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High expression of PI3K core complex genes is associated with poor prognosis in chronic lymphocytic leukemia Louise Kristensen a , Thomas Kristensen a , Niels Abildgaard b , Mads Thomassen c , Mikael Frederiksen d , Torben Mourits-Andersen e , Michael Boe Møller a,∗ a
Department of Pathology, Odense University Hospital, Odense, Denmark Department of Hematology, Odense University Hospital, Odense, Denmark c Department of Genetics, Odense University Hospital, Odense, Denmark d Department of Hematology, Haderslev Hospital, Haderslev, Denmark e Department of Hematology, Hospital of Southwestern Jutland, Esbjerg, Denmark b
a r t i c l e
i n f o
Article history: Received 12 September 2014 Received in revised form 13 February 2015 Accepted 18 February 2015 Available online xxx Keywords: Chronic lymphocytic leukemia Autophagy BECN1 PIK3C3 PIK3R4 Prognosis
a b s t r a c t Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in the Western world. Autophagy is a highly conserved process in eukaryotic cells. In CLL autophagy is involved in mediating the effect of chemotherapy but the role of autophagy in CLL pathogenesis remains unknown. In the present study, we used real-time RT-PCR to analyze expression of the PIK3C3, PIK3R4, and BECN1 genes. These genes encode the components of the PI3K core complex, which is central to initiation of autophagy. A consecutive series of 149 well-characterized CLL cases from Region of Southern Denmark were included in the study. All three genes were observed to be independent markers of prognosis in CLL with high expression being associated with more aggressive disease. With this clear association with outcome in CLL, these genes thereby represent promising candidates for future functional studies on the role of autophagy in CLL, and they may further represent targets of treatment. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in the Western world [1]. The prognosis is highly variable with a median survival of 7–8 years [2]. Some patients live with the diagnosis without treatment while others have a more aggressive course. Treatment is not curative but longer periods of remission are achievable [1,3,4]. The PI3K (phosphoinositide 3-kinase) pathway plays an important role in the cell cycle and cancer development. PI3K’s are divided into three subclasses (classes I–III) according to their regulation and function [5]. Class I is the most studied class, and kinases participates in multiple cellular pathways central to cell regulation. They inhibit autophagy through the PI3K/AKT/mTOR pathway [6]. One of these class I kinases, PIK3C␦, is the target for treatment with Idelalisib in CLL patients [7]. Class III predominantly regulates membrane trafficking and has an important role in the autophagy pathway as it participates in the initiation core complex [5,6].
∗ Corresponding author at: Department of Pathology, Odense University Hospital, Winsløwsparken 15, 3rd Floor, 5000 Odense C, Denmark. Tel.: +45 6541 4806. E-mail address: [email protected] (M.B. Møller).
Autophagy is a highly conserved process in eukaryotic cells. Double membrane vesicles, autophagosomes, are formed to enclose organelles such as mitochondria and endoplasmatic reticulum. Autophagosomes fuse with lysosomes and their content is degraded by the lysosomal enzymes. The process of autophagy was initially described as a strategy for nutrition supply under stressed conditions but it may also lead to programmed cell death by an excessive activation of its self-degrading system. Autophagy takes place in both healthy and sick cells [8–11]. Autophagy can be divided into three phases: sequestration of cellular compounds, transport of the autophagosome to the lysosome, and degradation. The PI3K complex plays an important role in the initiation of autophagy were it controls vesicle nucleation [12]. In mammals the core complex is composed of BECN1 (beclin 1), PIK3C3 (VPS34), and PIK3R4 (VPS15). This complex serves as binding site for several proteins that are either promoters or inhibitors of autophagy [12–14]. In cancer, autophagy can participate in both tumor suppression and progression. Dependent on cellular context, it enables tumor cells to survive chemotherapy-mediated stress. On the other hand, it can maintain cellular homeostasis by removing damaged organelles and prevent the genomic damage that leads to cancer [9]. In leukemia, several studies have shown that autophagy
