MLL-Aberrations, Associated to T-AML, Are Induced by Chemotherapy In Vivo.

Background: Therapy related acute myelogenous leukemias (t-AML) are frequently observed following previous chemotherapy with alkylating agents and topoisomerase II-inhibitors (topo II-i.). Their incidence ranges between 5 – 15% in non-myeloablative and myeloablative treatment protocols respectively. T-AML after topo II-i. therapy often exhibit balanced MLL/11q23 translocations, e.g. t(9;11) and t(11;19). It is thought that the first step to induce MLL translocations is breakage and illegitimate recombination at the MLL breakpoint cluster region (bcr), which can be detected by genomic MLL-inverse PCR in vitro (Libura et al. 2004, Blood). The aim of this study was to detect the induction of MLL-aberrations in vivo after topo II-i. containing treatment regimens, using RT- and genomic inverse PCR (iPCR) and evaluate this information for early recognition of t-AML.

Material and Methods: Patients with non-Hodgkin’s lymphoma, treated by intermediate or high dose chemotherapy, (MegaCHOEP protocol of the DSNHL) were enrolled. 216 samples of 75 patients were taken as peripheral blood (PB) after informed consent according to the convention of Helsinki. Peripheral blood of healthy adult donors were used as control. Blood samples were subjected either to a nested t(9;11) and t(11;19) RT- PCR or the genomic MLL-iPCR allowing detection of non specific aberrations in the MLL breakpoint cluster region. iPCR was carried out as described (Libura et al.). Processing of samples was carried out according to GLP guidelines for PCR.

Results: 215 samples of 76 patients, taken at different time points of therapy have been investigated. The incidence of the t(9;11) and t(11,19) ranges between 0,5% (1/216) and 9,7% (21/216) of all samples and 1,3% (1/76) and 27,6% (21/76) of all patients. There was a tendency towards a higher incidence of these aberrations in patients which have completed therapy. Pretherapeutic samples were always PCR-negative and none of the patients has developed a t-AML so far. A total of 36 samples (18 healthy subjects, 18 patients) were subjected to MLL-iPCR. Surprisingly, the incidence of MLL-aberrations (deletions, insertions) in the healthy cohort was comparable to that of the patients’ samples (14/18 vs.12/18). We speculated that MLL-aberrations ex vivo might correlate with spontaneous apoptosis. We studied 10 samples of healthy donors at 0, 24 and 48h after having taken the sample. MLL-aberrations were not observed at 0 hrs, but emerged in 5/10 and 7/10 samples after 24 and 48h, respectively.

Conclusion: These results show, that chemotherapy seems to induce the emergence of the t-AML associated MLL-translocations t(9;11) and t(11;19). Since their incidence exceeds the incidence of t-AML, characterized by these translocations, most of the positive PCR-results seem to represent a transient chemotherapy induced genetic instability and not a t-AML in a preleukemic phase. The genomic MLL iPCR is currently used for the detection of chemotherapy-induced MLL-genomic aberrations. Our results indicate, that apoptosis might induce aberrations in the MLL bcr, which need to be discerned from those which are provoked by chemotherapy.