Association of Recurrence with a Tumor-informed Personalized ctDNA Detection Approach in Resectable Colorectal Cancer: Results of a Prospective Observational Study.

275 Background: While detection of MRD using ctDNA is prognostic for recurrence in CRC, some patients still recur prior to MRD detection. VICTORI is prospectively investigating NeXT Personal, an ultra-sensitive NGS-based MRD assay, to profile patients with resected CRC. Methods: Patients with CRC treated with curative intent (all stages) are tested for MRD using NeXT Personal, a bespoke assay with up to ~1,800 tumor-informed single nucleotide variants (SNVs) identified from whole-genome sequencing. Plasma is collected prior to surgery, every 2 weeks post-surgery up to week 8 (MRD landmark window), and every 3 months for up to 3 years (surveillance). We present preliminary results on 397 samples from the first 62 patients. Results: A total of 62 patients (N=36 rectal [58%], N=26 colon [42%]; N=46 stage I-III [74%], N=16 stage IV [26%]) were included in our analysis. Baseline pre-surgical sensitivity (treatment naive) was 93.5% [N=29/31]. Pre-surgical positivity rate in patients who had received neoadjuvant therapy and had residual cancer at the time of surgery was 67% [N=16/24]. 60 patients were evaluable for clinical outcomes. At a median follow-up of 355 days, 15 patients (25%) had a recurrence. Of these, all patients were ctDNA-positive prior to recurrence (100%, 14/14; 1 pt excluded due to lack of samples prior to recurrence). ctDNA detection preceded clinical relapse by a median of 194 days [range: 5-397]); ctDNA for 78.6% (N=11/14) were first detected in the MRD landmark window. All landmark-positive recurrences occurred within one year of surgery. The 14 recurrent cancers with samples were first detected at a median ctDNA concentration of 28.7 parts per million (PPM) (range 2.4-111,120), with 64.3% (9/14) of those detections in the ultra-low range of <100 PPM. MRD detection at week 4 and week 8 had the greatest reduction in RFS (HR 12.86 [2.74-60.28], p=0.0012 week 4; HR 16.14 [3.52-74.09], p=0.0004 week 8), with weeks 4, 6 and 8 having similar higher prevalence of ctDNA detection (36.0% [18/50], 35.3% [18/51], 38.5% [20/52] respectively). Week 2 detection rate was 17.0% (8/46). cfDNA concentration was highest at week 2 (4.63ng/ml vs. 2.22 at baseline, p=0.00028) and 4 (3.68 vs. 2.22, p=0.0081), returning to baseline levels at week 6 (2.28 vs. 2.22, p=0.67) and 8 (2.48 vs. 2.22, p=0.64). Conclusions: In this interim report after a median ~1 year follow-up, NeXT Personal detected MRD for all patients prior to disease recurrence. Most initial MRD detection was in the ultra-sensitive range <100ppm and detection at 4-8 weeks after surgery was highly prognostic for recurrence.

448 Background: There is compelling rationale that detection of MRD following curative therapy may identify patients at high risk of relapse requiring intensified adjuvant therapy. Combining MRD detection with CGP creates an opportunity to offer MRD-guided treatment with precision cancer therapeutics. Here we analyze the observation arm of the IMvigor-010 study to understand the genomics of resected early stage bladder cancer and to validate CGP-informed personalized MRD detection in circulating tumor DNA (ctDNA). Methods: Using the resected tumor, tissue CGP was performed retrospectively with a 300+ gene assay, followed by MRD detection using FoundationOne Tracker (F1T). Briefly, coding, synonymous, and non-coding variants were selected from tumor tissue sequencing using an optimized algorithm that filters out non-tumor derived variants (germline, clonal hematopoiesis derived, sequencing artifacts). Tumor-informed personalized multiplex PCR-next generation sequencing (Natera) assay was designed and used to detect and quantify variant allelic frequency (VAF) in ctDNA from 182 patients. ctDNA levels were reported in mean tumor molecules per mL of plasma. F1T, a tissue-informed personalized monitoring assay, was performed on plasma samples collected at an MRD timepoint a median of 11 weeks post-surgery. Results: At the MRD timepoint, ctDNA was detected in 66/182 (36%). Focusing on the 66 ctDNA-positive patients, 58 had relapsed (88% PPV) at time of analysis. Median disease-free survival (DFS) from randomization was 3 months in ctDNA-positive vs not reached in ctDNA-negative population (HR = 5.7, 95% CI: 3.8-8.6, p <.0001). Median overall survival (OS) was 13 months in ctDNA-positive vs not reached in ctDNA-negative (HR = 5.7, 95% CI: 3.4-9.7, p <.0001). Potentially actionable CGP findings included FGFR2/3 short variants (SVs) and fusions (13%), ERBB2 SVs and amplifications (13%), PIK3CA SVs (20%), CDKN2A SVs and losses (41%) and tumor mutational burden (TMB) ≥10 mutations/Mb (35%). Conclusions: Tissue CGP-informed personalized MRD detection can detect low levels of residual ctDNA in patients with resected early stage bladder cancer, identifying a population with inferior DFS and OS. This technologic approach, synergizing regulatory-grade actionable CGP with ctDNA-based MRD detection, creates new opportunities for precision adjuvant therapy across a range of high-risk cancer types. Clinical trial information: NCT02450331.

9 Background: ctDNA-based post-surgical detection of molecular residual disease (MRD) is known to be predictive of a high risk of recurrence. Here, we report the first results of BESPOKE CRC, a multicenter, prospective, observational study evaluating the ability of a tumor-informed ctDNA assay to inform ACT treatment decisions in stage II/III CRC patients (pts). Methods: Of the 1792 pts enrolled between 2020-07-02 and 2022-08-25, plasma samples from the first 350 pts with stage II-III CRC were analyzed. ctDNA was detected and quantified using a personalized, tumor-informed assay (Signatera, Natera, Inc.). Following curative resection, 232 pts received ACT and 118 underwent observation. Results: The cohort included 154 stage II and 196 stage III CRC pts; the median follow-up was 24.8 months. ctDNA results at the post-op MRD time point (tp) were available for 295 pts; 15.6% (46/295; stage II: 9/130=6.9%; stage III: 37/165=22.4%) of pts were ctDNA positive (ctDNA+) at MRD tp (MRD+). MRD-positivity was significantly associated with inferior disease-free survival (DFS) in stages II-III combined (HR=20.8, 95% CI: 10.0-43.4, p<0.0001) and in stage-stratified subgroups (stage II: HR=25.7, 95% CI 6.8-96.7; stage III: HR=18.1, 95% CI 7.3-45.1). Within the MRD+ group, pts receiving ACT had longer DFS compared to those in the observation group (median DFS: 18.7 vs 6.7 months; HR=3.9, 95% CI: 1.3-11.5, p=0.01). In contrast, no benefit of ACT was observed in MRD- pts (HR=1.1, 95% CI: 0.3-3.9, p=0.89). Of the MRD+ pts, 39.1% (18/46) had ctDNA clearance at 12-weeks post-surgery tp. Pts with ctDNA clearance had longer DFS compared to those who remained positive (median DFS: 24.2 vs 13.8 months; HR=0.4, 95% CI 0.1-1.0, p=0.045), however, had worse DFS than pts who were ctDNA- at both 4- and 12-weeks (HR=22.5, 95% CI: 6.8-75.0, p<0.0001). Notably, 44.4% (8/18) pts with ctDNA clearance recurred; all 8 turned back ctDNA+ before radiological detection of relapse. ctDNA results during surveillance were available for 339 pts, of whom 8.3% (58/339) were ctDNA+ and had significantly worse DFS compared to serially ctDNA- pts (HR=124.3, 95% CI: 29.8-518.7, p<0.0001). Conclusions: ctDNA-based MRD detection of MRD was highly prognostic of recurrence in an early representative subset of BESPOKE CRC cohort. Data from the expanded cohort will be presented at the meeting. ctDNA MRD results were also predictive: significant benefit from ACT was observed in MRD+ but not in MRD- pts. Additionally, early ctDNA clearance in response to adjuvant therapy and ctDNA status during surveillance were prognostic of pt outcomes. Our results highlight the potential utility of ctDNA-guided adjuvant therapy in pts with stage II/III CRC. The results of BESPOKE CRC herein, as one of the first ctDNA-based prospective studies, will be further validated by ongoing ctDNA-directed randomized clinical trials. Clinical trial information: NCT04264702 .

