SEARCH

SEARCH BY CITATION

Keywords:

  • chronic lymphocytic leukaemia;
  • proliferation;
  • immunophenotype

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

The proliferating component in chronic lymphocytic leukaemia (CLL) is usually small (<1%) and restricted to a specific micro-environmental niche. To characterize the proliferating component, CLL cells from bone marrow or lymph nodes of 23 patients were assessed for expression of up to 66 surface antigens in combination with nuclear Ki-67/MCM6. Ki-67 expression was associated with step-wise increases in CD23/CD95/CD86/CD39/CD27 and decreases in CD24/CD69/CXCR4/CXCR5. Ki-67+ cells showed increased CD38 expression, but with considerable inter-patient variability: in some cases Ki-67 expression was only detectable in CD38 CLL cells. The results suggest continuous re-entry into the cell cycle as no distinct stem cell pool was detectable.

Chronic lymphocytic leukaemia (CLL) is often considered an accumulative disease as the majority of cells are arrested in the G0 stage of cell cycle (Hamblin & Oscier, 1997). Recent in vivo studies of the cellular kinetics of CLL demonstrate that a small proportion of CLL cells, typically around 1%, are in the active stages of cell cycle (Messmer et al, 2005). Immunohistochemical studies showed that the proliferating cells in CLL are restricted to specific micro-enviromental structures, known as pseudofollicles or proliferation centres, found in bone marrow and lymph nodes (Schmid & Isaacson, 1994; Soma et al, 2006). The cells within proliferation centres have a different immunophenotype to peripheral CLL cells, with increased expression of CD23 and CD38 in addition to the proliferation-associated markers, such as CD71 and Ki-67 (Schmid & Isaacson, 1994; Granziero et al, 2001; Soma et al, 2006). Current laboratory methods used to identify proliferating CLL cells often rely upon immunohistochemical staining, which requires tissue processing that inhibits the extraction of material for further molecular or proteomic analysis (Soma et al, 2006).

Ki-67 is a nuclear and nucleolar protein which is centrally involved in cell proliferation (Endl & Gerdes, 2000; Brown & Gatter, 2002). Phosphorylation and dephosphorylation of the protein in vivo coincides with the transit of cells into cell cycle, under the control of the Cyclin-B/cdc-2 complex. Expression of Ki-67 is seen during the late G1 phase and throughout S, G2 and M phases (Schrader et al, 2005). Minichromosome maintainance protein-6 (MCM-6) belongs to a group of proteins involved in the initiation of DNA replication and is detectable throughout G1 phase prior to expression of Ki-67 (Gonzalez et al, 2005). This study aimed to assess the cell surface immunophenotypic profile in combination with Ki-67 and MCM6, to determine whether there were specific changes in protein expression associated with entry into and progression through the cell cycle.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

Bone marrow (n = 19) and fresh lymph node biopsies (n = 5) were assessed. All samples had a confirmed diagnosis of CLL and neoplastic cells had completely replaced normal B-lymphopoiesis. Analysis was performed on anonymized surplus diagnostic material according to a protocol approved by the local ethical review committee. All patients provided consent for investigation. Investigations were performed on either whole bone marrow or on cells prepared by perfusing FACSflow (BD Biosciences, Oxford, UK) through a small segment of a fresh lymph node.

Initially, cells from a single case were incubated with combinations of CD19 phycoerythrin cyanin 5·5 (PE-Cy5·5; Invitrogen, Paisley, UK) together with a PE and allophycocyanin (APC)-conjugated antibody from a series of 66 antibodies which had been reported to be expressed on CLL or other B-cell abnormalities (see Rawstron et al, 2006 for complete list). Cells were then fixed and permeabilized using either FIX and PERM (Invitrogen, Paisley, UK) or INTRASure (BD Biosciences) according to manufacturers’ instructions and incubated with 10 μl of the fluorescein isothicyanate (FITC)-conjugated antibody to MCM-6 or Ki-67 (BD Biosciences).

