Nuclear factor-κB pathway-activating gene aberrancies in primary cutaneous large B-cell lymphoma, leg type.

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 (ABC-DLBCL), including strong expression of known targets of the NF-κB pathway, such as IRF4 (Davis et al., 2001; Hoefnagel et al., 2005). In nodal ABC-DLBCL, increased NF-κB 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-κB signaling in nodal ABC-DLBCL (Compagno et al., 2009). The genes most frequently affected by these NF-κB-activating genetic aberrations are TNFAIP3 (A20), CD79B, CARD11 (CARMA1), and MYD88, which we investigated in 10 cases of PCLBCL-LT (Supplementary Table S1 online). TNFAIP3 (A20) is a tumor suppressor gene, downregulating NF-κB 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 high-density 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 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 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 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-κB 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-κB pathway activation through this mutation is called chronic active B-cell receptor signaling, and occurs in cases with wild-type 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-κB 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 potentially relevant in constitutive activation of the NF-κB 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-κB signaling (Figure 1). Nodal ABC-DLBCL showed L265P mutations in 29% of cases, and the consequent amino-acid substitution in the βD sheet of MYD88 has been shown to be oncogenically active, leading to aberrant activation of NF-κB 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-κB signaling by the inhibition of PKC-β, which is required for CARD11-dependent activation of the NF-κB pathway (Sommer et al., 2005). In a mouse model, sotrastaurin was selectively toxic for CD79-mutant 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 ABC-DLBCL, 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). In summary, 7 out of 10 cases of PCLBCL-LT showed genetic alterations in the NF-κB pathway (Table 1). Although precise representation is difficult in our relatively small study cohort, for pathway-activating 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-κB pathway in PCLBCL-LT, and provide a rationale to develop therapy targeted at components of the NF-κB pathway in this type of lymphoma. The authors state no conflict of interest. We thank Wim Corver for his excellent technical assistance. This study was supported in part by the Polish National Science Center (GKP). SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper