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.

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.

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.

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.

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 prognostic value of TP53 mutations in chronic lymphocytic leukemia is independent of Del17p13: implications for overall survival and chemorefractoriness.

PURPOSE: Del17p13 predicts poor outcome and chemorefractoriness in chronic lymphocytic leukemia (CLL). Conversely, it is unknown whether TP53 mutations carry any prognostic value independent of del17p13. We tested the independent prognostic value of TP53 mutations in CLL. EXPERIMENTAL DESIGN: The study was based on a consecutive series of 308 CLL. DNA sequencing of TP53 exons 2 to 10 and del17p13 interphase fluorescence in situ hybridization were done at CLL diagnosis. Study end points were survival and chemorefractoriness. RESULTS: At diagnosis, TP53 mutations (n = 32) occurred in 31 of 308 (10.0%) patients. Of all CLL showing TP53 disruption by either mutation and/or deletion (n = 44), 10 cases (22.7%) showed TP53 mutations in the absence of del17p13. Multivariate analysis selected TP53 mutations (hazard ratio, 3.20; P = 0.002) as an independent predictor of overall survival after adjustment for del17p13. Also, multivariate analysis selected TP53 mutations (hazard ratio, 3.97; P < 0.001) as an independent predictor of chemorefractoriness after adjustment for del17p13. Compared with cases without TP53 alterations, CLL harboring any type of TP53 disruption (mutation only, del17p13 only, or both mutation and del17p13) uniformly displayed a high prevalence of unfavorable prognosticators and poor outcome. Analysis of sequential CLL samples showed the acquisition of new or additional TP53 alterations at the time of chemorefractoriness. CONCLUSIONS: These data show that (a) TP53 mutations are an independent predictor of short survival and chemorefractoriness, and (b) that CLL presenting with TP53 mutations without del17p13 fare as poorly as CLL carrying del17p13. Because CLL harboring TP53 mutations without del17p13 are currently not recognized by conventional diagnostic strategies, these results may be relevant for a comprehensive prognostic characterization of CLL.

Biological and clinical risk factors of chronic lymphocytic leukaemia transformation to Richter syndrome.

Predictors of chronic lymphocytic leukaemia (CLL) transformation to Richter syndrome (RS) are not established and were investigated in 185 consecutive CLL cases. Actuarial incidence of RS (n = 17; all diffuse large B-cell lymphomas) at 10 years was 16.2% (95% confidence interval: 8.0-24.4%). At CLL diagnosis, prognosticators of RS by univariate analysis were IGHV homology >/=98% (P = 0.006), IGHV4-39 usage (P < 0.001), del13q14 absence (P = 0.004), expression of CD38 (P < 0.001) and ZAP70 (P = 0.004), size (P < 0.001) and number (P < 0.001) of lymph nodes, advanced Binet stage (P = 0.002), and lactate dehydrogenase (P < 0.001). Multivariate analysis, performed separately for biological and clinical variables, identified CD38 expression [Hazard ratio (HR) = 4.26; P = 0.018], IGHV4-39 usage (HR = 4.29; P = 0.018), and lymph node size >/=3 cm (HR = 9.07; P < 0.001) as independent RS prognosticators. A multivariate model simultaneously analysing biological and clinical variables identified lymph node size >/=3 cm (HR = 6.51; P = 0.001) and del13q14 absence (HR = 4.08; P = 0.031) as independent RS prognosticators. Risk factors of CLL transformation differed from risk factors of CLL progression. These results suggest that CD38 and del13q14 may identify biological subsets of CLL with different RS predisposition. Predominant nodal disease, CD38 expression, IGHV4-39 usage, and absence of del13q14 may help in predicting RS at CLL diagnosis. Close monitoring and a careful biopsy policy are needed in patients carrying transformation risk factors.

CD49d expression is an independent risk factor of progressive disease in early stage chronic lymphocytic leukemia.

Identification of prognosticators for Binet A chronic lymphocytic leukemia is important for selecting patients with dismal prognosis. We analyzed CD49d expression in 140 consecutive Binet A chronic lymphocytic leukemia. At diagnosis, CD49d >or=30% (54/140, 38.6%) associated with proliferation markers, namely CD38 >or=30% (p=3.9 x 10(-6)), LDH (p=0.007) and beta2-microglobulin (p=0.020). Univariate log-rank analysis identified CD49d >or=30% as a risk factor of treatment free survival (p=8.3 x 10(-5)), time to progression to a more advanced stage (p=4.7 x 10(-4)), and time to lymphocyte doubling (p=0.009). Multivariate analysis selected CD49d >or=30% as an independent treatment free survival predictor after adjustment for biological (HR 2.28; 95% CI 1.71-4.45, p=0.015) and both biological and clinical variables analyzed together (HR 3.33, 95% CI 1.61-6.90, p=0.001). Within Binet A subgroups harboring favorable biological variables (IGHV homology <98%, favorable karyotype, CD38 <30%, ZAP70 <20%) or clinical variables, CD49d >or=30% consistently identified a subset of patients with short treatment free survival. Our observations indicate CD49d >or=30% as a new marker for the initial prognostic assessment of Binet A chronic lymphocytic leukemia.

5-hydroxytryptamine1B receptor and triptan response in migraine, lack of association with common polymorphisms.

Triptans mediate vasoconstriction of meningeal vessels via stimulation of vascular 5-hydroxytryptamine (5-HT)(1B) receptors. These drugs are recommended for acute treatment in patients with moderate-to-severe migraine attacks and in those patients with mild-to-moderate headache that are not controlled adequately by other agents. Yet, approximately 25% of all migraine users and 40% of all attacks do not respond to triptan treatment. Among the hypothesis to explain this is the possibility that genetic single nucleotide polymorphisms that alter the receptor, for example changing the transcriptional rate and therefore the amount of target protein might change the clinical response to these drugs. In the present contribution, we therefore decided to evaluate whether single nucleotide polymorphisms on the 5-HT(1B) gene might contribute to inter-individual variability in clinical responses to triptans. Two polymorphisms in the promoter region of the 5-HT(1B) receptor (T-261G and A-161T) and the synonymous variation G861C in the coding region were genotyped by restriction fragment length polymorphism in 105 migraine patients. In our sample population, 71% of patients responded to triptans. Allelic and diplotype frequencies were not significantly different between responders and non-responders. On the other hand, extrapolation of in vitro data on promoter activity would suggest that patients with higher copy number of receptors respond slightly better. Our data therefore do not support the involvement of 5-HT(1B) single nucleotide polymorphisms in mediating the inter-individual variability to triptans.

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

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.

Saliva is a reliable and practical source of germline DNA for genome-wide studies in chronic lymphocytic leukemia.

High-throughput genomics requires tumor DNA matched to germline DNA, that cannot be easily obtained in the context of leukemia. Using chronic lymphocytic leukemia as a model, saliva DNA was frequently devoid of tumor DNA also during overt disease, and passed quality controls for SNP-array (77/102, 75.4%) and next generation sequencing (71/102, 69.6%). Compared to saliva, urine provides germline DNA of similar quality but in lower amounts. Saliva DNA was successfully run on SNP 6.0 arrays, and passed quality control call rate thresholds. On these bases, saliva represents a useful source of germline DNA for high-throughput genomic studies of hematologic neoplasia.

Analysis of the chronic lymphocytic leukemia coding genome: role of NOTCH1 mutational activation.

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.