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.

Background: Inflammatory breast cancer (IBC) is a rare and aggressive form of breast cancer, accounting for 1-3% of cases and more frequent in North of Africa (7-11%). A major challenge in treating IBC is the lack of specific therapeutic targets. Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) was found overexpressed and associated to poor prognosis in IBC vs. non-IBC (nIBC). As a an activator of inflammation in different types of cancer, our study aimed to evaluate in vitro and in clinical samples the role of MARCKS in inflammation and tumor microenvironment in IBC samples compared to nIBC. Methods: Using siRNA; we knocked-down MARCKS expression in IBC (SUM149) and nIBC (MDA-MB-231) cell lines, then evaluated the inhibitory effect on migration, invasion and mechanism of action in both single and co-culture with M2 macrophages. Inflammation was induced by LPS in vitro, and the clinical relevance of protein expression of MARCKS, CD163, CD68, LPS and NFkB was assessed in a large series of IBC vs. nIBC patients using immunohistochemistry (IHC). Results: Using IHC, our results showed that in IBC samples only, the presence of M2 macrophages (CD163) was associated with obesity and that obese patients presenting M2 macrophage infiltration had a poorer 5-years overall survival. We also found an overexpression of NFκB in IBC patients correlated to MARCKS and M2 macrophages infiltration. We also showed in vitro that MARCKS-knockdown impaired cell migration and invasion in SUM149 (IBC) compared to MD-MB-231 (nIBC) cells. Furthermore, using western blot, only in IBC, MARCKS inhibition regulated several proteins involved in IBC inflammation and tumor microenvironment (TME) particularly NFκB and EGFR known to be directly implicated in IBC aggressiveness with better regulation of the different pathways in co-culture with macrophages M2. Conclusions: Our study suggests an important role of MARCKS as a potential target modulating the TME and inflammation in IBC that needs further investigation using in vivo models. Citation Format: Maroua Manai, Pascal Finetti, Nadia Bayou, Yoldez Houcine, Wissal Ben Taher, Balssem Mosbahi, Rihab Benhassen, Jeannine Donahue, Valerie Fraser, Carolina Reduzzi, Maha Driss, Hamouda Boussen, François Bertucci, Massimo Cristofanilli. Role of MARCKS in regulating the tumor microenvironment 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 P1-06-27.

Background: Circulating tumor (ct) DNA testing is a standard of care approach to evaluate patients (pts) with advanced or metastatic breast cancer (BC) for the detection of resistant and actionable somatic (s) alterations. ctDNA testing could identify incidental germline (g) mutations. While the prognostic and predictive role of known gBRCA1/2 mutation is well established, the significance of sBRCA1/2 alterations is still debated. We aimed to establish a variant allele frequency (VAF) threshold to distinguish germline from somatic mutations and to explore the impact of BRCA1/2 alterations detected by ctDNA on survival outcomes. Methods: A retrospective multi-institutional cohort of pts with BC and at least one BRCA1/2 mutation detected by standard clinical ctDNA next-generation sequencing (Guardant 360®) for metastatic disease was included in the analysis. The incidence of BRCA1/2 mutation detected by ctDNA and germline testing was analyzed to assess VAF threshold to predict the likelihood of detecting germline mutations. Receiving operating characteristic (ROC) curves were generated to determine the VAF cut-off; differences in survival were tested using the log-rank test. Results: 294 pts were included in the analysis. BRCA1 and BRCA2 mutations were detected in 104 (35.4%) and 166 (56.5%) pts respectively, while 24 (8.2%) had a co-mutation in BRCA1 and BRCA2 genes. The median age at diagnosis was 50 years (interquartile range [IQR] 44-62), and family history of cancer was known for 45.9% of pts. The most represented subtype was hormone receptor positive/HER2 negative (HR+/HER2-) (70.4%), followed by triple negative (TN) (15.7%) and HER2 positive (13.9%). At the time of the first BRCA1/2 detection in ctDNA, bone (67.1%) and visceral (56.2%) sites were mainly involved. The mean VAF was 10.0% (standard deviation [SD] 19.7%, range 0.02%-84.3%) for BRCA1 and 12.1% (SD 20.1%, range 0.06%-80.3%) for BRCA2 alterations. The most co-mutated genes were TP53 (58.1%), PIK3CA (35.8%), EGFR (25.1%), ESR1 (23.7%), and ERBB2 (17.2%). The germline testing was available for 157 (53.4%) pts, detecting a gBRCA1 mutation in 16 (10.2%) pts and a gBRCA2 mutation in 37 (23.6%) pts. Comparing the ctDNA and germline testing, 13/16 pts had concordant BRCA1 mutation. In 3 discordant cases, only BRCA2 alteration with different VAF (0.30-80.1%) was detected by ctDNA. All pts with gBRCA2 mutations (37) also had a BRCA2 mutation in ctDNA. An optimal VAF cut-off of 38.4% (AUC 0.99) for BRCA1 and 16.1% (AUC 0.96) for BRCA2 was established as the threshold for the likelihood of a germline mutation detected by ctDNA analysis. For 18 pts with TNBC and 91 pts with HR+/HER2- BC, the detection of BRCA1/2 mutations was at the ctDNA baseline test, before starting a new treatment, with a median of 2 (range 