The Pathology of Chronic Lymphocytic Leukemia / Small Lymphocytic Lymphoma (CLL/SLL)

Chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL/SLL) is a neoplastic B cell process. It accounts for approximately 90 percent of chronic lymphoid leukemias in the US and Europe. The majority of patients diagnosed with CLL are older adults; the median age at the time of diagnosis is 65 years. In addition, more men than women are diagnosed with this disease. All patients with CLL, by definition, have bone marrow and peripheral blood (PBL) involvement; the lymphocyte count is >10 x 10⁹; however, the diagnosis can still be made even though the lymphocyte count is less than 10 x 10⁹ if the morphology and phenotype is typical of CLL.

Morphologically, the CLL cells are small round and monotonous in most cases, although in some cases they may show some nuclear irregularities. These cells usually look very much like normal lymphocytes that circulate in the peripheral blood. Hence, immunophenotypic analysis is often required to identify the malignant cells and is extremely important in those cases where the lymphocyte count is low.


Picture of CLL cells (Diff Quik; cytospin 60x original magnification)	Picture of a CLL cell clot (hematoxylin and eosin; 60x original magnification)

Characteristically CLL/SLL involves multiple anatomic sites at presentation including the peripheral blood, lymph nodes, spleen, etc. This is because the proliferation is relatively slow growing and hence most people are not ill at presentation. In many cases the disease is diagnosed serendipitously; the clinician notes an abnormal white blood count on a routine complete blood count (CBC). In other cases, patients may present with fatigue, easy bruising and/or infection due to anemia, thrombocytopenia and/or neutropenia secondary to replacement of normal bone marrow elements (erythroid and myeloid precursors and megakaryocytes) by the malignant process. Infrequently they may present with symptoms of what is known as a mass effect i.e. an enlarged lymph node or tissue that is compressing or compromising some other organ or tissue. Occasionally patients may present with symptoms of autoimmune abnormalities as a few cases are associated with immunoglobulin production that may bind to other tissues and cells causing problems, like thrombocytopenia, for the patient.

Although the diagnosis of CLL/SLL is usually made by examining the peripheral blood, lymph nodes and bone marrow biopsy specimens are often involved and may be obtained for pathologic examination to either diagnosis the disease, stage the disease or to investigate any changes in the patient’s “normal” disease state (i.e. identification of a rapidly enlarging lymph node). The lymph nodes involved by CLL/SLL usually show a diffuse pattern of involvement with effacement of the normal architecture. In otherwords, the follicles, sinuses, interfollicular area and medullary areas of the lymph node are replaced by small, round bland appearing lymphocytes. Although CLL/SLL is a diffuse process, at low power collections of pale cells are seen. These collections are known as proliferation centers. The cells in these collections are also malignant cells, but are somewhat larger and are the mitotically active (i.e. they are dividing or proliferating). These latter cells usually only account for only a small percentage of the neoplastic cell population.


Lymph node replaced by CLL (hematoxylin and eosin; 2x original magnification)	Proliferation center cells in CLL: note they are larger than the standard CLL cells (40x original magnification).

When CLL/SLL involves the bone marrow it can exhibit a variety of patterns: nodular, mixed nodular and diffuse, and diffuse. Only occasionally does one identify proliferation centers in the bone marrow.


Bone marrow involved by CLL: Nodular pattern (hematoxylin and eosin; 4x original magnification).	Bone marrow involved by CLL: Diffuse pattern (hematoxylin and eosin; 4x originalmagnification).

CLL/SLL exhibits a characteristic phenotype or pattern of antigen expression. However, to understand the characteristic phenotype of CLL/SLL, one needs to step back and review normal lymphoid cell antigen expression.

Antigens are proteins or glycoproteins that are expressed by the cell which we use to identify the cell and the cell uses to function. Normal B cells express a variety of antigens depending on their stage of development, state of activation, type of function, etc. However, most B cells express one, and usually more than one, “pan-B” cell antigen. The pan-B cell markers that are commonly used for flow cytometry (the most common way to evaluate peripheral blood samples for CLL/SLL) are CD19, CD20 (the antigen to which the drug Rituxan^® binds) and CD22. Other B cell antigens that are more variably used for diagnostic purposes are CD79a and PAX-5. In addition each individual B cell expresses either kappa or lambda immunoglobulin, but not both. When a B cell population consists of a mixture of both kappa and lambda positive cells it is known as a polytypic population. When a B cell population only expresses one light chain (only kappa or only lambda), it is known as a monotypic population.

The lymphoid cells not only consist of B cells, but they also consist of T-cells. The T-cells also express a number of “pan-T” antigens including CD2, CD3, CD5 and CD7; they also express CD4 or CD8 or CD4 with CD8 or no CD4 or CD8 (hence they are different than B-cells which express only kappa or only lambda, but not both). In most peripheral blood samples the percentage of T-cells is much greater than the percentage of B- cells. Normal ratio of T-cells to B-cells is in the range of 70-80 percent T-cells to about 10-20 percent B-cells.

