| Abstract|| |
Background/Aims: Human epididymal secretory protein 4 (HE4) is originally described as an epididymis-specific protein but more recently suggested to be a putative serum tumor marker for some tumors, including breast carcinomas. In this study, we aimed to investigate the interactions between HE4 expression and molecular subtypes of breast carcinomas. Methods: HE4 expressions were studied in 242 formalin-fixed, paraffin-embedded breast carcinoma specimens and their association with different pathological and clinical parameters was evaluated. Results: Immunohistochemical (IHC) staining for HE4 was negative in 3 (1.2%) cases, weakly positive (1+) in 7 (2.9%) cases, moderately positive (2+) in 58 (24.0%) cases, and strongly positive (3+) in 174 (71.9%) cases. A correlation between IHC HE4 staining grade and molecular groups was detected (P = 0.005). Furthermore, it was found that HE4 expression was strongly associated with histological tumor grade, c-erbB2 expression, and positive fluorescence in situ hybridization test results that detect human epidermal growth factor receptor 2 (HER2)/neu amplification (P = 0.022, P = 0.014, and P = 0.011). Conclusion: This study showed that HE4 expression is associated with HER2/neu amplification in breast cancers. These results may be commented as HE4 expression rises in patients with HER2/neu amplification. As is known, HER2/neu amplification is a poor diagnostic factor in breast cancer and HE4 expression is possibly associated with poor prognosis.
Keywords: Breast cancer, human epididymal secretory protein 4, immunohistochemistry, molecular groups
|How to cite this article:|
Akoz G, Diniz G, Ekmekci S, Ekin ZY, Uncel M. Evaluation of human epididymal secretory protein 4 expression according to the molecular subtypes (luminal A, luminal B, human epidermal growth factor receptor 2-positive, triple-negative) of breast cancer. Indian J Pathol Microbiol 2018;61:323-9
|How to cite this URL:|
Akoz G, Diniz G, Ekmekci S, Ekin ZY, Uncel M. Evaluation of human epididymal secretory protein 4 expression according to the molecular subtypes (luminal A, luminal B, human epidermal growth factor receptor 2-positive, triple-negative) of breast cancer. Indian J Pathol Microbiol [serial online] 2018 [cited 2021 Jun 13];61:323-9. Available from: https://www.ijpmonline.org/text.asp?2018/61/3/323/236613
| Introduction|| |
Human epididymal secretory protein 4 (HE4) is a member of whey acidic protein (WAP) family, which is also termed as WAP-4 disulfide core domain (WFDC2). It consists of two WAP chains and eight cysteine residues which constitute four disulfide bonds. It is localized on chromosome 20., HE4 is a secretory protein which was first described in epididymal duct and thought to have a function in the spermatogenesis. Later on it has been detected to be an antiprotease expressed in normal epithelium of the reproductive tract and respiratory epithelium of the proximal airways. HE4 has been reported to be a tumor marker for serous and endometrioid ovarian carcinomas.
In 2003, HE4 was approved by the US Food and Drug Administration (FDA) as a serum tumor marker to be used in the diagnosis ovarian cancer.
Increased HE4 expression has been demonstrated in many cancer types, especially in gynecologic and pulmonary cancers.,, HE4 is also expressed in ductal breast carcinoma; however, the role of this protein in breast carcinoma has not been precisely known.,
Clinically, gene expression studies for breast cancers have been divided into three basic molecular subtypes as for their prognosis, and treatment targets as estrogen receptor (ER)-positive luminal (A and B) group, human epidermal growth factor receptor 2 (HER2)-amplified group, and triple-negative group. In addition to the currently used methods, it has been thought that new parameters with a significant prognostic value which will contribute to the treatment are needed. The aim of this study is to investigate the correlations between different clinicopathologic variables, including mainly HE4 expression and molecular subtypes of breast carcinoma.
| Methods|| |
Patient characteristics and evaluated clinicopathologic parameters
A total of 297 patients who underwent mastectomy and excisional breast biopsy between 2011 and 2014 and histopathologically diagnosed as breast carcinoma in the Pathology Laboratory of Izmir Tepecik Training and Research Hospital were included in the study.
