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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: Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) in combination with endocrine therapy (ET) provide significant clinical benefit in patients with HR+/HER2- metastatic breast cancer (MBC) and have become a standard of care treatment. Prior insights from tumor profiling and preclinical analyses suggest that AKT1 activation can induce CDK4/6i resistance. We hypothesized that targeting AKT1 following CDK4/6i progression may be an effective therapeutic strategy and conducted a clinical trial to evaluate both doublet (ET+AKTi) and triplet (ET+AKTIi+CDK 4/6i) therapy in the ≥ 2nd line MBC setting. Methods: TAKTIC is an open-label phase Ib clinical trial (clinicaltrials.gov NCT03959891) evaluating the combination of the AKT inhibitor ipatasertib (ipat) with fulvestrant (Arm A), an aromatase inhibitor (Arm B), or the triplet combination (Arm C) with fulvestrant + palbociclib (palbo). The primary objective is to evaluate the safety (NCI CTCAE 5.0) and tolerability of ipat in combination with endocrine therapy +/- CDK4/6i. Secondary objectives include clinical efficacy, as determined by objective response rate (RECIST v1.1), clinical benefit rate (CBR), progression-free survival (PFS), and overall survival (OS). Key inclusion criteria include unresectable HR+/HER2- MBC; at least 1 prior therapy for MBC including any CDK4/6i; up to 2 prior lines of chemotherapy for MBC (no limit on prior endocrine therapy). Here, we present an updated interim analysis from all study arms. Results: The trial completed accrual with 77 pts enrolled from June 2019 – February 2022, including 19 on Arm A, 16 on Arm B, and 42 on Arm C. Median age was 62 (range 32-88) and 65/77 pts (84%) received prior CDK4/6i (median no. of prior lines = 3, range 1-13). 56/77 pts (73%) had measurable disease at baseline and 50/77 pts (65%) had visceral metastases in the liver/lung (68% Arm A, 44% Arm B, 71% Arm C). Pts enrolled on Arms A and B received ipat at 400mg in combination with fulvestrant or an aromatase inhibitor, respectively. In Arm C, 27/42 pts enrolled into the dose escalation phase and received ipat + palbo at varying doses in combination with fulvestrant. Two DLTs were observed in the 300mg ipat + 125mg palbo cohort (grade 4 neutropenia ≥ 7 days). ET+400mg ipat + 100mg palbo was determined to be the recommended phase 2 dose (R2PD), and the remaining 15/42 pts on Arm C were treated at this dose level in the expansion phase. Treatment was well tolerated in all arms. Grade 3 and 4 toxicities included neutropenia (39/77, 50.6%), leukopenia (15/77, 19.5%), diarrhea (11/77, 14/3%), transaminitis (7/77, 9.1%), lymphopenia (6/77, 7.8%), rash (6/77, 7.8%), and thrombocytopenia (3/77, 3.9%). As of 6/28/2022, 16/77 pts remain on treatment. The median treatment duration for all pts is estimated at 6 months (range 0.5-39). Among the 56 pts with measurable disease, 11 had partial response (PR) and 32 had stable disease (SD) as the best response. CBR, defined as percentage of pts who achieved PR or SD > 6 months, was 48% across the study (53% Arm A, 31% Arm B, 57% Arm C). The median PFS was 5.5 months (95% confidence interval [CI]: 3.8 – 7.4) and the median OS was 24.5 months (95% CI: 17.1 – 33.9). Conclusions: The combination of ipat with endocrine therapy +/- palbo is well tolerated in heavily pre-treated pts, with preliminary evidence of clinical activity. This trial demonstrates how molecular insights related to CDK4/6i resistance inform potential therapy combinations. Further studies are needed to evaluate AKTi-based combinations in pts with HR+ MBC. Citation Format: Seth A. Wander, Jennifer C. Keenan, Andrzej Niemierko, Dejan Juric, Laura M. Spring, Jeffrey Supko, Neelima Vidula, Steven J. Isakoff, Lianne Ryan, Sarah Padden, Elizabeth Fisher, Amber Newton, Beverly Moy, Leif Ellisen, Douglas S. Micalizzi, Aditya Bardia. PD13-07 Combination therapy with the AKT inhibitor, ipatasertib, endocrine therapy, and a CDK4/6 inhibitor for hormone receptor positive (HR+)/HER2 negative metastatic breast cancer (MBC): results from the phase I TAKTIC trial [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 PD13-07.

Background: Clinical outcomes in breast cancer differ across racial and ethnic populations. We have previously demonstrated that receipt of genotype-matched therapy targeted to an actionable mutation may potentially improve patient outcomes (Vidula, CCR, 2021). We evaluated the impact of race on clinical, socioeconomic, and genomic characteristics, clinical trial participation, and receipt of genotype-matched therapy among patients with metastatic breast cancer (MBC). Methods: We conducted a retrospective study of patients with MBC at an academic institution who underwent cell-free DNA testing (cfDNA, Guardant360, 74 gene panel) as part of routine clinical care from 11/29/2016-11/2/2020. Patient demographics (including self-reported race and ethnicity) and clinical trial enrollment (at same institution) were determined by retrospective data collection. Mutations identified in cfDNA were characterized as actionable based on the variant interpretation performed by Guardant360 using vetted genomic databases, and receipt of genotype-matched therapy targeted to an actionable mutation was determined as previously described (Vidula, CCR, 2021). Pearson’s chi-squared and Wilcoxon rank-sum tests were used to compare categorical and continuous variables between groups, with p< 0.05 indicating statistical significance. Results: Four hundred and twenty-five patients with MBC and cfDNA results were identified, of which 369 were White (87%), 27 Black (6.4%), 15 Hispanic (3.5%), and 14 Asian (3.3%). There were no significant differences in median age at MBC diagnosis (p=0.064), disease subtype distribution (p=0.74), proportions of de-novo/recurrent MBC (p=0.95), presence of visceral metastases (p=0.84), Charleston comorbidity index (p=0.93), menopausal status (p=0.3), and level of education (p=0.44) across racial groups. Higher proportions of non-primary English speakers were seen in Hispanic (80%) and Asian (29%) races (p< 0.001). Median distance traveled to the institution also varied based on race, with White patients traveling further (White: 39.1 miles, Black: 21.8 miles, Hispanic 9.4 miles, Asian 9.1 miles, p< 0.001). In addition, type of insurance varied based on race, with White patients having the highest rates of commercial insurance and Medicare, Black patients having the highest rate of state-supported insurance, and Asian patients having the highest uninsured rates (p< 0.001). Clinical trial enrollment rates did not significantly differ by race (White: 44%, Black: 37%, Hispanic: 47%, and Asian 21%, p=0.34), but patients without insurance were significantly less likely to be enrolled on a trial than those with commercial insurance (p=0.03). The proportion of patients with ≥1 actionable mutation in cfDNA did not vary significantly by race (White: 78%, Black: 56%, Hispanic: 73%, Asian 86%, p=0.18) and the median number of actionable mutations found in cfDNA was similar across races (p=0.31). However, receipt of genotype-matched therapy targeted to an actionable mutation varied by race, with the highest rates of matched therapy in White patients (White: 28%, Black: 11%, Hispanic 13%, Asian 14%, p< 0.001). After multivariable logistic regression adjusting for subtype, commercial insurance versus other insurance types, and proximity to the center, White patients remained significantly more likely to receive matched therapy (p=0.029). Conclusions: We observed significant race-based differences in non-English speaking status, insurance type, and median distance traveled to the institution. Racial/ethnic minority patients were less likely to receive genotype-matched therapy than White patients. Further research is needed to identify barriers and reduce disparities in access to precision medicine. Citation Format: Rupali Sood, Lianne Ryan, Andrzej Niemierko, Laura M. Spring, Dejan Juric, Steven J. Isakoff, Seth A. Wander, Jennifer Shin, Naomi Ko, Leif Ellisen, Beverly Moy, Aditya Bardia, Neelima Vidula. Impact of Race on Clinical, Socioeconomic, and Genomic Characteristics, Clinical Trial Participation, and Receipt of Genotype-matched Therapy Among Patients with Metastatic 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 PD1-10.

Background: CDK4/6 inhibitors (CDK4/6i) are standard of care for the management of HR+/HER2- metastatic breast cancer (MBC). Genomic alterations that drive resistance to CDK4/6i are diverse, and while the molecular landscape is heterogeneous, several mechanisms of CDK4/6i resistance converge on the RAS/MAPK and PI3K/AKT/mTOR signaling pathways. NF1 downregulates RAS and dampens cellular proliferation. Laboratory-based models demonstrate that loss of NF1 is associated with resistance to endocrine therapy (ET), and emergence of NF1 mutations (NF1m) are correlated with progressive disease (PD) in circulating tumor DNA (ctDNA). While NF1m may diminish CDK4/6i susceptibility, a clear relationship has not been elucidated. The primary objective of this study was to characterize patient (pt) response to CDK4/6i in NF1m HR+/HER2- MBC. Methods: We identified 47 pts with NF1m via a database with one or more ctDNA samples sequenced at variable time-points as part of routine care for MBC. NF1m were categorized as pathogenic (p)NF1m or variants of uncertain significance (VUS) based on their associated Guardant report. We identified 27 pts with HR+/HER2- MBC and NF1m that received at least 1 line of CDK4/6i in the metastatic setting. Intrinsic resistance was defined as PD < 6 months on a CDK4/6i regimen, and acquired resistance was defined as PD >6 months. Pts with intrinsic resistance or acquired resistance and NF1m detected post-PD were categorized as having a resistance phenotype potentially driven by NF1m. Pts with NF1m detected prior to therapy and >6 months clinical response on a CDK4/6i were categorized as having NF1m tumors sensitive to CDK4/6i. Results: The NF1m cohort (n = 27) had 9 pts with pNF1m, while 18 pts expressed VUS. The median age at MBC diagnosis was 54 years, and 67% had visceral metastasis at ctDNA collection. Pts received a median of 1 prior line (range: 0 - 6) of ET or chemotherapy in the metastatic setting before CDK4/6i. Amongst pts with pathogenic variants (n = 9), we found 3 pts with pNF1m were intrinsically resistant to CDK4/6i. Acquired resistance was seen in 1 pt with pNF1m detected post-PD, and 2 pts had evidence of both acquired and subsequent intrinsic resistance to a later line of CDK4/6i. Overall, 67% (6/9) of pNF1m pts demonstrated a CDK4/6i resistance phenotype; mutant allele fraction (AF) ranged from 0.2% - 29.9%, and the mean maximum allele fraction (MAF) was 6.0%. Pre- and post-treatment samples were available on 3 pts with pNF1m, and 1 of these pts had an AF rise from 2.7% to 12.3% when comparing ctDNA pre- and post-CDK4/6i. ctDNA from 4 of 6 resistant tumors harbored other putative drivers including alterations in FGFR, KRAS, PTEN, and RB. We identified 2 counter-examples of pNF1m tumors sensitive to CDK4/6i. These pts expressed relatively low NF1m AF, ranging 0.1% - 0.5% with a mean MAF 0.3%. Another pNF1m pt had intrinsic resistance to initial CDK4/6i but was sensitive to later-line CDK4/6i. In the subgroup of pts with VUS-NF1m (n = 18), a more mixed picture of resistance and sensitivity was seen. 8 pts had intrinsic or acquired resistance, 8 pts had NF1m tumors sensitive to CDK4/6i, and 1 pt had evidence of both; 61% (n = 11) of pts expressed alterations in other resistance mediating genes. 1 pt stopped therapy due to toxicity rather than PD. Conclusions: Our work demonstrates that tumor expression of pNF1m may be associated with CDK4/6i resistance in pts with HR+/HER2- MBC, and allele fraction could be predictive of drug susceptibility. Tumors harboring VUS had varied sensitivity, suggesting that some of these mutations may not be pathogenic, and counter-examples of pNF1m MBC benefiting from CDK4/6i plus ET highlight the complexities in predicting drug response based on single gene alteration. Future effort is warranted to explore the potential impact of NF1 on CDK4/6i resistance, as well as the potential role for therapies targeting the MAPK pathway in this patient population. Citation Format: Maxwell R. Lloyd, Lianne Ryan, Arielle J. Medford, Jennifer C. Keenan, Laura M. Spring, Neelima Vidula, Beverly Moy, Dejan Juric, Leif Ellisen, Aditya Bardia, Seth A. Wander. Investigating NF1 Mutations in Circulating Tumor DNA of Patients with Hormone-receptor Positive (HR+) Breast Tumors Resistant to CDK4/6 Inhibition (CDK4/6i): A Retrospective Clinical 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 P1-13-07.

