Abstract Background HER2-targeted therapies have transformed the trajectory of HER2-positive (HER2+) metastatic breast cancer (MBC), with a subgroup of responders remaining on first line (1L) therapy for many years (yrs). Despite these promising outcomes, the paradigm remains palliative, with some patients (pts) receiving therapy indefinitely. Opportunities to interrupt therapy are controversial and anecdotal in the setting of a limited understanding of long-term responders and lack of predictive biomarkers. Methods We identified prevalent pts with HER2+ MBC seen and consented at Dana-Farber Cancer Institute between 2010 and 2023, regardless of the original date of MBC diagnosis. Exceptional responders (ExRes) were pts without evidence of progressive disease (PD) 3 yrs from initiation of 1L therapy for MBC. Conventional responders (ConRes) were pts who experienced PD within 3 yrs of 1L treatment initiation. We compared clinicopathological characteristics and treatment patterns between ExRes and ConRes using Chi-square or Wilcoxon test. We analyzed median time to treatment switch due to PD (TTS-PD) – i.e., time from metastatic diagnosis to 1L treatment end due to PD - and overall survival, via the Kaplan-Meier method and with a landmark analysis at year 3 (Y3). Our primary aim was to identify predictors of exceptional response to 1L anti-HER2 therapy. Results Of 635 pts with HER2+ MBC, we identified 147 ExRes and 370 ConRes and excluded 118 pts due to follow up ≤ 3 yrs and no PD events. Median follow up was 7.1 yrs (IQR 5.5-11.0) for ExRes and 7.1 yrs (IQR 4.0-11.4) for ConRes. Median age at MBC diagnosis was 50.7 yrs (range 21.9-91.9) for ExRes and 49.8 yrs (26.9-82.3) for ConRes. ExRes presented more often with de novo MBC than ConRes (52.1 vs 30.6%, p< 0.0001). On metastatic samples, more ExRes than ConRes had HER2 3+ tumors by immunohistochemistry (IHC) (93.7 vs 81.0%, p=0.002), whereas the proportion of ER-positive disease was similar between the two groups (45.6 vs 53.4%, p=0.1). Most pts received (neo)adjuvant anti-HER2 agents in both groups (62.5 vs 72.7%, p=0.1). More ExRes than ConRes (55.8 vs 42.7% p=0.007) received 1L chemotherapy (CT) plus trastuzumab (H)/pertuzumab (P). Alternative 1L treatments were CT plus H (26.5 vs 25.7%), H +/- P +/- endocrine therapy (8.2 vs 10.5%), T-DM1 (2 vs 9.2%), tyrosine kinase inhibitors-based regimens (7.5 vs 10.8%). For pts with recurrent MBC, disease-free interval was longer for ExRes than ConRes (median 4.7 vs 3.4 yrs, p=0.01). Visceral involvement at MBC relapse was similar for ExRes and ConRes (78.2 vs 77.8%, p=0.9). Brain metastases at any timepoint were less frequent among ExRes than ConRes (42.9 vs 55.1%, p=0.01). Among ExRes, 70 (47.6%) pts experienced PD after Y3, with a median TTS-PD of 4.6 (4.1-5.1) yrs. In a landmark analysis at Y3, the 2-year TTS-PD was 68.5% (95% CI: 59.8%-75.7%) (5 yrs from treatment start), and 4-year overall survival was 86.5% (95% CI: 78.1%-91.9%) (7 yrs from treatment start). ConRes received a median of 5 (1-19) treatment regimens for MBC, with a median 1L TTS-PD of 12 (10.8-13) mo. A total of 89 (60.5%) ExRes and 214 (57.8%) ConRes underwent tumor sequencing. Data comparing genomic features between ExRes and ConRes will be presented. Conclusions In this prospective cohort, 28% of pts with HER2+ MBC had exceptional response to 1L anti-HER2 therapy. Consistent with prior data, ExRes present more frequently with de novo disease and HER2 IHC 3+ tumors. In our cohort, ExRes had a median follow-up of 7.1 years and 52.4% never experienced PD. Although a significant proportion of pts with HER2+ MBC derive long-term benefit from anti-HER2 therapy, the treatment paradigm remains palliative, and it is unknown whether biomarkers may reliably predict exceptional response and guide future treatment. Ongoing work will explore whether genomic features vary between ExRes and ConRes. Citation Format: Stefania Morganti, Tianyu Li, Katheryn Santos, Melissa Hughes, Nolan Priedigkeit, Yvonne Li, Marla Lipsyc-Sharf, Gregory Kirkner, Janet Files, Julie Kasparian, Elizabeth Grant, Gunjan Gupta, Giuseppe Curigliano, Sara Tolaney, Andrew Cherniack, Nabihah Tayob, Nancy Lin, Heather Parsons. Predictors of exceptional response to first line HER2-targeted therapy for metastatic 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 PO2-04-01.

