Pathology of hairy-cell leukaemia

Transcription

1 Best Practice & Research Clinical Haematology Vol. 16, No. 1, pp , 2003 doi: /ybeha , 2 Pathology of hairy-cell leukaemia Kelly J. Bethel* MD Director Robert W. Sharpe MD Department of Pathology, Scripps Clinic, La Jolla, CA 92037, USA Hairy-cell leukaemia (HCL) is a low grade B-cell lymphoproliferative process that presents either with lymphocytosis or neutropenia/monocytopenia. It is a disease predominantly of bone marrow and spleen, although it can also involve other organs and sites. Recent advances including multiparameter flow cytometry and the development of antibodies with high specificity for HCL have permitted precise distinction of typical HCL from other lymphoproliferative diseases that can morphologically mimic the appearance of HCL. Although there is a commonly held belief that HCL is associated with a significant increase in second neoplasms, several recent studies have not supported this notion. The development of extremely effective therapy for HCL results in a high incidence of complete remission. However, a significant percentage of patients continue to harbour minimal residual disease that can be revealed with immunohistochemical and flow cytometric studies. Key words: antigens, CD; immunohistochemistry; immunophenotyping; leukaemia, hairy cell; leukaemia, hairy cell pathology; leukaemia, hairy cell immunology; leukaemia, B-cell; lymphoma; lymphoma, B-cell; neoplasm; residual; receptors, interleukin-2. Hairy-cell leukaemia (HCL) has a characteristic morphological appearance in the peripheral blood, bone marrow and spleen. Coupled with distinctive immunophenotypic and cytochemical characteristics, HCL has features that permit precise classification and distinction from other B-cell lymphoproliferative processes. This unique profile is of critical clinical importance because of the specific treatment protocols utilized in the management of HCL and, in particular, the striking responsiveness of the disease to therapy with cladibrine. A subset of patients with HCL has atypical morphological, immunophenotypic or cytochemical features. Usually designated hairy-cell leukaemia-variant, this lymphoproliferative process poses both diagnostic and management challenges that are distinct from those of typical HCL. This entity, and the differential diagnosis raised by its atypical features, is the subject of a subsequent chapter. This chapter discusses the morphology and ancillary diagnostic findings of typical HCL. Emphasis is placed on the immunophenotypic profile characteristic of HCL and * Corresponding author. Tel.: þ ; Fax: þ address: [email protected] (K.J. Bethel) /03/$ - see front matter Q 2003 Elsevier Science Ltd. All rights reserved.

2 16 K. J. Bethel and R. W. Sharpe the evolving role of immunohistochemical and flow cytometry studies in the diagnosis of HCL. Transformations of HCL to other lymphoproliferative disorders and associated malignancies are also reviewed. Finally, the approach to evaluation for residual disease following treatment is addressed. MORPHOLOGY Peripheral blood The morphological presentation of HCL in the peripheral blood is variable. Some patients present with the appearance of leukaemic peripheral blood, including a moderate lymphocytosis composed of morphologically identifiable hairy cells. In other cases, the peripheral blood smear is notable only for monocytopenia or neutropenia, with scant to absent circulating hairy cells. A hairy cell is a cell of small to intermediate size with an inconspicuous appearance under low-power microscopy consistent with a mature lymphoid cell. At higher power, however, characteristic features can be identified. The sharply delineated nuclear contour is generally oval and sometimes contains an indentation, giving the nucleus a kidney-shaped appearance. Occasional cases may have nuclei that are monocytoid, folded or even bilobed in shape. The chromatin stains medium blue with Romanowskytype stains (e.g. Wright stain), is finely granular, evenly distributed, and may contain a small, centrally located, often inconspicuous nucleolus. The generous cytoplasm appears pale blue on Wright-stained peripheral smears. It has a textured appearance at high magnification not quite foamy, but vaguely flocculent. The edges of the cytoplasmic domain are often indistinct, and the pale blue colour of the cytoplasm merges imperceptibly with the unstained background of the blood smear. Many hairy cells have a cytoplasmic border best described as frayed, although the most typical cells have a visible circumferential halo of long slender villous cytoplasmic projections that produce the classic hairy appearance (see Figure 1). Distinction from the villous projections present in the circulating lymphoma cells of splenic lymphoma with villous lymphocytes (SLVL) can be challenging. One of the most useful distinguishing features is the circumferential distribution of the hairs in a hairy cell, which is different from the longer, often focally clustered projections in SLVL. Bone marrow Because HCL induces bone marrow fibrosis, it is often difficult to get cytological preparations from studies of bone marrow. Dry taps are common when attempting to collect aspirate material, and even touch preparations of the core biopsy may lack evaluable cells. However, careful scrutiny of even paucicellular cytology preparations can be useful because of the fairly characteristic morphology of the cells, which is the same as that described above in the peripheral blood. In histological sections of the core biopsy, HCL occurs as an interstitial, patchy, or diffuse non-paratrabecular lymphoid infiltrate with a well-spaced appearance due to the large cytoplasmic domain of the cells (see Figure 2). There is usually an accompanying background of reticulin fibrosis. It has been demonstrated that hairy cells synthesize and assemble fibronectin, which may contribute to the reticulin fibrosis. 1 Characteristically, the infiltrates are accompanied by extravasated red cells. Occasional blood lakes, similar to those seen in the spleen, may be observed in

