Dendritic cell type 3 arises from Ly6C+ monocyte-dendritic cell progenitors.

Conventional dendritic cells (cDCs) are professional antigen-presenting cells that control the adaptive immune response. Their subsets and developmental origins have been intensively investigated but are still not fully understood as their phenotypes, especially in the DC2 lineage and the recently described human DC3s, overlap with monocytes. Here, using LEGENDScreen to profile DC vs. monocyte lineages, we found sustained expression of FLT3 and CD45RB through the whole DC lineage, allowing DCs and their precursors to be distinguished from monocytes. Using fate mapping models, single-cell RNA sequencing and adoptive transfer, we identified a lineage of murine CD16/32+CD172a+ DC3, distinct from DC2, arising from Ly6C+ monocyte-DC progenitors (MDPs) through Lyz2+Ly6C+CD11c- pro-DC3s, whereas DC2s develop from common DC progenitors (CDPs) through CD7+Ly6C+CD11c+ pre-DC2s. Corresponding DC subsets, developmental stages, and lineages exist in humans. These findings reveal DC3 as a DC lineage phenotypically related to but developmentally different from monocytes and DC2s.

IntestLine: a shiny-based application to map the rolled intestinal tissue onto a line.

SUMMARY: To allow the comprehensive histological analysis of the whole intestine, it is often rolled to a spiral before imaging. This Swiss-rolling technique facilitates robust experimental procedures, but it limits the possibilities to comprehend changes along the intestine. Here, we present IntestLine, a Shiny-based open-source application for processing imaging data of (rolled) intestinal tissues and subsequent mapping onto a line. The visualization of the mapped data facilitates the assessment of the whole intestine in both proximal-distal and serosa-luminal axis, and enables the observation of location-specific cell types and markers. Accordingly, IntestLine can serve as a tool to characterize the intestine in multi-modal imaging studies. AVAILABILITY AND IMPLEMENTATION: Source code can be found at Zenodo (https://doi.org/10.5281/zenodo.7081864) and GitHub (https://github.com/SchlitzerLab/IntestLine).

Clonal evolution mechanisms in NT5C2 mutant-relapsed acute lymphoblastic leukaemia.

Relapsed acute lymphoblastic leukaemia (ALL) is associated with resistance to chemotherapy and poor prognosis. Gain-of-function mutations in the 5'-nucleotidase, cytosolic II (NT5C2) gene induce resistance to 6-mercaptopurine and are selectively present in relapsed ALL. Yet, the mechanisms involved in NT5C2 mutation-driven clonal evolution during the initiation of leukaemia, disease progression and relapse remain unknown. Here we use a conditional-and-inducible leukaemia model to demonstrate that expression of NT5C2(R367Q), a highly prevalent relapsed-ALL NT5C2 mutation, induces resistance to chemotherapy with 6-mercaptopurine at the cost of impaired leukaemia cell growth and leukaemia-initiating cell activity. The loss-of-fitness phenotype of NT5C2+/R367Q mutant cells is associated with excess export of purines to the extracellular space and depletion of the intracellular purine-nucleotide pool. Consequently, blocking guanosine synthesis by inhibition of inosine-5'-monophosphate dehydrogenase (IMPDH) induced increased cytotoxicity against NT5C2-mutant leukaemia lymphoblasts. These results identify the fitness cost of NT5C2 mutation and resistance to chemotherapy as key evolutionary drivers that shape clonal evolution in relapsed ALL and support a role for IMPDH inhibition in the treatment of ALL.

BTK inhibition sensitizes acute lymphoblastic leukemia to asparaginase by suppressing the amino acid response pathway.