Over half of hormone receptor-positive (HR+) breast cancer recurrences occur >5 years from diagnosis, however, little is known about well-being or breast cancer risk perceptions and knowledge in long-term HR+ breast cancer survivors. From 1/2021 to 1/2022, we surveyed patients with a history of stage II/III, HR+ breast cancer, ≥5 years from diagnosis, without recurrence about concerns and perceptions related to their diagnosis and recurrence risk, physical and emotional health, knowledge, and risk reduction. Logistic regression identified factors associated with overestimation of 5–10 year distant recurrence risk. Among 166 women, median age at diagnosis was 51, 2.4

Though there has been substantial progress in the development of anti-HER2 therapies to treat HER2-positive metastatic breast cancer (MBC) within the past two decades, most patients still experience disease progression and cancer-related death. HER2-directed tyrosine kinase inhibitors (TKIs) can be highly effective therapies for patients with HER2-positive MBC, however, an understanding of resistance mechanisms is needed to better inform treatment approaches. We performed whole exome sequencing on 111 patients with 73 tumor biopsies and 120 cell-free DNA (cfDNA) samples to assess mechanisms of resistance. In 11/26 patients with acquired resistance, we identified alterations in previously characterized genes, such as PIK3CA and ERBB2 that could explain treatment resistance. Mutations in growing subclones identified potential novel mechanisms of resistance in 5/26 patients and included alterations in ESR1, FGFR2, and FGFR4. Additional studies are needed to assess the functional role and clinical utility of these alterations in driving resistance.

PURPOSE Clonal hematopoiesis of indeterminate potential (CHIP) is frequent in patients with solid tumors. Prospective data about CHIP prevalence at breast cancer diagnosis and its dynamic evolution under treatment selective pressure are limited. PATIENTS AND METHODS We performed targeted error-corrected sequencing on 614 samples from 380 patients with breast cancer. We investigated the dynamics of CHIP on prospectively collected paired samples from patients with early breast cancer (eBC) receiving chemotherapy (CT) or endocrine therapy (ET). We assessed the correlation of CHIP with survival in patients with metastatic triple-negative breast cancer (mTNBC). We estimated the risk of progression to treatment-related myeloid neoplasms (t-MN) according to the clonal hematopoiesis risk score (CHRS). In exploratory analyses, we considered clonal hematopoiesis (CH) with variant allele fraction (VAF) ≥0.005. RESULTS CHIP was identified in 15% of patients before treatment. Few CHIP emerged after treatment, and the risk of developing new mutations was similar for patients receiving CT versus ET (odds ratio [OR], 1.16; P = .820). However, CT increased the risk of developing new CH with VAF ≥0.005 (OR, 3.45; P = .002). Five TP53-mutant CH with VAF ≥0.005 emerged among patients receiving CT. Most patients had low risk of t-MN according to the CHRS score. CHIP did not correlate with survival in mTNBC. CONCLUSION CHIP is frequent in patients with breast cancer. In this study, CT did not lead to emergence of new CHIP, and most patients had low risk of developing t-MN. This finding is reassuring, given long life expectancy of patients with eBC and the association of CHIP with morbidity and mortality. However, TP53-mutant CH with VAF ≥0.005 emerged with CT, which carries high risk of t-MN. Evolution of these small clones and their clinical significance warrant further investigation.

