Clinicoprognostic implications of increased serum levels of vascular endothelial growth factor and basic fibroblastic growth factor in early B-cell chronic lymphocytic leukaemia.

To assess the relative merit of increased serum levels of vascular endothelial growth factor and basic fibroblastic growth factor in predicting the risk of disease progression of patients with early B-cell chronic lymphocytic leukaemia we analyzed 81 Binet stage A patients whose sera were taken at the time of diagnosis and evaluated for the presence of vascular endothelial growth factor and basic fibroblast growth factor using an enzyme-linked immunosorbent assay. Serum levels of vascular endothelial growth factor positively correlated with Rai sub-stages (P=0.03), peripheral blood lymphocytosis (P=0.03), bone marrow histology (P=0.04) and beta2-microglobulin (beta2-m) (P=0.006). When dealing with basic fibroblast growth factor only a correlation with Rai sub-stages (P=0.02) could be found. Different cut-offs set on the basis of a stratification in quartiles, failed to demonstrate any correlation between serum levels of basic fibroblast growth factor and disease progression. In contrast, patients with increased serum levels of vascular endothelial growth factor (above median value, 203 pg ml(-1)) had a three times increased risk of disease progression, although, in multivariate analysis only Rai sub-stages (P=0.0001) and lymphocyte doubling time (P=0.002) retained their prognostic significance. Low levels of vascular endothelial growth factor were indicative of good clinical outcome in the subgroup of patients with either low (P=0.02) or high (P=0.03) beta2-m concentration. Finally, the highest prognostic power was obtained when serum vascular endothelial growth factor and beta2-m were examined in combination. Median of progression-free survival of patients who had both serum vascular endothelial growth factor and beta2-m higher than median value was only 13 months, in contrast median progression-free survival of patients with one marker increased (i.e. above the 50th percentile) was 40 months. Patients with both markers below the median experienced the best clinical outcome (median progression-free survival not reached at 40 months). In conclusion, serum levels of either vascular endothelial growth factor or basic fibroblast growth factor are high in patients with early chronic lymphocytic leukaemia, however, only vascular endothelial growth factor predicts behaviour of disease and helps to refine the prognosis of stage A patients.

Elevated serum levels of soluble CD44 can identify a subgroup of patients with early B-cell chronic lymphocytic leukemia who are at high risk of disease progression.

BACKGROUND: Although soluble CD44 (sCD44) is considered a reliable marker of both tumor burden and disease activity, to the authors' knowledge, its predictive and prognostic value in B-cell chronic lymphocytic leukemia (CLL) has not been addressed to date. METHODS: The authors studied 94 previously untreated CD5-positive B-cell CLL patients whose sera was taken at the time of diagnosis, stored at - 70 degrees C, and analyzed for the presence of standard sCD44 (sCD44(std)) using a commercial enzyme-linked-immunoadsorbent-assay. The impact of the sCD44 level on the clinical outcome of the disease was assessed in 74 patients with early CLL (61 Binet Stage A patients and 13 asymptomatic Stage B patients). Because the time to disease progression appears to predict the survival time of patients with CLL, it was used as a surrogate endpoint in the current study. RESULTS: Patients with higher than median sCD44 levels (i.e., 642 ng/mL) had a more advanced clinical disease stage (P = 0.04), higher peripheral blood lymphocytosis (P = 0.006), and increased circulating levels of either lactate dehydrogenase (P = 0.01) or beta(2)-microglobulin (P < 0.0001). In univariate analysis, seven of the nine parameters investigated predicted progression-free survival (PFS). In a stepwise multiple regression analysis, only 2 parameters provided independent prognostic information regarding PFS: Rai substages (0 vs. I-II) (P = 0.002) and serum sCD44 levels > 642 ng/mL (P = 0.01). When added to the classification of smoldering CLL versus nonsmoldering CLL, the sCD44 level distinguished two groups within the group of nonsmoldering Stage A patients; patients with a sCD44 level > 642 ng/mL had a median PFS of 36 months, whereas patients with a sCD44 level < 642 ng/mL experienced a longer PFS (median had not been reached at 8 years of follow-up). Furthermore, serum levels of sCD44 defined two different patterns of PFS within the group of patients with Rai disease Stages I-II (P = 0.01). CONCLUSIONS: An increased serum level of sCD44 can be considered to be a promising parameter for predicting the risk of disease progression in patients with early CLL. Furthermore, sCD44 helps to refine the prognostic stratification of patients with either nonsmoldering CLL or Rai Stage I-II disease, thus enabling the identification of different prognostic subgroups in patients with early CLL.

