Research ArticleCancer

Genomic profiling of ER+ breast cancers after short-term estrogen suppression reveals alterations associated with endocrine resistance

See allHide authors and affiliations

Science Translational Medicine  09 Aug 2017:
Vol. 9, Issue 402, eaai7993
DOI: 10.1126/scitranslmed.aai7993
  • Fig. 1. A subset of ER+ breast cancers remains highly proliferative despite letrozole-mediated estrogen deprivation.

    (A) Schema of clinical trial of 143 patients with ER+/HER2 breast cancer treated for 10 to 21 days with letrozole. Arrows indicate general time points at which a biopsy was taken or surgery was performed. (B) Heat map displaying pre- and post-letrozole treatment IHC (by AQUA) scores for Ki67, ER, and PR in tumor specimens stratified by Ki67 response to letrozole. Molecular subtype, recurrence score by IHC4, and histologic type are also noted. (C) Paired pre- and post-letrozole treatment tumor specimens from the trial were stratified into sensitive, intermediate, or resistant response categories based on posttreatment Ki67 scores. BrCa, breast cancer; pts, patients; QD, once daily; ln, natural log; NST, invasive carcinoma of no special type; ILC, invasive lobular carcinoma.

  • Fig. 2. WES identifies CNAs associated with endocrine therapy resistance.

    (A) Tile plot of variants identified in significantly mutated genes detected by WES in 54 tumor samples. Samples are listed by response category (13 resistant, 8 intermediate, 30 sensitive, and 3 unknown). Genes were considered significantly mutated if their associated q value was ≥0.1 [−log10 (q value) ≥1.0, delineated by the solid red line in the histogram on the right]. (B) Heat map showing log2 copy number ratios for genomic regions with recurrent gains (red) or losses (green) by GISTIC. Available for CNV analysis were 12 resistant, 8 intermediate, 35 sensitive, and 4 unknown tumors.

  • Fig. 3. CCND1 and FGFR1 amplifications detected by FISH are associated with resistance to estrogen deprivation.

    (A) Representative FISH images from our cohort displaying samples with CCND1 amplification (patient 7629), FGFR1 amplification (patient 7670), CCND1 and FGFR1 coamplification (patient 7657), and a patient negative for CCND1 and FGFR1 amplification (patient 1213). Magnification = ×100 for each image, representative scale bar shown in FGFR1/7629. (B) Graphical summary of CCND1 amplification, FGFR1 amplification, and FGFR1/CCND1 coamplification across letrozole-sensitive, letrozole-intermediate, and letrozole-resistant patients as per their posttreatment Ki67 categorization. Numbers for this analysis are shown in Table 3.

  • Fig. 4. Gene expression analysis reveals multiple pathways that strongly correlate with resistance to estrogen deprivation.

    (A) Principal components analysis of gene expression shows that tumors separate by response to estrogen. PC, principal component. (B) This separation is maintained even after removal of proliferation-associated genes. (C) With the removal of proliferation-associated genes, differential gene expression analysis using RNAseq data for response to letrozole shows enrichment for SH2 domain– and SH3 domain–containing genes (highlighted in red text). (D and E) Pathway analyses performed using RNAseq data from letrozole-treated breast cancers reveal an up-regulation of genes involved in the cell cycle (D), particularly in tumors with FGFR1 and CCND1 coamplification, as well as ECM proteins (E). ES, enrichment signature. (F) Pathway analysis after removal of proliferation genes shows up-regulation of secretome-related signaling. (G and H) Analysis of the RB1 loss gene expression signature shows increased expression in the resistant tumors (G) and tumors coamplified for CCND1 and FGFR1 (H). Bars, mean ± SD. P values represent results of a one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (*P < 0.05 and ***P < 0.001).

  • Fig. 5. RNAseq identifies ESR1 fusion transcripts associated with resistance to antiestrogen therapy.

