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Acute myeloid leukemia (AML) is a hematological malignancy derived from neoplastic myeloid progenitor cells characterized by abnormal clonal proliferation and differentiation. Although novel therapeutic strategies have recently been introduced, the prognosis of AML remains unsatisfactory. So far, the efficacy of chimeric antigen receptor (CAR)-T therapy in AML has been hampered by several factors including the poor accumulation of the blood-injected cells in the leukemia bone marrow (BM) niche, where chemotherapy-resistant leukemic stem cells reside. Thus, we hypothesized that overexpression of CXCR4, whose ligand CXCL12 is highly expressed by BM stromal cells within the niche, could improve T cell homing to the BM and consequently enhance their intimate contact with BM-resident AML cells facilitating disease eradication. Specifically, we engineered conventional CD33.CAR-cytokine induced killer cells (CIKs) with the wild-type CXCR4 and the variant CXCR4R334X, responsible for leukocyte sequestration in the BM of WHIM syndrome patients. Overexpression of both CXCR4wt and CXCR4mut in CD33.CAR-CIKs resulted in significant improvement of chemotaxis toward recombinant CXCL12 or BM stromal cell conditioned medium with no observed impairment of cytotoxic potential in vitro. Moreover, CXCR4-overexpressing CD33.CAR-CIKs showed enhanced in vivo BM homing, associated with a prolonged retention for the CXCR4R334X variant. However, only CD33.CAR-CIKs co-expressing CXCR4wt but not CXCR4mut exerted a more sustained in vivo antileukemic activity and extended animal survival, suggesting a non-canonical role for CXCR4 in modulating CAR-CIK functions independent of BM homing. Taken together, these data suggest that arming CAR-CIKs with CXCR4 may represent a promising strategy for increasing their therapeutic potential for AML.

Artificial intelligence is getting a foothold in medicine for disease screening and diagnosis. While typical machine learning methods require large labeled datasets for training and validation, their application is limited in clinical fields since ground truth information can hardly be obtained on a sizeable cohort of patients. Unsupervised neural networks – such as Self-Organizing Maps (SOMs) – represent an alternative approach to identifying hidden patterns in biomedical data. Here we investigate the feasibility of SOMs for the identification of malignant and non-malignant regions in liquid biopsies of thyroid nodules, on a patient-specific basis. MALDI-ToF (Matrix Assisted Laser Desorption Ionization - Time of Flight) mass spectrometry-imaging (MSI) was used to measure the spectral profile of bioptic samples. SOMs were then applied for the analysis of MALDI-MSI data of individual patients’ samples, also testing various pre-processing and agglomerative clustering methods to investigate their impact on SOMs’ discrimination efficacy. The final clustering was compared against the sample's probability to be malignant, hyperplastic or related to Hashimoto thyroiditis as quantified by multinomial regression with LASSO. Our results show that SOMs are effective in separating the areas of a sample containing benign cells from those containing malignant cells. Moreover, they allow to overlap the different areas of cytological glass slides with the corresponding proteomic profile image, and inspect the specific weight of every cellular component in bioptic samples. We envision that this approach could represent an effective means to assist pathologists in diagnostic tasks, avoiding the need to manually annotate cytological images and the effort in creating labeled datasets.

Background Allogeneic hematopoietic stem-cell transplantation is the standard of care for Hurler syndrome (mucopolysaccharidosis type I, Hurler variant [MPSIH]). However, this treatment is only partially curative and is associated with complications. Methods We are conducting an ongoing study involving eight children with MPSIH. At enrollment, the children lacked a suitable allogeneic donor and had a Developmental Quotient or Intelligence Quotient score above 70 (i.e., none had moderate or severe cognitive impairment). The children received autologous hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with an alpha-L-iduronidase (IDUA)-encoding lentiviral vector after myeloablative conditioning. Safety and correction of blood IDUA activity up to supraphysiologic levels were the primary end points. Clearance of lysosomal storage material as well as skeletal and neurophysiological development were assessed as secondary and exploratory end points. The planned duration of the study is 5 years. Results We now report interim results. The children's mean (+/- SD) age at the time of HSPC gene therapy was 1.9 +/- 0.5 years. At a median follow-up of 2.10 years, the procedure had a safety profile similar to that known for autologous hematopoietic stem-cell transplantation. All the patients showed prompt and sustained engraftment of gene-corrected cells and had supraphysiologic blood IDUA activity within a month, which was maintained up to the latest follow-up. Urinary glycosaminoglycan (GAG) excretion decreased steeply, reaching normal levels at 12 months in four of five patients who could be evaluated. Previously undetectable levels of IDUA activity in the cerebrospinal fluid became detectable after gene therapy and were associated with local clearance of GAGs. Patients showed stable cognitive performance, stable motor skills corresponding to continued motor development, improved or stable findings on magnetic resonance imaging of the brain and spine, reduced joint stiffness, and normal growth in line with World Health Organization growth charts. Conclusions The delivery of HSPC gene therapy in patients with MPSIH resulted in extensive metabolic correction in peripheral tissues and the central nervous system. (Funded by Fondazione Telethon and others; ClinicalTrials.gov number, ; EudraCT number, .)Hematopoietic Gene Therapy for Hurler Syndrome Eight patients with Hurler syndrome who lacked suitable allogeneic donors received autologous hematopoietic stem and progenitor cells transduced ex vivo with an alpha-L-iduronidase-encoding lentiviral vector. This therapy resulted in extensive metabolic correction in peripheral tissues and the central nervous system.

