Abstract
Background/Aim: Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that is deeply involved in the pathogenesis of lung cancer. We evaluated the metabolic characteristics of non-small cell lung cancer (NSCLC) in patients with and without expression of EZH2 using 18F-fluorodeoxyglucose positron-emission tomography/ computed tomography (18F-FDG PET/CT). Materials and Methods: The EZH2 protein expression of 268 patients with resected NSCLC, whose preoperative 18F-FDG PET/CT information was available, was evaluated by immunohistochemistry with clone 6A10 antibody; cases with Allred score ≥3 were considered positive. Results: Maximized standard uptake values in cases of EZH2-positive NSCLC and adenocarcinoma were significantly higher in comparison to EZH2-negative cases (p<0.001, and p<0.001, respectively). The expression of EZH2 in NSCLC and adenocarcinoma was associated with significantly poorer survival. Conclusion: In patients with NSCLC and adenocarcinoma, EZH2 positivity was associated with a high uptake of FDG, suggesting that EZH2 might define metabolically malignant lung cancer.
Throughout the world, lung cancer remains a deadly neoplasm; however, the elucidation of the genetic pathogenesis has paved the way for a new era in the management of lung cancer (1). Alterations in the epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) genes are representative driver oncogenes in non-small cell lung cancer (NSCLC), and specific inhibitors, such as gefitinib and crizotinib, have dramatically prolonged the survival of patients with oncogene-driven lung cancer (2, 3). Furthermore, newly-emerged immune checkpoint inhibitors against programmed death-1 and programmed death-ligand 1, such as nivolumab and atezolizumab, have attracted a great deal of attention due to the superior antitumor response that they afford in comparison to standard chemotherapy (4-6). Thus, the clinical course of patients with lung cancer has been changing dramatically.
In addition to the above-mentioned mechanisms, epigenetic dysregulation, including DNA methylation, histone modifications and non-coding RNA, have been shown to be involved in the pathogenesis of various types of cancer, including lung cancer (7). Among histone modifications, histone methylation is an important process that mainly regulates the transcription of the genes associated with the pathogenesis of cancer (8). Thus far, approximately 65 histone methyltransferases have been identified; some of these can be targeted by specific inhibitors (9).
Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase, which has a catalytic SET domain as a conserved domain among species. EZH2 methylates lysine residue 27 on histone H3 (H3K27), which results in repression of transcription (10). EZH2 was shown to be associated with murine embryonic development through the regulation of H3K27-methylation-related transcription (10). Furthermore, a series of reports has shown the deep involvement of EZH2 in aggressive features, such as the proliferation and survival of various types of cancer, including lung cancer, bladder cancer and melanoma (11-14). Our previous report demonstrated that EZH2 is overexpressed in lung and bladder cancer in comparison to normal organ tissues, and that the down-regulation of EZH2 by siRNA significantly suppresses proliferation in lung cancer cell lines (11). Specific inhibitors targeting EZH2 have been developed and are currently under evaluation in clinical trials (9, 13). Thus, much attention has been paid to the significance of EZH2 in lung cancer.
The metabolic activity (the glucose metabolism) of cancer cells can be measured by 18F-fluorodeoxyglucose positron-emission tomography/computed tomography (18F-FDG PET/CT), and the uptake of FDG has been reported to be associated with the biological features of cancer cells such as proliferation, the histological type, and the clinical stage (15-20). In addition, 18F-FDG PET/CT is an important tool in predicting the prognosis of lung cancer (21, 22). Furthermore, several reports have demonstrated the correlation between oncogenic alterations in EGFR and ALK genes and the metabolic tumor burden on 18F-FDG PET/CT, suggesting a possible role of 18F-FDG PET/CT in predicting the presence of such oncogenes (23-25). However, the relationship between glucose metabolism and the expression of EZH2 has yet to be investigated. Given the aforementioned aggressive feature of EZH2-expressing lung cancer, we hypothesized that the uptake of FDG might be associated with the expression of EZH2 in patients with lung cancer.
The current study was conducted in order to clarify the association between the expression of EZH2 and the metabolic tumor burden in NSCLC, as measured by the maximum standardized uptake value (SUVmax) on preoperative 18F-FDG PET/CT.
