Actinin-4 splice variant - a complementary diagnostic and prognostic marker of pancreatic neuroendocrine neoplasms

Abstract

Introduction: For pathological diagnosis of pancreatic neuroendocrine neoplasms (pNENs) the routinely used immunohistochemical markers are chromogranin A (CgA) and synaptophysin (Syn). Their ability as prognostic markers is not well established. A splice variant of actinin-4 (Actn-4sv) was recently found to be an excellent biomarker of neuroendocrine neoplasms of the lung. We aimed to investigate the expression of Actn-4sv in pNENs and evaluate its quality as a biomarker of pNENs. Methods: Paraffin-embedded and frozen tissues specimens from 122 pNENs were analyzed. Western blots were performed to prove and compare the relative amount of Actn-4sv expression in pNENs tissue homogenates. For comparison pancreatic ductal adenocarcinoma (PDAC) and normal pancreatic tissues were analyzed in parallel. Immunohistochemistry (IHC) of paraffin sections of pNENs for Actn-4sv were performed and compared to the classic neuroendocrine markers CgA and Syn. Correlations were calculated between the staining intensity and distribution of Actn-4sv and staging, grading and afflicted lymph nodes respectively. Results: Actn-4sv was expressed in 88.5% (108/122) of pNENs, but not in normal pancreatic tissues (0/14) or PDAC (0/14). Compared to CgA and Syn, Actn-4sv was not detectable in islet cells of the normal pancreas. Staining intensity of Actn-4sv on pNENs negatively correlated to the histological grading (Spearman r=-0.4990, p<0.0001) and staging (r = -0.2581, p = 0.0041) but no correlation to afflicted lymph nodes was found. A significantly better overall survival was observed for pNEN patients with higher expression of Actn-4sv (hazard ratio 2.7; log-rank test p= 0.0349). Conclusions: The expression of Actn-4sv may be an important prognostic factor for patients with pNENs. Its expression correlates with the grading and staging of the tumors.

Keywords: actinin-4; actinin-4 splice variant; pNEN; survival.

Figure 1

Expression of Actinin-4 and actinin4-splice variant (Actn-4sv) in pancreatic tissue extracts from chronic pancreatitis (CP), pancreatic ductal adenocarcinoma (PDAC), pancreatic neuroendocrine neoplasia (pNEN), and normal tissue using western blot. A: Lanes 1-2: CP-, lanes 3-4: PDAC-, lanes 5-6: pNEN- and lanes 7-8: normal-pancreatic (CO) tissue extracts. B: Detection of Actn-4sv in three pNENs but not in the corresponding adjacent normal tissues (donor). Moderate to strong expression of Actn-4sv was detectable in all pNEN tissue extracts but not detectable in normal pancreatic tissue, CP and PDAC extracts. Using a pan-actinin-4 antibody, detecting both the wild type actinin-4 and Actn-4sv, positive staining was found in all lysates. Immunoblots for β-actin were used as loading control reference.

Figure 2

Expression of Actinin-4 and Actn4sv proteins in human PDAC cell lines and human pNEN cell line BON-1. A: Proteins were extracted from A549 cells that had been transfected (mock) with plasmid pEGFP (lane 1) or pEGFP-ACTN-4-SV (lane 2) and PDAC cell lines (lane 3-7) and blotted with 15H2 (Actn-4sv), 13G9 (pan-ACTN-4) and anti-β-actin (loading control) antibodies. B: Actn-4sv expression was only detectable in BON-1 cells. Lanes 1- 2: MiaPaca-2; lane 3-4: T3M4; lane 5: BON-1 (M); lane 6: BON-1after 72 h starvation (low glucose, serum free), lane 7, BON-1 (L). (L= low glucose 5.5µM and M = medium glucose 22 µM).

Figure 3

IHC of FFPE tissue on consecutive slides of normal pancreatic tissue (panel A-C) and pNEN (panel D-F), small inserts show the corresponding negative controls. Staining for Actn-4sv (A, D), CgA (B, E), and Syn (C, F). In normal pancreas the islet cells are stained for CgA and Syn but not for Actn-4sv. In contrast in pNEN a strong staining for the three markers is visible. (Magnification x 200)

Figure 4

A: Survival of pNEN patients in regard to grading. B: Correlation of Actn-4sv staining intensity vs. grading.

Figure 5

Postoperative survival of pNEN patients in regard to Actn-4sv expression (Kaplan-Meier estimates). Significantly better survival for patients with median to strong Actn-4sv staining intensity (log-rank test, p = 0.0349).

Figure 6

IHC on tumor tissues for expressed hormones (positive/negative): insulin (27/75), gastrin (11/77), glucagon (21/78) and somatostatin (21/74) versus intensity (Low High) of Actinin-4 splice variant staining.

Figure 7

Abundance of Actn-4 and Actn-4sv mRNA variants in pNEN transcriptomes. RNAseq datasets from TCGA (left) and generated in-house were analyzed for incorporation of exon 8 (Actn-4, dark grey) or the alternative exon 8' (Actn-4sv, light grey). All samples analyzed express Actn-4sv at RNA level. For comparability between samples, expression was normalized as described in the Materials & Methods section. The black horizontal bar denotes those samples for which also Actn-4sv IHC has been analyzed in this study.

Figure 8

Abundance of Actn-4 and Actn-4sv mRNA variants in PDAC cell lines. RNAseq datasets of pancreatic ductal adenocarcinoma cell lines were obtained from the TCGA Legacy Archive and analyzed analogously to the pNEN transcriptomes. ACTN4 expression varies widely between cell lines while Actn-4sv (light grey) expression is generally low, ranging between 1 and 5% of total ACTN4 mRNA.