| Abstract|| |
Background: Transurethral resection of tumor is the main treatment of non-muscle-invasive urothelial carcinoma, but it is associated with high rate of recurrence and/or progression and this arouses the need for adjuvant therapy. Topoisomerase II (Top II), KI-67, and P53 are proliferation and cell cycle regulation markers that may predict tumor response to therapy. Aim: This study aimed to assess Top II, KI-67, and P53 expression and their effect on clinical outcome and response to therapy of non-muscle-invasive urothelial carcinoma. Materials and Methods: Fifty cases of non-muscle invasive urothelial carcinoma were collected; Top II, KI-67, and P53 expression was evaluated. Patients received treatment then tumor recurrence was correlated with the expression of previous markers. Results: There was a significant association between high Top II score, P53, and KI-67 and high tumor grade (P = 0.0001, 0.001, and 0.0001), submucosal infiltration (P = 0.0001 and 0.01), and recurrence (P = 0.01, 0.001, and 0.001). Conclusion: Top II, P53, and KI-67 may predict tumor response to therapy and the clinical outcome in non-muscle-invasive urothelial carcinoma.
Keywords: KI-67, non-muscle-invasive urothelial carcinoma, P53, recurrence, topoisomerase II
|How to cite this article:|
Elkady N, Sultan M, Elkhouly E. Evaluation of topoisomerase II, ki-67, and P53 expression in non-muscle-invasive urothelial carcinoma and their clinical significance. Indian J Pathol Microbiol 2018;61:526-31
|How to cite this URL:|
Elkady N, Sultan M, Elkhouly E. Evaluation of topoisomerase II, ki-67, and P53 expression in non-muscle-invasive urothelial carcinoma and their clinical significance. Indian J Pathol Microbiol [serial online] 2018 [cited 2019 Mar 21];61:526-31. Available from: http://www.ijpmonline.org/text.asp?2018/61/4/526/242996
| Introduction|| |
One of the major causes of morbidity and mortality in the United States is bladder cancer where 75,000 new cases and 16,000 deaths occur yearly, most of these tumors are urothelial carcinoma.
In Egypt, it accounts for 10.1% of all tumors according to the last National Cancer Institute Registry 2003–2004. Salem and Mahfouz, 2012, reported 30,000 new cases annually. About 30.83% of bladder cancer occurred between 61 and 70 year-old with a median age of 60. The male-to-female ratio is 3.5:1.
About 60% of bladder cancer present as superficial (PTa or PTl) urothelial carcinomas, where tumors are confined to the epithelium or lamina propria.
The initial treatment of superficial tumors is transurethral resection of the bladder tumor (TURBT). Unfortunately, TURBT may be followed by tumor recurrence in 40%–80% and by tumor progression with the invasion of muscularis propria in 20%–30% of cases. The risk of tumor recurrence or progression after transurethral resection of tumor (TURT) is affected by many factors; some of them are tumor size, its grade, the depth of tumor invasion, presence of multiple tumor foci, and presence of carcinoma in situ (CIS). That is why, additional therapy (either chemotherapy or immunotherapy) was used after TURT according to the level of tumor risk.
The most commonly used drugs, which are effective intravesically, are doxorubicin, epirubicin, mitomycin, mitoxantrone, thiotepa, and Bacillus Calmette–Guerin (BCG). All drugs have shown a beneficial effect, and there is no best efficient one. The risk needed to be decreased is specified first (either progression and or recurrence) to choose between immunotherapy and chemotherapy. Recurrence risk only is reduced by chemotherapy.
Doxorubicin was the first chemotherapeutic drug to be used as an intravesical agent. It has been used for more than 20 years as a comparative agent in treating superficial bladder cancer.
BCG instillation as well is used as intravesical adjuvant therapy. After its use, the recurrence rates have decreased by 20%–30%. However, the side effects are still prominent including granulomatous and ulcerative cystitis, hepatitis, and lung infections. In case of persistent disease following BCG therapy, cystectomy can be done or chemotherapeutic agents such as mitomycin C, thiotepa, doxorubicin, epirubicin, and valrubicin can be used intravesically. However, the use of these drugs is limited due to toxicity that follows systemic absorption.
Although tumor stage and grade are crucial for stratifying patients for treatment, more accurate prediction of tumor behavior, especially of superficial bladder cancers, is needed to choose the optimal treatment of individual cases.
Excess unregulated proliferation and ability for invasion and metastasis are two biological features of cancer cells that affect the biological behavior of human malignancies. In addition, tumor response to chemotherapy depends on tumor growth fraction and apoptosis because several anticancer drugs cause cell death by apoptosis.
