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International Journal of PharmTech Research CODEN (USA): IJPRIF, ISSN: 0974-4304, ISSN(Online): 2455-9563 Vol.9, No.10, pp 299-308, 2016
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Occurrence of RB- tumor suppressor gene overexpression in woman suffering with cervical carcinomain mid Euphrates
Elhamjwad kadhem1, Azhar Emran Althahab2
1College of education of Girl,University of Kufa, Kufa, Iraq
2College of science, Department of biology, University of Babylon, Hilla, Iraq
Abstract : The retrospective research was designed as immunohistochemical study to demonstrate the expression state of tumor suppressor genes (RB) in (56) formalin-fixed, paraffin embedded cervical tissues were included: 27 with malignant cervical carcinoma (CC) and 16 with benign cervical tumors, 13 apparently healthy cervical tissues were used as a control group. The age of these individuals (patients and control groups) ranged between 23 and 73 years. They were collected from the pathological archives of teaching laboratories at many different private histopathological laboratories in Babylon, AL-Najaf, Kerbela, AL-Qadisiya, during the period from April 2015 to January 2016. After sectioning of these cervical biopsies and staining by hematoxyline and eosin, a final definitive histopathological diagnosis was done by a consultant histopathologist. The obtained results are summarized as follows: The mean age of the patients with cervical carcinoma (54 + 11.22years) was higher than the mean age of the benign group (38.8+9.85 years) and the mean age of those females in the group of healthy control (35.5+8.64years). There are significant differences ( p< 0.05) between different groups according to age. The well differentiated grade of CC constituted 52%, whereas those with moderately and poorly differentiated grades CC constituted 37%and 11% respectively. Over expression of RB - TSG was detected by IHC in 59.3 % of cervical cancer cases and in 43.8% of benign cervical tumor group, while none of control group showed RB- over expression. A high percentage 43.6% showed of cervical cancer have a moderate score (score II). Flowed by 37.5% and 18.8% were found to have low score (score I) and strong score (score III), respectively. While, in benign cervical tumor group, the highest percentage 57.1% were found to have moderate score (score II) and 28.6%, 14.3% have strong score (score III) and low score (score I) respectively.
The highest percentage of RB protein expression it was found within poorly differentiated that have 100% (3 out of 3 cases). Followed by 60% (6 out of 10cases), 57.1 %( 8 out of 14 cases) within moderately, well differentiated respectively.
Keywords: RB- tumor, gene overexpression, cervical carcinomain, mid Euphrates.
Introduction:
The retinoblastoma (Rb) gene mapped on chromosome 13q14.2, encodes a 110 kDa nuclear phosphoprotein, and regarded as important regulator of the cell cycle at G1-S transition, that is at the convergence of several positive and negative regulatory pathways and are often referred to collectively as the Rb pathway1. Disruption of the Rb pathway function through direct Rb mutation or mutation of upstream regulator of Rb, such as p16INK4a, is thought to occur in the vast majority of human cancer including cervical cancer2,3.
The cancer is a second greatest common disease in females worldwide, it is the main cancer of women in most developing countries, wherever 80% of cases occur4.
The risk of cervical cancer is more in women aging 40 years of age or older and those who smoke, take contraceptive drugs for more than 5 years, have the history of multiple sex partners or immunosuppression5. However, infection by certain types of human Papilloma virus (HPV) has been considered as the most significant risk factor for the development of cervical cancer6,7.
Objective: To determine the frequency of RB-overexpression in uterine cervical biopsies with or without dysplasia. Therefore, RB-Immunohistochemistry (IHC) provides valuable additional information in the interpretation of cervical histology with resultant improvement in definitive identification of dysplastic lesions and reduction of inter-observer disagreement in conventional histology.
