Breast Cancer and Healthy Control Subjects
Postmenopausal women with newly diagnosed histologically-confirmed breast cancer that was estrogen receptor (ER) positive and/or progesterone receptor (PR) positive and human epidermal growth factor receptor 2 (HER2) negative with resected stage I to III disease were eligible, prior to adjuvant endocrine therapy. For healthy controls, postmenopausal women without a history of malignancy were eligible. Women were defined as postmenopausal if they were at least 60 years of age, had undergone bilateral oophorectomy, or were younger than 60 years of age with cessation of regular menses for at least twelve consecutive months and plasma levels of estradiol and follicle-stimulating hormone in the postmenopausal range. Exclusion criteria included medical illnesses with potential suppressive or activating impact on immune and bowel function as determined by the treating investigator, systemic antibiotic or probiotic use within six months, use of hormone-replacement therapy within the past twelve months, or a history of gastric or intestinal surgery.
This was a prospective single institution case-control pilot study. Postmenopausal women with breast cancer were recruited from breast medical oncology practices at the NYU Langone Health’s Perlmutter Cancer Center (PCC) during routine office visits with their oncologists to discuss and/or initiate adjuvant endocrine therapy with an aromatase inhibitor. Postmenopausal women without breast cancer (controls) were recruited from three subject pools: (a) relatives and/or friends of patients; (b) women undergoing routine mammographic screening at PCC’s facilities; and (c) female faculty or staff members. All subjects with breast cancer were planned to receive adjuvant endocrine therapy with an aromatase inhibitor (i.e., anastrozole, letrozole, or exemestane). Enrolled breast cancer subjects provided serial plasma, stool, and urine samples at baseline (prior to initiation of endocrine therapy for the breast cancer patients), and then at 1, 3, 6, and 12 months while on endocrine therapy. Samples were collected at identical time points for the control subjects, of which none received endocrine therapy while enrolled on study. Analyses are reported on the baseline samples in this publication. The study was approved by the NYU Grossman School of Medicine Institutional Review Board (IRB). Written informed consent was obtained from all subjects prior to enrollment in the study. Subjects who completed the study received a one hour-massage at the NYU Integrative Health Center in appreciation for their participation.
Sample And Data Collection
Sample collection (plasma, urine, and stool) followed standardized procedures. After providing informed consent, study subjects were provided with a specimen collection kit with instructions for collecting a fecal sample at home, and materials to transport the specimens to NYU PCC. Fecal specimens were collected in four 20-mL screw top Sarstedt tubes, including two preloaded with 5 mL RNAlater (QIAGEN) and two with 5 mL sterile PBS. After specimen collection, the fecal tubes were secured, and two frozen gel packs were added. Urine was collected in a screw-top container, without preservative in the clinic. Blood samples were obtained by venipuncture in the clinic and collected in heparin containing vacutainer tubes. All specimens were transported to the NYU biorepository, where they were processed and stored at − 80°C. The urine and plasma specimens were shipped on dry ice in batches to the laboratory (A.F.) at University of Hawaii and stored at − 80°C until analysis.
Subjects in both groups also completed a questionnaire at baseline requesting information about age, height and weight, ethnicity, medical history of immune or gastrointestinal disorders, medication use including antibiotics within past the six months, diet, alcohol use, and smoking history (Online Resource 1). Follow-up questionnaires were completed at each subsequent time point inquiring about any changes in the above. Subjects were also followed for development of breast cancer recurrence or new malignancies.
Fecal Microbiome Analysis
The bacterial composition of the gut microbiome was assessed using 16S ribosomal RNA gene sequencing of subject fecal samples. FASTQ files were preprocessed with QIIME2 (v. 2018.11) . Following the data input, sequences underwent an error correction step with qiime dada2 denoise-paired (parameters: --p-trunc-len-f 0, --p-trunc-len-r 0, --p-trim-left-f 20, --p-trim-left-r 20) command. We then assigned taxonomy to the sequences by training a Naïve Bayes classifier on the V4 region of the 16S rRNA gene with qiime feature-classifier fit-classifier-I-bayes command based on the GreenGenes database (v 13_8). Following the taxonomy assignment, the abundance table was rarefied to the depth of 20,000 sequences and collapsed on both genus and species taxonomic levels. All downstream analysis including α-diversity, β-diversity and microbial composition was performed in R (v. 3.5.2). LefSe (Linear discriminant analysis Effect Size)  was used to find bacteria that explain differences between breast cancer and healthy control fecal samples with the following parameters: 0.05 alpha value and an LDA score threshold of 2.
Sex Hormone Analysis
The 11 most predominant steroidal estrogens in women, namely estrone (E1), estradiol (E2), 2-hydroxyestrone (2-OHE1), 2-hydroxyestradiol (2-OHE2), 2-methoxyestrone (2-MeOE1), 2-hydroxy-3-O-methylestrone (2OH-3MeO-E1), 4-hydroxyestrone (4-OHE1), 4- hydroxyestradiol (4-OHE2), 16α-hydroxyestrone (16α-OHE1), 16-ketoestradiol (16keto-E2), and estriol (E3)  as well as progesterone, and testosterone were measured from plasma and urine by our validated orbitrap liquid chromatography-mass spectrometry (LCMS) (model Q-Exactive, Thermo Scientific, Waltham, MA) assay, as described in detail previously .
In brief, plasma or urine were mixed with ascorbic acid as preservative and deuterated or 13C labeled analytes as internal standards followed by enzymatic hydrolysis with beta-glucuronidase and sulfatase for total analyte levels (conjugated plus unconjugated analytes) or without hydrolysis for unconjugated analytes followed by extraction with methyl tertiary butyl ether. The dried extract was derivatized with 1-methylimidazole sulfonyl chloride in sodium bicarbonate followed by analysis of the tagged analytes with high-resolution accurate-mass mass spectrometry in positive mode after electrospray ionization using monoisotopic protonated analyte masses ±5ppm to account for measurement inaccuracies as detailed previously . Urinary creatinine levels were determined using a clinical autoanalyzer (Roche-Cobas MiraPlus CC) and a kit from Randox Laboratories (cat. No. CR 510, Crumlin, UK) based on the Jaffé reaction with a lower limit of quantitation of < 15 µM. In this study, we found inter- and intra assay cv values of 0.8% at 187 µM. Urinary concentrations were adjusted for creatinine levels to account for differences in urine volume.
Circulating sex hormone binding globulin (SHBG) levels were measured with a well validated enzyme linked immunosorbent assay kit from R&D Systems, Inc. (Minneapolis, MN, kit# DSHBG0B, lot# P248238) following exactly the manufacturer’s protocol.
Differences in measured analyte levels between cases and controls were evaluated by the unpaired t-test with common variance (Microsoft Excel version 16.54).