Mitotic Count by Phosphohistone H3 Immunohistochemical Staining Predicts Survival and Improves Interobserver Reproducibility in Well-differentiated Neuroendocrine Tumors of the Pancreas

Well-differentiated neuroendocrine tumors (WDNETs) of the pancreas are graded on the basis of mitotic count or Ki67 index. Mitotic count has a narrow cutoff; its assessment is time consuming and carries poor interobserver reproducibility. Phosphohistone H3 (PHH3) is a mitosis-specific marker whose value has been validated in several tumor types. We sought to assess the utility of PHH3 in histologic grading of pancreatic WDNETs. Sixty-three cases of surgically resected primary pancreatic WDNETs were retrieved, and immunohistochemical analysis for PHH3 and Ki67 was performed. Mitotic rate was independently assessed by 4 pathologists on hematoxylin and eosin (HE; in 50 high-power fields [HPFs], expressed as mitoses/10 HPF) and PHH3 stains (in 50 HPFs, one 10x, and one 20x hotspot). PHH3 and Ki67 labeling indices were determined on a single 20x hotspot and expressed as the percentage of positive cells to total cells. We found that mitotic counts by various methods significantly correlated with each other and also with PHH3 and Ki67 indices, with the best correlation seen within the 3 different PHH3 counts (in 50 HPFs, one 10x and one 20x hotspot). Moreover, mitotic count on PHH3 was less time consuming than that on HE (1.68 vs. 3.67 min for 50 HPFs, P < 0.0001). Histologic grade determined by PHH3 significantly correlated with disease-specific and disease-free survivals, with the best cutoffs of >= 4 mitoses/10HPF (2mm(2)), >= 7 mitoses/10x hotspot, >= 5 mitoses/20x hotspot (log rank test, P < 0.0001), and >= 0.16% for PHH3 labeling index (log rank test, P < 0.0006). Tumor grades based on PHH3 stain also showed significant correlation with patient survivals in multivariate Cox proportional hazards models (P < 0.05). Histologic grades by mitotic counts on PHH3 demonstrated high concordance and kappa agreement with grades determined by mitotic count on HE. PHH3 stain also showed improved interobserver agreement in both original mitotic count (intraclass correlation 0.98 vs. 0.79) and final grade assignment (Fleiss kappa 0.69 vs. 0.46) as compared with HE. Thus, our data confirmed that histologic grading by PHH3 stain has practical and prognostic values and offers reduced time and improved interobserver reproducibility in mitotic rate assessment and grade assignment. Although larger series are needed for validation, mitotic rate can potentially be determined by counting 1 hotspot, which will greatly facilitate the assessment of histologic grade in pancreatic WDNETs.