Clinical presentation, pathologic features, diagnosis, and differential diagnosis of chronic lymphocytic

leukemia
INTRODUCTION Chronic lymphocytic leukemia (CLL) is one of the chronic lymphoproliferative disorders
(lymphoid neoplasms). It is characterized by a progressive accumulation of functionally incompetent
lymphocytes, which are usually monoclonal in origin.
CLL is considered to be identical (ie, one disease with different manifestations) to the mature (peripheral) B cell
neoplasm small lymphocytic lymphoma (SLL), one of the indolent non-Hodgkin lymphomas.
EPIDEMIOLOGY CLL is one of the most common leukemia in adults in Western countries, accounting for
approximately 25 to 30 percent of all leukemias in the United States [1]. The disorder is more common in men,
with a male to female ratio of approximately 1.3:1 to 1.7:1 [1,2]. The incidence rates among men and women in
the United States are approximately 6.75 and 3.65 cases per 100,000 population per year, respectively [3]. In
Europe, these incidence rates are 5.87 and 4.01 cases per 100,000 population per year, respectively [4].
CLL is considered to be mainly a disease of older adults, with a median age at diagnosis of 70 years [5];
however, it is not unusual to make this diagnosis in younger individuals from 30 to 39 years of age [2]. The
incidence increases rapidly with increasing age. It is estimated that 18,960 new cases of CLL will be diagnosed
in 2016: 10,830 in males and 8,130 in females [1].
The incidence of CLL varies by race and geographic location. In the United States, there is a higher incidence
among Caucasians as compared with African Americans or Asian Pacific Islanders [2,3]. The incidence of CLL
is extremely low in Asian countries such as China and Japan, where it is estimated to occur at a frequency that
is approximately 10 percent of that seen in Western countries [6-9]. The incidence of CLL in Africa is not as low
as it is in Asia [10,11].
Genetic rather than environmental factors are the most likely explanation for these differences. A genetic effect
on incidence was initially suggested by observational studies that have shown that Japanese who settled in
Hawaii do not have a higher incidence of CLL than native Japanese [12,13]. Further support for a genetic effect
was provided by a genotyping study in African Americans that demonstrated that this population had a lower
frequency of single nucleotide polymorphisms associated with an increased incidence of CLL in other
populations [14]. Furthermore, the cytogenetic and molecular genetic characteristics of CLL appear to be
similar throughout the world, although one study suggests that the clinical course may be more aggressive in
Japan [15,16].
There are no clearly discernible occupational or environmental risk factors that predispose to CLL [17,18].
Although there has been an increase in all other types of leukemias among atomic bomb survivors, there has
been no increase in the incidence of CLL [19,20]. In addition, despite a few reports of an excess risk of CLL
among farmers [17,18], those with benzene and heavy solvent exposure [17,18,21,22], rubber manufacturing
workers [22,23], or those with multiple episodes of pneumonia [24], these associations have not been proven
[25].
Family studies CLL and other lymphoid, hematologic, and solid tumors occur with higher than expected
frequency among first-degree family members of patients with CLL [26-31]. In addition, one study
demonstrated that up to 17 percent of first-degree family members of patients with CLL were found by flow
cytometry to have monoclonal B cell lymphocytosis (MBL) [32]. Of note, while virtually all cases of CLL are
preceded by MBL, only a small percentage of persons with MBL will ultimately develop CLL.

A study of multi-generational familial cases of CLL noted that the median ages at onset in the child (51 years)
and parent generations (72 years) were significantly different (ie, genetic anticipation) [33]. Although there is no
available proof of genetic transmission of this disease, certain genetic polymorphisms may predispose patients
to familial cancer, including CLL [34-37]. DNA analysis in a set of monozygotic twins with CLL suggested that
the transforming events in CLL occur late in life and result, even in monozygotic twins, in genetically distinct
malignant cells [38].
A number of candidate chromosomal regions are currently being explored for the presence of susceptibility
genes for familial CLL [30,39,40].
CLINICAL PRESENTATION
Symptoms Patients with CLL may have a wide range of symptoms and physical and laboratory
abnormalities at the time of diagnosis. Some patients consult a physician because they have noted painless
swelling of lymph nodes, often in the cervical area, which spontaneously wax and wane, but do not altogether
disappear.
