Clinical significance of integrin-linked kinase in laryngeal squamous cell carcinoma

G Model

ANL-2186; No. of Pages 6 Auris Nasus Larynx xxx (2017) xxx–xxx Contents lists available at ScienceDirect

Auris Nasus Larynx journal homepage: www.elsevier.com/locate/anl

Clinical significance of integrin-linked kinase in laryngeal squamous cell carcinoma Ping-An Wu a,b, Shi-Sheng Li b, Qing-Lai Tang b, Bing-Bing Liu b, Xin-Ming Yang b,* a Department of Otolaryngology, Head and Neck Surgery, The University of Hong Kong-Shenzhen Hospital, Shenzhen 518053, Guangdong, PR China b Department of Otolaryngology, Head and Neck Surgery, The Second Xiangya Hospital, Central South University, Changsha 410011, Hunan, PR China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 2 November 2015 Accepted 8 September 2016 Available online xxx

Objective: To investigate the expression of integrin-linked kinase (ILK) and its relationship with clinicopathological parameters in laryngeal squamous cell carcinoma (LSCC). Methods: 116 patients who had previously undergone complete resection of tumor for LSCC were studied retrospectively. The level of ILK expression in tumor tissues and adjacent nontumor tissues were determined by immunohistochemistry. Results: Increased expression of ILK was found in 65.5% of cases. The expression of ILK protein was significantly associated with tumor grade (p = 0.046), lymph node metastasis (p = 0.020), and pTNM stage (p = 0.019). Kaplan–Meier survival estimates showed a significant correlation between ILK expression and patient survival rate (log-rank p < 0.05). The multivariate survival analysis revealed that N status was statistically significant prognostic factor (p < 0.001). Other parameters, such as ILK expression, cannot predict disease prognosis separately. Conclusion: Increased expression of integrin-linked kinase is associated with lymph node metastases and patient survival rate in laryngeal squamous cell carcinoma. However, it does not appear to be an independent prognostic predictor in LSCC. ß 2016 Elsevier Ireland Ltd. All rights reserved.

Keywords: Laryngeal carcinoma Integrin-linked kinase Immunohistochemistry Metastases Prognosis

1. Introduction Laryngeal carcinoma, classified as squamous cell carcinoma (LSCC) in 90% of the cases, is a common cause of morbidity and mortality worldwide, accounting for 5% of all human malignancies [1]. Despite tremendous development in surgery, radiotherapy, and chemotherapy, the long-term prognosis for patients with LSCC remains unsatisfactory [2,3]. Locoregional recurrence, cervical lymph nodes metastases, and distant metastases are the factors that significantly affect the prognosis

* Corresponding author. Fax: +86 731 85533525. E-mail address: [email protected] (X.-M. Yang).

in LSCC patients [4]. Recognition and identification of tumor marker associated with recurrence and/or metastasis have great significance in the prediction of tumor biological behavior and direction of therapeutic strategy. Integrin-linked kinase (ILK) was discovered in 1996 as an b1-integrin subunit cytoplasmic domain interactor [5], is implicated in the regulation of anchorage-dependent cell growth and survival, cell cycle progression, epithelial– mesenchymal transition (EMT), invasion and migration, cell motility and contraction, and vascular development [6– 8]. Increased ILK expression has also been reported in many types of tumors, such as melanoma, colon, prostate, ovarian cancer, non-small cell lung cancer, and head and neck squamous cell carcinomas [6,9–11]. Overexpression of ILK

http://dx.doi.org/10.1016/j.anl.2016.09.002 0385-8146/ß 2016 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: Wu P-A, et al. Clinical significance of integrin-linked kinase in laryngeal squamous cell carcinoma. Auris Nasus Larynx (2017), http://dx.doi.org/10.1016/j.anl.2016.09.002

