|Ahead of print
Serum sex hormone levels and hormone receptor status in identifying breast cancer risk in women
Kasuni H M Akalanka1, Sagarika Ekanayake1, Kamani Samarasinghe2
1 Department of Biochemistry, Faculty of Medical Sciences, University of Sri Jayewardenepura, Nugegoda, Sri Lanka
2 Department of Pathology, Faculty of Medical Sciences, University of Sri Jayewardenepura, Nugegoda, Sri Lanka
|Date of Submission||05-May-2019|
|Date of Decision||05-Jun-2019|
|Date of Acceptance||30-Jun-2020|
|Date of Web Publication||02-Nov-2020|
Department of Biochemistry, Faculty of Medical Sciences, University of Sri Jayewardenepura, Nugegoda
Source of Support: None, Conflict of Interest: None
Background: The association of sex hormones with receptor status and breast cancer (BC) incidence is studied with inconclusive results. The present work assessed the serum estrogen, progesterone, and testosterone concentrations and estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) status of newly diagnosed Sri Lankan BC patients and studied the possibility of risk assessment for BC using these parameters.
Methods: Serum estrogen, progesterone, and testosterone concentrations of newly diagnosed BC patients (n = 155) were assessed and compared with apparently healthy age-matched women (n = 75). Hormone concentrations were assessed with an enzyme immunoassay competition method with fluorescent detection (Biomerieux, France). Hormone receptor statuses were recorded from histopathology reports.
Results: Estrogen and progesterone concentrations were not significantly different according to the menstrual phase of premenopausal BC or healthy women or according to the menopausal status. Testosterone concentration of BC patients was significantly (P = 0.001) lower than in apparently healthy women. Estrogen and progesterone concentrations were not significantly different according to ER and PR status. However, progesterone concentrations of postmenopausal were significantly different (P = 0.021) among HER2 overexpressed women when compared to HER2-negative women. Postmenopausal women with progesterone level below 0.25 ng/mL were more likely to have HER2 overexpressed (P = 0.002).
Conclusions: Serum estrogen and progesterone concentrations of BC patients were not significantly different when compared with apparently healthy women. Women with testosterone level below 0.26 ng/mL demonstrated a higher risk of having BC. Serum progesterone concentrations of BC patients were significantly higher among HER2 overexpressed women compared to HER2-negative women.
Keywords: Breast cancer, estrogen, hormone receptor status, progesterone, testosterone
Key Message Low serum testosterone levels in women may be indicative of breast cancer development and progression.
|How to cite this URL:|
M Akalanka KH, Ekanayake S, Samarasinghe K. Serum sex hormone levels and hormone receptor status in identifying breast cancer risk in women. Indian J Cancer [Epub ahead of print] [cited 2020 Nov 24]. Available from: https://www.indianjcancer.com/preprintarticle.asp?id=299721
| » Introduction|| |
Long-term exposure to estrogen is associated with increased risk of breast cancer (BC). Increased concentrations of endogenous estrogen are strongly associated with increased risk of BC in postmenopausal women. Estrogen concentration in breast tissue of such patients is higher when compared with apparently healthy women. Estradiol (E2), acting through estrogen receptor (ER) alpha (ERα), stimulates cell proliferation and initiates mutations from replication errors during pre-mitotic DNA synthesis. The promotional effects of E2 subsequently support the growth of cells harboring mutations, which with time accumulate and induce neoplastic transformation.
Progesterone, essential for normal breast development, preparation for lactation, and breastfeeding, is significant in the development of BC. The actions are primarily mediated by its high-affinity receptors (progesterone receptor [PR]-A and -B). However, progesterone action in BC is grossly studied and remains controversial due to the difficulty in studying progesterone in isolation from other hormones (growth factors, prolactin), which could also contribute to BC biology. According to in-vitro studies, progesterone at a concentration similar to that of the third trimester of pregnancy exhibited strong antiproliferative effect. Progesterone levels are also associated with lower proliferation and lower ER expression by the tumors in postmenopausal patients. Thus, cyclical addition of progesterone to postmenopausal estrogen therapy is believed to protect against BC and improve prognosis.
