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Background: Reconstruction of mandibular bone defects is a surgical challenge, and microvascular reconstruction is the current gold standard. The field of tissue bioengineering has been providing an increasing number of alternative strategies for bone reconstruction.Methods: In this preclinical study, the performance of two bioengineered scaffolds, a hydrogel made of polyethylene glycol-chitosan (HyCh) and a hybrid core-shell combination of poly (L-lactic acid)/poly (ε-caprolactone) and HyCh (PLA-PCL-HyCh), seeded with different concentrations of human mesenchymal stromal cells (hMSCs), has been explored in non-critical size mandibular defects in a rabbit model. The bone regenerative properties of the bioengineered scaffolds were analyzed by in vivo radiological examinations and ex vivo radiological, histomorphological, and immunohistochemical analyses.Results: The relative density increase (RDI) was significantly more pronounced in defects where a scaffold was placed, particularly if seeded with hMSCs. The immunohistochemical profile showed significantly higher expression of both VEGF-A and osteopontin in defects reconstructed with scaffolds. Native microarchitectural characteristics were not demonstrated in any experimental group.Conclusion: Herein, we demonstrate that bone regeneration can be boosted by scaffold- and seeded scaffold-reconstruction, achieving, respectively, 50% and 70% restoration of presurgical bone density in 120 days, compared to 40% restoration seen in spontaneous regeneration. Although optimization of the regenerative performance is needed, these results will help to establish a baseline reference for future experiments.

Human mesenchymal stromal cells (hMSCs), whether used alone or together with three-dimensional scaffolds, are the best-studied postnatal stem cells in regenerative medicine. In this study, innovative composite scaffolds consisting of a core–shell architecture were seeded with bone-marrow-derived hMSCs (BM-hMSCs) and tested for their biocompatibility and remarkable capacity to promote and support bone regeneration and mineralization. The scaffolds were prepared by grafting three different amounts of gelatin–chitosan (CH) hydrogel into a 3D-printed polylactic acid (PLA) core (PLA-CH), and the mechanical and degradation properties were analyzed. The BM-hMSCs were cultured in the scaffolds with the presence of growth medium (GM) or osteogenic medium (OM) with differentiation stimuli in combination with fetal bovine serum (FBS) or human platelet lysate (hPL). The primary objective was to determine the viability, proliferation, morphology, and spreading capacity of BM-hMSCs within the scaffolds, thereby confirming their biocompatibility. Secondly, the BM-hMSCs were shown to differentiate into osteoblasts and to facilitate scaffold mineralization. This was evinced by a positive Von Kossa result, the modulation of differentiation markers (osteocalcin and osteopontin), an expression of a marker of extracellular matrix remodeling (bone morphogenetic protein-2), and collagen I. The results of the energy-dispersive X-ray analysis (EDS) clearly demonstrate the presence of calcium and phosphorus in the samples that were incubated in OM, in the presence of FBS and hPL, but not in GM. The chemical distribution maps of calcium and phosphorus indicate that these elements are co-localized in the same areas of the sections, demonstrating the formation of hydroxyapatite. In conclusion, our findings show that the combination of BM-hMSCs and PLA-CH, regardless of the amount of hydrogel content, in the presence of differentiation stimuli, can provide a construct with enhanced osteogenicity for clinically relevant bone regeneration.

Extracellular vesicles (EVs) are various sets of cell-derived membranous structures containing lipids, nucleic acids, and proteins secreted by both eukaryotic and prokaryotic cells. It is now well recognized that EVs are key intercellular communication mediators, allowing the functional transfer of bioactive chemicals from one cell to another in both healthy and pathological pathways. It is evident that the condition of the producer cells heavily influences the composition of EVs. Hence, phenotypic changes in the parent cells are mirrored in the design of the secreted EVs. As a result, EVs have been investigated for a wide range of medicinal and diagnostic uses in different hematological diseases. EVs have only recently been studied in the context of Chronic Myeloid Leukemia (CML), a blood malignancy defined by the chromosomal rearrangement t(9;22) and the fusion gene BCR-ABL1. The findings range from the impact on pathogenesis to the possible use of EVs as medicinal chemical carriers. This review aims to provide for the first time an update on our understanding of EVs as carriers of CML biomarkers for minimal residual disease monitoring, therapy response, and its management, as well as the limited reports on the use of EVs as therapeutic shuttles for innovative treatment approaches.

The introduction of imatinib in 2000 opened the era of tyrosine kinase inhibitors (TKIs) for CML therapy and has revolutionized the life expectancy of CML patients, which is now quite like the one of the healthy aged population. Over the last 20 years, both the TKI therapy itself and the objectives have undergone evolutions highlighted and discussed in this review. The main objective of the CML therapy in the first 10 years after TKI introduction was to abolish the disease progression from the chronic to the blastic phase and guarantee the long-term survival of the great majority of patients. In the second 10 years (from 2010 to the present), the main objective of CML therapy moved from survival, considered achieved as a goal, to treatment-free remission (TFR). Two phenomena emerged: no more than 50–60% of CML patients could be candidates for discontinuation and over 50% of them molecularly relapse. The increased cumulative incidence of specific TKI off-target side effects was such relevant to compel to discontinue or reduce the TKI administration in a significant proportion of patients and to avoid a specific TKI in particular settings of patients. Therefore, the treatment strategy must be adapted to each category of patients. What about the patients who do not get or fail the TFR? Should they be compelled to continue the TKIs at the maximum tolerated dose? Alternative strategies based on the principle of minimal effective dose have been tested with success and they are now re-evaluated with more attention, since they guarantee survival and probably a better quality of life, too. Moving from treating the disease to treating the patient is an important change of paradigm. We can say that we are entering a personalized CML therapy, which considers the patients’ age, their comorbidities, tolerability, and specific objectives. In this scenario, the new techniques supporting the monitoring of the patients, such as the digital PCR, must be considered. In the present review, we present in deep this evolution and comment on the future perspectives of CML therapy.

