570 Background: Detection of molecular residual disease (MRD), using circulating tumor DNA (ctDNA), following treatment for early-stage TNBC is associated with a high risk of recurrence. ctDNA clearance in response to NAC has shown potential for predicting pathologic compete response (pCR) and improving the prognostic ability of pCR status. PARTNER is a prospective, phase II-III, randomized controlled clinical trial, which recruited early-stage basal TNBC BRCA1/2 wild-type patients (Nature April 2024). Control regimen was neoadjuvant carboplatin–paclitaxel followed by anthracycline-based NAC. Experimental arms added olaparib to the platinum-taxane backbone. A sub-study of serial blood samples for ctDNA analysis during NAC and post-op were analyzed with 2 tumor informed MRD assays using primary tumor and germline sequencing: 1) whole-exome sequencing (WES) to select ≤200 variants and 2) whole-genome sequencing (WGS) to select 400-5000 variants for the two bespoke MRD assays, respectively. Both assays were independently used to assess available plasma samples collected for the presence or absence of ctDNA. Methods: This prospective sub study included TNBC patients enrolled within PARTNER with serial blood collections at baseline (prior to NAC), mid-NAC, post-NAC, 2-4 weeks post-op, 3 months post-op and 12 months post-op. Germline and somatic DNA were provided via the Personalised Breast Cancer Program. The primary objective was to determine the association of ctDNA positivity post-op with distant recurrence-free interval (DRFI). The distribution of DRFI by ctDNA status was compared using the log-rank test. The Cox proportional hazards regression model was used to estimate the strength of the relationship between ctDNA positivity and DRFI. Results: Median WES MRD assay panel size was 159 variants (range 47 – 200). At baseline ctDNA was detected in 50 of 55 patients (91%). After NAC, 4 of 63 patients had detectable ctDNA. 24 of 28 non-pCR patients were ctDNA negative and 0 of 35 pCR patients were ctDNA positive. Of 61 post-op patients available to assess DRFI distant recurrences developed in 8 patients (13.1%) within a median follow up of 5 years. Post-op 5 patients were ctDNA positive and 3 developed distant recurrences (log-rank p < 0.0001, HR = 20.2, 95% CI = 4.3, 95.1). In addition, 56 were ctDNA negative and 51 (91%) were distant recurrence free. Median WGS MRD assay panel size was 2962 variants (range 552 - 5000). At baseline ctDNA was detected in 46 of 47 patients (98%). Additional WGS MRD results are being generated and will be presented at the meeting. Conclusions: Post-op detection of ctDNA using a WES MRD assay was highly prognostic for distant recurrence in TNBC patients following NAC. WGS MRD improved baseline detection.

568 Background: Detection of circulating tumor DNA (ctDNA) is strongly associated with recurrence across a variety of cancers. The use of ctDNA testing to measure molecular residual disease (MRD) is still in its infancy, and understanding sample characteristics may help set expectations for oncologists and their patients. Methods: For the first thousand patients with MRD testing in Exact Sciences’ commercial laboratory, the tumor and first plasma sample were used to record tumor type, tumor sample type (biopsy/surgical), tumor stage, MRD assay panel size (up to 200 variants), cfDNA levels, and ctDNA status (qualitative and quantitative), and, when data were available, presence of targetable alterations (TAs) were recorded. Results: A total of 1088 samples with valid results were included. Patients were mostly female (N=703, 64.6%) with a median age of 64 (range 17–97) years, and most had either breast (N=380, 34.9%) or colorectal (N=335, 30.8%) cancer. Most (N=632, 58.1%) tumor samples were surgical, and average number of variants for MRD assays from 86 in ovarian cancer to 189 in melanoma. Plasma cfDNA yield ranged from 15 ng (the minimum required) to 10,709 ng and was significantly higher in 412 ctDNA+ (median 86.1 ng, mean 239.7 ng) compared to ctDNA– (median 73.5 ng, mean 114.6 ng) samples (p<0.001). In 13 cancers with at least 10 patient samples, only breast cancer showed a difference in the average number of identified variants between sample types (biopsy: 129.1 versus surgical: 101.5), stage IV cancers had more cfDNA than stage I, range of ctDNA+ samples was from 27% in melanoma to 65% in gastroesophageal cancers, and ctDNA+ proportion tended to increase with stage: 26 (14.2%) of 183 stage I, 84 (32.1%) of 262 stage II, 140 (38.9%) of 360 stage III, and 140 (65.1%) of 215 stage IV. ctDNA+ samples had levels between 2.6 and ≥352,000 parts per million (PPM), with 27 (6.6%) samples below 15 PPM, 40 (9.7%) between 15 and 50 PPM, and 345 (83.7%) above 50 PPM. Genomic profiling was performed on 158 samples, most from breast (N=50, 31.6%) and colorectal (N=17, 10.8%) cancer patients. The mean number of TAs per sample was 4.6, with little difference between the 114 ctDNA+ patients (mean = 4.4, range 0–24) and 44 ctDNA– patients (mean = 5.1, range 1–29). TAs were present in 83 (72.8%) and 36 (81.8%) tumors in ctDNA+ and ctDNA– patients, respectively. Of note, both high microsatellite instability and high tumor mutational burden tumors had numerically higher proportion in ctDNA– (15.9% and 9.1%, respectively) compared to ctDNA+ (5.3% in both) patients. Conclusions: ctDNA+ frequency varied by cancer type and cfDNA yields and ctDNA+ proportion increased numerically with stage, though where patients were in their treatment cycles was unknown. Of patients with profiling results, a large proportion of patients had TAs regardless of ctDNA status, indicating a potential for immediate consideration of matched therapies in ctDNA+ patients.

