Diagnosis Of Pancreatic Masses And RAd-p53 Gene Therapy For Pancreatic Cancer Using EUS

Pancreatic carcinoma is a kind of malignant tumor with poor prognosis. In recent years, the morbidity of pancreatic cancinoma has a trend to increase all over the world. About 28000 patients suffering pancreatic cancer were newly diagnosed in each year in USA. Pancreatic cancer is the fourth leading death cause of malignant tumor and is the second leading cause of tumor of gut in USA. The statistics data from China indicated that the incidence of pancreatic cancer has been the seventh leading cause of death of malignant tumors. This tumor became one of the common malignant tumors in Chinese population.The clinical symptoms of pancreatic cancer in early stage are not usually obvious. As their clinical symptoms presents, most of pancreatic cancers are already in advanced stage with poor prognosis. Therefore, the correct diagnosis of pancreatic masses is always in difficulty. Despite the great improvement in that imaging modalities such as computerized tomography and ultrasound guided fine needle aspiration have recently been developed and applied to diagnosis of pancreatic cancer, which had high sensitivity and specificity and accuracy. Endoscopic harvesting of cell from the duct of pancreas is also a convenient diagnosis method. However, the useful information of biopsy of cells or tissues for diagnosis of the pancreatic micro masses was not able to be obtained through these techniques.In recent decade, as endoscopic ultrasound was increasingly used in digestive systemic disease, the addition of fine needle aspiration used for the diagnosis of pancreatic cancer has archived great progress. Endoscopic ultrasound-guided fine needle aspiration characters with high accuracy, less injury to tissues, and shorter distance to biopsy the masses. This technique for diagnosis and therapy has developed rapidly all around the world. As there are more helpful experimental measurements, the using of EUS-FNA will be helpful to early diagnosis of pancreatic masses with great advantages.The p53 gene is a tumor suppressor gene. The p53 mutations, which cause the p53 protein accumulation and less or no wild p53 protein expression in the tumors, have been found in 60%～80% of pancreatic carcinoma. The mutation p53 protein in cells causes the disorder of differentiation, uncontrolled of cell growth, finally resulting in cancer formation. By delivery of wild p53 gene to the pancreatic cancer cell line, the apoptosis of cancer cell enhanced and the growth of cancer masses slowed down. This kind of biological therapy can inhibit the cancer growth in animal model and small clinical trials.EUS-FNA was firstly used in our hospital in 1999, and the early diagnosis of pancreatic masses through FNA increased and the number of patients whom the EUS-FNA were performed on accumulated to 180. This study was performed to investigate the application of EUS-FNA in diagnosis of the pancreatic masses, and the cytomorphological assessment and p53 mutation protein expression in the pancreatic cells or tissues. According to the evidence-based laboratory medicine, the information of EUS-FNA and clinical diagnosis after operation were analyzed.With the development of endoscopic ultrasound, the EUS-FNI has been to a powerful weapon for the endoscopist to cure the advanced pancreatic cancer. Local delivery of rAd-p53 wild gene is a new therapy for the later stage of pancreatic cancer. We designed and performed a small clinical trial to investigate the safety and the efficiency of the rAd-p53 gene therapy delivered by EUS-FNI.Aims of this study1. Analyzed the application of EUS-FNA combination with cytomorphological assessment and p53 mutation protein expression for the diagnosis of pancreatic masses.2. Evaluation the safety and efficiency of the rAd-p53 gene therapy delivered by EUS-FNI.Patients and settingsFrom November 1999 to January 2007, 163 patients with pancreatic masses were performed EUS-FNA in the centre of endoscope of Changhai hospital. The patients containing 64 female, and 99 male, was 18 to 87 years old, the mean age 56.5 years old. All patients were treated routinely by computerized tomography or magnetic resonance imaging evaluation of the pancreatic masses before EUS-FNA biopsy, and those patients with masses but not in pancreas were excluded in this study. Eight patients, who suffered from later stage pancreatic cancer without operation chance according to the CT or MRI and clinical situations, were involved in the gene therapy trial. 2 ml of rAd-p53 were injected into the cancer guided by EUS once a week, and total injections of 4-5 times. 15 patients were given routine chemotherapy as control group.EUS-FNA procedureAll patients in this study were performed ordinary ultrasound, CT, MRI detection of pancreas before the EUS-FNA biopsy. Under anesthesia EUS-FNA was performed by a well-trained endoscopist. FNA was performed by a 22-gauge, 10-cm long needle. The aspirated samples were expelled onto slides, and two smears were made for each aspiration pass.Cytomorphological assessmentCells specimen were fixed 95% alcohol. Multiple passes, if necessary, were done in order to obtain diagnostic material. If cell block material was obtained by a separate pass and was fixed in 10% buffered neutral formalin. Thin sections (4 lm) from paraffin-embedded cell block were cut on the following day and were stained with haematoxylin and eosin. The expression of p53 protein was measured by routine immunohistochemistry method.The delivery into pancreatic cancer of rAd-p53 by EUS-FNIAfter the EUS-FNA was finished and the cytomorpholical assessment verified that there are cancer cells in specimen. The rAd-p53 was injected into the cancer masses of 8 patients. The immunological responses were measured by detection of cytokines and CD3/CD4/CD8 of white blood cells for three times during 3 months after the gene treatment.ResultsEUS-FNA were performed successfully in 163 patients with pancreatic diseases. The positive rate of cytologic examination was 48%, and the cytologic examination was negative in patients with benign pancreatic diseases. Sensitivity, specificity, diagnostic accuracy, positive predictive values and negative predictive values of HE staining for pancreatic cancer is 48%, 100%, 70.4%, 100% and 59%.During 1999 to 2002, there is only one pancreatic cancer biopsy by EUS-guided FNA; detection of the sample did find the cancer cells.In 2004, the positive of detection of pancreatic cancer was 17.6 percent (3/17). The positive rate increased to 30 percent(9/30) in 2005, and 47.4 percent in 2006. During last three years(2004-2006), the consistent rate of the FNA and cytomorphological assessment and clinical diagnosis were 15/29(51.7%),29/50(58%),54/85(63.5%) respectively.Mutation protein of p53 was detected in 29 pancreatic FNA cytologic samples. The sensitivity, specificity, diagnostic accuracy, positive predictive values and negative predictive values of detection p53 protein for pancreatic cancer is 59%,100%,76%,100% and 63% respectively.The delivery of rAd-p53 by EUS-FNI was safe, convenient, less complications. The data showed that the rAd-p53 improved the immune response, such as the higher production of interleukins (IL-2, IL-6, and IL-8) compared with the cytokine levels before gene therapy. The effect of gene therapy in this study did not show statistically difference compared with the chemotherapy groups. However, in special cases, the effect of rAd-p53 had greatly improved the condition of patients.ConclusionsEUS-guided FNA biopsy of pancreatic masses, which proved to be safe, less complication in patients suffering from pancreatic disease, were performed more and efficiently during recent years in our hospital. With enough cell or tissue sample, the cytomorphological evaluation have high specificity, but a little lower sensitivity. The mutation protein of p53 assessment was helpful for correct diagnosis, and the positive predictive value and diagnostic accuracy increased significantly after combining with the cytomorphology.The gene therapy by EUS-FNI characterized with safety, less injury, little complication, and enhanced the immune function. The condition of patients with later stage pancreatic cancer could be improved, the reduction of cancer masses was also observed in special cases.