http://dx.doi.org/10.1016/j.leukres.2015.02.008 0145-2126/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kristensen L, et al. High expression of PI3K core complex genes is associated with poor prognosis in chronic lymphocytic leukemia. Leuk Res (2015), http://dx.doi.org/10.1016/j.leukres.2015.02.008
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is important for the death of leukemic cells. In most of these studies, autophagy was affected by treatment with different chemotherapeutic agents [15]. In CLL, some drugs have an autophagy-mediated effect on CLL cells. An example is APO866, an inhibitor of nicotinamide phosphoribosyl transferase, which contributes to autophagic cell death [16]. Also dexamethasone, which is used clinically for CLL treatment, kills the malignant lymphocytes through induction of autophagy before the onset of apoptosis [17]. Another clinically useful agent in CLL treatment, fludarabine, also acts partly through autophagy. In sensitive CLL cells, a BECN1 signaling mechanism mediates autophagic cell death in response to fludarabine, whereas in resistant cells, basal autophagy prevents the cells from dying [18]. Another study shows that fludarabine and CAL-101, the PIK3Cıinhibitor now known as Idelalisib [7], induces autophagy, whereas neither chlorambucil or rituximab produced this effect [19]. The histone deacetylase inhibitor, MGCD0103, inhibits autophagy in CLL cells by activating the PI3K/AKT/mTOR pathway and down regulating the key regulators of autophagy [20]. Other studies have shown that Tenovin-6 (tnv-6), an antineoplastic molecule, is cytotoxic to CLL cells through dysregulated autophagy [21]. Autophagy thus appears to represent an important cellular mechanism involved in mediating the effect of chemotherapy in CLL, but the role of autophagy in CLL pathogenesis is unknown, and whether the level of autophagic activity is associated to outcome has not been studied. Because of the central role of the PI3K core complex in autophagy initiation, we therefore measured the mRNA expression levels of its components: BECN1, PIK3C3, and PIK3R4 in a cohort of untreated CLL patients in the present study. The expression levels were correlated to time to treatment (TTT) and overall survival (OS). All three genes were each observed to be independently associated with prognosis in CLL. 2. Material and methods 2.1. Patient material Patients diagnosed with CLL, according to the WHO classification, at the Department of Pathology, Odense University Hospital, Denmark, from January 2005 to June 2011, were included in the present study. A total of 418 patients fulfilled this criterion. Of these 418 cases, patients were excluded if lymphocytes comprised less than 70% of blood leucocytes (201 cases excluded), if the patient had received prior prednisone treatment or any other treatment for CLL (12 cases excluded), if flow cytometry analysis for Zap70 was not performed at the time of diagnosis (54 cases excluded), or if fluorescence in situ hybridization (FISH) analysis was not performed at the time of diagnosis (2 cases excluded). The final study population comprised 149 well-characterized CLL cases. As normal control samples (N = 9) we used CD19+ cells that were isolated by positive selection from peripheral blood mononuclear cells (PBMNCs) from healthy blood donors at the Blood Bank of Odense University Hospital using MACS Micro Beads, human (Miltenyi Biotech, Gladbach, Germany). The median age of normal controls was 61 years with a range of 54–66 years. The appropriate local scientific ethics committee approvals were obtained. 2.2. Real-time RT-PCR gene expression analysis Total RNA was extracted from 106 PBMNCs and eluted in a volume of 50 l elution buffer using the MagNA Pure LC RNA Isolation Kit High Performance with the MagNA Pure LC Instrument according to the manufacturer’s recommendations (Roche Applied Science, Mannheim, Germany). First strand complementary DNA
(cDNA) was then synthesized from 12.5 l RNA using the M-MLV Reverse Transcriptase kit (Invitrogen, Foster City, CA). The gene expression analysis was performed using the ABI PRISM 7900HT Sequence detecting system (Applied Biosystems, Foster City, CA) using a custom made Taqman Array (Applied Biosystems, Foster city, CA) according to the manufacturer’s instructions. qPCR thermal cycling conditions were: 2 min at 50 ◦ C, 10 min at 94.5 ◦ C followed by 50 cycles of 30 s at 97 ◦ C, and 1 min at 59.7 ◦ C. The SDS software version 2.4 was used for the data collection. If no PCR product was present, the Ct value was set to 50 (N = 20, for PIK3R4). Gene expression levels, measured as the threshold cycle (Ct) values, were normalized to the three reference genes GAPDH, GUSB, and TBP using the normalization tool GeNorm in the StatMiner software (Integromics, Madrid, Spain) [22]: dCt = Cttarget − Ctreference,average. Relative gene expression levels were calculated using the 2−ddCt method. 