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In spite of leukemia therapy improvements obtained over the last decades, therapy is not yet effective in all cases. Current approaches in Acute Lymphoblastic Leukemia (ALL) research focus on identifying new molecular targets to improve outcome for patients with a dismal prognosis. In this light phosphoproteomics seems to hold great promise for the identification of proteins suitable for targeted therapy. We employed Reverse Phase Protein Microarrays to identify aberrantly activated proteins in 118 pediatric B-cell precursor (BCP)-ALL patients. Signal transduction pathways were assayed for activation/expression status of 92 key signalling proteins. We observed an increased activation/expression of several pathways involved in cell proliferation in poor clinical prognosis patients. MLL-rearranged tumours revealed BCL-2 hyperphosphorylation through AMPK activation, which indicates that AMPK could provide a functional role in inhibiting apoptosis in MLL-rearranged patients, and could be considered as a new potential therapeutic target. Second, in patients with poor clinical response to prednisone we observed the up-modulation of LCK activity with respect to patients with good response. This tyrosine-kinase can be down-modulated with clinically used inhibitors, thus modulating LCK activity could be considered for further studies as a new additional therapy for prednisone-resistant patients. Further we also found an association between high levels of CYCLIN E and relapse incidence. Moreover, CYCLIN E is more expressed in early relapsed patients, who usually show an unfavourable prognosis. We conclude that functional protein pathway activation mapping revealed specific deranged signalling networks in BCP-ALL that could be potentially modulated to produce a better clinical outcome for patients resistant to standard-of-care therapies.

Circular RNAs (circRNAs) are abundantly expressed in the haematopoietic compartment, but knowledge on their diversity among blood cell types is still limited. Nevertheless, emerging data indicate an array of circRNA functions exerted through interactions with other RNAs and proteins, by translation into peptides, and circRNA involvement as regulatory molecules in many biological processes and cancer mechanisms. Interestingly, the role of specific circRNAs in leukemogenesis has been disclosed by a few studies, mostly in acute myeloid leukemia. In this study, circRNA expression in B-cells, T-cells and monocytes of healthy subjects is described, including putative new circRNA genes. Expression comparison considered 6,228 circRNAs and highlighted cell population-specific expression and exon usage patterns. Differential expression has been confirmed by qRT-PCR for circRNAs specific of B-cells (circPAX5, circAFF3, circIL4R, and circSETBP1) or T-cells (circIKZF1, circTNIK, circTXK, and circFBXW7), and for circRNAs from intronic (circBCL2) and intergenic regions that were overexpressed in lymphocytes. Starting from this resource of circRNA expression in mature blood cell populations, targeted examination identified striking and generalized upregulated expression of circPAX5, circPVT1 and circHIPK3 in pediatric B-precursor acute lymphoblastic leukemia, and disclosed circRNAs with variable expression across cytogenetic subtypes.

Direct antiviral agents (DAAs) have excellent efficacy against chronic hepatitis C virus (HCV) infection. Despite this strength, recent studies raised concerns about an unexpected hepatocellular carcinoma (HCC) occurrence rate after DAA therapy. In this exploratory case-control study, we evaluated the potential use of miRNAs as serum biomarkers for the detection of early HCC in DAA-treated patients. In the discovery phase, the circulating miRNome was assessed in 10 matched patients with (HCC+) or without HCC (HCC−) occurrence. Microarray analysis was performed before (T0) and after one month of the DAA therapy (T1). MiRNAs discriminating HCC+ and HCC− patients were validated in 60 samples by means of RT-qPCR. We estimated the time-averaged difference of a given miRNA between HCC+ and HCC− patients using a bootstrapped random-effect generalized least square regression model (RE-GLS). At T0, miR-1207-5p, miR-1275, miR-3197, miR-4443, miR-3178, miR-483-5p, miR-4706, miR-4793-3p and miR-1246 discriminated HCC+ from HCC− patients (p < 0.05). At T1, only miR-1180-3p, miR-1228-3p, miR-4329 and miR-4484 (p < 0.05) discriminated HCC+ from HCC− patients. The subsequent validation phase identified miR-3197 as changing with both disease and time. Our results suggest that patients might be already committed to HCC occurrence before DAA therapy. MiR-3197 shows some potential for the identification of patients at risk of HCC during DAA treatments.

