Expression of choline kinase alpha to predict outcome in patients with early-stage non-small-cell lung cancer: a retrospective study

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Expression of choline kinase alpha to predict outcome in patients with early-stage non-small-cell lung cancer: a retrospective study
Transcript Published online September 11, 2007 DOI:10.1016/S1470-2045(07)70279-6 1 ArticlesExpression of choline kinase alpha to predict outcome in patients with early-stage non-small-cell lung cancer: a retrospective study  Ana Ramírez de Molina, Jacinto Sarmentero-Estrada, Cristóbal Belda-Iniesta, Miquel Tarón, Victor Ramírez de Molina, Paloma Cejas, Marcin Skrzypski, David Gallego-Ortega, Javier de Castro, Enrique Casado, Miguel Angel García-Cabezas, Jose Javier Sánchez, Manuel Nistal, Rafael Rosell, Manuel González-Barón, Juan Carlos Lacal Summary Background   Adequate prognostic markers to predict outcome of patients with lung cancer are still needed. The aim of this study was to assess whether choline kinase alpha (ChoKα) gene expression could identify patients with different prognoses. ChoKα is an enzyme involved in cell metabolism and proliferation and has a role in oncogene-mediated transformation in several human tumours, including lung cancer. Methods   60 patients with non-small-cell lung cancer (NSCLC) who had undergone surgical resection in a single centre were enrolled into the study as the training group. We used real-time reverse-transcriptase PCR (RT-PCR) to measure ChoKα gene expression and analyse the association between ChoKα expression and survival in evaluable patients. Additionally, a second group of 120 patients with NSCLC from a different hospital were enrolled into the study as the validation group. We did an overall analysis of all 167 patients who had available tissue to confirm the cut-off point for future studies. The primary endpoints were lung-cancer-specific survival and relapse-free survival. Findings   Seven of the 60 patients in the training group were not evaluable due to insufficient tissue. In the 53 evaluable patients, the cut-off for those with ChoKα overexpression was defined by receiver operator under the curve (ROC) methodology. 4-year lung-cancer-specific survival was 54·43% (95% CI 28·24–80·61) for 25 patients with ChoKα expression above the ROC-defined cut-off compared with 88·27% (75·79–100) for 28 patients with concentrations of the enzyme below this cut-off (hazard ratio [HR] 3·14 [0·83–11·88], p=0·07). In the validation group, six of the 120 enrolled patients were not evaluable due to insufficient tissue. For the other 114 patients, 4-year lung-cancer-specific survival was 46·66% (32·67–59·65) for those with ChoKα expression above the ROC-defined cut-off compared with 67·01% (50·92–81·11) for patients with concentrations of ChoKα below the cut-off (HR 1·87 [1·01–3·46], p=0·04). A global analysis of all 167 patients further confirmed the association between ChoKα overexpression and worse clinical outcome of patients with NSCLC: 4-year lung-cancer-specific survival for ChoKα expression above the ROC-defined cut-off was 49·00% (36·61–60·38) compared with 70·52% (59·80–76·75) for those with concentrations of ChoKα below the cut-off (HR 1·98 [1·14–3·45], p=0·01). The overall analysis confirmed the cut-off for ChoKα expression should be 1·91-times higher than concentrations noted in healthy tissues when ChoKα is used as an independent predictive factor of relapse-free and lung-cancer-specific survival in patients with early-stage NSCLC. Interpretation   ChoKα expression is a new prognostic factor that could be used to help identify patients with early-stage NSCLC who might be at high risk of recurrence, and to identify patients with favourable prognosis who could receive less aggressive treatment options or avoid adjuvant systemic treatment. New treatments that inhibit ChoKα expression or activity in patients with lung cancer should be studied further. Introduction Lung cancer is the leading cause of cancer-related death, accounting for one-third of all deaths from cancer worldwide. 