Increase In ctDNA Research Articles

Early circulating tumor DNA (ctDNA) changes during treatment with immune checkpoint inhibitors (ICI) as a predictor of clinical outcomes in patients (pts) with advanced stage melanoma/skin cancer.

225 Background: ctDNA monitoring has shown promising results in predicting relapse in resected solid tumors. Its role in treatment response assessment and predicting survival outcomes in the unresectable or metastatic disease setting merits further investigation. In our study, we attempt to assess the role of early ctDNA changes in predicting disease response, progression, and overall survival outcomes in pts with advanced stage melanoma/skin cancer treated with ICI therapy. Methods: A retrospective analysis using a personalized, tumor-informed ctDNA assay (Natera) on prospectively collected plasma samples from pts with unresectable stage III/IV melanoma/skin cancer treated with anti-PD-1 based therapy at the University of Wisconsin (Madison) was performed. Baseline ctDNA levels were assessed prior to the start of treatment and at 3-4 weeks (i.e. prior to the second treatment dose). A logistic regression model was used to evaluate the odds of overall disease control [complete response + partial response + stable disease, per RECIST version 1.1] based on the change in ctDNA levels (decrease vs increase) between both time points. Cox proportional hazard models were used to investigate the effects of ctDNA level change on progression free survival (PFS) and overall survival (OS). Results: 46 pts were evaluated. 82% melanoma, 14% Merkel cell carcinoma, 2% skin adenocarcinoma and 2% squamous cell carcinoma. 77% were treated with dual ICI (anti-PD-1 based) therapy and 23% with anti-PD-1 monotherapy. Median follow up was 12.1 months. Median change in ctDNA levels from baseline were -4.83 MTM/mL among pts with ctDNA decrease and +37.39 MTM/mL among pts with ctDNA increase. A qualitative decrease in ctDNA level was associated with overall disease control (OR 65.00, 95% CI 11.88-587.89, p<0.0001), longer PFS (HR 0.08, 95% CI 0.03-0.22, p<0.0001), and longer OS (HR 0.17, 95% CI 0.05-0.54, p=0.0008) compared to an increase in ctDNA level from baseline to 3-4 weeks after starting ICI therapy. Median PFS was not reached (NR) and 1.63 months; and median OS was NR and 5.57 months among pts with ctDNA decrease and ctDNA increase, respectively. Conclusions: We found that early ctDNA dynamics after 3-4 weeks of ICI initiation in pts with advanced melanoma/skin cancer appears to be a candidate strategy to predict treatment response, risk of progression, and potentially long-term survival. Larger prospective studies are warranted to validate the utility of ctDNA in treatment monitoring.

Safety and anti-tumor activity of BAY 2927088 in patients with HER2-mutant NSCLC: Results from an expansion cohort of the SOHO-01 phase I/II study.

LBA8598 Background: HER2 ( ERBB2) mutations have been reported in approximately 2-4% of patients (pts) with NSCLC, with exon 20 insertions being the most common. BAY 2927088 is an oral, reversible tyrosine kinase inhibitor that potently inhibits mutant HER2 and mutant EGFR in preclinical models. Encouraging objective responses were observed in pts with NSCLC harboring a HER2-activating mutation and treated with BAY 2927088 in the dose-escalation/backfill part of the Phase I/II SOHO-01 trial (NCT05099172). More recently the FDA has granted Breakthrough Therapy designation for BAY 2927088 for previously treated pts with advanced NSCLC and activating HER2 mutations. Here we report the safety, anti-tumor activity, and longitudinal circulating tumor DNA (ctDNA) data in a cohort of pts treated with BAY 2927088 from the expansion part of this trial. Methods: Pts with advanced NSCLC harboring a HER2-activating mutation and who experienced disease progression after at least 1 systemic therapy, but naïve to HER2-targeted therapy, were enrolled and received BAY 2927088 at 20 mg twice daily. Plasma samples were collected at baseline and several on-treatment time points for longitudinal ctDNA profiling using next-generation sequencing (NGS; Oncomine Precision Assay). Results: As of February 19, 2024, 34 pts were treated with BAY 2927088, with a median follow-up of 8 months. Median age was 62 years, 68% were female, 74% had never smoked, and 53% had received ≥2 lines of systemic anti-cancer therapy. Median duration of treatment with BAY 2927088 was 7.1 months (range 0.2-9.2). Treatment was ongoing in 17 pts (50%). Ten pts had a dose reduction, 8 had dose interruptions, and 3 discontinued study treatment due to a drug-related adverse event. The most common adverse events were diarrhea (85%; mainly grade 1-2) and rash (47%; grade 1-2). In 33 pts evaluable for efficacy, responses were observed in 23 (objective response rate 70%; 95% CI 51.3, 84.4) and 5 (15%) had stable disease for a disease control rate of 82% (95% CI 64.5, 93.0). Responses were rapid (median time to response 5.7 weeks) and durable (median duration of response not reached). Median progression-free survival was 8.1 months (95% CI 4.4, not evaluable). In a subset of 20 pts with successful paired (baseline, on-treatment) blood NGS and detectable HER2 ctDNA at baseline, 19/20 (95%) had a decrease in ctDNA and 1 pt with progressive disease had an increase in ctDNA; 15/20 (75%) had no detectable ctDNA after 6 weeks of treatment (including 3/4 pts with stable disease). Conclusions: BAY 2927088 led to rapid, substantial, and durable responses in pts with pretreated HER2-mutant NSCLC. The safety profile was consistent with previously reported data. These data support the further clinical development of BAY 2927088 in pts with HER2-mutant NSCLC. Clinical trial information: NCT05099172 .

A randomized study evaluating tailoring of advanced/metastatic colorectal cancer (mCRC) therapy using circulating tumor DNA (ctDNA): TACT-D.

