Ikaros sets the threshold for negative B-cell selection by regulation of the signaling strength of the AKT pathway.

Inhibitory phosphatases, such as the inositol-5-phosphatase SHIP1 could potentially contribute to B-cell acute lymphoblastic leukemia (B-ALL) by raising the threshold for activation of the autoimmunity checkpoint, allowing malignant cells with strong oncogenic B-cell receptor signaling to escape negative selection. Here, we show that SHIP1 is differentially expressed across B-ALL subtypes and that high versus low SHIP1 expression is associated with specific B-ALL subgroups. In particular, we found high SHIP1 expression in both, Philadelphia chromosome (Ph)-positive and ETV6-RUNX1-rearranged B-ALL cells. As demonstrated by targeted knockdown of SHIP1 by RNA interference, proliferation of B-ALL cells in vitro and their tumorigenic spread in vivo depended in part on SHIP1 expression. We investigated the regulation of SHIP1, as an important antagonist of the AKT signaling pathway, by the B-cell-specific transcription factor Ikaros. Targeted restoration of Ikaros and pharmacological inhibition of the antagonistic casein kinase 2, led to a strong reduction in SHIP1 expression and at the same time to a significant inhibition of AKT activation and cell growth. Importantly, the tumor suppressive function of Ikaros was enhanced by a SHIP1-dependent additive effect. Furthermore, our study shows that all three AKT isoforms contribute to the pro-mitogenic and anti-apoptotic signaling in B-ALL cells. Conversely, hyperactivation of a single AKT isoform is sufficient to induce negative selection by increased oxidative stress. In summary, our study demonstrates the regulatory function of Ikaros on SHIP1 expression in B-ALL and highlights the relevance of sustained SHIP1 expression to prevent cells with hyperactivated PI3K/AKT/mTOR signaling from undergoing negative selection.

An artificial intelligence-assisted clinical framework to facilitate diagnostics and translational discovery in hematologic neoplasia.

BACKGROUND: The increasing volume and intricacy of sequencing data, along with other clinical and diagnostic data, like drug responses and measurable residual disease, creates challenges for efficient clinical comprehension and interpretation. Using paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) as a use case, we present an artificial intelligence (AI)-assisted clinical framework clinALL that integrates genomic and clinical data into a user-friendly interface to support routine diagnostics and reveal translational insights for hematologic neoplasia. METHODS: We performed targeted RNA sequencing in 1365 cases with haematological neoplasms, primarily paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) from the AIEOP-BFM ALL study. We carried out fluorescence in situ hybridization (FISH), karyotyping and arrayCGH as part of the routine diagnostics. The analysis results of these assays as well as additional clinical information were integrated into an interactive web interface using Bokeh, where the main graph is based on Uniform Manifold Approximation and Projection (UMAP) analysis of the gene expression data. At the backend of the clinALL, we built both shallow machine learning models and a deep neural network using Scikit-learn and PyTorch respectively. FINDINGS: By applying clinALL, 78% of undetermined patients under the current diagnostic protocol were stratified, and ambiguous cases were investigated. Translational insights were discovered, including IKZF1plus status dependent subpopulations of BCR::ABL1 positive patients, and a subpopulation within ETV6::RUNX1 positive patients that has a high relapse frequency. Our best machine learning models, LDA and PASNET-like neural network models, achieve F1 scores above 97% in predicting patients' subgroups. INTERPRETATION: An AI-assisted clinical framework that integrates both genomic and clinical data can take full advantage of the available data, improve point-of-care decision-making and reveal clinically relevant insights promptly. Such a lightweight and easily transferable framework works for both whole transcriptome data as well as the cost-effective targeted RNA-seq, enabling efficient and equitable delivery of personalized medicine in small clinics in developing countries. FUNDING: German Ministry of Education and Research (BMBF), German Research Foundation (DFG) and Foundation for Polish Science.

