​School of Medicine and Surgery

This record contains raw data related to article "Targeted Mutational Analysis of Circulating Tumor DNA to Decipher Temporal Heterogeneity of High-Grade Serous Ovarian Cancer". We have previously demonstrated that longitudinal untargeted analysis of plasma samples withdrawn from patients with high-grade serous ovarian cancer (HGS-EOC) can intercept the presence of molecular recurrence (TRm) earlier than the diagnosis of clinical recurrence (TRc). This finding opens a clinical important temporal window to acquire through plasma sample analysis a real-time picture of those emerging molecular lesions that will drive and sustain the growth of relapsed disease and ultimately will confer resistance. In this proof of principle study, the same genomic libraries obtained at the diagnosis (T0), TRm and TRc were further analyzed by targeted resequencing approach to sequence the coding region of a panel of 65 genes to provide longitudinal analysis of clonal evolution as a novel strategy to support clinical decisions for the second-line treatment. Experiments were performed on plasma and tumor tissues withdrawn on a selection of previously analyzed cohorts of cases (i.e., 33 matched primary and synchronous lesions and 43 plasma samples from 18 patients). At T0, the median concordance of mutations shared by each tumor tissue biopsy and its matched plasma sample was 2.27%. This finding confirms the limit of a single tumor biopsy to be representative of the entire disease, while plasma analysis can recapitulate most of the main molecular lesions of the disease. A comparable scenario was observed during longitudinal analysis, where, with the exception of the TP53 gene and germline mutations in BRCA1/2 genes, no other gene shared the same locus specific gene mutation across T0, TRm and TRc time points. This high level of temporal heterogeneity has important implications for planning second-line treatment. For example, in three out of 13 cases, plasma ctDNA analysis at TRm or TRc reported acquired novel variants in the TP53BP1 gene not present at T0. In particular, patient 21564, potentially eligible for PARP-inhibitor (PARPi) treatment at the time of diagnosis (BRCA1 c.5182delA mutation), would unlikely respond to these drugs in second-line therapy due to the presence of eight distinct TP53BP1 variants in plasma samples collected TRc. This study demonstrates that liquid biopsy provides a real-time molecular picture to intercept those actionable genetic vulnerabilities or drug resistance mechanisms that could be used to plan a more rational second-line treatment.

