Waldenström macroglobulinemia: From biology to treatment

Ilyas Sahin1,*, Houry Leblebjian2,*, Steven P. Treon1, Irene M. Ghobrial1

1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard

Medical School, Boston, MA, USA

2Pharmacy Department, Dana-Farber Cancer Institute, Harvard Medical School,

Boston, MA, USA

* These authors have equal contribution

Corresponding author:

Irene M. Ghobrial, MD

Medical Oncology, Dana-Farber Cancer Institute,

450 Brookine Ave Boston, MA, 02115

Phone: (617)-632-4198

Fax: (617)-632-4862

Email: Irene_ghobrial@dfci.harvard.edu

Key words: biology, Waldenström macroglobulinemia, MYD88, bruton tyrosine kinase, treatment

Running title: Waldenström macroglobulinemia

Abstract: 117; Text: 6127; Table: 2; References: 116

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Abstract

Waldenström macroglobulinemia (WM) is distinct B-cell lymphoproliferative

disorder primarily characterized by bone marrow infiltration of lymphoplasmacytic

cells along with production of a serum monoclonal immunoglobulin M (IgM). In

this review, we describe the biology of WM, the diagnostic evaluation for WM with

a discussion of other conditions that are in the differential diagnosis and clinical

manifestations of the disease as well as current treatment options. Within the

novel agents discussed are everolimus, perifosine, enzastaurin, panobinostat,

bortezomib and carfilzomib, pomalidomide, and ibrutinib. Many of the novel

agents have shown good responses and have a better toxicity profile compared

to traditional chemotherapeutic agents, which makes them good candidates to be

used as primary therapies for WM in the future.

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Introduction

Waldenström macroglobulinemia (WM) is a B-cell lymphoproliferative disorder

characterized by bone marrow infiltration of lymphoplasmacytic cells along with

production of a serum monoclonal immunoglobulin M (IgM)[1,2]. WM first

described by Jan Gosta Waldenström in 1944 by reporting two patients

presented with oronasal bleeding, anemia, high serum viscosity,

hepatosplenomegaly and infiltration of the BM by lymphoid cells. Unlike multiple

myeloma, the patients had lymphocytic bone marrow infiltration rather than

plasmacytic and no bone lesions or symptoms [3,4].

WM is classified as a lymphoplasmacytic lymphoma (LPL) according to World

Health Organization (WHO) and the Revised European American Lymphoma

(REAL) classificiations [4,5]. WM is a rare cancer with an incidence rate of about

3 cases per million people per year in the United States.1,500 new cases are

diagnosed with WM each year in the United States [6,7]. The median age at

diagnosis varies between 63 and 68 years [1]. Men are twice more commonly

affected compared to women and, incidence rates for white population are 2-3

fold higher than in black population [8].

Biology of WM

The cell of origin

WM is thought to originate from an arrested B cell that has undergone somatic

hypermutation in germinal center without differentiating to plasma cells [9]. It has

been shown that WM may originate from an IgM+ and/or IgM+IgD+ memory B

cell without initiating switching events. WM cells are characterized by a specific

immunophenotype (positive for surface IgM, CD19, CD20, CD22, CD25, CD27,

FMC7; negative for CD103, and variable CD5, 10, 23, CD138 expression), which

help in differentiating from other NHL and multiple myeloma that appear

morphologically similar.

Cytogenetic findings and copy number variations

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The most common cytogenetic abnormality in WM patients is the deletion of the

long arm of chromosome 6, which was reported in about 40-50% in recent

studies [10-12]. Other studies demonstrated the presence of other cytogenetic

abnormalities, including trisomy 4, trisomy 5, monosomy 8, and deletion of the

long arm of chromosome 20 [10].

It has been reported that 83% of patients with WM have various chromosomal

abnormalities [13]. Gain of 6p was the second most common abnormality (17%),

which was always concomitant with 6q loss. B lymphocyte-induced maturation

protein-1 (BLIMP-1) and tumor necrosis factor α-induced protein 3 (TNFAIP3 or

A20) are the two candidate tumor suppressor genes mapping two distinct

minimal deleted regions on the long arm of chromosome 6 [14,15]. BLIMP-1 is a

transcription factor acting mainly as a repressor, which plays a crucial role in

orchestrating the differentiation of B cells into plasma cells [16]. TNFAIP3 is an

ubiquitin-modifying enzyme that acts as a negative regulator of TNF and toll-like

receptor (TLR) mediated responses. Inactivating mutations were observed in

TNFAIP3 and TNF receptor-associated factor 3 (TRAF3) resulting in activation of

the nuclear factor kappa B (NF-kB) signaling pathways. Moreover, a minimal

deleted region, including MIRN15A and MIRN16-1, was demonstrated on 13q14

in about 10-13% of patients [13,17].

Whole genome sequencing

Treon et al has recently reported the presence of MYD88 L265P somatic

mutation (in chromosome 3p22.2) in over 90% of WM patients via whole genome

sequencing [18]. High frequency of this mutation in WM patients (70-100%) was

also confirmed by other studies via Sanger sequencing, polymerase chain

reaction (PCR) or allele specific polymerase chain reaction (AS-PCR) [19-23].

