research paper
MYD88 L265P and CXCR4 mutations in lymphoplasmacytic lymphoma identify cases with high disease activity
Janine Schmidt, Birgit Federmann, Natalie Schindler, Julia Steinhilber, Irina Bonzheim, Falko Fend and Leticia Quintanilla-Martinez Institute of Pathology and Comprehensive Cancer Centre, Eberhard-Karls-University, University Hospital T€ ubingen, T€ ubingen, Germany Received 2 October 2014; accepted for publication 20 January 2015 Correspondence: Leticia Quintanilla-Martinez, Institute of Pathology, University Hospital of T€ ubingen, Liebermeisterstrasse 8, T€ ubingen 72076, Germany. E-mail: Leticia.quintanilla-fend@med. uni-tuebingen.de FF and LQ-M contributed equally to this study.
Summary Recurrent mutations in MYD88 have been identified in >90% of lymphoplasmacytic lymphoma (LPL). Recently, WHIM (warts, hypogammaglobulinaemia, infections, myelokathexis) syndrome-like mutations in CXCR4 have been described in 28% of LPL cases, and seem to impact clinical presentation and response to therapy. We investigated the presence of the MYD88 L265P mutation in 90 decalcified, formalin-fixed, paraffin-embedded (FFPE) bone marrow (BM) biopsies, including 51 cases of LPL, 14 cases of B-cell chronic lymphocytic leukaemia (CLL), 13 cases of marginal zone lymphoma (MZL) and 12 normal controls. In addition, the C-terminal domain of CXCR4 was sequenced in LPL cases. MYD88 L265P was found in 49/51 (96%) LPL cases and in 1/13 (76%) MZL (splenic type), whereas all CLL samples remained negative. The two MYD88 wild type LPL cases were associated with cold agglutinin disease. Mutations in CXCR4 were detected in 17/47 (362%) LPL cases, which showed a higher extent of BM infiltration and lower leucocyte counts (P = 002), haemoglobin (P = 005) and platelet counts (P = 001). In conclusion the detection of MYD88 L265P mutation in FFPE samples is reliable and useful for subtyping small B-cell lymphomas in BM biopsies. In addition, the presence of CXCR4 mutations identifies a subgroup of LPL patients with higher disease activity. Keywords: lymphoplasmacytic lymphoma, WHIM, MYD88 L265P, CXCR4, cold agglutinin disease.
Lymphoplasmacytic lymphoma (LPL) is a rare B-cell lymphoproliferative disorder composed of lymphocytes, plasmacytoid cells and plasma cells usually infiltrating the bone marrow (BM) and sometimes the lymph nodes and spleen, which does not fulfil the criteria for other small B-cell non-Hodgkin lymphomas (B-NHL). Waldenstr€ om macroglobulinaemia (WM) is diagnosed when patients with LPL, in addition to BM infiltration, show a serum immunoglobulin (Ig)M paraprotein of any concentration. Although most patients show a paraproteinaemia of IgM type, other Ig isotypes may occur (Owen et al, 2003; Swerdlow et al, 2008). Autoimmune phenomena or cryoglobulinaemia has been noted in up to 20% of WM patients. Until recently, separation of LPL from other low grade B-NHL was difficult due to the lack of a specific immunophenotype and characteristic genetic aberrations. Recently, using whole genome sequencª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 169, 795–803
ing, a recurrent point mutation in the myeloid differentiation factor 88 gene (MYD88), resulting in a single nucleotide substitution changing a leucine at position 265 of the MYD88 coding region into a proline (L265P) (Ngo et al, 2011), was discovered in 67–100% of LPL/WM (Treon et al, 2012; Gachard et al, 2013; Varettoni et al, 2013a; Xu et al, 2013). MYD88 is an adaptor protein involved in Toll-like receptor (TLR) as well as interleukin (IL) 1 and IL18 signalling (Akira & Takeda, 2004; Kawai & Akira, 2010). Stimulation of MYD88 by TLRs happens through homophilic binding of the Toll-IL1 receptor (TIR; also termed TIRAP) domains of both the adaptor protein and the TLR. Activated MYD88 recruits IL1 receptor-associated kinase 1 (IRAK1) and IRAK4, as well as Bruton tyrosine kinase (BTK) (Yang et al, 2013), ultimately leading to activation of NF-jB (NFKB1). Inhibition of the MYD88 signalling pathway blocks
First published online 29 March 2015 doi: 10.1111/bjh.13361
J. Schmidt et al the translocation of NF-jB into the nucleus in cells expressing MYD88 L265P, and it was shown that WM cells depend on NF-jB signalling for growth and survival (Leleu et al, 2008; Treon et al, 2012; Poulain et al, 2013). The MYD88 L265P is not exclusive to WM/LPL, but can also be identified in a subset of diffuse large B-cell lymphomas (DLBCL) of the activated B-cell type, whereas mutations in other small B-cell lymphomas are rare, including small fractions of splenic marginal zone lymphomas (SMZL) (4–13%), mucosa-associated lymphoid tissue (MALT) lymphomas (7–9%) (Ngo et al, 2011; Gachard et al, 2013) and rare cases of chronic lymphocytic leukaemia (CLL) (Martinez-Trillos et al, 2014). This mutation is absent in nodal MZLs and multiple myeloma (MM) (Je et al, 2012; Gachard et al, 2013; Xu et al, 2013). Given the difficulties in separating SMZL and MALT-lymphomas from LPL/WM with unusual clinical presentations, the true frequency and relevance of MYD88 mutations in these entities is not yet clear. In the pathogenesis of WM/LPL, this mutation seems to be an early event, because it can also be identified in 50–80% of IgM monoclonal gammopathy of undetermined significance (MGUS), a precursor lesion for WM. This indicates that secondary genetic alterations are necessary to acquire the fully malignant phenotype. In addition to the MYD88 L265P, a variety of other recurrent genetic alterations have been identified in LPL, most notably mutations in CXCR4, which are partly identical to germline mutations in WHIM (warts, hypogammaglobulinaemia, infections, myelokathexis) syndrome (Hernandez et al, 2003). In WHIM syndrome, these mutations prolong G-protein signalling upon receptor stimulation due to loss of inhibitory serines in the cytoplasmic tail of the protein. CXCR4 stimulation by its ligand CXCL12/SDF1 activates downstream signalling in WM cells, increasing migration and adhesion to the BM microenvironment (Ngo et al, 2008). It has been suggested that mutations in CXCR4, which virtually always co-exist with the MYD88 L265P, are associated with significantly higher serum IgM levels, more BM involvement and symptomatic disease. Very recent data indicate that this might be mainly restricted to cases carrying nonsense, rather than frameshift CXCR4 mutations (Treon et al, 2014). Of interest, despite their association with higher disease burden, CXCR4 mutations do not have a negative impact on overall survival. In addition to modulating disease presentation, CXCR4 mutations seem to have an impact on response to novel treatments, such as the BTK inhibitor ibrutinib (Cao et al, 2015). To date, several different methods have been published for the detection of MYD88 L265P, but these studies used mainly DNA from fresh BM or peripheral blood mononuclear cells. We therefore designed a sensitive and reliable assay for the detection of MYD88 L265P in decalcified formalin-fixed, paraffin-embedded (FFPE) BM biopsies and tested its diagnostic utility for classification of small B-cell lymphomas. Given the potential clinical relevance of WHIMlike CXCR4 mutations in LPL, we additionally studied their 796
presence in LPL cases and correlated them with clinico-pathological findings.