Acute Myeloid Leukemia (AML) still represents an unmet clinical need for adult and pediatric patients. Adoptive cell therapy by chimeric antigen receptor (CAR)-engineered T cells demonstrated a high therapeutic potential, but further development is required to ensure a safe and durable disease remission in AML, especially in elderly patients. To date, translation of CAR T cell therapy in AML is limited by the absence of an ideal tumor-specific antigen. CD123 and CD33 are the two most widely overexpressed LSCs biomarkers but their shared expression with endothelial and hematopoietic stem and progenitor cells (HSPCs) increases the risk of undesired vascular and hematologic toxicities. To counteract this issue, we established a balanced Dual CAR strategy aimed at reducing off-target toxicities while retaining full functionality against AML. Cytokine-Induced Killer (CIK) cells, co-expressing a first-generation low affinity anti-CD123 IL3-zetakine and an anti-CD33 as costimulatory receptor (CCR) without activation signaling domains, demonstrated a powerful antitumor efficacy against AML targets without any relevant toxicity on HSPCs and endothelial cells. The proposed optimized Dual CAR CIK strategy could offer the opportunity to unleash the potential of specifically target CD123+/CD33+ leukemic cells while minimizing toxicity against healthy cells.

Colon adenocarcinoma (COAD) has a limited range of diversified, personalized therapeutic opportunities, besides DNA hypermutating cases; thus both new targets or broadening existing strategies for personalized intervention are of interest. Routinely processed material from 246 untreated COADs with clinical follow-up was probed for evidence of DNA damage response (DDR) by multiplex immunofluorescence and immunohistochemical staining for DDR complex proteins (ℽH2AX, pCHK2, pNBS1) and for type I interferon response, T-lymphocyte infiltration (TILs), mutation mismatch repair defects (MMRd). FISH analysis for chromosome 20q copy number variations was obtained. 33.7% of COAD display a coordinated DDR on quiescent, non-senescent, non-apoptotic glands, irrespective of TP53, chromosome 20q abnormalities, type I IFN response. Clinicopathological parameters did not differentiate DDR + cases from the other cases. TILs were equally present in DDR and non-DDR cases. DDR + MMRd cases were preferentially retaining wild-type MLH1. The outcome after 5FU-based chemotherapy was not different in the two groups. DDR + COAD represents a subgroup not aligned with known diagnostic, prognostic or therapeutic categories, with potential new targeted treatment opportunities.

The podocyte injury, and consequent proteinuria, that characterize the pathology of idiopathic membranous nephropathy (IMN) is mediated by an autoimmune reaction against podocyte antigens. In particular, the activation of pathways leading to abundant renal deposits of complement is likely to involve the binding of mannose-binding lectin (MBL) to aberrant glycans on immunoglobulins. To obtain a landscape of circulatory IgG Fc glycosylation characterizing this disease, we conducted a systematic N-glycan profiling study of IgG1, 2, and 4 by mass spectrometry. The cohort included 57 IMN patients, a pathological control group with nephrotic syndrome (PN) (n = 20), and 88 healthy control subjects. The effect of sex and age was assessed in all groups and controlled by rigorous matching. Several IgG Fc glycan traits were found to be associated with IMN. Interestingly, among them, only IgG4-related results were specific for IMN and not for PN. Hypo-galactosylation of IgG4, already shown for IMN, was observed to occur in the absence of core fucose, in line with a probable increase of pro-inflammatory IgG. In addition, elevated levels of fucosylated IgG4, along with low levels of hybrid-type glycans, were detected. Some of these IgG4 alterations are likely to be more pronounced in high PLA2R (phospholipase A2 receptor) patients. IgG Fc glycosylation patterns associated with IMN warrant further studies of their role in disease mechanisms and may eventually enrich the diagnostic spectrum regarding patient stratification.

