Diagnosis And Prognosis Of Pancreatic Cancer By A Novel Serum MicroRNA Expression Profile-based Biomarker

Pancreatic cancer (PaC) is the tourth leading cause of cancer-related deaths in western countries and has the poorest survival rate (<5%) among the common malignancies.Although surgical resection shows promise as an effective treatment for PaC, lack of effective tool for diagnosis at the early stage results in5-year survival rate from more than50%in patients with stage Ⅰ rapidly dropping to less than5%in patients with more advanced stage. Both imaging techniques and serological markers, such as carbohydrate antigen19-9(CA19-9) and carcinoembryonic antigen (CEA), are less sensitive and specific. Molecular markers in pancreatic juice, pancreatic duct brushings, duodenal aspirates, or duodenal tissue have also been used for PaC classification. These tests, however, are less practical for screening or routine clinical checkups since sample collection is invasive and difficult. In addition, it is still difficult to distinguish chronic pancreatitis (CP), a high-risk population of PaC, from PaC. Therefore, to improve the prognosis of PaC, it is urgent to develop specific and non-invasive biomarkers for PaC diagnosis, especially for early stage tumors. Detection of pancreatic cancer (PaC) especially at early stages remains a great challenge due to lack of specific biomarker. The aim of this study was to identify PaC-specific serum miRNA (miRNAs) expression profile and test its specificity and sensitivity as a biomarker for PaC diagnosis and prognosis.The present study enrolled157patients who had been clinically classified as PaC at the time of participation, between2005and2009. In the initial biomarker screening stage, pooled serum samples from25PaC cases (Tianjin Medical University Cancer Institute and Hospital) and25matched controls(Jinling Hospital) were subjected to Solexa sequencing to select miRNAs whose expression was altered in PaC cases compared to that in controls. Subsequently, the number of serum miRNAs included as the PaC signature was refined by a two-phase experimental procedure using TaqMan probe-based qRT-PCR assay. The training phase used serum samples from the25PaC cases and25controls that had been employed for Solexa sequencing, whereas the validation phase used serum samples from an additional95PaC cases (Changhai Hospital, Ruijin Hospital, and Beijing Cancer Hospital&Institute) and81controls (Jinling Hospital). The panel of serum miRNAs selected as the PaC biomarker was further examined in the second control group comprising82CP cases (Changhai Hospital and Ruijin Hospital). Furthermore, additional37.PaC cases (First Affiliated Hospital of Nanjing Medical University) and32controls (Jinling Hospital) were analyzed in a blind fashion, in which the investigators performing the molecular analysis on the blood samples were blinded to the patients’ clinical diagnosis. Finally, additional55suspicious PaC cases (Jinling Hospital) based on preliminary diagnosis were analyzed for the seven miRNA levels using the same methods. All the protocols, including the diagnosis procedure and serum collection manner, are identical in these hospitals. Written informed consent was obtained from all patients and volunteers prior to the study, and the study was approved by the ethics committee of each participating institution.All the157patients enrolled in the present study were clinically and pathologically diagnosed with pancreatic ductal adenocarcinoma. There was no significant difference in the distribution of smoking (p=0.8704), alcohol consumption (p=0.5200), age (p=0.3406), and gender (p=0.8906) between the cancer patients and the normal control subjects. In the initial screening phase by Solexa sequencing, serum samples were pooled from25PaC patients or25healthy donors. A genome-wide expression profiling of serum miRNAs obtained by Solexa sequencing showed that PaC serum had44miRNAs upregulated and19miRNAs downregulated compared to the controls. After the selection and validation process,7miRNAs were found to have significantly different expression levels in the PaC compared to the control. This7-serum miRNA-based biomarker distinguished various stages of. PaC from cancer-free controls with high sensitivity and specificity, and also accurately discriminated PaC patients from chronic pancreatitis (CP) patients. Among the7miRNAs, level of miR-21in serum was significantly associated with overall survival of PaC. PaC patients with high levels of serum miR-21exhibited a lower survival rate compared to those with low levels of serum miR-21(p<0.05; log-rank test). Subsequently, a univariate and multivariate Cox proportional hazard regression model was performed to determine the influence of serum miRNA level as well as clinicopathological characteristics (gender, age, TNM stage, etc) on patient survival. The results showed that the miR-21expression level could serve as an independent indicator for predicting the survival rate of PaC patients.7-miRNA signature could be used to separate PaC from CP cases. Interestingly, no significant differences were observed in the levels of these seven miRNAs between CP patients and normal controls. The ROC curve also indicated that this7-miRNA-based biomarker could accurately discern CP cases from PaC cases (AUC=0.993) but not from normal controls (AUC=0.644). Furthermore, the forecast accuracy rate of the7-serum miRNA as a biomarker in discriminative diagnosis of55clinical suspicious PaC was83.64%.The present study has identified a seven-serum miRNA-based biomarker for accurately discerning PaC cases from cancer-free controls and CP cases. The7-serum miRNA-based biomarker may serve as a novel non-invasive approach for PaC diagnosis and prognosis. Discovery of the serum miRNAs as potential biomarkers overcomes the obstacle in invasive process of tissue sample collection.