Selective homing of CAR-CIK cells to the bone marrow niche enhances control of the acute myeloid leukemia burden.

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 is still unsatisfactory. So far, the efficacy of chimeric antigen receptor (CAR)-T-cell 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 in which chemotherapy-resistant leukemic stem cells reside. Thus, we hypothesized that overexpression of CXCR4, whose ligand CXCL12 is highly expressed by BM stromal cells within this 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 (wt) CXCR4 and the variant CXCR4R334X, responsible for leukocyte sequestration in the BM of patients with warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis syndrome. 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 coexpressing CXCR4wt but not CXCR4mut exerted a more sustained in vivo antileukemic activity and extended animal survival, suggesting a noncanonical 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.

Hematopoietic Stem- and Progenitor-Cell Gene Therapy for Hurler Syndrome.

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 α-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, NCT03488394; EudraCT number, 2017-002430-23.).

A DNA damage response-like phenotype defines a third of colon cancers at onset.

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), that is, the gathering of DDR-associated molecules at discrete nuclear spots, by multiplex immunofluorescence and immunohistochemical staining for DDR complex proteins (γH2AX, pCHK2, and pNBS1). We also tested the cases for type I interferon response, T-lymphocyte infiltration (TILs), and mutation mismatch repair defects (MMRd), known to be associated with defects of DNA repair. FISH analysis for chromosome 20q copy number variations was obtained. A total of 33.7% of COAD display a coordinated DDR on quiescent, non-senescent, non-apoptotic glands, irrespective of TP53 status, chromosome 20q abnormalities, and 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, exploiting the DNA damage repair pathways.

Intracranial Pressure Monitoring Practice, Treatment, and Effect on Outcome in Aneurysmal Subarachnoid Hemorrhage.

BACKGROUND: Intracranial pressure (ICP) monitoring and its management in aneurysmal subarachnoid hemorrhage (aSAH) is variable worldwide. The present study aimed to explore the practice of ICP monitoring, its variability across countries, and the association with 6-month outcomes in aSAH. METHODS: This was a preplanned subanalysis of SYNAPSE-ICU, a multicenter, international, prospective, observational cohort study focused on patients diagnosed with aSAH. We evaluated the variability in ICP monitoring across countries through a logistic regression model adjusted for case-mix and considered countries as a random effect. The association between ICP probe insertion and 6-month mortality and a poor neurological outcome, defined as an Glasgow Outcome Score Extended ≤ 4, was assessed by using a propensity score approach. RESULTS: A total of 423 patients with aSAH from 92 centers across 32 countries were included in this analysis. ICP monitoring was used in 295 (69.7%) patients. Significant between-country variability in ICP insertion was observed, with an incidence ranging between 4.7% and 79.9% (median odd ratio 3.04). The median duration of ICP monitoring was 12 days (first quartile [Q1] through third quartile [Q3] range 8-18), with an overall daily median ICP value of 14 mm Hg (Q1-Q3 10-19) and a median maximum value of 21 mm Hg (Q1-Q3 16-30). Patients monitored with ICP received more aggressive therapy treatments compared with non-monitored patients (therapy intensity level, TIL, score 10.33 [standard deviation 3.61] vs. 6.3 [standard deviation 4.19], p < 0.001). In more severe patients, ICP monitoring was significantly associated with better 6-month outcome (poor neurological outcome: odds ratio 0.14, 95% confidence interval 0.02-0.53, p = 0.0113; mortality: hazard ratio 0.25, 95% confidence interval 0.13-0.49, p < 0.0001). However, no significant effect was observed in patients with both reactive pupils. CONCLUSIONS: Our cohort demonstrated high variability in ICP insertion practice among countries. A more aggressive treatment approach was applied in ICP-monitored patients. In patients with severe aSAH, ICP monitoring might reduce unfavorable outcomes and mortality at 6 months.

Spatially Resolved Molecular Approaches for the Characterisation of Non-Invasive Follicular Tumours with Papillary-like Features (NIFTPs).

Noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP) are low-risk thyroid lesions most often characterised by RAS-type mutations. The histological diagnosis may be challenging, and even immunohistochemistry and molecular approaches have not yet provided conclusive solutions. This study characterises a set of NIFTPs by Matrix-Assisted Laser Desorption/Ionisation (MALDI)-Mass Spectrometry Imaging (MSI) to highlight the proteomic signatures capable of overcoming histological challenges. Archived formalin-fixed paraffin-embedded samples from 10 NIFTPs (n = 6 RAS-mutated and n = 4 RAS-wild type) were trypsin-digested and analysed by MALDI-MSI, comparing their profiles to normal tissue and synchronous benign nodules. This allowed the definition of a four-peptide signature able to distinguish RAS-mutant from wild-type cases, the latter showing proteomic similarities to hyperplastic nodules. Moreover, among the differentially expressed signals, Peptidylprolyl Isomerase A (PPIA, 1505.8 m/z), which has already demonstrated a role in the development of cancer, was found overexpressed in NIFTP RAS-mutated nodules compared to wild-type lesions. These results underlined that high-throughput proteomic approaches may add a further level of biological comprehension for NIFTPs. In the future, thanks to the powerful single-cell detail achieved by new instruments, the complementary NGS-MALDI imaging sequence might be the correct methodological approach to confirm that the current NIFTP definition encompasses heterogeneous lesions that must be further characterised.

Dissecting the histological features of lupus nephritis highlights new common patterns of injury in class III/IV.

OBJECTIVE: The 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. METHODS: 353 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. RESULTS: Segmental 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). CONCLUSIONS: This 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.

One-year pulmonary impairment after severe COVID-19: a prospective, multicenter follow-up study.

BACKGROUND: Long-term pulmonary sequelae following hospitalization for SARS-CoV-2 pneumonia is largely unclear. The aim of this study was to identify and characterise pulmonary sequelae caused by SARS-CoV-2 pneumonia at 12-month from discharge. METHODS: In this multicentre, prospective, observational 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 12 months from discharge. Pulmonary function tests and diffusion capacity for carbon monoxide (DLCO), 6 min walking test, high resolution CT (HRCT) scan, and modified Medical Research Council (mMRC) dyspnea scale were collected. RESULTS: Out of 287 patients hospitalized with SARS-CoV-2 pneumonia and followed up at 1 year, DLCO impairment, mainly of mild entity and improved with respect to the 6-month follow-up, was observed more frequently in the "oxygen only" and "IMV" group (53% and 49% of patients, respectively), compared to 29% in the "CPAP" group. Abnormalities at chest HRCT were found in 46%, 65% and 80% of cases in the "oxygen only", "CPAP" and "IMV" group, respectively. Non-fibrotic interstitial lung abnormalities, in particular reticulations and ground-glass attenuation, were the main finding, while honeycombing was found only in 1% of cases. Older patients and those requiring IMV were at higher risk of developing radiological pulmonary sequelae. Dyspnea evaluated through mMRC scale was reported by 35% of patients with no differences between groups, compared to 29% at 6-month follow-up. CONCLUSION: DLCO alteration and non-fibrotic interstitial lung abnormalities are common after 1 year from hospitalization due to SARS-CoV-2 pneumonia, particularly in older patients requiring higher ventilatory support. Studies with longer follow-ups are needed.

Cytomolecular Classification of Thyroid Nodules Using Fine-Needle Washes Aspiration Biopsies.

Fine-needle aspiration biopsies (FNA) represent the gold standard to exclude the malignant nature of thyroid nodules. After cytomorphology, 20-30% of cases are deemed "indeterminate for malignancy" and undergo surgery. However, after thyroidectomy, 70-80% of these nodules are benign. The identification of tools for improving FNA's diagnostic performances is explored by matrix-assisted laser-desorption ionization mass spectrometry imaging (MALDI-MSI). A clinical study was conducted in order to build a classification model for the characterization of thyroid nodules on a large cohort of 240 samples, showing that MALDI-MSI can be effective in separating areas with benign/malignant cells. The model had optimal performances in the internal validation set (n = 70), with 100.0% (95% CI = 83.2-100.0%) sensitivity and 96.0% (95% CI = 86.3-99.5%) specificity. The external validation (n = 170) showed a specificity of 82.9% (95% CI = 74.3-89.5%) and a sensitivity of 43.1% (95% CI = 30.9-56.0%). The performance of the model was hampered in the presence of poor and/or noisy spectra. Consequently, restricting the evaluation to the subset of FNAs with adequate cellularity, sensitivity improved up to 76.5% (95% CI = 58.8-89.3). Results also suggest the putative role of MALDI-MSI in routine clinical triage, with a three levels diagnostic classification that accounts for an indeterminate gray zone of nodules requiring a strict follow-up.

