Human Pathology (2015) 46, 707–715
www.elsevier.com/locate/humpath
Original contribution
ORAOV1 overexpression in esophageal squamous cell carcinoma and esophageal dysplasia: a possible biomarker of progression and poor prognosis in esophageal carcinoma☆,☆☆ Man Li MD a,b,1 , Xiaobin Cui MD a,b,c,1 , Yaoyuan Shen MD a , Hongchao Dong MD a , Weihua Liang BS a,b , Yunzhao Chen MD, PhD a,b , Jianming Hu MD a,b,c , Shugang Li MD, PhD d , Jing Kong MS a , Hongan Li BS a,b , Jin Zhao MD a,b,⁎, Feng Li MD, PHD a,b,c,⁎ a
Department of Pathology and Key Laboratory for Xinjiang Endemic and Ethnic Diseases, Shihezi University School of Medicine, Shihezi 832002, Xinjiang, China b Department of Pathology, The First Affiliated Hospital, Shihezi University School of Medicine, Shihezi 832002, Xinjiang, China c Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China d Department of Preventive Medicine, Shihezi University School of Medicine, Shihezi 832002, Xinjiang, China Received 30 June 2014; revised 11 January 2015; accepted 21 January 2015
Keywords: Esophageal squamous cell cancer; Esophageal squamous intraepithelial neoplasia; Oral cancer overexpressed 1; Overexpression; Prognosis
Summary Oral cancer overexpressed 1 (ORAOV1) has been reported to exhibit high amplification levels in esophageal squamous cell cancer (ESCC) and in premalignant lesions. However, ORAOV1 protein expression levels in ESCC and esophageal squamous intraepithelial neoplasia (ESIN) have not yet been reported. We have explored the relationship of ORAOV1 protein expression with ESCC and ESIN by immunohistochemically analyzing tissue microarrays containing esophageal samples from patients with various clinical features and prognoses. The percentage of ESCC, high-grade ESIN (HGESIN), low-grade ESIN (LGESIN), and nontumoral control patients overexpressing ORAOV1 were 70.63% (101/143), 77.36% (41/53), 48.96% (47/96), and 5.79% (7/121), respectively. ORAOV1 overexpression also appears to be significantly higher in ESCC, HGESIN, and LGESIN than in the controls (all P b .001), and the levels observed for ESCC and HGESIN were also significantly higher than that in LGESIN (both P = .001). These results corresponded to high sensitivity and specificity values in ESCC, HGESIN, and LGESIN tissues. Furthermore, the increased expression of ORAOV1 is significantly associated with lymph node metastasis
☆
Competing interests: The authors declare that they have no competing interests. Funding/Support: This work was supported by grants from the Ministry of Science and Technology of China (2010DFB34100 and 2012AA02A503), the National Natural Science Foundation of China (no. 81160301, 81360358, 81260301), and a joint foundation for nurturing the outstanding young scientists of Shihezi University (no. 2013ZRKXYQ-YD19). ⁎ Corresponding authors at: Department of Pathology and Key Laboratory for Xinjiang Endemic and Ethnic Diseases, Shihezi University School of Medicine, North 4th Road, Shihezi 832002, Xinjiang, China. E-mail addresses: [email protected] (J. Zhao), [email protected] (F. Li). 1 Authors contributed equally to this work. ☆☆
http://dx.doi.org/10.1016/j.humpath.2015.01.009 0046-8177/© 2015 Elsevier Inc. All rights reserved.
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M. Li et al. (P = .001) and an advanced TNM stage (III + IV) (P = .014), and patients with ORAOV1 overexpression experienced shorter overall survival time compared with those with lower ORAOV1 (χ2 = 11.505, P = .001). This study provides the first evidence of ORAOV1 overexpression in ESCC and ESIN and demonstrates a potential role in tumor progression and metastasis. ORAOV1 overexpression could, therefore, be used as a novel biomarker of poor prognosis in patients with ESCC. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Esophageal squamous cell cancer (ESCC) is one of the most common malignancies detected and treated worldwide [1,2]. In 2008, the incidence and mortality rates associated with esophageal cancer in China alone were 16.7 and 13.4 per 100 000 persons, respectively. Because most patients with ESCC present with advanced metastasis at diagnosis [3], the efficacy of ESCC therapy is far from optimal, and current treatment options only yield a 5-year survival rate of 17% [4]. Therefore, it is crucial to identify novel biomarkers that are both sensitive and specific for ESCC to detect the disease earlier as well as increase the efficiency and number of treatment options available for these patients. Unfortunately, although our understanding of ESCC pathogenesis is continually expanding, recent research in this field has yet to significantly improve upon the current clinical techniques used to diagnose ESCC. Esophageal squamous cell cancer progression is a multistage process, whereby a normal esophagus will first develop basal cell hyperplasia, followed by intraepithelial neoplasia, carcinoma in situ, and, finally, invasive carcinoma [5,6]. Esophageal squamous intraepithelial neoplasia (ESIN) has been shown to be a histologic precursor of ESCC [7] and can be divided into 2 types, low-grade ESIN (LGESIN) and high-grade ESIN (HGESIN), based on the severity of cytologic and architectural atypia [8]. Many reports have suggested that ESIN has prognostic significance for esophageal carcinoma because dysplastic lesions are frequently encountered in cancerous esophagus tissue [9,10]. Therefore, the protein expression abnormalities observed during ESIN are likely the most promising candidates for predicting the development of ESCC. However, very few studies using ESIN models have focused on protein expression changes in relationship to squamous cell carcinogenesis. Notably, previous studies suggest that one of the most frequent genomic changes associated with ESCC development is the amplification of chromosome 11q13.3 [11,12]. Many oncogenes have been identified within this region, indicating that they may play important roles in ESCC tumorigenesis. These genes include cyclin D1 (CCND1) and cortactin (CTTN), which have been found to be overexpressed in various cancers, including ESCC [13,14]. Oral cancer overexpressed 1 (ORAOV1), also known as tumor amplified and overexpressed sequence 1 (TAOS1), has also been mapped to this chromosomal region, being found only approximately
12 kb away from the CCND1 gene locus. Not surprisingly, ORAOV1 has also been associated with a variety of human cancers, including oral cancer [15-18], laryngeal cancer [19], cervical cancer [20], gastric cancer [21], and ESCC [22]. Oral squamous cell carcinoma (OSCC), in particular, appears to have a wide range of overlap with ESCC in terms of gene abnormalities, including changes in CCND1 and CTTN expression as well as progression pattern after lesion formation in the squamous epithelium. Importantly, in OSCC tumors and lesions, amplification of the ORAOV1 gene [16] as well as overexpression of ORAOV1 messenger RNA (mRNA) have been observed [15,17], suggesting a relationship between this specific gene and cancer progression in this cellular context. Furthermore, ORAOV1 gene amplification and mRNA overexpression have also both been reported in ESCC tumor tissues [22,23]. Thus, we hypothesize that ORAOV1 may also play a similarly significant role in ESCC. However, to our knowledge, the expression and potential function of the ORAOV1 protein in ESCC tissues and esophageal precancerous lesions have not been reported in the literature. In this study, we have investigated ORAOV1 protein expression by analyzing surgically resected primary ESCC specimens as well as ESIN specimens of various degrees and adjacent morphologically normal tissue using immunohistochemical techniques. In doing so, we have determined that ORAOV1 protein expression is up-regulated during the development and progression of ESCC. Our findings suggest that ORAOV1 overexpression may serve as a reliable biomarker of poor prognosis in patients with ESCC in the early stages of the disease. We suspect that clinical diagnosis using ORAOV1 early on will enhance the efficiency and options for ESCC treatment and warrants further investigation.
2. Materials and methods 2.1. Patients and samples Primary tumor samples were obtained from patients with ESCC during tumor resection at the pathology department of the First Affiliated Hospital of Shihezi University School of Medicine between 1997 and 2014. The research protocol used in this study was approved by the medical ethics and human clinical trial committee of the Shihezi University School of Medicine, and all recruited subjects were enrolled
Overexpression of ORAOV1 protein in ESCC and ESIN with written informed consent. A total of 143 ESCC samples were obtained, fixed for 24 hours in formalin, and embedded in paraffin. None of the patients in this study had received preoperative chemotherapy or radiotherapy, and the clinical and pathologic data were available for all patients. The TNM stage was evaluated for each patient in accordance with the Cancer Staging Manual of 2010 [24]. Follow-up information was collected by phone or other methods. In addition, 121 matched nontumoral esophageal tissues, 96 LGESIN specimens, and 53 HGESIN specimens were also analyzed in this study.
2.2. Tissue microarray construction Each paraffin-embedded sample was sectioned and stained for hematoxylin and eosin. The various regions of each tumor sample were identified from these hematoxylin and eosin–stained slides. Subsequently, the fields corresponding to these selected regions were located in the remaining paraffin block for tissue microarray (TMA) construction. Tissue cylinders with a diameter of 1.0 mm were then punched from these areas of each donor tissue block and inserted into a recipient paraffin block using a tissue arrayer (Alphelys, Plaisir, France). The area of each tissue cylinder was reviewed to ensure that at least 70% of it represented the specified region of interest in that sample. Finally, serial sections 4-μm thick were then prepared from the TMA blocks for immunohistochemical staining.
