- Email: [email protected]
Nuclear Factor-κB Pathway–Activating Gene Aberrancies in Primary Cutaneous Large B-Cell Lymphoma, Leg Type
L Koens et al. NF-kB-Activating Gene Aberrancies in PCLBCL-LT
Abdlsattar Zebary1, Katarina Omholt1, Remco van Doorn2, Paola Ghiorzo3, Katja Harbst4, Carolina Hertzman Johansson1, Veronica Ho¨iom1, Go¨ran Jo¨nsson4, Dace Pjanova5, Susana Puig6, Giovanna B. Scarra3, Mark Harland 7, Ha˚kan Olsson4, Suzanne Egyhazi Brage1, Jane Palmer 8, Lena Kanter-Lewensohn1, Ismini Vassilaki 9, Nicholas K. Hayward 8, Julia Newton-Bishop7, Nelleke A. Gruis2, Johan Hansson1 and the Melanoma Genetics Consortium (GenoMEL) 1
Department of Oncology-Pathology, Cancer Center Karolinska, Karolinska Institutet, Stockholm, Sweden; 2Department of Dermatology, Leiden University Medical Centre, Leiden, The Netherlands; 3Department of Internal Medicine and Medical Specialties, University of Genoa and Laboratory of Genetics of Rare Hereditary Cancers, IRCCS San MartinoIST Research Hospital, Genoa, Italy; 4 Department of Oncology, Clinical Sciences,
Lund University, Lund, Sweden; 5Latvian Biomedical Research and Study Centre, Riga, Latvia; 6Melanoma Unit, Department of Dermatology, Hospital Clinic, University of Barcelona, IDIBAPS and CIBER de Enfermedades Raras, Barcelona, Spain; 7Section of Epidemiology and Biostatistics, Cancer Research UK Clinical Centre, Leeds Institute of Molecular Medicine, University of Leeds, Leeds, UK; 8Queensland Institute of Medical Research, Brisbane, Queensland, Australia and 9Karolinska University Hospital, Dermatologic Diagnostic Centrum Solna, Stockholm, Sweden E-mail: [email protected]
SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid
REFERENCES Eskandarpour M, Hashemi J, Kanter L et al. (2003) Frequency of UV-inducible NRAS mutations in melanomas of patients with germline CDKN2A mutations. J Natl Cancer Inst 95: 790–8
Fecher LA, Amaravadi RK, Flaherty KT (2008) The MAPK pathway in melanoma. Curr Opin Oncol 20:183–9 Goldstein AM, Chan M, Harland M et al. (2006) High-risk melanoma susceptibility genes and pancreatic cancer, neural system tumors, and uveal melanoma across GenoMEL. Cancer Res 66:9818–28 Hansson J (2008) Familial melanoma. Surg Clin North Am 88:897–916 Hayward NK (2003) Genetics of melanoma predisposition. Oncogene 22:3053–62 Jovanovic B, Egyhazi S, Eskandarpour M et al. (2010) Coexisting NRAS and BRAF mutations in primary familial melanomas with specific CDKN2A germline alterations. J Invest Dermatol 130:618–20 Lee JH, Choi JW, Kim YS (2011) Frequencies of BRAF and NRAS mutations are different in histological types and sites of origin of cutaneous melanoma: a meta-analysis. Br J Dermatol 164:776–84 Omholt K, Grafstro¨m E, Kanter-Lewensohn L et al. (2011) KIT pathway alterations in mucosal melanomas of the vulva and other sites. Clin Cancer Res 17:3933–42
Nuclear Factor-jB Pathway–Activating Gene Aberrancies in Primary Cutaneous Large B-Cell Lymphoma, Leg Type Journal of Investigative Dermatology (2014) 134, 290–292; doi:10.1038/jid.2013.265; published online 18 July 2013
TO THE EDITOR Primary cutaneous large B-cell lymphoma, leg type (PCLBCL-LT) is an aggressive cutaneous lymphoma with a 5-year overall survival of approximately 40%. Because of its aggressive nature, the treatment of first choice is anthracyclin-based chemotherapy combined with rituximab (Senff et al., 2008). However, patients often show a progressive disease course despite treatment with polychemotherapy. Furthermore, owing to age and comorbidity, not all patients are eligible for this treatment. Therefore, new and additional therapies for PCLBCL-LT are necessary, with a focus on more specific, targeted therapies with fewer side effects than conventional chemotherapy. At the mRNA expression level, PCLBCL-LT
resembles nodal activated B-cell-type diffuse large B-cell lymphoma (ABCDLBCL), including strong expression of known targets of the NF-kB pathway, such as IRF4 (Davis et al., 2001; Hoefnagel et al., 2005). In nodal ABC-DLBCL, increased NF-kB activity has a role in its pathogenesis through transcriptional activation of genes involved in cellular survival mechanisms. Previous studies demonstrated that mutations in multiple genes can cause deregulation of NF-kB signaling in nodal ABC-DLBCL (Compagno et al., 2009). The genes most frequently affected by these NF-kB-activating genetic aberrations are TNFAIP3 (A20), CD79B, CARD11 (CARMA1), and MYD88, which we investigated in 10 cases of PCLBCLLT (Supplementary Table S1 online).
