Abstract

Seventy percent of breast cancers express the estrogen receptor (ER), and agents that target the ER are the mainstay of treatment. However, virtually all people with ER+ breast cancer develop resistance to ER-directed agents in the metastatic setting. Beyond mutations in the ER itself, which occur in 25–30% of people treated with aromatase inhibitors14, knowledge about clinical resistance mechanisms remains incomplete. We identified activating HER2 mutations in metastatic biopsies from eight patients with ER+ metastatic breast cancer who had developed resistance to aromatase inhibitors, tamoxifen or fulvestrant. Examination of treatment-naive primary tumors in five patients showed no evidence of pre-existing mutations in four of five patients, suggesting that these mutations were acquired under the selective pressure of ER-directed therapy. The HER2 mutations and ER mutations were mutually exclusive, suggesting a distinct mechanism of acquired resistance to ER-directed therapies. In vitro analysis confirmed that the HER2 mutations conferred estrogen independence as well as—in contrast to ER mutations—resistance to tamoxifen, fulvestrant and the CDK4 and CDK6 inhibitor palbociclib. Resistance was overcome by combining ER-directed therapy with the irreversible HER2 kinase inhibitor neratinib.

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Data availability

Tumor and germline whole-exome sequencing data generated and analyzed for this study have been deposited in the access-controlled public repository dbGaP with accession code phs001285. RNA-seq data are available through GEO under accession GSE121411. Additional data generated in this study, including tumor exome analysis, are available within the paper and in the supplementary information.

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Acknowledgements

We thank Q. Quartey and P. Ram for technical assistance, F. Luo, R. Jeselsohn, C. Strathdee, I. Leshchiner, D. Rosebrock, D. Livitz and G. Getz for technical advice, and B. Kaplan, H. Greulich, C. Strathdee, F. Luo, E. Goetz and L. Garraway for providing reagents. We thank C. Johannessen, M. Brown, M. Meyerson and R. Bose for helpful discussions and comments on the manuscript. We are grateful to all the patients who volunteered for our tumor biopsy protocol and generously provided the tissue analyzed in this study. This work was supported by the Department of Defense W81XWH-13-1-0032 (N.W.), AACR Landon Foundation 13-60-27-WAGL (N.W.), National Cancer Institute Breast Cancer SPORE at DF/HCC P50CA168504 (N.W.), Susan G. Komen CCR15333343 (N.W.), the V Foundation (N.W.), the Breast Cancer Alliance (N.W.), the Cancer Couch Foundation (N.W.), the MBC Collective (N.W.), Breast Cancer Research Foundation (N.U.L. and E.P.W.), ACT NOW (to Dana-Farber Cancer Institute Breast Oncology Program), Fashion Footwear Association of New York (to Dana-Farber Cancer Institute Breast Oncology Program), Friends of Dana-Farber Cancer Institute (to N.U.L.), the Klarman Family Foundation and HHMI (to A.R.) and Dana-Farber/Harvard Cancer Center SPORE grant P50CA168504.

Author information

Author notes

  1. These authors contributed equally: U. Nayar, O. Cohen.

Affiliations

  1. Center for Cancer Precision Medicine, Dana-Farber Cancer Institute, Boston, MA, USA

    • Utthara Nayar
    • , Ofir Cohen
    • , Christian Kapstad
    • , Adrienne G. Waks
    • , Seth A. Wander
    • , Lori Marini
    • , Karla Helvie
    • , Nelly Oliver
    •  & Nikhil Wagle
  2. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    • Utthara Nayar
    • , Ofir Cohen
    • , Christian Kapstad
    • , Adrienne G. Waks
    • , Seth A. Wander
    • , Samuel Freeman
    • , Lori Marini
    • , Karla Helvie
    • , Nelly Oliver
    • , Eric P. Winer
    • , Nancy U. Lin
    •  & Nikhil Wagle
  3. Harvard Medical School, Boston, MA, USA

