**3.1 Clinico-pathological characteristics of recruited patients**

The clinico-pathological characteristics of EC patients included in the analysis are summarised in **Table 1**. Median age was 65 years (range, 30 – 79 years). There were 28 males and 6 females with male-to-female ratio of 5.7:1. Of those 34 patients, 28 patients had EAC and 6 patients had ESCC. One-year disease specific survival was approximately 73.5%. Twenty-five (74%) patients received neoadjuvant therapy.

## **3.2 Sex hormones profiles**

The median (Inter quartile range) (IQR) of the serum sex hormones levels (34 patients) against their corresponding laboratory reference ranges (Derby Hospital biochemistry department) is summarised in **Table 2**. Median serum levels of FSH and LH hormone were higher than normal. However, the median of serum levels of E2, progesterone, testosterone, and SHBG were within the normal ranges **Table 2**.


*Serum Sex Hormone Profiles in Potentially Resectable Esophageal Cancer DOI: http://dx.doi.org/10.5772/intechopen.95030*

#### **Table 1.**

*Patient Characteristics.*

In a gender-based analysis, there were significant higher median (IQR) levels of LH and FSH in female patients compared to LH and FSH levels in male patients. In contrast, women had significantly lower median levels E2, testosterone compared to men. There was no significant difference in serum levels of progesterone and SHBG between men and women (**Table 3**).

### **3.3 Correlation between serum sex hormones level and estrogen receptor expression**

The results of correlation between serum sex hormones and ERα and ERβ expression at mRNA are summarised in **Tables 4** and **5**. There was no significant correlation between the expression of ERα or ERβ mRNA in normal mucosa or tumour samples and serum level of LH, FSH, E2, Testosterone, FAI, or SHBG. In analysis of the correlation of hormones levels with estrogen receptors expression in both genders, we found that there was significant inverse correlation between testosterone level and ERβ mRNA expression in normal mucosa from male patients (r = - 0.41, *p* = 0.03) (**Table 6**). In female patients, there was significant inverse correlation between progesterone level and ERα mRNA expression in EC samples (r = - 0.87, *p* = 0.04) level (**Table 7**). No correlation was demonstrated between the levels of rest of sex hormones and ERs expression in either gender (**Tables 6** and **7**).


*LH, luteinizing hormone; FSH, follicular stimulating hormone; E2, 17*β*-Oestradiol; SHBG, serum hormone binding globulin; FHI, free androgen index.*

#### **Table 2.**

*The median (IQR) of serum sex hormones levels of patients with EC (n = 34).*


*LH, luteinizing hormone; FSH, follicular stimulating hormone; E2, 17*β*-Oestradiol; SHBG, serum hormone binding globulin; FHI, free androgen index.*

*\*Calculated using two-tail Mann Whitney U test.*

#### **Table 3.**

*Gender-based comparison of serum sex hormones levels in patients with EC.*


*LH, luteinizing hormone; FSH, follicle stimulating hormone; E2, 17*β*-Oestradiol; SHBG, serum hormone binding globulin; FHI, free androgen index; r, the correlation coefficient. \*Calculated using two-tail Spearman correlation.*

#### **Table 4.**

*Correlation between serum sex hormones levels and ER*α *mRNA expression.*

#### *Serum Sex Hormone Profiles in Potentially Resectable Esophageal Cancer DOI: http://dx.doi.org/10.5772/intechopen.95030*


*LH, luteinizing hormone; FSH, follicle stimulating hormone; E2, 17*β*-Oestradiol; SHBG, serum hormone binding globulin; FHI, free androgen index; r, the correlation coefficient.*

*\* Calculated using two-tail Spearman correlation.*

#### **Table 5.**

*Correlation between serum sex hormones levels and ER*β *mRNA expression.*


*LH, luteinizing hormone; FSH, follicle stimulating hormone; E2, 17*β*-Oestradiol; SHBG, serum hormone binding globulin; FHI, free androgen index; r, the correlation coefficient. \*Calculated using two-tail Spearman correlation.*

#### **Table 6.**

*Correlation between serum sex hormones levels and estrogen receptors mRNA expression in male patients (n = 28).*


*LH, luteinizing hormone; FSH, follicle stimulating hormone; E2, 17*β*-Oestradiol; SHBG, serum hormone binding globulin; FHI, free androgen index; r, the correlation coefficient. \*Calculated using two-tail Spearman correlation.*

#### **Table 7.**

*Correlation between serum sex hormones levels and estrogen receptors mRNA expression in female patients (n = 6).*


*LH, luteinizing hormone; FSH, follicle stimulating hormone; E2, 17*β*-Oestradiol; SHBG, serum hormone binding globulin; FHI, free androgen index; r, the correlation coefficient. \*Calculated using two-tail Spearman correlation.*

