Acta Oto-Laryngologica. 2014; 134: 1101–1108
ORIGINAL ARTICLE
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SOX2 as prognostic factor in head and neck cancer: a systematic review and meta-analysis
WEIMIN LI1, BAOPING LI2, RONGGUANG WANG1, DONGYAN HUANG1, WEI JIN1 & SHIMING YANG1 1
Department of Otolaryngology, Head and Neck Surgery, Institute of Otolaryngology, Chinese PLA General Hospital, Chinese PLA Medical School, Beijing and 2Department of Otolaryngology, Head and Neck Surgery, Chinese PLA 264 Hospital, Taiyuan, PR China
Abstract Conclusion: SOX2-positive head and neck cancer patients had a worse prognosis, and this was associated with common clinicopathological poor prognostic factors. Objective: To investigate the correlation between SOX2-positive head and neck cancer and clinicopathological features and its impact on survival. Methods: A search in PubMed and Chinese CNKI (up to 1 July 2013) was performed. Only articles in which SOX2 antigen was detected in situ localization by immunohistochemical staining were included. This meta-analysis was done using RevMan 5.2 software. Outcomes included overall survival and various clinicopathological features. Results: A total of 926 gastric cancer patients from 9 studies were included. Meta-analysis showed that patients with SOX2 expression had a significantly worse 5-year overall survival compared with those with low expression (relative risk (RR) = 2.38, 95% confidence interval (CI) = 1.10–5.15, p = 0.03, random-effect). With respect to clinicopathological features, SOX2 overexpression as assessed by the immunohistochemistry method was closely correlated with tumor T stage, lymph node metastasis, and TNM stage.
Keywords: Cancer stem cells, prognosis, overall survival, clinicopathological features
Introduction Head and neck cancer is the sixth most common cancer worldwide, with an estimated annual burden of 563 826 incident cases (including 274 850 oral cavity cancers, 159 363 laryngeal cancers, and 52 100 oropharyngeal cancers, and 301 408 deaths [1]. Despite significant improvements in surgery, radiation therapy, and chemotherapy, long-term survival rates in patients with advanced stage head and neck squamous cell carcinoma (HNSCC) have not significantly increased in the past 30 years [2]. It is, therefore, desirable to develop a deeper understanding of the biology of this disease to adapt current therapeutic strategies and to develop therapies that are more effective. Evidence has recently been accumulating to support the hypothesis that solid tumors
contain a small subpopulation of cells called cancer stem-like cells (CSCs), which exhibit self-renewing capacities and are responsible for tumor maintenance and metastasis [3], and possibly for resistance towards chemotherapy and radiation therapy. Thus, it is of major importance to investigate CSCs associated with cancer progression as they may be important factors in determining the clinical outcomes of cancer. Sox2, a member of the SOX (SRY-related high mobility group box) family, was originally discovered to be involved in the maintenance of embryonic stem cell pluripotency [4]. Lately, Sox2 has been used widely as a marker to identify CSCs and as a prognostic factor for different types of tumors [5–8]. With respect to head and neck cancer, the correlation between SOX2 and clinicopathological features of head and neck cancer and its prognostic
Correspondence: Shiming Yang, Department of Otolaryngology, Head and Neck Surgery, Institute of Otolaryngology, Chinese PLA General Hospital, Beijing 100853, PR China. Tel: +86 010 66938219. Fax: +86 010 66938019. E-mail: [email protected]; [email protected]
(Received 17 February 2014; accepted 1 April 2014) ISSN 0001-6489 print/ISSN 1651-2251 online 2014 Informa Healthcare DOI: 10.3109/00016489.2014.913311
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value is relatively unclear. Thus a systematic review of published literature was conducted to clarify the relationship between CSC marker SOX2 and head and neck cancer based on current evidence. Material and methods
Acta Otolaryngol Downloaded from informahealthcare.com by National Silicosis Library on 10/28/14 For personal use only.