Hematoxylin and eosin (H and E)-stained archived slides were reevaluated based on 2012 Breast Tumor Classification of the World Health Organization. Pathology records were analyzed in consideration of the parameters listed in [Table 1] and [Table 2].
|Table 1: Comparisons between human epididymal secretory protein 4 staining intensities and other variables-I|
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|Table 2: Comparisons between human epididymal secretory protein 4 staining intensities and other variables-II|
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Immunohistochemical evaluation of human epididymal secretory protein 4
For immunohistochemical (IHC) studies, H and E staining was used to select appropriate paraffin blocks and to identify the viable tumor areas. IHC was performed using streptavidin-biotin peroxidase method (Invitrogen, Camarillo, CA, USA). Serial 5-μm sections were obtained, and these slides were baked overnight at 60°C, dewaxed in xylene, and hydrated with distilled water through decreasing concentrations of alcohol. All slides were treated with heat-induced epitope retrieval in the microwave (in 10 mm/L citrate buffer, pH 6.0, for 20 min, followed by cooling at room temperature for 20 min) and blocked for endogenous peroxidase and biotin. The purified monoclonal mouse antibodies against human epididymal protein 4 (anti-HE4 Ab, Abcam, ab24480) were used at a dilution of 1:40.
Stained slides were evaluated by three observer pathologists under a light microscope. Intensity of cytoplasmic staining (0, negative; 1+, weakly positive; 2+, moderately positive; 3+, strongly positive) and percentage of stained cells (0, 1 [1%–24%], 2 [25%–49%], 3 [50%–74%], 4 [75%–100%] were evaluated and graded. As an HE4-positive control, normal terminal ductal lobular unit was used [Figure 1].,
|Figure 1: Normal terminal duct lobular unit was used for human epididymal secretory protein 4 positive control (DAB, ×200)|
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For the analysis of variables, SPSS 22.0 (IBM Corporation, Armonk, NY, USA) program was used. Fitness of data to normal distribution was evaluated using Shapiro–Wilk test, and homogeneity of variance was assessed with Levene test. For one-to-one comparison of quantitative data of more than two independent groups, one-way ANOVA test and Kruskal–Wallis H-test were employed. For one-to-one comparison of categorical data, Pearson's Chi-square, linear-by-linear association tests, and Monte–Carlo simulation technique were used. For the investigation of their effects on HE4 staining status, mortality, and lifespan, Kaplan–Meier (product limit method)-log rank (Mantel-Cox) analysis was employed. Quantitative variables were expressed in tables as mean ± standard deviation and median, range (maximum–minimum); categorical variables were indicated as n (%) variables were analyzed within 95% confidence interval, and P < 0.05 was accepted as statistically significant.
| Results|| |
HE4 expression was IHC studied in 242 cases; cytoplasmic staining intensity (0, negative; 1+, weak; 2+, moderate; 3+, strong) and percentage of positively stained cells (0, 1 [1%–24%], 2 [25%–49%], 3 [50%–74%], 4 [75%–100%]) were evaluated and graded., Accordingly, 3 (1.2%) cases could not be stained (negative), while the other specimens were weakly (1+; n = 7, 29%), moderately (2+; n = 58, 24.0%), and strongly (3+, n = 174, 71.9%) stained with cytoplasmic HE4 dyes [Figure 2], [Figure 3], [Figure 4]. Still, among 239 (97.5%) cases with HE4 positivity, median percentages of stained areas in respective number of cases were as follows: n = 1; 0.4%; range, 1%–24%; n = 2: 0.8%; range 50%–74%, and n = 236: 97.5%; range 75%–100%. In none of the cases, percentage of stained area ranged between 25% and 49%. As a result of these data, since data were not sufficient to evaluate patients based on the percentages of the stained areas, analysis of the cases based on percentage of HE4 staining could not be performed. All parameters were compared with HE4 staining intensity.
