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Introduction: The current standard of care for patients with inoperable stage III non-small cell lung cancer includes chemoradiotherapy (CRT) followed by 1 year of checkpoint inhibitor (CPI) therapy. Nevertheless, the optimal duration of consolidation CPI remains unknown. Here, we characterized the relationship between circulating tumor DNA (ctDNA) minimal residual disease (MRD) and clinical outcomes of patients with unresectable locally advanced non-small cell lung cancer treated on a phase 2 trial of short-course consolidation immunotherapy after CRT, with the goal of testing whether ctDNA may be able to identify patients who do not require a full year of treatment., Methods: Plasma samples for ctDNA analysis were collected from patients on the Big Ten Cancer Research Consortium LUN 16-081 trial after completion of CRT, before day 1 of cycle 2 (C2D1) of CPI (i.e., 1 mo after treatment start), and at the end of up to 6 months of treatment. Tumor-informed ctDNA MRD analysis was performed using cancer personalized profiling by deep sequencing. Levels of ctDNA at each time point were correlated with clinical outcomes., Results: Detection of ctDNA predicted significantly inferior progression-free survival after completion of CRT (24-mo 29% versus 65%, p = 0.0048), before C2D1 of CPI (24-mo 0% versus 72%, p < 0.0001) and at the end of CPI (24-mo 15% versus 67%, p = 0.0011). In addition, patients with decreasing or undetectable ctDNA levels after 1 cycle of CPI had improved outcomes compared with patients with increasing ctDNA levels (24-mo progression-free survival 72% versus 0%, p < 0.0001). Progression of disease occurred within less than 12 months of starting CPI in all patients with increasing ctDNA levels at C2D1., Conclusions: Detection of ctDNA before, during, or after 6 months of consolidation CPI is strongly associated with inferior outcomes. Our findings suggest that analysis of ctDNA MRD may enable personalizing the duration of consolidation immunotherapy treatment., Competing Interests: Disclosure Dr. Durm receives support from Bristol-Myers Squibb; grants or contracts from AstraZeneca, Merck, and Mirati; consulting fees from Cook Biotech; payments or honoraria from Curio Science, DAVA Oncology, and AstraZeneca; and participates on an advisory board for Curio Science, DAVA oncology, and AstraZeneca. Dr. Ganti receives research grants or contracts from VA Office of Research and Development, Merck, TAB Biosciences, NEKTAR Therapeutics, Mirati Therapeutics, IOVANCE Therapeutics, and Apexigen; royalties or licenses from Oxford University Press; consulting fees from AstraZeneca, Flagship Biosciences, G1 Therapeutics, Jazz Pharmaceuticals, Cardinal Health, Mirati Therapeutics, Beigene Ltd., Sanofi Genzyme, Blueprint Medicines, and Regeneron Pharmaceuticals; payments or honoraria from MedLearning Group and Plexus Communications; participates on a data safety monitoring board for YmAbs Therapeutics; holds leadership or fiduciary roles at Academic and Community Cancer Research United and A Breath of Hope for Lung Cancer; and has received equipment or materials from Takeda Pharmaceuticals and Chimerx. Dr. Alizadeh receives research grants or contracts from the National Cancer Institute, National Heart, Lung and Blood Institute, National Institutes of Health, Celgene, Bristol-Myers Squibb, and Pfizer; consulting fees from ADCT, Celgene, Chugai, Genentech, Gilead, Janssen, Pharmacyclics, and Roche; has patents planned, issued, or pending with FortySeven Inc., Foresight Diagnostics, CiberMed Inc., and Roche; participates on a data safety monitoring board for Lymphoma Research Foundation; holds leadership or fiduciary roles at the American Society of Hematology, American Society of Clinical Oncology, American Society of Clinical Investigation, and Leukemia & Lymphoma Society; and has equity ownership interests in CiberMed Inc., Foresight Diagnostics, FortySeven Inc., and CARGO Therapeutics. Dr. Hanna receives grants or contracts from Merck, Genentech, Bristol-Myers Squibb, and Natera. Dr. Diehn receives grants or contracts from AstraZeneca and Varian Medical Systems; royalties or licenses from Roche and Foresight Diagnostics; consulting fees from AstraZeneca, Boehringer Ingelheim, Genentech, Illumina, Gritstone Bio, and Regeneron; payments or honoraria from Bristol-Myers Squibb, Japan, and Novartis; support for attending meetings or travel from Foresight Diagnostics and Regeneron; has patents planned, issued, or pending with Stanford University; holds leadership or fiduciary roles at Foresight Diagnostics; and holds stock or stock options in CiberMed Inc., Foresight Diagnostics, and Gritstone Bio. The remaining authors declare no conflict of interest., (Copyright © 2024 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Purpose: Small cell lung cancer (SCLC) is characterized by rapid progression after platinum resistance. Circulating tumor (ctDNA) dynamics early in treatment may help determine platinum sensitivity., Materials and Methods: Serial plasma samples were collected from patients receiving platinum-based chemotherapy for SCLC on the first 3 days of cycle one and on the first days of subsequent cycles with paired samples collected both before and again after infusions. Tumor-informed plasma analysis was carried out using CAncer Personalized Profiling by deep Sequencing (CAPP-Seq). The mean variant allele frequency (VAF) of all pretreatment mutations was tracked in subsequent blood draws and correlated with radiologic response., Results: ctDNA kinetics were assessed in 122 samples from 21 patients. Pretreatment VAF did not differ significantly between patients who did and did not respond to chemotherapy (mean 22.5% v 4.6%, P = .17). A slight increase in ctDNA on cycle 1, day 1 immediately post-treatment was seen in six of the seven patients with available draws (fold change from baseline: 1.01-1.44), half of whom achieved a response. All patients who responded had a >2-fold decrease in mean VAF on cycle 2 day 1 (C2D1). Progression-free survival (PFS) and overall survival (OS) were significantly longer in patients with a >2-fold decrease in mean VAF after one treatment cycle (6.8 v 2.6 months, log-rank P = .0004 and 21.7 v 6.4 months, log rank P = .04, respectively)., Conclusion: A >2-fold decrease in ctDNA concentration was observed by C2D1 in all patients who were sensitive to platinum-based therapy and was associated with longer PFS and OS.

