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Originally published as JCO Early Release 10.1200/JCO.2005.02.7938 on January 23 2006 © 2006 American Society of Clinical Oncology. Elevated Serum B-Lymphocyte Stimulator Levels in Patients With Familial Lymphoproliferative DisordersFrom the Division of Hematology and Internal Medicine, Department of Immunology, and Divisions of Epidemiology and Biostatistics, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, MN; and Zymogenetics, Seattle, WA Address reprint requests to Stephen M. Ansell, MD, PhD, Division of Hematology and Internal Medicine, Mayo Clinic, 200 First St SW, Rochester, MN 55905; e-mail: ansell.stephen{at}mayo.edu
PURPOSE: Serum B-lymphocyte stimulator (BLyS) levels have been found to be elevated in a number of immune disease models. Therefore, we sought to establish whether BLyS levels were elevated in patients with B-cell lymphoproliferative disorders and to determine whether elevated BLyS levels correlated with clinical characteristics of the disease. PATIENTS AND METHODS: Specimens were collected from the peripheral blood of individuals diagnosed with B-cell chronic lymphocytic leukemia (B-CLL; n = 70) or from age- and sex-matched patients seen at the same institution (n = 41). Serum BLyS levels were determined by enzyme-linked immunosorbent assay, and sequencing of the BLyS promoter was performed by conventional methods and confirmed by restriction fragment length polymorphism analysis. RESULTS: We found that elevated BLyS levels were more common in patients with familial B-CLL than individuals with sporadic B-CLL or normal controls. Because of this association, we sequenced the BLyS promoter in patients with B-CLL and normal controls and identified a polymorphic site, –871 C/T. We found that the wild-type sequence was significantly underrepresented in patients with familial B-CLL (4%) compared with patients with sporadic B-CLL (30%; P = .01) or controls (24%; P = .04). Furthermore, using a luciferase reporter under control of the BLyS promoter containing either a C or a T at position –871, we found that the reporter construct containing a T at –871 had a 2.6-fold increase in activity (P = .004). CONCLUSION: Our data suggest serum BLyS levels are elevated in patients with familial B-CLL and that elevated BLyS levels correlate with the presence of a T at –871 in the BLyS promoter.
B-lymphocyte stimulator (BLyS) is a tumor necrosis factor family member critical for maintenance of normal B-cell development and homeostasis.1-5 Transgenic overexpression of BLyS in mice results in elevated numbers of mature B cells6,7 and development of autoimmune-like manifestations reminiscent of systemic lupus erythematosus and Sjögren's syndrome.6,8 Additionally, there is increasing evidence suggesting a role for BLyS in the growth and survival of malignant B cells.9-14 BLyS is expressed by monocytes, macrophages, dendritic cells, neutrophils, and malignant B cells.9,10,12,14-18 However, the mechanism underlying its expression remains to be clearly defined. BLyS expression has been shown to be regulated by interferon gamma, granulocyte-macrophage colony-stimulating factor, and interleukin-10,15,16 and BLyS levels have been found to be elevated in a number of immune disease models.9-14 Therefore, we sought to determine whether BLyS levels were elevated in patients with lymphoproliferative disorders.
Patient Selection The study group included 70 untreated patients diagnosed with B-cell chronic lymphocytic leukemia (B-CLL; Table 1); 24 patients had familial CLL, and 46 patients had sporadic CLL. Familial CLL patients were classified as those patients who had a first- or second-degree relative diagnosed with a B-cell lymphoproliferative malignancy including B-CLL, non-Hodgkin's lymphoma, multiple myeloma, or Hodgkin's lymphoma. All other B-CLLs were considered to be sporadic. For patients with CLL, Rai stage, CD38 expression, immunoglobulin gene mutation status, and chromosome analysis by fluorescent in situ hybridization were evaluated.19,20 Control specimens were obtained from 41 age- and sex-matched patients who were seen at the same institution for other medical issues and who did not have a personal or family history of a B-cell malignancy. Written informed consent was provided for all specimens.
BLyS Enzyme-Linked Immunosorbent Assay Enzyme-linked immunosorbent assay plates were coated with 1 µg/mL of anti-BLyS (Zymogenetics, Seattle, WA), and BLyS was detected with 1 µg/mL of biotinylated anti-BLyS (Zymogenetics) followed by strepavidin-horseradish peroxidase and 3,3',5,5'-tetramethyl benzidine substrate.11 Patient serum samples were diluted 1:5 and 1:25 in triplicate, and BLyS serum levels were calculated from a standard curve generated with recombinant human BLyS (Zymogenetics) in 20% human sera. The detection limit of purified BLyS was 300 pg/mL. Statistical comparisons of BLyS levels between patient groups were based on the Kruskal-Wallis test or the unpaired t test.