#### **Table 8.**

*Correlation of serum sex hormones level & 1-year disease specific survival.*


*LH, luteinizing hormone; FSH, follicle stimulating hormone; E2, 17*β*-Oestradiol; SHBG, serum hormone binding globulin; FHI, free androgen index; T-stage, tumour depth; LN-stage, lymph node involvement; VI, vascular invasion; BM, barrett's metaplasia; CRM, circumferential resection margin; r, the correlation coefficient. \*Calculated using two-tail Spearman correlation.*

#### **Table 9.**

*Correlation between serum sex hormones levels and pathological features in male patients (n = 28).*


*Serum Sex Hormone Profiles in Potentially Resectable Esophageal Cancer DOI: http://dx.doi.org/10.5772/intechopen.95030*

*LH, luteinizing hormone; FSH, follicle stimulating hormone; E2, 17*β*-Oestradiol; SHBG, serum hormone binding globulin; FHI, free androgen index; T-stage, tumour depth; LN-stage, lymph node involvement; VI, vascular invasion; BM, barrett's metaplasia; CRM, circumferential resection margin; r, the correlation coefficient. \*Calculated using two-tail Spearman correlation.*

#### **Table 10.**

*Correlation between serum sex hormones levels and pathological features in female patients (n = 6).*

#### **3.4 Correlation between serum sex hormones levels and clinico-pathological features**

Gender-based analysis of correlation between serum hormones levels and survival outcome at one year and pathological features after intended curative resection was performed using two-tail Spearman test. There was highly significant positive correlation between 1-year disease survival outcome and serum E2 level in female patients (r = 1, p < 0.0001) (**Table 8**). However, no significant association was determined between sex hormones levels and 1-year disease specific survival in male patients (**Table 7**).

No correlation was demonstrated between serum hormonal levels and pathological features of EC from either male or female patients (**Tables 9** and **10**).

#### **4. Discussion**

In this study, we investigated sex steroid hormones profiles in patients with established EC. Our results demonstrated that there was no correlation between serum level of LH, FHS, E2, SHBG, or FAI and ER expression in normal esophageal mucosa or EC from both genders. Whilst 1-year disease survival is significantly correlated with high E2 level in female patients, no significant correlation is identified between survival outcome and sex hormones level in male patients.

The disparity in the incidence of EC between males and females has always raised the question regarding the role of sex hormones and sex EC in esophageal carcinogenesis. In an *in vitro* study, Matsuoka et al. were first to investigate the

effect of E2 and testosterone on the growth of newly established cancer cell lines of ESCC origin, which expressed both ER and androgen receptor (AR). They found that the growth rate of the cell lines was inhibited by estrogen and enhanced by testosterone [29]. In a similar way, Utsumi et al. suggested that the presence of ER arbitrates the inhibitory effect of estrogen when it was noted that the growth of ER positive transplanted tumour was significantly greater in male than it was in female nude mice. Such a difference was not observed for ER negative tumour. Also, the growth rate of ER positive tumours was enhanced in oophorectomised female mice and significantly suppressed with a physiological dose of E2 compared to ER negative cancer [30, 31]. Likewise, Ueo et al studied sex hormone dependency and hormone responsiveness on both ER positive (KSE-1) and ER negative (KSE-2) ESCC cell lines transplanted in male and female nude mice. They found that the administration of E2 significantly inhibited the growth of KSE-1 tumours in both males and females in conjunction with an increase in E2 levels. No similar influence on KSE-2 growth was identified. Therefore, they suggested that the growth of human EC cells with sex hormone receptor is influenced by circulating hormone levels and can be manipulated by systemic E2 administration [32].

EAC often expresses ER, which suggests a possible biological involvement of steroid hormones. Wang et al. suggested that ER and their signalling could have a role in EC development when he found that 43.75% (21/48) and 20.83% (10/48) of EC cases were positive for ER and progesterone receptor (PR) respectively, while these receptors were negative in esophageal tissues taken from normal subjects [33]. Similarly, Tiffin et al suggested that the role of ER may warrant further investigation when they identified mild to moderate ER staining in most of their esophageal tissue samples. However, the author did not discriminate between the ER subtypes detected [34]. Interestingly, Nozoe et al found that ERα expression was significantly higher in male patients with ESCC in comparison to females. Also, a positive expression of ERα plus negative expression of ERβ was found to be an unfavourable independent prognostic indicator in patients with ESCC. Therefore, they suggested that hormonal therapy using estrogen may have a role in improving the outcome in this type of cancer and its possible anti-tumour effect requires more in depth both laboratory and clinical based investigations [35]. Akgun et al. studied the expression of ERβ in Barrett's metaplasia and associated EAC. They concluded that all EAC and most precursor lesions, Barrett's metaplasia with or without dysplasia, express ERβ in a significantly high percentage of the cells. These findings raise the possibility that EAC may benefit from treatment and/or chemoprevention by SERM [36]. Likewise, Liu et al. found that ERβ1, ERβ2, ERβ3 and ERβ5 are overexpressed in EAC compared to its precursor lesion Barrett's metaplasia negative for dysplasia, suggesting a significant biological role [37]. In contrast, Kalayarasan et al. studied the expression of ERα and ERβ as well as Progesterone Receptors (PR) in both tumour tissue and adjacent normal mucosa samples of 45 cases of EC (ESCC = 30 cases, EAC= 15 cases). They found neither tumours nor normal mucosa expressed ERα or PR. However, all cases of EAC, irrespective of their stage/grade, were strongly positive for ERβ and the intensity of staining in tumours was significantly higher than in normal adjacent mucosa. Therefore, they suggested that estrogen may have an effect on the growth of EAC and this effect may be mediated by ERβ [38]. Despite numerous studies assessing the role of sex hormones in AEC development, the findings are still featured by some inconsistency. It seems therefore important that the molecular mechanisms of ER in esophageal carcinogenesis are further clarified.

