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Purpose:Plasma genotyping may identify mutations in potentially “actionable” cancer genes, such as BRCA1/2, but their clinical significance is not well-defined. We evaluated the characteristics of somatically acquired BRCA1/2 mutations in patients with metastatic breast cancer (MBC).Experimental Design:Patients with MBC undergoing routine cell-free DNA (cfDNA) next-generation sequencing (73-gene panel) before starting a new therapy were included. Somatic BRCA1/2 mutations were classified as known germline pathogenic mutations or novel variants, and linked to clinicopathologic characteristics. The effect of the PARP inhibitor, olaparib, was assessed in vitro, using cultured circulating tumor cells (CTCs) from a patient with a somatically acquired BRCA1 mutation and a second patient with an acquired BRCA2 mutation.Results:Among 215 patients with MBC, 29 (13.5%) had somatic cfDNA BRCA1/2 mutations [nine (4%) known germline pathogenic and rest (9%) novel variants]. Known germline pathogenic BRCA1/2 mutations were common in younger patients (P = 0.008), those with triple-negative disease (P = 0.022), and they were more likely to be protein-truncating alterations and be associated with TP53 mutations. Functional analysis of a CTC culture harboring a somatic BRCA1 mutation demonstrated high sensitivity to PARP inhibition, while another CTC culture harboring a somatic BRCA2 mutation showed no differential sensitivity. Across the entire cohort, APOBEC mutational signatures (COSMIC Signatures 2 and 13) and the “BRCA” mutational signature (COSMIC Signature 3) were present in BRCA1/2-mutant and wild-type cases, demonstrating the high mutational burden associated with advanced MBC.Conclusions:Somatic BRCA1/2 mutations are readily detectable in MBC by cfDNA analysis, and may be present as both known germline pathogenic and novel variants.

Purpose: Metastatic HR+ breast cancer initially responds to serial courses of endocrine therapy but ultimately becomes refractory Elacestrant, a new generation oral selective estrogen receptor degrader (SERD) and antagonist, has demonstrated efficacy in a subset of women with advanced HR+ breast cancer (Bidard FC, et al J Clin Oncol 2022;40:3246-3256), but there are few patient-derived models to characterize its effect in advanced cancers with diverse treatment histories and acquired mutations In this translational study, we tested the efficacy of elacestrant on ex vivo circulating tumor cells (CTCs) and patient-derived xenograft (PDX) mouse models with serial exposures to fulvestrant and conducted a reanalysis of the EMERALD trial to evaluate the efficacy of elacestrant vs standard endocrine therapy among patients who had received prior fulvestrant. Methods: Using PDX models and cultured CTCs isolated from metastatic breast cancer patients, we analyzed sensitivity to elacestrant, compared with the currently approved SERD, fulvestrant We further analyzed clinical responses to elacestrant, compared with endocrine therapy, among women who had previously been treated with a fulvestrant-containing regimen from the recent phase 3 EMERALD Study (NCT03778931), Results: In a patient-derived HR+ breast cancer PDX model, we demonstrate that elacestrant, at a clinically relevant dose, is highly effective in tumors extensively pretreated with fulvestrant In cultured CTCs isolated from HR+ breast cancer patients who had received multiple prior treatments, including fulvestrant, elacestrant is active, independent of mutations in the estrogen receptor (ESR1) or Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA) genes In these cells, elacestrant suppresses estrogen-receptor (ER) signaling at concentrations below those required for protein degradation Post-hoc analysis of the EMERALD clinical trial confirms that elacestrant is effective in patient populations previously treated with fulvestrant-containing regimens, irrespective of ESR1 mutation status. Conclusion: In the EMERALD study, elacestrant showed significantly prolonged PFS compared with SOC (including fulvestrant) Using a PDX model and CTCs isolated from patients with MBC from the EMERALD study, this translational investigation demonstrates that elacestrant retains efficacy in breast cancer cells that have acquired resistance to currently available ER-targeting therapies Elacestrant may be an option for patients with HR+/HER2- breast cancer whose disease progressed while receiving fulvestrant in the metastatic setting. Citation Format: Taronish D. Dubash, Aditya Bardia, Brian Chirn, Brittany A. Reeves, Joseph A. LiCausi, Risa Burr, Ben S. Wittner, Sumit Rai, Hitisha Patel, Teeru Bihani, Heike Arlt, Francois-Clement Bidard, Virginia G. Kaklamani, Philippe Aftimos, Javier Cortés, Simona Scartoni, Monica Binaschi, Nassir Habboubi, A. John Iafrate, Mehmet Toner, Daniel A. Haber, Shyamala Maheswaran. Efficacy of novel oral SERD elacestrant in fulvestrant-refractory hormone receptor-positive (HR+) breast cancer: a translational investigation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT073.

