Received: 21 July 2017 | Revised: 30 October 2017 | Accepted: 6 November 2017

DOI: 10.1111/cas.13450

ORIGINAL ARTICLE

Clinical significance of disease-specific MYD88 mutations in

circulating DNA in primary central nervous system lymphoma

Keiichiro Hattori1,2 | Mamiko Sakata-Yanagimoto1,2 | Yasuhito Suehara1,2 |

Yasuhisa Yokoyama1,2 | Takayasu Kato1,2 | Naoki Kurita2 | Hidekazu Nishikii1,2 |
Naoshi Obara1,2 | Shingo Takano3 | Eiichi Ishikawa3 | Akira Matsumura3 |
Yuichi Hasegawa2 | Shigeru Chiba1,2

1
Department of Hematology, Graduate
School of Comprehensive Human Sciences,
Recent sequencing studies demonstrated the MYD88 L265P mutation in more than
University of Tsukuba, Tsukuba, Japan 70% of primary central nervous system lymphomas (PCNSL), and the clinical signifi-
2
Department of Hematology, Faculty of cance of this mutation has been proposed as diagnostic and prognostic markers in
Medicine, University of Tsukuba, Tsukuba,
Japan PCNSL. In contrast, mutational analyses using cell-free DNAs have been reported in
3
Department of Neurosurgery, Faculty of a variety of systemic lymphomas. To investigate how sensitively the MYD88 L265P
Medicine, University of Tsukuba, Tsukuba,
mutation can be identified in cell-free DNA from PCNSL patients, we carried out
Japan
droplet digital PCR (ddPCR) and targeted deep sequencing (TDS) in 14 consecutive
Correspondence
PCNSL patients from whom paired tumor-derived DNA and cell-free DNA was
Mamiko Sakata-Yanagimoto, Department of
Hematology, Faculty of Medicine, University available at diagnosis. The MYD88 L265P mutation was found in tumor-derived
of Tsukuba, Tsukuba, Ibaraki, Japan.
DNA from all 14 patients (14/14, 100%). In contrast, among 14 cell-free DNAs eval-
Email: sakatama-tky@umin.net
and uated by ddPCR (14/14) and TDS (13/14), the MYD88 L265P mutation was
Shigeru Chiba, Department of Hematology,
detected in eight out of 14 (ddPCR) and in 0 out of 13 (TDS) samples, implying
Faculty of Medicine, University of Tsukuba,
Tsukuba, Ibarak, Japan. dependence on the detection method. After chemotherapy, the MYD88 L265P
Email: schiba-tky@umin.net
mutation in cell-free DNAs was traced in five patients; unexpectedly, the mutations
Funding information disappeared after chemotherapy was given, and they remained undetectable in all
Leukemia Research Fund and Takeda
patients. These observations suggest that ddPCR can sensitively detect the MYD88
Science Foundation for Cancer Research to
S.C. Grant/Award Number: ‘n/a’, Grants-in- L265P mutation in cell-free DNA and could be used as non-invasive diagnostics, but
Aid for Scientific Research from the Ministry
may not be applicable for monitoring minimal residual diseases in PCNSL.
of Education, Culture, Sports, and Science of
Japan (Grant/Award Numbers: ‘15H01504’,
‘16H02660’, ‘25112703’). KEYWORDS
cell-free DNA, droplet digital PCR, MYD88 L265P, non-invasive diagnosis, primary central
nervous system lymphoma

1 | INTRODUCTION appropriate diagnosis. However, histological diagnosis of PNCSL is

sometimes difficult because of deep brain structure involvement.
Primary central nervous system lymphoma (PCNSL) is a rare subtype Therefore, there is a need for a more reliable and minimally invasive
of diffuse large B-cell lymphoma (DLBCL) that arises within the brain biomarker detection method aiding the diagnosis of PCNSL.
or eyes. PCNSL accounts for up to 2%-3% of primary malignant brain Recently, circulating tumor DNAs, which are fragmented DNAs
tumors and less than 1% of non-Hodgkin lymphomas (NHL) in adults.1 released through the apoptotic process of tumor cells, in plasma and
Almost all PCNSL patients undergo invasive surgical procedures for serum have received attention as materials for non-invasive