The scarcity of malignant Hodgkin and Reed-Sternberg cells hampers tissue-based comprehensive genomic profiling of classic Hodgkin lymphoma (cHL). By contrast, liquid biopsies show promise for molecular profiling of cHL due to relatively high circulating tumour DNA (ctDNA) levels 1-4 . Here we show that the plasma representation of mutations exceeds the bulk tumour representation in most cases, making cHL particularly amenable to noninvasive profiling. Leveraging single-cell transcriptional profiles of cHL tumours, we demonstrate Hodgkin and Reed-Sternberg ctDNA shedding to be shaped by DNASE1L3, whose increased tumour microenvironment-derived expression drives high ctDNA concentrations. Using this insight, we comprehensively profile 366 patients, revealing two distinct cHL genomic subtypes with characteristic clinical and prognostic correlates, as well as distinct transcriptional and immunological profiles. Furthermore, we identify a novel class of truncating IL4R mutations that are dependent on IL-13 signalling and therapeutically targetable with IL-4Rα-blocking antibodies. Finally, using PhasED-seq 5 , we demonstrate the clinical value of pretreatment and on-treatment ctDNA levels for longitudinally refining cHL risk prediction and for detection of radiographically occult minimal residual disease. Collectively, these results support the utility of noninvasive strategies for genotyping and dynamic monitoring of cHL, as well as capturing molecularly distinct subtypes with diagnostic, prognostic and therapeutic potential., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Purpose: Clinical outcomes of patients with CNS lymphomas (CNSLs) are remarkably heterogeneous, yet identification of patients at high risk for treatment failure is challenging. Furthermore, CNSL diagnosis often remains unconfirmed because of contraindications for invasive stereotactic biopsies. Therefore, improved biomarkers are needed to better stratify patients into risk groups, predict treatment response, and noninvasively identify CNSL., Patients and Methods: We explored the value of circulating tumor DNA (ctDNA) for early outcome prediction, measurable residual disease monitoring, and surgery-free CNSL identification by applying ultrasensitive targeted next-generation sequencing to a total of 306 tumor, plasma, and CSF specimens from 136 patients with brain cancers, including 92 patients with CNSL., Results: Before therapy, ctDNA was detectable in 78% of plasma and 100% of CSF samples. Patients with positive ctDNA in pretreatment plasma had significantly shorter progression-free survival (PFS, P < .0001, log-rank test) and overall survival (OS, P = .0001, log-rank test). In multivariate analyses including established clinical and radiographic risk factors, pretreatment plasma ctDNA concentrations were independently prognostic of clinical outcomes (PFS HR, 1.4; 95% CI, 1.0 to 1.9; P = .03; OS HR, 1.6; 95% CI, 1.1 to 2.2; P = .006). Moreover, measurable residual disease detection by plasma ctDNA monitoring during treatment identified patients with particularly poor prognosis following curative-intent immunochemotherapy (PFS, P = .0002; OS, P = .004, log-rank test). Finally, we developed a proof-of-principle machine learning approach for biopsy-free CNSL identification from ctDNA, showing sensitivities of 59% (CSF) and 25% (plasma) with high positive predictive value., Conclusion: We demonstrate robust and ultrasensitive detection of ctDNA at various disease milestones in CNSL. Our findings highlight the role of ctDNA as a noninvasive biomarker and its potential value for personalized risk stratification and treatment guidance in patients with CNSL., [Media: see text].

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., Competing Interests: Disclosure GA reports receipt of honoraria for participation in Advisory Board from Astellas, AstraZeneca, Bayer, Janssen, Novartis, Orion, Pfizer, Sanofi, Sapience, receipt of honoraria as invited speaker from Astellas, AstraZeneca, Janssen, receipt of royalties for licensing fees from Janssen, receipt of institutional research grants from Astellas and Janssen, non-remunerated activities as a PI in Astellas, Janssen, and non-remunerated advisory role in AstraZeneca and Janssen; FCB reports receipt of honoraria for participation in Advisory Board from Archer, BioNTech, Lilly, Novartis, Pfizer, receipt of honoraria to institution for participation in Advisory Board from AstraZeneca, receipt of honoraria as invited speaker from AstraZeneca, Novartis, Pfizer, Roche, Seagen, receipt of honoraria to institution as invited speaker from Pfizer, Sanofi, receipt of honoraria for expert testimony from Hikma, institutional non-financial interest for research as a coordinating PI from AstraZeneca, Pfizer, ProLynx, Saga and Seagen; GC reports receipt of honoraria for participation in Advisory Board from AstraZeneca, Bristol Myers Squibb (BMS), Daiichi Sankyo, Ellipsis, Exact Sciences, Lilly, Merck, Pfizer, Roche, Veracyte, receipt of honoraria as invited speaker from AstraZeneca, Daiichi Sankyo, Novartis, Pfizer, Roche, receipt of honoraria for writing engagement from Pfizer, institutional research grant for investigator initiated clinical trial from Merck, institutional funding for conduct of phase I studies from Astellas, AstraZeneca, Blueprint Medicine, BMS, Daiichi Sankyo, Kymab, Novartis, Philogen, Roche, Sanofi, non-remunerated activities as an officer of the Italian National Health Council—Advisor for Ministry of Health, non-remunerated advisory role as a member of the Scientific Council of Europa Donna, non-remunerated advisory role in Fondazione Beretta, and non-remunerated member of Board of Directors of the Lega Italiana Lotta ai Tumori; LDMA reports receipt of honoraria as invited speaker, participation in speaker bureau from Roche, institutional research collaboration grant from NanoString and receipt of education grant from BMS; MD reports receipt of honoraria for participation in Advisory Board from AstraZeneca, Boehringer Ingelheim, Genentech, Gritstone Oncology, Illumina, receipt of honoraria for consultancy from BioNTech, Novartis, RefleXion, Roche, has ownership interest with CiberMed, Foresight Diagnostics, receipt of royalties for licensing fees from Foresight Diagnostics, Roche Diagnostics, receipt of institutional financial interest as a coordinating PI from AstraZeneca, Varian, receipt of funding from Genentech, non-financial interest for receipt of reagents from Illumina, non-remunerated activities as a member of Board of Directors of Foresight Diagnostics; AI reports receipt of honoraria for participation in Advisory Board from Bayer, Chugai Pharmaceutical Co, GlaxoSmithKline, Philips, Roche, institutional non-financial interest as a coordinating PI from AstraZeneca, Bayer, Ipsen, Merck, Merck Sharp & Dohme (MSD) and Roche; JL reports receipt of honoraria to institution as invited speaker from Roche; JDM reports receipt of honoraria for participation in Advisory Board from Illumina, BMS, receipt of honoraria for consultancy from PierianDx, receipt of royalties for licensing fees administered by Stanford University from the United States Patent Office, non-remunerated activities as a member of Board of Directors of the Association for Molecular Pathology, non-remunerated leadership role as a Chair of the Informatics Subdivision of the Association for Molecular Pathology, non-remunerated leadership role as a Vice Chair of the CLSI MM23—Molecular Diagnostic Methods for Solid Tumors Committee of the Clinical and Laboratory Standards Institute (CLSI), non-remunerated activities in the US NIH/NCI as a PI for the NIH 1-UG1-CA233333-01, UNITS: The UNC/UT National Clinical Trials Network Group Integrated Translational Science Production and Consultation Center; CM reports receipt of honoraria for participation in Advisory Board from Biocartis, Merck Serono, receipt of honoraria as invited speaker from Amgen, Guardant Health, Merck Serono, and Pierre Fabre, Roche, receipt of royalties for licensing fees administered by Institut Investigació Hospital del Mar; NN reports receipt of honoraria for participation in Advisory Board from AstraZeneca, Bayer, Biocartis, Incyte, Novartis, Qiagen, Roche, receipt of honoraria as invited speaker from BMS, Eli Lilly, Illumina, Merck, MSD, Sanofi, Thermo Fisher, receipt of institutional research grants from AstraZeneca, Biocartis, Blueprint, Illumina, Incyte, Merck, Qiagen, Roche, Thermo Fisher, leadership non-remunerated role as a President of the International Quality Network for Pathology (IQN Path) and President of the Italian Cancer Society (SIC); KP reports receipt of honoraria for participation in Advisory Board from MSD, Menarini, Hello Healthcare, Sanofi, receipt of honoraria as invited speaker from Abcam, Ipsen Pharma, Medac, Agena, institutional financial interest from Angle plc, European Liquid Biopsy Society, Böhringer Ingelheim and for participation in IMI JT ID Cancer from EU/IMI Cancer-ID consortium; SP reports receipt of honoraria for participation in Advisory Board from Amgen, AstraZeneca, Bayer, BeiGene, Blueprint, BMS, Boehringer Ingelheim, Daiichi Sankyo, Guardant Health, Janssen, Eli Lilly, Merck KGaA, Novartis, Roche, Takeda, institutional financial interest for research as a coordinating PI from Ariad, Boehringer Ingelheim, Celgene, Daiichi Sankyo, Takeda, Turning Point Therapeutics, as a local PI from AstraZeneca, GlaxoSmithKline, Roche, Trizell, as a sub-investigator from Amgen, MSD, non-remunerated advisory role in ALK Positive UK, International Association for the Study of Lung Cancer, Lung Cancer Europe, Ruth Strauss Foundation, non-remunerated leadership role in the British Thoracic Oncology Group as a Chair of Steering Committee, European Thoracic Oncology Platform as a Foundation Council Member, non-remunerated member of Thoracic malignancy Faculty in the European Society for Medical Oncology, non-remunerated member of Board of Directors in the Mesothelioma Applied Research Foundation; JSRF reports consultancy fees from Goldman Sachs, Repare Therapeutics, Paige.AI and Eli-Lilly, membership of the Board of Directors Group Oncoclinicas, stock ownership of Repare Therapeutics, and honoraria for ad hoc participation in the Scientific Advisory Board of Repare Therapeutics, Paige.AI, Roche Tissue Diagnostics, Novartis, Roche/Genentech, Invicro and Personalis; JT reports consultancy fee from Haystack Oncology, receipt of honoraria for participation in Advisory Board from AstraZeneca, BMS, MSD, Inivata, Pierre Fabre, receipt of honoraria as invited speaker from Merck Serono, Amgen and Servier; JS reports ownership interest as a co-founder of Mosaic Biomedicals SL, member of Board of Directors of Northern Biologics Inc., receipt of institutional research grants from Roche Glycart AG, Mosaic Biomedicals SL, Hoffmann-La Roche Ltd, Northern Biologics Inc., Ridgeline Therapeutics, non-remunerated activities as a member of Board of Directors of Asociación Española Contra el Cáncer and a member of Board of Directors, Secretary General of the European Association for Cancer Research (AACR); TY reports receipt of honoraria as invited speaker from Bayer, Chugai, Eli Lilly, Merck Biopharma, Ono, Taiho, receipt of institutional research grants from Amgen, MSD, Ono, Taiho, receipt of institutional financial interest as a local PI from Chugai, Daiichi Sankyo, MSD, Ono, Parexel International, Sanofi and Sumitomo Dainippon; NT reports receipt of honoraria for participation in Advisory Board from Arvinas, AstraZeneca, BMS, GlaxoSmithKline, Lilly, MSD, Novartis, Pfizer, Repare Therapeutics, Roche/Genentech, Zentalis Pharmaceuticals, institutional funding for research from AstraZeneca, MSD, Pfizer, Roche/Genentech, institutional non-financial interest for provision of material for research from BioRad and for provision of assays from Guardant Health. JS is co-founder of Mosaic Biomedicals and has ownership interests from Mosaic Biomedicals and Northern Biologics and reports receipt of grant/research support from Mosaic Biomedicals, Northern Biologics, Roche/Glycart and Hoffmann la Roche. All other authors have declared no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Circulating tumor-derived DNA (ctDNA) is an emerging biomarker for many cancers, but the limited sensitivity of current detection methods reduces its utility for diagnosing minimal residual disease. Here we describe phased variant enrichment and detection sequencing (PhasED-seq), a method that uses multiple somatic mutations in individual DNA fragments to improve the sensitivity of ctDNA detection. Leveraging whole-genome sequences from 2,538 tumors, we identify phased variants and their associations with mutational signatures. We show that even without molecular barcodes, the limits of detection of PhasED-seq outperform prior methods, including duplex barcoding, allowing ctDNA detection in the ppm range in participant samples. We profiled 678 specimens from 213 participants with B cell lymphomas, including serial cell-free DNA samples before and during therapy for diffuse large B cell lymphoma. In participants with undetectable ctDNA after two cycles of therapy using a next-generation sequencing-based approach termed cancer personalized profiling by deep sequencing, an additional 25% have ctDNA detectable by PhasED-seq and have worse outcomes. Finally, we demonstrate the application of PhasED-seq to solid tumors., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Growing evidence demonstrates that circulating tumor DNA (ctDNA) minimal residual disease (MRD) following treatment for solid tumors predicts relapse. These results suggest that ctDNA MRD could identify candidates for adjuvant therapy and measure response to such treatment. Importantly, factors such as assay type, amount of ctDNA release, and technical and biological background can affect ctDNA MRD results. Furthermore, the clinical utility of ctDNA MRD for treatment personalization remains to be fully established. Here, we review the evidence supporting the value of ctDNA MRD in solid cancers and highlight key considerations in the application of this potentially transformative biomarker., Significance: ctDNA analysis enables detection of MRD and predicts relapse after definitive treatment for solid cancers, thereby promising to revolutionize personalization of adjuvant and consolidation therapies., (©2021 American Association for Cancer Research.)

Most circulating tumor DNA (ctDNA) assays are designed to detect recurrent mutations. Pediatric sarcomas share few recurrent mutations but rather are characterized by translocations and copy-number changes. We applied Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) for detection of translocations found in the most common pediatric sarcomas. We also applied ichorCNA to the combined off-target reads from our hybrid capture to simultaneously detect copy-number alterations (CNA). We analyzed 64 prospectively collected plasma samples from 17 patients with pediatric sarcoma. Translocations were detected in the pretreatment plasma of 13 patients and were confirmed by tumor sequencing in 12 patients. Two of these patients had evidence of complex chromosomal rearrangements in their ctDNA. We also detected copy-number changes in the pretreatment plasma of 7 patients. We found that ctDNA levels correlated with metastatic status and clinical response. Furthermore, we detected rising ctDNA levels before relapse was clinically apparent, demonstrating the high sensitivity of our assay. This assay can be utilized for simultaneous detection of translocations and CNAs in the plasma of patients with pediatric sarcoma. While we describe our experience in pediatric sarcomas, this approach can be applied to other tumors that are driven by structural variants., (©2021 The Authors; Published by the American Association for Cancer Research.)

Purpose: Patients with Diffuse Large B-cell Lymphoma (DLBCL) in need of immediate therapy are largely under-represented in clinical trials. The diagnosis-to-treatment interval (DTI) has recently been described as a metric to quantify such patient selection bias, with short DTI being associated with adverse risk factors and inferior outcomes. Here, we characterized the relationships between DTI, circulating tumor DNA (ctDNA), conventional risk factors, and clinical outcomes, with the goal of defining objective disease metrics contributing to selection bias., Patients and Methods: We evaluated pretreatment ctDNA levels in 267 patients with DLBCL treated across multiple centers in Europe and the United States using Cancer Personalized Profiling by Deep Sequencing. Pretreatment ctDNA levels were correlated with DTI, total metabolic tumor volumes (TMTVs), the International Prognostic Index (IPI), and outcome., Results: Short DTI was associated with advanced-stage disease ( P < .001) and higher IPI ( P < .001). We also found an inverse correlation between DTI and TMTV ( R S = - 0.37; P < .001). Similarly, pretreatment ctDNA levels were significantly associated with stage, IPI, and TMTV (all P < .001), demonstrating that both DTI and ctDNA reflect disease burden. Notably, patients with shorter DTI had higher pretreatment ctDNA levels ( P < .001). Pretreatment ctDNA levels predicted short DTI independent of the IPI ( P < .001). Although each risk factor was significantly associated with event-free survival in univariable analysis, ctDNA level was prognostic of event-free survival independent of DTI and IPI in multivariable Cox regression (ctDNA: hazard ratio, 1.5; 95% CI [1.2 to 2.0]; IPI: 1.1 [0.9 to 1.3]; -DTI: 1.1 [1.0 to 1.2])., Conclusion: Short DTI largely reflects baseline tumor burden, which can be objectively measured using pretreatment ctDNA levels. Pretreatment ctDNA levels therefore have utility for quantifying and guarding against selection biases in prospective DLBCL clinical trials., Competing Interests: David M. KurtzStock and Other Ownership Interests: Foresight DiagnosticsConsulting or Advisory Role: Roche Molecular Diagnostics, GenentechPatents, Royalties, Other Intellectual Property: Patent filings on ctDNA detection and methods for treatment selection based on statistical frameworks, assigned to Stanford University Ulrich DührsenHonoraria: Roche Pharma AG, Janssen-Cilag, Novartis, Amgen, CPT Cellex Patient Treatment GmbHConsulting or Advisory Role: Takeda, Gilead Sciences, AbbvieResearch Funding: CelgeneTravel, Accommodations, Expenses: Abbvie, Janssen-Cilag Andreas HüttmannHonoraria: TakedaConsulting or Advisory Role: TakedaTravel, Accommodations, Expenses: Takeda, Roche Pharma AG Christine SchmitzResearch Funding: NovartisTravel, Accommodations, Expenses: Abbvie Brian J. SworderStock and Other Ownership Interests: Allogene TherapeuticsPatents, Royalties, Other Intellectual Property: Dr Sworder has patents related to methods of resistance to immunotherapy for lymphoma Barzin Y. NabetEmployment: Roche/GenentechStock and Other Ownership Interests: Roche/GenentechPatents, Royalties, Other Intellectual Property: Dr Nabet has a patent pending related to immunomodulatory nucleic acids, Provisional patent related to noninvasive means to predict immune checkpoint blockade outcomes Olivier CasasnovasHonoraria: Roche/Genentech, Takeda, Gilead Sciences, Bristol-Myers Squibb, Merck, Abbvie, Celgene, Janssen, AmgenConsulting or Advisory Role: Roche/Genentech, Takeda, Gilead Sciences, Bristol-Myers Squibb, Merck, Abbvie, Celgene, Janssen, IncyteResearch Funding: Roche/Genentech, Gilead Sciences, TakedaTravel, Accommodations, Expenses: Roche/Genentech, Takeda, Gilead Sciences, Janssen Jason R. WestinConsulting or Advisory Role: Novartis, Celgene, Genentech/Abbvie, Kite/Gilead, Janssen Scientific Affairs, Amgen, MorphoSys, Juno Therapeutics, CurisResearch Funding: Celgene, Janssen, Genentech, Novartis, Kite/Gilead, Bristol-Myers Squibb, Curis Gianluca GaidanoHonoraria: Janssen, Abbvie, Sunesis Pharmaceuticals, AstraZenecaConsulting or Advisory Role: Janssen, Abbvie, AstraZeneca, Sunesis PharmaceuticalsSpeakers' Bureau: Janssen, AbbvieTravel, Accommodations, Expenses: Janssen, Amgen Davide RossiHonoraria: Abbvie, Janssen, AstraZenecaResearch Funding: Abbvie, Janssen, AstraZeneca Michel MeignanHonoraria: RocheTravel, Accommodations, Expenses: Roche Maximilian DiehnStock and Other Ownership Interests: CiberMed, Foresight DiagnosticsConsulting or Advisory Role: Roche, AstraZeneca, Illumina, Reflexion Medical, Gritstone Oncology, BioNTech AG, Novartis, GenentechResearch Funding: Varian Medical Systems, IlluminaPatents, Royalties, Other Intellectual Property: Patent filings on ctDNA detection assigned to Stanford University, Patent filings on tumor treatment resistance mechanisms assigned to Stanford UniversityTravel, Accommodations, Expenses: Roche, Varian Medical Systems, AstraZeneca Ash A. AlizadehLeadership: Lymphoma Research FoundationStock and Other Ownership Interests: CiberMed, CAPP Medical, Forty Seven, Foresight DiagnosticsHonoraria: Roche, Janssen OncologyConsulting or Advisory Role: Celgene, Roche/Genentech, Gilead Sciences, Cibermed, Foresight DiagnosticsResearch Funding: CelgenePatents, Royalties, Other Intellectual Property: Patent filings on ctDNA detection, assigned to Stanford UniversityTravel, Accommodations, Expenses: Roche, Gilead SciencesNo other potential conflicts of interest were reported.

Because cancer is caused by an accumulation of genetic mutations, mutant DNA released by tumors can be used as a highly specific biomarker for cancer. Although this principle was described decades ago, the advent and falling costs of next-generation sequencing have made the use of tumor DNA as a biomarker increasingly practical. This review surveys the use of cellular and cell-free DNA for the detection of cancer, with a focus on recent technological developments and applications to solid tumors. It covers ( a ) key principles and technology enabling the highly sensitive detection of tumor DNA; ( b ) assessment of tumor DNA in plasma, including for genotyping, minimal residual disease detection, and early detection of localized cancer; ( c ) detection of tumor DNA in body cavity fluids, such as urine or cerebrospinal fluid; and ( d ) challenges posed to the use of tumor DNA as a biomarker by the phenomenon of benign clonal expansions.

An increasing number of studies are describing potential uses of circulating tumour DNA (ctDNA) in the care of patients with colorectal cancer. Owing to this rapidly developing area of research, the Colon and Rectal-Anal Task Forces of the United States National Cancer Institute convened a panel of multidisciplinary experts to summarize current data on the utility of ctDNA in the management of colorectal cancer and to provide guidance in promoting the efficient development and integration of this technology into clinical care. The panel focused on four key areas in which ctDNA has the potential to change clinical practice, including the detection of minimal residual disease, the management of patients with rectal cancer, monitoring responses to therapy, and tracking clonal dynamics in response to targeted therapies and other systemic treatments. The panel also provides general guidelines with relevance for ctDNA-related research efforts, irrespective of indication.

Although treatment of non-small cell lung cancer (NSCLC) with immune checkpoint inhibitors (ICIs) can produce remarkably durable responses, most patients develop early disease progression. Furthermore, initial response assessment by conventional imaging is often unable to identify which patients will achieve durable clinical benefit (DCB). Here, we demonstrate that pre-treatment circulating tumor DNA (ctDNA) and peripheral CD8 T cell levels are independently associated with DCB. We further show that ctDNA dynamics after a single infusion can aid in identification of patients who will achieve DCB. Integrating these determinants, we developed and validated an entirely noninvasive multiparameter assay (DIREct-On, Durable Immunotherapy Response Estimation by immune profiling and ctDNA-On-treatment) that robustly predicts which patients will achieve DCB with higher accuracy than any individual feature. Taken together, these results demonstrate that integrated ctDNA and circulating immune cell profiling can provide accurate, noninvasive, and early forecasting of ultimate outcomes for NSCLC patients receiving ICIs., Competing Interests: Declaration of Interests J.J.C. reports paid consultancy from Lexent Bio Inc. A.A.C. reports speaker honoraria and travel support from Roche Sequencing Solutions (RSS), Varian, and Foundation Medicine; a research grant from RSS; and has served as a paid consultant for Oscar Health. T.M. is a co-founder of Imvaq. J.W.N. reports research support from Genentech (GNE)/Roche, Merck, Novartis, Boehringer Ingelheim, Exelixis, Takeda Pharmaceuticals, Nektar Therapeutics, Adaptimmune, and GSK, and has served in a consulting or advisory role for AstraZeneca (AZ), GNE/Roche, Exelixis Inc., Jounce Therapeutics, Takeda Pharmaceuticals, and Eli Lilly. H.A.W. has received honoraria from Novartis and AZ and has participated on the advisory boards of Xcovery, Janssen, and Mirati. S.K.P. reports grant support from EpicentRx, Forty Seven, Bayer, and Boehringer Ingelheim and serves in a consulting or advisory role for AZ, AbbVie, G1 Therapeutics, and Pfizer. M. Das reports grant support from AbbVie, United Therapeutics, Varian, and Celgene and serves in a consulting role for AZ and Bristol-Myers Squibb (BMS). A.M.N. has patent filings related to expression deconvolution and cancer biomarkers and has served as a consultant for Roche, Merck, and CiberMed. M.D.H. reports paid consultancy from BMS, Merck, GNE, AZ/MedImmune, Nektar, Syndax, Janssen, Mirati Therapeutics, Shattuck Labs, and Blueprint Medicines; travel/honoraria from BMS and AZ; research funding from BMS; and a patent has been filed by MSK related to the use of tumor mutation burden to predict response to immunotherapy (PCT/US2015/062208), which has received licensing fees from Personal Genome Diagnostics. A.A.A. reports ownership interest in CiberMed and FortySeven, patent filings related to cancer biomarkers, and paid consultancy from GNE, Roche, Chugai, Gilead, and Celgene. M. Diehn reports research funding from Varian, ownership interest in CiberMed, patent filings related to cancer biomarkers, paid consultancy from Roche, AZ, and BioNTech, and travel/honoraria from Reflexion. M.Diehn, A.A.A., B.Y.N., and M.S.E. are co-inventors on a provisional patent application filed by Stanford University relating to this manuscript., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Purpose: Treatment with PD-(L)1 blockade can produce remarkably durable responses in patients with non-small cell lung cancer (NSCLC). However, a significant fraction of long-term responders ultimately progress and predictors of late progression are unknown. We hypothesized that circulating tumor DNA (ctDNA) analysis of long-term responders to PD-(L)1 blockade may differentiate those who will achieve ongoing benefit from those at risk of eventual progression., Experimental Design: In patients with advanced NSCLC achieving long-term benefit from PD-(L)1 blockade (progression-free survival ≥ 12 months), plasma was collected at a surveillance timepoint late during/after treatment to interrogate ctDNA by Cancer Personalized Profiling by Deep Sequencing. Tumor tissue was available for 24 patients and was profiled by whole-exome sequencing ( n = 18) or by targeted sequencing ( n = 6)., Results: Thirty-one patients with NSCLC with long-term benefit to PD-(L)1 blockade were identified, and ctDNA was analyzed in surveillance blood samples collected at a median of 26.7 months after initiation of therapy. Nine patients also had baseline plasma samples available, and all had detectable ctDNA prior to therapy initiation. At the surveillance timepoint, 27 patients had undetectable ctDNA and 25 (93%) have remained progression-free; in contrast, all 4 patients with detectable ctDNA eventually progressed [Fisher P < 0.0001; positive predictive value = 1, 95% confidence interval (CI), 0.51-1; negative predictive value = 0.93 (95% CI, 0.80-0.99)]., Conclusions: ctDNA analysis can noninvasively identify minimal residual disease in patients with long-term responses to PD-(L)1 blockade and predict the risk of eventual progression. If validated, ctDNA surveillance may facilitate personalization of the duration of immune checkpoint blockade and enable early intervention in patients at high risk for progression., (©2020 American Association for Cancer Research.)

Radiologic screening of high-risk adults reduces lung-cancer-related mortality 1,2 ; however, a small minority of eligible individuals undergo such screening in the United States 3,4 . The availability of blood-based tests could increase screening uptake. Here we introduce improvements to cancer personalized profiling by deep sequencing (CAPP-Seq) 5 , a method for the analysis of circulating tumour DNA (ctDNA), to better facilitate screening applications. We show that, although levels are very low in early-stage lung cancers, ctDNA is present prior to treatment in most patients and its presence is strongly prognostic. We also find that the majority of somatic mutations in the cell-free DNA (cfDNA) of patients with lung cancer and of risk-matched controls reflect clonal haematopoiesis and are non-recurrent. Compared with tumour-derived mutations, clonal haematopoiesis mutations occur on longer cfDNA fragments and lack mutational signatures that are associated with tobacco smoking. Integrating these findings with other molecular features, we develop and prospectively validate a machine-learning method termed 'lung cancer likelihood in plasma' (Lung-CLiP), which can robustly discriminate early-stage lung cancer patients from risk-matched controls. This approach achieves performance similar to that of tumour-informed ctDNA detection and enables tuning of assay specificity in order to facilitate distinct clinical applications. Our findings establish the potential of cfDNA for lung cancer screening and highlight the importance of risk-matching cases and controls in cfDNA-based screening studies.

Background & Aims: Biomarkers are needed to risk stratify after chemoradiotherapy for localized esophageal cancer. These could improve identification of patients at risk for cancer progression and selection of additional therapy., Methods: We performed deep sequencing (CAncer Personalized Profiling by deep Sequencing, [CAPP-Seq]) analyses of plasma cell-free DNA collected from 45 patients before and after chemoradiotherapy for esophageal cancer, as well as DNA from leukocytes and fixed esophageal tumor biopsy samples collected during esophagogastroduodenoscopy. Patients were treated from May 2010 through October 2015; 23 patients subsequently underwent esophagectomy, and 22 did not undergo surgery. We also sequenced DNA from blood samples from 40 healthy control individuals. We analyzed 802 regions of 607 genes for single-nucleotide variants previously associated with esophageal adenocarcinoma or squamous cell carcinoma. Patients underwent imaging analyses 6-8 weeks after chemoradiotherapy and were followed for 5 years. Our primary aim was to determine whether detection of circulating tumor DNA (ctDNA) after chemoradiotherapy is associated with risk of tumor progression (growth of local, regional, or distant tumors, detected by imaging or biopsy)., Results: The median proportion of tumor-derived DNA in total cell-free DNA before treatment was 0.07%, indicating that ultrasensitive assays are needed for quantification and analysis of ctDNA from localized esophageal tumors. Detection of ctDNA after chemoradiotherapy was associated with tumor progression (hazard ratio, 18.7; P < .0001), formation of distant metastases (hazard ratio, 32.1; P < .0001), and shorter disease-specific survival times (hazard ratio, 23.1; P < .0001). A higher proportion of patients with tumor progression had new mutations detected in plasma samples collected after chemoradiotherapy than patients without progression (P = .03). Detection of ctDNA after chemoradiotherapy preceded radiographic evidence of tumor progression by an average of 2.8 months. Among patients who received chemoradiotherapy without surgery, combined ctDNA and metabolic imaging analysis predicted progression in 100% of patients with tumor progression, compared with 71% for only ctDNA detection and 57% for only metabolic imaging analysis (P < .001 for comparison of either technique to combined analysis)., Conclusions: In an analysis of cell-free DNA in blood samples from patients who underwent chemoradiotherapy for esophageal cancer, detection of ctDNA was associated with tumor progression, metastasis, and disease-specific survival. Analysis of ctDNA might be used to identify patients at highest risk for tumor progression., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Circulating tumor DNA (ctDNA) molecular residual disease (MRD) following curative-intent treatment strongly predicts recurrence in multiple tumor types, but whether further treatment can improve outcomes in patients with MRD remains unclear. We applied CAPP-Seq ctDNA analysis to 218 samples from 65 patients receiving chemoradiation therapy (CRT) for locally advanced NSCLC, including 28 patients receiving consolidation immune checkpoint inhibition (CICI). Patients with undetectable ctDNA after CRT had excellent outcomes whether or not they received CICI. Among such patients, one died from CICI-related pneumonitis, highlighting the potential utility of only treating patients with MRD. In contrast, patients with MRD after CRT who received CICI had significantly better outcomes than patients who did not receive CICI. Furthermore, the ctDNA response pattern early during CICI identified patients responding to consolidation therapy. Our results suggest that CICI improves outcomes for NSCLC patients with MRD and that ctDNA analysis may facilitate personalization of consolidation therapy.

Purpose.—: Clinical use of analytical tests to assess genomic variants in circulating tumor DNA (ctDNA) is increasing. This joint review from the American Society of Clinical Oncology and the College of American Pathologists summarizes current information about clinical ctDNA assays and provides a framework for future research., Methods.—: An Expert Panel conducted a literature review on the use of ctDNA assays for solid tumors, including preanalytical variables, analytical validity, interpretation and reporting, and clinical validity and utility., Results.—: The literature search identified 1338 references. Of those, 390, plus 31 references supplied by the Expert Panel, were selected for full-text review. There were 77 articles selected for inclusion., Conclusions.—: The evidence indicates that testing for ctDNA is optimally performed on plasma collected in cell stabilization or EDTA tubes, with EDTA tubes processed within 6 hours of collection. Some ctDNA assays have demonstrated clinical validity and utility with certain types of advanced cancer; however, there is insufficient evidence of clinical validity and utility for the majority of ctDNA assays in advanced cancer. Evidence shows discordance between the results of ctDNA assays and genotyping tumor specimens, and supports tumor tissue genotyping to confirm undetected results from ctDNA tests. There is no evidence of clinical utility and little evidence of clinical validity of ctDNA assays in early-stage cancer, treatment monitoring, or residual disease detection. There is no evidence of clinical validity or clinical utility to suggest that ctDNA assays are useful for cancer screening, outside of a clinical trial. Given the rapid pace of research, reevaluation of the literature will shortly be required, along with the development of tools and guidance for clinical practice.

Purpose: Outcomes for patients with diffuse large B-cell lymphoma remain heterogeneous, with existing methods failing to consistently predict treatment failure. We examined the additional prognostic value of circulating tumor DNA (ctDNA) before and during therapy for predicting patient outcomes., Patients and Methods: We studied the dynamics of ctDNA from 217 patients treated at six centers, using a training and validation framework. We densely characterized early ctDNA dynamics during therapy using cancer personalized profiling by deep sequencing to define response-associated thresholds within a discovery set. These thresholds were assessed in two independent validation sets. Finally, we assessed the prognostic value of ctDNA in the context of established risk factors, including the International Prognostic Index and interim positron emission tomography/computed tomography scans., Results: Before therapy, ctDNA was detectable in 98% of patients; pretreatment levels were prognostic in both front-line and salvage settings. In the discovery set, ctDNA levels changed rapidly, with a 2-log decrease after one cycle (early molecular response [EMR]) and a 2.5-log decrease after two cycles (major molecular response [MMR]) stratifying outcomes. In the first validation set, patients receiving front-line therapy achieving EMR or MMR had superior outcomes at 24 months (EMR: EFS, 83% v 50%; P = .0015; MMR: EFS, 82% v 46%; P < .001). EMR also predicted superior 24-month outcomes in patients receiving salvage therapy in the first validation set (EFS, 100% v 13%; P = .011). The prognostic value of EMR and MMR was further confirmed in the second validation set. In multivariable analyses including International Prognostic Index and interim positron emission tomography/computed tomography scans across both cohorts, molecular response was independently prognostic of outcomes, including event-free and overall survival., Conclusion: Pretreatment ctDNA levels and molecular responses are independently prognostic of outcomes in aggressive lymphomas. These risk factors could potentially guide future personalized risk-directed approaches.

Circulating tumor DNA (ctDNA) shed from cancer cells into the peripheral blood can be non-invasively collected and tested for the presence of tumor-specific mutations. Mutations identified in ctDNA can predict responses to targeted therapies and emerging evidence suggests that changes in ctDNA levels over time can be used to monitor response to therapy and detect disease recurrence. Given the emergence of targeted therapies in advanced non-small cell lung cancer (NSCLC), liquid biopsies utilizing ctDNA testing represent a powerful approach to genotype tumors and monitor for the development of resistance. Here, we review current and potential future clinical applications of ctDNA testing for patients with advanced NSCLC., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Identifying molecular residual disease (MRD) after treatment of localized lung cancer could facilitate early intervention and personalization of adjuvant therapies. Here, we apply cancer personalized profiling by deep sequencing (CAPP-seq) circulating tumor DNA (ctDNA) analysis to 255 samples from 40 patients treated with curative intent for stage I-III lung cancer and 54 healthy adults. In 94% of evaluable patients experiencing recurrence, ctDNA was detectable in the first posttreatment blood sample, indicating reliable identification of MRD. Posttreatment ctDNA detection preceded radiographic progression in 72% of patients by a median of 5.2 months, and 53% of patients harbored ctDNA mutation profiles associated with favorable responses to tyrosine kinase inhibitors or immune checkpoint blockade. Collectively, these results indicate that ctDNA MRD in patients with lung cancer can be accurately detected using CAPP-seq and may allow personalized adjuvant treatment while disease burden is lowest. Significance: This study shows that ctDNA analysis can robustly identify posttreatment MRD in patients with localized lung cancer, identifying residual/recurrent disease earlier than standard-of-care radiologic imaging, and thus could facilitate personalized adjuvant treatment at early time points when disease burden is lowest. Cancer Discov; 7(12); 1394-403. ©2017 AACR. See related commentary by Comino-Mendez and Turner, p. 1368 This article is highlighted in the In This Issue feature, p. 1355 ., (©2017 American Association for Cancer Research.)

Patients with diffuse large B cell lymphoma (DLBCL) exhibit marked diversity in tumor behavior and outcomes, yet the identification of poor-risk groups remains challenging. In addition, the biology underlying these differences is incompletely understood. We hypothesized that characterization of mutational heterogeneity and genomic evolution using circulating tumor DNA (ctDNA) profiling could reveal molecular determinants of adverse outcomes. To address this hypothesis, we applied cancer personalized profiling by deep sequencing (CAPP-Seq) analysis to tumor biopsies and cell-free DNA samples from 92 lymphoma patients and 24 healthy subjects. At diagnosis, the amount of ctDNA was found to strongly correlate with clinical indices and was independently predictive of patient outcomes. We demonstrate that ctDNA genotyping can classify transcriptionally defined tumor subtypes, including DLBCL cell of origin, directly from plasma. By simultaneously tracking multiple somatic mutations in ctDNA, our approach outperformed immunoglobulin sequencing and radiographic imaging for the detection of minimal residual disease and facilitated noninvasive identification of emergent resistance mutations to targeted therapies. In addition, we identified distinct patterns of clonal evolution distinguishing indolent follicular lymphomas from those that transformed into DLBCL, allowing for potential noninvasive prediction of histological transformation. Collectively, our results demonstrate that ctDNA analysis reveals biological factors that underlie lymphoma clinical outcomes and could facilitate individualized therapy., (Copyright © 2016, American Association for the Advancement of Science.)

Circulating tumour DNA (ctDNA) analysis facilitates studies of tumour heterogeneity. Here we employ CAPP-Seq ctDNA analysis to study resistance mechanisms in 43 non-small cell lung cancer (NSCLC) patients treated with the third-generation epidermal growth factor receptor (EGFR) inhibitor rociletinib. We observe multiple resistance mechanisms in 46% of patients after treatment with first-line inhibitors, indicating frequent intra-patient heterogeneity. Rociletinib resistance recurrently involves MET, EGFR, PIK3CA, ERRB2, KRAS and RB1. We describe a novel EGFR L798I mutation and find that EGFR C797S, which arises in ∼33% of patients after osimertinib treatment, occurs in <3% after rociletinib. Increased MET copy number is the most frequent rociletinib resistance mechanism in this cohort and patients with multiple pre-existing mechanisms (T790M and MET) experience inferior responses. Similarly, rociletinib-resistant xenografts develop MET amplification that can be overcome with the MET inhibitor crizotinib. These results underscore the importance of tumour heterogeneity in NSCLC and the utility of ctDNA-based resistance mechanism assessment.

B A. A Alizadeh b Foresight Diagnostics: Consultancy, Current equity holder in private company, Patents & Royalties; Gilead: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties; Syncopation: Current equity holder in private company, Patents & Royalties; Roche: Consultancy; Adaptive Biotechnologies: Consultancy; Karyopharm: Consultancy; BMS: Consultancy, Research Funding; Genentech: Consultancy; Cibermed Inc: Consultancy, Current equity holder in private company, Patents & Royalties. B Conflicts of interests pertinent to the abstract b B D. M. Kurtz b Other remuneration: Roche: Consultancy; Adaptive Biotechnologies: Consultancy; Foresight Diagnostics: Consultancy, Current equity holder in private company, Patents & Royalties; Genentech: Consultancy. B W. Wilson b Other remuneration: Foresight Diagnostics: Consultancy, Current equity holder in private company, Patents & Royalties; Gilead: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties; Syncopation: Current equity holder in private company, Patents & Royalties; Roche: Consultancy; Adaptive Biotechnologies: Consultancy; Karyopharm: Consultancy; BMS: Consultancy, Research Funding; Genentech: Consultancy; Cibermed Inc: Consultancy, Current equity holder in private company, Patents & Royalties. [Extracted from the article]

Distinct molecular determinants of treatment-failure in elderly Hodgkin lymphoma identified by cell-free DNA profiling: A LYSA Study Interestingly, pre-treatment ctDNA levels did not differ between EBV+ and EBV- tumors ( I p i = 0.92), while specific genetic aberrations were significantly associated with respective EBV status (Figure C). Older HL pts, especially those with high baseline ctDNA levels and/or EBV+ tumors, have an exceedingly high risk of treatment failure with conventional chemotherapy, and likely require future risk-adapted strategies. [Extracted from the article]

Trimodality therapy with chemoradiation (CRT) followed by esophagectomy is the standard of care for locally advanced esophageal cancer. An unresolved question is whether pathologic complete response (pCR) can be assessed non-invasively for patients post-CRT. In this study, we assessed whether diffusion-weighted imaging (DWI) with MRI or PET can be used as predictors of pCR and other clinical outcomes after CRT. Patients were enrolled on a single-arm institutional trial (PA13-0380) assessing the role of imaging in predicting outcomes in potentially resectable esophageal patients undergoing trimodality therapy. All patients received neoadjuvant CRT, and 29 patients had subsequent surgery. DWI MRI and PET scans were obtained at baseline, 2 weeks after the start of CRT (interim) and 4 to 6 weeks after completion of CRT (follow up). Apparent diffusion coefficients (ADCs) were calculated based on DWI images. Circulating tumor DNA was obtained for 27 patients post-radiation using CAPP-Seq. Mann-Whitney tests compared imaging changes associated with pCR. Discrimination of pCR by imaging changes was quantified by received operating characteristics. Youden's index was applied to select optimal thresholds. Kaplan-Meier analysis was performed to assess differences in overall survival (OS) and progression-free survival (PFS) by changes in DWI, PET, and ctDNA parameters. Our cohort of 60 patients had a median follow up of 42.7 months, age of 65.4 yrs, and ECOG of 1 at completion of CRT. 90% were male, 58% had a history of smoking, and 85% were white. 83% had adenocarcinoma with the rest squamous cell carcinoma. Stages of the patients ranged from IIA to IIIB. All had moderately (47%) or poorly (53%) differentiated disease. All received 41.4-50.4 Gy in 1.8 Gy fractions with the majority receiving 50.4 Gy (95%). 29 patients underwent surgery after CRT of which 8 (27.6%) had pCR. Mean ΔADC from baseline to mid-treatment was most associated with pCR (AUC = 0.98, p<0.001) for patients undergoing surgery. Max ΔADC from baseline to first follow-up was most associated with OS (p = 0.002) and PFS (p<0.001) for the whole cohort. 27 patients had ctDNA analyzed after RT with the presence of ctDNA significantly associated with worse OS (HR = 0.12, p = 0.05) and PFS (HR = 0.10, p = 0.002). Combining ctDNA and max ΔADC generated a model that was more predictive of OS and PFS than either alone. We found that neither the PET parameters of TLG or SUV max at baseline or changes in these parameters from baseline to mid-treatment or first follow-up were as predictive as DWI. We show that changes in DWI is associated with pCR, OS, and PFS in resectable esophageal cancer patients undergoing CRT. DWI was more predictive than PET and a model combining DWI and ctDNA was the most predictive of clinical outcomes. This study shows the significant promise of using DWI in potentially guiding treatment decisions in esophageal cancer patients and will require validation in a larger cohort. [ABSTRACT FROM AUTHOR]