Chronic Lymphocytic Leukemia



Chronic Lymphocytic Leukemia


James B. Johnston

Matthew Seftel

Spencer B. Gibson



Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature-appearing lymphocytes in the blood, marrow, lymph nodes, and spleen.1, 2, 3, 4 Small lymphocytic lymphoma (SLL) is the same disease, but primarily there is involvement of lymph nodes and spleen. Both CLL and SLL are antedated by monoclonal B-cell lymphocytosis (MBL) in which small numbers of CLL cells can be detected in the blood of asymptomatic individuals.1, 2, 3, 4 The CLL cells are monoclonal B lymphocytes that express CD19, CD5, and CD23 with weak or no expression of surface immunoglobulin (Ig), CD20, CD79b, and FMC7.1, 2, 3, 4 Recent evidence suggests that CLL cells have a similar gene expression profile as memory B-cells, and there is considerable heterogeneity in CLL in terms of cellular morphology, phenotype, biology, molecular genetics, and prognosis.1, 2, 3, 4, 5, 6, 7 The incidence of CLL varies throughout the world, being highest in North America and rare in the Far East. In North America, CLL/SLL accounts for one-quarter of all leukemias, with the incidence reported by the Surveillance, Epidemiology and End Results (SEER) Program being 5.13/100,000 for the years 1993 to 2004.8 A total of 75% of cases were CLL and 25% SLL. However, the incidence is likely much higher, as many patients are diagnosed by flow cytometry and are not included in tumor registries.9 By combining data from flow cytometry and the cancer registry the incidence of CLL/SLL is 7.99/100,000, with the median age at diagnosis being 71.5 years.9 The median age at diagnosis is younger for males (70 years) than for females (73 years), with the male:female ratio being 1.3:19 (Fig. 90.1). One third of patients are less than 65 years and 10% less than 50 years. Using SEER data, it has been shown that relative survival is poorest for those >80 years, and worse for men.10 Interestingly, the 10-year relative survival has increased over the last 20 years for all age groups except for those 80 years of age or older at the time of diagnosis (Fig. 90.2).10 Moreover, relative survival has improved for men, diminishing the difference previously seen between the sexes.






FIGURE 90.1. Age distribution of 351 males and 265 females diagnosed through the provincial cancer registry and a centralized flow cytometry facility in Manitoba over a 5-year period (1998 to 2003). The median age at diagnosis was 70 years for men and 73 years for women (P = 0.0281). Overall median age was 71.5 years. From Seftel MD, Demers AA, Banerji V, et al. High incidence of chronic lymphocytic leukemia (CLL) diagnosed by immunophenotyping: a population-based Canadian cohort. Leuk Res 2009;33:1463-1468, with permission.

Although the median age at diagnosis for CLL patients in the population is 71.5 years, it is 64 years in CLL clinics and as low as 58 years in specialized clinics.11, 12, 13 Thus, it is likely that many older patients are not being referred for assessment and therapy.




CLINICAL FINDINGS

Most CLL patients in the general population are elderly (median age 71.5 years). As a result of referral bias the median age of patients seen in the specialist clinic is 64 years, with 20% to 25% of patients being <55 years old.11, 12, 243, 244 However, the presenting features are similar regardless of age.243, 244 Nowadays, 70% to 80% of patients are diagnosed incidentally when they have a routine blood count and will have early-stage (Rai 0 or I) disease.245 Alternatively, lymphadenopathy, splenomegaly, or both may be detected during a regular physical examination. When symptomatic, the most frequent complaint is fatigue or a vague sense of being unwell. Less frequently, enlarged nodes or the development of an infection is the initial complaint, and the most frequent infections are bacterial pneumonias. Fever and weight loss are uncommon at presentation but may occur with advanced and drug-resistant disease.

