The authors have declared that no competing interests exist.
Conceived and designed the experiments: CZ. Performed the experiments: QW XL. Analyzed the data: QW XL SR NC. Contributed reagents/materials/analysis tools: MZ YZ JL WC W. Cai CZ W. Cao. Wrote the paper: QW XL.
POTEE (POTE ankyrin domain family, member E) is a newly identified cancer-testis antigen that has been found to be expressed in a wide variety of human cancers including cancers of the colon, prostate, lung, breast, ovary, and pancreas.
To measure the serum levels of POTEE in patients with non-small-cell lung cancer (NSCLC) and to explore the clinical significance of POTEE in NSCLC.
104 NSCLC patients, 66 benign lung disease patients and 80 healthy volunteers were enrolled in this study from May 2013 to February 2014. Serum POTEE levels were measured using enzyme-linked immunosorbent assay (ELISA). Numerical variables were recorded as means ± standard deviation (SD) and analyzed by independent
Serum POTEE levels were significantly higher in NSCLC patients than in benign lung disease patients and healthy controls (mean ± SD [pg/ml], 324.38± 13.84 vs. 156.93 ± 17.38 and 139.09 ± 15.80,
Serum POTEE level in NSCLC patients is associated with TNM stage and is a potential prognostic factor.
Lung cancer is the leading cause of cancer-related deaths worldwide, with non-small-cell lung cancer (NSCLC) representing approximately 80% of these cases [
Cancer testis (CT) antigens are a type of protein that is expressed in normal adult tissue only in the testis as well as a variety of tumors of different histological origins. In view of their pattern of expression and high immunogenicity, CT antigenss are considered very attractive candidates as cancer biomarkers and vaccines [
However, the functions and roles of POTEE in lung cancer remain unclear. In this study, we preliminarily explored the relationship between serum POTEE level and clinical characteristics and survival in NSCLC patients.
Peripheral blood was drawn from patients and healthy donors at Shanghai Pulmonary Hospital from May 2013 to February 2014. Sera were obtained from a study cohort of 250 participants as follows: 104 patients with NSCLCs, including adenocarcinoma, squamous cell carcinoma, and large-cell anaplastic carcinoma; 66 benign lung disease patients; and 80 healthy donors. The inclusion criteria for the NSCLC patients were as follows: (1) pathologically confirmed NSCLC; (2) age ≥18 years old; (3) had not received any anti-tumor therapy. Tumor staging was determined according to the 2009 TNM staging classification system. Demographic and pathological data, including age, gender, and smoking history, were collected. The inclusion criteria for benign lung disease patients were as follows: (1) no history of cancer; (2) benign lung disease patients confirmed by pathogen or pathology detection; (3) the diagnosis of benign lung disease patients were bronchiectasis, chronic obstructive pulmonary disease, tuberculosis, pneumonia, chronic bronchitis and lung abscess, the characteristics and demographics of these patients are included in
Serum POTEE levels were determined by enzyme-linked immunosorbent assay (ELISA) using an immunoassay kit (Wuhan Huamei Biotech, China; Cat. no.CSB-EL740863 HU) according to the manufacturer’s protocol. The optical density (OD) at 450 nm was measured, and the standard curves were established with OD 450 as the Y axis; these curves were used to determine protein expression levels. Results are reported as the concentration of POTEE (pg/ml) in the serum sample.
Serum tumor markers, including carcinoeyonmbric antigen (CEA), cytokeratin (CYFRA21-1) and carbohydrate antigen 19–9 (CA19-9) were determined by radioimmunoassay (RIA) using CEA, CYFRA21-1(CIS Bio International) and CA19-9 (China Institute of Atomic Energy) RIA kits according to the manufacturer’s protocol. Results were gained by 1470 WIZA RDTM counter instruments.
