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Entinostat, nivolumab and ipilimumab for women with advanced HER2-negative breast cancer: a phase Ib trial

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Abstract

We report the results of 24 women, 50% (N = 12) with hormone receptor-positive breast cancer and 50% (N = 12) with advanced triple-negative breast cancer, treated with entinostat + nivolumab + ipilimumab from the dose escalation (N = 6) and expansion cohort (N = 18) of ETCTN-9844 (NCT02453620). The primary endpoint was safety. Secondary endpoints were overall response rate, clinical benefit rate, progression-free survival and change in tumor CD8:FoxP3 ratio. There were no dose-limiting toxicities. Among evaluable participants (N = 20), the overall response rate was 25% (N = 5), with 40% (N = 4) in triple-negative breast cancer and 10% (N = 1) in hormone receptor-positive breast cancer. The clinical benefit rate was 40% (N = 8), and progression-free survival at 6 months was 50%. Exploratory analyses revealed that changes in myeloid cells may contribute to responses; however, no correlation was noted between changes in CD8:FoxP3 ratio, PD-L1 status and tumor mutational burden and response. These findings support further investigation of this treatment in a phase II trial.

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Fig. 1: Study schema.
Fig. 2: Clinical efficacy.
Fig. 3: Survival data.
Fig. 4: CyTOF of peripheral blood samples.
Fig. 5: Peripheral cytokine profiling.
Fig. 6: Mutational evaluation.

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Data availability

All of the datasets generated during the current study that can be shared are available as Supplementary Tables. Whole-exome sequencing data are not publicly available due to lack of consent for data sharing. Source data are provided with this paper and are also available at https://github.com/FertigLab/J15221_trial_paper/tree/main/input_tables.

Code availability

All code used for analyses is available on GitHub at https://github.com/FertigLab/J15221_trial_paper.

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Acknowledgements

We thank the individuals who volunteered to participate in this study, NCI CTEP, Bristol Myers Squibb and Syndax Pharmaceuticals for the supply of study drugs via NCI and the research teams and physicians at participating sites. We thank C. Thornburn from the Johns Hopkins Immune Monitoring Core, G. Rosner and M. Carducci for valuable input during the study and S. Do from the University of Southern California for help finalizing the paper for publication. The following sources provided funding for the current study: NCI’s Experimental Therapeutics Clinical Trials Network (UM1CA186689, UM1CA186690, UM1CA186691, UM1CA186717 and U24CA247648 to E.T.R.T., R.M.C., V.S., E.J.F. and E.M.J.), Bloomberg Kimmel Institute for Immunotherapy (E.T.R.T. and E.M.J.), Analytical Pharmacology Core of the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins (P30CA006973, UL1TR 001079 and 1S10RR026824-01 to M.A.R.) and the Clinical Protocol and Data Management facilities (P30CA006973, P30CA014089 and P30CA047904 to A.O. and M.D.). The Cancer Therapy Evaluation Program supplied entinostat, nivolumab and ipilimumab. Support was also provided by the Commonwealth Foundation Grant (2018-2020 to E.T.R.T. and NCCN Young Investigator Award 2015 to R.M.C.), Mary Kay Foundation (Cancer Research Grant Program 2015 to V.S.), V Foundation (Translational Award 2017 to R.M.C. and V.S.), Tower Cancer Research Foundation (Career Development Award 2019–2021 to E.T.R.T.), Concern Foundation (Conquer Cancer Now Award 2020–2022 to E.T.R.T.), METAVivor (Young Investigator Award 2022–2024 to E.T.R.T.), Dana and Albert ‘Broccoli’ Professorship and the Broccoli Foundation and the Breast Cancer Research Foundation (to E.M.J.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NCI or the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA

    Evanthia.T. Roussos Torres, Won J. Ho, Ludmila Danilova, Joseph A. Tandurella, James Leatherman, Christine Rafie, Chenguang Wang, Melinda Downs, Ashley O’Connor, Sarah M. Shin, Alexei Hernandez, Elizabeth L. Engle, Michelle A. Rudek, Qingfeng Zhu, Robert A. Anders, Ashley Cimino-Mathews, Elana J. Fertig, Elizabeth M. Jaffee, Vered Stearns & Roisin M. Connolly

  2. Department of Medicine, Division of Medical Oncology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    Evanthia.T. Roussos Torres

  3. University of Miami Miller School of Medicine, Miami, FL, USA

    Christine Rafie

  4. University of Pittsburgh Cancer Institute and UPMC Cancer Center, Pittsburgh, PA, USA

    Adam Brufsky

  5. Yale Cancer Center, New Haven, CT, USA

    Patricia LoRusso

  6. City of Hope, Duarte, CA, USA

    Vincent Chung

  7. Cedars-Sinai Cancer, Los Angeles, CA, USA

    Yuan Yuan

  8. Cancer Therapy Evaluation Program (CTEP), National Cancer Institute, Bethesda, MD, USA

    Richard Piekarz & Howard Streicher

  9. Division of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, OH, USA

    Zahra Talebi

  10. Cancer Research @UCC, College of Medicine and Health, University College Cork, Cork, Ireland

    Roisin M. Connolly

Authors
  1. Evanthia.T. Roussos Torres
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  2. Won J. Ho
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  3. Ludmila Danilova
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  6. Christine Rafie
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  7. Chenguang Wang
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  11. Yuan Yuan
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Contributions

First corresponding author E.T.R.T. and senior corresponding author R.M.C. equally contributed to data collection and analysis along with other members of the team and formulated the whole paper. R.M.C. served as the lead principal investigator on the clinical trial until she moved to Ireland in 2019. E.T.R.T. served as the translational principal investigator. W.J.H. performed liquid-based CyTOF, analyzed the data and provided feedback on the manuscript. L.D. and J.A.T. provided statistical analyses for clinical and correlative results, provided text for statistical sections and edited the paper. J.L. and C.R. helped process, organize, prepare and deliver specimens for analyses. C.W. provided statistical support for initial clinical analysis. A.B., P.L.R., V.C. and Y.Y. served as site principal investigators during the clinical trial and provided feedback on the paper. M.D. served as the primary clinical research coordinator for the study. A.O. served as the main clinical regulatory supervisor for the trial. S.M.S. and A.H. provided technical assistance in running liquid-based CyTOF. E.L.E. optimized and performed IHC staining of tumor samples. R.P. and H.S. served as primary contacts with NCI/CTEP and provided input on the paper. Z.T. and M.A.R. performed the pharmacokinetic analyses. Q.Z. analyzed CD8 and FoxP3 staining, R.A.A. and A.C.-M. served as the primary study pathologists, and A.C.-M. scored PD-L1 and TILs on all samples and provided input on paper writing. E.J.F. provided senior input for statistical analysis and genomic correlative data. E.M.J. provided input on the clinical trial design and at all stages of enrollment, analysis and paper preparation. V.S. took over as the primary clinical principal investigator when R.M.C. moved to Ireland and also provided input on clinical trial design and at all stages of enrollment, analysis and paper preparation.

Corresponding authors

Correspondence to Evanthia.T. Roussos Torres or Roisin M. Connolly.

Ethics declarations

Competing interests

R.M.C. has received an unrestricted educational grant from Pfizer and research funding for clinical trials from MSD Ireland, Pfizer, Daiichi Sankyo and AstraZeneca, all to her institution. R.M.C. has consulted for AstraZeneca/Daichii, Gilead and Seagen and has received travel support from Novartis. M.A.R. has received research grants to the institution from Celgene, Cullinan Apollo and RenovoRx and is a cofounder and board member and holds equity in Geminus Therapeutics. M.A.R.’s spouse is employed by GlaxoSmithKline. V.S. received research grants to their institution from Abbvie, Biocept, Pfizer, Novartis and Puma Biotechnology and is a member of the Data Safety Monitoring Board at Immunomedics and AstraZeneca. A.C.-M. has received research grants to their institution from HeritX, Genentech and Bristol Myers Squibb and serves as a consultant to Bristol Myers Squibb. A.B. is a paid consultant for Lilly, Novartis, Pfizer, Astrazeneca, Eisai, Roche and Sanofi. P.L.R. reports personal fees from Abbvie, Agios, Five Prime, GenMab, Halozyme, Roche-Genentech, Genentech, Cytomx, Takeda, SOTIO, Cybrexa, Agenus, Tyme, IQVIA, TRIGR, Pfizer, ImmunoMet, Black Diamond, Glaxo-Smith Kline, QED Therapeutics, AstraZeneca, EMD Serono, Shattuck, Astellas, Salarius, Silverback, MacroGenics, Kyowa Kirin, Kineta, Zentalis, Molecular Templates, ABL Bio, SK Life Science and STCube; these are all outside the submitted work. E.M.J. reports ongoing collaboration with Roche on new approaches to Precision Medicine Tumor Boards. V.C. is part of the speaker’s bureau for Ipsen and Coherus and is a Perthera and Pfizer consultant. Y.Y. is part of the speaker’s bureau for Gilead, AstraZeneca, Daiichi Sankyo and Merck, is a paid consultant for Gilead, Pfizer, Stemline and AstraZeneca and has received research support from Genentech, Merck, Pfizer, Imugene, Agenus and Gateway Foundation. R.A.A. has received research support from Bristol Myers Squibb, Merck, StandUp2Cancer and FLXbio and is a paid consultant for Bristol Myers Squibb, Merck, Incyte, AstraZeneca and FLXbio. E.J.F. is on the Scientific Advisory Board of Resistance Bio/Viosera Therapeutics and is a paid consultant for Merck and Mestag Therapeutics. E.M.J. is a paid consultant for Adaptive Biotech, CSTONE, Achilles, DragonFly and Genocea. E.M.J. receives funding from Lustgarten Foundation and Bristol Myers Squibb and is the Chief Medical Advisor for Lustgarten and a Scientific Advisory Board member for Parker Institute for Cancer Immunotherapy and C3 Cancer Institute. E.M.J. reports other support from Abmeta and Adventris, personal fees from Achilles, Dragonfly, Mestag, The Medical Home Group and Surgtx, other support from Parker Institute and grants and other support from the Lustgarten Foundation, Genentech, Bristol Myers Squibb and Break Through Cancer outside the submitted work. E.T.R.T. is a consultant for Synaptical and is a shareholder in this company. All other authors have nothing to disclose.

Peer review

Peer review information

Nature Cancer thanks Dominic Edelmann, Heather McArthur and Hatem Soliman for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Survival evaluation in all 24 patients.

A. Progression-free Survival (PFS) including all 24 patients. B. Overall survival (OS) including all 24 patients. Dotted lines at 50% survival probability represent mean values listed for each graph and indicate PFS/ OS and 95% CIs. Abbreviations: CI, confidence interval; NA, not applicable.

Source data

Extended Data Fig. 2 Peripheral CyTOF for Immune Evaluation.

A. CyTOF expression of markers used to determine immune cell subsets. Cell sub types listed on the right were assigned based on differential expression of the markers listed across the bottom. Neu- neutrophil, GMDSC- granulocytic myeloid derived suppressor cell, MMDSC_I-III monocytic myeloid derived suppressor cell types I-III, Mono I-III- monocytes types I-III, cDC1- classical dendritic cell type 1, cDC2-classic dendritic cell type 2, pDC-plasmacytoid dendritic cell, Baso-basophil, CD4_T- CD4 positive T cell, CD8_T- CD8 positive T cell, DNT- double negative T cells (neither CD4 nor CD8 expressing T cells), DPT-double positive T cells (both CD4 and CD8 positive), NK_like-natural killer cell, B- B cell, UA- unassigned cell type. B. Expression of CCR5 at baseline and Post Run-in. C. Expression of CD141 at baseline and Post Run-in. D. Expression of HLADR at baseline and Post Run-in. All P values listed in associated tables represent two-sided paired Wilcoxon’s rank sum testing. *Denotes significant change sustained at Week 8. N = 17 at baseline, N = 17 at Post Run-in, and N = 10 at Week 8 biologically independent samples. Box plots show the median, upper, and lower quartiles, and whiskers extend to 1.5 times the interquartile range.

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Extended Data Fig. 3 Peripheral CyTOF lymphocyte marker evaluation.

No significant differences in proportions of CD4 and CD8 T cell, B cell and NK cell populations across the treatment groups and in association with response (A). Significant changes in expression in markers on CD4 T cells (A) and CD8 T cells (B) in responders and non-responders (by RECIST). Values in orange represent a mean increase of marker expression at Post Run-In calculated as difference between baseline and Post Run-In (delta). Values in blue represent a mean decrease in at Post Run-In. *Indicates markers with sustained significant change between Run-In and Week 8. P values represent pairwise Wilcoxon rank sums. C. Expression of markers on B cells from peripheral CyTOF. Non-responders by RECIST (red), responders (blue). P values are indicative of two-sided pairwise Wilcoxon’s rank sum testing and compares the differences in value from baseline to Post Run-in in responders vs. non-responders listed in table. N = 17 at baseline, n = 17 at Post Run-in, and n = 10 at Week 8 biologically independent samples. Box plots show the median, upper, and lower quartiles, and whiskers extend to 1.5 times the interquartile range.

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Extended Data Fig. 4 Evaluation of tumor biopsies for T cell infiltration.

Ratio of CD8/FoxP3. A. Log-transformed ratios of CD8 density to FoxP3 density at different timepoints. N = 12 at baseline, n = 10 at Post Run-in, and n = 4 at Week 8 biologically independent samples. B. Average TIL percentage change over time and by response. *In these cases, only tumor bed was noted (no tumor visualized) consistent with complete tumor regression. N = 17 at baseline, n = 17 at Post Run-in, and n = 8 at Week 8 biologically independent samples. P-value is from pairwise two-sided Wilcoxon’s rank sum test. Box plots show the median, upper, and lower quartiles, and whiskers extend to 1.5 times the interquartile range.

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Extended Data Fig. 5 Evaluation of PD-L1 status.

A. Average percentage of immune cells demonstrating PD-L1 staining by response and by breast cancer subtype at baseline (n = 14 biologically independent samples). B. Average percentage of tumor cells demonstrating PD-L1 staining by response and by breast cancer subtype at baseline (n = 14 biologically independent samples). P-value is from pairwise two-sided Wilcoxon’s rank sum test. Red dots indicate HR-positive patients and blue indicate TNBC. Box plots show the median, upper, and lower quartiles, and whiskers extend to 1.5 times the interquartile range.

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Extended Data Fig. 6 Mutational type analysis.

A. Summary of incidence of variant classifications across all samples. B. Summary of types of variants; triple nucleotide polymorphism (TNP), single nucleotide polymorphism (SNP), oligo-nucleotide polymorphism (ONP), insertion (INS), double nucleotide polymorphism (DNP), deletion (DEL). C. Single nucleotide variant (SNV) classes at baseline across all samples. T-thymine, G-guanine, A-adenosine, C-cytosine. For example thymine altered to guanine (T > G). D. Top 10 mutated genes in responders. E. Top 10 mutated genes in non-responders. Percentages listed next to each mutated gene indicates number of samples with mutations in given gene.

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Roussos Torres, E., Ho, W.J., Danilova, L. et al. Entinostat, nivolumab and ipilimumab for women with advanced HER2-negative breast cancer: a phase Ib trial. Nat Cancer (2024). https://doi.org/10.1038/s43018-024-00729-w

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