Transcriptome Sequence Analysis of Pediatric Acute Megakaryoblastic Leukemia Identifies An Inv(16)(p13.3;q24.3)-Encoded CBFA2T3-GLIS2 Fusion Protein As a Recurrent Lesion in 39% of Non-Infant Cases:  A Report From the St. Jude Children’s Research Hospital – Washington University Pediatric Cancer Genome Project. Acute Megakaryoblastic Leukemia (AMKL) accounts for ~10% of childhood acute myeloid leukemia (AML).  Although AMKL patients with down syndrome (DS-AMKL) have an excellent 5 year event-free survival (EFS), non-DS-AMKL patients have an extremely poor outcome with a 3 year EFS of less than 40%. With the exception of the t(1;22) translocation seen in infant non-DS-AMKL, little is known about the molecular genetic lesions that underlie this leukemia subtype. To define the landscape of mutations that occur in non-DS-AMKL, we performed transcriptome sequencing on diagnostic blasts from 14 cases (discovery cohort) using the illumina platform. Our results identified chromosomal rearrangements resulting in the expression of novel fusion transcripts in 12/14 cases. Remarkably, in 7/14 cases we detected an inversion on chromosome 16 [inv(16)(p13.3;q24.3)] that resulted in the juxtaposition of the CBFA2T3, a member of the ETO family of transcription factors, next to GLIS2 resulting in a CBFA2T3-GLIS2 chimeric gene encoding an in frame fusion protein. 6 cases in the discovery cohort fused exon 10 of CBFA2T3 to exon 3 of GLIS2, while 1 case carried a larger product that fused exon 11 of CBFA2T3 to exon 1 of GLIS2.  Both products retain the 3 CBFA2T3 N-terminal nervy homology regions that mediate protein interactions, and the 5 GLIS2 C-terminal zinc finger domains that bind the Glis DNA consensus sequence, along with one of its N-terminal transcriptional regulatory domains.  GLIS2 is a member of the GLI super family of transcription factors and has been demonstrated to play a role in regulating expression of GLI target genes as well as inhibiting WNT signaling through the binding of beta catenin.  Although GLIS2 is not normally expressed in hematopoietic cells, the translocation results in high level expression of the CBFA2T3-GLIS2 fusion protein. In addition to CBFA2T3-GLIS2, chimeric transcripts were detected in 6/7 cases that lacked evidence of the inv(16)(p13.3;q24.3).  Specifically, we detected GATA2-HOXA9, MN1-FLI1, NIPBL-HOXB9, NUP98-KDM5A, GRB10-SDK1 and C8orf76-HOXA11AS, each in an individual case.  Importantly, several of the genes involved in these translocations either play a direct role in normal megakaryocytic differentiation (GATA2 and FLI1), or have been previously shown to be involved in leukemogenesis (HOXA9, MN1, HOXB9).  Evaluation of a recurrency cohort of 42 samples including 14 additional pediatric cases and 28 adult cases by RT-PCR revealed 4 additional pediatric samples carrying CBFA2T3-GLIS2 for an overall frequency of 39% in pediatric AMKL.  In addition to these somatic structural variations, we also identified mutations in genes previously shown to play a role in megakaryoblastic leukemia including activating mutations in JAK2 and MPL (36%).  To gain insight into the mechanism whereby CBFA2T3-GLIS2 promotes leukemogenesis, we introduced the fusion into murine hematopoietic cells and assessed its effect on in vitro colony replating as a surrogate measure of self-renewal. Hematopoietic cells transduced with a mCherry expressing retroviral vector failed to form colonies after the second replating. By contrast, expression of either wild-type GLIS2 or the CBFA2T3-GLIS2 fusion resulted in a marked increase in the self-renewal capacity, with colony formation persisting through eight replatings.  Immunophenotypic analysis of the CBFA2T3-GLIS2 expressing colonies revealed evidence of megakaryocytic differentiation. Importantly, the CBFA2T3-GLIS2 cells remained growth factor dependent suggesting that cooperating mutations in growth factor signaling pathways are required for full leukemic transformation. Taken together these data identify a novel cryptic inv(16)-encoded CBFA2T3-GLIS2 fusion protein as a recurrent driver mutation in approximately 40% of non-infant pediatric non-DS-AMKLs. Moreover, the majority of pediatric cases that lacked this lesion were shown by transcriptome sequence analysis to contain other chromosomal rearrangements that encoded fusion proteins that directly alter megakaryocytic differentiation and/or myeloid cell growth. The alteration of a key transcriptional regulator within the hedgehog signaling pathways in a substantial percentage of pediatric AMKL raises the possibility that inhibition of this pathway may have a therapeutic benefit in this aggressive form of AML. Gene expression profiling was performed on 29 single diagnosis tumor samples

Bone marrow Mesenchymal Stem Cells (BM-MSCs), due to their strong protective and anti-inflammatory abilities, have been widely investigated in the context of several diseases for their possible therapeutic role, based on the release of a highly proactive secretome composed of soluble factors and Extracellular Vesicles (EVs). BM-MSC-EVs, in particular, convey many of the beneficial features of parental cells, including direct and indirect β-amyloid degrading-activities, immunoregulatory and neurotrophic abilities. Therefore, EVs represent an extremely attractive tool for therapeutic purposes in neurodegenerative diseases, including Alzheimer's disease (AD). We examined the therapeutic potential of BM-MSC-EVs injected intracerebrally into the neocortex of APPswe/PS1dE9 AD mice at 3 and 5 months of age, a time window in which the cognitive behavioral phenotype is not yet detectable or has just started to appear. We demonstrate that BM-MSC-EVs are effective at reducing the Aβ plaque burden and the amount of dystrophic neurites in both the cortex and hippocampus. The presence of Neprilysin on BM-MSC-EVs, opens the possibility of a direct β-amyloid degrading action. Our results indicate a potential role for BM-MSC-EVs already in the early stages of AD, suggesting the possibility of intervening before overt clinical manifestations.

