This month we hear from Young Scientist, Guido Trezeguet. Guido is pursuing his PhD at the Paediatric Hospital Dr. Juan P Garrahan in Buenos Aires, Argentina. We met in person at the recent Prague congress, and I was very impressed with his work related to cell free DNA for the detection of acute graft rejection. Although the technique he is working on may enable wider clinical application due to reduced cost compared to earlier approaches, there are particular challenges in the paediatric setting. Looking forward to hearing developments arising from Guido’s work! Read on!
After graduating as a biochemist, I started the Ph.D. program at Hospital de Pediatría Garrahan, Buenos Aires, Argentina. My work is related to assessing the immunological risk in paediatric liver transplant patients through the evaluation of HLA molecular mismatch and to studying donor- and recipient-derived cell-free DNA (cfDNA) as a biomarker of graft dysfunction.
I am also involved in pharmacokinetic and safety studies of immunosuppressant drugs used in paediatric liver transplantation to optimize the therapeutic regimen for individual recipients.
A typical day at the hospital would include laboratory work and close interaction with all the professionals involved in transplant-patient care. To collect all the data and samples required in the protocol that we are performing, I need to routinely maintain close follow-up of include patients, which are more than 150 to date. To do this, I assist with the daily medical update performed both in the intensive and intermediate care units, where I decide if the patient meets the inclusion criteria for the required samples, collect additional samples if required, and record important data including modifications in the immunosuppressive scheme or relevant clinical events.
I also collect samples during surgeries, including transplantations and liver biopsies. When a liver transplant is performed, I collect samples from donor and recipient to obtain their DNA, and recipients’ plasma for histocompatibility and sensitization evaluation, respectively. During for-cause and protocol biopsies, I collect a plasma sample before the intervention to evaluate donor- and recipient-derived cfDNA, a buccal swab sample if pre-transplant recipient’s genomic DNA is not available and a fragment of the biopsied tissue to perform transcriptomic analysis.
To evaluate the performance and characterize the kinetics of cfDNA in paediatric liver transplant patients, we are using a commercial kit validated for kidney recipients. Considering the high costs of those kits, we are developing a methodology to differentiate and quantify donor- and recipient-derived cfDNA through digital-droplet PCR, which would be affordable for Latin-American countries. We collect information regarding the genomic target variants reported in our population, perform in-silico analysis, and we are now advancing with the technical aspects of the qPCR performance and its analysis through High Resolution Melting.
I think that all the research we are performing in a paediatric liver transplant cohort is innovative, as for a long time the liver was considered a non-immunogenic organ, and the risk of de-novo immunological activation based on donor and recipient HLA mismatches has not been considered for organ allocation or for the individualization of the immunosuppressive scheme.
There is now evidence demonstrating that donor and recipient HLA mismatches are not innocuous, but impact on the development of donor-specific antibodies (DSA) and on the incidence of acute and chronic rejection, which affects long-term graft survival.
In liver transplant patients, DSA evaluation is not performed in the clinical setting worldwide as their impact on graft survival is probably underestimated, considering the limited data published in this specific cohort. Our approach is to evaluate donor and recipient HLA molecular mismatch, which demonstrated superiority over antigen mismatch.
We are performing a prospective, longitudinal study that includes the quantification of donor and recipient HLA molecular mismatch at transplantation, the kinetics of donor- and recipient-derived cfDNA during the first-year post-transplant and clinical outcomes including de-novo DSA formation, histopathology findings from for-cause and protocol biopsies and its transcriptomic analysis.
The evaluation of cfDNA as a biomarker of graft injury has been validated in kidney transplant recipients but its application to liver transplantation, and more specifically to paediatrics, is innovative. Moreover, the development of an affordable methodology to use it as a graft injury monitoring tool is necessary for our region.
The change of paradigm regarding liver allograft immunogenicity with the consequent incorporation of the evaluation of immunological parameters (DSA in serum and C4d deposits in the biopsied tissue) in our guidelines for the follow-up of liver transplant patients. From the research that we are performing, if the immunological risk stratification is proven to be feasible through the HLA molecular mismatch in liver transplant patients, it would permit a radical evolution in practice as the immunosuppressive scheme could be individualized.
The development of molecular methodologies to evaluate graft health would be an evolution in practice as it would i) reduce the necessity of invasive interventions by its monitoring through cfDNA in peripheral blood and ii) predict the evolution of certain diagnosis through the evaluation of gene expression (transcriptomics) when a biopsy is required.
I became interested in transplantation since coming across transplant immunology during my degree, especially since it combines immunology and genomics. When I started working on my thesis, the multidisciplinary and dynamic nature of the work caught my attention as I am constantly learning from professionals in different areas, including pharmacists, biochemists, biotechnologists, surgeons, nurses, etc. I am particularly interested in TDM as the correlation between variability in the immunosuppressants trough concentrations and clinical outcomes were proven to be elemental.
I think that with the availability of biomarkers, PK/PD modelling will be feasible to be applied to transplant patients. Also, during the IATDMCT congress (Prague), work related to microbiome and pharmacokinetics caught my attention, as liver transplant patients usually have several complications in the post-transplant setting, with abdominal collections that get infected and require all kinds of antibiotics, affecting the microbiome of each patient differently and probably, the pharmacokinetics of concomitant medications. Another work presented that sounded futuristic to me was the development of patches to monitor levels for several drugs in real-time.
In my opinion, biomarkers will gain weight in the future to i) predict immunological risk (HLA molecular mismatch) allowing immunosuppression; and ii) exclude the diagnosis of rejection (cfDNA), reducing the number of biopsies required (due to a high negative predictive value). Further in the future, perhaps the incorporation of transcriptomics to predict injury events through evaluation of activated immunological pathways, or if rejection/injury events are worsening or improving.
The first feeling I had during Dario Cattaneo’s talk at the IATDMCT Congress was that every physician involved in the care of patients that require chronic treatment should be involved in TDM. I think that a major challenge is to transmit the importance of TDM to them.
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