University of Virginia Health System

Small Lymphocytic B Cell Lymphoma (SLL)/ Small B Cell Chronic Lymphocytic Leukemia
(B Cell CLL, CLL)

(a) Small B cell CLL- Peripheral blood smear, Wright-Giemsa stain, 1000x

(b) Small B cell CLL demonstrating an interstitial pattern of involvement- Bone marrow biopsy, H&E stain, 100x

(c) Small B cell CLL demonstrating a diffuse pattern of involvement- Bone marrow biopsy, H&E stain, 100x

Description:

The clonal lymphocytes in SLL/small B-CLL are small, approximately the same size as normal small lymphocytes in the peripheral blood, and characteristically have a monotonous appearance (a). The nuclear:cytoplasmic ratio is high, and the cytoplasm is scant and grey to light blue in color without granules. The nucleus is frquently eccentric in location, the nuclear chromatin is clumped, and nucleoli are not seen. Smudge cells (naked lymphocyte nuclei), which are the result of smear artifact, are frequently seen, and increase as the level of lymphocytosis increases. Scattered prolymphocytes are seen, and also increase as the level of lymphocytosis increases. When the percentage of prolymphocytes is less than 50%, the impact on clinical course is less. When the percentage of prolymphocytes is >50% of the lymphocytes, the criteria for prolymphocytic transformations is met and the course tends to be more aggressive. Prolymphocytes are larger, approximately 1 1/2 times that of the small lymphocytes and have more basophilic cytoplasm. The nuclear chromatin is more immature, but still clumped, and prominent nucleoli are often visible.

The bone marrow is always involved in small B-CLL, and the pattern may be interstitial, nodular (b) or diffuse (c). The interstitial or nodular pattern requires immunohistochemistry to differentiate from normal B cell follicles or polyclonal T cell aggregates. 

Immunophenotype:

The clonal B cells express a very distinct profile: CD19+, sIg+, CD5+, and CD23+, but CD10- and FMC7-. The intensity of the sIg is weak, unlike most other B cell leukemias, and is usually IgM and IgD.

Again, this immunologic profile is not absolute. Cases of CLL that meet other laboratory and clinical criteria for the diagnosis of B-CLL may be C5 and/or CD23 negative. In some cases clonality of CLL may require the demonstration of IgV(H) gene rearrangement by pcr-DNA studies.

The expression of the T cell marker, CD5 (an activation marker,) is not the only T cell marker expressed on the clonal B cells in B-CLL. A majority of the unmutated B cell CLL cases express the CD3 zeta chain-associated protein (ZAP-70), a finding that identifies a very poor prognostic subset.

Pathobiology:

In 1967, William Dameshek (Blood 29 (Suppl): 566, 1967) proposed a pathogenetic model for chronic lymphocytic leukemia (CLL) that was adopted for decades. In this model, the clinical and laboratory features of CLL were thought to result from the slow accumulation of morphologically mature and immunoincompetent lymphocytes due to faulty apoptosis. Several studies over the past decade or so have clearly demonstrated that this concept does not hold. In no particular chronological order these observations include:

• The clonal B cells in B-CLL are antigen-experienced cells. Somatic hypermutation of IgV(H) genes, an event that occurs after antigenic stimulation in the germinal center (T cell dependent) or in the marginal zone (T cell independent), can be demonstrated in most cases. The presence or absence of somatic hypermutation has evolved as the single most important prognostic factor. In mutated cases the prognosis is much better (Hamblin TJ, et al. Blood 94:1848, 1999).
• Although defective apoptosis is an important pathogenetic mechanism for the accumulation of the clonal B cells, a significant, but variable, proliferation of the clonal cells can be demonstrated in most cases (Messmer BT, et al. J Clin Invest 115: 755, 2005.) This observation is supported by the observance of "growth centers" (pseudofollicles) on paraffin fixed specimens of lymph nodes and bone marrows of patients with B-CLL (Pizzolo G, et al: Blood 62:1289, 1993.) These "growth centers" appear as light areas and represent proliferation of the clonal B cells. Clonal cells ranging from immunoblast-like to small cleaved cells are present. In the study by Messmer BT, et al, those cases with the highest proliferation rate tended to be more aggressive. This proliferative process could serve to replenish the cells that die by apoptosis.
• The continued proliferation and thus expansion of the B cell clone could be contributed to by several other mechanisms:
• Constant antigenic stimulation through their B cell receptor (BCR). The antigens appear to be mostly T cell-independent ones, and include self-antigens and microbial-derived ones that are polysaccharides or lipopolysaccharides. In this regard these clonal B cells resemble normal marginal zone B cells, a subset of which express CD5 (B1a B cells) (see under normal lymphocyte). A similar process could occur with germinal center derived clonal, memory B cells, some of which exit the germinal center and migrate to the marginal zones. 
• Interaction with CD4+ T cells, stromal cells and dendritic cells in the marginal zones and bone marrow 
• Response to various cytokines, released mostly from activated CD4+ T cells and chemokines released from stromal and dendritic cells. These soluble molecules are thought to inhibit apoptosis of the clonal B cells and thus promote clonal expansion. 
• Other factors- There are certainly other mechanisms involved in the survival and expansion of the clonal B cells in B-CLL . The role of the various cytogenetic abnormalities, which appear to be relatively late events, in sustaining the clonal proliferation remains unclear. No cytogenetic abnormalities or other events have been identified to be responsible for the clonal transformation. It is now well documented that there is a familial form of B cell CLL, and hopefully studies of these families will reveal some of the mechanisms involved in the transforming events (Sellick GS, et al. Semin Oncol 33:195, 2006).
• Monoclonal B-cell lymphocytosis with a phenotypic profile of B-CLL and a normal peripheral blood absolute lymphocyte count has been reported to occur in 3.5% of normal adults above the age of 40 years (Rawstrom AC, et al. Blood 100:635, 2002). The entity has been referred to as a "monoclonal B-cell lymphocytosis of uncertain clinical significance." In a recent follow-up, these individuals did progress very slowly (rate of 1.1% per year) to CLL requiring treatment (Rawston AC, et al. N Engl J Med 359: 575, 2008). Further study of these individuals also could shed some light on the early transforming events in B-CLL. 

The cell of origin of the clonal B cells in CLL remains elusive (Caligaris-Cappio F, et al. Hematol/Oncol Clin 18:849, 2004). Much evidence points to the B1 lymphocytes, specifically the B1a subset which is CD5+; the B1b subset is CD5-. The B1 designation serves to distinguish these normal B cells from the mantle zone B cells (virgin B cells) that are referred to as B2 cells (conventional B cells). Many features (e.g. immunophenotype, mutational status) indicate that B cell CLL cells are not derived from mutations in the mantle zone B cells. The mantle zone B cells are the cell of origin of mantle cell lymphoma/leukemia.

Prognostic Factors:

Included are stage at diagnosis, cytogenetic profiles, mutational status of the IgV(H) genes, immunophenotypic profiles, and other factors.

Prior to the demonstration of recurrent cytogenetic abnormalities employing the flourescence in situ hybridization (FISH) technology and the ability to determine the mutational status of the IgV(H) genes of the clonal B cells, the only prognostic indicator available was the stage of the disease (disease burden) at diagnosis. Although stage at diagnosis was an important indicator of the subsequent course of the disease, a horizontal look at the pace of the disease in an individual patient was necessary to determine prognosis, and this would not infrequently require a few years to assess.

• Stage at diagnosis (tumor burden) and prognosis:
•  In 1975, Rai KR, et al. (Blood 46:219, 1975) proposed the first staging system for CLL (the immunophenotype was not known at that time.) This initial staging system identified 5 stages based mainly on tumor burden. Dr. Rai later modified this system to identify 3 risk categories (Rai KR (eds). CLL: Recent progress and future directions. vol 59, UCLA symposium on molecular and cellular biology. New York: Alan T Liss; 1987, p 253). In Table 1 is an adaptation from the modified Rai classification system.

