Next Generation Sequencing to Detect Acute Rejection in Lung Transplant Patients.

Study Overview
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Brief Summary
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Lung transplantation is a consolidated treatment in selected patients with end-stage respiratory diseases; however, acute rejection remains an important cause of lung allograft loss and a risk factor for chronic allograft dysfunction. Histopathological examination of lung tissue is the gold standard for the diagnosis of acute rejection, therefore recipients undergo surveillance transbronchial biopsy and bronchoalveolar lavage after transplantation. However, the obtained tissue is sometimes inadequate for histopathology, and the endoscopic procedure can lead to complications (bleeding, pneumothorax). The quantification of donor-derived cell-free DNA (ddcfDNA) in the recipient plasma has shown to be increased in case of acute rejection, and could represent an early and non-invasive diagnostic marker to detect acute rejection. We planned to enroll all patients aged 18 to 65 years old enlisted for lung transplantation at our centre. Patients undergoing retransplantation and patients with a history of prior solid organ transplantation were excluded. The quantification of donor-derived cell-free DNA was performed 15 days and 3, 6, and 12 months after transplantation, concurrently with the routine surveillance bronchoscopies as per our protocol; the same analysis was also conducted in case of suspected clinical rejection.

Detailed Description
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The purpose of the study is the identification of a non-invasive post-transplantation acute lung rejection monitoring method, by identification of donor derived cell free DNA (ddcf-DNA) in the recipient's plasma. At the time of transplantation, the ddcf-DNA derived from lymphocytes present in the peripheral blood of the donor and recipient will be genotyped by analysis of about 300 single nucleotide polymorphisms (SNPs) with a high probability of being differently homozygous between donor and recipient, according to the frequencies in the population reported on genomic databases. During the post-transplant follow-up, the presence of ddcf-DNA will be monitored by identification of the donor's SNP in the free DNA extracted from the recipient's plasma using the next-generation sequencing (NGS) method. Levels of ddcfDNA in the recipient will be correlated with clinical and histological data obtained from routine 3, 6 and 12 month post-transplant surveillance lung biopsies. All patients enrolled in the study will sign a study-specific written informed consent. -Timeline: T-1 (before lung transplantation): DNA genotyping of the recipient; T0 (lung transplantation): genotyping of donor DNA; T1, T2, T3, T4 (15 days, 3, 6, 12 months after lung transplantation): ddcfDNA research on recipient serum; Tr: in any case of clinical suspicion of acute cellular rejection. At T2, T3, T4 and Tr, surveillance trans-bronchial lung biopsies (TBBs) are also performed, as per our centre's standard post-transplant lung follow-up protocol. -Sample size and statistical analysis: Proportion of ddcfDNA/recipient DNA: stable patients (pA): 0.01, patients with graft damage (pB): 0.30, proportion nA/nB: 3, Power (1-beta): 0.80, Alpha: 5%, Margin of non inferiority δ: 0.05. Population (A+B) : 48; for possible exit from the study we add 7 cases for a total of 55 patients.

Official Title
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Next Generation Sequencing for the Diagnosis of Acute Rejection in Patients Undergoing Lung Transplantation. Pilot Study for Comparative Longitudinal Descriptive Trial.

Conditions
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Lung Transplant Rejection

Intervention / Treatment
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* Diagnostic Test: Quantification of donor-derived cell-free DNA


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Patients undergoing lung transplantation at our centre Patients aged 18 to 65 years old Written informed consent to partecipate in the study Exclusion Criteria: Patients undergoing retransplantation Patients with a history of prior solid organ transplantation

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 65 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Lung transplantation recipients<br>Recipients aged 18 to 65 years old undergoing lung transplantation at Fondazione IRCCS Ospedale Policlinico Milano undergoing quantification of donor-derived cell-free DNA on serial plasma samples | Diagnostic Test: Quantification of donor-derived cell-free DNA<br>* Quantification of ddcfDNA on serial plasma samples during follow-up after lung transplantation<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Proportion ddcfDNA | Proportion of the ddcfDNA on the recipient's plasma cell free DNA | 1 year | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| ddcfDNA-clinical data correlation | Clinical data on trans-bronchial lung biopsies, pulmonary functional tests, culture of bronchoalveolar lavage fluid, donor-specific anti-human leukocyte antigen antibodies. | 1 year | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Acute rejection, Donor-derived cell-free DNA, Lung transplantation, Next-generation sequencing