Clinico-prognostic relevance of quantitative immunophenotyping in B-cell chronic lymphocytic leukemia with emphasis on the expression of CD20 antigen and surface immunoglobulins.

Expression of CD20, evaluated as antibody binding capacity (ABC) (i.e. absolute number of molecules of antibody per cell), was analyzed using flow cytometry on leukemic cells of 93 previously untreated patients, all fulfilling strict criteria of "immunologically typical" (i.e. CD5+, CD23+) B-cell chronic lymphocytic leukemia (CLL). Although changes of CD20 antigen density did not correlate with clinical parameters representative of either tumor mass (i.e. clinical stage, histological pattern of bone marrow involvement, absolute peripheral blood lymphocytosis) or disease progression (i.e. lymphocyte doubling time), a trend toward a better life-expectancy was observed in the low CD20 expression group compared with the high CD20 expression group (p = 0.05; relative risk of death, 0.51, 95% confidence interval, 0.24-1.04). Given the correlation between CD20 ABC and mean fluorescence intensity (MFI) of light chain (LC) surface immunoglobulins (Sm Ig) (r = 0.481, p < 0.0001), as well as the impact of MFI of Sm Ig LC on overall survival (p = 0.01; relative risk of death 0.44; 95% confidence interval, 0.10 to 0.76), we tried to verify whether a combination of B-cell markers, evaluated in a quantitative manner, could have additive prognostic properties. To this purpose we gave a value of 1 or 0 to each B-cell marker according to whether it was expressed at a low (i.e. CD20 ABC < 17.9 x 10(3) molecules/cell, MFI of LC Sm Ig < 100) or high (i.e. CD20 ABC > or = 17.9 x 10(3) molecules/cell, MFI of LC Sm Ig > or = 100) level thus allowing patient stratification into two groups with scores of 2 and 0-1, respectively. Survival of patients who scored 2 was significantly longer respectively. Survival of patients who scored 2 was significantly longer than that of patients who scored 0-1 (p = 0.02; relative risk of death, 0.44; 95% confidence interval, 0.22-0.72). However, when quantitative changes of CD20 antigen and LC Sm Ig expression, either alone or in combination, were simultaneously analyzed in a Cox model which included usual clinico-hematological features, only absolute peripheral blood lymphocytosis (p = 0.0001) and Binet clinical stages (p = 0.0001) maintained their prognostic power unmodified. Although variability of CD20 and Sm Ig expression make it possible to appreciate biological heterogeneity of B-cell CLL better, however, they cannot substitute well-established clinico-hematological features in the prognostic assessment of B-CLL patients.

Comparative flow cytometric evaluation of bcl-2 oncoprotein in CD5+ and CD5- B-cell lymphoid chronic leukemias.

BACKGROUND AND OBJECTIVES: Levels of intracellular bcl-2 oncoprotein have been found to be increased in leukemic cells of CD5+ B-chronic lymphocytic leukemia (CLL) patients. However, it is not clear whether bcl-2 overexpression is a peculiar feature of CD5+ B-CLL. Based on this background we carried out a quantitative flow cytometric evaluation of intracellular bcl-2 levels on leukemic cells of CD5+ and CD5- B-CLL. METHODS: We assessed in flow cytometry levels of bcl-2 protein using a quantitative indirect immunofluorescence assay (QIFI kit) on samples from 46 previously untreated CD5+ B-CLL patients. Results were compared with those obtained on either normal peripheral blood B-lymphocytes or leukemic cells from 7 CD5- B-CLL patients intentionally selected for statistical comparison. RESULTS: A relatively homogeneous amount of bcl-2 protein which did not reflect either clinical-biological features at the time of diagnosis nor in vivo response to therapy was found. Results expressed as antibody binding capacity (ABC) accounted for a mean value of 12.2 +/- 1.5 x 10(3) molecules/cell (range, 6.4-13 x 10(3) molecules/cell). Levels of bcl-2 detected on CD5+ B-CLL leukemic cells were significantly lower than those of B peripheral blood lymphocytes from healthy donors (p = 0.0001). The same applied when comparing CD5+ and CD5- B-CLL patients (bcl-2 ABC, 8.07 +/- 0.26 x 10(3) molecules/cell vs. 12.2 +/- 1.5 x 10(9) molecules/cell; p = 0.0001). INTERPRETATION AND CONCLUSIONS: According to the role of bcl-2 in preventing apoptosis, our results indicate that differences in the pattern of expression of such an oncoprotein, might, at least in part, explain the more aggressive clinical course of CD5- B-CLL forms.

Differential expression of BCL-2 oncoprotein and Fas antigen on normal peripheral blood and leukemic bone marrow cells. A flow cytometric analysis.

