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Background: CDK4/6 inhibitors (CDK4/6i) paired with endocrine therapy (ET) are considered first-line (1L) therapy for patients (pts) with HR+ HER2- advanced breast cancer (aBC). A minority of pts will demonstrate primary resistance to CDK4/6i, as characterized by early progression. Thymidine kinase 1 (TK1) is a cell-cycle regulated enzyme downstream of CDK4/6 and involved in nucleotide metabolism during DNA synthesis. Prior studies have shown TK1 may serve as a biomarker of response to CDK4/6i, with early TK1 activity (TK1a) suppression after initiation of CDK 4/6i therapy associated with improved PFS. Lack of TK1a suppression may be associated with primary resistance to CDK4/6i. In this study, we aim to analyze response to subsequent lines of therapy and overall survival (OS) of pts with early progression on 1L CDK4/6i. Methods: Pts with HR+ HER2- aBC from a phase II trial of an alternative schedule of palbociclib (palbo alt dosing trial NCT 3007979) and from a retrospective palbociclib study were included in this analysis. Pts in the palbo alt dosing trial underwent baseline and C1D15 TK1a analysis after initiation on CDK4/6i. C1D15 TK1a suppression was defined at TK1a < 30 Du/L. Pts in the retrospective palbociclib study included pts receiving palbo as part of their standard of care 1L therapy for HR+ HER2- aBC at Washington University in Saint Louis from 2016 to 2021. Clinical information, including treatment start and stop dates on each of the next-line therapies, were collected from the electronic medical record. PFS was estimated by the treatment duration on a specified treatment regimen. Early progression on CDK4/6i was defined as PFS < 6 mo. Best response was defined as next line of therapy with the numerically longest PFS. OS was defined as time to death from the initiation of CDK4/6i. Results: Of the 54 pts enrolled on the palbo alt dosing trial, 51 pts were evaluable for clinical benefit and 46 pts were evaluable for TK1a suppression rate at C1D15. 7 pts (15.2%) were found without TK1a suppression at C1D15. This lack of TK1a suppression on palbo was associated with a significantly shorter PFS (median PFS=3.1 mo) compared to not reached in pts with TK1a suppression at C1D15. We conducted clinical analysis on N=26 pts who exhibited early progression on CDK4/6i which included 10 pts from the palbo alt dosing trial and 16 from the retrospective study. The average subsequent line of therapies in this cohort was 3, with the most common second line (2L) therapy being chemotherapy (N=17, 65.4%) and ET (N=8, 30.8%). The median PFS for pts receiving 2L chemotherapy and ET was 4.09 mo and 3.64 mo, respectively. 10 pts received both chemotherapy and ET with 7 (70.0%) achieving best response with chemotherapy compared to 3 pts (30.0%) who achieved best response with ET. The median OS for the cohort was 14.6 mo. Conclusions: Early progression on CDK4/6i is associated with a particularly poor prognosis. In our cohort, the median OS was far below the expected median OS for pts receiving 1L palbo as reported in the PALOMA-2 trial (14.6 mo vs 53.9 mo). Early progression on CDK4/6i is associated with more aggressive disease which may respond more favorably to chemotherapy, as demonstrated by best response to therapy. Further prospective studies are warranted to explore this treatment approach. Citation Format: Katherine K. Clifton, Shana N. Thomas, Jingqin Luo, Jing Xi, Nusayba A. Bagegni, Foluso O. Ademuyiwa, Rama Suresh, Ashley Frith, Andrew A. Davis, Ron Bose, Katherine Weilbaecher, Whitney L. Hensing, Timothy Pluard, Massimo Cristofanilli, Hyo S. Han, Adam M. Brufsky, Kevin Kalinsky, Shom Goel, Seth A. Wander, Lindsay L. Peterson, Cynthia Ma. PD13-09 Clinical outcomes of patients with HR+ HER2- advanced breast cancer with early progression on CDK4/6 inhibitors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD13-09.