LBA3557 Background: Identifying non-responders to expensive salvage therapies with modest benefits and substantial treatment related adverse events (TRAEs) (e.g. regorafenib [Reg] or TAS102 [Tas] in mCRC) is key to precision care. Retrospective studies suggest that ctDNA changes at timepoints (4-10 weeks [wks] into therapy) before radiographic assessment may predict treatment outcomes. However, prospective studies assessing early ctDNA changes are lacking and clinical utility remains uncertain. Methods: TACT-D is a randomized study to validate early dynamic changes in ctDNA (ΔctDNA: change in either maximum variant allele frequency (maxVAF) or mean VAF in predicting treatment response/resistance. Patients (pts) with mCRC clinically eligible for Reg/Tas were randomly assigned 2:1 to either standard of care (SOC) or ctDNA arm. All pts had ctDNA sequencing by Guardant360 CDx assay on cycle 1 day 1 (C1D1) and C1D15. On SOC arm, pts were restaged at 8 wks. On ctDNA arm, ΔctDNA (C1D15 - C1D1) was run in real-time and increase in ctDNA (ΔctDNA > 0) triggered early radiographic restaging. Therapy was continued for responders (RECISTv1.1 stable disease/response) and stopped for progression (non-responders). Co-primary endpoints were: 1) comparison of TRAEs among study arms and 2) association of ΔctDNA and objective response rate (RR). Key secondary endpoints were progression free (PFS) and overall (OS) survival. Study was powered (82%; 2-sided α = .05) to detect 30% decrease in toxicity. Results: Between 4/2019 and 8/2023, 100 pts were randomized; 80 evaluable had median age of 56 years, 46% were females, 44% and 56% received Tas and Reg, respectively. Baseline ctDNA levels (ρ 0.90) and ΔctDNA (ρ 0.68) using maxVAF (reported below) and mean VAF showed strong correlation (P < .001). Median ΔctDNA for entire cohort was -47% with no significant difference by treatment arms (SOC -45% v Exp -58%, P = .79) and therapy (Tas -71 v Reg -44, P = .19). ΔctDNA increased in 18% pts. Grade 3/4 TRAEs (32% v 40%, P = .62) did not differ significantly between arms. No significant association was seen between ΔctDNA and RR (OR .88, P = 1.0), PFS (HR .99, P = .88) and OS (HR 1.00, P = .64). Notably, higher baseline maxVAF was strongly associated with response (median maxVAF: 21.8% in non-responders v 3.4% in responders), PFS (HR 1.02) and OS (HR 1.03) (all P < .001). After adjusting for baseline maxVAF, ΔctDNA was found to be associated with both PFS (HR 1.87, P = .038) and OS (HR 3.55, P = .001). Conclusions: In the first prospective study of clinical utility of monitoring ctDNA in mCRC, baseline ctDNA was strongly prognostic for clinical benefit from salvage therapies in mCRC. Adjusted for this prognostic impact, ΔctDNA between C1D1 and C1D15 was predictive of clinical outcomes. Efforts are needed to establish novel signatures, optimal cutoffs/intervals for assessing ctDNA response in mCRC, tailored to pts and their therapy. Clinical trial information: NCT03844620 .

Abstract 6558: Ultra-sensitive ctDNA detection predicts response to immune checkpoint inhibition in advanced melanoma patients

Abstract Immune checkpoint inhibition (ICI) provides clinical benefit to a subset of cancer patients. Monitoring circulating tumor DNA (ctDNA) in peripheral blood may allow clinicians to predict ICI response or resistance earlier than imaging and track response more frequently. In this study, melanoma patients receiving ICI were profiled using an ultra-sensitive, tumor-informed ctDNA detection platform, and the findings were correlated with clinical outcome. In this retrospective study, 188 plasma samples were collected from 23 advanced melanoma patients receiving immune checkpoint inhibitor (ICI) treatment, spanning a period of up to 40 months. Levels of ctDNA were then profiled using the NeXT Personal® assay. NeXT Personal is a liquid biopsy platform that utilizes whole-genome sequencing of tumor and normal samples to create a customized panel for each patient, encompassing up to 1,800 genetic somatic variants. This enables the detection of molecular residual disease (MRD) down to a limit of detection (LOD) of 1-3 parts per million (PPM). We subsequently compared the results obtained through NeXT Personal with RECIST-defined measurements and clinical outcomes. Ultra-sensitive ctDNA detection was achieved across a wide dynamic range, spanning from 2.3 PPM to over 130,000 PPM (median LOD = 2 PPM). A significant number of detections were attained at low PPM levels, with 30% (31/105) occurring below 100 PPM and 25% (26/105) occurring below 50 PPM. 94% (17/18) of baseline samples were ctDNA+ among patients without recent second-line therapy, including all patients who progressed. All ctDNA+ detections aligned with imaging results that indicated the presence of tumor. Likewise, all plasma timepoints corresponding to complete responses (CR) assessed via RECIST were ctDNA-. Early increase in ctDNA from baseline to treatment cycle 3 significantly predicted PFS (log-rank 0.003; Wald p=0.01, HR=8.35, 95% CI 1.62-43.15). Survival was predicted by both imaging CR (log-rank p=0.018) and ctDNA clearance (log-rank p=0.003). Molecular progression, defined as a 20% increase in ctDNA, significantly predicted OS (log-rank p=0.023), whereas imaging PD status did not predict OS (log-rank p=0.09). We achieved detection of ctDNA with an LOD down to 1.3 PPM in this cohort, with nearly a third of detections occurring below 100 PPM, suggesting the importance of an ultra-sensitive ctDNA approach even in late stage patients. Our findings suggest that ctDNA changes arepredictive of ICI response in late stage melanoma. These results highlight the potential for patient-specific ctDNA monitoring to inform patient management and for clincal trial design. Citation Format: Christoffer Gebhardt, Laura Keller, Isabel Heidrich, Julian Koett, Glenn Geidel, Daniel J. Smit, Ronald Simon, Stefan W. Schneider, Jason Pugh, Charles W. Abbott, Sean Boyle, Richard O. Chen, Klaus Pantel. Ultra-sensitive ctDNA detection predicts response to immune checkpoint inhibition in advanced melanoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6558.