Methods:
Among fifty six (56) formalin-fixed, paraffin-embedded uterine cervical tissue blocks from patients who had undergone hysterectomy or punch biopsy from the cervix , (43) malignant and benign blocks were collected from the archives of histopathology laboratories of different general hospital in mid - Euphrates as well as many private laboratories. With thirteen cervical tissue blocks without any significant pathological changes have been obtained from patients sustained hysterectomies for uterine bleeding and were included as apparently healthy control group. Sections of 4-μm thickness were cut from the paraffin blocks for hematoxylin-eosin staining and a detailed histopathological classification was assigned according to the criteria of the WHO (2011)8.
Immunohistochemical staining was performed using the primary mouse monoclonal antibodies against RB-TSG, dilution 1:40 (clone E6H4, dakocytomation), immunostaining for RB-TSG: After routine de paraffinization in xylene and rehydration through serial dilutions of alcohol the sections were subjected to heat-mediated antigen retrieval for 15 minutes in citrate buffer (pH 6.0). To minimize nonspecific binding, blocking was performed with1% BSA at RT for 30 minutes. The primary antibodies were applied overnight at 4 °C followed by envision visualization mouse system (dakocytomation). 3,3-diaminobenzidine (DAB) was used as the chromogen for 5 minutes and haematoxylin, as a counterstain. Stained sections were dehydrated and mounted in xylene. The percentage of immunopositive cells was evaluated. The immunoreactivity for all cell cycle regulatory proteins investigated in this study was evaluated as strong, moderately and weak according to the values of median. Statistical analysis was performed by SPSS statistical software package version 20, using the x2 test t- test and ANOVA for comparing and finding any relations between HPV positivity and other variations. Statistical significance was assumed at the P<0.05 level.
Results:
The archival specimen collected in this study was related to cervical tumor patients whom ages were ranged from twenty three years to seventy three years. In malignant cervical tumors, the mean age was constituted (54 + 11.22 years). While in benign cervical tumor and healthy control groups were(38.8 + 9.85 years) control (35.5+ 8.64 years, significant differences (p< 0.05)revealed between different groups according to age (table1).
Table (1): Distribution of cervical tumor patients according to their age
Maximum |
Minimum |
S.E |
S.D |
Mean age |
N |
The Patients
|
73 |
23 |
2.1 |
11.22 |
54 |
27 |
Malignant cervical tumors |
53 |
25 |
2.4 |
9.85 |
38.8 |
16 |
Benign cervicaltumors |
48 |
23 |
2.3 |
8.64 |
35.5 |
13 |
Healthy cervical tissues control |
( P <0.05) |
Statistical analysis |
the results of present study show that well differentiated grade cervical carcinomas constituted 52% (14 of total 27 cases) , whereas cases with moderately and poorly differentiated grades constituted 37% (10 out of 27 cases) and 11% (3 out of 27 cases) respectively. Fig (1)
Fig (1) :Tumor grading of cervical carcinoma groups
The statistical analysis of grading distribution of cervical carcinoma shows significant differences (p<0.05) between well and poorly differentiated grade, while non-significant difference was noticed between well and moderately differentiated cervical carcinomas . Over expression of mutated RB- protein was detected as a brownish discoloration at nuclear localization (Figure 2). Over expression of RB-TSG was detected in 59.3% (16 out of 27) cells with cervical cancers and in 43.8% (7 out of 16) cases with benign cervical tumor, while non-cases of control group showed RB- over expression. A highest percentage 57.1 % (4 out of 7 cases), 43.6 %( 7 out of 16 case) was involving cases with benign, malignant cervical tumor that have moderately score (score II) while none of control group showed positive signal, significant differences( p<0.05).