|Figure 2: Weak (1+) cytoplasmic human epididymal secretory protein 4 staining intensity (DAB, ×200)|
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|Figure 3: Moderate (2+) cytoplasmic human epididymal secretory protein 4 staining intensity (DAB, ×200)|
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|Figure 4: Strong cytoplasmic human epididymal secretory protein 4 (3+) staining intensity (DAB, ×200)|
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Demographic and histopathological parameters
The results of the comparisons between HE4 staining intensities and demographic and histopathological parameters are shown in [Table 1] and [Table 2]. Among these parameters, only between HE4 staining intensities and tumor grades, a statistically significant difference was detected (P = 0.022); however, since numerical distribution among groups was not even, the result was not found to be reliable.
Comparison between HE4 staining intensity and other IHC parameters is summarized in [Table 3]. A correlation between HE4 expression and ER, progesterone receptor (PR), and Ki67 was not detected; however, HE4 expression was significantly correlated with c-erbB2 (P = 0.014). As HE4 staining intensity increased, number of c-erbB2 3+ stained cases increased; in other words, staining intensities increased in parallel. When the results of fluorescence in situ hybridization (FISH) test performed to evaluate HER2/neu amplification and HE4 expressions were compared, the results significantly confirmed the parallelism between staining intensities (P = 0.011).
|Table 3: Comparisons between human epididymal secretory protein 4 staining intensities and other immunohistochemical variables|
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Correlations between molecular subtypes and HE4 staining intensities were analyzed. Most of 1+ and 2+ cases were included in luminal B group, and as HE4 staining intensities increased, number of HER2-positive cases also increased. A statistically significant difference was found between these results (P = 0.005).
The survival analysis which considered mortality and lifespans of the patients based on HE4 staining intensities could not demonstrate any significant difference between median lifespans of the patients (P = 0.351).
| Discussion|| |
Breast cancer is the most frequently seen malignancy in women worldwide. According to surveillance, epidemiology, and end results program database, it is ranked third among cancer-related causes of death in women. Breast cancers consist of heterogenous group of tumors and demonstrate variations as for pathologic characteristics and clinical presentation. In the past, breast cancers were classified as for their histological types and grades; similarities in their gene expression profiles occurring with time were used to develop molecular classification. In the practice of pathology, these molecular subtypes which were also determined using IHC markers as ER, PR antibodies, Ki67, HER2/neu, CK5/6, and epidermal growth factor receptor had significant effects on the development of new treatment approaches in breast cancer.,, When incidence and mortality rates were taken into consideration, nowadays to be able to categorize breast cancer patients into different risk groups more accurately, new markers are needed in addition to clinicopathologic factors used. Thus, patients in the lower risk groups are saved from adverse effects of unnecessary treatments, and more aggressive treatment modalities may be applied for previously identified high-risk patients.
The expression of gene product HE4 (also termed as WFDC2) protein was first detected in epididymis and respiratory tract. It contains two WAP chains and four disulfide bonds composed of eight cysteine residues. It is localized on chromosome 20., HE4 which is a secretory protein contains a signal peptide and a short (10 kDa) and acidic (pH = 4.3) polypeptide rich in cysteine. Since it was first identified in the distal epididymal duct, it was thought to have a function in spermatogenesis. However, currently, many functions of this protein as antibacterial activity and anti-inflammatory effects just such as antiproteinases, leukocyte protease 1, and elafin have been also recognized.
