Pathologic and Molecular Genetic Features of Chronic Lymphocytic Leukemia

Pathologic and Molecular Genetic Features of Chronic Lymphocytic Leukemia Eric D. Hsi Chronic lymphocytic leukemia (CLL) is an indolent B-cell leukemia. While many patients may not require therapy, some patients will suffer a progressive course and die of their disease. This clinical heterogeneity is reflected in the molecular genetic heterogeneity that is becoming apparent through studies of immunoglobulin heavy chain gene mutational status, chromosomal numerical abnormalities, microRNA abnormalities, and genetic abnormalities identified by whole genome sequencing. Indeed, many of these studies are becoming routine in the assessment of patients with CLL or being incorporated into clinical trials to further stratify patients for appropriate therapies. Here, we will review the morphologic, immunophenotypic, and molecular genetic features of CLL and touch upon the concept of monoclonal B-cell lymphocytosis. Semin Oncol 39:74-79 © 2012 Elsevier Inc. All rights reserved.

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hronic lymphocytic leukemia (CLL) is the most common leukemia in the Western hemisphere and is currently defined by the presence of circulating CLL cells at levels ⱖ5 ⫻ 109/L. Lymph node involvement is common in the form of small lymphocytic lymphoma. By definition in the 2008 World Health Organization (WHO) classification, the designation SLL is used when there is tissue involvement by lymphocytes with the morphology and immunophenotype of CLL but there are ⬍ 5 ⫻ 109/L circulating leukemia cells present.1 Here, we will review the pathologic and molecular genetic features of CLL. The pathologic features of SLL are not in scope of this review. CLL is a malignant neoplasm of mature B cells characterized by an indolent yet generally incurable clinical course. The median age of presentation is in the seventh decade with a male predominance of 1.5 to 2:1.1 Patients may present with an incidentally discovered lymphocytosis, which upon further investigation is shown to be CLL. Others may present with signs/ symptoms related to tumor burden such as fatigue, infection, organomegaly, or lymphadenopathy. The diagnosis of CLL is usually straightforward after morphologic and immunophenotypic analysis. We have learned much about the molecular genetics of CLL in the past 10 to 15 years that has informed us about the Department of Clinical Pathology, Cleveland Clinic, Cleveland, OH. Address correspondence to Eric D. Hsi, MD, Cleveland Clinic, L-11, 9500 Euclid Ave, Cleveland, OH 44195. E-mail: [email protected]. 0270-9295/ - see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1053/j.seminoncol.2011.11.007

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diagnosis, prognosis, and biology of this disease. Here, we will review the pathologic and molecular genetic features of CLL, integrating recent findings that have implications on our understanding of the pathogenesis of CLL.

MORPHOLOGY After initial screening by automated hematology analyzers, examination of the peripheral blood smear is the first step in the pathologic evaluation. Red blood cell changes, although not specific, will reflect the abnormalities that may be related to the patient’s disease. For example, presence of anemia, spherocytes, and polychromasia may suggest the presence of a CLLrelated immune-mediated hemolytic anemia. Thrombocytopenia or neutropenia may be evident and could be a manifestation of marrow replacement by CLL. Left shift and/or toxic granulation will suggest the possibility of bacterial infection. Most important, however, is assessment of the lymphocytes. The absolute lymphocyte count may vary widely, from mildly elevated to absolute counts well over 100 ⫻ 109/L. The lymphocytes are small with scant to moderate amounts of cytoplasm. Nuclei are generally round with a hyper-condensed chromatin pattern alternating with lighter areas forming a “cracked” pattern likened to the pattern of a soccer ball (Figure 1). Nucleoli are absent. Occasional prolymphocytes are always seen but vary from less than 1% up to 55% of the lymphocytes. These are intermediately sized cells with a more open chromatin pattern compared to typical CLL cells and a small nucleolus is present that is usually centrally located.2 Occasional cases will show an “atypical” or variant Seminars in Oncology, Vol 39, No 1, February 2012, pp 74-79

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older literature6; however, this has not been rigorously examined with modern therapy.