Abbreviations: ABC-DLBCL, activated B-cell-type diffuse large B-cell lymphoma; cDNA, complementary DNA; PCLBCL-LT, primary cutaneous large B-cell lymphoma, leg type Accepted article preview online 13 June 2013; published online 18 July 2013
290
Journal of Investigative Dermatology (2014), Volume 134
TNFAIP3 (A20) is a tumor suppressor gene, downregulating NF-kB signaling by targeting several proteins involved in the activation of this pathway. By quantitative PCR, transcriptional levels of TNFAIP3 varied in our PCLBCL-LT cases (Supplementary Figure S1 online), and therefore we investigated potential underlying mechanisms for this variation. Four cases with relatively low mRNA expression levels showed heterozygous deletion of the gene region by highdensity fine-tiling comparative genomic hybridization. Homozygous deletions as observed in 10% of nodal ABC-DLBCL (Compagno et al., 2009) were not found. In cases without deletion of TNFAIP3, but displaying relatively low levels of mRNA, we could not detect epigenetic silencing through promoter hypermethylation, as was encountered in approximately 40% of nodal ABC-DLBCL (Honma et al., 2009). Possibly, in these cases, reduced mRNA expression was regulated by altered transcription factor
L Koens et al. NF-kB-Activating Gene Aberrancies in PCLBCL-LT
demonstrated to influence proliferation and survival in normal B cells in a gene dose–dependent manner (Chu et al., 2011), the relatively low expression of TNFAIP3 in most of the PCLBCL-LT samples can still be functionally relevant and might suggest a role for this
L IG L IG
IG IG L L
activity. One-third of cases of nodal ABC-DLBCL display genetic alterations in intron 3, leading to transcripts with a premature stop codon, giving rise to truncated nonfunctional proteins. Again, we could not detect these transcripts in PCLBCL-LT. As TNFAIP3 was
IGH IGH IL-1R
TLR
CD79B CD79A
Activating mutation first ITAM domain:20% SYK
IRAK4 IRAK1
BTK
Cytoplasm
MYD88
LYN
Activating mutation L265P: 30%
TRAF6 PKCβ BCL10
Activating mutation coiled-coil domain:10%
CARD11
MALT1 NEMO
Heterozygous deletion:40%
A20
IKKα IKKβ
NF-κB Nucleus NF-κB Survival proliferation
Figure 1. Pathway overview and genetic aberrancies in primary cutaneous large B-cell lymphoma, leg type. The suggested relations between the different components of B-cell receptor signaling and MYD88 signaling are represented (Yang et al., 2012). The molecules affected by genetic aberrancies in PCLBCL-LT are indicated in red, and the percentages of cases affected by these aberrancies are indicated. Molecules that can be therapeutically targeted for inhibition of the NF-kB pathway are represented in green.
tumor suppressor in the pathogenesis of PCLBCL-LT. CD79B is a component of the B-cell receptor, and has a combined function with CD79A in activating the NF-kB pathway (Figure 1; Dal Porto et al., 2004). Recurrent somatic mutations in the first immunoreceptor tyrosine-based activation motif tyrosine Y196 of CD79B were detected in 18% of nodal ABC-DLBCL. NF-kB pathway activation through this mutation is called chronic active B-cell receptor signaling, and occurs in cases with wildtype CARD11. In our series, concordant with nodal ABC-DLBCL, in 20% an Y196 mutation was detected in DNA and complementary DNA (cDNA), in the absence of mutations in CARD11, suggesting that chronic active B-cell receptor signaling is a mechanism involved in these cases of PCLBCL-LT. CARD11 is a signaling scaffold protein that functions as a critical component in constitutive NF-kB activation in ABC-DLBCL (Ngo et al., 2006), downstream of the B-cell receptor (Figure 1). In one sample, we found a missense mutation in codon 415 of exon 9 (D415E), and a R423W mutation in the same exon in both DNA and cDNA. The functional relevance of these D415E and R423W mutations is not known. However, mutations in the coiled-coil domain of CARD11, as encountered in 10% of nodal ABC-DLBCL, are
Table 1. Overview of genetic aberrancies TNFAIP3/A20 Splicing site Case Del. Hypermethylation qPCR (DDCq) Mut. Splice variant
CD79B
CARD11
MYD88
Y196 ITAM mutation
Mutation coiled-coil domain
L265P mutation
DNA
cDNA
Exons 5–8 and 10
Exon 9
DNA
cDNA
1
No
No
0.50
No
No
No
No
No
No
L265P
L265P
2
No
No
0.51
No
No
No
No
No
No
No
No
3
No
No
0.08
No
No
No
No
No
No
No
No
4
±
No
0.11
No
No
No
No
No
No
L265P
L265P
5
±
No
0.13
No
No
No
No
No
D415E, R423W
No
No
6
No
No
NA
NA
NA
No
No
No
No1
No
No
7
No
NA
0.03
No
No
Y196H
Y196H
No
No
L265P
No
8
No
No
0.17
No
No
Y196F
Y196F
No
No
L265P
L265p
9
±
NA
0.01
No
No
NA
No
No
No
NA
No
10
±
No
1
No
No
No
0.01
No
No
No
No
No
Abbreviations: cDNA, complementary DNA; Del., deletion; Mut., mutation; NA, not assessed; qPCR, quantitative PCR; ±, heterozygous deletion. 1 A silent mutation (D415D) was delected.
www.jidonline.org
291
L Koens et al. NF-kB-Activating Gene Aberrancies in PCLBCL-LT
potentially relevant in constitutive activation of the NF-kB pathway, as changes in this domain have the potential to disrupt autoinhibition of CARD11 signaling, leading to receptor-independent activation (Lamason et al., 2010). MYD88 is a Toll-like receptor–associated adaptor protein, among others involved in the activation of NF-kB signaling (Figure 1). Nodal ABC-DLBCL showed L265P mutations in 29% of cases, and the consequent amino-acid substitution in the bD sheet of MYD88 has been shown to be oncogenically active, leading to aberrant activation of NF-kB signaling (Ngo et al., 2011), a mechanism most likely not related to (chronic active) B-cell receptor signaling (Figure 1). Somatic MYD88 L265P mutations in PCLBCL-LT have been previously described in 69% of cases (Pham-Ledard et al., 2012). We encountered a 40% mutation rate in our series, concordant with nodal ABC-DLBCLs. Furthermore, we showed that not all mutations in the DNA seem to be transcribed to mRNA, as for one sample the mutation was not detected in cDNA. The relevance of the above-described mutations for novel therapeutic strategies has already been explored. For example, sotrastaurin can interfere with NF-kB signaling by the inhibition of PKC-b, which is required for CARD11-dependent activation of the NF-kB pathway (Sommer et al., 2005). In a mouse model, sotrastaurin was selectively toxic for CD79mutant DLBCL (Naylor et al., 2011) as compared with unmutated DLBCL, but only in the presence of wild-type CARD11. In a phase I trial, BTK-inhibitor ibrutinib generated a clinical (partial) response in 40% of refractory nodal ABCDLBCL, both in patients with tumors bearing CD79 mutations or a combination of CD79 and MYD88 mutations. However, patients with only MYD88 mutations were refractory to this treatment, reflecting B-cell receptor signaling independency of MYD88-mutated tumors (Wilson et al., 2012).
292
In summary, 7 out of 10 cases of PCLBCL-LT showed genetic alterations in the NF-kB pathway (Table 1). Although precise representation is difficult in our relatively small study cohort, for pathwayactivating mutations in CD79B, MYD88, and CARD11 the percentages of tumors affected strikingly resemble those present in nodal ABC-DLBCL (Figure 1, Supplementary Table S2 online). These findings strongly suggest a role for constitutive activation of the NF-kB pathway in PCLBCL-LT, and provide a rationale to develop therapy targeted at components of the NF-kB pathway in this type of lymphoma. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS We thank Wim Corver for his excellent technical assistance. This study was supported in part by the Polish National Science Center (GKP).
Lianne Koens1, Willem H. Zoutman2, Passorn Ngarmlertsirichai3, Grzegorz K. Przybylski3,4, Piotr Grabarczyk3, Maarten H. Vermeer2, Rein Willemze2, Patty M. Jansen1, Christian A. Schmidt3 and Cornelis P. Tensen2 1
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands; 2 Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands; 3 Clinic for Internal Medicine C, University Greifswald, Greifswald, Germany and 4Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland E-mail: [email protected] SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid
REFERENCES Chu Y, Vahl JC, Kumar D et al. (2011) B cells lacking the tumor suppressor TNFAIP3/A20 display impaired differentiation and hyperactivation and cause inflammation and autoimmunity in aged mice. Blood 117:2227–36 Compagno M, Lim WK, Grunn A et al. (2009) Mutations of multiple genes cause
Journal of Investigative Dermatology (2014), Volume 134
deregulation of NF-kappaB in diffuse large B-cell lymphoma. Nature 459:717–21 Dal Porto JM, Gauld SB, Merrell KT et al. (2004) B cell antigen receptor signaling 101. MolImmunol 41:599–613 Davis RE, Brown KD, Siebenlist U et al. (2001) Constitutive nuclear factor kappaB activity is required for survival of activated B cell-like diffuse large B cell lymphoma cells. J Exp Med 194:1861–74 Hoefnagel JJ, Dijkman R, Basso K et al. (2005) Distinct types of primary cutaneous large B-cell lymphoma identified by gene expression profiling. Blood 105:3671–8 Honma K, Tsuzuki S, Nakagawa M et al. (2009) TNFAIP3/A20 functions as a novel tumor suppressor gene in several subtypes of nonHodgkin lymphomas. Blood 114:2467–75 Lamason RL, McCully RR, Lew SM et al. (2010) Oncogenic CARD11 mutations induce hyperactive signaling by disrupting autoinhibition by the PKC-responsive inhibitory domain. Biochemistry 49:8240–50 Naylor TL, Tang H, Ratsch BA et al. (2011) Protein kinase C inhibitor sotrastaurin selectively inhibits the growth of CD79 mutant diffuse large B-cell lymphomas. Cancer Res 71: 2643–53 Ngo VN, Davis RE, Lamy L et al. (2006) A loss-offunction RNA interference screen for molecular targets in cancer. Nature 441:106–10 Ngo VN, Young RM, Schmitz R et al. (2011) Oncogenically active MYD88 mutations in human lymphoma. Nature 470:115–9 Pham-Ledard A, Cappellen D, Martinez F et al. (2012) MYD88 somatic mutation is a genetic feature of primary cutaneous diffuse large B-cell lymphoma, leg type. J Invest Dermatol 132:2118–20 Senff NJ, Noordijk EM, Kim YH et al. (2008) European Organization for Research and Treatment of Cancer and International Society for Cutaneous Lymphoma consensus recommendations for the management of cutaneous B-cell lymphomas. Blood 112:1600–9 Sommer K, Guo B, Pomerantz JL et al. (2005) Phosphorylation of the CARMA1 linker controls NF-kappaB activation. Immunity 23:561–74 Wilson WH, Gerecitano JF, Goy A et al. (2012) The Bruton’s tyrosine kinase (BTK) inhibitor, ibrutinib (PCI-32765), has preferential activity in the ABC subtype of relapsed/refractory de novo diffuse large B-cell lymphoma (DLBCL): interim results of a multicenter, open-label, phase 2 study. Blood (ASH Annual Meeting Abstracts) 120:4039 (abstract) Yang Y, Shaffer AL, Tolga Emre MC et al. (2012) Exploiting synthetic lethality for the therapy of ABC diffuse large B-cell lymphoma. Cancer Cell 21:723–37
Copyright of Journal of Investigative Dermatology is the property of Nature Publishing Group and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
Abdlsattar Zebary1, Katarina Omholt1, Remco van Doorn2, Paola Ghiorzo3, Katja Harbst4, Carolina Hertzman Johansson1, Veronica Ho¨iom1, Go¨ran Jo¨nsson4, Dace Pjanova5, Susana Puig6, Giovanna B. Scarra3, Mark Harland 7, Ha˚kan Olsson4, Suzanne Egyhazi Brage1, Jane Palmer 8, Lena Kanter-Lewensohn1, Ismini Vassilaki 9, Nicholas K. Hayward 8, Julia Newton-Bishop7, Nelleke A. Gruis2, Johan Hansson1 and the Melanoma Genetics Consortium (GenoMEL) 1
Department of Oncology-Pathology, Cancer Center Karolinska, Karolinska Institutet, Stockholm, Sweden; 2Department of Dermatology, Leiden University Medical Centre, Leiden, The Netherlands; 3Department of Internal Medicine and Medical Specialties, University of Genoa and Laboratory of Genetics of Rare Hereditary Cancers, IRCCS San MartinoIST Research Hospital, Genoa, Italy; 4 Department of Oncology, Clinical Sciences,
Lund University, Lund, Sweden; 5Latvian Biomedical Research and Study Centre, Riga, Latvia; 6Melanoma Unit, Department of Dermatology, Hospital Clinic, University of Barcelona, IDIBAPS and CIBER de Enfermedades Raras, Barcelona, Spain; 7Section of Epidemiology and Biostatistics, Cancer Research UK Clinical Centre, Leeds Institute of Molecular Medicine, University of Leeds, Leeds, UK; 8Queensland Institute of Medical Research, Brisbane, Queensland, Australia and 9Karolinska University Hospital, Dermatologic Diagnostic Centrum Solna, Stockholm, Sweden E-mail: [email protected]
SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid
REFERENCES Eskandarpour M, Hashemi J, Kanter L et al. (2003) Frequency of UV-inducible NRAS mutations in melanomas of patients with germline CDKN2A mutations. J Natl Cancer Inst 95: 790–8
Fecher LA, Amaravadi RK, Flaherty KT (2008) The MAPK pathway in melanoma. Curr Opin Oncol 20:183–9 Goldstein AM, Chan M, Harland M et al. (2006) High-risk melanoma susceptibility genes and pancreatic cancer, neural system tumors, and uveal melanoma across GenoMEL. Cancer Res 66:9818–28 Hansson J (2008) Familial melanoma. Surg Clin North Am 88:897–916 Hayward NK (2003) Genetics of melanoma predisposition. Oncogene 22:3053–62 Jovanovic B, Egyhazi S, Eskandarpour M et al. (2010) Coexisting NRAS and BRAF mutations in primary familial melanomas with specific CDKN2A germline alterations. J Invest Dermatol 130:618–20 Lee JH, Choi JW, Kim YS (2011) Frequencies of BRAF and NRAS mutations are different in histological types and sites of origin of cutaneous melanoma: a meta-analysis. Br J Dermatol 164:776–84 Omholt K, Grafstro¨m E, Kanter-Lewensohn L et al. (2011) KIT pathway alterations in mucosal melanomas of the vulva and other sites. Clin Cancer Res 17:3933–42
Nuclear Factor-jB Pathway–Activating Gene Aberrancies in Primary Cutaneous Large B-Cell Lymphoma, Leg Type Journal of Investigative Dermatology (2014) 134, 290–292; doi:10.1038/jid.2013.265; published online 18 July 2013
TO THE EDITOR Primary cutaneous large B-cell lymphoma, leg type (PCLBCL-LT) is an aggressive cutaneous lymphoma with a 5-year overall survival of approximately 40%. Because of its aggressive nature, the treatment of first choice is anthracyclin-based chemotherapy combined with rituximab (Senff et al., 2008). However, patients often show a progressive disease course despite treatment with polychemotherapy. Furthermore, owing to age and comorbidity, not all patients are eligible for this treatment. Therefore, new and additional therapies for PCLBCL-LT are necessary, with a focus on more specific, targeted therapies with fewer side effects than conventional chemotherapy. At the mRNA expression level, PCLBCL-LT
resembles nodal activated B-cell-type diffuse large B-cell lymphoma (ABCDLBCL), including strong expression of known targets of the NF-kB pathway, such as IRF4 (Davis et al., 2001; Hoefnagel et al., 2005). In nodal ABC-DLBCL, increased NF-kB activity has a role in its pathogenesis through transcriptional activation of genes involved in cellular survival mechanisms. Previous studies demonstrated that mutations in multiple genes can cause deregulation of NF-kB signaling in nodal ABC-DLBCL (Compagno et al., 2009). The genes most frequently affected by these NF-kB-activating genetic aberrations are TNFAIP3 (A20), CD79B, CARD11 (CARMA1), and MYD88, which we investigated in 10 cases of PCLBCLLT (Supplementary Table S1 online).
Abbreviations: ABC-DLBCL, activated B-cell-type diffuse large B-cell lymphoma; cDNA, complementary DNA; PCLBCL-LT, primary cutaneous large B-cell lymphoma, leg type Accepted article preview online 13 June 2013; published online 18 July 2013
290
Journal of Investigative Dermatology (2014), Volume 134
TNFAIP3 (A20) is a tumor suppressor gene, downregulating NF-kB signaling by targeting several proteins involved in the activation of this pathway. By quantitative PCR, transcriptional levels of TNFAIP3 varied in our PCLBCL-LT cases (Supplementary Figure S1 online), and therefore we investigated potential underlying mechanisms for this variation. Four cases with relatively low mRNA expression levels showed heterozygous deletion of the gene region by highdensity fine-tiling comparative genomic hybridization. Homozygous deletions as observed in 10% of nodal ABC-DLBCL (Compagno et al., 2009) were not found. In cases without deletion of TNFAIP3, but displaying relatively low levels of mRNA, we could not detect epigenetic silencing through promoter hypermethylation, as was encountered in approximately 40% of nodal ABC-DLBCL (Honma et al., 2009). Possibly, in these cases, reduced mRNA expression was regulated by altered transcription factor
L Koens et al. NF-kB-Activating Gene Aberrancies in PCLBCL-LT
demonstrated to influence proliferation and survival in normal B cells in a gene dose–dependent manner (Chu et al., 2011), the relatively low expression of TNFAIP3 in most of the PCLBCL-LT samples can still be functionally relevant and might suggest a role for this
L IG L IG
IG IG L L
activity. One-third of cases of nodal ABC-DLBCL display genetic alterations in intron 3, leading to transcripts with a premature stop codon, giving rise to truncated nonfunctional proteins. Again, we could not detect these transcripts in PCLBCL-LT. As TNFAIP3 was
IGH IGH IL-1R
TLR
CD79B CD79A
Activating mutation first ITAM domain:20% SYK
IRAK4 IRAK1
BTK
Cytoplasm
MYD88
LYN
Activating mutation L265P: 30%
TRAF6 PKCβ BCL10
Activating mutation coiled-coil domain:10%
CARD11
MALT1 NEMO
Heterozygous deletion:40%
A20
IKKα IKKβ
NF-κB Nucleus NF-κB Survival proliferation
Figure 1. Pathway overview and genetic aberrancies in primary cutaneous large B-cell lymphoma, leg type. The suggested relations between the different components of B-cell receptor signaling and MYD88 signaling are represented (Yang et al., 2012). The molecules affected by genetic aberrancies in PCLBCL-LT are indicated in red, and the percentages of cases affected by these aberrancies are indicated. Molecules that can be therapeutically targeted for inhibition of the NF-kB pathway are represented in green.
tumor suppressor in the pathogenesis of PCLBCL-LT. CD79B is a component of the B-cell receptor, and has a combined function with CD79A in activating the NF-kB pathway (Figure 1; Dal Porto et al., 2004). Recurrent somatic mutations in the first immunoreceptor tyrosine-based activation motif tyrosine Y196 of CD79B were detected in 18% of nodal ABC-DLBCL. NF-kB pathway activation through this mutation is called chronic active B-cell receptor signaling, and occurs in cases with wildtype CARD11. In our series, concordant with nodal ABC-DLBCL, in 20% an Y196 mutation was detected in DNA and complementary DNA (cDNA), in the absence of mutations in CARD11, suggesting that chronic active B-cell receptor signaling is a mechanism involved in these cases of PCLBCL-LT. CARD11 is a signaling scaffold protein that functions as a critical component in constitutive NF-kB activation in ABC-DLBCL (Ngo et al., 2006), downstream of the B-cell receptor (Figure 1). In one sample, we found a missense mutation in codon 415 of exon 9 (D415E), and a R423W mutation in the same exon in both DNA and cDNA. The functional relevance of these D415E and R423W mutations is not known. However, mutations in the coiled-coil domain of CARD11, as encountered in 10% of nodal ABC-DLBCL, are
Table 1. Overview of genetic aberrancies TNFAIP3/A20 Splicing site Case Del. Hypermethylation qPCR (DDCq) Mut. Splice variant
CD79B
CARD11
MYD88
Y196 ITAM mutation
Mutation coiled-coil domain
L265P mutation
DNA
cDNA
Exons 5–8 and 10
Exon 9
DNA
cDNA
1
No
No
0.50
No
No
No
No
No
No
L265P
L265P
2
No
No
0.51
No
No
No
No
No
No
No
No
3
No
No
0.08
No
No
No
No
No
No
No
No
4
±
No
0.11
No
No
No
No
No
No
L265P
L265P
5
±
No
0.13
No
No
No
No
No
D415E, R423W
No
No
6
No
No
NA
NA
NA
No
No
No
No1
No
No
7
No
NA
0.03
No
No
Y196H
Y196H
No
No
L265P
No
8
No
No
0.17
No
No
Y196F
Y196F
No
No
L265P
L265p
9
±
NA
0.01
No
No
NA
No
No
No
NA
No
10
±
No
1
No
No
No
0.01
No
No
No
No
No
Abbreviations: cDNA, complementary DNA; Del., deletion; Mut., mutation; NA, not assessed; qPCR, quantitative PCR; ±, heterozygous deletion. 1 A silent mutation (D415D) was delected.
www.jidonline.org
291
L Koens et al. NF-kB-Activating Gene Aberrancies in PCLBCL-LT
potentially relevant in constitutive activation of the NF-kB pathway, as changes in this domain have the potential to disrupt autoinhibition of CARD11 signaling, leading to receptor-independent activation (Lamason et al., 2010). MYD88 is a Toll-like receptor–associated adaptor protein, among others involved in the activation of NF-kB signaling (Figure 1). Nodal ABC-DLBCL showed L265P mutations in 29% of cases, and the consequent amino-acid substitution in the bD sheet of MYD88 has been shown to be oncogenically active, leading to aberrant activation of NF-kB signaling (Ngo et al., 2011), a mechanism most likely not related to (chronic active) B-cell receptor signaling (Figure 1). Somatic MYD88 L265P mutations in PCLBCL-LT have been previously described in 69% of cases (Pham-Ledard et al., 2012). We encountered a 40% mutation rate in our series, concordant with nodal ABC-DLBCLs. Furthermore, we showed that not all mutations in the DNA seem to be transcribed to mRNA, as for one sample the mutation was not detected in cDNA. The relevance of the above-described mutations for novel therapeutic strategies has already been explored. For example, sotrastaurin can interfere with NF-kB signaling by the inhibition of PKC-b, which is required for CARD11-dependent activation of the NF-kB pathway (Sommer et al., 2005). In a mouse model, sotrastaurin was selectively toxic for CD79mutant DLBCL (Naylor et al., 2011) as compared with unmutated DLBCL, but only in the presence of wild-type CARD11. In a phase I trial, BTK-inhibitor ibrutinib generated a clinical (partial) response in 40% of refractory nodal ABCDLBCL, both in patients with tumors bearing CD79 mutations or a combination of CD79 and MYD88 mutations. However, patients with only MYD88 mutations were refractory to this treatment, reflecting B-cell receptor signaling independency of MYD88-mutated tumors (Wilson et al., 2012).
292
In summary, 7 out of 10 cases of PCLBCL-LT showed genetic alterations in the NF-kB pathway (Table 1). Although precise representation is difficult in our relatively small study cohort, for pathwayactivating mutations in CD79B, MYD88, and CARD11 the percentages of tumors affected strikingly resemble those present in nodal ABC-DLBCL (Figure 1, Supplementary Table S2 online). These findings strongly suggest a role for constitutive activation of the NF-kB pathway in PCLBCL-LT, and provide a rationale to develop therapy targeted at components of the NF-kB pathway in this type of lymphoma. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS We thank Wim Corver for his excellent technical assistance. This study was supported in part by the Polish National Science Center (GKP).
Lianne Koens1, Willem H. Zoutman2, Passorn Ngarmlertsirichai3, Grzegorz K. Przybylski3,4, Piotr Grabarczyk3, Maarten H. Vermeer2, Rein Willemze2, Patty M. Jansen1, Christian A. Schmidt3 and Cornelis P. Tensen2 1
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands; 2 Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands; 3 Clinic for Internal Medicine C, University Greifswald, Greifswald, Germany and 4Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland E-mail: [email protected] SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid
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