G Model

ANL-2186; No. of Pages 6 P.-A. Wu et al. / Auris Nasus Larynx xxx (2017) xxx–xxx

2

participates in a panel of processes involved in tumor progression, including lymphangiogenesis [6], invasion and migration [9,10], indicating the significance of ILK as a potential target for cancer therapy. Small-molecule inhibitors of ILK activity have been identified and shown to inhibit tumor growth, invasion, and angiogenesis [12,13]. However, there were few published reports evaluating the role of ILK protein expression in LSCC. In order to gain better insight into the clinical relevance of ILK in LSCC, the present study was carried out to investigate ILK expression in a larger number of LSCC tissue samples and to further assess whether ILK expression was correlated with clinicopathological parameters and prognosis in patients with LSCC. 2. Methods 2.1. Patient characteristics A total of 116 patients who were diagnosed as LSCC at the Department of Otolaryngology, Head and Neck Surgery, the Second Xiangya Hospital of Central South University between March 2002 and January 2007 were studied retrospectively. Patients involved in this study signed the informed consent. The entire study was approved by the local Ethics Committee. Specimens of primary tumor and the corresponding histological nontumor tissues from the same patients were separated by two experienced pathologists. In this study, there were 110 male and 6 female patients with an average age of 63.4 years (age range: 47–75 years). All patients had no history of previous malignancies, no history of radiotherapy or chemotherapy. Patients with distant metastasis were excluded from this study. Tumors staging was performed according to TNM staging system of the International Union Against Cancer (UICC, 2002). Histological types of LSCC were determined according to the system of World Health Organization. Patients included in our study underwent different types of surgeries according to the size of primary site. In 116 laryngeal carcinoma patients, 23 patients underwent laser resection, 44 patients underwent partial laryngectomy, and 49 patients underwent total laryngectomy. Patients with lymph nodes metastases confirmed by preoperative evaluations or with supraglottic tumor underwent neck dissection. In the postoperative histological examination, 63 patients with locally advanced tumors, cervical lymph node metastasis with extranodal spread or positive surgical excision margins received post-surgical radiotherapy and chemotherapy. 2.2. Immunohistochemistry The specimens of formalin-fixed, paraffin-embedded tissues were cut into 4 mm sections by a microtome mounted on polyL-lysine coated glass slides and air-dried overnight at 37 8C. Paraffin tissue sections were deparaffinized in xylene and rehydrated in descending ethanol series and water according to standard protocols. Endogenous peroxidase activity was blocked by immersion for 20 min in 3% hydrogen peroxidase, after which the slides were rinsed in phosphate-buffered saline (PBS, pH 7.2–7.4). Then the sections were boiled for 10 min in

a 0.01 M citrate buffer (pH 6.0) for antigen retrieval. After preincubation with 1% bovine serum albumin (Sigma Chemical Co., St. Louis, MO) for 30 min, tissue sections were incubated overnight at room temperature with rabbit polyclonal IgG of ILK (1:500, Santa Cruz Biotechnology, USA). After incubation, the sections were rinsed in PBS and incubated with the secondary biotinylated goat-anti-rabbit IgG (1:200, Santa Cruz Biotechnology, USA) for 30 min at room temperature. After washing in PBS, the slides were incubated with streptavidin conjugated with horse radish peroxidase (Sigma). The peroxidase reaction was developed with 3,30 -diaminobenzidine (DAB, Sigma). Finally, the sections were counterstained with hematoxylin, dehydrated, cleared in xylene and finally embedded in Entellan. Negative control sections were treated using PBS in the primary antibody. All the immunostained tissue sections were reviewed and scored under a microscope for expression and localization of ILK protein by two pathologists independently and blindly. The scores of each section were compared and if there was a discrepancy, the two pathologists reviewed them again and reached a consensus. Briefly, five high-powered fields under the microscope were randomly chosen and 100 cells in each field were counted. The staining scores (IHS) were calculated by combining an estimate of the percentage of immunoreactive cells (quantity score) with an estimate of the staining intensity (staining intensity score). For the percentage of staining, score 0 indicated no staining; score 1, 1–10% of cells stained; score 2, 11–50%; score 3, 51– 80%; score 4, 81–100%. Staining intensity scores were as follows: score 0, no staining; score 1, light yellow (weak); score 2, yellow (moderate); and score 3, deep yellow (strong). We defined the IHC staining score as the multiply of the intensity and percentage scores. Patients were classified into two groups according to the staining score of tumor tissues as follows: samples with scores 0–6 of tumor stained as negative staining and samples with scores 7–12 of tumor stained were considered as positive [14,15]. 2.3. Follow-up After the completion of treatment, patients underwent routine surveillance every 1–3 months. Recurrence and metastasis were diagnosed by physical examination, imaging evaluation, operation, and postoperative pathological examinations. Follow-up rate was 100% (116/116). Overall survival (OS) and disease-free survival (DFS) were calculated from the day of surgery to the date of death or that of tumor relapse. Deaths from other causes were treated as censored cases. The follow-up time ranged from 23 to 60 months, with a median follow-up time of 44 months (SD = 11.466). 2.4. Statistical analysis The association between ILK expression and clinicopathological parameters was statistically evaluated by using Fisher’s exact test or the chi-square test. Survival curves were calculated by the Kaplan–Meier method and were compared using the logrank test. The correlation of variables with survival was analyzed by multivariate analysis using a Cox proportional