SHIP1 Is Present but Strongly Downregulated in T-ALL, and after Restoration Suppresses Leukemia Growth in a T-ALL Xenotransplantation Mouse Model.

Acute lymphoblastic leukemia (ALL) is the most common cause of cancer-related death in children. Despite significantly increased chances of cure, especially for high-risk ALL patients, it still represents a poor prognosis for a substantial fraction of patients. Misregulated proteins in central switching points of the cellular signaling pathways represent potentially important therapeutic targets. Recently, the inositol phosphatase SHIP1 (SH2-containing inositol 5-phosphatase) has been considered as a tumor suppressor in leukemia. SHIP1 serves as an important negative regulator of the PI3K/AKT signaling pathway, which is frequently constitutively activated in primary T-ALL. In contrast to other reports, we show for the first time that SHIP1 has not been lost in T-ALL cells, but is strongly downregulated. Reduced expression of SHIP1 leads to an increased activation of the PI3K/AKT signaling pathway. SHIP1-mRNA expression is frequently reduced in primary T-ALL samples, which is recapitulated by the decrease in SHIP1 expression at the protein level in seven out of eight available T-ALL patient samples. In addition, we investigated the change in the activity profile of tyrosine and serine/threonine kinases after the restoration of SHIP1 expression in Jurkat T-ALL cells. The tyrosine kinase receptor subfamilies of NTRK and PDGFR, which are upregulated in T-ALL subgroups with low SHIP1 expression, are significantly disabled after SHIP1 reconstitution. Lentiviral-mediated reconstitution of SHIP1 expression in Jurkat cells points to a decreased cellular proliferation upon transplantation into NSG mice in comparison to the control cohort. Together, our findings will help to elucidate the complex network of cell signaling proteins, further support a functional role for SHIP1 as tumor suppressor in T-ALL and, much more importantly, show that full-length SHIP1 is expressed in T-ALL samples.

The Prognostic Effect of IKZF1 Deletions in ETV6::RUNX1 and High Hyperdiploid Childhood Acute Lymphoblastic Leukemia.

IKZF1 deletions are an established prognostic factor in childhood acute lymphoblastic leukemia (ALL). However, their relevance in patients with good risk genetics, namely ETV6::RUNX1 and high hyperdiploid (HeH), ALL remains unclear. We assessed the prognostic impact of IKZF1 deletions in 939 ETV6::RUNX1 and 968 HeH ALL patients by evaluating data from 16 trials from 9 study groups. Only 3% of ETV6::RUNX1 cases (n = 26) were IKZF1-deleted; this adversely affected survival combining all trials (5-year event-free survival [EFS], 79% versus 92%; P = 0.02). No relapses occurred among the 14 patients with an IKZF1 deletion treated on a minimal residual disease (MRD)-guided protocols. Nine percent of HeH cases (n = 85) had an IKZF1 deletion; this adversely affected survival in all trials (5-year EFS, 76% versus 89%; P = 0.006) and in MRD-guided protocols (73% versus 88%; P = 0.004). HeH cases with an IKZF1 deletion had significantly higher end of induction MRD values (P = 0.03). Multivariate Cox regression showed that IKZF1 deletions negatively affected survival independent of sex, age, and white blood cell count at diagnosis in HeH ALL (hazard ratio of relapse rate [95% confidence interval]: 2.48 [1.32-4.66]). There was no evidence to suggest that IKZF1 deletions affected outcome in the small number of ETV6::RUNX1 cases in MRD-guided protocols but that they are related to higher MRD values, higher relapse, and lower survival rates in HeH ALL. Future trials are needed to study whether stratifying by MRD is adequate for HeH patients or additional risk stratification is necessary.

The cracking of Scots pine (Pinus sylvestris) cones.

