Changes In ctDNA Research Articles

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.

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 2490: Quantification of tumor fraction and outcomes association in a real-world non-small cell lung cancer (NSCLC) cohort using a tissue agnostic epigenomic circulating tumor DNA (ctDNA) assay

Abstract Background: The validity of ctDNA assays for evaluating molecular response to therapy by measuring changes in variant allele fraction (VAF) while on treatment is well established, but they are prone to limitations such as low ctDNA levels and interference from copy number variation and clonal hematopoeisis (CH), which may be overcome by methylation-based quantification. ctDNA levels may be monitored throughout a patient's (pts) journey to indicate when relapse or progression is present, often sooner than current methods (RECIST). Here we describe the performance of a methylation-based ctDNA assay to quantify ctDNA levels and correlate changes with outcomes in a real-world NSCLC cohort. Methods: RADIOHEAD is a pan-tumor observational study of 1200 patients on standard of care ICI treatment regimens with plasma samples collected prospectively for retrospective analysis. We randomly selected 241 NSCLC pts that have a combined total of 761 samples (236 baseline, 191 C3D1, and 128 at 6 months post first dose). Samples were analyzed using a commercial NGS assay, which quantifies circulating tumor fraction (cTF) via interrogating thousands of methylation sites from ctDNA, validated with LoB, LoD, LoQ, and linearity studies. Cox proportional hazards (CPH) were used for comparison of real-world progression free survival (rwPFS). Gender, age, disease stage, and blood-assessed tumor mutational burden (bTMB) were included as covariates. Median rwPFS was calculated using Kaplan Meier analysis. rwPFS ordinal groups were defined as PFS event within 3mo, 3-6mo, 6-12mo, and greater than 12mo, and association with early ctDNA changes was assessed with a chi-squared test. Results: In this cohort, 4, 11, 83 and 142 pts were stage I to IV, respectively (1 unknown). Methylation based detection of ctDNA at baseline or C3D1 was associated with shorter rwPFS (baseline: mPFS 12.7 mo [9.3-19.5] vs NR [16.4-NR]; HR=2.2 [1.3-3.7] p<0.005, C3D1: mPFS 12.4 mo [9.3-16.1] vs NR [19.5-NR] HR= 2.2 [1.3-3.5], p<0.005) independent of stage, gender, or age. Longer rwPFS was associated with >95% reduction in cTF from baseline to C3D1 or low ctDNA at both timepoints (rwPFS <3mo = 12.5%, 3-6 mo = 30.8%, 6-12 = 37.1%, ≥12 mo = 55.3%; N=153, p=0.015). For pts with ctDNA not detected at C3D1, ctDNA detection at 6 months post first dose was strongly associated with shorter rwPFS (mPFS 13.5 mo [9.5-NR] vs. NR [19.5-NR]; HR= 6.2 [1.9-20.1], p<0.005). Conclusions: These data demonstrate a significant association of ctDNA detection via methylation-based cTF and on-treatment changes in cTF with rwPFS. Furthermore, subsequent cTF detection 6-months on-treatment, without cTF detected early on-treatment was associated with worse outcomes, suggesting the value of serial monitoring to improve identification of patients likely to progress. Citation Format: Sara Wienke, Sean Gordon, Samantha Liang, Jing Wang, Reagan Barnett, Kyle Chang, Shile Zhang, Carin R. Espenschied, Katie Quinn, Kimberly Banks. Quantification of tumor fraction and outcomes association in a real-world non-small cell lung cancer (NSCLC) cohort using a tissue agnostic epigenomic circulating tumor DNA (ctDNA) assay [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 2490.

Abstract 1191: Personalized neoantigen DNA vaccine GNOS-PV02 and pembrolizumab as second-line treatment for advanced hepatocellular carcinoma