Background: Addition of PI3K/mTOR inhibitor after progression on CDK4/6 inhibitor (CDK4/6i) and endocrine therapy (ET) can potentially restore sensitivity to CDK4/6i and prevent adaptive activation of the PI3K/mTOR pathway. To evaluate this hypothesis, we conducted a Phase Ib study of gedatolisib (G), a dual inhibitor of PI3K/mTOR, palbociclib (P) a CDK4/6i, and ET (with letrozole [LET] or fulvestrant [FUL]) in women with hormone receptor positive (HR+)/HER2- advanced breast cancer (ABC). Manageable toxicity and preliminary antitumor activity were observed in 35 patients(pts) enrolled in the dose escalation portion of the study (Forero-Torres, ASCO 2018) and 103 pts enrolled in the expansion portion of the study (Layman, SABCS 2021). Here, we report updated efficacy and safety data and sub-group analysis by PIK3CA mutation status in the four expansion study arms with a March 3, 2022, data cut-off. Methods: Pts with HR+/HER2- ABC were treated in four expansion arms: A) G+P+LET as first-line treatment, B) G+P+FUL as 2nd line treatment in pts without prior CDK4/6i; C & D) G+P+FUL as 2nd or 3rd line therapy in pts with prior CDK4/6i. P, LET, and FUL were administered at standard doses. G 180 mg was intravenously administered weekly in Arms A, B, and C and three weeks on/one week off in Arm D. The primary endpoint was investigator assessed objective response rate (ORR). Secondary endpoints included safety, duration of response and progression free survival (PFS). Results: Of the 103 pts treated with G+P+ ET in the expansion arms (A-D), 100% had measurable disease at baseline, 71% (73/103) lacked PIK3CA mutations (wild type; WT), 27% (28/103) had PIK3CA-mutations (MT), 70% (72/103) had evidence of bone metastases, and 59% (61/103) had liver metastases. The most frequent grade 3 and 4 treatment related AEs (TRAE) with G+P+ET included neutropenia (63%), stomatitis (27%), rash (20%), fatigue (11%) and hyperglycemia (7%). Treatment discontinuation due to TRAEs was 6.5% in Arm A, 15.4% in Arm B, 9.4% in Arm C and 3.7% in Arm D. Efficacy data for each arm is presented in Table 1. Promising ORR and PFS were seen in all arms regardless of PIK3CA mutation status. In Arm D, ORR was 63% overall, 73% in PIK3CA-MT pts, and 60% in PIK3CA-WT pts. Median PFS in Arm D was 12.9 months with a median follow up of 29 months. 60% and 48% of pts in the PIK3CA-MT and PIK3CA-WT Arm D sub-groups, respectively, were progression free at 12 months. Conclusions: These preliminary data demonstrate promising activity of G+P+ET combination in pts who were CDK4/6i-naïve and in those whose disease progressed on or after CDK4/6i therapy regardless of PIK3CA mutation status. Encouraging results in CDK4/6i treatment naïve patients warrant further evaluation of gedatolisib in combination with CDK4/6i treatment in the front-line setting. Arm D results provide a strong basis for the initiated Phase 3 study (VIKTORIA-1) in pts whose disease progressed on or after CDK4/6i therapy. Table 1. Efficacy Data by Expansion Arms Citation Format: Robert Wesolowski, Hope Rugo, Erica Stringer-Reasor, Hyo S. Han, Jennifer M. Specht, E. Claire Dees, Peter Kabos, Ulka Vaishampayan, Seth A. Wander, Janice Lu, Keerthi Gogineni, Alexander I. Spira, Anne F. Schott, Maysa Abu-Khalaf, Pratima Nayak, Brian F. Sullivan, Igor Gorbatchevsky, AND Rachel M. Layman. PD13-05 Updated results of a Phase 1b study of gedatolisib plus palbociclib and endocrine therapy in women with hormone receptor positive advanced breast cancer: Subgroup analysis by PIK3CA mutation status [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 PD13-05.

Background: CDK 4/6 inhibitors have demonstrated substantial efficacy in treating ER+/HER2- metastatic breast cancer. Therefore, there is great interest in exploring their ability to reduce recurrence risk in early breast cancer. However, conflicting results were observed in the large adjuvant phase 3 clinical trials investigating combination of endocrine therapy and CDK 4/6 inhibitor (PALLAS, MONARCH-E). While these adjuvant clinical trials evaluated upfront use of CDK 4/6 inhibitor, the optimal timing of adding CDK 4/6 inhibitor for HR+/HER2- breast cancer remains unknown. We conducted a prospective phase II clinical trial to evaluate the addition of a CDK 4/6 inhibitor, ribociclib, in patients who were already on adjuvant endocrine therapy. Methods: In part 1 of the clinical trial, eligibility included patients with localized stage I-III ER+ (≥ 10%), HER2- breast cancer; completed surgery; and were on adjuvant endocrine therapy (any number of years) with at least one year or more of treatment remaining. Patients were randomized to two different ribociclib schedules: continuous (400 mg daily of 28-day cycle; arm 1) or intermittent (600 mg daily on days 1-21 of 28-day cycle; arm 2) for one year. Patients were concurrently treated with an aromatase inhibitor (plus GnRH agonist if premenopausal). Tolerance was evaluated via CTCAE version 4.03 and proportion of subjects who discontinued CDK 4/6 treatment early. Stratification factors for statistical analysis included: disease stage (III vs lower), duration of prior endocrine therapy (within 2 years; 2-5 years vs > 5 years), and whether the patient received prior chemotherapy or not. Baseline characteristics and risk factors for recurrence and for early discontinuation were compared between the arms of the study using Pearson's chi-squared test. Actuarial analysis of time to recurrence was done using the Kaplan-Meier estimator. The primary objective of part 1 was to estimate adherence to ribociclib treatment in the adjuvant setting. Results: In total, 81 patients were enrolled between February 2018 and September 2019, and 25 (31%) discontinued ribociclib treatment early, with no significant difference between study arms. The most common grade 3 or greater adverse events (AEs) leading to study discontinuation were neutropenia (44%), alanine aminotransferase increase (28%), and aspartate aminotransferase increase (16%). Among patients who discontinued early, neutropenia was more frequent in the 600 mg arm, 9 of 12 patients (75%), versus 2 of 13 patients (15%) in the 400 mg arm. No patients discontinued early due to prolonged QTc. Ribociclib was dose reduced for 22 patients (27%), with no significant difference between study arms (p = 0.12). After a median follow-up of 20 months, two patients have experienced disease recurrence with recurrence-free survival of 100% at 1 year and 97% (95% CI 88-99%) at 2 years. Biomarker (ctDNA) results will be reported at the meeting. Conclusions: Results demonstrate that while serious AEs with one year of adjuvant ribociclib are low, a substantial number of patients discontinued adjuvant CDK 4/6 inhibitor within 1 year. Overall, with limited follow-up, only two patients had recurrent disease since completion of ribociclib treatment. Tolerability and identifying patient subsets who will most benefit need to be carefully considered with CDK 4/6 inhibitors in the adjuvant setting. Citation Format: Laura M Spring, Lauren Scarpetti, Andrzej Niemierko, Steven J Isakoff, Beverly Moy, Seth A Wander, Elisabeth Smith, Elizabeth Abraham, Jennifer Shin, Jaymin M Patel, Amy Comander, Therese Mulvey, Aditya Bardia. Phase II study of adjuvant endocrine therapy with CDK 4/6 inhibitor, ribociclib, for localized ER+/HER2- breast cancer (LEADER, part 1) [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 P1-14-02.

Background: CDK 4/6 inhibitors have transformed the landscape of breast oncology. A CDK 4/6 inhibitor in combination with endocrine therapy is recommended as 1st line therapy for patients with metastatic hormone receptor positive breast cancer. CDK 4/6 inhibitors have purported immunomodulatory effects and while effective, myelosuppression is a common adverse effect of CDK 4/6 inhibitor treatment of breast cancer. The impact of CDK 4/6 inhibitor therapy on immunogenicity of vaccines is not known. In this study, we evaluated the spike antibody response to SARS-CoV-2 vaccines among patients with breast cancer receiving endocrine therapy with or without CDK 4/6 inhibitors.Methods: In the Cancer COVID and Vaccine (CANVAX) study eligible patients included patients with breast cancer who had completed all scheduled doses of SARS-CoV-2 vaccines. Chart review was conducted to identify patients who had received endocrine therapy with or without CDK 4/6 inhibitor. We used validated assays to measure anti-SARS-CoV-2 total IgA/M/G spike antibodies and virus neutralization. We evaluated the magnitude of antibody response based on geometric mean concentrations (GMCs) as well as the % of patients with inadequate seroconversion (defined as levels Citation Format: Elyssa Denault, Erika Nakajima, Vivek Naranbhai, Alejandro Balazs, Lindsey Mortensen, Elizabeth Niehoff, Caroline Barabell, Jennifer A. Hutchinson, Seth A. Wander, Aron S. Rosenstock, Leif W. Ellisen, Beverly Moy, Steven J. Isakoff, Justin F. Gainor, A. John Iafrate, Aditya Bardia, Laura M. Spring. Immunogenicity of SARS-CoV-2 vaccines in patients with breast cancer receiving CDK 4/6 inhibitors [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 P3-23-02.

Background: Endocrine therapy with CDK 4/6 inhibitors (ET/CDK4/6i) represents the 1st line therapy for ER+/HER2- ABC. While majority of patients derive clinical benefit with combination therapy, a subset have refractory disease with progression within 6 months. However, predictive biomarkers for rapid progression are lacking. In this study, we evaluated genomic profiles associated with rapid disease progression on ET/CDK4/6i. Methods: We identified 77 patients who received 1st line ET/CDK4/6i combination therapy (AI or SERD with one of the 3 approved CDK4/6is) and had ctDNA analysis performed via plasma based genotyping utilizing the commercially available Guardant360 assay at three sites: Washington University in St. Louis, MO, Northwestern University (Chicago, IL), and Massachusetts General Hospital (Boston, MA). We aimed to look at the differences in patient characteristics and genomic profiles of the tumors assessed from baseline ctDNA specimens between the patients with rapid progression (time to progression TTP Citation Format: Marko Velimirovic, Lorenzo Gerratana, Andrew A Davis, Whitney L Hensing, Katherine Clifton, Ami N Shah, Paolo D'Amico, Charles S Dai, Elyssa N Denault, Cynthia X Ma, Seth A Wander, Dejan Juric, Massimo Cristofanilli, Bruce A Chabner, Aditya Bardia. Genomic predictors of rapid progression to first line endocrine and CDK4/6 inhibitor combination therapy in patients with estrogen receptor positive (ER+) HER-2 negative (HER2-) advanced breast cancer (ABC) [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 P2-07-02.

Background: The cyclin-dependent kinase 4/6 inhibitors, with endocrine therapy (ET), have become the standard of care for patients with HR+/HER2- MBC. Prior insight from tumor biopsies and preclinical analyses suggest that AKT1 activation can provoke CDK4/6i resistance, highlighting a potential therapeutic role for AKT inhibition (AKTi) in this setting. However, combinatorial inhibition can be associated with significant toxicity and identification of the optimal biological dose is often challenging. In this translational co-clinical study, we evaluated escalating doses of AKTi combination with CDK 4/6i in parallel patient-derived pre-clinical models as well as a phase 1b clinical trial. Methods: In an open-label phase Ib dose-escalation clinical trial (TAKTIC, NCT03959891), we evaluated the safety, tolerability and efficacy of escalating doses of the AKT1 inhibitor ipatasertib (ipat) in combination with palbociclib (palbo) and fulvestrant (fulv) for patients with HR+/HER2- MBC. Inclusion criteria include unresectable or metastatic disease, at least 1 prior therapy for MBC including any CDK4/6i, and up to 2 prior lines of chemotherapy for MBC (no limit on prior endocrine therapy). In addition, response to escalating doses of ipat and palbo (with fulv) were explored in vitro via an ATP-based viability assay in tumor cell lines derived from circulating tumor cells (CTC) isolated from patients with endocrine-refractory HR+ MBC. Results: In the dose-escalation portion of the phase 1b clinical trial, 23 patients received the triplet combination of ipat, palbo, and fulv (median number of prior lines = 4.3, range 1-7; 100% with prior CDK4/6i): 3 pts received ipat at 200mg + 125mg palbo, 15 pts received 300mg + 125mg palbo, and 5 pts received 400mg + 100mg palbo, all with fulv (500 mg). Among the 23 patients, 20 patients (86.9%) had disease control (4 partial response and 16 stable disease) as the best response, per RECIST. Grade 3/4 toxicities included neutropenia (n=20), lymphopenia (n=3), diarrhea (n=3), thrombocytopenia (n=2), transaminitis (n=2), and rash (n=2). Two DLTs were observed in the 300mg ipat + 125mg palbo cohort (grade 4 neutropenia ≥ 7 days), but none at 400mg + 100mg palbo. The combination of ipat and palbo demonstrated an additive effect in vitro, with increased sensitivity to lower doses of palbo in the presence of ipat. Based on the totality of data, 400mg ipat + 100mg palbo + fulv 500 mg was selected as the recommended phase II dose (RP2D) in the post-CDK4/6i setting. Conclusions: The triplet combination of endocrine therapy with AKTi and lower dose CDK4/6i appears to be well tolerated in heavily pre-treated pts, with preliminary evidence of clinical activity. Further study is needed to evaluate biomarkers associated with higher AKTi benefit in order to guide rational development of combination therapy for patients with HR+/HER2- MBC in the post-CDK4/6i setting. Overall, this translational study demonstrates how insight into the molecular mechanisms of CDK4/6i resistance and combinatorial modeling can be leveraged to develop actionable therapeutic regimens for patients with MBC. Citation Format: Seth A. Wander, Douglas S. Micalizzi, Taronish Dubash, Dejan Juric, Laura M. Spring, Neelima Vidula, Jennifer Keenan, Maureen Beeler, Elene Viscosi, Dante Che, Elizabeth L. Fisher, Rachel A. Hepp, Beverly Moy, Steven J. Isakoff, Leif W. Ellisen, Jeffrey G. Supko, Shyamala Maheswaran, Daniel A. Haber, Aditya Bardia. AKT inhibition in combination with endocrine therapy and a CDK4/6 inhibitor (CDK4/6i) in patients with hormone receptor positive (HR+)/HER2 negative metastatic breast cancer (MBC) and prior CDK4/6i exposure: A translational investigation [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 P1-18-22.

