Keywords platelet - Waldenström macroglobulinemia - receptor - bleeding
Introduction
Waldenström macroglobulinemia (WM) is the clinical manifestation of lymphoplasmacytic
lymphoma, which is a rare, low-grade, B cell lymphoma. WM is characterized by bone
marrow infiltration with malignant cells and hypersecretion of immunoglobulin (Ig)
M paraprotein. It constitutes less than 5% of all non-Hodgkin lymphomas, with an incidence
of approximately 0.3/100,000 cases/year.[1 ] Many patients are asymptomatic at the time of their initial diagnosis and do not
require treatment.[2 ]
[3 ] However, approximately 30% of these patients are likely to need therapy within 2
years of diagnosis, and 80% within 10 years.[4 ] Indications for treatment are varied, but most commonly include constitutional symptoms,
bone marrow or organ dysfunction, hyperviscosity and neuropathy. With an evolving
treatment landscape in WM involving Bruton's tyrosine kinase (BTK) inhibitors, proteasome
inhibitors and newer agents targeting BCL2 and CXCR4, understanding potential mechanisms
of bleeding is important, as some of these therapies increase bleeding risk and necessitate
the use of alternative agents in a personalized medicine approach.
Molecular Basis of WM
WM patients carry one or more somatic genetic mutations within malignant lymphoplasmacytoid
cells ([Fig. 1 ]), which have been reported to be present in less mature lymphoid and hematopoietic
progenitor cells in some cases.[5 ] Whether common WM mutations can be detected in megakaryocytes and platelets remains
an open research question. Understanding the WM genomic landscape is important, not
only because specific mutations can influence disease presentation and treatment options,[3 ]
[6 ] but also because they can potentially affect platelet function (see below). The
most common genetic defect in WM is a gain-of-function mutation within the myeloid
differentiation factor (MYD ) 88 gene resulting in a leucine-265 to proline (L265P) substitution within the cytoskeletal
adaptor protein Myd88, detected in lymphoplasmacytoid cells in over 90% of WM patients.[7 ]
[8 ]
[9 ]
[10 ]
[11 ]
[12 ]
[13 ] Of note, the MYD88L265P transcript has also been detected in WM plasma cells, mature B lymphocytes, phenotypically
normal B cell precursors and CD34+ hematopoietic precursor cells.[5 ] There are several other less common mutations now identified[14 ]
[15 ] and at least two distinct WM signature DNA methylation profiles specific for memory
B cells or plasma cells.[16 ]
Fig. 1 Pathways of development of lymphoplasmacytoid B cells in healthy individuals and
in WM patients. In WM patients, mutations and loss of DNA methylation occur to the
lymphoplasmacytoid cells, with enrichment in B memory cells at an earlier stage of
differentiation. The most common mutation, MYD88L265P , results in increased BTK phosphorylation and faster MYD88 complexing with IRAK1/4
in response to low or absent TLR or IL-1R stimulus, which results in increased NF-κB
translocation to the nucleus, increased target gene transcription, uncontrolled proliferation
of WM lymphoplasmacytoid cells and overproduction of IgM. BTK, Bruton's tyrosine kinase;
IgM, immunoglobulin M; IL-1R, interleukin-1 receptor; IRAK, IL-1 receptor-associated
kinase; MYD88, myeloid differentiation primary response 88; NF-κB, nuclear factor
kappa-light-chain-enhancer of activated B cells; TLR, toll-like receptor; WM, Waldenström
macroglobulinemia.
Through association with toll-like receptors (TLR) and the interleukin-1 receptor
(IL-1R), Myd88 has an important role in coordinating innate immune cell responses.
Cells expressing Myd88L265P protein exhibit constitutive activation of TLR and IL-1R pathways, leading to nuclear
translocation of transcription factor nuclear factor (NF)-κB and enhanced B cell proliferation
and survival.[17 ]
[18 ]
MYD88L265P
has also been detected in approximately 61% of IgM monoclonal gammopathy of undetermined
significance (IgM-MGUS) patients.[9 ]
[10 ]
[12 ]
[19 ] This mutation is likely to be an early oncogenic event in WM development, with IgM-MGUS
acting as a precursor condition. However, additional genetic mutations likely contribute
to WM onset.[14 ]
[20 ] Of note, Myd88 is expressed in platelets and megakaryocytes and is essential for
appropriate TLR-driven platelet responses to viremia.[21 ] A link between Myd88 activation and platelet function will be discussed below.
Gain-of-function mutations in the gene encoding the C-X-C chemokine receptor (CXCR ) 4 also occur in approximately 30% of WM patients.[22 ]
[23 ]
[24 ] CXCR4 engages with stromal-derived factor (SDF) 1 to mediate the homing of cells
to the bone marrow. A common activating mutation involving serine-338 in the C-terminal
region of CXCR4 prevents CXCR4 internalization following SDF-1 stimulation.[25 ] This leads to persistent CXCR4 activation and signaling via AKT, ERK and BTK pathways,
and bone marrow myeloid cell migration, adhesion, proliferation, and survival.[26 ]
[27 ]
The mutations in WM lymphoplasmacytoid cells, within genes involved in cell proliferation
and survival, cause an over-proliferation of these cells, resulting in overproduction
of IgM ([Fig. 1 ]). Increased IgM contributes to the neurological and bleeding symptoms observed in
WM, as correction of blood IgM levels often resolves symptoms.[24 ] WM malignant cell infiltration of the bone marrow leads to cytopenias, which increase
bleeding risk and predispose to infections. While some of these mutations have been
detected in lymphoid and hematopoietic precursors, it is not evident that the mutations
arise in platelet-producing megakaryocytes or contribute to a bleeding phenotype.
This review will explore the multifactorial nature of bleeding encountered in WM and
explore considerations that may aid clinical decisions around therapy for these patients.
