Background: ctDNA dynamics are associated with treatment response, and ctDNA detection following treatment is associated with disease recurrence in early breast cancer (EBC). Highly sensitive assays may permit effective risk stratification and guide interventional strategies. RaDaR, a bespoke assay using deep sequencing of tumour-specific variants, has been shown to detect recurrence in the postoperative setting. We quantified ctDNA using RaDaR in serial samples from a large prospective cohort of EBC patients who received standard neoadjuvant chemo- (+/- HER2- targeted) therapy. Methods: Patients with EBC of all receptor subtypes receiving neoadjuvant therapy were enrolled in the TRACER cohort from 2015. Plasma samples (Streck) were collected at baseline, during treatment, perioperatively, and during follow-up. RaDaR was performed on all available timepoints for patients with tissue available for exome sequencing (assay requirement). Clinical and pathologic characteristics, treatment, and recurrence outcomes were collected. Results: 145 patients were recruited as of April 2021, and over 700 plasma samples were collected through December 2021 (patient characteristics, Table 1). 115 (79%) tissue samples were retrieved for assay design. Data are presented for the initial 43 patients and 265 samples analyzed, including 82 post-surgical time points. Median time since diagnosis was 3.5 years (range, 1.5-5.0). Exome sequencing and assay generation were successful in all patients (n=43), yielding assays targeting a median of 48 (range 22-50) variants. 88% (38/43 patients) had ctDNA detected at baseline, with median variant allele frequency (eVAF) in positive patients of 0.15% (range, 0.0019-4.9%). ctDNA levels fell rapidly with treatment: 19/37 (51.3%; median eVAF in positive patients: 0.0098%, range 0.001-0.156%) had ctDNA detected prior to cycle 2, and 4/32 (12.5%) had ctDNA detected at cycle 4 or 5 (mid treatment; median eVAF in positive patients: 0.001%, range, 0.0007-0.011%). In the perioperative period, 17/18 (94%) of patients with available pre-operative specimens and 27/28 (96%) of patients with available post-operative samples had clearance of ctDNA. In adjuvant follow up, ctDNA was detected in 4 of the 43 analyzed patients, with ctDNA clearance observed following planned switch of endocrine therapy in one. Clinical follow-up continues, and analysis of the remaining samples (72 patients and over 425 samples) is underway. Conclusion: RaDaR demonstrated high sensitivity for ctDNA prior to treatment in patients receiving neoadjuvant therapy for EBC, permitted dynamic monitoring of treatment effect, and identified patients with persistent ctDNA after curative-intent therapy, as well as ctDNA emergence prior to clinical recurrence. Full results for the TRACER cohort and analysis of clinical covariates will be presented at the meeting. ClinicalTrials.gov NCT03702309. Table 1. Patient Baseline Characteristics Citation Format: Mitchell J. Elliott, Zachary Veitch, Philippe Bedard, Eitan Amir, Aaron Dou, Jesus Fuentes Antras, Michelle Nadler, Nicholas Meti, Nancy Gregorio, Elizabeth Shah, Helen Chow, Nathan Campbell, Samantha Terrell, Charlene Knape, Karen Howarth, Lillian Siu, Hal Berman, David W. Cescon. Circulating Tumour DNA (ctDNA) Detection and Dynamics in Patients with Early Breast Cancer (EBC): Results of the Neoadjuvant TRACER cohort [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 P6-01-16.

