Relationship between in vitro drug sensitivity and clinical response of patients to treatment in chronic lymphocytic leukemia
- Authors:
- Published online on: January 8, 2015 https://doi.org/10.3892/ijo.2015.2823
- Pages: 1259-1267
Abstract
Introduction
Chronic lymphocytic leukemia (CLL) belongs to the group of hematological neoplasms with unknown etiology. Great progress in cancer diagnostics and treatment has been made, but this type of leukemia remains incurable. The simultaneous coexistence of two populations of quiescent and cycling cells in CLL treatment represents a special challenge (1–3).
It is accepted that an accumulation of genetic aberrations or epigenetic modifications in neoplastic lymphocytes could be related to heterogeneity in the clinical course of CLL and transferred on response to therapy. Recently, a large number of studies have been focused on the identification and evaluation of factors which have an impact on treatment and reflect prognostic value, e.g., genomic aberrations, alterations in miRNA level, and epigenetic modifications (4–6). Differences in the expression of factors regulating apoptosis and cell signaling, as well as the microenvironment of malignant cells, may also be responsible for the heterogeneity of leukemic cells and their response to therapy (7–9).
At present, there is an increasing number of treatment options, and a large number of agents with anticancer potential are undergoing preclinical and clinical studies (10–12). Among the new therapies for CLL, much attention is paid to agents with the potential to turn on apoptosis (13). In light of the new therapeutic options (e.g., immunochemotherapy, immunomodulators, kinase inhibitors), and disease heterogeneity, one of the most pressing issues is an elaboration of effective methods for determining the individual sensitivity of CLL patients to potential therapeutic(s), and the selection of optimal treatment for each patient (14,15).
The special importance of such approaches has been confirmed by the data showing a profound immunological defect reflected by hypogammaglobulinemia and elevated vulnerability to patient infections associated with fludarabine and cyclophosphamide administration (16). Therefore, personalized therapy needs to be particularly addressed towards ‘refractory’ CLL patients (17).
The results of two single cases given in our earlier report (18), together with the combined results of 28 other cases presented in the current report, confirm an association between the results of in vitro testing (cytotoxicity and pro-apoptotic ability) of the tested drugs and the subsequent clinical response to the applied treatment.
Differential scanning calorimetry (DSC) is a relatively fast thermal technique that provides data on physical and energetic properties of cellular structures/compounds (19–21). Earlier, it was documented that this method could be useful for the monitoring of native chromatin in cell nuclei (19).
It has been reported by our laboratory that an additional thermal transition occurs at 95±5°C in the DSC profiles of nuclear fraction preparations obtained from the mononuclear cells of patients in an advanced stage of CLL, along with the main transition at 83±3°C characteristic for healthy donors (22,23). Moreover, the obtained results also revealed that the decrease (or even loss) of thermal transition at 95±5°C in thermal scans of nuclear preparations of CLL cells, after both in vivo and in vitro drug administration, are attributable to chromatin fragmentation during apoptosis (23,24).
In the current study, the comparative cytometric analysis of cell viability, apoptosis rate, DSC profiles of nuclear preparations, and PARP-1 expression by western blotting in the group of 28 patients were applied to examine the ability of leukemic cells to enter apoptosis after their exposure to cladribine or fludarabine combined with mafosfamide.
As described, a significant decrease or even complete loss of thermal transition at 95±3°C was observed in DSC scans of nuclear preparations when therapy was effective (22,24). Moreover, our results reveal that a comparison of the DSC profiles of nuclear preparations with the results of cell viability, apoptosis rate, and proteolytic degradation of the PARP-1 display a good predictive value for CLL cell sensitivity to anti-cancer drugs. This prediction is of importance as it allows an opportunity to choose the optimal therapy for patient avoiding ineffective anticancer therapy.
Materials and methods
Ethics statement
The study was approved by the Local Ethics Committee of the Medical University of Lodz (Lodz, Poland) (no. RNN/143/10/KE); all patients signed a declaration of consent.
Patients, response criteria
In the current study the peripheral blood samples from 28 randomized, untreated previously progressive CLL patients (16 men, 12 women) with white blood cell counts ranging from 45 to 600×109/l were included (Table I). The immunophenotypic characteristics of leukemic cells (CD5+/CD19+/CD23+, presence on cell surface immunoglobin κ or λ chains) were determined cytometrically. The diagnosis of CLL and clinical staging determination were established according to standard clinical, immunological and cytological IWCLL criteria (25). The group included eligible patients who underwent randomization (26).
 | Table IPatients’ characteristic features, comparative results of in vivo and in vitro response to treatment. |
Blood samples from the patients were collected before administration with cladribine + cyclophosphamide (CC), or fludarabine + cyclophosphamide (FC). The choice of therapeutic schedules was made on accepted ECOG standards and as a result of prognostic factor analysis. Drug combination was applied the next day. Clinical response to the treatment after six cycles of drug administration was evaluated by NCI-sponsored Working Group criteria (25). Complete response (CR), partial response (PR) or non-responder (NR) criteria have been explained before (24,26).
