Immunohistochemical WWOX Expression and Association with Angiogenesis, p53 Expression, Cell Proliferation and Clinicopathological Parameters in Cervical Cancer

Abstract

Objective The current study evaluated the expression of WW domain-containing oxidoreductase (WWOX), its association with clinicopathological features and with p53, Ki-67 (cell proliferation) and CD31 (angiogenesis) expression in patients with invasive cervical squamous cell carcinoma (ICSCC). To the best of our knowledge, no other study has evaluated this association.

Methods Women with IB stage-ICSCC (n = 20) and women with uterine leiomyoma (n = 20) were prospectively evaluated. Patients with ICSCC were submitted to type B-C1 radical hysterectomy and pelvic lymphadenectomy. Patients in the control group underwent vaginal hysterectomy. Tissue samples were stained with hematoxylin and eosin for histological evaluation and protein expression was detected by immunohistochemistry studies.

Results The WWOX expression was significantly lower in the tumor compared with the expression in the benign cervix (p = 0.019). The WWOX expression was inversely associated with the CD31 expression in the tumor samples (p = 0.018). There was no association between the WWOX expression with the p53 expression (p = 0.464) or the Ki-67 expression (p = 0.360) in the samples of invasive carcinoma of the cervix. There was no association between the WWOX expression and tumor size (p = 0.156), grade of differentiation (p = 0.914), presence of lymphatic vascular invasion (p = 0.155), parametrium involvement (p = 0.421) or pelvic lymph node metastasis (p = 0.310) in ICSCC tissue samples.

Conclusion The results suggested that WWOX may be involved in ICSCC carcinogenesis, and this marker was associated with tumor angiogenesis.

Resumo

Objetivo O presente estudo avaliou a expressão do WWOX, sua associação com características clinicopatológicas e com a expressão do p53, ki-67 (proliferação celular) e CD31 (angiogênese) em pacientes com carcinoma invasivo de células escamosas do colo uterino, ou simplesmente câncer do colo uterino (CCE).

Métodos Foram avaliadas prospectivamente pacientes com CCE no estágio IB (n = 20) e mulheres com mioma uterino, no grupo controle (n = 20). As pacientes com CCE foram submetidas à histerectomia radical e à linfadenectomia pélvica do tipo B-C1. As mulheres no grupo-controle foram submetidas à histerectomia vaginal. As amostras de tecido foram coradas com hematoxilina e eosina para avaliação histológica e a expressão das proteínas foi detectada por imuno-histoquímico.

Resultados A expressão do WWOX foi significativamente menor no tumor quando comparada com sua expressão no colo do útero benigno (p = 0,019). A expressão tumoral de CD31 foi inversamente associada à expressão de WWOX (p = 0,018). Sua expressão não foi associada à expressão tumoral de p53 e Ki-67 em pacientes com CCE (p = 0,464 e p = 0,360, respectivamente). Não houve associação entre a expressão de WWOX e o tamanho do tumor (p = 0,156), grau de diferenciação (p = 0,914), presença de invasão vascular linfática (p = 0,155), comprometimento do paramétrio (p = 0,421) ou metástase dos linfonodos pélvicos (p = 0,310) em pacientes com CCE.

Conclusão Os resultados sugeriram que o WWOX pode estar envolvido na carcinogênese do CICECU e esse marcador foi associado à angiogênese tumoral.

Keywords

cervical neoplasia - immunohistochemistry - tumor suppressor gene - WWOX

Palavras-Chave

câncer de colo uterino - imunohistoquímica - gene supressor tumoral - WWOX

Introduction

Cervical cancer is the most common gynecological neoplasia in the developing world.[1] Developing countries account for two-thirds of the cases and for more than 85% of all deaths due to cervical cancer.[2] [3] In 2016, 16,630 new cases are expected in Brazil alone, representing the third most common malignancy and the fourth leading cause of death among women.[3]

Studies have shown that persistent infection with human papillomavirus (HPV) plays a critical role in cervical carcinogenesis.[4] [5] However, HPV infection alone is not sufficient to induce malignant transformation, and additional genetic or epigenetic changes in tumor cells are required for tumorigenesis.[6] [7] [8] The development and progression of cervical cancer are likely to be associated with the loss of growth suppression, increased cell growth rates and angiogenesis.[9] [10] These combinations of genetic abnormalities generate cells that divide more rapidly or evade cell death, liberating them from growth control and cell cycle checkpoints.[11]

