CD200, CD22, CD23, CD24, CD25, CD27, CD31, CD38, CD39, CD43,

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1 Cytometry Part B (Clinical Cytometry) 78B (Suppl. 1):S42 S46 (2010) Chronic Lymphocytic Leukaemia (CLL) and CLL- Type Monoclonal B-Cell Lymphocytosis (MBL) Show Differential Expression of Molecules Involved in Lymphoid Tissue Homing Andy C. Rawstron, 1,2 * Jane Shingles, 1 Ruth de Tute, 1 Fiona Bennett, 1 Andrew S. Jack, 1,2 and Peter Hillmen 3 1 HMDS, St. James s Institute of Oncology, Leeds, United Kingdom 2 HYMS, University of York, Heslington, York, United Kingdom 3 Department of Hematology, St. James s Institute of Oncology, Leeds, UK Introduction: The aim of this study was to screen for cell surface markers that could discriminate CLLtype MBL from CLL or identify CLL cases likely to have stable disease. Methods: Six color flow cytometry was performed on CLL-type MBL (n 5 94) and CLL (n 5 387) at diagnosis or relapse; 39 cases had poor-risk chromosomal abnormalities (17p and/or 11q deletion). Expression of 30 markers was analysed: CCR6, CD10, CD103, CD11c, CD138, CD200, CD22, CD23, CD24, CD25, CD27, CD31, CD38, CD39, CD43, CD49d, CD5, CD52, CD62L, CD63, CD79b, CD81, CD86, CD95, CXCR5, HLADR, IgD, IgG, IgM, LAIR1. Results: There was no difference in expression between CLL-type MBL and CLL for the majority of markers. Differential expression was observed for several markers, mainly between MBL and CLL cases with adverse-risk chromosomal abnormalities. These differences included lower expression of CD38 (9.4-fold lower, P ) and CD49d (3.2-fold lower, P ) and higher expression of LAIR-1 (3.7-fold higher, P ), CXCR5 (1.25-fold higher, P ), and CCR6 (1.9-fold higher P < 0.001) on CLL-type MBL compared to CLL with adverse chromosomal abnormalities. CD62L (L-selectin) which mediates lymphocyte adhesion to endothelial venules of lymphoid tissue, was expressed at a significantly different level between CLL-type MBL and both CLL sub-groups, with 1.3-fold lower (P ) expression levels on the MBL cases. However, there was broad overlap in expression levels. Conclusions: CLL-type MBL is phenotypically identical to CLL for a very broad range of markers. Differential expression is predominantly related to known prognostic markers and proteins involved in homing to lymphoid tissue. VC 2010 International Clinical Cytometry Society Key terms: MBL; CLL; lymphoid tissue homing How to cite this article: Rawstron AC, Shingles J, de Tute R, Bennett F, Jack AS, Hillmen P. Chronic lymphocytic leukaemia (CLL) and CLL-type monoclonal B-cell lymphocytosis (MBL) show differential expression of molecules involved in lymphoid tissue homing. Cytometry Part B 2010; 78B (Suppl. 1): S42 S46. Chronic lymphocytic leukaemia (CLL) is a disorder of malignant B-cells with a unique phenotype and an annual incidence of 4.5 per 100,000 person-years (1). The majority of individuals with CLL do not require treatment at presentation and active monitoring is undertaken until there is evidence of progressive marrow failure, massive lymphadenopathy/splenomegaly, or progressive lymphocytosis with a rapid doubling time (2). Despite a generally good prognosis, the diagnosis of Grant sponsor: ACR, ASJ and PH received funding support from Leukaemia and Lymphoma Research ( *Correspondence to: Andy C. Rawstron, St. James s Institute of Oncology, Level 3 Bexley Wing, Beckett Street, Leeds LS9 7TF, UK. andy.rawstron@nhs.net Received 26 February 2010; Revision 6 April 2010; Accepted 6 May 2010 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: /cyto.b VC 2010 International Clinical Cytometry Society

