HER 2 Expression in Ovarian Mucinous Carcinomas in Tunisia

The ovary cancer is the sixth most common cancer among Tunisian women (Missaoui et al., 2010). Mucinous carcinoma accounts for 2% to 5% of all epithelial ovarian cancers. This cancer continues to be diagnostically challenging, and many mucinous tumors involving the ovaries are metastases from other sites. It has a poor prognosis in the advanced stages and responds poorly to conventional chemotherapy (paclitaxel/carboplatin) (Kurman and Seidman, 2000; Hess et al., 2004; Gurung et al., 2013). An efficient treatment is not yet available (Pectasides et al., 2005). The Human epidermal growth factor receptor 2 (HER2, c-erb-B2) gene, located on chromosome 17q11, encodes the HER2 protein, which is a 185-kDa transmembrane receptor tyrosine kinase with no identified physiological ligand. The HER2 gene is involved in intracellular signaling transduction pathways leading to cell growth and differentiation (Di Fiore et al., 1987; Kauraniemi et al., 2006; Unal et al., 2013). Studies have suggested that abnormal expression of HER2 gene by mutation or amplification may play an important role in tumor genesis (Auersperg et al., 2001; Garcia-Velasco et al., 2008; Cuadros and Villegras, 2009; Moatter et al., 2011; Duman et al., 2012; Pazhoomand et al., 2013; Unal et al., 2013). HER2 has been reported to be overexpressed or amplified in a variety of human tumors (Nicholson et al., 2001; Rubin and Yarden, 2001; McAlpine et al., 2009; Ghaffari et al., 2011; Wang et al., 2011; Sardari et al., 2012; Duman et al., 2012; Panagiotou et al., 2012;


Introduction
The ovary cancer is the sixth most common cancer among Tunisian women (Missaoui et al., 2010).Mucinous carcinoma accounts for 2% to 5% of all epithelial ovarian cancers.This cancer continues to be diagnostically challenging, and many mucinous tumors involving the ovaries are metastases from other sites.It has a poor prognosis in the advanced stages and responds poorly to conventional chemotherapy (paclitaxel/carboplatin) (Kurman and Seidman, 2000;Hess et al., 2004;Gurung et al., 2013).An efficient treatment is not yet available (Pectasides et al., 2005).

HER2 Expression in Ovarian Mucinous Carcinomas in Tunisia
Adjuvant therapy using a monoclonal antibody against HER2 protein (Trastuzumab) is effective alone and in combination with conventional cytotoxic chemotherapy in patients whose breast carcinomas have amplification of HER2 (Goldenberg, 1999;Shak et al., 1999;Piccart-Gebhart et al., 2005;Romond et al., 2005;Duman et al., 2012;Tai et al., 2013;Unal et al., 2013).However, the significance of HER2 overexpression and amplification in the initiation and progression of ovarian mucinous carcinoma is less well understood (Weroha et al., 2011), and the treatment with HER2 antagonists has been disappointing (Bookman et al., 2003;Frederick et al., 2009).
In the current study, we investigated the HER2 protein expression by immunohistochemistry in formalin-fixed, paraffin embedded tissues from 27 Tunisian women with mucinous tumors of the ovary.

Tissue samples
Twenty-eight specimens were retrieved from the surgical pathology files of Pathology Department, Farhet Hached University Hospital of Sousse, Tunisia.The cases studied were distributed into the following groups, according to the World Health Organization (WHO) Classification of Tumors of the Breast and Female Genital Organs, 2003 (Tavassoli and Devilee, 2003): ovarian mucinous carcinoma (n=14) and ovarian mucinous borderline tumor (n=13).In this study, a positive staining breast ductal carcinoma was used as positive control case.
Medical records were reviewed for data on age, marital status, tumor size, histopathology, and tumor stage and grade.
All tissues been routinely fixed in 4% buffered formalin and paraffin embedded.The cases were selected randomly, and the slides were reviewed by two pathologists.The study was approved by the Human Ethics Committee at the Farhet Hached University Hospital of Sousse (Tunisia) and it conformed to the provisions of the Declaration of Helsinki.
One or two paraffin blocks containing representative portions of the tumors were selected for each case, and 4-μm-thick sections were obtained.

