With the aim of describing the long-term follow-up and to define the prognostic role of the clinical/pathological/molecular characteristics at diagnosis for childhood, adolescent and young adults affected by anaplastic large cell lymphoma (ALCL), we analyzed 420 patients aged up to 22 years homogeneously treated within the international ALCL99 trial. The 10-year progression free survival (PFS) was 70% and overall survival was 90%, rare late relapses occurred but no secondary malignancies were reported. Among clinical/pathological characteristics, only patients presenting a small cell/lymphohistiocytic (SC/LH) pattern were independently associated with risk of failure (hazard ratio = 2.49). Analysis of minimal disseminated disease (MDD), available for 162 patients, showed that both SC/LH pattern (hazard ratio = 2.4) and MDD positivity (hazard ratio = 2.15) were significantly associated with risk of failure in multivariate analysis. Considering MDD and SC/LH results, patients were separated into three biological/pathological (bp) risk groups: a high-risk group (bpHR) including MDD-positive patients with SC/LH pattern; a low-risk group (bpLR) including MDD-negative patients without SC/LH pattern; and an intermediate-risk group (bpIR) including remaining patients. The 10-year PFS was 40%, 75% and 86% for bpHR, bpIR and bpLR, respectively (p < 0.0001). These results should be considered in the design of future ALCL trials to tailor individual treatments.

Plasma exosomal microRNAs (miRNAs) are considered as valid circulating biomarkers for cancer diagnosis and prognosis. Quantitative real-time polymerase chain reaction (qRT-PCR), the most commonly used technique to assess circulating miRNA levels, requires a normalization step involving uniformly expressed endogenous miRNAs. However, there is still no consensus on reference miRNAs for plasma exosomal miRNA abundance normalization. In this study, we identified a panel of miRNAs with stable abundance by analyzing public plasma exosome RNA-seq data and selected miR-486-5p, miR-26a-5p, miR-423-5p and miR191-5p as candidate normalizers. Next, we tested the abundance variation of these miRNAs by qRT-PCR in plasma exosomes of healthy donors and pediatric patients with anaplastic large cell lymphoma, Burkitt lymphoma, Hodgkin lymphoma and mature B-cell acute lymphoblastic leukemia. MiR-486-5p and miR-26a-5p showed the most stable levels, both between healthy controls and patients and among the malignancies analyzed. In light of previous reports on miRNA stability in different exosome isolation methods, our data indicated that miR-26a-5p is a bona fide reference miRNA for qRT-PCR normalization to evaluate miRNA abundance from circulating plasma exosomes in studies of hematological malignancies.

To describe incidence, causes, and outcomes related to pediatric intensive care unit (PICU) admission for patients undergoing hematopoietic stem cell transplantation (HSCT), we investigated the risk factors predisposing to PICU admission and prognostic factors in terms of patient survival. From October 1998 to April 2015, 496 children and young adults (0 to 23 years) underwent transplantation in the HSCT unit. Among them, 70 (14.1%) were admitted to PICU. The 3-year cumulative incidence of PICU admission was 14.3%. The main causes of PICU admission were respiratory failure (36%), multiple organ failure (16%), and septic shock (13%). The overall 90-day cumulative probability of survival after PICU admission was 34.3% (95% confidence interval, 24.8% to 47.4%). In multivariate analysis, risk factors predisposing to PICU admission were allogeneic HSCT (versus autologous HSCT, P = .030) and second or third HSCT (P = .018). Characteristics significantly associated with mortality were mismatched HSCT (P = .011), relapse of underlying disease before PICU admission (P < .001), acute respiratory distress syndrome at admission (P = .012), hepatic failure at admission (P = .021), and need for invasive ventilation during PICU course (P < .001). Our data indicate which patients have a high risk for PICU admission after HSCT and for dismal outcomes after PICU stay. These findings may provide support for the clinical decision-making process on the opportunity of PICU admission for severely compromised patients after HSCT.

Minimal disseminated and residual disease (MDD/MRD) analyzed by qualitative PCR for NPM-ALK fusion transcripts are validated prognostic factors in pediatric ALK-positive anaplastic large cell lymphoma (ALCL). Although potentially promising, MDD quantification by quantitative real-time PCR in international trials is technically challenging. Quantification of early MRD might further improve risk stratification. We aimed to assess droplet digital PCR for quantification of minimal disease in an inter-laboratory setting in a large cohort of 208 uniformly treated ALCL patients. Inter-laboratory quality control showed high concordance. Using a previously described cut-off of 30 copies NPM-ALK/10⁴ copies ABL1 (NCN) in bone marrow and peripheral blood, MDD quantification allowed identification of very high-risk patients (5-year PFS% 34 ± 5 for patients with ≥30 NCN compared to 74 ± 6 and 76 ± 5 for patients with negative or <30 NCN, respectively, p < 0.0001). While MRD positivity was confirmed as a prognostic marker for the detection of very high-risk patients in this large study, quantification of MRD fusion transcripts did not improve stratification. PFS% was 80 ± 5 and 73 ± 6 for MDD- and MRD-negative patients, respectively, versus 35 ± 10 and 16 ± 8 for MRD-positive patients with <30 and ≥30 NCN, p < 0.0001. Our results suggest that MDD quantification by dPCR enables improved patient stratification in international clinical studies and patient selection for early clinical trials already at diagnosis.

Over the past 15 years, several biological and pathological characteristics proved their significance in pediatric anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma (ALCL) prognostic stratification. However, the identification of new non-invasive disease biomarkers, relying on the most important disease mechanisms, is still necessary. In recent years, plasmatic circulating small extracellular vesicles (S-EVs) gathered great importance both as stable biomarker carriers and active players in tumorigenesis. In the present work, we performed a comprehensive study on the proteomic composition of plasmatic S-EVs of pediatric ALCL patients compared to healthy donors (HDs). By using a mass spectrometry-based proteomics approach, we identified 50 proteins significantly overrepresented in S-EVs of ALCL patients. Gene Ontology enrichment analysis disclosed cellular components and molecular functions connected with S-EV origin and vesicular trafficking, whereas cell adhesion, glycosaminoglycan metabolic process, extracellular matrix organization, collagen fibril organization and acute phase response were the most enriched biological processes. Of importance, consistently with the presence of nucleophosmin (NPM)-ALK fusion protein in ALCL cells, a topological enrichment analysis based on Reactome- and Kyoto Encyclopedia of Genes and Genomes (KEGG)-derived networks highlighted a dramatic increase in proteins of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway in ALCL S-EVs, which included heat shock protein 90-kDa isoform alpha 1 (HSP90AA1), osteopontin (SPP1/OPN) and tenascin C (TNC). These results were validated by Western blotting analysis on a panel of ALCL and HD cases. Further research is warranted to better define the role of these S-EV proteins as diagnostic and, possibly, prognostic parameters at diagnosis and for ALCL disease monitoring.