2.3. Assessment for IGHV mutation status IGHV gene mutational status was analyzed using cDNA and leader primers as recommended by the European Research initiative on CLL [23]. In cases, where leader primers did not result in successful amplification of a PCR product, a second PCR using FR1 primers was performed. Sequencing was performed by direct sequencing of the PCR reaction using both forward and reverse primers with the BigDye Terminator v1.1 Cycle Sequencing Reaction Kit (Applied Biosystems, Foster City, CA). Alignment was performed using IgBLAST and sequences with less than 98% homology compared to the corresponding germline IGHV were considered mutated. 2.4. Fluorescence in situ hybridization (FISH) FISH analyses were performed on May Grünwald Giemsa (MGG) stained blood cells. The FISH panel (Vysis, Abbott Molecular, Abbott Park, IL) included specific probes for ATM (11q22), 13q14.3, TP53 (17p13) and the centromeric region of chromosome 12. 2.5. Flowcytometry assessment of Zap70 Data were acquired and analyzed using a FACSCanto II (BD Biosciences, San Jose, CA) instrument with FACSDiva 6.1.2 software (BD Biosciences, San Jose, CA). The cells were analyzed for Zap70, CD45, CD10, CD19, CD5, CD20, CD23, kappa, and lambda. CLL cells were defined as CD19+CD20(dim)CD23+CD5+ and the cut point for Zap70 positivity was 20% [24]. 2.6. Data analysis and statistical methods A Wilcoxon (Mann–Whitney U) test was used for comparison of gene expression levels. The Benjamini Hochberg approach was used to adjust for false discovery rate. TTT was the primary outcome endpoint, and was calculated from date of diagnosis until first day a patient received treatment. OS was a secondary endpoint and was calculated from date of diagnosis until death from any cause. Patients who did not receive treatment or did not die during the follow up period were censored by last follow up date. Survival curves were calculated by the Kaplan and Meier method and were compared using the log-rank test. Multivariate Cox regression analysis, either with TTT or OS as dependent values, was performed. Categorical data were compared by Pearson Chi-square. Stata ver. 13.1 (StataCorp LP, TX,) and SPSS ver. 21 (SPSS Ltd., Chicago, IL) were used for the statistical analysis. P values < 0.05 were considered significant.
Please cite this article in press as: Kristensen L, et al. High expression of PI3K core complex genes is associated with poor prognosis in chronic lymphocytic leukemia. Leuk Res (2015), http://dx.doi.org/10.1016/j.leukres.2015.02.008
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3. Results The main characteristics of the 149 patients are listed in Table 1. All data are from time of diagnosis. The median age was 71 years, with a range of 48–92 years. Sixty five percent were males and the majority of patients had Binet stage 1 disease (66%). Most of the patients had favorable prognostic characteristics, such as mutated IGHV (60%), low Zap70 expression (54%), or absence of both 11qand 17p- (86%). Median follow-up time was 60 months (range 0.4–95 months). Median TTT was 51 months (range 0–95 months). When treated, the patients were treated with either fludarabine based chemotherapy (n = 29), R-CHOP (n = 2), bendamustine (n = 2), leukeran (n = 33), rituximab monotherapy (n = 1) or prednisone (n = 3). Seventy (47%) of the patients received treatment during the follow-up period. The 1- and 3-year OS were 93.5% and 79.8% respectively, with a median OS of 82 months. The relative gene expression levels, measured as dCt values, for PIK3C3, PIK3R4, and BECN1 in the 149 patient samples were distributed as shown in Fig. 1. dCt values had a bell shaped distribution for BECN1 and PIK3C3. The median value was chosen as cut point for high and low expression for these genes. PIK3R4 expression (dCt) distributed into two groups, one group with high expression, and one group with expression below the limit of detection. The latter group comprised all the samples not producing a Ct value during the 50 cycles of PCR. The expression levels of the patient group were measured as fold increase/decrease relative to the average expression levels of the normal control group. For BECN1, the expression level was lower in CLL patients than in normal controls, with an overall threefold reduction (p < 0.01). Overall there was a 33-fold reduction in PIK3R4 