The Influence of Secretin Stimulated Pancreatic Secretion on ctDNA Expression in Patients With Pancreatic Ductal Adenocarcinoma: The Role of Biomarkers in Determining the Prognosis of Pancreatic Ductal Adenocarcinoma

Background Pancreatic ductal adenocarcinoma (PDAC) is an aggressive oncological disease with a poor prognosis and low survival rate. Due to its biological nature, it is generally challenging to accomplish early diagnostics and therefore to achieve successful therapeutic results. Recently, many trials and studies worldwide aim at better understanding of tumor biology and providing a novel tool for diagnostics, disease monitoring and developing a targeted therapy. One of the subjects of interest happen to be the so-called liquid biopsies. Isolation of biomarkers such as miRNA, tumor suppressor genes or amino acids from these biopsies, their analysis, quantification and incorporation into clinical oncology represents a complex issue where further research is highly required. Subjects, materials, methods A crucial prerequisite for biomarker analysis is a sufficient amount of genetic information. In our study, the effect of intravenous secretin administration on ctDNA release in patients with PDAC will be analysed. More than 90% KRAS mutation rate in patients with PDAC has been scientifically proven, therefore patients with positive KRAS mutation in their peripheral blood plasma and scheduled for surgical therapy will be included in the study. Peripheral blood plasma of 36 patients with PDAC diagnosis will then be collected in specific intervals during the first two hours of the operation. Plasmatic levels of ctDNA in the samples will be quantified. A standardized panel of biomarkers will be designed and tested in patients with PDAC prior to surgery and in specific intervals after surgery. The results will be analyzed in correlation to minimal residual disease, survival rate and other indicators to determine the prognosis of the disease. Statistic evaluation will be done in cooperation with specialists experienced in bioinformatics and biomedical statistics. Introduction: Pancreatic ductal adenocarcinoma (PDAC) ranks among the most serious and aggressive malignant diseases with an unfavorable prognosis and high mortality \[1,2\]. Five year survival for patients with PDAC is 10 %, and ten-year survival is 6 % \[3\]. According to the Institute of Health Information and Statistics of the Czech Republic, incidence of PDAC in Czechia is 21.9/100000 inhabitants, prevalence is 23,2/100000 inhabitants. In addition to insufficient response to systemic chemotherapy, this statistic also contributes significantly to the difficulty of early detection of the disease due to its often asymptomatic period, while treatment methods may be significantly limited at the time of diagnosis. The indication of surgical treatment as a potentially curative method clearly depends on the established extent of the disease \[4\]. Currently, the worldwide monitored marker is the level of Ca19-9 in the serum, also in relation to the determination of preoperative and postoperative prognosis \[5\]. However, due to the lack of sufficient sensitivity and specificity for PDAC, Ca19-9 cannot be classified as a specific marker with sufficient predictive value \[6\]. More than three hundred markers have been proposed in various biological materials (blood, urine, feces, saliva, bile, pancreatic juice, etc.) for screening, early diagnosis, response to treatment and prognosis, but none of the biomarkers currently has a wider application in daily clinical practice \[6,7\]. According to recent studies, several biomarkers \[6\] have been identified with a high potential for further investigation in correlation with the assessment of prognosis and future use in screening in the general population \[6,7,8\] Early diagnosis is a key part of treatment options and the prognosis improvement in PDAC, therefore a set of biomarkers with significant potential for practical clinical use was chosen for this project to be analyzed. One of those is KRAS protooncogene from the RAS family, which is frequently mutated in PDAC \[9, 10, 11, 12\]. The mutated KRAS gene is about to be detected from the ctDNA isolated from the patient's plasma before and after intravenous stimulation with secretin, a hormone responsible also for pancreatic secretion \[13\]. We suppose that the secretin stimulation is going to potentiate the release of ctDNA. Analysis of mutant KRAS, mRNA, miRNA, siRNA, exoRNA, S100 proteins and amino acids could therefore become an efficient hallmark in PDAC monitoring during the diagnostic - therapeutic process. Objectives: The aim of this project is to prove the potentiating influence of intravenous secretin stimulation on ctDNA release in patients with PDAC and thereby mediate higher plasmatic levels of ctDNA potentially usable for further analysis. Besides that, the role of KRAS mutation in ctDNA, mRNA, miRNA, siRNA, exoRNA, S100 proteins and amino acids is about to be analyzed throughout the therapeutic process to monitor the correlation of the marker levels to the standardized indicators of life expectancy, quality of life etc. at a time. Zero hypothesis: The plasmatic level of ctDNA after intravenous secretin administration will not be significantly higher than before the administration. The plasmatic levels of mRNA, miRNA, exogenic RNA and S100 proteins will not be significantly lower after surgery than prior to surgery and they will not show any correlation with postoperative course of the disease. Methodology and statistic evaluation: In the first phase, both male and female patients in any age-range diagnosed with PDAC and indicated for surgical therapy will be selected. Considering multiple blood sample collections in a short period of time, physiological effects of secretin and logistic optimization of the process, we have decided to involve the patients indicated for surgery. Patients with tumor duplicity will be excluded. Only the patients with signed consent of participation in the study will be included. These patients' blood plasma will be collected from a peripheral vein during an outpatient visit prior to surgery and tested for the positivity of mutated KRAS gene with heteroduplex analysis using denaturing capillary electrophoresis. 36 patients with KRAS positive plasma sample will then be routinely prepared for surgery. In the first two hours of the surgical procedure, 16 micrograms of intravenous secretin will be applied. During this period of time, the intravenous secretin administration to the patient is safe and will not interfere with the surgical phase of pancreatic resection. In the interval of 3 minutes and 3x 5 minutes from the secretin application, blood samples will be collected from the peripheral vein as well by the anesthesia personnel. All blood samples will be collected to the prepared sampling kits with no need for further technical processing, transported to the laboratory and subjected for the analysis and quantification of ctDNA ant other candidate markers. In the second phase of the project, the most promising marker panels will be established based on the current knowledge and preliminary results. Blood plasma of the patients diagnosed with PDAC, indicated for surgical therapy will then be tested for the marker levels preoperatively and postoperatively before dismissal, 3, 6 and 12 months after surgery during outpatient examination. The results will then be analyzed and evaluated statistically in correlation with minimal residual disease, survival rate, quality of life and other standardized indicators to monitor the prognosis of the disease. Statistic evaluation will be realized in cooperation with specialists with a broad experience in bioinformatics and biomedical statistics. Outcomes and significance: Our project will provide a systematic literature review regarding liquid biopsy and biomarker analysis and its incorporation to clinical oncology in patients diagnosed with PDAC. The effect of intravenous administration of secretin on ctDNA release to patients with PDAC as an innovative method will be analyzed, potentially procuring a significantly larger amount of genetic information for biomarker analysis in these patients. Moreover, the results and outcome will help to design a panel of biomarkers and to evaluate their clinical implication in disease monitoring and prognosis evaluation.