Aberrant Kif2a and Ki67 expression predicts poor survival in laryngeal squamous cell carcinoma

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Aberrant Kif2a and Ki67 expression predicts poor survival in laryngeal squamous cell carcinoma Qicheng Zhang a, Wei Zhang a, Jie Zhang a, Hongli Xu b, Yiwen You a,* a b

Department of Otorhinolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, China Department of Otorhinolaryngology Head and Neck Surgery, The Third People’s Hospital of Nantong, Nantong, Jiangsu Province, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 22 August 2015 Accepted 20 October 2015 Available online xxx

Objective: Previous studies have demonstrated that aberrant Kif2a expression is associated with tumor aggressiveness and poor prognosis in various human cancers. However, the clinicopathological significance of Kif2a in laryngeal squamous cell carcinoma (LSCC) is unknown. The aim of this study is to analyze the prognostic significance of Kif2a, alone and in combination with Ki67 protein expression, in LSCC patients. Methods: We examined the expression status of Kif2a and Ki67 protein in 165 laryngeal tissues (137 primary tumors and 28 cases of matched normal surgical margins) by immunohistochemistry (IHC) analysis using tissue microarrays. Results: We found that expression correlated with clinical parameters and overall survival (OS). Our results revealed that Kif2a and Ki67 protein expression was significantly higher in cancerous tissues compared to normal surgical margins. High Kif2a protein expression (Kif2a+) was significantly associated with tumor stage (P = 0.026) and cervical lymph node metastasis (P = 0.034). A significant correlation between Kif2a and Ki67 protein expression was observed (P = 0.040). Multivariate analysis revealed that patients with Kif2a+ (hazard ratio [HR] 3.640, 95% confidence interval [CI] 1.253–10.570; P = 0.018) and high Ki67 (Ki67+) expression (HR 3.086, 95%CI 1.016–9.371; P = 0.047) had poor OS. Conclusion: We speculate that Kif2a and Ki67 can be used as prognostic markers for LSCC patients. ß 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Kif2a Ki67 Prognosis Laryngeal squamous cell carcinoma Tissue microarrays

1. Introduction Laryngeal squamous cell carcinoma (LSCC), which accounts for 3% of all cancers, ranks as the second most common malignancy of the head and neck region [1–3]. Worldwide, an estimated 3650 laryngeal cancer-related deaths occurred in 2012, and the morbidity of LSCC is increasing annually [1]. Surgical resection is the mainstay treatment for these patients. Despite many advanced means of diagnosis and treatment, the overall survival (OS) of LSCC patients has not significantly improved

* Corresponding author. Tel.: +86 13585229333. E-mail address: [email protected] (Y. You).

over the past few decades [4]. Cumulative clinical evidence shows that tumor stage, primary site, pathological differentiation, and cervical lymph node metastases are correlated with prognosis [5]. These parameters, however, are not sufficient for evaluating the malignancy of these tumors [6]. Therefore, it is urgent to find promising biomarkers that can be used to precisely predict prognosis in patients with LSCC. Microtubules (MTs), as fundamental parts of the cytoskeleton, are not only essential for the cellular mitotic activity of tumor cells, but also for invading normal neighboring tissues and for developing distant metastasis. Members of the kinesin13 family, including Kif2a, kif2b, and kif2c/MCAK, are classified as MT depolymerases that depolymerize MTs at their ends [7–9]. Among these proteins, Kif2a is regarded as an

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Please cite this article in press as: Zhang Q, et al. Aberrant Kif2a and Ki67 expression predicts poor survival in laryngeal squamous cell carcinoma. Auris Nasus Larynx (2015), http://dx.doi.org/10.1016/j.anl.2015.10.012

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important regulator of microtubule dynamics during mitosis progression. In addition, Kif2a is necessary for bipolar spindle assembly [10–12]. Cells lacking Kif2a form monopolar spindles instead of bipolar spindles, which are MT-based apparatuses, and block cell cycle progression [13–15]. Previous studies have shown that overexpression of Kif2a indicates poor prognosis in patients with several human cancers, such as breast and oral cavity cancer [16,17]. However, the roles of Kif2a in LSCC have been unknown to date. To determine whether Kif2a can be used as a prognostic marker in patients with LSCC, we examined the expression status of Kif2a by immunohistochemistry (IHC) analysis using tissue microarrays (TMAs). We also examined the proliferation index of Ki67. Furthermore, we correlated Kif2a and Ki67 expression levels with various clinicopathological features and OS in LSSC patients. 2. Materials and methods 2.1. Tissue sample collection and clinical data collection A total of 137 patients with primary LSCC were enrolled from the Department of Otolaryngology and Head and Neck Surgery, Affiliated Hospital of Nantong University, Jiangsu, China, between 2000 and 2009. LSCC diagnosis was made according to the World Health Organization (WHO) criteria [18] and TNM classification [UICC, 2009]. Detailed clinical data, including patient age, gender, histological grade, cervical lymph node metastasis status, TNM classification, and alcohol and tobacco consumption were documented in all cases. 135 of the 137 patients were male and 2 were female; the mean age of these cases at surgery was 61.2 years (range 29–87 years). 50 (36.5%) of the 137 patients had a supraglottic localization, 74 (54.0%) had a glottic localization and 13 (9.5%) had a subglottic localization. The T status was T1–T2 in 87 and T3–T4 in 50 patients. The TNM clinical stage was I–II in 76 cases and III–IV in 61 cases. 20 patients had cervical lymph nodes metastasis (N+) while 117 did not (N0). The histopathological grade was high in 54 cases, and moderate/low in 83 cases. 19 patients were treated with carbon dioxide (CO2) laser surgery, 97 had a partial laryngectomy, and 21 underwent a total laryngectomy. 66 cases had unilateral neck dissection and 27 underwent bilateral neck dissection (functional or radical neck dissection based on clinical and operative findings). The date of operation was used as the initial time of follow-up. Complete follow-up data were obtained up to May 2010. The median follow-up duration of these patients was 34 months (range 2–104 months). None of these patients received preoperative adjuvant therapy, such as chemotherapy or radiotherapy. Tissue samples from surgically resected LSCC were prepared for TMAs. Written informed consent was obtained from all patients or their guardians before enrollment. The Affiliated Hospital of Nantong University Ethics Committee approved the study protocol. 2.2. TMA construction and IHC analysis We prepared 165 formalin-fixed, paraffin-embedded tissues resected from LSCC patients for this study. These included 137 laryngeal cancer samples and 28 matched normal surgical

margins. A total of three TMAs were constructed as previously described using the manual Tissue Microarrayer System Quick Ray (UT06, UNITMA, Korea) in the Department of Pathology, the Affiliated Hospital of Nantong University [19]. Mouse monoclonal anti-human Kif2a antibody (dilution 1:100) (Abcam, ab55383) and rabbit monoclonal anti-human Ki67 antibody (dilution 1:100) (Abcam, ab16667) were used for IHC staining [19]. Tissue sections were counterstained with Mayer’s hematoxylin. All cases were examined by two independent pathologists who were blinded to clinical information. Kif2a and Ki67 expression was quantified using the semiquantitative H-score method, taking both staining intensity and percentage of positive tumor cells into account [20]. The staining intensity was graded as follows: 0 (absent), 1 (weak staining), 2 (moderate staining), and 3 (intense staining). The percentage of positive tumor cells stained and intensity were multiplied to obtain the intensity percentage score. Finally, the staining scores ranged from 0 (no staining) to 300 (100% of cells with a score of three for staining intensity) [19,21]. 2.3. Statistical analysis Before statistical analysis, we converted the continuous scores of Kif2a and Ki67 protein expression from IHC results into a twolevel grading system (low or high) according to specific cutoff values, which were determined to be significant in terms of OS using the X-tile software (The Rimm Lab at Yale University; http://www.tissuearray.org/rimmlab) [22,23]. Next, all statistical analyses were performed using SPSS 20.0 version software package for Windows (IBM Corp, Armonk, NY, USA). A Pvalue of less than 0.05 was deemed statistically significant. The relationship between Kif2a or Ki67 expression and clinicopathological parameters was calculated by Pearson Chi-square test. The correlation between Kif2a and Ki67 protein expression was calculated using the Chi-square test. The survival curves compared using the log-rank test were plotted using the Kaplan– Meier method. Univariate and multivariate analyses were calculated to examine the relationship between overall survival time and variables using a Cox’s proportional hazards regression model. 3. Results 3.1. IHC results of Kif2a and Ki67 in laryngeal tissues Kif2a protein was detected in the cytoplasm, while Ki67 protein was localized in the nucleus (Fig. 1). At the initial stage of the statistical analysis, we applied the X-tile software program for TMA data and first identified a significant cutoff point in terms of OS in LSCC [22,23]. Consequently, the cutoff point of 80 was selected for both Kif2a and Ki67 protein: a score of 0–80 was identified as low expression, while 80–300 was considered high expression. For all subsequent analyses, Kif2a and Ki67 protein expression status were merely considered as either ‘‘low’’ or ‘‘high’’ according to this specific cutoff value [22,23]. As shown in Table 1, 80 of 137 LSCC cases (58.39%) expressed high Kif2a levels (Kif2a+), while 79 cases (57.66%) overexpressed Ki67 proteins (Ki67+), and 53 cases (38.69%)