The effective and sensitive monitoring of Minimal Residual Disease or Measurable Residual Disease (MRD) is a very important aspect in the management of patients affected by hematologic malignancies. The recent availability of new technologies has opened to the improvement of MRD monitoring. It is particularly relevant in patients affected by Chronic Myeloid Leukemia (CML). MRD monitoring is key in the management of CML patients thanks to the efficacy of TKIs therapy. Moreover, the policies of TKIs discontinuation aimed at treatment free remission are strongly based on the good selection of patients eligible for stopping TKIs therapy. The recently described application of digital PCR in CML patients monitoring seems to improve the accuracy and precision in the identification of optimal responders. The present review reports an overview on the application of digital PCR in the monitoring of MRD in CML and its impact on TKIs discontinuation trials and, consequently, on TFR success.

BackgroundThe COVID-19 pandemic has had a significant impact on the management and care of onco-hematological patients, particularly those with lymphoproliferative disorders who are at higher risk for COVID-19 associated bacterial and fungal superinfections.Case presentationWe present the successful treatment of a 44-year-old male patient with refractory mantle cell lymphoma treated with chimeric antigen receptor T (CAR-T) cell therapy, despite concurrent COVID-19 infection. The patient developed grade II cytokine release syndrome, requiring admission to the intensive care unit. The CAR-T cells expanded effectively, and the patient achieved complete metabolic remission. During the treatment course, the patient experienced complications including COVID-19-associated pulmonary aspergillosis and a co-infection with Stenotrophomonas maltophilia and the SARS-CoV-2 omicron variant. Prompt antifungal and antibacterial therapy, along with appropriate COVID-19 treatment, led to the resolution of these infections. Dexamethasone was also administered to reduce inflammation and aid hematologic recovery. Despite the presence of multiple infections, the patient achieved complete remission of lymphoma, highlighting the effectiveness of CAR-T cell therapy in this high-risk patient.ConclusionDespite the challenges posed by concurrent infections, the decision to proceed with CAR-T cell therapy in this patient proved to be successful, resulting in complete remission of lymphoma. Early initiation of supportive therapies and the use of dexamethasone contributed to the resolution of complications. This case underscores the importance of individualized decision-making and the potential benefits of CAR-T cell therapy in similar high-risk patients.

This paper presents tegdet, a new Python library for anomaly detection in unsupervised approaches. The input of the library is a univariate time series, representing observations of a given phenomenon. Then, tegdet identifies anomalous epochs, i.e., time intervals where the observations differ in a given percentile of a baseline distribution. Epochs are represented by time evolving graphs and the baseline distribution is given by the dissimilarities between a reference graph and the graphs of the epochs. Currently, the library implements 28 dissimilarity metrics, i.e., 28 different anomaly detection techniques, and its extensible design allows to easily introduce new ones. tegdet exposes a complete functionality to carry out the anomaly detection, through a straightforward designed API. Summarizing, to the best of our knowledge, tegdet is the first publicly available library, based on time evolving graphs, for anomaly detection in time series. Our experimentation shows promising results. For example, Clark and Divergence techniques can achieve an accuracy of 100%, while the time to build the model and predict lasts for few hundreds milliseconds.

BackgroundMinimal residual disease (MRD) monitoring is an important tool to optimally address post-transplant management of acute myeloid leukemia (AML) patients.MethodsWe retrospectively analyzed the impact of bone marrow CD34+ molecular chimerism and WT1 on the outcome of a consecutive series of 168 AML patients submitted to allogeneic stem cell transplantation.ResultsThe cumulative incidence of relapse (CIR) was significantly lower in patients with donor chimerism on CD34+ cells ≥ 97.5% and WT1 < 213 copies/ABL x 10^4 both at 1st month (p=0.008 and p 213 showed intermediate prognosis. 12 of these patients fell in this category because of molecular chimerism < 97.5% at a time-point in which WT1 was < 213.ConclusionsOur results confirm that lineage-specific molecular chimerism and WT1 after allo-SCT (1st and 3rd month) are useful MRD markers. When considered together at 3rd month, CD34+ molecular chimerism could represent an earlier predictor of relapse compared to WT1. Further studies are necessary to confirm this preliminary observation.

