YEXMP-03752; No of Pages 6 Experimental and Molecular Pathology xxx (2015) xxx–xxx
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Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
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Hui-Qiang Liu, Yong-Hong Wang, Lan-Lan Wang, Min Hao ⁎
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Department of Obstetrics and Gynecology, The Second Hospital of Shanxi Medical University, Taiyuan 030001, PR China
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a r t i c l e
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Article history: Received 16 April 2015 Accepted 18 April 2015 Available online xxxx
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Keywords: P16INK4A Survivin Cervical cancer Cervical intraepithelial neoplasia Cervical squamous cell carcinoma Prognosis
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Objective: To explore the roles of p16INK4A (p16) and survivin expressions in cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma. Methods: The p16 and survivin expressions were detected in 50 cervical squamous cell carcinoma tissues, 150 various grades of CIN tissues and 30 normal cervical tissues using immunohistochemistry. All data were analyzed applying SPSS 17.0 software. Results: The p16 and survivin expressions showed the presence of statistical significance in cervical cancer, CINI, CINII, CINIII and normal cervical tissues (P b 0.05), and the comparison also revealed statistical significance among groups (all P b 0.05); the p16 and survivin expressions were positively correlated with the grade of cervical diseases (both P b 0.05). Moreover, p16 protein was associated with CIN grade and lymph node metastases in cervical cancer (all P b 0.05); survivin protein was also related with clinical stages, CIN grade and lymph node metastases (all P b 0.05); the p16 and survivin expressions were positively correlated with cervical cancer (r = 0.854, P b 0.001), and associated with poor prognosis of cervical cancer. Conclusion: Briefly, p16 and survivin expression may be correlated with the pathogenesis and prognosis of cervical cancer. © 2015 Published by Elsevier Inc.
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1. Introduction
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P16INK4A and survivin: Diagnostic and prognostic markers in cervical intraepithelial neoplasia and cervical squamous cell carcinoma
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Cervical cancer is recognized as a multistage progression model for 38 continuous infections with carcinogenic human papillomavirus (HPV) 39 types (Luhn et al., 2013). There are two clinical variants including squa40 mous cell carcinomas occupying 85%–90% and adenocarcinoma carcino41 mas comprising 10%–25% of cases (Yeasmin et al., 2012). More recent 42 reports have suggested that higher cervical intraepithelial neoplasia 43 (CIN) grades associate with greater risks that lesions will advance to in44 vasive cervical cancer (Wei et al., 2014). CIN, also named cervical dys45 plasia as a precancerous condition, is the dysplasia and premalignant 46 Q11 transformation of squamous cells on the surface of the cervix (Martin 47 et al., 2011). Recently, new biomarkers have been estimated for their 48 potential role to improve the diagnostic accuracy of cervical biopsy 49 (Alshenawy, 2014). 50 P16INK4a (P16) protein, whose gene locates on 9p21, is a negative 51 regulator of the cell cycle and a tumor suppressor (Salehinejad et al., 52 2014). The p16 inhibits cyclin-dependent kinase (CDK) by inactivating 53 CDK4/6 as well as impeding the phosphorylate retinoblastoma (pRb) 54 Q12 thus inhibiting cell cycle from G1 to S phase (Abrahao et al., 2011; ⁎ Corresponding author at: Department of Obstetrics and Gynecology, The Second Hospital of Shanxi Medical University, No. 382, Wuyi Road, Yingze District, Taiyuan 030001, PR China. E-mail address: [email protected] (M. Hao).
Montebugnoli et al., 2011). Moreover, overexpression of p16 protein by immunohistochemistry has been discovered in HPV, which is closely correlated with cervical cancer (Subhawong et al., 2009). In routine clinical practice, p16 protein has been used as a marker for high-risk HPV infection in cervical cancer including squamous cell carcinomas (Bohn et al., 2010; Subhawong et al. 2009). The BIRC5 gene encodes the survivin protein, also called inhibitor of apoptosis repeat-containing-5; survivin is known as the smallest member of the inhibitor of apoptosis (IAP) family (Rauch et al., 2014). There is evidence showing that survivin acts a pivotal role in cell survival and cell division promotion (Athanasoula et al., 2014). Furthermore, survivin has been upregulated in some human cancers and discovered to be correlated with decreased patient survival as well as increased aggressiveness indicating that apoptosis inhibition by survivin could be a crucial predictive and prognostic marker of poor outcomes in some human cancers (Nigam et al., 2015). In this regard, the purpose of this study was to assess the correlations of p16 and survivin with the pathogenesis and prognosis of cervical cancers.
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2. Patients and methods
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2.1. Ethics statement
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The present study was performed with the approval of the Ethics 75 Committee of the Second Hospital of Shanxi Medical University. This 76
http://dx.doi.org/10.1016/j.yexmp.2015.04.004 0014-4800/© 2015 Published by Elsevier Inc.
Please cite this article as: Liu, H.-Q., et al., P16INK4A and survivin: Diagnostic and prognostic markers in cervical intraepithelial neoplasia and cervical squamous cell carcinom..., Exp. Mol. Pathol. (2015), http://dx.doi.org/10.1016/j.yexmp.2015.04.004
t1:6 t1:7 t1:8 t1:9 t1:10 t1:11
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Normal cervical tissue CIN tissue CINI CINII CINIII Cervical squamous cell cancer tissue
30 150 50 50 50 50
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+
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29 66 35 22 9 4
1 34 10 9 18 6
0 31 3 15 12 21
0 19 2 4 11 19
Positive rate (%) 3.3 56.0 30.0 56.0 82.0 92.0
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research was conducted in line with the Helsinki Declaration. All patients signed informed consent before the beginning of the research.
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2.2. Sample collection
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Between January 2009 and August 2011, 230 women aged from 23 to 71 years with an average age of 41.26, received cervical biopsy and surgical removal from the Second Hospital of Shanxi Medical University, 83 were enrolled in our study. The medical records of all patients were 84 complete; any chemotherapy was not accepted before operation. Fifty 85 of 230 patients with cervical squamous cell carcinoma were classified 86 into stage I (n = 36) and stage II (n = 14) based on The International 87 Federation of Gynecology and Obstetrics (FIGO); it was divided into 88 high differentiation group (n = 8), moderate differentiation group 89 (n = 31) and low differentiation group (n = 11) according to WHO his90 tological grading criteria; the presence of lymph node metastasis was 91 found in 26 of them; other 24 patients without lymph node metastasis; 92 the depth of myometrial invasion (≥1/2) was shown in 23 of them, and 93 the depth of myometrial invasion (b 1/2) was discovered in other 27 in94 dividuals. In our study, 150 individuals were diagnosed with CIN, in95 cluding CINI (n = 50), CINII (n = 50) and CINIII (n = 50). Thirty 96 Q13 normal cervixes were obtained from uterine total resection for uterine 97 fibroids. All sections from patients were checked by two pathologists. 98 All patients should have not received radiotherapy, chemotherapy or 99 hormone therapy. This research was agreed by the Second Hospital of 100 Shanxi Medical University Ethics Committee; all patients signed in101 formed consent.