2.3. Determination of ORAOV1 protein expression Immunohistochemistry was performed using the Envision system (Dako, Carpinteria, CA) to detect ORAOV1 protein expression. Briefly, sections of each specimen were prepared on glass slides and baked at 65°C for 30 minutes. The tissue sections were then deparaffinized by 3 consecutive treatments with xylene and rehydrated by sequential immersion in graded alcohol. After deparaffinization, the sections were incubated in methanol hydrogen peroxide (3%) for 10 minutes to remove endogenous peroxidase and subsequently washed with tap water. Antigen retrieval was then performed by heating the samples in a microwave oven under high power for approximately 8 minutes in a citrate buffer solution and then cooled for 30 to 40 minutes at room temperature. Slides were washed 3 times in phosphate-buffered saline (PBS) and then incubated with anti-ORAOV1 antibody (ab64843; Abcam, Cambridge, MA; dilution 1:500). Each section was incubated with 150 μL of the diluted antibody at 4°C overnight, whereas negative control sections were incubated with PBS. After allowing the slides to warm at room temperature for 20 minutes, they were washed 3 times in PBS and then incubated with 150 μL of secondary antibody for 30 minutes at 37°C. After a final wash with PBS and treatment with fresh diaminobenzidine solution, these sections were lightly stained with hematoxylin to highlight cell nuclei and dehydrated with graded alcohol and
709 xylene. Antifade mounting medium (ZSGB-Bio, Beijing, China) and glass cover slips were then added. Slides were stored at room temperature until analysis. All of the immunostained slides were blindly evaluated by 2 pathologists under a 5-headed microscope (Olympus Optical, Tokyo, Japan) and were assigned a consensus score. The slides were categorized as ORAOV1 positive when brownish-yellow particles appeared in the cytoplasm. The staining intensity was scored as 0 (no staining), 1 (weak staining), 2 (medium staining), or 3 (strong staining). The percentage of stained cells was scored as 0 (b5% of the cells), 1 (6%-25% of the cells), 2 (26%-50% of the cells), 3 (51%-75% of the cells), or 4 (≥76% of the cells). The product of both scores was used to identify 4 categories of expression: +++ (a score of 9-12), ++ (a score of 5-8), + (a score of 2-4), and − (a score of 0-1) [25]. In cases where the 2 pathologists disagreed on the immunostaining results, a third pathologist was consulted to analyze the staining.
2.4. Statistical methods Statistical analysis of the experimental data was performed using SPSS 17.0 (SPSS Inc, Chicago, IL). Categorical data were analyzed using χ2 tests to determine the statistically significant differences in ORAOV1 protein expression in the ESCC and ESIN samples compared with the nontumoral control tissues. The correlations between ORAOV1 expression and the clinicopathological factors were explored by Mann-Whitney U and Kruskal-Wallis H tests. Receiver operating characteristic (ROC) curves and the area under the curve (AUC) were used to evaluate the specificity and sensitivity of ESCC and ESIN prediction using a 95% confidence interval (CI). The survival rates were calculated using the Kaplan-Meier method, and the survival curves were analyzed using the log-rank test. The univariate and multivariate hazard ratios for different variables were calculated using the Cox proportional hazards model. In all of the statistical analyses, a P b .05 was considered statistically significant.
3. Results 3.1. Clinicopathological demographics of the patients with ESCC The present study included 143 patients with ESCC, of which 108 (75.52%) were men and 35 (24.48%) were women. The age of the patients at the time of surgery ranged from 36 to 81 years, with a median age of 62 years. Of these 143 ESCC cases, 41 (28.67%) were well differentiated, 73 (51.05%) were moderately differentiated, and 29 (20.28%) were poorly differentiated [26]. Lymph node metastasis was present in 49 (34.27%) of the patients. Furthermore, 95 (66.43%) of the patients were at I or II clinical stage, whereas 48 (33.57%) were at stage III or IV.
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3.2. ORAOV1 protein expression is increased in ESCC and ESIN compared with nontumoral squamous epithelium tissue Immunohistochemical staining of ESCC, HGESIN, LGESIN, and nontumoral squamous epithelium tissues demonstrated that ORAOV1 is localized to the cytoplasm. However, the distribution of ORAOV1 staining was significantly different among these 4 groups, with strong or medium ORAOV1 staining observed in the ESCC, HGESIN, and LGESIN tissues and weak or no ORAOV1 staining in the nontumoral squamous epithelium (Fig. 1). Furthermore, the overall percentages of ESCC, HGESIN, LGESIN, and nontumoral tissue samples expressing ORAOV1 were 100% (143/143), 100% (53/53), 97.92% (94/96), and 90.91% (110/121), respectively. The percentages of patients overexpressing ORAOV1 (categorized as strong or medium staining) in each of these groups were
M. Li et al. 70.63% (101/143), 77.36% (41/53), 48.96% (47/96), and 5.79% (7/121) for the ESCC, HGESIN, LGESIN, and nontumoral tissues, respectively (Table 1). Furthermore, we observed an increasing trend in the rate of ORAOV1 expression/overexpression as the cell type progressed from normal (nontumoral) to increasing levels of metastasis (LGESIN followed by HGESIN and ESCC), with the level of ORAOV1 overexpression in the ESCC, HGESIN, and LGESIN tissues being significantly higher compared with the controls (70.63%, 77.36%, and 48.96% versus 5.79%, respectively; all P b .001). Furthermore, these data also indicate that ORAOV1 overexpression was significantly higher in ESCC and HGESIN compared with LGESIN (both P = .001). Interestingly, of the 4 tissues, ORAOV1 overexpression was found to be the highest in HGESIN, not ESCC; however, the difference between these 2 groups was not significant (P = .349) (Fig. 2).
Fig. 1 Immunohistochemical staining of ORAOV1 expression in ESCC (A), original magnification ×40; HGESIN (B), ×40; LGESIN (C), ×40; and nontumoral esophageal tissue (D), ×40. Immunohistochemical staining for ORAOV1 demonstrated strong cytoplasmic expression in ESCC (E), ×200, and HGESIN (F), ×200, and medium expression in LGESIN (G), ×200; whereas ORAOV1 staining indicated weak expression in nontumoral esophageal tissue (H), ×200. Hematoxylin and eosin staining is shown for ESCC (I), ×200; HGESIN (J), ×200; LGESIN (K), ×200; and nontumoral esophageal tissue (L), ×200.
Overexpression of ORAOV1 protein in ESCC and ESIN Table 1
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ORAOV1 expression in ESCC, HGESIN, LGESIN, and control tissues
Group
n
ESCC HGESIN LGESIN control
143 53 96 121
Pa
ORAOV1 expression (n/%) −
+
++
0 0 2 (2.08) 11 (9.09)
42 (29.37) 12 (22.64) 47 (48.96) 103 (85.12)
70 28 47 7
+++ (48.95) (52.83) (48.96) (5.79)
31 (21.68) 13 (24.53) 0 0
b.001
Abbreviations: ORAOV1, oral cancer overexpressed 1; ESCC, esophageal squamous cell cancer; HGESIN, high-grade esophageal squamous intraepithelial neoplasia; LGESIN, lowgrade esophageal squamous intraepithelial neoplasia. a P b .05 indicates a significant association among the variables.
3.3. ORAOV1 may be a potential diagnostic biomarker of ESCC and ESIN
3.4. ORAOV1 overexpression is associated with lymph node metastasis and advanced TNM stage in ESCC
We used ROC curves to evaluate the ORAOV1 immunohistochemical scores determined for each of the ESCC, HGESIN, LGESIN, and control tissues. We found that the ESCC, HGESIN, and LGESIN tissues were easily distinguished from the controls, with ROC AUC values of 0.901 (95% CI, 0.863-0.939), 0.906 (95% CI, 0.850-0.963), and 0.811 (95% CI, 0.748-0.875), respectively (Fig. 3). We next determined sensitivity and specificity values for each cancerous tissue type using cutoff levels that optimized the diagnostic accuracy rate, while minimizing the false-negative and false-positive rates (Table 2). The sensitivity and specificity values for ESCC were 89.5% and 79.3% (cutoff score of 4). High-grade ESIN and LGESIN were determined to have sensitivity and specificity values of 77.4% and 94.2% (cutoff score of 5) and 83.3% and 79.3% (cutoff score of 4), respectively. Taken together, these results support the notion that ORAOV1 may be a potential diagnostic biomarker of ESCC and ESIN.
To investigate the potential functional implications of aberrant ORAOV1 expression during ESCC, we evaluated the relationship between ORAOV1 expression and the clinicopathological characteristics of ESCC, such as age, sex, histologic grade, lymph node metastasis status, and TNM stage. Notably, the increased expression of ORAOV1 appears to be significantly associated with lymph node metastasis (P = .001) and advanced TNM stage (P = .014). From this result, we can infer that the up-regulated expression of ORAOV1 may contribute to tumor metastasis and aggressiveness. Other parameters such as age, sex, and tumor differentiation had no significant relationship with ORAOV1 expression (Table 3).
Fig. 2 Box plot showing significantly higher overexpression of ORAOV1 in ESCC, HGESIN, and LGESIN tissues than that in nontumoral esophageal tissue, and the ORAOV1 overexpression was significantly higher in ESCC and HGESIN than in LGESIN. *P = .001. **P b .001.
3.5. ORAOV1 overexpression predicts poor prognosis in ESCC The overall survival of patients with ESCC in relationship to ORAOV1 expression was the primary end point of interest in the present study; thus, we examined the correlation between ORAOV1 protein expression and the postoperative survival of patients who underwent curative surgery. Of the 143 ESCC cases examined, clinical follow-up information was available for 75 patients. The deadline of follow-up was the date of final contact (December 25, 2013) or death. The mean follow-up period of postoperative survival was 18 months (ranging from 1-96 months after treatment). Univariate analysis of the 75 enrolled patients with ESCC revealed a tendency for patients with higher ORAOV1 expression to have poorer outcomes than those with lower expression. The median survival time of patients with ORAOV1 overexpression was 14 months (range, 1-90 months), which was significantly lower than the median survival of 27 months (range, 1-96 months) in patients with lower ORAOV1 expression. Furthermore, using a Kaplan-Meier analysis, we determined that this difference in survival time, with patients overexpressing ORAOV1 experiencing a shorter overall survival time than patients with lower ORAOV1 expression, was significant (χ2 = 11.505, P = .001, Fig. 4A). Moreover, the postoperative mortality rate was higher in
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Fig. 3 Receiver operating characteristic curve analysis of the ORAOV1 immunohistochemical scores for detecting ESCC (A), HGESIN (B), and LGESIN (C) tissues from the controls. The AUC in ESCC, HGESIN, and LGESIN is 0.901, 0.906, and 0.811, respectively.
patients with ORAOV1 overexpression than in those with weak or no expression (P = .001, Fig. 4B). Finally, to determine whether the inclusion of the ORAOV1 immunohistochemical analysis provided additional prognostic information, multivariate Cox regression analysis was performed for the expression of ORAOV1 and all clinicopathological factors included in the univariate analysis. Statistically, it appears that ORAOV1 overexpression is a significant factor in the multivariate Cox regression model (P = .007; hazard ratio, 3.329; Table 4). Together, these data indicate that ORAOV1 overexpression is a significant independent prognostic factor for poor prognosis in ESCC and could thus be used as a potential biomarker for prognosis evaluation in patients with ESCC.
levels of ORAOV1 protein compared with nontumoral tissues, suggesting that ORAOV1 plays an important role in ESCC and its related precancerous lesions. These findings are consistent with those of a previous study conducted by Komatsu et al [22] on ORAOV1 mRNA expression, which found that ORAOV1 was frequently overexpressed in ESCC. Similarly, the expression of ORAOV1 mRNA in OSCC tissues was also found to be significantly higher compared with normal oral tissues [17,27], indicating that a similar mechanism may exist in multiple cancers. Moreover, we have revealed that HGESIN and LGESIN tissues also exhibited significantly higher ORAOV1 expression Table 3 Association between ORAOV1 expression and clinicopathological factors in ESCC Characteristics
4. Discussion
n
ORAOV1 expression (n/%) +
The ORAOV1 gene, identified in 2002 by Huang et al [15], is 414 nucleotides long and includes 5 known exons; however, the function of the encoded protein is currently unknown. ORAOV1 has been reported to be associated with a variety of cancers. In the present study, we have demonstrated that overexpression of the ORAOV1 protein was positively associated with the development and progression of ESCC. To the best of our knowledge, this is the first study to demonstrate that ESCC tissues exhibit significantly higher Table 2 The high sensitivity, specificity, and AUC values of ORAOV1 in ESCC, HGESIN, and LGESIN Comparison
AUC
Sensitivity (%)
Specificity (%)
Cutoff value a
ESCC vs control HGESIN vs control LGESIN vs control
0.901 0.906 0.811
89.5 77.4 83.3
79.3 94.2 79.3
4 5 4
Abbreviations: AUC, area under the curve; ORAOV1, oral cancer overexpressed 1; ESCC, esophageal squamous cell cancer; HGESIN, high-grade esophageal squamous intraepithelial neoplasia; LGESIN, low-grade esophageal squamous intraepithelial neoplasia. a Cutoff level was set to provide optimal sensitivity and specificity.
Age (y) b60 50 ≥60 93 Sex Male 108 Female 35 Differentiation a Well 41 Moderate 73 Poor 29 L/N metastasis Negative 94 Positive 49 TNM stage b I + II 95 III + IV 48
++
P
+++
13 (26.0) 23 (46.0) 14 (28.0) .243 29 (31.18) 47 (50.54) 17 (18.28) 28 (25.92) 60 (55.56) 20 (18.52) .854 14 (40.0) 10 (28.57) 11 (31.43) 10 (24.39) 18 (43.90) 13 (31.71) .244 c 22 (30.14) 37 (50.68) 14 (19.18) 10 (34.48) 15 (51.72) 4 (13.80) 37 (39.36) 40 (42.55) 17 (18.09) .001 5 (10.20) 30 (61.22) 14 (28.58) 35 (36.84) 42 (44.21) 18 (18.95) .014 7 (14.58) 28 (58.33) 13 (27.09)
Abbreviations: ESCC, esophageal squamous cell cancer; L/N metastasis, lymph node metastasis; TNM, tumor, node, metastasis. a Histologic grade was based on World Health Organization classification published in 2010 [26]. b TNM stage was based on the American Joint Committee on Cancer criteria published in 2010 [24]. c Kruskal-Wallis H test.
Overexpression of ORAOV1 protein in ESCC and ESIN
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Fig. 4 Kaplan-Meier survival curves showing patients with ESCC with overexpressed ORAOV1 (2+/3+) and low ORAOV1 levels (−/1+). A, Patients with overexpressed ORAOV1 experienced a significantly shorter postoperative survival time than those with low ORAOV1 levels (P = .001). B, Patients with ORAOV1 overexpression had a higher risk of death than those with lower ORAOV1 levels (P = .001).
compared with nontumoral tissues and that ESCC and HGESIN overexpress ORAOV1 at a higher level than LGESIN. These results indicate that ORAOV1 may be overexpressed immediately after transformation of the esophageal squamous epithelium. Xia et al [16] reported increased ORAOV1 amplification beginning at the onset of mild epithelial dysplasia, whereas no amplification was observed in normal mucosa and hyperplasia. These findings are consistent with our observation of increasing ORAOV1 expression during the progression of ESCC, as the ORAOV1 gene copy number is strongly correlated with its expression level [15,17]. Clinical observations have suggested that ESIN can be categorized as a precancerous dysplastic lesion from which squamous cell carcinoma can arise [28]. Furthermore, several studies have also shown an increased risk of developing invasive ESCC in patients with mild dysplasia, moderate dysplasia, severe dysplasia, and carcinoma in situ [28-30]. In the present study, we observed an increase in the expression of ORAOV1 in HGESIN and ESCC compared with the LGESIN tissue samples, whereas the difference between ESCC and HGESIN was not significant. From these data, we can infer
Table 4
that ORAOV1 may play an important role in the development and progression of ESCC. ORAOV1 is located approximately 12 kb from the CCND1 gene, and both genes have been implicated as driving forces behind 11q13 gene amplification [15,16]. Consistent with this notion, Jiang et al [20] observed that, in HeLa cells, the expression of CCND1 was reduced in ORAOV1-silenced cells. These data suggest some form of interaction between these 2 genes, and it has been proposed that CCND1 may be a downstream effector of ORAOV1 [20]. Thus, it is likely that the functions of ORAOV1 and CCND1 are related. This hypothesis is further supported by Shamma et al [31], who previously reported that CCND1 overexpression begins early in ESCC dysplasia, which, as shown in the present study, mirrors the expression pattern of ORAOV1 in different esophageal tissues. Further research is required to fully characterize the relationship between ORAOV1 and CCND1 and to determine the functional mechanisms involved in the development of ESCC. To examine the feasibility of applying ORAOV1 as a clinical biomarker of ESCC and ESIN, we constructed ROC
Univariate and multivariate survival analyses of clinicopathological characteristics
Characteristics
Univariate analysis HR
95% CI
Multivariate analysis P
ORAOV1 overexpression 3.673 1.614 8.362 .002 ⁎ Age (≥60 y) 1.043 0.529 2.057 .903 Sex (female) 1.541 0.789 3.009 .205 Differentiation a (moderate/poor) 0.881/0.828 0.354/0.365 2.197/1.887 .787/.651 L/N metastasis (positive) 2.038 1.083 3.834 .027 ⁎ TNM stage b (III + IV) 2.138 1.134 4.033 .019 ⁎
HR
95% CI
3.329 1.116 1.321 0.708/0.681 1.109 1.617
1.385 0.537 0.622 0.272/0.269 0.326 0.484
Abbreviations: L/N, lymph node; CI, confidence interval; HR, hazard radio. a Histologic grade was based on World Health Organization classification published in 2010 [26]. b TNM stage was based on the American Joint Committee on Cancer criteria published in 2010 [24]. ⁎ Significant difference that 95% CI was not including 1.
P 8.002 2.319 2.808 1.843/1.721 3.778 5.406
.007 ⁎ .768 .469 .479/.417 .869 .435
714 curves and the AUC to evaluate the specificity and sensitivity of this marker in each tissue type. We obtained high sensitivity and specificity values, which highlight the potential usefulness of ORAOV1 as a novel clinical diagnostic biomarker and therapeutic target for ESCC and ESIN treatment. Furthermore, our results were obtained via immunohistochemical analysis, which has been established as a highly sensitive and costeffective method for evaluating protein expression [32]. Indeed, this method has been used to identify several genes as potential candidates for the development of novel diagnostic markers, therapeutic drugs, and immunotherapies [33,34]. Additional work using other protein analysis techniques is necessary to validate the immunohistochemistry results reported here. One of the most important findings of this study was the discovery that the high expression of ORAOV1 in ESCC tissues is associated with lymph node metastasis and an advanced TNM stage (III + IV). This suggests that ORAOV1 overexpression may be one of the causative factors of esophageal mucosal disorders and could potentially contribute to the metastasis and aggressiveness of ESCC. Our results are corroborated by many other reports investigating ORAOV1 in other types of tumors. For example, one study reported that overexpression of ORAOV1 in ESCC tissues was significantly associated with lymph node metastasis and an advanced TNM stage [22], whereas others have observed abnormal amplification of ORAOV1 in OSCC and gastric adenocarcinoma with lymph node metastasis and an advanced TNM stage [16,21]. ORAOV1 has also been reported to play a crucial role in tumor progression and angiogenesis in OSCC [18], and the average intratumoral microvessel density of ORAOV1-silenced tumors was found to be much lower than that of the controls, suggesting that silencing of ORAOV1 has an antiangiogenic effect in OSCC tumors. Additional studies have also revealed that the expression of vascular endothelial growth factor, an important factor in tumor angiogenesis, is much lower in ORAOV1-silenced tumors than in the controls [18]. Therefore, it is possible that ORAOV1 plays a role in mediating the proangiogenic effects of vascular endothelial growth factor in cancerous oral tissues, thereby contributing to tumor growth, lymph node metastasis, and distant metastasis. In addition to the TNM stage of the patient, the level of cellular differentiation in the ESCC is another important clinicopathological characteristic; however, we found no significant correlation between differentiation and ORAOV1 overexpression, although the well-differentiated ESCC samples did show a tendency for higher ORAOV1 expression. These observations are consistent with a study by Xavier et al [17] that focused on ORAOV1 mRNA expression in OSCC tissues but are contradicted by a study by Xia et al [16] in which ORAOV1 amplification was associated with poorly differentiated OSCC. The discrepancies between these results may be attributed to the diversity of tumor types analyzed, the heterogeneity of methods used, and the limitations of the small sample sizes. Further investigation using larger sample sizes is required to clarify the relationship between ORAOV1 expression and ESCC differentiation.
M. Li et al. Because all of the clinicopathological characteristics assessed in this study were prognostic factors in ESCC, it was essential to conduct a systematic analysis to confirm the potential influence of ORAOV1 protein expression on the prognosis of patients with ESCC. Our results show that patients with ESCC with higher ORAOV1 expression had a shorter postoperative survival time than those with lower ORAOV1 expression. Cox analysis also revealed that ORAOV1 overexpression could serve as a significant independent prognostic factor to predict the risk of death, with a high hazard ratio of 3.329. The relationship between ORAOV1 amplification/expression and overall survival rates has been analyzed previously [17], and it has been hypothesized that ORAOV1 amplification could predict poor prognosis [16]. We support this theory with the first report demonstrating an association between ORAOV1 expression and prognosis of ESCC. These results further expand our understanding of ORAOV1 function in terms of patient health at the cellular, tissue, and organismal levels. Although this study does not directly test the functional mechanism of ORAOV1, previous reports have indicated that a complex signaling cascade may be regulated by this protein during tumor pathogenesis. For instance, ORAOV1 silencing has been shown to lead to a distinct S-phase arrest through the suppression of DNA synthesis and down-regulation of the expression of several cell cycle regulators, including cell division control 2 (Cdc2), cyclin A, and cyclin B1 [18,20]. Furthermore, ORAOV1 silencing also induces apoptosis by increasing the expression of p53 and decreasing the expression of Bcl-2 [20]. This process appears to involve the activation of caspases 3, 8, and 9, indicating that the intrinsic and extrinsic apoptotic pathways are activated by ORAOV1 silencing. In light of our data, these findings suggest that ORAOV1 may promote tumor growth by regulating cell cycle progression and apoptosis, resulting in a poor outcome/prognosis of the patient with ESCC. In conclusion, this study is the first to show that ORAOV1 protein is overexpressed in ESCC and ESIN tissues and that such overexpression is correlated with lymph node metastasis and advanced TNM stages (III/IV) of ESCC. Accordingly, overexpression of ORAOV1 may be useful in identifying patients with ESCC who will likely experience shorter postoperative survival times. Our data suggest that ORAOV1 is useful as a prognostic and diagnostic biomarker and may also be a novel therapeutic target for ESCC. Further research is required to validate these concepts and fully elucidate the functional mechanism of this complex protein.
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Overexpression of ORAOV1 protein in ESCC and ESIN [3] Layke JC, Lopez PP. Esophageal cancer: a review and update. Am Fam Physician 2006;73:2187-94. [4] Ma X, Chen K, Huang S, et al. Frequent activation of the hedgehog pathway in advanced gastric adenocarcinomas. Carcinogenesis 2005; 26:1698-705. [5] Wang LD, Yang HH, Fan ZM, et al. Cytological screening and 15 years' follow-up (1986-2001) for early esophageal squamous cell carcinoma and precancerous lesions in a high-risk population in Anyang County, Henan Province, Northern China. Cancer Detect Prev 2005;29:317-22. [6] Shimizu M, Nagata K, Yamaguchi H, Kita H. Squamous intraepithelial neoplasia of the esophagus: past, present, and future. J Gastroenterol 2009;44:103-12. [7] Hamilton SR, Aaltonen LA, editors. Pathology and genetics of tumours of the digestive system. Lyon: IARC Press, World Health Organization; 2000. [8] Nagamatsu M, Mori M, Kuwano H, Sugimachi K, Akiyoshi T. Serial histologic investigation of squamous epithelial dysplasia associated with carcinoma of the esophagus. Cancer 1992;69:1094-8. [9] Morita M, Kuwano H, Yasuda M, et al. The multicentric occurrence of squamous epithelial dysplasia and squamous cell carcinoma in the esophagus. Cancer 1994;74:2889-95. [10] Ying J, Shan L, Li J, et al. Genome-wide screening for genetic alterations in esophageal cancer by aCGH identifies 11q13 amplification oncogenes associated with nodal metastasis. PLoS One 2012;7:e39797. [11] Shi ZZ, Jiang YY, Hao JJ, et al. Identification of putative target genes for amplification within 11q13.2 and 3q27.1 in esophageal squamous cell carcinoma. Clin Transl Oncol 2014;16:606-15. [12] Shi ZZ, Liang JW, Zhan T, et al. Genomic alterations with impact on survival in esophageal squamous cell carcinoma identified by array comparative genomic hybridization. Genes Chromosom Cancer 2011; 50:518-26. [13] Luo ML, Wang MR. CTTN (EMS1): an oncogene contributing to the metastasis of esophageal squamous cell carcinoma. Cell Res 2007;17: 298-300. [14] Sunpaweravong P, Sunpaweravong S, Puttawibul P, et al. Epidermal growth factor receptor and cyclin D1 are independently amplified and overexpressed in esophageal squamous cell carcinoma. J Cancer Res Clin Oncol 2005;131:111-9. [15] Huang X, Gollin SM, Raja S, Godfrey TE. High-resolution mapping of the 11q13 amplicon and identification of a gene, TAOS1, that is amplified and overexpressed in oral cancer cells. Proc Natl Acad Sci 2002;99:11369-74. [16] Xia J, Chen Q, Li B, Zeng X. Amplifications of TAOS1 and EMS1 genes in oral carcinogenesis: association with clinicopathological features. Oral Oncol 2007;43:508-14. [17] Xavier FCA, Rodini CO, Paiva KBS, et al. ORAOV1 is amplified in oral squamous cell carcinoma. J Oral Pathol Med 2012;41:54-60. [18] Jiang L, Zeng X, Yang H, et al. Oral cancer overexpressed 1 (ORAOV1): a regulator for the cell growth and tumor angiogenesis in oral squamous cell carcinoma. Int J Cancer 2008;123:1779-86.