e15625 Background: Detection of molecular residual disease (MRD) with ctDNA is highly prognostic for recurrence in CRC. However, many cancers still recur without detectable ctDNA and increased lead time before clinical recurrence may create a window of opportunity to intervene. Here we apply an ultra-sensitive assay, NeXT Personal, to profile patients in the VICTORI study, a prospective cohort of patients with CRC managed with curative-intent treatment. Methods: To date, the first 33 patients with CRC have undergone panel creation. NeXT Personal was employed to construct personalized liquid biopsy panels for each patient with up to ~1,800 single nucleotide variants (SNV) identified via whole-genome sequencing. For each patient, plasma is collected before curative intervention (baseline), every 2 weeks for 2 months (MRD window), and every 3 months for up to 3 years (surveillance). VICTORI will enroll > 175 patients and include a health economics analysis. Results: Of 33 patients enrolled to date, 25 (76%) are stage I-III and 8 are stage IV treated with curative interventions. 18 colon cancers and 15 rectal cancers are included. Pre-operative baseline sensitivity was 90.6% (29/32; 1 patient excluded as pCR at the time of baseline blood draw). Of the MRD- patients at baseline, 2 had prior neoadjuvant treatment and 1 was stage 1. Preliminary results (median 7.2 month follow-up; 5 recurrences) show 80% (4/5) of recurrences are MRD+ at the 4-week landmark and 50% (2/4; 1 missing sample) were MRD+ at the 2-week landmark. In 75% (3/4) stage II-III recurrences, the first MRD+ detection was in the ultra-low range of < 100 PPM ( < 0.01% tumor fraction). MRD detection at week 4 and 8 trended towards poorer recurrence-free survival (RFS) with similar separation of patients (log rank p = 0.062 and p = 0.011, respectively). Conclusions: Preliminary results from our prospective study demonstrate strong rates of ctDNA detection at week 4 landmark analysis, potentially due to detection of very low levels of ctDNA with an ultra-sensitive MRD assay. The majority of the first detections for MRD were in the ultra-low ctDNA range, indicating the importance of high sensitivity MRD testing. [Table: see text]

Molecular Residual Disease Detection Via Cancer Personalized Profiling By Deep Sequencing in Patients with PTCL Undergoing Autologous Stem Cell Transplantation

187 Background: Detection of MRD following metastatic liver resection in advanced CRC patients is associated with poor prognosis with high rate of relapse. Nevertheless, there is currently no standard of care to guide further therapy after curative intent surgery. MRD detection has the promise to be implemented into standard of care and guide treatment decision making. Here we establish feasibility of MRD detection using Foundation Medicine’s novel tissue-informed personalized monitoring assay, FoundationOne Tracker (F1T), in mCRC patients undergoing surgical resection with curative intent. Methods: Tissue-based CGP was performed retrospectively on a cohort of 72 patients from the PREDATOR trial. Trackable patient-specific single nucleotide variants were selected using a novel computational approach negating the need for buffy coat sequencing to filter germline variants. Personalized multiplex PCR was used to detect and evaluate prognostic value of ctDNA from plasma collected at MRD timepoint post-surgery (median 27 days, range 8-99.5). Median follow-up of patients in the overall population was 10.7 months (range: 0.9-53.8 months). Survival analyses were performed using the Kaplan-Meier Estimator and Cox regression. Results: Post-surgical F1T analysis was successful on 96% of cases (69/72). CGP analysis revealed at least one driver mutation in 57% of samples (41/72) including KRAS/NRAS (46%) and BRAF mutations (3%). MRD was detected in 45% (31/69) of patients, of which 94% (29/31) had progressed at the time of the data cut. Median progression -free survival (PFS) was 3.2 months (2.1-7.1) in ctDNA-positive vs 28 months (20.9-NA) in ctDNA-negative population (HR 5, CI 2.7-9.3, p<0.001). Median overall survival (OS) was 31.6 months in ctDNA-positive vs not reached in ctDNA-negative group (HR 27, CI 3.6-205, p<0.001). Conclusions: CGP-informed post-operative MRD detection is a strong prognostic biomarker and correlates with survival outcomes in patients with resected mCRC. F1T is a novel and convenient technological approach to MRD detection utilizing highly validated FMI testing to reveal potentially targetable mutations and inform personalized ctDNA monitoring. These results demonstrate the ability of F1T to accurately detect MRD in mCRC patients following surgical resection, without the need for germline sequencing.

