Predictive Value For Disease Progression Of Thrombin Generation and D-Dimer Levels In Melanoma Patients Receiving BRAF Inhibitor Therapy
Authors: Verzeroli Cristina , Marchetti Marina , Merelli Barbara , Mandala Mario , Falanga Anna 
Published: October 2019

Introduction
Abnormalities of markers of blood clotting activation characterize the development of most cancers, including malignant melanoma, which still remains a largely incurable disease. Recently, the advent of therapy targeting the oncogene BRAF V600E mutation, expressed by about 50% of melanoma patients, has opened new perspectives for this disease outcome, with response rates in metastatic melanoma of >50%. In this study we wanted to evaluate the impact of a direct inhibitor of BRAF V600E, i.e. Vemurafenib, on the hemostatic profile of patients with melanoma, and analyze the relation of hemostatic parameters to the disease outcome.

Methods
Twenty BRAF V600E mutation-positive metastatic melanoma patients (10M/10F, age: 23-74 years) receiving Vemurafenib (960 mg twice daily) were enrolled at our Oncology Division upon informed consent. Blood was collected before (T0), and after one (T1) and four weeks (T2) of therapy. Thrombin generation (TG) was assessed by the calibrated automated thrombogram assay (CAT, Stago) in plasma samples, both in the absence and the presence of exogenous tissue factor (TF, 1 and 5 pM). TG curves were described in terms of lag-time, endogenous thrombin potential (ETP), peak height and time to peak (ttpeak). Plasma D-Dimer levels were measured by a turbidimetric immunoassay by D-Dimer-HS (Instrumentation Laboratory) and results expressed in ng/ml. Thirty-four healthy subjects acted as a control group.

Results
Before therapy, TG performed in the absence of exogenous TF was significantly (p<0.01) increased in plasma from melanoma patients compared to controls, as determined by a higher thrombin peak (135±110 vs 51±35 nM), and ETP (1,474±984 vs 699±510 nM*min) and a shorter lag-time (15.8±4.2 vs 23.2±6.2 min) and ttpeak (20.2±4.6 vs 28.2±6.3 min). Significantly (p<0.05) higher thrombin peaks were also found in TG induced by 1 (304±122 nM) and 5 pM TF (329±72 nM) versus controls (1 pM TF: 232±96 nM; 5 pM TF: 284±78 nM). During BRAF V600E inhibitor therapy, TG in the absence of TF remained statistically unchanged. Differently, TG induced by 1pM TF increased in terms of ETP and thrombin peak, reaching the statistical significance at T2 (T0 vs T2: ETP: 1,587±475 vs 1,780±522 nM*min, p<0.01; thrombin peak: 304±122 vs 336±132 nM, p<0.05), simultaneously to a significant (p<0.01) prolongation of the lag-time (5.2±1 vs 6.3±1.1 min) and ttpeak (8.5±1.2 vs 9.5±1.4 min). Similar results were observed with 5 pM TF. D-dimer levels, significantly (p<0.01) elevated at T0 in patients (325±296 ng/ml) compared to controls (83±50 ng/ml), further increased at T1 (431±240 ng/ml, p=n.s.), and returned to pre-treatment values at T2 (286±221 ng/ml). The tumor response to therapy was evaluated by computed tomography after 2 months treatment. Five patients achieved >85% disease remission, 5 patients had between 85 and 25% remission, and 10 were non-responders. In patients with >85% remission response compared to the other patients, TG in the absence of TF showed a significant increase from T0 to T2 in the lag-time (17% vs 8%), and ttpeak (18% vs 6%) together with a decrease in ETP (14% vs 2%) and thrombin peak (-44% vs 7%). Accordingly, the same patients showed at T0 and T2 lower levels of D-dimer values compared to the other patients (T0: 152±101 vs 383±393 ng/ml; T2: 224±168 vs 307±296).

Conclusions
Before therapy the study group of melanoma patients presented with a hypercoagulable state as determined by high levels of TG and D-dimer. In patients who responded >85% to BRAF V600E inhibitor therapy a down-regulation of these markers was observed as compared to patients who responded <85% or did not respond. The data suggest a possible association between hypercoagulation, BRAF V600E mutation, and response to therapy. Therefore, further research is worth in this field to establish whether thrombotic markers, namely TG and D-dimer, may be a useful tool to select patients who may benefit from BRAF V600E inhibitor therapy.

Disclosures:
No relevant conflicts of interest to declare.