Metastatic Breast Cancer Cohort Research Articles

Abstract PO1-10-07: A Video Intervention to Improve Patient Understanding of Tumor Genomic Testing in Patients with Metastatic Breast Cancer: Primary Results of a Prospective Intervention Trial

Abstract Background: Tumor genomic testing (TGT) has become standard-of-care for all patients with metastatic breast cancer (MBC). American Society of Clinical Oncology (ASCO) and American College of Medical Genetics (ACMG) guidelines for patient education prior to TGT are not widely followed. We have previously demonstrated disparities in general genomic knowledge across race and income. The purpose of this study was to develop a concise (3-4 minute) video for patient education prior to TGT and evaluate the video’s impact in a prospective interventional trial. We report the results of the primary endpoint of the MBC cohort (ClinicalTrials.gov NCT05215769). Methods: We previously published our internal quality improvement cycle involving provider surveys, patient focus groups, and adult learning theory-based content development for TGT educational videos. An animated video incorporating culturally diverse images available in English and Spanish was created to be applicable to any cancer type, with MBC-specific content included for patients with breast cancer. A total of 150 participants were enrolled at a single tertiary academic institution, of whom 53 were diagnosed with MBC. Participants completed validated survey instruments immediately prior to video viewing (T1), immediately post-viewing (T2) and 60-90 days later, after TGT results were documented (T3). Instruments included: 1) 10-question objective genomic knowledge/understanding (GKU); 2) 10-question video message-specific knowledge/recall (VMSK); 3) 11-question Trust in Physician/Provider (TIPP); 4) attitudes regarding TGT. The primary objective was to assess change in VMSK between T1 and T2 and a cohort of 50 participants provided 90% power to detect an effect size of 0.47 from pre- to post-video using two-sided Wilcoxon signed-rank test with alpha of 0.05. Associations of VMSK, GKU, and TIPP with categorical demographic variables were explored with Kruskal-Wallis test. Results: From April 2022 to May2023, a total of 150 participants were enrolled (MBC n=53, lung cancer n=38, metastatic cancer of any type n=59). The MBC cohort analysis is presented. The MBC cohort had a median age of 59; all were female; majority Caucasian (48/53, 91%); most were married/in domestic partnership (35/53, 66%). For the primary endpoint, there was a significant increase in video message-specific knowledge (Wilcoxon signed rank p< 0.0001) but there was no significant change in general genomic knowledge (p=0.89) or trust in provider (p=0.59). Improvement of video message-specific knowledge was consistent across demographic groups, including age, income, and education. Of the 10-questions in the VMSK survey, results for four questions significantly improved after viewing the video, including questions informing the likelihood of TGT impact on treatment decision, incidental germline findings, and cost of testing (Table 1). Baseline genomic knowledge was significantly associated with income (nominal p=0.028), with higher income associated with higher baseline knowledge. Conclusions: A concise, 3-4 minute, broadly applicable video incorporating culturally diverse images administered prior to TGT significantly improved video message-specific knowledge across all demographic groups. Ongoing work includes analysis of additional cohorts (lung, any type) and evaluation in community oncology setting with a goal to provide a paradigm to efficiently educate and empower patients while addressing ASCO/ACMG guidelines within the flow of clinical practice. Table 1. Response to video message-specific questions before versus after tumor genomic testing educational video intervention Citation Format: Daniel Stover, Deloris Veney, Lai Wei, Amanda Toland, Carolyn Presley, Tasleem Padamsee, Clara Lee, Heather Hampel, William Irvin, Jawad Francis, Michael Bishop, Shelly Hovick, Leigha Senter. A Video Intervention to Improve Patient Understanding of Tumor Genomic Testing in Patients with Metastatic Breast Cancer: Primary Results of a Prospective Intervention Trial [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 PO1-10-07.

Abstract LB391: Deep learning AI predicts HRD and platinum response from histologic slides

Abstract Background: Cancers with homologous recombination deficiency (HRD) can benefit from platinum salts and PARP inhibitors. Standard diagnostic tests, including FDA-approved companion diagnostics, for detecting HRD require molecular profiling, which is not universally available with global testing rates lowest among minority, rural, and other underserved populations. Methods: We trained DeepHRD, a deep-learning platform for predicting HRD from hematoxylin and eosin (H&E)-stained histopathological slides, using primary breast (n=1,008) and ovarian (n=459) cancers from The Cancer Genome Atlas (TCGA). DeepHRD was compared to four standard HRD molecular tests using breast (n=349) and ovarian (n=141) cancers from multiple external and independent datasets, including clinical cohorts with platinum complete response, progression-free survival (PFS) and overall survival (OS) endpoints. Results: DeepHRD detected HRD from held-out H&E-stained breast cancer slides in TCGA with an AUC of 0.81 ([0.77-0.85]; 95% confidence interval). This performance was confirmed in two independent primary breast cancer cohorts (AUC=0.76; [0.71-0.82]). In an external platinum-treated metastatic breast cancer cohort, samples detected as HRD had a higher complete response (AUC=0.76; [0.54-0.93]) with 3.7-fold increase in median PFS (14.4 versus 3.9 months; p-value=0.0019) and hazard ratio (HR) of 0.45 (p=0.0047) after correcting for PAM50 molecular subtype and age at diagnosis. This deep-learning classifier outperformed four genomic HRD tests used in the clinic, including standard HRD score, BRCA1/2, 26-HR gene panel and single-base substitution signature 3 (SBS3). Multiresolution spatial mapping identified morphological features utilized by DeepHRD for detecting HRD, notably enriched for neoplastic and necrotic tissues, and a higher macrophage density. Through transfer learning to high-grade serous-ovarian cancer, DeepHRD-positive samples exhibited better overall survival after TCGA first-line (HR=0.46; p=0.030) and an external neoadjuvant (HR=0.49; p=0.015) platinum-treated cohorts. Conclusion: In summary, DeepHRD exhibits consistent hazard ratios ranging from 0.45 to 0.49 across the three clinical cohorts and captures 1.8- to 3.1-fold more HRD-positive breast and ovarian cancer patients. DeepHRD-positive breast cancer patients that received platinum exhibited better complete response and PFS. Similarly, DeepHRD-positive platinum-treated ovarian cancer patients had a better OS. DeepHRD’s ability to detect HRD from digital H&E slides provides an important precision oncology tool that can be utilized in resource-constrained and underserved areas where genomic testing is generally not existent. Citation Format: Erik N. Bergstrom, Ammal Abbasi, Marcos Diaz-Gay, Loïck Galland, Sylvain Ladoire, Scott M. Lippman, Ludmil B. Alexandrov. Deep learning AI predicts HRD and platinum response from histologic slides [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 LB391.