Were found on comparing the results (according to score) between cervical cancers, benign tumors and control group, (table 2)
Table (2): Distribution of immunohistochemistry results for Rb protein according to signal scoring
Chi-square &p value |
Healthy cervical tissues (n=13) |
Benign cervical tumors (n=16) |
Malignant cervical cancers (n=27) |
Rb over expression |
||||
1.3* Sig. |
% |
N |
% |
N |
% |
N |
||
100 |
13/13 |
56.3 |
9/16 |
40.7 |
11/27 |
Negative |
||
0.0 |
0/13 |
43.7 |
7/16 |
59.3 |
16/27 |
Positive |
||
0.0 |
0/0 |
14.3 |
1/7 |
37.5 |
6/16 |
I |
Scoring |
|
0.0 |
0/0 |
57.1 |
4/7 |
43.6 |
7/16 |
II |
||
0.0 |
0/0 |
28.6 |
2/7 |
18.8 |
3/16 |
III |
Among (27) malignant cervical tumors, 16 cases (59.3%) showed positive RB –IHC reactions, a percentage of 56.3% (9 cases out of 16 ) was found to have moderate signal intensity while each weak and strong signals intensities constituted 25% (4 out of 16),18.8% (3 out of 16) respectively. In benign cervical tumor 43.7% (7 out of 16 cases) showed positive RB –IHC reactions, were 42.9% (3 cases out of 7) has strong signals intensity, and 28.6% (2 out of 7) showed in both weak and moderately differentiated. None of healthy cervical tissues showed RB—IHC reaction, significant differences (p <0.05) were found on comparing studded group of cervical tumors with the control group table (3)
Table (3): Frequency distribution of immunohistochemistry results for RB protein according to signal intensity
Chi-square &p value |
Healthy cervical tissues (n=13) |
Benign cervical tumors (n=16) |
Malignant cervical cancers (n=27) |
Tissue Rb intensity |
||||
1.3*Sig |
% |
N |
% |
N |
% |
N |
||
100 |
13/13 |
56.3 |
9/16 |
40.7 |
11/27 |
Negative |
||
0.0 |
0 |
43.7 |
7/16 |
59.3 |
16/27 |
Positive |
||
0.0 |
0 |
28.6 |
2/7 |
25 |
4/16 |
Weak |
Intensity |
|
0.0 |
0 |
28.6 |
2/7 |
56.3 |
9/16 |
Moderate |
||
0.0 |
0 |
42.9 |
3/7 |
18.8 |
3/16 |
High |
A
B
C
D
Fig. (2): Cervical carcinoma showing the results of immunohistochemical staining of Rb protein expression usingBiotinylated -Labeled anti-Rb protein antibody, stained by DAB-Chromogen (Brown) and counter stained by mayer's hematoxyline (Blue). A. cervical cancer with negative Rb –ICH reactions (40X).B .positive Rb –ICH reaction with strong score and high signal intensity (40X).C .positive Rb –ICH reaction with moderate score and high signal intensity (40X). D. Positive Rb –ICH reaction with low score and high signal intensity (40X).
Our result revealed, the highest percentage of positive RB-IHC reactions it was found within poorly differentiated that have 100% (3 out of 3 cases). Followed by 60% (6 out of 10 cases), 57.1 %( 8 out of 14 cases) within moderately, well differentiated respectively. The overall Rb –IHC scoring according to tumor grading showed significant (p<0.05) differences table (4).
Table (4): Relationship of RB-IHC scoring with grading of cervical carcinoma
Rb signal |
Tumor grade |
Chi-square & p value |
||||
Well differentiated n=(14) |
Moderately differentiated N= 10 |
Poorly differentiated N=(3) |
||||
Rb signal |
Positive |
Count |
8 |
6 |
3 |
1.5*Sig.
|
% within tumor grade |
57.1% |
60% |
100% |
|||
negative |
Count |
6 |
4 |
0 |
||
% within tumor grade |
42.9% |
40% |
0.0% |
|||
Total |
Count |
14 |
10 |
3 |
||
% within tumor grade |
100% |
100% |
100% |
Discussion:
Invasive cancer of the uterine cervix is one of the major killer's cancers that affect women across the world, also its lines as the most significant of all the 10 greatest common female neoplasms in developing countries, whereas it defenses as the fifth in western developed nations, however the disease continues to be the second most common cancer in women world-wide, The tragedy behind the thousands of deaths occurring each year from the disease is the lost opportunity of them being prevented10.
