Metastasis is the leading cause of death in patients with breast cancer (BC), but the mechanisms underlying metastasis formation are still poorly understood. Circulating tumor cells (CTCs) are considered the main seed of metastasis with demonstrated prognostic impact in patients with BC. They are conventionally identified as cells positive for epithelial markers and lacking leukocyte markers. Nonetheless, circulating cells expressing both markers [dual-positive cells (DPcells)] have been reported but poorly investigated. Here, we evaluated, in a cohort of 340 patients with advanced BC, the prognostic impact of DPcells, showing their association with worse survival, particularly in patients with less than five CTCs. Their prognostic value varied among BC subtypes, with greater relevance observed in triple-negative and HER2-positive BC. Moreover, by performing single-cell genomic profiling of DPcells isolated from patients, we detected genomic aberrations in 28 and 93% of analyzed DPcells and CTCs, respectively. In vivo, DPcells were detected only in the blood of immunocompetent but not immunodeficient mice and no differences in the lung metastatic colonization ability of DPcells versus control cancer cells were observed. Our findings highlight the importance of studying this overlooked subpopulation of CTCs as a prognostic biomarker in BC, which might be particularly important in specific BC subtypes. Moreover, our results support the malignancy and metastasis-forming capability of DPcells and underline the need for future studies better defining the origin of these cells.

BACKGROUND:CDK4/6 inhibitors (CDK4/6i) in combination with endocrine therapy (ET) represent the standard of care for hormone receptor-positive, HER2-negative (HR+/HER2-) metastatic breast cancer (MBC) patients. However, no head-to-head randomized trials have directly compared palbociclib, ribociclib, and abemaciclib. Moreover, predictive biomarkers of resistance to CDK4/6i remain largely undefined. This study aimed to evaluate circulating tumor DNA (ctDNA)-based epigenetic and fragmentomic biomarkers as potential predictors of response and resistance in patients receiving CDK4/6i. METHODS:We conducted a biomarker-driven analysis within the prospective, multicenter MAGNETIC.1 study, enrolling 149 patients with HR+/HER2- MBC treated with first-line endocrine therapy and a CDK4/6 inhibitor. Plasma samples were collected at baseline and during treatment (3 and 6 months). Droplet digital PCR was used to assess ESR1 promoter methylation and ACTB fragmentomic profiles. Progression-free survival (PFS) and overall survival (OS) were evaluated, and molecular dynamics were compared between treatment groups. RESULTS:After a median follow-up of 34.8 months, no statistically significant differences in PFS or OS were observed between ribociclib and palbociclib treated patients, although ribociclib was associated with numerically longer PFS and higher survival rates. At the molecular level, palbociclib treatment was characterized by transient increases in ESR1 promoter methylation at the first evaluation and a rebound in ACTBshort fragment levels at six months relative to baseline. These dynamic patterns were not observed among patients receiving ribociclib. CONCLUSIONS:ctDNA-based methylation and fragmentomic profiling revealed exploratory, treatment specific molecular dynamics, highlighting biological differences between CDK4/6 inhibitors. These findings support the feasibility of liquid biopsy-based biomarker studies in this setting, although their potential clinical relevance remains preliminary and requires validation in larger cohorts with earlier and more granular on-treatment timepoints.

