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Phosphatidylinositol 3-kinase (PI3K) pathway plays a key role in resistance to the drugs targeting human epidermal growth factor receptor 2 (HER2). Activating mutations in the gene encoding alpha catalytic subunit of PI3K (PIK3CA) are present in approximately 30% of HER2+ tumors. PIK3CA mutations are linked to drug resistance and decreased survival in patients with HER2+ breast cancer. To overcome this resistance mechanism, we designed a phase IB/II clinical trial to evaluate the combination of HER2 small molecule inhibitor tucatinib with PI3K inhibitor alpelisib in patients with HER2+ metastatic breast cancer (NCT05230810). This multicenter clinical trial is conducted through the Academic Breast Cancer Consortium (ABRCC), with the University of Colorado Cancer Center as the lead site. Target enrollment: 40 patients. This is a run-in phase IB/roll-over phase II study. Phase IB will follow Time-to-Event Bayesian Optimal Interval design and enroll from 9 to 19 patients to find the maximum tolerated doses (MTDs) of tucatinib and alpelisib. From 21 to 31 patients will be enrolled in phase II part, for a total of 40 patients in the final efficacy analysis. Main inclusion criteria: 1. Women and men ≥ 18 years old 2. Eastern Cooperative Oncology Group (ECOG) performance status 0-1 3. Presence of activating PIK3CA mutation in the tumor 4. Patients with HR-/HER2+ or HR+/HER2+ breast cancer may enroll; ovarian suppression is mandatory for premenopausal patients with HR+/HER2+ disease 5. HR+/HER2+ patients should be agreeable to concomitant treatment with fulvestrant 6. Prior treatment with at least two FDA-approved HER2-targeted agents 7. Measurable or evaluable disease. Bone only disease is allowed. 8. Subjects with untreated central nervous system (CNS) metastases not needing immediate local therapy, and subjects with previously treated stable or progressive brain metastases may enroll, provided that there is no indication for immediate re-treatment. For patients with treated CNS metastases: time from treatment of CNS disease until the first dose of study drugs should be as follows: WBRT ≥ 21 days, surgical resection ≥ 14 days, SRS ≥ 7 days. 9. Adequate organ and marrow function Main exclusion criteria: 1. Contraindications to undergo contrast brain MRI 2. Leptomeningeal disease 3. Poorly controlled seizures 4. Diabetes mellitus type I, or uncontrolled diabetes mellitus type II 5. Acute pancreatitis within 1 year of screening, or history of chronic pancreatitis 6. History of severe cutaneous hypersensitivity reactions 7. Toxicities of prior cancer therapies that have not resolved to grade 1 or less, except peripheral neuropathy, which must have resolved to grade 2 or less, and alopecia 8. Previous treatment with EGFR or HER2 tyrosine kinase inhibitors, or PI3K/mTOR/AKT inhibitors. 9. Systemic anti-cancer therapy, palliative radiation to extracranial sites, or surgery within 2 weeks of the first dose of study drugs 10. Active bacterial, fungal, or viral infections, hepatitis B, C, or HIV 11. Clinically significant cardio-vascular disease Primary objectives: • Phase IB: safety and tolerability of combination therapy • Phase II: efficacy by progression free survival Exploratory assessment of biomarkers will be performed in the liquid biopsy samples. Study contact: Elena Shagisultanova, MD, PhD, elena.shagisultanova@cuanschutz.edu Citation Format: Elena Shagisultanova, Kari B. Wisinski, Chelsea D. Gawryletz, Farrah M. Datko, Diana Medgyesy, Jennifer R. Diamond, Virginia F. Borges, Peter Kabos. Clinical Trial of Alpelisib and Tucatinib in Patients with PIK3CA-Mutant HER2-Positive 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 OT3-09-01.

Background: Triple negative breast cancer (TNBC) represents the most aggressive breast cancer subtype, and despite recent treatment advances, clinical outcomes remain poor, particularly in the metastatic setting. TNBC is a biologically heterogeneous entity, and up to 50% of TNBC express the androgen receptor (AR). Gene expression subtyping has identified a subset of AR-TNBC with a luminal AR-driven (LAR) phenotype. Consequently, there has been great interest in translating androgen receptor signaling inhibitors (ARSIs) approved for use in prostate cancer such as enzalutamide and bicalutamide into the management of LAR-TNBC. However, early phase clinical trials of anti-androgens in metastatic AR-TNBC have had modest results. While multiple mechanisms of anti-androgen resistance have been identified in prostate cancer, including AR amplification and over-expression, emergence of AR splice variants (AR-Vs), and transition to AR independent growth, molecular determinants of anti-androgen resistance in TNBC remain poorly understood, and pre-clinical models to study acquired ARSI resistance in LAR-TNBC have been limited. Methods: A novel pre-clincial model of acquired ARSI resistance was derived from the LAR subtype MDA-MB-453 cell line through serial passaging with increasing concentrations of enzalutamide. Celltiter Blue viability assays were used to determine enzalutamide IC50 of parental and enzalutamide-resistant (EnzR) cell lines. RNA was extracted from the parental and resistant cells, reverse transcribed and expression of AR, splice-variants and canonical AR target genes were evaluated using RT-qPCR. Immunofluorescence was used to investigate the amount and nuclear localization of the AR protein within the parental and enzalutamide resistant cells. Results: EnzR MDA-MB-453 cells were derived through culture in increasing concentrations of enzalutamide for >6 months, and found to have a significant increase in enzalutamide IC50 compared to the parental cell line. EnzR MDA-MB-453 cells had a 4.5 fold increase in full-length AR expression at the transcriptional level compared to the parental cell line. AR protein expression and nuclear localization were also increased in EnzR cells compared to the parental cell line. Canonical AR targets including NKX3.1 were downregulated in response to enzalutamide in parental but not EnzR cells. While pathogenic AR splice variants (AR-Vs) implicated in ARSI resistance in prostate cancer were detected at low levels in MDA-MB-453 cells, no increase in AR-V expression was observed in the EnzR cell line. Conclusions: We have developed a novel pre-clinical model of anti-androgen/ARSI resistance in LAR-TNBC, and identified AR overexpression and persistent AR signaling associated with acquired enzalutamide resistance in LAR-TNBC. While pathogenic AR splice variants have been implicated in ARSI resistance in metastatic prostate cancer and are detected in the MDA-MB-453 cell line model, enzalutamide resistance was not associated with increased AR splice variant expression in this model. Future work will investigate mechanisms leading to AR overexpression as well as combination therapeutic strategies to overcome acquired ARSI resistance. Citation Format: Hannah Krause, Marina N. Sharifi, Serena K. Wolfe, Jamie M. Sperger, Kari B. Wisinski, Ruth O’Regan, Joshua Lang. Increased androgen receptor expression as a mechanism of acquired anti-androgen resistance in androgen receptor-positive triple negative breast cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-14.

