ctDNA Levels Research Articles

Efficacy and safety of XELOX combined with anlotinib and penpulimab vs XELOX as adjuvant therapy in ctDNA-positive gastric and esophagogastric junction adenocarcinoma: A protocol for a randomized, controlled, multicenter phase II clinical trial—EXPLORING study.

TPS486 Background: The clinical benefit of current postoperative adjuvant chemotherapy for gastric adenocarcinoma/gastroesophageal junction adenocarcinoma (GA/GEJA) leaves much to be desired. CtDNA could serve as a potential marker to identify patients (pts) who are most at risk of recurrence. Reinforcing standard adjuvant chemotherapy with immunotherapy has already been indicated to significantly boost clinical benefit, albeit such evidence is rare in GA/GEJA. Here, we intend to prove the clinical benefit of the reinforcement of adjuvant chemotherapy with immunotherapy in patients who are deemed high risk of recurrence by ctDNA analysis, in hope of shedding light on further improvements in adjuvant therapy for GA/GEJA. Methods: This study is designed as a prospective, randomized, controlled, multicenter phase II study in patients histologically or cytologically diagnosed with CG/GEJC (Siewert III), 18-75 years, ECOG PS 0-1, underwent D2 gastrectomy and achieved R0 or R1 resection. Beginning from February 2022, a total of 270 stage III patients will be enrolled and subjected to ctDNA sequencing, and those with positive results will subsequently be randomized 1:1 to arm A or B. CtDNA-negative patients will be assigned to arm C and pts who had ctDNA positive but failed to be randomized will be assigned to arm D. Pts in arm A will receive anlotinib, penpulimab and XELOX for 6-8 cycles, maintained with anlotinib and penpulimab for up to 1 year, while pts in arm B will receive XELOX alone for 6-8 cycles. Pts in arms C&D will receive the investigator’s choice of therapy. Arms A&B will be monitored for ctDNA levels on a 3-month cycle for 1 year. Survival data, laboratory findings, imaging reports and nutritional status will be regularly followed-up in all arms. The primary endpoint is the median disease-free survival (DFS) of arm A versus arm B determined via CT/MRI imaging. Secondary endpoints include the median DFS of arm B versus arm C, the 2- and 3-year DFS rates, overall survival (OS), the impact of hallmark molecules (presumably ctDNA) on the treatment response, adverse events (AEs), and the impact of nutrition status and exercise on recurrence. Clinical trial information: NCT05494060 .

PP01.15 Circulating Tumor DNA (ctDNA) Level: a Prognostic Biomarker for Clinical Outcomes in Non-Small Cell Lung Cancer (NSCLC) Patients Across Diverse Therapy Classes

PP01.15 Circulating Tumor DNA (ctDNA) Level: a Prognostic Biomarker for Clinical Outcomes in Non-Small Cell Lung Cancer (NSCLC) Patients Across Diverse Therapy Classes

Clinical correlations with EGFR circulating tumor DNA testing in all-stage lung adenocarcinoma.

Information on genetic alterations, notably EGFR mutations, is important for guiding non-small-cell lung cancer (NSCLC) treatment. Circulating tumor DNA (ctDNA) analysis represents a less invasive alternative to tissue biopsy for analyzing mutation status, but its clinical value may vary across disease stages. To explore clinical correlates of ctDNA and tissue/plasma-based EGFR mutation (EGFRm) status across all NSCLC stages. Ninety patients were analyzed, representing three cohorts: newly-diagnosed early-stage, advanced-stage, and recurrent NSCLC. Relationships among clinical/surgical parameters, ctDNA, EGFRm status, and survival outcomes were analyzed. Plasma/tissue EGFRm concordance was lower in early-stage (58.6%) than in advanced-stage patients (87.5%). In early-stage patients, ctDNA levels were variable and not significantly associated with clinical/surgical parameters. In advanced-stage patients, time to EGFR-TKI treatment failure (TTF), but not overall survival (OS), was significantly longer in EGFRm-positive vs. EGFRm-negative patients. In patients with recurrent disease, 40% of plasma samples were EGFRT790M-positive at recurrence. In T790M-positive patients, we noted slight trends toward longer OS with vs. without osimertinib treatment and longer OS and TTF with second-line vs. later-line osimertinib. Our results affirm the use of ctDNA testing in advanced-stage and recurrent NSCLC. Further studies on osimertinib as early-line therapy, clinical correlates and the utility of plasma-based testing in early-stage NSCLC are warranted.

Comprehensive ctDNA Measurements Improve Prediction of Clinical Outcomes and Enable Dynamic Tracking of Disease Progression in Advanced Pancreatic Cancer.

Circulating tumor DNA (ctDNA) has emerged as a promising tumor-specific biomarker in pancreatic cancer, but current evidence of the clinical potential of ctDNA is limited. In this study, we used comprehensive detection methodology to explore the utility of longitudinal ctDNA measurements in patients with advanced pancreatic cancer. A targeted eight-gene next-generation sequencing panel was used to detect point mutations and copy-number aberrations (CNA) in ctDNA from 324 pre-treatment and longitudinal plasma samples obtained from 56 patients with advanced pancreatic cancer. The benefit of ctDNA measurements to predict clinical outcome and track disease progression was assessed. We detected ctDNA in 35/56 (63%) patients at baseline and found that it was an independent predictor of shorter progression-free survival (PFS) and overall survival (OS). After initiation of treatment, ctDNA levels decreased significantly before significantly increasing by the time of progression. In some patients, ctDNA persistence was observed after the first chemotherapy cycles, and it was associated with rapid disease progression and shorter OS. Longitudinal monitoring of ctDNA levels in 27 patients for whom multiple samples were available detected progression in 19 (70%) patients. The median lead time of ctDNA measurements on radiologically determined progression/time of death was 19 days (P = 0.002), compared with 6 days (P = 0.007) using carbohydrate antigen 19-9. ctDNA is an independent prognostic marker that can be used to detect treatment failure and disease progression in patients with advanced pancreatic cancer.