Distribution of cervical cancer patients according to their age
The experimental management pattern of cervical cancer will help us to recognize the appropriate age for cervical cancer screening11.
On reviewing the 56 cases which were included in this study, it was found the age of the patients with cervical tumors was ranging between 23-73 years with mean age was (48.5+ 12.9 years).Among cervical cancer, benign tumor cases the mean age (54± 11.22),(38.8± 9.85) respectively. While the mean age among control group was (35.5 ± 8.64) (Table 1).These results are consistent with those reported world-wide where these cervical tumors usually affecting females over forty years of age12.
Also, on reviewing (table 1), it was noticed that the percentages of CC cases are increased with the proceeding of age of patients. The present results could have their importance when realizing that the age of the cervical cancer patients is an important factor both for the occurrence and management of the disease13.
However, the result relatively in agreement with other studying In Indonesia where showed that the highest incidence was in the age group of 55 - 64 years ,were it was reported that malignant tumors have increased with the proceeding of age of patients14.
Likewise, the mean age in our study compatible to the mean age found by Missaoui et al (2010) 15 in Tunisia , Badaret al (2007) 16 At Muslim – community, were the mean age of these studies 52.1 and 49.2 years old respectively. Moreover, Sando et al.(2014)17 reported the mean age was (52.73 ± 3.82 years), Engbang et al.(2015) 18 in his research revealed the mean age was (51.22 ± 11.93 years old), and Elmajjawi et al. Morocco, in his series of 696 cases that had an average age of 50 years19.
Also, the result found that cervical cancer was higher at elderly ages. This might be caused by deficiencies in screening programs that has been very recently used in Iraq These findings of high percentage of cervical cancer with the increasing of the patients age could be associated by many other factors which enhance the appearance of malignant cervical tumor in young age group through the proceeding of age such as genetic predisposition, hormonal factors and changes in life style which characterized by a highly caloric diet-rich in fat (11). Aging is also a risk factor for persistent infection. The rate of persistent high-risk infection for women older than age 55 is 50%, while the persistent rate of women younger than 25 years old is 20% , Cervical cancer is extremely rare in women younger than age 2020.
However, in contrast to our result many other studies at developed countries showed the increase in cervical cancer incidence at younger age21,22. England and Wales reported a decrease cervical cancer prevalence in the age group of 45 - 64 years and 55 - 64 years and a significantly increase in younger age groups, 35 - 39 years and 40- 44 years. Moreover, Switzerland another country with successful screening programs, especially at 20 - 44 years old, reported a decrease in cervical cancer occurrence in 30 - 64 years old group. So the incidence of cervical cancer at younger age may be contributed by early exposure of sexual activity, early menarche, multiplexes partner, sexual promiscuity, HPV infection, a high incidence of sexual transmitted disease, smoking, and oral contraception23,24.
Furthermore, other factor that donates the earlier finding is screening programs in developed countries which have been carried out sufficiently, allowing early detection of cervical cancer at early stage25. On over view the dissimilarity in the mean age and the highest age group prevalence of cervical cancer patients among our study and studies in countries with sufficient screening programs revealed that the screening programs in Iraq have not been conducted sufficiently. The presence of cervical cancer diagnosed in post-menopausal age indicated that cervical cancer screening should not be stopped in menopausal age. So a report that women who
are screened every three years before 50 years, did not develop cervical cancer, but women who did not undergo regular screening, may still develop cervical cancer after the age of 5026.
Histopathological grading of cervical carcinoma
This study has depended on that popular grading system to fix or document the histopathology grading of our series of cervical cancers. Grading of the presented malignant cases were assessed according to the WHO grading system of cervical carcinoma , revealing that grade I(well differentiated grade) was reported in 14 (52%) cases, grade II(moderately differentiated grade) in 10 (37%), while those of grade III (poorly differentiated grade) were 3 (11%) cases (Figure 1).
