Serotypes and Clonal Composition of Streptococcus pneumoniae Isolates Causing IPD in Children and Adults in Catalonia before 2013 to 2015 and after 2017 to 2019 Systematic Introduction of PCV13

We found that with the incorporation of the PCV13 vaccine, the numbers of IPD cases caused by serotypes included in this vaccine decreased in all of the age groups. Still, there was an unforeseen increase of the serotypes not included in this vaccine causing IPD, especially in the >65-year-old group.

SECEDO: SNV-based subclone detection using ultra-low coverage single-cell DNA sequencing

Recently developed single-cell DNA sequencing technologies enable whole-genome, amplifi-cation-free sequencing of thousands of cells at the cost of ultra-low coverage of the sequenced data(<0.05x per cell), which mostly limits their usage to the identification of copy number alterations(CNAs) in multi-megabase segments. Aside from CNA-based subclone detection, single-nucleotide vari-ant (SNV)-based subclone detection may contribute to a more comprehensive view on intra-tumorheterogeneity. Due to the low coverage of the data, the identification of SNVs is only possible whensuperimposing the sequenced genomes of hundreds of genetically similar cells. Here we present SingleCell Data Tumor Clusterer (SECEDO, lat. 'to separate'), a new method to cluster tumor cells basedsolely on SNVs, inferred on ultra-low coverage single-cell DNA sequencing data. The core aspects ofthe method are an efficient Bayesian filtering of relevant loci and the exploitation of read overlapsand phasing information. We applied SECEDO to a synthetic dataset simulating 7,250 cells and eighttumor subclones from a single patient, and were able to accurately reconstruct the clonal composition,detecting 92.11% of the somatic SNVs, with the smallest clusters representing only 6.9% of the totalpopulation. When applied to four real single-cell sequencing datasets from a breast cancer patient,SECEDO was able to recover the major clonal composition in each dataset at the original sequencingdepth of 0.03x per cell, an 8-fold improvement relative to the state of the art. Variant calling on theresulting clusters recovered more than twice as many SNVs with double the allelic ratio compared tocalling on all cells together, demonstrating the utility of SECEDO. SECEDO is implemented in C++ and is publicly available at https://github.com/ratschlab/secedo.

Chronic Lymphocytic Leukemia Cells with Mutated Nfkbie Are Positively Selected By Microenvironmental Signals and Display Reduced Sensitivity to Ibrutinib Treatment