Selected Articles from This Issue

Shoushtari and colleagues report a pilot trial of ONCOS-102, a novel oncolytic adenovirus encoding GM-CSF, administered intratumorally either sequentially or in combination with pembrolizumab in patients with melanoma resistant to prior PD-1 blockade. Treatment was well tolerated. Objective responses were seen in 7 of 20 patients, and size reductions in noninjected lesions suggested local delivery of ONCOS-102 can drive a systemic anti-tumor effect. Serial biopsies of injected tumors at baseline, Week 3 (following ONCOS-102 and prior to pembrolizumab), and Week 9 (following ONCOS-102 and pembrolizumab) indicated that while most tumors experience CD8+ and CD4+ infiltration after ONCOS-102 injection, sustained infiltration at Week 9 was associated with clinical benefit. Future trials of ONCOS-102 and checkpoint inhibition are warranted in anti-PD-1-resistant melanoma. These findings suggest trials using viral agents in anti-PD-1-resistant disease should not solely rely on early onset of cytotoxicity to predict clinical response.Wainberg and colleagues report on the combination of very low doses of chemotherapy (10% of the usual dose) with a full dose of talazoparib (TAL). The study design reports preferentially increasing the dose of TAL before increasing the chemotherapy to improve the hematologic profile as well as exploring efficacy signals among non-BRCA patients. This phase I study demonstrates that TAL with low-dose temozolomide or irinotecan shows an improved hematologic toxicity profile than most prior chemo/PARP inhibitor combinations and has signals of clinical activity in small cell lung cancer and ovarian cancer. The results of this phase I study are supportive of ongoing trials with this combination.Obesity has been unexpectedly associated with improved outcomes with immune checkpoint inhibitors and BRAF-targeted therapies in metastatic melanoma; however, the biology underlying this observation is unknown. Hahn and colleagues examined associations between body mass index (BMI) and molecular, immune, and metabolic features of melanoma tumors as well as host microbiome in 782 patient specimens. Host BMI was associated with altered tumor metabolism, with downregulation of oxidative phosphorylation (OXPHOS) and other metabolic pathways in tumors from high BMI patients. OXPHOS has been associated with resistance to targeted and immune therapies, suggesting a potential mechanism underlying the “obesity paradox” in metastatic melanoma.Circulating tumor DNA (ctDNA) levels are associated with tumor volume and minimal residual disease in lymphoid malignancies. Fernández-Miranda and colleagues suggest that pre-treatment ctDNA levels in follicular lymphoma patients could be useful to predict response to treatment and early progression as an independent predictive biomarker or in combination with clinical variables. Circulating tumor DNA levels and cell-free DNA genotyping at mid-treatment and end-of-treatment are associated with treatment response and early progression follicular lymphoma. Circulating tumor DNA measurement at diagnosis and follow-up is a valuable tool that might feasibly be used in clinical trials and, eventually, in daily clinical practice.

Response prediction by mutation- or methylation-specific detection of ctDNA dynamics in pretreated metastatic colorectal cancer.

Serial analysis of circulating tumor DNA (ctDNA) levels is a promising tool for both relapse prediction in the curative setting, as well as predicting clinical benefit from systemic treatment in metastasic colorectal cancer (mCRC). Most data in this context are derived from treatment naive patients. To predict progressive disease (PD) as early as possible through monitoring of changes in ctDNA levels during systemic treatment in pretreated patients with mCRC. A prospective, single-center, observational study. Patients treated beyond first-line were prospectively included between February 2020 and September 2021. Blood for ctDNA detection was taken before every treatment cycle from start of treatment until first restaging by CT-scan. ctDNA was detected by mutation- (mut-ctDNA) and methylation-specific ddPCR. Receiver Operating Characteristic (ROC)-analysis was used to describe sensitivity and specificity for prediction of PD at restaging for all time points. A total of 42 patients were included who all carried a mutation in tumor tissue. Detection rate of mut-ctDNA was 88.1% and 74.4% for meth-ctDNA. Absolute ctDNA levels before treatment were prognostic in terms of overall survival. Levels of ctDNA were significantly higher in patients with PD at restaging. Median time from start of treatment to restaging was 93 days (95% CI 88.8-96). After a median of 19 days of treatment (95% CI 16.1-20.2), a decline of either mutation- or methylation-specific ctDNA levels of ⩽58% predicted PD at restaging with a sensitivity/specificity of 92.9/85.7% and 85.7/100%, respectively. Median time to restaging was 66 days (95% CI 56.8-75.2). There was no significant increase of sensitivity/specificity at later time points of ctDNA measurements. Monitoring early changes of ctDNA levels either by mut- or meth-ctDNA allows for early prediction of PD in pretreated patients with mCRC. This has the potential to complement RECIST-based treatment assessment with the aim to switch potentially insufficient treatments as early as possible, which is of particular interest in higher treatment lines.

Circulating Tumor DNA correlates with Lactate Dehydrogenase, CYFRA 21-1, and CRP levels in patients with advanced NSCLC.

Purpose: To investigate potential association between selected tumor markers and laboratory parameters (lactate dehydrogenase [LDH], neutrophils, hemoglobin, neutrophils, lymphocytes, C-reactive protein, albumin, carcinoembryonic antigen, and cytokeratin 19 fragment 21-1 [CYFRA 21-1]) and circulating tumor DNA (ctDNA) with survival in patients with advanced non-small cell lung cancer (NSCLC). Patients and Methods: The study encompassed 82 patients from a single center. All patients had (localy-) advanced adenocarcinomas. ctDNA was determined before starting therapy and at 6 weeks follow-up. Laboratory parameters were measured before each cycle of therapy and oncomarkers before starting the therapy as standard clinical practice. Mann-Whitney U test, Cox proportional hazards model, Fisher's exact test, and Kaplan-Meier survival estimation with Gehan-Wilcoxon test were used for statistical analysis of the corresponding variables. Results: We have confirmed predictive or prognostic significance for some of the selected laboratory markers and oncomarkers. Above all, we demonstrate a significant relationship between the levels of LDH and the oncomarker CYFRA 21-1 and the presence or absence of ctDNA at the time of diagnosis. We also demonstrate significantly lower CRP levels in patients within whom the ctDNA disappeared during treatment. A similar but statistically insignificant trend was observed for LDH. Conclusions: CYFRA 21-1, LDH and probably CRP correlate with ctDNA levels in NSCLC. Repeated measurement of these markers could thus help in early detection of disease progression in the same way as does ctDNA monitoring.

Narrative review: blood and tumor biomarker testing in non-small cell lung cancer without an oncogenic driver.

For patients with metastatic non-small cell lung cancer (NSCLC) without an oncogenic driver, systemic therapy with immune checkpoint inhibitors (ICIs) alone or in combination with chemotherapy have significantly improved the outcomes. However, the majority of patients do not have a durable response, and there is a need for additional predictive biomarkers. The objective of this narrative review is to describe potential biomarkers for immunotherapy. Narrative overview of the literature synthesizing the findings of literature reporting retrospective, prospective, and subset analyses of studies investigating potential predictive biomarkers for ICI. Tumor expression of programmed death ligand-1 (PD-L1) is the only clinically available biomarker for patients receiving ICI-based therapy. However, PD-L1 has significant limitations and studies have investigated the predictive value of higher PD-L1 expression levels. There has been interest in tumor mutation burden (TMB) based on the premise that a higher TMB would be associated with a more neoantigens, which would increase the likelihood of an immune response. The studies to date have not revealed a consistent association with TMB level and survival benefit. Kelch-like ECH Associated Protein 1 (KEAP1) and serine/threonine kinase 11 (STK11) mutations have been associated with worse outcomes with ICI but these mutations appear to be associated with a worse prognosis, and not predictive for ICI. Tumor infiltrating lymphocytes (TIL's) are the mechanism of immune response, and there is interest in further investigating the presence, type and distribution of TIL's to predict immune benefit. Circulating tumor deoxyribonucleic acid (ctDNA) levels, at baseline and on treatment samples, are being investigated to assess response to therapy and long-term benefit of ICI. None of the current biomarkers in development are validated for use in routine clinical care. Given the complexity of NSCLC biology and immune response to ICI most likely a composite biomarker using multiple biomarkers will need to be develop.

Abstract PR007: A liposomal priming agent increases the sensitivity of liquid biopsies

Abstract Liquid biopsy measurements, such as analysis of circulating tumor DNA (ctDNA) shed by cancer cells, have garnered significant attention for their potential to empower the field of precision oncology. Given that ctDNA tests could enable minimally invasive monitoring and molecular profiling of disease, they are being investigated for use in earlier detection via pan-cancer screening tests, for tracking tumor evolution to inform therapy selection, and for making treatment decisions during minimal residual disease surveillance. However, a typical blood draw carries ultra-low levels of ctDNA (as low as 1.7 copies of the tumor genome in 15mL of blood for a 1cm lung tumor), and this fundamentally limits the sensitivity and the clinical utility of ctDNA-testing in many settings. To push beyond current ctDNA detection limits, we present a first-in-class liquid biopsy priming agent that is given prior to a blood draw to increase the abundance of ctDNA in circulation. Our priming agent consists of liposomes that transiently block the uptake of cell free DNA (cfDNA) by macrophages in the liver, resulting in increased cfDNA available for diagnostic analysis in blood. Using an in vitro 2D assay, we first identified a DSPE-based liposomal formulation that inhibits the uptake of cfDNA by two independent murine macrophage cell lines. Next, we injected our liposomal agent into healthy or tumor-bearing mice and collected blood for further analysis. We found that in healthy mice priming increases the half-life and the recovery of cfDNA from a blood draw, assayed via qPCR. In tumor-bearing mice, the priming agent increases the recovery of ctDNA by up to 60-fold (P = 0.0103) and improves the sensitivity of a ctDNA diagnostic assay from 0% to 75% at low tumor burden. Importantly, cfDNA levels in mice return to baseline within five hours of agent administration and repeated dosing shows no evidence of toxicity. Our priming strategy should be of interest for precision oncology applications, including early detection, longitudinal monitoring of therapeutic response, and surveillance for minimal residual disease. This tumor-agnostic priming agent should also improve the performance of ctDNA analytical techniques other than mutational profiling and may even increase the recovery of cfDNA from other body fluids beyond plasma. Moreover, we believe that this work sets a precedent for the potential development of priming agents for liquid biopsy at large across other analytes. In summary, here we present a first-in-class liquid biopsy priming agent capable of improving the sensitivity and the robustness of ctDNA testing in tumor-bearing mice by modulating liver cfDNA clearance. We envision that further development of liquid biopsy priming agents will signify a major step forward for the successful deployment of precision oncology tools across all stages of cancer management. Citation Format: Carmen Martin-Alonso, Shervin Tabrizi, Kan Xiong, Ahmet Bekdemir Bekdemir, Sahil Patel, Zhenyi An, Timothy Blewett, Sainetra Sridhar, Douglas Shea, Ava Amini, Jesse D. Kirkpatrick, Jin Bae, Eugenia Roberts, Ruolin Liu, Justin Rhoades, Todd Golub, J. Christopher Love, Viktor A. Adalsteinsson, Sangeeta N. Bhatia. A liposomal priming agent increases the sensitivity of liquid biopsies. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr PR007.

Feasibility of Longitudinal ctDNA Assessment in Patients with Uterine and Extra-Uterine Leiomyosarcoma.

Background: Leiomyosarcomas (LMS) are aggressive malignancies with a propensity for early relapse. Current surveillance modalities include physical exam and imaging; however, radiological response to therapy may only manifest after 4-6 cycles of treatment. Herein, we evaluated the feasibility of longitudinal circulating tumor DNA (ctDNA) assessment in LMS patients to identify disease progression. Methods: We performed a retrospective review of patients with LMS who underwent treatment at Stanford Cancer Center between September 2019 and May 2022. ctDNA detection was performed using a personalized, tumor-informed ctDNA assay. Genomic analysis was conducted to characterize tumor mutation burden (TMB) and known driver mutations. Results: A total of 148 plasma samples were obtained from 34 patients with uterine (N = 21) and extrauterine (N = 13) LMS (median follow-up: 67.2 (19-346.3) weeks] and analyzed for ctDNA presence. Nineteen patients had metastatic disease. The most frequently mutated driver genes across sub-cohorts were TP53, RB1, and PTEN. Patients were stratified into four sub-cohorts (A-D) based on ctDNA kinetics. ctDNA levels tracked longitudinally with progression of disease and response to therapy. Conclusion: Our results indicate that while undetectable ctDNA may suggest a lower likelihood of relapse, ctDNA positivity may indicate progressive disease, enabling closer monitoring of patients for early clinical intervention.

Using Tumor-Informed Circulating Tumor DNA (ctDNA)-Based Testing for Patients with Anal Squamous Cell Carcinoma.

Anal squamous cell carcinoma (SCCA) is an uncommon malignancy with a rising incidence that has a high cure rate in its early stages. There is an unmet need for a reliable method to monitor response to treatment and assist in surveillance. Circulating tumor DNA (ctDNA) testing has shown great promise in other solid tumors for monitoring disease progression and detecting relapse in real time. This study aimed to determine the feasibility and use of personalized and tumor-informed ctDNA testing in SCCA. We analyzed real-world data from 251 patients (817 plasma samples) with stages I-IV SCCA, collected between 11/5/19 and 5/31/22. The tumor genomic landscape and feasibility of ctDNA testing was examined for all patients. The prognostic value of longitudinal ctDNA testing was assessed in patients with clinical follow-up (N = 37). Whole-exome sequencing analysis revealed PIK3CA as the most commonly mutated gene, and no associations between mutations and stage. Anytime ctDNA positivity and higher ctDNA levels (MTM/mL) were associated with metastatic disease (P = .004). For 37 patients with clinical follow-up, median follow-up time was 21.0 months (range: 4.1-67.3) post-diagnosis. For patients with stages I-III disease, anytime ctDNA-positivity after definitive treatment was associated with reduced DFS (HR: 28.0; P = .005). Our study demonstrates the feasibility of personalized and tumor-informed ctDNA testing as an adjunctive tool in patients with SCCA as well as potential use for detection of molecular/minuteimal residual disease, and relapse during surveillance. Prospective studies are needed to better evaluate the use of ctDNA testing in this indication.

Detectability of Plasma-Derived Circulating Tumor DNA Panel in Patients Undergoing Primary Treatment for Uveal Melanoma.

To investigate the presence of plasma circulating tumor DNA (ctDNA) in patients with uveal melanoma during and after primary tumor treatment. Detectability and variant allele frequency of ctDNA were assessed using a 129-oncogene panel using next-generation deep sequencing and hybridization capture in 69 patients with uveal melanoma undergoing primary treatment with enucleation (n = 8, during surgery) or plaque brachytherapy (n = 61; postoperative day 0, 1, 2, or 3). Follow-up assessments were performed in 39 patients over a median of 21 months (range, 3.2-31.9 months) of follow-up. Correlations between genomic data and disease parameters were performed. Overall, ctDNA was detectable in 20 of 69 patients with uveal melanoma (28.9%) during the perioperative period. On the day of enucleation, ctDNA was detected in two of eight patients (25%). In patients undergoing brachytherapy, ctDNA was significantly more detectable on postoperative days 2 or 3 compared with postoperative day 0 or 1 (32.4% vs 0.0%; P = 0.0015). Patients with follow-up ctDNA that became detectable or had an increased variant allele frequency were significantly more likely to develop metastasis compared with patients with follow-up ctDNA that became undetectable or decreased variant allele frequency (P = 0.04). In patients with detectable vs. undetectable ctDNA, there was no significant difference in tumor size, stage or location. ctDNA is significantly more detectable at 48 to 72 hours after plaque brachytherapy compared with less than 48 hours. ctDNA can be detected during enucleation. Relative increases in ctDNA levels may herald the development of clinically apparent metastases.

A phase II trial of GSK2256098 and trametinib in patients with advanced pancreatic ductal adenocarcinoma

Mitogen-activated protein kinase kinase (MEK) is activated by mutated KRAS in >90% of pancreatic ductal adenocarcinoma (PDAC). MEK and focal adhesion kinase (FAK) are frequently co-activated in PDAC providing a rationale for combining trametinib, an oral allosteric MEK1/2 inhibitor, with GSK2256098, an oral FAK inhibitor. Advanced PDAC patients whose disease progressed after first line palliative chemotherapy were treated with GSK2256098 250 mg twice daily and trametinib 0.5 mg once daily orally. The primary endpoint was clinical benefit (CB; complete response, partial response, or stable disease ≥24 weeks). Twenty-four patients were planned to enroll using a 2-stage minimax design (P0=0.15, P1=0.40; alpha =0.05, power 0.86). The combination would be considered inactive if 2/12 or fewer patients achieved CB at the end of stage 1, and would be considered active if >7/24 response-evaluable patients achieved CB by the end of stage 2. Serial blood samples were collected for circulating tumor DNA (ctDNA) mutation profiling. Sixteen patients were enrolled and 11 were response evaluable. Of those 11, 10 had progressive disease as best tumor response and one had stable disease for 4 months. No treatment related grade ≥3 adverse events (AEs) were observed. The median progression free survival (PFS) was 1.6 (95% CI: 1.5-1.8) months and the median overall survival (OS) was 3.6 (95% CI: 2.7-not reached) months. One response-inevaluable patient achieved clinical stability for 5 months with reduction in CA19-9 and ctDNA levels with a MAP2K1 treatment resistance mutation detected in ctDNA at clinical progression. The combination of GSK2256098 and trametinib was well tolerated but was not active in unselected advanced PDAC.

Analysis of Circulating Tumor DNA to Predict Risk of Recurrence in Patients With Esophageal and Gastric Cancers.

Circulating tumor DNA (ctDNA) analyses allow for postoperative risk stratification in patients with curatively treated colon and breast cancers. Use of ctDNA in esophagogastric cancers (EGC) is less characterized and could identify high-risk patients who have been treated with curative intent. In this retrospective analysis of real-world data, ctDNA levels were analyzed in the preoperative, postoperative, and surveillance settings in patients with EGC using a personalized multiplex polymerase chain reaction-based next-generation sequencing assay. Plasma samples (n = 943) from 295 patients at > 70 institutions were collected before surgery, postoperatively, and/or serially during routine clinical follow-up from September 19, 2019, to February 21, 2022. ctDNA detection was annotated to clinicopathologic features and recurrence-free survival. A total of 295 patients with EGC were analyzed, and 212 patients with stages I-III disease were further explored. Pretreatment ctDNA was detected in 96% (23/24) of patients with preoperative time points. Postoperative ctDNA was detected in 23.5% (16/68) of patients with stage I-III EGC within 16 weeks (molecular residual disease window) after surgery without receiving systemic therapy. ctDNA detection at any time point after surgery (hazard ratio [HR], 23.6; 95% CI, 10.2 to 66.0; P < .0001), within the molecular residual disease window (HR, 10.7; 95% CI, 4.3 to 29.3; P < .0001), and during the surveillance period (HR, 17.7; 95% CI, 7.3 to 50.7; P < .0001) was associated with shorter recurrence-free survival. In multivariable analysis, ctDNA status and clinical stage of disease were independently associated with outcomes. Using real-world data, we demonstrate that postoperative tumor-informed ctDNA detection in EGC is feasible and allows for enhanced patient risk stratification and prognostication during curative-intent therapy.

Risk Profiling of Patients with Previously Untreated Diffuse Large B-Cell Lymphoma (DLBCL) By Measuring Circulating Tumor DNA (ctDNA): Results from the POLARIX Study

Background: Retrospective studies suggest that change in ctDNA levels after 1-2 cycles of induction therapy in patients (pts) with previously untreated DLBCL is associated with event-free survival and overall survival (OS), using a log-fold change (LFC) cutoff in ctDNA levels of 2.0 (Kurtz DM, et al. J Clin Oncol 2018). The prognostic value of ctDNA has not been prospectively validated; this is a prespecified exploratory analysis of the prognostic value of ctDNA in the POLARIX study (NCT03274492). Methods: Pts in POLARIX had previously untreated DLBCL and were randomized to receive Pola-R-CHP or R-CHOP (Tilly H, et al. N Engl J Med 2022). Plasma ctDNA levels were measured at baseline and Cycle 2 Day 1 (C2D1) using the AVENIO NHL CAPP-Seq assay and are reported as mean mutant molecules per mL (MMPM; Herrera AF, et al. Blood Adv 2022). Plasma-depleted whole blood at baseline was used as a source of germline DNA to filter non-tumor-specific variants. Change in ctDNA levels was characterized as the log10 ratio between baseline and C2D1 MMPM (LFC); a LFC cutoff of 2.0 was used in this analysis. ctDNA clearance was determined with a detection cutoff of p=0.005 (Scherer F, et al. Sci Transl Med 2016). Univariate and multivariate Cox regression were used to identify relationships between ctDNA and progression-free survival (PFS) and OS. Hazard ratios (HR) are reported with 95% confidence intervals. Results: Results for paired ctDNA samples at baseline and C2D1 were available for 319/440 (72.5%; Pola-R-CHP) and 299/439 (68.1%; R-CHOP) pts. A median 135 (range: 1-644) variants were detected per pt. Baseline ctDNA levels were not statistically different between the two arms (median MMPM: Pola-R-CHP, 356; R-CHOP, 237; p=0.2); high baseline MMPM was associated with high baseline IPI score (p<0.001), ABC DLBCL (p<0.001), ECOG performance status >1 (p<0.001), and presence of bulky disease (>7.5cm; p<0.001). In both arms, pts with baseline ctDNA levels above the median MMPM had shorter PFS (Pola-R-CHP: HR, 1.96 [1.21-3.18]; R-CHOP: HR, 1.43 [0.92-2.20]) and OS (Pola-R-CHP: HR, 2.17 [1.07-4.37]; R-CHOP: HR, 2.10 [1.05-4.21]) than pts with baseline ctDNA levels below the median MMPM. Change in ctDNA levels from baseline to C2 as a continuous fold-change value was associated with PFS (HR, 0.75 [0.65-0.87]) and OS (HR, 0.78 [0.63-0.96]) in both arms. Pts with greater reductions in ctDNA levels after one treatment cycle had longer PFS and OS than pts with lower ctDNA reductions after one cycle. LFC ≥2.0 or undetectable ctDNA at C2D1 were achieved by 66.8% (213/319) of Pola-R-CHP-treated pts and 65.6% (196/299) of R-CHOP-treated pts. Stratifying pts at this LFC threshold was strongly prognostic of survival outcomes by arm (LFC <2.0 vs LFC ≥2.0, PFS HR: Pola-R-CHP 1.86 [1.18-2.92], R-CHOP 2.21 [1.43-3.41], OS HR: Pola-R-CHP 1.48 [0.77-2.81], R-CHOP 2.67 [1.34-5.30]). To define an optimal risk cutoff for pts receiving Pola-R-CHP, pts were grouped into training and validation cohorts. A LFC threshold of 2.5 (LFC ≥2.5 Pola-R-CHP: 54.2% [173/319]; R-CHOP: 54.8% [164/299]) was better able to identify pts at risk of poorer outcomes (LFC <2.5 vs LFC ≥2.5, PFS HR 2.89 [1.78-4.69], 24-month estimates 65.7% [58.3-74.0] vs 87.0% [82.0-92.2]; OS HR 1.87 [0.98-3.58], 24-month estimates 86.2% [80.8-92.0] vs 91.8% [87.7-96.0]; Figure A). In the Pola-R-CHP arm, pts with ctDNA clearance at C2D1 had superior outcomes vs pts with detectable ctDNA at C2D1 (not cleared vs cleared, PFS HR 2.98 [1.53-5.80]; OS HR 2.74 [1.07-7.02]). LFC 2.5 and ctDNA clearance were evaluated in separate multivariate analyses; it was found that both LFC 2.5 and ctDNA clearance are prognostic for PFS and OS in Pola-R-CHP treated pts, independent of key baseline risk factors (Figure B). LFC 2.5 and ctDNA clearance were also prognostic in R-CHOP-treated pts. Lastly, the top five commonly mutated genes identified in baseline ctDNA were HIST1H variants, TP53, PIM1, MYD88, and BCL2; analysis of clonal change on- and post-treatment is ongoing. Conclusions: Based on this prespecified exploratory analysis from the POLARIX study, ctDNA analysis has prognostic value for pts with previously untreated DLBCL. Pts who did not achieve ≥2.5 LFC and/or did not have ctDNA clearance following one cycle of Pola-R-CHP had inferior outcomes than those who did. Early changes in ctDNA levels may be of use in risk-adapted trial designs to identify pts in need of alternative treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular Clustering on Ctdna May Improve Prognostic Stratification of DLBCL Patients

