Early-life infection depletes preleukemic cells in a mouse model of hyperdiploid B-cell acute lymphoblastic leukemia.

Epidemiological studies report opposing influences of infection on childhood B cell acute lymphoblastic leukemia (B-ALL). Although infections in the first year of life appear to exert the largest impact on leukemia risk, the effect of early pathogen exposure on the fetal preleukemia cells (PLC) that lead to B-ALL has yet to be reported. Using cytomegalovirus as a model early-life infection, we show that virus exposure within one week of birth induces profound depletion of transplanted B-ALL cells in two mouse models and of in situ-generated PLC in Eu-ret mice. The age-dependent depletion of PLC results from an elevated STAT4-mediated cytokine response in neonates, with high levels of IL-12p40-driven IFN-g production inducing PLC death. Similar PLC depletion can be achieved in adult mice by impairing viral clearance. These findings provide mechanistic support for an inhibitory effect of early-life infection on B-ALL progression and could inform development of therapeutic or preventative approaches.

A cross-standardized flow cytometry platform to assess phenotypic stability in precursor B-cell acute lymphoblastic leukemia (B-ALL) xenografts.

With the continued poor outcome of relapsed acute lymphoblastic leukemia (ALL), new patient-specific approaches for disease progression monitoring and therapeutic intervention are urgently needed. Patient-derived xenografts (PDX) of primary ALL in immune-deficient mice have become a powerful tool for studying leukemia biology and therapy response. In PDX mice, the immunophenotype of the patient's leukemia is commonly believed to be stably propagated. In patients, however, the surface marker expression profile of the leukemic population often displays poorly understood immunophenotypic shifts during chemotherapy and ALL progression. We therefore developed a translational flow cytometry platform to study whether the patient-specific immunophenotype is faithfully recapitulated in PDX mice. To enable valid assessment of immunophenotypic stability and subpopulation complexity of the patient's leukemia after xenotransplantation, we comprehensively immunophenotyped diagnostic B-ALL from children and their matched PDX using identical, clinically standardized flow protocols and instrument settings. This cross-standardized approach ensured longitudinal stability and cross-platform comparability of marker expression intensity at high phenotyping depth. This analysis revealed readily detectable changes to the patient leukemia-associated immunophenotype (LAIP) after xenotransplantation. To further investigate the mechanism underlying these complex immunophenotypic shifts, we applied an integrated analytical approach that combined clinical phenotyping depth and high analytical sensitivity with unbiased high-dimensional algorithm-based analysis. This high-resolution analysis revealed that xenotransplantation achieves patient-specific propagation of phenotypically stable B-ALL subpopulations and that the immunophenotypic shifts observed at the level of bulk leukemia were consistent with changes in underlying subpopulation abundance. By incorporating the immunophenotypic complexity of leukemic populations, this novel cross-standardized analytical platform could greatly expand the utility of PDX for investigating ALL progression biology and assessing therapies directed at eliminating relapse-driving leukemic subpopulations.

IFN-γ directly inhibits murine B-cell precursor leukemia-initiating cell proliferation early in life.

The early-life immune environment has been implicated as a modulator of acute lymphoblastic leukemia (ALL) development in children, with infection being associated with significant changes in ALL risk. Furthermore, polymorphisms in several cytokine genes, including IL-10 and IFN-γ, are associated with leukemia development. However, the mechanisms and timing of these influences remain unknown. Here, we use the Eµ-ret transgenic mouse model of B-cell precursor ALL to assess the influence of IFN-γ on the early-life burden of leukemia-initiating cells. The absence of IFN-γ activity resulted in greater numbers of leukemia-initiating cells early in life and was associated with accelerated leukemia onset. The leukemia-initiating cells from IFN-γ-knockout mice had reduced suppressor of cytokine signaling (SOCS-1) expression, were significantly more sensitive to IFN-γ, and exhibited more rapid expansion in vivo than their wild-type counterparts. However, sensitivity to this inhibitory pathway was lost in fully transformed IFN-γ-knockout leukemia cells. These results demonstrate that the influence of IFN-γ on ALL progression may not be mediated by selection of nascent transformed cells but rather through a general SOCS-mediated reduction in B-cell precursor proliferation. Thus, while cytokine levels may influence leukemia at multiple points during disease progression, our study indicates a significant early influence of basal, infection-independent cytokine production on leukemogenesis.