Women with highest free testosterone concentrations had 3.3 times BC risk when compared against women with least concentrations. An increased risk was witnessed in postmenopausal and premenopausal women with higher serum testosterone concentrations., In contrast, some studies revealed that endogenous bioavailable testosterone concentrations were lower in women with BC compared to women in control group with prominence among postmenopausal women. Similarly, pre- and postmenopausal BC women with hormone deficiency when treated with subcutaneous testosterone implants or testosterone-combined aromatase inhibitor anastrozole had reduced incidence of BC. The higher bioavailability of testosterone counteracts the proliferative effects of estrogens on mammary tissue and inhibits cancer development and/or tumor growth.
BC cells express different types of receptors. The three major types are the ER, PR, and human epidermal growth factor receptor 2 (HER2) with some cells expressing none of these receptors (basal-like or triple negative). A study in Sri Lanka reported 31.7% of ER+ expression in women above 35 years of age. A higher ER+ expression (44.6%) with a similar PR+ (40.4%) expression was reported in India. In addition, 63% of ER+/PR+ tumors with lower incidence of other (ER+/PR− (13%), ER−/PR+ (3%), and ER−/PR− (21%)) tumors is reported. Older women were more likely to be diagnosed with ER+/PR+ tumors, whereas more than one-third of women of 30–39 years were diagnosed with ER−/PR− tumors.
Present study compared the serum estrogen, progesterone, and testosterone concentrations in newly diagnosed BC patients with apparently healthy age-matched women. The hormone receptor status of newly diagnosed BC patients was assessed and correlated with the hormone concentrations to study their associations as no such data are available in relation to Sri Lankan BCs.
| » Subjects and Methods|| |
A prospective cross-sectional study was conducted from 2013 to 2015.
Sample size was calculated considering the BC prevalence in Sri Lanka (19/100,000 population) with a standard normal variant at 5% type 1 error (1.96). Calculated sample size was 31. However, 155 newly diagnosed BC patients were enrolled for the study. Age-matched apparently healthy women (n = 75) were enrolled for the comparative study covering the minimum sample size.
Patient and control recruitment
Newly diagnosed BC patients (n = 155) who had not undergone any treatment (chemotherapy, radiotherapy, surgery, etc.) for BC were selected from National Cancer Institute Maharagama (Apeksha Hospital), Sri Lanka. The controls comprised a group of apparently healthy age-matched (25–75 years) women (n = 75).
The date of the first day of last menstrual cycle was recorded to approximate premenopausal women into follicular, ovulatory, and luteal phases. Average length of a menstrual cycle was considered 28 days. Accordingly, women in days 1–13, 14, and 15–28 were considered to be in follicular, ovulation, and luteal phases, respectively. If a woman had not had naturally occurring menstrual cycles during past 12 months or if they had undergone a hysterectomy without complete oophorectomy before menopause was considered as postmenopausal.
Serum hormone analyses
Venous blood samples from patients who have not undergone treatment for cancer and apparently healthy women (AHW) were collected and serum separated. Serum estrogen, progesterone, and testosterone concentrations (VIDAS® kits) were measured using enzyme immunoassay competition method with final fluorescent detection using Mini-Vidas analyzer (Biomerieux, France).
Hormone receptor status
The hormone receptor status (ER, PR, and HER2) of BC patients enrolled in the study (n = 155) was recorded from histopathology reports.
Statistical data analysis was carried out using SPSS version 16.0 (2007, SPSS for Windows, SPSS Inc., Chicago, IL, USA) package. The quantitative data with skewed distribution were presented as median (interquartile range [IQR]). The qualitative data were expressed by calculating the frequency and percentage. P value of less than 0.05 (P < 0.05) was considered to be significant. Nonparametric significances were analyzed by Mann–Whitney U test. Correlations of parametric and nonparametric data were analyzed by the Pearson and Spearman test, respectively. Receiver operative characteristic curve was plotted for determination of cutoff values of some selected biochemical parameters.
Ethical approval and informed consent
All procedures performed in the study involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Ethical clearance for the study was obtained from Ethics Review Committee, Faculty of Medical Sciences, University of Sri Jayewardenepura, Sri Lanka (652/12, 13/48). The approval for registering patients and accessing histopathology data were obtained from the Director, National Cancer Institute, Maharagama, Sri Lanka. Informed written consent was obtained prior to enrolling participants.
| » Results|| |
The study sample and the controls each consisted 33% premenopausal and 67% postmenopausal BC patients. Median sex hormone concentrations with IQR of premenopausal BC patients and AHW are illustrated in [Table 1]. Serum estrogen and progesterone concentrations at each phase among premenopausal BC patients were not significantly different when compared with age-matched controls. However, serum testosterone concentrations of BC patients were significantly lower (P = 0.001) than apparently healthy women.