Background. The conditioning regimens with different alkylators at different doses can influence the outcome of allogeneic stem cell transplantation (SCT), but conclusive data are missing. Methods. With the aim to analyze real-life allogeneic SCTs performed in Italy between 2006 and 2017 in elderly patients (aged >60 y) with acute myeloid leukemia or myelodysplastic syndrome, we collected 780 first transplants data. For analysis purposes, patients were grouped according to the type of alkylator included in the conditioning (busulfan [BU]-based; n = 618; 79%; treosulfan [TREO]-based; n=162; 21%). Results. No significant differences were observed in nonrelapse mortality, cumulative incidence of relapse, and overall survival, although in the TREO-based group, we observed a greater proportion of elderly patients (P

BackgroundChimeric antigen receptor (CAR) T-cell therapy represents the most advanced immunotherapy against relapsed/refractory B cell malignancies. While cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome are distinctive, known CAR T-cell acute adverse events, hematological toxicity has been increasingly reported. Cytopenia following CAR T-cell treatment is attributed in most cases to lymphodepletion regimens, bridging chemotherapy, or radiotherapy. However, when cytopenia becomes prolonged, the development of myelodysplastic syndrome (MDS) should be considered.Case presentationWe report a case of high risk (HR)-MDS following CAR T-cell therapy in a patient with relapsed diffuse large B cell lymphoma. Eight months after CAR T-cell infusion, the blood count showed progressive, worsening cytopenia and the bone marrow biopsy revealed multilineage dysplasia without excess of blasts associated with chromosome 7 deletion and RUNX1 mutation. Next generation sequencing analysis, retrospectively performed on stored samples, showed a germ line CSF3R mutation, CEBPA clonal hematopoiesis, but no RUNX1 lesion.ConclusionWe describe a case of HR-MDS, with deletion of chromosome 7 and acquisition of RUNX1 mutation, developing after CAR T-cell therapy in a patient with clonal hematopoiesis (CH). Previous chemotherapy favored MDS onset; however, we could not exclude the fact that the impairment of immunosurveillance related to either lymphodepletion or CAR T-cell infusion may play a role in MDS development. Thus, we designed a multicenter prospective study (ClonHema-CAR-T-Study) to investigate if cytopenia after CAR T-cell treatment may be due to underling CH as well as the presence of secondary myeloid malignancies.

Abstract Background Intermittent treatment with TKIs is an option for the great majority (70%–80%) of CML patients who do not achieve a stable deep molecular response and are not eligible for treatment discontinuation. For these patients, the only alternative is to assume TKI continuously, lifelong. Methods The Italian phase III multicentric randomized OPTkIMA study started in 2015, with the aim to evaluate if a progressive de‐escalation of TKIs (imatinib, nilotinib, and dasatinib) is able to maintain the molecular response (MR3.0) and to improve Health Related Quality of Life (HRQoL). Results Up to December 2018, 166/185 (90%) elderly CML patients in stable MR3.0/MR4.0 completed the first year of any TKI intermittent schedule 1 month ON and 1 month OFF. The first year probability of maintaining the MR3.0 was 81% and 23.5% of the patients who lost the molecular response regained the MR3.0 after resuming TKI continuously. Patients’ HRQoL at baseline was better than that of matched peers from healthy population. Women was the only factor independently associated with worse baseline HRQoL (p > 0.0001). Overall, global HRQoL worsened at 6 (p

BackgroundInfectious complications are a significant cause of morbidity and mortality in patients undergoing allogeneic haematopoietic stem cell transplantation (Allo-SCT). The BATMO (Best-Antimicrobial-Therapy-TMO) is an innovative program for infection prevention and management and has been used in our centre since 2019. The specific features of the BATMO protocol regard both prophylaxis during neutropenia (abandonment of fluoroquinolone, posaconazole use in high-risk patients, aerosolized liposomal amphotericin B use until engraftment or a need for antifungal treatment, and letermovir use in CMV-positive recipients from day 0 to day +100) and therapy (empirical antibiotics based on patient clinical history and colonization, new antibiotics used in second-line according to antibiogram with the exception of carbapenemase-producing K pneumoniae for which the use in first-line therapy is chosen).MethodsData on the infectious complications of 116 transplant patients before BATMO protocol (Cohort A; 2016 - 2018) were compared to those of 84 transplant patients following the introduction of the BATMO protocol (Cohort B; 2019 - 2021). The clinical and transplant characteristics of the 2 Cohorts were comparable, even though patients in Cohort B were at a higher risk of developing bacterial, fungal, and CMV infections, due to a significantly higher proportion of myeloablative regimens and haploidentical donors.ResultsNo change in the incidence of infections with organ localization was observed between the two Cohorts. A significant reduction in Clostridioides difficile infections by day +100 was observed in Cohort B (47% vs. 15%; p=0.04). At day +30, a higher incidence of Gram-negative bloodstream infections (BSIs) was observed in Cohort B (12% vs. 23%; p=0.05). By day +100 and between days +100 and +180, the incidence of BSIs and of the various etiological agents, the mortality from Gram-negative bacteria, and the incidence of invasive fungal infections were not different in the two Cohorts. The incidence of CMV reactivations by day +100 dropped drastically in patients of Cohort B, following letermovir registration (51% vs. 15%; p=0.00001).DiscussionThe results of this study suggest that the BATMO program is safe. In particular, the choice to avoid prophylaxis with fluoroquinolone was associated with an increase in Gram-negative BSIs by day +30, but this did not translate into higher levels of mortality. Moreover, this strategy was associated with a significant reduction of Clostridiodes difficile infections. The efficacy of anti-CMV prophylaxis with letermovir was confirmed by a significant reduction in CMV reactivations. Even though patients in Cohort B were at higher risk of developing fungal infections (more haploidentical transplants with more myeloablative regimens), the extensive use of posaconazole for prophylaxis balanced this risk, and no increase in the incidence of fungal-associated complications was observed.

