Cristofanilli, Massimo

e15060 Background: In metastatic Breast Cancer (mBC) Circulating Tumor Cells (CTCs) are an established prognostic indicator of patients (pts) with highly aggressive disease, poor clinical outcomes, and poor response rates to therapy. However, CTCs are typically found in < 20% of mBC pts and many pts without CTCs also progress. Cancer associated macrophage-like cells (CAMLs) are inflammatory tumor macrophages also found in blood, in > 90% of mBC, and are indicators of poor clinical outcomes independent of CTCs. We conducted a prospective study to model CTC & CAML subtypes from 212 mBC pt blood samples prior to induction of new systemic therapies to optimize and validate risk models based on 2-year outcomes of Progression-free survival (PFS) & Overall survival (OS). Methods: An observational multi-institutional prospective study was conducted on 212 mBC pts prior to induction of a new line of therapy who were progressing on their current therapy. Anonymized (7.5ml) whole blood samples were taken prior to therapy induction and filtered by CellSieve filtration. Quantities and subtypes of CTCs & CAMLs were categorized by their expression of CK+/CD45+/CD14+ for CAMLs and CK+/CD45-/CD14- for CTCs. PFS (RECIST v1.1) and OS hazard ratios (HRs) were calculated by censored univariate & multivariate analysis at 2 years. Results: Pt median age was 51 years (range 25-92); TNBC (44%), ER/PR+ (30%), HER2+ (15%). Pts were treated with chemotherapy alone (18%), immune checkpoint inhibitor (46%), targeted (40%), hormone (8%) and unknown (5%). 38% (81/212) pts had ≥1 CTCs which correlated with significantly poorer PFS (HR = 1.9; 95%CI 1.3-2.7; p = 0.0016) and OS (HR = 2.0; 95%CI, 1.3-3.0; p = 00025), with the most significant risk at ≥8 CTCs (14% pts), PFS (HR = 3.8; 95%CI, 2.1-6.9; p < 0.0001) and OS (HR = 7.2; 95% CI, 3.4-14.9; p < 0.0001). Independently, 93% (n = 196/212) of pts had ≥1 CAML, with a threshold of >40um size (84% pts) had significantly poorer PFS (HR = 2.0; 95% CI, 1.2-3.1; p = 0.0032), but not OS (HR = 1.5; 95%CI, 0.9-2.7; p = 0.1169). However, a threshold of >125um size CAML (43% pts) also had significantly poorer PFS (HR = 1.8; 95% CI, 1.2-2.7; p = 0.0013) and OS (HR = 1.6; 95% CI, 1.0-2.5; p = 0.0233). By combining models, the best outcomes were seen in pts with 0 CTCs & 0 CAMLs (mPFS = 12.6 & mOS = 21.3), followed by 0 CTCs & ≥40um CAMLs (mPFS = 5.2 & mOS = 17.5) and 0 CTCs & ≥125um CAMLs (mPFS = 4.9 & mOS = 15.2). Poorer outcomes were seen with ≥1 CTCs (mPFS = 3.3 & mOS = 13.5) and the worst outcomes with ≥8 CTCs (mPFS = 2.5 & mOS = 4.8). Conclusions: CTCs were uncommon in mBC pts but correlated with very poor clinical outcomes. In parallel analysis, CAMLs were common with CAML size correlating with increasingly poorer outcomes. By combining CTC & CAML subtypes, mBC pts were more accurately stratified by risk of progression and death. Additional multivariate studies correlating treatment class and tumor response rates are ongoing.

