ctDNA Monitoring Research Articles

Feasibility of disease recurrence monitoring in liver post-transplantation for patients with hepatocellular carcinoma via personalized and tumor-informed ctDNA test.

e16123 Background: Hepatocellular carcinoma (HCC) is an aggressive malignancy for which liver transplantation can be curative. Unfortunately, ̃8-20% of HCC patients will go on to relapse post-transplantation. Personalized and tumor-informed circulating tumor DNA (ctDNA) testing (Signatera, bespoke mPCR NGS assay) has been validated to accurately predict relapse across solid tumors, ahead of radiological imaging. Here, we demonstrate the feasibility of ctDNA testing for monitoring relapse in HCC patients who underwent liver transplantation with curative intent. Methods: A total of 10 HCC patients, stage I-IV, who underwent curative liver transplantation with longitudinal ctDNA monitoring were included in the analysis. Alpha-fetoprotein (AFP) levels and images were measured during surveillance in a subset of patients. Results: In this cohort of 10 patients, 2 (20%) tested ctDNA positive during surveillance, both of whom relapsed. Of these, one tested ctDNA positive two months prior to imaging. Of the 8 patients who did not test ctDNA positive during surveillance, all remained disease-free by imaging. Two patients had elevated AFP, neither of whom relapsed. Of the 2 patients who relapsed, AFP levels were available for one patient and fell within the normal range. Conclusions: Our study illustrated the feasibility of performing longitudinal ctDNA assessment in patients with HCC (post-transplantation) during surveillance. ctDNA status but not AFP was associated with recurrence and was able to inform disease status ahead of imaging. To facilitate clinical decision-making, specifically with adjuvant therapy and immunosuppression management, additional studies with larger patient cohorts will be needed to validate the clinical utility of ctDNA testing in HCC.

P-283 Performance characteristics of a tumor-informed circulating tumor DNA (ctDNA) minimal residual disease (MRD) assay in stage 1-3 colorectal cancer (CRC) in clinical practice

Detection of MRD has emerged as a tool for risk stratification in patients with early stage CRC with potential implication for adjuvant therapy. Recently, the sensitivity of MRD testing for early detection of tumor recurrence/ metastasis compared with conventional imaging has been questioned. We sought to evaluate the diagnostic performance characteristics of the Signatera™ ctDNA assay in patients with stage 1-3 CRC at West Cancer Center and Research Institute (WCCRI). Patients with CRC who received ctDNA MRD testing 3-8 weeks after curative surgical resection for stage 1-3 disease from 12/2019 to 02/2022 were evaluated. Both patients with single testing and serial ctDNA assays were included. Individual chart review was performed to collect demographic information, verify diagnosis, stage, pathology, ctDNA and imaging results, and treatment course. ctDNA positivity rates were calculated and analyzed by diagnosis, stage, and clinical course. MRD results from 104 patients were available, 64 of which had sequential results. Positivity rate at initial testing was associated with stage: stage 1 0/9 pts, stage 2 5/40 (12.5%), stage 3 15/55 (27%). In the 64 patients with sequential data, conversion from negative to positive occurred in 7/50 (14%), 8/14 patients initially positive results converted to negative after adjuvant therapy, 5 of which developed ctDNA positivity in further monitoring after 3-14 months (range) with to date 4 recurrences confirmed by imaging. 27/104 patients were found to be ctDNA positive at any time point of which 2 patients were initially ctDNA negative with presence of overt disease (1 small pulmonary metastases, 1 incidentally found peritoneal nodule). In sequential ctDNA monitoring, both patients eventually showed positive ctDNA results. Only 1 patient had a false positive result at low Mean Tumor Molecule level (0.06 MTM/mL). Tumor-informed ctDNA MRD testing has very high sensitivity and specificity, but a one-time negative test result does not preclude the presence of metastatic disease, in particular with regard to pulmonary and peritoneal tumor manifestations. Prospective randomized and non-randomized studies are underway to further assess the clinical utility of ctDNA MRD testing in early stage CRC.

Colon adjuvant chemotherapy based on evaluation of residual disease (CIRCULATE-US): NRG-GI008.

