Study population

Seven hundred and twenty-six patients diagnosed with breast cancer by specialist breast surgeons and histopathological sections were recruited from Chung-Ho Memorial Hospital of Kaohsiung Medical University (KMU) (a medical center in southern of Taiwan) from September 2013 to June 2018 and follow-up of these patients was continued until July 2019. Patients under the age of 20, non-citizens (n ​​= 1), with benign breast disease ( n = 11), patients with any other cancer (n = 1) and patients with incomplete data (n = 11) were excluded. Since 66 patients had no urine samples, only 636 breast cancer patients had urine phthalate metabolite tests.

In this cohort study of patients, 636 patients with breast cancer were recruited in this study and 47 patients already in recurrence were excluded from this study and the patients were followed every 6 months until the end. of this study. For recurrence-free survival analyses, breast cancer recurrence and metastasis were study outcomes (recurrent patients, including patients who died of breast cancer), while cancer patients non-recurrent breast cancer were censored at loss to follow-up, withdrawal, and last follow-up date. Finally, a total of 589 patients with non-recurrent breast cancer with 1576.68 person-years were followed and 45 new cases of recurrent breast cancer were diagnosed. Prospective follow-up was performed to explore the association between phthalate exposure and risk of breast cancer recurrence in new recurrent patients (n=45) vs non-recurrent patients (n=544) (Fig. 1 ). The researchers regularly followed the patients for breast cancer recurrence, metastasis, and survival every 6 months by medical record, and extracted the patient’s medical history, including breast density, treatment, TNM stage ( TNM classification of malignant tumors), ER, PR, human epidermal growth factor receptor 2 (HER2), etc. Immunohistochemistry (IHC) was used to test for hormone receptors.

All of these patients provided morning blood and urine samples and completed questionnaires with trained interviewers to obtain information regarding sociodemographic characteristics, alcohol consumption, smoking status, dietary habits, reproductive history , family history of breast cancer, etc. According to the standard classification of the World Health Organization (WHO), BMI was classified as non-obese (BMI 2) and overweight or obesity (BMI ≥ 25 kg/m²). Written informed consents were obtained from all participants prior to the collection of data and biological samples. The study protocol was approved by the Institutional Review Board (IRB) of Chung-Ho Memorial Hospital of Kaohsiung Medical University (KMU) (IRB no. KMUHIRB-20120104, KMUHIRB-20140055 and KMUHIRB-G (I)-20150026). The study was carried out in accordance with the Declaration of Helsinki.

Phthalate reagents

Nine phthalate analytical standards were purchased from Cambridge Isotope Laboratories (CIL) (Andover, MA, USA); namely, MEP (product code: ULM-4585-MT-1.2), MnBP (ULM-6148-MT-1.2), MiBP (ULM-7919-MT-1.2), MEHP (ULM-4583-MT-1.2), mono -(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) (ULM-4662-MT-1.2), mono-(2-ethyl-5-carboxypentyl) phthalate (MECPP) (ULM-8149-MT-1.2), MEOHP (ULM-4663-MT-1.2), MMP (ULM-6697-MT-1.2) and MBzP (ULM-6149-MT-1.2). Nine internal phthalate standards were purchased from Toronto Research Chemicals (TRC) (North York, Ontario, Canada) and Cambridge Isotope Laboratories (CIL) respectively; namely, MEP-d4 (Cat. No.: M542582), MnBP-d4 (M525102), MiBP-d4 (M547702), MEHP-d4 (M542492), MEHHP-d4 (M542512), MECPP-¹³C4 (product code: CLM-8148-MT-1.2), MEOHP-¹³C4 (CLM-6640-MT-1.2), MMP-d4 (M566542) and MBzP-d4 (M524902).

Sample preparation

First morning urine samples were collected in a sterile brown glass bottle from all breast cancer participants and then frozen at -20°C until analysis. For analysis, urine samples were thawed at 4°C, then 1 mL of urine sample was transferred to a glass tube, followed by the addition of 10 μL of internal standards solution of mixed isotopes and 250 μL of ammonium acetate (1 M, pH 6.5). After adding 3 μL of beta-glucuronidase from E.coli K12 (Roche Diagnostics GmbH, Mannheim, Germany), the mixture was shaken and incubated for 90 min at 37°C in a water bath, then placed for 10 min at room temperature. The mixture was mixed with 2 mL of phosphate buffer (pH 2) for 30 s and centrifuged at 3500 rpm for 10 min at 4°C. The supernatant was purified with a solid phase extraction cartridge (Agilent ABS Elut-NEXUS) (Agilent Technologies, Palo Alto, CA, USA). After sample loading, 2 mL of formic acid and 1 mL of water were added to the cartridge, then eluted with 2 mL of acetonitrile and 2 mL of ethyl acetate and dried with nitrogen gas at 55℃. The residue was reconstituted by adding 200 μL of water and taken for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Evaluation of urinary metabolites of phthalates by LC-MS/MS

Concentrations of nine urinary phthalate metabolites: MEP, MnBP, MiBP, MEHP, MEHHP, MECPP, MEOHP, MMP and MBzP were measured by the same double-blind technician using the LC-MS/MS method, which was a Waters ACQUITY UPLC system (Waters Corporation, Milford, MA, USA) coupled to a Finnigan TSQ Quantum Ultra triple quadrupole MS (Thermo Fisher Scientific, San Jose, CA, USA) equipped with an electrospray ionization (ESI) source ) in negative ion mode and combined with Xcalibur software (Thermo Fisher Scientific).