3590 Background: CRC is a leading cause of cancer-related mortality globally. Detection of molecular residual disease (MRD) is an early indicator of recurrence and may allow for timely intervention. Here, we evaluated an analytically validated tumor-informed ctDNA MRD assay (Oncodetect) in a cohort of patients with CRC. Methods: This retrospective study utilized data and specimens from 468 patients with Stage II, III or resectable Stage IV CRC consecutively enrolled from June 1, 2020 through November 30, 2022 in the GALAXY study with available residual samples. Tumor tissue underwent whole-exome sequencing to identify up to 200 tumor-specific variants for designing a personalized MRD test. The test was used to assess ctDNA status in plasma at three timepoints: post-surgical (PS), post-definitive therapy (PDT) and in the surveillance period, which included the PDT and subsequent timepoints. The primary endpoint was the association of ctDNA status during surveillance with disease-free survival (DFS). An analysis of the association between RNA-seq expression and DFS was also planned. Results: Analysis included a total of 1648 ctDNA results from 417 patients with ≥1ctDNA result from ≥1 timepoint. Among these patients, 296 (71.0%) had colon and 121 (29.0%) had rectal cancer, 141 (33.8%) had Stage II, 249 (59.7%) Stage III and 27 (6.5%) Stage IV disease, and 255 (61.2%) received adjuvant chemotherapy. Median follow-up was 1.9 years. The median ctDNA level among detections, measured in mean tumor molecules per ml (MTM/ml) was 1.187 (range 0.006-3180.5). During surveillance, ctDNA detection was strongly prognostic for DFS (HR 36.6; CI 21.9 – 61.2; ctDNA status as a time-dependent variable). Similarly, ctDNA detection was strongly associated with DFS at the PS and PDT timepoints (Table). Multivariable analysis showed ctDNA status remained strongly associated with DFS while other clinicopathological factors did not. The median lead time between ctDNA detection and clinical recurrence was 97 days (95% CI: 51-114). RNA-seq analysis is ongoing. Conclusions: In a cohort of 468 patients who underwent curative-intent surgery for stage II-IV CRC, a tumor-informed quantitative ctDNA assay using up to 200 variants was strongly prognostic for DFS at all timepoints. The prognostic ability of RNA-seq expression analysis for ctDNA status and outcome in this cohort is currently being determined. Association of ctDNA status with DFS. Statistic Result Surveillance (n= 398) HR (95% CI) 36.6 (21.9 – 61.2), p<0.0001 Sensitivity (95% CI) 64.8% (53.2 - 74.9%) Specificity (95% CI) 98.8% (96.8 - 99.5%) PS (n = 241) HR (95% CI) 7.5 (4.3 – 13.1), p<0.0001 Sensitivity (95% CI) 44.2% (31.6% - 57.7%) Specificity (95% CI) 95.6% (91.6% - 97.8%) PDT (n = 367) HR (95% CI) 24.0 (13.8 – 41.7), p<0.0001 Sensitivity (95% CI) 45.5% (34.0% - 57.4%) Specificity (95% CI) 99.0% (97.1% - 99.7%)

PURPOSE:To summarize clinically relevant genomic alterations in solid tumor samples from over 10,000 patients. METHODS:Descriptive statistics were used to summarize findings of retrospectively analyzed OncoExTra assay data from solid tumor samples. RESULTS:The analysis cohort included 11,091 solid tumor samples from 10,768 patients. Therapeutically actionable alterations were present in 92.0% of patient samples. Biomarkers associated with on- or off-label FDA-approved therapies were detected in 29.2% and 28.0% of samples, respectively. The prevalence of hotspot alterations detected at variant allele frequency (VAF) <5% was analyzed among 7,481 samples (67.5%) harboring ≥1 of these events: 13.7% (1,022 of 7,481) had ≥1 alteration detected at VAF <5%, and 9.8% (558 of 5,690) of hotspot alterations associated with an on- or off-label FDA-approved therapy were detected at VAF <5%. Common and rare mutations in the TERT promoter were found in 8.4% (933) of samples. Whole transcriptome sequencing detected clinically relevant fusions in 7.5% of samples, with highest frequencies in prostate cancer (42.0%). The METe14 transcript was found in 14 NSCLC samples (2.7%). CONCLUSIONS:The broad capabilities of the OncoExTra assay detected therapeutically actionable and other clinically relevant genomic events that can inform clinical decision-making for patients with advanced solid tumors.

302 Background: Patients with stage III CRC undergo surgery and adjuvant chemotherapy. Evaluation of MRD using a highly sensitive and specific, tumor-informed ctDNA assay may help predict which patients will have a recurrence. Methods: Patients with stage III CRC were enrolled after surgery in an observational study across 19 Pennsylvanian hospitals between 2016-2020. Tumor tissue and serial blood samples (every 3 months for years 1-3, biannually for years 4-5) were collected. Following whole exome sequencing (WES) of the tumor and selection of 50 – 200 somatic variants, a personalized MRD assay was used to measure ctDNA status of each sample. Three timepoints were used to analyze the association of ctDNA with recurrence: the single sample post-surgical (PS) and post-definitive therapy (PDT) timepoints, and the surveillance period, which included the PDT and subsequent samples. Primary endpoint was the association of ctDNA status during surveillance with recurrence-free survival (RFS). Results: A total of 124 patients with 1029 ctDNA results were analyzed. Almost all patients (96.0%) received adjuvant chemotherapy, primarily with an oxaliplatin-based regimen. On average, patients had 10 (SD = 4.5) blood draws and the median follow-up was 4.8 years. WES identified 172 (range 50 – 200) somatic variants per patient on average. Patients who were ctDNA positive during surveillance had a significantly higher likelihood of recurrence than patients who remained ctDNA negative (HR 49.6, 95% CI: 16.6 – 148.3; ctDNA status as time-dependent variable). At all timepoints, ctDNA detection was strongly associated with recurrence (Table). In multivariable analyses, ctDNA was strongly prognostic for recurrence but CEA was not. The median lead time between positive ctDNA result and a clinical diagnosis of recurrence was 10.4 months. Conclusions: In this cohort of patients with stage III CRC with long-term follow-up, a tumor-informed ctDNA assay using up to 200 variants was strongly prognostic for recurrence at all timepoints. Hazard ratios (HR), sensitivity, and specificity, with 95% confidence intervals (95% CI), for the association of ctDNA status with recurrence-free survival (RFS) at the surveillance, postsurgical (PS), and post-definitive therapy (PDT) timepoints (three-year RFS for the PS and PDT timepoints are also shown). Timepoint Statistic Result Surveillance(N = 111) HR(95% CI) 49.6**(16.6 – 148.3) Sensitivity(95% CI) 20/22 = 90.9%(72.2 – 97.5%) Specificity(95% CI) 82/87 = 94.3%(87.2 – 97.5%) PS(N = 88) HR(95% CI) 9.6**(3.2 – 29.5) Sensitivity(95% CI) 14/18 = 77.8%(54.8 – 91.0%) Specificity(95% CI) 53/66 = 80.3%(69.2 – 88.1%) RFS at 3 yrsctDNA(+), ctDNA(-) 54.5%, 96.1% PDT(N = 97) HR(95% CI) 16.7**(6.9 – 40.3) Sensitivity(95% CI) 10/21 = 47.6%(28.3 – 67.6%) Specificity(95% CI) 75/76 = 98.7%(92.9 – 99.8%) RFS at 3 yrsctDNA(+), ctDNA(-) 18.2%, 90.0% *P< 0.001, **P<0.0001.

