We performed WGS in six cases of SMZL and identified NOTCH2 mutations in half of these cases. Sanger sequencing of 93 additional SMZLs and 103 other types of B cell lymphoma or leukemia or reactive lymphoid hyperplasia showed NOTCH2 mutations in 22 additional SMZL patients, yielding an overall frequency of 25.3%. No mutations were identified in other non-MZL B cell lymphomas and leukemias analyzed. Moreover, in 19 patients with NOTCH2-mutated SMZL, constitutional DNA was available for assessment and was confirmed to be wild-type, thus indicating somatic acquisition of NOTCH2 mutation in SMZL.
In total, we identified 26 NOTCH2
mutations in 25 SMZL patients. These mutations represented six unique types of nonsense mutations, five unique types of frameshift mutations, and three unique types of missense mutations. 25 of these mutations affected the TAD or PEST domains, with 23 predicted to yield protein truncation at or upstream of the PEST domain. The remaining case harbored a somatic p.V1667I mutation in the HD. All of these mutations were identified in the same protein domains as have been reported for NOTCH1
in T-ALL, CLL/SLL, and MCL. However, NOTCH1
mutations in T-ALL are more prevalent in the HD than the TAD and PEST domain (Fig. S3). Disruption of the C-terminal PEST domain renders NOTCH less susceptible to regulation by ubiquitin-mediated proteolysis, and thus results in increased activation of the NOTCH pathway (Gupta-Rossi et al., 2001
; Oberg et al., 2001
; Wu et al., 2001
). Using reporter assays for assessment of NOTCH activation, we confirmed that representative mutations affecting either the PEST or HD indeed resulted in NOTCH2 transcriptional hyperactivation (not depicted).
Interestingly, in contrast to the activating effect in SMZL and other lymphoid malignancies, recent studies have suggested that the NOTCH pathway can also function as a tumor suppressor. Loss-of-function mutations in the NOTCH pathway (NCSTN, MAML1, APH1A, and NOTCH2) were recently identified in chronic myelomonocytic leukemia (Klinakis et al., 2011
). Other studies have identified oncogenic mutations within the epidermal growth factor repeat region of NOTCH1 in head and neck cancer (Agrawal et al., 2011
; Stransky et al., 2011
). Loss-of-function mutations affecting NOTCH family and pathway genes have also been implicated in skin and lung cancers (Wang et al., 2011
). Finally animal and in vitro studies suggest a tumor suppressor role in hepatocellular carcinoma (Viatour et al., 2011
), pancreatic carcinoma (Mazur et al., 2010
), and neuroblastoma (Zage et al., 2012
). In contrast, mutations identified in lymphoid malignancies (T-ALL, B-CLL/SLL, mantle cell, and diffuse large B cell lymphoma) have all been gain-of-function mutations confined to the C-terminal region extending from exon 25 to exon 34, as was the case in our initial study.
Pathogenic germline mutations in the TAD/PEST domain of NOTCH2
have been reported in Hajdu-Cheney syndrome (HCS), a rare autosomal dominant skeletal disorder characterized by facial anomalies, acroosteolysis, and osteoporosis (Isidor et al., 2011
; Simpson et al., 2011
). The NOTCH2
mutations in HCS include one report of a transmitted p.R2400X mutation (Simpson et al., 2011
; ). No predilection for lymphoma or B lymphocyte dysfunction in HCS patients has been reported to date. Interpretation of the significance of this is confounded by the extreme rarity of this disease. Nonetheless, it is reasonable to speculate that additional genetic alterations may be required for SMZL development.
