The n e w e ng l a n d j o u r na l of
original article
From the Dana–Farber Cancer Institute
(S.P.T., L.X., G.Y., Y.Z., X.L., Y.C., P.S.,
R.J.M., C.J.P., C.T., Z.R.H.), Brigham and
Women’s Hospital (G.S.P., S.J.R.), and
Massachusetts General Hospital (A.R.S.,
N.L.H.), Harvard Medical School; and the
Department of Pathology and Laboratory
Medicine, Boston University School of
Medicine (Z.R.H.) — all in Boston; the
Department of Hematology Oncology,
University of Pavia Medical School and
Fondazione Istituto Di Ricovero e Cura a
Carattere Scientifico (IRCCS) Policlinico
San Matteo, Pavia, Italy (L.A.); and Com-
plete Genomics, Mountain View, CA
(J.M.L., D.A.S., S.E.L.). Address reprint
requests to Dr. Treon at the Bing Center
for Waldenström’s Macroglobulinemia,
Dana–Farber Cancer Institute, M547, 450
Brookline Ave., Boston, MA 02115, or at
steven_treon@dfci.harvard.edu.
N Engl J Med 2012;367:826-33.
DOI: 10.1056/NEJMoa1200710
Copyright © 2012 Massachusetts Medical Society.
826
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m e dic i n e
MYD88 L265P Somatic Mutation
in Waldenström’s Macroglobulinemia
Steven P. Treon, M.D., Ph.D., Lian Xu, M.S., Guang Yang, Ph.D.,
Yangsheng Zhou, M.D., Ph.D., Xia Liu, M.D., Yang Cao, M.D.,
Patricia Sheehy, N.P., Robert J. Manning, B.S., Christopher J. Patterson, M.A.,
Christina Tripsas, M.A., Luca Arcaini, M.D., Geraldine S. Pinkus, M.D.,
Scott J. Rodig, M.D., Ph.D., Aliyah R. Sohani, M.D., Nancy Lee Harris, M.D.,
Jason M. Laramie, Ph.D., Donald A. Skifter, Ph.D., Stephen E. Lincoln, Ph.D.,
and Zachary R. Hunter, M.A.
A bs t r ac t
Background
Waldenström’s macroglobulinemia is an incurable, IgM-secreting lymphoplasmacytic
lymphoma (LPL). The underlying mutation in this disorder has not been delineated.
Methods
We performed whole-genome sequencing of bone marrow LPL cells in 30 patients with
Waldenström’s macroglobulinemia, with paired normal-tissue and tumor-tissue
sequencing in 10 patients. Sanger sequencing was used to validate the findings in
samples from an expanded cohort of patients with LPL, those with other B-cell
disorders that have some of the same features as LPL, and healthy donors.
Results
Among the patients with Waldenström’s macroglobulinemia, a somatic variant
(T→C) in LPL cells was identified at position 38182641 at 3p22.2 in the samples
from all 10 patients with paired tissue samples and in 17 of 20 samples from pa-
tients with unpaired samples. This variant predicted an amino acid change (L265P)
in MYD88, a mutation that triggers IRAK-mediated NF-κB signaling. Sanger se-
quencing identified MYD88 L265P in tumor samples from 49 of 54 patients with
Waldenström’s macroglobulinemia and in 3 of 3 patients with non–IgM-secreting
LPL (91% of all patients with LPL). MYD88 L265P was absent in paired normal tissue
samples from patients with Waldenström’s macroglobulinemia or non-IgM LPL and
in B cells from healthy donors and was absent or rarely expressed in samples from
patients with multiple myeloma, marginal-zone lymphoma, or IgM monoclonal gam-
mopathy of unknown significance. Inhibition of MYD88 signaling reduced IκBα and
NF-κB p65 phosphorylation, as well as NF-κB nuclear staining, in Waldenström’s
macroglobulinemia cells expressing MYD88 L265P. Somatic variants in ARID1A in
5 of 30 patients (17%), leading to a premature stop or frameshift, were also identified
and were associated with an increased disease burden. In addition, 2 of 3 patients
with Waldenström’s macroglobulinemia who had wild-type MYD88 had somatic
variants in MLL2.