Background. Diffuse large B-cell lymphoma (DLBCL) displays a high degree of molecular heterogeneity and may be classified into molecular clusters harboring specific lesions and therapeutic vulnerabilities. To date, DLBCL molecular clusters have been identified on the tissue biopsy and do not consider mutations deriving from different anatomical compartments. Currently, DLBCL prone to become long-term survivors after R-CHOP or, alternatively, destined to relapse early after treatment initiation cannot be unequivocally identified a priori. The quantification of circulating tumor DNA (ctDNA) on the liquid biopsy at the time of diagnosis allows to stratify the outcome of DLBCL patients but its integration with molecular clustering has so far not been evaluated. Purpose. The aims of this study were: i) to evaluate the potential contribution of liquid biopsy to the identification of DLBCL molecular clusters; and ii) to evaluate whether the assignment to a specific molecular cluster may improve patients’ stratification in combination with ctDNA load. Methods. A multicenter cohort of newly diagnosed DLBCL patients provided with ctDNA from the plasma and genomic DNA (gDNA) from the lymph node (LN) biopsy represented the basis of this study. gDNA extracted from granulocytes was analyzed for comparative purposes. The LyV4.0 CAPP-Seq assay comprising a panel of 59 genes relevant to B-cell malignancies was used. The LymphGen tool was utilized for cluster analysis. Results. The cohort included 77 newly diagnosed DLBCL patients treated prospectively with R-CHOP. The median age of the studied cohort was 67 years, 68.8% presented with stage III-IV and 22.1% had a high-risk IPI. After a median follow-up of 33.9 months, the 40-month progression-free survival (PFS) and overall survival (OS) were 66.5% and 80.2%, respectively. Mutational analysis identified at least one somatic non-synonymous mutation in 92.2% of patients (71/77) in the LN biopsy and in 87.0% (67/77) in the ctDNA. The most frequently mutated genes in both compartments and in more than 20% of patients were KMT2Dfollowed by PIM1 and HIST1H1E. To assign patients to molecular clusters, we applied the LymphGen tool to both ctDNA and LN. This tool allowed the cluster assignment in 40.3% (27/67) DLBCL on ctDNA and in 46.5% (33/71) on the LN biopsy. On ctDNA, 9 patients were classified as MCD, 5 as ST2, 7 as EZB, 5 as BN2 and 1 as molecular composite (BN2/ST2). By evaluating the clinical impact of molecular clusters, patients assigned to the ST2 and BN2 clusters in both ctDNA (p=0.032) and in LN biopsy (p=0.007), displayed a very good PFS compared to other patients. By recursive partitioning for PFS, patients with a baseline ctDNA load <2Log10hGE (N=17) had a 40-month PFS of 94.1% compared to 54.6% for patients (N=49) with higher ctDNA levels (p=0.003) and were not further stratified. Conversely, patients with ctDNA levels >2Log10hGE were further stratified by the assignment to the ST2 and/or BN2 clusters. Patients assigned to cluster ST2 or BN2 (N=10), despite having ctDNA levels >2Log10hGE, had a 40-month PFS of 100% compared to 44.4% for patients (N=39) classified as MCD or EZB or not classified (p=0.004). Therefore, by combining ctDNA levels and molecular clusters identified on the liquid biopsy, we identified 2 different groups of patients with a significantly different PFS and OS (both p<0.001). Patients with a <2Log10hGE and/or assigned to cluster BN2 and ST2 presented a 40-month PFS and OS of 96.2% and 100%, respectively. Conversely, patients with a ctDNA level >2Log10hGE and not assigned to cluster ST2 and BN2 presented a 40-month PFS and OS of 44.4% and 61.5%, respectively (Figure 1). To evaluate the ctDNA dynamics according to a specific molecular cluster, we analyzed the ctDNA of 48 patients after one cycle of R-CHOP. Ten were classified as ST2 or BN2 at the time of diagnosis of which, after 1 cycle of R-CHOP, eight (80%) reached an early molecular response (EMR). Interestingly, the 2 ST2/BN2 patients who did not achieve an EMR have remained in CR and have not progressed at the last follow-up. Conclusions. Molecular clustering on the liquid biopsy reflects in most cases the prognostic impact of clusters identified in the tissue biopsy. If validated, the combination of ctDNA levels <2 Log10hGE and ST2/BN2 molecular clusters on the liquid biopsy represents a powerful biomarker to identify patients who can benefit most from R-CHOP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Circulating Tumor DNA As a Biomarker for Molecular Residual Disease Assessment in Patients with Diffuse Large B-Cell Lymphoma

Introduction: PET/CT (Positron Emission Tomography/Computed Tomography) scans are the preferred modality of treatment response monitoring and surveillance in patients with diffuse large B-cell lymphoma (DLBCL). However, the interpretation of PET/CT results can be challenging due to low sensitivity of 18F-fluorodeoxyglucose (FDG) tracers, leading to high rates of false positivity and false negativity at the interim and end-of-treatment scans. Circulating tumor DNA (ctDNA) has been demonstrated as an independent prognostic biomarker across numerous cancer types including lymphoma. To date, studies examining the use of ctDNA have mostly employed targeted gene panels, which may not appropriately capture the genetic heterogeneity of the disease. Here, we examined the feasibility of utilizing personalized and tumor-informed ctDNA testing in patients with DLBCL. Methods: In a real-world study, 51 patients diagnosed with systemic DLBCL were enrolled from September 20, 2020 to May 5, 2022. ctDNA analysis was conducted using a personalized, tumor-informed assay designed to detect the top 16 clonal mutations identified in a patient's tissue specimen (SignateraTM bespoke mPCR NGS assay). For newly diagnosed and relapsed/refractory patients, blood samples were collected and analyzed at diagnosis (for newly diagnosed DLBCL), prior to each cycle of therapy, and every 3 months thereafter during surveillance. Our primary objective was to determine the feasibility of developing a bespoke ctDNA assay for individual patients and evaluate concordance rates between ctDNA levels at baseline with the presence of active disease on PET/CT scans. Secondary objectives were to evaluate ctDNA kinetics during treatment and investigate correlation with PET/CT results. Exploratory analyses included correlation of baseline ctDNA levels at diagnosis with high-risk features, such as double/triple hit disease, Ki67, and revised International Prognostic Index (R-IPI), as well as survival outcomes. Clinical follow-up is available for all patients and longitudinal ctDNA assessment is ongoing. Results: For all patients enrolled in the study, staging information and patient characteristics are outlined in Table 1. Of the 51 patients analyzed for ctDNA, a personalized assay with 16 tumor-specific mutations was successfully designed for 49 patients (96.1%). Of those, 47 were starting upfront or second line therapy and 2 were being monitored during surveillance. Prior to treatment initiation, ctDNA was detected in 97.9% (46/47) of patients with levels ranging from 0.1-5957.8 mean tumor molecules (MTM)/ml of plasma. Of the 46 ctDNA-positive patients, longitudinal assessment of ctDNA revealed concordance with radiologic findings at the restaging PET scan. ctDNA levels at baseline were prognostic of patient outcomes. Patients with ctDNA levels above the median (249.37 MTM/ml) had inferior overall survival compared to patients with ctDNA levels below the median (p=0.0009) (Figure 1). Lastly, we plan to present analyses on the correlation of ctDNA levels with clinical risk features and ctDNA dynamics with patient outcomes. Conclusion: Measurement of ctDNA with a personalized and tumor-informed assay is feasible in patients with DLBCL, with a baseline detection rate of 97.9%. Baseline ctDNA levels were prognostic with higher MTM/ml correlating with inferior OS. Our results suggest that ctDNA is a useful biomarker and should be considered when evaluating patient response to treatment. Taken together, longitudinal ctDNA monitoring is feasible in DLBCL and could provide further insights into disease progression. Additional follow-up and clinical studies are needed to evaluate the utility of ctDNA testing for clinical decision making in this setting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ultrasensitive Circulating Tumor DNA (ctDNA) Dynamics after Autologous CD30.CAR-T Cell Therapy for Relapsed or Refractory (r/r) Classical Hodgkin Lymphoma (CHARIOT Trial)