168 Background: Identifying molecular residual disease (MRD) using circulating tumor DNA (ctDNA) analysis after curative surgery can potentially stratify the recurrence risk and facilitate personalization of adjuvant treatment in patients with colorectal cancer (CRC). We conducted a prospective study “COSMOS-CRC-01” to evaluate the utility of a plasma-only ctDNA assay integrating genomic and epigenomic signatures. Methods: Patients with resectable clinical stage 0–III colorectal cancer were eligible. Plasma samples were collected at structured pre- and post-surgical timepoints and analyzed using Guardant Reveal, a plasma-only ctDNA assay that detects the presence of MRD by identifying somatic alterations and methylation signatures of cell-free DNA associated with colorectal cancer. Results: As of April 2021, 501 patients were enrolled in the COSMOS-CRC-01, of which 496 patients had their post-operative 4-week ctDNA status. In this analysis, we included the first 100 patients enrolled with clinical stage II or more CRC. Seven patients were excluded due to non-curative resection or pathological stage (pStage) IV. The assay was able to produce a result for all 93 samples analyzed (failure rate of 0%). MRD was detected in 23 (25%) patients 4 weeks after surgery. The MRD detection rate was 20% in pStage II disease and 29% in pStage III disease. Patients with positive MRD were older than those with negative MRD ( p = 0.0001). Across all MRD positive samples, 30%, 9%, and 61% were positive by both genomic and epigenomic, only genomic, and only epigenomic calls. Genomic signatures included APC, BRAF, KRAS, and TP53 mutations with the lowest variant allelic fraction of 0.04%. With a median follow-up time of 12.2 months (range 8-18 months), 9 of 93 patients recurred (9.7%). ctDNA was detected from a single 4-week post-surgical sample in 55% of patients who recurred (5 of 9). 1-year disease-free survival was 81.2% in patients with positive MRD and 93.9% in those with negative MRD (hazard ratio 3.49, 95% CI 0.93–13.10, p = 0.049). Multivariate analysis including baseline characteristics associated with recurrence risk showed that MRD status had the strongest association with the recurrence. Post-operative 4-week serum CEA level was not associated with risk for recurrence. Conclusions: While follow-up in this cohort is currently limited, the results suggest that a single post-surgery sample run on a plasma-only assay that integrates genomic and epigenomic signatures can more accurately stratify patients with CRC by recurrence risk than previously known clinical factors. Data from baseline and longitudinal timepoints will be reported as available along with longer term clinical follow-up. Clinical trial information: UMIN000037765.