Waldenström Macroglobulinemia Patients Can Present with a Bleeding Phenotype
Waldenström Macroglobulinemia Patients Can Present with a Bleeding Phenotype
When Jan Waldenström first described WM in 1944, the symptoms in the two patients
included oronasal bleeding.[28 ] While bleeding is often a feature of the initial presentation of WM, bleeding symptoms
usually resolve after therapeutic intervention, implying that WM disease etiology
does not directly affect platelet production and function. However, four studies have
evaluated the frequency of bleeding in patients with WM before treatment intervention
([Table 1 ]). When considered together, approximately 17% of patients displayed bleeding symptoms,[145 ]
[146 ]
[2 ]
[30 ] with limited description of magnitude. Most other studies describe easy bruising
and chronic oronasal bleeding as a common symptom of WM. It is worth noting that due
to a paucity of large studies on this issue, evidence-based guidelines and standard
of care management for treatment of bleeding in these patients are lacking. Thus,
reported treatment approaches are variable, with inconsistent reporting of key diagnostic
information. Standardized reporting of laboratory findings and outcomes is also lacking.
Table 1
Studies reporting the frequency of bleeding in treatment-naïve WM patients
Study
Sample size
% Bleeding
Nature of bleeding
Perkins et al 1970[145 ]
62
36
Not specified
Merlini et al 2003[146 ]
215
7
Hemorrhagic manifestations
García-Sanz et al 2001[2 ]
217
23
Not specified
Merchionne et al 2011[30 ]
121
12.3
Epistaxis and gum bleeding
Abbreviation: WM, Waldenström macroglobulinemia.
Laboratory Test Abnormalities Associated with Bleeding in WM
The cause of the WM bleeding phenotype is unknown, but laboratory studies have identified
several vascular and platelet abnormalities that often co-occur ([Table 2 ]). All of these sequelae are likely to contribute to bleeding symptoms in WM patients
prior to initiation of treatment; however several of these findings could occur as
a result of IgM protein binding to and/or inhibition of coagulation factor(s) function.
Table 2
Laboratory test abnormalities associated with bleeding in WM patients
Reference
Sample size
Finding
Comments and observations that may explain bleeding symptoms
Hivert et al 2012[133 ]
43/72 (59%)
Reduced VWF levels
- Possibly due to IgM-mediated inhibition.[48 ]
- Possibly due to specific autoantibody-mediated destruction, absorption onto malignant
cells, altered blood rheology resulting in VWF multimer proteolysis by ADAMTS-13.[36 ]
- Results in reduced VWF-GPIb-IX-V-mediated platelet activation and bleeding.
Gavriatopoulou et al 2019[134 ]
Hivert et al 2012[133 ]
6/42 (14%)
10/72 (14%)
Increased VWF levels
- Correlated with poor prognosis and low circulating ADAMTS-13 levels.[134 ]
- Reflects greater engagement between WM lymphoplasmacytoid cells and endothelium.[133 ]
[134 ]
- Could trigger thrombosis due to increased VWF-GPIb-IX-V-mediated platelet activation.
- Could result in increased bleeding due to low numbers of VWF multimers.[135 ]
Castillo et al 2019[38 ]
49/320 (15%)
Reduced FVIII levels
- FVIII is produced in the liver. Low FVIII levels possibly caused by liver dysfunction.[136 ]
- FVIII is bound to VWF in plasma. Low FVIII levels possibly coincide with AVWS.
- Possibly due to IgM-mediated inhibition of FVIII or VWF.[48 ]
[49 ]
[50 ]
- Results in decelerated FX activation and bleeding.[137 ]
Saraya et al 1972[138 ]
Kasturi et al 1978[139 ]
3/3 (100%)
4/4 (100%)
Reduced platelet adhesion
- Reduced in vivo platelet adhesion to a wound.[138 ]
- Results in bleeding
Kasturi et al 1978[139 ]
4/4 (100%)
Reduced platelet activation
- Platelets take up microparticles containing tissue factor. Platelet activation measured
via release of tissue factor in response to ADP.[140 ]
[141 ]
- Results in bleeding
Saraya et al 1972[138 ]
Kasturi et al 1978[139 ]
2/3 (67%)
3/4 (75%)
Reduced platelet aggregation
- Reduced aggregation in response to ADP and adrenaline. Within two standard deviations
for noradrenaline, thrombin, and collagen.[138 ]
- Results in bleeding
Abbreviations: ADAMTS-13; a disintegrin and metalloprotease; ADP, adenosine diphosphate;
aPTT; activated partial thromboplastin time; AVWS; acquired von Willebrand syndrome;
FVIII, factor VIII; PT; prothrombin time; TF; tissue factor; VWF; von Willebrand factor.
Note: Sample size indicates number of patients and percentage of cohort with clinically
significant bleeding.
Thrombocytopenia
Thrombocytopenia is a common occurrence in WM, often coincident with anemia. One large
WM study (n = 454) classified 18% of WM patients with thrombocytopenia.[32 ] Mechanisms by which thrombocytopenia arises are likely to be complex, potentially
resulting from combinations that are autoimmune- and drug-mediated, as well as marrow
infiltration which can cause overcrowding of hematopoietic stem cells and progenitors,
resulting in disturbed megakaryopoiesis and thrombopoiesis.[33 ] Occasionally thrombocytopenia is secondary to peripheral platelet sequestration
within the spleen (splenomegaly).[34 ] In isolation, thrombocytopenia rarely explains the observed bleeding, and the degree
of bleeding is often out of step with the platelet count.