Background: Despite evidence of substantial breast cancer risk reduction, few high-risk women adopt chemopreventive medications such as selective estrogen receptor modulators (SERMs) or aromatase inhibitors (AIs). Women with benign breast disease, such as atypical hyperplasia (AH) or lobular carcinoma in situ (LCIS), have an increased risk of developing breast cancer and derive a greater benefit from antiestrogens compared to other high-risk women. Reasons for low uptake of chemoprevention include insufficient patient and clinician knowledge about antiestrogens, time constraints during the clinical encounter, and concerns about side effects. To address these barriers, we have developed patient and provider web-based decision support tools to improve informed choice about breast cancer chemoprevention among women with AH or LCIS. Study design: We are conducting a cluster-randomized controlled trial of clinical decision support to improve chemoprevention informed choice among women with AH or LCIS and their treating providers. Twenty-six U.S. sites through the SWOG Cancer Research Network were randomly assigned 1:1 to standard educational materials alone or in combination with the patient-centered decision aid (RealRisks) and provider decision support tool (BNAV). A total of 415 patients and 200 healthcare providers will be recruited from these sites. RealRisks consists of interactive modules to calculate personalized breast cancer risk and elicit preferences on chemoprevention. The modules are available in English and Spanish. BNAV is comprised of self-directed case-based learning modules on breast cancer risk assessment and chemoprevention. Patients complete questionnaires at baseline, 6 and 12 months. Providers complete surveys at baseline and after their enrolled patient’s 6-month clinical encounter. The primary endpoint is chemoprevention informed choice at 6 months, using a measure combining knowledge, attitude, and intention scales. Secondary endpoints include perceived breast cancer risk/worry, chemoprevention knowledge/intention, decision conflict/regret, shared decision-making, and chemoprevention uptake. For patients who begin chemoprevention, adherence and reasons for discontinuation are assessed annually for up to 5 years. Barriers and facilitators to implementing RealRisks and BNAV into clinic workflow will be assessed by conducting patient and provider interviews at baseline and mid-implementation. Eligibility criteria: Eligible patients include women, age 35-74 years, with AH or LCIS, no history of breast cancer, no prior use of SERMs or AIs, no bilateral mastectomies, English or Spanish-speaking, and access to the internet. Eligible providers include breast surgeons, medical oncologists, primary care providers, and physician extenders who see patients with AH or LCIS. Statistical methods: We have 90% power to detect a 15% increase in the frequency of chemoprevention informed choice with a 1-sided 0.025 level test, assuming an intraclass correlation (ICC) of 0.02 to account for clustering, roughly equal accrual at each site, 10% loss to follow-up, and ≤10% event rate in the control arm. Current/target accrual: The trial was activated on 9/1/2020. As of 7/7/2022, all 26 sites have been randomized, 157/200 providers and 184/415 patients have been enrolled. Discussion: Our hybrid effectiveness/implementation study seeks to evaluate the effectiveness of a multi-level intervention in promoting informed decision-making about breast cancer chemoprevention. Study results will provide valuable insights on how the decision support tools are integrated in diverse clinical settings. Citation Format: Katherine D. Crew, Garnet Anderson, Kathryn Arnold, Andrew Stieb, Jacquelyn N. Amenta, Cynthia Law, Ana Sandoval-Leon, Sarah Colonna, Tari King, Debra Mangino, Sandhya Pruthi, Maria Grosse Perdekamp, Christa Braun-Inglis, Stacy Krisher, Lisa Yee, Danielle Bertoni, Samantha Seaward, Kari B. Wisinski, Justin Floyd, Corrine Zarwan, Tarah J. Ballinger, Lindi VanderWalde, Masey M. Ross, Preston Steen, Shelly Lo, Alison Conlin, Kathleen Yost, John Ellerton, Erin Lin, Holly J. Pederson, Sagar Sardesai, Cheryl Jernigan, Dawn Hershman, Marian L. Neuhouser, Banu K. Arun, Rita Kukafka. SWOG 1904: Cluster-randomized controlled trial of patient and provider decision support to increase chemoprevention informed choice among women with atypical hyperplasia or lobular carcinoma in situ (MiCHOICE) [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 OT1-15-01.

PURPOSE National Cancer Institute–Molecular Analysis for Therapy Choice is a multicohort trial that assigns patients with advanced cancers to targeted therapies on the basis of central tumor genomic testing. Arm B evaluated afatinib, an ErbB family tyrosine kinase inhibitor, in patients with ERBB2-activating mutations. METHODS Eligible patients had selected ERBB2 single-nucleotide variants or insertions/deletions detected by the National Cancer Institute–Molecular Analysis for Therapy Choice next-generation sequencing assay. Patients had performance status ≤ 1, left ventricular ejection fraction > 50%, grade ≤ 1 diarrhea, and no prior human epidermal growth factor receptor 2 (HER2) therapy. Patients received afatinib 40 mg once daily in 28-day cycles. The primary end point was objective response rate (ORR). Secondary end points were 6-month progression-free survival, overall survival, toxicity, and molecular correlates. RESULTS A total of 59 patients were assigned and 40 were enrolled. The median age was 62 years, 78% were female, 68% had performance status = 1, and 58% had received > 3 prior therapies. The confirmed ORR was 2.7% (n = 1 of 37; 90% CI, 0.14 to 12.2), and 6-month progression-free survival was 12.0% (90% CI, 5.6 to 25.8). A confirmed partial response occurred in a patient with adenocarcinoma of extra-mammary Paget disease of skin who progressed after cycle 6. Two unconfirmed partial responses were observed (low-grade serous gynecological tract and estrogen receptor–positive/HER2-negative immunohistochemistry breast ductal carcinoma). Of 12 patients with breast cancer, 1 additional patient with lobular carcinoma (estrogen receptor–positive/HER2 fluorescent in situ hybridization) had a 51% reduction in target lesions but progressed because of a new lesion at cycle 6. The most common (> 20%) treatment-related adverse events were diarrhea (68%), mucositis (43%), fatigue (40%), acneiform rash (30%), dehydration (27%), vomiting (27%), nausea (27%), anemia (27%), and anorexia (22%). Four patients (11%) discontinued because of adverse events. CONCLUSION Although afatinib did not meet the prespecified threshold for antitumor activity in this heavily pretreated cohort, the response in a rare tumor type is notable. The safety profile of afatinib was consistent with prior studies.

PURPOSE Cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) with endocrine therapy (ET) improves progression-free survival (PFS) and overall survival (OS) in hormone receptor–positive (HR+), human epidermal growth factor receptor 2–negative (HER2–) metastatic breast cancer (MBC). Although preclinical and clinical data demonstrate a benefit in changing ET and continuing a CDK4/6i at progression, no randomized prospective trials have evaluated this approach. METHODS In this investigator-initiated, phase II, double-blind placebo-controlled trial in patients with HR+/HER2– MBC whose cancer progressed during ET and CDK4/6i, participants switched ET (fulvestrant or exemestane) from ET used pre-random assignment and randomly assigned 1:1 to the CDK4/6i ribociclib versus placebo. PFS was the primary end point, defined as time from random assignment to disease progression or death. Assuming a median PFS of 3.8 months with placebo, we had 80% power to detect a hazard ratio (HR) of 0.58 (corresponding to a median PFS of at least 6.5 months with ribociclib) with 120 patients randomly assigned using a one-sided log-rank test and significance level set at 2.5%. RESULTS Of the 119 randomly assigned participants, 103 (86.5%) previously received palbociclib and 14 participants received ribociclib (11.7%). There was a statistically significant PFS improvement for patients randomly assigned to switched ET plus ribociclib (median, 5.29 months; 95% CI, 3.02 to 8.12 months) versus switched ET plus placebo (median, 2.76 months; 95% CI, 2.66 to 3.25 months) HR, 0.57 (95% CI, 0.39 to 0.85); P = .006. At 6 and 12 months, the PFS rate was 41.2% and 24.6% with ribociclib, respectively, compared with 23.9% and 7.4% with placebo. CONCLUSION In this randomized trial, there was a significant PFS benefit for patients with HR+/HER2– MBC who switched ET and received ribociclib compared with placebo after previous CDK4/6i and different ET.

Supplementary Figure S5. Mena isoform expression does not affect Integrin alphav, beta1 or FAK levels.

PDF - 3969K, Supplemental Figure 1. Therapeutic inhibition of SFKs can block nEGFR translocation in colorectal tumor models.

This file contains Supplementary Table 1 and provides hazard ratios for recurrence free survival and overall survival for polyploidy and clinical variables using a Cox proportional hazard model.

Supplementary Figure Legends

Multinucleated H2B-RFP Tub-GFP MDA-MB-231 Multipolar Division

PDF - 69K, Legend for Supplementary Figure 1.

Mononucleated H2B-RFP Tub-GFP MDA-MB-231 Bipolar Division

This file contains supplementary figures S1-S4. These illustrate correlation of ploidy from chromosome 17 FSIH versus 6-chromosome FISH, chromosome spreads of cell lines, additional dose-response curves, and data demonstrating DPBQ does not operate by binding DNA or by inhibiting topoisomerase II.

