Cristina López

Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer.

NOTCH1 mutations identify a genetic subgroup of chronic lymphocytic leukemia patients with high risk of transformation and poor outcome

NOTCH1 has been found recurrently mutated in a subset of patients with chronic lymphocytic leukemia (CLL). To analyze biological features and clinical impact of NOTCH1 mutations in CLL, we sequenced this gene in 565 patients. NOTCH1 mutations, found in 63 patients (11%), were associated with unmutated IGHV, high expression of CD38 and ZAP-70, trisomy 12, advanced stage and elevated lactate dehydrogenase. Sequential analysis in 200 patients demonstrated acquisition of mutation in one case (0.5%) and disappearance after treatment in two. Binet A and B patients with NOTCH1-mutated had a shorter time to treatment. NOTCH1-mutated patients were more frequently refractory to therapy and showed shorter progression-free and overall survival after complete remission. Overall survival was shorter in NOTCH1-mutated patients, although not independently from IGHV. NOTCH1 mutation increased the risk of transformation to diffuse large B-cell lymphoma independently from IGHV, with this being validated in resampling tests of replicability. In summary, NOTCH1 mutational status, that was rarely acquired during the course of the disease, identify a genetic subgroup with high risk of transformation and poor outcome. This recently identified genetic subgroup of CLL patients deserves prospective studies to define their best management.

DNA methylome analysis in Burkitt and follicular lymphomas identifies differentially methylated regions linked to somatic mutation and transcriptional control

Although Burkitt lymphomas and follicular lymphomas both have features of germinal center B cells, they are biologically and clinically quite distinct. Here we performed whole-genome bisulfite, genome and transcriptome sequencing in 13 IG-MYC translocation–positive Burkitt lymphoma, nine BCL2 translocation–positive follicular lymphoma and four normal germinal center B cell samples. Comparison of Burkitt and follicular lymphoma samples showed differential methylation of intragenic regions that strongly correlated with expression of associated genes, for example, genes active in germinal center dark-zone and light-zone B cells. Integrative pathway analyses of regions differentially methylated in Burkitt and follicular lymphomas implicated DNA methylation as cooperating with somatic mutation of sphingosine phosphate signaling, as well as the TCF3-ID3 and SWI/SNF complexes, in a large fraction of Burkitt lymphomas. Taken together, our results demonstrate a tight connection between somatic mutation, DNA methylation and transcriptional control in key B cell pathways deregulated differentially in Burkitt lymphoma and other germinal center B cell lymphomas.

Different distribution of NOTCH1 mutations in chronic lymphocytic leukemia with isolated trisomy 12 or associated with other chromosomal alterations.

Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in Western countries. Chromosomal abnormalities commonly found using conventional cytogenetics and FISH are del(11)(q22‐23), trisomy 12, del(13)(q14), and del(17)(p13). Trisomy 12 is the most frequent numerical abnormality in CLL. It can appear isolated or associated with other chromosomal aberrations, including t(14;18)(q32;q21) and trisomy 18. The aim of this study was to determine whether CLL patients with isolated trisomy 12 or associated with other chromosomal alterations have different clinico‐pathological features, including a different distribution NOTCH1 mutation. Patients were classified into four groups: Group 1, isolated trisomy 12 (n = 14); Group 2, trisomy 12 plus trisomy 18 (n = 4); Group 3, trisomy 12 plus t(14;18) (n = 8); and Group 4: patients with trisomy 12 plus other abnormalities not involving BCL2 (n = 28). The Binet stage and expression of ZAP70 were significantly different among cytogenetic groups. NOTCH1 mutations were detected in 6/12 (50%) patients from Group 1, 4/25 (16%) patients from Group 4, and in no patient from groups 2 and 3 (P = 0.020). Patients in Group 2 had a more rapid disease progression (median Treatment‐free Survival 2 months) as against patients from Groups 1 (50 months), 3 (69 months), or 4 (68 months; P = 0.001). These findings indicate that the distribution of NOTCH1 mutations in CLL with trisomy 12 is heterogeneous and that the presence of additional chromosomal abnormalities such as trisomy 18 could change the prognosis of these patients.

Recurrent mutation of JAK3 in T-cell prolymphocytic leukemia.