expression in CLL patients compared to normal controls. For the PIK3R4 high group, the reduction was eightfold (p < 0.01) compared to normal controls. A fold change could not be calculated for the PIK3R4 low group, as the expression level was below the detection limit. There was no significant difference for PIK3C3 between CLL patients and normal controls (1.3 fold reduction, p = 0.13). The gene expression levels were correlated with the parameters listed in Table 1. The PIK3C3 expression level correlated to mutational status of IGHV (p = 0.01) whereas the expression level of BECN1 and PIK3R4 did not correlate to any of the prognostic markers of CLL. Fig. 2 shows the TTT and OS according to high versus low gene expression in all patients. The primary endpoint, TTT, was significantly longer in patients with low gene expression for all three genes. Patients with PIK3C3 high had a median TTT of 24 months whereas the median TTT was not reached for PIK3C3 low (p < 0.01). After 3 years, 57% of PIK3C3 high patients had received treatment compared with 30% of PIK3C3 low patients (p < 0.01). Patients with BECN1 high had a median TTT of 27 months compared with 77 months for BECN1 low (p = 0.01). At 3 years 55% of BECN1 high patients had received treatment compared with 33% of BECN1 low patients (p < 0.01). Patients with PIK3R4 high had a median TTT of 42 months whereas the median TTT was not reached for PIK3R4 low (p = 0.02). At 3 years 47% of PIK3R4 high patients had received treatment compared with 23% of PIK3R4 low patients (p = 0.03). Of the known prognostic parameters, Binet stage 1 (p < 0.01), mutated IGHV (p < 0.01), low Zap70 expression (p < 0.01), and presence of 13q- (p < 0.01) were associated with longer TTT. Presence of 11q(p = 0.02) and trisomy 12 (p = 0.02) were associated with shorter TTT. The number of 17p- cases was too low for meaningful statistical survival analysis. Analysis of the secondary endpoint, OS, showed that PIK3R4 low and BECN1 low both were associated with longer OS. Patients with BECN1 high had a median OS of 72 months whereas the median OS was not reached for BECN1 low (p < 0.01). Patients with PIK3R4 high had a median OS of 82 months whereas the median OS was not
Fig. 1. Distribution of dCt values for patients (N = 149). The dCt is inversely related to the gene expression level, meaning that a low dCt is equal to a high relative gene expression level. For PIK3R4, samples with a dCt above 10 represent the 22 samples with undetectable expression. A Ct value of 50 was assigned to these samples in order to calculate a dCt value.
Please cite this article in press as: Kristensen L, et al. High expression of PI3K core complex genes is associated with poor prognosis in chronic lymphocytic leukemia. Leuk Res (2015), http://dx.doi.org/10.1016/j.leukres.2015.02.008
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Fig. 2. Kaplan–Meier plots of TTT and OS for 149 CLL patients according to gene expression levels.
Please cite this article in press as: Kristensen L, et al. High expression of PI3K core complex genes is associated with poor prognosis in chronic lymphocytic leukemia. Leuk Res (2015), http://dx.doi.org/10.1016/j.leukres.2015.02.008
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Table 1 Distribution of gene expression levels and prognostic factors.
Age (years) median Gender Male (%) Female (%) Binet stage Stage 1 (%) Stage 2 (%) Stage 3 (%) Zap70 expression Low (%) High (%) IGHV hypermutation Mutated-CLL (%) Unmutated-CLL (%) FISH abnormalities 13q- (%) Normal (%) Trisomy 12 (%) 11q- (%) 17p- (%)
Total
PIK3C3
N = 149
High N = 75 (50%)
Low N = 74 (50%)
High N = 127 (85%)
PIK3R4 Low N = 22 (15%)
BECN1 High N = 75 (50%)
Low N = 74 (50%)
71
71
71
71
67
73
70
97(65) 52 (35)
48 (64) 27 (36)
49 (66) 25 (34)
84 (66) 43 (34)
13 (59) 9 (41)
52 (69) 23 (31)
45 (61) 29 (39)
99 (67) 21 (14) 28 (19)
47 (64) 10 (14) 17 (22)
52 (70) 11 (15) 11 (15)
84 (67) 19 (15) 23 (18)
15 (68) 2 (9) 5 (23)
48 (65) 11 (15) 15 (20)
51 (69) 10 (14) 13 (17)
80 (54) 69 (46)
36 (48) 39 (52)
44 (59) 30 (41)
64 (50) 63 (50)
16 (73) 6 (27)
36 (48) 39 (52)
44 (59) 30 (41)
90 (60) 59 (40)
38 (51)* 37 (49)
52 (70) 22 (30)
76 (60) 51 (40)
14 (64) 8 (36)
46 (61) 29 (39)
44 (59) 30 (41)
77 (52) 32 (22) 18 (12) 16 (11) 5 (3)
39 (51) 16 (50) 9 (50) 10 (63) 1 (20)
38 (49) 16 (50) 9 (50) 6 (37) 4 (80)
63 (82) 27 (84) 17 (94) 15 (94) 5 (100)
14 (18) 5 (16) 1 (6) 1 (6) 0 (0)
41 (53) 14 (44) 12 (67) 7 (44) 1 (20)
36 (47) 18 (56) 6 (33) 9 (56) 4 (80)
Binet stage was available for 148 patients. * Indicates a significance level of
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Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
High expression of PI3K core complex genes is associated with poor prognosis in chronic lymphocytic leukemia Louise Kristensen a , Thomas Kristensen a , Niels Abildgaard b , Mads Thomassen c , Mikael Frederiksen d , Torben Mourits-Andersen e , Michael Boe Møller a,∗ a
Department of Pathology, Odense University Hospital, Odense, Denmark Department of Hematology, Odense University Hospital, Odense, Denmark c Department of Genetics, Odense University Hospital, Odense, Denmark d Department of Hematology, Haderslev Hospital, Haderslev, Denmark e Department of Hematology, Hospital of Southwestern Jutland, Esbjerg, Denmark b
a r t i c l e
i n f o
Article history: Received 12 September 2014 Received in revised form 13 February 2015 Accepted 18 February 2015 Available online xxx Keywords: Chronic lymphocytic leukemia Autophagy BECN1 PIK3C3 PIK3R4 Prognosis
a b s t r a c t Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in the Western world. Autophagy is a highly conserved process in eukaryotic cells. In CLL autophagy is involved in mediating the effect of chemotherapy but the role of autophagy in CLL pathogenesis remains unknown. In the present study, we used real-time RT-PCR to analyze expression of the PIK3C3, PIK3R4, and BECN1 genes. These genes encode the components of the PI3K core complex, which is central to initiation of autophagy. A consecutive series of 149 well-characterized CLL cases from Region of Southern Denmark were included in the study. All three genes were observed to be independent markers of prognosis in CLL with high expression being associated with more aggressive disease. With this clear association with outcome in CLL, these genes thereby represent promising candidates for future functional studies on the role of autophagy in CLL, and they may further represent targets of treatment. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in the Western world [1]. The prognosis is highly variable with a median survival of 7–8 years [2]. Some patients live with the diagnosis without treatment while others have a more aggressive course. Treatment is not curative but longer periods of remission are achievable [1,3,4]. The PI3K (phosphoinositide 3-kinase) pathway plays an important role in the cell cycle and cancer development. PI3K’s are divided into three subclasses (classes I–III) according to their regulation and function [5]. Class I is the most studied class, and kinases participates in multiple cellular pathways central to cell regulation. They inhibit autophagy through the PI3K/AKT/mTOR pathway [6]. One of these class I kinases, PIK3C␦, is the target for treatment with Idelalisib in CLL patients [7]. Class III predominantly regulates membrane trafficking and has an important role in the autophagy pathway as it participates in the initiation core complex [5,6].
∗ Corresponding author at: Department of Pathology, Odense University Hospital, Winsløwsparken 15, 3rd Floor, 5000 Odense C, Denmark. Tel.: +45 6541 4806. E-mail address: [email protected] (M.B. Møller).
Autophagy is a highly conserved process in eukaryotic cells. Double membrane vesicles, autophagosomes, are formed to enclose organelles such as mitochondria and endoplasmatic reticulum. Autophagosomes fuse with lysosomes and their content is degraded by the lysosomal enzymes. The process of autophagy was initially described as a strategy for nutrition supply under stressed conditions but it may also lead to programmed cell death by an excessive activation of its self-degrading system. Autophagy takes place in both healthy and sick cells [8–11]. Autophagy can be divided into three phases: sequestration of cellular compounds, transport of the autophagosome to the lysosome, and degradation. The PI3K complex plays an important role in the initiation of autophagy were it controls vesicle nucleation [12]. In mammals the core complex is composed of BECN1 (beclin 1), PIK3C3 (VPS34), and PIK3R4 (VPS15). This complex serves as binding site for several proteins that are either promoters or inhibitors of autophagy [12–14]. In cancer, autophagy can participate in both tumor suppression and progression. Dependent on cellular context, it enables tumor cells to survive chemotherapy-mediated stress. On the other hand, it can maintain cellular homeostasis by removing damaged organelles and prevent the genomic damage that leads to cancer [9]. In leukemia, several studies have shown that autophagy
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Please cite this article in press as: Kristensen L, et al. High expression of PI3K core complex genes is associated with poor prognosis in chronic lymphocytic leukemia. Leuk Res (2015), http://dx.doi.org/10.1016/j.leukres.2015.02.008
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is important for the death of leukemic cells. In most of these studies, autophagy was affected by treatment with different chemotherapeutic agents [15]. In CLL, some drugs have an autophagy-mediated effect on CLL cells. An example is APO866, an inhibitor of nicotinamide phosphoribosyl transferase, which contributes to autophagic cell death [16]. Also dexamethasone, which is used clinically for CLL treatment, kills the malignant lymphocytes through induction of autophagy before the onset of apoptosis [17]. Another clinically useful agent in CLL treatment, fludarabine, also acts partly through autophagy. In sensitive CLL cells, a BECN1 signaling mechanism mediates autophagic cell death in response to fludarabine, whereas in resistant cells, basal autophagy prevents the cells from dying [18]. Another study shows that fludarabine and CAL-101, the PIK3Cıinhibitor now known as Idelalisib [7], induces autophagy, whereas neither chlorambucil or rituximab produced this effect [19]. The histone deacetylase inhibitor, MGCD0103, inhibits autophagy in CLL cells by activating the PI3K/AKT/mTOR pathway and down regulating the key regulators of autophagy [20]. Other studies have shown that Tenovin-6 (tnv-6), an antineoplastic molecule, is cytotoxic to CLL cells through dysregulated autophagy [21]. Autophagy thus appears to represent an important cellular mechanism involved in mediating the effect of chemotherapy in CLL, but the role of autophagy in CLL pathogenesis is unknown, and whether the level of autophagic activity is associated to outcome has not been studied. Because of the central role of the PI3K core complex in autophagy initiation, we therefore measured the mRNA expression levels of its components: BECN1, PIK3C3, and PIK3R4 in a cohort of untreated CLL patients in the present study. The expression levels were correlated to time to treatment (TTT) and overall survival (OS). All three genes were each observed to be independently associated with prognosis in CLL. 2. Material and methods 2.1. Patient material Patients diagnosed with CLL, according to the WHO classification, at the Department of Pathology, Odense University Hospital, Denmark, from January 2005 to June 2011, were included in the present study. A total of 418 patients fulfilled this criterion. Of these 418 cases, patients were excluded if lymphocytes comprised less than 70% of blood leucocytes (201 cases excluded), if the patient had received prior prednisone treatment or any other treatment for CLL (12 cases excluded), if flow cytometry analysis for Zap70 was not performed at the time of diagnosis (54 cases excluded), or if fluorescence in situ hybridization (FISH) analysis was not performed at the time of diagnosis (2 cases excluded). The final study population comprised 149 well-characterized CLL cases. As normal control samples (N = 9) we used CD19+ cells that were isolated by positive selection from peripheral blood mononuclear cells (PBMNCs) from healthy blood donors at the Blood Bank of Odense University Hospital using MACS Micro Beads, human (Miltenyi Biotech, Gladbach, Germany). The median age of normal controls was 61 years with a range of 54–66 years. The appropriate local scientific ethics committee approvals were obtained. 2.2. Real-time RT-PCR gene expression analysis Total RNA was extracted from 106 PBMNCs and eluted in a volume of 50 l elution buffer using the MagNA Pure LC RNA Isolation Kit High Performance with the MagNA Pure LC Instrument according to the manufacturer’s recommendations (Roche Applied Science, Mannheim, Germany). First strand complementary DNA
(cDNA) was then synthesized from 12.5 l RNA using the M-MLV Reverse Transcriptase kit (Invitrogen, Foster City, CA). The gene expression analysis was performed using the ABI PRISM 7900HT Sequence detecting system (Applied Biosystems, Foster City, CA) using a custom made Taqman Array (Applied Biosystems, Foster city, CA) according to the manufacturer’s instructions. qPCR thermal cycling conditions were: 2 min at 50 ◦ C, 10 min at 94.5 ◦ C followed by 50 cycles of 30 s at 97 ◦ C, and 1 min at 59.7 ◦ C. The SDS software version 2.4 was used for the data collection. If no PCR product was present, the Ct value was set to 50 (N = 20, for PIK3R4). Gene expression levels, measured as the threshold cycle (Ct) values, were normalized to the three reference genes GAPDH, GUSB, and TBP using the normalization tool GeNorm in the StatMiner software (Integromics, Madrid, Spain) [22]: dCt = Cttarget − Ctreference,average. Relative gene expression levels were calculated using the 2−ddCt method. 