Ellis, Matthew J.

LBA513 Background: Neoadjuvant aromatase inhibitor (AI) therapy is a rational and effective approach to improving the breast conservation surgery (BCS) rate for postmenopausal patients with large, estrogen receptor (ER) rich breast cancers. Barriers to adopting this strategy include lack of experience in this management approach in the US and uncertainty regarding the comparative effectiveness of the three approved aromatase inhibitors for this indication. Methods: ACOSOG Z1031 is a multicenter, open-label, neoadjuvant phase III screening study that randomized postmenopausal women with clinical stage II/III ER rich (Allred score 6-8) breast cancer to 16 weeks of either exemestane (EXE) 25 mg daily, letrozole (LET) 2.5 mg daily, or anastrozole (ANA) 1 mg daily. At baseline study participants were either marginal for BCS (MBCS), candidates for mastectomy only (MO), or inoperable (IO). Planned enrolment was 125 patients per arm in order that the likelihood of the treatment with the “best” 16-week clinical response rate (based on caliper measurements) by WHO criteria (cRR) was included among the subset of treatments with “similar” cRR (90% power). Secondary endpoints included: extent of surgery, radiologic and pathologic response rates. Results: From 4/1/2006 to 10/1/2009, 377 postmenopausal women with clinical stage II or III ER rich breast cancer were enrolled. 374 women began treatment and were included in an intent-to-treat analysis. Median age was 66 yrs (range: 43-90 yrs), Median tumor size was 4.0 cm (range: 2-13 cm). The 16-week cRR was 60.5% (95%CI: 51.3-69.1%) for EXE; 70.9% (95% CI: 62.2-78.6%) for LET, and 66.7% (95% CI: 57.6-74.9%) for ANA. Seventeen patients did not have surgery due to refusal (12 pts), progression (3 pts) or other medical conditions (2 pts). The BCS rate was 78% (163/207) in MBCS group; 42% (77/163) in MO group; and 75% in IO group (3/4). Surgeons made the decisions regarding procedure choice 75% of the time in both the MBCS and the MO categories. Conclusions: This large multicenter screening trial selected non-steroidal AIs for further development due to their higher observed cRR. The study demonstrates that high response and breast conservation rates and low rates of disease progression can be achieved through patient selection based on high ER expression. We are currently refining our approach for early detection of poor response to AIs through an assessment of the tumor Ki67 proliferation index at 2 to 4 weeks (Z1031 Cohort B). [Table: see text]

Purpose To determine the pathologic complete response (pCR) rate in estrogen receptor (ER) –positive primary breast cancer triaged to chemotherapy when the protein encoded by the MKI67 gene (Ki67) level was > 10% after 2 to 4 weeks of neoadjuvant aromatase inhibitor (AI) therapy. A second objective was to examine risk of relapse using the Ki67-based Preoperative Endocrine Prognostic Index (PEPI). Methods The American College of Surgeons Oncology Group (ACOSOG) Z1031A trial enrolled postmenopausal women with stage II or III ER-positive (Allred score, 6 to 8) breast cancer whose treatment was randomly assigned to neoadjuvant AI therapy with anastrozole, exemestane, or letrozole. For the trial ACOSOG Z1031B, the protocol was amended to include a tumor Ki67 determination after 2 to 4 weeks of AI. If the Ki67 was > 10%, patients were switched to neoadjuvant chemotherapy. A pCR rate of > 20% was the predefined efficacy threshold. In patients who completed neoadjuvant AI, stratified Cox modeling was used to assess whether time to recurrence differed by PEPI = 0 score (T1 or T2, N0, Ki67 < 2.7%, ER Allred > 2) versus PEPI > 0 disease. Results Only two of the 35 patients in ACOSOG Z1031B who were switched to neoadjuvant chemotherapy experienced a pCR (5.7%; 95% CI, 0.7% to 19.1%). After 5.5 years of median follow-up, four (3.7%) of the 109 patients with a PEPI = 0 score relapsed versus 49 (14.4%) of 341 of patients with PEPI > 0 (recurrence hazard ratio [PEPI = 0 v PEPI > 0], 0.27; P = .014; 95% CI, 0.092 to 0.764). Conclusion Chemotherapy efficacy was lower than expected in ER-positive tumors exhibiting AI-resistant proliferation. The optimal therapy for these patients should be further investigated. For patients with PEPI = 0 disease, the relapse risk over 5 years was only 3.6% without chemotherapy, supporting the study of adjuvant endocrine monotherapy in this group. These Ki67 and PEPI triage approaches are being definitively studied in the ALTERNATE trial (Alternate Approaches for Clinical Stage II or III Estrogen Receptor Positive Breast Cancer Neoadjuvant Treatment in Postmenopausal Women: A Phase III Study; clinical trial information: NCT01953588).

