Detection Of Molecular Residual Disease Research Articles

Quantification of Tumor-Associated Cell-Free HPV DNA in Surgical Drain Fluid Equates to Molecular Residual Disease and Correlates with High-Risk Pathology

<h3>Purpose/Objective(s)</h3> Multi-institutional efforts aimed at safely de-escalating treatment in human papillomavirus (HPV) associated oropharyngeal squamous cell carcinoma (OPC) patients rely on subjective pathology for risk stratification. While plasma HPV assays are emerging for post-treatment disease surveillance, they have poor sensitivity to detect postoperative molecular residual disease (MRD). Here we present a liquid biopsy assay of the serosanguinous fluid removed from surgical drains, proximal to the surgical site, to sensitively measure postoperative MRD. We hypothesize that HPV quantified in surgical drain fluid (SDF), 1) represents post-operative regional MRD, and 2) could improve patient risk-stratification to guide precision radiotherapy. <h3>Materials/Methods</h3> We collected SDF at 24-48 hours post-surgery for cell-free (cf) DNA extraction. Our 87-patient head and neck cancer cohort included 74 HPV(+) and 8 HPV(-) OPCs, and 5 benign tumors. Paired plasma and tumor samples were collected from approximately half of all patients. We used the HPV16 E6T2aE7 plasmid to compare our Taqman quantitative PCR (qPCR) to digital droplet PCR (ddPCR). We validated HPV(+) OPC PCR results using next-generation HPV sequencing (NGS) in 9 patients. Then we correlated SDF HPV copies to extranodal extension (ENE), AJCC 8^th edition pathological stage, smoking pack-years, recurrence-free survival (RFS), and composite risk scores derived from OPC de-escalation clinical trials. Statistical methods included Fisher's Exact test, Mann-Whitney (MW) test, Kruskal-Wallis (KW) test, and Kaplan-Meier (KM) analysis. <h3>Results</h3> Our HPV qPCR assay's limit of detection was 9.7 copies, similar to what we saw with ddPCR. HPV genotyping was concordant between NGS and PCR, and all SDF cfDNA samples with nondetectable (ND) HPV via PCR were ND via NGS. HPV was detected in 63 of 74 (85%) SDF samples compared to only 7 of 48 (15%) paired plasma samples. Median HPV copies in ENE+ patients' SDF was 15-fold higher than SDF from ENE- patients (P = 0.002). HPV copies also increased with increasing nodal (P = 0.01) but not primary tumor stage (P = 0.4), suggesting post-operative SDF HPV load reflects invasive nodal disease and is a proxy for regional MRD. When we stratified our cohort according to high-risk and low-risk scores drawn from four clinical trials (RTOG 9501, EORTC 22931, ECOG 3311, MC1273), SDF HPV load was consistently higher in patients with trial-defined high-risk pathology (mean AUC = 0.73). Lastly, KM RFS analysis of HPV-high and HPV-low patients, defined by an RTOG 9501-derived SDF classifier, showed HPV-high patients trended toward worse RFS (HR = 3.1; P = 0.1). <h3>Conclusion</h3> Liquid biopsy assessment of SDF, the biofluid most proximal to the nodal environment, enables postoperative regional MRD detection in HPV(+) OPC, and correlates with high-risk nodal pathology and worse RFS. If confirmed, SDF cfDNA analysis could aid traditional pathology and imaging to personalize adjuvant radiotherapy for surgically treated HPV(+) OPC.

Comprehensive Genomic Profiling (CGP)-Informed Personalized Molecular Residual Disease (MRD) Detection: An Exploratory Analysis from the PREDATOR Study of Metastatic Colorectal Cancer (mCRC) Patients Undergoing Surgical Resection.

A majority of patients with metastatic colorectal cancer (mCRC) experience recurrence post curative-intent surgery. The addition of adjuvant chemotherapy has shown to provide limited survival benefits when applied to all patients. Therefore, a biomarker to assess molecular residual disease (MRD) accurately and guide treatment selection is highly desirable for high-risk patients. This feasibility study evaluated the prognostic value of a tissue comprehensive genomic profiling (CGP)-informed, personalized circulating tumor DNA (ctDNA) assay (FoundationOne®Tracker) (Foundation Medicine, Inc., Cambridge, MA, USA) by correlating MRD status with clinical outcomes. ctDNA analysis was performed retrospectively on plasma samples from 69 patients with resected mCRC obtained at the MRD and the follow-up time point. Tissue CGP identified potentially actionable alterations in 54% (37/69) of patients. MRD-positivity was significantly associated with lower disease-free survival (DFS) (HR: 4.97, 95% CI: 2.67–9.24, p < 0.0001) and overall survival (OS) (HR: 27.05, 95% CI: 3.60–203.46, p < 0.0001). Similarly, ctDNA positive status at the follow-up time point correlated with a marked reduction in DFS (HR: 8.78, 95% CI: 3.59–21.49, p < 0.0001) and OS (HR: 20.06, 95% CI: 2.51–160.25, p < 0.0001). The overall sensitivity and specificity at the follow-up time point were 69% and 100%, respectively. Our results indicate that MRD detection using the tissue CGP-informed ctDNA assay is prognostic of survival outcomes in patients with resected mCRC. The concurrent MRD detection and identification of actionable alterations has the potential to guide perioperative clinical decision-making.