715 [19] Jarmuz-Szymczak M, Pelinska K, Kostrzewska-Poczekaj M, et al. Heterogeneity of 11q13 region rearrangements in laryngeal squamous cell carcinoma analyzed by microarray platforms and fluorescence in situ hybridization. Mol Biol Rep 2013;40:4161-71. [20] Jiang L, Zeng X, Wang Z, et al. Oral cancer overexpressed 1 (ORAOV1) regulates cell cycle and apoptosis in cervical cancer HeLa cells. Mol Cancer 2010;9:20. [21] Kang JU, Koo SH. ORAOV1 is a probable target within the 11q13.3 amplicon in lymph node metastases from gastric adenocarcinoma. Int J Mol Med 2012;29:81-7. [22] Komatsu Y, Hibi K, Kodera Y, Akiyama S, Ito K, Nakao A. TAOS1, a novel marker for advanced esophageal squamous cell carcinoma. Anticancer Res 2006;26:2029-32. [23] Shi ZZ, Shang L, Jiang YY, et al. Consistent and differential genetic aberrations between esophageal dysplasia and squamous cell carcinoma detected by array comparative genomic hybridization. Clin Cancer Res 2013;19:5867-78. [24] Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A, editors. AJCC cancer staging manual. 7th ed. New York, NY: Springer; 2010. [25] Cui XB, Pang XL, Li S, et al. Elevated expression patterns and tight correlation of the PLCE1 and NF-κB signaling in Kazakh patients with esophageal carcinoma. Med Oncol 2014;31:791. [26] Bosman FT, Carneiro F, Hruban RH, Theise ND, editors. WHO classification of tumours of the digestive system. Lyons: World Health Organization; 2010. [27] Jiang L, Yang HS, Wang Z, et al. ORAOV1-A correlates with poor differentiation in oral cancer. J Dent Res 2009;88:433-8. [28] Dawsey SM, Lewin KJ, Wang GQ, et al. Squamous esophageal histology and subsequent risk of squamous cell carcinoma of the esophagus. A prospective follow-up study from Linxian, China. Cancer 1994;74:1686-92. [29] Wang G, Abnet C, Shen Q, et al. Histological precursors of oesophageal squamous cell carcinoma: results from a 13 year prospective follow up study in a high risk population. Gut 2005;54:187-92. [30] Limburg PJ, Wei W, Ahnen DJ, et al. Randomized, placebo-controlled, esophageal squamous cell cancer chemoprevention trial of selenomethionine and celecoxib. Gastroenterology 2005;129:863-73. [31] Shamma A, Doki Y, Shiozaki H, et al. Cyclin D1 overexpression in esophageal dysplasia: a possible biomarker for carcinogenesis of esophageal squamous cell carcinoma. Int J Oncol 2000;16:261-6. [32] Falini B, Bigerna B, Fizzotti M, et al. ALK expression defines a distinct group of T/null lymphomas ("ALK lymphomas") with a wide morphological spectrum. Am J Pathol 1998;153:875-86. [33] Ishikawa N, Takano A, Yasui W, et al. Cancer-testis antigen lymphocyte antigen 6 complex locus K is a serologic biomarker and a therapeutic target for lung and esophageal carcinomas. Cancer Res 2007;67:11601-11. [34] Yamabuki T, Takano A, Hayama S, et al. Dikkopf-1 as a novel serologic and prognostic biomarker for lung and esophageal carcinomas. Cancer Res 2007;67:2517-25.
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Original contribution
ORAOV1 overexpression in esophageal squamous cell carcinoma and esophageal dysplasia: a possible biomarker of progression and poor prognosis in esophageal carcinoma☆,☆☆ Man Li MD a,b,1 , Xiaobin Cui MD a,b,c,1 , Yaoyuan Shen MD a , Hongchao Dong MD a , Weihua Liang BS a,b , Yunzhao Chen MD, PhD a,b , Jianming Hu MD a,b,c , Shugang Li MD, PhD d , Jing Kong MS a , Hongan Li BS a,b , Jin Zhao MD a,b,⁎, Feng Li MD, PHD a,b,c,⁎ a
Department of Pathology and Key Laboratory for Xinjiang Endemic and Ethnic Diseases, Shihezi University School of Medicine, Shihezi 832002, Xinjiang, China b Department of Pathology, The First Affiliated Hospital, Shihezi University School of Medicine, Shihezi 832002, Xinjiang, China c Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China d Department of Preventive Medicine, Shihezi University School of Medicine, Shihezi 832002, Xinjiang, China Received 30 June 2014; revised 11 January 2015; accepted 21 January 2015
Keywords: Esophageal squamous cell cancer; Esophageal squamous intraepithelial neoplasia; Oral cancer overexpressed 1; Overexpression; Prognosis
Summary Oral cancer overexpressed 1 (ORAOV1) has been reported to exhibit high amplification levels in esophageal squamous cell cancer (ESCC) and in premalignant lesions. However, ORAOV1 protein expression levels in ESCC and esophageal squamous intraepithelial neoplasia (ESIN) have not yet been reported. We have explored the relationship of ORAOV1 protein expression with ESCC and ESIN by immunohistochemically analyzing tissue microarrays containing esophageal samples from patients with various clinical features and prognoses. The percentage of ESCC, high-grade ESIN (HGESIN), low-grade ESIN (LGESIN), and nontumoral control patients overexpressing ORAOV1 were 70.63% (101/143), 77.36% (41/53), 48.96% (47/96), and 5.79% (7/121), respectively. ORAOV1 overexpression also appears to be significantly higher in ESCC, HGESIN, and LGESIN than in the controls (all P b .001), and the levels observed for ESCC and HGESIN were also significantly higher than that in LGESIN (both P = .001). These results corresponded to high sensitivity and specificity values in ESCC, HGESIN, and LGESIN tissues. Furthermore, the increased expression of ORAOV1 is significantly associated with lymph node metastasis
☆
Competing interests: The authors declare that they have no competing interests. Funding/Support: This work was supported by grants from the Ministry of Science and Technology of China (2010DFB34100 and 2012AA02A503), the National Natural Science Foundation of China (no. 81160301, 81360358, 81260301), and a joint foundation for nurturing the outstanding young scientists of Shihezi University (no. 2013ZRKXYQ-YD19). ⁎ Corresponding authors at: Department of Pathology and Key Laboratory for Xinjiang Endemic and Ethnic Diseases, Shihezi University School of Medicine, North 4th Road, Shihezi 832002, Xinjiang, China. E-mail addresses: [email protected] (J. Zhao), [email protected] (F. Li). 1 Authors contributed equally to this work. ☆☆
http://dx.doi.org/10.1016/j.humpath.2015.01.009 0046-8177/© 2015 Elsevier Inc. All rights reserved.
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M. Li et al. (P = .001) and an advanced TNM stage (III + IV) (P = .014), and patients with ORAOV1 overexpression experienced shorter overall survival time compared with those with lower ORAOV1 (χ2 = 11.505, P = .001). This study provides the first evidence of ORAOV1 overexpression in ESCC and ESIN and demonstrates a potential role in tumor progression and metastasis. ORAOV1 overexpression could, therefore, be used as a novel biomarker of poor prognosis in patients with ESCC. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Esophageal squamous cell cancer (ESCC) is one of the most common malignancies detected and treated worldwide [1,2]. In 2008, the incidence and mortality rates associated with esophageal cancer in China alone were 16.7 and 13.4 per 100 000 persons, respectively. Because most patients with ESCC present with advanced metastasis at diagnosis [3], the efficacy of ESCC therapy is far from optimal, and current treatment options only yield a 5-year survival rate of 17% [4]. Therefore, it is crucial to identify novel biomarkers that are both sensitive and specific for ESCC to detect the disease earlier as well as increase the efficiency and number of treatment options available for these patients. Unfortunately, although our understanding of ESCC pathogenesis is continually expanding, recent research in this field has yet to significantly improve upon the current clinical techniques used to diagnose ESCC. Esophageal squamous cell cancer progression is a multistage process, whereby a normal esophagus will first develop basal cell hyperplasia, followed by intraepithelial neoplasia, carcinoma in situ, and, finally, invasive carcinoma [5,6]. Esophageal squamous intraepithelial neoplasia (ESIN) has been shown to be a histologic precursor of ESCC [7] and can be divided into 2 types, low-grade ESIN (LGESIN) and high-grade ESIN (HGESIN), based on the severity of cytologic and architectural atypia [8]. Many reports have suggested that ESIN has prognostic significance for esophageal carcinoma because dysplastic lesions are frequently encountered in cancerous esophagus tissue [9,10]. Therefore, the protein expression abnormalities observed during ESIN are likely the most promising candidates for predicting the development of ESCC. However, very few studies using ESIN models have focused on protein expression changes in relationship to squamous cell carcinogenesis. Notably, previous studies suggest that one of the most frequent genomic changes associated with ESCC development is the amplification of chromosome 11q13.3 [11,12]. Many oncogenes have been identified within this region, indicating that they may play important roles in ESCC tumorigenesis. These genes include cyclin D1 (CCND1) and cortactin (CTTN), which have been found to be overexpressed in various cancers, including ESCC [13,14]. Oral cancer overexpressed 1 (ORAOV1), also known as tumor amplified and overexpressed sequence 1 (TAOS1), has also been mapped to this chromosomal region, being found only approximately
12 kb away from the CCND1 gene locus. Not surprisingly, ORAOV1 has also been associated with a variety of human cancers, including oral cancer [15-18], laryngeal cancer [19], cervical cancer [20], gastric cancer [21], and ESCC [22]. Oral squamous cell carcinoma (OSCC), in particular, appears to have a wide range of overlap with ESCC in terms of gene abnormalities, including changes in CCND1 and CTTN expression as well as progression pattern after lesion formation in the squamous epithelium. Importantly, in OSCC tumors and lesions, amplification of the ORAOV1 gene [16] as well as overexpression of ORAOV1 messenger RNA (mRNA) have been observed [15,17], suggesting a relationship between this specific gene and cancer progression in this cellular context. Furthermore, ORAOV1 gene amplification and mRNA overexpression have also both been reported in ESCC tumor tissues [22,23]. Thus, we hypothesize that ORAOV1 may also play a similarly significant role in ESCC. However, to our knowledge, the expression and potential function of the ORAOV1 protein in ESCC tissues and esophageal precancerous lesions have not been reported in the literature. In this study, we have investigated ORAOV1 protein expression by analyzing surgically resected primary ESCC specimens as well as ESIN specimens of various degrees and adjacent morphologically normal tissue using immunohistochemical techniques. In doing so, we have determined that ORAOV1 protein expression is up-regulated during the development and progression of ESCC. Our findings suggest that ORAOV1 overexpression may serve as a reliable biomarker of poor prognosis in patients with ESCC in the early stages of the disease. We suspect that clinical diagnosis using ORAOV1 early on will enhance the efficiency and options for ESCC treatment and warrants further investigation.