e14535 Background: Anti-programmed cell death ligand PD-L1/PD-1 immune checkpoint inhibitors (ICIs) are approved for metastatic breast cancer (mBC) patients (pts) with a tumor combined positive score (CPS) ≥10% in their tissue. However, many pts with < 10% CPS may also respond to ICIs. Recent studies in lung cancer have shown that circulating tumor cells (CTCs) and tumor-macrophage fusion cells (TMFCs) both express, and may upregulate, PD-L1 which is predictive for ICI responses. We initiated a prospective study to monitor PD-L1 in CTCs & TMFCs in mBC pts prior to receiving systemic ICIs to correlate their PD-L1 expression to progression free survival (PFS) and overall survival (OS) at 24 months. Methods: We conducted a multi-institutional prospective pilot study of n = 102 pts with pathologically confirmed mBC, having failed ≥1 prior lines of therapy, prior to receiving ICIs (n = 52 Pembrolizumab, n = 39 Retifanlimab, n = 5 Atezolizumab, and n = 1 Nivolumab). Anonymized blinded peripheral blood samples (7.5ml) were procured prior to ICI induction (T0) and (~40 days after ICI induction (T1). TMFCs & CTCs were isolated using Cellsieve microfilters with average PD-L1 expressions measured then categorized as negative/low or high. Tumor CPS was categorized as negative/low (< 10%) or high (> 10%). We compared TMFC PD-L1 to tissue CPS by linear regression. TMFC/CTC PD-L1 expression and tissue PD-L1 CPS were compared to pts’ PFS and OS by Cox proportional univariate and multivariate analysis over 24 months. Results: Of n = 102 pts, median age = 59 years (range 32-77); 42% (43/102) were ER+, 33% (33/102) were PR+, 5% (5/102) were HER2+, and 56% (57/102) were triple-negative. 32% (32/102) had an ECOG score of 0, 25% (26/102) had ECOG 1, and 43% of scores were unavailable. N = 42 pts had available tumor CPS data, which did not correlate to TMFC/CTC PD-L1 at T0 or T1. Pts with high CPS did not significantly predict for PFS (HR = 0.78; 95% CI, 0.3-1.9; P = 0.7627) nor OS (HR = 0.54; 95% CI, 0.2-1.7; P = 0.4471). Pts with high TMFC/CTC PD-L1 at T0 also did not significantly predict for PFS (HR = 0.74; 95% CI, 0.5-1.2; P = 0.2799) nor OS (HR = 0.84; 95% CI, 0.4-1.6; P = 0.7220). However, of the n = 82 pts who provided T1 samples, high CTC/TMFC PD-L1 at T1 was significant for better PFS (HR = 2.55; 95% CI, 1.6-4.2; P = 0.0004) and better OS (HR = 2.06; 95%CI, 1.1-3.8; P = 0.0309). Conclusions: In this study of mBC pts treated with PD-L1 ICIs, tissue CPS did not correlate with PD-L1 expression in CTCs or TMFCs. Further, neither CPS nor baseline PD-L1 in CTC/TMFCs predicted for clinical outcomes in ICI therapies. However, PD-L1 in CTC/TMFCs ~40 days post-induction of ICI did predict for better response rates. While this study suggests predictive value of monitoring PD-L1 in blood during ICI therapies, further studies are required to refine and validate these findings.