Abstract Background: PD-1 inhibitors have modest efficacy as monotherapy in hepatocellular carcinoma (HCC). A personalized therapeutic cancer vaccine (PTCV) tailored against neoantigens identified in an individual’s tumor may enhance responses to PD-1 inhibitors through the induction of tumor-specific immunity. Here, we present results from a single-arm Phase Ib/2a trial evaluating a DNA plasmid (GNOS-PV02) encoding up to 40 neoantigens co-administered with plasmid-encoded IL-12 (pIL12) in combination with pembrolizumab (PEMBRO) in patients (pts) with advanced HCC.Methods: Pts were eligible for study therapy upon progression or intolerance with a 1L tyrosine kinase inhibitor (TKI). The PTCV [GNOS-PV02 (1mg) and pIL12 (0.34mg)] was administered intradermally via in vivo electroporation Q3w x 4 doses, and Q9w thereafter. PEMBRO was administered at 200mg IV Q3w. The primary endpoints were safety and immunogenicity. To evaluate the secondary endpoint of ORR per RECIST 1.1 by investigator review with a null hypothesis of an ORR of 16.9% (KN-240, Finn et al ASCO 2019), 36 pts were included to provide 80% power to reject the null hypothesis at the one-sided 0.10 level, assuming the true ORR rate of 33.1%. The data cut date was August 18, 2023.Results: Among the 36 enrolled pts who received at least one dose of treatment, there were no DLTs or treatment-related grade ≥3 events. The most common treatment-related adverse events were injection site reactions, observed in 41.6% of pts. ORR (mITT) per RECIST 1.1 was 30.6% (11/36; 9 confirmed and 2 unconfirmed) with 8.3% (3/36) of pts achieving a CR. This achieved statistical significance with a one-sided p-value = 0.031 (1-sided 90% CI 20.4%-100%) The mOS was 19.9 months. ctDNA changes correlated with radiographic responses and preceded them. A complete molecular response (100% ctDNA clearance) was detected in 7 pts including the 3 radiographic CRs, and 4 additional pts who continue to show durable tumor control (3PR, 1 SD). Immunological analyses confirmed the induction of neoantigen-specific T cell responses by IFNγ-ELISpot in 19/22 (86.4%) evaluable pts, and pts with a larger ELISpot response showed a trend towards longer OS. Multi-parametric cellular profiling and single-cell analysis revealed active, proliferative, and cytolytic vaccine-specific CD4+ and CD8+ effector T cells in the blood of immunized pts. In 14/14 (100%) of pts with paired pre- and on-treatment blood and tumor biopsies, we identified by TCRβ bulk sequencing expanded T cell clones in the peripheral blood that also trafficked into the tumor. Conclusions: Our results show that a PTCV plus PEMBRO is well tolerated and has clinical activity in pts with advanced HCC, and support the PTCV mechanism of action based on the induction of anti-tumor T cells in peripheral blood and tumor. A confirmatory phase 3 clinical study assessing OS is planned. Citation Format: Mark Yarchoan, Edward J. Gane, Thomas U. Marron, Renzo Perales-Linares, Jian Yan, Neil Cooch, Daniel Shu, Elana J. Fertig, Luciane T. Kagohara, Gabor Bartha, Josette Northcott, John Lyle, Sarah Rochestie, Joann Peters, Jason Connor, Elizabeth Jaffee, Alfredo Perales-Puchalt, David B. Weiner, Ildiko Csiki, Niranjan Y. Sardesai. Personalized neoantigen DNA vaccine GNOS-PV02 and pembrolizumab as second-line treatment for advanced hepatocellular carcinoma [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 1191.

Fulvestrant and everolimus efficacy after CDK4/6 inhibitor: a prospective study with circulating tumor DNA analysis

In a prospective study (NCT02866149), we assessed the efficacy of fulvestrant and everolimus in CDK4/6i pre-treated mBC patients and circulating tumor DNA (ctDNA) changes throughout therapy. Patients treated with fulvestrant and everolimus had their ctDNA assessed at baseline, after 3–5 weeks and at disease progression. Somatic mutations were identified in archived tumor tissues by targeted NGS and tracked in cell-free DNA by droplet digital PCR. ctDNA detection was then associated with clinicopathological characteristics and patients’ progression-free survival (PFS), overall survival (OS) and best overall response (BOR). In the 57 included patients, median PFS and OS were 6.8 (95%CI [5.03–11.5]) and 38.2 (95%CI [30.0-not reached]) months, respectively. In 47 response-evaluable patients, BOR was a partial response or stable disease in 15 (31.9%) and 11 (23.4%) patients, respectively. Among patients with trackable somatic mutation and available plasma sample, N = 33/47 (70.2%) and N = 19/36 (52.8%) had ctDNA detected at baseline and at 3 weeks, respectively. ctDNA detection at baseline and PIK3CA mutation had an adverse prognostic impact on PFS and OS in multivariate analysis. This prospective cohort study documents the efficacy of fulvestrant and everolimus in CDK4/6i-pretreated ER + /HER2- mBC and highlights the clinical validity of early ctDNA changes as pharmacodynamic biomarker.

Characterizing longitudinal molecular changes in ctDNA in patients (pts) with metastatic castration-resistant prostate cancer (mCRPC).