Abstract 1209: Tumor informed circulating tumor DNA monitoring for early treatment response and survival outcomes on trastuzumab + pertuzumab

Abstract Background: Advances in cancer treatment have led to the rise of targeted therapies as practice-changing modalities, demonstrating significant efficacy in molecularly selected patients. However, depth of response and survival benefit is variable. Limitations in radiographic imaging create challenges in determining a clinical response. Circulating tumor DNA (ctDNA) is a promising minimally-invasive, predictive biomarker. Here we explore the utility of tumor-informed, on-treatment ctDNA dynamics for early response monitoring and prognostication in patients with HER2-positive tumors of various histologies receiving trastuzumab + pertuzumab on the phase II basket trial, MyPathway (NCT02091141). Methods: In this retrospective study, 58 patients were included based on availability of tissue comprehensive genomic profiling (CGP) results and baseline/on-treatment plasma. FoundationOne® Tracker was utilized to detect and quantify ctDNA, selecting 2-16 tumor-derived variants from tissue CGP and monitoring the corresponding patient’s plasma with multiplex PCR. ctDNA was assessed at baseline and Cycle 3 Day 1 (C3D1) of therapy. Correlations between ctDNA changes and objective response, progression-free survival (PFS) and overall survival (OS) were made using Kaplan Meier analyses and landmarked at C3D1. Patients ctDNA- at baseline and C3D1 were excluded. Results: Of the 58 patients, personalized ctDNA assays were successfully designed for 52 (89.7%) representing 14 unique tumor histologies. Of 52, 48 (92.3%) had ctDNA results available for baseline analysis with a median of 100.7 mean tumor molecules per mL (MTM/mL); 89.6% were ctDNA+ with a median of 119.5 MTM/mL. A total of 39 patients had both baseline and C3D1 results for ctDNA response analyses. On-treatment ctDNA decline >90% (N=10) was associated with longer survival compared to <90% decline (N=16) or any increase (N=13) in ctDNA (OS: NR, 9.4 months (mo); P= 0.007). CtDNA dynamics remained predictive in a disease specific analysis of colorectal cancer (N=18; OS: NR, 10.2 mo; P= 0.04). Similar results were seen when stratifying patients by baseline ctDNA level (above/below median MTM/mL) or HER2 (ERBB2) mutation vs amplification. ctDNA increase at C3D1 preceded radiographic progression by a median of 1.3 mo. In patients with stable disease, >90% decline in ctDNA was associated with longer OS compared to <90% decline or any increase (NR, 9.4 mo; P= 0.01). Conclusion: Tumor-informed ctDNA monitoring provides insight into early treatment response and survival outcomes in patients with diverse HER2 amplified or mutated tumors receiving HER2 targeted therapy. On-treatment ctDNA dynamics refine and can potentially detect response/progression ahead of standard of care response assessments, suggesting early ctDNA monitoring as a valuable complementary tool for real-time treatment response monitoring. Citation Format: Razelle Kurzrock, Merrida Childress, Wenshu Li, Anna Muse, Julia Malato, Yong Wang, Minetta Liu, Alexey Aleshin, Richard S. Wang, Julia Thierauf, Christopher J. Sweeney, John Hainsworth, Tania Szado, Amanda Young, Katja Schulze, David Spigel, Funda Meric-Bernstam, Charles Swanton, Howard A. Burris. Tumor informed circulating tumor DNA monitoring for early treatment response and survival outcomes on trastuzumab + pertuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1209.

Circulating Tumor DNA Dynamics Fail to Predict Efficacy of Poly(ADP-ribose) Polymerase/VEGFR Inhibition in Patients With Heavily Pretreated Advanced Solid Tumors.

Cell-free circulating tumor DNA (ctDNA) has shown its potential as a quantitative biomarker for longitudinal monitoring of response to anticancer therapies. However, ctDNA dynamics have not been studied in patients with heavily pretreated, advanced solid tumors, for whom therapeutic responses can be weak. We investigated whether changes in ctDNA could predict clinical outcomes in such a cohort treated with combined poly(ADP-ribose) polymerase/vascular endothelial growth factor receptor inhibitor therapy. Patients with metastatic pancreatic ductal adenocarcinoma (PDAC), triple-negative breast cancer (TNBC), small-cell lung cancer (SCLC), or non-small-cell lung cancer (NSCLC) received up to 7 days of cediranib 30 mg orally once daily monotherapy lead-in followed by addition of olaparib 200 mg orally twice daily. Patients had progressed on a median of three previous lines of therapy. Plasma samples were collected before and after cediranib monotherapy lead-in and on combination therapy at 7 days, 28 days, and every 28 days thereafter. ctDNA was quantified from plasma samples using a multigene mutation-based assay. Radiographic assessment was performed every 8 weeks. ctDNA measurements were evaluable in 63 patients. The median baseline ctDNA variant allele fractions (VAFs) were 20%, 28%, 27%, and 34% for PDAC, TNBC, SCLC, and NSCLC, respectively. No association was observed between baseline VAF and radiographic response, progression-free survival, or overall survival (OS). Similarly, no association was found between ctDNA decline and radiographic response or survival. However, an increase in ctDNA at 56 days of combination therapy was associated with disease progression and inferior OS in a landmark analysis. ctDNA levels or dynamics did not correlate with radiographic response or survival outcomes in patients with advanced metastatic malignancies treated with olaparib and cediranib.

Early On-treatment Circulating Tumor DNA Measurements and Response to Immune Checkpoint Inhibitors in Advanced Urothelial Cancer

