A Research Protocol for Collection of Peripheral and Portal Vein Blood From Patients With Pancreatic Adenocarcinoma for Translational Research

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.