T‐cell prolymphocytic leukemia (T‐PLL) is an aggressive post‐thymic T‐cell malignancy characterized by the recurrent inv(14)(q11q32)/t(14;14)(q11;q32) or t(X;14)(q28;q11) leading to activation of either the TCL1 or MTCP1 gene, respectively. However, these primary genetic events are insufficient to drive leukemogenesis. Recently, activating mutations in JAK3 have been identified in other T‐cell malignancies. Since JAK3 is essential for T‐cell maturation, we analyzed a cohort of 32 T‐PLL patients for mutational hot spots in the JAK3 gene using a step‐wise screening approach. We identified 14 mutations in 11 of 32 patients (34%). The most frequently detected mutation in our cohort was M511I (seen in 57% of cases) previously described as an activating change in other T‐cell malignancies. Three patients carried two mutations in JAK3. In two patients M511I and R657Q were simultaneously detected and in another patient V674F and V678L. In the latter case we could demonstrate that the mutations were on the same allele in cis. Protein modeling and homology analyses of mutations present in other members of the JAK family suggested that these mutations likely activate JAK3, possibly by disrupting the activation loop and the interface between N and C lobes, increasing the accessibility of the catalytic loop. In addition, four of the 21 patients lacking a JAK3 point mutation presented an aberrant karyotype involving the chromosomal band 19p13 harboring the JAK3 locus. The finding of recurrent activating JAK3 mutations in patients with T‐PLL could enable the use of JAK3 inhibitors to treat patients with this unfavorable malignancy who otherwise have a very poor prognosis.

Chronic lymphocytic leukaemia with 17p deletion: a retrospective analysis of prognostic factors and therapy results.

Patients with chronic lymphocytic leukaemia (CLL) whose tumour cells harbour a 17p deletion (17p‐) are universally considered to have a poor prognosis. The deletion can be detected at diagnosis or during the evolution of the disease, particularly in patients who have received chemotherapy. We sought to evaluate the natural history of patients with 17p‐ CLL, identify predictive factors within this prognostic subgroup, and evaluate the results of different therapeutic approaches. Data from 294 patients with 17p‐ CLL followed up at 20 different institutions was retrospectively collected and analysed. Median age was 68 (range 27–98) years at the time of fluorescence in situ hybridization analysis. After 17p‐ documentation, 52% received treatment, achieving an overall response rate of 50%. Median overall survival was 41 months, and was significantly shorter in patients with elevated beta2‐microglobulin concentration (P < 0·001), B symptoms (P = 0·016), higher percentage of cells with deletion (P < 0·001), and acquired deletions (P = 0·012). These findings suggest that patients with 17p‐ CLL have a variable prognosis that can be refined using simple clinical and laboratory features, including 17p‐ clone size, beta2‐microglobulin concentration, presence of B symptoms and type of deletion (de novo versus acquired).

Chronic lymphocytic leukemia in the elderly: clinico-biological features, outcomes, and proposal of a prognostic model

We investigated the clinico-biological features, outcomes, and prognosis of 949 patients with chronic lymphocytic leukemia according to age. No biological differences (cytogenetics by fluorescent in situ hybridization, IGHV, ZAP-70, CD38, NOTCH1, SF3B1) were found across age groups. Elderly patients (>70 years; n=367) presented more frequently with advanced disease (Binet C/Rai III-IV: 10/12% versus 5/5%; P<0.001), were treated less frequently (23.8% versus 41.9% at 3 years; P<0.001) and in most cases did not receive highly effective regimens and thus had a lower overall response rate (49% with 14% having complete responses versus 69% with 31% having complete responses; P<0.001). The elderly patients also had a shorter overall survival (6.6 versus 13.3 years; P<0.001) and higher disease-unrelated mortality (34.9% versus 6.9% at 10 years; P<0.001). However, disease-attributable mortality was not significantly different between younger and older patients. A combination of Binet stage, ZAP-70 level, β2-microglobulin concentration and comorbidity identified two risk groups (low-risk: 0–1 parameters; high-risk: 2–4 parameters) with different overall survivals (median: 6.8 versus 11.4 years, P<0.001). In patients requiring treatment, comorbidity at treatment (Cumulative Illness Rating Scale-T>4; hazard ratio 2.2, P<0.001) and response (treatment failure versus response: hazard ratio 1.60, P<0.04) were the most important prognostic factors for overall survival. In conclusion, in our series, elderly patients with chronic lymphocytic leukemia did not present with any biological features distinct from those of younger patients, but did have a poorer clinical outcome. This study highlights the importance of comprehensive medical care, achieving response to therapy, and specific management strategies for elderly patients with chronic lymphocytic leukemia.

