Cell Cycle Protein Expression in Neuroendocrine Tumors: Association of CDK4/CDK6, CCND1, and Phosphorylated Retinoblastoma Protein With Proliferative Index

Abstract

Objectives: Dysregulation of the cell cycle has been observed and implicated as an etiologic factor in a range of human malignancies, but remains relatively unstudied in neuroendocrine tumors (NETs). We evaluated expression of key proteins involved in cell cycle regulation in a large cohort of NETs.

Methods: We evaluated immunohistochemical expression of CDKN1B, CDKN1A, CDKN2A, CDK2, CDK4, CDK6, cyclin D1, cyclin E1, and phosphorylated retinoblastoma protein (phospho-RB1) in a cohort of 267 patients with NETs. We then explored associations between cell cycle protein expression, mutational status, histologic features, and overall survival.

Results: We found that high expression of CDK4, CDK6, CCND1, and phospho-RB1 was associated with higher proliferative index, as defined by MKI67. We additionally observed a trend toward shorter overall survival associated with low expression of CDKN1B. This association seemed strongest in SINETs (multivariate hazards ratio, 2.04; 95% confidence interval, 1.06-3.93; P = 0.03). We found no clear association between CDKN1B mutation and protein expression.

Conclusions: Our results suggest that dysregulation and activation of the CDK4/CDK6-CCND1-phospho-RB1 axis is associated with higher proliferative index in NETs. Investigation of the therapeutic potential of CDK4/CDK6 inhibitors in higher grade NETs is warranted.

FIGURE 1

Cell cycle protein expression in NETs. A, Representative expression of cell cycle protein in NETs. a: High CDKN1B expression; b: Low CDKN1B expression; c: High phospho-RB1 expression; d: High CDK4 expression; e: High CDK6 expression; f: High CCND1 expression (×400). B, Expression of inhibitory cell cycle proteins according to tumor subtype. Low expression of CDKN1B was more common in PNETs (42.3%) and other NETs (48.7%) than in SINETs (23.8%). Low expression of CDKN1A was more common in SINETs (62.8%) than in other NETs (34.2%) and PNETs (12.0%). C, Expression of activating cell cycle proteins according to tumor subtype. High expression of phospho-RB1 was more common in PNETs (46.2%) than in other NETs (30.7%) and SINETs (19.6%). High expression of CDK2 and CCND1 was also more common in PNETS (30.8% and 38.5%, respectively) than in SINETs (8.5% and 1.2%, respectively). NETs indicates neuroendocrine tumors; OTHERs, other neuroendocrine tumors including appendix, colon, lung, ovary, paraganglioma, parathyroid, pheochromocytoma, rectum, stomach, thymus, and unknown primary; p-, phospho; P, phosphorylated; PNETs, pancreatic neuroendocrine tumor; SINETs, small intestinal neuroendocrine tumors.

FIGURE 2

Association between expression of cell cycle proteins in NETs according to pathway. Raw immunoreactivity scores were used for correlation analysis. CCND1 expression was positively correlated with expression of CDK4 (R = 0.12, P = 0.05) and phospho-RB1 (R = 0.21, P = 0.0007). CDK6 expression was positively correlated with expression of CDK4 (R = 0.26, P < 0.0001). NETs indicates neuroendocrine tumors; P, phosphorylated.

FIGURE 3

Overall survival in patients with SINETs according to CDKN1B expression. Survival curves were estimated using the Kaplan-Meier method, and statistical significance was defined as a two-sided P < 0.05. Low expression of CDKN1B was associated with shorter survival in SINETs (P = 0.02). SINETs indicates small intestinal neuroendocrine tumors.