⫾ 22.7

0.8–52.4 0.4–88.5

23 34

32.0 ⫾ 18.6 26.3 ⫾ 24.0

1.9–61.2 0.4–88.5

30 27

18.3 ⫾ 16.5 40.1 ⫾ 21.9

0.4–61.2 9.8–88.5

26 15 16

20.1 ⫾ 17.0 33.7 ⫾ 22.5 37.7 ⫾ 24.8

0.4–53.5 1.9–81.5 5.1–88.5

0.022

31 20

24.0 ⫾ 17.0 37.7 ⫾ 26.6

0.4–52.2 1.5–88.5

0.051

26 18

27.0 ⫾ 20.7 38.4 ⫾ 25.1

0.4–72.8 4.0–88.5

0.125

28 16

24.2 ⫾ 20.8 40.9 ⫾ 24.3

0.4–65.3 5.1–88.5

0.029

29 7 14

24.8 ⫾ 23.5 48.7 ⫾ 16.2 32.3 ⫾ 18.0

0.4–88.5 24.7–72.8 6.5–65.3

0.033

p-Value 0.228

0.886

0.027

0.321 ⬍ 0.001

CLL, chronic lymphocytic leukemia; WBC, white blood cell count; IgVH, immunoglobulin variable heavy chain; SD, standard deviation.

population was defined by gating of the lymphoid population on the side scatter (SSC)-CD45 plot, followed by gating of the CD19 ⫹ CD160 ⫹ population. CD160 expression was defined as the percentage of B cells that expressed CD160. The cut-off point for ZAP-70 positivity in CLL cells was 20% and for other antigens was 30% [11]. Of 57 patients with CLL, 43 were males and 14 were females (male:female ratio ⫽ 3.1). The median age at diagnosis was 71 years (range 35–90 years). According to the Binet clinical staging system, 26 (45.6%) patients were in Binet A, 15 (26.3%) in Binet B and 16 (28.1%) in Binet C. The CD160⫹ ⬍ 30% group included 32 cases and the CD160⫹ ⬎ 30% group included 25 cases. The expression levels of CD19⫹, CD19 ⫹ CD5⫹, CD19 ⫹ CD23⫹, CD20⫹, CD38 ⫹ and CD19 ⫹ CD200 ⫹ were elevated significantly in patients in the CD160⫹ ⬎ 30% group. The possibility of other correlative factors was analyzed, and results are presented in Table I and Figure 1. There was no significant difference in the percentage of CD160 ⫹ cells in terms of gender (male 30.4 ⫾ 23.1% vs. female 23.1 ⫾ 17.9%; p ⫽ 0.228), mean age (⬍ 70 28.1 ⫾ 23.3% vs. ⱖ 70 29.0 ⫾ 21.1%; p ⫽ 0.886), β2-microglobulin (⬍ 3.0 mg/L 24.0 ⫾ 17.0% vs. ⱖ 3.0 mg/L 37.7 ⫾ 26.6%; p ⫽ 0.051) and IgVH (mutated 27.0 ⫾ 20.7% vs. unmutated 38.4 ⫾ 25.1%; p ⫽ 0.125). The percentage of CD160 ⫹ cells was significantly increased in patients with high WBC counts (⬍ 35 ⫻ 109/L 18.3 ⫾ 16.5% vs. ⱖ 35 ⫻ 109/L 40.1 ⫾ 21.9%; p ⬍ 0.001). The percentage of

CD160 ⫹ cells was significantly increased in patients with Binet C stage compared to earlier stages (p ⫽ 0.022). The percentage of CD160 ⫹ cells was significantly increased in patients with higher expression levels of ZAP-70 (p ⫽ 0.029) and trisomy 12 abnormality (p ⫽ 0.033) compared to patients with lower expression levels of ZAP-70 and other cytogenetics, respectively. In this study, 57 cases of CLL were divided into the CD160 ⬍ 30% group (32 cases) and the CD160 ⬎ 30% group (25 cases). The percentage of CD19 ⫹ CD5⫹ CD23 ⫹ CD160 ⫹ among CD19 ⫹ cells was 31.1 ⫾ 24.9%. The percentage of CD19 ⫹ cells was significantly increased in the CD160 ⬎ 30% group (84.4 ⫾ 11.7% vs. 58.2 ⫾ 25.7%; p ⬍ 0.001). Furthermore, the percentage of CD19 ⫹ CD5⫹ cells and CD19 ⫹ CD23⫹ cells was also significantly increased in the CD160 ⬎ 30% group (74.4 ⫾ 16.8% vs. 34.9 ⫾ 25.5% and 69.5 ⫾ 19.5% vs. 42.4 ⫾ 23.9%; p ⬍ 0.001). CD19 ⫹ CD5⫹ cells and CD19 ⫹ CD23⫹ cells have been identified as tumor cells in CLL. Thus, it may be the case that in patients with CLL, CD160 expression is correlated with higher counts of B lymphocytes (especially CLL cells) in vivo. This result is similar to that found in previous studies [9], and demonstrates that expression of CD160 may also correlate with CLL cell survival and proliferation in vivo. Moreover, we observed that CD160 is correlated with CD20 in CLL (p ⬍ 0.001). In previous studies CD20, which is known to be a specific antigen of lymphocytes, has been recognized as an important and effective target for CLL. Monoclonal antibodies (McAb) directed against the CD20 antigen on B cells have altered the treatment landscape dramatically for patients with CLL [12]. These cells are recognized and killed through multiple mechanisms, including direct induction of apoptosis, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and clearance by the reticuloendothelial system. Thus high expression of CD20 in CLL may be associated with a good prognosis [13]. CD160 is also expressed specifically on CLL cells, and this correlation suggests that CD160 McAb may well become a new method of treatment for CLL. Further, the CD160 ⬎ 30% and CD160 ⬍ 30% groups showed a significant difference in expression of CD200 (71.8 ⫾ 17.3% vs. 34.4 ⫾ 23.4, p ⬍ 0.001). CD200 has been identified as one of the antigens up-regulated on the B-CLL cell surface compared with normal B cells. Recent studies from different centers have demonstrated that CD200 expression may help in differential diagnosis between mantle cell lymphoma and B-cell CLL [14,15]. CD160, therefore, may also be a potential diagnostic marker of CLL. Moreover, as CD160 may be involved in the pathogenesis of CLL due to its immunosuppressive properties, it may serve in the future as a potential target for antitumor therapeutics. In this study, we analyzed correlations between clinical and biological parameters and the expression of CD160 (Table I). There were significant correlations between CD160 expression on CLL lymphocytes and lymphadenopathy, WBC count, Binet stage, ZAP-70 expression and cytogenetic abnormalities. However, there was no significant relationship between the expression of CD160 and IgVH or β2-microglobulin. Therefore, we suggest that