The analysis of tumor suppressor genes expression in human cancer is very important to gain a better insight in the process of tumorigenesis and for the early diagnosis of malignant transformation.[12] The WW domain-containing oxidoreductase (WWOX) gene is located at the site of chromosome 16 (16q23.3–24.1), specifically the FRA16D site.[13] [14] This region displays profound chromosomal instability and is the second most active fragile site in the human genome.[15] [16] The WWOX expression is altered in several types of tumor, including breast cancer, prostate and esophageal cancer, and also seems to be involved in the progression and prognosis of these cancers.[17] [18] [19] [20] [21] [22] [23] [24]

Despite the potential relevance of the WWOX gene in carcinogenesis, surprisingly, little research has focused on its role in the development of cervical cancer.[8] Given the high incidence of this cancer, the current study evaluated the immunohistochemical expression of WWOX in women with invasive cervical squamous cell carcinoma (ICSCC) and its association with the expression of genes p53, CD31 and Ki-67, which are involved in important stages of carcinogenesis, such as angiogenesis and cell proliferation. In addition, we also investigated the potential association between the WWOX expression and clinicopathological parameters.

Methods

The study protocol was approved by the local Research and Ethics Committee, and all patients signed an informed consent form before being included in this study.

The study group consisted of 20 women with stage IB ICSCC and the control group was composed of 20 women with uterine myoma. The mean age of the patients was prospectively evaluated at 49.1 ± 1.7 years (mean ± standard errorof the mean [SEM], range 27–78 years).

The patients with cervical cancer were submitted to Piver-Rutledge class III radical hysterectomy and pelvic lymphadenectomy.[25] This was the primary treatment for all patients because none had previously been submitted to radiotherapy and/or chemotherapy. The clinical stage was defined preoperatively by pelvic examination under general anesthesia, according to the recommendations of the International Federation of Gynecology and Obstetrics (FIGO) Vaginal hysterectomy was performed for the uterine myomas, according to the modified Heaney technique.[26] [27]

The cervix tissue samples were fixed in 10% neutral-buffered formalin immediately after the surgery. Then they were embedded in paraffin and stained with hematoxylin and eosin for histological evaluation. The pathological specimens were analyzed by two pathologists, according to the recommendations of the American Society of Pathologists.[28] The clinicopathological characteristics, such as tumor size, differentiation grade, lymphatic vascular invasion, parametrial involvement and status of pelvic lymph nodes, were recorded.

Immunohistochemistry

Tissue sections from ICSCC and normal cervices samples were stained with WWOX (Upstate, NY, USA), p53 (clone DO7, DAKO), Ki-67 (clone MIB-1, DAKO) and CD31 (clone JC/70A, DAKO) antiserum. Briefly, 4-µm paraffin-embedded sections were dewaxed in xylene and hydrated with graded ethanol. Endogenous peroxidase activity was blocked with 3% H₂O₂ in water for 10 minutes. Heat-induced antigen retrieval was performed with 1 mM EDTA buffer at pH 8.0 for 30 minutes in a steamer at 96°C.

Primary polyclonal rabbit antiserum was used at 1:100, 1:100, 1:100 and 1:40 dilutions for WWOX, p53, Ki-67 and CD31, respectively, for 18 hours at 4°C. This was followed by incubation with the labeled streptavidin-biotin NovoLink Max Polymer Detection System (Leica Biosystems, Nussloch, Germany). The peroxidase activity was developed with DAB (Sigma, St Louis, MI, USA) with timed monitoring using a positive control sample. The sections were then counterstained with hematoxylin, dehydrated and mounted.

Analysis of WWOX, p53, Ki-67 and CD31 staining

All slides were examined under light microscopy. The staining for WWOX, p53, Ki-67 and CD31 was evaluated according to the number of positive stained cells by two pathologists blinded to the clinical information of each patient. The WWOX protein is expressed in the cytoplasm ([Fig. 1]). The p53 and Ki-67 proteins demonstrated only nuclear reactivity, while the CD31 showed both nuclear and cytoplasmic staining in the cells. For each protein, epithelium cells for WWOX, p53 and Ki-67 and endothelium cells for CD31 presenting any expression were considered positive and counted, regardless of the staining intensity.