Acquired von Willebrand Syndrome
Von Willebrand factor (VWF) is a biorheological shear sensitive glycoprotein (GP)
produced by the vascular endothelium and megakaryocytes. VWF plays a vital role in
primary hemostasis by triggering GPIb-IX-V-mediated platelet activation and formation
of an adhesive bridge between platelets and the vasculature at sites of endothelial
injury. VWF is also a carrier protein for factor VIII (FVIII) and contributes to fibrin
clot formation.[35 ] Acquired von Willebrand syndrome (AVWS) is an uncommon disorder caused by a loss
of high-molecular-weight VWF multimers, either by specific autoantibody-mediated destruction,
absorption onto malignant cells, or increased fluid shear stress resulting in VWF
multimer unfolding and proteolysis by ADAMTS-13.[36 ] In hematological cancer, AVWS is reported in lymphoproliferative neoplasms and several
myeloproliferative disorders.[37 ] AVWS occurs in 6% of WM patients, where incidence is strongly correlated with elevated
IgM levels (30–60 g/L).[38 ] Symptoms include mucosal and GI bleeding, which generally resolve following WM therapy.[38 ] To mitigate bleeding, specific therapeutic approaches aim to increase VWF antigen
levels (treatment with desmopressin and/or transfusion of FVIII/VWF concentrate),
remove an offending autoantibody (plasmapheresis), or disturb destructive autoantibody
functions, via transfusion of intravenous immunoglobulin (IVIg).[39 ]
[40 ] IVIg has been reported to successfully increase VWF/FVIII levels and reduce bleeding
times in AVWS linked to IgGMGUS.[41 ]
[42 ] Although the mechanism of action of IVIg is unclear, isolated case reports clearance
of VWF in the setting of IgM-MGUS may also be ablated by IVIg therapy, implying that
IVIg could be a favourable therapeutic option for AVWS associated with WM.[39 ]
[41 ]
Hyperviscosity
Hyperviscosity, caused by the accumulation of large (approximately 925 kDa), pentameric,
positively charged IgM paraprotein in the blood, is a classic manifestation of WM.
The IgM proteins electrostatically interact with sialic acid-rich red blood cells,
resulting in an agglutinating effect and contributing to increased viscosity.[43 ] Healthy individuals have around 1.5 g/L of IgM, of which 80% is intravascular,[44 ] and hyperviscosity emerges when IgM levels exceed 50 to 60 g/L.[44 ] Hyperviscosity causes the physical tearing of small venules from increased rheological
drag,[45 ] and the suspected inhibitory coating of platelets by IgM protein, resulting in reduced
platelet adhesion and aggregation.[46 ] Symptoms include bleeding, vision problems, and neurological symptoms, which occur
in 13% of WM patients.[47 ] Acute management of hyperviscosity involves plasmapheresis, while longer term management
with chemo-immunotherapy or targeted agents works by depleting the IgM-producing cells
in the marrow.
Hemostasis-Inhibiting Paraproteins
Circulating paraproteins have been reported to have VWF and FVIII-inhibitory activity
in WM in vivo, caused by IgM[48 ]
[49 ]
[50 ] or IgG[51 ]
[52 ] antibodies. This has also been observed in other paraproteinemias, including multiple
myeloma, MGUS, lymphoma, chronic lymphocytic leukemia, and amyloidosis.[53 ]
[54 ] Reports showed that the monoclonal IgM isolated from a WM patient demonstrated antiplatelet
activity and immune thrombocytopenia in vivo,[55 ]
[56 ] implying a derangement causing autoimmunity. Additionally, WM IgM cryoglobulins
can suppress erythroid and granulocyte progenitor cells grown in culture in vitro,[57 ] and possibly megakaryocyte progenitor cell maturation, which could alter platelet
quality and function.
Cryoglobulinemia
Cryoglobulinemia, where temperature-sensitive Igs form concentration-dependent insoluble
aggregates that precipitate below 37°C, can occur in WM.[58 ] These cryoglobulins may form immune complexes, where monoclonal IgM antibodies bind
to the Fc region of polyclonal IgG antibodies.[44 ] The symptoms of cryoglobulinemia occur at varied cryoglobulin concentrations depending
on the individual. These include purpura and mucosal bleeding, caused by the tearing
of small blood vessels by the aggregates, and are observed in approximately 5% of
WM patients.[44 ]
Amyloidosis
Amyloidosis can be associated with potentially life-threatening hemorrhage, by causing
coagulation factor deficiency, hyperfibrinolysis, platelet dysfunction, angiopathy,
and/or vascular fragility.[59 ] Amyloidosis is characterized by the production of misfolded proteins, often monoclonal
Ig light and/or heavy chains, which form insoluble amyloid fibrils, that accumulate
and form plaques, leading to tissue and organ dysfunction. IgM amyloidosis occurs
in 7.5% of WM patients and is associated with a dramatic reduction in overall survival,
from 12.1 to 2.5 years.[60 ] Bleeding occurs in 5 to 41% of amyloidosis patients, ranging from ecchymoses and
purpura, to gastrointestinal (GI) and postprocedural bleeding.[61 ]
[62 ]
[63 ]
[64 ]
[65 ] Bleeding results from increased vessel-wall fragility from perivascular amyloid
deposition, and/or from acquired factor X (FX) deficiencies. Acquired FX deficiencies
occur in 5 to 10% of amyloidosis patients and are caused by the absorption of FX and
pentraxin-2 onto amyloid fibrils, particularly in the spleen, resulting in direct
FX removal from circulation and indirect FX internalization by macrophages.[66 ] This can be corrected by splenectomy, chemotherapy treatment, or autologous hematopoietic
cell transplant.[66 ]
To summarize, WM patients display significantly elevated concentrations of serum IgM
and one or more symptoms of blood hyperviscosity, cryoglobulinemia, coagulation irregularities,
thrombocytopenia, amyloidosis and bleeding. The factor(s) responsible for bleeding
in untreated WM remains to be fully defined, but bleeding is likely to be the result
of a combination of all of the above observations.