What is changing in the natural history of chronic lymphocytic leukemia?

BACKGROUND AND OBJECTIVES: In the last few years there has been a trend towards an improvement in overall survival of patients with chronic lymphocytic leukemia (CLL). Studies based on tumor registries of the general population or including patients referred to hematologic institutions have analyzed reasons for these changes. However, results need to be validated on independent series. DESIGN AND METHODS: We retrospectively evaluated 518 CLL patients diagnosed at our institution between January 1970 and December 1998. In this cohort of patients we looked at characteristics affecting natural history such as age and sex distribution, stage at diagnosis, survival probability and impact of the disease status on the actuarial survival. Trends in these variables were analyzed after splitting the whole series into three groups according to the period in which the diagnosis was made. Group I consisted of 75 patients diagnosed between 1970 and 1979, group II consisted of 149 patients diagnosed in the period 1980--1989, group III was composed of 293 patients diagnosed between 1991 and 1998. RESULTS: Age and sex distribution did not reflect different periods of diagnosis. The proportion of patients in whom diagnosis was established in low clinical stage (stage A) was higher in the group III (72%) than in groups I or II (26.3% and 50.3%, respectively) (p < 0.0001). Differences in the stage distribution affected life-expectancy which was longer for patients diagnosed in the nineties (median survival, 93 months) than in those diagnosed in the eighties (median survival, 54 months) or in the seventies (median survival, 38 months) (p < 0.0001). Finally, survival analyses by stage showed an improvement of life-expectancy when dealing with patients of high risk category (p =0.005). INTERPRETATION AND CONCLUSIONS: CLL patients diagnosed in the last decade enjoy the best clinical outcome, mostly as a result of a greater proportion of patients in the low-risk clinical stage and a relatively longer survival of the high risk group. It is not clear whether these changes represent true modifications of the natural history of CLL. At the beginning of the third millennium CLL continues to be a fatal disease with a significant impact on life-expectancy.

Surface CD14 positivity in B-cell chronic lymphocytic leukaemia is related to clinical outcome.

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 x 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 x 10(9)/l. A significant 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 x 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 significant 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 x 10(9)/l and 136 months for those with a CD14+ cell count < 5 x 10(9)/l. In the multivariate Cox regression model, Rai stage, age and CD14+ cell count were independent significant factors for the prediction of OS. Finally, when the same analysis was restricted to Rai stages 0-II, CD14+ cell count was the only significant independent parameter influencing OS, with a relative death risk of 3.8. In conclusion, these data reveal that CD14+ represents 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.

Increased serum levels of vascular endothelial growth factor predict risk of progression in early B-cell chronic lymphocytic leukaemia.