Serena Capici,1,* Luca Sala,1,* Stefania Galimberti,2 Maria Grazia Valsecchi,2 Nicola Squillace,3 Giulia Gustinetti,3 Marina Elena Cazzaniga,1,4 Paolo Bonfanti3,4 1Phase 1 Research Unit, ASST Monza, Monza, Italy; 2Bicocca Bioinformatics Biostatistics and Bioimaging B4 Center, School of Medicine and Surgery, University of Milano - Bicocca, Monza, Italy; 3Infectious Disease Unit, ASST Monza, Monza, Italy; 4School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy*These authors contributed equally to this workCorrespondence: Marina Elena CazzanigaPhase 1 Research Unit, ASST Monza, Monza, ItalyTel +390392339037Email marina.cazzaniga@asst-monza.itAbstract: The severe acute respiratory syndrome coronavirus 2 pandemic has dominated the global health scenario from the beginning of 2020 and still represents a major health emergency. Cytokine inhibitors as tocilizumab have been used to treat COVID-19 severe pneumonia with conflicting results. We performed a retrospective study whose results can contribute to the general overview regarding the role of these agents in severe COVID-19 pneumonia, suggesting an interesting, even not statistically significant evidence of the effectiveness of tocilizumab treatment in this disease and sow a seed of reflection about their use in future waves of pandemic. We compared two cohorts of patients treated with local standard of care and with tocilizumab in the experimental one. With a median follow-up of 92 days, deaths were 6 and 16 in the tocilizumab and the standard of care group, respectively. With a longer follow-up than previous studies, a trend in difference with regards to mortality of the groups was observed.Keywords: Sars-Cov-2, COVID-19, pneumonia, tocilizumab

AimsIdentification and characterisation of monoclonal gammopathies of renal significance (MGRS) is critical for therapeutic purposes. Amyloidosis represents one of the most common forms of MGRS, and renal biopsy remains the gold standard for their classification, although mass spectrometry has shown greater sensitivity in this area.MethodsIn the present study, a new in situ proteomic technique, matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI), is investigated as an alternative to conventional laser capture microdissection MS for the characterisation of amyloids. MALDI-MSI was performed on 16 cases (3 lambda light chain amyloidosis (AL), 3 AL kappa, 3 serum amyloid A amyloidosis (SAA), 2 lambda light chain deposition disease (LCDD), 2 challenging amyloid cases and 3 controls). Analysis began with regions of interest labelled by the pathologist, and then automatic segmentation was performed.ResultsMALDI-MSI correctly identified and typed cases with known amyloid type (AL kappa, AL lambda and SAA). A ‘restricted fingerprint’ for amyloid detection composed of apolipoprotein E, serum amyloid protein and apolipoprotein A1 showed the best automatic segmentation performance (area under the curve >0.7).ConclusionsMALDI-MSI correctly assigned minimal/challenging cases of amyloidosis to the correct type (AL lambda) and identified lambda light chains in LCDD cases, highlighting the promising role of MALDI-MSI for amyloid typing.

BACKGROUND: Chimeric antigen receptor (CAR) T cell immunotherapy has resulted in complete remission (CR) and durable response in highly refractory patients. However, logistical complexity and high costs of manufacturing autologous viral products limit CAR T cell availability. METHODS: We report the early results of a phase I/II trial in B cell acute lymphoblastic leukemia (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT) using donor-derived CD19 CAR T cells generated with the Sleeping Beauty (SB) transposon and differentiated into cytokine-induced killer (CIK) cells. RESULTS: The cellular product was produced successfully for all patients from the donor peripheral blood (PB) and consisted mostly of CD3(+) lymphocytes with 43% CAR expression. Four pediatric and 9 adult patients were infused with a single dose of CAR T cells. Toxicities reported were 2 grade I and 1 grade II cytokine-release syndrome (CRS) cases at the highest dose in the absence of graft-versus-host disease (GVHD), neurotoxicity, or dose-limiting toxicities. Six out of 7 patients receiving the highest doses achieved CR and CR with incomplete blood count recovery (CRi) at day 28. Five out of 6 patients in CR were also minimal residual disease negative (MRD(–)). Robust expansion was achieved in the majority of the patients. CAR T cells were measurable by transgene copy PCR up to 10 months. Integration site analysis showed a positive safety profile and highly polyclonal repertoire in vitro and at early time points after infusion. CONCLUSION: SB-engineered CAR T cells expand and persist in pediatric and adult B-ALL patients relapsed after HSCT. Antileukemic activity was achieved without severe toxicities. TRIAL REGISTRATION: ClinicalTrials.gov NCT03389035. FUNDING: This study was supported by grants from the Fondazione AIRC per la Ricerca sul Cancro (AIRC); Cancer Research UK (CRUK); the Fundación Científica de la Asociación Española Contra el Cáncer (FC AECC); Ministero Della Salute; Fondazione Regionale per la Ricerca Biomedica (FRRB).

