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

Pancreatic endocrine tumors (PETs) are clinically challenging neoplasms. The incidence of these tumors in the United States is estimated to be 4 cases per million persons per year, with a 5-year survival of 44% to 71%.1–3 Based on the presence or absence of metastases and/or gross invasion of adjacent organs, they are classified as pancreatic endocrine carcinomas (PECAs) or tumors PETs (World Health Organization, 2004).4 Clinical behavior of these neoplasms ranges from benign to highly aggressive. In the presence of negative metastatic workup at the time of first tissue diagnosis, it is difficult to predict which of these tumors will become metastatic (“malignant”) or remain localized to the pancreas (“benign”). Conventional prognostic criteria include the presence of a functional tumor, larger tumor size, presence of metastases, higher mitotic rate, tumor necrosis, vascular invasion, perineural invasion, and higher proliferation rate, conventionally assessed as Ki-67 index.4–6 To further refine prognostic evaluation of these neoplasms, a number of histological grading and staging schemes have been proposed.5,7–8 However, it has been difficult to establish reliable prognostic models for these neoplasms, due mainly to their rarity and complex biology. From a practical standpoint, histological tumor characteristics such as cytological atypia and angiovascular and perineural invasion are either not evident or, if present, fail to distinguish between indolent and aggressive tumors. Other frequently evaluated pathological criteria, including tumor size, mitotic count/histological grade, and Ki-67 index, often fail to predict malignant behavior.9 Because patients who develop distant metastases from pancreatic endocrine neoplasms are rarely cured and 5-year survival rates diminish significantly,10 identification of molecular markers of prognosis is vitally important to quantify the risk of (1) future metastases in patients with clinically localized pancreatic endocrine neoplasms and (2) metastatic recurrence in patients with resected pancreatic endocrine primaries. Such molecular markers may therefore identify patients who could benefit from adjuvant therapies and also provide objective criteria to determine the need for postoperative surveillance. Recently, we and others11–17 have carried out gene expression profiling in PETs. To identify candidate progression genes in pancreatic endocrine neoplasms, we used Affymetrix 2.0 gene chip on fresh-frozen metastatic primary PECAs (MP PECAs) that already had metastasized to the liver at the time of resection of the pancreatic primary and a set of frozen non-MP PETs that remained free of clinically detectable metastases until the last patient follow-up. All of the pancreatic endocrine primaries used in this experiment were nonfunctional. We applied rigorous criteria, taking into consideration the P values and fold changes in the differentially expressed genes, including gene function/class and pathway analysis using Metacore from GeneGo, to select our leading “candidate progression genes” that were differentially expressed between the 2 sets of pancreatic primaries (metastatic and nonmetastatic). Among these, CD24 antigen, insulin receptor, TMPRSS6, SERPINA1, SMURF1, RNF43, and AKR1C2 were notably up-regulated, whereas RUNX1T1, protocadherin 9, RASSF5, RERG, ST14, glucagon, and PDGFRL were notably down-regulated. The down-regulation of RUNX1T1, represented by 2 different probe sets, is shown in metastatic as compared with nonmetastatic pancreatic endocrine primaries from our original Affymetrix 2.0 gene chip analysis on RNA extracted from frozen tumor tissues (Fig. 1). From our “candidate progression gene list,” we validated underexpression of several leading candidate progression genes including RUNX1T1 and overexpression of TMPRSS6, SERPINA1, and others on an independent test set of archival PECAs (with liver metastases) using quantitative real-time polymerase chain reaction. 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 ... In this study, we have further validated the differential expression of RUNX1T1 in pancreatic endocrine neoplasms with and without synchronous liver metastases at the protein level. Furthermore, we found RUNX1T1 protein expression in the primary tumor tissue to be more informative than other conventional pathological criteria as predictors of liver metastases. This is an important finding with potential clinical and therapeutic implications that merits further evaluation in larger-scale clinical validation studies.