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Neuron-Specific Enolase Is an Independent Prognostic Factor in Resected Lung Adenocarcinoma Patients with Anaplastic Lymphoma Kinase Gene Rearrangements

Background An extensive body of research reveals the clinical value of serum tumor markers in lung cancer patients, including carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCCA), cytokeratin-19 fragments (Cyfra21-1), and neuron-specific enolase (NSE), but little is known about the clinical properties of these serum tumor markers in anaplastic lymphoma kinase (ALK)-positive lung cancer patients. Matreial/Methods We retrospectively analyzed 54 patients harboring ALK rearrangements and 520 patients without ALK rearrangements, and all these patients were treated exclusively by surgery between 2011 and 2016. Results NSE level (P=0.007 for OS) was identified as an independent prognostic factor among patients with resected ALK-positive adenocarcinoma of the lung. Conclusions A high level of NSE is associated with worse outcome among resected lung adenocarcinoma patients harboring ALK rearrangements.


Background
The prognostic factors of lung cancer might be important in daily clinical practice due to its high prevalence and mortality rates. In recent years, the efficacy of cancer treatment has achieved a qualitative leap since many oncogenic drivers have been discovered, such as anaplastic lymphoma kinase (ALK) genes, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF). These developments have improved molecular diagnosis and accurate therapy for lung cancer patients.
The ALK gene was first described as NPM-ALK fusion protein, mainly in anaplastic large-cell lymphomas [1]. It accounts for about 6.7% of NSCLCs [2] and 11% of lung adenocarcinoma [3]. Although a low proportion of lung cancer patients have ALK rearrangements, the total number of the cases is considerable and these patients would benefit from molecular-targeted treatment. Thus, it is important to explore predictive factors among lung cancer patients that harbor ALK rearrangements in clinical practice.
In this study, we carried out a retrospective clinical research of a total 574 operable lung adenocarcinoma patients in which 54 patients had ALK rearrangements. We covered clinical factors between serum tumor markers and the clinical features in patients of stage I, II, and III who underwent partial or complete surgical operation. We explored the clinical features in ALK-positive lung adenocarcinoma and assessed whether these serum tumor markers are independent indicators for prognosis in these patients.

Patient selection
We retrospectively analyzed a database of consecutive lung adenocarcinoma patients treated by surgery between March 2011 and August 2016 at Tianjin Medical University Cancer Institute and Hospital. Approval by the Research Ethics Committee of Tianjin Medical University Cancer Institute was gained and its purpose was to explore the clinical significance of serum tumor markers at the beginning of diagnosis as prognostic factors in the subset of lung adenocarcinoma patients with ALK rearrangements. Examinations prior to the operation included physical examination, level of serum tumor biomarkers, blood chemistry analysis, CT scan, fiberoptic bronchoscopy, brain MRI, and bone scanning. All patients underwent partial or complete surgical resection exclusively. The selection criteria were as follows: (a) the pathological diagnosis was lung adenocarcinoma; (b) ALK-rearrangement positive (by the Ventana ALK [D5F3] IHC Kit, Roche, Switzerland); (c) clinical stages I, II, IIIA, or IIIB disease defined by the tumor node metastasis (TNM) criteria of NSCLC (AJCC,7th edition) and the TNM stage assessment was according to CT scans, brain MRI, and bone emission CT scans; (d) patients were untreated at the time routine blood analyses were performed; and (e) preoperative evaluation showed that surgery was appropriate for patients. Adjuvant treatment included chemotherapy, radiotherapy, or targeted therapy. Follow-up data were gathered directly from the clinical record or telephone follow-up. CT scans were checked every 3 or 4 months to monitor recurrence and then annually after 3 years. Disease-free survival (DFS) was from the date of surgery to recurrence, or the date of final follow-up. Overall survival (OS) was measured to the date to death caused by disease or to the date of final follow-up.

Tumor-associated antigens
The concentrations of serum tumor biomarkers were detected by immunoenzymometric assay (Roche Diagnostics, China) within 1 week before the operation. The cut-off values of serum tumor markers were as follows: CEA 5.0 ng/ml, SCCA 1.5 ng/ml, Cyfra21-1 3.3 ng/ml, and NSE 15.2 ng/ml, using the reference values provided by the manufacturer.