8005 Background: Osimertinib (osi) is a third-generation, central nervous system-active EGFR-TKI, that potently and selectively inhibits EGFR-TKI sensitizing and EGFR T790M resistance mutations. Adj osi (3 years [yrs]) is recommended for resected EGFRm stage IB–IIIA NSCLC, based on significant improvements in disease-free survival (DFS) and overall survival (OS) in the Phase III ADAURA study (NCT02511106). A trend towards an increased DFS event rate beyond 3 yrs suggests some pts may benefit from longer adj osi treatment (tx). We explored if plasma ctDNA-based, tumor-informed MRD could predict disease recurrence. Methods: Eligible pts (≥18 yrs [≥20 in Japan/Taiwan], WHO PS 0/1 with completely resected EGFRm [Ex19del/L858R] stage IB, II or IIIA [AJCC 7th edition] NSCLC) were randomized 1:1 to osi 80 mg once daily or placebo (pbo) until disease recurrence, tx completion (up to 3 yrs), or a discontinuation criterion was met. Personalized MRD panels (RaDaR, NeoGenomics) were used, comprised of ≤50 tumor-specific variants, based on whole exome sequencing of resected tumor tissue (variants identified in germline DNA removed). Plasma sample collection: baseline (BL; at randomization surgery and adj Ctx, if received), on tx (every 12 weeks [wks]), tx discontinuation, and post-tx completion (wk 12, wk 24, then every 24 wks until 5 yrs). Plasma samples were analyzed for detection of ctDNA (MRD+). Molecular recurrence or DFS (MRD/DFS) was defined as time from randomization to post-BL MRD+, disease recurrence, or death and was compared across arms. DFS was investigator-assessed. Results: Of 682 pts randomized, 245 (36%) had samples required to produce MRD panels, which were evaluable for 220 (32%) pts across both arms. In the osi vs pbo arms, 5/112 (4%) vs 13/108 (12%) pts were MRD+ at BL; 4/5 pts became MRD– during osi tx vs 0/13 pts on pbo. On tx, MRD detection had clinical sensitivity of 65% (62 MRD+/96 DFS+), specificity of 95% (118 MRD–/124 DFS–) and preceded a DFS event by a median (95% CI) of 4.7 (2.2, 5.6) months (mos) across both arms. Median follow-up time from randomization was 44.2 (95% CI 42.4, 49.1) and 19.1 (95% CI 11.1, 28.3) mos for osi and pbo arms, respectively. Overall, 86% (95% CI 78, 92) vs 36% (95% CI 27, 45) pts in the osi vs pbo arms were MRD/DFS event free at 36 mos (HR: 0.23; 95% CI 0.15, 0.36). MRD/DFS events in the osi arm were detected at any time post-BL in 28 (25%) pts, of whom 19/28 (68%) had events post-adj osi. Most (11/19 [58%]) MRD/DFS events detected occurred within 12 mos of osi tx completion. Conclusions: MRD+ preceded DFS events in most pts with a median lead time of 4.7 mos across both arms. MRD– was maintained for most pts during adj osi tx with the majority of MRD/DFS events occurring after osi tx completion. MRD detection could potentially identify a subset of pts likely to benefit from longer adj osi. Clinical trial information: NCT02511106 .

