Serial Liquid Biopsies Research Articles

Evaluation of blood MSI burden dynamics to trace immune checkpoint inhibitor therapy efficacy through the course of treatment.

Analysis of serial liquid biopsy (LB) samples has been found to be a promising approach for the monitoring of tumor dynamics in the course of therapy for patients with colorectal cancer (CRC). Currently, somatic mutations are used for tracing the dynamics of the tumor via LB. However, the analysis of the dynamic changes in the molecular signatures such as microsatellite instability (MSI) is not currently used. We hypothesized that changes in blood MSI burden (bMSI) could be registered using serial LB sampling in the course of immune checkpoint inhibitors (ICI), and that its changes could potentially correlate with treatment outcomes. We report the preliminary findings of the observational trial launched to study (NCT06414304) the dynamics of bMSI in 9 MSI-positive CRC patients receiving ICI. NGS-based MSI testing was performed on both pre-treatment FFPE and serial LB samples. For patients who had detectable bMSI burden in any of the LB samples (n = 8, 89%), median bMSI was 1.4% (range, 0.01-40%). Among patients with detectable MSI in available FFPE samples, median MSI burden was 29.3% (range, 10-40%). bMSI detected in baseline LB and FFPE samples were positively correlated (Pearson's R 0.47). Maximal variant allele frequencies of driver mutations observed in LB were also positively correlated with bMSI burden (Pearson's R 0.7). Patients who had clinical benefit had undetectable bMSI burden at follow-up. Our results provide the rationale for further validation of bMSI as a predictive biomarker of ICI in MSI-positive patients.

Utility of circulating tumor DNA (ctDNA) to inform treatment of patients with metastatic breast cancer.

1042 Background: Circulating tumor DNA (ctDNA) can overcome limitations of tissue biopsy (tbx) in patients with metastatic breast cancer (mBC). While tbx provides a snapshot of a tumor’s molecular profile from one site and time, ctDNA provides a more comprehensive landscape of the tumor genome. Serial liquid biopsies (lbx) can be easily obtained for monitoring clinical response and resistance over time. The Individualized Molecular Analyses Guide Efforts in Breast Cancer (IMAGE) II study evaluates tbx and serial lbx methods for tumor genomic profiling in mBC. Methods: IMAGE II (NCT02965755) is a prospective, multi-center trial to evaluate the clinical utility of lbx versus tbx. Eligible individuals had mBC of any subtype, with progression requiring therapy change. Tbx sequencing per standard of care was required unless biopsy was not technically feasible. Upon enrollment, we collected baseline plasma for tissue-agnostic comprehensive genomic profiling (CGP) by Foundation Medicine. Both lbx and tbx sequencing results were reviewed in real time by the Johns Hopkins Molecular Tumor Board to provide treatment recommendations. Clinical data was recorded. We collected serial lbx samples 1-2 weeks post initiation of next line of therapy, at first restaging, and at progression. Here we present the analysis of data collected at enrollment, and serial lbx results will be reported at a later date. Results: Total enrollment was 199 pts, of whom 194 were women (median age = 57 years, range 27-86 years). There were 140 HR+HER2-, 4 HR-HER2+, 20 HR+HER2+ and 35 triple negative (TNBC) cases. Metastases were found in bone only in 19 pts, or included lung in 99 pts, liver in 97 pts, and brain in 14 pts. Median prior lines of metastatic therapy was 2, with 44 pts (22.1%) having been on 4 or more, including antibody-drug conjugates (14 pts), immunotherapy (2), cytotoxic chemotherapy (74), endocrine (88), and targeted therapy (12). 9 pts were newly diagnosed with mBC at the time of enrollment. 110 pts (55%) had tbx sequencing. In lbx-based CGP results from 190 patients, the genes most frequently altered were TP53(90 patients), PIK3CA (63), ESR1 (54). Copy number amplifications were found in FGFR1 (21 patients) and FGFR3 (12), and copy number losses were found in PTEN in 3 patients. Of the 87 patients whose baseline lbx was profiled using FoundationOne Liquid CDx which allows calculation of ctDNA tumor fraction (TF), 69 (79.3%) pts had detectable ctDNA TF. 60 patients (69.0%) had ctDNA TF at least 1%. We will present the frequency of clinically actionable mutations detected via lbx vs tbx genomic profiling, accounting for sites of metastases, receptor subtype and prior therapy exposure, as well as the association between ctDNA TF and sites of disease. Conclusions: A high rate of ctDNA TF detection in pts with mBC highlights the value of lbx for CGP in this setting for detecting multiple alteration classes across mBC subtypes and metastatic sites. Clinical trial information: NCT02965755 .

Molecular profiling of serial liquid biopsy specimens utilizing cell free DNA (cfDNA) and circulating tumor cells (CTCs) in TBCRC041: A phase II study of alisertib in endocrine resistant metastatic breast cancer (MBC).

1037 Background: TBCRC041, a randomized phase 2 trial of alisertib (A) ± fulvestrant (F) in women with HR+/HER2- MBC demonstrated promising clinical activity for alisertib (1). Interrogation of baseline (PreC1) and end of cycle 1 (EOC1) cfDNA and CTCs may identify biomarkers associated with response to A±F. Methods: Plasma cfDNA was sequenced using the Guardant INFINITY platform, which includes genomic and methylated tumor fraction (mTF) analysis. Pathogenic variants were determined using publicly available databases and Mayo Clinic annotation pipelines. CTCs were identified as nucleated, EpCAM+/ cytokeratin+/ CD45- cells and assessed for ER/HER2 staining (RareCyte, Seattle). PFS was defined as the time from randomization to progression or death. EOC1-PFS differs from PFS as time starts at C2D1. Stratified log-rank tests and conditional Cox modeling were used to assess whether PFS or EOC1-PFS differed by mutations in ESR1 or PIK3CA, pre-CTC ≥ 5, or mTF>1%. Results: Of the 91 eligible pts, 84 progressed, and 73 were deceased. In the 86 pts with PreC1-cfDNA results, variants were identified in ESR1 (42; 48.8%), PIK3CA (36; 41.9%), AKT1 (7; 8.1%), PTEN(7; 8.1%), BRCA1 (4; 4.7%), and BRCA2 (7; 8.1%). Pts with a PIK3CAmut had significantly decreased PFS (HR 1.8; 95%CI: 1.1 -2.9, p=0.014) but not by ESR1mut status (p=0.583). PreC1 and EOC1-mTF were determined for 78 and 76 pts, respectively. PreC1-mTF was 0-1% in 21; 1.1-10.0% in 27; and 10.1-100% in 30. mTF levels remained between 0-1% in 17 pts fell to 0-1% in 11 pts, and was 0-1% in 5 pts without a preC1-mTF determination. PFS did not differ with respect to PreC1-mTF (p=0.210), but EOC1-PFS was worse in pts with mTF >1% (HR 3.0; 95%CI: 1.6 -5.2, p<0.001). PreC1 and EOC1-CTC were determined for 78 and 76 pts. 36 (46.2%) pts had a PreC1 CTC count ≥ 5. EOC1-CTC levels remained at (n=17) or increased to (n=1) ≥ 5 and was ≥ 5 in 2 pts without a preC1-CTC determination. EOC1-PFS was not significantly different for EOC1-CTC ≥ 5 (p=0.065), but was significantly decreased in pts with PreC1-CTC≥5 (HR=1.8; 95%CI: 1.1-3.0; p=0.018). Broader cfDNA NGS panel and CTC-derived ER/HER2 staining results will be presented at the meeting. Conclusions: Among patients receiving A±F, PFS varied based on the PIK3CAmutation status. cfDNA and CTC assessment PreC1 and EOC1 provided complementary information. Further validation in larger datasets is needed. 1. JAMA Oncol 2023;9:815. Clinical trial information: NCT02860000 .

Abstract LB449: Strategies to overcome resistance to PI3Kalpha-selective inhibitors mediated by acquired alterations in the PI3K/AKT pathway

Abstract PIK3CA is among the most frequently mutated kinases in multiple types of cancer, including in 40% of hormone receptor-positive, HER2-negative breast cancers. The PI3Kα-selective orthosteric inhibitor alpelisib is the only drug targeting PIK3CA currently approved in this patient population, in combination with fulvestrant. As commonly observed with other targeted therapies, resistance often develops during treatment with PI3Kα-selective inhibitors. In one of the largest patient cohorts to date, including serial liquid biopsies and rapid autopsies in 39 patients, our group has observed genomic alterations within the PI3K/AKT pathway as one of the major mechanisms of resistance to these agents. Among them, we have identified for the first-time secondary mutations in PIK3CA (W780R and Q859K) that decrease the affinity of the PI3Kα-selective inhibitors alpelisib and inavolisib, leading to acquired resistance. Multiple small molecule inhibitors designed to suppress signaling through the PI3K/AKT axis are currently in pre-clinical development. These include AKT inhibitors, isoform-selective as well as pan-PI3K inhibitors, and a novel subclass of allosteric PI3Kα-selective inhibitors designed to preferentially inhibit both kinase (H1047R) and helical (E545K) domain mutant PI3Kα activity. We have introduced all the emergent alterations observed in our patient cohort into breast cancer models and screen how they modify the effect of a structurally diverse array of PI3K/AKT inhibitors. While some acquired mutations compromise the effect of specific drugs, we have determined that the W780R mutation could drive universal resistance to ATP-competitive PI3K inhibitors that bind the catalytic pocket. Importantly, vertical pathway inhibition or allosteric inhibition with pan-mutant-selective PI3Kα inhibitors such as RLY-2608 or STX-478 could overcome resistance induced by all emerging secondary PIK3CA mutations.Dose-limiting toxicity in the form of hyperglycemia, rash or gastrointestinal issues are common in patients treated with orthosteric PI3Kα inhibitors. While mutant selective PI3Kα inhibitors induce less adverse effects in patients, they have a decreased potency targeting wildtype PIK3CA. We have identified wildtype PIK3CA-mediated feedback pathway reactivation as a potential mechanism of resistance to this type of inhibitors. Our work suggests that combining orthosteric and allosteric PI3Kα inhibitors as well as downstream pathway deactivation with AKT inhibitors could result in an increased therapeutic index and delay the emergence of resistance in patients. This work provides an insightful characterization of the current knowledge on clinical acquired resistance to PI3Kα inhibitors and proposes a detailed strategy to overcome resistance mediated by most PI3K/AKT alterations described to date. Citation Format: Ferran Fece de la Cruz, Andreas Varkaris, Elizabeth E. Martin, Bryanna L. Norden, Nicholas Chevalier, Allison M. Kehlmann, Ignaty Leshchiner, Haley Barnes, Sara Ehnstrom, Parasvi Patel, Janice S. Kim, Haley Ellis, Ioannis Sanidas, Kayao T. Lau, Aditya Bardia, Laura M. Spring, Steven J. Isakoff, Jochen K. Lennerz, Gad Getz, Ryan B. Corcoran, Dejan Juric. Strategies to overcome resistance to PI3Kalpha-selective inhibitors mediated by acquired alterations in the PI3K/AKT pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB449.

