Relationship between chromosomal aberrations and gene expressions in the p53 pathway in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a neoplasia that occurs with the accumulation of CD5 +B cells. These neoplastic cells infiltrate peripheral blood, lymph nodes, bone marrow and lymphoid tissues. The prognosis of CLL disease is variable and the average age of diagnosis is 70 years [1]. Chronic lymphocytic leukemia disease is widely seen in Western societies. The CLL cells consist of monoclonal B lymphocytes expressing the CD23, CD 19, CD5 antigens on their surface; in addition CD20, CD79b, FMC7 and Ig antigens are expressed in minute amounts [2]. The CELL patients have a high survival rate and 33.0% of patients do not need treatment. The initial phase of indolent follows the progression of the disease and the aggressive form of the disease that requires treatment [3]. More than 50.0% of CLL cases are diagnosed in the early stage of the disease, usually with lymphocytosis on routine blood screening. Repetitive gene mutations, including NOTCH1 and SF 3B1, elevation of serum thymidine kinase (TK) or β2-microglobulin (β2-MG) levels and downregulation of immunoglobulin heavy chain genes (IGHV) have prognostic significance [4].

Currently, two well-known clinical staging systems are used to classify patients according to risk groups based on clinicopathological characteristics [5]. Two widely accepted methods of hematologically based staging, Rai and Binet systems, have been used in both routine patients and in clinical trials, and have some similarities. The Rai classification is based on the presence or absence of lymphadenopathy and peripheral blood lymphocytosis, platelet count, hemoglobin (Hb) level. In the Binet staging system, CLL is categorized according to the number of lymphoid tissues, the presence of anemia and thrombocytopenia [6]. Fluorescence in situ hybridization (FISH) detects chromosomal anomalies in more than 80.0% of CLL cases, most of which are chromosomal deletions [7]. The most frequent chromosomal abnormalities are deletions of the TP53 gene located on 17p13 (8.0%), trisomy 12 (16.0%), 11q22.3-q23.1 deletions (19.0%) and 13q14 deletions (53.0%). Patients with 17p deletion (17p-) and 11q deletion (11q-) are associated with poor prognosis and short survival. The 13q deletion (13q-) is associated with a slower progressive form of the disease and a better prognosis [8].

The P53 gene protects the integrity of the genome. The p53 gene is functionally ineffective in all cancers and has a role in preventing tumorigenesis [9]. The P53 pathway responds to stresses in cell division and replication. Stress signals are also transmitted to the p53 protein [10]. In response to the P53 activation, P53-mediated down-stream events consist of cell cycle arrest and apoptosis. DNA damage can activate p53 with the inclusion of DNA double-strand break and DNA repair intermediates following chemical damage to DNA. In this case, there is an increase in p53 level of the cell and activation of p53 as a transcription factor [11].

Determining the prognosis of CLL patients is important for survival and remission. Prognostic parameters should be considered in the genetic evaluation of the process of the disease. By determining the differences in gene expression in CLL cases, it will be possible to use them as markers. There are few studies in the literature regarding the overall evaluation of genes in the p53 pathway. Thus, we designed the current study to perform expression profiling of 22 genes for the first time in CLL. We aimed to obtain new data with respect to the pathogenesis of the disease and infer new molecular markers related to prognosis of CLL. Our results are possibly significance for clarifying the currently unknown molecular pathogenesis of CLL. In this study, the most frequently selected two housekeeping genes (HKG), were used as candidates for normalizing RT² Profiler polymerase chain reaction (PCR) Array data in CLL. These genes are β-actin (ACTE) and β2-microglobulin (β2-A/G).

In the present study, we performed expression profiling of 22 genes (ATM, ATR, BAX, CASP9, CDK4, CD-KN1A, CDKN2A, CHEK1, CHEK2, E2F1, GADD45A, MDM2, MDM4, PCNA, RB1, TP53, BCL2, APAF1, E2F3, MCL1, PTEN, PTX3) in the p53 pathway, which are related to apoptosis, proliferation, cell cycle, cell growth, differentiation and DNA repair processes. We also looked for a possible relation between gene expression alterations and cytogenetic abnormalities.

Patient Specimens. Peripheral blood samples were collected from 30 patients with CLL and 15 healthy subjects at the İstanbul Medical Faculty Hospital (Istanbul, Turkey). Demographical characteristics, physical findings, laboratory results (complete blood count, peripheral smear, immunoglobulin values, FISH and flow cytometry results), Rai and Binet staging, medications and treatment indications were recorded. The diagnosis of CLL was defined by the criteria of the International Workshop on CLL (IWCLL) 2008. Sixteen male (53.3%) and 14 female (46.6%) patients, whose median age was 63.8 ± 9.48 years (range 46-86 years), were included in the analyses. Table 1 shows the patients’ diagnostic data and FISH results. There were seven males (46.6%) and eight females (53.3%) included as controls whose median age was 56.53 + 6.80 years (range 44-70 years). Our study was approved by the local ethics committee (İstanbul University, İstanbul Medical Faculty Ethic Committee of Clinical Research Approval, number: 2015/920; date: May 7 2015).

Cytogenetic evaluation with respect to 13q and 17p deletions and trisomy 12 was performed with the FISH technique as a part of routine studies. Patients both with and without chromosomal aberrations (deletion of 13q, 17p regions, also trisomy 12 [+]) were included in the study. We evaluated the prognosis according to good and bad prognoses. The 17p or 11q deletions of prognostic factors were examined to determine treatment approach and survival. We selected the 17p deletion from two deletions because it was suggested to investigate that deletion in patients with active or progressive disease symptoms and treatment indication.

Interphase Fluorescence In Situ Hybridization (I-FISH). Interphase FISH analyses were done on peripheral blood obtained from CLL patients. The CLL panel contained commercial FISH probes for the detection of deletions at 13ql4, 13q34, 17p13 and trisomy 12 (Cyto-cell, Cambridge, Cambridgeshire, UK). Gibco PB-MAX™ Karyotyping Medium (Gibco Life Technologies, Carlsbad, CA, USA) was used on cultured peripheral blood cells fixed in 3:1 Carnoy’s fixative. Standard cytogenetic procedures were applied for the samples. Interphase nuclei were counterstained with DAPI (Cytocell). Fluorescence in situ hybridization signals were evaluated under a fluorescence microscope (Zeiss Axioskop 2 plus; Carl Zeiss CMP GmbH, Göttingen, Germany). The images were captured on a computer with karyotyping and FISH software (MetaSystems ISIS FISH imaging system; MetaSystems GmbH, Altlussheim, Germanay). Two hundred interphase nuclei were analyzed for each sample. The cutoff values were 5.0% for D13S319 (13ql4.2, red)/13qter (13q34, blue)/12cen (trisomy 12, green) deletion. Enumeration probe have two red, two blue and two green signals in a normal cell and 5.0% for 17p13.1 (P53, red) probe has two red and two green signals in a normal cell.

RNA Extraction and cDNA Synthesis. Total RNA was isolated from whole blood samples using QiaAmp RNA Blood Mini Kit (cat #52304; Qiagen GmbH, Hilden, Germany), according to the manufacturer’s protocol. RNA quality and quantity were checked using a NanoDrop 2000c (Thermo Fisher Scientific Inc., Wilmington, DE, USA). The A260/A280 ratio of RNA was 1.8-2.0. The same amount of total RNA was used for reverse transcription of each sample in the study. It was started with 0.5 pg (500 ng) total RNA in accordance with a 96-well plate format. The cDNA was synthesized from 0.5 μg of mRNA for the reverse transcription and quantitative PCR (RT-qPCR), using an RT2 First Strand Kit (cat #330404; Qiagen GmbH) following the manufacturer’s protocol. The RT2 Profiler PCR Array protocol was continued.

RT2 Profiler PCR Array. Expression of 22 genes from isolated RNA related to p53-mediated signal transduction detected in DNA repair, apoptosis, cell cycle, proliferation and differentiation processes, were profiled using RT² Profiler PCR Array (Qiagen; Custom cat #CAPH 13446). Real-time PCR-Custom RT² Profiler PCR Array’s F format-96-well (CAPH_133446F; cat #330131/12 plate) was used in this study. Custom RT2 Profiler PCR Array was performed to measure the expression levels of 22 genes beside the reference genes (ACTB and β2-GM) for each well of the patient or control group; a total of 25 (μL of 12.5 μL SYBR green master mix, 10.5 μL ddH₂O and 1 μL cDNA were prepared to using RT2 SYBR Green qPCR Mastermix kit (cat #330502; Qiagen) on the Light Cycler 480 II RT-PCR Instrument (Roche Diagnostics GmbH, Mannheim, Germany). The 12.5 μL SYBR green master mix and 12.5 μL RNase-free water were added for each of the HKG (ACTB and β2-MG) and positive PCR control.

Data Analysis. For the analysis of data with Light Cycler 480 system (Roche Diagnostics GmbH), the Second Derivative Max Method was used. This method was defined as the crossing point of the PCR reaction and this crossing point value is the maximum value of the second derivative of the amplification curve. We used commercially available primers for all genes (Qiagen GmbH). Threshold cycle (Ct) values for each sample were evaluated using web-based RT2 Profiler PCR Array Data analysis 3.5 [12].

The 2^-ᐃᐃCt method is used as a relative quantification strategy for qPCR data analysis. This method directly measures relative gene expression levels between different samples using the Ct values produced by the qPCR system. Gene expression levels were also defined as fold change. Relative expressions were calculated in accordance with the 2^-ᐃᐃCt method. Relative quantification related the PCR signal of the target transcript in a patient group to the signal of another sample such as a control group. The values of positive PCR control (Ct^PPC) wells were examined. The mean Ct^PPC ratio in each RT2 Profiler PCR Array should be 20 ± 2. The ACt was used for each gene in all plate using Ct values for the gene of interest (GOI) and the HKG (ᐃCt: Ct_GOI-Ct_HKG). When replication was performed, the mean ACt values of each gene (each well) were calculated.

Statistical Analysis. In the evaluation of the data, Qiagen Custom RT² Profiler PCR Array data analysis methods were used. Student’s t-test analysis was performed by a web-based program. Significant differences in expression levels by Student t-test analysis are shown as log-transforming values (logFC) to identify fold regulation. A p value of <0.05 was considered to indicate a statistically significant result.

Clinical Characteristics of CLL Patients. Thirty patients with chronic lymphocytic leukemia (with and without chromosomal aberrations) and 15 healthy adults as control group were included in the study. Fourteen CLL patients were female (46.6%) and 16 were male (53.3%); the male/female ratio was 1.14. Eight individuals in the control group were female (53.3%) and seven were male (46.6%). The mean ages of the CLL and control groups were 63.8 ± 9.4 and 56.5 ± 6.8, respectively. Median and minimum-maximum ages were 63.8 (46-86) and 56.5 (44-70), respectively. The age of diagnosis was 58.1 ± 10.2 in the CLL group. There was no statistically significant difference between the two groups with respect to gender distribution (p >0.05). According to the Rai staging, 11 patients were in stage 0 (36.6%), one in stage I (3.33%), 10 in stage II (33.3%) and two were in stage III (6.66%). In the Binet staging system, 17 patients were in stage A (56.6%), eight in stage B (26.6%), and five in stage C (16.6%).

All patients were evaluated by the FISH technique and RT2 Profiler PCR Array method. The distribution of the patients with respect to prognosis (based on FISH findings) and complete blood count results in the CLL group are shown in Table 1. The mean difference values of the patient and control group according to average ᐃCt and fold change are shown in Table 2.

Results of FISH. Patients with both deletions and trisomy (del 17p, del 13q and trisomy 12) and normal [nondel(13q), del(17p), trisomy 12] were included in the study. As a result of the FISH assessment in the CLL group: 11 had 13q [-] (36.7%), five had 17p [-] (16.7%), nine without deletion (normal) (3.00%), five had trisomy 12 (16.7%).

We evaluated the prognosis according to good and bad prognoses. Good prognosis was found in 20 patients and poor prognosis in 10 patients. The 17p or 11 q deletions of prognostic factors were examined to determine treatment approach and survival. We selected the 17p deletion from two deletions because it was suggested to investigate that deletion in patients with active or progressive disease symptoms and treatment indication. According to the interphase-FISH results, the cutoff value was higher than 5.0% in 21/30 CLL patients. These findings were considered clonal when the percent of cells with any given chromosome aberration exceeded the normal cutoff value (5.0%).