Abstract Genetic risks in breast cancer remain only partly understood. Here, we report the results of a genome-wide association study of germline DNA from 4,658 women, including 252 women experiencing a breast cancer recurrence, who were entered on the MA.27 adjuvant trial comparing the aromatase inhibitors (AI) anastrozole and exemestane. Single-nucleotide polymorphisms (SNP) of top significance were identified in the gene encoding MIR2052HG, a long noncoding RNA of unknown function. Heterozygous or homozygous individuals for variant alleles exhibited a ∼40% or ∼63% decrease, respectively, in the hazard of breast cancer recurrence relative to homozygous wild-type individuals. Functional genomic studies in lymphoblastoid cell lines and ERα-positive breast cancer cell lines showed that expression from MIR2052HG and the ESR1 gene encoding estrogen receptor-α (ERα) was induced by estrogen and AI in a SNP-dependent manner. Variant SNP genotypes exhibited increased ERα binding to estrogen response elements, relative to wild-type genotypes, a pattern that was reversed by AI treatment. Further, variant SNPs were associated with lower expression of MIR2052HG and ERα. RNAi-mediated silencing of MIR2052HG in breast cancer cell lines decreased ERα expression, cell proliferation, and anchorage-independent colony formation. Mechanistic investigations revealed that MIR2052HG sustained ERα levels both by promoting AKT/FOXO3-mediated ESR1 transcription and by limiting ubiquitin-mediated, proteasome-dependent degradation of ERα. Taken together, our results define MIR2052HS as a functionally polymorphic gene that affects risks of breast cancer recurrence in women treated with AI. More broadly, our results offer a pharmacogenomic basis to understand differences in the response of breast cancer patients to AI therapy. Cancer Res; 76(23); 7012–23. ©2016 AACR.

Abstract Purpose: PI3K/AKT pathway activation is an important endocrine resistance mechanism in estrogen receptor–positive (ER+) breast cancer. After promising preclinical modeling of MK-2206, an allosteric pan-AKT inhibitor, with either estrogen deprivation or fulvestrant, we conducted a phase I trial in patients with metastatic ER+HER2− breast cancer to determine the recommended phase II treatment dose (RPTD) of MK-2206 when combined with either anastrozole, fulvestrant, or anastrozole/fulvestrant. Experimental Design: ER+ breast cancer cell lines were exposed in vitro to MK-2206 plus estrogen deprivation with or without fulvestrant and monitored for apoptosis. A standard 3+3 design was employed to first determine the maximum tolerated dose (MTD) of MK-2206 plus anastrozole based on cycle 1 toxicity. Each cycle was 28 days. The RPTD was determined on the basis of toxicities observed at MTD level during the first 3 cycles. Subsequent patients received MK-2206, at the RPTD determined above, plus fulvestrant or anastrozole/fulvestrant to define RPTD for these additional regimens. Results: MK-2206 induced apoptosis in parental ER+ but not in long-term estrogen-deprived cell lines, for which fulvestrant was required for apoptosis induction. Thirty-one patients enrolled. The RPTD was defined as MK-2206 150 mg orally weekly with prednisone prophylaxis for each combination. Grade 3 rash was dose limiting. 42% (95% CI, 23%–63%) patients derived clinical benefit without progression within 6 months. Response was not associated with tumor PIK3CA mutation. Conclusions: MK-2206 plus endocrine treatments were tolerable. MK-2206 in combination with anastrozole is being further evaluated in a phase II neoadjuvant trial for newly diagnosed ER+HER2− breast cancer. Clin Cancer Res; 22(11); 2650–8. ©2016 AACR. See related commentary by Jansen et al., p. 2599