Asparaginase (ASNase) therapy has been a mainstay of acute lymphoblastic leukemia (ALL) protocols for decades and shows promise in the treatment of a variety of other cancers. To improve the efficacy of ASNase treatment, we used a CRISPR/Cas9-based screen to identify actionable signaling intermediates that improve the response to ASNase. Both genetic inactivation of Bruton's tyrosine kinase (BTK) and pharmacological inhibition by the BTK inhibitor ibrutinib strongly synergize with ASNase by inhibiting the amino acid response pathway, a mechanism involving c-Myc-mediated suppression of GCN2 activity. This synthetic lethal interaction was observed in 90% of patient-derived xenografts, regardless of the genomic subtype. Moreover, ibrutinib substantially improved ASNase treatment response in a murine PDX model. Hence, ibrutinib may be used to enhance the clinical efficacy of ASNase in ALL. This trial was registered at www.clinicaltrials.gov as # NCT02884453.

Clonal dynamics in pediatric B-cell precursor acute lymphoblastic leukemia with very early relapse.

INTRODUCTION: One-quarter of the relapses in children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) occur very early (within 18 months, before completion of treatment), and prognosis in these patients is worse compared to cases that relapse after treatment has ended. METHODS: In this study, we performed a genomic analysis of diagnosis-relapse pairs of 12 children who relapsed very early, followed by a deep-sequencing validation of all identified mutations. In addition, we included one case with a good initial treatment response and on-treatment relapse at the end of upfront therapy. RESULTS: We observed a dynamic clonal evolution in all cases, with relapse almost exclusively originating from a subclone at diagnosis. We identified several driver mutations that may have influenced the outgrowth of a minor clone at diagnosis to become the major clone at relapse. For example, a minimal residual disease (MRD)-based standard-risk patient with ETV6-RUNX1-positive leukemia developed a relapse from a TP53-mutated subclone after loss of the wildtype allele. Furthermore, two patients with TCF3-PBX1-positive leukemia that developed a very early relapse carried E1099K WHSC1 mutations at diagnosis, a hotspot mutation that was recurrently encountered in other very early TCF3-PBX1-positive leukemia relapses as well. In addition to alterations in known relapse drivers, we found two cases with truncating mutations in the cohesin gene RAD21. CONCLUSION: Comprehensive genomic characterization of diagnosis-relapse pairs shows that very early relapses in BCP-ALL frequently arise from minor subclones at diagnosis. A detailed understanding of the therapeutic pressure driving these events may aid the development of improved therapies.

Multiclonal complexity of pediatric acute lymphoblastic leukemia and the prognostic relevance of subclonal mutations

Genomic studies of pediatric acute lymphoblastic leukemia (ALL) have shown remarkable heterogeneity in initial diagnosis, with multiple (sub)clones harboring lesions in relapse-associated genes. However, the clinical relevance of these subclonal alterations remains unclear. We assessed the clinical relevance and prognostic value of subclonal alterations in the relapse-associated genes IKZF1, CREBBP, KRAS, NRAS, PTPN11, TP53, NT5C2, and WHSC1 in 503 ALL cases. Using Molecular Inversion Probe sequencing and breakpoint-spanning PCR we reliably detected alterations below 1% allele frequency. We identified 660 genomic alterations in 285 diagnosis samples of which 495 (75%) were subclonal. RAS pathway mutations were common, particularly in minor subclones, and comparisons between RAS hotspot mutations revealed differences in their capacity to drive clonal expansion in ALL. We did not find an association of subclonal alterations with unfavorable outcome. Particularly for IKZF1, an established prognostic marker in ALL, all clonal but none of the subclonal alterations were preserved at relapse. We conclude that, for the genes tested, there is no basis to consider subclonal alterations detected at diagnosis for risk group stratification of ALL treatment.

Integration of genetic and clinical risk factors improves prognostication in relapsed childhood B-cell precursor acute lymphoblastic leukemia.

