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Importance Adding fulvestrant to anastrozole (A+F) improved survival in postmenopausal women with advanced estrogen receptor (ER)–positive/ERBB2 (formerly HER2)–negative breast cancer. However, the combination has not been tested in early-stage disease. Objective To determine whether neoadjuvant fulvestrant or A+F increases the rate of pathologic complete response or ypT1-2N0/N1mic/Ki67 2.7% or less residual disease (referred to as endocrine-sensitive disease) over anastrozole alone. Design, Setting, and Participants A phase 3 randomized clinical trial assessing differences in clinical and correlative outcomes between each of the fulvestrant-containing arms and the anastrozole arm. Postmenopausal women with clinical stage II to III, ER-rich (Allred score 6-8 or >66%)/ERBB2-negative breast cancer were included. All analyses were based on data frozen on March 2, 2023. Interventions Patients received anastrozole, fulvestrant, or a combination for 6 months preoperatively. Tumor Ki67 was assessed at week 4 and optionally at week 12, and if greater than 10% at either time point, the patient switched to neoadjuvant chemotherapy or immediate surgery. Main Outcomes and Measures The primary outcome was the endocrine-sensitive disease rate (ESDR). A secondary outcome was the percentage change in Ki67 after 4 weeks of neoadjuvant endocrine therapy (NET) (week 4 Ki67 suppression). Results Between February 2014 and November 2018, 1362 female patients (mean [SD] age, 65.0 [8.2] years) were enrolled. Among the 1298 evaluable patients, ESDRs were 18.7% (95% CI, 15.1%-22.7%), 22.8% (95% CI, 18.9%-27.1%), and 20.5% (95% CI, 16.8%-24.6%) with anastrozole, fulvestrant, and A+F, respectively. Compared to anastrozole, neither fulvestrant-containing regimen significantly improved ESDR or week 4 Ki67 suppression. The rate of week 4 or week 12 Ki67 greater than 10% was 25.1%, 24.2%, and 15.7% with anastrozole, fulvestrant, and A+F, respectively. Pathologic complete response/residual cancer burden class I occurred in 8 of 167 patients and 17 of 167 patients, respectively (15.0%; 95% CI, 9.9%-21.3%), after switching to neoadjuvant chemotherapy due to week 4 or week 12 Ki67 greater than 10%. PAM50 subtyping derived from RNA sequencing of baseline biopsies available for 753 patients (58%) identified 394 luminal A, 304 luminal B, and 55 nonluminal tumors. A+F led to a greater week 4 Ki67 suppression than anastrozole alone in luminal B tumors (median [IQR], −90.4% [−95.2 to −81.9%] vs −76.7% [−89.0 to −55.6%]; P < .001), but not luminal A tumors. Thirty-six nonluminal tumors (65.5%) had a week 4 or week 12 Ki67 greater than 10%. Conclusions and Relevance In this randomized clinical trial, neither fulvestrant nor A+F significantly improved the 6-month ESDR over anastrozole in ER-rich/ERBB2-negative breast cancer. Aromatase inhibition remains the standard-of-care NET. Differential NET response by PAM50 subtype in exploratory analyses warrants further investigation. Trial Registration ClinicalTrials.gov Identifier: NCT01953588