Pine cones show functionally highly resilient, hygroscopically actuated opening and closing movements, which are repeatable and function even in millions of years old, coalified cones. Although the functional morphology and biomechanics behind the individual seed scale motions are well understood, the initial opening of the cone, which is often accompanied by an audible cracking noise, is not. We therefore investigated the initial opening events of mature fresh cones of Scots pine (Pinus sylvestris) and their subsequent motion patterns. Using high-speed and time lapse videography, 3D digital image correlation techniques, force measurements, thermographic and chemical-rheological resin analyses, we are able to draw a holistic picture of the initial opening process involving the rupture of resin seals and very fast seed scale motion in the millisecond regime. The rapid cone opening was not accompanied by immediate seed release in our experiments and, therefore, cannot be assigned to ballistochory. As the involved passive hydraulic-elastic processes in cracking are very fine-tuned, we hypothesize that they are under tight mechanical-structural control to ensure an ecologically optimized seed release upon environmental conditions suitable for wind dispersal. In this context, we propose an interplay of humidity and temperature to be the external "drivers" for the initial cone opening, in which resin works as a crucial chemical-mechanical latch system.

Amsacrine combined with etoposide and methylprednisolone is a feasible and safe component in first-line intensified treatment of pediatric patients with high-risk acute lymphoblastic leukemia in CoALL08-09 trial.

BACKGROUND: The prognosis of children with acute lymphoblastic leukemia (ALL) has improved considerably over the past five decades. However, to achieve cure in patients with refractory or relapsed disease, novel treatment options are necessary. METHODS: In the multicenter trial Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia (CoALL)08-09, one additional treatment element consisting of the rarely used chemotherapeutic agent amsacrine combined with etoposide and methylprednisolone (AEP) (amsacrine 2 × 100 mg/m2 , etoposide 2 × 500 mg/m2 , and methylprednisolone 4 × 1000 mg/m2 ) was incorporated into the first-line treatment of pediatric patients with poor treatment responses at the end of induction (EOI), measured by minimal residual disease (MRD). These patients were stratified into a high-risk intensified arm (HR-I), including an AEP element at the end of consolidation. Patients with induction failure (IF), that is, with lack of cytomorphological remission EOI, were eligible for hematopoietic stem cell transplantation (HSCT) after remission had been reached. These patients received AEP as a part of their MRD-guided bridging-to-transplant treatments. RESULTS: A significant improvement in probability of overall survival (pOS) was noted for the CoALL08-09 HR-I patients compared to MRD-matched patients from the preceding CoALL07-03 trial in the absence of severe or persistent treatment-related toxicities. Relapse rate and probability of event-free survival (pEFS) did not differ significantly between trials. In patients with IF, stable or improved MRD responses after AEP were observed without severe or persistent treatment-related toxicities. CONCLUSION: In conclusion, AEP is well tolerated as a component of the HR treatment and is useful in bridging-to-transplant settings.

Facing the Green Threat: A Water Flea's Defenses against a Carnivorous Plant.

Every ecosystem shows multiple levels of species interactions, which are often difficult to isolate and to classify regarding their specific nature. For most of the observed interactions, it comes down to either competition or consumption. The modes of consumption are various and defined by the nature of the consumed organism, e.g., carnivory, herbivory, as well as the extent of the consumption, e.g., grazing, parasitism. While the majority of consumers are animals, carnivorous plants can also pose a threat to arthropods. Water fleas of the family Daphniidae are keystone species in many lentic ecosystems. As most abundant filter feeders, they link the primary production to higher trophic levels. As a response to the high predatory pressures, water fleas have evolved various inducible defenses against animal predators. Here we show the first example, to our knowledge, in Ceriodaphnia dubia of such inducible defenses of an animal against a coexisting plant predator, i.e., the carnivorous bladderwort (Utricularia x neglecta Lehm, Lentibulariaceae). When the bladderwort is present, C. dubia shows changes in morphology, life history and behavior. While the morphological and behavioral adaptations improve C. dubia's survival rate in the presence of this predator, the life-history parameters likely reflect trade-offs for the defense.

