Published OnlineFirst May 16, 2016; DOI: 10.1158/1078-0432.CCR-16-0148 Personalized Medicine and Imaging Clinical Cancer Research TransCONFIRM: Identification of a Genetic Signature of Response to Fulvestrant in Advanced Hormone Receptor–Positive Breast Cancer Rinath Jeselsohn1,2, William T. Barry1,3, Ilenia Migliaccio4, Chiara Biagioni4,5, Jin Zhao2, Jonas De Tribolet-Hardy2, Cristina Guarducci4, Martina Bonechi4, Naomi Laing6, Eric P. Winer1, Myles Brown1,2, Angelo Di Leo4,5, and Luca Malorni4,5 Abstract Purpose: Fulvestrant is an estrogen receptor (ER) antago- Results: Pathway analysis demonstrated that increased EGF nist and an approved treatment for metastatic estrogen pathway and FOXA1 transcriptional signaling is associated with þ receptor–positive (ER ) breast cancer. With the exception of decreased response to fulvestrant. Using a multivariate Cox mod- ER levels, there are no established predictive biomarkers of el, we identified a novel set of 37 genes with an expression that is response to single-agent fulvestrant. We attempted to identify independently associated with progression-free survival (PFS). a gene signature of response to fulvestrant in advanced breast TFAP2C, a known regulator of ER activity, was ranked second in cancer. this gene set, and high expression was associated with a decreased Experimental Design: Primary tumor samples from 134 response to fulvestrant. The negative predictive value of TFAP2C patients enrolled in the phase III CONFIRM study of patients expression at the protein level was confirmed by IHC. þ with metastatic ER breast cancer comparing treatment with Conclusions: We identified biologic pathways and a novel gene þ either 250 mg or 500 mg fulvestrant were collected for signature in primary ER breast cancers that predicts for response genome-wide transcriptomic analysis. Gene expression pro- to treatment in the CONFIRM study. These results suggest poten- filing was performed using Affymetrix microarrays. An explor- tial new therapeutic targets and warrant further validation as atory analysis was performed to identify biologic pathways predictive biomarkers of fulvestrant treatment in metastatic breast and new signatures associated with response to fulvestrant. cancer. Clin Cancer Res; 22(23); 5755–64. Ó2016 AACR. Introduction fulvestrant was shown to have activity in metastatic disease after progression on an AI (2). In a phase II prospective study in Fulvestrant is a highly selective estrogen receptor alpha (ER) which postmenopausal patients received fulvestrant after pro- ligand devoid of agonist activity. When bound to ER, fulvestrant gression on an AI, 30% of the patients derived clinical benefitat leads to ER degradation resulting in decreased cellular ER levels 24 weeks, and previous response to an AI did not predict and to the inactivation of both transactivation domains, AF1 response to fulvestrant (3). More recently, the phase II FIRST and AF2. This ability to induce ER degradation has led to its study indicated that first-line therapy with fulvestrant at 500 mg classification as a selective ER degrader (SERD). The early has superior activity compared with an AI (4). Currently, there is clinical trials of fulvestrant showed comparable activity to an ongoing phase III study (FALCON) comparing fulvestrant aromatase inhibitors (AI) in advanced breast cancer after pro- (500 mg) to an AI for first-line treatment of metastatic disease gression on first-line endocrine treatment (1). In addition, (www.clinicaltrials.gov identifier NCT TO1602380), which could lead to the approval of fulvestrant as first-line treatment. 1Susan F. Smith Center for Women's Cancers, Dana Farber Cancer These studies and other studies demonstrate that not all patients Institute, Boston, Massachusetts. 2Center for Functional Caner Epige- benefit from single-agent fulvestrant, but there is a subgroup of 3 þ netics, Dana Farber Cancer Institute, Boston, Massachusetts. Depart- patients with metastatic ER breast cancer that gain substantial ment of Biostatistics and Computational Biology, Dana Farber Cancer fi Institute, Boston, Massachusetts. 4Translational Research Unit, Hos- and durable clinical bene t from single-agent fulvestrant even pital of Prato, Istituto Toscano Tumori, Prato, Italy. 5"Sandro Pitigliani" after progression on previous endocrine treatment. However, Medica Oncology Unit, Istituto Toscano Tumori, Prato, Italy. 