Abstract Introduction: The ALTERNATE trial randomized postmenopausal women with ER Allred 6-8 HER2- breast cancer to 6 months of NET with anastrozole (A), fulvestrant (F) or the combination (A+F). Biopsies were taken preNET and after 4-weeks(wks). Patients with Ki67 values >10% at 4-wks were offered triage to neoadjuvant chemotherapy. Patients with on-treatment Ki67 ≤ 10% who completed NET underwent surgery and Ki67 was reassessed. The primary endpoint was endocrine-sensitive disease rate (ESDR). ESD is defined as pCR or PEPI-0 residual disease (pT1-2, pN0, Ki67 ≤ 2.7%). We previously reported that the ESDR difference between the F-containing arms and the A arm was not >10% (ASCO 2020) and that baseline RNA-seq-based intrinsic subtypes predicted outcomes overall (SABCS 2021). Herein we describe relationships between PAM50 intrinsic subtype and Ki67 values by treatment arm because comparative drug effectiveness in adjuvant endocrine therapy studies in ER+ HER2- breast cancer can be predicted by the degree of Ki67 suppression (PMC3518447). Methods: 743 of the 1297 eligible patients (A: 264; F: 231; A+F: 248) had RNA extracted from preNET frozen tumor biopsies with >50% tumor content and subjected to RNA seq. Intrinsic subtypes were then assigned as LumA, LumB, and NonLum (Basal or HER2-E) using open-source PAM50-based informatics. Differences in the proportion with wk4 Ki67 > 10%, % change in wk4 ki67, and surgical CCCA (Ki67 ≤ 2.7%) rate (sxCCCA) between treatments and by intrinsic subtype was assessed using stratified logistic regression, Wilcoxon rank sum test, and Fisher’s exact test, respectively. Analysis of sxCCCA excluded those who failed to complete NET for reasons other than disease progression or early Ki67 >10%. Results: Amongst the 358 LumA cases there were no significant differences in Ki67-based endpoints between treatments. Among the 292 LumB cases, the wk4 ki67 > 10% rate was lower with A+F (19.4%) than A (43%) (P=0.0002) and was somewhat lower in F (31%) versus A (P=0.076). The % change in wk4 Ki67 in LumB cases, adjusted for baseline Ki67, showed markedly superior suppression for A+F versus A (-90% vs. -77%; P=<0.0001) and versus F (-90% vs. -80%; P=0.0026). Furthermore sxCCCA rates were significantly higher with A+F than A (53% vs. 25% P = <0.0001) and somewhat higher for F (37%) than A (p=0.068), indicating that superior antiproliferative effects for A+F persist after 6 months on therapy. Lack of Ki67 suppression in response to treatment was observed in the majority of 43 NonLum samples regardless of treatment. Conclusion: The combination of A+F was significantly more effective than either drug alone for the control of LumB breast cancer cell proliferation. This suggests that A+F may be a more effective adjuvant endocrine therapy than A alone in LumB disease. The lower Ki67 suppression with A alone also suggests that poorer outcome in some LumB tumors may be due to insufficient ER targeting rather than ER-independent tumor growth Support: U10CA180821, U10CA180882, U24CA196171, UG1CA189856, U10CA180868 (NRG), NCI BIQSFP, BCRF, Genentech, AstraZeneca. https://acknowledgments.alliancefound.org. (MJE) CPRIT RR140033, P50-CA186784, P50-CA58223, U01-CA214125, U24-CA210954, Gift from Ralph and Lisa Eads, McNair Scholarship. ClinicalTrials.gov Identifier: NCT01953588 Citation Format: Matthew J. Ellis, Meenakshi Anurag, Jeremy Hoog, Aranzazu Fernandez-Martinez, Cheng Fan, Richard Gibbs, Souzan Sanati, Kiran Vij, Mark Watson, Travis Dockter, Olwen Hahn, Joseph Guenther, Abigail Caudle, Erica Crouch, Amy Tiersten, Monica Mita, Wajeeha Razaq, Tina J. Hieken, Yang Wang, A. Marilyn Leitch, Gary W. Unzeitig, Eric Winer, Anna Weiss, Kelly Hunt, Ann H. Partridge, Charles M. Perou, Vera Suman, Cynthia X. Ma, Lisa A. Carey. The effect of intrinsic subtype on inhibition of tumor growth by anastrozole vs. fulvestrant vs. the combination: Results from the Alliance neoadjuvant endocrine therapy (NET) ALTERNATE trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT026.

