Abstract
The ETV6/TEL gene is a member of the ETS family of transcription factors that has been mainly studied in hematological diseases. This study provides the first investigation of ETV6 expression in non-small cell lung cancer (NSCLC). In this study, ETV6 expression was immunohistochemically studied in 170 consecutive patients with NSCLC. The association between ETV6 expression and clinicopathological parameters was evaluated. Kaplan-Meier survival analysis and Cox proportional hazards models were used to estimate the effect of ETV6 expression on survival. ETV6 expression was observed in 135 of the 170 (79.4%) patients. ETV6 expression was positive for nuclear staining. From the clinicopathological standpoint, the expression of ETV6 was significantly correlated with age (P = 0.014). The overall survival was significantly enhanced in the group with a low expression of ETV6 compared with the group with a high expression of ETV6 (five-year survival rates, 56.53% versus 29.88%; P = 0.002), and the same finding was obtained for disease-free survival (five-year survival rates, 52.24% versus 30.47%; P = 0.001). Multivariable analysis confirmed that ETV6 expression increased the hazard of death after adjusting for other clinicopathological factors (hazard ratio, 2.002; 95% confidence interval, 1.303-3.074; P = 0.002). Our study demonstrated that ETV6 was markedly involved in the development of NSCLC and could serve as a potential prognostic marker for this deadly disease.
Keywords: ETV6, non-small cell lung cancer, prognosis
Introduction
Lung cancer (LC) remains the most frequent human malignancy and a major public health problem and represents the most common cancer because more than a million people in the world die from this disease each year [1]. Several independent prognostic factors for the survival of patients with NSCLC have been identified: performance status, disease stage, age, sex, and amount of weight lost [2]. To improve the outcome and quality of life of patients with NSCLC, many recent studies have focused on finding new therapeutic targets and identifying biomarkers to facilitate individualized treatment [3]. The better understanding of the molecular biology of NSCLC obtained through these studies has led to a revolution in the work-up of these patients. The epidermal growth factor receptor (EGFR) gene and the fusion of the echinoderm microtubule-associated protein-like 4 gene (EML4) with the anaplastic lymphoma kinase gene (ALK) and variants has become routine in many centers because patients with tumors harbouring such alterations may benefit from tyrosine kinase inhibitors as part of their treatment regimen [4,5]. Over the past several decades, hundreds of papers have discussed molecular markers or proteins that have an association with some clinical outcome, and these typically include overall or disease-free survival in NSCLC.
The ETV6 gene is a member of the ETS family of transcription factors. It is a leukemia-associated gene located on 12p13 and is involved in different chromosomal translocations found in myeloid and lymphoid malignancies and the normal development of neural and mesenchymal tissues. ETV6 is a strong transcriptional repressor that acts through its HLH and internal domains. It mainly promotes tumor through its fusion with partner genes. At present, 48 chromosomal bands have been identified to be involved in ETV6 translocations, insertions or inversions, and 30 ETV6 partner genes have been molecularly characterized [6-8]. Five potential mechanisms of ETV6-mediated leukemogenesis have been identified: constitutive activation of the kinase activity of the partner protein, modification of the original functions of a transcription factor, loss of function of the fusion gene, which affects ETV6 and the partner gene, activation of a proto-oncogene in the vicinity of a chromosomal translocation, and a dominant negative effect of the fusion protein due to transcriptional repression mediated by wild-type ETV6 [9]. ETV6 has been implicated in congenital fibrosarcoma [10] and prostate carcinoma [11]. However, ETV6 has not been detected in NSCLC. This gene was an attractive candidate for NSCLC molecular research at this locus.
Methods and materials
Patients and clinical tissue samples
In this study, 230 specimens of NSCLC were collected at the Department of Pathology of The Sun Yat-Sen University Cancer Centre in China, between July 2002 and May 2007. The cases were selected based on a distinctive pathological diagnosis of NSCLC in patients who underwent primary and curative tumor resection without preoperative chemotherapy or radiotherapy, the availability of resection tissue, and follow-up data. Formalin-fixed paraffin-embedded tissue blocks and the corresponding hematoxylin and eosin-stained (H&E-stained) slides were overlaid for TMA sampling. The slides were reviewed by a senior pathologist (Q.N.W.) to determine and mark out representative tumor areas. Duplicates of 1.0-mm-diameter cylinders were punched from representative tumor areas of individual tissue blocks and re-embedded into a recipient paraffin block at a defined position using a tissue-arraying instrument (Beecher Instruments, Silver Spring, MD, USA).