Statistical analysis
SPSS Statistics 24 (IBM Corporation, NY, USA) was used for all statistical analyses. Univariate analyses of the comparisons of baseline clinical characteristics between cohorts were carried out using the chi-squared test. Survival was conducted using Cox regression analysis. To examine differences in survival between different cohorts, the log-rank test was used. Cox proportional hazard method was applied to calculate hazard ratio (HR) and the 95% confidence intervals (CI), and the multivariate Cox method was performed using the backward selection method. In all analyses, a p-value less than 0.05 was regarded as significant.

Patient characteristics
Our study cohort included 574 lung adenocarcinoma patients who underwent partial or complete surgical resection between 2011 and 2016. As shown in Table 1 Table 3).

Univariate and multivariate analysis of all patients
Univariate analysis in all patients showed that clinical stage, surgical resection, metastasis in regional lymph node, tumor size, CEA level, and Cyfra21-1 level were associated with DFS, while OS was associated with clinical stage, surgical resection, metastasis in regional lymph node, CEA level, and Cyfra21-1 level (  (Table 2).

Univariate and multivariate analysis of ALK-positive and -negative patients
For ALK-positive patients, by univariate analysis, we found that DFS was related to smoking history, clinical stage, metastasis in regional lymph node, CEA level, and Cyfra21-1 level, while clinical stage, CEA, NSE, and Cyfra21-1 were correlated with OS (Table 3 For ALK-negative patients, by univariate analysis, we found that DFS was related to clinical stage, surgical resection, maximum size of tumor, metastasis in regional lymph node, CEA, and Cyfra21-1, while clinical stage, surgical resection, adjuvant treatment, metastasis in regional lymph node, CEA level, and Cyfra21-1 level were correlated with OS (Supplementary    Figure 1A).
For ALK-positive patients, the average NSE level was 13.86 ng/ml. Patients were also stratified into lower (n=44) and higher (n=10) NSE groups ( Table 3). The average DFS in high-NSE patients was 13.89 months, as compared with 19.51 months for low-NSE patients. The average OS for high-NSE patients was 19.26 months as compared with 26.83 months for low-NSE patients. The survival analysis revealed that high serum NSE level was significantly relevant to decreased OS (P=0.005) ( Figure 1B). Multivariate Cox regression analysis showed that NSE was an independent prognostic factor for OS (P=0.007).
For ALK-negative patients, the survival analysis revealed that high serum NSE level was not associated with OS (P=0.189) ( Figure 1C).

Discussion
Anaplastic lymphoma kinase is a receptor protein-tyrosine kinase of the insulin receptor protein-tyrosine kinase superfamily. EML4-ALK fusion was recently reported as a neoteric targeted therapy molecular in lung cancer and has become increasingly important in the diagnosis of lung cancer. Several studies have reported that more ALK-positive cases could be seen in light-or never-smoking patients [8], type of adenocarcinoma [9], and females and young patients [10]. ALK rearrangement has also been reported to be more common in males (P=0.039) [11], but other studies reported no sex difference [8,12]. In our study, consistent with the abovementioned studies, ALK expression was strongly correlated with age and smoking history and was more common in never-smoking and young patients. However, no sex difference was found in our study cohort. Due to the low positive rate or other reasons, the prognostic factors in ALK-positive lung adenocarcinoma patients are unclear and no prognostic models have been developed. Therefore, it is necessary to find effective prognostic factors among these patients.
A recent study including 173 ALK-positive NSCLC patients reported that neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) were closely related to survival, and PLR was a significant prognostic factor in ALK-positive NSCLC [13]. In the study by Suzuki et al. [14], CD56 + was reported to be associated with poor prognosis (P=0.002) in both ALK-negative and ALK-positive subgroups. Further research is needed to better predict the prognosis of ALK-positive patients. Serum tumor makers are routinely used in examinations after admission to help guide diagnosis, classification, and treatment of these patients. To the best of our knowledge, the present study is the first to report that the level of NSE in blood is an independent prognostic factor in ALK-positive lung adenocarcinoma patients.