1036 Background: Activating PIK3CA mutations are established oncogenic drivers and predictive biomarkers in MBC. In contrast, PIK3CA amplifications are rare, poorly characterized, and of unclear clinical significance. Tissue-based studies report a prevalence of 1–7%, but their detection and relevance in ctDNA are largely unexplored. Moreover, whether amplifications interact with co-occurring mutations to define a distinct high-risk subset is unknown. We therefore aimed to characterize the impact of PIK3CA amplifications detected in ctDNA through a multi-institutional cohort of patients (pts) with MBC. Methods: This retrospective study analyzed a multi-institutional cohort of 1579 pts with MBC and baseline ctDNA testing with the Guardant360 NGS panel within a large academic consortium (PMAC). Hormone Receptor positivity (HR+) and HER2 status were defined based on the most recent biopsy. Associations between SNVs, CNVs, and clinical characteristics were assessed using multivariable logistic regression. The impact of prognosis, adjusted for the number of prior treatment lines, was evaluated through Cox regression for overall survival (OS), defined from time of baseline ctDNA collection. Results: Among 1,579 pts, 1,121 (71%) were HR+/HER2–, 214 (13.5%) HER2+, and 244 (15.5%) had triple-negative breast cancer (TNBC). PIK3CA CNVs were detected in 7.7% of pts, and 44.7% harbored a concomitant PIK3CA SNVs. PIK3CA CNVs were significantly enriched in TNBC compared with other subtypes (Odds Ratio [OR] 2.02, p=0.011). In the overall population, PIK3CA CNVs were associated with significantly worse OS (HR 2.46, p<0.001), an effect observed across all subtypes, including HR+/HER2– (HR 2.21, p<0.001), HER2+ (HR 5.23, p<0.001), and TNBC (HR 1.81, p=0.018). In multivariable analysis, PIK3CA CNVs remained independently associated with inferior OS in the overall cohort (HR 1.61, p=0.015) and in HR+/HER2– disease (HR 1.38, p=0.048). Notably, the coexistence of a PIK3CA CNV and mutation identified a subset with particularly poor prognosis. Compared with patients harboring PIK3CA SNVs alone, those with concurrent SNVs and CNVs had significantly worse OS in the overall population (HR 1.80, p=0.002), in HR+/HER2– disease (HR 1.51, p=0.037), and in TNBC (HR 5.72, p<0.001). Conclusions: PIK3CA amplifications detected in ctDNA are relatively frequent in MBC and are associated with significantly worse survival across molecular subtypes. The coexistence of PIK3CA amplification and mutation identifies a distinct high-risk subset with particularly poor prognosis, beyond the effect of mutations alone. Further mechanistic understanding and real-world outcome analyses are needed to determine whether PIK3CA amplifications, alone or in combination with mutations, have predictive relevance for response or resistance to PI3K-pathway inhibitors.

Supplementary Figure 1 Shows the distribution of ERBB2 mutations among metastatic cancer patients, survival outcomes according to metastatic sites (bone vs visceral), and detailed CTC counts from HR+/Her2- MBC patients. Supplementary Figure 2 Shows the therapeutic effect of Tras-ALN on metastasis spread from bone metastasis models of HR+ breast cancer via uCT, live and ex vivo bioluminescence profiling of metastatic organs. Supplementary Figure 3 shows the increased expression of ERBB2 in MSC 3D coculture and anti-ER treatment conditions. Supplementary Figure 4 shows the immune-independent effect of Tras-ALN in blocking metastasis progression. Supplementary Figure 5 shows that direct cell-cell interaction is not required for MSC-induced Her2 expression based on 3D transwell culture conditions. Supplementary 1 of Figure 6 shows genetic modulations of Her2 (ERBB2) in MCF7 and ZR75-1 breast cancer models. Supplementary 2 of Figure 6 shows that genetic alteration of Her2 impacts multiorgan metastasis seeding in HR+ breast cancer models. Supplementary 1 of Figure 7 shows that miRNAs with Her2 targeting potential are associated with overall survival in breast cancer. Supplementary 2 of Figure 7 shows that miR-133a/b abrogates brain metastasis seeding from bone-derived ZR75-1 cells.

Recent advances in mass spectrometry-based single-cell proteomics (SCP) technologies have revolutionized the SCP field. However, current SCP approaches generally employ sub-μL to 1 μL processing volumes for effective single-cell sample preparation using either ultralow-volume specialized devices or a 384-well plate by frequently adding water to compensate for evaporation, which limits their broad accessibility and analytical robustness. Here, we report a robust and convenient SCP method termed iSOP (improved surfactant-assisted one-pot processing) for the processing of single cells at a low μL processing volume using a 384-well plate with tight sealing to avoid sample drying loss. After systematic optimization, 3 μL was selected for iSOP as the processing volume, with a mixture of trypsin and Lys-C enzymes (2 ng of each enzyme) in terms of robustness, sensitivity, and operation convenience. With a commonly accessible LC-MS platform, iSOP-MS can detect and quantify ∼1200-1800 protein groups from single HeLa or MCF7 cells. Application of iSOP-MS to two neuroblastoma cell lines enabled reliable identification of an average of ∼1700 and ∼2050 protein groups from single BE2-C and SK-N-SH cells, respectively, and precise characterization of cellular heterogeneity. When compared to other available SCP methods, iSOP-MS is more robust and convenient for routine, cost-effective, quantitative SCP analysis.