TPS3643 Background: Currently, there are no biomarkers validated prospectively in randomized studies for resected colon cancer (CC) to determine need for adjuvant chemotherapy (AC). However, circulating tumor DNA (ctDNA) represents a highly specific and sensitive approach (especially with serial monitoring) for identifying minimal/molecular residual disease (MRD) post-surgery in CC patients (pts), and may outperform traditional clinical and pathological features in prognosticating risk for recurrence. CC pts who do not have detectable ctDNA (ctDNA-) are at a much lower risk of recurrence and may be spared the toxicities associated with AC. Furthermore, for CC pts with detectable ctDNA (ctDNA+) who are at a very high risk of recurrence, the optimal AC regimen has not been established. We hypothesize that for pts whose CC has been resected, ctDNA status may be used to risk stratify for making decisions about AC. Methods: In this prospective phase II/III trial, up to 1,912 pts with resected stage III A, B (all pts) and stage II, IIIC (ctDNA+ only) CC will be enrolled. Based on the post-operative ctDNA status using personalized and tumor informed assay (SignateraTM, bespoke assay), those who are ctDNA- (Cohort A) will be randomized to immediate AC with fluoropyrimidine (FP) + oxaliplatin (Ox) for 3-6 mos per established guidelines vs . serial ctDNA monitoring. Patients who are ctDNA+ post-operatively or with serial monitoring (Cohort B) will be randomized to FP+Ox vs . more intensive AC with addition of irinotecan (I) for 6 mos. The primary endpoints for Cohort A are time to ctDNA+ status (phase II) and disease-free survival (DFS) in phase III in the immediate vs . delayed AC arms. The primary endpoint for Cohort B is DFS in the FP+Ox vs FP+Ox+I arms for both phase II and phase III portions of the trial. Secondary endpoints include prevalence of detectable ctDNA post-operatively, time-to-event outcomes (overall survival and time to recurrence) by ctDNA status, and the assessment of compliance to adjuvant therapy. Biospecimens including archival tumor tissue, post-operative and serial matched/normal blood samples will be collected for exploratory correlative research. Active enrollment across the NCTN started in early 2022. Support: U10-CA-180868, -180822; UG1CA-189867; Clinical trial information: NCT05174169.

Serial postoperative ctDNA monitoring of breast cancer recurrence.

562 Background: Up to 30% of patients with breast cancer relapse after primary treatment. There are no sensitive or reliable tests to monitor these patients and detect distant metastases before overt recurrence. Here, we demonstrate the use of personalized circulating tumour DNA (ctDNA) profiling performed postoperatively, postadjuvantly, and serially for detection of recurrence in breast cancer. Methods: Patients with primary breast cancer (n=188) were recruited following surgery and adjuvant therapy and were followed-up for up to 10 years with semi-annual blood sampling for ctDNA analysis. Patients (n=29) with insufficient residual tumour for whole exome sequencing (WES) were excluded from the analysis. Tumour WES profiles were generated for 159 patients; samples from 2 patients failed WES QC requirements and a personalised ctDNA panel could not be generated for 1 patient. In 156 patients, plasma samples (n=1141) were retrospectively tested for the presence of ctDNA using personalized Signatera assays (mPCR-NGS) targeting up to 16 somatic single nucleotide variants selected from primary tumour WES. Results: Plasma ctDNA was detected ahead of clinical or radiologic relapse in 30 of the 34 relapsed patients (sensitivity of 88%). Metastatic relapse was predicted with a lead interval of up to 2 years (median: 10 months, range: 0-39 months); median lead intervals for HR+/HER2- were 15 (2 - 39); for HR-/HER2+ 6 (0.5 – 12) for HR+/HER2+ 8 (5 – 14) and 9 (0-20) for TNBC. Patients with a positive ctDNA test had poorer relapse-free-survival (RFS) (HR=47.5; 95% CI 18.5-161.4; p <0.001) from surgery and all four breast cancer subgroups showed a similarly reduced RFS. Overall survival was also significantly reduced for patients who were ctDNA positive (HR=84.15; 95%CI 16.43-1538; p <0.001). The number of variants, mean VAF and MTM/mL varied between patients, with significantly higher values at the time closest to relapse than in the first ctDNA positive sample (p =0.0002). Among the 4 relapsed patients not detected in the study all were HR+/HER2-, 1 had a local recurrence, 2 had bone recurrence (1 with axillary LN involvement) and 1 had cancer cells in pleural fluid. Of the remaining 122 patients, only 5 developed ctDNA-positivity, all with low VAF, none of them have relapsed by the follow-up census date (31 December 2021). However, follow-up for some of these patients limits definitive assessment. Lastly, 4 patients developed a second primary cancer (2 breast, 2 lung) all of whom were ctDNA-negative. Conclusions: This study demonstrates that serial post-operative ctDNA analysis has strong prognostic value. More importantly, earlier detection of metastatic disease provides a possible window for therapeutic intervention, while repeated negative ctDNA tests can provide reassurance to patients. Future interventional studies may assess the clinical utility of ctDNA-based risk-stratification.

Dynamic changes in longitudinal circulating tumour DNA profile during metastatic colorectal cancer treatment.

Circulating tumour DNA (ctDNA) has been spotlighted as an attractive biomarker because of its easy accessibility and real-time representation of tumour genetic profile. However, the clinical utility of longitudinal ctDNA monitoring has not been clearly defined. Serial blood samples were obtained from metastatic colorectal cancer patients undergoing first-line chemotherapy. ctDNA was sequenced using a targeted next-generation sequencing platform which included 106 genes. Changes in ctDNA profile and treatment outcome were comprehensively analysed. A total of 272 samples from 62 patients were analysed. In all, 90.3% of patients had detectable ctDNA mutation before treatment. ctDNA clearance after chemotherapy was associated with longer progression-free survival which was independent of radiological response (adjusted hazard ratio 0.22, 95% confidence interval 0.10-0.46). Longitudinal monitoring was able to detect ctDNA progression which preceded radiological progressive disease (PD) in 58.1% (median 3.3 months). Diverse resistant mutations (34.9%) and gene amplification (7.0%) at the time of PD were discovered. For 16.3% of the PD patients, the newly identified mutations could be potential candidates of targeted therapy or clinical trial. ctDNA profile provided a more accurate landscape of tumour and dynamic changes compared to radiological evaluation. Longitudinal ctDNA monitoring may improve personalised treatment decision-making.

