Adopted from Hunter, Yang, Xu, Liu, Castillo, and Treon. Bing Center for Waldenstrom’s Macroglobuliemia, Journal of Clinical Oncology 2016

Waldenström’s macroglobulinemia (WM) is a distinct B-cell malignancythat results from the accumulation, predominantly in the bone marrow, of clonally related B type lymphocytes, lymphoplasmacytic cells and plasma cells which secrete a monoclonal IgM protein.¹ This condition is considered to correspond to the lymphoplasmacytic lymphoma (LPL) as defined by the World Health Organization classification system.² Most cases of LPL are WM, with less than 5% of cases made up of IgA, IgG and non-secreting LPL

Predisposition

WM is an uncommon disease, with a reported age-adjusted incidence rate of 3.4 per million among males and 1.7 per million among females in the USA, and a geometrical increase with age.³ The incidence rate for WM is higher among Caucasians, with African descendants representing only 5% of all patients. The incidence of WM may be higher for individuals of Askenazi Jewish decent.⁴ Genetic factors appear to be an important to the pathogenesis of WM. A common predisposition for WM with other malignancies has been raised,^4,5 with numerous reports of familiar clustering of individuals with WM alone, and with other B-cell derived malignancies.^6-9 In a study from the Bing Center for WM, 26% of 924 consecutive patients with WM had a first or second degree relative with either WM or another B-cell malignancy.⁴ Several rare inherited gene variants as well as the over-expression of the BCL2 protein (a protein which blocks cell death) have been proposed as predisposition events, however further confirmation and characterization of these findings is needed.^9,10 The presence of familial WM predispositionwas associated with inferior treatment response and progression free survival with the notable exception of regimens containing the proteasome inhibitor bortezomib.¹¹ An increased risk of death associated with familial versus sporadic WM has also been reported in a Swedish registry study.¹² While further confirmatory studies are needed to establish the importance of familial predisposition in WM, the data support the collection of familial history in WM patients.

Cytogenetics

Chromosome 6q deletions encompassing 6q21-25 have been observed in up to half of WM patients, and at a comparable frequency amongst patients with and without a familial history.^6,13-15 The presence of 6q deletions have been suggested to distinguish WM patients from those with the precursor condition IgM monoclonal gammopathy of unknown significance (MGUS) and to serve as a prognostic marker, though the latter remains controversial.^13-15 Gains in chromosome 6p are frequently present in cases where chromosome 6q is deleted.^13,14 Other chromosome abnormalities detected by cytogenetic or FISH analyses include deletions in 13q14, 17p, and 11q, trisomy 4, 12, and 18.^16,17 IgH rearrangements (where translocations cause genes to fuse to the IgH locus that codes for IgM production) are uncommon in WM, and may be helpful in discerning cases of WM from IgM myeloma wherein IgH switch region rearrangements are a prominent feature.¹⁸

Somatic Mutations

Next generation sequencing studies have identified highly recurrent mutations in MYD88, CXCR4, ARID1A, and CD79, and other genes, as well as copy number alterations effecting important regulatory genes in chromosome 6q, and elsewhere (Figure 1). Transcriptional changes (i.e. coding of RNA that directs cell growth and survival), disease presentation, therapeutic outcome, and overall survival are impacted by mutations in MYD88 and/or CXCR4.

Figure 1. Summary of genome and transcriptome findings in Waldenstrom’s Macroglobulinemia, and relevance to clinical presentation and treatment outcome.

MYD88

A highly recurrent mutation in MYD88 (MYD88 L265P) was first identified in WM patients by the Bing Center using paired tumor cell/normal cell whole genome sequencing, and subsequently confirmed by multiple groups using Sanger sequencing and allele specific polymerase chain reaction assays.^19-24 MYD88 L265P is expressed in up to 90-100% of WM cases when allele-specific PCR has been employed using both CD19 sorted WM cells and unsorted bone marrow cells.^21-24 Non-L265P MYD88 mutations have also rarely been identified in WM patients, including S219C, M232T, and S243N, all of which have been observed in other MYD88 mutated B-cell malignancies.²⁵ By comparison, MYD88 mutations are absent or expressed in low frequency in other B-cell malignancies that share similar morphological and clinicopathological features as WM, including IgM secreting myeloma (0%), marginal zone lymphoma (6-10%), and chronic lymphocytic leukemia (3-8%) thereby enabling molecular discrimination.²⁰ The presence of mutated MYD88 in cerebrospinal fluid (fluid that bathes the brain and spinal column), as well pleuritic fluid (fluid that bathes the lining of the lungs) in WM patients has also permitted diagnostic and treatment implementation in patients with symptomatic disease outside of the bone marrow and lymph nodes.^26,27 Structural chromosome events causing duplication of DNA material are also known to occur on chromosome 3p(where MYD88 resides) that result in added mutated MYD88 gene presence in 12-13% of untreated patients, and up to 25% of previously treated patients, and occurmore often in previously treated patients with CXCR4 mutations.^20,28-30 The mechanisms and clinical significance of these structural changes remains to be determined, though the presence of increased mutated MYD88 expression due to structural alterations may oddly confer a favorable treatment outcome in patients undergoing ibrutinib therapy.³⁰

MYD88 mutations encompass the entire WM clone, and are detectable in 50-80% of IgM but not IgG or IgA MGUS suggesting an early oncogenic role for WM pathogenesis.^20-22 IgM MGUS patients with mutated MYD88 appear to be at higher risk of progression to WM.³¹ The presence or absence of MYD88 mutations also appears to distinguish two distinct populations of WM patients. Patients lacking MYD88 mutations show similar disease morphology (i.e. WM cells look similar under the microscope) to MYD88 mutated patients but they present with significantly lower bone marrow (BM) disease involvement and serum IgM levels.³² Despite the presentation of MYD88 wild-type (WT) WM patients with lower BM disease burden and serum IgM levels, overall survival may be shorter for these patients with a ten-fold increased risk of death versus MYD88 mutated WM patients.³²

MYD88 is an adaptor protein that normally interacts with the Toll like receptor (TLR) whose function is to sense for “infection” and IL1 receptor families whose function is to detect inflammation. Upon receptor activation, normal MYD88 undergoes homodimerization (binding to other MYD88 molecules). Thehomodimerizationof MYD88 acts as a scaffold for recruitment of other proteins resulting in the assembly of a “Myddosome” that can trigger downstream signaling leading to activation of nuclear factor kappa-B (NFkB), powerful stimulant of growth and survival of WM cells.³³ Mutations in MYD88 were first described in activated B-cell (ABC) subtyped diffuse large B-cell lymphoma (DLBCL), and were shown to trigger constitutive MYD88 homodimeriztionand NFkB activation through the IRAK1/IRAK4 kinase signaling proteins.³⁴ In WM, NFkB is activated through IRAK1/IRAK4 as well as by Bruton’s Tyrosine Kinase (BTK), a target of ibrutinib, which triggers NFkB independently of IRAK4/IRAK1 (Figure 2).^{20,23,24,34,35} Recruitment and activation of IRAK1/IRAK4, as well as BTK can be blocked through either genetic knockdown of the MYD88 gene, use or expressionof peptides (mini proteins) that block MYD88 homodimerization and induce cell death of MYD88 mutated WM cells.^20,23,35,36 Mutated MYD88 can also transactivate Hematopoietic Cell Kinase (HCK), a protein that is a member of the SRC family that is activated by interleukin-6 that is elevated in the serum of WM patients. IL6 is also triggered by mutated MYD88 (Figure 2).³⁷ Activated HCK contributes to the growth and survival signaling of mutated WM cells through multiple growth and survival pathways that include BTK, PI3K/AKT and MAPK/ERK1/2 signaling.³⁷

Figure 2. Mutated MYD88 related signaling in Waldenstrom’s Macroglobulinemia. Mutated MYD88 transactivates NFkB through divergent pathways that include IRAK1/IRAK4 and BTK.³⁵ Mutated MYD88 also triggers transcription and activation of the SRC family member HCK. Activated HCK can then trigger BTK, AKT, and ERK1/2 mediated pro-growth and survival signaling in WM cells.³⁷

CXCR4

Somatic activating mutations in the C-terminal domain of the CXCR4 gene are also present in up to 40% of WM patients, and are nearly always observed in conjunction with MYD88 mutations in WM patients.^28,32,38 CXCR4 mutations are essentially unique to WM, as they have not been described so far in other diseases with the exception of a few MZL cases.^29,39,40 Inherited (germline) mutations that closely resemble those found in WM cells are also present in patients with WHIM (autosomal dominant warts, hypogammaglobulinemia, infection and myelokathexis) syndrome.^41,42 In patients with WHIM syndrome, activation of CXCR4 by its ligand CXCL12 causes extended chemotactic signaling that results in sequestration of neutrophils in the bone marrow (myelokathexis) and impaired lymphocyte development.⁴² In WM, over 30 different nonsense (mutations that result in part of the protein not being coded) and frame shift mutations (mutations that cause part of the protein to be scrambled) in the C-terminal domain of CXCR4 have been described.^32,38,39,40 Mutations in the C-terminal domain of CXCR4 result in loss of regulatory serine amino acids, which undergo phosphorylation (activation) following CXCR4 receptor activationby CXCL12, a protein that is released by other bone marrow cells and triggers CXCR4.⁴³

Unlike MYD88, the number of cells that are mutated within the WM clone is highly variable, and multiple CXCR4 mutations can be present within individual patients that reside in separate clones or are present with a cell (compound heterozygous events).^38,39 The subclonal nature of CXCR4 mutations relative to MYD88 suggests that these mutations occur after MYD88, though could occur early in WM pathogenesis given their detection in IgM MGUS patients.^39,40 Like MYD88, the presence of CXCR4 mutations can impact disease presentation in WM. Patients with CXCR4 mutations present with a significantly lower rate of lymph node enlargement, and those with CXCR4 nonsense mutations have an increased bone marrow disease burden, serum IgM levels, and/or risk of symptomatic hyperviscosity.^32,38,40 Despite differences in clinical presentation, CXCR4 mutations do not appear to adversely impact overall survival in WM.^32,38

Using in vitro models, WM cells engineered to express mutated CXCR4 showed increased drug resistance in the presence of CXCL12 to multiple therapeutics including bendamustine, fludarabine, bortezomib, idelalisib, and ibrutinib.^44-46 The above studies also showed that CXCL12 mediated drug resistance in mutant CXCR4 transduced WM cells could be reversed by use of CXCR4 blocking agents.

ARID1A

Somatic mutations in ARID1A are present in 17% of WM patients, including single-nucleotide variants leading to premature protein truncation and frameshift changes. Patients with both ARID1A and MYD88 L265P mutations, as compared with patients who did not have ARID1A mutations, had greater bone marrow disease involvement, and lower hemoglobin and platelet count. ARID1A and its frequently deleted homologue ARID1B (discussed below) are found on chromosome 6q are members of the switch/sucrose non-fermentable (SWI/SNF) family. The SWI/SNF family members regulate chromatin remodeling, and can modulate gene regulation. While still poorly understood in the context of hematological malignancies, ARID1A can modulate TP53, and is thought to act as a epigenetic tumor suppressor in ovarian cancer wherein mutations in ARID1A have been more thoroughly evaluated.^47,48

CD79A/CD79B

CD79A and CD79B are components of the B-cell receptor (BCR) pathway. CD79A plays diverse roles in B-cell ontogeny, and forms a heterodimer with CD79B. The CD79A/B complex associates with the immunoglobulin heavy chain which is required for cell surface expression of BCR, and BCR induced signaling.⁴⁹ Activating mutations in the immunotyrosine-based activation motif (ITAM) part of CD79A and CD79B have been reported in the ABC subtype of DLBCL, and activate BCR growth and survival signaling through a cascade that includes SYK, PLCy2, and BTK.³⁴ Dual MYD88 and CD79B mutations occur in ABC DLBCL, and are associated with ibrutinib response.⁵⁰ The role of BCR in triggering WM growth and survival signaling remains to be clarified, though aberrantly enhanced BCR signaling was observed in WM cells stimulated with BCR activating agents.⁵¹ Deletions of LYN that are found in 70% of WM patients could contribute to hyper-responsive BCR signaling as informed by lyn-/- transgenic mice.⁵² Mutations in both CD79A and CD79B have been observed in WM in 8-12% of patients, and though are mainly found in MYD88 mutated patients, a CD79B mutation was observed in a MYD88 wild-type WM patient.^28,38,53 In one study, CD79A and CD79B were nearly exclusive to CXCR4 mutations, suggesting that two distinct MYD88 mutated populations may exist with WM.³⁸ In a small series of WM patients, the presence of CD79B along with MYD88 mutations were associated with disease transformation.⁵⁴ The contribution of LYN deletions, as well as CD79A and CD79B mutations to aberrant BCR triggered growth and survival signaling, clinical presentation, disease transformation and treatment outcome remain to be more clearly defined in WM.

Other mutations

Other recurrent somatic mutations have been identified in MYBBP1A, TP53, MLL2, HIST1H1E, and HIST1H1B in WM patients.^28,53 MLL2 is a histone methyltransferase that methylates Lys-4 of histone H3 (H3K4me). MLL2 is a frequent target of somatic mutations in follicular lymphomas (89%) and diffuse large B-cell lymphomas (32%).^55,56 Mutations in MLL2 were identified in 2 of 3 WM patients with wild-type MYD88 that included a single-nucleotide variant, and a deletion resulting in a frameshift mutation. None of the 27 MYD88 mutated WM patients who underwent whole genome had MLL2 mutations.¹⁹

Copy number alterations

Copy number alterations of genes are common in WM patients, and impact genes with important regulatory functions in both chromosome 6q, as well as non-chromosome 6q regions (Figure 1).²⁸ In chromosome 6q, loss of genes that modulate NFkB activity (TNFAIP3, HIVEP2), BCL2 family of proteins (BCLAF1), apoptosis (FOXO3), BTK (IBTK), plasmacytic differentiation (PRDM1) and ARID1B are observed. Non-chromosome 6q genes that arecommonly deleted include ETV6, a transcription repressor; BTG1, thatoftenis deleted in DLBCL,and associated with glucocorticoid resistance in acute lymphocytic leukemia; as well as LYN, a kinase that plays a regulatory role for BCR signaling. PRDM2 and TOP1 that participate in TP53-related signalingare also frequently deleted in WM patients.²⁸

Gene Expression Profiling

Earlier gene expression profiling studies that reflect levels of RNA coding showed over-expression of IL6, as well as a gene profile that closely resembled chronic lymphocytic leukemia (CLL).^57-59 More recently, next generation transcriptome studies (RNASeq) have permitted analysis of gene expression in context of gene mutations (Figure 3). Comparison of WM cells derived from the BM of patients with WM with healthy donor (HD) B-cells showed increased expression of genes involved in how DNA is spliced and repaired such as DNTT, RAG1, and RAG2, as well as members of the CXCR4 pathway genes CXCL12, VCAM1, and CXCR4 itself.⁶⁰ These latter findings may indicate a role for CXCR4 signaling regardless of CXCR4 mutation status in WM. Dysregulation of BCL2 family members that function as blockers of cell death, including up-regulation of BCL2 and BCL2L1 was also observed. Among MYD88 mutated patients, those with CXCR4 mutations showed silencing of RNA expression for tumor suppressor genes associated with acquisition of mutated MYD88 including WNK2, TP53I11, PRDM5 and CABLES1. Based both on expression and pathway analysis, modulation of MYD88 signaling in context of CXCR4 mutations was associated with the down-regulation of TLR4, and increased transcription of the IRAK4/IRAK1 inhibitor, IRAK3. WM cells derived from patients with MYD88 WT, as well asmutated CXCR4 show impaired B-cell differentiation signaling versus those derived from MYD88 mutated CXCR4 WT patients. Moreover, BM disease involvement was impacted by transcriptional activity of MYD88, CXCR4, as well as CXCL13.⁶⁰ Serum CXCL13 levels were found to impact BM disease involvement and hemoglobin levels in an independent cohortof WM patients supporting the latter finding.⁶¹

Figure 3. Summary of findings for gene losses in Waldenstrom’s Macroglobulinemia by chromosome location.

Genomic based treatment approach to WM

Ibrutinib was recently approved by the U.S. Food and Drug Administration and the European Medicines Agency for the treatment of WM, and was adopted into National Comprehensive Cancer Network (NCCN) guidelines (www.nccn.org) and WM Consensus Guidelines for the treatment of symptomatic WM patients.⁶² Patients with wild-type MYD88 showed absence of major responses (partial response or better) and inferior PFS to ibrutinib versus those patients with mutated MYD88 including non-L265P mutations.^25,63 Moreover among mutated MYD88 patients, the presence of CXCR4 mutations resulted in lower major (61.9% versus 91.7%) response rate versus wild-type CXCR4 patients. Major response attainment was also delayed among CXCR4 mutated patients that improved with prolonged (>6 months) therapy.⁶³ Delayed response attainment was also reported among CXCR4 mutated patients in another multicenter study that administered single agent ibrutinib to rituximab refractory WM patients.⁶⁴ Major response attainment was also adversely impacted by wild-type MYD88 and mutated CXCR4 mutation status among previously untreated patients who received single agent everolimus.⁶⁵ Among patients receiving treatment with carfilzomib, rituximab and dexamethasone (CaRD), no major response differences were observed between wild-type and mutated CXCR4 WM patients.⁶⁶ However, in an ongoing study of ixazomib, dexamathasone and rituximab (IDR) delays in response were observed among CXCR4 mutated patients.⁶⁷

While treatment choice should take into account specific goals of therapy, necessity for rapid disease control, risk of treatment-related neuropathy, immunosuppression and secondary malignancies, and planning for future autologous stem cell transplantation, MYD88 and CXCR4 mutation status may be useful in treatment selection for symptomatic patients. A guide recommended by the authors for the use of MYD88 and CXCR4 mutation status in the treatment approach of symptomatic untreated and previously treated patients are presented in Figures 4,5.

Figure 4. Author recommended guide to the use of MYD88 and CXCR4 mutation status in the management of symptomatic, previously untreated patients with Waldenstrom’s Macroglobulinemia. If symptomatic hyperviscosity, severe cryoglobulinemia, cold agglutinemia, or rapidly progressing moderate to severe IgM demyelinating peripheral neuropathy, plasmapheresis should be considered, then systemic therapy. If not, proceed to systemic therapy.BDR, bortezomib, dexamethasone, rituximab; Benda-R, bendamustine, rituximab. For patients selected to receive rituximab, consider giving chemotherapy alone until IgM^61,70

Figure 5. Author recommended guide to the use of MYD88 and CXCR4 mutation status in the management of symptomatic, previously treated patients with Waldenstrom’s Macroglobulinemia. If symptomatic hyperviscosity, severe cryoglobulinemia, cold agglutinemia, or rapidly progressing moderate to severe IgM demyelinating peripheral neuropathy, plasmapheresis should be considered, then systemic therapy. If not, proceed to systemic therapy. BDR, bortezomib, dexamethasone, rituximab; Benda-R, bendamustine, rituximab. For patients selected to receive rituximab, consider giving chemotherapy alone until IgM2 prior therapies, nucleoside analogues in non-autologous transplant candidates, and autologous transplant can be considered in patients with multiple relapses and chemosenstitive disease.^62,71

Novel Targets

Investigational therapies under development for WM include agents that target MYD88, CXCR4, and BCL2 signaling. IRAK1/IRAK4 kinases mediate mutated MYD88 directed NFkB signaling, and their inhibition triggers apoptosis in mutated MYD88 expressing malignant cells.^28,34,35 Moreover, combined BTK and IRAK1/4 inhibition induces synergistic killing of MYD88L265P malignant cells.³⁵ Compounds that inhibit IRAK signaling are under intense preclinical investigation for use in MYD88 mutated diseases. HCK is a SRC family member that is transactivated by mutated MYD88, and along with BTK is a target of ibrutinib.³⁷ Ibrutinib partially attenuates HCK activity in MYD88 mutated WM and ABC DLBCL cells, and the use of a more potent toolbox HCK inhibitor triggered higher levels of apoptosis in MYD88 mutated WM cell lines and primary cells. In pre-clinical studies, the CXCR4 antagonists plerixafor and ulocuplomab blocked CXCL12 rescue of apoptosis mediated by ibrutinib, idelalisib, and other therapeutics.^44-46 Delayed responses and lower major response rates were also observed among CXCR4 mutated patients on ibrutinib.^63,64 A clinical trial examining ibrutinib with ulocuplomab in symptomatic CXCR4 mutated WM patients is being planned. The anti-apoptotic factor BCL-2 is over-expressed in WM cells including those derived from MYD88 wild-type and mutated patients.^58,60 The BCL-2 inhibitor venetoclax induces apoptosis, and shows at least additive anti-apoptotic activity against WM cells co-treated with either ibrutinib or idelalisib, regardless of CXCR4 mutation status.⁶⁸ In a prospective clinical study that included multiple B-cell malignant histologies, 3 of 4 WM previously treated WM patients responded, that included one CR.⁶⁹ A dedicated clinical trial examining the activity of venetoclax in previously treated WM patients is underway (NCT02677324). The presence of ARID1A, HIST1H1B and HIST1H1E mutations, along with recurrent ARID1B deletions suggests that epigenetic dysregulationis likely to be present in WM, and further investigation is therefore warranted. EZH2 inhibitors may be a particularly effective in the context of ARID1A mutations in WM and preclinical evaluation of such strategies should also be considered.⁷⁰

Summary

Next generation sequencing has revealed recurring somatic mutations in WM that include MYD88, CXCR4, ARID1A, CD79B, as well as loss of genes with important regulatory functions. Diagnostic discrimination of WM from overlapping B-cell malignancies is aided by MYD88 mutation status, while disease presentation and treatment outcome is impacted by both MYD88 and CXCR4 mutation status. MYD88 and CXCR4 mutation status may be helpful in treatment selection for symptomatic patients. Novel therapeutic approaches under investigation include therapeutics targeting MYD88, CXCR4 and BCL2 signaling.

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