The present study is the first to evaluate serum levels of vascular endothelial growth factor (VEGF) in B-cell chronic lymphocytic leukaemia (CLL). All 68 B-cell CLL patients and 31 control subjects analysed had detectable serum levels of VEGF, with no statistically significant difference between two proups. An aberrant increase of circulating levels of VEGF was found in only 17.6% of cases. B-cell CLL patients whose serum VEGF levels were higher than the median (i.e. 194.8 pg/ml) or 75th percentile (i.e. 288.5 pg/ml) values were more frequently at an advanced clinical stage. In contrast, no correlation with other clinico-biological features representative of either tumour mass [bone marrow (BM) histology, peripheral blood (PB) lymphocytosis, beta-2 microglobulin (beta-2m), LDH, interleukin-6 (IL-6)] or disease-progression (DP) [lymphocyte doubling time (LDT)] was found. Serum levels of VEGF predicted the risk of DP in early CLL. Among 41 patients in Binet stage A, progression-free survival (PFS) was significantly shorter in those patients whose VEGF serum concentrations were above the median value. Interestingly, characteristics of stage A patients stratified according to the median value of VEGF were similar with respect to many clinico-biological features, thus suggesting a possible independent prognostic role for such a marker. Finally, when added to the Rai subclassification, VEGF serum levels identified two groups with different PFS within stages I-II. We conclude that increased serum levels of VEGF can be considered useful for predicting the risk of DP and add prognostic information to the Rai subclassification of stage A CLL.

Natural history of early chronic lymphocytic leukemia. A single institution study with emphasis on the impact of disease-progression on overall survival.

BACKGROUND AND OBJECTIVE: Criteria for identifying patients with early chronic lymphocytic leukemia (CLL) who are likely to progress to a more advanced clinical stage rely on results of prospective clinical trials. Is not clear whether these same criteria apply to patients followed-up in the setting of clinical practice. With the aim of addressing this issue we investigated the clinical outcome of a series of patients with Binet stage A CLL. DESIGN AND METHODS: Two hundred and four Binet stage A CLL patients observed at a single institution over an 18-year period form the basis of this study. Different proposals for subclassifying Binet stage A were validated by using our patients as test-set cases. RESULTS: The survival of patients with early CLL (i.e., Binet stage A and Rai stage 0) was significantly different from that of an age- and sex-matched population. Three of 4 different criteria for subclassifying stage A (Rai substaging, Montserrat criteria, French Group proposal), when applied to our patients, gave similar results in terms of sample size, death rate and disease progression (DP) risk. The French Group proposal, based exclusively on blood counts and hemoglobin levels, was not effective in predicting the risk of DP. Forty-nine (23.5%) patients progressed to a more advanced clinical stage (30 to stage B and 19 to stage C); the risk of DP was 32.8% at 5 years and 49.6% at 10 years. When analyzed as a time-dependent variable (Mantel-Byar method), DP had a clear cut-impact on overall survival (p < 0.0001). Finally, outlook for survival of patients who experienced a change of clinical stage was similar to that of patients in that stage at the time of diagnosis. INTERPRETATION AND CONCLUSIONS: As many patients are now being diagnosed while asymptomatic and at a younger age than previously, an accurate evaluation of prognosis is mandatory in early CLL. How prognostic information translates into a policy of early or delayed therapy is still unclear. Our results further support a conservative approach for CLL in early stage; patients who progress into a more advanced stage have similar survival to those in that stage at diagnosis.

Fludarabine treatment in B-cell chronic lymphocytic leukemia: response, toxicity and survival analysis in 47 cases.