DNA topoisomerase II (Top II) is an important enzyme in cell cycle; it plays an essential role in new chromosome segregation after replication, condensation of chromosomes, and DNA superhelicity alteration. It exists in two isoforms, α and β; Top IIα is a key enzyme in DNA metabolism and replication. Top IIβ may cause drug resistance through different mechanisms.
Top IIα binds to DNA creating a breakthrough which another DNA can pass, this is followed by religation of the cleaved DNA strand that finally leads to reduction of DNA twisting and supercoiling. This helps untangling of DNA which allows DNA to engage in transcription, replication, or repair processes.
Many cytotoxic drugs target Top IIα; the most important are doxorubicin, mitoxantrone, and etoposide. These drugs induce apoptosis through binding to Top IIα –DNA complexes inhibiting DNA religation which leads to stabilization of the breaks. The accumulation of double-stranded DNA breaks is lethal to the cell causing apoptosis. The level of Top II determines tumor sensitivity Top IIα inhibitors. High proliferation is associated with high concentrations of Top IIα and more sensitivity to Top II poisons. Therefore, Top IIα assessment may be of value for the choice of adjuvant therapy in superficial bladder cancer.
The research that investigated the prognostic and predictive significance of Top IIα showed controversial results. Some revealed that high rate of recurrence, progression, and worse prognosis in non-muscle-invasive bladder cancer was associated with Top IIα protein overexpression or gene amplification. Others showed that Top IIα overexpression or amplification predicts better response to anthracycline-based chemotherapy with better prognosis.
KI-67 is one of the proliferation markers. Its level is high in tumors with rapidly dividing cells and high growth fraction. Tumors with high proliferation rate have a better response to chemotherapy, so detection of KI-67 level can predict the response to chemotherapy and further liability for recurrence.
P53 is the guardian of the genome; it acts as a tumor suppressor gene that sensitizes the cells to growth inhibitory or apoptotic-inducing signals, so cells with abnormal P53 can escape growth inhibitory and apoptotic stimuli, so they have unrestrained proliferation potential and progressive genomic instability., P53 is one of the prognostic and predictive factors of urothelial carcinoma.
This study aimed at evaluation of the predictive role of Top II, KI-67, and P53 expression in non-muscleinvasive urothelial bladder carcinoma.
| Materials and Methods|| |
A selected fifty cases of non-muscle-invasive urothelial carcinoma collected from Menoufia University Hospital during the period between January 2013 and September 2015. The clinical data of the patients were collected from the hospital records including age and sex.
These cases underwent TURT. All patients were stratified according to tumor risk level. Low-risk patients received immediate dose of doxorubicin, it was followed by intravesical instillation of BCG in high-risk patients. Cystoscopic follow-up was done 3 months later. We excluded patients with coincident CIS from the study.
Hematoxylin- and eosin-stained sections were obtained from TURT specimens for evaluation of the histopathological features of tumors such as tumor type, grading, depth of invasion, mitotic count and tumor recurrence.
Four paraffin sections from each case, 4 mm in thickness, were stained by the immunohistochemical method. Streptavidin-biotin amplified system was used. Three primary antibodies were used; a purified mouse monoclonal antibody against Top IIα (Ab-4), (Cat#Ms-1819) (7 ml ready to use), a purified rabbit monoclonal antibody against KI-67, clone (Sp6), (Cat# RM 9106s), (7 ml ready to use), and a purified mouse monoclonal antibody against P 53, clone (PAb 2040), (Cat# Ms-104-P0), 100 μL, Thermo Scientific Lab Vision Corporation, Fremont, USA. The detection kit was formed of ready-to-use antipolyvalent antibody and HRP/DAB (Lab-vision).
Slides were submitted for subsequent steps of deparaffinization and rehydration. Boiling the slides in citrate buffer (6 pH) was done for antigen retrieval, followed by cooling to room temperature. Adding the primary antibodies and incubation overnight at room temperature was done followed by secondary antibody, DAB as a chromogen substrate, then Mayer's hematoxylin as a counterstain.
Top II expression was evaluated in urothelial carcinoma cells. Positive Top II expression was considered when nuclear staining was detected in any number of cells.
The semi-quantitative immunoreactive score (IRS) was used for evaluation of Top II expression. In each case, the intensity of staining and percentage of positive cases were evaluated at high power (×400). Staining intensity was evaluated as follows: negative = 0, low = 1; moderate = 2, and strong = 3. The percentages of stained cells were classified as follows: negative = 0%; 1 <10%; 2 = 10%–50%; 3 = 51%–80%; and 4, >80%. If IRS score ranged from 0 to 2, it was considered negative, and if it ranged between 2 and 3, it was considered mild, whereas moderate IRS was considered when score ranged between 4 and 6 and strong when IRS ranged between 7 and 12.