BackgroundImmune checkpoint inhibitors (ICIs) can induce durable disease control in metastatic urothelial cancer (mUC), but only 20–25% of patients respond. Early identification of a nondurable response will improve management strategies. ObjectiveTo investigate whether on-treatment circulating tumor DNA (ctDNA) measurements can predict ICI responsiveness in mUC patients. Design, setting, and participantsThis study consists of a discovery cohort of 40 mUC patients and a prospective multicenter validation cohort of 16 mUC patients. Plasma cell-free DNA was collected at baseline and after 3 and 6 wk on ICIs. The ctDNA levels were calculated from targeted sequencing. Outcome measurements and statistical analysisOutcome measurements were progression-free survival (PFS), overall survival (OS), and nondurable response (PFS ≤6 mo). Relationships with ctDNA were assessed using Cox regression. Changes in ctDNA level at 3 and 6 wk were categorized by an increase or decrease relative to baseline. Results and limitationsIn the discovery cohort, ctDNA was detected in 37/40 (93%) of patients at baseline. A ctDNA increase was observed in 12/15 (80%) and ten of 12 (83%) patients with a nondurable response at 3 and 6 wk, respectively. Of patients with a durable response (PFS >6 mo), 94% showed a decrease. A ctDNA increase at 3 wk was associated with shorter PFS (hazard ratio [HR] 7.8, 95% confidence interval [CI] 3.1–19.5) and OS (HR 8.0, 95% CI 3.0–21.0), independent of clinical prognostic variables. Similar results were observed at 6 wk. The 3-wk association with PFS was validated in a prospective cohort (HR 7.5, 95% CI 1.3–42.6). Limitations include the limited number of patients. ConclusionsEarly changes in ctDNA levels are strongly linked to the duration of ICI benefit in mUC and may contribute to timely therapy modifications. Patient summaryBenefit from immunotherapy can be predicted after only 3 wk of treatment by investigating cancer DNA in blood. This could help in timely therapy changes for urothelial cancer patients with limited benefit from immunotherapy.

Utilization of circulating tumor DNA (ctDNA) analysis to detect minimal residual disease post-surgery and disease progression in metastatic thymic tumors.

8598 Background: Thymic epithelial tumors (TETs) are rare and tend to have an indolent disease course. Follow-up consists of physical examinations and routine imaging (CT scans) with no currently available biomarkers to complement surveillance. This study aimed to assess the utility of ctDNA in MRD detection and disease monitoring in TETs. Methods: From Nov 2020 till Feb 2022, ctDNA analysis using a tumor-informed mPCR-NGS (Signatera) assay was performed on 29 patients (pts) with primary (pTETs) or metastatic TETs (mTETs). For pts with early-stage disease, the baseline ctDNA was measured 4-8 w post-surgery and repeated q3m. For mTETs, ctDNA was measured at the first visit, and then q3m with interval CT scan q3-4 m. ctDNA was quantified as mean tumor molecules (MTM)/mL of plasma. All pts had at least one measurement (up to 4). GraphPad was used for statistical analysis. Results: Of 29 pts overall, 9 pts were excluded at the time of analysis for various reasons; a) insufficient specimen (n=5), b) presence of concurrent active cancer (n=2), and c) final report delay (n=2). Of 20 evaluable pts, 7 pts had pTETs and 13 mTETs. The median age was 50 yrs (range, 17-72), and WHO histology distribution of AB=3, B1=5, B2=9, B3=12, TC=3. History of autoimmune disease and second malignancy (not active at the time of obtaining ctDNA) was reported in 33% (n=9) and 40% (n=8) of evaluable pts, respectively. Of 13 metastatic pts, 6 had stage IVA with pleural-only metastasis, 7 pts had Stage IVB with 2 extra-thoracic (liver, adrenal), and 5 with intrathoracic (lung, chest wall, and LNs) involvement. From 7 pTETs, 6 pts had R0 resection with undetectable post-operative ctDNA, while R1 resection with detectable ctDNA was reported (0.47 MTM/mL) in one pt. In mTETs, the mean level of ctDNA in pts with the stable disease was 0.18 MTM/mL (0.1-0.2), pleural-only progression 1.26 MTM/mL (0.1-1.9), and progressive disease (primary and/or pleural) 7.67 MTM/mL(5.04-9.5). Comparing the mean level of ctDNA in clinically stable metastatic pts with those who had progression, pts with pleural-only progression did not have a significantly higher level of ctDNA (p=0.51). In contrast, pts with primary and/or pleural progression had significantly higher levels of ctDNA (p=0.02). The level of ctDNA correlated with tumor size. An increase of 4-6mm in tumor size was associated with an approximate 0.2 MTM/mL increase in ctDNA or making the undetectable status detectable. Conclusions: The ctDNA level correlates well with the tumor volume in mTETs. In pTETs, ctDNA was negative following R0 resection but not in the R1, supporting its potential use as a surrogate biomarker for MRD. This is a pilot study, and the expansion of these data is ongoing to validate the utility of ctDNA testing in these settings. If validated, ctDNA may augment or minimize the routine use of CT scans for disease monitoring in resected and mTETs.

MO-0873 Rapid increase in ctDNA at commencement of curative-intent radiotherapy for NSCLC

MO-0873 Rapid increase in ctDNA at commencement of curative-intent radiotherapy for NSCLC

Abstract 1052: Background variability in plasma ctDNA levels in patients with advanced lung cancer in the absence of treatment

Abstract Introduction: Molecular response (MR) is defined as a reduction in circulating tumor DNA (ctDNA) levels between baseline and early on-treatment timepoint(s). MR is predictive of long-term outcomes in several advanced cancers treated with different therapeutic modalities. Common MR thresholds include reductions in ≥50%, or 100% (clearance) in ctDNA levels. A better understanding of the background variability in measured plasma ctDNA levels in the absence of therapy is required to enable improved interpretation of MR, especially as its use increases in drug development and clinical management. Methods: In the largest study of double baseline pre-treatment ctDNA samples reported to date, paired screening and pre-treatment cycle 1 day 1 (C1D1) samples (IQR: 8-24, median 17 days apart) from 360 patients with advanced EGFRm non-small cell lung cancer (NSCLC) from the FLAURA (1L, N=132) and AURA3 (2L, N=228) trials were compared to characterize background variability in ctDNA. ctDNA was quantified using NGS (Guardant Health G360 and OMNI) and ddPCR (Biodesix) assays. Various potential drivers of background variability, such as ctDNA quantification platforms, coverage, and number of variants at baseline were evaluated to better understand any underlying confounding effects. Results: Pre-treatment background variability was consistently observed in both AURA3 and FLAURA cohorts. Most of the variability reflected any increase in pre-treatment ctDNA in 28% (64/228) of cases in AURA3, and 13% (17/132) of cases in FLAURA. Variability reflecting a ≥50% decrease in pre-treatment ctDNA was observed in 8% (18/228) of cases in AURA3 and 9% (12/132) of cases in FLAURA, with only 2% of cases showing ctDNA clearance in AURA3 and 0% in FLAURA. Testing technical replicates sequenced a year apart and comparing ctDNA levels quantified with NGS v. ddPCR for potential sources of technical variability did not show large variability. Moreover, sources like cell-free DNA input, allele frequency levels, and days in between screening and C1D1 were tested but could not fully account for the variability observed. Conclusion: Although pre-treatment increases in ctDNA levels were expected in patients with advanced cancer, background variability also included reductions in ctDNA that may be confounded as MR. Understanding the impact of factors on the transient nature of ctDNA is key, but as momentum mounts to use ctDNA as a biomarker of therapeutic response, it is crucial to test and account for background variability rates to improve MR interpretability. Additional work is warranted to elucidate the background variability rate in other cancer types and settings. Citation Format: Diana M. Vega, Aleksandra Markovets, Christopher Abbosh, Katie Quinn, Kyle Chang, Sara Wienke, Ryan Hartmaier, Matthew Hellmann, J Carl Barrett, Darren Hodgson. Background variability in plasma ctDNA levels in patients with advanced lung cancer in the absence of treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1052.