    • Utthara Nayar
    • , Ofir Cohen
    • , Adrienne G. Waks
    • , Seth A. Wander
    • , Samuel Freeman
    • , Eric P. Winer
    • , Nancy U. Lin
    •  & Nikhil Wagle
  4. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    • Utthara Nayar
    • , Ofir Cohen
    • , Christian Kapstad
    • , Adrienne G. Waks
    • , Seth A. Wander
    • , Corrie Painter
    • , Samuel Freeman
    • , Nicole S. Persky
    •  & Nikhil Wagle
  5. Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    • Michael S. Cuoco
    • , Orit Rozenblatt-Rosen
    •  & Aviv Regev
  6. Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA

    • Adrienne G. Waks
    • , Seth A. Wander
    • , Eric P. Winer
    • , Nancy U. Lin
    •  & Nikhil Wagle
  7. Division of Oncology, Department of Medicine, Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO, USA

    • Cynthia X. Ma
  8. Howard Hughes Medical Institute and Koch Institute of Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

    • Aviv Regev

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Contributions

U.N., O.C. and N.W. conceived and designed the study; U.N., C.K. and M.S.C. performed experiments; O.C. performed the computational analyses; O.C. and S.F. evaluated the evolutionary trajectories; A.G.W., S.A.W. and N.W. performed clinical data abstraction and annotation; C.P. and N.S.P. performed kinase structural modeling; O.R. and A.R. supervised the RNA-seq experiment; L.M., K.H. and N.O. assisted with acquisition and annotation of clinical samples; C.X.M. is the principal investigator on the clinical trial of fulvestrant/neratinib on which patient 315 was treated; E.P.W., N.U.L. and N.W. oversaw patient enrollment and sample collection on the metastatic biopsy protocol; U.N., O.C. and N.W. wrote the manuscript with input from all authors; N.W. supervised the study.

Competing interests

N.W. was previously a stockholder in Foundation Medicine, was previously a consultant for Novartis, and has received sponsored research support from Novartis and Puma Biotechnology. N.U.L. has received research funding from Genentech, Cascadian Therapeutics, Array Biopharma, Novartis and Pfizer. C.X.M. receives consulting fees from Puma Biotechnology, Novartis and Pfizer, and research funding from Puma Biotechnology and Pfizer. S.A.W. is a consultant for Foundation Medicine and InfiniteMD. E.P.W. is a consultant for InfiniteMD, Genentech and Eli Lilly. A.R. is a scientific advisory board member of ThermoFisher Scientific, Syros Pharmaceuticals and Driver Group and a founder of Celsius Therapeutics. None of these entities had any role in the conceptualization, design, data collection, analysis, decision to publish or preparation of the manuscript.

Corresponding author

Correspondence to Nikhil Wagle.

Integrated supplementary information

  1. Supplementary Figure 1 Spectrum of HER2 mutations observed in MBC across several studies.

    HER2 mutations in MBC identified in this study as well as in several recent studies are depicted along the length of the protein. In addition to the current study, we aggregated HER2 mutations observed in MBC in Lefebvre et al. (PLoS Med. 2016) and Yates et al. (Cancer Cell 2017), as well as the MBC subset from the MET500 study of Robinson et al. (Nature 2017), the MBC subset from the MSK-IMPACT compendium of Zehir et al. (Nat. Med. 2017) and the MBC subset from the AACR Project GENIE Consortium V1.0.15 (Cancer Discov. 2017), excluding duplicate patients from MSK-IMPACT. The mutations identified in the current study are highlighted. Among these, P1074L, I628M, R143G, and S653C (seen in two patients) have not been previously described (colored in purple). G727A, Leu755Ser, V777L, L869R, and R1153L have been observed in MBC in other studies. A more detailed list of the mutations can be found in Supplementary Table 1.

  2. Supplementary Figure 2 Clinical timelines for the four patients with uncharacterized HER2 mutations, as well as detailed clinicopathological features.

    Patient histories are shown from breast cancer diagnosis until metastatic biopsy sequenced in this study; arrows represent distinct therapies and durations, described in the key (upper right). Asterisks demarcate the diagnosis of metastatic disease in each case.