Please cite this article in press as: Wu P-A, et al. Clinical significance of integrin-linked kinase in laryngeal squamous cell carcinoma. Auris Nasus Larynx (2017), http://dx.doi.org/10.1016/j.anl.2016.09.002

G Model

ANL-2186; No. of Pages 6 P.-A. Wu et al. / Auris Nasus Larynx xxx (2017) xxx–xxx

hazards model. The SPSS 13.0 software package (SPSS, Inc., Chicago, IL) was used for statistics and p values of <0.05 was considered to be statistically significant.

Table 1 Correlations of ILK expression and clinicopathological parameters. Clinicopathologic variables

Number of patients

Immunohistochemical staining for ILK was performed in 116 tumors and adjacent nonneoplastic laryngeal tissue. Immunoreactivity for ILK was observed in the cytoplasm and/or the nucleus of cancer cells (Fig. 1). None or weak expression of ILK was detected in most adjacent nonneoplastic laryngeal tissue and positive expression was detected in 20.7% (24 of 116) of the cases. In contrast, tumor cells expressed ILK protein in most LSCC cases, and positive expression was detected in 65.5% (76 of 116) of the cases. This result showed that ILK was over-expressed in LSCC compared to tumoradjacent normal tissues suggests that ILK is up-regulated in LSCC cells (p = 0.00). 3.2. Correlations between ILK expression levels and clinicopathological parameters As shown in Table 1, there was no statistically significant correlation between ILK expression status and clinical factors of age, gender, tumor site, and tumor size. However, the expression of ILK protein was significantly associated with tumor grade (p = 0.046), lymph node metastasis (p = 0.020), and pTNM stage (p = 0.019).

Gender Male Female Age (years) 60 >60 Tumor site Supraglottic Glottic Subglottic pTNM stage I–II III–IV Tumor size T1-T2 T3-T4 Tumor grade G1 G2 G3 Lymph node N0 N+

p (x2)

ILK protein expression Negative

Positive

110 6

38 2

72 4

1.000 (0.000)

48 68

19 21

29 47

0.440 (0.597)

38 68 10

11 26 3

27 42 7

0.598 (1.028)

44 72

21 19

23 53

0.019 (5.504)

53 63

20 20

33 43

0.631 (0.230)

58 40 18

26 11 3

32 29 15

0.046 (6.140)

51 65

24 16

27 49

0.020 (5.417)

3. Results 3.1. Expression of ILK in LSCC tissue and clinicopathologic parameters

3

3.3. Relationship between ILK expression and survival In the Kaplan and Meier patient survival estimates, the 5-year overall survival rate and disease-free survival rate of positive expression group were significantly decreased than the

Fig. 1. Immunohistochemical studies of ILK in tumor tissue of LSCC. (A) Light yellow staining in cytoplasmatic expression pattern indicated weak positive expression of ILK. (B) Yellow staining in cytoplasmatic expression pattern indicated moderate positive expression of ILK. (C) Deep yellow staining in cytoplasmatic and nuclear expression pattern indicated strong positive expression of ILK. (D) No staining in cytoplasmatic and nuclear expression pattern indicated negative staining of ILK. Original magnification: 200 in A–D.

Please cite this article in press as: Wu P-A, et al. Clinical significance of integrin-linked kinase in laryngeal squamous cell carcinoma. Auris Nasus Larynx (2017), http://dx.doi.org/10.1016/j.anl.2016.09.002

G Model

ANL-2186; No. of Pages 6 P.-A. Wu et al. / Auris Nasus Larynx xxx (2017) xxx–xxx

4

Fig. 2. Kaplan–Meier survival curves showing 5-year overall survival rate (A) and disease-free survival rate (B) of patients with positive or negative ILK expression. Patients with negative ILK expression have a significantly better 5-year overall survival rate (62.2% vs 34.5%, p = 0.016) and disease-free survival rate (54.6% vs 28.6%, p = 0.016) than those with positive ILK expression.