170 informative cases were collected from the all cases. They included 128 (75.3%) men and 42 (24.7%) women and were with a mean age of 59 years. The average follow-up time was 49.64 months (median, 36.83 months; range, 0.93-115.77 months). Patients whose cause of death remained unknown were excluded from our study. The clinicopathological characteristics of these patients, including gender, age, smoking, histology type, T stage, clinical stage, metastasis, death, and follow-up time, are detailed in Table 1. We reclassified the lung adenocarcinomas using the newest guideline [12]. The tumor stage was defined according to the American Joint Committee on Cancer/International Union against Cancer Tumor-Node-Metastasis (TNM) classification system [13]. The institute research medical ethics committee of Sun Yat-Sen University granted approval for this study.
Table 1.
Characteristics of non-small cell lung carcinoma patients
| Characteristic | Non-small cell lung cancer patients (N = 170)% |
|---|---|
| Gender | |
| Female | 42 (24.7) |
| Male | 128 (75.3) |
| Age (years) | |
| Median (range) | 59 (38-78) |
| Mean ± SD | 58.55±9.98 |
| Smoking | |
| Yes | 104 (61.2) |
| No | 66 (38.8) |
| Histology type | |
| ACC | 130 (76.5) |
| SCC | 40 (23.5) |
| T stage | |
| 1, | 18 (10.6) |
| 2, 3, 4 | 152 (89.4) |
| Clinical stage | |
| I, II | 99 (58.2) |
| III | 71 (41.8) |
| Metastasis | |
| No | 82 (48.9) |
| Yes | 88 (51.1) |
| Death | |
| No | 62 (37.9) |
| Yes | 108 (62.1) |
| Follow-up time (months) | |
| Median (range) | 36.83 (0.93-115.77) |
| Mean + SD | 49.64 ± 33.41 |
Immunohistochemistry (IHC) and evaluation
IHC was performed to examine ETV6 expression in NSCLC tissue specimens. Primary antibodies against ETV6 (1:50 dilution, ab54705, Abcam, USA) were used in this study, and these were incubated for 2 h at room temperature. The slides were sequentially incubated with a secondary antibody (Envision; Dako, Glostrup, Denmark) for 1 h at room temperature and stained with DAB (3,3- diaminobenzidine). Finally, the sections were counterstained with Mayer’s hematoxylin, dehydrated, and mounted. A negative control was obtained by replacing the primary antibody with a normal murine immunoglobulin G. Known immunostaining-positive slides were used as positive controls.
The IHC results were evaluated and scored independently by three pathologists without prior knowledge of the clinicopathological outcomes of the patients. A semi-quantitative estimation was made using a composite score obtained by adding the values of the staining intensity and the relative abundance of the positive cells. The intensity was graded as 0 (negative), 1 (weakly positive), 2 (moderately positive) or 3 (strongly positive). The abundance of the positive cells was graded from 0 to 4 according to their percentages (0, < 5%; 1, 5-25%; 2, 26-50%; 3, 51-75%; 4, 76-100%). The concordance rate of 83.4% of the evaluated results from the pathologists demonstrated that this scoring method was highly reproducible. The value was selected until at least two pathologists reported consistent results. In cases in which the scoring was completely different, all of the pathologists worked together to confirm the score.
Selection of cutoff score
Receiver operating characteristic (ROC) curve analysis was performed to determine the cutoff score for a “high expression” designation with the 0, 1-criterion implemented in the SPSS software. For the ETV6 score, the sensitivity and specificity for each outcome under study was plotted, thus generating various ROC curves. The clinicopathological characteristics were dichotomized into the following groups: T classification (T1 versus T2-T4), N classification (N0 versus N1-N3), clinical stage (I-II versus III-IV), cancer progression (Yes versus No), and survival status (death due to lung cancer versus censored). The cutoff score is the point on the curve that has both maximum sensitivity and specificity [14].