BACKGROUND AND OBJECTIVE: Fludarabine monophosphate (FAMP) is a purine analog with specific therapeutic activity in B-cell chronic lymphocytic leukemia (CLL). Its current use as front-line therapy of CLL is still a matter of debate both because of the controversial results of the clinical trials so far reported and because of the toxicity profile of the drug. In order to contribute to clarifying the possible role of FAMP, we report a retrospective analysis of the results obtained with the purine analog in CLL patients in different phases of the disease. DESIGN AND METHODS: Forty-seven patients affected by advanced CLL, 36% untreated, 31.9% relapsed and 31.9% resistant, were treated with FAMP 25 mg/m2/day, either for 4 days every 3 weeks in 29 cases, or for 5 days every 4 weeks in 18. The median number of FAMP cycles was 6 (range 2-11). Response was defined according to total tumor mass (TTM) score reduction and toxicity was expressed according to WHO grading criteria. The median follow-up of the series was 13 months from the beginning of FAMP therapy. RESULTS: Out of 47 evaluable patients the response rate was 74.4%, with 34% complete response (CR). The overall response rate was 94%, 80% and 46.6% in untreated, relapsed and resistant cases, respectively; a significantly higher number of responses was associated with no previous treatment and number of FAMP cycles. Fifty-three percent of all cases and 58.8% of untreated ones did not experience any toxicity. Treatment-related side effects were mainly autoimmune phenomena in untreated patients and infectious complications in treated ones. One heavily pre-treated patient died because of neurologic complications. Median time to re-treatment was18 months (range 1-30) and was influenced by age and previous treatment. The overall median survival was 35.7 months with a significantly higher proportion of surviving cases among RAI 0-II stages, responders and patients receiving more than 5 FAMP cycles. INTERPRETATION AND CONCLUSIONS: The present report confirms the high efficacy of FAMP in previously pre-treated cases with acceptable toxicity and encourages its use as front-line treatment provided that the results of randomized trials demonstrate its superiority over conventional chemotherapy. The possible development of autoimmune phenomena should, however, be considered seriously.

Clinico-biological implications of increased serum levels of interleukin-8 in B-cell chronic lymphocytic leukemia.

BACKGROUND AND OBJECTIVE: Constitutive cellular expression and serum release of biologically active interleukin-8 (IL-8) has been reported in B-cell chronic lymphocytic leukemia (CLL). Given the autocrine role played by IL-8 in the process of cell accumulation characteristic of this disease we tried to investigate clinico-biological implications of increased serum levels of this cytokine in an unselected series of B-cell CLL patients. DESIGN AND METHODS: Serum levels of IL-8 were determined at the time of diagnosis in 58 previously untreated B-CLL patients using an immunoenzyme assay. Results were correlated with main clinico-hematologic features as well as with the risk of disease progression. Finally, we looked for associations between IL-8 and molecules directly involved in apoptosis, such as intracellular bcl-2 and soluble APO-1/Fas. RESULTS: Increased serum levels of IL-8 were found in 15 out of 58 (25.8%) B-cell CLL patients. Serum levels of IL-8 did not reflect clinico-biological features representative of tumor mass such as clinical stage, histopathologic pattern of bone marrow (BM) involvement, b2-microglobulin, sCD23 and sCD27 titers. Interestingly, circulating levels of IL-8 paralleled those of intracellular bcl-2 (r = 0.522; p = 0.01), thus confirming that the antiapoptotic effect of IL-8 can be exerted through a bcl-2 dependent pathway. Levels of IL-8 did not match those of soluble Apo-1/Fas (r = -0.013; p = 0.943). Finally, stage A patients with levels of IL-8 above the median value (i.e. 4.5 pg/mL) were more likely to progress to a more advanced clinical stage than those with levels below the median value (p < 0.05). INTERPRETATION AND CONCLUSIONS: IL-8 is an interesting marker in B-cell CLL, closely involved in the pathogenesis of disease. Furthermore, it is useful for predicting the pace of disease progression in early clinical stages.

Clinico-prognostic implications of simultaneous increased serum levels of soluble CD23 and beta2-microglobulin in B-cell chronic lymphocytic leukemia.

Soluble CD23 (sCD23) and beta-2 microglobulin (beta2-m) are reliable prognostic parameters in B-cell chronic lymphocytic leukemia (CLL); however, their merit over well-established clinical variables such as clinical stages, bone marrow (BM) histology and lymphocyte doubling time (LDT) remains to be defined. Furthermore, information dealing with the impact on overall survival of the simultaneous increase of either beta2-m or sCD23 are lacking. In this prospective study based on 106 B-cell CLL patients, we propose a combination of beta2-m and sCD23 as a strong prognostic system whose statistical significance was mainly due to an excess of deaths in the subgroup displaying increased serum levels of either beta2-m or sCD23. Multivariate survival analysis confirmed the important dominant role of such a finding, thus excluding features with a high degree of codependence (i.e. clinical stages, LDT) and including variables with low association (i.e. BM histology) in the final regression model. The presence of increased serum levels of beta2-m/sCD23 and diffuse BM histology signified high-risk disease, whereas the absence of any adverse variable was associated with prolonged survival; in between there was a subgroup with only 1 characteristic which displayed an intermediate pattern of survival. Finally, on the basis combined increased serum levels of beta2-m and sCD23, a better stratification of low- and intermediate-risk patients could be obtained, thus allowing the formulation of a clinico-biological staging for CLL.