B-cell-specific conditional expression of Myd88(p.L252P) leads to the development of diffuse large B-cell lymphoma in mice

The adaptor protein MYD88 is critical for relaying activation of Toll-like receptor signaling to NF-κB activation. MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Twenty-nine percent of activated B-cell-type DLBCL (ABC-DLBCL), which is characterized by constitutive activation of the NF-κB pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2 Here, we generated a novel mouse model in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88(p.L252P) (the orthologous position of the human MYD88(p.L265P) mutation) from the endogenous locus. These mice develop a lymphoproliferative disease and occasional transformation into clonal lymphomas. The clonal disease displays the morphologic and immunophenotypical characteristics of ABC-DLBCL. Lymphomagenesis can be accelerated by crossing in a further novel allele, which mediates conditional overexpression of BCL2 Cross-validation experiments in human DLBCL samples revealed that both MYD88 and CD79B mutations are substantially enriched in ABC-DLBCL compared with germinal center B-cell DLBCL. Furthermore, analyses of human DLBCL genome sequencing data confirmed that BCL2 amplifications frequently co-occurred with MYD88 mutations, further validating our approach. Finally, in silico experiments revealed that MYD88-mutant ABC-DLBCL cells in particular display an actionable addiction to BCL2. Altogether, we generated a novel autochthonous mouse model of ABC-DLBCL that could be used as a preclinical platform for the development and validation of novel therapeutic approaches for the treatment of ABC-DLBCL.

Coordination features of bis(N-heterocyclic carbenes) and bis(oxazolines) with 1,3-alkylidene-2,4,6-trimethylbenzene spacers. Synthesis of the ligands and silver and palladium complexes

The synthesis of simple imidazolium-based ligand precursors containing a 1,3-alkylidene-2,4,6-trimethylbenzene spacer was examined and different synthetic protocols were applied depending on the nature of the alkylidene arm. For a methylene arm, simple dications 5a,b.2CI were obtained directly. The higher homologue counterparts were conveniently prepared by general multistep routes following a five-step sequence for ethylene dications 6a,b.2Br or a six-step sequence for propylene dications 7a,b.2Br in > or = 52% overall yield. Imidazolium salts based on the shorter methylene spacer were used to prepare palladium complexes (17-20) with N-heterocyclic carbenes via transmetallation from well-defined silver compounds or directly in basic conditions. In order to facilitate spectroscopic characterisation of the palladium species two [Pd(allyl)(bis-oxazoline)]+ (25-26) complexes with the same ligand bridge were synthesized. [PdX2bisL] complexes appeared in solution as mixtures of species, mononuclear with cis- or trans-geometry or oligomeric compounds. The reaction of [PdCl(allyl)]2 and micro-bis(carbene)(AgX)2 complexes in 1 : 1 or in 0.5 : 1 ratio leads to binuclear compounds [Pd2Cl2(allyl)2(micro-bis-carbene)] (19a,19b) and to very labile monomeric [Pd(allyl)(bis-carbene)]+ (20a,20b) compounds, respectively. The preparation of analogous [Pd(allyl)(bis-oxazoline)]+ complexes showed the formation of one of the four possible isomers. [Pd(allyl)(bis-oxazoline)]PF6 complexes were inactive as catalytic precursors in the allylic substitution reaction.

Genes encoding members of the JAK-STAT pathway or epigenetic regulators are recurrently mutated in T-cell prolymphocytic leukaemia.

T‐cell prolymphocytic leukaemia (T‐PLL) is an aggressive leukaemia. The primary genetic alteration in T‐PLL are the inv(14)(q11q32)/t(14;14)(q11;q32) leading to TRD/TRA‐TCL1A fusion, or the t(X;14)(q28;q11) associated with TRD/TRA‐MTCP1 fusion. However, additional cooperating abnormalities are necessary for emergence of the full neoplastic phenotype. Though the pattern of secondary chromosomal aberrations is remarkably conserved, targets of the changes are largely unknown. We analysed a cohort of 43 well‐characterized T‐PLL for hotspot mutations in the genes JAK3, STAT5B and RHOA. Additionally, we selected a subset of 23 T‐PLL cases for mutational screening of 54 genes known to be recurrently mutated in T‐cell and other haematological neoplasms. Activating mutations in the investigated regions of the JAK3 and STAT5B genes were detected in 30% (13/43) and 21% (8/39) of the cases, respectively, and were mutually exclusive. Further, we identified mutations in the genes encoding the epigenetic regulators EZH2 in 13% (3/23), TET2 in 17% (4/23) and BCOR in 9% (2/23) of the cases. We confirmed that the JAK‐STAT pathway is a major mutational target, and identified epigenetic regulators recurrently mutated in T‐PLL. These findings complement the mutational spectrum of secondary aberrations in T‐PLL and underscore the potential therapeutical relevance of epigenetic regulators in T‐PLL.