For KI-67 and P53, the regions with high immune-reactive areas were chosen followed by counting 1000 cells at high power (×400). The index used for evaluation stratified their expression into low and high expression according to the percentage of positive nuclei. The cutoff point for KI-67 was 13% and 6% for P53%.
Statistical analysis of tabulated data was done using SPSS program version 11, IBM incorporation, USA. Chi-square and Fisher's exact tests were used in comparison between qualitative variables. P < 0.05 was considered statistically significant.
| Results|| |
The age of the patients presented with superficial urothelial carcinoma ranged between 49 and 70 years, with a mean age ± standard deviation (SD) of 60.4 ± 4.73 years and a median of 60 years. Urothelial carcinoma predominately affects males (62% of cases) more than females (38% of cases).
Twenty-four of the studied cases (48%) were high-grade tumors, 32 cases (64%) were Ta, and 18 cases (36%) were T1. Mitotic count ranged from 4 to 18 with mean ± SD of 10.6 ± 4.6. Recurrence was experienced in 19 (38%) cases. Recurrence was significantly associated with high-grade tumors (P = 0.01), tumors with deeper invasion (P = 0.05), and those with high mitotic count (P = 0.001) [Table 1].
|Table 1: Relationship between recurrence and clinicopathological parameters|
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As regard immunostaining results, Top II expression was found in all cases where 20 (40%) of the studied cases showed mild expression [Figure 1]a, 21 (42%) cases showed moderate expression, and 9 (18%) cases showed high expression [Figure 1]b. P53 expression was positive in 47 (94%) cases, 29 (58%) of them showed low expression, while the other 18 (36%) cases showed high expression [Figure 1]d. Twenty-six cases (52%) revealed low KI-67 expression and 24 cases (48%) showed high expression [Figure 1]c.
|Figure 1: (a) Low nuclear expression of topoisomerase II in low-grade papillary urothelial carcinoma (IHC, ×100). (b) High-grade urothelial carcinoma (T1) showing strong nuclear expression of topoisomerase II in 90% of tumor cell nuclei (IHC, ×100). (c) High proliferation rate in high-grade urothelial carcinoma indicated by high KI-67 expression in tumor cells (IHC, ×200). (d) P53 is highly expressed in nuclei of tumor cells in case of high-grade urothelial carcinoma (IHC ×200)|
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There was a significant association between high score of Top II expression and high tumor grade (P = 0.0001), submucosal infiltration (P = 0.0001), and tumor recurrence (P = 0.01) [Table 2]. High expression of P53 was significantly associated with high tumor grade (P = 0.001) and tumor recurrence (P = 0.001). While no significant association was found between P53 expression and depth of tumor invasion (P = 0.32) [Table 2]. High-tumor grade, submucosal infiltration, and tumor recurrence were significantly associated with high KI-67 score (P = 0.0001, 0.01, and 0.001) [Table 2].
|Table 2: Relationship between topoisomerase II, P53, and KI 67 expression and the most important clinicopathological prognostic parameters|
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There was a positive correlation between H score of Top II expression and that of P53 and it was statistically significant [Table 3].
|Table 3: Correlation between H score of topoisomerase II expression and P53 expression|
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| Discussion|| |
This study included fifty patients with superficial urothelial carcinomas (transitional cell carcinomas) of the urinary bladder. Slides from included specimens were H and E stained for the evaluation of pathological findings then immunohistochemically stained for evaluation of Top II, KI-67, and P53 followed by investigation of the relationship between the findings and various clinicopathological parameters.
The results showed that urothelial carcinoma is more in old age, with mean ± SD of 60.4 ± 4.73 and it showed male prominence (62% of cases) which is matched to results of Chaux et al., 2012.
Thirty-eight percent of the cases experienced recurrence after receiving treatment. Recurrence was associated with high-grade tumors and deeper invasion. These results were the same like Feng et al., 2014, who concluded that tumor differentiation and extent of invasion are important factors that determine tumor liability for recurrence.
These findings can be explained by the fact that tumor progression is a multistep process as by time the tumor cells experience accumulation of multiple genetic abnormalities that make the tumor develop new subclones having more malignant potential and aggressive features such as rapid proliferation, ability to invade and metastasize, and resistance to therapy with subsequent recurrence. High proliferation is due to acquiring self-sufficiency of growth with resistance to growth inhibiting signals, while invasion can be achieved through the acquired ability of cells to detach from each other, degrade extracellular matrix (ECM), and to move to another site.
Recurrence may be explained by genetic evolution and selection where the subclones of tumor cells that have genetic alteration allowing them to resist drugs can survive and regrow the tumor after therapy and this is also a part of tumor progression.
DNA topoisomerases are enzymes which are important for DNA replication, transcription, translation, and DNA recombination through changing DNA shape without interfering with its structural units. They act also as coenzymes for other enzymes such as DNA and RNA polymerases through relaxing the double helix.
