Pathology of chronic lymphocytic leukemia: an update

Annals of Diagnostic Pathology 11 (2007) 363 – 389

Review Article

Pathology of chronic lymphocytic leukemia: an update Kedar V. Inamdar, MD, PhD, Carlos E. Bueso-Ramos, MD, PhD⁎ Department of Hematopathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA

Abstract

Keywords:

Chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) is a clonal lymphoproliferative disorder characterized by proliferation of morphologically and immunophenotypically mature lymphocytes. CLL/SLL may proceed through different phases: an early phase in which tumor cells are predominantly small in size, with a low proliferation rate and prolonged cell survival, and a transformation phase with the frequent occurrence of extramedullary proliferation and an increase in large, immature cells. Although some patients with CLL have an indolent disease course and die after many years of unrelated causes, others have very rapidly disease progression and die of the disease within a few years of the diagnosis. In the past few years, considerable progress has been made in our ability to diagnose and classify CLL accurately. Through cytogenetics and molecular biology, it has been shown that CLL and variants are associated with a unique genotypic profile and that these genetic lesions often have a direct bearing on the pathogenesis and prognosis of the disease. Similarly, the development of antibodies to new biologic markers has allowed the identification of a unique immunophenotypic profile for CLL and variants. Moreover, accumulating evidence suggests that CLL cells respond to selected microenvironmental signals and that this confers a growth advantage and an extended survival to CLL cells. In this article, we will review the progress in the pathobiology of CLL and give an update on prognostic markers and tools in current pathology practice for risk stratification of CLL. © 2007 Elsevier Inc. All rights reserved. Chronic lymphocytic leukemia; Cytogenetics; Molecular; Immunophenotype

1. Incidence and epidemiology Chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) is the most common type of leukemia in older adults in the West, representing approximately 16% to 30% of all leukemias [1-3]. Australia, the continental United States, Italy, Switzerland, and Ireland have some of the highest reported incidence rates amongst the developed nations. In contrast to other low-grade B-cell lymphomas, the incidence of CLL/SLL is gradually declining [2,4]. According to the data collected from 12 Surveillance, Epidemiology and End Result (SEER) registries between 1992 and 2001, the rates of CLL/SLL declined an average of 2.1% per year [2]. As with most other leukemias, CLL is found in twice as many males as females and is slightly more

⁎ Corresponding author. Tel.: +1 713 792 6328; fax: +1 713 792 8438. E-mail address: [email protected] (C.E. Bueso-Ramos). 1092-9134/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.anndiagpath.2007.08.002

common in whites than in blacks [3]. The role of genetic factors in the etiology of CLL and other B-cell lymphoproliferative disorders is now well established [5]. Significant familial aggregation of CLL has been demonstrated, but the mode of inheritance is unknown.

2. Clinical features Chronic lymphocytic leukemia is primarily a disease of older adults, with more than 90% of the cases occurring in persons older than 50 years; however, CLL has also been described in young adults [6,7]. Unlike the signs and symptoms of acute leukemia, those of CLL develop gradually, and the onset of the disease is difficult to pinpoint. In fact, it is not unusual for the disease to be accidentally discovered because of an elevated lymphocyte count during the course of a routine visit to a physician [5]. Asymptomatic disease is seen in about 25% patients, and the duration of the relatively asymptomatic phase of CLL is extremely variable.

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Table 1 Typical features of chronic lymphocytic leukemia Morphology

PBS: Mature-appearing lymphocytes with high nuclear to cytoplasmic ratios, with scant agranular cytoplasm and homogeneously condensed chromatin without nucleoli. Characteristic “soccer ball’ chromatin pattern. Numerous smudge cells. AIHA. BM: Nodular, interstitial, and diffuse pattern of infiltration. LN: Vaguely nodular pattern with alternating dark zones of mature CLL cells and light zones (proliferation centers) of PL and paraimmunoblasts. Immunophenotype Dim expression of sIg (IgM/IgM +IgD/IgG) with kappa or lambda light chain restriction and expression of CD19 (dim), CD20 (dim), and CD79a CD5, CD22, CD23, CD43, CD11c. Negative for CD10 and FMC7 Prognosis Median survival 7.5 y; 50% 5-y survival; 30% N10-y survival Differential ALL, PLL, MCL, FL, HCL, reactive lymphocytosis, diagnosis monoclonal B-lymphocytosis AIHA indicates autoimmune hemolytic anemia; PBS, peripheral blood smear; BM, bone marrow; LN, lymph node; SCCL, small cleaved cell lymphoma; LGL, large granular lymphocytosis; ATLL, adult T-cell leukemia/lymphoma; PL, prolymphocytes.

Most of the patients have unexplained absolute and persistent lymphocytosis; mild lymphadenopathy of the cervical, supraclavicular, and/or axillary nodes; and splenomegaly as the earliest signs of CLL. Mild anemia and thrombocytopenia are also common in the earlier stages of the disease and are seen in approximately 50% and 25% of CLL patients, respectively. Cutaneous invasion occurs in 5% of patients in the form of nodular and diffuse skin infiltrations, erythroderma, exfoliative dermatitis, and secondary skin infections [8,9]. With disease progression, organ infiltration occurs, which can lead to massive adenopathy with splenomegaly, hypersplenism, and subsequent peripheral cytopenias. These patients present with B symptoms including fever, weight loss, and night sweats. Later in the disease, the bone marrow becomes more extensively infiltrated by the neoplastic cells, which results in more severe anemia, thrombocytopenia, and neutropenia due to extensive marrow replacement by the tumor cells. An increased tendency for bleeding, probably due to thrombocytopenia, further worsens the anemia and compromises hemostasis. Patients with CLL have significantly impaired immunologic activity. Hypogammaglobulinemia is found in approximately 50% of patients with CLL [10]. The immunoglobulin deficiency increases susceptibility to developing a variety of infections. Bacterial infections, especially of the respiratory tract, urinary tract, and skin, as well as viral infections such as herpes zoster and herpes simplex, are common and dramatically contribute to patient morbidity and mortality. Autoimmunity is frequently seen in CLL, with up to 25% of patients developing Coombs' positive autoimmune hemolytic anemia at some time during the course of the disease [11,12]. Antibodies produced against red blood cells and detected with the direct antiglobulin (Coombs') test may

precede, simultaneously occur with, or follow the development of CLL. Red cell aplasia is a rare occurrence [13]. Autoantibodies to platelets and neutrophils may also develop and lead to immune thrombocytopenic purpura and neutropenia. Bence Jones paraproteinemia has been reported in up to 65% of patients with CLL [14], and heavy-chain paraproteins, either IgM or IgG, can be detected. Table 1 summarizes the typical features of CLL. The hematologic abnormalities of CLL are characterized by a peripheral blood and bone marrow lymphocytosis. Anemia, when it occurs, is usually normochromic and normocytic, with a normal or low reticulocyte count (Fig. 1).

3. Criteria for diagnosis Guidelines for the diagnosis of CLL were originally proposed by the National Cancer Institute–Sponsored Working Group in 1988 and followed a year later by the International Working Group on CLL [15,16]. The current diagnosis of CLL is based on the revised guidelines of the criteria originally proposed by National Cancer Institute– Sponsored Working Group [17]. Diagnosis requires a persistent (N1 month) peripheral blood lymphocytosis (N5 × 109 cells/L) of mature-appearing lymphocytes in the absence of other causes and circulating lymphocytes with CLL immunophenotype (dim CD20 + , CD19 + /CD5 + , CD23+, FMC7−, and weak-intensity surface immunoglobulin [Ig]), as shown by flow cytometric immunophenotypic analysis of peripheral blood (Fig. 2). Lymphocyte infiltration of the bone marrow (more than 30% lymphocytes of all nucleated cells) is no longer required for diagnosis, but examination of bone marrow is essential in staging and follow-up cases to monitor the response to therapy. Morphologically, the lymphocytes are small or slightly

Fig. 1. Peripheral blood from a patient with CLL (original magnification ×1000). Note the lymphocytes with characteristically clumped, “soccer ball” chromatin.

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Fig. 2. Peripheral blood flow cytometric analysis of chronic lymphocytic leukemia. Leukemic bone marrow lymphocytes are gated by CD45-scattered analysis. The plot of CD5 vs CD19 is used to demonstrate dual-positive neoplastic lymphocytes with weak CD19 fluorescence intensity. The plot of CD23 vs FMC7 illustrates positive staining of the cells for CD23 but no staining of FMC7. The cells are then plotted for both CD19 vs κ and CD19 vs λ, showing κ light-chain clonality with a 15:1 ratio of kappa to lambda for the dual CD19+, CD5+ cells.

larger than normal and have a relatively mature, welldifferentiated appearance with a hypercondensed, almost “soccer ball”–appearing nuclear chromatin pattern (Fig. 1). Bare nuclei called smudge cells are common. Morphologic heterogeneity in CLL does exist and has been addressed by the French-American-British group [18]. Bone marrow examination, although not necessary for the diagnosis, is still valuable for prognostication based on a diffuse or nondiffuse pattern of infiltration (Fig. 3) [19]. Several reports document the prognostic significance of the pattern of bone marrow infiltration by CLL, with worse prognosis and shorter survival associated with diffuse involvement [20,21]. Although the morphological characteristics of the lymphocytes involved in most cases of CLL are quite distinctive, the membrane immunophenotype of the neoplastic cells must be determined for a definitive diagnosis [22]. A typical CLL case would demonstrate neoplastic cells that express CD5, CD11c, CD19 (dim), CD20 (dim), CD23, CD22 (weak

or negative), and monoclonal kappa or lambda Ig light chain. They are negative for FMC7 and CD79b (Fig. 2). 4. Morphological features 4.1. Peripheral blood examination Peripheral blood examination characteristically reveals an absolute lymphocytosis usually greater than 5000/μL with a predominance of a monotonous lymphoid cell population on the blood smear. About 6% of CLL cases present with absolute lymphocyte counts (ALC) of less than 5000/μL and with no clinical manifestations of CLL, and in such cases, a relative lymphocytosis of greater than or equal to 50% of the total differential count can be used as a clue to further workup of CLL by immunophenotyping and bone marrow examination [23]. They are small, and some are indistinguishable from normal mature lymphocytes when examined

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with Wright stain. They have high nuclear to cytoplasmic ratios with scant agranular cytoplasm and homogeneously condensed chromatin without nucleoli (Fig. 1). Numerous smudge cells, which represent degenerated leukemic cells, are also present in the blood smear. The lymphocytes of CLL are morphologically variable. Approximately 15% of patients with CLL will have atypical morphological features characterized by an increased (N10%) number of circulating prolymphocytes (PL), designated as CLL/PL, or an increased (N15%) number of circulating lymphoplasmacytic and cleaved cells, designated as “atypical” CLL [24,25] (Fig. 4). Prolymphocytes are larger than the small leukemic lymphocytes (9-10 μm) and have more cytoplasm, a rounded nucleus with less condensed chromatin, and a prominent central nucleolus. Alternatively, they may show features of activated proliferating lymphocytes, be larger in size (greater than 2 red blood cells), have exaggerated nuclear irregularities, cleaved or folded nuclei (greater than 15% cleaved, “reider cells”), open chromatin, 1-2 nucleoli, and moderate amounts of cytoplasm [26]. Cases with the latter features have been referred to as mixed-type CLL in the French-American-British classification. A variety of intracytoplasmic inclusions can be found in the leukemic lymphocytes of CLL, with a reported incidence ranging from 3% to 18% [27,28]. These include rod-like crystals, vacuoles, Ig molecules, and filamentous inclusions [29-31]. The current view is that CLL is a disease of clonal B lymphocytes that replicate at a normal to higher-than-normal rate and do not appear to have an inherent apoptotic defect [32].