Materials and Methods
Patients. We retrospectively examined patients with primary NSCLC who underwent complete surgical resection at the Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University. Among them, we selected the patients whose preoperative 18F-FDG PET/CT information was available. In total, 223 patients with adenocarcinoma (ADC) and 45 patients with squamous cell carcinoma (SCC), who underwent surgical resection from December 2003 to December 2012, were eligible for inclusion in our study. A total of 268 paraffin-embedded specimens were retrieved from the registry of the Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University. Patients with a history of SCC of the head and neck or esophagus were excluded from this study because the possibility of metastatic squamous cell carcinoma from these cancers could not be excluded. The clinicopathological features, including the age at surgery, sex, smoking status, pathological tumor-node-metastasis (TNM) stage (seventh edition of the lung cancer staging system), pleural or lymphovascular invasion and SUVmax were examined. Tumor differentiation, the histological subtype of ADC (World Health Organization Classification 2015) (26), and the EGFR mutation status were also examined in a subset analysis of the cases of ADC. The EGFR status had been determined in 191 tumor tissue specimens using the peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp method (Mitsubishi Chemical Medience, Tokyo, Japan) (27). The clinical information and follow-up data were obtained from the patients' medical records. This study was approved by our Institutional Review Board (approval number: 28-380).
18F-FDG PET/CT. For each patient, 185 MBq of FDG was intravenously injected after fasting for at least 4 h. Scans were conducted from the middle of the thigh to the top of the skull at 60 min after the administration of FDG. FDG-PET/CT images were obtained using an integrated PET/CT scanner, Discovery STE (GE Medical Systems, Milwaukee, WI) or Biograph mCT (Siemens Medical Solutions, Erlangen, Germany). All emission scans were performed in the three-dimensional mode. The acquisition time per bed position was 3 min for Discovery STE and 2 min for Biograph mCT. We reconstructed the PET images using the ordered-subset expectation-maximization method (VUE Point Plus; GE Healthcare, Hino city, Tokyo, Japan) with two full iterations of 28 subsets for Discovery STE and the iterative True-X algorithm and TOF (Ultra HD-PET) with two full iterations of 21 subsets. The True-X algorithm incorporates an additional specific correction for the point-spread function. The full-width at half-maximum values of Discovery STE and Biograph mCT were 5.2 and 4.4 mm, respectively. A low-dose 16-slice CT (tube voltage, 120 kV; effective tube current, 30-250 mA, Discovery STE) and a low-dose 32-slice CT (tube voltage. 120 kV; the use of angular and longitudinal dose modulation, CAREDose4D®, Biograph mCT) from the vertex to the proximal thigh were performed for attenuation correction, and for determining the precise anatomic location of the lesions before the acquisition of the PET image. The CT scan was reconstructed by filtered back projection into 512×512 pixel images with a slice thickness of 5 mm to match the PET scan. The uptake of FDG in the lesion was evaluated based on the SUVmax, which was calculated by a dedicated workstation for each scanners.
Immunohistochemical analysis. Immunohistochemistry was performed in 268 cases of surgically resected primary NSCLC using formalin-fixed tissue sections. Sections were cut (4 μm thickness), dewaxed with xylene, and rehydrated through a graded series of ethanol. After inhibition of endogenous peroxidase activity for 30 min with 3% H2O2 in methanol, the sections were pretreated with Target Retrieval Solution (Dako, Glostrup, Denmark) in a decloaking chamber at 110°C for 15 min and then incubated with monoclonal antibodies at 4°C overnight. Immune complexes were detected with a DAKO EnVision Detection System (Dako). The sections were finally reacted in 3,3’-diaminobenzidine, counterstained with hematoxylin, and mounted. The primary antibody was a mouse monoclonal antibody to human EZH2 (clone 6A10, dilution 1:100; Leica Biosystems, Newcastle-upon-Tyne, UK). Carcinoma cells showing nuclear staining for EZH2 were considered to be positively stained cells. The evaluations were performed independently by three investigators (G.T., K.T. and M.K.). When the independent assessments did not agree, the slides were reviewed together to achieve consensus. The consensus judgments were adopted as the final results. Allred scores were applied to discriminate between the positive (≥3) and negative (<3) cases (28). Human tonsil sections were used as positive controls.