Most symptomatic patients have enlarged lymph nodes, as well as splenomegaly. Enlargement of the cervical and supraclavicular nodes occurs more frequently than axillary or inguinal lymphadenopathy. The lymph nodes are usually discrete, freely movable, and nontender. Painful enlarged nodes usually indicate superimposed bacterial or viral infection. There is usually only mild to moderate enlargement of the spleen, and splenic infarction is uncommon. Less common manifestations are enlargement of the tonsils, abdominal masses due to mesenteric or retroperitoneal lymphadenopathy, and skin infiltration. Skin lesions in CLL may be caused by squamous and basal cell carcinomas which are thought to be related to immunosuppression (see section “Second Malignancies”).246 Other common cutaneous manifestations include shingles and recalcitrant warts. Direct involvement of the skin by CLL typically affects the face and the features can be quite variable, from macules or papules, which may be vascular or to more extensive involvement that may simulate rhinophyma.247 Patients can also present with symptomatic anemia, which may be related to marrow replacement or, more rarely, to autoimmune hemolysis or aplasia. Alternatively, patients may have bruising or bleeding, most commonly related to thrombocytopenia and rarely to acquired von Willebrand disease, or factor VIII inhibitors. Rarely, patients may present with a paraneoplastic syndrome, such as nephrotic syndrome, paraneoplastic pemphigus, or angioedema (see section “Autoimmune Manifestations”).


Peripheral Blood

The median lymphocyte count at diagnosis is 20 to 30 × 109/L, and in most patients, there is a continuous increase in the lymphocyte count over time.13, 243, 244 In half the patients, it takes more than 12 months for the lymphocyte count to double; cyclic fluctuations of up to 50 × 109/L can occur in the lymphocyte counts of untreated patients, and in others, the count may remain stable for years.244 The CLL cells are small to medium-sized lymphocytes with clumped chromatin, inconspicuous nucleoli, and a small ring of cytoplasm. Cytoplasmic inclusions occasionally may be observed in CLL cells and may be crystalline, globular, tubular, or rod-shaped.248 Smudge cells (basket cells or shadow cells of Gumprecht) are commonly seen in the peripheral blood smear in CLL, but not in other lymphoid disorders, and are caused by a decrease in the cellular content of vimentin, a cytoskeletal protein required for maintaining cell structure.249, 250 The number of smudge cells can vary from 1% to 75% (median 28%) and appears to be remarkably consistent in individual patients.250 One study suggests that patients with ≥30% smudge cells are more likely to have mutated IgVH and have a better prognosis than those with <30% smudge cells.250 There can be variations in cell morphology, with some cells being PL, whereas others are larger with abundant cytoplasm, and some are plasmoid or cleaved.204, 251 The French/American/British classification system divides patients into 3 groups depending on the percentage of abnormal cells.251 In classical CLL, >90% of cells are small, and when 11% to 54% of the cells are PL, it is termed CLL/PLL. When >15% of the lymphocytes are plasmoid or cleaved and <10% are PL, it is termed atypical CLL.203, 204, 251 Approximately 80% of patients have classical CLL, and 20% have CLL/PLL or atypical CLL. If ≥55% of the cells are PL, the patient has PLL.


Bone Marrow and Lymph Nodes

Marrow infiltration in CLL may be interstitial, nodular, mixed (nodular and interstitial), or diffuse, with mixed being the most common and nodular the least common.3, 252 Diffuse involvement, in which there is effacement of the fat spaces by tumor, carries the worst prognosis.3, 252 The marrow involvement is random and contrasts with follicular lymphomas, in which paratrabecular involvement is the rule. In contrast to marrow, involvement of the lymph node is diffuse. Proliferation centers with PL and paraimmunoblasts are typically seen in both marrow and lymph nodes.


Immunophenotyping

CLL can usually be readily differentiated from other disorders by immunophenotyping1, 2, 3, 253, 254, 255 (Table 90.2). The leukemic cells have the B-cell markers CD19, CD20 (low), CD43, and CD79b (low) and must be CD5+. In addition, the cells show clonal light chain restriction, weak expression of sIgM and sIgD, and are CD23+ and CD10. The cells are also CD27+,43 consistent with being memory B-cells.5 Alternatively, the presence of CD27, CD5, and CD23 could reflect the activated nature of the CLL cell.42 Matutes et al.254, 256 recommend 5 markers to differentiate CLL from other B-cell malignancies (Table 90.3). Typical CLL should be surface Ig (weak), CD5+, CD23+, CD79b or CD22 (weak), and FMC7.