Data were acquired using a FACSort flow cytometer, and analysed using CELLQuest software (BD Biosciences). Initially, a region was set around cells with high expression of CD19 and low-side scatter characteristics (low granularity); a second region was set on physical characteristics of the CD19+ cells to exclude debris and non-specific binding. Expression of the nuclear antigen was assessed for cells gated in both of these regions. Ki-67 expression was poorly detectable with the Caltag FIX and PERM reagent (Fig 1A) and the BD INTRASure reagent was used in all subsequent experiments. The geometric mean fluorescence intensity of the surface-bound antibodies was recorded for the Ki-67 or MCM-6 positive and negative cell fractions. The aim was to identify antigens that reproducibly differentiate proliferating CLL cells; antibodies that showed equivalent or no binding were excluded from further analysis. The remaining 24 antigens were tested on a further four cases (i.e. five in total).

image

Figure 1.  Chronic lymphocytic leukaemia cells in cell cycle have highly regulated expression of several membrane proteins. (A) Shows normal bone marrow B cells, gated using CD19 expression and light scatter characteristics, from the same patient. B-progenitors, identified by very high levels of CD38 expression in the upper quadrants of each plot, are highly proliferative. However, Ki-67 expression is only poorly detectable using a standard approach with Caltag Fix and Perm (panel A). The manufacturer's proposed modification for detection of Ki-67 incorporates methanol, which abrogates the ability to assess surface antigens. Ki-67 expression is more readily detectable using the BD INTRASure reagent (panel B). (B) Shows representative plots of CD38 (top row), CD23 (middle row) and CD39 (bottom row) in comparison to Ki-67 expression on CLL cells pregated according to CD19 expression and light scatter characteristics. Ki-67+ cells show relatively increased levels of CD38 expression but the pattern is heterogeneous. In some patients the Ki-67+ cells show no CD38 expression, even if there is a small fraction of CD38+ CLL cells (see patient 1, left column). The majority of patients have a spectrum of CD38 expression on both Ki-67+ and Ki-67 cells (see patient 2, middle column), while in the remaining patients the Ki-67+ cells exclusively show strong CD38 expression (see patient 3, right column). Independent of the variability in CD38 expression, Ki-67+ cells uniformly show the strongest expression of CD39 and CD23.

Download figure to PowerPoint

The dChip analysis program (Li & Wong, 2001) was used to identify antigens that showed differential expression between the proliferating and the resting CLL cells (minimum reproducible >1·1 or <0·9-fold change in expression level). These antigens were tested on a further 18 cases (i.e. 23 in total). The antibodies tested on five or more cases are shown in Table I.

Table I.   Relative fold change in membrane protein expression in Ki-67+ chronic lymphocytic leukaemia (CLL) cells compared with Ki-67 CLL cells.
AntibodyNo. casesAverage fold difference in expression (Ki-67+ vs. Ki-67)Minimum fold differenceMaximum fold difference P-value
  1. *BD Pharmingen (Oxford, UK). †Becton Dickinson (Oxford, UK). ‡R&D systems (Abingdon, UK). §Caltag (Buckingham, UK). ¶Serotec (Oxford, UK). NS, not significant (P > 0·05); PE, phycoerythrin; APC, allophycocyanin.

CD24 PE*23−2·20−1·80−4·100·028
CD69 PE†23−1·59−1·11−2·410·111
CXCR4 PE*23−1·580·73−2·930·042
CXCR5 PE‡23−1·38−1·08−3·680·095
CD84 PE*50·650·550·69NS
CD82 PE*50·770·610·88NS
IL-4R PE50·860·621·18NS
CD40 PE*50·860·600·94NS
LAIR-1 PE*50·860·511·06NS
CCR6 APC*50·930·791·16NS
CD25 APC†231·281·131·420·022
CCR7 PE*51·361·082·28NS
CD5 APC†231·541·132·290·004
CD63 PE*231·651·222·000·017
CD43 APC§232·011·072·720·005
CD38 APC†232·081·412·640·036
CD27 APC¶232·211·594·100·004
CD71 PE*232·511·315·310·083
CD39 PE*232·692·093·030·001
CD86 PE*232·872·193·860·003
CD95 PE*232·982·174·500·002
CD23 APC†233·011·694·500·002

Results

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

Of the 66 antibodies used on the initial test case, 42 showed equivalent levels of binding on proliferating and resting CLL cells and were excluded from further analysis. The expression of the remaining 22 antibodies was tested in a further four cases and compared using dChip software. Of these, 15 antibodies showed a difference in expression greater that 50 units of fluorescence or a fold difference of <0·5 or >2·0 and were selected for further analysis in 23 cases. Table I lists the proteins that showed a highly specific and reproducible change in expression level in proliferating CLL cells compared with their resting counterpart fold-increase in expression of antigens in Ki-67+ CLL cells relative to Ki-67 CLL cells.

Several reproducible patterns of protein expression were seen in the proliferating and resting fractions in CLL (see Fig 1B). The majority of proteins tested showed increased levels of expression in the proliferating fraction of CLL. Antigens, such as CD43 and CD39 were homogenously expressed at a high level in the Ki-67+ cell fraction while the resting CLL cells showed heterogenous but consistently weaker expression (Fig 1B, bottom row). CD23 showed a similar profile but with more heterogeneous expression in the proliferating cells (Fig 1B, middle row). The relationship between CD38 and Ki-67 showed much greater inter-patient variability than was observed for other antigens. In cases where most of the CLL cells expressed CD38 above control levels, the Ki-67+ fraction usually showed higher levels of expression than the resting CLL cells. However, in cases where only a proportion of total CLL cells showed CD38 expression, the Ki-67+ fraction often showed only a modest increase in CD38 expression relative to the resting CLL cells. In such cases, the cells with the strongest CD38 expression were frequently lacking Ki-67 expression (Fig 1B, top row).

Expression of CD24, CD69, CXC chemokine receptors CXCR4 and CXCR5, was relatively lower in the Ki-67+ cell fraction. The majority of changes in protein expression were present on MCM-6 + Ki-67 cells, i.e. at the initiation of cell cycle, with the exception of CD24, which remained high on CLL cells initiating cell cycle (MCM-6 + Ki-67) and then decreased a median twofold as cells entered S-phase (MCM-6 + Ki-67+).

Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

The proteins CD39, CD86, CD95 and CD23 were uniformly increased during cell cycle in CLL cells from all patients tested. CD39 is a dual-spanning transmembrane NTPDase that hydrolyses extracellular ATP and ADP, eliciting various cellular responses such as proliferation, differentiation, chemotaxis and cytokine release (Qawi & Robson, 2000). CD86 (B7-2) is one of the molecules involved in the cognate interaction between T cell and B cells and is critical for germinal centre formation (Lenschow et al, 1996). CD95 is involved in the regulation of apoptosis and is normally present in B cells during progenitor and germinal centre stages of differentiation associated with high rates of proliferation (Kuppers, 2003). CD23 is a low-affinity receptor for immunoglobulin E and is already known from immunohistochemical studies to be more strongly expressed in proliferation centres (Soma et al, 2006).

Other than CD23, these molecules have rarely been considered to be associated with proliferation in CLL whereas much attention has focussed on the relationship between CD38 and Ki-67 expression. Although the level of CD38 expression was generally increased in proliferating CLL cells compared with resting cells, there was no direct relationship between CD38 and Ki-67 expression. Ki-67 expression was predominantly restricted to the CLL cells expressing the highest levels of CD39, CD86, CD95 and CD23 but was frequently absent in CLL cells with the highest levels of CD38 expression.

The CXC chemokine receptors, CXCR4 and CXCR5, as well as CD24 and CD69 were downregulated during the cell cycle. CD24 expression induces proliferation in combination with signals generated by antigen receptors in normal B cells and expression was only downregulated when CLL cells entered S-phase. CXCR4 binds stromal cell-derived factor-1 (SDF-1) and these molecules are centrally involved in the chemoattraction of CLL cells to the stromal cells responsible for their survival. Small peptide CXCR4 inhibitors antagonize activation and survival in CLL (Burger et al, 2005). Our data suggest that resting CLL cells express high levels of CXCR4 and migrate to stromal cells that secrete SDF-1. On contact, CXCR4 expression is decreased as the CLL cells enter into cell cycle. Therefore CXCR4 inhibitors may be best applied in combination with agents that can target CLL cells that are already bound to stromal support.

It was not possible to compare the expression of K-67 and ZAP-70 directly in individual patients because the only Ki-67 and ZAP-70 reagents that generated optimal results available at the time of study were all FITC-conjugated. Further studies in this area are being undertaken with the development of suitable PE-conjugated ZAP-70 antibodies. There is no evidence for a discrete stem cell population. Proliferating cells have increased or decreased expression of several specific markers, but the levels of these markers on proliferating cells are within the same range of expression found in resting cells. The ability to identify protein expression profiles that enrich for proliferating cells opens the possibility of purify these cellular fractions and performing comparative analyses to further investigate the molecular mechanisms underlying CLL cell proliferation. In particular, the relationship between CD38 expression and proliferation requires further investigation. The data supports the theory that CLL cells are cycling into and out of the proliferation centres; therefore targeting the proliferating cells would be expected to stop the accumulation of the CLL clone. Understanding the specific changes in expression of therapeutic targets, such as CXCR4 and CD23, will facilitate the development of strategies for inhibiting disease progression and eradicating minimal residual disease.