Abstract Background: In metastatic cancer, Circulating Tumor cells (CTCs) are established prognostic indictors of patients (pts) less likely to respond to new lines of systemic therapy, with poor clinical outcomes, such as shorter progression free survival (PFS) and overall survival (OS). However, CTCs are typically found in specific malignancies (breast, prostate & colon), often in &lt;20% of pts with metastatic disease, and pts without CTCs may also rapidly progress. Recently, an inflammatory pro-tumorigenic macrophage emanating from tumor stroma (i.e. Cancer associated macrophage-like cell [CAML]) was found in &gt;90% of metastatic cancer pts, and whose phagocytic engorgement appears to correlate with poor outcomes, independent of CTCs. As CTCs and CAMLs are isolated in conjunction from a single blood sample, and both are prognostic for outcomes, we evaluated their utilization prior to induction of new systemic therapy in 6 types of metastatic cancer to model pt risk stratification based on 2 year outcomes. Methods: A prospective 2 year blind multi-institutional study was undertaken to model CTCs and CAMLs in prognosticating outcomes prior to induction of a new line of systemic therapy (n=233) in metastatic: Breast (n=60), Prostate (n=40), Pancreas (n=25), Colon (n=28), Renal Cell Carcinoma (RCC) (n=39), and Lung (n=40). Blood was filtered by CellSieveTM filters with subtypes of CTCs & hyper-enlarged CAMLs (≥100µm) enumerated. A machine learning algorithm was trained on this initial data set to develop predictive models which could stratify pt populations by risk for likelihood of PFS & OS over 2 years, including known clinical variables. Results: CTCs were absent in 80% (n=185/233) of pts, and their absence was prognostic for better PFS (HR=1.6, p=0.046), but not OS (HR=1.3, p=0.2045). In parallel, enlarged CAMLs (≥100µm) were found in 28% (n=51/185) of pts without CTCs, and were also prognostic for worse PFS (HR=2.0, p=0.0082) and OS (HR=1.9, p=0.0412). Specifically, CTCs were found in 55% (n=33) breast, 20% (n=8) prostate, 12% (n=3) pancreas, 11% (n=3) colon, 0% (n=0) RCC, and 0% (n=0) lung pts. Enlarged CAMLs were found in 45% (n=27) breast, 20% (n=8) prostate, 44% (n=11) pancreas, 44% (n=17) colon, 18% (n=7) RCC and 20% (n=8) lung pts. Overall, models indicated that ≥1 CTC (n=47) had mPFS=3.9 & mOS=14.9, while pts with 0 CTCs and ≥100µm CAMLs (n=51) had a mPFS=6.9 & mOS=13.8, and pts with 0 CTCs and &lt;100µm CAMLs (n=134) had a mPFS=10.7 & mOS&gt;24 months. Conclusions: These initial models confirm that both CTCs and enlarged CAMLs are prognostic indicators of worse PFS & OS. The simultaneous quantification both CTCs and CAMLs allows for more accurate pan-cancer risk stratification in an array of cancer pt populations. Additional clinical variables incorporated into the models may allow better risk subtyping and possibly forecast optimal treatment regimens. Citation Format: Daniel L. Adams, Steven H. Lin, Massimo Cristofanilli, Carolina Reduzzi, Saranya Chumsri, Martin J. Edelman, Susan Tsai, Raymond C. Bergan, Mohammed Aldakkak, Thai H. Ho, Cha-Mei Tang. Combining circulating tumor cells and cancer associated macrophage-like cells enhances risk stratification models in pan-cancer metastatic disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1068.

e15045 Background: Increased cancer cell mitosis in tumor tissue is widely regarded as an indicator of aggressive disease, a predictor of shorter survival, and an indicator of response to specific anthracycline based chemotherapies. Circulating tumor cells (CTCs) are cancer cells that detach from primary tumors, enter the bloodstream, and seed metastatic sites, which in patients (pts) with metastatic breast cancer (mBC) are also predictors of poor prognosis and reduced therapy response. Furthermore, small preliminary studies have identified a specific subtype of mitotic CTCs that appears to correlate with poor survival rates compared to CTC detection alone. However, larger studies have yet to be conducted to validate the clinical utility of mitotic CTCs. In this large multi-institutional prospective study, we isolated CTCs from the blood of n = 155 mBC pts to subtype mitotic CTCs to assess progression-free survival (PFS) and overall survival (OS) over 2 years. Methods: Peripheral blood samples (7.5ml) were prospectively collected from n = 155 mBC pts progressing on systemic therapies and prior to new systemic therapy induction (i.e., chemotherapy n = 90, hormone therapy n = 13, immunotherapy n = 51, targeted therapy n = 62). Samples were processed using CellSieve microfilters to isolate CTCs via size exclusion and fluorescently stained for CD45, Cytokeratin, and DAPI. CTCs were