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The expression of p16 in cervical tissues was detected using immunohistochemistry streptavidin–perosidase (SP) method. All specimens of patients were procured from 10% formalin fixed, embedded by paraffin, cut continuously in 4 μm sections and dewaxed. After alcoholic dehydrated, 3% hydrogen peroxide was added to block the endogenous peroxidase activity. Then, the antigen retrieval (AR) method was applied, and we added the monoclonal antibody against human p16 protein and survivin monoclonal antibody (Labvision, USA) with a 1:50 dilution; after being developed for 1 h at 37 °C, biotinylated rabbit anti-mouse immunoglobulin G (IgG) was added as the secondary antibodies; after being developed for 30 min at 37 °C, 3′-diaminobenzidine (DAB) was added, 1– 2 min after color reaction, and washed using phosphate-buffered saline (PBS) buffer solution 3 times (2 min per time). Sections then were stained with hematoxylin (HE) for 1 min; after being dehydrated, it became transparent and was then covered by general clarity gum. Positive prostate tissues were used as a positive control, and PBS were used as a negative control instead of the first antibody. Immunohistochemical reaction product of p16 showed yellow or brown located in cytoplasm and/or nucleus. The proportion and distribution of the positive cells were as follows: negative (−): single cells were stained (the number of the positive cells: b5%); weakly positive (+): sporadic or small cell clusters were stained (the number of the positive cells: 5%–24%); positive (++): schistose or clusters of cells were dyed (the number of the positive cells: 25%–50%); strong positive (+++): disperse cells were dyed (the number of the positive cells: N50%). Survivin positive cells expressed as brown-yellow particles in the cytoplasm and/or nucleus after staining. A combination of the rate of the positive cells in similar cells and tinctorial strength of the positive cells was taken into consideration; semiquantitative method was applied to estimate the results. Positive intensity was divided into 4 scales based on the degree of coloration: no coloration (negative, 0 point), light yellow (weak positive, 1 point), brown-yellow (moderate positive, 2 points) and chocolate brown (strong positive, 3 points). Other scores were based on the proportion of the positive cells: less than 5% (0 point), 5%–25% (1 point), 26%–50% (2 points), 51%–75% (3 points) and more than 75% (4 points). The comprehensive evaluation was calculated by two kinds of scales: 0 point is negative (−); 1–4 points are
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Groups
2.3. Immunohistochemistry
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Table 1 Expressions of P16 in normal cervical tissue, cervical intraepithelial neoplasia and cervical squamous cell cancer.
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H.-Q. Liu et al. / Experimental and Molecular Pathology xxx (2015) xxx–xxx
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Fig. 1. The expressions of p16 in normal cervical tissue, cervical intraepithelial neoplasia and cervical squamous cell carcinoma (A: p16 was negatively expressed in normal cervical tissues (IHE DAB coloration, 200×); B: p16 was negatively expressed in CINI tissues (IHE DAB coloration, 200×); C: p16 positive expression was weak in CINII tissues (IHE DAB coloration, 200×); D: p16 was positively expressed in CINIII tissues (IHE DAB coloration, 200×); E: p16 positive expression was strong in cervical squamous cell carcinoma tissues (IHE DAB coloration, 200×)).
Please cite this article as: Liu, H.-Q., et al., P16INK4A and survivin: Diagnostic and prognostic markers in cervical intraepithelial neoplasia and cervical squamous cell carcinom..., Exp. Mol. Pathol. (2015), http://dx.doi.org/10.1016/j.yexmp.2015.04.004
105 106 107 Q14 108 109 110 111 112 113 114 Q15 115 116 Q16 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139
H.-Q. Liu et al. / Experimental and Molecular Pathology xxx (2015) xxx–xxx
t2:6 t2:7 t2:8 t2:9 t2:10 t2:11
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Normal cervical tissue CIN tissue CINI CINII CINIII Cervical squamous cell cancer tissue
30 150 50 50 50 50
−
+
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28 74 42 25 7 10
1 23 8 10 5 9
1 32 0 12 20 12
0 21 0 3 18 19
Positive rate (%) 3.7 50.7 16.0 50.0 86.0 80.0
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2.4. Follow-up
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Clinical, pathological and discharge data were established once all patients were admitted to hospital. The period of follow-up was from the discharge after systemic treatment to December, 2013. After discharge, patients were followed-up once every 3 months the first year, every 3–6 months the second year, every half a year the third year and/or the fourth year. The survival time was calculated by months.
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2.5. Statistical analysis
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Data were analyzed by applying the SPSS 17.0 software, and using chi-square test and fourfold table. Spearman's rank-correlation test (α = 0.05, two side) was used to analyze the correlation. Survival analysis was conducted by using Kaplan–Meier analysis; P b 0.05 was used for determining the significance.
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3. Results
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3.1. The expressions of p16 in normal cervical tissue, cervical intraepithelial neoplasia and cervical squamous cell carcinoma
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As shown in Table 1 and Fig. 1, the expressions of p16 in normal cervical tissue, CIN and cervical squamous cell carcinoma were 0.0%, 56.0%
The expressions of survivin in normal cervical tissue, CIN and cervical squamous cell carcinoma were 0.0%, 50.7% and 80.0%, respectively; the comparison among groups revealed the existence of statistical significance (χ2 = 26.31, P b 0.001; χ2 = 48.00, P b 0.001; χ2 = 10.94, P b 0.001) (Table 2 and Fig. 2). The expressions of survivin in CINI, CINII, and CINIII were 16.0%, 50.0% and 86.0%, respectively, and the comparison in three groups revealed the presence of statistical significance; moreover, the comparison among groups also indicated statistical significance (χ2 = 13.07, P b 0.001; χ2 = 49.02, P b 0.001; χ2 = 14.89, P b 0.001). The results of correlation analysis indicated that the expression of survivin was positively correlated with the grade of cervical diseases (r = 0.449, P b 0.001).
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The expressions of p16 in poorly, moderately and highly differentiated cervical cancer were 63.6%, 96.8% and 100.0%, respectively; the differences evidenced statistical significance (P = 0.018) (as shown in Table 3). Moreover, the expression of p16 protein was associated with lymph node metastases in cervical cancer. The expression of p16 was 100.0% in cervical lymph node metastases and 83.3% without metastases; the comparison revealed statistical differences (P = 0.046).
