Abstract 3942: Increased frequency of proliferating peripheral white blood cells in the blood of hepatocellular carcinoma patients compared with noncancer controls
Authors: Hutton Claire , Uitrakul Suriyon , Ogle Laura F. , Reeves Helen L. , Greystoke Alastair , Veal Gareth J. , Jamieson David 
Published: July 2016

Abstract
Introduction
Proliferation of peripheral blood cells, including activated CD8+ T-cells, has been reported in numerous pathologies including haematopoietic cancers and solid tumours. During development of a method to detect and characterise circulating tumour cells by imaging flow cytometry it became apparent that peripheral proliferating lymphocytes, while still rare, are substantially more common than CTCs in all cancer patients and are present in everybody. We investigated the frequency of these cells in patients with hepatocellular carcinoma (HCC) and healthy volunteers, and went on to assess their utility as a surrogate tissue for proof of mechanism studies of anti-proliferative agents.
Methods
Whole blood samples (4ml) from 7 patients with hepatocellular carcinoma and 14 healthy volunteers were collected into EDTA tubes. After RBC lysis and fixation WBCs were stained with fluorochrome conjugated antibodies to Ki67 and CD45 and the nuclei were stained with DAPI to assess relative DNA content. For each sample images of 100,000 cells were collected by imaging flow cytometry. Putative G2 cells were identified as single cells with DNA content equivalent to twice the amount of the majority of cells and the same as images of doublets. The frequency of cells with nuclear localised Ki67 reactivity was assessed independently of DNA content. Whole blood from healthy volunteers was also treated by ex vivo incubation with PMA ± the CDK inhibitor Dinaciclib.
Results
The mean frequency of Ki67 positive G1 peripheral WBCs in blood from patients with HCC was 0.44%, as compared to a frequency of 0.24% in the blood from healthy volunteers (p = 0.007). Incubation of healthy volunteer blood with PMA for 20 minutes at 37°C resulted in a small but reproducible increase in the number of Ki67 positive cells and this increase was abrogated by simultaneous incubation with Dinaciclib (141 ± 41% versus 92 ± 39%, expressed as a percentage of no PMA control, p = 0.0007). Additional staining of the ex vivo samples with an antibody to pMCM2, as a potential proof of mechanism marker for CDC7 inhibitor efficacy, was carried out. Results showed that pMCM2 expression was associated with ki67 and the frequency of pMCM2 positive cells also increased with exposure to PMA
Conclusion
The frequency of proliferating WBCs is increased in patients with HCC. These cells may have utility as a predictive/prognostic biomarker, or possibly in the pharmacodynamic monitoring of anti-proliferative pharmacological agents.
Citation Format: Claire Hutton, Suriyon Uitrakul, Laura F. Ogle, Helen L. Reeves, Alastair Greystoke, Gareth J. Veal, David Jamieson. Increased frequency of proliferating peripheral white blood cells in the blood of hepatocellular carcinoma patients compared with noncancer controls. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3942.