Serial multiomic analysis in advanced breast cancer: A novel platform to inform therapeutic decisions.

e13001 Background: We designed and implemented a platform wherein patients with metastatic treatment-refractory cancer would receive individually tailored treatment informed by multi-omic data and iteratively adapted in real-time. Here we describe the workflow of this platform and quantify operational characteristics for a metastatic breast cancer cohort. Methods: Participants with metastatic breast cancer progressing after standard-of-care therapy were enrolled. Sites demonstrating disease progression were targeted for biopsy. Resultant tissue was analyzed by a next-generation sequencing (NGS) solid tumor panel, whole exome sequencing, whole transcriptomic sequencing, multiplex protein analysis of a panel of cancer-relevant proteins and phosphoproteins, and focused immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH) analysis. Results: Between 1/1/2017 to 12/31/2021, 74 participants were consented and 55 were enrolled. Median age at enrollment was 54 years, most were ECOG 0-1, and received a median of 2 lines of therapy in the metastatic setting. There were 95 biopsies collected from 55 individual participants and analytics were successfully generated in 94% of the cases. At the provider’s request, an integrated clinical, molecular, and cancer biology tumor board (TB) was convened for 25 participants. A total of 21 participants received matched therapy based on clinical parameters coupled to a consideration of biologically informed therapeutic vulnerabilities and 6 had a PFS2/PFS1 > 1.3. Twenty-one participants had a median of 2 repeat progression biopsies (range 2-5) and new alterations with potential therapeutic implications were identified. Repeat biopsies could have led to a new treatment in 11 of the participants, though only 3 received a therapy recommended by the tumor board owing to either rapid disease progression, clinical deterioration, or lack of access to drug. Conclusions: We demonstrated the practical feasibility and initial operating characteristics of a platform designed to comprehensively understand and integrate the clinical and molecular characteristics of a cohort with metastatic progressive breast cancer as they evolve on therapy and to use this information to deliver iteratively tailored therapy in real time. Further, clinical responses were observed in a subset of participants. These findings suggest an opportunity to incorporate this platform earlier in a disease course or to design adaptive clinical trials with access to therapies aimed at targeting new biological alteration.

Abstract 3433: Global proteomic analysis of urine using a label-free LC-MS/MS approach identifies potential biomarkers of metastatic breast cancer

Abstract Introduction: Breast cancer (BC) is a complex heterogenous disease and is a leading cause of death in women. Following primary BC therapy ~40% of patients will develop metastatic BC (MBC) and 60-70% of the recurrent disease will occur at distant sites. Median survival of MBC patients with distant recurrence is ~2-3 yr. While the early detection and monitoring of BC progression are essential to improve prognosis and reduce BC-related mortality, there is a lack of surveillance strategies for monitoring patients for distant recurrence of MBC. The aim of our study was to identify urinary biomarkers for detection and monitoring of MBC. Method: We have conducted a comparative label-free LC-MS/MS analysis of the urinary proteome of patients with MBC and healthy age-matched controls (HC). A hybrid quadrupole time of flight (Q-Tof™) mass spectrometer was used for urine analysis via liquid chromatography (LC) with tandem mass spectrometry (MS/MS). Three different database search algorithms were used for peptide identification including MASCOT, Protein Lynx Global Server and Ion Accounting software. Only peptides detected in at least two out of three replicated LC-MS experiments were counted towards protein identification. Identified candidate biomarkers were validated via ELISA assays. Results: Using this approach, we identified ~400 urinary proteins of which ~104 and 89 were unique to the MBC and HC groups, respectively. Upregulated proteins in the MBC cohort were associated with angiogenesis, apoptosis, proteolysis, ECM regulation, and cell adhesion pathways. Using bioinformatic and in silico approaches, we identified a specific metastasis signature comprised of CALB1, S100A8, ZAG, VTN and TN. Diagnostic accuracies of these candidate markers were validated using independent training and validation sets according to the REMARK criteria. Urinary VTN (uVTN) and uTN levels correlated significantly with disease status in MBC patients when compared to patients with locally invasive BC and HC. Interestingly, a small subset of patients with bone metastasis also had significantly higher uVTN levels. A multiplexed marker panel of uVTN and uTN could discriminate between HC and MBC groups (AUC=0.831, P<0.001). Blinded testing indicated that uVTN levels (cutoff>1018 ng/ml) could discriminate (AUC=0.782, P<0.006) between MBC and HC. Longitudinal analysis of samples from MBC patients indicated a strong correlation between uVTN levels and disease status. Conclusions: Our findings suggest that uVTN and uTN have the potential to serve as biomarkers for the detection of MBC. While validation in larger cohorts is necessary, these results may be useful in the development of noninvasive diagnostic tests for monitoring BC progression and recurrence. [Supported by: the Advanced Medical Research Foundation, the Jo Ann Webb Fund for Angiogenesis Research and the S. Elizabeth O’Brien Trust] Citation Format: Roopali Roy, Elisa Schunkert, Petra Olivova, Martin Gilar, Scott Geromanos, Guo-Zhong Li, John Gebler, Adelle Dagher, Andrew El-Hayek, Rama Aldakhlallah, David Zurakowski, Susan Pories, Marsha A. Moses. Global proteomic analysis of urine using a label-free LC-MS/MS approach identifies potential biomarkers of metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3433.

Abstract 5459: Association between stomatitis and treatment efficacy with novel TROP2-directed antibody drug conjugate (ADC), datopotamab deruxtecan, in patients with metastatic cancer

Abstract Background: Incidence of toxicity with immunotherapy has been correlated with likelihood of treatment response (Hussaini, Cancer Treat Rev 2021; Schuell, Br J Cancer 2005). However, this phenomenon has not yet been demonstrated with antibody-drug conjugates. Datopotamab deruxtecan (Dato-DXd) is a novel ADC composed of an anti-Trop2 monoclonal antibody, a stable tetrapeptide-based cleavable linker, and a topoisomerase-I inhibitor payload (Bardia, Annals of Onc 2021). Early phase studies have demonstrated significant activity in heavily pretreated patients with metastatic non-small cell lung cancer (NSCLC) and metastatic breast cancer (MBC). Stomatitis was a common treatment-emergent adverse event (TEAE) observed in clinical trials. We conducted a study to evaluate the association between incidence of stomatitis and treatment response with Dato-DXd and evaluate the impact of steroid mouthwash. We hypothesized that presence and grade of mucositis would correlate with observed treatment efficacy. Methods: We included all patients treated at one academic institution (Massachusetts General Hospital) with Dato-DXd. Stomatitis was assessed by NCI-CTCAE V5.0. Objective Response Rate (ORR) was assessed by RECIST criteria with independent radiology review. Use of mouthwash was evaluated by secondary review and categorized as primary or secondary prophylaxis. A time-to-event analysis was performed to evaluate the time to onset of stomatitis. Results: 60 patients, including 19 with MBC and 41 with NSCLC, were treated with Dato-DXd between June 2018-September 2022. The median age of patients was 64 and 41/60 patients were female. The median time to onset of mucositis was 14 days (95% CI 12-42 days). There was no significant difference in incidence of stomatitis between the MBC and NSCLC cohorts. Compared to patients who had progressive disease (PD), patients who had a partial response (PR) or stable disease (SD) were 11 times more likely to have stomatitis (OR 11.0, 95% CI 3.61-44.01, p<0.001). Later in the trial (January 2020 onward), 28 patients received primary steroid prophylaxis - excluding these patients, those who were found to have PR/SD were 8.8 times more likely to develop stomatitis compared to patients who had PD (OR 8.8, 95% CI 1.5-73.3, p=0.023). Conclusion: The development of stomatitis may be associated with higher likelihood of therapeutic efficacy. Given that these findings are a hypothesis-generating post-hoc exploratory analysis from a single institution, this requires validation in larger studies before drawing strong conclusions and impacting clinical decision making. Further research is also needed evaluate the relationship between TEAEs and therapeutic efficacy with other ADCs. Citation Format: Rachel O. Abelman, Laura M. Spring, Phoebe K. Ryan, Geoffrey Fell, Dejan Juric, Rebecca S. Heist, Aditya Bardia. Association between stomatitis and treatment efficacy with novel TROP2-directed antibody drug conjugate (ADC), datopotamab deruxtecan, in patients with metastatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5459.

