ctDNA Clearance Research Articles

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 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.

Abstract 976: Detection of circulating tumor DNA predicts survival in advanced HCC patients treated with personalized therapeutic DNA cancer vaccine in combination with immune checkpoint blockade

Abstract Circulating tumor DNA (ctDNA) has enabled the non-invasive monitoring of molecular residual disease (MRD) which reflects therapeutic response/resistance prior to conventional imaging approaches. However, little is known about the applications of ctDNA in the neoantigen-targeted personalized cancer vaccine setting, potentially due to the limited sensitivity of current ctDNA assays. NeXT Personal®, an ultra-sensitive tumor-informed ctDNA assay was used to longitudinally track ctDNA in advanced hepatocellular carcinoma (HCC) patients treated with GNOS-PV02 (a personalized therapeutic DNA cancer vaccine) in combination with pembrolizumab. Advanced unresectable or metastatic HCC patients that progressed on, or were intolerant to, first-line TKI therapy were enrolled into the Phase 1b/2a GT-30 study [NCT04251117]. Whole exome/transcriptome tumor sequencing was used for the design of GNOS-PV02. Patients were treated with GNOS-PV02 (1mg; Q3W x 4, Q9W) and plasmid-encoded IL-12 (0.3mg; Q3W x 4, Q9W) in combination with pembrolizumab (200mg; Q3W). Clinical Response was evaluated by RECIST 1.1 at baseline and Q9W. The ctDNA analysis and tracking for progression was performed as an exploratory objective. Over 200 prospective baseline and on-treatment plasma samples from 31 patients were collected and analyzed using NeXT Personal. WGS was performed to identify up to ~1,800 selected tumor variants to create a personalized panel for MRD detection. NeXT Personal has been analytically validated to detection thresholds down to 1-3 parts per million (PPM) of ctDNA with >99.95% specificity. ctDNA was detected in 152 of 207 plasma samples with a dynamic range of 1.76 - 166,968 PPM. The limit of ctDNA detection ranged down to 1.05 PPM. 96% (24/25) of patients were baseline ctDNA+. The percentage change of ctDNA relative to baseline at week 9 (C4D1) significantly correlated with best overall response (CR/PR/SD vs. PD: p = 0.0076). The ctDNA change from baseline to week 9 was predictive of OS (P=0.008), with 31.2% 2-yr OS (95% CI: 13.2 - 73.7%) for molecular non-responders vs 100% 2-yr OS (95%CI: 100 - 100%) for molecular responders. In addition, the ctDNA levels at week 9 were predictive of best overall response (P = 0.017). ctDNA burden at week 9 was significantly correlated with overall survival (HR = 5.46, 95%CI: 1.79 - 16.65; P=0.003; C-index = 0.886). ctDNA clearance was observed in all 3 CR patients with a lead time of 483, 126 and 105 days compared with MRI. This ultra-sensitive ctDNA assay shows significant association between ctDNA change and clinical response and survival, and can be used to accurately predict clinical outcome. The convenience of non-invasive liquid biopsy and rapid availability of data could enable the use of ctDNA to allow real-time monitoring of personalized cancer immunotherapy. Citation Format: Jian Yan, Bailiang Li, Josette Northcott, Charles W. Abbott, Rachel M. Pyke, Renzo Perales-Linares, Neil Cooch, Sarah Rochestie, Joann Peters, Edward J. Gane, Mark Yarchoan, Thomas U. Marron, Sean M. Boyle, Ildiko Csiki, Richard Chen, Niranjan Y. Sardesai. Detection of circulating tumor DNA predicts survival in advanced HCC patients treated with personalized therapeutic DNA cancer vaccine in combination with immune checkpoint blockade [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 976.

Abstract 970: Liquid biopsy-informed precision oncology clinical trial to evaluate the utility of ctDNA comprehensive genomic profiling