  3. Supplementary Figure 3 Clonal structure and evolutionary dynamics of uncharacterized HER2 (ERBB2) mutations.

    Clonal dynamics are shown for three metastatic samples with uncharacterized HER2 (ERBB2) mutations by comparing the metastatic clonal cell fraction/CCF (y axis) to the matched primary CCF (x axis). ERBB2 mutations are mapped to metastatic acquired clones in all three patients (colored in red, upper left corner) while not detected in the matched primary (CCF ~ 0). 'Truncal’ mutations that are shared between metastatic and primary tumors are found in all patients, demonstrating that the primary and metastatic samples are clonally related (colored in gray, upper right corner; CCF ~ 1 in both primary and metastatic samples). Primary-specific mutations are found in clones that were dominant in the primary tumor but not observed in the metastatic tumor (colored in blue). The phylogenetic relationships among clones are reconstructed for each patient starting from the normal cell (white circle) connected to the ancestral cancer cells (gray trunk). The phylogenetic divergence of the primary clones and subclones is depicted with blue edges, and that of the metastatic clones and subclones in red. Selected mutations in cancer genes are marked on the corresponding branches of the cancer phylogeny.

  4. Supplementary Figure 4 HER2 kinase domain mutants confer resistance to anti-estrogen agents in MCF7 cells.

    MCF7 HER2-mutant and control cells were compared on the basis of sensitivity to estrogen deprivation and the anti-ER agents tamoxifen and fulvestrant. a, MCF7 cells expressing the indicated HER2 mutants were serum starved for 2 d, followed by treatment with vehicle or 10 nM estradiol (E2) as indicated. After a week, relative viability compared to cells grown in complete medium was analyzed by CellTiter-Glo. Results shown are the mean +/− s.e.m. of three technical replicates and are representative of six independent experiments. bd, MCF7 control or HER2-mutant cells were plated in complete medium containing several concentrations of tamoxifen (b), fulvestrant (c), or GDC-0810 (d). Viability was determined after a week and normalized to untreated wells. Results shown are the mean +/− s.e.m. of three technical replicates and are representative of six independent experiments.

  5. Supplementary Figure 5 HER2 extracellular and signaling domain mutations do not confer resistance to anti-estrogen agents.

    ad, T47D HER2-mutant and control cells were compared on the basis of sensitivity to estrogen deprivation and the anti-ER agents tamoxifen, fulvestrant, and GDC-0810. a, T47D cells expressing the indicated HER2 mutants or controls were serum starved for 2 d, followed by treatment with vehicle or 10 nM estradiol (E2) as indicated. After a week, relative viability compared to cells grown in complete medium was analyzed by CellTiter-Glo. Results shown are the mean +/− s.e.m. of three technical replicates and are representative of eight independent experiments. bd, Cells were plated as in a and switched to full medium containing several concentrations of tamoxifen (b), fulvestrant (c), or GDC-0810 (d) after 2 d. Cells were retreated after 3 d. Viability was determined by CellTiter-Glo assay after a week and normalized to untreated wells. Results shown are the mean +/− s.e.m. of three technical replicates and are representative of eight independent experiments. e,f, Levels of the HER2 activation markers phospho-ERK and phospho-AKT were examined by western blotting in T47D HER2-mutant and control cells. T47D HER2-mutant and control cells were plated in estrogen-deprived medium for 48 h and then switched to fresh medium supplemented with CSS (e) or complete medium containing DMSO or 1 μM fulvestrant (f) for 24 h. Whole-cell extracts were analyzed by western blotting using the indicated antibodies. Results shown are representative of five independent experiments. g, Levels of ER downstream target transcripts were examined by qPCR in T47D HER2-mutant and control cells. T47D HER2-mutant and control cells were plated in estrogen-deprived medium for 48 h, followed by RNA extraction and qRT–PCR using primers for ESR1, PGR, GREB1, or TFF1. Results shown are the mean +/− s.e.m. of three independent experiments.

