Detection Of Tumor DNA Research Articles

Clinical significance of circulating tumor DNA in localized non-small cell lung cancer: a systematic review and meta-analysis.

Circulating tumor DNA (ctDNA) detection holds promise for genetic analyses and quantitative assessment of tumor burden. A systematic review and meta-analysis were conducted to investigate the clinical relevance of ctDNA among patients with localized non-small cell lung cancer (NSCLC). PubMed, EMBASE, and the Cochrane Library were searched for eligible studies published from January 2001 to April 2022. After quality assessments and data extraction, diagnostic accuracy variables and prognostic data were calculated and analyzed by Meta-Disc 1.4, Review Manager 5.4.1, and STATA 17.0. Eight prospective studies and one retrospective study including 784 patients with localized NSCLC were used in our meta-analysis. The pooled sensitivity and specificity of ctDNA for minimal residual disease (MRD) detection were 0.58 and 0.93, respectively. The pooled positive and negative likelihood ratios were 7.57 (95% confidence interval (CI) 2.84-20.20) and 0.45 (95% CI 0.37-0.55), respectively. The area under the summary receiver operating characteristic curve was 0.8967, and the diagnostic odds ratio was 32.26 (95% CI 14.63-71.12). In addition, both precurative-treatment and postcurative-treatment ctDNA positivity was associated with worse recurrence-free survival (hazard ratio (HR), 3.82 and 8.32, respectively) and worse overall survival (HR, 3.82 and 4.73, respectively). The findings suggested that ctDNA detection has beneficial utility regarding MRD detection specificity; moreover, positive ctDNA was associated with poor prognosis in patients with localized NSCLC.

Circulating tumor DNA detection in MRD assessment and diagnosis and treatment of non-small cell lung cancer.

Circulating tumor DNA (ctDNA) has contributed immensely to the management of hematologic malignancy and is now considered a valuable detection tool for solid tumors. ctDNA can reflect the real-time tumor burden and be utilized for analyzing specific cancer mutations via liquid biopsy which is a non-invasive procedure that can be used with a relatively high frequency. Thus, many clinicians use ctDNA to assess minimal residual disease (MRD) and it serves as a prognostic and predictive biomarker for cancer therapy, especially for non-small cell lung cancer (NSCLC). Advanced methods have been developed to detect ctDNA, and recent clinical trials have shown the rationality and feasibility of ctDNA for identifying mutations and guiding treatments in NSCLC. Here, we have reviewed recently developed ctDNA detection methods and the importance of sequence analyses of ctDNA in NSCLC.

Multimodal Urine Cell-Free DNA-Based Residual Disease Detection and Prognostication in Muscle-Invasive Bladder Cancer Patients

<h3>Purpose/Objective(s)</h3> Our group recently showcased a liquid biopsy assay (uCAPP-Seq) based on the detection of single nucleotide variants (SNVs) in urine cell-free DNA (cfDNA) from patients with muscle-invasive bladder cancer (MIBC). We found that assay positivity corresponded with the presence of residual cancer and a higher risk of disease recurrence despite curative-intent therapy (Chauhan et al., PLOS Medicine, 2021). Here, we explored multimodal urine-based genomics by integrating ultra-low-pass whole genome sequencing (ULP-WGS) and uCAPP-Seq for improving the sensitivity of residual disease detection and survival prediction among MIBC patients. <h3>Materials/Methods</h3> Urine samples from 74 MIBC patients were collected preoperatively on the day of radical cystectomy to assess urine tumor DNA (utDNA). We performed ULP-WGS of urine cfDNA from all 74 patients as well as 15 healthy adults. Tumor fraction (TF) based on genome-wide copy number alterations in urine cfDNA was estimated using ichorCNA. Variant allele frequency (VAF) based on SNVs was estimated by uCAPP-Seq. Pathologic complete response (pCR) was determined by surgical pathology. Multivariate logistic regression and random forest models were utilized to predict disease status based on VAF and TF levels, along with clinical variables (smoking, age, sex, race, and histology). Kaplan-Meier (KM) analysis was used to assess overall survival (OS) and progression-free survival (PFS). <h3>Results</h3> Mean utDNA VAF and TF levels were both significantly higher in patients without pCR compared to those who achieved pCR (11.9% vs. 4.0% VAF, p=0.044; 12.7% vs. 5.2% TF, p = 0.019) (Table 1). Our multimodal logistic regression model identified utDNA VAF (p < 0.001) and smoking status (p = 0.034) as significant predictors of disease status. Random forest modeling demonstrated that utDNA VAF and TF were both significant predictors. It achieved a sensitivity of 83% and a specificity of 79% for detecting residual disease. Strikingly, among the 15 patients with negative findings by both ULP-WGS and uCAPP-Seq, none experienced cancer progression (median follow-up time = 7.2 months). In contrast, the 59 patients with positive utDNA by ULP-WGS or uCAPP-Seq had significantly worse PFS (median = 6.0 months; HR = 3.54, p = 0.027). Moreover, utDNA positivity trended towards predicting significantly worse OS (HR = 3.43, p = 0.15). <h3>Conclusion</h3> Integration of ULP-WGS with uCAPP-Seq enabled robust detection of residual urine tumor DNA with high sensitivity and predicted progression-free survival in muscle-invasive bladder cancer patients. In the future, this type of multimodal urine-based genomic analysis may lead to more precise risk stratification and nonoperative clinical decision-making for muscle-invasive bladder cancer patients.

Applications of Focused Ultrasound for the Treatment of Glioblastoma: A New Frontier.