Abstract P2-26-07: Alteration of gut microbiota signatures and its association with diarrhea during abemaciclib treatment: A multicenter prospective cohort study (KBCRN-A002 study)

Abstract Background: Abemaciclib is a selective CDK4 and CDK6 inhibitor with demonstrated efficacy in hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer. The most common adverse event across previous trials was early-onset diarrhea, affecting the patients’ quality of life and necessitating dose reductions. However, the exact mechanism for the lower rate of diarrhea in the other CDK4 and CDK6 inhibitors compared with abemaciclib is unknown. Ample evidence indicates that the gut microbiome is a tumor-extrinsic factor associated with the anti-tumor response; however, reported microbial signatures associated with adverse events by anti-cancer agent are inconsistent. To determine the underlying mechanism, we evaluated the correlation between diarrhea with abemaciclib and microbiota signatures in a metastatic breast cancer cohort. Methods: The KBCRN-A002 study is a multicenter, prospective cohort study, which aims to evaluate the association between gut microbiota signatures and abemaciclib-induced diarrhea in breast cancer patients. Patients with metastatic breast cancer who were receiving abemaciclib were eligible. The primary objective of this study is the correlation between diarrhea and the microbiota signatures and immune profile. Incidence and severity of diarrhea were evaluated by the Bristol stool scale at baseline, from day 1 to day 14, and at day 90 of treatment. Stool samples were collected at baseline and at day 90 after the start of abemaciclib treatment. The gut microbiota signature was evaluated by 16S rRNA analysis. Blood samples were collected at baseline and at days 14 and 90 after starting abemaciclib to evaluate the correlation between the gut microbiota signatures and the systemic immune profile in peripheral blood mononuclear cells (PBMCs). The immune profile was evaluated by mass cytometry, multi-plex cytokines assay, and RNA-sequencing of bulk PBMCs. We characterized the gut microbiota signatures, immune cell composition, immune cell signature, comprehensive cytokines, and severity of diarrhea in all patients. Results: We analyzed 39 patients, 77 stool samples, and 117 blood samples. In the preplanned interim analysis, among the 39 patients, 90% experienced diarrhea. Depleted gut microbiome α-diversity was positively associated with abemaciclib treatment and the severity of diarrhea. The relative abundances of 10 intestinal bacteria species increased and those of 18 intestinal bacteria decreased significantly after abemaciclib treatment, including bacteria known to be involved in diarrhea severity and anti-tumor immunity, such as Faecalibacterium (Table). The immune cell and cytokine profiles in PBMCs were also associated with the gut microbiota signatures. Conclusions: Gut microbiota signatures are associated with abemaciclib-induced diarrhea and the immune profile in metastatic breast cancer patients. These findings can help to elucidate the mechanism of diarrhea caused by abemaciclib and offer strategies for its management and prevention. Intestinal Microbiota Altered by Abemaciclib Citation Format: Kosuke Kawaguchi, Yurina Maeshima, Hiroshi Ishiguro, Kazuhiko Yamagami, Sachiko Takahara, Hirofumi Suwa, Masae Torii, Shigenori Nagai, Yasuaki Sagara, Wakako Tsuji, Hiroyasu Yamashiro, Takeshi Kotake, Shinji Fukuda, Kuniaki Saito, Yasuko Yamamoto, Masako Kataoka, Yuki Himoto, Atsushi Yonezawa, Yukiko Fukui, Yuki Nakamura, Wei Li, Sunao Tanaka, Satoshi Morita, Masakazu Toi. Alteration of gut microbiota signatures and its association with diarrhea during abemaciclib treatment: A multicenter prospective cohort study (KBCRN-A002 study) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-07.

Abstract P5-05-03: Characterization of the genomic landscape of breast carcinoma patients with NF1 alterations using comprehensive cell-free tumor DNA next-generation sequencing

Abstract Background: NF1 (neurofibromin type 1) encodes neurofibromin, and is commonly altered in many cancers, including breast cancer. NF1 suppresses breast cancer by not only negatively regulating RAS signaling but also by independently acting as a transcriptional co-repressor of the estrogen receptor (ER). In this study, we analyzed the genomic landscape of patients with NF1 alterations from a large genomic database to define what unique patient characteristics were associated with NF1 alterations. Methods: Retrospective analysis of the Guardant Health database based on samples from the commercially available Guardant360® plasma-based circulating tumor DNA (ctDNA) assay. Samples were queried between June 2020-June 2022 for patients with any detected NF1 alteration and breast cancer diagnosis. NF1 synonymous alterations were excluded from this study. Statistics were conducted using a two-sided Fisher’s exact test. Results: NF1 alterations were found in 895 patients with breast cancer over 1156 samples, typically in female patients (98.2%) diagnosed with breast carcinoma (99.4%). The average age of patients was 66 years old (23-93), with a median of 1.4 serial tests (1-19). The common nonsynonymous NF1 alterations are missense mutations (56.5%), nonsense mutations (23.5%), indels (22.3%), and aberrant splicing mutations (8.2%). There were significant differences in NF1 alteration frequency between younger (< 55 y/o) vs. older (≤55 y/o) patients, with older patients demonstrating an increase in NF1 alterations (p< 0.0001) across all mutation types except for splice mutations. There was also a significant difference in NF1 alterations between female vs. male patients, with male patients trending toward a higher frequency in NF1 missense alterations. Mutations affecting genes encoding the receptor tyrosine kinase (e.g., HER2) and the Ras-MAP kinase pathways (e.g., several RAS and RAF genes) co-occur with NF1 mutations. In contrast, there is no evidence of co-occurrence with mutations in the ESR1 gene, which encodes ER. The blood tumor mutational burden (bTMB) score was evaluable in 848 patients with an average score of 26.1 mut/Mb (range 1.16-447.7). In addition, mutations affecting genes controlling the cell cycle were also found to co-occur with NF1 mutations. Conclusions: Plasma-based liquid biopsy via G360 can efficiently identify NF1 alterations illustrating that such genetic alterations are common in this metastatic breast cancer cohort. Analysis of co-occurrent mutations supports our model that a key role of NF1 is to act as an ER transcriptional co-repressor, such that its loss is functionally redundant with acquiring ESR1 mutations. Oncogenic activation of the RTK-Ras pathways is needed for efficient progression to metastasis by additional mutations in this pathway. Mutational co-occurrence may also identify collaborating molecular events that collaborate with NF1 loss to promote treatment relapse and metastasis. Citation Format: Eric Chang, Jill Tsai, Bora Lim. Characterization of the genomic landscape of breast carcinoma patients with NF1 alterations using comprehensive cell-free tumor DNA next-generation sequencing [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-05-03.

