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

Abstract

Background: The transcription factors ARX and PDX1, and alternative lengthening of telomeres (ALT) were recently described as prognostic markers for resected non-functional pancreatic neuroendocrine tumors (PanNETs). ALT positive tumors with ARX expression relapse most often. Currently, tumor size is the only preoperative marker used to decide whether or not to operate, thus additional preoperative prognostic markers are needed. Therefore, it is critical to assess the performance of these biomarkers on preoperative cytologic specimens.

Methods: Endoscopic fine-needle aspiration cellblock material and corresponding surgical specimens of 13 patients with PanNETs were assessed for histology, immunohistochemical staining of ARX, PDX1, Synaptophysin, Ki67, and telomere-specific fluorescence in situ hybridization to detect ALT, and then associated with clinicopathological features. Scoring for ARX and PDX1 was performed blinded by two independent observers.

Results: Of the 13 surgical specimens, 8 were ARX+/PDX1-, 2 ARX-/PDX1+, and 3 ARX+/PDX1+. Concordance between cytologic and surgical specimens for ARX protein expression was 100%, whereas concordance for PDX1, ALT, and WHO tumor grade was 85%, 91%, and 73%, respectively. There was a perfect inter-observer agreement in ARX and PDX1 scoring.

Conclusion: ARX can reliably be determined in cytologic specimens and has low inter-observer variability. For cytology, false-positive PDX1 expression was observed, possibly due to contamination or sampling, while ALT had a false-negative case due to incomplete sampling. As previously observed, tumor grade is underestimated in cytologic specimens. Thus, ARX and ALT are the most promising markers to predict metastatic behavior in PanNETs, thereby warranting further validation in larger studies.

Keywords: cytology; endoscopic ultrasound; neuroendocrine tumor; pancreas; prognostic markers.

Figure 1

Immunohistochemistry and telomere‐specific fluorescence in situ hybridization of cytologic and surgical specimens. Representative images of cytologic and surgical specimens. A, patient 13; ARX positive, PDX1 negative tumor, ALT positive. B, patient 6; ARX negative, PDX1 positive tumor, without ALT. C, patient 10, discordant case for PDX1 expression; ARX positive, PDX1 negative, ALT positive surgical specimen. PDX1 positive cells are present in the surgical specimen but are below the defined cut‐off. Also note the cytoplasmic background fluorescence in the telomere FISH cytologic specimen. IHC at ×40, 50 μm scale bar. Telomere FISH at ×100, nucleus visible as DAPI blue and ultrabright telomeric signals in the red channel [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 2

Comparison of matched cytologic and surgical specimens. Thirteen patients for which the cytologic specimen (gray bar and circle) was compared to the surgical specimen (blue bar and circle). Differences in transcription factor subtype (text within circle), telomere phenotype (circle fill), and Ki67 labeling index (y‐axis) can be observed between cytologic and surgical specimens. For patients 2 and 5, the telomere phenotype and Ki67 index were not interpretable. Primary tumor size and behavioral characteristics are given per patient. Comparison of continuous Ki67 labeling index is shown on the right side with a paired Wilcoxon test. A, ARX positive; B, PDX1 positive; DP, double (ARX/PDX1) positive; LN, lymph nodes; N.I., not interpretable; U, unknown [Color figure can be viewed at http://wileyonlinelibrary.com]