Table 1:
Modified Rai Staging System at Diagnosis for B Cell CLL

*The original definition for lymphocytosis was ≥15,000/mm³, but was later decreased to ≥5,000/mm³ if immunophenotyping demonstrated a clonal B cell process with the characteristic B cell CLL profile (CD19+, CD5+, and CD23+.) The diagnosis is now made with less than 5000/mm³ lymphocytes if the cells are clonal and are CD19+, CD5+, and CD23+.
**Anemia is defined as a hemoglobin <11 grams/dl
***Thrombocytopenia is defined as a platelet count <100,000/mm³

The criteria used to document the prescence of lymphadenopathy and hepatosplenomegaly depended on the physical examination. CT and MRI findings were not a part of the staging. The etiology of the anemia and thrombocytopenia was not factored into the formula. An autoimmune mechanism for the anemia or thrombocytopenia does not have the same significance as anemia or thrombocytopenia due to a "crowding out effect" in the bone marrow by the clonal B cells. Cytopenias due to sequestration in an enlarged spleen also was not considered in the staging process.

In the Binet staging system, published in 1981 (Binet JL, et al. Cancer 48:198, 1981), three stages also were identified (Table 2), and depended more on the extent of lymphadenopathy.

Table 2:
Binet Staging System at Diagnosis for B Cell CLL

*Splenomegaly or hepatomegaly is counted as one lymph node site

Cytogenetics:

These findings, as determined by FISH analyses, have evolved as very important prognostic indicators in newly diagnosed cases of B-CLL. In Table 3 are the most frequent cytogenetic findings and their impact on survival.

Table 3:
Incidence and Prognostic Significance of Cytogenetic Findings as Determined by FISH in Chronic B-CLL. Adapted from Dohner H, et al. A Retrospective Study of 325 Cases. N Engl J Med 243:1920, 2000

In this retrospective study, only a single abnormality was found in 54%, 2 in 20%, and >2 in only 8%. In this and subsequent studies the 13q abnormality tended to occur as an early event while others tended to appear later in the course of the disease. A clear survival advantage was observed between those with only a 13q and those with the 17p abnormality. Unlike the prognostic significance of cytogenetics found in acute myeloid leukemia, in which a good cytogenetic abnormality trumps that of a bad cytogenetic finding in the same patient, bad cytogenetics trump the good in B-CLL. If a 13q coexisted with the 17p, the prognosis was similar to those with an isolated 17p abnormality. The same pattern was observed with the 11q abnormality, although to a lesser degree. Normal cytogenetics and the presence of the 12q trisomy were similar as to prognostic significance.

The pathogenetic significance of these various cytogenetic abnormalities remain unclear. Even though the retinoblastoma tumor suppressor gene is located in band 13q, it is probably not the relevant pathogenetic gene. The fact that this mutation is an early event suggests a possible pathogeineic role in the initial transforming event (Stelgenbauer S. Dohner H. Hematol/Oncol Clin N Am 18(4):827, 2004).

The ataxia telangiectasia mutated (ATM) gene is found in the band 11q23 region, but it is mutated in less than 25% of cases.

No relevant mutated gene has been identified in the 12q13 region of patients with trisomy 12.

In the region of the 17p13 deletion is the p53 tumor suppressor gene, but again only a minority demonstrate deletions in the p53 gene. Resistance to therapy with purine analogs and rituximab is associated with the 17p deletion, but the mechanism is unclear.

Mutational status of the IgV(H) genes:

Patients who demonstrate IgV(H) mutations have a significant survival advantage when compared to unmutated cases (Dample RN, et al. Blood 94:1840, 1999 and Hamblin TJ, et al. Blood 94:1848, 1999). In the mutated cases the median survival was >24 years compared to 8-9 years for unmutated cases. ZAP-70 is frequently used as a surrogate marker for mutational status, and even though correlation is not 100%, it serves as an important prognostic study. ZAP-70 also has an independent significance; ZAP-70 positivity in mutated cases confers a poor prognosis as does absence of IgV(H) mutation in ZAP-70 negative cases (Rassenti LZ, et al. N Engl J Med 351:893, 2004). The cut off for ZAP-70, negative versus positive, is <20%. The expression of CD38 (cut off <30% for negative versus positive) also has been used as a surrogate marker for mutational status, but the correlation is not as good as with ZAP-70. The presence or absence of CD38 expression also is an independent prognostic factor. If positive the prognosis is worse whether the clonal cells are mutated or not (Del Poeta G, et al. Blood 98: 2633, 2001).

