Absence of CXCR4 mutations but high incidence of double mutant in CD79A/B and MYD88 in primary central nervous system lymphoma.

Abstract

Primary central nervous system lymphoma (PCNSL) is a rare form of extra nodal diffuse large B-cell lymphoma (DLBCL) confined to the central nervous system (CNS). Genomic analyses are challenging in PCNSL, due to its low incidence and limited access to samples (Deckert et al, 2014). Most PCNSL samples have an activated B-cell-like phenotype (ABC) with a constitutive activation of the NF-ĸB and JAK-STAT pathways (Deckert et al, 2014). Although the underlying genetic basis remains unclear, some evidence suggests a higher incidence of both somatic mutations affecting the immunoreceptor tyrosine-based activation motif (ITAM) signalling modules of CD79A and CD79B (the two B-cell receptor subunits) and activating mutations of MYD88 (MYD88 L265P). These mutations can induce sustained survival in ABC-DLBCL cell-lines by promoting the activation of the NF-ĸB pathway (Davis et al, 2010; Ngo et al, 2011). There are still two main unanswered questions regarding PCNSL: Why do lymphoma cells involve the CNS in the first place? and Why is PCNSL usually confined to the CNS? The expression of chemokine receptors on malignant cells plays an important role in their tropism (Deckert et al, 2014). One key B-cell migration regulator is the chemokine receptor CXCR4 that regulates migration to lymph nodes and others organs. CXCR4 expression has been described in PCNSL cells (Smith et al, 2007). Whole genome sequencing has identified activating somatic mutations in CXCR4 in Waldenström macroglobulinemia (WM) (Treon et al, 2014). The latter are similar to the nonsense and frameshift germline mutations found in WHIM syndrome (Warts, hypogammaglobulinemia, infections and myelokathexis). Further delineating differences in terms of disease tropism based on CXCR4 status have been described in WM (Roccaro et al, 2014). The most frequently described variant, CXCR4 C1013G, results in a stop codon leading to the truncation of the C-terminal domain of CXCR4. Rare CXCR4 C1013G mutations have been described in DLBCL (Roccaro et al, 2014). The aim of the present study was to analyse the mutational status of CXCR4 and to correlate it with MYD88, CD79A and CD79B mutational patterns in PCNSL. We analysed 23 Epstein-Barr virus (EBV)- and human immunodeficiency virus (HIV)-negative PCNSL patients diagnosed between 1999 and 2013. B-cell differentiation sub-classification was assessed by immunohistochemistry (IHC) using CD10, MUM1 and BCL6 antibodies. Mutational studies of CXCR4, MYD88L265P, CD79A and CD79B genes were performed as previously described on either frozen or fixed embedded paraffin sections using Sanger sequencing or allele-specific polymerase chain reaction (Poulain et al, 2013, 2014). Patients’ characteristics are shown in Table 1. Ninety percent of patients received either high dose methotrexate and/or cytarabine-containing regimens. We did not find any CXCR4 mutations in our patients. Overall, 13 PCNSL patients (56%) harboured a total of 26 mutations in either exon 5 of MYD88 or CD79A/B (Table 2). CD79B mutations were observed in nine cases (39%). CD79B mutations involving the first ITAM tyrosine (Y196) were found in eight patients and were associated with other mutations in two cases. Mutations of CD79A leading to truncated protein were observed in two patients. However, CD79A and CD79B mutations appeared to be mutually exclusive. Twelve (52%) patients harboured the MYD88 L265P mutation and three patients were homozygotes (13%). In the remaining patients, we detected both a normal and a mutated allele, consistent with a heterozygous pattern. We did not observe any other mutation in exon 5 of MYD88 in our cohort. Interestingly, associations between CD79A/B and MYD88 L265P mutations were observed in our cohort (9/23, 39% of cases, P = 0·004), although no particular biological or clinical characteristic was associated with this genomic pattern. Overall survival was 11·03 months in the subset of patients bearing a CD79A/B mutation and 4·67 months in those without this aberration. However, this did not reach statistical significance, probably due to the size of this series (data not shown). Larger studies are therefore required to draw any firm conclusions on the prognostic impact of breakpoint cluster region (BCR) mutations. CD79A/B mutations in the ITAM domains, which reduce its negative regulation by LYN kinase, have previously been reported in DLBCL and WM (Davis et al, 2010; Poulain et al, 2013). We found a higher frequency of CD79B (mostly at the Y196 residue) and CD79A mutations than in previous series (Montesinos-Rongen et al, 2011; Kraan et al, 2013). In the latter study, only a hot spot of mutations at Y196 was found. Other CD79A/B mutations were here described. The frequency of MYD88 L265P mutations in our cohort was consistent with previous reports (Montesinos-Rongen et al, 2011; Kraan et al, 2013). The association between CD79A/B and MYD88 L265P reported here is higher than previously described in nodal DLBCL and WM, including Bing-Neel syndrome patients, suggesting a specific genomic landscape in PCNSL patients (Ngo et al, 2011; Kraan et al, 2013; Poulain et al, 2014). Conceivably, the association of mutations in MYD88 signalling and BCR pathway may potentially endow lymphoma-initiating cells with a selective growth advantage in immuno-privileged sites, such as the CNS. Given the pivotal role of adhesion and chemokine receptors in lymphoma dissemination, we investigated the mutational status of CXCR4, which is rarely mutated in DLBCL (Deckert et al, 2014). When considering extranodal DLBCL, the mutational landscape may differ from nodal DLBCL. The CXCR4 C1013G-WHIM associated mutations were initially related to extramedullary spread in WM and ibrutinib drug resistance in lymphoma (Roccaro et al, 2014; Treon et al, 2014). In our study, we did not observe any CXCR4 mutation. Similarly, there was no genetic alteration of CXCR4 in Bing-Neel Syndrome (Poulain et al, 2014). Further studies in understanding the molecular mechanisms that govern extranodal involvement in DLBCL are required. In this study, we failed to identify any CXCR4 mutations but instead found a high frequency of double mutants CD79A/B and MYD88 L265P among PCNSL patients, suggesting that alterations in both the BCR and the toll-like receptor (TLR) signalling pathways are central in the pathogenesis of PCNSL. Therefore, the crosstalk between the TLR and BCR pathways could be the basis for novel treatment approaches for these patients. We would like to thank Anne Sophie Blanchis, Valérie Grandieres, Claudine Delsault, Axelle Séghir for their technical assistance. Authors disclose no potential conflicts of interest. Conception and design: FM, SP; Collection and assembly of data: SP, EMB, ST, ED, HD,CR, FM; Data analysis and interpretation: SP, CR, CAM, CP, PD, FM; Manuscript writing: FM, SP, EMB; All authors critically reviewed and approved the final version of the manuscript.