Chronic Lymphocytic Leukemia: Proliferative and Apoptotic Profile on Lymph Node, Studied by Immunohistochemistry Including the Proliferation Centers

Abstract 2538

Chronic lymphocytic leukemia (CLL) has a proliferation rate higher than previously recognized. Proliferation centers (PC) play an important role in the biology of CLL given the fact that they constitute its proliferative compartment. The complexity of the microenvironment of PC, as well as the molecular events taking place in the PC and their clinical significance remain to be elucidated.

The aim was to identify the presence of PC in lymph node and other tissues, except the bone marrow; to analyze their proliferative status; to evaluate the expression of molecules implicated in the apoptotic process in PC, compared to their expression in the non-PC areas; and to correlate the aforementioned findings with the clinical and laboratory features in a series of CLL patients.

Fifty patients, fulfilling the diagnostic criteria of CLL/SLL and in whom lymph node biopsy or other tissue material (44 lymph nodes, 5 spleens, 1 skin) were available, were enrolled in this study. Twenty five biopsies were performed at diagnosis while the rest prior to treatment initiation. All necessary clinical and biological data were recorded on each patient, including Binet stage, lymphocyte doubling time, bone marrow infiltration pattern, CD 38 expression, IgVH mutational status and FISH analysis for genetic abnormalities. PC were defined as pale areas containing large cells so called paraimmunoblasts and prolymphocytes, surrounded by a dark background of small lymphocytes. Immunohistochemical detection of the following molecules participating in the apoptotic process were studied in all tissue sections: Bcl-2, Fas, FasL, c-FLIP (in the PC and the non PC areas) as well as cleaved caspase 3 that was evaluated in the entire tumor area. ZAP-70 was also studied by immunohistochemistry while proliferation status was assessed by the Ki-67 immunostaining.

The median age of our patients at diagnosis was 55 years (36–77). Twenty-nine (58%) had disease stage A, 17(34%) B and 4(8%) C, while 12% had B-symptoms, 26% elevated LDH levels and 25% presented with rapid lymphocyte doubling time. 39% needed treatment immediately after diagnosis.

Proliferations centers were present and easily identified after staining with haematoxylin-eosin on lymph node and splenic sections with their numbers varying from case to case. Proliferation assessment revealed that median Ki67 proliferation index per PC was 10% (1–20%), while median Ki67 in the whole tissue section was 3% (1–8%) (p<0,0001).

Apoptotic molecules evaluation disclosed that the expression of cleaved-caspase 3 was very low (median 0.0046%). Fas, FasL and cFLIP activity was expressed in various percentages in the PC and in the non-PC areas as shown in Table 1. Further on, in cases of overexpression of Fas and FasL, an analogous increase of cFLIP expression was not observed. Patients with elevated Ki67 proliferation index in PC area tended to express more Fas and FasL in the same areas. Strong homogenous Bcl-2 expression was observed both in the PC and in the non PC areas. Weaker Bcl-2 expression in PC compared to non PC areas was observed in 10 patients in whom a higher Fas (p=0.054) and FasL (p=0.005) was present. 48% of the cases were ZAP-70 positive. ZAP70 positivity was correlated with increased expression of cFLIP in PC (p=0.0216). The only statistically significant correlation between apoptotic molecule profile and clinical features was between an increased FasL expression in PC and B-symptoms (p: 0.0263). In univariate survival analysis, coexpression of Fas/FasL and cFLIP at increased levels in PC correlated with poor disease-free and overall survival (p=0.0031, p=0.0521 respectively).

Proliferative centers in lymph node and splenic sections of CLL patients present particular proliferative and apoptotic profile. Inhibition of Fas mediated apoptosis in PC may not be attributable to cFLIP expression. Increased Fas,FasL and cFlip coexpression in PC was correlated with disease-free and overall survival.