CD27 in B-cell chronic lymphocytic leukemia. Cellular expression, serum release and correlation with other soluble molecules belonging to nerve growth factor receptors (NGFr) superfamily.

BACKGROUND AND OBJECTIVE: CD27, a transmembrane homodimer belonging to the nerve growth factor (NGF) receptor superfamily, is typically expressed on leukemic CD5+ cells in B-cell chronic lymphocytic leukemia (CLL) and found in soluble form in the serum of CLL patients. Therefore, we investigated clinico-biological implications of increased serum levels of sCD27 in an unselected series of B-CLL patients. DESIGN AND METHODS: Serum CD27 (sCD27) levels were determined at the time of diagnosis in 82 previously untreated B-CLL patients using a sandwich enzyme-linked immunoassay (ELISA). Results were correlated with either clinico-hematological or biological features. Finally, quantitative flow cytometric analyses of membrane CD27 (mCD27) expression were carried out on peripheral blood (PB) cells of 22 B-CLL patients and 5 healthy controls, respectively. RESULTS: CD27 was found to be expressed on the surface of both resting normal and leukemic B cells. sCD27 levels were significantly higher in B-CLL patients (median value 2150 U/mL) than in healthy controls (median value 220 U/mL) (p < 0.0001). There was a close relationship between sCD27 and soluble TNF-alpha, another molecule belonging to the NGF receptor superfamily. Changes in sCD27 level correlated with clinical stage, beta 2 microglobulin and LDH. INTERPRETATION AND CONCLUSIONS: These findings indicate that sCD27 is a reliable marker of tumor mass in B-CLL. Its potential prognostic value should be tested in prospective studies.

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.

Increased bcl-2/bax ratio in B-cell chronic lymphocytic leukemia is associated with a progressive pattern of disease.

In order to evaluate clinical implications of altered expression of bcl-2 and bax proteins in B-cell chronic lymphocytic leukemia (CLL) we studied 27 patients with this disease. Cytofluorometric levels of bcl-2 did not reflect the status of disease. In contrast bax expression was lower in progressive than in non-progressive disease, therefore leading to a higher bcl-2/bax ratio in patients of the former group. If confirmed in longitudinal studies, quantitative cytofluorometric evaluation of bcl-2 and bax protein might help to identify patients with progressive disease who could possibly benefit from early therapy.

Clinical and prognostic evaluation of bone marrow infiltration (biopsy versus aspirate) in early chronic lymphocytic leukemia. A single center study.

BACKGROUND AND OBJECTIVE: Recently published studies dealing with chronic lymphocytic leukemia (CLL) patients in early clinical stage reported that bone marrow (BM) biopsies and aspirates can be considered complementary methods of evaluating the extent of BM involvement. Consequently, we designed the present study to investigate the clinical and prognostic implications of BM biopsies and aspirates in a series of stage A CLL patients followed-up in a single center. PATIENTS AND METHODS: BM biopsy sections and aspirate smears obtained at the time of diagnosis from 102 CLL stage A patients were retrospectively evaluated. Results were correlated with clinical and hematological features as well as with survival and disease-progression risk. RESULTS: Diffuse (D) BM histology was detected in 10 patients (9.8%) while 21 (20.5%) displayed lymphocyte infiltration (LI) > 80%. Twenty-six patients (25.4%) died with a 5- and 10-year survival probability of 85% and 50%, respectively. The survival of patients with D-BM histology was significantly shorter than that of patients with non-diffuse (non-D) histology (p < 0.05). Interestingly, when considering only CLL-related deaths (i.e. leukemia progression, infections) were considered, there was an increase in the statistical significance of BM histology (p = 0.01). There was no difference in life expectancy in cases with LI either using different cut-off levels (i.e. 70% and 80%) or excluding non-CLL related deaths. According to our experience, disease progression could only be predicted by BM histology (p = 0.008), while LI was not useful for forecasting progression to more advanced stages (p = NS). INTERPRETATION AND CONCLUSIONS: In patients with early CLL, BM histology provides more reliable information regarding the clinical outcome of the disease than LI.