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This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any
medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Cancer Science. 2017;1–6. wileyonlinelibrary.com/journal/cas | 1

2 | HATTORI ET AL.

diagnosis.2 Many genetic alterations identified in tumors have been of an 8-channel disposable droplet generator cartridge (Bio-Rad).
used to detect circulating tumor DNAs in patients with both solid Additional 70 lL droplet generation oil (Bio-Rad) was loaded into
cancers and hematological malignancies.2-4 the oil well for each channel. After droplet generation, droplets were
Whole exome and targeted sequencing studies have determined transferred carefully into a 96-well PCR plate. The plate was heat-
the genetic profiles of PCNSL. Notably, mutations in genes associ- sealed with the PX1 PCR Plate Sealer (Bio-Rad) and proceeded to
ated with the nuclear factor kappa B (NF-jB) and B-cell receptor sig- thermal cycling. Amplification was carried out on the 20 lL reaction
naling pathways are highly frequent in PCNSL.5-12 In particular, the mixture on the QX200 Droplet digital PCR system (Bio-Rad).
MYD88 L265P mutation was found in 38%-85.4% of PCNSL patients After PCR, the 96-well PCR plate was subjected to the QX-200
but never in those with non-hematological brain tumors, suggesting droplet reader (Bio-Rad). ddPCR data were analyzed by QuantaSoft
that this mutation is useful for differential diagnosis of PCSNL analysis software (Bio-Rad) that accompanied the droplet reader. In
among central nervous system (CNS) tumors.5-12 our analyses, MYD88 L265P mutation-specific signals were gener-
Herein, we examined how sensitively the MYD88 L265P muta- ated in the FAM channel, whereas MYD88 wild-type signals were in
tion is detected in cell-free DNAs in PCNSL patients at diagnosis the HEX channel.
and during the disease course.

2.4 | Targeted deep sequencing

2 | MATERIALS AND METHODS
Targeted deep sequencing (TDS) was carried out on Ion Torrent PGM
platform (Thermo Fisher Scientific, Waltham, MA, USA) for MYD88
2.1 | Patient characteristics
L265P mutation as previously described with minor modifications.13
From October 2014 to June 2017, a total of 30 consecutive patients The Ion Plus Fragment Library Kit (Thermo Fisher Scientific) was used
diagnosed with PCNSL were identified in the clinical records at the to prepare the libraries according to the recommended protocol for
University of Tsukuba Hospital. Sixteen patients were excluded from libraries of short amplicons.
this study because CNS tumor or serum samples before treatment Primers were designed to amplify the genomic regions encoding
were not available. The remaining 14 patients were analyzed in a codon 265 in MYD88 (Table S2). PCR was carried out in a final vol-
retrospective way (Table S1). ume of 10 lL with tumor-derived DNAs and cell-free DNAs,
This retrospective study was approved by the institutional review 5 nmol/L primers, and KOD Neo reagents (Toyobo, Osaka, Japan).
board of University of Tsukuba Hospital. Samples were provided in The PCR protocol was as follows: 10 minutes at 95°C, followed by
accordance with the “Guidelines for clinical measurement and diag- 39 cycles of 15 seconds at 95°C and 60 seconds at 60°C. Then,
nosis technology improvement project”. The guidelines are promoted PCR amplicons were ligated to barcode adapters and P1 adapters,
by Tsukuba Medical Laboratory of Education and Research Center and amplified to generate libraries. Quantification of the amplified
and University of Tsukuba Hospital, Japan. libraries was done by quantitative PCR with the Ion Library Quantifi-
cation kit according to the manufacturer’s instructions (Thermo
Fisher Scientific). The libraries were then subjected to deep
2.2 | Serum and plasma collection
sequencing on Ion Torrent PGM with the Ion 318 chip according to
Serum samples were collected before first-line therapy in all 14 the standard protocol for 300 base pair single-end reads (Thermo
patients. A plasma sample before treatment was available in one Fisher Scientific). The data were analyzed by Variant caller 5.2
patient only (TP103). Serial serum samples were preserved during the (Thermo Fisher Scientific). The sequencing variations were adopted
clinical courses from five patients (TP87, TP89, TP90, TP92, and as mutations when frequencies were higher than 2% for tumor-
TP99). Cell-free DNAs were extracted from 700 to 3000 lL serum or derived DNA and 0.2% for cell-free DNA.
plasma using the QIAmp Circulating Nucleic Acid Kit (Qiagen, Hilden,
Germany). Genomic DNAs were also extracted from 14 formalin-fixed,
2.5 | Statistical analysis
paraffin-embedded (FFPE) CNS tumor samples using the FFPE tissue
Kit (Qiagen). Final DNAs were dissolved in 30-50 lL AVE buffer (Qia- Statistical analysis was conducted using the EZR software.14 We
gen, Hilden, Germany) and were stored at 20°C until use. used two-sided tests in all calculations. P-value ≤ .05 was considered
statistically significant.