With regard to neoplasia, isolated NOTCH2
mutations have been reported in a single case of SMZL and a single case of extranodal MZL in a previous study (Trøen et al., 2008
), as well as in a small proportion of cases of diffuse large B cell lymphoma (Lee et al., 2009
) or Richter’s transformation (Fabbri et al., 2011
), but no evidence for prognostic implications for NOTCH2
mutations was presented in any of these studies. NOTCH2 shares significant homology with NOTCH1, and transforming capacity has been demonstrated for truncated alleles of both proteins (Ellisen et al., 1991
; Rohn et al., 1996
; Capobianco et al., 1997
). Intriguingly, loss-of-function mutations affecting NOTCH family and pathway genes have recently been implicated in the pathogenesis of myeloid (Klinakis et al., 2011
) and epithelial malignancies (Mazur et al., 2010
; Agrawal et al., 2011
; Stransky et al., 2011
; Viatour et al., 2011
; Wang et al., 2011
), as well as in neuroblastoma (Zage et al., 2012
). These studies highlight the context-dependent roles of NOTCH and its signaling partners, which upon mutation, may contribute to the pathogenesis of neoplasia via different mechanisms in diverse cell types. Altogether, these findings suggest that the 26 NOTCH2
mutations we identified are likely to be pathogenic events contributing to aberrant NOTCH2 signaling in malignant SMZL cells.
Examination of NOTCH2
mutational status in nonsplenic MZLs revealed mutation in ~5% of cases analyzed. The NOTCH2
mutation identified in a single case of extranodal MZL of the breast was a p.R2400X nonsense mutation. This mutation was also identified in 9 of 99 (9.1%) SMZL cases. The selectivity of NOTCH2
mutations for malignancies of marginal zone B cells is in keeping with the known role of NOTCH2 in marginal zone cell fate determination (Saito et al., 2003
; Witt et al., 2003
). It is noteworthy that NOTCH1 dictates T cell fate and that supraphysiological NOTCH1 signaling induces T-ALL (Weng et al., 2004
). We speculate that because NOTCH2 specifies marginal zone B cell fate, supraphysiological NOTCH2 signaling may analogously play a role in the pathogenesis of MZL.
Somatic mutations affecting specific genes that impact SMZL prognosis are largely unknown. Although previous studies have implicated a role for mutations targeting genes in the NF-κB pathway in a subset of SMZL (Rossi et al., 2011
), only TP53 alterations affecting a small minority of cases have been demonstrated to impact SMZL prognosis (Salido et al., 2010
; Rinaldi et al., 2011
). TP53 mutations were not identified in our initial WGS screen of six index cases of SMZL. We have found that the presence of NOTCH2
mutations in SMZLs at time of diagnosis predicted an adverse disease course characterized either by refractoriness to therapy, histological transformation to higher grade disease, or an otherwise aggressive clinical course. Assessment of NOTCH2
mutation status in cases of SMZL may thus be useful to predict the risk of aggressive disease. This finding may also inform clinical decision-making at diagnosis, with the presence of NOTCH2
mutation being an indication for more aggressive therapy. By analogy with pathogenetic mechanisms of NOTCH1
mutation in T-ALL, it is tempting to speculate that similar downstream targets promoting proliferation, survival and deregulated metabolic pathways are also deregulated in SMZL. In addition to predicting a more aggressive disease course with an increased tendency to relapse, there is a trend toward reduced overall survival (i.e., time to death) among patients with NOTCH2
-mutated SMZL (). However, this trend in overall survival did not reach the level of statistical significance, presumably because of the small sample size in this study (P = 0.16).
In summary, we used WGS to reveal high-frequency recurrent somatic mutations involving NOTCH2 in SMZL. NOTCH2 mutations appear to be specific for marginal zone lymphomas as compared with other B cell leukemias and lymphomas. Additionally, our initial studies indicate an adverse outcome for patients with NOTCH2-mutated SMZL. Therefore, we have identified a biomarker specific for a subset of SMZL patients that may have value in both diagnosis and prognosis of patients with SMZL. Our findings therefore expand the spectrum of recurrent genetic alterations affecting genes in the NOTCH pathway in human malignancy and suggest potential therapies targeting NOTCH2 in the treatment of SMZL. Our studies further underscore the ability of unbiased large-scale screening approaches to uncover novel molecular mechanisms in neoplasia.