Endocrine Therapy Resistance Research Articles

Acetate utilization promotes hormone therapy resistance in prostate cancer through neuroendocrine differentiation

AimsTumor fatty acid (FA) metabolic plasticity plays a pivotal role in resistance to therapy and poses limitations to anticancer strategies. In this study, our aim is to uncover the role of acetate metabolism in neurodifferentiation (NED)-mediated castration-resistant prostate cancer (CRPC). MethodsWe conducted analyses using LC-MS/MS on clinical prostate cancer tissue before and after hormone therapy. We established tumor xenograft mouse models, primary tumor cells, and human-derived organoids to detect the novel mechanism of NED and to identify potential therapies. ResultsThe hormone therapy-induced upregulation of acetate metabolism was mediated by acyl-CoA synthetase short-chain family member 2 (ACSS2), which increased c-MYC expression for NED induction. Notably, combined treatment with an ACSS2 inhibitor and enzalutamide significantly reduced the xenograft tumor volume. ConclusionOur findings uncovered the critical role of acetate metabolism in NED-mediated CRPC and suggest that ACSS2 inhibitors may represent a novel, low-toxicity strategy when combined with hormone therapy for treating patients with NED-mediated CRPC.

7881 Using Selective Estrogen Receptor Modulators to Reveal Therapeutic Vulnerabilities in ESR1 Mutant Breast Cancer

Abstract Disclosure: S.W. Fanning: Grant Recipient; Self; Olema Oncology. Hotspot activating somatic mutations to ESR1, the gene for estrogen receptor alpha (ER), enable hormone therapy resistance and drive breast cancer metastasis. The Y537S missense mutation within the ER ligand binding domain is the most activating, therapy resistant, and increases metastatic potential. Next generation antiestrogens including lasofoxifene (laso) and elacestrant (RAD1901) show improved progression-free survival over fulvestrant (fulv), but further progress is needed to achieve durable response. To fully address Y537S ESR1, it is critically important to understand how the mutation boosts metastasis. We have developed a small molecule antiestrogen, T6I-29, that uniquely impacts transcriptional pathways related to cell-cell adhesions and morphology. Two allele-specific phenotypes identified by the Oesterreich Laboratory enhance the metastasis of Y537S ESR1 breast cancer cells. T6I-29-specific pathway effects were driven by a significant downregulation of dickkopf-1 (DKK1), which was also observed but to a lesser extent with fulv, laso, and RAD1901. DKK1 is an immunosuppressive, tumor-secreted glycoprotein and its overexpression correlates with poor outcomes in multiple cancers. However, DKK1 acts as a tumor-suppressor in colorectal cancer, which does not rely on ER. Our preliminary studies suggest that DKK1 plays a role in ER+ breast cancer progression. We measured significantly elevated DKK1 protein levels in the blood plasma of 108 diverse ER+ breast cancer patients compared to 100 matched healthy female donors and levels increase coincident with tumor stage. Unfortunately, we lack ESR1 mutation status and other critical details to make further insights on these patients. DKK1 gene expression and protein secretion is stimulated in WT cells by the pathogenic hormone 17β-estradiol (E2), while it is constitutively expressed in Y537S ESR1 cells. DKK1 is characterized as a WNT antagonist but can also induce PI3K/Akt signaling. Interestingly, DKK1 is elevated and ERα responsive in WT not mutant PI3KCA cell lines. DKK1 knockdown also reduced proliferation of Y537S ESR1 breast cancer cells where elevated DKK1 was observed. Presentation: 6/3/2024

Induction of the TEAD Co-activator VGLL1 by Estrogen Receptor-Targeted Therapy Drives Resistance in Breast Cancer.

Resistance to endocrine therapies (ET) is common in estrogen receptor (ER) positive breast cancer, and most relapsed patients die with ET-resistant disease. While genetic mutations provide explanations for some relapses, mechanisms of resistance remain undefined in many cases. Drug-induced epigenetic reprogramming has been shown to provide possible routes to resistance. By analyzing histone H3 lysine 27 acetylation (H3K27ac) profiles and transcriptional reprogramming in models of ET resistance, we discovered that selective ER degraders (SERDs), such as fulvestrant, promote expression of VGLL1, a co-activator for TEAD transcription factors. VGLL1, acting via TEADs, promoted expression of genes that drive growth of fulvestrant-resistant breast cancer cells. Pharmacological disruption of VGLL1/TEAD4 interaction inhibited VGLL1/TEAD-induced transcriptional programs to prevent growth of resistant cells. EGFR was among the VGLL1/TEAD-regulated genes, and VGLL1-directed EGFR upregulation sensitized fulvestrant-resistant breast cancer cells to EGFR inhibitors. Taken together, these findings identify VGLL1 as a transcriptional driver in ET resistance and advance therapeutic possibilities for relapsed ER+ breast cancer patients.

Neuroendocrine differentiation (ND) in sensitivity of neuroendocrine tumor (NET) cells to ONC201/TIC10 cancer therapeutic.