HIF-1α has been suggested to interplay with Wnt signaling components in order to activate a neuronal differentiation process in both normal brain and glioblastoma (GBM). Based on these data, we explored the molecular mechanisms underlying the observed capability of GBM cells to acquire a neuronal phenotype upon Wnt signaling stimulation and how the microenvironment, particularly hypoxia, modulates this process. : here, the employment of ChIP-seq techniques together with co-immunoprecipitation approaches allowed to reconstruct the molecular interactions responsible for activating specific pro-differentiating transcriptional programs in GBM cells. Moreover, gene silencing/over-expression approaches coupled with the functional analysis of cell phenotype were applied to confirm ChIP-driven hypotheses. Finally, we combined the use of publicly available gene expression datasets with protein expression data by immunohistochemistry to test the clinical relevance of obtained results. : our data clearly suggest that HIF-1α is recruited by the β-catenin/TCF1 complex to foster neuronal differentiation gene transcription in hypoxic GBM cells. Conversely, at higher oxygen levels, the increased expression of TCF4 exerts a transcriptional inhibitory function on the same genomic regions, thus counteracting differentiation. Moreover, we demonstrate the existence of a positive correlation between the expression levels of HIF-1α, TCF1 and neuronal phenotype in GBM tumors, accompanied by the over-expression of several Wnt signaling components, finally affecting patient prognosis. : we unveiled a peculiar mechanism by which TCF1 and HIF-1α can induce a reminiscent neuronal differentiation of hypoxic GBM cells, which is hampered, in normoxia, by high levels of TCF4, thus not only controlling the balance between differentiation and stemness, but also impacting on intra-tumoral heterogeneity and eventually patient outcome.

The KMT2A/AFF1 rearrangement is associated with an unfavorable prognosis in infant acute lymphocytic leukemia (ALL). Discordant ALL in monozygotic twins is uncommon and represents an attractive resource to evaluate intrauterine environment–genetic interplay in ALL. Mutational and epigenetic profiles were characterized for a discordant KMT2A/AFF1-rearranged infant monozygotic twin pair and their parents, and they were compared to three independent KMT2A/AFF1-positive ALL infants, in which the DNA methylation and gene expression profiles were investigated. A de novo Q61H NRAS mutation was detected in the affected twin at diagnosis and backtracked in both twins at birth. The KMT2A/AFF1 rearrangement was absent at birth in both twins. Genetic analyses conducted at birth gave more insights into the timing of the mutation hit. We identified correlations between DNA methylation and gene expression changes for 32 genes in the three independent affected versus remitted patients. The strongest correlations were observed for the RAB32, PDK4, CXCL3, RANBP17, and MACROD2 genes. This epigenetic signature could be a putative target for the development of novel epigenetic-based therapies and could help in explaining the molecular mechanisms characterizing ALL infants with KMT2A/AFF1 fusions.

To induce and sustain the leukaemogenic process, MLL-AF4+ leukaemia seems to require very few genetic alterations in addition to the fusion gene itself. Studies of infant and paediatric patients with MLL-AF4+ B cell precursor acute lymphoblastic leukaemia (BCP-ALL) have reported mutations in KRAS and NRAS with incidences ranging from 25 to 50%. Whereas previous studies employed Sanger sequencing, here we used next generation amplicon deep sequencing for in depth evaluation of RAS mutations in 36 paediatric patients at diagnosis of MLL-AF4+ leukaemia. RAS mutations including those in small sub-clones were detected in 63.9% of patients. Furthermore, the mutational analysis of 17 paired samples at diagnosis and relapse revealed complex RAS clone dynamics and showed that the mutated clones present at relapse were almost all originated from clones that were already detectable at diagnosis and survived to the initial therapy. Finally, we showed that mutated patients were indeed characterized by a RAS related signature at both transcriptional and protein levels and that the targeting of the RAS pathway could be of beneficial for treatment of MLL-AF4+ BCP-ALL clones carrying somatic RAS mutations.