Background: Over half of HR+ BC recurrences occur >5 years (y) from diagnosis (dx). While the risk of late recurrence is constant and extends for at least 20y, little is known about concerns, perceptions, knowledge, and interest in risk reduction in longer-term HR+ BC survivors. Methods: From 1/2021-1/2022, we prospectively identified patients (pts) at Dana-Farber Cancer Institute with a history of stage II/III, HR+/HER2- BC, ≥5y from dx, without recurrence. Pts were invited to participate in a study investigating circulating tumor DNA and risk of recurrence as well as a separate, 1-time survey that assessed physical/mental health (PROMIS), dx/treatment concerns (Brief Illness Perception Questionnaire), risk perceptions, knowledge, and interest in risk reduction. “Overestimation” was defined as estimating ≥20% risk based on the response to the question: “If 100 women with HR+ BC are treated according to recommended guidelines, about how many will have BC come back in the 5-10y following completion of active treatment.” Descriptive statistics included medians and proportions. Logistic regression identified factors associated with overestimation of 5-10y metastatic recurrence risk. Results: Among 166 women (of 209 sent surveys, 79%), median age at dx was 51 (range 21-76), 4% were Hispanic and/or Black; 19% did not have a college degree. Approximately 30% had stage III disease, most received chemotherapy (72%) and radiation (81%) and over half (57%) a mastectomy. Median time from dx was 10 y (range: 5-23). Almost all (97%) reported prior (44%) or current hormonal therapy (14% tamoxifen, 39% AI). Median PROMIS anxiety (53; range: 37-73), physical (51, range: 32-68), and mental (51, range: 25-68) scores were similar to population norms (score of 50). On a 0 (not at all)-10 (extremely) scale, the median rating for concern about dx/treatment was 5; for emotional impact of dx/treatment, the median rating was 9. Regarding risk perceptions, participants estimated that on average, a median of 15 and 10 women (of 100 women) would develop a loco-regional or distant recurrence, respectively, in the 5-10y interval; 43% and 40% estimated the risk of loco-regional and distant recurrence as ≥20%, respectively, for this interval. Pts without a college degree were more likely to overestimate 5-10y distant recurrence risk (multivariable OR: 3.66, 95% CI: 1.56, 8.59); age, chemotherapy receipt, surgery type, stage, and grade were not associated with overestimation. When asked, on average, which women have a higher chance of BC returning after 5y, 17% correctly responded HR+; 42% responded triple negative and 41% responded the risk was the same for both. While >1/3 responded they believed alcohol in moderation may decrease the risk of BC coming back, most also responded that having a healthy weight, eating ≥5 fruits/vegetables a day, and exercise may decrease this risk, with over half reporting engagement in these behaviors (Table). Conclusion: While most longer-term stage II/III HR+ BC survivors report mental and physical health commensurate to population norms, inaccurate knowledge and perceptions about recurrence are common. Strategies to effectively communicate risk (e.g., pictograms, decision/conversation aids) and risk reduction information can promote an accurate understanding of risk in the setting of longer-term HR+ BC survivorship, potentially mitigating emotional concerns which are prevalent ≥5y post-dx. The association between lower educational attainment underscores the importance of attention to literacy and numeracy when developing interventions to improve risk communication. Table. Perceived impact of health behaviors on recurrence risk. Citation Format: Shoshana Rosenberg, Yue Zheng, Katheryn Santos, Elizabeth Riley, Hugh Meadows, Craig Snow, Melissa E. Hughes, Elizabeth Frank, Nancy U. Lin, Ann Partridge, Eric Winer, Heather A. Parsons. Patient-reported outcomes, perceptions, and knowledge about recurrence in women with high-risk hormone receptor-positive (HR+) breast cancer (BC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-05-05.

Cell-free DNA (cfDNA) has the potential to inform tumor subtype classification and help guide clinical precision oncology. Here we developed Griffin, a new method for profiling nucleosome protection and accessibility from cfDNA to study the phenotype of tumors using as low as 0.1x coverage whole genome sequencing (WGS) data. Griffin employs a novel GC correction procedure tailored to variable cfDNA fragment sizes, which improves the prediction of chromatin accessibility. Griffin achieved excellent performance for detecting tumor cfDNA in early-stage cancer patients (AUC=0.96). Next, we applied Griffin for the first demonstration of estrogen receptor (ER) subtyping in metastatic breast cancer from cfDNA. We analyzed 254 samples from 139 patients and predicted ER subtype with high performance (AUC=0.89), leading to insights about tumor heterogeneity. In summary, Griffin is a framework for accurate clinical subtyping and can be generalizable to other cancer types for precision oncology applications.

