No abstract

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<17.9times103 molecules/cell, MFI of LC Sm Ig<100) or high (i.e. CD20 ABC≥17.9times103 molecules/cell, MFI of LC Sm Ig≥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 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.

The expression of myelomonocytic (My+) associated antigens on lymphocytes from B-chronic lymphocytic leukemia (B-CLL) was studied in 62 patients. Most of them expressed at least a My(+) antigen: CD11b in 40 cases (64.2%), CD13 in 31 (50%), CD14 in 18 (29%), CD33 in 41 (66%), CD36 in 6 (9.6%). The relationship between the clinical features of the disease and My(+) antigen status was studied. No significant correlation was found between Rai's clinical stages and the average percent value of CD11b, CD13, CD33 and CD36. In contrast, patients in Rai's stage 0 had a significant lower value of CD14 positive cells than those with more advanced disease (P < 0.001). Interestingly, patients with a diffuse bone marrow histology had a higher average percent value of CD33-positive cells (76.3% ± 29.3) than those with a non-diffuse one (50.4% ± 37.5). Furthermore, 90% of My(-) patients fulfilled Montserrat's criteria of "smouldering" CLL while only 48.3% of My(+) cases did (P < 002). My(+) lineage antigens on B-CLL cells may provide another criterion to characterize patient subgroups with a poor prognosis.

CD 10-positive lymphoid cells are of interest because they are the most immature recognizable B cells and because they are usually present in acute lymphocytic leukemia (ALL) [l]. Although the presence of CD10-positive lymphoid cells has been considered an early marker of relapse in off-therapy ALL children [2], studies dealing with CDlO antigen intensity in such patients are virtually absent. In fact, fluorescence intensity of CDlO-positive cells is heterogeneous. It has previously been shown that normal CD 10-positive cells display a lower mean CDlO antigen density than do cells from most cases of CDIO-positive ALL [3]. This separation, however, is far from absolute.Ten off-therapy ALL children in continued remission formed the basis of this study aimed at investigating the presence of bone marrow (BM) CDlO-positive cells. There were 4 males and 6 females. Median age at the diagnosis of ALL was 7 years (range: 5-12 years). As far as antileukemic therapy is concerned, all patients were treated according to the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) protocols. At the time of the BM study, all had remained disease-free for 1-12 months (median: 7 months) from the discontinuation of therapy. In each patient, BM aspirates were analyzed by flow cytometry with fluorescein-labeled monoclonal antibodies.The results of BM cell phenotyping were as follows [mean % (range)]: CD3 = 28.9 (16.9-56); CD4 = 12.6 (8.4-11.6); CD8 = 16 (6.8-30); CD2 = 26.3 (5.9-66.6); HLA-DR = 62.9 (44-74.3); CD19 = 49.2 (1 1.4-72.7); CD20 = 43.2 (11-67.6); CDlO (weakly) = 40 (32.1-58.5); CDlO (strongly) = 2.1 (1.5-3.2); CD34 = 9.7 (4.5-16. I).The FACS profile from a representative patient stained for CDlO is shown in Figure 1. The histogram suggests the presence of cell subpopulations with weak and strong fluorescence. The inflection point between the peaks occurred approximately at the value of 100 in a scale of 250 channels of fluorescence intensity. By setting a cutoff at this value we defined two subpopulations of CD1O-positive cells that differ with respect to their fluorescence intensity, with mean specific fluorescence 0 1991 Wiley-Liss, Inc. 119.7 2 12.6 and 86.5 -t 10.9 for cells displaying strong and weak CDlO antigen density, respectively.Our results demonstrate that strongly CD10-positive BM subpopulation, similar to that shown by CD10-positive ALL, can be found in off-therapy ALL children without clinical or hematological evidence of relapse. This feature suggests the immaturity of the strongly CD1O-positive lymphoid cells. Further studies should address the proper role of this observation with regard to the outcome of disease.

Summary. The aberrant expression of the myelomonocytic antigen CD14 was investigated in 128 untreated patients diagnosed with B-cell chronic lymphocytic leukaemia (B-CLL). A cut-off value of 5´10 9 /l CD14-positive cells was chosen for statistical analysis because it showed the best discriminating power among patients with different clinical features. 56 cases had a CD14 cell count >5´10 9 /l. A signi®cant correlation was found between Rai and Binet stages and total tumour mass (TTM) score on one hand, and the absolute CD14 cell cut-off, on the other. This relationship was more evident in Rai 0±II and Binet A±B stages, where a CD14 cell count >5´10 9 /l was preferentially distributed among patients with a higher tumoral mass. In univariate analysis the survival probability at 5 and 10 years showed a signi®cant correlation with Rai and Binet stages, TTM score, CD14 absolute cell count and median age. The median overall survival (OS) was 63 months for patients with a CD14 cell count >5´10 9 /l and 136 months for those with a CD14 cell count < 5´10 9 /l. In the multivariate Cox regression model, Rai stage, age and CD14 cell count were independent signi®cant factors for the prediction of OS. Finally, when the same analysis was restricted to Rai stages 0±II, CD14 cell count was the only signi®cant independent parameter in¯uencing OS, with a relative death risk of 3´8. In conclusion, these data reveal that CD14represents an important marker for predicting OS in B-CLL patients and, therefore, we suggest that it should be included in the immunological characterization of B-CLL.