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Table of Contents
Year : 2013  |  Volume : 1  |  Issue : 1  |  Page : 17-21

Immunohistochemical expression of HER2 in urothelial bladder carcinoma and its correlation with p53 and p63 expression

1 Department of Pathology, CHU Habib Bourguiba, Sfax, Tunisia
2 Department of Urology, CHU Habib Bourguiba, Sfax, Tunisia

Date of Web Publication24-Jan-2018

Correspondence Address:
Slim Charfi
Laboratoire d'anatomie et de cytologie pathologiques, CHU Habib Bourguiba, Route ElAin Km 0,5, 3029 Sfax
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Source of Support: None, Conflict of Interest: None

DOI: 10.1016/j.jmau.2013.06.001

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This study assessed the human epidermal growth factor receptor-2 (HER2) protein expression in urothelial bladder carcinoma and its relationship with p53 and p63 expression. In 151 patients, paraffin-embedded tissues of transurethral resection or cystectomy were evaluated by immunohistochemistry (IHC), using antibodies against HER2, p53 and p63. HER2 overexpression (score 3+), p53 overexpression and decreased expression of p63 were detected in 14 (9.3%), 48 (31.8%) and 58 (38.4%) tumors, respectively. HER2 overexpression, p53 overexpression and decreased expression of p63 were associated with high tumor grade (p = 0.0002, p = 0.0002 and p = 0.046, respectively) and advanced TNM stage (p = 0.017, p = 0.012 and p = 0.001, respectively) of urothelial bladder carcinoma. In univariate analysis HER2 overexpression was significantly associated with decreased expression of p63 (p = 0.037). In multivariate analysis, only tumor grade was correlated with HER2 overexpression (p = 0.006). In this study, HER2 is overexpressed in 9.3% of urothelial bladder carcinomas. HER2 overexpression was strongly correlated with tumor grade but not with TNM stage, p53 and p63. The development of target therapies using anti-HER2 and the identification of patients which who benefit from those therapies need further studies.

Keywords: HER2, p53, p63, Immunohistochemistry, Urothelial bladder carcinoma

How to cite this article:
Charfi S, khabir A, Mnif H, Ellouze S, Mhiri MN, Boudawara-Sellami T. Immunohistochemical expression of HER2 in urothelial bladder carcinoma and its correlation with p53 and p63 expression. J Microsc Ultrastruct 2013;1:17-21

How to cite this URL:
Charfi S, khabir A, Mnif H, Ellouze S, Mhiri MN, Boudawara-Sellami T. Immunohistochemical expression of HER2 in urothelial bladder carcinoma and its correlation with p53 and p63 expression. J Microsc Ultrastruct [serial online] 2013 [cited 2022 Dec 4];1:17-21. Available from: https://www.jmau.org/text.asp?2013/1/1/17/223893

  1. Introduction Top

Urothelial bladder carcinoma represents in Tunisia the first urological cancer. Its incidence is estimated at 13.1–19 per 100,000 [1],[2]. Several factors are involved in determining prognosis and treatment selection. Tumor grade and TNM stage are two independent factors which determine the treatment strategy. The human epidermal growth factor receptor-2 (HER2) is known to contribute to physiologic mechanisms of cell proliferation by an intrinsic tyrosine kinase activity. The assessment of the HER2 status is crucial for the management of breast cancer, for both prognosis and prediction of the response to targeted therapies. In urothelial bladder carcinoma, immunohistochemical HER2 expression varies among different studies, ranging from 9 to 81% [3],[4],[5],[6],[7],[8],[9],[10],[11],[12],[13]. Recently a large multicenter series investigating invasive bladder carcinoma has shown HER2 overexpression in 9.2% [12]. Several studies have demonstrated that p53 and p63 are two prognostic markers for urothelial carcinoma associated to tumor progression and poor clinical outcome [15],[16],[17],[18],[19]. This study explored whether immunohistochemical HER2 expression is related to these two markers (p53 and p63).

  2. Materials and methods Top

2.1. Study population and tissue specimens

A series of 151 tumors from 151 patients (130 men and 21 women; mean age 65.94 ± 12.8 years, range 26–101 years) diagnosed with primary urothelial carcinoma of the urinary bladder was selected from the Habib Bourguiba Pathology Department files from 2006 to 2008. Paraffin-embedded tissues were obtained after transurethral resection for bladder tumor or cystectomy. Histological examination on hematoxylin–eosin (H&E) stained slides was carried out confirming the diagnosis urothelial cell carcinoma. The TNM classification was used for tumor staging and the 2004 WHO classification for tumor grading [20],[21]. Tumor grade and TNM stage of these tumors were summarized in [Table 1].
Table 1: Histopathological characteristics of the bladder tumors.

