CYP19A1 gene expression in the peripheral blood of Brazilian women with breast cancer relapse

Background The CYP19A1 gene, which encodes the enzyme responsible for androgen aromatization into estrogens, may play an important role in breast cancer aggressiveness. However, no study has evaluated CYP19A1 gene expression in the peripheral blood of women with relapsed breast cancer. Methods In this cross-sectional study, CYP19A1 gene expression was quantified by RT-PCR in the peripheral blood of 146 women with breast cancer who were first divided into two groups according to the expression of CYP19A1 (low and high); each group had 73 patients. Subsequently, women were divided into two groups: those without recurrence (control, n = 85) and those with recurrence (study, n = 61). Statistical analysis of the data was performed using ANOVA, the Mann-Whitney, Chi-square or Fisher’s exact test (p <  0.05). Results There were no significant differences between the relative expression of CYP19A1 mRNA in the low expression group and the high expression group according to the variables studied. There were no significant differences in CYP19A1 gene expression in the study and control groups (p = 0.8461). In the relapse group, CYP19A1 gene expression was significantly higher in the hybrid luminal subtype than in the triple-negative subtype (p = 0.0321), whereas it was significantly lower in HER2-negative cases than in HER2-positive cases (p <  0.0376). Women with locoregional recurrence showed higher expression than women with distant recurrence (p <  0.0001). Conclusions The present study found no significant differences between women with high and low expression of the CYP19A1 gene mRNA or between those in the study group and the control group. However, in women with recurrence, there was increased expression of CYP19A1 mRNA in those who had the luminal hybrid subtype and locoregional relapse and decreased expression in those negative for HER2.


Background
Breast cancer is the most common malignancy affecting women worldwide, and in 2018, the global estimate of impacted women was approximately 2,089,000, with a mortality rate of nearly 627,000 [1,2]. Its incidence is higher in the most developed regions of the world compared to developing and underdeveloped regions [3].
In Brazil, which is a developing country, breast cancer is the second most common malignancy in women after non-melanoma skin cancer, with an estimated 59,700 new cases and 15,403 cases of death from the disease in 2018 [4]. Additionally, approximately 40% of patients who develop disease recurrence die, especially in the first 2 to 3 years, when the risk of recurrence is higher [5][6][7]. Although physical examination and mammography are important to ensure early diagnosis of the disease and to reduce mortality, breast cancer is still frequently diagnosed in advanced stages in Brazil, resulting in high mortality rates, even with the current therapeutic strategies [8].
It has been suggested that the most appropriate therapeutic and prognostic strategies for breast cancer may be developed using genes that are associated with the development, growth and aggressiveness of breast cancer as biomarkers [9,10]. This includes the CYP19A1 gene that encodes the aromatase enzyme, which is involved in estrogen biosynthesis, as it promotes androgen aromatization in estrogens [11,12]. The CYP19A1 gene has been studied as a prognostic marker of breast cancer due to its genetic control in estrogen biosynthesis [13,14]. This gene has tissue-specific promoters, and principally, normal breast adipose tissue maintains low levels of aromatase expression primarily via the I.4 distal promoter. However, in breast cancer, an exchange between the I.4 and I.3 promoters and the I.7 and II promoters occurs, leading to increased production of aromatase and local estrogen [15,16].
Some studies have examined CYP19A1 gene expression in breast cancer using quantitative reverse transcription polymerase chain reaction (RT-PCR), which is considered a standard method for the quantitative measurement of gene expression; however, many of these studies have shown controversial results [16,17]. Miyoshi et al. found no significant association between CYP19A1 expression levels and breast cancer [18]. On the other hand, Friesenhengst et al. evaluated CYP19A1 expression in tumors of women with breast cancer, and the results showed a significant association between high CYP19A1 gene expression and estrogen receptor expression, menopausal status, metastasis-free survival, overall survival, disease-free survival and local and distant recurrence [11]. Thus, the controversies surrounding the gene expression of CYP19A1 in breast tumor studies and, to the best of our knowledge, the absence of studies analyzing the peripheral blood of women with recurrent breast cancer led to the design of this study.

Patients
This cross-sectional study involved 146 women from 34 to 80 years of age who had breast cancer and received care at the Mastology Clinic of Perola Byington Hospital (Sao Paulo, Brazil) between July and September 2018. The Internal Review Board of the Federal University of Piauı and Perola Byington Hospital approved the study under number CAAE: 43447015.8.0000, and all the patients signed an informed consent form prior to admission. The women were first divided into two groups, with low and high expression of CYP19A1 with 73 patients each. Subsequently, women were divided into two groups, without recurrence (control, n = 85) and with recurrence (study, n = 61). Women who were over 18 years of age, with and without breast cancer recurrence in the operable stage, who were diagnosed and treated in the past 10 years and had histologically confirmed diagnoses (disease at diagnosis) were included in the study. Women with a history of another neoplasm, a serious concomitant disease or an initial diagnosis of metastatic breast cancer were excluded from the study.