BACKGROUND: Fas antigen (Ag) has recently been identified as the putative surface molecule capable of transducing apoptotic signals into cells. Alterations in the expression of proto-oncogene bcl-2 have been implicated in the regulation of apoptosis. MATERIALS AND METHODS: By employing a monoclonal antibody to bcl-2 protein (124 clone) and to Fas Ag (UB2 clone) the expression of these molecules was analyzed at flow cytometry on bone marrow (BM) and peripheral blood (PB) samples from patients suffering from different lymphoid and myeloid leukemic diseases (27 acute non-lymphocytic leukemia [ANLL]; 14 acute lymphocytic leukemia [ALL]; 19 B-cell chronic lymphocytic leukemia [CLL]; 2 Ph1+ chronic myeloid leukemia [CML]; one CD8+ T-cell chronic lymphoproliferative disorders). Results were compared with those observed on normal PB leukocytes and BM B-cell precursors from patients with non-neoplastic hematological disorders. RESULTS: Fas Ag was constitutively expressed by both monocytes and neutrophils, while lymphocytes expressed bcl-2 with no difference between B and T cell subsets. Interestingly, bcl-2 expression was always absent on neutrophils. When dealing with ANLL patients, a relatively low bcl-2 and high Fas Ag phenotype characterized subtypes with granulocytic (M2) or promyelocytic (M3) differentiation. This observation was confirmed in a small number of patients for whom bcl-2 levels were quantified as antibody binding capacity (ABC) in molecules/cell. Leukemic cells from patients with ALL constitutively expressed bcl-2, the pattern of this expression being quantitatively lower than that of immature B-cell precursors. Finally, high bcl-2 and low Fas Ag expression represented a crucial part of the B-cell CLL immunophenotype. CONCLUSIONS: Although based on a small number of patient and control samples, our results suggest that bcl-2 and Fas Ag are coordinately expressed on normal PB leukocytes. Fas Ag is expressed at low levels on B-CLL cells, generally considered long-surviving cells. The relatively lower bcl-2-expression detected in both M2 and M3 subtypes may explain, at least in part, the higher remission rates obtained in these forms of ANLL than in other less differentiated morphological variants.

Intercellular adhesion molecules (ICAMs) 2 and 3 are frequently expressed in B cell chronic lymphocytic leukemia.

Using different monoclonal antibodies (moAbs) from the 5th International Workshop on Leukocyte Differentiation Antigens we studied the expression of intercellular adhesion molecules (ICAMs) 2 and 3 on a homogeneous group of 23 B cell chronic lymphocytic leukemia (CLL) patients. Our results show that either ICAM-2 or ICAM-3 are constitutively expressed on CD5+ B-CLL cells. Owing to the role of ICAM molecules in governing the migration and traffic of lymphocytes to lymph nodes, our findings need to be validated in a more consistent patient series to understand clinico-prognostic implications of such an expression.

Expression on leukemic cells and serum circulating levels of intercellular adhesion molecule-1 (ICAM-1) in B-cell chronic lymphocytic leukemia: implications for prognosis.

Expression of intercellular adhesion molecule-1 (ICAM-1) has been correlated clinical and laboratory characteristics of 62 patients with untreated CD5+ chronic lymphocytic leukemia (CLL). ICAM-1 was detected on B-CLL cells from 19 out of 62 patients (30.6%) and its expression did not correlate with the majority of immunological markers. An important finding of this study was the association between ICAM-1 expression and mean fluorescence intensity (MFI) of Slg (r = 0.507; P < 0.001). As far as correlation with clinical parameters is concerned, a statistically significant association between Binet clinical stages and ICAM-1 expression was found (P < 0.05). Furthermore, an atypical CLL morphology was more frequently encountered among patients who expressed ICAM-1 (P < 0.005). To obtain more information on the role of ICAM-1 in CLL we measured serum levels with a sandwich enzyme immunoassay. In 47 B-cell CLL patients studied, the mean value of circulating ICAM-1 levels was significantly higher (561 +/- 201 ng/ml) than that observed in 25 normal controls (353 +/- 162 ng/ml; P < 0.005); a clear correlation being found with Binet clinical stages (P = 0.026) and bone marrow (BM) histology (P < 0.005). We conclude that circulating ICAM-1 is elevated in CLL and such an increase reflects tumor mass as defined by clinical stages and BM histology, rather than peripheral blood lymphocytosis (r = 0.240). Even if soluble ICAM-1 appears to be less specifically increased that soluble CD23 serum levels, these circulating molecules were not completely independent of each other (r = 0.512; P < 0.001).

CD11c expression in B-cell chronic lymphocytic leukemia. A comparison of results obtained with different monoclonal antibodies.

In order to obtain more information on the pattern of CD11c-positivity in otherwise typical B-cell chronic lymphocytic leukemia (CLL), we analyzed immunological and clinico-pathological features of 99 such patients studied with two different monoclonal antibodies (MoAbs). Fifty-two out of 70 (74.2%) patients stained with IOM-11C MoAb and 3 out of 29 (10.3%) patients stained with Leu-M5 MoAb had more than 30% CD11c positive cells (P < 0.0002). The two groups were similar with regard to the expression of B-cell CLL-related antigens (CD5, CD20, CD23), as well as clinico-pathological features (i.e. Binet clinical stage and pattern of bone marrow involvement), thus suggesting that differences in CD11c expression were due to different reactivity patterns of the MoAbs utilized. In our experience, the use of different reagents may affect immunophenotyping results, thus providing conflicting data at times.