Statistical analysis. The associations between the expression of EZH2 protein and patient characteristics were analyzed using Fisher's exact test. The univariate and multivariate analyses of the relationship between the expression of EZH2 and other patient characteristics were performed by using logistic regression with a backward elimination method. Receiver operating characteristic (ROC) curve analyses were used to determine the SUVmax cut-off values. We examined the association between the EZH2 expression frequency and the SUVmax in preoperative 18F-FDG PET/CT scans using Student's t-test. Overall survival (OS) was defined as the time from the initial surgery until death from any cause, while the disease-free survival (DFS) was defined as the time from the initial surgery until recurrence. The Kaplan–Meier method was used to estimate the probability of survival. The curves of the two groups were compared using the log-rank test. All of the statistical analyses were conducted using JMP version 12 software (SAS Institute, Cary, NC, USA). p-Values of less than 0.05 were considered to indicate a statistically significant difference.
Results
Association between the expression of EZH2 and SUVmax in NSCLC patients. A total of 268 patients with NSCLC who underwent complete surgical resection, including 223 with ADC and 45 with SCC, were included in the present study (Table I). The median age of the patients was 68 years (range: 37-86 years) and 149 (55.6%) patients were male. One hundred and thirty-six patients (50.7%) had a history of smoking. Patients with stage I, II and III NSCLC comprised 72.4%, 17.9%, and 9.7%, respectively. Table II and III show the characteristics of the ADC and SCC patients.
Table IV shows a summary of the frequency of EZH2 positivity and the SUVmax on PET/CT. The expression of EZH2 was positively detected in 117 (43.7%) patients (all NSCLC), 43 (34.5%) with ADC, and 40 (88.9%) with SCC. The mean of SUVmax in the patients overall was 6.32. The SUVmax in EZH2-positive patients was significantly higher in comparison to EZH2-negative patients in the analyses of overall NSCLC (p<0.001; Table II and Figure 1A), and ADC (p<0.001; Table II and Figure 1B); there was no significant difference in SUVmax between the EZH2-positive and EZH2-negative SCC cases [data not shown; SCC: 10.67 (2-31.05) and 10.99 (5.6-17.62), respectively, p<0.918).
Univariate and multivariate analyses of the association between the expression of EZH2 and other patient characteristics. The association between the expression of EZH2 and the other patient characteristics was also analyzed. The optimal cut-off SUVmax values were determined using ROC curves. The cut-off SUVmax values for NSCLC overall, ADC, and SCC were 4.4, 4.1, and 9.3, respectively (Figures 2 and 3). A multivariate analysis revealed that the SUVmax, as well as the presence of vascular invasion and SCC histology, were independent predictors of EZH2 positivity in patients with NSCLC (Table III). Furthermore, the SUVmax was an independent predictor of EZH2 positivity in patients with ADC (Table V). The presence of vascular invasion and an invasive histological subtype also independently predicted the expression of EZH2 (data not shown). In contrast, the SUVmax was not a predictor of the EZH2 positivity in SCC patients (data not shown).
DFS and OS according to the expression of EZH2. The DFS and OS of patients with EZH2-positive NSCLC were significantly worse in comparison to those of EZH2-negative patients (Figure 4A and B; log-rank: p<0.001 and p<0.001, respectively). There was a significant difference in the DFS and OS of EZH2-positive and EZH2-negative patients with ADC (Figure 4C and D; log-rank: p<0.001 and p=0.004, respectively). In contrast, there was no significant difference in the DFS or OS of EZH2-positive and EZH2-negative SCC patients (data not shown; log-rank: p=0.418 and p=0.305, respectively).