Fig. 1 WWOX cytoplasmic immunostaining. Normal cervical epithelium (A) and invasive squamous cell carcinoma (ISCC) (B). Less intense expression of WWOX can be observed in the ISCC compared with the benign tissue. (original magnification x200).

The immunostaining was analyzed semiquantitatively. At least 1,000 epithelium cells were analyzed in 10 fields, at 200x magnification. In other words, for each field, at least 100 epithelium cells were checked. With the data obtained from all the analyzed fields, the positive index was calculated using the following formula:

Therefore, the positive index indicated the percentage of positive cells over the number of total epithelial cells analyzed. For the WWOX, p53, Ki-67 and CD31 proteins, the following grades were considered:

• Grade 1: 0 to 25% of immunopositive cells;

• Grade 2: 26 to 50% of immunopositive cells;

• Grade 3: 51 to 75% of immunopositive cells;

• Grade 4: 76 to 100% of immunopositive cells.

Statistical Analysis

The statistical analysis was performed with SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). The data were analyzed using the chi-squared test to evaluate significant differences between the groups. The level of significance was set at p < 0.05. Power calculations showed that the sample size (n = 20) provided a minimal detectable difference of 35% between the two prevalence rates, with a power of 80% and a type I error of 5%.

Results

The clinical stage (FIGO) was IB1 in 14 patients (70%) and IB2 in 6 patients (30%). The average tumoral volume was 18.4 ± 19.1 cm³ (0.3–140.0 cm³). The tumor was well-differentiated (G1) in 1 case (5%), moderately differentiated (G2) in 15 cases (75%) and poorly differentiated in 4 (20%) cases. Lymphatic vascular invasion (LVSI) was present in four patients (20%). The average number of dissected lymph nodes was 17.9 ± 5.1 (range 10–28). Parametrium involvement was noted in 8 patients (40%). Pelvic lymph node metastasis was observed in 9 patients (45%) and the average number of pelvic lymph nodes affected by the tumor at the time of the pathological examination was 1.3 ± 2.3 (range 0–9 nodes).

The WWOX immunostaining was lower in the tumor compared with the benign cervix (p = 0.019). In 100% of the controls (n = 20) the WWOX expression was grade 4. This high expression was observed in 65% (n = 13) of the study group ([Fig. 2]). However, WWOX lower grades expression, grade 3 (n = 6) and grade 1 (n = 1), occurred only in the cervical squamous cell carcinoma samples.

Fig. 2 Expression of WWOX in the benign cervix samples (control group = 20 samples) and in the squamous cell carcinoma samples (study group = 20 samples). The higher WWOX expression was observed in all control group samples. Note: Differences between groups were assessed by χ2 test (two groups). Grade 1: less than 25% of cells showed positivity; grade 2: 26–50% expression; grade 3: 51–75% expression; grade 4: greater than 75% expression.

There was no association between tumor WWOX expression and tumor size (p = 0.156), differentiation grade (p = 0.914), presence of lymphatic vascular invasion (p = 0.155), parametrium involvement (p = 0.421) and pelvic lymph node metastasis (p = 0.310) in patients with invasive carcinoma of the cervix (study group) ([Table 1]).

Table 1 Association of tumor size, grade of differentiation, presence of lymphatic vascular invasion, parametrium involvement and pelvic lymph node metastasis with tumor WWOX expression in patients with invasive squamous cell carcinoma of the cervix

Variables	WWOX expression			p
	Grade 1	Grade 3	Grade 4
Tumor size				0.156
< 4 cm	1 (7.7%)	2 (15.4%)	11 (76.9%)
≥ 4 cm	0	4 (66.7%)	2 (33.3%)
Grade of differentiation				0.914
G1	0	0	1 (100%)
G2	1 (7.7%)	5 (30.8%)	9 (61.5%)
G3	0	1 (25%)	3 (75%)
Lymphatic vascular invasion	1 (25%)	1 (25%)	2 (50%)	0.155
Parametrial invasion	1 (12.5%)	2 (25%)	5 (62.5%)	0.421
Lymph node metastasis	1 (12.5%)	3 (37.5%)	4 (50%)	0.310

Note: Differences between groups were assessed by χ2 test (two groups). Grade 1: when less than 25% of cells showed positivity; grade 2: with 26–50% expression; grade 3: with 51–75% expression; grade 4: with greater than 75% expression.