3 Pathology of HCL 17 Figure 1. Peripheral blood, 100 (oil). At high power, this circulating hairy cell has a sharply circumscribed oval nucleus, textured -appearing cytoplasm and nearly circumferential villous cytoplasmic extensions, or hairs. Figure 2. Bone marrow, 20. At low power, the hairy cell infiltrate in the bone marrow is interstitial, wellspaced, and accompanied by vascular tenting from the associated reticulin fibrosis.

4 18 K. J. Bethel and R. W. Sharpe Figure 3. Bone marrow, 40. At high power, the oval-to-reniform nuclear shape of the hairy cells can be appreciated, along with the voluminous cytoplasm that yields a fried-egg appearance to the individual cells. the bone marrow. At high power, the individual cells have a fried egg appearance, with the cleared out non-staining cytoplasm surrounding the small, dark, central nucleus (see Figure 3). Minimal involvement of the bone marrow can be extremely subtle, as the cells are small and tend to intercalate interstitially in a nearly invisible manner. Careful highpower inspection of haematoxylin and eosin (H&E) stained sections under oil immersion may reveal small numbers of characteristic cells, which can be further demonstrated by using immunostains (see discussion below). Although not generally obvious in diagnostic bone marrow biopsies, abnormal bone remodelling has been described in areas of hairy-cell infiltrate, including excessive bone resorption, which can result in lytic-appearing lesions on imaging studies. 2 Spleen HCL involves the red pulp of the spleen, with expansion by the well-spaced mononuclear infiltrate, and marked diminution of the white pulp areas (see Figure 4). Within the red pulp, a characteristic feature is the formation of blood lakes, which are blood-filled areas surrounded by hairy cells. These are probably formed by disruption of splenic sinusoidal vascular architecture by the neoplastic hairy cells. The red pulp pattern of involvement in the spleen is in contrast to the pattern displayed by other low-grade B-cell processes and is a useful histopathological distinguishing feature. Lymph node HCL can involve lymph nodes, although clinically significant lymphadenopathy is usually present only in advanced disease. The pattern of involvement is interfollicular.