Abstract Background: Neoadjuvant ET (NET) offers an opportunity to assess ET sensitivity for ER+ HER2- BC and potentially to tailor therapy. Ki67 >10% on biopsy after 2-4 weeks (wks) of NET identifies patients (pts) with intrinsic ET resistance; while pathologic complete response (pCR) and modified preoperative endocrine prognostic index of 0 (mPEPI 0: pT1-2N0, Ki67 ≤2.7%) at surgery indicates sensitivity to ET. However, on-NET biopsy is not always acceptable or feasible and delays the ET sensitivity determination. PAM50 ROR score and intrinsic subtypes by tumor RNA profiling are prognostic in pts with early stage ER+ HER2- BC, and predict pCR rates to neoadjuvant chemotherapy (NCT) (PMC2667820). We therefore hypothesized that PAM50 analysis on pre-NET biopsies could predict the likelihood of a) a high on-NET Ki67, b) mPEPI-0 or pCR at surgery and, c) pCR for pts triaged to NCT. Methods: The ALTERNATE trial is a phase III study that randomized postmenopausal pts with clinical stage II/III ER+ (Allred score 6-8) HER2- BC to receive neoadjuvant anastrozole, fulvestrant, or both for 6 months before surgery. Research biopsy was required at pre-NET and wk 4, then optional at wk 12. Pts with Ki67 >10% on biopsy at wk 4 or 12 discontinued NET and were offered NCT. PAM50 intrinsic subtype and ROR-P values were generated from mRNA sequencing (RNASeq) analysis on pre-NET biopsies using open-source informatics (PMC7723687) and evaluated for prediction of on-NET Ki67 >10% at wk 4 or 12, pCR or mPEPI-0 post NET, and pCR post NCT. Results: 749 of 1,297 eligible trial pts were included in the analyses, after excluding 548 pts due to insufficient pre-NET tumor for RNASeq (n=511) or PAM50 normal subtype (n=37). Similar to the entire ALTERNATE population, the rate of Ki67 >10% at wk 4 or 12 was 24.4% (95% CI: 21.4-27.7%) and the rate of mPEPI-0/pCR post NET was 19.8% (95% CI: 17.0-22.8%). There were 393 (52.5%) Lum A, 302 (40.3%) Lum B, and 54 (7.2%) non-Lum (9 Basal, 45 HER2-E) BCs. These included 196 (26.2%) ROR-P low, 354 (47.3%) ROR-P medium and 199 (26.6%) ROR-P high BCs. Both the rates of Ki67 >10% at wk 4 or 12 and mPEPI-0/pCR differed significantly with respect to PAM50 subtype or ROR-P category, such that Lum A or ROR-P low BCs were least likely to have a Ki67 >10% at wk 4 or 12 and most likely to achieve mPEPI-0/pCR (Table). 93 of 168 (55.4%) pts triaged to NCT had RNA-seq results, yielding 26 Lum A, 49 Lum B, 4 Basal and 14 HER2-E, with the pCR rates of 0%, 6.1%, 0%, and 21.4%, respectively. There were 10 ROR-P low, 39 medium, and 44 high tumors, with a pCR rate of 0%, 5.1% and 9.1%, respectively. Conclusion: These data indicate that both baseline ROR-P and intrinsic subtype are predictive of early on-NET Ki67 > 10% and mPEPI 0/pCR at surgery after NET. For pts triaged to NCT based on an early on-NET Ki67 >10%, the HER2-E group had the highest pCR rate (20%) and no pCRs were observed in Lum A. These data may be useful for directing neoadjuvant therapy in postmenopausal pts with ER+ HER2- BC. Support: U10CA180821, U10CA180882, U24CA196171, UG1CA189856, U10CA180868 (NRG), NCI BIQSFP, BCRF, Genentech, AstraZeneca. https://acknowledgments.alliancefound.org. (MJE) CPRIT RR140033, P50CA186784, P50-CA58223, U01 CA214125, U24CA210954, Gift from Ralph and Lisa Eads, McNair Scholarship. Trials.gov Identifier: NCT01953588. Table 1.Rates of Ki67 >10% and mPEPI-0/pCR post NET