Search strategy A comprehensive literature search of electronic databases PubMed and Chinese CNKI was performed up to July 1, 2013.The following search terms were used: (SOX2 or sex-determining region Y or SRY-box 2) and (outcome or survival or prognosis) and (oral or buccal or mouth or tongue or ‘head and neck’ or pharyngeal or pharynx or oropharyngeal or laryngeal) and (cancer or neoplasms or tumor or carcinoma) and (neoplastic stem cells or cancer stem cell or tumorinitiating cell). The citation lists associated with all the studies retrieved in the search were used to identify other potentially relevant publications. Review articles were also scanned to find additional eligible studies. The title and abstract of each study identified in the search were scanned to exclude any clearly irrelevant ones. The remaining articles were browsed to determine whether they contained information on the topic of interest. Selection criteria Diagnosis of head and neck cancer was proven by histopathological methods. Studies of SOX2 expression based on primary head and neck cancer tissue (via either surgery or biopsy), rather than serum or any other kinds of specimen were included. All studies on the correlation of SOX2 overexpression with clinicopathological markers and the association of SOX2 overexpression with overall survival (OS) of head and neck cancer were included. There was no limitation on the minimum patients for every single study. When there were multiple articles by the same group based on similar patients and using the same detection methods, only the largest or the most recent article was included. Data extraction Data tables were made to extract all relevant data from texts, tables, and figures of each included study, including author, publication year, patient’s country, tumor stage, number of patients, research technique used, cut-off value of SOX2, and tumor site. In case the prognosis was only plotted as Kaplan–Meier curve
in some articles, the software GetData Graph Digitizer 2.24 was applied to digitize and extract the data. Statistical analysis For ease of analysis, the following data on SOX2 expression and clinicopathological factors were combined into single categories: I and II stage; III and IV stage; T1 and T2 stages; T3 and T4 stages; and well differentiated and moderate differentiation. Odds ratios (ORs) with 95% confidence interval (CI) were used to evaluate the association between SOX2 expression and clinicopathological factors, including tumor TMN staging, lymph node status, tumor grade, and tumor differentiation. The relative risk (RR) was used for assessing the association of SOX2 and the survival outcome combined over studies. The heterogeneity assumption was calculated by the Q test and p values > 0.05 indicated a lack of heterogeneity among studies, so the OR and RR were calculated by a fixed-effects model (the Mantel– Haenszel method and chi-squared tests). Otherwise, a random-effects model (the DerSimonian–Laird method) was used. The influence of individual studies on the summary effect estimate was displayed using the sensitivity analysis. In addition, funnel plots and the Egger’s test were used to estimate possible publication bias. Cochrane Review Manager, version 5.2 (Cochrane Library, Oxford, UK) was used to calculate the available data from each investigation. Results Description of studies A total of nine publications met the criteria for this analysis [9–17]. The total number of patients was 926, ranging from 45 to 162 patients per study. The main characteristics of the eligible studies are summarized in Table I. Nine articles dealt with clinicopathological factors. Five studies determined OS. Four studies only reported the association between SOX2 expression and clinicopathological factors without OS analysis. There was one kind of method used to evaluate SOX2 expression in head and neck cancer specimens: immunohistochemistry (IHC). Correlation of SOX2 expression with clinicopathological parameters The association between SOX2 and several clinicopathological parameters is illustrated in Figure 1. The expression of SOX2 correlated with more T3/T4 category patients (pooled OR = 2.73, 95% CI = 1.28–5.82, p = 0.009, random-effect). Moreover,
SOX2 as prognostic factor in head and neck cancer
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Table I. General characteristics of included studies. Study
Patient’s country
Year
Site
Tumor stage (UICC)
Technique
Züllig [9]
Switzerland
2013
Oral
I–III
IHC
120
Luo [10]
China
2013
Nasopharyngeal
I–IV
IHC
122
Du [11]
China
2011
Oral tongue
I–III
IHC
82
ND ND
No. of patients
Cut-off (IHC) Staining scores ‡8 Staining scores ‡6
Ye [12]
China
2013
Laryngeal
I–IV
IHC
45
Wang [13]
China
2009
Esophageal
I–IV
IHC
162
Staining scores ‡6
Ge [14]
China
2010
Hypopharyngeal
I–IV
IHC
85
Staining scores ‡4
Michifuri [15]
Japan
2012
Oral
I–IV
IHC
80
ND
Tang [16]
China
2013
Laryngeal
I–IV
IHC
161
Tang [17]
China
2013
Laryngeal
I–IV
IHC
69
Staining scores ‡6 >5%
ND, not documented.
SOX2 expression was also associated with stage III/IV TNM staging and lymph node metastasis, leading to a risk difference of 4.29 (95% CI = 1.73–10.62, p = 0.002, random-effect) and 2.59 (95%
CI = 1.70–3.93, p < 0.0001, random-effect), respectively. However, SOX2 was not associated with tumor differentiation (pooled OR = 1.54, 95% CI = 0.47– 5.01, p = 0.48, random-effect).
A Study or Subgroup Du Ge Michifuri Tang Tang Wang Ye Züllig
Experimental Control Risk ratio Events Total Events Total Weight M-H. Random, 95% CI 5 6 6 7 3 19 19 5
51 67 80 88 38 58 34 26
1 6 0 1 1 7 9 13
31 18 0 73 31 104 11 94
11.8% 16.1% 12.1% 11.3% 17.7% 14.2% 16.8%
Risk ratio M-H. Random, 95% CI
3.26 [0.36, 29.30] 0.20 [0.05, 0.71] Not estimable 6.22 [0.75, 51.80] 2.57 [0.25, 26.04] 6.75 [2.63, 17.33] 0.28 [0.05, 1.50] 1.48 [0.48, 4.63]
442 362 100.0% 1.54 [0.47, 5.01] Total (95% CI) 70 38 Total events Heterogeneity. Tau2 = 1.83; Chi2 = 25.36, df = 6 (P = 0.0003); I2 = 76% Test for overall effect: Z = 0.71 (P = 0.48)
0.01 0.1 1 10 100 Favors [experimental] Favors [control]
B Study or Subgroup Luo Du Wang Ge Michifuri Tang Tang
Experimental Control Risk ratio Events Total Events Total Weight M-H. Random, 95% CI 37 1.24 [0.88, 1.76] 58 19.5% 64 27 31 10.0% 51 5 2.43 [1.02, 5.82] 20 41 58 72 104 22.0% 1.02 [0.83, 1.26] 51 67 18 20.0% 13 1.05 [0.77, 1.45] Not estimable 24 80 0 0 58 23 73 19.1% 2.09 [1.44, 3.03] 88 29 38 4 31 9.3% 5.91 [2.33, 15.01]
446 315 100.0% 1.57 [1.09, 2.26] Total (95% CI) 260 144 Total events Heterogeneity. Tau2 = 0.14; Chi2 = 25.06, df = 5 (P = 0.0001); I2 = 80% Test for overall effect: Z = 2.44 (P = 0.01)
Risk ratio M-H. Random, 95% CI
0.01 0.1 1 10 100 Favors [experimental] Favors [control]
Figure 1. Forest plot of odds ratio was assessed for association between SOX2 and clinicopathologic features, such as tumor differentiation (A), T stage (B), TNM staging (C), and lymph node status (D).