5 Pathology of HCL 19 Figure 4. Spleen, 10. The splenic infiltrate occupies the red pulp compartment, with atrophy of the white pulp. Here the trabeculum is lacking the normal cuff of white pulp lymphocytes and there is a diffuse red pulp infiltrate of hairy cell leukaemia. Architectural features similar to those seen in spleen and bone marrow are seen. The typical appearance is patchy areas of well-spaced small lymphoid cells with voluminous clear cytoplasm, dotted with extravasated red blood cells, and focal blood lake formation (see Figure 5). Other sites Although predominantly a disease of peripheral blood, bone marrow and spleen, HCL can involve other body sites. Hepatic involvement may be seen in HCL, and is generally portal and sinusoidal in distribution. A distinctive feature of HCL in the liver, in contrast to other low-grade B-cell processes, is the finding of so-called angiomatous lesions, which are areas of focal hairy-cell infiltrate within hepatic lobules. The foci form small cavities, which appear lined by tumour cells and are filled with tumour cells and red blood cells. 3 The features are analogous to the blood lakes described in the spleen. Ultrastructural studies demonstrate disruption of the hepatic sinusoidal lining cells with extravasation of blood cells and other changes similar to those of peliosis hepatis. 4 As noted above, HCL can involve lymph nodes, although generally this is a finding associated with high tumour burden. Abdominal lymphadenopathy has been correlated with a more immature morphological appearance and a more aggressive clinical course. Whether this clinical evolution represents part of the natural history of HCL or is a form of transformation of hairy-cell leukaemia is uncertain. 5,6 Skin involvement has been reported, including an interesting case of transient leukaemia cutis in a patient with HCL who had systemic perturbation by pulmonary

6 20 K. J. Bethel and R. W. Sharpe Figure 5. Lymph node, 10. Hairy cell leukaemia occurs in an interfollicular and paracortical pattern. Here, it nearly entirely occupies the nodal paracortex, with several residual peripheral germinal centres. tuberculosis. 7 Also described in the literature is a case of HCL discovered in a herniorrhaphy specimen. 8 ANCILLARY DIAGNOSTIC STUDIES Acid phosphatase stain (TRAP) A useful cytochemical stain for the identification of HCL is tartrate-resistant acid phosphatase (TRAP). TRAP staining can be performed on air-dried cytological preparations, such as peripheral blood smears or bone marrow aspirate smears. Positivity appears as cytoplasmic granularity, and when such staining is present in cells that are otherwise morphologically suspect as hairy cells the specificity of the cytochemical stain is high. While TRAP positivity has been reported in the cells of several lymphoid malignancies, such as B-CLL, T-CLL (now T-PLL), ATCL/L and HCL 9,it is nevertheless a helpful stain for identifying HCL as the cells in these other processes are usually morphologically distinct from hairy cells. Immunoperoxidase staining of paraffin-embedded tissue As in other lymphoproliferative diseases, immunohistochemical stains have become a mainstay in the diagnosis and monitoring of patients with HCL. Both generic B-cell antibodies and markers with specificity for HCL are of utility. The B-cell markers CD20 and CD79a are useful in the quantification of disease, uncovering a morphologically indistinct infiltrate, and cytological characterization of positive cells (see Figure 6.). DBA.44 and antibodies reactive with TRAP provide a moderate degree of specificity for

7 Pathology of HCL 21 Figure 6. Bone marrow, CD20 immunostain, 100 (oil). The immunostain demonstrates a strong and coarse membrane pattern staining of the interstitially distributed cells. This technique is very useful to highlight a minimal infiltrate, which can be very subtle and hard to appreciate on an H&E-stained section. HCL and are of use in confirming the diagnosis or in ensuring that a small residual population of B-cells following chemotherapy is indicative of HCL. DBA.44 is an antibody, originally raised against B lymphocytes, that was found to be a sensitive marker of HCL and was expressed in greater than 95% of cases. 10 While this antibody, positive in less than 20% of other low-grade B-cell lymphoproliferative disease, appears to be useful in distinguishing HCL, it marks up to 80% of cases of SLVL, an entity which is often a primary differential diagnostic consideration in suspected HCL. 11 Cytochemical staining for TRAP requires cytological material and cannot be performed on paraffin-embedded tissue. This limitation has been circumvented by the development of an antibody, 9C5, with high specificity for acid phosphatase isoenzyme 5 responsible for the cytochemical TRAP positivity of HCL. 12 This antibody marks greater than 90% of HCL and has a sensitivity slightly less than that of cytochemical TRAP. As with DBA.44, this antibody marks only a subset of the total neoplastic population and is also dimly expressed in some cases of SLVL. 13 Immunohistochemical analysis assists in the assessment of biopsies from patients with HCL in a number of ways. Both DBA.44 and 9C5, because of their moderately high specificity, are of utility in confirming suspected HCL, particularly in the absence of fresh or cytological material for flow cytometric and cytochemical studies. CD20, which shows uniform bright expression in HCL, is an excellent antibody for quantifying the neoplastic infiltrate and almost always reveals a greater degree of marrow involvement than suspected from standard histological stains. This is particularly important following treatment when there is no morphological evidence of residual HCL (see discussion of residual disease that follows). DBA.44 and 9C5 are poor substitutes for quantification because a significant portion of the neoplastic infiltrate is often not marked by these