5. Bone marrow The morphology of peripheral blood lymphocytes in CLL is duplicated in the bone marrow aspiration and biopsy specimens. Bone marrow aspirate smears reveal a lympho-

Fig. 3. Bone marrow involvement by CLL. (A) Photomicrograph of bone marrow biopsy section, showing involvement by CLL, with nodular and interstitial patterns, characterized by distinct, randomly distributed aggregates of small lymphocytes. (B) Photomicrograph of a bone marrow biopsy section, showing involvement by CLL, with a diffuse pattern. The entire bone marrow space between bone trabeculae is replaced by small lymphocytes. (C) Bone marrow aspirate smear from a patient with CLL (original magnification ×1000). Note the monotonous appearance of the cell population and lack of megakaryocytes.

Fig. 4. A case of CLL with atypical cytomorphological features, bone marrow aspirate smear (original magnification ×1000). Note the binucleate forms and the cleaved nuclei in some neoplastic cells.

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cytosis of ≥30% of all nucleated cells in the bone marrow differential count. There is a variation in the extent of medullary space involvement by the tumor cells, ranging from patchy infiltration of lymphocytes to diffuse involvement of the entire medullary cavity. The three main patterns of bone marrow involvement by CLL include nodular, interstitial, and diffuse (Fig. 3). These can occur as isolated patterns, but more often, combinations of these types occur [20]. The nodular pattern (Fig. 3A) is characterized by discrete, nonparatrabecular aggregates of small lymphocytes scattered throughout the marrow space. In contrast to benign lymphoid aggregates in the bone marrow, the nodular aggregates of CLL are less compact and have irregular borders with neoplastic lymphoid cells infiltrating into the surrounding space. Formation of proliferation centers, also known as pseudofollicular growth centers, may be seen cases with extensive involvement. In the interstitial pattern (Fig. 3A) the lymphocytes are admixed with the normal hematopoietic elements without much effacement of the architecture. In the diffuse growth pattern, neoplastic lymphoid cells infiltrate as diffuse sheets, virtually replacing the entire marrow space including the fat cells. The nodular and interstitial patterns are usually accompanied by preservation of normal hematopoiesis. In the diffuse pattern (Fig. 3B) the entire bone marrow space between bone trabeculae is replaced by small lymphocytes. Evidence suggests that patients with the diffuse pattern of involvement have a significantly shorter life expectancy, compared with those with a nodular or interstitial growth pattern [20,33]. An interfollicular pattern, more commonly seen in the lymph nodes than in the bone marrow, has been reported [34]. In this pattern, large paratrabecular and interstitial reactive germinal centers (GCs) devoid of peripheral mantle zones are usually seen. The diffuse vs nondiffuse patterns correlate well with ζ-associated protein 70 (ZAP-70) expression and Ig variable heavy chain (IgVH) mutation status. Chronic lymphocytic leukemia cases with nodular pattern of infiltration are somatically hypermutated and do not express ZAP-70, whereas those with diffuse involvement have an unmutated IgVH gene and show ZAP-70 expression [35].

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B-lymphocytes. Small lymphocytic lymphoma remains localized to the lymph node in over two thirds of cases, but some cases eventually progress to peripheral blood involvement, and then the disease is referred to as CLL/SLL. The two entities are now considered as a single disease with different tissue expressions [36,37]. Small lymphocytic lymphoma is the nodal counterpart of CLL [22]. The cellular components of SLL in the lymph node include small lymphocytes, PL, and paraimmunoblasts. The PL and paraimmunoblasts are usually concentrated in proliferation centers, also known as pseudofollicular growth centers, that are characteristic of this neoplasm [22,38] (Fig. 5). Sometimes this characteristic pseudofollicular pattern may be difficult to discern in more diffuse involvement when the PL and paraimmunoblasts are more uniformly scattered throughout the lymph node admixed with the smaller lymphocytes [39,40]. Plasmacytoid differentiation, although uncommon, has been reported in SLL/CLL, and these patients often present with serum monoclonal paraprotein, usually IgM [38,41-43]. Such cases have all the morphological and immunophenotypic features of CLL, which distinguishes them from lymphoplasmacytic lymphoma (LPL)/Waldenstrom's macroglobulinemia. Sometimes Reed-Sternberg–Hodgkin–like cells can be seen in the background of an otherwise typical case of SLL/ CLL [44]. These cells are not present in the usual background of Hodgkin's disease but are admixed with the neoplastic cells of SLL/CLL. Such cases are distinct from the Hodgkin variant of Richter transformation as well as from true composite lymphomas with coexistent SLL/CLL and Hodgkin's lymphoma. 7. Other organs After the bone marrow and lymph node, the spleen is the next most common site of involvement by CLL. In the

6. Lymph nodes Although CLL primarily involves the peripheral blood and the bone marrow, lymph node involvement is also very common. The lymph nodes are diffusely infiltrated by neoplastic lymphocytes that morphologically resemble the neoplastic cells in the peripheral blood and the bone marrow [36]. In some cases, however, tissue involvement occurs without an overt leukemic phase, and in these circumstances, the term small lymphocytic lymphoma is used [36]. Small lymphocytic lymphoma involves mainly lymph nodes and extranodal tissues and, to a lesser extent, peripheral blood and bone marrow. It is a diffuse non-Hodgkin's lymphoma (NHL) characterized by neoplastic transformation of small

Fig. 5. Chronic lymphocytic leukemia/SLL in a lymph node. Note the alternating dark and light zones. The light zones comprise proliferation centers formed by PL and paraimmunoblasts, whereas the smaller neoplastic CLL cells form the outer rim of dark zones.

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spleen, CLL is predominantly a white pulp disease; however, frequent and prominent infiltration of the red pulp is seen at presentation [45-47]. Diffuse infiltration of red pulp is common in diffuse involvement. Rare cases of isolated splenic involvement in the absence of nodal or bone marrow disease have been reported, and these patients tend to have a favorable prognosis [48]. Liver infiltration by CLL cells is primarily concentrated around the portal tracts, with sparing of the sinuses. Other extranodal and extramedullary sites of involvement include the tonsils, skin [49], gastrointestinal tract [50], and pleura [51]. Occlusion of coronary arteries by CLL as a cause of myocardial infarction was reported in a single case [52].

8. Immunophenotypic features A typical case of CLL can usually be diagnosed and subclassified based on morphological features alone. An immunophenotypic characterization of the neoplasm as either B cell using monoclonal antibodies confirms the diagnosis. The characteristic immunophenotype for CLL is dim expression of surface Ig (sIg), which is usually IgM or IgM with IgD or IgG with κ or λ light chain restriction and expression of B-cell–associated antigens (CD19, CD20, and CD79a), CD5, CD22, CD23, CD43, and CD11c (Table 1). A CD5+ and CD23+ phenotype is essentially diagnostic of CLL. A typical example of a CLL immunophenotype is shown in Fig. 2. Dim expression of CD20 is a useful feature that supports the diagnosis of CLL [53]. To facilitate the distinction of CLL from other B-lymphoproliferative disorders, an international scoring system based on expression patterns of immunophenotypic markers in CLL has been established. According to this system, 5 immunophenotypic markers are assessed in CLL: sIg, CD5, CD23, FMC7, and CD79b (or CD22). The score is based on expression of CD5 (1 point), CD23 (1 point), low-level (dim) surface Ig (1 point), and lack of CD79b (or CD22) (1 point), or FMC7 (1 point). Cases with scores of 4 or 5 are considered typical for CLL. Scores of 3 or less are considered atypical for CLL [54]. Of particular interest is the aberrant expression of CD5 in CLL. CD5 is normally a marker of T lymphocytes and B cells of early ontogeny, such as cord blood cells. In mouse models, normal CD5+ B cells are localized at the periphery of GC or in the mantle zones of secondary reactive follicles and have phenotypic and functional properties of circulating B cells. The precise functional role of the CD5 molecule is unknown. It has been proposed that CLL may arise from these normal CD5+ B cells as a result of aberrant antibody production [55]. This view has become less accepted in light of the findings that CD5 expression can be induced on surface of B cells by mitogenic stimulation [56]. Furthermore, identification of a distinct subtype of CLL with a somatically hypermutated IgVH gene, a characteristic of post-GC cell, makes the CD5+ naïve B-cell a less attractive candidate [57]. Approximately 7% to 20% of CLL lack

