Deployment and development of the Precision Pathology Platform of the Paris Transplant Group
The Paris Transplant Group (PTG) Precision Pathology Platform specializes in RNA/DNA extraction from all types of samples (FFPE, frozen tissues, plasma, etc….) and its analysis (transcriptomic or genomic) using various tools such as the NanoString® nCounter™ or, more recently, the CareDx® AlloSeq™ assay detecting the presence of circulating donor-derived DNA (dd-cfDNA).
More generally, the main activity of the platform is dedicated to multiplexed direct measurements of gene expression with high levels of accuracy and sensitivity. In close collaboration with the PTG team of bioinformaticians and statisticians, the platform aims to develop risk stratification algorithms based on individual immune monitoring to improve long-term patient survival and optimize graft allocation policies.
More specifically, the PTG Precision Pathology platform seeks to increase the level of phenotyping of cohorts by studying invasive (biopsy) and non-invasive (plasma dd-cfDNA) biomarkers to better characterize rejection activity, stage and response to treatment.
Capable of performing these analyses on all types of research concerning solid organ transplantation (including heart, lung, liver, kidney) and open to any type of collaboration in this sense, the platform is referenced by the “University of Paris” and certified “IDEX” since 2021. It is currently involved in a dozen transplantation projects, some of which are part of clinical trials and are part of the clinical routine of hospitals.
1- Transcriptome analysis of biopsies
On the invasive component, the platform has investigated technologies allowing the transcriptomic study of biopsies, in particular a method already proven with NGS (for Next Generation Sequencing) which allows the study of the entire transcriptome (several tens of thousands of transcripts).
In addition to the existing work in “microarray”, NGS allows the identification of potential unknown transcripts in the pathological mechanisms of transplant rejection. This technique has several disadvantages: a complex data analysis that requires teams of highly qualified bioinformaticians, a relatively high cost and the need to perform this analysis on an additional biopsy different from the one used for clinical routine.
To overcome these drawbacks, the platform uses another, more innovative method of transcriptomic analysis: the NanoString® nCounter™, which can generate high-quality results from RNA-compromised samples such as the FFPE biopsies used in clinical routine. Beyond the advantage of being performed on the same sample used for light microscopy, the nCounter™ offers the possibility of analyzing large cohorts of retrospective and longitudinal samples, often archived in pathology departments, in decentralized multicenter studies.
This equipment has the particularity of being based on a gene panel specially developed for transplantation: the Banff Human Organ Transplant panel (B-HOT) which is the result of the work of the Banff Molecular Diagnostic Working Group (MDWG). As a reminder, the Banff classification is a nomenclature and classification scheme for renal transplant pathology established in 1991 and updated every 2 years. This consortium of experts had been looking since 2013 to add a molecular diagnosis to its classification. It is in this sense that the platform has been and still is strongly involved in the international Banff consortium :
- Platform members actively participate in all recent Banff conferences and have an important impact on the annual conference report with Pr Alexandre Loupy as 1st author on several major publications of the consortium.
- Platform members have been an important element in the development of the B-HOT panel (notably the publication in the AJT 2019 dedicated to it). The B-HOT panel includes validated and informative genes from major peer-reviewed “microarray” and NanoString® studies on renal, cardiac, pulmonary, and liver allograft biopsies identified by the MDWG through an extensive literature review.
The complete platform equipment (nCounter™, Maxwell® and all materials), funded by Prof. Alexandre Loupy’s RHU KTD-innov, started its activity in January 2019 and was installed in the PARCC research center in late 2020. The platform has already processed over 2500 samples and collaborated with internal and external teams on various projects.
2- Analysis of dd-cfDNA in the plasma of transplant patients
The platform is also constantly looking for new approaches that can significantly improve the monitoring of transplant patients through the use of molecular approaches.
Pursuing this goal, it is now equipped with a brand new AlloSeq™ platform to detect dd-cfDNA. The AlloSeq™ assay is a blood-based, and therefore non-invasive, test that measures the amount of dd-cfDNA for transplant monitoring through NGS sequencing. Briefly, it allows the quantification of donor-derived circulating DNA in a plasma sample from the transplanted patient. Indeed, circulating DNA (cfDNA) is a fragmented DNA found in the bloodstream and comes from apoptotic or necrotic cells. In the case of rejection, the graft cells will typically go into apoptosis or necrosis releasing cfDNA into the bloodstream. The detection of this graft-derived cfDNA is therefore an earlier indication of graft rejection than a biopsy for cause.
3- Digital Pathology
The third component uses artificial intelligence methods (Machine Learning) to automatically identify microscopic structures on histopathology slides to assist pathologists in their diagnosis.