BACKGROUND:There are currently no molecular tests to identify individual breast cancers where radiotherapy (RT) offers no benefit. Profile for the Omission of Local Adjuvant Radiotherapy (POLAR) is a 16-gene molecular signature developed to identify low-risk cancers where RT will not further reduce recurrence rates. METHODS:An individual participant data meta-analysis was performed in 623 patients of node-negative estrogen receptor-positive and HER2-negative early breast cancer enrolled in 3 RT randomized trials for whom primary tumor material was available for analysis. A Cox proportional hazards model on time to locoregional recurrence was used to test the interaction between POLAR score and RT. RESULTS:A total of 429 (69%) patients' tumors had a high POLAR score, and 194 (31%) had a low score. Patients with high POLAR score had, in the absence of RT, a 10-year cumulative incidence of locoregional recurrence (20%, 95% confidence interval [CI] = 15% to 26%, vs 5%, [CI] 2% to 11%) for those with a low score. Patients with a high POLAR score had a large benefit from RT (hazard ratio [HR] for RT vs no RT = 0.37, 95% CI = 0.23 to 0.60; P < .001). In contrast, there was no evidence of benefit from RT for patients with a low POLAR score (HR = 0.92, 95% CI = 0.42 to 2.02; P = .832). The test for interaction between RT and POLAR was statistically significant (P = .022). CONCLUSIONS:POLAR is not only prognostic for locoregional recurrence but also predictive of benefit from RT in selected patients. Patients aged 50 years and older with estrogen receptor-positive and HER2-negative disease and a low POLAR score could consider omitting adjuvant RT. Further validation in contemporary clinical cohorts is required.

We compared 21-gene recurrence score (RS) distribution and expression of the single-gene/gene groups within this assay between BC patients with pathogenic variants (PV) in BRCA1/2 vs the general 21-gene-tested BC population. This retrospective study included consecutive 21-gene-tested female ER + HER2-negative BC patients with germline PVs in BRCA1/2. RS/gene expression data were compared to a previously described commercial use database (CDB, N = 799,986). Chi-square and 1-sample t test were used to compare RS distribution and single-gene/gene group scores between the study group and the CDB. Study group patients (N = 81) were younger and their RS results were higher compared to the CDB (age: median [IQR], 56 [47–61.5] vs 60 [51–67] years; p < 0.001; proportion of patients with RS ≥ 26: 49.4

3620 Background: The role of adjuvant chemotherapy (CT) in stage II CC is debated. The validated 12-gene Oncotype DX Colon Recurrence Score test provides a Recurrence Score (RS) result (range, 0-100) which estimates recurrence risk (RR) in stage II/III pts. We studied treatment and clinical outcomes in CC pts in whom treatment decisions incorporated the RS result. Methods: This prospectively designed cohort study included all stage II, MMR-P, CC pts who underwent the 12-gene Oncotype DX testing through Clalit between 1/2011 and 12/2016 and had available data with minimum 3-yr follow-up. Kaplan-Meier (KM) estimates and log-rank tests were used to compare RR and CC specific mortality (CCSM) between RS categories. Multivariable analysis (MVA) identified variables associated with RR/CCSM. Results: The analysis included 938 pts. Median age, 68 (IQR, 60-76) yrs; 96% had T3 tumors, and 89% had ≥12 nodes examined. Median RS was 26 (IQR, 19-33). The 3 RS categories (0-29, 30-40, and 41-100) included 65%, 24%, and 11% of pts, respectively. The overall CT use rate was 24%, with a significant difference between the 3 categories (14%, 36%, and 60%, respectively, P< .0001). Pts with very low RS (0-15) comprised 14% of the cohort (CT use rate, 11%). Younger pts, and those with invasion/perforation/obstruction were more likely to receive CT. Clinical outcomes with a median follow up of 6.9 (IQR, 5.5-8.6) yrs are presented (Table). Among untreated pts, KM estimates for RR and CCSM differed significantly between the 3 RS categories ( P< .0001). Outcome of untreated RS 0-15 pts was excellent. In an MVA model, male sex, presence of invasion/perforation/obstruction and higher RS category (RS 41-100 vs 0-29 and vs 30-40, but not RS 30-40 vs 0-29) were associated with increased RR. For CCSM, the results were similar, but this time age ≥70 yrs replaced sex as a significant prognostic variable. Clinical outcomes within each RS group did not differ significantly between treated and untreated pts, but were numerically better with CT in the RS 41-100 group. Conclusions: This real-world analysis showed that the RS results provide independent prognostic information in stage II CC. Further studies are needed to investigate the potential role of the RS result as a predictor of CT benefit, but the data suggest that this benefit may be limited to pts with high RS results. [Table: see text]