Background optimal imaging intervals for patients with hormone receptor-positive/HER2-negative metastatic breast cancer (MBC) remains undefined. Aim of this study was to analyze the temporal patterns of disease progression to identify high risk subgroups that may benefit from intensified monitoring. Methods we analyzed 149 hormone receptor-positive/HER2-negative MBC patients prospectively enrolled in the MAGNETIC.1 trial (NCT05814224) and treated with first line endocrine therapy. Hazard rates (HR) for disease progression were determined according to clinico-pathological and liquid biopsy features. Results in the overall population, two distinct progression-risk peaks emerged at 2–3 months (32.9/1000 person-months) and at 24 months (28.0/1000). Higher risk of progression was observed in lobular carcinoma (61.1) [HR 61.12 per 1000 person month (pm)], progesterone receptor-negative status (HR 39.07), fulvestrant-based treatment (HR 46.88), liver metastases (HR 59.00), and presence of ≥ 3 metastatic sites (HR 40.10). Conclusions Hazard distribution in hormone receptor-positive/HER2-negative MBC is biphasic and modulated by readily available clinical variables. High-risk subgroups may benefit from intensified radiologic and liquid-biopsy surveillance during the first three months and around two years after treatment start.

Inflammatory breast cancer (IBC) is a rare, aggressive disease with poorly understood biology. To provide a comprehensive characterization of blood-based biomarkers in IBC, we retrospectively analyzed 341 metastatic breast cancer patients (83 IBC, 258 non-IBC). Circulating tumor cells (CTCs), CTC clusters, cytokeratin + /CD45+ cells (DPcells), and tumor-derived extracellular vesicles (tdEVs) were evaluated. Matched circulating tumor DNA (ctDNA) data were available for 59 IBC and 110 non-IBC. Analyte distributions and overall survival (OS) association were compared by IBC status. Circulating cellular analytes showed similar distributions, except for lower tdEVs in IBC. CTCs and tdEVs were associated with OS irrespective of IBC status, whereas DPcells only in IBC. TP53 (63%), PIK3CA (29%), and MYC (17%) were the most frequently altered genes in IBC. TP53 sequence variants (SVs; SNVs/indels) and CCNE1/MYC CNVs were enriched in IBC, but after adjusting for confounders, only PIK3CA SVs remained significantly less frequent. Network analysis identified TP53 SVs as key node in IBC, co-occurring with a CNV module (MYC/CCNE1/PIK3CA/FGFR1) with higher centrality in IBC than non-IBC. In multivariable analyses, PIK3CA and MYC CNVs were associated with worse OS. A clinical–ctDNA model showed strong prognostic performance, validated in an independent IBC cohort (n = 39). Overall, integrated liquid biopsy profiling unveiled distinct IBC features and identified prognostic biomarkers.

Background. Inflammatory breast cancer (IBC) is a rare and aggressive type of BC with a very poor prognosis and that accounts for 10% of BC-related deaths. Diagnosis and treatment of IBC are particularly challenging, as its symptoms resemble mammary infection, and the tumor has already metastasized at the time of diagnosis. Compared to non-IBC (nIBC), IBC tumors are enriched in pro-tumorigenic, M2-polarized tumor-associated macrophages (TAM) with immunosuppressive functions. Despite growing body of work, the mechanisms controlling IBC development and immune landscape are largely unknown. In particular, we still do not understand (a) how tumor cells drive infiltration of TAMs in the tumor microenvironment; (b) whether IBC intra-tumor TAMs dampen T cell responses as in other types of cancers, thus allowing tumor growth; (c) how to identify patients at high risk of developing IBC and, once diagnosed, the individuals at risk of disease progression. Exosomes are small circulating extracellular vesicles that mediate cell-to-cell communication that are released by cancer cells locally in the primary tumor and in the blood circulation, thus potentially serving as predictive biomarkers and therapeutic targets for the systemic effects of the disease. We have previously shown that exosomes are functional determinants of BC progression and prepare distant sites for metastatic seeding by establishing favorable pre-metastatic niches, but their role in IBC outcomes is not known. Methods. We have analyzed the proteomic profile of exosomes from IBC (MDA-IBC3, SUM-149, SUM-190, KPL4, and FC-IBC-02) and nIBC cell lines (MCF-7 and MDA-MB-231) as well as plasma exosomes from IBC and nIBC patients and healthy controls by liquid chromatography mass spectrometry. The functional role of cell line- or patient-derived exosomes was tested via incubation with THP-1 cells. Results. We have identified a signature of IBC-specific proteins that were enriched more than 2-fold in IBC cell exosomes compared to nIBC cell exosomes, regardless of estrogen receptor (ER) or human epidermal growth factor receptor 2 (HER2) status. Among them, polypeptide N-acetylgalactosaminyltransferase 7 (GALNT7) was found up-regulated 5-fold in IBC exosomes. GALNT7 is an O-glycosylating enzyme that is highly expressed in cancer cells of BC patient biopsies. O-glycosylation in TAMs is associated with polarization towards a pro-tumorigenic, M2-like phenotype and significantly increases tumor growth. Moreover, interaction of macrophages with membrane O-glycans expressed on GALNT7-expressing tumor cells or other macrophages receiving GALNT7-enriched exosomes triggers the expression of activation markers found in IBC-associated TAMs, including CD206, CD163, and PD-L1. We also detected a specific, TAM-related protein signature in exosomes from plasma of IBC patients compared to nIBC or healthy controls that may help with stratification and treatment of IBC patients. Conclusions. Based on our preliminary data, we propose that IBC cell-derived exosomes promote reprogramming of TAMs to shape an immune-suppressive tumor microenvironment and support disease progression, and as such may serve as “liquid biopsy” biomarkers for IBC diagnosis and prognosis. Citation Format: Serena Lucotti, Mara Serena Serafini, Amanda Kaylan Strickland, Maroua Manai, Elisabetta Molteni, Letizia Pontolillo, Eleonora Nicolo’, Caterina Gianni, Nadia Bayou, Valerie Fraser, Carolina Reduzzi, Massimo Cristofanilli, David Lyden. Exosomes as drivers and biomarkers of TAM recruitment and immune evasion in inflammatory breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-04-17.