Absolute lymphocyte counts at end of induction correlate with distinct immune cell compartments in pediatric B cell precursor acute lymphoblastic leukemia.

Several retrospective studies in children with B cell precursor (BCP) acute lymphoblastic leukemia (ALL) provided clinical evidence that higher absolute lymphocyte counts (ALC) early into treatment significantly correlated with improved relapse-free and overall survival. It still remains unknown, however, whether the predictive role of higher ALCs reflects general bone marrow recovery or a more specific attribute of immune function. To investigate this question, we implemented a prospective observational cohort study in 20 children with BCP ALL on day 29 (D29) of induction chemotherapy and immunophenotyped their lymphoid (T, B and natural killer cells) and myeloid (neutrophils, monocytes, dendritic cells) compartments. In a first evaluation of a cohort treated with Children's Oncology Group-based induction chemotherapy, the immune cell compartments were differentially depleted at D29. Neither gender, risk status, minimal residual disease, nor bone marrow recovery markers correlated with D29 ALC. In contrast, both CD3+ T cell and dendritic cell compartments, which did not correlate with age, significantly correlated with D29 ALC (p < 0.0001). In addition, subset complexity of cellular immune compartments was preserved at D29. This study reveals that D29 ALC significantly correlates with distinct immune cell compartments but not with bone marrow recovery markers, suggesting that higher D29 ALCs may contribute to leukemia control by inducing specific host immune activity.

Quantification of fetal myocardial function in pregnant women with diabetic diseases and in normal controls using speckle tracking echocardiography (STE).

Background The purpose of our study was to quantify the fetal myocardial function in pregnant women with diabetic diseases (FDM) and in normal controls (FC) using speckle tracking echocardiography (STE). Methods In this prospective study, the myocardial strain and dyssynchrony were analyzed using STE in a transversal four-chamber view in 180 fetuses (53 FDM, 127 FC) between 19 and 39 weeks of gestation. The measurements of the global and segmental longitudinal strain of both chambers (2C) and of the single left chamber (1C) were executed offline via QLab 10.5 (Philips Medical Systems, Andover, MA, USA). We assessed dyssynchrony as the time difference between peaks in strain in the mid segments of both chambers (interventricular dyssynchrony, 2C_DYS) and of the single left chamber (intraventricular dyssynchrony, 1C_DYS). Results Measurements were feasible with a high median frame rate of 199 frames/s (1st quartile: 174, 3rd quartile: 199). The global and segmental myocardial longitudinal strain of 2C and 1C were decreased and 2C_DYS and 1C_DYS were increased in pregnancies with diabetes compared to normal controls. Conclusion Our study demonstrates that fetal hearts affected by maternal diabetes mellitus (DM) show low myocardial strain values and high interventricular dyssynchrony. Two-chamber interventricular dyssynchrony has the potential to become a diagnostic marker for DM.

Heterodimer-specific TLR2 stimulation results in divergent functional outcomes in B-cell precursor acute lymphoblastic leukemia.

Reports of spontaneous acute lymphoblastic leukemia (ALL) remissions following severe bacterial infections suggest that bacterial components may trigger elimination of ALL. To date, TLR2, which recognizes a broad range of bacterial pathogens through TLR1 or TLR6 heterodimerization, has not been fully evaluated for direct effects on ALL. Studies investigating TLR2 signaling in other tumor cell types utilizing single ligands have yielded contradictory results, and comparative, heterodimer-specific analyses of TLR2 stimulation are lacking. In this study, we report that two well-characterized heterodimer-specific TLR2 ligands, Pam3 CSK4 (TLR2/1), and Pam2 CSK4 (TLR2/6), induce ALL cell lines and primary ALL samples to upregulate CD40 expression. However, only Pam3 CSK4 triggers Caspase-8-mediated apoptosis and sensitizes cells to vincristine-mediated cytotoxicity. Consistent with this result, stimulation of ALL cells through TLR2/1 or TLR2/6 activates Mal, p38 and the NF-κB and PI3K signaling pathways with divergent kinetics that may underlie their distinct downstream effects. Our results reveal a novel branching in downstream responses to heterodimer-specific TLR2 stimulation in ALL cells and emphasize the need for comparative studies to determine differential biological effects observed in specific tumor cells. Based on our results, TLR2/1 ligand Pam3 CSK4 possesses potential for generating anti-ALL activity through its direct effects on leukemic blasts.

Recommendations for using artificial intelligence in clinical flow cytometry.