0-9) previous lines of therapy. The median progression-free survival (mPFS) was 7.3 months (mos) (CI 3.9-10.7) for TNBC pts and 8.4 mos (CI 4.5-12.3) for HR+/HER2- pts; no significant survival differences were observed between BRCA1 and BRCA2 mutations. The 2-year overall survival (OS) rate was 69% and 78% in the TN and HR+/HER2- subgroups. Exploring the outcome according to therapy in the HR+/HER2- subgroup, no significant differences were assessed between chemotherapy, CDK4/6 inhibitors plus endocrine therapy, or PARP inhibitors, although a longer mPFS for the latter was observed (4.5 vs. 8.9 vs. 14 mos). Conclusions: The VAF cut-off identified for the likelihood of a germinal mutation detected by ctDNA resulted lower than expected, underlining the importance of a larger germline BC screening, with considerable impact on therapeutic decision making and germline testing of other family members. Further analysis to explore the interplay of different co-mutations with BRCA1/2 will be performed and validation in additional dataset is needed. Citation Format: Letizia Pontolillo, Carolina Reduzzi, Andrew A. Davis, Arielle J. Medford, Emily Podany, Lorenzo Gerratana, Annika Putur,, Surbhi Warrior, Caterina Gianni, Eleonora Nicolò, Katherine Clifton, Whitney L. Hensing, Marko Velimirovic, Laura Munoz-Arcos, Mara S. Serafini, Elisabetta Molteni, Marla Lipsyc-Sharf, Jeannine Donahue, Neelima Vidula, Nadia Bayou, Charles S. Dai, Jennifer C. Keenan, Amir Behdad, William J. Gradishar, Emilio Bria, Cynthia X. Ma, Diana Giannarelli, Aditya Bardia, Massimo Cristofanilli. BRCA1/2 alterations in circulating tumor DNA: correlation with germline origin and impact on survival in 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 P2-05-21.

Abstract Background: The use of circulating tumor (ct) DNA testing allows to detect resistant and actionable somatic alterations and it could be associated with the incidental identification of germline mutations. An univocal variant allele frequency (VAF) threshold to distinguish germinal form somatic mutations has not been assessed yet. We aimed to correlate BRCA1/2 ctdNA detected mutations with germline mutational status to determine potential VAF cutoff. Materials and methods:We retrospectively analyzed the incidence of both somatic (s) and germinal (g) BRCA1/2 alterations in a multiinstitutional retrospective breast cancer (BC) cohort to assess the VAF threshold for the likelihood of germline mutations’ detection by ctDNA next-generation sequencing (Guardant 360) testing. Clinical variables were analyzed using descriptive analyses and receiving operating characteristic (ROC) curves were generated to determine the VAF cutoff. Results: Two hundred and fourteen patients (pts) with sBRCA1/2 mutations (including variant of uncertain significant and synonymous) detected by ctDNA testing, referred to the enrolling centers between January 2015 to May 2023 were included in the analysis. The median age at diagnosis was 50 years old (Interquartile range (IQR)43,5-61.5), 43% had a family history of cancer. Hormone receptor positive/HER2 negative was the most represented subtype (68%) followed by the HER2 positive (16%) and triple negative (16%) ones. At ctDNA baseline, 95.8% of pts had a metastatic disease; the main sites of metastases were bone (67.4%) and visceral (59.3%). Ninetyfour (43.9%) and 137 (64%) pts had a sBRCA1 and sBRCA2 alterations respectively, while 17 (7.9%) had a co-mutation in BRCA1 and BRCA2. The mean VAF value for sBRCA1 alterations was 10.1% (standard deviation (SD) 19.5%, range [0.04%-84.3%]) while for sBRCA2 alterations was 10.8% (SD 18.9%, range [0.21%-80.3%]). The germinal testing, performed per standard of care, was available for 100 pts (46.7%). Among these, 42 (42%) had at least a pathogenic variant detected. A gBRCA1 mutation was present in 11 (11%) pts while 25 (25%) had a gBRCA2 mutation. Comparing the somatic and germinal testing, 9/100 pts had a concordance for BRCA1 detection, the 2 discordant cases had both a low allele frequency sBRCA2 alterations; the concordance for BRCA2 detection was instead 100%. An optimal cutoff of 38.4% (AUC 0.98) for BRCA1 and 19.5% (AUC 0.96) for BRCA2 was assessed by ROC analysis as the likelihood of a germline meaning of a somatic mutation detected by ctDNA analysis. Conclusion: The identification of BRCA1/2 alterations in ctDNA could guide the use of germline testing. The VAF cutoff identified for the likelihood of a germinal mutation is lower than expected, suggesting that a wider population should be screened for germinal mutation with relevant impact on the therapeutic choices and family screening. Citation Format: Letizia Pontolillo, Carolina Reduzzi, Andrew A. Davis, Arielle J. Medford, Annika Putur, Lorenzo Gerratana, Katherine Clifton, Whitney L. Hensing, Marko Velimirovic, Surbhi Warrior, Mara S. Serafini, Eleonora Nicolò, Laura Munoz-Arcos, Jeannine Donahue, Charles S. Dai, Jennifer C. Keenan, Amir Behdad, William J. Gradishar, Diana Giannarelli, Emilio Bria, Cynthia X. Ma, Aditya Bardia, Massimo Cristofanilli. Germline BRCA1/2 mutations detected by circulating tumor DNA testing in breast cancer patients: A retrospective mutiinstitutional analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 977.

