Alessio Bruscaggin

Integrated mutational and cytogenetic analysis identifies new prognostic subgroups in chronic lymphocytic leukemia.

The identification of new genetic lesions in chronic lymphocytic leukemia (CLL) prompts a comprehensive and dynamic prognostic algorithm including gene mutations and chromosomal abnormalities and their changes during clonal evolution. By integrating mutational and cytogenetic analysis in 1274 CLL samples and using both a training-validation and a time-dependent design, 4 CLL subgroups were hierarchically classified: (1) high-risk, harboring TP53 and/or BIRC3 abnormalities (10-year survival: 29%); (2) intermediate-risk, harboring NOTCH1 and/or SF3B1 mutations and/or del11q22-q23 (10-year survival: 37%); (3) low-risk, harboring +12 or a normal genetics (10-year survival: 57%); and (4) very low-risk, harboring del13q14 only, whose 10-year survival (69.3%) did not significantly differ from a matched general population. This integrated mutational and cytogenetic model independently predicted survival, improved CLL prognostication accuracy compared with FISH karyotype (P < .0001), and was externally validated in an independent CLL cohort. Clonal evolution from lower to higher risk implicated the emergence of NOTCH1, SF3B1, and BIRC3 abnormalities in addition to TP53 and 11q22-q23 lesions. By taking into account clonal evolution through time-dependent analysis, the genetic model maintained its prognostic relevance at any time from diagnosis. These findings may have relevant implications for the design of clinical trials aimed at assessing the use of mutational profiling to inform therapeutic decisions.

Mutations of NOTCH1 are an independent predictor of survival 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.

Mutations of the SF3B1 splicing factor in chronic lymphocytic leukemia: association with progression and fludarabine-refractoriness

The genetic lesions identified in chronic lymphocytic leukemia (CLL) do not entirely recapitulate the disease pathogenesis and the development of serious complications, such as chemorefractoriness. While investigating the coding genome of fludarabine-refractory CLL, we observed that mutations of SF3B1, encoding a splicing factor and representing a critical component of the cell spliceosome, were recurrent in 10 of 59 (17%) fludarabine-refractory cases, with a frequency significantly greater than that observed in a consecutive CLL cohort sampled at diagnosis (17/301, 5%; P = .002). Mutations were somatically acquired, were generally represented by missense nucleotide changes, clustered in selected HEAT repeats of the SF3B1 protein, recurrently targeted 3 hotspots (codons 662, 666, and 700), and were predictive of a poor prognosis. In fludarabine-refractory CLL, SF3B1 mutations and TP53 disruption distributed in a mutually exclusive fashion (P = .046). The identification of SF3B1 mutations points to splicing regulation as a novel pathogenetic mechanism of potential clinical relevance in CLL.

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

Notch2 mutations represent the most frequent lesion in splenic marginal zone lymphoma.

Disruption of BIRC3 associates with fludarabine chemorefractoriness in TP53 wild-type chronic lymphocytic leukemia

The genetic lesions identified to date do not fully recapitulate the molecular pathogenesis of chronic lymphocytic leukemia (CLL) and do not entirely explain the development of severe complications such as chemorefractoriness. In the present study, BIRC3, a negative regulator of noncanonical NF-κB signaling, was investigated in different CLL clinical phases. BIRC3 lesions were absent in monoclonal B-cell lymphocytosis (0 of 63) and were rare in CLL at diagnosis (13 of 306, 4%). Conversely, BIRC3 disruption selectively affected 12 of 49 (24%) fludarabine-refractory CLL cases by inactivating mutations and/or gene deletions that distributed in a mutually exclusive fashion with TP53 abnormalities. In contrast to fludarabine-refractory CLL, progressive but fludarabine-sensitive patients were consistently devoid of BIRC3 abnormalities, suggesting that BIRC3 genetic lesions associate specifically with a chemorefractory phenotype. By actuarial analysis in newly diagnosed CLL (n = 306), BIRC3 disruption identified patients with a poor outcome similar to that associated with TP53 abnormalities and exerted a prognostic role that was independent of widely accepted clinical and genetic risk factors. Consistent with the role of BIRC3 as a negative regulator of NF-κB, biochemical studies revealed the presence of constitutive noncanonical NF-κB activation in fludarabine-refractory CLL patients harboring molecular lesions of BIRC3. These data identify BIRC3 disruption as a recurrent genetic lesion of high-risk CLL devoid of TP53 abnormalities.