Background: Detection of circulating tumour DNA (ctDNA) in patients (pts) who have completed treatment for early-stage breast cancer is associated with a high risk of future relapse. Identifying those at high risk of subsequent relapse may allow tailoring of further therapy to delay or prevent recurrence. Previous analysis of this cohort showed that tools capable of detecting ctDNA at lower concentrations are needed to increase sensitivity and lengthen the lead time between ctDNA detection and relapse. We compared ctDNA detection via a personalised sequencing assay to dPCR in patients from the cTRAK TN clinical trial. Methods: The cTRAK-TN trial recruited 161 pts into prospective ctDNA surveillance with dPCR, with ctDNA positive pts randomised to 1) CT staging plus pembrolizumab therapy for patients without relapse or 2) observation. Pts had serial post-treatment surveillance plasma samples collected every 3 months for up to 2 years. Whole exome sequencing (WES) was performed on tumor DNA from FFPE samples to design personalised Residual Disease and Recurrence (RaDaR®) multiplex PCR based NGS assays. Retrospectively, plasma DNA extracted from a minimum of 2mls banked plasma, was sequenced with personalised RaDaR assays, and ctDNA detection identified with a proprietary algorithm. dPCR assays tracked 1-2 mutations, as previously described. Primary endpoint was rate of positive ctDNA detection by 12 months from start of surveillance in both assays. Secondary endpoints were agreement in ctDNA detection between RaDaR and dPCR assays and lead-time between ctDNA detection and disease recurrence. Results: Overall, 147 pts and 241 tissue samples were subject to WES, and RaDaR assays were developed for 142 pts with sufficient plasma for testing. RaDaR assays tracked a median of 47 variants (range 33-56) per patient, and a total of 907 timepoints were analysed (median 6 timepoints per pt, range 1-11). With RaDaR, 39.4% (56/142) patients tested ctDNA positive during follow-up, with a median ctDNA detected level of 0.081% estimated variant allele fraction (eVAF). With dPCR, 35.2% (50/142) pts tested ctDNA positive. The ctDNA detection rate by 12 months from the start of ctDNA surveillance was 36.2% (95% CI; 27.6% – 43.7%) with RaDaR and 29.9% (95%CI; 21.6% – 37.3%) with dPCR. The overall test agreement between RaDaR and dPCR assays was 92.7% (95%CI; 90.7% – 94.4%). From a patient perspective, 58.7% pts were ctDNA negative for both assays, 32.9% ctDNA were positive for both assays and 8.6% presented discrepancies. ctDNA was detected by RaDaR but not by dPCR in 9 pts and it was detected by dPCR but not by RaDaR in 3 pts. Among ctDNA positive pts, 55.2% were first detected positive by RaDaR, 5.2% by dPCR, and 39.6% were detected at the same time-point (test of proportions, p< 0.001). The median lead time from ctDNA detection to relapse was 7.1 months (95% CI 5.9 – 15.9%) with RaDaR and 5.7 months (95% CI 3.2% – 7.4%) with dPCR. Conclusion: The RaDaR personalised multi-mutation sequencing assay detected MRD with a longer median lead time prior to relapse, and with higher sensitivity, than dPCR mutation tracking assays. These findings have implications for the choice of ctDNA assay in clinical trials designed to treat patients at the point of MRD detection. Citation Format: Maria Coakley, Prithika Sritharan, Guillermo Villacampa, Claire Swift, Kathryn Dunne, Lucy Kilburn, Katie Goddard, Patricia Rojas, Andy Joad, Warren Emmett, Charlene Knape, Karen Howarth, Peter S. Hall, Catherine Harper-Wynne, Tamas Hickish, Iain Macpherson, Alicia F. Okines, Andrew M. Wardley, Duncan Wheatley, Simon Waters, Rosalind Cutts, Isaac Garcia-Murillas, Judith Bliss, Nicholas Turner. PD5-03 Comparison of a personalized sequencing assay and digital PCR for circulating tumor DNA based Molecular Residual Disease detection in early-stage triple negative breast cancer in the cTRAK-TN trial [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 PD5-03.

