Introduction: Breast cancer is the leading cause of cancer mortality among women. Some biomarkers and clinical features are used for the diagnosis and prognosis of this tumor, but no prognostic or predictive marker is routinely available specifically for hormone receptor positive tumors. Homocysteine is well known as a risk factor in atherosclerotic vascular diseases, but its participation in cancer biology is still unclear. The aim of this study was to evaluate serum Homocysteine and Cysteine as biomarkers of disease progression in breast tumor. As a secondary objective, the effect of a short course (one month) of hormonal treatment on Homocysteine, Cysteine and DNA methylation levels was also evaluated. Methods: Blood samples, tumor samples and normal adjacent tissue were collected during the initial biopsy (pre-treatment) and after one month of hormonal therapy (post-treatment). Serum Homocysteine and Cysteine were analyzed by HPLC and tissue global DNA methylation was determined by the Methylation-Sensitive Restriction Enzyme (MSRE) technique. Results: Variations in Homocysteine levels were significantly correlated with Disease-Free Survival. Cox proportional risk model demonstrated that nodal status and Homocysteine levels were independent prognostic factors for disease-free survival (DFS). A significant difference was observed between pre-and post-treatment levels of Homocysteine and Cysteine in advanced tumors, suggesting a prognostic role in patients with poor clinical characteristics. Conclusion: Although more studies are needed to confirm these results, our research suggests that Hcy might be used as a prognostic biomarker for breast cancer.
Breast cancer is the leading cause of cancer death among women worldwide [
Estrogen receptor positive (ER+) tumors have a better prognosis than most breast tumors, partly due to their response to hormonal therapies [3,4]. These drugs may modulate the estrogen receptor itself or inhibit estrogen production in post-menopausal women. Although it’s very effective, hormonal therapies cannot prevent relapse in about 30% of patients with estrogen receptor positive breast tumors, and no prognostic or predictive marker of response is routinely available for this group of patients [
Homocysteine (Hcy) is an extensively studied prognostic marker in atherosclerotic vascular diseases [
This work evaluates blood levels of homocysteine and its metabolite cysteine as biomarkers of survival and disease progression in post-menopausal women with non-metastatic breast cancer. We also studied the effect of a short-course of pre-operative (neoadjuvant) endocrine therapy on Homocysteine and Cysteine metabolism in a subgroup of these patients.
Ninety-seven post-menopausal breast cancer patients from two different institutions (Hospital São Paulo and Hospital Pérola Byington in São Paulo-Brazil) were evaluated. Sixty-seven of those patients participated in a randomized study of one month neoadjuvant endocrine therapy (tamoxifen or anastrozol) versus placebo.
The present study was approved by both Universidade Federal de São Paulo and Pérola Byington Hospital Institutional Review Boards, and all patients provided written informed consent. All patients were submitted to a diagnostic biopsy, and only patients with invasive breast carcinomas were included. Tumor samples were also obtained during definitive surgery for the removal of tumor tissue, for those patients participating in the randomized study. Serum and plasma were collected from all patients at the time of the initial biopsy (pre-treatment) and at the day of surgery (post-treatment) for patients in the randomized study.
For experimental purposes, clinical and pathological data were obtained from patients’ medical records. Observed clinical parameters were tumor size (T), lymph node positivity (N0, negative lymph node and N1; positive lymph node), clinical Stage (CS, initial stages = I and II; late stages = III), local relapse, distant relapse, date of last event (relapse, death or last visit) and cause of death. Initial tumor size was defined by clinical examination or ultrasound, whichever was greater. Final tumor size was defined by pathological gross examination.
Immunohistochemical characteristics such as estrogen receptor and progesterone receptor status were also recorded.
The total serum Hcy (tHcy) and Cys (tCys) levels were measured according to the method described by Pfeiffer and co-authors [
One microgram of DNA extracted from tumor sample or adjacent tissue was digested with either MspI or HpaII restriction enzymes (Fermentas, St. Leon-Rot, Germany). Non-digested DNA, and MspI and HpaII digested DNA were visualized in 0.8% agarose gel. Observed bands were quantified with ImageJ and methylation ratio was calculated subtracting HpaII digested DNA quantification from the MspI quantification, divided by nondigested DNA quantification (HpaII-MspI/non-digested DNA).
Kruskal-Wallis test was used to compare Hcy, Cys and pathological tumor size values, and these variables were correlated using Spearman correlation test when adequate [
Disease-free survival (DFS) was calculated from the date of diagnosis until date of first relapse or death, and presented using the Kaplan-Meyer method [
Multivariate analyses tested all variables found in the univariate analysis with a p-value lower than 0.05, and excluded patients with one or more missing data. All statistical tests were two-sided, and the level of significance was set at 5%. Analyses were done using the Statistical Package for Social Science SPSS (release 15.0, Chicago, Illinois, USA). The analysis adhered to the reporting recommendations for tumor marker prognostic studies.
Patients’ characteristics are listed in
Homocysteine (Hcy) and cysteine (Cys) levels were measured at the time of the initial biopsy in all patients (entire group). Patients that did not participate in the trial received therapy according to the discretion of their physicians, and had no Hcy or Cys measured after the biopsy.
Patients participating in the randomized trial (trial group) had Hcy and Cys measured again at the time of the surgery.