Conclusions
MYD88 L265P is a commonly recurring mutation in patients with Waldenström’s
macroglobulinemia that can be useful in differentiating Waldenström’s macro-
globulinemia and non-IgM LPL from B-cell disorders that have some of the same
features. (Funded by the Peter and Helen Bing Foundation and others.)
n engl j med 367;9  nejm.org  august 30, 2012
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Copyright © 2012 Massachusetts Medical Society. All rights reserved.
 MYD88 L265P Mutation in Waldenström’s Macroglobulinemia
W
aldenström’s macroglobulin-
emia is an IgM-secreting lymphoplas-
macytic lymphoma (LPL).1,2 Clinical
manifestations of Waldenström’s macroglobu-
linemia include cytopenia resulting from bone
marrow infiltration by lymphoplasmacytic cells,
paraprotein-related cryoglobulinemia, the cold
agglutinin syndrome, demyelinating neuropathy,
and symptomatic hyperviscosity.3 The oncogenic
basis of Waldenström’s macroglobulinemia has
not been defined. Familial clustering of Walden-
ström’s macroglobulinemia and other B-cell dis-
orders suggests that genetic factors play a role in
the pathogenesis of Waldenström’s macroglobu-
linemia in certain patients.4-6 IgM monoclonal
gammopathy of unknown significance (MGUS)
is characterized by the presence of a monoclonal
IgM protein and the absence of bone marrow dis-
ease involvement on histologic examination.1
IgM MGUS can progress to Waldenström’s mac-
roglobulinemia or other B-cell lymphoprolifera-
tive disorders, with an estimated probability of
progression of 1.5 to 3.0% per year.7-9 The onco-
genic events responsible for the progression of
IgM MGUS to Waldenström’s macroglobulin-
emia are unknown. Knowledge of the genetic
events responsible for the pathogenesis of
Waldenström’s macroglobulinemia may permit
advancements in diagnostic testing and the de-
velopment of targeted therapies.
Me thods
Patients and Cell Samples
We performed whole-genome sequencing in 30 pa-
tients who met the diagnostic criteria for Walden-
ström’s macroglobulinemia.1 Their participation
was approved by the institutional review board at
the Dana–Farber Cancer Institute, and we have
applied for deposition of their whole-genome-
sequencing data in the database of Genotypes and
Phenotypes (dbGaP) of the National Center for
Biotechnology Information (NCBI). The first and
last authors vouch for the accuracy and complete-
ness of the data and analyses. All the participants
provided written informed consent. The charac-
teristics of the patients are summarized in Table
S1 in the Supplementary Appendix, available with
the full text of this article at NEJM.org. Two pa-
tients had a blood relative with Waldenström’s
macroglobulinemia, and 7 patients had a blood
relative with a different B-cell cancer. Bone mar-
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Copyright © 2012 Massachusetts Medical Society. All rights reserved.
row and peripheral-blood mononuclear cells
were sorted with the use of magnetic beads
(Miltenyi). The purity of isolated B cells (CD19+)
was more than 90%, and the median clonal B-cell
population, as assessed by light-chain-restriction
analysis, was 96%. CD19-depleted peripheral-blood
mononuclear cells were used as normal paired
samples.
Whole-Genome Sequencing
High-molecular-weight DNA was isolated with the
use of the AllPrep DNA/RNA Mini Kit (Qiagen).
Library construction and whole-genome sequenc-
ing of paired-end clones were performed by Com-
plete Genomics, as described previously.10-12 Read
sequences were aligned to reference genome
NCBI Build 37. The Complete Genomics Analysis
Tool (CGAT), version 1.3, was used to identify
high-confidence somatic variants as those with a
somatic score of 0.1, indicating an estimated one
false somatic single-nucleotide variant per 17.7 Mb
of DNA.13,14 The copy number was estimated on
the basis of the percent guanine–cytosine (GC)
normalized read-depth, and acquired uniparental
disomy was identified as copy-number–neutral loss
of heterozygosity. Allele imbalance was deter-
mined according to the percentage of reads that
mapped to the minor allele at heterozygous single-
nucleotide polymorphisms and was averaged over
500 Kb.
Validation by Sanger Sequencing
Polymerase-chain-reaction primers were designed
to amplify a 726-bp fragment covering myeloid dif-
ferentiation primary response gene (88) (MYD88)
L265P (forward primer, 5′-GGGATATGCTGA­A­C­
T­A AGTTGCCAC3′; reverse primer, 5′-GAC­GTG­
TC­TGTGAAGTTGGCATCTC3′). Amplified frag-
ments were isolated with the use of the QIAquick
Gel Extraction Kit (Qiagen) and were sequenced
with the use of the forward primer 5′GCTG­T­T­
GTTAACCCTGGGGTTGAAG3′ and the reverse
primer 5′-GACGTGTCTGTGAAGTTGGCA­TC­T­C3′.
