Usefulness of Differential Scanning Calorimetry for Monitoring Ex Vivo the Changes In Responses of CLL Cells to Anti-Cancer Drugs: Development of Personalized Therapy

Chronic lymphocytic leukemia (CLL) is the most frequent type of leukemia among elderly people in Europe and North America. CLL is considered as a cancer involving mainly deregulated apoptosis. A strong difference between patients in the disease progression, response to anti-cancer therapy, and outcome is a major challenge in elaboration of efficacious treatment. Therefore, it is important to develop personalized therapeutic protocols based on the individual patient's sensitivity to medication. The aim of our ex vivo studies was to monitor the changes in responses of leukemic cells to anti-cancer agent(s) and correlate results obtained by DSC and other supplementary techniques with patients clinical response.

Leukemic cells were obtained from peripheral blood of 14 patients prior to the onset of CC (cladribine + cyclophosphamide) therapy. Primary tumour cell samples were exposed ex vivo to CM or FM, as well as to ROSC alone for 48 hrs. We monitored the induction and progress of apoptosis in CLL cells exposed ex vivo to purine analogs combined with mafosfamide i.e. CM (cladribine + mafosfamide), FM (fludarabine + mafosfamide), and additionally to R-roscovitine (ROSC), an inhibitor of cyclin-dependent kinases, by a few techniques, i.e. cell viability test, apoptosis assay, differential scanning calorimetry (DSC), immunoblotting and determination of caspase-3/7 activity in the culture medium. The characteristic changes in chromatin complex induced upon the above treatments were detected by DSC, a simple thermal technique. This study received approval of the Medical University of Lodz Ethical Committee (RNN/196/07/KE). Informed consent was obtained from all the patients.

Marked differences in the individual patients’ sensitivity to the agents used were registered. A decrease or even loss of transition at 95±5°C in thermal scans of nuclear fraction preparations occurred in the treated cells in which apoptosis was induced. Remarkably, the changes in thermal profiles coincided with an accumulation of apoptotic cells, a decrease of the number of viable cells, and the changes in cellular levels as well as functional status of apoptosis-related proteins (caspase-9, Mcl-1) and CLL prognostic factor – kinase Zap-70. However, no significant changes were observed in thermal profiles of nuclei originating from CLL cells resistant to the treatment. Our studies revealed that the ex vivo exposure of CLL cells to CM combination, equivalent to clinically used CC regimen, was of prognostic value. Among the DSC profiles of nuclear preparations isolated from leukemic cells treated ex vivo with CM 70% of the cases examined indicated the decrease or loss of transition at about 95 ± 5°C. Moreover, we noticed a statistically significant dependence between the patient's response to CC treatment and thermal transition reduction at 95 ± 5°C (p=0.034). Interestingly, the results of the examined group did not show any correlation between disease advance (Rai stage) or cytogenetic abnormalities versus the clinical patient's response to the used treatment.

Our results demonstrate the advantage of DSC in the evaluation of the treatment efficacy and indicate that its application could facilitate the choice of highly effective therapy for individual patients.

No relevant conflicts of interest to declare.