BACKGROUNDDetection of minimal residual disease (MRD) using circulating tumor DNA (ctDNA) represents an attractive alternative to imaging, currently considered the gold standard in routine surveillance of early breast cancer (BrCa) following primary therapy. ctDNA has the potential to identify patients who may eventually develop distant metastatic disease and, as such, its implementation in the routine clinical follow-up setting may offer the means for earlier intervention for patients with oligometastatic disease and improved overall survival. However, due to the highly heterogeneous nature of the genomic alterations in BrCa, ultra-sensitive ctDNA assays are required for follow-up surveillance. Here we evaluate RaDaR™, a personalised liquid biopsy-based sequencing assay for the detection of residual disease and monitoring after standard treatment in early-stage BrCa. METHODS38 early-stage BrCa patients recruited through the BRandO BiO registry study were included (18% TNBC, 74% HR+/HER2-, 8% HER2+). 21 patients experienced clinical recurrence (13 distant and 8 local), with a median time to progression of 18.9 months. The remaining 17 case-control patients had no disease recurrence at the time of 3-year follow-up. Whole exome sequencing (WES) was performed on formalin-fixed, paraffin-embedded (FFPE) tumor tissue from curative-intent surgery and selected variants were used to design personalised RaDaR panels (38-54 variants/panel: median 49). In total, 52 plasma samples were analyzed using RaDaR. This included samples taken at the time of recurrence and at 12-months post-diagnosis where available (33 samples from 21 patients), or in the case of no recurrence, samples taken at 3-year follow up, including one control case that had two additional samples analyzed at 12-months and 4-years of follow up (19 samples from 17 patients). RESULTSIn total, ctDNA was detected in 12/13 (92%) patients with distant recurrence and 3/8 (38%) patients with local recurrence at an estimated median variant allele frequency (VAF) of 0.827% (range: 0.0029% - 38%). The lowest levels were seen in the 3 patients with local recurrence (0.0029%, 0.0146% and 0.0248% VAF). Of the 6 patients negative for ctDNA, 5 had local and one distant recurrence of unusual histology, indicating a possible alternative origin or second primary tumor. Only one of the 17 control cases was positive for ctDNA (0.0085% VAF), from a patient with a Luminal A, stage I tumor, potentially indicating the presence of early molecular recurrence that precedes clinical progression. Two additional time points from this patient also showed positive ctDNA results, which could be indicative of residual disease remaining dormant. Of the 12 patients with disease recurrence for which an earlier plasma sample was available, 4 patients (3 with distant and one with local recurrence) had ctDNA detected at the earlier time point, a median of 92 days (range 42 – 308 days) prior to clinical recurrence. 3 patients had ctDNA detected only at the time of recurrence (one distant and 2 local). None of the 5 patients with ctDNA negative results at the time of recurrence had detectable ctDNA levels at the earlier timepoint. CONCLUSIONIn this real-world pilot study, the RaDaR assay detected the presence of ctDNA in plasma to levels as low as 0.0029% VAF. We found that ctDNA detection was strongly associated with distant recurrence in early-stage BrCa, with a sensitivity of 92% (12 of 13 cases detected). In a limited number of cases where samples were available prior to recurrence, ctDNA could be detected ahead of clinical progression, potentially offering the opportunity for earlier intervention. Citation Format: Wolfgang Janni, Jens Huober, Sophia Huesmann, Christodoulos Pipinikas, Tatjana Braun, Volkmar Müller, Giovanni Marsico, Angelina Fink, Paula Freire-Pritchett, Karin Koretz, Charlene Knape, Amelie deGregorio, Brigitte Rack, Thomas WP Friedl, Lisa Wiesmueller, Peter Möller, Karen Howarth, Klaus Pantel, Nitzan Rosenfeld. Detection of early-stage breast cancer recurrence using a personalised liquid biopsy-based sequencing approach [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-07.