Materials and methods Sample collection and DNA extraction Fifty-one EDTA-decalcified FFPE BM trephine biopsies involved by LPL diagnosed between December 2000 and September 2014 at the Institute of Pathology, University Hospital of T€ ubingen, were included in the study. Additionally, FFPE BM biopsies of 13 cases of MZL (eight splenic MZL, four nodal MZL and one extranodal MZL) and 14 cases of CLL diagnosed in the same period were included in the study. All cases included were newly diagnosed without previous treatment. Cases were re-evaluated using haematoxylin and eosin (H&E), Giemsa, AS-D chloroacetate esterase and reticulin stains and standard diagnostic immunohistochemistry (see Data S1). Classification of the cases as LPL, MZL or CLL was according to the 2008 World Health Organization (WHO) classification and the Second International Workshop on WM (Owen et al, 2003; Swerdlow et al, 2008). In addition, 12 reactive BM biopsies without lymphoma involvement were included as controls. Paraffin-embedded cell blocks of the cell lines TMD8, HBL1 (MYD88 L265P heterozygous) and OCI-Ly19 (wild type, WT), provided by Alexander Weber (University of T€ ubingen, Germany), were used for preparation of dilution series for sensitivity testing. TMD8 and HBL1 are DLBCL cell lines with an activated B-cell (ABC) gene expression profile, whereas OCI-Ly19 has a germinal centre B-cell (GCB) gene expression profile. DNA was isolated from whole tissue sections (unless stated differently) and cell blocks, respectively, after protease digestion with a standard phenol-chloroform-extraction protocol (Sotlar et al, 2003).
Detection of MYD88 L265P by locked-nucleic-acid (LNA) -clamped polymerase chain reaction (PCR) with melting curve analysis Amplification of both WT and mutant DNA was performed on a LightCycler 480 instrument (Roche Diagnostics, Mannheim, Germany) using primers (forward primer 50 -TG CAGGGGTTGGTGTAGT-30 ; reverse primer 50 -GTTGTTAA CCCTGGGGTTG-30 ) and FRET hybridization probes (L265P mut 50 -CAGAAGCGACCGATCCCC-30 ; L265P Anc 50 -CAAG TACAAGGCAATGAAGAAAGAGTTCCC-30 ) from TIB Molbiol (Berlin, Germany). The hybridization probe L265P mut was mutant-specific, therefore resulting in a higher melting temperature for samples carrying the mutation as compared to WT samples. In order to detect minimal quantities of mutant DNA we added 001 lM of a WT-specific lockednucleic-acid (LNA) clamping oligomere (50 -AAGCGACTGATCCC-30 ; TIB Molbiol) for suppression of the WT signal and enhancement of the sensitivity (see Data S1). ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 169, 795–803
MYD88 L265P and CXCR4 Mutations in LPL
Sequencing of MYD88 and CXCR4 Forward and reverse primers designed for LNA-clamped PCR were used to amplify a 189 bp fragment covering a section of exon 5 of the MYD88 gene, including L265 for subsequent Sanger sequencing. For analysis of the CXCR4 gene, M13-tailed primers (forward: 50 - TGTAAAACGACGGCCAG TACATTGGGATCAGCATCGAC-30 ; reverse: 50 - CAGGAAAC AGCTATGACCATCTGTGTTAGCTGGAGTG-30 ; TIB Molbiol) were designed to amplify a 336 bp fragment covering a part of the carboxyl-terminal (c-terminal) domain (see Data S1). Purification of PCR products and subsequent analysis of sequencing reactions was performed as previously described (Adam et al, 2013).
(median = 3974%). LPL showed a diffuse or nodular, interand peritrabecular infiltration pattern with relatively small, monotonous lymphoid cells, lymphoplasmacytic cells and plasma cells accompanied by an increased number of reactive mast cells (Fig 1A–D). Immunohistochemically, LPL cases showed expression of pan-B-cell markers and a variably prominent plasma cell component with light chain restriction and IgM expression (Fig 1E–F). Only one patient in this study showed an IgG paraprotein. The age and sex ratio at presentation was similar among LPL, MZL and CLL patients. However, patients with CLL tended to have a higher BM tumour burden than patients with LPL or MZL infiltration and to be younger at first diagnosis.
Assessment of MYD88 L265P in LPL and other small B-NHLs
Clinical features Clinical data were collected from the files of the Department of Internal Medicine II (Haematology and Oncology) or obtained by contacting the primary physicians. The study was approved by the local ethics committee (105/2013BO2).
Statistical analysis Statistical analysis of the clinical data was calculated with JMP software, version 10 (SAS Institute GmbH, B€ oblingen, Germany). Clinical parameters were compared depended on the mutation status of MYD88 and CXCR4 using Wilcoxon or Chi2 test for comparison of nominal data; P < 005 were considered significant.
Results Clinical and histological features A total of 51 LPL, 13 MZL and 14 CLL cases were included in the study. The clinical findings are summarized in Table I. The median age of patients with LPL was 692 years (range 46–91) with a slight male predominance. The mean IgM level in serum at diagnosis was 287 g/l (range 095–79). The extent of BM infiltration ranged from 3% to 100%
The sensitivity of the assay was first assessed by a dilution series using DNA from the MYD88 L265P heterozygous ABC DLBCL cell lines, TMD8 and HBL1, and from the GCB DLBCL cell line, OCI-Ly19, which has WT MYD88. The melting curve analysis following amplification of the DNA resulted in two different melting peaks, at ~54°C for the WT allele and ~62°C for the mutant allele. In the dilution series of the two mutant cell lines with addition of the LNA oligomer, 25–5% mutant DNA was reliably detected (Fig 2A). The MYD88 L265P mutation was detected in 49/51 (96%) LPL patients whereas all 12 reactive controls remained negative (Fig 2B1–B2). The two LPL patients with WT MYD88 had a BM infiltration of ~10%. To investigate whether the negativity was due to the low tumour cell content, macrodissection of the lymphoma areas was performed. However, the two cases remained negative for MYD88 L265P (Fig 2 B3–B4). The single LPL case with paraprotein of IgG isotype carried the MYD88 L265P mutation. Of the other entities included in the study, only one of 13 (77%) MZL, a splenic MZL, was positive for the MYD88 L265P mutation, and all CLL samples remained negative. For confirmation, a total of 20 cases were subjected to Sanger sequencing. Of 10 examined LPL cases, seven were clearly
Table I. Clinical data of patients with WM/LPL, MZL or B-CLL.
Age (years), median (range) Sex (male/female) BM involvement, mean % (range) Paraprotein IgM (g/l), (range) Paraprotein IgG (g/l) (n = 1) Haemoglobin (g/l) Adenopathy Splenomegaly
WM/LPL (n = 51)*
MZL (n = 13)*
B-CLL (n = 14)*
692 (46–91) 33/15 3974 (3-100) 287 (095-790) 94 111 (74–160) 14 (3181%) 6 (1333%)
695 (55–84) 7/6 3523 (10-90) – – 122 (87–171) 1 (11%) 6 (667%)
633 (50–83) 8/6 625 (15-100) – – 93.4 (50–153) 3 (273%) 6 (545%)
*Not all clinical data were available for every patient. BM, bone marrow; IgM, Immunoglobulin M; IgG, Immunoglobulin G. ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 169, 795–803
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positive for the MYD88 L265P mutation, and three revealed a WT sequence, including the two negative cases, with the melting curve analysis. Only one case that was positive with the melting curve analysis showed a WT sequence. This case had an infiltration volume of 10–15%, in keeping with the detection limit for Sanger sequencing. The five sequenced CLL cases and four of five MZL cases analysed remained negative for the MYD88 L265P mutation.