imaged, enumerated, and subtyped based on the presence of ≥1 mitotic event, using established visual indicators. PFS and OS were assessed over 2 years by censored univariate and multivariate analyses. Results: CTCs were detected in ~45% (n = 70/155) of pts, while mitotic CTCs were identified in a subset of this group, ~23% (n = 35/155). The presence of any CTC was associated with poor outcomes (PFS HR = 2.16, 95%CI = 1.4-3.2, p = 0.0002 and OS: HR = 2.51, 95%CI = 1.5-4.0, p = 0.0002). The presence of mitotic CTCs correlated with significantly poorer outcomes (PFS: HR = 5.6, 95%CI = 3.1-10.2, p < 0.0001 and OS: HR = 7.3, 95%CI = 3.7-14.5, p < 0.0001). Overall, pts with no CTCs had a median PFS (mPFS) of 7 months and median OS (mOS) of 24 months, pts with non-mitotic CTCs had a mPFS of 6 months and mOS of 15 months; pts with both CTCs and mitotic CTCs had a mPFS of 3 months and mOS of 5 months. Notably, 97% of pts with a mitotic CTC progressed within 6 months, regardless of systemic therapy type. Interestingly at 2 years, a sole non-progressive pt with a mitotic CTC was receiving alpelisib (a specialized PI3KCA inhibitor). Conclusions: Overall, the detection of CTCs indicated poor outcomes in pts, with mitotic CTCs correlating with significantly more aggressive disease and further reduced survival rates regardless of systemic therapy type. This study highlights the need for larger, more comprehensive studies to expand upon and validate these findings.

Abstract Circulating tumor cells (CTCs) are cancer cells that detach from tumors, enter the blood and seed distant sites. CTCs spread as single cells, or cluster aggregates, and can be heterogenous with differing biological phenotypes, i.e. epithelial, mesenchymal, progenitor, & stem cell. Three clinically relevant CTC subtypes are often described in patients (pts) 1) CTCs with normal epithelial traits (strong filamentous cytokeratin (CK+ & CD45-/CD14-), 2) Epithelial-mesenchymal CTCs (EMTs), cancer cells with downregulated epithelial & upregulated mesenchymal traits (low diffused CK+, CD45-/CD14-) and 3) tumor-macrophage fusion cells (TMFCs), cancer cells that fuse with macrophages (polynucleated DAPI+, diffuse CK+ and CD14+/CD45+). To better elucidate the biological and clinical importance of the 3 CTC subtypes, we isolated CTCs by a marker agnostic method from the blood of n=49 metastatic cancer pts. We then enumerated and phenotypically subtyped CTCs comparing clinical utility of clustering and CTC heterogeneity over 2 years. Peripheral blood samples were collected from 49 metastatic cancer pts, breast (n=38), pancreatic (n=9), colon (n=3), lung (n=1), and prostate (n=1). Whole blood (7.5mL) was filtered by size exclusion using CellSieve microfilters and fluorescently stained for CD45, CD14, CK, and DAPI to identify CTCs, EMTs and TMFCs. Cells were imaged, enumerated and fluors quenched by the QUAS-R technique; followed by re-stained for CD44, CD24, CD133, CD144, N-Cadherin, EpCAM, and Vimentin. Markers were categorized as negative, low, medium, or high. A heterogeneity biomarker score was developed as high, 2-3 log deviations between marker expression, or low 0-1. Pt progressive free survival (PFS) and overall survival (OS) were analyzed over 2 years. CTCs were found in 40% of pts, EMTs in 51%, & TMFCs in ∼93%. For subtyping, CD44 was in CTCs 88%, EMTs 59%, & TMFCs 91%; CD24 in CTCs 40%, EMTs 41%, & TMFCs 6%; CD133 in CTCs 100%, EMTs 8%, & TMFCs 100%; CD144 in CTCs 100%, EMTs 77%, & TMFCs 82%; N-cadherin in CTCs 100%, EMTs 0%, & TMFCs 45%; Vimentin in CTCs 51%, EMTs 100%, & TMFCs 83%; EpCAM in CTCs 74%, EMTs 12%, & TMFCs 20%. CTCs and EMTs were found to have high heterogeneity in 30% of samples in both individual cells and clusters, with a high biomarker heterogeneity significantly correlated with poorer outcomes (PFS: HR=15.4, p=0.0014, CI=3.4-68.9; OS: HR=12.4, p=0.0056, CI=2.6-59.1). TMFCs were found to have high heterogeneity, 40% of samples, but this was not correlated with outcomes. In this pilot study, it appeared that isolated CTC, EMTs and TMFCs subtypes have differing heterogenous expressions of epithelial, mesenchymal, progenitor, and stem cell markers. Further, high rates biomarker heterogeneity in CTCs and EMTs, but not TMFCs, correlated with faster rates of progression & death. Larger validation experiments with biomarker stratification is ongoing. Citation Format: Alexis B. Duffy, Massimo Cristofanilli, Carolina Reduzzi, Cha-Mei Tang, Saranya Chumsri, Susan Tsai, Mohammed Aldakkak, Daniel L. Adams. Heterogeneity of epithelial, mesenchymal, progenitor, and stem cell phenotypes on CTCs correlates to clinical outcomes in metastatic cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1989.