IntroductionCirculating tumor DNA (ctDNA) testing may identify patients at high risk for recurrence following chemoradiation (CRT) for locally advanced non-small cell lung cancer (LA-NSCLC). We evaluated the feasibility of ctDNA testing on a readily available commercial fixed-gene panel to predict outcomes in patients with LA-NSCLC.MethodsPlasma of 43 patients was collected at CRT initiation (pre-CRT), completion (post-CRT1), quarterly follow up for 12 months (post-CRT2, 3, 4, 5 respectively) after CRT, and at disease progression. ctDNA analysis was performed using InVisionFirst®-Lung to detect mutations in 36 cancer-related genes. ctDNA clearance was defined as absence of pre-CRT variants at post-CRT1. Patients without detectable pre-CRT variants or no post-CRT1 samples were excluded.ResultsTwenty eight of 43 patients (65%) had detectable variants pre-CRT. Nineteen of 43 patients (44%) had detectable pre-CRT variants and post-CRT1 samples and were included in analysis. Median age at diagnosis was 65 years (43-82), and most patients had stage IIIB disease (10/19, 53%). Two patients died from non-cancer related causes before post-CRT2 and were excluded from further analysis. All three patients who did not clear ctDNA had tumor relapse with a median time to relapse of 74 days (30-238), while 50% (7/14) of those who cleared ctDNA have remained disease free. Progression free survival was longer in patients who cleared ctDNA compared to those who did not (median 567 vs 74 d, p = 0.01).ConclusionsAlthough it is feasible to use ctDNA testing on a limited gene panel to identify patients with LA-NSCLC who are at high risk for disease recurrence following CRT, further studies will be necessary to optimize these assays before they can be used to inform clinical care in patients with lung cancer.

6052 Background: Immuno-oncology agents (IO) have become standard-of-care in the treatment of R/M HNSCC, but only a subset of patients (pts) benefit. Highly sensitive quantification of plasma circulating tumor DNA (ctDNA) may permit real time assessment of disease under selective pressures of treatment. Methods: R/M HNSCC pts treated with platinum-based chemotherapy (CT) or IO (anti-PD1/L1 +/- second IO) underwent serial ctDNA collection pre-cycles 1/2/3 and at disease progression, corresponding to timepoints (T) 1-4. T1 was considered baseline. Whole exome sequencing of pt tumor tissue identified patient specific somatic variants which were used as targets for RaDaR, a personalized multiplexed PCR-based NGS assay. Matched buffy coat DNA was sequenced to filter germline mutations and identify confounding CHIP. RaDaR was applied at each available T, an estimated variant allele frequency (eVAF) was calculated and correlated with progression free- (PFS) and overall- survival (OS). Findings were compared against prior (ESMO 2021) data generated using a fixed 580 gene CAPP-seq (CAncer Personalized Profiling by deep Sequencing) panel designed specifically against squamous cell carcinoma. Results: A total 114 plasma samples from 38 pts were analyzed. Of 35 pts with ctDNA detected at T1 and/or T2, 26 received IO and 9 CT. Median age was 62 (20-84), 77% were male, 69% prior smokers and 26% HPV positive. Median PFS and OS, for all 35 pts, was 2.57 mo (95% CI 0.48-4.66) and 8.37 mo (95% CI 5.42-11.32) respectively. For IO treated pts, median PFS was 2.45 mo (95% CI 0-5.18) and median OS 7.38 mo (95% CI 3.84-10.93). RaDaR panels targeted a median 48 variants (17-50). ctDNA was detected in 35/38 (92%) patients at baseline, with median eVAF 0.345% (range 0.0004% - 43.37%). ctDNA abundance at baseline did not correlate with PFS or OS. A decrease in Δ eVAF from T1 to T2, by > 30%, or > 50% identified pts with improved PFS, with HR 0.45 (0.21, 0.96) p = 0.04, 0.31 (0.14, 0.70) p < 0.01, and 0.23 (0.10, 0.56) p < 0.01, respectively. Similar results were observed for the 26 IO pts, with HR 0.40 (0.16, 1.03) p = 0.06, 0.19 (0.05, 0.66) p < 0.01, and 0.06 (0.01, 0.47) p < 0.01, respectively. A similar, but non-significant, trend was seen in median OS for pts with a decrease vs. increase in Δ eVAF (T1 to 2), 8.8 mo vs 7.3 mo (HR = 0.87 (0.42, 1.79)). For 31 pts, a comparison of Δ ctDNA levels, based on personalized RaDaR vs. CAPP-seq assays, from T1 to T2 demonstrated a correlation coefficient of R = 0.57, P < 0.01. Conclusions: In pts with R/M HNSCC, a decrease in ctDNA eVAF after first treatment correlated with improved PFS. There was a significant correlation between fixed CAPP-seq and personalized RaDaR assays when comparing Δ in ctDNA levels. Clinical Trial: NCT03712566.