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C Study or Subgroup Luo Ye Wang Ge Tang Tang
Experimental Control Risk ratio Events Total Events Total Weight M-H, Random, 95% CI 59 23 41 13 67 29
68 34 58 14 88 38
30 2 72 65 30 4
54 11 104 71 73 31
19.4% 13.0% 20.8% 9.9% 21.0% 16.0%
5.24 [2.17, 12.68] 9.41 [1.73, 51.11] 1.07 [0.53, 2.16] 1.20 [0.13, 10.82] 4.57 [2.32, 8.99] 21.75 [5.99, 78.95]
300 344 100.0% 4.29 [1.73, 10.62] Total (95% CI) 203 232 Total events Heterogeneity. Tau2 = 0.90; Chi2 = 22.26, df = 5 (P = 0.0005); I2 = 78% Test for overall effect: Z = 3.15 (P = 0.002) Acta Otolaryngol Downloaded from informahealthcare.com by National Silicosis Library on 10/28/14 For personal use only.
Risk ratio M-H, Random, 95% CI
0.01 0.1 1 10 100 Favors [experimental] Favors [control]
D Study or Subgroup Züllig Ye Wang Ge Michifuri Tang Tang
Experimental Control Events Total Events Total 2 26 4 55 1 14 11 34 33 58 104 24 16 67 3 18 25 80 0 0 10 31 88 73 1 16 31 38
Risk ratio Weight M-H, Random, 95% CI 1.06 [0.18, 6.21] 8.3% 7.00 [0.80, 61.07] 3.1% 48.7% 1.52 [0.78, 2.96] 12.7% 1.57 [0.40, 6.12] Not estimable 3.43 [1.54, 7.61] 24.9% 2.2% 21.82 [2.69, 177.08]
391 292 100.0% Total (95% CI) 52 128 Total events Heterogeneity. Chi2 = 9.23, df = 5 (P = 0.10); I2 = 46% Test for overall effect: Z = 4.45 (P < 0.00001)
Risk ratio M-H, Random, 95% CI
2.59 [1.70, 3.93]
0.01 0.1 1 10 100 Favors [experimental] Favors [control]
Figure 1. (Continued).
SOX2 overexpression and 5-year overall survival The 5-year OS rate was extracted from five studies, all of which relied on IHC solution. The accumulative 5-year OS rates of SOX2-positive and SOX2-negative head and neck patients were 38.6% (100/259) and 73.1% (190/260), respectively. Meta-analysis indicated that patients with SOX2-positive expression suffered with a significant poor prognosis in comparison with patients with SOX2-negative expression
Study or Subgroup Luo Du Ge Tang Tang
Experimental Control Events Total Events Total 22 37 27 85 51 4 31 25 52 67 15 18 35 66 22 95 38 2 31 25
(RR = 2.38, 95% CI = 1.10–5.15, p = 0.03, random-effect). In fact, three of five studies also concluded that SOX2 overexpression was a poor prognostic factor in head and neck cancer patients (Figure 2). Sensitivity analysis A single study involved in the meta-analysis was deleted each time to reflect the influence of the
Risk ratio Weight M-H. Random, 95% CI 22.6% 1.87 [1.24, 2.82] 17.6% 3.80 [1.46, 9.89] 23.6% 0.93 [0.73, 1.19] 2.29 [1.49, 3.52] 22.4% 10.20 [2.62, 39.73] 13.8%
Risk ratio M-H. Random, 95% CI
2.38 [1.10, 5.15] 259 Total (95% CI) 260 100.0% 159 70 Total events Heterogeneity. Tau2 = 0.65; Chi2 = 48.86, df = 4 (P < 0.00001); I2 = 92% 0.01 0.1 1 10 100 Test for overall effect: Z = 2.20 (P = 0.03) Favors [experimental] Favors [control] Figure 2. Forest plot of relative risk (RR) for overall survival among included studies.
SOX2 as prognostic factor in head and neck cancer A
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Begg’s funnel plot with pseudo 95% confidence limits
0
–2 0
.5
1
1.5
s.e. of: logrr B
Begg’s funnel plot with pseudo 95% confidence limits 2
Logrr
1
0
–1 0
.5
1
1.5
s.e. of: logrr C
Begg’s funnel plot with pseudo 95% confidence limits 2
1 Logrr
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Logrr
2
0
–1 0
.2
.4 s.e. of: logrr
.6
.8
Figure 3. Begg’s funnel plots for assessing the publication bias risk. (A) Tumor differentiation, (B) T stage, (C) TNM staging, (D) lymph node status, (E) overall survival.