PURPOSE NCI-MATCH is a precision medicine trial using genomic testing to allocate patients with advanced malignancies to targeted treatment subprotocols. This report combines two subprotocols evaluating trametinib, a MEK1/2 inhibitor, in patients with Neurofibromatosis 1 ( NF1[S1] or GNA11/Q [S2]) altered tumors. METHODS Eligible patients had tumors with deleterious inactivating NF1 or GNA11/Q mutations by the customized Oncomine AmpliSeq panel. Prior MEK inhibitor treatment was excluded. Glioblastomas (GBMs) were permitted, including malignancies associated with germline NF1 mutations (S1 only). Trametinib was administered at 2 mg once daily over 28-day cycles until toxicity or disease progression. Primary end point was objective response rate (ORR). Secondary end points included progression-free survival (PFS) at 6 months, PFS, and overall survival. Exploratory analyses included co-occurring genomic alterations and PTEN loss. RESULTS Fifty patients were eligible and started therapy: 46 with NF1 mutations (S1) and four with GNA11 mutations (S2). In the NF1 cohort, nonsense single-nucleotide variants were identified in 29 and frameshift deletions in 17 tumors. All in S2 had nonuveal melanoma and GNA11 Q209L variant. Two partial responses (PR) were noted in S1, one patient each with advanced lung cancer and GBM for an ORR of 4.3% (90% CI, 0.8 to 13.1). One patient with melanoma in S2 had a PR (ORR, 25%; 90% CI, 1.3 to 75.1). Prolonged stable disease (SD) was also noted in five patients (four in S1 and one in S2) with additional rare histologies. Adverse events were as previously described with trametinib. Comutations in TP53 and PIK3CA were common. CONCLUSION Although these subprotocols did not meet the primary end point for ORR, significant responses or prolonged SD noted in some disease subtypes warrants further investigation.

The therapeutic options for patients with noninvasive or invasive breast cancer are complex and varied. These NCCN Clinical Practice Guidelines for Breast Cancer include recommendations for clinical management of patients with carcinoma in situ, invasive breast cancer, Paget disease, phyllodes tumor, inflammatory breast cancer, and management of breast cancer during pregnancy. The content featured in this issue focuses on the recommendations for overall management of ductal carcinoma in situ and the workup and locoregional management of early stage invasive breast cancer. For the full version of the NCCN Guidelines for Breast Cancer, visitNCCN.org.

Inhibitors of poly(ADP-ribose) polymerase (PARP) proteins potentiate antitumor activity of platinum chemotherapy. This study sought to determine the safety and tolerability of PARP inhibitor talazoparib with carboplatin and paclitaxel.We conducted a phase I study of talazoparib with carboplatin AUC5-6 and paclitaxel 80 mg/mForty-three patients were treated. The MTD for both schedules was talazoparib 250mcg daily. The main toxicity was myelosuppression including grade 3/4 hematologic treatment-related adverse events (TRAEs). Dose modification occurred in 87% and 100% of patients for schedules A and B, respectively. Discontinuation due to TRAEs was 13% in schedule A and 10% in B. Ten out of 22 evaluable patients in schedule A and 5/16 patients in schedule B had a complete or partial response. Twelve out of 43 patients received ≥6 cycles of talazoparib after induction, with a 13-month median duration of maintenance.We have established the recommended phase II dose of Talazoparib at 250mcg on a 3- or 7-day schedule with carboplatin AUC6 and paclitaxel 80 mg/m

Supplemental Tables 1 and 2

Supplementary Figure from A Phase I Study Investigating AZD8186, a Potent and Selective Inhibitor of PI3Kβ/δ, in Patients with Advanced Solid Tumors

Supplementary Figure Legends S1-S5

Purpose:To characterize safety and tolerability of the selective PI3Kβ inhibitor AZD8186, identify a recommended phase II dose (RP2D), and assess preliminary efficacy in combination with abiraterone acetate or vistusertib.Patients and Methods:This phase I open-label study included patients with advanced solid tumors, particularly prostate cancer, triple-negative breast cancer, and squamous non–small cell lung cancer. The study comprised four arms: (i) AZD8186 monotherapy dose finding; (ii) monotherapy dose expansion; (iii) AZD8186/abiraterone acetate (with prednisone); and (iv) AZD8186/vistusertib. The primary endpoints were safety, tolerability, and identification of the RP2D of AZD8186 monotherapy and in combination. Secondary endpoints included pharmacokinetics (PK), pharmacodynamics, and tumor and prostate-specific antigen (PSA) responses.Results:In total, 161 patients were enrolled. AZD8186 was well tolerated across all study arms, the most common adverse events being gastrointestinal symptoms. In the monotherapy dose-finding arm, four patients experienced dose-limiting toxicities (mainly rash). AZD8186 doses of 60-mg twice daily [BID; 5 days on, 2 days off (5:2)] and 120-mg BID (continuous and 5:2 dosing) were taken into subsequent arms. The PKs of AZD8186 were dose proportional, without interactions with abiraterone acetate or vistusertib, and target inhibition was observed in plasma and tumor tissue. Monotherapy and combination therapy showed preliminary evidence of limited antitumor activity by imaging and, in prostate cancer, PSA reduction.Conclusions:AZD8186 monotherapy had an acceptable safety and tolerability profile, and combination with abiraterone acetate/prednisone or vistusertib was also tolerated. There was preliminary evidence of antitumor activity, meriting further exploration of AZD8186 in subsequent studies in PI3Kβ pathway–dependent cancers.

This file contains additional technical detail (antibodies used, gating strategy for FACS) and additional experimental data to complement that shown in the main figures.

Although antiestrogen therapies are successful in many patients with estrogen receptor alpha-positive (ERα+) breast cancer, 25% to 40% fail to respond. Although multiple mechanisms underlie evasion of these treatments, including tumor heterogeneity and drug-resistant cancer stem cells (CSC), further investigations have been limited by the paucity of preclinical ERα+ tumor models. Here, we examined a mouse model of prolactin-induced aggressive ERα+ breast cancer, which mimics the epidemiologic link between prolactin exposure and increased risk for metastatic ERα+ tumors. Like a subset of ERα+ patient cancers, the prolactin-induced adenocarcinomas contained two major tumor subpopulations that expressed markers of normal luminal and basal epithelial cells. CSC activity was distributed equally across these two tumor subpopulations. Treatment with the selective estrogen receptor downregulator (SERD), ICI 182,780 (ICI), did not slow tumor growth, but induced adaptive responses in CSC activity, increased markers of plasticity including target gene reporters of Wnt/Notch signaling and epithelial–mesenchymal transition, and increased double-positive (K8/K5) cells. In primary tumorsphere cultures, ICI stimulated CSC self-renewal and was able to overcome the dependence of self-renewal upon Wnt or Notch signaling individually, but not together. Our findings demonstrate that treatment of aggressive mixed lineage ERα+ breast cancers with a SERD does not inhibit growth, but rather evokes tumor cell plasticity and regenerative CSC activity, predicting likely negative impacts on patient tumors with these characteristics.Significance: This study suggests that treatment of a subset of ERα+ breast cancers with antiestrogen therapies may not only fail to slow growth but also promote aggressive behavior by evoking tumor cell plasticity and regenerative CSC activity. Cancer Res; 78(7); 1672–84. ©2018 AACR.

Purpose: Hyperactivation of AKT is common and associated with endocrine resistance in estrogen receptor–positive (ER+) breast cancer. The allosteric pan-AKT inhibitor MK-2206 induced apoptosis in PIK3CA-mutant ER+ breast cancer under estrogen-deprived condition in preclinical studies. This neoadjuvant phase II trial was therefore conducted to test the hypothesis that adding MK-2206 to anastrozole induces pathologic complete response (pCR) in PIK3CA mutant ER+ breast cancer.Experimental Design: Potential eligible patients with clinical stage II/III ER+/HER2− breast cancer were preregistered and received anastrozole (goserelin if premenopausal) for 28 days in cycle 0 pending tumor PIK3CA sequencing. Patients positive for PIK3CA mutation in the tumor were eligible to start MK-2206 (150 mg orally weekly, with prophylactic prednisone) on cycle 1 day 2 (C1D2) and to receive a maximum of four 28-day cycles of combination therapy before surgery. Serial biopsies were collected at preregistration, C1D1 and C1D17.Results: Fifty-one patients preregistered and 16 of 22 with PIK3CA-mutant tumors received study drug. Three patients went off study due to C1D17 Ki67 >10% (n = 2) and toxicity (n = 1). Thirteen patients completed neoadjuvant therapy followed by surgery. No pCRs were observed. Rash was common. MK-2206 did not further suppress cell proliferation and did not induce apoptosis on C1D17 biopsies. Although AKT phosphorylation was reduced, PRAS40 phosphorylation at C1D17 after MK-2206 persisted. One patient acquired an ESR1 mutation at surgery.Conclusions: MK-2206 is unlikely to add to the efficacy of anastrozole alone in PIK3CA-mutant ER+ breast cancer and should not be studied further in the target patient population. Clin Cancer Res; 23(22); 6823–32. ©2017 AACR.