Platelet Dysfunction in WM
Platelet Dysfunction in WM
Platelets circulate throughout the vasculature and are the primary mediators of hemostasis
([Fig. 2 ]). These are produced in the bone marrow by megakaryocytes, via controlled endomitosis.
A healthy individual generally has a very stable platelet count; however, the numbers
of circulating platelets can range from 150 to 400 × 109 platelets/L. Thrombocytopenic individuals (<100 × 109 platelets/L) can have a heightened risk of bleeding, which significantly increases
if the platelet count falls below 20 × 109 platelets/L. However, as mentioned earlier, bleeding can also occur in the absence
of thrombocytopenia. Thus, the prediction of an imminent bleeding event should not
rely solely on a low platelet count.[67 ]
[68 ]
[69 ] Besides the platelet count, platelet quality and functionality are critical components
of an effective hemostatic response. This involves detection of injury-exposed collagen
and other matrix proteins, and sensing alterations to local blood rheology. Platelets
respond by adhering to a site of vascular injury and undergoing platelet activation.
Platelet surface receptors coordinate this response[70 ] as well as the subsequent formation of a thrombus (platelet aggregate or blood clot),
which acts to seal the blood vessel, reduce blood loss and begin the process of wound
repair ([Fig. 2 ]).[71 ]
[72 ]
Fig. 2 Simplified hemostasis. (A ) Platelet plug formation. Following an injury, GPIb-IX-V and GPVI bind exposed extracellular
matrix ligands such as von Willebrand factor (vWF) and collagen respectively to enable
platelet adhesion to the endothelium. Engagement of these receptors triggers platelet
signaling, and platelets undergo changes to the cytoskeleton and release granules.
The platelet-specific integrin, αIIbβ3, becomes active and binds fibrinogen, bridging
adjacent platelets. Platelets aggregate forming a platelet plug at the site of the
injury. (B ) Blood coagulation and the securing of the platelet plug. Tissue factor (TF) is exposed
at the site of the injury triggering the extrinsic coagulation cascade, resulting
in thrombin production. Thrombin converts fibrinogen into insoluble fibrin which secures
the platelet plug in place. Blood hyperviscosity and high levels of IgM are likely
to interfere with these hemostatic pathways, and potentially underpin bleeding events.
GPVI, glycoprotein VI; IgM, immunoglobulin M.
Platelet Function Is Controlled by Surface Receptors and Signaling Pathway Proteins
Levels of platelet receptors and their attendant surface densities mediate platelet
responsiveness to molecular cues in the vasculature. Low receptor numbers and densities
have been associated with bleeding in patients receiving mechanocirculatory support[73 ] and in trauma patients.[74 ] Further, a loss of platelet receptors, including GPIbα and GPVI,[75 ] and a diminution of platelet function prior to therapy[76 ] have been demonstrated in leukemia patients. The molecular explanation for these
losses remains undefined, but may be linked to disturbances in bone marrow cellularity
and megakaryocyte maturation.[77 ] As GPIb-IX-V and the collagen/fibrin receptor GPVI also contribute to efficient
thrombus generation by binding thrombin and other coagulation proteins,[78 ]
[79 ]
[80 ]
[81 ] any alteration to normal levels of platelet adhesion receptors due to changes in
megakaryocyte maturity could disrupt efficient thrombin generation at the platelet
surface.
Bleeding has been observed in WM patients who do not have hyperviscosity, thrombocytopenia,
cryoglobulinemia or AVWS,[30 ]
[82 ] implying that there are other potential causes of bleeding in WM. It is possible
that many WM patients do not display chronic bleeding symptoms but possess an underlying
platelet lesion. When these patients experience vascular and hemostatic challenges
such as surgery or trauma, or when the platelet lesion is present in combination with
one or more common bleeding causes as outlined above, the platelet lesion can become
more evident and unexplained bleeding complications ensue.[83 ]
[84 ] Studies specifically evaluating platelet function in WM would help identify patients
with reduced hemostatic capacity and enhanced bleeding risk, and this information
could aid in clinical decisions regarding therapeutic approaches.
Standard WM Therapies May Accentuate the Bleeding Phenotype
Standard WM Therapies May Accentuate the Bleeding Phenotype
WM is an incurable disease where the treatments aim to alleviate the symptoms and
achieve prolonged remissions.[1 ]
[2 ]
[3 ] Treatment decisions in this regard are generally based on the symptoms, diagnostic
laboratory profile and the availability of drugs and clinical trials.[85 ] For asymptomatic patients, a “wait and watch” approach is routinely implemented.[3 ] Options for treatment of symptomatic WM patients include alkylating chemotherapy
agents (bendamustine, cyclophosphamide), proteosome inhibitors (bortezomib, carfilzomib,
ixazomib), or the first-generation BTK inhibitor (ibrutinib), alone or in combination
with rituximab. Newer therapeutic options include new, irreversible and more selective
BTK inhibitors (acalabrutinib, zanubrutinib) administered as a monotherapy, and emerging
options include BCL2 antagonists (venetoclax).[85 ] Many of these treatments carry an attendant bleeding risk which can be enhanced
in WM patients and will be discussed in this context below.
Rituximab
Rituximab is an anti-CD20 monoclonal antibody that specifically causes B cell depletion
and thus acts to reduce IgM production.[86 ] Although rituximab is commonly used in combination with chemotherapy agents, if
used as monotherapy, it can be associated with >25% rises in IgM (an IgM flare), which
can exacerbate hyperviscosity-related bleeding symptoms.[87 ] This risk is reduced when used in combination with other drugs. Alternatively, rituximab
can be associated with acute thrombocytopenia, linked with cytokine release syndrome
and complement activation.[88 ]
Alkylating Chemotherapies
Alkylating chemotherapies, such as bendamustine and cyclophosphamide, have been used
effectively as a frontline therapy in WM to kill rapidly dividing cells.[89 ] However, hematopoietic stem cells and their progenitor lineages are also sensitive
to these therapies, resulting in cytopenias, particularly thrombocytopenia, and increasing
the risk of bleeding.
