Intracellular cytarabine triphosphate in circulating blasts post-treatment predicts remission status in patients with acute myeloid leukemia

•Intracellular ara-CTP is quantifiable in circulating AML blasts post-treatment.•Intracellular ara-CTP 4 hours post-treatment correlates with day 28 burden decrease.•Sampling for analysis can be integrated into a busy hemato-oncology ward. Cytarabine remains the backbone of therapy in acute myeloid leukemia (AML). The ability to assess intracellular cytarabine triphosphate (ara-CTP) levels in patients receiving cytarabine represents a major goal in the prediction of treatment response. This study, conducted within a clinical setting, aimed to assess ara-CTP levels in circulating peripheral blasts from non-M3 AML patients receiving cytarabine at one of three dosing levels, using a novel biosensor assay. Results from the initial 72 hours post-commencement were correlated with day 28 remission status, with feasibility parameters concurrently assessed. Intracellular ara-CTP was detectable in ex vivo blasts post-treatment for standard-dose (SD) and high-dose (HD) patients (p < 0.05), and quantification revealed a 27-fold increase in intracellular steady-state concentration between the two dosing levels. For low-dose cytarabine, high rates of patient discharge and low intracellular concentrations limited analysis; however, assessment of intracellular ara-CTP concentration was achievable in a dwindling population of blasts for SD and HD treatment cohorts, with 4 hours post-treatment commencement potentially being most predictive of clinical response (r = –0.912, p = 0.0113). Concurrent assessment of peripheral leukemia-associated immunophenotype (LAIP)-positive cells revealed a decline in burden (0–72 hours), which correlated with remission status (p < 0.05). Unexpectedly high rates of night sampling led to challenges associated with sampling rates, but did not have an impact on patient compliance. Additional training of night staff improved feasibility substantially. Multiple peripheral sampling during the initial 72 hours of treatment is feasible in newly diagnosed patients, and ara-CTP is detectable over the initial 24 hours, facilitating prediction of chemosensitivity of leukemic blasts to cytarabine. Cytarabine remains the backbone of therapy in acute myeloid leukemia (AML). The ability to assess intracellular cytarabine triphosphate (ara-CTP) levels in patients receiving cytarabine represents a major goal in the prediction of treatment response. This study, conducted within a clinical setting, aimed to assess ara-CTP levels in circulating peripheral blasts from non-M3 AML patients receiving cytarabine at one of three dosing levels, using a novel biosensor assay. Results from the initial 72 hours post-commencement were correlated with day 28 remission status, with feasibility parameters concurrently assessed. Intracellular ara-CTP was detectable in ex vivo blasts post-treatment for standard-dose (SD) and high-dose (HD) patients (p < 0.05), and quantification revealed a 27-fold increase in intracellular steady-state concentration between the two dosing levels. For low-dose cytarabine, high rates of patient discharge and low intracellular concentrations limited analysis; however, assessment of intracellular ara-CTP concentration was achievable in a dwindling population of blasts for SD and HD treatment cohorts, with 4 hours post-treatment commencement potentially being most predictive of clinical response (r = –0.912, p = 0.0113). Concurrent assessment of peripheral leukemia-associated immunophenotype (LAIP)-positive cells revealed a decline in burden (0–72 hours), which correlated with remission status (p < 0.05). Unexpectedly high rates of night sampling led to challenges associated with sampling rates, but did not have an impact on patient compliance. Additional training of night staff improved feasibility substantially. Multiple peripheral sampling during the initial 72 hours of treatment is feasible in newly diagnosed patients, and ara-CTP is detectable over the initial 24 hours, facilitating prediction of chemosensitivity of leukemic blasts to cytarabine. Prediction of response to therapy and risk stratification is a major goal in the early treatment of acute myeloid leukemia (AML). Whilst complete remission (CR) rates have greatly improved in younger patients with AML, high relapse rates and poor disease-free survival still pose a significant challenge [1Bose P Vachhani P Cortes JE Treatment of relapsed/refractory acute myeloid leukemia.Curr Treat Options Oncol. 2017; 18: 17Crossref PubMed Scopus (145) Google Scholar]. The backbone of AML therapy is cytarabine (ara-C), given at one of three dosing levels, depending on stage of disease (induction or relapse) and patient fitness (intensive or non-intensive therapy). Because of the rapid commencement of therapy after diagnosis, often prior to prognostic information being available, early assessment predicting response to cytarabine could be an important clinical decision-making tool. The relationship between intracellular ara-CTP accumulation and disease response has long been controversial, with some studies advocating a link [2Karp JE Donehower RC Dole GB Burke PJ Correlation of drug-perturbed marrow cell growth kinetics and intracellular 1-B-D-arabinofuranosylcytosine metabolism with clinical response in adult acute myelogenous leukemia.Blood. 1987; 69: 1134-1140PubMed Google Scholar, 3Rustum YM Preisler HD Correlation between leukemic cell retention of 1-beta-D-arabinofuranosylcytosine 5′-triphosphate and response to therapy.Cancer Res. 1979; 39: 42-49PubMed Google Scholar] and others dismissing it [4Gruber A Liliemark E Tidefelt U et al.Pharmacokinetics of mitoxantrone, etoposide and cytosine arabinoside in leukemic cells during treatment of acute myelogenous leukemia: Relationship to treatment outcome and bone marrow toxicity.Leukemia Res. 1995; 19: 757-761Crossref PubMed Scopus (8) Google Scholar, 5Koehl U Hollatz G Rohrbach E et al.Pharmacology of intracellular cytosine-arabinoside-5′-triphosphate in malignant cells of pediatric patients with initial or relapsed leukemia and in normal lymphocytes.Cancer Chemother Pharmacol. 2007; 60: 467-477Crossref PubMed Scopus (4) Google Scholar]. This single-centre, observational study used a previously validated biosensor technology [6Anderson E Conway M Alloush H et al.Investigation and verification of a bioluminescent biosensor for the quantitation of ara-CTP generation: A biomarker for cytosine arabinoside sensitivity in acute myeloid leukaemia.Biosensors Bioelectronics. 2014; 52: 345-353Crossref PubMed Scopus (4) Google Scholar] to assess intracellular ara-CTP concentration in circulating blasts over the initial 72 hours post-treatment. This was used to predict remission status in AML patients (n = 26) after treatment with low-, standard-, and high-dose (LD, SD, HD) cytarabine. Samples were gathered under informed consent (15/WM/0415) from non-M3 AML participants (≥18 years) receiving cytarabine therapy (n = 26) (Table 1). Patients received one of three dosing levels as judged by the consultant hematologist according to current UK guidelines (SD: 200 mg/m2 infused in divided doses twice daily; HD: 1.5–3 g/m2 infused in divided doses twice daily; LD: 20 mg twice daily by subcutaneous injection) [7Milligan DW Grimwade D Cullis JO et al.Guidelines on the management of acute myeloid leukaemia in adults.Br J Haematol. 