Background. Patients (pts) with early triple negative breast cancer (eTNBC) are at increased risk of breast cancer recurrence and death. Recent studies have focused on escalation of therapy, with current treatment standard of at least five drugs – and associated toxicities - for eTNBC. Though presence of residual disease after neoadjuvant therapy (NAT) as measured by residual cancer burden (RCB) helps guide addition of adjuvant treatment, more effective tools to tailor therapy are limited. Persistence of circulating tumor DNA (ctDNA) in the setting of residual disease is associated with high risk of distant recurrence. However, more sensitive minimal residual disease (MRD) assays are needed to potentially guide optimization of systemic therapy. Methods. TBCRC 030 is a phase II randomized study of 12 weeks of NAT single agent cisplatin or paclitaxel for stage II-III TNBC, followed by surgery. The primary objective of the parent study was to correlate baseline biomarker for homologous recombination deficiency and RCB by study arm. From this group, responders (RCB 0/1) and non-responders (RCB 2/3) from both study arms who did not receive additional NAT prior to surgery were selected for analysis from the study cohort, matched on baseline nodal status and tumor size. As a post hoc study amendment, available pts were followed for event free survival (EFS). Plasma samples were collected prior to treatment initiation (W0), at three weeks (W3), and at twelve weeks, prior to surgery (W12). Whole genome sequencing (WGS) was performed on primary tumor tissue to identify somatic mutations and design for each pt a tumor-informed, ctDNA assay tracking up to 1000 mutations to detect MRD. Detection limit was computed for each tested sample as previously described. For each sample assayed, we report tumor fraction (TFx) when MRD was detected and the detection limit at 90% power when MRD was not detected. Results. Of 139 study pts, 68 had complete tissue and plasma samples and no receipt of additional NAT. Of these, 22 were responders. These responders, and 22 matched non-responders were identified for analysis. Data from 22 pts – 11 responders, 11 non-responders - are described here; full analysis on all 44 pts will be presented at the meeting. Personalized ctDNA assays were designed targeting 434 to 1000 variants (median 1000) and applied to 66 plasma samples. At W0, 100% (22/22) were positive for ctDNA; 73% (16/22) and 55% (12/22) were positive at W3, and W12, respectively. In pts with T1-T2 tumors median TFx was 4.1e-3(7.8e-6, 3.4e-2) and 4.7e-1(4.3e-2, 9.0e-1) in pts with T3-T4 tumors. TFx decreased from W0 to W3 and from W0 to W12 in responders (Table 1). By W12, ctDNA had cleared in 7/8 pts with RCB 0, 1/3 with RCB 1, 2/8 with RCB 2, and 0/3 with RCB 3. Overall, ctDNA levels were broad with median TFx of 1.5e-3 (range 2.9e-6 to 0.90). Detection limit at 90% power for all tested samples was a median of 8.8e-6 (range 9.9e-7 to 6.8e-3). To investigate whether ctDNA persistence after NAT was associated with BC recurrence, we analyzed a separate group of all 8 pts with known recurrence and with complete data and samples. All pts had persistent ctDNA at W12 (median TFx 6.8e-3, [2.9e-6 to 6.6e-2]). Conclusions. After 3 weeks of NAT for eTNBC, ctDNA TFx decreased, with a 3900-fold change in responders and 18-fold change in non-responders. By W3, TFx for most pts with RCB 0/1 were below the 1 in 10,000 limit of detection for many currently available assays, emphasizing the need for sensitive tests to potentially guide therapy. Additional studies will determine if ctDNA-guided approaches in eTNBC can improve pt outcomes. Table 1: Tumer Fraction and Tumer Fraction Fold Change by Response to Neoadjuvant Therapy Citation Format: Heather A. Parsons, Timothy Blewett, Xiangying Chu, Sainetra Sridhar, Katheryn Santos, Kan Xiong, Vandana Abramson, Ashka Patel, Ju Cheng, Adam M. Brufsky, Justin Rhoades, Jeremy Force, Ruolin Liu, Tiffany A. Traina, Lisa Carey, Mothaffar Rimawi, Ahmed Elkhanany, Vered Stearns, Jennifer M. Specht, Harold Burstein, Antonio C. Wolff, Eric Winer, Nabihah Tayob, Ian Krop, Todd Golub, Erica L. Mayer, Viktor Adalsteinsson. PD11-06 Circulating tumor DNA association with residual cancer burden after neoadjuvant therapy in triple negative breast cancer in TBCRC 030 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD11-06.

