Quantitative real time PCR analysis of apoptotic gene expression in chronic lymphocytic leukemia patients and their relationships with chemosensitivity

Chronic Lymphocytic leukemia (CLL) is a disease of morphologically mature, but immunologically less mature lymphocytes manifested by progressive accumulation of these cells within the blood, bone marrow, and lymphatic tissues poignant in the main older population [1].

Hematological malignancies have been reported in high rates among countries of Asia including Kurdistan as a region of Iraq [2]. While within the Western World, CLL have been rated as the most prevalent adult malignant neoplastic disease with an incidence of 4.2/100 000/year [3].

Apoptosis is a cellular mechanism used to remove cells that are damaged, infected, or have reached the end of their lifecycle [4]. Interrupted control of apoptosis can cause many diseases, including cancer, autoimmune diseases, and degenerative disorders [5]. The ultimate role of the anti-apoptotic protein BCL2 is to intercept the action of pro-apoptotic proteins responsible for stoma formation within the mitochondria [6, 7]. The vital equilibrium that exists between anti-apoptotic members such as BCL2, and pro-apoptotic members aids to dictate whether the cell initiates apoptosis [8, 9].

Preclinical studies demonstrates that BCL2 has a mechanistic part in many conventional modes of cancer treatment [10–14] and its knockdown may potentiate the chemotherapy [15, 16]. BCL2 has also been involved in promoting resistance to traditional therapies in preclinical models [8, 17, 18]. Constitutively higher expression of the BCL2 gene makes CLL cells more resistant to apoptosis, resulting in the accumulation of long-lived, clonal lymphocytes which characterizes the disease [19]. The treatment of relapsed chronic lymphocytic leukemia (CLL) has recently changed with the introduction of drugs that inhibit intracellular B-cell receptor signaling [20].

Selective targeting of BCL2 with ABT-199/venetoclax provided an acceptable safety profile and induced valuable outcomes in patients with relapsed CLL or SLL, including those with poor prognostic features [21].

Having a major role in cellular response to stress, p53 perform as the major barrier for tumorogenesis [22]. This impediment should be eliminated in order to allow tumor development. Indeed, nearly more or less than half of human cancers bear p53 gene mutations; in the majority of remaining cancer patients, p53 task is compromised by different mechanisms [23].

The current study aimed to analyze, BCL2 and p53 genes expression in CLL patients regarding chemotherapeutic regimen, number of treatment courses and disease states watch & wait (WW), newly diagnosed (ND) and chemotherapy (CHT).

The study was conducted on 55 CLL patients visiting Hiwa Hospital for Hematology & Oncology in Sulaimaniyah, Iraq, either for diagnosis, to continue the CHT courses or changes in their disease state, over three-months period from 1st October to 30th December-2013. Diagnosis and staging were based on standard clinical, morphological and immunophenotyping criteria. Detailed information was taken directly from the patient, or from the data in computer-based patient records.

The present study was carried out in four steps: Step I: Collecting blood samples from CLL patients and 40 healthy individuals as control. Step II: Total RNA was extracted from peripheral blood by total RNA isolation kit from Geneaid™ (Geneaid Biotech Ltd., Taiwan) according to manufacturer’s instruction in Kurdistan Institution for Strategic Studies and Scientific Research (KISSR) in Sulaimaniyah. Step III: cDNA was synthesized in Shiraz Institute for Cancer Research/Shiraz Medical College/Iran by Thermo Scientific Maxima H Minus First Strand cDNA Synthesis Kit with dsDNAse according to manufacturer’s instruction. Step IV: qRT-PCR analysis was processed in 48 well StepOne™ Real-Time PCR System from Applied Biosystems (Thermo Fisher Scientific, Waltham, MA USA) in Shiraz Institute for Cancer Research/Shiraz Medical College/Iran. TaqMan master mix was used for BCL2 and SYBR green for p53; using B-actin as internal control normalized gene expression.