205 Background: Next-generation sequencing (NGS) of ctDNA is a novel technology that can complement tumor tissue NGS and has the potential to inform prognosis, treatment (tx) selection, and real time evolution of tumor molecular alterations. While tx options for pts with mCRPC have advanced, tx resistance is inevitable. Our real-world evidence study aims to evaluate longitudinal molecular changes in ctDNA following systemic tx to inform sequential tx strategies for pts with mCRPC. Methods: We interrogated data from GuardantINFORM, a clinical-genomics database with ctDNA molecular information and structured commercial payer-claims data. We conducted genetic mutations analysis in pts diagnosed with prostate cancer both before and after Androgen Receptor Signaling Inhibitors (ARSi), Poly(ADP‐ribose) Polymerase Inhibitors (PARPi), and taxanes. The post-tx analysis was performed on samples collected within 3 months of tx discontinuation prior to initiation of a subsequent tx as a proxy of disease progression. Mann-Whitney test was conducted to assess the differences in mutations observed pre and post tx. We also examined the association between ctDNA burden and overall survival (OS). Results: Overall, from a database of 21,682 patients with prostate cancer, following the above filters, we analyzed 145 pts with paired pre / post-ARSI, 54 with paired pre / post-PARPi, and 115 with paired pre / post-taxane samples. Table highlights the most commonly altered genes prevalent in post-tx samples. The most frequent (%pre/%post) AR alterations post-ARSI included AR amplifications (11%/23%), AR T878A (5%/9%), AR L702H (2%/6%), AR H875Y (0.7%/1.4%), and AR F877L (0%/1.4%). The most frequent HRR alterations post PARPi included ATM (25%/25%), BRCA2 (25%/18%), BRCA1 (3%/5%), CDK12 (6%/5%), and PALB2 (3%/2%). All three tx exhibited a significantly higher prevalence of mutations post-tx (mean increase in alterations 0.3-1.3, p<0.001 for all tx). Pts with higher ctDNA burden in pre-tx samples had worse OS compared to pts with low or undetectable burden. Conclusions: Our study revealed dynamic shifts in genetic mutations in pts with mCRPC following ARSi, PARPi and taxanes. Additionally, we highlight the prognostic significant of ctDNA burden in mCRPC. These data can help inform personalized sequential tx strategies for pts with mCRPC. [Table: see text]

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.

Predictive value of dynamic changes in ctDNA and baseline biomarkers with neoadjuvant atezolizumab in operable urothelial carcinoma in the ABACUS trial.

534 Background: Circulating tumour DNA (ctDNA) is being explored in the neoadjuvant setting in multiple prospective trials. Dynamic changes may be a surrogate marker of pathological complete response. ABACUS was a multi-centre, single-arm phase II trial investigating two cycles of atezolizumab before cystectomy in patients with muscle-invasive urothelial cancer who were ineligible for or refused neoadjuvant cisplatin-based chemotherapy (NCT02662309). Results of primary and secondary endpoints have been previously published. Here we perform exploratory biomarker analysis using different definitions of ctDNA response and assess correlation with tissue response at time of cystectomy in the ABACUS trial. We also assess how these definitions of response correlate with baseline biomarker expression. Methods: Patients with baseline PDL-1, CD8, TMB and sequential ctDNA measurements (baseline and at cystectomy) were included. ctDNA analysis was performed using the Signatera assay, PD-L1 positivity was defined as ≥5% of immune cell staining, TMB was assessed using the FoundationOne CDx assay and CD8 measurement was performed via immunohistochemistry analyses. Two definitions of ctDNA response were used—ctDNA clearance and a 50% reduction (or greater) in ctDNA variant allele frequency (VAF). Results were correlated with pathological complete response (pCR) rate at time of cystectomy and relapse-free survival. Results: The 2-yr DFS and OS rates in ABACUS were 68% and 77%, respectively (N=95). 40 patients had sequential DNA analysis. 43% (17/40) had pathological complete response and 20% (8/40) experienced relapse. 63% (25/40) patients were ctDNA+ at baseline, with 40% (10/25) having ctDNA response of 50% VAF reduction and 8% (3/40) achieving ctDNA clearance. 30% (3/10) of patients who had VAF reduction experienced relapse and 40% (4/10) achieved pCR. All patients with ctDNA clearance achieved pCR none relapsed. There was no association between VAF reduction of 50% and tissue response (pCR) (p=0.24). Additional analysis showed no association between a ctDNA reduction of 75% (rather than 50%) and pCR (p=0.24). Baseline PDL-1, TMB and not predictive of pathological complete response (p=0.18, p=0.77, p=0.10, respectively) or ctDNA response (p=0.54, p=0.77, p=0.74) to neoadjuvant atezolizumab. There was no difference in relapse rates between ctDNA/PD-L1+ve vs ctDNA+ve/PD-L1-ve patients (16% vs 16%, respectively). Conclusions: ctDNA clearance is rare but appears more accurate than 50% reduction in VAF to predict response/relapse. This is relevant for ongoing neoadjuvant trials planning to use this as an endpoint. Combining immune tissue based biomarkers with ctDNA does not appear to improve biomarker accuracy.

Early molecular assessment to predict prognosis in patients who respond to chemotherapy on initial imaging in pancreatic cancer.

683 Background: It is helpful to predict early the response to anticancer drugs in non-resectable solid tumors. The purpose of this study is to evaluate whether circulating tumor DNA (ctDNA) helps predict prognosis in patients with non-resectable pancreatic cancer who have received first-line FFX or GnP therapy and have responded to treatment (CR, PR, SD groups) on initial imaging. Methods: A total of 61 patients with non-resectable pancreatic cancer treated with FFX (N=22) and GnP (N=39) as first-line therapy were included in the study. Before and after chemotherapy treatment (median 42 days, after two courses), KRAS-mutated ctDNA was measured using droplet digital PCR with CA19-9. We defined the Responder group as those with CR, PR, or SD other than PD on initial imaging and the molecular assessment as the change in KRAS-mutated ctDNA before and after treatment. (1) No appearance of KRAS-mutated ctDNA before and after treatment was defined as molecular negative (mNT); (2) disappearance of KRAS-mutated ctDNA or decrease in copy number after treatment was defined as molecular response (mR); (3) new appearance of KRAS-mutated ctDNA or increase in copy number after treatment was defined as molecular progressive disease (mPD). We investigated whether the increase or decrease of CA19-9 before and after treatment (Increase group, decrease group) and the molecular assessment were useful as prognostic factors in the Responder group. Results: Male: female = twenty-nine: thirty-two. There were fourteen patients in the locally advanced group and forty-seven patients in the distant metastatic group, both of whom were considered non-resectable. Thirty-eight patients received any second-line treatment. The prognosis of fourteen patients with PD on initial imaging was significantly worse than that of the Responder group (N=47) (Responder: PD=18.4: 9.2 months, p=0.000108). The molecular assessment in the responder group showed 30 patients in mNT group, 10 patients in mR group, and 7 patients in mPD group. The MST was 21.7, 13.2, and 13.2 months. In univariate analysis, increased CA19-9 (Increase group) and mPD were associated with worse prognosis (Increase: Decrease=13.6: 18.4 months, p=0.0487), (mPD: mNT+mR=13.2: 21.7 months, p=0.0194) and the only independent prognostic factor in multivariate analysis was mPD (Hazard ratio=3.005, p=0.0361). Conclusions: The prognosis of patients with molecular negative or molecular response after chemotherapy is relatively good (MST; 21.7 months), and efforts should be made to continue GnP and FFX. In contrast, the prognosis of patients with molecular progressive disease in the responder group is worse. Therefore, strict follow-up and change of anticancer drugs, including palliation, should be considered.

Non-covalent binding interaction of bioactive coumarin esculetin with calf thymus DNA and yeast transfer RNA: A detailed investigation to decipher the binding affinities, binding location, interacting forces and structural alterations at a molecular level

Esculetin is a well-known coumarin derivative found abundantly in nature possessing an extensive array of pharmacological and therapeutic properties. Consequently, to comprehend its molecular recognition mechanism, our objective is to conduct a complete investigation of its interactions with the nucleic acid, specifically ct-DNA, and t-RNA, using spectroscopic and computational techniques. The intrinsic fluorescence of esculetin is quenched when it interacts with ct-DNA and t-RNA, and this occurs through a static quenching mechanism. The thermodynamic parameters demonstrated that the interaction is influenced by hydrogen bonding and weak van der Waals forces. CD and FT-IR results revealed no conformational changes in ct-DNA and t-RNA structure on binding with esculetin. Furthermore, competitive displacement assay with ethidium bromide, melting temperature, viscosity measurement, and potassium iodide quenching experiments, reflected that esculetin probably binds to the minor groove of ct-DNA. The molecular docking results provided further confirmation for the spectroscopic findings, including the binding location of esculetin and binding energies of esculetin complexes with ct-DNA and t-RNA. Molecular dynamics simulation studies demonstrated the conformational stability and flexibility of nucleic acids.

Circulating Tumor DNA Is a Variant of Liquid Biopsy with Predictive and Prognostic Clinical Value in Breast Cancer Patients

This paper introduces the reader to the field of liquid biopsies and cell-free nucleic acids, focusing on circulating tumor DNA (ctDNA) in breast cancer (BC). BC is the most common type of cancer in women, and progress with regard to treatment has been made in recent years. Despite this, there remain a number of unresolved issues in the treatment of BC; in particular, early detection and diagnosis, reliable markers of response to treatment and for the prediction of recurrence and metastasis, especially for unfavorable subtypes, are needed. It is also important to identify biomarkers for the assessment of drug resistance and for disease monitoring. Our work is devoted to ctDNA, which may be such a marker. Here, we describe its main characteristics and potential applications in clinical oncology. This review considers the results of studies devoted to the analysis of the prognostic and predictive roles of various methods for the determination of ctDNA in BC patients. Currently known epigenetic changes in ctDNA with clinical significance are reviewed. The possibility of using ctDNA as a predictive and prognostic marker for monitoring BC and predicting the recurrence and metastasis of cancer is also discussed, which may become an important part of a precision approach to the treatment of BC.