Real-time quantitative PCR (RT-qPCR) is the gold standard to quantify the BCR-ABL1 transcript for molecular response monitoring in chronic myeloid leukemia (CML) patients, and it plays a pivotal role in clinical decision-making process, even if it presents technical limits. Increasing data suggest that digital PCR (dPCR) is more accurate and reliable than RT-qPCR in CML minimal residual disease monitoring and in patients’ selection for treatment discontinuation. But what about the identification of treatment discontinuation failures? We present the case of a CML patient enrolled both in a study aiming to comparatively assess molecular response by RT-qPCR and dPCR and in the progressive arm of the OPTkIMA trial. This is a phase III trial including CML patients randomized to receive a fixed versus a progressive intermittent tyrosine kinase inhibitor regimen. At 24 months, because of two consecutive detections of MR2.0 by RT-qPCR, the patient resumed daily treatment. Conversely, dPCR revealed a stability of molecular response and even a slight decreasing of transcript over time. An additional specimen was sampled one month after the first MR2.0 detection because of clinical decision: RT-qPCR resulted MR3.0 and dPCR confirmed the transcript’s stability. Nowadays, the resumption of therapy is RT-qPCR-driven despite its limits in detection and robustness. In this case, according to dPCR, the patient could have continued intermittent treatment and the stability of response was then confirmed by RT-qPCR. So, dPCR could be able to better identify peculiar clinical response to therapy.

Abstract Treatment‐free remission (TFR) by tyrosine kinase inhibitors (TKI) discontinuation in patients with deep molecular response (DMR) is a paramount goal in the current chronic myeloid leukemia (CML) therapeutic strategy. The best DMR level by real‐time quantitative PCR (RT‐qPCR) for TKI discontinuation is still a matter of debate. To compare the accuracy of digital PCR (dPCR) and RT‐qPCR for BCR‐ABL1 transcript levels detection, 142 CML patients were monitored for a median time of 24 months. Digital PCR detected BCR‐ABL1 transcripts in the RT‐qPCR undetectable cases. The dPCR analysis of the samples, grouped by the MR classes, revealed a significant difference between MR4.0 and MR4.5 (P = 0.0104) or MR5.0 (P = 0.0032). The clinical and hematological characteristics of the patients grouped according to DMR classes (MR4.0 vs MR4.5‐5.0) were superimposable. Conversely, patients with dPCR values

In pediatric rhabdomyosarcoma (RMS), elevated Akt signaling is associated with increased malignancy. Here, we report that expression of a constitutively active, myristoylated form of Akt1 (myrAkt1) in human RMS RD cells led to hyperactivation of the mammalian target of rapamycin (mTOR)/70-kDa ribosomal protein S6 kinase (p70S6K) pathway, resulting in the loss of both MyoD and myogenic capacity, and an increase of Ki67 expression due to high cell mitosis. MyrAkt1 signaling increased migratory and invasive cell traits, as detected by wound healing, zymography, and xenograft zebrafish assays, and promoted repair of DNA damage after radiotherapy and doxorubicin treatments, as revealed by nuclear detection of phosphorylated H2A histone family member X (γH2AX) through activation of DNA-dependent protein kinase (DNA-PK). Treatment with synthetic inhibitors of phosphatidylinositol-3-kinase (PI3K) and Akt was sufficient to completely revert the aggressive cell phenotype, while the mTOR inhibitor rapamycin failed to block cell dissemination. Furthermore, we found that pronounced Akt1 signaling increased the susceptibility to cell apoptosis after treatments with 2-deoxy-D-glucose (2-DG) and lovastatin, enzymatic inhibitors of hexokinase, and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), especially in combination with radiotherapy and doxorubicin. In conclusion, these data suggest that restriction of glucose metabolism and the mevalonate pathway, in combination with standard therapy, may increase therapy success in RMS tumors characterized by a dysregulated Akt signaling.

Malnutrition is a common problem after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and could impair immune function. Immune dysfunction after allo-HSCT may be linked with infections, GVHD, and relapse and negatively affect the outcome. Aim of this review was to identify malnutrition biomarkers, potentially useful for immune-system monitoring, in the setting of allo-HSCT. After a systematic search, no satisfying biomarker was found, except for citrulline. Citrulline could be useful in monitoring gastrointestinal function after allo-HSCT and its role in the complex relationship with immune-system function ought to be better explored. A multi-omics approach, including biomarkers and PRO (patient reported outcomes) is, in our opinion, the optimal way to study the relationship between malnutrition and transplant outcomes.