Blood sampling
Peripheral blood was collected by a specialized technician using a disposable syringe and needle after medical consultation. The first 1 mL of peripheral blood was discarded to prevent contamination by epidermal cells. A 1 mL sample of total peripheral blood from each patient was preserved in 3 mL TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) and stored at − 80°C until RNA extraction.
Total RNA extraction and cDNA synthesis RNA extraction was performed using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. RNA concentration, integrity and purity were analyzed using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Inc.) and agarose gel electrophoresis. Complementary DNA (cDNA) was synthesized from 2000 ng RNA using SuperScript III First-Strand Synthesis System (Invitrogen; Thermo Fisher Scientific, Inc.) with a total reaction volume of 20 μL containing 50 μM Oligo (dT) 20, 10 mM DNTP, 1 mL 10X RT buffer, 0.1 M DTT, 40 U/μL RNaseOUT and 200 U/μl SuperScript III RT. The incubation conditions for reverse transcription (RT) were 50°C for 60 min and 70°C for 15 min. The samples were placed in long-term storage at 4°C. The cDNA was kept at − 20°C and was diluted 10-fold prior to use in the quantitative RT-PCR.
Quantitative RT-PCR CYP19A1 mRNA expression was determined by quantitative RT-PCR using Power SYBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) and an ABI 7500 detection system equipped with SDS v1.4 software. The following primers were used for detection and quantitation of CYP19A1 mRNA: sense primer, 5′-CACATCCTCAATACCAGGTCC-3′; antisense primer, 5′-CAGAGATCCAGACTCGCATG-3′. BETA-ACTIN (ACTB) was used as an endogenous normalization control. The following primers were used for ACTB: sense primer, 5′-CACTGTGTTGGCGT ACAGGT-3′ and antisense primer, 5′-AAATCTGGCA CCACACCTTC-3′. Reactions were performed in a final volume of 13 μL, containing 3 μL DNA sample, 6.4 μL SYBR Green Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.), 0.4 μL primers (Custom TaqMan Gene Expression Assays, Applied Biosystems; Thermo Fisher Scientific, Inc.), and 2.9 μL of ultrapure sterile water, in 96-well plates using the StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). After initial denaturation for 10 min at 95°C, the samples were subjected to 40 amplification cycles, consisting of two steps: 15 s at 95°C and 1 min at 60°C. Samples were evaluated in duplicate, and two negative controls were added to each plate containing the same reaction compounds, but the DNA sample was replaced with water. Relative quantitation of CYP19A1 mRNA expression as a target was performed using the 2^-ΔCT method using the mean values obtained from the threshold cycle (CT) of 146 samples and the ACTB CT values as an endogenous control.

Statistical analysis
To evaluate the associations between CYP19A1 expression and clinical and histopathologic variables, the Mann-Whitney, Chi-square or Fisher's exact test and unidirectional ANOVA with multiple comparisons were performed using the Bonferroni post-test method. The values of p < 0.05 were interpreted as statistically significant. All statistical analyses were performed with Graph-Pad Prism software 6.0 (GraphPad Software, San Diego, CA, USA).

Correlations between CYP19A1 mRNA levels and histopathologic features
Using the median as the cut-off point, the patients were divided in low expression group and high expression group of CYP19A1. There were no significant differences between the relative expression of CYP19A1 mRNA in the low expression group and the high expression group according to the variables studied (Table 1). Patients were classified according to their sensitivity to endocrino therapy (ET) based on 2nd international consensus guidelines for advanced breast cancer, developed by European School of Oncology and European Society of Medical Oncology [19], responsive to ET, when relapses occur after 2 years of adjuvant ET or resistant when a relapses occurs in the first 2 years of adjuvant ET. In the current study, 44.27% of patients were considered responsive to endocrine therapy and 55.73% considered unresponsive.
Association of relative expression of CYP19A1 mRNA with the clinical and histopathologic features There were no significant differences in the relative expression of CYP19A1 mRNA in the study group compared to the control, p = 0.8461 (Fig. 1). In the study group, CYP19A1 mRNA expression was significantly higher in patients with a hybrid luminal molecular subtype than in patients with a triple-negative subtype, p = 0.0321 (Fig. 2). There were no significant differences in the relative expression of CYP19A1 mRNA in women with locoregional recurrence in the pre or post menopause (p = 0.116). No other associations were observed between the relative expression of CYP19A1 mRNA and the other variables studied, such as age, use of tobacco, menopausal status, grade, nodal status (N), tumor stage, estrogen and progesterone receptor, HER2 and histological type.
Correlations between the median expression levels of CYP19A1 mRNA and histopathologic features according to recurrence Using the median as the cut-off point, the median expression of CYP19A1 mRNA was classified as high or low and then analyzed to determine its association with clinical and histopathological features ( Table 2). The group of women with recurrence of breast cancer and negative HER2 receptor expression showed reduced CYP19A1 mRNA levels compared to those with positive HER2 receptor expression (p < 0.0376). Further, the mRNA expression of CYP19A1 was significantly higher in women with locoregional recurrence than in women with distant recurrence (p < 0.0001). There was no significant difference between the expression of CYP19A1 and the other variables studied.