Circulating Tumor DNA Monitoring Reveals Molecular Progression before Radiologic Progression in a Real-life Cohort of Patients with Advanced Non-small Cell Lung Cancer.

The clinical potential of liquid biopsy in patients with advanced cancer is real-time monitoring for early detection of treatment failure. Our study aimed to investigate the clinical validity of circulating tumor DNA (ctDNA) treatment monitoring in a real-life cohort of patients with advanced non-small cell lung cancer (NSCLC). Patients with advanced or noncurative locally advanced NSCLC were prospectively included in an exploratory study (NCT03512847). Selected cancer-specific mutations were measured in plasma by standard or uniquely designed droplet digital PCR assays before every treatment cycle during first-line treatment until progressive disease (PD). Correlation between an increase in ctDNA (= molecular progression) and radiologic PD was investigated, defined as lead time, and the corresponding numbers of likely futile treatment cycles were determined. Utility of ctDNA measurements in clarifying the results of nonconclusive radiologic evaluation scans was evaluated. Cancer-specific mutations and longitudinal plasma sampling were present in 132 of 150 patients. ctDNA was detectable in 88 (67%) of 132 patients treated by respectively chemotherapy (n = 41), immunotherapy (n = 43), or combination treatment (n = 4). In 66 (90%) of 73 patients experiencing PD, a ctDNA increase was observed with a median lead time of 1.5 months before radiologic PD. Overall, 119 (33%) of 365 treatment cycles were administered after molecular progression. In addition, ctDNA measurements could clarify the results in 38 (79%) of 48 nonconclusive radiologic evaluations. ctDNA monitoring leads to earlier detection of treatment failure, and clarifies the majority of nonconclusive radiologic evaluations, giving the potential of sparing patients from likely futile treatments and needless adverse events. Treatment monitoring by ctDNA has the clinical potential to reveal PD before radiologic evaluation and consequently spare patients with advanced cancer from likely ineffective, costly cancer treatments and adverse events.

Circulating DNA in the neoadjuvant setting of early stage colon cancer

Background While circulating tumour (ct)DNA is an indicator of minimal residual disease and negative prognostic factor in stage II-III colon cancer, no study has ever analysed the value of this biomarker in colon cancer patients treated with neoadjuvant chemotherapy. We sought to fill this gap by using prospectively collected plasma samples from 80 stage III colon cancer patients, receiving one cycle of neoadjuvant FOLFOX followed by surgery +/− adjuvant FOLFOX in the PePiTA trial. Material and Methods Samples were collected at baseline, 2 weeks and surgery. NPY and WIF1 were selected as universal methylation markers for ctDNA, and analysed with ddPCR technology. ROC curves were applied for cut-off points, and outcome measures included 5-year disease-free survival (DFS) and 6-year overall survival (OS). Results After a median follow-up of 52.5 months, baseline circulating-free (cf) DNA was an independent prognostic factor for DFS (HR 3.35, 95% CI: 1.15–9.77, p = .03), and a trend towards a similar association was observed for relative cfDNA changes between baseline and surgery (HR 2.57, 95% CI: 0.94–7.05, p = .07). Among 60 ctDNA assessable patients, 25 (42%) had detectable ctDNA at baseline. While detection of ctDNA at any pre-operative timepoint was not associated with outcome, patients with ctDNA increase (change of the worst trending methylation marker ≥11%, or mean ctDNA change of NPY and WIF1 ≥ 0%) between baseline and surgery showed a trend towards worse 5-year DFS (HR 3.66, 95% CI: 0.81–16.44, p = .09). Conclusion This is the first study of ctDNA in the neoadjuvant setting of early-stage colon cancer. Results are hypothesis-generating and should be confirmed in larger series.

Monitoring efficacy of neoadjuvant sunitinib in metastatic renal cell carcinoma using a personalized and tumor informed ctDNA assay.

4525 Background: Approximately 30% of renal cell carcinoma (RCC) cases present as stage IV at the time of diagnosis. Metastatic RCC (mRCC) is associated with poor outcomes, with a five-year survival rate below 50%. Tools to evaluate the efficacy of novel systemic regimens are needed. Biomarkers like ctDNA can accurately and non-invasively assess molecular residual disease (MRD) in response to therapy and monitor disease status over time to predict disease progression. Here, we aimed to determine the feasibility of performing personalized and tumor-informed ctDNA testing in mRCC patients enrolled on a study integrating systemic therapy with surgical cytoreduction, as well as to measure patient response to sunitinib by assessing ctDNA dynamics. Methods: We analyzed a cohort of 21 mRCC patients with median age of 59.5 (43-76) years who were treated with sunitinib, and had planned cytoreductive nephrectomy during their second cycle of therapy. Baseline and post-first cycle ctDNA levels were measured using a personalized and tumor-informed ctDNA assay (SignateraTM bespoke mPCR NGS assay). Changes in ctDNA levels from baseline to the best response time point were correlated with disease status as assessed by radiological imaging. Results: In this cohort, baseline ctDNA was detected in 81% (17/21) of patients, with higher ctDNA concentrations observed in patients who presented with multiple distant metastases (n = 11) compared to the 10 cases with a single metastatic mass (median 5.8 vs 1.3 mean tumor molecules/mL, not statistically significant). Of those with baseline ctDNA measurement, 12% (2/17) cleared their ctDNA, 24% (4/17) had a decrease in ctDNA, and 59% (10/17) had an increase in ctDNA after 4 weeks of sunitinib. During the course of treatment, patients whose ctDNA concentration increased from baseline were more likely to experience a disease progression (HR: 3.9 95% CI 1.13-13.7; p = 0.032) compared to those whose ctDNA decreased. In addition, higher ctDNA concentration before surgery after initial treatment with sunitinib correlated with shorter time to progression (p = 0.04). Conclusions: Our results demonstrate the feasibility and prognostic value of personalized and tumor-informed ctDNA testing for determining response to systemic therapy in patients with mRCC. Early signs of unfavorable ctDNA kinetics can provide rationale for modification of systemic therapy in order to enhance response. Future work exploring the clinical utility of ctDNA testing in larger mRCC cohorts is warranted.

Cell-Free Tumor DNA Dominant Clone Allele Frequency Is Associated With Poor Outcomes in Advanced Biliary Cancers Treated With Platinum-Based Chemotherapy.

This investigation sought to evaluate the prognostic value of pretreatment of circulating tumor DNA (ctDNA) in metastatic biliary tract cancers (BTCs) treated with platinum-based first-line chemotherapy treatment. We performed a retrospective analysis of 67 patients who underwent ctDNA testing before platinum-based chemotherapy for first-line treatment for metastatic BTC. For analysis, we considered the detected gene with highest variant allele frequency as the dominant clone allele frequency (DCAF). Results of ctDNA analysis were correlated with patients' demographics, progression-free survival (PFS), and overall survival (OS). The median age of patients was 67 (27-90) years. Fifty-four (80.6%) of 67 patients evaluated had intrahepatic cholangiocarcinoma; seven had extrahepatic cholangiocarcinoma, and six gallbladder cancers. Forty-six (68.6%) of the patients were treated with cisplatin plus gemcitabine, and 16.4% of patients received gemcitabine and other platinum (carboplatin or oxaliplatin) combinations, whereas 15% of patients were treated on a clinical trial with gemcitabine and cisplatin plus additional agents (CX4945, PEGPH20, or nab-paclitaxel). TP53, KRAS, FGFR2, ARID1A, STK11, and IDH1 were the genes with highest frequency as DCAF. The median DCAF was 3% (0%-97%). DCAF > 3% was associated with worse OS (median OS: 10.8 v 18.8 months, P = .032). Stratifying DCAF in quartiles, DCAF > 10% was significantly related to worse PFS (median PFS: 3 months, P = .014) and worse OS (median OS: 7.0 months, P = .001). Each 1% increase in ctDNA was associated with a hazard ratio of 13.1 in OS when adjusting for subtypes, metastatic sites, size of largest tumor, age, sex, and CA19-9. DCAF at diagnosis of advanced BTC can stratify patients who have worse outcomes when treated with upfront platinum-based chemotherapy. Each increase in %ctDNA decreases survival probabilities.

Abstract P1-18-05: Early changes in circulating tumor DNA and its effect on clinical outcomes in patients with advanced breast cancer receiving the CDK4/6 inhibitor palbociclib: Genotyping results from POLARIS

Abstract Background: POLARIS is a prospective, real-world study of palbociclib in patients with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2–) advanced breast cancer (ABC) in the United States and Canada. These analyses evaluated the difference in tumor mutation profiles of patients receiving palbociclib in the first line versus second line or greater and examined the applicability of circulating tumor DNA (ctDNA) monitoring in a real-world setting. Methods: The clinical database cut-off date was March 16, 2021. Patients in the biomarker analysis group provided consent for serial blood sample collections, received ≥1 dose of initial palbociclib combination treatment, and had ≥1 ctDNA measurement available. The Guardant360 platform with somatic single-nucleotide variants in complete or critical exons of 73 genes was used. The association between early changes in ctDNA mutation allele fractions at Cycle 2 Day 1 (C2D1) and disease progression at Week 24 was evaluated using univariate and multivariable logistic regression analysis adjusting for line of therapy. Results: Among patients with HR+/HER2– ABC (N=347), 93.9% (n=326) had ctDNA measured at baseline, of which 85.9% (n=280) had ≥1 ctDNA alteration detected; 66.6% of patients received palbociclib treatment in the first line and 33.4% as second line or greater ABC setting. The frequencies of gene mutations at baseline were generally higher in patients receiving palbociclib as a second-line or greater therapy compared to those who received it as first-line therapy, particularly for ESR1 mutations (30% vs 15%) and FGFR1 mutations (13% vs 9%). With a median (range) follow-up duration of 18.5 (0.1-46.3) months, patients with total ctDNA increase at C2D1 (log ratio change >0) were 3.74 times more likely to have disease progression at Week 24 (odds ratio, 3.74 [95% CI, 1.71-8.17]; P=0.001) compared with those without change or with a decrease in ctDNA at C2D1 (log ratio change ≤0). The observed significant association remained after adjusting for line of therapy in the multivariable regression analysis (odds ratio, 2.62 [95% CI, 1.14-6.04]; P=0.023). Conclusions: Among patients with HR+/HER2- ABC receiving palbociclib, early changes in ctDNA mutations were significantly associated with disease progression. Further studies are needed to confirm these findings and to evaluate the clinical utility of ctDNA-guided “adaptive” early therapeutic interventions to improve outcomes in patients with metastatic breast cancer. Pfizer; NCT03280303 Citation Format: Debu Tripathy, Zhe Zhang, Joanne L. Blum, Meghan S. Karuturi, Steven L. McCune, Bijoy Telivala, Shailendra Lakhanpal, Kamal Patel, Richard C. Frank, Kit Lu, Chetan Deshpande, Yao Wang, Yuan Liu, Aditya Bardia. Early changes in circulating tumor DNA and its effect on clinical outcomes in patients with advanced breast cancer receiving the CDK4/6 inhibitor palbociclib: Genotyping results from POLARIS [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 P1-18-05.

