Hairy-cell leukemia (HCL) is a well-defined clinicopathological entity whose underlying genetic lesion is still obscure. We searched for HCL-associated mutations by performing massively parallel sequencing of the whole exome of leukemic and matched normal cells purified from the peripheral blood of an index patient with HCL. Findings were validated by Sanger sequencing in 47 additional patients with HCL. Whole-exome sequencing identified five missense somatic clonal mutations that were confirmed on Sanger sequencing, including a heterozygous mutation in BRAF that results in the BRAF V600E variant protein. Since BRAF V600E is oncogenic in other tumors, further analyses were focused on this genetic lesion. The same BRAF mutation was noted in all the other 47 patients with HCL who were evaluated by means of Sanger sequencing. None of the 195 patients with other peripheral B-cell lymphomas or leukemias who were evaluated carried the BRAF V600E variant, including 38 patients with splenic marginal-zone lymphomas or unclassifiable splenic lymphomas or leukemias. In immunohistologic and Western blot studies, HCL cells expressed phosphorylated MEK and ERK (the downstream targets of the BRAF kinase), indicating a constitutive activation of the RAF–MEK–ERK mitogen-activated protein kinase pathway in HCL. In vitro incubation of BRAF-mutated primary leukemic hairy cells from 5 patients with PLX-4720, a specific inhibitor of active BRAF, led to a marked decrease in phosphorylated ERK and MEK. The BRAF V600E mutation was present in all patients with HCL who were evaluated. This finding may have implications for the pathogenesis, diagnosis, and targeted therapy of HCL. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.)

The pathogenesis of chronic lymphocytic leukemia (CLL), the most common leukemia in adults, is still largely unknown. The full spectrum of genetic lesions that are present in the CLL genome, and therefore the number and identity of dysregulated cellular pathways, have not been identified. By combining next-generation sequencing and copy number analysis, we show here that the typical CLL coding genome contains <20 clonally represented gene alterations/case, including predominantly nonsilent mutations, and fewer copy number aberrations. These analyses led to the discovery of several genes not previously known to be altered in CLL. Although most of these genes were affected at low frequency in an expanded CLL screening cohort, mutational activation of NOTCH1, observed in 8.3% of CLL at diagnosis, was detected at significantly higher frequency during disease progression toward Richter transformation (31.0%), as well as in chemorefractory CLL (20.8%). Consistent with the association of NOTCH1 mutations with clinically aggressive forms of the disease, NOTCH1 activation at CLL diagnosis emerged as an independent predictor of poor survival. These results provide initial data on the complexity of the CLL coding genome and identify a dysregulated pathway of diagnostic and therapeutic relevance.

Key Points: The integration of mutations and cytogenetic lesions improves the accuracy of survival prediction in chronic lymphocytic leukemia.

Analysis of the chronic lymphocytic leukemia (CLL) coding genome has recently disclosed that the NOTCH1 proto-oncogene is recurrently mutated at CLL presentation. Here, we assessed the prognostic role of NOTCH1 mutations in CLL. Two series of newly diagnosed CLL were used as training (n = 309) and validation (n = 230) cohorts. NOTCH1 mutations occurred in 11.0% and 11.3% CLL of the training and validation series, respectively. In the training series, NOTCH1 mutations led to a 3.77-fold increase in the hazard of death and to shorter overall survival (OS; P < .001). Multivariate analysis selected NOTCH1 mutations as an independent predictor of OS after controlling for confounding clinical and biologic variables. The independent prognostic value of NOTCH1 mutations was externally confirmed in the validation series. The poor prognosis conferred by NOTCH1 mutations was attributable, at least in part, to shorter treatment-free survival and higher risk of Richter transformation. Although NOTCH1 mutated patients were devoid of TP53 disruption in more than 90% cases in both training and validation series, the OS predicted by NOTCH1 mutations was similar to that of TP53 mutated/deleted CLL. NOTCH1 mutations are an independent predictor of CLL OS, tend to be mutually exclusive with TP53 abnormalities, and identify cases with a dismal prognosis.

Immune dysregulation is a cardinal feature of chronic lymphocytic leukemia (CLL) from its early stage and worsens during clinical observation, even in absence of disease progression. Although the mechanisms remain unclear, new insights are emerging into the complex relationship between the CLL clone and its immune environment. T cells are increased in early-stage disease and show progressive accumulation and exhaustion. The mechanisms that drive this expansion may include auto-antigens involved in the original clonal expansion. In addition, chronic viral infections such as cytomegalovirus generate huge virus-specific immune responses, which are further expanded in CLL. Attention is now focused largely on the direct immunosuppressive properties of the tumor. Remarkably, CLL clones often have features of the recently described regulatory B cells producing immunosuppressive IL-10. Better knowledge of the regulatory properties intrinsic to CLL cells may soon become more important with the switch from chemotherapy-based treatments, which trade control of CLL with further impairment of immune function, to the new agents targeting CLL B-cell receptor–associated signaling. Treatment with these new agents is associated with evidence of immune recovery and reduced infectious complications. As such, they offer the prospect of immunologic rehabilitation and a platform from which to ultimately replace chemotherapy.

Richter syndrome (RS) represents the development of diffuse large B-cell lymphoma in the context of chronic lymphocytic leukemia. The scarcity of biologic information about RS has hampered the identification of molecular predictors of RS outcome. We addressed this issue by performing a comprehensive molecular characterization of 86 pathologically proven RS. TP53 disruption (47.1%) and c-MYC abnormalities (26.2%) were the most frequent alterations, whereas common genetic lesions of de novo diffuse large B-cell lymphoma were rare or absent. By multivariate analysis, lack of TP53 disruption (hazard ratio, 0.43; P = .003) translated into significant survival advantage with 57% reduction in risk of death. An algorithm based on TP53 disruption, response to RS treatment, and Eastern Cooperative Oncology Group performance status had 80.9% probability of correctly discriminating RS survival (c-index = .809). RS that were clonally unrelated to the paired chronic lymphocytic leukemia phase were clinically and biologically different from clonally related RS because of significantly longer survival (median, 62.5 months vs 14.2 months; P = .017) and lower prevalence of TP53 disruption (23.1% vs 60.0%; P = .018) and B-cell receptor stereotypy (7.6% vs 50.0%; P = .009). The molecular dissection of RS into biologically distinct categories highlights the genetic heterogeneity of this disorder and provides clinically relevant information for refining the prognostic stratification of patients.