CD5 expression [58,59]. Patients with CD5− CLL usually have a milder form of disease lacking splenomegaly, anemia, and lymph node involvement and thus tend to have a favorable prognosis [60]. The subjectivity in the interpretation of CD5 negativity has led to a lack of consensus among investigators regarding the clinical significance of this particular subgroup. CD23 expression by CLL cells is also of interest. CD23 is a 45-kd transmembrane glycoprotein that functions as a low-affinity receptor of IgE. It is expressed at low levels in normal B cells, but upon activation, high expression is seen on B cells, and yet, it is characteristically expressed in CLL in which the neoplastic lymphocytes are believed to be dormant. High levels of soluble CD23 are found in sera from CLL patients, which directly correlate with disease activity [61,62]. FMC7 is a 105-kd glycoprotein that binds to a multimeric CD20 complex. There is a strong correlation between CD20 and FMC7 expression in normal and malignant B cells [63]. FMC7 is usually not expressed in most CLL cases, whereas other B-cell disorders consistently express FMC7. A minority of CLL cases are positive for FMC7; however; these cases also demonstrate atypical morphology with bright CD20 and sIg expression. Clinically they have a more aggressive course compared with FMC7-negative cases [64,65]. CD79b, also known as B29, is another B-cell marker that is consistently negative or underexpressed in CLL cases, and its level of expression directly correlates with the level of sIg expression in CLL [66]. Its dimerization with CD79a is critical in the assembly of the B-cell receptor complex and its subsequent expression on the surface [67,68]. The lack or underexpression of CD79b and consequently sIg has been attributed to the development of mutations in the coding sequences of the B29 gene in CLL patients that produce a truncated form of the protein, which, in turn, are responsible for reduced expression of sIg [69-71]. In contrast to CLL, most other B-cell neoplasms are CD79b-positive. In view of its diagnostic utility as an important negative marker of CLL, it has been incorporated in flow cytometric antibody panels for immunophenotyping of CLL as well as in the assessment of minimal residual disease. Neoplastic cells in a subset of CLL cases, however, express CD79b with reported frequencies of CD79b expression in CLL in the literature, ranging from 5% to 27% [66,72,73]. Evidence regarding the significance of this, however, is limited. It has been reported in the past that CD79b expression is associated with atypical morphological features, [66,74] but this data is controversial because other studies report no such association in their independent observations [73,75].

9. Other markers The expression of CD38 and ZAP-70 has more of a role in the prognostication of CLL than in the diagnosis of CLL and

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will be discussed later under the section of prognostic markers. Several additional markers, including members of the bcl-2 family, T-cell antigens, adhesion molecules, and antigens specific for myeloid and monocytic cells, have been reported in CLL, but the significance of their expression and use in the diagnosis or detection of minimal residual disease remains to be investigated. Apoptosis resistance is the hallmark of neoplastic cells in CLL, and in that regard, expression of the bcl-2 family of proteins in CLL is important. bcl-2 is overexpressed in almost all CLL, although the mechanisms underlying this are still undetermined [76]. Unlike in follicular lymphoma (FL), in which bcl-2 overexpression has been associated with t (14;18) (in which the bcl-2 open reading frame fuses with Ig regulatory sequences that puts it under direct influence of the Ig promoter, leading to overexpression of bcl-2), this mechanism is not seen in CLL, although there are rare reports of t(14;18) in CLL [77]. In addition to bcl-2, Bax and bcl-xL are also overexpressed, overall giving rise to apoptosis resistance in CLL cells. More recently, mantle cell lymphoma (MCL)–1, another antiapoptotic member of the bcl-2 family, was shown to be overexpressed in CLL and to contribute to cytotoxic therapy failure [78]. Abnormalities of expression of a variety of cell adhesion molecules including β1-integrins, β2-integrins, selectins, and the Ig supergene family of proteins have been investigated in CLL in order to understand the biologic behavior of CLL and also to determine the prognostic significance of different cell adhesion molecules phenotypic profiles [79-83]. β-Integrins are generally underexpressed in CLL compared with other B-cell NHLs, and the expression of CD49d/CD29, in particular, correlates with advanced clinical stage. Intense CD49d expression has been observed in CLL patients with massive lymphadenopathy and splenomegaly [80,84]. Low expression of CD11a/CD18 is a characteristic feature of CLL that allows its distinction from other B-cell NHLs, which are frequently CD11a/ CD18–positive [84-86]. In addition, other members of the integrin family including CD11c/CD18, CD31, CD48, and CD58 are also underexpressed in CLL patients, especially in those with chromosome 11q23 abnormalities [87]. Serum levels of intercellular adhesion molecule 1 (CD54), a 90-kd protein belonging to the Ig supergene family, are significantly elevated in CLL patients compared with healthy subjects and positively correlate with tumor burden and hepatosplenomegaly in the advanced clinical stage of CLL [80,88,89]. Similarly, strong expression of CD44 and CD62L (L-selectin) also correlate with progression of CLL and shorter survival [90-92].

cells that accumulate primarily through prolonged survival due to defects in apoptosis [93,94]. The small number of dividing leukemic cells in CLL makes conventional cytogenetic testing problematic, and mitogen stimulation of CLL B-cells is required to achieve an adequate number of metaphases for analysis. Genetic abnormalities, including the most common chromosomal abnormalities and gene rearrangements, are listed in Table 2.The presence of multiple chromosomal abnormalities in CLL has been implicated as an indicator of poor prognosis. Chromosomal aberrations encountered in CLL are, in general, deletions and/or amplifications of involved chromosomal regions; translocations are rare. None of the proto-oncogenes involved in the pathogenesis of other mature B-cell malignancies, including bcl-1, bcl-2, bcl-6, Pax-5, and c-Myc, show primary alterations in CLL [77,95]. Chromosome abnormalities can be detected in up to 50% of patients with CLL using conventional cytogenetic analysis, and early studies identified trisomy 12, 11q−, and 17p− as markers of poor prognosis [96,97]. By conventional Gbanding chromosomal analysis, the most common chromosomal abnormality in CLL is an extra chromosome 12, called “trisomy 12” (in 15%-20% of cases), followed by structural abnormalities of chromosomes 11, 13, and 14, which may occur alone or together with deletions or translocations of chromosome 13q14 [98-100]. In 1990, a pooled analysis reported on the prognostic significance of chromosome analysis using conventional cytogenetics in 433 patients with CLL [100]. Patients with a normal karyotype or 13q− abnormalities had a better survival than patients with trisomy 12 or a complex karyotype. A high percentage of cells in metaphase with chromosomal abnormalities, indicating highly proliferative leukemic cells, were associated with poor survival. After controlling for age and stage, the Table 2 Chromosomal abnormalities in CLL and their prognostic effect Chromosome Abnormality 13

11

17 12

10. Chromosomal abnormalities Chromosomal abnormalities in CLL are difficult to detect by conventional cytogenetic analysis, primarily due to the low proliferation rate in CLL. Most CLL cells are G0 phase

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6 14

del 13q14

Gene(s) affected

RB1, Leu-1, Leu-2, Leu-5, CLLD6CLLD8, KPNA3, miR15, miR16 del 11q22-23 FDX, ATM, MLL, PZLF, Mre11, RDX, NPAT, CUL5, PPP2R1B del 17p13 p53 +12 CDK2, CDK4, STAT6, APAF-1, MDM-2, CCLU1 del 6q t(14;19) bcl-3 (q32;q13)

Prognosis References Good

[99-108]

Poor

[95-117]

Poor Poor

[118-128] [91,130-137]

Poor Not known

[96,138-142] [145-148]

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prognostic significance of chromosome abnormalities by conventional cytogenetic testing remained significant. With the development of interphase fluorescence in situ hybridization (FISH) techniques, it has become possible to detect selected chromosome abnormalities in nondividing cells. Although more sensitive and specific than conventional cytogenetics, FISH does not completely evaluate all chromosomes. Studies using FISH probes in CLL found that chromosomal abnormalities were more common than those detected using conventional cytogenetics and had a different distribution. By interphase cytogenetics, the most common chromosomal abnormalities in CLL include deletions involving chromosome band 13q14, followed by deletion of 11q22.3-q23.1 and trisomy 12 [98,101]. The prognostic significance of chromosome analysis by FISH was studied using a comprehensive set of probes in 325 patients with CLL [101]. Chromosome abnormalities were identified in 82% of patients; 55% of patients had a 13q−; 18%, 11q−; 16%, trisomy 12; 7%, 17p−; 6%, 6q−; and 29%, more than 1 chromosome abnormality. On multivariate analysis, 17p− and 11q− were identified as variables associated with shorter overall survival.

11. Abnormalities of chromosome 13 Chromosome 13 abnormalities in CLL are characterized by interstitial deletion of band 13q14 or deletion of a larger region of the chromosome. Most of the 13q14 deletions are undetectable at the cytogenetic level but are detectable in more than 50% of the CLL cases with the use of molecular probes for the 13q14 region. The 13q14 deletions represent early clonal aberrations and suggest the presence of a tumor suppressor gene the loss or inactivation of which may be crucial to development of CLL. The 13q14 region harbors the well-characterized retinoblastoma (RB1) gene. Monoallelic loss of RB1 gene is seen in approximately 30% of CLL patients [102], but disruption of both alleles is rare. Subsequent studies identified additional loci D13S25 and D13S319 located distal to the 13q14 band, which are more frequently deleted in CLL, postulating a role for novel tumor suppressor genes located in these regions [103-105]. Several groups have since attempted a detailed characterization of this region and have narrowed it to a 1-Mb DNA fragment that has been sequenced. Up to 8 genes located in this region, including Leu-1, Leu-2, Leu-5, CLLD6-CLLD8, KPNA3, and LOC511131 have been examined for aberrations at the DNA or RNA level to implicate them in the pathogenesis of CLL, but none so far has yielded promising results [105-109]. More recently, deletion or down-regulation of micro-RNAs located in the 13q14 region has been implicated in the pathogenesis of CLL. Allelic loss of 2 micro-RNA genes, miR15 and miR16, located on 13q14 in more than 65% of CLL cases correlates with down-regulation of miR15 and miR16 expression in CLL cases with 13q14 deletions [110]. In addition, these genes are also known to harbor germline

mutations in CLL patients that are absent from patients with other types of cancers, thus implicating a functional role for these genes in the pathogenesis of CLL. Furthermore, a distinct micro-RNA expression profile has been identified for CLL that clearly identifies prognostic subgroups and correlates with IgVH mutation status, ZAP-70 expression, and disease progression [111].

12. Chromosome 11 abnormalities Structural aberrations of chromosome 11 are, by far, the most common recurring genetic event in a variety of lymphoproliferative disorders. Its incidence in CLL reported in various studies ranges from 12% to 25% [98,112,113]. The region most frequently deleted in CLL involves the long arm of chromosome 11 between bands q22 and q23. This region is rich in multiple genes that are involved in various hematologic malignancies, some of which are tumorsuppressor genes, including the FDX, ataxia telangiectasia mutated (ATM), MLL, PZLF, Mre11, and RDX genes [112,114]. The del 11q22.3 group of patients often has a poor prognosis because of early-onset advanced disease, extensive bulky lymphadenopathy, rapid lymphocyte doubling times, and shorter treatment-free survival. In one study, the acquisition of 11q deletion was a secondary event often following the acquisition of the 13q14 abnormality and was associated with CLL with increased PL rather than with typical CLL. In addition, acquisition of this abnormality was associated with clonal evolution and disease progression [115]. Tumor cells carrying 11q22.3 deletions often show up-regulation of genes that control cell cycle progression and signaling pathways [112,116,117]. Amongst the candidate tumor suppressor genes, ATM mutated in an autosomal recessive genetic syndrome called ataxia telangiectasia (characterized by neurologic abnormalities, infertility, increased sensitivity to radiation-induced DNA damage and increased susceptibility for developing various forms of cancer) has been implicated in the pathogenesis of CLL. The ATM gene frequently shows germline as well as somatic mutations in CLL patients, thus predisposing ATM heterozygotes to CLL [118]. More than two thirds of CLL cases have monoallelic loss of ATM. The other nondeleted allele often bears point mutations, most frequently in the PI-3 kinase domain [119]. More recently, a search for other disease-related genes in the 11q22-23 region identified 3 new candidate genes, NPAT (cell-cycle regulation), CUL5 (ubiquitin-dependent apoptosis regulation), and PPP2R1B (component of the cellcycle and apoptosis regulating PP2A) that were significantly down-regulated in CLL cases with 11q deletion [120].

13. Abnormalities of chromosome 17 band p13 Loss of genetic material from chromosome 17 in CLL most often involves the short arm, more specifically the

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region at band 17p13. The p53 gene, located on the short arm of chromosome 17 at band 13, plays an integral role in inducing apoptosis or cell cycle arrest after DNA damage. In CLL, a functional p53 pathway is an important indicator of responsiveness to purine nucleoside analogues, perhaps explaining the prognostic importance of 17p− abnormalities [121,122]. Multiple mechanisms by which p53 dysregulation may occur include direct p53 mutation due to abnormalities of chromosome 17p and inactivation by regulator genes, some of which reside on chromosome 11q [123]. p53 mutations occur in about 10% to 15% of patients with CLL [121,123,124]. Acquisition of p53 mutations is a late event in CLL, detected in about 10% of early CLL cases but in more than 30% of CLL patients in Richter transformation [123,125]. p53 abnormalities in CLL are associated with advanced stage at presentation, resistance to fludarabine and alkylating agent–based therapies, high incidence of transformation and disease progression in 1 to 2 years, and worst clinical outcome [125-127]. Detection of p53 abnormalities in CLL is possible at the protein level by immunohistochemistry (IHC) or flow cytometry and at the DNA level by FISH or direct DNA sequencing. The clinical effect and sensitivity of different methods to detect abnormalities have been assessed in various studies, and all techniques equally identify p53 as a marker of bad prognosis and define high-risk subpopulations in CLL. p53 deletions are detected by FISH in 10% to 20% cases and independently predict poor survival and resistance to chemotherapy in CLL patients [125]. Expression at the protein level has been assessed by immunohistochemical and immunocytochemical techniques. Immunohistochemical assessment of p53 protein expression may also have prognostic significance in CLL. Wild-type p53 protein is targeted to MDM-2–mediated ubiquitination and subsequent degradation and, thus, has a short half-life in early-stage tumor cells in CLL [128]. The protein product of mutated p53 has a prolonged survival in cells, and it is thus possible to detect p53 expression by IHC or immunocytochemistry using monoclonal antibody against p53 [129-131]. p53 positivity, as assessed by either IHC and immunocytochemistry, serves as an independent prognostic variable in CLL and is associated with p53 gene mutations, advanced disease, resistance to therapy, and poor survival [130,131]. Recent analysis of p53 expression by flow cytometric immunophenotypic analysis found that flow cytometry was more sensitive and specific than FISH in predicting p53 mutations as assessed by direct DNA sequencing and that flow cytometry was an effective screening technique for CLL patients to identify subgroups with disease likely to progress to an advanced stage [132].

14. Trisomy 12 Trisomy 12 is the most common genetic abnormality identified in CLL when assessed by conventional cytoge-

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netic analysis using banding techniques. Its reported incidence in various studies is between 10% and 20%, either as an isolated finding or in combination with other genetic aberrations such as del6q, del13q14, and others. When assessed by interphase FISH, its incidence seems to be slightly higher, in the range of 20% to 40% [94,133-135]. The occurrence of trisomy 12 is more common in CLL with atypical morphological and immunophenotypic features, including cleaved cytomorphology, lymphoplasmacytoid differentiation, bright CD20 expression, FMC7 positivity, and high frequency of CD38 expression [135-137]. In addition, CLL cases with trisomy 12 often have unmutated IgVH, portending poor prognosis. Numerous studies have evaluated the prognostic effect of trisomy 12 in CLL and found that acquisition of this abnormality is associated with advanced-stage disease, shorter time to progression, and significantly poor median survival rates [134-137]. Detailed structural analyses of chromosome 12 identified minimally gained region limited to bands 12q13-q15. A number of genes including CDK2, CDK4, STAT6, APAF-1, and MDM-2 are located in this region and play critical roles in the regulation of oncogenesis, cell cycle control, and apoptosis; however, the role of these genes in the pathogenesis of CLL has not been well characterized. RNA expression analyses of the candidate genes by microarray and quantitative polymerase chain reaction (PCR) showed up-regulation of genes mapping to region 12q in CLL cases with trisomy 12 compared with those without trisomy 12 [138]. However, protein expression analysis of 12 candidate genes in CLL cases with and without trisomy 12 did not reveal significant differences in protein expression between the 2 groups, suggesting postranslational mechanisms of deregulated expression [139]. More recently, the CCLU1 gene, the product of which resembles interleukin 4, was identified as a CLLspecific gene located on chromosome 12q22 that may be involved in the pathogenesis of CLL [140]. 15. Other chromosomal aberrations 15.1. del 6q Deletions of the long arm of chromosome 6 are detected in 4% to 6% of CLL cases by conventional cytogenetics, whereas by FISH analysis, up to 9% cases show 6q21 deletions [99,141]. Deletions of 6q are more common in SLL, the tissue counterpart of CLL [142], and are often associated with features that overlap with CLL with increased PL, including high white cell counts, extensive lymphadenopathy, and splenomegaly [142,143]. Morphologically, cases with 6q− often show atypical features, including large lymphocytes, immunoblast-like cells, prolymphocytoid cells, and cleaved lymphocytes [144,145]. A recent study analyzed 217 cases of CLL and found that CLL with 6q− is characterized by a high incidence of atypical morphology, classic immunophenotype with CD38

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positivity, and intermediate incidence of IGVH somatic hypermutation [144].

groups has become important for proper management of patients with CLL (reviewed by Ghia et al [155]).

16. Abnormalities of chromosome 14

18. Clinical factors of prognostic value in CLL

Abnormalities of chromosome 14 in CLL, although rare, are seen in about 6% to 14% of CLL cases and generally involve band 14q32, which harbors the Ig heavy chain (IgH) gene [100,146]. Translocations rather than deletions or gains are frequently reported involving this region. Over 50% of these translocations were previously reported to involve the CCND1 gene on chromosome 11 band q13, giving rise to t(11;14)(q13;q32), which is now considered a hallmark of MCL [147]. In retrospect, cases that exhibit this translocation are now categorized as MCL in the current 2001 World Health Organization classification of hematopoietic neoplasms [148]. The IgH locus may be involved in translocations with other partners, although their mention in the literature is limited to case reports. One such translocation involves chromosome 19 band q13 that involves the BCL3 gene, giving rise to t(14;19) (q32;q13). This translocation juxtaposes the BCL3 gene at chromosome 19q13 with the Ig heavy chain gene locus at 14q32. Since its first description in 2 CLL patients in 1983, many literature reports have described this translocation in CLL [149-152]. Translocation (14;19) (q32;q13) cases are associated with atypical lymphocyte morphology composed of a mixture of small and larger paraimmunoblast-like cells, increased PL (usually b10%), and cleaved/indented nuclei. In addition, such patients present at a younger age and with rapid disease progression [151-153]; however, prognostic studies assessing the effect of this abnormality on survival are lacking.

In the group of clinical factors that better help prognosticate CLL, clinical stage, lymphocyte doubling time, and the pattern of bone marrow infiltration by the neoplastic cells are, by far, the most critical factors that predict the clinical course of the disease. Indolent CLL disease is associated with a Rai stage of 0, I, or II, blood lymphocyte doubling time (LDT) greater than 12 months [156], and a nondiffuse pattern of bone marrow lymphocyte infiltration [20]. A Rai stage of III or IV, short LDT (b12 months) and a diffuse lymphocyte infiltration of the bone marrow characterize the aggressive form of CLL.

17. Clinical course, prognostic factors, and staging The clinical course of CLL is extremely variable due to the heterogeneity of the disease, with survival ranging from months to several years and largely dependent on the clinical stage. The overall median survival for patients with CLL is currently 7.5 years; 50% of patients are alive 5 years after diagnosis, whereas 30% have a 10-year survival [154]. Chronic lymphocytic leukemia can be an indolent disease with an asymptomatic presentation and a stable or slowly rising peripheral lymphocyte count, and it may not require any treatment. The disease may progress as late as 10 to 15 years after the initial diagnosis. In contrast, approximately 20% of patients with CLL have a very aggressive clinical course, presenting with progressive lymphocytosis of the peripheral blood and marrow, lymphadenopathy, splenomegaly, anemia, neutropenia, thrombocytopenia, autoimmune phenomena, and infection that progress rapidly from initial diagnosis, and death can occur within 1 to 2 years. The wide variation seen among patients is not fully understood, but a search for clinical and biologic factors to predict the CLL patient's prognosis and identify various stages and risk

18.1. Clinical staging The clinical staging systems proposed by Rai et al [157], Binet et al [158], and the International Workshop on CLL system [15] are the 3 major staging systems that are the strongest predictors of clinical outcome in CLL; however, only the Rai system is widely used in the United States. The Rai and Binet staging systems, along with median survival for each system by stage, are shown in Table 3. In his initial proposal, Rai categorized CLL into 5 stages that correlated Table 3 Staging systems for chronic lymphocytic leukemia Stage

Clinical features

Rai staging system [158] Original Modified 0 Low Persistent lymphocytosis (N1 mo) in PB and BM (≥5 × 109 lymphs/L in PB) If b5 × 109 lymphocytes/L) then ≥30% lymphocytes in BM) I Intermediate Lymphocytosis and lymphadenopathy II Lymphocytosis and hepatomegaly with or without lymphadenopathy III High Lymphocytosis and anemia (Hgb b11 g/dL) IV Lymphocytosis and thrombocytopenia (platelets b100 × 109/liter) Binet staging system [157] A Lymphocytosis with lymphadenopathy a in b2 LN regions B Lymphadenopathy at N3 sites in the absence of anemia or thrombocytopenia C Anemia (Hgb b10 g/dL) and/or thrombocytopenia (platelets b100 × 106/dL) with or without lymphadenopathy

Median survival (y)

N10

6-8

b2

5

2

PB indicates peripheral blood; BM, bone marrow; Hgb, hemoglobin. a Survival equivalent to that of age- and sex-matched (French) population.

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with survival. Patients with Rai stage 0 disease have the longest survival, exceeding 20 years in some and with approximately 75% of patients alive after 10 years. Patients in Rai stage I have a median survival of 8 to 10 years, whereas for Rai stage II the survival drops to 5 to 8 years. It was later modified to a simpler version that combined the different stages into 3 risk categories. Stage 0 cases were included in the low-risk category with median survival over 10 years, stages I and II were categorized as intermediaterisk group with median survival of 7 years, and stages III and IV with median survival less than 2 years were considered high-risk CLL [159]. Staging systems for CLL do not consistently predict whether a patient's clinical course is more likely to be indolent or progressive. 18.2. Lymphocyte doubling time One calculation that may be associated with meaningful differences in disease kinetics is the LDT. The LDT is calculated by determining the number of months it takes the ALC to double in number. One study reported patients with a LDT of 12 months or less had a median survival of 61 months, whereas median survival was not reached for patients with a LDT of more than 12 months (median follow-up, 118 months) [160]. For patients with early-stage disease (Binet stage A or B), the estimated median survival was 66 months for those with a LDT of ≤12 months, whereas no patients with an LDT of more than 12 months had died with a median follow-up 48 months. Although other reports have confirmed the usefulness of the LDT for patients with early-stage disease [156,161], there are significant problems with this tool. The LDT is inherently retrospective, and its temporal change can be quite variable. It is also confounded by a host of factors that may transiently affect the ALC. Treatment decisions based on this variable may delay treatment for some patients with characteristics of aggressive disease. 18.3. Pattern of bone marrow infiltration Several groups have demonstrated the prognostic value of histologic patterns of bone marrow infiltration by CLL cells. Gray et al. [162] first described the significance of bone marrow infiltration patterns in CLL. They categorized lymphocytic infiltration of bone marrow by CLL into nodular, mixed (nodular and diffuse), and diffuse patterns and correlated these patterns with survival. An interstitial pattern was later described by Rywlin [163], and thus, 4 patterns of infiltration were recognized that were evaluated by several investigators in various combinations to better assess the prognostic effect of bone marrow histology in CLL [157,163-167]. In a study of 115 patients by Gray et al [162] that included patients with lymphosarcoma cell leukemia, patients with a diffuse pattern of involvement had a median survival of 2 years, compared with patients with the nodular and mixed patterns, who did relatively well with a median survival of 9 years. Rozman et al [19] evaluated 63 cases of CLL and identified 4 patterns of bone marrow infiltration

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(interstitial, nodular, mixed, and diffuse) that correlated well with different clinical staging systems in CLL. Furthermore, they found that CLL patients with interstitial and nodular involvement of bone marrow did relatively better in terms of life expectancy compared with those with diffuse involvement. In a multivariate analysis of 329 CLL patients, Rozman et al [20] demonstrated that the bone marrow pattern of infiltration was a significant independent prognostic variable in CLL and remained markedly significant in the final regression model over other prognostic variables, including anemia and hepatosplenomegaly. Interestingly, Binet et al [168] failed to find a significant difference in survival according to the bone marrow histologic pattern in their study of 129 CLL patients; however, the presence of marked lymphocytosis with diffuse bone marrow infiltration in their stage II and IV CLL patients is in keeping with the results of other investigators. 19. Biologic factors of prognostic value in CLL The most extensively studied biologic prognostic factors include serum thymidine kinase, serum β2 microglobulin, soluble CD23, greater than 10% PL on peripheral blood, and p53 expression. In addition, many new biologic markers have emerged in the past decade in an attempt to identify prognostic subgroups in CLL. Only a few of these have been uniformly accepted to have an independent prognostic value in risk stratification of CLL. These include mutational status of IgVH gene, surface expression of CD38, cytoplasmic expression of ZAP-70, and the presence of chromosomal abnormalities associated with a more progressive clinical course and shorter survival duration (reviewed in Ghia et al [155] and Faguet [169]). 19.1. Serum thymidine kinase Thymidine kinase (TK) is a cellular enzyme involved in a salvage pathway for DNA synthesis. The predominant form of TK is present in dividing cells and absent in nondividing cells, and thus, this enzyme is a potentially useful marker of proliferative activity. Assays for TK are feasible and commercially available, making it a potentially widely available and useful prognostic marker. Early studies reported that elevated TK levels correlated with advanced Rai stage and progressive disease [67] and, on retrospective analysis, untreated patients with indolent disease could be separated into 2 prognostic groups based on their initial serum TK. In a more recent study of 113 patients with CLL, TK was an independent prognostic variable on multivariate analysis; however, this study did not include assessment of IgVH mutation status, CD38, or chromosome analysis by FISH [170]. Later, these same investigators reported on the significance of TK in 122 untreated CLL patients [171]. When Binet stage A patients were stratified by TK level, median progression-free survival was 9 months (range, 5-13 months) for patients with

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high serum TK levels (N7.1 U/L), compared with 49 months (range, 24-74 months) for patients with low serum TK levels (P b .001). Another recent study reported on the relationship of TK and mutation status and found that a TK level of N15 U/L was a strong and independent predictor of nonmutated IgVH genes that allowed identification of prognostic subgroups in CLL [172]. Others have reported a relationship between TK levels and LDT, markers of disease burden (ALC), and markers of cell turnover (lactate dehydrogenase [LDH], serum β2 serum, microglobulin) [173,174]. In one retrospective review of 188 patients, serum TK was a significant predictor of both survival and response to treatment [173]. In summary, elevated TK levels in CLL patients correlate with unmutated IgVH genes, CD38 and ZAP-70 expression, and intermediate to high-risk chromosomal abnormalities including del11q23, trisomy 12 and 17p13. In addition, patients with VH3-21 gene usage tend to have higher TK levels. Moreover, the significance of TK as a prognostic variable is retained in CLL cases with progression, unlike ZAP-70 and mutational status of IgVH, which tend to show no significant differences in survival in advanced Binet B and C stages compared with Binet A stage of CLL [174,175].

as a predictor of clinical outcome, particularly in early stages of CLL [179-182]. 19.4. Immunoglobulin variable heavy chain mutation status

The prognostic significance of serum β2 microglobulin (B2M) levels in CLL was initially reported by Di Giovanni et al [176]. Serum B2M is a marker that positively correlates with tumor burden and disease stage in patients with CLL [170,176]. A series of studies have found B2M to be the strongest predictor of 5-year survival on multivariate analysis when controlling for age, stage, and performance status in previously treated or untreated CLL patients. One prospective trial [177] of 106 untreated patients, however, did not find the use of B2M as a single marker to be a significant predictor of survival in multivariate analysis that controlled for stage and LDT. Thus, while B2M has some prognostic relevance in CLL, additional studies are needed to demonstrate the independence of this marker from stage and LDT and to define its role in the management of early-stage CLL patients.

Historically, CLL B cells were believed to represent a leukemic transformation of naïve B-lymphocytes that had not undergone GC antigen exposure and subsequent somatic mutation of their Ig genes. Several recent studies have found that approximately 50% of CLL clones exhibit somatic mutation of their Ig chains, which suggests that some CLL clones arise from post-GC, “memory” B cells [57]. In 1999, 2 publications simultaneously reported on the prognostic significance of IgVH gene mutational status, and significant survival differences were observed [183,184]. The median survival was 8 to 9 years for patients with nonmutated IgVH genes (germline cells) but greater than 24 years for patients with somatically mutated IgVH genes [183]. Patients with nonmutated IgVH genes had aggressive disease, advanced clinical stage, unfavorable cytogenetic abnormalities, and shorter survival than those with somatically mutated genes [184]. In addition, CLL patients with nonmutated IgVH genes had a higher risk of relapse after stem cell transplant [185]. Others have used specific IgVH sequences (VH3-21) to identify subgroups of patients that perform poorly regardless of the mutational status of VH gene [186,187]. This finding implies that response to specific antigens that select the VH3-21 sequence may confer different biologic behavior among patients with mutated IgVH genes [188]. The percentage of nucleotide mutation in the CDR3 region of the IgVH gene that optimally defines a “mutated” type of clones is currently not standardized because IgVH gene mutation testing is expensive, technically difficult, and not widely available for clinical use; however, data from several studies indicate that cases with more than 2% deviation of their IgVH sequence from germline are mutated, whereas those with 2% or less deviation are considered germline [183,184,189,190]. An unmutated IgVH gene sequence is associated with much more aggressive disease and shorter survival compared with a somatically mutated IgVH gene sequence [183,184].