Background The 21-gene recurrence score (RS) assay (Oncotype DX) is used to guide adjuvant chemotherapy use for patients with hormone receptor-positive, HER2 (human epidermal growth factor receptor 2)-negative, axillary node-negative breast cancer. Its role, however, in providing prognostic information for late distant recurrence when added to clinicopathologic prognostic factors is unknown. Methods A patient-specific meta-analysis including 10,004 women enrolled in three trials was updated using extended follow-up data from TAILORx, integrating the RS with histologic grade, tumor size, and age at surgery for the RSClin tool. Cox models integrating clinicopathologic factors and the RS were compared by using likelihood ratio (LR) tests. External validation of prognosis for distant recurrence in years 0 to 10 and 5 to 10 was performed in an independent cohort of 1098 women in a real-world registry. Results RSClin provided significantly more prognostic information than either the clinicopathologic factors (Delta LR chi-square, 86.2; P<0.001) or RS alone (Delta LR chi-square, 131.0; P<0.001). The model was prognostic in an independent cohort for distant recurrence by 10 years after diagnosis (standardized hazard ratio, 1.56; 95% confidence interval, 1.25 to 1.94), was associated with late distant recurrence risk between 5 and 10 years after diagnosis (standardized hazard ratio, 1.78; 95% confidence interval, 1.25 to 2.55), and approximated the observed 10-year distant recurrence risk (Lin concordance, 0.87) and 5- to 10-year distant recurrence risk (Lin concordance, 0.92). Conclusions The 21-gene RS is prognostic for distant recurrence and overall survival in early breast cancer. A model integrating the 21-gene RS and clinicopathologic factors improved estimates of distant recurrence risk compared with either used individually and stratified late distant recurrence risk. (Funded by the National Cancer Institute, National Institutes of Health [U10CA180820, U10CA180794, UG1CA189859, U10CA180868, and U10CA180822] and others.)

Abstract Background: RSClin® (Sparano et al J Clin Oncol 2020, PMID 33306425) is an educational tool that estimates the 10-year risk of distant recurrence (DR) and the absolute benefit of chemotherapy in HR-positive, HER2-negative, node-negative early breast cancer. The RSClin tool integrates the Recurrence Score® (RS) result with the clinical-pathological features tumor grade, tumor size and patient age using patient-specific meta-analysis (PSMA) applied to the NSABP B-14, TAILORx and NSABP B-20 studies. Approximately 3 years of additional follow-up data have accumulated in the TAILORx study since the RSClin tool was developed, including 50% more DR events in the RS 11-25 group (375 vs. 250), and 46% more DR events in the overall population (561 vs. 384). Methods: The RSClin tool estimates were updated to include the extended follow-up data from TAILORx. As before, the PSMA included 10,004 women with hormone-receptor positive, HER2-negative, node-negative breast cancer from the NSABP B-14 (n=577) trial receiving endocrine therapy (ET) alone and the TAILORx trial receiving ET alone (n=4854) or chemotherapy (CT) plus ET (n=4573). The baseline risk estimate used TAILORx event rates with ET alone. Since there was evidence of differing hazard ratios in years 0-5 versus 5+, models using time-varying effects were used. A patient-specific estimator of absolute CT benefit was computed by combining a PSMA estimate of the individualized relative CT effect from the TAILORx and NSABP B-20 (n=550 HER2-negative) trials with risk estimates for ET alone. Similar methods were used to develop and validate a new tool for assessing the risk of late DR (from year 5 to 10) in patients who survive DR-free for 5 years on ET alone after surgery. External validation of DR risk estimation was performed in an independent cohort of 1098 women enrolled in the Clalit Health Service registry. Results: With the updated data, the model integrating RS result with clinical-pathological features was more informative for DR risk than either RS result alone or the clinical-pathological features alone, both for 10-year DR risk and for late DR risk (likelihood ratio tests, each p≤.001). The updated 10-year RSClin risk estimates are similar to the existing RSClin tool risk estimates with slightly lower risk estimates for very high RS results and more precise estimates for mid-range RS results. The updated chemotherapy effect estimates are slightly lower than for the existing RSClin. The updated RSClin tool 10-year risk estimates were strongly associated with DR risk in the independent Clalit registry (Cox regression p< .001) and closely approximated the observed DR risk (Lin concordance 0.87). The new tool’s late DR risk estimates beyond 5 years were strongly associated with late DR risk in the Clalit registry (Cox regression p=.002) and closely approximated the observed late DR risk (Lin concordance 0.92). Conclusions: The RSClin tool 10-year DR risk estimates have been updated to incorporate extended follow-up data from the TAILORx study. A new tool for assessing late DR risk has been developed and validated that provides more prognostic information than either clinical-pathological features or genomic risk used individually. Citation Format: Joseph Sparano, Michael Crager, Robert Gray, Gong Tang, Jess Hoag, Frederick Baehner, Charles Geyer, Steven Shak, Norman Wolmark, Salomon Stemmer. Update of RSClin with extended TAILORx follow-up and development and validation of a new tool for risk of late distant recurrence [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 PO1-02-02.

