Haemostatic defects in B-cell malignancies

Abstract

Optimal platelet, coagulation and vascular bed functions are crucial for haemostasis, with impairments resulting in bleeding and thrombosis. Waldenstrom Macroglobulinaemia (WM) is a rare lymphoma, characterised by the myeloid differentiation primary response (MYD)88L265P mutation in 92% of patients. Clinical features include bone marrow (BM) infiltration and immunoglobulin (Ig)M paraprotein hypersecretion by malignant lymphoplasmacytoid B cells, and hyperviscosity and bleeding symptoms. WM is treated with Bruton's tyrosine kinase inhibitors (BTKis), which can exacerbate bleeding. Haemostatic functions are understudied in WM. This thesis aimed to evaluate platelet and coagulation functions in untreated (n=13) and BTKi-treated (n=5) WM patients, compared to healthy donors (HDs, n=15). Distinct phenotypes were identified using multivariate analyses. The effects of IgM paraprotein on haemostasis and the presence of MYD88L265P in the platelet-producing megakaryocytes (MKs) were also assessed. Untreated WM patients displayed mildly reduced platelet counts (151+/-79x109/L), which correlated with 3.8-fold increased thrombopoietin (TPO) levels. Platelets from untreated but not BTKi-treated WM patients displayed reduced reticulation and desialylation, possibly indicating deranged age and lifespan. These platelets displayed normal levels of adheso-signalling surface receptors and responses to physiological agonists, however contributions to the acceleration of thrombin generation were decreased by 74% relative to HD platelets. Despite untreated WM patients being stable and not displaying bleeding symptoms, multivariate analyses distinguished their platelet phenotypes from HDs with relative accuracy, based on reduced platelet counts, reticulation and fibrinogen binding to unstimulated platelets, and elevated TPO levels. These analyses also accurately distinguished BTKi-treated from untreated WM patient platelets based on reduced glycoprotein (GP)VI-mediated platelet activation response and GPVI, CD9 and soluble GPVI levels. WM patient plasma samples displayed delayed and reduced thrombin generation compared to HDs. Coagulation factor contributions to thrombus amplitude were increased in WM patient whole blood samples, possibly indicating increased fibrinogen contribution. Multivariate analyses accurately distinguished coagulation phenotypes in WM patients with cardiovascular comorbidities from those without, based on accelerated and increased thrombin generation measurements. IgM paraprotein was enriched from WM plasma to a purity of 83%. When included in platelet assays, 50-60 mg/mL IgM (equivalent to levels observed in hyperviscosity patients) reduced HD platelet adhesion, spreading and aggregatory responses to physiological agonists. IgM bound to activated platelets, possibly electrostatically. IgM reduced plasma thrombin generation potential by 6%. Haematopoietic cells were sorted from WM patient BM aspirates. MYD88L265P was identified in isolated B cells in 67% (6/9) patients, and intriguingly, also in 50% (3/6) lymphoid progenitors, 33% (2/6) stem cells and 20% (2/10) MKs. Platelets from a WM patient with MYD88L265P in the BM displayed 38% increased Toll-like receptor-mediated platelet activation, upstream of Myd88, compared to a WM patient without the mutation. This novel finding suggests that constitutively active Myd88L265P may be present in WM platelets and may alter inflammatory signalling responses to relevant pathogens. This is the first study to investigate haemostasis in a WM cohort of this size, using modern, sensitive, research techniques. This work identified platelet and coagulation defects in stable, well-managed WM patients, which were exacerbated by BTKi therapy or inclusion of IgM paraprotein. These defects may underpin bleeding in symptomatic WM patients. Monitoring of platelet and coagulation properties using multivariate analyses may help stratify patients for bleeding risk and personalise treatments accordingly.