Molecular And Immunohistochemical Characteristics And Their Clinical Associations Of Intraductal Papillary Mucinous Tumor Of The Pancreas (IPMN)

An intraductal papillary mucinous tumor(IPMN)is a common precancerous lesion of pancreatic cancer.In order to elucidate the molecular and clinical significance of IPMN,we have conducted the following studies:(1)KRAS,GNAS and RNF43 mutations were detected in 61 IPMN cases by Sanger sequencing and the next generation sequencing.We analyzed the correlations among mutations,clinicopathological features and prognostic data.(2)KRAS,GNAS,RNF43 and CTNNB1 mutations were detected in 9 IOPN cases.Meanwhile,the expression of anti-mitochondrial antibody,HepPar-1,β-catenin,CD117,Nestin,SMAD4 and P53 were detected by immunohistochemistry.The similarities and differences of clinicopathological and molecular characteristics of oncocytic and non-oncocytic(gastric,intestinal,pancreatobiliary)IPMN were compared.(3)The mutations of KRAS and GNAS mutations,the expression of MUC1,MUC2 and MUC6 were detected in 124 cases of non-oncocytic IPMN.The similarities and differences of clinicopathological features,MUC protein expression and molecular characteristics between the intestinal and non-intestinal subtype(gastric and pancreatic bile duct type),the gastric subtype and the pancreatobiliary subtype were analyzed.The results showed that:(1)The mutation rate of KRAS in IPMN samples was 57%,and there was no significant correlation between the mutation status of KRAS and the clinicopathological features or prognosis(P>0.05).The mutation rate of GNAS was 66%(40/61).The mutation status was significantly correlated with the histological type(P=0.000).The mutation rate in the intestinal type was the highest(93%),while the GNAS mutation was not detected in the oncocytic type.The mutation rate of RNF43 was 13%(8/61),and there was a correlation between RNF43 mutation and tumor dysplasia(P=0.010).3 cases of RNF43 mutation occurred in high-grade dysplasia IPMN,the other5 cases of RNF43 mutation were in invasive IPMN,no mutation was detected in low-grade dysplasia.However,the RNF43 mutation was not related to other clinicopathological features,nor did it affect the prognosis(P>0.05).Through correlation analysis,we found that there was a correlation between GNAS mutation and RNF43 mutation(P=0.042),and all 8 cases of RNF43 mutation had GNAS mutation at the same time.(2)The mutation rates of KRAS,RNF43 and CTNNB1 in IOPN cases were 22%(2/9),11%(1/9)and 22%(2/9)respectively.The immunohistochemical results showed that the positive rates of anti-mitochondrial antibody,HepPar-1,-catenin,CD 117,Nestin,SMAD4 and P53 were 100%(9/9),67%(6/9),11%(1/9),67%(6/9),11%(1/9),11%(1/9)and 22%(22%)respectively.The mutation rates of KRAS,GNAS and CTNNB1 in oncocytic and non-oncocytic IPMN were significantly different.The mutation rate of KRAS in oncocytic IPMN was 22%,significantly lower than that in non-oncocytic IPMN(61%).There was no GNAS mutation in oncocytic IPMN,but the mutation rate of GNAS in non-oncocytic IPMN was 71%.The mutation rate of CTNNB1 in oncocytic IPMN was 22%,while there was no CTNNB1 mutation in non-oncocytic IPMN.However,the clinicopathological features and prognosis between oncocytic and non-oncocytic IPMN were not different;(3)51 cases of intestinal IPMN were strongly positive for MUC2 expression.Of the 34 gastric IPMN cases,28(82%)were MUC6 positive,and 13(38%)were accompanied by MUC1 positive.In 39 cases of pancreatobiliary IPMN,37 cases(95%)were MUC1 positive,and 25 cases(64%)expressed MUC6 simultaneously.Intestinal type and non-intestinal type IPMN were significantly different in gender composition ratio,proportion of pancreatitis,imaging classification,dysplasia,differentiation of invasive cancer,frequency of invasive cancer,lymph node metastasis rate,and GNAS mutation rate.The gastric and pancreatobiliary IPMN were significantly different among