A phase II randomized therapeutic optimization trial for subjects with refractory metastatic colorectal cancer using circulating tumor DNA (ctDNA): Rapid 1 trial.

TPS223 Background: Patients with advanced colorectal cancer after progressing through first line therapy, have several FDA-approved systemic therapies that are associated with clinical benefit for a substantive minority of patients. Current clinical practice is to trial these various treatments in a step-wise fashion using CT scans every 3 months to evaluate effectiveness. This process requires 3-4 months between therapeutic interventions from which the patient may ultimately derive no clinical benefit, may have a performance status decline; limiting the number of possible interventions and increases risk for physical and financial toxicity. An alternative to the traditional CT-scan guided approach for disease assessment is a circulating tumor DNA (ctDNA) intervention. The Signatera ctDNA assay which utilizes 16 truncal mutations derived from a patient’s tumor, can be assessed every 2 weeks for a rapid determination of the effectiveness of a systemic therapy. This may allow patients to be exposed to many treatments during a short time, limiting toxicity, allowing for a quicker determination of clinical benefit and personalization of treatment. The aim of this study will be to compare the traditional scan-driven approach vs an intervention guided by ctDNA assessments, both arms using a pre-specified order of chemotherapy treatments. Methods: This is a phase 2 randomized study of patients with refractory metastatic adenocarcinoma of the colon or rectum. Participants are eligible after progression or intolerance to first line chemotherapy or recurrence within 6 months of adjuvant oxaliplatin based chemotherapy. They must have RECIST measurable metastatic disease that is not eligible for definitive management. Tissue from the primary and/or metastatic deposit is required for Signatera NGS analysis and subjects must have measurable ctDNA at sampling. Participants must be ≥18 years old without major organ dysfunction and have an ECOG performance status of 0 to 2. Subjects with Microsatellite High, deficient in DNA mismatch repair genes, or BRAF V600E mutations are excluded. Subjects will be randomized 1:1 to Arm A (ctDNA guided intervention) or Arm B (scan-guided control group). Patients in both arms will undergo systemic treatments in a standardized pre-specified order. Arm A will have ctDNA assessments every 2 weeks until an intervention shows a significant decrease, then every 4 weeks until Progressive Disease (PD) by scan or significant ctDNA increase. CT imaging will be performed every 12 weeks. Those in Arm B, will have CT imaging every 12 weeks and blood collected for post-hoc analysis every 4 weeks until PD by scan. The primary endpoint is overall survival. Secondary endpoints include progression free survival and overall response. Exploratory analysis with be performed of the microbiome. Enrollment continues to a maximum of 78 patients. Clinical trial information: NCT04786600.

Early HPV ctDNA Kinetics and Imaging Biomarkers Predict Therapeutic Response in p16+ Oropharyngeal Squamous Cell Carcinoma.

In locally advanced p16+ oropharyngeal squamous cell carcinoma (OPSCC), (i) to investigate kinetics of human papillomavirus (HPV) circulating tumor DNA (ctDNA) and association with tumor progression after chemoradiation, and (ii) to compare the predictive value of ctDNA to imaging biomarkers of MRI and FDG-PET. Serial blood samples were collected from patients with AJCC8 stage III OPSCC (n = 34) enrolled on a randomized trial: pretreatment; during chemoradiation at weeks 2, 4, and 7; and posttreatment. All patients also had dynamic-contrast-enhanced and diffusion-weighted MRI, as well as FDG-PET scans pre-chemoradiation and week 2 during chemoradiation. ctDNA values were analyzed for prediction of freedom from progression (FFP), and correlations with aggressive tumor subvolumes with low blood volume (TVLBV) and low apparent diffusion coefficient (TVLADC), and metabolic tumor volume (MTV) using Cox proportional hazards model and Spearman rank correlation. Low pretreatment ctDNA and an early increase in ctDNA at week 2 compared with baseline were significantly associated with superior FFP (P < 0.02 and P < 0.05, respectively). At week 4 or 7, neither ctDNA counts nor clearance were significantly predictive of progression (P = 0.8). Pretreatment ctDNA values were significantly correlated with nodal TVLBV, TVLADC, and MTV pre-chemoradiation (P < 0.03), while the ctDNA values at week 2 were correlated with these imaging metrics in primary tumor. Multivariate analysis showed that ctDNA and the imaging metrics performed comparably to predict FFP. Early ctDNA kinetics during definitive chemoradiation may predict therapy response in stage III OPSCC.

Detection of clinical progression through plasma ctDNA in metastatic melanoma patients: a comparison to radiological progression

BackgroundThe validity of circulating tumour DNA (ctDNA) as an indicator of disease progression compared to medical imaging in patients with metastatic melanoma requires detailed evaluation.MethodsHere, we carried out a retrospective ctDNA analysis of 108 plasma samples collected at the time of disease progression. We also analysed a validation cohort of 66 metastatic melanoma patients monitored prospectively after response to systemic therapy.ResultsctDNA was detected in 62% of patients at the time of disease progression. For 67 patients that responded to treatment, the mean ctDNA level at progressive disease was significantly higher than at the time of response (P < 0.0001). However, only 30 of these 67 (45%) patients had a statistically significant increase in ctDNA by Poisson test. A validation cohort of 66 metastatic melanoma patients monitored prospectively indicated a 56% detection rate of ctDNA at progression, with only two cases showing increased ctDNA prior to radiological progression. Finally, a correlation between ctDNA levels and metabolic tumour burden was only observed in treatment naïve patients but not at the time of progression in a subgroup of patients failing BRAF inhibition (N = 15).ConclusionsThese results highlight the low efficacy of ctDNA to detect disease progression in melanoma when compared mainly to standard positron emission tomography imaging.

Dynamics of circulating tumor DNA in patients with advanced solid tumors treated with cediranib and olaparib.