Abstract Background: PARP inhibitors (PARPi) are a standard of care for breast cancer (BC) patients (pts) with germline BRCA (gBRCA) mutation, with benefit demonstrated also in patients with somatic (s) BRCA1/2 mutations. The use of circulating tumor (ct) DNA testing allows to detect resistant and actionable alterations in BC, but it could be associated with the incidental identification of germinal mutations. The aim of this study was to describe the incidence of both, somatic and germinal BRCA1/2 alterations, and the impact on the therapeutic outcomes in a retrospective BC cohort with clinical ctDNA testing for their BC. Materials and methods: A retrospective cohort of 245 BC pts that underwent ctDNA analysis (Guardant 360) between August 2014 and May 2023 at Weill Cornell Medical College was identified. Pts with at least a BRCA1/2 alteration, including variant of uncertain significant (VUS) and synonymous, were enrolled in the study. A descriptive analysis was performed. Results: Among 35 patients included, 34 had metastatic disease, the median age was 61 years old (IQR 40-67), and 65.7% were post-menopausal at diagnosis. 57.1% of pts had hormone receptor positive/HER2 negative (HR+/HER2-) BC, 20% had HER2 positive disease and 22.9% were triple-negative (TN) BC. Ten (28.6%) and 20 (57.1%) pts had a sBRCA1 or a sBRCA2 alteration on ctDNA, respectively; 5 pts (14.3%) had a coexisting sBRCA1/2 mutation. The median variant allele frequency (VAF) was 1.2% for BRCA1 and 2.7% for BRCA2 alterations. Furthermore, 53.3% and 6.7% of sBRCA1 and 40% and 8% of sBRCA2 mutations were identified as VUS and synonymous, respectively. The most common associated genomic alterations included TP53 (62.9%), ESR1 (20%) and PI3KCA (52%) with a median number of 4 variants (IQR 2-7). Next generation sequencing (NGS) testing on tissue was available for 3 patients: two demonstrated concordance with BRCA alterations detected by ctDNA, whereas no alteration in BRCA1/2 genes was found for the third patient despite the tissue analysis was performed at the same time of the ctDNA. Germline testing was available for 24 (69%) pts; a corresponding gBRCA1 mutation was found for 3 pts (VAF 48.6%-84.3%), and a gBRCA2 for 8 pts (VAF 26.7%-49.5%). Due to the small sample size, a univocal VAF cut-off to detect gBRCA1/2 mutations could not be calculated. Twenty-nine pts had a sBRCA1/2 mutation detected by ctDNA test before starting a new therapy; of these 55.2% had HR+/HER2- disease, while 17.2% and 27.6% were HER2 positive and TN BC, respectively. The median progression free survival (mPFS) was 10.3 (3.2-17.4) months (ms) and the 1-year and 2-year overall survival (OS) rates were 82.3% and 70.5%, respectively. The mPFS was 10.3 ms (0-25.0) for patients (n=16) that underwent chemotherapy, 5.6 ms (0-15.1) for those who had received endocrine therapy (n=9), and not reached for patients treated with PARPi (n=4). Conclusions: ctDNA analysis allows the detection of sBRCA1/2 mutations that could be missed by tissue-based testing. The identification of BRCA alterations on ctDNA could guide the use of germline testing even when these are present at a lower VAF than expected, with relevant impact on the therapeutic choices and family screening. Further evaluation to establish the impact of sBRCA1/2 detected by ctDNA on the therapeutic algorithm decision are needed. Citation Format: Letizia Pontolillo, Eleonora Nicolò, Laura Munoz Arcos, Carolina Reduzzi, Mara Serena Serafini, Amanda Kaylan Strickland, Nadia Bayou, Jeannine Donahue, Elisabetta Molteni, Lorenzo Gerratana, Diana Giannarelli, Eleni Andreopoulou, Emilio Bria, Massimo Cristofanilli. Association between BRCA alterations detected by circulating tumor DNA and germline mutations in breast cancer patients: a retrospective mono-institutional analysis [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 PO3-14-11.