Most patients feel entirely well with no symptoms when a routine blood count reveals an absolute
lymphocytosis, leading to a diagnosis of CLL. Five to 10 percent of patients present with the typical "B"
symptoms of lymphoma which include one or more of the following [41]:
Unintentional weight loss 10 percent of body weight within the previous six months.
Fevers of >100.5F (>38C) for 2 weeks without evidence of infection.
Drenching night sweats without evidence of infection.
Extreme fatigue (ie, ECOG Performance status 2 or worse; cannot work or unable to perform usual
activities) (table 1).

Occasionally, the presenting features are those of an acquired immunodeficiency disorder, manifested by
infections, autoimmune complications such as hemolytic anemia, thrombocytopenia or pure red cell aplasia, or
exaggerated reactions to insect stings or bites (especially mosquito).
Signs
Lymphadenopathy The most common abnormal finding on physical examination of the patient with CLL is
lymphadenopathy, present in 50 to 90 percent of patients among various series [42,43]. Lymph node
enlargement may be generalized or localized, and individual lymph nodes can vary greatly in size. The most
commonly affected sites are cervical, supraclavicular, and axillary.
Characteristically, enlarged nodes in CLL are firm, rounded, discrete, nontender, and freely mobile upon
palpation. Exceptions to these generalizations are encountered, particularly when the nodes have grown
rapidly. Occasionally, several enlarged nodes in the same anatomical site (eg, cervical triangle, axilla or
femoral-inguinal areas) may become confluent, forming large spherical lymphoid masses. In addition, new

lymph nodes may appear in places other than the usual lymph node-bearing sites, such as over the sacrum or
the thorax.
Splenomegaly The spleen is the second most frequently enlarged lymphoid organ, being palpably enlarged
in 25 to 55 percent of cases [42,43]. As is the case with enlarged lymph nodes, an enlarged spleen in CLL is
usually painless and nontender to palpation, with a sharp edge and a smooth firm surface. Painful and
infarcted splenic enlargement is an unusual presenting feature.
Hepatomegaly Enlargement of the liver may be noted at the time of initial diagnosis in 15 to 25 percent of
cases [42,43]. The liver is usually only mildly enlarged, ranging from 2 to 6 cm below the right costal margin,
with a span of dullness to percussion of approximately 10 to 16 cm. Upon palpation, the liver is usually
nontender and firm with a smooth surface.
Skin Infiltration with CLL cells may occur in any organ, but, at the time of diagnosis, the skin (leukemia
cutis) is the most commonly involved non-lymphoid organ. These lesions most commonly involve the face and
can manifest as macules, papules, plaques, nodules, ulcers, or blisters [44]. Diagnosis is made based upon
biopsy of the involved skin. Leukemia cutis is seen in fewer than 5 percent of cases and may not significantly
affect overall prognosis unless they represent foci of Richter's transformation.
Nonspecific secondary cutaneous lesions may be due to infection, bleeding, vasculitis, or paraneoplastic
pemphigus [45]. An exaggerated reaction to insect bites, especially mosquito bites, has been reported, and
may alert the clinician to the diagnosis of CLL [46].
Other organ involvement Virtually any lymphoid tissue may be enlarged at diagnosis, including Waldeyer's
ring in the pharynx. In contrast to other lymphomas, clinically relevant gastrointestinal mucosal involvement is
rarely seen in CLL. Similarly, meningeal leukemia is unusual at the time of initial presentation.
Membranoproliferative glomerulonephritis (MPGN) has occasionally been described in CLL and appears to be
a paraneoplastic phenomenon mediated by deposition and possibly processing of cryoprecipitating or
noncryoprecipitating M-components [47-49].
LABORATORY ABNORMALITIES
Lymphocytosis The most noteworthy laboratory abnormality found in CLL is lymphocytosis in the
peripheral blood and bone marrow. Although the absolute blood lymphocyte threshold for diagnosing CLL has
been placed at >5000/microL [5 x 109/L] B lymphocytes [41], a significant proportion of patients present with
counts as high as 100,000/microL [100 x 109/L].