Therapeutic plans (TPs) were introduced in Italy in 2004 in order to ensure the continuity in the prescription of new drugs between specialist physicians and general practitioners (GPs). Over the years this prescription tool was updated several times: starting from a paper form without any template (\"paper TP\") to a template defined by AIFA to collect specific clinical information, up to a web-based form to collect all information into a database. Over time-critical issues concerning its usefulness have been raised, especially when AIFA established several extensions for TP validity to ensure the social distancing required by the covid-19 pandemic. Therefore, after several years from their establishment, pending adoption of necessary implementing of Ministerial Decree of 25th March 2020, a check of the actual impact of TPs is required, in order to plan their review. This study provide a detailed overview of all TPs active in Italy at the 11th May 2020. From Farmadati database, all drugs reimbursed by the National Health Service (class A drugs) and requiring TP were selected. The consumption of these drugs has been derived from OsMed Reports that make available data of medicines consumption and expenditure in the general population in Italy. The analysis showed that TP is required for the prescription of 935 medicinal products (9.6% of class-A drugs) and 147 different active substances (belonging to 34 different Therapeutic Groups and 66 subgroups). Out of these, 67 (46%) required a paper TP without any template, 72 (49%) a paper TP on AIFA template, and 8 (5%) a web-based TP. The Therapeutic Group with the largest number of active ingredients with TP were antidiabetics (19.7%), followed by immunomodulating and immunosuppressants (9.5%) and medications for asthma and COPD (6.8%). Consumption analysis of drugs with TP showed that this prescription tool covers 943,899,598 DDD per year, equal to 2,586,026 DDD/day. This means that TPs have a very high impact in terms of the prevalence of patients treated on the entire care process. Of all annual DDDs prescribed on TP, 46.8% concerned drugs with TP on template AIFA, 34.5% drugs with web-based TP, while the remaining 18.7% drugs with paper TP without a template. This analysis may provide the basis for an analytical case-by-case review of TP maintenance needs, trying to maximize the benefits of this tool and to reduce its possible adverse effects. This review could be helpful to ensure the appropriateness in the drug uses, to enhance the role of general medicine, and to simplify the pathways of millions of patients ensuring the continuity of their care.

I Piani Terapeutici (PT) sono stati introdotti nel 2004 con l’obiettivo di assicurare, per i nuovi farmaci, una continuità prescrittiva e assistenziale tra specialista e medico di medicina generale (MMG). Nel corso degli anni questo strumento ha subìto diversi aggiornamenti, passando da un modulo senza alcun template “PT cartaceo”, a un PT su template AIFA con raccolta di specifiche informazioni cliniche, fino a giungere al PT web-based con la raccolta di queste informazioni in appositi database. Non sono mancate nel tempo le critiche circa la sua utilità, ancora più quando, a seguito delle necessità di distanziamento imposto dalla pandemia da covid-19, l’ente regolatorio ha disposto diverse proroghe per la sua validità. Pertanto, dopo diversi anni dalla loro istituzione, nelle more dell’attuazione del DM 25 marzo 2020, è opportuna una verifica degli impatti e degli esiti di questi strumenti per programmare una loro eventuale revisione. Si fornisce qui una fotografia dettagliata e analitica di tutti i PT attivi in Italia all’11 maggio 2020. Lo studio ha previsto la ricerca in Farmadati di tutti i farmaci in fascia A con prescrizione su PT AIFA, i cui consumi sono stati ricavati dal flusso OsMed. L’analisi ha evidenziato che il PT riguarda 935 specialità medicinali, corrispondenti al 9,6% dei farmaci in fascia A, 147 differenti principi attivi, rientranti in 34 Gruppi Terapeutici e 66 sottogruppi. Dei 147 principi attivi, 67 (46%) prevedevano un PT cartaceo (senza alcun template), 72 (49%) un PT cartaceo su template AIFA e 8 (5%) un PT web-based. I Gruppi Terapeutici con il maggior numero di principi attivi con PT sono risultati gli antidiabetici (19,7%), seguiti da immunomodulanti e immunosoppressori (9,5%) e dai farmaci per asma e BPCO (6,8%). Dall’analisi del consumo dei farmaci sottoposti a PT risulta che questa modalità prescrittiva assorbe circa 943.899.598 DDD annue, pari a 2.586.026 DDD/die. Ciò significa che i PT hanno anche un impatto molto rilevante in termini di prevalenza di pazienti trattati sull’intero processo assistenziale. Di tutte le DDD annue prescritte su PT, il 46,8% riguardava farmaci con PT template AIFA, il 34,5% farmaci con PT web-based, mentre il restante 18,7% farmaci con PT cartacei senza template. Questa analisi può costituire la base da cui intraprendere una revisione caso per caso delle necessità dello strumento PT, cercando di massimizzare il più possibile i vantaggi e le potenzialità di questo strumento e ridurre i suoi possibili effetti sfavorevoli. Tutto ciò al fine di garantire l’appropriatezza di impiego dei farmaci, potenziare la medicina generale e semplificare i percorsi di milioni di assistiti, assicurando loro la continuità assistenziale.