by PAM50 subtype and ROR-P categoryKi67 >10% at wk 4 or 12mPEPI 0/pCR post NETPAM50 SubtypenYes, n (%)PnNo, n (%)PLum A37251 (13.7%) 95% CI: 10.4-17.6%<0.0001393104 (26.5%) 95%CI: 22.2-31.1%<0.0001Lum B29394 (32.1%) 95% CI: 26.8-37.8%30243 (14.2%) 95%CI: 10.5-18.7%Non-luminal (Basal and HER2-E)5338 (71.7%) 95%CI: 57.6-83.2%541 (1.9%) 95%CI: 0.05-9.9%ROR-P CategorynYes, n (%)PnNo, n (%)PLow18018 (10.0%) 95%CI: 6.0-15.3%<0.000119660 (30.6%) 95%CI: 24.2-37.6%<0.0001Intermediate34474 (21.5%) 95%CI: 17.3-26.2%35471 (20.1%) 95%CI: 16.0-24.6%High19491 (46.9%) 95%CI: 39.7-54.2%19917 (8.5%) 95%CI: 5.1-13.3% Citation Format: Cynthia X Ma, Meenakshi Anurag, Travis Dockter, Jeremy Hoog, Aranzazu Fernandez-Martinez, Cheng Fan, Richard Gibbs, Souzan Sanati, Kiran Vij, Mark Watson, Olwen Hahn, Joseph Guenther, Abigail Caudle, Erika Crouch, Amy Tiersten, Monica Mita, Wajeeha Razaq, Tina J Hieken, Yang Wang, A. Marilyn Leitch, Gary W Unzeitig, Anna Weiss, Eric P Winer, Kelly Hunt, Ann H Partridge, Lisa A Carey, Charles M Perou, Matthew J Ellis, Vera Suman. Pam50 intrinsic subtype and risk of recurrence score (ROR) for the prediction of endocrine (ET) sensitivity and pathologic response to chemotherapy in postmenopausal women with clinical stage II/III estrogen receptor positive (ER+) and HER2 negative (HER2-) breast cancer (BC) in the alternate trial (Alliance A011106) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD9-03.

PURPOSE CALGB 40603 ( NCT00861705 ), a 2 × 2 randomized phase II trial, demonstrated that adding carboplatin or bevacizumab to weekly paclitaxel (wP) followed by doxorubicin and cyclophosphamide significantly increased the pathologic complete response (pCR) rate in stage II-III triple-negative breast cancer. We now report long-term outcomes (LTOs) and correlative science end points. PATIENTS AND METHODS The Kaplan-Meier method was used to estimate LTOs in 443 patients who initiated study treatment. Log-rank tests and Cox proportional hazards models evaluated the impact of clinical characteristics, pathologic response, calculated residual cancer burden (RCB) in patients with residual disease (RD), treatment assignment, and dose delivery during wP on LTOs, including event-free survival (EFS). Genomic predictors of treatment response and outcomes were assessed on pretreatment tumor samples by mRNA sequencing. RESULTS Among baseline characteristics, only the clinical stage was associated with LTOs. At a median follow-up of 7.9 years, LTOs were not significantly improved with either carboplatin or bevacizumab, overall or in patients with basal-like subtype cancers by genomic analysis. Patients with pCR (n = 205, 46.3%) had significantly higher 5-year EFS (85.5% v 56.6%, log-rank P < .0001) and overall survival (87.9% v 63.4%, P < .0001) rates compared with patients with RD, even those with RCB class I. Among clinical and genomic features, evidence of immune activation, including tumor-infiltrating lymphocytes and low B-cell receptor evenness, was associated with pCR and improved EFS. CONCLUSION Despite higher pCR rates, neither carboplatin nor bevacizumab appeared to improve LTOs although the study was not powered to assess these secondary end points. pCR was associated with superior LTOs even when compared with minimal RD. Markers of immune activation in pretreatment tumor biopsies were independently associated with higher pCR rates and improved survival.