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2.2. Immunohistochemistry

Before immunostaining, two pathologists (SC and AK) reviewed hematoxylin–eosin stained slides from the original paraffin-embedded blocks in each case. One section representing the most invasive areas of each tumor was selected. Briefly, 4 μm-thick sections were cut from each paraffin block, mounted on poly-L-lysinecoated slides, fixed in acetone for 10 min, and left to dry overnight at 37°C. Slides were deparaffinized in xylene followed by absolute ethanol and subsequent rehydration in graded ethanol. The sections were then pre-treated with 3% hydrogen peroxide for 10 min to inactivate endogenous peroxides and washed in phosphate-buffered saline (PBS) solution. Heat-induced antigen retrieval was performed using epitope retrieval solution (DAKO) at 95 °C for 40 min. After heating, slides were allowed to cool down to room temperature and were briefly washed with PBS. Blocking solution (Protein Block Serum: 0.25% Casein in PBS containing Carrier Protein and NaN3; DAKO) was used for 5 min to prevent nonspecific immunostaining. Immunohistochemical staining was performed using the streptavidin-biotin peroxidase system. Slides were incubated for 30 min at room temperature with anti-HER2 (clone CB11, Novocastra, Newcastle upon Tyne, UK; dilution, 1/40), anti-p53 (Clone: DO-7, code: M7001, DAKO; dilution, 1/50) and anti-p63 (Clone: A4A, code: M7247, DAKO; dilution, 1/40), then washed with PBS before applying the biotinylated secondary antibody (anti-rabbit, DAKO) for 5 min. Sections were incubated with the streptavidin-biotin complex reagent (Universal Quick Kit, DAKO) for 15 min and developed with 3,3’-diaminobenzidine tetrahydrochloride (DAB) for 30 min. Finally, tissues sections were counterstained by Mayer's hematoxylin, dehydrated, and mounted (DAKO). Internal/external, positive/negative controls for each antibody were used for validation of the reactions.

2.3. Evaluation of immunohistochemical staining

HER2 positivity was assessed using the ASCO scoring system, evaluating only membranous staining [22]. The level of HER2 protein expression was assessed semiquantitatively by the intensity and percentage of staining and scored on a scale of 0–3+. Only tumors with score 3+ are categorized as positive. A score of 1+ was defined as barely perceptible membrane staining in >10% of cells, a score of 2+ was defined as weak-to-moderate complete membrane staining present in >10% of tumor cells, and a score of 3+ was defined as strong complete membrane staining in >30% of tumor cells. A cytoplasmic staining was considered nonspecific. Only nuclear immunostaining for p53 and p63 was considered positive. Regarding p53, tumors showing immunoreactivity of more 10% of tumor cell nuclei were considered positive (p53 overexpression), in accordance with the practice used in previous studies because it has been shown that accumulation of p53 protein in 10% or more of the tumor cell nuclei strongly correlates with mutations in the p53 gene [18],[19],[23]. For p63 immunoreactivity, a cut off of 90% was used considering tumors with immunoreactivity less than 90% of cell as decreased expression of p63 and staining of more than 90% of cells as normal [23]. All fields of the section has been analysed by two pathologists (S.C. and A.K.) using multi viewing microscope. When the results were different, agreement was reached with careful discussion.

2.4. Statistical analysis

Data available for statistical evaluation was performed with Statistical Package for the Social Sciences SPSS (version 17.0, IBM, Chicago, IL, USA) for windows software. Initially, a descriptive analysis of all collected variables was performed. The correlation between expression of HER2, p53, p63 and tumor grade and TNM stage were assessed with Fisher's exact and chi-square tests. The same tests were used for the correlation between p53, p63 and HER2 expression. For multivariate analysis, binary logistic regression was performed incorporating significant parameters by the univariate analysis. All differences were deemed significant at the level of p <0.05.

  3. Results Top

3.1. Expression of HER2 and association with pathologic characteristics

Strong protein expression (3+) was observed in 14 of 151 tumors (9.3%) [Figure 1]. An immunohistochemistry 0/1+ score was detected in respectively 55 (36.4%) and 61 (40.4%). 21 cases (13.9%) were scored as 2+. HER2 overexpression was significantly associated with the tumor grade (p = 0.0002) and TNM stage (p = 0.017) [Table 2].
Figure 1: Strong and diffuse HER2 immunostaining surrounding the entire cell membrane (score 3+) (HER2 400×).

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Table 2: Association of HER2, p53 and p63 immunostaining with histopathological parameters.