Abstract Background: Invasive lobular carcinoma (ILC) has distinctive clinical and molecular features compared with invasive ductal carcinoma (IDC). CTCs and tdEVs are independent prognostic factors in MBC, however, liquid biopsy studies focusing on ILC are to date scarce. Higher CTC levels have been observed in ILC than IDC. One study including 28 ILC pts suggested the use of higher CTC cutoff for prognosis stratification. The clinical significance of tdEVs in ILC is unexplored. This study aimed to assess differences in the distribution and prognostic value of CTCs and tdEVs between ILC and IDC. Methods: Blood samples were collected from 304 pts with MBC before starting a new line of therapy at Northwestern University (Chicago, IL) between 2016 and 2021 (NU16B06 trial). Blood was processed with the CellSearch® system. The ACCEPT software was applied to CellSearch® images to automatically enumerate CTCs and tdEVs. Association of CTCs and tdEVs with overall survival (OS) was tested in ILC and IDC pts. A count of CTCs ≥ 5 was considered high. The cutoff levels for tdEVs were < 20 (low), 20-79 (intermediate), and ≥ 80 (high), as previously reported. Additional exploratory cutoffs for CTCs (≥ 20, ≥ 50, ≥ 80, and ≥ 100) and tdEVs (based on quartiles of tdEV distribution in the overall population and ILC) were also evaluated. Results: Of the 304 pts, 47 had ILC while 257 had IDC. 56% of pts received first-line therapy. Median CTC count was 8 [interquartile range (IQR) 2-35] in ILC and 1 (IQR 0-8) in IDC pts (P <.001). A significantly higher median tdEVs count was observed in ILC (36; IQR 18-115) than in IDC (11; IQR 2-86) pts (P =.002). High levels of CTCs (≥5) were not associated with OS in ILC (P =.88), even when higher cutoffs (≥ 20, ≥ 50, and ≥ 80) were used. Only the detection of ≥ 100 CTCs was significantly associated with OS in ILC pts (median OS 36 vs 10 months; HR 3.4; P =.028). In contrast, the prognostic effect of CTCs ≥ 5 was strong in IDC and maintained for all cutoffs. Similarly, while the prognostic value for tdEVs was confirmed for IDC using both 20-80 and exploratory cutoffs (3-105 and 18-155), no association between tdEV levels and OS was observed in ILC regardless of the cutoffs used. Conclusion: ILC is associated with higher CTCs and tdEVs compared to IDC, probably due to the impaired cell-cell adhesion that characterizes ILC. Unlike IDC, the prognostic effect for the established cutoff of ≥ 5 CTC was not observed in ILC. A higher CTC cutoff could be considered for this subtype of BC. For tdEV no association with OS was observed, regardless of the cutoff used. These findings emphasize that ILC is a distinct entity also in the liquid biopsy features. Because of the low prevalence of ILC, research efforts should be directed at combining data from ILC pts to better characterize this subtype of BC and understand the reason behind the different associations with OS as compared to IDC. Citation Format: Eleonora Nicolo, Lorenzo Gerratana, Lorenzo Foffano, Laura Munoz-Arcos, Mara S. Serafini, Maroua Manai, Letizia Pontolillo, Nadia Bayou, Elisabetta Molteni, Amanda K. Strickland, Caterina Gianni, Youbin Zhang, Paolo D'Amico, Andrew A. Davis, Jeannine Donahue, Huiping Liu, William J. Gradishar, Ami N. Shah, Giuseppe Curigliano, Carolina Reduzzi, Massimo Cristofanilli. Distribution and prognostic significance of circulating tumor cells (CTCs) and tumor-derived extracellular vesicles (tdEVs) in patients (pts) with lobular metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7500.

Patient advocates, referring to those individuals that have been diagnosed with the disease for which they advocate, are essential stake holders in healthcare. For those facing the stages of being diagnosed with Inflammatory Breast Cancer (IBC), the "call to advocate" is an immediate response to being diagnosed with a rare and aggressive disease that progresses rapidly, often in a matter of weeks or months. There is a great stigma and bias in the medical community that has inhibited the education and study of IBC. A lack of understanding of the disease, how it presents and how to treat it leaves many IBC patients facing misdiagnosis. Communication is a cornerstone of healthcare; this goes beyond the patient-provider dynamic. Education of IBC must be a grassroots initiative. There should be no barrier to care in the diagnosis, treatment, study and survivorship of inflammatory Breast Cancer. It is not just an oncologist's lesson to learn, but that of all providers in healthcare. In this chapter you will hear how 4 women who were diagnosed with IBC faced the difficult tasks of navigating through the healthcare system on their own and came out on the other side using their experience to help others. In conclusion, in defining the evolving roles of Patient Advocacy in IBC over the past 25 years, we examine what has been done, along with its challenges, and what work still remains from the perspectives of different patient advocates.