Abstract Background: The expanding role of next-generation sequencing (NGS) in precision oncology has led to a paradigm shift towards genotype-targeted therapies, with liquid biopsies (LBs) offering a minimally invasive means of comprehensive genomic profiling. Methods: This is an observational LB-informed precision oncology clinical trial with primary endpoints to assess feasibility and determine the prevalence of actionable mutations in LB informing genotype-targeted therapeutic recommendations by the Johns Hopkins Molecular Tumor Board (JH MTB; NCT05585684). Secondary endpoints are progression-free and overall survival, ctDNA dynamics assessment, and concordance between plasma and tumor NGS. Target enrollment is 150 patients with advanced solid tumors who have progressed on at least one line of systemic therapy. Serial LB-derived genotypes are obtained at baseline, early on therapy, and upon progression using a CLIA ultra-sensitive 33-gene panel NGS assay (Elio Plasma Resolve, Personal Genome Diagnostics/Labcorp). Variant actionability is assessed by an ensemble knowledgebase, registry, and computational characterization approach, and results are reviewed by the JH MTB, followed by treatment recommendations. Results: The trial’s pre-specified interim analysis included 30 enrolled patients with evaluable LBs; 30 baseline, 13 on-therapy, and 6 progression LBs were analyzed. Twenty-five patients had non-small cell lung cancer among which 9 were EGFR-mutated, 1 patient had small cell lung cancer, 3 patients had gastro-esophageal cancer, and 1 patient had a neuroendocrine tumor. In total, 74 variants were identified across all time points; these were cross-referenced in somatic and germline registries, variant knowledgebases, and the biomedical literature, together with computational characterization of functional consequence to assign mutation actionability. Tumor-confirmed variants comprised 36% of baseline and 42% of both on-therapy and progression variants. At baseline, 20% of the variants were putatively clonal hematopoiesis-derived with similar findings at the on-therapy timepoint. Thirty percent of baseline variants and 33% of variants at progression were deemed actionable. Upon JH MTB review, 29% of patients received a genotype-matched therapeutic recommendation based on LB results, with MTB recommendations followed in 38% of the patients. In analyzing ctDNA dynamics, of the 13 patients with on-therapy samples, 23% showed ctDNA clearance, 31% showed persistence, while 46% had undetectable ctDNA at both time points. Among patients with LBs at the time of progression, a third showed ctDNA emergence, a third had ctDNA persistence, and the remainder had undetectable ctDNA throughout the follow-up period. Conclusion: Our findings highlight the clinical relevance of liquid biopsies in guiding treatment selection and monitoring. Citation Format: Maria Fatteh, Michael Conroy, Timsy Wanchoo, Selina Shiqing Teh, Jaime Wehr, Archana Balan, Rachel Karchin, Ellen Verner, Katerina Karaindrou, Rena Xian, Christopher Gocke, Ming-Tseh Lin, Peggy Fitzpatrick, Christine L. Hann, Joseph C. Murray, Vincent K. Lam, Josephine L. Feliciano, Julie R. Brahmer, Kristen A. Marrone, Patrick M. Forde, Katie Fiallos, Dana Petry, Taxiarchis Botsis, Mark Sausen, the Johns Hopkins Molecular Tumor Board Investigators, Jenna Canzoniero, Jessica Tao, Valsamo Anagnostou. Liquid biopsy-informed precision oncology clinical trial to evaluate the utility of ctDNA comprehensive genomic profiling [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 970.

Neoadjuvant nivolumab or nivolumab plus LAG-3 inhibitor relatlimab in resectable esophageal/gastroesophageal junction cancer: a phase Ib trial and ctDNA analyses

Gastroesophageal cancer dynamics and drivers of clinical responses with immune checkpoint inhibitors (ICI) remain poorly understood. Potential synergistic activity of dual programmed cell death protein 1 (PD-1) and lymphocyte-activation gene 3 (LAG-3) inhibition may help improve immunotherapy responses for these tumors. We report a phase Ib trial that evaluated neoadjuvant nivolumab (Arm A, n = 16) or nivolumab–relatlimab (Arm B, n = 16) in combination with chemoradiotherapy in 32 patients with resectable stage II/stage III gastroesophageal cancer together with an in-depth evaluation of pathological, molecular and functional immune responses. Primary endpoint was safety; the secondary endpoint was feasibility; exploratory endpoints included pathological complete (pCR) and major pathological response (MPR), recurrence-free survival (RFS) and overall survival (OS). The study met its primary safety endpoint in Arm A, although Arm B required modification to mitigate toxicity. pCR and MPR rates were 40% and 53.5% for Arm A and 21.4% and 57.1% for Arm B. Most common adverse events were fatigue, nausea, thrombocytopenia and dermatitis. Overall, 2-year RFS and OS rates were 72.5% and 82.6%, respectively. Higher baseline programmed cell death ligand 1 (PD-L1) and LAG-3 expression were associated with deeper pathological responses. Exploratory analyses of circulating tumor DNA (ctDNA) showed that patients with undetectable ctDNA post-ICI induction, preoperatively and postoperatively had a significantly longer RFS and OS; ctDNA clearance was reflective of neoantigen-specific T cell responses. Our findings provide insights into the safety profile of combined PD-1 and LAG-3 blockade in gastroesophageal cancer and highlight the potential of ctDNA analysis to dynamically assess systemic tumor burden during neoadjuvant ICI that may open a therapeutic window for future intervention. ClinicalTrials.gov registration: NCT03044613.

Circulating Tumor DNA (ctDNA) Clearance May Predict Treatment Response in Neoadjuvant Colorectal Cancer Management.

Background: Circulating tumor DNA (ctDNA) is extracellular DNA released by tumors and has been proposed as a marker of residual disease as well as a predictor of disease recurrence in the adjuvant setting. However, data are lacking on the utility of this biomarker in the neoadjuvant setting. Methods: We performed a retrospective study of stage III and IV colorectal cancer patients receiving neoadjuvant treatment at a single institution. Results: Seventeen patients converted from a positive pre-neoadjuvant ctDNA to a negative ctDNA prior to surgery. Five patients remained persistently positive despite systemic treatment. ctDNA conversion was found to be associated with a higher incidence of favorable treatment effect scores on final surgical pathology. There was no difference in recurrence-free survival in this small population. Furthermore, no added benefit was identified for patients receiving additional neoadjuvant therapy after the time of positive to negative ctDNA conversion. Conclusions: This study highlights the potential utility of ctDNA and the need for prospective trials in the neoadjuvant setting to monitor treatment response and guide decisions on treatment duration.

Circulating Tumor DNA Profiling in Liver Transplant for Hepatocellular Carcinoma, Cholangiocarcinoma, and Colorectal Liver Metastases: A Programmatic Proof of Concept.