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Begg’s funnel plot with pseudo 95% confidence limits
0
–2 0
E
.5 s.e. of: logrr
1
Begg’s funnel plot with pseudo 95% confidence limits 1
0 Logrr
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Logrr
2
–1
–2 0
.2
.4
.6
s.e. of: logrr Figure 3. (Continued).
individual data set on the pooled ORs, and the corresponding pooled ORs were not materially altered (data not shown), indicating that the results were statistically robust. Publication bias Begg’s funnel and Egger’s test were performed to access the publication bias in this meta-analysis. The shape of the funnel plots did not reveal obvious evidence of asymmetry (Figure 3). Egger’s test showed no significant publication bias for degree of differentiation (p = 0.881), T stage (p = 0.091), TNM staging (p = 0.188), lymph node metastasis (p = 0.188), or OS (p = 0.624). Discussion The present meta-analysis is the first study to systematically estimate the association between the stem cell
marker SOX2 and head and neck cancer survival. The presence of both significant and non-significant studies addressing the importance of stem cells in head and neck cancer made it necessary to perform a quantitative aggregation of the survival results. The results of the present study indicate that the stem cell marker SOX2 was significantly associated with tumor T stage, TNM staging, and lymph node metastasis, as well as OS. The results suggest that this marker could be developed for clinical applications. SOX2, a transcription factor-coding gene located on 3q26.3–27, encodes a transcription factor involved in the determination of cell fate and regulation of embryonic development. After creating a protein complex with other proteins it acts as a transcriptional activator that is essential to maintain self-renewal of undifferentiated embryonic stem cells [18,19]. It is an important cell surface marker for both stem cells and CSCs in various tissues. The role of SOX2 as a CSC marker has been documented in lung, gastric, and
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SOX2 as prognostic factor in head and neck cancer thyroid cancers [8,20]. As regards the prognostic value of SOX2, a few papers reported significant associations with head and neck cancer, and that SOX2 might be considered as a new marker to determine the stage and management of SOX2 [10,13]. It is notable that this association was observed in our meta-analysis of SOX2 phenotype and tumor T stage, TNM staging, and lymph node metastasis, as well as OS, suggesting that this marker can be developed for clinical applications. For future studies, co-expression of head and neck CSC markers associated with patient survival may be more meaningful for clinical application in head and neck cancer. Several studies have shown that CSCrelated factors, including ALDH1 and CD133, are associated with head and neck cancer progression. In addition, CSCs have major phenotypic and functional heterogeneity, which may help distinguish them from cancer cells, and may be of potential benefit in the future. There were some potential limitations in this study. First, in meta-analyses of prognostic factors, variability in definitions, outcomes, measurements, and experimental procedures might contribute to between-study heterogeneity. In the current metaanalysis, despite the fact that we tried to optimize standardization, some remaining variability in definitions was unavoidable. Second, as reported above, potential publication bias was a concern. We restricted our review to articles published in English or Chinese because other languages were not accessible to the readers. This selection could favor the positive studies that are more often published in English while the negative ones tend to be more often reported in native languages. In summary, this meta-analysis indicated that SOX2 expression was associated with common clinical parameters of head and neck cancer, such as tumor T stage, TNM staging, depth of invasion, and lymph node metastasis. Moreover, positive SOX2 expression was associated with a worse outcome than SOX2-negative expression, and SOX2 was an independent factor associated with reduced survival. Further studies of SOX2 and its potential as a marker for head and neck prognosis in clinic are warranted.
Acknowledgments The project was supported by subject Clinical scientific research foundation of Chinese PLA General Hospital, grant No. 2012FCZHCG1007 and Otology diseases of tissue repair and functional reconstruction research, grant No. 2012BAI12B01.