    (A) Diagram of chromosome 6 highlighting the 6q25 locus and showing that ESR1 is recurrently rearranged with other genes in 6q25, including AKAP12, c6orf211, and CCDC170. (B) Heat map of ESR1 fusion results from NanoString nCounter analysis. Left panels represent counts resulting from analysis of probes covering each exon of 5′ 6q25 genes; right panels represent counts resulting from probes covering each exon of ESR1 (3′ on 6q25). See tables S6 and S7 for validation results. E2/C2, cells transduced with a construct encoding the ESR1 ex2–CCDC170 ex2 fusion; GFP, cells transduced with a construct encoding green fluorescent protein.

  • Fig. 6. FGFR1 and CCND1 amplification–mediated resistance to estrogen deprivation is therapeutically actionable.

    (A) FGFR1 and CCND1 coamplified CAMA1 breast cancer cells were seeded in estrogen-free medium supplemented with FGF3/19 (100 ng/ml) and treated with 500 nM of the CDK4/6 inhibitor, palbociclib (Palbo), and/or 1 μM of the FGFR1 inhibitor, lucitanib (Luc). When the untreated control cells reached 50 to 70% confluence (day 12), the cells were fixed and stained with crystal violet, and two-dimensional (2D) growth was quantified. The combination of palbociclib + lucitanib was sufficient to overcome resistance to estrogen withdrawal better than either single agent. ns, not significant. Further highlighting this effect, (B) presents the CI values calculated based on the average of the fold change in 2D growth with increasing combination doses of lucitanib (0 to 1000 nM) and palbociclib (0 to 500 nM) relative to untreated controls from three independent experiments. CI < 1 represents synergism, CI = 1 represents an additive effect, and CI > 1 represents antagonism. (C) S phase analysis of CAMA1 cells treated with FGF3/19 (100 ng/ml), ±1 μM lucitanib, and ±500 nM palbociclib reveals that treatment with palbociclib or the combination of lucitanib + palbociclib can overcome FGF-induced persistence of CAMA1 cells under estrogen-deprived conditions. Individual plots for each treatment condition can be found in fig. S7A. (D) Finally, immunoblot analysis of CAMA1 whole-cell lysates shows that only combined inhibition of FGFR1 and CDK4/6 can simultaneously decrease extracellular signal–regulated kinase 1/2 (ERK1/2) and Rb phosphorylation. P values represent results of two-way Student’s t test. Lucitanib = 1 μM and palbociclib = 500 nM. Comb, combination of lucitanib and palbociclib.

  • Fig. 7. Profiling of serial breast tumor biopsies suggests 8p11-12 and 11q13 amplifications and ESR1 mutations as markers of endocrine therapy resistance.

    (A) Schema of the treatment course of seven patients with ER+ breast cancer from which pretreatment, postneoadjuvant chemotherapy, and metastatic recurrence biopsies were collected. Dashed lines represent time points at which biopsies were taken. A/C, adriamycin and cyclophosphamide. (B) Diagram of the landscape of genomic alterations for the seven patients as per targeted next-generation profiling by FoundationOne, including time to recurrence and previous treatment. The ESR1 mutations that were identified are specifically named.

  • Table 1. Baseline clinical characteristics of the 143 study patients (n = 155 tumors).

    n, number of patients or number of tumors for which data were available.

    Age (n = 140 patients)Years
      Range45–87
      Mean64
    Tumor type (n = 155)n (%)
      NST (no special type)84 (54)
      Lobular20 (13)
      NST with special features (lobular, mucinous,
    tubular, and cribriform)
    44 (28)
      Special type7 (5)
    Tumor histological grade (n = 155)n (%)
      Low54 (35)
      Intermediate83 (54)
      High17 (11)
    Pathological stage* (n = 149)n (%)
      IA74 (50)
      IB6 (4)
      IIA39 (26)
      IIB18 (12)
      IIIA4 (2.5)
      IIIB1 (0.75)
      IIIC6 (4)
      Not available1 (0.75)
    Tumor size (n = 154)n (%)
      T1a: ≤ 5.0 mm7 (5)
      T1b: 5.1–10.0 mm30 (19)
      T1c: 10.1–20.0 mm70 (45)
      T2: 20.1–50.0 mm42 (27)
      T3: ≥50.1 mm5 (3)
    Nodal status (n = 146)n (%)
      Negative98 (67)
      1–337 (25)
      4–95 (3)
      ≥106 (4)
    Tumor laterality (n = 143)n (%)
      Unilateral
        Unifocal133 (93)
        Multifocal4 (3)
      Bilateral6 (4)
    Hormone receptor positivity%
      Estrogen receptor
        Range14–100
        Mean89.5
      Progesterone receptor
        Range0–100
        Mean52.5
    Allred score (% positive range, n = 155)ER+, n (%)PR+, n (%)
      0 (0)0 (0)17 (11)
      1 (<1)0 (0)0 (0)
      2 (1–10)0 (0)25 (16)
      3 (11–33)5 (3)17 (11)
      4 (34–66)6 (4)21 (13.5)
      5 (≥67)142 (92)72 (46.5)
      Unknown2 (1)3 (2)

