The biology of hairy cells

Best Practice & Research Clinical Haematology Vol. 16, No. 1, pp. 1–13, 2003 doi:10.1053/ybeha.2003.235

1 The biology of hairy cells M. Zuzel

MD

Reader in Haematology

J. C. Cawley*

MD, PhD, FRCP, FRCPath

Professor of Haematology Department of Haematology, Royal University of Liverpool Hospital, Duncan Building, Daulby Street, Liverpool L69 3GA, UK

The evidence that hairy cells are activated clonal late B cells is presented. The largely non-specific (i.e. not confined to hairy-cell leukaemia) chromosome and genetic abnormalities are then described. Next, the features of malignant-cell activation are considered, including the distinctive morphology of hairy cells and their expression of activation-related antigens and activated adhesion receptors. Also, signalling and cytokine production are discussed in the context of malignant-cell activation. It is then demonstrated that many of the distinctive clinicopathological features of hairy-cell leukaemia can be explained in terms of the interaction of the activated malignant cells with other types of cell and tissue matrix. Finally, the biological basis of the hairy cell’s unusually high sensitivity to IFN-a and nucleoside analogues is discussed. Key words: adhesion receptors; B cells; cell activation; cell signalling; cytogenetics; cytokines; fibrosis; hairy cells; hairy-cell leukaemia; homing; IFN-a; pseudosinuses; surface immunoglobulin; T cells.

As in other haematological malignancies, many if not all of the clinicopathological features of hairy-cell leukaemia (HCL) can be related to the distinctive biological properties of the malignant cells.1 Thus, hairy cells (HCs) are abnormal clonal mature B cells with a phenotype of highly activated cells arrested at an advanced stage of maturation. Despite their apparent activation, HCs have a low proliferative rate, and the disease, therefore, usually has a very protracted course. Although many cytogenetic abnormalities have been described, the underlying oncogenic event(s) responsible for the disease are not yet clear. However, it is likely that one or more of these abnormalities are responsible for the intrinsic cell activation. This activation then, in turn, greatly influences aspects of the in vivo behaviour of HCs such as their tissue distribution and their influence on the tissue microenvironment, including the extracellular matrix and other resident cells. It is * Corresponding author. Tel.: þ44-115-706-2000; Fax.: þ 44-151-706-5810. E-mail address: [email protected] 1521-6926/03/$ - see front matter Q 2003 Elsevier Science Ltd. All rights reserved.

2 M. Zuzel and J. C. Cawley

also likely that intrinsic signals play a role in the distinctive sensitivity of HCs and HCL to treatment.

HCS ARE ABNORMAL CLONAL LATE B CELLS Following the first detailed description in 19582 of what has become known as hairy-cell leukaemia, there was a period of uncertainty regarding the exact origin of the malignant cells. The very peculiar morphology of HCs did not suggest any obvious normal counterpart, and the pattern of tissue involvement in the disease did not clearly indicate the lymphoid origin of the malignant cells. However, by the late 1970s it was unequivocally established that HCL is a clonal expansion of late B cells expressing light-chain-restricted surface immunoglobulin (SIg) of either IgG or multiple heavy-chain isotypes.3 Further studies demonstrated the unusual predominance of IgG34, and the expression of multiple heavy-chain isotypes at the molecular level.5 The latter study confirmed that clonally related multiple heavy-chain isotypes co-exist in single HCs, with individual isotypes presumably generated via RNA splicing. It was suggested that these cells are arrested at a point of isotype switching where RNA processing may precede deletional recombination.5 It is not clear why many HC clones do not undergo further isotype switching, but retain IgG3 expression. However, it has been suggested that this reflects an abnormality of HC/T-cell interaction (see the final section in this chapter). Most HC clones contain mutated VH genes, without intraclonal heterogeneity and without preferential usage of particular VH gene families.5,6 Because it is now known that VH hypermutation can occur in B cells developing along very different routes where they are exposed to a variety of different stimuli7, the presence of VH mutation in HCs provides little information concerning their route of development. Moreover, the possibility that VH hypermutation is driven by intrinsic activation signal(s) mimicking environmental stimuli for this process should also be considered. Following the introduction of monoclonal antibodies, a large number of studies in the 1980s confirmed the notion that HCs are mature B cells with little or no propensity to differentiate into plasma cells either in vivo or upon in vitro stimulation.8 The features identifying HCs as clonal late B cells are listed below: † strong expression of light-chain-restricted SIg; † multiple clonally related heavy-chain isotypes often expressed by single clone (IgG3 most frequent); † VH genes mutated; no intraclonal variation and no preferential VH family usage; † CD markers of mature (CD102 20þ, FMC7þ), but not terminally differentiated (SIgþ CD19þ) B cells; † little or no propensity for plasmacytoid differentiation.