Definition of IgG Subclass-Specific Glycopatterns in Idiopathic Membranous Nephropathy: Aberrant IgG Glycoforms in Blood.

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.

Comparison of tumor microenvironment in primary and paired metastatic ER+/HER2- breast cancers: results of a pilot study.

BACKGROUND: Tumor microenvironment (TME) is a dynamic setting and changes in TILs and their subpopulations are potential candidates to influence the metastatic process. Aim of this pilot study is to describe the changes occurring between primary breast cancers and their paired metastases in terms of TILs composition. To assess if these changes influence the process of metastasis development, we used a control group of patients. METHODS: We retrospectively identified 18 Luminal patients, for whom primary and metastatic tissue were available (cases) and 18 paired-matched patients (controls), not relapsed after at least 9 years of follow-up, and we quantified TILs and their composition (i.e. T CD8+ and CD4+/FOXP3+). The presence of TILs was defined as ≥10%. RESULTS: Our results showed that the microenvironment composition of relapsed patients was poor of TILs (median = 5%, I-III quartiles = 0.6-5%), CD8+ (2.5%, 0-5%) and CD4+/FOXP3 + (0%, 0-0.6%) in the primary tumor. Comparable results were observed in their related metastases (TILs 3.8%, 0.6-5%; CD8+ 0%, 0-1.3%; CD4+/FOXP3+ 0%,0-1.9%). On the contrary, the microenvironment in the control group was richer of TILs (5%, 5-17.5%) in comparison to cases, both in primary tumor (p = 0.035) and related metastases (p = 0.018). Although CD8+ in controls were similar to cases at primary tumor (p = 0.6498), but not at metastasis (p = 0.0223), they expressed only one part on the TILs subpopulations (p = 0.0060), while TILs in the cases at primary tumor were almost completely CD8+ (p = 0.5034). CONCLUSIONS: These findings suggest that the lack of activation of immune system in the primary tumor might influence the multifactor process of cancer progression.

Ex vivo thyroid fine needle aspirations as an alternative for MALDI-MSI proteomic investigation: intra-patient comparison.

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.

Targeting CD33 in Chemoresistant AML Patient-Derived Xenografts by CAR-CIK Cells Modified with an Improved SB Transposon System.

The successful implementation of chimeric antigen receptor (CAR)-T cell therapy in the clinical context of B cell malignancies has paved the way for further development in the more critical setting of acute myeloid leukemia (AML). Among the potentially targetable AML antigens, CD33 is insofar one of the main validated molecules. Here, we describe the feasibility of engineering cytokine-induced killer (CIK) cells with a CD33.CAR by using the latest optimized version of the non-viral Sleeping Beauty (SB) transposon system "SB100X-pT4." This offers the advantage of improving CAR expression on CIK cells, while reducing the amount of DNA transposase as compared to the previously employed "SB11-pT" version. SB-modified CD33.CAR-CIK cells exhibited significant antileukemic activity in vitro and in vivo in patient-derived AML xenograft models, reducing AML development when administered as an "early treatment" and delaying AML progression in mice with established disease. Notably, by exploiting an already optimized xenograft chemotherapy model that mimics human induction therapy in mice, we demonstrated for the first time that CD33.CAR-CIK cells are also effective toward chemotherapy resistant/residual AML cells, further supporting its future clinical development and implementation within the current standard regimens.

Six-Month Pulmonary Impairment after Severe COVID-19: A Prospective, Multicentre Follow-Up Study.

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.

OMICS in Chronic Kidney Disease: Focus on Prognosis and Prediction.