A benign B-cell population consists of mixture of kappa and lambda positive B cells that express pan-B cell antigens and do not express T-cell antigens (although there are rare exceptions). In contrast a malignant B-cell population is usually monotypic (expressing either kappa light chain or lambda light chain) and may exhibit aberrant (abnormal) antigen expression. In the case of CLL/SLL, the malignant B-cells express the “pan” T- cell antigen CD5. In addition, the malignant CLL cells are usually also CD23 positive.

The “phenotype” or antigen expression profile of lymphoid cells is usually determined by either immunohistochemistry, which is performed on tissue sections, or by flow cytometry which is used for fluid samples, such as peripheral blood and bone marrow aspirates. Lymph nodes are tissues can be put into a fluid suspension for flow cytometry and cells in fluids can be put into tissue blocks so that all materials can be analyzed by both techniques. This is important because the different immunophenotypic techniques give the physicians different information. However, in order to use these different techniques need sufficient numbers of cells are necessary.

With respect to CLL, flow cytometry is the preferred method of initial analysis, although the diagnosis can also be made by immunohistochemical staining of tissue sections. Because CLL cells tend to only faintly express surface immunoglobulin, flow cytometry which is much more sensitive than our eyes, can detect low levels of immunoglobulin expression. Thus, the flow cytometer is important in identifying monotypic B-cell populations in CLL. In addition, flow cytometry can easily show co-expression of B--cell antigens with the T-cell antigen CD5 (although this can be determined in tissue sections as well).

FLOW CYTOMETRY

Furthermore, CD23 expression is also easily determined by flow cytometry (as well as by immunohistochemistry). Flow cytometry, in addition, is the best means of determining the level of CD38 expression on the malignant cells. CD38 is a prognostic marker in CLL. Although there are antibodies that will detect CD38 antigen expression in tissue sections, they are not as nearly as sensitive or reliable as those that are available for flow cytometry. The best method of detection of expression of another prognostic marker, ZAP-70, is still controversial. In some laboratories ZAP-70 expression is detected by flow cytometry, however, in many laboratories ZAP-70 expression is determined by immunohistochemical staining of cell clot block sections. In addition, immunohistochemistry of cell clot blocks can be helpful ruling out an important disease in the differential diagnosis of CLL. Specifically, immunohistochemical staining for BCL-1, positive in mantle cell lymphoma and negative in CLL, is important as mantle cell lymphoma in general has a much poorer prognosis than CLL.

IMMUNOHISTOCHEMISTRY OF TISSUE SECTIONS


Immunostaining for PAX-5 (B cell)		Immunostaining for CD3 (T cell)

Immunostaining for CD23		Immunostaining for CD5 (T cell antigen abnormally expressed in CLL)

Although all cases of CLL contain clonal rearrangements of the immunoglobulin heavy and light chain genes, consistent with their malignant nature, it has been recently found that there are two types of CLL as determined by the presence or absence of somatic hypermutations in the immunoglobulin gene. The patients with somatic hypermutations in the immunoglobulin gene of their CLL cells have a relatively good prognosis (reports of median survival of 25 years) while those whose cells contain the germline configuration of the immunoglobulin gene (i.e. no somatic hypermuatations) have a worse prognosis with a significantly shorter survival. Although the presence and absence of somatic hypermutations correlate well with prognosis, the studies necessary to determine the configuration (make-up) of the DNA is time consuming and expensive. Surrogate phenotypic markers, thus, have been identified that correlate with somatic hypermutations and survival. The two most important surrogate markers are CD38 and ZAP-70. Although CD38 correlates with the DNA configuration in about two-thirds of cases, ZAP-70 expression correlates with the presence or absence of somatic hypermutations in approximately 90 percent of CLL cases. Specifically, expression of ZAP-70 by the malignant CLL cells correlates with the lack of somatic hypermutations and a poor prognosis and the lack of ZAP-70 correlates with the presence of somatic hypermuations and a good prognosis.

IMMUNOHISTOCHEMISTRY OF CELL CLOTS FOR ZAP-70 EXPRESSION


PAX-5 (B cells)	CD3 (T cells)	ZAP-70 negative (matches CD3)

PAX-5 (B cells)	CD3 (T cells)	ZAP-70 positive (matches PAX-5 + CD3)

Approximately 80 percent of CLLs have abnormal cytogenetics when examined by FISH (fluorescent in situ hybridization). Trisomy 12 has been reported to occur in about 20 percent of cases; this abnormality is seen predominately in cases of CLL without somatic hypermutations. In contrast 13q14 deletions that are found in approximately 50 percent of cases are seen more often in cases containing somatic hypermutations in the immunoglobulin gene. Deletions of 11q22-23 are present in 20 percent of cases; additional abnormalities include 6q21 and 17p13 deletions.

For additional information, the reader is advised to consult the WHO (World Health Organization Classification of Tumors): Tumors of Haematopoietic and Lymphoid Tissues, edited by E.S. Jaffe, N.L Harris, H. Stein and J.W. Vardiman.