BACKGROUND:In the context of hormone receptor positive, HER2 negative Metastatic breast cancer (MBC), CDK 4/6 inhibitors (CDK4/6i) combined with endocrine therapy represent the standard first-line treatment, improving Progression-Free Survival (PFS) and Overall Survival (OS). Despite these benefits, resistance to treatment develops, necessitating early risk classification to guide clinical management. This study explores the potential of cell-free DNA (cfDNA) fragmentomics, specifically ACTB fragments, in predicting tumor dynamics and treatment outcomes in luminal MBC, based on the principle that shorter DNA fragments are generally indicative of circulating tumor DNA (ctDNA) from tumor cells, while longer fragments are associated with leukocyte lysis. METHODS:In the MAGNETIC.1 study, 141 women with luminal-like MBC were enrolled between January 2018 and January 2023. Blood samples were collected at baseline (BL), and after 3 (T3) and 6 (T6) months of treatment. cfDNA was extracted and analyzed using droplet digital PCR (ddPCR) to quantify ACTB fragments (136 bp, 420 bp, and 2,000 bp). Continuous variables were compared using the Mann-Whitney test and Kruskall Wallis test depending on data distribution and number of groups. Categorical variables were compared using the Chi-square test or Fischer's exact test whenever appropriate. Differences in survival were tested by log-rank test and uni- and multivariable Cox regression. RESULTS:By categorizing the values of actinic fragments into interquartiles (Q1, Q2, and Q3), ACTBshort Q3 at baseline was significantly associated with negative PR expression (RRR 0.27, P = 0.012) and a higher frequency of liver metastasis (RRR = 3.75, P = 0.009). In terms of clinical outcomes, regarding PFS a significant role was observed for baseline ACTBshort Q3 (HR 1.92, P = 0.041) and ACTBmedium Q3 (HR 0.47, P = 0.043), the latter maintaining significance in multivariable analysis (HR 0.33, 95 %, P = 0.012). For OS, ACTBshort Q3 demonstrated a significant impact in both univariable (HR 3.94, P = 0.003) and multivariable analyses (HR 3.25, P = 0.023). CONCLUSIONS:This study demonstrates the feasibility of employing a fragmentomics mutation agnostic approach in luminal MBC. Baseline and longitudinal changes in ACTB fragments were significantly associated with clinical outcomes, suggesting their potential as non-invasive biomarkers for early risk classification and monitoring tumor dynamics.