IMMUNOPHENOTYPE The immunophenotype of CLL has been well characterized by flow cytometry and all cases of suspected CLL should undergo this analysis to confirm the diagnosis and separate it from other B-cell leukemias. CLL cells express CD19, CD20, CD23, and CD5. Subtle phenotypic features such as dim CD20 compared to normal B cells and dim or a lack of expression of FMC7 and CD79b are seen.7,8 Surface immunoglobulin is also usually dimly expressed compared to normal B cells and may be undetectable in some cases (Figure 2). A scoring system has been proposed that makes use of CD19, CD22, CD23, FMC7, and sIg characteristics that may aid in diagnosis (Table 1).8 CD38 and ZAP-70 have been shown to be of prognostic value in CLL, with expression of these markers associated with poor outcome in multiple studies.9 –12

Figure 1. Morphology of typical CLL (A) and mixed-type or atypical CLL (B). The cells of CLL are small with round nuclei and condensed chromatin. In (B) there are cells with nuclear irregularities and heterogeneity of size with an occasional prolymphocyte containing a visible nucleolus.

cytology with slightly irregular nuclei and/or mildly increased prolymphocytes. In some instances, plasmacytic differentiation may be seen that can be associated with an IgM paraprotein. Such cases should be distinguished from lymphoplasmacytic lymphoma through immunophenotypic studies and clinical correlation. Cases of atypical CLL defined by 10% to 15% atypical cells as described above have also been variably termed “mixed cell type” of CLL and are more likely to have an immunophenotype differing from the classical features of CLL (see below) and less favorable outcome.3,4 Such cases of CLL have a molecular genetic correlate with the presence of trisomy 12.5 Bone marrow involvement is present in virtually all cases of CLL and patterns of involvement include interstitial, nodular (non-paratrabecular), and diffuse. The diffuse pattern of involvement has been associated with poor outcome compared to other patterns in the

Figure 2. Bone marrow trephine of CLL showing an interstitial pattern (A) with preservation of adipocytes and a diffuse pattern (B) with sheets of CLL cells.

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Table 1. Immunophenotypic Scoring System

for CLL

Score

CLL cases (%)

Other B-Cell Leukemias (%)

B-NHL (%)

5 4 3 2 1 0

52 35 10 3 0.2 0.2

0 0 1 10 33 56

1 0.5 4 23 39 33

Point system: Surface Ig: weak ⫽ 1, moderate/strong ⫽ 0. CD5: positive ⫽ 1, negative ⫽ 0. CD23: positive ⫽ 1, negative ⫽ 1. FMC7: negative ⫽ 1, positive ⫽ 0. CD22: weak/negative ⫽ 1, moderate/strong ⫽ 0. Abbreviation: NHL, non-Hodgkin lymphoma. Adapted from Matutes et al.8

These markers, in particular ZAP-70, are associated with unmutated immunoglobulin heavy chain variable region genes (IGVH).13–15 However, they are imperfectly correlated and thus neither can be used as a surrogate for IGVH mutational status. Studies in a large cohort of patients assessed for these three parameters point to the relative importance of these markers. In a multivariable analysis of 705 patients analyzed for IGVH mutational status, CD38, and ZAP-70, ZAP-70 was the primary discriminator for time to first treatment and IGVH status was only predictive of time to first treatment in the ZAP-70 –negative patient population with low- to intermediate-risk disease.14 While ZAP-70 and CD38 do yield prognostic information in CLL, methodologic variability between laboratories and variable cutoffs have limited the widespread clinical utility of these tests.16 As ZAP-70 is an intracellular antigen with relatively weak expression, standardization is technically difficult.

MOLECULAR GENETICS Immunoglobulin Gene Rearrangements and Mutational Analysis Molecular genetic studies are generally not required to establish the diagnosis of CLL. If performed, immunoglobulin (Ig) gene rearrangement studies will demonstrated monoclonality. More detailed analysis of the specific Ig gene rearrangement has shown that molecular heterogeneity that has provided great insight and raised many questions about the nature of CLL. Sequence analysis of CLL IGVH genes revealed that a subset of CLL patients (35%– 45%) have unmutated genes (⬍2% variation from the germline published sequences). These patients have been shown in numerous studies to have an unfavorable prognosis.12,17,18