Although very unusual, it should be noted that when blood leukocyte counts are greatly in excess
of 400,000/microL [400 x 109/L], the increased number of cellular elements may result in abnormally high whole
blood viscosity.
Cytopenias Neutropenia, anemia, and thrombocytopenia may be observed at the time of initial diagnosis,
and are usually not severe. These can be related to autoimmune hemolytic anemia, pure red cell aplasia,
autoimmune thrombocytopenia, or agranulocytosis
Patients with CLL have an increased incidence of autoimmune hemolytic anemia (AIHA). The direct
antiglobulin (Coombs) test (DAT) may be positive at some time during the course of the disease in up to
35 percent of cases; overt AIHA occurs in 11 percent of cases [50]. The positive and negative predictive
value of the DAT was reported as part of a prospective clinical trial in patients with CLL [51]. The DAT

status was able to correctly predict the development of AIHA in 83 percent of cases with a positive
predictive value (chance that a DAT-positive patient will develop AIHA) of 28 percent and a negative
predictive value (chance that a DAT-negative patient will remain free of AIHA) of 93 percent.
Pure red cell aplasia (PRCA) is rare, occurring in approximately 0.5 percent of patients. However, if this
disorder is specifically sought for via bone marrow aspiration and absolute reticulocyte count, PRCA may
be found in up to 6 percent of patients with CLL [50]. Unlike AIHA, PRCA may occur early in the course of
CLL.
(Auto)immune thrombocytopenia is suggested when a bone marrow biopsy shows adequate numbers of
megakaryocytes but the peripheral blood has an abnormally low platelet count. This complication occurs
in 2 to 3 percent of patients with CLL and may be the event that initially brings the patient to medical
attention [50]. Retrospective studies have reported varying impacts of immune thrombocytopenia on the
clinical course [52,53].
Rarely, agranulocytosis may be encountered in CLL (approximately 0.5 percent).
The presence of anemia and/or thrombocytopenia has prognostic implications that are discussed separately.
Immunoglobulin abnormalities Hypogammaglobulinemia is present in approximately 8 percent of patients
at the time of initial diagnosis and may develop in up to two-thirds of patients later in the course of the disease.
Usually all three immunoglobulin classes (IgG, IgA, and IgM) are decreased, but in some patients only one or
two may be low. Significant degrees of hypogammaglobulinemia and neutropenia, when present, result in
increased vulnerability of CLL patients to major bacterial infections.
Polyclonal increases in gamma globulins can be seen in up to 15 percent of patients, while a monoclonal
protein is present in up to 5 percent of patients [54,55].
In a study of 109 persons who had developed CLL and had serially collected pre-diagnostic serum samples,
the prevalences of an abnormal free light chain ratio, monoclonal protein (M-protein), and
hypogammaglobulinemia before CLL diagnosis were 38, 13, and 3 percent, respectively [54]. M-proteins and
an abnormal free light chain ratio were detected up to 9.8 years before CLL diagnosis in 48 of the 109
subjects.
Other abnormal findings There are no characteristic abnormalities in blood chemistry, but elevated levels
of serum lactate dehydrogenase (LDH) and beta-2 microglobulin were found in approximately 60 percent in
one series of patients with progressive or advanced CLL entering a therapeutic trial [56]. Elevations of uric
acid, hepatic enzymes (ALT or AST) and, rarely, calcium may also be observed.
PATHOLOGIC FEATURES The diagnostic evaluation of a patient suspected of having CLL should include a
complete blood count with differential, examination of the peripheral smear, and an immunophenotypic analysis
of the circulating lymphocytes. While a bone marrow aspirate and biopsy and lymph node biopsy are not
among the required elements of the diagnostic work-up, abnormalities seen on these examinations are
included in this discussion of pathologic features. Chromosomal changes seen in CLL are also not diagnostic
features of the disease and are presented separately
Morphology The peripheral blood smear of patients with CLL demonstrates a lymphocytosis. The leukemic
cells are typically small, mature appearing lymphocytes with a dense nucleus, partially aggregated (clumped)
chromatin, and without discernible nucleoli. There is a narrow border of clear to slightly basophilic cytoplasm
(picture 1 and picture 2) [41].

