Venetoclax-based therapies in pediatric advanced MDS and relapsed/refractory AML: a multicenter retrospective analysis

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Selezione del donatore alternativo nel trapianto allogenico di cellule staminali ematopoietiche nel paziente pediatrico: donatore non correlato, unità di sangue cordonale o familiare aploidentico? Esperienza del centro trapianti dell'U.O oncoematologia pediatrica di Pisa

La selezione del donatore è un punto cruciale nel trapianto di cellule staminali ematopoietiche (TCSE), essendo uno dei maggiori determinanti degli esiti dello stesso. In assenza di un fratello HLA-identico (MSD), in ambito pediatrico, le linee guida internazionali suggeriscono una ricerca gerarchica partendo da un donatore non familiare da banca (MUD) altamente compatibile per arrivare alle unità di sangue cordonale (UCB) ed infine alla scelta del donatore aploidentico (APLO). Numerosi studi retrospettivi suggeriscono come MUD 10/10 o 12/12 siano simili a MSD in termini di risultati mentre MUD con mismatch, APLO e UCB sono comparabili tra loro. Tali evidenze in ambito pediatrico sono limitate. Il nostro scopo è quello di contribuire allo sviluppo di un nuovo algoritmo di selezione del donatore attraverso la comparazione dei TCSE APLO, MUD e UCB. Sono stati selezionati 85 pazienti sottoposti ad un primo TCSE allogenico con donatore MUD, APLO o UCB tra gennaio 2003 e novembre 2016, affetti da patologie oncologiche e di età inferiore ai 18 anni. Sono stati considerati solo MUD “well-matched” ovvero ≥9/10 o ≥10/12. Non si è discriminato in base al grado di compatibilità MUD e UCB. La tecnica di Kaplan-Meier è stata utilizzata per stimare sopravvivenza globale (OS), sopravvivenza libera da malattia (DFS) e graft-versus-host disease free, relapse free survival (GRFS). Il metodo delle incidenze cumulative è stato utilizzato per valutare recidiva, graft-versus-host disease acuta (aGVHD) 2-4, aGVHD3-4, GVHD cronica (cGVHD), mortalità non dovuta a recidiva (NRM), attecchimento di polimorfonucleati (PMN) e piastrine (PLT). I tassi di primary graft failure (PGF) sono stati confrontati col metodo del chi-quadro. È stata poi eseguita un’analisi multivariata col modello di regressione di Cox considerando come variabile dipendente GRFS e come variabili indipendenti cellularità del graft, patologia e stato della patologia al TCSE, tempo diagnosi-TCSE, presenza di infezione CMV dopo il TCSE, fonte di cellule staminali ematopoietiche utilizzata, il tipo di regime di condizionamento e il tipo di profilassi farmacologica della GVHD. I risultati principali sono stati, rispettivamente per APLO, MUD e UCB: OS (62.2%, 59.2%, 50.0% p>0.05), DFS (55.6%, 56.0%, 37.5% p>0.05), aGVHD 2-4 (22.2%, 15.8%, 0% p>0.05), aGVHD 3-4 (11.1%, 3.3%, 0% p>0.05), cGVHD 2-4 (50.0%, 29.7%, 31.8% p>0.05), recidiva (33.3%, 22.6%, 31.3% p>0.05), NRM (0%, 9.1%, 18.8% p>0.05), GRFS (44.4%, 50.7%, 37.5% p>0.05), PGF (22.2%, 1.7%, 6.7% p0.05), giorno medio di attecchimento PLT (16.3, 26.8, 61.4 p<0.05). L’analisi multivariata evidenzia non evidenza associazioni statisticamente significative. Al di là della significatività statistica, APLO appare non inferiore rispetto a MUD e UCB se utilizzato come primo TCSE in termini di OS, DFS, aGVHD2-4, aGVHD3-4, cGVHD, recidiva, GRFS, PGF, tempo di attecchimento di PMN e PLT. Per DFS, GRFS e ricostituzione immunologica APLO risulta paragonabile a MUD mentre UCB è associato ad una performance peggiore. I tassi di aGVHD2-4, aGVHD3-4 e cGVHD sono simili tra APLO e MUD e maggiori rispetto al gruppo UCB, in accordo con la letteratura internazionale. La scelta di un donatore aploidentico risulta una valida opzione in assenza di un MSD o di un MUD 10/10 o 12/12 nell’ambito del TCSE pediatrico eseguito per patologie oncologiche, tanto da poter ipotizzare di essere equiparato a MUD con mismatch. Si noti che la pronta disponibilità di APLO rappresenta un elemento molto importante da considerare. Ulteriori indagini sono necessarie al fine di avvalorare la proposta di un cambiamento nell’algoritmo di selezione del donatore

SINGLE CELL BIOLOGY AS A NOVEL TOOL IN OSTEOSARCOMA RESEARCH: APPLICATION TO CHILDREN, ADOLESCENTS AND YOUNG ADULTS

Osteosarcoma is a rare and malignant bone tumor, primarily affecting adolescents during their pubertal growth phase. It is characterized by a grim prognosis when accompanied by metastatic disease. Circulating tumor cells (CTCs) may play a significant role in the pathogenesis of osteosarcoma, but their specific function remains poorly understood. Objectives: our primary objective was to implement a method for isolating CTCs in osteosarcoma patients using the DEPArray™ technology. We have then conducted downstream molecular analysis to elucidate their relationship with the primary tumor, metastasis, and clinical outcomes. Our secondary goal was to assess the efficacy of a droplet sequencing technique for single-cell RNA sequencing (scRNAseq) in identifying CTCs. Methods: We enrolled pediatric patients from the prospective TOSCANO cohort, obtaining peripheral blood samples at the time of diagnosis (PRE) and prior to surgery (POST). Clinical data were collected, and patients were categorized as either good responders (GR) or poor responders (PR) based on histological necrosis (≥90% and < 90%, respectively). We isolated peripheral blood mononuclear cells (PBMC) and performed physical-based enrichment using the microfluidic system Parsortix™. Subsequently, the cells were fixed and stained with DAPI (as a positive control) and antibodies against CD45 (as a negative control), cytokeratins 8/18/19, and EPCAM (indicative of an epithelial phenotype), as well as TWIST and vimentin (indicative of a mesenchymal phenotype). Single cells were then sorted using DEPAarray™. Whole genome amplification was performed on the individually sorted cells, followed by analysis of copy number alterations (CNAs). In a landmark experiment, we gather data from a previous next-generation sequencing (NGS) custom panel applied on the diagnostic tissue specimen of OS4. We compared the revealed FLT4 point mutation among primary tumor, metastatic tissue and representative mesenchymal and epithelial CTCs with Sanger sequencing. Furthermore, in a pilot experiment, we attempted to investigate CTCs within the PBMC collection using single-cell RNA sequencing (scRNAseq). We counted PBMCs (PRE) from OS4, obtained a small aliquot of 3000 cells, and enriched the remaining cells with Parsortix™. These two groups of cells were pooled and analyzed. Primary patient-derived osteosarcoma cells were analyzed in a separate run. Results: enrollment is still open, preliminary data from 6 pediatric patients were considered. CTCs were efficiently isolated and both mesenchymal (M-CTCs) and epithelial circulating cells (E-CTCs) were found. A significant negative correlation was observed between E-CTCs(PRE) and maximum standard uptake value (SUVmax) at the diagnosis (r= -0.84; p=0.02) in PR patients. A border-line significant positive correlation was observed for POST-PRE variations (∆) in M-CTCs and progression free survival (PFS) duration (r= 0.80; p= 0.08). CNAs were only partially shared among primary tumors/metastasis and CTCs. In the landmark experiment with OS4 samples, we have confirmed that CTCs shared the known FLT4 point mutation (c.3971G>C) with the metastasis and the primary tumor. Heterozygous mutations was identified in all the sample with the exception of the representative M-CTC in which also a wild type karyotype was noted in contrast to OS4 primary tumor, metastasis and representative E-CTC. In relation to the secondary endpoint, scRNAseq revealed a possible cluster of tumor-educated platelets. Conclusion: we successfully isolated and characterized CTCs in young osteosarcoma patients. The enumeration of CTCs proved valuable for clinical correlations, and the analysis of CNAs and point mutations indicated the shared characteristics of CTCs with the corresponding primary tumor or metastasis, although some differences of unknown significance were observed. In-droplet scRNAseq did not identify CTCs but provided additional information. Collectively, these observations underscore the efficiency of single-cell platforms in studying mesenchymal/epithelial plasticity in osteosarcoma, offering precious insights in clinical settings. While further studies are necessary to validate these preliminary findings, they hold the potential to shed new light on osteosarcoma pathogenesis and may have implications for therapeutic interventions

Effective treatment of late-onset noninfectious pulmonary complication with ruxolitinib in an 8-year-old boy

Ruxolitinib could be considered as an option in the treatment of LONIPCs in children when other treatments are ineffective. Spirometry is a valuable tool for both diagnosis and follow-up of LONIPCs in children

The role of gut and lung microbiota in susceptibility to tuberculosis

Tuberculosis is one of the most common infectious diseases and infectious causes of death worldwide. Over the last decades, significant research effort has been directed towards defining the understanding of the pathogenesis of tuberculosis to improve diagnosis and therapeutic options. Emerging scientific evidence indicates a possible role of the human microbiota in the pathophysiology of tuberculosis, response to therapy, clinical outcomes, and post-treatment outcomes. Although human studies on the role of the microbiota in tuberculosis are limited, published data in recent years, both from experimental and clinical studies, suggest that a better understanding of the gut–lung microbiome axis and microbiome–immune crosstalk could shed light on the specific pathogenetic mechanisms of Mycobacterium tuberculosis infection and identify new therapeutic targets. In this review, we address the current knowledge of the host immune responses against Mycobacterium tuberculosis infection, the emerging evidence on how gut and lung microbiota can modulate susceptibility to tuberculosis, the available studies on the possible use of probiotic–antibiotic combination therapy for the treatment of tuberculosis, and the knowledge gaps and future research priorities in this field

Chloroquine treatment enhances regulatory T cells and reduces the severity of experimental autoimmune encephalomyelitis.

BACKGROUND: The modulation of inflammatory processes is a necessary step, mostly orchestrated by regulatory T (Treg) cells and suppressive Dendritic Cells (DCs), to prevent the development of deleterious responses and autoimmune diseases. Therapies that focused on adoptive transfer of Treg cells or their expansion in vivo achieved great success in controlling inflammation in several experimental models. Chloroquine (CQ), an anti-malarial drug, was shown to reduce inflammation, although the mechanisms are still obscure. In this context, we aimed to access whether chloroquine treatment alters the frequency of Treg cells and DCs in normal mice. In addition, the effects of the prophylactic and therapeutic treatment with CQ on Experimental Autoimmune Encephalomyelitis (EAE), an experimental model for human Multiple Sclerosis, was investigated as well. METHODOLOGY/PRINCIPAL FINDINGS: EAE was induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein (MOG35-55) peptide. C57BL/6 mice were intraperitoneally treated with chloroquine. Results show that the CQ treatment provoked an increase in Treg cells frequency as well as a decrease in DCs. We next evaluated whether prophylactic CQ administration is capable of reducing the clinical and histopathological signs of EAE. Our results demonstrated that CQ-treated mice developed mild EAE compared to controls that was associated with lower infiltration of inflammatory cells in the central nervous system CNS) and increased frequency of Treg cells. Also, proliferation of MOG35-55-reactive T cells was significantly inhibited by chloroquine treatment. Similar results were observed when chloroquine was administrated after disease onset. CONCLUSION: We show for the first time that CQ treatment promotes the expansion of Treg cells, corroborating previous reports indicating that chloroquine has immunomodulatory properties. Our results also show that CQ treatment suppress the inflammation in the CNS of EAE-inflicted mice, both in prophylactic and therapeutic approaches. We hypothesized that the increased number of regulatory T cells induced by the CQ treatment is involved in the reduction of the clinical signs of EAE

Analysis of the cellular infiltration of the CNS show reduced IFN-γ and IL-17 producing cells in CQ treated EAE mice.

<p>(A) CQ treated-mice presented reduced infiltration of inflammatory cells. (B) The percentage of IFN-γ- and IL-17-producing cells infiltrating the brain was reduced while the frequency of IL-10- producing cells was found augmented in brain of mice treated with CQ. (C, D and E) Gene expression of IFN-γ, IL-17 and IL-10 in the CNS followed the same pattern, respectively. (F) The expression of FOXP3 was evaluated in the CNS by RT-PCR. (G) The frequency of CD25⁺Foxp3⁺ cells was evaluated in spleens of mice. Results are representative of two independent experiments and are expressed as mean ± SEM for at least five animals. p<0,05 (*) and p<0.01 (**).</p

Transfer of CQ-elicited Treg cells reduces the severity of ongoing EAE.

<p>(A) Naïve C57BL/6 mice were treated with chloroquine (5 mg/kg/day) for five consecutive days. Three days after the last dose of the treatment, splenic CD4⁺CD25⁺ cells were isolated using magnetic beads and cells (5×10⁵ cells per mouse) were transferred into mice with ongoing EAE (10 days after immunization). As controls, mice received the same number of CD4⁺CD25⁻ cells. (B) The clinical course of the disease was evaluated routinely. (C) The brains and spinal cords were collected and the enriched infiltrating cells were counted. The frequency of IL-17-, IL-10- and IFN-γ-producing cells was analyzed by flow cytometry as well. (D) The spleens were collected and CFSE-stained cells were cultivated in the presence of MOG_35–55 peptide for 96 h. Dye decay and cytokine production were analyzed by flow cytometry. Results are expressed as mean ± SEM for at least five animals. p<0,05 (*), p<0,01 (**) and p<001 (***).</p

Chloroquine administration alters the frequency of regulatory T (Treg) cells and dendritic cells (DCs), but not the proliferative capability of T cells.

<p>Briefly, mice were treated with chloroquine via i.p. for five consecutive days. Three days after the last dose mice were killed and splenic cells were analyzed by flow cytometry. Increased numbers of Treg cells (A) and reduced frequency of DCs (B) was found in mice treated with chloroquine when compared to the control group. In addition, splenic T cells proliferative response was not altered in the presence of concanavalin-A (C). Subpopulations of leukocytes showed slight changes when compared to control subjects (D). Results are representative of three independent experiments.</p