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: CDK4/6 inhibitors have emerged as the standard of care for HR+ MBC. However, there is limited insight into the potential benefit of abemaciclib following prior progression on palbociclib or ribociclib. Based on a multi-center cohort of patients with HR+ MBC who had received abemaciclib after prior palbociclib progression (Wander SA et al ASCO 2019), we have previously reported that abemaciclib after prior CDK4/6i progression was well tolerated and that a subset of patients derived durable clinical benefit. Identifying molecular predictors of sensitivity to abemaciclib after prior CDK4/6i progression constitutes an important area of research. Given the high frequency of ESR1 mutations in HR+ MBC with antiestrogen resistance, we evaluated the translational impact of ESR1 mutations in mediating response to abemaciclib in this setting. Methods: To evaluate abemaciclib sensitivity in ESR1 mutant cell lines, T47D HR+ breast cancer cells were modified to over-express multiple mutant ESR1 isoforms via lentiviral infection and antibiotic selection. These isoforms included ESR1 Y537S, Y537N, and D538G. In an additional T47D cell line, RB1 expression was knocked down via CRISPR. The resulting derivative cell lines were grown in the absence of estrogen (via charcoal-stripped serum, CSS) or in escalating doses of abemaciclib. Cell viability was measured via cell-titer-glo assay. For clinical validation, we identified patients with MBC who had ESR1 mutations detected by targeted sequencing of cell-free DNA (cfDNA), via CLIA certified Guardant assay, and had abemaciclib exposure following prior progression on palbociclib or ribociclib in the existing multi-center cohort from six US institutions. Results: All ESR1 mutant derivative cells demonstrated enhanced growth in estrogen deprivation compared to GFP controls, as expected, and were similarly sensitive to escalating doses of abemaciclib monotherapy in vitro, suggesting that ESR1 mutations do not confer resistance to abemaciclib. Interestingly, two patients with ESR1 mutations (in the absence of concurrent driver alterations in RB1, FGFR, CCNE2, and ERBB2) demonstrated progression on palbociclib and sensitivity to abemaciclib. In one patient, cfDNA obtained prior to palbociclib and fulvestrant exposure failed to reveal any ESR1 alteration. Following progression on palbociclib, and prior to sequential exposure to abemaciclib, an ESR1 Y537N alteration was identified. The patient went on to receive 16 months of abemaciclib monotherapy. In a second patient, an ESR1 D538G alteration was identified following progression on palbociclib and fulvestrant. The patient had several intervening regimens, and subsequently went on to receive abemaciclib and fulvestrant for 16 months. RB1-null T47D cells were resistant to abemaciclib monotherapy in vitro, as expected and, in the clinical dataset, the presence of alterations in previously identified genomic mediators of CDK4/6i resistance, such as RB1, were associated with progression on both palbociclib and abemaciclib. Conclusions: HR+ breast cancer cells expressing mutant ESR1 isoforms were resistant to estrogen deprivation but retained sensitivity to abemaciclib in vitro. Furthermore, patients harboring ESR1 mutations via targeted sequencing of cfDNA, in the absence of other known mediators of CDK4/6i resistance, were shown to derive clinical benefit from abemaciclib following prior progression on palbociclib. These results suggest that patients with HR+ MBC, ESR1 mutation, and clinical resistance to anti-estrogen treatment and palbociclib may be candidates for abemaciclib treatment. Further research is warranted to confirm these novel translational observations. Citation Format: Seth A. Wander, Hyo S. Han, Gabriela N. Johnson, Maxwell R. Lloyd, Pingping Mao, Utthara Nayar, Kailey Kowalski, Casey R. Stein, Veronica Mariotti, Leslie SL Kim, Maren Levin, Jing Xi, Apurva Pandey, Siobhan Dunne, Azadeh Nasrazadani, Adam Brufsky, Kevin Kalinsky, Cynthia X Ma, Joyce O’Shaughnessy, Nikhil Wagle, Aditya Bardia. Esr1 mutation as a potential predictor of abemaciclib benefit following prior cdk4/6 inhibitor (cdk4/6i) progression in hormone receptor-positive (hr+) metastatic breast cancer (mbc): A translational investigation [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-10.

Background: BM in MBC patients cause significant morbidity and mortality. BRCA1 germline mutations have previously been shown to be associated with an increased risk of developing BM (Lee et al, 2011), with an incidence as high as 15% (Zavitsanos et al, 2016). We previously reported that a subset of MBC patients may have somatic BRCA mutations in the absence of germline BRCA mutations (Vidula N, SABCS, 2017). In this study, we evaluated the incidence and clinical characteristics of BM in MBC patients with somatic BRCA mutations detected by cfDNA. Methods: MBC patients with somatic BRCA1 or 2 mutations detected by cfDNA (Guardant360TM, next generation sequencing, 73 gene panel; mutations classified as somatic by Guardant360TM) with at least 4 months of follow-up post-testing at an academic institution were identified. From this cohort, we identified patients who developed BM post cfDNA testing. A retrospective review of medical records and Guardant360TM reports was conducted to identify demographics, tumor subtype, type of cfDNA BRCA mutation, whether the BRCA mutation was known to be pathogenic, germline BRCA mutation status, mutant allele fraction (MAF), clonality (MAF ratio of BRCA mutation/gene mutation with highest MAF ≥ 0.25 for clonal, and Results: Of 36 MBC patients with somatic BRCA mutations, 9 (25%) developed BM and 27 (75%) did not have BM (non-BM). The median time to development of BM was 6.7 months after cfDNA testing. Of the BM patients, 5 (56%) had triple-negative (TN) and 4 (44%) had hormone receptor positive (HR+)/HER2- MBC in comparison with the non-BM cases where 5 (19%) had TN, 19 (70%) had HR+/HER2-, and 3 (11%) had HER2+ MBC. Very few patients (1 BM and 2 non-BM) had known co-existing separate germline BRCA mutations (rest not known BRCA carriers confirmed by negative germline testing and/or absence of family history suggestive of a germline BRCA phenotype). The median age at MBC diagnosis was similar for BM and non-BM patients (57 years). PIK3CA and TP53 mutations were commonly seen in both BM and non-BM cases. Additionally, MYC, EGFR, and CCNE1 mutations were commonly seen in BM cases. As outlined in Table 1, among patients with BM, the somatic BRCA mutations were commonly BRCA1, clonal, known to be pathogenic (56%), and present at a higher MAF, but these findings did not reach statistical significance possibly due to the small sample size. Brain tumor tissue mutation status in BM patients and correlation with cfDNA results will be presented at the meeting. Conclusions: We observed a relatively high incidence (25%) of BM in MBC patients with somatic BRCA mutations detectable by cfDNA, which were often known to be pathogenic mutations (56%), and often associated with co-existing MYC, EGFR, and CCNE1 mutations. Further research using a larger cohort with adequate statistical power is needed to validate these findings, and may help identify MBC patients at risk for BM using a liquid biopsy. Table 1.Characteristic BMNon-BMPrior anthracycline and/or platinum6 (67%)16 (59%)Type of somatic BRCA mutationBRCA16 (67%)11 (41%)BRCA22 (22%)15 (56%)BRCA1 and 21 (11%)1 (4%)Median BRCA MAF0.40.17ClonalityClonal6 (67%)13 (48%)Subclonal3 (33%)14 (52%)Mutation known to be pathogenic5 (56%)7 (26%)Common co-existing mutationsPIK3CA5 (56%)12 (44%)TP535 (56%)15 (56%)MYC5 (56%)8 (30%)EGFR5 (56%)8 (30%)CCNE15 (56%)6 (22%)KIT3 (33%)5 (19%) Citation Format: Neelima Vidula, Andrzej Niemierko, Giuliana Malvarosa, Priscilla Brastianos, Erica Blouch, Kristen Shannon, Steven Isakoff, Seth Wander, Laura Spring, Jerry Younger, Kristin Price, Beverly Moy, Dejan Juric, Leif Ellisen, Aditya Bardia. Brain metastases (BM) in patients with metastatic breast cancer (MBC) and circulating cell-free DNA (cfDNA) somatic BRCA mutations [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-09-06.

Background: The AKT1 oncogene represents a central node in the well-characterized PI3K/AKT/mTOR signal transduction pathway involved in the regulation of cellular proliferation as well as therapeutic resistance. With the emergence of multiple targeted therapies for HR+ MBC, we sought to explore the prevalence of AKT1 alterations, the spectrum of co-occurring mutations, and any potential impact on response to endocrine-based therapy, including CDK 4/6 and PIK3CA inhibitors. Methods: Presence or absence of AKT1 mutation was interrogated via cell-free DNA (cfDNA) analysis (Guardant assay) among patients with HR+/HER2- MBC receiving treatment at the Massachusetts General Hospital who underwent routine clinical cfDNA testing. Genomic sequencing results were then compared to detailed clinical annotation based upon retrospective chart review. In the laboratory, HR+/HER2- T47D breast cancer cells were transfected with AKT1, under the control of a doxycycline-inducible promoter, to induce AKT1 overexpression. Sensitivity to escalating doses of fulvestrant and palbociclib were interrogated via cell-titer-glo viability assay in vitro. Results: Among 272 patients with HR+/HER2- MBC, we identified 13 patients with AKT1 mutation (4.7%). 12/13 (92%) patients had a canonical AKT1E17K alteration, one patient (8%) had an AKT1 E49K alteration. AKT1 mutations occurred concurrently with alterations in ESR1 (31%), PIK3CA (23%), TP53 (23%), ERBB2 (15%), and RB1 (8%). All patients had been exposed to some form of anti-estrogen prior to sequencing, either via (neo)adjuvant therapy and/or in the metastatic setting; 5/13 (38%) had exposure to a CDK4/6 inhibitor prior to cfDNA sequencing. Following the identification of an AKT1 alteration, examples of rapid clinical progression on fulvestrant, CDK4/6i, and/or everolimus were identified, however the presence of an AKT1 alteration was not sufficient to predict pan-resistance to these agents, as counter-examples of clinical benefit were also identified. In an index patient with an AKT1 mutation and prior progression on letrozole and palbociclib, subsequent treatment with fulvestrant and abemaciclib did not yield clinical benefit, highlighting the potential role of AKT1 in mediating clinical resistance to endocrine therapy and CDK4/6i. In the pre-clinical model, HR+/HER2- breast cancer cells demonstrated resistance to both fulvestrant and palbociclib following upregulation of exogenous AKT1 expression. Updated clinical data with additional patients and response to regimens incorporating an anti-estrogen, a CDK4/6 inhibitor, an mTOR inhibitor, and/or a PIK3CA inhibitor will be presented at the meeting. Conclusions: Activating AKT1 mutations can be identified via targeted sequencing of cfDNA in patients with HR+/HER2- MBC, and may play an important role in mediating resistance to anti-estrogens, CDK4/6 inhibitors, and other emerging targeted therapies. Heterogeneity in response to endocrine-based therapy following the identification of an AKT1 alteration may be related to tumor-extrinsic factors, the AKT1 allelic fraction, or cooperativity between multiple resistance drivers. Further research is needed to confirm these findings and guide optimal therapeutic sequencing strategies for patients with HR+/HER2- AKT1 mutant MBC. Citation Format: Seth Wander, Pingping Mao, Maxwell Lloyd, Kailey Kowalski, Gabriela N. Johnson, Giuliana Malvarosa, Beverly Moy, Leif W. Ellisen, John Iafrate, Nikhil Wagle, Aditya Bardia. AKT1 alterations following exposure to endocrine-based therapy in patients with hormone-receptor positive (HR+) metastatic breast cancer (MBC): Clinical and functional implications [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-10-08.

