Prognostication by multigene proliferative marker panel compared with Ki-67 in small-intestinal NENs.

242 Background: Ki-67 is the major proliferative marker in clinical use to determine neuroendocrine neoplasm (NEN) prognosis. Ki-67 is unable to predict the outcome of SI-NENs, as the majority have a low (≤2) Ki-67%. Therefore, we aimed to identify a sensitive panel of proliferative markers using qRT-PCR to more accurately define the proliferation of these slow growing tumors. Methods: We identified genes with a mechanistic function in cell cycle progression that were over-expressed in RNA microarrays of SI-NENs (n=8) compared to adjacent normal tissue (n=4) (dCHIP, annotation databases). Timing of marker gene expression (qRT-PCR) in proliferating cell-cycle phases (S, G 2 , M) was determined in flow-sorted SI-NEN cell lines (KRJ-1, H-STS) after propidium iodide staining. RNA expression of candidate proliferative markers was then investigated using an in vivo model and two independent tumor datasets, and transcript level compared to Ki-67% protein expression (immunohistochemical staining). Results: Twenty genes with a mechanistic role in proliferation were identified and 17 confirmed to be expressed in proliferating cell cycle phases. Each tumor expressed a unique profile of the 17 proliferative markers. Both Ki-67 protein and Ki-67 RNA transcript levels failed to differentiate in vivo SI-NEN models or patient samples despite variable proliferative capacity (e.g., WDNETs versus WDNECs). Although most tumors showed low levels of Ki-67 expression, the tumors expressed high levels of select alternative proliferative markers. Hierarchical clustering provided a novel and clinically meaningful prognostic classification. Conclusions: Proliferation of individual SI-NENs is regulated by unique combinations of multiple genes with a mechanistic role in cell-cycle progression. Regulation of proliferation in SI-NENs is therefore complex and cannot accurately be defined by Ki-67 as a single marker. A panel of proliferative RNA markers has potential to significantly improve prognostication in patients with SI-NENs.