Simple SummaryGlioblastoma (GBM) is a common primary brain tumor with a short median overall survival despite aggressive treatment with resection, chemotherapy, and radiation therapy. Focused ultrasound (FUS) represents a promising new therapeutic approach for treatment of GBM. Unlike imaging forms of ultrasound, FUS can successfully penetrate the skull surrounding the brain, allowing for non-invasive ablation of tumor tissue. FUS can also temporarily disrupt the blood–brain barrier, a microvascular network that prohibits diffusion of most therapeutic agents, allowing chemotherapeutic drugs to penetrate the tumor. Other modalities are under investigation and include means of stimulating the immune system and sensitizing tumors to radiation therapy. The feasibility and safety of transcranial FUS has been illustrated in animal models and clinical trials. Precise results can be obtained under guidance from magnetic resonance imaging, limiting side effects. Successful outcomes from clinical trials will likely continue to motivate investigation and innovation of FUS technology for GBM.Glioblastoma (GBM) is an aggressive primary astrocytoma associated with short overall survival. Treatment for GBM primarily consists of maximal safe surgical resection, radiation therapy, and chemotherapy using temozolomide. Nonetheless, recurrence and tumor progression is the norm, driven by tumor stem cell activity and a high mutational burden. Focused ultrasound (FUS) has shown promising results in preclinical and clinical trials for treatment of GBM and has received regulatory approval for the treatment of other neoplasms. Here, we review the range of applications for FUS in the treatment of GBM, which depend on parameters, including frequency, power, pulse duration, and duty cycle. Low-intensity FUS can be used to transiently open the blood–brain barrier (BBB), which restricts diffusion of most macromolecules and therapeutic agents into the brain. Under guidance from magnetic resonance imaging, the BBB can be targeted in a precise location to permit diffusion of molecules only at the vicinity of the tumor, preventing side effects to healthy tissue. BBB opening can also be used to improve detection of cell-free tumor DNA with liquid biopsies, allowing non-invasive diagnosis and identification of molecular mutations. High-intensity FUS can cause tumor ablation via a hyperthermic effect. Additionally, FUS can stimulate immunological attack of tumor cells, can activate sonosensitizers to exert cytotoxic effects on tumor tissue, and can sensitize tumors to radiation therapy. Finally, another mechanism under investigation, known as histotripsy, produces tumor ablation via acoustic cavitation rather than thermal effects.

S328 Detection of Mutated Tumor DNA From Colorectal Cancer Using Real Time-PCR

S328 Detection of Mutated Tumor DNA From Colorectal Cancer Using Real Time-PCR

Abstract A13: Evolutionary history of transformation from chronic lymphocytic leukemia to Richter syndrome

Abstract Richter syndrome (RS), an aggressive lymphoma that develops in patients with chronic lymphocytic leukemia (CLL), is a striking example of histologic transformation. While recent therapeutic advances have transformed the treatment landscape of CLL and lymphoma, RS remains associated with dismal overall survival. Despite an advanced genomic and molecular characterization of CLL over the past decade, the current understanding of the genetic factors driving evolution of CLL to RS is limited. To decipher the genetics underlying this transformation, we have performed an integrative analysis of exome, genome and transcriptome data generated from matched RS and CLL samples from a discovery cohort of 53 patients with newly diagnosed RS of DLBCL histology. Through computational deconvolution of CLL and RS clones, we constructed phylogenetic relationships and traced evolution of CLL to RS, confirming both clonal related (87%) and unrelated cases (13%). In addition to identifying recognized RS-risk genetic lesions, we discovered novel RS-specific alterations, including 5 putative somatic driver genes (IRF2BP2, SRSF1, B2M, DNMT3A and EZH2), frequent copy number alterations beyond del(9p21)(CDKN2A/B), (including amp(7q21.2) (CDK6), amp(9p24) (PDL1/L2), and amp(1q23)(MCL1)), and recurrent whole genome duplication and chromothripsis. Integration of exome and genome sequencing data led to the identification of distinct molecular subtypes of RS with prognostic importance. To confirm these molecular subtypes, a validation cohort of 47 RS cases has been assembled with paired exome and transcriptome data. To further investigate the stepwise clonal evolution of CLL to RS, we performed single-cell RNA-sequencing on biopsy samples obtained at diagnosis from 5 individuals with clonally related transformation. Using a novel tool, CNVSingle, we inferred allele specific single-cell copy number alterations that enabled identification of the single-cell clusters representing distinct CLL and RS genetic subclones as well as intermediate, or transitional, evolutionary states. RS cells displayed gene expression enriched in pathways of MYC targets and cell cycle, in line with similar analysis on bulk transcriptomes. Finally, by ultra-low pass (ULP)-WGS sequencing of plasma samples from RS patients, we demonstrate detection of RS tumor DNA in plasma months prior to initial clinical diagnosis (n=3 of 6) or post-allogeneic stem cell transplant relapse (n=2 of 2). cfDNA is thus a promising tool for early detection of emerging RS and RS relapse as well as for non-invasive detection surrounding diagnosis. Altogether, our study defines RS-specific alterations and provides a molecular definition of RS, identifies distinct genetic subtypes of RS with prognostic significance, traces the evolutionary path to RS and suggests future strategies for improved detection. Citation Format: Erin M Parry, Ignaty Leshchiner, Romain Guieze, Connor Johnson, Eugen Tausch, Sameer A Parikh, Camilla K Lemvigh, Conor Messer, Filippo Utro, Chaya Levovitz, Kahn Rhrissorrakrai, Matthew S Davids, Julien Broseus, Shuqiang Li, Ziao Lin, Binyamin A Knisbacher, Christof Schneider, Laura Z Rassenti, Thomas J Kipps, Nitin Jain, William Wierda, Florence Cymbalista, Neil E Kay, Kenneth J Livak, Brian P Danysh, Chip Stewart, Donna Neuberg, Jennifer R Brown, Laxmi Paridi, Stephan Stilgenbauer, Gaddy Getz, Catherine Wu. Evolutionary history of transformation from chronic lymphocytic leukemia to Richter syndrome [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A13.

788O Association of pre-treatment ctDNA with disease recurrence and clinical and translational factors in patients with stage IIIB-D/IV melanoma treated with adjuvant immunotherapy (CheckMate 915)

In patients (pts) with resectable melanoma, circulating tumor DNA (ctDNA) detection post-resection may be useful to inform disease state and recurrence risk. Post-resection pre-treatment plasma from 1127 pts with stage IIIB-D/IV resected melanoma treated with nivolumab + ipilimumab vs nivolumab in the phase 3 CheckMate 915 study (NCT03068455) was retrospectively evaluated for ctDNA status and level, using a tumor-guided, pt-specific panel of up to 200 variants (Invitae Personalized Cancer MonitoringÔ). Kaplan-Meier and Cox regression models were used to evaluate the association between ctDNA status and recurrence-free survival (RFS), alone and combined with baseline clinical factors and biomarkers, including interferon-γ, TMB, tumor PD-L1, CD8+ T cells, tumor thickness, ulceration, and lymph node involvement. Overall pre-treatment ctDNA prevalence was ∼16% (95% CI: 14%–18%) and similar across most baseline demographics. A trend of greater ctDNA+ prevalence in higher stage III substages of melanoma was observed (IIIB = 11% [35/333], IIIC = 18% [110/596], IIID = 41% [13/32]). Pre-treatment ctDNA was associated with an increased risk of recurrence (HR 1.87, 95% CI: 1.48–2.36; Table). No significant interaction between ctDNA status and treatment arm was seen (ratio of hazard ratios: 0.99, 95% CI: 0.63–1.57). Patients with ctDNA present exhibited a greater rate of recurrence, seen as early as week 13 of therapy (Table). Distant metastasis-free survival results were similar. In composite analyses, improved RFS prediction was observed after combining ctDNA with clinical factors and biomarkers.Table: 788ORFS probability by pre-treatment ctDNA status in CheckMate 915 treated patientsStatusNumber of pts (n)13-week RFS (95% CI)12-month RFS (95% CI)24-month RFS (95% CI)ctDNA+18372.3% (65–78.3)58.4% (50.7–65.3)45.5% (37.9–52.8)ctDNA-94491.7% (89.7–93.3)75.1% (72.1–77.8)65.4% (62.1–68.4) Open table in a new tab Pre-treatment ctDNA was associated with increased risk of early recurrence across treatment arms. ctDNA is a useful biomarker for combined analyses predicting outcome for adjuvant melanoma.

Comparing single-target and multitarget approaches for postoperative circulating tumour DNA detection in stage II-III colorectal cancer patients.

Circulating tumour DNA (ctDNA) detection for postoperative risk stratification in cancer patients has great clinical potential. However, low ctDNA abundances complicates detection. Multitarget (MT) detection strategies have been developed to increase sensitivity. Yet, empirical evidence supporting performance gains of MT vs. single‐target (ST) strategies in a postoperative setting is limited. We compared ctDNA detection in 379 paired plasma samples from 112 stage II–III colorectal cancer patients by ST digital PCR and MT sequencing of 16 patient‐specific variants. The strategies exhibited good concordance (90%, Cohen's Kappa 0.79), with highly correlated ctDNA quantifications (Pearson r = 0.985). A difference was observed in ctDNA detection preoperatively (ST 72/92, MT 88/92). However, no difference was observed immediately after surgery in recurrence (ST 11/22, MT 10/22) or nonrecurrence (both 2/34) patients. In serial samples, detection was similar within recurrence (ST 13/16, MT 14/16) and nonrecurrence (ST 3/49, MT 1/49) patients. Both approaches yielded similar lead times to standard‐of‐care radiology (ST 4.0 months, MT 4.1 months). Our findings do not support significant performance gains of the MT strategy over the ST strategy for postoperative ctDNA detection.

Clinical implementation of plasma cell-free circulating tumor DNA quantification by digital droplet PCR for the monitoring of Ewing sarcoma in children and adolescents.

BackgroundTreatment stratification and response assessment in pediatric sarcomas has relied on imaging studies and surgical/histopathological evidence of vital tumor cells. Such studies and evidence collection processes often involve radiation and/or general anesthesia in children. Cell-free circulating tumor DNA (ctDNA) detection in blood plasma is one available method of so-called liquid biopsies that has been shown to correlate qualitatively and quantitatively with the existence of vital tumor cells in the body. Our clinical observational study focused on the utility and feasibility of ctDNA detection in pediatric Ewing sarcoma (EWS) as a marker of minimal residual disease (MRD).Patients and methodsWe performed whole genome sequencing (WGS) to identify the exact breakpoints in tumors known to carry the EWS-FLI1 fusion gene. Patient-specific fusion breakpoints were tracked in peripheral blood plasma using digital droplet PCR (ddPCR) before, during, and after therapy in six children and young adults with EWS. Presence and levels of fusion breakpoints were correlated with clinical disease courses.ResultsWe show that the detection of ctDNA in the peripheral blood of EWS patients (i) is feasible in the clinical routine and (ii) allows for the longitudinal real-time monitoring of MRD activity in children and young adults. Although changing ctDNA levels correlated well with clinical outcome within patients, between patients, a high variability was observed (inter-individually).ConclusionctDNA detection by ddPCR is a highly sensitive, specific, feasible, and highly accurate method that can be applied in EWS for follow-up assessments as an additional surrogate parameter for clinical MRD monitoring and, potentially, also for treatment stratification in the near future.

