IN CHRONIC LYMPHOCYTIC LEUKEMIA THE AMOUNT OF PROLIFERATION CENTERS IN TISSUE-BIOPSIES CORRELATES WITH UNFAVORABLE CYTOGENETIC ABNORMALITIES

Background. In chronic lymphocytic leukemia (CLL) molecular cytogenetic abnormalities may identify specific disease entities and may be associated with distinct prognosis. Currently, peripheral blood is the preferred source to assess the biological risk. However, fluorescence in-situ hybridization (FISH) on paraffin-embedded fixed tissues (PEFT) may also be used to study cytogenetic abnormalities in CLL lymph-node where the “proliferation center” (PC) is the histopatologic hallmark. PCs probably represent the sites where T-dependent immune responses to unknown antigens promote clonal B cell selection and expansion. Aims. i) to analyze the sensitivity and reproducibility of FISH on PEFT, ii) to correlate specific chromosome lesions to PC extension; iii) to estimate the frequency of chromosome lesions on lymph node samples. Method: 183 CLL cases (including 20 Richter syndrome, RS) arranged in 5 tissue macro-arrays (TMAs) were analyzed by FISH for deletions involving 11q23/ATM, 13q14 and 17p13/p53; trisomy 12; and translocations involving the immunoglobulin gene (IgH) on band 14q32. Cytogenetic features were correlated to 2 histologic patterns: 1) PC-rich group including CLL cases with confluent and large PCs and RS cases and 2) typical group with scattered, well-distinct PCs. Results. Assessable data with the complete 5 probe-panel were obtained in 101/183 cases (55,1%); in 58 cases the results were partial and in 24 cases no data were collected for all the probes mainly because of insufficient numbers of cells on TMA samples. Chromosomal aberrations were detected in 79/101 cases (78.2%). The most frequent abnormality was 13q- (36.7% of the cases), followed by 14q32 translocations (30.8%), 11q- (24.7%), +12 (19.5%) and 17p- (15.6%). Ten cases showed 14q32/IgH gene extra-copies (3-5 signals in 10-30% of the nuclei). 17p-, +12 and 14q32/IgH translocations were more frequently encountered in the PCs-rich group (P<.001, .030 and .043 respectively). The PC-rich group was significantly associated to “high-risk”cytogenetic abnormalities (11q- and/or 17p-; P=.001) and to a higher number of abnormalities (≥ 2; P=.001). The difference persists even if RS cases were excluded. Conclusions. FISH on TMAs is a reproducible and a feasible tool for the evaluation of cytogenetic abnormalities in CLL allowing for a decreased both time consumption and experimental variability. The incidence of cytogenetic abnormalities in our series resembles that of historical series with exception of 14q translocation and 17p- possibly due to a higher number of patients with resistant or progressive disease. The higher occurrence of unfavourable cytogenetic features in PC-rich lymph nodes suggest that PCs may be sites where, due to an antigenic stimuli, B cells are induced to proliferate thus conferring genetic instability. Further studies will clarify if cytogenetic abnormalities occurring in lymph-node and bone marrow PCs precede their appearance in the peripheral blood thus supporting the idea that at tissue level PC is crucial for triggering clonal proliferation nourishing the accumulation compartment.