BTK Inhibitors
The B cell receptor (BCR) signaling pathway is a central determinant of B cell fate
and function. This pathway is activated in WM, particularly in patients bearing the
Myd88 mutation.[14 ] When an antigen binds to the BCR, this triggers BCR clustering and initiation of
signal transduction via phosphorylation of BCR cytoplasmic tyrosine-based activation
motifs (ITAMs). ITAM clustering enables recruitment of Src-family kinases, which serve
to phosphorylate Syk and activate phosphoinositide 3-kinase (PI3K) δ. PI3Kδ mediates
the conversion of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-triphosphate,
which engages BTK. BTK phosphorylates phospholipase C (PLC) γ2, activating NF-κB,
NF of activated T cells, and mitogen-activated protein (MAP) kinase pathways ([Fig. 3 ]). These are all key elements of survival, development and cell proliferation pathways.
Fig. 3 Platelets and B lymphocytes utilize common signaling pathways. BTK is differentially
involved in the downstream signaling pathways triggered by ligand engagement of the
major platelet adhesion/signaling receptors (αIIbβ3, GPVI, and GPIbα of the GPIb-IX-V
complex) and the BCR. In receptors with ITAMs, following ligand binding and receptor
clustering, phosphorylation of the cytoplasmic ITAMs and recruitment of SFKs ensue,
resulting in phosphorylation of Syk and activation of PI3K. PI3K mediates the conversion
of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-triphosphate,
which engages BTK, resulting in phosphorylation of PLCγ. In platelets, this leads
to activation and aggregation. In B cells, activation of MAPKs, NF-κB, and NFAT leads
to B cell proliferation, development, and survival. BCR and GPVI ITAM signaling are
more reliant on the BTK pathway compared with GPIbα and αIIbβ3, thus signaling downstream
of these receptors is more sensitive to BTK inhibition. BCR, B cell receptor; BTK,
Bruton's tyrosine kinase; GP, glycoprotein; ITAM, immunoreceptor tyrosine-based activation
motif; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor κB; NFAT, nuclear
factor of activated T cells ; PI3K, phosphoinositide 3-kinase; PLCγ, phospholipase
C γ; SFK, Src-family kinases.
The clinical use of BTK inhibitors for the treatment of B cell malignancies has grown
remarkably, resulting in improved outcomes. At present, three different covalent irreversible
BTK inhibitors are approved for clinical use. These inhibitors (ibrutinib, zanubrutinib
and acalabrutinib) all bind cysteine 481 within the ATP-binding pocket of BTK with
different avidities and selectivities, and serve to inhibit the phosphorylation of
downstream kinases in the BCR signaling pathway, blocking B cell activation. As many
as 20 new BTK inhibitors are under development.[90 ] Nonetheless, bleeding remains a significant side effect that is associated with
the use of these therapies.[91 ]
Platelet activation requires intra-platelet signaling, triggered by platelet receptor–ligand
interactions, leading to enhanced platelet aggregation. Several of these pathways
involve activation of BTK[92 ]
[93 ] ([Fig. 3 ]), which is important in receptor signaling. Importantly there is redundancy in this
pathway, as studies of genetically engineered mice or patients with X-linked agammaglobulinemia
with loss-of-function mutations to the BTK gene did not reveal any bleeding propensity, most likely due to compensatory signaling
by Tec and other kinases.[94 ] BTK inhibitors interfere with BTK activity by irreversibly and covalently binding
to the kinase domain,[95 ] preventing autophosphorylation of BTK and phosphorylation of PLCγ2 and MAP kinases.
Ibrutinib is less specific for BTK and can inhibit other tyrosine kinases such as
TEC (tyrosine kinase expressed in hepatocellular carcinoma), ITK (interleukin-2-inducible
T cell kinase), TXK (T and X cell expressed kinase), and EGFR (epidermal growth factor
receptor), while the second-generation therapeutics zanubrutinib and acalabrutinib
are more specific to BTK ([Table 3 ]). As BTK is critical for BCR signaling, there is a level of specificity achieved
by targeting BTK. However, because of the broad kinase target spectrum of most tyrosine
kinase inhibitors, they inevitably have off-target adverse events ([Table 3 ]). Platelets rely on tyrosine kinase activity for their activation. BTK is involved
in platelet signaling pathways, mediated by GPVI, CLEC-2, GPIb, and αIIbβ3, that enable
platelet adhesion in flowing blood.[96 ] Platelet function has been shown to be inhibited in patients being treated with
ibrutinib,[97 ]
[98 ] as well as several other tyrosine kinase inhibitors.[99 ] Platelets from ibrutinib- but not zanubrutinib-treated patients showed reduced levels
of GPIb-IX-V and αIIbβ3 and an ablation of platelet aggregate formation.[100 ]
Table 3
Inhibited kinases and adverse events associated with various Bruton's tyrosine kinase
inhibitors
BTK inhibitor
Number of studies evaluated[142 ]
Total number of patients[142 ]
Type of hematological malignancies included[142 ]
Inhibited kinases:
IC50 ± SD (nM)[143 ]
% Adverse events:
grade 1–2 (≥ grade 3)[142 ]
Ibrutinib
12
1,263
TN + R/R CLL/SLL with P53 aberrations or del(17)p
TN + pretreated WM
R/R MCL