Prostate cancer (PCa) neuroendocrine tumor (NET)-like cells with low or absent androgen receptor (AR) signaling cause hormone therapy resistance and poor prognosis. Small cell lung carcinoma (SCLC), a high-grade NET, presents with metastasis early and has poor survival. ONC201/TIC10 is a first-in-class cancer therapeutic with clinical activity in diffuse gliomas and neuroendocrine tumors. We hypothesized that markers of neuroendocrine differentiation, activation of the integrated stress response (ISR) and the TRAIL pathway, as well as the expression of ClpP, contribute to neuroendocrine tumor cell death and sensitivity to ONC201. We show that PCa and SCLC cell lines (N=6) are sensitive to ONC201, regardless of the extent of neuroendocrine differentiation. Endogenous levels of some NET markers (CgA, FoxO1, ENO2, PGP9.5, SOX2) are present in a spectrum in PCa and SCLC cell lines. Overexpression of neural transcription factor BRN2 in DU145 PCa cells does not increase expression of NET differentiation markers FoxO1, ENO2, PGP9.5, and CgA at 48 hours. However, the transient BRN2 overexpression showed slight decreases in some NET markers on the spectrum while maintaining sensitivity of PCa cells to ONC201 before any phenotypic change related to NET differentiation. Our results show that ONC201 has preclinical activity against PCa including those without NET markers or in PCa cells with transient overexpression of neural transcription factor BRN2. Our results have relevance to activity of ONC201 in PCa where most castrate-resistant androgen-independent cancers are not therapy resistant due to NET differentiation. Importantly, NET differentiation does not promote resistance to ONC201 supporting further clinical investigations across the spectrum of PCa.

Real-world Comparison of P53 Immunohistochemistry and TP53 Mutation Analysis Using Next-generation Sequencing.

TP53 mutation in breast cancer (BC) is associated with chemoresistance, endocrine therapy resistance, and late recurrence, resulting in poor prognosis. Nuclear accumulation of p53 in immunohistochemistry (IHC) is a surrogate marker of TP53 mutation. This study analyzed the frequency, type, and distribution of TP53 mutations in BCs and assessed the efficacy of p53 IHC as a surrogate marker of TP53 mutation. We collected data from 112 BC cases, including the results of p53 IHC and next-generation sequencing (NGS). Over-expression of p53 IHC was observed in 36 patients (32.1%), complete absence in 19 patients (17.0%), aberrant cytoplasmic staining in 1 patient (0.9%), and wild-type in 56 (50.0%) patients. The concordance rate between TP53 mutation and p53 IHC was 88.4% in all BCs, 89.9% in luminal BCs, and 86.0% in triple-negative BCs (TNBC). TNBC, abnormal p53 IHC pattern, p53 IHC over-expression, neoadjuvant chemotherapy (NAC) history, TP53 mutation, and high pre-treatment ki-67 labeling index (≥50%) were significantly associated with worse distant metastasis-free survival (DMFS) and overall survival (OS) (p<0.05). Pre-NAC clinical stage III was associated with worse DMFS but not OS. Multivariate analysis showed that NAC history, TNBC, and p53 IHC over-expression were independent predictors of worse DMFS. An abnormal p53 IHC pattern and NAC history were independent predictors of worse OS. P53 IHC is a valid surrogate marker of TP53 mutation in BC. Accumulation of abnormal p53 alone, regardless of TP53 mutation, was associated with worse DMFS and can be used as an easily accessible biomarker to predict chemoresistance.

A Perspective of PI3K/AKT/mTOR Pathway Inhibitors to Overcome Drug-resistance in Breast Cancer Therapy.

The heterogeneous disease, breast cancer (BC), is a frequently detected cancer today, including hormone receptor-positive (HR+), human epidermal growth factor receptor-2-positive (HER2+), and triple-negative (ER-, PR-, HER2-) BC. Advanced endocrine therapies could improve about 85% HR+ BC patient survival. Still, 20% - 30% of cases of endocrine therapy resistance are observed. For all kinds of breast cancer, drug resistance is a common and dangerous phenomenon, comprised of two types: de novo resistance and acquired resistance (prolonged exposure). According to recent works of literature, the PI3K/AKT/mTOR pathway has become an emerging target for overcoming drug resistance in BC therapy due to its close association with tumour growth and resistance from current therapies. Activation of the PI3K/AKT/mTOR pathway was found to promote multidrug resistance by elevating drugs' outflow. The first orally active PI3K inhibitor, Alpelisib (BYL-719) in fulvestrant combination, was approved for treating HR+/ HER2- metastatic BC. Therefore, utilizing PI3K/mTOR/AKT inhibitors in combination with currently available strategies could be an optimistic approach to overcoming drug resistance and resensitizing drug-resistant tumor cells of BC. Here, in this perspective, BC cancer therapies related to drug resistance, the involvement of PI3K/AKT/mTOR pathway in drug resistance and multi-drug resistance, and the role of PI3K/AKT/mTOR inhibitors in getting rid of drug resistance have been illuminated.

Real-world experience with CDK4/6 inhibitors in hormone receptor-positive metastatic and recurrent breast cancer: findings from an Asian population

PurposeCyclin-dependent kinase 4/6 inhibitors (CDK4/6i) combined with endocrine therapy have demonstrated significant clinical benefits in progression-free and overall survival. This study investigates the outcomes associated with two kinds of CDK4/6i in patients with hormone receptor (HR)-positive metastatic and relapsed breast cancer to inform real-world evidence of treatment strategies.MethodsThis retrospective study included 340 Taiwanese patients with HR-positive advanced breast cancer from the Taipei Veterans General Hospital, between 2018 and 2023. We analyzed patient characteristics, treatment strategies and outcomes associated with two CDK4/6i. The efficacy of patients who experienced economic burden and interrupted CDK4/6i treatment after 2 years of National Health Insurance (NHI) reimbursement was also investigated.ResultsPatients receiving ribociclib and palbociclib showed no significant differences in age, histology, body mass index(BMI), or pathologic status. The distribution of disease status and endocrine therapy partners was comparable between the two groups. Dose reduction was similar, while patients with palbociclib tended to discontinue CDK4/6i usage, and those with ribociclib tended to switch to the other CDK4/6i or endocrine partners. There was no significant difference in progression-free survival (PFS) between the two CDK4/6i in the first-line setting. Adverse prognostic factors were increasing HER2 IHC score, higher Ki-67 levels, visceral and liver metastasis, prior chemotherapy, and endocrine therapy resistance, while higher BMI, bone-only metastasis, and letrozole treatment were associated with a lower risk of progression. The limited follow-up time in our study was insufficient to assess the outcomes of patients treated with interrupted CDK4/6i for up to two years under the NHI reimbursement policy.ConclusionTreatment outcomes between the two types of CDK4/6i did not differ significantly, indicating the safety and efficacy of CDK4/6i for the Asian population. Ribociclib and palbociclib showed similar efficacy in PFS in the real-world setting.

Genomic alterations associated with resistance and circulating tumor DNA dynamics for early detection of progression on CDK4/6 inhibitor in advanced breast cancer.

Combined CDK4/6 inhibitor (CDK4/6i) and endocrine therapy significantly improves outcome for patients with estrogen receptor-positive (ER+) metastatic breast cancer, but drug resistance and thus disease progression inevitably occur. Herein, we aimed to identify genomic alterations associated with combined CDK4/6i and endocrine therapy resistance, and follow the levels of specific mutations in longitudinal circulating tumor DNA (ctDNA) for early detection of progression. From a cohort of 86 patients with ER+ metastatic breast cancer we performed whole exome sequencing or targeted sequencing of paired tumor (N = 8) or blood samples (N = 5) obtained before initiation of combined CDK4/6i and endocrine therapy and at disease progression. Mutations in oncogenic genes at progression were rare, while amplifications of growth-regulating genes were more frequent. The most frequently acquired alterations observed were PIK3CA and TP53 mutations and PDK1 amplification. Longitudinal ctDNA dynamics of mutant PIK3CA or private mutations revealed increased mutation levels at progression in 8 of 10 patients (80%). Impressively, rising levels of PIK3CA-mutated ctDNA were detected 4-17 months before imaging. Our data add to the growing evidence supporting longitudinal ctDNA analysis for real-time monitoring of CDK4/6i response and early detection of progression in advanced breast cancer. Further, our analysis suggests that amplification of growth-related genes may contribute to combined CDK4/6i and endocrine therapy resistance.

Bayesian network analysis of drug treatment strategies for thyroid associated ophthalmopathy.

The first line treatment for moderate to severe active thyroid associated ophthalmopathy is glucocorticoid pulse therapy, but for patients with contraindications to hormone therapy or hormone resistance, it is urgent to find a suitable treatment plan. To find a reliable alternative to hormone pulse therapy for thyroid associated ophthalmopathy by comparing the efficacy with first-line treatment regimens. Search PubMed, Ovid, Web of science, Cochrane library, and Clinical Trials.gov for randomized controlled trials on the treatment of thyroid associated ophthalmopathy published as of July 7, 2024. Quality evaluation and Bayesian network analysis were conducted using RevMan 5.3 software, STATA15.0 software, and ADDIS 1.16.8 software. A total of 666 patients were included in 11 studies and 8 interventions. Network analysis showed that the three interventions of mycophenolate mofetil combined with glucocorticoids, Teprotumumab and 99Tc-MDP were superior to glucocorticoid pulse therapy in improving clinical activity scores and proptosis. The regimen of glucocorticoids combined with statins can improve the quality of life score and diplopia score of patients. Neither methotrexate combined with glucocorticoids nor rituximab alone showed additional advantages when compared with glucocorticoid pulse therapy. Mycophenolate mofetil combined with glucocorticoid therapy is very beneficial for moderate to severe active thyroid associated ophthalmopathy. Mycophenolate mofetil may be a good choice when patients have contraindications to hormone use or hormone resistance. Teprotumumab is very promising and may be able to avoid patients undergoing orbital decompression surgery. The durability and safety of its long-term efficacy need to be further observed.

Evaluation of the Mammalian Aquaporin Inhibitors Auphen and Z433927330 in Treating Breast Cancer.

