Search

Simple Summary: Detection of minimal disease in blood or bone marrow is associated with high relapse risk in children with anaplastic large cell lymphoma (ALCL). The persistence of minimal residual disease after one course of chemotherapy indicates a relapse risk of 80%. While quantification of minimal disease might further improve the identification of high-risk patients, the assays used for quantification currently are not transferable between multiple laboratories. We aimed to test a digital PCR method (dPCR) for comparison of minimal disease quantification between two laboratories and the usefulness of quantification for risk stratification of children with ALCL. Quantification of minimal disease by dPCR was concordant between laboratories and allowed identification of patients at very high risk for relapse. Qualitative detection of minimal residual disease after one course of chemotherapy sufficed to identify children at the highest risk of treatment failure. International dissemination of this assay will allow patient selection for new targeted treatment approaches. 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. [ABSTRACT FROM AUTHOR]

The unsatisfactory cure rate of relapsing ALK-positive Anaplastic Large-Cell Lymphoma (ALCL) of childhood calls for the identification of new prognostic markers. Here, the small RNA landscape of pediatric ALK-positive ALCL was defined by RNA sequencing. Overall, 121 miRNAs were significantly dysregulated in ALCL compared to non-neoplastic lymph nodes. The most up-regulated miRNA was miR-21-5p, whereas miR-19a-3p and miR-214-5p were reduced in ALCL. Characterization of miRNA expression in cases that relapsed after first line therapy disclosed a significant association between miR-214-5p down-regulation and aggressive non-common histology. Our results suggest that miR-214-5p level may help to refine the prognostic stratification of pediatric ALK-positive ALCL. [ABSTRACT FROM AUTHOR]

Emerging evidence indicates that extracellular vesicles, particularly exosomes, play a role in several biological processes and actively contribute to cancer development and progression, by carrying and delivering proteins, transcripts and small RNAs (sRNAs). There is high interest in studying exosomes of cancer patients both to develop non-invasive liquid biopsy tests for risk stratification and to elucidate their possible involvement in disease mechanisms. We profiled by RNA-seq the sRNA content of circulating exosomes of 20 pediatric patients with Anaplastic Large Cell Lymphoma (ALCL) and five healthy controls. Our analysis disclosed that non-miRNA derived sRNAs constitute the prominent fraction of sRNA loaded in exosomes and identified 180 sRNAs significantly more abundant in exosomes of ALCL patients compared to controls. YRNA fragments, accounting for most of exosomal content and being significantly increased in ALCL patients, were prioritized for further investigation by qRT-PCR. Quantification of RNY4 fragments and full-length sequences disclosed that the latter are massively loaded into exosomes of ALCL patients with more advanced and aggressive disease. These results are discussed in light of recent findings on the role of RNY4 in the modulation of tumor microenvironment. [ABSTRACT FROM AUTHOR]

Simple Summary: Despite success of targeted therapy, cancer cells very often find a way to survive treatment; this eventually causes a tumor to relapse. In a particular type of lymphoma carrying a specific chromosomal rearrangement named anaplastic large-cell lymphoma (ALCL), selective drugs targeting the cause of the disease can induce spectacular remission of chemotherapy-resistant cancer. However, the lymphoma relapses again in about half of the cases, leaving no therapeutic options. We studied the possibility to combine two simultaneous treatments in order to prevent the relapse, starting from the hypothesis that acquiring resistance to two drugs at the same time is statistically very unlikely. We demonstrate that treating lymphoma cells with drug combinations has superior efficacy in comparison with single drug treatments, both in cell cultures and in mice. Anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL) is a subtype of non-Hodgkin lymphoma characterized by expression of the oncogenic NPM/ALK fusion protein. When resistant or relapsed to front-line chemotherapy, ALK+ ALCL prognosis is very poor. In these patients, the ALK inhibitor crizotinib achieves high response rates, however 30–40% of them develop further resistance to crizotinib monotherapy, indicating that new therapeutic approaches are needed in this population. We here investigated the efficacy of upfront rational drug combinations to prevent the rise of resistant ALCL, in vitro and in vivo. Different combinations of crizotinib with CHOP chemotherapy, decitabine and trametinib, or with second-generation ALK inhibitors, were investigated. We found that in most cases combined treatments completely suppressed the emergence of resistant cells and were more effective than single drugs in the long-term control of lymphoma cells expansion, by inducing deeper inhibition of oncogenic signaling and higher rates of apoptosis. Combinations showed strong synergism in different ALK-dependent cell lines and better tumor growth inhibition in mice. We propose that drug combinations that include an ALK inhibitor should be considered for first-line treatments in ALK+ ALCL. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

[Display omitted] Anaplastic large cell lymphoma (ALCL) is a CD30-positive lymphoma accounting for 20% of all pediatric T-cell lymphomas. Current first line treatment can cure most of ALCL patients but 10–30% of them are resistant or relapse. In this context, liquid biopsy has the potential to help clinicians in disease screening and treatment response monitoring. Small-RNA-sequencing analysis performed on plasma small-extracellular vesicles (s-EVs) from 20 pediatric anaplastic lymphoma kinase positive (ALK +) ALCL patients at diagnosis revealed a specific miRNAs cargo in relapsed patients compared to non-relapsed, with seven miRNAs enriched in s-EVs of relapsed patients. MiR-146a-5p and miR-378a-3p showed a negative prognostic impact both in univariate and multivariate analysis, possibly representing, together with let-7 g-5p, a miRNA panel for the early identification of high-risk patients. Among them, miR-146a-5p is known to modulate tumor supporting-M2 macrophages differentiation, but the role of these cells in pediatric ALK + ALCL is still unknown. To elucidate the role of miR-146a-5p and M2 macrophages in pediatric ALCL disease, THP-1-derived macrophages were treated with s-EVs from ALK + ALCL cell lines, showing increased miR-146a-5p intracellular expression, migrating capability and M2-markers CD163 and Arginase-1 upregulation. In turn, conditioned media from M2 macrophages or miR-146a-5p-transfected THP-1 increased ALCL cells' aggressive features and were enriched in interleukin-8. Overall, these data suggest a role of miR-146a-5p in promoting macrophage infiltration and M2-like polarization in ALCL. Our findings incite further investigation on the role of M2 macrophages in ALCL aggressiveness and dissemination, also considering the novel treatment options targeting tumor associated macrophages. [ABSTRACT FROM AUTHOR]