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Results:In the training set, high PKM2 mRNA levels were associated with decreased progression-free survival (PFS; 4.9 months vs 6.4, P=0.006), overall survival (OS; 10.1 vs 17.0 months, P=0.01) and disease control rate (DCR; 57.7% vs 74.3% P=0.021) compared to patients with low PKM2 levels. In the validation set, high PKM2 mRNA levels were also associated with deceased PFS (3.7 vs 5.9 months, P=0.006), OS (8.3 vs 16.8 months, P=0.003) and DCR (57.7% vs 70.9% P=0.049) compared to those with low PKM2 mRNA levels.
There was no correlation between the PKM2 mRNA levels and the PFS (5.6 vs 5.9, P=0.43) or the OS (9.8 vs 10.1, P=0.51) in the control group. Multivariate analysis revealed high PKM2 mRNA expression as an independent predictive factor for the poor patients' outcome. Systemic platinum-based chemotherapy remains the mainstay for the treatment of advanced non-small-cell lung cancer (NSCLC) since it improves survival, symptom control and quality of life compared to best supportive care.
Despite these advances, response to front-line chemotherapy remains poor since patients experience disease progression on an average of 4–6 months from the treatment initiation, and 1-year survival rate is. Patient populationFormalin-fixed, paraffin-embedded (FFPE) tissues from 148 consecutive patients with histologically confirmed stage IIIB (with pleural effusion) and IV NSCLC who were treated with front-line platinum-based chemotherapy were retrospectively collected and analysed (training set).
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The validation set consisted of 157 NSCLC patients with unresectable stage IIIB (with pleural effusion) or IV from an independent cohort of patients receiving platinum-based chemotherapy doublets in the front-line setting. Furthermore, 85 NSCLC patients who were treated with front-line non-platinum-based doublets were enrolled (control group). The used platinum-based regimens in the three cohorts of patients are presented in. All the above mentioned patients received front-line treatment in the context of two randomised trials conducted by the Hellenic Oncology Research Group. The study was approved by the Ethics and Scientific Committees of the University General Hospital of Heraklion. All patients gave their written informed consent for the use of their tissue for translational research.
TrainingValidationControlFeatureN%N%N%14815785GenderMale7891FemaleMedian age (range) years60 (34–78)61 (31–80)62 (37–79)Tumour typeSquamous20Non-squamous6880ECOG PS0–211456StageIIIB (wet)28IV6172Platinum-based first-linePlatinum+docetaxelNAPlatinum+docetaxel+Avastin18122214NAPlatinum+gemcitabine15101711NAPlatinum+pemetrexed5474NANon-platinum-based first-lineDocetaxel+gemcitabineNANA85100Post-progression treatment5565EGFR mutational statusEGFR wt273410EGFR mut441EGFR uknown11711974PKM2 mRNA expressionMedian (range)12.77 (0.34–71.88)NA12.73 (0.36–70.34)High expression49Low expression51. Specimen selection and RNA extractionFormalin-fixed, paraffin-embedded tumour sections were examined by a pathologist (EL) in order to identify the most appropriate tumour areas for dissection.
Serial sections of 5 μm were prepared and stained with Nuclear Fast Red (Sigma-Aldrich, St Lious, MO, USA). In the case of samples with. MRNA expression analysisReverse transcription and RT–qPCR have been described elsewhere. Briefly, 200 ng of total RNA were used for cDNA synthesis using SuperScript III reverse transcriptase (Invitrogen).
Relative cDNA quantification for PKM2 and β-actin and phosphoglycerate kinase 1 ( PGK) as internal controls was performed using the ABI Prism 7900 HT Sequence Detection System (AB, Foster City, CA, USA).The primers and 5′-labelled fluorescent reporter dye (6FAM) probe sets were designed using the Primer Express 2.0 Software (AB) according to the Ref Seq for PKM2 and were as follows: PKM2, 5′-GCCATAATCGTCCTCACCAAGT-3′ (forward), 5′-GCACGTGGGCGGTATCTG-3′ (reverse) and 5′-CAGGTCTGCTCACCAGG-3′ (probe). The primers and probe sequences for both housekeeping genes, β-actin and PGK have been reported elsewhere. Comparative Ct method was used for gene expression analysis using both β-actin and PGK as reference genes and commercial RNA (Stratagene, La Jolla, CA, USA) as calibrators. Final expression values were determined as follows: 2 −(ΔCT sample−ΔCT calibrator), where ΔCT values of the calibrator and sample were determined by subtracting the CT value of the target gene from the mean value of both reference genes.