7 Flexible probabilistic methods to unlock the clinical potential of liquid biopsy sampling

OBJECTIVES/GOALS: Decoding the origins of cell-free DNA (cfDNA) released from dying cells in a liquid biopsy sample (e.g. blood) offers the potential to provide insight into the dynamic, organism-wide changes reflective of health and disease. Thus, making cfDNA an ideal target for serial, minimally invasive monitoring of disease-related changes. METHODS/STUDY POPULATION: We develop a probabilistic method that leverages the co-regulation of neighboring CpG sites on individual methylome-wide sequencing (WGBS) reads to more flexibly model cell-specific methylation compared to prior methods that focus on the methylation rate of a single CpG site. We then extend our cross-sectional model to account for sequential sampling within the same subject. The increased sampling frequency is critical to identifying the evolutionary dynamics of disease progression influencing treatment response and resistance, and disease recurrence. We utilize Bayesian inference techniques to model patient-specific longitudinal profiles of cell-type turnover in simulated serial samples. RESULTS/ANTICIPATED RESULTS: We found our model more effective at capturing a range of methylation patterns on cfDNA fragments with lower Root Mean Square Error across simulations compared to a single CpG model. We apply our model to detect significant (p < 0.05, Friedman’s test) increases in cellular contributions from lung and cardiac tissue in breast cancer patients (n=15) undergoing radiation therapy compared to baseline. We also identify signals of radiation induced toxicity to the liver in right-sided breast cancer patients (n=8) receiving radiation treatment compared to left-sided breast cancer patients (n=7). Finally, we show our extended model results in more efficient estimates of simulated cell-type turnover profiles compared to analyzing serial samples cross-sectionally, ignoring the longitudinal nature of the data. DISCUSSION/SIGNIFICANCE: Here we address an unmet need in developing novel statistical methodologies to decode the origins of methylated cfDNA obtained from liquid biopsy samples. We demonstrate the far-ranging clinical utility of serial liquid biopsy sampling to complement and advance the standards of clinical care in oncology and other pathologies.

Abstract 2390: Longitudinal assessment of circulating tumor DNA in patients with advanced colorectal cancer: A proposed general statistical framework and visualization tool

Abstract Background: Understanding how ctDNA levels change over time can act as a surrogate marker for disease progression. Since ctDNA evolution is complicated—exhibiting variability within and between patients—we propose a rigorous and flexible statistical framework that comprehensively represents these types of time-varying biomarkers. We propose employing a hierarchal random effects cubic spline model due to its advantages over traditional longitudinal modeling approaches such as the former’s ability to incorporate patient characteristics and create patient-specific results. Visualization of individual patient trajectories may provide clinical utility in precision oncology settings. Methods: 167 patients with CRC were selected from GuardantINFORM, a real-world database linking genomic and claims data. All patients received chemotherapy and had at least three serial liquid biopsy tests completed via Guardant360. To meet model assumptions, ctDNA levels, measured by maximum variant allele frequency on each test, were transformed into logits. Due to the model’s hierarchal structure, an unconditional cubic spline model was fit first, producing an estimated response pattern for the cohort. Next, as patient-level results are of interest, the unconditional model was built upon by fitting a conditional model that incorporated covariates consisting of demographic, health status, and mortality information, which provided numerous patient-level response patterns. The best fitting conditional model was guided by Akaike’s information criteria. Results: Since model parameter estimates are uninterpretable, and because numerous patient-level projections are generated (each covariate value combination produces a unique projection), an R-Shiny application was developed to visually present and compare results in an intuitive interactive fashion. Additionally, to enhance the understanding of patient response patterns, velocity plots, which provides the instantaneous rate of change in ctDNA levels at different time points, are also provided. Conclusions: We demonstrate that the proposed method can successfully be applied to genomic data to describe and explore complex patient-level temporal ctDNA patterns while accounting for the impact of covariate values have on these patterns. We implemented the proposed methodology as a visualization tool that can be used in a wide variety of settings, ranging from hypothesis testing in clinical trials to patient monitoring. Results from the model can further our basic conceptualization of ctDNA dynamics and enhance our ability to integrate these results into targeted, patient centric, clinical decision-making. Citation Format: Christopher R. Pretz, Jiemin Liao, Leylah Drusbosky, Amar Das. Longitudinal assessment of circulating tumor DNA in patients with advanced colorectal cancer: A proposed general statistical framework and visualization tool [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 2390.

Circulating microRNA Analysis in a Prospective Co-clinical Trial Identifies MIR652-3p as a Response Biomarker and Driver of Regorafenib Resistance Mechanisms in Colorectal Cancer.