Abstract Tumor associated macrophages are known to fuse with cancer cells resulting in the formation of a cellular phenomenon called tumor-macrophage fusion cells (TMFCs). TMFCs are a highly aggressive cell subtype that may move into circulation, evade immune surveillance, and possibly aid in seeding of metastatic sites. The formation of TMFCs involves CD47, a ubiquitous surface receptor that acts as a “don’t eat me” signal, interacting with the macrophage protein SIRPa, a down regulator of phagocytosis, and syncytin-1, a mediator of hyperploidy cancer fusion. In this analysis, we isolated circulating TMFCs from the blood of n=39 metastatic cancer patients (pts) to evaluate the expressions of CD47, SIRPa and syncytin-1 as it relates to stable or progressive disease via 30 day imaging by PET scan. We conducted a prospective pilot study of n=39 pts with pathologically confirmed metastatic breast (n=17), prostate (n=7), pancreas (n=7), or colon (n=8) cancer receiving standard of care systemic therapy. We isolated TMFCs from 7.5ml peripheral blood using a CellSieve microfilter, and stained with CD47, SIRPa, & syncytin-1. TMFCs were identified based on large cell size (&gt;30 µm) & multinucleated structure, and were categorized into 4 distinct subtypes: A) &gt;2 distinct non-contacting nuclei within a TMFC, B) 2 distinct non-contacting nuclei, C) 2 contacting nuclei, D) a single multinucleated nuclei. CD47, SIRPa, and syncytin-1 expressions were categorized as negative, low, or high. TMFCs subtypes and marker expressions were compared to pts’ clinical status (i.e. progressive, stable or responding) at first available PET/CT scans after blood draw (∼45 days). TMFCs were identified in 72% of pts (n=28/39), average 8 cells/sample, and ranged 1-77 cells. Of the TMFCs, 12% were subtype A, 11% as B, 11% as C, and 66% as D. CD47 expression was seen in 100% of TMFs, with low expression in 39% of subtype A cells, 58% subtype B, 39% subtype C, & 62% subtype D. Notably, CD47 was significantly lower (p=0.010) in subtype B than other groups. Further, the expression of syncytin-1 in subtype B cells was significantly lower (p=0.02) than in other subtypes, and subtype D cells had 75% higher syncytin-1 localized to the nuclear regions. No correlations were found in the SIRPa expression and subtypes. Interestingly, progressing pts had 200% more TMFCs, more subtype B (83%), and significantly lower (p&lt;0.001) CD47 expressing TMFCs than non-progressing pts. In this pilot study on the expression profiles of circulating TMFCs, it appears that specific subtypes of TMFC with low CD47 expression may be associated with progressive disease. Interestingly, this contrasts existing studies on primary tumors which often correlate higher CD47 with more aggressive cancers. This finding suggests that low CD47 cancer cells circulate blood in some pts with progressing disease and may be pertinent to efforts targeting CD47 in cancer. Citation Format: Sonia Muthuraj, Massimo Cristofanilli, Saranya Chumsri, Susan Tsai, Raymond C. Bergan, Cha-Mei Tang, Daniel L. Adams. Low CD47 expression in specific populations of tumor macrophage fusion cells in blood correlates with progressive disease in metastatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1979.

