Measurable residual disease monitoring for patients with acute myeloid leukemia following hematopoietic cell transplantation using error corrected hybrid capture next generation sequencing

Autoři: Vidya Balagopal aff001;  Andrew Hantel aff002;  Sabah Kadri aff001;  George Steinhardt aff001;  Chao Jie Zhen aff001;  Wenjun Kang aff003;  Pankhuri Wanjari aff001;  Lauren L. Ritterhouse aff001;  Wendy Stock aff002;  Jeremy P. Segal aff001
Působiště autorů: Department of Pathology, Division of Genomic and Molecular Pathology, The University of Chicago, Chicago, Illinois, United States of America aff001;  Department of Medicine, Section of Hematology/Oncology, The University of Chicago, Chicago, Illinois, United States of America aff002;  Center for Research Informatics, The University of Chicago, Chicago, Illinois, United States of America aff003
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224097


Improved systems for detection of measurable residual disease (MRD) in acute myeloid leukemia (AML) are urgently needed, however attempts to utilize broad-scale next-generation sequencing (NGS) panels to perform multi-gene surveillance in AML post-induction have been stymied by persistent premalignant mutation-bearing clones. We hypothesized that this technology may be more suitable for evaluation of fully engrafted patients following hematopoietic cell transplantation (HCT). To address this question, we developed a hybrid-capture NGS panel utilizing unique molecular identifiers (UMIs) to detect variants at 0.1% VAF or below across 22 genes frequently mutated in myeloid disorders and applied it to a retrospective sample set of blood and bone marrow DNA samples previously evaluated as negative for disease via standard-of-care short tandem repeat (STR)-based engraftment testing and hematopathology analysis in our laboratory. Of 30 patients who demonstrated trackable mutations in the 22 genes at eventual relapse by standard NGS analysis, we were able to definitively detect relapse-associated mutations in 18/30 (60%) at previously disease-negative timepoints collected 20–100 days prior to relapse date. MRD was detected in both bone marrow (15/28, 53.6%) and peripheral blood samples (9/18, 50%), while showing excellent technical specificity in our sample set. We also confirmed the disappearance of all MRD signal with increasing time prior to relapse (>100 days), indicating true clinical specificity, even using genes commonly associated with clonal hematopoiesis of indeterminate potential (CHIP). This study highlights the efficacy of a highly sensitive, NGS panel-based approach to early detection of relapse in AML and supports the clinical validity of extending MRD analysis across many genes in the post-transplant setting.

Klíčová slova:

Acute myeloid leukemia – Bone marrow – Disease surveillance – Flow cytometry – Mutation detection – Next-generation sequencing – Point mutation – Polymerase chain reaction


1. Scott BL, Pasquini MC, Logan BR, Wu J, Devine SM, Porter DL, et al. Myeloablative versus reduced-intensity hematopoietic cell transplantation for acute myeloid leukemia and myelodysplastic syndromes. J Clin Oncol. 2017;10;35(11):1154–1161. doi: 10.1200/JCO.2016.70.7091 28380315

2. Hantel A, Stock W, Kosuri S. Molecular Minimal Residual Disease Testing in Acute Myeloid Leukemia: A Review for the Practicing Clinician. Clinical Lymphoma, Myeloma and Leukemia. 2018; 18(10):636–647. doi: 10.1016/j.clml.2018.06.017 30006258

3. Mosna F, Capelli D, Gottardi M, Mosna F, Capelli D, Gottardi M. Minimal Residual Disease in Acute Myeloid Leukemia: Still a Work in Progress? J Clin Med. 2017 Jun 3;6(6):57.

4. Paietta E. Should minimal residual disease guide therapy in AML? Best Pract Res Clin Haematol. 2015 Jun 1;28(2–3):98–105. doi: 10.1016/j.beha.2015.10.006 26590765

5. Schuurhuis GJ, Heuser M, Freeman S, Béne MC, Buccisano F, Cloos J, et al. Minimal/measurable residual disease in AML: a consensus document from the European LeukemiaNet MRD Working Party. Blood. 2018; 131(12):1275–1291. doi: 10.1182/blood-2017-09-801498 29330221

6. Sufliarska S, Minarik G, Horakova J, Bodova I, Bojtarova E, Czako B, et al. Establishing the method of chimerism monitoring after allogeneic stem cell transplantation using multiplex polymerase chain reaction amplification of short tandem repeat markers and Amelogenin. Neoplasma. 2007; 54(5):424–30. 17688372

7. Thiede C, Bornhäuser M, Ehninger G. Evaluation of STR informativity for chimerism testing—Comparative analysis of 27 STR system in 203 matched related donor recipient pairs. Leukemia. 2004; 18(2):248–54. doi: 10.1038/sj.leu.2403212 14671648

8. Manasatienkij C, Ra-ngabpai C. Clinical application of forensic DNA analysis: A literature review. Journal of the Medical Association of Thailand. 2012; 95(10):1357–63. 23193753

9. Matsuda K, Yamauchi K, Tozuka M, Suzuki T, Sugano M, Hidaka E, et al. Monitoring of hematopoietic chimerism by short tandem repeats, and the effect of CD selection on its sensitivity. Clin Chem. 2004; 50(12):2411–4. doi: 10.1373/clinchem.2004.037580 15563497

10. Rautenberg C, Germing U, Haas R, Kobbe G, Schroeder T. Relapse of Acute Myeloid Leukemia after Allogeneic Stem Cell Transplantation: Prevention, Detection, and Treatment. Int J Mol Sci. 2019;20(1):228.

11. Patkar N, Kodgule R, Kakirde C, Raval G, Bhanshe P, Joshi S, et al. Clinical impact of measurable residual disease monitoring by ultradeep next generation sequencing in NPM1 mutated acute myeloid leukemia. Oncotarget. 2018 Nov 27;9(93):36613–24. doi: 10.18632/oncotarget.26400 30564301

12. Jongen-Lavrencic M, Grob T, Hanekamp D, Kavelaars FG, al Hinai A, Zeilemaker A, et al. Molecular Minimal Residual Disease in Acute Myeloid Leukemia. N Engl J Med. 2018 Mar 29;378(13):1189–99. doi: 10.1056/NEJMoa1716863 29601269

13. Roloff G, Lai C, Hourigan C, Dillon L. Technical Advances in the Measurement of Residual Disease in Acute Myeloid Leukemia. J Clin Med. 2017; 19:6(9):E87. doi: 10.3390/jcm6090087 28925935

14. Meacham F, Boffelli D, Dhahbi J, Martin DI, Singer M, Pachter L. Identification and correction of systematic error in high-throughput sequence data. BMC Bioinformatics. 2011 Nov 21;12(1):451.

15. Liang RH, Mo T, Dong W, Lee GQ, Swenson LC, McCloskey RM, et al. Theoretical and experimental assessment of degenerate primer tagging in ultra-deep applications of next-generation sequencing. Nucleic Acids Res. 2014; 42(12):e98. doi: 10.1093/nar/gku355 24810852

16. Young AL, Wong TN, Hughes AEO, Heath SE, Ley TJ, Link DC, et al. Quantifying ultra-rare pre-leukemic clones via targeted error-corrected sequencing. Leukemia. 2015; 29,1608–11. doi: 10.1038/leu.2015.17 25644247

17. Kinde I, Wu J, Papadopoulos N, Kinzler KW, Vogelstein B. Detection and quantification of rare mutations with massively parallel sequencing. Proc Natl Acad Sci. 2011;108(23):9530–5. doi: 10.1073/pnas.1105422108 21586637

18. Ivey A, Hills RK, Simpson MA, Jovanovic J V., Gilkes A, Grech A, et al. Assessment of Minimal Residual Disease in Standard-Risk AML. N Engl J Med. 2016 Feb 4;374(5):422–33. doi: 10.1056/NEJMoa1507471 26789727

19. Thol F, Gabdoulline R, Liebich A, Klement P, Schiller J, Kandziora C, et al. Measurable residual disease monitoring by NGS before allogeneic hematopoietic cell transplantation in AML. Blood. 2018 Oct 18;132(16):1703–13. doi: 10.1182/blood-2018-02-829911 30190321

20. Kim T, Moon JH, Ahn J-S, Kim Y-K, Lee S-S, Ahn S-Y, et al. Next-generation sequencing-based posttransplant monitoring of acute myeloid leukemia identifies patients at high risk of relapse. Blood. 2018 Oct 11;132(15):1604–13. doi: 10.1182/blood-2018-04-848028 30108064

21. Kadri S, Long BC, Mujacic I, Zhen CJ, Wurst MN, Sharma S, et al. Clinical Validation of a Next-Generation Sequencing Genomic Oncology Panel via Cross-Platform Benchmarking against Established Amplicon Sequencing Assays. In: Journal of Molecular Diagnostics. 2017; (1):43–56. doi: 10.1016/j.jmoldx.2016.07.012 27836695

22. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009 Aug 15 [cited 2019 Apr 11];25(16):2078–9. doi: 10.1093/bioinformatics/btp352 19505943

23. Genomic and Epigenomic Landscapes of Adult De Novo Acute Myeloid Leukemia. N Engl J Med. 2013; 368(22):2059–74. doi: 10.1056/NEJMoa1301689 23634996

24. Costello M, Pugh TJ, Fennell TJ, Stewart C, Lichtenstein L, Meldrim JC, et al. Discovery and characterization of artifactual mutations in deep coverage targeted capture sequencing data due to oxidative DNA damage during sample preparation. Nucleic Acids Res. 2013 Apr 1;41(6):e67–e67. doi: 10.1093/nar/gks1443 23303777

25. Zeijlemaker W, Kelder A, Oussoren-Brockhoff YJM, Scholten WJ, Snel AN, Veldhuizen D, et al. Peripheral blood minimal residual disease may replace bone marrow minimal residual disease as an immunophenotypic biomarker for impending relapse in acute myeloid leukemia. Leukemia. 2016 Mar 16;30(3):708–15. Available from: doi: 10.1038/leu.2015.255 26373238

26. Weinkauff R, Estey EH, Starostik P, Hayes K, Huh YO, Hirsch-Ginsberg C, et al. Use of peripheral blood blasts vs bone marrow blasts for diagnosis of acute leukemia. Am J Clin Pathol. 1999 Jun;111(6):733–40. doi: 10.1093/ajcp/111.6.733 10361507

27. Corbacioglu A, Scholl C, Schlenk RF, Eiwen K, Du J, Bullinger L, et al. Prognostic impact of minimal residual disease in CBFB-MYH11-positive acute myeloid leukemia. J Clin Oncol. 2010 Aug 10;28(23):3724–9. Available from: doi: 10.1200/JCO.2010.28.6468 20625124

28. Cheung AMS, Chow HCH, Liang R, Leung AYH. A comparative study of bone marrow and peripheral blood CD34 + myeloblasts in acute myeloid leukaemia. Br J Haematol. 2009 Feb;144(4):484–91. doi: 10.1111/j.1365-2141.2008.07431.x 19055666

29. Debarri H, Lebon D, Roumier C, Cheok M, Marceau-Renaut A, Nibourel O, et al. IDH1/2 but not DNMT3A mutations are suitable targets for minimal residual disease monitoring in acute myeloid leukemia patients: a study by the Acute Leukemia French Association. Oncotarget. 2015 Dec 8;6(39):42345–53. doi: 10.18632/oncotarget.5645 26486081

30. Pløen GG, Nederby L, Guldberg P, Hansen M, Ebbesen LH, Jensen UB, et al. Persistence of DNMT3A mutations at long-term remission in adult patients with AML. Br J Haematol. 2014 Nov 1;167(4):478–86. doi: 10.1111/bjh.13062 25371149

31. Genovese G, Kähler AK, Handsaker RE, Lindberg J, Rose SA, Bakhoum SF, et al. Clonal Hematopoiesis and Blood-Cancer Risk Inferred from Blood DNA Sequence. N Engl J Med. 2014 Dec 25;371(26):2477–87. doi: 10.1056/NEJMoa1409405 25426838

32. Shlush LI, Zandi S, Mitchell A, Chen WC, Brandwein JM, Gupta V, et al. Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia. Nature. 2014 Feb 12;506(7488):328–33. doi: 10.1038/nature13038 24522528

33. Hjortholm N, Jaddini E, Hałaburda K, Snarski E. Strategies of pain reduction during the bone marrow biopsy. Ann Hematol. 2013 Feb 6;92(2):145–9. doi: 10.1007/s00277-012-1641-9 23224244

34. Ommen HB, Schnittger S, Jovanovic J V., Ommen IB, Hasle H, Ostergaard M, et al. Strikingly different molecular relapse kinetics in NPM1c, PML-RARA, RUNX1-RUNX1T1, and CBFB-MYH11 acute myeloid leukemias. Blood. 2010 Jan 14;115(2):198–205. doi: 10.1182/blood-2009-04-212530 19901261

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2019 Číslo 10