Abstract PD10-03: PD10-03 Super-enhancer-oriented integrative bioinformatics identifies aberrant KLF4 signaling in endocrine-resistant breast cancer (BC)

Abstract Background: Recent studies suggest that enhancer reprogramming underlies heterogeneity and disease progression in estrogen receptor-positive (ER+) BC. Cell-type/state specific transcription is governed by high-order assemblies of master transcription factors (TFs) and epigenetically defined regulatory regions including super-enhancers (SEs). We previously showed that aberrant activation of the pioneer TF FOXA1 promotes enhancer and transcriptional reprogramming in endocrine-resistant BC, involving the ER and the AP-1 FRA1 and c-JUN TFs. As SEs maintain a robust cell-type/state specific core transcriptional regulatory circuitry (CRC) in developmental and tumorigenic processes, we sought to identify key additional TFs in SE/FOXA1-driven CRCs in endocrine resistance, which could serve as attractive therapeutic targets. Methods: TF binding motif at the shared SEs (mapped by H3K27ac ChIP-seq) between MCF7-parental (P) cells with ectopic FOXA1 overexpression (OE) and the endogenous FOXA1-amplifed tamoxifen-resistant (TamR) cells was analyzed by HOMER. ER-bound SEs distinguishing TamR vs. P cells were defined by integrating the SEs with our prior ER ChIP-seq data (PMID 28507152). KLF4 motif within these ER-bound SEs was scanned using FIMO and linked to nearby genes by intersection with the previously defined promoter-tethered regions (PTRs) (PMID 24141950). Differential gene expression in MCF7-TamR cells upon KLF4 knockdown (KD) by 3 unique siRNAs was analyzed using limma from edgeR. The biological and clinical significance of the KLF4-dependent genes was analyzed using Gene Ontology and survival modeling with METABRIC and the ER+ metastatic BC cohort (SABCS19-GS2-02). Cell migration was assessed by the wound-healing assay. Results: We identified KLF4 among the top enriched TF binding motifs at the shared SEs in FOXA1-overexpressing MCF7-P cells and the FOXA1-amplified TamR cells. Analysis of our prior RNA-seq data of MCF7-P and TamR cells upon OE or siRNA KD of FOXA1, FRA1, or c-JUN (PMIDs 27791031, 31826955, 32424275, SABCS21-PD1-05) revealed KLF4, the Yamanaka factor for induced pluripotent stem cells, as a common target activated by the FOXA1/FRA1/c-JUN axis. We next identified 44 genes commonly down-regulated upon KLF4 KD in the MCF7-TamR cells. This KLF4-dependent 44-gene set was enriched in biological processes of embryonic development and tumor progression, preferentially dependent on ER in MCF7-TamR vs. P cells, highly elevated in ER+ metastases vs. primary tumors, and associated with poor outcome in ER+ BC treated with endocrine therapy. KLF4 KD, using the 2 siRNAs that generated similar pathway perturbations in MCF7-TamR cells, reduced TamR cell migration. Notably, among the genes co-dependent on KLF4 and ER in TamR cells, PYGB was the only gene with a PTR residing in an ER-bound SE established in TamR but not P cells. Glycogen phosphorylase B, encoded by PYGB, is the rate-limiting enzyme in glycogen degradation and plays a role in the progression of various tumors. Expression of KLF4, FOXA1, and FRA1 are commonly activated during the differentiation of human embryonic stem cells into foregut endoderm and in the inner core of fibroblasts of first-trimester human placenta villi, suggesting a unique role of KLF4 in mediating lineage-specific CRC, possibly by engaging PYGB and the glycogen metabolic pathway in advanced ER+ disease. Conclusions: Using SE-oriented integrative bioinformatics, we identified KLF4 as a potential novel target in the FOXA1/AP-1 transcriptional axis. As KLF4 binding motif resides in the unique ER-bound SEs of TamR cells, KLF4 likely forms an auto-regulated loop amplifying CRC in transcriptional reprogramming, among which the PYGB/glycogen metabolic pathway merits further investigation in endocrine-resistant ER+ disease. Citation Format: Chia Chia Liu, Lanfang Qin, Shanunak Sathe, Sarmistha Nanda, Jamunarani Veeraraghavan, Ofir Cohen, Nikhil Wagle, Mothaffar Rimawi, C. Kent Osborne, Xiaoyong Fu, Rachel Schiff. PD10-03 Super-enhancer-oriented integrative bioinformatics identifies aberrant KLF4 signaling in endocrine-resistant breast cancer (BC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-03.

Abstract P6-10-09: Mutational landscape of breast cancer patients in ROME trial: preliminary results

Abstract BACKGROUND: The Rome Trial is a randomized phase II trial (NCT04591431). The aim is to evaluate efficacy and safety of a tailored treatment (TT) compared to standard of care (SoC) in patients with solid tumors. Here we report the preliminary results of the molecular alterations, microsatellite status (MS) and tumor mutational burden (TMB) in metastatic breast cancer (mBC) cohort. METHODS: MBC patients who received at least 1 and no more than 2 systemic treatments were enrolled. Tissue samples were collected within 6 months from the screening. Centralized Next Generation Sequencing (NGS) was performed on both tissue and liquid biopsy. Molecular alterations were evaluated by the Molecular Tumor Board (MTB) using COSMIC, ClinVar, OncoKB and VarSome datasets. Genes with at least 10% frequency of mutation, MS and TMB are reported. RESULTS: From Oct 2020 to June 2022, 980 pts with solid tumors were enrolled. Complete screening mutational data are available for sixty-two pts from the mBC cohort (63% HR+/HER2-, 35% triple negative, 2% HR-/HER2+). NGS was available both on tissue and liquid biopsy in 48 (77%) pts, 14 had only liquid biopsy available due to tissue test failure. 328 genes resulted altered with a median of 7 alteration per pts (0-31). Some pathways were frequently altered: PIK3CA/AKT/MTOR (60%), TP53 (60%), Cell cycle/cycline (35%), FGF/FGFR (26%), BRCA1/2 (17%). The most frequent altered genes were: TP53 (61%), PIK3CA (50%), ESR1 (27%), CCND1 (27%), FGF19 (24%), FGF3 (24%), FGF4 (22%), MYC (22%), FGFR1 (21%), PTEN (21%), EMSY (16%), RB1 (14%), RAD21 (14%), TET2 (13%), BRCA2 (11%), GATA3 (11%), KRAS (10%). No pts with MSI status were reported. Eight (13%) had a high TMB (>10) and the overall median TMB was 5.5 (0-24). Median TMB was similar in tissue and liquid samples (5 and 5.3 mut/mb, p= 0.8). Actionable mutations were detected in 34 pts (54%). Twenty-eight (45%) pts were assigned to a specific TT after the MTB discussion: ipatasertib (16), pemigatinib (5), ipilimumab plus nivolumab (4), lapatinib plus trastuzumab, TDM1 and everolimus (1). MTB requested a germline test for 6 pts: 4 were confirmed (66%; 2 BRCA, 1 PALB2, 1 BRIP1). CONCLUSIONS: The extensive NGS analysis performed in the ROME trial shown that several pathways are commonly mutated in mBC, with target drug potentially available. About 15% of pts had a high TMB but MSI is confirmed as a rare event in breast cancer. Germline mutations have been identified in patients with no prior indication for germline testing. Citation Format: Andrea Botticelli, Simone Scagnoli, Pierfranco Conte, Chiara Cremolini, Paolo Antonio Ascierto, Federico Cappuzzo, Massimo Aglietta, Federica Mazzuca, Ettore Capoluongo, Giovanni Blandino, Umberto Malapelle, Marianna Nuti, Giulia D’Amati, Bruna Cerbelli, Giancarlo Pruneri, Mauro Biffoni, Giuseppe Giannini, Francesco Cognetti, Giuseppe Curigliano, Paolo Marchetti. Mutational landscape of breast cancer patients in ROME trial: preliminary results [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-09.