There are other prognostic factors that are helpful with management decisions:

• Lymphocyte doubling time- If the time for doubling of the peripheral blood lympyhocyte count is <1 year, prognosis is poor. These cases usually have other poor prognostic factors, e.g. bad cytogenetics, unmutated status.
• Short telomere length- not used clinically.
• Transformation to large cell B cell lymphoma (Richter's transformation) or, to a lesser degree, transformation to prolymphocytic B cell leukemia.

Clinical and Laboratory Manifestations and Management

The vast majority of patients with B-CLL at diagnosis are asymptomatic, and those with the isolated 13q deletion remain so for many years, and a significant number, 30% or more, never require therapeutic intervention. Because of this observation together with the fact that treatment of asymptomatic patients has not been shown to prolong survival, and the fact that no therapeutic regimen, with the exception of allogeneic bone marrow transplant (Gribbon JG, et al. Best Prac Res Clin Haematol 20:513, 2007), is curative, the approach to any individual patient should be a very conservative one.

This conclusion does not mean that presently available therapies are not effective in the management of symptomatic patients. The author is firmly convinced that therapy with presently available agents in patients with advanced and symptomatic disease prolongs survival. The author has observed many patients with advanced disease, and some in a near-terminal state, achieve an excellent remission with several different therapies, and survive for many years. The choice of which therapeutic regimen to use should, in general, be based on the ease of administration, and the immediate and long term adverse effects of the therapy used.

Another important factor in deciding on the need for therapeutic intervention is whether the clinical or laboratory manifestations in any individual patient are a direct result of disease progression. If a patient is symptomatic from autoimmune hemolytic anemia, which occurs in up to 25% of cases during the course of the disease, treatment should be directed at the hemolytic anemia not to the leukemia. Other cytopenias such as thrombocytopenia and neutropenia may be on an immune basis, but are much less frequent (<5%) than autoimmune hemolytic anemia (Rozman C, et al. N Engl J Med 333:1052, 1995).

Splenomegaly which may be massive in some patients also results in a variable pancytopenia due to sequestration of peripheral blood elements and hemodilution due to plasma volume expansion, and other manifestations of "big spleen" (Hess CE, et al. Blood 47:629, 1976). Splenectomy in these patients often results in resolution of the cytopenias and related symptoms for long periods of time without the need for other therapeutic interventions.

Recurrent infections involving the upper and lower respiratory tract are probably the most frequent clinical manifestations. The mechanism of this apparant mucosal immunodeficiency state is unclear. There is not a good correlation with the degree of hypogammaglobulinemia, which is also a common feature with an incidence of 50% or more during the course of the disease. Prophylactic IVIg often does not alter the frequencies of these infections. Prompt antibiotic therapy, in the author's experience, not only shortens the duration of symptoms, but also serves to prevent the development of pulmonary disorders such as bronchiectasis.

Transformation to large cell B cell lymphoma (Richter's transformation) which occurs in about 5% of cases should be treated as de novo large cell B cell lymphoma. The large cell lymphomatous component often resolves for long periods leaving only the small B cell CLL component. Transformation to B cell prolymphocytic leukemia occurs in 10% or more, and has less of an impact on survival, but may be a reason to treat (Melo JV, et al. B J Haematol 63:377, 1986). Transformation to acute lymphoblastic leukemia or the development of multiple myeloma is quite rare, occurring in less than 1% of cases. 

Epithelial cancers (e.g. skin, lung, GI tract) occur at an increased rate in patients with chronic B cell CLL (Molica S. Leukemia and Lymphoma 46(1):49, 2005), as does an increased incidence of familial cancers (Linet MS, et al. Am J Epidemiol 130:655, 1989). Patient education to include the avoidance of smoking and sunlight exposure is an important aspect of management. Careful monitoring for the development of these neoplasms is an important aspect of follow-up.