TPS98 Background: Despite standard of care management with surgery ± adjuvant chemotherapy treatment (ACT), >30% of patients (pts) with resectable CRC recur. The presence of ctDNA in plasma after resection and after ACT, has been shown to be a strong prognostic factor for the risk of recurrence, suggesting that the presence of ctDNA in blood is molecular evidence of residual disease. As such, ctDNA analysis may enable post-surgical risk stratification for ACT decision-making, as well as detection of molecular recurrence during surveillance and subsequent early intervention. The CORRECT-I study aims to validate the association of post-definitive therapy and pre-recurrence follow-up ctDNA positivity with recurrence-free interval (RFI) in pts who have undergone complete surgical resection for stage II or III CRC. Methods: This study is a prospective, observational, multicenter study, which is open in Israel, Italy, Japan, Spain, and the UK. Pts must have pathologically confirmed stage II or III CRC, have undergone complete surgical resection, and have tissue available from the primary resection. Pts may not have started ACT prior to baseline blood draw post-surgery. Pts are asked to provide serial whole blood specimens for ctDNA analysis at a) post-surgical baseline, b) pre-recurrence follow-up visits (max 15 blood draws over max 5 years), and c) clinical recurrence. ctDNA will be analyzed with an NGS-based tumor-informed MRD assay that identifies somatic genomic alterations from DNA derived from the patient’s tumor tissue and detects a selected subset of tumor-specific (bespoke) ctDNA in their blood. The primary objective of this study is to validate the association of post-definitive therapy and serial pre-recurrence follow-up ctDNA positivity with RFI, using a Cox proportional hazards regression analysis. Further objectives include the assessment of sensitivity and specificity of the ctDNA assay, the association between ctDNA at individual timepoints and RFI, ctDNA dynamics and RFI, and the time from ctDNA positivity to clinical recurrence. A multivariable model, including ctDNA, clinicopathological risk features, serial serum CEA assessments, and recurrence risk as determined by the Oncotype Colon Recurrence Score will be fit with RFI as endpoint. The primary analysis will be conducted when ≥30 histologic and/or radiographic confirmed cases of clinical recurrence have been observed. As of April 2, 2024, 158 pts of 400 have been enrolled.

Abstract Introduction: Immune checkpoint inhibitors (ICI) are an important therapeutic option for patients with triple negative breast cancer (TNBC). However, identification of patients most likely to respond is challenging. PD-L1 positivity by immunohistochemistry is the standard biomarker used for ICI therapy selection in TNBC. However, other biomarkers, such as analysis of the tumor microenvironment (TME) may be more accurate in predicting response. The XernaTM TME Panel uses RNA sequencing data and machine learning to analyze the TME, utilizing the angiogenic and immunogenic biology of the TME to classify tumors into four TME subtypes. In this study, the distribution of Xerna TME subtypes and associated genomic alterations in TNBC were investigated for their potential use in therapy selection. Methods: A total of 203 TNBC patient samples underwent tumor-normal whole-exome, whole-transcriptome sequencing testing with the OncoExTraTM assay. The whole-transcriptome expression data were analyzed using the Xerna TME Panel to assign each sample to one of four subtypes: Immune Active (IA), Immune Suppressed (IS), Immune Desert (ID) and Angiogenic (A). The IA and IS subtypes both have high immune scores that may be particularly sensitive to ICI therapy. Actionable alterations, defined as those with FDA-approved matched therapies in any cancer, with matched clinical trials, or with evidence in cancer guidelines or the literature for possible matched therapies, were also identified and associations across Xerna subtypes were explored. Results: Approximately half (100 of 203; 49.3%) of the patient samples had high (IA+IS) immune subtypes (Table 1). Targetable alterations associated with an FDA-approved therapy were present in 114 (56.2%) patients. No biomarkers were significantly associated (p < 0.05) with high (IA+IS) versus low (ID+A) immune scores. Biomarkers associated with ICI response, namely mismatch repair gene alterations (MSH2/3/6, MLH1/3, PMS1/2), high tumor mutational burden (TMB-high) and microsatellite instability were detected in only 6 (3.0%), 3 (1.5%) and 1 (0.5%) patient samples respectively, and all but 1, an MSH6 alteration, were in high immune subtype samples. Conclusions: The Xerna TME Panel classified 49.3% of TNBC patient tumors to IA or IS, suggesting they may respond to ICI therapy. Many (56.2%) patient tumors harbored alterations associated with FDA-approved therapies, providing the potential for novel combination therapies. These findings warrant further study and clinical validation in TNBC patients treated with ICI therapy. Table 1. Frequency of actionable biomarkers that were present in at least 10 (5%) TNBC patient samples. Citation Format: Gargi Basu, Janine Lobello, Snehal Thakkar, Jessica Aldrich, Matthew Halbert, Patrick Eimerman, Cynthia Flannery, Nishitha Therala, David Hall, Daniel Pointing, Lea Vohar, Roman Luštrik, Luka Ausec, Mark Uhlik, Seema Iyer, Laura Benjamin, Frederick Baehner. Prevalence of genomic alterations in Xerna tumor microenvironment subtypes in triple negative 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 PO2-06-10.