2 PROTEIN EXPRESSION PROFILE IN CLL AND MBL S43 Table 1 Markers Used for Investigation of Cell Surface Phenotype in CLL and CLL-Type MBL Pac Blue FITC PE PE-Cy5.5 PE-Cy7 APC 1 CD20 CD14 CD56 CD19 CD3 CD8 2 CD20 IgG IgD CD19 CD27 IgM 3 CD20 CD81 CD79b CD19 CD5 CD43 4 CD20 CD52 CD200 CD19 CD5 CD23 5 CD20 CD49d LAIR-1 CD19 CD5 CXCR5 6 CD20 CD31 CD95 CD19 CD38 CD10 7 CD20 CD24 CD86 CD19 CD38 CD22 8 CD20 CD103 CD25 CD19 CD10 CD11c 9 CD20 HLA-DR CD62L CD19 CD27 CCR6 10 CD20 CD39 CD63 CD19 CD38 CD138 CLL carries a significant psychological burden with emotional well-being scores significantly lower than the general population as well as other individuals with cancer, which may be attributable to the fact that nothing is being done about their malignancy (3). CLL-type Monoclonal B-cell Lymphocytosis (MBL) is defined as the presence of a monoclonal B-cell population with a phenotype consistent with CLL but with a B- cell count below 5,000/lL and without lymphadenopathy, splenomegaly, immune-mediated cytopenias, or other features of CLL (4). Individuals with this condition have a low risk, typically 1% per year, of developing progressive disease that requires treatment (5). The risk is significantly lower than for individuals with Rai stage 0 CLL (6). Prognostic factors such as IGHV mutation status, chromosomal abnormalities, CD38, and ZAP-70 expression, which are associated with a poor prognosis in CLL, may also be predictive of outcome in CLL-type MBL but there are relatively few cases with poor-risk prognostic factors and prognostication is not straightforward (5 8). The fact that most individuals with CLL-type MBL will have a stable condition may allow characterization of other prognostic factors which could not otherwise be identified without long-term follow-up. The aim of this study was to determine whether any immunophenotypic features differentiate CLL-type MBL from CLL and so could be candidate prognostic markers and potentially assist in the diagnostic process. METHODS Patient Samples The study includes samples from 481 individuals (median age 71.4 years, range , male:female ratio 1.3:1) referred for investigation of a lymphoproliferative disorder and shown to have a CLL-phenotype B-cell population. CLL-type MBL was diagnosed from 94 peripheral blood samples (median B-cell count /L, range ; median lymphocyte count /L, range ). CLL was diagnosed in 387 cases from 226 peripheral blood samples (median B-cell count / L, range 5 350, median lymphocyte count /L, range 7 358) and 161 bone marrow aspirate samples (median B-cell percentage of leucocytes 71%, range 5 95%). Within the CLL group, 39 were demonstrated to have chromosomal abnormalities associated with a poor outcome (see ifish section below). In all cases, informed written consent was provided to undertake diagnostic investigations. Ethical approval was granted to perform biological studies on anonymized waste material (REC 04/Q1107/40). Flow Cytometry Leucocytes were prepared by incubation of whole blood with a four-fold excess of ammonium chloride (Vickers, 0.86% in distilled H 2 O) at 37 C for 5 min and washed twice with FACSFlow (BD Biosciences) containing 0.1% Bovine Serum Albumin (Sigma) and cells were aliquoted into microtitre plate wells and centrifuged at 2,000 rpm for 2 min. The supernatant was discarded and antibodies were added to each well. Cells were incubated for 30 min in the dark at 4 C, washed twice with FACSFlow/BSA, resuspended in 200 ll FACS- Flow and acquired immediately using a FACSCanto II cytometer. First, samples were assessed with Lambda FITC, Kappa PE, CD19 PE-Cy5.5, CD5 PE-Cy7, CD20 APC, CD45 APC- Cy7. Only samples containing CD19þ B-cells with expression of CD5, weak CD20 and monoclonal light chain expression were included. Samples were then assessed with a panel of 10 tubes shown in Table 1. Antibody sources were as follows: CD3 PE-Cy7 Becton Dickinson (Cat. No ), CD5 PE-Cy7 Becton Dickinson (Cat. No ), CD8 APC Becton Dickinson (Cat. No ), CD10 APC Becton Dickinson (Cat. No ), CD10 PE-Cy7 Becton Dickinson (Cat. No ), CD11c APC Becton Dickinson (Cat. No ), CD14 FITC In-House, CD19 PE-Cy5.5 Caltag (Cat. No. MHCD1918), CD20 Pac Blue E-Biosciences (Cat. No ), CD20 APC Becton Dickinson (Cat. No ), CD22 APC Becton Dickinson (Cat. No ), CD23 APC Becton Dickinson (Cat. No ), CD24 FITC BD Pharmingen (Cat. No ), CD25 PE Becton Dickinson (Cat. No ), CD27 PE- Cy7 E-Biosciences (Cat. No ), CD31 FITC BD Pharmingen (Cat. No ), CD38 PE-Cy7 Becton Dickinson (Cat. No ), CD39 FITC Serotec (Cat. No. MCA1268F), CD43 APC Caltag (Cat. No. MHCD4305), CD45 APC-Cy7 Becton Dickinson (Cat. No ), CD49d FITC Serotec (Cat. No. MCA923F), CD52 FITC Serotec (Cat. No. SFL1642F), CD56 PE