In all experiments, only triplicates with a s.d. Of the CT value. Immunohistochemistry and staining evaluationApproximately, 4 μm-thick FFPE tissue sections were stained with haematoxylin and eosin and histopathologically verified by a pathologist. The primary antibody used for PKM2 staining was directed against the specific sequence of exon 9 that is unique to PKM2 (rabbit polyclonal Ab, cat. 3198, Cell Signaling, Danvers, MA, USA; dilution 1: 600). Immunostaining was performed using Ultra Vision LP Quanto Detection System HRP Polymer (Thermo Fisher Scientific, Fremont, CA, USA).
Sections stained for PKM2 were previously treated in Tris-EDTA buffer (pH 9.0) for 13 min. Staining evaluation was performed by two independent pathologists (EL and AK) blinded to each other's scores and to each patient's clinical information. Staining of PKM2 was scored as the product of the intensity on a scale of 0–5: low and 5.1–8: high expression, as described elsewhere. Study design and statistical analysisProgression-free survival (PFS) and overall survival (OS) were calculated from the start of treatment to the first documented disease progression or death, respectively. Objective responses were recorded according to the RECIST criteria. Cutoff points were calculated according to the median value for the mRNA expression. Samples with mRNA expression above or equal to the median were considered as samples with high expression, while those with value below the median as samples with low expression.
All the laboratory analyses were performed blinding to the clinical data.The potential association between baseline characteristics, response and gene expression levels were compared with either the two-sided Fisher's exact test or the chi-square test for categorical variables and the Kruskal–Wallis test for continuous variables. The normality of continuous variables was verified with the Kolmogorov–Smirnov test. The association of risk factors with time-to-event end points was analysed with the log-rank test and the Kaplan–Meier method was used to plot the corresponding time-to-progression and survival curves. A univariate Cox regression analysis, with hazard ratios (HRs) and 95% confidence intervals (CIs), was used to assess the association between each potential prognostic factor and survival and time to progression. These factors were then included in a multivariate Cox proportional hazards regression model with a stepwise procedure (both forward and backward) to evaluate the independent significance of different variables on survival and time to progression. Statistical significance was set at P=0.05.
PKM2 mRNA expression and patients' outcomeIn total, 390 NSCLC patients were treated with front-line platinum and/or non-platinum containing doublets in the context of two randomised trials conducted by the Hellenic Oncology Research Group. Among the 305 patients treated with platinum containing doublets, 148 and 157 of them were analysed as the training and the validation set, respectively.
The remaining 85 platinum-naive patients constituted the control set. PKM2 mRNA expression was successfully assessed in all of the samples analysed and the level of expression was associated with PFS and OS. Flow chart of NSCLC patients analysed for PKM2 mRNA expression.The median mRNA expression level was 12.77 (minimum, maximum: 0.34, 71.88) for the training set and the same cutoff was used for the analysis of the validation and control sets. In 30 tumour specimens, which were randomly selected from the training and the validation sets that were stained for PKM2 protein expression by immunohistochemistry, there was no correlation between PKM2 mRNA and protein expression (Spearman's test, P=0.275; data not shown). Moreover, there was no significant correlation between the PKM2 mRNA expression and the patients' age, gender, tumour histology, stage and PS (all P-values 0.05).In the training set, patients with high tumoural PKM2 mRNA levels had significantly shorter median PFS (4.9 vs 6.4 months; P=0.006; ) compared to patients with low tumoural PKM2 mRNA levels. Similarly, patients with high mRNA expression of PKM2 were significantly associated with decreased median OS (10.1 vs 17.0; P=0.01; ) compared to the patients with low expression levels. On the contrary, there was no significant correlation between the PKM2 mRNA levels and the objective response to cisplatin-based chemotherapy ( P=0.497; ).
However, when the analysis was performed according to the clinical benefit (DCR) a significant correlation was observed between PKM2 mRNA expression levels and DCR (; 74.3% and 57.7% DCR for patients with low and high PKM2 mRNA expression, respectively; P=0.021). Abbreviations: CR=complete response; DCR=disease control rate; ORR=objective response rate; PD=progressive disease; PR=partial response; SD=stable disease.Results in the validation set were similar to those in the training set. Median PFS was significantly decreased in patients with high mRNA expression of PKM2 (3.7 vs 5.9 months; P=0.006; ) in comparison with those with low mRNA levels. Furthermore, patients with high PKM2 mRNA levels had significantly decreased median OS (8.3 vs 16.8; P=0.003; ) as compared with those whose tumours had low PKM2 mRNA levels.
