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The Combined Role of EN2, CD73 and KI67 in Predicting Recurrence and Progression of Non-muscle Invasive Bladder Cancer

Yomna A. Zamzam , Aymen M. El-Saka, Ahmed M. El-Saka, Hassan El-Tatawy
Journal of Cancer Research and Treatment. 2017, 5(3), 92-99. DOI: 10.12691/jcrt-5-3-4
Published online: August 04, 2017

Abstract

Objectives: The survival rate for the most of patients with non- muscle invasive bladder cancer (NMIBC) is favorable; however, the rates of recurrence and progression to muscle invasive bladder cancer (MIBC) are the major principles of long- term outcome. There is an essential need to find a panel of markers to predict tumor recurrence and progression to be used in regular clinical practice and treat the disease appropriately. Therefore, we investigated in this study, the immunohistochemical expression of EN2, CD73, p63 and Ki-67 in NMIBC in order to evaluate the ability of this panel of markers to predict tumor recurrence and progression. Methods: In all, 122 patients diagnosed with NMI transitional cell carcinoma of the bladder in a Urology and Pathology Department from January 2014 to December 2016, were included in the study. Paraffin-embedded specimens were obtained by transurethral resection of the bladder tumors. After grading and staging of the sections stained with haematoxylin and eosin, they were evaluated using EN2, CD73 p63 and KI67 immunohistochemical staining before and after immunotherapy with bacille Calmette-Guerin (BCG), and patients were followed up for 3 years. Results: High CD73 expression is significantly associated with lower stage (p= 0.01) and lower grade (p= 0.02). There was no significant relationship between EN2, p63 and KI67 expression and tumor staging or grading. There was a significant relation between EN2, Ki67 positivity and CD73 negativity with recurrence, progression and death of the studied cases. Conclusions: Patients with TCC who are selected for BCG treatment should preferably be positively immunoreactive for CD73 but negative for both EN2 and Ki67. These patients are likely to be less susceptible to recurrence and/or progression after BCG adjuvant therapy. On the other hand different immunological profile necessitates closer follow up for early detection of tumor recurrence and progression.

1. Introduction

Despite being a potentially curable malignancy, the management of non- muscle-invasive bladder cancer is still a major problem for clinicians. This could be attributed to the association between muscle-invasive bladder cancer (MIBC stages T2+) with the high morbidity and mortality of the patients, besides the high costs of non-muscle-invasive bladder cancer (NMIBC, stages Ta/T1) observation. Furthermore, some of these superficial tumors recur and progress into MIBC 1, 2. Thus the choice of further intravesical adjuvant therapy after transurethral resection should depend on the patient’s risk of recurrence and progression 3.

More accurate markers are needed in addition to clinicopathological factors to determine the further need for postoperative follow-up programs 4. In spite of plentiful studies have evaluated the predictive value of different biomarkers regarding the progression of NMIBC 5, 6, 7, 8, none of them has a confirmed role in regular clinical practice.

The transcription factor Engrailed-2 (EN2), is expressed in both breast cancer and prostate cancer. Some studies reported that EN2 has an oncogenic role, as its expression in a non-malignant mammary epithelial line associated with increased cell proliferation, survival and invasion 9. Recently EN2 is observed as tumor specific urinary biomarker for the early diagnosis of prostate cancer and bladder cancer 10, 11.

CD73 is a glycosylphosphatidylinositol- (GPI-) anchored cell-surface enzyme that plays an essential role in the purinergic signaling pathway by dephosphorylating AMP (adenosine monophosphate) into adenosine, having enzymatic function as adhesive and signaling molecule that mediates cancer invasion and metastasis 12, 13, 14.

High CD73 expression has been recognized in many types of malignancy. Moreover, it has prognostic and diagnostic value for patients with colon cancer and papillary thyroid carcinoma respectively. The role of CD73 in BC is still debatable 15, 16.