Sequencing of BLyS Promoter
Restriction Fragment Length Polymorphism Analysis
Luciferase Reporter Assay HL60 cells were transiently transfected with 10 µg of pGL3, pGL3 –871C, or –871T BLyS promoter plasmid along with 40 ng of pGL4.75[hRluc/CMV] Renilla plasmid (Promega) by electroporation (300 V, 10 msec) using a gene pulser (Bio-Rad Laboratories, Richmond, CA). Forty-eight hours after electroportion, Dual-Luciferase Reporter Assays (Promega) were performed according to the manufacturer's instructions. Firefly luciferase activity was normalized to Renilla activity.
In our initial studies, we examined the serum BLyS levels in patients with a lymphoproliferative disorder and compared them with normal age-matched controls. We used patients diagnosed with B-CLL as our model system (Fig 1A). In the normal controls (n = 41), the mean serum BLyS level was 6.68 ng/mL (± 7.0 ng/mL, three specimens below detection), and in CLL patients, the mean serum BLyS level was 11.93 ng/mL (± 15.86 ng/mL, nine specimens below detection), a nearly two-fold increase. However, the levels were not found to be significantly different (P = .057). Elevated BLyS levels (> 20 ng/mL; control mean plus two standard deviations) were found in two (5%) of 41 controls compared with 13 (19%) of 70 CLL patients (P = .04; Fig 1B). When we examined the clinical characteristics of the CLL patients with elevated BLyS levels, we found that six (46%) of 13 of the individuals with elevated BLyS levels had a familial history of B-cell malignancies, which is higher than what was expected (9%).22 Therefore, we compared serum BLyS levels between controls (n = 41) and sporadic (n = 46) and familial (n = 24) CLL cohorts and found that the mean serum BLyS level was 10.36 ng/mL in the sporadic CLL patients; seven (15%) of 46 sporadic CLL patients had BLyS levels exceeding 20 ng/mL (Figs 1C and 1D). In the familial CLL patients, the mean serum BLyS level was elevated compared with controls (14.73 ng/mL; P = .03), and we found that six (25%) of 24 familial CLL patients (P = .01) had BLyS levels exceeding 20 ng/mL (Figs 1C and 1D). The frequency of elevated BLyS in the familial CLL cohort relative to the normal controls was increased five-fold, suggesting that elevated serum BLyS levels may correlate with familial CLL.
To determine whether elevated BLyS levels correlated with clinical features other than familial incidence, we next evaluated the relationship between clinical characteristics and BLyS expression in control, sporadic, and familial CLL patients. We saw no significant difference in age, sex, Rai stage, CD38 expression, immunoglobulin gene mutation status, or chromosomal abnormalities (Table 1). Zap-70 expression analysis has not been determined on the specimens used in this study. In summary, these data suggest that elevated serum BLyS levels do not correlate with any of the currently studied risk factors for B-CLL other than a family history of B-cell malignancy.
Because of the correlation between BLyS levels and familial incidence of B-cell malignancies, we next wanted to determine whether there was a common underlying genetic event influencing BLyS expression. Although the BLyS promoter region remains to be fully characterized, reports indicate the presence of a number of polymorphisms.21,23 We began by sequencing the BLyS promoter in patients with B-CLL and normal controls (data not shown). We identified a polymorphic site, –871 C/T (dbSNP ID: rs9514828), that was previously reported to be expressed at increased frequency in systemic lupus erythematosus patients of Japanese descent with anti-Sm antibody and was associated with elevated monocyte BLyS mRNA levels.21 The change in gene sequence from C To determine the significance of the –871 polymorphism, we transfected HL60 cells, which express BLyS RNA, with a luciferase reporter construct under control of the BLyS promoter region containing either C or T at position –871. In support of our previous findings, we found that the reporter construct containing a T at –871 had a 2.6-fold (n = 6, P = .004) increase in luciferase activity compared with the promoter construct containing a C at –871 (Fig 1F). This region of the BLyS promoter contains a consensus transcription factor binding site for myeloid zinc finger protein (MZF1), a transcription factor expressed by myeloid lineage cells, a prominent source of BLyS.16,24 It is possible that alterations in MZF1 binding may influence BLyS expression and, therefore, contribute to elevated BLyS levels. A detailed analysis of this promoter region as well as a large-scale analysis of the presence of this polymorphism in the general population and individuals with a B-cell lymphoproliferative disorder is currently underway. In summary, our data suggest that serum BLyS levels are elevated in patients with familial CLL relative to both normal controls and individuals with sporadic CLL and that elevated BLyS levels may correlate with the presence of a T at –871 in the BLyS promoter.
Although all authors completed the disclosure declaration, the following authors or their immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
Dollar Amount Codes (A) < $10,000 (B) $10,000-99,999 (C)
We thank Renee Tschumper and Nancy Bone for their help in determining B-cell chronic lymphocytic leukemia immunoglobulin mutation status and levels of CD38 expression.
Supported in part by Grants No. CA92104 and CA97274 from the National Institutes of Health and a Translational Research Grant from the Leukemia and Lymphoma Society.