The WWOX expression was not associated with tumor expression of p53 and Ki-67 in patients with ICSCC (p = 0.464 and p = 0.360, respectively) ([Fig. 3A] and [B]). The tumor expression of CD31 was associated inversely with the WWOX expression (p = 0.018). Tumor samples with higher grades of WWOX expression presented lower grades of CD31 expression ([Fig. 3C]).

Fig. 3 Graphs demonstrate the association between the grades of WWOX immunostaining and the p53 (A), Ki-67 (B) and CD31 (C) grades of expression in patients with invasive squamous cell carcinoma (ISCC) (n = 20). In (C), the higher CD31 expression was significantly associated with a reduced WWOX expression (p = 0.018). Note: Differences between groups were assessed by the χ2 test (two groups). Grade 1: less than 25% of cells showed positivity; grade 2: 26–50% expression; grade 3: 51–75% expression; grade 4: greater than 75% expression.

Discussion

The WWOX is a tumor suppressor gene and its genomic location in a common fragile site makes this gene attractive for several research groups.[13] [22] The altered expression of WWOX is usually caused by previous carcinogenic exposure; in cervical cancer, the HPV infection could play this role. However, studies investigating WWOX expression involved in the development of invasive primary cervical cancer remain incipient.[29]

This study aimed to evaluate the WWOX expression in the squamous cervical carcinoma. The association between the WWOX expression with clinicopathological features, p53 expression, Ki-67 expression (cell proliferation biomarker) and CD31 expression (angiogenesis biomarker) in patients ICSCC was also investigated. To the best of our knowledge, no other study has evaluated this association, and this is the strength of our study.

The WWOX expression was decreased in samples of invasive cervical squamous cell carcinoma compared with the benign cervix. These results are consistent with the only previous study that evaluated WWOX expression in cervical cancer, which identified a reduction or absence of WWOX protein expression in 69% of the patients with invasive cervical cancer. In preinvasive lesions, they observed that the expression was low or undetectable in 43.1% of cervical intraepithelial neoplasia (CIN) grade 1 and in 50% of CIN grades 2/3. The authors also detected a greater loss of WWOX expression from CIN2/3 to ICSCC than from CIN 1 to CIN2/3, suggesting that the WWOX protein is more important during cervical cancer progression than in the initial process of carcinogenesis.[8] Our study did not investigate preinvasive lesions and cannot confirm these results for CIN.

The current study also showed that the WWOX expression is inversely associated with the CD31 expression. Angiogenesis is a prerequisite for tumor growth and is also correlated with the potential for solid tumor metastasis.[30] The CD31 expression is related to neovascularization and it seems to be associated with the clinical course of cervical cancer.[31] The results suggest that decreased WWOX expression in ICSCC can allow CD31 overexpression. Since the WWOX is a tumor suppressor gene, it may suppress tumor angiogenesis,[32] inhibiting the expression of CD31; so, it has potential to be a prognostic marker in ICSCC. The inverse association observed between the expressions of WWOX and CD31 strengthens this hypothesis. However, further research will be required to validate these findings and establish this link. Different pathways implicated in cervical cancer angiogenesis should be evaluated.

No significant association was found between the WWOX expression and the p53 and Ki-67 expressions in ICSCC, which may suggest that the loss of WWOX expression occurs earlier than the alteration in the p53 and Ki-67 expression. This result may be explained by the fact that the samples used in this study were obtained from women with ICSCC classified as IB1 and IB2 stages, according to the FIGO classification. The p53 gene has been extensively studied to explain the oncogenicity of high-risk HPV types in cervical cancer and mutations occur very rarely in early stages of the tumor.[33] [34] In more advanced stages of cervical carcinoma, the p53 expression may be greater either due to increased abnormalities in the control of its expression or degradation, or due to an increased incidence of p53 mutations.[35] [36] If the study had included women with advanced stages of cervical cancer, an association between the WWOX and p53 expressions would possibly have been established.

