RC6.1 - ECE_3949 - Combining bulk, single nuclei and single cell RNA sequencing of phaeochromocytomas and paragangliomas to gain insights into metastatic potential

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Abstract Background Phaeochromocytomas and paragangliomas (PPGLs) often display a unique pattern of metastatic spread whereby, following initial tumour resection, there is a prolonged period of remission before recurrence arises with metastatic spread. Treatment options for metastatic PPGLs are limited and outcomes are poor. Current tools used to predict metastatic risk underperform. A biomarker is urgently needed to identify patients at high risk of metastasis. This is especially relevant given the genetic drivers involved in PPGLs and the ever-increasing pool of patients identified as carries of these variants through cascade screening. RNA-sequencing has shown significant utility in increasing our understanding of PPGLs. Bulk RNA sequencing (RNA-seq) has been used to describe PPGLs in clusters and single nuclei RNA sequencing (SnRNA-seq) has helped to refine this classification even further. With this work we will combine bulk, single nuclei and single cell RNA sequencing (ScRNA-seq) to identify transcriptomic markers of metastatic disease in PPGLs. Methods We have collated bulk RNA-seq datasets from publicly available sources (n = 326) with data from our own, local tumour collections (n = 27) to compare the transcriptome of metastatic to non-metastatic PPGLs focusing on SDHx mutations. We have performed a differentially expressed gene (DEG) analysis of metastatic and non-metastatic PPGLs to identify genes that may have a role in metastatic disease. We have also performed Sn and ScRNA-seq on 8 samples including one that had metastasized at the time of resection and another that metastasized following a period of remission. Results Our bulk RNA-seq analyses identified the following overexpressed genes in metastatic PPGLs: FN1, VEGFA, CDK1, PTTG1, ABCA12 and MMP9. Both ABCA12 and MMP9 were consistently overexpressed when our analyses isolated SDHx samples. ScRNA-seq provided a detailed picture of the tumour microenvironment (TME) of PPGLs when compared to SnRNA-seq. The cellular populations seen in PPGLs across the spectrum of genotypes were comparable. There were differences in the expression of PTTG1, MMP9 and ABCA12 in the chromaffin cells and TME of pre-metastatic and metastatic PPGLs that warrant further investigation. Discussion We have demonstrated that bulk RNA-seq can be used to identify genes that are overexpressed in metastatic PPGLs. Sn and ScRNA-seq allow us to explore the expression profile of tumour cells and the TME. PTTG1 and MMP9 have a defined role in the development of metastasis in a number of cancers. By combining these state-of-the-art techniques we show that their overexpression in the TME of PPGLs may have a role in metastatic spread.

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