2.3. Assessment for IGHV mutation status IGHV gene mutational status was analyzed using cDNA and leader primers as recommended by the European Research initiative on CLL [23]. In cases, where leader primers did not result in successful amplification of a PCR product, a second PCR using FR1 primers was performed. Sequencing was performed by direct sequencing of the PCR reaction using both forward and reverse primers with the BigDye Terminator v1.1 Cycle Sequencing Reaction Kit (Applied Biosystems, Foster City, CA). Alignment was performed using IgBLAST and sequences with less than 98% homology compared to the corresponding germline IGHV were considered mutated. 2.4. Fluorescence in situ hybridization (FISH) FISH analyses were performed on May Grünwald Giemsa (MGG) stained blood cells. The FISH panel (Vysis, Abbott Molecular, Abbott Park, IL) included specific probes for ATM (11q22), 13q14.3, TP53 (17p13) and the centromeric region of chromosome 12. 2.5. Flowcytometry assessment of Zap70 Data were acquired and analyzed using a FACSCanto II (BD Biosciences, San Jose, CA) instrument with FACSDiva 6.1.2 software (BD Biosciences, San Jose, CA). The cells were analyzed for Zap70, CD45, CD10, CD19, CD5, CD20, CD23, kappa, and lambda. CLL cells were defined as CD19+CD20(dim)CD23+CD5+ and the cut point for Zap70 positivity was 20% [24]. 2.6. Data analysis and statistical methods A Wilcoxon (Mann–Whitney U) test was used for comparison of gene expression levels. The Benjamini Hochberg approach was used to adjust for false discovery rate. TTT was the primary outcome endpoint, and was calculated from date of diagnosis until first day a patient received treatment. OS was a secondary endpoint and was calculated from date of diagnosis until death from any cause. Patients who did not receive treatment or did not die during the follow up period were censored by last follow up date. Survival curves were calculated by the Kaplan and Meier method and were compared using the log-rank test. Multivariate Cox regression analysis, either with TTT or OS as dependent values, was performed. Categorical data were compared by Pearson Chi-square. Stata ver. 13.1 (StataCorp LP, TX,) and SPSS ver. 21 (SPSS Ltd., Chicago, IL) were used for the statistical analysis. P values < 0.05 were considered significant.
Please cite this article in press as: Kristensen L, et al. High expression of PI3K core complex genes is associated with poor prognosis in chronic lymphocytic leukemia. Leuk Res (2015), http://dx.doi.org/10.1016/j.leukres.2015.02.008
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3. Results The main characteristics of the 149 patients are listed in Table 1. All data are from time of diagnosis. The median age was 71 years, with a range of 48–92 years. Sixty five percent were males and the majority of patients had Binet stage 1 disease (66%). Most of the patients had favorable prognostic characteristics, such as mutated IGHV (60%), low Zap70 expression (54%), or absence of both 11qand 17p- (86%). Median follow-up time was 60 months (range 0.4–95 months). Median TTT was 51 months (range 0–95 months). When treated, the patients were treated with either fludarabine based chemotherapy (n = 29), R-CHOP (n = 2), bendamustine (n = 2), leukeran (n = 33), rituximab monotherapy (n = 1) or prednisone (n = 3). Seventy (47%) of the patients received treatment during the follow-up period. The 1- and 3-year OS were 93.5% and 79.8% respectively, with a median OS of 82 months. The relative gene expression levels, measured as dCt values, for PIK3C3, PIK3R4, and BECN1 in the 149 patient samples were distributed as shown in Fig. 1. dCt values had a bell shaped distribution for BECN1 and PIK3C3. The median value was chosen as cut point for high and low expression for these genes. PIK3R4 expression (dCt) distributed into two groups, one group with high expression, and one group with expression below the limit of detection. The latter group comprised all the samples not producing a Ct value during the 50 cycles of PCR. The expression levels of the patient group were measured as fold increase/decrease relative to the average expression levels of the normal control group. For BECN1, the expression level was lower in CLL patients than in normal controls, with an overall threefold reduction (p < 0.01). Overall there was a 33-fold