6 Background: Our previous analysis of data from the prospective, observational GALAXY study (UMIN000039205) reported post surgical detection of molecular residual disease (MRD) to be prognostic of patient (pt) outcomes and the most significant risk factor for recurrence regardless of BRAF V600E status. Here, we present an updated analysis and correlation of ctDNA dynamics with outcomes in pts with radically resected, stage II-IV CRC from the GALAXY study. Methods: A personalized, tumor-informed assay (Signatera, Natera, Inc.) was used for the detection and quantification of ctDNA in serial plasma samples collected at 1, 3, 6, 9, 12, 18, and 24 months post surgery until recurrence. CT scans of chest/abdomen/pelvis were conducted every 6 months. Post curative-intent surgery, pts underwent either treatment with adjuvant chemotherapy (ACT; N = 1,000) or observation (N = 1,518). The primary endpoint was disease-free survival (DFS), defined as the time between the date of surgery and date of detection of relapse/death due to any cause. Results: Of the 3,034 CRC pts enrolled between May 2020 and November 2022 in GALAXY study, 2,518 pts met the inclusion criteria and were analyzed in this substudy. The median follow-up was 16.3 months (range 0.1-37 mos). During the post-op MRD window, ctDNA results were available for 2,093 pts, 309 (14.8%) of whom were ctDNA+ and 1,784 (85.2%) were ctDNA-. Pts who were ctDNA+ during the MRD window (MRD+) had a significantly inferior DFS compared to MRD- pts (HR: 15.75, 95%CI: 12.59-19.68, p < 0.0001). Within the MRD+ group, a landmark analysis of ctDNA dynamics from MRD detection to 3-month time point revealed that pts who remained ctDNA+ were over 5-times more likely to recur compared to those who had ctDNA clearance (HR: 5.4, 95%CI: 3.58-7.67%, p < 0.0001). Among the 309 MRD+ pts, 181 received adjuvant therapy, 72.9% (132/181) of whom had ctDNA clearance. Notably, for those pts with subsequent ctDNA time points available, 68/126 (54%) had sustained clearance vs. 58/126 (46%) pts eventually returned ctDNA+. Pts with sustained clearance had remarkably better outcomes compared to those with transient ctDNA clearance (HR: 32.57, 95% CI: 9.94-106.76, p < 0.0001). Furthermore, we observed that among MRD+ pts treated with ACT, a 50% or greater decrease in ctDNA levels (mean tumor molecules/mL) at 6 months was associated with better DFS compared to pts with <50% decrease or increase in ctDNA levels (HR: 2.39, 95% CI: 1.32-4.34, p = 0.004). Conclusions: ctDNA-based detection of MRD as well as ctDNA dynamics in response to ACT were highly prognostic of pt outcomes. Ongoing randomized VEGA and ALTAIR studies in the CIRCULATE-Japan will establish clinical utility of ctDNA-guided adjuvant treatment. Clinical trial information: UMIN000039205.

A majority of patients with metastatic colorectal cancer (mCRC) experience recurrence post curative-intent surgery. The addition of adjuvant chemotherapy has shown to provide limited survival benefits when applied to all patients. Therefore, a biomarker to assess molecular residual disease (MRD) accurately and guide treatment selection is highly desirable for high-risk patients. This feasibility study evaluated the prognostic value of a tissue comprehensive genomic profiling (CGP)-informed, personalized circulating tumor DNA (ctDNA) assay (FoundationOne®Tracker) (Foundation Medicine, Inc., Cambridge, MA, USA) by correlating MRD status with clinical outcomes. ctDNA analysis was performed retrospectively on plasma samples from 69 patients with resected mCRC obtained at the MRD and the follow-up time point. Tissue CGP identified potentially actionable alterations in 54% (37/69) of patients. MRD-positivity was significantly associated with lower disease-free survival (DFS) (HR: 4.97, 95% CI: 2.67–9.24, p < 0.0001) and overall survival (OS) (HR: 27.05, 95% CI: 3.60–203.46, p < 0.0001). Similarly, ctDNA positive status at the follow-up time point correlated with a marked reduction in DFS (HR: 8.78, 95% CI: 3.59–21.49, p < 0.0001) and OS (HR: 20.06, 95% CI: 2.51–160.25, p < 0.0001). The overall sensitivity and specificity at the follow-up time point were 69% and 100%, respectively. Our results indicate that MRD detection using the tissue CGP-informed ctDNA assay is prognostic of survival outcomes in patients with resected mCRC. The concurrent MRD detection and identification of actionable alterations has the potential to guide perioperative clinical decision-making.