Abstract Patients with metastatic breast cancer (MBC) typically have short survival times and their successful treatment represents one of most challenging aspects of patient care. This poor prognostic behavior is in part due to molecular features including increased tumor cell clonal heterogeneity, multiple drug resistance mechanisms, and alterations of the tumor microenvironment. The AURORA US Metastasis Project was established with the goal to identify molecular features specifically associated with metastasis. We therefore collected and molecularly characterized specimens from 55 metastatic breast cancer (BC) patients representing 51 primary cancers and 102 metastases. The 153 unique tumors were assayed using RNAseq, tumor/germline DNA exomes and low pass whole genome sequencing, and global DNA methylation microarrays. We found intrinsic molecular subtype differences between primary tumors and their matched metastases to be rare in triple negative breast cancer (TNBC)/Basal-like subtype tumors. Conversely, tumor subtype changes were relatively frequent in estrogen receptor positive (ER+) cancers where ~30% of Luminal A cases switched to Luminal B or HER2-enriched (HER2E) subtypes. Clonal evolution studies identified changes in expression subtype coincident with DNA clonality shifts, especially involving HER2 amplification and/or the HER2E expression subtype. We further found evidence for ER-mediated downregulation of genes involved in cell-cell adhesion in metastases. Microenvironment differences varied according to tumor subtype where ER+/Luminal metastases had lower fibroblast and endothelial cell content, while TNBC/Basal-like metastases showed a dramatic decrease in B cells and T cells. In 17% of metastatic tumors, we identified DNA hypermethylation and/or focal DNA deletions near HLA-A that were associated with its’ significantly reduced expression, and with lower immune cell infiltrates. We also identified low immune cell features in brain and liver metastases when compared to other metastatic sites, even within the same patient. These findings have direct implications for the treatment of metastatic breast cancer patients with immune- and HER2-targeting therapies and suggest potential novel therapeutic avenues for the improvement of outcomes for some patients with MBC.

Alpha‐1 antitrypsin deficiency (AATD) liver disease is characterized by marked heterogeneity in presentation and progression, despite a common underlying gene mutation, strongly suggesting the involvement of other genetic and/or epigenetic modifiers. Variation in clinical phenotype has added to the challenge of detection, diagnosis, and testing of new therapies in patients with AATD. We examined the contribution of DNA methylation (5‐methylcytosine [5mC]) to AATD liver disease heterogeneity because 5mC responds to environmental and genetic cues and its deregulation is a major driver of liver disease. Using liver biopsies from adults with early‐stage AATD and the ZZ genotype, genome‐wide 5mC patterns were interrogated. We compared DNA methylation among patients with early AATD, and among patients with normal liver, cirrhosis, and hepatocellular carcinoma derived from multiple etiologic exposures, and linked patient clinical/demographic features. Global analysis revealed significant genomic hypomethylation in AATD liver–impacting genes related to liver cancer, cell cycle, and fibrosis, as well as key regulatory molecules influencing growth, migration, and immune function. Further analysis indicated that 5mC changes are localized, with hypermethylation occurring within a background of genome‐wide 5mC loss and with patients with AATD manifesting distinct epigenetic landscapes despite their mutational homogeneity. By integrating clinical data with 5mC landscapes, we observed that CpGs differentially methylated among patients with AATD disease are linked to hallmark clinical features of AATD (e.g., hepatocyte degeneration and polymer accumulation) and further reveal links to well‐known sex‐specific effects of liver disease progression. Conclusion: Our data reveal molecular epigenetic signatures within this mutationally homogeneous group that point to ways to stratify patients for liver disease risk.