Post-treatment Plasma Samples Research Articles

Abstract PO4-13-09: Paclitaxel affects the efficacy of PD-1 blockade by interfering with lipid metabolism reprogramming in patients with breast cancer

Abstract Background: The question of how chemotherapeutic drugs affect the efficacy of PD-1/PD-L1 antibodies in patients with breast cancer is still pending. In a previously single-cell sequencing study, we have demonstrated that paclitaxel reduces the level of CXCL13+ T cells and other immune cells critical for atezolizumab to work, leading to inferior efficacy of atezolizumab in triple negative breast cancer (TNBC). How paclitaxel affects these important immune cells remains obscure. Recently, plasma metabolomic and proteomics analysis have shown that patients’ systemic metabolic signatures is correlated with the response of PD-1/PD-L1 antibody. As such, understanding the differential metabolic influences of the diverse chemotherapeutic drugs that comprise an immune response to TNBC offers an opportunity to appropriately select chemotherapeutic backbones for ICIs. Methods: We collected pre- and post-treatment plasma samples from breast cancer patients for metabolomic and proteomic analysis, including four patient cohorts: two independent cohorts (discovery cohort and validation cohort) in which patients were treated with ICIs, one cohort in which patients were treated with paclitaxel, and one cohort of patients treated with nab-paclitaxel. Patients were divided into responder group (R, tumor shrinkage) or non-responder group (NR, tumor increase) according to the change in tumor size after treatment relative to baseline. This was followed by integrating metabolomics and proteomics with single-cell transcriptome, along with in vitro experiments, to shed light on the mechanisms of action leading to ICIs resistance in breast cancer patients. Results: In the discovery cohort, the metabolites that mostly differed between the R and NR groups at baseline were lysoglycerophospholipids. These results were further validated in the validation cohort of patients. The dynamic changes of lysoglycerophospholipids were still significantly different between the R and the NR groups after ICIs treatment in the discovery cohort. It’s worth noting that the level of lysophosphatidylcholines (LPCs), the most prominent lysoglycerophospholipids, increased significantly in the R group, while no significant changes among LPCs were found in the NR group after ICIs treatment. In the validation cohort, the median PFS of LPClow patients was 1.8 months versus 6.8 months in the LPChigh group (P = 0.0068). After one cycle of paclitaxel treatment, the metabolic status of patients changed dramatically, especially the levels of lipids. It is notable that most LPCs were significantly downregulated after paclitaxel treatment. However, in patients who received nab-paclitaxel, few significantly changed lipids were detected after one cycle nab-paclitaxel treatment compared with their baseline conditions. Interestingly, the levels of LPCs were upregulated after one cycle nab-paclitaxel treatment, but decreased after paclitaxel treatment. Single-cell omics results showed that in patients' PBMCs, CXCR4+ NK cells were the most closely related to plasma LPC levels, while FGFBP2+ NK cells were in the tumor microenvironment. We isolated and expanded NK cells from PBMCs for in vitro experiments, and found that LPC stimulation can significantly upregulate the expression of CXCR4+ in NK cells, and further increase the chemotaxis and cytotoxicity of NK cells. Conclusions: In breast cancer patients receiving ICIs, the systemic lipid metabolism status, especially lysoglycerophospholipids, is closely related to the efficacy of immunotherapy. The key molecule LPC, the most prominent lysoglycerophospholipids, may promote the efficacy of immunotherapy by activating NK cells in breast cancer patients. Paclitaxel may impair the efficacy of immunotherapy by affecting the systemic lipid metabolism status, especially by reducing plasma LPC levels, so it is not a suitable partner for ICIs. Citation Format: Hongnan Mo, Jiashu Han, Jingtong Zhai, Xiaoying Sun, Xiuwen Guan, Haili Qian, Fei Ma. Paclitaxel affects the efficacy of PD-1 blockade by interfering with lipid metabolism reprogramming in patients with breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-13-09.

Proteomic, single-cell and bulk transcriptomic analysis of plasma and tumor tissues unveil core proteins in response to anti-PD-L1 immunotherapy in triple negative breast cancer

BackgroundAnti-PD-1/PD-L1 treatment has achieved durable responses in TNBC patients, whereas a fraction of them showed non-sensitivity to the treatment and the mechanism is still unclear. MethodsPre- and post-treatment plasma samples from triple negative breast cancer (TNBC) patients treated with immunotherapy were measured by tandem mass tag (TMT) mass spectrometry. Public proteome data of lung cancer and melanoma treated with immunotherapy were employed to validate the findings. Blood and tissue single-cell RNA sequencing (scRNA-seq) data of TNBC patients treated with or without immunotherapy were analyzed to identify the derivations of plasma proteins. RNA-seq data from IMvigor210 and other cancer types were used to validate plasma proteins in predicting response to immunotherapy. ResultsA random forest model constructed by FAP, LRG1, LBP and COMP could well predict the response to immunotherapy. The activation of complement cascade was observed in responders, whereas FAP and COMP showed a higher abundance in non-responders and negative correlated with the activation of complements. scRNA-seq and bulk RNA-seq analysis suggested that FAP, COMP and complements were derived from fibroblasts of tumor tissues. ConclusionsWe constructe an effective plasma proteomic model in predicting response to immunotherapy, and find that FAP+ and COMP+ fibroblasts are potential targets for reversing immunotherapy resistance.

Abstract 2427: The development of a tissue-agnostic genome-wide methylome enrichment MRD assay for applications across the cancer care continuum for head and neck malignancies

Abstract Background: Plasma-based tests to quantify circulating cell-free DNA cancer signal have emerged as viable applications across the cancer continuum, from early detection to optimal disease management. Here we demonstrate the feasibility of a tissue-agnostic, genome-wide methylome enrichment platform based on cell-free methylated DNA immunoprecipitation and high throughput sequencing (cfMEDIP-seq) for cancer detection, cancer signal quantification, and prognostication in head and neck cancer (HNC). Methods: Pre-treatment plasma samples from individuals with newly diagnosed stage I-IV HPV+ or HPV- HNC were analyzed with a bisulfite-free, non-degradative, genome-wide methylome enrichment platform using 5-10 ng of cell-free DNA. For cancer detection, a machine learning classifier used differentially methylated regions to distinguish cancers from non-cancer controls. The area under the receiver operating characteristic curve (AUC) and 95% confidence intervals were calculated. Cancer signals were quantified from average normalized counts across informative methylated regions and a 95% specificity threshold. For prognostication, events were defined as recurrence, progression, or death due to HNC, whichever occurred earliest. Time to event was compared for samples with cancer signal quantities above versus below the threshold. Post-treatment and longitudinal plasma samples from individuals with Stage I-IVB HNC (HPV+ and HPV- included) will be analyzed for recurrence prediction and detection of relapse. More than 100 patients and 300 samples will be analyzed. Results: For cancer detection, 92 pre-treatment plasma samples from HNC cases were distinguished from 674 controls with an AUC of 0.96 (0.94, 0.98). The AUC was 0.93 (0.86, 1.0) for Stage I, 0.93 (0.83, 1.0) for Stage II, 0.96 (0.94, 0.99) for Stage III, and 0.97 (0.96, 0.99) for Stage IV. For prognostication, 91 pre-treatment samples were included (7 stage I, 16 stage II, 23 stage III, 45 stage IV). Median follow-up time was 50.6 months with 27 events. Likelihood of recurrence or progression was significantly higher in samples with cancer signal above the threshold [hazard ratio 5.4 (95% CI 2.25, 12.95), log-rank P<0.001]. In the upcoming analysis, data will be reported on the ability to predict recurrence and relapse in post-treatment samples. Conclusions: The cfMeDIP-seq approach demonstrated robust detection of HNC, across all stages and subtypes, and the ability to predict recurrence and progression from pre-treatment samples. We will report training data with cross validation to predict recurrence and relapse using post-treatment and longitudinal sampling. Collectively, data from these studies indicate that genome wide methylome enrichment has multiple use cases across the care continuum for patients with HNC. Citation Format: Geoffrey Liu, Jun Min, Yarong Wang, Justin Burgener, Ben Brown, Karen Budhraja, Junjun Zhang, Owen Hall, Shu Yi Shen, Martha Pienkowski, Shao Hui Huang, Laurie Ailles, Katrina Rey-McIntyre, Jeremy B. Provance, Eduardo Sosa, Cynthia Frye, Scott Bratman, Brian Allen, Joshua T. Jones, Abel Licon, Jing Zhang, Anne-Renee Hartman, Daniel D. De Carvalho. The development of a tissue-agnostic genome-wide methylome enrichment MRD assay for applications across the cancer care continuum for head and neck malignancies [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 2427.