Likewise Abdul-Hussein A., (2011)27 reported similar percentages in the Iraqi patients were 67. 44% found in grade I, 25.58% in grade II and 6.98% in grade III respectively, this may be due to early detection of cervical neoplasm change. However, Gichangiet al., (2002)28 of their studied CC grading revealed 13% as well-differentiated, 28% as moderately-well differentiated and 48% of the histological subtypes were reported as poorly differentiated this result are incompatible to our results. Also our study dissimilar to the result finding byKlaeset al. (2002)29, Bergeron et al. (2010)30, Galganoet al. (2010)31 Which reported the poorly differentiated constituted 72%,76.6%,75% respectively.
Viladiuet al. (1977) 32 identified clinical grad as the only independent prognostic factor by contrast advanced grad was identified as a prognostic factor in our study. The survival rate of advanced grad patients is also less. The prognostic models of age and grad are powerful in predicting death and relapse. Longest survival was for patients with early stage disease, younger patients and after primary surgery. We found International Federation of Gynecology and Obstetrics (FIGO) stage, grade and lymph node metastases of significant prognostic value for survival in cervical adenocarcinoma33.
The evaluation of RB-tumor suppressor genes among malignant and benign cervical tumors:
The definition of immunhistochemical staining an altered Rb expression differs greatly between studies. Various investigators considered that all nuclei in tumor cells must be negative to classify a tumor as having an altered Rb expression20. While, others regarded tumors with areas of positively and negatively stained tumor cell nuclei as having an altered Rb expression34. Deletion and Mutations of the RB gene are established in retinoblastoma moreover, loss of RB function has been described in a variety of human malignancies The tumor suppressor gene retinoblastoma is functionally deactivated in a large fraction of human cancers35-37.
In the present study, Rb protein was detected by IHC test in 59.3% of malignant cervical tumors, 43.8% of benign cervical tumors but no signal was reported in the tissues of control group, our result showed overexpression of pRb in majority of malignant and benign cervical tumors as compared with normal cervical squamous epithelium. Statistical analysis showed significant differences in pRb expression between normal and cervical tumor (Table 2).
In a study carried by Wang and Lu (2004)43, found that the loss of nuclear Rb expression in small cell carcinoma is not evident may suggest that Rb protein is deregulate through two different mechanisms in HPV related tumors. Rb deregulation is achieved through accelerated protein degradation as evidenced by a negative nuclear staining or Rb is inactivated by the HPV E7 oncoprotein at the functional level and thus the nuclear Rb protein detectable immunohistochemically. In present study, the majority of the Rb positive cases showed a heterogeneous staining pattern. The intensity of the nuclear staining varied from cells to cells with variable staining proportion of cells having an unstained nucleus. Similar finding have been observed and reported by
Zur Hausen, (2002)44. This variation in staining probably resulted from asynchronous progression of the cells through the cell cycle39.
Moreover, the result revealed significant association could be detected with different grades of malignant tumors. These results are in contrary with other studies such as Liu et al., 199445 and Lie et al, 201046 who found that no- significant relation between the alteration of RB-gene and grading of ovarian cancers. Moreover, The E6and E7 its two early HPV genes, play essential role in tumor formation. There is integration of viral DNA into host DNA which is the serious step in cervical carcinogenesis. As E6 and E7 are the viral oncoproteins, uncontrolled expression of these proteins are seen when there is inactivation of host tumor suppressor genes (p53 & pRb). This will result in loss of normal maturation sequence, causing persistent, proliferative HPV infection. This will further result in transformation to high grade dysplasia and progresses to invasive carcinoma47,48.
Conclusion:
We establish that highest age – specific frequency was noticed in elderly aged- patients. Alsoa majority cervical carcinomas are pronounced by strong expression RB-TSG proteins, although in need of corroboration on a larger pool of cases, show that Rb-TSG may represent promising tool toward the identification of patients with poorer prognosis who may benefit from more aggressive therapy and HPV screening.
References:
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