Clinical impact of small TP53 mutated subclones in chronic lymphocytic leukemia

TP53 mutations are strong predictors of poor survival and refractoriness in chronic lymphocytic leukemia (CLL) and have direct implications for disease management. Clinical information on TP53 mutations is limited to lesions represented in >20% leukemic cells. Here, we tested the clinical impact and prediction of chemorefractoriness of very small TP53 mutated subclones. The TP53 gene underwent ultra-deep-next generation sequencing (NGS) in 309 newly diagnosed CLL. A robust bioinformatic algorithm was established for the highly sensitive detection of few TP53 mutated cells (down to 3 out of ∼1000 wild-type cells). Minor subclones were validated by independent approaches. Ultra-deep-NGS identified small TP53 mutated subclones in 28/309 (9%) untreated CLL that, due to their very low abundance (median allele frequency: 2.1%), were missed by Sanger sequencing. Patients harboring small TP53 mutated subclones showed the same clinical phenotype and poor survival (hazard ratio = 2.01; P = .0250) as those of patients carrying clonal TP53 lesions. By longitudinal analysis, small TP53 mutated subclones identified before treatment became the predominant population at the time of CLL relapse and anticipated the development of chemorefractoriness. This study provides a proof-of-principle that very minor leukemia subclones detected at diagnosis are an important driver of the subsequent disease course.

Alteration of BIRC3 and multiple other NF-κB pathway genes in splenic marginal zone lymphoma

Splenic marginal zone lymphoma (SMZL) is one of the few B-cell lymphoma types that remain orphan of molecular lesions in cancer-related genes. Detection of active NF-κB signaling in 14 (58%) of 24 SMZLs prompted the investigation of NF-κB molecular alterations in 101 SMZLs. Mutations and copy number abnormalities of NF-κB genes occurred in 36 (36%) of 101 SMZLs and targeted both canonical (TNFAIP3 and IKBKB) and noncanonical (BIRC3, TRAF3, MAP3K14) NF-κB pathways. Most alterations were mutually exclusive, documenting the existence of multiple independent mechanisms affecting NF-κB in SMZL. BIRC3 inactivation in SMZL recurred because of somatic mutations that disrupted the same RING domain that in extranodal marginal zone lymphoma is removed by the t(11;18) translocation, which points to BIRC3 disruption as a common mechanism across marginal zone B-cell lymphomagenesis. Genetic lesions of NF-κB provide a molecular basis for the pathogenesis of more than 30% of SMZLs and offer a suitable target for NF-κB therapeutic approaches in this lymphoma.

Association between molecular lesions and specific B-cell receptor subsets in chronic lymphocytic leukemia.

Genetic lesions and B-cell receptor (BCR) signaling are both oncogenic drivers in chronic lymphocytic leukemia (CLL). However, scant data are available on preferential associations between specific genetic alterations and stereotyped BCR subsets. By analyzing 1419 cases, 2 CLL subsets (2 and 8) harboring stereotyped BCR are enriched in specific molecular alterations influencing disease course. SF3B1 mutations are the genetic hallmark of IGHV3-21-CLL belonging to subset 2 (52%) but are evenly represented in nonstereotyped IGHV3-21-CLL. Trisomy 12 (87%) and NOTCH1 mutations (62%) characterize IGHV4-39-CLL belonging to subset 8 but occur with the expected frequency in IGHV4-39-CLL with heterogeneous BCR. Clinically, co-occurrence of SF3B1 mutations and subset 2 BCR configuration prompts disease progression in IGHV3-21-CLL, whereas cooperation between NOTCH1 mutations, +12, and subset 8 BCR configuration invariably primes CLL transformation into Richter syndrome. These findings provide a proof of concept that specific stereotyped BCR may promote or select molecular lesions influencing outcome.

Different impact of NOTCH1 and SF3B1 mutations on the risk of chronic lymphocytic leukemia transformation to Richter syndrome

Richter syndrome (RS) represents the development of an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL), in the context of chronic lymphocytic leukaemia (CLL). At least two types of RS exist: (i) transformation of CLL into a clonally related DLBCL, that accounts for ~80% of cases; and (ii) development of a DLBCL unrelated to the CLL clone. Clonally related RS and clonally unrelated RS are distinct disorders (Rossi et al, 2011a). Clinically, transformation into a clonally related RS is frequently lethal with an expected survival of a few months, while CLL patients developing a clonally unrelated RS display a survival probability in the range of de novo DLBCL (Rossi et al, 2011a). Biologically, clonally related RS frequently acquire genetic lesions of TP53, MYC and NOTCH1, which are otherwise absent or exceptional in clonally unrelated RS (Rossi et al, 2011a). NOTCH1 and SF3B1 mutations are predictors of poor outcome in CLL (Puente et al, 2011; Rossi et al, 2011b, 2012; Wang et al, 2011; Quesada et al, 2012). Here, we investigated a large multi-institutional series of 605 CLL patients to verify whether NOTCH1 and SF3B1 mutations at diagnosis may help in the identification of CLL at high risk of RS transformation. The study was approved by the Ethical Committee of the Ospedale Maggiore della Carità di Novara associated with the Amedeo Avogadro University of Eastern Piedmont (Protocol Code 59/CE; Study Number CE 8/11). Patients provided informed consent in accordance with local Institutional Review Board requirements and the Declaration of Helsinki. CLL was diagnosed according to National Cancer Institutes-International Workshop on CLL criteria (Hallek et al, 2008). RS diagnosis was histologically-proven (Müller-Hermelink et al, 2008). NOTCH1, SF3B1, TP53 and IGHV mutations were analysed by Sanger sequencing (Rossi et al, 2012). Probes (Abbott) used for fluorescent in situ hybridization analysis were: LSI13 and LSID13S319, CEP12, LSIp53 and LSIATM (Rossi et al, 2011a). CD38 expression was analysed by flow cytometry, utilizing a cut-off of 30% to define positivity (Rossi et al, 2009). VH CDR3 subset analysis and nomenclature were according to Murray et al (2008). Clonal relationship between CLL/DLBCL paired samples was established by comparing IGHV-D-J sequences. Time-toevent analysis utilized the Kaplan–Meier method. The cumulative incidence of RS development was calculated accounting for death as a competing risk, and was compared across groups with the Gray’s test. The association between exposure variables and outcome was estimated by univariate and multivariate Cox regression analysis. Further details are reported in Supplementary Methods. Features of the CLL cohort are reported in Table SI. At CLL presentation, NOTCH1 mutations occurred in 12·2% patients, being mostly represented (82·4%) by a recurrent two bp frameshift deletion (c.7544_7545delCT). SF3B1 mutations occurred in 5·8% cases and were mainly represented (50·0%) by the K700E missense substitution. After a median follow-up of 6·4 years, 30/605 (4·9%) CLL had transformed into a clonally related RS, while 10/605 (1·6%) had developed a clonally unrelated DLBCL. NOTCH1 mutations at disease presentation recurred in 12/30 (40·0%) CLL that subsequently transformed into a clonally related RS, whereas they were consistently absent in the 10 CLL that developed a clonally unrelated RS. SF3B1 mutations were absent in all CLL that subsequently transformed to a RS, both clonally related or unrelated. The close relationship between NOTCH1 mutations and clonally related RS was reinforced by the following observations: (i) NOTCH1 mutations were the second most frequent genetic lesion (40·0%) at transformation after TP53 disruption (60·0%); and (ii) NOTCH1 mutations observed in the RS phase were already present at CLL presentation in all patients, either at the clonal (n = 5) or subclonal (n = 7) level, suggesting a preferential selection of NOTCH1 mutated clones during disease progression and transformation. Notably, none of the clonally related RS acquired SF3B1 mutations at transformation. NOTCH1 mutations were associated with a ~5·8-fold increase in the crude hazard of transformation into a clonally related RS (HR: 5·81; P < 0·001) (Table I). Consistently, CLL harbouring NOTCH1 mutations had a significantly higher cumulative probability of transforming into a clonally related RS (45·0% at 15 years) compared to CLL without NOTCH1 mutations (4·6% at 15 years) (P < 0·001) (Fig. 1A). The association between NOTCH1 mutations and transformation into a clonally related RS also remained significant after correcting for death as a competing risk (P < 0·001). Conversely, NOTCH1 mutations had no impact on the development of a clonally unrelated DLBCL (P = 0·494). Correspondence

The NOTCH pathway is recurrently mutated in diffuse large B cell lymphoma associated with hepatitis C virus infection

Hepatitis C virus has been found to be associated with B-cell non-Hodgkin lymphomas, mostly marginal zone lymphomas and diffuse large B-cell lymphoma. Deregulation of signaling pathways involved in normal marginal zone development (NOTCH pathway, NF-κB, and BCR signaling) has been demonstrated in splenic marginal zone lymphoma. We studied mutations of NOTCH pathway signaling in 46 patients with hepatitis C virus-positive diffuse large B-cell lymphoma and in 64 patients with diffuse large B-cell lymphoma unrelated to HCV. NOTCH2 mutations were detected in 9 of 46 (20%) hepatitis C virus-positive patients, and NOTCH1 mutations in 2 of 46 (4%). By contrast, only one of 64 HCV-negative patients had a NOTCH1 or NOTCH2 mutation. The frequency of the NOTCH pathway lesions was significantly higher in hepatitis C virus-positive patients (P=0.002). The 5-year overall survival was 27% (95%CI: 5%–56%) for hepatitis C virus-positive diffuse large B-cell lymphoma patients carrying a NOTCH pathway mutation versus 62% (95%CI: 42%–77%) for those without these genetic lesions. By univariate analysis, age over 60 years, NOTCH2 mutation, and any mutation of the NOTCH pathway (NOTCH2, NOTCH1, SPEN) were associated with shorter overall survival. Mutation of the NOTCH pathway retained an independent significance (P=0.029). In conclusion, a subset of patients with hepatitis C virus-positive diffuse large B-cell lymphoma displays a molecular signature of splenic marginal zone and has a worse clinical outcome.