Abstract
Objectives
A combined digital droplet PCR (ddPCR)/pyrosequencing assay system was developed that demonstrated advantages applicable to multiple qualitative and quantitative molecular genetic diagnostic applications. Data for characterizing this combined approach for hematologic stem cell transplantation (HSCT) and allele quantification from graft-derived cell-free (cf) DNA in solid organ transplantation (SOT) is presented.
Methods
ddPCR and pyrosequencing assays targeting 32 SNPs/markers were established. ddPCR results from 72 gDNAs of 55 patients after allogeneic HSCT and 107 plasma-cfDNAs of 25 liver transplant recipients were compared with established methods/markers, i.e. short-tandem-repeat PCR and ALT, respectively.
Results
The ddPCR results were in good agreement with the established marker. The limit of detection was 0.02 % minor allele fraction. The relationship between ddPCR and STR-PCR was linear with R2=0.98 allowing to transfer previously established clinical STR-PCR cut-offs to ddPCR; 50-fold higher sensitivity and a variation coefficient of <2 % enable the use of low DNA concentrations (e.g. pre-sorted cells). ddPCR detected liver allograft injury at least as sensitive as ALT suggesting that ddPCR is a reliable method to monitor the transplant integrity, especially when other biomarkers are lacking (e.g. kidney).
Conclusions
Combining pyrosequencing for genotyping and ddPCR for minor allele quantification enhances sensitivity and precision for the patient after HSCT and SOT. The assay is designed for maximum flexibility. It is expected to be suitable for other applications (sample tracking, prenatal diagnostics, etc.).
Acknowledgments
The authors would like to thank Regina Meuser for critically revising the statistical part of this report. This report includes work performed by Lilli Greulich during the preparation of her doctoral theses.
-
Research funding: None declared.
-
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
-
Competing interests: Authors state no conflict of interest.
-
Informed consent: Informed consent was obtained from all individuals included in this study.
-
Ethical approval: Research involving human subjects complied with all relevant national regulations, institutional policies and is in accordance with the tenets of the Helsinki Declaration (as revised in 2013), and has been approved by the local Ethics Committee (Ethik Kommission der Landesärztekammer Rheinland-Pfalz) and is part of the patient admission agreement (§14 Abs. 3) of the University Medical Center Mainz (https://www.unimedizin-mainz.de/patientenmanagement/allgemeine-vertragsbedingungen.html).
-
Data availability: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
References
1. Lindahl, H, Vonlanthen, S, Valentini, D, Björklund, AT, Sundin, M, Mielke, S, et al.. Lineage-specific early complete donor chimerism and risk of relapse after allogeneic hematopoietic stem cell transplantation for acute myeloid leukemia. Bone Marrow Transplant 2022;57:753–9. https://doi.org/10.1038/s41409-022-01615-8.Search in Google Scholar PubMed PubMed Central
2. Blouin, AG, Askar, M. Chimerism analysis for clinicians: a review of the literature and worldwide practices. Bone Marrow Transplant 2022;57:347–59. https://doi.org/10.1038/s41409-022-01579-9.Search in Google Scholar PubMed PubMed Central
3. Lion, T, Watzinger, F, Preuner, S, Kreyenberg, H, Tilanus, M, Weger, RD, et al.. The EuroChimerism concept for a standardized approach to chimerism analysis after allogeneic stem cell transplantation. Leukemia 2012;26:1821–8. https://doi.org/10.1038/leu.2012.66.Search in Google Scholar PubMed
4. Clark, JR, Scott, SD, Jack, AL, Lee, H, Mason, J, Carter, GI, et al.. Monitoring of chimerism following allogeneic haematopoietic stem cell transplantation (HSCT): technical recommendations for the use of short tandem repeat (STR) based techniques, on behalf of the United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping Chimerism Working Group. Br J Haematol 2015;168:26–37. https://doi.org/10.1111/bjh.13073.Search in Google Scholar PubMed
5. Jacque, N, Nguyen, S, Golmard, J-L, Uzunov, M, Garnier, A, Leblond, V, et al.. Chimerism analysis in peripheral blood using indel quantitative real-time PCR is a useful tool to predict post-transplant relapse in acute leukemia. Bone Marrow Transplant 2015;50:259–65. https://doi.org/10.1038/bmt.2014.254.Search in Google Scholar PubMed
6. Sellmann, L, Rabe, K, Bünting, I, Dammann, E, Göhring, G, Ganser, A, et al.. Diagnostic value of highly-sensitive chimerism analysis after allogeneic stem cell transplantation. Bone Marrow Transplant 2018;53:1457–65. https://doi.org/10.1038/s41409-018-0176-7.Search in Google Scholar PubMed
7. Knight, SR, Thorne, A, Lo Faro, ML. Donor-specific cell-free DNA as a biomarker in solid organ transplantation. A systematic review. Transplantation 2019;103:273–83. https://doi.org/10.1097/TP.0000000000002482.Search in Google Scholar PubMed
8. Schütz, E, Fischer, A, Beck, J, Harden, M, Koch, M, Wuensch, T, et al.. Graft-derived cell-free DNA, a noninvasive early rejection and graft damage marker in liver transplantation: a prospective, observational, multicenter cohort study. PLoS Med 2017;14:e1002286. https://doi.org/10.1371/journal.pmed.1002286.Search in Google Scholar PubMed PubMed Central
9. Cilloni, D, Petiti, J, Rosso, V, Andreani, G, Dragani, M, Fava, C, et al.. Digital PCR in myeloid malignancies: ready to replace quantitative PCR? Int J Mol Sci 2019;20:2249–62. https://doi.org/10.3390/ijms20092249.Search in Google Scholar PubMed PubMed Central
10. Hindson, CM, Chevillet, JR, Briggs, HA, Gallichotte, EN, Ruf, IK, Hindson, BJ, et al.. Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods 2013;10:1003–5. https://doi.org/10.1038/nmeth.2633.Search in Google Scholar PubMed PubMed Central
11. Beck, J, Bierau, S, Balzer, S, Andag, R, Kanzow, P, Schmitz, J, et al.. Digital droplet PCR for rapid quantification of donor DNA in the circulation of transplant recipients as a potential universal biomarker of graft injury. Clin Chem 2013;59:1732–41. https://doi.org/10.1373/clinchem.2013.210328.Search in Google Scholar PubMed
12. Waterhouse, M, Pfeifer, D, Follo, M, Duyster, J, Schäfer, H, Bertz, H, et al.. Early mixed hematopoietic chimerism detection by digital droplet PCR in patients undergoing gender-mismatched hematopoietic stem cell transplantation. Clin Chem Lab Med 2017;55:1115–21. https://doi.org/10.1515/cclm-2016-0900.Search in Google Scholar PubMed
13. Bio-Rad Laboratories, Inc. Rare mutation detection best practices guidelines: bulletin# 6628. Hercules, CA: Bio-Rad Laboratories, Inc; 2017. Retrieved from https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6628.pdf Search in Google Scholar
14. George, D, Czech, J, John, B, Yu, M, Jennings, LJ. Detection and quantification of chimerism by droplet digital PCR. Chimerism 2013;4:102–8. https://doi.org/10.4161/chim.25400.Search in Google Scholar PubMed PubMed Central
15. Stahl, T, Böhme, MU, Kröger, N, Fehse, B. Digital PCR to assess hematopoietic chimerism after allogeneic stem cell transplantation. Exp Hematol 2015;43:462–8.e1. https://doi.org/10.1016/j.exphem.2015.02.006.Search in Google Scholar PubMed
16. Kliman, D, Castellano-Gonzalez, G, Withers, B, Street, J, Tegg, E, Mirochnik, O, et al.. Ultra-sensitive droplet digital PCR for the assessment of microchimerism in cellular therapies. Biol Blood Marrow Transplant 2018;24:1069–78. https://doi.org/10.1016/j.bbmt.2017.12.802.Search in Google Scholar PubMed
17. Campomenosi, P, Gini, E, Noonan, DM, Poli, A, D’Antona, P, Rotolo, N, et al.. A comparison between quantitative PCR and droplet digital PCR technologies for circulating microRNA quantification in human lung cancer. BMC Biotechnol 2016;16:60. https://doi.org/10.1186/s12896-016-0292-7.Search in Google Scholar PubMed PubMed Central
18. Santurtún, A, Riancho, JA, Arozamena, J, López-Duarte, M, Zarrabeitia, MT. Indel analysis by droplet digital PCR: a sensitive method for DNA mixture detection and chimerism analysis. Int J Leg Med 2017;131:67–72. https://doi.org/10.1007/s00414-016-1422-4.Search in Google Scholar PubMed
19. Kim, SY, Jeong, MH, Park, N, Ra, E, Park, H, Seo, SH, et al.. Chimerism monitoring after allogeneic hematopoietic stem cell transplantation using quantitative real-time PCR of biallelic insertion/deletion polymorphisms. J Mol Diagn 2014;16:679–88. https://doi.org/10.1016/j.jmoldx.2014.06.005.Search in Google Scholar PubMed
20. Pettersson, L, Vezzi, F, Vonlanthen, S, Alwegren, K, Hedrum, A, Hauzenberger, D. Development and performance of a next generation sequencing (NGS) assay for monitoring of mixed chimerism. Clin Chim Acta: Int J Med Chem 2021;512:40–8. https://doi.org/10.1016/j.cca.2020.10.034.Search in Google Scholar PubMed
21. Guo, X, Dai, X, Zhou, T, Wang, H, Ni, J, Xue, J, et al.. Mosaic loss of human Y chromosome: what, how and why. Hum Genet 2020;139:421–46. https://doi.org/10.1007/s00439-020-02114-w.Search in Google Scholar PubMed
22. Ganster, C, Kämpfe, D, Jung, K, Braulke, F, Shirneshan, K, Machherndl-Spandl, S, et al.. New data shed light on Y-loss-related pathogenesis in myelodysplastic syndromes. Genes Chromosomes Cancer 2015;54:717–24. https://doi.org/10.1002/gcc.22282.Search in Google Scholar PubMed
23. Valero-Garcia, J, González-Espinosa, MDC, Barrios, M, Carmona-Antoñanzas, G, García-Planells, J, Ruiz-Lafora, C, et al.. Earlier relapse detection after allogeneic haematopoietic stem cell transplantation by chimerism assays: digital PCR versus quantitative real-time PCR of insertion/deletion polymorphisms. PLoS One 2019;14:e0212708. https://doi.org/10.1371/journal.pone.0212708.Search in Google Scholar PubMed PubMed Central
24. Koreth, J, Kim, HT, Nikiforow, S, Milford, EL, Armand, P, Cutler, C, et al.. Donor chimerism early after reduced-intensity conditioning hematopoietic stem cell transplantation predicts relapse and survival. Biol Blood Marrow Transplant 2014;20:1516–21. https://doi.org/10.1016/j.bbmt.2014.05.025.Search in Google Scholar PubMed PubMed Central
25. Liesveld, JL, Rothberg, PG. Mixed chimerism in SCT: conflict or peaceful coexistence? Bone Marrow Transplant 2008;42:297–310. https://doi.org/10.1038/bmt.2008.212.Search in Google Scholar PubMed
26. Spyridonidis, A, Bertz, H, Waterhouse, M, Finke, J. Long-term lymphoid-restricted split chimerism after myeloablative allogeneic BMT for bcr-abl+ ALL. Bone Marrow Transplant 2008;42:829–31. https://doi.org/10.1038/bmt.2008.256.Search in Google Scholar PubMed
27. Beck, J, Oellerich, M, Schulz, U, Schauerte, V, Reinhard, L, Fuchs, U, et al.. Donor-derived cell-free DNA is a novel universal biomarker for allograft rejection in solid organ transplantation. Transplant Proc 2015;47:2400–3. https://doi.org/10.1016/j.transproceed.2015.08.035.Search in Google Scholar PubMed
28. Grskovic, M, Hiller, DJ, Eubank, LA, Sninsky, JJ, Christopherson, C, Collins, JP, et al.. Validation of a clinical-grade assay to measure donor-derived cell-free DNA in solid organ transplant recipients. J Mol Diagn 2016;18:890–902. https://doi.org/10.1016/j.jmoldx.2016.07.003.Search in Google Scholar PubMed
29. Sigdel, TK, Archila, FA, Constantin, T, Prins, SA, Liberto, J, Damm, I, et al.. Optimizing detection of kidney transplant injury by assessment of donor-derived cell-free DNA via massively multiplex PCR. J Clin Med 2018;8:19–35. https://doi.org/10.3390/jcm8010019.Search in Google Scholar PubMed PubMed Central
30. Zou, J, Duffy, B, Slade, M, Young, AL, Steward, N, Hachem, R, et al.. Rapid detection of donor cell free DNA in lung transplant recipients with rejections using donor-recipient HLA mismatch. Hum Immunol 2017;78:342–9. https://doi.org/10.1016/j.humimm.2017.03.002.Search in Google Scholar PubMed PubMed Central
31. Oellerich, M, Christenson, RH, Beck, J, Schütz, E, Sherwood, K, Price, CP, et al.. Donor-derived cell-free DNA testing in solid organ transplantation: a value proposition. J Appl Lab Med 2020;5:993–1004. https://doi.org/10.1093/jalm/jfaa062.Search in Google Scholar PubMed
32. Rabkin, JM, Corless, CL, Rosen, HR, Olyaei, AJ. Immunosuppression impact on long-term cardiovascular complications after liver transplantation. Am J Surg 2002;183:595–9. https://doi.org/10.1016/S0002-9610(02)00826-7.Search in Google Scholar
Supplementary Material
This article contains supplementary material (https://doi.org/10.1515/cclm-2023-0198).
© 2023 Walter de Gruyter GmbH, Berlin/Boston