Abstract Purpose: The aim of PREDICT was to confirm clinical validity and the potential for clinical utility of serial circulating tumor cell (CTC) enumeration in patients with metastatic breast cancer, focusing on its prognostic value in different breast cancer subtypes and clinical settings. Experimental Design: In total, 4,436 individual patient-level data with CTC results from both baseline and one follow-up (CellSearch; Menarini Silicon Biosystems) were analyzed to evaluate the association between CTC detection and overall survival (OS) in the full patient cohort and separately for tumor and treatment types. Results: Using the cutoff ≥1 CTC for CTC positivity, 913 (20.6%) patients had 0 CTCs at both time points (neg/neg) and 325 (7.3%) and 1,189 (26.8%) patients converted from CTC negative to CTC positive (neg/pos) or vice versa (pos/neg), whereas 2,009 (45.3%) patients had at least one CTC at both time points (pos/pos). The median OS for the neg/neg, neg/pos, pos/neg, and pos/pos group was 45.6, 26.1, 32.3, and 17.3 months, respectively (P &lt; 0.0001, global log-rank test). CTC responders (pos/neg) showed a lower risk of death compared with CTC nonresponders (pos/pos; HR, 0.48; 95% confidence interval, 0.44–0.53). Similar results were obtained in subgroup analyses according to hormone receptor and HER2 subtype, treatment type, and with a ≥5 CTC cutoff for CTC positivity. Conclusions: Follow-up CTC assessments strongly predict OS independently from tumor subtype and treatment. New randomized trials to define the clinical utility of CTC monitoring for risk stratification and as an early response marker in metastatic breast cancer are urgently needed.

TPS1137 Background: BriaCell (BC) human immortalized cell lines have been engineered to trigger robust immune responses and deliver efficacy through diverse mechanisms, including adaptive (involving T-cells) and innate responses (dendritic and NK cells). BC can match patients’ HLA antigens for optimal effectiveness. BC works by several mechanisms, including direct antigen presentation and activation of CD4+ T-cells. When combined with an immune checkpoint inhibitor, the BC regimen has demonstrated clinical benefit in 54 heavily treated MBCs. Most pts whose disease has progressed after checkpoint inhibitor (CPI) therapy showed similar or improved PFS compared to the patient's last treatment regimen. Disease control after prior antibody-drug conjugates (ADCs) has been observed in 40% of patients, and clinical benefit has been seen in 5/7 patients with untreated intracranial metastases. CD8+ Immuno PET shows an increase in CD8+ tumor infiltrating lymphocytes suggesting systemic activation and infiltration into lymphoid organs and metastatic sites. Sequencing of CPI with BC and the latest phase 3 formulation are associated with improved clinical outcomes, including OS (median 13.4 months), PFS, and CBR, which are reflected in the design of this ongoing pivotal registration enabling Phase 3 study [doi:10.1158/1538-7445.sabcs22-p3-07-12]. Methods: Multicenter randomized, open label Bria-IMT regimen (with SV-BR-1-GM cells) plus CPI vs Treatment of Physicians' Choice (TPC) in MBCs with no approved curative therapies available. ECOG <2 allowed with no limit on prior therapies. Eligibility encompasses all MBC subtypes including pts with CNS metastases. Prior immune therapy is allowed if administered more than 21 days before the first cycle of Bria-IMT; treatments within 21 days preclude participation. Initial randomization will be 1:1:1 to the Bria-IMT regimen + CPI (combination), TPC, and the Bria-IMT regimen alone (monotherapy). There will be 100 sites across the US, Canada, and ex-NA for 404 total evaluable pts. The Bria-IMT regimen includes: Day -2 Cyclophosphamide 300mg/m2, Day 0 20 million irradiated SV-BR-1-GM cells intradermally, and 0.1mcg peg α interferon intra-dermally (Day 2 or 3) into each Bria-IMT inoculation site. After the first 150 pts have enrolled, the monotherapy arm will be discontinued. Bria regimen + CPI arms treatment cycles occur q3w. CPI infusion is given every cycle between Day –3 to Day 3 TPC cycle details will be according to the site's SOC. Imaging assessment will be q6w x2 then q8w. The primary endpoint is overall survival with an interim analysis at 144 events targeting a hazard ratio of 0.6. Key secondary endpoints will include PFS, ORR, CBR, CNS event free survival, and TWiST (time without symptoms or toxicities). Safety analysis will be ongoing. Patient-reported outcomes will be incorporated to assess subjective treatment effects. Clinical trial information: NCT06072612 .

