Journal of Surgical Oncology 2015;111:834–839
Elevated Expression of CCAT2 is Associated with Poor Prognosis in Esophageal Squamous Cell Carcinoma XUELIN ZHANG, MD,1 YUNHUA XU, MD,2 HE CHUNYA MS,3 XIAOWEI GUO, MD,3 JIAN ZHANG, MD,4 CHENGZHI HE, PhD,5 LING ZHANG, MD,6 MIN KONG, MD,4 BAOFU CHEN, PhD,4* 4 AND CHENGCHU ZHU, PhD * 1 Department of Thoracic Surgery, Taizhou Central Hospital, Taizhou, Zhejiang, China Department of Shanghai Lung Cancer Center, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai, China 3 Department of Surgical Oncology, Taizhou Central Hospital, Taizhou, Zhejiang, China 4 Department of Thoracic Surgery, Taizhou Hospital, Linhai, Taizhou, Zhejiang, China 5 Department of Gastroenterology, Institute of Digestive Diseases, Tongji Hospital Affiliated to Tongji University, Shanghai, China 6 Department of Gynaecology and Obstetrics, Taizhou Central Hospital, Taizhou, Zhejiang, China 2
Background and Objectives: CCAT2, a novel long non-coding RNAs (lncRNAs), is found to promote the metastasis and invasion of colon, lung, and breast cancers. This study aimed to investigate the level of CCAT2 in esophageal squamous cell carcinoma (ESCC) and to elucidate its clinical significance. Methods: The expression level of CCAT2 and the status of MYC amplification were examined in 229 ESCC samples using quantitative real- time PCR. Results: CCAT2 was upregulated in ESCC tissues, especially in cases with lymph node metastasis (LNM), advanced TNM stages, and MYC amplification. Furthermore, the level of CCAT2 was positively correlated with TNM stages, LNM, and the number of positive lymph nodes. High CCAT2 expression and MYC amplification were significantly associated with TNM stages and LNM. Survival analyses revealed that high CCAT2 expression and MYC amplification were significantly associated with poorer overall survival in ESCC patients. Furthermore, patients with high CCAT2 expression and MYC amplification had a 2.199-fold increased risk of death compared with those with low CCAT2 expression and MYC non-amplification. Conclusions: Our study provides the first evidence associating CCAT2 expression and poor survival in ESCC. CCAT2 may be a prognostic biomarker and therapeutic target for ESCC.
J. Surg. Oncol. 2015;111:834–839. ß 2015 Wiley Periodicals, Inc.
KEY WORDS: Long non-coding RNA; CCAT2; esophageal squamous cell carcinoma; MYC; gene amplification
INTRODUCTION Esophageal carcinoma is a common malignant tumor worldwide and is the sixth leading cause of cancer death[1]. China is one of countries with high incidence of esophageal carcinoma. The annual incidence and mortality rates of esophageal carcinoma rank fifth and fourth, respectively[2], and the incidence and mortality rates have remained high for the past years. Among the pathological types of esophageal carcinoma, 90% are esophageal squamous cell carcinoma (ESCC)[1]. The survival rate of ESCC patients has been increased through a variety of therapy methods, including surgery, new adjuvant chemotherapy, and biotherapy. However, most ESCC patients are diagnosed at an advanced stage. Since ESCC are not sensitive to radiotherapy and chemotherapy, the prognosis of ESCC patients remains poor, with the 5-year survival rate up to 30%[3,4]. To date, the factors that influence the occurrence, development and prognosis of ESCC remain unclear. Therefore, identification of the biomarkers that influence the development and progression of ESCC and predict the prognosis of ESCC will be meaningful. In addition to numerous protein-coding messenger RNAs in the human transcriptome, a large number of non-coding transcripts are also present, including microRNA (miRNAs) and long non-coding RNAs (lncRNAs). LncRNA is a kind of RNA with transcripts longer than 200 nt. LncRNAs were initially regarded as spurious transcriptional noise, without biological functions. However, recent
ß 2015 Wiley Periodicals, Inc.