TABLE 90.2 IMMUNOPHENOTYPES OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) AND OTHER CHRONIC B-CELL DISORDERS



























































































































































Condition


smIg


CD5


CD10


CD11c


CD19


CD20


CD22


CD23


CD25


CD43


CD79b


CD103′


FMC7


CLL


Dim


++



-/+


++


Dim


-/+


++


+/-


+




-/+


Prolymphocytic


leukemia


+++


-/+


-/+


-/+


++


+++


++


++


-/+


+


++



+


Splenic marginal


zone lymphoma


++


-/+


-/+


+/-


++


++


++


+/-


-/+


+


++


-/+


++


Marginal zone


lymphomas


++




+/-


++


++


+/-


+/-



-/+


++



+


Mantle cell


lymphoma


++


++


-/+



++


++


++




+


++



++


Follicular


lymphoma


++


-/+


++



++


++


++


-/+




++



++


HCL


+++




++


+++


+++


+++



+++


+


+


+++


+++


HCL variant


+++




++


+++


+++


+++




+


+


+++


+++


Waldenström


macroglobulinemia


++




-/+


++


++


+



-/+


+/-


+



+


-, not expressed; -/+, usually is not expressed; +/-, usually is expressed; + to +++, varying degrees of strength of expression; HCL, hairy cell leukemia; smIg, surface membrane Ig. Adapted from References 254-257.


One of the typical features of the CLL cell is the overexpression of membrane CD23, which is related to deregulation of NOTCH2 signaling and may play a role in the decreasing apoptosis.257, 258 The presence of CD23 is useful to differentiate CLL from mantle cell lymphoma, which is also CD5+.257 Cell surface CD23 undergoes spontaneous proteolysis, producing elevated serum levels of CD23, the level of which is a marker of disease stage and progression.257

The BCR complex is required for the proliferation of B-cells after immune stimulation and is a complex formed by sIg and Igα/Igβ (CD79a/CD79b). CLL cells lack CD79b, which is related to overexpression of an alternatively spliced form of the gene.259 The FMC7 antibody identifies an epitope of CD20 and usually strongly stains hairy cell leukemia and PLL; however, only 16% of CLL cases stain positively, presumably because CD20 is only weakly expressed in typical stable CLL.253, 260 In general, those patients who are FMC7+ have high levels of surface IgM, low expression of CD23, and poor prognosis.253 Although the myelomonocytic antigens (CD11b, CD13) may be expressed in multiple myeloma, acute lymphoblastic leukemia, and in the CD5- chronic lymphoid leukemias, they are not expressed in CLL.261








TABLE 90.3 SCORING SYSTEM FOR DIAGNOSIS OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)













































Marker


Marker Intensity


Score


Marker Intensity


Score


Surface Ig


Weak


1


Strong


0


CD5


+


1



0


CD23


+


1



0


CD22/CD79b


Weak


1


Strong


0


FMC7



1


+


0


+, present; -, absent.


Note: Diagnosis of chronic lymphocytic leukemia requires a score of 4 or 5.


From Matutes E, Polliack A. Morphological and immunophenotypic features of chronic lymphocytic leukemia. Rev Clin Exp Hematol 2000;4:22-47.


The CD5 antigen is most commonly associated with mature T-cells and is expressed weakly on thymocytes.2, 262 However, normal B-cells carrying the CD5 marker are located in the mantle zone of the lymph node, and small numbers of these cells are also present in the peripheral blood.262 The CD5 molecule has been cloned and appears to be involved in the activation of T lymphocytes. The function of CD5 on the B-cell remains unknown, but its induction has been shown to be inhibited by the T-cell-derived cytokine IL-4. CD5+ B-cells can be stimulated to secrete anti-DNA antibodies and rheumatoid factors in vitro, and increased numbers are found in the peripheral blood of patients with rheumatoid arthritis, systemic lupus erythematosus, and Sjögren syndrome.262 It has been suggested that the normal counterpart of the CLL cell is the CD5+ B-cell,19 although detailed immunophenotyping demonstrated significant differences between the two groups.263 Thus, the CD5+ B-cell appears to be a resting antigen-naive cell, whereas the CLL cell is activated and antigen-experienced. Moreover, the specific activation marker expression pattern varies between cells that have IgVH gene mutations and those that do not, with the unmutated group resembling B-cells at an earlier state of activation.41 Older studies suggested that about 5% of cases of CLL could be CD5 and were more likely to be FMC7+, CD23, CD11b+, and CD13+ and have a poor prognosis.261 However, these CD5 cases should be reclassified, as CD5+ is essential for the diagnosis of CLL.1, 3