INTRODUCTION:No definitive answers currently exist regarding optimal first-line therapy for HER2-mutant NSCLC. Access to rapid tissue sequencing is a major barrier to precision drug development in the first-line setting. ctDNA analysis has the potential to overcome these obstacles and guide treatment. METHODS:We retrospectively analyzed patients with metastatic HER2-mutant NSCLC who underwent prospective clinical ctDNA sequencing and received systemic therapy at Memorial Sloan Kettering Cancer Center (MSK) from January 2016 to September 2022. HER2 mutations were identified by next-generation sequencing through MSK-IMPACT, MSK-ACCESS or Resolution ctDx LungTM assay. Primary endpoints were time to the next treatment (TTNT) and overall survival (OS). RESULTS:Sixty-three patients were included in the primary analysis. Chemoimmunotherapy (33/63, 52.4 %) was the predominant first-line treatment with a median TTNT of 5.1 months (95 %CI 4.1 - 6.1) whereas 55.0 % (22/40) of patients who received second-line T-DXd obtained a median TTNT of 9.2 m (95 % CI, 0-22.2). Plasma ctDNA was tested before first-line therapy in 40 patients with a median OS of 28.0 months (95 % CI 21-34), in whom 31 patients (78.0 %) had detectable ctDNA. HER2 mutations were detected on ctDNA with a median turnaround time of 13 days, occasionally co-occurred with EGFR and MET alterations and were tracked longitudinally correlating with treatment response. Patients with detectable baseline ctDNA had significantly shorter OS (hazard ratio (HR), 5.25; 95 % CI, 1.2-23.9; p = 0.019). CONCLUSION:Chemoimmunotherapy remains a major treatment option for metastatic HER2-mutant NSCLC. ctDNA can rapidly detect HER2 and co-mutations, and it has the potential to guide and monitor optimal first-line therapy. As a negative prognostic biomarker, detectable ctDNA at baseline would need to be taken into account for patient selection in future studies.

Circulating tumor DNA (ctDNA) holds promise as a biomarker for predicting clinical responses to therapy in solid tumors, and multiple ctDNA assays are in development. However, the heterogeneity in ctDNA levels prior to treatment (baseline) across different cancer types and stages and across ctDNA assays has not been widely studied. Friends of Cancer Research formed a collaboration across multiple commercial ctDNA assay developers to assess baseline ctDNA levels across five cancer types in early- and late-stage disease. This retrospective study included eight commercial ctDNA assay developers providing summary-level de-identified data for patients with non-small cell lung cancer (NSCLC), bladder, breast, prostate, and head and neck squamous cell carcinoma following a common analysis protocol. Baseline ctDNA levels across late-stage cancer types were similarly detected, highlighting the potential use of ctDNA as a biomarker in these cancer types. Variability was observed in ctDNA levels across assays in early-stage NSCLC, indicative of the contribution of assay analytical performance and methodology on variability. We identified key data elements, including assay characteristics and clinicopathological metadata, that need to be standardized for future meta-analyses across multiple assays. This work facilitates evidence generation opportunities to support the use of ctDNA as a biomarker for clinical response.

533 Background: In randomized studies (NSABP B-20, SWOG 8814, TAILORx, RxPONDER), the predictive effect of the 21-gene Breast Recurrence Score assay (RS) on chemotherapy (CT) benefit has been evaluated in patients with node-negative (N0) and node-positive (N+) breast cancer. Retrospective analyses of TAILORx and RxPONDER demonstrated differences in outcome by racial/ethnic groups (RG); however, there were no differences in CT benefit for any of the clinical outcome measures. In this study we characterized the association between the RS result and breast cancer-specific mortality (BCSM) and chemotherapy benefit in BCSM in RG in the recently updated, population-based SEER registries. Methods: Eligible patients were from the SEER 17 registries, November 2022 submission, and had non-metastatic, hormone receptor-positive, HER2-negative, node negative (N0) breast cancer diagnosed between 2006 and 2019 with a RS result. Analyses were weighted using the inverse of estimated propensity of using CT (yes vs. none/unknown). Prognosis was ascertained by estimating breast cancer specific survival (BCSS) at 9 years among patients with CT recorded as none/unknown by RG. The log-rank test was used to compare BCSM by RG. Multivariable Cox models, including RS result (26-100 vs. 0-25) x CT interaction, were used to assess whether RS result was predictive of CT benefit. Analyses were performed in the overall cohort and in Hispanic (H), non-Hispanic Asian and Pacific Islander (NHAPI), non-Hispanic Black (NHB), and non-Hispanic White (NHW) RGs. Results: There were 145,642 eligible patients and 3,212 breast cancer deaths. Median follow-up was 68 months. NHB patients had higher median RS result and CT usage than other groups (p < .001). In N0 patients, higher RS result was associated with lower 9-year BCSS, both overall and by race/ethnicity (p < .001). In Cox multivariable models (Table), the interaction between RS result by CT was significant among all N0 patients (p < .001) and in NHB patients (p = 0.005), NHW patients (p = 0.002), and H patients (p = 0.019), but not in NHAPI patients (p = 0.766). Analyses of patients with N+ disease and more detailed analysis of CT benefit according to RS results by RG are underway and will also be presented. Conclusions: Real-world evidence from the SEER registries in over 145,000 patients confirms the 21-gene assay is prognostic for BCSS in all groups by race and ethnicity. The RS result was predictive of CT benefit in N0 patients overall and in Hispanic, NHB, and NHW patients, but not in NHAPI patients in this analysis. [Table: see text]