The multi-kinase inhibitor (mKi) regorafenib has demonstrated efficacy in chemorefractory patients with metastatic colorectal cancer (mCRC). However, lack of predictive biomarkers and concerns over significant toxicities hamper the use of regorafenib in clinical practice. Serial liquid biopsies were obtained at baseline and monthly until disease progression in chemorefractory patients with mCRC treated with regorafenib in a phase II clinical trial (PROSPECT-R n = 40; NCT03010722) and in a multicentric validation cohort (n = 241). Tissue biopsies collected at baseline, after 2 months and at progression in the PROSPECT-R trial were used to establish patient-derived organoids (PDO) and for molecular analyses. MicroRNA profiling was performed on baseline bloods using the NanoString nCounter platform and results were validated by digital-droplet PCR and/or ISH in paired liquid and tissue biopsies. PDOs co-cultures and PDO-xenotransplants were generated for functional analyses. Large-scale microRNA expression analysis in longitudinal matched liquid and tissue biopsies from the PROSPECT-R trial identified MIR652-3p as a biomarker of clinical benefit to regorafenib. These findings were confirmed in an independent validation cohort and in a "control" group of 100 patients treated with lonsurf. Using ex vivo co-culture assays paired with single-cell RNA-sequencing of PDO established pre- and post-treatment, we modeled regorafenib response observed in vivo and in patients, and showed that MIR652-3p controls resistance to regorafenib by impairing regorafenib-induced lethal autophagy and by orchestrating the switch from neo-angiogenesis to vessel co-option. Our results identify MIR652-3p as a potential biomarker and as a driver of cell and non-cell-autonomous mechanisms of resistance to regorafenib.

Allosteric PI3Kα Inhibition Overcomes On-target Resistance to Orthosteric Inhibitors Mediated by Secondary PIK3CA Mutations.

In one of the largest patient cohorts analyzed to date, this study defines the clinical landscape of acquired resistance to PI3Kα inhibitors. Genomic alterations within the PI3K pathway represent a major mode of resistance and identify a novel class of secondary PIK3CA resistance mutations that can be overcome by an allosteric PI3Kα inhibitor. See related commentary by Gong and Vanhaesebroeck, p. 204 . See related article by Varkaris et al., p. 240 . This article is featured in Selected Articles from This Issue, p. 201.

Detecting ALK, ROS1, and RET fusions and the METΔex14 splicing variant in liquid biopsies of non-small-cell lung cancer patients using RNA-based techniques.

ALK, ROS1, and RET fusions and MET∆ex14 variant associate with response to targeted therapies in non-small-cell lung cancer (NSCLC). Technologies for fusion testing in tissue must be adapted to liquid biopsies, which are often the only material available. In this study, circulating-free RNA (cfRNA) and extracellular vesicle RNA (EV-RNA) were purified from liquid biopsies. Fusion and MET∆ex14 transcripts were analyzed by nCounter (Nanostring) and digital PCR (dPCR) using the QuantStudio® System (Applied Biosystems). We found that nCounter detected ALK, ROS1, RET, or MET∆ex14 aberrant transcripts in 28/40 cfRNA samples from positive patients and 0/16 of control individuals (70% sensitivity). Regarding dPCR, aberrant transcripts were detected in the cfRNA of 25/40 positive patients. Concordance between the two techniques was 58%. Inferior results were obtained when analyzing EV-RNA, where nCounter often failed due to a low amount of input RNA. Finally, results of dPCR testing in serial liquid biopsies of five patients correlated with response to targeted therapy. We conclude that nCounter can be used for multiplex detection of fusion and MET∆ex14 transcripts in liquid biopsies, showing a performance comparable with next-generation sequencing platforms. dPCR could be employed for disease follow-up in patients with a known alteration. cfRNA should be preferred over EV-RNA for these analyses.

Liquid biopsy-informed precision oncology study to evaluate utility of plasma genomic profiling for therapy selection.

TPS1616 Background: Liquid biopsies have enabled a transformation in the management of patients with cancer as they have the potential to detect, characterize, and monitor tumor burden. However, the increasing complexity involved in the integration of genomic data into clinical practice has presented challenges with the practical implementation of liquid biopsies in clinical cancer care. To address these challenges, multidisciplinary teams comprised of experts within oncology, genetics, pathology, and informatics have created Molecular Tumor Boards (MTBs), which can improve clinical outcomes for cancer patients by efficiently utilizing the technologies created by the rapidly expanding precision oncology industry. Nevertheless, there remains a gap in the optimal implementation and interpretation of these diagnostic findings to maximize patient outcomes and access to standard of care therapies and clinical trials. Methods: We instigated a prospective observational liquid biopsy-informed precision oncology study, in which patients with metastatic solid tumors will be matched with genotype-driven therapies via serial liquid biopsies (NCT05585684). Liquid biopsy-derived genotypes will be generated through a clinical NGS assay (PGDX Plasma Focus) and will be annotated by an automated ensemble approach for variant actionability assessment, developed by the Johns Hopkins MTB, followed by a treatment recommendation. Key eligibility criteria include patients ≥18 years old, with ECOG performance status of 0-1 and with metastatic disease that have relapsed or progressed after standard of care first-line therapies or patients for which first-line standard of care therapeutic options are not expected to yield a clinical response. The primary objectives of the trial focus on feasibility of the approach and evaluating the fraction of patients with actionable mutations, for which the MTB recommendation is enacted upon. Unique to this study, a recommendation to not pursue genotype-targeted therapy is equally considered for personalized clinical decision making. Secondary objectives are to investigate whether enacting upon MTB recommendations improves progression-free and overall survival and perform a comparative analysis with tumor-based NGS. Exploratory objectives are to determine if serial molecular profiling after initiation of systemic treatment is sufficient to determine therapeutic response, compare with RECIST radiographic response and determine whether changes in liquid biopsy dynamics correlate with progression-free and overall survival. This study will enroll 150 patients with solid tumors, including lung, esophageal, breast, and head and neck cancer and mesothelioma to evaluate the clinical utility of liquid biopsy genomic profiling. Patient accrual for this study initiated in February 2023. Clinical trial information: NCT05585684 .

