Evaluation of coagulopathy before and during induction chemotherapy for acute lymphoblastic leukaemia, including assessment of global clotting tests

Patients with acute lymphoblastic leukaemia (ALL) are at high venous thromboembolism (VTE) risk (affecting between 1.5 and 37%) with up to 50% affecting the cerebral veins. Thrombosis is a potentially avoidable source of morbidity and mortality in ALL patients, particularly during the early phases of chemotherapy. Prevention and treatment of thrombosis with antithrombotic drugs may be complicated by co-existent bleeding risk due to factors such as thrombocytopenia and the need for procedures. Conventional clotting tests (platelet count (PLT), prothrombin time (PT), partial thromboplastin time (PTT), Clauss fibrinogen) only measure isolated components of haemostasis with no measure of cellular/plasma interactions, platelet function, natural anticoagulants, Von Willebrand factor (VWF) and fibrinolysis. Therefore, when complex, multiple reciprocal changes occur, these tests are poor at predicting overall balance. Consistent with this, in patients with ALL, conventional tests are often abnormal, implying a bleeding tendency when in fact, thrombosis is the greater risk. Global clotting tests are an alternative approach measuring the net effect of multiple pathways. Thromboelastography (for example, ROTEM) tests whole-blood clot formation and fibrinolysis, activated by intrinsic (INTEM) or extrinsic pathways (EXTEM). FIBTEM tests fibrinogen in isolation and thrombin generation reflects the net balance of proand anticoagulant proteins in plasma. In other similar complex coagulopathies (for example, liver failure, trauma and cardiac surgery), global tests are clinically useful and may be more reflective of haemostatic balance. To comprehensively evaluate the coagulopathy in ALL patients before and during induction chemotherapy (the highest VTE risk period for current UK treatment protocols), we undertook a prospective, single centre longitudinal cohort study (GlobALL study REC approval ref 14/EM/1315) in which blood samples at multiple time points were analysed in parallel using global, conventional clotting tests, specific factor and anticoagulant levels. Newly diagnosed adults and children with ALL commencing intensive chemotherapy at UHBristol NHS Foundation trust were eligible for study enrolment. Participants gave informed written consent. VTE and bleeding events were recorded for the first 90 days of treatment. ALL treatment was with UKALL2011 (patients o25 y, Peg-Asparaginase d4 and d18), UKALL14 (patients 25–65, PegAsparaginase D18 and d4 if o40 y) and UKALL60+ protocols (patients 465 no Peg-Asparaginase). Adult patients (418 y) routinely received low molecular weight heparin (LMWH) thromboprophylaxis in hospital if no contraindications but not on discharge. There was no routine thromboprophylaxis in children o18 y. Samples from 20 healthy adult controls were also obtained. Blood samples were collected into EDTA and trisodium citrate at d0 (pre-treatment), d4, d5 (only if d4 Peg-Asparaginase was administered), d10, d18, d19 (only if d18 Peg-Asparaginase was administered), d24, d29 and d36. Each blood sample underwent analysis using standard laboratory methods for PLT, PT, activated PTT, Clauss fibrinogen, D dimer level, activities of antithrombin, protein C, coagulation factor VIII, antigenic levels of VWF:Ag and protein S. Global coagulation analysis of whole blood was performed within 4 h of venepuncture using a ROTEM analyser and the EXTEM, INTEM and FIBTEM tests. Plasma samples were analysed by calibrated automated thrombography to measure thrombin generation using an Ascent fluorometer (Thermo-Lab systems, Helsinki, Finland) and Stago PPP LOW (1pM tissue factor) reagent. Statistical analysis was performed using GraphPad Prism (La Jolla, CA, USA) and SAS (Buckinghamshire, UK). One-way analysis of variance (ANOVA) comparison between groups using Tukey’s multiple comparisons test was used. Longitudinal distribution of the data over the study period was measured with a repeated measures ANOVA using an autoregressive variance matrix for normally distributed data and Kruskal–Wallis test for non-parametric data. A P-value of less than 0.05 was considered significant. Thirty-five patients were recruited between January 2015 and January 2016 (24 male, 31B, 4T lineage, median age 12 y, range 18 months 67 y). Twenty-nine patients received d4 and d18 PegAsparaginase, three received d18 only and three did not receive Asparaginase (as BCR-ABL positive). Thirty-four patients had central venous line for induction chemotherapy. Six patients received LMWH thromboprophylaxis during induction for a median duration of 15 days (range 4–42). Out of 232 blood samples collected, 221 were of sufficient quality to process. The overall VTE incidence was 17% (6 out of 35) (median age 32 y, range 14–67 y), including 2 patients on preceding LMWH thromboprophylaxis. Two life-threatening cerebral VTE occurred, three line-associated clots and one deep vein thrombosis (latter patient did not receive Peg-Asparaginase). Thrombosis was diagnosed at a median 35 days after chemotherapy initiation (range day 10–50). All events occurred when PLTs 450 × 10/l. Two adults had clinically relevant non-major bleeding and overt consumptive coagulopathy prior to treatment. Compared to 20 healthy controls (Table 1) and appropriate reference ranges for age and gender, before induction chemotherapy (d0), patients with ALL showed multiple abnormalities in the panel of haemostasis tests (Table 1). The tests which showed the highest proportion of values outside reference interval were reduced PLT, PTT, thrombin generation (ETP (endogenous thrombin potential) and peak) and EXTEM maximal clot firmness (MCF) (425% of results below reference intervals), and increased Clauss fibrinogen, D dimer, VWF:Ag, FVIII, FIBTEM MCF (425% of results above reference intervals; Table 1). Over the first 36 days of induction chemotherapy, (Table 1), there were dynamic changes in haemostasis tests. By d29, the tests which showed the highest proportion of values outside reference interval were reduced Clauss Fibrinogen, thrombin generation (ETP and peak), antithrombin, FIBTEM MCF and EXTEM MCF (425% of results below reference intervals) and increased PT, PTT, VWF:Ag, FVIII and EXTEM clotting time (CT) (425% of results above reference intervals; Table 1). The most clinically and statistically significant changes (d29 compared to d0) were Citation: Blood Cancer Journal (2017) 7, e574; doi:10.1038/bcj.2017.54