Immunocytochemical detection of deoxycytidine kinase in haematological malignancies and solid tumours

Abstract

Background: Deoxycytidine kinase (dCK) is responsible for the activation of several clinically important deoxynucleoside analogues used for the treatment of haematological and solid malignancies.

Aim: To measure dCK expression in tumour cells from different origins.

Method: A rabbit antihuman dCK antibody was used for the immunocytochemical detection of dCK expression in three leukaemic cell lines (HL60, U937, and CCRF-CEM) and 97 patient samples (paediatric acute myeloid leukaemia (AML) and lymphoid leukaemia (ALL), retinoblastoma, paediatric brain tumours, and adult non-small cell lung cancer (NSCLC)).

Results: CCRF-CEM, U937, and HL60 cells stained positively for dCK and the degree of expression correlated with dCK activity. dCK expression varied between tumour types and between individual patients within one tumour type. dCK was located predominantly in the cytoplasm. The staining intensity was scored as negative (0), low (1+), intermediate (2+), or high (3+). Expression of dCK was high in AML blasts. In contrast, brain tumour samples expressed low amounts of dCK. dCK staining ranged from low (1+) to high (3+) in ALL blasts, retinoblastoma, and NSCLC tissue samples. Staining was consistent (interobserver variability, 88%; kappa = 0.83) and specific. Western blotting detected the dCK protein appropriately at 30 kDa, without additional bands.

Conclusions: Immunocytochemistry is an effective and reliable method for determining the expression of dCK in patient samples and requires little tumour material. This method enables large scale screening of dCK expression in tumour samples.

Figure 1

Immunocytochemical staining of deoxycytidine kinase in the leukaemic cell lines. (A) U937 phosphate buffered saline negative control, (B) U937, (C) CCRF-CEM, and (D) HL60.

Figure 2

Immunocytochemical staining of deoxycytidine kinase (dCK) in patient samples from different tumour types. (A) An acute myeloid leukaemia sample and (B) a retinoblastoma sample with strong expression of dCK (3+). (C) An acute lymphoid leukaemia sample with weak staining (1+); (D) A brain tumour sample negative for dCK (0). Non-small cell lung cancer tissue stained for dCK: (E) phosphate buffered saline negative control and (F) a sample with high dCK expression (3+)

Figure 3

Expression of the deoxycytidine kinase (dCK) protein in different tumour types. The acute myeloid leukaemia (AML) and retinoblastoma samples showed high expression of dCK (predominantly 3+). In contrast, the brain tumour samples showed low expression of dCK (0–2+) and in acute lymphoid leukaemia (ALL) samples dCK expression was intermediate (2+). NSCLC, non-small cell lung cancer.

Figure 4

Western blot using the anti-deoxycytidine kinase (dCK) antibody (1/5000 dilution) on the leukaemic cell lines HL60 and U937, purified dCK protein (dCK-his), and two AML patient samples. Samples (20 µg protein) were loaded in duplicate and β actin was used for normalisation. The band for dCK was located at 30 kDa, as expected. The purified dCK protein band was located slightly higher because of the histidine tag. No additional bands were seen. The intensity of the band for the CCRF-CEM cells was intermediate, as described previously.