3609 Background: The role of biomarker-guided therapy for resectable colorectal cancer (CRC) remains unestablished. Circulating tumor DNA (ctDNA) has emerged as a minimally invasive biomarker for detecting molecular residual disease (MRD) and predicting recurrence. Tumor-informed ctDNA testing offers the advantage of tumor sequencing and MRD detection in a single workflow, suggesting its potential for facilitating integration of biomarker-guided therapy into perioperative treatment. Methods: GALAXY, a large-scale prospective observational study, monitored post-surgical ctDNA MRD status in patients with clinical stage II to IV or relapsed CRC (UMIN000039205). Tumor tissue and matched normal DNA were processed for whole-exome sequencing (WES) to identify and track patient-specific somatic single nucleotide variants (SNVs), for ctDNA detection in the associated patients’ plasma samples. Actionable biomarkers having OncoKB therapeutic levels 1-3 were explored and identified separately from the Signatera test (through WES data). Results: Of the 4,590 patients enrolled between May 2020, and October 2022, 2,695 who were not enrolled in randomized trials and had available WES data were included in this analysis. Identified actionable biomarkers included RAS/ BRAF wild-type in 1,369 (50.8%) patients, tumor mutational burden (TMB) high in 256 (10.3%), MSI high in 236 (9.5%), BRAF V600E in 176 (6.5%), KRAS G12C in 61 (2.3%), ERBB2 amplification in 47 (1.7%), TP53 Y220C in 33 (1.2%), NTRK1/ 2/ 3 fusion in 1 (0.04%), and RET fusion in 1 (0.04%). ctDNA positivity during the MRD window (2 to 10 weeks post-surgery) varied from 5.1% in those with TMB high and MSI high to 29.8% in those with ERBB2 amplification. ERBB2 amplification was the only actionable biomarker significantly associated with ctDNA positivity during the MRD window (odds ratio 2.20). Positive ctDNA MRD status correlated with significantly worse DFS across all actionable biomarkers, with hazard ratios ranging from 8.46 for TP53 Y220C to 5.53e+8 for BRAF V600E. In MSI high disease, recurrence was observed in only 3 of 225 (1.3%) patients with negative ctDNA compared to 5 of 12 (41.7%) with positive ctDNA during the MRD window. Adjustment by the propensity score revealed that adjuvant chemotherapy provided survival benefits in ctDNA-positive patients with pathological stage II and III disease across various biomarkers. In patients with one or more actionable mutations selected among the16-plex of the ctDNA MRD assay, those variants were detected at time of disease recurrence. Conclusions: Our study highlights the importance of ctDNA-based MRD as both a prognostic and predictive tool for CRC across different actionable biomarkers. These findings suggest the potential of MRD-driven targeted therapeutic strategies for CRC with specific actionable biomarkers. Clinical trial information: UMIN000039205.

156 Background: Despite receiving curative resection, a considerable percentage of colorectal cancer (CRC) patients still experience disease recurrence. The early detection of molecular residual disease (MRD) has the potential to improve risk assessment. Methods: A total of 104 patients with stage Ⅰ-Ⅳ CRC were enrolled, of which 259 serial plasma samples were collected. Multi-gene targeted sequencing was conducted on tumor and plasma samples to identify somatic variants. Results: Among the 104 patients, 14 cases had positive landmark MRD and 32 cases had positive longitudinal MRD. Patients with positive landmark MRD had significantly higher recurrence risk compared to those with negative landmark MRD (Hazard Ratio [HR]: 7.325; 95% CI, 2.877-18.647; P &lt; 0.001). Similarly, positive longitudinal MRD was associated with increased recurrence risk (HR: 9.385; 95% CI: 3.085-28.556; P &lt; 0.001), with a higher HR value. Positive MRD detection preceded CT confirmed recurrence in 85.7% of patients, with a median lead time of 198.5 days. In multivariate analysis, positive-MRD was the most significant prognostic factor for DFS. The combination of KRAS variant and MRD status improved the efficiency of prognostic prediction, the area under the curve value for the combination of MRD and KRAS was higher than MRD and KRAS. By analyzing the characteristic of cfDNA fragments, we found that mutation sites tended to be enriched in shorter cell free DNA (cfDNA) fragments. Conclusions: Our study demonstrated an excellent prognostic prediction potential of circulating tumor DNA-based MRD detection among postoperative CRC patients, especially the combination of KRAS variant and MRD improved the efficiency of risk stratification through MRD. This study may facilitate the incorporation of MRD and KRAS into prognostic prediction. Our study revealed that mutation sites tended to be enriched in shorter DNA fragments, providing valuable insights for future ctDNA detection method.