Supplementary Data from A Phase I Study Investigating AZD8186, a Potent and Selective Inhibitor of PI3Kβ/δ, in Patients with Advanced Solid Tumors

Fig S1 Study Flowchart Fig S2 PIK3CA somatic mutation pattern Fig S3 Lack of apoptotic effect by anastrozole monotherapy (C1D1) and in combination with MK-2206 (C1D17 and surgery) Fig S4 CDH1 somatic mutation pattern Fig S5 ESR1 somatic mutation pattern

Background Antiprogestins, including selective progesterone receptor (PgR) modulators (SPRMs) that act as PgR antagonists, are a promising class of therapeutics for overcoming endocrine resistance including in patients who develop activating estrogen receptor 1 (ESR1) mutations after prior endocrine therapy (ET). The SMILE study (NCT04738292) is a multi-institutional phase II clinical trial to determine the efficacy and safety of an antiprogestin, onapristone in combination with fulvestrant as second-line therapy for patients with ER+,PgR+, HER2 -metastatic breast cancer (MBC). Methods Patients with locally advanced or MBC who progress on ≥2 lines of prior therapy with ET as a single agent or in combination with CDK4/6 inhibitors, mTOR inhibitors and one line of chemotherapy in the metastatic setting are eligible. Other criteria include ECOG performance status ≤ 2, measurable disease per RECIST 1.1 criteria, adequate organ function, availability of an archived tumor biopsy block confirming the diagnosis of MBC, optional biopsy at disease progression on trial, and optional 18F-fluorofuranylnorprogesterone (18F-FFNP) PET/CT imaging. Ovarian suppression is allowed. Prior ET with tamoxifen or aromatase inhibitor therapy in the adjuvant or metastatic setting is allowed. Patients with prior exposure to fulvestrant, antiprogestins, or CDK inhibitors in the adjuvant setting are ineligible. Following consent, all subjects will receive fulvestrant at the recommended dosing schedule plus onapristone 50 mg orally, twice daily, until disease progression, unacceptable toxicity, or discontinuation for any other reason. Using the Simon Two-Stage design, a total of 39 patients will be enrolled over two years from 6-8 sites within Wisconsin Oncology Network (WON). In the first stage of the trial, 21 subjects will be enrolled; if ≥2 responses are observed, then 18 additional subjects will be enrolled in the second stage. This design yields a one-sided type I error rate of 5% if the ORR is 7% and power of 80% when the combination's true response rate is 20%. The primary objective is to evaluate the objective response rate; secondary objectives include safety and tolerability, progression-free survival, disease control rate, and duration of response. Other correlates include optional functional imaging of PgR binding with 18F-FFNP PET/CT and biomarker analysis (ER, total PgR, HER2, Ki67, CD24, CD44, LDH1, KLF4, CK 5/6, PhosphoSer294-PgR on tissue samples, ESR1 mutations, and circulating tumor DNA analysis). The study was approved in January 2021, and enrollment is active at the time of this submission. Citation Format: Sailaja Kamaraju, Amy Fowler, Chung Yee Cheng, Lubna N Chaudhary, John Burfeind, Janet Retseck, Amye J Tevaarwerk, Mark Burkard, Elisavet Paplomata, Amanda M Parkes, Julie M Jorns, Sergey Tarima, Douglas Yee, Hallgeir Rui, Carol Lange, Tarek Sahmoud, Kari B Wisinski. The SMILE study: A phase 2 trial of onapristone in combination with fulvestrant for patients with ER+ and HER2- metastatic breast cancer after progression on endocrine therapy and CDK4/6 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 OT2-16-01.

Subunits of the chromatin remodeler SWI/SNF are the most frequently disrupted genes in cancer. However, how post-translational modifications (PTM) of SWI/SNF subunits elicit epigenetic dysfunction remains unknown. Arginine-methylation of BAF155 by coactivator-associated arginine methyltransferase 1 (CARM1) promotes triple-negative breast cancer (TNBC) metastasis. Herein, we discovered the dual roles of methylated-BAF155 (me-BAF155) in promoting tumor metastasis: activation of super-enhancer-addicted oncogenes by recruiting BRD4, and repression of interferon α/γ pathway genes to suppress host immune response. Pharmacological inhibition of CARM1 and BAF155 methylation not only abrogated the expression of an array of oncogenes, but also boosted host immune responses by enhancing the activity and tumor infiltration of cytotoxic T cells. Moreover, strong me-BAF155 staining was detected in circulating tumor cells from metastatic cancer patients. Despite low cytotoxicity, CARM1 inhibitors strongly inhibited TNBC cell migration in vitro, and growth and metastasis in vivo. These findings illustrate a unique mechanism of arginine methylation of a SWI/SNF subunit that drives epigenetic dysregulation, and establishes me-BAF155 as a therapeutic target to enhance immunotherapy efficacy.

Authors: Apoorva Anandan, MD (1,2), Trinetri Ghosh (3), Jiwei Zhao (3), Kari Wisinski, MD (1,2) (1) Department of Medicine, Section of Hematology/Oncology, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI (2) University of Wisconsin Carbone Cancer Center, Madison, WI (3) University of Wisconsin Department of Biostatistics and Medical Informatics Madison, WI Background: A combined analysis of the SOFT/TEXT trials demonstrated an overall survival (OS) benefit to endocrine therapy in addition to OFS compared to endocrine therapy alone, further establishing the importance of adequate OFS in premenopausal women with HR+ breast cancers. Factors associated with inadequate OFS are ambiguous, but younger age, type of chemotherapy, high body mass index (BMI), and time from treatment completion seem to be important. Additionally, data remains limited regarding whether monitoring of OFS should be routinely performed and optimal timing of hormone levels. We sought to identify predictors of inadequate OFS among an AYA population of hormone receptor positive breast cancer receiving OFS at our institution. Methods: A retrospective, descriptive study was conducted looking at AYA patients (pts) aged 18-39 with a diagnosis of HR+ breast cancer who previously and/or currently received oncologic care for management of their breast cancer at the institution. Data was collected from pts diagnosed between 2000-2022. Patients who had previously received or are currently receiving OFS in the adjuvant and/or metastatic setting were included in the study. Data was collected regarding age, BMI, chemotherapy regimen received in the neoadjuvant and/or adjuvant setting, type, dose, and frequency of ovarian suppression, number of times estradiol was monitored, and frequency of estradiol levels >20. This was used as the cut off based on a comprehensive review of data consistently categorizing levels >20 as not to be postmenopausal. Results: 74 AYA patients who received OFS were included with median age of 28 (range 20-39) and average BMI 27.7 (range 15-45). 70% of the population was Caucasian, 10% African American, and 20% Hispanic. Estradiol levels were monitored in 46 of the 74 pts (62%). The frequency of estradiol monitored ranged from 1-22 times. 16 out of the 46 (35%) pts had estradiol checked only once, 10 (22%) twice, and 16 (35%) four or more times. 22 out of 46 pts (48%) had estradiol levels >20 when checked at least once. 9 out of 22 (41%) had estradiol >20 when checked more than four times. Out of 74 pts, 36 received OFS every month (49%), 32 received OFS every three months (43%). Only 4 out of 74 pts (5%) switched from monthly to every three months, meanwhile only 1 (1%) switched from every three months to monthly. 18 out of the 36 pts (50%) receiving monthly OFS had estradiol levels checked with 9 (50%) having estradiol >20. Meanwhile, 20 out of the 32 pts who received OFS every 3 months (63%) had estradiol levels checked with 10 (50%) having estradiol >20. The average age of pts with estradiol >20 was 33.6 while the average BMI of those with estradiol >20 was 29.2. Finally, 64 out of 74 pts (86%) received chemotherapy in the neoadjuvant and/or adjuvant setting. 41 out of 64 (64%) had estradiol levels checked. 18 out of 41 (44%) had estradiol >20. Discussion: Our data indicates a high degree of clinician variability in monitoring estradiol levels in AYA pts treated with OFS. Lack of adequate OFS was seen in nearly half of pts in this cohort. Higher BMI and young age may be predictors of lack of adequate ovarian suppression, supporting the findings of other studies. An algorithm for routine monitoring of estradiol may improve outcomes with OFS especially in young pts or those with a high BMI. Citation Format: Apoorva Anandan, Trinetri Ghosh, Jiwei Zhao, Kari B. Wisinski. Predictors of Incomplete Ovarian Function Suppression (OFS) in Adolescent and Young Adult (AYA) Women with Hormone Receptor Positive (HR+) 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 P2-03-24.