8 22 K. J. Bethel and R. W. Sharpe antibodies. Finally, and particularly in the setting of minimal residual disease, CD20 serves to highlight small populations of B-lymphocytes that can be examined for cytological features of HCL. In our institution, CD20 is routinely employed in the assessment of all HCL bone marrows, primarily for quantification, while other antibodies are used infrequently to support a morphological impression of HCL in the absence of cytochemical or flow cytometry studies. Immunophenotype Immunophenotypic analysis of HCL has played a pivotal role in ascertaining the lineage and probable normal counterpart of the neoplastic cells in this disorder. Until recently, these studies played a relatively small role in the clinical diagnosis of HCL. However, in the past 10 years the development of (i) antibodies of high specificity and (ii) multiparameter flow cytometry have facilitated the identification of minor populations of abnormal cells and established a utility for immunophenotypic analysis in the diagnosis of HCL comparable to other lymphoproliferative disorders. The emergence of readily available immunophenotypic analysis by flow cytometry for the evaluation of haematolymphoid malignancies in the 1990s significantly altered the approach to the diagnosis of these neoplasms. Immunophenotypic profiles rapidly assumed equal footing with morphological findings in the classification of disease. However, flow cytometry requires fresh tissue. Patients with HCL frequently manifest a dearth of circulating malignant cells. As well, the consistent presence of reticulin fibrosis usually causes an inaspirable bone marrow. For these reasons, large numbers of neoplastic cells for immunophenotypic analysis are rarely available in patients with HCL. Two recent developments have altered this equation and ushered immunophenotypic analysis to the forefront in the evaluation of patients with suspected HCL. First, late generation flow cytometers are capable of simultaneous analysis of four labelled antibodies in addition to assessment of forward and side-angle light scatter. This multiparameter capability enables an approach to flow cytometry known as population analysis that can permit the identification of extremely small subsets of cells. The second and synergistic advance was the identification of antibodies to antigens with a high specificity for HCL, specifically CD103, CD25 and bright CD11c. These two advances permit the reliable identification of HCL even with leukaemic populations of less than 0.5%. Because small populations of circulating hairy cells are present in nearly all untreated patients with HCL, immunophenotypic analysis of the peripheral blood or bone marrow has become a mainstay of diagnosis, relegating acid phosphatase stains to a supporting role. Prior to the identification of cell-surface antigens with high specificity for HCL, it was recognized that the distinctive topology of the neoplastic cells resulted in characteristic light scatter properties that could be exploited as a diagnostic parameter. 14 The irregular cytoplasmic projections that impart the characteristic hairy appearance of the leukaemic cells cause an increase in right-angle light scatter similar to that of monocytes and distinct from other benign and neoplastic lymphocytes. Coupling of a lineage-specific marker with right-angle light scatter permits the rapid identification of an abnormal lymphocytic population suspect for HCL. This characteristic finding is exploited in many laboratories as part of strategies for performing cost-effective immunophenotypic analysis, serving as a trigger for the assessment of antigens specific to hairy cell leukaemia. CD11c is a cell-surface antigen that is expressed on granulocytes, monocytes, natural killer cells and small populations of normal Tand B lymphocytes. This antigen is