Background: The cyclin-dependent 4/6 inhibitors (CDK4/6i), in combination with an anti-estrogen, have emerged as the standard of care for patients with HR+/HER2- MBC. While only limited insight exists into the molecular factors that drive progression, emerging evidence suggests that activation of AKT1 may provoke resistance in a subset of patients. Overexpression of AKT1 also conveys resistance in HR+/HER2- breast cancer cells in vitro. AKT1 plays a well-established role in promoting tumor progression and metastasis. Targeting AKT1 following progression on CDK4/6i may provide durable clinical benefit in HR+/HER2- MBC. Trial Design: This is an open-label phase Ib trial with three arms - Arm A includes fulvestrant + ipatasertib and Arm B includes an aromatase inhibitor (AI) + ipatasertib. Arm C includes fulvestrant + ipatasertib + palbociclib. Arm A and B are dose-expansion cohorts while Arm C is a dose-escalation (standard 3+3 design) followed by subsequent dose-expansion. Eligibility Criteria: Key inclusion criteria include the presence of locally advanced/unresectable and metastatic HR+/HER2- breast cancer; postmenopausal status or pre/peri-menopausal status s/p oophrectomy or GNRH agonist; at least one prior therapy for MBC including any CDK4/6i; adequate performance status (ECOG 0-2); and adequate bone marrow and hepatic function. Stable CNS metastatic disease and up to two prior lines of chemotherapy for MBC are allowed (no limit on number of prior lines of endocrine therapy). Key exclusion criteria include prior use of fulvestrant (for Arm A); any prior intolerable toxicity with CDK4/6i (for Arm C); prior exposure to an AKT inhibitor in any setting; active CNS disease; concurrent use of specific CYP3A4 inhibitors or inducers; poorly controlled intercurrent illness; and pregnancy or active child-bearing potential. Specific Aims: The primary objective of this study is to evaluate the safety and tolerability of ipatasertib in combination with endocrine therapy (aromatase inhibitor or fulvestrant) with or without CDK4/6i in patients with HR+/HER2- MBC who have received prior CDK 4/6i. Secondary objectives include assessment of objective response rate (ORR), clinical benefit rate (CBR), and progression free survival (PFS). Exploratory objectives include next generation sequencing of solid tumor biopsies and cfDNA along with immunohistochemical analysis of tumor biopsies to identify genomic and protein-based predictors of response and resistance. Statistical Methods: Arms A and B constitute dose-expansion cohorts to confirm the safety/tolerability of ipatasertib use with anti-estrogens and evaluate preliminary efficacy. A dose-limiting toxicity (DLT) rate of Present and Target Accrual: Anticipated accrual for Arms A+B will be n=15 (each). Arm C enrollment will be n=15-30 pending establishment of RP2D/MTD prior to dose expansion. Current enrollment n=4, since study activation in May 2019. Citation Format: Seth A. Wander, Dejan Juric, Laura M. Spring, Neelima Vidula, Maureen Beeler, Karleen Habin, Elene Viscosi, Lauren Scarpetti, Elizabeth Tripp, Rachel Hepp, Beverly Moy, Steven J. Isakoff, Leif W. Ellisen, Aditya Bardia. A phase 1b trial to evaluate safety and anti-tumor activity of the AKT inhibitor, ipatasertib, in combination with endocrine therapy with/without CDK 4/6 inhibitor for patients with hormone receptor positive (HR+)/HER2 negative metastatic breast cancer (MBC) (TAKTIC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT2-08-01.

Background: The CDK4/6 inhibitors have emerged as standard first- or second-line regimens in combination with an antiestrogen for patients with HR+/HER2- MBC. While these agents convey significant clinical benefit in many patients, intrinsic resistance can occur and, in patients who respond, acquired resistance is unfortunately inevitable. Despite their widespread use, we have limited insight into the molecular mechanisms governing response and resistance to these agents. Methods: Whole exome sequencing (WES) was performed on metastatic tumor biopsies from 58 patients (pts) with HR+/HER2- MBC who received a CDK4/6 inhibitor with or without an antiestrogen at the Dana-Farber Cancer Institute, including 7 pts with pre/post-exposure biopsy pairs. Among these biopsies, 69.5% were characterized as resistant (intrinsic or acquired) and 30.5% were characterized as sensitive. To validate putative resistance mediators identified in patient samples, HR+/HER2- breast cancer cells were modified via CRISPR knockout or lentiviral overexpression. Sensitivity of these cells to antiestrogens and CDK4/6i was interrogated via cell-titer-glo assay. In parallel, HR+/HER2- breast cancer cells were cultured to resistance in the presence of an escalating dose of CDK4/6i. Derivative cell lines were subjected to western blotting in an effort to interrogate the putative resistance mediators identified in pts. Novel dependencies were identified in these derivative cell lines via treatment with targeted therapeutic agents in vitro. Results: WES of tumors with CDK4/6i exposure revealed candidate mechanisms of resistance including biallelic RB1 disruption (n=4, 10%) and activating events in AKT1 (n=5, 12.5%), RAS (n=4, 10%), aurora kinase A (AURKA, n=11, 27.5%), and cyclin E2 (CCNE2, n=6, 15%). Convergent evolution toward biallelic RB1 disruption was identified in a single patient with one pre- and two post-exposure biopsies, while acquisition of AKT1 mutation and amplification was identified in two separate instances. Knockout of RB1 and overexpression of AKT1, KRAS G12D, AURKA, and CCNE2 provoked CDK4/6i and antiestrogen resistance in vitro. Breast cancer cells cultured to resistance in CDK4/6i demonstrated concordant acquisition of RB1 downregulation, RAS/ERK activation, AURKA overexpression, and CCNE2 overexpression. Derivative resistant cell lines with RB1 loss or AURKA gain demonstrated enhanced sensitivity to a novel AURKA inhibitor (LY3295668), while cells with RAS activation were highly sensitive to ERK inhibition (via LY3214996). CCNE2-overexpressing cells were highly sensitive to prexasertib, a CHEK1 inhibitor. Conclusions: The genomic landscape of resistance to CDK4/6i is heterogeneous with multiple potential mediators that play well-established roles in cell division and oncogenic signal transduction. We present novel mechanisms of clinical resistance including activation of AKT1 and RAS family oncogenes as well as amplification of AURKA and CCNE2. These drivers were able to provoke resistance to CDK4/6i in vitro. Finally, in each case, a novel dependency was identified which is readily translatable into the clinic. These results underscore the potential of next-generation sequencing as a critical tool to enable identification of resistance mediators, while also suggesting that the presence of specific genomic alterations may define new therapeutic opportunities in CDK4/6i-resistant HR+ MBC. Citation Format: Seth A. Wander, Ofir Cohen, Xueqian Gong, Gabriela N. Johnson, Jorge Buendia-Buendia, Maxwell Lloyd, Dewey Kim, Flora Luo, Pingping Mao, Karla Helvie, Kailey Kowalski, Utthara Nayar, Stephen Parsons, Ricardo Martinez, Lacey Litchfield, Xiang Ye, Chun Ping Yu, Valerie Jansen, Levi A. Garraway, Eric P. Winer, Sara M. Tolaney, Nancy U. Lin, Sean Buchanan, Nikhil Wagle. The genomic landscape of intrinsic and acquired resistance to cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) in patients with hormone receptor-positive (HR+)/HER2- metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD2-09.

Background: Serial cell free DNA (cfDNA) analysis in metastatic breast cancer (MBC) is a noninvasive method for tracking tumor evolution and the emergence of new somatic alterations through the course of the disease. The detection of new actionable alterations may impact treatment selection; thus, serial cfDNA collection is increasingly being performed in the clinic. However, it is not well-understood how often serial cfDNA testing identifies new actionable alterations. We evaluated changes in the genomic spectrum of actionable alterations in serial cfDNA analysis of HER2 negative (HER2-) MBC. Method: Patients with HER2- MBC who underwent plasma-based cfDNA testing (Guardant360, 74-gene assay) between February 2015 and February 2021 at an academic institution were included. A retrospective review of records was conducted to identify subtype, demographics, lines of therapy, and cfDNA results. At baseline cfDNA testing, all cfDNA alterations were considered new. For patients with serial draws, new cfDNA alterations in each draw, compared to previous draws, were quantified and characterized. The pathogenicity of new alterations was determined using the OncoKB precision oncology database. New pathogenic alterations were further classified as actionable alterations (AA) using the ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT). Alterations that met the ESCAT I (alteration-drug match was associated with improved outcomes in clinical trials) or ESCAT II (alteration-drug match was associated with antitumor activity) criteria were considered as AA. Results: 344 patients with hormone receptor positive (HR+) MBC and 95 patients with triple negative (TN) MBC had a baseline cfDNA draw. Among the HR+ cohort, 139, 79 and 48 patients had 2nd, 3rd, and 4th serial cfDNA draws, respectively. Among the TN cohort, 33 and 18 patients had 2nd and 3rd serial cfDNA draws, respectively. Table 1 depicts the prevalence of AA found in each of these draws, as well as the median number of prior therapies at each point of collection and the mean time between serial draws. The median number of new genomic alterations was lower in subsequent draws compared to the baseline, regardless of subtype (HR+: 4 vs. 2, TN: 4 vs. 1.5-3, at baseline vs. subsequent draws, respectively). In the HR+ cohort the proportion of patients with new AA (ESCAT I/II) decreased from 63% at baseline to 27-33% in the 2nd-4th draws. While some of the new AA in subsequent draws were new actionable variants in the same genes that were known to be altered in previous draws (in 19%, 68% and 57% of patients with new AA in the 2nd, 3rd and 4th draws, respectively), the remainder were new AA in previously unaltered genes. In the TN cohort 25% of patients had new AA at baseline and this proportion of TN patients with new AA continued to decrease in subsequent draws (9% and 0% in the 2nd and 3rd draws, respectively). PIK3CA and ESR1 were the most frequent genes with new AA in the ER+ cohort, regardless of draw number. In the TN cohort, PIK3CA was the most frequent gene with new AA in the first draw, and in the second draw, new AA in PIK3CA, BRCA2, and ERBB2 were present at an equal frequency. Conclusion: While the proportion of patients with HER2- MBC identified to have new AA in serial cfDNA decreased with time for both HR+ and TNBC, new alterations continue to emerge, particularly for patients with HR+ MBC. Further research is needed to determine the impact of serial cfDNA testing on the selection of genotype-matched therapy and outcomes. Table 1. Citation Format: Yael Bar, Jennifer C. Keenan, Lianne Ryan, Dejan Juric, Jennifer Shin, Seth A. Wander, Laura M. Spring, Beverly Moy, Leif Ellisen, Steven J. Isakoff, Aditya Bardia, Neelima Vidula. Changes in the Genomic Spectrum of Actionable Alterations in HER2 Negative Metastatic Breast Cancer in Serial Cell Free DNA (cfDNA) 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 P4-01-14.

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.