studies have demonstrated that lncRNAs have important roles in cellular biological processes and diseases[5]. LncRNAs regulate the expression levels of target genes at epigenetic, transcriptional, and post-transcriptional levels[6–9]. Abnormal levels of lncRNAs affect the expression of downstream genes, which promote the occurrence and development of diseases including cancer [6,7,10–12]. Increasing evidence has demonstrated that lncRNAs can function as oncogenes and tumor suppressors[12]. To date, only a few lncRNAs have been
Grant sponsor: Research Fund of the Taizhou Science and Technology Agency; Grant number: 131KY14-04; Grant sponsor: Research Fund of the Department of Science and Technology of Zhejiang Province; Grant number: 2014C33154 and 2011C13039- 2. Xuelin Zhang, Yunhua Xu, and Chunya He contributed equally to this work. Conflict of interest: none * Correspondence to: Chengchu Zhu, MD, PhD or Baofu Chen, MD, PhD, Department of Thoracic Surgery, Taizhou Hospital, No. 150 Ximen Road, Linhai, Zhejiang 317500, China. Fax: þ861-576-88526012, E-mail: [email protected] (C.Z.) E-mail: [email protected] (B.C) Received 12 November 2014; revised manuscript received 29 December 2014; Accepted 31 December 2014 DOI 10.1002/jso.23888 Published online in Wiley Online Library (wileyonlinelibrary.com).
CCAT2 and ESCC characterized functionally, while the functions of most of lncRNAs remain unknown. Single nucleotide polymorphism rs6983267 on chromosome 8q24 is associated with risk of several cancers, such as colorectal, lung, gastric, and prostate cancers[13–16]. Recent study revealed that rs6983267 is located in a novel lncRNA, CCAT2, and affects its expression[7]. CCAT2 overexpression can promote the metastasis and invasion of breast, lung, and colon cancer cells[7,17,18]. The expression level of CCAT2 influences the prognosis of colon and breast cancers[7,18]. However, no study has been conducted to examine the role of CCAT2 in ESCC. In addition, Redis et al. found that the expression level of CCAT2 in breast cancer was affected by 8q24 amplification[18]. Since chromosome 8q24 is often amplified in ESCC[19–21] and CCAT2 is approximately 335 kb away from MYC gene, it is most likely that amplification of MYC and CCAT2 occur simultaneously. MYC amplification therefore indirectly reflects the status of 8q24.2 amplification. Previous study revealed that CCAT2 regulated MYC- related pathway to promote chromosomal instability [7]. In the present study, we investigated the levels of CCAT2 and the status of MYC amplification in ESCC, and further analyzed the relationship between CCAT2 expression and MYC amplification, and clinical features and prognosis of ESCC patients to determine the effects of CCAT2 in the development and progression of ESCC. The results may provide a theoretical basis for the diagnosis, treatment, and prognosis of ESCC.
835
MATERIALS AND METHODS Clinical specimens A total of 229 pairs of ESCC and matched paracancerous tissues were obtained from patients who received surgical resection of ESCC between 2007 and 2014 at four hospitals. The distance from adjacent tissue to the edge of tumor tissue was more than 3 cm. The resected samples were pathologically examined by H&E staining. All patients did not receive anti-cancer treatment prior to surgery. Written informed consents were obtained from all patients before sample collection. The study protocol was approved by the hospital ethical committees.
Quantitative real- time PCR (qRT-PCR) Total RNA was extracted using RecoverAllTM Total Nucleic Acid Isolation Kit (Ambion, Austin, Texas) according to the manufacturer’s protocol. After xylene dewaxing, ethanol washing, and protease digestion, total RNA was extracted from FFPE sections. Nanodrop 2000 was used to detect the concentration and purity of total RNA, and then was stored at –80 °C for later use. PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Dalian, China) was used to remove genomic DNA and synthesize cDNA. Quantitative real-time PCR was done using SYBR1 Premix Ex Taq II (Tli RNaseH Plus) (Takara, Dalian, China) on ABI 7900HT (Applied Biosystems, California). The reaction was performed
Fig. 1. CCAT2 was upregulated in ESCC tissues. A, CCAT2 expression in ESCC and adjacent noncancerous tissues. B, CCAT2 expression in LNM positive and negative patients. C, CCAT2 expression at different TNM stages. D, CCAT2 expression in MYC amplified and non-amplified patients. Journal of Surgical Oncology
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Fig. 2. Kaplan–Meier estimates of overall survival for ESCC patients according to CCAT2 expression (A), MYC amplification (B), and the combination of CCAT2 expression and MYC amplification (C).