|Table 1: Sex hormone concentrations of premenopausal breast cancer patients and healthy women|
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Median concentrations of postmenopausal sex hormones are illustrated in [Table 2] with IQRs. Serum estrogen and progesterone concentrations of postmenopausal BC patients were not significantly different to that of AHW but estrogen was noticeably lower. However, when compared with premenopausal women, postmenopausal women had significantly lower (P = 0.000) serum estrogen and progesterone concentrations. Serum testosterone concentrations of all BC patients irrespective of menopausal status were significantly low (P = 0.001) when compared with healthy women. The odds ratio in predicting risk association of low serum testosterone with BC risk was studied with a receiver-operating characteristic (ROC) curve and the area under the curve (85%; confidence interval (CI) 0.683–0.829) and testosterone cutoff value (0.26 ng/mL) determined with 69% specificity and 72% sensitivity [Figure 1].
|Table 2: Sex hormone concentrations of postmenopausal (BC and apparently healthy) women|
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|Figure 1: ROC curve for testosterone concentrations among breast cancer and apparently healthy women|
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ER, PR, and HER2 status of BC patients are illustrated in [Table 3]. Positive estrogen and PR expressions (ER+ PR+) were observed in 37.4% of BC patients, while 38.1% of patients were ER− PR−.
|Table 3: Frequency of hormone receptor expression among breast cancer patients (n=155)|
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The highest incidence (25.2%) of hormone receptor expression was observed to be ER+ PR+ HER2− tumors followed by ER− PR− HER2+ tumors (21.2%). The next highest incidence was triple negative tumors (17%) with ER− PR+ HER2 − tumors being least common.
Significant differences (P > 0.05) in serum estrogen, progesterone, or testosterone concentrations were not observed with respect to ER expression or PR expression among postmenopausal BC patients. Median sex hormone concentrations with IQRs are illustrated in [Table 4].
|Table 4: Sex hormone concentration according to receptor status among newly diagnosed postmenopausal breast cancer patients (n=97)|
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An increase in median estrogen level was observed among ER− PR− patients when compared with ER+ PR+ patients. However, any significant changes in sex hormones were not observed among women with above receptor expressions. Serum estrogen and testosterone concentrations were not different according to HER2 expression (P > 0.05). However, progesterone concentrations were significantly low (P = 0.021) among HER2 overexpressed postmenopausal BC patients when compared with HER2-negative patients. The odds ratio in predicting risk association of progesterone concentrations of HER2+, HER2− with BC risk was studied with ROC curve [Figure 2]. Area under the cure was 0.81 (95% CI 0.677–0.942). A progesterone cutoff value of 0.25 ng/mL was obtained with 76% sensitivity and 66% specificity.
|Figure 2: ROC curve for progesterone concentrations among HER2−and HER2+|
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| » Discussion|| |
Mean serum estrogen and progesterone concentrations among premenopausal BC women and premenopausal AHW were not significantly different (P > 0.05) at each phase of the menstrual cycle. However, the above values could vary as the estrogen concentration varies over the menstrual cycle and blood was collected without relating to menstrual cycle. The length of a menstrual cycle could vary from 25 to 35 days.
Likewise, the estrogen and progesterone concentrations were not significantly different among postmenopausal BC patients and controls. However, 92% of postmenopausal patients had serum estrogen below 58 pg/mL and 28% among them had serum estrogen closer to the lower limit of detection (9 pg/mL) but still within the recommended level. Increased concentrations of endogenous estrogen are strongly associated with increased risk of BC among postmenopausal women. However, it was not apparent in the present group of patients. Progesterone concentrations of 59% postmenopausal patients were closer to the lowest limit of detection (0.25 ng/mL). According to cell line studies, concentration of progesterone similar to third trimester of pregnancy exhibits strong antiproliferative effect. In addition, progesterone levels are associated with lower proliferation and lower ER expression by the tumors in postmenopausal patients. Postmenopausal estrogen therapy with cyclical addition of progesterone protects against BC. Thus, individualizing the treatment process specially among BC patients with hormone deficiencies will be beneficial in reducing the BC risk.