Sanger sequencing was used to validate the re-
sults of whole-genome sequencing and to evalu-
ate MYD88 L265P expression in tumor samples
from 24 additional patients with Waldenström’s
macroglobulinemia, 3 patients with non-IgM LPL
(2 with IgG LPL and 1 with IgA LPL), 10 patients
with myeloma, and 46 patients with marginal-zone
lymphoma (21 with a splenic subtype, 20 with an
extranodal subtype, and 5 with a nodal subtype).
827
 The n e w e ng l a n d j o u r na l
CD19+ bone marrow mononuclear cells isolated
from 21 patients with IgM MGUS were also se-
quenced, with cloning and sequencing of at least
100 clones performed by Genewiz for 7 patients
with IgM MGUS in whom direct Sanger sequenc-
ing did not reveal MYD88 L265P. Samples with
sufficient DNA from 11 patients with IgM MGUS
were evaluated by means of an IgH chain-­
rearrangement assay (In Vivo Scribe Technolo-
gies). CD19+ peripheral-blood mononuclear cells
from 15 healthy donors and from BCWM.1,
MWCL-1, WM-WSU, Ramos, MM1.S, RPMI 8226,
MEC-1, and U266 cell lines were also sequenced
for MYD88 L265P.
Inhibitors of MYD88 Signaling
BCWM.1 and MWCL-1 Waldenström’s macro-
globulinemia cell lines expressing MYD88 L265P,
bone marrow cells from a patient with Wald­
enström’s macroglobulinemia, and Ramos,
MM1.S, RPMI 8226, and U266 cell lines ex-
pressing wild-type MYD88 were incubated over-
night with 100 μM of a control peptide, 100 μM
of an inhibitor of MYD88 homodimerization
(IMG-2005-5, ­IMGENEX), a dimethylsulfoxide
vehicle control, or 10 μM of a dual interleukin-1
receptor–associated kinase (IRAK) 1 and IRAK4
kinase inhibitor (407601, EMD).15,16
Western Blot Analysis
and Immunofluorescence Staining
Western blotting was performed with the use of
total protein and phosphospecific antibodies for
nuclear factor of kappa light polypeptide gene en-
hancer in B-cells inhibitor, alpha (IκBα) (Ser32)
(Cell Signaling), nuclear factor κB (NF-κB) p65
(Ser529) (BD Biosciences), and glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) (Santa Cruz
Biotechnology). We examined the nuclear and cy-
toplasmic expression of NF-κB p65 by means of
immunofluorescence staining,17 using a rabbit
polyclonal antihuman NF-κB p65 antibody (Cell
Signaling), followed by donkey antirabbit IgG-
DyLight 488 secondary antibody (Abcam).
Statistical Analysis
Categorical variables were compared with the use
of Fisher’s exact probability test, and ordinal vari-
ables with the use of the Mann–Whitney U test.
P values were adjusted for multiple comparisons
according to the method of Benjamini and Hoch-
berg. P values of 0.05 or less were considered to
828 n engl j med 367;9  nejm.org  august 30, 2012
The New England Journal of Medicine
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Copyright © 2012 Massachusetts Medical Society. All rights reserved.
of m e dic i n e
indicate statistical significance. Calculations were
performed with the use of the R statistical pack-
age (R Foundation for Statistical Computing).
R e sult s
Identification of MYD88 L265P as the Most
Common Somatic Alteration
Tumor and normal genomes were sequenced to
an average of 66-times coverage (range, 60 to 91)
of mapped individual reads. The average gross
mapped yield for these genomes was 186.89 Gb
(range, 171.56 to 262.03). In all 10 patients with
paired tissue samples, we identified a variant at
position 38182641 in chromosome 3p22.2 result-
ing in a single nucleotide change, T→C, in MYD88.