1. Moore PA, Belvedere O, Orr A, et al: BLyS: Member of the tumor necrosis factor family and B lymphocyte stimulator. Science 285:260-263, 1999 2. Schneider P, MacKay F, Steiner V, et al: BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth. J Exp Med 189:1747-1756, 1999 3. Mukhopadhyay A, Ni J, Zhai Y, et al: Identification and characterization of a novel cytokine, THANK, a TNF homologue that activates apoptosis, nuclear factor-kappaB, and c-Jun NH2-terminal kinase. J Biol Chem 274:15978-15981, 1999 4. Shu HB, Hu WH, Johnson H: TALL-1 is a novel member of the TNF family that is down-regulated by mitogens. J Leukoc Biol 65:680-683, 1999[Abstract] 5. Gross JA, Johnston J, Mudri S, et al: TACI and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease. Nature 404:995-999, 2000[CrossRef][Medline] 6. Mackay F, Woodcock SA, Lawton P, et al: Mice transgenic for BAFF develop lymphocytic disorders along with autoimmune manifestations. J Exp Med 190:1697-1710, 1999 7. Do RK, Hatada E, Lee H, et al: Attenuation of apoptosis underlies B lymphocyte stimulator enhancement of humoral immune response. J Exp Med 192:953-964, 2000 8. Khare SD, Sarosi I, Xia XZ, et al: Severe B cell hyperplasia and autoimmune disease in TALL-1 transgenic mice. Proc Natl Acad Sci U S A 97:3370-3375, 2000 9. Novak AJ, Bram RJ, Kay NE, et al: Aberrant expression of B-lymphocyte stimulator by B chronic lymphocytic leukemia cells: A mechanism for survival. Blood 100:2973-2979, 2002 10. Novak AJ, Darce JR, Arendt BK, et al: Expression of BCMA, TACI, and BAFF-R in multiple myeloma: A mechanism for growth and survival. Blood 103:689-694, 2004 11. Novak AJ, Grote DM, Stenson M, et al: Expression of BLyS and its receptors in B-cell non-Hodgkin lymphoma: Correlation with disease activity and patient outcome. Blood 104:2247-2253, 2004 12. Kern C, Cornuel JF, Billard C, et al: Involvement of BAFF and APRIL in the resistance to apoptosis of B-CLL through an autocrine pathway. Blood 103:679-688, 2004 13. Briones J, Timmerman JM, Hilbert DM, et al: BLyS and BLyS receptor expression in non-Hodgkin's lymphoma. Exp Hematol 30:135-141, 2002[CrossRef][Medline] 14. Moreaux J, Legouffe E, Jourdan E, et al: BAFF and APRIL protect myeloma cells from apoptosis induced by interleukin 6 deprivation and dexamethasone. Blood 103:3148-3157, 2004 15. Scapini P, Nardelli B, Nadali G, et al: G-CSF-stimulated neutrophils are a prominent source of functional BLyS. J Exp Med 197:297-302, 2003 16. Nardelli B, Belvedere O, Roschke V, et al: Synthesis and release of B-lymphocyte stimulator from myeloid cells. Blood 97:198-204, 2001 17. Craxton A, Magaletti D, Ryan EJ, et al: Macrophage- and dendritic cell–dependent regulation of human B-cell proliferation requires the TNF family ligand BAFF. Blood 101:4464-4471, 2003 18. He B, Chadburn A, Jou E, et al: Lymphoma B cells evade apoptosis through the TNF family members BAFF/BLyS and APRIL. J Immunol 172:3268-3279, 2004 19. Shanafelt TD, Geyer SM, Kay NE: Prognosis at diagnosis: Integrating molecular biologic insights into clinical practice for patients with CLL. Blood 103:1202-1210, 2004 20. Rai KR, Sawitsky A, Cronkite EP, et al: Clinical staging of chronic lymphocytic leukemia. Blood 46:219-234, 1975 21. Kawasaki A, Tsuchiya N, Fukazawa T, et al: Analysis on the association of human BLYS (BAFF, TNFSF13B) polymorphisms with systemic lupus erythematosus and rheumatoid arthritis. Genes Immun 3:424-429, 2002[CrossRef][Medline] 22. Catovsky D: The search for genetic clues in chronic lymphocytic leukemia. Hematol Cell Ther 39:S5-S11, 1997 (suppl 1) 23. Jiang Y, Ohtsuji M, Abe M, et al: Polymorphism and chromosomal mapping of the mouse gene for B-cell activating factor belonging to the tumor necrosis factor family (Baff) and association with the autoimmune phenotype. Immunogenetics 53:810-813, 2001[CrossRef][Medline] 24. Morris JF, Hromas R, Rauscher FJ III: Characterization of the DNA-binding properties of the myeloid zinc finger protein MZF1: Two independent DNA-binding domains recognize two DNA consensus sequences with a common G-rich core. Mol Cell Biol 14:1786-1795, 1994 Submitted May 24, 2005; accepted December 8, 2005.
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
T results in loss of a BsrB1 restriction site, allowing us to screen for the polymorphism by restriction digest (
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