TABLE I. Association of CCAT2 Expression and Myc Amplification With Clinicopathologic Features CCAT2 expression Clinicopathological features Age (%) 60 >60 Sex (%) Male Female Tumor size 4 >4 Grade (%) Well Moderate Poor General classification Medullary type Ulcerative type Others type TNM (%) I þ II III þ IV LNM (%) No Yes
Journal of Surgical Oncology
MYC amplification
Low
High
P value
Positive
Negative
P value
41 (936.0) 73 (64.0)
47 (40.9) 68 (59.1)
0.498
40 (42.1) 55 (57.9)
48 (35.8) 86 (64.2)
0.339
86 (75.4) 28 (24.6)
84 (73.0) 31 (27.0)
0.763
75 (78.9) 20 (21.1)
95 (70.9) 39 (29.1)
0.220
53 (47.3) 59 (52.7)
60 (52.6) 54 (47.4)
0.506
45 (47.9) 49 (52.1)
68 (51.5) 64 (48.5)
0.686
18 (15.8) 75 (65.8) 21 (18.4)
18 (15.7) 81 (70.4) 16 (13.9)
0.637
15 (15.8) 63 (66.3) 17 (17.9)
21 (15.7) 93 (69.4) 20 (14.9)
0.826
25 (23.4) 62 (57.9) 20 (18.7)
36 (33.0) 63 (57.8) 10 (9.2)
0.070
29 (31.9) 54 (59.3) 8 (8.8)
32 (25.6) 71 (56.8) 22 (17.6)
0.155
71 (62.8) 42 (37.2)
54 (48.2) 58 (51.8)
0.032
41 (44.1) 52 (55.9)
84 (63.6) 48 (36.4)
0.004
65 (57.5) 48 (42.5)
49 (43.0) 65 (57.0)
0.034
37 (39.4) 57 (60.6)
77 (57.9) 56 (42.1)
0.007
CCAT2 and ESCC 1
in a final volume of 20 mL containing 2 SYBR Premix Ex Taq II (Tli RNaseH Plus), 0.4 mM of each primer and 2 mL of cDNA. The reaction conditions were as follow: 95°C for 30 s, followed by 40 cycles at 95 °C for 5 s, and then 60 °C for 34 s. Relative expression of CCAT2 was normalized to reference RNU6B and was calculated using the 2- DDCt equation.
Quantitative real- time genomic PCR analysis Genomic DNA was purified using QIAamp DNA FFPE Tissue Kit (Qiagen, Germany) according to the manufacturer’s protocol. MYC amplification was detected using TaqMan Copy Number Assay as previously described[19]. In addition to MYC, RPPH1 on chromosome 14q11.2 was used as a single-copy control gene.
Statistical analyses Comparison of the levels of CCAT2 between ESCC and adjacent tissues was performed using Student’s paired t-test. Mann–Whitney U test was used to evaluate significant differences between two independent groups of samples. The status of MYC amplification was determined as previously described[19]. Spearman’s rho test was used to analyze the correlation between the expression level of CCAT2 and MYC amplification. Patients were divided into high and low expression groups based on the median expression level of CCAT2. Chi-square or Fisher’s exact test was used to analyze the correlation between CCAT2 expression and MYC amplification and clinicopathological features, as appropriate. The correlations between overall survival, and CCAT2 expression levels and MYC amplification were examined by using the Kaplan–Meier method and log- rank test. Hazard ratios (HRs) and 95% confidence intervals (95%CIs) were calculated using Cox proportional hazard regression model. All statistical analyses were performed using SPSS software (version 19.0, SPSS, Chicago, Illinois) and Graphpad Prism 5.0 (GraphPad Software Inc., CA, USA).
