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Original article
Accurate quantification of chromosomal lesions via short tandem repeat analysis using minimal amounts of DNA
  1. Johann-Christoph Jann1,
  2. Daniel Nowak1,
  3. Florian Nolte1,
  4. Stephanie Fey1,
  5. Verena Nowak1,
  6. Julia Obländer1,
  7. Jovita Pressler1,
  8. Iris Palme1,
  9. Christina Xanthopoulos1,
  10. Alice Fabarius1,
  11. Uwe Platzbecker2,
  12. Aristoteles Giagounidis3,
  13. Katharina Götze4,
  14. Anne Letsch5,
  15. Detlef Haase6,
  16. Richard Schlenk7,
  17. Gesine Bug8,
  18. Michael Lübbert9,
  19. Arnold Ganser10,
  20. Ulrich Germing11,
  21. Claudia Haferlach12,
  22. Wolf-Karsten Hofmann1,
  23. Maximilian Mossner1
  1. 1 III Medizinische Klinik, Hämatologie und Onkologie, Universitätsmedizin Mannheim, Mannheim, Germany
  2. 2 Medizinische Klinik und Poliklinik I, Universitatsklinikum Carl Gustav Carus, Dresden, Germany
  3. 3 Klinik für Hämatologie, Onkologie und Palliativmedizin, Marienhospital, Düsseldorf, Germany
  4. 4 III. Medizinischen Klinik des Klinikums rechts der Isar, Technische Universitat Munchen, Munchen, Germany
  5. 5 Medizinische Klinik für Hämatologie, Onkologie, Campus Benjamin Franklin, Charite Universitatsmedizin Berlin, Berlin, Germany
  6. 6 Klinik für Hämatologie und Medizinische Onkologie, Georg-August-Universitat Gottingen Universitatsmedizin, Gottingen, Germany
  7. 7 NCT Trial Center, Nationales Centrum für Tumorerkrankungen (NCT), Heidelberg, Gemany
  8. 8 Medizinische Klinik II, Abteilung für Hämatologie/Onkologie, Klinikum der Johann Wolfgang Goethe-Universitat Frankfurt, Frankfurt am Main, Germany
  9. 9 Abteilung für Innere Medizin I, Hämatologie und Onkologie, Universitatsklinikum Freiburg, Freiburg, Germany
  10. 10 Abteilung für Hämatologie, Hämostaseologie, Onkologie und Stammzelltransplantation, Medizinische Hochschule Hannover, Hannover, Germany
  11. 11 Abteilung für Hämatologie, Onkologie und klinische Immunologie, Heinrich-Heine-Universitat Dusseldorf Medizinische Fakultat, Dusseldorf, Germany
  12. 12 Münchner Leukämie Labor, München, Germany
  1. Correspondence to Maximilian Mossner, Department of Hematology and Oncology, University Hospital Mannheim, Pettenkoferstraße 22, Mannheim 68169, Germany; maximilian.mossner{at}medma.uni-heidelberg.de; m.mossner{at}qmul.ac.uk

Abstract

Background Cytogenetic aberrations such as deletion of chromosome 5q (del(5q)) represent key elements in routine clinical diagnostics of haematological malignancies. Currently established methods such as metaphase cytogenetics, FISH or array-based approaches have limitations due to their dependency on viable cells, high costs or semi-quantitative nature. Importantly, they cannot be used on low abundance DNA. We therefore aimed to establish a robust and quantitative technique that overcomes these shortcomings.

Methods For precise determination of del(5q) cell fractions, we developed an inexpensive multiplex-PCR assay requiring only nanograms of DNA that simultaneously measures allelic imbalances of 12 independent short tandem repeat markers.

Results Application of this method to n=1142 samples from n=260 individuals revealed strong intermarker concordance (R²=0.77–0.97) and reproducibility (mean SD: 1.7%). Notably, the assay showed accurate quantification via standard curve assessment (R²>0.99) and high concordance with paired FISH measurements (R²=0.92) even with subnanogram amounts of DNA. Moreover, cytogenetic response was reliably confirmed in del(5q) patients with myelodysplastic syndromes treated with lenalidomide. While the assay demonstrated good diagnostic accuracy in receiver operating characteristic analysis (area under the curve: 0.97), we further observed robust correlation between bone marrow and peripheral blood samples (R²=0.79), suggesting its potential suitability for less-invasive clonal monitoring.

Conclusions In conclusion, we present an adaptable tool for quantification of chromosomal aberrations, particularly in problematic samples, which should be easily applicable to further tumour entities.

  • Chromosomal deletion
  • short tandem repeats
  • PCR
  • deletion 5q
  • myelodysplastic syndrome

This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. See: http://creativecommons.org/licenses/by/4.0/

https://doi.org/10.1136/jmedgenet-2017-104528

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    Footnotes

    • Contributors J-CJ, DN, W-KH and MM designed the study, analysed data and wrote the manuscript; J-CJ and MM conducted experimental design and most of the experiments including assessment of short tandem repeat patterns for lesion quantification; W-KH supervised the whole study and provided research infrastructure; AF, DH and CH provided metaphase cytogenetics and interphase-FISH analyses; SF, VN, JO, JP, IP and CX provided assistance for molecular analyses and clinical sample workup; FN, UP, AG, KG, AL, DH, RS, GB, ML, AG, UG and DN provided patient material and clinical data within the LEMON-5 trial.

    • Competing interests CH declares part ownership of the MLL Munich Leukemia Laboratory GmbH.

    • Ethics approval Institutional Ethics Review Board II of the Medical Faculty Mannheim, University of Heidelberg.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Correction notice This paper has been amended since it was published Online First. Owing to a scripting error, some of the publisher names in the references were replaced with ‘BMJ Publishing Group’. This only affected the full text version, not the PDF. We have since corrected these errors and the correct publishers have been inserted into the references.