Introduction: The Human Epidermal Growth Factor Receptor 2 (HER2) plays a central role in breast cancer (BC). Nowadays, the assessment of HER2 status and the selection of patients eligible for anti-HER2 therapy rely on immunohistochemistry (IHC) and in situ hybridization on tissue biopsy, an invasive approach, unable to capture intratumor heterogeneity and dynamic of HER2 expression. Circulating tumor cells (CTCs) offer an alternative material to evaluate HER2 expression in real-time, through a simple blood draw. Limitations to the use of CTCs for HER2 assessment derive from limited sensitivity in detection methods and lack of standardization.To overcome these limits, we developed a semiautomated pipeline combining label-independent CTC enrichment and HER2 expression quantification and we compared it to the gold-standard CellSearch®, in a cohort of patients (pts) with metastatic BC (mBC). Methods: Blood samples (7.5 mL) were collected in EDTA tubes from mBC pts enrolled at Weill Cornell Medicine and processed within 1 hour for CTC analysis. CTCs were captured using Parsortix™ and stained for epithelial (EpCAM, cytokeratins [CK]), leukocyte (CD45) and HER2 markers as well as for nuclear staining. CTCs were identified as nucleated cells, EPCAM/CK+ and CD45-. For each CTC, 4 color digital images were processed with the automated post-processing developed tool: a combination of an open-source image analysis software CellProfiler and a custom MATLAB code allowing for CTC identification and HER2 expression categorization into high, low and no expression. A second aliquot for each sample was processed in parallel with CellSearch® for comparison. Results: A total of 16 whole blood samples from mBC pts were collected: 5 at baseline (31%), 4 at progression (25%) and 7 at restaging (44%). The study cohort included 5 pts (31%) with HER2+ BC, 5 pts (31%) with hormone receptor-positive (HR+)/HER2- BC, and 6 pts (3%) with HR-/ HER2- BC.By using our pipeline, CTCs were identified in all the samples, whereas CellSearch® analysis detected CTCs in 77% of samples (10/13 of evaluable samples).A total of 320 CTCs were detected in the 16 CTC-positive samples by our pipeline, versus 368 CTCs detected by CellSearch® in 10 CTC-positive samples. We were able to detect HER2+ CTCs in all the 16 processed samples (100%), while CellSearch® only did in 4/13 of the evaluable samples (31%). Among all detected CTCs, 142/320 were HER2+ (44.4%) by using our pipeline, compared to 40/368 (10.9%) identified by CellSearch®. Of the CTCs detected with our pipeline, 81.7% (116/142) had a high HER2 expression and 18.3% (26/142) a low expression. Our pipeline was therefore able to identify HER2+ CTCs in 11/11 (100%) of pts with a HER2 negative status on tissue as opposed to 2/9 (22.2%) of the samples processed by CellSearch®. Interestingly, in addition to EPCAM+/CK+ CTCs, with our pipeline we were also able to identify 83 cells with low/negative EPCAM/CK expression. These cells were CD45-negative and 12% of the cases presented a HER2 expression. Conclusion: The current study showed the feasibility of a real-time HER2 assessment on CTCs enriched from mBC pts. The developed pipeline was able to count and identify HER2-positive CTCs with higher efficiency than the gold-standard CellSearch® overall and interestingly, also in the HER2-negative subgroup. This is a preliminary analysis that should be confirmed in larger cohorts. HER2 marker is a therapeutic target and an accurate and real-time assessment of the HER2 status could be used to better guide treatment in a larger cohort of patient with advanced breast cancer with any detectable HER2 expression that can be effectively treated with antibody-drug conjugates. Citation Format: Nadia Bayou, Sarah Henretta, Laura Arcos Munoz, Elisabetta Molteni, Caterina Gianni, Mara Serena Serafini, Amanda Strickland, Eleonora Nicolo`, Letizia Pontillo, Jyothi Manohar, Olivier Elemento, Massimo Cristofanilli, Carolina Reduzzi. A novel, semi-automated Pipeline for HER2 Quantification on CTCs in breast cancer patients. Is cytopathology of peripheral blood a new diagnostic option? [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-01-20.

Background: Invasive lobular carcinoma (ILC) comprises 10-15% of all breast cancers (BC), predominantly presenting as low grade and hormone receptor positive (HRpos). Triple-negative ILC (TN-ILC) is rare, accounting for approximately 2% of all triple-negative BC (TNBC) and 0.1% of all BC. Due to its rarity, TN-ILC is poorly understood and lacks well-established treatment standards. This study aims to investigate the genomic characteristics of TN-ILC through circulating tumor DNA (ctDNA) profiling within a large multi-center consortium. Methods: The study analyzed a retrospective cohort of 750 patients (pts) with HER2 negative metastatic BC with ctDNA testing using the Guardant360 NGS panel within a large multicenter academic consortium. HR and HER2 status were defined based on the most recent biopsy. Associations across single nucleotide and copy number variations (SNVs and