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  EARLY B-CLL CLONES: TARGETING THE CCLLA FOR DIAGNOSIS   GB FAGUET   ABSTRACT  
    Chronic Lymphocytic Leukemia (CLL) is a clonal lymphoproliferative disorder of B-lineage in >95% of cases. The clinical diagnosis is based on a sustained lymphocytosis of blood (>5.0 x 10 9 /l) and bone marrow (>30% replaced) with or without infiltration of lymphoid-bearing organs. In cases with blood lymphocytes < 5.0 x 10 9 /l, the marrow criterion is frequently not met. In such cases, the diagnosis hinges on confirming clonality by Ig gene restriction or rearrangement, or more readily by immunophenotype, particularly expression of differentiation antigens CD5, CD19, CD20 and low density monotypic surface immunoglobulins, or of the cCLLa, a non-differentiation CLL-associated antigen. CLL clones in the earliest phase of development exhibit only sustained relative or borderline absolute blood lymphocytosis with no demonstrable marrow lymphocytosis. In such cases, kappa/lambda analysis, coexpression of CLL markers, the clonal excess assay or expression of cCLLa associated with characteristic cytomorphology are sufficient to confirm the diagnosis. The universal expression of cCLLa by CLL clonal cells and its non-expression by normal B- or T-lymphocytes confer a diagnostic advantage to this marker, particularly in patients only partially meeting accepted diagnostic criteria.
  DATA PRESENTATION & DISCUSSION  
    A diagnosis of CLL for individuals presenting with generalized lymphadenopathy and/or splenomegaly associated with an absolute lymphocytosis (ALC) >10 x 10 9 /l is seldom in doubt (1). However, with the advent of automated and inexpensive blood count determinations obtained routinely, increasing numbers of individuals exhibit milder degrees of lymphocytosis without clinical correlates that are of uncertain significance. While in some of these patients reactive T-cells account for the increased ALC (2), a significant patient subset has demonstrable clonal proliferations, mainly CLL (3). Therefore, minimum diagnostic criteria have been developed to ensure diagnostic accuracy and to facilitate comparative assessment of clinical trial results. Two major CLL working groups have complementary criteria: the International Workshop on CLL (IW-CLL) requires an ALC >10 x 10 9 /l of predominantly mature-looking cells, and either >30% replacement of marrow cellularity by these cells, or demonstration of clonality by immunophenotype (4). According to the National Cancer Institute-sponsored CLL Working Group (NCI-CLL), an ALC >5.0 x 10 9 /l is acceptable provided the IW-CLL marrow and clonality criteria are both met (5). However, because degree of marrow lymphocytosis parallels extent of disease, the diagnostic marrow criterion is seldom met in patients with blood lymphocytes <5.0 x 10 9 /l (3) and demonstration of clonality is imperative (3). Clonality can be confirmed by demonstrating Ig gene restriction or rearrangement (6), or by immunophenotype (7). The latter has become the de facto preferred method due to its reliability and wide availability. This test is predicated on detecting surface markers following patterns of distribution characteristic for the various lymphoproliferative disorders. In CLL, markers of diagnostic value include: CD5, CD19, CD20, CD23 and low density monotypic surface immunoglobulin (MosIg) heavy or light chains. As the size of the clone progresses, so does the percentage of circulating cells expressing these markers. This is exemplified in Figure 1 where expression of CD19, MosIg and cCLLa by blood lymphocytes from 121 CLL patients are plotted as a function of each patient’s ALC. As shown, in CLL patients with advanced stages (reflected in ALC >30 x 10 9 /l), the fraction of cells expressing CLL markers can reach 99% of the ALC greatly facilitating the immunophenotypic diagnosis. However, at the other end of the spectrum, clonal dilution by remnant normal B- and T-lymphocytes in patients with early disease results in clones representing a few percentages (<10% in the example) of the ALC. Clonal dilution can be extreme in patients with emerging CLL clones or following chemotherapy-induced hematologic remissions (8) in whom the clone size often approaches the sensitivity limit of routine immunophenotyping assays (9). In such cases, several assay modifications designed to quantify the clonal excess or detection of the novel, differentiation-independent CLL antigen (cCLLa) provide powerful diagnostic tools (3,10). The figure also demonstrates that the three markers shown are co-expressed by clonal cells of each individual patient (intra-marker correlation r >0.994).
        In this report, the immunophenotypes of blood lymphocytes in patients exhibiting different stages of CLL (from emerging clones (3) to advanced stages (11)) will be presented to illustrate the value and limitations of each approach (Table 1). As shown in the table, disease progression is associated with increasing blood and marrow lymphocytosis (from 20 x 10 9 /l and 43.3% in low-risk (11) cases to 54.3 x 10 9 /l and 82.7% in high-risk (11) patients, respectively). Given the fact that disease progression is due to the accumulation of clonal cells, it is not surprising that the fraction of these cells relative to total lymphocytes in blood and marrow rose from 62% and 64.7% in low-risk patients to 79% and 85.4% in high risk cases. Under these circumstances the IW- and NCI-CLL diagnostic criteria (ALC, monoclonal phenotype and marrow lymphocytosis) are easily met. However, patients with emerging CLL (3) (“Emerging” column on the table) whose ALC and marrow lymphocytosis average 6.0 x 10 9 /l and 13.9%, respectively fail to meet the >30% marrow lymphocytosis criterion. Progression of the B cell clone to overt B-CLL occurred in 8 out of 25 of the emerging patients (3). Of the nine patients in the low risk patient group (Rai stage 0), three showed progressive disease. Our data (3) have shown that these patients’ clinical, hematologic and disease progression pattern profiles are indistinguishable from that of patients with overt CLL, except for the size of their neoplastic clone. Thus, the advent of clonal cell recognition via surface marker detection by flow cytometry has considerably reduced the importance of the quantitative marrow diagnostic criterion. This technique, particularly the kappa/lambda analysis, coexpression of CLL markers and the clonal excess assay, is helpful for diagnosing emerging CLL clones and for detecting remnant CLL cells in blood or marrows of patients exhibiting complete hematologic remissions. However, because these tests target differentiation antigens also shared by normal lymphocytes diagnostic uncertainty occurs as the clonal size approaches the sensitivity limit of the assays (9). In contrast, our data have shown that targeting the differentiation-independent, disease-restricted cCLLa greatly enhanced the discriminating power of phenotypic assays for detecting emerging B-CLL clones at pre-clinical stages of development (3) and suggest that cCLLa should be included in epidemiological studies of B-cell proliferative disorders.
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