ObjectiveThe International Society of Nephrology/Renal Pathology Society classification is the gold standard for the characterisation of lupus nephritis (LN) on renal biopsy, with therapeutic repercussions. Its recent revision simplified the current class subdivisions, eliminating the S/G forms of class IV, although data on a possible pathogenetic/clinical value of this subdivision are still contradictory.Methods353 renal biopsies from Belimumab International Study in LN were assessed through central pathology review. Univariate logistic models and a decision tree were performed on 314 adequate biopsies to evaluate the impact of histological features on focal/diffuse classes. Removing class I/II (n=6) and ‘pure’ class V (n=34), principal component analysis (PCA) and heatmap were used to explore similarities among III, IVS and IVG biopsies either incorporating or not the mixed classes (+V, n=274). Finally, a method aimed at partitioning the cases into k clusters based on their similarity (KMeans), was used to study features from the cohort of ‘pure’ class III/IVS/IVG cases (n=214) to determine alternative subdivisions based on phenotypic data.ResultsSegmental endocapillary hypercellularity (EH) was prevalent in class III, global EH, wire loops, hyaline thrombi and double contours were hallmarks of class IVG, with IVS cases showing intermediate characteristics. Heatmap and PCA confirmed the segregation of these features among classes, showing better segregation for focal/diffuse LN as compared with the mixed classes (+V). KMeans revealed the presence of two main clusters, membranoproliferative-like (n=83) or vasculitis-like (n=131).ConclusionsThis study reveals new phenotypic forms of LN surpassing the traditional classes as determined by the current classification. Future validation and confirmation are required to confirm these findings.

Background One cause of poor outcomes in children of low-income countries affected by acute lymphoblastic leukaemia (ALL) is loss to follow-up due to abandonment of treatment. Assuming this type of loss to follow-up as independent censoring, as in standard Kaplan–Meier estimates, ignores the likely association of abandonment with biologic and socio-economic factors related to outcome. Moreover, when comparing treatment protocols adopted in different time periods, possible imbalances in patients’ characteristics must be considered. We aim to compare the outcome of children enrolled in two subsequent protocols for ALL treatment (2000–2007 and 2008–2015) in Honduras, taking both dependent censoring due to abandonment of treatment and imbalances between patient characteristics into account. Methods Marginal structural models based on inverse probability of treatment and censoring (IPTC) weighting allow the estimation of potential event-free survival (EFS) as if no abandonment of treatment occurred and the whole cohort was exposed, or not, to both protocols. An Aalen additive model and a logistic-regression model were used to build abandonment and treatment weights, respectively. Results The two protocols recruited 514 and 717 patients. Measured baseline covariates in both protocols were gender, age, white blood cell count, central nervous system involvement, tumour histology and socio-economic status. The potential EFS is slightly higher under the more recent protocol in the first 3 years but no difference is estimated in the long period [survival difference at 5 years (95% confidence interval) = 0.1% (−0.97%; 1.13%)]. Both protocols would allow reducing the event rate by 12–13% if there was no abandonment of treatment. Conclusions Using IPTC weighting, we found a similar potential effect of the two treatment protocols if the imbalance due to the different distribution of potential confounders and to abandonment of therapy was removed.

