Serum Biomarkers to Characterize the Effects of Therapy on Ovarian Reserve in Premenopausal Women With Early-stage Breast Cancer or BRCA Mutations

Inclusion Criteria:

For Premenopausal Women with Early Stage Breast Cancer

• Premenopausal female patients age 18-44 with breast cancer as defined as: a. AJCC Stage 0-III breast cancer, regardless of hormone-receptor status orHER2-overexpression, before the start of planned adjuvant or neoadjuvantchemotherapy and/or hormonal therapy. If chemotherapy is planned, the regimen mustbe either CMF, anthracycline containing, or taxane containing. If hormonal therapyis planned, the regimen must be limited to tamoxifen. All biologics (e.g.bevacizumab, trastuzumab, lapatinib, etc.) are allowed in addition to the abovetherapies.

• Have regular menstrual cycles; patients can have no more than 1 irregular cycle (tooearly or too late) within the past year or were pregnant in the past 12 months,and/or at least 10 spontaneous cycles within the past year.

Subject Inclusion:

For Unaffected High Risk Premenopausal Women with BRCA mutations

• Premenopausal Women ages 25-45 with known BRCA mutations

• Have regular menstrual (21-35 days); patients can have no more than 1 irregularcycle (too early or too late) within the past year and/or at least 10 spontaneouscycles within the past year.

• No history of breast or ovary cancer.

Subject Inclusion:

For affected BRCA1 and BRCA2 mutation carriers, affected women with non-BRCA mutations, and BC patients with no mutations (control)

• Premenopausal women age 21-45 with stage 0-3 breast cancer

• No prior ovarian surgery or ovarian disease

• No prior chemotherapy

• Regular menstrual periods (21-35 days), no PCOS

• No hormonal contraception within the prior 4 weeks

• Mutation testing decision based on NCCN Guidelines V1.2021: according to theseGuidelines, both centers test all pre-menopausal women with breast cancer for BRCAand non-BRCA mutations which are the subject of this proposal

• Receiving an anthracycline (typically doxo/Adriamycin) and Cy (AC)-basedchemotherapy protocol

Exclusion Criteria:

For Cohort of Premenopausal Women with Early Stage Breast Cancer

• Prior therapy for breast cancer or any other cancer, such as chemotherapy, hormonaltherapy or immunotherapy.

• Ovarian resection, unilateral or bilateral oophorectomy, hysterectomy or radiationto pelvic region.

• Plans for risk-reducing bilateral oophorectomy within one year of completion ofchemotherapy.

• Prior known infertility; infertility is defined as one year of inability to conceivedespite unprotected intercourse and/or the need to use medication (e.g clomiphene)or assisted reproductive technology (e.g. intrauterine insemination or in vitrofertilization) to attempt pregnancy.

• Family history of a first-degree relative with non-surgical menopause < age 40

• Current pregnancy.

Subject Exclusion Criteria:

For Unaffected High Risk Premenopausal Women with BRCA mutation

• Prior chemotherapy or immunotherapy for breast cancer or any other cancer

• Ovarian resection, unilateral or bilateral oophorectomy, hysterectomy or radiationto pelvic region or ovarian disease (e.g. polycystic ovarian syndrome).

• Plans for risk-reducing bilateral oophorectomy within one year

• Prior known infertility; (except women who have undergone voluntary tubal ligation);infertility is defined as one year of inability to conceive despite unprotectedintercourse and/or the need to use medication (e.g clomiphene) or assistedreproductive technology (e.g. intrauterine insemination or in vitro fertilization)to attempt pregnancy.

• Family history of a first-degree relative with non-surgical menopause < age 40

• Current pregnancy Subject Exclusion Criteria For affected BRCA1 and BRCA2 mutationcarriers, affected women with non-BRCA mutations, and BC patients with no mutations (control)

• Subjects who did not undergo mutation testing, as well as those who tested positivefor more than one family (BRCA1, BRCA2, non-BRCA) of ATM-Pathway gene mutations,will be excluded.

• Women aged >42 years will be excluded as they carry a very high probability ofchemotherapy-induced OI49. It is important to select an age range where immediate OIis less likely to occur because if most women become menopausal post-chemotherapy,the comparison of ovarian reserve decline will not be feasible.

• In our prior grant period, we showed that the AC-based and CMF regimens similarlycompromise ovarian reserve (Fig. 1)9 Currently, the AC-based chemotherapy isutilized in >90% breast ca cases, and CMF protocol is rarely administered9. For thisreason and to enhance uniformity, rare non-AC-based protocols will be excluded.

• A previous study showed that smoking and BRCA mutations may have additive negativeeffects on the age of menopause27, consequently, we will exclude current smokers (smoked 100 cigarettes lifetime and currently smokes, per CDC). Based on our paststudies6,9, the smoking incidence is low in our study population (<20%) and shouldnot significantly limit accrual.

• BMI: In our recent publication9 (Fig. 1), the mean BMI was 24.22 ±0.4 (median 23.2;range 17-42); hence the extreme BMI values are rare in our population. Nevertheless,those with BMI of <18.5 and >40 will be excluded.