Please cite this article in press as: Zhang Q, et al. Aberrant Kif2a and Ki67 expression predicts poor survival in laryngeal squamous cell carcinoma. Auris Nasus Larynx (2015), http://dx.doi.org/10.1016/j.anl.2015.10.012

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Fig. 1. Representation of Kif2a and Ki67 protein expression in laryngeal cancers and normal tissues (matched normal surgical margins) on TMA sections. (A) LSCC with high Kif2a expression and high Ki67 expression; (B) LSCC with no Kif2a expression and no Ki67 expression; (C) Matched normal surgical margins with no Kif2a expression and no Ki67 expression; columns 1 and 2 are Kif2a staining with 40 (bar 500 mm) and 400 (bar 50 mm) magnification, respectively, and columns 3 and 4 are Ki67 staining with 40 (bar 500 mm) and 400 (bar 50 mm) magnification, respectively.

co-expressed both high Kif2a and high Ki67 levels (Kif2a+/ Ki67+). Although high Kif2a (Kif2a+) or Ki67 (Ki67+) expression was also detected in normal surgical margins, their frequency was significantly higher in cancer tissue (P = 0.011 and P = 0.034, respectively) compared to normal surgical margins. Similarly, the frequency of both high Kif2a and high Ki67 coexpression (Kif2a+/Ki67+) was also significantly higher in LSCC (P = 0.004; Table 1). 3.2. Association of Kif2a and Ki67 expression with clinical pathological parameters in LSCC Next, we examined the correlation between Kif2a or Ki67 protein expression and various clinical pathological parameters

among LSCC patients by Chi-square test. As indicated in Table 2, high Kif2a expression was significantly associated with cervical lymph node metastasis (P = 0.034) and TNM stage (P = 0.026); conversely, no statistically significant correlations were calculated between age, gender, alcohol consumption, tobacco consumption, T classification and tumor location. Similarly, high Ki67 expression was significantly associated with cervical lymph node metastasis (P = 0.029) and TNM stage (P = 0.018). Interestingly, high Kif2a and Ki67 co-expression (Kif2a+/Ki67+) also showed a positive association with cervical lymph node metastasis (P = 0.003) and advanced stage (TNM stage; P = 0.002). In addition, a statistical correlation between Kif2a and Ki67 protein expression was detected using the Chi-square test (P = 0.040; Table 2).

Table 1 kif2a and ki67 expression in laryngeal tissues. Groups

No

Kif2a Kif2a+ expression (%)

Normal surgical margin Laryngeal cancer

Ki67 Pearson x2

P value

6.449

0.011*

Ki67+ expression (%)

Kif2a/Ki67 Pearson x2

P value

4.508

0.034*

Kif2a+/Ki67+ (%)

28

9 (32.14)

10 (35.71)

3 (10.71)

137

80 (58.39)

79 (57.66)

53 (38.69)

Pearson x2

P value

8.113

0.004*

Kif2a+ represents high Kif2a expression; Ki67+ represents high Ki67 expression; Kif2a+/Ki67+ represents both high Kif2a and high Ki67 coexpression. * P < 0.05.

