Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts.

One of the most frequent genes affected in pediatric ALL is the CDKN2A/2B gene, acting as a secondary cooperating event and playing an important role in cell-cycle regulation and chemosensitivity. Despite its inclusion in combined CNA (copy-number alterations) classifiers, like the IKZF1plus entity and the UKALL CNA profile, the prognostic impact of the individual gene deletions outside the context of a combined CNA evaluation remains controversial. Addressing the CDKN2A/2B deletions’ additive prognostic effect in current risk-stratification algorithms, we present a retrospective study of a Greek pediatric ALL cohort comprising 247 patients studied over a 24-year period (2000–2023). Herein, we provide insight regarding the correlation with disease features, MRD clearance, and independent prognostic significance for this ALL cohort treated with contemporary BFM-based treatment protocols. Within an extended follow-up time of 135 months, the presence of the CDKN2A/2B deletions (biallelic or monoallelic) was associated with inferior EFS rates (65.1% compared to 91.8% for the gene non-deleted subgroup, p < 0.001), with the relapse rate accounting for 22.2% and 5.9%, respectively (p < 0.001). The presence of the biallelic deletion was associated with the worst outcomes (EFS 57.2% vs. 89.6% in the case of any other status, monoallelic or non-deleted, p < 0.001). Survival differences were demonstrated for B-ALL cases (EFS 65.3% vs. 93.6% for the non-deleted B-ALL subgroup, p < 0.001), but the prognostic effect was not statistically significant within the T-ALL cohort (EFS 64.3 vs. 69.2, p = 0.947). The presence of the CDKN2A/2B deletions clearly correlated with inferior outcomes within all protocol-defined risk groups (standard risk (SR): EFS 66.7% vs. 100%, p < 0.001, intermediate risk (IR): EFS 77.1% vs. 97.9%, p < 0.001, high risk (HR): EFS 42.1% vs. 70.5% p < 0.001 for deleted vs non-deleted cases in each patient risk group); additionally, in this study, the presence of the deletion differentiated prognosis within both MRD-positive and -negative subgroups on days 15 and 33 of induction. In multivariate analysis, the presence of the CDKN2A/2B deletions was the most important prognostic factor for relapse and overall survival, yielding a hazard ratio of 5.2 (95% confidence interval: 2.59–10.41, p < 0.001) and 5.96 (95% confidence interval: 2.97–11.95, p < 0.001), respectively, designating the alteration’s independent prognostic significance in the context of modern risk stratification. The results of our study demonstrate that the presence of the CDKN2A/2B deletions can further stratify all existing risk groups, identifying patient subgroups with different outcomes. The above biallelic deletions could be incorporated into future risk-stratification algorithms, refining MRD-based stratification. In the era of targeted therapies, future prospective controlled clinical trials will further explore the possible use of cyclin-dependent kinase inhibitors (CDKIs) in CDKN2A/2B-affected ALL pediatric subgroups.

Data regarding the quantitative expression of TCR Vβ subpopulations in children with autoimmune diseases provided interesting and sometimes conflicting results. The aim of the present study was to assess by comparative flow cytometric analysis the peripheral blood CD4+ TCR Vβ repertoire of children with an organ-specific autoimmune disorder, such as type 1 diabetes mellitus (T1DM), in comparison to children with a systemic autoimmune disease, such as Systemic Lupus Erythematosus (SLE) in comparison to healthy age-matched controls of the same ethnic origin. The CD4+ TCR Vβ repertoire was analysed by flow cytometry in three groups of participants: a) fifteen newly diagnosed children with T1DM (mean age: 9.2 ± 4.78 years old), b) nine newly diagnosed children with SLE, positive for ANA and anti-dsDNA, prior to treatment (mean age: 12.8 ±1.76 years old) and c) 31 healthy age-matched controls (mean age: 6.58 ± 3.65 years old), all of Hellenic origin.

We aimed to assess the lipopolysaccharide (LPS), or heat shock (HS) induction, and glutamine-modulating effects on heat shock protein-90α (HSP90α) and cytokines in an ex vivo model using peripheral blood mononuclear cells (PBMCs). The PBMCs of patients with septic shock, trauma-related systemic inflammatory response syndrome (SIRS), and healthy subjects were incubated with 1 μg/mL LPS at 43 °C (HS). Glutamine 10 mM was added 1 hour before or after induction or not at all. We measured mRNA HSP90α, monocyte (m) and lymphocyte (l) HSP90α proteins, interleukin (IL)-1b, -6, -8, -10, tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein-1 (MCP-1) supernatant levels. Heat shock increased the HSP90α mRNA and mHSP90α in all groups (10-fold in sepsis, p < 0.001 and p = 0.047, respectively). LPS induced the mHSP90α and lHSP90α in healthy (p < 0.001) and mHSP90α in SIRS (p = 0.004) but not in sepsis. LPS induced the cytokines at 24 and 48 h in all groups, especially in trauma (p < 0.001); HS only induced the IL-8 in healthy (p = 0.003) and septic subjects (p = 0.05). Glutamine at 10 mM before or after stimulation did not alter any induction effect of LPS or HS on HSP90α mRNA and mHSP90α protein in sepsis. In SIRS, glutamine before LPS decreased the mHSP90α but increased it when given after HS (p = 0.018). Before or after LPS (p = 0.049) and before HS (p = 0.018), glutamine decreased the lHSP90α expression in sepsis but increased it in SIRS when given after HS (p = 0.003). Regarding cytokines, glutamine enhanced the LPS-induced MCP-1 at 48 h in healthy (p = 0.011), SIRS (p < 0.001), and sepsis (p = 0.006). In conclusion, glutamine at 10 mM, before or after LPS and HS, modulates mHSP90α and lHSP90α in sepsis and SIRS differently and unpredictably. Although it does not alter the stimulation effect on interleukins, glutamine enhances the LPS induction effect on supernatant MCP-1 in all groups. Future research should seek to elucidate better the impact of glutamine and temperature modulation on HSP90α and MCP-1 pathways in sepsis and trauma.

Pneumococcal vaccination schedules are traditionally assessed based on the antibody response. The Memory B Cell (MBC) response has been less studied, despite its role in the magnitude and longevity of protection. We compared the immune response to different vaccination schedules with the 13-valent Pneumococcal Conjugate Vaccine (PCV13) and investigated the relationship between MBCs and the antibody response. Total and pneumococcal serotype (PS)-specific MBCs, their subsets and PS-specific IgG antibodies induced by a 3 + 0 (group A), 2 + 1 (group B) or 3 + 1 (group C) schedule in healthy infants were studied before and 1 month after the last PCV13. The relatively immature IgM+IgD+ MBC subset was the predominant subset in all groups but was larger in group A compared to group B and group C, indicating that age might be a significant parameter of the composition of the MBC pool. PS-specific MBCs at baseline were higher in group A, but they increased significantly only in the groups receiving the booster schedules (groups B and C). PS-specific IgM-only MBCs at baseline positively corelated with the antibody response and the PS-specific swIg MBCs post-immunization. Our findings illustrate the importance of a booster dose for the enrichment of PS-specific immunological memory. IgM-only MBCs and swIg MBCs may serve as additional correlates of vaccine-induced protection.