The Structural and Mechanical Basis for Passive-Hydraulic Pine Cone Actuation.

The opening and closing of pine cones is based on the hygroscopic behavior of the individual seed scales around the cone axis, which bend passively in response to changes in environmental humidity. Although prior studies suggest a bilayer architecture consisting of lower actuating (swellable) sclereid and upper restrictive (non- or lesser swellable) sclerenchymatous fiber tissue layers to be the structural basis of this behavior, the exact mechanism of how humidity changes are translated into global movement are still unclear. Here, the mechanical and hydraulic properties of each structural component of the scale are investigated to get a holistic picture of their functional interplay. Measurements of the wetting behavior, water uptake, and mechanical measurements are used to analyze the influence of hydration on the different tissues of the cone scales. Furthermore, their dimensional changes during actuation are measured by comparative micro-computed tomography (µ-CT) investigations of dry and wet scales, which are corroborated and extended by 3D-digital image correlation-based displacement and strain analyses, biomechanical testing of actuation force, and finite element simulations. Altogether, a model allowing a detailed mechanistic understanding of pine cone actuation is developed, which is a prime concept generator for the development of biomimetic hygromorphic systems.

An alternative CYB5A transcript is expressed in aneuploid ALL and enriched in relapse.

BACKGROUND: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a genetically heterogenous malignancy with poor prognosis in relapsed adult patients. The genetic basis for relapse in aneuploid subtypes such as near haploid (NH) and high hyperdiploid (HeH) BCP-ALL is only poorly understood. Pathogenic genetic alterations remain to be identified. To this end, we investigated the dynamics of genetic alterations in a matched initial diagnosis-relapse (ID-REL) BCP-ALL cohort. Here, we firstly report the identification of the novel genetic alteration CYB5Aalt, an alternative transcript of CYB5A, in two independent cohorts. METHODS: We identified CYB5alt in the RNAseq-analysis of a matched ID-REL BCP-ALL cohort with 50 patients and quantified its expression in various molecular BCP-ALL subtypes. Findings were validated in an independent cohort of 140 first diagnosis samples from adult BCP-ALL patients. Derived from patient material, the alternative open reading frame of CYB5Aalt was cloned (pCYB5Aalt) and pCYB5Aalt or the empty vector were stably overexpressed in NALM-6 cells. RNA sequencing was performed of pCYB5Aalt clones and empty vector controls followed by differential expression analysis, gene set enrichment analysis and complementing cell death and viability assays to determine functional implications of CYB5Aalt. RESULTS: RNAseq data analysis revealed non-canonical exon usage of CYB5Aalt starting from a previously undescribed transcription start site. CYB5Aalt expression was increased in relapsed BCP-ALL and its occurrence was specific towards the shared gene expression cluster of NH and HeH BCP-ALL in independent cohorts. Overexpression of pCYB5Aalt in NALM-6 cells induced a distinct transcriptional program compared to empty vector controls with downregulation of pathways related to reported functions of CYB5A wildtype. Interestingly, CYB5A wildtype expression was decreased in CYB5Aalt samples in silico and in vitro. Additionally, pCYB5Aalt NALM-6 elicited a more resistant drug response. CONCLUSIONS: Across all age groups, CYB5Aalt was the most frequent secondary genetic event in relapsed NH and HeH BCP-ALL. In addition to its high subgroup specificity, CYB5Aalt is a novel candidate to be potentially implicated in therapy resistance in NH and HeH BCP-ALL. This is underlined by overexpressing CYB5Aalt providing first evidence for a functional role in BCL2-mediated apoptosis.

Clofarabine increases the eradication of minimal residual disease of primary B-precursor acute lymphoblastic leukemia compared to high-dose cytarabine without improvement of outcome. Results from the randomized clinical trial 08-09 of the Cooperative Acute Lymphoblastic Leukemia Study Group.