Similarly to the training set, although there was no significant correlation between PKM2 mRNA levels and objective response rate ( P=0.390; ) in the validation set, a marginal correlation with the DCR (70.9% vs 57.7% P=0.049; ) was observed.Finally, the analysis of the whole group of patients (after combining the training and the validation sets) clearly revealed that high PKM2 mRNA expression was associated with decreased PFS (3.9 vs 6.3 months; P=0.001), OS (9.6 vs 16.8 months; P. Univariate and multivariate analysisUnivariate analysis in the whole group of patients enrolled in both the training and validation sets revealed that patients with high tumoural PKM2 mRNA expression levels (HR: 1.89, 95% CI: 1.48–2.27; P=0.003), PS of 2 (HR: 2.64, 95% CI: 1.89–3.17; P=0.001) as well as stage IV (HR: 1.77, 95% CI: 1.11–2.31; P=0.03) were significantly associated with decreased PFS, whereas age 70 years ( P=0.77), gender ( P=0.51), histology ( P=0.61) and tumour differentiation ( P=0.14) did not show any significant correlation with the PFS. Similarly, high PKM2 mRNA expression (HR: 1.93, 95% CI: 1.54–2.46; P=0.002) and PS of 2 (HR: 2.87, 95% CI: 1.92–3.44; P70 years ( P=0.23), gender ( P=0.48), stage IV ( P=0.17), histology ( P=0.94) and tumour differentiation ( P=0.31) were not significantly associated with decreased OS.
Abbreviations: CI=confidence interval; OS=overall survival; PFS=progression-free survival; PS=performance status.Multivariate Cox regression analysis revealed that high tumoural PKM2 mRNA expression (HR: 1.81, 95% CI: 1.40–2.38; P=0.002) as well as PS of 2 (HR: 3.97, 95% CI: 2.77–4.16; P. DiscussionIn the current study, we investigated the role of tumoural PKM2 mRNA expression levels as a predictive factor in the outcome of metastatic NSCLC patients treated with front-line platinum-based chemotherapy. In a training set of 148 patients, those with tumours having low mRNA levels of PKM2 presented significantly higher PFS ( P=0.006) and OS ( P=0.01) and DCR ( P=0.021) as well. Our results were confirmed in an independent cohort of 157 patients who have also been treated with cisplatin-based chemotherapy in the first-line setting. Patients with low PKM2 mRNA levels attained statistically significant increase of PFS ( P=0.006) and OS ( P=0.003) and higher DCR ( P=0.049) as well.
Unlike the results in the training and validation set, in the control group of 85 patients, who did not received platinum-based chemotherapy, PKM2 mRNA levels were not correlated with PFS ( P=0.43) and OS ( P=0.51). This observation clearly suggests that the predictive value of PKM2 mRNA levels is mainly related to the platinum compounds. This association could not be attributed to a possible effect of tyrosine kinase inhibitors administered to our patients since the number of patients who received this anti-EGFR treatment was very low.
Furthermore, multivariate analysis revealed that high PKM2 mRNA expression was an independent predictive factor for shorter PFS and decreased OS in both the training and validation sets.PKM2 is one of the four isoforms of pyruvate kinase, the enzyme that catalyses the formation of pyruvate and ATP from PEP and ADP. The four isoforms of pyruvate kinase are encoded by two genes that are expressed in a cell- and tissue-specific manner. The L and R isoenzymes, derived from the PKLR gene are expressed in the liver and red blood cells, respectively. The PKM gene encodes the M1- and M2-type isoenzymes. It consists of 12 exons, of which 9 and 10 are alternatively spliced in a mutually exclusive manner to give rise to the PKM1 and PKM2 isoforms, respectively. Alternative splicing in PKM gene is regulated by heterogeneous nuclear ribonucleoproteins hnRNP, under the control of c-Myc (; ). From the four isoforms of pyruvate kinase, cancer cells exclusively express the M2 isoform.
PKM2 but not PKM1 is necessary for aerobic glycolysis since the replacement of PKM2 by PKM1 reduced the capacity of tumour cell lines to develop into a tumour. PKM2 is negatively regulated in response to growth factors by binding to tyrosine-phosphorylated proteins (; ). Phosphorylation of PKM2 results in the formation of its inactive dimeric form that enables the diversion of glycolytic intermediates into anabolic pathways (; ). Also, PKM2 enzymatic activity can be modulated by a variety of post-translational modifications such as acetylation, sumoylation, ubiquitination and oxidation (;;; ). The lack of significant correlation between PKM2 mRNA and protein expression observed in the current study could be explained on the basis of post-translational modifications mentioned above.
It is obvious that IHC and mRNA expression analysis in a larger cohort of patients would confirm this observation.The role of PKM2 to modulate the cytotoxicity of cisplatin and its derivatives is as yet not fully explored. Proteomic analysis showed that PKM2 is downregulated in oxaliplatin-resistant cell lines, while high mRNA expression was associated with higher response rate in oxaliplatin-treated colorectal cancer patients.