Statistical analyses were performed using SPSS 17.0 software (Chicago, IL, USA) and GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA). Numerical variables were recorded as means ± SD and analyzed using independent
Our study cohort of 250 participants comprised 104 NSCLC patients, 66 benign lung disease patients, and 80 healthy subjects. Mean age was 61.3 ± 9.5 yr in the NSCLC patients group, 58.5 ± 11.5 yr in the benign lung disease patients group, and 57.9 ± 13.6 yr in the healthy control group (
Variables | NSCLC (n, %) | Benign (n, %) | Controls (n, %) | |
---|---|---|---|---|
Age | 0.097 | |||
≥65 yr | 33 (31.7) | 21 (31.8) | 26 (32.5) | |
<65 yr | 71 (68.3) | 45 (68.2) | 54 (67.5) | |
Sex | 0.410 | |||
Male | 59 (56.7) | 43 (65.2) | 52 (65.0) | |
Female | 45 (43.3) | 23 (34.8) | 28 (35.0) | |
Smoking status | 0.232 | |||
Yes | 66 (63.5) | 34 (51.5) | 43 (53.7) | |
No | 38 (36.5) | 32 (48.5) | 37 (46.3) |
Serum POTEE levels were significantly higher in NSCLC patients than in benign lung disease patients and healthy control subjects. The mean serum POTEE level was 324.38 ± 13.84 pg/ml for the NSCLC patient group, 156.93 ± 17.38 pg/ml for the benign lung disease group, and 139.09 ± 15.80 pg/ml for the healthy control group (
A ROC curve analysis was carried out to assess the value of POTEE in NSCLC diagnosis. ROC curve is to reflect the sensitivity and specificity of continuous variable comprehensive index, the area under the curve the greater the diagnosis accuracy is higher. The AUC was 0.793 (95% confidence interval [CI], 0.7–0.9). We chose the point with highest sensitivity and specificity, 205.27 pg/ml, as the cutoff value. With the cutoff value, which was defined as the normal value based on the mean value (plus two standard deviations) obtained from healthy controls, serum POTEE achieved a diagnostic sensitivity of 68.3% and a specificity of 82.9% (
As shown in
Group | Categories | n (%) | POTEE levels (pg/ml) | |
---|---|---|---|---|
Gender | Male | 59 (56.7) | 327.30 ± 25.83 | 0.864 |
Female | 45 (43.3) | 320.55 ± 29.58 | ||
Age, yr | ≥65 | 33 (31.7) | 311.64 ± 34.51 | 0.656 |
<65 | 71 (68.3) | 330.30 ± 23.53 | ||
Histological type | Adenocarcinoma | 64 (61.5) | 294.03 ± 25.02 | 0.907 |
Squamous | 29 (27.9) | 312.62 ± 37.17 | ||
Adenosquamous | 11 (10.6) | 309.67 ± 60.35 | ||
TNM stage |
I–IIIA | 25 (24.0) | 252.60 ± 38.85 | 0.036 |
IIIB–IV | 79 (76.0) | 347.09 ± 21.85 | ||
PS score | 0–1 | 94 (90.4) | 313.13 ± 20.54 | 0.228 |
2 | 10 (9.6) | 229.85 ± 59.75 | ||
Family history | Yes | 11 (10.6) | 262.06 ± 80.08 | 0.495 |
No | 93 (89.4) | 305.45 ± 19.73 | ||
Smoking history | Yes | 66 (63.5) | 338.63 ± 28.19 | 0.142 |
Never | 38 (36.5) | 279.12 ± 25.78 |
PS, performance status;
#, 2 patients in stage I, 5 patients in stage II, 18 patients in stage IIIA, 34 patients in stage IIIB, 45 patients in stage IV;
*, serum POTEE level was associated with TNM stage.
We analyzed whether serum POTEE level is related to tumor response to chemotherapy. Among the 104 patients with NSCLC, 76 patients were at TNM stage IIIb–IV and received doublet chemotherapy. Among 76 patients, 33 patients had been treated with gemcitabine plus cisplatin, 23 had been treated with vinorelbine plus cisplatin, 11 had been treated with paclitaxel plus cisplatin and 9 had been treated with vinorelbine plus carboplatin. The POTEE serum level cutoff value of 205.27 pg/ml with highest sensitivity and specificity was selected to categorize patients as high POTEE (n = 43) and low POTEE (n = 33) groups. We analyzed patient responses to platinum-based chemotherapy (
High POTEE expression (≥205.27 pg/ml) | Low POTEE expression (<205.27 pg/ml) | ||
---|---|---|---|
PR, n | 7 | 13 | |
SD, n | 12 | 9 | |
PD, n | 24 | 11 | |
ORR, % | 16.3 | 39.3 | 0.023 |
DCR, % | 44.2 | 66.6 | 0.043 |
ORR, objective response rate; DCR, disease control rate; PR, partial response; PD, progression disease; SD, stable disease.