TPS1127 Background: Patients with estrogen receptor-positive (ER+) metastatic breast cancer (mBC) may develop resistance to endocrine therapy (ET), particularly following treatment with a cyclin-dependent kinase 4/6 inhibitor (CDK4/6i), potentially driven by a mutation in the ERa-coding gene, ESR1. Lasofoxifene (LAS), an oral, next-generation ET and ER breast antagonist, was evaluated in two phase 2 studies of women with ER+/HER2- mBC and an ESR1 mutation who had disease progression on previous ET and CDK4/6i. The ELAINE 1 trial showed numerically greater progression-free survival (PFS, median ~6 vs 4 mos; P=0.138), objective response rate (ORR, 13% vs 3%; P=0.124), and clinical benefit rate (CBR, 37% vs 22%; P=0.117) with LAS monotherapy versus the ER degrader fulvestrant (fulv), with a favorable safety profile (1). The single-arm, ELAINE 2 trial demonstrated that LAS plus abemaciclib (Abema) was well tolerated with a median PFS of ~13 mos, ORR of 56%, and CBR of 66% (2). Based on these data, the phase 3, registrational, ELAINE 3 trial was initiated. Methods: ELAINE 3 (NCT05696626) is an open-label, phase 3, multicenter study evaluating the efficacy, safety, and tolerability of LAS plus Abema versus fulv plus Abema. Key inclusion criteria are pre- and postmenopausal women and men aged ≥18 yrs; ER+/HER2-, locally advanced and/or mBC (measurable and/or non-measurable disease); ≥1 acquired ESR1 mutation; progression on an aromatase inhibitor plus palbociclib or ribociclib as their first hormonal treatment for advanced/mBC; and ≤1 line of chemotherapy in the advanced/metastatic setting. Patients will be randomized 1:1 to receive LAS 5 mg/day plus Abema 150 mg BID, or fulv 500 mg IM on days 1, 15, and 29, then once monthly plus Abema 150 mg BID. Treatment will continue until progression, death, unacceptable toxicity, or withdrawal from the study. The primary endpoint is PFS; key secondary endpoints are ORR, overall survival, CBR, duration of response, and time to response. Time to cytotoxic chemotherapy, quality of life, and safety will also be evaluated. Blood samples for circulating tumor DNA (ctDNA) will be collected for genomic analyses at screening, at weeks 4 and 8 and every 8 weeks thereafter, and at the final visit. Outcomes with LAS/Abema and fulv/Abema will be compared using a stratified Cox proportional hazards model and stratified logrank test. Expected PFS is ≥10.3 mos for LAS/Abema and 7 mos for fulv/Abema (PFS hazard ratio of 0.68 at final analysis). The target sample size is 400, to achieve 90% power with a one-sided type I error rate of 0.025 after 285 PFS events. Full recruitment is expected to occur over 18 mos at approximately 125 global sites. 1. Goetz MP, et al. Ann Oncol. 2023;34:1141-1151. 2. Damodaran S, et al. Ann Oncol. 2023;34:1131-1140. Clinical trial information: NCT05696626 .

Abstract Introduction: Estrogen receptor-positive (ER+)/HER2-negative (HER2-) breast cancers commonly acquire constitutively active mutations in the ERα-encoding gene (ESR1), resulting in endocrine therapy (ET) resistance. In the phase 2, ELAINE 2 study (NCT04432454), LAS (a breast ER antagonist) combined with Abema provided a median progression-free survival (PFS) of ~13 months (mos) and clinical benefit rate (CBR) of 66% in patients with endocrine-resistant, ER+/HER2- mBC and ESR1 mutations (mESR1; Damodaran. J Clin Oncol 2023;41:suppl16;1057). Our objective was to describe baseline genomic alterations co-occuring with mESR1 and treatment responses to LAS plus Abema in patients with these concurrent genomic alterations. Methods: Women (age ≥18 yrs) with ER+/HER2- mBC that progressed after prior ET and CDK4/6 inhibitor (CDK4/6i) with an mESR1 detected in blood using the Sysmex-Inostics SafeSeq circulating tumor DNA (ctDNA) test were enrolled in ELAINE 2. Oral LAS 5 mg/day and Abema (provided by Eli Lilly and Co) 150 mg BID were taken until disease progression, death, unacceptable toxicity, or withdrawal. The Guardant360 CDx test was used to identify and describe baseline genomic alterations in ctDNA (including mESR1). Median PFS and CBR were assessed in patients with mESR1 and co-existing baseline genomic alterations. For patients who withdrew before disease progression, their last observation time was used as the progression time for PFS estimation. Data were summarized descriptively with no formal hypothesis testing. Results: Of the 29 patients (median age 60 yrs) enrolled, mutations in ESR1 were identified in 26 by Guardant360; all these 26 patients had received prior CDK4/6i, 21 (81%) prior fulvestrant, and 12 (46%) prior chemotherapy for mBC. Alterations in 39 genes were concurrently identified with mESR1, including TP53 mutations (n=11), PIK3CA mutations (n=8), CCND1 amplifications (n=6), and FGFR1 amplifications (n=5); co-amplification of CCND1 and FGFR1 was identified in 3 patients. LAS plus Abema treatment was associated with a CBR of 73% and a median PFS of 12.9 mos in the 26 patients with mESR1 (Table). CBRs were 63% in the co-occurring PIK3CA mutation subgroup and 64% in the TP53 mutation subgroup; median PFS times were 7.8 and 8.3 mos, respectively. All patients in the CCND1 and FGFR1 amplification subgroups achieved clinical benefit (CBR 100%) and the median PFS was 16.6 mos for both subgroups. In patients with mESR1 and co-alterations in ≥2 other genes of interest (PIK3CA, FGFR1, CCND1, TP53, ERBB2, CCNE1, or RB1), CBR was 83% and median PFS was 16.6 mos. Conclusion: Using Guardant360 ctDNA profiling from patients in ELAINE 2, we demonstrate that other baseline genomic alterations are frequently detected concurrently with mESR1 in the endocrine resistant setting, but without apparent compromise on the efficacy of LAS plus Abema. These results should be interpreted with caution considering the small numbers of patients and the exploratory nature of the analysis. The ELAINE 2 study suggests the potential of LAS plus Abema for treating ESR1-mutated, ER+/HER2- mBC in the post-CDK4/6i setting. This will be further evaluated in the larger, registrational, phase 3, ELAINE 3 study (NCT05696626) that compares LAS plus Abema with fulvestrant plus Abema. Table. Treatment response by genomic alterations concurrent with ESR1 mutations (n=26). aFor patients who withdrew before disease progression, the last observation time was used as the progression time for PFS estimation. bOther genes of interest include PIK3CA, FGFR1, CCND1, TP53, ERBB2, CCNE1, or RB1. CBR, clinical benefit rate; mPFS, median progression-free survival. Citation Format: Senthil Damodaran, Massimo Cristofanilli, Matthew Goetz, Ciara O'Sullivan, Gary Riordan, Hope Rugo, Daniel Stover, Seth Wander, Dominic Carroll, Paul V. Plourde, David J Portman, Einav Nili-Gal Yam. Baseline genomic alterations and the activity of lasofoxifene (LAS) plus abemaciclib (Abema) in patients with ER+/HER2- metastatic breast cancer (mBC): the ELAINE 2 study [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-14-09.