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3.2. Expression of p53 and p63 and association with pathologic characteristics [Table 2]

Overexpression of p53 was observed in 48 of 151 tumors (31.8%). Decreased expression of p63 expression was found in 58 of 151 tumors (38.4%) [Figure 2] and [Figure 3]. p53 overexpression and decreased expression of p63 were correlated with high tumor grade (p = 0.0002 and 0.046, respectively) and TNM stage (p = 0.017 and 0.012, respectively) [Table 2].
Figure 2: p53 immunohistochemical expression. (A) diffuse nuclear staining of tumor cells (p53 200×). (B) p53 immunostaining in less than 10% of tumor cells (p53 200×).

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Figure 3: p63 immunohistochemical expression. (A) strong and diffuse nuclear staining of neoplastic urotlelial cells in low grade tumor (p63 200×). (B) heterogenous immunoreactivity in high grade tumor (p63 200×).

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3.3. Association of HER2 expression with p53 and p63 expression

In a statistical analysis of the association between HER2 expression and p53/p63 expression, HER2 overexpression was correlated to a decreased expression of p63 (p = 0.037) but not with p53 overexpression (p = 0.350) [Table 3].
Table 3: Association of immunohistochemical HER2 expression with p53 and p63 expression.

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3.4. Multivariate analysis of combined variables incorporating pathologic characteristics

In a multivariate analysis considering tumor grade, decreased expression of p63 and TNM stage as co-variables, HER2 overexpression was correlated with tumor grade (p = 0.006) but not with decreased expression of p63 and TNM stage.

  4. Discussion Top

In this study, 151 primary urothelial bladder carcinomas from Tunisian population were screened for HER2 expression and its correlation with p53 and p63 expression. To our knowledge, this is the first study to analyse immunohistochemical correlations of Her-2 with p53 and p63. HER2 is overexpressed in 9.3% of tumor samples which is comparable to rates of 9.2 (tumors 2+ and 3+) and 5.1% (tumors 3+) reported by Laé et al. [14]. But this rate is lower than reported in several previous studies ranging from 21.7% to 80% [3],[4],[6],[7],[8],[9],[10],[12],[13]. Several hypotheses could explain these wide variations, as well as the relatively low rate of HER2 overexpression reported in our series. One of the major issues is the variability in immunohistochemistry assays, related to the heterogeneity between kits, antibodies, protocols, interpretations of staining or cut-off values. Scoring systems are variable and there is no consensus on the definition of HER2 overexpression in bladder carcinoma. Most authors used criteria of Wolff et al. reported for breast carcinoma (ASCO scoring system) [22].

In our study only tumors scored 3+ were considered overexpressing HER2. We consider that tumors scored 2+ should not be considered as overexpressing Her-2 without demonstration of HER2 gene amplification by in situ hybridization technique. In fact, literature data showed that the majority of cases scored 2+ are not amplificated by in situ hybridisation. In the study of Laé et al., all cases scored 2+ (n = 42) were negative by fluorescent in situ hybridization (FISH) technique [14]. Moreover, Caner et al. noted that only 2 among 9 cases were amplificated by FISH and 4 among 12 cases were amplificated by real-time quantitative PCR for HER2 status [4].

P53 overexpression in urothelial carcinoma was reported in 30.8–60% [24],[25]. Some investigators noted a prognostic significance for p53 staining in both univariate and multivariate analysis [24],[26],[27],[28]. Shariat et al. [16] demonstrate that p53 is the strongest predictor marker of bladder carcinoma outcome in patient undergoing radical cystectomy. In our series p53 was overexpressed in 31.8% of tumors and was correlated to higher tumor grade and TNM stage. p63 is a homologue of the p53 tumor suppressor gene, that encodes multiple proteins that may either transactivate p53 responsive genes (Tap63) or act as a dominant negative factor toward p53 (deltaNp63) [19]. p63 is constitutively expressed in the basal cell compartment of the stratified epithelium, including the urothelium, in which deltaNp63 is the predominant isoform expressed [15]. p63 is decreased in most invasive cancers whereas papillary superficial tumors maintain its p63 expression [19]. In urothelial carcinoma, decreased deltaNp63 expression is associated with more advanced disease and poorer prognosis [15],[17],[19],[23]. In our series decreased expression of p63 was associated with higher grade and TNM stage.

In our study, HER2 overexpression was correlated with decreased expression of p63 in univariate analysis. This correlation was lost on multivariate analysis. In fact HER2 overexpression was significantly related to tumor grade but not with TNM stage and p63 expression.

  5. Conclusion Top

In conclusion, HER2 was overexpressed in 9.3% of urothelial carcinoma in the current study, and was strongly correlated with high tumor grade. The development of target therapies using anti-HER2 and the identification of patients who may benefit from those therapies need further studies.

Conflict of interest

We have no conflict of interest to declare.

  References Top

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3]

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