Abstract Background: Several prospective, randomized clinical trials showed that the CDK4/6 inhibitor palbociclib in combination with either letrozole or fulvestrant significantly improves progression-free survival (PFS) in patients with ER+/HER2- metastatic breast cancer (MBC). However, in some cases, there is development of endocrine resistance and ultimate disease progression. Molecular analysis performed in tissue specimens collected in the PALOMA-3 study, comparing fulvestrant and Palbociclib versus fulvestrant placebo, demonstrated that one of the genes associated with an increased clinical benefit is SIRT3 (Sirtuin 3) which is thought to control mitochondrial integrity and metabolism (Cristofanilli M. et al., Lancet Oncol. 2016). This study aimed to evaluate in preclinical models the predictive role of SIRT3 in ER+/HER2- breast tumors. Methods: Using lentiviruses we generated MCF-7 and T47D cells stably expressing either control shRNA (sh ctr) or shRNA targeting SIRT3 (shSIRT3). With these models, we studied cell viability, tumor growth, apoptosis, and autophagy in MCF-7 cells treated with fulvestrant and palbociclib as a single treatment or in combination, these either alone or in combination with shSIRT3 (knockdown). Nude mice were used for our in vivo studies, and sh ctr or shSIRT3 MCF7 cells were injected. Results: We showed in vitro that the response to palbociclib was significantly associated with levels of SIRT3 expressionas high or low in ER+ cells (p< 0.0001). Furthermore, we found that irreversible cell growth inhibition mediates the beneficial effect of palbociclib in SIRT3 high expressing cellscompared to low expression (p< 0.001).Then, we evaluated the mechanistic activity of SIRT3. We showed thatSIRT3-low expression cells induced enhanced sensitivity to palbociclib by targeting the reactive oxygen species (ROS)/autophagy axis:(i) SIRT3 regulated the mitochondrial homeostasis (e.g. glucose, lactate, pyruvate, succinates), (ii) ROS levels where lower in sh crt comparing and shSIRT3 in treated and untreated cells (p< 0.001), (iii) in terms of senescence,a higher level of positive cells for β-gal activity was found (p≤0.05) and (iv) for autophagy, our western blot analysis showed cleaved LC3 proteins. Our in vivo studies showed that SIRT3 regulated treatment response to palbociclib with a significant decrease in tumor weight and tumor volume (p< 0.01) and importantly, after immunohistochemistry analysis, we validated the role of SIRT3 in regulating the proteins involved in the autophagy/senescence balance, was mainly in epithelial cells compared to stromal cells. Conclusion: Our preclinical studies demonstrated that elevated SIRT3 expression levels sensitizes ER+/HER2- breast tumors to palbociclib treatment. This study suggested that SIRT3 expression could represent a potential predictive biomarker in HR+ MBC patients treated with Palbociclib. Citation Format: Maroua Manai, Ghada Sahraoui, Raoudha Doghri, Lorenzo Gerratana, Paolo D’Amico, Youbin Zhang, Jeannine Donahue, Ami Shah, Carolina Reduzzi, Wenan Qiang, Massimo Cristofanilli. SIRT3 as a new potential predictive biomarker for response to CDK4/6 inhibition in ER+/HER2- metastatic breast cancer patients [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 PO3-14-12.

549 Background: Tumor-informed ctDNA-based detection of minimal residual disease (MRD) in the adjuvant setting for patients (pts) with early breast cancer (EBC) is strongly associated with recurrence. However, the clinical impact of early ctDNA detection and potential therapeutic intervention remains unclear. In this multi-institution retrospective analysis, we investigated whether ctDNA detection in the adjuvant setting impacted the care of pts with EBC. Methods: This multisite (4 geographically distinct regions in the US) study included ptswith stage I-III EBC who had adjuvant MRD testing between 11/2020 - 01/2024. A personalized, tumor-informed 16-plex PCR assay (Signatera) was used for plasma ctDNA detection. All tests were ordered in the real-world clinical setting. Data were obtained by retrospective review of electronic medical records. Results: As of 01/2024, 464 EBC pts had tumor-informed ctDNA testing to monitor MRD. Of these, 58 pts (13%) were ctDNA+ at ≥1 timepoint post-surgery: 7 (12%) with stage I EBC, 25 (43%) stage II, 26 (45%) with stage III. Tumor subtypes included HR+/HER2- (38; 65%), HR-/HER2- (17; 30%), and HER2+ (3; 5%). Of the ctDNA+ group, most pts (47; 81%) had testing at >1 timepoint (median 3; range 1-20 timepoints). Overall, 28 (48%) had ctDNA+ at all timepoints, 11 (19%) were persistently ctDNA+ after initial ≥1 negative result (ctDNA-), and 19 (33%) cleared ctDNA after initial ≥1 ctDNA+ result. Adjuvant ctDNA+ results impacted the care plan in 53/58 (91%) pts. 45/58 (78%) had restaging imaging within 1 year of a ctDNA+ result, and 25/45 (56%) had radiographic evidence of recurrence at that time: 23 distant and 2 local recurrences. 10/25 (40%) of these recurrences were in pts with HR-/HER2- EBC. Of 20 pts that were ctDNA+ despite no radiographic evidence of recurrence (MRD+ corresponding to molecular recurrence), 17/20 (85%) had HR+/HER2- EBC. At least 5/20 (25%) had increased imaging surveillance after ctDNA+ results, and at least 14/20 (70%) had repeat ctDNA testing. In 9/20 (45%) pts with molecular recurrence, providers changed therapy based on the ctDNA+ result, including enrollment into a clinical trial. 5/9 (56%) of these pts had a subsequent ctDNA clearance, 4 (80%) of whom remained without radiologic evidence of disease (median follow-up 12.53 (range 6.08-21.6) months from time of ctDNA+). 3/9 pts had persistent ctDNA+ despite change in therapy and all 3/3 (100%) of these pts had a radiographic recurrence. Conclusions: In this multi-institutional study, ctDNA detection impacted clinical care in most pts. A subset of pts had a change in systemic therapy with subsequent ctDNA clearance which was associated with better prognosis compared to those without ctDNA clearance. Further validation of these findings in additional datasets and clinical trial(s) are needed to inform incorporation of ctDNA testing into routine care.

Abstract Background: Liquid biopsy provides a real-time assessment of metastatic breast cancer (MBC). Recently, the complementary prognostic value of tumor-derived extracellular vesicles (tdEVs) and circulating tumor cells (CTCs) has been reported. We have previously confirmed the strong prognostic significance of CTCs and tdEVs in inflammatory breast cancer (IBC). While previous studies have reported the association of CTCs with circulating tumor DNA (ctDNA) alterations in MBC, no evidence is available for tdEVs. This study aimed to analyze the association of tdEVs with ctDNA alterations, to investigate the molecular pathways underlying their presence. Moreover, we explored potential differences of this association in patients (pts) with IBC to provide a comprehensive liquid biopsy-based portrait of this aggressive subtype of BC. Methods: Blood samples were collected from 355 pts with MBC before starting a new line of therapy at Northwestern University (Chicago, IL) between 2016 and 2021 (NU16B06 trial). For CTCs and tdEVs analysis, 7.5 mL of blood was processed with the CellSearch® system. The ACCEPT software was applied to CellSearch® images to automatically enumerate CTCs and tdEVs. Positivity cutoff was ≥5 for CTCs, while tdEV cutoff points were < 20 (low), 20-79 (intermediate), and ≥80 (high). For ctDNA analysis, matched plasma samples (± 1 month) were analyzed with the Guardant360™ NGS platform for the detection of somatic single nucleotide variants (SNVs) and copy number variations (CNVs), which were classified into oncogenic pathways based on defined profiles generated on the Cancer Genome Atlas database (p53, PI3K, ER, RTK, RAS, RAF, WNT, MYC, cell cycle, notch). Associations between ctDNA pathways alterations and tdEVs were tested in the overall population by multinomial logistic regression and corrected for significant clinical characteristics. Differences in liquid biopsy features between IBC and non-IBC subgroups were analyzed through Fisher's exact test. Results: Of the 355 MBC pts, 210 (62%) had HR+ BC, 61 (17%) had HER2+ BC, and 68 (20%) had triple-negative BC. Eighty-three (23%) had a diagnosis of IBC and had a numerically lower tdEV count. Also, lower tdEVs were detected in HER2+ BC as compared with HR+. Significantly higher tdEVs were observed among pts with lobular histology, liver, and bone metastases. CTC count was significantly associated with tdEV number. A matched plasma sample for ctDNA analysis was available for 175 pts (62 IBC and 113 non-IBC). In the overall population, SNVs in ER pathway were associated with intermediated/high levels of tdEVs (p=0.004 and 0.008). A similar association was observed for CNVs in the cell cycle pathway (p=0.008 and 0.007). Moreover, associations with ≥80 tdEVs were observed for PI3K SNVs and CNVs (p=0.039 and 0.004, respectively), RTK CNVs (p=0.023), and MYC CNVs (p=0.001). In multivariable analysis, clinical characteristics associated with higher tdEVs were lobular histology (p=0.014 for 20-79 and < 0.001 for ≥80) and bone metastases (p=0.019 for 20-79 and 0.002 for ≥80). When considering clinical variables of interest, only ER SNVs and MYC CNVs were significantly associated with intermediate (p=0.031) and high (p=0.022) tdEV counts, respectively. While the association with ER SNVs and cell cycle CNVs was significant both in the IBC and non-IBC subgroups, others were specific for a certain subgroup: higher tdEVs were significantly associated with PI3K SNVs only in non-IBC pts whereas the significant association with CNVs in PI3K and MYC pathways was observed only in pts with IBC. Conclusion: Detection of tdEVs was associated with particular genomic profiles. These alterations seem to be different in IBC further underlying a different biology of this BC subtype. Additional studies are needed to explore how to integrate different liquid-biopsy based biomarkers in the management of pts with MBC. Citation Format: Eleonora Nicolò, Lorenzo Gerratana, Lorenzo Foffano, Laura Munoz Arcos, Mara Serena Serafini, Maroua Manai, Letizia Pontolillo, Nadia Bayou, Elisabetta Molteni, Amanda Kaylan Strickland, Youbin Zhang, Paolo D’Amico, Andrew Davis, Jeannine Donahue, Huiping liu, William Gradishar, Ami Shah, Giuseppe Curigliano, Carolina Reduzzi, Massimo Cristofanilli. Association of tumor-derived extracellular vesicles with circulating tumor DNA alterations in metastatic breast cancer patients: exploring differences in inflammatory breast cancer [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 PO3-06-05.

Abstract Background: Limited data are available to determine the best therapeutic strategy for hormone-receptor positive (HR+) HER2 negative (HER2-) advanced breast cancer (ABC) after progression on cyclin dependent kinase 4/6 inhibitors (CDK4/6i). The aim of this study was to characterize the genomic and prognostic profile of patients (pts) that experienced disease progression after first-line therapy with a CDK4/6i. Methods: The study retrospectively analyzed a multi-institutional cohort of 75 patients (pts) with HR+/HER2- ABC after experiencing progression on first-line endocrine therapy (ET) with CDK4/6i and characterized by circulating tumor DNA (ctDNA) next-generation sequencing (Guardant 360). Oncogenic pathways (i.e., RTK, RAS, RAF, MEK, NRF2, ER, WNT, MYC, p53, cell cycle, notch, and PI3K) were defined based on previous research (Sanchez-Vega et al. Cell, 2018). Associations across single nucleotide variations (SNVs), copy number variations (CNVs), and pathway classification were tested by uni- and multivariable logistic regression with respect to a CDK4/6i naïve group comprising 247 patients with HR+/HER2- ABC (control). Prognosis was analyzed through Cox regression for overall survival (OS). Result: Our study cohort included 75 ABC pts who experienced progression after first line CDK4/6i plus ET: 43 pts (57.3%) were treated with ET while 31 (41.3%) with a non-ET- second line therapy. The most common histology was invasive ductal carcinoma (70.6%), 37.3% of pts were negative for progesterone expression and 26.7% had de novo metastatic disease. The PI3K (33.3%), p53 (28%) and ER (25.3%) pathways were the most mutated in the CDK4/6i-treated cohort. Comparing our population with a CDK4/6i naïve cohort (N=247), the multivariate analysis showed a higher prevalence in the study cohort of SNVs mutations in: i) ESR1 gene [Odds ratio (OR) 2.93; p=0.005]; ii) cell-cycle pathway (OR 4.24; p=0.033); iii) ER pathway (OR 2.04; p= 0.034). Moreover, in the study cohort a numerical but not significant increase of RB1 SNVs was also observed. Multivariable analyses of the 43 pts that received ET second line therapy showed a negative prognostic impact with TP53 SNVs in both progression-free survival (PFS) [Hazard ratio (HR) = 3.34; 95% CI: 1.15-9.66, p=0.026] and OS (HR = 3.85; 95% CI: 1.52-9.77, p=0.005). The prognostic role of p53 pathway mutations was also confirmed for both PFS (HR = 4.71, 95% CI: 1.64-13.50; p=0.004) and OS (HR = 3.25; 95% CI: 1.27-8.3; p=0.014). The potential interaction between the prognostic oncogenic pathways and the outcome was investigated according to the treatment strategy (ET vs non-ET). A consistent impact was observed across the above prognostic pathways both for PFS and OS. A numerical difference was observed in terms of PFS in pts with p53 pathway mutations that underwent non-ET second line. Conclusions: The genomic landscape of progressive disease after CDK4/6i exposure is significantly different from the genomic alterations detectable in treatment-naïve pts, which could potential impact future treatment strategies for patients with disease progression after adjuvant CDK4/6i exposure. Our data suggest that the choice of second-line treatment could potentially be guided by the identification of actionable mutation by ctDNA, although further prospective studies are needed to validate the clinical utility of this approach. Citation Format: Letizia Pontolillo, Carolina Reduzzi, Andrew Davis, Lorenzo Gerratana, Arielle Medford, Katherine Clifton, Whitney L. Hensing, Marko Velimirovic, Ami N. Shah, Jeannine Donahue, Laura Munoz Arcos, Charles S. Dai, Jennifer Keenan, Amir Behdad, William Gradishar, Emilio Bria, Cynthia Ma, Aditya Bardia, Massimo Cristofanilli. Genomic landscape characterization after exposure to cyclin dependent kinase 4/6 inhibitors: a retrospective multi-institutional consortium analysis [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 PO4-15-06.