Novel treatment strategies are needed to improve cure for all children with acute lymphoblastic leukemia (ALL). To this end, we investigated the therapeutic potential of clofarabine in primary ALL in trial CoALL 08-09 (clinicaltrials gov. identifier: NCT01228331). The primary study objective was the minimal residual disease (MRD)- based comparative assessment of cytotoxic efficacies of clofarabine 5x40 mg/m2 versus high-dose cytarabine (HIDAC) 4x3g/m2, both in combination with PEG-ASP 2,500 IU/m2 as randomized intervention in early consolidation. The secondary objective was an outcome analysis focused on treatment arm dependence and MRD after randomized intervention. In B-cell precursor (BCP)-ALL, eradication of MRD was more profound after clofarabine compared to cytarabine, with 93 versus 79 of 143 randomized patients per arm reaching MRD-negativity (c2 test P=0.03, leftsided P [Fisher's exact test]=0.04). MRD status of BCP-ALL after randomized intervention maintained its prognostic relevance, with a significant impact on event-free survival (EFS) and relapse rate. However, no difference in outcome regarding EFS and overall survival (OS) between randomized courses was observed (5-year EFS: clofarabine 85.7, SE=4.1 vs. HIDAC 84.8, SE=4.7 [P=0.96]; OS: 95.7, SE=1.9 vs. 92.2, SE=3.2 [P=0.59]), independent of covariates or overall risk strata. Severe toxicities between randomized and subsequent treatment elements were also without significant difference. In conclusion, clofarabine/PEG-ASP is effective and safe, but greater cytotoxic efficacy of clofarabine compared to HIDAC did not translate into improved outcomes indicating a lack of surrogacy of post-intervention MRD at the trial level as opposed to the patient level, which hampers a broader implementation of this regimen in the frontline treatment of ALL.

Thwarting predators? A three-dimensional perspective of morphological alterations in the freshwater crustacean Daphnia.

Predation is a major selective agent, so that many taxa evolved phenotypically plastic defensive mechanisms. Among them are many species of the microcrustacean genus Daphnia, which respond to an increased predation risk by developing inducible morphological alterations. Some of these features are obvious and easily recognized, e.g., crests in D. longicephala, while others are rather hidden, such as the bulkier shape of D. magna induced by the presence of the tadpole shrimp Triops. In this study we investigated the extraordinary diversity of morphological adaptations in the presence of predators with different foraging strategies in six predator-prey systems. For the first time we were able to analyze the unexposed and predator-exposed morphs comprehensively using three-dimensional scanning and reconstruction. We show that morphological changes are manifold in appearance between species and predators, and go beyond what has been known from previous 2D analyses. This further demonstrates the enormous trait flexibility of Daphnia. Interestingly, we found that among this variety some species share morphological strategies to counter a predator, while others use a different strategy against the same predator. Based on these intra- and interspecific comparisons, we discuss the mechanisms by which the respective defense might operate. These data therefore contribute to a deeper understanding of the inducible defenses' morphology as well as their diversified modes of operation in Daphnia, being a cornerstone for subsequent investigations, including the determination of costs associated with morphological change.

Snapshot prey spectrum analysis of the phylogenetically early-diverging carnivorous Utricularia multifida from U. section Polypompholyx (Lentibulariaceae).

Utricularia multifida is carnivorous bladderwort from Western Australia and belongs to a phylogenetically early-diverging lineage of the genus. We present a prey spectrum analysis resulting from a snapshot sampling of 17 traps-the first of this species to our knowledge. The most abundant prey groups were Ostracoda, Copepoda, and Cladocera. The genus cf. Cypretta (Cyprididae, Ostracoda) was the predominant prey. However, a high variety of other prey organisms with different taxonomic backgrounds was also detected. Our results indicate that U. multifida may potentially be specialized in capturing substrate-bound prey. Future approaches should sample plants from different localities to allow for robust comparative analyses.