AQPs contribute to breast cancer progression and metastasis. We previously found that genetic inhibition of Aqp7 reduces primary tumor burden and metastasis in breast cancer. In this study, we utilized two AQP inhibitors, Auphen and Z433927330, to evaluate the efficacy of therapeutic inhibition of AQPs in breast cancer treatment. The inhibitors were evaluated in breast cancer for both cytotoxicity and metabolic stability assays across both murine and human breast cancer cell lines. Both AQP inhibitors also affected the expression of other AQP transcripts and proteins, which demonstrates compensatory regulation between AQP family members. As a single agent, Auphen treatment in vivo extended overall survival but did not impact primary or metastatic tumor burden. However, Auphen treatment made cells more responsive to chemotherapy (doxorubicin) or endocrine treatment (tamoxifen, fulvestrant). In fact, treatment with Tamoxifen reduced overall AQP7 protein expression. RNA-seq of breast cancer cells treated with Auphen identified mitochondrial metabolism genes as impacted by Auphen and may contribute to reducing mammary tumor progression, lung metastasis, and increased therapeutic efficacy of endocrine therapy in breast cancer. Interestingly, we found that Auphen and tamoxifen cooperate to reduce breast cancer cell viability, which suggests that Auphen treatment makes the cells more susceptible to Tamoxifen. Together, this study highlights AQPs as therapeutic vulnerabilities of breast cancer metastasis that are promising and should be exploited. However, the pharmacologic results suggest additional chemical refinements and optimization of AQP inhibition are needed to make these AQP inhibitors appropriate to use for therapeutic benefit in overcoming endocrine therapy resistance.

Targeting Unique Ligand Binding Domain Structural Features Downregulates DKK1 in Y537S ESR1 Mutant Breast Cancer Cells.

Resistance to endocrine therapies remains a major clinical hurdle in breast cancer. Mutations to estrogen receptor alpha (ERα) arise after continued therapeutic pressure. Next generation selective estrogen receptor modulators and degraders/downregulators (SERMs and SERDs) show clinical efficacy, but responses are often non-durable. A tyrosine to serine point mutation at position 537 in the ERα ligand binding domain (LBD) is among the most common and most pathogenic alteration in this setting. It enables endocrine therapy resistance by superceding intrinsic structural-energetic gatekeepers of ER hormone-dependence, it enhances metastatic burden by enabling neomorphic ER-dependent transcriptional programs, and it resists SERM and SERD inhibiton by reducing their binding affinities and abilities to antagonize transcriptional coregulator binding. However, a subset of SERMs and SERDs can achieve efficacy by adopting poses that force the mutation to engage in a new interaction that favors the therapeutic receptor antagonist conformation. We previously described a chemically unconventional SERM, T6I-29, that demonstrates significant anti-proliferative activities in Y537S ERα breast cancer cells. Here, we use a comprehensive suite of structural-biochemical, in vitro, and in vivo approaches to better T6I-29's activities in breast cancer cells harboring Y537S ERα. RNA sequencing in cells treated with T6I-29 reveals a neomorphic downregulation of DKK1, a secreted glycoprotein known to play oncogenic roles in other cancers. Importantly, we find that DKK1 is significantly enriched in ER+ breast cancer plasma compared to healthy controls. This study shows how new SERMs and SERDs can identify new therapeutic pathways in endocrine-resistant ER+ breast cancers.

Reciprocal regulation between RACGAP1 and AR contributes to endocrine therapy resistance in prostate cancer

BackgroundEndocrine resistance driven by sustained activation of androgen receptor (AR) signaling pathway in advanced prostate cancer (PCa) is fatal. Characterization of mechanisms underlying aberrant AR pathway activation to search for potential therapeutic strategy is particularly important. Rac GTPase-activating protein 1 (RACGAP1) is one of the specific GTPase-activating proteins. As a novel tumor proto-oncogene, overexpression of RACGAP1 was related to the occurrence of various tumors.MethodsBioinformatics methods were used to analyze the relationship of expression level between RACGAP1 and AR as well as AR pathway activation. qRT-PCR and western blotting assays were performed to assess the expression of AR/AR-V7 and RACGAP1 in PCa cells. Immunoprecipitation and immunofluorescence experiments were conducted to detect the interaction and co-localization between RACGAP1 and AR/AR-V7. Gain- and loss-of-function analyses were conducted to investigate the biological roles of RACGAP1 in PCa cells, using MTS and colony formation assays. In vivo experiments were conducted to evaluate the effect of RACGAP1 inhibition on the tumor growth.ResultsRACGAP1 was a gene activated by AR, which was markedly upregulated in PCa patients with CRPC and enzalutamide resistance. AR transcriptionally activated RACGAP1 expression by binding to its promoter region. Reciprocally, nuclear RACGAP1 bound to the N-terminal domain (NTD) of both AR and AR-V7, blocking their interaction with the E3 ubiquitin ligase MDM2. Consequently, this prevented the degradation of AR/AR-V7 in a ubiquitin-proteasome-dependent pathway. Notably, the positive feedback loop between RACGAP1 and AR/AR-V7 contributed to endocrine therapy resistance of CRPC. Combination of enzalutamide and in vivo cholesterol-conjugated RIG-I siRNA drugs targeting RACGAP1 induced potent inhibition of xenograft tumor growth of PCa.ConclusionIn summary, our results reveal that reciprocal regulation between RACGAP1 and AR/AR-V7 contributes to the endocrine resistance in PCa. These findings highlight the therapeutic potential of combined RACGAP1 inhibition and enzalutamide in treatment of advanced PCa.