CHROMOSOME ABNORMALITIES AND GENE MUTATIONS IN HCS Karyotypic and mutational analysis has so far given little insight into the oncogenic event(s) responsible for the development of the disease. A wide range of chromosomal abnormalities has been reported9, but none are consistently present and most, if not all, are found in other haematological malignancies.

Biology of hairy cells 3

However, abnormalities involving chromosome 5 are common in HCL (30 – 40% of cases)10, although they are rare in other mature B-lymphoid malignancies. These abnormalities may be either numerical (most commonly trisomy 5) or structural. Moreover, it has been suggested that, in some patients, loss of a tumour suppressor located in 5q13.3 may be involved in the pathogenesis of the disease.10 With regard to genetic abnormalities of potential pathogenetic importance in HCL, only a few have been described. The most commonly observed are monoallelic deletion11 or mutations of p53.12 The character of these mutations is different from that seen in other haematological malignancies and their impact on disease progression is clearly much less pronounced than in other chronic lymphoid leukaemias such as chronic lymphocytic and prolymphocytic leukaemias (CLL and PLL).12 Mutations of Bcl-6 have also been reported in , 25% of cases, but their significance is unclear.13 Interestingly, HCL is, in addition to mantle-cell lymphoma, the only other chronic B-cell disorder in which the malignant cells overexpress cyclin D1 (BCL-1 or PRAD-1).14 However, in contrast to the overexpression in mantle-cell lymphoma, that in HCL is not the result of 11:14 translocation. Presumably, therefore, high cyclin D1 expression in HCL is the result of either intrinsic or extrinsic stimulation (see the following section). Genetic and related abnormalities in HCL are listed below:

† † † †

no consistent karyotypic abnormality; a wide range of non-specific chromosome changes; abnormalities of 5q may be significant; mutations of p53 and Bcl-6 in about one-third of cases; functional significance unclear; † cyclin D1 over-expressed for reasons other than 11:14 translocation – ?activation.

Table 1. Features of HC activation. Feature

Comments

Morphology

Appearance of metabolically active cells. Surface ruffles and microvilli. Active cytoskeleton

Activation antigens

Express surface structures associated with activation of normal B cells and of certain other cell types

Adhesion receptors

Constitutively active and, as a result, HCs interact with various adhesive ligands without need for further activation

Cytokine secretion

A range of cytokines produced and enhanced by adhesion

Activation signals

Activated signalling components include Src family kinases, Rho-family GTPases, PKC and MAP kinases

4 M. Zuzel and J. C. Cawley

HCS ARE INTRINSICALLY ACTIVATED B CELLS The morphological appearance of HCs suggests that they are activated cells. The demonstration of surface-activation markers and activated intracellular signalling components supports this notion (Table 1). Morphology The morphology of HCs at the level of both light and electron microscopes corresponds to that of highly metabolically active, rather than quiescent, cells.1 Thus, HCs display a relative lack of chromatin condensation and possess abundant cytoplasm with plentiful mitochondria and ribosomes, together with scattered strands of rough endoplasmic reticulum. The ribosome – lamellar (R/L) complexes so distinctive of the disease15 may be related to the latter structures, but their nature and function are still unclear. The cortical cytoskeleton is clearly in a state of active re-organization as shown by the presence of distinctive membrane ruffling and microvilli formation. Much of the actin within HCs is polymerized into F-actin which supports membrane ruffles and extends into the microvilli.16 In contrast, in CLL and normal B cells, F-actin is less prominent and its distribution is punctate or confined to the centre of the cell.16 Among malignant lymphoid cells, HCs are unique in their strong expression of tartrate-resistant acid phosphatase (TRAP).17 This expression is readily detected cytochemically and forms part of the routine evaluation of HCL patients. The full biological significance of TRAP expression remains unknown, but it is clearly an activation feature because other lymphocytes can also express this acid phosphatase isoenzyme after stimulation.18 Activation antigens Cell-surface structures which are associated with normal B-cell activation (e.g. CD22, CD25, CD72 and CD40 ligand) are strongly expressed by HCs.19 Furthermore, markers normally lost after B-cell activation (e.g. CD21 and CD24) are expressed only at low levels.19 Other diagnostically important ‘HC-restricted’ antigens, such as CD11c, CD103 (B-ly7) and HC2, have each been associated with the activation of certain lymphoid and non-lymphoid types of cell.19,20 In addition, HCs express CD95 (Fas), which is found on normal activated B cells and provides apoptotic signals for elimination of cells not selected by antigen. However, when engaged by Fas ligand, the CD95 of HCs does not generate a proapoptotic signal.21 This suggests a failure of death-inducing-signalling complex (DISC) formation in HCs which may protect them from elimination by activated T cells. Adhesion receptors HCs possess a distinctive range of adhesion receptors (Table 2), and the increased expression of some of these (e.g. CD11c, CD49d, CD51) is a feature of cell activation. In vitro cell-adhesion studies have shown that a4/a5b1 integrins, avb3 integrin and CD44 readily interact with their specific ligands—fibronectin (FN), vitronectin (VN) and hyaluronan (HA), respectively.22,23 Moreover, ligand binding causes a spectrum of cellular responses such as spreading on FN and motility on VN and HA.22,23 In addition, HCs assemble soluble FN into an insoluble matrix, a process known to be mediated by

