Advanced Estrogen Receptor Positive Breast Research Articles

Abstract LB394: Tumor evolution upon treatment pressure with aromatase inhibitors in locally advanced estrogen receptor positive breast cancer

Abstract BackgroundAromatase inhibitors (AIs) are commonly used in the management of estrogen receptor-positive (ER+) breast cancer as they effectively reduce estrogen levels which inhibits tumor growth and recurrence. Aromatase inhibitors are increasingly used in selected patients as neoadjuvant therapy. Clinical trialThe NEOLETEXE is a neoadjuvant, randomized, open-label, intra-patient, cross-over, single-center phase II clinical trial, aimed to treat postmenopausal patients with locally advanced breast cancer. Most patients presented large T3/T4 tumors and/or N2/N3 involvement. Patients were randomly assigned to neoadjuvant therapy with either letrozole (2.5 mg daily) or exemestane (25 mg daily) for 3 months followed by a cross-over to the alternative AI for another 3 months followed by surgery. A total of 102 patients were enrolled in the NEOLETEXE trial. Aim of the studyWe aimed to compare clones derived from bulk exome sequencing with single-cell analyses at DNA and RNA level from the same tumors at different time points of treatment in order to characterize evolutionary trajectories of cancer and immune cells in 23 patients at scRNA level and 16 patients at scDNA level from the NEOLETEXE trial. MethodsTumor biopsies taken before treatment (baseline) at crossover (after 3 months) and end of neoadjuvant therapy (after 6 months), were analyzed by single-cell DNA (MissionBio) and scRNA (10xGenomics), T cell receptor (TCR), and B cell receptor (BCR) sequencing. Results We employed the The Mission Bio Tapestri Platform, which enables targeted scDNAseq of nuclei isolated from fresh frozen tumor tissue using microfluid droplet technology. A custom panel of 497 amplicons covering 528 sequence variants was designed to study treatment response selected by the following criteria: 1) Up to 10 mutations from each sublonal clusters defined by analysing 32 samples (3 time points) from 11 NeoLetExe patients with whole exome sequencing (WES) data prioritizing mutations in genes part of the Cancer Gene Census (COSMIC), 2) known mutations covering all chromosomes to enable CNV analysis and 3) “Hand-picked” mutations with significance for breast cancer (e.g. hotspot ESR1 mutations). We performed SNV and CNV analysis and reconstruction of mutational lineages with user developed tools. On average, 81% of the clusters defined by WES was validated by scDNAseq and copy number profiles matching between the two data sets. Fishplot with variants defining cell clusters in WES using data from scDNAseq show clonal development during treatment.The scRNA analyses revealed cell types were identified through clustering of the scRNA data. We focused on the epithelial cells to first distinguish between the normal and malignant epithelial cells using the InferCNV algorithm. We next examined CD4, CD8, NK cells, and macrophages, using RNA velocity and diffusion pseudotime using CellRank and scVelo to delineate the differentiation trajectories of immune cell types. Conclusions We created a detailed map that provides a high-resolution view of the diverse clones and cell types present in tumor biopsies from the NEOLETEXE trial. We characterize the impact of therapies on the evolutionary dynamics of both cancer and immune cells. Our analyzes sought insights into the underlying factors that contribute to the sensitivity and resistance to aromatase inhibitors. Citation Format: Vessela N. Kristensen, Grethe I. Alnaes, Patrik Vernhoff, Leonard Schmiester, Salim Ghannoum, Marie Fongaard, Paal M. Bjornstad, Knut Selsaas, Stephanie Geisler, Manouchehr Seyedzadeh, Unn-Cathrin Buvarp, Torben Luders, Diether Lambrechts, Marianne Lyngra, Arnoldo Frigessi, Xavier Tekpli, Jurgen Geilser. Tumor evolution upon treatment pressure with aromatase inhibitors in locally advanced estrogen receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB394.

247P Efficacy and safety of ribociclib (RIB) in combination with letrozole (LET) in patients with estrogen receptor–positive advanced breast cancer (ABC): Secondary and exploratory results of phase 3b RIBECCA study

RIBECCA was a phase 3b, multicentre, open-label study of RIB + LET in patients (pts) of any menopausal status with HR+, HER2–ABC, who were not amenable to curative treatment (tx). The study investigated RIB + LET in pt settings broader than those considered in MONALEESA (ML) studies. Data on primary endpoint, clinical benefit rate (CBR) at week 24 in cohorts A (tx naive postmenopausal pts) and B (comprising cohort B1 [tx naive pre- and perimenopausal pts] and B2 [pre-, peri- and postmenopausal pts pretreated with ≤1 prior chemotherapy and ≤2 lines of endocrine therapy]) were reported previously.

Abstract PS12-25: Patterns of treatment and outcomes in real world patients with advanced estrogen receptor positive breast cancer receiving palbociclib and endocrine therapy