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164 165 166 167 168 169 170 171
174 175 176 177 178 179 180 181 182 183 184 185
3.3. Correlations of p16 and survivin expressions with clinico-pathological 186 characteristics in cervical cancer 187
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3.2. The expressions of survivin in normal cervical tissue, cervical 172 intraepithelial neoplasia and cervical squamous cell carcinoma 173
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regarded as weak positive (+); 5–8 points are taken as moderate positive (++); 9–12 points are recognized as strong positive (+++).
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and 92.0%, respectively; the comparison among groups revealed the existence of statistical significance (χ2 = 64.94, P b 0.001; χ2 = 21.36, P b 0.001; χ2 = 31.50, P b 0.001). The expressions of p16 demonstrated the presence of statistical significance in cervical squamous cell carcinoma, CINI, CINII, CINIII and normal cervical tissues (P b 0.05). The expressions of p16 in various grades were 30.0%, 56.0% and 82.0%, respectively, and the comparison in three groups demonstrated the presence of statistical significance; moreover, the comparison among groups also showed statistical significance (χ2 = 6.90, P = 0.008; χ2 = 7.90, P = 0.005; χ2 = 27.44, P b 0.001). The results of correlation analysis indicated that the expression of p16 were positively correlated with the grade of cervical diseases (r = 0.556, P b 0.001).
Table 2 Expressions of survivin in normal cervical tissue, cervical intraepithelial neoplasia and cervical squamous cell cancer.
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Fig. 2. The expressions of survivin in normal cervical tissue, cervical intraepithelial neoplasia and cervical squamous cell carcinoma (A: survivin was negatively expressed in normal cervical tissues (IHE DAB coloration, 200×); B: survivin was negatively expressed in CINI tissues (IHE DAB coloration, 200×); C: survivin positive expression was weak in CINII tissues (IHE DAB coloration, 200×); D: survivin was positively expressed in CINIII tissues (IHE DAB coloration, 200×); E: survivin positive expression was strong in cervical squamous cell carcinoma tissues (IHE DAB coloration, 200×)).
Please cite this article as: Liu, H.-Q., et al., P16INK4A and survivin: Diagnostic and prognostic markers in cervical intraepithelial neoplasia and cervical squamous cell carcinom..., Exp. Mol. Pathol. (2015), http://dx.doi.org/10.1016/j.yexmp.2015.04.004
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4. Discussion
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As mentioned before, cervical cancers are preceded by CINs; moreover, higher CIN grades show greater potential of progressing to cervical cancers as compared to low CIN grades. Previous studies have suggested that high-risk HPV infection is the most crucial cause of malignant transformation of cervical epithelium (Mandigo et al., 2015; Mittal et al., 2014). E6 and E7 of HPV, which could inactivate chief cellular regulatory proteins, have the function of controlling cell survival, growth, differentiation and apoptosis, as well as genome stability (Milrot et al., 2012). This study showed that the expressions of p16 in normal cervical tissue, CIN and cervical squamous cell carcinoma were gradually increased; moreover, with the developing of CIN grades, the p16 expressions were also statistically increased. Besides, the results
t3:1 t3:2
Table 3 Correlations of p16 expression with clinico-pathological characteristics in cervical cancer.
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t3:3 t3:4 t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 Q7 t3:13 t3:14 t3:15 t3:16 t3:17 t3:18 t3:19 t3:20 t3:21 t3:22 t3:23
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Clinical pathological characteristics
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n
Age ≤40 N40 Clinical stage (FIGO) I II Grade of differentiation Well-differentiated Moderately-differentiated Poorly-differentiated Lymph node involvement + − Interstitium infiltration ≥1/2 b1/2 Tumor diameter ≤4 N4
P16
Positive rate (%)
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22 24
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91.7 92.3
1.000
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91.7 92.9
1.000
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8 30 7
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100.0 96.8 63.6
0.018
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26 20
0 4
100 83.3
0.046
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21 25
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91.3 92.6
1.000
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1.000
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Positive rate (%) P
Age ≤40 N40 Clinical stage (FIGO) I II Grade of differentiation Well-differentiated Moderately-differentiated Poorly-differentiated Lymph node involvement + − Interstitium infiltration ≥1/2 b1/2 Tumor diameter ≤4 N4
24 20 26 20
t4:4 t4:5
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83.3 76.9
0.728
36 26 14 14
10 72.2 0 100.0
0.045
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4 50.0 6 80.6 0 100.0
0.027
26 24 24 16
2 8
92.3 66.7
0.035
23 19 27 21
4 6
82.6 77.8
0.736
37 30 13 10
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81.1 76.9
0.707
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Fifty patients with cervical cancer were followed-up by telephone and registering outpatients' reexamination. The follow-up time ranged 8–70 months with the medium time of 46 months and a rate of 100%. The 3 year survival rates were 84.0%; eight patients died for tumor recurrence. As shown in Kaplan–Meier curves (Fig. 3), the 3 year survival rates of low expression of p16 protein (90.0%) were obviously higher than those of high expression of p16 protein (82.5%); the comparison among groups demonstrated statistical significance (P b 0.05). The 3 year survival rates of low expression of survivin (94.7%) were also significantly higher than those of high expression of survivin (77.4%); the comparison among groups showed statistical significance (P b 0.05) (Fig. 4).
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Survivin +–+++ −
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Clinical pathological characteristics n
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3.4. Correlations of p16 and survivin with prognosis in cervical cancer
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t4:6 t4:7 t4:8 t4:9 Q8 t4:10 t4:11 t4:12 t4:13 t4:14 t4:15 t4:16 t4:17 t4:18 t4:19 t4:20 t4:21 t4:22 t4:23 t4:24
demonstrated that the expressions of p16 were gradually increased in poorly, moderately and highly differentiated cervical cancer tissues, and it was also positively associated with lymph node metastases in cervical carcinoma. The above data suggests that the p16 protein could be an important clinical pathological biomarker for cervical squamous cell carcinoma. The 3 year follow-up results indicated that the expression of p16 protein was negatively correlated with the survival rates of patients with cervical cancer suggesting that p16 protein also could be a prognostic biomarker for cervical cancer patients. As mentioned in the Introduction section, p16 is known as a tumor suppressor and a CDK inhibitor, which could slow down the cell cycle; its overexpression has been documented to be representative of neoplastic and dysplastic epithelium of cervix (Yildiz et al., 2007). The expression of viral oncogenes HPV (E6 and E7) has the capability to bind and inactivate pRb, whose status has a notable effect on the p16 expression, and also is correlated with the malignant transformation of cervical epithelial cells (van de Putte et al., 2003). Thus we hypothesize that p16 expression is caused owing to the functional inactivation of pRb by HPV proteins (E6 and E7) in cervical lesions. In the present study, we also assessed the correlation between survivin and cervical cancer. The overexpression of survivin has been reported to be related with worsened patient survival in numerous cancers, while survivin is not expressed or at a low level in normal or undifferentiated tissues; moreover, survivin expressions are closely correlated with tumor development, infiltration as well as prognosis (Nigam et al. 2015; Waligorska-Stachura et al., 2012). The results showed that the survivin expressions in normal cervical tissue, CIN and cervical squamous cell carcinoma were gradually increased; moreover, the survivin expressions in CINI, CINII and CINIII were also significantly increased indicating that survivin could be a credible marker for the diagnosis and differentiating CIN grades. Furthermore, survivin expressions were also correlated with clinical stages, differentiated cervical carcinomas and metastases suggesting that survivin protein could
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Table 5 Correlations of p16 and survivin in cervical cancers.
