Exploring circulating biomarkers in patients with hepatocellular cancer

Abstract

Hepatocellular cancer (HCC) is the sixth commonest cancer worldwide and the second leading cause of cancer mortality. The majority of patients present with advanced disease, where cure is not an option and palliative therapies are limited. What is more, biomarkers to stratify available therapies effectively are lacking. Tissue is not routinely obtained as diagnosis of HCC is largely reliant on imaging, as this is sensitive and specific in the majority of patients with cirrhosis and liver biopsy carries small but recognised risks - including tumour seeding and bleeding. Serum AFP lacks HCC diagnostic sensitivity and specific, but remains the only clinically useful serum biomarker – employed despite its limitations in surveillance programmes as well as to monitor HCC progression and to stratify patients for therapy. This highlights the need for alternative biomarkers for HCC management. Sampling patient blood is a quick, non-invasive and inexpensive method and may be regarded as a ‘liquid biopsy’ if it could deliver clinically relevant molecular information about the tumour or its microenvironment. Several liquid biopsy methods have been explored: circulating tumour cell (CTC) detection and characterisation; circulating tumour DNA (ctDNA) KRAS mutation detection; circulating immune cell counts and gene expression signatures of peripheral blood mononucleocytes (PBMCs). CTCs can be detected in patients with cancer and have the potential to provide diagnostic, prognostic and treatment stratifying information. A method for CTC detection using the Imagestream – an imaging fluorescent activated cells sorter with multi-channel immunofluorescence - was developed and optimised. A CD45-immunomagnetic depletion resulted in ≥95% reduction in WBCs with the maintenance of a recovery rate of 51.3-65.37% of CTCs. The remaining cell suspension was labelled with a panel of fluorescent antibodies to enable cell characterisation prior to running through the Imagestream. Between 1 and 1642 CTCs were detected in 65% of HCC patients. The presence of CTCs indicated a worse median overall survival (OS) in HCC patients (24.5 months vs 12 months, Log-Rank p>0.0001). Expression of biomarkers on CTCs was heterogeneous with CK being the most commonly detected biomarker, followed by DNA- ii PK. In some patients, CTCs observed were negative for all of the biomarkers in the panel but detectable on the basis of size and morphology. Clusters of CTCs and leucocyte interactions with CTCs were also observed. Studies on plasma ctDNA detected KRAS mutation in 2/38 (5%) of patients with HCC. Retrospective patient blood data analysis demonstrated that circulating neutrophils were the key circulating immune cell driving HCC progression. In two patient cohorts – Newcastle (n=583) and Hong Kong (n=585), circulating neutrophil counts were an independent predictor of prognosis. Furthermore, in the combined cohort (n=1168), the neutrophil count, either alone or combined with platelets and lymphocytes, was associated with significant differences in patients survival. In a small pilot cohort, gene expression analysis of PBMCs identified NFAT in the immune response as being the top altered pathway in patients with NAFLD/NASH-HCC. DNA extracts prepared from PBMCs in patients with NAFLD-NASH-HCC were more similar to samples derived from patients with HCV-HCC compared to non-cancerous NAFLD/NASH controls. These pilot data were encouraging, supporting a potential role for future application of liquid biopsy tools in the clinical setting for patients with HCC.