Background: Patients (pts) treated for early stage breast cancer have a 30% lifetime risk of developing incurable, distant metastatic disease. Current models suggest that this occurs through escape of cells from the primary tumor into the circulation and subsequent sequestration of “disseminated tumor cells” (DTCs), in the bone marrow and other sequestration sites, where they enter dormancy. DTCs identified by immunohistochemistry (IHC) are associated with poor prognosis in longitudinal studies and meta-analyses, increasing odds of recurrence by approximately 2 to 5-fold. However, little is known about the test characteristics of the DTC-IHC assay, clinical DTC detection rates over time, and patient and disease risk factors that can identify pts harboring these cells. Methods: The PENN-SURMOUNT Screening Study (NCT 02732171) is a prospective, longitudinal study examining bone marrow and blood biomarkers of recurrence among pts within 5 years of diagnosis who have completed therapy for primary breast cancer (with the exception of endocrine therapy). Pts with positive lymph nodes, triple negative receptors, ER-positivity with RS ≥ 25 and/or high-risk MammaPrint (MP), or residual disease after neoadjuvant chemotherapy were screened with bone marrow aspirate (BMA) for presence of DTCs. A positive DTC-IHC result is defined by the presence of at least one pancytokeratin-DAB positive cell utilizing the methods of Naume et al. Cytospin slides prepared from the BMA are independently reviewed by two pathologists with adjudication for the presence of DTCs; Pts who screen negative for DTCs are offered repeat screening annually. Pts who screen positive are referred to an interventional clinical trial (CLEVER, NCT 03032406). Results: A total of 194 pts screened eligible for enrollment on PENN-SURMOUNT between 6/2016 and 3/2020. Of these, 158 consented and 151 underwent BMA with successful IHC analysis on 100%. Pts came from 22 U.S. states; ≥ 1/3 traveled over 50 miles to the study center. At baseline BMA, 36/151 (24%) had at least 1 measurable DTC by IHC. Patient characteristics and DTC distribution among subpopulations are shown in Table 1. Of the 78/115 who were initially DTC negative and continued to be eligible for repeat screening, as of 3/2020, 46 (59%) returned for at least one repeat BMA. 13/46 (28%) had at least 1 detectable DTC on 1 of up to 3 subsequent follow up assessments for a total DTC positivity rate of 32.5% (49/151). 48 (98%) DTC+ pts have subsequently enrolled on the CLEVER trial. Conclusions: BMA assessment for DTCs is feasible in pts with high risk, early stage breast cancer. DTCs are detected in up to a third of breast cancer survivors with repeat assessment during the surveilance period. DTC positivity rates are relatively similar across all receptor subtypes, and after both neoadjuvant and adjuvant chemotherapy. Pts harboring DTCs are highly likely to enroll on interventional trials designed to reduce recurrence risk. Table 1. Patient characteristics and distribution of % DTC positivity among subpopulationsDTC+ (N=49)DTC- (N=102)Total (N=151)DTC+ Rate (Overall: 32.5%)DEMOGRAPHICSMedian Age at BMA (yrs)51.9 (43.9-60.6*)50.5 (42.9-58.1)50.5 (43.8-58.8)N/ARaceCaucasian44 (89.8%)91 (89.2%)135 (89.4%)32.6%African American5 (10.2%)9 (8.8%)14 (9.3%)35.7%Other0 (0%)2 (2.0%)2 (1.3%)0%Menopausal StatusPre-15 (30.6%)34 (33.3%)49 (32.5%)30.6%Post-34 (69.4%)68 (66.7%)102 (67.5%)33.3%BMI at BMA (kg/m2)24.2 (21.9-28.9*)26.9 (23.4-31.4)26.1 (22.8-30.4)N/ARECEPTOR STATUSER/PR+ HER2neg (by ASCO/CAP)24 (49.0%)51 (50.0%)75 (49.7%)32.0%HER2+ (any ER/PR)9 (18.4%)12 (11.8%)21 (13.9%)42.9%ER/PRneg HER2neg23 (46.9%)48 (47.1%)71 (47.0%)32.4%RISK CRITERIALymph Node Positive24 (49.0%)65 (63.7%)89 (58.9%)27.0%Triple Negative (ER/PR<10%)27 (55.1%)50 (49.0%)77 (51.0%)35.0%Non-pCR11 (22.4%)25 (24.5%)36 (23.8%)30.6%RS ≥ 25 and/or High Risk MP6 (12.2%)8 (7.8%)14 (9.3%)42.9%Median T size (cm) -excluding NACT2.1 (1.5-2.9*)1.8 (1.3-2.8)1.8 (1.3-2.9)N/APRIOR THERAPYAdjuvant Chemo25 (51.0%)60 (58.8%)85 (56.3%)29.4%Neoadjuvant Chemo22 (44.9%)41 (40.2%)63 (41.7%)34.9%Endocrine Therapy19 (38.8%)47 (46.1%)66 (43.7%)28.8%XRT29 (59.2%)75 (73.5%)104 (69.3%^)27.9%*Ranges represent interquartile range^ XRT data not available on 1 patient; n=150 was used to figure percentage Citation Format: Lauren Bayne, Isoris Nivar, Brooke Goodspeed, Shannon Deluca, E. Paul Wileyto, Natalie Shih, Anupma Nayak, Michael D Feldman, Joshua Edwards, Kevin Fox, Jennifer M. Matro, Susan Domchek, Hayley Knollman, Rachel Jankowitz, Angela Bradbury, Payal D. Shah, Jewell Graves, George Woodfield, Elizabeth Chislock, Jianping Wang, George Belka, Lewis A. Chodosh, Amy S. Clark, Angela DeMichele. Identifying breast cancer survivors with dormant disseminated tumor cells: The PENN-SURMOUNT screening study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD9-11.