Background: KEYNOTE-522 was a randomized, double-blind, placebo-controlled phase 3 trial which resulted in the FDA approval of pembrolizumab with neoadjuvant chemotherapy for patients (pts) with newly diagnosed, high-risk, early-stage triple negative breast cancer (TNBC). Given the improvement in pathological complete response (pCR) and event-free survival rates, this regimen has emerged as standard-of-care (SOC) therapy. Adverse events in pts on this treatment regimen in clinical practice is unknown and understanding the real-world toxicity of this regimen is critical. Methods: In this IRB approved retrospective, single-center study we examined pts with early-stage TNBC who received planned treatment per KEYNOTE-522 per SOC from 2021-present. This regimen includes a year of pembrolizumab combined with 4 cycles of neoadjuvant carboplatin/paclitaxel followed by 4 cycles of doxorubicin/cyclophosphamide. Number and length of treatment delays, treatment related toxicities of all grades, and pCR rate were collected from the electronic medical record. Results: Of the 87 identified pts, 2 were excluded due to locally recurrent or metastatic disease and 6 did not receive immunotherapy due to concerns for toxicity or patient preference. Of the 79 pts who initiated treatment with chemotherapy and immunotherapy, median age of the cohort was 52 (27-77). 9 pts had a BRCA1 mutation and 1 pt had a BRCA2 mutation. 41 (51.9%) had T1-2 disease and 38 (48.1%) had T3-4 disease. 37 (46.8%) pts had N0 disease and 42 (53.2%) had N1-3 disease. 15 pts had baseline comorbidities, including heart disease, kidney disease, type II DM, and/or peripheral neuropathy. 68 pts (86.1%) had baseline ECOG 0, 9 (11.4%) had ECOG 1, and 2 (2.5%) had ECOG 2. At the time of analysis, 70 pts (88.6%) were receiving active treatment, of which 47 (67.1%) had completed ≥50% of the planned neoadjuvant therapy. Of pts completing ≥50% of planned neoadjuvant therapy and pts off therapy (N=56), 31 (55.4%) had 1 or more hospitalizations and 23 (41.1%) had 1 or more emergency room visits. 30 pts had treatment delays (53.6%) and 21 pts (37.5%) had dose reductions. Rates of adverse events are presented in Table 1. Of the 79 analyzed pts, 35 have undergone surgery. pCR rate was 45.7% (N=16). 8 (22.9%) pts had RCB-I, 4 (11.4%) pts had RCB-II, 3 (8.6%) pts had RCB-III, and 4 (11.4%) pts had residual disease without RCB calculation. Updated analysis will be included at time of presentation. Conclusions: In this single-center retrospective study of pts receiving chemoimmunotherapy for TNBC, we found higher rates of grade 3 toxicity, including nausea, fatigue, neutropenia, diarrhea, peripheral neuropathy, and hypothyroidism, and lower pCR rate than was reported in the KEYNOTE-522 trial. This may reflect a more heterogeneous population of pts treated in routine clinical practice who are typically less fit than pts on clinical trials. Additionally, pts in this study had higher T stages (48.1% with T3-4 disease vs 26.0% in trial) and node positive disease (53.7% with N1-3 disease vs 48.8% in trial). Limitations include immaturity of data and small sample size; however, these data warrant validation through longer-term follow-up and multicenter validation. Adverse Events in pts receiving Keynote-522 regimen as SOC and on clinical trial Citation Format: Mara Hofherr, Jennifer Hedgecorth, Foluso O. Ademuyiwa, Lindsay L. Peterson, Nusayba A. Bagegni, Rama Suresh, Ashley Frith, Ron Bose, Katherine Weilbaecher, Cynthia Ma, Andrew A. Davis, Katherine K. Clifton. Real-world analysis of adverse events of patients with triple negative breast cancer receiving therapy per KEYNOTE-522 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-06-06.