Flow cytometry is a key clinical tool in the diagnosis of many hematologic malignancies and traditionally requires close inspection of digital data by hematopathologists with expert domain knowledge. Advances in artificial intelligence (AI) are transferable to flow cytometry and have the potential to improve efficiency and prioritization of cases, reduce errors, and highlight fundamental, previously unrecognized associations with underlying biological processes. As a multidisciplinary group of stakeholders, we review a range of critical considerations for appropriately applying AI to clinical flow cytometry, including use case identification, low and high risk use cases, validation, revalidation, computational considerations, and the present regulatory frameworks surrounding AI in clinical medicine. In particular, we provide practical guidance for the development, implementation, and suggestions for potential regulation of AI-based methods in the clinical flow cytometry laboratory. We expect these recommendations to be a helpful initial framework of reference, which will also require additional updates as the field matures.

The Eµ-Ret mouse is a novel model of hyperdiploid B-cell acute lymphoblastic leukemia.

The presence of supernumerary chromosomes is the only abnormality shared by all patients diagnosed with high-hyperdiploid B cell acute lymphoblastic leukemia (HD-ALL). Despite being the most frequently diagnosed pediatric leukemia, the lack of clonal molecular lesions and complete absence of appropriate experimental models have impeded the elucidation of HD-ALL leukemogenesis. Here, we report that for 23 leukemia samples isolated from moribund Eµ-Ret mice, all were characterized by non-random chromosomal gains, involving combinations of trisomy 9, 12, 14, 15, and 17. With a median gain of three chromosomes, leukemia emerged after a prolonged latency from a preleukemic B cell precursor cell population displaying more diverse aneuploidy. Transition from preleukemia to overt disease in Eµ-Ret mice is associated with acquisition of heterogeneous genomic abnormalities affecting the expression of genes implicated in pediatric B-ALL. The development of abnormal centrosomes in parallel with aneuploidy renders both preleukemic and leukemic cells sensitive to inhibitors of centrosome clustering, enabling targeted in vivo depletion of leukemia-propagating cells. This study reveals the Eµ-Ret mouse to be a novel tool for investigating HD-ALL leukemogenesis, including supervision and selection of preleukemic aneuploid clones by the immune system and identification of vulnerabilities that could be targeted to prevent relapse.

Erythroid side scatter: A parameter that improves diagnostic accuracy of flow cytometry myelodysplastic syndrome scoring.

BACKGROUND: Flow cytometry immunophenotyping (FCM) is a benchmark test for integrated diagnosis of myelodysplastic syndromes (MDS). Our department's FCM-MDS-score follows international guidelines and additionally includes the maturing erythroid (mEry) side scatter (SSC)/lymphocyte SSC ratio (mErySSCr), often increased in MDS patients. A recent exploratory computational flow analysis study highlighted mErySSC as the top feature for separating MDS from non-MDS. Thus, we sought to systematically evaluate the diagnostic accuracy of mErySSCr in conventional diagnostic FCM as used currently in-house. METHODS: Historical MDS (n = 93), chronic myelomonocytic leukemia (CMML; n = 27) and non-neoplastic cytopenia (n = 57) cohorts were created. Differences between these cohorts and LG-MDS entities were mapped and the mErySSCr cut-off was refined. Prospective bone marrows (n = 213) received for marrow failure work-up were used to determine the sensitivity and specificity of mErySSCr, both as a sole parameter and as a component of the MDS-score. RESULTS: Low-grade (LG)-MDS mErySSCr differed more prominently from controls (p = <0.0001) than high-grade (HG)-MDS (p = 0.024). CMML and controls had a similar mErySSCr. As sole parameter, mErySSCr specificity was 91.1% (n = 112 non-MDS diagnoses) and sensitivity was 36% for LG-MDS (n = 36) and 25% for new HG-MDS diagnoses (n = 16). The specificity of the MDS-score was similar if mErySSCr was omitted (81.3% with and 82.1% without). The MDS-score sensitivity for new HG-MDS diagnoses and CMML (n = 17) was 100%, and was not affected by mErySSCr. The score sensitivity for LG-MDS however, dropped from 86.1% to 72.2% when mErySSCr was excluded. CONCLUSION: mErySSCr increases the diagnostic accuracy of flow-based MDS scoring in our setting, particularly for LG-MDS.

Age and ligand specificity influence the outcome of pathogen engagement on preleukemic and leukemic B-cell precursor populations.

Common infections have long been proposed to play a role in the development of pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, epidemiologic studies report contradictory effects of infection exposure on subsequent B-ALL risk, and no specific pathogen has been definitively linked to the disease. A unifying mechanism to explain the divergent outcomes could inform disease prevention strategies. We previously reported that the pattern recognition receptor (PRR) ligand Poly(I:C) exerted effects on B-ALL cells that were distinct from those observed with other nucleic acid-based PRR ligands. Here, using multiple double-stranded RNA (dsRNA) moieties, we show that the overall outcome of exposure to Poly(I:C) reflects the balance of opposing responses induced by its ligation to endosomal and cytoplasmic receptors. This PRR response biology is shared between mouse and human B-ALL and can increase leukemia-initiating cell burden in vivo during the preleukemia phase of B-ALL, primarily through tumor necrosis factor α signaling. The age of the responding immune system further influences the impact of dsRNA exposure on B-ALL cells in both mouse and human settings. Overall, our study demonstrates that potentially proleukemic and antileukemic effects can each be generated by the stimulation of pathogen recognition pathways and indicates a mechanistic explanation for the contrasting epidemiologic associations reported for infection exposure and B-ALL.

Targetable lesions and proteomes predict therapy sensitivity through disease evolution in pediatric acute lymphoblastic leukemia.

Childhood acute lymphoblastic leukemia (ALL) genomes show that relapses often arise from subclonal outgrowths. However, the impact of clonal evolution on the actionable proteome and response to targeted therapy is not known. Here, we present a comprehensive retrospective analysis of paired ALL diagnosis and relapsed specimen. Targeted next generation sequencing and proteome analysis indicate persistence of actionable genome variants and stable proteomes through disease progression. Paired viably-frozen biopsies show high correlation of drug response to variant-targeted therapies but in vitro selectivity is low. Proteome analysis prioritizes PARP1 as a pan-ALL target candidate needed for survival following cellular stress; diagnostic and relapsed ALL samples demonstrate robust sensitivity to treatment with two PARP1/2 inhibitors. Together, these findings support initiating prospective precision oncology approaches at ALL diagnosis and emphasize the need to incorporate proteome analysis to prospectively determine tumor sensitivities, which are likely to be retained at disease relapse.

PDX models reflect the proteome landscape of pediatric acute lymphoblastic leukemia but divert in select pathways.

BACKGROUND: Murine xenografts of pediatric leukemia accurately recapitulate genomic aberrations. How this translates to the functional capacity of cells remains unclear. Here, we studied global protein abundance, phosphorylation, and protein maturation by proteolytic processing in 11 pediatric B- and T- cell ALL patients and 19 corresponding xenografts. METHODS: Xenograft models were generated for each pediatric patient leukemia. Mass spectrometry-based methods were used to investigate global protein abundance, protein phosphorylation, and limited proteolysis in paired patient and xenografted pediatric acute B- and T- cell lymphocytic leukemia, as well as in pediatric leukemia cell lines. Targeted next-generation sequencing was utilized to examine genetic abnormalities in patients and in corresponding xenografts. Bioinformatic and statistical analysis were performed to identify functional mechanisms associated with proteins and protein post-translational modifications. RESULTS: Overall, we found xenograft proteomes to be most equivalent with their patient of origin. Protein level differences that stratified disease subtypes at diagnostic and relapse stages were largely recapitulated in xenografts. As expected, PDXs lacked multiple human leukocyte antigens and complement proteins. We found increased expression of cell cycle proteins indicating a high proliferative capacity of xenografted cells. Structural genomic changes and mutations were reflected at the protein level in patients. In contrast, the post-translational modification landscape was shaped by leukemia type and host and only to a limited degree by the patient of origin. Of 201 known pediatric oncogenic drivers and drug-targetable proteins, the KMT2 protein family showed consistently high variability between patient and corresponding xenografts. Comprehensive N terminomics revealed deregulated proteolytic processing in leukemic cells, in particular from caspase-driven cleavages found in patient cells. CONCLUSION: Genomic and host factors shape protein and post-translational modification landscapes differently. This study highlights select areas of diverging biology while confirming murine patient-derived xenografts as a generally accurate model system.

Clinical and laboratory phenotypes associated with the aspirin-like defect: a study in 17 unrelated families.