Acknowledgement

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

This work was supported by the Leukaemia Research Fund.

References

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
  • Brown, D.C. & Gatter, K.C. (2002) Ki67 protein: the immaculate deception? Histopathology, 40, 211.
  • Burger, M., Hartmann, T., Krome, M., Rawluk, J., Tamamura, H., Fujii, N., Kipps, T.J. & Burger, J.A. (2005) Small peptide inhibitors of the CXCR4 chemokine receptor (CD184) antagonize the activation, migration, and antiapoptotic responses of CXCL12 in chronic lymphocytic leukemia B cells. Blood, 106, 18241830.
  • Endl, E. & Gerdes, J. (2000) The Ki-67 protein: fascinating forms and an unknown function. Experimental Cell Research, 257, 231237.
  • Gonzalez, M.A., Tachibana, K.E., Laskey, R.A. & Coleman, N. (2005) Control of DNA replication and its potential clinical exploitation. Nature reviews. Cancer, 5, 135141.
  • Granziero, L., Ghia, P., Circosta, P., Gottardi, D., Strola, G., Geuna, M., Montagna, L., Piccoli, P., Chilosi, M. & Calligaris-Cappio, F. (2001). Survivin is expressed on CD40 stimulation and interfaces proliferation and apoptosis in B- cell chronic lymphocytic leukemia. Blood, 97, 27772783.
  • Hamblin, T.J. & Oscier, D.G. (1997) Chronic lymphocytic leukaemia: the nature of the leukaemic cell. Blood Reviews, 11, 119128.
  • Kuppers, R. (2003) Somatic hypermutation and B cell receptor selection in normal and transformed human B cells. Annals of the New York Academy of Sciences, 987, 173179.
  • Lenschow, D.J., Walunas, T.L. & Bluestone, J.A. (1996) CD28/B7 system of T cell costimulation. Annual Review of Immunology, 14, 233258.
  • Li, C. & Wong, W.H. (2001) Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proceedings of the National Academy of Science of the United States of America, 98, 3136.
  • Messmer, B.T., Messmer, D., Allen, S.L., Kolitz, J.E., Kudalkar, P., Cesar, D., Murphy, E.J., Koduru, P., Ferrarini, M., Zupo, S., Cutrona, G., Damle, R.N., Wasil, T., Rai, K.R., Hellerstein, M.K. & Chiorazzi, N. (2005) In vivo measurements document the dynamic cellular kinetics of chronic lymphocytic leukemia B cells. The Journal of Clinical Investigation, 115, 755764.
  • Qawi, I. & Robson, S.C. (2000) New developments in anti-platelet therapies: potential use of CD39/vascular ATP diphosphohydrolase in thrombotic disorders. Current Drug Targets, 1, 285296.
  • Rawstron, A.C., De Tute, R., Jack, A.S. & Hillmen, P. (2006) Flow cytometric protein expression profiling as a systematic approach for developing disease-specific assays: identification of a chronic lymphocytic leukaemia-specific assay for use in rituximab-containing regimens. Leukemia, 20, 21022110.
  • Schmid, C. & Isaacson, P.G. (1994) Proliferation centres in B-cell malignant lymphoma, lymphocytic (B CLL): an immunophenotypic study. Histopathology, 24, 445451.
  • Schrader, C., Janssen, D., Klapper, W., Siebman, J.U., Meusers, P., Brittinger, G., Kneba, M., Tiemann, M. & Parwaresch, R. (2005) Minichromosome maintenance protein 6, a proliferation marker superior to Ki-67 and independent predictor of survival in patients with mantle cell lymphoma. British Journal of Cancer, 93, 939945.
  • Soma, L.A., Craig, F.E. & Swerdlow, S.H. (2006) The proliferation center microenvironment and prognostic markers in chronic lymphocytic leukemia/small lymphocytic lymphoma. Human Pathology, 37, 152159.