A feasibility study characterising tumour microenvironment of longitudinally collected biopsies of pancreatic tumours upon endoscopic ultrasound guided radiofrequency ablation

Abstract BackgroundMost patients with pancreatic ductal adenocarcinoma (PDAC) are metastatic at presentation with dismal prognosis warranting improved systemic therapy options. Longitudinal sampling for assessment of treatment response poses a challenge for validating novel therapies. In this feasibility study, we evaluate the role of endoscopic ultrasound (EUS)-guided serial fine-needle aspiration biopsies (FNABs) to study the molecular changes associated with radiofrequency ablation (RFA).MethodsTwo stage III inoperable gemcitabine-treated PDAC patients were recruited in the treatment arm of ARDEO (ethically approved Phase-II prospective randomized clinical study). Post examination, targeted RFA was delivered thrice, and sequential FNABs of tumour were taken before and after treatment and analysed using a custom NanoString panel (144 genes) consisting of cancer and cancer-associated fibroblast (CAFs) subtypes and immune changes. CAF culture was established from one FNAB and characterised by immunofluorescence and immunoblotting.ResultsSerial EUS-RFA treatments were well tolerated with no complications. Both patients had stable disease immediately after EUS-RFA, however, with different survival outcomes. Two-course RFA led to upregulation of CD1E gene (involved in antigen presentation) in both, patient 1 and 2 (4.5 and 3.9-fold) compared to baseline. Patient 1 showed increased T cell genes (CD4 – 8.7-fold, CD8 – 35.7-fold), cytolytic function (6.4-fold) and inflammatory response (8-fold). Greater than 2-fold upregulation of immune checkpoint genes was observed post 2nd RFA in patient 1, patient 2 or both. Further, two-course RFA led to increased PDGFRa (4.5-fold) and CAF subtypes B and C genes in patient 1 and subtypes A, B and D genes in patient 2. Patient2-derived CAFs post 1st RFA showed expression of PDGFRα, POSTN and MYH11 proteins. Finally, RFA led to downregulation of classical PDA subtype in both patients. ConclusionsFor the first time, this feasibility study validates longitudinal sampling by EUS-FNABs as an appropriate research tool to study tumour microenvironmental changes associated with local PDAC immunomodulatory treatments like RFA.