Inactivating mutations in NF-kB pathway genes, such as the NF-kB inhibitor NFKBIE, are among the more frequent genetic lesions in chronic lymphocytic leukemia (CLL). However, the role of these genetic lesions in CLL pathogenesis and treatment resistance is still largely unknown and requires further study in in vivo models of the disease. To this end, we generated transplantable murine leukemias with inactivating NFKBIE mutations and investigated their impact on leukemia growth and response to ibrutinib (IBR) treatment. The NFKBIE mutations were introduced by CRISPR/Cas9 editing in two recently established autoreactive leukemia lines derived from the Eμ-TCL1 murine CLL model. These cell lines proliferate spontaneously in vitro in a BCR-dependent manner, but also respond with increased proliferation to certain microenvironmental signals, such as those generated by Toll-like receptor (TLR) stimulation (Chakraborty S et al, Blood 2021). To investigate whether NFKBIE mutations can affect the proliferation of these cell lines in vitro, we performed competition experiments with mixed cultures of cells with wild type and mutated NFKBIE. Analysis of the clonal composition after 2 weeks showed no change in the mutant allele frequency (MAF), suggesting that NFKBIE mutations do not affect the spontaneous in vitro growth of the immortalized leukemia cells. However, repeated TLR or BCR stimulation of these cells with CpG-DNA, LPS, anti-IgM or autoantigen resulted in a 2-3 fold increase in MAF, suggesting that NFKBIE mutations provide a growth advantage when the cells are exposed to certain microenvironmental signals (n=3 experiments/condition, P&lt;0.05 for each condition). To investigate the impact of NFKBIE mutations on leukemia growth in vivo, the same cells were transplanted by intraperitoneal injection in wild type mouse recipients (n=8) and the clonal composition was determined 3 weeks later by MAF analysis of cells isolated from peritoneal cavity (PC), blood and spleen. A significant increase in MAF was observed only in leukemia cells isolated from the spleen (P&lt;0.05), suggesting that microenvironmental signals that positively select NFKBIE-mutated cells are available only in certain tissue compartments. Because mutations in other NF-kB pathway genes have been associated with resistance to IBR in mantle cell lymphoma, we next investigated whether NFKBIE mutations can also affect the response to IBR treatment. In vitro BrdU-incorporation experiments showed that IBR inhibits the proliferation of cells with mutated NFKBIE to a significantly lesser extent compared to cells with wild type NFKBIE (% proliferating cells with wild type and mutated NFKBIE, respectively, cultured without IBR: 90% vs 88%, P=n.s., with 0.2 μM IBR: 57% vs 73%, P&lt;0.001, with 1.0 μM IBR: 28% vs 53%, P&lt;0.001). Consistent with this finding, positive selection of NFKBIE-mutated cells was observed in the presence of IBR after 14 days in mixed culture competition experiments (mean MAF without IBR 47%, with 0.2 μM IBR 61%, p=0.032, with 1.0 μM IBR 64%, p=0.034). The greater resistance of NFKBIE-mutated cells to IBR was further validated by in vivo competition experiments showing a significantly greater increase in MAF in mice treated with IBR compared to controls in all three investigated compartments (n=4 mice/group, PC: P=0.029, blood P=0.029, spleen: P=0.001). To validate these findings in the clinical setting, we investigated the presence of NFKBIE mutations in a cohort of 84 IBR-treated CLL patients. Mutations of NFKBIE were detected at pre-treatment in 10/84 patients, 7/10 with &gt;10% VAF values. Kaplan Meier analysis showed a trend towards reduced progression-free and overall survival from the beginning of IBR treatment for NFKBIE-mutated cases (Figure 1A). Analysis of an extended cohort of over 200 cases is ongoing and will be presented at the meeting. Finally, to investigate whether leukemic cells with mutated NFKBIE remain sensitive to other BCR inhibitors, we tested their growth in the presence of the PI3K inhibitor idelalisib or SYK inhibitor fostamatinib (Figure 1B). In contrast to IBR, both drugs inhibited the proliferation of NFKBIE-mutated cells in vitro, with a greater effect observed with idelalisib. Collectively, these data demonstrate that NFKBIE mutations can reduce the response to IBR treatment and suggest that such cases may benefit more from treatment with a PI3K inhibitor. Figure 1 Figure 1. Disclosures Marasca: Janssen: Honoraria, Other: Travel grants; AstraZeneca: Honoraria; AbbVie: Honoraria, Other: Travel grants. Tafuri: Roche: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Laurenti: Janssen: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria; BeiGene: Honoraria. Gattei: abbVie: Research Funding; Janssen: Research Funding; Menarini: Research Funding.

Neutral competition within a long-lived population of symmetrically dividing cells shapes the clonal composition of cerebral organoids

Cerebral organoids model the development of the human brain and have become an indispensable tool for studying neural development and neuro-developmental diseases. Comprehensive whole-organoid lineage tracing has revealed the fates of the lineages arising from each initial stem cells to be highly diverse, with lineage sizes ranging from one to more than 20,000 cells. This variability exceeds what can be explained by existing stochastic models of corticogenesis, which indicates that an additional source of stochasticity must exist. We propose the quantitative SAN model in which this additional source of stochasticity is neutral competition within a long-lived population of symmetrically dividing cells. In this model, the eventual size of a lineage is determined by its survival time within this population of symmetrically dividing cells, which due to neutral competition varies widely between individual lineages. We demonstrate the SAN model to explain the experimentally observed variability of lineage sizes and use it to derive a formula that captures the quantitative relationship between survival time and lineage size. Finally, we show that our model implies the existence of a mechanism which keeps the size of the population of symmetrically diving cells approximately constants, and that it enables this mechanism to be probed experimentally.