Abstract Background: Advancement in the treatment of metastatic estrogen receptor positive (ER+) breast cancer has led to the introduction of CDK4/6 inhibitors such as Palbociclib, which are associated with reversal of endocrine resistance and delayed requirement for chemotherapy. Clinical trials to date have demonstrated improved survival outcomes for patients on these agents. The objective of this study was to evaluate their role in a real-world setting. Methods: We performed a retrospective multicentre analysis of patients (pts) with metastatic ER+ breast cancer who were commenced on Palbociclib (PAL) between January 2010 and September 2019. Data extracted included demographics, disease characteristics, treatments, toxicities, response rates and survival outcomes. Statistical analysis was performed using Cox proportional hazard model for univariate analysis and Kaplan Meier curves for survival data. Results: We identified 271 pts. The median age was 60 years (31-88) and 18% (n=48) pts were premenopausal. PAL was combined with the following ET partners: Aromatase inhibitor (AI) (38%, n=103), Fulvestrant (FUL) (38%; n=103), Tamoxifen (4%, n=10), FUL & AI combination (16%, n=44) and other (4%, n=11). Among 71 pts treated in the 1st line, overall response rate (ORR) was 56% (n=40). The median PFS (progression free survival) was 35 months (95% confidence interval [CI], 17.2-52.7) in all pts and 25 months (mo) in those not treated with FUL. In 1st line pts with de novo disease (37%, n=26), there appeared to be a trend towards improved PFS in the FUL vs non FUL group - not reached vs 25 mo (HR 0.21; 95% CI 0.02-1.79, p=0.15). There was no significant difference in PFS between the FUL vs non FUL group in relapsed pts (63%, n=45) treated in the 1st line - 22 vs 20 mo (HR 1.0, 95%CI 0.35-2.9; p=0.96). The median OS was 59 mo (95% CI, 9.5 to 108). Of 74 pts treated in the 2nd line, ORR was 24% (n=18), median PFS was 10 mo (95% CI, 5.8-14.1) & OS was 25 mo (95% CI, 18.3-31.6). Among 126 3rd line pts, ORR was 16% (n=20), median PFS was 5 mo (95% CI, 3.5-6.4) and OS was 20 mo (95% CI, 12.8-27.1). 3 of 9 pts achieved >6 mo of stable disease after switching ET and continuing Palbociclib beyond progression. The most frequent grade 3 toxicities were neutropenia (40%), anaemia (4%), fatigue (3%) & thrombocytopenia (2.5%). The rate of febrile neutropenia was 2.5%. Dose reductions occurred in 40%, with the most common reason being neutropenia. Treatment was discontinued in 3% due to toxicity. Premenopausal status or dose reductions were not associated with poorer survival outcomes. Conclusions: Palbociclib appears to be safe and tolerable in a real-world population and is associated with favourable survival outcomes comparable to that seen in a clinical trial setting. Combining Palbociclib with Fulvestrant as opposed to other endocrine therapies may delay progression in the 1st line setting in patients with de novo metastatic ER+ breast cancer but larger studies are needed to explore this hypothesis further. Citation Format: Lisa Prior, Darko Skrobo, Hazel Murray, Abdul Rehman Farooq, Anne Horgan, Paula Calvert, Emmet Jordan, John McCaffrey, Lillian Smyth, Miriam O'Connor, Michaela Higgins, Desmond Carney, Janice Walshe, Giuseppe Gullo, John Crown, Catherine M Kelly. Patterns of treatment and outcomes in real world patients with advanced estrogen receptor positive breast cancer receiving palbociclib and endocrine therapy [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 PS12-25.

Survival rates of advanced estrogen-receptor positive breast cancer. Analysis of 211 cases.

About 80% of breast cancer (BC) cases express estrogen receptor (ER), which has been correlated with good prognosis and response to estrogen deprivation Aim: To characterize ER positive advanced BC (ABC) patients treated at our institution assessing the impact of clinical pre-sentation (stage IV, de novo disease at diagnosis versus systemic recurrence) and BC subtype on survival rates. We evaluated 211 ER+ advanced BC (ABC) patients, treated between 1997 and 2017. The median overall survival (OS) was 37 months. Median OS for the period 1997/2006 and 2007/2017 were 33 and 42 months, respectively (p = 0.47). Luminal A, ABC stage IV disease at diagnosis displayed better OS rates than Luminal B stage IV tumors (100 and 32 months respectively, p < 0.01). Clinical presentation (stage IV vs. systemic recurrence) and tumor subtype are key determinants of OS in ABC.

Abstract PD2-04: Different mechanism of CDK4/6 inhibitor resistance between ribociclib and abemaciclib

Abstract Background: CDK4/6 inhibitors—palbociclib, ribociclib, and abemaciclib—result in prolonged progression free survival for advanced estrogen receptor positive breast cancer patients. However, the optimum CDK4/6 inhibitor for each patient remains unclear until the emergence of side effects, which are the key differentiators between the two treatments. Furthermore, an optimum treatment after acquisition of resistance is poorly understood. Therefore, we elucidated the difference in CDK4/6 inhibitor resistance mechanisms using ribociclib- and abemaciclib-resistant cell lines in vitro.Methods: We established hormone-resistant cell lines. Estrogen deprivation-resistant (EDR) cell lines (EDR1:ER-positive, EDR2:ER-negative) and fulvestrant-resistant (MFR) cell lines were established from the MCF-7 cell line. We established ribociclib-resistant cell lines (RIBR) and abemaciclib-resistant cell lines (ABER) from EDR1 by long-term culture (7 months for RIBR and 12 months for ABER) with ribociclib and abemaciclib, respectively. We also established CDK6 overexpressed cell lines (MCF7-C6) from MCF-7 cell line using the stably transfected CDK6 expression vector.Results: First, we identified the efficacies of ribociclib and abemaciclib in several cell lines. Luminal cell lines (MCF-7 and T-47D) exhibited high sensitivity to ribociclib and abemaciclib compared to non-luminal cell lines (MDA-MB-231, BT-20, and SKBR3). The hormone-resistant cell lines (EDR1, EDR2, and MFR) exhibited similar sensitivity to ribociclib and abemaciclib as that of MCF-7; this was independent of hormone resistant mechanism.Subsequently, we compared RIBR and ABER, which are the models of acquired ribociclib- and abemaciclib-resistant cell lines, respectively. We confirmed that RIBR and ABER decreased ribociclib and abemaciclib sensitivity, respectively. RIBR showed low sensitivity to abemaciclib. Similarly, ABER exhibited low sensitivity to ribociclib. Immunoblot analysis showed that compared to EDR1, RIBR and ABER had extremely low levels of total Retinoblastoma (RB). Interestingly, compared to EDR1, CDK6 levels were upregulated in ABER and remained unaltered in RIBR. On the other hand MCF7-C6 reduced sensitivity not only abemaciclib but also ribociclib compared to MCF-7 cell line. Furthermore, compared to EDR1, RIBR exhibited lower levels of p21 and p27 compared to EDR1 (P21 levels were extremely reduced, but p27 levels were slightly reduced). Additionally, ABER showed markedly lower levels of p27; however, the p21 level was slightly increased compared to EDR1. PI3K inhibitor and mTOR inhibitor suppressed cell growth in RIBR and ABER to the same extent as EDR1, indicating that resistance to CDK4/6 inhibitors was still dependent on the PI3K/Akt/mTOR pathway. Chemotherapeutic drugs were effective both in RIBR and ABER.Discussion &amp; Conclusion: There are various mechanisms of CDK4/6 inhibitor resistance, such as loss of RB, CDK6 amplification, and high CDK2 activity. However, the mechanism of resistance between ribociclib and abemaciclib might be different because the CDK6 level was different, although RB level was decreased in both RIBR and ABER. Furthermore, both p21 and p27 are considered as the internal inhibitors of CDK2, although more factors in RIBR and ABER were different. CDK4/6 inhibitor resistant cell lines acquired cross resistance to another CDK4/6 inhibitor. Although the mechanism of resistance between ribociclib and abemaciclib might be different, use of drugs targeting the PI3K/Akt/mTOR pathway and chemotherapeutic drugs would be an effective strategy following the development of resistance. Our findings suggest that the mechanism of resistance differs depending on the type of CDK4/6 inhibitors used, and provide some insights into the treatment strategies after acquisition of CDK4/6 inhibitor resistance. Citation Format: Masafumi Iida, Daichi Toyosawa, Misato Nakamura, Emi Tokuda, Toshifumi Niwa, Ryuichi Yoshida, Takanori Ishida, Shin-ichi Hayashi. Different mechanism of CDK4/6 inhibitor resistance between ribociclib and abemaciclib [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD2-04.