CXCR4 mutation status in WM Mutations in the c-terminal cytoplasmic tail of CXCR4 were found in 17 of 47 (362%) WM/LPL cases (Table II). For the four remaining cases no more material was available for analysis. Of the mutated cases, six (353%) had a frameshift mutation (FS) and 11 (647%) a nonsense mutation (NS). All of the CXCR4-mutated cases were also positive for MYD88 L265P. Table III summarizes the clinical data of the LPL patients at diagnosis according to the presence or absence of the MYD88 and CXCR4 mutations. Comparison of the clinical data of the MYD88L265P/CXCR4WT and the 798
Fig 1. Representative morphology of lymphoplasmacytic lymphoma. (A–B) Haematoxylin and eosin (H&E) staining of a MYD88-mutated lymphoplasmacytic lymphoma case (A: original magnification 50x, B: original magnification 630x). (C) Giemsa stain demonstrates the monotonous small cell infiltration with increased numbers of reactive mast cells (original magnification 630x). (D) Chloroacetate esterase staining highlighting the mast cells (original magnification 400x). (E) CD20 (immunoperoxidase: original magnification 50x), (F) CD138 (immunoperoxidase: original magnification 200x) and (G) IgM stain (immunoperoxidase: original magnification 400x).
MYD88L265P/CXCR4WHIM mutated cases revealed lower levels of leucocytes (P = 002), haemoglobin (P = 005) and platelets (P = 001) as indicators of more active disease (Fig 3). Additionally, although not reaching statistical significance, BM involvement was higher in the MYD88L265P/CXCR4WHIM mutated group (4838% vs. 3437%) whereas lymphadenopathy was more common in the CXCR4WT group (385% vs. 20%). Patients with CXCR4FS showed a significant lower level of leucocytes (P = 005), and patients with CXCR4NS showed a significant lower level of platelets (P = 001) and lower levels of leucocytes (P = 008) when compared to patients with CXCR4WT (Table IV and supplemental Fig 1). BM involvement was higher in both CXCR4FS/NS patients but this did not reach statistical significance. However, no significant differences were observed between CXCR4FS and CXCR4NS mutated cases, most probably due to the low case number in this study. The two cases with MYD88WT and CXCR4WT had very low BM infiltration burden (10–15%), relatively low IgM levels (32 and 103 g/l), no organomegaly at diagnosis, and a history of cold agglutinin disease (CAD) associated with autoimmune haemolytic anaemia. ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 169, 795–803
MYD88 L265P and CXCR4 Mutations in LPL
(A1)
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Fig 2. Sensitivity assessment and analyses of controls and LPL/MZL samples with the melting curve assay. (A) Assessment of the sensitivity of the melting curve assay by a dilution series in the MYD88 L265P heterozygous ABC DLBCL cell lines HBL1 (A1) and TMD8 (A2). DNA was mixed with different amounts of MYD88 wild type DNA (OCI-Ly19). 1: 100% TMD8/HBL1, 2: 50% TMD8/HBL1, 3: 20% TMD8/HBL1, 4: 10% TMD8/HBL1, 5: 5% TMD8/HBL1, 6: 25% TMD8/HBL1, 7: 0% TMD8/HBL1, 8: negative control. (B1) Analysis of the reactive controls without addition of LNA (red) and with 001 lM LNA (blue). (B2) Representative melting curve analysis of a MYD88 L265P-positive case. (B3) Representative melting curve analysis of a MYD88 L265P-negative case. (B4) Analysis of the two MYD88 L265P-negative LPLs after macrodissection. Pos: positive.
Discussion Since the first description of mutations in the gene encoding for the adaptor protein MYD88, especially the L265P missense mutation, in DLBCL, ABC type (Ngo et al, 2011), several groups have identified MYD88 mutations in other B-cell disorders. The MYD88 L265P has been reported to be positive in 70–100% of LPL/WM cases (Treon et al, 2012; Gachard et al, 2013; Varettoni et al, 2013a; Xu et al, 2013). The variation in the positivity in the different studies most probably reflects the different techniques used to detect the mutation. With more sensitive techniques, the L265P mutation is now identified in >90% of the LPL cases and has become a genetic hallmark of the disease. Accordingly, using a melting curve assay with addition of a LNA-clamping oligomere for suppression of the WT signal, we reliably reached a relative detection limit of 5–25% of mutant allele present in EDTA-decalcified, paraffin-embedded BM samples. The high frequency of MYD88 L265P mutation in WM/LPL cases ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 169, 795–803
was confirmed in this study, with 96% of our samples carrying the mutation. The two negative LPL cases had a tumour infiltration of ~10% but remained negative after macrodissection and re-analysis, making a technical failure unlikely. Besides the low BM infiltration volume and the relatively low IgM level at presentation, these patients had a CAD with autoimmune haemolytic anaemia and no organomegaly. Interestingly, a recent study has shown that the lymphoplasmacytic infiltrates found in patients with CAD are always MYD88WT, suggesting that CAD-associated lymphoproliferative disease is a distinct entity different from LPL (Randen et al, 2014). Although our findings in two cases seem to support this contention, more studies are necessary to confirm whether these are two different diseases or if patients with CAD represent a specific subgroup of LPL with MYD88WT. Of note, none of the 49 LPL cases carrying the MYD88 L265P mutation were associated with CAD. The single case of LPL with IgG isotype in our study was positive for the MYD88 L265P mutation. So far, only very few cases of 799
J. Schmidt et al Table II. Somatic variants in CXCR4 detected by Sanger sequencing. Patient
Mutation type
Protein change
Nucleotide change
MYD88 L265P status
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Non-sense Non-sense Non-sense Non-sense Non-sense Non-sense Non-sense Non-sense Non-sense Non-sense Non-sense Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift
p.S338Ter p.S338Ter p.S338Ter p.S338Ter p.S338Ter p.S338Ter p.S338Ter p.S338Ter p.S338Ter p.S338Ter p.S338Ter p.S341 fs p.S341 fs p.T318 fs p.S338 fs p.G336 fs p.S338 fs
c.1013C>G c.1013C>G c.1013C>G c.1013C>G c.1013C>G c.1013_1015insTAA c.1013C>G c.1000C>T c.1013C>G c.1013C>A c.1013C>G c.1021delT c.1021dupT c.950_951dupTC c.1012_1015delTCAT/c.1009_1012delCATT c.1005dupT c.1012_1015delTCAT/c.1009_1012delCATT
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive
WT, wild type; Ter, termination; fs, frameshift; ins, insertion; del, deletion; dup, duplication. Table III. Comparison of clinical data dependent on MYD88 and CXCR4 mutation status.
N Gender (Male/Female) Age (years) BM involvement (%) Leucocytes ( 9 109/l) Haemoglobin (g/l) Platelets ( 9 109/l) Paraprotein IgG (g/l) Paraprotein IgM (g/l) Lymphadenopathy Splenomegaly Leukaemic involvement of PB
MYD88WT/CXCR4WT
MYD88L265P/CXCR4WT
MYD88L265P/CXCR4WHIM
P-value†
2 2/0 765 (70–83) 125 (10–15) 726 (7–751) 106 (95–117) 281 (269–293) – 679 (328–103) 0 0 2 (100%)
28* 20/8 6879 (48–89) 3437 (3–100) 724 (31–119) 1166 (74–160) 29169 (72–499) 94 2642 (61–790) 10 (3846%) 5 (1923%) 5 (1852%)
17* 11/6 7035 (46–91) 4838 (10–100) 552 (25–10) 1017 (75–154) 20387 (47–450) – 3457 (095–729) 3 (20%) 1 (625%) 2 (125%)
074 063 011 002 005 001 na 014 03 038 07
*Not all clinical data were available for every patient. †MYD88L265P/CXCR4WT and MYD88L265P/CXCR4WHIM were compared by Wilcoxon or Chi2 test and a P < 005 was considered significant. The MYD88WT/CXCR4WT group was not included in statistical analyses because of the low case number. BM, bone marrow; PB, peripheral blood; IgM, Immunoglobulin M; IgG, Immunoglobulin G; na, not analysed.
non-IgM LPL have been analysed for this mutation, and both positive and negative results have been reported (Manasanch et al, 2013). Interestingly, the MYD88 L265P mutation seems to represent an early event in the pathogenesis of the disease, because it is also identified in a significant proportion of IgM MGUS, a putative precursor lesion of LPL (Varettoni et al, 2013b; Xu et al, 2013). In contrast, it is only rarely present in splenic MZL (Yan et al, 2012; Gachard et al, 2013; Varettoni et al, 2013a; Xu et al, 2013), MALT lymphoma (Ngo et al, 2011; Gachard et al, 2013) and CLL and completely absent in multiple myeloma (Je et al, 2012; Gachard et al, 2013), entities which might pose a problem in the differential diagnosis of WM/LPL. Although it was not our primary aim to establish the frequency of this mutation in 800
other small B-NHL, our series confirms that it is absent from most cases of CLL and MZL, confirming MYD88 mutational analysis as a valuable diagnostic tool. Of note, a recent study has shown that MYD88 L265P mutations in CLL, although rare (39%), identify a group of young patients with favourable outcome (Martinez-Trillos et al, 2014). To further characterize our LPL cases, the presence of WHIM-like mutations in CXCR4 was also investigated. CXCR4 mutations represent the second most common alteration identified in LPL/WM, and seem to have an impact on clinical presentation and possibly treatment response (Treon et al, 2014). The 362% incidence of CXCR4 mutations observed in our LPL series is higher compared to the 27– 29% published previously (Hunter et al, 2014; Treon et al, ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 169, 795–803
MYD88 L265P and CXCR4 Mutations in LPL
Fig 3. Statistical analysis of BM involvement, serum IgM, haemoglobin, leucocyte and platelet levels of LPL patients at diagnosis with MYD88L265P and CXCR4WT/WHIM mutations. (A) MYD88L265P/CXCR4WT (n = 22), MYD88L265P/CXCR4WHIM (n = 16). (B) MYD88L265P/CXCR4WT (n = 27), MYD88L265P/ CXCR4WHIM (n = 17). (C) MYD88L265P/ CXCR4WT (n = 26), MYD88L265P/CXCR4WHIM (n = 15). (D) MYD88L265P/CXCR4WT (n = 27), MYD88L265P/CXCR4WHIM (n = 16). (E) MYD88L265P/CXCR4WT (n = 26), MYD88L265P/ CXCR4WHIM (n = 15). Data were analysed by Wilcoxon test. * = indicates a P < 005 that was considered significant. Box plots are shown overlaying with the single data points.
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Table IV. Comparison of clinical data dependent on MYD88 and CXCR4 WHIM FS or NS mutation status.
N Gender (Male/Female) Age (years) BM involvement (%) Leucocytes ( 9 109/l) Haemoglobin (g/l) Platelets ( 9 109/l) Paraprotein IgG (g/l) Paraprotein IgM (g/l) Lymphadenopathy Splenomegaly Leukaemic involvement of PB
MYD88L265P/CXCR4WT
MYD88L265P/CXCR4WHIM/FS
MYD88L265P/CXCR4WHIM/NS
P-value†
28* 20/8 6879 (48–89) 3437 (3–100) 724 (31–119) 1166 (74–160) 29169 (72–499) 172 2642 (61–790) 10 (3846%) 5 (1923%) 5 (1852%)
6* 4/2 6383 (46-83) 4583 (20–80) 521 (31–75) 1002 (75–127) 2586 (64–450) – 3274 (095–522) 2 (40%) 1 (1667%) 1 (20%)
11* 7/4 7391 (53–91) 4977 (10–100) 573 (25–10) 1026 (80–154) 1765 (47–360) – 3566 (56–729) 1 (10%) 0 1 (91%)
089 032 029 006 016 003 na 034 024 033 075
*Not all clinical data were available for every patient. †Comparison of all three mutation status together by Wilcoxon test or Chi2 test. A P < 005 was considered significant. BM, bone marrow; PB, peripheral blood; IgM, Immunoglobulin M; IgG, Immunoglobulin G; na, not analysed.
2014). This is most probably not due to the sensitivity of the methods used because Treon et al (2014) used Sanger sequencing, like in the present study, and whole exome ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 169, 795–803
sequencing was used by Hunter et al (2014), which displays a higher sensitivity. In contrast to MYD88 mutations, CXCR4 mutations seem to be acquired later on in the disease and 801
J. Schmidt et al associated with higher disease activity. Accordingly, we confirmed the difference in clinical presentation between patients with mutated or WT CXCR4 gene. CXCR4-mutated LPL cases showed a higher extent of BM infiltration (4838% vs. 3437%), and lower levels of leucocytes (P = 002), haemoglobin (P = 005) and platelets (P = 001). Although failing to reach statistical significance, we observed a higher incidence of lymphadenopathy in the CXCR4WT group (385% vs. 20%), in keeping with recent results, indicating a lower propensity for homing to the BM microenvironment due to the lack of prolonged CXCL12-induced signalling present in CXCR4-mutated cases (Treon et al, 2014). Despite the fact that our analysis did not identify a significant clinical difference at diagnosis between patients with CXCR4WHIM/NS and CXCR4WHIM/FS, probably due to the low patient numbers in the present study, our data show that patients carrying the CXCR4WHIM/NS mutation have higher IgM levels and lower platelet levels at diagnosis. To date, the presence of CXCR4 mutations in MYD88mutated cases of small B-NHL subtypes other than LPL, mainly SMZL, CLL and, perhaps, MALT-lymphoma has not been investigated, and it remains to be seen whether this might aid in discrimination of these entities. In conclusion, molecular examination of EDTA-decalcified BM biopsies for MYD88 L265P is a valuable tool for the differential diagnosis of small B-cell proliferations. The additional examination of CXCR4 in LPL/WM provides important clinical and possibly therapeutically relevant information. The presence of CAD-associated lymphoplasmacytoid infiltrates in BM seems to be associated with a MYD88WT, suggesting a distinct entity or a distinct group within LPL cases.
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Acknowledgements The authors would like to thank Claudia Kloss and Sema Colak for their excellent technical assistance. J.S. was supported, in part, through an internal grant from the Interdisciplinary Centre for Clinical Research (IZKF) of the University of T€ ubingen. This project is in part supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 685) to LQ-M and FF.
Author contributions LQ-M and FF designed the study, supervised the experimental work, analysed data and wrote the manuscript. JaS performed the experimental work and helped writing the manuscript. BF requested and provided clinical information. NS and JuS helped performing the experimental work. IB analysed data and supervised the experimental work.
Competing interest statement The authors declare no conflict of interest.
Supporting Information Additional Supporting Information may be found in the online version of this article: Data S1. Methods. Fig S1. Statistical analysis of BM involvement, serum IgM, leucocytes, haemoglobin and platelet levels of LPL patients at diagnosis with MYD88L265P compared to CXCR4WHIM/FS and CXCR4WHIM/NS mutations.
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MYD88 L265P and CXCR4 mutations in lymphoplasmacytic lymphoma identify cases with high disease activity
Janine Schmidt, Birgit Federmann, Natalie Schindler, Julia Steinhilber, Irina Bonzheim, Falko Fend and Leticia Quintanilla-Martinez Institute of Pathology and Comprehensive Cancer Centre, Eberhard-Karls-University, University Hospital T€ ubingen, T€ ubingen, Germany Received 2 October 2014; accepted for publication 20 January 2015 Correspondence: Leticia Quintanilla-Martinez, Institute of Pathology, University Hospital of T€ ubingen, Liebermeisterstrasse 8, T€ ubingen 72076, Germany. E-mail: Leticia.quintanilla-fend@med. uni-tuebingen.de FF and LQ-M contributed equally to this study.
Summary Recurrent mutations in MYD88 have been identified in >90% of lymphoplasmacytic lymphoma (LPL). Recently, WHIM (warts, hypogammaglobulinaemia, infections, myelokathexis) syndrome-like mutations in CXCR4 have been described in 28% of LPL cases, and seem to impact clinical presentation and response to therapy. We investigated the presence of the MYD88 L265P mutation in 90 decalcified, formalin-fixed, paraffin-embedded (FFPE) bone marrow (BM) biopsies, including 51 cases of LPL, 14 cases of B-cell chronic lymphocytic leukaemia (CLL), 13 cases of marginal zone lymphoma (MZL) and 12 normal controls. In addition, the C-terminal domain of CXCR4 was sequenced in LPL cases. MYD88 L265P was found in 49/51 (96%) LPL cases and in 1/13 (76%) MZL (splenic type), whereas all CLL samples remained negative. The two MYD88 wild type LPL cases were associated with cold agglutinin disease. Mutations in CXCR4 were detected in 17/47 (362%) LPL cases, which showed a higher extent of BM infiltration and lower leucocyte counts (P = 002), haemoglobin (P = 005) and platelet counts (P = 001). In conclusion the detection of MYD88 L265P mutation in FFPE samples is reliable and useful for subtyping small B-cell lymphomas in BM biopsies. In addition, the presence of CXCR4 mutations identifies a subgroup of LPL patients with higher disease activity. Keywords: lymphoplasmacytic lymphoma, WHIM, MYD88 L265P, CXCR4, cold agglutinin disease.
Lymphoplasmacytic lymphoma (LPL) is a rare B-cell lymphoproliferative disorder composed of lymphocytes, plasmacytoid cells and plasma cells usually infiltrating the bone marrow (BM) and sometimes the lymph nodes and spleen, which does not fulfil the criteria for other small B-cell non-Hodgkin lymphomas (B-NHL). Waldenstr€ om macroglobulinaemia (WM) is diagnosed when patients with LPL, in addition to BM infiltration, show a serum immunoglobulin (Ig)M paraprotein of any concentration. Although most patients show a paraproteinaemia of IgM type, other Ig isotypes may occur (Owen et al, 2003; Swerdlow et al, 2008). Autoimmune phenomena or cryoglobulinaemia has been noted in up to 20% of WM patients. Until recently, separation of LPL from other low grade B-NHL was difficult due to the lack of a specific immunophenotype and characteristic genetic aberrations. Recently, using whole genome sequencª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 169, 795–803
ing, a recurrent point mutation in the myeloid differentiation factor 88 gene (MYD88), resulting in a single nucleotide substitution changing a leucine at position 265 of the MYD88 coding region into a proline (L265P) (Ngo et al, 2011), was discovered in 67–100% of LPL/WM (Treon et al, 2012; Gachard et al, 2013; Varettoni et al, 2013a; Xu et al, 2013). MYD88 is an adaptor protein involved in Toll-like receptor (TLR) as well as interleukin (IL) 1 and IL18 signalling (Akira & Takeda, 2004; Kawai & Akira, 2010). Stimulation of MYD88 by TLRs happens through homophilic binding of the Toll-IL1 receptor (TIR; also termed TIRAP) domains of both the adaptor protein and the TLR. Activated MYD88 recruits IL1 receptor-associated kinase 1 (IRAK1) and IRAK4, as well as Bruton tyrosine kinase (BTK) (Yang et al, 2013), ultimately leading to activation of NF-jB (NFKB1). Inhibition of the MYD88 signalling pathway blocks
First published online 29 March 2015 doi: 10.1111/bjh.13361
J. Schmidt et al the translocation of NF-jB into the nucleus in cells expressing MYD88 L265P, and it was shown that WM cells depend on NF-jB signalling for growth and survival (Leleu et al, 2008; Treon et al, 2012; Poulain et al, 2013). The MYD88 L265P is not exclusive to WM/LPL, but can also be identified in a subset of diffuse large B-cell lymphomas (DLBCL) of the activated B-cell type, whereas mutations in other small B-cell lymphomas are rare, including small fractions of splenic marginal zone lymphomas (SMZL) (4–13%), mucosa-associated lymphoid tissue (MALT) lymphomas (7–9%) (Ngo et al, 2011; Gachard et al, 2013) and rare cases of chronic lymphocytic leukaemia (CLL) (Martinez-Trillos et al, 2014). This mutation is absent in nodal MZLs and multiple myeloma (MM) (Je et al, 2012; Gachard et al, 2013; Xu et al, 2013). Given the difficulties in separating SMZL and MALT-lymphomas from LPL/WM with unusual clinical presentations, the true frequency and relevance of MYD88 mutations in these entities is not yet clear. In the pathogenesis of WM/LPL, this mutation seems to be an early event, because it can also be identified in 50–80% of IgM monoclonal gammopathy of undetermined significance (MGUS), a precursor lesion for WM. This indicates that secondary genetic alterations are necessary to acquire the fully malignant phenotype. In addition to the MYD88 L265P, a variety of other recurrent genetic alterations have been identified in LPL, most notably mutations in CXCR4, which are partly identical to germline mutations in WHIM (warts, hypogammaglobulinaemia, infections, myelokathexis) syndrome (Hernandez et al, 2003). In WHIM syndrome, these mutations prolong G-protein signalling upon receptor stimulation due to loss of inhibitory serines in the cytoplasmic tail of the protein. CXCR4 stimulation by its ligand CXCL12/SDF1 activates downstream signalling in WM cells, increasing migration and adhesion to the BM microenvironment (Ngo et al, 2008). It has been suggested that mutations in CXCR4, which virtually always co-exist with the MYD88 L265P, are associated with significantly higher serum IgM levels, more BM involvement and symptomatic disease. Very recent data indicate that this might be mainly restricted to cases carrying nonsense, rather than frameshift CXCR4 mutations (Treon et al, 2014). Of interest, despite their association with higher disease burden, CXCR4 mutations do not have a negative impact on overall survival. In addition to modulating disease presentation, CXCR4 mutations seem to have an impact on response to novel treatments, such as the BTK inhibitor ibrutinib (Cao et al, 2015). To date, several different methods have been published for the detection of MYD88 L265P, but these studies used mainly DNA from fresh BM or peripheral blood mononuclear cells. We therefore designed a sensitive and reliable assay for the detection of MYD88 L265P in decalcified formalin-fixed, paraffin-embedded (FFPE) BM biopsies and tested its diagnostic utility for classification of small B-cell lymphomas. Given the potential clinical relevance of WHIMlike CXCR4 mutations in LPL, we additionally studied their 796
presence in LPL cases and correlated them with clinico-pathological findings.
Materials and methods Sample collection and DNA extraction Fifty-one EDTA-decalcified FFPE BM trephine biopsies involved by LPL diagnosed between December 2000 and September 2014 at the Institute of Pathology, University Hospital of T€ ubingen, were included in the study. Additionally, FFPE BM biopsies of 13 cases of MZL (eight splenic MZL, four nodal MZL and one extranodal MZL) and 14 cases of CLL diagnosed in the same period were included in the study. All cases included were newly diagnosed without previous treatment. Cases were re-evaluated using haematoxylin and eosin (H&E), Giemsa, AS-D chloroacetate esterase and reticulin stains and standard diagnostic immunohistochemistry (see Data S1). Classification of the cases as LPL, MZL or CLL was according to the 2008 World Health Organization (WHO) classification and the Second International Workshop on WM (Owen et al, 2003; Swerdlow et al, 2008). In addition, 12 reactive BM biopsies without lymphoma involvement were included as controls. Paraffin-embedded cell blocks of the cell lines TMD8, HBL1 (MYD88 L265P heterozygous) and OCI-Ly19 (wild type, WT), provided by Alexander Weber (University of T€ ubingen, Germany), were used for preparation of dilution series for sensitivity testing. TMD8 and HBL1 are DLBCL cell lines with an activated B-cell (ABC) gene expression profile, whereas OCI-Ly19 has a germinal centre B-cell (GCB) gene expression profile. DNA was isolated from whole tissue sections (unless stated differently) and cell blocks, respectively, after protease digestion with a standard phenol-chloroform-extraction protocol (Sotlar et al, 2003).
Detection of MYD88 L265P by locked-nucleic-acid (LNA) -clamped polymerase chain reaction (PCR) with melting curve analysis Amplification of both WT and mutant DNA was performed on a LightCycler 480 instrument (Roche Diagnostics, Mannheim, Germany) using primers (forward primer 50 -TG CAGGGGTTGGTGTAGT-30 ; reverse primer 50 -GTTGTTAA CCCTGGGGTTG-30 ) and FRET hybridization probes (L265P mut 50 -CAGAAGCGACCGATCCCC-30 ; L265P Anc 50 -CAAG TACAAGGCAATGAAGAAAGAGTTCCC-30 ) from TIB Molbiol (Berlin, Germany). The hybridization probe L265P mut was mutant-specific, therefore resulting in a higher melting temperature for samples carrying the mutation as compared to WT samples. In order to detect minimal quantities of mutant DNA we added 001 lM of a WT-specific lockednucleic-acid (LNA) clamping oligomere (50 -AAGCGACTGATCCC-30 ; TIB Molbiol) for suppression of the WT signal and enhancement of the sensitivity (see Data S1). ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 169, 795–803
MYD88 L265P and CXCR4 Mutations in LPL
Sequencing of MYD88 and CXCR4 Forward and reverse primers designed for LNA-clamped PCR were used to amplify a 189 bp fragment covering a section of exon 5 of the MYD88 gene, including L265 for subsequent Sanger sequencing. For analysis of the CXCR4 gene, M13-tailed primers (forward: 50 - TGTAAAACGACGGCCAG TACATTGGGATCAGCATCGAC-30 ; reverse: 50 - CAGGAAAC AGCTATGACCATCTGTGTTAGCTGGAGTG-30 ; TIB Molbiol) were designed to amplify a 336 bp fragment covering a part of the carboxyl-terminal (c-terminal) domain (see Data S1). Purification of PCR products and subsequent analysis of sequencing reactions was performed as previously described (Adam et al, 2013).
(median = 3974%). LPL showed a diffuse or nodular, interand peritrabecular infiltration pattern with relatively small, monotonous lymphoid cells, lymphoplasmacytic cells and plasma cells accompanied by an increased number of reactive mast cells (Fig 1A–D). Immunohistochemically, LPL cases showed expression of pan-B-cell markers and a variably prominent plasma cell component with light chain restriction and IgM expression (Fig 1E–F). Only one patient in this study showed an IgG paraprotein. The age and sex ratio at presentation was similar among LPL, MZL and CLL patients. However, patients with CLL tended to have a higher BM tumour burden than patients with LPL or MZL infiltration and to be younger at first diagnosis.
Assessment of MYD88 L265P in LPL and other small B-NHLs
Clinical features Clinical data were collected from the files of the Department of Internal Medicine II (Haematology and Oncology) or obtained by contacting the primary physicians. The study was approved by the local ethics committee (105/2013BO2).
Statistical analysis Statistical analysis of the clinical data was calculated with JMP software, version 10 (SAS Institute GmbH, B€ oblingen, Germany). Clinical parameters were compared depended on the mutation status of MYD88 and CXCR4 using Wilcoxon or Chi2 test for comparison of nominal data; P < 005 were considered significant.
Results Clinical and histological features A total of 51 LPL, 13 MZL and 14 CLL cases were included in the study. The clinical findings are summarized in Table I. The median age of patients with LPL was 692 years (range 46–91) with a slight male predominance. The mean IgM level in serum at diagnosis was 287 g/l (range 095–79). The extent of BM infiltration ranged from 3% to 100%
The sensitivity of the assay was first assessed by a dilution series using DNA from the MYD88 L265P heterozygous ABC DLBCL cell lines, TMD8 and HBL1, and from the GCB DLBCL cell line, OCI-Ly19, which has WT MYD88. The melting curve analysis following amplification of the DNA resulted in two different melting peaks, at ~54°C for the WT allele and ~62°C for the mutant allele. In the dilution series of the two mutant cell lines with addition of the LNA oligomer, 25–5% mutant DNA was reliably detected (Fig 2A). The MYD88 L265P mutation was detected in 49/51 (96%) LPL patients whereas all 12 reactive controls remained negative (Fig 2B1–B2). The two LPL patients with WT MYD88 had a BM infiltration of ~10%. To investigate whether the negativity was due to the low tumour cell content, macrodissection of the lymphoma areas was performed. However, the two cases remained negative for MYD88 L265P (Fig 2 B3–B4). The single LPL case with paraprotein of IgG isotype carried the MYD88 L265P mutation. Of the other entities included in the study, only one of 13 (77%) MZL, a splenic MZL, was positive for the MYD88 L265P mutation, and all CLL samples remained negative. For confirmation, a total of 20 cases were subjected to Sanger sequencing. Of 10 examined LPL cases, seven were clearly
Table I. Clinical data of patients with WM/LPL, MZL or B-CLL.
Age (years), median (range) Sex (male/female) BM involvement, mean % (range) Paraprotein IgM (g/l), (range) Paraprotein IgG (g/l) (n = 1) Haemoglobin (g/l) Adenopathy Splenomegaly
WM/LPL (n = 51)*
MZL (n = 13)*
B-CLL (n = 14)*
692 (46–91) 33/15 3974 (3-100) 287 (095-790) 94 111 (74–160) 14 (3181%) 6 (1333%)
695 (55–84) 7/6 3523 (10-90) – – 122 (87–171) 1 (11%) 6 (667%)
633 (50–83) 8/6 625 (15-100) – – 93.4 (50–153) 3 (273%) 6 (545%)
*Not all clinical data were available for every patient. BM, bone marrow; IgM, Immunoglobulin M; IgG, Immunoglobulin G. ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 169, 795–803
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positive for the MYD88 L265P mutation, and three revealed a WT sequence, including the two negative cases, with the melting curve analysis. Only one case that was positive with the melting curve analysis showed a WT sequence. This case had an infiltration volume of 10–15%, in keeping with the detection limit for Sanger sequencing. The five sequenced CLL cases and four of five MZL cases analysed remained negative for the MYD88 L265P mutation.
CXCR4 mutation status in WM Mutations in the c-terminal cytoplasmic tail of CXCR4 were found in 17 of 47 (362%) WM/LPL cases (Table II). For the four remaining cases no more material was available for analysis. Of the mutated cases, six (353%) had a frameshift mutation (FS) and 11 (647%) a nonsense mutation (NS). All of the CXCR4-mutated cases were also positive for MYD88 L265P. Table III summarizes the clinical data of the LPL patients at diagnosis according to the presence or absence of the MYD88 and CXCR4 mutations. Comparison of the clinical data of the MYD88L265P/CXCR4WT and the 798
Fig 1. Representative morphology of lymphoplasmacytic lymphoma. (A–B) Haematoxylin and eosin (H&E) staining of a MYD88-mutated lymphoplasmacytic lymphoma case (A: original magnification 50x, B: original magnification 630x). (C) Giemsa stain demonstrates the monotonous small cell infiltration with increased numbers of reactive mast cells (original magnification 630x). (D) Chloroacetate esterase staining highlighting the mast cells (original magnification 400x). (E) CD20 (immunoperoxidase: original magnification 50x), (F) CD138 (immunoperoxidase: original magnification 200x) and (G) IgM stain (immunoperoxidase: original magnification 400x).
MYD88L265P/CXCR4WHIM mutated cases revealed lower levels of leucocytes (P = 002), haemoglobin (P = 005) and platelets (P = 001) as indicators of more active disease (Fig 3). Additionally, although not reaching statistical significance, BM involvement was higher in the MYD88L265P/CXCR4WHIM mutated group (4838% vs. 3437%) whereas lymphadenopathy was more common in the CXCR4WT group (385% vs. 20%). Patients with CXCR4FS showed a significant lower level of leucocytes (P = 005), and patients with CXCR4NS showed a significant lower level of platelets (P = 001) and lower levels of leucocytes (P = 008) when compared to patients with CXCR4WT (Table IV and supplemental Fig 1). BM involvement was higher in both CXCR4FS/NS patients but this did not reach statistical significance. However, no significant differences were observed between CXCR4FS and CXCR4NS mutated cases, most probably due to the low case number in this study. The two cases with MYD88WT and CXCR4WT had very low BM infiltration burden (10–15%), relatively low IgM levels (32 and 103 g/l), no organomegaly at diagnosis, and a history of cold agglutinin disease (CAD) associated with autoimmune haemolytic anaemia. ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 169, 795–803
MYD88 L265P and CXCR4 Mutations in LPL
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(B4)
Fig 2. Sensitivity assessment and analyses of controls and LPL/MZL samples with the melting curve assay. (A) Assessment of the sensitivity of the melting curve assay by a dilution series in the MYD88 L265P heterozygous ABC DLBCL cell lines HBL1 (A1) and TMD8 (A2). DNA was mixed with different amounts of MYD88 wild type DNA (OCI-Ly19). 1: 100% TMD8/HBL1, 2: 50% TMD8/HBL1, 3: 20% TMD8/HBL1, 4: 10% TMD8/HBL1, 5: 5% TMD8/HBL1, 6: 25% TMD8/HBL1, 7: 0% TMD8/HBL1, 8: negative control. (B1) Analysis of the reactive controls without addition of LNA (red) and with 001 lM LNA (blue). (B2) Representative melting curve analysis of a MYD88 L265P-positive case. (B3) Representative melting curve analysis of a MYD88 L265P-negative case. (B4) Analysis of the two MYD88 L265P-negative LPLs after macrodissection. Pos: positive.
Discussion Since the first description of mutations in the gene encoding for the adaptor protein MYD88, especially the L265P missense mutation, in DLBCL, ABC type (Ngo et al, 2011), several groups have identified MYD88 mutations in other B-cell disorders. The MYD88 L265P has been reported to be positive in 70–100% of LPL/WM cases (Treon et al, 2012; Gachard et al, 2013; Varettoni et al, 2013a; Xu et al, 2013). The variation in the positivity in the different studies most probably reflects the different techniques used to detect the mutation. With more sensitive techniques, the L265P mutation is now identified in >90% of the LPL cases and has become a genetic hallmark of the disease. Accordingly, using a melting curve assay with addition of a LNA-clamping oligomere for suppression of the WT signal, we reliably reached a relative detection limit of 5–25% of mutant allele present in EDTA-decalcified, paraffin-embedded BM samples. The high frequency of MYD88 L265P mutation in WM/LPL cases ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 169, 795–803
was confirmed in this study, with 96% of our samples carrying the mutation. The two negative LPL cases had a tumour infiltration of ~10% but remained negative after macrodissection and re-analysis, making a technical failure unlikely. Besides the low BM infiltration volume and the relatively low IgM level at presentation, these patients had a CAD with autoimmune haemolytic anaemia and no organomegaly. Interestingly, a recent study has shown that the lymphoplasmacytic infiltrates found in patients with CAD are always MYD88WT, suggesting that CAD-associated lymphoproliferative disease is a distinct entity different from LPL (Randen et al, 2014). Although our findings in two cases seem to support this contention, more studies are necessary to confirm whether these are two different diseases or if patients with CAD represent a specific subgroup of LPL with MYD88WT. Of note, none of the 49 LPL cases carrying the MYD88 L265P mutation were associated with CAD. The single case of LPL with IgG isotype in our study was positive for the MYD88 L265P mutation. So far, only very few cases of 799
J. Schmidt et al Table II. Somatic variants in CXCR4 detected by Sanger sequencing. Patient
Mutation type
Protein change
Nucleotide change
MYD88 L265P status
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Non-sense Non-sense Non-sense Non-sense Non-sense Non-sense Non-sense Non-sense Non-sense Non-sense Non-sense Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift
p.S338Ter p.S338Ter p.S338Ter p.S338Ter p.S338Ter p.S338Ter p.S338Ter p.S338Ter p.S338Ter p.S338Ter p.S338Ter p.S341 fs p.S341 fs p.T318 fs p.S338 fs p.G336 fs p.S338 fs
c.1013C>G c.1013C>G c.1013C>G c.1013C>G c.1013C>G c.1013_1015insTAA c.1013C>G c.1000C>T c.1013C>G c.1013C>A c.1013C>G c.1021delT c.1021dupT c.950_951dupTC c.1012_1015delTCAT/c.1009_1012delCATT c.1005dupT c.1012_1015delTCAT/c.1009_1012delCATT
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive
WT, wild type; Ter, termination; fs, frameshift; ins, insertion; del, deletion; dup, duplication. Table III. Comparison of clinical data dependent on MYD88 and CXCR4 mutation status.
N Gender (Male/Female) Age (years) BM involvement (%) Leucocytes ( 9 109/l) Haemoglobin (g/l) Platelets ( 9 109/l) Paraprotein IgG (g/l) Paraprotein IgM (g/l) Lymphadenopathy Splenomegaly Leukaemic involvement of PB
MYD88WT/CXCR4WT
MYD88L265P/CXCR4WT
MYD88L265P/CXCR4WHIM
P-value†
2 2/0 765 (70–83) 125 (10–15) 726 (7–751) 106 (95–117) 281 (269–293) – 679 (328–103) 0 0 2 (100%)
28* 20/8 6879 (48–89) 3437 (3–100) 724 (31–119) 1166 (74–160) 29169 (72–499) 94 2642 (61–790) 10 (3846%) 5 (1923%) 5 (1852%)
17* 11/6 7035 (46–91) 4838 (10–100) 552 (25–10) 1017 (75–154) 20387 (47–450) – 3457 (095–729) 3 (20%) 1 (625%) 2 (125%)
074 063 011 002 005 001 na 014 03 038 07
*Not all clinical data were available for every patient. †MYD88L265P/CXCR4WT and MYD88L265P/CXCR4WHIM were compared by Wilcoxon or Chi2 test and a P < 005 was considered significant. The MYD88WT/CXCR4WT group was not included in statistical analyses because of the low case number. BM, bone marrow; PB, peripheral blood; IgM, Immunoglobulin M; IgG, Immunoglobulin G; na, not analysed.
non-IgM LPL have been analysed for this mutation, and both positive and negative results have been reported (Manasanch et al, 2013). Interestingly, the MYD88 L265P mutation seems to represent an early event in the pathogenesis of the disease, because it is also identified in a significant proportion of IgM MGUS, a putative precursor lesion of LPL (Varettoni et al, 2013b; Xu et al, 2013). In contrast, it is only rarely present in splenic MZL (Yan et al, 2012; Gachard et al, 2013; Varettoni et al, 2013a; Xu et al, 2013), MALT lymphoma (Ngo et al, 2011; Gachard et al, 2013) and CLL and completely absent in multiple myeloma (Je et al, 2012; Gachard et al, 2013), entities which might pose a problem in the differential diagnosis of WM/LPL. Although it was not our primary aim to establish the frequency of this mutation in 800
other small B-NHL, our series confirms that it is absent from most cases of CLL and MZL, confirming MYD88 mutational analysis as a valuable diagnostic tool. Of note, a recent study has shown that MYD88 L265P mutations in CLL, although rare (39%), identify a group of young patients with favourable outcome (Martinez-Trillos et al, 2014). To further characterize our LPL cases, the presence of WHIM-like mutations in CXCR4 was also investigated. CXCR4 mutations represent the second most common alteration identified in LPL/WM, and seem to have an impact on clinical presentation and possibly treatment response (Treon et al, 2014). The 362% incidence of CXCR4 mutations observed in our LPL series is higher compared to the 27– 29% published previously (Hunter et al, 2014; Treon et al, ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 169, 795–803
MYD88 L265P and CXCR4 Mutations in LPL
Fig 3. Statistical analysis of BM involvement, serum IgM, haemoglobin, leucocyte and platelet levels of LPL patients at diagnosis with MYD88L265P and CXCR4WT/WHIM mutations. (A) MYD88L265P/CXCR4WT (n = 22), MYD88L265P/CXCR4WHIM (n = 16). (B) MYD88L265P/CXCR4WT (n = 27), MYD88L265P/ CXCR4WHIM (n = 17). (C) MYD88L265P/ CXCR4WT (n = 26), MYD88L265P/CXCR4WHIM (n = 15). (D) MYD88L265P/CXCR4WT (n = 27), MYD88L265P/CXCR4WHIM (n = 16). (E) MYD88L265P/CXCR4WT (n = 26), MYD88L265P/ CXCR4WHIM (n = 15). Data were analysed by Wilcoxon test. * = indicates a P < 005 that was considered significant. Box plots are shown overlaying with the single data points.
(A)
(B)
(C)
(D)
(E)
Table IV. Comparison of clinical data dependent on MYD88 and CXCR4 WHIM FS or NS mutation status.
N Gender (Male/Female) Age (years) BM involvement (%) Leucocytes ( 9 109/l) Haemoglobin (g/l) Platelets ( 9 109/l) Paraprotein IgG (g/l) Paraprotein IgM (g/l) Lymphadenopathy Splenomegaly Leukaemic involvement of PB
MYD88L265P/CXCR4WT
MYD88L265P/CXCR4WHIM/FS
MYD88L265P/CXCR4WHIM/NS
P-value†
28* 20/8 6879 (48–89) 3437 (3–100) 724 (31–119) 1166 (74–160) 29169 (72–499) 172 2642 (61–790) 10 (3846%) 5 (1923%) 5 (1852%)
6* 4/2 6383 (46-83) 4583 (20–80) 521 (31–75) 1002 (75–127) 2586 (64–450) – 3274 (095–522) 2 (40%) 1 (1667%) 1 (20%)
11* 7/4 7391 (53–91) 4977 (10–100) 573 (25–10) 1026 (80–154) 1765 (47–360) – 3566 (56–729) 1 (10%) 0 1 (91%)
089 032 029 006 016 003 na 034 024 033 075
*Not all clinical data were available for every patient. †Comparison of all three mutation status together by Wilcoxon test or Chi2 test. A P < 005 was considered significant. BM, bone marrow; PB, peripheral blood; IgM, Immunoglobulin M; IgG, Immunoglobulin G; na, not analysed.
2014). This is most probably not due to the sensitivity of the methods used because Treon et al (2014) used Sanger sequencing, like in the present study, and whole exome ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 169, 795–803
sequencing was used by Hunter et al (2014), which displays a higher sensitivity. In contrast to MYD88 mutations, CXCR4 mutations seem to be acquired later on in the disease and 801
J. Schmidt et al associated with higher disease activity. Accordingly, we confirmed the difference in clinical presentation between patients with mutated or WT CXCR4 gene. CXCR4-mutated LPL cases showed a higher extent of BM infiltration (4838% vs. 3437%), and lower levels of leucocytes (P = 002), haemoglobin (P = 005) and platelets (P = 001). Although failing to reach statistical significance, we observed a higher incidence of lymphadenopathy in the CXCR4WT group (385% vs. 20%), in keeping with recent results, indicating a lower propensity for homing to the BM microenvironment due to the lack of prolonged CXCL12-induced signalling present in CXCR4-mutated cases (Treon et al, 2014). Despite the fact that our analysis did not identify a significant clinical difference at diagnosis between patients with CXCR4WHIM/NS and CXCR4WHIM/FS, probably due to the low patient numbers in the present study, our data show that patients carrying the CXCR4WHIM/NS mutation have higher IgM levels and lower platelet levels at diagnosis. To date, the presence of CXCR4 mutations in MYD88mutated cases of small B-NHL subtypes other than LPL, mainly SMZL, CLL and, perhaps, MALT-lymphoma has not been investigated, and it remains to be seen whether this might aid in discrimination of these entities. In conclusion, molecular examination of EDTA-decalcified BM biopsies for MYD88 L265P is a valuable tool for the differential diagnosis of small B-cell proliferations. The additional examination of CXCR4 in LPL/WM provides important clinical and possibly therapeutically relevant information. The presence of CAD-associated lymphoplasmacytoid infiltrates in BM seems to be associated with a MYD88WT, suggesting a distinct entity or a distinct group within LPL cases.
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Acknowledgements The authors would like to thank Claudia Kloss and Sema Colak for their excellent technical assistance. J.S. was supported, in part, through an internal grant from the Interdisciplinary Centre for Clinical Research (IZKF) of the University of T€ ubingen. This project is in part supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 685) to LQ-M and FF.
Author contributions LQ-M and FF designed the study, supervised the experimental work, analysed data and wrote the manuscript. JaS performed the experimental work and helped writing the manuscript. BF requested and provided clinical information. NS and JuS helped performing the experimental work. IB analysed data and supervised the experimental work.
Competing interest statement The authors declare no conflict of interest.
Supporting Information Additional Supporting Information may be found in the online version of this article: Data S1. Methods. Fig S1. Statistical analysis of BM involvement, serum IgM, leucocytes, haemoglobin and platelet levels of LPL patients at diagnosis with MYD88L265P compared to CXCR4WHIM/FS and CXCR4WHIM/NS mutations.
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