XPO1 mutations identify early-stage CLL characterized by shorter time to first treatment and enhanced BCR signalling.

Here we evaluated the epigenomic and transcriptomic profile of XPO1 mutant chronic lymphocytic leukaemia (CLL) and their clinical phenotype. By ATAC-seq, chromatin regions that were more accessible in XPO1 mutated CLL were enriched of binding sites for transcription factors regulated by pathways emanating from the B-cell receptor (BCR), including NF-κB signalling, p38-JNK and RAS-RAF-MEK-ERK. XPO1 mutant CLL, consistent with the chromatin accessibility changes, were enriched with transcriptomic features associated with BCR and cytokine signalling. By combining epigenomic and transcriptomic data, MIR155HG, the host gene of miR-155, and MYB, the transcription factor that positively regulates MIR155HG, were upregulated by RNA-seq and their promoters were more accessible by ATAC-seq. To evaluate the clinical impact of XPO1 mutations, we investigated a total of 957 early-stage CLL subdivided into 3 independent cohorts (N = 276, N = 286 and N = 395). Next-generation sequencing analysis identified XPO1 mutations as a novel predictor of shorter time to first treatment (TTFT) in all cohorts. Notably, XPO1 mutations maintained their prognostic value independent of the immunoglobulin heavy chain variable status and early-stage prognostic models. These data suggest that XPO1 mutations, conceivably through increased miR-155 levels, may enhance BCR signalling leading to higher proliferation and shorter TTFT in early-stage CLL.

Chemical and Microstructural Nanoscale Homogeneity in Superconducting YBa2Cu3O7-x Films Derived from Metal-Propionate Fluorine-free Solutions.

Research involved in developing alternative energy sources has become a necessity to face global warming. In this context, superconductivity is an appealing solution to enhance clean electrical energy provided that lower production costs can be attained. By implementation of chemical solution deposition techniques and high-throughput growth methods, low-cost nanostructured epitaxial cuprate superconductors are timely candidates. Here, we present a versatile and tunable solution method suitable for the preparation of high-performance epitaxial cuprate superconducting films. Disregarding the renowned trifluoroacetate route, we center our focus on the transient liquid-assisted growth (TLAG) that meets the requirement of being a greener chemical process together with ultrafast growth rates beyond 100 nm/s. We developed a facile, fast, and cost-effective method, starting from the synthesis of metal-propionate powders of Y, Ba, and Cu of high purity and high yields, being the precursors of the fluorine-free solutions, which enable the chemical and microstructural nanoscale homogeneity of YBa2Cu3O7-x (YBCO) precursor films. These solutions present endured stability and enable precise tunability of the composition, concentration, porosity, and film thickness. Homogeneous precursor films up to thicknesses of 2.7 µm through eight layer multidepositions are demonstrated, thus establishing the correct basis for epitaxial growth using the fast kinetics of the TLAG process. YBCO films of 500 nm thickness with a critical current density of 2.6 MA/cm2 at 77 K were obtained, showing the correlation of precursor film homogeneity to the final YBCO physical properties.

Kinetic Control of Ultrafast Transient Liquid Assisted Growth of Solution-Derived YBa2 Cu3 O7 -x Superconducting Films.

Transient liquid assisted growth (TLAG) is an ultrafast non-equilibrium growth process mainly governed by kinetic parameters, which are only accessible through fast in situ characterizations. In situ synchrotron X-ray diffraction (XRD) analysis and in situ electrical resistivity measurements are used to derive kinetic diagrams of YBa2 Cu3 O7- x (YBCO) superconducting films prepared via TLAG and to reveal the unique peculiarities of the process. In particular, diagrams for the phase evolution and the YBCO growth rates have been built for the two TLAG routes. It is shown that TLAG transient liquids can be obtained upon the melting of two barium cuprate phases (and not just one), differentiated by their copper oxidation state. This knowledge serves as a guide to determine the processing conditions to reach high performance films at high growth rates. With proper control of these kinetic parameters, films with critical current densities of 2-2.6 MA cm-2 at 77 K and growth rates between 100-2000 nm s-1 are reached. These growth rates are 1.5-3 orders of magnitude higher than those of conventional methods.