9 Pathology of HCL 23 consistently expressed in HCL. 15 CD11c is expressed in other lymphoproliferative disorders, including B-cell chronic lymphocytic leukaemia/small lymphocytic lymphoma and splenic marginal zone lymphoma, seeming to limit the diagnostic utility of this antigen. 16 However, the intensity of expression of CD11c in HCL is 30-fold greater than B-CLL/SLL and SMZL. Similar brightness of CD11c expression is encountered only in prolymphocytic leukaemia. 17 Hence, the bright expression of CD11c retains utility in the diagnosis of HCL. The IL-2 receptor CD25 is expressed in greater than 90% of HCL. 18 However, this marker is also expressed in nearly half of B-CLL and other types of B-cell lymphoproliferative disorders. As with CD11c, CD25 is typically brighter in HCL than in other lymphoproliferative diseases. Unlike CD11c, this difference in intensity is small and does not permit reliable distinction between HCL and other B-cell processes. 17 Nevertheless, CD25 retains utility in the immunophenotypic analysis of HCL. Because of the reliable expression of this antigen in HCL, absence of CD25 can be a pertinent finding in excluding typical HCL. CD103 is a cell surface antigen initially identified as a marker of intraepithelial T lymphocytes. This antigen is expressed in greater than 95% of cases of HCL. 19 Except for in a minority of splenic marginal zone lymphomas, it is rarely expressed in other B-cell lymphoproliferative diseases. 17 Hence, expression of this antigen is the most specific immunophenotypic marker of HCL. The immunophenotypic profile of typical HCL is remarkably consistent. Pan B- cell antigens are expressed, with high intensity of CD20 and CD22. In addition, the cells express CD11c, CD25 and CD103. Surface immunoglobulin is usually brightly positive. This characteristic phenotype will be expressed in greater than 90% of cases of HCL. Occasionally, otherwise typical HCL will manifest immunophenotypic aberrations from the characteristic pattern. CD10 expression is the most common variation, present in 25% of cases in one large series. 17 CD5 is expressed in less than 5% of cases of HCL. Lastly, antigens commonly expressed in HCL, including CD11c, CD25 and CD103, may occasionally be absent. These phenotypic variations, while not excluding a diagnosis of typical HCL, raise additional diagnostic considerations, including variant HCL, splenic marginal zone lymphoma, follicular lymphoma and atypical B-CLL. 20 The evolution of flow cytometry during the past decade has led to an important shift in data analysis. With a limited number of parameters that could be simultaneously assessed, interpretation relied predominantly on the percentage of cells expressing evaluated antigens and co-expression by populations of cells of two antigens. The availability of flow cytometers capable of analysing five or six parameters simultaneously, coupled with the burgeoning knowledge of patterns of antigen expression and intensity in haematolymphoid malignancies, have permitted through the use of sophisticated gating strategies comprehensive analysis of specific abnormal populations of cells. This approach to interpretation of immunophenotypic data has greatly enhanced the sensitivity of flow cytometry in the detection of abnormal cellular populations. The distinctive and consistent immunophenotype observed in HCL has established population analysis by flow cytometry as a primary diagnostic tool. HCL can be reliably detected and characterized in the peripheral blood, bone marrow, and other body tissues even when the neoplastic cells comprise less than 1% of the analysed population. 21 When neoplastic cells are present in large numbers, the distinctive phenotype of typical HCL usually is easily distinguished from other B-cell lymphoproliferative diseases. In the setting of a very small number of leukaemic cells