This change predicted a switch of leucine to pro-
line at amino acid position 265 (L265P). A medi-
an of 3419 somatic variants (range, 2540 to 4011)
were identified, and MYD88 L265P was the most
common. It was also expressed in 16 of 20 pa-
tients with Waldenström’s macroglobulinemia
who had unpaired samples. MYD88 L265P was
therefore expressed in 26 of the 30 patients in
whom whole-genome sequencing was performed.
The median percentage of reads supporting the
MYD88 L265P variant for these 26 patients was
46.3% (range, 23.4 to 95.7); this percentage may
have reflected the presence of polyclonal B cells
or contaminating non–CD19+ cells in the sam-
ples sequenced or, alternatively, the presence of
MYD88 L265P in subpopulations of clonal lym-
phoplasmacytic cells in some patients. In con-
trast, MYD88 L265P was not expressed in any
reads from paired normal tissue samples sub-
jected to whole-genome sequencing.
The frequency of the MYD88 L265P mutation
among patients with a positive family history
was 100% (9 of 9 patients), and the frequency
among patients with sporadic cases was 86% (18
of 21) (P = 0.60). In 22 of the 26 patients with
MYD88 L265P expression, the variant was hetero-
zygous, and in the other 4 patients, an acquired
uniparental disomy event (median, 49.5 MB
[range, 48.5 to 50.0]) at 3p22.2 resulted in ho-
mozygous MYD88 L265P expression in at least a
subset of tumor cells. These uniparental disomy
events were absent in paired normal samples.
No distinguishing clinical or laboratory features
were observed in patients who were homozygous
for MYD88 L265P, as compared with patients who
were heterozygous, though patients who were ho-
 MYD88 L265P Mutation in Waldenström’s Macroglobulinemia
mozygous for MYD88 L265P had a longer mean
interval since the diagnosis of Waldenström’s
macroglobulinemia (56.4 months vs. 11.1 months,
P = 0.21). Among the 4 patients who were homo-
zygous for MYD88 L265P, 2 had a positive family
history (accounting for 22% of all patients with
a positive family history), and 2 had sporadic
cases (accounting for 11% of all patients with
sporadic cases) (P = 0.84).
No recurring somatic variants were identified in
other genes encoding toll-like receptor or NF-κB
signaling pathways in the 10 patients with paired
samples. The next most common somatic vari-
ants, after MYD88, occurred in the AT-rich inter-
active domain 1A gene (ARID1A) and the histone
cluster 1, H1e gene (HIST1H1E), with variants
occurring in each of these genes in 2 of 10 pa-
tients with paired samples (20%). Two nonrecur-
ring single-nucleotide variants were identified by
whole-genome sequencing in each gene and were
validated by Sanger sequencing (see Table S2 in
the Supplementary Appendix). Three additional
patients with unpaired samples had variants in
ARID1A, and the somatic status was confirmed by
Sanger sequencing of normal tissue. Therefore,
5 of 30 patients (17%) had a mutation in ARID1A,
including three single-nucleotide variants leading
to premature protein truncation (one each at R173,
Q547, and Q934), and two frameshift changes (a
deletion at position 27057944 and an insertion at
position 27107136 in chromosome 1). Patients
with both ARID1A and MYD88 L265P mutations, as
compared with patients who did not have ARIDIA
mutations, had greater involvement of bone mar-
row disease (90% vs. 50%, P = 0.08), a lower hema-
tocrit (26% vs. 32%, P = 0.03), and a lower platelet
count (168,000 per cubic millimeter vs. 207,000 per
cubic millimeter, P = 0.08). No somatic variants in
HIST1H1E were identified in any of the patients
with unpaired samples. Additional somatic vari-
ants were identified in nonrecurring genes and
occurred only in single patients (see Table S2 in
the Supplementary Appendix).