508 Background: The RSClin tool was developed to provide estimates of recurrence risk and absolute chemotherapy benefit for patients with HR+/HER2- node-negative breast cancer using the 21-gene recurrence score (RS) and clinicopathologic factors. For patients with node-positive disease, we developed a new tool (RSClin N+) to increase the prognostic and predictive utility of risk estimates by combining RS with clinicopathologic factors and menopausal status. Methods: We used Cox regression to estimate 5-year risk of invasive disease or death (iDFS) and likelihood ratio (LR) tests to compare fit of RSClin N+ with RS alone and clinicopathologic factors (tumor grade, tumor size, positive lymph nodes [N1/N2], age) alone in 5283 node-positive patients treated with chemoendocrine therapy (CET) vs endocrine therapy alone (ET) in the S8814 (n=367: N1=227, N2=140, all postmenopausal) and RxPONDER (n/N1=4916) trials. Data from both studies were pooled and stratified by study for postmenopausal models; premenopausal models were fit only on RxPONDER data. Absolute CET benefit across combination of covariate values was estimated as the difference between ET and CET risk estimates. Validation of RSClin N+ was performed in 592 Nmic/N1 patients in the Clalit registry real-world dataset. Results: For pre- and postmenopausal patients, RSClin N+ provided significantly more prognostic information for iDFS than RS or clinicopathologic factors alone (LR Chi-square p<0.05). In postmenopausal patients, RS and clinicopathologic factors were independently prognostic, but only RS showed interaction with CET benefit (p=0.016). Absolute CET benefit for iDFS ranged from <0.1% to 21.5% as the RS increased from 0 to 50. Table shows examples of risk stratification and predicted benefit by RS within clinical low and high risk patients. In premenopausal patients, both RS and clinicopathologic factors remained prognostic but no interaction was observed between RS and CET.In external validation, RSClin N+ risk estimates were concordant with (Lin’s concordance=0.92) and prognostic for observed risk (HR 1.75; 95% CI 1.38 to 2.20). Conclusions: The RSClin N+ model provides improved estimates of prognostic risk and absolute CET benefit than clinical or genomic data alone in node-positive, HR+/HER2- breast cancer and could be used in patient counseling. [Table: see text]

Background and Purpose Clinico-pathological factors alone are insufficient to select patients at low risk of recurrence after breast conserving surgery and systemic therapy for the omission of radiotherapy (RT). We conducted a study to validate the prognostic and predictive value of the 16-gene signature, Profile for the Omission of Local Adjuvant Radiation (POLAR), for radiation response in the Scottish Conservation Trial (SCT). Materials and Methods Transcriptome-wide profiling was performed on formalin fixed paraffin embedded (FFPE) samples from patients from the SCT with invasive BC randomised to ± RT following breast conserving surgery (BCS). Patients with follow-up data for locoregional recurrence and gene expression data were included in the analysis of POLAR. Results 224 patients were included in the analysis. The continuous standardised POLAR score was prognostic for LRR in the no RT arm after adjusting for relevant covariates (HR=1.78 [1.20-2.64], p=0.003). POLAR was the only factor to remain significant in multivariable Cox PH models. In the subgroup of node-negative patients with ER+/HER2-negative tumours (N=137), there was a statistically significant RT benefit for patients with cancers with a POLAR high score (HR=0.31 [0.11-0.88], p=0.028) but not for patients with cancers with a POLAR low score (HR=0.5 [0.1-2.4], p=0.39). Conclusion For patients with early-stage invasive BC, treated with BCS but without RT, POLAR is prognostic for LRR. Additionally, in women with ER+, HER2-negative, node-negative BC, POLAR may identify a group of women with low risk of LR that do not benefit from adjuvant RT .