CtDNA for Risk of Recurrence Assessment in Patients Treated with Neoadjuvant Treatment: A Systematic Review and Meta-analysis.

We wanted to investigate the association between circulating tumor DNA (ctDNA) detection at baseline, during and after neoadjuvant treatment, after surgery, and recurrence, in patients with nonmetastatic cancer. In this systematic review and meta-analysis, we included studies that investigated patients undergoing neoadjuvant treatment for nonmetastatic cancer and provided recurrence indices stratified for ctDNA status at the following timepoints: baseline, during treatment, posttreatment, and postsurgery. Study quality was reported with the Newcastle-Ottawa scale, REMARK checklist, and GRADE approach. PubMed, Embase, Cochrane Library, and Web of Science were our data sources (inception to 3 June 2021). The main outcome was risk of recurrence. We identified ten studies including 727 patients with rectal, breast, gastric, and bladder cancer. All studies reported posttreatment ctDNA analysis, while seven, four, and six reported baseline, during treatment, and postsurgery ctDNA analysis, respectively. ctDNA detection was associated to recurrence across all timepoints [baseline: risk ratio (RR) 2.86, 95% confidence interval (CI) 1.33-6.14, during treatment: RR 3.81, 95% CI 2.09-6.92, posttreatment: RR 4.29, 95% CI 2.79-6.60, postsurgery: RR 8.03, 95% CI 3.16-20.43]. Heterogeneity was low to moderate. This meta-analysis of observational studies found that ctDNA detection in patients undergoing neoadjuvant treatment for nonmetastatic cancer was associated with recurrence. A stronger association was evident in posttreatment and postsurgery timepoints. However, some studies reported low negative predictive value (NPV) of pathological complete response, showing that ctDNA-detection-guided escalation and de-escalation studies following neoadjuvant treatment regimens are needed before its role as a treatment guidance can be affirmed.

TP53 mutations in Pap test DNA are a potential biomarker of ovarian cancer risk in high-risk women (226)

<b>Objectives:</b> The use of Pap tests as a molecular diagnostic for high-grade serous ovarian carcinoma (HGSOC) is a promising concept, but studies to detect tumor DNA report limited sensitivity. <i>TP53</i> somatic mutations from clonal expansions in non-neoplastic tissue can be detected in Pap tests and blood and could potentially be utilized for cancer risk assessment. We aimed to determine whether <i>TP53</i> somatic mutations in blood and Pap test DNA are associated with HGSOC in women with and without germline <i>BRCA1</i> or <i>BRCA2 (BRCA)</i> mutations. <b>Methods:</b> Peripheral blood and Pap samples were collected from 30 women with HGSOC (10 with a germline <i>BRCA</i> mutation) and 40 women without cancer (20 with a germline <i>BRCA</i> mutation) who underwent gynecologic surgery at the University of Washington. Genomic DNA from buffy coats and Pap cell pellets was used for ultra-deep sequencing of the <i>TP53</i> coding region (mean depth ~3300x) using duplex sequencing for error correction. Mutations were characterized based on pathogenicity and presence in the UMD <i>TP53</i> Cancer Database. Mutation frequencies (total coding mutations divided by nucleotides sequenced) were compared between groups using t-tests. Fitted linear regression models were used to explore age-related trends, and logistic regression models were used to predict cancer with adjustments for age. <b>Results:</b> Total 436 coding <i>TP53</i> mutations were identified in DNA from Pap tests, and 262 mutations were identified in the DNA from blood. Pap tests demonstrated an overall higher <i>TP53</i> mutation frequency in patients with HGSOC than in those without cancer (2.0x10^-6 vs 1.4x10^-6, p=0.03), but this effect was driven by and limited to patients with <i>BRCA</i> mutations (2.4x10^-6 vs 1.4x10^-6, p=0.01). Women with <i>BRCA</i> mutations and HGSOC also demonstrated a greater frequency of mutations affecting the DNA-binding domain, predicted to be pathogenic, and frequently found in cancer (referred to as ‘cancerlike') (all p<0.05). Cancer-like mutations increased with age in Pap test samples from women without cancer (p<0.01, R²=0.23) but not in patients with HGSOC. Age-adjusted logistic regression confirmed that cancer-like <i>TP53</i> mutation frequency was associated with HGSOC in patients with germline <i>BRCA</i> mutations (OR: 4.2, 95% CI: 1.2-14.3). There were no significant differences between HGSOC and non-cancer cases when examining <i>TP53</i> mutations in blood samples. Among 22 cases with known <i>TP53</i> tumor mutations, the tumor mutation was identified in six Pap samples (27%) and zero blood samples. <b>Conclusions:</b> The identification of HGSOC-specific mutations in Pap tests, even with highly accurate DNA sequencing methods, has limited sensitivity for cancer detection. However, Pap tests can detect abundant low-frequency cancer-like <i>TP53</i> mutations at a higher rate in women with germline <i>BRCA</i> mutations and HGSOC compared to women without cancer. Somatic <i>TP53</i> mutation frequency using age-related thresholds should be further explored as a biomarker for ovarian cancer risk in high-risk women.

Clinical Validity of Tumor-Informed Circulating Tumor DNA Analysis in Patients Undergoing Surgery of Colorectal Metastases.

Accurate biomarkers to monitor tumor load and response in metastatic colorectal cancer patients undergoing surgery could optimize treatment regimens. This study explores the clinical validity of tumor-informed quantification of circulating tumor DNA in blood using ultra-deep sequencing. Resection specimens from 53 colorectal cancer patients were analyzed for tumor-specific mutations in 15 genes. These mutations were used to measure the presence of circulating tumor DNA in preoperatively collected plasma samples using hybrid capture-based sequencing. Additional postoperative measurements were performed one week after surgery in sixteen patients. The study was conducted at the Radboud university medical center. A total of 53 colorectal cancer patients undergoing surgery of metastases were included. The detection of circulating tumor DNA. At least one tumor-specific mutation was detected in all tumor samples. In preoperative plasma samples circulating tumor DNA was detected in 88% (37/42) of systemic treatment-naïve patients and in 55% (6/11) of patients who received preoperative chemotherapy. More specifically, in 0% (0/3) of cases with a subtotal or partial pathological response and in 75% (6/8) of cases without a pathological response in the resection specimen (p = 0.06). In postoperative plasma samples circulating tumor DNA was detected in 80% (4/5) of patients with an incomplete resection and in 0% (0/11) of those with a complete resection (p = 0.003). The study was limited by the heterogeneity of the cohort and the small number of postoperative plasma samples. These data indicate that tumor-informed circulating tumor DNA detection in plasma of patients undergoing surgery for metastatic colorectal cancer is feasible and may have clinical value in response monitoring and predicting residual disease. Prospective studies are needed to establish the clinical utility of circulating tumor DNA analysis to guide treatment decisions in these patients. See Video Abstract at http://links.lww.com/DCR/B990.

Spatiotemporal genomic analysis reveals distinct molecular features in recurrent stage I non-small cell lung cancers.

Stage I non-small cell lung cancer (NSCLC) presents diverse outcomes. To identify molecular features leading to tumor recurrence in early-stage NSCLC, we perform multiregional whole-exome sequencing (WES), RNA sequencing, and plasma-targeted circulating tumor DNA (ctDNA) detection analysis between recurrent and recurrent-free stage I NSCLC patients (CHN-P cohort) who had undergone R0 resection with a median 5-year follow-up time. Integrated analysis indicates that the multidimensional clinical and genomic model can stratify the prognosis of stage I NSCLC in both CHN-P and EUR-T cohorts and correlates with positive pre-surgical deep next generation sequencing (NGS) ctDNA detection. Increased genomic instability related to DNA interstrand crosslinks and double-strand break repair processes is significantly associated with early tumor relapse. This study reveals important molecular insights into stage I NSCLC and may inform clinical postoperative treatment and follow-up strategies.

Abstract 2232: Preliminary results for a novel single extracellular vesicle assay for early lung cancer: The power of co-localized detection of surface biomarkers

Abstract Introduction: Screening for lung cancer (LC), the leading cause of cancer deaths, with helical computerized tomography lowers mortality but uptake is poor. Investigations into new approaches such as using circulating tumor cells and circulating tumor DNA for LC detection have soared in the last decade. However, the low abundance of these targets has limited the performance of these approaches as screening tools. We hypothesize that co-localization of biomarkers on the surface of individual extracellular vesicles (EVs), which are shed into the circulation by cancer cells, may lead to development of a blood test for early stage LC. We evaluated the potential of our approach in detecting early stage LC in clinical samples. Methods: EVs were purified from plasma using size-exclusion chromatography and immunoaffinity capture, and biomarkers co-localized on the EV surface were detected with proximity ligation qPCR. We used antibody combinations comprising 1 capture antibody and 2 oligonucleotide-tagged detection antibodies, recognizing 1, 2 or 3 unique biomarkers. We evaluated this approach by testing plasma samples from early stage I/II lung adenocarcinoma (LUAD) patients (15 smokers, 19 non-smokers), late stage III/IV LUAD patients (16 smokers, 18 non-smokers), and healthy donors (34 smokers, 33 non-smokers). Samples were from one vendor, processed using a standardized protocol. LUAD samples were sourced from a cancer research center and healthy samples from a primary care facility. PCR cycle threshold (Ct) values were generated for each combination and data was evaluated using univariate analysis. Results: Combinations recognizing 3 biomarkers were better in detecting all stages of LUAD (AUC=0.83, 95% CI 0.77-0.90), as compared to combinations recognizing 2 biomarkers (AUC=0.71, 95% CI 0.63-0.80) or 1 biomarker (AUC=0.50, 95% CI 0.35-0.55), demonstrating greater accuracy with an increasing number of co-localized biomarkers. In detecting LUAD (all stages) at a specificity of 0.80 (95% CI 0.69-0.88), sensitivity improved as the number of co-localized biomarkers increased from 1 (0.08, 95% CI 0.03-0.18) to 2 (0.60, 95% CI 0.48-0.72) to 3 (0.76, 95% CI 0.65-0.86). In detecting early stage I/II LUAD, the most effective combination used 3 biomarkers (STn, MUC1, CEACAM6) and had a sensitivity of 0.56 (95% CI 0.38-0.73). Conclusions: These preliminary data highlight the potential of detecting biomarkers co-localized on the surface of single EVs as an effective tool for early stage LC detection, and the benefit of using 3 biomarkers simultaneously. Despite inherent challenges associated with commercial samples, our finding that detection of co-localized EV surface biomarkers distinguished LUAD is promising. Additional studies with LC cohorts beyond LUAD are underway to refine combinations and independently validate our assay for early stage LC detection. Citation Format: Daniel P. Salem, Laura T. Bortolin, Sanchari Banerjee, Kelly M. Biette, Delaney M. Byrne, Anthony D. Couvillon, Peter A. Duff, Jonian Grosha, Daniel Gusenleitner, MacKenzie Sadie King, Christopher R. Sedlak, Ibukunoluwapo O. Zabroski, Karen Copeland, Emily S. Winn-Deen, Eric K. Huang, Christine D. Berg, Joseph C. Sedlak. Preliminary results for a novel single extracellular vesicle assay for early lung cancer: The power of co-localized detection of surface biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2232.

Abstract 5114: Ultra-sensitive detection of minimal residual disease (MRD) through whole genome sequencing (WGS) using an AI-based error suppression model in resected early-stage non-small cell lung cancer (NSCLC)

Abstract Background: Early detection of recurrence and monitoring of MRD post-surgery is critical for clinical decision-making to tailor adjuvant therapy. In early-stage NSCLC, circulating tumor DNA (ctDNA) detection is especially challenging, requiring highly sensitive and specific assays. Therefore, we used a WGS approach (MRDetect) for ultra-sensitive ctDNA detection in NSCLC patients (pts) undergoing curative surgery. Methods: We conducted a pilot study to evaluate the MRDetect approach in serial plasma samples (including pre-surgery, post-surgery and follow-up [f/u] timepoints) from resected stage IB-IIIA NSCLC pts. Pts underwent routine surveillance by computed tomography scans. ctDNA was extracted from ~1mL plasma. MRDetect uses WGS by a tumor-informed approach (sequencing coverage 40x for tumor, 20x for plasma DNA) combined with AI-based error suppression models (trained and calibrated with a non-cancer cohort, n=17) to increase the signal to noise ratio for precise ctDNA detection, and improve the accuracy of readouts especially for low tumor burden scenarios. The assay reports the detection and quantification of ctDNA burden in blood with a prognostic value for risk of recurrence. The ability of the assay to predict recurrence from a single sample, taken at the clinical landmark point (median 1.6 mths post-surgery, range 0.1-6.5) was evaluated. Results: Overall, 52 NSCLC pts were enrolled (n=88 plasma samples) with median clinical f/u of 32.6 mths (range 3.1-98.6). There were 43 pts with post-surgery landmark samples, with median age 62 years, 70% were male, 79% were adenocarcinoma and 49% were EGFR mutated. 26% were stage IB and 37% each were stage II and III. There were 15/18 (sensitivity 83%) pts with confirmed radiological recurrence in which MRDetect was positive, including 6/7 (86%) EGFR mutated pts. The median RFS in MRDetect positive pts was 15.2 mths (range 3.7-33.4). Among 25 pts with no recurrence (median f/u 25.6 mths), MRDetect reported 4 pts to be MRD positive (specificity 84%). These results were consistent between EGFR mutated (sensitivity 86%, specificity 86%) and wildtype pts (sensitivity 82%, specificity 82%). For longitudinal samples (n=17 pts), negative ctDNA was associated with absence of recurrence in 14/15 pts (specificity 93%). At the AACR meeting, results from a planned larger validation study will be presented. Conclusion: Using a robust WGS implemented AI-based computational platform (MRDetect), we demonstrate high sensitivity and specificity detection of MRD in both EGFR mutated and wildtype NSCLC. With an increasing number of therapeutic options in the adjuvant setting for NSCLC, an ultra-sensitive MRD assay has the potential to facilitate personalized clinical decision-making for tailoring both the need and choice of adjuvant therapies. Citation Format: Aaron C. Tan, Stephanie P. Saw, Gillianne G. Lai, Kevin L. Chua, Angela Takano, Boon-Hean Ong, Tina P. Koh, Amit Jain, Wan Ling Tan, Quan Sing Ng, Ravindran Kanesvaran, Tanujaa Rajasekaran, Sunil Deochand, Dillon Maloney, Danielle Afterman, Tomer Lauterman, Noah Friedman, Imane Bourzgui, Nidhi Ramaraj, Zohar Donenhirsh, Ronel Veksler, Jonathan Rosenfeld, Ravi Kandasamy, Iman Tavassoly, Boris Oklander, Asaf Zviran, Wan-Teck Lim, Eng-Huat Tan, Anders J. Skanderup, Mei-Kim Ang, Daniel S. Tan. Ultra-sensitive detection of minimal residual disease (MRD) through whole genome sequencing (WGS) using an AI-based error suppression model in resected early-stage non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5114.

Abstract 2144: Holistic sampling of clonal dynamics using cfDNA in lung TRACERx

Abstract Background: Minimal residual disease (MRD) detection using liquid biopsy has the potential to improve patient outcomes in non-small cell lung cancer (NSCLC). Liquid biopsy may also provide representative clonal sampling through the disease course, but current clonal deconvolution methods are ineffective in plasma samples with &amp;lt;1% tumor content, common in the localised or MRD setting. Methods: We analysed 1071 plasma samples from 198 TNM I-III NSCLC patients in TRACERx who underwent multiregion exome sequencing of primary tumor and relapse tissue, with 416 standard of care surveillance scans. Seventy-four patients suffered a relapse. We targeted a median of 200 tumor-specific mutations per patient consisting of clonal and subclonal variants and sequenced cell-free DNA (cfDNA) using anchored multiplex PCR to a median unique depth of 2230X with 5 supporting duplicates. We used library-specific trinucleotide background models to call MRD. We developed ECLIPSE (Extraction of CLonality from LIquid bioPSiEs), to perform formal clonal deconvolution in &amp;lt;1% purity plasma samples by leveraging copy number and mutation clone identities from tumor tissue. Results: Median MRD lead time was 119 days (range 0-1137) in patients with pre-operative circulating tumor DNA (ctDNA) detection. In the 13% of relapse patients lacking pre-operative ctDNA detection the median MRD lead time was 0 days (range 0-589). MRD lead times positively correlated with clonal ctDNA fraction doubling times (DTs). Cancer cell fractions (CCFs) of subclones estimated in plasma with ECLIPSE and tissue collected concurrently were proportional (P &amp;lt; 0.001, R2 = 0.6). Subclonal mutations that would appear clonal in single biopsies could be separated from true clonal mutations using their CCF in plasma (P &amp;lt; 0.001, OR = 0.44), distinguishing clonal from subclonal mutations, where the latter are unlikely to represent effective therapeutic targets. Where cfDNA and tissue was available at relapse we detected 28/29 metastatic tissue subclones in cfDNA with an additional 8 cfDNA-unique subclones. These subclones were more frequently estimated as present in only a subset of metastatic cells using cfDNA (P = 0.008, OR = 5.5) consistent with localisation to unsampled metastatic sites. Metastatic competent subclones had higher CCFs in pre-operative plasma (P &amp;lt; 0.001, OR = 4.5). Shifts in clonal dynamics were concurrent with treatment. Finally, patients with cfDNA-detected polyphyletic metastatic seeding had shorter disease-free survival than those with monophyletic seeding (HR = 2.89, 95% CIs 1.46-5.73). Conclusions: Tumor-informed anchored multiplex PCR most commonly detected MRD before clinical relapse and allowed determination of clonal ctDNA DT. Using ECLIPSE, plasma samples of &amp;lt;1% purity allow formal measurements of clonal dynamics from diagnosis to relapse, which impacts patient outcome and has the potential to guide personalised medicine. Citation Format: Alexander Mark Frankell, Christopher Abbosh, Aaron Garnett, Judit Kisistok, Thomas Harrison, Morgan Weichert, Abel Licon, Selvaraju Veeriah, Bob Daber, Mike Moreau, Aamir Shahpurwalla, Aaron Odell, Adrian Chesh, Kevin Litchfield, Emilia Lim, Daniel E. Cook, Clare Puttick, Maise Al-Bakir, Fabio Gomes, Akshay Patel, Lizi Manzano, Paula Roberts, Ariana Huebner, Nicolas Carey, Joan Riley, Todd Druley, Jacqui A. Shaw, Nicholas McGranahan, Mariam Jamal-Hanjani, Josh Stahl, Nicolai Birkbak, the Lung TRACERx consortium, Charles Swanton. Holistic sampling of clonal dynamics using cfDNA in lung TRACERx [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2144.

The EHA Research Roadmap: Malignant Lymphoid Diseases.

The EHA Research Roadmap: Malignant Lymphoid Diseases.

Leveraging personalized circulating tumor DNA (ctDNA) for detection and monitoring of molecular residual disease in high-risk melanoma.

9579 Background: High-risk melanoma has variable prognosis. Adjuvant immuno- (IO) and targeted therapy (TT) are approved for stage III-IV resected disease. However, a significant proportion of patients (pts) are cured by local treatment alone or relapse despite adjuvant therapy. Liquid biopsy with ctDNA assays have been used to predict response to treatment and identify pts at higher risk of progression/death. Personalized ctDNA assays are a highly sensitive approach that may enhance upfront risk stratification and early detection of relapse. Methods: Serial ctDNA Monitoring as a predictive Biomarker in advanced neoplAsms (SAMBA) is a Princess Margaret prospective ctDNA kinetics study (NCT03702309) in high-risk melanoma pts. Plasma is collected pre-op (pre-local treatment, if feasible), post-op (after surgery), and every 3-6 months (m) until radiological progressive disease (rPD). Personalized amplicon based NGS assays by Inivata (RaDaR) were used to detect somatic variants in ctDNA identified through whole-exome sequencing of matched tumor tissue. Progression free survival (PFS) and overall survival (OS) from the time of surgery were estimated with the Kaplan Meier and compared with the log-rank test. Results: As of December 2021, 82 of 100 planned pts have been enrolled. A total of 191 samples from 47 pts have been analyzed. Median age was 66 years (27-87), 33 were male (70%). Seven (15%), 30 (64%) and 10 (21%) were stage II/III/IV respectively. All pts had surgery and 8 (17%) adjuvant radiation. No systemic therapy was given to 11 pts (23%); 30 (64%) had IO and 6 (13%) TT. rPD occurred in 13 pts (28%). Median follow up was 24 months. A median of 48 variants were included in the personalized ctDNA panel design (35-52). ctDNA was detected (ctDNA+) at any time point in 12/47 pts (26%), of which 5/12 (42%) were BRAF and NRAS wt on tissue. Median PFS was 4.9 months (m) for ctDNA+ pts and not reached (NR) for ctDNA- pts at post-op (HR = 2.71 CI 0.60-12.31, p = 0.179). Median OS was 23.1 m vs NR in ctDNA+ vs ctDNA- pts (HR = 8.9, CI 1.45-54.77, p = 0.004). Two ctDNA+ pts had neoadjuvant IO and became ctDNA- before surgery. One, free of disease after 12 m, had ctDNA- in 4 follow up samples. The other pt was ctDNA+ in the post-op sample and relapsed within 3 m. Four of 45 (9%) pts had ctDNA+ at post-op. Two of them, including a pt who had neoadjuvant IO, did not receive adjuvant therapy and had rPD within 3 m. The other 2 pts received adjuvant IO; ctDNA cleared and pts remain free of disease at 12 and 34 m. Three pts with rising ctDNA over time experienced rPD after a median of 4 m (2-7). Conclusions: Personalized ctDNA analysis with RaDaR may improve risk of death stratification and selection of pts who could benefit from adjuvant treatment. Detection of ctDNA may precede rPD. Follow-up will continue in pts with rising ctDNA who have not yet had rPD. Pts accrual and sample collection are ongoing, and additional data will be presented. Clinical trial information: NCT03702309.

Clinician utilization of a plasma-only, multiomic minimal residual disease (MRD) assay in 2,000 consecutive patients with colorectal cancer (CRC).

e15586 Background: Circulating tumor DNA (ctDNA) for detection of MRD in resected CRC is a prognostic factor for recurrence. However, current utilization of available commercial tests has not been investigated. We report real world clinician use of a validated, plasma-only, multiomic MRD assay (Guardant Reveal) in a large unselected CRC cohort. Methods: Results from Guardant Reveal MRD testing ordered for clinical care in the US were retrospectively queried for the first 2,000 consecutive CRC cases. Pts could be enrolled in a post-operative program to inform adjuvant treatment (PostOP, up to 3 tests within 3-16 weeks post-resection) or a surveillance program (SP) for recurrent tests post-treatment. Pts could have tests across both programs and/or one-time tests. All subsequent tests for the first 2,000 patients were included for analysis (data cut-off: 1/15/2022). Recurrent programs were analyzed for stage (stg) II/III only. Clinical factors were derived from test requisition forms. Results: 2681 tests from 1993 pts were analyzed; 7 pts were excluded due to missing cancer stage. Median age was 64 years (range: 21-65), 55% were male, most (94%) had stg II/III disease (Table). ctDNA was detected in 25% of all pts; detection increased with stage (Table) as expected. Among stg II/III pts, 330 (21%) had only PostOP test/s, 950 (51%) SP only, 54 (3%) had both PostOP and SP; the remainder had one-time test/s outside defined programs. In Stg II/III pts with &gt;1 PostOP test (26%/17% respectively), ctDNA was detected in 102/384 (27%), of whom 72% had it detected on the first test. The median time from surgery to first result was 5 weeks (10 weeks for a second result). 95% of all stg II/III pts had results from PostOP testing before week 12 post-resection. In Stg II/III pts with &gt;1 SP test (47%/54% respectively), ctDNA was detected in 244/1024 (24%), of whom 87% had it detected on a first test. The average time from date of surgery to first surveillance test was 305 days (median: 489, range:51-4618). Conclusions: ctDNA detection rates by a plasma-only multiomic MRD assay in this large CRC clinical cohort are similar to published rates. ctDNA orders by clinicians were most frequent in Stg II/III surveillance settings followed by PostOp, consistent with the population size of eligible patients for PostOp vs. Surveillance use-case. Importantly, nearly all pts tested PostOP had results prior to 12 weeks post-resection, which may inform adjuvant therapy decisions. These findings should be correlated with clinical outcomes to improve the utilization and utility of MRD testing in adjuvant management of CRC. [Table: see text]

Circulating tumor DNA (ctDNA) detection of molecular residual disease (MRD) as a potential biomarker in localized soft tissue sarcoma (STS).

11547 Background: Surgery and (neo)adjuvant radiotherapy are the mainstay curative treatments for localized STS. Despite treatment, approximately 50% of STS patients (pts) experience metastatic relapse and routine use of adjuvant systemic therapy (AST) remains controversial. The presence of ctDNA following curative treatment of STS is a potential biomarker for MRD and may identify patients who benefit from AST. Given the genomic heterogeneity of STS, a histology-agnostic approach to ctDNA detection in this population is desirable. Methods: Pts with localized, high risk (size ≥ 5cm, grade ≥ 2) disease were enrolled prior to (neo) adjuvant radiotherapy and surgery. Blood for ctDNA was collected at diagnosis; post-radiotherapy, post-surgery and every 3 months for up to 2 years. Whole exome sequencing (WES) of archival tumor- and matched buffy coat-DNA were carried out to identify somatic variants. Personalized and tumor-informed, multiplex PCR next generation sequencing-based ctDNA assay (Signatera™ assay) was performed on plasma obtained at the serial timepoints. A sample level positive call required ≥ 2 variants above a confidence calling threshold. Absolute ctDNA levels were expressed as mean tumor molecules per milliliter (MTM/ml) of plasma, based on variant allele frequencies and quantity of cell free DNA. Standard radiologic surveillance (every 3 months) was performed following surgery. The primary endpoint was a ctDNA detection rate of 70% at diagnosis. Secondary endpoints included MRD detection and correlation of ctDNA levels with disease relapse. Results: Seventy-six plasma samples from 10 pts [8 males and 2 females; median age 64 years (range 46–84)] were obtained prospectively. STS subtypes were undifferentiated pleomorphic sarcoma (n = 4), myxofibrosarcoma (n = 2), dedifferentiated liposarcoma (n = 2), myxoid liposarcoma (n = 1), and pleomorphic liposarcoma (n = 1). All tumors successfully underwent WES with adequate data quality for Signatera™ assay design. The personalized ctDNA assay was performed on a median of 7 plasma samples per patient (range: 5 – 10). ctDNA was detected in 7 pts (70%) at diagnosis, with median ctDNA level of 1.6 MTM/ml (range: 0.2 – 137.8), achieving the study primary endpoint. Immediate post-surgery samples were negative in all pts. However, ctDNA was detected in 2 out of 2 pts who developed metastatic disease during follow-up. Conclusions: Personalized tumor-informed ctDNA assays in localized high-risk STS at diagnosis are feasible. In this series, all patients had undetectable levels of ctDNA post-surgery and patients who experienced disease relapse demonstrated a detectable rise in ctDNA levels. Further interrogation of this approach for detection of post-treatment MRD as a possible biomarker of benefit from AST is ongoing.