These data suggested that some CLL cases may arise from naïve B cells (unmutated CLL), while others derive from post-germinal center or memory B cells (mutated CLL). Work involving sequence comparison and assignment to specific IGVH family genes has shown additional complexity. For example, patients expressing the IGVH3–21 gene have a poor prognosis regardless of mutation status.19,20 Patients expressing the IGVH3–72 gene have a favorable immunophenotypic profile (CD38- and ZAP70 –negative) and a stable disease course.21,22 Usage of IGVH1– 69 is the most frequent finding in CLL and these cases are usually unmutated. Besides the prognostic value associated with IGVH analysis, comparison of structure of the B-cell receptor has led to the notion that CLL cells derive from antigen-experienced cells, in keeping with both the phenotype (expression of CD27, a memory B-cell marker, in CLL) and gene expression profiling studies showing similarities to memory B cells, regardless of IGVH mutational status.23,24 Sequence analysis of IGVH genes has shown that there is a bias toward certain IGVH gene family members and that this bias was not a reflection of the normal IGVH gene repertoire, even when taking into account the restriction associated with aging.25–27 Furthermore, it has been recognized that CLL cells from unrelated patients from different geographic locations have very similar antigen recognition motifs in their antigen-binding regions. This information was gleaned from comparison of IGVH gene usage, heavy chain complementarity determining region (CDR) 3, and combinations of heavy and light chain CDR3 regions.28 –31 Indeed in one study, 22% of CLL cases were found to carry these “stereotyped” receptors.30 Thus, it appears that CLL cells derive from antigen-experienced cells and a model in which CLL results from antigenselected cells through T-cell– dependent (germinal center) and non–T-cell– dependent paths to account for hypermutated and non-hypermutated CLL, respectively. Alternative models are under investigation and include ones in which a marginal zone B-cell represents the cell of origin.32 The implications of these findings are that chronic B-cell receptor antigen stimulation through external or autoantigens may play a role in constitutive activation and contribute a growth advantage for emerging clones that may then develop additional genetic abnormalities associated with CLL.

Karyotypic Analysis and Identification of Affected Genes Karyotypic abnormalities have long been known to occur in CLL; however, until the application of interphase fluorescence in situ hybridization (FISH), the incidence and clinical significance of such abnormalities were unclear. In decreasing order of frequency, del 13q14 (55%), del 11q (18%), trisomy 12 (16%), del 17p

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Whole Genome Sequencing

Figure 3. Overall survival based on genetic abnormalities detected by FISH. Reprinted with permission.33 Copyright 2000 Massachusetts Medical Society.

(7%), and del 6q (6%) were seen, with some cases harboring more than one abnormality.33 Importantly, outcome differences were seen, with del 13q having the best outcome and del 17p the worst (Figure 3).33 Indeed, these latter patients have a very short time to treatment and poor response to conventional therapy. Interphase FISH tests for these abnormalities are now available and commonplace in patients with CLL. In the search for the genes involved in the minimally deleted region (MDR) of chromosome 13q14 in CLL, it was discovered that microRNAs (miRs) were included in the MDR. miR15a and miR16 were deleted or downregulated in the majority of CLL cases (68%) and it was found that these miRs negatively regulated BCL2, pointing to a pathogenetic role in CLL.34,35 Subsequent murine studies in which the MDR or the specific miR15a/ 16 –1 cluster were deleted have resulted in development of monoclonal B-cell lymphocytosis and CLL with low penetrance.36 Interestingly, the MDR deleted mice more frequently developed CLL than the miRs deleted mice alone and also showed a more aggressive clinical course, suggesting secondary genetic abnormalities in the region of chromosome 13q14 can modulate the behavior of the disease.37 Further work in understanding the role of miRs in the pathogenesis of CLL will likely link previously “unrelated” pathways and provide new insights for potential therapeutic targets. For example, loss of the miR15a/16 cluster is now linked to upregulation of TP53, decreased transactivation of the miR15a/16 cluster, and reduced repression of BCL2 and MCL1, as well as P53-mediated transactivation of miR34a, miR34b, and miR34c with inhibition of target ZAP70 RNA expression.38