Combining cyclin-dependent kinase (CDK) inhibitors with endocrine therapy improves outcomes for patients with metastatic estrogen receptor-positive breast cancer but its value in earlier-stage patients is unclear. We examined evolutionary trajectories of early-stage breast cancer tumors, using single-cell RNA sequencing of serial biopsies from the FELINE clinical trial of endocrine therapy (letrozole) alone or combined with the CDK inhibitor ribociclib. Despite differences in subclonal diversity evolution across patients and treatments, common resistance phenotypes emerged. Resistant tumors treated with combination therapy showed accelerated loss of estrogen signaling with convergent upregulation of JNK signaling through growth factor receptors. In contrast, cancer cells maintaining estrogen signaling during mono- or combination therapy showed potentiation of CDK4/6 activation and ERK upregulation through ERBB4 signaling. These results indicate that combination therapy in early-stage estrogen receptor-positive breast cancer leads to emergence of resistance through a shift from estrogen to alternative growth signal-mediated proliferation. Bild and colleagues identify convergent clonal evolution mechanisms through single-cell genomic analyses, explaining therapy resistance in patients with early-stage breast cancer treated with endocrine and CDK4/6 therapy in the FELINE trial.

The NCCN Guidelines for Breast Cancer include up-to-date guidelines for clinical management of patients with carcinoma in situ, invasive breast cancer, Paget disease, phyllodes tumor, inflammatory breast cancer, male breast cancer, and breast cancer during pregnancy. These guidelines are developed by a multidisciplinary panel of representatives from NCCN Member Institutions with breast cancer–focused expertise in the fields of medical oncology, surgical oncology, radiation oncology, pathology, reconstructive surgery, and patient advocacy. These NCCN Guidelines Insights focus on the most recent updates to recommendations for adjuvant systemic therapy in patients with nonmetastatic, early-stage, hormone receptor–positive, HER2-negative breast cancer.

Radiotherapy is a critical tool for reducing locoregional recurrence, extending survival, and palliating symptoms in patients with breast cancer. With an ever-expanding armamentarium of systemic agents available, and an increasing trend toward the use of hypofractionated radiation regimens, it can be difficult to determine the safety of concurrent therapy. In particular, new targeted agents in both the adjuvant and metastatic setting have limited prospective or long-term data demonstrating safety when delivered concurrently with radiotherapy. Other systemic agents, including chemotherapy and endocrine therapy, are also important components of the overall treatment strategy for localized and metastatic breast cancer, and are often delivered concurrently with radiation in certain clinical scenarios. This review explores the safety, efficacy, and pitfalls of delivering radiation in conjunction with systemic therapies for breast cancer.

Background: The CALGB 40603 and BrighTNess studies found that adding high dose carboplatin every 3 weeks to the standard neoadjuvant DD AC - paclitaxel regimen in TNBC increased the pathologic complete response (pCR) rate to 54-58% but with the cost of increased treatment related toxicities resulting in decreased completion of the full course of treatment. Our hypothesis is by changing the high dose carboplatin every 3 weeks to low dose weekly carboplatin, it will retain the same benefit in pathologic response rate and minimize the treatment related toxicities, which in turn permits the full course of neoadjuvant treatment given to patients. Patients and Methods: This multi-center study was conducted through the WON (NCT03301350). Eligible TNBC patients for neoadjuvant chemotherapy received weekly carboplatin (AUC=2) and paclitaxel 80 mg/m2 for 12 doses, followed by dose dense doxorubicin 60 mg/m2 and cyclophosphamide 600 mg/m2 every 2 weeks with granulocyte growth factor support for 4 cycles. A one-week break was added before DD AC after the first 22 patients in order to minimize prolonged cytopenia. Primary end point was pCR. Secondary endpoints were frequency of dose modification and treatment related toxicities. Results: Accrual target was 50 with 80% power to detect a 20% difference using a one-sided binomial test at an alpha significance level of 0.025, but study was terminated earlier. Twenty-nine eligible patients consented for the study from November 2017 to February 2020. Median age was 52 year-old (range, 33-80). Twenty-eight patients received the study regimen. Three were removed due to early study termination and one (3.8%) died from grade 5 neutropenic sepsis after the last cycle of DD AC before surgery. Thus, 24 patients were evaluable for the primary outcome and 26 were evaluable for dose modification and toxicity outcomes. Eight patients (33%) achieved pCR and another 4 patients (17%) had minimal (ypT1aN0) residual disease. Of 26 patients, 24 (92%) were able to receive the full course of treatment +/- dose modification. One patient required the discontinuation of weekly carboplatin/paclitaxel completely due to severe infusion reaction and one did not finish the treatment before the study was terminated. Eight patients (31%) required dose delay with 5 of them during the weekly carboplatin/paclitaxel treatment. Seven patients (27%) also required dose reduction with 5 of them during the weekly carboplatin/paclitaxel treatment. Seventeen patients (65%) experienced severe adverse event (grade 3 or 4) with the majority of events (12/17) relating to grade 3-4 neutropenia or neutropenic fever. Conclusion: Although our study showed a lower pCR rate of 33% in compare to the previous CALGB 40603 (54%) and BrighTNess (58%) studies, we demonstrated that the majority of patients (92%) were able to receive the full course of study treatment. This weekly carboplatin with paclitaxel - AC regimen was used in the recent Keynote 522 study as the backbone chemotherapy to combine with check-point inhibitor immunotherapy in TNBC. However, any dosing schedules of carboplatin, weekly or every 3 weeks, add substantially to grade 3 or 4 toxicity of paclitaxel - AC. Aggressive supportive care management is needed when carboplatin is used. Citation Format: Yee Chung Cheng, Ruth O'Regan, Lubna N Chaudhary, Sailaja Kamaraju, Harvey Einhorn, Emmanuel Sampene, Marialane Pigsley, Mark E Burkard, Christopher R Chitambar, Kari B Wisinski. A phase II study of neoadjuvant weekly carboplatin/paclitaxel followed by dose-dense doxorubicin/cyclophosphamide (DD AC)in patients with triple negative breast cancer (TNBC): Wisconsin oncology network (WON) study [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 PS13-51.

Background: Somatic genomic alterations that activate the phosphatidylinositol-3-kinase (PI3K) pathway signaling are found in 30-50% of breast cancers, and the p110a-specific inhibitor alpelisib is approved for use in combination with fulvestrant in PIK3CA mutated hormone receptor-positive (HR+) metastatic breast cancer. However, preclinical and clinical data has demonstrated significant variability in response to alpelisib and other PI3K inhibitors even in the presence of hotspot PIK3CA activating mutations, likely reflecting functional differences between somatic alterations, bypass signaling mechanisms, and intratumor clonal heterogeneity. Thus, there is an ongoing need for novel predictive biomarkers to help guide patient selection for these therapies. Circulating tumor cells (CTCs) represent an accessible source of tumor derived analytes that allow for the interrogation of protein level readouts of PI3K pathway activation, may better reflect the biologic heterogeneity of metastatic disease than single site solid tumor biopsy, and are amenable to longitudinal analysis. Here, we report the development of an assay to evaluate the expression of activated AKT (AKT pS473) in CTCs as a putative biomarker of sensitivity to PI3K inhibitor therapies in metastatic breast cancer. Methods: Peripheral blood for CTC isolation was collected serially from patients with metastatic breast cancer and known somatic PI3K pathway mutation status. Samples were processed using VERSA (Versatile Exclusion-based Rare Sample Analysis), a microfluidic technology that integrates CTC capture and downstream analysis. Following fixation, CTCs were captured immunomagnetically with antibodies for EpCAM and Trop2, permeabilized and stained on chip for pan-AKT plus AKT pS473. Quantitative fluorescent microscopy was used to enumerate CTCs, defined as cytokeratin positive cells with intact nuclei and negative for a group of normal blood cell markers (CD45/CD11b/CD34/CD66b), and to quantify activated AKT protein expression in CTCs and matched peripheral blood mononuclear cells (PBMCs). Results: 100% of patients with somatic PIK3CA mutations in our pilot cohort had CTCs with detectable phospho-AKT expression, and in 50% of patients, the median phospho-AKT/pan AKT ratio was higher in CTCs compared to matched peripheral blood cells, suggestive of increased PI3K pathway activity. All patients demonstrated heterogeneity in phospho-AKT expression and phospho-AKT/pan-AKT ratio among individual CTCs at a single timepoint and across timepoints in longitudinal analysis. Conclusion: We report here the feasibility of quantitative and longitudinal detection of activated AKT in EpCAM/Trop2 captured CTCs from metastatic breast cancer with somatic PIK3CA mutations. Future work will prospectively evaluate multiple clinical applications of this assay in patients receiving alpelisib plus endocrine therapy for PIK3CA mutated HR+ metastatic breast cancer, as well as expanding the assay to include the detection of additional phospho-protein readouts of PI3K pathway activity in CTCs. In addition to the potential to complement solid biopsy PIK3CA mutation status as a predictive biomarker of sensitivity to PI3K therapies, this assay has the unique potential to provide a pharmacodynamic assessment of PI3K inhibitor activity in CTCs while on treatment, which may serve as an early biomarker of clinical response or resistance. Citation Format: Marina N Sharifi, Jamie M Sperger, Cole Gilsdorf, Serena K Wolfe, Amanda Parkes, Kari B Wisinski, Ruth M O'Regan, Joshua M Lang. Detection of PI3K pathway activation in circulating tumor cells in PIK3CA mutated metastatic breast cancer as a putative predictive biomarker for PI3K inhibitor therapies [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 PS5-35.