Comprehensive genomic profiling of ESR1, PIK3CA, AKT1, and PTEN in HR(+)HER2(−) metastatic breast cancer: prevalence along treatment course and predictive value for endocrine therapy resistance in real-world practice

BackgroundThe treatment landscape for HR(+)HER2(−) metastatic breast cancer (MBC) is evolving for patients with ESR1 mutations (mut) and PI3K/AKT pathway genomic alterations (GA). We sought to inform clinical utility for comprehensive genomic profiling (CGP) using tissue (TBx) and liquid biopsies (LBx) in HR(+)HER2(−) MBC.MethodsRecords from a de-identified breast cancer clinicogenomic database for patients who underwent TBx/LBx testing at Foundation Medicine during routine clinical care at ~ 280 US cancer clinics between 01/2011 and 09/2023 were assessed. GA prevalence [ESR1mut, PIK3CAmut, AKT1mut, PTENmut, and PTEN homozygous copy loss (PTENloss)] were calculated in TBx and LBx [stratified by ctDNA tumor fraction (TF)] during the first three lines of therapy. Real-world progression-free survival (rwPFS) and overall survival (rwOS) were compared between groups by Cox models adjusted for prognostic factors.Results ~ 60% of cases harbored 1 + GA in 1st-line TBx (1266/2154) or LBx TF ≥ 1% (80/126) and 26.5% (43/162) in LBx TF < 1%. ESR1mut was found in 8.1% TBx, 17.5% LBx TF ≥ 1%, and 4.9% LBx TF < 1% in 1st line, increasing to 59% in 3rd line (LBx TF ≥ 1%). PTENloss was detected at higher rates in TBx (4.3%) than LBx (1% in TF ≥ 1%). Patients receiving 1st-line aromatase inhibitor + CDK4/6 inhibitor (n = 573) with ESR1mut had less favorable rwPFS and rwOS versus ESR1 wild-type; no differences were observed for fulvestrant + CDK4/6 inhibitor (n = 348).ConclusionOur study suggests obtaining TBx for CGP at time of de novo/recurrent diagnosis, followed by LBx for detecting acquired GA in 2nd + lines. Reflex TBx should be considered when ctDNA TF < 1%.

Targeting aberrant fatty acid synthesis and storage in endocrine resistant breast cancer cells.

Lipid metabolic reprogramming is an emerging characteristic of endocrine therapy (ET) resistance in estrogen receptor-positive (ER+) breast cancer. We explored changes in lipid metabolism in ER+ breast cancer cell lines following acquired resistance to common endocrine treatments and tested efficacy of an inhibitor in current clinical trials. We derived ER+ breast cancer cell lines resistant to Tamoxifen (TamR), Fulvestrant (FulvR), and long-term estrogen withdrawal (EWD). Parental and ET resistant cells were subjected to global gene expression and unbiased lipidomic profiling. Lipid storage changes were assessed via neutral lipid staining with Oil Red O (ORO). The impact of the fatty acid synthase (FASN) inhibitor TVB-2640 on the growth and lipid storage of these cell lines was evaluated. Additionally, 13 C 2 -acetate tracing was used to examine FASN activity in parental and ET resistant cells in the absence or presence of TVB-2640. Compared to parental cells, lipid metabolism and processing pathways were notably enriched in ET resistant cells, which exhibited distinct lipidomes characterized by increased triglyceride and polyunsaturated FA (PUFA) species. ET-resistant cells displayed enhanced cytoplasmic lipid droplets. Increased FASN protein levels were observed in ET-resistant cells, and TVB-2640 effectively inhibited FASN activity. FASN inhibition reduced cell growth in some but not all cell lines and ET resistance types and did not correlate to lipid storage reduction. 13 C 2 -acetate tracing confirmed reduced palmitate synthesis and enhanced PUFA synthesis in ET-resistant cells, especially when combined with FulvR. ET resistant breast cancer cells exhibit a shift towards enhanced triglyceride storage and complex lipids enriched with PUFA acyl chains. While targeting FASN alongside ET may not fully overcome ET resistance in our models, focusing on the unique lipid metabolic dependencies, such as PUFA pathways, may present a promising alternative strategy for treating ET resistant breast cancer.

Comprehensive clinical characteristics and ctDNA mutational profile analysis of endocrine resistance/sensitivity to adjuvant ET therapy in luminal breast cancer from the GEICAM/2014-03_RegistEM registry.

1011 Background: Patients (pts) with hormone receptor-positive (HR) early breast cancer (BC) may develop resistance to adjuvant endocrine therapies (ET), with relapse occurring months (m) to decades after surgery. Our study explores clinical characteristics, prognosis, and oncogenic drivers in circulating tumor DNA (ctDNA) at relapse in HR+ BC pts, based on their sensitivity to adjuvant ET. Methods: In the REGISTEM study, we identified 805 primary HR-positive BC pts who developed metastatic relapse. These cases were classified based on their sensitivity to adjuvant ET, according to the 5th ESO-ESMO ABC Guidelines: 1ET-resistance (1ET-R, relapse within the first 2 years of adj ET; n=138), 2ET-resistance (2ET-R, relapse after the first 2 years or &lt;12 m after completing adj ET; n=284), and ET-sensitive (ET-S, relapse after &gt;12 m of completing adj ET; n=383). Out of this cohort, 139 individuals had plasma samples obtained prior to the initiation of the first-line treatment for advanced disease, and these samples were analyzed using the AVENIO ctDNA assay. Results: Pts with 1ET-R were significantly more likely to have grade 3, stage III, and highly proliferative tumors (Ki67&gt;20%). The median time to distant relapse was 24 m for 1ET-R, 52.8 m for 2ET-R, and 124 m for ET-S cases. For first-line treatment, the median PFS was 8.1 m for 1ET-R, 13 m for 2ET-R, and 21.2 m for ET-S relapse (p&lt;0.0001). With a median follow-up in the advanced setting of 34 m, median OS was 28 m for 1ET-R, 46 m for 2ET-R, and 55 m for ET-S relapses (p&lt;0.0001). Pts with ET resistance had more mutations (mut) and higher mut allele frequency than ET-sensitive relapse. Pts with ET resistance showed higher incidence of mut in TP53, ESR1, PTEN, andhigh copy number in EGFRand ERBB2 than ET-sensitive, but similar incidence of mut in PIK3CA.Mut in ESR1 and high copy number in EGFR were correlated with poorer PFS, while ESR1 mut were additionally associated with worse OS, although these associations were not independent of the ET relapse groups (Table). Furthermore, mut in PIK3CA were associated to worse PFS only in ET-R pts. Conclusions: In HR-positive BC, both primary and secondary resistance to adjuvant ET represents a highly aggressive disease with enriched actionable genetic alterations detected at the time of diagnosis of metastatic relapse. Addressing these alterations has the potential to mitigate the unfavorable prognosis associated with these cases. Clinical trial information: NCT02819882 .[Table: see text]

A phase 2, open-label, single-arm, prospective, multicenter study of nab-sirolimus plus letrozole in advanced or recurrent endometrioid endometrial cancer.

TPS5640 Background: This clinical trial is designed to evaluate the antitumor activity of nab-sirolimus in combination with letrozole in patients with advanced or recurrent endometrioid endometrial carcinoma (EEC). Despite recent pivotal data demonstrating improved outcomes with immunotherapy plus chemotherapy, regardless of mismatch repair status, alternative nonchemotherapy-based treatment options for patients with advanced or recurrent EEC remain necessary. Dysregulation of mTOR signaling is implicated in the pathology of EEC, in which &gt;80% harbor PTEN or PI3K/Akt/mTOR pathway alterations. Moreover, crosstalk between mTOR and estrogen receptor signaling pathways is associated with endocrine therapy resistance. GOG-3007 and other phase 2 studies have demonstrated that the combination of conventional mTOR inhibitors (mTORis) and endocrine therapy provides clinical benefit in patients with EEC. nab-Sirolimus is an mTOR inhibitor that uses albumin-bound sirolimus nanoparticles given by IV and is approved by the US FDA for malignant perivascular epithelioid cell tumor. Preclinical data of nab-sirolimus demonstrated improved tumor accumulation, mTOR inhibition, and tumor growth suppression compared with conventional mTORis. We hypothesize that the simultaneous inhibition of mTOR and ER with nab-sirolimus and letrozole, respectively, may provide a signal of synergistic antitumor activity in patients with recurrent EEC. Methods: In this phase 2, open-label, single-arm, multicenter study (NCT05997017), nab-sirolimus (100 mg/m2, intravenous, on days 1 and 8 of each 21-day cycle) and letrozole (2.5 mg, oral, daily) are administered to patients (~29 planned, Simon’s 2-stage design) with clinically confirmed, advanced, or recurrent EEC. Eligibility criteria include age ≥18 years, ≤1 prior chemotherapy-based regimen in the advanced, metastatic, or recurrent setting, prior adjuvant chemotherapy is permitted, Eastern Cooperative Oncology Group performance status score 0 or 1, naive to mTORi, and measurable disease (per Response Evaluation Criteria in Solid Tumors [RECIST] v1.1). The primary endpoint is best overall response rate by RECIST v1.1. Key secondary endpoints include duration of response, disease control rate (defined as complete response plus partial response plus stable disease of ≥12 weeks after start of treatment), progression-free survival, overall survival, and safety. Exploratory endpoints include correlation of baseline molecular biomarkers with clinical outcomes. The trial is open for enrollment. Clinical trial information: NCT05997017 .

Neoadjuvant targeted therapy in early resectable HER2-mutant lobular breast cancer.