ASTX660-01 Phase 2: A Case Study of Tolinapant-Induced Pseudoprogression in CTCL

Background: Cutaneous T-Cell Lymphomas (CTCL) are rare malignancies that comprise <10% of Non-Hodgkin Lymphoma. Limited treatment options exist for these patients, especially after progression following front-line therapy. Tolinapant is an oral non-peptidomimetic antagonist of the inhibitor of apoptosis proteins (IAPs), cIAP1/2 and XIAP. It plays a role in regulated tumor cell death and immunomodulation (Ferrari et al., Blood Adv. 2021) and has been evaluated in the clinic (ASTX660-01 trial, NCT02503423). Previously, we reported an overall response rate of 28% in CTCL patients treated with tolinapant, with a median duration of response of 8.8 months (Michot et al., EHA 2022). During this trial, in which the patients were given tolinapant on Weeks 1 and 3 in 4-week cycles, clinical investigators have described multiple instances of tumor flare and pseudoprogression, consistent with the described immunomodulatory effects associated with tolinapant treatment. Here, we present one such case, a 58-year-old male, who was diagnosed with mycosis fungoides in 2010, which had previously transformed despite multiple therapies. This subject was selected for a more detailed study due to their clinical course with multiple blood samples and biopsies collected, permitting longitudinal histological and molecular characterization. At enrollment in March 2021, the subject presented with red, scaly plaques and cutaneous tumors scattered on the extremities and trunk. At one-month follow up, the subject had an excellent initial symptomatic response with the visual resolution of several tumors. Moreover, the itch reported as “moderate” at baseline had improved with tolinapant treatment. However, during continued therapy, the remaining plaques and tumors progressively swelled, appearing pink and indurated on dosing, indicative of flare. During off-dosing weeks, these signs of flare were reduced. The subject also reported increased itch during treatment that partly resolved during off-dosing weeks. Considering the disappearance of some tumors and the improved appearance during off-dosing weeks, this was pseudoprogression. Methods: Cell surface phenotype of the lymphoma was determined by flow cytometry (Standard Leukemia/Lymphoma and T-Cell Therapy Panel, NeoGenomics Laboratories) on blood samples collected at screening and during Cycles 1, 3 and 5. To characterize the clinical observations, skin biopsies including tumor tissues were collected at baseline and during Cycles 1, 2, 6, 7 and 10. We used multiplex immunofluorescence analysis (COMET™, Lunaphore Technologies SA), gene expression profiling (PanCancer IO 360™ and Pathways panels, NanoString Technologies, Inc.), genomic profiling (FoundationOne™ Heme, Foundation Medicine, Inc.) and cytokine/chemokine measurements (Human ExplorerMAP™ v. 1.0, xMAP® Technology, Luminex Corporation). Results: Blood samples collected at screening and post-treatment confirmed the presence of lymphoma cells that were CD3 +, CD4 +, CD7 -, CD8 - and CD26 -. Multiplex immunofluorescence analysis of the biopsies was able to detect lymphoma cells as CD4 +, CD7 - and CD8 -. Sparsely distributed CD8 + cells were observed at baseline (screening sample). In the biopsy taken on Cycle 2 Day 1, the level of CD8 + cells was markedly increased. Representative images are shown below (left: baseline; right: post-treatment). Analysis of genes associated with CD8 + T cells, cytotoxic cells, B cells and NK cells showed an increase in gene probe signals for CD8A, CD8B, GZMA, GZMH and PRF1. Post-treatment plasma samples showed an elevation in chemokines involved in leukocyte recruitment, such as, CXCL1, CXCL10 and CCL25. Similarly, consistent systemic changes in CXCL10, the chemokine directing recruitment of CXCR3 + cytotoxic T cells, were also detected in tolinapant-treated peripheral TCL patients in the same trial. Conclusions: Our data offer a cellular and molecular insight into a case of clinical pseudoprogression observed during tolinapant treatment. Understanding pseudoprogression is important for clinical investigators and patients to ensure treatment with tolinapant, or other IAP antagonists, are not discontinued prematurely.

Methylated Whole Genome Cell-Free DNA Sequencing before Chimeric Antigen Receptor T-Cell Therapy for Large B-Cell Lymphoma Predicts Treatment Outcomes