The results are expressed as mean ± SEM. Independent student’s t-test was used to analyze statistical differences between the means of gene expressions in CLL patient (treated and untreated) and control. One-way analysis of variance (ANOVA) test was used for comparison the difference in BCL2 and p53 gene expressions among patients with different disease states (WW, ND and CHT), with different age, disease duration and frequency of treatment course. When appropriate, the LSD (least significant difference) test was used for multiple comparisons between groups. The threshold for significant difference was set at P < 0.05. SPSS® 21 software (SPSS Inc., Chicago, IL, USA) was used to carry out the statistical analyses.

The present study showed that in healthy subjects BCL2 and p53 expression by B-lymphocytes seem to be inversely related. Conversely, anti apoptotic activity expressed by BCL2 gene and tumor suppressor activity represented by p53 gene expression, were in a balanced state. A three-fold increase of BCL2 expression and a 50% decrease in p53 expression was reported in CLL patients. The finding is in agreement with Klobusická et al. [24], indicating an overexpression of that P53 protein and down regulation of BCL2 expression in a subgroup of B-CLL patients.

In ND patients the BCL2 and p53 gene expression were higher than that in patients who were underwent treatment with CHT. This is in line with Lazaridou et al. [25] suggesting an early overexpression of BCL2 and expression of p53 during advanced stages of the disease.

In contrary, patients in WW state showed lower BCL2 expression than ND patients and CHT patients and this partly explain why WW patients were asymptomatic. A relative expression of p53 with BCL2 gives evidence the cells still living in a balanced state, which may become disrupted in future by several predisposing factors.

Age has an effect on p53 gene expression, younger patients showed normal levels with a decrease at the start of the treatment and an increase in older patients. This is in agreement with the study carried out by Stoklosa and Golab [26] which involved investigating several types of cancer with the finding that p53 gene is a potentially useful biomarker for predicting prognosis and patient’s response to therapy

Gender has an effect on apoptosis gene expression, so that expression of BCL2 gene was reported to be higher and p53 was lower in female compared to male. Dissimilarities in gene expression between genders this result is similar to study results done by Klobusická M. and colleagues[27] which they reported that the expression of BCL2 protein was significantly higher in female patients under 50 years and inverse relationship between a higher accumulation of p53 and repressed BCL2 expression and vice versa was observed.

BCL2 gene expression in CLL and CLL/SLL was higher than healthy subject. Comparison of individual expression indicated that gene expression was higher in many CLL/SLL patients than CLL patients, which might explain the resistance to CHT. Although CLL and SLL do vary slightly, the cell of origin in CLL and CLL/SLL is identical and they have the same genetic pattern, morphology and prognostic markers. However, their survival and response to treatment tend to be very similar [28]. Expression of p53 gene was lower in CLL and CLL/SLL compared to the control group, however, the difference was not statistically significant. Similarly, Santos et al. [29] reported that both CLL and CLL/SLL are currently considered different manifestations of the same entity in the fourth edition of the World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues. It is suspected that differential expression of chemokine receptors (e.g., reduced expression of R1 and CCR3 in SLL cells), integrins (e.g., CLL cells have lower expression of integrin αLβ2), and genetic abnormalities (a higher incidence of trisomy 12 and lower incidence of del(13q) is found in CLL/SLL) may explain some of the clinical differences between these two disorders [29].

The study showed proportional increase in BCL2 gene expression with patients’ treatment regimens, whereas treatment courses were not directly affecting patients’ gene expression. In contrary, this increase gives more imbalances between pro and anti-apoptotic activity to B-cells. Expression of p53 gene was not changed during treatment courses except for patients who took about 21–25 courses and this might be due to the high proliferation activity of the B-cells at this stage of the disease that gives little higher gene expression in comparison with other treatment courses. The results of high expression of BCL2 in patients with high treatment courses or with long disease durations is similar to study results done by Reed JC [30] he reported that the overexpression of BCL2 is associated with tumor development or progression, and resistance to anticancer chemo- and radiotherapy and disagrees with studies done by Aguilar-Santelises et al. [31] they reported an early overexpression of BCL2 and expression of p53 during advanced stages of the disease.