There were no statistical differences in basic characteristics , as well as type of post-operative treatment, between those patients in the randomized trial and those not participating in the trial, and among groups in the randomized trial (data not shown). Because there were no statistical differences between groups, we treated the data as belonging to the same group.
The entire group had biochemical characteristics measured at the time of the initial biopsy, but variations (∆) were calculate only for trial patients, which had samples collected both at the time of the initial biopsy and at the time of surgery. Results are shown in
Homocysteine and Cyste ine l evels, both initial and at surgery, and their variations (∆) were weak or moderately correlated between themselves (r = 0.579, r = 0.756 and r = 0.621, respectively, with p < 0.001 for all tests). Initial Cysteine levels were weakly and inversely correlated with initial global DNA methylation levels (r = −0.366, p = 0.028). These results are expected in light of the metabolic relationships among Homocysteine, Cysteine and DNA methylation in the methionine cycle [
anastrozol, tamoxifen or placebo (trial group). Homocysteine levels decreased significantly in patients with advanced disease (
There was no significant statistical correlation regarding Hcy or Cys levels variations and tumor size (data not shown). Interestingly, one patient in the anastrozole group presented a residual tumor with less than 1 mm of size, and other showed no residual tumor after surgery. In the tamoxifen group, one patient as well had no detectable tumor mass after surgery.
Hcy is a crucial component of the methionine cycle, which is linked to DNA methylation [
Regarding the initial biopsy, we found no significant statistical differences between serum levels of Hcy (r = −0.013; p = 0.938), or Cys (r = −0.069; p = 0.672), and
DNA methylation for normal and tumor tissue (r = −0.214; p = 0.645 and r = 0.405; p = 0.62) for the entire group or the trial group.
We also did not find any significant statistical correlation between Hcy and Cys variations (∆Hcy and ∆Cys) and the use of antiestrogens or placebo on a multivariate analysis (data not shown).
Global survival was not significantly correlated with any clinical (data not shown) or isolated biochemical parameters, including Hcy or Cys (
We found a significant inverse correlation between increased Homocysteine levels, and disease-free survival (
Increased Homocysteine levels have long been studied as a risk factor for cardiovascular disease, and more recently for some neurological problems and pregnancy complications [
and as a risk factor for breast cancer [
Breast cancer is a very common type of cancer, and has been studied extensively. Although several biomarkers are described in the literature [18-20], there are many efforts to increase the biomarker list and to personalize cancer diagnosis and treatment. However, most of these efforts look at molecular profiles and depend on molecular genetics facilities or tissue sampling processing, which are expensive and not largely available.
Hcy metabolism can be disrupted by defects in transulfuration or remethylation pathways, which may cause increases in Hcy levels. The accumulation of Hcy levels can result in Cys levels augmentation [17,21]. As expected, our results show a correlation between Hcy and Cys levels, both before and after antiestrogens therapy. A correlation between Hcy and Cys variations (∆Hcy/ΔCys) was also observed.
DNA methylation is a well-studied epigenetic mechanism involved in gene expression control [
Soares and co-authors [
We also correlated Hcy and Cys levels variations with clinical-pathological features of breast tumors, as nodal status (N) and clinical stage (CS). Our results show a significant reduction of the Hcy and Cys levels after antiestrogens or placebo in patients with poor clinical characteristics (Figures 1(a), (c) and (d)). Although we could not find a statistically significant difference for nodal status and Hcy levels (
At this moment, few data are available regarding Hcy role in tumor biology. Some efforts have been done to evaluate the relationship between Hcy and Cys levels with the risk of developing breast tumors. Some studies observed that an increased breast cancer risk was associated with high levels of Hcy or Cys in women with low levels of folate [17,21,26]. In addition, high plasma or serum levels of Hcy were also observed in patients with other cancer types [16,27-31].
Although most authors analyzed Hcy levels and other methionine cycle components as factors for developing cancer, only a few studies discussed the possible role of Hcy as a prognostic factor. Bobe and co-workes [
To check the possibility that the Hcy and/or Cys present prognostic or predictive value, we evaluated patients with resectable breast tumors, followed for a period of four to seven years. Due to a small number of fatal events, the study lacked power to determine the contribution of the Hcy and Cys as prognostic factors in overall survival (OS). Nevertheless, our results show that an increase in Hcy levels after a short course of neoadjuvant therapy is significantly correlated with a longer disease-free survival (DFS) (
The short-term course of tamoxifen or anastrozol had little influence on overall survival (OS) or disease free survival (DFS) (data not shown). A study from Washington University [
High Hcy level is a well-known cardiovascular risk factor and Hcy levels have also been suggested as a potential tumor marker [
The authors thank Dr. Andre Mattar for providing patient samples, Pérola Byington Clinical Research Department and São Paulo Hospital Serviço de Arquivo Médico e Estatística for patients’ clinical data. The authors would like to thank Universidade Federal de São Paulo, Fundação de Amparo à Pesquisa do Estado de São Paulo (2011/12306-1, to M.G.J.; 2008/56564-1, to C.S.P.; 2010/ 04585-5, to G. Aguiar; 2010/00245-5 to B.F.A.C), Conselho Nacional de Desenvolvimento Científico e Tecnológico (151578/2009-5, to D.F.I.; Research felowship 306485/2008-7 to M.G.J. and to 301742/2010-3 V.D’A.) and Coordenação de Aperfeiçoamento Pessoal de Nível Superior-CAPES (2745/2010, to D.F.I.).