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Short-chain organic acids (SCOAs) are the intermediates in the anaerobic fermentation process, and can be used in food, textile, and pharmaceutical industries to produce different end use products. SCOAs can be separated, purified, and concentrated by different processes, such as distillation, extraction or membrane-based systems. SCOAs production adds more profitable possibilities to an acidic fermentation process by integration these marketable acids as highly concentrated mixtures with other refinery processes. The present study investigated two approaches for recovering of SCOAs: i) the production of clarified SCOAs liquid by microfiltration (MF) and then performing their concentration by reverse osmosis (RO) and ii) the recovery and concentration by the so-called integrated neutralization and acidified reaction method. The results of MF showed that some SCOAs were retained in the retentate together with the solids. However, in the following RO treatment, SCOAs could be successfully concentrated with a yield retention of over 90 % from the SCOAs liquid. In the latter method, a color-free SCOAs liquid was obtained with an increase in the total SCOAs concentration from 23 g/L to 146 g/L., Competing Interests: Declaration of competing interest Hereby, I confirm that there is no conflict of interest in submitted manuscript., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Biogas production is seen as one of the key measures in circular economy providing several benefits for the environment. In practice, however, these benefits may not be achieved if the production is not implemented and managed in ways that reduce gaseous emissions. Thus, this study aimed at highlighting how different management practices impact the climate during the life cycle of biogas production in comparison to management without biogas production (reference). Advanced, more emission-reducing practices resulted in 97-107% and conventional practices in 57-75% less emissions when biogas was utilized as transport fuel. If biogas was utilized in CHP (combined heat and power production), the emission reductions were 67-74% and 13-30%, respectively. This reflects the fact that inefficient practices can lead to minimal emission reduction without achieving the desired climate benefit in comparison to the reference. On the European level, this may also mean that the emission reduction demands of RED II (Renewable Energy Directive) regulation are not met. Therefore, when supporting biogas production with public funds, assurance of using emission-reducing practices should be made a prerequisite., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2023 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

This study focuses on the feasibility of the dry anaerobic digestion of solid agricultural biomass for efficient renewable-energy production and nutrient recycling. Methane production and the amount of nitrogen in the digestates were measured in pilot- and farm-scale leach-bed reactors. In the pilot scale, with a digestion time of 133 days, the methane production of a mixture of whole crop fava bean and horse manure corresponded to 94% and 116%, respectively, of the methane potentials of the solid substrates. The mono-digestion of fava beans resulted in relatively low methane production (production/potential ratios of 59% and 57%). In two full-scale experiments, the methane production of mixtures of clover-grass silage, chicken manure, and horse manure corresponded to 108% and 100% of their respective methane potentials with digestion times of 117 and 185 days. In co-digestion, the production/potential ratios were similar in the pilot and farm experiments. High nitrogen loss was observed in the farm scale when the digestate was stored in a stack covered with a tarpaulin during summertime. Thus, although the technology seems promising, attention needs to be paid to management practices to minimise nitrogen losses and greenhouse gas emissions.

A decline in the carbon content of agricultural soils has been reported globally. Amendments of forest industry side-streams might counteract this. We tested the effects of industrial conifer bark and its cascade process materials on the soil microbiome under barley (Hordeum vulgare L.) in clay and silt soil microcosms for 10 months, simulating the seasonal temperature changes of the boreal region. Microbial gene copy numbers were higher in clay soils than in silt. All amendments except unextracted bark increased bacterial gene copies in both soils. In turn, all other amendments, but not unextracted bark from an anaerobic digestion process, increased fungal gene copy numbers in silt soil. In clay soil, fungal increase occurred only with unextracted bark and hot water extracted bark. Soil, amendment type and simulated season affected both the bacterial and fungal community composition. Amendments increased bacteria originating from the anaerobic digestion process, as well as dinitrogen fixers and decomposers of plant cells. In turn, unextracted and hot water extracted bark determined the fungal community composition in silt. As fungal abundance increase and community diversification are related to soil carbon acquisition, bark-based amendments to soils can thus contribute to sustainable agriculture., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

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 YBa 2 Cu 3 O 7- 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/cm 2 at 77 K were obtained, showing the correlation of precursor film homogeneity to the final YBCO physical properties.