AbstractBACKGROUNDFor many years, basic and clinical researchers have taken advantage of the analytical sensitivity and specificity afforded by mass spectrometry in the measurement of proteins. Clinical laboratories are now beginning to deploy these work flows as well. For assays that use proteolysis to generate peptides for protein quantification and characterization, synthetic stable isotope–labeled internal standard peptides are of central importance. No general recommendations are currently available surrounding the use of peptides in protein mass spectrometric assays.CONTENTThe Clinical Proteomic Tumor Analysis Consortium of the National Cancer Institute has collaborated with clinical laboratorians, peptide manufacturers, metrologists, representatives of the pharmaceutical industry, and other professionals to develop a consensus set of recommendations for peptide procurement, characterization, storage, and handling, as well as approaches to the interpretation of the data generated by mass spectrometric protein assays. Additionally, the importance of carefully characterized reference materials—in particular, peptide standards for the improved concordance of amino acid analysis methods across the industry—is highlighted. The alignment of practices around the use of peptides and the transparency of sample preparation protocols should allow for the harmonization of peptide and protein quantification in research and clinical care.

Abstract Background. We recently identified an in-frame ESR1 translocation in a tumor and PDX pair from a patient with endocrine therapy resistant advanced disease. The fusion gene preserved the N-terminal 365aa of ESR1 containing the intact activation function 1 (AF1), DNA-binding domain (DBD) and hinge, followed by the C-terminal transactivation domain of YAP1. The ESR1-365&gt;YAP1 fusion drove estrogen-independent and anti-estrogen-resistant tumor cell growth (Cell Reports 4:1116, 2013). We therefore sought to further investigate the role of ESR1 fusion genes in breast cancer. Methods. 711 cases from TCGA, 82 ER+ cases from neoadjuvant endocrine therapy trials and 25 ER+ systemic relapse samples were screened for fusion genes using RNAseq. Fusion genes involving ESR1 were subsequently assayed for estrogen response element (ERE)-mediated transcriptional activity and ability to drive estradiol independent breast cancer cell growth. Results. Multiple ESR1 fusions were identified in mostly luminal B cancers. ESR1 fusions resulted most frequently from rearrangements involving another gene on Chromosome (Chr) 6 but also fusions with genes on other chromosomes. The fusions involving partners on Chr6 that retained an intact N terminal AF1 and DNA binding domain (DBD) of ESR1 (≥1-253aa) included an Out-of-Frame (OF) fusion event with CCDC170 (a gene immediately centromeric to ESR1), POLH (in-frame or IF), AKAP12 (IF), PCMT1 (OF), SYNE1 (OF) and GPR126 (OF). Also identified were inter-chromosomal translocations involving Chr12p-NOP2 (IF) and ChrX-PCDH11X (IF) and Chr7q-AKR1D1 (OF). The ESR1 fusions with transcriptional potential (because of a retained DNA binding domain) included variable 5’ ESR1 exons that preserved 253aa, 365aa, 412aa or 458aa of ESR1 sequence: in all cases disrupting ligand binding though loss of C terminal sequence. The effect of the IF and OF 3’ sequences on the transcriptional activity of the relevant ESR1 fragment was highly variable. The most transcriptionally active fusion (more active than the relevant ESR1 fragment alone) was the ESR1-365&gt;YAP1 as well as ESR1-365&gt;PCDH11X and ESR1-253&gt;CCDC170. This is remarkable since PCDH11X is not considered a transcription factor and the CCDC170 sequence was OF – in both cases suggesting the activity of the fusion was due to "neomorphic" properties. Two fusions, NOP2 and POLH were inactive in the ERE reporter assay, yet both stimulated estradiol independent growth. In these cases we suggest that the ESR1 locus can function as a "promoter trap" which allows the identification of genes with previously unknown functions in endocrine therapy resistance. Conclusions: In luminal-type breast cancer the ESR1 gene was fused to multiple 3’ partners with remarkably heterogeneous functions. The two most transcriptionally active, ESR1-365&gt;YAP1 and ESR1-365&gt;PCDH11X, were both identified in endocrine therapy refractory advanced disease – suggesting a role in fatal disease progression. Citation Format: Jieya Shao, Jin Zhang, Robert J Crowder, Rodrigo Goncalves, Chanpheng Phommaly, Breast AWG and Network, Charles M Perou, Christopher A Maher, E Aubrey Thompson, Matthew J Ellis. ESR1 gene fusions implicated in endocrine therapy resistance of ER+ breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD6-4.