Analysis of Differentially Expressed Genes. The RT2 Profiler PCR Array data were expressed as fold change. The 2^-Ct method is the normalized gene expression in the patient sample divided by 2^-Ct in the control sample. The average ACt values of ATM, ATR, BAX, CASP9, CDK4, CDKN2A, CHEK1, CHEK2, E2F3, MCL1, MDM2, MDM4, PCNA, RB1, P53 and BCL2 genes were found to be statistically different than the controls. However, APAF1, CDKN1A, E2F1, GADD45A, PTEN and PTX3 genes were not statistically significant (Table 2). The RT² Profiler PCR Array data analysis showed that ATM, ATR, BAX, CASP9, CDK4, CDNKN1A, CDKN2A, CHEK1, CHEK2, E2F1, MDM2, MDM4, PCNA, RB1, P53, GADD 45A and BCL2 genes were upregulated. On the other hand, APAF1, E2F3, MCL1, PTEN, CDKN2A and PTX3 genes were downregulated (Table 2).

Normalized expression of genes was compared between CLL patients and healthy controls (Figure 1). CHEKA, CDKN2A, CDK4, PCNA are known as cell cycle and cell growth, proliferation and differentiation genes; CHEK2, ATR, ATM, RB1 are cell cycle genes; the MDM4 gene is cell growth, proliferation and differentiation gene; the CASP9 gene is an apoptotic gene; the BCL2 gene is apoptotic (anti-apoptosis) and cell growth, proliferation and differentiation gene; P53 and BAX are apoptotic (induction of apoptosis) and cell cycle genes. The p values were calculated using a Student’s t-test on the 2^-ᐃCt values. The RT2 Profiler PCR Array data analysis version 3.5 was used for comparisons between experimental groups.

Figure 1

String v10 analysis (https://string-db.org) was used to create gene-gene and network interactions. Our initial results using RT² Profiler PCR Array analysis in CLL patients revealed 18 differently expressed genes. Network analysis used for the probable interactions of these 18 genes through the String web-mediated tool showed that the interactions of the resulting network were enriched (p <1.0e-16) (Figure 2). A significant enrichment was detected in a subgroup of genes involved in the mitotic cell cycle G1/S transition regulation [GO: 2000045], cell cycle negative regulation [GO: 0045786], mitotic cell cycle Gl/S transition negative regulation [GO: 2000134], cell cycle phase transition regulation [GO: 1901987] and cell cycle phase transition negative regulation [GO: 1901988] in the biological process by Gene Ontology (GO) (http://www.geneontology.org) enrichment analysis. In the analysis used for molecular function, enrichment of a gene subset was determined in specific binding to the protein domain [GO: 0019904], specific binding to the disordered domain [GO: 0097718], binding to the BH3 domain [GO: 0051434], binding to the identical protein [GO: 0042802] and protein binding [GO: 0005515]. In the cellular component analysis, it was revealed that the nuclear part [GO: 0044428], nucleoplasm [GO: 0005654], intracellular organelle lumen [GO: 0070013], nucleus [GO: 0005634] and chromosome [GO: 0005694] demonstrated a significative enrichment in a subset of genes.

Figure 2

Chronic lymphocytic leukemia is a neoplastic disease characterized by the accumulation of small lymphocytes in blood, bone marrow and lymphoid tissues. The CLL disease is the most common form of leukemia in older persons in Western countries [13]. Recent advances allowed clinicians to have a relatively better understanding of the genetic mechanisms related to CLL development and their clinical use as new biomarkers. In 2000, Döhner et al. [14] described a hierarchical prognostic model based on FISH findings. Accordingly, five risk categories were defined: those patients with the 17p53 deletion had the worst prognosis followed by 11q22-q23del, tril2, 13q14deland normal karyotype [14]. Indeed, the TP53 gene defects (deletion or mutation) is one of the two genetic parameters used in the international prognostic index for CLL (the other is the mutational status of the IGHV gene) [15].

In 80.0% of CLL cases with 17pdel, a mutation with complete loss of function is observed in the remaining allele. However, 4.0-5.0% of cases have a mutation without a deletion. Such sole monoallelic mutations at the gene level also effect outcome [16,17]. However, the TP53 aberrations are seen in only 3.0-8.0% of de novo cases and 40.0-50.0% of refractory cases. Therefore, in this study, we hypothesized that not only mutations, but also expression defects of other genes in the TP53 pathway may be playing a role in disease pathogenesis [18]. We used Qiagen RT2 Profiler PCR Array for the amplification and expression detection of 22 genes in the TP53 pathway for 30 patients and 15 healthy controls. We also compared the level of gene expression in patients with good and poor prognosis (grouped according to FISH findings). Genes that differed more than HKG (ACTS and β2-MG) between the two groups were considered significant. When we compared the CLL group with the control group, we found that there were significant differences in mRNA levels. In our study, we determined 22 mRNAs that were differentially expressed by more than one-fold up or down after RT2 Profiler PCR Array. Many of these mRNAs encode apoptotic, cell cycle, cell proliferation, growth and differentiation proteins.

For 16 genes (ATM, ATR, BAX, CASP9, CDK4, CDKN 2A, CHEK1, CHEK2, E2F3, MCL1, MDM2, MDM4, PCNA, RB1, P53 and BCL2) the Student’s /-test corrected p value was statistically significant (p <0.001). The Student’s t-test corrected p value of APAF1, CDKN1A, E2F1, GADD45A, PTEN and PTX3 genes was not statistically significant. We observed upregulation of 17 mRNA levels, including ATM, ATR, BAX, CASP9, CDK4, CDKN1A, CDKN2A, CHEK1, CHEK2, MDM2, MDM4, PCNA, RB1, P53, E2F1, GADD45A and BCL2 in our study. Two of them, CDKN2A (p <0.001) and BCL2 (p <0.001), were upregulated by more than 10.22- and 8.51-fold (Figure 1).

The alteration of gene expression included the down-regulation of E2F3, MCL1, APAF1, PTEN and PTX3 mRNA expression. The highest downregulation, that was about 1.84-fold, was observed for the PTX3 mRNA expression in CLL patients. We can especially say that these three genes (CDNK2A, BCL2 and PTX3) in the p53 pathway are linked to the mechanism of the disease and may have a significant effect on the diagnosis and prognosis of the disease.

The BCL2 gene has been shown to be overexpressed in the absence of gene rearrangement in most CLL cases. BCL2, is a protooncogen, has apoptosis regulatory function. BAX, the opposite of BCL2 protein activity, is a homologous protein that accelerates cell death rate. An increase in BCL2/BAX ratios was detected in CLL patients compared to the control group. It has been reported that drug-induced apoptosis in B-CLL cells is associated with reversing the BCL2/BAX ratio. Increased BCL2 mRNA level and increased BCL2/BAX ratio were also reported by Pepper et al. [19].

Vucicevic et al. [20] reported marked overexpression of BCL2 mRNA in a cohort of CLL. In addition, BAX mRNA and BCL2/BAX ratio were determined to be significantly higher in CLL samples than the controls. The authors pointed out that abnormal BCL2 and BAX expression may have an impact on the pathogenesis and clinical course of CLL [20]. The increased BCL2/BAX ratio (CLL/ control fold change: 8.51/2.55 = 3.33) in our study is in parallel with the studies mentioned above. Given that BCL2 has an anti-apoptotic effect, while BAX is implicated in apoptosis, the altered ratio of expression of the two genes as described above favors cell survival, which is in agreement with the nature of CLL where the life-span of the cells are abnormally long. The p16^Inka (CDKN2A) is a tumor suppressor gene, which is inactivated in human neoplasms by mutations, hypermethylations and homozygous deletions [21].

The CDKN2A gene methylation has been identified in multiple myeloma patients (61.0%), B cell precursor acute lymphoblastic leukemia (ALL) patients (75.0%), B cell non-lymphomas patients (14.0%). In studies, the p16 promoter methylation was not observed in non-Hodgkin lymphomas.

Tsirigotis et al. [21] revealed that genetic changes in the CDKN2A gene are rare in CLL patients. Further studies are needed to explain the role of the CDKN2A gene promoter methylation in the pathogenesis of CLL.

Ogawa et al. [22] aimed to determine the exact behavior and frequency of inactivation of the p16 gene in various hematopoietic tumor types and to reveal the importance of human hematopoietic malignancies in development. The p16 gene inactivation triggers tumor formation. The p16 gene deletion is frequently observed in lymphoid malignancies. Although pl6 deletions were observed in all subtypes of ALL, in the study of Ogawa et al. [22], the p16 deletion ratio appears to be lower in T-ALL than in B-ALL.

The CDKN2A gene might be upregulated in CLL patients compared to controls in our study due to the higher number of patients in the early stage and the limited number of patients included in the study. Although decreased expression of the CDKN2A gene is associated with an increased risk in CLL and the other human cancers in the literature. According to the study of Romagosa et al. [23], p16Inka protein also plays a role in apoptosis and angiogenesis; therefore, these functions may be associated with overexpression of cancer. They observed p16Inka as over-expressed in endometrial, colorectal and basal cell carcinoma. Accordingly, our study is consistent with the study of Romagosa et al. [23], showing that the CDKN2A gene may facilitate differential diagnosis of tumors that progressively increase the CDKN2A expression. Overexpression of the CDKN2A gene suggests that it may have a prognostic role in the tumors in which the irregular CDKN2A gene is overexpressed.

Pentraxin 3 (pTX3) is an essential component of congenital immunity and acts as a prognostic biomarker to control infections. The PTX3 gene has been known as an extrinsic oncosuppressor in cancer and plays an important role in hematological malignancies and allogeneic hematopoietic stem cell transplantation [24]. It appears that expression of PTX3 in CLL was not investigated in previous studies. There are studies on the PTX3 gene in other cancer types. The PTX3 gene induces tumor progression in malignancies and also functions as a tumor suppressor in human neoplasms. Ke et al. [25] found that low PTX3 immuno-histochemical expression is associated with good prognosis in gliomas.

Liu et al. [26] analyzed the prognostic significance of serum PTX3 levels in patients with colorectal cancer (CRC). An increase in serum PTX3 levels correlates with poor prognosis in postoperative CRC patients. Serum PTX3 level can be used as an independent prognostic biomarker in colorectal cancers [26].

The PTX3 gene has been shown to act as an extrinsic oncosuppressor gene of cancer-related inflammation (CRI) and reduce CRI. Loss of PTX3 was observed in cancer types such as CRC and leiomyosarcoma other than CLL [27]. The PTX3 gene has biological roles such as apoptosis and immune defense. Therefore, in our study, the down-regulation of the PTX3 gene in CLL patients compared to controls may slow down apoptosis of leukemic cells in patients and thus increase the CLL cells. These results may shed light on the pathogenesis of CLL disease.

We discussed genes with significantly different expression based on ACt value in CLL patients. At the last stage of the our study, based on the results obtained from RT² Profiler PCR Array Data analysis, gene expressions were evaluated by the 2^-Ct method. According to RT² Profiler PCR Array Data analysis, PTX3 and CDNK2A genes can be used to guide CLL pathogenesis. The CDNK2A gene, which had the highest upregulation by more 10.22-fold [95% confidence interval (95% CI): 7.14-13.3] and the PTX3 gene expression had the highest downregulation, which was 1.84-fold (95% CI: 0.37-0.71) in CLL patients/ control group. We focused first on the expression profile of 22 key genes to the p53 pathway in this study. On the other hand, other gene(s) associated with the p53 pathway, together with the genes investigated in our study may shed light on the genetic mechanism of the p53 pathway. In this study, the effects of gene expression changes in the p53 pathway in the prognosis and molecular pathogenesis of CLL were investigated. In conclusion, our results imply that there is marked expression disturbances of many genes in the p53 pathway. This suggests that mechanisms other than genomic alterations affecting the TP53 gene per se may be operative in the disease pathogenesis.

As a continuation of this study, changes in protein levels associated with our genes in the p53 pathway will be investigated. In future studies, it is important to evaluate the localization and function of proteins in the cell by researching RNA transcripts on this pathway with various protein analysis methods. Advanced researches are necessary to explain by which mechanisms such expression abnormalities occur, such as abnormal methylation and up- or downregulation of particular miRNAs.