Clonally unrelated Richter syndrome are truly de novo diffuse large B-cell lymphomas with a mutational profile reminiscent of clonally related Richter syndrome.

Richter syndrome (RS) is mostly due to the direct transformation of the chronic lymphocytic leukaemia (CLL) clone, as documented by the same immunoglobulin heavy-chain variable region (IGHV) rearrangement in both CLL and RS cells. In rare cases characterized by a better outcome, the RS clone harbours a different IGHV rearrangement compared to the CLL phase. We investigated the CLL phase of clonally unrelated RS to test whether the RS clone was already identifiable prior to clinicopathologic transformation, albeit undetectable by conventional approaches. CLL cells of eight patients with unrelated RS were subjected to an ultra-deep next-generation sequencing (NGS) approach with a sensitivity of 10-6 . In 7/8 cases, the RS rearrangement was not identified in the CLL phase. In one case, the RS clone was identified at a very low frequency in the CLL phase, conceivably due to the concomitance of CLL sampling and RS diagnosis. Targeted resequencing revealed that clonally unrelated RS carries genetic lesions primarily affecting the TP53, MYC, ATM and NOTCH1 genes. Conversely, mutations frequently involved in de novo diffuse large B-cell lymphoma (DLBCL) without a history of CLL were absent. These results suggest that clonally unrelated RS is a truly de novo lymphoma with a mutational profile reminiscent, at least in part, of clonally related RS.

Multiregional sequencing and circulating tumour DNA analysis provide complementary approaches for comprehensive disease profiling of small lymphocytic lymphoma.

We aimed at molecularly dissecting the anatomical heterogeneity of small lymphocytic lymphoma (SLL), by analysing a cohort of 12 patients for whom paired DNA from a lymph node biopsy and circulating cells, as well as plasma-circulating tumour DNA (ctDNA) was available. Notably, the analyses of the lymph node biopsy and of circulating cells complement each other since a fraction of mutations (20·4% and 36·4%, respectively) are unique to each compartment. Plasma ctDNA identified two additional unique mutations. Consistently, the different synchronous sources of tumour DNA complement each other in informing on driver gene mutations in SLL harbouring potential prognostic and/or predictive value.

An update on: molecular genetics of high-risk chronic lymphocytic leukemia.

Introduction: During the past few years, new genomic approaches have elucidated the molecular genetics of chronic lymphocytic leukemia (CLL) to a large extent. As a consequence, specific high-risk genetic features of the disease, e.g. TP53 disruption, have become the backbone of the treatment algorithm for CLL and serve as robust biomarkers for a precision medicine approach to this leukemia.Areas covered: This review covers the genetics of CLL and highlights the translational implications of molecular biomarkers that characterize patients with a high risk of disease progression. Knowledge of the genetic landscape of CLL has allowed the identification of the main molecular features associated with chemo-refractoriness, as well as resistance to BCR inhibitors and BCL2 inhibitors. The molecular basis of Richter transformation has also been characterized.Expert opinion: The term 'high risk CLL' has been changing over time, and might be subject to further changes in the future. With the advent of new therapeutic strategies targeting pathogenetic pathways of the disease, the definition is shifting from the historical view of refractoriness to chemo-immunotherapy, to refractoriness to BCR inhibitors and/or to BCL2 inhibitors. Patients failing these novel medicines are those for whom new therapeutic approaches are still highly needed.

Biological and clinical implications of BIRC3 mutations in chronic lymphocytic leukemia.