1  Lung cancer consists of a number of diseases of diverse aetiology, divided broadly into small-cell lung cancer (SCLC), which comprises 20% of all lung cancers, and non-small-cell lung cancer (NSCLC), which accounts for the remaining 80%. 2  NSCLC is thought to srcinate in lung epithelial cells and includes diverse histological subtypes, including adenocarcinoma, bronchioloalveolar, squamous, anaplastic and large-cell carcinoma. 2  Despite intensive research over the past decades, 5-year survival of patients with lung cancer is less than 15%. 3  Currently, the most accurate prognostic factor for patients with NSCLC is tumour, node, metastasis (TNM) staging. 3,4   However, patients with early-stage NSCLC have a broad spectrum of survival, which indicates the need for additional prognostic parameters to better predict disease outcome. 3  In the past decade, efforts have focused on the identification of molecular markers that could identify patients with a high risk of relapse after curative surgical resection. 5  To improve global survival, several immunohisto-chemical markers have been proposed as prognostic indicators, including KRAS, P53, PRb, Ki-67, and the excision repair cross-complementation protein ERCC1. 6–9  Additionally, other molecules, such as cyclooxygenase-2, E-cadherin, vascular endothelial growth factor (VEGF), Published  Online September 11, 2007 DOI:10.1016/S1470-2045(07)70279-6 Translational Oncology Unit, CSIC-UAM-La Paz, National Center of Biotechnology, Madrid, Spain  (A Ramírez de Molina PhD,  J Sarmentero-Estrada MSc, C Belda-Iniesta MD, P Cejas PhD, D Gallego-Ortega MSc,  J de Castro MD, E Casado MD, M A García-Cabezas PhD, Prof M Nistal PhD, Prof M González-Barón MD, Prof J C Lacal PhD) ; Medical Oncology Division (C Belda-Iniesta, P Cejas, J de Castro, E Casado, Prof M González-Barón) and Pathology Department ( M A García-Cabezas, Prof M Nistal),  La Paz University Hospital, Madrid, Spain; Catalan Institute of Oncology, Hospital Germans Trias i Pujol, Badalona, Spain (M Tarón PhD, Prof R Rosell MD) ; Preventive Medicine, San Carlos Hospital, Madrid, Spain (V Ramírez de Molina MD) ; Medical University of Gdansk, Gdansk, Poland (M Skrzypski MD) ; and Department of Statistics, Autonoma University, Madrid, Spain (Prof J J Sánchez PhD)Correspondence to: Prof Juan Carlos Lacal, National Center of Biotechnology, CSIC, Darwin 3, Campus de Cantoblanco, 28049 Madrid, Spain  Articles 2 Published online September 11, 2007 DOI:10.1016/S1470-2045(07)70279-6 and epidermal-growth-factor receptor (EGFR), have been analysed at the mRNA level for their prognostic usefulness. 10–12  These developments validate the use of techniques based on mRNA measurements as useful and highly reproducible methods for the classification and prognosis of various types of cancers. 13–16  However, given that the 5-year overall survival of patients with lung cancer is still poor, a better understanding of the molecular biology of tumours, new biological prognostic markers, and new approaches for treatment are still needed to improve clinical diagnosis, therapeutic treatment and, consequently, patient outcome.Choline kinase alpha (ChoKα), the enzyme responsible for the generation of phosphorylcholine, is involved in metabolic processes and cell proliferation. 17,18  Studies have shown a role for ChoKα in oncogene-mediated trans-formation 19,20  and in the generation of human tumours, including lung cancer. 21–25  Also, ChoKα has been described as a new oncogene that mediates human cell transform-ation and induces in-vivo tumsrcenesis. 26 Furthermore, ChoKα-specific inhibitors have been shown to have in-vitro antiproliferative and in-vivo antitumoral activity against human xenografts. 27–32  In this study, we aimed to assess whether ChoKα gene expression could identify patients with different prognoses. Methods Patients 60 patients were enrolled initially in the training group and we analysed frozen specimens of cancer tissue from 53   patients for whom we had tissue, who had NSCLC, and who had undergone surgical resection for NSCLC between February, 2001, and December, 2004, and who were followed up by the Medical Oncology Division at La Paz University Hospital, Madrid, Spain. Inclusion criteria were: age 18 years or older; completely resected NSCLC; intraoperative mediastinal-node dissection for reliable mediastinal staging or biopsy of nodes at N3; and, in accordance with our hospital’s criteria for surgery, Eastern Cooperative Oncology Group (ECOG) scores of 0 or 1. Patients who fulfilled the inclusion criteria were included in the analysis if enough good-quality frozen tissue was available for the analysis. All evaluable patients had had a perioperative CT scan that confirmed localised disease. Exclusion criteria were: involvement of tracheobronchial angle nodes (station 10), mixed histological features, and other cancers in the previous 5 years. Follow-up was done according to the criteria used in the Thoracic Surgery Department, La Paz University Hospital, and included clinical assessments and CT of thorax every 3 months for 2 years, and every 6 months thereafter. Clinical and radiological data on clinical history were confirmed by the oncologist at the corresponding institution and were recorded by an independent observer at La Paz University Hospital. We validated data from the training group by comparing them with data from a separate group of patients from Hospital Germans Trias i Pujol, Badalona, Spain,   and who had undergone surgical resection of NSCLC between May, 2002, and September, 2005 (in the validation group, 120 patients were enrolled initially for whom we had tissue for 114 patients). All patients gave written informed consent. This study was approved by the institutional review board of the hospitals involved. Clinical data were retrieved and revised by CBI and JDC (La Paz University Hospital), and MS and RR (Hospital Germans Trias i Pujol, Badalona). Procedures All cell lines used in this study were maintained under standard conditions of temperature (37ºC), humidity (95%), and carbon dioxide (5%). Human primary bronchial epithelial cells (BEC; CC-2541; Cambrex, Walkersville, MD, USA) were grown in bronchial epithelial cell growth media (BEGM; CC-3170; BulletKit (Cambrex). Human primary mammary epithelial cells (HMEC; CC-2551; Clonetics, San Diego, CA, USA) were grown in mammary epithelial basal medium (MEBM) supplemented with a bullet kit as recommended by the manufacturer (CC-3150; Clonetics). Epithelial NSCLC cells H460 and H1299, and SCLC cell lines H510 and H82, were maintained in Roswell Park Memorial Institute (RPMI) medium supple-mented with 10% fetal bovine serum (FBS; Life Technologies, Grand Island, NY, USA). Cells were propagated twice a week, and new nitrogen stocks were used for all cell lines after eight rounds of propagation. The specific ChoK inhibitor MN58b 17,27–30 was provided by TCD Pharma SL (Madrid, Spain) free of charge. Frozen tissue samples were homogenised and lysed in buffer that contained 1·5 mmol/L magnesium chloride, 0·2 mmol/L EDTA, 0·3 M sodium chloride, 25 mmol/L 4-2-hydroxyethyl-1-piperazinethanesulphonic acid (HEPES) at pH 7·5, 20 mmol/L β-glycerophosphate, and 0·1% Triton X-100 (Merck, Darmstadt, Germany). Cells were lysed in ice-cold lysis buffer (50 mmol/L Tris, pH 7·4, 0·25% NP-40, 0·25% sodium dodecyl sulphate, 150 mmol/L   sodium chloride, 15 mmol/L β-glycero-phosphate, 10 mmol/L sodium pyrophosphate, 50 mmol/L sodium fluoride, 10 mg/mL aprotinin, and 1 mmol/L   phenylmethylsulfonyl fluoride [PMSF]). Nuclei and detergent-insoluble material were removed by centrifugation at 13 000 rpm [A1] for 20 min at 4ºC. Western blot analysis of equal amounts of cell lysates (30 µg) was done by the use of each corresponding antibody. Proteins were resolved by electrophoresis onto 10% dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to nitro-cellulose. Blots were blocked for 2 h in 5% non-fat dried milk in Tris-Tween buffered saline (T-TBS). Measurement of ChoKα was done by the use of a monoclonal antibody against human ChoKα. 33  Blots were washed in T-TBS three times  Articles Published online September 11, 2007 DOI:10.1016/S1470-2045(07)70279-6 3 for 10 min each and incubated for 1 h with ChoKα anti-body (ratio of 1:5000). As loading control, blots were assayed against α-tubulin (dilution ratio of 1:4000; T9026; Sigma-Aldrich, St Louis, MO, USA).Choline kinase assays were done by use of homogenised cells or tissues (as described above) with the source of ChoK in buffer containing 100 mmol/L Tris-HCl at pH 8·0, 100 mmol/L magnesium chloride, and 10 mmol/L ATP. Choline at a physiological concentration of 200 µmol/L was used as a substrate in the presence of 14  C-methylcholine chloride (50–60 Ci per mmol/L, Amersham International, Didcot, UK). Reactions were done at 37°C for 30 min and stopped with ice-cold   trichloroacetic acid (TCA)   at a final concentration of 16%. Hydrophilic derivatives of choline (ie, choline, phosphorylcholine [PCho]) were resolved on thin-layer chromatography (TLC) plates (60 A silica gel, Whatman, Clifton, NJ, USA) with use of 0·9% sodium chloride:methanol:ammonium hydroxide (50mL:70mL:5mL; v:v:v) as the liquid phase. Radioactivity corresponding to PCho was quantified automatically by an electronic radiographic system (Instantimager; Packard, Meriden, CT, USA).For cell-proliferation assays, cells were seeded in 24-well plates at a density of 30×10³ cells/well, and incubated for 24 h under standard conditions, as described earlier. Cells were then treated with different concentrations of the ChoK inhibitor, MN58b, and maintained for the times indicated. Quantification of the number of cells remaining in each well was carried out by staining with crystal violet, as described previously. 19   Gene expression assays ChoKα mRNA concentrations were analysed by quantitative RT-PCR by the use of Taqman probes (Applied Biosystems, Foster City, CA, USA). Total RNA was extracted from cell lines by use of RNAeasy mini kit (Qiagen Inc, Valencia, CA, USA) and from clinical samples with Trizol Reagent (Invitrogen, Carisbad, CA, USA) in accordance with the manufacturer’s instructions. 1 µg of total RNA was reverse transcribed by High Capacity cDNA Archive Kit (Applied Biosystems), for 2 h at 37ºC. Gene-expression assays of cDNAs were done in triplicate by use of the ABI PRISM 7700 Sequence Detector (Applied Biosystems). GAPDH and 18S ribosomal mRNA were amplified as internal controls. Probes (Taqman Gene Expression Assays; CHKA Hs00608045_m1, Applied Biosystems) were used for the amplification of ChoKα. ChoKα expression in healthy tissue was measured by analysing a pool of RNA from nine different samples of healthy human lung tissues, and from commercial RNA from human lung tissue (catalogue number 540019, lot 0750468, Stratagene, La Jolla, CA, USA). [A2] Statistical analyses Two independent groups of patients were used for this study. Patients were also stratified in to pathological ChoKαα-tubulin200     P    C    h   o    (   c   o   u   n    t   s   p   e   r   m    i   n   u    t   e    )    /   µ   g   p   r   o    t   e    i   n 120010008006004000 BAC     L   o   g     1    0     (   r   e    l   a    t    i   v   e   q   u   a   n    t    i    t   y    ) 2·01·51·00·50·0–0·5BECH82H510H1299H460 Cell line Figure 1 : ChoKα overexpression in human lung cancer-derived cells (A) RT-PCR for ChoKα in four different human lung-tumour cell lines in relation to  [A3]  human primary BEC normalised to the endogenous control 18S; three independent experiments were done, and all samples were amplified in triplicate; means and SD are shown. (B) Protein expression analysed by western blot analysis of same cells shown in (A); concentrations of α-tubulin were used as loading control. (C) ChoK enzymatic activity in the same cells as (A); data are means and 95% CI of three independent experiments, each done in duplicate. PCho=phosphorylcholine.  Articles 4 Published online September 11, 2007 DOI:10.1016/S1470-2045(07)70279-6 stages for further analysis. Quantity of ChoKα expression normalised with the endogenous control (AQ) was calculated by the 2 -∆Ct  method for both healthy and tumour tissues (Applied Biosystems). Time to death was obtained for the analysis of lung-cancer-specific survival, and time to progression (defined as any relapse—local, distant, or local and distant) for the analysis of relapse-free survival. Both parameters were defined from the time of surgical procedure. Receiver operating characteristic (ROC) curves were obtained to show the relation between sensitivity and false-positive rate at different cut-off values of ChoKα expression for lung-cancer-specific survival and relapse-free survival. The cut-off