Pretreated GvHD
BTK: 1.5 ± 0.2
TEC: 10 ± 12
ITK: 4.9 ± 1.2
TXK: 2.0 ± 0.3
EGFR: 5.3 ± 1.3
Bleeding: 35 (4.4)[144 ]
AF: 12.4 (5.9)
Hypertension: 19.6 (15.6)
Rash: 15.8 (1.1)
Diarrhea: 47.3 (3.8)
Acalabrutinib
9
937
TN + R/R + ibrutinib-intolerant CLL/SLL
TN + R/R WM
R/R MCL
BTK: 5.1 ± 1.0
TEC: 126 ± 11
ITK: >1,000
TXK: 368 ± 141
EGFR: >1,000
Bleeding: 40.7 (2.5)
AF: 4.3 (1.1)
Hypertension: 8.6 (3.9)
Rash: 16.1 (0.3)
Diarrhea: 37.7 (1.9)
Zanubrutinib
6
756
TN + R/R CLL/SLL with del(17)p
TN + R/R + symptomatic WM
R/R MCL
BTK: 0.5 ± 0.0
TEC: 44 ± 19
ITK: 50 ± 5
TXK: 2.2 ± 0.6
EGFR: 21 ± 1
Bleeding: 39.2 (3.9)
AF: 2.0 (0.7)
Hypertension: 10.9 (2.8)
Rash: 17.7 (0)
Diarrhea: 20.8 (1.4)
Tirabrutinib
5
178
R/R CLL
TN + R/R WM
R/R MCL
R/R B cell malignancies or NHLs
BTK: 5.6 ± 1.0
TEC: 77 ± 7
ITK: >1,000
TXK: 116 ± 35
EGFR: >1,000
Bleeding: 17.8 (1.8)
AF: 0 (0)
Hypertension: 0 (0)
Rash: 24.7 (3.7)
Diarrhea: 19.8 (1.1)
Abbreviations: AF, atrial fibrillation; BTK, Bruton's tyrosine kinase; CLL, chronic
lymphocytic leukemia; EGFR, epidermal growth factor receptor; GvHD, graft-versus-host
disease; ITK, interleukin-2-inducible T cell kinase; MCL, mantle cell lymphoma; NHL,
non-Hodgkin lymphoma; R/R, relapsed/refractory; SD, standard deviation; SLL, small
lymphocytic lymphoma; TEC, tyrosine kinase expressed in hepatocellular carcinoma;
TN, treatment-naïve; TXK, T and X cell expressed kinase; WM, Waldenström mcroglobulinemia.
Consistent with an off-target effect of BTK inhibitors on platelets, a meta-analysis
by Brown and colleagues found that approximately 40% of patients with B cell malignancies
receiving ibrutinib experienced bleeding, with 4.4% experiencing major hemorrhage
(> grade III) ([Table 3 ]).[101 ] Reports of bleeding in WM patients as a result of ibrutinib treatment were slightly
lower than calculated for all B cell malignancy patients,[96 ] where approximately 23% of WM patients on ibrutinib experienced bleeding ([Table 4 ]). While differences in study design and definition of what constitutes a major or
minor bleed may account for some of the disparity, it is likely that differences in
disease pathogenesis may also contribute to incidence of bleeding across these different
malignancies. Ibrutinib discontinuation reverses major toxicities observed in WM patients
(bleeding, GI toxicity), despite causing IgM rebound in 73% and withdrawal symptoms
(fever, body aches, night sweats, arthralgia, headaches) in 19%.[102 ]
[103 ]
Table 4
Reports of bleeding frequency in Waldenström macroglobulinemia patients treated with
ibrutinib
Reference
Sample size
Number of WM patients
Study type
n , minor bleeding
(% grade 1–2)
n , major bleeding (% grade 3–5)
Thrombocytopenia
Bleeding description
Abeykoon et al 2020
80
80 (100%)
Nonclinical trial (84% previously treated, 16% treatment naïve)
3 (4%) hemorrhage,
1 (1%) petechiae
2 (3%) hematuria
1 (1%)
8 (10%)
Unspecified hemorrhage, petechiae, hematuria; intracranial hemorrhage due to CNS involvement
Ali et al 2017
45
8 (18%)
Retrospective observational cohort analysis
Previously treated
14 (30.5%)
0 (0%)
NA
Bruising, epistaxis; gastrointestinal (rectal) bleeding
Dimopoulos et al 2017
31
31 (100%)
Previously treated
12 (39%) bleeding
7 (23%) bruising
0 (0%)
5 (16%)
Grade 1–2 bleed, bruising
Dimopoulos et al 2018
75
75 (100%)
Phase 3 trial of ibrutinib plus rituximab
51% in ibrutinib + rituximab cf. 31% placebo + rituximab
4% incidence in each arm
0 (0%)
Unspecified low-grade bleeding; unspecified major hemorrhage
Treon et al 2018
30
30 (100%)
Treatment-naïve
2 (7%) bruising,
1 (3) postprocedural hemorrhage
1 (3%)
1 (3%)
Bruising, postprocedural hemorrhage; rectal bleeding
Treon et al 2015
63
63 (100%)
Previously treated
2 (3%)
0 (0%)
9 (14%)
Epistaxis, postprocedural hemorrhage
Abbreviations: CNS, central nervous system.
Note: n : number of individuals; minor bleeding consisted of grade 1–2 bleeds; major bleeding
comprised grade 3–5 bleeds.
Antiplatelet and Anticoagulant Therapies
WM affects an older demographic, many of whom have existing comorbidities. Treatments
of these comorbidities can include antiplatelet or anticoagulant drugs, such as aspirin,
clopidogrel, warfarin or direct oral anticoagulants, which carry an attendant bleeding
risk. Treatment of WM patients with antiplatelet or anticoagulant therapies in combination
with ibrutinib is common because of a significantly increased risk (approximately
10%) of atrial fibrillation with ibrutinib.[24 ]
[104 ]
[105 ]
[106 ]
[107 ]
[108 ]
[109 ]
[110 ] The use of these therapeutics concomitantly elevates the bleeding risk. In one study
of B cell malignancy patients, major bleeding occurred in 3.7% of patients receiving
ibrutinib monotherapy compared with 5.1% receiving ibrutinib in combination with antiplatelet
reagents or anticoagulants.[101 ]
Taken together, WM treatments increase bleeding risk, and treatments for WM comorbidities
can enhance this risk. Therefore, it is important to strengthen our understanding
of the molecular basis underlying the WM bleeding phenotype to accurately estimate
bleeding risk, to adjust clinical management plans accordingly, minimize bleeding
potential and improve patient quality of life.
Evaluating the Bleeding Phenotype in the Diagnostic Laboratory
Evaluating the Bleeding Phenotype in the Diagnostic Laboratory
Current approaches to WM patients with bleeding include coagulation testing, measurement
of plasma viscosity and specialized blood and platelet testing ([Fig. 4 ]). Further platelet quality and function tests utilizing research tools are emerging
([Fig. 4 ]). However, these tests present several challenges which will be discussed below.
Fig. 4 Recommended pathway to evaluate bleeding phenotypes in Waldenström macroglobulinemia,
with treatment options. Blue: asymptomatic WM; red: symptomatic WM; green: therapy
recommendations[1 ] (Castillo et al 2019). ADAM10, a disintegrin and metalloproteinase 10; Ag, antigen;
aPTT, activated partial thromboplastin time; BM, bone marrow; CBA, collagen binding
assay; DOAC, direct oral anticoagulant; FBC, full blood count; FVIII, factor VIII;
IgM, immunoglobulin M; LTA, light transmission aggregometry; PFA, platelet function
analyzer; PT, prothrombin time; RBC, red blood cell; R:Co: ristocetin cofactor assay;
ROTEM, rotational thromboelastometry; SE, side effect; sGPVI, soluble glycoprotein
VI; TEG, thromboelastography; TKI, tyrosine kinase inhibitor; VWD, von Willebrand
disease; VWF, von Willebrand factor.
Laboratory Tests Assessing Blood Coagulation
Coagulation assays measuring prothrombin time (PT)/international normalized ratio
and activated partial thromboplastin time (aPTT) are routinely performed.[15 ] It should be noted that these tests are influenced by several preanalytical variables
which can result in considerable intra- and inter-laboratory variation.[111 ] Variables include phlebotomy technique, anticoagulant volume based on patient hematocrit,
sample mixing and centrifugation, sample transport conditions, delays in transport
(over 4 hours), patient age, gender (females have increased levels of certain coagulation
factors and antithrombin, and reduced Protein S), physiological states (postsurgery,
pregnancy), and drugs (anticoagulants, antiplatelets, anti-inflammatories).
VWF antigen and cofactor binding assays have value in the appropriate clinical context
(bleeding phenotype).[15 ] VWF antigen assays measure VWF levels using a monoclonal antibody in a sandwich-based
enzyme-linked immunosorbent assay (ELISA). The original VWF ristocetin cofactor binding
assay (VWF:RCo) remains the gold standard method to measure VWF activity in plasma,
by evaluating donor platelet agglutination following ristocetin-mediated VWF unfolding
and binding to platelet-GPIbα, using light transmission aggregometry (LTA). Unfortunately,
this method is insensitive at VWF levels below 20 U/dL, and subject to variation based
on the source of ristocetin, variation in donor platelets, and the presence of VWF
A1 domain mutations resulting in poor ristocetin–VWF binding.[112 ] Newer versions of the VWF:RCo assay address sensitivity limitations, for example
through a chemiluminescence-based method which directly evaluates VWF binding to magnetic
particles coated with recombinant GPIbα in an active configuration (removing the requirement
for ristocetin). Additionally, the VWF collagen-binding assay analyses VWF multimers
by measuring the preferential binding of high-molecular-weight VWF multimers to collagen,
using an ELISA.[113 ] This assay has been shown to be more sensitive, reproducible, and less variable
than the VWF:RCo assay, improving discrimination between functional and nonfunction
VWF.[114 ] VWF multimer analysis by gel electrophoresis is clinically informative but challenging
to perform and not widely available as a diagnostic assay.
Thrombin generation assays measure the rate and “hemostatic potential” of plasma for
thrombin generation, via the cleavage of a quenched synthetic fluorogenic or chromogenic
substrate. Assays require calibration, and although several commercial kits exist,
limitations including a lack of standardization and reference values across laboratories
have been highlighted and are being addressed.[115 ]
[116 ] Thrombin generation assays are becoming more prevalent, but will require additional
clinical trials with well-defined endpoints to fully determine utility. To date, these
assays have been used to monitor anticoagulant or antiplatelet therapy,[117 ] and abnormal measurements have been associated with bleeding in patients with rare
inherited coagulation disorders such as hemophilia[118 ] and the risk of venous thromboembolism recurrence.[119 ] Capturing the contribution of blood cells to thrombin potential, however, remains
a clear gap in clinical applications of this assay.[120 ] Platelets accelerate the initiation of thrombin production via provision of membranes
bearing phosphatidylserine and receptors that interact with coagulation proteins (GPIb-IX-V
complex, GPVI and αIIbβ3 amongst others),[72 ] as well as the release of granule contents. Platelets, erythrocytes, leukocytes
and the endothelium are all likely to modulate thrombin potential in vivo. Further,
in the setting of pathologies with high paraprotein levels such as WM, the degree
to which high levels of plasma proteins interfere with normal production of thrombin
remains to be determined.