Circulating tumor DNA (ctDNA) is emerging as a promising, non-invasive diagnostic and surveillance biomarker in solid organ malignancy. However, its utility before and after liver transplant (LT) for patients with primary and secondary liver cancers is still underexplored. Patients undergoing LT for hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), and colorectal liver metastases (CRLM) with ctDNA testing were included. CtDNA testing was conducted pre-transplant, post-transplant, or both (sequential) from 11/2019 to 09/2023 using Guardant360, Guardant Reveal, and Guardant360 CDx. 21 patients with HCC (n = 9, 43%), CRLM (n = 8, 38%), CCA (n = 3, 14%), and mixed HCC/CCA (n = 1, 5%) were included in the study. The median follow-up time was 15 months (range: 1-124). The median time from pre-operative testing to surgery was 3 months (IQR: 1-4; range: 0-5), and from surgery to post-operative testing, it was 9 months (IQR: 2-22; range: 0.4-112). A total of 13 (62%) patients had pre-transplant testing, with 8 (62%) having ctDNA detected (ctDNA+) and 5 (32%) not having ctDNA detected (ctDNA-). A total of 18 (86%) patients had post-transplant testing, 11 (61%) of whom were ctDNA+ and 7 (33%) of whom were ctDNA-. The absolute recurrence rates were 50% (n = 5) in those who were ctDNA+ vs. 25% (n = 1) in those who were ctDNA- in the post-transplant setting, though this difference was not statistically significant (p = 0.367). Six (29%) patients (HCC = 3, CCA = 1, CRLM = 2) experienced recurrence with a median recurrence-free survival of 14 (IQR: 6-40) months. Four of these patients had positive post-transplant ctDNA collected following diagnosis of recurrence, while one patient had positive post-transplant ctDNA collected preceding recurrence. A total of 10 (48%) patients had sequential ctDNA testing, of whom n = 5 (50%) achieved ctDNA clearance (+/-). The remainder were ctDNA+/+ (n = 3, 30%), ctDNA-/- (n = 1, 10%), and ctDNA-/+ (n = 1, 11%). Three (30%) patients showed the acquisition of new genomic alterations following transplant, all without recurrence. Overall, the median tumor mutation burden (TMB) decreased from 1.23 mut/Mb pre-transplant to 0.00 mut/Mb post-transplant. Patients with ctDNA positivity experienced recurrence at a higher rate than the ctDNA- patients, indicating the potential role of ctDNA in predicting recurrence after curative-intent transplant. Based on sequential testing, LT has the potential to clear ctDNA, demonstrating the capability of LT in the treatment of systemic disease. Transplant providers should be aware of the potential of donor-derived cell-free DNA and improved approaches are necessary to address such concerns.

Analysis of the effect of baseline detection and early clearance of ct-DNA, on survival outcomes among patients with advanced EGFR-mutant non-small cell lung cancer.

To determine if circulating tumor DNA (ct-DNA) dynamics of epidermal growth factor receptor (EGFR) mutation in plasma can identify asubset of patients with EGFR-mutant (EGFR- m) non-small cell lung cancer (NSCLC) with inferior survival outcomes, we analyzed and compared survival outcomes among patients with and without baseline presence and early clearance of EGFR ct-DNA in plasma. For 66patients newly dia-gnosed with EGFR- m NSCLC, plasma samples were collected at baseline and 1st response assessment at 12-24weeks for extraction of ct-DNA. Estimation of ct-DNA (EGFR exons 18, 19, 20and 21) was done using droplet digital polymerase chain reaction (dd-PCR) on the QX200ddPCR system (BioRad, USA). Patients with detectable EGFR ct-DNA at baseline (sample 1), with either undetectable or persistent detectable ct-DNA in sample 2were classified as clearers and non-clearers, respectively. Fifty-three patients received 1st/ 2nd generation EGFR tyrosine kinase inhibitors (TKIs) and 13received either 3rd generation TKI (osimertinib) or chemotherapy plus gefitinib. The baseline ct-DNA-positive group had more patients with extra thoracic disease (60.4vs. 48.5%). For the entire cohort, there was no difference in median progression-free survival (PFS) among baseline ct-DNA-negative (13.57months) vs. ct-DNA-positive patients (12.32months) (HR 0.74). There was asignificant improvement of PFS among early ct-DNA clearers vs. non-clearers (12.32vs. 9.92months; HR 0.57). For those treated with 1st/ 2nd generation EGFR TKIs, this improvement in median PFS among early ct-DNA clearers vs. non-clearers was more apparent (11.76vs. 6.8months; HR 0.34). Baseline detection of the presence of ct-DNA of EGFR mutation in plasma was not predictive of first-line PFS, but is associated with extra thoracic disease. Patients with EGFR mutation and persistence of ct-DNA at first follow-up have worse PFS and overall survival (OS) in comparison to those clearing the same in plasma, especially among those treated with 1st/ 2nd generation EGFR TKIs.

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.

Longitudinal circulating tumor DNA monitoring for detection of molecular residual disease in patients with penile squamous cell carcinoma.

11 Background: Penile squamous cell carcinoma (SCC) is an extremely rare, aggressive disease, representing 0.4-0.6% of all malignancies in males in the U.S. and Europe. Early diagnosis is imperative, as lymphatic spread is associated with a dismal prognosis. The estimated 5-year survival rate in patients with pelvic lymph node involvement is 0%. Moreover, there are limited treatment options that carry significant toxicity risks. Accurate biomarkers to evaluate treatment response and guide treatment to improve outcomes are needed. Molecular residual disease monitoring with circulating tumor DNA (ctDNA) may be helpful in these scenarios. Methods: This is a retrospective analysis of a cohort of 7 patients with advanced HPV-unassociated penile SCC treated at Rush University Medical Center who monitored closely for molecular residual disease (MRD). Signatera (Natera Inc.), a personalized tumor-informed ctDNA assay, was utilized for detection and quantification of ctDNA in patients’ plasma samples, measured at regular intervals. Progression of disease was based on radiographic imaging. Results: In our cohort, personalized, tumor-informed ctDNA assays were designed for 100% (7/7) of the patients. ctDNA was detectable in at least one time point in all of the patients. One patient underwent partial penectomy and lymph node (LN) dissection with 1/11 LN involved. He is being monitored without adjuvant treatment and has sustained ctDNA clearance. Another patient underwent surgery, and had positive post-operative ctDNA. He was treated with adjuvant TIP (paclitaxel, ifosfamide, and cisplatin) with subsequent ctDNA clearance. In 3 of the patients, ctDNA was checked at regular intervals and rising ctDNA prompted imaging studies that showed progression. These patients proceeded to second line treatment options. The remaining 2 patients are non-surgical candidate with LN positivity, who will begin treatment with TIP and monitoring of ctDNA after each cycle of chemotherapy. Twenty-eight percent (2/7) of patients remain alive with sustained ctDNA clearance. Twenty-eight percent (2/7) of patients remain alive with ctDNA positivity, planned to initiate treatment and monitoring of ctDNA at regular intervals. Forty-three percent (3/7) of patients who had persistent ctDNA positivity are now deceased. Conclusions: Our data suggests that a personalized, tumor-informed ctDNA assay is not only useful for MRD monitoring, but also highly predictive of treatment response and imaging-based progression in patients with penile SCC. In our sample, MRD status correlated with both radiographic progression and clinical outcomes. Prospective studies are needed to better understand how ctDNA can be used to guide neoadjuvant and adjuvant approaches, including treatment intensification or de-escalation, in patients with penile SCC.

Circulating tumor DNA (ctDNA) for informing adjuvant chemotherapy (ACT) in stage II/III colorectal cancer (CRC): Interim analysis of BESPOKE CRC study.

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

A phase II trial of TAS-102 in patients with colorectal cancer with ctDNA-defined minimal residual disease post-adjuvant therapy: Results from the MD Anderson INTERCEPT Program.

136 Background: Circulating tumor DNA (ctDNA) has emerged as a novel marker with high specificity and positive predictive value for identifying radiographically occult microscopic or minimal residual disease (MRD) in colorectal cancer (CRC) patients (pts). An additional widely acknowledged unmet need is the development of therapeutic strategies to eradicate MRD and increase the cure rate of the disease. Ongoing trials are evaluating MRD as a biomarker for determining the intensity of post-operative adjuvant therapy. However, the feasibility and efficacy of therapy in pts who are MRD+ during surveillance (after all curative intent treatments including adjuvant chemotherapy) is unknown. Methods: In this proof-of-concept phase II study, pts with stages II-IV CRC noted to be MRD+ during surveillance (ctDNA+ but no radiographic evidence of disease on imaging in last 30 days) received 6 months (m) of TAS-102. Pts were identified through the MD Anderson INTERCEPT program that integrates MRD testing with a tissue informed assay (Signatera) into standard of care for CRC pts. The primary endpoint was 6-m ctDNA clearance and secondary objectives included 3-m ctDNA clearance, and disease-free survival (DFS). Assuming H0 = 5% spontaneous, HA = 30% ctDNA clearance with TAS 102 & 1-sided α = 0.05, 15 patients are required for β = 85%. Results: Between 7/22/22 and 9/7/23,15 pts with MRD were enrolled. Baseline characteristics included a median age of 62 years (range: 37-73), Caucasian (77%), male (77%), resected Stage IV (92%) and median ctDNA level of 1.55 MTM/ml (range 0.04-28.72). At data cutoff, 11 pts had 3-m ctDNA levels and 10 had a decrease in their ctDNA level (91%) including 5 with undetectable levels (45%). At 6-m, 60% of patients had ctDNA levels at or below prior levels; three maintained undetectable ctDNA at 6-m: of those, two pts had subsequent radiographic progression following therapy completion. Median DFS was 9.4-m in the full cohort. Conclusions: We demonstrate the feasibility of identifying and enrolling MRD+ pts through screening during surveillance onto therapeutic trials and show the ability of an experimental therapy to convert patients to MRD negative status, a necessary but not sufficient step for disease eradication. Preliminary results suggest that treatment with TAS-102 is effective in inducing ctDNA clearance in the short term in most pts, but with no evidence of persistent clearance. Updated results including 6-m ctDNA levels and survival data will be presented. Clinical trial information: NCT05343013 .

Phase II results of circulating tumor DNA as a predictive biomarker in adjuvant chemotherapy in patients with stage II colon cancer: NRG-GI005 (COBRA) phase II/III study.