Early and Sustained Circulating Tumor DNA Response Dynamics after Loncastuximab Tesirine for Relapsed/Refractory Diffuse Large B-Cell Lymphoma

Introduction: Circulating tumor DNA (ctDNA) has emerged as a tool to characterize tumors and track minimal residual disease (MRD) in many malignancies, including diffuse large B-cell lymphoma (DLBCL). ctDNA levels have been shown as prognostic after first-line treatment and CD19-targeted chimeric antigen receptor (CAR19) T cell therapy in DLBCL. Loncastuximab tesirine (loncastuximab tesirine-lpyl [Lonca]), an antibody-drug conjugate comprising an antibody targeting CD19 and a pyrrolobenzodiazepine dimer cytotoxin, is approved for relapsed/refractory (R/R) DLBCL. The ultrasensitive ctDNA-MRD detection method, phased variant enrichment and detection sequencing (PhasED-Seq) was applied to evaluate molecular response and mutational genotypes in patients (pts) undergoing Lonca treatment. Methods: LOTIS-2 study (NCT03589469) evaluated the efficacy of Lonca in R/R DLBCL after ≥ 2 lines of prior systemic therapy. Samples from 33 LOTIS-2 pts were profiled by PhasED-Seq (Foresight Diagnostics), representing a range of best responses to therapy, including 8 complete response (CR), 14 partial response (PR), and 11 progressive disease (PD). Pts received a median of 4 treatment cycles with Lonca (range 2-22). Baseline plasma and peripheral blood mononuclear cells were used to identify tumor-specific phased variants (PVs), which were used to monitor ctDNA-MRD after 1 cycle of treatment (cycle 2, day 1 [C2D1]) and at the end of treatment (EOT). Samples were reported quantitatively with levels of ctDNA-MRD and qualitatively as ctDNA-MRD positive or negative. Absolute ctDNA levels and log-fold change (LFC) in ctDNA were compared with outcomes including best overall response, progression-free survival (PFS), and overall survival (OS), as determined by the independent review committee. The ctDNA mutational profile of each pt was evaluated at baseline and EOT to assess emerging clonal alterations with emphasis on CD19. Results: PVs were successfully genotyped from pretreatment plasma in 31/33 pts (94%) with sufficient material for analysis. The median pretreatment ctDNA level was 141 haploid genome equivalents /mL (hGE/mL) (range 0.4-3608), similar to previous DLBCL studies in first-line and relapsed settings. Interestingly, pretreatment ctDNA levels were not predictive of outcomes to treatment (hazard ratio [HR] 0.99, P=0.96). The prognostic value of ctDNA levels was assessed at C2D1. Absolute ctDNA levels and the change from baseline levels were significantly prognostic for the response to treatment. Pts achieving either a CR or a PR had significantly greater reduction in their ctDNA levels than those failing to respond (CR vs PD, median LFC -1.5 vs 0.1, P=0.001; PR vs PD, median LFC -1.1 vs 0.1, P=0.006) (Fig 1A). Interestingly, pts achieving a CR or a PR did not have significantly different changes in ctDNA at C2D1 ( P=0.15). When dividing pts at the median into those with high vs low ctDNA levels at C2D1, lower levels of ctDNA were significantly associated with superior PFS and OS (PFS: HR 4.4, P=0.009; OS: HR 3.2, P=0.01) (Fig 1B). At the EOT, 5 of 6 CR pts had cleared their ctDNA-MRD to undetectable levels vs 1 of 6 at C2D1. The CR pt who did not show deepening of molecular response was the only CR pt who ended Lonca treatment for radiographic disease progression; all other CR pts ended treatment for other reasons (e.g., toxicity, transplant, or persistent complete remission). To assess for emergent clonal mutations, the mutational profiles of pts were evaluated before Lonca treatment and at EOT. Prior studies in DLBCL after CAR19 T-cell therapy have revealed recurrent, although infrequent, mutations in CD19 as a mechanism of resistance. In this cohort, no emergent alterations in CD19 at the EOT (0/31 pts) were observed. One pt was identified with a baseline mutation in CD19 (R363C); this pt achieved a PR followed by PD after 3 cycles of treatment with persistence of the mutation in CD19. Conclusions: ctDNA molecular response assessment using PhasED-Seq is prognostic for outcomes in pts receiving Lonca monotherapy. ctDNA levels as early as C2D1 can predict outcomes and are indicative of a fast response to Lonca. Furthermore, molecular responses can deepen with additional cycles. CD19 alterations do not appear to be a common emergent mechanism of resistance to Lonca. In this exploratory study, ctDNA-MRD predicts Lonca efficacy and outcomes and should be further considered as a universal biomarker in DLBCL.

Pharmacodynamic Biomarkers and CtDNA Support the Mechanism of Action and Clinical Efficacy of Golcadomide (CC-99282) Combined with R-CHOP in Previously Untreated Aggressive B-Cell Lymphoma

