Alternative lengthening of telomeres and ATRX/DAXX loss can be reliably detected in FNAs of pancreatic neuroendocrine tumors

Abstract

Purpose Pancreatic neuroendocrine tumors (PanNETs) frequently utilize the Alternative Lengthening of Telomeres (ALT) pathway for telomere maintenance. ALT is strongly correlated with ATRX and DAXX alterations and a poor prognosis in primary PanNETs. Since fine needle aspiration (FNA) is a non-invasive way of sampling tumors, we evaluated whether we could accurately detect ALT and loss of ATRX/DAXX in a primary PanNET cohort of FNAs. Experimental design All pre-operative FNA cytology cases (2005–2016) with adequate remnant FNA cell block material were assessed for ALT by telomere-specific fluorescent in situ hybridization (FISH) and ATRX and DAXX protein expression by immunohistochemistry (IHC). For 21 patients undergoing tumor resection, the resected case was also assessed to determine the concordance between the FNA and surgical specimen. Results In the primary PanNET cohort of 65 FNAs, ALT was detected in 15 cases (23%). While all ATRX-negative and DAXX-negative tumors were ALT-positive, 3 of 14 (21%) ALT-positive cases did not show nuclear loss of either ATRX or DAXX. The ALT-positive tumors were associated with larger radiographic size (4.9 v. 2.4 cm, on average, p<0.05) and higher grade (p<0.05). Overall, there was 100% concordance in ALT status and ATRX/DAXX IHC results between the FNA and surgical specimen. Conclusions Both ALT and loss of ATRX/DAXX can be accurately performed on FNA specimens with adequate material. Since ALT is a fundamental mechanism of pathogenesis, the ability to determine ALT in small biospecimens has implications for the design of clinical trials.

Assessment of ARX expression, a novel biomarker for metastatic risk in pancreatic neuroendocrine tumors, in endoscopic ultrasound fine‐needle aspiration

“…ARX expression was found to be perfectly concordant between preoperative cytologic specimens and postoperative surgical specimens and there was no inter‐observer variation. ALT was confirmed to be a reliable marker with very good concordance between cytologic and surgical specimens . Furthermore, the presence of ALT was the best predictor of metastatic tumor behavior.…”

“…Protein loss, detected by immunohistochemistry (IHC), can be used as a surrogate for somatic inactivating mutations in ATRX and DAXX . Recently, promising results in determining these markers in preoperative cytologic specimens have been published . Nevertheless ATRX/DAXX IHC poses some difficulties, such as the need for positive internal controls, intratumoral heterogeneity, and presence of mutations (eg, missense) that alter function, but not protein expression or localization.…”

Advances in the cytologic diagnosis of gastroenteropancreatic neuroendocrine neoplasms

“…Some immunohistochemical stains are available that can be useful to distinguish grade 3 NET from NEC by interrogating for loss of protein expression resulting from genetic mutations. Specifically, the loss of nuclear immunohistochemical staining with retinoblastoma (Rb), death‐domain–associated protein (DAXX), and adenosine triphosphatase‐dependent helicase ATRX (ATRX) correlate with mutation status in histologic and cytologic studies . NECs of any primary site can have RB1 gene mutations, whereas NETs have not been reported with this mutation .…”

“…DAXX and ATRX mutations are correlated with loss of expression of their respective nuclear proteins by immunohistochemistry, as discussed above. Although heterogeneous expression for these markers has been demonstrated in resected tumors, DAXX/ATRX immunohistochemistry and ALT fluorescent in situ hybridization have revealed complete concordance between resection specimens and formalin‐fixed cell‐block material prepared from FNAs . ALT also can be detected in tumors with retained DAXX and ATRX nuclear protein expression …”

“…Specifically, the loss of nuclear immunohistochemical staining with retinoblastoma (Rb), death-domain-associated protein (DAXX), and adenosine triphosphatase-dependent helicase ATRX (ATRX) correlate with mutation status in histologic and cytologic studies. [31][32][33][34] NECs of any primary site can have RB1 gene mutations, whereas NETs have not been reported with this mutation. 32,35 Retained Rb staining does not exclude NEC, because this mutation is present only in a subset of NECs.…”