Abstract P5-02-24: Whole genome sequencing of long-term, never relapse exceptional responders HER2+ advanced metastatic breast cancer

Abstract Background: Anti-HER2 therapies such as trastuzumab used for the treatment of patients with HER2+ metastatic breast cancer (MBC) have led to significant improvements to disease progression. We previously identified cases from the “Thousand Patient HER2 database” project at Saint Vincent’s University Hospital (SVUH) Dublin, of HER2+ MBC long-term durable complete responders to trastuzumab, and reported that Copy Number Aberration (CNA) burden may represent a novel prognostic predictor to trastuzumab response from the exome analysis of in HER2+ MBC “exceptional responders’’ (ExRs). However, whole-genome sequencing (WGS) allows a better understanding of how CNA affects the MBC genome and to-date, the complete genome of this “exceptional” cohort has never been described. Methods: We performed WGS analysis to characterise the CNA profiles of 9 ExRs from our HER2+ MBC cohort treated with trastuzumab. Samples were obtained from patients who never progressed/relapsed for more than 5 years (OS > 60 months). DNA was sequenced from tumours (primary or metastases) and matching control (blood or normal tissue) at a mean depth of 60X and 30X, respectively (18 samples). Somatic single nucleotide variants (SNV) were detected using GATK4 Mutect2 and CNA were identified using Control-FREEC. Results: Eighty-five HER2+ MBC were identified with OS > 60 months, of which 28 were ExRs with bone, lung, liver and lymph metastasis who responded exceptionally to trastuzumab, with a mean OS of 108 months (range 61-236 months). This cohort includes patients who were diagnosed between 31 and 80 years old (median=51). WGS analysis revealed CNA in chr6p21 with amplification of CCND3 and in chr17q12 with amplification of RAD51D. SNV were identified in genes involved in the DNA damage repair (DDR) pathway such as ATM, BRCA2, RAD50 and FANCA. On-going analysis will allow the CNA profiles of all ExRs to be presented and their CNA burden calculated in order to investigate the relationship between whole genome CNA burden and HER2+ MBC patient survival. Conclusion: To our knowledge, this is the first study to sequence the whole genome of HER2+ MBC, never relapse exceptional responders. The identification of the genomic aberrations of these metastatic patients increases our understanding of the mechanisms involved in MBC progression. CNA burden may represent a novel prognostic predictor to trastuzumab response and new outcomes for patients, particularly as MBC is generally termed incurable. Citation Format: Charlotte Andrieu, Laura P. Ivers, Jose Javier Berenguer Pina, Darko Skrobo, Jo Ballot, Alex J. Eustace, Cecily Quinn, Giuseppe Gullo, Naomi Walsh, John P. Crown. Whole genome sequencing of long-term, never relapse exceptional responders HER2+ advanced metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-24.

Abstract P2-03-14: Tumor-agnostic measurement of ctDNA levels in first line ER+, HER2- metastatic breast cancer patients as a marker for survival

Abstract INTRODUCTION: Circulating tumor cell (CTC) count and levels of circulating tumor DNA (ctDNA) have proven their individual prognostic value in metastatic breast cancer (MBC). Whereas CTCs can be quantified by enumeration, levels of ctDNA are often expressed as the variant allele frequency of the dominant mutation. The modified fast aneuploidy screening test-sequencing system (mFAST-SeqS) estimates ctDNA-load by measuring chromosomal aneuploidy instead of mutations. The mFAST-SeqS method is cheap and provides a comprehensive overview of the tumor genome without prior knowledge on genomic tumor aberrations. Previously, we showed in a small MBC cohort (n=50), heterogeneous with regard to subtype and line of treatment, that the mFAST-SeqS-derived genome-wide aneuploidy score (GWA-score) was an independent prognostic biomarker (1). Our current aim was to provide evidence for the prognostic value of GWA-score in a homogeneous cohort of estrogen receptor positive (ER+), HER2 negative (HER2-) MBC patients, starting first line treatment with an aromatase inhibitor. MATERIALS & METHODS: CTCs were enumerated using the CellSearch-system (Menarini) and screened for ctDNA fraction using mFAST-SeqS in ER+, HER2- MBC patients, starting with first line aromatase inhibitors. CTC-count was divided into high (CTChigh) and low (CTClow) using the validated cut-off of 5 CTCs/7.5 ml blood; similarly GWA-score was divided in high (GWAhigh)and low (GWAlow) based on the previously described cut-off of 5 (2). PFS and OS were estimated using Kaplan-Meier. Cox regression was used for uni- and multivariable analysis. RESULTS: One hundred and thirty-four patients were included in this analysis. Mean age was 68 (range 40-90). Median follow up was 26 months. Sixty-five samples had both scores low and 30 both high, 25 samples were CTChigh/GWAlow and 14 were CTClow/GWAhigh. PFS and OS were not significantly different between CTChigh versus CTClow groups (p=0.07 and p=0.26, median PFS 15 vs 24 months, median OS 45 vs 49 months). However, PFS and OS were significantly different between GWAhigh versus GWAlow groups (p < 0.01 for both, median PFS 11 vs 24 months, median OS 31 vs 51 months). In multivariate analysis, GWA-score was a significant prognostic factor for both PFS and OS while presence of visceral disease was also prognostic for OS (table 1). CONCLUSIONS: In patients treated with aromatase inhibitors in the first line of treatment, GWA-score was an independent prognostic marker for a worse PFS and OS and may have added value beyond CTC-count. 1. Verschoor, N., et al., Abstract P2-01-17: Circulating tumor cell count and levels of circulating tumor DNA are complementary prognostic biomarkers in metastatic breast cancer - A pilot study. Cancer Research, 2022. 82(4_Supplement): p. P2-01-17-P2-01-17. 2. Belic, J., et al., mFast-SeqS as a Monitoring and Pre-screening Tool for Tumor-Specific Aneuploidy in Plasma DNA. Adv Exp Med Biol, 2016. 924: p. 147-155. Table 1: Uni- and multivariate Cox regression for PFS and OS Citation Format: Noortje Verschoor, Jaco Kraan, Vanja de Weerd, Ngoc M Van, Agnes Jager, Stefan Sleijfer, John WM Martens, Saskia M Wilting. Tumor-agnostic measurement of ctDNA levels in first line ER+, HER2- metastatic breast cancer patients as a marker for survival [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-03-14.

Long-Term Results with Everolimus in Advanced Hormone Receptor Positive Breast Cancer in a Multicenter National Real-World Observational Study

Everolimus is the first oral targeted therapy widely used in advanced HR+/HER2- breast cancer. We sought to evaluate the impact of everolimus-based therapy on overall survival in the ESME-MBC database, a national metastatic breast cancer cohort that collects retrospective data using clinical trial-like methodology including quality assessments. We compared 1693 patients having received everolimus to 5928 patients not exposed to everolimus in the same period. Overall survival was evaluated according to treatment line, and a propensity score with the inverse probability of treatment weighting method was built to adjust for differences between groups. Crude and landmark overall survival analyses were all compatible with a benefit from everolimus-based therapy. Adjusted hazard ratios for overall survival were 0.34 (95% CI: 0.16-0.72, p = 0.0054), 0.34 (95% CI: 0.22-0.52, p < 0.0001), and 0.23 (95% CI: 0.14-0.36, p < 0.0001) for patients treated with everolimus in line 1, 2, and 3 and beyond, respectively. No clinically relevant benefit on progression-free survival was observed. Causes for everolimus discontinuation were progressive disease (56.2%), adverse events (27.7%), and other miscellaneous reasons. Despite the limitations inherent to such retrospective studies, these results suggest that adding everolimus-based therapy to the therapeutic sequences in patients with advanced HR+/HER2- breast cancer may favorably affect overall survival.

1725P Metastasis-resident bacteria in advanced hormone receptor-positive breast cancer are related to primary tumor microbiota and show distinct composition

Tumor-resident bacteria are an emerging component of the tumor microenvironment. Recent studies have shown their presence over multiple cancer types. Lately, mechanistic evidence in a murine breast cancer model indicates that these bacteria promote the metastatic process. However, the presence and conformation of the microbiome of human metastatic tumors have not been determined. Here, we characterized tumor-resident bacteria in a cohort of metastatic hormone receptor-positive breast cancer (MHRBC) patients with matched primary tumors. We performed bacterial 16S rDNA sequencing targeting hypervariable regions V2-4 and V6-9 on FFPE tissues from 40 patients with MHRBC and their matched primary tumors. Sequence data were processed using high resolution sample inference with DADA2. Controls included normal breast tissue, paraffin from all blocks and a simulated bacterial community, comprising known intra and extracellular bacteria. Taxonomy was assigned using the SILVA database v138. Feature selection was used to determine amplicon sequences in primary tumors related to their metastasis. A machine learning classifier was generated to predict the metastatic site from selected amplicons. α-diversity was similar among sample types, while β-diversity showed segregation between metastatic and primary tumors. Alphaproteobacteria, Gammaproteobacteria and Bacilli had increased relative abundance across metastatic and primary tumors. Differential abundance of Proteobacteria and Firmicutes species was identified in metastatic tumors. A machine learning classifier using the 5 top ranked amplicons in primary tumors was capable of 100% precision and high recall for prediction of bone and liver metastatic site, but not lung metastases. We identified and categorized the tumor-resident bacteria of MHRBC. To our knowledge, this is the first study looking at the composition of metastatic breast cancer microbiome. While insights have been gained on primary tumor microbiota, the role of metastasis-resident bacteria including local immunity and treatment resistance warrants further study.

Clinical subtype, treatment response, and survival in De Novo and recurrent metastatic breast cancer.

This study evaluated whether patients with de novo metastatic breast cancer (MBC) have superior outcomes compared to those with recurrent MBC in a contemporary treatment era and examined factors related to outcome differentials. Using an institutional database, we examined patient and tumor characteristics, treatment response, and outcome among 232 patients with de novo and 612 patients with recurrent MBC diagnosed between 2011 and 2017. De novo MBC had 9-month (m) longer overall survival (OS) than recurrent MBC (36.4 vs 27.4m, p < 0.001). Contributions to this difference included nearly twofold more HER2-positive (29.3% vs 15.2%) and significantly fewer triple-negative breast cancers (20.3% vs 32.4%, both p < 0.001) in de novo compared with recurrent MBC cohorts. Stratified by clinical subtype, progression-free survival (PFS) on first-line therapy was significantly longer in de novo MBC in all but the triple-negative subtype, 25.5 vs 11.6m (p < 0.001) among 390 patients with hormone receptor-positive, HER2-negative, 11.4 vs 5.4m (p = 0.002) among 142 patients with HER2-positive, and 4.0 vs 3.0m (p = 0.121) among 162 with triple-negative MBC. In multivariable analysis, de novo status remained independently associated with improved OS (hazard ratio 0.63, 95% CI 0.49-0.80), regardless of subtype and other features. Patients with de novo MBC have better outcomes than those with recurrent MBC. Differences in clinical subtype and response to therapy in the metastatic setting contribute to, but do not fully explain, this difference. Longer PFS to first-line therapy in de novo MBC suggests biologic differences compared to recurrent MBC, which may be intrinsic or due to acquired resistance from treatment for prior localized breast cancer in recurrent disease.

Primary results from TROPiCS-02: A randomized phase 3 study of sacituzumab govitecan (SG) versus treatment of physician’s choice (TPC) in patients (Pts) with hormone receptor–positive/HER2-negative (HR+/HER2-) advanced breast cancer.

LBA1001 Background: HR+/HER2– disease is the most common subtype of metastatic breast cancer (MBC). Treatment includes sequential endocrine therapy combined with targeted agents followed by single-agent chemotherapy, with increasingly shorter durations of benefit. SG is an anti–Trop-2 antibody-drug conjugate approved for metastatic triple-negative breast cancer with ≥2 prior therapies (≥1 for MBC). The HR+/HER2– MBC cohort of the phase 1/2 IMMU-132-01 study (n = 54) had an objective response rate (ORR) of 31.5%, median progression-free survival (PFS) of 5.5 mo, median overall survival (OS) of 12 mo, and a manageable safety profile with SG. TROPiCS-02 is a phase 3 randomized study (NCT03901339) to confirm SG outcomes in HR+/HER2– advanced breast cancer. Methods: Adults with locally determined, HR+/HER2– unresectable locally advanced or MBC, ECOG performance status of 0 or 1, and 2-4 prior chemotherapy regimens for MBC were eligible; 1 prior therapy for MBC was allowed if disease progressed ≤12 mo after (neo)adjuvant therapy. Pts must have received ≥1 prior taxane, CDK4/6 inhibitor, and endocrine therapy in any setting. Pts were randomized 1:1 to receive SG (10 mg/kg IV on d1 and 8, every 21d) or TPC (capecitabine, eribulin, vinorelbine, or gemcitabine) until disease progression/unacceptable toxicity. Primary endpoint was PFS per RECIST v1.1 by blinded independent central review (final analysis) with key secondary endpoint of OS (1st planned interim analysis). Results: At data cutoff on Jan 3, 2022, 272 vs 271 pts were randomized to receive SG vs TPC, respectively. Pt characteristics in the SG vs TPC arms were similar (3 median prior chemotherapy regimens for MBC [range, 0-8]; 95% had visceral metastases, 86% had prior endocrine therapy for MBC for ≥6 mo, 60% and 38% received prior CDK4/6 inhibitors for ≤12 and &gt; 12 mo, respectively). SG (vs TPC) improved median PFS (5.5 vs 4.0 mo; HR, 0.66; 95% CI, 0.53-0.83; P= 0.0003); PFS rates at 6 and 12 mo were 46% vs 30% and 21% vs 7%, respectively. SG vs TPC showed a numeric but nonsignificant difference in OS (13.9 vs 12.3 mo; HR, 0.84; P= 0.143); ORR (21% vs 14%) and clinical benefit rate (34% vs 22%) were higher with SG vs TPC and median duration of response was 7.4 vs 5.6 mo, respectively. Overall, 74% vs 60% of patients (SG vs TPC) had grade ≥3 treatment-emergent adverse events (AEs); neutropenia (51% vs 39%) and diarrhea (10% vs 1%) were most common. AEs leading to discontinuation of SG vs TPC were low (6% vs 4%). There was 1 treatment-related death in the SG arm; none in the TPC arm. Conclusions: SG had a statistically significant, clinically meaningful PFS benefit over single-agent chemotherapy and a manageable safety profile in pts with heavily pre-treated HR+/HER2– endocrine-resistant, unresectable locally advanced or MBC, who have limited treatment options. Clinical trial information: NCT03901339.