2.3 | ddPCR assay

3 | RESULTS
Droplet digital polymerase chain reaction (ddPCR) reagents and
Primer/probe mix for MYD88 L265P were purchased from Bio-Rad
3.1 | Validation of the ddPCR assay and TDS for
(Hercules, CA, USA). The 20 lL PCR mix, composed of 10 lL of 29
detecting the MYD88 L265P mutation
ddPCR Supermix for Probes (No dUTP) (Bio-Rad), 1 lL ddPCR Muta-
tion Assay (Bio-Rad) that used an amplicon of 65 nt, 5 lL ultra pure The signal emitted by the MYD88 L265P mutation was clearly distin-
distilled water and 4 lL cell-free DNA was loaded into sample wells guished from that by wild-type DNA in a 2D fluorescent amplitude
HATTORI ET AL. | 3

plot (Figure S1). Lower limits of detection (LOD) and quantification

3.2 | MYD88 L265P mutation in tumor-derived
(LOQ) in ddPCR were determined using serial dilutions of DNA with
DNA
the MYD88 L265P mutation. We chose a frozen tissue-derived DNA
in which AmpliSeq measured 24.2% variant allele frequency (VAF) at We examined the MYD88 L265P mutation in tumor-derived DNA
the depth of 1920. This sample was serially diluted at 1/4 each prepared from 14 PCNSL patients by ddPCR and TDS (Table 1). In
down to (1/4)7, by mixing with the copy number-adjusted DNA this cohort, MYD88 L265P mutation was found in all patients (14/
without MYD88 L265P mutation. All dilutions were then applied to 14, 100%; range of VAF, 10.5%-87.0%) and mean depths for the
ddPCR. Mutation ratio was calculated for each sample by Quanta- mutation in tumor-derived DNAs were 16868x to 19993x (Table 1).
Soft and plotted (Figure 1).
In the 1/4 (corresponding to 6.05% by calculation), (1/4)2
3.3 | Detection of the MYD88 L265P mutation in
(1.5125%), (1/4)3 (0.378125%), (1/4)4 (0.09453125%), (1/4)5
cell-free DNAs at diagnosis
(0.0236328125%), dilutions, fractional abundance (referring to the pro-
portion of mutated DNA by QuantaSoft) measured by ddPCR was Amounts of cell-free DNA, prepared from 1 mL serum or plasma
8.3%, 3.1%, 1.0%, 0.2% and 0.1%. After being converted to log-log that was collected before treatment, ranged from 107 ng to
scale, data showed precision and linearity down to a dilution of 0.024%; 2410 ng (Figure 3).
this material corresponded to a fractional abundance of 0.1% (coeffi- The MYD88 L265P mutation was detected by ddPCR in eight
cient of determination [R2] was .994). The positive control diluted to (1/ out of 14 serum-derived cell-free DNAs at diagnosis (Figure 1;
4)6 (0.005908203125%) was judged as positive by ddPCR, whereas the Figure S1). Among six negative subjects, we prepared plasma-derived
fractional abundance was 0.07%, implying that the quantification was cell-free DNA from only 1; the mutation was also undetectable in
inaccurate (Figure 1). All dilutions less than (1/4)6 showed 0%. this preparation (TP103, Table S3). VAF of the MYD88 L265P muta-
Based on this result, the LOQ of ddPCR was defined as 0.024% tion in cell-free DNAs was significantly lower than those of the
with a fractional abundance of 0.1%. The LOD could be lowered to tumors (mean, 0.34% [range, 0.1%-0.69%] vs 48.5% [10.5%-87%],
0.0059% with a fractional abundance of 0.07%. P < .001). Thirteen samples, except for one sample of TP100
The same dilution series was also applied to amplicon-based because the amount of DNA in this sample was not sufficient, were
sequencing at the approximate depth of 20 000 reads and the muta- also evaluated by TDS. Mean depths for MYD88 L265P in cell-free
tion ratio was plotted (Figure 2). In the 1/4 (corresponding to 6.05% DNA were 12 560x to 19 996x. In this method, the mutations were
by calculation), (1/4)2 (1.5125%), and (1/4)3 (0.378125%) dilutions, not detected at all irrespective of whether or not they were detect-
VAF measured by amplicon-based sequencing was 7.2%, 2.7%, and able by ddPCR.
3
0%. All dilutions less than (1/4) showed 0%. Based on this, the cut- Presence of mutations in cell-free DNAs was not significantly
off value was determined at 1.51%. After being converted to log-log associated with laboratory data, such as lactate dehydrogenase
scale, data showed precision and linearity down to a dilution of (LDH) (P = .360) and soluble interleukin-2 receptor (sIL2R) (P = .354)
1.51% (R2 was 1; Figure 2). levels, concentrations of cell-free DNAs (P = .105), tumor sizes
Therefore, LOQ and LOD of TDS were defined as 1.51% with a (P = .611) and clinical outcomes (OS, P = .386; PFS, P = .629).
VAF of 2.7%.