Introduction More than half of patients with relapsed or refractory large B-cell lymphoma (R/R LBCL) will experience progressive disease after CD19 chimeric antigen receptor T-cell therapy (CART). Circulating tumor DNA profiling has been used to prognosticate patients before CART, but mainly has focused on identification of somatic genomic aberrations. Epigenetic changes, specifically aberrant DNA methylation, can alter transcriptional regulation of genes important for treatment resistance. Altered methylation features can be detected from cell-free DNA (cfDNA) in patients with LBCL and are associated with outcomes after frontline immunochemotherapy. In the current study, we used whole genome bisulfite sequencing (WGBS) to analyze the methylation landscape in cfDNA from patient plasma before CART and identified novel methylation signatures associated with poor treatment outcomes. Methods Patients with R/R LBCL who received standard-of-care CART with plasma samples drawn before lymphodepletion (LD) or at cell infusion were identified. The primary endpoint was 3-month overall response after CART. cfDNA was extracted from 3-5 ml of plasma and cfDNA libraries were prepared using a Zymo-Seq cell free WGBS kit. Each library was sequenced to 300 - 600 million read pairs at a read length of 150 bp, enabling an average CpG coverage of 7-13X per detected CpG. Unique alignment rate among all libraries were about or above 82%. Reads were adapter trimmed with Trim Galore and aligned to reference genome hg19 using Bismark. Methylation calling was performed by MethylDackel. Differentially methylated cytosines (DMC, FDR ≤0.05) and regions (DMR) from WGBS data with an absolute methylation difference of ≥ 0.1 between 3-month responders and non-responders were identified using DSS. Functional enrichment analysis of genes overlapped by DMRs was performed by gprofiler2. Results A total of 22 patients with plasma collected before LD (n=21) or at cell infusion (n=1) who received CART were identified. Median follow-up was 21.5 months (95% CI 12.5-NR). Three-month response was evaluable in 21 patients (1 pending evaluation). Overall response rate (ORR) was 77% (8 CR, 9 PR) and 55% (9 CR, 2 PR) at one- and 3-months post-CART, respectively. Median PFS was 6.5 months (95% CI 3.0-NR), and median OS was 57.7 months (95% CI 7.7-NR). Patient and disease characteristics are described in Table 1. Differential methylation analysis identified 22,815 DMCs, of which 1,043 were hypomethylated and 21,772 were hypermethylated in non-responders. The global average methylation values across 25 million cytosines were 0.807 and 0.786 for non-responders and responders, respectively (p=0.265). A total of 582 DMRs were identified, of which 34 were hypomethylated and 548 were hypermethylated in non-responders (Figure 1). Of these, 281 (48%) overlapped intergenic regions, 265 (46%) gene bodies, and 33 (6%) promoter regions. DMRs overlapping DNA repair gene PARP1 (areaStat 88.2), B-cell receptor signaling genes SYK (27.2), FYN (21.4), and PTPN6 (20.9), polycomb repressor complex genes JARID2 (25.2) and BCOR (21.4), tumor suppressor WT1 (47.1), immune mediators NRP1 (80.0), TRAF3 (56.7), PAK3 (28.4), IRAK2 (28.7), and TNFS14 (23.6), and non-coding RNAs MIR4520 (95.8) and VTRNA-2 (23.1) were hypermethylated in non-responders and IL17RA (-33.2) hypomethylated. Functional enrichment analysis on overlapped genes also identified common molecular functions involving guanyl nucleotide exchange factors (p=0.0007), GTPase regulation (p=0.001), and cGMP binding (p=0.007). Discussion This proof-of-concept study is the first to perform WGBS on pretreatment plasma from patients with R/R LBCL to associate methylation signatures with post-CART outcomes. We identified that non-responders were enriched in hypermethylated DMRs compared to responders, overlapping with genes important to lymphomagenesis, immune function, and GTP energy metabolism. Further work will expand this cohort, compare with matched-tissue to find tumor-specific markers, and identify candidate DMR targets for targeted bisulfite sequencing from pre- and post-treatment plasma samples to assess response. By identifying high-risk methylation signatures in the plasma of CART recipients, we may be able to identify patients to be prioritized for novel treatment strategies such as combination with hypomethylating agents to improve outcomes.

406 Tapinarof cream 1% once daily for the treatment of extensive atopic dermatitis in adolescents and children: outcomes from the 4-week maximal usage trial

Abstract Tapinarof is a first-in-class, nonsteroidal, topical, aryl hydrocarbon receptor agonist approved by the Food and Drug Administration for the treatment of plaque psoriasis in adults, and under investigation for the treatment of atopic dermatitis (AD) in adults and children. Tapinarof cream 1% once daily (QD) demonstrated significant efficacy vs. vehicle and was well tolerated in adults and adolescents with moderate to severe AD in a previously reported phase 2b trial. The pharmacokinetic (PK) profile of tapinarof across psoriasis and AD trials in adults is characterized by no-to-minimal systemic absorption and decreasing plasma concentrations over the course of treatment. Here, we report PK, tolerability and safety of tapinarof cream 1% QD in adolescents and children down to age 2 years with extensive AD. Adolescents and children with a Validated Investigator Global Assessment scale for Atopic Dermatitis™ (vIGA-AD™) score ≥3 (moderate or worse) and body surface area (BSA) involvement ≥25% (ages 12–17 years) or ≥35% (ages 2–11 years) were enrolled into three age cohorts (2–6, 7–11 and 12–17 years) and were treated with tapinarof cream 1% QD for 4 weeks. Primary endpoints included PK parameters, investigator-assessed Local Tolerability Scale (LTS) scores by visit (overall and for sensitive areas) and treatment-emergent adverse events (TEAEs). Overall, 36 patients (12 per cohort) were enrolled. Patients’ mean age was 8.9 years and 66.7% were male. At baseline, 66.7%, 75% and 91.7% of patients had a vIGA-AD score of 3 for cohorts 2–6, 7–11 and 12–17 years, respectively. Mean BSA (range) affected was 52.4% (36.0–90.0%), 42.0% (35.0–72.0%) and 33.9% (26.0–54.5%) for the three age cohorts, respectively. Mean Eczema Area and Severity Index scores were 30.2, 21.0 and 20.3, respectively. No-to-minimal tapinarof systemic exposure was observed, consistent with previous trials in adults. Mean tapinarof maximum plasma concentration (Cmax) at Day 1 was 2.4 ng/mL, and the median time to Cmax was approximately 3 h, for all patients. Approximately 25% of post-treatment plasma samples were below the quantifiable limit of the highly sensitive assay [<50 pg (10–12)/mL]. There was no correlation between tapinarof exposure (Cmax on Day 1) and baseline %BSA affected. Mean overall investigator-assessed LTS score was 0.1 (no irritation) at Weeks 1 and 4. Investigators assessed that the majority of patients had no irritation (LTS score of 0), including sensitive and intertriginous skin. The TEAEs were reported by eight patients (22%) and were all mild or moderate; one patient discontinued due to two unrelated TEAEs. One case of mild folliculitis and no contact dermatitis occurred. Most patients (87.5%) chose to enroll in the 48-week long-term extension trial. This is the first trial evaluating tapinarof in children down to 2 years of age. Tapinarof cream 1% QD demonstrated no-to-minimal systemic exposure in children with extensive AD, even when measured with a highly sensitive assay. There was a low incidence of TEAEs in patients with up to 90% BSA affected. Tapinarof cream was well tolerated, including sensitive and intertriginous skin areas.

Abstract 3375: Ultrasensitive ctDNA minimal residual disease monitoring in early NSCLC with PhasED-Seq