Whole genome sequencing of HER2+ metastatic breast cancer and CNA comparison between long term survivor and short-term survivor.

e13019 Background: The introduction of anti-HER2 therapies such as trastuzumab for HER2+ metastatic breast cancer (MBC) has led to significant improvements to disease progression. We and others have reported long-term durable complete response to trastuzumab in patients with HER2+ MBC. We hypothesise that genomic copy number alteration (CNA) and tumour mutational burden (TMB) may act as a prognostic measure of predicting response to trastuzumab in long-term survivors (LTS) compared to short-term survivors (STS). Using whole-genome sequencing (WGS) to detect these alterations, we aim to identify the molecular characteristics of primary and metastatic lesions in patients with metastatic HER2+ breast cancer. Methods: WGS was performed on samples from two patients from a HER2+ MBC cohort; a of long-term survivor (PFS 27 months; OS &gt; 60 months) and a short-term survivor (PFS 4 months; OS &lt; 14 months). Primary tumours, adjacent normal tissue and metastases were sequenced at a mean depth of 60X. Reads were aligned on the hg38 reference genome using Burrows-Wheeler Aligner (bwa), CNA were detected using Control-FREEC to evaluate total CNA burden. Single nucleotide variants were detected using GATK Mutect2 to calculate TMB. Results: Analysis of DNA chromosome disruption (fraction of the genome amplified/deleted) revealed a higher overall fraction of disrupted genome in the LTS primary tumour and metastasis compared with the STS (0.061 and 0.064 vs 0.031 and 0.047, respectively). Further delineation of the distribution of CNA burden in all genomes identified chr1 as the most altered in the STS samples whereas chr17 was the most disrupted in the LTS samples. HER2 and CDK12 were amplified in both LTS and STS samples (4 copies in LTS primary tumour and metastasis, 18 and 14 copies in STS primary tumour and metastasis respectively). A high TMB was estimated in both samples but was higher in the STS primary tumour than in the LTS one (161 vs 142, respectively). TMB was increased in LTS metastatic lesions. NRAS mutation was found in LTS primary tumour but was lost in the metastasis. Conclusions: This pilot study highlights the potential for whole-genome sequencing to identify CNA and TMB in HER2+ MBC in patients who display long-term and short-term survival. Further samples from this cohort need to be studied to investigate the relationship between CNA burden and HER2+ MBC patient survival.

Treatment patterns and their impact on the outcome of patients with HR+/HER2+ metastatic breast cancer in a large real-world cohort.

1043 Background: The place of endocrine therapy (ET) in the treatment of hormone receptor-positive (HR+), HER2+ metastatic breast cancer (MBC) is still not clearly defined. Data suggest that blocking both HR and HER2 signaling pathways could be an efficacious strategy to overcome secondary resistance. Methods: We aimed to retrospectively evaluate the impact of first line (L1) therapy for HR+/HER2+ MBC patients (pts) included between 2008- 2017 in the French real-world ESME MBC database (NCT03275311). Our primary endpoints were median overall survival (mOS) and median first progression-free survival (mPFS1). We used descriptive statistics and the Kaplan-Meier method to report patient characteristics and outcomes. Cox proportional hazards models and a propensity score were constructed to report and adjust for prognostic factors. Results: From the 23,501 female pts in the ESME MBC cohort, 1,790 pts had HR+/HER2+ MBC treated with Trastuzumab (T, n=1,089) or Trastuzumab-Pertuzumab (TP, n=701) during L1. Among them, 1,584 pts received antiHER2 therapy+CT+/-ET and 206 pts, antiHER2+ET only. Pts with antiHER2+CT+/-ET had more often ECOG performance status 0 (29.5% vs 15.8%, p&lt;0.001), grade III tumors (36.6% vs 25.6%, p=0.007), time to MBC &lt; 6 mo (51.6% vs 29.1%, p&lt;0.001), TP as antiHER2 therapy (43.2% vs 9.4%, p&lt;0.001), ≥3 metastatic sites (23.2% vs 14.8%, p=0.007), visceral metastasis (56.5% vs 42.4%, p&lt;0.001), and less often bone-only disease (18.4% vs 35%, p&lt;0.001) than pts with antiHER2+ET. In multivariable analysis, antiHER2+CT+/-ET was not superior to antiHER2+ET (Table), while TP was superior to Trastuzumab, and this result was confirmed by matching pts using a propensity score ( p=0.76 for mOS and p=0.85 for mPFS1). Using the time-dependent ET variable among pts with antiHER2+CT, pts with maintenance ET had significantly better PFS1 and OS than those without (adjusted HR for PFS1=0.70 [95%CI 0.60-0.82], adjusted HR for OS=0.47 [95%CI 0.39-0.57], p&lt;0.001). Conclusions: These data suggest that endocrine therapy could be an interesting less toxic alternative to chemotherapy in combination with antiHER2 therapy as first line treatment for HR+/HER2+ MBC pts.[Table: see text]

Abstract P2-01-01: Genetic alterations detected by circulating tumor DNA (ctDNA) in HER2-low metastatic breast cancer (MBC)