Interestingly, our results did not show any statistically significant association between the clinicopathological features and the WWOX expression in ICSCC. Previous studies have shown that carcinogenesis markers are related to clinicopathological features. For example, in studies with advanced tumor stages, the p53 expression was reported to correlate with stage, tumor size and grade.[33] [37] On the other hand, another study did not show an association between the p53 expression and other prognostic histological variables (tumor grade, depth, lymphovascular space invasion) in early-stage cervical carcinomas.[38] [39] The same behavior can be observed when evaluating the WWOX expression and the clinicopathological parameters. These unexpected findings could be attributed to the evaluated cervical cancer stages and to our limited sample size. To better evaluate this association, further studies are warranted, with a greater number of patients and including ICSCC samples of other stages.

Studies have shown that multiple mechanisms may be responsible for reducing the WWOX expression in carcinomas. The most common mechanism for the decreased WWOX expression is hemizygous deletions.[40] Another mechanism that reduces WWOX transcriptional level is hypermethylation of its promoter and coding regions. This mechanism may play a role in the downregulation of WWOX expression in several cancer cell lines by silencing the gene.[41] [42] The mechanism involved in the inactivation of WWOX expression in cervical tumors was not evaluated in this study. For this reason, future studies should focus on the methylation status of the promoter region of WWOX or other epigenetic alterations which may influence the WWOX expression.

Conclusion

In conclusion, the present study suggested that the WWOX gene may be involved in ICSCC carcinogenesis and it is associated with tumor angiogenesis. A better characterization of the WWOX expression is necessary in normal, preneoplastic and ICSCC to more fully understand how the loss of the WWOX gene expression contributes to the carcinogenesis of cervical cancer. A detailed definition of WWOX functions may lead to the identification of new targets for intervention in tumor development and progression.

Conflict of Interests

The authors declare that there was no conflict of interests.

Contributors

Seabra M. A. L., Cândido E. B., Vidigal P. V. T., Lamaita R. M., Rodrigues A. N. and Silva Filho A. L. contributed with the project and the interpretation of data, writing of the article, critical review of the intellectual content and final approval of the version to be published.

