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% H2 O2 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.