This is the first study in Sri Lanka that reports significantly lower (P = 0.001) testosterone concentrations among BC patients when compared to age-matched controls. Patients in the study sample with testosterone below 0.26 ng/mL had a risk of 3.64 (CI 2.04–6.52) times of having BC. Present study data agree with reported lower endogenous bioavailability of testosterone concentrations in women with BC.
Subcutaneous testosterone implants or testosterone combined with aromatase inhibitor anastrozole reduced the incidence of BC among pre- and postmenopausal women presenting symptoms of hormone deficiency. The higher bioavailability of testosterone counteracts the proliferative effects of estrogens on mammary tissue and thereby exerts a protective role to the breast, inhibiting cancer development and/or tumor growth. Further studies are needed to observe if this treatment could be applied to Sri Lankan women in preventing the incidence of BC.
The hormone receptor status of breast carcinoma could predict the response to adjuvant endocrine therapy. In the present study, ER expression was more common (55%) than PR expression (45%). Compared to previous Sri Lankan data where the ER+ were 32% and 46%, our results indicate a higher ER+ expression indicating a rise in prevalence through the years. The ER+ expression was also higher than reported Indian study (44.6%). The estrogen and PR expression (ER+PR+) was observed in 37.4% of BC women, while 38.1% of BC women were ER− PR−. The least common (7%) receptor expression was ER− PR+ in the study population. Higher percentage occurrence (63%) of ER+PR+ tumors and lower percentage occurrence (21%) of ER− PR− tumors were observed in a study in the USA. Considering the age at diagnosis and receptor status of the study sample, majority of women (54%) in the age group of 41–50 years were ER+ PR+, while majority of women above 61 years were ER−PR−. Among women in the age group of 31–40 years, a similar percentage of women were having ER+PR+ and ER−PR− carcinomas. However, with regard to the ER, PR status, and the age, contrasting observations had been reported. According to previous findings older women are more likely to be diagnosed with ER+PR+ tumors and younger BC patients are less likely to be ER+PR+.
Among the present study sample, HER2 overexpression was observed among 23%, while 43.4% showed a borderline expression. These results are in agreement with studies that reveal 25–30% of BCs are HER2+. Majority of women in all age categories had borderline HER2 expression. The HER2 gene encodes for HER2 receptor proteins on breast cells that control the growth of breast cells. In some BCs, the HER2 gene is amplified increasing the HER2 receptors causing uncontrolled growth of breast cells. Thus, HER2 overactivity is associated with adverse biological characteristics and poor clinical outcomes, and more than one-third (35%) of women in the age group of 31–40 showed HER2 overexpression. A negative association between HER2 receptor expression and serum progesterone concentration was observed. A BC patient having progesterone concentration lower than 0.25 ng/mL was more likely to be diagnosed with HER2 overexpressed status with high sensitivity and specificity. However, studies with larger sample sizes of hormone concentrations and hormone receptor status will aid in identifying the possibility of predicting the HER2 receptor status with a simple blood test instead of a biopsy, which would facilitate personalized treatment and management procedures.
Seventeen percent of women with BC in the study sample were identified as triple negative (ER− PR− HER2−) and the incidence was higher when compared with earlier studies that reported 8.1% of triple-negative BCs. Women in the age categories 41–50 and 51–60 were observed to have higher incidence of triple-negative cancer. Thus, present findings contrast the report that states women with triple-negative BCs are significantly more likely to be under the age of 40. Due to the absence of the hormone receptors, women with triple-negative BC are unlikely to respond to hormonal therapies.
| » Conclusions|| |
Serum estrogen and progesterone concentrations of BC patients were not significantly different when compared with AHW, irrespective of the menopausal status. However, serum testosterone concentration was significantly low among BC patients and women with testosterone concentrations below 0.26 ng/mL demonstrated higher risk of having BC compared to AHW. ER+ PR+ HER2− were found as the highest reported receptor expression followed by ER+ PR+ HER2+. Triple negative BC incidence was higher than reported. Serum progesterone concentrations of postmenopausal BC patients were significantly lower among HER2 overexpressed women compared to HER2-negative women. Further studies with large sample sizes are needed to conclude the association of sex hormone concentrations and hormone receptor status and if testosterone can be used in assessing the BC risk.
Financial support and sponsorship
The authors disclose receipt of the following University of Sri Jayewardenepura, Sri Lanka, grants ASP/06/RE/MED/2012/20 and ASP/06/RE/MED/2013/30 as financial support for the research.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]