A. Kroeze and A. S. Cornelissen are first authors, M. F. Pascutti and M. Verheij are second authors, and C. Voermans and Sacha S. Zeerleder are senior authors.

Funding information: Landsteiner Foundation for Blood Transfusion Research, Grant/Award Numbers: 1101, 1719; Sanquin Research, Grant/Award Number: PPOC 13-027

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Abstract

Background

Cell-free DNA (cfDNA) and nucleosomes, consisting of cfDNA and histones, are markers of cell activation and damage. In systemic inflammation these markers predict severity and fatality. However, the role of cfDNA in acute Graft-versus-Host Disease (aGvHD), a major complication of allogeneic hematopoietic stem cell transplantation (HSCT), is unknown.

Objective

The aim of this study is to investigate the role of cfDNA as a marker of aGvHD.

Methods

We followed nucleosome levels in 37 allogeneic HSCT patients and an established xenotransplantation mouse model. We determined the origin of cfDNA with a species-specific polymerase chain reaction.

Results

In the plasma of aGvHD patients, nucleosome levels significantly increased around the time of aGvHD diagnosis compared to pretransplant, concurrently with a significant increase of known aGvHD markers ST2 and REG3α. In mice, we confirmed that nucleosomes were elevated during clinically detectable aGvHD. We found cfDNA to be mainly of human origin and to a lesser extent of mouse origin, indicating that cfDNA is released by (proliferating) human xeno-reactive PBMC and damaged mouse cells.

Conclusion

We show increased cfDNA both in an aGvHD mouse model and in aGvHD patients. We also demonstrate that donor hematopoietic cells and to a lesser degree (damaged) host cells are the cellular source of cfDNA in aGvHD. We propose that nucleosomes and cfDNA might be an additive marker for aGvHD.

CONFLICT OF INTEREST

The authors declare no competing financial interests.

Open Research

DATA AVAILABILITY STATEMENT

The original data of this study are available on reasonable request from the corresponding author.

Supporting Information

Filename

Description

ejh13806-sup-0001-FigureS1.jpgimage/jpp, 911.2 KB

FIGURE S1 Nucleosomes can be used as a measure for cfDNA and are increased post aHSCT in both no-aGvHD and aGvHD patients. (A,B) Levels of nucleosomes within the plasma measured by ELISA, and cfDNA isolated from the plasma and measured by either real-time PCR on the Albumin gene (A) or digital droplet PCR on the β-actin gene(B) correlate highly in 39 longitudinal samples of nine aHSCT patients. (C,D) Nucleosomes increase significantly in the plasma obtained 1 month after aHSCT compared to baseline, in both patients that develop aGvHD (n = 17) and patients that do not develop aGvHD (n = 13). Spearman correlation (A,B), Wilcoxon matched-pairs signed rank test (C,D).

ejh13806-sup-0002-FigureS2.jpgimage/jpp, 841.9 KB

FIGURE S2 IL6 and IL12p40 are not relevantly increased in aGVHD patients. (A,B) IL6 (A) and IL12 p40 (B) levels are low before aHSCT in both non-aGVHD patients (n = 27) and aGVHD patients (n = 19). (C) IL6 levels remain low and do not differ between non-aGVHD patients (n = 25) and aGVHD patients (n = 17) 1 month after aHSCT. (D) ILp12p40 levels are significantly increased in aGVHD patients (n = 17) compared to non-aGVHD patients (n = 25) 1 month after aHSCT; however, this increase seems not as a relevant increase, since most of the data points are under the detection limit. Data are medians ± IQR. Mann–Whitney test.

Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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Volume109, Issue3

September 2022

Pages 271-281

Cell-free DNA levels are increased in acute graft-versus-host disease

Abstract

Background

Objective

Methods

Results

Conclusion

CONFLICT OF INTEREST

Open Research

DATA AVAILABILITY STATEMENT

Supporting Information

REFERENCES

References

Information

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Cell-free DNA levels are increased in acute graft-versus-host disease

Abstract

Background

Objective

Methods

Results

Conclusion

CONFLICT OF INTEREST

Open Research

DATA AVAILABILITY STATEMENT

Supporting Information

REFERENCES

References

Related

Information