Purpose: Plasma genotyping may identify mutations in potentially “actionable” cancer genes, such as BRCA1/2, but their clinical significance is not well-defined. We evaluated the characteristics of somatically acquired BRCA1/2 mutations in patients with metastatic breast cancer (MBC). Experimental Design: Patients with MBC undergoing routine cell-free DNA (cfDNA) next-generation sequencing (73-gene panel) before starting a new therapy were included. Somatic BRCA1/2 mutations were classified as known germline pathogenic mutations or novel variants, and linked to clinicopathologic characteristics. The effect of the PARP inhibitor, olaparib, was assessed in vitro, using cultured circulating tumor cells (CTCs) from a patient with a somatically acquired BRCA1 mutation and a second patient with an acquired BRCA2 mutation. Results: Among 215 patients with MBC, 29 (13.5%) had somatic cfDNA BRCA1/2 mutations [nine (4%) known germline pathogenic and rest (9%) novel variants]. Known germline pathogenic BRCA1/2 mutations were common in younger patients (P = 0.008), those with triple-negative disease (P = 0.022), and they were more likely to be protein-truncating alterations and be associated with TP53 mutations. Functional analysis of a CTC culture harboring a somatic BRCA1 mutation demonstrated high sensitivity to PARP inhibition, while another CTC culture harboring a somatic BRCA2 mutation showed no differential sensitivity. Across the entire cohort, APOBEC mutational signatures (COSMIC Signatures 2 and 13) and the “BRCA” mutational signature (COSMIC Signature 3) were present in BRCA1/2-mutant and wild-type cases, demonstrating the high mutational burden associated with advanced MBC. Conclusions: Somatic BRCA1/2 mutations are readily detectable in MBC by cfDNA analysis, and may be present as both known germline pathogenic and novel variants.

Summary Deregulation of oncogenic signals in cancer triggers replication stress. Immediate early genes (IEGs) are rapidly and transiently expressed following stressful signals, contributing to an integrated response. Here, we find that the orphan nuclear receptor NR4A1 localizes across the gene body and 3′ UTR of IEGs, where it inhibits transcriptional elongation by RNA Pol II, generating R-loops and accessible chromatin domains. Acute replication stress causes immediate dissociation of NR4A1 and a burst of transcriptionally poised IEG expression. Ectopic expression of NR4A1 enhances tumorigenesis by breast cancer cells, while its deletion leads to massive chromosomal instability and proliferative failure, driven by deregulated expression of its IEG target, FOS. Approximately half of breast and other primary cancers exhibit accessible chromatin domains at IEG gene bodies, consistent with this stress-regulatory pathway. Cancers that have retained this mechanism in adapting to oncogenic replication stress may be dependent on NR4A1 for their proliferation.

Plasma genotyping identifies potentially actionable mutations at variable mutant allele frequencies, often admixed with multiple subclonal variants, highlighting the need for their clinical and functional validation. We prospectively monitored plasma genotypes in 143 women with endocrine-resistant metastatic breast cancer (MBC), identifying multiple novel mutations including HER2 mutations (8.4%), albeit at different frequencies highlighting clinical heterogeneity. To evaluate functional significance, we established ex vivo culture from circulating tumor cells (CTCs) from a patient with HER2-mutant MBC, which revealed resistance to multiple targeted therapies including endocrine and CDK 4/6 inhibitors, but high sensitivity to neratinib (IC50: 0.018 μM). Immunoblotting analysis of the HER2-mutant CTC culture line revealed high levels of HER2 expression at baseline were suppressed by neratinib, which also abrogated downstream signaling, highlighting oncogenic dependency with HER2 mutation. Furthermore, treatment of an index patient with HER2-mutant MBC with the irreversible HER2 inhibitor neratinib resulted in significant clinical response, with complete molecular resolution of two distinct clonal HER2 mutations, with persistence of other passenger subclones, confirming HER2 alteration as a driver mutation. Thus, driver HER2 mutant alleles that emerge during blood-based monitoring of endocrine-resistant MBC confer novel therapeutic vulnerability, and ex vivo expansion of viable CTCs from the blood circulation may broadly complement plasma-based mutational analysis in MBC.

PURPOSE: Plasma genotyping may identify mutations in potentially “actionable” cancer genes, such as BRCA1/2, but their clinical significance is not well defined. We evaluated the characteristics of somatically acquired BRCA1/2 mutations in patients with MBC. METHODS: Patients with MBC undergoing routine cell-free DNA (cfDNA) next generation sequencing (73 gene panel) before starting a new therapy were included. Somatic BRCA1/2 mutations were classified as known germline-pathogenic mutations or novel variants, and linked to clinicopathological characteristics. The effect of the PARP inhibitor olaparib was assessed in vitro, using cultured circulating tumor cells (CTCs) from a patient with a somatically acquired BRCA1 mutation and a second patient with an acquired BRCA2 mutation. RESULTS: Among 215 MBC patients, 29 (13.5%) had somatic cfDNA BRCA1/2 mutations (nine (4%) known germline-pathogenic and rest (9%) novel variants). Known germline-pathogenic BRCA1/2 mutations were common in younger patients (p=0.008), those with triple-negative disease (p=0.022), and they were more likely to be protein-truncating alterations and be associated with TP53 mutations. Functional analysis of a CTC culture harboring a somatic BRCA1-mutation demonstrated high sensitivity to PARP inhibition, while another CTC culture harboring a somatic BRCA2 mutation showed no differential sensitivity. Across the entire cohort, APOBEC mutational signatures (COSMIC Signatures 2 and 13) and the “BRCA” mutational signature (COSMIC Signature 3) were present in BRCA1/2 mutant and wild-type cases, demonstrating the high mutational burden associated with advanced MBC. CONCLUSION: Somatic BRCA1/2 mutations are readily detectable in MBC by cfDNA analysis, and may be present as both known germline-pathogenic and novel variants.

Plasma genotyping identifies potentially actionable mutations at variable mutant allele frequencies, often admixed with multiple subclonal variants, highlighting the need for their clinical and functional validation. We prospectively monitored plasma genotypes in 143 women with endocrine-resistant metastatic breast cancer (MBC), identifying multiple novel mutations including HER2 mutations (8.4%), albeit at different frequencies highlighting clinical heterogeneity. To evaluate functional significance, we established ex vivo culture from circulating tumor cells (CTCs) from a patient with HER2-mutant MBC, which revealed resistance to multiple targeted therapies including endocrine and CDK 4/6 inhibitors, but high sensitivity to neratinib (IC50: 0.018 μM). Immunoblotting analysis of the HER2-mutant CTC culture line revealed high levels of HER2 expression at baseline were suppressed by neratinib, which also abrogated downstream signaling, highlighting oncogenic dependency with HER2 mutation. Furthermore, treatment of an index patient with HER2-mutant MBC with the irreversible HER2 inhibitor neratinib resulted in significant clinical response, with complete molecular resolution of two distinct clonal HER2 mutations, with persistence of other passenger subclones, confirming HER2 alteration as a driver mutation. Thus, driver HER2 mutant alleles that emerge during blood-based monitoring of endocrine-resistant MBC confer novel therapeutic vulnerability, and ex vivo expansion of viable CTCs from the blood circulation may broadly complement plasma-based mutational analysis in MBC.