Table 2 Cox proportional hazards regression model analysis: correlations of clinicopathological parameters, ILK expression and prognosis. Clinicopathologic variables

Gender Male Female Age (years) 60 >60 Tumor site Supraglottic Glottic Subglottic Tumor size T1-T2 T3-T4 Tumor grade G1 G2 G3 Lymph node N0 N+ ILK Low expression High expression

Univariate analysis

Multivariate analysis

Exp(B)

p

Exp(B)

p

0.388

0.350

–

–

0.974

0.233

–

–

1.435

0.081

–

–

2.145

0.017

0.760

0.488

1.468

0.073

–

–

4.957

0.000

4.957

0.000

2.197

0.021

1.638

0.155

negative expression group (log-rank p < 0.05, Fig. 2). The univariate survival analysis revealed that T status (p = 0.017), N status (p < 0.001) and ILK expression (p = 0.021) all were statistically significant prognostic factors (Table 2). However, age, gender, tumor site, and tumor grade were not significant factors. The multivariate survival analysis revealed that N status was a statistically significant prognostic factor (p < 0.001) (Table 2). Other parameters, such as T status and ILK expression, cannot predict disease prognosis separately. 4. Discussion ILK has been implicated in various aspects of the process of carcinogenesis particularly those concerning tumor progression. The recent identification of small molecule ILK inhibitors

that show encouraging results in suppression of tumor growth and invasion renders ILK a promising target for novel cancer therapies [7,12,13]. In the present study, the ILK in LSCC and tumor-adjacent normal tissues were detected by immunohistochemistry. It showed that ILK was highly expressed in LSCC compared to tumor-adjacent normal tissues. These results agreed with previous report which showed that ILK was overexpressed in LSCC carcinoma cells compared with non-tumor cells [16]. This finding suggests a potential role for ILK in LSCC. Increased ILK expression may be a result of a malfunctioning of upstream components in the integrin and growth factor signaling pathways. Overexpression of ILK in epithelial cells results in anchorage-independent cell growth with increased cell cycle progression, and constitutive up-regulation of cyclin D and cyclin A expression [17]. Moreover, we found that the high expression of ILK in LSCC was related to tumor grade, pTNM stage, lymph node metastasis, and 5-year survival. These results suggest that ILK expression is correlated with tumor progression in LSCC. Cases with strong ILK expression were reported to be significantly more frequent in advanced colorectal cancer [18] and advanced clear cell renal carcinoma [19]. ILK-mediated pathway that may enhance tumor progression is its regulation on MMP expression [20,21]. During tumor progression, MMPs facilitate the pathological processes of tumor invasion, angiogenesis, and metastasis by breaking down the extracellular matrix. Recent studies have also linked ILK expression to tumor grade of prostate [22], clear cell renal carcinoma [19], chondrosarcoma [23], colon cancer [24], and non-small cell lung cancer [25]. We also demonstrated that the expression of ILK correlated with tumor grade. Kaplan–Meier analysis revealed that ILK expression was significantly associated with worse disease-free survival (DFS) and overall survival (OS) rates in patients with LSCC (both p = 0.016). The overall prognosis of patients with positive ILK expression was significantly poorer than that of patients with negative expression. Univariate analysis showed that not only ILK expression, but also the tumor size and lymph node metastases were correlated with life span of LSCC patients. The size of primary tumor significantly affected prognosis. Primary tumor with large size may have more extensive invasion to the adjacent tissues and

Please cite this article in press as: Wu P-A, et al. Clinical significance of integrin-linked kinase in laryngeal squamous cell carcinoma. Auris Nasus Larynx (2017), http://dx.doi.org/10.1016/j.anl.2016.09.002