Application of next generation sequencing technology has identified previously unknown recurrent mutations in NOTCH1, XPO1, MYD88, and KLHL6 in 12.2%, 2.9%, 2.4%, and 1.8% of CLL cases, respectively. NOTCH1 and XPO1 mutations are associated with IGVH unmutated CLL while MyD88 and KHL6 are associated with IGVH mutated CLL. Both NOTCH1 and MYD88 mutations appear to be activating mutations. NOTCH1 mutated cases have been shown to overexpress NOTCH1 pathway genes and are associated with unfavorable prognosis. Thus, in addition to a potential therapeutic target, this mutation appears to confer prognostic information.39 Investigation of the coding genome of fludarabine-refractory CLL has also revealed a recurrent mutation in a component of the spliceosome, SF3B1. These somatic mutations were missense mutations that are clustered in hotspot codons 662,666, and 700 and predicted poor prognosis. The mutations were found in 17% of fludarabine refractory cases but in only 5% of CLL at diagnosis. This gene has also been shown to be mutated in myelodsyplastic syndromes (refratory anemia with ring sideroblasts).40

MONOCLONAL B-CELL LYMPHOCYTOSIS CLL-phenotype monoclonal B-cell lymphocytosis (MBL) is related to CLL and defined as ⬍5,000 cells/␮L with the immunophenotype of CLL in otherwise asymptomatic patients without lymphadenopathy or defining abnormality or other well-characterized lymphoproliferative disorder (Figure 4).1,41 Recent studies have shown that 3% to 5% of adults have CLL-type cells present in the blood at levels over 1/␮L while up to 20% of adults over 60 years of age have CLL type cells when high-sensitivity phenotyping is performed. The natural history of MBL is being elucidated. In a seminal study, a large series of 185 MBL patients with median follow-up of 6.7 years showed that progressive CLL developed in 15%, with 7% requiring chemotherapy, and only four patients died of CLL. The estimated risk of developing CLL requiring treatment in MBL patients with a lymphocytosis was 1.1%.42 This is in keeping with subsequent studies.43 The bone marrow histopathology of MBL patients appears to show nodular or interstitial involvement with retention of adequate hematopoietic reserves. Thus, while bone marrow examination in not recommended for MBL patients, it is advisable in patients with cytopenias as this may reflect unrelated disease.43 Investigation of the molecular abnormalities seen in high-count MBL showed good-risk CLL abnormalities such as mutated IGVH and del13q14.43,44 Poor-risk abnormalities such as del 17p are only rarely seen and usually are in a minority of cells.43 Of note, the IGVH usage in very low-level MBL may be different than in high-count MBL, which resembles that of typical CLL.

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addition to immunophenotyping to establish a diagnosis and separate CLL from other mimics, interphase FISH testing has now become widely performed to aid in prognosis and management. Advances in our understanding of the B-cell receptor structure in CLL provide insight not only into the potential cell of origin for CLL but in the role of B-cell signaling and activation in development of CLL. MicroRNAs are emerging as important epigenetic contributors in the development or progression of CLL. In addition to contributing to our understanding of the biology of CLL, these advances may result in new therapies for selected/targeted subsets of CLL patients requiring therapy.

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Figure 4. Monoclonal B-cell lymphocytosis. Peripheral blood morphology (A) and flow cytometry dot plots (B) from an asymptomatic patient with an incidentally discovered mild lymphocytosis of 4.5 ⫻ 109/L (normal range, 1.0 – 4.0 ⫻ 109/L). The hemoglobin and platelet count were normal. Immunophenotyping showed a population of B cells expressing CD5, CD19, dim CD20, and CD23 but lacking detectable surface immunoglobulin. The cells were also negative for FMC7 (not shown). The absolute lymphocyte count of the abnormal B cells was 3.7 ⫻ 109/L.

This suggests that B-cell receptor signals may play a role in progression from MBL to CLL.43– 45 Recent detailed studies at the single-cell level suggest that many instances of MBL are, in fact, oligoclonal.46,47

SUMMARY Our understanding of CLL has advanced in the last decade to the point where our concepts of this disease now include a common precursor lesion and genetic analyses suggest an antigen-experienced B-cell that is more than an “inert” cell accumulating over time. In

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