Abstract P2-11-08: ESR1 mutations are not a mechanism of primary resistance to aromatase inhibitors in ER-positive breast cancer treated with neoadjuvant endocrine therapy

Abstract Introduction: Mutations in the ESR1 gene (ESR1m) are important mechanisms of resistance to endocrine therapy (ET) in estrogen receptor-positive (ER+) advanced breast cancer and have been recognized as a prognostic and predictive biomarker as well as a potential therapeutic target. ESR1m prevalence has been described as 9-45% in cohorts of ET-resistant metastatic tumors in a variety of publications. We recently reported a prevalence of 25% in a cohort of ER+ breast cancer patients with visceral metastasis. However, the role of ESR1m as a mechanism of resistance to ET used in early-stage disease is not well studied. Neoadjuvant endocrine therapy (NET) is being increasingly explored, not only to allow less extensive surgery but also as a scientific tool, exploring biomarkers to predict outcomes. The preoperative endocrine prognostic index (PEPI) combining Ki67 score, ER Allred score, tumor size, and nodal status after NET is a surrogate of endocrine sensitivity and can identify a subgroup of patients with primary resistance to ET. In this study, we evaluated the prevalence of ESR1m in tumor samples from patients with ER+ breast cancer that were primarily resistant to neoadjuvant aromatase inhibitor (AI) therapy. Methods: We followed a prospective cohort of postmenopausal patients with ER+ HER2- stages II-III breast cancer treated with neoadjuvant endocrine therapy (NET). Patients were treated with anastrozole for a recommended period of at least three months. Tumor samples from patients with a pattern of primary endocrine-resistant tumors (defined as a PEPI Score higher than 3) were selected and analyzed for the presence of ESR1m. ESR1m were evaluated in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue using real-time quantitative polymerase chain reaction (RT-qPCR). Mutations in codons 380, 537, and 538 of the ESR1 gene were analyzed. Results: 127 patients were included in the cohort, of which 100 (79%) had completed NET and had surgery. Among these patients, the PEPI Score ranged from 0 to 3 in 70% (70/100), and 30% (30/100) had a PEPI Score of 4 or more and were selected. 23 patients were included in the analysis (6 did not consent or were lost to follow-up, and one was HER2-positive). Patients characteristics are summarized in Table 1. The median duration of NET was 22 weeks. All samples of tumor tissue from the surgical specimens after NET were evaluable. Quantification of DNA extraction and reference gene cycle threshold values confirmed that the material was adequate for the analysis. We compared these findings with a study from our group using the same methodology in patients with advanced disease where ESR1 mutations were detected in 25% (n=32) of patients with visceral metastasis of ER+ breast cancer resistant to endocrine therapy. ESR1 mutations were not identified in any of the 23 patients with early-stage ER+ breast cancer resistant to NET (p 0.01, Fisher´s exact test) Discussion: Growing evidence supports the notion that there are different mechanisms of primary and secondary endocrine resistance. Our study suggests that ESR1 mutations do not evolve rapidly and do not represent a common mechanism of primary endocrine resistance in the neoadjuvant setting. Therefore, ESR1m should be considered a mechanism of acquired endocrine resistance in the context of advanced disease. Further research should be conducted to identify factors associated with intrinsic resistance to ET. Citation Format: Tomas Reinert, Susana Ramalho, Vivian CA Vasconcelos, Leonardo R Silva, Ana Elisa R Silva, Camila A Andrade, Maria Beatriz PL Kraft, Guilherme P Coelho, Jovana Mandelli, Monique Binotto, Cesar Cabello, Geisilene RP Silva, Jose Bines, Carlos H Barrios, Matthew J Ellis, Marcia S Graudenz. ESR1 mutations are not a mechanism of primary resistance to aromatase inhibitors in ER-positive breast cancer treated with neoadjuvant endocrine therapy [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 P2-11-08.

