Background: Our team previously published (1) results at one month after lung transplantation (LTx) that donor-derived cfDNA (dd-cfDNA) alone is not specific for discriminating acute rejection (AR) from other early events such as infection (INF). We demonstrated the importance of a multi-parametric cfDNA study by an algorithm that considers the %dd-cfDNA to differentiate stable from non-stable patients, and the fragmentome study of cfDNA size to discriminate the injury type (INF, AR).
The aims of the study are to confirm these results and adjust the diagnosis threshold of the algorithm previously published, using a larger cohort.
Materials and Methods: This was a prospective study involving 92 patients (62 first patients and 30 additional patients) from the Marseille Hospital. %dd-cfDNA was determined by NGS (AlloSeq cfDNA assay, CareDx) and size profile was assessed by BIABooster (Adelis). A biopsy at day 30 (D30) established the following groups among patients: stable and non-stable (AR, INF and AR+INF).
Results: The results of this new analysis are consistent with our previous results. % dd-cfDNA was significantly higher in non-stable patients at D30 (p=0.001). The threshold previously identified as 1.72% was adjusted to 2.19%, yielding satisfactory analytical performance (sensitivity=72.7%, specificity=87.2%, PPV=76.2%), and notably, an NPV=85.0% for discriminating stable patients. Among the "non-stable" group, the results confirmed that patients with INF had significantly higher percentages of small cfDNA fragments (80-120bp) compared to AR patients (p=0.028) and stable patients (p=0.044). The initial threshold of 3.7%, adjusted to 3.4%, facilitated the identification of INF patients among non-stable patients (PPV=88.9%).
Conclusion and Discussion: The combination of the two analyses effectively differentiates the type of allograft injury. This study, conducted on a larger cohort confirms the previous results and adjust the previously established cut-off values to increase the test reliability.

References
1. Pedini P, Coiffard B, Cherouat N, Casas S, Fina F, Boutonnet A, Baudey JB, Aho P, Basire A, Simon S, et al. Clinical relevance of cell-free DNA quantification and qualification during the first month after lung transplantation. Front Immunol (2023) 14:1183949. doi: 10.3389/fimmu.2023.1183949