3 S44 RAWSTRON ET AL. FIG. 1. Protein expression profile in CLL and MBL demonstrating very similar levels of expression for all markers assessed. Becton Dickinson (Cat. No ), CD62L PE Becton Dickinson (Cat. No ), CD63 PE BD Pharmingen (Cat. No ), CD79b PE Coulter (Cat. No. IM1612), CD81 FITC Becton Dickinson (Cat. No ), CD86 PE BD Pharmingen (Cat. No ), CD95 PE BD Pharmingen (Cat. No ), CD103 FITC IQ Products (Cat. No. IQP-111F), CD138 APC Miltenyi (Cat. No ), CD200 PE Serotec (Cat. No. MCA1960PE), CCR6 AF647 BD Pharmingen (Cat. No ), CXCR5 AF647 BD Pharmingen (Cat. No ), IgD PE BD Pharmingen (Cat. No ), IgG FITC BD Pharmingen (Cat. No ), IgM APC BD Pharmingen (Cat. No ), HLA-DR FITC Becton Dickinson (Cat. No ), Kappa PE Becton Dickinson (Cat No ), Lambda FITC Becton Dickinson (Cat No ), LAIR-1 PE BD Pharmingen (Cat. No ). Fluorescence In-Situ Hybridization Cytospins were fixed in methanol acetic acid (MAA, 3:1) and pretreated in 2xSSC/0.1% NP-40 and dehydrated in an ethanol series. Five microliter of probe in 50% formamide hybridization buffer was applied. Probes were either 13q14/13q34 ( ) or the CLL multicolor probe set ( , Abbott/Vysis, Maidenhead, UK). Cells and probe were codenatured at 73 C for 3 min and hybridized overnight at 37 C. Post hybridization stringency wash was carried out at 69 C for 2 2 min in 0.4xSSC/0.3%NP-40, followed by 1 2 min in 2xSSC/ 1%NP-40. DAPI-counterstained cells were examined using Zeiss Axioplan fluorescent microscope; images were captured using a Sony CCD camera and processed using ISIS3 software (MetaSystems, Germany). Statistical Analysis Differences in median fluorescence intensity between groups were assessed using the Wilcoxon-Mann-Whitney U test in STATA 9.0 software (Statacorp, TX), corrected for multiple testing (Bonferroni correction). All P-values are two-sided. RESULTS Figure 1 shows the expression profile for 30 markers in CLL compared to CLL-type MBL cases. There was no significant difference in expression for the majority of markers. Modest differences in expression were seen for known prognostic markers CD38 and CD49d. In addition, there were also minor differences in expression of CD62L, CXCR5, CCR6, and LAIR1. The CLL cases were subdivided into those with 17p and 11q deletions which are associated with a poor outcome, and the remainder with normal-risk abnormalities (deletion 13q14, trisomy 12 or no abnormalities). Figure 2 shows the expression profile for markers with differential expression, comparing MBL with normal-risk and poor-risk CLL. For the majority of markers, CLL cases without adverse-prognosis chromosomal abnormalities showed the same protein expression profile as MBL and any differential expression was predominantly between MBL and poor-risk CLL. CD38 and CD49d were 9.4-fold lower (P ¼ 0.007) and 3.2-fold lower (P ¼ 0.008), respectively on CLL-type MBL compared to CLL with adverse chromosomal abnormalities. Levels of the inhibitory receptor LAIR-1, which is downregulated during the normal germinal centre reaction, was 3.7-fold higher (P ¼ 0.003) in CLL-type MBL compared to adverse prognosis CLL. Also there was 1.9-fold higher (P < 0.001) CCR6 expression and