Background: Cancer treatment decisions are often made without specific and representative information that can inform personalized treatment. The aim of this study was to determine if we can predict, based on clinical features, which treatment regimen may maximize real-world time-to-treatment discontinuation (rwTTD) after a hormone-receptor-positive (HR+) metastatic breast cancer (MBC) patient stops responding to a first CDK4/6 inhibitor in any line. Methods: We used patient reported data (PRD) about diagnosis and treatment and medical records from 1,777 patients across the U.S. and Canada from Count Me In’s Metastatic Breast Cancer Project (MBCproject). We interviewed 17 people, academic and community based medical oncologists and MBC patients, to inform the analysis plan. Patient eligibility criteria were prior HR+ MBC diagnosis, received exactly one prior CDK4/6 inhibitor (CDK4/6) containing regimen, start date of any subsequent regimen within four months of the end date of the CDK4/6-containing regimen, and completion of MBCproject’s follow-up questionnaire at least one month after the start date of the subsequent regimen. We processed RWD from the follow-up questionnaire, performed chart review in ambiguous cases of patient eligibility, performed conformance, completeness, and plausibility verification checks to determine the dataset’s fit-for-use, and described treatment variation seen in real-world settings. We designed a new user, active-comparator cohort study with rwTTD as the continuous outcome measure, used known and hypothesized confounders to control for treatment-by-indication bias, assessed covariate balance across cohorts, and conducted Cox proportional hazards (PH) outcome regressions to identify clinically relevant associations and estimate treatment effects across regimens. The analysis plan was publicly registered with the Center for Open Science prior to performing the analysis. Results: 261 eligible HR+ MBC patients were identified, with 110 unique pairs of CDK4/6-containing and subsequent regimens. The most common CDK4/6-containing regimen was Letrozole and Palbociclib (n=98) and subsequent regimen was Capecitabine (n=63). Three mutually exclusive and clinically relevant groupings of subsequent regimens chosen for analysis were chemotherapy only (n=99), fulvestrant-containing (n=53), and everolimus-containing (n=42). Among patients in these three groups, 93.9%+ are white race, 95%+ are non-hispanic, 2.7-9.4% live in a medically underserved area, 7.1-13.1% have HR+/HER2+ MBC, mean age at subsequent treatment was 52.6-53.8 years, 17-36% had bone-only metastasis and 14.3-25.3% had liver metastasis at MBC diagnosis, median number of past treatment regimens was one, and median time on CDK4/6-containing regimen was 9-14 months. The median rwTTD was 9, 15, and 5 months in the three groups, respectively. Out of 11 covariates, nine covariates failed to reject the null hypothesis that the distribution of values are the same across the three cohorts (p>0.05). Outcome regression Cox PH revealed rwTTD hazard ratio (HR) of 2.52 [1.53-4.15; 95% confidence interval (CI)] for presence of liver metastasis, HR of 1.09 [0.63-1.89; 95% CI] for presence of bone-only metastasis, HR of 2.00 [1.20-3.33; 95% CI] for everolimus-containing regimen vs. chemotherapy only, HR of 0.85 [0.50-1.46; 95% CI] for fulvestrant-containing regimen vs. chemotherapy only, and HR of 0.82 [0.65-1.00; 95% CI] for every six months rwTTD on previous CDK4/6-containing regimen. Conclusion: In this cohort, chemotherapy was the most common treatment regimen following CDK4/6 even in second and third line settings and in patients with bone-only metastasis, which is a deviation from guideline-based treatment for many HR+ MBC patients. PRD helps develop hypotheses about patient response to treatment following CDK4/6 that can be further evaluated in larger, more diverse observational studies and clinical trials. Table 1. Characteristics of eligible patients who received chemotherapy only, everolimus containing, or fulvestrant containing regimens. Citation Format: Ariel B. Carmeli, Seth A. Wander, Mary McGillicuddy, Caroline Block, Nikhil Wagle. Real-World Time-to-Treatment Discontinuation in Hormone-Receptor-Positive Metastatic Breast Cancer Patients following CDK4/6 Inhibitor Treatment, Based on Observational Data Collected Through Patient-Partnered Research [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 P3-02-02.

Background: For patients with hormone receptor-positive (HR+) metastatic breast cancer (MBC) and progression on combination endocrine therapy plus palbociclib/ribociclib, abemaciclib can be effective and well-tolerated, conferring durable clinical benefit in a subset of patients (Wander SA et al. JNCCN 2021). We previously reported that, even in patients with ESR1-mutant (ESR1-MUT) HR+ MBC, some - but not all - still achieve long-term disease control with abemaciclib (Wander SA et al. SABCS 2020). How to identify which patients are likely to benefit from abemaciclib after progression on combination endocrine therapy and CDK4/6 inhibition is not known and, more generally, predicting tumor response to CDK4/6 inhibition has been an area of ongoing research. Because resistance to CDK4/6 inhibition can occur through multiple mechanisms, we hypothesized that a comprehensive resistance panel, rather than single genetic markers or immunohistochemical readouts, would provide an effective predictive tool. Methods: To determine which patients with ESR1-MUT MBC and progression on endocrine therapy plus palbociclib/ribociclib benefit from abemaciclib, we examined a multicenter cohort of such patients who had genomic profiling by standard commercially available assays, the majority of which were via plasma-based cell-free DNA (cfDNA) genotyping assays. We generated a curated list based upon prior literature of CDK4/6i resistance drivers that had been validated in tumor biopsy specimens and in laboratory models: these genes were involved in cell cycle regulation (CCNE1/2, RB1, AURKA) and growth factor signaling pathways (ERBB2, FGFR1/2, AKT1, PTEN, KRAS, FAT1). Progression-free survival (PFS) was defined as time from abemaciclib initiation to time of discontinuation due to disease progression or death; patients who discontinued due to toxicity were right-censored. To examine the cellular effects of different mutations, we also studied the impact of ESR1-MUT, RB1 loss, and KRAS activation on the growth and survival (using an ATP abundance-based assay) of patient-derived circulating tumor cell (CTC) lines treated with palbociclib and abemaciclib in vitro. Results: Among patients with ESR1-MUT MBC with disease progression on endocrine therapy plus palbociclib/ribociclib (n=28), absence of co-existing genomic alterations in our curated panel was associated with greater clinical benefit with subsequent abemaciclib. Patients lacking a mutation in this resistance panel (n=17) had a median PFS of 7.0 months (95% CI: 4.1-13.2); patients with at least one mutation in this panel (n=11) had a median PFS of 3.5 months (95% CI: 2.1-5.4). The difference in PFS was statistically significant (p=0.02, log-rank test). On univariable Cox regression the hazard ratio for patients with a mutation in the resistance panel was 2.8 (95% CI: 1.1-7.1, p=0.03). In vitro, two out of three patient-derived cell lines with ESR1-MUT remained sensitive to abemaciclib, while those with mutation in RB1 or KRAS were less sensitive to abemaciclib. Conclusions: In our study, absence of co-existing genomic alterations in a curated panel was associated with greater clinical benefit with subsequent abemaciclib among patients with ESR1-MUT MBC with prior disease progression on endocrine therapy plus palbociclib/ribociclib. While a small dataset, this is the first demonstration of a genomic panel associated with continued CDK4/6 inhibitor sensitivity. Future directions include testing this panel outside of ESR1-MUT MBC and refining the panel in additional datasets with increased sample size, to guide therapy selection for patients with HR+ MBC. Citation Format: Jamie O Brett, Taronish D Dubash, Andrzej Niemierko, Veronica Mariotti, Leslie SL Kim, Jing Xi, Apurva Pandey, Siobhan Dunne, Azadeh Nasrazadani, Maxwell R Lloyd, Laura M Spring, Douglas Micalizzi, Maristela Onozato, Dante Che, Adam Brufsky, Kevin M Kalinsky, Cynthia X Ma, Joyce O’Shaughnessy, Hyo S Han, A. John Iafrate, Shyamala Maheswaran, Daniel A Haber, Aditya Bardia, Seth A Wander. Association between co-existing genomic alterations and abemaciclib benefit in patients with metastatic hormone receptor-positive breast cancer with ESR1 mutations following disease progression on prior endocrine therapy plus palbociclib or ribociclib [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 PD2-03.

Introduction: The advent of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) has transformed the clinical practice of HR+/HER2- metastatic breast cancer. Palbociclib, ribociclib, and abemaciclib have been approved in conjunction with anti-estrogens, while abemaciclib has also been approved as monotherapy based on single agent activity in the MONARCH-1 trial (objective response rate/ORR: 19.7% and median progression-free-survival/PFS = 6.0 months; Dickler M et al CCR 2017). However, there is limited insight into the mechanisms governing resistance to CDK 4/6i and the potential utility of continued CDK4/6i after progression on a prior CDK 4/6i-based therapy. Methods: We evaluated the clinical outcomes of patients with metastatic HR+/HER2- breast cancer who had received abemaciclib following an initial course of palbociclib-based therapy at our institution. In addition, we conducted genomic analysis utilizing next-generation sequencing of tissue samples and blood (cell-free DNA/cfDNA analysis) where available. Results: From June, 2014 through July, 2018, a total of 49 patients received abemaciclib, and 14 patients had prior palbociclib exposure. One patient was deceased shortly after initiating abemaciclib and one patient was lost to follow-up. Among the 12 remaining patients, eight had sequential courses of CDK4/6-based therapy, while four patients had at least one intervening non-CDK 4/6i based regimen. At data-cutoff of 8/15/2018, five patients (41.7%) had early progression on abemaciclib (PFS equal to or less than 120 days) while three (25%) patients had ongoing benefit (PFS greater than 120 days, two of three actively on therapy). Three additional patients had recently initiated abemaciclib therapy (less than 120 days prior to current analysis). Preliminary analysis of baseline cfDNA results in patients with early progression on abemaciclib therapy after prior CDK4/6i revealed the presence of RB1 mutation, FGFR1 amplification, and TP53 mutation, among others. Additional analyses with mature clinical data (including updated PFS and ORR), toxicity assessment during secondary CDK4/6i exposure, and further analysis of genomic sequencing results will be provided at the meeting. Conclusions: The majority of patients had early disease progression on abemaciclib after prior exposure to CDK4/6i suggesting potential cross-resistance to CDK4/6i mediated by common drivers. However, a subset of patients derived clinical benefit with continued exposure to CDK4/6i, highlighting the need for additional research to evaluate potential predictive biomarkers and guide rational utilization of continued CDK4/6 blockade in metastatic HR+/HER2- breast cancer. Citation Format: Wander SA, Spring LM, Stein CR, Yuen M, Zangardi M, O'Shaughnessy J, Bardia A. Abemaciclib after prior palbociclib exposure in patients with metastatic hormone-receptor positive (HR+)/HER2- breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-18-39.

Background For HR+ MBC, ESR1 point mutations (ESR1-MUT) are a common mechanism of acquired resistance to aromatase inhibition (AI); ESR1 fusions (ESR1-FUS) are rare and promote intrinsic resistance to ER-targeting drugs. Retrospective analyses of CDK4/6i trials suggest ESR1-MUT does not cause CDK4/6i resistance, but whether CDK4/6i is effective for ESR1-MUT, or for ESR1-FUS, in the real-world setting is unknown. Methods Real-world evidence was sourced from the GuardantInform database of commercial payer claims and ctDNA tests from 170,000+ individuals. Patients with MBC who started CDK4/6i within 30 days of ctDNA testing were categorized as ESR1-MUT vs. ESR1-WT and analyzed for time-to-next-treatment (TTNT). Separately, cases with ESR1-FUS detected by tissue RNA-Seq were extracted from a clinicopathologic database at an academic cancer center. Results There was no significant difference in TTNT on CDK4/6i for ESR1-MUT vs. ESR1-WT. As expected, ESR1-MUT had shorter overall survival (OS), even after adjustment for age, CDK4/6i drug, and prior treatment (HR 0.58 (0.42-0.82), p=0.002, multivariable Cox). Endocrine partner analysis was limited by lack of clinical annotation to 27% of cases: AI was given to 55% of ESR1-WT and 25% of ESR1-MUT; fulvestrant was given to 39% of ESR1-WT and 68% of ESR1-MUT. Additional stratified analyses will be presented. In the clinicopathologic database, we identified 4 ESR1-FUS cases, and all received CDK4/6i. Progression-free survival durations on CDK4/6i were 4, 10, 11, and 33+ months. Conclusions Using real-world evidence, we demonstrate that CDK4/6i is effective in both ESR1-MUT and ESR1-WT HR+ MBC, supporting the use of CDK4/6i in this setting. CDK4/6i may be additionally beneficial for patients with ESR1-FUS. Future directions include expanding the ESR1-FUS cohort and deciphering the heterogeneity of CDK4/6i responses in this patient population. ESR1-WT ESR1-MUT p-value n=612 n=145 TTNT, median days (95% CI) 99 (85-121) 102 (85-152) 0.84 (log-rank) OS, median years (95% CI) 5.1 (4.5-NA) 2.2 (2.0-NA) Citation Format: Jamie O. Brett, Caroline M. Weipert, Lauren L. Ritterhouse, Nicole Zhang, Junhua Yu, Lianne Y. Ryan, Laura M. Spring, Miguel N. Rivera, Jochen K. Lennerz, Dora Dias-Santagata, Leif W. Ellisen, Aditya Bardia, Seth A. Wander. CDK4/6 inhibition (CDK4/6i) is effective in the real-world setting for hormone receptor-positive metastatic breast cancer (HR+ MBC) with ESR1 mutations and fusions [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 5248.