G Model

ANL-2186; No. of Pages 6 P.-A. Wu et al. / Auris Nasus Larynx xxx (2017) xxx–xxx

the malignant cells may detach from their original site, leading to the tumor metastases and poor prognosis. Lymph node metastases are widely accepted as most important prognostic factor in LSCC [26,27]; patients with lymph node metastases have 5-year survival rate below 50% [28]. The status of ILK expression might be dependent on the status of lymph node metastasis or other variables. So the multivariate analysis for survival was performed. Multivariate analysis revealed that the lymph node metastases could be considered as an independent factor for poor prognosis of LSCC patients, other parameters, such as T-stage, ILK expression, cannot predict disease prognosis separately. In our study, the influence of clinicopathological parameters and ILK expression on the disease prognosis may be achieved by their relationship with lymph node metastases; when these parameters and ILK expression were analyzed together with nodal metastases, lymph node metastases may act as the strongest prognostic predictor. Interestingly, previous study that overexpression of ILK in human laryngeal squamous cell carcinomas suggested a possible role for ILK in the development of these tumors. However, in contrast to studies in other human malignancies, ILK expression was not correlated with tumor progression parameters such as grade and TNM stage [16]. Our present data are inconsistent with this study. These conflicting results may be due to the differences in the pathological samples, the antibodies, the experimental methods, or evaluation system. In the present study, 62.1% (72/116) of patients were diagnosed as tumor stages III–IV; these may be an important reason for the higher lymph node metastases rate and lower 5-year survival rate than published data. In conclusion, we demonstrated ILK in human laryngeal squamous cell carcinomas suggesting a possible role for ILK in the development of these tumors. It is possible that neck dissection or postoperative adjuvant treatment modality may be important for patients whose tumors have strong ILK expression. In the future, small molecule antagonists of ILK may be used to interfere with recurrence, led to an increase in overall survival of LSCC patients. Further studies are needed to confirm the prognostic and therapeutic value of ILK for LSCC. Conflict of interest

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

The authors declare that they have no conflict of interest. References [1] Sanderson RJ, Ironside JA. Squamous cell carcinomas of the head and neck. BMJ 2002;325:822–7. [2] Bezerra de Souza DL, Jerez Roig J, Bernal MM. Laryngeal cancer survival in Zaragoza (Spain): a population-based study. Clin Transl Oncol 2012;14:221–4. [3] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin 2013;63:11–30. [4] Cosetti M, Yu GP, Schantz SP. Five-year survival rates and time trends of laryngeal cancer in the US population. Arch Otolaryngol Head Neck Surg 2008;134:370–9. [5] Hannigan GE, Leung-Hagesteijn C, Fitz-Gibbon L, Coppolino MG, Radeva G, Filmus J, et al. Regulation of cell adhesion and

[21]

[22]

[23]

[24]