Patient advocates, referring to those individuals that have been diagnosed with the disease for which they advocate, are essential stake holders in healthcare. For those facing the stages of being diagnosed with Inflammatory Breast Cancer (IBC), the "call to advocate" is an immediate response to being diagnosed with a rare and aggressive disease that progresses rapidly, often in a matter of weeks or months. There is a great stigma and bias in the medical community that has inhibited the education and study of IBC. A lack of understanding of the disease, how it presents and how to treat it leaves many IBC patients facing misdiagnosis. Communication is a cornerstone of healthcare; this goes beyond the patient-provider dynamic. Education of IBC must be a grassroots initiative. There should be no barrier to care in the diagnosis, treatment, study and survivorship of inflammatory Breast Cancer. It is not just an oncologist's lesson to learn, but that of all providers in healthcare. In this chapter you will hear how 4 women who were diagnosed with IBC faced the difficult tasks of navigating through the healthcare system on their own and came out on the other side using their experience to help others. In conclusion, in defining the evolving roles of Patient Advocacy in IBC over the past 25 years, we examine what has been done, along with its challenges, and what work still remains from the perspectives of different patient advocates.

Patient advocates, referring to those individuals that have been diagnosed with the disease for which they advocate, are essential stake holders in healthcare. For those facing the stages of being diagnosed with Inflammatory Breast Cancer (IBC), the "call to advocate" is an immediate response to being diagnosed with a rare and aggressive disease that progresses rapidly, often in a matter of weeks or months. There is a great stigma and bias in the medical community that has inhibited the education and study of IBC. A lack of understanding of the disease, how it presents and how to treat it leaves many IBC patients facing misdiagnosis. Communication is a cornerstone of healthcare; this goes beyond the patient-provider dynamic. Education of IBC must be a grassroots initiative. There should be no barrier to care in the diagnosis, treatment, study and survivorship of inflammatory Breast Cancer. It is not just an oncologist's lesson to learn, but that of all providers in healthcare. In this chapter you will hear how 4 women who were diagnosed with IBC faced the difficult tasks of navigating through the healthcare system on their own and came out on the other side using their experience to help others. In conclusion, in defining the evolving roles of Patient Advocacy in IBC over the past 25 years, we examine what has been done, along with its challenges, and what work still remains from the perspectives of different patient advocates.

1098 Background: Circulating tumor cells (CTCs) are an independent prognostic factor in metastatic breast cancer (MBC). The prognostic value of CTCs and the optimal cutoff for patients (pts) with inflammatory breast cancer (IBC), one of the most aggressive types of BC, has not been fully established. Recent evidence showed the complementary prognostic value of tumor-derived extracellular vesicles (tdEVs) to CTCs in MBC. The significance of tdEVs in IBC is unexplored. This study aimed to assess the prognostic value of CTCs and tdEVs in metastatic IBC. Methods: This study retrospectively analyzed 308 pts with MBC enrolled at Northwestern University (Chicago, IL) before starting a new line of therapy between 2016 and 2021 (NU16B06 trial). Blood samples were processed for CTCs using the CellSearch system. We applied the open source ACCEPT software to archived CellSearch images to enumerate CTCs and tdEVs. TdEVs cutoff levels were <20 (low), 20-79 (intermediate), and ≥80 (high), as previously reported. The association of CTCs and tdEVs with overall survival (OS) was tested in the overall population (OvP) and IBC pts. Results: Of the 308 pts, 69 were diagnosed with IBC. 51% of pts received first-line therapy. CTCs enumerated by ACCEPT were strongly correlated with manual count (r=0.86) and hence used for this analysis. Median CTC count was 1 [interquartile range (IQR) 0-7] in IBC pts and 3 (IQR 0-14) in non-IBC (p=0.11). A significantly lower median tdEVs count was observed in IBC (7; IQR 3-45) than in non-IBC (22; IQR 4-137) pts (p=0.03). In IBC, higher CTC and tdEV counts were seen in the triple negative subtype (p=0.03) and non-visceral involvement (p=0.02), respectively. In the OvP median OS (mOS) was worse among pts with ≥5 CTCs (HR 2.6; p=0.001) and with elevated tdEVs (HR 3.3; p<0.001). Only tdEVs were independently associated with poorer OS in multivariable analysis. In pts with <5 CTCs there was a stepwise decrement in OS with increased tdEVs count (p=0.3); in pts with ≥5 CTCs, elevated tdEVs levels were associated with significantly worse OS (p=0.03). IBC pts with ≥5 CTCs had shorter mOS (8 vs 47 months; HR 3.6; p<0.001); this association was weaker using ≥1 as CTC cutoff (HR 2.4; p=0.01). OS was adversely associated with increasing tdEVs levels (33 vs 23 vs 7 months for low, intermediate, and high subgroups, respectively; HR 3.7; p=0.001). Furthermore, elevated tdEVs were associated with shorter mOS in pts with ≥1 (p=0.055) and ≥5 CTCs (p=0.6). Among pts with <5 CTCs no significant difference emerged among tdEVs subgroups (p=0.6). Conclusions: This study confirmed the strong prognostic significance of CTCs and tdEVs in MBC, including IBC. Pts with IBC had fewer CTCs and tdEVs compared to non-IBC, probably due to the predominant lymphatic spread of IBC. tdEVs offer the possibility to better define prognosis of IBC pts. Further studies are needed to evaluate their complementarity to CTCs.