the tumor size,the degree of dysplasia and the proportion of invasive cancer,but there are no difference between the other clinicopathological and molecular characteristics.In conclusion,our findings suggest that:(1)GNAS and RNF43 mutations may play a synergistic role in the development of IPMN.According to the fact that the mutation rate of RNF43 in high-grade dysplasia IPMN and invasive IPMN is significantly higher,we speculate that RNF43 can be used as a molecular marker to distinguish between low-grade and high-grade dysplasia IPMN,which may affect clinical diagnosis and treatment decision.(2)Compared with non-oncocytic IPMN,the oncocytic type has distinct histomorphological features:the cytoplasm is eosinophilic,habouring significant nucleolus and more complex papillary structures,but no mucus in the cyst;and the mutation frequency of KRAS,GNAS and CTNNB1 is different from that of other types of IPMN.Therefore,we believe that oncocytic IPMN should be considered as an independent type of pancreatic intraductal neoplasm.(3)According to histological morphology,molecular mutation characteristics and MUC protein expression,intestinal IPMN is easy to be identified.The characteristics of gastric type and pancreatobiliary type IPMN are the same as most of the clinicopathological features,the expression of MUC1 and MUC6 protein is sometimes overlaping,and the dysplasia grade and histological features is also overlapped.Therefore,the differential diagnosis of gastric and pancreatobiliary subtype is sometimes very difficult.So we suggest to use the gastric/pancreatobiliary subtype with the classification of low/high-grade dysplasia to replace the original classification of gastric type and pancreatobiliary subtype.Pancreatic cancer is a highly lethal malignant tumor.Studies have shown that the expression of Transcobalamin 1(TCN1)was increased in many tumor tissues and patients’blood,but its role in pancreatic cancer is still unclear.In this study,we preliminarily discussed the function of TCN1 in pancreatic cancer and its effect on the biological behavior of human pancreatic cancer cell line.By immunohistochemistry,we detected the expression of TCN1 in 146 cases of pancreatic cancer FFPE samples,and analyzed the correlation between TCN1 expression and prognosis.Meanwhile,we detected the expression of TCN1 in human pancreatic cancer cell lines by RT-qPCR and Western blot.After transfecting human pancreatic cancer cell lines with PCMV6-Entry/TCN 1 overexpressed plasmids and TCN1 siRNA,we identified the expression of TCN1 in the cells from mRNA and protein levels.At the same time,we used CCK-8,Transwell,wound healing assay and flat clone formation to detect cell growth,migration and formation of cloning.Through the BALB/c mouse model,we further examined the effect of TCN1 on the ability of pancreatic cancer cell to form tumor in vivo.In addition,in order to study the molecular mechanism of TCN1,we also carried out transcriptome sequencing of TCN1 Knock down BxPC-3 cells,and analyzed the differentially expressed genes by KEGG and GO.Our results showed that TCN1 was highly expressed in pancreatic cancer tissue compared with the normal pancreatic ductal epithelium adjacent to the carcinoma,and the expression level was negatively correlated with the prognosis of the patients.Compared with normal pancreatic duct cell lines,the expression of TCNl in human pancreatic cancer cell lines(MIA PaCa-2,AsPC-1 and BxPC-3)was also significantly increased.Upregulation/knockdown of TCN1 expression can increase/decrease the proliferation,migration and cloning ability of pancreatic cancer cell lines.Stable overexpression of TCN1 MIA PaCa-2 cells can significantly increase the ability of subcutaneous tumor formation in BALB/c nude mice.Our findings suggest that TCN1 is an oncogene in the development of pancreatic cancer and may become a new target for the treatment of pancreatic cancer.