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Targeting dormant tumor cells to prevent recurrent breast cancer: a randomized phase 2 trial

Nature Medicine (2025)Cite this article

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Abstract

Breast cancer recurrence may arise from dormant disseminated tumor cells (DTCs) that persist in bone marrow and other sites. Clinically, DTCs are independently associated with breast cancer recurrence and death. Preclinical studies in mouse models identified autophagy and mammalian target of rapamycin (mTOR) signaling as critical mechanisms of tumor dormancy and escape. We subsequently tested the effects of transient versus chronic inhibition of autophagy with chloroquine or hydroxychloroquine (HCQ) and mTOR signaling with rapamycin (RAPA) or everolimus (EVE) on residual tumor cell (RTC) burden and recurrence-free survival (RFS). In mice harboring dormant RTCs, inhibition of mTOR alone or in combination with autophagy inhibition decreased RTC burden and improved RFS in a duration-dependent manner. RTC number was strongly and inversely correlated with RFS, suggesting that RTC reduction mediated an improvement in RFS. To translate findings clinically, we performed a randomized phase 2 trial (CLEVER) of HCQ, EVE or their combination in breast cancer survivors within 5 years of diagnosis who had detectable DTCs on bone marrow aspirate. Primary endpoints were feasibility and safety; secondary endpoints included DTC reduction/clearance and RFS. In total, 51 DTC+ patients initiated HCQ (n = 15), EVE (n = 15) or HCQ + EVE (n = 21). Treatment was feasible and tolerable; only one patient discontinued early for grade 3 toxicity. At 42 months median follow-up, landmark 3-year RFS for HCQ, EVE and HCQ + EVE was 91.7%, 92.9% and 100%, respectively, and was greater in those who cleared DTCs versus those who did not (hazard ratio (HR) = 0.21 (95% confidence interval 0.01–3.4)). Posterior probabilities were 98–99.9% that three cycles of HCQ, EVE or HCQ + EVE led to reduced or undetectable DTCs compared to observation alone, with estimated DTC reductions of 80%, 78% and 87%, respectively. These findings provide proof-of-concept that targeting dormant RTCs with HCQ, EVE or their combination in breast cancer survivors or mouse models depletes minimal residual disease, warranting a definitive human randomized controlled trial. ClinicalTrials.gov registration: NCT03032406.

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Fig. 1: RFS outcomes and impact on the number of RTCs in the mouse CLEVER preclinical study.
Fig. 2: CLEVER human trial CONSORT and impact of treatment on DTC number and outcomes.
Fig. 3: Changes in DTC number, probability of DTC clearance and RFS by treatment and DTC status in the CLEVER trial.

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Data availability

All data supporting the findings of this study are available within the paper. The data used and/or analyzed during the current study are available in the Supplementary Information or from the corresponding author(s) on request, recognizing that certain patient-related data not included in the paper were generated as part of the clinical trial and may be subject to patient confidentiality. It is estimated that the corresponding authors will respond to external data requests within 2 weeks of receipt of the request to verify whether the request is subject to any intellectual property or confidentiality obligations. Uncropped original western blots corresponding to Extended Data Fig. 2e,f,k,l are provided. The authors do not have IRB approval or patient consent to share identifying or sensitive data on CLEVER clinical trial participants and therefore cannot report data in a public repository. Source data are provided with this paper.

Code availability

This study used custom code for Bayesian data modeling, which will be made available upon request. It is estimated that the corresponding authors will respond to requests for code within 2 weeks of receipt of the request.

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Acknowledgements

We thank the Abramson Cancer Center and Penn Medicine for their support of the 2-PREVENT Translational Center of Excellence, as well as the staff and members of the 2-PREVENT TCE for their efforts in support of the CLEVER study. We acknowledge support from the following funders: the National Cancer Institute (R01CA208273 to A.D. and L.C.), Department of Defense (BC160784 to A.D. and L.C.), V Foundation, Breast Cancer Research Foundation (to A.D. and L.C.), QVC ‘Shoes on Sale’ (to A.D.), Jerry S. Rosenbloom (to A.D. and L.C.), Sara and Jim Gowing (to A.D. and L.C.), Avon Foundation (to A.D. and L.C.), Raynier Institute & Foundation (to A.D. and L.C.), Rhoda Polly Danziger and Michael Danziger (to L.C.), Andrea Orsher and Robert Orsher (to A.D.), the Dietz & Watson Family (to A.D.), and Novartis for providing Everolimus for the CLEVER trial. We appreciate the important contribution of the 2-PREVENT TCE Patient Advocate Board—J. Perlmutter, J. LaScala, C. Abi-Khattar, J. Bowles, M. Ayres, L. Mikulski and S. Axler. Finally, we are indebted to the CLEVER trial participants and their families, without whom this research would not be possible. This study was presented at the European Society of Clinical Oncology on 23 October 2023.