Background. Despite substantial progress in the treatment of HER2+ MBC, most patients (pts) still experience disease progression and cancer-related death. HER2-directed TKIs are highly effective therapies for pts with HER2+ MBC; however, an understanding of resistance mechanisms is needed. Pts receiving HER2-directed TKIs with cell-free DNA (cfDNA) sampling across the treatment spectrum present a unique opportunity to examine genomic alterations. Methods. Pts with biopsy-proven HER2+ MBC were selected from DF/HCC approved protocols for ultra-low pass whole genome sequencing if ≥1 cfDNA and/or tissue sample had been collected prior to and after at least six weeks of TKI treatment. Only pts with ≥ 1 sample with tumor fraction (TFx) ≥ 9.5% and ≥ 1 additional sample with TFx ≥ 4.5% were analyzed. Whole exome sequencing (WES) was performed on 19 tumor biopsies and 64 cfDNA samples from 25 pts. WES samples were analyzed, variants called and annotated, copy number profiles inferred, and TFx estimated. PhylogicNDT was used to detect the phylogenetic architecture, and clones were identified as growing, stable, shrinking, or truncal; mutational signatures were called using SignatureAnalyzer. After filtering likely benign variants, mutations were labeled likely resistance mechanisms if there was known evidence of resistance to anti-HER2 therapy in a human breast cancer cell line, breast cancer mouse models, or in vivo. Findings were correlated with pt clinicopathologic data. Results. In 4/8 pts with acquired resistance (TKI treatment ≥ 180 days), we identified alterations in previously characterized pathways and genes that could explain treatment resistance (e.g. PIK3CA, ERBB2, FGFR2). Mutations in growing subclones identified potential novel mechanisms of resistance and included inactivating mutations in CDK12, KMT2D, KMT2C, CHEK2, BRCA2, and FAT1 genes. In 7/17 pts with intrinsic resistance, we identified mutations in overlapping pathways and genes such as ERBB2 and PIK3CA, which were present in both growing subclonal and truncal clones. Four pts with hormone receptor positive disease - two of whom had not received prior aromatase inhibitors - had an activating ESR1 hotspot mutation (D538G). This raises the possibility that activating ESR1 mutations may be involved in resistance in these cases. Mutational signature analysis revealed a subset of samples with widespread APOBEC activation (with and without hyper mutation), subclonal HR/MMR-related signature, and capecitabine-related 5FU signature. Conclusions. Genomic analysis of paired samples from pts with HER2+ MBC identifies candidate resistance mechanisms to anti-HER2 TKIs and clonal evolution over time in the context of heterogeneity in treatment and sample timing. Additional studies will determine the functional role and clinical utility of assessing these alterations to overcome resistance. Citation Format: Heather A. Parsons, Conor Messer, Katheryn Santos, Brian P. Danysh, Melissa E. Hughes, Ashka Patel, Raquel A. Jacobs, Kara Slowik, Julian Hess, Chip Stewart, Kristy Schlueter-Kuck, Kahn Rhrissorrakrai, Filippo Utro, Chaya Levovitz, Nikhil Wagle, Jose Pablo Leone, Rachel Freedman, Laxmi Parida, Ian E. Krop, Gad Getz, Nancy U. Lin. Genomic mechanisms of resistance to tyrosine kinase inhibitors (TKIs) in HER2+ metastatic breast cancer (HER2+ MBC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3874.

Clonal hematopoiesis of indeterminate potential after (neo)adjuvant chemotherapy versus endocrine therapy for early breast cancer: the CIRCE-eBC prospective cohort study Background: Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related condition associated with higher risk of hematologic malignancies, cardiovascular disease, and all-cause mortality. Patients (pts) with early breast cancer (eBC) receiving chemotherapy (CT) have increased risk of treatment-related myeloid neoplasms (tMN); the benefit of CT in eBC pts is often limited. Little is known about the prevalence and dynamics of CHIP in eBC pts. Methods: We prospectively identified two cohorts of pts with eBC: cohort A – pts receiving (neo)adjuvant CT with or without adjuvant endocrine therapy (ET); cohort B – pts receiving ET only. Blood was collected prior to initiation of treatment (T1) and after either (neo)adjuvant CT or 6-18 months of adjuvant ET (T2). We performed targeted sequencing of cryopreserved peripheral blood mononuclear cell (PBMC)-derived genomic DNA and defined CHIP as the presence of ≥1 pathogenic somatic mutation at variant allele fraction (VAF) ≥0.02. In additional analyses we used VAF≥0.005. Results: We enrolled 118 and 116 pts in cohorts A and B, respectively. Pts in cohort A were younger (median age 51 vs 57, p< 0.001), less frequently Caucasian (83.9 vs 96.6%; p=0.005) and former/current smokers (28.0 vs 43.1%, p=0.038). All pts in cohort B had hormone receptor-positive (HR+) eBC; in cohort A 50% of pts had HR+/HER2-, 16% had HR+/HER2+, 8.5% had HR-/HER2+ and 25.4% had HR-/HER2- eBC (p< 0.001). Pts in cohort A had higher stage (stage II-III 68.7 vs 27.6%; p< 0.001) and grade (grade 3 65.3 vs 15.5%; p< 0.001) tumors. Genetic testing was more frequently performed in pts receiving CT (88.1 vs 68.1%, p< 0.001), though the rate of germline pathogenic variants was similar (13.5 vs 17.7%, p=0.079). In cohort A, 38% received anthracyclines, 7% platinum and 57% anthracycline/platinum-sparing CT. Median time between T1 and T2 was 189.5 (150,406) and 280 (147, 425) days in cohort A and B (p< 0.001). The prevalence of CHIP, defined by mutations at VAF ≥ 0.02, was similar at T1 in cohort A (14.4%) vs B (18.1%) (p=0.556). Number of pts with new CHIP variants at T2 was also similar (A 3.4% vs B 6.0%) (p=0.373). After adjusting for age and stage, odds ratio (OR) of developing new CHIP variants in cohort B vs A was 1.28 (95% CI 0.32 – 5.68, p=0.733). Age correlated with baseline prevalence of CHIP (p< 0.001). Most frequent new CHIP variants at T2 in cohort A were DNMT3A (3), PPM1D (1), NF1 (1). To investigate whether pts receiving CT were more likely to have emergence of small hematopoietic clones, we assessed pathogenic variants present at VAF ≥0.005. These were detected at T1 in 55 (46.6%) and 61 (52.6%) pts in cohort A and B, respectively. Few pts without pathogenic variants at T1 developed them at T2 (3 pts in cohort A and 4 in cohort B). 21 pts (27 variants) in cohort A and 11 pts (12 variants) in cohort B had new variants at T2. Pts with new variants vs not (32 vs 202 pts) had similar characteristics, excepting age (median 60.5 vs 54.0, p=0.011). After correcting for age and stage, OR of developing new pathogenic variants given ET vs CT was 0.25 (95% CI 0.10-0.62, p=0.003). Most frequent newly detected variants were in DNMT3A (14), PPM1D (5), TET2 (4) and TP53 (2) in cohort A; DNMT3A (3), TET2 (3) and ZNF318 (2) in cohort B. Conclusions: In the CIRCE-eBC study, CT administration did not lead to emergence of CHIP over a 6-9 month period vs ET alone. This finding is reassuring in the setting of long life-expectancy for eBC pts and the association of CHIP with significant morbidity and mortality. However, consistent with known risk of development of MN, CT was associated with emergence of low frequency pathogenic variants in PPM1D and TP53, which have been associated with elevated risk of tMN. The evolution and prognostic role of these small clones is unclear and warrants additional investigation. Citation Format: Stefania Morganti, Qingchun Jin, Katheryn Santos, Christopher Gibson, Ashka Patel, Alex Wilson, Margaret Merrill, Julie Vincuilla, Samantha Stokes, Marla Lipsyc-Sharf, Tonia Parker, Tari King, Elizabeth A. Mittendorf, Giuseppe Curigliano, Melissa E. Hughes, Nabihah Tayob, Nancy U. Lin, Peter Miller, Sara Tolaney, Judy Garber, Heather A. Parsons. Clonal hematopoiesis of indeterminate potential after (neo)adjuvant chemotherapy versus endocrine therapy for early breast cancer: the CIRCE-eBC prospective cohort study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-14-03.

Purpose: To examine circulating tumor DNA (ctDNA) and its association with residual cancer burden (RCB) using an ultrasensitive assay in patients with triple-negative breast cancer (TNBC) receiving neoadjuvant chemotherapy (NAT). Patients and Methods: We identified responders (RCB-0/1) and matched non-responders (RCB-2/3) from the phase II TBCRC 030 prospective study of neoadjuvant paclitaxel vs. cisplatin in TNBC. We collected plasma samples at baseline, three weeks, and twelve weeks (end of therapy). We created personalized ctDNA assays utilizing MAESTRO mutation enrichment sequencing. We explored associations between ctDNA and RCB status and disease recurrence. Results: Of 139 patients, 68 had complete samples and no additional NAT. Twenty-two were responders and 19 of those had sufficient tissue for whole-genome sequencing. We identified an additional 19 non-responders for a matched case-control analysis of 38 patients using a MAESTRO ctDNA assay tracking 319-1000 variants (median 1000) to 114 plasma samples from 3 timepoints. Overall, ctDNA positivity was 100% at baseline, 79% at week 3, and 55% at week 12. Median tumor fraction (TFx) was 3.7 x 10-4 (range: 7.9 x 10-7 to 4.9 x 10-1). TFx decreased 285-fold from baseline to week 3 in responders and 24-fold in non-responders. Week 12 ctDNA clearance correlated with RCB: clearance was observed in 10/11 patients with RCB-0, 3/8 with RCB-1, 4/15 with RCB-2, and 0/4 with RCB-3. Among 6 patients with known recurrence five had persistent ctDNA at week 12. Conclusion: NAT for TNBC reduced ctDNA TFx by 285-fold in responders and 24-fold in non-responders. In 58% (22/38) of patients, ctDNA TFx dropped below the detection level of a commercially available test, emphasizing the need for sensitive tests. Additional studies will determine if ctDNA-guided approaches can improve outcomes.