2. Materials and methods 2.1. Patients and samples Primary tumor samples were obtained from patients with ESCC during tumor resection at the pathology department of the First Affiliated Hospital of Shihezi University School of Medicine between 1997 and 2014. The research protocol used in this study was approved by the medical ethics and human clinical trial committee of the Shihezi University School of Medicine, and all recruited subjects were enrolled
Overexpression of ORAOV1 protein in ESCC and ESIN with written informed consent. A total of 143 ESCC samples were obtained, fixed for 24 hours in formalin, and embedded in paraffin. None of the patients in this study had received preoperative chemotherapy or radiotherapy, and the clinical and pathologic data were available for all patients. The TNM stage was evaluated for each patient in accordance with the Cancer Staging Manual of 2010 [24]. Follow-up information was collected by phone or other methods. In addition, 121 matched nontumoral esophageal tissues, 96 LGESIN specimens, and 53 HGESIN specimens were also analyzed in this study.
2.2. Tissue microarray construction Each paraffin-embedded sample was sectioned and stained for hematoxylin and eosin. The various regions of each tumor sample were identified from these hematoxylin and eosin–stained slides. Subsequently, the fields corresponding to these selected regions were located in the remaining paraffin block for tissue microarray (TMA) construction. Tissue cylinders with a diameter of 1.0 mm were then punched from these areas of each donor tissue block and inserted into a recipient paraffin block using a tissue arrayer (Alphelys, Plaisir, France). The area of each tissue cylinder was reviewed to ensure that at least 70% of it represented the specified region of interest in that sample. Finally, serial sections 4-μm thick were then prepared from the TMA blocks for immunohistochemical staining.
2.3. Determination of ORAOV1 protein expression Immunohistochemistry was performed using the Envision system (Dako, Carpinteria, CA) to detect ORAOV1 protein expression. Briefly, sections of each specimen were prepared on glass slides and baked at 65°C for 30 minutes. The tissue sections were then deparaffinized by 3 consecutive treatments with xylene and rehydrated by sequential immersion in graded alcohol. After deparaffinization, the sections were incubated in methanol hydrogen peroxide (3%) for 10 minutes to remove endogenous peroxidase and subsequently washed with tap water. Antigen retrieval was then performed by heating the samples in a microwave oven under high power for approximately 8 minutes in a citrate buffer solution and then cooled for 30 to 40 minutes at room temperature. Slides were washed 3 times in phosphate-buffered saline (PBS) and then incubated with anti-ORAOV1 antibody (ab64843; Abcam, Cambridge, MA; dilution 1:500). Each section was incubated with 150 μL of the diluted antibody at 4°C overnight, whereas negative control sections were incubated with PBS. After allowing the slides to warm at room temperature for 20 minutes, they were washed 3 times in PBS and then incubated with 150 μL of secondary antibody for 30 minutes at 37°C. After a final wash with PBS and treatment with fresh diaminobenzidine solution, these sections were lightly stained with hematoxylin to highlight cell nuclei and dehydrated with graded alcohol and
709 xylene. Antifade mounting medium (ZSGB-Bio, Beijing, China) and glass cover slips were then added. Slides were stored at room temperature until analysis. All of the immunostained slides were blindly evaluated by 2 pathologists under a 5-headed microscope (Olympus Optical, Tokyo, Japan) and were assigned a consensus score. The slides were categorized as ORAOV1 positive when brownish-yellow particles appeared in the cytoplasm. The staining intensity was scored as 0 (no staining), 1 (weak staining), 2 (medium staining), or 3 (strong staining). The percentage of stained cells was scored as 0 (b5% of the cells), 1 (6%-25% of the cells), 2 (26%-50% of the cells), 3 (51%-75% of the cells), or 4 (≥76% of the cells). The product of both scores was used to identify 4 categories of expression: +++ (a score of 9-12), ++ (a score of 5-8), + (a score of 2-4), and − (a score of 0-1) [25]. In cases where the 2 pathologists disagreed on the immunostaining results, a third pathologist was consulted to analyze the staining.
2.4. Statistical methods Statistical analysis of the experimental data was performed using SPSS 17.0 (SPSS Inc, Chicago, IL). Categorical data were analyzed using χ2 tests to determine the statistically significant differences in ORAOV1 protein expression in the ESCC and ESIN samples compared with the nontumoral control tissues. The correlations between ORAOV1 expression and the clinicopathological factors were explored by Mann-Whitney U and Kruskal-Wallis H tests. Receiver operating characteristic (ROC) curves and the area under the curve (AUC) were used to evaluate the specificity and sensitivity of ESCC and ESIN prediction using a 95% confidence interval (CI). The survival rates were calculated using the Kaplan-Meier method, and the survival curves were analyzed using the log-rank test. The univariate and multivariate hazard ratios for different variables were calculated using the Cox proportional hazards model. In all of the statistical analyses, a P b .05 was considered statistically significant.
3. Results 3.1. Clinicopathological demographics of the patients with ESCC The present study included 143 patients with ESCC, of which 108 (75.52%) were men and 35 (24.48%) were women. The age of the patients at the time of surgery ranged from 36 to 81 years, with a median age of 62 years. Of these 143 ESCC cases, 41 (28.67%) were well differentiated, 73 (51.05%) were moderately differentiated, and 29 (20.28%) were poorly differentiated [26]. Lymph node metastasis was present in 49 (34.27%) of the patients. Furthermore, 95 (66.43%) of the patients were at I or II clinical stage, whereas 48 (33.57%) were at stage III or IV.
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3.2. ORAOV1 protein expression is increased in ESCC and ESIN compared with nontumoral squamous epithelium tissue Immunohistochemical staining of ESCC, HGESIN, LGESIN, and nontumoral squamous epithelium tissues demonstrated that ORAOV1 is localized to the cytoplasm. However, the distribution of ORAOV1 staining was significantly different among these 4 groups, with strong or medium ORAOV1 staining observed in the ESCC, HGESIN, and LGESIN tissues and weak or no ORAOV1 staining in the nontumoral squamous epithelium (Fig. 1). Furthermore, the overall percentages of ESCC, HGESIN, LGESIN, and nontumoral tissue samples expressing ORAOV1 were 100% (143/143), 100% (53/53), 97.92% (94/96), and 90.91% (110/121), respectively. The percentages of patients overexpressing ORAOV1 (categorized as strong or medium staining) in each of these groups were
M. Li et al. 70.63% (101/143), 77.36% (41/53), 48.96% (47/96), and 5.79% (7/121) for the ESCC, HGESIN, LGESIN, and nontumoral tissues, respectively (Table 1). Furthermore, we observed an increasing trend in the rate of ORAOV1 expression/overexpression as the cell type progressed from normal (nontumoral) to increasing levels of metastasis (LGESIN followed by HGESIN and ESCC), with the level of ORAOV1 overexpression in the ESCC, HGESIN, and LGESIN tissues being significantly higher compared with the controls (70.63%, 77.36%, and 48.96% versus 5.79%, respectively; all P b .001). Furthermore, these data also indicate that ORAOV1 overexpression was significantly higher in ESCC and HGESIN compared with LGESIN (both P = .001). Interestingly, of the 4 tissues, ORAOV1 overexpression was found to be the highest in HGESIN, not ESCC; however, the difference between these 2 groups was not significant (P = .349) (Fig. 2).