19.3. Serum-soluble CD23

19.5. CD38 expression in CLL

The association between CD23 expression and proliferation in CLL became evident from the observations of Fournier et al [178], who showed that increased expression of CD23 promoted entry of resting CLL cells into G1 and S phases of the cell cycle in the absence of other stimuli [62]. They found that CD23 levels in sera of CLL patients were 3- to 500-fold higher than those in control sera as well as sera from other B-lymphoproliferative disorders. Several studies have established the importance of sCD23 as a powerful predictor of disease activity and progression and, thus, prognosis in CLL [61,177,179,180]. The ability of sCD23 to predict disease activity underscores its importance

Because of the technical complexity of testing for IgVH mutations, the lack of wide availability of testing facilities, and the associated cost, a search for a surrogate marker of mutational status led to the discovery of CD38, which is expressed on the surface of neoplastic cells in CLL. One early report found that CLL cells with higher CD38 expression were more likely to have nonmutated IgVH genes and that CD38 status possessed a prognostic value similar to that of IgVH mutation status with regard to median survival [183]. The median survival for patients with intermediate Rai stage with 30% or more of cells expressing CD38 was 10 years, whereas no patient with less than 30% CD38 cells

19.2. β2 Microglobulin

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died during the follow-up period. Thus, CD38 status was proposed as a possible surrogate for IgVH mutation status. Several investigators subsequently corroborated the observations made by Damle et al [183] and further substantiated the independent prognostic significance of both markers [132,189,191-193]. In one study of 168 CLL patients, most of whom had low or intermediate Rai stage disease, progression-free survival 5 years after diagnosis was 75% for CD38-negative patients but only 37% for patients who were CD38 positive. Eight-year survival was 92% for CD38negative patients and 50% for CD38-positive patients, and on multivariate analysis, CD38 status was a significant prognostic variable for overall survival [194]. Some studies, however, did not confirm the predictive power of CD38 as a surrogate for IgVH status [126,189,195-197]. In another study of 145 patients with predominantly Binet stage A disease, the predictive value of IgVH status, CD38 status, and disease stage was evaluated and all 3 variables were significant on multivariate analysis. Furthermore, the combination of CD38 and IgVH mutational status had an even greater prognostic power than either marker alone [198]. The pattern of CD38 expression in CLL identifies 3 groups of CLL patients: those that are homogenously negative for CD38, those who are homogenously CD38 positive, and a third group with a bimodal pattern of CD38 expression with distinct subsets of CD38-positive and CD38-negative cells in the same population [199]. Furthermore, the presence of any CD38-positive clone, rather than a numerically defined cutoff, identified patients who developed progressive disease and had worse overall survival independent of stage [199]. The change in CD38 expression over the course of disease in CLL and its possible link to disease progression is an interesting issue. Although some studies have reported no change or infrequent alteration in CD38 status through the course of disease, [183,200] other studies reported changes from CD38 positivity to CD38 negativity or from CD38 negativity to acquisition of CD38 expression in as many as 10% to 25% of cases. The switch from a CD38-negative to a CD38-positive phenotype signals evolution to a more aggressive disease [192,195,201]. The optimal threshold by which to classify CD38 status varies between 20% and 30%, a range based on statistical comparisons between CD38positive and CD38-negative groups. Using these cutoffs, several studies have been able to identify 2 distinct subgroups in CLL that have significant differences in terms of survival and disease progression [126,191,193,194,197,199,200]. Some investigators have proposed lower cutoffs of 7% that allow the best separation of the 2 prognostic groups, [132,189] whereas one report found that the presence of a CD38-positive population of neoplastic cells (regardless of its proportion) was enough to significantly change the outcome of disease [199]. On balance, expression of CD38 by flow cytometry appears to be of prognostic significance in CLL. Confirmation of the prognostic significance of bimodal C38 expres-

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sion may further refine the ability of this marker to identify patients with aggressive disease. 19.6. ζ-Associated protein 70 Expression in CLL ζ-Associated protein 70 is a tyrosine kinase that is present mainly in T and natural killer cells and functions as a tyrosine kinase actively involved in T-cell receptor signaling and required for T-cell activation [202,203]. Its role in B-cell receptor signaling was first suggested from gene expression profiling studies in CLL in which the gene for ZAP-70 was differentially expressed between patients with mutated and unmutated IgVH genes [204]. The ability of ZAP-70 to discriminate between IgVH mutated and unmutated cases was further confirmed by several studies [205-209]. A recent report of gene expression profiling in 107 patients with CLL confirmed that, of the genes studied, ZAP-70 was the single gene best able to distinguish IgVH mutation status, correctly predicting mutation status in 93% of the patients [209]. The ability of ZAP-70 to predict a shorter time to treatment was also similar to that of IgVH gene mutation status. Thus, ZAP-70 became a surrogate marker for IgVH gene mutational status in CLL. Regarding the clinical significance of this prognostic marker, Durig et al [206] analyzed ZAP-70 mRNA by semiquantitative PCR and protein expression by flow cytometry and showed that high mRNA and protein levels correlated with significantly shorter progression-free survival and unfavorable clinical outcome. With advances in flow cytometric techniques, some groups analyzed the ability of ZAP-70 by flow cytometry as a surrogate marker for IgVH mutational status to discriminate between CLL cases. Flow cytometric analysis of ZAP-70 remains a technical challenge for many, owing to ZAP-70's intracellular location and its weak expression in CLL cells and to the requirement that the cell membrane be permeabilized. Nevertheless, it is more useful than CD38 as a prognostic marker, as it gives a homogeneous staining pattern for the neoplastic cells on the scattergram, as opposed to CD38, which frequently seems to show a spectrum of staining. In addition, the expression of ZAP-70 remains constant over the course of the disease, unlike that of CD38 [205-207]. The cutoff to classify cases into ZAP-70–positive vs ZAP-70– negative is arbitrary and varies between 10% and 20% in various studies [205,207,208], with the optimal cutoff remaining to be determined. In a retrospective series, ZAP70 expression, as determined by flow cytometry, was evaluated as a surrogate for IgVH mutation status. Using an arbitrary cutoff of 20% or more for classifying CLL patients as ZAP-70 positive or ZAP-70 negative, ZAP-70 expression correctly identified 32 (91%) of 35 patients with nonmutated IgVH genes. No patient with mutated IgVH genes was ZAP-70 positive and, on multivariate regression, ZAP-70 was found to correlate with IgVH gene mutation status. Binet stage A patients who were ZAP-70 positive showed rapid disease progression with shorter median survival compared with ZAP-70–negative patients [205].

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Immunohistochemistry is also a reliable tool for evaluating ZAP-70 expression and its prognostic significance in CLL and can be used as an alternative method in cases in which flow cytometry is difficult (Fig. 6). One recent study showed a good correlation between ZAP-70 expression and unmutated IgH variable genes in CLL/SLL and demonstrated that ZAP-70 by IHC on routinely fixed, paraffinembedded tissue sections is a specific (92%) and relatively sensitive (78%) marker of unmutated IgH variable genes [210]. Wiestner et al [209] also demonstrated that ZAP-70 IHC was as good as western blotting and reverse transcriptase–PCR techniques in predicting the IgVH mutational status in CLL patients. In previous studies, flow cytometric and IHC evaluation of ZAP-70 in CLL has mostly involved the peripheral blood and lymph node tissue. More recently, Zanotti et al [211] assessed the usefulness of ZAP-70 as a prognostic marker by IHC analysis of bone marrow trephine biopsies and showed a good concordance between ZAP-70 analysis by IHC and flow cytometry as well as between ZAP-70 expression by IHC and IgVH mutational status. Thus, ZAP-70 is a promising prognostic marker and an important prognostic variable for CLL patients, but standardization of its measurement either at the mRNA or protein level have not yet met with great success. Furthermore, the role of ZAP-70 in the pathogenesis of CLL needs further investigation. 19.7. Transformation of CLL The terms transformation and progression are applied in a rather loose fashion and many times used interchangeably. For the purpose of this review, transformation is the development of an aggressive lymphoma that appears morphologically different from the low-grade indolent lymphoma in the background, as defined by Kroft [212]. Traditionally CLL is considered an indolent disease, in that

Fig. 6. ζ-Associated protein 70 expression in CLL cells by immunohistochemistry. The expression is nuclear and cytoplasmic. ZAP-70 expression in CLL is associated with poor prognosis.

many patients live for many years with the diagnosis and die of secondary unrelated causes and not of leukemia [213]. Transformation occurs in about 5% to 10% of cases and is accompanied by disease progression, resistance to therapy, and an exceptionally poor prognosis [169,214]. This transformation is often accompanied by the appearance of complex chromosomal changes either that were not present earlier or in addition to the commonly present trisomy 12 (reviewed in Robak [215] and Tsimberidou et al [216]). Several types of transformation in CLL have been described. The onset of the terminal transformation of CLL is suggested when there is a proliferation of a new population of lymphoid cells, specifically larger cells with immature-appearing morphological features, a finer nuclear chromatin pattern, and a prominent nucleolus, termed prolymphocytic transformation, which is relatively low-grade and slowly progressive (Fig. 7A). It occurs in approximately 2% of all CLL cases and is characterized by an increasing proportions of PL in the peripheral blood ranging from 11% to 55% and a median survival of 2 years. Such transformation is accompanied by B symptoms, a rapid rise in the peripheral blood counts, splenomegaly, and elevated LDH. The other form of transformation in CLL characterized by development of a high-grade NHL, most frequently diffuse large B-cell lymphoma (DLBCL), is referred to as “Richter syndrome” (RS) or Richter transformation [131,216,217]. Maurice N. Richter originally described this syndrome in 1928 as a rapid development of large cell lymphoma in a patient with CLL [218]. Subsequently, Lortholary et al [219], in 1964, described similar presentations in 4 patients with CLL and coined the term Richter syndrome. The incidence of RS is between 3% and 10% in the western world, with rare cases described on other continents [130,131,220-222]. The disease is rapidly progressive with a median survival of 4 to 5 months. Richter transformation is clinically characterized by a rapid onset of B-symptoms, progressive enlargement of lymph nodes, hepatosplenomegaly, markedly elevated LDH levels, paraproteinemia, hypercalcemia, and the presence of circulating immunoblasts in the peripheral blood [223,224]. A biopsy of the involved tissues is required to establish the diagnosis. The most common morphological picture of transformation is that of an immunoblastic variant of DLBCL, composed of large cells with round to oval nuclei with minimal nuclear membrane irregularities, vesicular chromatin, and single prominent nucleoli with variable eosinophilic cytoplasm. Such cells either completely efface the nodal architecture or are admixed with CLL/SLL neoplastic cells in the background. Diffuse large B-cell lymphoma with centroblastic cytomorphology is also seen, although this variant is less common than the former (Fig. 7B). Most cases of DLBCL transformation of CLL/SLL reported in the literature tend to retain most of the immunophenotype of CLL/SLL except for downregulation of CD5 and IgD expression with transformation [214,225-228]. In some cases, however, CD5 positivity may

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be retained [214,227,229,230], and such cases may be difficult to differentiate from CD5+ de novo DLBCL, which is a distinct entity that differs from Richter transformation or CD5− DLBCL cases in terms of genotypic and immunophenotypic features as well as clinical behavior. Cases of

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DLBCL transformation of CLL/SLL that retain CD5 usually also express CD23, a feature not commonly observed with CD5+ de novo DLBCL. In addition, they usually lack the bcl-6 gene rearrangements of CD5+ de novo DLBCL [229]. Whether the DLBCL of RS occurs as a transformation of the original CLL clone or whether it represents an independent, immunophenotypically and genotypically distinct neoplasm seems to be a matter of debate. Molecular gene rearrangement and surface Ig isotype analyses have shown that the large cells of RS and the CLL cells have the same surface Ig isotype and identical gene rearrangements, supporting a common clonal origin in approximately 70% to 80% of the cases [227,230,231]. However, a subset of cases have different patterns of antigen expression or gene rearrangements, supporting a hypothesis that RS may represent a different malignant clone [214,227,232-236]. The molecular mechanisms underlying the transformation of CLL to RS are not yet fully elucidated. It has been proposed that acquisition of complex genetic abnormalities may be responsible for transformation [237], and in that regard, RS cases have been examined for the presence of chromosome 11, 12, 13, and 17 abnormalities, which are commonly observed in CLL (reviewed in Tsimberidou and Keating [216]). Deletions of 11q23 and acquisition of trisomy 12 that are associated with atypical morphology and immunophenotype as well as high rates of proliferation and disease progression in CLL are also found at a high frequency in RS and, hence, are implicated in the pathogenesis of RS [238,239]. In addition, p53, a tumor suppressor gene that is frequently mutated in a variety of human cancers, has been discovered in 15% of CLL patients and in 40% of patients with Richter's syndrome [121-123]. Abnormalities of other tumor suppressor genes implicated in the pathogenesis of RS include deletion of Rb [240,241] and p27 [242] mutations of p16INK4A [243] and p21, increased copy numbers of C-MYC [244], decreased expression of A-MYB gene [245], and hypermethylation of hMLH1 promoter [246]. Patients with RS also show high levels of microsatellite stability and loss of hetorozygosity (LOH) at chromosomes 11, 17, and 20, suggesting that genomic instability rather than a single chromosome abnormality, as evidenced by frequent complex karyotypic abnormalities may Fig. 7. Transformation of CLL. (A) CLL with prolymphocytic transformation (original magnification ×100). Note the larger cells in the background of typical CLL cells. The large cells have features of PL, with immatureappearing morphological features, a finer nuclear chromatin pattern, a prominent nucleolus, and abundant pale cytoplasm. (B) A case of typical RS. Note the large B-cell component of the RS. The large cells have a centroblastic cytomorphology with round to oval nuclei with minimal nuclear membrane irregularities, vesicular to slightly coarse chromatin, and membrane bound nuclei. (Courtesy of Dr L Jeffrey Medeiros, Department of Hematopathology, University of Texas, MD Anderson Cancer Center, Houston, Tex). (C) Hodgkin variant of RS. Characteristic HRS cells are scattered in the background of a typical CLL. The HRS cells are large, mononuclear, and sometimes binucleated with enlarged vesicular nuclei, single prominent eosinophilic nucleoli, and abundant eosinophilic cytoplasm. They demonstrate the characteristic CD15+, CD30+, CD20−, CD45− immunophenotype, supporting the diagnosis of CHL.

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Table 4 Differential diagnosis of CLL Disorder Features

Common phenotype

Associated genetic abnormalities

CLL/ SLL

CD19+, CD20+, CD5+, CD22(−) CD23+, CD10(−), CD11c−/+, CD43+, clonal sIgM and sIgD weak

del 13q14 , del 11q23, trisomy 12, del 6q

PLL

MCL

FL

MZL

HCL

PBS: Persistent lymphocytosis, small cells with “soccer-ball” chromatin, scant agranular cytoplasm, smudge cells LN: Proliferation centers, PL, and paraimmunoblasts PBS: Increasing white blood cell counts, larger cells with less-mature chromatin and single prominent nucleoli PBS: Medium-sized cells with homogeneously condensed chromatin, nuclear clefts LN: Monotonous cell population, no proliferation centers, presence of epithelioid histiocytes and hyalinized venules PBS: Irregular clefts, notches, or folds that may traverse the entire width of the nucleus; centrocytes and centroblasts PBS: Plasmacytoid cells with eccentrically located nuclei, abundant pale cytoplasm LN: regressed GCs, follicular colonization, monocytoid cells with abundant pale cytoplasm and irregular nuclear contours PBS: Oval to beanshaped nuclei, abundant pale cytoplasm, and fine, hair-like, irregular cytoplasmic projections BM: Fried-egg appearance in BM, TRAP+

CD19+, CD20+, No CD5(−) CD22+, consistent CD23(−), CD10(−), alteration bright clonal sIg

CD19+, CD20+, CD5+, CD22+, CD23(−) CD10(−), CD43+, moderate clonal sIg (IgM N IgD)

t(11;14) (q13;q32)

CD19+, CD20+, CD5(−), CD22+, CD10+, CD11c(−), CD43(−), bright clonal sIg

t(14;18) (q32;q21)

CD19+, CD20+, Trisomy 3 CD5(−), CD22+, CD23(−), CD10(−), CD11c+, CD25(−), CD103(−), moderate clonal sIg

Fig. 8. Acute lymphoblastic leukemia, bone marrow aspirate smear (original magnification ×1000). The lymphoblasts are larger than the CLL cells and have immature chromatin pattern, scant cytoplasm, and single nucleoli.

being reported in 0.4% to 0.5% of CLL cases [247-249]. Morphologically, 2 forms of RS with CHL features can be recognized. In the first form, there are scattered Hodgkin and Reed-Sternberg (HRS) cells in the background of neoplastic CLL cells (Fig. 7C). In the other form, it appears that CHL coexists with CLL in that the HRS cells are present in a typical polymorphous background of eosinophils, plasma cells, and neutrophils distinct from the CLL cells. In addition to the morphological resemblance, the large cells also demonstrate immunophenotypic features (CD15+, CD30+, CD45− , and CD20−/+) of typical HRS cells. The clonal relationship between HRS cells and CLL cells was analyzed by PCR-based single-cell analysis of the IgH

CD19+, CD20+, No consistent CD5(−), CD22+, alteration CD23(−), CD10(−), CD11c+, CD25+, CD103+, moderate clonal sIg

+/− indicates variable, more often positive; −/+, variable, more often negative; (−), negative; cIg: cytoplasmic Ig.

be responsible for RS transformation of CLL [216,246]. A small subset of CLL patients develops classic Hodgkin lymphoma (CHL) during the course of their disease, which is termed the Hodgkin variant of Richter transformation. This form of transformation is quite rare,

Fig. 9. Prolymphocytic leukemia, peripheral blood (original magnification ×1000). Prolymphocytes are larger, less mature-appearing cells. The chromatin is less condensed than in a typical CLL lymphocyte but less dispersed than that of a lymphoblast. A large prominent vesicular nucleolus is a characteristic feature of PL. Cases with more than 55% PL are diagnosed as PLL.

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CDRIII region. Both CLL and HRS cells share identical IgH CDRIII sequences, suggesting that the HRS and CLL cells are a part of the same clone in some cases of RS with CHL features [250]. DNA sequencing and IHC studies also demonstrated the shared clonal origin of these 2 diseases [249]. The median survival of patients with the Hodgkin variant of RS is inferior to that of patients with CHL alone or those who develop concurrent CHL and CLL, but these patients fare better than the classic RS patients [247]. In addition to RS and the Hodgkin variant of RS, secondary lymphoid malignancies including hairy cell leukemia (HCL), multiple myeloma, plasmablastic lymphoma, T-cell lymphoma, and acute lymphoblastic leukemia

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(ALL) as unusual variants of CLL transformation have been documented in the literature but are beyond the scope of this review. Readers are directed to excellent reviews that describe these and other transformations in CLL in great detail [215,216,221].

20. Differential diagnosis The differential diagnosis of CLL is broad and encompasses a spectrum of entities that include ALL; prolymphocytic leukemia (PLL); NHLs in leukemic phase, especially MCL and FL; HCL; Waldenstrom's macroglobulinemia

Fig. 10. Mantle cell lymphoma. (A) Morphologic features of MCL involving a lymph node. The proliferation of a monotonous population of medium-sized lymphoid cells with irregular nuclear contours, the lack of pseudofollicles/proliferation centers, and the presence of epithelioid histiocytes and hyalinized venules in the background are some of the morphological clues that favor MCL over CLL. (B) Flow cytometric analysis of MCL in leukemia phase. Leukemic marrow lymphocytes are gated by CD45-scattered analysis. The plot of CD5 versus CD19 demonstrates dual positive neoplastic lymphocytes. In contrast to CLL, the neoplastic lymphocytes in MCL show positive staining for FMC7 but no staining of CD23.

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Fig. 11. Follicular lymphoma. (A) A peripheral blood smear from a patient with FL in leukemic phase (original magnification ×1000). The circulating cells of FL are small with nuclei that demonstrate irregular clefts, notches, or folds that may traverse the entire width of the nucleus and scant cytoplasm. (B) A case of FL in the lymph node showing a predominantly follicular pattern and composed of a mixture of centrocytes (small-cleaved cells) and centroblasts (large noncleaved cells) in varying proportions.