• References

• 1 Waggoner SE. Cervical cancer. Lancet 2003; 361 (9376): 2217-2225 . Doi: 10.1016/S0140-6736(03)13778-6
• 2 Parkin DM, Ferlay J, Curado MP. , et al. Fifty years of cancer incidence: CI5 I-IX. Int J Cancer 2010; 127 (12) 2918-2927 . Doi: 10.1002/ijc.25517
• 3 Ministério da Saúde. Instituto Nacional de Câncer José de Alencar Gomes da Silva. Estimativa 2016: Incidência de Câncer no Brasil . Rio de Janeiro: INCA; 2016. http://www.inca.gov.br/estimativa/2016/estimativa-2016-v11.pdf . Accessed May 15, 2017
• 4 Spriggs AI, Boddington MM. Progression and regression of cervical lesions. Review of smears from women followed without initial biopsy or treatment. J Clin Pathol 1980; 33 (06) 517-522 . Doi: 10.1136/jcp.33.6.517
• 5 Stensen S, Kjaer SK, Jensen SM. , et al. Factors associated with type-specific persistence of high-risk human papillomavirus infection: A population-based study. Int J Cancer 2016; 138 (02) 361-368 . Doi: 10.1002/ijc.29719
• 6 Silva-Filho AL, Traiman P, Triginelli SA. , et al. Expression of p53, Ki-67, and CD31 in the vaginal margins of radical hysterectomy in patients with stage IB carcinoma of the cervix. Gynecol Oncol 2004; 95 (03) 646-654 . Doi: 10.1016/j.ygyno.2004.07.059
• 7 Burger EA, Kim JJ, Sy S, Castle PE. Age of acquiring causal human papillomavirus (HPV) infections: leveraging simulation models to explore the natural history of HPV-induced cervical cancer. Clin Infect Dis 2017; 65 (06) 893-899 . Doi: 10.1093/cid/cix475
• 8 Giarnieri E, Zanesi N, Bottoni A. , et al. Oncosuppressor proteins of fragile sites are reduced in cervical cancer. Cancer Lett 2010; 289 (01) 40-45 . Doi: 10.1016/j.canlet.2009.07.017
• 9 Ratovitski EA. Anticancer natural compounds as epigenetic modulators of gene expression. Curr Genomics 2017; 18 (02) 175-205 . Doi: 10.2174/1389202917666160803165229
• 10 Pavel AB, Sonkin D, Reddy A. Integrative modeling of multi-omics data to identify cancer drivers and infer patient-specific gene activity. BMC Syst Biol 2016; 10: 16 . Doi: 10.1186/s12918-016-0260-9
• 11 Del Mare S, Husanie H, Iancu O. , et al. WWOX and p53 dysregulation synergize to drive the development of osteosarcoma. Cancer Res 2016; 76 (20) 6107-6117 . Doi: 10.1158/0008-5472.CAN-16-0621
• 12 Iliopoulos D, Guler G, Han SY. , et al. Fragile genes as biomarkers: epigenetic control of WWOX and FHIT in lung, breast and bladder cancer. Oncogene 2005; 24 (09) 1625-1633 . Doi: 10.1038/sj.onc.1208398
• 13 Paige AJ, Zucknick M, Janczar S. , et al. WWOX tumour suppressor gene polymorphisms and ovarian cancer pathology and prognosis. Eur J Cancer 2010; 46 (04) 818-825 . Doi: 10.1016/j.ejca.2009.12.021
• 14 Bednarek AK, Laflin KJ, Daniel RL, Liao Q, Hawkins KA, Aldaz CM. WWOX, a novel WW domain-containing protein mapping to human chromosome 16q23.3-24.1, a region frequently affected in breast cancer. Cancer Res 2000; 60 (08) 2140-2145
• 15 Hezova R, Ehrmann J, Kolar Z. WWOX, a new potential tumor suppressor gene. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2007; 151 (01) 11-15
• 16 Kuroki T, Yendamuri S, Trapasso F. , et al. The tumor suppressor gene WWOX at FRA16D is involved in pancreatic carcinogenesis. Clin Cancer Res 2004; 10 (07) 2459-2465 . Doi: 10.1158/1078-0432.CCR-03-0096
• 17 Karras JR, Schrock MS, Batar B, Huebner K. Fragile genes that are frequently altered in cancer: players not passengers. Cytogenet Genome Res 2016; 150 (3-4): 208-216 . Doi: 10.1159/000455753
• 18 Nunez MI, Rosen DG, Ludes-Meyers JH. , et al. WWOX protein expression varies among ovarian carcinoma histotypes and correlates with less favorable outcome. BMC Cancer 2005; 5: 64 . Doi: 10.1186/1471-2407-5-64
• 19 Gao G, Smith DI. Very large common fragile site genes and their potential role in cancer development. Cell Mol Life Sci 2014; 71 (23) 4601-4615 . Doi: 10.1007/s00018-014-1753-6
• 20 Zhu B, Wang D, Zhang Q, Wu S, Yu L, Tao Y. [Expressions of WWOX and CD133 in colorectal cancer and their clinical significance]. Nan Fang Yi Ke Da Xue Xue Bao 2015; 35 (11) 1586-1590
• 21 Salah Z, Aqeilan R, Huebner K. WWOX gene and gene product: tumor suppression through specific protein interactions. Future Oncol 2010; 6 (02) 249-259 . Doi: 10.2217/fon.09.152