TPS630 Background: Invasive lobular carcinoma (ILC) accounts for approximately 10-15% of all breast cancers and is characterized by hormone receptor positivity (HR+), low Ki-67 score, and loss of cell adhesion receptors such as E-cadherin. While these tumors tend to be indolent initially, late recurrences and distant metastasis are common, and ILC has very low rates of pathologic complete response (pCR) to both neoadjuvant chemotherapy and endocrine therapy (ET). HER2 ( ERBB2) mutations are enriched in about 10% of unselected ILC, with some reports showing the prevalence as high as 27% in pleomorphic and higher grade ILCs. HER2 mutations in ILC are associated with ET resistance and a worse prognosis. Neratinib is a HER2 tyrosine kinase inhibitor (TKI) currently FDA-approved for adjuvant treatment of HER2-amplified (HER2+) early-stage breast cancer. It has shown safety and efficacy in combination with ET in metastatic HER2-mutant HR+ breast cancer. We hypothesize that neoadjuvant treatment with neratinib and ET will improve pathologic responses in newly diagnosed HER2-mutant HR+ ILC. Methods: This study (NCT05919108) is an open label multi-site Phase II clinical trial for 30 patients with Stage I-III ILCs and a HER2 activating mutation. Patients will be identified by next generation sequencing (NGS) of the diagnostic biopsy and randomized to 4 weeks of ET +/- neratinib; a biopsy will be done at the end of the lead-in phase. All patients will then receive ET and neratinib for 20 weeks prior to proceeding to surgery. To optimize tolerability, the first cycle of neratinib will include a dose escalation and antidiarrheal prophylaxis. The primary objective of this study will be the pre-operative endocrine prognostic index (PEPI) score, which will be compared to a historical PEPI-0 rate for patients with HR+ breast cancer treated with neoadjuvant ET. Secondary objectives will include pCR, residual cancer burden, and rates of breast conserving surgery. Correlative studies will include biomarkers of tumor cell cycle arrest, apoptosis, HER2 pathway activation, and gene expression on the pre- and on-treatment (4 week) biopsy from the lead-in phase to document molecular synergy between neratinib and ET. Patient-derived organoids from the 4-week biopsies will be established and subjected to single-cell RNA, ATAC seq, and DNA sequencing to study tumor evolution and elucidate epigenetic programs and somatic alterations that allow breast cancer survival despite continuous HER2-directed therapy. This is a first-in-kind, mechanism-based, molecularly targeted combination of a HER2 TKI and ET in HER2-mutant ILC. We posit results from this study may generate a clear signal of clinical activity leading to a randomized phase III trial aimed at incorporating HER2-directed therapies as a treatment for operable, early stage ILC. This signal may also suggest for the first time the added value of NGS in patients with newly diagnosed HR+ breast cancer. Clinical trial information: NCT05919108 .

ESR1 mutations kit performance with SensID ESR1 reference set 1% AF.

e13087 Background: Oestrogen receptor 1 (ER/ ESR1) mutations have arisen as crucial biomarkers for endocrine therapy resistance in ER positive metastatic breast cancer. Detecting these mutations is key for understanding acquired resistance during treatment. Current clinical trials, exploring ESR1 mutation prevalence during various treatments, often use expensive and time-consuming next-generation sequencing (NGS)-based assays or digital PCR (dPCR) assays that require specialist equipment. The APIS ESR1 Mutations Kit offers a cost effective, rapid, and highly sensitive (≤ 1% MAF) alternative to NGS, and dPCR assays. This targeted, qPCR-based assay detects eleven ESR1 mutations across ESR1 exons 5 (E380Q), 7 (S463P) and 8 (P535H, L536R/Q/H/P, Y537C/S/N and D538G). Designed for circulating-free DNA (cfDNA) samples, it reduces the need for invasive procedures in clinical settings, providing researchers in both centralised and decentralised laboratory settings the flexibility to explore ESR1 mutations with precision. Here, we demonstrate the performance of the APIS ESR1 Mutations Kit using the first commercially available cfDNA reference set (SensID ESR1 Reference Set 1% AF cfDNA) for nine ESR1 mutations (Table). Methods: The SensID ESR1 standards were assessed with the APIS ESR1 Mutations Kit. All vials were diluted with nuclease-free water to 3 ng/µL, resulting in 15 ng total input per reaction. Samples were run according to the APIS ESR1 Mutations Kit handbook. All PCR runs were performed using a QuantStudio 5 Dx (ThermoFisher) instrument. Results: The APIS ESR1 Mutations Kit successfully detected all ESR1 mutations, with expected target calls for the nine mutations at 1% MAF. No false positive results for the remaining mutation targets were observed. Similarly, no false positives for any mutation targets were reported for vial 5, containing the wildtype (WT) sample, reaffirming the kit’s accuracy in distinguishing WT and mutant samples. Conclusions: The ESR1 Mutations Kit demonstrated highly sensitive and specific detection of the SensID ESR1 Reference Set 1% AF cfDNA, confirming the kit’s LoD of ≤ 1% MAF with externally validated samples. [Table: see text]

A Functional Survey of the Regulatory Landscape of Estrogen Receptor-Positive Breast Cancer Evolution.

Only a handful of somatic alterations have been linked to endocrine therapy resistance in hormone-dependent breast cancer, potentially explaining ∼40% of relapses. If other mechanisms underlie the evolution of hormone-dependent breast cancer under adjuvant therapy is currently unknown. In this work, we employ functional genomics to dissect the contribution of cis-regulatory elements (CRE) to cancer evolution by focusing on 12 megabases of noncoding DNA, including clonal enhancers, gene promoters, and boundaries of topologically associating domains. Parallel epigenetic perturbation (CRISPRi) in vitro reveals context-dependent roles for many of these CREs, with a specific impact on dormancy entrance and endocrine therapy resistance. Profiling of CRE somatic alterations in a unique, longitudinal cohort of patients treated with endocrine therapies identifies a limited set of noncoding changes potentially involved in therapy resistance. Overall, our data uncover how endocrine therapies trigger the emergence of transient features which could ultimately be exploited to hinder the adaptive process. Significance: This study shows that cells adapting to endocrine therapies undergo changes in the usage or regulatory regions. Dormant cells are less vulnerable to regulatory perturbation but gain transient dependencies which can be exploited to decrease the formation of dormant persisters.