Statistical analysis
Statistical analysis was performed using the SPSS statistical software package (standard version 16.0; SPSS, Chicago, IL, USA). ROC curve analysis was applied to determine the cutoff score for high expression of ETV6. The correlation between ETV6 expression and the clinicopathological features of patients with lung cancer was analyzed by the X2-test. For univariate survival analysis, survival curves were obtained using Kaplan-Meier analyses. In the multivariate statistical analysis, we treated the overall survival time as “time”, the overall survival status as “status”, and “death” as the reference (with a value of 1), whereas the expression of ETV6 and other significant clinicopathological features were treated as the comparing parameters (with a relative value). The Cox proportional hazards regression model was used to identify independent prognostic factors. The predictive accuracy was quantified using the Harrell concordance index. Differences were considered significant if the P value from a two-tailed test was < 0.05.
Results
Expression of ETV6 in non-small cell lung cancer tissues
The ETV6/Tel IHC staining in NSCLC tissues revealed immunoreactivity primarily in the nucleus within tumor cells. The ETV6/Tel expression level could be evaluated informatively in 170 lung cancers by the TMA. The immunoreactivity scores of ETV6/Tel in NSCLC ranged from 1 to 7. According to the ROC curve analysis, the immunoreactivity score for ETV6/Tel above the cutoff value of 2.5 was defined as high expression (Figure 1A and 1B), whereas scores below or equal to the cutoff value were considered low expression (Figure 1C and 1D). ETV6/Tel was detected in 135 of 170 tissues (79.4%). High ETV6/Tel expression in tumor tissue was found in 110 of 170 (64.7%) patients.
Figure 1.
Distinct patterns of ETV6 immunostaining in NSCLC. (A, B) A High expression of ETV6 was observed in a NSCLC Specimen in which the score of ETV6 immunostaining in cytoplasm was ≥ 2.5 (A. Original magnification *40; B. Original magnification *100). (C, D) Low expression of ETV6 was observed in a NSCLC specimen in which the score of ETV6 immunostaining in cytoplasm was < 2.5 (C. Original magnification *40; D. Original magnification *100).
Selection of cutoff scores for ETV6 expression
ROC curve analysis showed that ETV6/Tel expression has some sort of predictive value in NSCLC, with the maximum area under the curve (AUC) reaching 0.637 (Table 2). Tumors with scores above the obtained cutoff value were considered to exhibit high ETV6/Tel expression, which led to the greatest number of tumors classified based on the presence or absence of a clinical outcome. For the survival analysis, the cutoff score for the expression of ETV6/Tel was 2.5 (Table 2).
Table 2.
Corresponding cutoff score of ETV6/Tel expression for each clinicopathological feature according to ROC curve analysis
| Feature | Cutoff score | P value |
|---|---|---|
| Histology type | 2.5 | 0.697 |
| T stage | 2.5 | 0.261 |
| N stage | 4.5 | 0.156 |
| Clinical stage | 4.5 | 0.038 |
| Survival status | 2.5 | 0.003 |
Relationship between ETV6 expression and clinicopathological parameters
The relationship between ETV6 expression and clinicopathological parameters was further analyzed. Significant correlations were found between ETV6 expression and age (P = 0.014). There were no statistical connections between EIF4A2 expression and the remaining clinicopathological parameters, such as gender, smoking, and lung membrane invasion, and histology type (P > 0.05) (Table 3).
Table 3.
Association of ETV6 expression and clinicopathological characteristics in non-small cell lung carcinoma patients
| ETV6 expression | ||||
|---|---|---|---|---|
|
|
||||
| Case | Low | High | P value | |
|
|
||||
| Variable | n = 170 | n = 60 | n = 110 | |
| Age (years) | ||||
| ≥ 49.5 | 137 | 42 | 95 | |
| < 49.5 | 33 | 18 | 15 | 0.014 |
| Gender | ||||
| Male | 128 | 45 | 83 | |
| Female | 42 | 15 | 27 | 1.000 |
| Smoking | ||||
| Yes | 104 | 33 | 71 | |
| No | 66 | 27 | 39 | 0.251 |
| Lung membrane invasion | ||||
| Yes | 78 | 22 | 56 | |
| No | 92 | 38 | 54 | 0.079 |
| Histology type | ||||
| ACC | 130 | 48 | 82 | |
| SCC | 40 | 12 | 28 | 0.456 |
| T stage | ||||
| 1 | 18 | 8 | 10 | |
| 2, 3, 4 | 152 | 52 | 100 | 0.438 |
| N stage | ||||
| 0, | 82 | 30 | 52 | |
| 1, 2, 3 | 88 | 30 | 58 | 0.751 |
| Clinical stage | ||||
| I, II | 99 | 38 | 61 | |
| III | 71 | 22 | 49 | 0.334 |
Association of high ETV6 expression in NSCLC with poor survival
The five-year overall and disease-free survival rate of the cohort of 170 NSCLC patients were 39.28% and 38.14%, respectively (Figure 2A and 2B). Of these 170 NSCLC patients, Kaplan-Meier and log-rank test analyses indicated that high ETV6 expression is significantly associated with poor overall survival (OS) (five-year survival rates, 29.88% vs. 56.53%, log-rank test, χ2 = 9.777, P = 0.002, Figure 2C) and disease-free survival (DFS) (five-year survival rates, 30.47% vs. 52.24%, log-rank test, χ2 = 10.370, P = 0.001, Figure 2D).