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Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References [1] Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics 2002. CA Cancer J Clin 2005;55:74–108. [2] Jones AS, Morar P, Phillips DE, Field JK, Husband D, Helliwell TR. Second primary tumors in patients with head and neck squamous cell carcinoma. Cancer 1995;75: 1343–53. [3] Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer 2008;8:755–68. [4] Wang J, Rao S, Chu J, Shen X, Levasseur DN, Theunissen TW, et al. A protein interaction network for pluripotency of embryonic stem cells. Nature 2006;444: 364–8. [5] Chen H-Y, Yu S-L, Chen C-H, Chang G-C, Chen C-Y, Yuan A, et al. A five-gene signature and clinical outcome in non-small-cell lung cancer. N Engl J Med 2007;356:11–20. [6] Wilbertz T, Wagner P, Petersen K, Stiedl A-C, Scheble VJ, Maier S, et al. SOX2 gene amplification and protein overexpression are associated with better outcome in squamous cell lung cancer. Mod Pathol 2011;24:944–53. [7] Otsubo T, Akiyama Y, Yanagihara K, Yuasa Y. SOX2 is frequently downregulated in gastric cancers and inhibits cell growth through cell-cycle arrest and apoptosis. Br J Cancer 2008;98:824–31. [8] Huang P, Qiu J, Li B, Hong J, Lu C, Wang L, et al. Role of Sox2 and Oct4 in predicting survival of hepatocellular carcinoma patients after hepatectomy. Clin Biochem 2011; 44:582–9. [9] Züllig L, Roessle M, Weber C, Graf N, Haerle S, Jochum W, et al. High sex determining region Y-box 2 expression is a negative predictor of occult lymph node metastasis in early squamous cell carcinomas of the oral cavity. Eur J Cancer 2013;49:1915–22. [10] Luo W, Li S, Peng B, Ye Y, Deng X, Yao K. Embryonic stem cells markers SOX2, OCT4 and Nanog expression and their correlations with epithelial-mesenchymal transition in nasopharyngeal carcinoma. PLoS ONE 2013;8:e56324. [11] Du L, Yang Y, Xiao X, Wang C, Zhang X, Wang L, et al. Sox2 nuclear expression is closely associated with poor prognosis in patients with histologically node-negative oral tongue squamous cell carcinoma. Oral Oncol 2011; 47:709–13. [12] Ye F, Huang GQ, Fu MY, Ma YZ, Kang HJ. Expression of SOX2 protein and its clinical significance in laryngeal carcinoma. J Clin Otorhinolaryngol Head Neck Surg 2013;27:136–9. [13] Wang Q, He W, Lu C, Wang Z, Wang J, Giercksky KE, et al. Oct3/4 and Sox2 are significantly associated with an unfavorable clinical outcome in human esophageal squamous cell carcinoma. Anticancer Res 2009;29:1233–41. [14] Ge N, Lin H-X, Xiao X-S, Guo L, Xu H-M, Wang X, et al. Prognostic significance of Oct4 and Sox2 expression in hypopharyngeal squamous cell carcinoma. J Transl Med 2010;8:4085–95. [15] Michifuri Y, Hirohashi Y, Torigoe T, Miyazaki A, Kobayashi J, Sasaki T, et al. High expression of ALDH1 and SOX2 diffuse staining pattern of oral squamous
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cell carcinomas correlates to lymph node metastasis. Pathol Int 2012;62:684–9. [16] Tang XB, Shen XH, Li L, Zhang YF, Chen GQ. SOX2 overexpression correlates with poor prognosis in laryngeal squamous cell carcinoma. Auris Nasus Larynx 2013;40:481–6. [17] Tang XB, Li L. Prognostic significance of Oct4 and Sox2 expression in laryngeal squamous cell carcinoma. Pract Med J 2013;29:751–3. [18] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131:861–72.
[19] Kashyap V, Rezende NC, Scotland KB, Shaffer SM, Persson JL, Gudas LJ, et al. Regulation of stem cell pluripotency and differentiation involves a mutual regulatory circuit of the NANOG, OCT4, and SOX2 pluripotency transcription factors with polycomb repressive complexes and stem cell microRNAs. Stem Cells Dev 2009;18:1093– 108. [20] Sholl LM, Barletta JA, Yeap BY, Chirieac LR, Hornick JL. Sox2 protein expression is an independent poor prognostic indicator in stage I lung adenocarcinoma. Am J Surg Pathol 2010;34:1193–8.
ORIGINAL ARTICLE
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SOX2 as prognostic factor in head and neck cancer: a systematic review and meta-analysis
WEIMIN LI1, BAOPING LI2, RONGGUANG WANG1, DONGYAN HUANG1, WEI JIN1 & SHIMING YANG1 1
Department of Otolaryngology, Head and Neck Surgery, Institute of Otolaryngology, Chinese PLA General Hospital, Chinese PLA Medical School, Beijing and 2Department of Otolaryngology, Head and Neck Surgery, Chinese PLA 264 Hospital, Taiyuan, PR China
Abstract Conclusion: SOX2-positive head and neck cancer patients had a worse prognosis, and this was associated with common clinicopathological poor prognostic factors. Objective: To investigate the correlation between SOX2-positive head and neck cancer and clinicopathological features and its impact on survival. Methods: A search in PubMed and Chinese CNKI (up to 1 July 2013) was performed. Only articles in which SOX2 antigen was detected in situ localization by immunohistochemical staining were included. This meta-analysis was done using RevMan 5.2 software. Outcomes included overall survival and various clinicopathological features. Results: A total of 926 gastric cancer patients from 9 studies were included. Meta-analysis showed that patients with SOX2 expression had a significantly worse 5-year overall survival compared with those with low expression (relative risk (RR) = 2.38, 95% confidence interval (CI) = 1.10–5.15, p = 0.03, random-effect). With respect to clinicopathological features, SOX2 overexpression as assessed by the immunohistochemistry method was closely correlated with tumor T stage, lymph node metastasis, and TNM stage.