Recently, Zuguchi et al found that there is increased nuclear ERβ reactivity in human ESCC in comparison to matched normal mucosa [39]. Moreover, increased ERβ expression seems to have unfavourable correlation to the histo-pathological stage of the disease [39]. Therefore, they concluded that EC is an estrogen dependant

#### *Serum Sex Hormone Profiles in Potentially Resectable Esophageal Cancer DOI: http://dx.doi.org/10.5772/intechopen.95030*

malignancy and ERβ might provide an additional therapeutic target for treatment of ESCC [39]. Another study by Sukocheva et al demonstrated that tamoxifen and raloxifen inhibited the growth of esophageal AC cell lines proliferation by inducing apoptosis and cell cycle arrest [40]. However, the study did not differentiate between the role of each receptor, especially when we know for fact that SERMs have different agonist and antagonist function of ER in different body tissues.

Several studies demonstrated that women undergoing curative EC resection have better long term survival outcome compared to men [6, 7]. In a case control study, Wang et al, found that there was a low serum E2 level from healthy controls from an area with a high incidence of ESCC compared to counterparts from a low incidence area in China [41]. Hence, they suggested that the discrepancy in incidence of ESCC may be explained by the lack of a protective E2 effect [41]. In another study, Petrick JL et al identified a high ratio of androgens to estrogens – particularly testosterone:estradiol ratio – was more common in EAC patients than controls, including after restriction to cases without weight loss in the previous 5 years [42]. The lack of association between circulating E2 and ER expression in this study may reflect the theoretical possibility that ER expression is influenced more by intra-tumor E2 rather than circulating hormone concentrations [43, 44]. For instance, Recchione et el found that E2 level in breast cancer samples is significantly higher compared to serum level of E2. Similarly, in a comparison of blood and breast cancer tissue concentrations sex steroids by Secreto et al showed that there was significantly higher level of E2 in the tumours than in the blood [45].

Recently, Xie, S-H et al found that an increased disease-specific mortality with lower SHBG levels and higher FSH levels in male EAC patients without surgical treatment. No clear associations were observed for dehydroepiandrosterone sulphate, LH, prolactin, testosterone, 17-OH-progesterone, progesterone, E2, androstenedione, testosterone:estradiol ratio or free testosterone index [46]. In our study, there was no correlation between sex hormones level and pathological features. However, we found higher serum E2 level had significant positive correlation with 1-year disease specific survival in women. This may indicate that E2 may play antiproliferative effect and in turn reduce the risk of developing distant micro metastases. For example, high dose estrogens, like diethylstilbestrol found to paradoxically inhibit breast cancer growth by activation of Fas/FasL apoptotic pathway [47]. In another in vitro study, Al-Khyatt et al found that ERs antagonists induced apoptosis in EC cell lines proliferation. Thus, they concluded that their findings may indicate that the ER system is involved in OC progression and thus may provide a novel target for the treatment of OC [25].

In a study by Awan et al [48], a raised testosterone level had a positive correlation with the presence of EC compared to control group. Furthermore, serum testosterone levels decreased after surgical resection of the tumour [48]. In this study it was demonstrated that testosterone had an inverse correlation with ERβ mRNA expression in normal mucosa from male patients. Several studies investigated the role of androgens in the pathogenesis of estrogen-dependant cancers like breast cancer [49]. Nevertheless, the significance of cross-talk between androgens and ER in EC is not clear and warrants further investigation [50].

The number of patients used in this study is relatively small, especially female patients. This could be the reason behind the lack of any association between serum sex hormones and ER expression or pathological features. Another limitation is there was no matched healthy control. The serum sex hormones levels of healthy controls could have been used for comparison as well as studying the correlation between hormones levels and risk factors profiles for EC.

In summary, in this study, there was no association between sex hormones profiles and the expression of ER or pathological features of EC in both genders.

#### *Reproductive Hormones*

Interestingly, survival outcome was better in women with increased serum level of E2. Current published evidence supports that sex hormones may play a role in esophageal carcinogenesis. Hence, future research work may include a populationbased study looking into the correlation of sex hormones and risk factors profiles for EC in healthy volunteers and patients is required. Likewise, a well-designed *in vivo* study to address the potential therapeutic role of ERα and ERβ in treatment of EC is warranted.