Laboratory Tests Assessing Platelet Function
Both LTA and the platelet function analyzer (PFA-100 and PFA-200)[121 ] are established diagnostic platelet function tests. Whole blood impedance aggregometry
(Multiplate) is an emerging tool,[122 ] and together with the PFA-100/200, these techniques enable rapid screening of platelet
responses to physiological agonists (adenosine diphosphate, thrombin, collagen). These
tests are influenced by the same preanalytical variables as the PT and aPTT tests
and require platelet counts to be above 100 × 109 /L, so are not suitable for thrombocytopenic patients. The LTA also requires a high
sample volume, and elevated levels of bilirubin and lipids increase plasma turbidity
and affect LTA data. The PFA-100/200 has the advantage of incorporating fluid shear
stress into the assay, and so provides a readout that is more physiological, but remains
dependent on an aperture closure time and does not evaluate platelet secretion defects.[123 ] All of these tests lack sensitivity to changes in receptor levels and can discriminate
only certain platelet function disorders.[124 ]
Laboratory Tests Assessing both Blood Coagulation and Platelet Function
Thromboelastography (TEG) and rotational thromboelastometry (ROTEM) are simple, automated,
highly sensitive, emerging viscoelastic tests that provide a global assessment of
hemostasis and are widely utilized in massive transfusion and acute bleeding scenarios.[125 ] Parameters including time to clot initiation, rate of clot formation, clot firmness
and strength and clot lysis time are quantified, and the contribution of platelets
to clot parameters can also be gleaned.[126 ] Oncologic diseases can cause coagulopathic states that may be identifiable by TEG
or ROTEM; however, more work is required to evaluate the utility of these tests for
assessing hemostasis beyond surgical bleeding.[125 ] Further, ROTEM and TEG are not yet standardized for evaluation of thrombocytopenic
samples, which may be relevant to patients with hematological diseases, particularly
those receiving treatment. There are no data to date specifically evaluating whole
blood clotting using TEG or ROTEM in WM.
In summary, evaluating platelet function and coagulation in WM patients presents several
challenges. First, none of the standardized platelet functional assays can evaluate
platelets in thrombocytopenic WM patients. Second, evaluation of platelet receptor
levels, which govern platelet function and are often diminished in hematological malignancies,[84 ] is not routinely evaluated by flow cytometry. This is due to lack of standardized
routine protocols for platelet flow cytometry and lack of clear understanding of its
clinical implications. Finally, none of these assays evaluate platelet function under
conditions that replicate vascular shear rates found in flowing blood.
Future Directions
In the research laboratory setting, several additional techniques can be used to evaluate
platelet function. The bone marrow microenvironment is disrupted in WM, contributing
to the initiation and propagation of WM lymphoplasmacytoid cells and likely disturbing
megakaryocyte maturation and platelet production. Therefore, megakaryocyte and platelet
flow cytometry can be used to evaluate the levels of receptors (αIIbβ3, GPIbα, GPVI),
as well as extent of platelet activation (P-selectin, active αIIbβ3) on circulating
platelets. Platelet flow cytometry has the advantage of remaining viable even when
the platelet count is extremely low, and useful data can be gathered on chemically
fixed samples, meaning samples can be stored for short periods. Levels of shed receptor
ectodomains can be quantified by ELISA[127 ] and microfluidic systems can be used to quantify platelet adhesion to immobilized
substrates (collagen, fibrinogen, fibrin) under shear, providing direct readouts of
platelet function under conditions that mimic a range of vascular rheological conditions.[128 ]
[129 ]
[130 ]
The generation of thrombin and fibrin as part of the coagulation pathway plays a crucial
role in the securing of platelet aggregates across sites of blood vessel injury. Insufficient
levels or defective coagulation factors can lead to formation of an unstable thrombus.
Whole blood coagulation, evaluated by ROTEM or TEG, assesses coagulation throughout
all phases of clot formation, triggered via extrinsic or intrinsic coagulation pathways.
While these parameters have not been previously evaluated in WM patients, it would
be of interest to compare samples from newly diagnosed individuals with those on different
therapies and ascertain whether a platelet defect can be determined. Additionally,
it might be of value to evaluate the effect of WM plasma, particularly from patients
with high levels of IgM and hyperviscosity, in mixing experiments using healthy donor
plasma-depleted blood to assess the effect of elevated IgM on whole blood coagulation.
Besides enhancing the availability of these tests, it will also be important to define
the situations in which these tests will be most helpful. As information on the platelet
lesion and more broadly the hemostatic defects in WM emerge, existing diagnostic tools
such as ROTEM or TEG may become more widely applied. While platelet flow cytometry
and testing for platelet activation markers using ELISAs remain distant from the diagnostic
laboratory, these additional new approaches can potentially be developed and incorporated
into diagnostic algorithms and may help guide therapy decisions in patients with compromised
hemostatic pathways.
Finally, evaluation of WM-related genes within the megakaryocytic progenitor populations
has not yet been explored. As platelets and megakaryocytes express both Myd88 and
CXCR4,[131 ] the prevalence of WM-related gene mutations in the megakaryocytes and megakaryocyte
progenitor populations should be evaluated. Platelet α-granules possess functional
membrane-bound CXCR4 as well as SDF-1. Activating CXCR4 mutations in megakaryocytes, like the CXCR4
S338X mutation, prevents CXCR4 internalization by platelets, following SDF-1 stimulation.[25 ] This may impair platelet aggregation, thromboxane A2 production and dense granule secretion,[132 ] and could contribute to a bleeding phenotype.
Concluding Remarks
With the advent of BTK inhibitors as efficacious and routine therapies for WM, it
is important that patients are evaluated and monitored continually for bleeding propensity.
By applying new approaches complemented by sensitive research-based techniques (flow
cytometry, thrombin generation assays, platelet spreading assays), in combination
with megakaryocyte-specific genetic approaches to evaluate common WM mutations, it
may be possible to stratify WM patients for bleeding risk based on platelet functionality.
One or more of these techniques could be integrated into routine testing for WM patients
at diagnosis and then during treatment, particularly in patients who present with
an elevated risk for bleeding.