Aspirin-like defect (ALD) is a rare, mostly autosomal dominant inherited dysfunction of the intraplatelet arachidonic acid (AA) pathway leading to impaired thromboxane A2 signalling. We aimed to establish diagnostic criteria for ALD diagnosis and present clinical and laboratory phenotypes of 52 individuals from 17 unrelated families. Platelet in vitro function was determined on the basis of platelet aggregation response (PAR) to AA, adenosine diphosphate, collagen and ristocetin as well as PFA-100 closure times (CT). Using impaired PAR to AA (< or =10%) as the mandatory diagnostic criterion, ALD could be confirmed in 17 patients. Subsequently, family members were investigated and among 35 individuals an additional 13 ALD patients as well as 4 individuals with mild ALD (PAR to AA: 19-32%) were identified. At least one bleeding symptom was reported by 25 (74%) ALD patients and prolonged CT was detected in 24 (71%) of the cases, both significantly correlated with impaired PAR to AA (P = 0.001 and P = 0.002, respectively). An estimated 0.6% prevalence was determined for ALD in our paediatric patients with suspected coagulation disorders. Due to the mild bleeding symptoms, ALD is probably underdiagnosed. If ALD is suspected, PAR to AA is suitable for the identification of individuals at risk of increased haemorrhage.

Genome-wide platelet RNA profiling in clinical samples.

Human blood platelets are anucleate cells that contain minute amounts of translational active mRNA. Investigation of the gene expression profile by microarray analysis has become an excellent tool for better understanding of normal and pathological platelet function. Its use, however, is often limited by the low yield of megakaryocytic-derived mRNA, the possible contamination with leukocytes during platelet preparation and the small platelet volume in clinical settings, especially in pediatric patients. In this chapter, we present a protocol for the isolation of leukocyte-depleted platelet samples in clinical settings and an optimized procedure for transcript profiling, using the Agilent oligo microarray technology. In particular, we discuss the special aspects of platelet purification when working with blood sample volumes not exceeding 3-5 ml, which is typical in pediatric patients and we furthermore provide detailed information for transcript profiling of extremely small amounts of platelet RNA.

Tumor Variant Identification That Accounts for the Unique Molecular Landscape of Pediatric Malignancies.

Precision oncology trials for pediatric cancers require rapid and accurate detection of genetic alterations. Tumor variant identification should interrogate the distinctive driver genes and more frequent copy number variants and gene fusions that are characteristics of pediatric tumors. Here, we evaluate tumor variant identification using whole genome sequencing (n = 12 samples) and two amplification-based next-generation sequencing assays (n = 28 samples), including one assay designed to rapidly assess common diagnostic, prognostic, and therapeutic biomarkers found in pediatric tumors. Variant identification by the three modalities was comparable when filtered for 151 pediatric driver genes. Across the 28 samples, the pediatric cancer-focused assay detected more tumor variants per sample (two-sided, P < .05), which improved the identification of potentially druggable events and matched pathway inhibitors. Overall, our data indicate that an assay designed to evaluate pediatric cancer-specific variants, including gene fusions, may improve the detection of target-agent pairs for precision oncology.

Y-box-binding protein 1 contributes to IL-7-mediated survival signaling in B-cell precursor acute lymphoblastic leukemia.

Y-box-binding protein 1 (YB-1) is a regulatory protein that is associated with drug resistance and relapse in solid tumors. As YB-1 mediates some of its activity through growth factor receptor signaling dysregulation, the present study compared the expression of YB-1 and interleukin 7 (IL-7) receptor α (IL-7Rα) in pediatric B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) and normal BCP cells. The expression levels of IL-7Rα and YB-1 were higher in relapsed vs. diagnostic samples of primary BCP ALL; however, co-expression was also observed in a minor BCP cell population in samples from healthy donors. Functional crosstalk between YB-1 and IL-7R was detected: Overexpression of YB-1 increased surface levels of IL-7R in B cells, and the stimulation of BCP ALL cell lines and primary samples by IL-7 activated YB-1 by phosphorylation at S102 in a phosphatidylinositol 3-kinase-independent and MEK1/2-dependent manner. Targeted knockdown of YB-1 reduced IL-7-mediated protection against rapamycin, and an inhibitor of MEK1/2 potentiated rapamycin-mediated killing in the presence of IL-7. These data establish a novel link between two well-characterized pro-survival factors in acute leukemia, and suggest that YB-1 inhibition may represent a novel therapeutic strategy for increasing sensitivity to chemotherapy in patients with refractory acute B-cell leukemia.