10 24 K. J. Bethel and R. W. Sharpe it is important to distinguish this population from small subsets of normal lymphocytes with unusual antigen expression, specifically CD103 and CD11c. The bright expression of CD20, CD22 and CD11c, coupled with immunoglobulin light-chain restriction, usually permits discrimination between HCL and small normal populations expressing CD103 and CD11c. A highly sensitive and specific technique, immunophenotypic profiling now occupies a primary role in the diagnosis of HCL. The presence of characteristic morphological and immunophenotypic findings in the peripheral blood and/or bone marrow is sufficient to establish a definitive diagnosis and supplants the need for cytochemical and immunohistochemical studies, although the latter is of considerable utility in quantification, assessment of response to therapy and exclusion of minimal residual disease. Electron microscopy evaluation Hairy cells have a characteristic appearance under the electron microscope. In the past, prior to the wide availability of reliable immunophenotyping, electron microscopy has been useful for diagnosis. The presence of ribosome-lamellar structures in the cytoplasm is the characteristic ultrastructural feature of HCL, although they can also be rarely seen in other neoplastic and non-neoplastic cells. 22 Furthermore, their presence and appearance can be affected by interferon therapy, which can result in a morphologically immature structure. 23 Ultrastructural evaluation of the hairs demonstrates thin microvilli with associated actin-containing intermediate filaments. With TPA induction, the cells sprout long dendritic processes rich in submembranous F-actin, which makes intertwined networks. 24 In practical terms, with the advent of widely available immunostaining and flow cytometry, electron microscopy is rarely useful in the diagnosis of HCL. TRANSFORMATIONS OF HAIRY-CELL LEUKAEMIA Transformation to aggressive B-cell neoplasms Very rarely, HCL transforms to an aggressive lymphoproliferative process with an immature cytological appearance that retains some morphological, cytochemical, immunophenotypic or molecular features of the antecedent HCL. This process is referred to as blastic transformation of HCL. 25,26 There are a few case reports of diffuse large B-cell lymphoma arising in patients with HCL However, none of these reports demonstrate a clonal relationship between the two neoplasms, and whether these cases represent transformation of HCL or an associated but different lymphoid malignancy is uncertain. Transformations to other low-grade B-cell processes There are rare case reports of transformations between HCL and other low-grade B-cell processes. In one case, a patient with HCL transformed after 9 years to a lymphoplasmacytic lymphoma with an accompanying circulating IgG monoclonal protein. 31 A second report describes transformation from B-CLL to HCL characterized by acquisition of CD11c and CD Both reports pre-date

11 Pathology of HCL 25 the recognition of CD103 as a specific marker of HCL and the description of SLVL, an entity that can closely mimic HCL. Hence, in the absence of recent confirmatory reports, the existence of transformation between HCL and other low-grade B-cell processes is speculative. ASSOCIATED MALIGNANCIES IN HAIRY-CELL LEUKAEMIA There is a widely held belief that patients with HCL have an increased risk of developing a variety of second malignancies. 33 This notion has been supported in several recent and relatively small series, one suggesting a link to therapy with alpha-interferon. 34,35 However, several recent large series, including one with over 1000 patients, failed to show a significant increase in second malignancies in patients with HCL or a relationship between secondary neoplasia and interferon therapy While it is likely that HCL confers, at most, only a very modest increase in the overall incidence of second neoplasms, there are several intriguing disease associations that warrant discussion, specifically multiple myeloma (MM) and large granular lymphocytic (LGL) leukaemia. Multiple myeloma There have been case reports of coexistent HCL and MM 39,40 and a small series of three patients with synchronous HCL and MM. 41 However, no studies document evidence of a clonal linkage between the neoplasms in these patients. Additionally, the large series of HCL patients described above document no increase in the incidence of MM. It can be concluded that patients with HCL are at no increased risk of MM. Whether HCL rarely transforms to MM is an unanswered question awaiting molecular evidence of clonal relationship. LGL (T/NK cell processes) There appears to be an association between HCL and lymphoproliferative disorders of large granular lymphocytes Two recent studies have investigated this apparent association. In one series of 32 HCL patients, 10 were found to have populations of clonal cytotoxic T cells. 45 A second study demonstrated in HCL patients oligoclonal T- cell populations with restricted and skewed TCRBV repertoires. 46 These findings suggest that aberrant T-cell proliferations are common in HCL and may reflect a response to immunological aberrations induced by HCL, or may be due to the same stem cell defect as the co-existent HCL. ASSESSMENT OF RESIDUAL DISEASE The assessment of patients with HCL for evidence of residual disease and the clinical significance of minimal residual leukaemia are vexed issues. While the sequential development of treatment strategies employing alpha-interferon, deoxycoformycin, and 2-chlorodeoxyadenosine (2-CdA) have proven remarkable therapeutic advances in the treatment of HCL 47, it has become evident that a significant percentage of patients in apparent remission have demonstrable residual disease using moderately sensitive techniques, including immunohistochemistry and flow cytometry. 48 Often, the degree

