Next Generation Flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma

[EN]Flow cytometry has become a highly valuable method to monitor minimal residual disease (MRD) and evaluate the depth of complete response (CR) in bone marrow (BM) of multiple myeloma (MM) after therapy. However, current flow-MRD has lower sensitivity than molecular methods and lacks standardization. Here we report on a novel next generation flow (NGF) approach for highly sensitive and standardized MRD detection in MM. An optimized 2-tube 8-color antibody panel was constructed in five cycles of design-evaluation-redesign. In addition, a bulk-lysis procedure was established for acquisition of ⩾107 cells/sample, and novel software tools were constructed for automatic plasma cell gating. Multicenter evaluation of 110 follow-up BM from MM patients in very good partial response (VGPR) or CR showed a higher sensitivity for NGF-MRD vs conventional 8-color flow-MRD -MRD-positive rate of 47 vs 34% (P=0.003)-. Thus, 25% of patients classified as MRD-negative by conventional 8-color flow were MRD-positive by NGF, translating into a significantly longer progression-free survival for MRD-negative vs MRD-positive CR patients by NGF (75% progression-free survival not reached vs 7 months; P=0.02). This study establishes EuroFlow-based NGF as a highly sensitive, fully standardized approach for MRD detection in MM which overcomes the major limitations of conventional flow-MRD methods and is ready for implementation in routine diagnostics.This work has been supported by the International Myeloma Foundation-Black Swan Research Initiative, the Red Temática de Investigación Cooperativa en Cáncer (RTICC); grant SA079U14 from the Consejería de Educación, Junta de Castilla y León, Valladolid, Spain and; grant DTS15/00119 from Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain

B-cell regeneration profile and minimal residual disease status in bone marrow of treated multiple myeloma patients

Simple Summary B-cell regeneration during therapy has been associated with the outcome of multiple myeloma (MM) patients. However, the effects of therapy and hemodilution in bone marrow (BM) B-cell recovery have not been systematically evaluated. Here, we show that hemodilution is present in a significant fraction of MM BM samples, leading to lower total B-cell, B-cell precursor (BCP), and normal plasma cell (nPC) counts. Among MM BM samples, decreased percentages (vs. healthy donors) of BCP, transitional/naive B-cell (TBC/NBC) and nPC populations were observed at diagnosis. BM BCP, but not TBC/NBC, increased after induction therapy. At day+100 post-autolo-gous stem cell transplantation, a greater increase in BCP with recovered TBC/NBC numbers but persistently low memory B-cell and nPC counts were found. At the end of therapy, complete response (CR) BM samples showed higher CD19(-) nPC counts vs. non-CR specimens with no clear association between BM B-cell regeneration profiles and patient outcomes. B-cell regeneration during therapy has been considered as a strong prognostic factor in multiple myeloma (MM). However, the effects of therapy and hemodilution in bone marrow (BM) B-cell recovery have not been systematically evaluated during follow-up. MM (n = 177) and adult (>= 50y) healthy donor (HD; n = 14) BM samples were studied by next-generation flow (NGF) to simultaneously assess measurable residual disease (MRD) and residual normal B-cell populations. BM hemodilution was detected in 41 out of 177 (23%) patient samples, leading to lower total B-cell, B-cell precursor (BCP) and normal plasma cell (nPC) counts. Among MM BM, decreased percentages (vs. HD) of BCP, transitional/naive B-cell (TBC/NBC) and nPC populations were observed at diagnosis. BM BCP increased after induction therapy, whereas TBC/NBC counts remained abnormally low. At day+100 postautologous stem cell transplantation, a greater increase in BCP with recovered TBC/NBC cell numbers but persistently low memory B-cell and nPC counts were found. At the end of therapy, complete response (CR) BM samples showed higher CD19(-) nPC counts vs. non-CR specimens. MRD positivity was associated with higher BCP and nPC percentages. Hemodilution showed a negative impact on BM B-cell distribution. Different BM B-cell regeneration profiles are present in MM at diagnosis and after therapy with no significant association with patient outcome

Tracing the fungal carbon metabolic roadmap

Plant biomass is one of the major sources of energy on Earth, used by living organisms and industry. However, its complete conversion remains a major industrial challenge. Fungi are the main degraders of plant biomass. They produce enzymes to degrade the polymers to mono- or short oligomers that can be taken up and metabolized by the cells. Aspergillus niger is considered an industrial workhorse due to its ability to produce different compounds and enzymes. The aim of this thesis was to reconstruct the metabolic network of A. niger focusing on carbon metabolism and predict metabolic capabilities in other species across the fungal kingdom. Genome-scale metabolic network reconstruction links genetic, metabolic and bibliomic information in a mathematical equation for a better estimation of the metabolism of an organism. We have updated and expanded the A. niger genome-scale metabolic network creating strain specific metabolic networks for three of the most common A. niger strains used in academia. By extending the model to other strains, we enable users to update and validate the model against the experimental data. During plant biomass degradation, activation of gene expression via specific inducers is balanced with gene repression via carbon catabolite repression (CCR) mediated by the CreA repressor protein. CCR prevents producing unnecessary proteins, when sufficient monosaccharides are present in the environment. We studied carbon utilization of A. niger WT and ΔcreA strain using a manually curated carbon catabolic network based on the A. niger NRRL 3 gold-standard genome. Starting with simple monosaccharide utilization, we showed pathway specific induction according to substrate and constitutive expression of the main carbon catabolic pathways (glycolysis, TCA and glyoxylic acid cycle), and identified CreA specific target genes. In nature, sugars do not occur as pure substrates, but in a heterogeneous mixture, after the crude substrates are depolymerized by enzymes. We explored carbon utilization by growing A. niger on a mixture of monosaccharides. This showed how in the presence of several carbon sources, the fungus utilized first high-energy content sugars and when they were finished, continued with less preferred sugars regardless of whether CreA was present or not. We went also further and studied gene expression during growth on different crude substrates and showed how substrate composition shapes gene expression over time. Moreover, our data showed that a ΔcreA strain needed more time to adapt to a new environment, after which gene expression became similar to the WT. A. niger is not the only fungus able to degrade plant biomass, and therefore we studied whether or not genome content can be used to predict metabolic abilities in other species. Our data showed that although genome content between closely related species was similar, regulatory systems and alternative pathways are important to understand their metabolic abilities. Taken together, this thesis depicts A. niger carbon metabolism and its further implementation in the fungal kingdom, focusing on plant biomass degradation and its industrial applications, using bioinformatics as driver

Tracing the fungal carbon metabolic roadmap

Plant biomass is one of the major sources of energy on Earth, used by living organisms and industry. However, its complete conversion remains a major industrial challenge. Fungi are the main degraders of plant biomass. They produce enzymes to degrade the polymers to mono- or short oligomers that can be taken up and metabolized by the cells. Aspergillus niger is considered an industrial workhorse due to its ability to produce different compounds and enzymes. The aim of this thesis was to reconstruct the metabolic network of A. niger focusing on carbon metabolism and predict metabolic capabilities in other species across the fungal kingdom. Genome-scale metabolic network reconstruction links genetic, metabolic and bibliomic information in a mathematical equation for a better estimation of the metabolism of an organism. We have updated and expanded the A. niger genome-scale metabolic network creating strain specific metabolic networks for three of the most common A. niger strains used in academia. By extending the model to other strains, we enable users to update and validate the model against the experimental data. During plant biomass degradation, activation of gene expression via specific inducers is balanced with gene repression via carbon catabolite repression (CCR) mediated by the CreA repressor protein. CCR prevents producing unnecessary proteins, when sufficient monosaccharides are present in the environment. We studied carbon utilization of A. niger WT and ΔcreA strain using a manually curated carbon catabolic network based on the A. niger NRRL 3 gold-standard genome. Starting with simple monosaccharide utilization, we showed pathway specific induction according to substrate and constitutive expression of the main carbon catabolic pathways (glycolysis, TCA and glyoxylic acid cycle), and identified CreA specific target genes. In nature, sugars do not occur as pure substrates, but in a heterogeneous mixture, after the crude substrates are depolymerized by enzymes. We explored carbon utilization by growing A. niger on a mixture of monosaccharides. This showed how in the presence of several carbon sources, the fungus utilized first high-energy content sugars and when they were finished, continued with less preferred sugars regardless of whether CreA was present or not. We went also further and studied gene expression during growth on different crude substrates and showed how substrate composition shapes gene expression over time. Moreover, our data showed that a ΔcreA strain needed more time to adapt to a new environment, after which gene expression became similar to the WT. A. niger is not the only fungus able to degrade plant biomass, and therefore we studied whether or not genome content can be used to predict metabolic abilities in other species. Our data showed that although genome content between closely related species was similar, regulatory systems and alternative pathways are important to understand their metabolic abilities. Taken together, this thesis depicts A. niger carbon metabolism and its further implementation in the fungal kingdom, focusing on plant biomass degradation and its industrial applications, using bioinformatics as driver

(Post-)genomics approaches in fungal research

To date, hundreds of fungal genomes have been sequenced and many more are in progress. This wealth of genomic information has provided new directions to study fungal biodiversity. However, to further dissect and understand the complicated biological mechanisms involved in fungal life styles, functional studies beyond genomes are required. Thanks to the developments of current -omics techniques, it is possible to produce large amounts of fungal functional data in a high-throughput fashion (e.g. transcriptome, proteome, etc.). The increasing ease of creating -omics data has also created a major challenge for downstream data handling and analysis. Numerous databases, tools and software have been created to meet this challenge. Facing such a richness of techniques and information, hereby we provide a brief roadmap on current wet-lab and bioinformatics approaches to study functional genomics in fungi

Metabolic modeling of fungi

Fungi have received special interest from the biotechnological sector focused on the production of active biomolecules and strain engineering. Genome-scale metabolic models (GEMs) are used to understand and improve their metabolism. GEMs can be obtained using computational methods, but they are susceptible to errors. Here, we describe the process to reconstruct a GEM and discuss several methodologies for analysis and validation of the model. We review different applications and explore the latest advances in GEM reconstruction to ease the process. With the new developments, we will be able to reconstruct and analyze high-quality GEMs for non-model species

In vivo functional analysis of L-rhamnose metabolic pathway in Aspergillus niger: a tool to identify the potential inducer of RhaR

Abstract Background The genes of the non-phosphorylative L-rhamnose catabolic pathway have been identified for several yeast species. In Schefferomyces stipitis, all L-rhamnose pathway genes are organized in a cluster, which is conserved in Aspergillus niger, except for the lra-4 ortholog (lraD). The A. niger cluster also contains the gene encoding the L-rhamnose responsive transcription factor (RhaR) that has been shown to control the expression of genes involved in L-rhamnose release and catabolism. Result In this paper, we confirmed the function of the first three putative L-rhamnose utilisation genes from A. niger through gene deletion. We explored the identity of the inducer of the pathway regulator (RhaR) through expression analysis of the deletion mutants grown in transfer experiments to L-rhamnose and L-rhamnonate. Reduced expression of L-rhamnose-induced genes on L-rhamnose in lraA and lraB deletion strains, but not on L-rhamnonate (the product of LraB), demonstrate that the inducer of the pathway is of L-rhamnonate or a compound downstream of it. Reduced expression of these genes in the lraC deletion strain on L-rhamnonate show that it is in fact a downstream product of L-rhamnonate. Conclusion This work showed that the inducer of RhaR is beyond L-rhamnonate dehydratase (LraC) and is likely to be the 2-keto-3-L-deoxyrhamnonate

Blocking hexose entry into glycolysis activates alternative metabolic conversion of these sugars and upregulates pentose metabolism in Aspergillus nidulans

Plant biomass is the most abundant carbon source for many fungal species. In the biobased industry fungi, are used to produce lignocellulolytic enzymes to degrade agricultural waste biomass. Here we evaluated if it would be possible to create an Aspergillus nidulans strain that releases, but does not metabolize hexoses from plant biomass. For this purpose, metabolic mutants were generated that were impaired in glycolysis, by using hexokinase (hxkA) and glucokinase (glkA) negative strains. To prevent repression of enzyme production due to the hexose accumulation, strains were generated that combined these mutations with a deletion in creA, the repressor involved in regulating preferential use of different carbon catabolic pathways. Phenotypic analysis revealed reduced growth for the hxkA1 glkA4 mutant on wheat bran. However, hexoses did not accumulate during growth of the mutants on wheat bran, suggesting that glucose metabolism is re-routed towards alternative carbon catabolic pathways. The creAΔ4 mutation in combination with preventing initial phosphorylation in glycolysis resulted in better growth than the hxkA/glkA mutant and an increased expression of pentose catabolic and pentose phosphate pathway genes. This indicates that the reduced ability to use hexoses as carbon sources created a shift towards the pentose fraction of wheat bran as a major carbon source to support growth. Blocking the direct entry of hexoses to glycolysis activates alternative metabolic conversion of these sugars in A. nidulans during growth on plant biomass, but also upregulates conversion of other sugars, such as pentoses

Blocking hexose entry into glycolysis activates alternative metabolic conversion of these sugars and upregulates pentose metabolism in Aspergillus nidulans

Plant biomass is the most abundant carbon source for many fungal species. In the biobased industry fungi, are used to produce lignocellulolytic enzymes to degrade agricultural waste biomass. Here we evaluated if it would be possible to create an Aspergillus nidulans strain that releases, but does not metabolize hexoses from plant biomass. For this purpose, metabolic mutants were generated that were impaired in glycolysis, by using hexokinase (hxkA) and glucokinase (glkA) negative strains. To prevent repression of enzyme production due to the hexose accumulation, strains were generated that combined these mutations with a deletion in creA, the repressor involved in regulating preferential use of different carbon catabolic pathways. Phenotypic analysis revealed reduced growth for the hxkA1 glkA4 mutant on wheat bran. However, hexoses did not accumulate during growth of the mutants on wheat bran, suggesting that glucose metabolism is re-routed towards alternative carbon catabolic pathways. The creAΔ4 mutation in combination with preventing initial phosphorylation in glycolysis resulted in better growth than the hxkA/glkA mutant and an increased expression of pentose catabolic and pentose phosphate pathway genes. This indicates that the reduced ability to use hexoses as carbon sources created a shift towards the pentose fraction of wheat bran as a major carbon source to support growth. Blocking the direct entry of hexoses to glycolysis activates alternative metabolic conversion of these sugars in A. nidulans during growth on plant biomass, but also upregulates conversion of other sugars, such as pentoses

Additional file 9: of Blocking hexose entry into glycolysis activates alternative metabolic conversion of these sugars and upregulates pentose metabolism in Aspergillus nidulans

Figure S3. Expression patterns and validation of RNA-sequencing analysis by qPCR. (PDF 325 kb