RESULTS ESCC tissues demonstrated significantly upregulated CCAT2 expression compared with adjacent normal tissues (P < 0.001) (Fig. 1A). The level of CCAT2 was positively correlated with TNM stages, LNM, and the number of positive lymph nodes (P < 0.05). The levels of CCAT2 in patients with lymph node metastasis (LNM) and advanced TNM stages were significantly higher than those with nonLNM and TNM stages I and II, respectively (P < 0.001) (Fig. 1B and 1 C). When cases were divided into low and high CCAT2 expression groups according the median levels of CCAT2, high CCAT2 expression
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was significantly related to LNM (P ¼ 0.034) (Table I). Although there was no association between CCAT2 expression and TNM stages (P ¼ 0.057), high CCAT2 was also related to advanced TNM stages (P ¼ 0.032) when the stages of disease were classified into two groups by early stages I-II and advanced stages III-IV. Furthermore, patients with high CCAT2 expression had more positive lymph nodes (2.00 2.63) than those with low CCAT2 expression (1.24 2.10) (P ¼ 0.012). No significant difference was found between CCAT2 expression and other clinical features. Among 229 ESCC cases, MYC amplification was detected in 95 cases (41.5%). MYC amplification was observed in any TNM stage, in particular in advanced stages, indicating positive relationship between MYC amplification and clinical aggressiveness of ESCC. We next examined the association of MYC amplification with clinical features in all cases. MYC amplification was associated with LNM and TNM stages (P < 0.05). The status of MYC amplification was positive correlated with the number of positive lymph nodes (P ¼ 0.001). MYC amplification was not associated with age, sex, tumor size, histological grade, and pathologic type. We further examined whether there was an association of MYC amplification with the expression level of CCAT2. Increased MYC copy number correlated with increased level of CCAT2 (P < 0.001). The levels of CCAT2 in MYC amplification group was significantly higher than that in MYC non-amplification group (P < 0.001) (Fig. 1D). During 66 months follow-up period, 139 ESCC patients died. The median survival time (MST) was 31.0 months, and the 5-year survival rate was 25.5%. The MST was found to be shorter in patients with high CCAT2 expressing than in those with low CCAT2 expressing, and in patients with MYC amplification than in those with MYC nonamplified individuals (Fig. 2). Multivariate Cox regression analysis revealed that high CCAT2 expression (HR ¼ 1.432, 95% CI: 1.005– 2.041, P ¼ 0.047), MYC amplification (HR ¼ 1.505, 95% CI: 1.051– 2.154, P ¼ 0.026), and TNM (HR ¼ 2.666, 95% CI: 1.074–6.618, P ¼ 0.035) were independent prognosis factors for ESCC (Table II). We further analyzed the outcome of patients according to combination of CCAT2 expression and MYC amplification. As shown in Table II, patients with 1 (high CCAT2 level or MYC amplification) and 2 risk factors (high CCAT2 level and MYC amplification) had 1.734-fold (95% CI: 1.127–2.669) and 2.199-(95% CI: 1.410–3.430) increased risk of death, respectively, when compared with those with zero risk factors (low CCAT2 level and MYC non-amplification) (Fig. 2C).