t5:1 t5:2
P
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197 198
Table 4 t4:1 Correlations of survivin expression with clinico-pathological characteristics in cervical t4:2 cancer. t4:3
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However, the expression of p16 protein was impendence of age, clinical stages, invasive depth and tumor size; no existence of statistical significance was found (all P N 0.05). In Table 4, the expressions of survivin in clinical stages I and II (P = 0.045), various differentiated cervical carcinomas (P = 0.027) and with/without metastases (P = 0.046) indicated significant differences. Howbeit, the expression of survivin found no statistical significance in ages, invasive depth and tumor size (all P N 0.05). As shown in Table 5, 40 patients showed the expressions of p16 and survivin, and 4 participators showed no expression of p16 and survivin. The result of Spearman's rank-correlation test demonstrated that the expression of p16 was positively correlated with the expression of survivin in patients with cervical cancer (r = 0.854, P b 0.001).
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p16 −
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3 6 12 0
0 3 0 16
t5:5 t5:6 t5:7 t5:8
Please cite this article as: Liu, H.-Q., et al., P16INK4A and survivin: Diagnostic and prognostic markers in cervical intraepithelial neoplasia and cervical squamous cell carcinom..., Exp. Mol. Pathol. (2015), http://dx.doi.org/10.1016/j.yexmp.2015.04.004
H.-Q. Liu et al. / Experimental and Molecular Pathology xxx (2015) xxx–xxx
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Survival Functions cell
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276 277
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relevant transcription of extracellular matrix protein fibronectin (Milrot et al. 2012; Nestal de Moraes et al., 2013). Accordingly, at the cell surface, the newly generated fibronectin engages β1 integrin activating cell motility kinases such as Src and focal adhesion kinase (FAK), thus notably promoting tumor cells migration, invasion as well as metastasis in vivo (Altieri, 2010). Consistent with our results, survivin could be a risk associated protein for disseminated disease, resistance to therapy as well as unfavorable disease outcomes. Moreover, our results demonstrated that the expression of p16 was positively correlated with the expression of survivin in patients with cervical cancer suggesting that they could complete each other to obtain an accurate diagnosis. On all accounts, p16 and survivin are useful markers for disease progression and prognosis in cervical cancer.
Survival Functions
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also be a vital clinical pathological biomarker for cervical squamous cell carcinoma. Besides, the 3 year follow-up results revealed that the survival rates of low survivin expression were significantly higher as compared with high survivin expression suggesting that survivin protein could also be an effective prognostic biomarker. The overexpression of survivin has been reported to be related with worsened patient survival in numerous cancers (Nigam et al. 2015; Waligorska-Stachura et al. 2012). As is known to all, survivin plays a key role in inhibition of cell death (Wu et al., 2012). Under the stimuli of cell-death, survivin is produced from mitochondria to cytosol, and generates complexes with Xlinked inhibitor of apoptosis (XIAP), which could inhibit and bind caspases-3, -7 and -9; the complexes could enhance XIAP stability against ubiquitin-dependent degradation involved in XIAP mediated intracellular signaling, especially NF-kB activation thereby causing
cell
1.0
low expression high expression low expression-censored
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Cum Survival
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Fig. 3. Kaplan–Meier curves for survival comparing patients with low expression and high expression of p16.
high expression-censored
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10.00
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30.00
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surtime Fig. 4. Kaplan–Meier curves for survival comparing patients with low expression and high expression of survivin.
Please cite this article as: Liu, H.-Q., et al., P16INK4A and survivin: Diagnostic and prognostic markers in cervical intraepithelial neoplasia and cervical squamous cell carcinom..., Exp. Mol. Pathol. (2015), http://dx.doi.org/10.1016/j.yexmp.2015.04.004
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Acknowledgments
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The authors wish to thank all women who have participated in this study.
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No potential conflicts of interest were disclosed.
Mittal, S., et al., 2014. Reproducibility of cervical intraepithelial neoplasia diagnosis on histological review of cervical punch biopsies from a visual inspection with acetic acid and HPV detection-based screening program. Int. J. Gynaecol. Obstet. 126, 227–231. Montebugnoli, L., et al., 2011. Immunohistochemical expression of p16(INK4A) protein in oral lichen planus. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 112, 222–227. Nestal de Moraes, G., et al., 2013. Doxorubicin induces cell death in breast cancer cells regardless of Survivin and XIAP expression levels. Eur. J. Cell Biol. 92, 247–256. Nigam, J., et al., 2015. Prognostic significance of survivin in resected gallbladder cancer. J. Surg. Res. 194, 57–62. Rauch, A., et al., 2014. Survivin and YM155: how faithful is the liaison? Biochim. Biophys. Acta 1845, 202–220. Salehinejad, J., et al., 2014. Immunohistochemical expression of p16 protein in oral squamous cell carcinoma and lichen planus. Ann. Diagn. Pathol. 18, 210–213. Subhawong, A.P., et al., 2009. Most basal-like breast carcinomas demonstrate the same Rb−/p16+ immunophenotype as the HPV-related poorly differentiated squamous cell carcinomas which they resemble morphologically. Am. J. Surg. Pathol. 33, 163–175. van de Putte, G., et al., 2003. Expression of p27, p21, and p16 protein in early squamous cervical cancer and its relation to prognosis. Gynecol. Oncol. 89, 140–147. Waligorska-Stachura, J., et al., 2012. Survivin-prognostic tumor biomarker in human neoplasms—review. Ginekol. Pol. 83, 537–540. Wei, Q., et al., 2014. IMP3 expression is associated with poor survival in cervical squamous cell carcinoma. Hum. Pathol. 45, 2218–2224. Wu, S.F., et al., 2012. Altered expression of survivin, Fas and FasL contributed to cervical cancer development and metastasis. Eur. Rev. Med. Pharmacol. Sci. 16, 2044–2050. Yeasmin, S., et al., 2012. Biological and clinical significance of NAC1 expression in cervical carcinomas: a comparative study between squamous cell carcinomas and adenocarcinomas/adenosquamous carcinomas. Hum. Pathol. 43, 506–519. Yildiz, I.Z., et al., 2007. Efficiency of immunohistochemical p16 expression and HPV typing in cervical squamous intraepithelial lesion grading and review of the p16 literature. Pathol. Res. Pract. 203, 445–449.
