Introduction

Urothelial neoplasm (UN) is the 9th most common cancer and one of the important leading causes of cancer-related death worldwide (1, 2). It is identified as the fourth and eleventh most common type of malignancy in males and females, respectively (3). UC risk factors are well known (4). At one end of the range, there are low-grade tumors which have a low developing rate and need preliminary endoscopic treatment and observation but rarely present a warning to the patient. At the other extreme, there are high-grade tumors which have a high malignancy potential associated with considerable development and high cancer mortality rates (5). Also 80% of UN cases are diagnosed as non-muscle invasive bladder cancer (NMIBC), in which up to 50% of the cases would experience recurrence and 20% would progress within 5 years (6, 7). So, UNs are categorized based on histopathologic grading and muscle invasion. The majority of them are non-invasive low-grade tumors (1). UN management is mostly based on grading and staging of the tumor, but they are not sufficient to predict the patients’ outcome (8). Standard methods are available for UN diagnosis and prognosis assessment (8). These methods are expensive and uncomfortable, so biochemical markers as alternative diagnostic methods, have been developed (9, 10). Some biochemical markers are considered to be more sensitive and specific in the target population compared with routine examination for the prognosis assessment. Also, evaluation of biochemical markers is safe, non-invasive, and easy to use; however, it is not cheap and available (8, 10). Some studies have suggested that Survivin is an apoptosis’ inhibitor protein which blocks caspase activation (6, 11, 12). In cancer cells, Survivin is expressed in the G2/M phase of the cell cycle and counteracts apoptosis induction during mitosis by interfering with the function of caspases (11). Survivin is over expressed in several human malignancies leading to tumor progression and metastasis. Likewise, in UN, Survivin expressed in tumoral tissue causes rapid progression of the disease and increased rates of recurrence (13). So Survivin, an apoptosis inhibitor protein, is a useful marker for prognostic assessments in invasive bladder tumors (14). Functionally, Survivin inhibits apoptosis, promotes cell proliferation, and induces/enhances angiogenesis. In transitional cell carcinoma of the urinary bladder, Survivin has been shown to be a promising biomarker for cancer diagnosis, prognosis, and prediction of possible response to intravesical or systemic therapies (15). Because of its expression in cancer but not in normal tissues, we investigated the potential suitability of Survivin immunostaining as a new molecular marker for prognosis evaluation of UN (16). Human epidermal growth factor receptor-2 (HER2) is a member of tyrosine kinase receptor family. The epidermal growth factor (EGF) family of receptor tyrosine kinases comprises four members: HER1 (EGF receptor 1 Human EGF Receptor/ErbB1), HER2 (neu/ErbB2), HER3 (ErbB3) and HER4 (ErbB4). EGF receptors are commonly active in a dimeric form and interaction between different EGF receptor pairs represents a signal diversification and amplification mechanism (1). Although it is low expressed in normal tissues, it has a significant role in the pathogenesis of some cancers (17). HER2 over-expression particularly characterizes aggressive cancer types of various origins with poor outcome (18). Only a few studies have been conducted regarding the importance of HER2 status in UN, so there is still controversy over this subject (19). Our study aims to evaluate HER2 and Survivin expressions using immunohistochemical (IHC) analysis and their association with histologic grade of UNs.

Sample Selection

A total number of 84 cases of transitional bladder tumor were selected from the pathology archive of Sina Hospital affiliated to Tehran University of Medical Sciences from 2014 to 2018. The specimens were obtained via transurethral resection (TUR) and cystectomy (radical or partial). Patients’ records were also considered for demographic data. This study was cross sectional. Cases with incomplete information were excluded. Slides were re-assessed by an expert pathologist who was unaware of the patients’ history. After confirmation of diagnosis, appropriate blocks of each sample were selected. The specimens were classified according to the grade of tumor progression elaborated by World Health Organization (WHO)/International Society of Urological Pathology (ISUP) criteria approved in 2016 (20).  Tumor staging was also performed according to the College of American Pathologists (CAP) guideline. Other assessed items included muscle invasion, perineural invasion, lymphatic invasion, blood vascular invasion, multifocality or unifocality of tumor and presence of in situ component (21).

Immunohistochemistry Staining

We prepared 2 mm thick paraffin blocks. Slides were coated with Poly-L-lysine and then deparaffined and rehydrated. IHC staining was performed on one representative slide per tumor which exhibited a maximum of tumoral tissue in order to detect HER2 and Survivin expression. A 1:250 dilution of primary antibody (Polyclonal rabbit anti-human C-erb B-2, Dako) was prepared according to the manufacturer’s protocol for HER2 staining. A 1:100 dilution of Survivin (clone12c4, Dako, USA-monoclonal mouse anti human) was also prepared according to the manufacturer’s protocol. Hematoxylin was used to stain the background. Breast carcinoma samples positive control was used to evaluate HER2. HER2 expression staining was graded according to Table 1 (22) (Figure 1). For Survivin, tumors which showed increased expression in more than 10% of the nuclear tumoral cells, were considered positive in our study (23, 24) (Figure 2).

Statistical Analysis

Statistical analysis of the results was performed usingSPSS-18 software (SPSS Inc., Chicago, IL., USA). The HER2 and Survivin expressions were evaluated in general as well as correlation with tumor grade of the disease and other variables including age, sex, and muscular layer invasion. T-test and Chi-square tests were used for data analysis. All statistical tests were two-sided, and P-value