Background: Frailty is known to be associated with poorer outcomes in individuals admitted to hospital for medical conditions requiring intensive care. However, little evidence is available for the effect of frailty on patients’ outcomes after traumatic brain injury. Many frailty indices have been validated for clinical practice and show good performance to predict clinical outcomes. However, each is specific to a particular clinical context. We aimed to develop a frailty index to predict 6-month outcomes in patients after a traumatic brain injury. Methods: A cumulative deficit approach was used to create a novel frailty index based on 30 items dealing with disease states, current medications, and laboratory values derived from data available from CENTER-TBI, a prospective, longitudinal observational study of patients with traumatic brain injury presenting within 24 h of injury and admitted to a ward or an intensive care unit at 65 centres in Europe between Dec 19, 2014, and Dec 17, 2017. From the individual cumulative CENTER-TBI frailty index (range 0–30), we obtained a standardised value (range 0–1), with high scores indicating higher levels of frailty. The effect of frailty on 6-month outcome evaluated with the extended Glasgow Outcome Scale (GOSE) was assessed through a proportional odds logistic model adjusted for known outcome predictors. An unfavourable outcome was defined as death or severe disability (GOSE score ≤4). External validation was performed on data from TRACK-TBI, a prospective observational study co-designed with CENTER-TBI, which enrolled patients with traumatic brain injury at 18 level I trauma centres in the USA from Feb 26, 2014, to July 27, 2018. CENTER-TBI is registered with ClinicalTrials.gov, NCT02210221; TRACK-TBI is registered at ClinicalTrials.gov, NCT02119182. Findings: 2993 participants (median age was 51 years [IQR 30–67], 2058 [69%] were men) were included in this analysis. The overall median CENTER-TBI frailty index score was 0·07 (IQR 0·03–0·15), with a median score of 0·17 (0·08–0·27) in older adults (aged ≥65 years). The CENTER-TBI frailty index score was significantly associated with the probability of an increasingly unfavourable outcome (cumulative odds ratio [OR] 1·03, 95% CI 1·02–1·04; p

Funder: European Commission 7th Framework program, Funder: Australian Health and Medical Research Council, Funder: Università degli Studi di Milano - Bicocca, Purpose: The effect of high arterial oxygen levels and supplemental oxygen administration on outcomes in traumatic brain injury (TBI) is debated, and data from large cohorts of TBI patients are limited. We investigated whether exposure to high blood oxygen levels and high oxygen supplementation is independently associated with outcomes in TBI patients admitted to the intensive care unit (ICU) and undergoing mechanical ventilation. Methods: This is a secondary analysis of two multicenter, prospective, observational, cohort studies performed in Europe and Australia. In TBI patients admitted to ICU, we describe the arterial partial pressure of oxygen (PaO2) and the oxygen inspired fraction (FiO2). We explored the association between high PaO2 and FiO2 levels within the first week with clinical outcomes. Furthermore, in the CENTER-TBI cohort, we investigate whether PaO2 and FiO2 levels may have differential relationships with outcome in the presence of varying levels of brain injury severity (as quantified by levels of glial fibrillary acidic protein (GFAP) in blood samples obtained within 24 h of injury). Results: The analysis included 1084 patients (11,577 measurements) in the CENTER-TBI cohort, of whom 55% had an unfavorable outcome, and 26% died at a 6-month follow-up. Median PaO2 ranged from 93 to 166 mmHg. Exposure to higher PaO2 and FiO2 in the first seven days after ICU admission was independently associated with a higher mortality rate. A trend of a higher mortality rate was partially confirmed in the OzENTER-TBI cohort (n = 159). GFAP was independently associated with mortality and functional neurologic outcome at follow-up, but it did not modulate the outcome impact of high PaO2 and FiO2 levels, which remained independently associated with 6-month mortality. Conclusions: In two large prospective multicenter cohorts of critically ill patients with TBI, levels of PaO2 and FiO2 varied widely across centers during the first seven days after ICU admission. Exposure to high arterial blood oxygen or high supplemental oxygen was independently associated with 6-month mortality in the CENTER-TBI cohort, and the severity of brain injury did not modulate this relationship. Due to the limited sample size, the findings were not wholly validated in the external OzENTER-TBI cohort. We cannot exclude the possibility that the worse outcomes associated with higher PaO2 were due to use of higher FiO2 in patients with more severe injury or physiological compromise. Further, these findings may not apply to patients in whom FiO2 and PaO2 are titrated to brain tissue oxygen monitoring (PbtO2) levels. However, at minimum, these findings support the need for caution with oxygen therapy in TBI, particularly since titration of supplemental oxygen is immediately applicable at the bedside.

