Alternative Lengthening of Telomeres and Differential Expression of Endocrine Transcription Factors Distinguish Metastatic and Non-metastatic Insulinomas

Abstract

Insulin-producing pancreatic neuroendocrine tumors (PanNETs)/insulinomas are generally considered to be indolent tumors with an excellent prognosis after complete resection. However, some insulinomas have a poor prognosis due to relapses and metastatic disease. Recently, studies in non-functional PanNETs indicated that behavior can be stratified according to alpha- and beta-cell differentiation, as defined by expression of the transcription factors ARX and PDX1, respectively. It is unknown whether similar mechanisms play a role in insulinomas. Therefore, we determined ARX and PDX1 expression in a cohort of 35 sporadic primary insulinomas and two liver metastases of inoperable primary insulinomas. In addition, WHO grade and loss of ATRX or DAXX were determined by immunohistochemistry, and alternative lengthening of telomeres (ALT) and CDKN2A status by fluorescence in situ hybridization. These findings were correlated with tumor characteristics and clinical follow-up data. In total, five out of 37 insulinoma patients developed metastatic disease. Metastatic insulinomas were all larger than 3 cm, whereas the indolent insulinomas were smaller (p value < 0.05). All three primary insulinomas that metastasized showed ARX expression, 2/3 showed ALT, and 1/3 had a homozygous deletion of CDKN2A as opposed to absence of ARX expression, ALT, or CDKN2A deletions in the 32 non-metastatic cases. The two liver metastases also showed ARX expression and ALT (2/2). The presence of ARX expression, which is usually absent in beta-cells, and genetic alterations not seen in indolent insulinomas strongly suggest a distinct tumorigenic mechanism in malignant insulinomas, with similarities to non-functional PanNETs. These observations may inform future follow-up strategies after insulinoma surgery.

Keywords: Insulinoma; Liver metastasis; Malignant insulinoma; Neuroendocrine cells; Pancreatic neuroendocrine tumor.

Fig. 1

ARX expression, tumor size and liver metastases-free survival in primary insulinomas. a–c Positive ARX expression in the three metastatic primary insulinomas (patient 1, 2, 3) and no ARX expression in a non-metastatic insulinoma (d). Photos of immunohistochemistry, white-balanced, with 50-μm scale bar. e Tumor size plot. Cases with liver metastases in the size plot are circled (green), WHO grade is given. f Kaplan Meier of liver metastases-free survival for ARX expression in the primary insulinoma cohort. P value was calculated with the log-rank test

Fig. 2

Tumorigenic mechanisms in clinically defined insulinomas. Hypothetic distinct pathways of tumorigenesis in clinical insulinomas based on previously published data in combination with current findings [1, 12, 26, 35]. 1 Typical small insulinomas characterized by recurrent YY1 mutations (25%), neutral, or amplified chromosomal copy numbers, and endocrine transcription factor expression consistent with normal beta-cell differentiation (PDX1+/ARX−). 2 Large insulinomas with distinct tumorigenic mechanisms often seen in non-functional PanNETs and endocrine transcription factor expression inconsistent with normal beta-cell differentiation (ARX+)