Abstract B006: Development of a NF1-MPNST-PDX liquid biopsy model using whole-genome sequencing and quantitative PCR of mouse-derived cell-free DNA

Abstract Background: Malignant Peripheral Nerve Sheath Tumors (MPNST) are aggressive, NF1-associated soft tissue sarcomas with a dismal 40% 5-year survival rate, high rates of disease relapse and metastasis, and limited treatment options. Our lab has generated a collection of patient-derived xenograft (PDX) models, which recapitulate the variety of genetic changes that occur in human NF1-MPNST. Longitudinal analysis of PDX tumor burden in preclinical studies is currently limited to imprecise tumor measurement or expensive and time-consuming imaging modalities such as MRI. These methods of analysis fail to allow for the early detection of tumor formation, the assessment of treatment-response dynamics, the formation of resistant subclones, or the detection of molecular residual disease. Liquid biopsies using plasma-derived cell-free DNA (cfDNA) have been successful in addressing these challenges in the context of multiple solid tumor types including MPNST by our group previously (Szymanski et al. 2021). However, liquid biopsy of cfDNA in the context murine model systems is quite limited, primarily due the challenge of collecting sufficient plasma for analysis. We hypothesize that we can detect and longitudinally track tumor burden in response to treatment using human-tumor-specific quantitative PCR (qPCR) and whole-genome sequencing of sequentially collected PDX-bearing murine plasma to develop a NF1-MPNST-PDX liquid biopsy model. Methods: To address this challenge, we sequentially collected ≈50 µL of murine plasma weekly from two independent groups of NF1-MPNST-PDX-bearing mice and corresponding PDX-free controls for up to six weeks. The first group consisted of six PDX-bearing mice who underwent whole-genome sequence of murine-derived plasma cfDNA and paired PDX tissue. The second group consisted of fifteen NF1-MPNST-PDX-bearing mice and five PDX-free controls; plasma cfDNA samples collected from this group underwent human-specific LINE-1 qPCR, which should only detect human DNA derived from implanted PDX and allow for longitudinal tracking of tumor burden. Results: We serially collected ≈50 µL of plasma weekly from NF1-MPNST-PDX mice with no obvious impact on mouse survival or weight. Mean total cfDNA yield from the terminally collected samples was 1.12 ± 1.02 ng per uL plasma (n=21) for PDX-bearing animals with cfDNA fragments canonically appearing between 70 and 450 bps as determined by electropherogram. Whole-genome sequencing of murine plasma cfDNA revealed broad genomic aneuploidy detectable in NF1-MPNST-PDX plasma which corresponded to alterations present in PDX tissue, including MPNST-specific chromosome 8q gain, corresponding with our earlier findings (Dehner et al. 2021). Human-specific LINE-1 qPCR showed specific enrichment of tumor-derived LINE-1 in NF1-MPNST-PDX-bearing mice compared to PDX-free mice which showed no human LINE-1 enrichment. Conclusion: Our data suggest that PDX plasma cfDNA may be an informative biomarker that can be non-invasively collected and used to track tumor burden in NF1-MPNST-PDX models. Citation Format: Paul A. Jones, Wenjia Feng, Xiaochun Zhang, Peter K. Harris, Taylor Sundby, Jeffrey J. Szymanski, Divya Srihari, Faridi Qaium, Jack F. Shern, Aadel A. Chaudhuri, Angela C. Hirbe. Development of a NF1-MPNST-PDX liquid biopsy model using whole-genome sequencing and quantitative PCR of mouse-derived cell-free DNA [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B006.

684P Liquid biopsy for detection of molecular residual disease and recurrence in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) remains a substantial health burden. Early relapse and recurrent metastasis are some of the primary reasons for the poor prognosis. The recent advent of personalised assays capable to detect circulating tumour DNA (ctDNA) has enabled detection of molecular residual disease and recurrence following curative therapy. We conducted LIONESS, a single-centre prospective cohort study to assess ctDNA in plasma and saliva from HNSCC patients (Stage I-IVb) receiving primary surgery with curative intent. Samples were collected pre- and postoperatively and during clinical follow-up. Whole exome sequencing was performed on formalin-fixed paraffin-embedded tumour tissue to a median depth of 250x. Tumour-specific variants for personalised assay design (RaDaR, Inivata) were selected to analyse serial samples for evidence of molecular residual disease. Based on staging computer tomography images, tumour volumes were correlated to ctDNA and clinical parameters. In 348 longitudinal plasma samples from 46 patients analysed to date, ctDNA was detected to levels ranging from 10.8% variant allele fraction (eVAF) to as low as 0.0005%. ctDNA was detected in 10/10 cases with confirmed clinical recurrences with lead times up to 265 days. ctDNA was also detected in baseline saliva samples from patients with tumours of various anatomical locations. This included a case with two synchronous tumours, one pT4a laryngeal tumour and a second pT1 tumour of the floor of the mouth. Postoperative plasma ctDNA detection originating from the laryngeal tumour was observed 108 days ahead of clinical recurrence. Plasma ctDNA from the small pT1 tumour was not detected preoperatively and remained undetected throughout the patient’s course of management, however, ctDNA was detected at a baseline saliva sample. Work is ongoing for analysis of the full cohort of plasma and saliva samples, which will be presented at the congress. The use of ctDNA measurements in this HNSCC patient cohort has significant potential to guide treatment decisions and improve disease outcome. In future, ctDNA analysis with subsequent ctDNA-guided treatment may reduce morbidity for HNSCC patients.

ESMO recommendations on the use of circulating tumour DNA assays for patients with cancer: a report from the ESMO Precision Medicine Working Group

Circulating tumour DNA (ctDNA) assays conducted on plasma are rapidly developing a strong evidence base for use in patients with cancer. The European Society for Medical Oncology convened an expert working group to review the analytical and clinical validity and utility of ctDNA assays. For patients with advanced cancer, validated and adequately sensitive ctDNA assays have utility in identifying actionable mutations to direct targeted therapy, and may be used in routine clinical practice, provided the limitations of the assays are taken into account. Tissue-based testing remains the preferred test for many cancer patients, due to limitations of ctDNA assays detecting fusion events and copy number changes, although ctDNA assays may be routinely used when faster results will be clinically important, or when tissue biopsies are not possible or inappropriate. Reflex tumour testing should be considered following a non-informative ctDNA result, due to false-negative results with ctDNA testing. In patients treated for early-stage cancers, detection of molecular residual disease or molecular relapse, has high evidence of clinical validity in anticipating future relapse in many cancers. Molecular residual disease/molecular relapse detection cannot be recommended in routine clinical practice, as currently there is no evidence for clinical utility in directing treatment. Additional potential applications of ctDNA assays, under research development and not recommended for routine practice, include identifying patients not responding to therapy with early dynamic changes in ctDNA levels, monitoring therapy for the development of resistance mutations before clinical progression, and in screening asymptomatic people for cancer. Recommendations for reporting of results, future development of ctDNA assays and future clinical research are made.

Abstract 5916: Tumor-informed patient-specific panel outperforms tumor-naïve and tumor-informed fixed panel for circulating tumor DNA (ctDNA)-based postoperative monitoring of non-small cell lung cancer (NSCLC)

Abstract Background: Patient-specific flexible gene panels designed based on whole-exome sequencing (WES) of resected tumor tissues is a promising strategy for ctDNA-based detection of molecular residual disease (MRD) in early-stage NSCLC. Flexible gene panels could potentially overcome the limitations of fixed panels by incorporating more unique genomic regions that might be absent in fixed panels; however, no study has reported a head-to-head comparison of these two approaches in postoperative disease monitoring. In this study, we investigated the clinical utility of a novel Patient-specific pROgnostic and Potential tHErapeutic marker Tracking (PROPHET) tumor-informed ctDNA assay. Using the same set of longitudinal blood samples, we further compared the performance of PROPHET assay with tumor-informed (TI) and tumor-naïve (TN) fixed panels for predicting MRD and prognosis in surgical NSCLC patients. Methods: Fifty-three patients with stage I-III resected NSCLC from the MEDAL study (NCT03634826) with adequate samples and median follow-up of 647 days were analyzed. Matched surgical tumor tissue and blood samples collected at various time points, including before surgery (baseline), 3-days (B) and 1-month (C) postoperative time points before any adjuvant therapy and subsequent follow-up time points (F) were analyzed. PROPHET assay involved four major steps: identify somatic mutations using WES, customized design of a patient-specific panel consisting of 50 single nucleotide variants, ultra-deep unique molecular-identifier-based next-generation sequencing (UMI-NGS) of serial blood samples using the patient-specific panel, and MRD risk prediction. Fixed panel assay of serial blood samples was performed using UMI-NGS with 168 gene panel spanning 273 kb of human genome. Results: At 1-month post-surgery, PROPHET assay accurately predicted MRD-positive cases among relapsed patients (50%, 13/26), whereas all disease-free patients were MRD-negative (100%, 21/21). Three-year prognostication with PROPHET assay at B+C yielded higher sensitivity (59% vs 26% vs 22%), negative predictive value (66% vs 51% vs 50%), and hazard ratio (7.15, 95%CI [3.2-15.9] vs 4.48 [1.9-10.9] vs 5.58 [2.1-14.7]) as compared with TI and TN fixed panel assays. Disease monitoring using PROPHET assay at B/C/F accurately predicted the MRD risk in 70% (21/30) of relapsed patients at a median lead time of 318 days (range: 20-751), whereas TI assay predicted 43% (13/30) at 282 days (range 20-716) and TN assay predicted 37% (11/30) at 282 days (range: 20-634). Conclusion: Patient-specific tumor-informed ctDNA-based postoperative monitoring enables risk stratification at early postoperative settings better than fixed panel, which paves an alternative strategy in the individualized management of surgical NSCLC patients. Citation Format: Kezhong Chen, Haifeng Shen, Shuailai Wu, Pengfei Zhu, Chenyang Wang, Analyn Lizaso, Guannan Kang, Yang Wang, Juan Lv, Shuai Fang, Wenjun Wu, Fujun Qiu, Yuan Sun, Qiang Lu, Heng Zhao, Shannon Chuai, Fan Yang, Zhihong Zhang. Tumor-informed patient-specific panel outperforms tumor-naïve and tumor-informed fixed panel for circulating tumor DNA (ctDNA)-based postoperative monitoring of 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 5916.

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.

Detection of molecular residual disease (MRD) for cancer management: Lessons from longitudinal profiling of NSCLC patients undergoing chemotherapy or targeted therapy.

e15020 Background: Identification of cancer patients who are at ongoing risk of relapse remains of high priority to direct effective adjuvant therapy. Plasma circulating cell-free DNA (cfDNA) analyses offer an unprecedented approach to identify patients with molecular residual disease (MRD) earlier than clinical or radiological evidence, detecting drug resistance, progression and relapse with relatively high sensitivity and specificity. Here we used longitudinal surveillance to profile cfDNA mutational landscape undergoing chemotherapy or EGFR-TKI. We demonstrated how cutting-edge NGS and digital qPCR technologies are defining new subgroups of patients with MRD for individually tailored treatment strategy. Methods: Plasma samples were collected from 8 subjects with lung adenocarcinoma, 5 patients were treated with EGFR-TKI and 3 cases by chemotherapy. Overall, 92 longitudinally collected specimens (duration of 31-143 weeks) were analyzed with a 520-gene ultra-deep NGS platform as well as a digital qPCR assay. Standard-of-care tests of CEA, CA19-9 biomarkers and CT radiography were also performed in parallel. Results: The number of somatic mutations detected ranging from 0 to 52 in a sample and correlated strongly with TMB (tumor mutational burden). The concordance of actionable EGFR mutations (L858R, T790M, Ex19Del) detected by NGS and digital qPCR assays was 100% (n = 49/49). NGS assay detected additional driver mutations in ∼20 non-EGFR genes. Majority of EGFR mutations showed directional decline in allelic fraction (AF) after treatment with EGFR-TKI. Patient progressed following EGFR-TKI were registered to undergo chemotherapy based on real-time mutational profiles and did show good responses and PFS. Longitudinal ultra-deep NGS analyses can help detect and stratify MRD, including non-druggable genetic information that could guide best clinical decision-making. Conclusions: Longitudinal ctDNA surveillance of patients treated with EGFR-TKI by a highly sensitive multi-gene NGS panel enables MRD monitoring and can potentially guide a selection of optimal adjuvant therapies earlier, and provide new surrogate endpoints for early registration of these therapies.

Longitudinal Undetectable Molecular Residual Disease Defines Potentially Cured Population in Localized Non-Small Cell Lung Cancer.

This study confirms the prognostic value of MRD detection in patients with NSCLC after definitive surgery, especially in those with longitudinal undetectable MRD, which might represent the potentially cured population regardless of stage and adjuvant therapy. Moreover, the risk of developing detectable MRD decreased stepwise after 18 months since landmark detection. This article is highlighted in the In This Issue feature, p. 1599.

Impact of Circulating Tumor DNA-Based Detection of Molecular Residual Disease on the Conduct and Design of Clinical Trials for Solid Tumors.

PURPOSEEarlier detection of cancer recurrence using circulating tumor DNA (ctDNA) to detect molecular residual disease (MRD) has the potential to dramatically affect cancer management. We review evidence supporting the use of ctDNA as a biomarker for detection of MRD and highlight the potential impact that ctDNA testing could have on the conduct of clinical trials.METHODSWe searched the literature using MEDLINE (via PubMed) for articles from January 1, 2000, focusing on studies that assessed ctDNA as a predictor of cancer recurrence. Broadly focused searches on ctDNA and cancer were also performed to provide additional background information. www.clinialtrials.gov was searched to identify trials that incorporate ctDNA testing.RESULTSNumerous studies across different cancer types indicate that ctDNA-based MRD detection predicts recurrence with high sensitivity and specificity, and with lead times that precede standard imaging by up to 12 months. Recently, ctDNA testing has started being used to enroll MRD-positive patients at high risk of recurrence into trials, promising gains in statistical power that allow clinical utility to be demonstrated with smaller cohorts. Trials where ctDNA testing based-MRD detection is used to stratify patients into low or high-risk categories for treatment assignment are also ongoing. In addition, there is increasing evidence supporting the use of ctDNA dynamics or clearance as a surrogate end point, which could significantly reduce trial duration.CONCLUSIONctDNA-based trial enrichment across many cancers seems likely to become increasingly common for cost- and time-reduction benefits. Trial efficiency could also benefit from using ctDNA as a surrogate end point, leading to accelerated approval of new therapeutics. A clear demonstration of efficacy from trials that use ctDNA-based MRD detection to assign treatment could transform clinical practice.

Molecular residual disease (MRD) detection with a tissue comprehensive genomic profiling (CGP)-informed personalized monitoring assay: An exploratory analysis of the IMvigor-010 observation arm.

448 Background: There is compelling rationale that detection of MRD following curative therapy may identify patients at high risk of relapse requiring intensified adjuvant therapy. Combining MRD detection with CGP creates an opportunity to offer MRD-guided treatment with precision cancer therapeutics. Here we analyze the observation arm of the IMvigor-010 study to understand the genomics of resected early stage bladder cancer and to validate CGP-informed personalized MRD detection in circulating tumor DNA (ctDNA). Methods: Using the resected tumor, tissue CGP was performed retrospectively with a 300+ gene assay, followed by MRD detection using FoundationOne Tracker (F1T). Briefly, coding, synonymous, and non-coding variants were selected from tumor tissue sequencing using an optimized algorithm that filters out non-tumor derived variants (germline, clonal hematopoiesis derived, sequencing artifacts). Tumor-informed personalized multiplex PCR-next generation sequencing (Natera) assay was designed and used to detect and quantify variant allelic frequency (VAF) in ctDNA from 182 patients. ctDNA levels were reported in mean tumor molecules per mL of plasma. F1T, a tissue-informed personalized monitoring assay, was performed on plasma samples collected at an MRD timepoint a median of 11 weeks post-surgery. Results: At the MRD timepoint, ctDNA was detected in 66/182 (36%). Focusing on the 66 ctDNA-positive patients, 58 had relapsed (88% PPV) at time of analysis. Median disease-free survival (DFS) from randomization was 3 months in ctDNA-positive vs not reached in ctDNA-negative population (HR = 5.7, 95% CI: 3.8-8.6, p &lt;.0001). Median overall survival (OS) was 13 months in ctDNA-positive vs not reached in ctDNA-negative (HR = 5.7, 95% CI: 3.4-9.7, p &lt;.0001). Potentially actionable CGP findings included FGFR2/3 short variants (SVs) and fusions (13%), ERBB2 SVs and amplifications (13%), PIK3CA SVs (20%), CDKN2A SVs and losses (41%) and tumor mutational burden (TMB) ≥10 mutations/Mb (35%). Conclusions: Tissue CGP-informed personalized MRD detection can detect low levels of residual ctDNA in patients with resected early stage bladder cancer, identifying a population with inferior DFS and OS. This technologic approach, synergizing regulatory-grade actionable CGP with ctDNA-based MRD detection, creates new opportunities for precision adjuvant therapy across a range of high-risk cancer types. Clinical trial information: NCT02450331.

Abstract P2-01-10: Detection of ctDNA following surgery predicts relapse in breast cancer patients receiving primary surgery

Abstract Introduction: Identification of Molecular Residual Disease (MRD) by circulating tumour DNA (ctDNA) analysis has the potential to transform the clinical management of patients with early breast cancer. We present results from a proof-of-principle study to assess ctDNA analysis following primary surgery to identify MRD and anticipate which patients are at risk of relapse. Methods: Early breast cancer patients receiving primary surgery for breast cancer (48 total), enrolled in the PlasmaDNA/ITH sample collection studies were included in the analysis. Tumour DNA from FFPE samples was whole exome sequenced to identify patient specific mutations and design personalized Signatera ctDNA assays. Plasma samples were collected pre-surgery (n=31), 1-14 weeks post-surgery and prior to adjuvant therapy (n=48), and following adjuvant chemotherapy (n=36). Cell free DNA was extracted from a total of 144 plasma samples (median volume 3.6ml, range 1.8-4.7ml) and sequenced with Signatera ctDNA assays. Primary objective was to assess whether relapse free survival (RFS) and distant metastasis free survival (DMFS) are worse in patients with ctDNA detected at the post-surgery timepoint compared to those without ctDNA detected. Results: Median age was 50.5 years, 34 had hormone receptor positive HER2 negative (HR+HER2-), 5 HER2 positive and 9 triple negative breast cancer (TNBC), 32 were stage 1-2 and 16 were stage 3-4. At a median follow-up of 60 months post-surgery, 8 patients had relapsed. ctDNA was detected in the single post-surgery timepoint in 29% (14/48) of patients, and detected in 62.5% (5/8) of patients who relapsed. RFS in patients with ctDNA detected at a single post-surgery timepoint was worse than those with no detected ctDNA although it was not statistically significant (Hazard Ratio (HR): 3.7; 95% CI, 0.9-15.6; P=0.07), while ctDNA detection associated with worse DMFS (HR: 5.6; 95% CI, 1.1-29-3; P=0.04). DMFS at 4 years follow-up in those with MRD ctDNA detection was 0.78 (95% CI 0.47-0.92) and those without MRD detection was 0.97 (95% CI 0.80-0.99). In patients with a pre-surgical timepoint (n=31), 64.5% (20/31) had ctDNA detected. Detection of ctDNA at either pre-surgery or post-surgery was associated with worse outcomes compared to no ctDNA detection at both RFS (HR: 7.9; 95% CI, 0.9-64.7; P=0.05) and DMFS (HR: 6.7; 95% CI, 0.8-55.8; P=0.07). Conclusions: In this proof-of-principle study of early-stage breast cancer patients, ctDNA-detected MRD at a single post-surgical timepoint was associated with distant metastasis free survival. The majority of patients with ctDNA detected MRD did not relapse, during the period of follow-up, possibly suggesting activity of adjuvant therapy in these patients. Further assessment is warranted on the prognostic impact of ctDNA MRD detection, and its possible role in adjuvant chemotherapy selection. Citation Format: Isaac Garcia-Murillas, Rosalind J Cutts, Lara Ulrich, Matthew Beaney, Marie Robert, Maria Coakley, Catey Bunce, Giselle WalshCrestani, Sarah Hrebien, Ekaterina Kalashnikova, Hsin-Ta Wu, Scott Dashner, Himanshu Sethi, Alexey Aleshin, Alistair Ring, Alicia Okines, Ian E Smith, Mitch Dowsett, Peter Barry, Nicholas C Turner. Detection of ctDNA following surgery predicts relapse in breast cancer patients receiving primary surgery [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-10.

Comprehensive genomic profiling (CGP)-informed personalized molecular residual disease (MRD) detection: An exploratory analysis from the PREDATOR study of metastatic colorectal cancer (mCRC) patients undergoing surgical resection.

187 Background: Detection of MRD following metastatic liver resection in advanced CRC patients is associated with poor prognosis with high rate of relapse. Nevertheless, there is currently no standard of care to guide further therapy after curative intent surgery. MRD detection has the promise to be implemented into standard of care and guide treatment decision making. Here we establish feasibility of MRD detection using Foundation Medicine’s novel tissue-informed personalized monitoring assay, FoundationOne Tracker (F1T), in mCRC patients undergoing surgical resection with curative intent. Methods: Tissue-based CGP was performed retrospectively on a cohort of 72 patients from the PREDATOR trial. Trackable patient-specific single nucleotide variants were selected using a novel computational approach negating the need for buffy coat sequencing to filter germline variants. Personalized multiplex PCR was used to detect and evaluate prognostic value of ctDNA from plasma collected at MRD timepoint post-surgery (median 27 days, range 8-99.5). Median follow-up of patients in the overall population was 10.7 months (range: 0.9-53.8 months). Survival analyses were performed using the Kaplan-Meier Estimator and Cox regression. Results: Post-surgical F1T analysis was successful on 96% of cases (69/72). CGP analysis revealed at least one driver mutation in 57% of samples (41/72) including KRAS/NRAS (46%) and BRAF mutations (3%). MRD was detected in 45% (31/69) of patients, of which 94% (29/31) had progressed at the time of the data cut. Median progression -free survival (PFS) was 3.2 months (2.1-7.1) in ctDNA-positive vs 28 months (20.9-NA) in ctDNA-negative population (HR 5, CI 2.7-9.3, p&lt;0.001). Median overall survival (OS) was 31.6 months in ctDNA-positive vs not reached in ctDNA-negative group (HR 27, CI 3.6-205, p&lt;0.001). Conclusions: CGP-informed post-operative MRD detection is a strong prognostic biomarker and correlates with survival outcomes in patients with resected mCRC. F1T is a novel and convenient technological approach to MRD detection utilizing highly validated FMI testing to reveal potentially targetable mutations and inform personalized ctDNA monitoring. These results demonstrate the ability of F1T to accurately detect MRD in mCRC patients following surgical resection, without the need for germline sequencing.

A randomized, double-blind, phase III study comparing trifluridine/tipiracil hydrochloride therapy versus placebo in resected colorectal cancer patients who are positive for blood circulating tumor DNA after standard adjuvant therapy (EPOC 1905): ALTAIR trial in CIRCULATE-Japan (trial in progress).

TPS215 Background: Circulating tumor DNA (ctDNA) can be used to predict the risk of recurrence by detecting molecular residual disease (MRD) in patients with colorectal cancer (CRC). Although patients with MRD positive status have extremely high risk of relapse, no standard treatment has been established for these patients after adjuvant chemotherapy. Trifluridine/tipiracil hydrochloride (FTD/TPI) is an oral anti-tumor agent combining thymidine-based nucleoside analogue with a thymidine phosphorylase inhibitor, which presents improved survival in patients with metastatic CRC refractory to fluoropyrimidines. Methods: The ALTAIR trial is a randomized, double-blind, phase III study designed to establish the superiority of FTD/TPI as compared with placebo in patients with resected CRC who show MRD positive status at any time after curative resection. ctDNA testing for screening patients with MRD positive is performed in the observational GALAXY study (UMIN000039205) that is a prospectively conducted large-scale nationwide registry designed to monitor ctDNA status for patient who can undergo curative resection. A personalized tumor-informed assay (Signatera bespoke multiplex-PCR NGS assay) is used for the detection and quantification of ctDNA-based postsurgical MRD. Key eligibility criteria are (a) having undergone radical resection of primary and/or metastatic tumors, (b) a history of standard adjuvant chemotherapy, (c) positive ctDNA status within the previous 3 months at any time postoperatively, and (d) no obvious relapse confirmed by chest, abdominal, and pelvic CT scans. Patients will be randomly assigned in a 1:1 ratio to receive either 6 months of oral FTD/TPI or a matching course of placebo. Randomization is stratified by age (&lt;70 vs. ≥70 years), stage (stage II or lower vs stage III vs. stage IV or M1), primary tumor location (right-sided vs left-sided colon vs rectum), ctDNA status at 1 month (positive vs negative or unmeasurable), and institution. The primary endpoint is disease free survival (DFS). Key secondary endpoints include rate of conversion from positive to negative ctDNA status, overall survival, adverse events, and quality of life. Assuming that the median DFS in the placebo group is approximately 8 months, a total of 240 patients (120 per arm) will provide 80% power to detect an expected DFS hazard ratio of 0.667 at two-sided significance level of 0.05, with an enrollment period of 2 years and a follow-up period of 1 year. This trial is actively accruing across 39 institutions in Japan and Taiwan and opened to recruitment in August 2018. By October 2021, a total of 67 patients have been enrolled. Clinical trial information: JapicCTI-205363/NCT04457297.

Association of circulating tumor DNA dynamics with clinical outcomes in the adjuvant setting for patients with colorectal cancer from an observational GALAXY study in CIRCULATE-Japan.

9 Background: Circulating tumor DNA (ctDNA)-based molecular residual disease (MRD) has the potential to select patients who may benefit more from standard-of-care (SOC) adjuvant chemotherapy (ACT) by accurately assessing recurrence-risk post-surgery and by evaluating ACT efficacy. Here we present an analysis from GALAXY study, an observational study monitoring MRD, to evaluating the association of ctDNA dynamics with a short-term clinical outcome and ACT efficacy. Methods: A personalized tumor-informed assay (Signatera bespoke multiplex-PCR NGS assay) was used for post-surgical MRD detection in colorectal cancer (CRC) patients. Six-month disease-free survival (6M-DFS) rates were analyzed excluding patients enrolled in associated phase III trials (VEGA and ALTAIR). Results: Total 1,365 CRC patients enrolled between June 2020 and April 2021 were included in this analysis; 116 pStage I, 478 pStage II, 503 pStage III, and 268 oligomet resectable pStage IV (16% [42/268] received neoadjuvant chemotherapy). 6M-DFS rate by ctDNA dynamics from 4w to 12w were 98% in ‘negative to negative’ group (N = 618), 59% in ‘negative to positive’ (N = 32), 100% in ‘positive to negative’ (N = 58), and 45% in ‘positive to positive’ (N = 78), with a significant difference between ‘positive to negative’ and ‘positive to positive’ groups with hazard ratio (HR) of 52.3 (95% CI: 7.2-380.5; p &lt; 0.001), with a median follow-up time of 6.6 months. Further, out of 188 patients who were MRD+ at 4w with available MRD status at 12w, 95 received SOC ACT (80/95 received fluoropyrimidine [FP] + oxaliplatin and 15 received FP alone) by an investigator’s decision. ctDNA clearance rate at 12w was significantly higher in ACT vs. non-ACT; 57% (54/95) vs. 8% (7/93) in pStage I-IV (p &lt; 0.001), and 58% (42/72) vs. 11% (4/37) in pStage II-III (p &lt; 0.001). In addition, ctDNA clearance rate at 24w was also significantly higher in ACT vs. non-ACT; 26% (7/27) vs. 0% (0/30) in pStage I-IV (p = 0.003), and 33% (7/21) vs. 0% (0/15) in pStage II-III (p = 0.03). Cumulative incidence of ctDNA clearance was significantly higher in ACT vs. non-ACT (67% vs. 7% by 24w; cumulative HR = 17.1; 95% CI: 6.7-43.4, p &lt; 0.001). Among 4w-MRD+ patients, 6M-DFS rate was significantly higher in ACT vs. non-ACT; 84% vs. 34% (HR = 0.15; 95% CI: 0.078-0.25; p &lt; 0.001), which was seen in all stages, including pStage II. Conclusions: This analysis from the GALAXY study, is the largest MRD study to date, demonstrating the association of ctDNA dynamics with improved clinical outcomes in MRD+ patients. Our study shows that stratifying post-surgical treatment decisions using the assay can identify patients likely to benefit from ACT across all stages, including pStage II. ctDNA-guided adjuvant strategy will further be established by ongoing randomized VEGA and ALTAIR studies and will be presented in the future conferences. Clinical trial information: jRCT1031200006.

Impact of postoperative integrated genomic and epigenomic signatures of circulating tumor DNA (ctDNA) on recurrence in resected colorectal cancer: Initial report of a prospective ctDNA monitoring study COSMOS-CRC-01.

168 Background: Identifying molecular residual disease (MRD) using circulating tumor DNA (ctDNA) analysis after curative surgery can potentially stratify the recurrence risk and facilitate personalization of adjuvant treatment in patients with colorectal cancer (CRC). We conducted a prospective study “COSMOS-CRC-01” to evaluate the utility of a plasma-only ctDNA assay integrating genomic and epigenomic signatures. Methods: Patients with resectable clinical stage 0–III colorectal cancer were eligible. Plasma samples were collected at structured pre- and post-surgical timepoints and analyzed using Guardant Reveal, a plasma-only ctDNA assay that detects the presence of MRD by identifying somatic alterations and methylation signatures of cell-free DNA associated with colorectal cancer. Results: As of April 2021, 501 patients were enrolled in the COSMOS-CRC-01, of which 496 patients had their post-operative 4-week ctDNA status. In this analysis, we included the first 100 patients enrolled with clinical stage II or more CRC. Seven patients were excluded due to non-curative resection or pathological stage (pStage) IV. The assay was able to produce a result for all 93 samples analyzed (failure rate of 0%). MRD was detected in 23 (25%) patients 4 weeks after surgery. The MRD detection rate was 20% in pStage II disease and 29% in pStage III disease. Patients with positive MRD were older than those with negative MRD ( p = 0.0001). Across all MRD positive samples, 30%, 9%, and 61% were positive by both genomic and epigenomic, only genomic, and only epigenomic calls. Genomic signatures included APC, BRAF, KRAS, and TP53 mutations with the lowest variant allelic fraction of 0.04%. With a median follow-up time of 12.2 months (range 8-18 months), 9 of 93 patients recurred (9.7%). ctDNA was detected from a single 4-week post-surgical sample in 55% of patients who recurred (5 of 9). 1-year disease-free survival was 81.2% in patients with positive MRD and 93.9% in those with negative MRD (hazard ratio 3.49, 95% CI 0.93–13.10, p = 0.049). Multivariate analysis including baseline characteristics associated with recurrence risk showed that MRD status had the strongest association with the recurrence. Post-operative 4-week serum CEA level was not associated with risk for recurrence. Conclusions: While follow-up in this cohort is currently limited, the results suggest that a single post-surgery sample run on a plasma-only assay that integrates genomic and epigenomic signatures can more accurately stratify patients with CRC by recurrence risk than previously known clinical factors. Data from baseline and longitudinal timepoints will be reported as available along with longer term clinical follow-up. Clinical trial information: UMIN000037765.

Circulating tumor DNA-based molecular residual disease detection and recurrence monitoring in patients with advanced or metastatic anal squamous cell carcinoma.

6 Background: For patients with anal squamous cell carcinoma (ASCC), the current standard of care involves curative-intent definitive chemoradiation. For those that recur locally, salvage surgery maybe a consideration. ASCC, however, still lacks a non-invasive blood-based biomarker, which can be of value in this patient population for monitoring recurrence and/or response to immunotherapy later. Circulating tumor DNA (ctDNA) is a promising non-invasive tool to assess molecular residual disease (MRD) and recurrence in ASCC. Here, we evaluated real-world utility of ctDNA status to identify MRD and recurrence in ASCC patients across all stages. Methods: This is a retrospective analysis of patients with any stage ASCC receiving SOC or/and immunotherapy with immune checkpoint inhibitors. A personalized tumor-informed PCR/NGS-based assay (Signatera) was used for ctDNA detection, in the pre/on/post-treatment and surveillance setting. Results: In this study, plasma samples (n=105) were collected from 25 ASCC patients (13 females, 12 males; median age 66 years) at various timepoints for a median follow-up of 315 days (range: 59-1717), post-diagnosis; 12 patients were HPV-positive, 1 patient was HPV-negative, and 12 had unknown HPV status; 88% (22/25) of the patients had serial timepoints (≥2) available. ctDNA-positivity rates, test results, and ctDNA quantification by stage are summarized in Table. The quantitated ctDNA values (mean tumor molecules (MTM)/mL) increased in concordance with the stage of the disease, with values trending higher in stage III and IV. In addition, complete clinical outcome information was available for 22/25 patients at the time of releasing this data. Of the 96 plasma samples drawn from these patients, 68 (70%) were in the surveillance setting (post-definitive therapy). No recurrences were observed among 15 patients who cleared ctDNA on treatment and/or tested negative post-treatment, whereas, 6/7 ctDNA-positive patients were confirmed to have disease recurrence and 1 was pending confirmatory imaging. This often predated recurrence on scans. Conclusions: Measuring and monitoring MRD in patients with ASCC is feasible and has the potential to impact clinical decision making. Our study is the first to set the benchmark for the feasibility of using a tumor-informed assay in ASCC. Larger prospective studies are needed to explore the clinical utility of ctDNA status to guide disease surveillance and management of ASCC.[Table: see text]

Application Value of ctDNA-based MRD Dedection in Early Stage Non-small Cell Lung Cancer after Radical Surgery

肺癌是世界上最常见的恶性肿瘤，其中非小细胞肺癌（non-small cell lung cancer, NSCLC）约占肺癌总数的85%。接受手术的NSCLC患者的5年总生存期（overall survival, OS）从Ⅰa1期的92%到Ⅲb期的26%，持续下降的生存期使其临床上有强烈的精准辅助治疗需求，以根除分子残留病灶（molecular residual disease, MRD）。目前循环肿瘤DNA（circulating tumor DNA, ctDNA）作为提示MRD的分子指标逐渐从实验室走向临床。最新共识提出在围手术期NSCLC患者外周血中可稳定检测出丰度≥0.02%的ctDNA，是基于ctDNA作为MRD指标的可能。MRD检测技术支持了NSCLC根治术后进行监测的可能性，而且ctDNA能比NSCLC治疗后的影像监测更早地预示着疾病的复发，为精准辅助治疗方案的制定提供了宝贵依据。在早期NSCLC术后辅助治疗研究中，不同指南对是否应该进行适合的辅助治疗存在分歧，而MRD可以作为一个更精准的预测指标，对术后辅助治疗进行指导，使患者能在疾病治疗中获益。

Perioperative ctDNA-Based Molecular Residual Disease Detection for Non-Small Cell Lung Cancer: A Prospective Multicenter Cohort Study (LUNGCA-1).

We assessed whether perioperative circulating tumor DNA (ctDNA) could be a biomarker for early detection of molecular residual disease (MRD) and prediction of postoperative relapse in resected non-small cell lung cancer (NSCLC). Based on our prospective, multicenter cohort on dynamic monitoring of ctDNA in lung cancer surgery patients (LUNGCA), we enrolled 950 plasma samples obtained at three perioperative time points (before surgery, 3 days and 1 month after surgery) of 330 stage I-III NSCLC patients (LUNGCA-1), as a part of the LUNGCA cohort. Using a customized 769-gene panel, somatic mutations in tumor tissues and plasma samples were identified with next-generation sequencing and utilized for ctDNA-based MRD analysis. Preoperative ctDNA positivity was associated with lower recurrence-free survival (RFS; HR = 4.2; P < 0.001). The presence of MRD (ctDNA positivity at postoperative 3 days and/or 1 month) was a strong predictor for disease relapse (HR = 11.1; P < 0.001). ctDNA-based MRD had a higher relative contribution to RFS prediction than all clinicopathologic variables such as the TNM stage. Furthermore, MRD-positive patients who received adjuvant therapies had improved RFS over those not receiving adjuvant therapy (HR = 0.3; P = 0.008), whereas MRD-negative patients receiving adjuvant therapies had lower RFS than their counterparts without adjuvant therapy (HR = 3.1; P < 0.001). After adjusting for clinicopathologic variables, whether receiving adjuvant therapies remained an independent factor for RFS in the MRD-positive population (P = 0.002) but not in the MRD-negative population (P = 0.283). Perioperative ctDNA analysis is effective in early detection of MRD and relapse risk stratification of NSCLC, and hence could benefit NSCLC patient management.

CtDNA MRD Detection and Personalized Oncogenomic Analysis in Oligometastatic Colorectal Cancer From Plasma and Urine.

We applied AVENIO targeted next-generation sequencing to plasma, tumor, and urine samples acquired on the day of curative-intent surgery from 24 prospectively enrolled patients with oligometastatic CRC. Age-related clonal hematopoiesis was accounted for by removing variants also present in white blood cells. Plasma and urine ctDNA MRD were correlated with tumor cells detected in the surgical specimen, and adjuvant treatment strategies were proposed based on ctDNA-inferred tumor mutational burden (iTMB) and targetable alterations. Seventy-one percent of patients were treated with neoadjuvant chemotherapy. Tumor-naive plasma ctDNA analysis detected MRD at a median level of 0.62% with 95% sensitivity and 100% specificity, and 94% and 77% sensitivity when only considering patients treated with neoadjuvant chemotherapy and putative driver mutations, respectively. In urine, ctDNA MRD detection specificity remained high at 100%, but sensitivity decreased to 64% with median levels being 11-fold lower than in plasma (P < .0001). Personalized ctDNA MRD oncogenomic analysis revealed 81% of patients might have been candidates for adjuvant immunotherapy based on high iTMB or targeted therapy based on actionable PIK3CA mutations. Tumor-naive plasma ctDNA analysis can sensitively and specifically detect MRD in patients with oligometastatic CRC after neoadjuvant chemotherapy. Urine-based ctDNA MRD detection is also feasible; however, it is less sensitive than plasma because of significantly lower levels. Oligometastatic patients with detectable MRD may benefit from additional personalized treatment based on ctDNA-derived oncogenomic profiling.