BIRC3 is a recurrently mutated gene in chronic lymphocytic leukemia (CLL) but the functional implications of BIRC3 mutations are largely unexplored. Furthermore, little is known about the prognostic impact of BIRC3 mutations in CLL cohorts homogeneously treated with first-line fludarabine, cyclophosphamide, and rituximab (FCR). By immunoblotting analysis, we showed that the non-canonical nuclear factor-κB pathway is active in BIRC3-mutated cell lines and in primary CLL samples, as documented by the stabilization of MAP3K14 and by the nuclear localization of p52. In addition, BIRC3-mutated primary CLL cells are less sensitive to flu-darabine. In order to confirm in patients that BIRC3 mutations confer resistance to fludarabine-based chemoimmunotherapy, a retrospective multicenter cohort of 287 untreated patients receiving first-line FCR was analyzed by targeted next-generation sequencing of 24 recurrently mutated genes in CLL. By univariate analysis adjusted for multiple comparisons BIRC3 mutations identify a poor prognostic subgroup of patients in whom FCR treatment fails (median progression-free survival: 2.2 years, P<0.001) similar to cases harboring TP53 mutations (median progression-free survival: 2.6 years, P<0.0001). BIRC3 mutations maintained an independent association with an increased risk of progression with a hazard ratio of 2.8 (95% confidence interval 1.4-5.6, P=0.004) in multivariate analysis adjusted for TP53 mutation, 17p deletion and IGHV mutation status. If validated, BIRC3 mutations may be used as a new molecular predictor to select high-risk patients for novel frontline therapeutic approaches.

Diffuse large B-cell lymphoma genotyping on the liquid biopsy.

Accessible and real-time genotyping for diagnostic, prognostic, or treatment purposes is increasingly impelling in diffuse large B-cell lymphoma (DLBCL). Cell-free DNA (cfDNA) is shed into the blood by tumor cells undergoing apoptosis and can be used as source of tumor DNA for the identification of DLBCL mutations, clonal evolution, and genetic mechanisms of resistance. In this study, we aimed at tracking the basal DLBCL genetic profile and its modification upon treatment using plasma cfDNA. Ultra-deep targeted next generation sequencing of pretreatment plasma cfDNA from DLBCL patients correctly discovered DLBCL-associated mutations that were represented in >20% of the alleles of the tumor biopsy with >90% sensitivity and ∼100% specificity. Plasma cfDNA genotyping also allowed for the recovery of mutations that were undetectable in the tissue biopsy, conceivably because, due to spatial tumor heterogeneity, they were restricted to clones that were anatomically distant from the biopsy site. Longitudinal analysis of plasma samples collected under rituximab-cyclophosphamide-doxorubicin-vincristine-prednisone (R-CHOP) chemotherapy showed a rapid clearance of DLBCL mutations from cfDNA among responding patients. Conversely, among patients who were resistant to R-CHOP, basal DLBCL mutations did not disappear from cfDNA. In addition, among treatment-resistant patients, new mutations were acquired in cfDNA that marked resistant clones selected during the clonal evolution. These results demonstrate that cfDNA genotyping of DLBCL is as accurate as genotyping of the diagnostic biopsy to detect clonally represented somatic tumor mutations and is a real-time and noninvasive approach to tracking clonal evolution and the emergence of treatment-resistant clones.

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.

Genetic lesions associated with chronic lymphocytic leukemia chemo-refractoriness.

Fludarabine refractoriness (FR) represents an unsolved clinical problem of chronic lymphocytic leukemia (CLL) management. Although next-generation sequencing studies have led to the identification of a number of genes frequently mutated in FR-CLL, a comprehensive evaluation of the FR-CLL genome has not been reported. Toward this end, we studied 10 FR-CLLs by combining whole-exome sequencing and copy number aberration (CNA) analysis, which showed an average of 16.3 somatic mutations and 4 CNAs per sample. Screening of recurrently mutated genes in 48 additional FR-CLLs revealed that ~70% of FR-CLLs carry ≥1 mutation in genes previously associated with CLL clinical course, including TP53 (27.5%), NOTCH1 (24.1%), SF3B1 (18.9%), and BIRC3 (15.5%). In addition, this analysis showed that 10.3% of FR-CLL cases display mutations of the FAT1 gene, which encodes for a cadherin-like protein that negatively regulates Wnt signaling, consistent with a tumor suppressor role. The frequency of FAT1-mutated cases was significantly higher in FR-CLL than in unselected CLLs at diagnosis (10.3% vs 1.1%, P = .004), suggesting a role in the development of a high-risk phenotype. These findings have general implications for the mechanisms leading to FR and point to Wnt signaling as a potential therapeutic target in FR-CLL.

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.

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.

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.