SYSTEMI - systemic organ communication in STEMI: design and rationale of a cohort study of patients with ST-segment elevation myocardial infarction

BackgroundST-segment elevation myocardial infarction (STEMI) still causes significant mortality and morbidity despite best-practice revascularization and adjunct medical strategies. Within the STEMI population, there is a spectrum of higher and lower risk patients with respect to major adverse cardiovascular and cerebral events (MACCE) or re-hospitalization due to heart failure. Myocardial and systemic metabolic disorders modulate patient risk in STEMI. Systematic cardiocirculatory and metabolic phenotyping to assess the bidirectional interaction of cardiac and systemic metabolism in myocardial ischemia is lacking.MethodsSystemic organ communication in STEMI (SYSTEMI) is an all-comer open-end prospective study in STEMI patients > 18 years of age to assess the interaction of cardiac and systemic metabolism in STEMI by systematically collecting data on a regional and systemic level. Primary endpoint will be myocardial function, left ventricular remodelling, myocardial texture and coronary patency at 6 month after STEMI. Secondary endpoint will be all-cause death, MACCE, and re-hospitalisation due to heart failure or revascularisation assessed 12 month after STEMI. The objective of SYSTEMI is to identify metabolic systemic and myocardial master switches that determine primary and secondary endpoints. In SYSTEMI 150–200 patients are expected to be recruited per year. Patient data will be collected at the index event, within 24 h, 5 days as well as 6 and 12 months after STEMI. Data acquisition will be performed in multilayer approaches. Myocardial function will be assessed by using serial cardiac imaging with cineventriculography, echocardiography and cardiovascular magnetic resonance. Myocardial metabolism will be analysed by multi-nuclei magnetic resonance spectroscopy. Systemic metabolism will be approached by serial liquid biopsies and analysed with respect to glucose and lipid metabolism as well as oxygen transport. In summary, SYSTEMI enables a comprehensive data analysis on the levels of organ structure and function alongside hemodynamic, genomic and transcriptomic information to assess cardiac and systemic metabolism.DiscussionSYSTEMI aims to identify novel metabolic patterns and master-switches in the interaction of cardiac and systemic metabolism to improve diagnostic and therapeutic algorithms in myocardial ischemia for patient-risk assessment and tailored therapy.Trial registrationTrial Registration Number: NCT03539133

Circulating tumour DNA kinetics in recurrent/metastatic head and neck squamous cell cancer patients

PurposeImmune checkpoint blockade (ICB) has become a standard of care in the treatment of recurrent/metastatic head and neck squamous cell cancer (R/M HNSCC). However, only a subset of patients benefit from treatment. Quantification of plasma circulating tumour DNA (ctDNA) levels and on-treatment kinetics may permit real-time assessment of disease burden under selective pressures of treatment. Patients and methodsR/M HNSCC patients treated with systemic therapy, platinum-based chemotherapy (CT) or ICB, underwent serial liquid biopsy sampling. Biomarkers tested included ctDNA measured by CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) and markers of host inflammation measured by neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR). ResultsAmong 53 eligible patients, 16 (30%) received CT, 30 (57%) ICB [anti-PD1/L1] monotherapy and 7 (13%) combination immunotherapy (IO). Median progression-free survival (PFS) and overall survival (OS) were 2.8 months (95% CI, 1.3–4.3) and 8.2 months (95% CI, 5.6–10.8), respectively. Seven (13%) patients experienced a partial response and 21 (40%) derived clinical benefit. At baseline, median ctDNA variant allele frequency (VAF) was 4.3%. Baseline ctDNA abundance was not associated with OS (p = 0.56) nor PFS (p = 0.54). However, a change in ctDNA VAF after one cycle of treatment (ΔVAF (T1–2)) was predictive of both PFS (p< 0.01) and OS (p< 0.01). Additionally, decrease in ΔVAF identified patients with longer OS despite early radiological progression, 8.2 vs 4.6 months, hazard ratio 0.44 (95% CI, 0.19–0.87) p = 0.03. After incorporating NLR and PLR into multivariable Cox models, ctDNA ∆VAF retained an association with OS. ConclusionsEarly dynamic changes in ctDNA abundance, after one cycle of treatment, compared to baseline predicted both OS and PFS in R/M HNSCC patients on systemic therapy.

Abstract 1045: RePhyNER: Overcoming limitations of access to matched germline for serial liquid biopsy applications

Abstract Background: Comparison of a blood or tissue sample with a matched germline control is a key step for accurate detection of somatic mutations. This is particularly important for tracking minimal residual disease (MRD), since alterations erroneously considered to be tumor derived may lead to false detection. Use of blood leukocytes as a germline control is critical not only for appropriate censoring of constitutional allelic variants, but also for addressing those associated with clonal hematopoiesis (CH). However, such matched germline is not always available, or may be suboptimal when contaminated with tumor cells. We hypothesized that accurate tumor genotyping might be feasible without a matched germline, using a dedicated algorithmic framework relying on clonal relationships of alleles of interest (i.e., phylons). Methods: Here, we introduce RePhyNER (Recursive Phylon Nomination, Enumeration, and Recovery), an algorithm for addressing this challenge in serial liquid biopsies. RePhyNER relies on the assumption that when comparing two specimens from the same individual (e.g., before and after therapy), the genomic variants of interests (e.g., tumor somatic mutations) behave differently in their allelic levels when compared to variants needing to be censored (e.g., germline constitutional alleles or CH). Using the change in mean allelic level of all variants in ≥2 samples, RePhyNER relies on a recursive approach to test each candidate somatic variant’s corresponding change gradient against this distribution using count-based statistics. Results: We used simulations to assess the importance of multiple parameters including fraction of contaminating variants or alleles and mean allelic fold-change. These simulations showed substantially superior performance of a recursive approach and revealed that for ≥2-fold changes in the mean VAF in a pair of samples, RePhyNER accurately removed &amp;gt;99% of contaminating alleles while preserving ~100% of true variants. We then applied RePhyNER to 108 plasma cell-free DNA (cfDNA) samples from 47 patients with classic Hodgkin Lymphoma (cHL) profiled by PhasED-Seq and compared MRD-detection performance with or without matched germline. RePhyNER substantially improved specificity by ~30% (68% vs 99%), and Precision by ~50% (46% vs 96%), while only modestly reducing sensitivity (100% vs 91%). We next applied PhasED-Seq to a cohort of cHL patients (n=65) without matched germline information. MRD positivity within the first 2 cycles of therapy was associated with inferior outcome when using RePhyNER (P&amp;lt;0.05 vs ns), correctly reclassifying 26% of detected patients to undetected. Conclusions: RePhyNER enables germline-free and accurate genotyping for MRD detection. Notably, RePhyNER obviates the need for additional germline profiling overcoming key limitations described above and avoids the additional associated costs of sequencing. Citation Format: Mohammad Shahrokh Esfahani, Stefan Alig, Emily Hamilton, Joseph Schroers-Martin, Brian Sworder, Jan Boegeholz, Mari Olsen, Chih Long Liu, David Kurtz, Maximilian Diehn, Ash Alizadeh. RePhyNER: Overcoming limitations of access to matched germline for serial liquid biopsy applications [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 1045.

35 Novel statistical methods to unlock the clinical potential of liquid biopsy sampling

OBJECTIVES/GOALS: Decoding the origins of cell-free DNA (cfDNA) released from dying cells in a liquid biopsy sample (e.g. blood) can provide insight into the dynamic, organism-wide changes reflective of health and disease state. Making cfDNA an ideal target for genomic monitoring of disease-related changes. METHODS/STUDY POPULATION: Methylome-wide sequencing (WGBS) data present unique statistical challenges. To this end, we developed a novel statistical method using an Expectation-Maximization algorithm to decode the cellular origins of cfDNA fragments in liquid biopsies. Our flexible, probabilistic method leverages the co-regulation of neighboring CpG sites on the individual sequencing read to facilitate tissue of origin analysis, as opposed to prior methods that focus on the methylation rate of a single CpG site. We assess the performance of our model in various simulated settings and apply our model to an important clinical example in which we are able to detect early off-target tissue damage from radiation therapy via minimally invasive blood draws. RESULTS/ANTICIPATED RESULTS: We found our model more effective at capturing the range of biologically plausible methylation patterns on cfDNA read fragments compared to prior models that use single CpG sites. We also show our model is robust to high levels of noise inherent with WGBS data. We demonstrate the accuracy of cell-type proportion estimation on in-silico mixed cfDNA samples from real WGBS data. Finally, we use our model in a clinical application. We detect significant (p &lt; 0.05) increases in cellular contributions from lung and cardiac tissue in breast cancer patients (n=15) undergoing radiation therapy compared to baseline. We also detect novel signals of radiation induced toxicity to the liver in right-sided breast cancer patients (n=8) receiving radiation treatment compared to matched left-sided breast cancer patients (n=7). DISCUSSION/SIGNIFICANCE: Here we address an unmet need in developing novel statistical methodologies that can handle the unique complexities of methylated cfDNA obtained from liquid biopsy samples. We also demonstrate the far-ranging clinical utility of serial liquid biopsy sampling to complement and advance standards of clinical care in oncology and other pathologies.

Bintrafusp alfa plus paclitaxel for advanced gastric cancer as a second-line treatment: An open label, single-arm, phase Ib/II study.

374 Background: Anti-PD-1 monotherapy did not improve overall survival compared with paclitaxel in second-line treatment for advanced gastric cancer (AGC) in KEYNOTE-061 trial. Recent studies suggest that inhibition of transforming growth factor beta (TGF-β) signaling reverses immunosuppressive tumor microenvironment into improved responses to cancer immunotherapy. Especially, genomically stable subtype of AGC showed the increased level of TGF-β pathway. Here, we report the results of phase Ib/II trial of bintrafusp alfa (M7824), a first-in-class bifunctional fusion protein composed of the extracellular domain of TGF-βRII fused to a human IgG1 mAb blocking PD-L1, in combination with paclitaxel as a 2nd line treatment for AGC. Methods: This investigator-initiated, single-arm, open-label, phase Ib/II study enrolled HER2-negative, histologically confirmed AGC patients with evaluable disease who failed prior palliative 1st line treatment. Prior immune checkpoint inhibitor was not allowed. Phase 1b was planned to enroll 3 to 12 patients in a 3+3 design (bintrafusp alfa 1200mg on days 1 and 15 plus paclitaxel 80 mg/m2 (level 1) or 70 mg/m2 (level -1) on days 1, 8 and 15) every 28 days). The primary endpoints were to determine recommended phase II dose (RP2D) for phase Ib, and progression-free survival (PFS) at 6 months for phase II. Secondary endpoints included PFS, OS, ORR, DCR, and safety. Exploratory endpoints included immune-related biomarker changes from serial liquid biopsies (NCT04835896). Results: In phase 1b, dose limiting toxicities were not observed, and the RP2D was determined as dose level 1. During phase II, enrollment was stopped after 30 patients were recruited, following negative readouts from bintrafusp alfa trials in other indications. With a median follow-up duration of 10.9 months (95% CI 9.5–12.6), 7 patients remained on treatment (treatment duration range: 1.0 to 15.1 months) as of September, 2022. Median PFS was 3.8 months (95% CI, 2.6-7.8) and PFS rate at 6 months was 36.7% (95% CI, 20.1-53.4). Median OS was 9.6 months (95% CI, 4.1-not reached). In patients with measurable lesions (n=25), ORR was 28% (PR, n=7) and DCR was 68%. No patient was EBV-related. Patients with MSI-H (n=3; mPFS, 7.8 months; mOS, not reached) or PD-L1-positive (CPS≥1) (n=9; mPFS, 7.8 months; mOS, not reached) showed durable efficacy. Treatment-related AEs (≥G3) occurred in 23 pts (76.7%), including anemia (G3, n=12, 40%) and neutropenia (G3, n=9, 30%). Immune-related AE included urticaria (G3, n=2, 6.7%; G2, n=4, 13.3%), rash (G3, n=1, 3.3%), and pneumonitis (G3, n=1, 3.3%). Conclusions: Combination of bintrafusp alfa and paclitaxel was feasible with modest efficacy as 2nd line treatment for AGC. There was subset of patients with durable responses. Analyses of targeted next-generation sequencing from serial ctDNA sampling are ongoing. Clinical trial information: NCT04835896 .

Clinical utility of liquid biopsy and integrative genomic profiling in early-stage and oligometastatic cancer patients treated with radiotherapy

BackgroundLiquid biopsy and Integrative Genomic Profiling (IGP) are yet to be implemented into routine Radiation Oncology. Here we assess the utility of germline, tumour and circulating cell-free DNA-based genomic analyses for the clinical management of early-stage and oligometastatic cancer patients treated by precision radiotherapy.MethodsWe performed germline, tissue- and liquid biopsy NGS panels on 50 early-stage/oligometastatic cancer patients undergoing radiotherapy. We also monitored ctDNA variants in serial liquid biopsies collected during radiotherapy and follow-up and evaluated the clinical utility of such comprehensive approach.ResultsThe integration of different genomic studies revealed that only 1/3 of the liquid biopsy variants are of tumour origin. Altogether, 55 tumour variants (affecting 3/4 of the patients) were considered potentially actionable (for treatment and prognosis), whereas potential follow-up biomarkers were identified in all cases. Germline cancer-predisposing variants were present in three patients, which would have not been eligible for hereditary cancer testing according to clinical guidelines. The presence of detectable ctDNA variants before radiotherapy was associated with progression-free survival both in oligometastatic patients and in those with early-stage.ConclusionsIGP provides both valuable and actionable information for personalised decision-making in Radiation Oncology.

Genetic features and therapeutic relevance of emergent circulating tumor DNA alterations in refractory non-colorectal gastrointestinal cancers

Acquired resistance to systemic treatments is inevitable in most cancers, but the genetic basis for this in many cancer types has remained elusive due to constraints in obtaining tissue specimens longitudinally. In the management of gastrointestinal cancers, molecular profiling is conventionally performed at a single time point, although serial evaluations may yield biological insights that inform treatment decisions. We characterize genetic changes in serial liquid biopsies which provide real-time snapshots of tumor genetics and heterogeneity in refractory non-colorectal gastrointestinal cancers, and determine the clinical utility of repeat circulating tumor DNA (ctDNA) testing. In a national cohort of 449 patients with pancreatic, biliary, esophagogastric, and hepatocellular cancers, resistance to conventional therapies is broadly associated with tumor evolution. Emergent ctDNA alterations only detectable at progression occurs in 63% of patients and are frequently associated with treatment actionability. Tumor mutation burden is dynamic in cancers undergoing treatment, but is not associated with time to progression. Objective tumor responses in a case series of patients receiving treatment matched to emergent alterations show that repeat liquid biopsies may have clinical benefit by expanding treatment options in advanced gastrointestinal cancers.

A Pilot, Prospective, Observational Study to Investigate the Value of NGS in Liquid Biopsies to Predict Tumor Response After Neoadjuvant Chemo-Radiotherapy in Patients With Locally Advanced Rectal Cancer: The LiBReCa Study.