[88]; and monoclonal and benign polyclonal B-cell lymphocytosis. The key differentiating features that help distinguish the above entities from CLL are presented in Table 4. Acute Lymphoblastic leukemia and CLL are easily distinguished on morphological grounds alone in most cases (Fig. 8). The lymphoblasts tend to have a smoother nuclear chromatin pattern compared with the heavy condensation of nuclear chromatin in the CLL lymphocyte. The distinction is easily made when examining an appropriate monolayer area or feather-like edge of a well-stained blood smear. In rare cases where lymphoblasts are smaller and especially of the L1 type, immunofluorescence or IHC with terminal deoxynucleotidyl transferase (TdT) is useful. Lymphoblasts show positive results for nuclear TdT, whereas CLL cells are TdT-negative.

B-Prolymphocytic leukemia is characterized by a predominance of circulating PL (greater than 55%, usually greater than 70%). Prolymphocytes are larger, less-matureappearing cells than the typical lymphocytes seen in CLL, with the chromatin less condensed than in a typical CLL lymphocyte but less dispersed than that of a lymphoblast. A large prominent vesicular nucleolus is a characteristic feature of PL (Fig. 9). Prolymphocytes may also be seen in patients with CLL but they account for less than 10% of the circulating cells. When 11% to 55% PL are present, a mixedcell type of CLL or CLL/PLL is diagnosed [251]. This category includes patients with prolymphocytoid transformation. Cases with PL above 55% are diagnosed as PLL [18]. Patients with de novo PLL usually present with rapidly increasing white blood cell counts (often greater than 100 × 109 cells/L) and prominent splenomegaly without lymphadenopathy [252-254]. The immunophenotypic features of PLL differ from those of CLL. In contrast to CLL, the neoplastic cells in PLL are intensely positive for sIg, CD19, and CD20; variably positive for CD5 and FMC7; and negative for CD23. Most cases of PLL are B cell in nature, as demonstrated by strong CD20 and sIg (in contrast to weak CD20/sIg in CLL) and reactivity with the B-cell markers CD19 and CD22 [255]. Mantle cell lymphoma frequently has peripheral blood involvement in up to 75% cases at presentation, and under these circumstances, morphological recognition of MCL and its distinction from CLL/SLL are a diagnostic challenge. Identification of these 2 entities is important because MCL is considered to have a poor prognosis, with a median survival of 3 to 5 years. The number of circulating lymphoma cells in MCL, however, is not as prominent as in CLL, and absolute lymphocytosis is not as frequent [256,257]. The circulating lymphoma cells are composed of a heterogeneous population of small to intermediate-sized neoplastic lymphoid cells, usually slightly larger than normal lymphocytes. The nuclear characteristics and cytology of the cells are variable: round to clefted nuclear contours, with clumped to dispersed and sometimes blastoid chromatin. In the lymph nodes, MCL usually exhibits a diffuse pattern with complete effacement of lymph node architecture [42]. It can also form a nodular or mantle zone pattern. The lack of pseudofollicles/proliferation centers, the presence of epithelioid histiocytes, and hyalinized venules in the background of a monotonous population of small to medium-sized neoplastic cells with cleaved nuclei are some of the morphological clues that favor MCL over CLL [258] (Fig. 10A). The common immunophenotype of this lymphoma is shown in Table 4. A typical case of MCL shows bright sIg expression and positivity for CD19, CD20, CD5, and FMC7. In contrast to CLL, the cells in MCL lack expression of the CD23 surface antigen [22,255] (Fig. 10B). The t(11;14)(q13;q32) involving BCL1/cyclin D1 and IgH genes, resulting in overexpression of the cyclin D1 (PRAD 1) mRNA and protein, is considered a hallmark of MCL [259-262]. This chromosomal translocation can be detected by Southern blot analysis in 70% of cases or by

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Fig. 12. Hairy cell leukemia. (A) Peripheral blood smear from a patient with HCL (original magnification ×1000). The neoplastic cells characteristically have oval to bean-shaped nuclei; abundant pale cytoplasm; and fine, hair-like, irregular cytoplasmic projections. (B) Tartrate-resistant acid phosphatase stain of peripheral blood. Hairy cells show bright and intense granular staining (original magnification ×1000). (C) Bone marrow biopsy demonstrating involvement by HCL. The neoplastic infiltrate has a loose interstitial distribution pattern. The neoplastic cells are separated from each other by a rim of clear cytoplasm, giving the characteristic fried-egg appearance. (D) Flow cytometric analysis of hairy cell leukemia. Large mononuclear cells in leukemic bone marrow are gated by CD45 side-scattered analysis. The plots of CD22 versus CD11c and CD20 vs CD103 demonstrate a predominance of dual positive B-cells. The plots for both CD20 and λ show lambda light chain clonality. The B-cells are also reactive with anti-CD25 (anti–interleukin-2 receptor).

PCR in 30% to 45% of cases [263]. By immunohistochemical staining, a cyclin D1–positive tumor essentially rules out CLL.

Follicular lymphoma can progress to a leukemic phase in 5% to 23% of cases [264,265] and can have some degree of lymphocytosis but not to the extent seen with CLL. The

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circulating cells of FL are small with scant cytoplasm and nuclei that are irregular in shape and that demonstrate irregular clefts, notches, or folds that may traverse the entire width of the nucleus (Fig. 11A). Nuclear clefts can be seen in a subset of CLL cases, especially those with atypical features (Fig. 4). In such cases, immunophenotyping can assist in differentiation. In lymph nodes or extranodal tissues, FL may be nodular (follicular) or diffuse in distribution and is composed of a mixture of centrocytes (small cleaved cells) and centroblasts (large noncleaved cells) in varying proportions (Fig. 11B). Compared with immunologic markers in CLL, FL shows strong sIg, CD22 positivity, CD5 negativity, and often CD10 positivity [22,255]. In addition, molecular and cytogenetic studies for assessment of bcl-2 gene rearrangements or t(14;18) (q32;q21) found in 70% to 95% of FL can further assist in differentiating FL from CLL. This translocation can be detected by Southern blot analysis in more than 80% or by FISH in more than 95% of cases of FL [263]. Hairy cell leukemia is distinct from CLL in its clinical presentation, as well as on morphological and immunophenotypic grounds [22]. Most patients with HCL present with peripheral pancytopenias (monocytopenia in particular), splenomegaly, fever, and diffuse infiltration of bone marrow at presentation. Lymphadenopathy is uncommon, as HCL rarely involves the lymph nodes. In peripheral blood or bone marrow aspirate smears, the neoplastic cells characteristically have oval to bean-shaped nuclei; abundant pale cytoplasm; and fine, hair-like, irregular cytoplasmic projections (Fig. 12A). Bone marrow fibrosis is common, which makes it difficult to obtain a bone marrow aspirate (the socalled dry tap). Nevertheless, bone marrow biopsy and biopsy touch imprints are essential for diagnosis. The bone marrow biopsy shows a loose interstitial lymphocytic infiltrate surrounded by a clear cytoplasm that separates one cell from another, creating a “fried-egg” appearance (Fig. 12B). The most characteristic, although not specific, cytochemical feature of HCL is a strong positivity of tumor cells for tartrate-resistant acid phosphatase (TRAP) stain (Fig. 12C). Immunophenotypically, HCL cells exhibit reactivity with B-cell–associated antigens (CD19, CD20, CD22, CD79a), CD11c (strong), CD25 (strong), FMC7, and CD103 [22,255] (Fig. 12D). Reactive lymphocytosis can be a transient or persistent process. The transient form is self-limiting and is most commonly caused by viral infections such as infectious mononucleosis, viral hepatitis, and cytomegalovirus in adults and Bordetella pertussis in children [18]. It can also be triggered by ingestion of certain drugs such as isoniazid or allopurinol or may be associated with a hyposplenic state, autoimmune diseases, or trauma. The reactive lymphocytes often appear to be slightly enlarged and have variable amounts of pale basophilic cytoplasm; some may show prominent nucleoli. Cytoplasmic vacuolization may be present. The predominant cell type is T-helper cells with a CD4+, CD8− phenotype, and they are polyclonal. Reactive polyclonal

B-lymphocytosis is relatively rare. Most reported cases have occurred in females and show a strong association with smoking [266-270]. The degree of lymphocytosis is mild in contrast to CLL, and lymphadenopathy and splenomegaly are unusual. Morphologically, the lymphocytes show a subpopulation of bilobed or binucleated forms. Immunophenotypically, the lymphocytes are CD19+, CD5−, CD23−, and CD10− with polytypic expression of Ig light chains. Monoclonal B-lymphocytosis is defined by flow cytometric (or molecular) detection of very low levels of circulating B-lymphocytes with features similar to CLL in apparently healthy individuals with no evidence of hematological disease (reviewed in Marti et al [271]). This entity has been previously recognized under various terms, including benign monoclonal B-cell lymphocytosis, idiopathic persistent chronic lymphocytosis, B-monoclonal lymphocytosis of undetermined significance, smoldering CLL, and subclinical CLL [17,271-278]. The recently proposed diagnostic criteria recommend that a diagnosis of monoclonal β-lymphocytosis should be made if the monoclonal B-cell population demonstrates a κ:λ ratio of above 3:1 or less than 0.3:1, if more than 25% of the B-cells show lack of or reduced sIg expression, and if the cells show a CLL immunophenotype in the absence of other clinical or laboratory characteristics of CLL [271]. In conclusion, tremendous progress has been made over the past decade in our understanding of CLL as a disease, some of which was briefly touched upon in this review article. A wealth of data on various prognostic markers in CLL has emerged in the recent past that has helped us to further refine treatment modalities based on better risk stratification of CLL made possible by some important prognostic markers, such as ZAP-70, CD38, IgVH mutational status, and the recently identified micro-RNA expression profiles. As our knowledge about this disease continues to expand, some questions regarding the pathogenesis and biology of CLL will be resolved over the coming years, and new questions will continue to arise but, nevertheless, take us to the next level in our quest of achieving a complete cure for this disease. Acknowledgments The authors thank ChaRhonda Chilton for her editorial suggestions and La Kisha Rodgers and Andrea Sedo for her secretarial support. References [1] Kalil N, Cheson BD. Chronic lymphocytic leukemia. Oncologist 1999;4:352-69. [2] Morton LM, Wang SS, Devesa SS, et al. Lymphoma incidence patterns by WHO subtype in the United States, 1992-2001. Blood 2006;107:265-76. [3] Redaelli A, Laskin BL, Stephens JM, et al. The clinical and epidemiological burden of chronic lymphocytic leukaemia. Eur J Cancer Care (Engl) 2004;13:279-87.

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