We propose a pilot, prospective, translational study with the aim of identifying possible molecular markers underlying metastatic prostate cancer (PC) evolution with the use of liquid biopsy. Twenty-eight castrate sensitive, oligometastatic PC patients undergoing bone and/or nodal stereotactic body radiotherapy (SBRT) were recruited. Peripheral blood samples were collected before the commencement of SBRT, then they were processed for circulating cell free DNA (cfDNA) extraction. Deep targeted sequencing was performed using a custom gene panel. The primary endpoint was to identify differences in the molecular contribution between the oligometastatic and polymetastatic evolution of PC to same-first oligo-recurrent disease presentation. Seventy-seven mutations were detected in 25/28 cfDNA samples: ATM in 14 (50%) cases, BRCA2 11 (39%), BRCA1 6 (21%), AR 13 (46%), ETV4, and ETV6 2 (7%). SBRT failure was associated with an increased risk of harboring the BRCA1 mutation (OR 10.5) (p = 0.043). The median cfDNA concentration was 24.02 ng/mL for ATM mutation carriers vs. 40.04 ng/mL for non-carriers (p = 0.039). Real-time molecular characterization of oligometastatic PC may allow for the identification of a true oligometastatic phenotype, with a stable disease over a long time being more likely to benefit from local, curative treatments or the achievement of long-term disease control. A prospective validation of our promising findings is desirable for a better understanding of the real impact of liquid biopsy in detecting tumor aggressiveness and clonal evolution.

CMV infection is a major challenge in allogeneic stem cell transplantation (allo-SCT). The changing landscape in CMV management includes the introduction of letermovir in prophylaxis of high-risk patients and the source of CMV DNA monitoring (plasma—PL vs. whole blood—WB), for pre-emptive therapy (PET) initiation. We report here how our real-life experience in CMV management evolved, following letermovir registration. We focus on: (i) the effects of systematic use of letermovir for CMV prophylaxis in high-risk patients, (ii) the results of a longitudinal comparison of CMV DNAemia monitoring in PL and WB. From December 2018 to April 2020, 60 allo-SCTs have been performed in our center (LET ERA), of whom 45 received letermovir in prophylaxis from day 0 to day + 100, because of recipient positivity of anti CMV IgG. These patients were compared with a cohort of 41 allo-SCTs performed between November 2017 and November 2018 (NO LET ERA). Firstly, the incidence of CMV clinically significant infections, CMV disease, bacterial infections, proven/probable fungal infections, hospital re-admissions after allo-SCT by day + 100 in the two ERA were 8 vs. 44% (p = 0.0006), 2 vs. 12% (p = 0.02), 37 vs. 56% (p = 0.05), 8 vs. 19% (p = 0.09), and 23 vs. 39% (p = 0.09), respectively. By day + 180 these differences were 17 vs. 68% (p < 0.00001), 2 vs. 12% (p = 0.02), 45 vs. 78% (p = 0.09), 8 vs. 22% (p = 0.05), and 40 vs. 66% (p = 0.01), respectively. Secondly, from February to May 2019, we comparatively measured CMV DNA from WB and PL and we confirmed that there is a linear correlation between CMV DNA level in WB and PL (Spearman’s test r = 0.86). Moreover, CMV DNAemia at the time of PET in the 12 patients with a clinically significant CMV infection was higher in WB vs. PL (5.202 vs. 4.981 copies/ml, p = 0.1). Our real-life experience confirms that: (i) letermovir is highly effective, leading to a significant drop in CMV clinically significant infections and CMV-related complications by day + 100 and + 180 after allo-SCT; (ii) WB may be an effective alternative to PL as a source for CMV DNA monitoring, as a linear correlation of DNAemia was confirmed between WB and PL, even if the CMV DNAemia at PET initiation was comparable in the two sources.

BackgroundMyelodysplastic syndromes and acute leukemias after allogeneic stem cell transplantation (allo-SCT) are mainly caused by recurrence of the primitive leukemic clones. More rarely, they originate from donor hematopoietic stem cells, developing the so-called donor cell leukemia (DCL) or myelodysplastic syndromes (DC-MDSs). DCL and DC-MDS can be considered as an in vivo model of leukemogenesis, and even if the pathogenetic mechanisms remain speculative, a genetic predisposition of donor progenitor cells, an altered host microenvironment, and the impairment of immune surveillance are considered the main causes.Case PresentationWe report a case of DC-MDS diagnosed 5 years after an allo-SCT from a matched related donor (patient’s sister) in a patient with Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (Ph+ B-ALL). The sex-mismatch allowed us to identify the donor cell origin. At the onset, the DC-MDS was characterized by chromosome seven monosomy and NRAS, RUNX1, and BCOR mutations. Because of a familiar history of colorectal neoplasia and the variant allele frequency (VAF) of NRAS mutation at the onset, this mutation was searched on germline DNA in both the donor and the recipient, but the result was negative. Moreover, after transplant (+4 months), the patient developed severe and long-lasting chronic graft-versus-host disease (cGVHD), requiring multiple lines of treatments. Because of the severe immunosuppression, recurrent infections occurred and, lately, the patient died due to septic shock.ConclusionThis case report highlights the need, whenever possible, to evaluate the donor origin of the posttransplant myelodysplasia and acute leukemias. The potential key role of the impaired immune surveillance and of long-lasting immunosuppression appears to be emerging in the development of this case of DC-MDS. Finally, this case reminds the importance to investigate the familiar genetic predisposition in donors with a familiar history of neoplasia.