In addition, a proportion of cells may be comprised of larger lymphocytes with large, somewhat notched nuclei,
lacy appearing nuclear chromatin, and prominent, visible nucleoli. These "prolymphocytes" usually account for
a minority of the overall population of lymphocytes but can comprise up to 55 percent. The smear also often
contains "smudge" cells (also called "basket cells") that are lymphocytes that appear to have been flattened or
smudged in the process of being spread on the glass slide [57].
Immunophenotype Immunophenotypic analysis, usually by flow cytometry, is a key component to the
diagnosis of CLL (figure 1) [58]. There are three major characteristic immunophenotypic findings [41]:
Expression of B cell associated antigens including CD19, CD20, and CD23. Expression of CD20 is
usually weak. Expression of CD21 and CD24 can be seen, but is not required for diagnosis.
Expression of CD5, an antigen commonly expressed by T cells.
Low levels of surface membrane immunoglobulin (ie, SmIg weak). The immunoglobulin is most often
IgM or both IgM and IgD, and only a single immunoglobulin light chain is expressed (ie, either kappa or
lambda but not both), confirming the clonal nature of these cells. In rare cases, several Ig clones may
coexist.
In addition, CLL cells are usually negative for cyclin D1 and CD10. FMC7, CD22, and CD79b are also
commonly negative or weakly expressed [59].
The vast majority of cases will demonstrate a single clone of abnormal circulating B lymphocytes by flow
cytometry. Rarely, flow cytometry will identify biclonal disease. In one large study, this was estimated to
represent 1.4 percent of cases [60].
Bone marrow aspirate and biopsy Bone marrow aspirate and biopsy are not required for the diagnosis of
CLL. If bone marrow biopsy and aspiration are performed at the time of initial diagnosis, they usually
demonstrate normal to increased cellularity, with lymphocytes accounting for >30 percent of all nucleated cells
(picture 3 and picture 4 and picture 2).
In addition to an increased percentage of mature-appearing lymphocytes in the smears of the bone marrow
aspirate, there are three main types of infiltrative patterns of lymphocytes that are recognized in trephine
biopsy specimens of the bone marrow: nodular, interstitial, and diffuse. Sometimes, in a given biopsy sample,
one may see a mixture of nodular and interstitial, or nodular and diffuse infiltrative, patterns. Historically,
patients with diffuse infiltration tend to have advanced disease and a poorer prognosis, whereas, for prognostic
purposes, nodular and interstitial patterns may be grouped together in the prognostically better "non-diffuse"
category [61-63]. However, with the discovery of additional prognostic markers in CLL, the prognostic value of
bone marrow biopsy has become less clear. Prognostic markers in CLL are discussed in more detail
separately.
Lymph node biopsy CLL and small lymphocytic lymphoma (SLL) are considered to be the same disease
with different clinically manifestations. Historically, the diagnosis of SLL was made via a lymph node biopsy in a
patient presenting with lymphadenopathy but without peripheral lymphocytosis, while CLL was diagnosed
through examination of the peripheral blood and bone marrow in patients with lymphocytosis. Currently, the
diagnosis of SLL is reserved for patients demonstrating lymph node pathology consistent with CLL/SLL but
with an absolute peripheral clonal lymphocyte count that does not exceed 5000/microL [5 x 109/L] and no
evidence of neutropenia, anemia, or thrombocytopenia related to the disease [41,59,64].
The histopathologic lymph node findings in SLL and CLL are identical and consist of a diffusely effaced nodal
architecture with occasional residual naked germinal centers [41,59]. The infiltrate is composed of mostly

mature-appearing, small lymphocytes, with an admixture of prolymphocytes and paraimmunoblasts (picture 5).
The pathologic features on lymph node biopsy and the diagnosis of SLL are discussed in more detail
separately.
EVALUATION AND DIAGNOSIS The diagnosis of CLL is based upon a complete blood count with
differential, flow cytometry of the peripheral blood to determine the immunophenotype of circulating
lymphocytes, and examination of the peripheral smear [41].