Background: CDK 4/6i have altered the therapeutic landscape of HR+, HER2- MBC, improving progression free and overall survival (PFS and OS) compared to endocrine therapy (ET) alone. Despite durable responses to CDK 4/6i in a large majority of patients, treatment response monitoring in this population has historically included numerous serial blood-based and imaging studies at frequent time points. There is a growing global interest in utilizing novel non-invasive biomarker-driven disease monitoring assessments to improve patient outcomes and reduce health care costs. Thymidine kinase 1 (TK1), a key cell-cycle regulated enzyme important for nucleotide metabolism during DNA synthesis, is regulated by the E2F pathway, downstream of CDK 4/6. Studies have shown that DiviTum® TK1 activity (TKa) may serve as both a prognostic and predictive biomarker of CDK 4/6i treatment response (McCartney et al, Clin Canc Res, 2020; Malorni et al, Eur J Cancer, 2022; Bagegni et al, Breast Cancer Res, 2017). Early TKa suppression within 2 weeks (wk) post CDK 4/6i therapy initiation is associated with improved PFS, suggesting a subgroup of patients who may be able to de-escalate imaging frequency. Elevated TKa at baseline and post CDK 4/6i may identify patients with CDK 4/6i-resistant disease and disease progression (PD) requiring early therapy modification. TK IMPACT is a prospective, single-arm trial designed to assess the impact of incorporation of DiviTum® TKa on a physician’s decision regarding subsequent timing of routine disease monitoring modalities in patients with advanced HR+, HER2- MBC receiving ET plus CDK 4/6i (NCT04968964). Methods: Blood sample collections will be analyzed using DiviTum® TKa at baseline (bl), wk 2, 4, 6, 8, and Q 4 wks thereafter beginning at wk 8 during the first 24-wk time period of study enrollment (+/- 3 days); followed by Q 12 wks thereafter, until PD or 36 months, whichever occurs first. Optional repeat TKa within 2-4 wks (+/-3 days) is permitted in case of rising TKa. Research blood (bl, wk 2, 12, 24, 48, and PD) and optional archival tumor tissue collection at diagnosis and PD will be obtained for correlatives. The investigator will record intended imaging modalities and timing prior to receipt of TKa, followed by documentation of any changes in imaging testing interval after receipt of TKa. Key eligibility criteria include postmenopausal women age ≥18 years with HR+, HER2- MBC, to initiate (Cohort 1) or are currently receiving (≤24 months, Cohort 2) any FDA approved first line ET plus CDK 4/6i with a life expectancy > 6 months. The primary endpoint is any physician-reported intended change in imaging testing interval post TKa by study cohort, within the first 48-wk period of study participation. Key secondary endpoints are concordance rate between TKa values and progression status at first on-study imaging and longitudinal TKa dynamics. Key exploratory endpoints include plasma and tumor tissue-based biomarkers of CDK 4/6i response and resistance. A total of 40 patients will be enrolled (n=20/Cohort). The expected change rate is 20% with a 95% Wilson confidence interval of 0.105~0.248 across all patients and if within each cohort, with a 95% Wilson confidence interval of 0.081~0.416 for N=20. N=40 allows the lower limit of the 95% CI > 10% and that of the N=20 in Cohort 1 to be ~10%, indicating some clinically meaningful influence of TKa progression on patient management. The study is open to accrual and has presently enrolled 5 patients. Citation Format: Nusayba A. Bagegni, Isabella Grigsby, Leslie Nehring, Jingqin Luo, Jennifer Powers Carson, David W. Gibson, Meghan Horvath, Katherine K. Clifton, Foluso O. Ademuyiwa, Rama Suresh, Ashley Frith, Andrew A. Davis, Lindsay L. Peterson, Ron Bose, Amy Williams, Mattias Bergqvist, Cynthia Ma. TK IMPACT: Treatment Monitoring of Hormone Receptor Positive (HR+), HER2 Negative (HER2-) Metastatic Breast Cancer (MBC) Patients Receiving CDK 4/6 Inhibitors (CDK4/6i) with DiviTum® Thymidine Kinase 1 Activity [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-11-01.