Abstract P5-11-11: Influence of the adjuvant hormonal therapy on hormone sensitivity and survival outcomes in ER+ and HER2−advanced breast cancer: A subgroup analysis of the JBCRG-C06 Safari study

Abstract Background: There are now many treatment options for estrogen receptor-positive (ER+) and human epidermal growth factor receptor 2-negative (HER2−) advanced breast cancer (ABC). However, there are few reports indicating the optimal treatment sequence for this disease. Information to predict response to treatment is vital for personalized therapy and determining the most beneficial approach for individual patients. In this study, we attempted to determine predictive factors of response to hormonal therapy and survival outcomes using the large-scale databases constructed in the Safari study (UMIN000015168), a retrospective, multicenter cohort study involving 1,072 Japanese patients receiving fulvestrant 500mg for ER+ ABC. We examined the association between clinicopathological factors and time to failure (TTF) of fulvestrant in Japanese ABC patients. Methods: Among 1072 patients, 247 patients were selected for this study. Inclusion criteria was as follows: 1) patients treated with either SERM or AI in the adjuvant setting (AS) with known starting and finishing date of the treatment (patients relapsed on adjuvant treatment were excluded), 2) patients treated with SERM, AI and SERD in the metastatic setting (MS) as the first and the second line treatment. Influence of the adjuvant hormonal therapy on the TTF of the first line and the second line treatment and overall survival (OS) was assessed. Cox proportional hazards model was used for this analysis. Results: Patients treated with SERM in AS had significantly longer TTF of 1st line and 1st+2nd line of hormonal therapy in MS (1st line: HR 1.519, 95% CI 1.04-2.218, p=0.0307, 1st+2nd line: HR 2.372, 95% CI 1.584-3.551, p&amp;lt;0.001) compared with patients treated with AI in AS. Same trend was observed in OS. SERM treated patients in AS had significantly longer OS (OS: HR 2.748, 95% CI 1.574-4.796, p=0.0004) compared with AI treated patients in AS. Since these results described above are the results analyzed by multivariate analysis, they are all statistically significant independent of other factors such as disease free interval in this cohort. Conclusions: Interestingly, once AI-treated patients in AS have relapsed, their hormone sensitivity is lower and survival outcomes are worse compared with SERM-treated patients in AS in this cohort. Our results have consistency with the results of ABCSG-12 trial showing a pronounced higher risk of death for anastrozole-treated patients (Annals of Oncology 26: 313–320, 2015). Since it is a cohort of selected patients who were successfully treated with fulvestrant, prospective analysis would be therefore warranted. Citation Format: Takahiro Nakayama, Hidetoshi Kawaguchi, Norikazu Masuda, Kenjiro Aogi, Keisei Anan, Yoshinori Ito, Shoichiro Ohtani, Nobuaki Sato, Shigehira Saji, Toshimi Takano, Eriko Tokunaga, Seigo Nakamura, Yoshie Hasegawa, Masaya Hattori, Tomomi Fujisawa, Satoshi Morita, Miki Yamaguchi, Hiroko Yamashita, Toshinari Yamashita, Yutaka Yamamoto, Daisuke Yotsumoto, Masakazu Toi, Shinji Ohno. Influence of the adjuvant hormonal therapy on hormone sensitivity and survival outcomes in ER+ and HER2−advanced breast cancer: A subgroup analysis of the JBCRG-C06 Safari study [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 P5-11-11.

CDK4/6 Inhibitor Biomarker Research: Are We Barking Up the Wrong Tree?

CDK4/6 inhibitors have emerged as a significant advance for the treatment of patients with advanced estrogen receptor-positive breast cancer. However, the identification of predictive markers that optimize their use is proving harder than expected. In this commentary we advocate for unbiased discovery and a collaborative approach across trials.See related article by Finn et al., p. 110.

Abstract 1272: Whole genome Crispr screening to identify potential SERD molecule resistance mechanisms

Abstract Metastatic breast cancer results in substantial morbidity and mortality for women afflicted with this disease. Approximately 80% of breast cancers express the estrogen receptor. The cancer cell survival and proliferation is driven by the activation of the estrogen receptor. SAR439859 is an effective endocrine therapy for breast cancer selectively and effectively degrading the estrogen receptor. A phase 1/2 study of SAR439859 alone and/or in combination with palbociclib in postmenopausal women with estrogen receptor positive advanced breast cancer was initiated last year by Sanofi. CRISPR/Cas9 is a recent and revolutionary technology for efficient and directed alterations of the genome. CRISPR allows users to introduce DNA double-strand breaks at precise locations in the genome using complementary guide RNAs (SgRNA). These double strand breaks can be repaired by non-homologous end joining DNA repair mechanism. This DNA repair mechanism is an error prone DNA repair process which can lead to gene invalidation. We aimed at investigating the potential SAR439859 resistance mechanisms. A better understanding of resistance mechanisms is needed to overcome this potential problem and to propose complementary therapeutic solutions. We conducted a whole genome CRISPR screening in a breast tumor cell line with a thermofisher commercial SgRNA library (ref: M04305) to identified genes involved in potential SAR439859 resistance phenomena. CRISPR screening steps will be presented from the cell line selections to results analysis. Citation Format: Christophe Marcireau, Karine Berthelot, Alice Williart, Hamida Fournet, Delphine Debono, Gilbert Thill, Helene Erasimus, Dorine Chassin, Christophe Lanneau, Veeanagouda Yaligara, Cecile Orsini, Michel Didier, Vincent Mikol, Monsif Bouaboula, Laurent Debussche. Whole genome Crispr screening to identify potential SERD molecule resistance mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1272.