Surrogate Markers for IgVH Mutational Status

Both CD38 and ZAP-70 have been evaluated as surrogate markers for IgVH mutational status, as these can be measured readily by flow cytometry. To define the stage of maturation of the CLL cell, Damle et al.42 measured CD38 levels in CLL. Approximately 50% of patients had >30% CD38+ cells, and these patients had unmutated IgVH and had a worse prognosis than those with <30% CD38+ cells. However, not all have found such a correlation.2 CD38 acts as a receptor and enzyme and can produce cell replication and survival with a variety of signals.264 Activation of CD38+ cells, but not CD38 cells, through sIgM induces apoptosis and through IgD prolongs cell survival and induces differentiation.264 In addition, recent evidence suggests that the replicating cells in CLL are the CD38+ cells and the number of CD38+ cells is higher
in lymph nodes, likely reflecting the higher rate of cell proliferation at that site.115, 265 Although CD38 can be measured easily by flow cytometry there is disagreement about the number of cells that are required to define positivity, with values ranging from 5% to 30%.266, 267, 268 Moreover, the number of CD38 positive cells can vary over time.266, 267, 268

ZAP-70 is a member of the Syk-ZAP-70 protein kinase family and is expressed in T and natural killer (NK) cells and is important in T-cell signaling.269, 270 However, recent evidence suggests that normal B-cells may also express ZAP-70, particularly when activated.269, 270 Gene expression studies in CLL have also demonstrated that cells with unmutated IgVH have an increased expression of ZAP-70 whereas those with mutations have low levels.7 Subsequent studies have shown a 70% to 90% correlation between ZAP-70 expression and IgVH mutational status, regardless of whether ZAP-70 was measured by flow cytometry (≥20% cells positive), Western blot analysis, or immunohistochemistry.271, 272, 273, 274 Kröber et al.275 have shown that discordant cases may have poor prognostic features including deletions of 17p13 or 11q22-q23, or IgVH3-21 expression. ZAP-70 positivity also correlates moderately with CD38 positivity and the presence of poor-risk cytogenetics, that is, deletion 11q22-q23, deletion 17p13, and trisomy 12.275, 276 The poor prognosis associated with ZAP-70 positivity has been related to the magnified signaling by the BCR in ZAP-70-positive CLL cells.277 A major difficulty presently in the routine use of ZAP-70 is the variability in the assay as a result of the sensitivity of different antibodies and the use of different internal controls.266, 267, 268 Moreover, although initial studies suggested that ZAP-70 differed from CD38 in remaining stable during the disease course, more recent evidence indicates that ZAP-70 may also change over time.267, 268


Immunoglobulin Production in Chronic Lymphocytic Leukemia

Apart from having either κ or λ light chains on the cell-surface membrane, clonality is confirmed by the presence of unique idiotypic specificities of the Igs produced by CLL cells278 and by IG gene rearrangements.2, 18 CLL cells have low to undetectable amounts of monoclonal polyreactive IgM autoantibodies, frequently of the rheumatoid factor type, on their surface.262, 263 Thus, the same monoclonal autoantibody produced by a leukemia cell may react with a variety of antigens (e.g., IgG, cardiolipin, histones, or single- or double-stranded DNA).262 Surface and cytoplasmic Igs contain either κ or λ light chains but never both. Most cells display a single heavy chain class, usually µ, although some display µ and δ. Less commonly, γ, α, or no heavy chain determinant is found. Although the CLL cell is believed to be frozen at a particular stage in maturation, up to 50% of CLL patients have IgM+ leukemia cells that are able to undergo isotype switching to IgA (usually) or to IgG.279, 280 Rearrangements of the IGH and IGL genes are seen in CLL and these remain constant over time.2 Interestingly, the T-cell receptor β gene can be aberrantly re-arranged in 6% of CLL cases and this is associated with chromosome 6q deletions.281, 282