The NCCN Guidelines for Genetic/Familial High-Risk Assessment: Breast, Ovarian, and Pancreatic focus primarily on assessment of pathogenic or likely pathogenic variants associated with increased risk of breast, ovarian, and pancreatic cancer and recommended approaches to genetic testing/counseling and management strategies in individuals with these pathogenic or likely pathogenic variants. This manuscript focuses on cancer risk and risk management for BRCA-related breast/ovarian cancer syndrome and Li-Fraumeni syndrome. Carriers of a BRCA1/2 pathogenic or likely pathogenic variant have an excessive risk for both breast and ovarian cancer that warrants consideration of more intensive screening and preventive strategies. There is also evidence that risks of prostate cancer and pancreatic cancer are elevated in these carriers. Li-Fraumeni syndrome is a highly penetrant cancer syndrome associated with a high lifetime risk for cancer, including soft tissue sarcomas, osteosarcomas, premenopausal breast cancer, colon cancer, gastric cancer, adrenocortical carcinoma, and brain tumors.

Background Antiprogestins, including selective progesterone receptor (PgR) modulators (SPRMs) that act as PgR antagonists, are a promising class of therapeutics for overcoming endocrine resistance including in patients who develop activating estrogen receptor alpha (ESR1) mutations after prior endocrine therapy (ET). The SMILE study is a multi-institutional phase II clinical trial to determine the efficacy and safety of an antiprogestin, onapristone in combination with fulvestrant as second-line therapy for patients with ER+,PgR+/-, HER2 -metastatic breast cancer (MBC). Methods Patients with locally advanced or MBC who progress on ≥2 lines of prior therapy with ET as a single agent or in combination with CDK4/6 inhibitors, mTOR inhibitors and one line of chemotherapy in the metastatic setting are eligible. Other criteria include ECOG performance status ≤ 2, measurable disease per RECIST 1.1 criteria, adequate organ function, availability of an archived tumor biopsy block confirming the diagnosis of MBC, optional biopsy at disease progression on trial, and optional 18F-fluorofuranylnorprogesterone (18F-FFNP) PET/CT imaging. Ovarian suppression is allowed. Prior ET with tamoxifen or aromatase inhibitor therapy in the adjuvant or metastatic setting is allowed. Patients with prior exposure to fulvestrant, antiprogestins, or CDK inhibitors in the adjuvant setting are ineligible. Following consent, all subjects will receive fulvestrant at the recommended dosing schedule plus onapristone 50 mg orally, twice daily, until disease progression, unacceptable toxicity, or discontinuation for any other reason. Using the Simon Two-Stage design, a total of 39 patients will be enrolled over two years from 6-8 sites within Wisconsin Oncology Network (WON); study enrollment began in November 2021. In the first stage of the trial, thus far, nine patients were accrued who are tolerating the trial regimen well with no reported toxicities or serious events. The safety monitoring is planned when the cohort size reaches eleven patients, and interim analysis at 21 patients; if ≥2 responses are observed, then 18 additional subjects will be enrolled in the second stage. This design yields a one-sided type I error rate of 5% if the ORR is 7% and power of 80% when the combination’s true response rate is 20%. The primary objective is to evaluate the objective response rate; secondary objectives include safety and tolerability, progression-free survival, disease control rate, and duration of response. Other correlates include optional functional imaging of PgR binding with 18F-FFNP PET/CT and biomarker analysis (ER, total PgR, HER2, Ki67, CD24, CD44, LDH1, KLF4, CK 5/6, PhosphoSer294-PgR on tissue samples, ESR1 mutations, and circulating tumor DNA analysis). Citation Format: Sailaja Kamaraju, Amy Fowler, Lubna N. Chaudhary, Mark Burkard, Thomas Giever, Robert N Hegeman, Michele Pipp-Dahm, Yee Chung Cheng, Carol Lange, Julie M. Jorns, Amy Stella, Nauman Siddiqui, Luke Zurbriggen, Saurabh Rajguru, Sergey Tarima, Deepika Sriram, Janet Retseck, Hallgeir Rui, Tarek Sahmoud, Kari B. Wisinski. The SMILE Study: A phase II trial of onapristone in combination with fulvestrant for patients with ER-positive and HER2-negative metastatic breast cancer after progression on endocrine therapy and CDK 4/6 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 OT2-01-02.

Background: A CDK4/6 inhibitor (CDK4/6i) in combination with endocrine therapy (ET) is standard first-line therapy for advanced, hormone receptor (HR)-positive, HER2-negative breast cancer (BC). However, not all patients respond and responders eventually develop drug resistance and disease progression. Exosomes are small extracellular vesicles that are secreted by both normal and tumor cells as a mechanism for intercellular communication. The protein cargo of exosomes reflects biological processes activated in cancer cells and may serve as predictive biomarkers to select patients most likely to benefit from treatment and to identify mechanisms of resistance. Methods: Whole blood was collected in Streck tubes at baseline and at time points during treatment from patients with advanced, HR+/HER2- BC enrolled in a single arm, phase 2 trial of first line therapy with palbociclib plus tamoxifen that was conducted by The Big Ten Cancer Research Consortium (NCT02668666). Plasma was separated and stored at -80C within 48 hours of collection. Different exosome protein isolation methods were evaluated and optimized to maximize protein recovery. Exosome and plasma proteins were extracted, purified, and digested with trypsin. Tryptic peptides were isotopically labeled with Tandem Mass Tag (TMT) 10plex for protein expression level quantitation. Triplicate samples from each patient were analyzed by LC-MS/MS with QExactive HF Orbitrap mass spectrometer. An unsupervised clustering method was used to classify patients based on exosomal proteomic profiles. Results: We developed a sensitive and efficient exosome extraction method to obtain exosome protein from minimal volumes of patient plasma. The optimized exosome isolation method quantitatively identified 800 proteins from a 100 µl plasma sample. Significant enrichment of exosome-specific markers was observed when comparing patient samples with healthy donor samples. A network model was developed to differentiate responders/stable disease patients from non-responders using exosome proteomics data generated from pretreatment plasma samples. Preliminary data from the first 22 patients analyzed (responders, n= 6; stable disease, n=12, and non-responder, n=4) identified a network of 45 proteins that predicted response/stable disease vs progressive disease with high specificity (95%) and sensitivity (89%). We also noted significant differences in the exosome proteomic profiles of patients with de novo vs. recurrent metastatic disease. A network of 22 proteins differentiated de novo vs recurrent metastatic disease with > 85% sensitivity and 78% specificity, providing molecular evidence differentiating the two disease states. This finding is relevant in light of the higher response rate and improved PFS in patients with de novo metastatic disease in this trial, and confirms that this approach may provide molecular insight into mechanisms of primary resistance to CDK4/6i. Results for the entire trial cohort of 46 patients will be presented, along with analysis of serial samples collected at various time points during treatment. Conclusion: This proof-of-concept study demonstrates that an ultrasensitive exosome proteomics platform combined with deep learning methods is ideally suited for developing predictive protein biomarkers and for exploring molecular mechanisms of drug resistance. If results are confirmed, this novel approach holds great promise for identifying protein biomarkers that could be used to select patients unlikely to respond to ET and CDK4/6i in order to spare them ineffective treatment and for selecting participants for clinical trials of novel agents. Additionally, exosome proteomics data generated from serially collected specimens can be used to identify mechanisms of resistance that emerge during therapy. This approach can be widely applied to other treatment regimens and disease sites. This study was funded by Pfizer. Citation Format: Ziwei Zhang, Xiuyuan Ma, Julia Ekiert, Gayatry Mohapatra, Louis Coleman, Cristina I. Truica, Anne Blaes, Jatin Rana, Tandra Pavankumar, Lauren Green, Menggang Yu, Deborah Toppmeyer, Ruth O’Regan, Kari B. Wisinski, Oana C. Danciu, Kent Hoskins, Yu Gao. Quantitative proteomic analysis of plasma exosomes from patients with advanced hormone receptor-positive/HER2-negative breast cancer receiving palbociclib and tamoxifen [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-47.