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 YBa 2 Cu 3 O 7- x at 77 K and growth rates between 100-2000 nm s -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., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

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., (© 2022 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Nutrients can be circulated back to agriculture from waste streams through anaerobic digestion and digestate processing. Digestate processing, however, is making slow progress as circulated nutrient products have not been cost competitive compared to fossil fertilizers and not designed from the farmer's perspective to truly match with the regional nutrient need. In this study, the aim is to assess apply mathematical optimization to the design of a cost-optimal processing route for a biogas plant's digestate to produce fertilizer products based on specified regional needs. Another aim is to analyze whether such a cost-optimal solution can fully exploit the nutrient recycling potential, that is, the efficiency of such a solution in returning nutrients to agriculture. The results indicate that mathematical optimization allows the design of a cost-optimal digestate production routes based on the region's nutrient need and characteristics. The true cost optimum was found for a design combining three processing technologies and producing four nutrient products, which when mixed, would fulfil farmer's fertilization needs. However, there seems to be a conflict between an optimal economic design and a full exploitation of recycling potential as only 25% of the digestate's phosphorus was utilized within the case region. This is because only 29% of the digestate mass was used and processed as fertilizer, as the concentration of required nutrients was deemed too low for economic use. The proposed mathematical model could be implemented as tool to assist in biogas plant investment decisions., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Due to the increasing demand for low carbon-footprint bioproducts in the markets, innovative processes technologies and products are needed. The objective of this study was to assess the quality and potential of food waste (FW) from the hospitality sector to produce volatile fatty acids (VFAs). A batch type acid fermentation system was used to study VFA production in different process conditions (a decreased pH and increased organic loading rate). The evolution of VFAs and long-chain fatty acids was followed. Amplicon sequencing of the 16S rRNA gene was used to investigate the bacterial and archaeal community, and elucidate microbial communities in different FW and process conditions. The results show that high VFA concentrations (of up to 18 g/L) were achieved in overloaded conditions, which were also affected by the activity and composition of the inoculum. FW played an important role in modulating microbial composition, especially the bacterial communities belonging to the lactic acid bacteria group., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Volatile fatty acids (VFAs) are intermediates in the methane formation pathway of anaerobic digestion and can be produced through the fermentation of organic wastes. VFAs have become an anticipated resource- and cost-effective way to replace fossil resources with higher added value and more versatile fuels and chemicals. However, there are still challenges in the production of targeted compounds from diverse and complex biomasses, such as urban biowastes. In this study, the aim was to modulate the microbial communities through inoculum treatment to enhance the production of green chemicals. Thermal and freeze-thaw treatments were applied to the anaerobic digester inoculum to inhibit the growth of methanogens and to enhance the performance of acidogenic and acetogenic bacteria. VFA fermentation after different inoculum treatments was studied in batch scale using urban biowaste as the substrate and the process performance was assessed with chemical and microbial analyses. Inoculum treatments, especially thermal treatment, were shown to increase VFA yields, which were also correlating with the dynamics of the microbial communities and retention times of the test. There was a strong correlation between VFA production and the relative abundances of the microbial orders Clostridiales (families Ruminococcaceae , Lachnospiraceae and Clostridiaceae ), and Lactobacillales. A syntrophic relationship of these taxa with members of the Methanobacteriales order was also presumed.

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.

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., (Copyright© 2020 Ferrata Storti Foundation.)