Background: Neoadjuvant immune checkpoint inhibition (ICI) in combination with chemotherapy is approved for patients with high-risk, early-stage triple-negative breast cancer (TNBC) based on improved outcomes in the KEYNOTE-522 trial. However, some patients have primary resistant disease and do not achieve a pathological complete response (pCR), while others experience significant toxicity. Tumor-associated macrophages (TAMs) are a potential resistance mechanism for ICIs and are dependent on colony-stimulating factor 1 receptor (CSF1R). Therefore, we examined the addition of cabiralizumab, a CSF1R inhibitor, to neoadjuvant paclitaxel, carboplatin, and nivolumab to assess the safety, tolerability, and changes in the tumor microenvironment (TME) in patients with early-stage TNBC. Methods: This is a phase Ib/II, single-institution, randomized controlled clinical trial (NCT04331067) in patients with newly diagnosed Stage II-III TNBC. The primary endpoints include: (1) to determine the safety of a 12-week neoadjuvant regimen of paclitaxel (80 mg/m2 IV q week) + carboplatin (AUC5 IV q3 weeks) + nivolumab (240 mg IV q2 weeks) with or without cabiralizumab (4 mg/kg IV q2 weeks) and (2) to evaluate the effect of cabiralizumab on TAMs and changes in tumor infiltrating lymphocytes (TILs) in the TME between baseline and an on-treatment biopsy after 4 weeks of therapy. Adjuvant treatment is per investigator’s choice. Secondary objectives include evaluation of pCR rate and recurrence-free survival. Paired tissue and bone marrow biopsies are collected for evaluation of the TME and disseminated tumor cells, respectively. The study was designed to enroll 50 patients, including a 12-patient safety lead-in cohort. Here, we report the planned interim analysis of the safety lead-in cohort. Results: Between December 2020 and May 2022, we enrolled 12 patients to the safety lead-in, including 6 patients in each arm. 5 of 12 patients (41.7%) enrolled are underrepresented minorities, including 4 Black patients and 1 Hispanic patient. 2 of 6 patients in the nivolumab arm experienced grade 3 severe toxicity, including 1 patient who developed sepsis and 1 who developed peripheral neuropathy. 3 of 6 patients in the nivolumab + cabiralizumab arm developed grade 3 severe toxicity including 2 patients who experienced myositis and 1 patient who developed periorbital edema. Of the first 10 patients enrolled, 5 had a pCR (2 pCR in cabiralizumab arm, 3 pCR in non-cabiralizumab arm) and 3 had non-pCR (1 RCB-1 and 1 RCB-3 in cabiralizumab arm, 1 RCB-1 in non-cabiralizumab arm). 2 patients came off study prior to surgery (1 due to toxicity and 1 due to missing study visits). Data from the final 2 patients still on treatment will be available at the time of presentation. Discussion: Full safety, pathologic, and clinical response data in the safety lead-in cohort for patients with early-stage TNBC receiving neoadjuvant chemotherapy + nivolumab with or without cabiralizumab, will be presented. Citation Format: Andrew A. Davis, Leonel Hernandez-Aya, Jingqin Luo, Mateusz Opyrchal, Foluso O. Ademuyiwa, Nusayba A. Bagegni, Katherine K. Clifton, Jill Anderson, Trish Hammerschmidt, Leslie Nehring, David DeNardo, Mark Watson, Rebecca Aft, Cynthia Ma, Katherine Weilbaecher. Phase Ib/II study to evaluate safety and tolerability of cabiralizumab in combination with nivolumab and neoadjuvant chemotherapy in patients with localized triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-06-07.