In recent years, gradually increasing number of studies have revealed upregulation of HE4 in various neoplasias and occasionally its increased blood levels. As a result, HE4 has been considered as a potential tumor marker. In gene expression profile studies, upregulation of HE4 has been definitely determined, and multiple number of studies analyzed HE4 protein expression in epithelial ovarian tissues and enabled its applicability in histopathological diagnosis., HE4 protein expression was detected in epithelial ovarian cancer, especially in serous and endometrioid type, while in normal ovarian tissue, its expression could not be detected. However, in benign and borderline ovarian neoplasias, lower levels of HE4 expression were observed relative to malignant types.,, This finding is important in that it indicates HE4 expression as a potential marker to be used in the diagnosis of epithelial ovarian cancers. Besides, epithelial ovarian cancers can be discriminated from nonepithelial ones because nonepithelial ovarian cancer tissues cannot be stained with HE4. In addition, later on, Hellström et al. compared HE4 levels in serum samples harvested from the patients with epithelial ovarian cancer and those retrieved from healthy individuals with benign ovarian neoplasias and detected a prominent increase in serum HE4 levels of the patients with epithelial ovarian cancer. In 2003, the US FDA approved HE4 as a serum tumor marker in ovarian cancer. When HE4 is used in combination with CA125, specificity and sensitivity of HE4 in the differentiation between benign gynecologic conditions and epithelial ovarian cancers explicitly increase. The studies investigating chemotherapeutic response and HE4 levels revealed that higher serum HE4 levels correlated with chemoresistance and decreased survival rates in epithelial ovarian cancer.,
In 2006, Galgano et al. investigated HE4 mRNA and protein expressions in normal and malignant human tissues in a larger sampling size and detected positive HE4 reactivities in various malignancies including ovarian cancer. Based on the outcomes of this study, serous ovarian carcinomas demonstrated HE4 gene expression at the highest rate when compared with other carcinomas, while lung adenocarcinomas took the second place. Transitional cell, renal, and pancreatic carcinomas and breast carcinomas were included in the moderate HE4 gene expression group. As has been demonstrated IHC in many other studies, apart from mucinous type, in most of the ovarian surface epithelial tumors, HE4 positivity was detected. Besides, marked staining intensities were detected in endometrial, pulmonary, breast, pancreas carcinomas; renal, thyroid, salivary gland neoplasias; and malignant mesothelioma.
Jiang et al. analyzed serum HE4 levels in nonsmall cell lung carcinoma and detected a marked increase when compared with the healthy groups. In addition, higher HE4 levels were found to be correlated with the presence tumor-node-metastasis (TNM) stage, lymph node metastasis, and distant metastases. Five-year survival rates of the patients with higher serum HE4 levels were found to be prominently lower relative to those with reduced serum HE4 levels, and the authors concluded that HE4 expression is an independent poor prognostic factor in nonsmall cell lung cancers.
Guo et al. investigated clinical significance in gastric carcinomas of HE4 and detected that HE4 expression was correlated with Lauren classification, TNM stage, and tumor size, and also, a markedly lower incidence of gastric carcinoma in patients with HE4 overexpression relative to those without any information about overall survival was demonstrated.
In a study published by Gündüz et al. in 2016, they demonstrated that serum HE4 levels in breast cancer were significantly higher when compared with healthy volunteers and detected a positive correlation between serum CA15-3 and HE4 levels. With these results, they concluded HE4 protein as a potential biomarker which could be used in the diagnosis of breast cancer.
In literature reviews, HE4 expression has been correlated with clinically unfavorable factors. Limited number of studies investigating the correlation between breast cancer and HE4 expression have encouraged us to conduct this study.
In our study, we investigated the correlation between various clinicopathologic parameters mainly molecular subtypes of cases of breast cancer diagnosed in our hospital between 2011 and 2014 and HE4 expression status.
In the literature, only the study performed by Kamei et al. in 2010 analyzed tissue HE4 expression status and various parameters in breast cancer. In their study, a significant correlation was not detected between patient's age, histological subtype, tumor size, ER, PR, and HER2 expression, Ki67 value, tumor grade, and IHC HE4 staining status of tumors. However, in our study, a significant correlation was detected between tumor grade, HER2 expression, and HE4 staining intensity (P = 0.022, P = 0.014). According to these results, a decrease in tumor grade was detected as HE4 staining intensity increased, and an inverse correlation was revealed between HE4 staining intensity and grade of the tumor.