reduction in PIK3R4 expression in CLL patients compared to normal controls. For the PIK3R4 high group, the reduction was eightfold (p < 0.01) compared to normal controls. A fold change could not be calculated for the PIK3R4 low group, as the expression level was below the detection limit. There was no significant difference for PIK3C3 between CLL patients and normal controls (1.3 fold reduction, p = 0.13). The gene expression levels were correlated with the parameters listed in Table 1. The PIK3C3 expression level correlated to mutational status of IGHV (p = 0.01) whereas the expression level of BECN1 and PIK3R4 did not correlate to any of the prognostic markers of CLL. Fig. 2 shows the TTT and OS according to high versus low gene expression in all patients. The primary endpoint, TTT, was significantly longer in patients with low gene expression for all three genes. Patients with PIK3C3 high had a median TTT of 24 months whereas the median TTT was not reached for PIK3C3 low (p < 0.01). After 3 years, 57% of PIK3C3 high patients had received treatment compared with 30% of PIK3C3 low patients (p < 0.01). Patients with BECN1 high had a median TTT of 27 months compared with 77 months for BECN1 low (p = 0.01). At 3 years 55% of BECN1 high patients had received treatment compared with 33% of BECN1 low patients (p < 0.01). Patients with PIK3R4 high had a median TTT of 42 months whereas the median TTT was not reached for PIK3R4 low (p = 0.02). At 3 years 47% of PIK3R4 high patients had received treatment compared with 23% of PIK3R4 low patients (p = 0.03). Of the known prognostic parameters, Binet stage 1 (p < 0.01), mutated IGHV (p < 0.01), low Zap70 expression (p < 0.01), and presence of 13q- (p < 0.01) were associated with longer TTT. Presence of 11q(p = 0.02) and trisomy 12 (p = 0.02) were associated with shorter TTT. The number of 17p- cases was too low for meaningful statistical survival analysis. Analysis of the secondary endpoint, OS, showed that PIK3R4 low and BECN1 low both were associated with longer OS. Patients with BECN1 high had a median OS of 72 months whereas the median OS was not reached for BECN1 low (p < 0.01). Patients with PIK3R4 high had a median OS of 82 months whereas the median OS was not
Fig. 1. Distribution of dCt values for patients (N = 149). The dCt is inversely related to the gene expression level, meaning that a low dCt is equal to a high relative gene expression level. For PIK3R4, samples with a dCt above 10 represent the 22 samples with undetectable expression. A Ct value of 50 was assigned to these samples in order to calculate a dCt value.
Please cite this article in press as: Kristensen L, et al. High expression of PI3K core complex genes is associated with poor prognosis in chronic lymphocytic leukemia. Leuk Res (2015), http://dx.doi.org/10.1016/j.leukres.2015.02.008
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Fig. 2. Kaplan–Meier plots of TTT and OS for 149 CLL patients according to gene expression levels.
Please cite this article in press as: Kristensen L, et al. High expression of PI3K core complex genes is associated with poor prognosis in chronic lymphocytic leukemia. Leuk Res (2015), http://dx.doi.org/10.1016/j.leukres.2015.02.008
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Table 1 Distribution of gene expression levels and prognostic factors.
Age (years) median Gender Male (%) Female (%) Binet stage Stage 1 (%) Stage 2 (%) Stage 3 (%) Zap70 expression Low (%) High (%) IGHV hypermutation Mutated-CLL (%) Unmutated-CLL (%) FISH abnormalities 13q- (%) Normal (%) Trisomy 12 (%) 11q- (%) 17p- (%)
Total
PIK3C3
N = 149
High N = 75 (50%)
Low N = 74 (50%)
High N = 127 (85%)
PIK3R4 Low N = 22 (15%)
BECN1 High N = 75 (50%)
Low N = 74 (50%)
71
71
71
71
67
73
70
97(65) 52 (35)
48 (64) 27 (36)
49 (66) 25 (34)
84 (66) 43 (34)
13 (59) 9 (41)
52 (69) 23 (31)
45 (61) 29 (39)
99 (67) 21 (14) 28 (19)
47 (64) 10 (14) 17 (22)
52 (70) 11 (15) 11 (15)
84 (67) 19 (15) 23 (18)
15 (68) 2 (9) 5 (23)
48 (65) 11 (15) 15 (20)
51 (69) 10 (14) 13 (17)
80 (54) 69 (46)
36 (48) 39 (52)
44 (59) 30 (41)
64 (50) 63 (50)
16 (73) 6 (27)
36 (48) 39 (52)
44 (59) 30 (41)
90 (60) 59 (40)
38 (51)* 37 (49)
52 (70) 22 (30)
76 (60) 51 (40)
14 (64) 8 (36)
46 (61) 29 (39)
44 (59) 30 (41)
77 (52) 32 (22) 18 (12) 16 (11) 5 (3)
39 (51) 16 (50) 9 (50) 10 (63) 1 (20)
38 (49) 16 (50) 9 (50) 6 (37) 4 (80)
63 (82) 27 (84) 17 (94) 15 (94) 5 (100)
14 (18) 5 (16) 1 (6) 1 (6) 0 (0)
41 (53) 14 (44) 12 (67) 7 (44) 1 (20)
36 (47) 18 (56) 6 (33) 9 (56) 4 (80)
Binet stage was available for 148 patients. * Indicates a significance level of