Sanger Sequencing for MYD88 L265P
Sanger sequencing confirmed MYD88 L265P ex-
pression in all 26 tumor samples in which it had
been revealed by whole-genome sequencing and
in 1 sample from an additional patient, in whom
lymphoplasmacytic cells had read-level evidence
for MYD88 L265P but the variant did not meet the
criterion for high confidence. In this patient, ap-
n engl j med 367;9  nejm.org  august 30, 2012
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Copyright © 2012 Massachusetts Medical Society. All rights reserved.
proximately 10% of bone marrow lymphoplas-
macytic cells expressed MYD88 L265P. Sanger
sequencing therefore identified MYD88 L265P in
tumor samples from 27 of 30 patients with
Waldenström’s macroglobulinemia (90%) and con-
firmed the absence of MYD88 L265P in normal
tissue from 10 patients with paired samples and
from 9 patients with unpaired samples for whom
normal tissue was available. For the 3 patients
with Waldenström’s macroglobulinemia who did
not have MYD88 L265P expression, a review of all
the map reads and sequencing of the entire cod-
ing region revealed no other MYD88 variants. In
addition, we sought to identify variants exclusive
to patients with wild-type MYD88. In 2 of 3 pa-
tients with wild-type MYD88, variants in the
mixed-lineage leukemia 2 gene (MLL2) were iden-
tified and were confirmed to be somatic after
Sanger sequencing of normal tissue was per-
formed in these patients. One of these 2 patients
had a single-nucleotide variant in chromosome
12 at position 49425599 (A→G), and the other
patient had a deletion at position 49448413 (C)
resulting in a frameshift mutation. No other vari-
ants in recurring genes were identified in these
patients.
Sanger sequencing also revealed MYD88 L265P
expression in 22 of 24 patients with Walden-
ström’s macroglobulinemia in a separate cohort,
including 2 with homozygous expression. MYD88
L265P was expressed in both CD19+ cells and
CD138+ cells from 12 of these patients for whom
both cell sorts were available, a finding that was
consistent with the known distribution of the ma-
lignant Waldenström’s macroglobulinemia clone.2
Three patients with LPL who had IgG-secreting
disease or IgA-secreting disease also had MYD88
L265P expression, signifying that MYD88 L265P
is expressed in non–IgM-secreting LPL. MYD88
L265P was thus expressed in 52 of the 57 pa-
tients with LPL (91%).
MYD88 L265P was also detected as a heterozy-
gous variant in BCWM.1 and MWCL-1 Walden-
ström’s macroglobulinemia cell lines and was
not expressed in IgM-secreting WSU and Ramos
cell lines, which carry an 8;14 translocation, nor
in any myeloma cell lines. MYD88 L265P was
absent in peripheral-blood B cells from 15
healthy donors and in tumor samples from 10
patients with myeloma, including 2 with IgM
myeloma (P<0.001 for the comparison of sam-
ples from both groups of patients with samples
829
 The n e w e ng l a n d j o u r na l
Control Peptide
A B
MYD88 Inhibitor
C D
Figure 1. Inhibition of Myeloid Differentiation Primary Response Gene (88)
(MYD88) Signaling and the Effect on Nuclear Factor κB (NF-κB) p65
Expression in the Nucleus of Waldenström’s Macroglobulinemia Cells.
Nuclear and cytoplasmic staining for NF-κB p65 is shown in cells expressing
MYD88 L265P from the bone marrow of a patient with Waldenström’s macro­
globulinemia after in vitro treatment with either a control peptide (Panels A
and B) or a peptide inhibiting MYD88 homodimerization (Panels C and D).
Red indicates staining for NF-κB p65 in the cells. The NF-κB p65 is found in
the nucleus when cells are treated with a control peptide (Panel A) and in the
cytoplasm when cells are treated with the MYD88 inhibitor (Panel C). Staining
with 4′,6-diamidine-2-phenylindole dihydrochloride (DAPI) (Panels B and
D) shows the nucleus as blue. In Panel B, the red NF-κB p65 staining colo-
calizes with the blue staining in the nucleus. In Panel D, treatment with the
MYD88 inhibitor results in NF-κB p65 red staining in the cytoplasm and
not in the blue nucleus.
from patients with Waldenström’s macroglobulin-
emia). Of the 46 patients with marginal-zone
lymphoma, 3 patients (1 with a splenic subtype,
1 with an extranodal subtype, and 1 with a
nodal subtype) had MYD88 L265P expression
(7%, P<0.001 for the comparison with Walden-
ström’s macroglobulinemia). All 3 patients were
heterozygous for MYD88 L265P, and 2 of them
(1 with a splenic subtype and 1 with a nodal sub-
type) had extensive bone marrow involvement, a
monoclonal IgM protein, and clinicopathological
features that overlapped with those of Walden-
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Copyright © 2012 Massachusetts Medical Society. All rights reserved.
of m e dic i n e
ström’s macroglobulinemia. As determined by
means of direct Sanger sequencing, MYD88 L265P
was present in CD19+ cells from 2 of 21 patients
with IgM MGUS (10%, P<0.001 for the compari-
son with Waldenström’s macroglobulinemia).