Invasive ductal carcinoma was the most frequently (85%) detected histopathological subtype of breast cancer. In our study, although 62.3% of the cases had the morphology of invasive ductal carcinoma when cases with dominant in situ components were included in the study, its incidence raised up to 84.5% similar to literature data. In our study, a significant correlation was not detected between histopathological type of the tumor and HE4 staining intensity.
When HER2 staining status was considered, we detected that the number of c-erbB2 3+-stained cases increased in parallel with increased HE4 staining intensity. When results of FISH test which is performed to evaluate HER2/neu amplification, HE4 expressions were compared, significant confirmatory results were obtained (P = 0.011). Accordingly, number of FISH-positive cases increased in parallel with increased HEA staining intensity. When these two results were evaluated in combination, in breast tumors, one can say that in breast tumors, HER2/neu amplification increases in parallel with HE4 staining intensity. Since the presence of HER2/neu amplification is known to be a poor prognostic factor for breast cancer, one can conclude that strong HE4 expression is also an unfavorable prognostic parameter in breast cancer.
Kamei et al. detected a strong correlation between HE4 expression and lymph node metastases in breast cancer and suggested this finding as a probable marker in the prediction of lymph node metastasis. In our study, a significant correlation was not detected between HE4 staining status and lymph node metastasis. In the same study, 5-year survival rates were found to be lower among HE4-positive cases when compared with HE4-negative cases; however, overall survival rates did not differ between HE4-positive and -negative groups. Furthermore, in our study, in survival analysis performed in consideration of mortality rates and lifespans of the patients based on HE4 staining intensity, median lifespans of the patients did not differ significantly.
In the literature, nearly 75% of breast cancers were in the luminal group, while incidence of Her2 expressing and triple-negative groups was reported as 15% each., When the tumors were classified as for their molecular groups, percentages of cases were categorized in luminal B (32.3%), luminal A (24.5%), triple-negative (14.1%), and HER2-positive (29%) groups. More number of cases were included in HER2-positive group when compared with literature data.
The relationship between molecular subtypes and HE4 staining status was investigated, and most of the HE4 1+ and 2+ cases were seen in luminal B group, and increase in HE4 staining intensity also led to the inclusion of greater number of cases in HER2-positive group. A statistically significant difference was detected between these results (P = 0.005). This result supported the presence of parallelism between HER2/neu amplification and HE4 staining intensity.
| Conclusion|| |
This study has demonstrated the correlation between increase in the intensity of HE4 expression in breast tumors and HER2/neu amplification. Although HER2/neu amplification is a poor prognostic criterion, in the treatment of tumors with HER2/neu amplification in addition to hormonal therapy and conventional chemotherapeutic agents, a monoclonal antibody trastuzumab has been used. From these perspectives, HE4 can be a strong predictive agent for identifying groups with poor prognosis among patients with breast carcinoma, and at the same time, it may lead to development of new agents for targeted treatment. Further studies related to HE4 may offer new perspectives on these possibilities.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Moore RG, Brown AK, Miller MC, Skates S, Allard WJ, Verch T, et al.
The use of multiple novel tumor biomarkers for the detection of ovarian carcinoma in patients with a pelvic mass. Gynecol Oncol 2008;108:402-8.
Tamakoshi K, Kikkawa F, Shibata K, Tomoda K, Obata NH, Wakahara F, et al.
Clinical value of CA125, CA19-9, CEA, CA72-4, and TPA in borderline ovarian tumor. Gynecol Oncol 1996;62:67-72.
Kirchoff C, Habben I, Ivell R, Krull N. A major human epididymis-specific cDNA encodes a protein with sequence homology to extracellular proteinase inhibitor. Biol Reprod 1991;45:350-7.
Bingle L, Cross SS, High AS, Wallace WA, Rassl D, Yuan G, et al.