Growth of Scots pine plus trees clones, selected by resin productivity in Nizhny Novgorod region

The taxational indicators of clones of plus trees of Scots pine, selected by resin productivity, were studied in comparison with similar characteristics of plants, selected by linear parameters of the trunk. They are represented They are presented in the assortment of the forest seed plantation No. 10 in the Semenovsky forestry of the Nizhny Novgorod region, created in 1984 on a plot with the type of forest growing conditions — B2, and the type of forest — maynikovo-lingonberry pine. In the organization of the work, the principle of the only logical difference was observed, as well as the requirements for the typicality, suitability and expediency of the experience. As a test marker for checking the purity of the clonal composition of the plantation, the value of the angle of attachment of the first-order lateral branches to the trunk was used. The height and diameter of the trunk are taken into account in 571 trees with a continuous list. The distribution of the average values of the analyzed indicators in the vegetative offspring of plus trees compared with each other is not uniform. The highest height (16,70 ± 0,43 m) observed in clones of the K-011 plus tree selected by resin productivity is 2,65 m or 1,19 times higher than the lowest value (14,05 ± 0,44 m) inherent in clones of the K-113 plus tree selected by the same criteria, and 2,02 m or 1,14 times higher than the lowest value (14,23 ± 0,31 m) inherent in clones of the K-171 plus tree selected by the same criteria taxational indicators of the trunk. Differences in the taxational indicators of clones in the group of plus trees distinguished by resin productivity, as well as in the group of trunks distinguished by characteristics, correspond to the level of significant ones, which indicates the specificity of their genotypes. The degree of similarity of the plus trees in terms of trunk parameters is not the same, which indicates a different level of individual non-identity of each of the plus trees in relation to the others in their considered population.

Exites in Cambrian arthropods and homology of arthropod limb branches

The last common ancestor of all living arthropods had biramous postantennal appendages, with an endopodite and exopodite branching off the limb base. Morphological evidence for homology of these rami between crustaceans and chelicerates has, however, been challenged by data from clonal composition and from knockout of leg patterning genes. Cambrian arthropod fossils have been cited as providing support for competing hypotheses about biramy but have shed little light on additional lateral outgrowths, known as exites. Here we draw on microtomographic imaging of the Cambrian great-appendage arthropod Leanchoilia to reveal a previously undetected exite at the base of most appendages, composed of overlapping lamellae. A morphologically similar, and we infer homologous, exite is documented in the same position in members of the trilobite-allied Artiopoda. This early Cambrian exite morphology supplements an emerging picture from gene expression that exites may have a deeper origin in arthropod phylogeny than has been appreciated.

The site of breast cancer metastases dictates their clonal composition and reversible transcriptomic profile

Intratumoral heterogeneity is a driver of breast cancer progression, but the nature of the clonal interactive network involved in this process remains unclear. Here, we optimized the use of optical barcoding to visualize and characterize 31 cancer subclones in vivo. By mapping the clonal composition of thousands of metastases in two clinically relevant sites, the lungs and liver, we found that metastases were highly polyclonal in lungs but not in the liver. Furthermore, the transcriptome of the subclones varied according to their metastatic niche. We also identified a reversible niche-driven signature that was conserved in lung and liver metastases collected during patient autopsies. Among this signature, we found that the tumor necrosis factor–α pathway was up-regulated in lung compared to liver metastases, and inhibition of this pathway affected metastasis diversity. These results highlight that the cellular and molecular heterogeneity observed in metastases is largely dictated by the tumor microenvironment.