Background: Recurrent breast cancers arise from minimal residual disease (MRD): the pool of disseminated and circulating tumor cells (DTCs and CTCs) that survive in their host following treatment of primary breast cancer. Detection of DTCs in the bone marrow (BM) after treatment is strongly associated with an increased risk of recurrence. Through the analysis of novel genetically-engineered mouse models, we have generated a substantial body of evidence that autophagy and mTOR signaling play key roles in the survival of DTCs. Moreover, administration of agents that block these pathways in mice harboring MRD reduces DTC burden and concomitantly reduces tumor recurrence, providing the rationale for translating these findings to patients (pts). Trial Design: The PENN-SURMOUNT screening study uses a clinically validated IHC assay (DTC-IHC) to identify at-risk pts who harbor DTCs. DTC+ pts are eligible for enrollment on the CLEVER trial, which will determine the feasibility, safety and efficacy of administering hydroxychloroquine (HCQ) and/or everolimus (EVE) in DTC+ patients to target MRD and prevent recurrence. PENN-SURMOUNT is single center, prospective cohort study of pts who have completed therapy for primary breast cancer, are within 5 yrs of diagnosis and are at increased risk for relapse by virtue of nodal positivity, triple negative disease, ER+/Oncotype DX RS ≥ 25, or residual disease after neoadjuvant therapy. Pts undergo screening BM aspirate to test for DTCs following completion of adjuvant chemo and radiotherapy. The primary objective of the study is to determine the incidence and frequency of MRD in pts who have completed primary treatment for breast cancer and to ascertain eligibility for the CLEVER recurrence prevention trial. CLEVER is a randomized, controlled, open label phase II pilot trial. Target enrollment is 60 pts, with 15 pts allocated to each of 4 treatment arms: HCQ (600 mg BID), EVE (10mg daily), combination HCQ/EVE, or control/observation. A cycle is 28 days of continuous dosing. After a 3-month observation period, control pts will be offered HCQ/EVE therapy for 6 cycles; thus, the control group is actually a delayed treatment group and all pts will receive treatment. Pts who demonstrate persistent DTCs after 6 cycles will continue on combination therapy for an additional 6 cycles. The primary endpoint is feasibility of administering HCQ, EVE or the combination in this population. Secondary objectives include safety, efficacy (DTC reduction), and 3-year RFS. The principal translational objective is to assess the utility of a novel DTC assay, "DTC-Flow", for more sensitive detection and response to study therapy, compared to DTC-IHC. Additional translational objectives include determining whether patient DTCs, CTCs, and cell-free circulating plasma tumor DNA (ptDNA) biologically reflect the primary tumor and predict response. As of 5/23/17, 58 patients have been enrolled to PENN SURMOUNT, with a DTC-positivity rate of 22.6%; CLEVER opened in 2/2017; 11 patients are currently enrolled. Contact information: angela.demichele@uphs.upenn.edu Key words: Recurrence, disseminated tumor cells, dormancy, minimal residual disease, autophagy, mTOR, Everolimus, hydroxychloroquine Citation Format: Bayne LJ, Nivar I, Goodspeed B, Wileyto P, Savage J, Shih NNC, Feldman MD, Edwards J, Clark AS, Fox KR, Matro JM, Domchek SM, Bradbury AR, Shah PD, Chislock EM, Belka GK, Wang J, Amaravadi R, Chodosh LA, DeMichele AM. Detection and targeting of minimal residual disease in breast cancer to reduce recurrence: The PENN-SURMOUNT and CLEVER trials [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT2-07-09.