3035 Background: Circulating tumor DNA (ctDNA) has emerged as a potential biomarker to monitor treatment response in solid tumors. Our group previously showed that changes in ctDNA levels were predictive of radiographic response and survival in NSCLC patients receiving immunotherapy. Here we evaluated whether ctDNA dynamics could similarly be used to assess response in a PARP inhibitor-based therapy. Methods: A total of 122 patients with NSCLC, TNBC, PDAC or SCLC received cediranib (C) 30mg daily and Olaparib (O) 200mg twice daily in a phase II study NCI9881. Using a multigene NGS assay, ctDNA was measured longitudinally at baseline (T0), after 3 to 7 days of C monotherapy (T1), after 1 week of C+O combination (T2), after 4 weeks of C+O (T3), and every 4 weeks (T4+) thereafter. The first radiographic assessment was done after 8 weeks of C+O and every 8-12 weeks thereafter. CtDNA was quantified by determining the allele fraction of cancer-associated somatic mutations in plasma. We defined an early ctDNA response (e-ctDNA-R) as a &gt;10% decrease in mutant allele fraction from T0 to T2, and an early ctDNA progression (e-ctDNA-P) as a &gt; 10% increase; otherwise, it was stable ctDNA (e-ctDNA-S). Results: In total, 493 samples were analyzed from 94 patients, and 40 unique patients had both T0 and T2 ctDNA measurements, as well as corresponding radiographic assessments. These included 10 NSCLC, 17 TNBC, 3 SCLC and 10 PDAC. Of these patients, 4, 21, and 15 patients had PR, SD, and PD as best overall radiographic response respectively. Twenty-three (57.5%) patients had either e-ctDNA-R (17, 42.5%) or e-ctDNA-S (6, 15.0%), 18 (78.3%) of whom subsequently had either radiographic partial response (PR) or stable disease (SD). Seventeen (42.5%) patients had e-ctDNA-P, 10 (58.8%) of whom then had PD. A fair agreement was observed between e-ctDNA-R/S or e-ctDNA-P and radiographic PR/SD or PD with Cohen’s k 0.38 (70% agreement). The correlation between early ctDNA changes and PFS/OS are summarized in the table. All 25 patients with PR/SD eventually progressed. Of these, 17 (68%) had &gt;10% increase in ctDNA from the nadir prior to disease progression (median 94.6%, 95%CI 38.2%-389.1%). The time between ctDNA progression and ctDNA nadir was significantly shorter (median 21 days, 95%CI 21-28) than the time between radiographic/clinical progression and initial PR/SD (median 107 days, 95%CI 56-204, P=0.0015). Conclusions: Longitudinal ctDNA measurements could enable early assessment of treatment response, resistance, and disease progression in patients treated with PARP inhibitor-based therapy. However, in this study, tumor responses and ctDNA changes were generally not as robust as have been observed with other classes of therapy.[Table: see text]

Prognostic value of baseline and early changes of circulating-free (cf) and circulating tumor (ct) DNA in the neoadjuvant (NA) setting of early stage colon cancer (CC).

3600 Background: ctDNA is an indicator of minimal residual disease and negative prognostic factor in stage II-III CC treated with surgery +/- adjuvant chemotherapy (CT). No study, however, has ever analysed the prognostic value of this biomarker in CC patients (pts) treated with NACT. We sought to evaluate the prognostic value of baseline and early changes of cf/ctDNA in stage II-III CC pts who were treated with one cycle of NA FOLFOX CT followed by surgery +/- adjuvant FOLFOX CT in the PePiTA trial. Methods: PePiTA was a multicentre, single-arm, prospective phase II trial testing in vivo tumour chemosensitivity of early stage CC (as assessed by 18F-FDG PET/CT-based metabolic response to one cycle of NA FOLFOX) and its association with long-term outcome (NCT00994864). Plasma samples were prospectively collected at baseline, 2 weeks after one cycle of NA FOLFOX CT, and before surgery. NPY and WIF1 were selected as universal methylation markers for ctDNA and analysed with digital droplet (dd)PCR technology. Data from ddPCR were processed with the QuantaSoft v1.6 software (Bio-Rad). Survival outcome measures were 5-year disease-free survival (DFS) and 6-year overall survival (OS). ROC curve analyses, Kaplan-Meier method, cox proportional hazards models and log-rank tests were used. Statistical analyses were carried out with SPSS v25.0 (SPSS Inc.). Results: 80 pts were included (44 ypStage I-II and 36 ypStage III). After a median follow-up of 52.5 months, 5-year DFS and 6-year OS were 68% (95%CI 52-84) and 84% (95%CI 74-94), respectively. Pts with high (≥1600 ng/ml) baseline cfDNA had worse 6-year OS (HR 6.45, 95%CI 1.61-25.84; p = 0.008). Early changes of cfDNA after one cycle of NA FOLFOX CT failed to predict survival (HR DFS 0.96, 95%CI 0.38-2.43; p = 0.92; HR OS 0.62, 95%CI 0.16-2.50; p = 0.50). At baseline, 25 out of 60 (42%) ctDNA-assessable patients were positive. Detectable ctDNA at baseline (HR DFS 2.06, 95%CI 0.65-6.49; p = 0.22; HR OS 3.11, 95%CI 0.57-16.99; p = 0.19) or at any timepoint before surgery (HR DFS 1.65, 95%CI 0.54-5.04; p = 0.38; HR OS 2.80, 95%CI 0.54-14.44; p = 0.22) was not significantly associated with survival. A trend toward a significant association between ctDNA increase at surgery and 5-year DFS was found (HR 3.66, 95%CI 0.81-16.44; p = 0.09). Data on the correlation between early changes of cf/ctDNA and 18F-FDG PET/CT-based metabolic response will be presented at the meeting. Conclusions: For the first time, we have shown that baseline cfDNA may predict survival outcome in early stage CC pts treated with NACT. Pending confirmation in larger series, testing for cfDNA at baseline could help select high-risk pts who may benefit from FOxTROT-like, NACT treatment strategies. While analysis of ctDNA in this setting did not appear useful to predict prognosis, these results might be secondary to the small sample size.