Please cite this article in press as: Zhang Q, et al. Aberrant Kif2a and Ki67 expression predicts poor survival in laryngeal squamous cell carcinoma. Auris Nasus Larynx (2015), http://dx.doi.org/10.1016/j.anl.2015.10.012

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Table 2 Association of Kif2a and ki67 expression with clinical pathological parameters in LSCC. Groups

No.

Kif2a High expression (%)

137 Total Age (years) 49 <60 years 60 years 88 Gender Male 135 2 Female Tobacco consumption Yes 73 32 No Unknown 32 Alcohol consumption Yes 51 54 No Unknown 32 T classification T1 + T2 87 50 T3 + T4 TNM stage 24 Stage I 52 Stage II 61 Stage III, IV Lymph node metastasis Yes 20 No 117 Histopathological grade 54 High 71 Moderate 12 Low Tumor location Supraglottic Glottic Subglottic Ki67 expression High Low *

50 74 13

Ki67 x2

P value

80 (58.39)

Kif2a+/Ki67+

High expression (%)

x2

P value

79 (57.66)

High expression (%)

x2

P value

53 (38.69)

28 (57.14) 52 (59.09)

0.0492

0.825

25 (51.02) 54 (61.36)

1.3793

0.24

17 (34.69) 36 (40.91)

0.5126

0.474

79 (58.52) 1 (50.00)

0.0589

0.808

77 (57.04) 2 (100)

1.4901

0.222

52 (38.52) 1 (50.00)

0.1095

0.741

44 (60.27) 23 (71.88) 13 (40.63)

1.2966

0.255

44 (60.27) 24 (75.00) 11 (34.38)

2.1141

0.146

30 (41.10) 18 (56.25) 5 (15.63)

2.0588

0.151

32 (62.75) 35 (64.81) 13 (40.63)

0.0487

0.825

32 (62.75) 36 (66.67) 11 (34.38)

0.1768

0.674

21 (41.18) 27 (50.00) 5 (15.63)

0.8229

0.364

47 (54.02) 33 (66.00)

1.8747

0.171

45 (51.72) 34 (68.00)

3.4454

0.063

29 (33.33) 24 (48.00)

2.8795

0.09

7 (29.17) 31 (59.62)

4.9503

0.026*

8 (33.33) 29 (55.77)

5.6383

0.018*

4 (16.67) 17 (32.69)

8.7937

0.003*

42 (68.85)

42 (68.85)

32 (52.46)

16 (80.00) 64 (54.70)

4.4997

0.034*

16 (80.00) 63 (53.85)

4.7858

0.029*

14 (70.00) 39 (33.33)

9.6811

0.002*

34 (62.96) 41 (57.75) 5 (41.67)

0.7658

0.382

27 (50.00) 42 (59.15) 10 (83.33)

2.1447

0.143

18 (33.33) 30 (42.25) 5 (41.67)

1.0767

0.299

31 (62.00) 43 (58.11) 6 (46.15)

1.0718

0.585

24 (48.00) 44 (59.46) 11 (84.62)

5.8786

0.053

15 (30.00) 32 (43.24) 6 (46.15)

2.5439

0.280

52 (65.82) 28 (48.28)

4.2385

0.04*

P < 0.05.