value was established according to the best combination of sensitivity and false-positive rate (1–specificity) based on the ROC curves that were constructed for both endpoints (lung-cancer-specific survival and relapse-free survival). Optimum values of cut-off points obtained after these considerations in both groups of patients were nearly twice the concentration of the enzyme noted in healthy tissues.The Kaplan-Meier method was used to estimate lung-cancer-specific survival and relapse-free survival. Only death from recurrence of lung cancer was considered in the study. The effect of the different prognostic factors on tumour-related recurrence and survival was assessed by the log-rank test for univariate analysis. To assess the effect of ChoKα expression on survival, with adjustment for potential confounding factors, proportional hazards Cox regression modelling was used. Hazard ratios (HR) and 95% CI were calculated from the Cox regression model. All reported p values were two-sided. Statistical significance was defined as p<0·05. The statistical analyses were done by use of SPSS software (version 13.0). Role of the funding source The sponsors of the study had no role in the study design, data collection, data analysis, data interpretation, or in the writing of this report. ARM, CBI, MT, VRM, JC, JJS, RR, and JCL had access to the raw data and participated in the interpretation of results. The corresponding author had full access to all data and had final responsibility for the decision to submit for publication. Results ChoKα expression and activity were first established in different cell lines derived from human lung cancer. ChoKα mRNA levels, in terms of their corresponding normal human primary BEC, were increased in all the tumour cell lines analysed in this study (figure 1). The noted increase in mRNA levels resulted in an increase in protein expression and ChoK enzymatic activity (figure 1). Therefore, protein concentrations and enzymatic activity correlated well with mRNA concentrations. These results show that ChoKα is overexpressed, with a consequent increase in PCho production in lung-cancer cells. Accordingly, tumour cells were more sensitive to the antiproliferative effect of ChoKα inhibition than their corresponding primary human epithelial cells (table 1). Similar results were obtained when we used specific genetic interference of ChoKα with siRNA (data not shown). These results suggest that human lung-cancer cells might be appropriate candidates for an antitumoral treatment based on ChoKα inhibition.Surgical resection is the most important treatment for patients with early-stage NSCLC. To study whether ChoKα has any role in the development of this disease, ChoKα mRNA concentrations were measured by use of quantitative RT-PCR in patients with NSCLC whose tumours had been surgically resected. 60 patients were enrolled for the training group. Of these, seven patients were not evaluable due to insufficient tissue. The remaining 53 patients had median follow-up of 22 months (2–61). We identified local, distant, or local and distant recurrence in 13 of these patients, of which 11 patients died of lung cancer. IC 50  48 hIC 50  72 hIC 50 144 h Primary BEC40·5 (6·2)18·3 (4·8)4·2 (0·8)Primary HMEC44·7 (4·95) 20·9 (2·7)3·4 (0·13)H460 7·03 (2·03) [6]2·6 (0·8) [7]1·1 (0·1) [4]H1299 10·3 (2·5) [4]2·7 (0·7) [7]0·9 (0·1) [5]H510 1·1 (0·1) [37]0·4 (0·05) [46]0·1 (0·03) [42]H821·9 (0·2) [21]0·8 (0·04) [23]0·27 (0·01) [16] IC 50 =concentration of an inhibitor that is needed for 50% inhibition of its target. Data for the IC 50  are expressed in µmol/L (SD) after addition of MN58b. Numbers in square brackets indicate times   of sensitivity for each cell line compared with mean values noted for primary BEC cells. Data are the mean of four independent experiments, each done in triplicate, except for BEC cells that represent one single experiment done in triplicate. At any time point analysed, differences were found to be statistically significant between primary cells and the other four human tumour-derived cell lines (Mann-Whitney test, p≤0·001). Table 1:  Differential sensitivity to ChoK-specific inhibition of human lung cancer-derived cells and normal human primary cells TotalLow ChoKα expressionHigh ChoKα expression Patients, n 532825Median age, years (range)64 (43–84)64 (48–84)64 (43–81)SexMen 472621Women624Tumour typeSquamous-cell carcinoma362412Adenocarcinoma1239Other514Tumour stageIA550IB221012IIA202IIB642IIIA954IIIB-IV725Missing data2 2 0 Table 2: Characteristics of patients in the training group  Articles Published online September 11, 2007 DOI:10.1016/S1470-2045(07)70279-6 5 Median lung-cancer-specific survival time and median time to relapse were not reached in this group of patients at the time of assessment. 