681
CEA is a kind of glycoprotein involved in programmed cell death and cell adhesion, and is normally secreted during embryonic development, but the secretion stops before birth. Its specialized sialofucosylated glycoforms serve as functional ligands that may be vital to cancer cell metastasis [15]. CEA was reported to be an unspecific serum tumor marker with abnormal expression in various solid tumors. No consensus has been reached on the prognostic value of pretreatment CEA levels in lung cancer. Tomita et al. [16] reported that CEA was a useful serum tumor marker in NSCLC and that CEA was an important prognostic factor in NSCLC patients (p<0.0001), but other studies did not find this [5] To date, there have been no related reports on the relationship between CEA and ALK status. In our study, increased level of CEA was correlated with decreased DFS and OS in ALK-positive patients. CEA is involved in adhesion between cells and extracellular matrix, and its overexpression can inhibit cell differentiation and damage the structure of tissues. This could also partially be attributed to glycosylation of glycoproteins, which promote metastasis and invasion. The elevated level of CEA is one of the manifestations of activation of glycosylated enzymes, and other relevant molecules include AFP (a-fetoprotein) and NSE (neuron-specific enolase). Glycosylase activation leads to glycosylation of glycoproteins, which can affect ALK phosphorylation and downstream signaling [17][18][19], and thus have an effect on the prognosis of ALK-positive patients. In the present study, our cases were all adenocarcinoma patients, and CEA has already been shown to be predict poor prognosis in patients with resected lung adenocarcinomas [20].
CYFRA 21-1 is a fragment of cytokeratin (CK) 19. CK is the principal structural element of the cytoskeleton (keratin filaments) of epithelial cells, including bronchial epithelial cells. It elevated in many cell types of lung cancer, but mainly in squamous cell carcinoma [21]. It is reported that preoperative serum CYFRA 21-1 was a poor independent prognostic factor for adenocarcinoma of lung after surgery [22], which is consistent with our study. However, CYFRA 21-1 was reported to be unrelated to cancer recurrence or survival by Matsuoka et al. and Blankenburg et al., respectively [23,24]. In our study, CYFRA 21-1 was found to be associated with decreased DFS and OS in ALK-positive patients. High serum CYFRA 21-1 level might reflect poor differentiation because multi-directional differentiation indicates tumor aggressiveness and is also considered as a poor prognostic factor in early-stage lung cancer.
NSE is a neuron-specific isoenzyme of enolase, also known as enolase-g. The clinical significance of NSE in small cell lung cancer (SCLC) had been well accepted, since NSE is reported to be frequently elevated in patients diagnosed with SCLC [25]. However, the value of NSE in NSCLC remains unclear. In the study of Pujol et al. [7], NSE was reported to be a prognostic factor for survival. Similar results were found by other studies, including Jacot et al. [26] and Ferrigno et al. [27]. However, in the study of Reinmuth et al. [28], preoperative serum NSE level was not found to be related to prognosis. To the best of our knowledge, no research has focussed on determining the predictive role of NSE in ALK-positive lung adenocarcinoma patients. As described in our study, NSE is an independent prognostic factor for survival in ALK-positive lung adenocarcinoma patients. Since mixed element of SCLC-NSCLC may arise in a small fraction of all lung cancer cases [29] and some NSCLC patients do have an elevated NSE level, we hypothesized that the association between poor prognosis and elevated pretreatment serum NSE level in NSCLC patients might be due a co-existing neuroendocrine or small cell component in the solid tumor or tumor glucose metabolism [30]. Recently, Choi et al. [31] reported that higher glucose metabolism was found in EML4-ALK-rearranged NSCLC than in EML4-ALK-negative NSCLC patients, revealing that EML4-ALK may play a role in regulating glucose metabolism. Ma et al. [32] reported that EML4-ALK activates aerobic glycolysis, while fast ATP and a carbon source for biosynthesis require a high glycolysis rate, and thus make cancer cells replicate faster. NSE is considered a key enzyme in glycolysis and it plays an important role in aerobic glycolysis; this could partially explain how NSE affects the prognosis of ALK-positive patients. However, the adverse prognostic significance of elevated NSE was also presumed to be more malignant in lung cancers with neuroendocrine differentiation.
Adjuvant chemotherapy is not generally recommended for earlystage patients in daily clinical work. As described in our study, an elevated serum NSE level is associated with poor prognosis in ALK-positive patients. Therefore, we suggest postoperative adjuvant therapy for resected ALK-positive patients with elevated serum NSE levels.
Our study has some limitations. First, it was a single-center, retrospective investigation, so it is possible that selection bias may have affected our findings. Second, the study included an insufficient number of ALK-positive to provide strong conclusions. Nevertheless, it is the first study to show that elevated NSE level is a potential biomarker of poor prognosis for ALKpositive patients with resected lung adenocarcinoma. Further research is needed on the clinical value of NSE in prognosis.

Conclusions
High serum NSE level is not relevant to decreased OS in ALKnegative patients, but NSE is an independent prognostic factor in ALK-positive patients.