5 Background: For patients (pts) with colon cancer (CC), the detection of circulating tumor DNA (ctDNA) is associated with persistent disease after resection and outperforms traditional clinical and pathological features in prognosticating recurrence risk. We hypothesized that for pts with low-risk stage II CC, a positive ctDNA status after resection may identify those who benefit from adjuvant chemotherapy. Methods: In this prospective phase II/III clinical trial, pts with resected stage II CC without traditional high risk features and whom the evaluating oncologist deems suitable for active surveillance (i.e., not needing adjuvant chemotherapy) were randomized 1:1 to two arms: standard-of-care/observation (Arm A), or ctDNA assay directed therapy (Arm B). Postoperative blood was analyzed for ctDNA with the Guardant LUNAR assay, covering CC-relevant mutations and CC-specific methylation profiling. Arm B pts with ctDNA detected were treated with 6 mos of adjuvant (CAPOX or FOLFOX) chemotherapy. Primary endpoint for the phase II study was clearance of ctDNA at the 6-mo timepoint. A 1-sided Fisher exact test was used to compare ctDNA clearance between Arm A and Arm B among the first 16 pts with ctDNA detected at baseline. If p>.35, the study would be stopped for futility of ctDNA clearance but would otherwise continue to phase III if p≤.35. We present the results of the pre-planned phase II analysis. Results: 635 pts were randomized (Arm A: 318; Arm B: 317). One pt with ctDNA detected in Arm B declined protocol-directed chemotherapy but was included in the intention to treat analysis. Among the first 16 pts with ctDNA detected at baseline for the primary endpoint analysis, clearance of ctDNA after 6 mos was observed in 3/7 pts (43%, 95% CI 10-82%) in the control arm and in 1/9 pts (11%, 95% CI 0.3-48%) in the experimental arm after chemotherapy (p=.98). There were no unanticipated toxicities in those treated with chemotherapy. Conclusions: The phase II endpoint was not met and further enrollment has been halted based upon pre-specified study stopping rules utilizing the original assay. No improvement in ctDNA clearance was observed after 6 mos of chemotherapy for pts with ctDNA detected following resection of stage IIA CC. Future trials evaluating ctDNA as an integral biomarker for minimal residual disease determination must account for assay specificity in this pt population. Support: U10CA180868, -180822; UG1CA189867; Guardant Health. Clinical trial information: NCT04068103 .

Early identification and treatment of occult metastatic disease in stage III colon cancer (SU2C ACT3 clinical trial).

148 Background: Despite adjuvant chemotherapy, nearly 30% of patients with Stage III colon cancer (CC) will recur. We are performing a randomized clinical trial using a plasma-only circulating tumor DNA (ctDNA) platform after adjuvant therapy for patients with ctDNA-positive Stage III CC. Methods: Patients (pts) with Stage III CC are eligible for prescreening, including tumor tissue profiling by next-generation sequencing. Pts are screened 3-6 weeks after adjuvant chemotherapy. At screening blood is collected for ctDNA analysis with a CLIA-certified sequencing panel (Reveal, Guardant Health). A baseline CT scan is required to confirm lack of metastases. Pts with undetectable ctDNA (-) at screening undergo surveillance with imaging, labs and ctDNA testing every 3 months (Arm 3). ctDNA-detectable (+) pts who are biomarker-negative are randomized to 1:1 to FOLFIRI (Arm 1) or active surveillance (Arm 2). ctDNA+ pts with an actionable biomarker (biomarker-positive) are given 6 months biomarker-directed therapy as follows: V600E/MSS- encorafenib, binimetinib and cetuximab (Arm 5), HER2 amplified trastuzumab/pertuzumab (arm 6), and MSI-H nivolumab (Arm 4). Primary outcome is ctDNA clearance in Arms 1 and 2, 1-month after completion of additional adjuvant therapy or surveillance. Secondary outcomes include RFS and OS in Arms 1 and 2, ctDNA clearance and RFS/OS in the other arms. An interim analysis is planned after 13 patients are enrolled in Arm 1 and primary outcome will be assessed after 50 patients’ complete treatment/surveillance in Arms 1 and 2. Results: From 4/16/2020 through 9/8/2023, 147 patients were screened, 66 screen failed. Pre-screening as well as active ongoing enrollment is underway. To date, 73 patients have enrolled (46 male and 27 female). 53 (72.6%) were white. 10 (13.7%) patients were noted to be ctDNA+ within 6 weeks of completion of adjuvant therapy: 4 in Arm 1; 5 in Arm 2; 1 in Arm 4. 63 pts were ctDNA- and in Arm 3. Conclusions: In this ctDNA guided trial, with single time-point testing, only 13.7% of pts were ctDNA+ after adjuvant chemotherapy. 90% of ctDNA+ patients did not have an actionable biomarker. Strict windows for eligibility and enrollment initially restricted eligibility but these were adjusted to increase eligibility. To mitigate the low positivity rate and allow us to meet our accrual goals in arm 1 and 2, the protocol was amended to allow for entry into the ctDNA+ arms at any time during the first 3 years after completion of adjuvant therapy as opposed to within 6 weeks of completion of adjuvant therapy. The lessons learnt would have implications for other ctDNA-driven clinical trials. Clinical trial information: NCT03803553. Patient distribution in the SU2C ACT3 study. Total Screened Pre-Screening Screen Fail Enrolled Off Study ctDNA Treatment Arm + - Arm 1 Arm 2 Arm 3 Arm 4 Arm 5 Arm 6 147 8 66 73 10 10 63 4 5 63 1 0 0

Circulating tumor DNA (ctDNA) dynamics in patients with colorectal cancer (CRC) with molecular residual disease: Updated analysis from GALAXY study in the CIRCULATE-JAPAN.