The achievement of high growth rates in YBa 2 Cu 3 O 7 epitaxial high-temperature superconducting films has become strategic to enable high-throughput manufacturing of long length coated conductors for energy and large magnet applications. We report on a transient liquid assisted growth process capable of achieving ultrafast growth rates (100 nm s -1 ) and high critical current densities (5 MA cm -2 at 77 K). This is based on the kinetic preference of Ba-Cu-O to form transient liquids prior to crystalline thermodynamic equilibrium phases, and as such is a non-equilibrium approach. The transient liquid-assisted growth process is combined with chemical solution deposition, proposing a scalable method for superconducting tapes manufacturing. Additionally, using colloidal solutions, the growth process is extended towards fabrication of nanocomposite films for enhanced superconducting properties at high magnetic fields. Fast acquisition in situ synchrotron X-ray diffraction and high resolution scanning transmission electron microscopy (STEM) become crucial measurements in disentangling key aspects of the growth process.

A process model based on hot water extraction (HWE), slow pyrolysis and anaerobic digestion (AD) were used for pine and spruce bark utilisation. First tannins (32 mg/g and 11.8 mg/g, respectively) and polyphenols were recovered via HWE. Then, the residue was pyrolysed to produce biochar (marketable quality), gas (energy source) and liquid fractions. The liquid fraction was further separated into aqueous acidic fraction and to tar fraction. Bark, extracted bark residue and acidic liquid fraction from pyrolysis were treated in AD to produce biomethane and digestate. The methane yields from pine and spruce bark and extracted bark residue were low (from 42 to 96 mLCH4/gVSadded) and showed only small differences. In conclusion, cascade processing can improve the performance of subsequent single processes and utilise biomass sources with higher efficiency. The best processing chain may vary in different cases and the overall energy balance of processing needs further research., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (© 2017 by The American Society of Hematology.)

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.

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 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.

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.

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.

Purpose: Preimplantation Genetic Diagnosis (PGD) has proven to be a useful reproductive option for carriers of some chromosome rearrangements. The data presented in this study compares the impact of one versus two blastomere biopsy on the likelihood of achieving a PGD result, as well as the effect on subsequent embryo development and clinical outcomes., Methods: IVF-PGD couples had either one or two blastomeres biopsied from all embryos with ≥7 blastomeres on day 3 post oocyte collection. These blastomeres were assessed for the specific chromosome rearrangement using Fluorescent In-situ Hybridisation (FISH). Further embryo development was monitored on days 4 and 5. Clinical outcomes were assessed retrospectively., Results: The data shows that statistically more embryos achieved a PGD result following two blastomere biopsy, compared with one blastomere biopsy (92 % versus 88 %, respectively). Furthermore it was found that embryo development and clinical outcomes were similar between the two biopsy groups., Conclusions: Based on this analysis it appears that the biopsy of two blastomeres from embryos with ≥7 blastomeres on day 3 is a valid and successful approach for couples presenting for IVF-PGD for a chromosome rearrangement.

Next-generation whole-exome sequencing has revealed two novel genes, namely NOTCH1 and SF3B1, whose mutations predict poor outcome and preferentially associate with chemorefractory chronic lymphocytic leukemia (CLL). Analysis of 539 CLL cases documents that NOTCH1 mutations i) represent one of the most frequent cancer gene mutations involved at presentation; ii) cluster with cases harboring trisomy 12 and tend to be mutually exclusive with TP53 disruption among genetic subgroups; iii) identify high-risk patients showing poor survival similar to that associated with TP53 abnormalities; and iv) exert a prognostic role independent of widely accepted clinical and genetic risk factors. Mutations of SF3B1, a splicing factor that is a critical component of the spliceosome, recurrently associate with fludarabine-refractory CLL, occur at a low rate at CLL presentation and have a minor role in Richter transformation, corroborating the notion that CLL histological shift is molecularly distinct from chemorefractory progression without the Richter transformation.