Background: Deciphering the molecular landscape of resistance to the CDK4/6 inhibitors represents a critically important question for patients with hormone-receptor positive (HR+) metastatic breast cancer (MBC). Emerging insights from sequencing efforts suggest that inactivating alterations in the RB1 tumor suppressor occur in a small minority of patients and that a variety of heterogeneous mediators provoke resistance in patient samples. Proteins implicated in CDK4/6i resistance include cell cycle regulators such as cyclin E1/2, CDK6, and aurora kinase as well as known oncogenic signal transduction mediators involved in activation of the RAS-MEK and AKT-mTOR pathways. The insulin-like growth factor 1 receptor (IGF1R) has been implicated in modulating anti-estrogen resistance, and IGF1R inhibitors are currently in various stages of pre-clinical and clinical development. Methods: We identified patients with amplification events in IGF1R from a database containing targeted sequencing of solid tumor samples obtained from patients with HR+ MBC enrolled on a research biopsy protocol. Tumor biopsies may have been obtained at various points during each patient’s clinical treatment course. HR+ T47D cells were modified to over-express IGF1R via lentiviral infection and selection. Derivative cell lines were treated with IGF-1 ligand and downstream activation of the PI3K/AKT and RAS/MEK pathways were assessed via western blotting. Control cells (expressing GFP) were mixed with IGF1R-expressing cells 1:1 and cultured in the presence of IGF-1 ligand and palbociclib or other drugs, for 1-3 weeks. At the timepoint of interest, cells were harvested and the relative proportion of GFP or IGF1R-expressing cells were interrogated via flow cytometry. Results: We identified seven patients with HR+ MBC and IGF1R amplifications via targeted sequencing of tumor biopsies. Five of these patients had exposure to CDK4/6i-based therapy in the metastatic setting. Three patients demonstrated intrinsic resistance to CDK4/6i treatment (with duration Conclusions: IGF1R amplification events were identified in tumor biopsy samples that reflect either intrinsic or acquired resistance to CDK4/6i-based therapy. HR+ breast cancer cells which over-express IGF1R demonstrate enrichment under palbociclib drug selection in a flow cytometry-based competition assay, which was abrogated by concurrent use of a MEK or IGF1R inhibitor. These results suggest that IGF1R may join the increasingly heterogeneous landscape of CDK4/6i resistance mediators. Further exploration of this possibility is warranted. A subset of patients with IGF1R-mediated CDK4/6i resistance could benefit from therapeutic strategies designed to downregulate MEK or IGF1R activity. Citation Format: Seth A. Wander, Pingping Mao, Maxwell R. Lloyd, Gabriela N. Johnson, Kailey Kowalski, Utthara Nayar, Lillian M. Guenther, Kimberly Stegmaier, Eric P. Winer, Nancy U. Lin, Nikhil Wagle. Igf1r mediates cdk4/6 inhibitor (cdk4/6i) resistance in tumor samples and in cellular models [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD7-08.

Background: Although the androgen receptor (AR) is frequently co-expressed with ER and PR in hormone receptor-positive (HR+)/HER2- breast cancer, the biological significance of detectable AR alterations (ARalt) in metastatic disease (MBC) remains poorly understood. The primary objective of this study was to evaluate the association of ARalt status with clinical outcomes among women with HR+/HER2- MBC. Methods: Retrospective review was performed on patients with HR+/HER2- MBC treated at an academic institution, for whom genotyping information was available. ARalt status was determined using Guardant360, a 73-gene next-generation sequencing assay that detects both AR mutations and amplifications in circulating tumor DNA. Women with positive or unknown HER2 status and triple-negative breast cancer were excluded from analysis, as were cases of male breast cancer. Time-to-progression on the therapy initiated immediately following Guardant testing was compared based on ARalt status, excluding patients treated with androgen-directed therapies given potential for confounding. Cumulative incidence plots were generated and analyzed by Gray’s test, and propensity score-adjusted competing risk models were generated with the probability of treatment as a function of age at metastatic diagnosis, presence of visceral metastasis, presence of de novo metastases, as well as number of prior therapies. Additional analysis was performed to assess progression stratified by treatment type (endocrine or non-endocrine based). Results: Among women with HR+/HER2- MBC (n=222), 16 patients (7%) had detectable ARalt (12 point mutations, 4 amplifications). No baseline differences were observed between women with ARalt and those without AR alterations (ARwt), with respect to age at primary or metastatic diagnosis, menopause status, time to onset of metastasis or de novo metastatic disease, presence of visceral metastases, or number of endocrine/chemotherapies received prior to Guardant testing. ARalt tumors had a higher frequency of detected mutations (14% vs. 5%, p Conclusions: ARalt tumors demonstrate a higher rate of progression on endocrine-based therapy as compared to ARwt tumors, highlighting a potential role of AR in mediating resistance to endocrine therapy in HR+/HER2- disease. Further translational investigations are warranted to determine whether ARalt/HR+/HER2- disease represents a unique biological subtype that predominantly relies on AR signaling and may thus benefit from blockade with AR antagonists. Table 1. Multivariable competing risks model for endocrine progression.CovariatePFSMultivariableHR95% CIP-valuePositive ARalt status4.172.43-7.17 Citation Format: Charles Dai, Andrzej Niemierko, Neelima Vidula, Laura M Spring, Seth A Wander, Arielle J Medford, Katherine A Hesler, Giuliana Malvarosa, Jeffrey Peppercorn, Dejan Juric, Steven J Isakoff, Beverly Moy, Leif W Ellisen, Aditya Bardia. Molecular alterations in the androgen receptor and associated clinical outcomes in hormone receptor-positive/HER2- metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-02.

While recent studies have begun characterizing the metastatic breast cancer (MBC) genomics, our understanding of mechanisms of acquired endocrine-resistance, their induced cell-state, and their altered drug-response profile, remains lacking. We collected biopsies from patients with MBC with detailed clinicopathologic features. To date we profiled 520 exomes, and 291 transcriptomes, with 126 patients having multiple biopsy exomes. Curated endocrine-relapse set include 60 patients with pre-treatment and post-relapse exomes. Characterization of candidate mechanisms of resistance (MOR) included 909 RNA-seq profiles of T47D cells with introduced MOR under various drugs. In the acquired endocrine resistance cohort, as expected, we found frequent ESR1 acquired mutations (13 pt, 22%). Additionally, we identified acquired activating SNVs and amplifications in oncogenic receptor tyrosine kinases (RTKs) in 18/60 (30%), including EGF family - HER2 (n=7), ERRB3 (R525Q), and EGFR (S116F), and FGF family - FGFR1 (n=5), FGFR2 (n=4), and FGF3 amplicon (n=4). RNA-seq of T47D cells overexpressing HER2 activating mutations revealed a distinct cell-state (HER2-ACT). Similarly, FGFR activation revealed a district FGFR-ACT state. The transcriptional signatures of these HER2 and FGFR states were remarkably similar (odd-ratio= 91, p= 2.64E-94). To characterize the cell-state common to HER and FGFR, we defined RTK-ACT with 358 overlapping marker genes (see table). Canonical (estradiol) ER signaling is slightly elevated in RTK-ACT, however this state is strongly associated with growth-factor driven ER signaling, suggesting reprogramming of ER from AF2, to AF1 signaling. Consistent with this, RTK-ACT had significantly higher MAPK activation. Additionally, RTK-ACT Induced stronger similarity to Basal-state, enrichment in motility/migration, mesenchymal, and stem-like features, with top genes including CDH3, MBP7, and S100, ETV, DUSP, SPRY families (see table). To study RTK-driven state in-vivo, we analyzed RNA-seq from our MBC biopsies, and compared tumors with activating RTK mutations (n=38) with WT (n=118), and inferred activated RTK in-vivo (RTK.ACT.iv). Our in-vitro and in-vivo states show significant overlap (OR=4.71, p=9.42E-20). Furthermore, characterization of RTK.ACT.iv, recapitulated all the cell-state features observed in RTK.ACT - including higher growth-factors ER signaling, MAPK, and basal-like state (see table). We further studied the viability and transcription of these RTKs under various drugs (12 treatments), including fulvestrant, palbociclib, and specific tyrosine kinase inhibitors (TKIs) - Neratinib (pan-HER inhibitor) and FIIN-3 (FGFR-i). We found that HER2-ACT and FGFR-ACT signatures remain robust when treated with fulvestrant, palbociclib, and their combinations, as compared to TKIs suppression (see table). in-line with our viability results - suggesting intrinsic resistance to CDK4/6i and sensitivity to TKIs. This study demonstrated that activating RTKs constitute of prevalent modality of acquired resistance to endocrine therapies, inducing a distinct state with clinical implications - suggesting the potential benefit of combination therapies with specific TKIs over CDK4/6i. The common MAPK activity and our preliminary results - suggests the potential of convergence-node targeting strategy with added MEK or SHP2 inhibition. TableGene set AGene set BOdds-ratioP-value (two-sided)Fisher''s Exact test matHER2-ACT -FGFR-ACT -912.64E-9469, 131, 131, 22704HER2 S653C, L755S, V777L, L869R vs. GFP, under DMSO, rank genes based on the logFC (top 200)FGFR1, FGFR2 (WT, N550K, M538I, K660N) vs. GFP, Parental, Under DMSO, rank genes based on the logFC (top 200)HER2-ACT_1000 -FGFR-ACT_1000 -18.98.28E-248358, 642, 642, 21758HER2 S653C, L755S, V777L, L869R vs. GFP, under DMSO, rank genes based on the logFC- top 1000FGFR1, FGFR2 (WT, N550K, M538I, K660N) vs. GFP, Parental, Under DMSO, rank genes based on the logFC - top 1000RTK-ACTHALLMARK_ESTROGEN_RESPONSE_EARLY, MSigDB3.030.004229, 191, 349, 22474RTK-ACTER driven by Growth Factors, PMID: 208897186.771.74E-059, 86, 349, 22585RTK-ACTMEK_UP.V1_UP (MAPK), MSigDB10.95.72E-1827, 169, 331, 22496RTK-ACTRAS ONCOGENE (MAPK), PMID: 162730928.445.77E-1220, 158, 338, 22553RTK-ACTWU_CELL_MIGRATION, PMID 187243907.648.96E-1119, 165, 339, 22502RTK-ACTHUPER_BREAST_BASAL_VS_LUMINAL_UP, PMID 17409405131.38E-079, 45, 349, 22621RTK-ACTHALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION, MSigDB3.770.00031411, 189, 347, 22477RTK-ACTLIM_MAMMARY_STEM_CELL_UP, PMID 203461513.119.56E-0622, 467, 336, 22205RTK-ACTRTK-ACT.iv -4.719.42E-2060, 940, 298, 21992In MBC biopsies, compare RTK mutations with WT, rank genes based on the logFC - top 1000RTK-ACT.iv (in-vivo)HALLMARK_ESTROGEN_RESPONSE_EARLY, MSigDB1.950.020316, 184, 984, 22088RTK-ACT.ivER driven by Growth Factors, PMID: 208897182.640.0076310, 85, 990, 22193RTK-ACT.ivMEK_UP.V1_UP (MAPK), MSigDB2.863.91E-0522, 174, 978, 22098RTK-ACT.ivRAS ONCOGENE (MAPK), PMID: 162730921.620.13212, 166, 988, 22152RTK-ACT.ivWU_CELL_MIGRATION, PMID 187243903.573.60E-0725, 159, 975, 22115RTK-ACT.ivHUPER_BREAST_BASAL_VS_LUMINAL_UP, PMID 174094059.515.43E-1016, 38, 984, 22235RTK-ACT.ivHALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION, MSigDB2.090.0073817, 183, 983, 22090RTK-ACT.ivLIM_MAMMARY_STEM_CELL_UP, PMID 203461511.420.089229, 460, 971, 21819HER2-ACTHER2-ACT - Fulv3006.53E-183109, 91, 91, 22750HER2-ACTHER2-ACT - Palbo2331.46E-163101, 99, 99, 22749HER2-ACTHER2.ACT - Fulv + Palbo2197.64E-15999, 101, 101, 22742HER2-ACTHER2.ACT - Neratinib63.19.78E-7257, 143, 143, 22707HER2-ACTHER2.ACT - Fulv + Neratinib59.33.53E-6855, 145, 145, 22724FGFR-ACTFGFR.ACT - Palbo19003.2e-319157, 43, 43, 22778FGFR-ACTFGFR.ACT - Fulv + Palbo12501.38E-290148, 52, 52, 22767FGFR-ACTFGFR.ACT - Fulv8661.72E-266140, 60, 60, 22764FGFR-ACTFGFR.ACT - Palbo + FIIN.31062.88E-10474, 126, 126, 22710FGFR-ACTFGFR.ACT - Fulv + Palbo + FIIN.378.41.14E-8464, 136, 136, 22705FGFR-ACTFGFR.ACT - FIIN.367.42.16E-7559, 141, 141, 22730FGFR-ACTFGFR.ACT - Fulv + FIIN.336.89.47E-4541, 159, 159, 22733 Citation Format: Ofir Cohen, Pingping Mao, Utthara Nayar, Jorge E Buendia-Bue, Dewey Kim, Esha Jain, Karla Helvie, Daniel Abravanel, Kailey J Kowalski, Christian Kapstad, Samuel Freeman, Victor Adalsteinsson, Seth A Wander, Adrienne G Waks, Gad Getz, Aviv Regev, Eric Winer P Winer, Nancy U Nancy U. Lin, Nikhil Wagle. Acquired activating mutations in RTKs confer endocrine resistance in ER+ metastatic breast cancer through ER-reprogramming, MAPK signaling, and an induced stem-like cell state [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS2-02.