Keywords: Cancer stem cells, prognosis, overall survival, clinicopathological features
Introduction Head and neck cancer is the sixth most common cancer worldwide, with an estimated annual burden of 563 826 incident cases (including 274 850 oral cavity cancers, 159 363 laryngeal cancers, and 52 100 oropharyngeal cancers, and 301 408 deaths [1]. Despite significant improvements in surgery, radiation therapy, and chemotherapy, long-term survival rates in patients with advanced stage head and neck squamous cell carcinoma (HNSCC) have not significantly increased in the past 30 years [2]. It is, therefore, desirable to develop a deeper understanding of the biology of this disease to adapt current therapeutic strategies and to develop therapies that are more effective. Evidence has recently been accumulating to support the hypothesis that solid tumors
contain a small subpopulation of cells called cancer stem-like cells (CSCs), which exhibit self-renewing capacities and are responsible for tumor maintenance and metastasis [3], and possibly for resistance towards chemotherapy and radiation therapy. Thus, it is of major importance to investigate CSCs associated with cancer progression as they may be important factors in determining the clinical outcomes of cancer. Sox2, a member of the SOX (SRY-related high mobility group box) family, was originally discovered to be involved in the maintenance of embryonic stem cell pluripotency [4]. Lately, Sox2 has been used widely as a marker to identify CSCs and as a prognostic factor for different types of tumors [5–8]. With respect to head and neck cancer, the correlation between SOX2 and clinicopathological features of head and neck cancer and its prognostic
Correspondence: Shiming Yang, Department of Otolaryngology, Head and Neck Surgery, Institute of Otolaryngology, Chinese PLA General Hospital, Beijing 100853, PR China. Tel: +86 010 66938219. Fax: +86 010 66938019. E-mail: [email protected]; [email protected]
(Received 17 February 2014; accepted 1 April 2014) ISSN 0001-6489 print/ISSN 1651-2251 online 2014 Informa Healthcare DOI: 10.3109/00016489.2014.913311
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value is relatively unclear. Thus a systematic review of published literature was conducted to clarify the relationship between CSC marker SOX2 and head and neck cancer based on current evidence. Material and methods
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Search strategy A comprehensive literature search of electronic databases PubMed and Chinese CNKI was performed up to July 1, 2013.The following search terms were used: (SOX2 or sex-determining region Y or SRY-box 2) and (outcome or survival or prognosis) and (oral or buccal or mouth or tongue or ‘head and neck’ or pharyngeal or pharynx or oropharyngeal or laryngeal) and (cancer or neoplasms or tumor or carcinoma) and (neoplastic stem cells or cancer stem cell or tumorinitiating cell). The citation lists associated with all the studies retrieved in the search were used to identify other potentially relevant publications. Review articles were also scanned to find additional eligible studies. The title and abstract of each study identified in the search were scanned to exclude any clearly irrelevant ones. The remaining articles were browsed to determine whether they contained information on the topic of interest. Selection criteria Diagnosis of head and neck cancer was proven by histopathological methods. Studies of SOX2 expression based on primary head and neck cancer tissue (via either surgery or biopsy), rather than serum or any other kinds of specimen were included. All studies on the correlation of SOX2 overexpression with clinicopathological markers and the association of SOX2 overexpression with overall survival (OS) of head and neck cancer were included. There was no limitation on the minimum patients for every single study. When there were multiple articles by the same group based on similar patients and using the same detection methods, only the largest or the most recent article was included. Data extraction Data tables were made to extract all relevant data from texts, tables, and figures of each included study, including author, publication year, patient’s country, tumor stage, number of patients, research technique used, cut-off value of SOX2, and tumor site. In case the prognosis was only plotted as Kaplan–Meier curve
in some articles, the software GetData Graph Digitizer 2.24 was applied to digitize and extract the data. Statistical analysis For ease of analysis, the following data on SOX2 expression and clinicopathological factors were combined into single categories: I and II stage; III and IV stage; T1 and T2 stages; T3 and T4 stages; and well differentiated and moderate differentiation. Odds ratios (ORs) with 95% confidence interval (CI) were used to evaluate the association between SOX2 expression and clinicopathological factors, including tumor TMN staging, lymph node status, tumor grade, and tumor differentiation. The relative risk (RR) was used for assessing the association of SOX2 and the survival outcome combined over studies. The heterogeneity assumption was calculated by the Q test and p values > 0.05 indicated a lack of heterogeneity among studies, so the OR and RR were calculated by a fixed-effects model (the Mantel– Haenszel method and chi-squared tests). Otherwise, a random-effects model (the DerSimonian–Laird method) was used. The influence of individual studies on the summary effect estimate was displayed using the sensitivity analysis. In addition, funnel plots and the Egger’s test were used to estimate possible publication bias. Cochrane Review Manager, version 5.2 (Cochrane Library, Oxford, UK) was used to calculate the available data from each investigation. Results Description of studies A total of nine publications met the criteria for this analysis [9–17]. The total number of patients was 926, ranging from 45 to 162 patients per study. The main characteristics of the eligible studies are summarized in Table I. Nine articles dealt with clinicopathological factors. Five studies determined OS. Four studies only reported the association between SOX2 expression and clinicopathological factors without OS analysis. There was one kind of method used to evaluate SOX2 expression in head and neck cancer specimens: immunohistochemistry (IHC). Correlation of SOX2 expression with clinicopathological parameters The association between SOX2 and several clinicopathological parameters is illustrated in Figure 1. The expression of SOX2 correlated with more T3/T4 category patients (pooled OR = 2.73, 95% CI = 1.28–5.82, p = 0.009, random-effect). Moreover,
SOX2 as prognostic factor in head and neck cancer
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Table I. General characteristics of included studies. Study
Patient’s country
Year
Site
Tumor stage (UICC)
Technique
Züllig [9]
Switzerland
2013
Oral
I–III
IHC
120
Luo [10]
China
2013
Nasopharyngeal
I–IV
IHC
122
Du [11]
China
2011
Oral tongue
I–III
IHC
82
ND ND
No. of patients
Cut-off (IHC) Staining scores ‡8 Staining scores ‡6
Ye [12]
China
2013
Laryngeal
I–IV
IHC
45
Wang [13]
China
2009
Esophageal
I–IV
IHC
162
Staining scores ‡6
Ge [14]
China
2010
Hypopharyngeal
I–IV
IHC
85
Staining scores ‡4
Michifuri [15]
Japan
2012
Oral
I–IV
IHC
80
ND
Tang [16]
China
2013
Laryngeal
I–IV
IHC
161
Tang [17]
China
2013
Laryngeal
I–IV
IHC
69
Staining scores ‡6 >5%
ND, not documented.