Discussion
The detection of circulating CYP19A1 mRNA by RT-PCR has been shown to be significantly increased in breast cancer compared to normal controls, and quantitative RT-PCR is considered a sensitive and reliable method for studying mRNA in a variety of sites, such as bone marrow, lymph nodes, tissues and blood [20][21][22][23]. However, according to a survey of the literature, the evaluation of CYP19A1 mRNA by quantitative RT-PCR in the total blood of women with breast cancer recurrence was not reported, but some studies solely involving normal, peritumoral and tumoral tissues are available [11,18]. Peripheral blood has been used as a clinical sample for gene expression analysis in breast cancer, since peripheral blood samples are readily available, their acquisition is minimally invasive and they can be collected at low cost, thus making them an attractive alternative modality for diagnostic and prognostic purposes in cancer research [24,25]. According to some authors, there is a correlation between CYP19A1 mRNA levels in peripheral blood leukocytes and target tissues [26,27].
In the present study, comparison of peripheral blood CYP19A1 gene expression levels by quantitative RT-PCR between women with low and high expression and women with nonrelapsed breast cancer (control) and women with relapsed cancer (study) showed no statistically significant differences. In the group with relapsed cancer, CYP19A1 gene expression was significantly higher in women with a hybrid luminal molecular subtype than in women with a triple-negative subtype. Regarding tumor characteristics, the group of women with breast cancer recurrence showed a significant reduction in CYP19A1 mRNA in women with HER2-negative tumors compared to those with HER2-positive tumors. Additionally, CYP19A1 mRNA expression was significantly higher in women with locoregional recurrence than in those with distant recurrence, and there was no difference in relation to the other variables studied.
On evaluating the expression of the CYP19A1 gene in breast cancer tissue, Friesenhengst et al. [11] detected an association between the high expression of CYP19A1 in breast tumors and the incidence of breast cancer recurrence. However, consistent with our results, Girault et al. [28] and Licznerska et al. [29] evaluated CYP19A1 gene expression in breast cancer tissue and found no associations between CYP19A1 mRNA levels and disease recurrence. Darlix et al. [30] showed longer survival in women with hybrid luminal breast cancer than in those with triplenegative breast cancer, while other authors, such as Friesenhengst et al. [11] and Brown et al. [31], showed no association between CYP19A1 mRNA expression levels and molecular subtypes of breast cancer. However, these studies were performed in tumor tissue and had a smaller sample size than the present study.
Some authors have not shown an association between CYP19A1 gene expression in women with breast cancer and HER2 receptor status [11,32,33]. However, findings similar to those in this study were found by Subbaramaiah et al. [34], who showed lower levels of aromatase enzyme and CYP19A1 activity in HER2-negative tumors than in HER2-positive tumors. Some authors have shown that HER2 overexpression is the main determinant of increased expression of cyclooxygenase-2 and synthesis of prostaglandin E2 in breast tumor cells, which in turn, leads to increased CYP19A1 gene expression and aromatase activity [34][35][36].
Bollet et al. [32] showed a significant association between low expression of the CYP19A1 gene and an increased risk of locoregional recurrence. On the other hand, other studies have shown an association between the complete absence of CYP19A1 gene expression and shorter relapse-free survival [29,37]. Furthermore, consistent with our results, Friesenhengst et al. [11] and Salhab et al. [33] reported that high expression of the CYP19A1 gene was associated with increased locoregional recurrence. Estrogen synthesis in situ is believed to be primarily catalyzed by the enzyme aromatase, which is often overexpressed in breast tumors, thus explaining the increased levels of CYP19A1 mRNA in patients with   locoregional breast recurrence compared with those in patients with recurrence in regions more distal to the tumor such as liver, brain and bones [15,38]. Friesenhengst et al. [11] showed that CYP19A1 mRNA levels were significantly elevated in postmenopausal breast cancer patients. Tüzüner et al. [39] showed that the expression levels of the CYP19A1 gene were significantly decreased in patients older than 50 years. However, in agreement with our results, many studies have not shown any association between CYP19A1 gene expression and variables such as age, tobacco use, menopausal status, grade, nodal status, tumor stage, estrogen receptor, progesterone and histological type [18,28,29,32,37].

Conclusion
The present study found no significant differences between women with high and low expression of the CYP19A1 gene mRNA or between those in the study and control groups. However, in women with recurrence, there was increased expression of CYP19A1 mRNA in those who had the luminal hybrid subtype and locoregional relapse and decreased expression in those negative for HER2; nevertheless, further studies should be performed to consolidate the findings of the present study.