Biology of hairy cells 5

Table 2. Expression of adhesion receptors by HCs. Adhesion receptor Integrin

(CD)

Comment

a4b1

(41d/29)

Highly expressed. Involved in binding to matrix (fibronectin, FN) and other cells (VCAM-1, CD106)

a5b1

(49e/29)

Highly expressed. Involved in binding to, and assembly of, FN matrix

avb1

(51/29)

Weakly expressed. Function in HCs unclear

aLb2

(11a/18)

Little or no expression

aMb2

(11b/18)

Weakly expressed. Function in HCs unclear

axb2

(11c/18)

Highly expressed. Diagnostically important. Receptor for a number of ligands, including ICAM-1 (CD54), but function in HCs unclear

avb3

(51/61)

Receptor for vitronectin (VN) and PECAM-1 (CD31). Important in HC motility

aEb7

(103/b7)

Highly expressed and diagnostically important. Receptor for E cadherin, but function in HCL unclear

Other adhesion receptors CD44 Highly expressed. HC receptor for hyaluronan L -selectin (62L) Little or no expression. Shed on cell activation

activated a5b1.24 All of these observations suggest that adhesion receptors are constitutively activated in HCs and are capable of readily generating outside-in signals resulting in a range of functional responses relevant for the interaction of HCs with the tissue microenvironment (see next section). Cytokine secretion In addition to direct communication with the microenvironment via adhesion receptors, there is also a two-way interaction between HCs and other resident tissue cells via cytokines. Table 3 summarizes the cytokines/cytokine receptors potentially involved in the pathogenesis of HCL. In general, the function of cytokines as biological response modifiers is a stimulation/differentiation-dependent process highly regulated at the level of both receptor expression and cytokine production. Thus, cytokine production and receptor expression by HCs demonstrated in vitro can be taken as an indication of either intrinsic or exogenous activation. Adhesion is known to be an important regulator of cytokine production. Because the adhesion receptors of HCs are clearly activated, it is likely that the production of some cytokines is stimulated as a consequence of in vivo adhesion-receptor engagement. For example, autocrine tumour necrosis factor (TNF) production, which undoubtedly plays an important role in both the pathogenesis and therapy of HCL (see below), is greatly enhanced by integrin engagement.21 Also, HC adhesion to HA via CD44 stimulates bFGF production which, in turn, plays a role in the fibrosis of the disease (manuscript in preparation).

6 M. Zuzel and J. C. Cawley

Table 3. HC biology and cytokines. Cytokine Receptors on HCs

Comment

TNF-a

TNFRI and RII present Autocrinely produced. Serum levels high. Potential role in HC survival and response to therapy

IL-1b

Not studied, but presumably present

Cytokine and receptor levels increased in serum; origin unclear, but levels correlate with disease bulk.25 Reported to increase expression of SIg and PCA-126

IL-2

IL-2R a, b and g chains expressed but a predominant

Expression of CD25 (IL-2R achain) is of diagnostic importance and soluble IL-2R levels correlate with disease activity.27Cytokine not produced by HCs and does not cause HC proliferation28

IL-3

Receptor present

Among lymphoproliferative disorders, expression of the achain (CD123) of the receptor is a feature of HCL29, but neither autocrine nor paracrine effects of IL-3 on HCs described

IL-4

Not studied, but presumably present

Not produced by HCs. Acting alone has no effect, but may synergize with other growth factors for increased DNA synthesis28

IL-6

Present

Production may be induced by TNF.30 May participate in the proliferative effects of TNF31

IL-10

Not studied

Produced by HCs and may suppress T-cell cytokine production32

IL-15

Receptor present

Cytokine induces HC proliferation in vitro33

GM-CSF

Receptor present

Autocrine cytokine inhibits HC motility and prolongs cell survival34

M-CSF

Receptor present

mRNA increased on HC stimulation (unpublished observation). Exogenous M-CSF induces HC motility35

bFGF

Both FGF receptor and CD44v3 co-receptor present

Autocrinely produced by HCs.36 Involved in FN production by HCs (manuscript in preparation)

