PDAC Peripheral and Portal Vein Sampling

Pancreatic Ductal Adenocarcinoma (PDAC) presents an urgent medical need. Approximately 9000 new cases of pancreatic cancer are diagnosed every year in the United Kingdom (UK) and worldwide it is projected to become the second most common cause of cancer related death by 2030. Currently the diagnostic pathway starts with pancreatic protocol Computed Tomgraphy (CT) scan which demonstrates sensitivity of 90% and specificity of 99% in diagnosis of pancreatic cancer. Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET) can assist with ambiguous masses. Despite the high sensitivity and specificity of imaging modalities, definitive diagnosis is routinely obtained only by endoscopic ultrasound (EUS)-guided fine-needle aspiration (FNA). During this procedure four "passes" (biopsies) on average are required to obtain sufficient tissue for diagnosis.

Patients usually present with locally advanced or metastatic disease and are offered standard of care chemotherapy, which has only a limited impact on overall survival. Consequently the 5-year overall survival (OS) is only 2% with no significant improvement over the last 40 years. Approximately only 20% of the patients are eligible for surgical resection which is followed by adjuvant chemotherapy. However, 66% of these patients will experience local or distant recurrence, leading to a median overall survival in the range of 28 months. Improved stratification of patients for existing treatments may thus avoid unnecessary overtreatment and associated side effects to improve quality of life; conversely, it may also enable selection for a more appropriate treatment.

A significant reason for the lack of progress in tackling metastatic disease is the poor understanding of the biology and pathophysiology of metastasis. Studying the biology of PDAC metastasis is hampered by the fact that it is difficult to obtain tissue from the primary and metastatic sites. Moreover, serial biopsies following progression during treatment is largely not feasible, further hindering insight into the mechanisms of development of treatment resistance. The only easily-accessible blood-borne biomarker that is currently used in clinic is the serum marker carbohydrate antigen (CA) 19-9. However, its clinical use is limited firstly by the fact that 5-10% of the population are Lewis blood group negative and do not express CA19-9. Furthermore it is elevated in non-malignant pancreatic and extra-pancreatic diseases. Finally, CA19-9 cannot provide information about the genetic landscape that drives PDAC metastasis.

Metastatic spread is primarily haematogenous whereby tumour cells enter the circulation to metastasize mainly to the liver and lung. Such tumour cells have been shown to travel either as single circulating tumour cells (CTCs) or as groups of cells termed Circulating Tumour Microemboli (CTM). Heterogeneous CTM composed of cells expressing either epithellial or mesenchymal markers were isolated from patients with pancreatic cancer using a filtration-based method, the Isolation by Size of Epithelial Tumour cells platform (ISET), in a study performed at the Christie NHS Foundation Trust. As such, isolation and characterisation of CTCs and CTM will provide new insight into the underlying biological dependencies of metastasizing tumour cells and may thus provide opportunities to develop better and more accurate diagnostic and prognostic assays as well as opportunities to treat metastatic disease.

Detection and isolation of CTCs and CTM is limited by the fact that they represent very rare events compared to other cellular elements in the circulation. To this end, methods exploiting characteristics that differentiate CTCs from blood cells have been developed to overcome this obstacle. These CTC workflows usually start by enriching CTCs from a blood sample utilizing either surface markers present exclusively on CTCs, or their unique physical properties such as size, inertia, dielectric charge and density. Following enrichment, CTCs are isolated and can be simply enumerated or they are used for more advanced downstream analysis including genomic, transcriptomic and proteomic analysis as well as in vivo applications in cell culture and mouse models, with the aim of gaining insight into their function as metastatic seeds and biomarker development.

In the clinical setting, CTCs can be used to aid decision making. Indeed, enumeration of CTCs detected by the CellSearch platform has been FDA approved for prognosis and monitoring of metastatic breast, colorectal and prostate cancer. However, isolation and enumeration of CTCs in pancreatic cancer has given inconsistent results between studies as to whether CTCs can be used as prognostic biomarkers. These studies analysed CTCs isolated from peripheral blood. However, as the main venous drainage of the pancreas is through the portal vein to the liver, it may be hypothesized that CTCs could be filtered and removed as they pass through the liver capillaries. Hence, in order to define the biological and prognostic role of CTCs in pancreatic adenocarcinoma, it is critical to initiate studies with blood isolated from the portal vein to bypass confounding effects. Indeed, detection of CTCs in portal vein (PV) blood acquired at operation was recently reported by Bissolati et al.. By obtaining peripheral vein samples at the same time, this study confirmed that CTC number was greater in portal compared to peripheral samples and also demonstrated significant correlation between CTC positivity in portal vein samples and frequency on liver metastasis. However, it failed to show significant correlation with OS or Disease Free Survival (DFS). PV sampling has been performed safely; in December 2015 Catenacci et al. reported collection of PV blood from 18 patients with pancreatobilliary cancers by EUS-FNA with no complications during the procedure. PV puncture has also been previously reported for islet cell transplantation in patients with Type 1 Diabetes Mellitus. This procedure has low morbidity and also, potential complications, such as portal vein thrombosis, perihepatic or intraparenchymal hepatic haemorrhage and haemothorax are reported at a low rate.

During EUS-FNA a curvilinear echoendoscope is advanced to the distal stomach or duodenal bulb to provide a window of access to a branch of the PV. After verifying venous flow by Doppler signal, a 19-gauge EUS-FNA needle is advanced transhepatically into the portal vein branch. With the needle in the portal vein, the stylet is removed and negative-pressure suction is applied to aspirate blood. A transhepatic approach for portal vein branch access is an absolute requirement of this technique in order to minimize the risk of bleeding. The puncture site is monitored under EUS for complications. As tumour tissue will be obtained at the same time, a potential risk is the introduction of cancer cells into the circulation as the needle is withdrawn from the tumour. This risk however is circumvented as the route of the needle into the portal vein would avoid the primary mass.

The primary reason for drug failure in PDAC is the failure to identify predictive biomarkers for treatment response which would allow enrichment of patient sub-groups most likely to benefit from an individual therapy. This is in contrast to other major cancer types, in which advances in development of new cancer therapeutics and resulting improvement in survival outcomes are mainly due to the use of predictive biomarkers to select patients who will derive differential benefit from a particular therapeutic agent. Trastuzumab in breast cancers over-expressing HER-2, cetuximab for KRAS wild-type colorectal cancer, and gefitinib and erlotinib for Epidermal Growth Factor Receptor (EGFR) mutant non-small cell lung cancer best exemplify this therapeutic paradigm. By providing greater numbers of CTCs and CTM, analysis of PV sample will enable studies of their biology as well as development of cell lines and CDX models to allow identification of novel biomarkers and targets for new treatments.

Considering current gaps in knowledge of tumour biology of PDAC and the lack of progress in therapeutics, there is an urgent clinical need to develop a research strategy that will allow the discovery and validation of new biomarkers in this group of patients, and inform on biomarker-directed clinical trials to ultimately improve survival. To initiate such a research strategy, there is a need for systematic, prospective collection and analysis of blood samples acquired from peripheral and portal vein circulation to identify blood-borne surrogate tumour markers that will enable physicians to obtain relevant information for making personalised therapeutic decisions, in a minimally-invasive way.