Blood, toil, and taxoteres: Biological determinants of treatment-induced ctDNA dynamics for interpreting tumor response.

Collection and analysis of circulating tumor DNA (ctDNA) is one of the few methods of liquid biopsy that measures generalizable and tumor specific molecules, and is one of the most promising approaches in assessing the effectiveness of cancer care. Clinical assays that utilize ctDNA are commercially available for the identification of actionable mutations prior to treatment and to assess minimal residual disease after treatment. There is currently no clinical ctDNA assay specifically intended to monitor disease response during treatment, partially due to the complex challenge of understanding the biological sources of ctDNA and the underlying principles that govern its release. Although studies have shown pre- and post-treatment ctDNA levels can be prognostic, there is evidence that early, on-treatment changes in ctDNA levels are more accurate in predicting response. Yet, these results also vary widely among cohorts, cancer type, and treatment, likely due to the driving biology of tumor cell proliferation, cell death, and ctDNA clearance kinetics. To realize the full potential of ctDNA monitoring in cancer care, we may need to reorient our thinking toward the fundamental biological underpinnings of ctDNA release and dissemination from merely seeking convenient clinical correlates.

Abstract A021: On-treatment ctDNA monitoring of pancreatic cancer holds promise for improved treatment stratification

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease characterized by rapidly occurring chemoresistance due to clonal evolution. In addition, since most cases are unresectable, tissue analyses are limited to diagnostic biopsy specimens which hampers molecular analyses and does not reflect the genetic tumor heterogeneity. Hence, there is an urgency to develop new complementary tools to detect, characterize and monitor PDAC. The aim of the ongoing prospective, observational study “Chemotherapy, Host Response and Molecular Dynamics in Periampullary Cancer (CHAMP)” (NCT03724994) is to gain new insights into the clonal evolution and parallel immune responses, under the pressure of different cytotoxic treatments, in PDAC and other periampullary cancers. Methods: In a pilot study comprising ten CHAMP patients, an in-depth genomic analysis of ctDNA in serial blood samples (3-4 samples/patient) and matched tumor tissue, either from resected specimens or diagnostic biopsies, was performed using a broad targeted pan-cancer panel. Within this small case series, three patients with resectable disease received adjuvant chemotherapy and seven patients received palliative chemotherapy. Detected ctDNA fluctuation patterns were correlated to tumor progression. Results: In the three patients receiving adjuvant treatment, ctDNA could not be detected in the blood at any time point. For the five patients receiving palliative treatment, varying quantities of ctDNA were detected before the start of treatment (baseline), followed by a decrease during the first month of therapy. Quantitative ctDNA fluctuations were also observed to largely follow the clinical disease course as signs of radiological tumor progression were supported by elevated ctDNA levels. The concordance of known driver mutations in ctDNA detected at baseline and matched tumor tissue was overall high. Yet, a few discrepancies were seen, such as an ARID1A mutation detected only in ctDNA at the time of radiological tumor progression in one patient. Of note, a potentially druggable KRAS p.G12C mutation was observed in both tumor tissue and ctDNA in the same patient. In two palliative patients, one of whom did not respond to treatment, no ctDNA could be detected at any time point. Conclusion: The results from this pilot study emphasize the value of ctDNA analyses, both for characterization and on-treatment monitoring of PDAC. This is of particular importance in cases where no tissue biopsies are available, and at disease relapse or progression. Studies on 50 additional CHAMP patients are now ongoing, wherein a novel ultra-sensitive ctDNA profiling assay is utilized to further detect minimal residual disease and clinically actionable targets. Studies comparing the clonal landscape in treatment naïve primary tumors and/or metastases, serial blood samples, and primary and/or metastatic tumor tissue obtained postmortem, are also in progress. Citation Format: Alexandra Petersson, Sofie Olsson Hau, Jakob Eberhard, Markus Heidenblad, David Gisselsson, Karin Jirström. On-treatment ctDNA monitoring of pancreatic cancer holds promise for improved treatment stratification [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A021.

PD58-03 DETECTION OF INDIVIDUALIZED MUTATION USING PLASMA AND URINARY CIRCULATING TUMOR DNA IN PATIENTS WITH UPPER TRACT UROTHELIAL CARCINOMA