CNVs), histology, and HR status were tested by multinomial logistic regression in terms of Relative Risk Ratio (RRR), adjusting for significant clinical characteristics (i.e., lines of treatment, metastatic sites). Oncogenic pathway analysis was defined based on Sanchez-Vega F et al, Cell. 2018. Prognosis was analyzed for overall survival (OS) defined from the time of ctDNA collection. Results: Among the 750 pts analyzed, the cohort consisted of TN-ILC (N:16, 2%), HRpos-ILC (N:91, 12%), TN-invasive ductal carcinoma (IDC) (N:186, 25%), and HRpos-IDC (N:457, 61%). In TN-ILC, the most frequently altered genes were PIK3CA (50%), TP53 (44%), ERBB2 (25%), and CDH1 (25%). Less frequent alterations included BRAF, ALK, ARID1A, MET, RB1, and SMAD4, each occurring in 12-19% of cases. PIK3CA (48%) and TP53 (39%) had a similar alteration frequency in HRpos-ILC, and ESR1alterations were also common (36%). Multivariable multinomial logistic regression, designed with TN-IDC as reference, investigated differences in oncogenic pathway alterations across histologies and subtypes. In TN-ILC, a significant association was observed for RAS SNVs (RRR = 7.9, p = 0.038) and PI3K SNVs (RRR = 4.32, P = 0.015). In HRpos-ILC, cell cycle CNVs (RRR = 0.3, P = 0.013) and P53 SNVs (RRR = 0.26, P < 0.001) had a significantly lower prevalence than in TN-IDC, while RAS SNVs (RRR = 4.94, P = 0.022) and PI3K SNVs (RRR = 2.6, P = 0.003) were more common. Single gene alterations were investigated across significantly altered pathways. PIK3CA SNVs were more common in TN-ILC (50%) compared to TN-IDC (16%), but similar in frequency to HRpos-ILC (42%) (P<0.001). PTEN SNVs were higher in HRpos-ILC (11%) than in TN-IDC and TN-ILC (respectively 0% and 6%). KRAS SNVs were more represented in HRpos- and TN-ILC (respectively 6% and 8%), with respect to TN-IDC (1%) (P= 0.017). TP53 SNVs were significantly more detected in TN-IDC (68%) than in HRpos and TN-ILC (respectively 44% and 35%, P<0.001). A similarly unfavorable outcome was observed for TN-ILC and TN-IDC (median OS 12 and 13 months, respectively), compared to their HRpos counterparts (median OS 31 and 33 months, respectively) (P<0.001). Conclusions: Our study highlights distinct genomic features of triple-negative ILC detectable through ctDNA. These findings reinforce the need for improved understanding of TN-ILC to define personalized treatment options for this aggressive and rare subtype. Citation Format: Lorenzo Gerratana,Andrew A. Davis, Lorenzo Foffano, Carolina Reduzzi, Emily Podany, Arielle J. Medford, Marko Velimirovic, Katherine Clifton, Annika Putur, Laura Munoz-Arcos, Letizia Pontolillo, Rachel O Abelman, Caterina Gianni, Shaili Tapiavala, Elisabetta Molteni, Marla D Lipsyc-Sharf, Eleonora Nicolò, Eleni Andreopoulou, William J Gradishar, Fabio Puglisi, Cynthia X. Ma, Aditya Bardia, Massimo Cristofanilli. Differential ctDNA-based genomic features of Triple-Negative Metastatic Lobular Breast carcinoma: insights into a rare and poorly understood disease [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-05-16.

Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) plus endocrine therapy (ET) are the mainstay of treatment for hormone receptor positive, HER2 negative (HR + /HER2−) metastatic breast cancer (MBC). However, disease progression is inevitable and unveiling resistance mechanisms is crucial to guide post-CDK4/6i therapeutic strategies. In this study, we retrospectively analyzed a real-world, multi-institutional cohort of patients with HR + /HER2- MBC characterized by circulating tumor DNA (ctDNA) through next-generation sequencing (NGS) before starting second-line treatment. Among 93 patients previously treated with CDK4/6i, PIK3CA (37.6

Abstract Bone metastases can disseminate to secondary sites and promote breast cancer progression, creating additional clinical challenges. The mechanisms contributing to secondary metastasis are barely understood. Here, we evaluate the prediction power of HER2-expressing (HER2E) circulating tumor cells (CTC) after analyzing over 13,000 CTCs from a cohort of 137 patients with metastatic breast cancer with initial HR+/HER2− status and use preclinical models of bone metastasis (BM) to validate the role of HER2E CTCs in multiorgan metastases. Although HER2 expression was higher in patients with BM, experimental analyses revealed that HER2E CTCs derived from bone lesions were more dependent on HER2 activity and more susceptible to anti-HER2 therapy. Targeting the bone-mediated HER2 induction reduces CTC detection and abrogates secondary metastasis from the bone. Overall, we elucidate that HER2E CTCs can serve as a noninvasive biomarker for BM formation with high therapeutic benefit for patients with HR+ metastatic breast cancer. Significance: Given the urgent need for alternative strategies to block metastasis progression, we demonstrate that blocking HER2-mediated secondary metastasis improves clinical outcome and establish HER2 as a biomarker for bone metastasis in patients with initial HR+/HER2− breast cancer, which represents ∼70% of all cases.