DISCUSSION Current studies on endogenous miRNA have achieved promising results. Similar to miRNAs, lncRNAs also represent another important
TABLE II. Univariate and Multivariate Cox Regression Analyzes for Overall Survival in Escc Patients Univariate analysis Factors
HR (95%CI)
age (year) (>60/60) 0.826(0.585–1.167) sex (male/female) 0.891 (0.602–1.318) tumor size (cm) (>4/4) 1.125(0.805–1.572) general classification (ulcerative type/others) 1.280(0.902–1.818) tumor differentiation (poor/well, moderate) 1.204(0.775–1.870) TNM stage (III, IV/I, II) 1.896(1.347–2.670) LNM (positive/negative) 1.632(1.160–2.296) CCAT2 (high/low) 1.641(1.170–2.301) MYC amplification (positive/negative) 1.827(1.306–2.556) Number of risk factors (high CCAT2 expression and MYC amplification) 0 1 2 2.361(1.531–3.643) 1 1.865(1.221–2.850)
Journal of Surgical Oncology
Multivariate analysis P value
HR (95%CI)
P value
0.279 0.563 0.490 0.167 0.410
Elevated Expression of CCAT2 is Associated with Poor Prognosis in Esophageal Squamous Cell Carcinoma XUELIN ZHANG, MD,1 YUNHUA XU, MD,2 HE CHUNYA MS,3 XIAOWEI GUO, MD,3 JIAN ZHANG, MD,4 CHENGZHI HE, PhD,5 LING ZHANG, MD,6 MIN KONG, MD,4 BAOFU CHEN, PhD,4* 4 AND CHENGCHU ZHU, PhD * 1 Department of Thoracic Surgery, Taizhou Central Hospital, Taizhou, Zhejiang, China Department of Shanghai Lung Cancer Center, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai, China 3 Department of Surgical Oncology, Taizhou Central Hospital, Taizhou, Zhejiang, China 4 Department of Thoracic Surgery, Taizhou Hospital, Linhai, Taizhou, Zhejiang, China 5 Department of Gastroenterology, Institute of Digestive Diseases, Tongji Hospital Affiliated to Tongji University, Shanghai, China 6 Department of Gynaecology and Obstetrics, Taizhou Central Hospital, Taizhou, Zhejiang, China 2
Background and Objectives: CCAT2, a novel long non-coding RNAs (lncRNAs), is found to promote the metastasis and invasion of colon, lung, and breast cancers. This study aimed to investigate the level of CCAT2 in esophageal squamous cell carcinoma (ESCC) and to elucidate its clinical significance. Methods: The expression level of CCAT2 and the status of MYC amplification were examined in 229 ESCC samples using quantitative real- time PCR. Results: CCAT2 was upregulated in ESCC tissues, especially in cases with lymph node metastasis (LNM), advanced TNM stages, and MYC amplification. Furthermore, the level of CCAT2 was positively correlated with TNM stages, LNM, and the number of positive lymph nodes. High CCAT2 expression and MYC amplification were significantly associated with TNM stages and LNM. Survival analyses revealed that high CCAT2 expression and MYC amplification were significantly associated with poorer overall survival in ESCC patients. Furthermore, patients with high CCAT2 expression and MYC amplification had a 2.199-fold increased risk of death compared with those with low CCAT2 expression and MYC non-amplification. Conclusions: Our study provides the first evidence associating CCAT2 expression and poor survival in ESCC. CCAT2 may be a prognostic biomarker and therapeutic target for ESCC.
J. Surg. Oncol. 2015;111:834–839. ß 2015 Wiley Periodicals, Inc.
KEY WORDS: Long non-coding RNA; CCAT2; esophageal squamous cell carcinoma; MYC; gene amplification
INTRODUCTION Esophageal carcinoma is a common malignant tumor worldwide and is the sixth leading cause of cancer death[1]. China is one of countries with high incidence of esophageal carcinoma. The annual incidence and mortality rates of esophageal carcinoma rank fifth and fourth, respectively[2], and the incidence and mortality rates have remained high for the past years. Among the pathological types of esophageal carcinoma, 90% are esophageal squamous cell carcinoma (ESCC)[1]. The survival rate of ESCC patients has been increased through a variety of therapy methods, including surgery, new adjuvant chemotherapy, and biotherapy. However, most ESCC patients are diagnosed at an advanced stage. Since ESCC are not sensitive to radiotherapy and chemotherapy, the prognosis of ESCC patients remains poor, with the 5-year survival rate up to 30%[3,4]. To date, the factors that influence the occurrence, development and prognosis of ESCC remain unclear. Therefore, identification of the biomarkers that influence the development and progression of ESCC and predict the prognosis of ESCC will be meaningful. In addition to numerous protein-coding messenger RNAs in the human transcriptome, a large number of non-coding transcripts are also present, including microRNA (miRNAs) and long non-coding RNAs (lncRNAs). LncRNA is a kind of RNA with transcripts longer than 200 nt. LncRNAs were initially regarded as spurious transcriptional noise, without biological functions. However, recent
ß 2015 Wiley Periodicals, Inc.