• 22 Ludes-Meyers JH, Bednarek AK, Popescu NC, Bedford M, Aldaz CM. WWOX, the common chromosomal fragile site, FRA16D, cancer gene. Cytogenet Genome Res 2003; 100 (1-4): 101-110 . Doi: 10.1159/000072844
• 23 Schrock MS, Huebner K. WWOX: a fragile tumor suppressor. Exp Biol Med (Maywood) 2015; 240 (03) 296-304 . Doi: 10.1177/1535370214561590
• 24 Lewandowska U, Zelazowski M, Seta K, Byczewska M, Pluciennik E, Bednarek AK. WWOX, the tumour suppressor gene affected in multiple cancers. J Physiol Pharmacol 2009; 60 (Suppl. 01) 47-56
• 25 Piver MS, Rutledge F, Smith JP. Five classes of extended hysterectomy for women with cervical cancer. Obstet Gynecol 1974; 44 (02) 265-272
• 26 Heaney NS. Additional data in the technic of vaginal hysterectomy. West J Surg, Obstet Gynecol 1948; 56 (07) 377-385
• 27 Benedet JL, Bender H, Jones III H, Ngan HY, Pecorelli S. ; FIGO Committee on Gynecologic Oncology. FIGO staging classifications and clinical practice guidelines in the management of gynecologic cancers. Int J Gynaecol Obstet 2000; 70 (02) 209-262 . Doi: 10.1016/S0020-7292(00)90001-8
• 28 Kamura T, Shigematsu T, Kaku T. , et al. Histopathological factors influencing pelvic lymph node metastases in two or more sites in patients with cervical carcinoma undergoing radical hysterectomy. Acta Obstet Gynecol Scand 1999; 78 (05) 452-457 . Doi: 10.1034/j.1600-0412.1999.780520.x
• 29 Gao G, Smith DI. WWOX, large common fragile site genes, and cancer. Exp Biol Med (Maywood) 2015; 240 (03) 285-295 . Doi: 10.1177/1535370214565992
• 30 Sauer G, Deissler H. Angiogenesis: prognostic and therapeutic implications in gynecologic and breast malignancies. Curr Opin Obstet Gynecol 2003; 15 (01) 45-49
• 31 Randall LM, Monk BJ, Darcy KM. , et al. Markers of angiogenesis in high-risk, early-stage cervical cancer: A Gynecologic Oncology Group study. Gynecol Oncol 2009; 112 (03) 583-589 . Doi: 10.1016/j.ygyno.2008.11.013
• 32 Wen J, Xu Z, Li J. , et al. Decreased WWOX expression promotes angiogenesis in osteosarcoma. Oncotarget 2017; 8 (37) 60917-60932 . Doi: 10.18632/oncotarget.17126
• 33 Yin CM, Yao YF, Yan ZL, Yang HY. [Correlation between mutation of p53 gene 2-4 exons from peripheral blood and HPV16 positive cervical cancer susceptibility and clinical significance]. Zhonghua Fu Chan Ke Za Zhi 2017; 52 (05) 320-326 . Doi: 10.3760/cma.j.issn.0529-567X.2017.05.006
• 34 Wang X, Lv W, Qi F. , et al. Clinical effects of p53 overexpression in squamous cell carcinoma of the sinonasal tract: A systematic meta-analysis with PRISMA guidelines. Medicine (Baltimore) 2017; 96 (12) e6424 . Doi: 10.1097/MD.0000000000006424
• 35 Advincula AP, Wang K. The evolutionary state of electrosurgery: where are we now?. Curr Opin Obstet Gynecol 2008; 20 (04) 353-358 . Doi: 10.1097/GCO.0b013e3283073ab7
• 36 McCluggage WG, Connolly LE, McGregor G, Hyland PL, Hall PA. A Strategy for defining biologically relevant levels of p53 protein expression in clinical samples with reference to endometrial neoplasia. Int J Gynecol Pathol 2005; 24 (04) 307-312 . Doi: 10.1097/01.pgp.0000167113.86586.65
• 37 Portari EA, Russomano FB, de Camargo MJ. , et al. Immunohistochemical expression of cyclin D1, p16Ink4a, p21WAF1, and Ki-67 correlates with the severity of cervical neoplasia. Int J Gynecol Pathol 2013; 32 (05) 501-508 . Doi: 10.1097/PGP.0b013e31826f5cf6
• 38 Garima PS, Pandey S, Pandey LK, Saxena AK, Patel N. The role of p53 gene in cervical carcinogenesis. J Obstet Gynaecol India 2016; 66 (Suppl. 01) 383-388 . Doi: 10.1007/s13224-015-0754-1
• 39 Xiao J, Zhou J, Fu M. , et al. Efficacy of recombinant human adenovirus-p53 combined with chemotherapy for locally advanced cervical cancer: A clinical trial. Oncol Lett 2017; 13 (05) 3676-3680 . Doi: 10.3892/ol.2017.5901
• 40 Richards RI, Choo A, Lee CS, Dayan S, O'Keefe L. WWOX, the chromosomal fragile site FRA16D spanning gene: its role in metabolism and contribution to cancer. Exp Biol Med (Maywood) 2015; 240 (03) 338-344 . Doi: 10.1177/1535370214565990
• 41 Samuel N, Wilson G, Id Said B. , et al. Transcriptome-wide characterization of the endogenous miR-34A-p53 tumor suppressor network. Oncotarget 2016; 7 (31) 49611-49622 . Doi: 10.18632/oncotarget.10417
• 42 Héninger E, Krueger TE, Lang JM. Augmenting antitumor immune responses with epigenetic modifying agents. Front Immunol 2015; 6: 29 . Doi: 10.3389/fimmu.2015.00029