Abstract Background: Circulating tumor DNA (ctDNA) minimal residual disease (MRD) detection is a promising approach for personalization of adjuvant therapy in non-small cell lung cancer (NSCLC). First generation ctDNA MRD assays that employ tumor-informed approaches to track single nucleotide variants (SNVs) have limits of detection (LOD95) of ~1E-4 and have high positive predictive values for recurrence. However, they have suboptimal clinical sensitivity, with false negative results at the completion of therapy in most patients who will ultimately recur. PhasED-Seq is a novel ctDNA MRD method that tracks multiple “phased” variants (PVs) within individual DNA fragments with a LOD95 ~100-fold better than first generation assays. Here we report PhasED-Seq ctDNA MRD results for the first prospective cohort of early stage NSCLC patients. Methods: Tumor tissues (n=46), PBMCs (n=46) and plasma samples (n=169) from 46 Stage I-III NSCLC patients treated with curative intent were prospectively collected at Memorial Sloan Kettering Cancer Center. All patients underwent resection and received neoadjuvant +/- adjuvant therapy (n=14), adjuvant therapy only (n=17), or neither (n=15). Samples were analyzed in Foresight Diagnostics' CLIA laboratory (Aurora, CO) using personalized PhasED-Seq. Briefly, PVs were identified via whole genome sequencing of tumors and matched blood germline DNA. Custom capture panels targeting PVs were synthesized and used to assess MRD status in pre-, on- and post-treatment plasma samples. Detection of ctDNA MRD was assessed at a post-treatment landmark, defined as the first post-therapy sample or when not available the last post-surgical sample taken during therapy. To enable comparisons, the same plasma samples were analyzed using an SNV-based ctDNA MRD approach. Results: PVs were identified in tumor tissue from all 46 patients. Across all plasma samples PhasED-Seq achieved a median LOD95 of 1.3E-6 and as low as 2.5E-7. Of 74 plasma samples with detectable ctDNA, 38 (51%) contained concentrations below 1E-4 and the lowest level of ctDNA MRD detected was 1.7E-7. For post-treatment landmark samples (n=45), the median time from end of therapy was 2 months. Cancer recurred in all patients (n=10) with detectable MRD at the landmark. Furthermore, PhasED-Seq better stratified freedom from recurrence (log-rank p=3E-8, Cox HR=10.8) than the SNV-based approach (log-rank p=0.08, Cox HR=2.5) and detected MRD at the landmark in more patients who ultimately recurred (56% vs 28%). PhasED-Seq also achieved longer lead times, including detecting MRD in 66% of samples collected 12 to 24 months prior to recurrence versus only 33% using SNV-based monitoring. Conclusion: PhasED-Seq achieves ctDNA detection below 1 part per million and appears to be significantly more sensitive than SNV-based MRD monitoring. These results suggest that PhasED-Seq is a promising approach for use in risk adapted trials in early stage NSCLC. Citation Format: James M. Isbell, Bob T. Li, Pedram Razavi, Jorge Reis-Filo, Si-Yang Liu, Pier Selenica, Prasad Adusumilli, Matthew Bott, David R. Jones, Valerie W. Rusch, Smita Sihag, Darren J. Buonocore, Justin Jee, Emily Lebow, Daniel Gomez, Andreas Rimner, Fernando C. Santini, Charles M. Rudin, Jordan E. Eichholz, Andres Martinez, Daphne Alerte, Gregory J. Hogan, Andre Schultz, Ronald P. Schuyler, Alanna Roff, Dustin Hite, Jacob J. Chabon, David M. Kurtz, Ash A. Alizadeh, Maximilian Diehn. Ultrasensitive ctDNA minimal residual disease monitoring in early NSCLC with PhasED-Seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3375.

Abstract 5864: An angiogenic target of immune exclusion with checkpoint blockade in melanoma

Abstract With the development of immune checkpoint blockade to treat a variety of malignancies, better predictive biomarkers as well as new targets to further improve anti-tumor immunity remains a need. Our understanding of the role of angiogenesis in immune regulation continues to improve, and combinations of anti-angiogenesis and immune checkpoint blockade demonstrate improved clinical efficacy, as well as mechanisms for improved immune cell trafficking. Tumor biopsies from a phase I study with 46 advanced melanoma patients treated with Ipilimumab and Bevacizumab combination (Ipi-Bev) therapy, revealed activated endothelium accompanied by lymphocyte infiltration. To discover possible targets involved in the observed clinical benefit, a serological screen was performed using human protein arrays with post-treatment plasma samples of long-term patients with clinical benefit to Ipi-Bev therapy (Wu et. al., 2017; Hodi et. al., 2014). EDIL3 (EGF Like Repeats and Discoidin Domains 3), a pro-angiogenic extracellular matrix protein, was identified. Humoral responses against EDIL3 were associated with favorable clinical outcomes including improved response rates (p=0.003) and overall survival (p=0.03) to Ipi-Bev therapy. The anti-EDIL3 response was also observed to be significantly (p=0.03) related to Ipi-Bev treatment when compared with patients treated with Ipilimumab alone, anti-PD1 therapy alone, or the combination of Ipilimumab and Nivolumab in a combined analysis. Transcriptome analyses of TCGA SKCM (n=469) and BMS Checkmate 064 trial (n=90) related upregulated EDIL3 expression with enrichment of EMT, TGFβ signaling in fibroblasts, and angiogenesis signatures. Interestingly, TIDE analysis demonstrated a role for EDIL3 in T cell exclusion and immunosuppressive cancer-associated fibroblasts (CAFs) as its source in the tumor microenvironment. EDIL3 was also predicted as a biomarker of non-response to immune checkpoint blockade treatment. In vitro studies confirmed the upregulation of EDIL3 expression and secretion by patient-derived CAFs along with its role in modulating TGFβ1 mediated EMT. Mechanistically, EDIL3 was found to interfere with leukocyte-endothelial interactions by disturbing LFA-1/ICAM1 mediated adhesion and inhibited T cell transmigration in 2-D and microfluidic 3-D coculture studies with patient-derived tumor endothelial cells. Our findings suggest that the humoral response against EDIL3 may serve as one of the underlying mechanisms involved in effectuating an excluded to an inflamed phenotype observed in responders of checkpoint blockade treatment. This study develops EDIL3 as a potential target to enhance cytotoxic immune responses and improve the outcomes of combination immune therapy. Studies are ongoing for targeted inhibition of EDIL3 with implications for therapeutic application to promote anti-tumor immunity. Citation Format: Saba Tabasum, Dinesh Thapa, Anita Giobbie-Hurder, Jason L. Weirather, Xinqi Wu, Marco Campisi, Xiaoyun Li, Jingjing Li, Michael P. Manos, David A. Barbie, F. Stephen Hodi. An angiogenic target of immune exclusion with checkpoint blockade in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5864.

Abstract PD5-03: PD5-03 Comparison of a personalized sequencing assay and digital PCR for circulating tumor DNA based Molecular Residual Disease detection in early-stage triple negative breast cancer in the cTRAK-TN trial