2006; 135: 450-474Crossref PubMed Scopus (123) Google Scholar]. Fresh peripheral blood samples (≤4 mL) were collected pre-treatment (t = 0) and post-treatment (t=2, 4, 8, 12, 24, 48, and 72 hours ± 30 min) commencement, with standard-of-care assessment of remission/non-remission status (CR/NR) performed at day 28 as per current UK guidelines. Where a sampling time point coincided with an infusion time point, the blood sample was removed pre-infusion.Table 1Patient characteristicsCharacteristicCytarabine regimenLow doseStandard doseHigh dosen7136Median age, y (range)77 (63-79)67 (47-76)41 (22-55)Gender Male6101 Female135Ethnicity White, British6114 White, non-British002 Not known120FAB M0–1322 M2143 M4–5031 M6–7000 MDS/therapy-related MDS210 Not known130Cytogenetic risk group Favorable113 Intermediate661 Adverse052 Not known010Prior MDS430Presenting WBC count, × 109/L8.89 (2.26–88.4)8.76 (0.63–118.52)9.175 (2.98–24.48)Dose, mg20 (20–20)200 (160–220)4100 (3900–4600)Relevant co-medication Daunorubicin13/13 Mylotarg12/135/6 FLAG-IDA6/6 Tosedostat2/7 Lenalidomide2/7Body surface area, m22.11 (1.74–2.26)2.02 (1.64–2.22)2.02 (1.74–2.31)Bone marrow burden at presentation, %24.7 (3.8–91.8)42 (21.3–90.0)65.3 (11.0–85.0)Peripheral blasts at presentation, %8.6 (0.2–97.1)25.3 (0.7–88.0)54.2 (30–78.4)Day 28 bone marrow burden, %10.75 (0.1–72) 6Anderson E Conway M Alloush H et al.Investigation and verification of a bioluminescent biosensor for the quantitation of ara-CTP generation: A biomarker for cytosine arabinoside sensitivity in acute myeloid leukaemia.Biosensors Bioelectronics. 2014; 52: 345-353Crossref PubMed Scopus (4) Google Scholar0.90 (0.12–60) 12Elliott MA Litzow MR Letendre LL et al.Early peripheral blood blast clearance during induction chemotherapy for acute myeloid leukemia predicts superior relapse-free survival.Blood. 2007; 110: 4172-4174Crossref PubMed Scopus (57) Google Scholar1.75 (0.4–2.5) 5Koehl U Hollatz G Rohrbach E et al.Pharmacology of intracellular cytosine-arabinoside-5′-triphosphate in malignant cells of pediatric patients with initial or relapsed leukemia and in normal lymphocytes.Cancer Chemother Pharmacol. 2007; 60: 467-477Crossref PubMed Scopus (4) Google ScholarValues are expressed as number of patients or median (range).FLAG-IDA=fludarabine. cytarabine, granulocyte–macrophage colony-stimulating factor, idarubicin; FAB=French–American–British classification; WBC, white blood cell; MDS=myelodysplastic syndrome. Open table in a new tab Values are expressed as number of patients or median (range). FLAG-IDA=fludarabine. cytarabine, granulocyte–macrophage colony-stimulating factor, idarubicin; FAB=French–American–British classification; WBC, white blood cell; MDS=myelodysplastic syndrome. Peripheral blood mononuclear cells (PBMC) fractions were isolated from whole blood samples by density gradient centrifugation (Histopaque-1077, Sigma-Aldrich, Gillingham, UK) within 1 hour of withdrawal. PBMCs were pelleted at 300g (5 min), washed in RPMI-1640 medium (10 mL), re-suspended in red cell lysis buffer (0.15 mmol/L ammonium chloride, 0.01 mmol/L potassium bicarbonate, 0.001 mmol/L EDTA, pH 7.2–7.4) for 5 min, and washed in RPMI-1640 medium (without phenol red) (10 mL). Lysates were prepared and stored at –80°C until biosensor analysis as per Alloush et al. [8Alloush HM Anderson E Martin AD et al.A bioluminescent microbial biosensor for in vitro pretreatment assessment of cytarabine efficacy in leukemia.Clin Chem. 2010; 56: 1862-1870Crossref PubMed Scopus (15) Google Scholar]. leukemia-associated immunophenotype (LAIP)-positive absolute counts were determined on baseline peripheral blood and every 24 hours thereafter for 72 hours. Clearance of peripheral blasts was calculated by conversion of daily blast count to logarithmic scale, and subtraction from baseline. Ara-CTP stock solution (10 mmol/L, Jena Biosciences, Jena, Germany) was diluted (0–0.5 µmol/L) in cell lysate as per Alloush et al. [8Alloush HM Anderson E Martin AD et al.A bioluminescent microbial biosensor for in vitro pretreatment assessment of cytarabine efficacy in leukemia.Clin Chem. 2010; 56: 1862-1870Crossref PubMed Scopus (15) Google Scholar]. Limits of detection (LOD) and quantitation (LOQ) were calculated from the standard deviation of the blank (n = 6) as per Shrivastava et al. [9Shrivastava A Saxena P Gupta VB Spectrophotometric estimation of tamsulosin hydrochloride by acid-dye method.Pharm Methods. 2011; 2: 53-60Abstract Full Text PDF PubMed Google Scholar]. Results indistinguishable from background were reported as