Introduction: Estrogen receptor (ER) loss occurs in about 20% of recurrent breast cancers (BC) and is associated with unresponsiveness to endocrine therapy (ET) and poor prognosis. Prior studies evaluating ER-loss included predominately patients with invasive ductal carcinoma (IDC), and therefore the impact of ER-loss in invasive lobular carcinoma (ILC) is unknown. In this retrospective analysis, using real-world data, we aimed to determine the prevalence and clinical significance of ER-loss in ILC. Methods: Advanced BC were molecularly profiled at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing of DNA (592-gene panel or whole-exome sequencing), RNA (whole transcriptome sequencing, WTS) and immunohistochemistry (IHC) of select markers. A large real-world evidence (RWE) database combining Caris’ molecular data with clinical information obtained from insurance claims data (CODEai) was interrogated and overall survival (OS) was calculated from time of tissue collection to last patient contact. A tumor was considered to have ER-loss if therapies approved only for ER-positive BC were prescribed prior to obtaining a negative ER IHC result. OS was compared using Kaplan-Meier estimates for defined patient cohorts; significance was determined as p values < 0.05. For molecular analyses, Fisher-Exact or Chi-Square tests were used to determine p values. Correction for multiple comparisons was performed using Benjamini-Hochberg to calculate q values. Results: The RWE database included 24,824 patients with advanced BC. At the time of tissue collection for molecular profiling, 6,786 advanced BC patients had been previously treated with ET (with or without mTOR or CDK4/6 inhibitors), of whom 1,338 had data available on histologic classification and ER IHC. The final analytical cohort included 263 patients with ILC and 1,075 with IDC. ER-loss was identified in 11.4% of ILC (n=30/263) and 19.6% (n=210/1075) of IDC (p=0.0017). In ILC, ER-loss was associated with significantly worse OS (HR: 1.75, 95%CI: 1.10-2.79, p=0.016) compared with no ER-loss. In the cohort of patients with ER-loss, patients with ILC had significantly worse OS compared with IDC (HR=2.03, 95% CI: 1.267-3.251, p=0.003). Further, when 1,016 tumors with ER-loss (regardless of histology) were stratified by the median OS (mOS=11mo), positive PD-L1 expression (34% vs. 22%, p=0.04, q=0.22), HER2 IHC positivity (16% vs. 7.8%, p=0.003, q=0.08) and HER2 amplification (16% vs. 4.7%, p=0.0006, q=0.04) were enriched in patients with longer mOS; while amplification of TEFB (0.38% vs. 2.6%, p=0.047, q=0.23) and MYB (0.38% vs. 2.6%, p=0.047, q=0.23) were enriched in patients with shorter mOS. WTS identified 197 differentially expressed genes, the majority of which were enriched in patients with longer mOS (q< 0.05). Conclusions: In this large real-word dataset, ER-loss likely occurred in 11.4% of ILC and was associated with worse OS compared to both patients with IDC and ER-loss and ILC without ER-loss. Our analysis had several limitations; notably, our definition of ER-loss was based on prior treatment, we could not distinguish between de novo or recurrent metastatic disease and time of tissue collection was not standardized during the course of treatment. Thus, additional studies are needed to confirm these findings. However, this study does suggest that ER-loss occurs in a subset of patients with ILC and has poor prognostic implications. Citation Format: Whitney L. Hensing, Joanne Xiu, W. Michael Korn, Stephanie L. Graff, Irene Kang, Evanthia T. Roussos Torres, Arielle L. Heeke, Andrew A. Davis, Nusayba A. Bagegni, Katherine K. Clifton, Ron Bose, Cynthia Ma, Foluso O. Ademuyiwa. Prevalence and prognosis of ER-loss in advanced invasive lobular carcinoma [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-08.

Triple-negative breast cancer (TNBC) is a biologically aggressive yet heterogeneous disease that disproportionately affects younger women and women of color compared to other breast cancer subtypes. The paucity of effective targeted therapies and the prevalence of chemotherapeutic resistance in high-risk, early-stage TNBC pose significant clinical challenges. Deeper insights into the genomic and immune landscape have revealed key features of TNBC, including intrinsic genomic instability, DNA repair deficiency, and potentially an immunogenic tumor microenvironment. These advances led to landmark trials with immune checkpoint inhibitors in the advanced-stage setting, which subsequently translated into immunotherapy-based clinical trials in the early-stage setting and recent promising results. Pembrolizumab, an anti-programmed death 1 (PD-1) monoclonal antibody, was investigated in combination with platinum-, taxane- and anthracycline-based neoadjuvant chemotherapy followed by adjuvant pembrolizumab monotherapy for patients with high-risk, early-stage TNBC in the randomized, double-blind, placebo-controlled phase 3 KEYNOTE-522 trial. In July 2021, the US Food and Drug Administration (FDA) granted approval for pembrolizumab based on marked improvement in pathologic complete response rate and 3-year event-free survival compared to neoadjuvant chemotherapy alone. This advance immediately altered the longstanding treatment paradigm. Here, we review the impact of pembrolizumab plus chemotherapy for the treatment of patients with high-risk, early-stage TNBC, and discuss immunotherapy-related toxicity considerations, key immunomodulatory biomarkers under active investigation, and remaining clinical questions for future research directions.