MYD88 L265P was also detected in non-IgM LPL patients, suggesting that this

mutation can be seen in LPL patients independent of isotype [18,24,25]. Recent

studies also showed that this mutation might be seen in up to 80% of patients

with IgM monoclonal gammopathy of undetermined significance (MGUS) [18-

20,22,26]. In order to assess the impact of this mutation on the risk of

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progression from IgM-MGUS to WM, Varettoni et al performed a case-control

approach in IgM-MGUS patients and found that the MYD88 L265P mutation was

associated with a significantly higher risk of progression to WM or to other

lymphoproliferative disorders [20]. Together, these findings suggest a key role of

MYD88 L265P somatic mutation for the development of WM. This

nonsynonymous mutation causes a leucine (L) to proline (P) substitution in

codon 265 (L265P) of MYD88, which is thought to be a crucial player in the

activation of the canonical NF-κB pathway, downstream of TLR and interleukin-1

receptor (IL-1R) signaling [18,27-29]. When combined with immunoglobulin

heavy chain variable gene (IGHV) mutation status, MYD88 L265P mutation

appears to be a unique genetic signature as a diagnostic tool that distinguishes

WM from other similar B cell malignancies [21]. The second most common

somatic gene mutation in WM are C-terminal mutations, which are similar to

those found in the germline of patients with WHIM syndrome [30]. Up to 30% of

WM patients harbor CXCR4 mutations which enhances tumor dissemination and

survival of WM cells [31,32].

Epigenetics

Epigenetic regulation of tumor suppressors and oncogenes play an important

role in the development of many tumor types [33,34]. The main epigenetic

changes include DNA methylation and histone modification [35]. Other regulators

include non-coding RNAs such as miRNAs.

Upregulation of miRs including 155, 184, 206, 363, 494 and 542-3p, and

downregulation of miR-9 are identified in WM cells. Among these, miRNA-155

was found to be a critical regulator of tumor growth and proliferation of WM cells

both in vitro and in vivo [36]. Increased expression of miRNA-206 and reduced

expression of miRNA-9 increased histone acetylation leading to enhanced gene

transcription [37]. In addition, MIRN15A and MIRN16-1 are believed to act as

tumor suppressors by preventing the transcription of mRNAs encoding for

proteins involved in proliferation, cell cycle and anti-apoptosis, such as BCL-2

[38].

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Gene Expression Profiling (GEP)

A study of GEP in Waldenström macroglobulinemia demonstrated that WM has a

homogenous expression profile similar to that of patients with chronic

lymphocytic leukemia (CLL) [39]. Another study showed that the genes including

ATXN1, FMOD, LEF1 (WNT/b-catenin pathway), MARCKS discriminated WM

cells from CLL cells [14]. Furthermore, a recent study performed GEP on WM BM

CD19 + versus IgM MGUS BM CD19 + cells showed that 151 genes were

differently expressed. PI3K/Akt/mTOR, JAK/STAT and MAPK signaling pathways

were found to be the most important signaling pathways in these two disorders

[40].

Proteomic analysis

Hatjiharissi et al employed protein expression profiling of untreated WM and

normal bone marrow controls using antibody-based protein microarrays to

demonstrate proteins that are dysregulated in WM. The study showed that Ras

family proteins such as Rab-4 and p62DOK, as well as Rho family proteins such

as CDC42GAP and ROK α were up-regulated by >2-fold in WM. Other proteins

including cyclin-dependent kinases, apoptosis regulators, and histone

deacetylases were up-regulated by >1.3-fold [41].

Deregulated molecular pathways

MYD88/IRAK

Recent findings of the existence of a mutation in MYD88 in most of WM patients

have indicated that MYD88/IRAK signaling pathway might have a crucial role in

pathogenesis of WM. In this pathway, upon TLR or IL-1R stimulation, interleukin-

1 receptor-associated kinase 4 (IRAK4) recruited to the receptor by the adaptor

MyD88, which then activates IRAK1 that ultimately resulting in activation of NF-

κB [42]. However, the signaling cascade(s) by which MYD88 L265P induce NF-

κB activation in WM remains unclear. Studies showed that targeting

MYD88/IRAK signaling attenuated NF-κB signaling and survival of MYD88

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L265P–expressing WM cells [18,23]. Moreover, Yang et al demonstrated

increased phosphorylated Bruton tyrosine kinase (BTK) in WM cells transduced

to overexpress L265P compared to wild-type MYD88 [43]. Taken together, these

findings would suggest that MYD88 L265P triggers NF-κB via dual, which signal

through BTK and/or IRAK1/IRAK4 [44]. Therefore, combined suppression of both

BTK and IRAK signaling may provide synergistic inhibition of NF-κB signaling.

PI3K/Akt/mTOR

PI3K/AKT signaling inhibits apoptosis and regulates cell growth, survival, and

proliferation [45]. Although there is no evidence that there are activating

mutations in the PI3K/Akt/mTOR pathway in WM, it has been shown that this

pathway is activated in WM [46,47]. Recently, Yang et al showed that MYD88

L265P can activate PI3K delta [48]. External stimulation through the bone

marrow microenvironment with cytokines such as insulin-like growth factor (IGF-

1) or stromal- derived factor (SDF-1) may also contribute to activation of this

pathway [46]. Gene and protein expression of PTEN, a negative regulator of

PI3K/Akt pathways, were found to be decreased which lead to consistent

activation of the PI3K/Akt pathway in WM [47]. Additionally, inhibition of Akt by

perifosine, mTOR by RAD001, or dual PI3K/ mTOR inhibitor (NVP-BEZ235)

display activity against WM even in the presence of bone marrow stromal cells

[46,49].