F I G U R E 1 Sensitivity of our droplet digital PCR (ddPCR) assay.

DNA with MYD88 L265P mutation was mixed with DNA without F I G U R E 2 Sensitivity of our targeted deep sequencing (TDS)
the mutation at the concentrations indicated on the horizontal axis. assay. DNA with MYD88 L265P mutation was mixed with DNA
All diluted samples were individually subjected to ddPCR. Fractional without the mutation at the concentrations indicated on the
abundance (mutation ratio calculated by QuantaSoft) for each horizontal axis. All diluted samples were individually subjected to
sample is plotted TDS assay. Variant allele frequency for each sample is plotted
4 | HATTORI ET AL.

T A B L E 1 Targeted sequencing of MYD88 L265P for 14 PCNSL samples

Mutation ratio of MYD88 L265P (%)

Tumors Cell-free DNAs in serum

NGS NGS
ddPCR ddPCR
Reads showing the Reads showing the
VAF (%) mutation/total reads FA (%) VAF mutation/total reads FA (%)
TP73 43.2 8584/19870 ND 0 0/19996 0
TP87 65.5 13070/19954 ND 0 0/19930 0.40
TP89 35.1 6979/19882 ND 0 0/19981 0.19
TP90 40.9 8137/19895 ND 0 0/19980 0.09
TP92 53.3 10534/19763 ND 0 0/19985 0.10
TP94 61.3 12233/19956 ND 0 0/19948 0.38
TP95 36.4 7230/19862 ND 0 0/19963 0.14
TP96 56.1 11122/19825 ND 0 0/19972 0.47
TP98 88.7 17736/19993 87 0 0/19987 0
TP99 56.6 11299/19974 55.9 0 0/19994 0.69
TP100 75.2 15025/19980 79 ND ND 0
TP101 9.2 1844/19988 10.5 0 0/12560 0
TP102 43 7260/16868 38.3 0 0/19991 0
TP103 19.7 3928/19973 17.1 0 0/19989 0

ddPCR, droplet digital PCR; FA, fractional abundance; ND, not done; NGS, next-generation sequencing; PCNSL, primary central nervous system lym-
phoma; VAF, variant allele frequency.