  6. Supplementary Figure 6 HER2 kinase domain mutant I628M does not confer resistance to anti-estrogen agents.

    T47D HER2-mutant and control cells were compared on the basis of sensitivity to estrogen deprivation and the anti-ER agents tamoxifen and fulvestrant. a, T47D cells expressing the indicated HER2 mutants were serum starved for 2 d, followed by treatment with vehicle or 10 nM estradiol (E2) as indicated. After a week, relative viability compared to cells grown in complete medium was analyzed by CellTiter-Glo. Results shown are the mean +/− s.e.m. of three technical replicates and are representative of four independent experiments. bd, T47D control or HER2-mutant cells were plated in complete medium containing several concentrations of tamoxifen (b), fulvestrant (c), or GDC-0810 (d). Viability was determined after a week and normalized to untreated wells. Results shown are the mean +/− s.e.m. of three technical replicates and are representative of four independent experiments.

  7. Supplementary Figure 7 HER2 kinase domain compound mutant G727A/V777L confers resistance to anti-estrogen agents.

    T47D HER2-mutant and control cells were compared on the basis of sensitivity to estrogen deprivation and the anti-ER agents tamoxifen and fulvestrant. a, T47D cells expressing the indicated HER2 mutants were serum starved for 2 d, followed by treatment with vehicle or 10 nM estradiol (E2) as indicated. After a week, relative viability compared to cells grown in complete medium was analyzed by CellTiter-Glo. Results shown are the mean +/− s.e.m. of three technical replicates and are representative of four independent experiments. bd, T47D control or HER2-mutant cells were plated in complete medium containing several concentrations of tamoxifen (b), fulvestrant (c), or GDC-0810 (d). Viability was determined after a week and normalized to untreated wells. Results shown are the mean +/− s.e.m. of three technical replicates and are representative of four independent experiments.

  8. Supplementary Figure 8 Activating HER2 mutants confer resistance to anti-estrogen agents at low-level expression in T47D cells.

    T47D cells stably expressing GFP, wild-type HER2 or HER2 mutants under the control of a tet-on promoter were generated as described in the supplementary methods. a, Inducible T47D cells were treated with vehicle or 250 ng/mL doxycycline, and western blotting was performed to compare HER2 construct expression levels across cell lines. T47D cells expressing HER2 mutants constitutively under the EF-1α promoter (pLX307 vector) are included for comparison. Results shown are representative of five independent experiments. b, Inducible T47D cells were serum starved for 2 d in medium containing 250 ng/mL doxycycline, followed by vehicle or 10 nM estradiol (E2) as indicated. After a week, relative viability compared to cells grown in complete medium was analyzed by CellTiter-Glo. Results shown are the mean +/− s.e.m. of three technical replicates and are representative of two independent experiments. c,d, Inducible cell lines were serum starved for 2 d in medium containing 250 ng/mL (c) or 100 ng/mL (d) doxycycline, followed by treatment with complete medium containing several concentrations of fulvestrant along with the appropriate concentration of doxycycline. After a week, relative viability compared to cells grown in complete medium with doxycycline was analyzed by CellTiter-Glo assay. Results shown are the mean +/− s.e.m. of three technical replicates and are representative of three independent experiments.

  9. Supplementary Figure 9 ERBB1/2 signatures and growth factor-driven ER signatures among HER2 activating mutants and controls.

    Gene expression analysis by RNA sequencing of T47D cells expressing the indicated mutants or controls was performed after 2 d of serum starvation followed by treatment with DMSO or 1 μM fulvestrant for 24 h. Six replicates were performed for each specific construct and drug condition. ERBB1/2 signaling (left panels) and growth factor−driven ER signaling (right panels) signature strengths are shown for each of the HER2 mutants and the controls, across all replicates, in cells treated with DMSO (a), fulvestrant (b), and palbociclib (c).