Figure 2.
Association between ETV6 expression and NSCLC patient survival. TMA analyses were conducted in a cohort of 170 NSCLC patients diagnosed at M0. A. The five-year overall survival (OS) rate was 39.28%. B. The five-year disease-free survival (DFS) rate was 38.14%. C, D. High ETV6 expression levels were significantly associated with OS (P = 0.002) and disease-free survival (P = 0.001) in all NSCLC patients.
When the clinical stages were taken as stratifications, high ETV6 expression was found to be significantly associated with poor overall survival (OS) for patients with histology types ACC (five-year survival rates, 27.8% vs. 56.1%, log-rank test, χ2 = 8.940, P = 0.003, Figure 3A) and DFS (five-year survival rates, 28.6% vs. 53.2%, log-rank test, χ2 = 9.941, P = 0.002, Figure 3B); High ETV6 expression has shorter overall survival (OS) (five-year survival rates, 47.5% vs. 68.3%, log-rank test, χ2 = 4.424, P = 0.35, Figure 3C) and DFS (five-year survival rates, 48.6% vs. 68.1%, log-rank test, χ2 = 4.553, P = 0.033, Figure 3D) for the early stages (stage I-II) patient. In addition, significant correlations were found in patients at the late stages (stage III), which patients with high ETV6 expression exhibited significantly shorter overall survival (OS) (five-year survival rates, 7.8% vs. 36.4%, log-rank test, χ2 = 6.754, P = 0.009, Figure 3E) and DFS (five-year survival rates, 8.2% vs. 23.1%, log-rank test, χ2 = 7.318, P = 0.007, Figure 3F).
Figure 3.
A, B. High ETV6 expression levels were significantly associated with OS (P = 0.003) and disease-free survival (P = 0.002) in NSCLC patients at the adenocarcinoma. C, D. High ETV6 expression levels were significantly associated with OS (P = 0.035) and disease-free survival (P = 0.033) in NSCLC patients at the early clinical stage (stage I-II). E and F. High ETV6 expression levels were significantly associated with OS (P = 0.009) and disease-free survival (P = 0.007) in NSCLC patients at the late clinical stage (stage III).
Univariate and multivariate analyses of prognostic variables in patients with NSCLC
A univariate Cox proportional hazard regression analysis showed that ETV6 expression (P = 0.002) and the well-known clinicopathological prognostic parameters, such as T stage (P = 0.010), N stage (P < 0.001), lung membrane invasion (P = 0.031) and clinical stage (P < 0.001), were significantly associated with survival (Table 4). Moreover, independency tests were performed among the prognostic factors, including the expression of ETV6 and other clinicopathological features (T stage, N stage, lung membrane invasion and clinical stage) under a multivariate Cox proportional hazards regression model. Compared with the above-mentioned significant clinicopathological features (T stage, N stage, lung membrane invasion and clinical stage), ETV6 was an independent and unfavorable factor for overall patient survival (hazards ratio: 2.002; 95% confidence interval: 1.303-3.074; P = 0.002). Moreover, the T stage and clinical stage were found to be independent prognostic predictors of overall survival (P < 0.05) (Table 4).
Table 4.