For treatment regimens, Clb showed its effect on the apoptotic activity by decreasing the BCL2 gene expression. The study results are in agreement with a Pepper C. and colleagues [32] who reported that high levels of BCL2 in B-CLL cells were down-regulated in apoptotic cells after Clb treatment, however, the mean BCL2 expression in viable cells was increased. This was explained to be the result of the loss of lower BCL2-expressing cells into the apoptotic compartment. Clb alone or plus p shows slightly increase in p53 gene expression. This result agrees with the study carried out by Begleiter et al. [33], which reported that Clb, similarly to purine nucleoside, might induce cell death in CLL by the p53-dependent pathway.

For other treatment regimens p53 gene expression showed no significant changes, which may indicate that such treatment, were not directly affecting the p53 cellular activity and/or FCR may not be the most appropriate therapy. This assumption is supported by Hillmen et al. [34], who reported that patients with abnormalities of the p53 pathway have a very poor response to FCR (5% complete remissions in 17p-deleted CLL compared with nearly 50% for those patients without 17p deletion). However, FCR regimen cause massive increase in BCL2 gene expression and this result disagrees with known mechanism of action of fludarabine, which triggers apoptosis and disagree with study carried out by Plosker and Figgitt [35]. They reported that rituximab sensitizes neoplastic cells to the effects of chemotherapeutic agents. Thus, supporting the use of combination of CHT with rituximab. So rituximab down regulates interleukin (IL)-10 and BCL2 expression, thus making neoplastic B-cells potentially more susceptible to fludarabine-induced apoptosis [36].

The combination Clb and P then FCR decreases BCL2 gene expression and this might be due to synergistic activity of the treatment combination, which triggers further apoptosis.

Results showed in ND patients the BCL2 gene expression is high. This decreases by starting CHT at low levels in first year of treatment due to effect of CHT compared with ND. The expression of BCL2 fluctuated during different stages of treatment courses till two years into treatment. BCL2 showed almost constant expression after that period and this might be due to resistance to CHT that makes this straight expression. In comparison, p53 gene expression showed a straight expression during all stages of treatment courses giving evidence that it was not regulated by treatment.

Study results showed no statistically significant correlation between type of treatment and number of treatment courses with BCL2 and p53 gene expressions p value > 0.05 this result is similar to study results done by Cordone et al., they reported that in CLL the expression of the p53 protein, is strongly associated with p53 gene mutations, a morphological variant (CLL with >10% prolymphocytes), advanced clinical stage, progressive disease, poor response to therapy, and short survival [37] and disagrees with Thomas A. and colleagues study results which reported by chlorambucil produced almost identical changes in BCL2 and Bax expression in normal T-cells and leukaemic B-cells triggered to die by apoptosis, which together with the correlation between BCL2 and chemosensitivity confirms a pivotal role for BCL2 in regulating a distal step in the apoptotic pathway following cytotoxic cellular damage [38] and also disagrees with data reported by Robertson LE and colleagues, their data suggests that BCL2 is important in the pathogenesis and progression of CLL and that quantitation of BCL2 protein may provide useful prognostic information [39].

BCL2 gene expression was almost equal between the patients newly diagnosed and those underwent chemotherapy treatment. In contrary, p53 gene was extremely expressed in patients treated with chemotherapy agents particularly after 24–30 months disease duration; suggesting an early overexpression of BCL2 and expression of p53 during advanced stages of the disease.

Treatment courses were not directly affected patients’ gene expression. However, a proportional change in BCL2 and p53 gene expression was reported with different treatment regimens; Clb decreased and FCR regimen increased BCL2 gene expression. Higher p53 gene expression reported with the Clb + (Clb + P) regimen.