Transcriptomic analysis by RNA sequencing characterises malignant progression of canine insulinoma from normal tissue to metastatic disease

Y Capodanno,F O Buishand,J Kirpensteijn,R Elders,J A Mol,L Y Pang,D J Argyle

doi:10.1038/s41598-020-68507-z

Abstract

Insulinomas (INS) are the most common human and canine functioning pancreatic neuroendocrine tumours. The long-term prognosis for malignant INS is poor, because micrometastases are frequently missed during surgery. As human and canine malignant INS share clinical and histopathological features, dogs have been proposed as models for INS research. Using RNA-sequencing, we conducted a pilot study to better understand the underlying molecular mechanisms of canine INS. Normal canine pancreas and lymph node control tissues were compared with primary INS and INS-metastatic lymph nodes, revealing more than 3,000 genes differentially expressed in normal pancreas compared to primary INS. Only 164 genes were differentially expressed between primary INS and INS-metastatic lymph nodes. Hierarchical clustering analysis demonstrated similar genetic profiles in normal pancreas and early clinical stage primary INS, whereas late clinical stage primary INS resembled the genetic profile of INS-metastatic lymph nodes. These findings suggest that markers of malignant behaviour could be identified at the primary site of the disease. Finally, using the REACTOME pathways database, we revealed that an active collagen metabolism, extracellular matrix remodelling, beta-cell differentiation and non-beta-cell trans-differentiation might cause disease progression and hyperinsulinism in INS, identifying major pathways worthy of future research in this currently poorly controlled disease.

Highlights

Insulinomas (INS) are the most commonly diagnosed functioning pancreatic neuroendocrine tumours in humans and d ogs[1,2]
Multidimensional analysis using Principal Component Analysis (PCA) plots showed that the normal pancreatic tissues clustered separately from metastatic and normal lymphatic tissues, whereas primary INS lesions had a more heterogeneous pattern (Fig. 1B–D)
To the authors’ knowledge, an RNA-seq approach has never before been used to study canine INS, whereas recent studies have examined the genetics of human INS in the much broader context of P NETs15

Summary

Introduction

Insulinomas (INS) are the most commonly diagnosed functioning pancreatic neuroendocrine tumours in humans and d ogs[1,2]. Still, grading and staging have been so far insufficient to fully assess the degree of malignancy of human and canine INS In both humans and dogs, these tumours can have heterogeneous microscopic findings and the presence of metastases, mainly located in the liver, represents so far the only definitive feature that characterises individual tumours as m alignant[3,8,9,10,11]. Since the completion of the canine genome sequencing[19], various RNA-sequencing (RNA-seq) studies have identified gene signature characteristics of malignancy in a variety of cancers occurring in dogs such as m eningioma[13], mammary c arcinoma16, melanoma[20] and bladder c arcinoma[18], providing additional information about the oncogenesis of human tumours. Our recent study comparing human and canine INS cell lines identified the Notch pathway as a key regulator of stemness and chemoresistance both in vitro and in vivo, providing a rationale for focused further research on this druggable target for INS in both species[2]

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