Objective: The American College of Radiology (ACR) and the European Thyroid Association (EU) have proposed two scoring systems for thyroid nodule classification. Here, we compared the ability of the two systems in triaging thyroid nodules for fine-needle aspiration (FNA) and tested the putative role of an approach that combines ultrasound features and cytology for the detection of malignant nodules. Design and Methods: The scores obtained with the ACR and EU Thyroid Imaging Reporting and Data Systems (TIRADS) from a prospective series of 480 thyroid nodules acquired from 435 subjects were compared to assess their performances in FNA triaging on the final cytological diagnosis. The US features that showed the highest contribution in discriminating benign nodules from malignancies were combined with cytology to improve its diagnostic performance. Results: FNA was recommended on 46.5% and 51.9% of the nodules using the ACR and EU-TIRADS scores, respectively. The ACR system demonstrated a higher specificity as compared to the EU-TIRADS (59.0% vs. 52.4%, p = 0.0012) in predicting &gt, TIR3A/III (SIAPEC/Bethesda) nodules. Moreover, specific radiological features (i.e., echogenic foci and margins), combined with the cytological classes improved the specificity (97.5% vs. 91%, p &lt, 0.0001) and positive predictive values (77.5% vs. 50.7%, p &lt, 0.0001) compared to cytology alone, especially in the setting of indeterminate nodules (TIR3A/III and TIR3B/IV), maintaining an excellent sensitivity and negative predictive value. Conclusions: The ACR-TIRADS system showed a higher specificity compared to the EU-TIRADS in triaging thyroid nodules. The use of specific radiological features improved the diagnostic ability of cytology.

Predicting the prognosis of colorectal cancer (CRC) patients remains challenging and a characterisation of the tumour immune environment represents one of the most crucial avenues when attempting to do so. For this reason, molecular approaches which are capable of classifying the immune environments associated with tumour infiltrating lymphocytes (TILs) are being readily investigated. In this proof of concept study, we aim to explore the feasibility of using spatial lipidomics by MALDI-MSI to distinguish CRC tissue based upon their TIL content. Formalin-fixed paraffin-embedded tissue from human thymus and tonsil was first analysed by MALDI-MSI to obtain a curated mass list from a pool of single positive T lymphocytes, whose putative identities were annotated using an LC-MS-based lipidomic approach. A CRC tissue microarray (TMA, n = 30) was then investigated to determine whether these cases could be distinguished based upon their TIL content in the tumour and its microenvironment. MALDI-MSI from the pool of mature T lymphocytes resulted in the generation of a curated mass list containing 18 annotated m/z features. Initially, subsets of T lymphocytes were then distinguished based on their state of maturation and differentiation in the human thymus and tonsil tissue. Then, when applied to a CRC TMA containing differing amounts of T lymphocyte infiltration, those cases with a high TIL content were distinguishable from those with a lower TIL content, especially within the tumour microenvironment, with three lipid signals being shown to have the greatest impact on this separation (p < 0.05). On the whole, this preliminary study represents a promising starting point and suggests that a lipidomics MALDI-MSI approach could be a promising tool for subtyping the diverse immune environments in CRC.

To provide an assessment of the safety of ex-vivo gene therapy (GT) with hematopoietic stem and progenitor cells (HSPC), we reviewed in a systematic manner the literature on monogenic diseases to describe survival, genotoxicity and engraftment of gene corrected HSPC, across vector platforms and diseases. From 1995 to 2020, 55 trials for 14 diseases met inclusion criteria and 406 patients with primary immunodeficiencies (55.2%), metabolic diseases (17.0%), haemoglobinopathies (24.4%) and bone marrow failures (3.4%) were treated with gammaretroviral vector (γRV) (29.1%), self-inactivating γRV (2.2%) or lentiviral vectors (LV) (68.7%). The pooled overall incidence rate of death was 0.9 per 100 person-years of observation (PYO) (95%CI = 0.37–2.17). There were 21 genotoxic events out of 1504.02 PYO. All these events occurred in γRV trials (0.99 events per 100 PYO, 95%CI = 0.18–5.43) for primary immunodeficiencies. Pooled rate of engraftment was 86.1% (95%CI = 66.9–95.0%) for γRV and 99.0% (95%CI = 95.1–99.8%) for LV HSPC-GT (p = 0.002). A comprehensive meta-analysis on HSPC-GT showed stable reconstitution of haematopoiesis in most recipients with superior engraftment and safer profile in patients receiving LV-transduced HSPC.