Fig. 1 Immunohistochemical staining of ORAOV1 expression in ESCC (A), original magnification ×40; HGESIN (B), ×40; LGESIN (C), ×40; and nontumoral esophageal tissue (D), ×40. Immunohistochemical staining for ORAOV1 demonstrated strong cytoplasmic expression in ESCC (E), ×200, and HGESIN (F), ×200, and medium expression in LGESIN (G), ×200; whereas ORAOV1 staining indicated weak expression in nontumoral esophageal tissue (H), ×200. Hematoxylin and eosin staining is shown for ESCC (I), ×200; HGESIN (J), ×200; LGESIN (K), ×200; and nontumoral esophageal tissue (L), ×200.
Overexpression of ORAOV1 protein in ESCC and ESIN Table 1
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ORAOV1 expression in ESCC, HGESIN, LGESIN, and control tissues
Group
n
ESCC HGESIN LGESIN control
143 53 96 121
Pa
ORAOV1 expression (n/%) −
+
++
0 0 2 (2.08) 11 (9.09)
42 (29.37) 12 (22.64) 47 (48.96) 103 (85.12)
70 28 47 7
+++ (48.95) (52.83) (48.96) (5.79)
31 (21.68) 13 (24.53) 0 0
b.001
Abbreviations: ORAOV1, oral cancer overexpressed 1; ESCC, esophageal squamous cell cancer; HGESIN, high-grade esophageal squamous intraepithelial neoplasia; LGESIN, lowgrade esophageal squamous intraepithelial neoplasia. a P b .05 indicates a significant association among the variables.
3.3. ORAOV1 may be a potential diagnostic biomarker of ESCC and ESIN
3.4. ORAOV1 overexpression is associated with lymph node metastasis and advanced TNM stage in ESCC
We used ROC curves to evaluate the ORAOV1 immunohistochemical scores determined for each of the ESCC, HGESIN, LGESIN, and control tissues. We found that the ESCC, HGESIN, and LGESIN tissues were easily distinguished from the controls, with ROC AUC values of 0.901 (95% CI, 0.863-0.939), 0.906 (95% CI, 0.850-0.963), and 0.811 (95% CI, 0.748-0.875), respectively (Fig. 3). We next determined sensitivity and specificity values for each cancerous tissue type using cutoff levels that optimized the diagnostic accuracy rate, while minimizing the false-negative and false-positive rates (Table 2). The sensitivity and specificity values for ESCC were 89.5% and 79.3% (cutoff score of 4). High-grade ESIN and LGESIN were determined to have sensitivity and specificity values of 77.4% and 94.2% (cutoff score of 5) and 83.3% and 79.3% (cutoff score of 4), respectively. Taken together, these results support the notion that ORAOV1 may be a potential diagnostic biomarker of ESCC and ESIN.
To investigate the potential functional implications of aberrant ORAOV1 expression during ESCC, we evaluated the relationship between ORAOV1 expression and the clinicopathological characteristics of ESCC, such as age, sex, histologic grade, lymph node metastasis status, and TNM stage. Notably, the increased expression of ORAOV1 appears to be significantly associated with lymph node metastasis (P = .001) and advanced TNM stage (P = .014). From this result, we can infer that the up-regulated expression of ORAOV1 may contribute to tumor metastasis and aggressiveness. Other parameters such as age, sex, and tumor differentiation had no significant relationship with ORAOV1 expression (Table 3).
Fig. 2 Box plot showing significantly higher overexpression of ORAOV1 in ESCC, HGESIN, and LGESIN tissues than that in nontumoral esophageal tissue, and the ORAOV1 overexpression was significantly higher in ESCC and HGESIN than in LGESIN. *P = .001. **P b .001.
3.5. ORAOV1 overexpression predicts poor prognosis in ESCC The overall survival of patients with ESCC in relationship to ORAOV1 expression was the primary end point of interest in the present study; thus, we examined the correlation between ORAOV1 protein expression and the postoperative survival of patients who underwent curative surgery. Of the 143 ESCC cases examined, clinical follow-up information was available for 75 patients. The deadline of follow-up was the date of final contact (December 25, 2013) or death. The mean follow-up period of postoperative survival was 18 months (ranging from 1-96 months after treatment). Univariate analysis of the 75 enrolled patients with ESCC revealed a tendency for patients with higher ORAOV1 expression to have poorer outcomes than those with lower expression. The median survival time of patients with ORAOV1 overexpression was 14 months (range, 1-90 months), which was significantly lower than the median survival of 27 months (range, 1-96 months) in patients with lower ORAOV1 expression. Furthermore, using a Kaplan-Meier analysis, we determined that this difference in survival time, with patients overexpressing ORAOV1 experiencing a shorter overall survival time than patients with lower ORAOV1 expression, was significant (χ2 = 11.505, P = .001, Fig. 4A). Moreover, the postoperative mortality rate was higher in
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Fig. 3 Receiver operating characteristic curve analysis of the ORAOV1 immunohistochemical scores for detecting ESCC (A), HGESIN (B), and LGESIN (C) tissues from the controls. The AUC in ESCC, HGESIN, and LGESIN is 0.901, 0.906, and 0.811, respectively.
patients with ORAOV1 overexpression than in those with weak or no expression (P = .001, Fig. 4B). Finally, to determine whether the inclusion of the ORAOV1 immunohistochemical analysis provided additional prognostic information, multivariate Cox regression analysis was performed for the expression of ORAOV1 and all clinicopathological factors included in the univariate analysis. Statistically, it appears that ORAOV1 overexpression is a significant factor in the multivariate Cox regression model (P = .007; hazard ratio, 3.329; Table 4). Together, these data indicate that ORAOV1 overexpression is a significant independent prognostic factor for poor prognosis in ESCC and could thus be used as a potential biomarker for prognosis evaluation in patients with ESCC.
levels of ORAOV1 protein compared with nontumoral tissues, suggesting that ORAOV1 plays an important role in ESCC and its related precancerous lesions. These findings are consistent with those of a previous study conducted by Komatsu et al [22] on ORAOV1 mRNA expression, which found that ORAOV1 was frequently overexpressed in ESCC. Similarly, the expression of ORAOV1 mRNA in OSCC tissues was also found to be significantly higher compared with normal oral tissues [17,27], indicating that a similar mechanism may exist in multiple cancers. Moreover, we have revealed that HGESIN and LGESIN tissues also exhibited significantly higher ORAOV1 expression Table 3 Association between ORAOV1 expression and clinicopathological factors in ESCC Characteristics
4. Discussion
n
ORAOV1 expression (n/%) +
The ORAOV1 gene, identified in 2002 by Huang et al [15], is 414 nucleotides long and includes 5 known exons; however, the function of the encoded protein is currently unknown. ORAOV1 has been reported to be associated with a variety of cancers. In the present study, we have demonstrated that overexpression of the ORAOV1 protein was positively associated with the development and progression of ESCC. To the best of our knowledge, this is the first study to demonstrate that ESCC tissues exhibit significantly higher Table 2 The high sensitivity, specificity, and AUC values of ORAOV1 in ESCC, HGESIN, and LGESIN Comparison
AUC
Sensitivity (%)
Specificity (%)
Cutoff value a
ESCC vs control HGESIN vs control LGESIN vs control
0.901 0.906 0.811
89.5 77.4 83.3
79.3 94.2 79.3
4 5 4
Abbreviations: AUC, area under the curve; ORAOV1, oral cancer overexpressed 1; ESCC, esophageal squamous cell cancer; HGESIN, high-grade esophageal squamous intraepithelial neoplasia; LGESIN, low-grade esophageal squamous intraepithelial neoplasia. a Cutoff level was set to provide optimal sensitivity and specificity.
Age (y) b60 50 ≥60 93 Sex Male 108 Female 35 Differentiation a Well 41 Moderate 73 Poor 29 L/N metastasis Negative 94 Positive 49 TNM stage b I + II 95 III + IV 48
++
P
+++
13 (26.0) 23 (46.0) 14 (28.0) .243 29 (31.18) 47 (50.54) 17 (18.28) 28 (25.92) 60 (55.56) 20 (18.52) .854 14 (40.0) 10 (28.57) 11 (31.43) 10 (24.39) 18 (43.90) 13 (31.71) .244 c 22 (30.14) 37 (50.68) 14 (19.18) 10 (34.48) 15 (51.72) 4 (13.80) 37 (39.36) 40 (42.55) 17 (18.09) .001 5 (10.20) 30 (61.22) 14 (28.58) 35 (36.84) 42 (44.21) 18 (18.95) .014 7 (14.58) 28 (58.33) 13 (27.09)
Abbreviations: ESCC, esophageal squamous cell cancer; L/N metastasis, lymph node metastasis; TNM, tumor, node, metastasis. a Histologic grade was based on World Health Organization classification published in 2010 [26]. b TNM stage was based on the American Joint Committee on Cancer criteria published in 2010 [24]. c Kruskal-Wallis H test.