IntroductionCirculating tumor DNA (ctDNA) correlates with the response to therapy in different types of cancer. However, in patients with locally advanced rectal cancer (LARC), little is known about how ctDNA levels change with neoadjuvant chemoradiation (Na-ChRT) and how they correlate with treatment response. This work aimed to explore the value of serial liquid biopsies in monitoring response after Na-ChRT with the hypothesis that this could become a reliable biomarker to identify patients with a complete response, candidates for non-operative management.Materials and MethodsTwenty-five consecutive LARC patients undergoing long-term Na-ChRT therapy were included. Applying next-generation sequencing (NGS), we characterized DNA extracted from formalin-fixed paraffin embedded diagnostic biopsy and resection tissue and plasma ctDNA collected at the following time points: the first and last days of radiotherapy (T0, Tend), at 4 (T4), 7 (T7) weeks after radiotherapy, on the day of surgery (Top), and 3–7 days after surgery (Tpost-op). On the day of surgery, a mesenteric vein sample was also collected (TIMV). The relationship between the ctDNA at those time-points and the tumor regression grade (TRG) of the surgical specimen was statistically explored.ResultsWe found no association between the disappearance of ctDNA mutations in plasma samples and pathological complete response (TRG1) as ctDNA was undetectable in the majority of patients from Tend on. However, we observed that the poor (TRG 4) response to Na-ChRT was significantly associated with a positive liquid biopsy at the Top.ConclusionsctDNA evaluation by NGS technology may identify LARC patients with poor response to Na-ChRT. In contrast, this technique does not seem useful for identifying patients prone to developing a complete response.

The Role of Serial Liquid Biopsy in the Management of Metastatic Non-Small Cell Lung Cancer (NSCLC)

Lung cancer is the leading cause of cancer-related deaths. Surgery remains the best option to treat lung cancer when feasible. However, many cases are diagnosed beyond the initial stages. There has been tremendous progress in the treatment of lung cancer over the last few years. Studies have shown that biomarker-driven targeted therapies lead to better outcomes. Due to the technical difficulties and significant procedural risk associated with repeated tissue biopsies, analysis of tumor constituents circulating in the blood, such as circulating tumor DNA (ctDNA) and various proteins, is becoming more widely recognized as an alternative method of tumor sampling, i.e., liquid biopsy. Liquid biopsy is superior to tissue biopsy, as it is minimally invasive and easily repeatable. Given the recent data on changes in mutations as the disease progresses or responds to treatment, liquid biopsies can help monitor the changes and guide us in giving targeted drugs. Here we present a case of advanced NSCLC who was initially started on Alectinib based on positivity for ALK gene rearrangement found in the FISH study. At the time of progression, molecular profiling liquid biopsy was obtained, which revealed KRAS-p.G12C mutation. Thus, the patient’s therapy was later on changed to sotorasib after the FDA approved a KRAS-p.G12C mutation inhibitor.

A phase II study of pembrolizumab for HPV-associated papilloma patients with laryngeal, tracheal, and/or pulmonary involvement.

2590 Background: Recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus (HPV) types 6 &amp; 11. RRP proliferates in the squamous epithelium lining the respiratory tract impacting breathing, swallowing, and voice and carries a 3-5% risk of malignant transformation. Given the multi-focality of the disease and tolerized host immune response against HPV, in part through upregulation of the PD1:PDL1 axis, the safety and efficacy of systemic pembrolizumab (pembro) as a novel treatment for this benign tumor patient (pt) population was evaluated in a phase II clinical trial. Methods: RRP pts &gt; 12 years of age were treated with pembro 200mg every 3 weeks. Primary endpoints were best overall response (ORR) (measured by endoscopic lesional burden) and safety. Greater than 5 pts with disease response out of 21 (assuming &gt; 1 of first n = 11 with disease in response) provided 86% power to distinguish between a 15% and a 38% ORR (one-sided 8% binomial test). HPV-specific CD8+ T cell frequency and functional states and biomarkers of response and immune resistance are being evaluated in serial tissue and liquid biopsies (up to 8 biopsies/patient over the 24 months of treatment). Results: The Simon two-stage, stage 1 criteria was met. A total of 21 patients were enrolled and all are now off treatment. Median age (range) was 45 (19-68), 57% (12/21) were male and 67% (14/21) were white. 48% (10/21) had Juvenile-onset (Jo)-RRP, 57% (12/21) had pulmonary RRP involvement, and 19% (4/21) had SCC derived from their RRP. 62% (13/21) completed 24 months of treatment. Reasons for discontinuation included disease progression (14%, 3/21), treatment related adverse event (TRAEs) (14%, 3/21), and study withdrawal (10%, 2/21). A partial response (≥25% reduction in endoscopic tumor burden score) was observed in 57% (12/21) (95% CI: 34%-78.2%) of pts (7 of 10 with Jo-RRP and 5 of 11 with Adult-onset (Ao)-RRP disease responded). Stable disease was observed in 33% (7/21). No complete responses were observed. Fatigue was the most frequent TRAEs; Grade 3 TRAEs included uveitis and hypophysitis, both of which were reversible upon pembro discontinuation and steroid use. At a median follow-up: 25.6 (6.2-38.1 months), the mean number of surgical interventions was reduced by 7 surgeries/year (p = 0.004) in pts treated on the trial for &gt; 12 months, and, upon treatment completion, durable clinical benefit was observed with no additional treatment needed for the duration of the clinical trial follow-up for some pts. Conclusions: Pembro reduces the need for routine surgical interventions based on the durable response rates being achieved. Further study of pembro +/- other agents is warranted to achieve and sustain complete responses in this population. Clinical trial information: NCT02632344.