Background and objectiveLong-term pulmonary sequelae following SARS-CoV-2 pneumonia are not yet confirmed, however preliminary observations suggests a possible relevant clinical, functional and radiological impairment. The aim of this study was to identify and characterise pulmonary sequelae caused by SARS-CoV-2 pneumonia at 6-month follow-up.MethodsIn this multicenter, prospective, observational cohort study, patients hospitalised for SARS-CoV-2 pneumonia and without prior diagnosis of structural lung diseases were stratified by maximum ventilatory support (“oxygen only”, “continuous positive airway pressure (CPAP)” and “invasive mechanical ventilation (IMV)”) and followed up at 6 months from discharge. Pulmonary function tests and diffusion capacity for carbon monoxide (DLCO), 6 minutes walking test, chest X-ray, physical exam and modified Medical Research Council (mMRC) dyspnoea score were collected.ResultsBetween March and June 2020, 312 patients were enrolled (83, 27% women; median [IQR] age 61.1 [53.4,69.3] years). The parameters that showed the highest rate of impairment were DLCO and chest-X-ray, in 46% and 25% of patients, respectively. However, only a minority of patients reported dyspnoea (31%), defined as mMRC ≥ 1, or showed a restrictive ventilatory defects (9%). In the logistic regression model, having asthma as comorbidity was associated with DLCO impairment at follow-up, while prophylactic heparin administration during hospitalisation appeared as a protective factor. Need for invasive ventilatory support during hospitalisation was associated with chest imaging abnormalities.ConclusionDLCO and radiological assessment appear to be the most sensitive tools to monitor patients with COVID-19 during follow-up. Future studies with longer follow-up are warranted to better understand pulmonary sequelae.Summary at a glanceDLCO and radiological assessment are the most sensitive tools to monitor COVID-19 patients at 6-month follow-up. Invasive ventilatory support is a risk factor for detection of radiological abnormalities, but not for DLCO impairment, at follow-up. Whileuse of prophylactic heparin acts as a protective factor on the development of DLCOimpairment.

Idiopathic nephrotic syndrome (INS) is the most frequent primary glomerular disease in children, displaying high grade proteinuria and oedema. The mainstay of therapy are steroids, and patients are usually classified according to the treatment response (sensitive vs. resistant). The mechanisms involved in INS pathogenesis and treatment responsiveness have not yet been identified. In this context, the analysis of urinary extracellular vesicles (UEv) is interesting, since they represent a molecular snapshot of the parental cells, offering a “fingerprint” for monitoring their status. Therefore, the aim of this study is to verify the feasibility of using UEv of INS patients as indicators of therapy response and its prediction. UEv were isolated from the urine of pediatric patients in remission after therapy, they showed characteristic electrophoresis profiles that matched specific patient subgroups. We then built a statistical model to interpret objectively each patient UEv protein profile: in particular, steroid-resistant patients cluster together with a very distinct pattern from other INS patients and controls. In conclusion, the evaluation of the UEv protein profile looks promising in the investigation of INS, showing a disease signature that might predict clinical evolution.

Fine needle aspiration (FNA) is the reference standard for the diagnosis of thyroid nodules. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been successfully used to discriminate the proteomic profiles of benign and malignant thyroid FNAs within the scope of providing support to pathologists for the classification of morphologically borderline cases. However, real FNAs provide a limited amount of material due to sample collection restrictions. Ex vivo FNAs could represent a valuable alternative, increasing sample size and the power of statistical conclusions. In this study, we compared the real and ex vivo MALDI-MSI proteomic profiles, extracted from thyrocyte containing regions of interest, of 13 patients in order to verify their similarity. Statistical analysis demonstrated the mass spectra similarity of the proteomic profiles by performing intra-patient comparison, using statistical similarity systems. In conclusion, these results show that post-surgical FNAs represent a possible alternative source of material for MALDI-MSI proteomic investigations in instances where pre-surgical samples are unavailable or the number of cells is scarce. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-020-03088-4.