Pathways leading to cardiovascular effects of particulate matter exposure have been linked to oxidative stress, pulmonary and systemic inflammation, endothelial cell dysfunction, atherosclerosis and altered cardiac autonomic function. PM2.5 levels in Milano are greater than those observed in urban sites in Europe, while its chemical composition is similar to those of other European cities. Winter fine particles (PM2.5win) are characterized by the presence of nitrate, organic carbon fraction (with a quite high polycyclic aromatic hydrocarbons content) and elements (Pb, Al, Zn, V, Fe, Cr and others), with a negligible endotoxin content. To elucidate the changes at molecular level, we examined the toxic effects elicited after repeated Milano PM2.5win exposure in BALB/c mice, focusing both on heart and lungs. In the lung parenchyma of PM2.5win-treated mice, ET-1, Hsp70 and cytochromes (Cyp1A1 and Cyp1B1) levels resulted increased, comparing to sham. Within the heart, PM2.5win treatment increased Hsp-70, HO-1 and MPO levels. PM2.5win had a strong impact on global gene expression of heart tissue (181 up-regulated and 178 down-regulated genes) but a less impact on lung global gene expression profiling (14 up-regulated genes and 43 down-regulated genes). Focusing on modulated genes, in lung tissue we found two-three fold changes of those related to polycyclic aromatic hydrocarbons exposure and calcium signalling. In heart tissue the most striking aspect is the twofold to threefold increase in collagen and laminin related genes, together with genes involved in calcium signaling. The current study extends our previous findings, showing that repeated instillations of PM2.5win trigger systemic adverse effect. PM2.5win likely poses an acute threat principally to susceptible people, such as the elderly and those with unrecognized coronary artery or structural heart disease. The study of genomic responses will improve understanding of disease mechanisms and enable future clinical testing of interventions against the toxic effects of air pollutant.

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.

Abstract Background Tocilizumab blocks pro-inflammatory activity of interleukin-6 (IL-6), involved in pathogenesis of pneumonia the most frequent cause of death in COVID-19 patients. Methods A multicenter, single-arm, hypothesis-driven trial was planned, according to a phase 2 design, to study the effect of tocilizumab on lethality rates at 14 and 30 days (co-primary endpoints, a priori expected rates being 20 and 35%, respectively). A further prospective cohort of patients, consecutively enrolled after the first cohort was accomplished, was used as a secondary validation dataset. The two cohorts were evaluated jointly in an exploratory multivariable logistic regression model to assess prognostic variables on survival. Results In the primary intention-to-treat (ITT) phase 2 population, 180/301 (59.8%) subjects received tocilizumab, and 67 deaths were observed overall. Lethality rates were equal to 18.4% (97.5% CI: 13.6–24.0, P = 0.52) and 22.4% (97.5% CI: 17.2–28.3, P

The study tested the construct validity and reliability of the Caregiver Contribution to Self-Care of Chronic Obstructive Pulmonary Disease (COPD) Inventory and the Caregiver Self-Efficacy in Contributing to Self-Care of COPD Scale. The two instruments were developed by modifying the Self-Care of COPD Inventory and Self-Care Self-Efficacy Scale in COPD into caregiver versions. The psychometric properties were tested in a convenience sample of 261 informal caregivers of COPD patients recruited in Italy in two cross-sectional studies. Structural validity was tested by confirmatory factor analysis, construct validity by posing several hypotheses, and internal consistency through factor score determinacy and global reliability index for multidimensional scales. In confirmatory factor analysis, the caregiver contribution to self-care maintenance, monitoring and management scales, composing the Caregiver Contribution to Self-Care of COPD Inventory, presented good fit indices. Global reliability indices ranged 0.75–0.88. The caregiver self-efficacy scale presented a comparative fit index of 0.96 and a global reliability index of 0.82. The caregiver contribution to self-care and the caregiver self-efficacy scales correlated moderately among themselves and with the patient versions of the scales, and scores were higher with caregiver-oriented dyadic care types and female caregivers. Our study provides evidence of the two instruments’ construct validity and internal consistency.