Clinico-prognostic implications of increased levels of soluble CD54 in the serum of B-cell chronic lymphocytic leukemia patients. Results of a multivariate survival analysis.

BACKGROUND AND OBJECTIVE: Although less specific than sCD23, sCD54 levels have clinico-prognostic relevance in B-cell chronic lymphocytic leukemia (CLL). Since serological markers are now emerging as potentially important in CLL, we tried to verify whether sCD54 might complement clinical stages. METHODS: Serum levels of sCD54 were determined at the time of diagnosis in 115 previously untreated CLL patients. Results were correlated with clinicobiological parameters as well as with survival. RESULTS: Life-expectancy was significantly shorter in patients with higher serum levels of sCD54 (p < 0.001); however, in a Cox's multivariate survival analysis, the only variables which entered the regression model at a significant level were bone marrow (BM) histology (p = 0.03) and lymphocyte doubling time (LDT) (p = 0.04). Interestingly, when LDT was excluded from analysis the only significant variables were clinical stages (p < 0.05) and sCD54 (p < 0.05). These results suggest that sCD54 and LDT give similar prognostic information. INTERPRETATION AND CONCLUSIONS: In CLL, sCD54 is a reliable prognostic parameter whose value is independent of clinical stages. When investigated in relation to clinical outcome, serum levels of sCD54 were able to predict progression to a more advanced clinical stage. On the basis of these data, an integrated clinico-biological classification which separates intermediate risk into two prognostic subgroups is proposed.

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.

Cellular expression and serum circulating levels of CD23 in B-cell chronic lymphocytic leukemia. Implications for prognosis.

BACKGROUND: CD23 is a functionally relevant molecule in B-cell chronic lymphocytic leukemia (CLL) which mediates growth and differentiation signals in B-cells. An intriguing feature of CD23 is its ability to be cleaved from the cell surface and released into the serum. MATERIALS AND METHODS: Serum levels of soluble CD23 (sCD23) were determined with a sandwich enzyme immunoassay at the time of diagnosis in 90 previously untreated CLL patients, in order to evaluate whether they reflected disease activity and tumor load. Results were correlated with those dealing with CD23 expression on leukemic cells to verify whether the cellular counterpart determines serum levels. RESULTS: CD23 was detected on peripheral blood mononuclear cells (PBMC) from 78 out of 90 (86.6%) B-CLL patients, without correlation with clinical stage. Circulating levels of sCD23 in the serum of patients with CLL were highly elevated in comparison to 15 normal controls (p < 0.0005); this increase reflected tumor mass as defined by either clinical stage (p < 0.0005) or bone marrow (BM) histology (p < 0.0005). Neither percentage nor absolute number of CD23+ cells correlated with circulating levels. Interestingly, life expectancy was significantly shorter in patients with higher serum levels of sCD23 (p < 0.0005). When integrated into the Binet clinical staging system, sCD23 led to isolation of two subgroups with different prognosis among intermediate-risk patients. Furthermore, longitudinal studies support the idea that sCD23 can be utilized as an indicator of disease progression. CONCLUSIONS: sCD23 is a highly sensitive and suitable marker with prognostic potential in B-CLL.

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.