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Please cite this article as: Liu, H.-Q., et al., P16INK4A and survivin: Diagnostic and prognostic markers in cervical intraepithelial neoplasia and cervical squamous cell carcinom..., Exp. Mol. Pathol. (2015), http://dx.doi.org/10.1016/j.yexmp.2015.04.004
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Contents lists available at ScienceDirect
Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
3Q10
Hui-Qiang Liu, Yong-Hong Wang, Lan-Lan Wang, Min Hao ⁎
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Department of Obstetrics and Gynecology, The Second Hospital of Shanxi Medical University, Taiyuan 030001, PR China
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a r t i c l e
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Article history: Received 16 April 2015 Accepted 18 April 2015 Available online xxxx
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Keywords: P16INK4A Survivin Cervical cancer Cervical intraepithelial neoplasia Cervical squamous cell carcinoma Prognosis
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Objective: To explore the roles of p16INK4A (p16) and survivin expressions in cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma. Methods: The p16 and survivin expressions were detected in 50 cervical squamous cell carcinoma tissues, 150 various grades of CIN tissues and 30 normal cervical tissues using immunohistochemistry. All data were analyzed applying SPSS 17.0 software. Results: The p16 and survivin expressions showed the presence of statistical significance in cervical cancer, CINI, CINII, CINIII and normal cervical tissues (P b 0.05), and the comparison also revealed statistical significance among groups (all P b 0.05); the p16 and survivin expressions were positively correlated with the grade of cervical diseases (both P b 0.05). Moreover, p16 protein was associated with CIN grade and lymph node metastases in cervical cancer (all P b 0.05); survivin protein was also related with clinical stages, CIN grade and lymph node metastases (all P b 0.05); the p16 and survivin expressions were positively correlated with cervical cancer (r = 0.854, P b 0.001), and associated with poor prognosis of cervical cancer. Conclusion: Briefly, p16 and survivin expression may be correlated with the pathogenesis and prognosis of cervical cancer. © 2015 Published by Elsevier Inc.
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1. Introduction
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P16INK4A and survivin: Diagnostic and prognostic markers in cervical intraepithelial neoplasia and cervical squamous cell carcinoma
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Cervical cancer is recognized as a multistage progression model for 38 continuous infections with carcinogenic human papillomavirus (HPV) 39 types (Luhn et al., 2013). There are two clinical variants including squa40 mous cell carcinomas occupying 85%–90% and adenocarcinoma carcino41 mas comprising 10%–25% of cases (Yeasmin et al., 2012). More recent 42 reports have suggested that higher cervical intraepithelial neoplasia 43 (CIN) grades associate with greater risks that lesions will advance to in44 vasive cervical cancer (Wei et al., 2014). CIN, also named cervical dys45 plasia as a precancerous condition, is the dysplasia and premalignant 46 Q11 transformation of squamous cells on the surface of the cervix (Martin 47 et al., 2011). Recently, new biomarkers have been estimated for their 48 potential role to improve the diagnostic accuracy of cervical biopsy 49 (Alshenawy, 2014). 50 P16INK4a (P16) protein, whose gene locates on 9p21, is a negative 51 regulator of the cell cycle and a tumor suppressor (Salehinejad et al., 52 2014). The p16 inhibits cyclin-dependent kinase (CDK) by inactivating 53 CDK4/6 as well as impeding the phosphorylate retinoblastoma (pRb) 54 Q12 thus inhibiting cell cycle from G1 to S phase (Abrahao et al., 2011; ⁎ Corresponding author at: Department of Obstetrics and Gynecology, The Second Hospital of Shanxi Medical University, No. 382, Wuyi Road, Yingze District, Taiyuan 030001, PR China. E-mail address: [email protected] (M. Hao).
Montebugnoli et al., 2011). Moreover, overexpression of p16 protein by immunohistochemistry has been discovered in HPV, which is closely correlated with cervical cancer (Subhawong et al., 2009). In routine clinical practice, p16 protein has been used as a marker for high-risk HPV infection in cervical cancer including squamous cell carcinomas (Bohn et al., 2010; Subhawong et al. 2009). The BIRC5 gene encodes the survivin protein, also called inhibitor of apoptosis repeat-containing-5; survivin is known as the smallest member of the inhibitor of apoptosis (IAP) family (Rauch et al., 2014). There is evidence showing that survivin acts a pivotal role in cell survival and cell division promotion (Athanasoula et al., 2014). Furthermore, survivin has been upregulated in some human cancers and discovered to be correlated with decreased patient survival as well as increased aggressiveness indicating that apoptosis inhibition by survivin could be a crucial predictive and prognostic marker of poor outcomes in some human cancers (Nigam et al., 2015). In this regard, the purpose of this study was to assess the correlations of p16 and survivin with the pathogenesis and prognosis of cervical cancers.
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2. Patients and methods
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2.1. Ethics statement
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56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72
The present study was performed with the approval of the Ethics 75 Committee of the Second Hospital of Shanxi Medical University. This 76
http://dx.doi.org/10.1016/j.yexmp.2015.04.004 0014-4800/© 2015 Published by Elsevier Inc.
Please cite this article as: Liu, H.-Q., et al., P16INK4A and survivin: Diagnostic and prognostic markers in cervical intraepithelial neoplasia and cervical squamous cell carcinom..., Exp. Mol. Pathol. (2015), http://dx.doi.org/10.1016/j.yexmp.2015.04.004
t1:6 t1:7 t1:8 t1:9 t1:10 t1:11
77
Normal cervical tissue CIN tissue CINI CINII CINIII Cervical squamous cell cancer tissue
30 150 50 50 50 50
−
+
++
+++
29 66 35 22 9 4
1 34 10 9 18 6
0 31 3 15 12 21
0 19 2 4 11 19
Positive rate (%) 3.3 56.0 30.0 56.0 82.0 92.0
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research was conducted in line with the Helsinki Declaration. All patients signed informed consent before the beginning of the research.