Introduction Golcadomide (GOLCA, CC-99282) is an orally available cereblon E3 ligase modulator (CELMoD ®) that potently degrades target proteins Ikaros and Aiolos, enhancing antiproliferative, apoptotic, and immunomodulatory activity and preferentially distributing to lymphoid organs. In a phase 1 study, GOLCA monotherapy previously demonstrated a manageable safety profile with promising clinical activity in relapsed/refractory non-Hodgkin lymphoma (Michot Blood 2021). In the ongoing CC-220-DLBCL-001 study (NCT04884035), GOLCA dose finding is underway in patients with previously untreated aggressive B-cell lymphoma (BCL), in combination with R-CHOP in 21-day (D) cycles (C), at a variety of GOLCA dose levels (DLs) and schedules: DL-1 = 0.2 mg D1-7, DL1 = 0.4 mg D1-7, and DL2 = 0.4 mg D1-10. Here, we report Ikaros degradation, immune modulation, circulating tumor DNA (ctDNA) assessment, and baseline tumor genomics. Methods Peripheral blood was collected to analyze Ikaros and Aiolos levels in T cells by flow cytometry (C1D1, C1D7, and C2D1). Immune phenotyping of T and natural killer (NK) cells was performed by flow cytometry (C1D1, C1D15, C2D1, and C3D1). ctDNA levels were analyzed by the PhasED-Seq assay (timepoints C1D1, C2D1, C3D1, and end of treatment [EOT]; Kurtz Nat Biotechnol 2021); somatic variants were filtered by performing PhasED-seq on buffy coats. Pretreatment biopsies were analyzed by whole genome sequencing and whole transcriptome sequencing. DL1 (0.4 mg D1-7) and DL2 (0.4 mg D1-10) data were pooled. Results Ikaros levels in T cells at 5 hours after the first dose had a 1.1% median increase with 0.2 mg and 44% median decrease with 0.4 mg; by C1D7 pretreatment, Ikaros substantially decreased with both DLs (median: 0.2 mg, 87% decrease; 0.4 mg, 83% decrease). Ikaros differences between 0.2 mg and 0.4 mg were not substantial. By C2D1, Ikaros levels recovered to baseline. Based on preclinical benchmarks, steady-state Ikaros degradation in T cells suggested that direct tumor cell killing by GOLCA was achieved. Changes in T and NK cell phenotypes in peripheral blood included activation, increased proliferation, increased T effector memory and regulatory cell levels, and decrease in naive T cells. Changes were seen by C1D15 and maintained at C2D1 and C3D1. Overall, immune changes between 0.2 mg and 0.4 mg dose levels were not substantially different. Phased variant data from ctDNA and buffy coats were evaluable for 34 patients (0.2 mg, n =15; 0.4 mg, n = 19). Baseline ctDNA levels were similar between the 0.2 mg and 0.4 mg cohorts and, consistent with prior studies, correlated with International Prognostic Index and cell of origin. Previously reported landmarks such as early molecular response (EMR; ≥ 2 log decrease) at C2D1 and major molecular response (MMR; ≥ 2.5 log decrease) at C3D1 were assessed (Kurtz J Clin Oncol 2018) . A high fraction of patients designated high risk by pretreatment ctDNA (≥ 2.5 log haploid genome equivalents [HGE]/mL; Kurtz J Clin Oncol 2018) were clinical responders. Patients who achieved minimal residual disease (MRD) negativity on treatment generally remained MRD negative at subsequent timepoints. At C2D1, the EMR and MRD negativity rates for 0.2 mg were 73% (11/15) and 20% (3/15), respectively; for 0.4 mg these rates were 83% (15/18) and 44% (8/18). At C3D1, the MMR and MRD negativity rates for 0.2 mg were 83% (10/12) and 42% (5/12), respectively; for 0.4 mg these were 83% (10/12) and 58% (7/12). Although ctDNA differences between 0.2 mg and 0.4 mg were not substantial, numerically greater ctDNA changes were seen with 0.4 mg GOLCA + R-CHOP vs 0.2 mg GOLCA + R-CHOP and vs R-CHOP and R-CHOP-like regimens from other cohorts ( Figure). Insufficient samples were available to draw conclusions about EOT, but additional ctDNA data will be presented. Whole genome and transcriptome sequencing data from GOLCA + R-CHOP-treated patients will be shown. Conclusions Pharmacodynamic biomarkers and ctDNA levels indicate that both tumor-intrinsic and immune-modulating mechanisms of action of GOLCA remain relevant in combination with R-CHOP in previously untreated aggressive BCL. ctDNA data during treatment are consistent with the efficacy noted on clinical restaging. Translational data demonstrate the potency of GOLCA and will support future frontline clinical trial designs in patients with untreated aggressive BCL. Study support: Celgene, a Bristol-Myers Squibb Company

Serial circulating tumor DNA profiling predicts tumor recurrence after liver transplantation for liver cancer.

Minimal residual disease (MRD) is proposed to be responsible for tumor recurrence. The role of circulating tumor DNA (ctDNA) to detect MRD, monitor recurrence, and predict prognosis in liver cancer patients undergoing liver transplantation (LT) remains unrevealed. Serial blood samples were collected to profile ctDNA mutational changes. Baseline ctDNA mutational profiles were compared with those of matched tumor tissues. Correlations between ctDNA status and recurrence rate (RR) and recurrence-free survival (RFS) were analyzed, respectively. Dynamic change of ctDNA was monitored to predict tumor recurrence. Baseline mutational profiles of ctDNA were highly concordant with those of tumor tissues (median, 89.85%; range 46.2-100%) in the 74 patients. Before LT, positive ctDNA status was associated with higher RR (31.7% vs 11.5%; p = 0.001) and shorter RFS than negative ctDNA status (17.8 vs 19.4months; p = 0.019). After LT, the percentage of ctDNA positivity decreased (17.6% vs 47.0%; p < 0.001) and patients with positive ctDNA status had higher RR (46.2% vs 21.3%; p < 0.001) and shorter RFS (17.2 vs 19.2months; p = 0.010). Serial ctDNA profiling demonstrated patients with decreased or constant negative ctDNA status had lower RR (33.3% vs 50.0%; p = 0.015) and favorable RFS (18.2 vs 15.0months, p = 0.003) than those with increased or constant positive ctDNA status. Serial ctDNA profiling predicted recurrence months ahead of imaging evidence and serum tumor biomarkers. ctDNA could effectively detect MRD and predict tumor recurrence in liver cancer patients undergone LT.