Background: ESR1 hotspot mutations (HS) (i.e. 380, 536, 537, and 538) are important drivers of resistance to aromatase inhibitors, but the differential impact of genomic variants (HS vs non-HS) on response to endocrine therapies (ET) under clinical development, such as novel oral Selective Estrogen Receptor Degraders and Modulators (SERDs and SERMs), is not known. The aim of the study was to evaluate the impact of non-HS ESR1 mutations on the pharmacodynamics of SERDs and SERMs as an additional ET resistance mechanism. Materials and Methods: The study analyzed a multi-institutional cohort of 1008 patients with hormone receptor positive metastatic breast cancer characterized by circulating tumor DNA (ctDNA). Pathway classification was defined based on previous work (i.e. RTK, RAS, RAF, MEK, NRF2, ER, WNT, MYC, p53, Cell Cycle, Notch, PI3K). Single nucleotide variations (SNVs) were annotated through OncoKB; co-occurrence was tested by Fisher’s exact test. A structure-based computational strategy was used to create 3D-models of ESR1 mutants and predict changes in binding affinity (dAff) across approved and experimental drugs. A positive dAff reflects a lower affinity of the drug for mutant ESR1 compared with wild type and thus a potential for a reduced response. Results: Among the total 680 detected ESR1 mutations, 633 were missense, and 631 were gain-of-function. The most frequent mutations were in codon 537 (N=305), followed by 538 (N=224). No significant MAF differences were observed across ESR1 variants (P=0.0829). The L391F mutation resulted in an increased binding affinity for Lasofoxifene (LAS) (dAff -0.34), Giredestrant (GIR) (dAff -0.18), Elacestrant (ELA) (dAff -0.08) and Amcenestrant (AMC) (dAff -0.41), while a decreased binding affinity was observed for 4OH-Tamoxifen (TAM) (dAff 0.01), Imlunestrant (IML) (dAff 0.15), Fulvestrant (FUL) (dAff 0.43), and Camizestrant (CAM) (dAff 0.02). V392F decreased binding affinity for TAM (dAff 0.05), LAS (dAff 0.13), IML (dAff 0.11), GIR (dAff 0.11), FUL (dAff 0.04), CAM (dAff 0.05), AMC (dAff 0.06) but not for ELA (dAff -0.01). F404L decreased binding affinity for FUL (dAff 0.07), ELA (dAff 0.73), and CAM (dAff 0.26), while it increased binding affinity for TAM (dAff -0.27), LAS (dAff -0.02), IML (dAff -0.05), GIR (dAff -0.69), and AMC (dAff -2.01). G415E increased binding affinity for LAS, (dAff -0.15) GIR (dAff -0.02) and ELA (dAff -0.08), while it decreased binding affinity for TAM (dAff 0.11), IML (dAff 0.09), FUL (dAff 0.29), CAM (dAff 0.19) and AMC (dAff 0.10). Mutations in codon 537 did not affect dAff for TAM, GIR, and ELA; a significant decrease in binding affinity was observed for FUL and AMC, whereas it was increased for LAS. Mutational co-occurrence was tested between ESR1 mutations in FUL docking sites and oncogenic pathways. Significant associations were observed for cell cycle SNVs (P=0.047), Notch SNVs (P=0.020), and ER SNVs (P< 0.001). Within these pathways, significant single-gene associations were observed for FBXW7 SNVs (P=0.020), ESR1 SNVs (P< 0.001), and GATA3 SNVs (P= 0.016). Given the highly significant co-occurrence of non-HS with other ESR1 mutations, combined models were examined. The Y537/F404 combination resulted in decreased binding affinity for FUL and increased binding affinity for LAS, while L536/F404 decreased binding affinity for TAM and increased binding affinity for IML, ELA, and AMC. Notably, L540/F404 restored the FUL-ESR1 interaction resulting in an increased binding affinity (dAff -2.1). Conclusions: The study suggests that genomic variability in drug targets detectable through ctDNA may modulate therapeutic response. Preclinical models are under development to investigate the combined endocrine resistance mechanism suggested by the significant co-occurrence between ESR1 mutations in SERDs/SERMs docking sites and ESR1 hotspot mutations and provide valuable additional insights for drug development and future treatment algorithms. Citation Format: Lorenzo Gerratana, Rossana Roncato, Mattia Sturlese, Andrew A. Davis, Marko Velimirovic, Carolina REDUZZI, Katherine K. Clifton, Whitney L. Hensing, Ami N. Shah, Charles S. Dai, Paolo D’Amico, Arielle J. Medford, Alessandra Franzoni, Linda Cucciniello, Firas Wehbe, Seth A. Wander, Barbara Belletti, William Gradishar, Amir Behdad, Giuseppe Damante, Cynthia Ma, Fabio Puglisi, Aditya Bardia, Massimo Cristofanilli. PD10-01 Impact of ESR1 mutations on Selective Estrogen Receptor Degraders and Modulators: an integrated liquid-biopsy and pharmacodynamics approach. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-01.