4 PROTEIN EXPRESSION PROFILE IN CLL AND MBL S45 FIG. 2. Differences in expression between CLL-type MBL and CLL separated into prognostic groups according to chromosomal abnormalities with * indicating a significant difference (P < 0.05) between a CLL subgroup and MBL fold higher (P ¼ 0.002) CXCR5 expression in CLLtype MBL compared to adverse-risk CLL. Both of these markers are normally responsible for B-cell homing to secondary lymphoid tissue. Only the expression of CD62L (L-selectin) which mediates lymphocyte adhesion to endothelial venules of lymphoid tissue, was expressed at a significantly different level between CLLtype MBL and both CLL subgroups, with 1.3-fold lower (P ¼ 0.04) expression levels on the MBL cases. However, there was broad overlap in expression levels. Although the differences were not significant between normal-risk CLL and MBL for the other markers, stepwise differences in median expression were seen for CD38, LAIR1, CCR6, and to a lesser extent CXCR5. CONCLUSIONS These results demonstrate the very close biological identity between CLL and CLL-type MBL not only for the common diagnostic markers but also for less studied molecules such as chemokine receptors. This data confirms previous studies which indicate that MBL cases from the general population, typically with very low CLL-phenotype cell counts, show the same extended phenotype as clinical CLL (9). Increased expression of CD38 and/or CD49d have consistently been shown to predict a poorer outcome in CLL (10 13) and it is unsurprising that there are differences between CLL and MBL with respect to these markers. However the profile for these markers is similar between CLL and MBL and it is only in cases with poor-risk chromosomal abnormalities that there is a consistent marked increase in these proteins, whilst other CLL cases and MBL cases show a similar spectrum of expression. LAIR1 is an inhibitory molecule which is not well characterized in B-lymphoproliferative disorders but preliminary data suggests it has prognostic value in CLL and the pattern of expression demonstrated here is consistent with this. CXCR5, CCR6, and CD62L are responsible for homing and entry into lymphoid tissue and recruitment of B- cells to lymphoid follicles (14 16). Expression of the chemokine receptor molecules is mildly reduced in CLL compared to MBL and significantly reduced in poorprognosis CLL. CD62L expression is increased in both CLL groups relative to MBL cases. Although the differences in median fluorescence intensity appear to be relatively small they are likely to be of biological relevance. For example, the level of CXCR5 expression seen in CLL-type MBL is similar to that of normal peripheral B- cells, whereas the level of CXCR5 expression in CLL cases with poor-risk chromosomal abnormalities is similar to that seen in germinal centre B-cells and most lymphomas (unpublished observation). It is possible that the weaker expression of CD62L by the CLL-type cells in MBL cases makes it more difficult for the cells to enter lymphoid tissue and obtain a micro-environmental survival signal (17). In summary, CLL-type MBL is phenotypically almost identical to CLL with some differences in known prognostic markers and small but probably biologically significant differences in expression of markers involved in lymphoid tissue trafficking. LITERATURE CITED 1. HMRN: Incidence. Available at: Incidence.aspx 2. Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Döhner H, Hillmen P, Keating MJ, Montserrat E, Rai KR, Kipps TJ. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: A report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood 2008;111: Shanafelt TD, Bowen D, Venkat C, Slager SL, Zent CS, Kay NE, Reinalda M, Sloan JA, Call TG. Quality of life in chronic lymphocytic leukemia: An international survey of 1482 patients. Br J Haematol 2007;139: Marti GE, Rawstron AC, Ghia P, Hillmen P, Houlston RS, Kay N, Schleinitz TA, Caporaso N. Diagnostic criteria for monoclonal B-cell lymphocytosis. Br J Haematol 2005;130:

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