CLL cells may secrete idiotypic IgM and in most cases can be induced by mitogens to secrete IgM that can react to a variety of autoantigens.283, 284 Using a sensitive immunoblotting technique, monoclonal proteins can be detected in the serum of virtually all patients although the light chain of the protein is only the same as that on the CLL in half the cases.285 These findings indicate that the monoclonal protein in many cases is not derived from the tumor cells. Serum electrophoresis detects a monoclonal protein in approximately 5% of CLL cases and an abnormal serum-free light chain ratio is observed in one third of patients.286, 287 The presence of an abnormal ratio correlates with advanced disease, markers of aggressive disease, and poor prognosis.287


Functional Immune Abnormalities

Patients with CLL are immunosuppressed, as a result of hypogammaglobulinemia, alterations in T-cell function and to abnormalties in complement activation and neutrophil/monocyte function.288, 289 Moreover, the immunosuppression is potentiated by chemotherapy or immunotherapy. As a result, about 50% of patients have recurrent infections, with sepsis being an important cause of death. Apart from typical bacterial infections, CLL patients are also susceptible to opportunistic infections, particularly if they have received nucleoside analogues, steroids, or monoclonal antibodies. The nucleoside analogues are highly toxic to T lymphocytes, whereas monoclonal antibodies may be cytotoxic to B-cells (e.g., rituximab) or to both B- and T-cells (alemtuzumab).

The CLL cell has no useful immune function and shows poor stimulatory activity in mixed lymphocyte culture290 and in response to B-cell mitogens, such as pokeweed mitogen, lipopolysaccharide, and the Epstein-Barr virus.291, 292 However, phorbol esters, Staphylococcus aureus protein A from Cowan I, anti-µ antibodies, anti-CD40, and loxoribine have been shown to be potent mitogens.293, 294 Moreover, these cells are poor antigen-presenting cells and, as discussed below, can interfere with normal B- and T-cell function.295

The risk of infection is closely related to the extent of hypogammaglobulinemia in CLL, and the severity increases with the duration and stage of disease.288, 289, 296 The Ig levels are all decreased, and within the IgG class, reduced levels of IgG2 and IgG4 correlate best with the risk of infection; however, the decline in IgA levels is the most important predictor of infection.296, 297 Interestingly, the correlation between Ig levels and infection rate is not absolute, and some patients with normal Ig will have repeated infections whereas others with hypogammaglobulinemia will remain infection-free.288 Patients with CLL have reduced primary and secondary responses to immunization.289 Although patients with higher levels of gammaglobulin usually show better responses than those with low levels, the responses of both groups are abnormal. The pathogenesis of the hypogammaglobulinemia is poorly understood. However, impaired B-cell function and regulatory abnormalities of T-cells (including the reversal of normal helper/suppressor cell ratios) probably play a role. In addition, CLL-derived NK-cells have been shown to suppress Ig secretion by normal B-cells in vitro.298

The absolute number of T-cells may be increased in untreated CLL, and there are marked abnormalities in the surface markers, including inversion of the T-helper to -suppressor cell ratio, suggesting perturbations in T-cell function.290, 299 In addition, the increase in the number of T-suppressor cells may correlate with the degree of hypogammaglobulinemia.300 The T-cells usually respond normally to mitogens, such as phytohemagglutinin, in vitro and produce IL-2 and γ-interferon.301 However, there is decreased T-helper function with reduced reactivity to allogeneic and autologous B-cells.302, 303 Spontaneous and antibody-dependent cytotoxicities are reduced, suggesting an abnormality in the large granular lymphocyte population, including NK-cells.304