Richter syndrome (RS) represents the transformation of chronic lymphocytic leukemia to aggressive lymphoma. We explored intraclonal diversification (ID) of immunoglobulin genes in order to (i) follow the evolutionary history of the RS clone (ii) compare the role of ID in clonally related RS vs. clonally unrelated cases. Most (10/11, 90.9%) clonally related RS stem from the predominant clone observed at CLL diagnosis. One single RS had a transformation pattern compatible with sequential evolution from a secondary CLL subclone. Once RS transformation had occurred, all secondary CLL subclones disappeared and were substituted by the dominant RS clone with its own descendants. These observations suggest that genetic lesions associated with RS transformation are acquired by a cell belonging to the original CLL clone, rather than being progressively accumulated by later CLL subclones. Accordingly, most (9/11, 81.1%) clonally related RS harbored a genetic lesion disrupting TP53 that was already present, though at subclonal levels, in 5/11 (45.5%) samples of the paired CLL phase. A fraction of clonally related RS switched off ID (4/11, 36.4%) or reduced the levels of ID (5/11, 45.4%) at transformation. Conversely, all clonally unrelated RS harbored ID and were characterized by a significantly higher mutation frequency compared to clonally related RS (median: 1.18 × 10(-3) vs. 0.13 × 10(-3); p =0.002). These data indicate that (i) clonally related RS stems from a cell that is already present within the initial CLL clone and (ii) clonally unrelated and clonally related RS are biologically distinct disorders also in terms of antigen affinity maturation., (Copyright © 2011 UICC.)

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.

Trisomy 12, the third most frequent chromosomal aberration in chronic lymphocytic leukemia (CLL), confers an intermediate prognosis. In our cohort of 104 untreated patients carrying +12, NOTCH1 mutations occurred in 24% of cases and were associated to unmutated IGHV genes (P=0.003) and +12 as a sole cytogenetic abnormality (P=0.008). NOTCH1 mutations in +12 CLL associated with an approximately 2.4 fold increase in the risk of death, a significant shortening of survival (P<0.01) and proved to be an independent predictor of survival in multivariate analysis. Analogous to +12 CLL with TP53 disruption or del(11q), NOTCH1 mutations in +12 CLL conferred a significantly worse survival compared to that of +12 CLL with del(13q) or +12 only. The overrepresentation of cell cycle/proliferation related genes of +12 CLL with NOTCH1 mutations suggests the biological contribution of NOTCH1 mutations to determine a poor outcome. NOTCH1 mutations refine the intermediate prognosis of +12 CLL.

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.

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.

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.

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

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.

Several drugs used for diffuse large B-cell lymphoma (DLBCL) treatment rely on DNA damage for tumor cell killing. We verified the prognostic impact of the host DNA repair genotype in 2 independent cohorts of DLBCL treated with R-CHOP21 (training cohort, 163 cases; validation cohort, 145 cases). Among 35 single nucleotide polymorphisms analyzed in the training series, MLH1 rs1799977 was the sole predicting overall survival. DLBCL carrying the MLH1 AG/GG genotype displayed an increased death risk (hazard ratio [HR] = 3.23; P < .001; q =0 .009) compared with patients carrying the AA genotype. Multivariate analysis adjusted for International Prognostic Index identified MLH1 AG/GG as an independent OS predictor (P < .001). The poor prognosis of MLH1 AG/GG was the result of an increased risk of failing both R-CHOP21 (HR = 2.02; P = .007) and platinum-based second-line (HR = 2.26; P = .044) treatment. Survival analysis in the validation series confirmed all outcomes predicted by MLH1 rs1799977. The effect on OS of MLH1, a component of the DNA mismatch repair system, is consistent with its role in regulating the genotoxic effects of doxorubicin and platinum compounds, which are a mainstay of DLBCL first- and second-line treatment.