5

anchorage-dependent growth by a new 1-integrin-linked protein kinase. Nature 1996;379:91–6. Hannigan G, Troussard AA, Dedhar S. Integrin-linked kinase: a cancer therapeutic target unique among its ILK. Nat Rev Cancer 2005;5: 51–63. Canel M, Serrels A, Frame MC, Brunton VG. E-cadherin-integrin crosstalk in cancer invasion and metastasis. J Cell Sci 2013;126: 393–401. Wong RP, Ng P, Dedhar S, Li G. The role of integrin-linked kinase in melanoma cell migration, invasion, and tumor growth. Mol Cancer Ther 2007;6:1692–700. Choi YP, Kim BG, Gao MQ, Kang S, Cho NH. Targeting ILK and b4 integrin abrogates the invasive potential of ovarian cancer. Biochem Biophys Res Commun 2012;427:642–8. Chen D, Zhang Y, Zhang X, Li J, Han B, Liu S, et al. Overexpression of integrin-linked kinase correlates with malignant phenotype in nonsmall cell lung cancer and promotes lung cancer cell invasion and migration via regulating epithelial-mesenchymal transition (EMT)related genes. Acta Histochem 2013;115:128–36. Zhao D, Tang XF, Yang K, Liu JY, Ma XR. Over-expression of integrinlinked kinase correlates with aberrant expression of Snail, E-cadherin and N-cadherin in oral squamous cell carcinoma: implications in tumor progression and metastasis. Clin Exp Metastasis 2012;29:957–69. Younes MN, Yigitbasi OG, Yazici YD, Jasser SA, Bucana CD, El-Naggar AK, et al. Effects of the integrin linked kinase inhibitor QLT0267 on squamous cell carcinoma of the head and neck. Arch Otolaryngol Head Neck Surg 2007;133:15–23. Li Q, Li C, Zhang YY, Chen W, Lv JL, Sun J, et al. Silencing of integrin-linked kinase suppresses in vivo tumorigenesis of human ovarian carcinoma cells. Mol Med Rep 2013;7:1050–4. Soslow RA, Dannenberg AJ, Rush D, Woerner BM, Khan KN, Masferrer J, et al. COX-2 is expressed in human pulmonary, colonic, and mammary tumors. Cancer 2000;89:2637–45. Wang S, Liang J, Lin Y, Yao R. Differential expression of the Na+/I symporter protein in thyroid cancer and adjacent normal and nodular goiter tissues. Oncol Lett 2013;5:368–72. Goulioumis AK, Bravou V, Varakis J, Goumas P, Papadaki H. Integrinlinked kinase cytoplasmic and nuclear expression in laryngeal carcinomas. Virchows Arch 2008;453:511–9. Radeva G, Petrocelli T, Behrend E, Leung-Hugesteijin C, Filmus J, Singerlard J, et al. Overexpression of the integrin-linked kinase promotes anchorage-independent cell cycle progression. J Biol Chem 1997;272:13937–44. Li R, Liu B, Yin H, Sun W, Yin J, Su Q. Overexpression of integrinlinked kinase (ILK) is associated with tumor progression and an unfavorable prognosis in patients with colorectal cancer. J Mol Histol 2013;44:183–9. Engelman Mde F, Grande RM, Naves MA, de Franco MF, de Paulo Castro Teixeira V. Integrin-linked kinase (ILK) expression correlates with tumor severity in clear cell renal carcinoma. Pathol Oncol Res 2013;19:27–33. Troussard AA, Costello P, Yaganathan TN, Kumagai S, Roskelly CD, Dedhar S. The integrin-linked kinase (ILK) induces an invasive phenotype via AP-1 transcription factor-dependent upregulation of matrix metalloproteinase 9 (MMP-9). Oncogene 2000;19:5444–52. Matsui Y, Assi K, Ogawa O, Raven PA, Dedhar S, Gleave ME, et al. The importance of integrin-linked kinase in the regulation of bladder cancer invasion. Int J Cancer 2012;130:521–31. Graff JR, Deddens JA, Konicek BW, Colligan BM, Hurst BM, Carter HW, et al. Integrin-linked kinase expression increases with prostate tumor grade. Clin Cancer Res 2001;7:1987–91. Papachristou DJ, Gkretsi V, Rao UN, Papachristou GI, Papaefthymiou OA, Basdra EK, et al. Expression of integrin-linked kinase and its binding partners in chondrosarcoma: association with prognostic significance. Eur J Cancer 2008;44:2518–25. Bravou V, Klironomos G, Papadaki E, Taraviras S, Varakis J. ILK overexpression in human colon cancer progression correlates with activation of beta-catenin, down-regulation of E-cadherin and activation of the Akt-FKHR pathway. J Pathol 2006;208:91–9.

Please cite this article in press as: Wu P-A, et al. Clinical significance of integrin-linked kinase in laryngeal squamous cell carcinoma. Auris Nasus Larynx (2017), http://dx.doi.org/10.1016/j.anl.2016.09.002

G Model

ANL-2186; No. of Pages 6 6

P.-A. Wu et al. / Auris Nasus Larynx xxx (2017) xxx–xxx

[25] Takanami I. Increased expression of integrin-linked kinase is associated with shorter survival in non-small cell lung cancer. BMC Cancer 2005;5:1. [26] Cabanillas R, Rodrigo JP, Astudillo A, Domı´nguez F, Sua´rez C, Chiara MD. P53 expression in squamous cell carcinomas of the supraglottic larynx and its lymph node metastases: new results for an old question. Cancer 2007;109:1791–8.

[27] Liu Y, Su Z, Li G, Yu C, Ren S, Huang D. Increased expression of metadherin protein predicts worse disease-free and overall survival in laryngeal squamous cell carcinoma. Int J Cancer 2013;133: 671–9. [28] Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, et al. Cancer statistics, 2008. CA Cancer J Clin 2008;58:71–96.

Please cite this article in press as: Wu P-A, et al. Clinical significance of integrin-linked kinase in laryngeal squamous cell carcinoma. Auris Nasus Larynx (2017), http://dx.doi.org/10.1016/j.anl.2016.09.002