Background: HER2, IGFBP-2, and IGF-IR are proteins that are overexpressed in breast cancer. These three proteins, when used as immunogens, provide broad antigenic coverage to all molecular breast cancer subtypes. The proteins are also up-regulated in pre-invasive and high-risk breast lesions which are associated with progression to invasive cancer. Therefore, generating protective immunity against these antigens could have the result of preventing cancer development in high risk patients. We identified epitopes derived from these proteins, termed Th1 selective epitopes, that specifically stimulated T-helper 1 immune responses in humans and mice. The tri-antigen vaccine was effective in preventing the development of breast cancer in a transgenic mouse model of neu-expressing mammary cancer. We conducted a dose finding study of a plasmid-based vaccine encoding three extended Th1 selective epitopes derived from these antigens in breast cancer patients. Methods: Patients with non-metastatic, node positive, HER2 negative breast cancer that are in remission and defined as no evidence of disease were enrolled sequentially to one of 3 dose arms: 150, 300, and 600 mcg of the tri-antigen vaccine plasmid with 10 evaluable patients per arm (NCT02780401). Vaccines were given monthly intradermally for three total doses with rhu-GM-CSF (100mcg) as an adjuvant. The primary endpoint was safety through the 6-month follow-up visit and the secondary endpoints were immunogenicity, persistence of the immune response after vaccination, and assessment of potential stimulation of T-regulatory (T-reg) cells or myeloid derived suppressor cells to the overexpressed non-mutated antigens as well as determining the recommended Phase II dose. Results: Thirty-two patients were enrolled and 97% received all three vaccinations. The mean age at enrollment was 51.9 years with 61% of patients being pre-menopausal and 39% post-menopausal. The majority of patients were Stage II/III. Eighty-eight percent of patients had hormone receptor positive tumors and twelve percent were triple negative disease. The majority of adverse events (AE) (>95%) were grade 1 or 2. The most common AE were injection site reactions, flu like symptoms, fatigue, chills, myalgia, and nausea. All doses were immunogenic with the greatest magnitude antigen specific Type I immune responses seen in the low and intermediate doses. Eighty percent of patients at the 300mcg dose retained high levels of antigen specific immunity at 1 and 6 months after immunization as compared to 57% and 50% at the 100 and 600mcg doses respectively. No antigen specific Th2 cells, MDSC or T-reg were generated with vaccination. Immunizations did not upregulate PD-1 on CD4 or CD8 T-cells. Conclusions: A plasmid-based vaccine encoding extended Th1 selective epitopes derived from HER2, IGFBP-2, and IGF-IR could be administered safely and generate high levels of antigen specific interferon gamma secreting T-cells (Th1). The 300mcg dose elicited significant immune responses in the majority of patients which persisted at least 6 months after the end of immunization. A Phase II study of the vaccine given in the neoadjuvant setting to patients with HER2 positive breast cancer is ongoing (NCT04329065). Citation Format: Sasha E. Stanton, Kari B. Wisinski, William R. Gwin, Andrew Coveler, John B. Liao, Mark Burkard, Howard Bailey, KyungMann Kim, Thomas Havinghurst, Katina DeShong, Jennifer S. Childs, Eileen Dimond, Margaret Wojtowicz, Brandy M. Heckman-Stoddard, Denise Cecil, Mary Disis. Phase I trial of the safety and immunogenicity of a tri-antigen vaccine targeting HER2, IGFBP-2, and IGF-IR in patients with non-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 P2-02-02.

Background: Palbociclib is a CDK4/6 inhibitor used to treat metastatic hormone receptor-positive (HR+) breast cancer (MBC) in combination with endocrine therapy. Tamoxifen is an effective treatment for HR+ MBC, with different toxicity profile compared with aromatase inhibitors (AI) and fulvestrant. Preclinical data demonstrated synergy for the combination of tamoxifen and palbociclib, being effective in a model of acquired tamoxifen resistance. Methods: We conducted a non-randomized, open-label, single-arm, multicenter, phase II trial of palbociclib in combination with tamoxifen in patients with HR+/HER2 - advanced BC, with no prior therapy for MBC. Ovarian suppression was recommended for pre-menopausal women. Primary objective was progression free survival, PFS. Secondary objectives: objective response rate, ORR (CR or PR) based on RECIST 1.1 or MDA Criteria (for patients with bone only disease); safety and tolerability (using CTCAE v4); clinical benefit rate, CBR (CR, PR or SD lasting min 24 weeks); 2-year overall survival. Correlative objectives: proteomic analysis of plasma exosomes to identify mechanisms of primary and secondary resistance to tamoxifen/palbociclib. Results: Between 6/30/2016 and 7/02/2019, we enrolled 49 patients (47 evaluable): 23 pts with de-novo metastatic disease and 24 pts with recurrent BC (12 pts were on adjuvant treatment with AI at time of recurrence and 12 pts on surveillance). As of 6/30/2022 data cut-off, 2 pts were still on treatment. This is an updated analysis with median follow-up time of 24 months (range 8-51). Median age was 60 (range 39-82). The median PFS was 19.8 months with 95% CI (8-41) for pts with de-novo MBC and 7 months (2-12) for pts with recurrent BC. The ORR was 30% overall, 39% for pts with de novo MBC, 21% for pts with recurrent BC. CBR was 64% overall, 77% for pts with de novo MBC and 50% for pts with recurrent BC. CBR was 65% for white pts and 55% for AA pts. Best response per RECIST1.1: 14 pts (34%) had PR, 18 pts (44%) had SD, 9 pts (22%) had PD. All 6 pts with bone only disease had SD. The most common drug related grade ≥ 3 AE was neutropenia (51%), transient and manageable by dose modifications, no cases of febrile neutropenia. Four patients (8%) developed thromboembolic events (grades 2, 3, 4) and discontinued treatment. One patient died while on treatment from PD. More than 800 exosome proteins were detected in the samples analyzed so far. Exosomal protein networks in pretreatment samples predicted treatment response with 89% sensitivity and 95% specificity in unsupervised clustering. Data on correlative objectives will be presented in a separate abstract. Conclusions: The combination of palbociclib and tamoxifen showed tolerable, expected safety profile. This may be an alternative approach for selected patients in first line treatment of HR+ MBC, especially those who present with de-novo metastatic disease and are intolerant to AI; although this small study indicates a lower PFS. Table 1: Baseline Demographics Citation Format: Oana C. Danciu, Kent Hoskins, Jennifer Weiss, Cristina I. Truica, Anne Blaes, Deimante Tamkus, Jatin Rana, Tandra Pavankumar, Lauren Green, Yu Gao, Menggang Yu, Qianqian Zhao, Deborah Toppmeyer, Ruth O’Regan, Kari B. Wisinski. A single arm phase II trial of Palbociclib in combination with Tamoxifen as first line therapy for metastatic hormone receptor positive 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 P4-01-38.