Genome-wide interference of ZNF423 with B-lineage transcriptional circuitries in acute lymphoblastic leukemia.

Aberrant expression of the transcriptional modulator and early B-cell factor 1 (EBF1) antagonist ZNF423 has been implicated in B-cell leukemogenesis, but its impact on transcriptional circuitries in lymphopoiesis has not been elucidated in a comprehensive manner. Herein, in silico analyses of multiple expression data sets on 1354 acute leukemia samples revealed a widespread presence of ZNF423 in various subtypes of acute lymphoblastic leukemia (ALL). Average expression of ZNF423 was highest in ETV6-RUNX1, B-other, and TCF3-PBX1 ALL followed by BCR-ABL, hyperdiploid ALL, and KMT2A-rearranged ALL. In a KMT2A-AFF1 pro-B ALL model, a CRISPR-Cas9-mediated genetic ablation of ZNF423 decreased cell viability and significantly prolonged survival of mice upon xenotransplantation. For the first time, we characterized the genome-wide binding pattern of ZNF423, its impact on the chromatin landscape, and differential gene activities in a B-lineage context. In general, chromatin-bound ZNF423 was associated with a depletion of activating histone marks. At the transcriptional level, EBF1-dependent transactivation was disrupted by ZNF423, whereas repressive and pioneering activities of EBF1 were not discernibly impeded. Unexpectedly, we identified an enrichment of ZNF423 at canonical EBF1-binding sites also in the absence of EBF1, which was indicative of intrinsic EBF1-independent ZNF423 activities. A genome-wide motif search at EBF1 target gene loci revealed that EBF1 and ZNF423 co-regulated genes often contain SMAD1/SMAD4-binding motifs as exemplified by the TGFB1 promoter, which was repressed by ZNF423 outcompeting EBF1 by depending on its ability to bind EBF1 consensus sites and to interact with EBF1 or SMADs. Overall, these findings underscore the wide scope of ZNF423 activities that interfere with B-cell lymphopoiesis and contribute to leukemogenesis.

Long-term effects of elevated pCO2 levels on the expression of Chaoborus-induced defences in Daphnia pulex.

Increased carbon dioxide from fossil fuel combustion results in an enrichment of CO2 in the global carbon cycle. Recent evidence indicates that rising atmospheric CO2 impacts the partial pressure of carbon dioxide (pCO2) in freshwaters. This affects freshwater biota by disrupting chemical communication between predator and prey. One such well-described predator-prey interaction is the phantom midge larva Chaoborus preying on the freshwater crustacean Daphnia pulex. To counter Chaoborus predation, D. pulex develops defensive neckteeth in response to chemical cues. The strength of neckteeth expression is reduced when D. pulex experience elevated pCO2 levels. This is discussed to directly impair predator perception and results in reduced defence expression. However, it is not known whether there are also long-term effects associated with continuous elevated pCO2. Here, we investigated the effect of long-term exposure of D. pulex to elevated pCO2 levels in a life-table experiment over three generations. Using a flow-through system, we continuously exposed D. pulex to cues released by the predatory larva Chaoborus and control or elevated pCO2 levels. We determined morphological defence expression in the 2nd juvenile instar and the number of neonates as a measure for life-history traits over three successive generations. We detected that elevated pCO2 significantly reduces the expression of predator-induced morphological defences (i.e. neckteeth) and life-history parameters (i.e. number of neonates) in successive generations. Our data clearly show that at least three generations become more vulnerable to predation without indications of transgenerational acclimation. As Daphnia is a keystone grazer of freshwater ecosystems, this may destabilise population growth rates. In conclusion, long-term effects of pCO2-induced reduction of predator-induced plasticity may significantly affect trophic interactions.

A validated novel continuous prognostic index to deliver stratified medicine in pediatric acute lymphoblastic leukemia.

Risk stratification is essential for the delivery of optimal treatment in childhood acute lymphoblastic leukemia. However, current risk stratification algorithms dichotomize variables and apply risk factors independently, which may incorrectly assume identical associations across biologically heterogeneous subsets and reduce statistical power. Accordingly, we developed and validated a prognostic index (PIUKALL) that integrates multiple risk factors and uses continuous data. We created discovery (n = 2405) and validation (n = 2313) cohorts using data from 4 recent trials (UKALL2003, COALL-03, DCOG-ALL10, and NOPHO-ALL2008). Using the discovery cohort, multivariate Cox regression modeling defined a minimal model including white cell count at diagnosis, pretreatment cytogenetics, and end-of-induction minimal residual disease. Using this model, we defined PIUKALL as a continuous variable that assigns personalized risk scores. PIUKALL correlated with risk of relapse and was validated in an independent cohort. Using PIUKALL to risk stratify patients improved the concordance index for all end points compared with traditional algorithms. We used PIUKALL to define 4 clinically relevant risk groups that had differential relapse rates at 5 years and were similar between the 2 cohorts (discovery: low, 3% [95% confidence interval (CI), 2%-4%]; standard, 8% [95% CI, 6%-10%]; intermediate, 17% [95% CI, 14%-21%]; and high, 48% [95% CI, 36%-60%; validation: low, 4% [95% CI, 3%-6%]; standard, 9% [95% CI, 6%-12%]; intermediate, 17% [95% CI, 14%-21%]; and high, 35% [95% CI, 24%-48%]). Analysis of the area under the curve confirmed the PIUKALL groups were significantly better at predicting outcome than algorithms employed in each trial. PIUKALL provides an accurate method for predicting outcome and more flexible method for defining risk groups in future studies.

Leukemia-induced dysfunctional TIM-3+CD4+ bone marrow T cells increase risk of relapse in pediatric B-precursor ALL patients.

Interaction of malignancies with tissue-specific immune cells has gained interest for prognosis and intervention of emerging immunotherapies. We analyzed bone marrow T cells (bmT) as tumor-infiltrating lymphocytes in pediatric precursor-B cell acute lymphoblastic leukemia (ALL). Based on data from 100 patients, we show that ALL is associated with late-stage CD4+ phenotype and loss of early CD8+ T cells. The inhibitory exhaustion marker TIM-3 on CD4+ bmT increased relapse risk (RFS = 94.6/70.3%) confirmed by multivariate analysis. The hazard ratio of TIM-3 expression nearly reached the hazard ratio of MRD (7.1 vs. 8.0) indicating that patients with a high frequency of TIM-3+CD4+ bone marrow T cells at initial diagnosis have a 7.1-fold increased risk to develop ALL relapse. Comparison of wild type primary T cells to CRISPR/Cas9-mediated TIM-3 knockout and TIM-3 overexpression confirmed the negative effect of TIM-3 on T cell responses against ALL. TIM-3+CD4+ bmT are increased in ALL overexpressing CD200, that leads to dysfunctional antileukemic T cell responses. In conclusion, TIM-3-mediated interaction between bmT and leukemia cells is shown as a strong risk factor for relapse in pediatric B-lineage ALL. CD200/TIM-3-signaling, rather than PD-1/PD-L1, is uncovered as a mechanism of T cell dysfunction in ALL with major implication for future immunotherapies.

Prey capture analyses in the carnivorous aquatic waterwheel plant (Aldrovanda vesiculosa L., Droseraceae).

We investigated the predator-prey interactions between an Australian ecotype of the carnivorous waterwheel plant (Aldrovanda vesiculosa, Droseraceae) and its potential natural prey, the water flea Daphnia longicephala (Daphniidae), which also occurs in Australia. A. vesiculosa develops snap-traps, which close within ~10-100 ms after mechanical triggering by zooplankton prey. Prey capture attempts (PCAs) were recorded via high-speed cinematography in the laboratory. From 14 recorded PCAs, nine were successful for the plant (the prey was caught), and five were unsuccessful (prey could escape), resulting in a capture rate of ~64%. The prey animals' locomotion behaviour (antenna beat frequency and movement type) in trap vicinity or inside the open traps is very variable. Traps were mainly triggered with the second antennae. During trap closure, the animals moved only very little actively. A flight response in reaction to an initiated trap closure was not observed. However, several animals could escape, either by having a "lucky" starting position already outside the triggered trap, by freeing themselves after trap closure, or by being pressed out by the closing trap lobes. According to our observations in the successful PCAs, we hypothesize that the convex curvature of the two trap lobes (as seen from the outside) and the infolded trap rims are structural means supporting the capture and retention of prey. Our results are discussed in a broader biological context and promising aspects for future studies are proposed.

Characterization of novel, recurrent genomic rearrangements as sensitive MRD targets in childhood B-cell precursor ALL.

B-cell precursor (BCP) ALL carry a variety of classical V(D)J rearrangements as well as genomic fusions and translocations. Here, we assessed the value of genomic capture high-throughput sequencing (gc-HTS) in BCP ALL (n = 183) for the identification and implementation of targets for minimal residual disease (MRD) testing. For TRδ, a total of 300 clonal rearrangements were detected in 158 of 183 samples (86%). Beside clonal Vδ2-Dδ3, Dδ2-Dδ3, and Vδ2-Jα we identified a novel group of recurrent Dδ-Jα rearrangements, comprising Dδ2 or Dδ3 segments fused predominantly to Jα29. For IGH-JH, 329 clonal rearrangements were identified in 172 of 183 samples (94%) including novel types of V(D)J joining. Oligoclonality was found in ~1/3 (n = 57/183) of ALL samples. Genomic breakpoints were identified in 71 BCP-ALL. A distinct MRD high-risk subgroup of IGH-V(D)J-germline ALL revealed frequent deletions of IKZF1 (n = 7/11) and the presence of genomic fusions (n = 10/11). Quantitative measurement using genomic fusion breakpoints achieved equivalent results compared to conventional V(D)J-based MRD testing and could be advantageous upon persistence of a leukemic clone. Taken together, selective gc-HTS expands the spectrum of suitable MRD targets and allows for the identification of genomic fusions relevant to risk and treatment stratification in childhood ALL.

Results of CoALL 07-03 study childhood ALL based on combined risk assessment by in vivo and in vitro pharmacosensitivity.

We conducted a clinical trial and report the long-term outcome of 773 children with acute lymphoblastic leukemia upon risk-adapted therapy accrued in trial CoALL 07-03 (from the Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia). In a 2-step stratification, patients were allocated to receive either low- or high-risk treatment, based on initial white blood cell count, age, and immunophenotype. A second stratification was performed according to the results of in vitro pharmacosensitivity toward prednisolone, vincristine, and asparaginase (PVA score) and in vivo response after induction therapy (minimal residual disease [MRD]). Therapy was reduced for both risk groups in patients with a low PVA score or negative MRD result, and intensified in patients with a high PVA score. Overall outcome improved significantly compared with the predecessor CoALL 06-97 trial, with identical therapy backbone despite treatment reduction in 15.8% of patients (10-year probability of event-free survival, 83.5% vs 73.9%; overall survival, 90.7% vs 83.8%). Outcome for patients in the reduced treatment arms was superior to that of patients in the standard arms, associated with a profound reduction in frequency and severity of infectious complications. Importantly, we observed a lack of correlation between in vitro and in vivo drug response, as well as a lower predictive value of in vitro drug testing, reflecting an intrinsic limitation of this methodology that prevents its use for treatment stratification in future trials. In conclusion, it might be possible to reduce chemotherapy in children with acute lymphoblastic leukemia selected by stringent in vivo measurement of MRD without jeopardizing overall outcome.