Discussion
EZH2 is a histone methyltransferase which is frequently overexpressed in cancer in comparison to normal tissues, and is deeply involved in the pathogenesis, proliferation, and survival in various types of cancer, including lung cancer (11-14). Recently, the EZH2-driven tumorigenesis of lung cancer was demonstrated to be distinct from KRAS- and EGFR-mutated lung cancer (13); however, other reports have shown a close association between EZH2 and KRAS-driven lung cancer (29, 30). In addition to the tumorigenic role of EZH2, the expression of EZH2 has been shown to be involved in the sensitivity to platinum-based chemotherapy, antiangiogenic therapy, and topoisomerase II inhibitors (31-33). Clinically, several clinicopathological factors, such as younger age, a history of smoking, and advanced tumor stage, were significantly associated with higher expression of EZH2 in patients with lung ADC (14). Several EZH2 inhibitors have been developed and are currently under investigation for use in the treatment of human malignancies (9, 13). Thus, the biological and clinical significance of EZH2-expressing lung cancer has been gradually clarified, and it is possible that inhibitors against lung cancer with expression of EZH2 protein might have clinical application; thus, it would be beneficial to clarify the clinicopathological characteristics of patients with the expression of EZH2 protein in greater detail. In this translational study, we focused on the relevance between the expression of EZH2 and the metabolic tumor burden measured by 18F-FDG PET/CT, which can reveal meaningful clinical information associated with proliferation, the histological type, and the clinical stage of lung cancer (15-20).
The SUVmax values of EZH2-positive patients were significantly higher than those of EZH2-negative patients in the analyses of overall NSCLC and ADC, suggesting that the expression of EZH2 is a marker of high metabolic activity in overall NSCLC and ADC. These findings correspond to the abovementioned aggressive features of EZH2-expressing cancer cells (11-14). Furthermore, in the present study, the expression of EZH2 was significantly associated with vascular invasion and the invasive subtypes of ADC (Table III and IV). Furthermore, patients with EZH2-positive NSCLC and ADC exhibited significantly poorer DFS and OS in comparison to EZH2-negative patients, which is in line with previous reports (11, 14, 34). To the best of our knowledge, the current study is the first to show a statistically significant association between the metabolic tumor burden and the expression of EZH2 protein in surgically resected NSCLC, specifically ADC.
The biological mechanism underlying the elevated metabolic activity in EZH2-positive lung cancer might involve the AKT signaling pathway. The report by Kaira et al. suggested that the uptake of FDG in lung cancer is regulated by molecules that are associated with the glucose metabolism, hypoxia, angiogenesis, and the AKT signaling pathway (15). Preclinical experiments demonstrated that AKT phosphorylates EZH2 and negatively regulates the methyltransferase activity of EZH2, which leads to tumorigenesis (33). In addition to EZH2 expression and phosphorylation of EZH2, STAT3 activity can be reduced via the depletion of PI3K/AKT in lung cancer cell lines harboring a KRAS mutation at codon 12 (29). Furthermore, EZH2 was reported to induce AKT-dependent genomic instability in breast cancer (36). These findings suggest the association between high uptake of FDG and the expression of EZH2 protein might reflect the activation of the AKT signaling pathway.
The current study may offer another important finding. It suggests the possible role of 18F-FDG PET/CT in predicting the expression of EZH2. The multivariate analysis revealed that a high SUVmax on PET/CT was a predictor of EZH2 positivity in patients with overall NSCLC and ADC. Previous reports have shown that oncogenic alterations in EGFR and ALK genes are associated with metabolic tumor burden on 18F-FDG PET/CT, suggesting the possible role of 18F-FDG PET/CT in predicting expression of such oncogenes (23, 24). In addition, the role of 18F-FDG PET/CT in predicting a T790M mutation in the EGFR gene, which mediates resistance to EGFR-tyrosine kinase inhibitors has been reported: the detection of T790M was associated with a lower SUVmax (25). There are no reports as far as we are aware showing a relevant association between the expression of EZH2 and the metabolic characteristics of lung cancer. Given the possibility that EZH2 inhibitors might be clinically applied in the treatment of lung cancer, predicting the expression of EZH2 by convenient and non-invasive tools, such as 18F-FDG PET/CT, might be beneficial; however, standardized methods for defining the cut-off SUVmax value should be established through further studies with a larger study population.
This study is associated with several limitations, including its retrospective nature, single-institutional design and limited sample size. Large-scale, multi-institutional, prospective studies to investigate the relationship between the expression of EZH2 and the metabolic tumor burden are warranted.
In conclusion, EZH2-expressing NSCLC, specifically ADC, was associated with high glucose metabolism. The SUVmax on preoperative 18F-FDG PET/CT was found to be a predictor of the expression of EZH2 in patients with NSCLC, specifically those with ADC.
Footnotes
Disclosure
The Authors declare no conflicts of interest in association with the present study.
- Received January 14, 2017.
- Revision received February 24, 2017.
- Accepted February 28, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved