RUNX1T1: a novel predictor of liver metastasis in primary pancreatic endocrine neoplasms

Abstract

Objectives: Using gene expression profiling on frozen primary pancreatic endocrine tumors (PETs), we discovered RUNX1T1 as a leading candidate progression gene. This study was designed (1) to validate the differential expression of RUNX1T1 protein on independent test sets of metastatic and nonmetastatic PETs and (2) to determine if RUNX1T1 underexpression in primary tumors was predictive of liver metastases.

Methods: Immunohistochemical expression of RUNX1T1 protein was quantified using Allred scores on archival metastatic (n = 13) and nonmetastatic (n = 24) primary adult PET tissues using custom-designed tissue microarrays. Wilcoxon rank sum/Fisher exact tests and receiver operating characteristic curves were used in the data analysis.

Results: Median RUNX1T1 scores were 2 (2-7) and 6 (3-8) in metastatic versus nonmetastatic primaries (P < 0.0001). Eleven of 13 metastatic and 1 of 24 nonmetastatic primaries exhibited RUNX1T1-scores of 4 or less (P < 0.0001). Low RUNX1T1 expression was highly associated with hepatic metastases (P < 0.0001), whereas conventional histological criteria (Ki-67 index, mitotic rate, necrosis) were weakly associated with metastases (P = 0.08-0.15). Considering RUNX1T1 expression (Allred) score of 4 or less to be predictive, the sensitivity to predict hepatic metastases was 85%, with a specificity of 96%.

Conclusions: RUNX1T1 protein is underexpressed in well-differentiated metastatic primary PETs relative to nonmetastatic primaries and emerges as a promising novel biomarker for prediction of liver metastases.

FIGURE 1

Box plots showing down-regulation of 2 of the probe sets for RUNX1T1 gene in the primary PECAs that had already metastasized to the liver (MP1 group) as compared with those that were nonmetastatic (clinically localized) at the time of their resection (NM1 group). Data from gene expression profiling experiment on frozen tumor tissues from metastatic and nonmetastatic pancreatic endocrine primaries.

FIGURE 2

A, A primary, well-differentiated PET that had no clinical evidence of liver metastasis (non-MP PET) at the time of resection of the pancreatic primary (hematoxylin-eosin stain, original magnification ×200). B, Same case as in Figure 2A: A non-MP (clinically localized) PET with distinct (2 to 3+) nuclear immunoreactivity for RUNX1T1 protein in the tumor cells (RUNX1T1 protein immunostain, original magnification ×200). C, A primary, well-differentiated PECA that had clinical evidence of synchronous liver metastasis at the time of resection of the pancreatic primary (hematoxylin-eosin stain, original magnification ×200). D, Same case as in Figure 2C: An MP PECA that shows lack of nuclear expression of RUNX1T1 protein in most of the tumor cells (RUNX1T1 protein immunostain, original magnification ×200). E, Section of paratumorous histologically normal pancreatic tissue featuring a pancreatic islet from an MP PECA. RUNX1T1 expression in the islet cell nuclei is lower (only scattered islet cells show nuclear positivity for RUNX1T1) as compared with the paratumorous islet from a non-MP PET in Figure 2F (RUNX1T1 protein immunostain, original magnification ×400). F, Section of paratumorous histologically normal pancreatic tissue featuring a pancreatic islet from a nonmetastatic PET. RUNX1T1 expression in the islet cell nuclei is higher (most islet cells show nuclear positivity for RUNX1T1) as compared with the paratumorous islet from an MP PECA (Fig. 2E) (RUNX1T1 protein immunostain, original magnification ×400).

FIGURE 3

RUNX1T1 protein expression scores in 37 primary PETs.

FIGURE 4

Receiver operating characteristic curve showing the relationship between sensitivity and specificity of RUNX1T1 expression in primary PET tissue as a prognostic test to predict the presence of hepatic metastases.