Background During the Coronavirus disease 2019 (COVID-19) pandemic, advanced health systems have come under pressure by the unprecedented high volume of patients needing urgent care. The impact on mortality of this “patients’ burden” has not been determined. Methods and findings Through retrieval of administrative data from a large referral hospital of Northern Italy, we determined Aalen-Johansen cumulative incidence curves to describe the in-hospital mortality, stratified by fixed covariates. Age- and sex-adjusted Cox models were used to quantify the effect on mortality of variables deemed to reflect the stress on the hospital system, namely the time-dependent number of daily admissions and of total hospitalized patients, and the calendar period. Of the 1225 subjects hospitalized for COVID-19 between February 20 and May 13, 283 died (30-day mortality rate 24%) after a median follow-up of 14 days (interquartile range 5–19). Hospitalizations increased progressively until a peak of 465 subjects on March 26, then declined. The risk of death, adjusted for age and sex, increased for a higher number of daily admissions (adjusted hazard ratio [AHR] per an incremental daily admission of 10 patients: 1.13, 95% Confidence Intervals [CI] 1.05–1.22, p = 0.0014), and for a higher total number of hospitalized patients (AHR per an increase of 50 patients in the total number of hospitalized subjects: 1.11, 95%CI 1.04–1.17, p = 0.0004), while was lower for the calendar period after the peak (AHR 0.56, 95%CI 0.43–0.72, p Conclusions The pressure of a high volume of severely ill patients suffering from COVID-19 has a measurable independent impact on in-hospital mortality.

BACKGROUND Bronchoalveolar lavage (BAL) in patients with severe coronavirus disease 2019 (COVID‐19) may provide additional and complementary findings for the management of these patients admitted to intensive care units (ICUs). This study addresses the cytological features of the infection and highlights the more influential inflammatory components. The correlation between pathological variables and clinical data is also analyzed. METHODS The authors performed a retrospective analysis of the cytopathological features of BAL in 20 COVID‐19 patients and 20 members of a matched cohort from a critical ICU who had acute respiratory distress syndrome caused by other pulmonary conditions. RESULTS A comparison of the controls (n = 20) and the COVID‐19 patients (n = 20) revealed that the latter had a higher neutrophil count (median, 63.8% of the cell count) with lower percentages of macrophages and lymphocytes. An increase in the expression of CD68‐positive, monocytic multinucleated giant cells (MGCs) was reported; megakaryocytes were not detected on CD61 staining. Perls staining showed isolated elements. In situ RNA analysis demonstrated scattered chromogenic signals in type II pneumocytes. An ultrastructural analysis confirmed the presence of intracytoplasmic vacuoles containing rounded structures measuring 140 nm in diameter (putative viral particles). In COVID‐19 patients, the clinicopathological correlation revealed a positive correlation between lactate dehydrogenase values and MGCs (r = 0.54). CONCLUSIONS The analysis of BAL samples might be implemented as a routine practice for the evaluation of COVID‐19 patients in ICUs in the appropriate clinical scenario. Additional studies using a larger sample size of patients who developed COVID‐19 during the second wave of the epidemic in the autumn of 2020 are needed to further support our findings., An analysis of the cytological features of bronchoalveolar lavage samples can provide useful information for the management of coronavirus disease 2019 patients in the intensive care unit.

Background: Long-term pulmonary sequelae following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia are not yet confirmed; however, preliminary observations suggest a possible relevant clinical, functional, and radiological impairment. Objectives: The aim of this study was to identify and characterize pulmonary sequelae caused by SARS-CoV-2 pneumonia at 6-month follow-up. Methods: In this multicentre, prospective, observational cohort study, patients hospitalized for SARS-CoV-2 pneumonia and without prior diagnosis of structural lung diseases were stratified by maximum ventilatory support (“oxygen only,” “continuous positive airway pressure,” and “invasive mechanical ventilation”) and followed up at 6 months from discharge. Pulmonary function tests and diffusion capacity for carbon monoxide (DLCO), 6-min walking test, chest X-ray, physical examination, and modified Medical Research Council (mMRC) dyspnoea score were collected. Results: Between March and June 2020, 312 patients were enrolled (83, 27% women; median interquartile range age 61.1 [53.4, 69.3] years). The parameters that showed the highest rate of impairment were DLCO and chest X-ray, in 46% and 25% of patients, respectively. However, only a minority of patients reported dyspnoea (31%), defined as mMRC ≥1, or showed restrictive ventilatory defects (9%). In the logistic regression model, having asthma as a comorbidity was associated with DLCO impairment at follow-up, while prophylactic heparin administration during hospitalization appeared as a protective factor. The need for invasive ventilatory support during hospitalization was associated with chest imaging abnormalities. Conclusions: DLCO and radiological assessment appear to be the most sensitive tools to monitor patients with the coronavirus disease 2019 (COVID-19) during follow-up. Future studies with longer follow-up are warranted to better understand pulmonary sequelae.