6Astra there are no well-defined clinicopathologic features or molec- fi Zeneca Pharmaceuticals, Maccles eld, United Kingdom. ular markers other than ER expression to predict response to Note: Supplementary data for this article are available at Clinical Cancer single-agent endocrine treatment as first-line or second-line Research Online (http://clincancerres.aacrjournals.org/). therapy in the metastatic setting. Corresponding Authors: Myles Brown, Dana Farber Cancer Institute, 450 A number of multi-gene expression–based assays have been Brookline Ave, D730, Boston, MA 02215. Phone: 617-632-3948; Fax: 617-582- developed to assess prognosis and predict response to endocrine 8501; E-mail: [email protected]; and Rinath Jeselsohn, treatments and chemotherapy in early-stage estrogen receptor [email protected] þ positive (ER ) breast cancer. The OncotypeDx recurrence score doi: 10.1158/1078-0432.CCR-16-0148 (Genomic Health Inc) is clinically available and widely employed Ó2016 American Association for Cancer Research. in the primary setting to inform decisions concerning the benefit www.aacrjournals.org 5755 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst May 16, 2016; DOI: 10.1158/1078-0432.CCR-16-0148 Jeselsohn et al. cancer. Patients either experienced relapse on adjuvant endocrine Translational Relevance therapy, within 1 year from completion of adjuvant therapy, had Endocrine therapy is the mainstay of treatment for meta- de novo advanced disease or recurrence more than 1 year after þ static ER breast cancer. However, not all patients have a durable completion of adjuvant endocrine therapy. For patients with response with single endocrine agents, and therefore new ther- de novo metastatic disease or recurrences after more than 1 year þ apies for ER disease are emerging. These new agents can add from completion of adjuvant treatment, eligibility criteria requir- toxicity and cost underscoring the need to identify the patients ed prior treatment with an antiestrogen or an AI as first-line whowillrespondwelltoendocrinetherapyaloneandcould treatment. Central confirmation of ER status was not performed. be spared the toxicities of the new agents and on the other hand, The trial had a double-blind, placebo-controlled design, and select patients resistant to existing endocrine therapy who are patients were randomized to fulvestrant 500 mg or 250 mg given candidates for novel combinations. Herein, we report biologic on days 0, 14, 28, and every 28 days thereafter. Treatment was pathways and a putative gene signature that predict response to continued until disease progression unless any criteria for early fulvestrant in a completed randomized phase III trial. discontinuation were met first. The primary study endpoint was progression-free survival (PFS) defined as the time elapsing between the date of random assignment and the date of the earliest evidence of objective disease progression as evaluated of the addition of adjuvant chemotherapy to adjuvant endocrine by RECIST criteria. therapy (5, 6). PAM50, which measures the expression of 50 genes that classify breast cancers into intrinsic subtypes and is applied TransCONFIRM tumor samples for the calculation of a risk of recurrence score (RORS), has also From the 736 patients enrolled in the CONFIRM study, 134 fi fi been shown to be prognostic in the early disease context. An formalin- xed, paraf n-embedded (FFPE) primary tumor sam- analysis of untreated patients with breast cancer and patients that ples were collected with patient consent and local Institutional received neoadjuvant chemotherapy showed that the intrinsic Review Board approval. Samples were centrally reviewed by subtype classification and RORs provide prognostic and predic- a breast pathologist, and tissue microarrays were created in þ À tive information in ER and ER negative early-stage breast cancer quadruplicate. The distribution of the main clinicopatho- (7). A molecular marker or gene set to predict response or logic variables was similar between the entire CONFIRM popu- þ resistance to endocrine treatment in the setting of ER metastatic lation and the transCONFIRM subpopulation (Supplement- disease would be helpful to stratify patients that are less likely to ary Table S1). derive significant benefit from a single-agent endocrine treatment and are candidates for treatments that combine endocrine treat- Gene expression microarrays ment with newer targeted treatments or chemotherapy. The need RNA was extracted from the FFPE tumor samples using RNeasy fi for these tools is becoming more apparent as new treatment FFPE kits (Qiagen) and ampli ed with the WT-Ovation FFPE þ combinations are emerging as effective treatments for ER met- System V2 (NuGen). cDNA was labeled using the FL-Ovation astatic disease. The combination of letrozole and palbociclib was cDNA Biotin Module V2 (Nugen). Samples were run on the þ approved for first-line treatment in ER metastatic disease (8), but Affymetrix Human Transcriptome Array (HTA) 2.0 following the fl it is not clear that all patients need to