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

AMPLIFY-7P: Phase 1 and randomized phase 2 study of amphiphile immunotherapy ELI-002 7P as adjuvant treatment for subjects with G12D, G12R, G12V, G12C, G12A, G12S and G13D Kirsten rat sarcoma (KRAS)-mutated pancreatic ductal adenocarcinoma.

TPS720 Background: KRAS mutations occur in 25% of human tumors, frequently in pancreatic where over 85% relapse after locoregional treatment. ELI-002 7P is a lymph node targeted immunotherapy comprised of Amphiphile (Amph)-modified G12D, G12R, G12V, G12C, G12A, G12S and G13D mutant KRAS peptides together with an Amph-modified CpG oligonucleotide adjuvant. In the first in human AMPLIFY-201 study, a two peptide G12D/G12R formulation increased KRAS-specific T cells in 87% of patients, and T cell responses 13-fold or higher over baseline correlated with outcomes including reduction in risk of relapse or death. The AMPLIFY-7P study hypothesizes that adjuvant administration of ELI-002 7P will be safe and will delay relapse compared to observation in patients with KRAS mutated pancreatic cancers who have completed curative intent locoregional treatment. Methods: This open-label, phase 1 and randomized phase 2 study is evaluating ELI-002 7P in patients with KRAS mutated pancreatic adenocarcinoma following standard of care chemotherapy and surgery. In phase 2, patients are randomized 2:1 to ELI-002 7P versus observation with crossover of the observation arm permitted at the time of confirmed radiographic relapse via iRECIST. Eligible patients must be within 6 months of completion of locoregional treatment for up-front resectable pathologically confirmed pancreatic cancer (Stage I-III) with radiographic NED (no evidence of disease). Phase 2 patients are included regardless of biomarker evidence of recurrent disease, however, those who have not recovered absolute lymphocytes to the normal range following cytotoxic therapy are excluded. The primary phase 2 objective is to compare the efficacy of ELI-002 7P versus observation, with primary endpoint disease-free survival per investigator. Secondary objectives compare biomarker response (reduction or clearance of ctDNA or if ctDNA was not detectable, serum tumor antigen CA19-9) in the subset with positive baseline values, OS, safety, and iRECIST response rate in the crossover subset. Exploratory endpoints include immunogenicity, the association of immunogenicity with high resolution HLA typing, and evaluation of tumor-infiltrating T cells and the tumor microenvironment in the event biopsy tissue is available. An Independent Data Monitoring Committee reviewed phase 1 data and recommended opening phase 2. Subsequent patients will receive up to 10 doses of Amph-peptides 7P 700 mcg each (4.9 mg total), together with Amph-CpG-7909 (10.0 mg) administered over a five-month treatment period. In Phase 2, approximately 135 patients will be enrolled in the United States. An interim analysis is planned using group sequential design for control of overall alpha 0.10. Clinical trial information: NCT05726864 .

Evaluating novel therapies and circulating tumor DNA (ctDNA) as a marker of response in curatively treated gastrointestinal cancers with microscopic residual disease.

TPS765 Background: ctDNA has emerged as a novel tool that can identify the earliest sign of relapse (MRD) after curative therapies with specificities of 93-100%, positive predictive values over 98%, and median lead times of 8-9 months before radiographic relapse. ctDNA has allowed us to identify a "new stage" of high-risk patients (pts) with no evidence of disease radiographically (rNED) and MRD who lack guideline-directed treatment (tx) strategies as, traditionally, they wait for radiographic progression of disease (POD) before starting therapies. Treating MRD is a promising strategy supported by observational studies where ctDNA clearance is linked with improved survival and on-tx ctDNA kinetics predict drug efficacy. We designed a pilot investigator-initiated trial to 1) determine the feasibility of using a surrogate marker, ctDNA, as a rapid signal for positive therapeutic activity and 2) obtain pilot data on the efficacy of a novel combination therapy (atezolizumab [A] + bevacizumab [B]) in eradicating MRD in previously curatively treated pts with GI cancers. Methods: We will enroll 20 pts with any-stage GI cancers divided over 4 cohorts (n=5 colorectal [CRC], n=5 hepatocellular/biliary tract carcinoma, n=5 gastric, n=5 pancreatic adenocarcinoma) who have a positive Signatera ctDNA test and rNED any time after completing standard curative-intent therapies. Pts are treated with A 1200 mg IV + B 15 mg/kg IV on day 1 of a 21-day cycle for up to 1 year with imaging every 12 weeks and serial ctDNA testing every 3 weeks. Co-primary endpoints are rates of pt enrollment in 12 months and rates of ctDNA responses at a 12-week landmark after tx initiation. Pts with ctDNA POD (ctDNA doubling on each of 2 sequential tests, any rate of rise in 3 sequential tests, or radiographic relapse) will stop while those with ctDNA complete response (CR; clearance on 2 sequential tests + ongoing rNED) or ctDNA partial/stable response (PR; not ctDNA CR or POD + ongoing rNED) will continue tx. Each cohort's Bayesian predictive probability of positive therapeutic activity will be calculated. If at least 60% of a cohort experiences ctDNA CR, it will be expanded by 5 pts. This scenario is equivalent to Simon's 2-stage Minimax design testing a null hypothesis of 20% CR vs. an alternative hypothesis of at least 60% CR at 5% alpha with 80% power. If at least 5/10 pts in an expanded cohort experience ctDNA CR, this will generate interest in developing a larger confirmatory trial to evaluate pt survival outcomes with A+B and ctDNA as a surrogate for survival. Secondary endpoints are toxicity and reasons for enrollment failure. Exploratory endpoints are associations between ctDNA conversion and disease-free survival, tumor whole exome sequencing data, and peripheral blood immune profiles collected at baseline and on tx. Enrollment began in Q1 2023. 4 pts are enrolled in the CRC cohort. Clinical trial information: NCT05482516 .