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2.2. Sample collection
80
Between January 2009 and August 2011, 230 women aged from 23 to 71 years with an average age of 41.26, received cervical biopsy and surgical removal from the Second Hospital of Shanxi Medical University, 83 were enrolled in our study. The medical records of all patients were 84 complete; any chemotherapy was not accepted before operation. Fifty 85 of 230 patients with cervical squamous cell carcinoma were classified 86 into stage I (n = 36) and stage II (n = 14) based on The International 87 Federation of Gynecology and Obstetrics (FIGO); it was divided into 88 high differentiation group (n = 8), moderate differentiation group 89 (n = 31) and low differentiation group (n = 11) according to WHO his90 tological grading criteria; the presence of lymph node metastasis was 91 found in 26 of them; other 24 patients without lymph node metastasis; 92 the depth of myometrial invasion (≥1/2) was shown in 23 of them, and 93 the depth of myometrial invasion (b 1/2) was discovered in other 27 in94 dividuals. In our study, 150 individuals were diagnosed with CIN, in95 cluding CINI (n = 50), CINII (n = 50) and CINIII (n = 50). Thirty 96 Q13 normal cervixes were obtained from uterine total resection for uterine 97 fibroids. All sections from patients were checked by two pathologists. 98 All patients should have not received radiotherapy, chemotherapy or 99 hormone therapy. This research was agreed by the Second Hospital of 100 Shanxi Medical University Ethics Committee; all patients signed in101 formed consent.
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The expression of p16 in cervical tissues was detected using immunohistochemistry streptavidin–perosidase (SP) method. All specimens of patients were procured from 10% formalin fixed, embedded by paraffin, cut continuously in 4 μm sections and dewaxed. After alcoholic dehydrated, 3% hydrogen peroxide was added to block the endogenous peroxidase activity. Then, the antigen retrieval (AR) method was applied, and we added the monoclonal antibody against human p16 protein and survivin monoclonal antibody (Labvision, USA) with a 1:50 dilution; after being developed for 1 h at 37 °C, biotinylated rabbit anti-mouse immunoglobulin G (IgG) was added as the secondary antibodies; after being developed for 30 min at 37 °C, 3′-diaminobenzidine (DAB) was added, 1– 2 min after color reaction, and washed using phosphate-buffered saline (PBS) buffer solution 3 times (2 min per time). Sections then were stained with hematoxylin (HE) for 1 min; after being dehydrated, it became transparent and was then covered by general clarity gum. Positive prostate tissues were used as a positive control, and PBS were used as a negative control instead of the first antibody. Immunohistochemical reaction product of p16 showed yellow or brown located in cytoplasm and/or nucleus. The proportion and distribution of the positive cells were as follows: negative (−): single cells were stained (the number of the positive cells: b5%); weakly positive (+): sporadic or small cell clusters were stained (the number of the positive cells: 5%–24%); positive (++): schistose or clusters of cells were dyed (the number of the positive cells: 25%–50%); strong positive (+++): disperse cells were dyed (the number of the positive cells: N50%). Survivin positive cells expressed as brown-yellow particles in the cytoplasm and/or nucleus after staining. A combination of the rate of the positive cells in similar cells and tinctorial strength of the positive cells was taken into consideration; semiquantitative method was applied to estimate the results. Positive intensity was divided into 4 scales based on the degree of coloration: no coloration (negative, 0 point), light yellow (weak positive, 1 point), brown-yellow (moderate positive, 2 points) and chocolate brown (strong positive, 3 points). Other scores were based on the proportion of the positive cells: less than 5% (0 point), 5%–25% (1 point), 26%–50% (2 points), 51%–75% (3 points) and more than 75% (4 points). The comprehensive evaluation was calculated by two kinds of scales: 0 point is negative (−); 1–4 points are
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t1:5
P16
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n
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Groups
2.3. Immunohistochemistry
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t1:4
Table 1 Expressions of P16 in normal cervical tissue, cervical intraepithelial neoplasia and cervical squamous cell cancer.
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Fig. 1. The expressions of p16 in normal cervical tissue, cervical intraepithelial neoplasia and cervical squamous cell carcinoma (A: p16 was negatively expressed in normal cervical tissues (IHE DAB coloration, 200×); B: p16 was negatively expressed in CINI tissues (IHE DAB coloration, 200×); C: p16 positive expression was weak in CINII tissues (IHE DAB coloration, 200×); D: p16 was positively expressed in CINIII tissues (IHE DAB coloration, 200×); E: p16 positive expression was strong in cervical squamous cell carcinoma tissues (IHE DAB coloration, 200×)).
Please cite this article as: Liu, H.-Q., et al., P16INK4A and survivin: Diagnostic and prognostic markers in cervical intraepithelial neoplasia and cervical squamous cell carcinom..., Exp. Mol. Pathol. (2015), http://dx.doi.org/10.1016/j.yexmp.2015.04.004
105 106 107 Q14 108 109 110 111 112 113 114 Q15 115 116 Q16 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139
H.-Q. Liu et al. / Experimental and Molecular Pathology xxx (2015) xxx–xxx
t2:6 t2:7 t2:8 t2:9 t2:10 t2:11
140
Normal cervical tissue CIN tissue CINI CINII CINIII Cervical squamous cell cancer tissue
30 150 50 50 50 50
−
+
++
+++
28 74 42 25 7 10
1 23 8 10 5 9
1 32 0 12 20 12
0 21 0 3 18 19
Positive rate (%) 3.7 50.7 16.0 50.0 86.0 80.0
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2.4. Follow-up
143 144
147 148
Clinical, pathological and discharge data were established once all patients were admitted to hospital. The period of follow-up was from the discharge after systemic treatment to December, 2013. After discharge, patients were followed-up once every 3 months the first year, every 3–6 months the second year, every half a year the third year and/or the fourth year. The survival time was calculated by months.
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2.5. Statistical analysis
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Data were analyzed by applying the SPSS 17.0 software, and using chi-square test and fourfold table. Spearman's rank-correlation test (α = 0.05, two side) was used to analyze the correlation. Survival analysis was conducted by using Kaplan–Meier analysis; P b 0.05 was used for determining the significance.
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3. Results
156 157
3.1. The expressions of p16 in normal cervical tissue, cervical intraepithelial neoplasia and cervical squamous cell carcinoma
158 159
As shown in Table 1 and Fig. 1, the expressions of p16 in normal cervical tissue, CIN and cervical squamous cell carcinoma were 0.0%, 56.0%
The expressions of survivin in normal cervical tissue, CIN and cervical squamous cell carcinoma were 0.0%, 50.7% and 80.0%, respectively; the comparison among groups revealed the existence of statistical significance (χ2 = 26.31, P b 0.001; χ2 = 48.00, P b 0.001; χ2 = 10.94, P b 0.001) (Table 2 and Fig. 2). The expressions of survivin in CINI, CINII, and CINIII were 16.0%, 50.0% and 86.0%, respectively, and the comparison in three groups revealed the presence of statistical significance; moreover, the comparison among groups also indicated statistical significance (χ2 = 13.07, P b 0.001; χ2 = 49.02, P b 0.001; χ2 = 14.89, P b 0.001). The results of correlation analysis indicated that the expression of survivin was positively correlated with the grade of cervical diseases (r = 0.449, P b 0.001).