studies have demonstrated that lncRNAs have important roles in cellular biological processes and diseases[5]. LncRNAs regulate the expression levels of target genes at epigenetic, transcriptional, and post-transcriptional levels[6–9]. Abnormal levels of lncRNAs affect the expression of downstream genes, which promote the occurrence and development of diseases including cancer [6,7,10–12]. Increasing evidence has demonstrated that lncRNAs can function as oncogenes and tumor suppressors[12]. To date, only a few lncRNAs have been
Grant sponsor: Research Fund of the Taizhou Science and Technology Agency; Grant number: 131KY14-04; Grant sponsor: Research Fund of the Department of Science and Technology of Zhejiang Province; Grant number: 2014C33154 and 2011C13039- 2. Xuelin Zhang, Yunhua Xu, and Chunya He contributed equally to this work. Conflict of interest: none * Correspondence to: Chengchu Zhu, MD, PhD or Baofu Chen, MD, PhD, Department of Thoracic Surgery, Taizhou Hospital, No. 150 Ximen Road, Linhai, Zhejiang 317500, China. Fax: þ861-576-88526012, E-mail: [email protected] (C.Z.) E-mail: [email protected] (B.C) Received 12 November 2014; revised manuscript received 29 December 2014; Accepted 31 December 2014 DOI 10.1002/jso.23888 Published online in Wiley Online Library (wileyonlinelibrary.com).
CCAT2 and ESCC characterized functionally, while the functions of most of lncRNAs remain unknown. Single nucleotide polymorphism rs6983267 on chromosome 8q24 is associated with risk of several cancers, such as colorectal, lung, gastric, and prostate cancers[13–16]. Recent study revealed that rs6983267 is located in a novel lncRNA, CCAT2, and affects its expression[7]. CCAT2 overexpression can promote the metastasis and invasion of breast, lung, and colon cancer cells[7,17,18]. The expression level of CCAT2 influences the prognosis of colon and breast cancers[7,18]. However, no study has been conducted to examine the role of CCAT2 in ESCC. In addition, Redis et al. found that the expression level of CCAT2 in breast cancer was affected by 8q24 amplification[18]. Since chromosome 8q24 is often amplified in ESCC[19–21] and CCAT2 is approximately 335 kb away from MYC gene, it is most likely that amplification of MYC and CCAT2 occur simultaneously. MYC amplification therefore indirectly reflects the status of 8q24.2 amplification. Previous study revealed that CCAT2 regulated MYC- related pathway to promote chromosomal instability [7]. In the present study, we investigated the levels of CCAT2 and the status of MYC amplification in ESCC, and further analyzed the relationship between CCAT2 expression and MYC amplification, and clinical features and prognosis of ESCC patients to determine the effects of CCAT2 in the development and progression of ESCC. The results may provide a theoretical basis for the diagnosis, treatment, and prognosis of ESCC.
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MATERIALS AND METHODS Clinical specimens A total of 229 pairs of ESCC and matched paracancerous tissues were obtained from patients who received surgical resection of ESCC between 2007 and 2014 at four hospitals. The distance from adjacent tissue to the edge of tumor tissue was more than 3 cm. The resected samples were pathologically examined by H&E staining. All patients did not receive anti-cancer treatment prior to surgery. Written informed consents were obtained from all patients before sample collection. The study protocol was approved by the hospital ethical committees.
Quantitative real- time PCR (qRT-PCR) Total RNA was extracted using RecoverAllTM Total Nucleic Acid Isolation Kit (Ambion, Austin, Texas) according to the manufacturer’s protocol. After xylene dewaxing, ethanol washing, and protease digestion, total RNA was extracted from FFPE sections. Nanodrop 2000 was used to detect the concentration and purity of total RNA, and then was stored at –80 °C for later use. PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Dalian, China) was used to remove genomic DNA and synthesize cDNA. Quantitative real-time PCR was done using SYBR1 Premix Ex Taq II (Tli RNaseH Plus) (Takara, Dalian, China) on ABI 7900HT (Applied Biosystems, California). The reaction was performed
Fig. 1. CCAT2 was upregulated in ESCC tissues. A, CCAT2 expression in ESCC and adjacent noncancerous tissues. B, CCAT2 expression in LNM positive and negative patients. C, CCAT2 expression at different TNM stages. D, CCAT2 expression in MYC amplified and non-amplified patients. Journal of Surgical Oncology
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Fig. 2. Kaplan–Meier estimates of overall survival for ESCC patients according to CCAT2 expression (A), MYC amplification (B), and the combination of CCAT2 expression and MYC amplification (C).