A randomized controlled trial of CAPEOX vs observation in patients with early-stage colorectal cancer with positive MRD after curative surgery (CAREME).

TPS225 Background: Most patients (pts) with early-stage colorectal cancer (CRC) have a good prognosis, but some still relapse shortly after surgery. Effective methods to assess the risk of recurrence in these pts and guide adjuvant chemotherapy (ACT) decision-making are lacking. Increasing studies have shown that circulating tumor DNA (ctDNA) can detect minimal residual disease (MRD) and identify pts with a high risk of recurrence. ACT could improve survival of MRD-positive pts with stage Ⅱ CRC suggested by recent studies, whether it is appropriate for those with stage Ⅰ or clinically low-risk stage Ⅱ CRC remains unknown. Methods: CAREME is a multicenter randomized controlled clinical trial aimed at investigating the benefit of chemotherapy for MRD-positive pts with early-stage CRC. Pts with resected stage Ⅰ or Ⅱ CRC will be evaluated by oncologists. Those who did not receive neoadjuvant therapy and are deemed suitable for active surveillance (i.e., ACT is not needed) will undergo a postoperative ctDNA test using MinerVa MRD assay (a tumor-informed assay covering 769 cancer-related genes). Pts with mismatch repair-deficient tumors will be excluded and stage Ⅱ pts should have no traditional high-risk features according to clinical practice guidelines. Pts with positive ctDNA (N = 38) will be equally randomized into two groups: treatment group (Arm A) or surveillance group (Arm B). Arm A will receive 8 cycles of CAPEOX therapy while pts in Arm B will not receive any ACT; both groups will undergo ctDNA tests at 6 months after randomization and be followed up every 3 months. The primary endpoint is 18-month recurrence-free survival, and a key secondary endpoint is ctDNA clearance at 6 months after randomization. Tumor samples from the pts will be collected for MRD assay and exploratory research. Accrual started in February 2023. Support: Genecast Biotechnology Company. Clinical trial information: NCT05699746 .

Abstract C092: T cell responses and clinical outcomes in pancreatic and colorectal cancer patients with minimal residual disease in AMPLIFY-201, a phase 1 trial of a first-in-class amphiphile lymph node targeted mutant KRAS vaccine

Abstract Background: Mutations in RAS occur in > 25% of solid tumors and > 90% of patients (pts) with PDAC and > 40% of pts with CRC. ELI-002 2P is a vaccine comprised of lymph-node targeted Amphiphile (Amph)-modified G12D and G12R mutant KRAS peptides together with an Amph-modified CpG oligonucleotide adjuvant designed to expand polyfunctional mutant KRAS-specific T cells. Interim phase 1 AMPLIFY-201 data (O’Reilly et al; ASCO, 2023, #2528) showed that adjuvant administration of ELI-002 was well-tolerated, with T cell responses in 87%, and tumor biomarker responses in 77% (a subset of 32% had ctDNA clearance). Here we report relapse free survival data and correlations between ELI-002 2P- induced T cell levels and outcomes in pts with PDAC and colorectal cancer (CRC) at high risk for relapse following locoregional treatment. Methods: Resected PDAC (n=20) and CRC (n=5) pts with tumors harboring KRAS G12D or G12R who had minimal residual disease positivity (MRD+) defined as elevated circulating tumor DNA (ctDNA) and/or serum tumor biomarker (CA19-9/CEA), received up to 6 priming doses and 4 booster doses separated by a 3-month rest period of subcutaneous ELI-002 2P vaccine monotherapy comprised of Amph-peptides (700 mcg each G12D/G12R), admixed with Amph-CpG-7909 at 0.1, 0.5, 2.5, 5.0 and 10.0 mg per cohort dose level. Primary endpoints included safety and recommended phase 2 dose (RP2D) of Amph-CPG-7909; secondary endpoints: biomarker reduction/clearance; exploratory endpoints: included relapse free survival (RFS) using immune Response Evaluation Criteria in Solid Tumors (iRECIST) and immunogenicity assessed by direct ex vivo Fluorospot and intracellular cytokine staining of peripheral blood mononuclear cells. Results: Direct ex vivo polyfunctional mKRAS-specific T cell responses to ELI-002 2P were observed in 20/23 (87%; 50% induced both CD4+ and CD8+ T cells, median 13-fold and mean 56-fold increase from baseline), with response in 9/9 (100%) pts treated at highest two dose levels including the 10 mg RP2D. Clinical efficacy correlated with T cell response in evaluable pts (n=22): median tumor biomarker reduction/clearance was -86.9% vs -4.5% in above vs below median T cell responders, respectively (p < 0.0124). At 25 weeks median follow-up, the median RFS was not reached compared to 17.1 weeks in above versus below median T cell responders (HR 0.11; 95% CI 0.027-0.446; p = 0.0108). The association of RFS with T cell response was not confounded by other baseline prognostic variables (including tumor stage, recovery from prior cytotoxic therapy as assessed by absolute neutrophil count, or immune system subsets such as %CD4+ or %CD8+of CD3+ lymphocytes). Conclusions: The association of ELI-002 2P vaccine T cell responses with clinical outcomes in a novel MRD+ trial design underscores the potential to reduce recurrence in the setting of microsatellite stable PDAC and CRC. A new phase 1/2 trial, AMPLIFY-7P (NCT05726864), is evaluating a new seven peptide (G12D, G12R, G12V, G12C, G12A, G12S, G13D) ELI-002 7P formulation. Citation Format: Zev A. Wainberg, Shubham Pant, Colin D. Weekes, Muhammad Furqan, Pashtoon M. Kasi, Craig E. Devoe, Alexis D. Leal, Vincent Chung, James R. Perry, Lochana Seenappa, Lisa K. McNeil, Esther Welkowsky, Peter C. DeMuth, Christopher M. Haqq, Eileen M. O'Reilly. T cell responses and clinical outcomes in pancreatic and colorectal cancer patients with minimal residual disease in AMPLIFY-201, a phase 1 trial of a first-in-class amphiphile lymph node targeted mutant KRAS vaccine [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C092.