T C
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151 152
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145 146
Q4 Q5 Q6
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The expressions of p16 in poorly, moderately and highly differentiated cervical cancer were 63.6%, 96.8% and 100.0%, respectively; the differences evidenced statistical significance (P = 0.018) (as shown in Table 3). Moreover, the expression of p16 protein was associated with lymph node metastases in cervical cancer. The expression of p16 was 100.0% in cervical lymph node metastases and 83.3% without metastases; the comparison revealed statistical differences (P = 0.046).
B
D
164 165 166 167 168 169 170 171
174 175 176 177 178 179 180 181 182 183 184 185
3.3. Correlations of p16 and survivin expressions with clinico-pathological 186 characteristics in cervical cancer 187
N C O
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A
162 163
3.2. The expressions of survivin in normal cervical tissue, cervical 172 intraepithelial neoplasia and cervical squamous cell carcinoma 173
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regarded as weak positive (+); 5–8 points are taken as moderate positive (++); 9–12 points are recognized as strong positive (+++).
160 161
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t2:4
and 92.0%, respectively; the comparison among groups revealed the existence of statistical significance (χ2 = 64.94, P b 0.001; χ2 = 21.36, P b 0.001; χ2 = 31.50, P b 0.001). The expressions of p16 demonstrated the presence of statistical significance in cervical squamous cell carcinoma, CINI, CINII, CINIII and normal cervical tissues (P b 0.05). The expressions of p16 in various grades were 30.0%, 56.0% and 82.0%, respectively, and the comparison in three groups demonstrated the presence of statistical significance; moreover, the comparison among groups also showed statistical significance (χ2 = 6.90, P = 0.008; χ2 = 7.90, P = 0.005; χ2 = 27.44, P b 0.001). The results of correlation analysis indicated that the expression of p16 were positively correlated with the grade of cervical diseases (r = 0.556, P b 0.001).
Table 2 Expressions of survivin in normal cervical tissue, cervical intraepithelial neoplasia and cervical squamous cell cancer.
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t2:1 t2:2 t2:3
3
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Fig. 2. The expressions of survivin in normal cervical tissue, cervical intraepithelial neoplasia and cervical squamous cell carcinoma (A: survivin was negatively expressed in normal cervical tissues (IHE DAB coloration, 200×); B: survivin was negatively expressed in CINI tissues (IHE DAB coloration, 200×); C: survivin positive expression was weak in CINII tissues (IHE DAB coloration, 200×); D: survivin was positively expressed in CINIII tissues (IHE DAB coloration, 200×); E: survivin positive expression was strong in cervical squamous cell carcinoma tissues (IHE DAB coloration, 200×)).
Please cite this article as: Liu, H.-Q., et al., P16INK4A and survivin: Diagnostic and prognostic markers in cervical intraepithelial neoplasia and cervical squamous cell carcinom..., Exp. Mol. Pathol. (2015), http://dx.doi.org/10.1016/j.yexmp.2015.04.004
188 189 190 191 192 193 194
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4. Discussion
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As mentioned before, cervical cancers are preceded by CINs; moreover, higher CIN grades show greater potential of progressing to cervical cancers as compared to low CIN grades. Previous studies have suggested that high-risk HPV infection is the most crucial cause of malignant transformation of cervical epithelium (Mandigo et al., 2015; Mittal et al., 2014). E6 and E7 of HPV, which could inactivate chief cellular regulatory proteins, have the function of controlling cell survival, growth, differentiation and apoptosis, as well as genome stability (Milrot et al., 2012). This study showed that the expressions of p16 in normal cervical tissue, CIN and cervical squamous cell carcinoma were gradually increased; moreover, with the developing of CIN grades, the p16 expressions were also statistically increased. Besides, the results
t3:1 t3:2
Table 3 Correlations of p16 expression with clinico-pathological characteristics in cervical cancer.
226 227 228 229 230 231 232 233
t3:3 t3:4 t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 Q7 t3:13 t3:14 t3:15 t3:16 t3:17 t3:18 t3:19 t3:20 t3:21 t3:22 t3:23
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225
E
223 224
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215 216
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Clinical pathological characteristics
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212 213
n
Age ≤40 N40 Clinical stage (FIGO) I II Grade of differentiation Well-differentiated Moderately-differentiated Poorly-differentiated Lymph node involvement + − Interstitium infiltration ≥1/2 b1/2 Tumor diameter ≤4 N4
P16
Positive rate (%)
P
+–+++
−
24 26
22 24
2 2
91.7 92.3
1.000
36 14
33 13
3 1
91.7 92.9
1.000
8 31 11
8 30 7
0 1 3
100.0 96.8 63.6
0.018
26 24
26 20
0 4
100 83.3
0.046
23 27
21 25
2 2
91.3 92.6
1.000
37 13
34 12
3 1
91.9 92.3
1.000
N
210 211
U
204 205
Positive rate (%) P
Age ≤40 N40 Clinical stage (FIGO) I II Grade of differentiation Well-differentiated Moderately-differentiated Poorly-differentiated Lymph node involvement + − Interstitium infiltration ≥1/2 b1/2 Tumor diameter ≤4 N4
24 20 26 20
t4:4 t4:5
4 6
83.3 76.9
0.728
36 26 14 14
10 72.2 0 100.0
0.045
8 4 31 25 11 11
4 50.0 6 80.6 0 100.0
0.027
26 24 24 16
2 8
92.3 66.7
0.035
23 19 27 21
4 6
82.6 77.8
0.736
37 30 13 10
7 3
81.1 76.9
0.707
F
219 220
Fifty patients with cervical cancer were followed-up by telephone and registering outpatients' reexamination. The follow-up time ranged 8–70 months with the medium time of 46 months and a rate of 100%. The 3 year survival rates were 84.0%; eight patients died for tumor recurrence. As shown in Kaplan–Meier curves (Fig. 3), the 3 year survival rates of low expression of p16 protein (90.0%) were obviously higher than those of high expression of p16 protein (82.5%); the comparison among groups demonstrated statistical significance (P b 0.05). The 3 year survival rates of low expression of survivin (94.7%) were also significantly higher than those of high expression of survivin (77.4%); the comparison among groups showed statistical significance (P b 0.05) (Fig. 4).
202 203
Survivin +–+++ −
O
209
201
Clinical pathological characteristics n
R O
3.4. Correlations of p16 and survivin with prognosis in cervical cancer
199 200
t4:6 t4:7 t4:8 t4:9 Q8 t4:10 t4:11 t4:12 t4:13 t4:14 t4:15 t4:16 t4:17 t4:18 t4:19 t4:20 t4:21 t4:22 t4:23 t4:24
demonstrated that the expressions of p16 were gradually increased in poorly, moderately and highly differentiated cervical cancer tissues, and it was also positively associated with lymph node metastases in cervical carcinoma. The above data suggests that the p16 protein could be an important clinical pathological biomarker for cervical squamous cell carcinoma. The 3 year follow-up results indicated that the expression of p16 protein was negatively correlated with the survival rates of patients with cervical cancer suggesting that p16 protein also could be a prognostic biomarker for cervical cancer patients. As mentioned in the Introduction section, p16 is known as a tumor suppressor and a CDK inhibitor, which could slow down the cell cycle; its overexpression has been documented to be representative of neoplastic and dysplastic epithelium of cervix (Yildiz et al., 2007). The expression of viral oncogenes HPV (E6 and E7) has the capability to bind and inactivate pRb, whose status has a notable effect on the p16 expression, and also is correlated with the malignant transformation of cervical epithelial cells (van de Putte et al., 2003). Thus we hypothesize that p16 expression is caused owing to the functional inactivation of pRb by HPV proteins (E6 and E7) in cervical lesions. In the present study, we also assessed the correlation between survivin and cervical cancer. The overexpression of survivin has been reported to be related with worsened patient survival in numerous cancers, while survivin is not expressed or at a low level in normal or undifferentiated tissues; moreover, survivin expressions are closely correlated with tumor development, infiltration as well as prognosis (Nigam et al. 2015; Waligorska-Stachura et al., 2012). The results showed that the survivin expressions in normal cervical tissue, CIN and cervical squamous cell carcinoma were gradually increased; moreover, the survivin expressions in CINI, CINII and CINIII were also significantly increased indicating that survivin could be a credible marker for the diagnosis and differentiating CIN grades. Furthermore, survivin expressions were also correlated with clinical stages, differentiated cervical carcinomas and metastases suggesting that survivin protein could
235 236
Table 5 Correlations of p16 and survivin in cervical cancers.
t5:1 t5:2
P
208
197 198
Table 4 t4:1 Correlations of survivin expression with clinico-pathological characteristics in cervical t4:2 cancer. t4:3
T
206 207
However, the expression of p16 protein was impendence of age, clinical stages, invasive depth and tumor size; no existence of statistical significance was found (all P N 0.05). In Table 4, the expressions of survivin in clinical stages I and II (P = 0.045), various differentiated cervical carcinomas (P = 0.027) and with/without metastases (P = 0.046) indicated significant differences. Howbeit, the expression of survivin found no statistical significance in ages, invasive depth and tumor size (all P N 0.05). As shown in Table 5, 40 patients showed the expressions of p16 and survivin, and 4 participators showed no expression of p16 and survivin. The result of Spearman's rank-correlation test demonstrated that the expression of p16 was positively correlated with the expression of survivin in patients with cervical cancer (r = 0.854, P b 0.001).
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Survivin
− + ++ +++
237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267
t5:3
p16 −
+
++
+++
t5:4
4 0 0 0
3 0 0 3
3 6 12 0
0 3 0 16
t5:5 t5:6 t5:7 t5:8
Please cite this article as: Liu, H.-Q., et al., P16INK4A and survivin: Diagnostic and prognostic markers in cervical intraepithelial neoplasia and cervical squamous cell carcinom..., Exp. Mol. Pathol. (2015), http://dx.doi.org/10.1016/j.yexmp.2015.04.004
H.-Q. Liu et al. / Experimental and Molecular Pathology xxx (2015) xxx–xxx
5
Survival Functions cell
1.0
low expression high expression low expression-censored
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0.6
0.4
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Cum Survival
high expression-censored
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0.00
10.00
20.00
30.00
40.00
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surtime
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280 Q17281
D
E
T
C
278 279
E
276 277
R
275
R
273 274
relevant transcription of extracellular matrix protein fibronectin (Milrot et al. 2012; Nestal de Moraes et al., 2013). Accordingly, at the cell surface, the newly generated fibronectin engages β1 integrin activating cell motility kinases such as Src and focal adhesion kinase (FAK), thus notably promoting tumor cells migration, invasion as well as metastasis in vivo (Altieri, 2010). Consistent with our results, survivin could be a risk associated protein for disseminated disease, resistance to therapy as well as unfavorable disease outcomes. Moreover, our results demonstrated that the expression of p16 was positively correlated with the expression of survivin in patients with cervical cancer suggesting that they could complete each other to obtain an accurate diagnosis. On all accounts, p16 and survivin are useful markers for disease progression and prognosis in cervical cancer.
Survival Functions
N C O
271 272
also be a vital clinical pathological biomarker for cervical squamous cell carcinoma. Besides, the 3 year follow-up results revealed that the survival rates of low survivin expression were significantly higher as compared with high survivin expression suggesting that survivin protein could also be an effective prognostic biomarker. The overexpression of survivin has been reported to be related with worsened patient survival in numerous cancers (Nigam et al. 2015; Waligorska-Stachura et al. 2012). As is known to all, survivin plays a key role in inhibition of cell death (Wu et al., 2012). Under the stimuli of cell-death, survivin is produced from mitochondria to cytosol, and generates complexes with Xlinked inhibitor of apoptosis (XIAP), which could inhibit and bind caspases-3, -7 and -9; the complexes could enhance XIAP stability against ubiquitin-dependent degradation involved in XIAP mediated intracellular signaling, especially NF-kB activation thereby causing
cell
1.0
low expression high expression low expression-censored
0.8
Cum Survival
269 270
U
268
P
Fig. 3. Kaplan–Meier curves for survival comparing patients with low expression and high expression of p16.
high expression-censored
0.6
0.4
0.2
0.0 0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
surtime Fig. 4. Kaplan–Meier curves for survival comparing patients with low expression and high expression of survivin.
Please cite this article as: Liu, H.-Q., et al., P16INK4A and survivin: Diagnostic and prognostic markers in cervical intraepithelial neoplasia and cervical squamous cell carcinom..., Exp. Mol. Pathol. (2015), http://dx.doi.org/10.1016/j.yexmp.2015.04.004
282 283 284 285 286 Q18 287 288 289 290 291 292 293 294 295
Acknowledgments
299 300
The authors wish to thank all women who have participated in this study.
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Please cite this article as: Liu, H.-Q., et al., P16INK4A and survivin: Diagnostic and prognostic markers in cervical intraepithelial neoplasia and cervical squamous cell carcinom..., Exp. Mol. Pathol. (2015), http://dx.doi.org/10.1016/j.yexmp.2015.04.004
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