Isolation of CLL cells
Peripheral blood mononuclear cell (PBMC) samples of CLL patients were collected on EDTA. Mononuclear cells from blood samples were separated using Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA) according to manufacturer’s instructions. The CLL cell pellets were washed with phosphate-buffered saline (PBS), resuspended in RPMI medium and divided for the planned experiments.
Fluorescence in situ hybridization (FISH)
FISH analysis was performed on the interphase nuclei of leukemic cells from the blood of CLL patients before the onset of treatment (27). The following commercially available probes were used: LSI D13S319 (13q14.3)/LSI 13q34/CEP 12 probe, LSI p53 (17p13.1), LSI ATM (11q22.3) probe (Vysis; Abbott Laboratories, Abbott Park, IL, USA). The estimated cut-off levels were as follows: 8% for del(13)(q14.3), del(11)(q22.3) and del(17)(p13.1), and 5% for trisomy 12. Signals were counted in 200 interphase nuclei for each sample.
In vitro treatment
PBMC samples from patients were resuspended at a final density of 2.5–3.5×106 cells/ml in RPMI-1640 medium with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and incubated with cladribine or fludarabine with an active form of cyclophosphamide-mafosfamide for 48 h or without any agents [controls (Ctr)] at concentrations described previously (28,29). Control leukemic cells, as well as the CLL cells were exposed to anticancer agents for 48 h at 37°C in an atmosphere of 5% CO2.
Cladribine (Biodrybin) was from the Bioton S.A./Institute of Biotechnology and Antibiotics (Warsaw, Poland), fludarabine from Bayer Schering Pharma AG (Berlin, Germany). The alkylating agent mafosfamide was donated by Baxter Oncology GmbH (Frankfurt, Germany) or purchased from Niomech IIT GmbH (Bielefeld, Germany).
Cell viability and determination of apoptotic cell number
PBMC samples were incubated in culture medium only (Ctr) or in drug supplementation as indicated. The level of viable, as well as apoptotic cells after 48 h of drug treatment, was assessed by flow cytometry using Vybrant Apoptosis Assay kit no. 4 (Molecular Probes, Inc., Eugene, OR, USA). PBMC samples were analysed on LSR II Becton-Dickinson cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA).
Preparation of nuclear fraction and whole cell lysates
Both, the pelleted control and drug-treated CLL cell samples were rinsed with cold PBS and then suspended in isotonic sucrose solution containing 5 mM MgCl2, 0.5% Triton X-100, 50 mM Tris-HCl (pH 7.4) and protease inhibitors as previously described (22). Cell samples were homogenized in a Potter homogenizer, and centrifuged at 800 × g for 8 min resulting in a crude nuclear pellet.
PBMC samples were lysed in buffer containing 10 mM Tris-HCl (pH 7.5), 300 mM NaCl, 1% Triton X-100, 2 mM MgCl2, 0.1 M dithiothreitol and protease inhibitor cocktail (22).
Protein electrophoresis and immunoblotting
Protein concentration was determined colorimetrically (30). Protein samples (40 μg) were separated on 8.0% SDS-polyacrylamide gels and blotted onto Immobilon-P membrane. Equal protein loading and protein transfer were confirmed by Ponceau S staining. To avoid non-specific protein binding sites membranes were saturated with 5% non-fat dry milk in TBS (10 mM Tris-HCl, pH 7.5, 150 mM NaCl) for at least 1 h at room temperature. After extensive washing in TBS containing 0.05% Tween-20 (TBST) blots were incubated with primary antibodies specific to PARP-1 (sc-7150, 1:2,000), and actin (sc-7210, 1:1,000) from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA. The immune complexes were detected using alkaline phosphatase (AP)-coupled secondary antibodies as previously described (28).
DSC
Samples of the nuclear fraction isolated from PBMCs incubated with or without anticancer agents were prepared for calorimetric tests by the Almagor and Cole procedure (21,22). The probes were transferred into sample pans and hermetically sealed. Calorimetric experiments were performed on a Setaram TG-DSC 111 calorimeter (Setaram Instrumentation, Caluire, France) from 20 to 120°C at a scanning rate R (5°C/min, 0.083°C/sec) as reported previously (24).
