Pre- and Postoperative Incidence and Prognostic Implication of Positive Peritoneal Lavage and Circulating Tumor DNA in Patients With Pancreatic Cancer

1. Introduction Approximately 1.000 new cases of pancreatic cancer (PC) are diagnosed each year in Denmark. Ductal adenocarcinoma is by far the most frequent histological subtype of cancer in the pancreas and accounts for more than 90% of all cases [1]. Thus, in this protocol the term PC refers to pancreatic ductal adenocarcinoma. PC is a devastating disease with an overall 5-year survival of 3-7% [2]. Less than one in four patients are candidates for intended curative resection, but even after microscopically radical resection (R0 resection) the majority of patients will have recurrent disease within 2 years [3]. The discrepancy between pathological assessment and clinical outcome indicates that most patients will harbour non-detected (i.e. occult) cancer cells at the time of resection.

It is of utmost importance to improve our ability for prognostication and prediction of PC, aiming at a tailored and personalized approach for treatment decisions regarding this aggressive type of cancer. This project aims to take an important step in this direction, by a 2-tiered approach: 1) Examination of the peripheral blood (detection of circulating tumor DNA), and 2) the peritoneal lavage fluid (detection of tumor DNA in the peritoneal space). 2. Background PC is a deadly disease and surgical resection of the tumor is the only hope of cure. Approximately 20-25% of the PC patients are candidates for intended curative resection at the time of diagnosis, but despite microscopically radical resection the majority of patients will have recurrent disease within 2 years [3]. This indicates that most patients will harbour non-detected (i.e. occult) cancer cells at the time of resection. Studies suggest that free tumor cells in the peritoneum and in the blood are part of this occult disease burden, and that patients with such findings should not be operated but treated as having metastatic disease. However, the exact incidence of these tumor cells in an unselected cohort of patients undergoing pancreatic resection is unknown, and the potential impact on postoperative survival is also uncertain. In recent years, molecular biomarkers are increasingly being regarded as both predictive and prognostic tools for cancer patients. This study will use the most optimal available methods to investigate the incidence of tumor cells in the peritoneum and blood in PC patients, and to relate these findings to the final outcome of the resected patients. This project has become highly relevant since new treatment methods (i.e. Pressurized IntraPeritoneal Aerosol Chemotherapy (PIPAC)) may be used to eradicate free tumor cells.

2.1 Positive peritoneal lavage indicates a poor prognosis in PC patients Occult malignant cells may be detected in fluid obtained by peritoneal lavage (PLF) during staging laparoscopy, open surgery or sampled from postoperative drain fluid in patients with PC. The malignant cells are diagnosed by conventional cytology combined with immunocytology, but real-time polymerase chain reaction (RT-PCR) of CEA can increase the detection rate of occult malignant cells in peritoneal lavage fluid [4]. The majority of data report on the use of CEA [4], but also evaluation of EpCAM have been subject of interest. A recent study reported the combination of CEA/EpCAM mRNA as an optimized set-up for detection of free intraperitoneal tumor cells of various origins with a high sensitivity and an excellent specificity [5]. Another marker, CA 19-9, has gained interest, however mostly in studies including gastric cancer patients and presenting the results in protein levels rather than mRNA. These studies suggest that elevated levels of CA 19-9 in PL is associated with more advance stages of disease, is a more reliable predictive factor for staging than serum CA 19-9 levels and is a more sensitive marker than CEA [6, 7].

While mRNA- and protein-based analyses have shown promising results, only casuistic data exists on the detection of KRAS mutations in PLF [8-10]. However, in a Norwegian study including patients resected for rectal cancer, KRAS mutations were found in PLF collected immediately after surgery in 19 out of 237 patients. These KRAS positive patients had significantly poorer survival [11], and similar results might be found in patients resected for PC as well.

A recent systematic review and meta-analysis demonstrated that PC patients with positive peritoneal cytology (Cy+) had a significant poorer survival than patients with negative peritoneal cytology (Cy-)(HR 3.18), and the authors concluded that Cy+ patients should not undergo surgery [12]. This conclusion was supported by a significant lower overall survival and a higher peritoneal recurrence rate after resection of Cy+ patients when compared to Cy- patients [13]. Agreement that Cy+ in resectable PDAC is a negative predictor of prognosis came from another recent meta-analysis and systematic review. However, this study also indicated that the median OS was worse in patients without than in those with resection among patients with Cy+, thereby emphasizing need of further careful assessment of indications for radical resection in Cy+ patients [14]. Additionally, it should be noted that manipulation of tumor during surgery significantly increases the rate of patients with positive PLF (based on EpCAM mRNA measurements) from 10% to 54%, although this study was unable to detect a worse prognosis in EpCAM positive patients [3].