Somatic genetic abnormalities are initiators and drivers of disease and have proven clinical utility at initial diagnosis. However, the genetic landscape and its clinical utility at relapse are less well understood and have not been studied comprehensively. We analyzed cytogenetic data from 427 children with relapsed B-cell precursor ALL treated on the international trial, ALLR3. Also we screened 238 patients with a marrow relapse for selected copy number alterations (CNAs) and mutations. Cytogenetic risk groups were predictive of outcome postrelapse and survival rates at 5 years for patients with good, intermediate-, and high-risk cytogenetics were 68%, 47%, and 26%, respectively (P < .001). TP53 alterations and NR3C1/BTG1 deletions were associated with a higher risk of progression: hazard ratio 2.36 (95% confidence interval, 1.51-3.70, P < .001) and 2.15 (1.32-3.48, P = .002). NRAS mutations were associated with an increased risk of progression among standard-risk patients with high hyperdiploidy: 3.17 (1.15-8.71, P = .026). Patients classified clinically as standard and high risk had distinct genetic profiles. The outcome of clinical standard-risk patients with high-risk cytogenetics was equivalent to clinical high-risk patients. Screening patients at relapse for key genetic abnormalities will enable the integration of genetic and clinical risk factors to improve patient stratification and outcome. This study is registered at www.clinicaltrials.org as #ISCRTN45724312.

Natural variation and genetic analysis of the tiller angle gene MsTAC1 in Miscanthus sinensis.

Biomass yield is an important target trait in Miscanthus breeding for desirable energy crops. Tiller angle is a key trait of plant architecture because it determines planting density and further influences biomass yield through affecting photosynthesis efficiency. TAC1, a major gene involved in tiller and leaf angle control in rice and maize, respectively, has been extensively studied. Nucleotide variation at this gene, MsTAC1, was investigated in 33 Miscanthus sinensis accessions collected from different areas in China, and one genotype of Miscanthus × giganteus. A total of 136 loci, including 129 single base substitutions and seven InDels, occurred within the MsTAC1 gene of 1,874 bp. The genetic diversity at MsTAC1 is characterized by high nucleotide diversity (π value) and high heterozygosity. Clustering analysis indicated that the phylogenetic tree of the 33 M. sinensis accessions was correlated with their geographical sites of origin. The neutrality test revealed no strong selection pressure on coding and non-coding region variations of the MsTAC1 gene in the accessions. Phenotype evaluations were conducted for tiller angle and five other traits in the Miscanthus panels in the first two growth years of 2009 and 2010. Analysis of variance showed significant phenotypic variations in the examined traits. Association analysis using 246 markers detected 88 loci associated with all the test traits in either 1 or 2 years, and 11 of the 88 were year reproducible and thus reliable. These associations indicate that the variation of MsTAC1 affects the phenotypic value of the tiller angle, tiller number and biomass yield, suggesting that allelic variation in MsTAC1 affects multiple traits and demonstrates its significance in Miscanthus breeding programs.

From Planning Stage Towards FAIR Data: A Practical Metadatasheet For Biomedical Scientists.

Datasets consist of measurement data and metadata. Metadata provides context, essential for understanding and (re-)using data. Various metadata standards exist for different methods, systems and contexts. However, relevant information resides at differing stages across the data-lifecycle. Often, this information is defined and standardized only at publication stage, which can lead to data loss and workload increase. In this study, we developed Metadatasheet, a metadata standard based on interviews with members of two biomedical consortia and systematic screening of data repositories. It aligns with the data-lifecycle allowing synchronous metadata recording within Microsoft Excel, a widespread data recording software. Additionally, we provide an implementation, the Metadata Workbook, that offers user-friendly features like automation, dynamic adaption, metadata integrity checks, and export options for various metadata standards. By design and due to its extensive documentation, the proposed metadata standard simplifies recording and structuring of metadata for biomedical scientists, promoting practicality and convenience in data management. This framework can accelerate scientific progress by enhancing collaboration and knowledge transfer throughout the intermediate steps of data creation.

Transferability of microsatellite markers from Brachypodium distachyon to Miscanthus sinensis, a potential biomass crop.

Miscanthus sinensis has high biomass yield and contributed two of the three genomes in M. x giganteus, a bioenergy crop widely studied in Europe and North America, and thus is a potential biomass crop and an important germplasm for Miscanthus breeding. Molecular markers are essential for germplasm evaluation, genetic analyses and new cultivar development in M. sinensis. In the present study, we reported transferability of simple sequence repeat (SSR) markers from Brachypodium distachyon to M. sinensis. A set of 57 SSR markers evenly distributed across the B. distachyon genome were deliberately designed. Out of these B. distachyon SSR markers, 86.0% are transferable to M. sinensis. The SSR loci amplified in M. sinensis were validated by re-sequencing the amplicons. The polymorphism information content (PIC) of the transferable SSR markers varied from 0.073 to 0.375 with a mean of 0.263, assessed based on 21 M. sinensis genotypes. Phylogenetic tree based on 162 alleles detected by 49 SSR markers could unambiguously distinguish B. distachyon from M. sinensis, and cluster 21 M. sinensis genotypes into three groups that are basically in coincidence with their geographical distribution and ecotype classifications. The markers developed by the comparative genomic approach could be useful for germplasm evaluation, genetic analysis, and marker-assisted breeding in Miscanthus.

A turn-off-on near-infrared photoluminescence sensor for sequential detection of Fe3+ and ascorbic acid based on glutathione-capped gold nanoclusters.

Many reports have suggested that near-infrared (NIR) fluorescent probes are one of the most promising molecules for improving the sensitivity of fluorescence sensing and imaging. Herein, gold nanoclusters with excellent near-infrared photoluminescence (PL) were synthesized by a simply hydrothermal treatment of hydrogen tetrachloroaurate(III) trihydrate and glutathione (GSH). The NIR PL of GSH-capped gold nanoclusters (GSH-AuNCs) can be significantly quenched by Fe3+, which follows a dynamic quenching mechanism. However, the NIR PL of the GSH-AuNCs/Fe3+ system can be recovered after the addition of ascorbic acid (AA). The decrease and increase of NIR PL intensities of GSH-AuNCs were linearly correlated with the concentration of Fe3+ and AA, respectively. Therefore, a turn-off-on NIR PL sensing strategy can be constructed for sequential detection of Fe3+ and AA with the linear range of 0.7-180 µM and 0.5-120 µM, respectively. The proposed NIR PL sensor exhibits excellent sensing performance and has been applied to the determination of Fe3+ and AA in real samples with satisfactory results.

Upfront Treatment Influences the Composition of Genetic Alterations in Relapsed Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia.

Genomic alterations in relapsed B-cell precursor acute lymphoblastic leukemia (BCP-ALL) may provide insight into the role of specific genomic events in relapse development. Along this line, comparisons between the spectrum of alterations in relapses that arise in different upfront treatment protocols may provide valuable information on the association between the tumor genome, protocol components and outcome. Here, we performed a comprehensive characterization of relapsed BCP-ALL cases that developed in the context of 3 completed Dutch upfront studies, ALL8, ALL9, and ALL10. In total, 123 pediatric BCP-ALL relapses and 77 paired samples from primary diagnosis were analyzed for alterations in 22 recurrently affected genes. We found pronounced differences in relapse alterations between the 3 studies. Specifically, CREBBP mutations were observed predominantly in relapses after treatment with ALL8 and ALL10 which, in the latter group, were all detected in medium risk-treated patients. IKZF1 alterations were enriched 2.2-fold (p = 0.01) and 2.9-fold (p < 0.001) in ALL8 and ALL9 relapses compared to diagnosis, respectively, whereas no significant enrichment was found for relapses that were observed after treatment with ALL10. Furthermore, IKZF1 deletions were more frequently preserved from a major clone at diagnosis in relapses after ALL9 compared to relapses after ALL8 and ALL10 (p = 0.03). These data are in line with previous studies showing that the prognostic value of IKZF1 deletions differs between upfront protocols and is particularly strong in the ALL9 regimen. In conclusion, our data reveal a correlation between upfront treatment and the genetic composition of relapsed BCP-ALL.

Clonal Relationship Between Lichen Sclerosus, Differentiated Vulvar Intra-epithelial Neoplasia and Non HPV-related Vulvar Squamous Cell Carcinoma.

BACKGROUND/AIM: Differentiated vulvar intraepithelial neoplasia (dVIN) and lichen sclerosus (LS) can give rise to vulvar squamous cell carcinoma (VSCC), but genetic evidence is currently still limited. We aimed to determine genetic abnormalities in VSCC and backtrack these abnormalities in the dVIN and LS lesions. MATERIALS AND METHODS: DNA from VSCC and patient-matched dVIN and LS samples of twelve patients was collected. High-resolution genome-wide copy number analysis was performed and subsequently, we sequenced TP53. RESULTS: Copy number alterations were identified in all VSCC samples. One dVIN lesion presented with three copy number alterations that were preserved in the paired VSCC sample. Targeted sequencing of TP53 identified mutations in five VSCCs. All five mutations were traced back in the dVIN (n=5) or the LS (n=1) with frequencies ranging from 3-19%. CONCLUSION: Our data provide genetic evidence for a clonal relationship between VSCC and dVIN or LS.

Mutational landscape and patterns of clonal evolution in relapsed pediatric acute lymphoblastic leukemia.

Relapse of acute lymphoblastic leukemia (ALL) remains a leading cause of childhood death. Prior studies have shown clonal mutations at relapse often arise from relapse-fated subclones that exist at diagnosis. However, the genomic landscape, evolutionary trajectories and mutational mechanisms driving relapse are incompletely understood. In an analysis of 92 cases of relapsed childhood ALL, incorporating multimodal DNA and RNA sequencing, deep digital mutational tracking and xenografting to formally define clonal structure, we identify 50 significant targets of mutation with distinct patterns of mutational acquisition or enrichment. CREBBP, NOTCH1, and Ras signaling mutations rose from diagnosis subclones, whereas variants in NCOR2, USH2A and NT5C2 were exclusively observed at relapse. Evolutionary modeling and xenografting demonstrated that relapse-fated clones were minor (50%), major (27%) or multiclonal (18%) at diagnosis. Putative second leukemias, including those with lineage shift, were shown to most commonly represent relapse from an ancestral clone rather than a truly independent second primary leukemia. A subset of leukemias prone to repeated relapse exhibited hypermutation driven by at least three distinct mutational processes, resulting in heightened neoepitope burden and potential vulnerability to immunotherapy. Finally, relapse-driving sequence mutations were detected prior to relapse using deep digital PCR at levels comparable to orthogonal approaches to monitor levels of measurable residual disease. These results provide a genomic framework to anticipate and circumvent relapse by earlier detection and targeting of relapse-fated clones.

Structure-activity relationship of taxol inferring from docking taxol analogues to microtubule binding site.

In order to find the minimal structural requirements to maintain microtubule binding, 12 taxol analogues have been docked to the taxol binding site of tubulin. By comparing the interactions of each analogue with beta-tubulin, the structure-activity relationships are summarized as follow: C-2 benzoyl and taxane ring systems are the essential groups for microtubule binding, the improvements of bioactivity and bioavailability are dependent on the substituents at positions C-1, C-4, C-7, C-9, C-10, and C-14, whereas the C-13 side chain mainly provides a specific binding.

Accurate detection of low-level mosaic mutations in pediatric acute lymphoblastic leukemia using single molecule tagging and deep-sequencing.

Pathogenic mutations in relapse-associated genes in pediatric acute lymphoblastic leukemia may improve risk stratification when detected at subclonal levels at primary diagnosis. However, to detect subclonal mutations upfront, a deep-sequencing approach with high specificity and sensitivity is required. Here, we performed a proof-of-principle study to detect low-level mosaic RAS pathway mutations by deep sequencing using random tagging-based single molecule Molecular Inversion Probes (smMIPs). The smMIP-based approach could sensitively detect variants with allele frequency as low as 0.4%, which could all be confirmed by other techniques. In comparison, with standard deep-sequencing techniques we reached a detection threshold of only 2.5%, which hampered detection of seven low-level mosaic mutations representing 24% of all detected mutations. We conclude that smMIP-based deep-sequencing outperforms standard deep-sequencing techniques by showing lower background noise and high specificity, and is the preferred technology for detecting mutations upfront, particularly in genes in which mutations show limited clustering in hotspots.