Tracking endocrine resistance in estrogen-receptor positive breast cancer in ctDNA.

e14550 Background: Endocrine therapy is the standard of care treatment for patients with estrogen-receptor positive advanced breast cancer, owing to improved tolerability and comparable efficacy to that of cytotoxic chemotherapy. Half of such cancers will progress through first line therapy (primary endocrine resistance) and half will progress after an initial period of disease control (secondary or acquired endocrine resistance). A significant challenge is to test for and identify biomarkers which can guide the likely success of endocrine therapy as a single agent or in combination with small molecule inhibitors.This is particularly challenging where metastatic deposits reside at sites where biopsy is difficult. Potential biomarkers indicative of resistance to endocrine therapy have been identified and can be detected in circulating tumour DNA (ctDNA). CtDNA is shed from tumours and is detectable in a cancer patient’s bloodstream. Information on mutational profiles can guide an oncologist in the selection of targeted therapy in addition to hormonal therapy. Methods: We have analysed formalin-fixed paraffin-embedded(FFPE) tumour samples and longitudinal liquid biopsies from 19 patients who were treated with fulvestrant in combination with a novel inhibitor of the PIK3CA/AKT pathway with next-generation sequencing using a targeted 44-gene panel. Mutations identified using this technique were tracked during the course of treatment using droplet-digital PCR(ddPCR). Results: 57 samples were tested using a 44-gene panel; 19 FFPE tumour samples and matched ctDNA samples were obtained prior therapy and ctDNA samples at disease progression. Mutations detected in PIK3CA, AKT1, ESR1and TP53 genes were trackable in longitudinal ctDNA samples using ddPCR. The association between ctDNA dynamics and outcome will be presented. Conclusions: Multiple mutations that enable the early detection of treatment failure and resistance can be tracked in ctDNA. Investigating the clinical utility of ctDNA is paramount.

Highlights of This Issue

Current biomarkers for predicting the response to anti-PD-1 therapy require tissue biopsy and have not shown sufficient predictive values. Kim and colleagues discovered, in anti-PD-1-treated patients with metastatic or refractory thymic epithelial tumors and in patients with non-small cell lung cancer, a higher fold-change in the percentage of Ki-67+ cells among PD-1+CD8+ T cells 7 days after the first dose predicts superior durable clinical benefit and survival rates. This result was validated in an independent cohort of anti-PD-1-treated patients with non-small cell lung cancer. In summary, the first-week proliferative response of peripheral blood PD-1+CD8+ T cells predicts the response to anti-PD-1 therapy in solid tumors.CDK4/6 inhibitors (palbociclib, ribociclib, and abemaciclib) are a new class of drugs that are approved alone or in combination with endocrine therapy for advanced estrogen receptor positive breast cancer. Clark and colleagues sought to combine palbociclib with paclitaxel using a novel dosing schedule taking advantage of the mechanism of action of both drugs to potentially increase the efficacy of single agent paclitaxel. Administering the drugs on an alternating schedule potentially enables G1-synchronization by palbociclib, a few days rest without drug allows cells to re-enter the cell cycle, increasing the percentage of cells in M phase when exposed to paclitaxel. Alternating sequential palbociclib/paclitaxel in patients with Rb+ ABC is feasible and safe, without evidence of additive toxicity.Nowicki and colleagues describe a clinical trial using dual cell immunotherapy, consisting of genetically modified lymphocytes with NY-ESO-1-specific T-cell receptor adoptively transferred together with NY-ESO-1 peptide-pulsed dendritic cell vaccination with or without CTLA-4 immune checkpoint blockade with ipilimumab. The authors report that these protocols are generally safe, feasible, and result in high antitumor activity in patients with advanced sarcoma and melanoma. However, further improvements in durability of these cellular therapies are needed because patients frequently relapse after an initially vigorous antitumor response.Cancer of the esophagus claims more than 400,000 lives annually year. Without efficient, reliable screening, diagnosis often occurs too late to save the patient. To this end, Wang and colleagues developed a minimally invasive, rapid test using a swallowable, retrievable sponge (EsophaCap) in conjunction with epigenetic biomarkers to detect precancerous changes (Barrett's esophagus) in cells lining the esophagus. The EsophaCap was administered to 94 people over the course of the study. The combination of p16, NELL1, AKAP12, and TAC1 methylation yielded high sensitivity and specificity in diagnosing Barrett's esophagus. These findings indicate the need for large prospective studies testing this early diagnostic modality.

Antiproliferative Effect of Trifolium Pratens L. Extract in Human Breast Cancer Cells

Breast cancer is currently the most common cancer in women worldwide. Tamoxifen is used for the treatment of both early and advanced estrogen receptor positive breast cancer. Trifolium pratense L. has been suggested for cancer treatment in traditional medicine. The effect of T. pratense extract on breast cell lines (MCF-7 and MDA-MB-231) was investigated here. The cells were treated with T. pratense extract and cell viability was evaluated using trypan blue staining, MTT assay and lactate dehydrogenase activity measurement. Apoptosis, autophagy cell death was detected using fluorescent dye staining. Griess assay was performed to nitric oxide (No) measurement. Change in expression level of some apoptotic and autophagic- related genes was detected using real-time PCR. The combination effect of exteract and tamoxifen was evaluated by calculating the combination index and dose reduction index (DRI) values. T. pratense extract reduced the cell viability in dose and time- dependent manner. Apoptosis and autophagy was increased after treatment with T. pratense extract. Nitric oxide production significantly reduced in cells and combination of tamoxifen and T. pratense extract had a synergistic cytotoxic effect on breast cancer cell lines. T. pratense as a dietary supplement can be beneficial for some patients with breast cancer who receive conventional chemotherapy.

Alpelisib Plus Fulvestrant in PIK3CA-Altered and PIK3CA-Wild-Type Estrogen Receptor–Positive Advanced Breast Cancer

The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in patients with estrogen receptor-positive (ER+), endocrine therapy-resistant breast cancers. To assess the maximum tolerated dose (MTD), safety, and activity of alpelisib, an oral, PI3Kα-specific inhibitor, plus fulvestrant in patients with ER+ advanced breast cancer (ABC). An open-label, single-arm, phase 1b study of alpelisib plus fulvestrant was conducted at 10 centers in 5 countries. Participants were 87 postmenopausal women with PIK3CA-altered or PIK3CA-wild-type ER+ ABC, whose cancer progressed during or after antiestrogen therapy. The study began enrolling patients October 5, 2010, and the data cutoff was March 22, 2017. Escalating doses of alpelisib were administered once daily, starting at 300 mg, plus fixed-dose fulvestrant, 500 mg, in the dose-escalation phase; alpelisib at the recommended phase 2 dose plus fulvestrant in the dose-expansion phase. The primary end point was determination of the MTD of once-daily alpelisib plus fulvestrant. Secondary end points included safety and preliminary activity. From October 5, 2010, to March 22, 2017, 87 women (median age: 58 years [range, 37-79 years]; median of 5 prior lines of antineoplastic therapy) received escalating once-daily doses of alpelisib (300 mg, n = 9; 350 mg, n = 8; 400 mg, n = 70) plus fixed-dose fulvestrant (500 mg). During dose escalation, dose-limiting toxic effects were reported in 1 patient (alpelisib, 400 mg): diarrhea (grade 2), vomiting, fatigue, and decreased appetite (all grade 3). The MTD of alpelisib when combined with fulvestrant was 400 mg once daily, and the recommended phase 2 dose was 300 mg once daily. Overall, the most frequent grade 3/4 adverse events with alpelisib, 400 mg, once daily (≥10% of patients), regardless of causality, were hyperglycemia (19 [22%]) and maculopapular rash (11 [13%]); 9 patients permanently discontinued therapy owing to adverse events. Median progression-free survival at the MTD was 5.4 months (95% CI, 4.6-9.0 months). Median progression-free survival with alpelisib, 300 to 400 mg, once daily plus fulvestrant was longer in patients with PIK3CA-altered tumors (9.1 months; 95% CI, 6.6-14.6 months) vs wild-type tumors (4.7 months; 95% CI, 1.9-5.6 months). Overall response rate in the PIK3CA-altered group was 29% (95% CI, 17%-43%), with no objective tumor responses in the wild-type group. Alpelisib plus fulvestrant has a manageable safety profile in patients with ER+ ABC, and data suggest that this combination may have greater clinical activity in PIK3CA-altered vs wild-type tumors. ClinicalTrials.gov identifier: NCT01219699.

Abstract P6-04-02: CDK6 might be a key factor for efficacy of CDK4/6 inhibitor and the hormone sensitivity following acquired resistance

Abstract Background: CDK4/6 inhibitors have received FDA breakthrough therapy designation as 1st line treatment for advanced estrogen receptor positive breast cancer patients. However, the benefit offered by CDK4/6 inhibitors is individually different and furthermore acquired resistance to the drugs is monumental challenges. It is urgent need to search for the biomarker and understand the drug sensitivity and its alteration after acquired resistance. Results: To identify the efficacy of CDK4/6 inhibitors, we assessed IC50 of ribociclib in several cell lines. Luminal cell lines (MCF-7, T-47D) exhibited lower ribociclib IC50 than HER-2 (SK-BR-3) and triple negative cell lines (MDA-MB-231, BT-20). Immunoblot analysis of Luminal cell lines showed extremely lower levels of CDK6 compared with others. CDK6-transfected MCF-7 by means of expression vector reduced the sensitivity equivalent to MDA-MB-231 not only to ribociclib but also to palbociclib and abemaciclib. Protein level of ERα in CDK6-transfected MCF-7 stayed unchanged and fulvestrant sensitivity was unaltered as well. Subsequently, we detect the efficacy of ribociclib in hormone resistant cell lines. Estrogen deprivation-resistant (EDR) cells (EDR1:ER-positive, EDR2:ER-negative) and fulvestrant resistance (MFR) cells (loss of ER expression) established from MCF-7 maintained ribocilib sensitivity to the same degree with MCF-7. No marked difference in IC50 was observed between EDR1/2 and MFR, and CDK6 expressions were comparable to MCF-7. These results suggest that high level of CDK6 expression weaken the sensitivity to CDK4/6 inhibitors. The inhibitors would provide more effective benefits to tumors expressing lower level of CDK6 than the higher, independent of hormone sensitivity. To understand the characteristics in acquired resistance, ribociclib resistant cell lines (RIBR1/2) were established from EDR1 by long-term culture with ribociclib. RIBR designed lower level of p21, p27 and ERα by immunoblot analysis. EDR1 were promoted cell growth by estrogen addition, while RIBR were not. Further, ER activity of RIBR was intensely decreased, and mRNA levels of the ER target genes, PgR and EGR3 were also decreased. Therefore, the responsiveness to tamoxifen and fulvestrant were lost. On the other hand, PI3K inhibitor and mTOR inhibitor suppressed cell growth to the same extent as EDR1, suggesting that RIBR were reduced ER dependence and remain reliant on PI3K/Akt/mTOR pathway. Conclusion: The possibility of CDK6 as a biomarker is corroborated by the finding that low level of CDK6 expression is positively correlated with the efficacy of CDK4/6 inhibitor. Further ER dependence had decreased after acquired CDK4/6 inhibitor resistance whereas the dependence on PI3K/Akt/mTOR pathway still remain, indicating the inhibition of PI3K/Akt/mTOR pathway would be amenable to therapeutic target. Citation Format: Iida M, Nakamura M, Tokuda E, Niwa T, Ishida T, Hayashi S-I. CDK6 might be a key factor for efficacy of CDK4/6 inhibitor and the hormone sensitivity following acquired resistance [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-04-02.