Background Postcancer work limitations may affect a substantial proportion of patients and contribute to the "financial toxicity" of cancer treatment. The degree and nature of work limitations and employment outcomes are poorly understood for cancer patients, particularly in the immediate period of transition after active treatment. We prospectively examined employment, work ability, and work limitations during and after treatment. Methods A total of 120 patients receiving curative therapy who were employed prior to their cancer diagnosis and who intended to work during or after end of treatment (EOT) completed surveys at baseline (pretreatment), EOT, and 3, 6, and 12 months after EOT. Surveys included measures of employment, work ability, and work limitations. Descriptive statistics (frequencies, percentages, means with standard deviations) were calculated. Results A total of 111 participants completed the baseline survey. On average, participants were 48 years of age and were mostly white (95%) and female (82%) with a diagnosis of breast cancer (69%). Full-time employment decreased during therapy (from 88% to 50%) and returned to near prediagnosis levels by 12-month follow-up (78%). Work-related productivity loss due to health was high during treatment. Conclusions This study is the first to report the effects of curative intent cancer therapy on employment, work ability, and work limitations both during and after treatment. Perceived work ability was generally high overall 12 months after EOT, although a minority reported persistent difficulty. A prospective analysis of factors (eg, job type, education, symptoms) most associated with work limitations is underway to assist in identifying at-risk patients.

The NCCN Guidelines for Genetic/Familial High-Risk Assessment: Breast, Ovarian, and Pancreatic provide recommendations for genetic testing and counseling for hereditary cancer syndromes, and risk management recommendations for patients who are diagnosed with syndromes associated with an increased risk of these cancers. The NCCN panel meets at least annually to review comments, examine relevant new data, and reevaluate and update recommendations. These NCCN Guidelines Insights summarize the panel’s discussion and most recent recommendations regarding criteria for high-penetrance genes associated with breast and ovarian cancer beyond BRCA1/2, pancreas screening and genes associated with pancreatic cancer, genetic testing for the purpose of systemic therapy decision-making, and testing for people with Ashkenazi Jewish ancestry.

Background: Advanced triple negative breast cancer (TNBC) has a poor prognosis, with a median survival of approximately 12 months. Due to a lack of receptor targets, chemotherapy remains the mainstay of treatment. A subset of TNBC has defects in homologous recombination (HR) DNA repair due to germline BRCA mutations. Poly(ADP-ribose) polymerase (PARP) inhibitors have been shown to be an effective therapy in this subgroup. The phosphoinositide-3-kinase (PI3K) pathway inhibitors are also active in some breast cancers, lowering nucleotide pools required for DNA synthesis and S-phase progression. Preclinical studies suggest that PI3K stabilizes and preserves double strand break repair by interacting with the HR complex. Inhibition of PI3K/mTOR could impede PI3K interaction with the HR complex, thereby increasing dependency on PARP enzymes for DNA repair, and making this dual inhibition a rational combination. This trial is studying the safety and efficacy of combining the PI3K/mTOR inhibitor, gedatolisib, and the PARP inhibitor, talazoparib. Single-agent talazoparib is currently approved in advanced HER2 negative breast cancer with a germline BRCA 1/2 mutation. Trial design: This trial is designed with a safety run-in to determine the recommended phase 2 doses (RP2D) of the talazoparib and gedatolisib combination. The safety run-in will be followed by a phase II single-arm study of the combination, with 2 patient cohorts. Cohort A will be comprised of patients with advanced TNBC with negative or unknown germline BRCA1/2 status. Cohort B will be used for an exploratory analysis, comprised of patients with advanced HER2 negative breast cancer and a germline BRCA1/2 mutation. Eligibility Criteria: For the safety run-in phase, patients ≥18 of age with advanced breast cancer (defined as metastatic or unresectable) are eligible if they meet criteria for either cohort A or cohort B. For cohort A, all patients are required to have at least one line of prior systemic therapy for advanced breast cancer but no more than 2 lines of prior chemotherapy are allowed. For cohort B, no more than 2 lines of prior chemotherapy are allowed, with no limit on prior endocrine or targeted therapies. Patients with type I or poorly controlled diabetes are excluded due to the common side effect of hyperglycemia with gedatolisib. Specific aims: Primary objective of the safety run-in: Determine the RP2D of talazoparib in combination with gedatolisib in patients with advanced HER2 negative (triple negative or BRCA1/2 deficient) breast cancer. Primary objective of the phase II study: Estimate the objective response rate (ORR) in patients with BRCA1/2 negative, or unknown, advanced TNBC - Cohort A. Correlative analysis will include correlating the HR deficiency (HRD) score with clinical response in patients with TNBC. Statistical Methods: In the safety run-in, a 3+3 design will be used for dose escalation. Maximum tolerated dose (MTD) is decided when 6 patients are treated at a dose level with < 2 DLTs. The MTD will be the RP2D. The safety run-in (dose escalation) component will require 9-18 subjects. In the Phase II study, the primary endpoint for sample size justification is ORR for cohort A (TNBC). The null response rate of 5% will be compared to the alternative hypothesis of 20% ORR with a 1-sided alpha level of 0.1. Based on the exact binomial test, we propose 21 subjects to target a power level of 80%. ORR in cohort B is a secondary endpoint. Target Accrual: Our target accrual for the safety run-in is 9-18 participants. Assuming 10% of subjects will be not be evaluable for the primary outcomes, then the number of patients accrued to cohort A will be 24. Cohort B will remain at 12 since it is exploratory. Contact information: The study is being administered through the Big Ten Cancer Research Consortium and can be identified as BTCRC-BRE18-337. Citation Format: Sneha Phadke, Menggang Yu, Kathy Miller, Ami Shah, Oana Danciu, Kari Wisinski. Phase 2 trial with safety-run in of gedatolisib plus talazoparib in advanced triple negative or BRCA1/2 positive HER2 negative breast cancer [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 OT2-06-02.

The NCCN Guidelines for Breast Cancer include up-to-date guidelines for clinical management of patients with carcinoma in situ, invasive breast cancer, Paget disease, phyllodes tumor, inflammatory breast cancer, male breast cancer, and breast cancer during pregnancy. These guidelines are developed by a multidisciplinary panel of representatives from NCCN Member Institutions with breast cancer-focused expertise in the fields of medical oncology, surgical oncology, radiation oncology, pathology, reconstructive surgery, and patient advocacy. These NCCN Guidelines Insights focus on the most recent updates to recommendations for adjuvant systemic therapy in patients with nonmetastatic, early-stage, hormone receptor-positive, HER2-negative breast cancer.

Combining cyclin-dependent kinase (CDK) inhibitors with endocrine therapy improves outcomes for metastatic estrogen receptor positive (ER+) breast cancer patients but its value in earlier stage patients is unclear. We examined evolutionary trajectories of early-stage breast cancer tumors, using single cell RNA sequencing (scRNAseq) of serial biopsies from the FELINE clinical trial (#NCT02712723) of endocrine therapy (letrozole) alone or combined with the CDK inhibitor ribociclib. Despite differences in subclonal diversity evolution across patients and treatments, common resistance phenotypes emerged. Resistant tumors treated with combination therapy showed accelerated loss of estrogen signaling with convergent up-regulation of JNK signaling through growth factor receptors. In contrast, cancer cells maintaining estrogen signaling during mono- or combination therapy showed potentiation of CDK4/6 activation and ERK upregulation through ERBB4 signaling. These results indicate that combination therapy in early-stage ER+ breast cancer leads to emergence of resistance through a shift from estrogen to alternative growth signal-mediated proliferation.

Paclitaxel (Taxol) is a cornerstone of cancer treatment. However, its mechanism of cytotoxicity is incompletely understood and not all patients benefit from treatment. We show that patients with breast cancer did not accumulate sufficient intratumoral paclitaxel to induce mitotic arrest in tumor cells. Instead, clinically relevant concentrations induced multipolar mitotic spindle formation. However, the extent of early multipolarity did not predict patient response. Whereas multipolar divisions frequently led to cell death, multipolar spindles focused into bipolar spindles before division at variable frequency, and maintaining multipolarity throughout mitosis was critical to induce the high rates of chromosomal instability necessary for paclitaxel to elicit cell death. Increasing multipolar divisions in paclitaxel resulted in improved cytotoxicity. Conversely, decreasing paclitaxel-induced multipolar divisions reduced paclitaxel efficacy. Moreover, we found that preexisting chromosomal instability sensitized breast cancer cells to paclitaxel. Both genetic and pharmacological methods of inducing chromosomal instability were sufficient to increase paclitaxel efficacy. In patients, the amount of pretreatment chromosomal instability directly correlated with taxane response in metastatic breast cancer such that patients with a higher rate of preexisting chromosomal instability showed improved response to taxanes. Together, these results support the use of baseline rates of chromosomal instability as a predictive biomarker for paclitaxel response. Furthermore, they suggest that agents that increase chromosomal instability or maintain multipolar spindles throughout mitosis will improve the clinical utility of paclitaxel.