Abstract A002: A phase II study of peri-operative modified FOLFIRINOX in localized pancreatic ductal adenocarcinoma (PDAC)

Abstract Introduction: Advances in adjuvant chemotherapy with 5-fluorouracil, irinotecan, and oxaliplatin (FFX) have improved survival for patients (pts) with resectable PDAC yet few are cured by the current standard of care. Neoadjuvant FFX may lead to early control of micrometastasis and enhance cure rates for this aggressive malignancy. Methods: We performed a single-arm phase-2 clinical trial for resectable PDAC evaluating 6 months of perioperative modified (m) FFX (NCT02047474). Patients underwent baseline pancreatic protocol CT and were reviewed for resectability by a multidisciplinary panel. Enrolled pts received 6 cycles of neoadjuvant mFFX followed by surgery and 6 cycles of adjuvant mFFX. The primary endpoint was a ≥ 66% 12-month progression-free survival (PFS). Additional endpoints included overall survival (OS), circulating tumor DNA (ctDNA), tumor molecular features and tumor Keratin 17 levels. Whole blood was prospectively collected and processed to stored plasma (median volume 5.4 mL, range: 2.6 – 10.2) for retrospective ctDNA analysis using a personalized, tumor-informed ctDNA assay (SignateraTM). Survival rates were estimated by Kaplan-Meier. Results: Forty-six pts enrolled with 63 months median follow up, median age of 65 [R 46–80], 36 (78%) were ECOG 0, 30 (65%) had an endobiliary stent and no pts had staging laparoscopy. All pts started mFFX, and 37 pts (80%) completed all 6 preop cycles. Thirty-three pts (72%) underwent surgery with 3 pts developing investigator-assessed progression during neoadjuvant mFFX. Six pts were unresectable intraoperatively, and 27 pts (59%) underwent resection per protocol (25 R0, 2 R1). Ten additional pts underwent surgery off protocol. The 12-month PFS was 78% (95% CI: 64.2–94.4), median PFS and OS were 36.5 months (95% CI: 16.6–74.5) and 37.2 months (95% CI 17.5–not reached), respectively. Two-year OS was 59% (95% CI: 45.8–74.7). Baseline ctDNA was detected in 16/22 (73%) pts, and after 6 cycles of mFFX ctDNA was detected in 3/17 (18%) of pts. Patients with a positive ctDNA test 4 weeks post-resection had worsened PFS (HR 34.0, 95% CI: 2.6-4758.6; P = 0.006) and OS (HR: 11.7, 95% CI: 1.5–129.9; P = 0.021) compared to ctDNA negative. Mutational signature analysis revealed predominant signatures to be COSMIC signatures SBS1 and SBS5 (age related), and SBS15 (mismatch repair deficiency associated) which was associated with improved OS. Using Keratin 17 as a biomarker of the basal molecular subtype, either in diagnostic needle aspirates or surgical specimens, we found that high K17 expression was associated with worse survival. Conclusion: The perioperative mFFX clinical trial for resectable PDAC met its primary endpoint with a promising survival rate, and R0 resection rate of 93%. These findings warrant further evaluation in a randomized clinical trial. Most pts with detectable ctDNA at baseline experience ctDNA clearance after 6 cycles of neoadjuvant mFFX. In the post-operative setting, ctDNA positivity was strongly associated with recurrence. Signature SBS15 and low K17 were associated with improved OS. Citation Format: Michael Cecchini, Ronald Salem, Marie Robert, Suzanne Czerniak, Moein Rajaei, Jeffrey Townsend, Ondrej Blaha, Daniel Zelterman, Jaykumar Thumar, Jeremy Kortmansky, Wajih Zaheer, Neal Fischbach, Justin Persico, Stacey Stein, Sajid Khan, Charles Cha, Kevin Billingsley, John Kunstman, Sumedha Chowdhury, Robert Tseng, Carlos Mauricio, Deanne Yugawa, Luisa Escobar-Hoyos, Kimberly Johung, Christina Wiess, Erik Spickard, Vasily N. Aushev, George Laliotis, Adham Jurdi, Minetta C. Liu, Jill Lacy. A phase II study of peri-operative modified FOLFIRINOX in localized pancreatic ductal adenocarcinoma (PDAC) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A002.