In estrogen-receptor positive (ER+) breast cancer, therapies that target the ER have led to reductions in recurrence and mortality. However, in the metastatic setting, resistance to ER-targeted therapies is near universal. Various resistance mechanisms have been proposed, including ESR1 activating mutations and upregulation of alternative signal transduction pathways, yet clinically relevant resistance mechanisms have not been clearly defined. We conducted a genome-scale gain-of-function screen in ER+ breast cancer cell lines, spanning 17,255 overexpressed open reading frames (ORFs), to investigate genes whose overexpression is sufficient to confer resistance to selective estrogen receptor degraders (SERDs). Among several validated resistance genes, the fibroblast growth factor receptor (FGF/FGFR) pathway was among the top pathways, with several FGF ligands scoring as top resistance genes in this screen. In parallel, we performed whole exome sequencing (WES) in paired pre-treatment and post-resistance biopsies from 60 patients with ER+ metastatic breast cancer (MBC) who had developed resistance to ER-targeted therapy. The FGFR pathway was altered in 40% (24 of 60) of the post-resistance biopsies; alterations included FGFR1 amplifications (9 patients), FGFR2 amplifications and activating mutations (4 patients), and FGF3 amplifications (17 patients). In 12 out of the 24 cases (5 FGFR1, 4 FGFR2, 3 FGF3), the FGF/FGFR pathway alterations were present in the resistant tumors and not detected in the pre-treatment tumors, suggesting that these alterations were acquired under the selective pressure of ER-directed therapy. Overexpression of wildtype FGFR1, FGFR2, FGF3 in ER+ breast cancer cells (T47D and MCF7) led to resistance to fulvestrant as well as the combination of fulvestrant and the CDK4/6 inhibitor palbociclib. This resistance phenotype was reversed by the FGFR inhibitor PD173074, and, to a lesser extent, the MEK inhibitor trametinib and mTOR inhibitor everolimus, suggesting several potential treatment strategies. The three FGFR2 mutations (M538I, N550K and K660N) identified in these patients induced hyperactive FGFR signaling and conferred resistance to fulvestrant, which was abrogated by the irreversible FGFR inhibitors FIIN-2 and FIIN-3. Together, these results offer new insights into endocrine resistance and suggest new clinical approaches to overcome or preempt therapeutic resistance. Citation Format: Pingping Mao, Ofir Cohen, Kailey Kowalski, Justin Kusiel, Jorge Buendia, Pedro Exman, Seth A. Wander, Adrienne G. Waks, Eric P. Winer, Nancy U. Lin, Nikhil Wagle. Acquired FGF and FGFR alterations confer resistance to estrogen receptor (ER) targeted therapy in metastatic ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 314.

Background: While great strides have been made in the treatment of ER+ MBC, therapeutic resistance is nearly universal. The genomic evolution of ER+ breast cancer in the metastatic setting under the selective pressure of multiple lines of therapies is not well understood. To address this, we analyzed the clonal dynamics of serial metastatic samples (mets) to evaluate how tumors evolve and to identify acquired resistance mechanisms. Methods: We performed WES on 462 clinically annotated samples from 208 patients (pts) with ER+ MBC, including 67 primary tumor biopsies, 229 metastatic biopsies and 160 blood samples (cfDNA). Pts with multiple mets included cases with temporally concordant metastatic tumor and blood samples (48 pts) and cases with serial mets obtained over the course of treatment in the metastatic setting (69 pts). Treatments given between the serial mets included CDK4/6 inhibitors (23 pts), and selective estrogen receptor degraders (19 pts), among others. Results: In the temporally-concordant mets, we found that cfDNA mutations (muts) largely overlap with muts found in tumor biopsies, capturing >85% of clonal tumor muts. However, we observed a higher level of heterogeneity in cfDNA compared to biopsies (p.value< 1.05e-19, Welch test) and a subset of high-confidence muts that were only detected in cfDNA, including in clinically important genes such as ESR1, PIK3CA, KRAS, and ERBB2. Analysis of serial mets was used to elucidate the evolutionary dynamics within the metastatic setting under the selective pressure of treatment. The median duration between mets was 112 days and the median number of inter-biopsy unique treatments was two. Most tumors continued to evolve within the metastatic setting, with 50 out of 69 pts (72%) acquiring a meaningful sub-clone (50% increase in relative cancer cell fraction) and 31 out of 69 (45%) acquiring muts in known cancer genes, including a subset acquiring a plausible resistance alteration such as alterations that dysregulate ER (5 out of 69 pts, 7%; ESR1 mut, FOXA1 amplification (amp), NCOR1 bi-allelic deletion (del)), ERBB (4%; ERBB2 amp, ERBB3 mut), RAS (4%; KRAS mut, NRAS amp, NF1 del), FGF/FGFR (12%; FGFR2 mut, FGFR1/2 amp, FGF3 amp), and cell cycle (13%; RB1 del, CDK4 amp, AURKA amp, CDKN2A del). Finally, in pts who had multiple mets, we observed several cases of evolutionary convergence toward equivalent resistance mechanisms including convergent RB1 loss as a mechanism of resistance to a CDK4/6 inhibitor and convergent BRCA2 reversion following resistance to a PARP inhibitor. Conclusions: This study demonstrates that ER+ MBC continues to evolve under the selective pressure of treatments in the metastatic setting. These findings elucidate the challenge of studying high complexity and heavily treated tumors, while also highlighting some commonalities in the evolutionary trajectories selected by these treatments. The multiplicity of clinically relevant genomic alterations acquired in these advanced stages highlights the need for serial biopsies and the potential to inform post-progression therapeutic choices through targeting the acquired dependencies in post-progression tumors. Citation Format: Cohen O, Buendia-Buendia J, Wander S, Nayar U, Mao P, Waks A, Kim D, Freeman S, Adalsteinsson V, Helvie K, Livitz D, Rosebrock D, Leshchiner I, Dellostritto L, Garrido-Castro A, Jain E, Periyasamy S, Mackichan C, Lloyd M, Marini L, Krop I, Garraway L, Getz G, Winer E, Lin N, Wagle N. Evolutionary analysis of 462 serial metastatic biopsies from 208 patients with estrogen receptor-positive (ER+) metastatic breast cancer (MBC) using whole exome sequencing (WES) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-02.

Despite the reduction in cancer recurrence and mortality provided by therapies that target the estrogen receptor (ER), resistant ER+ breast cancer remains the most common cause of breast cancer death. Beyond mutations in ER in 25-30% of patients treated with aromatase inhibitors, which deplete circulating estrogen, our understanding of clinical mechanisms of resistance to agents that target the ER remains incomplete. We used whole exome sequencing (WES) of metastatic biopsies to identify mechanisms of resistance in patients with ER+ metastatic breast cancer (MBC) who had developed resistance to ER-directed agents, including aromatase inhibitors, tamoxifen, and fulvestrant. We noted mutations in human epidermal growth factor receptor 2 (HER2) in metastatic biopsies from 12 patients out of 168. Of these, eight mutations had previously been described as activating. Examination of the treatment-naïve primary tumors in the five patients with activating mutants where WES from the matched treatment-naïve primary tumor was available revealed no evidence of pre-existing mutations in four of the five patients, suggesting that these four mutations were acquired under the selective pressure of ER-directed therapy. These acquired HER2 mutations were mutually exclusive with ER mutations, suggesting a distinct mechanism of resistance to ER-directed therapies. In vitro analysis through expression in ER+/HER2- cell lines confirmed that these mutations conferred estrogen independence. In addition, and in contrast to ER mutations, these HER2 mutations resulted in resistance to tamoxifen, fulvestrant, and the CDK4/6 inhibitor palbociclib. The mutants result in increased AKT and ERK phosphorylation in ER+ cell lines, while simultaneously repressing ER levels and ER-dependent transcriptional targets. One mutation observed in two patients, S653C, occurring in the transmembrane domain and not previously observed in breast cancer, was shown to likely function through constitutive dimerization, a mechanism of activation not previously described for this mutation. Resistance caused by all four mutations was overcome by combining ER-directed therapy with the irreversible HER2 kinase inhibitor neratinib, suggesting a novel effective treatment strategy in these patients. Thus, we have shown that acquired activating HER2 mutations can confer endocrine resistance in ER+/HER2- breast cancer, and that such resistance can be effectively reversed by combination therapies that include an anti-HER2 inhibitor. Citation Format: Utthara Nayar, Ofir Cohen, Christian Kapstad, Adrienne Waks, Seth A. Wander, Corrie Painter, Samuel Freeman, Priyanka Ram, Nicole Persky, Lori Marini, Karla Helvie, Nelly Oliver, Cynthia X. Ma, Eric P. Winer, Nancy U. Lin, Nikhil Wagle. Acquired HER2 mutations in ER+ metastatic breast cancer confer resistance to ER-directed therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4952.

Consequences of the obesity epidemic on cancer morbidity and mortality are not fully appreciated. Obesity is a risk factor for many cancers, but the mechanisms by which it contributes to cancer development and patient outcome have yet to be fully elucidated. Here, we examined the effects of coculturing human-derived adipocytes with established and primary breast cancer cells on tumorigenic potential. We found that the interaction between adipocytes and cancer cells increased the secretion of proinflammatory cytokines. Prolonged culture of cancer cells with adipocytes or cytokines increased the proportion of mammosphere-forming cells and of cells expressing stem-like markers in vitro. Furthermore, contact with immature adipocytes increased the abundance of cancer cells with tumor-forming and metastatic potential in vivo. Mechanistic investigations demonstrated that cancer cells cultured with immature adipocytes or cytokines activated Src, thus promoting Sox2, c-Myc, and Nanog upregulation. Moreover, Sox2-dependent induction of miR-302b further stimulated cMYC and SOX2 expression and potentiated the cytokine-induced cancer stem cell-like properties. Finally, we found that Src inhibitors decreased cytokine production after coculture, indicating that Src is not only activated by adipocyte or cytokine exposures, but is also required to sustain cytokine induction. These data support a model in which cancer cell invasion into local fat would establish feed-forward loops to activate Src, maintain proinflammatory cytokine production, and increase tumor-initiating cell abundance and metastatic progression. Collectively, our findings reveal new insights underlying increased breast cancer mortality in obese individuals and provide a novel preclinical rationale to test the efficacy of Src inhibitors for breast cancer treatment. Citation Format: Picon-Ruiz M, Pan C, Drewes-Elger K, Jang K, Besser A, Zaho D, Morata-Tarifa C, Kim M, Ince TA, Azzam D, Wander S, Cote RJ, Guy HA, El-Ashry D, Torne-Poyatos P, Marchal JA, Slingerland JM. Interactions between adipocytes and breast cancer cells stimulate cytokine production and drive Src/SOX2/miR-302b mediated malignant progression [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-05-01.

p27 is a cell cycle regulator that acts largely to restrain normal cell growth. It is rarely mutated or deleted in human cancers, but is often degraded or mislocalized to the cytoplasm in aggressive tumors. Phosphorylation at T157 or T198 by different PI3K effector kinases leads to p27 cytoplasmic accumulation and enhanced cell motility and invasion in part via effects on the actin cytoskeleton, which are independent of its cell cycle role. However, the functional contributions of C-terminally phosphorylated, cytoplasmic p27 to cancer progression remain poorly understood. Targeted inhibition of PI3K/mTOR impaired tumor cell motility and metastasis via modulation of p27 in a bone metastatic model (Wander, S. et al. BCRT 2013). We observed that p27 knockdown in highly metastatic cancer lines with high levels of cytoplasmic p27pT157pT198 reverted EMT and impaired tumor cell invasion in vitro and metastasis in vivo. This led us to investigate whether PI3K-activated, phosphorylated p27 may function as a novel EMT regulator. A cell cycle defective (CK-) and double phosphomimetic p27 mutant (T157D/T198D or DD) was introduced into immortal human mammary epithelial cells with low PI3K activity. This revealed a novel, oncogenic function of p27 in regulating tumor progression, in that p27CK-DD induced epithelial-mesenchymal transition (EMT) and soft agar colony formation. p27CK-DD also increased motility, invasion and formation of metastasis in bladder and breast cancer models. A RT-PCR screen for potential p27-induced EMT mediators revealed a 20-fold increase in expression of the Twist1 transcription factor in p27CK-DD-transduced cells. Knockdown of Twist reversed the p27CK-DD-mediated increase in EMT marker expression. p27 knockdown rapidly attenuated Twist1-promoter activity and reduced Twist1 mRNA levels and also increased E-cadherin expression in metastatic cells, suggesting that Twist1 may play a critical role in p27CK-DD-induced EMT. These data extend our understanding of p27 function in human cancer and suggest that PI3K deregulated p27 may promote EMT and tumor metastasis through transcriptional activation of TWIST1. Citation Format: Dekuang Zhao, Alexandra H. Besser, Wen Zhou, Seth A. Wander, Jun Sun, Michael Durante, Feng Hong, Bin Wang, Tan Ince, Karoline Briegel, Joyce M. Slingerland. Cytoplasmic p27 promotes epithelial-mesenchymal transition and tumor progression via Twist1 upregulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1145. doi:10.1158/1538-7445.AM2014-1145

