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. 2012 Aug 27;209(9):1537-51.
doi: 10.1084/jem.20120904. Epub 2012 Aug 13.

The coding genome of splenic marginal zone lymphoma: activation of NOTCH2 and other pathways regulating marginal zone development

Davide Rossi  1 , Vladimir Trifonov, Marco Fangazio, Alessio Bruscaggin, Silvia Rasi, Valeria Spina, Sara Monti, Tiziana Vaisitti, Francesca Arruga, Rosella Famà, Carmela Ciardullo, Mariangela Greco, Stefania Cresta, Daniela Piranda, Antony Holmes, Giulia Fabbri, Monica Messina, Andrea Rinaldi, Jiguang Wang, Claudio Agostinelli, Pier Paolo Piccaluga, Marco Lucioni, Fabrizio Tabbò, Roberto Serra, Silvia Franceschetti, Clara Deambrogi, Giulia Daniele, Valter Gattei, Roberto Marasca, Fabio Facchetti, Luca Arcaini, Giorgio Inghirami, Francesco Bertoni, Stefano A Pileri, Silvia Deaglio, Robin Foà, Riccardo Dalla-Favera, Laura Pasqualucci, Raul Rabadan, Gianluca Gaidano
Affiliations

The coding genome of splenic marginal zone lymphoma: activation of NOTCH2 and other pathways regulating marginal zone development

Davide Rossi et al. J Exp Med. .

Abstract

Splenic marginal zone lymphoma (SMZL) is a B cell malignancy of unknown pathogenesis, and thus an orphan of targeted therapies. By integrating whole-exome sequencing and copy-number analysis, we show that the SMZL exome carries at least 30 nonsilent gene alterations. Mutations in NOTCH2, a gene required for marginal-zone (MZ) B cell development, represent the most frequent lesion in SMZL, accounting for ∼20% of cases. All NOTCH2 mutations are predicted to cause impaired degradation of the NOTCH2 protein by eliminating the C-terminal PEST domain, which is required for proteasomal recruitment. Among indolent B cell lymphoproliferative disorders, NOTCH2 mutations are restricted to SMZL, thus representing a potential diagnostic marker for this lymphoma type. In addition to NOTCH2, other modulators or members of the NOTCH pathway are recurrently targeted by genetic lesions in SMZL; these include NOTCH1, SPEN, and DTX1. We also noted mutations in other signaling pathways normally involved in MZ B cell development, suggesting that deregulation of MZ B cell development pathways plays a role in the pathogenesis of ∼60% SMZL. These findings have direct implications for the treatment of SMZL patients, given the availability of drugs that can target NOTCH, NF-κB, and other pathways deregulated in this disease.

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Figures

Figure 1.
Figure 1.
SMZL coding genome complexity. (a) Number and type of nonsilent mutations identified in the 8 discovery genomes. (b) The pattern of nucleotide substitutions in the discovery genomes revealed a predominance of transitions over transversions (121:67, ratio of 1.8) and a preferential targeting of G and C nucleotides (66.0% affecting G/C compared with 34.0% affecting A/T nucleotides). (c) Mutation frequency at specific dinucleotides (red bars). A significant bias toward alterations at 5′-CpG-3′ dinucleotides, which accounted for 15.4% of all missense and nonsense changes, was documented. The expected frequencies (gray bars) correspond to the dinucleotide sequence composition of the Consensus CDS. Asterisks denote statistically significant differences in overrepresented changes, as assessed by a Poisson distribution after correction for multiple hypotheses. (d) Fraction of sequencing reads reporting individual somatic nonsilent variants (gray circles) in the discovery genomes. The large majority of the nonsilent mutations (82.1%) were present in at least 20% of the reads. (e) Overall number and frequency of somatically acquired CNAs. Losses of whole chromosomal arms were not observed and are thus not reported in the figure. (f) Combined load of somatically acquired genetic lesions in the discovery genomes, including nonsilent mutations and CNAs.
Figure 2.
Figure 2.
Recurrently targeted pathways in SMZL. Percentage of SMZL cases harboring mutations in selected genes belonging to cellular pathways that are recurrently altered in SMZL. Numbers at the bottom indicate the actual number of mutated cases over the total samples analyzed. Asterisks denote genes that are also implicated in Toll-like receptor responses. BCR, B cell receptor.
Figure 3.
Figure 3.
NOTCH2 is frequently mutated in SMZL. (a) Schematic representation of the human NOTCH2 gene (bottom) and protein (top), with its key functional domains (EGF, epithelial growth factor; LNR, LIN-12/NOTCH repeats; HD, heterodimerization; TM, transmembrane; RAM, regulation of amino acid metabolism; TAD, transactivation domain). Color-coded symbols indicate the type and position of the mutations. (b) Prevalence of NOTCH2 mutations among mature B cell tumors (EMZL, extranodal MZ lymphoma; NMZL, nodal MZ lymphoma; HCL, hairy cell leukemia; CLL, chronic lymphocytic leukemia; MCL, mantle cell lymphoma; FL, follicular lymphoma; MM, multiple myeloma). Numbers on the top indicate the actual number of mutated cases over the total samples analyzed.
Figure 4.
Figure 4.
NOTCH2 expression in SMZL. Western blot analysis of NOTCH2 protein expression in purified primary tumor cells from 5 SMZL cases carrying WT or mutated (M) NOTCH2, and in the SMZL cell line Karpas 1718, also WT for NOTCH2 (left); the specificity of the antibody was validated by using the BJAB cell line, which lacks NOTCH2 mRNA expression (right; asterisk indicates nonspecific band). Arrow indicates the intact NOTCH2 protein; a band of lower molecular weight, consistent with the predicted size of the NOTCH2 protein encoded by the mutant allele, can be detected in all NOTCH2-mutated patients, but not in NOTCH2 WT samples. Where available, the relative NOTCH2 mRNA levels in the same samples are quantified by qRT-PCR (bottom; na, not available).
Figure 5.
Figure 5.
NOTCH2 mutations are associated with better OS. Kaplan-Meier estimates of OS in SMZL patients (n = 94), according to NOTCH2 mutation status.
Figure 6.
Figure 6.
Mutations of genes belonging to the NOTCH, migration/adhesion, NF-κB, and B cell receptor pathways in SMZL. Schematic diagram of the proteins targeted by mutations in SMZL, with their key functional domains (a, NOTCH pathway; b, NF-κB pathway). Symbols indicate the type of mutation.
Figure 7.
Figure 7.
Mutually exclusive involvement of genes implicated in MZ development. In the heatmap, rows correspond to genes and columns represent individual patients. Color coding is based on gene mutation status (white, WT; red, mutated). Asterisks denote genes that also modulate Toll-like receptor responses. BCR, B cell receptor.
Figure 8.
Figure 8.
Mutations of genes involved in chromatin remodeling and transcriptional regulation. Schematic diagram of the indicated proteins, with their key functional domains. Symbols indicate the type of mutation.
Figure 9.
Figure 9.
Integrated analysis of mutations affecting chromatin remodeling genes in SMZL. In the heatmaps, rows correspond to mutated genes and columns represent individual patients. Color coding is based on gene mutation status (white, WT; red, mutated). Analysis of chromatin remodeling genes was extended to the screening panel only (in total, 40 cases), and is thus shown separately.
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