Abstract Introduction: Approximately 40-50% of breast cancers are characterized by low HER2 expression (HER2-low), defined as immunohistochemistry (IHC) 1+ or 2+ and HER2 fluorescence in situ hybridization (FISH) unamplified, encompassing a large and heterogeneous subgroup that may confer benefit from novel HER2 directed therapies. Circulating tumor DNA (ctDNA) has emerged as a minimally invasive technique to detect cancer-specific gene aberrations. Genetic alterations in ctDNA of HER2-low MBC have not been well described, and we hypothesized that HER2-low MBC may have a distinct genomic profile, beyond standard histopathologic features. Methods: This retrospective cohort study included patients with MBC treated at Washington University in St. Louis, Northwestern University (Chicago, IL) and Massachusetts General Hospital (Boston, MA) who had undergone ctDNA analysis during the course of treatment using the commercially available Guardant360® assay. HER2 expression was evaluated by IHC/FISH according to ASCO/CAP guidelines on metastatic tissue biopsies (or primary breast tumor tissue if a metastatic site biopsy was not available). Tumors were classified as HER2-low (IHC 1+ or 2+/FISH negative), HER2-0 (IHC 0) or HER2-positive (IHC 3+ or IHC 2+/FISH amplified). Clinicopathologic characteristics and ctDNA genetic alterations for HER2-low MBC were described and compared with the HER2-0 and HER2-positive subgroups. Chi-square and Fisher’s exact tests were used for categorical variables. Logistical regression was performed for multivariable analyses. Results: A total of 991 patients with MBC were analyzed, including 160 (16.1%) HER2-positive, 351 (35.4%) HER2-0, and 480 (48.4%) HER2-low MBC. The majority (89.2%) of HER2-low MBC were estrogen-receptor positive (ER+). Compared with HER2-0 MBC, HER2-low MBC had a significantly higher incidence of PIK3CA mutations (OR 1.54, p=0.027). PDGFRA and MYC amplifications were also more common among HER2-low MBC (2.3% vs 0.28% and 8.1% vs 4.6%, respectively), although not significantly associated with this subtype in multivariable analysis. Within the ER+ MBC cohort, those with HER2-low also had higher rates of PIK3CA mutations (OR 1.66, p=0.012) and MYC amplification (OR 2.29, p=0.034), as compared to HER2-0. Compared with HER2-positive, HER2-low MBC had significantly lower rates of ERBB2 alterations (OR 0.26, p=0.0076 for ERBB2 mutations and OR 0.022, p&amp;lt;0.001 for ERBB2 amplification). ESR1, AKT1, and RB1 mutations were more common in HER2-low compared with HER2-positive MBC (14.0% vs 6.9%; 3.1% vs none; 3.1% vs none, respectively), but were not significant in multivariable analysis. Conclusions: Among patients with ER+ MBC, HER-low had a higher incidence of PIK3CA mutations and MYC amplification compared to HER2-0 MBC, and both of these alterations have been implicated as mechanisms of endocrine resistance. We did not demonstrate a high incidence of ERBB2 alterations in patients with HER2-low MBC. To our knowledge, this is the first study to describe genetic alterations detected by ctDNA in patients with HER2-low MBC. Given the emergence of novel HER2-targeted antibody drug conjugates with clinical activity in HER2-low MBC, these findings may guide combination treatment strategies and patient selection for future studies. Further studies are needed to confirm whether HER2-low MBC represents a truly unique biologic subtype. Citation Format: Whitney L Hensing, Lorenzo Gerratana, Katherine Clifton, Marko Velimirovic, Ami Shah, Paolo D'Amico, Carolina Reduzzi, Qiang Zhang, Charles S Dai, Nusayba A Bagegni, Mateusz Opyrchal, Foluso O Ademuyiwa, Bose Ron, Amir Behdad, Cynthia X Ma, Aditya Bardia, Massimo Cristofanilli, Andrew A Davis. Genetic alterations detected by circulating tumor DNA (ctDNA) in HER2-low metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-01.

Abstract P3-09-12: Breast cancer derived GATA3 mutations disrupt luminal transcriptional network

Abstract GATA3 has been identified as one of the most frequently mutated genes in breast cancer. In the METABRIC cohort, among 1,980 patient cases, 230 breast cancers harbored GATA3 mutations (~12%). 75% of the GATA mutations were observed in luminal breast tumors (47% in luminal A, 28% in luminal B). The recent genomic data in metastatic breast cancer also showed that the frequency of GATA3 somatic mutations was even higher in the metastatic breast cancer cohort. Lung, lymph nodes, and brain metastases were observed in the GATA3 mutant breast cancer patients. Based on these patient genomic data, GATA3 mutations have been considered as cancer drivers, yet the functional consequences of GATA3 mutations are underexplored. We and other groups previously identified that patients carrying GATA3 mutations have diverse clinical features. More than 70% of cases are small nucleotide deletions or insertions (indel), while less than 30% are missense mutations. By classifying the GATA3 indel mutations into 4 groups, we observed distinct clinical features. Somatic mutations found in the GATA3 second zinc-finger domain (ZnFn2) are significantly associated with poorer patient outcomes including worse patient survival. ZnFn2 mutations are predominantly found in luminal B breast tumors, while splice site mutations are frequently found in luminal A breast tumors and associated with better patient survival. These distinct clinical features clearly suggest the differential impacts of GATA3 mutations on breast cancer cells. To dissect the function of GATA3 mutants, we developed a GATA3 mutant cell line (R330fs/WT T47D cells) by CRISPR, which endogenously expresses a heterozygous R330 frame-shift (R330fs) mutant. R330fs mutations are found in multiple data cohorts. The majority (&amp;gt;90%) of GATA3 mutations are heterozygous. Therefore, this R330fs mutant T47D cell line mimics the type of alteration found in patients. Importantly, we found that the R330fs mutation induces transcriptional reprogramming in T47D luminal breast cancer cells leading to more aggressive phenotypes such as faster tumor growth. In the mutant cells, many epithelial marker genes (such as progesterone receptor and TFF1) were down-regulated while mesenchymal marker genes (such as TWIST1 and SNAI2) are up-regulated suggesting that R330fs induces a partial EMT in T47D cells. We also found that transcriptional changes in the R330fs mutant cells were strongly associated with redistribution of GATA3, Estrogen Receptor alpha, and FOXA1. Gene expression profiles in the other GATA3 mutant breast cancer cell lines also suggest similar genomic alterations. These results suggest the active roles of GATA3 mutations during breast cancer development. Citation Format: Motoki Takaku, Mika Saotome, Renju Nair. Breast cancer derived GATA3 mutations disrupt luminal transcriptional network [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-12.

Clinical outcomes and metastatic behavior between de novo versus recurrent HER2-positive metastatic breast cancer: A 17-year single-institution cohort study at Taipei Veterans General Hospital.

To assess the clinical outcomes and metastatic behavior between de novo versus recurrent human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer (MBC) based on a single-institution database in Taiwan. We retrospectively identified patients diagnosed between January 2000 and December 2017 with de novo stage IV or recurrent HER2-positive MBC. Several variables were recorded in patients with recurrent disease: age at diagnosis, metastatic site, hormone receptor (HR) status, HER2 status, and disease-free interval (DFI). Treatments and metastatic patterns were compared between de novo stage IV and recurrent MBC cohorts. Post-metastasis survival (PMS) was estimated using the Kaplan-Meier method with log-rank tests. Hazard ratios and 95% CIs were estimated using Cox regression analysis. In total, 1360 patients were diagnosed with breast cancer with HER2 overexpression. At baseline, de novo stage IV patients were older than recurrent MBC patients (median age 58 vs 53). The majority of the de novo stage IV patients were diagnosed after 2010, while most of the recurrent MBC patients were diagnosed during 2000-2009. An increased number of de novo stage IV patients underwent targeted therapy than recurrent MBC patients was also noted. PMS in patients with de novo stage IV and recurrent MBC was 79.2 months and 61.8 months, respectively, which indicated significant better survival in de novo stage IV than those with recurrent MBC disease. Longer survival was also noted in de novo stage IV and recurrent MBC with DFI >24 months than in those with recurrent MBC with DFI <24 months and in patients receiving HER2-targeted therapy after MBC diagnosis than in those not receiving the therapy. However, median PMS showed no significant difference between patients with the luminal B2 (HR-positive, HER2-negative) and HER2-enriched (HR-negative, HER2-positive) subtypes. After adjustment in multivariate analysis, a low risk of BC-specific death was observed in patients aged >50 years, those receiving HER2-targeted therapy for MBC, and those with oligometastasis, while patients with first metastases to the liver or brain showed a higher risk of BC-specific death than those without metastases. De novo and recurrent MBC have distinct characteristic, metastatic patterns and outcomes in Asian HER2-positive breast cancer patients. The age distribution and survivals between HR+/- status were different to non-Asian group. These differences should be further investigated in the future considering ethnic factor.