Immunohistochemistry
Immunohistochemistry staining of HER2 protein was performed using HerceptTest kit (Dako, code K5204).Formalin-fixed, paraffin-embedded tissues were deparaffinized in xylene, rehydrated through serial dilutions of alcohol and washed in phosphate -buffered saline (ph7.2).After pretreatment with Dako antigen retrieval solution in 10 mM citrate buffer (ph6.0) at 95°C for 40 minutes in a streamer, endogenous peroxidase activity was blocked in 3% hydrogen peroxide.Slides were then incubated with the rabbit anti-human HER2 protein antibody at room temperature (20-25°C) for 30 min.Diaminobenzidine was used as the chromogen for the immunostaining.Finally, sections were counterstained with hematoxylin and mounted.Negative controls were obtained by excluding the primary antibody.The images were captured by the Olympus microscopic digital camera system for study comparison.

Immunohistochemistry interpretation
In 2007, the American Society of Clinical Oncology and the College of American Pathologists (ASCO-CAP) established guidelines for the HER2 scoring algorithm for breast cancers.Although there is neither consensus nor valid data that correlate to clinical outcomes for ovarian cancers, we applied these current guideline recommendations to interpret the results of HER2 immunohistochemistry in this study (Wolff et al., 2007;Lin et al., 2011).The immunohistochemistry results was evaluated by two pathologists (Dr.Hmissa and Dr. Yacoubi).
Only membrane staining was evaluated for HER2 immunostaining in this study.We adopted the 2007 ASCO-CAP guideline recommendations for immunohistochemistry result categories instead of the Dako Hercep-Test recommendations (Dako, Carpinteria, California).Therefore, "positive" is defined as strong, complete, homogenous membrane staining in >30% of tumor cells; "equivocal" is defined as either strong, complete, homogeneous membrane staining in <30% or weak, moderate heterogeneous complete membrane staining in >10% of tumor cells, and "negative" is defined as no staining (score 0 points), or weak, incomplete membrane staining (score 1+ points) in any percentage of cells.

Clinicopathological features
Overall, the mean age at diagnosis was 52 years (ranging from 20 to 80 years).The mean age was 49.3 years among patients with ovarian mucinous carcinoma (20-72 years) and 55.5 years among women with ovarian mucinous borderline tumor (27-80 years).
At the time of their ovarian tumor diagnosis, 88.9% of patients with mucinous carcinomas and 85.7% of patients with mucinous borderline tumors were married.All patients had no records regarding personal or family history of cancers.
The tumor size of ovarian mucinous carcinoma varied from 1.5 cm to 30 cm with a mean of 17.5 cm.For the mucinous borderline tumors, the tumor size varied from 10 to 28 cm with a mean tumor size of 17 cm.
According to the International Federation of Gynecology and Obstetrics (FIGO, 1989) staging system, stage I was the most frequent (66.7%) followed by stages II and III with 11.1% and 22.2% respectively.All mucinous borderline tumors were localized.

