The First Part Of PHLDA3 Gene Inactivation Leads To Tumorigenesis And Clinical Significance. The Second Part Of The Pancreatic Neuroendocrine Tumor YY1 Gene Hotspot Mutation

Background and Aim:Our previous studies find the frequent loss of heterozygosity(LOH)of 1q31-32 in pancreatic neuroendocrine tumors(PNETs),PHLDA3 gene located in this region,the Japanese study observe islet cell hyperplasia in PHLDA3 gene knockout mice.So,the purpose of our study is to expore whether PHLDA3 gene knockout rat develop PNETs,to find whether human PNETs exist PHLDA3 gene abnormal and protein expression decrease,then explain the molecular mechanism of sporatic PNETs development.Elucidate the prognostic value of PHLDA3 expression at the same time.Methods:We detected the expression of PHLDA3 protein in 222 PNETs and 109 para-tumor tissues by immunohistochemical method,then analysis the clinical value of PHLDA3 expression.Methylation status of PHLDA3 gene promoter is determined by methylation specific PCR.Microsatellite markers are used to detect the presence of PHLDA3 LOH.Knockout of PHLDA3 gene in SD rats by CRISPR/Cas9 technique,reproduction and identify the genotype,the rats are killed at the age of 6 months,12 months and 18 months to observe whether there is tumor formation,then obtain the pancreatic tissue for paraffin embed and HE staining,analysis the changes of islet number and size between PHLDA3 knockout rats and wild rat,detect the rat serum sugar level and insulin level,analyse the correlation of PHLDA3 and glycometabolism.Results:PHLDA3 expression is more common in non-metastatic PNETs(28.5%vs.17.9%,P = 0.078),patients with positive expression of PHLDA3 protein have a better prognosis(overall survival:N=187,P=0.026;disease-free survival:N=187,P=0.024),and have a longer survival time(overall survival:N=187,Log RankP=0.017;disease-free survival:N=182,Log RankP=0.041).There is a high proportion of PHLDA3 gene promoter methylation in PNETs as well as the para-tissues(12/19 and 3/6,respectively),however,we find no correlation between promoter methylation and protein expression.25.7%PNETs exist PHLDA3 LOH,tumors with PHLDA3 LOH have a lower expression rate of PHLDA3(21%vs.52%,P = 0.098).PHLDA3 knockout rats could form PNETs,there's no obvious difference of islet size and morphology between PHLDA3 knockout rats and wild type rats,but the serum sugar level is lower in PHLDA3 knockout male rats(6.23 mmol/L vs.11.07 mmol/L,P =0.018).Conclusion:PHLDA3 knockout rats develop PNETs and wild control rats have no PNETs formed,which indicate PHLDA3 is a tumor suppressor gene of PNETs.Human PNETs exist PHLDA3 LOH and gene promoter methylation,as well as PHLDA3 protein loss or decrease of expression,which indicate the correlation of human PNETs and PHLDA3 inactivation.Background and Aim:Pancreas neuroendocrine tumors(PNETs)is a rare group of heterogeneous tumors,the annual incidence of PNETsis 0.94-1.27/10 million and increases year by year.Previous studyreport 30%of insulinomasexist YY1T372R hotspot mutation,however,the clinical significance of this mutation and the relationship between the mutation and protein expression are unclear,besides,this hotspot mutation in other PNET subtypes has not been reported.Methods:We perform the first generation sequencing in 75 insulinomas,19 functional but non-insulinoma PNETs and 44 non-functional PNETs about the YY1 hot spot mutation.The expression of YY1 protein is detected by immunohistochemistry in 103 PNETs and 29 paired para-tumor tissues,additional validation by western bolt is performed in 4 PNETs,1 para-tumor tissue and 3 normal pancreatic tissues.Then analysisthe clinical significance of YY1 mutation and expression.Results:There are 19%(14/75)of insulinomas and 1.5%(1/67)of non-functional PNETs exist YY1T372R heterozygous mutation,which indicate YY1T372R mutation specially exist in insulinomas(P = 0.001).Patients with YY1T372Rmutation are usually older(57.5 vs.43,P=0.006).There's no relationship between YY1T372R mutation withthe malignancy of insulinoma as well as the stage and grade of insulinoma,suggesting that YY1T372R mutation may be the early eventof tumorigenesis.Our study also found that the insulin levels and insulin release index decreased in patients with YY1T372R mutation(N = 69,P = 0.015and N = 69,P = 0.073,respectively);insulinoma patients with blood type Ohave a higher proportion of YY1 hot spot mutation than that of patients with other blood types(38.9%vs.13.2%,P = 0.018).Howere,the expression of YY1 protein in tumor tissues has no correlation with YY1T372R mutation.YY1 protein is widely expressed in the cell nucleus of PNETs and para-tumor tissues,but the expression intensity in PNETs is significantly increased compared to para-tumor tissues.IncreasedYY1 expression is more frequently seen in men patients and begin tumors(88.1%vs.72.1%,P=0.052and 82.7%vs.63.6%,P=0.053,respectively),patients with higher expression of YY1 have a lower body weight index(27.9 vs.29.3,P = 0.010).Kaplan-Meiersurvival analysis show that patients with high expression of YY1 have a longer overall survival as well as disease free survival(overall survival:N=71,Log Rank P =0.006;disease free survival:N=68,Log Rank P =0.009).Cox modles show that patients with higher expression of YY1 have a better prognosis(overall survival:HR15.643,95%Cl1.635-149.685,P =0.017;disease free survival:HR3.162,95%CI0.830-12.047,P =0.092).Conclusion:YY1T372R mutation is specifically present in insulinomas,and it ismore common seen in patients with blood type O.YY1T372R mutation is likely to be an early event during tumorigenesis and may affect insulin secretion.YY1T372R mutation have no correlation with protein expression.Increased expression of YY1 in PNETs suggesta better prognosis.