Richter syndrome (RS) represents the transformation of chronic lymphocytic leukaemia (CLL) to aggressive lymphoma. Risk factors of CLL transformation to RS are only partly known. We explored the role of the host genetic background as a risk factor for RS occurrence. Forty-five single nucleotide polimorphisms (SNPs) known to be relevant for CLL prognosis were genotyped in a consecutive cohort of 331 CLL, of which 21 had transformed to RS. After correcting for multiple testing and adjusting for previously reported RS risk factors, the LRP4 rs2306029 TT variant genotype was the sole SNP independently associated with a higher risk of RS transformation (Hazard Ratio: 4·17; P = 0·001; q = 0·047). The enrichment of LRP4 TT genotype in RS was confirmed in an independent series (n = 44) used for validation purposes. The LRP4 protein was expressed in CLL (n =66). Bioinformatic analysis scored LRP4 rs2306029 as a variant with possible deleterious and damaging variant of LRP4. LRP4 genotyping may help the recognition of patients with increased risk of RS at the time of CLL diagnosis., (© 2010 Blackwell Publishing Ltd.)

Richter's syndrome (RS) represents the transformation of chronic lymphocytic leukaemia (CLL) to aggressive lymphoma and is mostly represented by diffuse large B-cell lymphoma (DLBCL), with a post-germinal centre (GC) phenotype, clonally related to the pre-existing CLL. RS has a very poor prognosis and its pathogenetic mechanisms are poorly understood. In order to gain additional hints in RS pathogenesis, we performed a genome-wide DNA profiling study of 13 RS phases and eight matched CLL phases using the Affymetrix Human Mapping 250K NspI SNP arrays. Individual genomic profiles were heterogeneous, with no individual lesions occurring in more than half of the cases. However, several observations suggest that MYC pathway might be involved in RS. The 13q13.3-qter region containing MIRHG1 (MIR-17-92), a cluster of microRNA interacting with c-MYC, was acquired at the time of transformation. The 13q gain was coupled with the gain of c-MYC and loss of TP53. Translocation of c-MYC was acquired at transformation in a fraction of cases and this event appeared mutually exclusive with gain of MIRHG1. MYCN, a c-MYC homologue, was also recurrently gained. By comparing RS with 48 de novo DLBCL, RS presented a significantly lower prevalence of deletions affecting the PRDM1 and TNFAIP3, genes on 6q, known to be associated with a post-GC phenotype. In conclusion, the genomic profile of RS seems to differ from what observed in de novo DLBCL and in other transformed DLBCL. Genomic lesions occurring in RS are heterogeneous suggesting the existence of different RS subsets, possibly due to different transforming mechanisms. A deregulation of MYC pathway might represent one of the main transformation events in the pathogenesis of a subset of RS clonally related to the previous CLL., ((c) 2009 John Wiley & Sons, Ltd.)

