Expression And Significance Of PHH3 And NKX6-1 In Pancreatic Neuroendocrine Neoplasms

Background:Pancreatic neuroendocrine neoplasms(PanNENs)is a complicated and heterogeneous group of neoplasm with low incidence.In the past,it was believed that PanNENs was derived from pancreatic islets endocrine cells,while now it may occur anywhere in the pancreas,such as endocrine cells in the periphery of the pancreatic duct and pancreatic acini,not only from pancreatic islets.The incidence of PanNENs is growing rapidly recently.However,PanNENs has the poorly prognosis and some patients have missed the chance of operation due to the particularity of anatomy of pancreas and tumor has no specific clinical manifestation in early stage.Although PanNENs has a better survival than pancreatic cancer(PC),once tumor metastasis has emerged in patients,usually with a high degree of malignancy and the prognosis is poor.Therefore,the standardized diagnosis and treatment of PanNENs have become a great focus of the present medical community.During a long period,the classification and nomenclature had made larger change of each WHO standards because the insufficient knowledge of this disease.In 1980,PanNENs divided into carcinoid,mucocarcinoid,mixed forms carcinoidadenocarcinoma and peudotumor lesions.WHO(2000)classification divided PanNENs into well-differentiated endocrine tumor,well-differentiated endocrine carcinoma,poorly differentiated endocrine carinoma/small-cell carcinoma,mixed exocrine-endocrine carcinoma and tumor-like lesions.As all the pancreatic neuroendocrine neoplasms have malignant potential,the PanNENs should be classified by morphology and proliferative activity.According to latest WHO 2010 classification,neuroendocrine tumors are now divided into five types by Ki-67 proliferation index and mitotic count: well-differentiated neuroendocrine tumors(NETs),poorly differentiated neuroendocrine cancers(NECs),mixed adenoneuroendocrine carcinoma(MANEC),hyperplastic and preneoplastic lesions.NETs are then divided according to a grading scheme based on mitotic count or Ki-67 index in NETs-G1 and NETs-G2.All NECs are graded G3.MANEC include mixed acinar neuroendocrine carcinoma,ductal neuroendocrine carcinoma and acinar ductal neuroendocrine carcinoma,which can be divided into two parts: exocrine portion and endocrine portion,both part accounted for more than 30%.The definition of NETs is highly differentiated tumor with low or moderate grade malignant in WHO(2010)digestive system tumor classification.NET G3 is not advised but called as NEC G3,since G3 is high malignancy.However,the aggressiveness of well-differentiated NETs is much less than poorly differentiated NECs according to the G3 grading classified by mitotic image and Ki-67.This phenomenon prompts that PanNENs G3 is in the characteristic of heterogeneity,and should be further classified as well-differentiated pancreatic neuroendocrine tumors(NETs G3)and poorly-differentiated pancreatic neuroendocrine cancers(NECs G3).At present,the knowledge of biological characteristics,immunophenotype,treatment and prognosis of this well-differentiated pancreatic neuroendocrine tumor(NET G3)is deficient in clinical.Biological behaviors of NETs and NECs are relative independence,and they are not progressive tumor.The biological behaviors of these two solid tumor,such as tumor staging,five years survival rate,overall survival and disease free survival,are different due to sharp distinction signal pathways between NETs G3 and NECs G3.It is the urgent affairs that provide clinic with abundant diagnostic and prognosis information by using a unified pathological grading standard because the treatment and prognosis of different pathological diagnosis patients are different.Although malignant degree of PanNENs can be classified according to the proliferative activity of tumor cells Ki-67 and mitotic counts has been widely accepted in domestic and overseas,it has obvious defect in tumor grading: difficult to calculate,lack of reproducibility,low accuracy and so on.These defects clinical staging in the past lead to misdiagnosis for many patients.Objective:To provide detailed and accurate information to guide clinical treatment and prognosis by standardized the current diagnostic criteria for PanNENs and summarize the histological characteristics of different grade tumors,and seek for more accurate,sensitive and practical immunohistochemical markers to assist Ki-67 in the diagnosis and grading.Methods:A retrospective analysis of "pancreatic occupying" pancreatic cancer radical surgery treatment,and postoperative pathology confirmed 283 cases of PanNENs patients with clinical and pathological data from December 2000 to May 2016 in the First Affiliated Hospital of Second Military Medical University.Two senior pathologists calculated proliferation index by reading pathological sections,mitotic count of HE sections and Ki-67 labeled sections repeatedly,reformulated diagnosis grading according to WHO 2010 pathological classification of digestive system tumor and Consensus on the diagnosis of gastrointestinal endocrine tumors in China(2013),and also provide statistical analysis of the proportion and clinicopathological data of NET G1,NET G2 NET G3 and NEC G3.The mixed adenoneuroendocrine carcinoma(MANEC)includes adenocarcinoma and neuroendocrine carcinoma,each components has different growth pattern and different proportion.Accordingly,during analysis the morphometric data of HE,MANEC was not included,since MANEC has great effect with biology because its complex pathological morphology.In the process of,we selected typical tumor region and made markers.Automatic slicing paraffin-embedded sections from the archive,all of which were IHC stained with HE and immunohistochemical-staining respectively.Antibodies with diagnostic and prognostic value were screened by analyzing experimental data statistically.All data were analyzed by the SPSS 22.0 software for windows.Results:1、The 283 patients,including 132 males and 151 females(male: female=1:1.14),had an age range of 16-78 years(mean age 50±1.3 years);44 cases were functional PanNENs and 239 cases were NF-PanNENs in the study;the median tumor diameter was 4.1±1.2cm(range 1.1-17).2、Pathologic grade: The percentage of NET G1,NET G2,NET G3 and NEC G3 tumors were 44.88%,40.99%,11.66% and 2.47%.3、Clinical stage: The percentage of TNM stage I,stage II,and stage IV tumors were 46.3%,48.4% and 5.3% respectively.4、Expect 34 cases lost(12%),249 cases were followed up with the follow-up rate of 88%,median follow-up was 42(range 4-166)months.5、There was correlation between pathologic grades of PanNENs(NET G1,NET G2,NET G3 and NEC G3)with tumor diameter,lymphatic metastasis and AJCC TNM staging(P<0.05).The patient’s age,sex,tumors function and location were not correlated with pathologic grades of Pan NENs(NET G1,NET G2,NET G3 and NEC G3)(P>0.05).6 、 Immunohistochemical markers: The positive rates of CgA and PHH3 expression in NET G1,NET G2,NET G3 and NEC G3 were significant(P<0.001).There were no meaningful differences between the expression of Syn(P=0.121).NKX6-1 had no statistically significant since the instability expression and nucleo-cytoplasmic location are inaccurate in NET G1,NET G2,NET G3 and NEC G3.7、The expression rates of PHH3 in NET G1,NET G2,NET G3 and NEC G3 of PanNENs were significant(P<0.001).PHH3 has obvious relevance with existing classification index Ki-67(r=0.941,P<0.001).Conclusion:1.According to pathological diagnosis,the most common PanNENs wasNET G1,127 cases(44.88%);followed by NET G2,116 cases(40.99%);and NEC G3 least,7 cases(2.47%).2.There was correlation between pathologic grades of PanNENs(NET G1,NET G2,NET G3 and NEC G3)with tumor diameter,lymphatic metastasis and AJCC TNM staging(P<0.05).The patient’s age,sex,tumors function and location were not correlated with pathologic grades of PanNENs(NET G1,NET G2,NET G3 and NEC G3)(P>0.05);NKX6-1 had no statistically significant since the instability expression and nucleo-cytoplasmic location are inaccurate inNET G1,NET G2,NET G3 and NEC G3.3.PHH3 is a new kind of highly specific markers of PanNENs,there was a significant correlation with Ki-67,even has more advantages than Ki-67,and can be applied to pathological grading of PanNENs.