    *One hundred forty-three unique tumors + six contralateral tumors.

    †Positive but unknown proportion.

    • Table 2. Categorization of study samples by post-letrozole Ki67 proliferative index.

      Category%Ki67IMPACT tertileSamples (%)
      Sensitive≤2.7≤1.078 (56)
      Intermediate2.8–7.31.1–1.932 (23)
      Resistant≥7.4≥2.030 (21)
      Total140
    • Table 3. Summary of FGFR1 and CCND1 FISH analysis in trial tumors.

      Bold text highlights resistant samples amplified for FGFR1 and/or CCND1.

      Sensitive (%)Intermediate (%)Resistant (%)Total
      Neg/equiv26 (74)10 (83)10 (50)46
      FGFR1 amp2 (6)1 (8.5)1 (5)4
      CCND1 amp6 (17)1 (8.5)3 (15)10
      Coamp1 (3)0 (0)6 (30)7
      No. of tumors35122067
      Total amp9/35 (26)2/12 (17)10/20 (50)
      Pearson χ2χ2 = 12.6664P = 0.0487

    Supplementary Materials

    • www.sciencesignaling.org/cgi/content/full/9/402/eaai7993/DC1

      Materials and Methods

      Fig. S1. Flowchart of tumor sample availability for study analyses.

      Fig. S2. Immunohistochemical response of ER+ breast tumors to short-term letrozole, assessed by Ki67 and PR expression.

      Fig. S3. Log2 copy number ratios on chromosomes 8 and 11 as per WES analysis.

      Fig. S4. Individual FISH analysis for FGFR1 or CCND1 amplification and comparison with WES log2 CNV values and with RNAseq transcript expression.

      Fig. S5. Principal components analysis, Database for Annotation, Visualization and Integrated Discovery (DAVID), Gene Set Enrichment (GSE), and iRegulon analyses based on RNAseq data from ER+, letrozole-treated breast tumors and TCGA breast tumors.

      Fig. S6. RNAseq fusion validation pipeline and comparison of ESR1 transcript expression.

      Fig. S7. Pharmacological inhibition of FGFR1 and/or CDK4/6 activity mediated by FGFR1 ± CCND1 amplification/overexpression.

      Fig. S8. ERα transcriptional reporter assays assessing the activity of ERα LBD mutations.

      Table S1. Statistical correlation of whole exome identified SNVs and CNVs with lack of response to letrozole.

      Table S2. List of proliferation-associated genes (Excel file).

      Table S3. Three hundred forty-six putative gene fusion transcripts identified by RNAseq in 50 tumors (Excel file).

      Table S4. Primer sets for validation of RNAseq-identified putative fusion transcripts (Excel file).

      Table S5. Summary and sequences of 26 RNAseq-identified, RT-PCR–validated fusion transcripts (Excel file).

      Table S6. ESR1 fusion transcripts identified from TCGA RNAseq data.

      Table S7. Validation and diagrams of putative ESR1 fusions in patient and cell line cDNA.

      Table S8. Sanger sequences from RT-PCR–validated, NanoString-identified ESR1 fusion transcripts.

      Table S9. Clinical characteristics of 10 breast cancer patients profiled by FoundationOne harboring the ESR1 c.1265_1267delTGG/ERα p.V422del alteration.