Background: Phenytoin is a common medication for seizure treatment and prophylaxis in the intensive care unit (ICU). The clinical utility of the Sheiner-Tozer equation for adjusting total phenytoin levels for hypoalbuminemia remains controversial. Objective: The purpose of this study was to evaluate the correlation of this formula in predicting phenytoin serum concentrations. Methods: A retrospective cohort study was conducted in the adult ICU between January 1, 2010, and June 21, 2013. Patients meeting the following study criteria were included: age ≥18 years, admission to the ICU, simultaneously drawn total and free serum phenytoin concentrations with albumin ≤48 hours of phenytoin draws. Study end points were the correlation as well as the level of agreement in the interpretation of the free and adjusted phenytoin concentrations using the Sheiner-Tozer formula in critically ill patients with hypoalbuminemia. Results: A total of 238 patients were analyzed. Mean adjusted total phenytoin and free levels were 16.1 ± 8.1 and 1.5 ± 0.8 µg/mL, respectively ( r = 0.817; P < 0.001). Absolute agreement with level interpretation between adjusted total phenytoin and free levels was 77% (κ = 0.633; P < 0.001). Adjusted phenytoin serum concentrations more frequently overestimated the free level. Conclusions: There is a significant correlation between free and adjusted total phenytoin levels using the Sheiner-Tozer equation in critically ill patients. However, disagreement was noted with interpretation, primarily because of the adjusted concentration overestimating the free level. This imprecision may lead to inaccurate decision making regarding the management of phenytoin in this patient population. Thus, free phenytoin levels should be utilized.

Women with metastatic hormone receptor positive (HR+) breast cancer benefit from therapies targeting the Estrogen Receptor (ER), but the tumors eventually acquire resistance leading to disease progression. Endocrine resistance involves multiple mechanisms, including deregulation of ER signaling due to activating mutations in the ligand-binding domain of the ESR1 gene. In addition, recent reports describe specific contexts of ESR1 mutations in the preclinical setting where tamoxifen and fulvestrant have demonstrated limited activity. These data together with the frequency of ESR1 mutations (~20-60%) observed in mBC patients in the clinic suggest a need for more effective agents. The activity of elacestrant (RAD1901), an oral selective estrogen receptor degrader (SERD) with demonstrated activity against tumors harboring ESR1 mutations, both in preclinical models and in patients with previously treated advanced breast cancer, was assessed in ex vivo cultures of circulating tumor cells (CTCs) derived from women with metastatic HR+ breast cancer who had received multiple prior hormone therapies. Cultured breast CTCs underwent apoptosis following exposure to elacestrant, and CTCs harboring mutant ESR1 were significantly growth inhibited (IC50: 17.65 ± 3.32 nM) by elacestrant compared to the standard-of-care (SOC) agent fulvestrant. Furthermore, elacestrant eliminated the fulvestrant-resistant CTC populations in vitro. Elacestrant treatment led to significant suppression of ER target genes, PGR and GREB1. Elacestrant is currently being investigated versus standard of care endocrine monotherapy in a Phase 3 clinical trial (EMERALD) in patients with ER+/HER2- advanced/metastatic breast cancer. A co-primary endpoint of the EMERALD trial is evaluation of progression-free survival in patients whose tumors harbor ESR1 mutations. Citation Format: Taronish Dorab Dubash, Aditya Bardia, Brittany A Reeves, Brian Chirn, Annamaria Szabolcs, Ben S Wittner, John Iafrate, David Ting, Hitisha K Patel, Teeru Bihani, Mehmet Toner, Daniel A Haber, Shyamala Maheswaran. Elacestrant (RAD1901) inhibits growth of ex vivo cultured circulating tumor cells derived from hormone receptor-positive metastatic breast cancer (mBC) patients including those harboring ESR1 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 P4-01-06.