Monitoring of apoptosis and necrosis
The appearance of apoptotic and necrotic cells was monitored under a fluorescence microscope (magnification, ×400, Olympus IX70; Olympus, Tokyo, Japan). The cells after drug exposure were washed and suspended in PBS at the concentration 1×106 cells/ml. Cell suspensions were incubated with YO-PRO-1 and propidium iodide, and transferred onto microscopic slides for examination. The fluorescent dyes vary in characteristics and ability to penetrate cells. YO-PRO-1 passes through the plasma membrane of apoptotic cells and labels them selectively with green fluorescence, while the red-fluorescent propidium iodide is permeant only to necrotic cells. Stained cells were classified on the basis of their morphological and staining characteristics as early, late apoptotic or necrotic (31).
Data analysis
Descriptive statistic analysis was used to summarise patients’ clinical, laboratory and in vitro cladribine + mafosfamide (CM)/fludarabine + mafosfamide (FM) treatment data. The changes in cell viability, apoptosis rate, thermal transition at 95±5°C or PARP-1 proteolytic cleavage in respect to the clinical response of patients were compared using Fisher’s exact test with Bonferroni correction. The details of the statistical analyses are included in the text and Fig. 1C.
Results
The results of in vitro leukemic cell exposure to anticancer agents indicate their susceptibility to anticancer drugs
Leukemic PBMCs obtained from blood of 28 randomized patients scheduled for the first chemotherapy cycle were incubated for 48 h with cladribine or fludarabine combined with mafosfamide (CM or FM). The clinical characteristic of CLL patients, type of treatment and response in vivo and in vitro are presented in Table I.
Exposure of PBMC samples to both anticancer drug combinations reflects the differences in cell viability, as well as apoptosis rate. Comparative analysis of the above studies showed a different personal sensitivity and response of examined cells to the used drug combinations (Figs. 1 and 2). Moreover, the exposure of CLL cells to both investigated combinations usually decreased the viable cells number, which was associated with an elevation in apoptotic rate, but to a different extent. Among the investigated patient samples, we did not obtain the same results for a single patient. In leukemic cells isolated from blood of some patients, apoptosis at high rate was rapidly induced, whereas in other cases it was delayed and much weaker.
The comparison of the combined average results is illustrated in Fig. 1A and B. The diagrams in Fig. 2A and B show the combined data of in vitro tests for CLL patients nos. 14 and 18, respectively. Patient no. 18 did not respond (NR) to CC therapy applied in the clinic, but the results of in vitro tests suggest that for this patient, FC would display a chance for better response. The results for patient no. 14, who achieved a CR in vivo, also in vitro tests show that this type of treatment will be more profitable for this patient in comparison to FM/FC. The results of in vitro tests demonstrate leukemic patients’ personal predispositions that could be transferred to treatment efficacy in vivo (compare Figs. 1 and 2).
Moreover, in the group of the studied blood samples from CLL patients with sensitive and reactive leukemic cells, the response to the treatment in vivo as well as to in vitro conditions usually occurred. The second group showed a weak response to the drugs used, which could be correlated with the unsatisfactory response or even resistance of patients to therapy in vivo. A third group of CLL cells displays a high sensitivity to in vitro condition, which was concomitant with high reduction of cell viability in control untreated cells incubated for 48 h, as well as PARP-1 cleavage in control untreated cells. For these cases, the additional analysis using supplementary techniques are suggested to demonstrate the course of apoptosis process.
Thermal transition at 95±5°C is characteristic for nuclear samples from advanced stage of CLL patients
A study from our laboratory has shown that in advanced stages of CLL, the thermal profile of nuclei with an additional thermal transition at 95±3°C is present in ~74% of cases in advanced/aggressive disease (22). In the results presented here, all control nuclear preparations of leukemic cells indicated thermal transition at ~95°C. Moreover, in the majority of nuclear samples isolated from PBMCs of advanced leukemia patients qualified to drug administration (advanced stage of disease), this thermal transition was usually dominant. Interestingly, the changes in thermal profiles of nuclear preparations from leukemic PBMCs exposed to studied drug combinations were correlated with viable cell reduction, reflecting the potential efficacy of treatment.
To show that results of complementary tests closely adhere to DSC profiles, we selected for presentation two exemplary cases with different therapeutic outcomes: one illustrating a positive response to CM and potential resistance to FM (patient no. 14; Table I, Figs. 1 and 2A), and the second one reflecting a weak sensitivity to CM in comparison to FM (patient no. 18; Table I, Figs. 1 and 2B).
The thermal transition of exemplary nuclear fraction (patient no. 14) at 95±5°C was reduced after leukemic cell exposure to drugs for 48 h; the cells of this patient were characterized by a higher apoptosis rate in respect to control cells (Fig. 2AI and II). No decrease of transition at 95±5°C was observed in the cells treated with CM (patient no. 18) that could be a reason of chromatin hyper-condensation during PBMC cell exposure to drugs (Fig. 2BIII). While the changes in thermal profiles directed towards decrease of thermal transition at 95±5°C seem to be attributable to a degradation of chromatin during apoptotic DNA fragmentation.
The comparative analyses of nuclear fraction DSC scans with cell viability and apoptosis marker PARP-1 expression reflect leukemic cell sensitivity to anticancer drugs
Using the complementary in vitro tests, we assessed some parameters that simultaneously performed could predict a potent efficacy of anticancer drugs in leukemic PBMCs. It must be emphasized that the treatment response strongly varied between individual patients (Fig. 1A and B). Interestingly, for all studied 28 patients, the changes in thermal profiles at 95±5°C of nuclear samples obtained from PBMCs exposed to anticancer drugs correlated with the reduction of viable cell percentage and elevation of apoptotic cell fraction accompanied by the proteolysis in apoptotic marker PARP-1 (Fig. 2). In the group of examined 28 patients, 8 of them display a positive reaction (CR) in vivo and strong reaction (SR) in vitro, while negative responses were observed for 5 patients (3 of them in vivo and in vitro). Similarly, in vivo and in vitro correlations were seen for 4 patients reflecting PR. It must be stated that sometimes patients did not respond the same in vitro and in vivo. For five cases, there was an SR in vitro followed by a PR in vivo, and for six others the reaction to drug administration in vivo (CR) was stronger than the reaction in vitro (MR) (Table I).
It must be underlined that we did not receive the same results of four complementary tests for two individuals among 28 studied CLL patients. From the obtained results we have selected two exemplary CLL patients, i.e., one responding to CM and resistant to FM (no. 14), and the second one (no. 18) displaying a weak response to CM treatment. As demonstrated in Figs. 1A and 2, personal differences and distinct cell responses to in vitro CM and FM treatment in terms of cell viability (I), and apoptosis rate (II) were observed. These data were confirmed by the decrease of thermal transition at 95±5°C after cell exposure to CM, and the even higher thermal profile in the case of cells incubated with FM (patient no. 14; Fig. 2AIII). The obtained data were also confirmed by strong proteolytic cleavage of PARP-1 in leukemic cells incubated with CM (Fig. 2AIV). For patient no. 18, only a slight decrease of the living cell numbers exposed to CM in comparison to control ones (Figs. 1A, B and 2BI, II) was observed (Figs. 1 and 2) and correlated with no changes in the thermal profiles of nuclear fraction preparations. A slight PARP-1 cleavage, even in control untreated cells of this patient, was observed (Fig. 2BIV).
The apoptotic and necrotic morphological changes in examined cells were monitored during cell incubations with anticancer drugs (Fig. 3). The cell suspensions were incubated with YO-PRO-1 and propidium iodide and analyzed under fluorescence microscope (Olympus IX70) at magnification ×400. Numerous shrunken cells with the typical morphological features of early (green arrow) and late (yellow arrow) apoptosis, i.e., chromatin condensation, marginalization (semilunar shape) and nuclear fragmentation were seen after CM and FM treatment. Some swollen enlarged cells typical for necrosis (red arrow) and the cells with two nuclei (white arrow) were also observed. In some cells, the beginning of nuclear changes were visible as a condensed ring/sphere along the nuclear envelope (blue arrow).
The stained cell areas observed after leukemic cell incubations with drugs (patient no. 14) for 48 h chosen as an example of anticancer drugs effect monitoring on leukemic cell viability is illustrated in Fig. 3.
Finally, we addressed the question of whether evaluation of thermal profile changes upon drug treatment of leukemic cells might possess prognostic value. Interestingly, the results of the in vitro experiments suggest that patient no. 14 should respond better to CM/CC treatment. During randomized therapy option this patient was administered with CC, and after six cycles of chemotherapy he reached complete remission. The results for patient no. 18 indicated his very weak in vitro response to CM in comparison with FM. This patient was administered with CC and he did not respond (NR) to scheduled therapy (Table I).
The comparative statistical analysis using Fisher’s exact test with Bonferroni correction between clinical patient response (after six courses of therapy) and in vitro test data reflects a statistical correlation (p=0.017) between in vivo and in vitro response to the applied treatment (Fig. 1C). Interestingly, we did not find such correlation between the response to treatment versus stage of disease (Rai staging), or cytogenetic aberration on 11q and 13q chromosome.
As is shown in Table I, among the 28 patients for whom cytogenetic assay was performed, 25 underwent CC therapy and 12 reached CR (48%). The deletion within chromosome 13 (13q14) was the most common genetic abnormality in the investigated group (11/28, 39.28%). Among the studied samples of CLL patients, only three cases of chromosomal 17 abnormality (17p13.1) were detected, and one of them reached CR.
These results suggest that in vitro response of leukemic cells to anticancer agents display predictive value and could be helpful in the optimal therapeutic strategy selection for individual patients in order to avoid ineffective therapy based on purine analog combined with alkylating agent.
Discussion
Inherited genetic predispositions to CLL has directed attention towards extensive studies on genetic alterations which could disturb gene expression (6,32). Studies looking for new genetic aberrations and epigenetic modifications in chromatin structure have been performed (4–6). The diversities in response to CLL therapy occur in respect to personal genetic differences, having impact on cell signal transduction, cell cycle alterations and disturbance of apoptosis. Sometimes, because of personal variations in expression of some genes even directed therapy towards cancer-related or specific markers could not be fully effective. Personal diversities in response to treatment or retreatment that introduce toxicity could direct into myelodysplasia or secondary neoplasms (33,34).
More than 95% of leukemic cells in peripheral blood of patients display hyper-condensed heterochromatin and are between G0/G1 phase of the cell cycle. In more aggressive cases of disease, the population of cycling cells induce another request for CLL treatment directions. According to the theoretical predictions, activity of drugs and their capability to induce apoptosis should be the most effective manner for elimination of leukemic cells (34,35). Standard therapies used in clinical routine are based on the combination of purine analogs with alkylating agent(s), which induce DNA damage (36). In light of published data, a proportion of patients acquires resistance to the treatment (14,37,38). Thus, the optimization of drug(s) before their administration and elaborating tailored therapy is a need for the group of non-responding patients or those with high expression of unfavorable prognostic factors.
In the current study, we evaluated in vitro cytotoxicity, apoptosis induction potential and the changes in chromatin conformation caused by cladribine or fludarabine combined with mafosfamide to evaluate their potency for leukemic cell elimination by apoptosis. As the consequence of two CLL cases published recently (18), we extended our study up to 28 patients. We used four experimental approaches for monitoring, i.e., cell viability, rate of apoptosis, DSC analysis of nuclear preparations, and expression of apoptotic marker PARP-1. The obtained data revealed that in vitro exposure of leukemic PBMCs to drug combinations before patient treatment might indicate prognostic value. In the examined cases we observed that patients whose PBMCs were insensitive to given drug combinations in vitro did not respond to the treatment in vivo either. The positive responses to drug combinations in vitro, as the result of cell elimination by apoptosis, were in most cases followed by CR to therapy in vivo. It must be underlined that for some CLL patients whose cells indicate special sensitivity to in vitro conditions individualized tumor response testing suggested by Matutes et al (39) or cytotoxic tests performed previously by Nagourney et al (40) could not be fully effective (compare Fig. 1A and B).
Finally, the disappearance of some of the CLL cells as a consequence of apoptosis induction after their exposure to drugs was also confirmed by the changes in expression of certain apoptosis-related proteins, for example PARP-1. A recently published report concerning anticancer drug bioactivity on library of approved anticancer drugs confirms CLL cell exceptionality, but direct studies towards tailoring therapy for other types of malignances in general are needed (41).
In summary, the results of our studies revealed that determination of the in vitro CLL cell sensitivity based on purine analogs and their combinations with alkylating agent would be instrumental in the development of personalized therapy. It should be stated that from our point of view in vitro study gives the opportunity for CLL status analysis, before and during drug application (18,22–24). It reflects a special importance for a subset of patients resistant to therapy, as well as for those heavily pre-treated or those in weak condition.
Our current results for 28 patients confirm studies published previously as a case report (18) suggesting that in vitro incubations of leukemia cells with anticancer drugs is of predictive value and would help to select the optimal therapeutic strategy for individual patient in order to avoid ineffective treatment.
Acknowledgements
Financial support: Polish National Science Centre (Cracow, Poland) (Grant 2011/01/B/NZ/0102). We would like to thank Wojciech Sobala for statistical analysis.
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