In the Kaplan-Meier survival curve analysis, the group of patients with low POTEE expression had a significantly longer median progression-free survival (PFS; 6.3 months; 95% CI, 5.494–7.172) than the group with high POTEE expression (PFS, 4.8 months; 95% CI, 3.870–5.756) (
Survival curves were analyzed by Kaplan-Meier method and log-rank test. Patients with high POTEE levels had a significantly poorer survival than those with low POTEE levels (
Univariate analysis | Multivariate analysis | |||||
---|---|---|---|---|---|---|
Variables | HR | 95% CI | HR | 95% CI | ||
POTEE level (low vs. high) | 1.913 | 1.030–3.551 | 0.040 |
2.440 | 1.252–4.757 | 0.009 |
Age (≥60 vs. <60, yr) | 1.171 | 0.653–2.097 | 0.597 | 1.168 | 0.633–2.154 | 0.620 |
Sex (male vs. female) | 0.728 | 0.399–1.328 | 0.301 | 0.591 | 0.314–1.113 | 0.103 |
Histology | 1.115 | 0.753–1.652 | 0.586 | 1.140 | 0.722–1.801 | 0.573 |
TNM stage (I-IIIA vs. IIIB-IV) | 4.089 | 1.818–9.199 | 0.001 |
4.789 | 2.082–11.014 | 0.001 |
Smoking history | 1.321 | 0.720–2.424 | 0.369 | 0.807 | 0.366–2.776 | 0.593 |
Family history | 0.557 | 0.172–1.798 | 0.328 | 1.927 | 0.504–7.376 | 0.338 |
CI, confidence interval; HR, hazard ratio;
*, PFS was associated with POTEE level and TNM stage.
To our knowledge, this study is the first to assess POTEE expression in serum by ELISA to examine its clinical significance in NSCLC. We observed that the expression of POTEE in sera was significantly higher in NSCLC patients compared with benign lung disease patients and healthy volunteers. Furthermore, we preliminarily analyzed the clinical significance of POTEE in NSCLC patients. Levels of serum POTEE were significantly correlated with TNM stage. Survival analysis revealed that patients with low serum POTEE levels had longer progression-free survival (PFS) than those with high POTEE levels. High POTEE expression levels were associated with poor response to chemotherapy in NSCLC patients. Moreover, multivariate analysis also showed that high POTEE expression was an independent factor for poor survival in NSCLC patients. These results suggest that POTEE expression may be associated with prognosis of NSCLC patients and could serve as a potential biomarker.
Up to date, few previous studies reported an association between POTEE and cancer prognosis because POTEE is a newly identified CT antigen. The gene family POTE is classified into three groups: group 1 includes only POTE 8; group 2 includes POTE15, -18, and -21; and group 3 includes POTE2, -14, and -22 [
In the last few years the researches for CT antigen have made some progress. The unique expression pattern of CT antigen makes them become candidates for use in early diagnosis and as a prognostic marker. The CT antigen MAGE has been found in 85% of NSCLC cases, 75% of early multiple myeloma cases, and 35% early breast cancer cases [
In the present study, there had some limits. Our study cohort size was small; to establish the clinical utility of POTEE in serum, further studies should be performed with larger cohorts. In order to improve the specificity of POTEE ROC curve, we consider the following several aspect work needs to be done: (1) to enlarge the sample size to further optimize the cutoff value and confirm our results; (2) further, the perspective study should be performed; (3) to try the available combination of serum tumor biomarkers. In addition, we also need to investigate the functional mechanisms of POTEE in NSCLC.
In conclusion, our results show that POTEE is over-expressed in NSCLC and that POTEE level in serum is associated with TNM stage and prognosis in NSCLC patients.
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We thank our volunteers for donating their blood and our collaborators for collecting blood samples and participant data.