A Rapid Assay of Cytosine Arabinoside Uptake and Metabolism by Acute Myeloid Leukaemic Cells Using a Bioluminescent Bacterial Biosensor.

Abstract

We have already reported the construction and evaluation of a self-bioluminescent reporter strain of E. coli with high sensititivity to the nucleoside analogue cytosine arabinoside (Ara-C) the mainstay of treatment in patients with Acute Myeloid Leukaemia (Blood , 106 (11), Abstr. 2473; 695a). This rapid assay, which uses a bioluminescent biosensor to measure intracellular levels of Ara-C and its active metabolite Ara-CTP, has been utilised in leukaemic cell lines KG1a (FAB type M0) which is sensitive to Ara-C and THP-1 (FAB type M5) which is partially insensitive to Ara-C due to over expression of cytidine deaminase (cdd). It has also been used in primary cultures of AML cells from six patients with M0 (n=1), M2 (n=2), M3 (n=1) and M4 (n=2). Intracellular concentrations of Ara-C as low as 0.05 μM can be detected by an increase in light output from the bacterial biosensor. The technology is suitable for the quantitation of response to clinical Ara-C doses (between 25–100μM) in less than 8 hours. Results from the KG1a cell line with known sensitivity to Ara-C showed correlation between the rapid assay and a 3 day cytotoxicity test of Ara-C sensitivity. Preliminary tests with the six clinical marrow samples showed 100% concordance with clinical outcome within eight hours of commencing the assay (vide infra: Table 1). In newly diagnosed AML patients, only 70% are sensitive to Ara-C therapy. Interestingly, in our hands, the M3 patient was Ara-C insensitive despite the fact that, most commonly, M3 patients do not express p-glycoprotein and therefore do not require Ara-C. Our small cohort of patients were disproportionately insensitive to Ara-C and therefore had a poor outcome overall. This assay system is potentially suitable for prescreening AML samples prior to commencement of Ara-C containing chemotherapy regimens. It may also give information on relative Ara-C sensitivity thus permitting dose adjustment and prevention of unnecessary toxicity to other organs. The assay could be repeated at relapse to document clonal evolution and decide whether Ara-C can be used to achieve a second remission. Work is currently underway with an industrial partner to look at commercialisation of this assay.TABLE 1:Correlation between light output and clinical response in six patients with primary AMLPatientDiagnosisClinical OutcomeDifferential Light Output: cytotoxicity testBioluminescent Assay: Ara-C sensitivity1M0No response5%no2M2No Response84%no3M2No Response79%no4M3Remission Death91%no5M4Complete Response99%yes6M4No Response62%no