The expression of TP63 is in normal human uroepithelial cells fades away in the process of tumorgenesis and cancer progression till complete loss in higher grades and more advanced stages. Several studies concluded that loss of p63 is associated with shorter survival in patients with bladder cancer 17, 18. On contrary, a recent study concluded that maintenance of p63 expression is correlated to adverse outcomes in MIBC patients, and maybe T1 patients 19.

Ki-67 is a nuclear protein present during different stages of the cell cycles and widely used to assess the activities of cell proliferation in various cancers by immunohistochemistry 5. Ki-67 is a preferably convenient biomarker in comparison to other molecular biomarkers, such as p53, survivin, for the proliferation status of tumor cells. Recently, many studies continuously reported that Ki-67 is an independent indicator to predict poor clinical outcomes of both no-muscle invasive and muscle invasive BC patients 7, 20. In the last few years, there is a growing interest to recognize new biomarkers to predict tumor recurrence and progression of NMIBC like EN2 and CD73 12, 15. Based on these studies we tried to find a combined panel of markers including EN2 and CD73 helping in more reliable results in prediction of recurrence and progression of NMIBC.

1.1. Aim of the Work

The aim of this work to investigate the immunohistochemical expression of EN2, CD73, p63 and Ki-67 in NMIBC in order to evaluate the ability of this panel of markers to predict tumor recurrence and progression.

2. Material and Methods: Case Selection

This prospective randomized study was conducted on 122 patients in Urology and Pathology departments, Tanta University, Egypt. During the period from January 2014 to December 2016, 122 patients with NMI bladder cancer were included. Patients with NMI transitional cell carcinoma (TCC) after a pathological examination were selected. Tumors were graded and staged according to WHO and TNM 21, 22. Patients were followed up in the Urology Department; BCG was instilled for six consecutive weeks. At 3–4 weeks after the last BCG instillation and diagnostic cystoscopy were performed, randomized cold biopsies were taken from the tumor site, and the surrounding areas. Patients were followed up every 6 months for the next 2 years. The assessment of the response depended on the results of histopathology and was considered a complete response if they were negative. A positive biopsy was considered to be tumor recurrence regardless of any stage progression. Tumor stage progression, muscle involvement was considered to be tumor progression, requiring a change in treatment regimen or a radical cystectomy 2.

This study was approved by the ethics committee of Faculty of Medicine, Tanta University and written informed consent was obtained from each participant.

2.1. Histopathological Evaluation

After transurethral resection bladder specimens were immediately fixed in 10% neutral buffered formalin for up to 12 h, then dehydrated in ascending grades of alcohol and embedded in paraffin. Patients with NMI bladder cancer (Ta, T1) were selected for the immunohistochemical staining and BCG instillation.

2.2. Immunohistochemistry Procedure

Paraffin-embedded sections were immunostained for EN2, CD73, p63, ki67. In brief, after dewaxing, inactivating endogenous peroxidase activity and blocking cross-reactivity with normal serum (Vectastain Elite Kit; Vector, Burlingame, CA, USA), the sections were incubated for 60 minutes at room temperature or overnight at 4C, with a diluted solution of the primary antibody. Location of the primary antibodies was achieved by subsequent application of a biotinylated anti-primary antibody, an avidin-biotin complex conjugated to horseradish peroxidase, and diaminobenzidine (Vectastain Elite Kit, Vector, Burlingame, CA). The slides were counterstained with hematoxylin. Negative controls were established by replacing the primary antibody with PBS and normal mouse or rabbit serum.

The antibodies used in the study are mentioned in the Table 1, including their clones, manufacturers, antigen retrieval method, buffer pH for the retrieval, positive control, incubation time, temperature and their localization in the tissue.