Background: Detecting minimal residual disease (MRD) in the adjuvant setting can help identify patients (pts) with early breast cancer (EBC) at a higher risk of recurrence. Circulating tumor DNA (ctDNA)-based MRD detection is strongly associated with recurrence. Typically, MRD tests require prior knowledge of the tissue genetic alterations. However, obtaining tissue samples can be challenging. This multi-institution retrospective analysis investigates the impact of a plasma-only genetic and epigenetic ctDNA testing on the care of pts with EBC. Methods: Our retrospective study included 73 pts with stage I-III EBC who had MRD testing after curative-intent treatment between 09/2022 - 06/2024 at Weill Cornell Medicine (New York) and University of California Los Angeles (Los Angeles, CA). ctDNA evaluation was performed using the Guardant Reveal® (GR) tissue-free assay (Guardant Health, Redwood City, CA), a next-generation sequencing panel. ctDNA presence was determined by a custom bioinformatics classifier identifying tumor-derived variants and epigenetic methylation profiles. All tests were ordered in the real-world clinical setting, and data were gathered through a retrospective review of electronic medical records with appropriate IRB approval. A descriptive analysis was performed. Results: By 06/2024, 73 EBC pts had plasma-only ctDNA testing for MRD monitoring post-surgery. At diagnosis 22% were stage I, 53% were stage II and 21% of pts were stage III. Tumor subtypes included HR+/HER2- (44; 60%), HR-/HER2- (11; 15%), HER2+ (17; 24%), 1 case was unknown. Fifty-five pts (75%) had received neo/adjuvant chemotherapy. Among these, 10 pts had pathological complete response. At the time of the analysis, 49 pts were still receiving adjuvant therapy. The median recurrence free survival (RFS) since surgery was 22.5 months (interquartile range [IQR] 13.3-36.8). With a median follow-up of 2.2 years (IQR, 1.6-3.2) since diagnosis, 6 pts had a distant recurrence. Among them, 3 were HR+/HER2-, 2 were HR-/HER2- and 1 was HER2+ EBC. From 09/2022 to 06/2024, 124 GR tests were performed, with a median of one test (IQR 1-2) per patient. Thirty-nine pts (53%) had one GR test, while 47% had ≥2 tests. The median time between subsequent tests was 119 days (IQR, 93-200). 16 tests (13%) were positive, with 10 pts having at least one GR+ result over a median time of 7 months (IQR 2.7-12.1) from first testing to last follow-up. The first GR test was ordered after a median time from surgery of 13.8 months (IQR 7.0-50.5). Five out of 6 relapses were preceded by MRD+ testing, except for one patient that developed a solitary brain lesion. Pts with ctDNA+ had a primary EBC stage II or III (7 and 2, respectively, 1 unknown). In 7/10 pts, after a first ctDNA+ result, an imaging scan was planned with detection of distant asymptomatic disease recurrence in 3 cases. In 3/10 patients, a follow-up test after 3 months led to further scans and detection of 2 additional relapses. At the time of the analysis 5 patients MRD+ were free of detectable metastases (clinically or radiologically). Overall, ctDNA detection was the only prognostic factor for RFS (p <.001). These data estimate a negative predictive value of 98.4% and a positive predictive value of 50% for this GR test, with a related sensitivity and specificity of 83.3% and 92.5%, respectively. Conclusions: Our analysis suggests the utility of a tissue-free ctDNA assay in diagnosing minimal residual disease. This could have clinical implications by enabling earlier diagnosis and possibly intervention to delay or prevent metastatic recurrence. We confirm prior studies showing high specificity for recurrence detection with multi-omic plasma-only MRD testing. Sensitivity limitations may be due to the small sample size and short follow-up. These findings warrant further study in larger cohorts and future clinical trials. Citation Format: Caterina Gianni, Eleonora Nicolo’, Marla Lipsyc-Sharf, Brian DiCarlo, Eleni Andreopoulou, Ashley Schreier, Jeannine Donahue, Letizia Pontolillo, Laura S. Muñoz-Arcos, Mara Serena Serafini, Elisabetta Molteni, Nadia Bayou, Kelly Eng, Amanda Kaylan Strickland, Marko Velimirovic, Lorenzo Gerratana, Andrew A. Davis, Arielle Medford, Olivier Elemento, Aditya Bardia, Ugo De Giorgi, Carolina Reduzzi, Massimo Cristofanilli. Clinical impact of MRD detection via ctDNA tumor-agnostic assay in early-stage breast cancer patients: a real-world experience [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-01-19.