Author information

Authors and Affiliations

  1. Division of Hematology/Oncology, Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Angela DeMichele, Amy S. Clark, Lauren J. Bayne, Killian Rohn, Isoris Nivar, Shannon Deluca, Igor Makhlin & Brooke L. Goodspeed

  2. Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA

    Angela DeMichele, Amy S. Clark, Jessica Savage, Igor Makhlin & Lewis A. Chodosh

  3. 2-PREVENT Translational Center of Excellence, University of Pennsylvania, Philadelphia, PA, USA

    Angela DeMichele, Amy S. Clark, Emily Shea, Lauren J. Bayne, Christopher J. Sterner, Killian Rohn, Tien-chi Pan, Yan Chen, Shannon Deluca, Jessica Savage, Igor Makhlin, Dhruv K. Pant, Heather Martin, Adetutu Egunsola, Nathan Mears, Brooke L. Goodspeed, Elizabeth M. Chislock, Jewell Graves, Jianping Wang, Natalie Shih, George K. Belka, Anupma Nayak, Michael Feldman & Lewis A. Chodosh

  4. Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA, USA

    Emily Shea, Christopher J. Sterner, Samantha Dwyer, Tien-chi Pan, Yan Chen, Dhruv K. Pant, Heather Martin, Adetutu Egunsola, Nathan Mears, Elizabeth M. Chislock, Jewell Graves, Jianping Wang, George K. Belka & Lewis A. Chodosh

  5. Department of Biostatistics, Epidemiology and Informatics, University of Pennsylvania, Philadelphia, PA, USA

    Paul Wileyto

  6. Berry Consultants, Austin, TX, USA

    Lindsay R. Berry & Don Berry

  7. Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Natalie Shih, Anupma Nayak & Michael Feldman

  8. Department of Pathology and Laboratory Medicine, Indiana University, Indianapolis, IN, USA

    Michael Feldman

  9. Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Lewis A. Chodosh

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  1. Angela DeMichele
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  2. Amy S. Clark
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Contributions

L.A.C. and A.D. conceived of the approach to prevent recurrence by depleting MRD, designed the overall study, oversaw its conduct and obtained funding to support it. A.D. wrote the CLEVER protocol and provided oversight of study conduct. A.D., A.S.C. and J.S. provided clinical care to CLEVER trial participants and charted source documentation of clinical research visits. A.D., A.S.C. and J.S. conducted study visits and clinical management of study patients. L.J.B. provided project management, supervision of staff, input on regulatory matters and design of case report forms. K.R. cleaned and prepared CLEVER data for analysis. K.R., L.R.B., D.B., L.J.B., L.A.C. and A.D. generated figures (Figs. 2 and 3 and Extended Data Fig. 4) describing CLEVER trial results. I.N. coordinated participant visits and data collection on the CLEVER trial and assisted with patient sample collection. P.W. analyzed CLEVER feasibility data. L.R.B. and D.B. analyzed and interpreted CLEVER recurrence-free survival and DTC-IHC data. S.D. collected and tracked patient samples and managed sample inventory. S.E.D. and L.A.C. generated, analyzed and interpreted mouse preclinical data on the effects of CQ and RAPA on RTC number and recurrence-free survival, on which the mouse study and CLEVER trial were based. C.J.S., N.M., G.K.B. and S.E.D. performed mouse studies. Y.C. and A.E. processed mouse tumor samples and performed ddPCR to enumerate RTCs. E.S., T.C.P., D.K.P., G.K.B. and L.A.C. analyzed and interpreted mouse CLEVER recurrence-free survival and residual disease data. H.M. and E.S. performed and quantified western blots on mouse samples. E.S., J.W. and G.K.B. performed and analyzed mouse immunofluorescence studies. E.S., T.C.P., G.K.B. and L.A.C. generated figures describing mouse preclinical study results. G.K.B. and L.A.C. provided project management. G.K.B. provided supervision, wrote animal protocols and provided input on regulatory matters. B.L.G. performed patient BMAs. J.W., E.M.C. and J.G. contributed to the processing of patient samples. E.M.C. and J.G. provided control cell lines for the DTC-IHC assay, assisted in transitioning the DTC-IHC assay to a Clinical Laboratory Improvement Amendments (CLIA) laboratory and performed hemodilution assessment of BMAs. E.M.C., N.S., L.J.B. and I.M. contributed to the design and reporting of DTC-IHC assay rescreen testing and the implementation of the dual readers and adjudication system. M.F. and N.S. oversaw the transition of the DTC-IHC assay to a CLIA laboratory. M.F. and A.N. evaluated patient bone marrow samples for the presence of DTCs by DTC-IHC, adjudicated discordant reads and generated source pathology reports documenting DTC-IHC results. N.S., L.J.B. and S.D. provided operational support and coordination for sample collection and processing for the DTC-IHC assay. A.D., L.A.C. and E.S. drafted the manuscript. All authors approved the final manuscript and contributed to critical revisions of its intellectual content.

Corresponding authors

Correspondence to Angela DeMichele or Lewis A. Chodosh.