2.3. Assessment of Immunostaining

The evaluation was analyzed according to the intensity of staining and the percentage of stained cells. For EN2, scoring estimations were stratified into four categories, as follows (0) less than 10 % of cells stained positive for EN2; (1+) 10–25 % of cells stained positive for EN2; (2+) 25–50 % of cells stained positive for EN2; and (3+) more than 50 % of cells stained positive for EN2 10. For CD73 staining scoring was as follows: (0) <10% positively stained cells; (1+), 10-19% weakly to moderately stained cells; (2+), 10-19% intensively stained cells or 20-49% weakly stained cells; (3+), 20-49% moderately to intensely stained cells or ≥50% positively stained cells 16. For both EN2 and CD73 immunostaining, cases with scores of 2+ or 3+ were designated as “positive”, whereas cases with scores of 0 or 1+ were designated as “negative” 10, 16.

For p63 staining was considered positive if nuclear percentage between 0 (stained cells < 10%), 1+ (>10% stained cells < 80%), and 2+ (stained cells > 80-100%) was assessed. For Ki-67 immunoreactivity was considered positive when samples showed >20% nuclear reactivity. Kaplan-Meier analyses revealed that the Ki-67cutoff of 20% was the best discriminator for both bladder cancer progression and survival 2.

2.4. Statistical Analysis

Statistical presentation and analysis of the present study were conducted, using the mean, standard deviation, and chi-square test, person correlation by SPSS (version 15.0; SPSS Inc., Chicago, Illinois, USA) software. Significant differences were considered at p < 0.05.

3. Results

The current study included 122 patients whom had NMI bladder TCC, The mean age was 64.6 and the predominant sex was males (85.2%). For tumour grading, 28 cases were grade 1 (23%), 55 grade 2 (45%) and 39 grade 3 (32%). As regards staging of the included cases before BCG 55 were Ta and 39 were T1. Nine patients were lost during the follow up processing, 33 out of 113 remaining patients had recurrence(29.2%), 20 patients had a recurrence before 6 months and 13 after 6 months; 26 had progression, eight of whom died later. The clinicopathological characteristics of the included 122 cases are mentioned in Table 2.

There was no significant relationship between EN2, p63 and KI67 expression and tumor staging or grading. On other hand, high CD73 expression is significantly associated with lower stage (p = 0.01) and lower grade (p= 0.02).

There was a significant relationship between EN2 and ki67 positivity and recurrence (p=0.001), progression (p= 0.001, p=0.005 respectively). EN2 positivity and death relationship was statistically significant (p=0.006) of the studied tumors, while Ki67 positivity and death relationship was statistically insignificant.

There was a significant relation between CD73 negativity and recurrence (p <0.001), progression and death (p=0.006) of the studied tumors. On the other hand p63 negativity was statistically insignificant associated with recurrence, progression and death of the studied tumors (Figure 1 & Figure 2). The relationship between the expression of studied markers and grading, staging, recurrence, progression and death in studied cases are illustrated in table (3) and diagram (1).There was no significant relationship between EN2, p63 and KI67 expression and tumor staging or grading. On other hand, high CD73 expression is significantly associated with lower stage (p = 0.01) and lower grade (p= 0.02).

There was a significant relationship between EN2 and ki67 positivity and recurrence (p=0.001), progression (p= 0.001, p=0.005 respectively). EN2 positivity and death relationship was statistically significant (p=0.006) of the studied tumors, while Ki67 positivity and death relationship was statistically insignificant.

There was a significant relation between CD73 negativity and recurrence (p <0.001), progression and death (p=0.006) of the studied tumors. On the other hand p63 negativity was statistically insignificant associated with recurrence, progression and death of the studied tumors (Figure 1 & Figure 2). The relationship between the expression of studied markers and grading, staging, recurrence, progression and death in studied cases are illustrated in Table 3 and Diagram 1.

The sensitivity, specificity and accuracy of EN2 positivity, CD73 negativity, P63 negativity and Ki67 positivity for prognosis recurrence, progression and death were summarized in Table 4. The sensitivity of EN2 positivity, CD73 negativity, P63 negativity and Ki67 positivity for recurrence were 90.9%, 78.8%, 33.3% & 93.9%, for progression were 92.3%, 76.9%, 38.5% & 92.3 % and for death 100%, 100%, 37.5% and 100% respectively.