Post-transplant lymphoproliferative disorders (PTLDs) represent a frequent complication of solid organ transplantation. Although most PTLDs arise from recipient lymphoid cells, a considerable fraction of cases may arise from donor B-cells. In an attempt to clarify the histogenesis and pathogenesis of PTLDs derived from donor B-cells, monoclonal PTLDs occurring in liver transplant recipients were chosen as a model to compare donor (D-PTLDs) versus recipient PTLDs (R-PTLDs). The tumour panel included nine D-PTLDs and six R-PTLDs. D-PTLDs were early-onset, EBV-infected lymphoproliferations classified as polymorphic PTLD (P-PTLD; n = 7) or diffuse large B-cell lymphoma (DLBCL; n = 2) with tumour localization confined to the hepatic hilum. All R-PTLDs were late-onset DLBCLs and showed extrahepatic localization. A BCL-6(-)/MUM1(+)/CD138(+/-) phenotype, consistent with a post-germinal centre (GC) stage of pre-terminal B-cell differentiation, was observed in all D-PTLDs and in 2/6 R-PTLDs, whereas a BCL6(+)/MUM1(-)/CD138(-) profile, reminiscent of GC B-cells, was detected in 4/6 R-PTLDs. The presence of somatic IGHV hypermutation was observed in 6/9 D-PTLDs and in 4/6 R-PTLDs, suggesting derivation from antigen-experienced B-cells. IGHV4-39 was the IGHV gene most frequently encountered, being rearranged in 3/9 D-PTLDs. Among IGHV-mutated PTLDs, a mutational profile suggesting antigen stimulation and/or selection was observed in 4/6 D-s and in 2/4 R-PTLDs. The presence of ongoing IGHV mutations was detected in 2/4 D-PTLDs. Aberrant SHM was detected in 10/15 (66.7%) PTLDs, including 6/9 D-PTLDs and 4/6 R-PTLDs. Our findings suggest that (i) D-PTLDs show a clinical presentation distinct from R-PTLDs; (ii) immunophenotypic and genetic features of D-PTLDs are consistent with mature, GC-experienced B-cells; (iii) transformed donor-derived B-cells may experience antigen-driven stimulation and selection, and may acquire genetic lesions during neoplastic expansion in the recipient environment; and (iv) EBV infection and expression of viral oncoproteins may be relevant in the pathogenesis of D-PTLDs., ((c) 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Purpose: Few biological prognosticators are useful for prediction of Richter syndrome (RS), representing the transformation of chronic lymphocytic leukemia (CLL) to aggressive lymphoma. Stereotyped B-cell receptors (BCR) may have prognostic effect in CLL progression. We tested the prognostic effect of stereotyped BCR for predicting RS transformation., Experimental Design: The prevalence of stereotyped BCR was compared in RS (n = 69) versus nontransformed CLL (n = 714) by a case-control analysis. Subsequently, the effect of stereotyped BCR at CLL diagnosis on risk of RS transformation was actuarially assessed in a consecutive CLL series (n = 753)., Results: RS (n = 69) displayed a higher prevalence of stereotyped BCR (P < 0.001) compared with nontransformed CLL. The actuarial risk of RS transformation was significantly higher in CLL carrying stereotyped BCR (P < 0.001). Among BCR subsets most represented in CLL, subset 8 using IGHV4-39/IGHD6-13/IGHJ5 carried the highest risk of RS transformation [hazard ratio (HR), 24.50; P < 0.001]. Multivariate analysis selected stereotyped BCR (HR, 3.33; P = 0.001) and IGHV4-39 usage (HR, 4.03; P = 0.004) as independent predictors of RS transformation. The combination of IGHV4-39 usage and stereotyped BCR in the same patient identified CLL with a very high risk of RS transformation (5-year risk, 68.7%). The risk carried by stereotyped BCR and IGHV4-39 usage was specific for RS transformation and had no effect on CLL progression without transformation., Conclusions: Analysis of BCR features may help identify CLL patients at risk of RS. A close monitoring and a careful biopsy policy may help early recognition of RS in CLL patients using stereotyped BCR, particularly if combined with IGHV4-39.

Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic monoclonal expansion of <5.0 x 10(9)/l circulating CLL-phenotype B-cells. The relationship between MBL and Rai 0 CLL, as well as the impact of biological risk factors on MBL prognosis, are unknown. Out of 460 B-cell expansions with CLL-phenotype, 123 clinical MBL (cMBL) were compared to 154 Rai 0 CLL according to clinical and biological profile and outcome. cMBL had better humoral immune capacity and lower infection risk, lower prevalence of del11q22-q23/del17p13 and TP53 mutations, slower lymphocyte doubling time, and longer treatment-free survival. Also, cMBL diagnosis was a protective factor for treatment risk. Despite these favourable features, all cMBL were projected to progress, and lymphocytes <1.2 x 10(9)/l and >3.7 x 10(9)/l were the best thresholds predicting the lowest and highest risk of progression to CLL. Although IGHV status, CD38 and CD49d expression, and fluorescence in situ hybridization (FISH) karyotype individually predicted treatment-free survival, multivariate analysis identified the presence of +12 or del17p13 as the sole independent predictor of treatment requirement in cMBL (Hazard ratio: 5.39, 95% confidence interval 1.98-14.44, P = 0.001). Overall, these data showed that cMBL has a more favourable clinical course than Rai 0 CLL. Given that the biological profile can predict treatment requirement, stratification based on biological prognosticators may be helpful for cMBL management.