The role of migration in mutant evolution in fragmented populations

Mutant evolution in fragmented populations has been studied extensively in evolutionary biology. With an increased focus on evolutionary dynamics in medical research, quantification of mutant load in fragmented populations with varying levels of migration has become especially important. Examples of fragmented populations are hematopoietic stem cell niches in the bone marrow where cells can re-circulate between niches through the blood, or colonic crypts where movement of cells across different crypts is not thought to be common. Here we use a combination of experiments and theory to investigate the role of migration in mutant distribution. In the case of neutral mutants, the experiments confirmed that while the mean number of mutants is not influenced by migration, the probability distribution is, which manifested itself in a change in the skewedness of the distribution of the mutant numbers in the demes. In the case of disadvantageous mutants, we investigated the phenomenon of the increase in the expected number of mutants compared to that of the selection-mutation balance. In a single deme, this increase is observed when the deme size is lower than the critical size, $N_c$. In a fragmented system that consists of connected demes with a probability of migration, the increase in mutant numbers above the selection-mutation balance can be maintained in small ($N<N_c$) demes as long as the migration rate is sufficiently small. The migration rate above which the mutants approach the selection-mutation balance decays exponentially with $N/N_c$. These findings are relevant in the context of the complex and poorly understood processes that may lead to changes in the clonal composition in tissues and tumors.

POS0004 T-CELL REPERTOIRE OF SYNOVIAL FLUID IN SPONDYLOARTHROPATHIES EXHIBITS HALLMARKS OF HLA-DEPENDENT CLONAL EXPANSIONS AND REMAINS STABLE OVER 1.5 YEARS

Background:Different studies show involvement of T-cells in pathogenesis of spondyloarthropathies (SpAs) - a group of rheumatic diseases strongly associated with presence of several MHC-I alleles (HLA-B*27, -B*38, -B*39, etc). Recently we and others identified a specific T-cell receptor motif in blood and synovial fluid of HLA-B*27+ AS patients that reinforces the “arthritogenic peptide” hypothesis of AS pathogenesis [1-2]. However, common characteristics of clonal T-cell repertoire of synovial fluid in SpAs remain poorly investigated.Objectives:We aimed to investigate synovial fluid T-cell repertoires of SpA patients with different HLA-genotypes and stability of the clonal composition in recurring flares of the disease.Methods:Mononuclear cells were isolated from paired peripheral blood (PB) and synovial fluid (SF) samples of SpA patients (ankylosing spondylitis and psoriatic arthritis, n=27). For 3 patients additional SF samples were collected during relapse of the synovitis (after 9-15 months). CD4 and CD8 T-cells were isolated by immunomagnetic separation. Deep sequencing of UMI-tagged TCR beta cDNA libraries was used to accurately reconstruct clonal T-cell repertoires. HLA class I and II were typed for each donor using an in-house NGS-based system.Results:We observed restricted T-cell clonal composition in synovial fluid: on average only 6% of PB T-cell clonotypes were detected in SF of the same donor. T-cell repertoires of both CD4 and CD8 SF subsets compared to PB were highly oligoclonal (index Gini PB vs SF: CD4 0.36±0.10 vs 0.68±0.08, CD8 0.57±0.17 vs 0.81±0.12) in all patients. Number of identical amino acid CDR3 sequences between two repertoires correlated with the number of identical HLA-alleles for the donors. This trend was exhibited more strikingly in SF compared to PB, suggesting that common antigens may play a role in accumulation of identical T-cell clonotypes in the inflamed joint. Using several bioinformatic approaches we identified groups of highly similar SF clonotypes linked to HLA-B*27 and/or HLA-B*38 genotype.Total SF repertoires of relapsing synovitis of the same donor showed huge clonal overlap, and the most frequent clonotypes remained almost unchanged (Morisita’s overlap index for total SF repertoires 0.69±0.26; for top 1000 clonotypes 0.79±0.19, n=3).Conclusion:We report HLA-dependent sharing of identical and similar T-cell clonotypes in SF of patients with ankylosing spondylitis and psoriatic arthritis and high stability of SF repertoire during several flares that support antigen-driven accumulation of T-cells in the site of inflammation.References:[1]Komech EA et al. Rheumatology (Oxford). 2018;57(6):1097-1104.[2]Faham M et al. Arthritis Rheumatol. 2016;11(10):300-308.Acknowledgements:The work is supported by Russian Science Foundation grant №20-75-00041.Disclosure of Interests:None declared.