Background: Invasive lobular carcinoma (ILC) has distinctive clinical and genomic features compared to invasive ductal carcinoma (IDC); however, for patients (pts) with ILC treatment is selected according to the same guidelines as IDC. Better characterization of ILC and development of specific approaches for ILC pts is an unmet need. Liquid biopsy (LB) is a useful tool to achieve this goal. Our group showed that, compared to IDC, ILC has specific circulating tumor DNA (ctDNA) alterations and higher CTC count. Another study reported higher detection of CTC clusters (CTC-CL), considered the main seed of metastasis, in ILC. This finding is paradoxical since cell-cell adhesion (a key feature of CTC-CL) is impaired in ILC. Interestingly, an association between CTC-CL counts and ctDNA CDH1 alterations (a hallmark of ILC) was reported, regardless of histology. To investigate whether different mechanisms are responsible for CTC clustering in ILC and IDC, in this study we characterized CTC-CL according to the breast cancer (BC) histotype. Methods: Blood samples were collected from 351 pts with stage IV BC before starting a new line of therapy at Northwestern University (Chicago, IL) between 2016 and 2021 (NU16B06 trial). Blood samples were processed with the CellSearch system for CTC and CTC-CL enumeration by a single expert operator. CTC-CL were defined as groups of ≥2 CTCs , or ≥1 CTC clustered with ≥2 white blood cells (WBCs). The number and size of CTC-CL, and the presence of WBCs in CTC-CL (heterotypic) were compared between ILC and IDC. Also, the association between CTC-CL and overall survival (OS) was tested. To further explore mechanisms of CTC-CL formations, we assessed potential differences in the association between CTC-CL presence and ctDNA alterations in ILC vs IDC. For ctDNA analysis, matched plasma samples were analyzed using Guardant360 and tested for the 10 most altered genes and CDH1. Results: Of the 351 pts included, 255 (73%) had IDC while 45 (13%) had ILC. Overall, CTC-CL were identified in 45 (13%) pts and only in those with ≥ 5 CTCs. The presence of CTC-CL was significantly higher among ILC pts (27% vs 11% in IDC, p=0.004) but the total number of clustered CTCs was significantly lower in ILC than IDC (median 4.5 vs 9.5, p=0.039), suggesting a smaller size of CTC-CL in ILC. Indeed, the median and maximum number of CTCs per CTC-CL was numerically lower in ILC than IDC. Heterotypic CTC-CL were identified in 6 (50%) and 10 (36%) of ILC and IDC pts, respectively. Among these pts, there was a trend for a higher median (0 vs 2, p=0.37) and maximum number of WBCs (2.5 vs 3.5, p=0.26) in CTC-CL in ILC than IDC. Overall, the presence of >3 CTC-CL was associated with shorter OS (6 vs 21 months, p=0.001). A matched plasma sample for ctDNA analysis was available for 129 IDC and 19 ILC pts. CDH1 alterations were detected in 2 IDC and 1 ILC pts and associated with CTC-CL (p=0.015) only in IDC. No significant association between ctDNA alterations and CTC-CL was observed in ILC possibly due to the small sample size; further analysis is ongoing. Conclusion: ILC is characterized by a higher number of CTC-CL than IDC. CTC-CL in ILC appear to be different from IDC, being smaller but more frequently associated with WBCs. This suggests a possible different biology for CTC-CL formation in ILC related to the impaired cell-cell adhesion and a specific role played by the CTC-CL microenvironment. Indeed, the interaction with immune cells in ILC may promote the survival in the bloodstream of smaller CTC-CL thus enhancing metastatic efficacy. Further studies including a larger number of pts are needed to validate and better elucidate these findings. The study of CTC-CL could shed light on the distinct pattern of metastatic spread of ILC, potentially offering therapeutic opportunities, and serving as a useful prognostic factor. Citation Format: Eleonora Nicolò, Elisabetta Molteni, Lorenzo Foffano, Lorenzo Gerratana, Mara S. Serafini, Letizia Pontolillo, Caterina Gianni, Laura Munoz-Arcos, Nadia Bayou, Kaylan Strickland, Hunter Gaudio, Brenno Pastò, Maroua Manai, Youbin Zhang, Paolo D’Amico, Andrew A. Davis, Jeannine Donahue, Huiping Liu, William J. Gradishar, Giuseppe Curigliano, Carolina Reduzzi, Massimo Cristofanilli. Investigating differences in the composition of circulating tumor cells (CTCs) clusters in invasive lobular and ductal carcinoma to decipher lobular breast cancer metastasis [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-09-23.