TGFb

Not studied

Produced by HCs and elevated in HCL serum and BM.36 May be involved in suppression of the production and function of normal haematological cells

IFN-a

Receptor present

Induces HC apoptosis in the absence of cell adhesion21 and may induce autocrine production37

Thus, although the intrinsic oncogenic event(s) in HCs is (are) not yet identified, it is likely that they influence cell behaviour not only through enhanced adhesion, but also through enhanced production of autocrine cytokines, stimulated either directly or indirectly by adhesion-generated signals. Activation signals As alluded to above, the signals responsible for the activation of HCs have a dual origin. Some originate from microenvironmental stimuli but these are likely to be influenced by constitutive signals arising from the oncogenic transformation.

Biology of hairy cells 7

The activated state of HCs is reflected in their characteristic profile of phosphorylated proteins. The first of these to be described was CD20, and it was suggested that phosphorylation of this membrane protein leads to increased Ca2þ influx, and that the protein is phosphorylated by CAM kinase II.38 Other early observations were the increased expression of Src39 and the high activity of protein tyrosine kinase in cell lysates.40 More recently, we have observed that HCs express high levels of activation of different members of the MAP kinase family, together with active Rac (manuscript in preparation). This may explain why HCs survive in vitro and why they readily adhere to, and move on, various adhesive ligands. The phosphorylations of some of these proteins disappear during in vitro culture, suggesting that they are induced by signals originating from in vivo stimulation. However, other signals persist in long-term culture in the absence of any external stimulus and are, therefore, truly constitutive. Moreover, some constitutive signals are primary, while others arise from continued in vitro secretion of autocrine cytokines. For example, among MAP kinase-family proteins, only phosphorylation of ERK1/2 persists, and is maintained by a pathway involving a constitutively active PKC. In contrast, the phosphorylations of JNK and p38 MAP kinase observed directly ex vivo become progressively reduced, but can be restored by plating cells on adhesive ligands. The adhesion-restored phosphorylation of p38 is largely mediated by increased production of TNF which also activates NFkB. This activation of NFkB, together with constitutively active ERK, is likely to be responsible for HC survival. Other truly constitutively active signalling components in HCs are the Rho-family GTPases Rac and Cdc42 (manuscript in preparation). Together, these GTPases may play an important role in the in vitro persistence of the distinctive surface appearance of HCs and in their spontaneous motility on certain ligands.

EFFECTS ON OTHER TYPES OF CELL AND TISSUE MATRIX Studies of the biological properties of HCs have demonstrated that most, if not all, of the distinctive features of HCL are directly attributable to specific interactions of the malignant cells with the tissue microenvironment. The distinctive features of the disease are listed in Table 4 and discussed below. Pancytopenia Anaemia, leukopenia and thrombocytopenia are characteristic of HCL, and pancytopenia is present in about 70% of cases. The cytopenias are due in part to marrow suppression by HCs and also the result of hypersplenism. The latter is a particular feature of HCL because the splenic vascular space is greatly enlarged as a result of pseudosinus formation (see below). Monocytopenia The almost complete absence of circulating monocytes and monocyte precursors in the bone marrow (BM) is a consistent feature of the disease and is, therefore, of diagnostic importance.41 Although the absence of monocyte production could in general be due to either growth factor deficiency or the production of suppression

8 M. Zuzel and J. C. Cawley

Table 4. Effects of HCs on other types of cell and tissue matrix. Effect

Comment

Pancytopenia

Caused by hypersplenism and BM invasion by HCs

Monocytopenia/reduced dendritic cells

Contributes to the immune defect of HCL, but specific cause(s) still unclear

T-cell abnormalities

Functional abnormality contributes to the immune defect of the disease. Skewed and restricted TCR repertoire expression may represent an ineffective autoreactive population

Specific tissue homing

HCs home to splenic red pulp, BM and portal tracts, but not lymph nodes; specific integrin–ligand interactions contribute to this