To diagnose CLL, the 2008 iwCLL update of the National Cancer Institute guidelines on the diagnosis and
treatment of CLL concluded that the following two criteria must be met [41]:
Absolute B lymphocyte count in the peripheral blood 5000/microL [5 x 109/L], with a preponderant
population of morphologically mature-appearing small lymphocytes.
Demonstration of clonality of the circulating B lymphocytes by flow cytometry of the peripheral blood. A
majority of the population should express the following pattern of monoclonal B cell markers: extremely
low levels of SmIg and either kappa or lambda (but not both) light chains; expression of B cell associated
antigens (CD19, CD20, and CD23); and expression of the T cell associated antigen CD5.
In the current system, patients with an absolute lymphocyte count less than 5000/microL [5 x 109/L] and no
evidence of other disease manifestations (eg, enlarged lymph node) are diagnosed with a monoclonal B cell
lymphocytosis rather than CLL even if they have cytopenias [64].
As mentioned earlier, CLL and small lymphocytic lymphoma (SLL) have identical pathologic and
immunophenotypic features and are therefore considered two clinical manifestations of the same disease.
Patients with lymphadenopathy and an absolute peripheral B lymphocyte count that is less than 5000/microL [5
x 109/L] are given the diagnosis of SLL rather than CLL. The diagnosis of SLL is discussed in more detail
separately.
Prior to the current diagnostic criteria, the diagnosis of CLL was based upon an absolute lymphocyte count
(ALC) equal to or greater than 5000/microL [5 x 109/L] in the setting of an appropriate immunophenotype. With
this system, patients with an absolute B lymphocyte count (B-ALC) less than 5000/microL and an ALC more
than 5000/microL represented an overlap between CLL and monoclonal B cell lymphocytosis. The switch to
using B-ALC for the diagnosis of CLL in the current system eliminated this overlap, but has sparked
controversy [65,66].
While the current definition uses a cutoff of 5000 B lymphocytes per microL, the ideal cutoff value is still to be
determined. A retrospective analysis of 459 consecutive patients diagnosed with Rai stage 0 CLL over a
seven-year period at one institution found that cutoff values for ALC and B-ALC of 12,000 and
11,000 cells/microL, respectively, were associated with reduced rates of treatment-free and overall survival
[67]. In contrast, a cutoff value of 5000 cells/microL for either ALC or B-ALC was not associated with outcome.
DIFFERENTIAL DIAGNOSIS The diagnosis of CLL is suspected whenever the peripheral blood in an adult
demonstrates an absolute lymphocytosis. However, blood lymphocytosis may also occur with non-neoplastic
conditions, such as viral or other infections (eg, infectious mononucleosis, pertussis, toxoplasmosis), as well as
in neoplastic conditions other than CLL (eg, the leukemic phase of lymphomas, hairy cell leukemia,
prolymphocytic leukemia, and large granular cell lymphocyte leukemia).
The task is therefore to distinguish between reactive causes of lymphocytosis and clonal (malignant) causes,
and, for the latter, to distinguish CLL from the other malignant lymphoproliferative disorders (table 2). As
described above, if the flow cytometric phenotypic features are consistent with the diagnosis of CLL in a patient

with peripheral blood lymphocytosis, the diagnosis of CLL is clearly established. Features distinguishing these
conditions with blood lymphocytosis from CLL are summarized below.
Infectious causes of lymphocytosis A transient lymphocytosis can be seen in the peripheral blood of
patients who have an infection. To be diagnosed with CLL, the lymphocytosis must be sustained over a period
of time. This effectively excludes those conditions, such as infectious mononucleosis, pertussis, and
toxoplasmosis, in which blood lymphocyte counts rise and then typically return to normal after a few weeks. In
addition, unlike in CLL, a lymphocytosis does not occur in the bone marrow of patients with these infectious
causes of lymphocytosis. Furthermore, the lymphocytosis is not clonal in those with a non-malignant cause
and does not show the characteristic immunophenotype described above.
Monoclonal B cell lymphocytosis The term monoclonal B cell lymphocytosis (MBL) is used to categorize
individuals who have an absolute increase in the number of clonal B lymphocytes in the peripheral blood that
does not exceed 5000/microL [5 x 109/L] and who have no other disease manifestations (eg, lymphadenopathy
or organomegaly) [64].