SOX2 expression was also associated with stage III/IV TNM staging and lymph node metastasis, leading to a risk difference of 4.29 (95% CI = 1.73–10.62, p = 0.002, random-effect) and 2.59 (95%
CI = 1.70–3.93, p < 0.0001, random-effect), respectively. However, SOX2 was not associated with tumor differentiation (pooled OR = 1.54, 95% CI = 0.47– 5.01, p = 0.48, random-effect).
A Study or Subgroup Du Ge Michifuri Tang Tang Wang Ye Züllig
Experimental Control Risk ratio Events Total Events Total Weight M-H. Random, 95% CI 5 6 6 7 3 19 19 5
51 67 80 88 38 58 34 26
1 6 0 1 1 7 9 13
31 18 0 73 31 104 11 94
11.8% 16.1% 12.1% 11.3% 17.7% 14.2% 16.8%
Risk ratio M-H. Random, 95% CI
3.26 [0.36, 29.30] 0.20 [0.05, 0.71] Not estimable 6.22 [0.75, 51.80] 2.57 [0.25, 26.04] 6.75 [2.63, 17.33] 0.28 [0.05, 1.50] 1.48 [0.48, 4.63]
442 362 100.0% 1.54 [0.47, 5.01] Total (95% CI) 70 38 Total events Heterogeneity. Tau2 = 1.83; Chi2 = 25.36, df = 6 (P = 0.0003); I2 = 76% Test for overall effect: Z = 0.71 (P = 0.48)
0.01 0.1 1 10 100 Favors [experimental] Favors [control]
B Study or Subgroup Luo Du Wang Ge Michifuri Tang Tang
Experimental Control Risk ratio Events Total Events Total Weight M-H. Random, 95% CI 37 1.24 [0.88, 1.76] 58 19.5% 64 27 31 10.0% 51 5 2.43 [1.02, 5.82] 20 41 58 72 104 22.0% 1.02 [0.83, 1.26] 51 67 18 20.0% 13 1.05 [0.77, 1.45] Not estimable 24 80 0 0 58 23 73 19.1% 2.09 [1.44, 3.03] 88 29 38 4 31 9.3% 5.91 [2.33, 15.01]
446 315 100.0% 1.57 [1.09, 2.26] Total (95% CI) 260 144 Total events Heterogeneity. Tau2 = 0.14; Chi2 = 25.06, df = 5 (P = 0.0001); I2 = 80% Test for overall effect: Z = 2.44 (P = 0.01)
Risk ratio M-H. Random, 95% CI
0.01 0.1 1 10 100 Favors [experimental] Favors [control]
Figure 1. Forest plot of odds ratio was assessed for association between SOX2 and clinicopathologic features, such as tumor differentiation (A), T stage (B), TNM staging (C), and lymph node status (D).
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C Study or Subgroup Luo Ye Wang Ge Tang Tang
Experimental Control Risk ratio Events Total Events Total Weight M-H, Random, 95% CI 59 23 41 13 67 29
68 34 58 14 88 38
30 2 72 65 30 4
54 11 104 71 73 31
19.4% 13.0% 20.8% 9.9% 21.0% 16.0%
5.24 [2.17, 12.68] 9.41 [1.73, 51.11] 1.07 [0.53, 2.16] 1.20 [0.13, 10.82] 4.57 [2.32, 8.99] 21.75 [5.99, 78.95]
300 344 100.0% 4.29 [1.73, 10.62] Total (95% CI) 203 232 Total events Heterogeneity. Tau2 = 0.90; Chi2 = 22.26, df = 5 (P = 0.0005); I2 = 78% Test for overall effect: Z = 3.15 (P = 0.002) Acta Otolaryngol Downloaded from informahealthcare.com by National Silicosis Library on 10/28/14 For personal use only.
Risk ratio M-H, Random, 95% CI
0.01 0.1 1 10 100 Favors [experimental] Favors [control]
D Study or Subgroup Züllig Ye Wang Ge Michifuri Tang Tang
Experimental Control Events Total Events Total 2 26 4 55 1 14 11 34 33 58 104 24 16 67 3 18 25 80 0 0 10 31 88 73 1 16 31 38
Risk ratio Weight M-H, Random, 95% CI 1.06 [0.18, 6.21] 8.3% 7.00 [0.80, 61.07] 3.1% 48.7% 1.52 [0.78, 2.96] 12.7% 1.57 [0.40, 6.12] Not estimable 3.43 [1.54, 7.61] 24.9% 2.2% 21.82 [2.69, 177.08]
391 292 100.0% Total (95% CI) 52 128 Total events Heterogeneity. Chi2 = 9.23, df = 5 (P = 0.10); I2 = 46% Test for overall effect: Z = 4.45 (P < 0.00001)
Risk ratio M-H, Random, 95% CI
2.59 [1.70, 3.93]
0.01 0.1 1 10 100 Favors [experimental] Favors [control]
Figure 1. (Continued).