Abstract Background: Detection of circulating tumour DNA (ctDNA) in patients (pts) who have completed treatment for early-stage breast cancer is associated with a high risk of future relapse. Identifying those at high risk of subsequent relapse may allow tailoring of further therapy to delay or prevent recurrence. Previous analysis of this cohort showed that tools capable of detecting ctDNA at lower concentrations are needed to increase sensitivity and lengthen the lead time between ctDNA detection and relapse. We compared ctDNA detection via a personalised sequencing assay to dPCR in patients from the cTRAK TN clinical trial. Methods: The cTRAK-TN trial recruited 161 pts into prospective ctDNA surveillance with dPCR, with ctDNA positive pts randomised to 1) CT staging plus pembrolizumab therapy for patients without relapse or 2) observation. Pts had serial post-treatment surveillance plasma samples collected every 3 months for up to 2 years. Whole exome sequencing (WES) was performed on tumor DNA from FFPE samples to design personalised Residual Disease and Recurrence (RaDaR®) multiplex PCR based NGS assays. Retrospectively, plasma DNA extracted from a minimum of 2mls banked plasma, was sequenced with personalised RaDaR assays, and ctDNA detection identified with a proprietary algorithm. dPCR assays tracked 1-2 mutations, as previously described. Primary endpoint was rate of positive ctDNA detection by 12 months from start of surveillance in both assays. Secondary endpoints were agreement in ctDNA detection between RaDaR and dPCR assays and lead-time between ctDNA detection and disease recurrence. Results: Overall, 147 pts and 241 tissue samples were subject to WES, and RaDaR assays were developed for 142 pts with sufficient plasma for testing. RaDaR assays tracked a median of 47 variants (range 33-56) per patient, and a total of 907 timepoints were analysed (median 6 timepoints per pt, range 1-11). With RaDaR, 39.4% (56/142) patients tested ctDNA positive during follow-up, with a median ctDNA detected level of 0.081% estimated variant allele fraction (eVAF). With dPCR, 35.2% (50/142) pts tested ctDNA positive. The ctDNA detection rate by 12 months from the start of ctDNA surveillance was 36.2% (95% CI; 27.6% – 43.7%) with RaDaR and 29.9% (95%CI; 21.6% – 37.3%) with dPCR. The overall test agreement between RaDaR and dPCR assays was 92.7% (95%CI; 90.7% – 94.4%). From a patient perspective, 58.7% pts were ctDNA negative for both assays, 32.9% ctDNA were positive for both assays and 8.6% presented discrepancies. ctDNA was detected by RaDaR but not by dPCR in 9 pts and it was detected by dPCR but not by RaDaR in 3 pts. Among ctDNA positive pts, 55.2% were first detected positive by RaDaR, 5.2% by dPCR, and 39.6% were detected at the same time-point (test of proportions, p< 0.001). The median lead time from ctDNA detection to relapse was 7.1 months (95% CI 5.9 – 15.9%) with RaDaR and 5.7 months (95% CI 3.2% – 7.4%) with dPCR. Conclusion: The RaDaR personalised multi-mutation sequencing assay detected MRD with a longer median lead time prior to relapse, and with higher sensitivity, than dPCR mutation tracking assays. These findings have implications for the choice of ctDNA assay in clinical trials designed to treat patients at the point of MRD detection. Citation Format: Maria Coakley, Prithika Sritharan, Guillermo Villacampa, Claire Swift, Kathryn Dunne, Lucy Kilburn, Katie Goddard, Patricia Rojas, Andy Joad, Warren Emmett, Charlene Knape, Karen Howarth, Peter S. Hall, Catherine Harper-Wynne, Tamas Hickish, Iain Macpherson, Alicia F. Okines, Andrew M. Wardley, Duncan Wheatley, Simon Waters, Rosalind Cutts, Isaac Garcia-Murillas, Judith Bliss, Nicholas Turner. PD5-03 Comparison of a personalized sequencing assay and digital PCR for circulating tumor DNA based Molecular Residual Disease detection in early-stage triple negative breast cancer in the cTRAK-TN trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-03.

CtDNA as a biomarker of progression in oesophageal adenocarcinoma

BackgroundThe incidence of oesophageal adenocarcinoma (OAC) is rapidly increasing and despite improvements in treatment, the 5-year survival rate remains poor. Prognostic biomarkers that address the genomic heterogeneity in this highly complex disease will aid the development of precision therapeutics and improve patient survival. The aim of this study was to determine whether circulating tumour DNA (ctDNA) has prognostic significance as a biomarker in OAC patients.Patients and methodsWe profiled 209 blood and tumour samples from 57 OAC patients. Using a panel of 77 cancer genes, we sequenced ctDNA in plasma samples (n = 127) which were taken at multiple time points before and after therapy. In parallel, we sequenced matched tumour samples from 39 patients using the same gene panel. To assess whether the ctDNA profile reflected the tumour heterogeneity, we sequenced additional multi-region primary tumour samples in 17 patients. In addition, we analysed whole-genome and whole-exome sequencing data from primary tumours for a subset of 18 patients.ResultsUsing a tumour-agnostic approach, we found that detectable ctDNA variants in post-treatment plasma samples were associated with worse disease-specific survival. To evaluate whether the ctDNA originated from the primary tumour, we carried out a tumour-informed analysis which confirmed post-treatment ctDNA variants were associated with worse survival. To determine whether ctDNA could be used as a clinical follow-up test, we assessed blood samples from multiple time points before and after treatment, in a subset of patients. Results showed that the variant allele frequency of ctDNA variants increased with disease recurrence.ConclusionThis study demonstrates that ctDNA variants can be detected in patients with OAC and this has potential clinical utility as a prognostic biomarker for survival.

Association between post-treatment circulating biomarkers of inflammation and survival among stage II-III colorectal cancer patients.

Biomarker studies on colorectal cancer (CRC) prognosis are limited to pre-diagnostic or pre-operative measures. Post-treatment biomarkers are not well understood for their associations with CRC survival. We included 306 eligible incident stage II-III CRC cases from the population-based Seattle Colon Cancer Family Registry. Concentrations of C-reactive protein (CRP), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), adiponectin, and leptin were measured using post-treatment plasma samples. Adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) for all-cause and CRC-specific mortality were calculated using Cox proportional hazard models. Elevated levels of CRP, IL-6, MCP-1, and adiponectin were significantly associated with a higher risk of all-cause mortality within 10 years post blood draw with HRs (95% CI) of 1.32 (1.10-2.59), 2.72 (2.07-3.56), 1.97 (1.18-3.28) and 1.71 (1.14-2.58), respectively. IL-6 and adiponectin had a dose-response effect (Ptrend < 0.0001). For CRC-specific mortality, we observed positive associations for CRP (HR = 1.75, 95% CI: 1.2-2.56), IL-6 (HR = 5.02, 95% CI: 2.92-8.59), MCP-1 (HR = 3.78, 95% CI: 1.41-10.08), and adiponectin (HR = 3.16, 95% CI: 1.27-7.86), and inverse association for leptin (HR = 0.44, 95% CI: 0.29-0.68) within the first year of blood draw, whereas the association for IL-6 remained statistically significant over 10 years. Our results support the role of chronic inflammation in CRC progression and suggested several post-treatment inflammatory biomarkers, particularly IL-6, are promising prognostic markers for stage II-III CRC patients.

Association of Circulating Tumor DNA With Disease-Free Survival in Breast Cancer

Fragmented DNA is continuously released into the circulation following apoptosis and necrosis of both cancerous and noncancerous cells; when it is released by cancer cells, it is specifically known as circulating tumor DNA (ctDNA). Previous studies have suggested that ctDNA can reflect tumor burden and guide potential therapeutic targets. To determine the association of ctDNA with breast cancer disease-free survival (DFS) and progression-free survival in early, locally advanced, and metastatic breast cancer. An electronic search was conducted using the Cochrane Library, ScienceDirect, PubMed, and Embase from July 30, 2019, to October 31, 2019; all languages were included. The following search terms were used: ctDNA OR circulating tumor DNA OR liquid biopsy AND breast cancer OR breast carcinoma OR breast tumor AND prognosis OR survival. All titles were screened, and the appropriate abstracts were reviewed. If any data were missing, the authors contacted the study authors for permission to access data and extrapolate hazard ratios (HRs). To be included in the analysis, the studies had to meet the following prespecified inclusion criteria: (1) a ctDNA blood sample was measured; (2) DFS, progression-free survival, or relapse-free survival was reported as an HR; and (3) the patient population only had breast cancer. Retrospective and prospective observational cohort studies were included. Two authors (C.C. and C.F.) independently reviewed the literature. All data were recorded independently by both authors and were compared at the end of the reviewing process to limit selection bias. Duplicates were removed and any disparities were clarified. Data were pooled using a fixed-effects or random-effects model according to the study heterogeneity. This study adhered to the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) and Meta-Analysis of Observational Studies in Epidemiology (MOOSE). The primary outcome was the association of ctDNA with DFS or relapse-free survival in breast cancer. Secondary outcomes focused on subgroup analysis in the setting of early breast cancer and metastatic breast cancer. From a total of 263 publications found using the predefined search terms, data from 8 studies (3.0%) reporting on 739 patients in total were suitable for inclusion. Circulating tumor DNA gene variation detection (both before and after treatment) was statistically significantly associated with shorter DFS (HR, 4.44; 95% CI, 2.29-8.61; P < .001). Detection of ctDNA was statistically significantly associated with a reduction in DFS in both the early breast cancer subgroup (HR, 8.32; 95% CI, 3.01-22.99; P < .001) and the metastatic or locally advanced subgroup (HR, 1.91; 95% CI, 1.35-2.71; P < .001). Pretreatment and posttreatment plasma sample collection was analyzed in both early and metastatic groups. The posttreatment group encompassed both surgical and oncologic therapy. Pretreatment plasma detection of ctDNA was statistically significantly associated with reduced DFS (HR, 3.30; 95% CI, 1.98-5.52; P < .001). Posttreatment sampling of ctDNA failed to achieve statistical significance (HR, 8.17; 95% CI, 1.01-65.89; P = .05). In this systematic review and meta-analysis, elevated plasma ctDNA was associated with a high risk of relapse. This finding suggests that plasma ctDNA may provide an excellent method to stratify risk and personalize patient follow-up.