JAK/STAT

It was shown that expression of both Jak1 and Stat3 proteins is significantly

higher in the WM patients as compared to healthy controls [41]. Although no

mutations associated with this constitutive Jak/Stat signaling have been

described in WM to date, cytokine-mediated stimulation of this pathway may be

involved in the dysregulation of IgM [50]. One important cytokine believed to play

pivotal role is IL-6. It is widely accepted that IL-6 activates MAPK/ERK pathway

and the transcription factor Stat3 via the tyrosine kinase receptor signaling of

JAK1 and 2 [51].

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The nuclear factor kappa B pathway

NF-kB comprises a family of transcription factors that serve as important

regulators of the transcription of many genes involved in inflammation, innate

immunity, cell growth, and apoptosis [52]. Inhibition of this pathway by

proteasome inhibitors such as bortezomib, NPI-0052 and Onyx0912 can inhibit

WM cells and overcome resistance induced by bone marrow stromal cells

[53,54]. Studies using bortezomib alone or in combination with rituximab in newly

diagnosed or even in relapsed WM patients have shown significant activity with

over 80% response rate [55-57].

Risk factors for developing WM

The underlying causes of WM are poorly understood. The main risk factor for the

development of WM is pre-existing IgM-MGUS. Other risk factors include familial

history of WM or other B-cell malignancies and immunological factors.

IgM-MGUS. IgM-MGUS patients are up to 46 times more likely to develop WM

than the general population [58]. IgM-MGUS progresses mostly to WM; Kyle et

al demonstrated the cumulative incidence of progression was 10% at 5 years,

18% at 10 years, and 24% at 15 years, respectively [58]. Abnormal serum free

light chain (FLC) ratio, the serum monoclonal protein and serum albumin

concentrations were shown to be risk factors for progression [58,59]. In addition,

recent work by Varettoni et al has shown that MYD88 L265P mutation is also an

independent predictor of progression [60]. IgM-MGUS patients are also at

increased risk of developing lymphoproliferative disorder, lymphoma, primary

amyloidosis and chronic lymphocytic leukemia [61]. We have previously

demonstrated that increased serum FLC (with a cut off of 60 mg/L) is a new

marker in WM disease and reflective of tumor burden. Therefore, it can be used

to differentiate IgM-MGUS from symptomatic WM [62]. Similarly, 6q deletion is

not seen in IgM- MGUS patients, whereas it is observed in about 30%-50% of

WM patients [63].

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Familial WM. Although WM represents a sporadic disease in most cases; various

reports reported strong familial predisposition. A study with clinicopathological

analysis from 257 unrelated WM patients showed that 48 (18.7%) patients had at

least one first-degree relative with WM or another B-cell disorder, including non-

Hodgkin lymphoma (NHL), Hodgkin lymphoma, MGUS, MM, chronic lymphocytic

leukemia (CLL) and acute lymphoblastic leukemia [64]. A large study with 2144

LPL/WM patients has demonstrated first-degree relatives of LPL/WM patients to

have 20-fold, 3.0-fold, 3.4-fold, and 5.0-fold increased risks of developing

LPL/WM, NHL, CLL, and MGUS, respectively [65]. The excess risks among

parents, siblings, and offspring were similar.

Immunological Risk Factors. It has been shown that history of certain

autoimmune and infectious diseases was associated with an increased risk of

LPL/WM [65].

Diagnosis

WM is primarily characterized by bone marrow infiltration of lymphoplasmacytic

cells which produce excessive amounts of a serum monoclonal IgM [1,2]. Of

note, an IgM paraprotein of any level is not sufficient for the diagnosis of WM,

since there are other lymphoproliferative disorders that produce IgM [66]. In

clinical practice, characteristic immunophenotype of high levels of surface CD19,

CD20, and Ig light chain expression in association with nonparatrabecular pattern

of BM infiltration is diagnostic for WM [1,4,5,67]. The intratrabecular pattern of

BM infiltration is mostly found in WM, whereas an exclusively paratrabecular

pattern is rare and should raise suspicion of follicular lymphoma. Dutcher bodies-

containing plasma cells and increased number of mast cells close to lymphoid

aggregates are commonly found in WM.

Immunophenotypic studies showed the following profile for WM cells: positive for

surface IgM, CD19, CD20, CD22, CD25, CD27, FMC7; negative for CD103, and

variable CD5, 10, 23, CD138 expression [29,68].

Clinical features

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The majority of WM patients present with signs and symptoms related to

elevated IgM levels and/or to infiltration of bone marrow and extramedullary sites

by malignant B cells. Cytopenia, which is associated with tumor cells

replacement in the BM, is the frequent clinical presentation of the disease.

Hyperviscosity resulting from high levels of proteins can lead to impairment of

microcirculation in the central nervous system. This may result in headache,

blurry vision, visual loss and epistaxis in WM patients. Some patients may

experience hepatosplenomegaly and lymphadenopathy, and some patients may

present with B symptoms including night sweats, fever, and weight loss.

Moreover, some patients with WM may present with other common

manifestations including neuropathy, cryoglobulinemia, skin rash (Schnitzler

syndrome), cold-agglutinin hemolytic anemia, and amyloidosis [69].