Overexpression of ORAOV1 protein in ESCC and ESIN
713
Fig. 4 Kaplan-Meier survival curves showing patients with ESCC with overexpressed ORAOV1 (2+/3+) and low ORAOV1 levels (−/1+). A, Patients with overexpressed ORAOV1 experienced a significantly shorter postoperative survival time than those with low ORAOV1 levels (P = .001). B, Patients with ORAOV1 overexpression had a higher risk of death than those with lower ORAOV1 levels (P = .001).
compared with nontumoral tissues and that ESCC and HGESIN overexpress ORAOV1 at a higher level than LGESIN. These results indicate that ORAOV1 may be overexpressed immediately after transformation of the esophageal squamous epithelium. Xia et al [16] reported increased ORAOV1 amplification beginning at the onset of mild epithelial dysplasia, whereas no amplification was observed in normal mucosa and hyperplasia. These findings are consistent with our observation of increasing ORAOV1 expression during the progression of ESCC, as the ORAOV1 gene copy number is strongly correlated with its expression level [15,17]. Clinical observations have suggested that ESIN can be categorized as a precancerous dysplastic lesion from which squamous cell carcinoma can arise [28]. Furthermore, several studies have also shown an increased risk of developing invasive ESCC in patients with mild dysplasia, moderate dysplasia, severe dysplasia, and carcinoma in situ [28-30]. In the present study, we observed an increase in the expression of ORAOV1 in HGESIN and ESCC compared with the LGESIN tissue samples, whereas the difference between ESCC and HGESIN was not significant. From these data, we can infer
Table 4
that ORAOV1 may play an important role in the development and progression of ESCC. ORAOV1 is located approximately 12 kb from the CCND1 gene, and both genes have been implicated as driving forces behind 11q13 gene amplification [15,16]. Consistent with this notion, Jiang et al [20] observed that, in HeLa cells, the expression of CCND1 was reduced in ORAOV1-silenced cells. These data suggest some form of interaction between these 2 genes, and it has been proposed that CCND1 may be a downstream effector of ORAOV1 [20]. Thus, it is likely that the functions of ORAOV1 and CCND1 are related. This hypothesis is further supported by Shamma et al [31], who previously reported that CCND1 overexpression begins early in ESCC dysplasia, which, as shown in the present study, mirrors the expression pattern of ORAOV1 in different esophageal tissues. Further research is required to fully characterize the relationship between ORAOV1 and CCND1 and to determine the functional mechanisms involved in the development of ESCC. To examine the feasibility of applying ORAOV1 as a clinical biomarker of ESCC and ESIN, we constructed ROC
Univariate and multivariate survival analyses of clinicopathological characteristics
Characteristics
Univariate analysis HR
95% CI
Multivariate analysis P
ORAOV1 overexpression 3.673 1.614 8.362 .002 ⁎ Age (≥60 y) 1.043 0.529 2.057 .903 Sex (female) 1.541 0.789 3.009 .205 Differentiation a (moderate/poor) 0.881/0.828 0.354/0.365 2.197/1.887 .787/.651 L/N metastasis (positive) 2.038 1.083 3.834 .027 ⁎ TNM stage b (III + IV) 2.138 1.134 4.033 .019 ⁎
HR
95% CI
3.329 1.116 1.321 0.708/0.681 1.109 1.617
1.385 0.537 0.622 0.272/0.269 0.326 0.484
Abbreviations: L/N, lymph node; CI, confidence interval; HR, hazard radio. a Histologic grade was based on World Health Organization classification published in 2010 [26]. b TNM stage was based on the American Joint Committee on Cancer criteria published in 2010 [24]. ⁎ Significant difference that 95% CI was not including 1.
P 8.002 2.319 2.808 1.843/1.721 3.778 5.406
.007 ⁎ .768 .469 .479/.417 .869 .435
714 curves and the AUC to evaluate the specificity and sensitivity of this marker in each tissue type. We obtained high sensitivity and specificity values, which highlight the potential usefulness of ORAOV1 as a novel clinical diagnostic biomarker and therapeutic target for ESCC and ESIN treatment. Furthermore, our results were obtained via immunohistochemical analysis, which has been established as a highly sensitive and costeffective method for evaluating protein expression [32]. Indeed, this method has been used to identify several genes as potential candidates for the development of novel diagnostic markers, therapeutic drugs, and immunotherapies [33,34]. Additional work using other protein analysis techniques is necessary to validate the immunohistochemistry results reported here. One of the most important findings of this study was the discovery that the high expression of ORAOV1 in ESCC tissues is associated with lymph node metastasis and an advanced TNM stage (III + IV). This suggests that ORAOV1 overexpression may be one of the causative factors of esophageal mucosal disorders and could potentially contribute to the metastasis and aggressiveness of ESCC. Our results are corroborated by many other reports investigating ORAOV1 in other types of tumors. For example, one study reported that overexpression of ORAOV1 in ESCC tissues was significantly associated with lymph node metastasis and an advanced TNM stage [22], whereas others have observed abnormal amplification of ORAOV1 in OSCC and gastric adenocarcinoma with lymph node metastasis and an advanced TNM stage [16,21]. ORAOV1 has also been reported to play a crucial role in tumor progression and angiogenesis in OSCC [18], and the average intratumoral microvessel density of ORAOV1-silenced tumors was found to be much lower than that of the controls, suggesting that silencing of ORAOV1 has an antiangiogenic effect in OSCC tumors. Additional studies have also revealed that the expression of vascular endothelial growth factor, an important factor in tumor angiogenesis, is much lower in ORAOV1-silenced tumors than in the controls [18]. Therefore, it is possible that ORAOV1 plays a role in mediating the proangiogenic effects of vascular endothelial growth factor in cancerous oral tissues, thereby contributing to tumor growth, lymph node metastasis, and distant metastasis. In addition to the TNM stage of the patient, the level of cellular differentiation in the ESCC is another important clinicopathological characteristic; however, we found no significant correlation between differentiation and ORAOV1 overexpression, although the well-differentiated ESCC samples did show a tendency for higher ORAOV1 expression. These observations are consistent with a study by Xavier et al [17] that focused on ORAOV1 mRNA expression in OSCC tissues but are contradicted by a study by Xia et al [16] in which ORAOV1 amplification was associated with poorly differentiated OSCC. The discrepancies between these results may be attributed to the diversity of tumor types analyzed, the heterogeneity of methods used, and the limitations of the small sample sizes. Further investigation using larger sample sizes is required to clarify the relationship between ORAOV1 expression and ESCC differentiation.
M. Li et al. Because all of the clinicopathological characteristics assessed in this study were prognostic factors in ESCC, it was essential to conduct a systematic analysis to confirm the potential influence of ORAOV1 protein expression on the prognosis of patients with ESCC. Our results show that patients with ESCC with higher ORAOV1 expression had a shorter postoperative survival time than those with lower ORAOV1 expression. Cox analysis also revealed that ORAOV1 overexpression could serve as a significant independent prognostic factor to predict the risk of death, with a high hazard ratio of 3.329. The relationship between ORAOV1 amplification/expression and overall survival rates has been analyzed previously [17], and it has been hypothesized that ORAOV1 amplification could predict poor prognosis [16]. We support this theory with the first report demonstrating an association between ORAOV1 expression and prognosis of ESCC. These results further expand our understanding of ORAOV1 function in terms of patient health at the cellular, tissue, and organismal levels. Although this study does not directly test the functional mechanism of ORAOV1, previous reports have indicated that a complex signaling cascade may be regulated by this protein during tumor pathogenesis. For instance, ORAOV1 silencing has been shown to lead to a distinct S-phase arrest through the suppression of DNA synthesis and down-regulation of the expression of several cell cycle regulators, including cell division control 2 (Cdc2), cyclin A, and cyclin B1 [18,20]. Furthermore, ORAOV1 silencing also induces apoptosis by increasing the expression of p53 and decreasing the expression of Bcl-2 [20]. This process appears to involve the activation of caspases 3, 8, and 9, indicating that the intrinsic and extrinsic apoptotic pathways are activated by ORAOV1 silencing. In light of our data, these findings suggest that ORAOV1 may promote tumor growth by regulating cell cycle progression and apoptosis, resulting in a poor outcome/prognosis of the patient with ESCC. In conclusion, this study is the first to show that ORAOV1 protein is overexpressed in ESCC and ESIN tissues and that such overexpression is correlated with lymph node metastasis and advanced TNM stages (III/IV) of ESCC. Accordingly, overexpression of ORAOV1 may be useful in identifying patients with ESCC who will likely experience shorter postoperative survival times. Our data suggest that ORAOV1 is useful as a prognostic and diagnostic biomarker and may also be a novel therapeutic target for ESCC. Further research is required to validate these concepts and fully elucidate the functional mechanism of this complex protein.
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