      Table S10. Primers for PCR and direct sequencing of ESR1 fusions identified by NanoString in patient and cell line cDNA.

      Table S11. Primer combination to validate NanoString-identified fusions in patient and cell line cDNA.

      Table S12. CCND1 and FGFR1 cloning primers.

      References (4453)

    • Supplementary Material for:

      Genomic profiling of ER+ breast cancers after short-term estrogen suppression reveals alterations associated with endocrine resistance

      Jennifer M. Giltnane, Katherine E. Hutchinson, Thomas P. Stricker, Luigi Formisano, Christian D. Young, Monica V. Estrada, Mellissa J. Nixon, Liping Du, Violeta Sanchez, Paula Gonzalez Ericsson, Maria G. Kuba, Melinda E. Sanders, Xinmeng J. Mu, Eliezer M. Van Allen, Nikhil Wagle, Ingrid A. Mayer, Vandana Abramson, Henry Gómez, Monica Rizzo, Weiyi Toy, Sarat Chandarlapaty, Erica L. Mayer, Jason Christiansen, Danielle Murphy, Kerry Fitzgerald, Kai Wang, Jeffrey S. Ross, Vincent A. Miller, Phillip J. Stephens, Roman Yelensky, Levi Garraway, Yu Shyr, Ingrid Meszoely, Justin M. Balko, Carlos L. Arteaga*

      *Corresponding author. Email: carlos.arteaga{at}vanderbilt.edu

      Published 9 August 2017, Sci. Transl. Med. 9, eaai7993 (2017)
      DOI: 10.1126/scitranslmed.aai7993

      This PDF file includes:

      • Materials and Methods
      • Fig. S1. Flowchart of tumor sample availability for study analyses.
      • Fig. S2. Immunohistochemical response of ER+ breast tumors to short-term letrozole, assessed by Ki67 and PR expression.
      • Fig. S3. Log2 copy number ratios on chromosomes 8 and 11 as per WES analysis.
      • Fig. S4. Individual FISH analysis for FGFR1 or CCND1 amplification and comparison with WES log2 CNV values and with RNAseq transcript expression.
      • Fig. S5. Principal components analysis, Database for Annotation, Visualization and Integrated Discovery (DAVID), Gene Set Enrichment (GSE), and iRegulon analyses based on RNAseq data from ER+, letrozole-treated breast tumors and TCGA breast tumors.
      • Fig. S6. RNAseq fusion validation pipeline and comparison of ESR1 transcript expression.
      • Fig. S7. Pharmacological inhibition of FGFR1 and/or CDK4/6 activity mediated by FGFR1 ± CCND1 amplification/overexpression.
      • Fig. S8. ERα transcriptional reporter assays assessing the activity of ERα LBD mutations.
      • Table S1. Statistical correlation of whole exome identified SNVs and CNVs with lack of response to letrozole.
      • Legends for tables S2 to S5
      • Table S6. ESR1 fusion transcripts identified from TCGA RNAseq data.
      • Table S7. Validation and diagrams of putative ESR1 fusions in patient and cell line cDNA.
      • Table S8. Sanger sequences from RT-PCR–validated, NanoString-identified ESR1 fusion transcripts.
      • Table S9. Clinical characteristics of 10 breast cancer patients profiled by FoundationOne harboring the ESR1 c.1265:1267delTGG/ERα p.V422del alteration.
      • Table S10. Primers for PCR and direct sequencing of ESR1 fusions identified by NanoString in patient and cell line cDNA.
      • Table S11. Primer combination to validate NanoString-identified fusions in patient and cell line cDNA.
      • Table S12. CCND1 and FGFR1 cloning primers.
      • References (4453)

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Table S2. List of proliferation-associated genes (Excel file).
      • Table S3. Three hundred forty-six putative gene fusion transcripts identified by RNAseq in 50 tumors (Excel file).
      • Table S4. Primer sets for validation of RNAseq-identified putative fusion transcripts (Excel file).
      • Table S5. Summary and sequences of 26 RNAseq-identified, RT-PCR–validated fusion transcripts (Excel file).

      [Download Tables S2 to S5]

    Navigate This Article