12 26 K. J. Bethel and R. W. Sharpe of persistent marrow infiltration is significant, comprising up to 10% of marrow cellularity. Hence, the term minimal residual disease in the context of HCL often reflects the difficulty in morphological assessment of bone marrow using routine histological and cytological stains rather than the utilization of extraordinarily sensitive techniques to ferret out rare residual neoplastic cells. Whether minimal residual disease in HCL predicts clinical relapse is uncertain. Existing studies, although difficult to compare because of varying criteria of residual HCL, suggest that the identification of MRD in HCL has no or only modest positive predictive value for relapse and is therefore of uncertain clinical utility. The counter-intuitive notion that a significant percentage of HCL patients in complete remission (CR) have readily demonstrable residual disease requires that the terms complete remission and minimal residual disease be clarified. In the context of HCL, CR following therapy is generally defined as: 1. absence of organomegaly; 2. normalization of peripheral blood counts; 3. absence of peripheral blood and bone marrow evidence of HCL using routine histological and cytochemical stains. Minimal residual disease, although specific criteria vary, is characterized as identification of residual HCL using either immunohistochemical or flow cytometric techniques in the absence of recognizable leukaemic infiltration of the marrow using routine histological and cytochemical stains. The vast majority of these patients with MRD will meet the other criteria of CR. Dramatic therapeutic advances in the treatment of HCL, culminating in the development of 2-CdA, have resulted in CR rates of 75 91%. 52 Although long-term follow-up studies have confirmed the durability of clinical response, the utilization of immunohistochemical stains and flow cytometry in the assessment of post-treatment peripheral blood and bone marrow specimens has demonstrated that many patients in CR have evidence of MRD. In contrast to the highly sensitive techniques utilizing polymerase chain reaction (PCR) and sophisticated cytometric analysis to detect minute populations of neoplastic cells ( %) in acute leukaemia and chronic myeloid leukaemia, relatively crude techniques will identify residual HCL in up to 50% of patients in CR. This reflects the insensitivity and inadequacy of routine morphological assessment in the evaluation of post-treatment bone marrows in HCL patients. The interstitial and single-cell infiltration pattern characteristic of HCL and the cytolog appearance of the cells that can mimic the appearance of granulocytic and myeloid precursors in histological sections stained with haematoxylin and eosin (H&E) combine to mask minor populations of residual hairy cells comprising up to 10% of marrow cellularity. Immunostains for CD20 and DBA.44 readily demonstrate residual HCL. However, there are several important caveats in the interpretation of immunostains. Both CD20 and DBA.44 are non-specific, and they stain up to 6 and 2% of non-neoplastic marrow lymphocytes. 53 Additionally, a subset of marrow mononuclear cells possesses an abundant cytoplasmic rim that can mimic the histological appearance of HCL. Hence, caution must be exercised in the interpretation of immunostains with careful evaluation of both the percentage and cytological appearance of the cells expressing CD20 and DBA.44. If the population of cells is less than 5%, a definitive diagnosis of residual HCL should be reserved for cases with additional supportive evidence such

13 as TRAP (cytochemical or immunohistochemical) positive cells or flow-cytometric evidence of HCL. Immunophenotypic analysis of the peripheral blood or bone marrow can also be employed in the assessment for MRD in HCL. Only a small number of neoplastic cells are typically present in the post-therapy peripheral blood and marrow aspirate specimens, the latter presumably because of the pericellular fibrosis induced by the HCL infiltrates. Because of the highly specific immunophenotypic profile and identification of light-chain restriction by population analysis, even very small populations (, 0.5%) can be definitively identified. Finally, while there has been a decline in utilization of cytochemical stains in the assessment of haematolymphoid malignancies caused by the higher specificity and general availability of immunohistochemical and flow cytometry studies, acid phosphatase stains for evaluating TRAP-positive cells retain their utility for evaluating MRD in HCL. The presence of even a few brightly TRAP-positive cells that are cytologically appropriate is strong presumptive evidence of MRD. It is valid to question the clinical relevance of performing additional studies to reveal MRD in bone marrow specimens with no morphological evidence of HCL. Since recent studies have demonstrated that MRD has positive predictive value for eventual clinical relapse, these studies are probably justifiable, although institution and provider-specific relevance should be established. If immunohistochemical studies are not performed, the pathological interpretation should specify that MRD cannot be excluded. Other important criteria when considering immunohistochemical studies are the experience of the interpreting pathologist with HCL, the quality of histological sections, and the adequacy of the core biopsy. In reviewing numerous post-treatment bone marrows from referring institutions, we have observed that a remarkable degree of immunohistochemically demonstrable HCL can be masked by poor-quality histological preparation. Additionally, even with well-prepared H&E sections, we are occasionally surprised at the degree of residual marrow infiltration by HCL revealed with immunostains. Hence, in our institution, immunostains for CD20 are routinely performed in all post-treatment marrows. SUMMARY Pathology of HCL 27 HCL is a B-cell lymphoproliferative disease with distinctive morphological, cytochemical and immunophenotypic features. An accurate diagnosis is of critical importance because of the unique approach to the management of HCL patients. The neoplastic cell in HCL is an abnormal lymphocyte with morphological features that permit distinction from other lymphoproliferations. The cells are small with round, oval and reniform nuclei that have smooth nuclear margins, granular chromatin and a single small nucleolus. The ample cytoplasm is grey blue and textured with margins that are often frayed with fine projections imparting a hairy appearance. While the appearance of the neoplastic cells in HCL is distinctive, there are similarities with other lymphoproliferative diseases, especially SLVL. Hence, the diagnosis should be confirmed with ancillary studies. The cytochemical demonstration of TRAP, the first such study developed, has retained a high degree of specificity and utility. Immunophenotypic analysis by flow cytometry has moved to the forefront of confirmatory studies in HCL owing to the recognition of an antigen expression profile that is nearly unique and the development of multi-parameter flow cytometers that can precisely characterize small cellular populations. The leukaemic cells in HCL express bright CD20, bright CD22, bright CD11c, CD25 and CD103.

14 28 K. J. Bethel and R. W. Sharpe HCL almost nevermorphologically transforms to a clinically more aggressive disease a feature unique among low-grade B-cell lymphoproliferative diseases. Additionally, although thereis a commonly held belief that HCL is associated with a significant increasein second neoplasms, several recent studies have not supported this notion. The subtle marrow infiltration pattern of HCL is a challenge to the interpretation of post-treatment bone marrow biopsies. Residual disease of less than 5% of marrow cellularity is generally not recognizable in routinely stained preparations. Hence, immunostains for CD20 or immunophenotypic analysis by flow cytometry are necessary to exclude minimal residual disease. Practice points HCL can present with abnormalities of the peripheral blood, including cytopenias or lymphocytosis the cytological and histological appearance of HCL is highly characteristic. However, because other lymphoproliferative processes can mimic this appearance, confirmatory studies should be performed traditional confirmatory testing for HCL consists of cytochemical staining for TRAP within the cytoplasm of the neoplastic cells immunohistochemical stains, including TRAP, CD20 and DBA.44, are useful both in confirming the diagnosis and quantifying the neoplastic infiltrate flow cytometry is the most specific confirmatory methodology, with typical cases showing expression of CD20, CD11c, CD25 and CD103 HCL rarely undergoes transformation to a morphologically and clinically more aggressive disease. Second malignancies are probably not increased ancillary diagnostic techniques, including immunohistochemical and flow cytometry studies, are important post-therapy to evaluate minimal residual disease that is often inapparent in routinely stained histological sections Research agenda the pathology of typical HCL is well-established. However, there is a need to focus attention on utilizing advances in immunophenotypic and molecular techniques to further clarify the characteristics of other lymphoproliferative disorders that mimic the appearance of HCL, and thus further delineate the distinctions between typical HCL, variant HCL, splenic marginal zone lymphoma and prolymphocytic leukaemia the nature and pathogenesis of the peculiar and consistent myelofibrosis that occurs in association with HCL needs additional characterization molecular evaluation of the occasional synchronous and metachronous association between HCL and MM will elucidate the relationship in individual patients, between these two terminally differentiated B-cell proliferations whether patients with no demonstrable post-therapy disease by routine and immunohistochemical evaluation of the bone marrow are cured of HCL or have minimal residual disease below the threshold of detection remains a vexed question. Sensitive molecular studies on unfixed bone marrow specimens from post-therapy HCL patients will assist in resolving this issue

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