The cause of these abnormalities is unclear, but the increase in T-cells has been ascribed to stimulation by CLL cells or chronic infection, such as cytomegalovirus (CMV).289, 305, 306 Alternatively, the B-, T-, and NK-cell functions may be suppressed by means of immunosuppressive factors produced by CLL B-cells.307, 308 The gene expression profile of CD4+ and CD8+ cells are different in CLL patients as compared to normal individuals.307 However, the abnormal expression could be induced in CD4+ and CD8+ cells from normal individuals by co-culturing them with CLL cells.307 Similar studies showed that CLL cells could affect the ability of normal T-cells to form normal immunologic synapses.308 One potential candidate that could be causing these changes is TGF-β, which is secreted by CLL cells and marrow stromal cells in CLL, and has been shown to be a potent inhibitor of normal B- and
T-cells.133, 134, 135, 136, 137 In addition, CLL cells express both CD40 and the CD40L (CD154), and these cells decrease CD154 expression by normal T-cells with resultant effects on normal B-cell differentiation and isotype switching.309

The levels of different complement components are decreased in CLL, particularly in patients with advanced disease.310 As well, multiple defects in neutrophil and monocyte function have been described in CLL, and these are associated with an increased risk of infection.288, 311


Autoimmune Manifestations

Despite being immune deficient, CLL patients have an increased incidence of autoimmune cytopenias secondary to autoantibody formation.312, 313, 314, 315, 316, 317, 318 The immune cytopenias may be present before or at the time of diagnosis in one third and during the course of disease in two-thirds.315 CLL is the most common cause of autoimmune hemolytic anemia (AIHA), causing 14% of cases, followed by systemic lupus erythematosus.312 Moreover, patients with AIHA have an increased incidence of MBL and subsequently developing CLL, but this is not true for patients with immune thrombocytopenia (ITP).314, 316 Overall, 4% to 10% of CLL patients develop AIHA, and this is usually associated with a warm-type antibody against the Rhesus system and a positive direct antiglobulin test (DAT).312, 313, 314, 315 Another 7% to 14% of cases may have a positive DAT without evidence of hemolysis. The incidence of this disorder is increased with male sex, older age, high lymphocyte count, advanced disease, and the presence of biologic markers of poor prognosis, such as high β2-microglobulin, high ZAP-70, and unmutated IgVH.314, 315, 317 The diagnosis of AIHA may be difficult in CLL, as multiple factors may produce anemia, a positive DAT can exist without hemolysis, and other diagnostic parameters may be influenced by the disease.314, 315, 317 Zent et al.317 have thus developed diagnostic criteria for the diagnosis of AIHA in CLL and include (a) hemoglobin <100 g/L; (b) ≥1 marker of hemolysis, i.e., reticulocytosis, increased indirect bilirubin without liver disease, increased lactate dehydrogenase (LDH) without another cause or increased marrow erythropoiesis; (c) positive DAT/cold agglutinins or ≥2 markers of hemolysis, without evidence of hypersplenism or bleeding. When fludarabine was introduced, AIHA was seen relatively frequently leading investigators to believe that this was a unique complication of the drug. However, in the LRF CLL4 trial the incidence of AIHA was similar in previously untreated patients who received chlorambucil (12%) as compared to fludarabine (11%), and the incidence was lowest in patients treated with a combination of fludarabine plus cyclophosphamide (5%) indicating that cyclophosphamide has a protective effect.318 The likelihood of developing AIHA was greater if the patient had a positive direct DAT before treatment, had advanced disease, or had a high β2-microglobulin test.318 The incidence of a positive DAT before treatment was 14% and approximately one third of patients receiving chlorambucil or fludarabine with a positive DAT developed AIHA. Conversely, the chance that a patient who was DAT-negative would develop AIHA was 7%. Thus, the high incidence of AIHA observed when fludarabine was first used was because patients receiving this drug usually had advanced and previously treated disease. The protective effect of cyclophosphamide has been confirmed in a subsequent study where the incidence of AIHA was 1%.319 Although the addition of rituximab to chemotherapy would be expected to reduce the incidence of AIHA further, it is reported to occur in <1% to 6.5% of patients receiving FCR.210, 319 The impact of the development of immune cytopenias on survival has been recently evaluated in 2 studies, which have demonstrated that the prognosis of these patients is no worse than those without cytopenias.315, 317

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Oct 21, 2016 | Posted by in HEMATOLOGY | Comments Off on Chronic Lymphocytic Leukemia

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