A Pilot Study Comparing the Efficacy of Lactate Dehydrogenase Levels Versus Circulating Cell-Free microRNAs in Monitoring Responses to Checkpoint Inhibitor Immunotherapy in Metastatic Melanoma Patients.

Simple SummaryImprovement in melanoma patients with metastatic disease is needed to better assess immunotherapies. Lactate dehydrogenase (LDH) is currently an accepted biomarker for stage IV, but it has limited utility for stage III melanoma patients. Thus, finding biomarkers for metastatic melanoma is important not only to identify progressive melanoma tumors, but also to monitor patients under checkpoint inhibitor immunotherapy (CII). The aim of this pilot study was to demonstrate the utility of circulating cell-free microRNAs (cfmiRs) as potential blood biomarkers for stage III and IV melanoma patients compared to LDH. To accomplish this aim, we profiled for cfmiR the plasma of metastatic melanoma patients before and during CII treatment, and compared them to normal healthy donors’ samples. The cfmiR profiling was performed using an NGS-based miRNA assay, which requires no extraction and a small volume input. We found specific cfmiR signatures in stage III and IV metastatic melanoma patients. As a proof of concept, our results showed that certain cfmiRs are associated with CII outcomes.Serum lactate dehydrogenase (LDH) is a standard prognostic biomarker for stage IV melanoma patients. Often, LDH levels do not provide real-time information about the metastatic melanoma patients’ disease status and treatment response. Therefore, there is a need to find reliable blood biomarkers for improved monitoring of metastatic melanoma patients who are undergoing checkpoint inhibitor immunotherapy (CII). The objective in this prospective pilot study was to discover circulating cell-free microRNA (cfmiR) signatures in the plasma that could assess melanoma patients’ responses during CII. The cfmiRs were evaluated by the next-generation sequencing (NGS) HTG EdgeSeq microRNA (miR) Whole Transcriptome Assay (WTA; 2083 miRs) in 158 plasma samples obtained before and during the course of CII from 47 AJCC stage III/IV melanoma patients’ and 73 normal donors’ plasma samples. Initially, cfmiR profiles for pre- and post-treatment plasma samples of stage IV non-responder melanoma patients were compared to normal donors’ plasma samples. Using machine learning, we identified a 9 cfmiR signature that was associated with stage IV melanoma patients being non-responsive to CII. These cfmiRs were compared in pre- and post-treatment plasma samples from stage IV melanoma patients that showed good responses. Circulating miR-4649-3p, miR-615-3p, and miR-1234-3p demonstrated potential prognostic utility in assessing CII responses. Compared to LDH levels during CII, circulating miR-615-3p levels were consistently more efficient in detecting melanoma patients undergoing CII who developed progressive disease. By combining stage III/IV patients, 92 and 17 differentially expressed cfmiRs were identified in pre-treatment plasma samples from responder and non-responder patients, respectively. In conclusion, this pilot study demonstrated cfmiRs that identified treatment responses and could allow for real-time monitoring of patients receiving CII.

Abstract 728: Early assessment of treatment effect in small cell lung cancer (SCLC) via ctDNA analysis

Abstract INTRODUCTION: Quantifying circulating tumor DNA (ctDNA) is an emerging method to non-invasively assess treatment effect for solid tumors. A ctDNA-based method for disease monitoring for small cell lung cancer (SCLC) would be beneficial to disease management. By employing plasma taken during early treatment cycles and NGS, we tested whether early response assessed by ctDNA level could predict treatment effect. METHODS: Using a 197-gene NGS assay, the AVENIO ctDNA Surveillance Kit (for Research Use Only, not for use in diagnostic procedures), ctDNA levels were assessed in post-treatment plasma samples based on variants identified at baseline. Mutant allele concentration or mutant molecules per milliliter-of-plasma (MMPM) was utilized to quantify ctDNA over all variants of all sequenced genomic regions. Samples collected from an observational German Lung Cancer Multi-Marker Study were analyzed retrospectively. In a cohort of 69 stage IV SCLC subjects, the association of survival with ctDNA-based monitoring classifier was evaluated in the first available post-treatment plasma sample after starting chemotherapy (median number of days after start of treatment = 25) by Kaplan-Meier methods and Cox proportional hazards model. RESULTS: The analysis demonstrated that subjects with mean MMPM less than the lower quartile (14.3) had longer progression free survival (PFS) compared to that of subjects with mean MMPM greater than or equal to the lower quartile (log-rank P = 0.0095; HR [95% CI] = 0.48 [0.27, 0.84]). The same classifier was applied for overall survival analysis, and subjects with mean MMPM less than the lower quartile had longer overall survival (OS) compared to that of subjects with mean MMPM greater than or equal to the lower quartile (log-rank P = 0.0095; HR [95% CI] = 0.46 [0.26, 0.84]). CONCLUSIONS: Changes in ctDNA levels in response to treatment may prove to be a valuable way of identifying subjects that either respond or fail to respond to therapy earlier than current standard of care methods such as computed tomography (CT) scan. Hypotheses will be validated in a separate cohort. Future prospective studies to confirm these results are needed. Citation Format: John Jiang, Christine Ju, Corinna Woestmann, Aarthi Balasubramanyam, Liu Xi, Stephanie J. Yaung, Sandeep Gattam, Bernd Hinzmann, Michael Thomas, Felix Lasitschka, Michael Meister, Marc Schneider, Felix Herth, Thomas Muley, Birgit Wehnl, John Palma. Early assessment of treatment effect in small cell lung cancer (SCLC) via ctDNA analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 728.

Drug Resistance Mutations and Associated Phenotypes Detected in Clinical Trials of Maribavir for Treatment of Cytomegalovirus Infection.

BackgroundIn separate phase 2 trials, 120 patients received maribavir for cytomegalovirus (CMV) infection failing conventional therapy (trial 202) and 119 received maribavir for asymptomatic infection (trial 203). Overall, 172 cleared their CMV infection (CMV DNA <200 copies/mL) within 6 weeks.MethodsBaseline and posttreatment plasma samples were tested for mutations in viral genes UL97, UL54, and/or UL27. Selected viral mutants were phenotyped for drug susceptibility.ResultsBaseline samples revealed UL54 mutations newly phenotyped as conferring resistance to standard DNA polymerase inhibitor(s), including K493N, P497S, K513T, L565V, V823A, A987V, and E989D. Of 29 patients (including 25 from trial 202) who cleared but later experienced recurrent CMV infection while on maribavir, 23 had available UL97 genotyping data; 17 had known resistance mutations (T409M or H411Y) and 5 additional had UL97 C480F alone. The newly phenotyped mutation C480F conferred high-grade maribavir resistance and low-grade ganciclovir resistance. Among 25 who did not respond to >14 days of therapy, 9 showed T409M or H411Y and 4 others showed C480F alone.ConclusionsAfter maribavir therapy (400–1200 mg twice daily), UL97 mutations T409M, H411Y, or C480F emerge to confer maribavir resistance in patients with recurrent CMV infection while on therapy or no response to therapy.Clinical Trials RegistrationNCT01611974 and EudraCT 2010-024247-32.

Integrated Assessment of Circulating Cell-Free MicroRNA Signatures in Plasma of Patients with Melanoma Brain Metastasis.

Primary cutaneous melanoma frequently metastasizes to distant organs including the brain. Identification of cell-free microRNAs (cfmiRs) found in the blood can be used as potential body fluid biomarkers for detecting and monitoring patients with melanoma brain metastasis (MBM). In this pilot study, we initially aimed to identify cfmiRs in the blood of MBM patients. Normal donors plasma (healthy, n = 48) and pre-operative MBM patients’ plasma samples (n = 36) were compared for differences in >2000 microRNAs (miRs) using a next generation sequencing (NGS) probe-based assay. A 74 cfmiR signature was identified in an initial cohort of MBM plasma samples and then verified in a second cohort of MBM plasma samples (n = 24). Of these, only 58 cfmiRs were also detected in MBM tissues (n = 24). CfmiR signatures were also found in patients who have lung and breast cancer brain metastasis (n = 13) and glioblastomas (n = 36) compared to MBM plasma samples. The 74 cfmiR signature and the latter cfmiR signatures were then compared. We found a 6 cfmiR signature that was commonly upregulated in MBM plasma samples in all of the comparisons, and a 29 cfmiR signature that distinguishes MBM patients from normal donors’ samples. In addition, we assessed for cfmiRs in plasma (n = 20) and urine (n = 14) samples collected from metastatic melanoma patients receiving checkpoint inhibitor immunotherapy (CII). Pre- and post-treatment samples showed consistent changes in cfmiRs. Analysis of pre- and post-treatment plasma samples showed 8 differentially expressed (DE) cfmiRs that overlapped with the 35 cfmiR signature found in MBM patients. In paired pre-treatment plasma and urine samples receiving CII 8 cfmiRs overlapped. This study identified specific cfmiRs in MBM plasma samples that may potentially allow for assessment of melanoma patients developing MBM. The cfmiR signatures identified in both blood and urine may have potential utility to assess CII responses after further validation.

LBA75 - 18-months relapse-free survival (RFS) and biomarker analyses of OpACIN-neo: A study to identify the optimal dosing schedule of neoadjuvant (neoadj) ipilimumab (IPI) + nivolumab (NIVO) in stage III melanoma

BackgroundPrimary analysis of the OpACIN-neo study testing 3 dosing schedules of neoadj IPI+NIVO identified 2 cycles IPI 1mg/kg + NIVO 3mg/kg (IPI1+NIVO3; arm B) as most favourable, with 20% grade 3-4 irAEs and a pathologic response rate (pRR) of 77%. After a median FU of 8.3mo none of the pts with a pathologic response versus 9/21 (43%) of the non-responders had relapsed. Here we present updated RFS and biomarker analyses. MethodsOpACIN-neo is a multicentre, randomized phase II trial in resectable stage III melanoma pts with ≥1 measurable lymph node metastasis (RECIST 1.1). 86 pts were randomized to arm A: 2x IPI3+NIVO1 Q3W (n=30); B: 2x IPI1+NIVO3 Q3W (n=30); or C: 2x IPI3 Q3W followed by 2x NIVO3 Q2W (n=26). Lymph node dissection was planned at wk 6. Primary endpoints were toxicity, radiologic RR and pRR; RFS and biomarker analyses were secondary endpoints. Mutational profiles, gene expression signatures (GES) and immune protein expression were examined in baseline biopsies by whole exome seq, RNA seq and digital spatial profiling (DSP) analysis. Pre- and post-treatment plasma samples were profiled for 92 proteins. ResultsAfter a median FU of 17.7mo, the median RFS was not reached in any of the arms. Estimated 18-mo RFS was 85% for all pts (95% CI 78%-93%), 90% for arm A (95% CI, 80%-100%), 82% for arm B (95% CI, 70%-98%) and 83% for arm C (95% CI, 70%-100%). Relapses were observed in 1/64 (2%) pathological responders versus 13/21 (62%) of the non-responders. High tumour mutational burden (TMB) and high interferon-y (IFN-y) signature were associated with pathologic response and favourable RFS. Cytokine and PD-1 levels in plasma increased post-treatment irrespective of response. Additional GES and DSP analysis will be presented. ConclusionsThe 18-mo FU confirms that durable RFS can be achieved with 2 cycles of neoadj IPI+NIVO without any additional adjuvant therapy. Pathologic response remains the strongest marker for RFS. TMB and IFN-y signature might serve as baseline markers identifying pts benefiting from neoadj IPI+NIVO. Neoadj 2 cycles IPI1+NIVO3 should be tested in a randomized phase III study versus adjuvant therapy. Clinical trial identificationNCT02977052. Legal entity responsible for the studyNetherlands Cancer Institute. FundingBMS. DisclosureE.A. Rozeman: Travel / Accommodation / Expenses: NanoString; Travel / Accommodation / Expenses: MSD. A.M. Menzies: Advisory / Consultancy: BMS; Advisory / Consultancy: MSD; Advisory / Consultancy: Roche; Advisory / Consultancy: Novartis; Advisory / Consultancy: Pierre Fabre. O. Krijgsman: Research grant / Funding (institution): BMS. T.M. Van: Travel / Accommodation / Expenses: NanoString. A.C.J. van Akkooi: Advisory / Consultancy, Research grant / Funding (institution): Amgen; Advisory / Consultancy, Research grant / Funding (institution): BMS; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy: MSD-Merck; Advisory / Consultancy: Merck-Pfizer; Advisory / Consultancy: 4SC. R.A. Scolyer: Advisory / Consultancy: MSD; Advisory / Consultancy: Neracare; Advisory / Consultancy: Novartis. J. Hansson: Advisory / Consultancy: BMS; Advisory / Consultancy: MSD; Advisory / Consultancy: Novartis. G.V. Long: Advisory / Consultancy: Aduro; Advisory / Consultancy: Amgen; Advisory / Consultancy: BMS; Advisory / Consultancy: Mass Array; Advisory / Consultancy: MSD; Advisory / Consultancy: Novartis; Advisory / Consultancy: Oncosec; Advisory / Consultancy: Pierre-Fabre; Advisory / Consultancy: Roche. C.U. Blank: Advisory / Consultancy, Research grant / Funding (institution): BMS; Advisory / Consultancy: MSD; Advisory / Consultancy: Roche; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy: Lilly; Advisory / Consultancy: Pfizer; Advisory / Consultancy: GSK; Advisory / Consultancy: GenMap; Advisory / Consultancy: Pierre Fabre; Research grant / Funding (institution): NanoString. All other authors have declared no conflicts of interest.