General References:

• Rozman C, Montserrat E. Chronic lymphocytic leukemia. N Engl J Med 333:1052, 1995
• Chiorazzi N, Rai KR, Ferrarini M: Chronic lymphocytic leukemia. N Engl J Med 352:804, 2005
• Müller-Hermelink HK, Montserrat E, Catovsky D, et al. Chronic lymphocytic leukemia/small lymphocytic lymphoma. In Swerdlow SH, Campo E, Harris NL, eds, Who classification of tumours of hematopoietic and lymphoid tissues. Lyon, France, 2008, p180
• Peinert S, Seymour JF. Indolent lymphomas other than follicular and marginal zone lymphomas. Hematol/Oncol Clin North Am 22(5):903, 2008
• Craig FE, Foon KA. Flow cytometric immunophenotyping for hematologic neoplasms. Blood 111:3941, 2008
• Dameshek W. Chronic lymphocytic leukemia- an accumulative disease of immunologically incompetent lymphocytes. Blood  29 (Suppl):566, 1967
• Hamblin TJ, Davis Z, Gardines A, et al. Unmutated IgV(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 94:1848, 1999
• Messmer BT, Messmer D, Allen SL, et al: In vivo measurements document the dynamic cellular kinetics of chronic lymphocytic leukemia B cells. J Clin Invest 115:755, 2005
• Pizzolo G, Chilosi M, Ambrosetti A et al: Immunohistologic study of bone marrow involvement in B cell lymphocytic leukemia. Blood 62:1289, 1993
• Rawston AC, Green MJ, Kuzmicki A, et al. Monoclonal B-lymphocytes with characteristics of "indolent" chronic lymphocytic leukemia are present in 3-5% of adults with normal blood elements. Blood 100:6351, 2002
• Sellick GS, Catovsky D, Houlston RS. Familial chronic lymphocytic leukemia. Semin Oncol 33:195, 2006
• Rawstron AC, Green MJ, Kuznicki A, et al. Monoclonal B-lymphocytes with characteristics of "indolent" chronic lymphocytic leukemia ar present in 3.5% of adults with normal blood counts. Blood 100:6351, 2002
• Rawston AC, Bennett FL, Sheila JM, et al. Monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia. N Engl J Med 35:575, 2008
• Caligaris-Cappio F, Ghia P: The nature and origin of the B-chronic lymphocytic leukemia cell: a tentative model. In chronic lymphocytic leukemia, Ferrarini M, Chiorazzi N (eds). Hematol/Oncol clin 18:849, 2004
• Rai KR, Saqitsky A, Cronkite EP, et al: Clinical staging of chronic lymphocytic leukemia. Blood 46:219, 1975
• Rai KR, Sawitsky A, Jagathambal K, et al. Chronic lymphocytic leukemia. In symposium on hematology and hematologic malignancies, Cassileth, PA (ed) Med Clin North Am 68:697, 1984
• Binet JL, Auguier A, Dighiero G, et al. A new prognostic classification of chronic lymphocytic leukemia derived from a multivariant survival analysis. Cancer 48: 198, 1981
• Dohner H, Stilgenbauer S, Benner A, et al: Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med 243:1910, 2000
• Stilgenbauer S, Dohner H: Molecular genetics and its clinical relevance. In Ferrarini M, Chiorazzi N (eds): Chronic lymphocytic leukemia. Hematol/Oncol Clin N Am. 18(4):827, 2004
• Rassenti LZ, Huynh L, Toy TL, et al. ZAP-70 compared with immunoglobulin gene mutation status as a predictor of disease progression 351:893, 2004
• Dample RN, Wasil T, Fais F, et al. IgV gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 94:1840, 1999
• Del Poeta G, Maurillo L, Venditti A, et al. Clinical significance of CD38 expression in chronic lymphocytic leukemia. Blood 98:2633, 2001
• Gribben JG. Stem-cell transplantation in chronic lymphocytic leukemia. Best Pract Res Clin Haematol 20:513, 2007
• Hess CE, Ayers CR, Sandusky WK, et al. Mechanism of dilutional anemia in massive splenomegaly. Blood 47:629, 1976
• Melo JV, Vatovsky D, Galton DA. The relationships between chronic lymphocytic leukemia and prolymphocytic leukemia. I. Clinical and laboratory features of 300 patients and characterization of an intermediate group. Br J Haematol 63:377, 1986
• Linet MS, VanNatta ML, Brookmeyer R, et al. Familial cancer history and chronic lymphocytic leukemia: a case-control study. Am J Epidemiol 130:655, 1989
• Molica S. Second neoplasms in chronic lymphocytic leukemia: incidence and pathogenesis with emphasis on the role of different therapies. Leukemia and Lymphoma 46(1):49, 2005

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