Abstract 2338: CDK4/6 and autophagy inhibitors synergize to induce senescence in cancers with an intact G1/S checkpoint

Abstract Deregulation of the cell cycle machinery is a hallmark of most cancers. The crucial role of the CDK4/6-CyclinD pathway in tumorigenesis has led to the successful development and FDA approval (palbociclib) of CDK4/6 inhibitors for the treatment of advanced estrogen receptor positive breast cancer. However, two major clinical challenges remain: i) lack of a reliable biomarker to predict treatment response and ii) adverse events leading to interruption or discontinuation of treatment which possibly thus curtailing therapeutic benefit. We found that treatment of ER+ breast cancer cell lines with the CDK4/6 inhibitor palbociclib resulted in a dose-dependent sustained growth inhibition and senescence. Interestingly, breast cancer cells activate autophagy in response to palbociclib, a stress response process that promotes cancer cell survival. Genetic ablation of crucial autophagic genes or pharmacological inhibition of autophagy increases the sensitivity of ER+ breast cancer cells to palbociclib and the other CDK4/6 inhibitors, ribociclib and abemaciclib. This was confirmed in vivo, where the combination of palbociclib and autophagy inhibitor, hydroxychloroquine (HCQ) resulted in a significantly improved and a sustained tumor shrinkage. To identify biomarkers that predict response to CDK4/6 inhibition, we examined the G1 checkpoint proteins and found that knockdown of Rb or overexpression of the oncogenic low molecular weight isoforms of Cyclin-E (LMW-E) mediates resistance to palbociclib and its combination with autophagy inhibitor. More significantly, immunohistochemically staining of pre-treatment biopsies from palbociclib treated patients strengthened the correlation between palbociclib efficacy and an intact G1/S checkpoint (Rb+ve /LMWE-ve), resulting in a significantly longer progression free survival compared to the other patient groups; thus solidifying Rb and LMW-E as reliable prognostic biomarkers for palbociclib treatment. Finally, we examined the biomarker driven synergy between CDK4/6 and autophagy inhibition in several other cancer cell lines. Several solid tumors (ovarian, lung, pancreatic, colon, prostate) and triple negative breast cancer (TNBC) cell lines exhibited a synergistic response to palbociclib/HCQ combination treatment dependent upon an intact G1/S transition (Rb+/LMWE-). This was also verified in a TNBC patient derived xenograft (PDX) model. Thus, this study addresses the aforementioned limitations and provides a novel and promising biomarker-driven combination therapeutic strategy to treat breast and other solid tumors. We predict that this combination of CDK4/6 and autophagy inhibitors would be more beneficial than standard dose palbociclib in patients, allowing us to lower the dose, minimize palbociclib mediated toxicities and potentially improve overall patient survival - a goal that has not yet been met with currently approved treatment combinations. Citation Format: Smruthi Vijayaraghavan, Cansu Karakas, Xian Chen, Iman Doostan, Akshara S. Raghavendra, Min Yi, Ravi Amaravadi, Kelly Hunt, Debu Tripathy, Khandan Keyomarsi. CDK4/6 and autophagy inhibitors synergize to induce senescence in cancers with an intact G1/S checkpoint [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2338. doi:10.1158/1538-7445.AM2017-2338

Abstract LB-069: ESR1 mutations in circulating tumour DNA predict outcome to endocrine treatment in patients with estrogen receptor positive advanced breast cancer; analysis of 521 patients in the SoFEA and PALOMA3 trials

Abstract Background. ESR1 mutations are selected by prior aromatase inhibitor (AI) therapy for advanced breast cancer. We addressed the impact of ESR1 mutations on sensitivity to standard therapies in SoFEA and PALOMA3, two phase III randomised trials that represent the trends in the current therapy for estrogen receptor positive advanced breast cancer. Methods. We assessed plasma DNA ESR1 mutations in the SoFEA trial that compared exemestane with fulvestrant-containing regimens in patients with prior sensitivity to non-steroidal AI, and in the PALOMA3 trial that compared fulvestrant plus placebo with fulvestrant plus palbociclib in patients with progression on prior endocrine therapy. We developed and validated multiplex digital droplet PCR assays to detect ESR1 mutations in circulating free DNA. Plasma had been immediately processed in PALOMA3 but there was a delay of up to 9 days before processing of EDTA samples in the SoFEA trial. Results. In SoFEA we demonstrated that delayed processing of plasma samples had no effect on detection rate of ESR1 mutations (p = 0.71), which were found in 39.1%(63/161) patients. Patients with ESR1 mutations had improved progression free survival (PFS) on fulvestrant compared to exemestane (HR = 0.52, 95%CI 0.30 to 0.92, p = 0.02), while patients with wild type ESR1 had similar PFS on either treatment (HR = 1.07, 95%CI 0.68 to 1.67, p = 0.77). In PALOMA3, ESR1 mutations were found in the plasma of 25.3%(91/360) patients. ESR1 mutations were almost exclusively found in patients with prior AI exposure with or without tamoxifen, and were very rare with prior tamoxifen exposure only (28.9% (90/311) versus 2.0% (1/49) respectively, p&amp;lt;0.001). ESR1 mutations were strongly associated with sensitivity to prior endocrine therapy (sensitive to prior endocrine therapy, 29.8% (85/285) versus resistant 8.0% (6/75), p&amp;lt;0.001), bone metastases (p = 0.001), prior lines of therapy for metastatic disease (p = 0.01), and with a trend towards ER and PR positive disease as opposed to ER positive but PR negative disease. Fulvestrant plus palbociclib improved PFS compared to fulvestrant plus placebo in both ESR1 mutant (HR = 0.43, 95%CI 0.25 to 0.74, p = 0.002) and ESR1 wild type patients (HR = 0.49 95%CI 0.35 to 0.70, p&amp;lt;0.001). ESR1 mutations were polyclonal in up to 49% of patients, with outcome on fulvestrant similar for polyclonal and monoclonal mutations. In an exploratory analysis there was no association between specific mutations and outcome on fulvestrant. Conclusions. ESR1 mutation analysis in plasma after progression on prior AI therapy has clinical utility in directing choice of further endocrine therapy. Citation Format: Ben O’Leary, Charlotte Fribbens, Lucy Kilburn, Sarah Hrebein, Isaac Garcia-Murillas, Matthew Beaney, Massimo Cristofanilli, Fabrice Andre, Sherene Loi, Sibylle Loibl, John Jiang, Cynthia Huang Bartlett, Maria Koehler, Mitch Dowsett, Judith Bliss, Stephen Johnston, Nicholas Turner. ESR1 mutations in circulating tumour DNA predict outcome to endocrine treatment in patients with estrogen receptor positive advanced breast cancer; analysis of 521 patients in the SoFEA and PALOMA3 trials. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-069.

