Abbas, S

Aleshin, A

Ang, Y

Bagwan, I

Beggs, A

Berrisford, R

Carroll, N

Chan, D

Chang, F

Cheah, C

Cheong, E

Ciccarelli, FD

Coleman, H

Coles, H

Contino, G

Crichton, C

Davies, A

Davies, J

Devonshire, G

Edwards, PAW

Eldridge, M

Fidziukiewicz, E

Fitzgerald, RC

Flint, C

Goh, V

Gossage, J

Grabowska, A

Grace, BL

Grantham, A

Grehan, N

Haidry, R

Hanna, GB

Hardwick, RH

Hayes, SJ

Hindmarsh, A

Hupp, TR

Jammula, S

Kaye, P

Kumar, B

Lagergren, J

Lishman, S

Lovat, L

Mahadeva, U

Malhotra, S

McManus, D

Miremadi, A

Moorthy, K

Northrop, A

Nutzinger, B

O'Donovan, M

O'Neill, JR

OCCAMS Consortium, .

Ococks, E

Parsons, SL

Peters, CJ

Petty, RD

Preston, SR

Puig, S

Redmond, AM

Safranek, P

Sanders, G

Saunders, J

Save, V

Scott, M

Secrier, M

Sharma, S

Sharrocks, A

Skipworth, RJE

Smyth, E

Smyth, EC

Soomro, I

Sothi, S

Sreedharan, L

Sujendran, V

Taniere, P

Tian Ng, AW

Tripathi, M

Tucker, O

Turkington, R

Underwood, TJ

Walker, RC

English

UCL Discovery

Gastroenterology 2021;161:1705–1708Serial Circulating Tumor DNA Detection Using a Personalized,Tumor-Informed Assay in Esophageal Adenocarcinoma PatientsFollowing ResectionHLETTERSdenocarcinoma of the esophagus is rapidly1Most current article© 2021 by the AGA Institute. Published by Elsevier Inc. This is an openaccess article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).0016-5085https://doi.org/10.1053/j.gastro.2021.07.011RESEARCAincreasing in incidence. Esophageal adenocarci-noma (EAC) is frequently advanced at presentation, andeven when treated with multimodality therapy, is cured inless than 50% of operated-on patients.2,3Circulating tumor DNA (ctDNA) has shown promise as aprognostic tool in multiple cancers and is a predictivebiomarker for treatment in non-small cell lung cancer.4,5We recently confirmed the prognostic value of ctDNA us-ing a non-EAC–specific panel in a large population ofresected EAC.6 In brief, patients who were ctDNA-positiveafter resection had worse survival than ctDNA-negativepatients (hazard ratio, 5.55; 95% confidence interval,2.42-12.71; P ¼ .0003).6 However, the sensitivity of a tu-mor-naïve panel for detecting recurrence was only 35%,implying many patients who recur are not detected.6 In thisstudy, we tested whether a personalized, tumor-informedassay would demonstrate superior sensitivity for detect-ing minimal residual disease (MRD) in patients withresected EAC.In this retrospective study, blood samples were collectedfrom 20 patients with EAC who underwent surgery orendoscopic mucosal resection (EMR). Blood samples werecollected before and after surgical treatment. This study wasconducted in accordance with the International Conferenceon Harmonization-Good Clinical Practice Guidelines andapproved by the United Kingdom National Ethics Frame-work (LREC, 10-H0305-1). All patients provided writteninformed consent.We identified tumor-specific variants using whole-genome sequencing data from our International CancerGenome Consortium project, mean coverage: 73x (tumor)and 37x (blood reference).7 We then used 16 of thesepatient-specific somatic single-nucleotide variants to designindividualized multiplex polymerase chain reaction-basedprimers for next-generation sequencing, used to identifyctDNA in patient plasma.For survival analysis, only patients who underwentsurgery were included. Patients who underwent EMR wereexpected to be cured and were excluded. Survival estimateswere calculated using the Kaplan-Meier method, and sur-vival plots were created using “survminer” R 0.4.4 software(R Foundation for Statistical Computing). Survival differ-ences were evaluated by univariate Cox regression analysisusing the “survival” R 2.44-1.1 package. P values weredetermined using the log-likelihood test.At least 1 sample was taken from all patients before andafter tumor removal (Figure 1A).Patient characteristics were consistent with those ex-pected in patients with EAC (median age, 62 years; 85%men) (Supplementary Table 1). Most (17 of 20 [85%]) weretreated with perioperative chemotherapy.Patients with deeper penetration of the gastroesopha-geal mucosa were more likely to have ctDNA identifiedpreoperatively (9 of 12 [75%] cT3 vs 2 of 5 [40%] T2);however, groups were similar with respect to cN, yN, andlymphovascular invasion (Supplementary Figure 1A). Allpatients that recurred were ctDNA-positive at baseline(100% sensitivity, P < .0001) (Supplementary Figure 1B).Patients who were ctDNA-positive before surgery hadsignificantly poorer disease-free survival (DFS) (P ¼ .042),with a median DFS of 32.0 months vs 63.0 months in ctDNA-negative preoperative patients. There was also a trend to-wards poorer cancer-specific survival (SupplementaryFigure 1C and D). None of the presurgical ctDNA-negativepatients relapsed after surgery (Supplementary Figure 1C).Of the 11 presurgical ctDNA-positive patients, 5 (45%)relapsed after surgery.Four patients were ctDNA-positive after surgery andrelapsed, 1 patient, who was ctDNA-negative, developedrecurrence 2.6 years after the last ctDNA testing, leading toa sensitivity of 80% (4 of 5) and specificity of 100% (12 of12). Median DFS was 14.2 months vs 51.2 months in ctDNA-positive vs ctDNA-negative in postoperative patients,respectively (Figure 1B), and median cancer-specific sur-vival was 18.0 months vs 53.4 months (Figure 1C). ctDNA-positivity at this time point was associated with inferiorDFS (P < .0001).When patients who did not have a plasmasample within 1 year of relapse were excluded, sensitivityand specificity were 100%. The median ctDNA variant allelefraction detected in positive samples after surgery was0.01% (range, 0.001%-15.9%). Response to neoadjuvantchemotherapy was reflected in the ctDNA fraction; a patientwith a complete response to neoadjuvant chemotherapywas ctDNA-negative after treatment (Figure 1D). In contrastresidual disease was detected in patients who had a poorresponse to neoadjuvant chemotherapy, including a patientwhere the ctDNA fraction increased during treatment(Figure 1E).To our knowledge, this study is the first to investigate theuse of a tumor-informed ctDNA assay to detect MRD inresected EAC. We demonstrate excellent sensitivity and spec-ificity of personalized ctDNA assays for the detection of ctDNAin patients after surgical resection. Recurrent disease devel-oped in all patients with ctDNA detected postoperatively. Thissensitive ctDNA assay provided amedian lead time of almost 1year before clinical or radiologic recurrence.One patient who was ctDNA-negative 6 months post-operatively developed a late potentially low ctDNA sheddingperitoneal recurrence >4 years after surgery; the last ctDNAsample available for this patient was >2 years beforeABDctDNA (Mean mutant molecules/mL)SurgeryctDNA AveragectDNA PositivectDNA NegativectDNA valuesPET/CT PositiveCisplatin/5’FUCarboplatin/TaxolPost surgery status Negative Positive Post surgery status Negative PositiveCP20P19P18P17P16P15P14P13P12P11P10P09P08P07P06P05P04P03P02P010 25 50 75Months from diagnosisStageIIIBIIIIVAClinical timepointEMRChemoSurgeryRecurrencectDNA statusNegativePositiveEnd pointIn_follow_upLost_to_follow_upFollow_up_endedDied_OACDied_not_OACDied_unknownP < .0001 P = .0027E-100 1000 200 300 400 500 6000110100Time Relative to Surgery (days)Patient 17ctDNA (Mean mutant molecules/mL)-100 0 100 200 3000110100Patient 16Time Relative to Surgery (days)Figure 1. (A) Sample timelines of the 20 patients (P) in the cohort. (B) Disease-free survival (DFS) in patients according tocirculating tumor (ct)DNA status post-surgery. (C) Cancer-related survival in patients according to postsurgical ctDNA status.(D) Patient who had a good response to chemotherapy, tumor regression grade 1, lead time on patient, 278 days. (E) Patientremained ctDNA-positive throughout treatment, and lead time was >500 days. CT, computed tomography; EMR, endoscopicmucosal resection; OAC, oesophageal adenocarcinoma; PET, positron emission tomography; 50FU, 50-fluorouracil.1706 Ococks et al Gastroenterology Vol. 161, No. 5RESEARCHLETTERSNovember 2021 ctDNA Detection After EAC Resection 1707RESEARCHLETTERSrelapse. This implies both temporal and anatomic reasonsfor the lack of a ctDNA-positive result predicting relapse forthis patient. Interestingly, ctDNA preoperatively wasmodestly prognostic, and this was also associated with tu-mor stage. Crucially, patients who were ctDNA-positivepreoperatively and became ctDNA-negative after surgeryhad a good prognosis, indicating that ctDNA is a valuabledynamic biomarker.In colorectal cancer, individualized ctDNA assessmentafter surgery can be considered a standard of care while thepredictive value of such assays is under investigation inlarge, randomised trials.8 In resected EAC, in part due tosurgical morbidity, fewer than half of the patients currentlyundergo the adjuvant component of perioperative chemo-therapy.3 The benefit of reserving adjuvant chemotherapyfor patients most likely to recur or switching to an alter-native regimen should be evaluated prospectively. In addi-tion, personalized ctDNA detection could also provideinsight on the most suitable treatment option for the patientbased on their ctDNA levels after neoadjuvant chemo-therapy. Our study also suggests that longitudinal moni-toring of ctDNA rather than a sample at a single time pointcould be valuable, because a minority of patients may havelate recurrences.This study is limited by modest sample size; however,given the robust, individualized methodology of ourapproach, we believe that these results are likely to begeneralizable.In summary, this study demonstrates that personalizedctDNA assays provide a tool with potential clinical applica-tion to predict relapse in patients with resected EAC. Thenext step will be to design prospective clinical trials thatrisk stratify adjuvant therapy based on MRD.Supplementary MaterialNote: To access the supplementary material accompanyingthis article, visit the online version of Gastroenterology atwww.gastrojournal.org, and at https://doi.org/10.1053/j.gastro.2021.07.011.EMMA OCOCKSMedical Research Council Cancer UnitHutchison/Medical Research Council Research CentreUniversity of CambridgeCambridge, United KingdomSHRUTI SHARMAResearch and Development DepartmentNatera, IncorporatedSan Carlos, CaliforniaALVIN WEI TIAN NGCancer Research UK Cambridge InstituteUniversity of CambridgeCambridge, United KingdomOCCAMS CONSORTIUMMedical Research Council Cancer UnitHutchison/Medical Research Council Research CentreUniversity of CambridgeCambridge, United KingdomALEXEY ALESHINMedical Affairs, OncologyNatera, IncorporatedSan Carlos, CaliforniaREBECCA C. FITZGERALDMedical Research Council Cancer UnitHutchison/Medical Research Council Research CentreUniversity of CambridgeCambridge, United KingdomELIZABETH SMYTHMedical OncologyCambridge University HospitalsNational Health Service Foundation TrustAddenbrooke’s HospitalCambridge, United KingdomReferences1. Edgren G, et al. Gut 2013;62:1406–1414.2. Cancer Research UK. https://www.cancerresearchuk.org/health-professional/cancer-statistics/incidence.3. Al-Batran SE, et al. Lancet 2019;393:1948–1957.4. Tie J, et al. Sci Transl Med 2016;8:346ra92.5. Abbosh C, Birkbak NJ, Wilson GA, Jamal-Hanjani M,Constantin T, Salari R, Le Quesne J, et al. Nature 2017;545:446–451.6. Ococks E, Frankell AM, Soler NM, et al. Ann Oncol2021;32:522–532.7. Frankell AM, et al. Nat Genet 2019;51:506–516.8. Tie J, Cohen JD, et al. JAMA Oncol 2019;5:1710–1717.Author names in bold designate shared co-first authorship.Received May 10, 2021. Accepted July 14, 2021.CorrespondenceAddress correspondence to: Rebecca C. Fitzgerald, MD, Medical ResearchCouncil Cancer Unit, Hutchison/Medical Research Council Research Centre,Box 197, Cambridge Biomedical Campus, Cambridge CB2 0XZ, UnitedKingdom. e-mail: rcf29@cam.ac.uk.Data Transparency StatementWhole-genome sequencing is available via the European Genome-phenomeArchive (EGAD00001007659). Code is available on request. Clinical data andcirculating tumor DNA results (mean tumor molecules/mL of plasma) at eachtime point can be made available upon request.Conflicts of interestThese authors disclose the following: Rebecca C. Fitzgerald received aneducational grant from Roche, is a share-holder and consultant for CytedLtd, and has received grant support from Medtronic. Emma Ococks hasreceived honoraria and/or travel and accommodation expenses from Roche.1708 Ococks et al Gastroenterology Vol. 161, No. 5RESEARCHLETTERSElizabeth Smyth has received an honorarium from Roche, Astellas,AstraZeneca, BMS, Merck, Celgene, Five Prime, Gritstone Oncology, andServier. Shruti Sharma and Alexey Aleshin are employees of Natera, Inc, withstock/options to own stock in the company. Alvin Wei Tian Ng discloses noconflicts.AcknowledgmentsThe authors thank Ginny Devonshire for data management and storage.CRediT Authorship ContributionsEmma Ococks, MSci (Formal analysis: Lead; Visualization: Lead; Writing –original draft: Equal). Shruti Sharma, PhD (Formal analysis: Equal;Visualization: Supporting; support with clinical interpretation: Equal). AlvinWei Tian Ng, PhD (Data curation: Lead). Oesophageal Cancer Clinical andMolecular Stratification Consortium, UK (Consortium: Supporting). AlexeyAleshin, MD, MBA (support with clinical interpretation: Supporting). RebeccaC. Fitzgerald, MD (Funding acquisition: Lead; Supervision: Lead; Writing –original draft: Equal). Elizabeth Smyth, MD (Supervision: Equal; Writing –original draft: Equal).FundingOesophageal Cancer Clinical and Molecular Stratification and whole genomesequencing of primary tumor samples was funded by a Cancer Research UKProgram Grant (RG66287). The laboratory of RCF is funded by a MedicalResearch Council Core Program Grant. Plasma sample library preparationand sequencing cost was covered by Natera.Oesophageal Cancer Clinical and Molecular Stratification (OCCAMS)ConsortiumRebecca C. Fitzgerald,1 Paul A.W. Edwards,1,2 Nicola Grehan,1 BarbaraNutzinger,1 Elwira Fidziukiewicz,1 Aisling M. Redmond,1 Alex Northrop,1Sujath Abbas,1 Elizabeth C. Smyth,3 Maria O’Donovan,1,4 AhmadMiremadi,1,4 Shalini Malhotra,1,4 Monika Tripathi,1,4 Amber Grantham,1Calvin Cheah,1 Hannah Coles,1 Connor Flint,1 Matthew Eldridge,2 MariaSecrier,2 Ginny Devonshire,2 Sriganesh Jammula,2 Jim Davies,5 CharlesCrichton,5 Nick Carroll,3 Richard H. Hardwick,3 Peter Safranek,3 AndrewHindmarsh,3 Vijayendran Sujendran,3 Stephen J. Hayes,6,7 Yeng Ang,6,8,9Andrew Sharrocks,9 Shaun R. Preston,10 Izhar Bagwan,10 Vicki Save,11Richard J.E. Skipworth,11 Ted R. Hupp,12 J. Robert O’Neill,3,11,12 OlgaTucker,13,14 Andrew Beggs,13,15 Philippe Taniere,13 Sonia Puig,13Gianmarco Contino,13 Timothy J. Underwood,16,17 Robert C. Walker,16,17Ben L. Grace,16 Jesper Lagergren,18,19 James Gossage,18,20 AndrewDavies,18,20 Fuju Chang,18,20 Ula Mahadeva,18 Vicky Goh,20 Francesca D.Ciccarelli,20 Grant Sanders,21 Richard Berrisford,21 David Chan,21 EdCheong,22 Bhaskar Kumar,22 L. Sreedharan,22 Simon L. Parsons,23 IrshadSoomro,23 Philip Kaye,23 John Saunders,6,23 Laurence Lovat,24 RehanHaidry,24 Michael Scott,25 Sharmila Sothi,26 Suzy Lishman,27 George B.Hanna,28 Christopher J. Peters,28 Krishna Moorthy,28 Anna Grabowska,29Richard Turkington,30 Damian McManus,30 Helen Coleman,30 and RussellD. Petty.311Medical Research Council Cancer Unit, Hutchison/Medical ResearchCouncil Research Centre, University of Cambridge, Cambridge, UnitedKingdom; 2Cancer Research UK Cambridge Institute, University ofCambridge, Cambridge, United Kingdom; 3Cambridge University HospitalsNational Health Service Foundation Trust, Cambridge, United Kingdom;4Department of Histopathology, Addenbrooke’s Hospital, Cambridge, UnitedKingdom; 5Department of Computer Science, University of Oxford, Oxford,United Kingdom; 6Salford Royal National Health Service Foundation Trust,Salford, United Kingdom; 7Faculty of Medical and Human Sciences,University of Manchester, United Kingdom; 8Wigan and Leigh NationalHealth Service Foundation Trust, Wigan, Manchester, United Kingdom; 9GIScience Centre, University of Manchester, United Kingdom; 10Royal SurreyCounty Hospital National Health Service Foundation Trust, Guildford, UnitedKingdom; 11Edinburgh Royal Infirmary, Edinburgh, United Kingdom;12Edinburgh University, Edinburgh, United Kingdom; 13University HospitalsBirmingham National Health Service Foundation Trust, Birmingham, UnitedKingdom; 14Heart of England National Health Service Foundation Trust,Birmingham, United Kingdom; 15Institute of Cancer and Genomic Sciences,University of Birmingham, Birmingham, United Kingdom; 16UniversityHospital Southampton National Health Service Foundation Trust,Southampton, United Kingdom; 17Cancer Sciences Division, University ofSouthampton, Southampton, United Kingdom; 18Guy’s and St Thomas’sNational Health Service Foundation Trust, London, United Kingdom;19Karolinska Institute, Stockholm, Sweden; 20King’s College London,London, United Kingdom; 21Plymouth Hospitals National Health ServiceTrust, Plymouth, United Kingdom; 22Norfolk and Norwich University HospitalNational Health Service Foundation Trust, Norwich, United Kingdom;23Nottingham University Hospitals National Health Service Trust,Nottingham, United Kingdom; 24University College London, London, UnitedKingdom; 25Wythenshawe Hospital, Manchester, United Kingdom;26University Hospitals Coventry and Warwickshire National Health ServiceTrust, Coventry, United Kingdom; 27Peterborough Hospitals National HealthService Trust, Peterborough City Hospital, Peterborough, United Kingdom;28Department of Surgery and Cancer, Imperial College, London, UnitedKingdom; 29Queen’s Medical Centre, University of Nottingham, Nottingham,United Kingdom; 30Centre for Cancer Research and Cell Biology, Queen’sUniversity Belfast, Northern Ireland, United Kingdom; 31Tayside CancerCentre, Ninewells Hospital and Medical School, Dundee, United Kingdom.A B CDPre surgery status Negative PositivePre surgery status Negative PositiveP = .093P = .042Supplementary Figure 1. (A) Presurgical detection of circulating tumor (ct)DNA across different stages. (B) Presurgicaldetection of ctDNA according to relapse status. (C) Disease-free survival (DFS) in patients according to ctDNA status atbaseline. (D) Cancer-related survival in patients according to baseline ctDNA status.November 2021 ctDNA Detection After EAC Resection 1708.e1Supplementary Table 1.Clinical Demographics of CohortVariableNo. or Median% or Range(N ¼ 20)SexMale 17 85Female 3 15Age, y 62.8 48.9–80.8T stageT1a 2 10T1 1 5T2 5 25T3 12 60N stageN0 9 45N1 6 30N2 2 10N3 1 5Nx 2 10TreatmentSurgery 17 85Endoscopic mucosalresection3 15ChemotherapyYes 17 85No 3 15Siewert’s classification1 12 602 5 253 3 151708.e2 Ococks et al Gastroenterology Vol. 161, No. 5

Serial ctDNA detection using a personalized, tumor-informed assay in esophageal adenocarcinoma patients following resection

https://discovery.ucl.ac.uk/id/eprint/10131919/7/Lovat_Serial%20ctDNA%20detection%20using%20a%20personalized%2C%20tumor-informed%20assay%20in%20esophageal%20adenocarcinoma%20patients%20following%20resection_VoR.pdf

Serial ctDNA detection using a personalized, tumor-informed assay in esophageal adenocarcinoma patients following resection

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