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Morphology: Morphologically, CLL cells are typically bland, small lymphocytes with clumped chromatin and scanty cytoplasm (Figure 9.2a). “Smudge,” “smear” or

“basket” cells are often present and may be prognostically important [3]. Increased numbers of prolymphocytes (Figure 2b) has been historically associated with aggres-sive disease: patients with >55% prolymphocytes are considered B-cell prolymphocytic leukemia (see below), whereas“typical CLL” is associated with <10% prolym-phocytes [4]. Patients with 10–55% prolymphocytes have intermediate disease features and are traditionally classi-fied as “CLL/PLL”; within the context of clinical trials and modern treatment algorithms these patients are generally managed as if they have typical CLL.

Atypical morphology in CLL is not uncommon and may include larger forms with less condensed chromatin and nuclear irregularities; these findings may be particularly common in patients with trisomy 12. Because of this variation in disease morphology, in modern practice, CLL is most commonly defined by its characteristic immunophenotype.

The bone marrow infiltrate in CLL may be inter-stitial, nodular or diffuse (“packed”). The cytological features of the neoplastic cells are comparable to that seen in the blood or bone marrow aspirate, i.e. small cells with a high nuclear : cytoplasmic ratio and con-densed nuclear chromatin. Larger cells may be present and these are the proliferation centers of the CLL (Figure 9.3). Immunocytochemistry can be performed on the bone marrow biopsy to demonstrate the CLL phenotype (see below).

Immunophenotype: Flow cytometry of the peripheral blood is the most common method for confirming

a diagnosis of CLL. The typical immunophenotypic profile for CLL is that of a mature B-cell (CD19-and CD79a-positive) with aberrant CD5 expression, CD23 positivity and dim expression of CD20 and surface immunoglobulin [5] (Figure 9.4). CD43 is usually positive. CD79b and FMC7 are absent or weakly expressed. CD23 expression is of importance as the major differential diagnosis of a CD5-positive mature B-cell leukemia is mantle cell lymphoma in leukemic phase (see below), which is typically CD23 negative.

Matuteset al. proposed the use of a scoring system to differentiate CLL from other mature B-cell leuke-mias [6]. This system initially usedfive markers for features typical of CLL:

1. CD5 positivity.

2. CD23 positivity.

3. FMC7 negativity.

4. Weak expression of surface immunoglobulin.

5. Weak or absent expression of CD22.

In a large cohort of cases with a variety of circulating B-leukemia or B-lymphoma cells, 87% of patients with CLL were found to have scores of 4 or 5, whereas 89%

and 72% of other B-leukemias and B-lymphomas had scores of 0 or 1, respectively [6]. A later refinement of this scoring system replaced CD22 with CD79b (typically absent in CLL), and broadened the criteria for defining CLL to 3–5 points [7]. This refinement improved diagnostic accuracy from 91.8–96.8% [7].

In CLL in transition to B-PLL (CLL/PLL), there may be two distinct populations onflow cytometry, with the B-PLL population being larger and showing brighter expression of CD20 and surface immunoglo-bulin. In contrast tode novo B-PLL, which is usually CD5-negative, transformed prolymphocytes in CLL/

PLL often retain CD5 expression [5].“Atypical” CLL (as defined by the “Matutes score”) may be CD23-negative or show increased expression of surface immunoglobulin, FMC7 and/or CD20; these cases may be associated with the trisomy 12 cytogenetic abnormality [8].

The typicalflow cytometry panel for CLL will include a tube assessing the co-expression of CD19 and CD38, an important prognostic indicator (see below).

Cytogenetic and molecular genetic features:

Cytogenetic studies in CLL have traditionally been hampered by the low mitotic activity of the tumor cells in vitro. In a large study of peripheral blood mononuclear cell karyotyping in 433 patients with

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d c

f e

Figure 9.2.Morphological features of mature B-cell neoplasms.

a. CLL: Tumor cells are small lymphocytes with clumped chromatin and scanty cytoplasm. Note the presence of“smudge” cells.

b. B-PLL: Prolymphocytes are twice the size of mature lymphocytes, with condensed chromatin and a single prominent nucleolus.

c. HCL: Hairy cells can be scanty in the blood, but have a distinctive appearance with abundant, weakly basophilic or gray cytoplasm with circumferential“hairy” projections and an oval or bean-shaped nucleus displaying homogeneous, moderately clumped chromatin.

d. Follicular lymphoma: Lymphoma cells are small cells, with a high nuclear : cytoplasmic ratio, condensed homogeneous chromatin and a characteristicfine nuclear cleft.

e. Mantle cell lymphoma. The lymphoma cells are pleomorphic with both small- and medium-sized forms present, and a variety of nuclear morphology.

f. Lymphoplasmacytic lymphoma. Circulating lymphoplasmacytoid cells and background rouleaux due to paraprotein.

Photographs courtesy of Dr. A George, St Vincent’s Pathology, Melbourne, Victoria, Australia.

CLL, clonal abnormalities were identified in only 50% [9]. Thus, the field of CLL genetics was slow to progress until the widespread availability of fluor-escentin situ hybridization (FISH) probes for com-mon cytogenetic aberrations [10]; this showed that approximately 80% of CLL samples harbor one or more karyotypic aberration. This high aberra-tion rate has since been confirmed by modern cultivation techniques (using an immuno-stimulatory

CpG-oligonucleotide and IL-2) capable of generating metaphases from > 98% of tested samples [11].

Cytogenetic features are most useful in prognosti-cation of CLL, and not in classification. One possible exception is isolated trisomy 12, which is common in CLL and uncommon in other mature B-cell leukemias [9]. The other“recurrent” CLL abnormalities (deletion of 11q22.3, deletion of 13q14 and deletion of 17p13) are non-specific and may be found in disparate tumors b

a

Figure 9.3.Chronic lymphocytic leukemia in a bone marrow trephine (H & E stain).

a. Low power view showing an interstitial nodular pattern of infiltration by CLL.

b. Higher power of a CLL nodule showing nucleolated larger cells in the center and peripherally located small mature CLL lymphocytes.

Figure 9.4.Immunophenotype of CLL (green arrows). Note expression of CD5, CD19, CD23; dim expression of CD20; and negative expression of FMC7 and CD79b. Surface immunoglobulin expression is weak and shows kappa light chain restriction.

Figure courtesy of Ms. I Cutter, St Vincent’s Pathology, Melbourne, Victoria, Australia.

such as mantle cell lymphoma and prolymphocytic leukemia.