Progression in WM: IgM-MGUS, asymptomatic WM, and symptomatic WM

Normally, WM is preceded by IgM-MGUS or smoldering WM [70]. IgM-MGUS is

described as an IgM<3 g/dL and <10% lymphoplasmacytic cells in the BM,

whereas smoldering WM is described as an IgM>3 g/dL or >10% LPL cells in the

BM in the absence of any symptoms attributable to WM. A recent study showed

that the cumulative probability of progression of smoldering WM to symptomatic

WM, amyloidosis, or lymphoma is 6% at 1 year, 39% at 3 years, and 59% at 5

years, respectively [71]. The percentage of lymphoplasmacytic cells in the bone

marrow, size of the serum M-spike, and the hemoglobin value were the major risk

factors for the progression. Although abnormal serum FLC ratio was shown as a

risk factor for progression in MGUS, it was not a predictive factor of progression

in patients with smoldering WM [71]. Since the criteria used for IgM-MGUS and

smoldering WM are not widely adopted in the WM literature, another

classification based on the consensus recommendation is more frequently used,

According to this classification, patients present with Ig protein but no LPL cells in

the BM should be diagnosed with Ig-MGUS, and patients with any level of IgM

monoclonal protein and presence of lymphoplasmacytic cells in the BM should

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be diagnosed with WM (asymptomatic or symptomatic) [4]. Patients diagnosed

with asymptomatic WM should be recognized and not treated because of the

stability of the disease for many years. In an analysis of 31 asymptomatic

patients, the median time to disease progression was 6.9 years [5].

Prognosis

In a study of 337 symptomatic WM patients, the median survival of patients was

6.4 years, and the median disease-specific survival was11.2 years [72]. Despite

indolent nature of the disease, WM prognosis shows wide variation, which makes

important to define prognostic factors. Cytopenias, high β2-microglobulin, low

albumin, serum IgM monoclonal protein, organomegaly and advanced age have

shown to be poor prognostic factors for WM patients [72-76]. International

Prognostic Scoring System (ISS-WM) is the most commonly used prognostic

system, which is based on 5 risk factors and the survival of patients at 5 years

[77]. Based on this system, high-risk WM patients were shown to have a 5-year

survival of 36%, whereas low-risk patients had a 5-year survival of 87%.

Diagnostic workup and differential diagnosis

Important points of workup for a patient with WM are summarized in Table 1.

Diseases that need to be considered for differential diagnosis are IgM-MM,

mantle cell lymphoma, marginal zone lymphoma (MZL), and other IgM-secreting

lymphomas.

Plasmacytic cell infiltration of BM and the presence of osteolytic lesions

differentiate IgM-MM from WM [1,5,69]. Renal insufficiency can be seen in IgM-

MM, whereas it is rare in WM. Some cytogenetic characteristics such as 13q

deletion, 11:14, or 4:14 translocations may also help to differentiate MM from

WM. In addition, MYD88 L265P mutation presents in over 90% of WM patients

but not MM patients [18-20]. This may also help to differentiate WM from not only

from MM but also from other B cell malignancies.

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Infiltration of BM by monomorphous, small medium lymphoid cells with irregular

nuclei may help to differentiate mantle cell lymphoma from WM. In addition to

the BM, involvement of extranodal sites and lymph nodes may be observed in

mantle cell lymphoma. Moreover, almost all cases present with t(11;14) (q13;q32)

[1,5,69].

Distinguishing WM from MZL can be difficult, particularly from splenic MZL

(SMZL). SMZL mostly involves the spleen and only rarely presents with

lymphadenopathy. It virtually always infiltrates the bone marrow with a

characteristic intertrabecular and intrasinusoidal pattern, which is not typical of

NMZL [78]. In immunophenotyping, some markers such as CD22 and CD11c are

overexpressed in patients with SMZL compared to patients with WM, whereas

CD25 is mostly seen in WM (88% vs 44%). Therefore, combination of CD25 and

CD22 may help to differentiate between WM and SMZL [69]. The CD103 Ag is

absent in WM patients, whereas it is present in 40% of SMZL patients. The most

common cytogenetic abnormality in WM patients is 6q deletion (30%-50% in

WM), whereas the most common abnormalities in SMZL are loss of 7q (19%)

along with +3q (19%) and +5q (10%). The use of MYD88 L265P may be

particularly helpful, as few if any patients with MZL demonstrate this mutation

whereas >90% of WM patients express this mutation [18].

Follicular lymphoma can be differentiated from WM with BM infiltration of small,

cleaved cells that are usually paratrabecular. In addition, rearrangement of Bcl-2

is the molecular hallmark in follicular lymphoma and seen in 70%-90% of the

cases [1,5,69].

Lastly, IgM myeloma may also share similar morphological features with WM,

including expression of CD20. Translocation involving chromosome 11 (t 11;14)

are common in IgM myeloma, but absent in WM [79].

Primary treatment options for WM

Despite the advances in the biology and treatment of WM, there is no standard of

care for the treatment of WM. Treatment decisions are based on the presence of

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symptoms, patient factors including age and functional status and disease factors

including presence of cytopenias, rate of disease progression, the level of IgM

protein, the presence of neuropathy, cryoglobulinemia, and hyperviscosity.

Current treatment options for WM include alkylating agents (e.g. chlorambucil,

and cyclophosphamide), nucleoseide analogue (fludarabine and cladribine), the

monoclonal antibody rituximab, and the proteasome inhibitor bortezomib [80-83].

Consensus-based response criteria for WM are found in table 2 [84].

Plasma exchange is a treatment technique used for hyperviscosity associated

with WM. Symptoms of hyperviscosity are generally present when IgM exceeds

4000mg/dL and includes epistaxis, gingival bleeding and visual changes due to

retinal hemorrhages. Plasma exchange is a temporary solution that is used for

short-term management of hyperviscosity until initiating systemic chemotherapy,

which treats the underlying cause of the hyperviscosity by reducing the IgM

[85,86].

Rituximab

One of the most widely available agents used for the treatment of WM is

rituximab, which is used either as a single agent or in combination regimes. It is a

chimeric monoclonal antibody that binds to the CD20 antigen on B-cell

lymphocytes. As a single agent it gives response rates of 35-48% [87-90], which

is inferior than response rates seen with combination regimens. In addition,

single agent rituximab can cause a “rituximab flare” phenomenon, which results

in a transient rise in IgM level producing symptoms of hyperviscosity and

requiring plasma exchange. This phenomenon is infrequently seen when

rituximab is used in combination with chemotherapy. Some of the combination

agents studied with rituximab include cyclophosphamide, bendamustine, and

bortezomib [55-57,91,92].

In a study by Dimopoulos et al, 72 patients with newly diagnosed WM received

rituximab, oral cyclophosphamide and dexamethasone. Overall response rate

(ORR), which includes minimal response or better, was 83%, including 7%

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complete response (CR), 67% partial remission (PR). The median time to

progression was 35 months with a median overall survival (OS) of 95 months.

Only 9% of patients experienced grade 3 or 4 neutropenia and none experienced

grade 3 or 4 thrombocytopenia [91,93].

A phase III trial conducted by the Study group Indolent Lymphomas (StiL)

included 549 patients with low grade lymphomas of which 162 were newly

diagnosed WM patients who were received 6 cycles of bendamustine and

rituximab (BR). Responding patients were randomized to rituximab every 2

months for 2 years or observation. In the 116 evaluable patients, ORR was 86%.

The results of the maintenance arm of this study remain to be reported. No

uncommon toxicities were observed during BR induction [94].

Maintenance therapy with rituximab is being used more commonly in patients

with WM. A retrospective study showed improvement in progression free and

overall survival among patients receiving maintenance rituximab when used at

375mg/m2 every 3 or 6 months over 2 years. A prospective study on

maintenance regimen of rituximab given every 2 months for 2 years is now being

studied [95].

Nucleoside analogues

Nucleoside analogs are conventional chemotherapeutic agents that inhibit DNA

synthesis through interfering with different steps in the process of DNA synthesis.

Fludarabine and cladribine are nucleoside analogues studied as single agent or

in combination with rituximab or cyclophosphamide.

A recent phase III study by Leblond et al showed the superiority of fludarabine

compared with chlorambucil in the treatment of WM. Three hundred and thirty

nine WM patients were included in this trial and the study showed no difference

in overall response rate between the two arms. However, the median PFS and

duration of response were significantly higher in the fludarabine arm then

chlorambucil with a median follow up of 36 months (PFS 36.3 vs 27.1 months

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p=0.012). Median overall survival was not reached in the fludarabine arm versus

69.8 months in the chlorambucil arm (p=0.014). Secondary malignancies were

more frequent in the chlorambucil arm with a 6-year cumulative incidence rate of

20.6% versus 3.7% in the fludarabine arm (p=0.001) [96]. This rate is

inconsistent with prior retrospective studies, which have shown higher rates of

secondary malignancies with fludarabine [97].

Adding rituximab to fludarabine can significantly enhance response without

added toxicity [98]. Combinations of fludarabine and rituximab or fludarabine,

cyclophosphamide and rituximab have shown response rates of 70-85%

[99,100].

New developments in WM

Many novel agents have promising results and are at various stages of study in

WM. These include everolimus, an mTOR inhibitor; perifosine, an AKT inhibitor;

enzastaurin, a PI3K/AKT inhibitor; panobinostat, a histone deacetylase inhibitor;

bortezomib, first in class of protasome inhibitor and carfilzomib, a new

irreversible proteasome inhibitor; pomalidomide, a new immunomodulatory

agent; and ibrutinib, a Bruton tyrosine kinase inhibitor [101,102].

Proteasome Inhibitors

Bortezomib is a reversible proteasome inhibitor that specifically acts on the 20S

of the proteasome. Blocking proteasomal degradation causes protein

accumulation, which can alter the normal progression of the cell cycle, which is

necessary for cell survival. Bortezomib in combination with rituximab with or

without dexamethasone is being commonly used in either newly diagnosed or

relapsed WM. The overall response rates with these combinations are more than

80%. Peripheral neuropathy is one of the adverse effects of bortezomib therapy,

which is less when bortezomib is given weekly instead of the twice-weekly

regimen [55-57].

