Endocrine Abstracts

Introduction: Phaeochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumours that are inherited in at least 40% of cases. The most common pathogenic germline variants (PGVs) implicated in the development of PPGLs are in genes coding for the subunits of succinate dehydrogenase (SDH). PPGLs are heterogenous tumours that have a variable clinical phenotype. Rates of metastatic disease varies from 5-40%. This variability is not currently understood. All patients identified with a PPGL related PGV are therefore offered screening that involves annual clinical review and a whole-body MRI every 2-3 years. With patient numbers increasing a more appropriate means of triage is needed. In this study we will use bulk and single cell RNA-sequencing (ScRNA-seq) to identify transcriptomic differences in metastatic vs non-metastatic SDH related PPGLs. Here we will discuss our approach to successfully extracting RNA from fresh as well as archived PPGL tissue samples for both bulk and ScRNA-seq.

Methods: 104 PPGL samples have been collected. Samples were provided in either formalin fixed paraffin embedded (FFPE) blocks or fresh frozen tissue (FFT) stored at -80°C. We have performed RNA extraction on 52 SDH related PPGLs (n = 39 FFPE, n = 13 FFT). RNA was successfully extracted from FFPE samples that were surgically resected up to 23 years previously (average = 11 years). A DV200 of >35% in these samples indicated sufficient RNA integrity for successful sequencing in cases of FFPE stored tissues. A subset of tumour samples collected directly from the operating theatre were processed for immediate single cell dissociation using a bespoke protocol that has resulted in successful ScRNA-seq in 2 PPGL samples.

Results: Bulk-sequencing data is now available for 32 SDH related PPGLs. Utilising archived tissues allows us to increase the number of confirmed metastatic cases for our downstream analysis. ScRNA-seq reveals the distinct cellular populations present in PPGLs.