A quantity of 10 μL of each sample was injected into an ACQUITY UPLC CSH Phenyl-Hexyl column (2.1 × 100 mm, 1.7 μm) (Waters Corporation, Milford, MA, USA) equipped with a BEH C18 column VanGuard pre-column (2.1 × 5 mm, 1.7 μm) (Waters Corporation) with a flow rate of 250 μL/min at 40℃ in the gradient of 30% solvent B (0.1% acetic acid in acetonitrile) for 0.0–5.0 min, then increased to 40% B for 10.0 min, 50% B for 12.0 min, and 100% B for 13.0 min, then reduced to 10% B in 13.1-15.0 min (solvent A: 0.1% acetic acid in water). The negative ion mode has been set as follows: ion sputtering voltage, 3000 V; sheath gas (N₂), 28psi; auxiliary gas (N₂), 10psi; and collision gas (Ar), 1.0 mTorr. Detection rates, precursor ion (m/z), product ion (m/z), retention times (min), and collision energy (V) are shown in Supplementary Table S1.

Method validation and sample determination

All experiments were performed in the laboratory of the KMU Research, Resource and Development Center with external quality assurance by the German external quality assessment system for toxicological analyses. A sample blank and quality control were performed simultaneously in each experiment with the analytical protocol. Urinary concentrations of phthalate metabolites were adjusted by creatinine for urine dilution. Creatinine adjusted units (μg/g) were calculated by dividing phthalate metabolite concentrations (μg/L) by creatinine concentrations (mg/dL) and multiplying by 100²².

The Σ₄DEHP was the sum of the urinary concentrations of MEHP, MEHHP, MECPP and MEOHP to represent exposure to a similar source²³. If the concentration values ​​were below the limit of detection (LOD), the values ​​were replaced by LOD/(square 2). The detection rates of MMP and MBzP were 0% and 68.9% separately; thus, the two metabolites were not considered in further analysis. The detection rates of the other seven urinary concentrations of phthalate metabolites were 98.4% to 100%. We calculated the intra- and inter-batch coefficient of variation (CV) for individual metabolites. Within-lot CV: MnBP (12.63%), MiBP (13.56%), MEHP (12.50%), MEHHP (7.89%), MECPP (13.85%) and MEOHP (9.97 %). Inter-batch CV: MnBP (8.18%), MiBP (6.34%), MEHP (7.83%), MEHHP (11.19%), MECPP (11.76%) and MEOHP (4.47 %).

statistical analyzes

Continuous and categorical variables of sociodemographic and clinical characteristics for the new recurrence and non-recurrence groups were compared using an independent sample. you-test and chi-square test or Fisher’s exact test. If the sociodemographic and clinical characteristics as risk factors associated with breast cancer were significantly different between the two groups, they were retained as potential confounding factors in the adjusted model. Creatinine-corrected urinary phthalate metabolite concentration values ​​were non-normal distributions, so the Mann-Whitney U or ln-transformed test for the independent sample you-test were used to analyze the differences between the two groups. Spearman’s correlation coefficients were used to assess correlations between individual concentrations of phthalate metabolites. Concentrations were categorized into quartiles based on the distribution of phthalate metabolite concentrations across all participants, with the first quartile defined as the reference group.

A multivariable Cox proportional hazards model was performed for the analysis of disease-free survival with new patients with recurrent breast cancer in relation to phthalate exposure. Hazard ratios (HR) and 95% confidence intervals (CI) were used to estimate the risk of breast cancer recurrence in relation to exposure to phthalates. Study patients were also stratified by ER (ER-negative vs. ER-positive), PR (PR-negative vs. PR-positive) and HER2 (HER2-negative vs. HER2-positive) status as well as the BMI (BMI 2 vs BMI ≥ 25 kg/m²) to estimate the interaction effect of phthalate exposure and hormone receptors as well as BMI on the risk of breast cancer recurrence. The interaction was estimated for the additive scale-based survival analysis in this prospective follow-up design.^24.25. The measure of the interaction effect as an additive scale was the relative excess risk due to interaction (RERI_TIME= e^β1+β2+β3−e^β1−e^β2+ 1)^24.26.

Since the associations between recurrent breast cancer and eight phthalate metabolites were assessed at the same time, we used Bonferroni’s correction (p-value multiplied by eight phthalate comparisons) to reduce potential issues (Type I error ) raised by multiple comparisons. Analyzes were performed using SPSS 22.0 and all tests were two-sided. p-values ​​

Ethical statements

Written informed consents were obtained from all participants prior to the collection of data and biological samples. The study protocol was approved by the Institutional Review Board (IRB) of Chung-Ho Memorial Hospital of Kaohsiung Medical University (KMU) (IRB no. KMUHIRB-20120104, KMUHIRB-20140055 and KMUHIRB-G (I)-20150026). The study was carried out in accordance with the Declaration of Helsinki.

Consent to participate/consent to publish

Written informed consents were obtained from all participants prior to the collection of data and biological samples. All authors have read and approved the article for publication.