You have accessJournal of UrologyCME1 May 2022PD58-03 DETECTION OF INDIVIDUALIZED MUTATION USING PLASMA AND URINARY CIRCULATING TUMOR DNA IN PATIENTS WITH UPPER TRACT UROTHELIAL CARCINOMA Daichi Tamura, Masakazu Abe, Renpei Kato, Yoichiro Kato, Shigekatsu Maekawa, Mitsugu Kanehira, Ryo Takata, Yutaka Suzuki, Hidewaki Nakagawa, Satoshi Nishizuka, and Wataru Obara Daichi TamuraDaichi Tamura More articles by this author , Masakazu AbeMasakazu Abe More articles by this author , Renpei KatoRenpei Kato More articles by this author , Yoichiro KatoYoichiro Kato More articles by this author , Shigekatsu MaekawaShigekatsu Maekawa More articles by this author , Mitsugu KanehiraMitsugu Kanehira More articles by this author , Ryo TakataRyo Takata More articles by this author , Yutaka SuzukiYutaka Suzuki More articles by this author , Hidewaki NakagawaHidewaki Nakagawa More articles by this author , Satoshi NishizukaSatoshi Nishizuka More articles by this author , and Wataru ObaraWataru Obara More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002643.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Upper tract urothelial carcinoma (UTUC) is an aggressive disease which have high recurrence after surgery. Current standard methods for the diagnosis and detection of recurrence are invasive or low sensitivity. Circulating tumor DNA (ctDNA) analysis using digital polymerase chain reaction (dPCR) has demonstrated promising results for monitoring as recurrence in several cancers. The aim of this study was to evaluate the validity of plasma and urinary ctDNA as a tumor biomarker for UTUC among a perioperative period. METHODS: This study included 23 patients who underwent radical nephroureterectomy (RNU) diagnosed as UTUC from January 2019 to December 2020. In each patient, whole exome sequencing (WES) of tumor DNA and corresponding peripheral blood mononuclear cell (PBMC) DNA were performed by next generation sequencer. Tumor specific mutations were identified by comparing the single nucleotide variants from tumors and PBMCs. To investigate ctDNA by dPCR analysis, we designed primers and probes for each mutation. We also obtained plasma and urine ctDNA from each patient. Longitudinal variant allele frequency (VAF) of ctDNA were plot on a perioperative period. RESULTS: The median age was 71 years old (range 57-82). 13 cases were renal pelvis cancer, and 10 cases were ureteral cancer. The median tumor mutation burden in all cases was 3.02/Mb (range 0.00-15.97). Tumor specific mutations such as TERT, PIK3CA and p53 gene were selected by individualized patient, and performed plasma and urinary ctDNA monitoring by dPCR in 7 patients. Tumor specific mutations were detected in preoperative samples and VAFs of ctDNA decreased quickly after RNU in 5 patients. Tumor specific mutations were not detected in 2 patients who were pT0 due to neoadjuvant chemotherapy. CONCLUSIONS: VAFs in plasma and urinary ctDNA were associated with tumor burden. Individualized ctDNA monitoring could be a useful biomarker for UTUC. Source of Funding: None © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e1012 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Daichi Tamura More articles by this author Masakazu Abe More articles by this author Renpei Kato More articles by this author Yoichiro Kato More articles by this author Shigekatsu Maekawa More articles by this author Mitsugu Kanehira More articles by this author Ryo Takata More articles by this author Yutaka Suzuki More articles by this author Hidewaki Nakagawa More articles by this author Satoshi Nishizuka More articles by this author Wataru Obara More articles by this author Expand All Advertisement PDF DownloadLoading ...

Individualized circulating tumor DNA monitoring in head and neck squamous cell carcinoma.

There is no useful biomarker to evaluate treatment response and early relapse in head and neck squamous cell carcinoma (HNSCC). Circulating tumor DNA (ctDNA) is a promising biomarker for detecting minimal residual diseases and monitoring treatment effect. We investigated whether individualized ctDNA analysis could help monitor treatment response and relapse in HNSCC. Mutation analysis of tumor and peripheral blood mononuclear cell (PBMC) DNAs of 26 patients with HNSCC was performed using a custom squamous cell carcinoma (SCC) panel. The identified individualized mutated genes were defined as ctDNA candidates. We investigated whether frequent ctDNA monitoring via digital PCR (dPCR) is clinically valid for HNSCC patients. TP53 was the most frequently mutated gene and was detected in 14 of 24 cases (58.2%), wherein two cases were excluded owing to the absence of tumor-specific mutations in the SCC panel. Six cases were excluded because of undesignable and unusable primer-probes for dPCR. Longitudinal ctDNA was monitored in a total of 18 cases. In seven cases, ctDNA tested positive again or did not test negative, and all seven cases relapsed after initial curative treatment. In 11 cases, after initial curative treatment, ctDNA remained negative and patients were alive without recurrence. Patients who remained negative for ctDNA during follow-up after initial curative treatment (n=11) had a significantly better prognosis than those who reverted to ctDNA positivity (n=7; p < 0.0001; log-rank test). Individualized ctDNA monitoring using SCC panel and dPCR might be a novel and promising biomarker for HNSCC.

Circulating tumor DNA as a predictive biomarker in Merkel cell carcinoma

Circulating tumor DNA as a predictive biomarker in Merkel cell carcinoma

Reporting on circulating tumor DNA monitoring in metastatic cancer-From clinical validity to clinical utility.

This commentary attempts to discuss the required standardization of circulating tumor DNA (ctDNA) analyses and thereby improve the clinical validity of ctDNA monitoring in the metastatic setting of solid tumors.

Circulating tumour DNA monitoring and early treatment for relapse: views from patients with early-stage melanoma

BackgroundCirculating tumour DNA (ctDNA) monitoring is a new technology that detects cancer DNA fragments in blood circulation. Regular monitoring with ctDNA has the potential to detect and treat cancer relapse earlier, but there is little evidence on patient acceptability. This study examines the views of patients with early-stage melanoma on the acceptability of the test and early treatment.MethodsA qualitative cross-sectional design using one-to-one semi-structured telephone interviews was employed. Twenty-five patients diagnosed with early-stage melanoma (Stage IA–IIC) were asked for their views on ctDNA monitoring and early treatment for relapse. Interviews were analysed using reflexive thematic analysis.ResultsTwo themes were generated: ctDNA monitoring would add service value where participants described regular ctDNA monitoring in follow-up care as more reassuring, more “scientific” than skin checks and preferable to scans. Test results provide opportunity and knowledge focuses on how participants wanted to know when to expect results to manage anxiety, with a positive result seen as an opportunity to receive treatment early.ConclusionsParticipants were positive about ctDNA monitoring and early treatment and would welcome extra surveillance, as well as trust ctDNA tests. This indicates the feasibility of the wider implementation of ctDNA tests, which have applicability for many tumour types and disease stages.

Circulating Tumor DNA-Based Genotyping and Monitoring for Predicting Disease Relapses of Patients with Peripheral T-Cell Lymphomas.

Plasma circulating tumor DNA (ctDNA) could reflect the genetic alterations present in tumor tissues. However, there is little information about the clinical relevance of cell-free DNA genotyping in peripheral T-cell lymphoma (PTCL). After targeted sequencing plasma cell-free DNA of patients with various subtypes of PTCL (n=94), we analyzed the mutation profiles of plasma ctDNA samples and their predictive value of dynamic ctDNA monitoring for treatment outcomes. Plasma ctDNA mutations were detected in 53 patients (56%, 53/94), and the detection rate of somatic mutations was highest in angioimmunoblastic T-cell lymphoma (24/31, 77%) and PTCL, not otherwise specified (18/29, 62.1%). Somatic mutations were detected in 51 of 66 genes that were sequenced, including the following top 10 ranked genes: RHOA, CREBBP, KMT2D, TP53, IDH2, ALK, MEF2B, SOCS1, CARD11, and KRAS. In the longitudinal assessment of ctDNA mutation, the difference in ctDNA mutation volume after treatment showed a significant correlation with disease relapse or progression. Thus, a ≥ 1.5-log decrease in genome equivalent (GE) between baseline and the end of treatment showed a significant association with better survival outcomes than a < 1.5-log decrease in GE. Our results suggest the clinical relevance of plasma ctDNA analysis in patients with PTCL. However, our findings should be validated by a subsequent study with a larger study population and using a broader gene panel.

Role of Circulating Tumor DNA Profiling in Patients with Non-Small Cell Lung Cancer Treated with EGFR Inhibitor

Objectives: During targeted therapy, tumor heterogeneity can drive the evolution of multiple tumor subclones harboring unique resistance mechanisms. Sequential profiling of plasma cell-free DNA (cfDNA) provides a noninvasive method for early detection of patient progression. We investigated whether the genetic dynamics detected in cfDNA during treatment can act as a predictive or prognostic marker of outcome. Methods: Patients with advanced EGFR-mutated non-small cell lung cancer (NSCLC) were included for consecutive blood sampling during EGFR-tyrosine kinase inhibitor (TKI) treatment. Blood samples were serially collected from patients at baseline, first follow-up, and progression. Extracted cfDNA was analyzed with next-generation sequencing. Results: Serial plasma samples (n = 187) from 63 patients were analyzed, and 44 patients showed circulating tumor DNA (ctDNA). EGFR mutations were detected in 36 of the 44 patients at baseline (81.8%). EGFR mutations were no longer detected in 19 of 36 shedders (52.8%) at 2 months after EGFR-TKI treatment and rebounded with resistant EGFR mutations (T790M or C797S) at progression. Other driver mutations such as KRAS G12D and BRAF V600E were found at baseline regardless of tissue EGFR status, suggesting tumor heterogeneity. Detection of ctDNA (shedder) at baseline associated with poor overall survival (p = 0.04) compared to nonshedder. Furthermore, in patients showing EGFR mutations in plasma at baseline, the clearing rate of those during the first 8 weeks of treatment served as a positive predictor for clinical outcome. Conclusion: Longitudinal liquid biopsies capture spatial and temporal heterogeneity underlining resistance to EGFR-TKIs in NSCLC. Thus, ctDNA monitoring during EGFR-TKI treatment is useful for detecting resistance mutations or predicting response. Dense serial monitoring using blood enables early prediction of treatment failure and provides a window of opportunity for well-timed intervention.

Abstract P2-01-13: Longitudinal circulating tumor DNA (ctDNA) monitoring by digital droplet PCR (ddPCR) in metastatic breast cancer

Abstract Background: A common challenge in treating patients with metastatic breast cancer (MBC) is ensuring adequate therapeutic efficacy, maintenance of quality of life, and avoidance of drug toxicity by preventing exposure to ineffective treatment. Critical to balancing these goals is the ability to quickly identify progression in order to minimize exposure to ineffective therapies. Imaging is the current standard-of-care for disease response evaluation, but high cost, relative low sensitivity, and tumor flare phenomenon remain significant issues. Circulating tumor DNA (ctDNA) testing based on next generation sequencing (NGS) is emerging as an alternative to imaging-based response assessment, but remains an expensive proposition. Here we evaluate a hybrid monitoring strategy using an initial baseline NGS-based ctDNA analysis to identify mutant alleles, followed by response monitoring with a low-cost, personalized digital droplet PCR (ddPCR) assay. Methods: Blood for ddPCR ctDNA analysis was collected at the same timepoint as a sample for commercial ctDNA analysis by NGS. PCR primer and probe sets were designed against clinically relevant mutations identified from each patient’s commercial ctDNA NGS report. ddPCR was then performed. Variant allele fractions (VAFs) detected by ddPCR were compared to allelic fractions reported by commercial NGS using Bland-Altman analysis and significance was determined by paired t-test. To demonstrate the potential for longitudinal ddPCR-based ctDNA monitoring, serial samples from the same patient were analyzed. Results: In our pilot validation set, 10 paired patient samples were analyzed by NGS and ddPCR. NGS-reported VAFs ranged from 0.30% to 16.20%. High comparative performance was observed between NGS and ddPCR platforms over expected mutant fractional abundance ranges. There was no significant quantitative difference between the two approaches (p=0.45). Longitudinal monitoring of ctDNA by ddPCR was performed on a metastatic hormone-positive breast cancer patient and revealed a significant rise in the clinically-relevant PIK3CA H1047L mutation that preceded imaging-based documentation of progression by 3 months. A final timepoint was taken several months before the patient’s death with a further significant increase in ctDNA burden. Conclusions: Here we demonstrate that highly-sensitive ddPCR-based ctDNA assays provide comparable results to next generation sequencing, but at considerably lower cost. These results indicate that bespoke tracking of select alleles by ddPCR offers an inexpensive and rapid approach to serial ctDNA monitoring in metastatic breast cancer patients. A larger prospective study has been designed and is underway. Citation Format: Nicole Higashiyama, LaTerrica Williams, Bryant McCue, Alphi Kuriakose, Tiffaney Tran, Stephanie Gonzalez, Mayra Licerio, Carol Chenault, Heidi Dowst, Susan Hilsenbeck, Matthew Ellis, Bora Lim, George Miles. Longitudinal circulating tumor DNA (ctDNA) monitoring by digital droplet PCR (ddPCR) in metastatic breast cancer [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 P2-01-13.

Abstract GS3-07: Circulating tumor DNA (ctDNA) dynamics in patients with hormone receptor positive (HR+)/HER2 negative (HER2-) advanced breast cancer (aBC) treated in first line with ribociclib (R) and letrozole (L) in the BioItaLEE trial