Introduction Liquid biopsies leveraging circulating tumor DNA (ctDNA) are rapidly emerging as minimally invasive tools to detect, genotype, and monitor diverse tumors. ctDNA dynamics and early responses are well-characterized in non-Hodgkin Lymphomas, especially during frontline immunochemotherapy. However, ctDNA responses and minimal residual disease (MRD) are less well-defined in classical Hodgkin Lymphomas (cHL), especially in the relapsed/refractory setting and after cellular immunotherapy, where significant unmet needs remain. We hypothesized that ultrasensitive ctDNA-based MRD detection by Phased Variant Enrichment & Detection Sequencing (PhasED-Seq; Kurtz et al, 2021 Nat Biotech) can accurately track response and predict progression after investigational therapy of r/r cHL with autologous CD30.CAR-T cells. Method This Phase 2 single arm, multicenter study (NCT04268706) investigates safety and efficacy of CD30.CAR-T cells in cHL patients experiencing progression after at least 3 lines of therapy. In the Pilot part of the study, ctDNA was analyzed as an exploratory biomarker using PhasED-Seq at multiple time-points, including at baseline (pre-treatment), Day 42 post-CD30.CAR-T infusion (D42), and upon progressive disease (PD). Exploratory analyses were performed to assess the predictive role of ctDNA and to compare metabolic tumor response by positron emission tomography (PET) scan with ctDNA-based minimal residual disease (MRD). We profiled 55 serial blood specimens (41 plasma and 14 buffy coat samples) from 14 patients with available samples. Cell-free DNA was profiled by PhasED-Seq in the Foresight Diagnostics CLIA laboratory (Aurora, CO). Baseline plasma and D42 buffy coat specimens were used to genotype each patient's tumor to identify Phased Variants (PVs), and these were used to monitor MRD in subsequent blood specimens. Patients/samples were reported as MRD positive when ctDNA levels exceeded an analytical detection threshold (~1 part per million cfDNA molecules), corresponding to 98% clinical specificity. Result As of 22 July 2022, 17 patients have been screened and 15 patients enrolled in the Pilot segment (median age: 35 years [21-57], 66.7% male). Overall response rate (ORR) after CD30.CAR-T single infusion was 73.3% (n=11), with complete response (CR) rate of 60% (n=9). ORR in 5 patients after the 2nd infusion was 100% (n=5), with CR of 60% (n= 3). ctDNA was successfully genotyped directly from pre-treatment plasma in 12 out of 14 patients where samples were available. Both patients where PV genotyping failed had received bridging cytoreductive therapy before baseline plasma collection. Out of 12 profiled patients, 7 patients achieved CR with median baseline ctDNA concentration of 44.9 hGE/mL. 3 patients had PD with median baseline ctDNA level of 94.0 hGE/mL. One patient with PR and one patient with SD had baseline ctDNA levels of 38.9 hGE/mL and 231.1 hGE/mL, respectively. These results suggest that pre-treatment ctDNA levels could have predictive value on patient response to CAR-T therapy. ctDNA responses largely mirrored radiographic responses. Specifically, among 7 patients achieving radiographic CR, 4 also achieved undetectable ctDNA, and the other 3 had ctDNA responses with low MRD levels (median: 0.2 hGE/mL) at D42. One patient with PR also had low ctDNA concentration at D42 (0.7 hGE/mL). In comparison, 3 patients experiencing PD had relatively higher ctDNA levels (median: 131.4 hGE/mL). Furthermore, ctDNA levels at D42 were significantly correlated with PET Deauville score (p=8.7e-4), and patients with a response had significantly lower ctDNA than those without a response (p=0.004) (Fig B). These results suggest that ctDNA-MRD levels are strongly correlated with metabolic responses determined by PET imaging. Similar results were observed after 2nd infusions of the CAR-T. 3 out of 3 patients achieving CR had low ctDNA concentrations at Day 42 post-2nd infusion (median: 0.2 hGE/mL). Among 2 patients with PR, 1 patient with available sample had 31.9 hGE/mL ctDNA. Conclusion Minimally invasive ctDNA analysis is a viable method to monitor responses, rapidly risk stratify and predict outcomes of patients with r/r cHL treated with CD30.CAR-T therapy. A single infusion of CD30.CAR-T therapy rapidly induces MRD negativity in a significant subset of patients at Day 42. ctDNA as exploratory biomarker will be further evaluated in Pivotal part of the study. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Determinants of Resistance to Engineered T-Cell Therapies Targeting CD19 in Large B-Cell Lymphomas

