Volná cirkulující DNA dárce jako marker pro sledování rejekce štěpu po transplantaci plic

• Thesis title in Czech: Volná cirkulující DNA dárce jako marker pro sledování rejekce štěpu po transplantaci plic
• Thesis title in English: Donor-derived Cell-free DNA in Lung Transplant Recipients as as Marker of Rejection
• Key words: volná cirukulující DNA, transplantace plic, dárce, příjemce, rejekce, štěp
• English key words: cell-free DNA, lung transplantation, donor, recipient, rejection, graft
• Academic year of topic announcement: 2021/2022
• Thesis type: dissertation
• Thesis language: čeština
• Department: Department of Pneumology (13-820)
• Supervisor: MUDr. Jan Havlín, Ph.D.
• Author: hidden - assigned and confirmed by the Study Dept.
• Date of registration: 04.10.2021
• Date of assignment: 04.10.2021
• Confirmed by Study dept. on: 08.12.2021
• Date and time of defence: 25.09.2025 11:15
• Date of electronic submission: 04.06.2025
• Date of proceeded defence: 25.09.2025
• Opponents: MUDr. Tereza Doušová, Ph.D.
• Advisors: doc. MUDr. Libor Fila, Ph.D.

References

1. Tsuji N, Agbor-Enoh S. Cell-free DNA beyond a biomarker for rejection: Biological trigger of tissue injury and potential therapeutics. J Hear Lung Transplant. 2021;40(6):405-413. doi:10.1016/j.healun.2021.03.007
2. Andargie TE, Tsuji N, Seifuddin F, et al. Cell-free DNA maps COVID-19 tissue injury and risk of death and can cause tissue injury. JCI Insight. 2021;6(7). doi:10.1172/jci.insight.147610
3. Keller M, Bush E, Diamond JM, et al. Use of donor-derived-cell-free DNA as a marker of early allograft injury in primary graft dysfunction (PGD) to predict the risk of chronic lung allograft dysfunction (CLAD). J Hear Lung Transplant. 2021;40(6):488-493. doi:10.1016/j.healun.2021.02.008
4. Agbor-Enoh S, Jackson AM, Tunc I, et al. Late manifestation of alloantibody-associated injury and clinical pulmonary antibody-mediated rejection: Evidence from cell-free DNA analysis. 2018. doi:10.1016/j.healun.2018.01.1305
5. Agbor-Enoh S, Wang Y, Tunc I, et al. Donor-derived cell-free DNA predicts allograft failure and mortality after lung transplantation. EBioMedicine. 2019;40(xxxx):541-553. doi:10.1016/j.ebiom.2018.12.029
6. Levine DJ, Ross DJ, Sako E. Single Center “Snapshot” Experience With Donor-Derived Cell-Free DNA After Lung Transplantation. Biomark Insights. 2020;15:53-55. doi:10.1177/1177271920958704
7. Dholakia S, De Vlaminck I, Khush KK. Adding insult on injury: Immunogenic role for donor-derived cell-free DNA? Transplantation. 2020;104(11):2266-2271. doi:10.1097/TP.0000000000003240

Preliminary scope of work

Odmítnutí štěpu (rejekce) je zásadní komplikací po transplantaci plic i přes velký rozvoj imunosupresivní terapie. Z důvodu absence vhodných biomarkerů zůstává stále nejspolehlivějším diagnostickým nástrojem k detekci akutní rejekce transbronchiální biopsie plic, která je však spojena s nezanedbatelnými komplikacemi.
Jedním z nových markerů rejekce je stanovení volné cirkulující dárcovské DNA (ddcfDNA) v krvi příjemce. Sekvenování nové generace (NGS) je vysoce senzitivní metoda umožňující odlišit ddcfDNA na základě stanovení rozdílů v jendonukleotidových polymorfismech mezi DNA dárce a příjemce. Cílem studie je zhodnotit vztah mezi hladinou ddcfDNA v krvi příjemce - pravidelně monitorovanou testem ProsperaTM (Natera Inc.) – a biopticky prokázanou rejekcí u pacientů po transplantaci plic.
V případě dostatečné senzitivity a specificity ve vztahu k rejekčním epizodám by monitorace ddcfDNA mohla v budoucnu umožnit vysoce žádoucí individualizaci udržovací imunosuprese, ev. nahradit rizikovou transbronchiální plicní biopsii u pacientů po transplantaci plic.

Preliminary scope of work in English

The current protocol for LuTx (lung transplantation) surveillance includes spirometry and bronchoscopy with transbronchial biopsy (TBBx) during the first year after LT to monitor for acute rejection. The invasive nature of biopsy and complicated TBBx protocol, as well as the requirements for patients to come to the healthcare facility for treatment, limit the ability of providers to assess graft status without incurring risk for the LT patients. In addition to the medical complications, the clinical utility of biopsy is further limited due to cost, inadequate sampling, and poor reproducibility.
Donor-derived cell-free DNA quantitation has been shown to be an efficient and accurate measure of allograft injury and a clinically useful method to monitor the status of the allograft in transplant patients. Previous studies have shown its value in detecting rejection, prompting closer follow up, and avoiding unnecessary biopsies.
Natera has previously demonstrated accurate quantification of cfDNA mixture proportions using a single-nucleotide polymorphism (SNP)-based massively multiplexed-PCR (mmPCR) methodology in the prenatal and oncology contexts. Leveraging this technology, Natera developed a non-invasive assay that estimates dd-cfDNA fraction (DF) in kidney transplant recipients by measuring the allele frequency at 13,926 SNPs chosen to maximize informative genotypes across ethnicities. A recent clinical validation study demonstrated the ability of this method to discriminate acute rejection (AR) from non-rejection with a sensitivity of 88.7%, specificity of 73.2%, and AUC of 0.87 using a DF cutoff of 1% in kidney transplant patients. Few such studies exist for lung however.
Plasma samples from patients who have undergone lung transplantation with clinical data, including laboratory data, biopsy results, and diagnoses will be used in this study. The use of these samples will enable the examination of the utility of novel rejection biomarkers such as dd-cfDNA in LT surveillance.
In the current study, we aim to assess the performance of the Prospera dd-cfDNA assay in detecting allograft rejection in LT biorepository samples with matched clinical data.