Abstract Background: The Preoperative Endocrine Prognostic Index (PEPI) is a method to predict outcome after neoadjuvant endocrine therapy that integrates Ki67, estrogen receptor (ER) analysis and pathological stage from the surgical specimen (Ellis, JNCI 100:1380, 2008). We sought to further develop the PEPI for use in clinical trials by: a) establishing an efficient SOP for Ki67 analysis, b) determining the effect of simplifying the score by removing the ER component (modified "mPEPI") and c) independent validation of mPEPI in the ACOSOG (Alliance) Z1031 neoadjuvant aromatase inhibitor trial (Ellis, M, JCO 29:234, 2011). Methods: Ki-67 assay development focused on reproducing a 2.7% Ki-67 cut point (CP) required for PEPI. Ventana Image analysis (IA) to replace labor-intensive visual point counting (VPC) was assessed to increase scoring efficiency. Discordant scores led to a triage approach where cases with complex histological features that could not be resolved by IA were flagged for VPC. The Ki-67 scoring approach was preliminarily validated on T1/2 N0 cases from the P024 and POL trials (SABCS 2013, abstract P3-05-190). Models with and without ER suggested ER was dispensable. A locked SOP for mPEPI was subsequently applied to the Z1031A trial. The primary endpoint was time from the date of surgery to local, regional, or distant recurrence in the mPEPI-0 group (T1/2 N0, Ki67 &lt;2.7%) versus the mPEPI&gt;0 group. Results. mPEPI by IA was evaluated on 202 of 377 (53%) patients enrolled into Z1031A (6% of IA results were triaged to VPC). All patients have been followed a minimum of 2 years (median: 5 years; max: 7 years). Only 10 patients in the mPEPI-0 group (22.7%) received adjuvant chemotherapy, versus 78 in the mPEPI&gt;0 group (49.4%). Time to breast cancer recurrence was decreased among those with mPEPI&gt;0 status relative to those with mPEPI-0 status (log rank p=0.012). Only one disease event among 44 (2%) cases with mPEPI-0 was observed versus 26 of 158 cases with mPEPI&gt;0 (16.5%) Conclusions. mPEPI-0 status can identify patients at low risk of relapse after neoadjuvant endocrine therapy: therefore mPEPI-0 status has operational characteristics similar to pCR after chemotherapy for ER-negative disease. mPEPI is undergoing prospective validation in the Alliance ALTERNATE trial that will assess whether Fulvestrant increases the mPEPI-0 rate and also will prospectively determine whether patients with mPEPI-0 status can safely be managed without adjuvant chemotherapy treatment. Citation Format: Souzan Sanati, Vera J Suman, Rodrigo Goncalves, Katherine DeSchryver, Cynthia X Ma, Jeremy Hoog, Erika Crouch, Michael Barnes, Gary Unzeitig, A Marilyn Leitch, Kelly K Hunt, Matthew J Ellis. Validation of the preoperative endocrine prognostic index in the ACOSOG (Alliance) Z1031 neoadjuvant aromatase inhibitor trial [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-11-13.