Background: Anti-CD19 chimeric antigen receptor (CAR19) T-cells have activity in patients with relapsed/refractory large B-cell lymphoma (rrLBCL), but over half of patients ultimately relapse. We originally applied cell-free DNA (cfDNA) analysis to a cohort (n=65) of patients receiving axicabtagene ciloleucel (axi-cel) to identify determinants of resistance and characterize molecular thresholds predictive of treatment failure. Here, we extend these analyses to an additional independent cohort (n=73) of patients to validate these thresholds and refine genomic alterations associated with resistance. Methods: We developed a novel hybrid capture strategy targeting 187 protein coding genes, all T-cell receptor (TCR) loci, and axi-cel retroviral vector. This approach allows for simultaneous evaluation of circulating tumor-derived DNA (ctDNA), CAR19-derived cfDNA (cfCAR) and T-cell receptor rearrangements (cfTCR) from blood plasma. We applied this platform to profile serial samples from discovery (n=65) and validation (n=73) cohorts of rrLBCL patients receiving CAR19 therapy (total n=128). Pretreatment and relapse tumor tissue was also profiled when available. A previously determined optimized pretreatment (Day 0) ctDNA threshold to stratify event free survival (EFS) was revalidated in the discovery cohort using bootstrap resampling (2.5 log10 haploid genome equivalents per milliliter). Results: The median follow-up was 36.4 months in the discovery cohort, and 12.5 months in the validation cohort. 55% (36/65) and 58% (42/73) of patients in the discovery and validation cohorts had progressed following CAR19, respectively. In both cohorts, patients with high pretreatment ctDNA demonstrated inferior EFS (Discovery: p<0.001, Validation, p=0.002). Similarly, a ~300-fold (2.5 log10) decrease in ctDNA levels at Day 28 (Major Molecular Response - MMR) post CAR19 infusion was identified as the optimal post-treatment change to stratify EFS, and patients in both cohorts who achieved MMR had significantly superior EFS (Discovery: p<0.001, Validation, p=0.028). When considering ctDNA, cfTCR and cfCAR as continuous variables in the discovery cohort, higher pre- and post-treatment ctDNA levels were prognostic of EFS at all assayed time-points, as were pre-treatment LDH, week 1 cfCAR19 and week 4 cfTCR levels (Fig. A). Forward stepwise multivariable regressions identified week 4 ctDNA level (HR=2.2, p<0.001) and week 1 cfCAR19 level (HR=0.6, p=0.017) as the optimal independent covariates for predicting outcomes (Fig. A). A Cox proportional hazards model incorporating these two variables was trained on the discovery cohort (CAR Outcome Score) and was validated to be prognostic for outcomes in the validation cohort (HR=2.7, p<0.001). Genomic alterations in several genes were significantly associated with inferior outcomes in both cohorts, including PAX5, IRF8, TMEM30A, PPM1D and TP53 (Fig. B). Additional, non-stereotyped, emergent alterations were noted in relapsing patients in other genes associated with immunological escape, including mutations in CD19 and amplifications in the PD-L1 locus. CIBERSORTx was used to characterize intratumoral cell type composition, which was found to be influenced by tumor genotypes. Further, CAR19 persistence in tumors at relapse was associated with specific changes in the tumor microenvironment, including regulatory T-cell expansion and B-cell depletion. Conclusions: Pre-treatment and dynamic ctDNA levels predict treatment failure of LBCL after CAR19 therapy, and a novel CAR outcome score incorporating week 4 ctDNA and week 1 cfCAR19 levels is strongly predictive of outcome. Genomic determinants of resistance to CAR19 therapy are diverse, including recurrent mutations in genes controlling B-cell identity (IRF8, PAX5), and those influencing the composition of the tumor microenvironment and heterotypic cell-cell interactions (TMEM30A). Additional immunological escape mechanisms arise under selective pressure from CAR19 cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Disease Characterization and Response Prediction in Myeloma Patients Undergoing Conventional and Cellular Therapies from Circulating Tumor DNA

Background: Multiple myeloma (MM) is characterized by dramatic biological and clinical heterogeneity. Prognostic biomarkers including genomic alterations and bone marrow (BM) minimal residual disease can resolve some of this heterogeneity; however, these rely on invasive tumor sampling which limits their utility. Here we develop and apply plasma cell-free DNA liquid biopsies via Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) to characterize tumors and quantify treatment response in patients with MM and explore its utility during anti-BCMA CAR T-cell therapy. Methods: We developed a 480 kb MM-specific CAPP-Seq panel, which covers 86 recurrently mutated coding genes as well as other recurrently mutated regions, for disease detection and quantification from circulating tumor DNA (ctDNA). Using this hybrid-capture panel, we profiled 214 plasma, germline and tumor DNA samples from 35 patients including 15 patients undergoing idecabtagene vicleucel (ide-cel) therapy. Samples were sequenced to ultra-high unique molecular depth as previously described (Newman et al, Nat Biotechnol 2016) to quantify ctDNA. Copy number variants (CNVs) were identified using our recently developed method, CANARy (Chabon et al, Nature 2020). Tumor variants and ctDNA levels were identified prior to and following therapy to assess ctDNA dynamics, and were correlated with clinical indices and patient outcomes. Results: We applied CAPP-Seq to samples from 35 patients with newly diagnosed (ND, n=10) or relapsed/refractory (RR, n=25) plasma cell disorders. A median of 87 single-nucleotide variants (SNVs, range 2-283) were detected per case. Among these, a median of 5 mutations/patient were in coding genes such as KRAS (25% of patients), TP53 (25%) and NRAS (19%). Most other SNVs occurred in genomic regions targeted by activation induced deaminase (AID), such as IGH, IGK and IGL. To validate tumor genotyping from plasma, we compared SNVs observed in plasma to paired BM samples in 18 cases. Here, we observed 84% (594/703) of all mutations identified in BM were also present in plasma, including 90% (26/29) of all coding alterations. Interestingly, we detected an additional 259 SNVs in the plasma that were not present in the BM, including KRAS, NRAS, and FAM46C mutations, suggesting ctDNA may better capture spatial tumor heterogeneity. We next utilized CAPP-Seq to quantify disease burden prior to treatment. We detected ctDNA in 97% (31/32) of pretreatment plasma with a median allele frequency of 0.91% (range 0.006 to 36%) and 3.82 log haploid genome equivalents per mL of plasma (hGE/mL). Levels of ctDNA were higher in RRMM compared to NDMM patients (4.26 vs 3.08 log hGE/mL, p=0.02) and correlated with clinical risk factors including R-ISS (stage 1: 2.83 vs stage 2/3: 4.18 log hGE/mL, p=0.01) as well as disease burden measurements including M-spike (rho=0.53, p=0.02) and free light chain ratio (rho=0.40, p=0.03). The fold change in ctDNA level at a median of 36 days from treatment initiation correlated with clinical response (median 0.060 for non-progressors vs 7.0 for progressors, p=0.04). Further, we applied CAPP-Seq to serial samples from 15 patients with RRMM receiving ide-cel. Notably, patients with longer response (>90 days) had significantly lower ctDNA levels at day 28 compared to those with early (<90 days) progression (0.6 vs 4.1 log hGE/mL; p=0.009, Fig 1). Lower ctDNA levels at day 28 predicted longer time to progression (proportional hazard model, p=0.02, HR=1.6). Finally, we assessed clonal evolution upon relapse after ide-cel. In multiple patients we observed emergence of new CNVs in ctDNA, including a case with an emergent loss of chr16, where TNFRSF17 (BCMA) resides (Fig 2). This copy number loss was observed 36 days prior to clinical relapse, was validated through WGS of the relapsed tumor, and resulted in loss of BCMA by immunohistochemistry. Conclusions: Assessment of ctDNA has significant potential utility in MM, as it allows for mutational genotyping, quantification of disease burden, and identification of tumor clonal evolution. Lower levels of ctDNA during treatment and its clearance are associated with improved outcomes, while detection of emergent alterations after targeted therapies may inform subsequent treatment strategies. Use of ctDNA may improve response and relapse prediction and reveal mechanisms of therapy resistance. *SS and DMK are co-senior authors Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal