Genomic Landscape of CXCR4 Mutations in Waldenström Macroglobulinemia.

PURPOSE: Whole-genome sequencing has revealed MYD88 L265P and CXCR4 mutations (CXCR4(mut)) as the most prevalent somatic mutations in Waldenström macroglobulinemia. CXCR4 mutation has proved to be of critical importance in Waldenström macroglobulinemia, in part due to its role as a mechanism of resistance to several agents. We have therefore sought to unravel the different aspects of CXCR4 mutations in Waldenström macroglobulinemia. EXPERIMENTAL DESIGN: We have scanned the two coding exons of CXCR4 in Waldenström macroglobulinemia using deep next-generation sequencing and Sanger sequencing in 98 patients with Waldenström macroglobulinemia and correlated with SNP array landscape and mutational spectrum of eight candidate genes involved in TLR, RAS, and BCR pathway in an integrative study. RESULTS: We found all mutations to be heterozygous, somatic, and located in the C-terminal domain of CXCR4 in 25% of the Waldenström macroglobulinemia. CXCR4 mutations led to a truncated receptor protein associated with a higher expression of CXCR4. CXCR4 mutations pertain to the same clone as to MYD88 L265P mutations but were mutually exclusive to CD79A/CD79B mutations (BCR pathway). We identified a genomic signature in CXCR4(mut) Waldenström macroglobulinemia traducing a more complex genome. CXCR4 mutations were also associated with gain of chromosome 4, gain of Xq, and deletion 6q. CONCLUSIONS: Our study panned out new CXCR4 mutations in Waldenström macroglobulinemia and identified a specific signature associated to CXCR4(mut), characterized with complex genomic aberrations among MYD88L265P Waldenström macroglobulinemia. Our results suggest the existence of various genomic subgroups in Waldenström macroglobulinemia.

MYD88 L265P mutation contributes to the diagnosis of Bing Neel syndrome.

Bing-Neel syndrome (BNS), a rare neurological syndrome associated with Waldenström macroglobulinaemia (WM), is a direct involvement of the central nervous system by lymphoplasmacytoid cells characterized with an adverse prognostic. The MYD88 L265P mutation has been identified in the vast majority of patients with WM. The diagnosis of BNS is often challenging because of the variety of clinical presentations associated with difficult histological techniques. We hypothesized that identification of MYD88 L265P mutation in the cerebrospinal fluid (CSF) would contribute to the diagnosis of BNS in addition to imaging, flow cytometry and cytology. We identified MYD88 L265P mutation in the CSF and the bone marrow of all cases of BNS using quantitative polymerase chain reaction qPCR and Sanger sequencing. Copy neutral loss of heterozygosity including MYD88 was observed in one case. No mutation of CXCR4, CD79A and CD79B was observed in parallel. We further showed that monitoring the quantitative expression of MYD88 L265P mutation might be a useful molecular tool to monitor response to chemotherapy using qPCR. In conclusion, identification of MYD88 L265P mutation might be a new molecular-based biomarker tool to add to the diagnostic and monitoring armamentarium for BNS.

Genome wide SNP array identified multiple mechanisms of genetic changes in Waldenstrom macroglobulinemia.

SNP array (SNPa) was developed to detect copy number alteration (CNA) and loss of heterozygosity (LOH) without copy number changes, CN-LOH. We aimed to identify novel genomic aberrations using SNPa in 31 WM with paired samples. Methylation status and mutation were analyzed on target genes. A total of 61 genetic aberrations were observed, 58 CNA (33 gains, 25 losses) in 58% of patients and CN-LOH in 6% of patients. The CNA were widely distributed throughout the genome, including 12 recurrent regions and identified new cryptic clonal chromosomal lesions that were mapped. Gene set expression analysis demonstrated a relationship between either deletion 6q or gain of chromosome 4 and alteration of gene expression profiling. We then studied methylation status and sought for mutations in altered regions on target genes. We observed methylation of DLEU7 on chromosome 13 in all patients (n = 12) with WM, and mutations of CD79B/CD79A genes (17q region), a key component of the BCR pathway, in 15% of cases. Most importantly, higher frequency of ≥3 CNA was observed in symptomatic WM. In conclusion, this study expands the view of the genomic complexity of WM, especially in symptomatic WM, including a potentially new mechanism of gene dysfunction, acquired uniparental disomy/CN-LOH. Finally, we have identified new potential target genes in WM, such as DLEU7 and CD79A/B.

MYD88 L265P mutation in Waldenstrom macroglobulinemia.

Mutation of the MYD88 gene has recently been identified in activated B-cell-like diffuse cell lymphoma and enhanced Janus kinase/signal transducer and activator of transcription (JAK-STAT) and nuclear factor κB (NF-κB) signaling pathways. A whole exome-sequencing study of Waldenstrom macroglobulinemia (WM) suggested a high frequency of MYD88 L265P mutation in WM. The genetic background is not fully deciphered in WM, although the role of NF-κB and JAK-STAT has been demonstrated. We analyzed MYD88 mutation in exon 5 and characterized the clinical significance of this genetic alteration in 67 WM patients. Clinical features; immunophenotypic markers; and conventional cytogenetic, fluorescence in situ hybridization, and single nucleotide polymorphism array data were analyzed. MYD88 L265P mutation was acquired in 79% of patients. Overall, we have identified alteration of the MYD88 locus in 91% of WM patients, including 12% with gain on chromosome 3 at the 3p22 locus that included the MYD88 gene. Patients with absence of MYD88 mutation were WM characterized with a female predominance, a splenomegaly, gain of chromosome 3, and CD27 expression. Importantly, inhibition of MYD88 signaling induced cytotoxicity and inhibited cell growth of cell lines issued from patients with WM. In conclusion, these results confirm a high frequency of MYD88 L265P mutation in WM. The discovery of MYD88 L265P mutation may contribute to a better understanding of the physiopathogeny of WM.

Genomic studies have identified multiple mechanisms of genetic changes in Waldenström macroglobulinemia.

The pathophysiology of Waldenström macroglobulinemia (WM), a lymphoproliferative disorder characterized by lymphoplasmacytic bone marrow infiltration associated with serum IgM paraprotein, is rather unclear; however, progress has been made in recent years to better determine the genetic profile of WM tumor cells. Studies based on high-throughput genomic analyses-including single-nucleotide polymorphism array (SNPa), array-based comparative genomic hybridization, and, recently, whole-genome sequencing--have improved deciphering some of the key molecular pathways associated with WM. Beyond the discovery of the myeloid differentiation primary response gene 88 (MYD88) L265P mutation, which will help greatly in the differential characterization of WM from other B-cell low-grade lymphomas, several other mechanisms of gene deregulation were identified and mapped that recurrently pointed out nuclear factor-kappa B (NF-κB), breakpoint cluster region (BCR), and Toll-like receptor (TLR) signaling pathways as potential targets for a better understanding of the physiopathology of WM and for future drug development. Herein, we summarize the current knowledge of the genomic patterns of WM to highlight its complexity.

High-throughput genomic analysis in Waldenström's macroglobulinemia.

Single-nucleotide polymorphism array (SNPa) and array-based comparative genomic hybridization (aCGH) are among the most sensitive genomic high-throughput screening techniques used in the exploration of genetic abnormalities in Waldenström's macroglobulinemia (WM). SNP and aCGH allow the identification of copy number abnormalities (CNA) at the kilobase level thus identifying cryptic genetic abnormalities unseen by lower-resolution approaches such as conventional cytogenetic or fluorescence in situ hybridization (FISH). CNA were identified in nearly 80% of cases by aCGH that delineated in addition minimal altered regions. At gene level, remarkable findings affecting genes involved in the regulation of the NF-kB signaling pathways were identified, such as biallelic inactivation of TNFAIP3 and TRAF3. SNPa also allowed characterization of copy neutral losses such as uniparental disomies (UPD), which is an important and frequent mechanism of gene alteration in cancer cells. Herein, we summarize the current knowledge of WM genomic basis using these high-throughput techniques.

Increased trisomy 12 frequency and a biased IgVH 3-21 gene usage characterize small lymphocytic lymphoma.

Small lymphocytic lymphoma (SLL) and chronic lymphocytic leukemia (CLL) are considered as similar entity by the WHO classification. We assessed the distribution of the four prognostic cytogenetic markers (deletion 11q23, 13q14, 17p13 and trisomy 12) and VH mutational status in 32 SLL and 119 CLL. Trisomy 12 was most frequent (36% vs 13%, p=0.014) and 13q14 deletion was less frequent (9% vs 44%, p=0.001) in SLL in comparison with CLL. An over representation of VH3-21 gene usage was found in SLL (17% vs 1%, p=0.011). In conclusion, SLL show specific genetic markers that distinguish them from classical CLL.

SDF1/CXCL12 (-801GA) polymorphism is a prognostic factor after treatment initiation in Waldenstrom macroglobulinemia.

The interaction of the chemokine CXCL12 with CXCR4 regulates homing of tumoral cells in bone marrow in Waldenstrom macroglobulinemia (WM). We assessed the distribution and the clinical influence of the CXCL12 (-801GA) polymorphism using PCR RFLP in a series of 114 WM patients. CXCL12 (-801AA) genotype was more frequent in WM patients compared with control subjects (p = 0.01). On the other hand, CXCL12 (-801GG) patients had a shorter median survival after initiation of first line therapy than remaining patients (p = 0.01). In conclusion, the CXCL12 (-801GA) polymorphism may either be associated with a high incidence of WM or influence clinical outcome.

Priming by tumor necrosis factor-alpha of human neutrophil NADPH-oxidase activity induced by anti-proteinase-3 or anti-myeloperoxidase antibodies.

Anti-proteinase-3 (anti-PR3) or anti-myeloperoxidase (anti-MPO) antibodies are capable of activating human neutrophils primed by TNF-alpha in vitro. We described previously the involvement of FcgammaRIIa and beta(2) integrins in this neutrophil activation. In the literature, the requirement of TNF priming has been attributed to an effect of TNF-alpha on the expression of PR3 or MPO on the cell surface. Under our experimental conditions, TNF-alpha (2 ng/ml) increased the binding of the antibody against PR3, whereas binding of the antibody against MPO could hardly be detected, not even after TNF-alpha treatment. The aim of this study was to consider (an)other(s) role(s) for TNF-alpha in facilitating the NADPH-oxidase activation by these antibodies. We demonstrate the early mobilization of the secretory vesicles as a result of TNF-induced increase in intracellular-free calcium ions, the parallel colocalization of gp91(phox), the main component of the NADPH oxidase with beta(2) integrins and FcgammaRIIa on the neutrophil surface, and the FcgammaRIIa clustering upon TNF priming. TNF-alpha also induced redistribution of FcgammaRIIa to the cytoskeleton in a dose- and time-dependent manner. Moreover, blocking CD18 MHM23 antibody, cytochalasin B, and D609 (an inhibitor of phosphatidylcholine phospholipase C) inhibited this redistribution and the respiratory burst in TNF-treated neutrophils exposed to anti-PR3 or anti-MPO antibodies. Our results indicate direct effects of TNF-alpha in facilitating neutrophil activation by these antibodies and further support the importance of cytoskeletal rearrangements in this priming process.

Increased expression of CD146, a new marker of the endothelial junction in active inflammatory bowel disease.

BACKGROUND: Crohn's disease (CD) and ulcerative colitis (UC), the 2 major forms of inflammatory bowel diseases (IBD), have been associated with disturbances in vascular physiology, including permeability and angiogenesis, that are in part regulated by the endothelial intercellular junctions. These junctions are composed of several adhesion molecules including the platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) and the more recently described CD146 (S-Endo1 Ag, MUC18). AIM: To study the expression of tissue and soluble form of CD146 in patients with CD or UC in relation to disease activity and location. This study was made in comparison with the soluble form of CD31 (sCD31). RESULTS: In active disease, a high expression of CD146 was observed on endothelial cells in intestinal biopsies from both CD and UC. In addition, we observed a decrease of sCD146 in relation to active disease and extensive location of CD and UC. Lower levels of sCD31 were also detected in active and extensive location of UC, but no difference could be observed in CD. CONCLUSION: sCD146 is a novel marker of the endothelial intercellular junction that reflects endothelial remodeling more effectively than soluble CD31. Further studies are warranted to determine whether sCD146 will provide a serological assay reflecting alterations in vascular permeability and vessel proliferation in the inflamed IBD intestine.

Pathogenesis of diseases associated with antineutrophil cytoplasm autoantibodies.

Little is known about the etiologies of diseases associated with circulating antineutrophil cytoplasm autoantibodies (ANCA), such as primary vasculitides and inflammatory bowel diseases. However, the understanding of immune mechanisms supposedly involved in the pathogenesis of these diseases is still growing. In the present review, we first focus on the mechanisms triggering the development of ANCA, including the potential role of microbial superantigens and the possible defect(s) in the progression of apoptosis or in the removal of apoptotic cells. We next concentrate on the contribution of ANCA to the clinical symptoms and on the pathogenic role of ANCA, including the accessibility of ANCA antigens as targets for circulating antibodies and the mode of action of ANCA. Mechanisms of neutrophil activation by ANCA include the engagement of Fcgamma receptors, the possible mechanisms of neutrophil-mediated tissue damage, and the neutrophil-endothelial interaction.

Expression of myeloperoxidase (MPO) by neutrophils is necessary for their activation by anti-neutrophil cytoplasm autoantibodies (ANCA) against MPO.

Anti-neutrophil cytoplasm autoantibodies (ANCA) directed against proteinase-3 and myeloperoxidase (MPO) activate tumor necrosis factor-alpha-primed neutrophils in vitro. We used neutrophils from one completely and one partially MPO-deficient donor to assess the requirement of MPO expression for neutrophil activation by anti-MPO antibodies. The MPO deficiencies were defined enzymatically, by immunocytochemistry and by immunoblotting. The mutations in the MPO genes of these donors were identified as a combination of a novel splice-site mutation at the 3' end of intron 11 (A-2-->C), a deletion of 14 nucleotides in exon 9 (A1555-C1568), and a novel C1907 --> T (636Thr-->Met) substitution in exon 11 in the completely MPO-deficient donor and as the same splice-site mutation and a novel C995 --> T (332Ala-->Val) substitution in exon 7 in the partially MPO-deficient donor. Monoclonal antibody 4.15 against MPO and MPO-ANCA-immunoglobulin G induced no superoxide anion production in these MPO-deficient neutrophils despite a normal production induced by other stimuli. Thus, the presence of MPO is a conditio sine qua non for neutrophil activation by anti-MPO antibodies. Moreover, we demonstrated that by means of these MPO-deficient cells, hydrogen peroxide may diffuse from neutrophils to surrounding cells, which may contribute to the damage induced by oxygen radicals in the pathology of systemic vasculitides.

Serological markers in inflammatory bowel diseases.

This chapter is an overview of the literature on serological markers of inflammatory bowel diseases (IBD), focusing on anti-neutrophil cytoplasm autoantibodies (ANCA) and anti- Saccharomyces cerevisiae mannan antibodies (ASCA). The methodology for ANCA and ASCA testing is first introduced. The value of these markers as diagnostic tools is then discussed. Other chapters are devoted to the potential role of ANCA and ASCA in disease monitoring, disease stratification and as subclinical markers in families. Finally reviewed are other antibodies recently tested in clinical trials such as pancreatic antibodies and antibodies directed against bacterial antigens. The role of these antibodies in the pathophysiology of IBD still needs to be assessed. We also need to identify the ASCA immunogen(s) eliciting the antibody response.