Abstract Neoadjuvant endocrine therapy (ET) in patients (pts) with locally advanced estrogen receptor positive (ER+) breast cancer (BC) improves breast conservation rate, and importantly offers an opportunity for individualized assessment of tumor responsiveness to ET to guide subsequent treatment. Previous neoadjuvant ET trials demonstrated that pathologic tumor size (pT), axillary lymph node status (N), and tumor Ki67 value at surgery predicted risk of relapse. Preoperative endocrine prognostic index (PEPI) was therefore developed to assign risk scores based on these factors. Modified PEPI 0, defined by pT1-2 N0 Ki67&lt; 2.7%, was associated with extremely low risk of recurrence without adjuvant chemotherapy. In addition, high Ki67 post 2-4 weeks (wks) of neoadjuvant ET identified resistant tumors with poor outcome. ALTERNATE trial is a three-arm phase III neoadjuvant/adjuvant ET trial designed to achieve two primary objectives. The first is to prospectively validate that modified PEPI 0 predicts &gt; 95% relapse-free survival (RFS). The second is to determine whether fulvestrant (F), or fulvestrant (F) plus anastrozole (A), is superior to A in inducing a higher rate of modified PEPI 0. Secondary objectives include assessing RFS for pts with endocrine resistant tumor, defined by Ki67 &gt; 10% at 4 or 12 wk, disease progression, or modified PEPI non-0, and pathologic responses of resistant tumors to neoadjuvant chemotherapy. Correlative studies include degrees of Ki67 suppression and ER level post treatment. In addition, ongoing research sequencing studies of DNA and RNA will be performed to contrast sensitive vs resistant tumors. During the first phase of the trial, 1200 pts are randomized 1:1:1 to the F, A or F/A. This provides an 82% chance, 1-sided alpha 0.025 chi-square test to detect at least 10% difference in modified PEPI 0 rate comparing F or F/A with A. While waiting for result of analysis, the A arm will continue for the 2nd phase enrollment. Only the F-containing arm(s) superior over A will be continued to the 2nd phase. During the 2nd phase, an additional 540 pts in each arm is estimated to obtain 317 pts with PEPI score 0. This will have a 90% chance, with a one-sided alpha=0.025 nonparametric Brookmeyer-Crowley type one sample survival test, rejecting that 5 year RFS rate is 95%. The maximum sample size is 2820 pts. Eligible pts include postmenopausal women with newly diagnosed clinical stage II or III ER+ (Allred score 6-8) HER2- BC. Tumor biopsy is required at baseline, 4 wk, and surgery for central Ki67 analysis. Treatment decision is individualized based on 4 wk Ki67 and modified PEPI score. Pts with Ki67 &gt; 10% at 4 wk are switched to chemotherapy. Pts with a modified PEPI score of 0 are recommended not to receive chemotherapy, but continue the assigned ET for 1.5 years followed by 3 years of A. This trial is currently accruing pts through CTSU. This research is supported in part by NCI BIQSFP, and grants from Breast Cancer Research Foundation, Genentech, and the Investigator-Sponsored Study Program of AstraZeneca. The Clinical Trials.gov Identifier: NCT01953588. Citation Format: Cynthia X Ma, Vera Suman, A Marilyn Leitch, Souzan Sanati, Katherine DeSchryver, Gary W Unzeitig, Paul Haluska, Mark Watson, Olwen Hahn, Jo Anne Zujewski, Kelly Hunt, Eric P Winer, Cliff A Hudis, Matthew J Ellis. Alliance A011106: ALTernate approaches for clinical stage II or III Estrogen Receptor positive breast cancer NeoAdjuvant TrEatment (ALTERNATE) in postmenopausal women: A phase III study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT2-1-04.

Purpose In patients with hormone-dependent postmenopausal breast cancer, standard adjuvant therapy involves 5 years of the nonsteroidal aromatase inhibitors anastrozole and letrozole. The steroidal inhibitor exemestane is partially non–cross-resistant with nonsteroidal aromatase inhibitors and is a mild androgen and could prove superior to anastrozole regarding efficacy and toxicity, specifically with less bone loss. Patients and Methods We designed an open-label, randomized, phase III trial of 5 years of exemestane versus anastrozole with a two-sided test of superiority to detect a 2.4% improvement with exemestane in 5-year event-free survival (EFS). Secondary objectives included assessment of overall survival, distant disease–free survival, incidence of contralateral new primary breast cancer, and safety. Results In the study, 7,576 women (median age, 64.1 years) were enrolled. At median follow-up of 4.1 years, 4-year EFS was 91% for exemestane and 91.2% for anastrozole (stratified hazard ratio, 1.02; 95% CI, 0.87 to 1.18; P = .85). Overall, distant disease–free survival and disease-specific survival were also similar. In all, 31.6% of patients discontinued treatment as a result of adverse effects, concomitant disease, or study refusal. Osteoporosis/osteopenia, hypertriglyceridemia, vaginal bleeding, and hypercholesterolemia were less frequent on exemestane, whereas mild liver function abnormalities and rare episodes of atrial fibrillation were less frequent on anastrozole. Vasomotor and musculoskeletal symptoms were similar between arms. Conclusion This first comparison of steroidal and nonsteroidal classes of aromatase inhibitors showed neither to be superior in terms of breast cancer outcomes as 5-year initial adjuvant therapy for postmenopausal breast cancer by two-way test. Less toxicity on bone is compatible with one hypothesis behind MA.27 but requires confirmation. Exemestane should be considered another option as up-front adjuvant therapy for postmenopausal hormone receptor–positive breast cancer.