Müllerian inhibiting substance (MIS), also known as anti-müllerian hormone, is produced by the granulosa cells of growing ovarian follicles. The level of MIS varies throughout a woman's lifetime. MIS is detectable from birth, but increases substantively at puberty. After puberty, MIS slowly decreases until it becomes undetectable after menopause [1]. MIS levels have recently been linked to breast cancer risk [2]. MIS, a member of the transforming growth factor (TGF)-β superfamily, is a 140 kDa dimeric glycoprotein that binds to the MIS type II receptor in breast tissue [3, 4]. MIS inhibits the growth of cultured breast cancer cells through G1 arrest and the induction of apoptosis [5, 6]. An eight-fold increase in the ratio of apoptotic cells in murine mammary tissue was observed after MIS injection [6]. Based on these observations, our hypothesis was that MIS would decrease breast cancer risk in premenopausal women, and that MIS serum levels would be different in women with benign breast disease vs. those with precancer or cancer. The objective of our study was to determine if MIS predicted the benign or malignant nature of a breast lesion requiring biopsy.
Material and methods
After Institutional Review Board approval, informed consent was obtained. Thirty blood samples were prospectively collected in serum separator tubes from premenopausal women 38-50 years of age scheduled to undergo diagnostic breast biopsy to determine the benign or malignant nature of a suspicious breast lesion identified on imaging or physical examination. Pregnant or lactating women, as well as women receiving chemotherapy or radiation therapy, were not eligible. The cohort of randomly collected samples included 14 from women diagnosed with cancer, 8 with precancer and 8 with benign lesions collected between November 2001 and May 2008. Participant samples were classified as "cancer" if the diagnostic biopsy demonstrated ductal carcinoma in situ or invasive breast cancer; "precancer" if the biopsy demonstrated atypical hyperplasia or lobular carcinoma in situ; or "benign" if the biopsy was benign. The blood was spun down, serum decanted and snap frozen at -80°C until analysis.
Samples were analyzed as a single batch by a scientist (WQ) blinded as to sample diagnosis using an MIS ELISA kit (catalog no. DSL-10-14400) from Diagnostic Systems Laboratories (Webster, TX), according to the manufacturer's instructions. The scientist analyzing the samples was blinded regarding the participant's diagnosis. The kit has a detection limit of 0.006 ng/mL. An intra-assay CV was calculated for all samples with all values that were at least three times the detection limit of the assay. The mean CV was 4.0% (range 0-16.1%). Values below the detection limit were assigned a value of half the detection limit, 0.003 ng/ml. The raw data were right-skewed and not normally distributed based on the Shapiro-Wilk test. Therefore, the data were logarithm transformed, checked using the same test, and found not to be significant. The transformed data were then analyzed. The distribution of MIS levels in the three risk groups (benign, precancer, and cancer) were assessed using analysis of variance (ANOVA). Pairwise differences between risk groups were analyzed using Fisher's LSD (least significant difference). Time from sample collection to analysis, participant age, age at menarche, body mass index (BMI), whether the participant had a first degree relative with breast cancer, current or ever use of oral contraceptive pills (OCPs) were assessed for their potential cofounding effects. All analyses were conducted using SAS JMP software.