Background: Patients (pts) treated for early stage breast cancer (BC) have a 30% lifetime risk of developing incurable, distant metastatic disease. Yet, standard monitoring after definitive therapy for primary disease is passive observation. Numerous studies have demonstrated that dormant bone marrow (BM) disseminated tumor cells (DTCs) are independently associated with recurrence, but assessment of DTCs is not performed in clinical practice, largely because of concerns about the acceptability and logistics of bone marrow aspiration (BMA) and lack of established therapies that target DTCs. As part of a large scale screening study for a clinical trial targeting DTCs, we examined pt attitudes about DTC screening and subsequently assessed feasibility and tolerability of BM DTC assessment. Methods: The PENN-SURMOUNT (Surveillance Markers of Utility for Recurrence after (Neo)adjuvant Therapy) Screening Study is a single center prospective, longitudinal cohort study examining BM and blood biomarkers of minimal residual disease (MRD) among pts within 5 years of BC diagnosis, who meet one of the following high risk criteria: positive axillary nodes, triple negative biology, ER+ with Oncotype Dx ≥ 25 and/or high risk Mammaprint, or residual disease (RD) after neoadjuvant chemotherapy (NACT). Consented pts undergo a baseline outpatient BMA; if negative, pts can repeat screening annually. During trial design, we surveyed 25 women with stage 2-3 BC at random from the breast clinic at the University of Pennsylvania to assess feasibility. On the SURMOUNT Study, we collected demographic and clinical characteristics of pts, and patient-centered survey data regarding feasibility and acceptability of the BMA that is administered within 48 hours of the procedure. Results: In the pre-trial feasibility survey, 21/25 (84%) pts indicated they were very/definitely interested in knowing if they harbored DTCs. Of those, 18 (86%) indicated moderate/definite interest in testing for DTCs with BMA after the BMA was described to them in detail. 20 (95%) of pts indicated moderate/definite interest in taking oral therapy to eradicate DTCs. 14 (67%) pts stated undergoing up to 3 additional BMA would not change their likelihood of undergoing the clinical trial; only 1 stated much less likely. In the subsequent SURMOUNT study, 361 pts have been referred to date; 167 were eligible, and 136 (81%) subsequently enrolled. 21 (13%) are still in screening. 130 pts have had at least 1 BMA with annual re-screens in 37 (year 1) and 8 (year 2). 39% traveled >50 miles to participate. Post-BMA symptoms were rare (bleeding 2%; redness 12%) though 59%/70% reported mild-moderate pain/tenderness. After BMA, 47%/29%/25% reported it was better/same/worse than expected. 30%/32%/22% reported minimal/moderate/high anxiety prior to the BMA. Afterward, only 20%/5%/4% reported minimal/moderate/high residual anxiety. In 128 pts with results, 38 (30%) have ≥ 1 DTC (30 initial, 8 on follow-up); by risk group: 20/77 (26%) node positive, 20/64 (31%) triple negative, 3/8 (38%) ER+/RS≥25, 7/29 (24%) with RD post-NACT. DTC+ pts were similar to DTC- in median age (50.4), race, distance traveled, menopausal status and BMI. 94% of DTC+ pts have entered the CLEVER clinical trial. Conclusion: A majority of BC survivors want to know DTC status; a majority of these are willing to have BMA and enroll on a clinical therapeutic trial, many are willing to travel to participate and are willing to undergo annual BMA assessment. The SURMOUNT study shows that screening for DTCs is feasible and effective in identifying pts for therapeutic intervention targeting MRD to reduce recurrence. Citation Format: Isoris Nivar, Tara Kauffman, Lauren Bayne, Paul Wiley, Brooke Goodspeed, Michael Feldman, Lewis Chodosh, Amy Clark, Angela DeMichele. Patient attitudes, experience and results of screening for minimal residual disease (MRD) for therapeutic intervention [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-12-01.