Abstract Background: ctDNA analysis is emerging as an attractive non-invasive approach to characterize tumor biology, describe its evolution over time, and predict treatment benefit. Here, we assessed the prognostic and predictive role of baseline and dynamic ctDNA analysis in HR+/HER2- aBC patients (pts) treated with R+L. Methods: 287 postmenopausal pts were enrolled in the BioItaLEE trial (NCT03439046). Liquid biopsies were collected at baseline (D0; n=263), day 15 of cycle 1 (D15; n=238), day 1 of cycle 2 (C2D1; n=241) and at first imaging (FI, at approximately 12 weeks; n=206). ctDNA analysis was carried out using a 533-amplicon Custom AmpliSeq HD Panel, with amplicons covering the coding exons of 39 BC-related genes (limit of detection: 0.1%). Target mutations were defined as single-nucleotide variant (SNV) or Insertion/Deletion detected at D0. When multiple target mutations were detected, the one with the highest variant allele frequency (VAF) was considered. The association between pre-treatment and on-treatment ctDNA dynamics with progression-free survival (PFS) was assessed using Multivariate Cox models. VAF clearance was defined as 100% decrease in a target mutation. Results: Median follow-up was 26.9 months and median PFS was 23.39 (20.8-NE) months. At baseline, target mutations were detected in 113 pts (43.0%), whereas 150 pts were wild-type (wt). Mean (SD) pre-treatment VAF at D0 was 11.3% (14.4). The absence of a target mutation at D0 was associated with good prognosis (HR: 0.41, 95% CI: 0.27–0.61; p&amp;lt;0.0001). Considering early ctDNA dynamics, a significant VAF reduction was observed at D15 and C2D1 with a mean (SD) change of -64.3% (55.9) and -68.6% (52.2), respectively. In pts with a target mutation detected at baseline, early VAF clearance was observed in 47.1% of pts at D15 and in 52.4% of pts at C2D1. Clearance at D15 or C2D1 was associated with improved PFS (D15, HR: 0.51, 95% CI: 0.28-0.91, p=0.0228; C2D1, HR: 0.44, 95% CI: 0.25-0.78, p=0.0052). Pts achieving clearance at D15, which was maintained at C2D1 (39.4%) had the lowest risk of progression compared to those who had no clearance at any or both timepoints (HR: 0.40, 95% CI: 0.20-0.79; p=0.084). Monitoring of the 150 pts without a detectable target mutation at baseline revealed a new, detectable mutation at later timepoints (D15, C2D1 and FI) in 34 pts (22.7%). The absence of new mutations was associated with a lower risk of progression (HR: 0.45, 95% CI: 0.24-0.85; p=0.0143). Considering all time points individually, D15 was the most informative of patient outcome. Indeed, pts without mutation at D15 (42.9%) had an extremely favorable outcome, either because they achieved early treatment-related clearance or maintained baseline absence of a target mutation (HR: 0.32, 95% CI: 0.20-0.51; p&amp;lt;0.0001). Notably, in pts with detectable target mutation at D15, a VAF below the median showed a trend for better prognosis versus high VAF (HR: 0.56, 95% CI: 0.30-1.04; p=0.065). Conclusions: The presence of a detectable mutation in baseline liquid biopsies appears to be a negative prognostic factor. Within this high-risk group, early VAF clearance during the first R+L cycle was informative of treatment benefit and associated with a lower risk of progression. Monitoring of ctDNA in patients without baseline mutations demonstrated that the detection of new mutations by FI assessment was associated with worse outcome. Overall, pre-treatment and early dynamics of ctDNA (assessed by NGS) represent promising prognostic and predictive biomarkers in patients with HR+/HER2- aBC treated with ribociclib/letrozole in the first-line. Further studies are warranted to validate the clinical utility of these biomarkers. Citation Format: Giampaolo Bianchini, Luca Malorni, Grazia Arpino, Alberto Zambelli, Fabio Puglisi, Lucia Del Mastro, Marco Colleoni, Filippo Montemurro, Giulia Bianchi, Ida Paris, Giacomo Allegrini, Marina Elena Cazzaniga, Michele Orditura, Claudio Zamagni, Stefano Tamberi, Daniela Castelletti, Matteo Benelli, Maurizio Callari, Angela Santoro, Michelino De Laurentiis. Circulating tumor DNA (ctDNA) dynamics in patients with hormone receptor positive (HR+)/HER2 negative (HER2-) advanced breast cancer (aBC) treated in first line with ribociclib (R) and letrozole (L) in the BioItaLEE trial [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 GS3-07.

Abstract P1-18-05: Early changes in circulating tumor DNA and its effect on clinical outcomes in patients with advanced breast cancer receiving the CDK4/6 inhibitor palbociclib: Genotyping results from POLARIS

Abstract Background: POLARIS is a prospective, real-world study of palbociclib in patients with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2–) advanced breast cancer (ABC) in the United States and Canada. These analyses evaluated the difference in tumor mutation profiles of patients receiving palbociclib in the first line versus second line or greater and examined the applicability of circulating tumor DNA (ctDNA) monitoring in a real-world setting. Methods: The clinical database cut-off date was March 16, 2021. Patients in the biomarker analysis group provided consent for serial blood sample collections, received ≥1 dose of initial palbociclib combination treatment, and had ≥1 ctDNA measurement available. The Guardant360 platform with somatic single-nucleotide variants in complete or critical exons of 73 genes was used. The association between early changes in ctDNA mutation allele fractions at Cycle 2 Day 1 (C2D1) and disease progression at Week 24 was evaluated using univariate and multivariable logistic regression analysis adjusting for line of therapy. Results: Among patients with HR+/HER2– ABC (N=347), 93.9% (n=326) had ctDNA measured at baseline, of which 85.9% (n=280) had ≥1 ctDNA alteration detected; 66.6% of patients received palbociclib treatment in the first line and 33.4% as second line or greater ABC setting. The frequencies of gene mutations at baseline were generally higher in patients receiving palbociclib as a second-line or greater therapy compared to those who received it as first-line therapy, particularly for ESR1 mutations (30% vs 15%) and FGFR1 mutations (13% vs 9%). With a median (range) follow-up duration of 18.5 (0.1-46.3) months, patients with total ctDNA increase at C2D1 (log ratio change &amp;gt;0) were 3.74 times more likely to have disease progression at Week 24 (odds ratio, 3.74 [95% CI, 1.71-8.17]; P=0.001) compared with those without change or with a decrease in ctDNA at C2D1 (log ratio change ≤0). The observed significant association remained after adjusting for line of therapy in the multivariable regression analysis (odds ratio, 2.62 [95% CI, 1.14-6.04]; P=0.023). Conclusions: Among patients with HR+/HER2- ABC receiving palbociclib, early changes in ctDNA mutations were significantly associated with disease progression. Further studies are needed to confirm these findings and to evaluate the clinical utility of ctDNA-guided “adaptive” early therapeutic interventions to improve outcomes in patients with metastatic breast cancer. Pfizer; NCT03280303 Citation Format: Debu Tripathy, Zhe Zhang, Joanne L. Blum, Meghan S. Karuturi, Steven L. McCune, Bijoy Telivala, Shailendra Lakhanpal, Kamal Patel, Richard C. Frank, Kit Lu, Chetan Deshpande, Yao Wang, Yuan Liu, Aditya Bardia. Early changes in circulating tumor DNA and its effect on clinical outcomes in patients with advanced breast cancer receiving the CDK4/6 inhibitor palbociclib: Genotyping results from POLARIS [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 P1-18-05.

Abstract OT2-24-02: ZEST: Randomized phase III study evaluating efficacy and safety of niraparib in patients with HER2-negative BRCA-mutated or triple-negative breast cancer with detectable circulating tumor DNA after definitive therapy

Abstract OT2-24-02: ZEST: Randomized phase III study evaluating efficacy and safety of niraparib in patients with HER2-negative BRCA-mutated or triple-negative breast cancer with detectable circulating tumor DNA after definitive therapy

Circulating tumor DNA predicts therapeutic outcome in mantle cell lymphoma

AbstractMantle cell lymphoma (MCL) is biologically and clinically heterogeneous and would benefit from prognostic biomarkers to guide management. Circulating tumor DNA (ctDNA) is a novel prognostic biomarker in diffuse large B-cell lymphoma that may have applicability in MCL. We analyzed ctDNA dynamics in previously untreated patients with MCL who received induction therapy with bortezomib and DA-EPOCH-R for 6 cycles followed by random assignment to observation or bortezomib maintenance in responding patients in a prospective phase 2 study. Most patients also underwent initial treatment window of bortezomib alone prior to induction. Serum was collected pretreatment, after the window, after cycles 1 and 2, at the end of induction, and at each follow-up visit along with restaging computed tomography scans. Next-generation sequencing was used to identify and quantify ctDNA encoding the immunoglobulin receptor sequences in serum as markers of minimal residual disease. Fifty-three patients were enrolled, with a median follow-up of 12.7 years. Patients without detectable ctDNA after 2 cycles of induction had longer progression-free survival (PFS) and overall survival (OS) compared with those with detectable ctDNA (median PFS, 2.7 vs 1.8 years; overall P = .005; median OS, 13.8 vs 7.4 years; overall P = .03). Notably, in vivo assessment of ctDNA dynamics during the bortezomib window was not prognostic, and there was no difference in PFS or OS with bortezomib maintenance. ctDNA monitoring after induction showed that molecular relapse preceded clinical relapse in some cases. In conclusion, interim ctDNA negativity strongly correlates with improved survival and supports the investigation of response-adapted strategies. This trial was registered at www.clinicaltrials.gov as #NCT00114738.

Longitudinal monitoring of circulating tumor DNA (ctDNA) during disease course of metastatic colorectal cancer (mCRC).

189 Background: ctDNA is an attractive alternative to tissue for its easy accessibility and real time representation of systemic tumor profile. We explored the utility of ctDNA profiling during the course of mCRC treatment. Methods: Serial blood samples were obtained from mCRC patients before and during first-line palliative chemotherapy at fixed intervals (after every four cycles) until confirmed disease progression. ctDNA was sequenced using targeted next-generation sequencing (NGS) platform with 106 genes. Changes of ctDNA profile and treatment outcome were comprehensively analyzed. Results: A total of 272 samples from 62 patients were analyzed. In the pre-treatment blood samples, 56 (90.3%) of patients had detectable ctDNA mutations including single nucleotide variants, short insertions/deletions and copy number changes (median 4.5 mutations/ patient, range 0 - 133). In 31 (50.0%) patients who had tissue NGS panel results performed in the clinic, overall concordance between mutations from ctDNA and tissue was 86.5%. In three patients, ctDNA mutational profiles were found to be completely different from tissue profiles. At further investigation, these patients were found to have a separate primary cancer in their colon. At the time of the first follow-up, most (98.0%) patients showed decrease of ctDNA from baseline, represented by average variant allele frequency (VAF) changes of all ctDNA mutations found. Clearance of ctDNA was achieved in 40 (78.4%) patients and was associated with longer progression-free survival (median PFS 11.8 moths in ctDNA clearance (+) vs. 4.7 months in ctDNA clearance (-), p &lt; 0.001). The ctDNA clearance at the same time point was able to further discriminate the patients in same category by RECIST 1.1. Serial follow-up monitoring revealed three patterns of ctDNA changes at the time of clinical progressive disease (PD): 1) re-emergence or re-increase of baseline ctDNA mutations, 2) emergence of new resistance mutations, 3) radiologic PD without evidence of ctDNA progression. In the patients with detectable ctDNA at PD, the ctDNA progression preceded radiologic progression in 25 (58.1%) patients by median of 3.3 months. The patients in clinical PD without ctDNA progression showed different patterns of metastasis having mainly extrahepatic spread, while 77.8% of the patients with ctDNA progression had their progression confirmed in liver metastasis. Diverse resistant mutations and gene amplifications in PD patients were discovered by ctDNA sequencing. For seven (16.3%) of the PD patients, the newly identified mutations could be potential candidates of targeted therapy or clinical trial. Conclusions: ctDNA profile provided additional information to conventional evaluation methods and reflected dynamic changes. ctDNA monitoring may improve precise treatment decision-making for individual patients.