SOX2 overexpression and 5-year overall survival The 5-year OS rate was extracted from five studies, all of which relied on IHC solution. The accumulative 5-year OS rates of SOX2-positive and SOX2-negative head and neck patients were 38.6% (100/259) and 73.1% (190/260), respectively. Meta-analysis indicated that patients with SOX2-positive expression suffered with a significant poor prognosis in comparison with patients with SOX2-negative expression
Study or Subgroup Luo Du Ge Tang Tang
Experimental Control Events Total Events Total 22 37 27 85 51 4 31 25 52 67 15 18 35 66 22 95 38 2 31 25
(RR = 2.38, 95% CI = 1.10–5.15, p = 0.03, random-effect). In fact, three of five studies also concluded that SOX2 overexpression was a poor prognostic factor in head and neck cancer patients (Figure 2). Sensitivity analysis A single study involved in the meta-analysis was deleted each time to reflect the influence of the
Risk ratio Weight M-H. Random, 95% CI 22.6% 1.87 [1.24, 2.82] 17.6% 3.80 [1.46, 9.89] 23.6% 0.93 [0.73, 1.19] 2.29 [1.49, 3.52] 22.4% 10.20 [2.62, 39.73] 13.8%
Risk ratio M-H. Random, 95% CI
2.38 [1.10, 5.15] 259 Total (95% CI) 260 100.0% 159 70 Total events Heterogeneity. Tau2 = 0.65; Chi2 = 48.86, df = 4 (P < 0.00001); I2 = 92% 0.01 0.1 1 10 100 Test for overall effect: Z = 2.20 (P = 0.03) Favors [experimental] Favors [control] Figure 2. Forest plot of relative risk (RR) for overall survival among included studies.
SOX2 as prognostic factor in head and neck cancer A
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Begg’s funnel plot with pseudo 95% confidence limits
0
–2 0
.5
1
1.5
s.e. of: logrr B
Begg’s funnel plot with pseudo 95% confidence limits 2
Logrr
1
0
–1 0
.5
1
1.5
s.e. of: logrr C
Begg’s funnel plot with pseudo 95% confidence limits 2
1 Logrr
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Logrr
2
0
–1 0
.2
.4 s.e. of: logrr
.6
.8
Figure 3. Begg’s funnel plots for assessing the publication bias risk. (A) Tumor differentiation, (B) T stage, (C) TNM staging, (D) lymph node status, (E) overall survival.
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Begg’s funnel plot with pseudo 95% confidence limits
0
–2 0
E
.5 s.e. of: logrr
1
Begg’s funnel plot with pseudo 95% confidence limits 1
0 Logrr
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Logrr
2
–1
–2 0
.2
.4
.6
s.e. of: logrr Figure 3. (Continued).
individual data set on the pooled ORs, and the corresponding pooled ORs were not materially altered (data not shown), indicating that the results were statistically robust. Publication bias Begg’s funnel and Egger’s test were performed to access the publication bias in this meta-analysis. The shape of the funnel plots did not reveal obvious evidence of asymmetry (Figure 3). Egger’s test showed no significant publication bias for degree of differentiation (p = 0.881), T stage (p = 0.091), TNM staging (p = 0.188), lymph node metastasis (p = 0.188), or OS (p = 0.624). Discussion The present meta-analysis is the first study to systematically estimate the association between the stem cell
marker SOX2 and head and neck cancer survival. The presence of both significant and non-significant studies addressing the importance of stem cells in head and neck cancer made it necessary to perform a quantitative aggregation of the survival results. The results of the present study indicate that the stem cell marker SOX2 was significantly associated with tumor T stage, TNM staging, and lymph node metastasis, as well as OS. The results suggest that this marker could be developed for clinical applications. SOX2, a transcription factor-coding gene located on 3q26.3–27, encodes a transcription factor involved in the determination of cell fate and regulation of embryonic development. After creating a protein complex with other proteins it acts as a transcriptional activator that is essential to maintain self-renewal of undifferentiated embryonic stem cells [18,19]. It is an important cell surface marker for both stem cells and CSCs in various tissues. The role of SOX2 as a CSC marker has been documented in lung, gastric, and
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SOX2 as prognostic factor in head and neck cancer thyroid cancers [8,20]. As regards the prognostic value of SOX2, a few papers reported significant associations with head and neck cancer, and that SOX2 might be considered as a new marker to determine the stage and management of SOX2 [10,13]. It is notable that this association was observed in our meta-analysis of SOX2 phenotype and tumor T stage, TNM staging, and lymph node metastasis, as well as OS, suggesting that this marker can be developed for clinical applications. For future studies, co-expression of head and neck CSC markers associated with patient survival may be more meaningful for clinical application in head and neck cancer. Several studies have shown that CSCrelated factors, including ALDH1 and CD133, are associated with head and neck cancer progression. In addition, CSCs have major phenotypic and functional heterogeneity, which may help distinguish them from cancer cells, and may be of potential benefit in the future. There were some potential limitations in this study. First, in meta-analyses of prognostic factors, variability in definitions, outcomes, measurements, and experimental procedures might contribute to between-study heterogeneity. In the current metaanalysis, despite the fact that we tried to optimize standardization, some remaining variability in definitions was unavoidable. Second, as reported above, potential publication bias was a concern. We restricted our review to articles published in English or Chinese because other languages were not accessible to the readers. This selection could favor the positive studies that are more often published in English while the negative ones tend to be more often reported in native languages. In summary, this meta-analysis indicated that SOX2 expression was associated with common clinical parameters of head and neck cancer, such as tumor T stage, TNM staging, depth of invasion, and lymph node metastasis. Moreover, positive SOX2 expression was associated with a worse outcome than SOX2-negative expression, and SOX2 was an independent factor associated with reduced survival. Further studies of SOX2 and its potential as a marker for head and neck prognosis in clinic are warranted.
Acknowledgments The project was supported by subject Clinical scientific research foundation of Chinese PLA General Hospital, grant No. 2012FCZHCG1007 and Otology diseases of tissue repair and functional reconstruction research, grant No. 2012BAI12B01.
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Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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