Tissue remodelling

BM fibrosis is a distinctive feature of HCL and is stimulated by CD44–HA interaction. Pseudosinus formation is diagnostic of HCL and the result of specific HC–endothelial cell interactions

factors, circulating levels of M-CSF are normal and factors that specifically suppress monocytes have not yet been identified in the disease (reviewed in Ref. 1). Dendritic cells Like monocytes, dendritic cells are virtually absent from the peripheral blood of HCL patients. This deficiency affects dendritic cells of both myeloid and lymphoid origin.42 Although the cause of this defect is unknown, it seems likely that similar mechanism(s) are involved in both the monocytopenia and dendritic-cell deficiency of the disease. Both slowly recover after successful treatment. T-cell abnormalities The absolute number of circulating T cells in HCL is relatively normal, as is the CD4:CD8 ratio.43 However, the proportion of CD4þ CD45 R0þ memory cells is significantly reduced in HCL patients as compared with healthy subjects.43 Furthermore, T cells are functionally abnormal, as are NK cells.44 Several factors may be responsible for this abnormal T-cell function. There is a disproportionate expansion of T cells lacking CD28, and these cells are unable to respond to HLA-class II-mismatched donor cells in mixed lymphocyte cultures.45 This absence of CD28, in combination with decreased antigen presentation due to reduced monocytes and dendritic cells may explain, at least in part, the T-cell dysfunction of HCL.

Biology of hairy cells 9

In contrast to the T cells in blood, those in the spleen of HCL patients show high percentages of CD45 R0þ cells within the CD4þ subset.32 Moreover, these cells appear activated and may represent an autoreactive population responding to the malignant HCs but unable to eliminate them (low expression of LFA-1 and ICAM by HCs may contribute to this ineffective elimination).32 The T-cell reaction to tumour antigens may be responsible for the skewed and very restricted TCR repertoire expression which, in turn, can further aggravate the immune deficiency of HCL.46 Specific tissue homing HCL has a very distinctive pattern of tissue involvement in that the malignant cells accumulate in the red pulp of the spleen without involving peripheral nodes in any major way. The BM is nearly always infiltrated and displays a fine reticulin fibrosis, which is absent from the spleen. The other organ consistently involved is the liver where the malignant cells are found along the hepatic sinusoids and in the portal tracts which are typically extensively fibrotic (reviewed in Ref. 1). For HCs to infiltrate each of these sites, specific adhesive interaction with matrix and resident cells, together with malignant-cell movement (perhaps stimulated by chemokines or paracrine/autocrine cytokines), are required. By studying the expression of adhesion receptors and their interaction with specific ligands, we have defined some of the mechanisms involved in these microenvironmental interactions. For example, we have proposed that localization in the vitronectin-rich splenic red pulp is related to avb3 expression by HCs31, while the absence of HC homing to peripheral nodes may be the consequence of low or absent L selectin on the malignant cells.47 Also, homing to marrow may be directed by the interaction of b1 integrins on HCs with VCAM on bone marrow endothelial and stromal cells.48 As regards chemokines and cytokines potentially involved in the tissue homing of HCs, relatively little is known. In a proportion of cases, HCs were found to express CXCR3, the receptor for MIG and IP-10 chemokines (CXCL9 and 10, respectively)49, and CXCR4, the receptor for SDF-1 (CXCL12).50 However, the precise roles of these receptors in tissue homing has not yet been established. We have shown that HCs express receptors for both M-CSF and GM-CSF and that M-CSF stimulates chemotaxis, while GM-CSF inhibits HC motility.34,35 Thus, M-CSF may be responsible for the frequent association of HCs with tissue macrophages, while GM-CSF may contribute to cell arrest at certain tissue sites. Tissue remodelling HCs also bind hyaluronan via CD44, and this may explain the tendency of the malignant cells to localize to areas rich in this glycosaminoglycan.23 Furthermore, the interaction with HA stimulates HCs to produce fibronectin (FN). The cells themselves then assemble the FN into a matrix to which they strongly adhere. This process is mediated by a4/a5b1 and is responsible for the reticulin fibrosis in HA-rich areas infiltrated by HCs (BM and portal tracts).23,24 Another distinctive feature of tissue remodelling by HCs is the formation of socalled pseudosinuses, especially in the spleen, but also sometimes in the BM.1 These are enlarged vascular spaces in which the endothelial lining is replaced by HCs. It is tempting to speculate that HCs adhere to endothelium via VCAM48 and then employ avb3 to penetrate between endothelial cells (where PECAM is the probable ligand)

10 M. Zuzel and J. C. Cawley

and then displace these cells by adhering to one or more components of the basement membrane.

BIOLOGICAL BASIS OF THE HIGH SENSITIVITY OF HCL TO TREATMENT HCL is unique among chronic lymphoproliferative disorders in being highly sensitive to IFN-a therapy (reviewed in Ref. 1). For many years the mechanism of this therapeutic effect remained obscure, mainly because the agent did not affect HC survival under conventional in vitro culture conditions. Although IFN therapy leads to the relatively rapid disappearance of HCs from the circulation, full remission is achieved only after prolonged administration of the agent. This suggests that the tissue microenvironment provides a degree of HC protection from the effects of the drug. We, therefore, cultured HCs in a serum-free medium on a non-adhesive surface to deprive them of stimulation by external growth factors and adhesion. Under these conditions, therapeutic concentrations of IFN-a caused HC apoptosis, which was prevented by blocking autocrine TNF. In addition, adhesion to either VN or FN blocked cell killing, despite an increase in autocrine TNF under these conditions. We, therefore, concluded that IFN kills HCs by sensitizing them to the apoptotic effects of autocrine TNF, and that cell adhesion generates signals which antagonize this effect.21 To gain further insight into the mechanisms involved, we have measured IAP (inhibitor of apoptosis) production during these experiments. This showed that IAP production stimulated by the NFkB pathway was reduced by IFN, and this reduction was completely abolished by cell adhesion.21 Because CLL cells are protected, rather than killed, by IFN under comparable in vitro conditions21, the unique sensitivity of HCs to killing by the drug is clearly an intrinsic property of HCs. We are currently investigating the role of pro-apoptotic p38 and JNK MAP kinases (which are activated in HCs, but not CLL lymphocytes) in the selective killing of the former cells by IFN.

Research agenda Major issues † the nature of the oncogenic event(s) responsible for the activated phenotype and developmental arrest of HCs † the relative contribution of intrinsic activation signals and various extrinsic stimuli to the clonal expansion and survival of HCs Other issues † the mechanism of bone marrow suppression † the nature of putative tumour antigens responsible for the apparent T-cell response to the HC clone † the functional importance of certain structures and proteins (e.g. R/L complexes, TRAP, axb2 and aEb7 integrins, the IL-3 receptor) selectively expressed by HCs

Biology of hairy cells 11

HCL is even more sensitive to nucleoside therapy than to IFN-a, but whether shared or different intracellular signals are involved in the response to both types of agent is unknown.

SUMMARY HCL is a clonal proliferation of abnormal activated B cells arrested at a late stage of maturation. HCs express light-chain-restricted SIg that can be either IgG (often IgG3) or of multiple heavy-chain isotypes. VH genes are usually mutated without intraclonal heterogeneity and without preferential usage of particular VH families. Numerous chromosomal abnormalities have been found in HCs, but none are consistently present or specific for HCL. The activated state of HCs is indicated by their very distinctive morphology, expression of a range of surface antigens associated with cell activation, activated adhesion receptors, and pronounced cytokine production (e.g. TNF-a). The activated state of adhesion receptors is evident from the ready interaction of HCs with a range of adhesive ligands without the need for additional stimulation. HC activation is also reflected in activated signalling components, including Src-family kinases, Rho-family GTPases, PKC and MAP kinases. Some of these signals are inherited from in vivo stimulation or from autocrine cytokine production, but others are truly constitutive and thus originate from the as yet unknown oncogenic event(s). The activated state of HCs is largely responsible for their unique interaction with, and effects on, the tissue microenvironment. Thus, HCs home to specific tissue sites (mainly red pulp of spleen and bone marrow), suppress resident cells, produce the fine reticulin fibrosis of BM and form pseudosinuses. Finally, the specific intrinsic pro-apoptotic signals of HCs are likely to be responsible for the ‘unique’ sensitivity of HCL to IFN-a and nucleosides.

REFERENCES 1. Burthem J & Cawley JC. Hairy-Cell Leukaemia. Berlin: Springer-Verlag, 1996. 2. Bouroncle BA, Wiseman BK & Doan CA. Leukemic reticuloendotheliosis. Blood 1958; 13: 609–630. 3. Burns GF, Cawley JC, Worman CP et al. Multiple heavy chain isotypes on the surface of the cells of hairycell leukemia. Blood 1978; 52: 1132–1147. 4. Kluin-Nelemans HC, Krouwels MM, Jansen JH et al. Hairy-cell leukaemia preferentially expresses the IgG3-subclass. Blood 1990; 75: 972 –975. * 5. Forconi F, Sahota SS, Raspadori D et al. Tumor cells of hairy cell leukemia express multiple clonally related immunoglobulin isotypes via RNA splicing. Blood 2001; 98: 1174–1181. 6. Maloum K, Magnac C, Azgui Z et al. VH gene expression in hairy cell leukaemia. British Journal of Haematology 1998; 101: 171–178. 7. Welleer S, Faili A, Garcia C et al. CD40-CD40L independent Ig gene hypermutation suggests a second B cell diversification pathway in humans. Proceedings of the National Academy of Sciences of the USA 2001; 98: 1166– 1170. 8. Pettitt AR, Zuzel M & Cawley JC. Hairy-cell leukaemia: biology and management. British Journal of Haematology 1999; 106: 2–8. 9. Sambani C, Trafalis DT, Mitsoulis-Mentzikoff C et al. Clonal chromosome rearrangements in hairy cell leukaemia: personal experience and review of literature. Cancer Genetics and Cytogenetics 2001; 129: 138–144. * 10. Wu X, Ivanova G, Merup M et al. Molecular analysis of the human chromosome 5q13.3 region in patients with hairy cell leukemia and identification of tumor suppressor gene candidates. Genomics 1999; 60: 161 –171. 11. Vallianatou K, Brito-Babapulle V, Matutes E et al. p53 gene deletion and trisomy 12 in hairy cell leukemia and its variant. Leukemia Research 1999; 23: 1041– 1045.

12 M. Zuzel and J. C. Cawley 12. Konig EA, Kusser WC, Day C et al. p53 mutations in hairy cell leukemia. Leukemia 2000; 14: 706–711. 13. Capello D, Vitolo U, Pasqualucci L et al. Distribution and pattern of BCL-6 mutations throughout the spectrum of B-cell neoplasia. Blood 2000; 95: 651–659. * 14. Brito-Babapulle V, Matutes E & Catovsky D. Cytogenetics, molecular genetics and immunophenotyping in hairy cell leukemia. In Tallman MS & Polliack A (eds) Hairy Cell Leukemia, pp 51 –52. Amsterdam: Harwood, 2000. 15. Katayama I, Li CY & Yam LT. Ultrastructural characteristics of the ‘hairy cells’ of leukemia reticuloendotheliosis. American Journal of Pathology 1972; 67: 361–370. 16. Caligaris-Cappio FG, Bergui L, Corbascio G et al. Cytoskeletal organisation is aberrantly rearranged in the cells of B chronic lymphocytic leukemia. Blood 1986; 67: 233–239. 17. Yam LT, Li CY & Lam KW. Tartrate-resistant acid phosphatase isoenzyme in the reticulum cells of leukemic reticuloendotheliosis. New England Journal of Medicine 1971; 284: 357–360. 18. Caligaris-Cappio F, Pizzolo G, Chilosi M et al. Phorbol ester induces abnormal chronic lymphocytic leukemia cells to express features of hairy cell leukemia. Blood 1985; 66: 1035–1042. 19. Burthem J, Zuzel M & Cawley JC. What is the nature of the hairy cell and why should we be interested? British Journal of Haematology 1997; 97: 511 –514. 20. van Der Vuurst De Vries AR & Logtenberg T. A phage antibody identifying an 80-kDa membrane glycoprotein exclusively expressed on a subpopulation of activated B cells and hairy cell leukemia B cells. European Journal of Immunology 1999; 29: 3898–3907. * 21. Baker PK, Pettitt AR, Slupsky JR et al. Response of hairy cells to IFNa involves induction of apoptosis through autocrine TNF-a and protection by adhesion. Blood 2002; 100: 647–653. * 22. Burthem J, Baker PK, Hunt JA & Cawley JC. Hairy cell interactions with extracellular matrix; expression of specific integrin receptors and their role in the cell’s response to specific adhesive proteins. Blood 1994; 84: 873– 882. * 23. Aziz KA, Till KJ, Zuzel M & Cawley JC. Involvement of CD44-hyaluronan interaction in malignant cell homing and fibronectin synthesis in hairy cell leukemia. Blood 2000; 96: 3161–3167. * 24. Burthem J & Cawley JC. The bone marrow fibrosis of hairy cell leukemia is caused by the synthesis and assembly of a fibronectin matrix by hairy cells. Blood 1994; 83: 497–504. 25. Barak V, Nisman B, Polliack A et al. Correlation of serum levels of interleukin-1 family members with disease activity and response to treatment in hairy cell leukaemia. European Cytokine Network 1998; 9: 33–39. 26. Takeuchi H & Katayama I. Interleukin-1 (IL-1 alpha and IL-1 beta) induces the differentiation/activation of B-cell chronic lymphoid leukaemia cells. Cytokine 1994; 6: 243–246. 27. Semenzato G, Trentin L, Zambello R et al. Origin of the soluble interleukin-2 receptor in the serum of patients with hairy cell leukemia. Leukemia 1988; 2: 788–792. 28. Barut BA, Cochran MK, O’Hara C & Anderson KC. Response patterns of hairy cell leukemia to B cell mitogens and growth factors. Blood 1990; 76: 2091–2097. 29. Munoz L, Nomdedeu JF, Lopez O et al. Interleukin-3 receptor achain (CD123) is widely expressed in haematological malignancies. Haematologica 2001; 86: 1261–1269. 30. Heslop HE, Bianchi AC, Cordingley FTet al. Effects of interferon alpha on autocrine growth factor loops in B lymphoproliferative disorders. Journal of Experimental Medicine 1990; 172: 1729– 1734. 31. Barak V, Nisman B & Foa R. Cytokines and cytokine receptors in hairy cell leukaemia: clinical relevance and correlation with disease activity. In Tallman MS & Polliack A (eds) Hairy Cell Leukemia, p 121. Amsterdam: Harwood, 2000. 32. Kluin-Nelemans HC, Kester MG, Oving I et al. Abnormally activated T lymphocytes in the spleen of patients with hairy-cell leukemia. Leukemia 1994; 8: 2095– 2101. 33. Trentin L, Zambello R, Facco M et al. Interleukin 16: a novel cytokine with regulatory properties on normal and neoplastic B lymphocytes. Leukemia & Lymphoma 1997; 27: 35–42. 34. Till KJ, Burthem J, Lopez A & Cawley JC. GM-CSF receptor: stage specific expression and function on late B cells. Blood 1996; 88: 479–486. 35. Burthem J, Baker PK, Hunt JA & Cawley JC. The action of M-CSF on B cells: M-CSF stimulates HC movement. Blood 1994; 83: 1381–1389. * 36. Gruber G, Schwarzmeier JD, Shehata M et al. Basic fibroblast growth factor is expressed by CD19/ CD11c-positive cells in hairy cell leukemia. Blood 1999; 94: 1077–1085. 37. Shehata M, Scwarzmeier JD, Nguyen STet al. Reconstitution of endogenous interferon-a by recombinant interferon in hairy cell leukemia. Cancer Research 2000; 60: 5420–5426. * 38. Genot EM, Meier KE, Licciardi KA et al. Phosphorylation of CD20 in cells from a hairy cell leukemia cell line. Evidence for involvement of calcium/calmodulin-dependent protein kinase II. Journal of Immunology 1993; 151: 71–82. 39. Lynch SA, Brugge JS, Fromowitz F et al. Increased expression of the src proto-oncogene in hairy cell leukemia and a subgroup of B-cell lymphomas. Leukemia 1993; 7: 1416– 1422.