PURPOSE To examine the prevalence and dynamics of circulating tumor DNA (ctDNA) and its association with metastatic recurrence in patients with high-risk early-stage hormone receptor–positive breast cancer (HR+ BC) more than 5 years from diagnosis. METHODS We enrolled 103 patients with high-risk stage II-III HR+ BC diagnosed more than 5 years prior without clinical evidence of recurrence. We performed whole-exome sequencing (WES) on primary tumor tissue to identify somatic mutations tracked via a personalized, tumor-informed ctDNA test to detect minimal residual disease (MRD). We collected plasma at the time of consent and at routine visits every 6-12 months. Patients were followed for clinical recurrence. RESULTS In total, 85 of 103 patients had sufficient tumor tissue; of them, 83 of 85 (97.6%) patients had successful whole-exome sequencing. Personalized ctDNA assays were designed targeting a median of 36 variants to test 219 plasma samples. The median time from diagnosis to first sample was 8.4 years. The median follow-up was 10.4 years from diagnosis and 2.0 years from first sample. The median number of plasma samples per patient was two. Eight patients (10%) had positive MRD testing at any time point. Six patients (7.2%) developed distant metastatic recurrence, all of whom were MRD-positive before overt clinical recurrence, with median ctDNA lead time of 12.4 months. MRD was not identified in one patient (1.2%) with local recurrence. Two of eight MRD-positive patients had not had clinical recurrence at last follow-up. CONCLUSION In this prospective study, in patients with high-risk HR+ BC in the late adjuvant setting, ctDNA was identified a median of 1 year before all cases of distant metastasis. Future studies will determine if ctDNA-guided intervention in patients with HR+ BC can alter clinical outcomes.

103 Background: Hormone receptor–positive breast cancer (HR+ BC) is the most common cause of BC-related death. Over half of metastatic recurrences occur ≥ 5 years (y) from diagnosis. Detection of minimal residual disease (MRD) via circulating tumor DNA (ctDNA) can identify cancer recurrence months to years in advance and may be an important tool to guide therapy. Little is known about ctDNA in the late adjuvant setting. We investigated ctDNA dynamics and clinical outcomes in pts ≥ 5 y from diagnosis of high-risk early-stage HR+ BC. Methods: Patients with high-risk HR+ BC (T3-4 or N2-3 or T1 with 3+ lymph nodes, or T2N1 [and Oncotype RS ≥ 26; grade 3; or Ki-67 ≥ 20%]) with no evidence of recurrence 5 y after diagnosis were prospectively identified and consented. Plasma samples were collected at time of consent and at routine visits every 6-12 mos. Whole-exome sequencing (WES) was performed on primary tumor tissue to identify somatic mutations and design for each patient a RaDaR assay, a tumor-informed liquid biopsy test to detect plasma ctDNA. Per current practice standards, pts did not undergo regular surveillance imaging. All pts were followed for development of local and/or distant metastatic recurrence, as determined by their clinical provider. Results: Of 103 pts enrolled, 85 had sufficient tumor tissue, and 83 pts had successful WES. Personalized RaDaR assays were designed targeting 12-51 variants (median, 36), and used to test 219 plasma samples from 83 pts. The number of plasma samples per patient ranged from 1-7 (median, 2). 57 pts (68.7%) had stage 3 disease, and most (75, 90.4%) received curative-intent chemotherapy. All pts received endocrine therapy (ET). 39 (47%) remained on adjuvant ET at time of last follow up. Of 44 pts who completed adjuvant ET, 41 (93.2%) received > 5 y of treatment. Time from diagnosis to first sample ranged from 4.9-20 y (median, 8.4 y). Median (range) follow up was 10.2 (6.7-22.3) y from diagnosis and 1.8 (0-3.6) y from first sample. 5 pts (6%) developed distant metastatic recurrence and 2 pts (2.4%) had locoregional recurrence. 4/83 (5%) pts were MRD+ at study entry and 8/83 (10%) pts were MRD+ at any time point. 5/5 (100%) pts with metastatic recurrence were MRD+, with ctDNA lead times up to 37.6 mos. ctDNA was detected at tumor fractions of 0.0027-26.84% (median, 0.396%). 2/8 (25%) MRD+ pts had not had clinical recurrence at latest follow up, one with no follow up since detection and one 15.4 mos from ctDNA detection (with ctDNA levels 0.045% and 26.84%). Conclusions: Here we report—to our knowledge—the first data on ctDNA detection in late adjuvant HR+ BC. 10% of pts had MRD at ≥ 5 y from diagnosis. ctDNA analysis identified MRD in all cases of distant recurrence. ctDNA was detected in 2 pts who have not yet experienced recurrence. Longer follow up is necessary for these pts at high risk. Additional studies will determine if ctDNA-guided intervention can alter clinical outcomes.