Discussion
HER2 gene amplification and expression have been extensively studied as the target for the cancer therapy.Normal ovarian surface epithelial cells express little or no HER2 (Wu et al., 2004).HER2 gene amplification and protein overexpression were reported in 18.3% to 35.3% mucinous carcinoma (Nicholson et al., 2001;Rubin and Yarden, 2001;McAlpine et al., 2009;Chay et al., 2013).In our study, HER2 expression was described in 14.3% of mucinous carcinomas.The variation in the rate of HER2 gene amplification and expression could be explained by several raisons including different detection methods used (immunohistochemistry, fluorescent in situ hybridization and chromogenic in situ hybridization), several types of biological material (blocs of tumors, tissue microarray), different immunohistochemical techniques (CB-11, HercepTest or non-commercial antibody), different staining protocols and subjective interpretation of stained samples.All these factors make difficult the comparison of HER2 status across research studies (Tuefferd et al., 2007).
In a previous study, McAlpine et al. investigated HER2 expression and amplification in 33 mucinous carcinomas and 16 mucinous borderline tumors of the ovary and they analyzed the potential use for trastuzumab therapy (McAlpine et al., 2009).Amplification of HER2 was observed in 18.2% mucinous carcinomas and 18.8% mucinous borderline tumors.HER2 amplification in primary mucinous carcinomas was not associated with an increased likelihood of recurrence.The prospectively identified recurrent mucinous carcinomas showed overexpression and amplification of HER2; one patient's tumor responded dramatically to trastuzumab in combination with conventional chemotherapy, while another patient experienced an isolated central nervous system recurrence after trastuzumab therapy.Thus, these authors considered that in ovarian mucinous carcinomas, HER2 amplification not necessarily of prognostic significance and trastuzumab therapy provides a treatment option for patients when the tumor has HER2 amplification and overexpression (McAlpine et al., 2009).
Recently, in a large Asian retrospective cohort from Singapore, Chay et al. analyzed HER2 expression in 113 cases by immunohistochemistry and dual in situ hybridization (Chay et al., 2013).HER2 protein expression was described in 27.4% of mucinous epithelial ovarian cancer.They observed a lower HER2 positivity among Chinese and a higher HER2 expression among Malays patients (Chay et al., 2013).Previously, these authors analyzed HER2 expression in only 17 primary mucinous carcinomas in Singapore also.They described HER2 amplification and expression in 35.3% and 29.4%, respectively (Yan et al., 2011).
In a previous study, Anglesio et al. have demonstrated amplification of HER2 in 18.2% of mucinous ovarian carcinomas and presented anecdotal evidence of response with HER2-targeted treatment in a small series of women with recurrent HER2-amplified mucinous carcinomas (Anglesio et al., 2013).More recently, these authors explored HER2 amplification in an independent cohort of 189 mucinous carcinomas and 199 mucinous borderline ovarian tumors by immunohistochemistry, fluorescent in situ hybridization (FISH), and chromogenic in situ hybridization (CISH), and interpreted per ASCO/CAP guidelines (Anglesio et al., 2013).HER2 overexpression/ amplification was reported in 18.8% of mucinous carcinomas and 6.2% of mucinous borderline tumors.In this study, there was excellent agreement between immunohistochemistry, FISH, and CISH assessment of HER2 status.HER2 amplification/overexpression was associated with decreased likelihood of disease recurrence.Thus, these authors supported the stratification of mucinous carcinomas for the testing of new treatments, with HER2-targeted therapy as a viable option for HER2+ advanced or recurrent disease (Anglesio et al., 2013).
HER2 expression has been analyzed across all histological subtypes of epithelial ovarian cancers and studies showed that HER2 status did not seem to be a significant event in nonmucinous epithelial ovarian cancers (Wu et al., 2004;Lin et al., 2011).Wu et al. showed previously that HER2 amplification is infrequent in early stage ovarian neoplasms: only 6.7% overall, including 3 serous carcinomas and one endometrioid carcinoma.None of the remaining 44 tumors (including clear cell carcinoma, transitional cell carcinoma, mixed epithelial carcinoma, carcinoma not otherwise specified) and 31 borderline tumors showed HER2 gene amplification or protein overexpression (Wu et al., 2004).However, Tueffred et al. showed that the decision algorithm usually used in breast cancer (immunohistochemistry as a screening test, with equivocal results confirmed by FISH) is appropriate in ovarian cancer (Tueffred et al., 2007).
In conclusion, HER2 expression is relatively common in mucinous epithelial carcinoma of the ovary, although not necessary of prognostic significance.Thus, trastuzumab therapy would be an option for patients with mucinous carcinoma when the tumor has HER2 overexpression.