Background: Emerging evidence demonstrates the effectiveness of targeted gene sequencing panels as a practical method for the diagnosis of inherited susceptibility to breast cancer. Sequencing of multiple high and moderate risk genes simultaneously accelerates the discovery of deleterious mutations (DM) or variants of unknown significance (VUS). However, a consequence of Multiplex Gene Panel (MGP) testing is the discovery of unexpected DMs in high or moderate risk genes other than BRCA1 or BRCA2 (BRCA1/2). The overall clinical utility and incremental gain of information conferred by MGP testing in hereditary cancer risk assessment is still unknown. Methods: We are conducting a multicenter prospective cohort study of patients undergoing cancer-risk assessment using a 25 gene sequencing panel, which includes APC, ATM, BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A, CHEK2, EPCAM, MLH1, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD51C, RAD51D, SMAD4, STK11, and TP53. Patients were recruited from August 2014 to June 2015 at three medical centers. Patients are enrolled if they meet standard criteria for genetic testing or are predicted to have a ≥ 2.5% probability of inherited susceptibility to cancer calculated by validated risk prediction models. We present a planned interim analysis after enrolling 500 of 2000 total participants. Results: HCP testing was performed for 332 patients referred for clinical suspicion of hereditary breast and ovarian cancer (HBOC). In this cohort, 96.7% were female (n=321) and the mean age was 50 years (standard deviation, SD=12.2); race/ethnicity was 43.1% Hispanic (n=143), 37% Non-Hispanic White (n=123), 4.2% Black (n=14), 10.5% Asian (n=35), and 1.8% other (n=6). Among this cohort, 37 tested positive for one deleterious mutation (DM) (11.1%: 95% confidence interval (CI), 8.2% to 15%) and 118 patients carried at least one variant of uncertain significance (VUS) (35.5%: 95% CI, 30.6% to 69%). Excluding BRCA1 or BRCA2, 14 patients (4.3%: 95% CI, 2.6% to 7.2%) have a DM in ATM (n=3), CHEK2 (n=2), MSH6 (n=1), MUTYH (n=3), PALB2 (n=1), PMS2 (n=1), RAD51C (n=2), and TP53 (n=2). In a patient with an unexpected PMS2 mutation, enhanced cancer surveillance based on Lynch Syndrome guidelines was recommended. Among 160 patients with a history of invasive breast cancer or breast DCIS, 19 patients carried a DM (11.8 %: 95 CI, 7.7% to 17.8%). Conclusion: In this multicenter prospective cohort study among a diverse group of participants undergoing 25-gene MGP testing, 11.1% of participants tested positive for a DM. Among participants testing negative for BRCA1 and BRCA2, MGP testing identified DMs in 4.3% of participants prompting clinically appropriate risk reduction recommendations and enhanced cancer surveillance. Ongoing recruitment and long-term follow-up are in progress. Citation Format: Idos GE, Kurian AW, Mcdonnell KJ, Ricker CN, Sturgeon DY, Culver JO, Lowstuter K, Hartman A-R, Allen B, Teeter C-R, Kingham KE, Koff R, Lebensohn A, Chun NM, Mills MA, Petrovchich I, Hong C, Ladabaum U, Ford JM, Gruber SB. Interim analysis of multiplex gene panel testing for inherited susceptibility to breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD7-01.