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Carfilzomib, an irreversible proteasome inhibitor, was approved by the FDA in

July of 2012 for the treatment of patients for relapsed/refractory myeloma and is

also being studied in WM. Antitumor activity of carfilzomib was validated in vivo

by Sacco et al who demonstrated that carfilzomib targeted the chymotrypsin-like

(CT-L) activity of both constitutive-(c20S) and immuno-(i20S) proteasome. This

led to the induction of toxicity in primary WM cells, as well as in other IgM-

secreting lymphoma cells [103].

The results recently reported from the CaRD trial by Treon et al in 31 patients.

Eighty seven percent of patients were previously untreated. Symptomatic

patients received carfilzomib in combination with rituximab and dexamethasone,

and were eligible for maintenance therapy with all three agents if they had stable

disease or better. The overall response rates and major response rates were

81% and 65% with 9 very good partial responses, 11 partial responses, and 4

minor responses. With a median follow-up of 9 months, 20 patients are free of

disease progression. Most common toxicities which were hyperglycemia related

to steroids; asymptomatic elevation of lipase and hypersensitivity to rituximab. All

toxicities were reversible, and no grade ≥2 peripheral neuropathy was observed

[104].

Oprozomib, an oral proteasome inhibitor, is in clinical development for MM and

other hematologic malignancies. A phase 1b/2 trial of once-daily, modified-

release oprozomib tablets was studied in 24 patients with hematologic

malignances. Dose escalation of oprozomib is currently being implemented using

two dosing schedules: oprozomib was given at 2 different dosing schedules;

once-daily, modified-release tablets (150 mg/d), on either days 1, 2, 8, and 9

(QDx2) or on days 1 to 5 (QDx5), both on a 14-day cycle. From the data that Dr.

Kaufman presented at the EHA meeting 2013, 24 patients were enrolled in the

study (16 with MM; 8 with WM) in 7 cohorts. WM patients on the QDx2 schedule

have shown no response while patients on the QDx5 schedule have shown 80%

ORR. The MTD of oprozomib had not been reached for either schedule. Most of

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the adverse effects seen were gastrointestinal. Dose escalation will continue in

this study until the MTD of oprozomib is reached. This data

Everolimus

Everolimus (RAD001) is an inhibitor of MTORC1, a component of the Akt-MTOR

pathway, which regulates growth, and survival of lymphoplasmacytic cells in WM.

A phase II trial of single agent everolimus was studied in 50 patients with

relapsed/ refractory WM. The overall response rate was 70% with a PR of 40%

and MR of 30%. The most common adverse events were cytopenias. Pulmonary

toxicity, which is a serious adverse effect of everolimus, was seen in 10% of

patients [105]. Another phase I/II study explored the use of everolimus in

combination with rituximab and/or bortezomib in 23 relapsed/refractory patients.

From the 19 patients who were evaluable for response, 1 (5%) patient very good

partial response (VGPR) and 9 (47%) minimal response (MR), for an overall

response rate including MR of 10/19 (53%) in this relapsed/refractory population

[106]. A prospective, multicenter study of everolimus as primary therapy was

conducted in 33 WM patients. The best overall response rate utilizing consensus

criteria was 72.2% (2 Very Good Partial Responses, 18 Partial Responses, 4

Minor Responses, and 9 Stable Disease), and the major response rate (PR or

better) was 60.6%. However, discordance between serum IgM levels upon which

consensus criteria for response are based, and BM disease response were

common and complicated response assessment. At 6-month assessments, 10 of

22 (45.5%) patients for whom both serum IgM and BM assessments were

performed, discordance between serum IgM and BM disease involvement were

observed. Among these patients, 2 had no change, and 8 had increased bone

marrow disease involvement despite decreases in serum IgM levels. Grade ≥2

hematologic and non-hematologic toxicities possibly, probably or definitively

associated with everolimus included anemia (39.4%), thrombocytopenia (12%),

neutropenia (18.2%), hyperglycemia (9.1%), oral ulcerations (27.3%), and

pneumonitis (15%) with a median follow-up of 9 months (range 1-45 months), 6

patients remain on study [107].

17

Enzastaurin

Enzastaurin is an oral serine/threonine kinase inhibitor that targets the PKC and

PI3/AKT pathway. A phase II trial of enzastaurin was studied in 42 patients with

WM who were previously treated with 1 to 5 regimens. Oral enzastaurin was

given at 250 mg twice daily (500 mg total) after a single loading dose (day 1,

cycle 1) of 375 mg 3 times daily (1,125 mg total) for 8 cycles of 28 days each or

until progressive disease. The objective response rate was 38.1%, with 2

patients achieving PR and 14 achieving minor response. Grade 3 adverse events

seen were leukopenia in one patient and septic shock in another one [107].

These results warrant further investigation of enzastaurin for the treatment of

WM.

Perifosine

Perifosine is an Akt inhibitor that belongs to a class of lipid-related compounds

called alkylphospholipids. A phase II trial was conducted with perifosine and

included 37 patients with WM who were previously treated with 1 to 5 regimens.

Oral perifosine was given at 150 mg daily until progression. The objective

response rate (MR or better) was 35% with 11% achieving PR and 24%

achieving MR. Progressive disease was observed in only 11% of patients since

54% had stable disease. Median PFS was 12.6 months. The most common

adverse events were GI toxicities including diarrhea, nausea and vomiting [108].

Immunomodulatory Drugs

Thalidomide and lenalidomide are immunomodulatory agents that are studied in

combination with Rituximab in symptomatic WM patient [109,110]. A phase 2

study included 25 patients, 20 of whom were previously untreated. They received

thalidomide (200 mg for 2 weeks, then 400 mg for a total of 1 year) and

Rituximab. Overall and major response rate were 72% and 64%, respectively,

with complete response (n = 1), partial response (n = 15), and major response (n

= 2). Median time to progression for all evaluable patients was 34.8 months.

Peripheral neuropathy was the most common adverse event (grade ≥2 in 44%),

18

and therefore limits the use of thalidomide in WM [109]. Lenalidomide was also

poorly tolerated in WM patients. It was tested in combination with rituximab in 16

patients, 12 of whom were previously untreated. The overall response and major

response (<50% decrease in serum IgM) rates were 50% and 25%, respectively,

with a median time to progression of 18.9 months. Premature discontinuation of

lenalidomide therapy occurred in 14 of 16 (88%) patients, which was most

commonly due to an acute decrease in hematocrit in 13 of 16 patients, which

resulted in 4 patients requiring hospitalizations due to anemia complications. This

was attributable to lenalidomide and led to cessation of further enrollment on this

study [110].

Pomalidomide, a 3rd generation immunomodulatory agent, has demonstrated

efficacy, as a single agent and with dexamethasone for the treatment of

relapsed/refractory multiple myeloma and as a single agent for myelofibrosis. A

phase I trial was conducted on 6 patients with relapsed and/or refractory WM.

Patients received daily oral pomalidomide (28 day cycles), starting with a dose of

1 mg. The median number of prior lines of therapy was 1-4. No dose limiting

toxicities (DLTs) were encountered at the 1mg dose. At the 2 mg dose level, 2

patients experienced DLTs (grade 4 dizziness and grade 3 syncope in 1 and

grade 4 neutropenia in the second); a 3rd withdrew from the study after

experiencing mild fever, headaches and blurred vision, which resolved after

plasma exchange and discontinuing pomalidomide. Other grade 3-4 adverse

events have been neutropenia and infection. The best response to date for the 5

evaluable patients was stable disease in 3 patients, and progressive disease in 2

[111]. A study looking into the combination of pomalidomide, dexamethasone and

rituximab was reported recently. Patients were enrolled on a dose escalating

Phase 1 study (3+3 design). Following amendment, dose level one was rolled

back to 0.5 mg daily, with intended escalation to 1, 2, 4 mg daily. Therapy

consisted of 52 weeks of daily POM with rituximab (375 mg/m2/wk) IV and

dexamethasone (40 mg) IV on weeks 1-4 and 12-15. Patients were required to

be on aspirin (325 mg) prophylaxis. Patients with IgM >4,000 mg/dL had

prophylactic pheresis to avoid symptomatic rituximab- related IgM flare. 7

19

patients were enrolled (3 at 0.5 mg, 4 at 1.0 mg of POM) before closure for

rituximab-related IgM flare events. The best overall response rate is 42.8% (1

VGPR; 2 PR). Grade ≥2 treatment related toxicities include neutropenia (43%),

symptomatic hyperviscosity from IgM flare with 3 patients (43%) all of whom had

a baseline IgM >4,000 requiring emergent pheresis for IgM flaring while on

therapy. Median response duration was 15.1 months, and all 3 patients continue

to respond [112].

Histone-Deacetylase Inhibitors (HDACi)

Preclinical studies have shown that epigenetic regulators, including histone

acetylation, regulate tumor progression. In vitro studies have confirmed the

antitumor activity of panobinostat (HDACi) in primary tumor cells and cell lines

[113] A phase II trial of panobinostat was studied in 36 patients with relapsed or

relapsed/refractory WM. Patients received oral panobinostat at 30 mg 3 times a

week. Overall response rate (MR or better) was achieved in 17 (47%) of patients,

with 22% partial remissions and 25% MR. In addition, 18 (50%) patients

achieved stable disease and none showed progression while on therapy. The

median progression-free survival was 6.6 months. Grade 3 and 4 toxicities

included thrombocytopenia, neutropenia, anemia, leukopenia, and fatigue [114].

Further studies are under way to better define the efficacy of HDAC inhibitors in

WM.

Bruton Tyrosine Kinase (BtK) inhibitor

Bruton’s tyrosine kinase (BtK) is an essential element of the BCR signaling

pathway, which regulates apoptosis, adhesion, and cell migration and homing.

BTK activation is facilitated by MYD88 pathway signaling in L265P expressing

WM cells, and participates in MYD88 downstream signaling. Ibrutinib (previously

PCI-32765) is an oral agent designed to specifically target and selectively inhibit

the enzyme Bruton's tyrosine kinase (BTK) leading to tumor cell killing of MYD88

L265P expressing WM cells [115].

20

A phase I study of ibrutinib was conducted in 56 patients with a variety of B-cell

malignancies including chronic lymphocytic leukemia/small lymphocytic

lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, follicular

lymphoma, Waldenstrom's macroglobulinemia and multiple myeloma. Objective

response rate in 50 evaluable patients was 60%, including complete response of

16%. Median progression-free survival in all patients was 13.6 months. Partial

responses were seen in 3 of the 4 patients with WM and the drug was well

tolerated [116].

63 patients including 17 with refractory disease were enrolled on a dedicated

ongoing phase II trial (ClinicalTrials.gov Identifier: NCT01614821) in patients with

relapsed/refractory WM disease. Patients had a median of 2 prior therapies,

hemoglobin of 10.5 g/dL, serum IgM of 3,610 mg/dL, and a median BM disease

involvement of 65%. At best response, median serum IgM level declined to

1,340 mg/dL, and median hemoglobin rose to 12.6 g/dL. With a median follow-up

of 6 (range 2-15 cycles), the best overall response rate i.e. minor response (MR)

or better using consensus criteria adapted from the 3rd International Workshop

on WM is 81% (4 VGPR; 32 PR, 15 MR), with a major response rate (PR or

better) of 57.1% and a median time to response of 4 weeks. 11 patients have

stable disease, and 1 patient was a non-responder. The most common grade >2

treatment related toxicities were thrombocytopenia (14.3%); neutropenia

(19.1%); stomatitis (1.6%); atrial fibrillation (1.6%); diarrhea (1.6%); herpes

zoster (1.6%); hematoma (1.6%); and epistaxis (1.6%). Attainment of major

responses was impacted by mutations in CXCR4. The major response rate was

77% for patients with wild-type CXCR4 vs. 30% in those with WHIM-like CXCR4

mutations.

Conclusion

Waldenstrom macroglobulinemia is a distinct lymphoproliferative disorder with

specific epigenetic/genetic aberrations, and characterized by production IgM

monoclonal secretion and lymphoplasmacytic cells in the BM. With the change in

understanding the pathogenesis of WM, many new targets and therapies have

21

emerged within the past couple of years. Current therapies used in the upfront or

relapsed setting include alkylating agents, bendamustine, nucleoside analogues,

bortezomib and the monoclonal antibody rituximab. Newer options include

everolimus and ibrutinib. However, targeted therapies are still warranted and

recent revelations from whole genome sequencing have provided opportunities

for their development.

Expert commentary & five-year view

To date there are no FDA approved therapeutic agents for treatment of WM.

Recently, whole genome sequencing helped us identify specific mutations in

subgroups of patients with WM (eg. MYD88 L265P mutation which is present in

over 90% of WM) which provides the framework for the investigation of BTK

inhibitors like ibrutinib. These newer agents show improved responses and lower

long term toxicities. In addition, many of the newer agents are orally administered

which makes them more convenient for patients. Some of the challenges for the

future of WM include combining of agents to achieve higher response rates, and

prolonged survival for patients, but with less toxicity.

Key Issues

Recent findings demonstrated that MYD88/IRAK signaling pathway might

have a critical role in pathogenesis of WM.

Somatic mutations in MYD88 are found in over 90% of WM patients,

which may help to differentiate WM from other B cell malignancies.

22

Current research efforts in inhibtion of BTK and IRAK signaling might

provide better treatment strategy for WM.

Many treatment options are available for WM patients and some of these

include rituximab, bortezomib, cyclophosphamide, and nucleoside

analogues fludarabine and cladribine which are less commonly used.

Novel agents under study are showing promising results as a single agent

or in combination. Some of these include everolimus, ibrutinib, as well as

the newer proteasome inhibitors carfilzomib and oprozomib.

There is a need for studies with PFS and OS as endpoints combined with

quality of life assessment as well as randomized phase III studies to

evaluate the benefit of different treatments for patients with WM.

23

Table : Workup for WM

Table 2: Response Definitions

Response Category Definition

Complete response (CR) Absence of serum monoclonal IgM protein by

immunofixation

Normal serum IgM level

Complete resolution of extramedullary disease*

Morphologically normal bone marrow aspirate

Very good partial response (VGPR) Monoclonal IgM protein is detectable

≥90% reduction in serum IgM level from baseline

24

Complete resolution of extramedullary disease*

No new signs or symptoms of active disease

Partial response (PR) Monoclonal IgM protein is detectable

≥50% but<90% reduction in serum IgM level from baseline

Reduction in extramedullary disease*

No new signs or symptoms of active disease

Minor response (MR) Monoclonal IgM protein is detectable

≥25% but<50% reduction in serum IgM level from baseline

No new signs or symptoms of active disease

Stable disease (SD) Monoclonal IgM protein is detectable

<25% reduction and <25% increase in serum

IgM level from baseline

No progression in extramedullary disease*

No new signs or symptoms of active disease

Progressive disease (PD) ≥25% increase in serum IgM level from

lowest nadir (requires confirmation) and/or

progression in clinical features attributable to the disease

*i.e. lymphadenopathy and splenomegaly

25

Financial and competing interests disclosure

IM Ghobrial is on the advisory board for Onyx, BMS and Celgene and receives research lab support from Genzyme and BMS. SP Treon receives research support from honoraria, and consulting fees from pharmacyclics, janssen pharmacuticals, and onyx pharmaceuticals. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

26

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115. Yang G XL, Zhou Y, Et Al. Participation of BTK in MYD88 signaling in malignant cells expressing the L265P mutation in Waldenstrom's macroglobulinemia, and effect on tumor cell with BTK-inhibitor PCI-32765 in combination with MYD88 pathway inhibitors [abstract]. J Clin Oncol suppl;abstract 8106(2012).

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• Phase I study of ibrutinib, BKT inihbitor, showed activity in patients with relapsed/refractory B-cell malignancies including Waldenström's macroglobulinemia.

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