3.3. Prognostic value of Kif2a and Ki67 protein expression in LSCC by univariate and multivariate survival analysis Univariate analysis revealed that parameters impacting prognosis in LSCC patients involved Kif2a expression level (hazard ratio [HR] 2.252, 95% confidence interval [CI] 1.178– 4.304; P = 0.014), as well as Ki67 expression level (HR 2.352, 95%CI 1.211–4.568; P = 0.012) and high Kif2a/high Ki67 (Kif2a+/Ki67+) co-expression (HR 2.236, 95%CI 1.206– 4.146; P = 0.011). Cervical lymph node metastasis (HR 4.517, 95%CI 2.309–8.837; P < 0.001) and TNM stage (HR 3.206, 95%CI 1.671–6.150; P < 0.001), as well as histological grade (HR 2.192, 95%CI 1.115–4.308; P = 0.023) were also significantly associated with poor OS. On multivariate analysis, the data showed that high Kif2a expression (Kif2a+, HR 3.640, 95%CI 1.253–10.570; P = 0.018) and high Ki67 expression (Ki67+, HR 3.086, 95%CI 1.016–9.371; P = 0.047) were independent prognostic factors of survival in patients with

LSCC (Table 3). Kaplan–Meier survival curves revealed that patients with high Kif2a and Ki67 expression had shorter survival time compared with patients with low Kif2a and Ki67 expression (Fig. 2A–C). 4. Discussion Despite several effective therapeutic strategies, the survival of LSCC patients, especially in patients with advanced disease, has not dramatically improved over the past three decades [4]. As with other neoplasms, primary tumor site, tumor stage, histological grade, and cervical lymph node metastasis are related to LSCC prognosis. Early detection, diagnosis, and prompt treatment (surgery or conservative) can improve overall survival. However, these clinical parameters are insufficient for precise evaluation of malignant potential [6]. Therefore, a thorough investigation of potential molecular biomarkers for predicting LSCC prognosis is of great significance [24].

Please cite this article in press as: Zhang Q, et al. Aberrant Kif2a and Ki67 expression predicts poor survival in laryngeal squamous cell carcinoma. Auris Nasus Larynx (2015), http://dx.doi.org/10.1016/j.anl.2015.10.012

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Table 3 Univariate and multivariate analysis of prognostic markers for overall survival in LSCC patients. Variable

Univariate analysis HR

Kif2a expression High vs Low Ki67 expression High vs Low Kif2a+/Ki67+ Kif2a+/Ki67+ vs non Kif2a+/Ki67+ Age (years) 60 years vs >60 years Tobacco consumption Yes vs No Alcohol consumption Yes vs No TNM stage Stage I, II vs Stage III, IV Lymph node metastasis Yes vs No Histopathological grade High vs Moderate vs Low Tumor location Glottic vs Supraglottic vs Subglottic T Grade T1+2 vs T3+4 *

P value

Multivariate analysis 95% CI

HR

P value

95% CI

2.252

0.014*

1.178–4.304

3.640

0.018*

1.253–10.570

2.352

0.012*

1.211–4.568

3.086

0.047*

1.016–9.371

2.236

0.011*

1.206–4.146

0.372

0.144

0.098–1.404

1.577

0.185

0.804–3.094

0.644

0.210

0.323–1.282

0.802

0.499

0.423–1.520

3.206

<0.001*

1.671–6.150

4.517

<0.001*

2.309–8.837

2.192

0.023*

1.115–4.308

2.118

0.037*

1.048–4.283

1.821

0.016*

1.118–2.967

2.373

0.006*

1.280–4.398

2.146

0.02*

1.126–4.093

P < 0.05.

Previous studies have shown that cytoskeletal reorganization plays an important role in the migration of neoplastic cells [25]. MTs, as important components of the cytoskeleton, are related to mitosis, trafficking, cell signaling, and cell migration [26]. Any interference with MTs will lead to significant changes in the proliferation and migration of tumor cells [16]. There are a variety of proteins, such as kinesin family, that regulate the status and function of MTs. Kif2a, a MT motor protein of the Kinesin13 family, has been demonstrated to have MTs depolymerizing activity in vitro [27,28]. Rather than direct involvement in MTbased motility, Kif2a functions by driving ATP-dependent depolymerization of MTs [29]. Either depletion of Kif2a by siRNA in human tumor cells or addition of Kif2a antibody to Xenopus eggs leads to the formation of monopolar spindles and uncoordinated cell division [30]. Consequently, identification