Background. Patients (pts) with metastatic triple negative breast cancer (mTNBC) receive serial cytotoxic chemotherapy regimens, often with cumulative myelosuppressive effects, impairing treatment tolerance. Clonal hematopoiesis of indeterminate potential (CHIP) refers to the detection of somatic mutations in genes recurrently mutated in hematologic malignancies in the blood of adults with no evident hematologic abnormalities. Little is known about the natural history of CHIP after breast cancer treatment. We sought to characterize CHIP in pts undergoing treatment for mTNBC. Methods. In this retrospective cohort study we identified 149 pts with biopsy-proven mTNBC at a single tertiary care institution with at least one blood sample collected within six months of metastatic diagnosis. We performed targeted sequencing of cryopreserved peripheral blood mononuclear cell (PBMC)-derived genomic DNA and defined CHIP as the presence of at least one pathogenic somatic mutation present at variant allelic fraction (VAF) of 0.02-0.35. We assessed the relationship between CHIP status and overall survival (OS), demographics, clinicopathologic features, germline mutation status, and type and timing of therapy. Results. We identified 27 unique CHIP variants across 22/149 pts (15%) within six months of metastatic diagnosis. Frequency of mutated genes were as follows: DNMT3A (n=15), PPM1D (n=4), TP53 (n=3), TET2 (n=2), SRCAP (n=1), ZBTB33 (n=1), ZNF318 (n=1). Median follow-up in the cohort was 37.9 months (IQR: 23.9-Not reached). The median age at time of blood draw was 55 years (IQR: 8.5) for pts with CHIP vs. 51 years (IQR: 16.5) for pts without CHIP. Ten (45%) pts with CHIP and 47 (37%) pts without CHIP were current or former smokers. Two (9%) pts with CHIP and 10 (7.9%) pts without CHIP were known germline mutation carriers of BRCA1, BRCA2 or PALB2. Twenty-two (100%) pts with CHIP and 124 (98%) pts without CHIP had received systemic chemotherapy for mTNBC prior to blood draw. There were no significant differences in type of chemotherapy regimen received between patients with or without CHIP. Twenty (90.9%) pts with CHIP vs. 96 (75.6%) of pts without CHIP had received radiation therapy prior to blood draw. Pts with CHIP had similar OS to those without CHIP (median OS 7.75 [2.20-31.7] vs. 9.33 [8.02-11.73] months). No pts developed therapy-related myeloid neoplasms (t-MN) or died of complications of cardiac disease. Conclusions. Pts with mTNBC had a higher frequency of CHIP than previously reported in age-matched healthy populations, but similar CHIP prevalence to what has been seen in cohorts of pts with solid tumors. Our study assessed for the presence of CHIP at only a single time point early in the metastatic course, but serial blood sampling later in treatment might reveal additional cases of CHIP. Though this cohort of patients with life-limiting mTNBC was small, presence of CHIP in the first six months of metastatic diagnosis was not associated with worse survival. Citation Format: Katheryn Santos, Qingchun Jin, Peter G. Miller, Ashka Patel, Gregory J. Kirkner, Janet L. Files, Melissa E. Hughes, Samantha M. Stokes, Nabihah Tayob, Daniel G. Stover, Christopher J. Gibson, Eric P. Winer, Nancy U. Lin, Judy E. Garber, Heather A. Parsons. Clonal hematopoiesis of indeterminate potential (CHIP) in metastatic triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-08-01.