8519 Background: Identifying localized non-small cell lung cancer (NSCLC) patients with residual disease following curative intent therapy is difficult due to normal tissue changes caused by surgery or radiation and an inability to detect microscopic disease. Analysis of circulating tumor DNA (ctDNA) might enable identification of molecular residual disease (MRD) and personalization of adjuvant treatment approaches but has not been explored in lung cancer. Methods: We applied CAPP-Seq, an ultra-sensitive next-generation sequencing based ctDNA quantitation method, to pre- and post-treatment blood samples from a cohort of 41 patients treated with chemoradiation, radiotherapy or surgery for stage I-III primary lung cancer. Detection of ctDNA at a single MRD time-point within 4 months of treatment completion was compared with surveillance by cross-sectional imaging. Furthermore, we developed an approach for identification of tumor mutation burden based on mutations detected in plasma, leveraging whole exome sequencing data from 1,177 NSCLCs sequenced by TCGA. Results: Median follow-up time was 35 months. Pre-treatment ctDNA was detected in 38 (93%) patients and 19 (46%) had detectable post-treatment ctDNA MRD. MRD+ patients displayed significantly inferior 3-year freedom from progression (0% vs. 92%; HR 38; P &lt; 0.0001) and 3-year overall survival (8% vs. 75%; HR 12; P &lt; 0.0001) than MRD- patients. Detection of ctDNA MRD had positive and negative predictive values for disease progression of 100% and 93%, respectively. Furthermore, we non-invasively identified activating EGFR mutations or high mutational burden (≥5 CAPP-Seq non-synonymous mutations, corresponding to &gt; 200 non-synonymous mutations per exome or &gt; 4 single nucleotide variants per megabase of exome) in 47% of patients with detectable ctDNA MRD, suggesting potentially favorable responses to TKIs and immune checkpoint inhibitors, respectively. Conclusions: Our results indicate that ctDNA analysis accurately detects MRD in localized lung cancer patients and could facilitate personalized adjuvant treatment at early time-points when disease burden is minimal.

Survival benefit of adjuvant chemotherapy based on molecular residual disease detection in resected colorectal liver metastases: subgroup analysis from CIRCULATE-Japan GALAXY

e15537 Background: Circulating tumor DNA (ctDNA) has emerged as a biomarker that can define the risk of recurrence after curative-intent surgery for patients with colorectal cancer (CRC). However, beyond the predictive power of postoperative ctDNA detection, the efficacy and potential limitations of ctDNA detection urgently need to be fully elucidated in a large cohort of CRC. Methods: We enrolled 309 patients with stages Ⅰ to Ⅳ CRC who underwent definitive surgery. Tumor tissues from those patients were sequenced by a custom-designed next-generation sequencing (NGS) panel to identify somatic mutations, and plasma was analyzed using ctDNA-based molecular residual disease (MRD) assay which integrated tumor genotype–informed and tumor genotype–naïve ctDNA analysis. Results: ctDNA was detected after surgery in 5.4%, 13.8%, 15% and 30% of patients with stage Ⅰ, Ⅱ, Ⅲ and Ⅳ disease, respectively, and in 17.5% of all longitudinal samples. Detection of MRD after surgery and in serial plasma samples during follow-up were associated with shorter disease-free survival (DFS) (hazard ratio [HR], 13.17; P &lt; 0.0001 and HR, 14.44; P &lt; 0.0001, respectively) independent of tumor site, stage, and MSI status. Within the population, only 13 patients (6.6%) with longitudinal undetectable MRD recurred, resulting in a negative predictive value of 93.4%. Correspondingly, the positive predictive value of longitudinal detectable MRD was 79.6%, with a median lead time of 10.1 months. Further subgroup analyses revealed that adjuvant therapy did not confer a superior survival for patients with undetectable or detectable MRD. Moreover, lung-only and peritoneal metastases were less detectable by MRD. Conclusions: Postoperative ctDNA status is a strong predictor of recurrence independent of stage and MSI status. Longitudinal undetectable MRD could be used to define the potentially cured population in CRC patients undergoing curative-intent surgery.