Abstract Background Adding cyclic short sustained inflations (sigh) to assisted ventilation yields optimizes lung recruitment, decreases heterogeneity and reduces inspiratory effort in patients with acute hypoxemic respiratory failure (AHRF). These findings suggest that adding sigh to pressure support ventilation (PSV) might decrease the risk of lung injury, shorten weaning and improve clinical outcomes. Thus, we conceived a pilot trial to test the feasibility of adding sigh to PSV (the PROTECTION study). Methods PROTECTION is an international randomized controlled trial that will be conducted in 23 intensive care units (ICUs). Patients with AHRF who have been intubated from 24 h to 7 days and undergoing PSV from 4 to 24 h will be enrolled. All patients will first undergo a 30-min sigh test by adding sigh to clinical PSV for 30 min to identify early oxygenation responders. Then, patients will be randomized to PSV or PSV + sigh until extubation, ICU discharge, death or day 28. Sigh will be delivered as a 3-s pressure control breath delivered once per minute at 30 cmH2O. Standardized protocols will guide ventilation settings, switch back to controlled ventilation, use of rescue treatments, performance of spontaneous breathing trial, extubation and reintubation. The primary endpoint of the study will be to verify the feasibility of PSV + sigh evaluated through reduction of failure to remain on assisted ventilation during the first 28 days in the PSV + sigh group versus standard PSV (15 vs. 22%). Failure will be defined by switch back to controlled ventilation for more than 24 h or use of rescue treatments or reintubation within 48 h from elective extubation. Setting the power to 80% and first-risk order to 5%, the computed size of the trial is 129 patients per arm. Discussion PROTECTION is a pilot randomized controlled trial testing the feasibility of adding sigh to PSV. If positive, it will provide physicians with an effective addition to standard PSV for lung protection, able to reduce failure of assisted ventilation. PROTECTION will provide the basis for a future larger trial aimed at verifying the impact of PSV + sigh on 28-day survival and ventilator-free days. Trial registration ClinicalTrials.gov, NCT03201263 . Registered on 28 June 2017.

Abstract Background Early after intubation, a layer of biofilm covers the inner lumen of the endotracheal tube (ETT). Cleaning the ETT might prevent airways colonization by pathogens, reduce resistance to airflow, and decrease sudden ETT obstruction. We investigated the effectiveness of a cleaning closed suction system in maintaining the endotracheal tube free from secretions. Methods We conducted a single center, randomized controlled trial, in the general intensive care unit of a tertiary-level university hospital. We enrolled 40 adult critically ill patients expected to remain intubated for more than 48 h, within 24 h from intubation. Patients were randomized to receive three ETT cleaning maneuvers/day using a novel device (Airway Medix Closed Suction System™, cleaning group) or to standard care (no ETT cleaning, standard closed suction, control group). After extubation, the amount of secretions in the ETTs was measured by micro-computed tomography. Results The volume of secretions in the ETTs from the cleaning group was lower than controls (0.081 [0.021–0.306] vs. 0.568 [0.162–0.756] mL, p = 0.001), corresponding to a cross-sectional area reduction six times lower (1[0–3] vs. 6 [2–10] %, p = 0.001). In a subset of 16 patients, the resistance to airflow tended to be lower after 1 day of treatment (p = 0.063) and was lower after 2 days (0.024), while no difference was present at enrollment (p = 0.922). ETT colonization did not differ between the two groups. Conclusions The use of a novel cleaning closed suction system proved to be effective in reducing secretions present in the ETT after extubation, possibly reducing resistance to airflow during intubation. Trial registration: clinicaltrials.gov NCT01912105