Univariate and multivariate Cox regression analyses of different prognostic variables in patients with NSCLC
| Variable | Subset | Hazard ratio (95% CI) | P value |
|---|---|---|---|
| Univariate analysis (N = 170) | |||
| Age | ≥ 49.5 vs. < 49.5 | 1.193 (0.726-1.958) | 0.486 |
| Smoking | Yes’s No. | 1.485 (0.998-2.211) | 0.051 |
| Gender | Male vs. Female | 0.926 (0595-1.439) | 0.731 |
| Histology type | ACC vs. SCC | 1.006 (0.643-1.573) | 0.980 |
| T stage | 1 vs. 2,3,4 | 2.938 (1.288-6.699) | 0.010 |
| N stage | 0 vs. 1,2,3 | 2.704 (1.803-4.055) | 0.000 |
| Clinical stage | I, II vs. III | 2.936 (1.990-4.330) | 0.000 |
| Lung membrane invasion | Yes’s No. | 1.515 (1.038-2.212) | 0.031 |
| ETV6 expression | Low vs. High | 1.952 (1.274-2.992) | 0.002 |
| Multivariate analysis (N = 170) | |||
| T stage | 1 vs. 2,3,4 | 2.448 (1.070-5.599) | 0.034 |
| Clinical stage | I, II vs. III | 2.862 (1.934-4.237) | 0.000 |
| ETV6 expression | Low vs. High | 2.002 (1.303-3.074) | 0.002 |
Discussion
In this study, we found that the overexpression of the ETV6 protein is likely correlated with poor prognosis of NSCLC patient. The ETV6 protein was expressed in 79.4% of 170 NSCLC patient specimens. ETV6 was identified as a fusion protein to platelet-derived growth factor receptor chain b in patients with chronic myelomonocytic leukemia [15]. ETV6 results in the formation of fusion proteins with oncogenic potential [16]. These findings suggested that ETV6 may play an important role in the tumorigenesis of NSCLC. Supportively, ETV6 expression and fusion with its partner gene are critical for establishing haematopoiesis [8], and are detected in congenital fibrosarcoma and prostate cancer as well as haematological malignancies [10,11]. However, the expression of ETV6 in NSCLC was not detected prior to this study.
The correlation of ETV6 with clinicopathological parameters in NSCLC was analyzed in our study. The expression ETV6 was significantly higher in the aged patients. This finding indicates that ETV6 expression is enhanced during degradation with aging. Moreover, higher expression of ETV6 is correlated with poorer disease-free and overall survival rates of NSCLC probability among stage III and Adenocarcinoma patients (P < 0.001). As reported, ETV6, which plays a role as a tumor suppressor gene, is frequently translocated in lymphoid and myeloid hematopoietic tumors and encodes a transcriptional repressor with an N-terminal pointed (PNT) homodimerisation domain and a C-terminal ETS DNA-binding domain [17]. In Pieter’s study for early immature human T cell leukemia, the expression of C-terminal (V345fs) and N-terminal (s105fs) ETV6 mutants abolished the transrepression effects of wild-type ETV6, suggesting that these mutant ETV6 alleles harbors dominant-negative activity, and these researchers also found that the ETV6-mutated cases exhibit a characteristic gene expression signature that is dominated by up-regulated transcripts [18]. These findings suggest that ETV6 may participate in the regulation of NSCLC progression.
Importantly, the results of the Kaplan-Meier survival analysis revealed that patients with high ETV6 expression to be highly at risk for cancer-related death, which means that the expression disorder of ETV6 may promote NSCLC progression. ETV6 acted as a suppressor gene in hematopoietic tumors, and our results may be explained by mutated ETV6 expression, which could abolish the function of wild-type ETV6 [18]. However, the mechanism of ETV6 expression in NSCLC needs further detailed research. Our further study will aim at to study ETV6 gene mutation in NSCLC.
The previous studies also found that an inhibitor of the ETV6 gene, such as PKC412, could serve as a therapeutic drug for the treatment of patients with ETV6-NTRK3 (EN) fusion [19], which suggests that ETV6 may become a therapeutic and prognostic biomarker.
In conclusion, the expression of ETV6 in NSCLC could be detected by IHC staining. A significant relationship was observed between ETV6 expression and patient age. The expression of ETV6 may be a good independent prognostic factor for both overall survival and disease-free survival in patients with NSCLC.
Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (No. 8120184).
Disclosure of conflict of interest
None.
References
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