The specificity of EN2 positivity, CD73 negativity, P63 negativity and Ki67 positivity for recurrence were 63.8%, 57.5%, 58.8% & 40%, for progression were 59.8%, 54%, 70% & 36.8 % and for death 51.4%, 50.5%, 61% and 32.4% respectively.

The accuracy of EN2 positivity, CD73 negativity, P63 negativity and Ki67 positivity for recurrence were 71.7%, 63.7%, 51.3% & 55.8%, for progression were 67.3%, 59.3%, 55.8% & 49.7 % and for death 54.9%, 54%, 59.3% and 37.2% respectively.

4. Discussion

The survival rate for the most of patients with NMIBC is favorable; however, the rates of recurrence and progression to MIBC are important for overall prognosis, as these are major principles of long-term outcome. The recurrence and progression probability rates rely upon several clinical and pathologic factors. Therefore, there is an essential need to find panel of markers to predict tumor recurrence and progression to treat the disease appropriately 23.

In the current study, expression of panel of markers (EN2, CD73, p63 and KI67) were studied in 122 NMI bladder TCC cases. High CD73 expression is significantly associated with lower stage (p = 0.01) and lower grade (p= 0.02), This result is in agreement with the results of Wettstein et al. 16 whom their study was the first in evaluating CD73 immunoreactivity in a large cohort of primary urothelial bladder carcinomas. They found that high CD73 expression is associated with lower stage, lower grade (P= 0,006).

On the other hand, there was no significant relationship between EN2, p63 and KI67 expression and tumor staging or grading. However, Charfia et al. 6, Wang et al. 7, Morgan et al. 11 showed a significant relationship between some of these markers tumor staging and grading, this may be attributed to in they assessed both NMI and MI bladder cancer in their studies .

The current study showed a significant relation between EN2 positivity and recurrence, progression and death of the studied tumors. The sensitivity and accuracy of EN2 positivity for recurrence were 90.9% and 71.7%, for progression 92.3% and 67.3 % and for death 100% and 55% respectively.

These results were in accordance with the results of Morgan et al. 10 that they had found EN2 was expressed in recurrent NMIBC. In patients with NMIBC, urinary EN2 was detected in most cases with an overall sensitivity of 82% and specificity of 75%. This suggests that EN2 expression may be an early event in bladder tumor progression. The molecular basis of the observed de novo EN2expression and physiology of EN2 secretion by bladder cancer cells is currently being investigated 9.

Li et al. 9 indicated that EN2 may promote cell proliferation, invasion, and metastasis in BC by activating the PI3K/Akt pathway and inhibiting PTEN. Importantly, EN2 knockdown significantly decreased tumor growth of BC through regulating the cell cycle, apoptosis and ECM-related proteins. Thus, EN2 may be a candidate oncogene in BC and may be used as a therapeutic target for the treatment of BC.

In the present study, there was a significant relation between CD73 negativity and recurrence, progression and death of the studied tumors. The sensitivity and accuracy of CD73 negativity for recurrence were 78.8% and 63.7%, for progression 76.9% and 59.3 %% and for death 100% and 54% respectively.

Only a few studies have evaluated the role of CD73 in BC. Similar findings were reported by Wettstein et al. 16 who stated that high CD73 immunoreactivity in NMIBC was associated with longer PFS in univariable Cox regression analysis.

Several studies have investigated CD73 immunohistochemistry in other solid tumors. Interestingly, similar results have been reported for ovarian and breast cancers. They found associations between overexpression of CD73 and better prognosis, lower stage, and better differentiation 24, 25.

Meanwhile, other studies of CD73 in different solid tumors found contradictory results; CD73 has been reported as a disadvantageous prognostic or predictive factor, namely, in colorectal, gastric, prostate, and some forms of breast cancer 26, 27, 28.

Taken together, conflicting results have been published for the role of CD73 in solid tumors. CD73 upregulation can be affected by several different mediators and conditions such as hypoxia. Presence of hypoxia in these tumors could partially explain the discrepancies 28. Besides, different tissues have very different enzymatic activity of CD73. This as well can potentially explain differences between different cancers 16.

For p63 expression, there was insignificant association between p63 negativity and recurrence, progression and death of the studied tumors. The sensitivity and accuracy of p63 negativity for recurrence were 33.3% and 51.3%, for progression 38.5% and 55.8 %% and for death 37.5% and 59.3% respectively.

Several studies concluded that loss of p63 is associated with shorter survival in patients with bladder cancer 17, 18. In contrast, however, a recent study claimed that maintenance of p63 expression is associated with adverse outcomes in MIBC patients, and maybe T1 patients 19. The insignificant association of P63 and recurrence and progression in this study may be explained by some studies used ΔNp63 which is an isoform of p63. Gaya et al. 29 investigated the prognostic potential of p63 and ΔNp63 expression levels in 134 patients with pT1 high-grade cancer (131 of 134 patients (97.8%) that were treated with adjuvant intravesical BCG therapy). They found that ΔNp63 expression was a significant predictive factor in both univariate and multivariate analyses, whereas p63 expression was not.

In the present study, there was a significant relation between Ki-67 positivity and recurrence, progression and death of the studied tumors. The sensitivity and accuracy of Ki-67 positivity for recurrence were 93.9% and 55.8 %%, for progression 92.3% and 49.7 % and for death 100% and 37.2 % respectively. Several studies likewise reported that Ki67 expression was independently associated with both disease recurrence and cancer-specific mortality in patients with organ-confined disease 2, 5, 7. Therefore, Ki67 expression may help us to identify such patients who are at increased risk of disease progression and may benefit from adjuvant therapies.

Moreover, 33 patients had recurrences (20 before 6 months and 13 after 6 months), 26 of whom had progression and 8 of whom eventually died. These patients had enhanced immunoreactivity for EN2 and ki67 but attenuated immunoreactivity for CD73. Notably, patients who will benefit from BCG should be negative for EN2 and KI67 and positive for CD73.

In conclusion, patients with TCC who are selected for BCG treatment should preferably be positively immunoreactive for CD73 but negative for both EN2 and Ki67. These patients are likely to be less susceptible to recurrence and/or progression after BCG adjuvant therapy. On the other hand different immunological profile necessitates closer follow up for early detection of tumor recurrence and progression.

Conflicts of Interest

There is no conflict to be declared.

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Cite this article:

Normal Style
Yomna A. Zamzam, Aymen M. El-Saka, Ahmed M. El-Saka, Hassan El-Tatawy. The Combined Role of EN2, CD73 and KI67 in Predicting Recurrence and Progression of Non-muscle Invasive Bladder Cancer. Journal of Cancer Research and Treatment. Vol. 5, No. 3, 2017, pp 92-99. http://pubs.sciepub.com/jcrt/5/3/4
MLA Style
Zamzam, Yomna A., et al. "The Combined Role of EN2, CD73 and KI67 in Predicting Recurrence and Progression of Non-muscle Invasive Bladder Cancer." Journal of Cancer Research and Treatment 5.3 (2017): 92-99.
APA Style
Zamzam, Y. A. , El-Saka, A. M. , El-Saka, A. M. , & El-Tatawy, H. (2017). The Combined Role of EN2, CD73 and KI67 in Predicting Recurrence and Progression of Non-muscle Invasive Bladder Cancer. Journal of Cancer Research and Treatment, 5(3), 92-99.
Chicago Style
Zamzam, Yomna A., Aymen M. El-Saka, Ahmed M. El-Saka, and Hassan El-Tatawy. "The Combined Role of EN2, CD73 and KI67 in Predicting Recurrence and Progression of Non-muscle Invasive Bladder Cancer." Journal of Cancer Research and Treatment 5, no. 3 (2017): 92-99.
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  • Figure 1. An immunohistochemistry panel for low grade TCC (Ta) [a, EN2 positive (+3); b, CD73 negative (0); c, Ki67 positive; and d, p63 positive (+1) X 200], showed recurrence before 6 months with progression to high grade TCC (T2)
  • Figure 2. An immunohistochemistry panel for low grade TCC (T1) a, EN2 negative (+1); b, CD73 positive (+3); c, Ki67 negative; and d, p63 positive (+2) X 200], showed no recurrence or progression in follow up
  • Table 4. Sensitivity, specificity and accuracy of EN2 positivity, CD73 negativity, P63 negativity and Ki67 positivity for prognosis recurrence, progression and death
[1]  Hall MC, Chang SS, Dalbagni G, Seigne JD, Skinner EC. The Bladder Cancer Clinical Guideline Update Panel. Guideline for the management of nonmuscle invasive bladder cancer (stages Ta, T1, and Tis). J Urol 2007; 2007(178): 2314-30, Update.
In article      View Article  PubMed
 
[2]  Hegazy R, kamel M, Salem EA, Salem NA, Fawzy A, Sakr A, et al. The prognostic significance of p53, p63 and her2 expression in non-muscle-invasive bladder cancer in relation to treatment with bacille Calmette–Guerin. Arab Journal of Urolog. 2015; 13(3), 225-230.
In article      View Article  PubMed
 
[3]  Feng C, Wang L, Ding G, Ding Q, Zhou Z, Jiang H, et al. Predictive value of clinicopathological markers for the metachronous bladder cancer and prognosis of upper tract urothelial carcinoma. Sci Rep. 2014; 4: 4015.
In article      View Article  PubMed
 
[4]  Stenzl A, Hennenlotter J, Schilling D. Can we still afford bladder cancer? Curr Opin Urol 2008; 18:488-492.
In article      View Article  PubMed
 
[5]  Wang L, Feng C, Ding G, Ding Q, Zhou Z, Jiang H, et al. Ki67 and TP53 expressions predict recurrence of non-muscle invasive bladder cancer. Tumour Biol. 2014; 35: 2989-2995.
In article      View Article  PubMed
 
[6]  Charfia S, Khabir A, Mnifa H, Ellouzea S, Mhirib M, Sellamia T. Immunohistochemical expression of HER2 in urothelial bladder carcinoma and its correlation with p53 and p63 expression. J Microsc Ultrastruct. 2013; 1: 17-21.
In article      View Article
 
[7]  Wang L, Zhou M, Feng C, Gao P, Ding G, Zhou Z, et al. Prognostic value of Ki67 and p63 expressions in bladder cancer patients who underwent radical cystectomy. Int Urol Nephrol. 2016 Apr; 48(4): 495-501.
In article      View Article  PubMed
 
[8]  Charfia S, Khabir A, Mnifa H, Ellouzea S, Mhirib M, Sellamia T. Immunohistochemical expression of HER2 in urothelial bladder carcinoma and its correlation with p53 and p63 expression. J Microsc Ultrastruct. 2013; 1: 17-21.
In article      View Article
 
[9]  Li Y, Liu H, Lai C, Su Z, Heng B, Gao S. Repression of engrailed 2 inhibits the proliferation and invasion of human bladder cancer in vitro and in vivo. Oncol Rep. 2015 May; 33(5): 2319-30.
In article      View Article
 
[10]  Morgan R, Bryan RT, Javed S, Launchbury F, Zeegers MP, Cheng KK, et al. Expression of Engrailed-2 (EN2) protein in bladder cancer and its potential utility as a urinary diagnostic biomarker. Eur J Cancer. 2013 Jun; 49(9): 2214-22.
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