As regard immunostaining results, Top II expression was found in all cases. There was a significant association between high score of Top II expression and high tumor grade (P = 0.0001) and submucosal infiltration (P = 0.0001). These results are in concordance with Nakopoulou et al., 2001, who found a significant association between Top II expression and high-grade tumors. He also stated that Top II is expressed in aggressive tumors proved by Top II expression in muscle-invasive urothelial carcinoma.
Due to its essential role in DNA replication and transcription, Top II can be used as a marker of proliferation. As mentioned before, during tumor progression, the cells acquire many features that make them more aggressive such as high proliferation and invasion of ECM. As high-grade tumors are associated with high proliferation rate, so Top II will be high in these tumors, and as long as high-grade tumors usually acquire more aggressive features that enable them to invade as a part of tumor progression as mentioned before. The same occurs in urothelial carcinoma where cellular proliferation and Top II expression increases with progression of tumor from superficial to muscle-invasive one.
This study has shown a significant association between tumor recurrence and high Top II expression (P = 0.01). This finding was the same like Nakopoulou et al., 2001.
Mutation in Top II is one of the mechanisms mediating atypical multidrug resistance that leads to tumor recurrence. These mutations are divided into two basic types: the first mutations lead to low levels of Top II enzyme which leads to reducing the activity of anti-Top II drug because these drugs create a permanent break in DNA double-strand through stabilization of Top II-DNA cleavage complex that finally derives cell death. The second mutation makes Top II less sensitive to the chemotherapy drugs due to qualitative changes.
P53 expression was positive in 47 cases (94%), 29 of them (58%) showed low expression, while the other 18 (36%) showed high expression. In this study, high expression of P53 was significantly associated with high-grade tumors (P = 0.001) and tumor recurrence (P = 0.001). No statistically significant association was detected as regards depth of tumor invasion (P = 0.32). Quintero et al., 2006, found a significant association between high P53 expression and tumor recurrence, but there was no association with grade or stage. Menéndez López et al., 2000, found a significant association between high P53 and tumor recurrence.
Twenty-six cases (52%) revealed low KI-67 expression and 24 revealed (48%) high expression. In this study, high tumor grade, submucosal infiltration, and tumor recurrence were significantly associated with high KI-67 score (P = 0.0001, 0.01, and 0.001), and these results are the same like Quintero et al., 2006.
One of the criteria of tumor progression is the insensitivity to growth inhibitory signals that allow excess proliferation of tumor cells and increase in tumor size. P53 is a tumor suppressor gene, any abnormality affects this gene will cause dysregulation of the cell cycle, and tumor cell will enter the cell cycle and continue proliferation without control.
P53 is also responsible for the activation of apoptosis pathway in case of the presence of irreversible cell damage. Most antineoplastic drugs act by inducing irreversible DNA damage with further cell death. In case of abnormal P53, the cells can resist treatment with subsequent recurrence.
This research showed a significant positive correlation between H score of Top II expression and Score of P53 (P = 0.0001). Wild-type (w-t) p53 negatively regulates the promoter for Top II. Top II mRNA decreased after the expression of w-t p53, but not mutant p53, suggesting that, after exposure to cytotoxic stresses, progression through the cell cycle may be regulated by the repression of Top II mRNA synthesis due to upregulation of p53 activity. Alterations of p53 (mutations) might result in increased transcription of Top IIα mRNA followed by elevation of protein concentrations that stimulates cell growth.
From all previous results, Top II can be a target for effective therapy (combined with other anticancer agents) to treat cancer and prevent recurrence. Anti-Top II is a cytotoxic drug that binds DNA-Top II complex stabilizing it and producing permanent break that induces cellular apoptosis. Despite their antineoplastic activity, they have unacceptable hematologic (myelosuppression) and non-hematologic (hemorrhagic cystitis) toxicity. New drugs were developed with modifying the structure to improve its antineoplastic effect and decrease the side effects.
There are a lot of Top II inhibitors that have been used in clinical practice such as in the treatment of lung cancer, laryngeal carcinoma, Kaposi sarcoma, and lymphocytic leukemia. Doxorubicin and camptothecin are the most widely used drugs; they can induce Top I-mediated DNA damage, which in turn induces apoptosis.
| Conclusion|| |
This study showed that Top II, P53, and KI-67 expression was significantly associated with higher chance of tumor recurrence. These findings declare the prognostic and predictive role of these markers, especially Top II, so investigating the level of Top II expression in superficial urothelial carcinoma may help us to use the optimal therapeutic strategies aiming at improving the prognosis and preventing tumor recurrence.
All gratitude is directed to all our colleagues and laboratory technicians who helped us in this work.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Department of Pathology, Faculty of Medicine, Menoufia University, Shibin El-Kom
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3]