The pleiotropic oncogenic effects of PI3K/mTOR have stimulated development of catalytic PI3K and mTOR inhibitors for the treatment of cancer. Present data provide a novel rationale for the use of dual PI3K/mTOR inhibitors to oppose tumor motility, invasion and metastasis. Cytoplasmic mislocalization of p27 is implicated as a key downstream driver of PI3K/mTOR-dependent tumor cell motility in vitro and bone metastatic outgrowth in vivo. Here, we report that PI3K/mTOR-kinase inhibition with PF-04691502 impairs breast cancer metastasis to bone, in part via effects on p27. Low-dose PF-04691502 impaired motility and invasion of highly PI3K/mTOR activated, bone-metastatic MDA-MB-1833 cells in vitro without affecting apoptosis or proliferation. Moreover, drug pre-treatment impaired bone metastasis in vivo. PF-04691502 decreased cytoplasmic p27pT157 and p27pT198, and p27T157D/T198D overexpression conferred resistance to inhibition of motility by PF-04691502. p27 knockdown in MDA-MB-1833 relieved RhoA-ROCK inhibition, decreased cell motility/invasion in vitro, and bone metastasis in vivo. p27 knockdown also reversed EMT phenotypic markers in MDA-MB-1833, as did PI3K/mTOR inhibition, demonstrating that p27 may be critical for maintenance of the EMT cellular program. Indeed, loss of cytoplasmic p27 reversed expression of an EMT gene profile supporting the notion that p27 acts as a key mediator of metastasis downstream of PI3K/mTOR. Cytoplasmic p27 in human cancers may prove to be a useful predictive marker to assess both PI3K/mTOR activation and the potential efficacy of catalytic PI3K/mTOR inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2224. doi:1538-7445.AM2012-2224

CD44 surface expression is associated with self renewal in several cancers. While CD44+/CD24neg/low/ESA+ breast cancer subpopulations are enriched for cancer stem cells (CSC), the relative contributions of CD24 negative versus low subpopulations to stemness and metastasis is poorly defined. Here we show that CD44+/CD24low+ CSC give rise to CD44+/CD24neg progeny with reduced tumorigenicity and altered drug sensitivities. CD44+/CD24low+ subpopulations in MDA-MB-231 and in primary dissociated ER negative breast cancers show greater sphere and soft agar colony formation, Notch1 activation, Sox2 and Nanog expression and chemo resistance compared to CD44+/CD24neg. CD44+/CD24low+ can self-renew and give rise to CD44+/CD24neg cells, while CD44+/CD24neg progeny are exclusively CD44+/CD24neg. Tumorigenicity was increased and metastasis arose exclusively from orthotopic CD44+CD24low+ xenografts. CD44+/CD24low+ had greater expression of metastasis- and embryonic stem cell- associated and Notch pathway activated genes than CD44+/CD24neg cells. Moreover, MDA-MB-231 cells overexpressing Notch1 intracellular domain (N1-ICD) had higher Sox2 expression, and higher indices of CSC self-renewal (higher % CD44+/CD24low+, ALDH1 activity, sphere and soft agar colony formation), all of which were abrogated by Sox2 knockdown. Gamma secretase inhibition reduced ES-TFs and self-renewal in CD44+CD24low+ progenitors, but had no effect on proliferation or survival of CD44+CD24neg cells supporting further clinical development of Notch targeting drugs for cancer treatment in humans. Thus, CD44+CD24low+ and CD44+CD24neg CSC in ER negative breast cancer have distinct properties. CD44+CD24low+ can self-renew and generate CD44+CD24neg progeny. Orthotopic xenograft CD44+CD24low+ tumors arose with reduced latency with higher frequent, were larger, and the sole source of metastasis. Notch1 activation of Sox2 drives CD44+CD24low+ self-renewal and is blocked by GSI providing a strong rationale for use of GSI as CSC targeting agents in ER negative breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3478. doi:1538-7445.AM2012-3478

While mutational loss of the p27 cell cycle regulatory protein is rare, oncogenic deregulation of p27 via accelerated degradation or mislocalization to the cytoplasm is commonly observed in human tumors. A growing body of evidence indicates that, when mislocalized to the cytoplasm, p27 promotes cell motility via RhoA-ROCK signaling inhibition. The present data explores the role of p27 in the regulation of both orthotopic mammary tumor growth and ectopic pulmonary metastasis in the MDA-MB-231-4175 model of malignant human breast cancer. The MDA-MB-231-4175 cell line (hereafter 4175) shows much greater metastasis to the lungs in murine xenograft models. Relative to the parental MDA-MB-231 cells, the metastatic 4175 derivative demonstrates activation of PI3K/mTOR signaling. These cells have elevated pmTOR, pAKTS473, pSGK1, and pRSK1. This results in increased total p27 and, when equal levels of total p27 are titrated, the highly metastatic 4175 cell line demonstrates extensive phosphorylation of p27 at the T157 and T198 sites. These sites have previously been implicated in the modulation of p27 localization and in the binding of p27 to RhoA. Thus, in the 4175 line, we demonstrate increased cytoplasmic p27, increased p27:RhoA binding, and impaired RhoA-ROCK signaling. This attenuation of RhoA activity correlates with increased migration (as measured by scratch assay) and increased invasion (as measured by modified transwell assay). Importantly, this hypermotility was abrogated by lentiviral knockdown of p27. Knockdown of p27 did not alter in vitro cell cycle profiles of either cell line as assessed by flow cytometry. The enhanced pulmonary tropism of the 4175 cell line was also abrogated in vivo following p27 knockdown: shRNAp27 reverts the lung metastatic phenotype of these cells back toward parental levels following tail vein injection in Balb/c nude female mice as assessed by IVIS imaging. Interestingly, orthotopic injection of these same lines, treated with the same lentivirus, into the mammary fat pad of Balb/c nude female mice produced a dramatically different result: shRNAp27 resulted in a significant increase in parental MDA-MB-231 tumor growth, while the metastatic derivative 4175 cells did not demonstrate a statistically meaningful change in orthotopic growth. These results indicate that, within the same cellular background, and in the same murine species, loss of p27 results in substantially different phenotypes depending upon the microenvironmental context: after tail vein injection cytoplasmic p27 plays a crucial role in promoting extravasation and colonization in the lung tissue; loss of p27 in this context impairs the metastatic process. Conversely, in the orthotopic environment, p27's pro-invasive role is not necessary to establish productive microenvironmental interactions and loss of p27 does not attenuate tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2333. doi:10.1158/1538-7445.AM2011-2333

Primary human breast cancer subpopulations with a CD44+ CD24NEG/LOW ESA+ phenotype exhibit self-renewal and generate xenograft tumors with as few as 100 cells in immunodeficient mice. While CD44 expression is strongly associated with self renewal in several cancers, the relative contributions of CD24 negative versus low subpopulations to stemness is poorly defined. Early reports have not distinguished negative from low CD24 expression in human lines and primary tumors, and both, together with CD44+ status have been linked to breast cancer stem cell phenotypes. In contrast, certain reports have associated CD24 expression with tumor progression and metastasis. Here, we show increased mitogenic kinase activation, expression of ESC and EMT genes in the CD24LOW subpopulation of CD44+ cells in both triple negative MDA-MB-231 breast cancer cells and primary dissociated breast cancers (DTs) compared to the CD24NEG cells. Moreover, tumorigenicity was increased and metastasis arose exclusively from orthotopic xeno-implantation of the CD44+CD24LOW cells. MDA-MB-231 and two different primary DTs were live sorted into CD44+CD24NEG and CD44+CD24LOW enriched subpopulations. Cells expressing other putative stem cell markers, ALDH1 and ESA were almost entirely CD44+CD24LOW. CD44+CD24LOWcells showed higher PI3-kinase/Akt, MAPK, and Src activites compared to CD44+ CD24NEG cells. While both subpopulations formed soft agar colonies and/or mammospheres, those arising from CD44+CD24LOW were greater in number and size than from CD44+CD24NEG cells. When cultured in 2D after sorting, CD44+ CD24LOW cells gave rise to both CD44+ CD24LOW and CD44+ CD24NEG progeny while CD44+ CD24NEG yielded only CD44+ CD24NEG progeny. CD44+CD24LOW expressed higher levels of ESC genes and EMT-associated genes than CD44+CD24NEG cells and uniquely expressed stem cell-associated miRNAs. On orthotopic injection into BalbC nude mice, both subpopulations initiated tumors with as few as 100 cells, however tumors arising from CD44+CD24LOW cells had a shorter latency and grew more rapidly than those arising from CD44+CD24NEG cells. In vivo imaging of luciferase positive cells showed CD44+CD24LOW cells yield metastasis while CD44+CD24NEG cells did not. Our data demonstrate, for the first time, distinct biological properties between the CD44+CD24LOW and CD44+CD24NEG enriched subpopulations of MDA-MB-231 and primary human breast cancer DTs. Low surface expression of CD24 is associated with ALDH1 and ESA+ status, increased self-renewal as shown by mammosphere and colony formation, tumorigenicity and metastatic potential of CD44+ cells. The ability to isolate and characterize such TSC-enriched subpopulations will be necessary for development of therapeutic strategies that preferentially target breast cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3335. doi:10.1158/1538-7445.AM2011-3335

The CDK inhibitor p27 plays a well-established role in cell cycle progression, regulating the G1-S phase transition. A growing body of evidence indicates a potential role for p27 as a mediator of tumor cell motility and invasiveness. The present data provides additional evidence for p27 as a mediator of cancer metastasis. These activities appear to require cytoplasmic mislocalization, and are independent of p27's action on CDKs and the cell cycle. p27 phosphorylation at T157 and T198 is mediated by the AGC kinases RSK, SGK, and AKT to promote cytoplasmic mislocalization and facilitate p27 binding to the RhoA cytoskeletal regulatory protein. p27-mediated inhibition of RhoA/ROCK signaling is associated with dynamic rearrangement of the actin cytoskeleton and increased cell migration and invasion. Overexpression of the PI3K effectors RSK, SGK, and mTOR all promote cytoplasmic mislocalization of p27, increased p27: RhoA binding, and all enhance directed cell migration as assayed by modified transwell assay. This increased tumor cell motility is dependent upon p27, as shRNA-mediated p27 knockdown reverts transwell invasion to parental levels. Overexpression of mTOR or these AGC kinases reduces cellular levels of RhoA-GTP and p-cofilin, both of which revert following shRNA p27 treatment. The increased p27: RhoA binding, decreased RhoA-GTP, and decreased p-cofilin correlate with dramatic loss of polymerized actin filaments as assayed by phalloidin staining, and all of these effects are rescued by shRNA p27. The in vivo relevance of these findings has been pursued in the highly metastatic human breast cancer sub-lines MDA-MB-231-1833 (bone-tropic) and MDA-MB-231-4175 (lung-tropic). Both highly metastatic variants demonstrate hyperactivation of the PI3K effectors SGK, RSK, and AKT relative to the parental MDA-MB-231 line and show markedly elevated p27 levels, with a shift in p27 protein localization to the cytoplasm, increased p27: RhoA binding, decreased p27-dependent levels of RhoA-GTP and p-cofilin, and increased p27-dependent invasiveness by modified transwell assay. The cell cycle profiles of MDA-MB-231-4175 and 1833 variants were unchanged by shRNA p27 supporting the notion that cytoskeletal effects of p27 in these lines are independent of cell cycle/Cdk regulatory activity. Of particular interest, the increase in lung tumor formation by MDA-MB-231-4175 following tail vein injection was strikingly reduced to near-parental levels after shRNA-mediated knockdown of p27. These results support a model whereby PI3K activation promotes p27 phosphorylation, mislocalization to the cytoplasm, enhanced RhoA binding and subsequent remodeling of the actin cytoskeleton. This cascade of events may be a major route whereby oncogenic PI3K activation mediates tumor cell acquisition of the invasive potential necessary for overt metastasis in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5137.