EP13.05-01 Early ctDNA Changes and Clinical Outcomes in Extensive Stage Small Cell Lung Cancer Patients on Nivolumab and Temozolomide

EP13.05-01 Early ctDNA Changes and Clinical Outcomes in Extensive Stage Small Cell Lung Cancer Patients on Nivolumab and Temozolomide

The Importance of Feasibility Assessment in the Design of ctDNA Guided Trials – Results From the OPTIPAL II Study

IntroductionBoth quantitative and molecular changes in ctDNA can hold important information when treating metastatic colorectal cancer (mCRC), but its clinical utility is yet to be established. Before conducting a large-scale randomized trial, it is essential to test feasibility. This study investigates whether ctDNA is feasible for detecting patients who will benefit from treatment with epidermal growth factor receptor inhibitors and the prognostic value of circulating tumor DNA (ctDNA) response. Materials and methodsPatients with mCRC, who were considered for systemic palliative treatment and were eligible for ctDNA analysis. Mutational testing on cell-free DNA (cfDNA) was done by ddPCR. ctDNA response from baseline to the third treatment cycle was evaluated in patients with detectable ctDNA at baseline. ctDNA maximum response was defined as undetectable ctDNA at the third treatment cycle, ctDNA partial response as any decrease in the ctDNA level, and ctDNA progression as any increase in the ctDNA level. ResultsForty-nine patients were included. The time to test results for mutational testing on cfDNA was significantly shorter than on tumor tissue (p < .001). Progression-free survival were 11.2 months (reference group), 7.5 months (HR = 10.7, p= .02), and 4.6 months (HR = 11.4, p= .02) in patients with ctDNA maximum response, partial response, and progression, respectively. Overall survival was 31.2 months (reference group), 15.2 months (HR = 4.1, p= .03), and 9.0 months (HR = 2.6, p= .03) in patients with ctDNA maximum response, partial response, and progression, respectively. ConclusionPretreatment mutational testing on cfDNA in daily clinic is feasible and can be applied in randomized clinical trials evaluating the clinical utility of ctDNA. Early dynamics in ctDNA during systemic treatment hold prognostic value.

Circulating Tumor DNA: A Promising Biomarker for Predicting Recurrence in Patients with BRAF-Negative Melanoma

For patients with BRAF wild-type stage III and IV melanoma, there is an urgent clinical need to identify prognostic biomarkers and biomarkers predictive of treatment response. Circulating tumor DNA (ctDNA) is emerging as a blood-based biomarker and has shown promising results for many cancers, including melanoma. The purpose of this study was to identify targetable, tumor-derived mutations in patient blood that may lead to treatment alternatives and improved outcomes for patients with BRAF-negative melanoma. Using a CAncer Personalized Profiling by deep Sequencing (CAPP-seq) pan-cancer gene panel, ctDNA from 150 plasma samples (n=106 patients) was assessed, including serial blood collections for a subset of patients (n=16). ctDNA variants were detected in 85% of patients, all in targetable pathways, such as vascular endothelial growth factor receptor, epidermal growth factor receptor, phosphatidylinositol 3-kinase/AKT, Bcl2/mammalian target of rapamycin (mTOR), ALK/MET, and cyclin-dependent kinase 4/6. Patients with stage IV melanoma with low ctDNA concentrations, <10 ng/mL, had significantly better disease-specific survival and progression-free survival. Patients with both a high concentration of ctDNA and any detectable ctDNA variants had the worst prognosis. In addition, these results indicated that longitudinal changes in ctDNA correlated with treatment response and disease progression determined by radiology. This study confirms that ctDNA may be used as a noninvasive liquid biopsy to identify recurrent disease and detect targetable variants in patients with late-stage melanoma.

Baseline mutations and ctDNA dynamics as prognostic and predictive factors in ER-positive/HER2-negative metastatic breast cancer patients.