P2.03-25 Assessing the Impact of Clonal Hematopoiesis in Disease Monitoring Using Targeted Cell-Free DNA (cfDNA) Sequencing Technology

Somatic variants found in plasma cell-free DNA (cfDNA) may derive from either solid tumors or clonal hematopoiesis (CH). Little is known about how this may impact plasma-based longitudinal disease monitoring using targeted sequencing of circulating tumor DNA (ctDNA). To assess the potential impact of CH in disease monitoring, we evaluated monitoring algorithms by targeted sequencing with and without matched peripheral blood mononuclear cells (PBMC). Samples were collected from a prospective observational study, where 62 late stage lung adenocarcinoma subjects were treated with first-line chemo or chemoradiation therapy. Pre-treatment plasma cfDNA and matched PBMC were analyzed with the AVENIO ctDNA Surveillance Kit (For Research Use Only, not for use in diagnostic procedures), a sequencing panel of 198 kilobases targeting cancer genes. Median input amounts of 25 ng cfDNA and 50 ng PBMC DNA were sequenced to median deduplicated depths of 4582 and 6134, respectively. A median of 120 single nucleotide variants were detected per cfDNA sample, with 93.1% of these identified in matched PBMC. Most PBMC-matched cfDNA variants were germline SNPs, with allele frequency (AF) at approximately 50% or 100%. A median of 1 (range 0-5) PBMC-matched cfDNA variants per sample were detected with an AF <10%, consistent with CH. The number of these variants was positively associated with age (p-value = 0.0039) and the most frequently mutated gene was TP53. The remaining somatic variants (i.e., in cfDNA and not PBMC) had an AF range 0.03-40.9%. These PBMC-informed variants (median of 7 per sample) were used in longitudinal monitoring in the first post-treatment plasma sample to assess early response to therapy. Association between ctDNA level and progression-free survival using the same monitoring algorithm yielded nearly identical results on somatic variants derived from filtering approaches independent of matched PBMC (HR 0.32; 95% CI 0.16 - 0.65; log-rank P = 0.0009) and the PBMC-informed method (HR 0.31; 95% CI 0.14 - 0.66; log-rank P = 0.0013). A targeted panel focused on solid tumors by design has limited impact from CH. For disease monitoring applications in a non-MRD setting, measuring multiple variants instead of a single variant further enables robust classifiers that can moderate the impact of variants, if any, from CH.

Early assessment of therapy response in non-small cell lung cancer (NSCLC) via longitudinal ctDNA analysis.

e20701 Background: Quantifying circulating tumor DNA (ctDNA) is an emerging method to non-invasively assess treatment effect for solid tumors. Despite disease heterogeneity in NSCLC, we set out to identify a broadly applicable ctDNA-based method for disease monitoring. By employing plasma taken during early treatment cycles, we tested whether early response assessed by ctDNA level could predict treatment effect. Methods: Using a 197-gene NGS assay, the AVENIO ctDNA Surveillance Kit (For Research Use Only, not for use in diagnostic procedures), we measured ctDNA levels in post-treatment plasma samples based on variants identified at baseline. We used samples from an observational German Lung Cancer Multi-Marker Study. In a cohort of 83 stage IV lung adenocarcinoma treated with first-line chemo or chemoradiation therapies, we evaluated the association between survival and ctDNA levels in the first available post-treatment plasma sample (median number of days after start of treatment = 23). We used a ctDNA-based monitoring algorithm, and applied it to an independent set of 22 late stage lung squamous cell carcinoma (SCC) that also underwent chemo or chemoradiation therapies to further evaluate the algorithm in different histology subtypes. Results: We divided the 83 adenocarcinoma cohort into training (n = 53) and test (n = 30) sets. We found that subjects with longer progression free survival (PFS) had mean allele fraction (AF) &lt; 1% in the training set. We applied the classifier to our validation set and found that subjects with mean AF &lt; 1% had longer PFS (HR 0.35; 95% CI 0.12 - 0.93; log-rank P = 0.028) and overall survival (OS) (HR 0.29; 95% CI 0.09 - 0.89; log-rank P = 0.021). Using cutoffs identified in adenocarcinoma, we applied the same algorithms to the SCC cohort. Subjects with mean AF &lt; 1% had longer PFS (HR 0.26; 95% CI 0.10 - 0.71; log-rank P = 0.005) and OS (HR 0.12; 95% CI 0.05 - 0.51; log-rank P = 0.001). Conclusions: Even in heterogeneous diseases such as NSCLC, changes in ctDNA levels in response to treatment may prove to be a valuable way of identifying subjects who may not benefit, which is earlier than current standard of care methods like computed tomography (CT) scan. Future prospective studies to confirm these results are warranted.

Cell-free DNA as a biomarker for taxane treatment in advanced prostate cancer.

5070 Background: Antitumor efficacy of chemotherapeutic drugs docetaxel and cabazitaxel against hormone-sensitive and castration-resistant prostate cancer (CRPC) is well recognized. The identification and validation of minimally invasive biomarkers of taxane response remain of paramount importance. Methods: Serial pre-, during and post-treatment plasma samples were collected prospectively from two large Phase III clinical trials, FIRSTANA (NCT01308567) and PROSELICA (NCT01308580). Chemotherapy-naïve patients (pts) were treated with docetaxel (75 mg/m2) or cabazitaxel (20 or 25 mg/m2) in FIRSTANA, and pts previously treated with docetaxel received cabazitaxel (20 or 25 mg/m2) as second-line chemotherapy in PROSELICA. Plasma cell-free DNA (cfDNA) was extracted, low-pass whole genome sequencing (lp-WGS) libraries were prepared using the QIAGEN QiaSeq FX DNA library kit, and samples sequenced on the Illumina NovaSeq 6000. Targeted next-generation sequencing (NGS) on a custom-made AmpliSeq panel of 30 genes, pre-selected for putative roles in taxane resistance, was also performed. lp-WGS copy number (CN) profiles were generated using ichorCNA (v0.1.0) and targeted NGS variant calls derived using IonReporter software. Results: Overall, lp-WGS data was generated from 265 samples (99 pts; 51 treated on the FIRSTANA study, 48 treated on the PROSELICA study), acquired at three time-points (pre-, during and at progression). Average cfDNA input was 10 ng and mean coverage achieved was ~2X (SD 1.2X). We observed changes in lp-WGS tumor purity over time as a result of therapy; samples from non-responding pts exhibited significantly higher tumor purity values post-treatment compared with samples from responding pts (p = 0.02). Targeted NGS results were available from 294 pts (153 from FIRSTANA, 141 from PROSELICA), and changes in tumor purity were also associated with treatment response (p = 0.04). CN frequency of key CRPC genes was similar to previously reported datasets; several aberrant loci associated with response to taxane therapy. Conclusions: cfDNA lp-WGS may have clinical utility in the management of lethal prostate cancer. Funding: Sanofi. Clinical trial information: NCT01308580, NCT01308567.