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Competing interests

A.D. has received institutional research funding from Novartis, Genentech, Pfizer and NeoGenomics. L.A.C. has received institutional research funding from Novartis, AstraZeneca and Merck Research Laboratories, and has served as an expert consultant to Teva Pharmaceuticals, Eisai, Sanofi, Takeda Pharmaceuticals, Eli Lilly, Whittaker, Clark and Daniels, Wyeth, Imerys, Becton Dickinson, Sterigenics and the U.S. Department of Justice in litigation. The other authors declare no competing interests.

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Nature Medicine thanks the anonymous reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: J. Nakhle and S. Sadanand, in collaboration with the Nature Medicine team.

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Presentation of the study European Society of Clinical Oncology, 23 October 2023.

Extended data

Extended Data Fig. 1 Recurrence-free survival outcomes in mouse CLEVER preclinical study following chronic treatment with study agents.

a, Schematic of orthotopic inducible Her2 mouse model and timing of rapamycin treatment in mice bearing dormant residual tumor cells. b, Recurrence-free survival of female nu/nu mice harboring dormant residual disease from fully regressed primary tumors treated with vehicle control (VEH) or 4 mg/kg RAPA (n = 20) initiated 28 d after Her2 downregulation. c–f, RFS outcomes for mice treated chronically with VEH, RAPA, EVE, CQ, HCQ or EVE + HCQ. c, RAPA or CQ compared to vehicle control (VEH); d, EVE or HCQ compared to VEH; e, RAPA or EVE compared to VEH; or f, HCQ or CQ compared to VEH. RFS outcomes shown in panels c–f and in Fig. 1 represent a single study with 16 treatment arms, where the results for different comparisons between treatment arms and treatment durations are shown as individual panels for clarity. Hazard ratios (HR) and unadjusted p values from the two-sided log-rank test are shown for the indicated comparisons.

Extended Data Fig. 2 Pharmacodynamic analysis of study agents.

a–d, Quantification of western blots for phospho-S6 (a,b) or LC3-II (c,d) performed on lysates of proliferative (a,c) or dormant (b,d) Her2-inducible primary tumor cells in vitro treated with vehicle (VEH), rapamycin (RAPA), everolimus (EVE), hydroxychloroquine [HCQ] or EVE + HCQ. A recurrent Her2 tumor cell line is shown as a control for proliferative samples. Proliferative and dormant VEH controls are shown for scale to the right. Representative western blots for proliferative (e) and dormant (f) tumor cells corresponding to a–d. g–j, Quantification of western blots for phospho-S6 (a,b) or LC3-II (c,d) performed on lysates of primary orthotopic tumors (g,i) or dormant residual orthotopic tumors (h,j) derived from Her2-inducible primary tumor cells collected from mice treated with VEH, RAPA, EVE, CQ, HCQ or EVE + HCQ. Primary tumor and dormant tumor VEH controls are shown for scale to the right. Representative western blots for primary tumor (k) and dormant (l) tumor lysates corresponding to g–j. Bars and error bars show mean ± s.e.m. Molecular weight markers are shown. For l, a dotted vertical line indicates where the molecular weight marker lanes were not immediately adjacent to the samples shown (which were otherwise contiguous).

Extended Data Fig. 3 Impact of treatment on residual tumor cell number in mouse CLEVER preclinical study.

a, Relative change in residual tumor cell number (RTC) count following 3 weeks or 9 weeks of treatment with the indicated therapies, shown as % change relative to vehicle control. b, Absolute RTC counts following 3 weeks or 9 weeks of treatment with the indicated therapies or vehicle control. Red boxes indicate residual tumor lesions that were classified as recurrent tumors based on progressive increases in tumor size. Purple triangles indicate RTC counts that were statistical outliers. MGRD, mammary gland residual disease. c, Plot of median RFS versus log10 median RTC counts following 3 weeks or 9 weeks of treatment with the indicated therapies or vehicle control shown as a combined cohort. The Pearson correlation coefficient is shown for the combined cohort and tested against the t distribution for estimating a two-sided p value.

Extended Data Fig. 4 Recurrence-free survival by receptor status and DTC status in the CLEVER trial.

a, Recurrence-free survival by receptor status. To avoid overlapping RFS curves, a small amount of vertical spacing has been added to the lines at 100% RFS to distinguish between the different receptor groups. b, Recurrence-free survival by DTC detection status ascertained after cycle 6 of treatment. Number of patients at risk at each time point is shown.

Extended Data Table 1 Feasibility assessment
Full size table
Extended Data Table 2 Dose modifications (cycles 1–6)
Full size table
Extended Data Table 3 CLEVER DTC clearance at cycle 6 (original arm assignment) and cycle 12 (additional HCQ + EVE) by treatment and receptor subtype
Full size table

Supplementary information

Source data

Source Data Extended Data Fig. 2, panel e

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Source Data Extended Data Fig. 2, panel l

Unprocessed western blots

Source Data Extended Data Fig. 2, panel l

Unprocessed western blots

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DeMichele, A., Clark, A.S., Shea, E. et al. Targeting dormant tumor cells to prevent recurrent breast cancer: a randomized phase 2 trial. Nat Med (2025). https://doi.org/10.1038/s41591-025-03877-3

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