Background. The identification of germline mutations in familial leukemia predisposition genes by next generation sequencing is of pivotal importance. Lately, some “blend pedigrees” characterized by both solid and hematologic malignancies have been described. Some genes were recognized as related to this double predisposition, while the involvement of others is still a matter of debate. ETV6 was associated with hematologic malignancies, in particular myeloid malignancies, and recently described as mutated also in oncologic patients. No clear evidences in its involvement in blend pedigrees are known. Case Presentation. We present our recent experience in the identification of an ETV6-mutated “blend pedigree,” suggesting the involvement of ETV6 in the predisposition to both solid and hematologic neoplasia. The pedigree recognition started with a MDS case enrolled in the NEXT-Famly protocol. The patient presented 9 relatives affected by solid tumors and hematological malignancies. Following the clinical trial protocol, the patient underwent NGS analysis, which confirmed the presence of a mutation on the noncoding region of ETV6 both on tumor and on germline DNA. The mutation resulted was shared by the still alive affected relatives. Conclusion. This evidence supports the involvement of ETV6 in the predisposition to both solid and hematologic neoplasia and the importance of the investigation of the noncoding regions of the genes as recently suggested by different expert groups.

Moving from indication to transplantation is a critical process in myelofibrosis. Most of guidelines specifically focus on either myelofibrosis disease or transplant procedure, and, currently, no distinct indication for the management of MF candidates to transplant is available. Nevertheless, this period of time is crucial for the transplant outcome because engraftment, non-relapse mortality, and relapse incidence are greatly dependent upon the pre-transplant management. Based on these premises, in this review, we will go through the path of identification of the MF patients suitable for a transplant, by using disease-specific prognostic scores, and the evaluation of eligibility for a transplant, based on performance, comorbidity, and other combined tools. Then, we will focus on the process of donor and conditioning regimens’ choice. The pre-transplant management of splenomegaly and constitutional symptoms, cytopenias, iron overload and transplant timing will be comprehensively discussed. The principal aim of this review is, therefore, to give a practical guidance for managing MF patients who are potential candidates for allo-HCT.

A role of endothelial cells (ECs) in Primary Myelofibrosis (PMF) was supposed since JAK2 mutation was found in endothelial precursor cells (EPCs) and in ECs captured by laser microdissection. By Cell Search method, the circulating endothelial cells (CECs) from 14 PMF patients and 5 healthy controls have been isolated and compared by NGS with CD34+Hematopoietic stem and progenitors cells (HSPCs) for panel of 54 myeloid-associated mutations. PMF patients had higher levels of CECs. No mutation was found in HSPCs and CECs from controls, while CECs from PMF patients presented several somatic mutations. 72% of evaluable patients shared at least one mutation between HSPCs and CECs. 2 patients shared the JAK2 mutation, together with ABL1, IDH1, TET2 and ASXL1, KMT2A, respectively. 6 out of 8 shared only NON MPN-driver mutations: TET2 and NOTCH1 in one case; individual paired mutations in TP53, KIT, SRSF2, NOTCH1 and WT1, in the other cases. In conclusion, 70% of PMF patients shared at least one mutation between HSPCs and CECs. These latter harbored several myeloid-associated mutations, besides JAK2V617F mutation. Our results support a primary involvement of EC in PMF and provide a new methodological approach for further studies exploring the role of the “neoplastic” vascular niche.

Abstract Background A normal number of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) is considered a biomarker for adequate new T- and B-cell production. In newborns, detection of TRECs and KRECs by real time PCR from dried blood spotted on filter paper is used for the screening of severe immunodeficiency. In adults, elderly and during diseases, where the number of TRECs is lower than in newborns and children, a large amount of DNA and a sensitive method of amplification are necessary to identify newly produced lymphocytes. Methods DNA was prepared from blood of 203 healthy adults (range: 18–91 years old) absorbed for 10 s on flocked swabs and let to dry, or from peripheral blood mononuclear cells. DNA was subjected to digital PCR and to well established conventional real time PCR-based method using TREC- and KREC-specific primers and probes. The number of TRECs and KRECs was expressed per mL of blood. Statistical analysis was performed by nested ANOVA, Pearson coefficient of determination, and by linear regression tests. Results The novel method for the storage of dried blood on nylon flocked swabs and the use of digital PCR allow quantification of TRECs and KRECs with high degree of sensitivity, specificity, accuracy, and precision. TRECs and KRECs were amplified by digital PCR in all tested blood samples, including those obtained from elderly individuals (>70 years old) and that were negative by real time PCR. Furthermore, values of TRECs and KRECs obtained by digital PCR were in the range of those acquired by real time PCR. Conclusions Our findings demonstrate that DNA isolation from dried blood on flocked swabs followed by digital PCR-based analysis represents a useful tool for studying new lymphocyte production in adults and elderly individuals. This suggests the potential use of the methodology when monitoring of clinical variables is limited by the number of molecules that can be amplified and detected, such as in patients with immunodeficiency or under immunosuppressive therapies.

Bone marrow and adipose tissue human mesenchymal stem cells were seeded in highly performing 3D gelatin–chitosan hybrid hydrogels of varying chitosan content in the presence of human platelet lysate and evaluated for their proliferation and osteogenic differentiation. Both bone marrow and adipose tissue human mesenchymal stem cells in gelatin–chitosan hybrid hydrogel 1 (chitosan content 8.1%) or gelatin–chitosan hybrid hydrogel 2 (chitosan 14.9%) showed high levels of viability (80%–90%), and their proliferation and osteogenic differentiation was significantly higher with human platelet lysate compared to fetal bovine serum, particularly in gelatin–chitosan hybrid hydrogel 1. Mineralization was detected early, after 21 days of culture, when human platelet lysate was used in the presence of osteogenic stimuli. Proteomic characterization of human platelet lysate highlighted 59 proteins mainly involved in functions related to cell adhesion, cellular repairing mechanisms, and regulation of cell differentiation. In conclusion, the combination of our gelatin–chitosan hybrid hydrogels with hPL represents a promising strategy for bone regenerative medicine using human mesenchymal stem cells.