4-year lung-cancer-specific   survival was 69·29% (95% CI 50·92–87·65), and 4-year relapse-free survival was 68·56% (52·93–84·18). Pathological and clinical parameters of the patients included in this study are summarised in table 2.Gene-expression analysis showed that ChoKα expres-sion was distributed differentially in the tumours, with normalised AQ values of mRNA copies oscillating between 0·72 and 13·88. To establish how ChoKα expres-sion in tumour samples compared with values in healthy tissues, ChoKα expression was analysed in a pool obtained from apparently healthy tissues adjacent to tumours from the patients in this study, and from commercial RNA obtained from healthy human lung tissue. AQ values of ChoKα expression were 1·73 in the healthy tissues from our patients, and 1·29 in the commercially obtained tissue. According to ROC methodology, an arbitrary cut-off point (close to twice the concentration found in healthy tissue) of 2·82 AQ   was established. Under these conditions, 25 out of the 53 tumour samples analysed for ChoKα overexpression were above this cut-off. Patients with ChoKα overexpression had worse survival from lung cancer than those with lower concentrations of this enzyme. 4-year lung-cancer-specific survival was 54·43% (95% CI 28·24–80·61) in patients with ChoKα expression above the cut-off versus 88·27% (75·79–100·00) in those who had expression below the cut-off; figure 2). Relapse-free survival was 53·33% (29·22–76·43) in patients who had ChoKα expression above the cut-off compared with 84·54% (70·56–98·51) in those who had expression below the cut-off (figure 2). HR of ChoKα overexpression and increased risk of relapse was 3·00 (0·92–9·77), p=0·06, and HR of ChoKα overexpres-sion and early mortality was 3·14 (0·83–11·88), p=0·07. These results suggest that ChoKα could be a prognostic factor in early-stage NSCLC because its overexpression is associated with worse clinical outcome. To verify this hypothesis, we assessed ChoKα expression in a validation group of patients. Of the 120 patients who were enrolled into this group, six did not have sufficient tissue and were excluded from analysis. Median follow-up of the remaining 114 patients was 29·3 months (95% CI 1·7–65·9). In this group of patients, 51 patients relapsed (one had local recurrence, 13 had distant metastasis, and 37 had local and distant metastasis) and 44 patients died due to disease [A4] . No patients received adjuvant treatment.Median lung-cancer-specific survival of these patients was not reached at the time of assessment, and median time to relapse was 38·57 months (34·70–45·20). 4-year TotalLow ChoKα expressionHigh ChoKα expression Patients, n 1145757Median age, years (range)64 (37–103)63 (37–103)62 (43–77)SexMen884444Women261313Tumour typeSquamous-cell carcinoma824339Adenocarcinoma291217Other321Median tumour size, mm45 (10–140)51 (10–130)52 (15–144) Tumour stageIA1495IB482919IIB321220IIIA20 713 Table 3: Characteristics of patients in the validation group 0·0     S   u   r   v    i   v   a    l    f   r   o   m     l   u   n   g   c   a   n   c   e   r 1·00·80·60·40·2 A p=0·070·0     R   e    l   a   p   s   e  -    f   r   e   e   s   u   r   v    i   v   a    l 1·00·80·60·40·2 B p=0·05Low ChoKα expressionHigh ChoKα expression Numbers at risk 504030201007060 High ChoKα expressionLow ChoKα expressionTime (months)237102428··1 147121825··0 504030201007060 Time (months)237102228··1 145111525··0 Figure 2 : Kaplan-Meier plots for ChoKα overexpression and survival in the training group (A) Lung-cancer-specific survival. (B) Relapse-free survival.
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