Abstract ES7-1: Neoadjuvant endocrine therapy

Abstract Neoadjuvant endocrine therapy has become a standard approach in clinical practice to improve the chance of breast conservation in postmenopausal women with locally advanced estrogen receptor positive breast cancer based on results from several neoadjuvant endocrine trials. Although data is limited, neoadjuvant endocrine therapy appears to as effective as chemotherapy in converting mastectomy to lumpectomy. In addition, neoadjuvant endocrine therapy allows an early assessement of tumor responsiveness to endocrine therapy so that adjuvant treatments could be tailored accordingly. Persistent cell proliferation on neoadjuvant endocrine therapy predicts a high risk of early relapse, for whom novel treatment strategies are in great need. In contrast, the Preoperative Endocrine Prognostic Index score of 0 (pathologic T2/3 N0 Ki67 &amp;lt;2.7% ER+) in response to neoadjuvant endocrine therapy is being prospectively evaluated in the ongoing Alliance A011106 (ALTERNATE) as a marker of endocrine sensitive disease, for whom chemotherapy could be avoided. Furthermore, neoadjuvant endocrine trials provide a unique research platform for biomarker studies and the testing of novel therapeutic hypothesis. Citation Format: Ma CX. Neoadjuvant endocrine therapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr ES7-1.

Abstract OT1-03-12: MANTA: A randomized phase II study of fulvestrant in combination with the dual mTOR inhibitor AZD2014 or everolimus or fulvestrant alone in estrogen receptor-positive advanced or metastatic breast cancer

Abstract Background: Resistance to endocrine therapy remains a major clinical challenge in ER+ breast cancer. Aberrant PI3K/AKT/mTOR pathway activation frequently occurs in ER+ breast cancer and is associated with resistance to endocrine therapy. Randomised clinical trials have demonstrated a substantial benefit of adding everolimus to endocrine treatment. However, there is increasing evidence that inhibition of only mTORC1 with rapalogues such as everolimus sets off a negative feedback mechanism that leads to increased AKT signalling and is linked with treatment resistance. AZD2014 is a dual inhibitor of both mTORC1 (rapamycin-sensitive) and mTORC2 (rapamycin insensitive); compared to rapalogues, AZD2014 has a broader range of growth inhibitory activity in preclinical models based on a more profound mTORC1 inhibition and the additional inhibition of mTORC2. AZD2014 is especially effective in ER+ breast cancer models, showing superior activity to everolimus both in hormone-sensitive and resistant models. Trial objectives: The main aims of this trial are (i) to determine whether dual inhibition of mTORC1 and mTORC2 with AZD2014 will increase the activity of endocrine therapy with fulvestrant in ER+ breast cancer, (ii) whether inhibition of mTORC1 and mTORC2 using AZD2014 will have superior anti-tumour activity compared to inhibition of mTORC1 alone with everolimus, and (iii) to compare the safety and efficacy of two schedules of AZD2014. Methods: MANTA is an international investigator led, sponsored, open-label, randomised phase II trial. Patients are randomised in a 2:3:3:2 ratio to receive either fulvestrant (500mg) alone, fulvestrant and AZD2014 at a continuous daily schedule (50mg BD), fulvestrant and AZD2014 at an intermittent schedule (2 days on, 5 days off; 125mg BD) or fulvestrant and everolimus (10mg OD). Patients are stratified by disease measurability and response to prior endocrine therapy. Treatment is given until disease progression (RECIST 1.1), intolerable toxicity or elective withdrawal. Eligibility criteria: This study enrols post-menopausal women with ER+, HER2-negative, advanced or metastatic, hormone refractory breast cancer. Patients must have at least one measurable lesion and no life-threatening visceral disease. Patients with significant pulmonary dysfunction, cardiovascular disease or uncontrolled diabetes are excluded. Patients must not have had previous treatment with fulvestrant, PI3K/Akt or mTOR inhibitors or no more than one line of chemotherapy for metastatic breast cancer. Endpoints: The primary endpoint is progression-free survival . Secondary endpoints include objective response rate, change in tumour size, clinical benefit rate, overall survival, duration of response, patient reported outcomes and pharmacokinetic parameters of AZD2014 and fulvestrant. Archival tumour tissue must be available to evaluate biomarkers associated with therapeutic response and resistance. Target accrual: Approximately 316 patients will be enrolled at ∼90 sites in the UK, Germany, Spain, Portugal, France, Hungary, Romania, Georgia, and South Korea. Recruitment opened in 2014 and 121 patients have been recruited to date. NCT02216786. Citation Format: Schmid P, Ferreira M, Dubey S, Zaiss M, Harper-Wynne C, Makris A, Brown V, Kristeleit H, Patel G, Perelló A, Jones A, Mithal N, Ruiz I, Kümmel S, Brunt AM, Guerra JA, Gonzalez Cao M, Saura C, Mousa K, Sarker S-J, Coetzee C, Swann R, Cortes J. MANTA: A randomized phase II study of fulvestrant in combination with the dual mTOR inhibitor AZD2014 or everolimus or fulvestrant alone in estrogen receptor-positive advanced or metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT1-03-12.

First-Line Fulvestrant for Advanced Breast Cancer

Estrogen receptor–positive advanced breast cancer is usually managed with endocrine therapy unless there is a heavy tumor burden (i.e., visceral crisis) or the tumor becomes refractory to endocrine manipulation. The optimal sequence of endocrine agents has been the subject of numerous clinical trials, though improvements in outcome with one sequence over another have often been difficult to discern. When fulvestrant was first approved, it was commonly used as second- or third-line therapy for advanced disease. However, clinical trials have demonstrated that it is …

Evaluating an ER Degrader for Breast Cancer.

Abstract Results from a phase I study of GDC-0810 show that this selective estrogen receptor degrader effectively engages its target, even in patients with ESR1 mutations, and may provide clinical benefit for postmenopausal women with advanced estrogen receptor–positive breast cancer.