chemotherapies in five patients who showed positivity of the

3.4 | MYD88 L265P mutation in cell-free DNAs
MYD88 L265P mutation in cell-free DNAs at diagnosis.
during the disease course
Patients TP92 and TP99 received the modified EORTC regimen15
To investigate whether the MYD88 L265P mutation in cell-free and the R-MPV regimen,16 respectively. They obtained complete
DNAs is a useful marker for estimating disease course, such as remission (CR) during the observation period. However, the mutations
remission and/or progression of disease (PD), we analyzed the were not detected at all throughout the treatment courses (Figure 4).
MYD88 L265P mutation in cell-free DNAs during and after In two patients (TP87 and TP89), once achieving CR, lymphomas
relapsed at 3 months (TP89) and 12 months (TP87) after completion
of the therapies. In TP89, relapse occurred in intraocular tissues
without reappearance of a tumor in the CNS. In TP87, tumor size at
the time of progression was smaller than that at diagnosis
(10 9 15 mm vs 42 9 55 mm). In contrast, TP90 was treated by
the modified EORTC regimen, but it was abandoned after induction
therapy because of PD. Tumor size at the time of progression was
bigger than that at diagnosis (56 9 70 mm vs 37 9 46 mm). In
these patients, the MYD88 L265P mutation was not detected in cell-
free DNAs despite disease progress after the relapse (Figure 4).

4 | DISCUSSION

The present study demonstrated that the MYD88 L265P mutation

was detected by the ddPCR assay in 57.1% of cell-free DNAs of
PCNSL patients at diagnosis. Further analysis is required to deter-
mine the actual detection rate of this mutation in cell-free DNAs of
F I G U R E 3 Concentrations of cell-free DNAs of primary central
nervous system lymphoma (PCNSL). Distribution of the cell-free PCNSL. Consistent with the previous report on liquid biopsies, our
DNA concentrations in serum in patients with PCNSL is shown by results confirmed that the ddPCR assay is a reliable method to
plots. The y-axis has a log scale detect mutations in cell-free DNAs and has an advantage over TDS
HATTORI ET AL. | 5

F I G U R E 4 Mutation ratio in cell-free DNAs and disease course. A, Patients who maintained complete remission (CR). B, Patients who
progressed during or after the therapy

in detecting very small numbers of mutation copies, avoiding biases studies have shown that cell-free DNA in several types of lym-
during the PCR procedures that happen in TDS. phomas could also be a surrogate marker for tumor burden.25-27 If
Nakamura et al reported that almost all PCNSL had MYD88 and the tumor burden in our cohort was less than in others because of
CD79B mutations, whereas they were not detected in glioblastomas the early setting of biopsies, the discordance might be explained.
(GBM).12,17 These mutations may serve as a genetic hallmark that Diagnosis and identifying minimal residual diseases (MRD) by
help a differential diagnosis among these two most common malig- using cell-free DNA have also been examined in DLBCL: cancer per-
nant brain tumors.12 sonalized profiling by deep sequencing (CAP-seq) and high-through-
In our analysis, concentration of cell-free DNAs in PCNSL was put sequencing identifying clonotypic Ig rearrangement using cell-free
107-2410 ng/mL. This estimation implies a much higher level than DNA were shown to be useful for early detection of relapse.21,25,26
that of cell-free DNA prepared from healthy volunteers, which were Recently, we showed that gene mutations specific to angioimmuno-
reported to be 1-10 ng/mL.18 Also, the estimated range appears to blastic T cell lymphoma in cell-free DNAs could be useful as sensitive
be higher than that of cell-free DNA amounts in patients with non- markers for circulating tumor DNAs and might be applicable for non-
hematological neoplasms, such as colorectal, lung, and breast cancer invasive monitoring of MRD.27 However, in the current study, the
(range 0.5-1980 ng/mL).19–21 However, this range of concentration MYD88 L265P mutation was not detected in cell-free DNAs even in
was lower than that of cell-free DNAs in patients with systemic lym- the PD state. Our results suggest that circulating tumor DNAs in
phomas (range 100-14 180 ng/mL).22,23 patients with PCNSL may not be applicable to monitoring the disease
In our analysis, the MYD88 L265P mutation was not detected in course of PCNSL, although those in patients with systemic lym-
cell-free DNAs by TDS. However, Fontanilles et al recently reported phomas were shown to be useful for monitoring of MRD.26,27 It
that a variety of somatic mutations specific to CNS tumors, including remains to be elucidated why the MYD88 L265P mutation was unde-
MYD88 L265P mutation, were detected in cell-free DNAs of PCNSL tectable in the PD state. Tumor size may not explain the reason, con-
patients by TDS.24 In their study, VAF of the MYD88 L265P muta- sidering that tumor size in the CNS at the time of disease progression
tion in cell-free DNAs was magnitudinally higher than those in our was bigger than that at diagnosis in a case (TP 90). Moreover, there
24
study (mean, 4.7% vs 0.34%). The higher VAFs in their study may was a possibility that the MYD88 L265P mutation-negative clone may
be a major reason why Fontanilles et al succeeded in the detection expand in relapsed tumors or that cell-free DNA may not circulate in
of mutations by TDS. In their study, materials used for extraction of peripheral blood after chemotherapy. Unfortunately, we analyzed the
cell-free DNAs were plasma, whereas ours were serum. This differ- course of MYD88 L265P mutation in cell-free DNAs in five cases
ence might explain the discordant VAF, despite the fact that our only. Further analysis will enable us to determine whether MYD88
analysis of one paired cell-free DNA prepared from serum and mutations are applicable for detecting MRD.
plasma failed to demonstrate it. Another potential cause of the dis- To our knowledge, this is the first study for the detection of gene
cordance could be the bias among the cohorts. Several previous mutations in cell-free DNAs in PCNSL patients using the ddPCR
6 | HATTORI ET AL.

assay. Ratio of detection of the MYD88 L265P mutation in cell-free peripheral T cell lymphoma in a case. Ann Hematol. 2015;94:1221-
DNA among the mutation-positive patients was 57%. The ddPCR- 1223.
14. Kanda Y. Investigation of the freely available easy-to-use software
based detection of the MYD88 L265P mutation in cell-free DNA may
‘EZR’ for medical statistics. Bone Marrow Transplant. 2013;48:452-
serve as a useful method for non-invasive diagnosis of PCNSL. 458.
15. Lee SY, Okoshi Y, Kurita N, et al. Prognosis factors in Japanese
elderly patients with primary central nervous system lymphoma trea-
ACKNOWLEDGMENTS ted with a nonradiation, intermediate-dose methotrexate-containing
regimen. Oncol Res Treat. 2014;37:378-383.
All authors have contributed significantly to the content of the 16. Hattori K, Sakata-Yanagimoto M, Okoshi Y, et al. A single institu-
manuscript. tional retrospective evaluation for younger patients with primary
central nervous lymphomas on a modified R-MPV regimen followed
by radiotherapy and high dose cytarabine. J Clin Exp Hematop. 2017;
CONFLICTS OF INTEREST 57: 41-46. https://doi.org/10.3960/jslrt.17012.
17. Brennan CW, Verhaak RG, McKenna A, et al. The somatic genomic
Authors declare no financial conflicts of interest for this article. landscape of glioblastoma. Cell. 2013;155:462-477.
18. Wan JC, Massie C, Garcia-Corbacho J, et al. Liquid biopsies come of
age: towards implementation of circulating tumour DNA. Nat Rev
ORCID Cancer. 2017;17:223-238.
19. Mouliere F, El Messaoudi S, Gongora C, et al. Circulating cell-free
Keiichiro Hattori http://orcid.org/0000-0002-0810-7887 DNA from colorectal cancer patients may reveal high KRAS or BRAF
mutation load. Transl Oncol. 2013;6:319-328.
20. Yoon KA, Park S, Lee SH, Kim JH, Lee JS. Comparison of circulating
plasma DNA levels between lung cancer patients and healthy con-
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