of Kif2a expression in tumor cells might lead to a better understanding of the regulation of tumor progression and will be beneficial for improving tumor therapy. In previous studies, higher Kif2a expression in squamous cell carcinoma of the tongue (70.5%) was demonstrated compared to paired normal adjacent tissues; a significant association of Kif2a expression with lymph node metastasis and tumor clinical stage was also found [17]. In primary breast cancer, 67% of cases overexpress Kif2A protein compared with adjacent epithelial tissue, and the follow-up results suggest that patients with Kif2a-overexpression have much poorer survival than patients without Kif2a-overexpressing. Moreover, knockdown of Kif2a expression by small interfering (si) RNA treatment led to decreased proliferation and migration of these cell lines [16].

Fig. 2. Survival curves of LSCC by the Kaplan–Meier method and the log-rank test. (A) Overall survival curves of kif2a+ (green line, 1) and kif2a (blue line, 0); (B) Overall survival curves of ki67+ (green line, 1) and ki67 (blue line, 0); (C) Overall survival curves of kif2a+/ki67+ (blue line, 1), kif2a+/ki67 or kif2a /ki67+ (green line, 2), kif2a /ki67 (yellow line, 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Our data are consistent with the previous studies mentioned above. In our study, we investigated Kif2a protein expression as well as its prognostic value in patients with LSCC. Clinical pathological analysis revealed that high Kif2a protein expression was associated with tumor clinical stage and cervical lymph node metastasis, suggesting that it is correlated with an aggressive phenotype. LSCC patients with high Kif2a expression were more likely to have significantly poorer OS by both univariate analysis and multivariate analysis. These findings suggest that Kif2a may play a crucial role in the progression of this malignant tumor. Tumor progression is a very complex process that includes cell transformation, angiogenesis, proliferation, invasion, dissemination, and subsequent adhesion and colonization of the secondary tissue or organ. Therefore, the survival of patients with malignancies is so complex that a single biomarker cannot predict all of these events. In other words, combined evaluation of various proteins should be used to precisely predict prognosis in patients with LSCC after resection. In our study, we simultaneously examined the expression of Ki67 in the same TMAs. Ki67, which is synthesized at the beginning of cell proliferation and expressed in all phases of the cell cycle except the G0 phase, is generally recognized as a proliferation-related nuclear protein used to characterize malignant lesions of multiple cancers including LSCC [31,32]. High Ki67 protein expression was also associated with tumor clinical stage and cervical lymph node metastasis in present study. Furthermore, IHC results showed a significant correlation between Kif2a and Ki67 protein expression, which means that the expression of Kif2a closely correlates with aggressive LSCC behavior. To the best of our knowledge, this is the first study that investigated Kif2a, alone or in combination with Ki67 protein expression, as well as their prognostic value in patients with LSCC. Our data demonstrate that Kif2a and Ki67 might be applied to the clinic to more precisely predict LSCC prognosis after operation. However, the underlying molecular mechanisms of Kif2a in tumorigenesis remain unknown and further in vitro and vivo analyses are warranted to investigate the exact mechanisms of Kif2a protein action. 5. Conclusion In summary, data from this study indicate that high Kif2a and Ki67 expression correlate with poor OS in LSCC. These results support the idea that Kif2a alone or in combination with Ki67 protein expression can be used as novel biomarkers to more precisely predict LSCC prognosis. Authors’ contribution You YW and Zhang QC designed research and wrote the paper; Zhang W, Zhang J, and Xu HL participated in the Clinical data collection; Zhang QC and Xu HL contributed to the final data analysis and figure preparation. All authors read and approved the final manuscript.

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Please cite this article in press as: Zhang Q, et al. Aberrant Kif2a and Ki67 expression predicts poor survival in laryngeal squamous cell carcinoma. Auris Nasus Larynx (2015), http://dx.doi.org/10.1016/j.anl.2015.10.012

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Please cite this article in press as: Zhang Q, et al. Aberrant Kif2a and Ki67 expression predicts poor survival in laryngeal squamous cell carcinoma. Auris Nasus Larynx (2015), http://dx.doi.org/10.1016/j.anl.2015.10.012