TABLE I. Association of CCAT2 Expression and Myc Amplification With Clinicopathologic Features CCAT2 expression Clinicopathological features Age (%) 60 >60 Sex (%) Male Female Tumor size 4 >4 Grade (%) Well Moderate Poor General classification Medullary type Ulcerative type Others type TNM (%) I þ II III þ IV LNM (%) No Yes
Journal of Surgical Oncology
MYC amplification
Low
High
P value
Positive
Negative
P value
41 (936.0) 73 (64.0)
47 (40.9) 68 (59.1)
0.498
40 (42.1) 55 (57.9)
48 (35.8) 86 (64.2)
0.339
86 (75.4) 28 (24.6)
84 (73.0) 31 (27.0)
0.763
75 (78.9) 20 (21.1)
95 (70.9) 39 (29.1)
0.220
53 (47.3) 59 (52.7)
60 (52.6) 54 (47.4)
0.506
45 (47.9) 49 (52.1)
68 (51.5) 64 (48.5)
0.686
18 (15.8) 75 (65.8) 21 (18.4)
18 (15.7) 81 (70.4) 16 (13.9)
0.637
15 (15.8) 63 (66.3) 17 (17.9)
21 (15.7) 93 (69.4) 20 (14.9)
0.826
25 (23.4) 62 (57.9) 20 (18.7)
36 (33.0) 63 (57.8) 10 (9.2)
0.070
29 (31.9) 54 (59.3) 8 (8.8)
32 (25.6) 71 (56.8) 22 (17.6)
0.155
71 (62.8) 42 (37.2)
54 (48.2) 58 (51.8)
0.032
41 (44.1) 52 (55.9)
84 (63.6) 48 (36.4)
0.004
65 (57.5) 48 (42.5)
49 (43.0) 65 (57.0)
0.034
37 (39.4) 57 (60.6)
77 (57.9) 56 (42.1)
0.007
CCAT2 and ESCC 1
in a final volume of 20 mL containing 2 SYBR Premix Ex Taq II (Tli RNaseH Plus), 0.4 mM of each primer and 2 mL of cDNA. The reaction conditions were as follow: 95°C for 30 s, followed by 40 cycles at 95 °C for 5 s, and then 60 °C for 34 s. Relative expression of CCAT2 was normalized to reference RNU6B and was calculated using the 2- DDCt equation.
Quantitative real- time genomic PCR analysis Genomic DNA was purified using QIAamp DNA FFPE Tissue Kit (Qiagen, Germany) according to the manufacturer’s protocol. MYC amplification was detected using TaqMan Copy Number Assay as previously described[19]. In addition to MYC, RPPH1 on chromosome 14q11.2 was used as a single-copy control gene.
Statistical analyses Comparison of the levels of CCAT2 between ESCC and adjacent tissues was performed using Student’s paired t-test. Mann–Whitney U test was used to evaluate significant differences between two independent groups of samples. The status of MYC amplification was determined as previously described[19]. Spearman’s rho test was used to analyze the correlation between the expression level of CCAT2 and MYC amplification. Patients were divided into high and low expression groups based on the median expression level of CCAT2. Chi-square or Fisher’s exact test was used to analyze the correlation between CCAT2 expression and MYC amplification and clinicopathological features, as appropriate. The correlations between overall survival, and CCAT2 expression levels and MYC amplification were examined by using the Kaplan–Meier method and log- rank test. Hazard ratios (HRs) and 95% confidence intervals (95%CIs) were calculated using Cox proportional hazard regression model. All statistical analyses were performed using SPSS software (version 19.0, SPSS, Chicago, Illinois) and Graphpad Prism 5.0 (GraphPad Software Inc., CA, USA).