Background GATA3 mutations (GATA3mut) have been reported in 10-20% of hormone receptor positive (HR+) breast cancers. It has been shown that targeting GATA3mut HR+ breast cancer with MDM2 inhibitors invokes synthetic lethality. MDM2 is an E3 ubiquitin ligase that targets p53 for degradation, and research suggests that restoring p53 by blocking MDM2 may be effective in treating GATA3mut HR+ breast cancer. One potential mechanism of this efficacy has been shown to be through the PI3K-AKT pathway. We thus sought to characterize the GATA3mut landscape in a multi-institutional cell-free DNA (cfDNA) analysis and to determine the association between GATA3mut and TP53 mutations, as well as alterations in the PI3K-AKT pathway and the impact of GATA3 on survival. Methods We analyzed cfDNA data collected at the Massachusetts General Hospital and at Washington University in St Louis via Guardant360, a next generation sequencing assay that analyzed up to 74 genes during the study period. The association of GATA3mut and co-mutations as well as number of prior therapies was estimated using Pearson’s chi-squared test for categorical variables, two-sample Wilcoxon rank-sum test for continues variables, and multivariable logistic regression. The impact of GATA3mut and GATA3 wildtype (WT) on progression-free survival (PFS) and overall survival (OS) was analyzed using multivariable Cox regression analysis, adjusting for age, number of prior therapies, visceral metastases, and de novo metastases. PFS and OS were evaluated in the overall study population, as well as in subgroups of patients that received endocrine monotherapy and chemotherapy. Results Out of 647 patients with HR+ MBC, 10% (n = 68) had non-synonymous GATA3 mutations. Among these 68 GATA3mut patients, 37% (n = 25) were mutations in exon 5, all but two of which were in the second zinc finger, and 62% (n = 42) were in exon 6. 62% (n = 42) were frameshift mutations, 20% (n = 14) were indels, and 18% (n = 12) were point mutations. Median mutant allele fraction (MAF) of GATAmut was 0.95% (range 0.03 – 30.5%). There was no statistically significant association of GATA3mut with the number of prior therapies, PR status, or the presence of ESR1, TP53, or PI3K-AKT pathway mutations. In the GATA3mut population, TP53 co-mutations (n = 21) were found with a median MAF of 0.6%. PI3K-AKT pathway alterations occurred in 47% (n=32) of GATA3mut patients (PIK3CA n = 27; AKT n = 2; PTEN n = 3). In the combined cohort, there was no significant difference in PFS or OS after adjusting for visceral metastases, de novo disease, number of prior therapies, and age. In a cohort of 80 patients that received endocrine monotherapy (GATA3 WT n = 74, GATA3mut n = 6), GATA3mut were associated with borderline worse PFS (HR 2.6; p = 0.061) and worse OS (HR 4.5; p = 0.009). There was no statistically significant difference in PFS or OS in a subgroup that received chemotherapy. Conclusions GATA3 mutations can be identified via cfDNA in patients with HR+ MBC. Co-mutations in TP53 occurred at overall low MAF. Further research is needed to characterize the functional impact of these low level TP53 co-mutations and develop therapeutic strategies to target GATA3 mutant MBC. Citation Format: Arielle J. Medford, Marko Velimirovic, Andrzej Niemierko, Whitney L. Hensing, Andrew A. Davis, Katherine K. Clifton, Jennifer C. Keenan, Charles S. Dai, Lesli A. Kiedrowski, Ami N. Shah, Lorenzo Gerratana, Laura M. Spring, Leif Ellisen, Robert C. Doebele, Massimo Cristofanilli, Aditya Bardia. Cell-free DNA detection of GATA3 mutations in metastatic hormone receptor positive breast cancer: a retrospective, observational multi-institutional analysis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-07.