Abstract PO2-16-03: ESR1 genomic alterations (GAs) and coexistent putative resistance alterations in comprehensive genomic profiling (CGP) of metastatic breast cancer (MBC)

Abstract Background: ESR1 mutations (ESR1mut) are an established biomarker of endocrine therapy (ET) resistance in patients (pts) with hormone receptor positive (HR+) MBC. Moreover, ESR1mut acquired in response to standard-of-care ET now confer access to novel ET recently approved by health authorities. However, the nuanced genomic landscape beyond the simple presence or absence of ESR1mut may be critically important. We report on detailed ESR1 GAs detected by tissue and liquid biopsies (TBx, LBx) and examine the prevalence of co-occurring GAs in other genes reported to confer intrinsic or acquired resistance to ET. Methods: CGP results from MBC biopsies were retrospectively analyzed (TBx 33,653 pts; LBx 7,134 pts) using FoundationOne® CDx and FoundationOne® Liquid CDx, hybrid-capture based sequencing platforms profiling 324 cancer-related genes. Foundation Medicine’s ctDNA tumor fraction (TF) is a composite algorithm prioritizing aneuploidy at higher levels to avoid germline signal and prioritizing variant allele frequency (VAF) of canonical alterations at lower levels to maximize dynamic range. Results: ESR1mut were detected in 3,755 (11.2%) TBx and 1,513 (21.2%) LBx. Among samples with ESR1mut, multiple ESR1mut were more common in LBx (19% 2, 12% 3+) than in TBx (6% 2, 0.5% 3+), with up to 10 different ESR1mut seen in one LBx. The most common mutations were D538G (39% and 52% of TBx and LBx with ESR1mut), Y537S (27%, 38%), Y537N (10%, 15%), and E380Q (10%, 14%). Activating ESR1 insertion-deletion mutations (indels) were detected in 81 TBx and 62 LBx (2% and 4%), representing 23 distinct variants at the protein level, including V422del (107), indels affecting codons 532-538 (29), G344_L345insC (7), and Y328_S329del (6). 28 fusions of the ESR1 DNA binding domain to 13 unique gene partners were detected; 10/28 (36%) were ESR1-CCDC170 fusions. ESR1 amplifications (ESR1amp) were detected in 704 (2.1%) TBx; 62 (8.8%) also harbored ESR1mut. ESR1amp were detected among 34 (0.5%) LBx; 8 (24%) also had ESR1mut. In TBx with ESR1mut and LBx collected from the same pt within 8 months (43 pairs), LBx sensitivity of detection of ESR1mut was 96% (26/27, 95% confidence interval CI [95%CI]: 79%-99%) when TF was ≥1%, but only 6% (1/16, 95%CI: 0.3%-32%) when LBx had TF &amp;lt; 1%. In 988/1,513 (65%) of LBx with ESR1mut, ≥1 mutations or fusions in genes associated with alternative ET resistance pathways were detected, including PIK3CA (46% of ESR1mut LBx), RB1 (12%), PTEN (10%), NF1 (7%), ARID1A (7%), AKT1 (5%), FGFR2 (4%), and KRAS (4%) and ERBB2 (3%). Prevalence of co-occurring GA associated with ET resistance was higher in LBx than in TBx (RB1, NF1, FGFR2, PTEN, EGFR, KRAS, PIK3CA, FDR &amp;lt; 0.001). Moreover, prevalence was higher in 240 LBx where ESR1 was a minor allele (VAF/TF &amp;lt; 10%) compared to 1,273 LBx were ESR1 was a major allele (FGFR2, PIK3CA, FGFR3, BRAF, ERBB2, RB1, FDR &amp;lt; 0.05). In pts with ESR1mut LBx and a historical TBx collected &amp;lt; 5 years earlier (182 pairs), GAs in RB1, FGFR2, KRAS, EGFR, and BRAF were detected primarily on the LBx but not the preceding TBx. Conclusions: CGP detects a wide spectrum of mutations in ESR1, including missense and indel mutations and fusions. When LBx TF is &amp;lt; 1%, sensitivity for ESR1mut is reduced and repeat testing should be considered. ESR1mut can coexist with complementary or competing resistance mechanisms, particularly when ESR1 is a minor allele, which could impact benefit from novel ET approved for patients with ESR1 mutations. Currently, CGP of LBx informs therapeutic recommendations for pts with HR+ MBC based on the binary presence or absence of ESR1mut; however, the clinical implications of the ESR1 mutation spectrum described herein and the potential impact of co-expression of other key pathway signals warrant further investigation. Citation Format: Heather McArthur, Hanna Tukachinsky, Alexa Schrock, Russell Madison, Oliver Holmes, Smruthy Sivakumar, Ethan Sokol, Ryon Graf, Julia Quintanilha, Kali Dougherty, Lincoln Pasquina, Geoffrey Oxnard, Mia Levy, Eric Winer. ESR1 genomic alterations (GAs) and coexistent putative resistance alterations in comprehensive genomic profiling (CGP) of metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-16-03.