Abstract

Abstract 4146

Introduction

The success of allogeneic stem cell transplantation (allo-SCT) relies on the result of the immune reactions between donor and recipient lymphohemopoietic systems. Chimerism quantification and monitoring post allo-SCT allows to estimate such immune reactions. In recent years, there has been a growing interest in the analysis of circulating cell-free nucleic acids in plasma. Although the ultimate origin of such nucleic acids is unknown, the most plausible hypothesis proposes cell disruption as the cause for the presence of nucleic acids in plasma. Analysis of chimerism in cell-free plasma DNA would complement the results of cell chimerism for a better prediction of different complications (engraftment failure, rejection, graft versus host disease…etc.).

Objective

Our goal was to quantitatively monitor chimerism in cell-free plasma DNA and to evaluate its usefulness for the prediction/early diagnosis of complications after allo-SCT that would favour early therapeutic intervention.

Patients and Methods

The study comprised 270 plasma samples from 29 allo-SCT patients. With a median follow up of 45,8 months (14,6– 74,7).

Whole blood (WB) (10 mL) was collected in EDTA containing tubes and was processed immediately after collection. Plasma was separated from the blood cells by centrifugation at 1600g for 10 min and cell-free plasma DNA was extracted according to the blood and body fluid protocol of the QIAamp DNA Blood Mini Kit (Qiagen). Quantitative chimerism analysis was performed by microsatellite PCR (STR; AmpFlSTR SGM plus, Applied Biosystems) in whole blood (WB), T-cells (CD3+) purified using immunomagnetic means (Miltenyi Biotec) and cell-free plasma DNA.

Chimerism was monitored at regular intervals post-transplant (+30,+60, +90, +180, +365). Sensitivity of the technique is 1% in WB and cell-free plasma DNA, and 5% in T-cells (derived from a 95% purity of enriched samples).

Results

During the first year of allo-SCT, the number of patients with mixed chimerism in cell-free plasma DNA was higher than in WB and T-cells (Table 1). Cell-free plasma DNA mixed chimerism was observed for a longer timer after transplant than “cellular” mixed chimerism, with a median of 105 vs. 40 and 27 days for cell-free plasma DNA, T-cells and WB, respectively.

Chimerism analysis revealed two groups of patients, one with mixed chimerism in both cell-free plasma DNA and “cellular DNA”, in which the percentage of recipient plasma DNA ranged between 10–30%, probably mostly derived from hematopoietic cells. The other group presented mixed chimerism in cell-free plasma DNA but complete “cellular” chimerism (100% donor) in which cells the percentage of recipient plasma DNA was lower than in the former group (1–10%).

A greater number of patients with graft versus host disease (GVHD),mainly acute but also chronic, presented mixed chimerism in cell-free plasma DNA and complete chimerism in WB and T-cells (Table 2). The presence of recipient cell-free plasma DNA could be derived by cell disruption in GVHD target organs.

Discussion

Quantitative follow-up of chimerism in cell-free plasma DNA, does not reproduce the results of “cellular” chimerism, as reported previously, and therefore, could not replace it for post-SCT follow-up. On the other hand, chimerism analysis in cell-free plasma DNA could be combined with the conventional follow-up of “cellular” chimerism for an improved diagnosis of life-threatening complications such as acute and chronic GVHD.

Table 1.

Percentage of patients in mixed chimerism during the first year post-allo-SCT and time to CC in whole blood, T-cells and cell free plasma DNA.

Days post-allo-SCTDays until CC (median)
+30+60+90+180+365
WB 35 24 20 27 
T cells 48 28 20 11 40 
Plasma 83 72 66 24 10 105 
Days post-allo-SCTDays until CC (median)
+30+60+90+180+365
WB 35 24 20 27 
T cells 48 28 20 11 40 
Plasma 83 72 66 24 10 105 
Table 2.

Pacients with mixed chimerism in whole bllod, T-cells and cell free plasma DNA in the moment of aGVHD and cGVHD developed.

acute GVHDchronic GVHD
Total 15/29 (51%) 15/29 (51%) 
WB 2/15 (13,3%) 0/15 (0%) 
T cells 4/15 (26,6%) 2/15 (13,3%) 
Plasma 11/15 (73,4%) 7/12 (58,3%) 
acute GVHDchronic GVHD
Total 15/29 (51%) 15/29 (51%) 
WB 2/15 (13,3%) 0/15 (0%) 
T cells 4/15 (26,6%) 2/15 (13,3%) 
Plasma 11/15 (73,4%) 7/12 (58,3%) 
Disclosures:

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.