In the same line of evidence it was shown that PKM2 protein and activity were lower in cisplatin-resistant human gastric carcinoma cell lines. In contrast, our results are in the opposite direction, since low PKM2 mRNA levels were associated with better outcome of NSCLC patients both in the training and validation set. This evidence is in agreement with previous results from our laboratory on SCLC patients treated with platinum-based chemotherapy, since patients with low expression levels of PKM2 attained significantly better PFS and OS. Accordingly, results from previous studies showed that inhibition of PKM2 mRNA expression by siRNA targeting in combination with chemotherapeutic agents, significantly increased apoptosis and decreased tumour volume in xenograft models.Several mechanisms of resistance to platinum compounds, either intrinsically or acquired, have been described. Decreased membrane transport, increased cytoplasmic detoxification, increased DNA repair activity and increased tolerance to DNA damage are the major mechanisms that can contribute to cisplatin resistance (; ). Excision repair complementation group 1 (ERCC1) and breast cancer susceptibility gene 1 (BRCA1) that participate in DNA-repair pathways have been considered as potential predictive factors, since their level of expression seems to be correlated and influence cisplatin efficacy in a variety of solid tumours.
It is also well established that platinum analogues interact with sulfur-containing thiomolecules such as glutathione and metallothionein leading to its inactivation and subsequent failure from binding to DNA (;; ). Also, studies in preclinical models and tumour samples suggest that elevated levels of glutathione or glutathione-related enzymes are associated with limited cisplatin efficacy (; ). Furthermore, glutathione as an antioxidant offers protection from ROS that are known to accumulate and induce apoptosis after radiotherapy and chemotherapeutic drug treatment (; ). Recent studies suggest that PKM2 might play a role in the control of glutathione and ROS concentrations, implying a possible role to cisplatin resistance. In human lung cancer cells, increased concentrations of ROS can inhibit PKM2 activity through oxidation of cysteine 358 (Cys 358; ). This resulted in the diversion of glucose intermediates into pentose phosphate pathway (PPP), which produces nicotinamide adenine dinucleotide phosphate (NADPH). Nicotinamide adenine dinucleotide phosphate provides reducing equivalents for the reduction of oxidised GSH (GSSG) to reduced GSH, thereby increasing ROS detoxification.
The above regulation mechanism of ROS concentrations by PKM2 seems to be specific since an oxidation-resistant mutant form of PKM2 failed to confer antioxidant response. Additionally, PKM2 has been described to regulate ROS accumulation by interacting with the cell-surface marker of stem cells, CD44 (, ). This interaction promotes glycolysis and increases the flux to PPP resulting to the production of NADPH and to the subsequent increase of reduced GSH and decrease ROS accumulation. The role of CD44 to inhibit ROS accumulation in cancer cells has also been addressed earlier.
It is questionable if the observed association between tumoural PKM2 mRNA expression and the poor patient's outcome in our study could be explained on the basis of the resistance mechanisms described above. Preliminary data from our laboratory strengthens this possibility since they demonstrate a strong positive correlation between PKM2 and CD44 expression in ovarian tumour specimens from patients treated with cisplatin in front line (data not shown).Tumour cells have multiple ways to regulate PKM2 both in transcriptional and post-transcriptional level, in order to ensure anabolic metabolism and survival under hypoxic conditions, PKM2 acting in a positive feedback loop interacts with HIF1α through the prolyl hydroxylase 3 and promotes the activation of HIF1α regulated genes. This positive feedback loop maintains expression of PKM2 and other glycolytic enzymes in high levels. Accordingly, the above mechanism seems to be biologically relevant since preliminary data from our laboratory have demonstrated that there is a strong positive correlation between PKM2 and HIF1α mRNA expression in NSCLC tumour samples (data not shown). A strong positive correlation also exist between PKM2 and c-Myc mRNA expression (data not shown). Therefore, as argued by our data, measuring PKM2 mRNA levels by RT–PCR in clinical samples seem to be reasonable.Although our results are retrospectively originated, in the best of our knowledge this study is the first one providing evidence for the predictive significance of a biomarker by validating the results in an independent cohort of patients.
Also, unpublished data from our laboratory have evaluated the predictive significance of PKM2 in other tumour types treated with cisplatin or its derivatives. However, this evidence has to be interpreted with caution and any clinical relevance of the tumoural PKM2 mRNA expression should be validated prospectively.
Furthermore, it is an interesting issue to investigate whether PKM2 mRNA are influenced by chemotherapy by using a rebiopsy in the metastatic tumour. In addition, it remains a challenge that has to be answered using in vitro models, to elucidate at which level of PKM2 regulation, either transcriptional or post-transcriptional, could modulate anticancer-drug cytotoxicity. Anastasiou D, Poulogiannis G, Asara JM, Boxer MB, Jiang JK, Shen M, Bellinger G, Sasaki AT, Locasale JW, Auld DS, Thomas CJ, Vander Heiden MG, Cantley LC. Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses. 2011; 334 (6060:1278–1283.
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