In recent years, the digital polymerase chain reaction has received increasing interest as it has emerged as a tool to provide more sensitive and accurate detection of minimal residual disease. In order to start the process of data alignment, we assessed the consistency of the BCR-ABL1 quantification results of the analysis of 16 RNA samples at different levels of disease. The results were obtained by two different laboratories that relied on The Qx100/Qx200 Droplet Digital PCR System (Bio-Rad) and Quant Studio 3D dPCR System (Thermofisher) platforms. We assessed the compatibility between the estimated values by linear regression, Bland–Altman bias-plot, and Mann–Whitney nonparametric test. The results confirmed the compatibility of the measures, allowing us tocompute an ‘alignment factor’ (AF), equal to 1.41, which was further validated by a different series of experiments. We conclude that the performed measurements by the two laboratories are comparable, and also equalized through the introduction of an alignment factor.

Extracellular vesicles (exosomes, in particular) are essential in multicellular organisms because they mediate cell-to-cell communication via the transfer of secreted molecules. They are able to shuttle different cargo, from nucleic acids to proteins. The role of exosomes has been widely investigated in solid tumors, which gave us surprising results about their potential involvement in pathogenesis and created an opening for liquid biopsies. Less is known about exosomes in oncohematology, particularly concerning the malignancies deriving from myeloid lineage. In this review, we aim to present an overview of immunomodulation and the microenvironment alteration mediated by exosomes released by malicious myeloid cells. Afterwards, we review the studies reporting the use of exosomes as disease biomarkers and their influence in response to treatment, together with the recent experiences that have focused on the use of exosomes as therapeutic tools. The further development of new technologies and the increased knowledge of biological (exosomes) and clinical (myeloid neoplasia) aspects are expected to change the future approaches to these malignancies.

Zebrafish has proven to be a versatile and reliable experimental in vivo tool to study human hematopoiesis and model hematological malignancies. Transgenic technologies enable the generation of specific leukemia types by the expression of human oncogenes under specific promoters. Using this technology, a variety of myeloid and lymphoid malignancies zebrafish models have been described. Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasia characterized by the BCR-ABL1 fusion gene, derived from the t (9;22) translocation causing the Philadelphia Chromosome (Ph). The BCR-ABL1 protein is a constitutively activated tyrosine kinas inducing the leukemogenesis and resulting in an accumulation of immature leukemic cells into bone marrow and peripheral blood. To model Ph+ CML, a transgenic zebrafish line expressing the human BCR-ABL1 was generated by the Gal4/UAS system, and then crossed with the hsp70-Gal4 transgenic line. The new line named (BCR-ABL1pUAS:CFP/hsp70-Gal4), presented altered expression of hematopoietic markers during embryonic development compared to controls and transgenic larvae showed proliferating hematopoietic cells in the caudal hematopoietic tissue (CHT). The present transgenic zebrafish would be a robust CML model and a high-throughput drug screening tool.

Intercellular communication is an essential hallmark of multicellular organisms and can be mediated through direct cell–cell contact or transfer of secreted molecules. In the last two decades, a third mechanism for intercellular communication has emerged that involves intercellular transfer of extracellular vesicles (EVs). EVs are membranous vesicles of 30–5000 nm in size. Based on their dimension and biogenesis, EVs can be divided into different categories, such as microvesicles, apoptotic bodies, ectosomes, and exosomes. It has already been demonstrated that protein changes, expressed on the surfaces or in the content of these vesicles, may reflect the status of producing cells. For this reason, EVs, and exosomes in particular, are considered ideal biomarkers in several types of disease—from cancer diagnosis to heart rejection. This aspect opens different opportunities in EVs clinical application, considering the importance given to liquid biopsy in the recent years. Furthermore, extracellular vesicles can be natural or engineered carriers of cytoprotective or cytotoxic factors and applied, as a therapeutic tool, from regenerative medicine to target cancer therapy. This is of pivotal importance in the so called “era of the 4P medicine”. This Editorial focuses on recent findings pertaining to EVs in different medical areas, from biomarkers to therapeutic applications.

We evaluated the advantages and the reliability of novel protocols for the enrichment of tumor extracellular vesicles (EVs), enabling a blood-based test for the noninvasive parallel profiling of multiple androgen receptor (AR) gene alterations. Three clinically relevant AR variants related to response/resistance to standard-of-care treatments (AR-V7 transcript, AR T878A point mutation and AR gene amplification) were evaluated by digital PCR in 15 samples from patients affected by Castration-Resistant Prostate Cancer (CRPC). Plasma was processed to obtain circulating RNA and DNA using protocols based on tumor EVs enrichment through immuno-affinity and peptide-affinity compared to generic extraction kits. Our results showed that immuno-affinity enrichment prior to RNA extraction clearly outperforms the generic isolation method in the detection of AR-V7, also allowing for a distinction between responder (R) and non-responder (NR) patients. The T878A mutation was detected, overall, in nine out of 15 samples and no approach alone was able to reveal mutations in all harboring samples, showing that the employed methods complement each other. AR amplification was detected in the majority of CRPC samples analysed using either cell-free DNA (cfDNA) or exosome isolation kits (80%). We demonstrated that selective isolation of a subset of circulating exosomes enriched for tumor origin, rather than analysis of total plasma exosomes, or total plasma nucleic acids, increases sensitivity and specificity for the detection of specific alterations.