Biology of hairy cells 13 40. Lower EE, Franco RS & Martelo OJ. Increased tyrosine protein kinase activity in hairy cell and monocytic leukemias. American Journal of Medical Sciences 1992; 303: 387 –391. 41. Seshadri RS, Brown EJ & Zipursky A. Leukemic reticuloendotheliosis: a failure of monocytic production. New England Journal of Medicine 1976; 295: 181–184. 42. Bourguin-Plonquet A, Rouard H, Roudot-Thoraval F et al. Severe decrease in peripheral blood dendritic cells in hairy cell leukaemia. British Journal of Haematology 2002; 116: 595–597. 43. van der Horst FAL, van der Marel A, den Ottolander GJ & Kluin-Nelemans HC. Decrease of memory T helper cells (CD4þCD45ROþ) in hairy cell leukemia. Leukemia 1993; 7: 46–50. 44. Trentin L, Zambello R, Agostini C et al. Mechanisms accounting for the defective natural killer activity in patients with hairy cell leukemia. Blood 1990; 75: 1525–1530. 45. van de Corput L, Falkenburg JH, Kester MG et al. Impaired expression of CD28 on T cells in hairy cell leukemia. Clinical Immunology 1999; 93: 256–262. * 46. Kluin-Nelemans HC, Kester MG, Melenhorst JJ et al. Persistent clonal excess and skewed T-cell repertoire in T cells from patients with hairy cell leukemia. Blood 1996; 87: 3795–3802. 47. Cawley JC, Zuzel M & Caligaris-Cappio F. Biology of the Hairy Cell. In Tallman MS & Polliack A (eds) Hairy Cell Leukemia, p 14. Amsterdam: Harwood, 2000. 48. Vincent AM, Burthem J, Brew R & Cawley JC. Endothelial interactions of hairy cells; the importance of a4b1 in the unusual tissue distribution of hairy cell leukemia. Blood 1996; 88: 3945–3952. 49. Trentin L, Agostini C, Facco M et al. The chemokine receptor CXCR3 is expressed on malignant B cells and mediates chemotaxis. Journal of Clinical Investigation 1999; 104: 115–121. 50. Durig J, Schmucker U & Duhrsen U. Differential expression of chemokine receptors in B cell malignancies. Leukemia 2001; 15: 752– 756.