The transcriptomic signature: an avenue for personalized monitoring of kidney transplant patients

Some of the main objectives of the KTD-innov project are to improve the graft rejection follow-up processes in order to perfect kidney transplantation and consequently promote patient comfort. This is why researchers are now aiming to find new reliable biomarkers to predict transplant rejection. The analysis of the transcriptomic signature appears to be a promising avenue for a personalized monitoring of kidney transplant patients. The focus is on this technique developed within the project.

The importance of biomarkers in monitoring kidney transplant patients

After a kidney transplant, personalized monitoring of post-transplant patients is a major challenge. It ensures that the graft substantially fulfils its biological functions for each patient while detecting possible signs of graft rejection, a decrease in efficiency of the kidney functions or the appearance of diseases.

At each follow-up consultation after the transplant, urine and blood tests are carried out, enabling the main biomarkers to be monitored, such as creatinine or proteinuria among others. As a protocol or in the event of an anomaly detected in a patient, tissue samples by biopsies are made in addition to conducting further analyses.

​In order to make precise diagnoses that would enable, in the event of a problem, the establishment of a more personalized and earlier therapeutic monitoring, research is now focusing on the expression of genes in biopsy samples.  In France, a consortium of experts from the KTD-innov project* has set up a technological platform that analyzes transcriptomic signatures from kidney biopsy samples. 

This team, led by professors Jean-Paul Duong Van Huyen and Alexandre Loupy, has been brought together around the necessary expertise for the proper implementation of such a platform. Blaise Robin, in management of the platform, is an engineer in molecular pathology. He works closely with Carmen Lefaucheur, professor of nephrology, Jessy Dagobert, engineer, Marion Rabant, nephropathologist, Daniel Yoo and Clément Coudereau, bioinformaticians, Maud Racapé, project manager, and many other collaborators. 

​The transcriptomics platform: an added value of the KTD-innov research project

Transcriptomics is the study of all the RNAs (RiboNucleic Acids) produced by cells, which reveal through their presence the production of certain proteins such as antibodies for example. These RNAs thus act as intermediate markers. By knowing the markers produced in the kidney, it is then possible to deduce the problems faced by the cells.

“This type of research isn’t new. There are many teams at the international level who are currently working on this approach,” Blaise Robin, transcriptomics project manager of the team, explains. “However, what is new, thanks to the KTD-innov project, is the scale that it’s deployed on with a cohort of over 800 patients involving 7 hospitals across France. This amount of patients brings more statistical power, which allows us to obtain more reliable results.”

Concretely, how does this happen? “The idea is to work from biopsies traditionally taken during the first year: in the 3rd and 12th month after kidney transplantation in hospital protocol. We retrieve the RNA from these samples to see if the genetic information contained in the biopsy of the transplant patient differs from a healthy patient’s,” explains Blaise Robin. “The differences that appear - also called transcriptomic signatures - could then allow to better diagnose a rejection. We even hope to detect it before histology.”

Already starting to process our RNA samples □ for @KTDInnov □ with #gentleMACS @miltenyibiotec noisy □ but so efficient □ combined with Maxwell @PromegaFR □ #NGS analysis ! pic.twitter.com/sNc96u0wrs

— Blaise Robin (@BlaiseRobin_PTG) April 25, 2019

​New transcriptomic tools for a more personalized patient monitoring

In the implementation of a protocol for the transcriptomic analysis of biopsies, the first issue concerns tests and processing samples. In a standard clinical routine, a biopsy is taken from the patient and then placed in formalin to preserve the cells. 24 hours later, the biopsy is transferred and then contained in an FFPE block (Formalin-Fixed Paraffin-Embedded). From this block, technicians take very thin slices of tissue for the pathologist so they can analyse the structure of the kidney under a microscope.

This is where the Parisian research team comes in. “From these same patient samples, we can now extract the RNA for analysis with what is known as NanoString technology," BLaise Robin said. “This innovation was made possible by the KTD-innov project. We were able to acquire a NanoString machine and go further in the analysis by testing this technique."

A fragment of the biopsy used during the routine is also placed in RNAlater, another fixator which, unlike the FFPE, substantially preserves the RNA but does less so for the kidney’s structure. “RNAlater enables a very high- quality RNA to be obtained while still maintaining a good conservation," specifies Blaise Robin. “The extracted RNA is then sent to our Parisian partners from the GENOM’IC platform at the Cochin Hospital to be analysed by NGS (Next Generation Genetic Information Sequencing). This analysis technique is even more advanced than NanoString and it allows more genes to be seen at the same time."

This double analysis in NanoString and NGS is a real asset to patient follow-up because it enables biomarkers to be reliably identified whether they are from biopsies traditionally taken in routine hospital practice or within the KTD-innov protocol framework.

​KTD-innov: putting cutting-edge technology at the service of transplant patients

Establishing the transcriptomic platform required significant preparatory work since it was necessary to optimize the way in which RNA is extracted – an essential step in the project - then set up the final protocol that is applied to all the biopsies of the 825 volunteer patients, the protocol being unmodifiable during the project.

“On the NanoString side, I observed the team’s work from the University of Edmonton, in Canada, and adapted their RNA extraction protocol to our needs," explains Blaise Robin, manager of the transcriptomics platform. “However, on the RNAlater side for the NGS, it was necessary to compare different RNA extraction kits, test protocols and modify and adapt incubation times in order to obtain, from the same amount of biopsy, the highest quality RNA in the greatest concentration possible. This work should enable to optimize the protocol to the max. “ Before adding that, “Thanks to KTD-innov, we can say that the French transcriptomics laboratory of Inserm U970 is now at the forefront of this precise type of NGS and NanoString analysis".

​And tomorrow?

Transcriptomic analysis promises to have a real impact on the field of transplantation, particularly in the improvement of post-transplant immune surveillance and the improvement of patient diagnoses during their follow-up. "We expect a lot from transcriptomic analysis. If viewed as a companion to histology, it could support complex cases and shed light on the type of diagnosis to be made and therefore the treatment to be followed," concludes professor Jean-Paul Duong Van Huyen. “In the longer term, we could even imagine that the transcriptomic signature could be one of the molecular parameters systematically used. In fact, testing the validity and use of molecular analyses in transplantation is one of the objectives of the pathologists and bioinformaticians in the Paris Transplant Group's research group. ”
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Find out more about the transcriptomic platform
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* The KTD-innov project aims to better understand the immunological mechanisms around the renal graft and to improve the diagnosis of graft rejection by collecting, centralizing and analysing clinical, biological and immunological data from thousands of kidney transplant patients in France. Research teams, clinical centres and industrial partners are currently collaborating on this project. Find out more.