For Diagnostic Purposes

Classically, the first step used for immunophenotypic identification of leukemic B-cells in a given sample is based on analysis of the restricted expression of surface (sIg) and/or cytoplasmic immunoglobulin (cIg) k or X light chains on B-cells (3,17-19). For many years, this approach has been used for the diagnostic screening of B-cell clonality in several different types of samples, whenever the presence of mature-appearing clonal leukemic B-cells was suspected or had to be ruled out. This is particularly relevant when cytomorphological and histopathological studies could not distinguish between leukemic and normal/reactive B-cells or when they could not establish the lineage and clonal nature of an expanded population of lymphoid cells.

The use of Ig light chain restriction as a marker for B-cell clonality relies on the fact that in different human samples containing normal/reactive polyclonal B-cells, both k+ and X+ B-cells are found at rather stable frequencies of around 1.5 k+ for each X+ cell (3,4,18,19) (Fig. 1A); infiltration by a population of clonal B-cells induces an imbalanced ratio between the number of k+ and X+ cells owing to the restricted use of either k or X Ig light chains by the population of clonal B-cells (Fig. 1B). The greater the proportion of clonal vs normal polyclonal B-cells is in the sample, the higher the probability of detecting an imbalanced k+/X+ ratio. Accordingly, k+/ X+ ratios either lower than 0.5 or higher than 3 indicate the presence of a population of clonal B-cells in the sample (17,19). Usually this strategy does not allow for a specific identification of neoplastic cells on the basis of uniquely aberrant phenotypic features. Even so in practice, such an approach, based on an imbalanced excess of either k+ or X+ B-cells, has proved to be highly valuable for the diagnostic screening of B-CLPD in PB, BM, lymph node, spleen, and other types of samples containing increased numbers of mature lymphocytes (3,4,18,19). In contrast, determination of the ratio between k+ and X+ B-cells has proved to be of limited value for the identification of low numbers of clonal B-cells among a major population of normal B-lymphocytes (indicating minimal disease levels) because of the relatively low sensitivity of this approach: between 10-1 and 10-2 in lymphoid tissue specimens and between 10-2 and 10-3 in PB and BM samples (18,19). Moreover, recent studies (20,21) show that in around 5% of leukemic B-cell CLPD, two or even more clones of neoplastic B-cells showing different and unique IgH gene rearrangements coexist in the same individual (20,21); in these patients, one clone is frequently k+ and the other X+, which may translate into a normal k+/X+ B-cell ratio, even in the presence of substantial tumor burden (Fig. 1C). All together, these observations pointed to the need for more specific immunophenotypic criteria for the identification of leukemic vs normal/reactive

Fig. 1. Representative dot plots of sIg k, sIg x, and CD5 expression on normal (A) and leukemic (B and C) PB B-cells. (B) A case with a monoclonal expansion of CD5+/sIg x+ B-cells. (C) A B-CLPD other than CLL carrying two different B-cell clones. One clone is CD5+/sIg x+ and the other of CD5-/sIg k+. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Fig. 1. Representative dot plots of sIg k, sIg x, and CD5 expression on normal (A) and leukemic (B and C) PB B-cells. (B) A case with a monoclonal expansion of CD5+/sIg x+ B-cells. (C) A B-CLPD other than CLL carrying two different B-cell clones. One clone is CD5+/sIg x+ and the other of CD5-/sIg k+. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin.

B-cells than an imbalanced K+/X+ ratio alone, particularly for cases with low tumor infiltration or two different clones of B-cells.

Following previous observations in acute leukemias (22-24), in recent years several different groups have clearly shown that not only in CLL patients (9,25-27), but also in most CLPD other than CLL, neoplastic B-cells frequently display aberrant phenotypes (25-28). For each disease entity, slight differences have been reported in the incidence of aberrant phenotypes, ranging from 80% in PLL up to virtually all (99%) cases in MCL, HCL, LPL, and SMZL (25). Interestingly, in most of these patients (90%), two or more aberrant phenotypes are detected (25). The combined analysis of aberrant phenotypes and restricted Ig light chain expression significantly increases the specificity and sensitivity of the second parameter, since light chain restriction would be specifically evaluated within the tumor cell compartment (those B-cells that display aberrant phenotypes) (Fig. 1). The sensitivity of this approach is between 10-4 and 10-5 (detection of at least one leukemic cell among 104 to 105) (25). Table 1 summarizes the most frequently observed pheno-typic aberrations in the different diagnostic groups of leukemic B-CLPD other than CLL.

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