Prognostic and predictive biomarkers to CDK4/6 inhibitors are lacking. Circulating tumour DNA (ctDNA) can be used to profile these patients and dynamic changes in ctDNA could be an early predictor of treatment efficacy. Here, we conducted plasma ctDNA profiling in patients from the PEARL trial comparing palbociclib+fulvestrant versus capecitabine to investigate associations between baseline genomic landscape and on-treatment ctDNA dynamics with treatment efficacy. Correlative blood samples were collected at baseline (C1D1) and prior to treatment (C1D15). Plasma ctDNA was sequenced with a custom error-corrected capture panel, with both univariate and multivariate Cox models used for treatment efficacy associations. A pre-specified methodology measuring ctDNA changes in clonal mutations between C1D1 and C1D15 was used for the on-treatment ctDNA dynamic model. 201 patients were profiled at baseline, with ctDNA detection associated with worse PFS/OS. Detectable TP53 mutation showed worse PFS and OS in both treatment arms, even after restricting population to baseline ctDNA detection. ESR1 mutations were associated with worse OS overall, which was lost when restricting population to baseline ctDNA detection. PIK3CA mutations confer worse OS only to patients on the palbociclib+fulvestrant treatment arm. ctDNA dynamics analysis (n=120) showed higher ctDNA suppression in the capecitabine arm. Patients without ctDNA suppression showed worse PFS in both treatment arms. We show impaired survival irrespective of endocrine or chemotherapy-based treatments for patients with HR+/HER2- MBC harbouring plasma TP53 mutations. Early ctDNA suppression may provide treatment efficacy predictions. Further validation to fully demonstrate clinical utility of ctDNA dynamics is warranted.

Early Clearance of Plasma Epidermal Growth Factor Receptor Mutations as a Predictor of Outcome on Osimertinib in Advanced Non-Small Cell Lung Cancer; Exploratory Analysis from AURA3 and FLAURA.

Plasma circulating tumor DNA (ctDNA) analysis is used for genotyping advanced non-small cell lung cancer (NSCLC); monitoring dynamic ctDNA changes may be used to predict outcomes. This was a retrospective, exploratory analysis of two phase III trials [AURA3 (NCT02151981), FLAURA (NCT02296125)]. All patients had EGFR mutation-positive (EGFRm; ex19del or L858R) advanced NSCLC; AURA3 also included T790M-positive NSCLC. Osimertinib (FLAURA, AURA3), or comparator EGFR-tyrosine kinase inhibitor (EGFR-TKI; gefitinib/erlotinib; FLAURA), or platinum-based doublet chemotherapy (AURA3) was given. Plasma EGFRm was analyzed at baseline and Weeks 3/6 by droplet digital PCR. Outcomes were assessed by detectable/non-detectable baseline plasma EGFRm and plasma EGFRm clearance (non-detection) at Weeks 3/6. In AURA3 (n = 291), non-detectable versus detectable baseline plasma EGFRm had longer median progression-free survival [mPFS; HR, 0.48; 95% confidence interval (CI), 0.33-0.68; P < 0.0001]. In patients with Week 3 clearance versus non-clearance (n = 184), respectively, mPFS (months; 95% CI) was 10.9 (8.3-12.6) versus 5.7 (4.1-9.7) with osimertinib and 6.2 (4.0-9.7) versus 4.2 (4.0-5.1) with platinum-pemetrexed. In FLAURA (n = 499), mPFS was longer with non-detectable versus detectable baseline plasma EGFRm (HR, 0.54; 95% CI, 0.41-0.70; P < 0.0001). For Week 3 clearance versus non-clearance (n = 334), respectively, mPFS was 19.8 (15.1 to not calculable) versus 11.3 (9.5-16.5) with osimertinib and 10.8 (9.7-11.1) versus 7.0 (5.6-8.3) with comparator EGFR-TKI. Similar outcomes were observed by Week 6 clearance/non-clearance. Plasma EGFRm analysis as early as 3 weeks on-treatment has the potential to predict outcomes in EGFRm advanced NSCLC.

Combining Solid and Liquid Biopsy for Therapy Monitoring in Esophageal Cancer

Esophageal cancer (EC) has one of the highest mortality rates among cancers, making it imperative that therapies are optimized and dynamically adapted to individuals. In this regard, liquid biopsy is an increasingly important method for residual disease monitoring. However, conflicting detection rates (14% versus 60%) and varying cell-free circulating tumor DNA (ctDNA) levels (0.07% versus 0.5%) have been observed in previous studies. Here, we aim to resolve this discrepancy. For 19 EC patients, a complete set of cell-free DNA (cfDNA), formalin-fixed paraffin-embedded tumor tissue (TT) DNA and leukocyte DNA was sequenced (139 libraries). cfDNA was examined in biological duplicates and/or longitudinally, and TT DNA was examined in technical duplicates. In baseline cfDNA, mutations were detected in 12 out of 19 patients (63%); the median ctDNA level was 0.4%. Longitudinal ctDNA changes were consistent with clinical presentation. Considerable mutational diversity was observed in TT, with fewer mutations in cfDNA. The most recurrently mutated genes in TT were TP53, SMAD4, TSHZ3, and SETBP1, with SETBP1 being reported for the first time. ctDNA in blood can be used for therapy monitoring of EC patients. However, a combination of solid and liquid samples should be used to help guide individualized EC therapy.