Prolymphocytic leukemia Both prolymphocytic leukemia (PLL) and CLL can present with lymphocytosis
and splenomegaly, and have circulating prolymphocytes in the blood. Prolymphocytes are morphologically
distinct from typical CLL cells. Compared with typical CLL cells, these are large cells with somewhat immatureappearing nuclear chromatin, a prominent vesicular nucleolus, and a moderate amount of cytoplasm (picture
6) [68,69]. In B-PLL, prolymphocytes are of B lineage, express normal amounts of SmIg and usually do not
express CD5. In contrast, typical CLL cells express only low amounts of SmIg and express CD5.
While prolymphocytes such as these can be seen in CLL, in B-PLL, by definition, more than 55 percent of the
circulating cells in the peripheral blood are prolymphocytes and, more typically, the percentage of
prolymphocytes is greater than 90 percent.
Mantle cell lymphoma Mantle cell lymphoma (MCL) can have a leukemic phase that may mimic CLL. Such
patients have circulating small lymphocytes, often with irregular or cleaved nuclei. Like the malignant cells of
CLL, MCL tumors coexpress CD5 and CD20. However, the neoplastic cells of MCL stain strongly for cyclin D1
and surface membrane immunoglobulin (SmIg), have a t(11;14) chromosomal abnormality, and are negative
for CD23 [70]. In contrast, the malignant cells in CLL are negative for cyclin D1 and are often CD23 positive.
Lymphoplasmacytic lymphoma Both lymphoplasmacytic lymphoma (LPL) and CLL are
lymphoproliferative disorders of small cells that usually have an indolent course. LPL is the lymphomatous
counterpart to Waldenstrm disease (ie, LPL leukemia). Peripheral blood involvement in LPL is usually less
prominent than in CLL; circulating malignant cells often have a plasmacytoid appearance (picture 7).
LPL can be distinguished from CLL by its lack of CD23 expression, the presence of strong staining for surface
IgM and CD20, and the presence of cytoplasmic Ig.
Hairy cell leukemia Both hairy cell leukemia (HCL) and CLL can be associated with an elevated
lymphocyte count in the peripheral blood, although leukocytosis is much less common in HCL, occurring in
only approximately 10 to 20 percent of cases. The diagnosis of HCL is often suspected based upon the
presence of circulating lymphocytes with cytoplasmic projections (hairy cells) (picture 8) [59]. HCL is frequently
distinguished from CLL based upon its immunophenotype that is usually CD5 negative, and positive for CD25,
CD11c, and CD103.
Follicular lymphoma Patients with follicular lymphoma (FL) can present in a similar fashion to those
with CLL/SLL with diffuse painless peripheral adenopathy, often waxing and waning over long periods of time.

Usually, these two disorders have distinct growth patterns on lymph node biopsy. Follicular lymphoma has a
nodular growth pattern which is not seen in CLL/SLL; however, on occasion, involved lymph nodes
in CLL/SLL tumors can have prominent proliferation centers that take on a mottled "pseudo-nodular"
appearance that mimics that of FL.
FL and CLL/SLL can be distinguished by immunophenotype. In contrast to the malignant cells
in CLL/SLL, most cells in FL demonstrate bright surface immunoglobulin (SmIg), express CD10 and lack CD5
expression. In contrast, CLL cells have extremely low levels of SmIg, express CD5, and lack CD10 expression.
In addition, FL characteristically has a t(14;18) translocation, which is rarely seen in CLL/SLL.
Splenic marginal zone lymphoma Both splenic marginal zone lymphoma (MZL) and CLL present with
splenomegaly and peripheral blood lymphocytosis. In addition, both CLL and splenic MZL can express CD23,
CD43, CD5, and IgD, although expression of these is much more typical of CLL. Unlike CLL/SLL, MZL may
have bright SmIg and CD20. In difficult cases, pathologic evaluation of the bone marrow, spleen, and lymph
nodes may be used in concert to determine the most likely diagnosis. Bone marrow morphology in MZL may
show notched nuclei. In addition, cytogenetic changes typically seen in CLL are not usually seen with MZL.