RESULTS ESCC tissues demonstrated significantly upregulated CCAT2 expression compared with adjacent normal tissues (P < 0.001) (Fig. 1A). The level of CCAT2 was positively correlated with TNM stages, LNM, and the number of positive lymph nodes (P < 0.05). The levels of CCAT2 in patients with lymph node metastasis (LNM) and advanced TNM stages were significantly higher than those with nonLNM and TNM stages I and II, respectively (P < 0.001) (Fig. 1B and 1 C). When cases were divided into low and high CCAT2 expression groups according the median levels of CCAT2, high CCAT2 expression
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was significantly related to LNM (P ¼ 0.034) (Table I). Although there was no association between CCAT2 expression and TNM stages (P ¼ 0.057), high CCAT2 was also related to advanced TNM stages (P ¼ 0.032) when the stages of disease were classified into two groups by early stages I-II and advanced stages III-IV. Furthermore, patients with high CCAT2 expression had more positive lymph nodes (2.00 2.63) than those with low CCAT2 expression (1.24 2.10) (P ¼ 0.012). No significant difference was found between CCAT2 expression and other clinical features. Among 229 ESCC cases, MYC amplification was detected in 95 cases (41.5%). MYC amplification was observed in any TNM stage, in particular in advanced stages, indicating positive relationship between MYC amplification and clinical aggressiveness of ESCC. We next examined the association of MYC amplification with clinical features in all cases. MYC amplification was associated with LNM and TNM stages (P < 0.05). The status of MYC amplification was positive correlated with the number of positive lymph nodes (P ¼ 0.001). MYC amplification was not associated with age, sex, tumor size, histological grade, and pathologic type. We further examined whether there was an association of MYC amplification with the expression level of CCAT2. Increased MYC copy number correlated with increased level of CCAT2 (P < 0.001). The levels of CCAT2 in MYC amplification group was significantly higher than that in MYC non-amplification group (P < 0.001) (Fig. 1D). During 66 months follow-up period, 139 ESCC patients died. The median survival time (MST) was 31.0 months, and the 5-year survival rate was 25.5%. The MST was found to be shorter in patients with high CCAT2 expressing than in those with low CCAT2 expressing, and in patients with MYC amplification than in those with MYC nonamplified individuals (Fig. 2). Multivariate Cox regression analysis revealed that high CCAT2 expression (HR ¼ 1.432, 95% CI: 1.005– 2.041, P ¼ 0.047), MYC amplification (HR ¼ 1.505, 95% CI: 1.051– 2.154, P ¼ 0.026), and TNM (HR ¼ 2.666, 95% CI: 1.074–6.618, P ¼ 0.035) were independent prognosis factors for ESCC (Table II). We further analyzed the outcome of patients according to combination of CCAT2 expression and MYC amplification. As shown in Table II, patients with 1 (high CCAT2 level or MYC amplification) and 2 risk factors (high CCAT2 level and MYC amplification) had 1.734-fold (95% CI: 1.127–2.669) and 2.199-(95% CI: 1.410–3.430) increased risk of death, respectively, when compared with those with zero risk factors (low CCAT2 level and MYC non-amplification) (Fig. 2C).
DISCUSSION Current studies on endogenous miRNA have achieved promising results. Similar to miRNAs, lncRNAs also represent another important
TABLE II. Univariate and Multivariate Cox Regression Analyzes for Overall Survival in Escc Patients Univariate analysis Factors
HR (95%CI)
age (year) (>60/60) 0.826(0.585–1.167) sex (male/female) 0.891 (0.602–1.318) tumor size (cm) (>4/4) 1.125(0.805–1.572) general classification (ulcerative type/others) 1.280(0.902–1.818) tumor differentiation (poor/well, moderate) 1.204(0.775–1.870) TNM stage (III, IV/I, II) 1.896(1.347–2.670) LNM (positive/negative) 1.632(1.160–2.296) CCAT2 (high/low) 1.641(1.170–2.301) MYC amplification (positive/negative) 1.827(1.306–2.556) Number of risk factors (high CCAT2 expression and MYC amplification) 0 1 2 2.361(1.531–3.643) 1 1.865(1.221–2.850)
Journal of Surgical Oncology
Multivariate analysis P value
HR (95%CI)
P value
0.279 0.563 0.490 0.167 0.410