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.

Abstract Introduction: Detection of circulating tumor DNA (ctDNA) presents a strategy to identify Molecular Residual Disease (MRD) in patients with breast cancer. Tools capable of detecting ctDNA at lower concentrations are needed to increase sensitivity and lengthen lead time between ctDNA detection and relapse. We present results from a highly sensitive personalized sequencing approach for ctDNA detection of MRD based on multiple patient specific mutations. Methods: 22 early breast cancer patients (12 hormone receptor positive HER2 negative (HR+HER2-), 7 HER2+ and 3 triple negative breast cancer (TNBC)) enrolled in the ChemoNEAR sample collection study were included. Tumor DNA from FFPE samples was Whole Exome Sequenced to identify patient specific mutations and design personalized Residual Disease and Recurrence (RaDaRTM) multiplex PCR assays. Cell free DNA was extracted from 147 plasma samples (median volume 4ml, range 0.5-5ml) and sequenced with RaDaR assays, with 10-61 variants (median 41) per panel, to 100,000x per locus. A matched single timepoint buffy coat was sequenced to identify confounding CHIP mutations. A proprietary algorithm was used to identify ctDNA. Tumor Sequencing of multiple biopsy timepoints was carried out for 14 patients (mean 2.8 samples per patient) and clonal populations estimated with Pyclone. For clusters of greater than 10 mutations, RaDaR panels were supplemented with additional variants for clonal tracking. Results: At a median follow-up of 24.6 months post-surgery, MRD was identified in 100% (17/17) of relapsed patients, and in none of the 54 time points in the 5 patients that did not relapse (p=0.0002, Log rank test). Detection of ctDNA levels ranged from 7.4 parts per million (ppm), equivalent to Allele Frequency (AF) of 0.0007%, to 13,195ppm (1.3%) (median 625ppm and 0.06% AF). Median lead-time from ctDNA detection to clinical relapse in patients with extracranial disease relapse was 12.89 months (range 3.72-26.04). In three patients with brain only relapse, ctDNA was detected prior to relapse in all patients (3/3, 100%) albeit with a reduced lead time over clinical relapse (3.85, 4.21 and 5.65 months), which was not previously achievable with single mutation dPCR MRD-detection assays. In 8/14 patients with multiple tumor samples sequenced, multiple clones (mean 3.4 clones/patient) were identified, with heterogenous polyclonal relapse in 4/8 patients, and a single clone detectable in 4/8 patients. Conclusions: In a retrospective, multi-center, proof-of-principle study of early stage breast cancer patients with personalized sequencing assays, ctDNA-detected MRD associates with relapse free survival and long lead time over clinical relapse. Sequencing based ctDNA testing can detect patients with brain-only relapses, with increased sensitivity over first generation dPCR-based ctDNA assays. Citation Format: Rosalind J. Cutts, Maria Coakley, Isaac Garcia-Murillas, Lara Ulrich, Karen Howarth, Warren Emmett, Malcolm Perry, Pete Ellis, Charlene Knape, Stephen R. Johnston, Alistair Ring, Simon Russell, Abigail Evans, Anthony Skene, Duncan Wheatley, Mitch Dowsett, Ian E. Smith, Nicholas C. Turner. Molecular residual disease detection in early stage breast cancer with a personalized sequencing approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 536.

Currently, routine surveillance after primary therapy of early Breast cancer (BrCa) is limited to breast imaging. Follow up surveillance using circulating tumour DNA (ctDNA) for the detection of molecular residual disease is a useful tool to identify patients who may eventually develop distant metastases and holds promise for earlier intervention and improved overall survival. However, genomic alternations in breast cancer are very heterogeneous and follow-up surveillance requires ultrasensitive ctDNA assays.