Background: CDK4/6 inhibitors (CDK4/6i) are standard first-line (1L) regimens for HR+/HER2- advanced breast cancer (aBC). Recent data from the randomized phase II MAINTAIN trial reported a PFS benefit for patients (pts) who received a new endocrine therapy plus ribociclib (ribo) versus new endocrine therapy alone) following progression on CDK4/6i as compared to pts who received endocrine therapy alone. However, second-line (2L) treatment patterns and patient outcomes following 1L CDK4/6i are relatively undescribed. Here we describe real-world 2L treatment patterns following 1L CDK4/6i and associated clinical outcomes from a large clinical genomics database. Methods: Real-world evidence (RWE) was sourced from the GuardantINFORM (Guardant Health) database which comprises aggregated commercial payer health claims and de-identified records from over 207,000 pts with comprehensive circulating tumor DNA (ctDNA) results via Guardant360 (G360) from 2014 to 2021. Pts with HR+/HER2- aBC with >1 claim of CDK4/6i and >1 claim for treatment after the index G360 test were included. Real-world time to treatment discontinuation (rwTTD) and real-world time to next treatment (rwTTNT) were assessed in months as proxies for progression-free survival. Real-world overall survival (rwOS) was also reported in months. Results: 2,795 pts met criteria for inclusion; 2,361 (84.5%) were treated with 1L palbociclib (palbo), 271 (9.7%) with 1L abemaciclib (abema) and 163 (5.8%) with 1L ribo. Chemotherapy (chemo,35.5%) and endocrine-only therapy (32.8%) were the most common 2L therapy regardless of the 1L CDK4/6i agent (Table 1). Other 2L agents included endocrine backbone change (14.7%) or CDK4/6i change (7.7%). Endocrine backbone changes were observed more frequently (15.6%) in pts receiving 1L palbo while CDK4/6i changes were more frequent in pts receiving abema (14.0%) or ribo (22.0%). Pts treated with 2L CDK4/6i had improved rwTTNT, rwTTD and rwOS compared to 2L chemo regardless of 1L agent [rwTTNT: 10.2 (95% CI: 7.2-11.7) vs. 7.2 (6.5-8.1); rwTTD: 6.8 (95% CI: 5.8-8.5) vs. 4.3 (95% CI:3.9-4.7); rwOS: NR (95% CI: 40.0-NR) vs. 34.8 (95% CI: 31.3-37.2)]; improvement in rwTTNT, rwTTD and rwOS were also observed for pts with 2L endocrine backbone changes compared to chemo [rwTTNT: 8.5 (95% CI: 7.2-9.6) vs. 7.2 (6.5-8.1); rwTTD: 6.9 (95% CI: 5.8-7.9) vs. 4.3 (95% CI:3.9-4.7); rwOS: 63.4 (95% CI: 51.2-NR) vs. 34.8 (95% CI: 31.3-37.2)]. Pts treated with 2L alpelisib had the shortest rwOS regardless of 1L CDK4/6i agent used [any 1L: NR (95% CI: 23.6-NR)]. Conclusions: A variety of 2L regimens following 1L CDK4/6i were observed, with an improvement in rwTTNT, rwTTD and rwOS in pts receiving 2L CDK4/6i or 2L endocrine backbone change only relative to 2L chemo. These data are hypothesis generating, and the observed improvement may be secondary to therapy choice versus pts who received 2L chemo having more aggressive disease. Larger randomized trials are ongoing to study sequencing and efficacy of 2L treatments following 1L CDK4/6i. Table 1. Distribution of 2L therapies by 1L CDK4/6i agent. Citation Format: Katherine K. Clifton, Seth A. Wander, Cynthia Ma, Andrew A. Davis, Caroline Weipert, Nicole Zhang, Leslie Bucheit. Real-world second-line treatment patterns and associated clinical outcomes for 2795 patients with advanced HR+ HER2- breast cancer treated with first-line CDK4/6 inhibitors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-01-18.