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Letter to the Editor 3

Figure 1. Correlation of CD160 expression with clinical factors of CLL.

CD160 may become a useful marker in the clinical diagnosis and prognosis of CLL. Unfortunately, our cohort was not suitable for analysis of the survival curve because only two patients died during follow-up. Greater sample sizes and a longer follow-up period are required to determine fully whether CD160 can be used to predict prognosis in patients with CLL.

In conclusion, this study demonstrated that expression of CD160 was up-regulated in patients with CLL and it was associated with counts of CLL cells, CD19 ⫹ CD5⫹ cells and CD19 ⫹ CD23⫹ cells. Consistent with the experiment in vitro, we speculated that CD160 could protect CLL cells against apoptosis and up-regulate the counts of CLL cells. Furthermore, this study investigated associations between

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the expression of CD160 and clinical parameters. The results showed that there were significant correlations between CD160 and most of these parameters, with the exception of IgVH and β2-microglobulin. Although more research is needed, we suggest that CD160 plays a critical role in the survival of CLL cells, which may become a potential significant marker for the clinical diagnosis and prognosis of CLL. Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal.

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[6] Chiorazzi N. Implications of new prognostic markers in chronic lymphocytic leukemia. Hematology Am Soc Hematol Educ Program 2012:76–87. [7] Giustiniani J, Bensussan A , Marie-Cardine A . Identification and characterization of a transmembrane isoform of CD160 (CD160-TM), a unique activating receptor selectively expressed upon human NK cell activation. J Immunol 2009;182:63–71. [8] Le Bouteiller P, Tabiasco J, Polgar B, et al. CD160: a unique activating NK cell receptor. Immunol Lett 2011;138:93–96. [9] Farren TW, Giustiniani J, Liu FT, et al. Differential and tumorspecific expression of CD160 in B-cell malignancies. Blood 2011;118: 2174–2183. [10] Liu FT, Giustiniani J, Farren T, et al. CD160 signaling mediates PI3K-dependent survival and growth signals in chronic lymphocytic leukemia. Blood 2010;115:3079–3088. [11] Damle RN, Wasil T, Fais F, et al. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 1999;94:1840–1847. [12] Le Garff-Tavernier M, Herbi L, de Romeuf C, et al. Antibodydependent cellular cytotoxicity of the optimized anti-CD20 monoclonal antibody ublituximab on chronic lymphocytic leukemia cells with the 17p deletion. Leukemia 2014;28:230–233. [13] Fang C, Zhuang Y, Wang L, et al. High levels of CD20 expression predict good prognosis in chronic lymphocytic leukemia. Cancer Sci 2013;104:996–1001. [14] Palumbo GA , Parrinello N, Fargione G, et al. CD200 expression may help in differential diagnosis between mantle cell lymphoma and B-cell chronic lymphocytic leukemia. Leuk Res 2009;33:1212–1216. [15] Pillai V, Pozdnyakova O, Charest K, et al. CD200 flow cytometric assessment and semiquantitative immunohistochemical staining distinguishes hairy cell leukemia from hairy cell leukemia-variant and other B-cell lymphoproliferative disorders. Am J Clin Pathol 2013;140:536–543.

High levels of CD160 expression up-regulated counts of chronic lymphocytic leukemia cells and were associated with other clinical parameters in Chinese patients with chronic lymphocytic leukemia.

High levels of CD160 expression up-regulated counts of chronic lymphocytic leukemia cells and were associated with other clinical parameters in Chinese patients with chronic lymphocytic leukemia. - PDF Download Free
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