Non-functional pancreatic neuroendocrine tumours: ATRX/DAXX and alternative lengthening of telomeres (ALT) are prognostically independent from ARX/PDX1 expression and tumour size

Abstract

Objective: Recent studies have found aristaless-related homeobox gene (ARX)/pancreatic and duodenal homeobox 1 (PDX1), alpha-thalassemia/mental retardation X-linked (ATRX)/death domain-associated protein (DAXX) and alternative lengthening of telomeres (ALT) to be promising prognostic biomarkers for non-functional pancreatic neuroendocrine tumours (NF-PanNETs). However, they have not been comprehensively evaluated, especially among small NF-PanNETs (≤2.0 cm). Moreover, their status in neuroendocrine tumours (NETs) from other sites remains unknown.

Design: An international cohort of 1322 NETs was evaluated by immunolabelling for ARX/PDX1 and ATRX/DAXX, and telomere-specific fluorescence in situ hybridisation for ALT. This cohort included 561 primary NF-PanNETs, 107 NF-PanNET metastases and 654 primary, non-pancreatic non-functional NETs and NET metastases. The results were correlated with numerous clinicopathological features including relapse-free survival (RFS).

Results: ATRX/DAXX loss and ALT were associated with several adverse prognostic findings and distant metastasis/recurrence (p<0.001). The 5-year RFS rates for patients with ATRX/DAXX-negative and ALT-positive NF-PanNETs were 40% and 42% as compared with 85% and 86% for wild-type NF-PanNETs (p<0.001 and p<0.001). Shorter 5-year RFS rates for ≤2.0 cm NF-PanNETs patients were also seen with ATRX/DAXX loss (65% vs 92%, p=0.003) and ALT (60% vs 93%, p<0.001). By multivariate analysis, ATRX/DAXX and ALT status were independent prognostic factors for RFS. Conversely, classifying NF-PanNETs by ARX/PDX1 expression did not independently correlate with RFS. Except for 4% of pulmonary carcinoids, ATRX/DAXX loss and ALT were only identified in primary (25% and 29%) and NF-PanNET metastases (62% and 71%).

Conclusions: ATRX/DAXX and ALT should be considered in the prognostic evaluation of NF-PanNETs including ≤2.0 cm tumours, and are highly specific for pancreatic origin among NET metastases of unknown primary.

Keywords: neuroendocrine tumors; pancreatic endocrine tumour; pancreatic islet cell; pancreatic pathology; pancreatic surgery.

Figure 1

A summary of the study design to include details of individual patient cohorts, biomarker staining with expected results and correlative clinicopathological findings on individual biomarker analysis. ALT, alternative lengthening of telomeres; ARX, aristaless-related homeobox gene; ATRX, alpha-thalassemia/mental retardation X-linked; DAXX, death domain-associated protein; PDX1, pancreatic and duodenal homeobox 1.

Figure 2

Representative examples of non-functional pancreatic neuroendocrine tumours (NF-PanNETs) that have been assessed by immunolabelling for ARX, PDX1, ATRX and DAXX, and telomere-specific fluorescence in situ hybridisation (FISH) for ALT. (A) NF-PanNET with positive expression for ARX (B) and negative expression for PDX1 (C), while both ATRX (D) and DAXX (E) exhibited preserved nuclear expression and an absence of alternative lengthening of telomeres (ALT) (F). (G) NF-PanNET with negative expression for ARX (H) and positive expression for PDX1 (I), while both ATRX (J) and DAXX (K) had preserved expression and an absence of ALT (L). (M) NF-PanNET with expression for both ARX (N) and PDX1 (O), but loss of nuclear ATRX (P) and preserved nuclear expression for DAXX (Q). the loss of ATRX expression correlated with the presence of large, ultrabright intranuclear foci by telomere-specific FISH, consistent with ALT (R). (S) NF-PanNET with positive ARX expression (T), negative PDX1 expression (U), preserved ATRX expression (V) and loss of DDAXX expression (W). Similar to loss of ATRX, DAXX-negative NF-PanNETs were associated with large, ultrabright telomere signals, consistent with ALT (X). ARX, aristaless-related homeobox gene; ATRX, alpha-thalassemia/mental retardation X-linked; DAXX, death domain-associated protein; PDX1, pancreatic and duodenal homeobox 1.

Figure 3

Kaplan-Meier curves comparing relapse-free survival (RFS) after surgical resection for patients with NF-PanNETs. (A) No statistically significant differences in RFS were identified between patients with NF-PanNETs (n=561) that were classified into four immunophenotypic groups: ARX-positive/PDX1-negative (ARX⁺), ARX-negative/PDX1-positive (PDX⁺), ARX-positive/PDX1-positive (DP) and ARX-negative/PDX1-negative (DN). (B) Similarly, no RFS difference was seen between patients with ARX⁺+DN NF-PanNETs and patients with PDX1⁺+DP NF-PanNETs. however (C) the RFS for patients with ALT-positive NF-PanNETs was significantly shorter than patients with ALT-negative NF-PanNETs. In addition, (D) the combination of ALT status and the immunophenotypic subgroups, ARX⁺+DN and PDX1⁺+DP, did not demonstrate any prognostic benefit over ALT status alone. ALT, alternative lengthening of telomeres; ARX, aristaless-related homeobox gene; NF-PanNETs, non-functional pancreatic neuroendocrine tumours; PDX1, pancreatic and duodenal homeobox 1.

Figure 4

Kaplan-Meier curves for RFS among patients with NF-PanNETs of ≤2.0 cm and without regional lymph node metastases (n=196). Based on the 2017/2019 WHO grading system for NF-PanNETs, (A) no statistically significant difference in RFS was identified; however (B) the presence of ALT was associated with shorter RFS. ALT, alternative lengthening of telomeres; NF-PanNETs, non-functional pancreatic neuroendocrine tumours; RFS, relapse-free survival.