The outcome of patients underwent to allogeneic stem cell transplantation (allo- SCT) is closely related to graft versus host disease (GvHD) and graft versus leukemia (GvL) effects which can be mediated by mHAgs. 23 mHAgs have been identified and reported to be differently correlated with GVHD or GVL and the aim of this work was develop a method to genotype the mHAgs described so far. For this study we used MALDI-TOF iPLEX Gold Mass Array technology. We tested 46 donor/recipient matched pairs that underwent allo-SCT because of Philadelphia positive (Ph+) chronic myeloid leukemia (n=29) or Ph+ acute lymphoblastic leukemia (n=17). Our data show that sibling pairs had a lesser number of mHAgs mismatches compared to MUD pairs. Notably, donor/recipient genomic mismatch on DPH1 was correlated with an increased risk of acute GvHD and LB-ADIR-1R mismatch on graft versus host direction was correlated with a better RFS with no increase of GvHD risk. Our work provides a simple, accurate and highly automatable method for mHAgs genotyping and suggest the role of mHAgs in addressing the immune reaction between donor and host.

T and B lymphocyte subsets have been not univocally associated to Graft-versus-host disease (GVHD) and relapse of hematological malignancies after stem cell transplantation (SCT). Their sequential assessment together with B and T cell neogenesis indexes has been not thoroughly analysed in relation to these changing and interrelated immunologic/clinic events yet. Lymphocyte subsets in peripheral blood (PB) and B and T cell neogenesis indexes were analysed together at different time points in a prospective study of 50 patients. Principal component analysis (PCA) was used as first step of multivariate analysis to address issues related to a high number of variables versus a relatively low number of patients. Multivariate analysis was completed by Fine-Gray proportional hazard regression model. PCA identified 3 clusters of variables (PC1-3), which correlated with acute GVHD: PC1 (pre-SCT: KRECs≥6608/ml, unswitched memory B 44%, CD8+TCM cells>4%; HR 1.9, p = 0.01), and PC3 (at aGVHD onset: CD4+TEMRA69%, switched memory CD19+ = 0 cells and KRECs

A human bone marrow-derived mesenchymal stromal cell (MSCs) and cord blood-derived CD34+ stem cell co-culture system was set up in order to evaluate the proliferative and differentiative effects induced by MSCs on CD34+ stem cells, and the reciprocal influences on gene expression profiles. After 10 days of co-culture, non-adherent (SN-fraction) and adherent (AD-fraction) CD34+ stem cells were collected and analysed separately. In the presence of MSCs, a significant increase in CD34+ cell number was observed (fold increase = 14.68), mostly in the SN-fraction (fold increase = 13.20). This was combined with a significant increase in CD34+ cell differentiation towards the BFU-E colonies and with a decrease in the CFU-GM. These observations were confirmed by microarray analysis. Through gene set enrichment analysis (GSEA), we noted a significant enrichment in genes involved in heme metabolism (e.g. LAMP2, CLCN3, BMP2K), mitotic spindle formation and proliferation (e.g. PALLD, SOS1, CCNA1) and TGF-beta signalling (e.g. ID1) and a down-modulation of genes participating in myeloid and lymphoid differentiation (e.g. PCGF2) in the co-cultured CD34+ stem cells. On the other hand, a significant enrichment in genes involved in oxygen-level response (e.g. TNFAIP3, SLC2A3, KLF6) and angiogenesis (e.g. VEGFA, IGF1, ID1) was found in the co-cultured MSCs. Taken together, our results suggest that MSCs can exert a priming effect on CD34+ stem cells, regulating their proliferation and erythroid differentiation. In turn, CD34+ stem cells seem to be able to polarise the BM-niche towards the vascular compartment by modulating molecular pathways related to hypoxia and angiogenesis.

Assessment of software nonfunctional properties (NFP) is an important problem in software development. In the context of model-driven development, an emerging approach for the analysis of different NFPs consists of the following steps: (a) to extend the software models with annotations describing the NFP of interest; (b) to transform automatically the annotated software model to the formalism chosen for NFP analysis; (c) to analyze the formal model using existing solvers; (d) to assess the software based on the results and give feedback to designers. Such a modeling→analysis→assessment approach can be applied to any software modeling language, be it general purpose or domain specific. In this paper, we focus on UML-based development and on the dependability NFP, which encompasses reliability, availability, safety, integrity, and maintainability. The paper presents the profile used to extend UML with dependability information, the model transformation to generate a DSPN formal model, and the assessment of the system properties based on the DSPN results.

Motor balance in developmental stuttering (DS) was investigated with Transcranial Magnetic Stimulation (TMS), with the aim to define novel neural markers of persistent DS in adulthood. Eleven DS adult males were evaluated with TMS on tongue primary motor cortex, compared to 15 matched fluent speakers, in a "state" condition (i.e. stutterers vs. fluent speakers, no overt stuttering). Motor and silent period thresholds (SPT), recruitment curves, and silent period durations were acquired by recording tongue motor evoked potentials. Tongue silent period duration was increased in DS, especially in the left hemisphere (P