WFDC2 (HE4): A potential role in the innate immunity of the oral cavity and respiratory tract and the development of adenocarcinomas of the lung. Respir Res 2006;7:61.
Drapkin R, von Horsten HH, Lin Y, Mok SC, Crum CP, Welch WR, et al.
Human epididymis protein 4 (HE4) is a secreted glycoprotein that is overexpressed by serous and endometrioid ovarian carcinomas. Cancer Res 2005;65:2162-9.
Hellström I, Raycraft J, Hayden-Ledbetter M, Ledbetter JA, Schummer M, McIntosh M, et al.
The HE4 (WFDC2) protein is a biomarker for ovarian carcinoma. Cancer Res 2003;63:3695-700.
Galgano MT, Hampton GM, Frierson HF Jr. Comprehensive analysis of HE4 expression in normal and malignant human tissues. Mod Pathol 2006;19:847-53.
O'Neal RL, Nam KT, LaFleur BJ, Barlow B, Nozaki K, Lee HJ, et al.
Human epididymis protein 4 is up-regulated in gastric and pancreatic adenocarcinomas. Hum Pathol 2013;44:734-42.
Kamei M, Yamashita S, Tokuishi K, Hashioto T, Moroga T, Suehiro S, et al.
HE4 expression can be associated with lymph node metastases and disease-free survival in breast cancer. Anticancer Res 2010;30:4779-83.
Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature 2012;490:61-70.
Howlader N, Noone AM, Krapcho M, Garshell J, Miller D, Altekruse SF. SEER cancer statistics review, 1975-2011. Based on November 2013 SEER Data Submission, Posted to the SEER Web Site. Bethesda (MD): National Cancer Institute; 2014.
Rosai J. Breast. In: Ackerman and Rosai Surgical Pathology. Philadelphia: Mosby; 2011. p. 1659-770.
Perou CM, Sørlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, et al.
Molecular portraits of human breast tumours. Nature 2000;406:747-52.
Sørlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, et al.
Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A 2001;98:10869-74.
Karlsen NS, Karlsen MA, Høgdall CK, Høgdall EV. HE4 tissue expression and serum HE4 levels in healthy individuals and patients with benign or malignant tumors: A systematic review. Cancer Epidemiol Biomarkers Prev 2014;23:2285-95.
Zhu L, Zhuang H, Wang H, Tan M, Schwab CL, Deng L, et al.
Overexpression of HE4 (human epididymis protein 4) enhances proliferation, invasion and metastasis of ovarian cancer. Oncotarget 2016;7:729-44.
Vallius T, Hynninen J, Auranen A, Carpén O, Matomäki J, Oksa S, et al.
Serum HE4 and CA125 as predictors of response and outcome during neoadjuvant chemotherapy of advanced high-grade serous ovarian cancer. Tumour Biol 2014;35:12389-95.
Jiang Y, Wang C, Lv B, Ma G, Wang L. Expression level of serum human epididymis 4 and its prognostic significance in human non-small cell lung cancer. Int J Clin Exp Med 2014;7:5568-72.
Guo YD, Wang JH, Lu H, Li XN, Song WW, Zhang XD, et al.
The human epididymis protein 4 acts as a prognostic factor and promotes progression of gastric cancer. Tumour Biol 2015;36:2457-64.
Gündüz UR, Gunaldi M, Isiksacan N, Gündüz S, Okuturlar Y, Kocoglu H, et al.
A new marker for breast cancer diagnosis, human epididymis protein 4: A preliminary study. Mol Clin Oncol 2016;5:355-60.
Donegan WL. History of breast cancer. In: Breast Cancer. Ontario: BC Decker Inc.; 2006. p. 1-14.
Vuong D, Simpson PT, Green B, Cummings MC, Lakhani SR. Molecular classification of breast cancer. Virchows Arch 2014;465:1-4.
Kibris Sehitleri Caddesi 51/11, Alsancak, Izmir, 35220
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]