Th237 - The Reverse Transcriptase Multiplex Ligation Dependent Probe Amplification (RT-MLPA) Assay as a Simple Molecular Tool for Diagnosis and Classification of Rejection in Formalin-fixed Paraffin-embedded Kidney Transplant Biopsies

Jack Beadle, MD, PhD student – Department of Immunology and Inflammation, Centre for Inflammatory Diseases – Imperial College, London, United Kingdom; Frederic Toulza, PhD – Department of Immunology and Inflammation, Centre for Inflammatory Diseases – Imperial College, London, United Kingdom; Philippe Ruminy – INSERM U1245 – Univ Rouen Normandie; Arnaud François, MD – Pathology Department – Rouen University Hospital; Dominique Bertrand, MD – Nephrology - kidney transplant unit – Rouen University Hospital; Dominique Guerrot, MD PhD – Nephrology - kidney transplant unit – Rouen University Hospital; Fanny Drieux, MD PhD – INSERM U1245 – Univ Rouen Normandie; Olivier Boyer, MD PhD – INSERM U1234 – Univ Rouen Normandie; Candice Roufosse – MD, PhD, Department of Immunology and Inflammation, Centre for Inflammatory Diseases, Imperial College, London, United Kingdom; Sophie Candon, MD PhD – INSERM U1234 – Univ Rouen Normandie

Abstract Text: Background
The Banff recommendations for the diagnosis of kidney transplant (KT) rejection includes molecular assessment of the transplant biopsy. However, implementation of molecular tools in clinical practice is still limited, partly due to the required expertise and the high cost. The reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) assay is a rapid and affordable assay that permits simultaneous evaluation of a restricted gene panel using paraffin-embedded tissue blocks. We aimed to develop and validate a gene panel and to propose an open-access tool for the diagnosis and classification of KT rejection.

Methods
A bicentric cohort of 220 KT biopsies, with 52 antibody-mediated rejection (AMR), 51 T cell-mediated rejection (TCMR) and 117 no-rejection controls was assessed. A 17-gene panel was identified, including genes significantly associated with at least one of these diagnoses. A support vector machine classifier (SVM) was developed. A subset of 109 biopsies was also assessed using the Nanostring B-HOT panel to compare the two assays.

Results
The SVM classifier train and test accuracy scores were 0.84 and 0.83, respectively. In the test cohort, the F1-score for AMR, control and TCMR were 0.88, 0.86 and 0.69, respectively. Gene expression levels assessed by RT-MLPA or Nanostring correlated: r=0.68, p< 0.0001, with a concordant molecular profile in 81% of the samples.

Conclusions
The 17-gene panel RT-MLPA assay, developed here for FFPE tissue blocks, is a simple molecular tool that can be used to diagnose kidney transplant rejection and classify it between humoral or cellular rejection, without the need for additional biopsy.