  10. Supplementary Figure 10 Combination therapeutic approaches in HER2-mutant cells.

    a,b, T47D HER2-mutant and control cells were compared on the basis of sensitivity to pan-HER kinase inhibitor neratinib alone or in combination with fulvestrant. a, T47D HER2-mutant or control cells serum starved for 2 d were switched to complete medium containing 100 nM fulvestrant, 32 nM neratinib, or the combination. Cells were retreated after 3 d, and viability was determined by CellTiter-Glo assay 1 week after the start of treatment. Results shown are the mean +/− s.e.m. of technical replicates and are representative of 13 independent experiments. b, Levels of the HER2 activation markers phospho-ERK and phospho-AKT were examined by western blotting in T47D HER2-mutant and control cells treated with neratinib. T47D HER-mutant and control cells serum starved for 2 d were switched to complete medium containing DMSO or 1 μM neratinib for 24 h. Whole-cell extracts were analyzed for the indicated proteins by immunoblotting. Results shown are representative of three independent experiments. c, T47D HER2-mutant and control cells serum starved for 2 d were switched to complete medium containing 320 nM neratinib. Cells were retreated after 3 d, and viability was determined by CellTiter-Glo assay a week after the start of treatment. Results shown are the mean +/− s.e.m. and are representative of 15 independent experiments. di, The effectiveness of inhibitors against CDK4 and CDK6 or the downstream HER2 targets MEK, ERK, PI3K, AKT or mTOR were examined in T47D HER2-mutant and control cells. Inhibitor monotherapy was compared to combination therapy with fulvestrant. Cells plated as in b were treated with 100 nM fulvestrant, 1 μM palbociclib (CDK4 and CDK6 inhibitor; d), 1 µM trametinib (MEK inhibitor; e), 3.2 μM VX-11e (ERK inhibitor; f), 320 nM BYL719 (PI3K inhibitor; g), 320 nM AZD5363 (AKT inhibitor; h), 10 nM everolimus (mTOR inhibitor; i), or the combination. Cells were retreated after 3 d, and viability was determined by CellTiter-Glo assay 1 week after the start of treatment. Results shown are the mean +/− s.e.m. of three technical replicates and are representative of six, four, six, two, nine, and two independent experiments, respectively.

  11. Supplementary Figure 11 ERBB1/2 signaling and growth factor-driven ER signaling signatures for HER2 activating mutants across multiple drugs.

    Gene expression analysis by RNA sequencing performed as described in Fig. 4 included T47D cells expressing the four activating HER2 mutants as indicated. Cells were serum starved then treated with 1 μM fulvestrant, 1 μM neratinib, 10 μM palbociclib, or combinations (1 µM fulvestrant + 1 µM neratinib, 1 µM fulvestrant + 10 µM palbociclib). Six replicates were performed for each specific construct and drug condition, as shown. ERBB1/2 signaling (a) and growth factor−driven ER signaling (b) signature strengths were compared in mutant cell lines under various treatment conditions.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–11 and Supplementary Note

  2. Reporting Summary

  3. Supplementary Table 1

    HER2 mutations observed in MBC across several studies

  4. Supplementary Tables 2–4

    Clinicopathological data for 12 patients with HER2 mutations

  5. Supplementary Table 5

    All SNVs from 12 patients with HER2 mutations

  6. Supplementary Table 6

    All CNAs from 12 patients with HER2 mutations

  7. Supplementary Table 7

    Differentially expressed genes comparing all mutant and control cell lines versus GFP under all drugs

  8. Supplementary Table 8

    Differentially expressed genes comparing activating HER2 mutants versus wild-type HER2 under all drugs

  9. Supplementary Table 9

    Gene set enrichment analysis for activating HER2 mutants versus GFP, under treatment with fulvestrant

  10. Supplementary Table 10

    Gene set enrichment analysis for activating HER2 mutants versus GFP, under treatment with DMSO

  11. Supplementary Table 11

    Gene set enrichment analysis for activating HER2 mutants versus wild-type HER2, under treatment with fulvestrant

  12. Supplementary Table 12

    Gene set enrichment analysis for activating HER2 mutants versus wild-type HER2, under treatment with fulvestrant

  13. Supplementary Table 13

    Primer sequences for qPCR

  14. Supplementary Data

    Uncropped western blots

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DOI

https://doi.org/10.1038/s41588-018-0287-5