Cloning and sequencing of at least 100 clones
showed that MYD88 L265P was not present in
the patients with IgM MGUS in whom direct se-
quencing did not reveal MYD88 L265P. In addi-
tion, a clonal IgH rearrangement was present in
at least a subset of transcripts in 7 of 11 patients
with IgM MGUS, none of whom had MYD88
L265P expression, indicating the presence of
clonal B cells in most samples used for Sanger
sequencing.
Inhibition of MYD88 Signaling in Cells
Expressing L265P
After culture with an inhibitor of MYD88 homodi-
merization, Waldenström’s macroglobulinemia
cells expressing MYD88 L265P showed a marked
decrease in nuclear staining of NF-κB p65 (Fig. 1),
as well as decreased IκBα and NF-κB p65 phos-
phorylation (see Fig. S1 and S2 in the Supplemen-
tary Appendix). Similar results were obtained when
cells expressing MYD88 L265P were incubated with
an IRAK 1/4 kinase inhibitor (see the Supplemen-
tary Appendix). In contrast, cells expressing wild-
type MYD88 either showed no IκBα, NF-κB p65 ac-
tivity, or nuclear NF-κB p65 staining or remained
unaffected by the inhibition of MYD88 signaling.
Discussion
We describe here the presence of a widely ex-
pressed somatic mutation (MYD88 L265P) in pa-
tients with Waldenström’s macroglobulinemia,
as identified by means of whole-genome sequenc-
ing and confirmed by means of Sanger sequencing.
Sanger sequencing also identified MYD88 L265P
in other patients with Waldenström’s macroglob-
ulinemia and in patients with non–IgM-secreting
LPL. In total, 91% of patients with LPL had MYD88
L265P expression. In contrast, MYD88 L265P was
absent in tissue samples from patients with my-
eloma, including samples from patients with IgM-
secreting myeloma, and was expressed in only a
small subgroup of patients with marginal-zone
lymphoma (7%), which included patients who had
features related to those of Waldenström’s mac-
roglobulinemia. MYD88 L265P may therefore be
useful in distinguishing LPL from marginal-zone
 MYD88 L265P Mutation in Waldenström’s Macroglobulinemia

Figure 2. MYD88-Directed NF-κB Signaling.

Extracellular space
After toll-like receptor or interleukin-1 receptor binds
to its ligand, the toll–interleukin-1 receptor (TIR) do-
mains activate MYD88 directly, or in the case of toll-
like receptor 4 trigger MYD88 through interactions
with TIR domain containing adaptor protein (TIRAP) Toll-like Interleukin-1
receptor 4 receptor
and Bruton’s tyrosine kinase (BTK). MYD88 then di-
merizes and triggers autophosphorylation of interleukin-1
receptor–associated kinase (IRAK) 4, with subsequent
recruitment and phosphorylation of IRAK1 and cyto-
solic release of membrane-bound tumor necrosis fac- Cell
membrane
tor receptor–associated factor 6 (TRAF6) from IRAK1.
The TGF-β–activated kinase 1 (TAK1) binding proteins TIRAP
(TAB1 and TAB2) promote binding of TRAF6 to TAK1,
thereby triggering phosphorylation of the heterotrimeric MYD88 MYD88
IκB kinase (IKK) complex (NEMO/IKKγ, IKKα, and IKKβ). BTK
K4

IR
P P A
The activation of this heterotrimeric complex leads to

A
IR

K4
IR
IκBα phosphorylation and release of NF-κβ p65 and Cytosol A K1 K1 P
P A
p50, which translocate to the nucleus and drive NF-κβ– IR
dependent prosurvival signaling. 2 TA
P TAB B1
TRAF6
TAK1 Activation by
P P phosphorylation
NEMO
lymphoma and multiple myeloma — an often
P IKKα IKKβ P
difficult task owing to overlapping morphologic,
immunophenotypic, cytogenetic, and clinical fea- P
tures.2,18,19 As compared with the levels of MYD88 Proteasomal IkBα
L265P expression in LPL, lower levels of MYD88 degradation P
p50 p65
P

L265P expression were reported in diffuse large-cell P

p50 p65
P
lymphoma of the activated B-cell type (14 to 29%),
mucosa-associated lymphoid-tissue lymphoma
(9%), and chronic lymphocytic leukemia (3%).20‑22
Of particular interest was the absence of MYD88
L265P in most, but not all, patients with IgM
MGUS as evaluated by Sanger sequencing. Only
2 of the 21 patients with IgM MGUS (10%) had
MYD88 L265P expression, both of whom had
Nuclear translocation
subcentimeter adenopathy but no bone marrow and signaling
infiltrate, which is required for the clinicopatho-
logical diagnosis of Waldenström’s macroglobu-
Nucleus
linemia.1,2 One of these patients had progressive
disease, as evidenced by serial increases in se-
rum IgM levels and a declining hematocrit; the be required to evaluate the contribution of MYD88
disease was diagnosed only recently in the other L265P to the progression to Waldenström’s mac-
patient, and the follow-up time has been short. roglobulinemia ratherDraft 2
than to other B-cell lympho-
COLOR FIGURE

07/30/2012
The significance of these findings for determin- proliferative disorders.
Author The absence of MYD88
Treon_oa1200710
Fig # 2
ing the relationship of IgM MGUS to Walden- L265P in most Title
of the patients with IgM MGUS
MYD88 L265P somatic
ström’s macroglobulinemia remains to be clari- could also reflect amutation lower frequency of clonal­
in Waldenstrom’s
Macroglobulinemia
fied. It is tempting to speculate that acquisition of B cells in theDEsamples Longowe used, which might not
MYD88 L265P represents a transforming event have been detected
ME
Artist
by Sanger sequencing. How-
Moskowitz
Williams
that facilitates the progression of IgM MGUS to ever, the presenceAUTHOR
of clonal IgH rearrangements
PLEASE NOTE:
Figure has been redrawn and type has been reset
Waldenström’s macroglobulinemia. Many, if not in most of the samples from these patients that
Please check carefully

most, cases of IgM MGUS progress to Walden- were evaluated suggests that the number of clonal
ström’s macroglobulinemia.7-9 A large prospec- B cells in the samples was probably sufficient.
tive study involving patients with IgM MGUS will The expression of MYD88 L265P occurred at

n engl j med 367;9  nejm.org  august 30, 2012 831

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Copyright © 2012 Massachusetts Medical Society. All rights reserved.
 The n e w e ng l a n d j o u r na l
a frequency that was independent of status with
respect to family history. Genetic variants asso-
ciated with familial Waldenström’s macroglobu-
linemia might therefore confer a predisposition
to IgM MGUS, but not Waldenström’s macroglobu-
linemia itself, particularly since cases of IgM
MGUS are present in affected families and prog-
ress over time to Waldenström’s macroglobulin-
emia.4-6 A larger study comprising familial and
sporadic cases of IgM MGUS and Waldenström’s
macroglobulinemia will be needed to clarify these
observations.
After MYD88, ARID1A had the most somatic vari-
ants, with variants occurring in 17% of patients.
ARID1A is a member of the SWI–SNF family of
proteins, which facilitate repositioning of nucleo-
somes for transcriptional regulation, DNA repair,
recombination, and chromosome segregation.23
ARID1A is a tumor-suppressor gene in breast, ovar-
ian, and gastric cancers, as well as in acute lym-
phoblastic leukemia, in which loss of expression
is associated with resistance to glucocorticoid
therapy.24-27 ARID1A mutations were present in
patients with sporadic cases and those with fa-
milial cases and were coexpressed with MYD88
L265P in all cases. Patients with both ARID1A
and MYD88 L265P mutations had more aggres-
sive disease features than did patients who did not
have ARID1A mutations. The presence of ARID1A
mutations in patients with Waldenström’s mac-
roglobulinemia may therefore have prognostic
as well as treatment implications, given the use
of glucocorticoids in many treatment regimens
for patients with Waldenström’s macroglobulin-
emia; however, we do not have sufficient data to
confirm these relationships.
Among the three patients with wild-type
MYD88, two had variants in MLL2, a gene encod-
ing the histone-modifying enzyme histone–lysine
N-methyltransferase.28 No patient with MYD88
L265P expression had MLL2 variants. Both pa-
tients with MLL2 variants had high levels of cir-
culating clonal B cells and CD23 expression,
features that are uncommon in Waldenström’s
macroglobulinemia.1-3 MLL2 is a frequent target
of somatic mutations in follicular lymphomas
(89%) and diffuse large B-cell lymphomas
(32%).28,29 A larger study involving patients with
Waldenström’s macroglobulinemia or non-IgM
LPL who have wild-type MYD88, as well as func-
tional studies, will be needed to validate the
significance of these findings with respect to
832 n engl j med 367;9  nejm.org  august 30, 2012
The New England Journal of Medicine
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Copyright © 2012 Massachusetts Medical Society. All rights reserved.
of m e dic i n e
the pathogenesis of Waldenström’s macroglobu-
linemia.
MYD88 is an adaptor molecule in toll-like re-
ceptor and interleukin-1 receptor signaling (Fig.
2).30,31 After stimulation of the toll-like receptor
or interleukin-1 receptor, MYD88 is recruited to
the activated receptor complex as a homodimer
and forms complexes with IRAK4, leading to ac-
tivation of IRAK1 and IRAK2.32,33 Tumor necrosis
factor receptor–associated factor 6 (TRAF6) is
then activated by IRAK1, leading to phosphory-
lation of IκBα and activation of NF-κB.34 Ngo et
al.20 found that inhibition of MYD88 signaling
decreased NF-κB activity and survival of activated
B-cell–type diffuse large-cell lymphoma cell lines
expressing MYD88 L265P. Our observation that
blocking of MYD88 signaling decreased NF-κB
activity is consistent with these findings. These
observations are of relevance to Waldenström’s
macroglobulinemia, since NF-κB signaling is
important for the growth and survival of Walden-
ström’s macroglobulinemia cells.17 Blockade of
IκBα by proteasome inhibitors is associated with
high rates of response in patients with Walden-
ström’s macroglobulinemia,35,36 and direct tar-
geting of tonic MYD88–IRAK signaling imposed
by MYD88 L265P provides a novel approach for the
treatment of Waldenström’s macroglobulinemia.
In conclusion, with the use of whole-genome
sequencing, we have identified a widely expressed
mutation (MYD88 L265P) that is present in more
than 90% of tumor samples from patients with
Waldenström’s macroglobulinemia or non-IgM
LPL. The presence of MYD88 L265P can help dif-
ferentiate Waldenström’s macroglobulinemia, as
well as non-IgM LPL, from B-cell disorders that
have some of the same features and provides in-
sight into the pathogenesis of Waldenström’s
macroglobulinemia. It remains to be seen wheth-
er MYD88 L265P signaling can be targeted for
the therapy of Waldenström’s macroglobulin-
emia and non-IgM LPL.
Presented in part at the Annual Meeting of the American So-
ciety of Hematology, Atlanta, December 10–13, 2011.
Supported by the Peter and Helen Bing Foundation, the Inter-
national Waldenström’s Macroglobulinemia Foundation, the Coy-
ote Fund for Waldenström’s Macroglobulinemia, the Walden-
ström’s Cancer Fund, the Bailey Family Fund for Waldenström’s
Macroglobulinemia, the D’Amato Family Fund for Genomic Dis-
covery, the Edward and Linda Nelson Fund for Waldenström’s
Macroglobulinemia Research, the Bauman Family Trust, and the
Tannenhauser Family Foundation.
Disclosure forms provided by the authors are available with
the full text of this article at NEJM.org.
 MYD88 L265P Mutation in Waldenström’s Macroglobulinemia

We thank the patients with Waldenström’s macroglobulin- Dana–Farber Cancer Institute) for providing samples from pa-
emia who provided samples for the study; and Drs. Yu-Tzu Tai tients with myeloma for these studies.
and Constantine Mitsiades (laboratory of Dr. Ken Anderson,

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