Chronic kidney disease (CKD) patients are characterized by a high residual risk for cardiovascular (CV) events and CKD progression. This has prompted the implementation of new prognostic and predictive biomarkers with the aim of mitigating this risk. The 'omics' techniques, namely genomics, proteomics, metabolomics, and transcriptomics, are excellent candidates to provide a better understanding of pathophysiologic mechanisms of disease in CKD, to improve risk stratification of patients with respect to future cardiovascular events, and to identify CKD patients who are likely to respond to a treatment. Following such a strategy, a reliable risk of future events for a particular patient may be calculated and consequently the patient would also benefit from the best available treatment based on their risk profile. Moreover, a further step forward can be represented by the aggregation of multiple omics information by combining different techniques and/or different biological samples. This has already been shown to yield additional information by revealing with more accuracy the exact individual pathway of disease.

An Italian multicenter retrospective-prospective observational study on neurological manifestations of COVID-19 (NEUROCOVID).

Neurological manifestations of COVID-19 have been described in both single case reports and retrospective scanty case series. They may be linked to the potential neurotropism of the SARS-COV-2 virus, as previously demonstrated for other coronaviruses. We report here the description of a multicenter retrospective-prospective observational study promoted by the Italian Society of Neurology (SIN), involving the Italian Neurological Departments, who will consecutively recruit patients with neurological symptoms and/or signs, occurred at the onset or as a complication of COVID-19. Hospitalized patients will be recruited either in neurological wards or in COVID wards; in the latter cases, they will be referred from other specialists to participant neurologists. Outpatients with clinical signs of COVID and neurological manifestations will be also referred to participating neurologists from primary care physicians. A comprehensive data collection, in the form of electronic case report form (eCRF), will register all possible neurological manifestations involving central nervous systems, peripheral nerves, and muscles, together with clinical, laboratory (including cerebrospinal fluid, if available), imaging, neurological, neurophysiological, and neuropsychological data. A follow-up at hospital discharge (in hospitalized patients), and for all patients after 3 and 6 months, is also planned. We believe that this study may help to intercept the full spectrum of neurological manifestations of COVID-19 and, given the large diffusion at national level, can provide a large cohort of patients available for future more focused investigations. Similar observational studies might also be proposed at international level to better define the neurological involvement of COVID-19.

Hematopoietic stem cell transplantation for CD40 ligand deficiency: Results from an EBMT/ESID-IEWP-SCETIDE-PIDTC study.

BACKGROUND: CD40 ligand (CD40L) deficiency, an X-linked primary immunodeficiency, causes recurrent sinopulmonary, Pneumocystis and Cryptosporidium species infections. Long-term survival with supportive therapy is poor. Currently, the only curative treatment is hematopoietic stem cell transplantation (HSCT). OBJECTIVE: We performed an international collaborative study to improve patients' management, aiming to individualize risk factors and determine optimal HSCT characteristics. METHODS: We retrospectively collected data on 130 patients who underwent HSCT for CD40L deficiency between 1993-2015. We analyzed outcome and variables' relevance with respect to survival and cure. RESULTS: Overall survival (OS), event-free survival (EFS), and disease-free survival (DFS) were 78.2%, 58.1%, and 72.3% 5 years after HSCT. Results were better in transplantations performed in 2000 or later and in children less than 10 years old at the time of HSCT. Pre-existing organ damage negatively influenced outcome. Sclerosing cholangitis was the most important risk factor. After 2000, superior OS was achieved with matched donors. Use of myeloablative regimens and HSCT at 2 years or less from diagnosis associated with higher OS and DFS. EFS was best with matched sibling donors, myeloablative conditioning (MAC), and bone marrow-derived stem cells. Most rejections occurred after reduced-intensity or nonmyeloablative conditioning, which associated with poor donor cell engraftment. Mortality occurred mainly early after HSCT, predominantly from infections. Among survivors who ceased immunoglobulin replacement, T-lymphocyte chimerism was 50% or greater donor in 85.2%. CONCLUSION: HSCT is curative in patients with CD40L deficiency, with improved outcome if performed before organ damage development. MAC is associated with better OS, EFS, and DFS. Prospective studies are required to compare the risks of HSCT with those of lifelong supportive therapy.

The management of haemoglobin interference for the MALDI-MSI proteomics analysis of thyroid fine needle aspiration biopsies.

MALDI-MSI represents an ideal tool to explore the spatial distribution of proteins directly in situ, integrating molecular and cytomorphological information, enabling the discovery of potential diagnostic markers in thyroid cytopathology. However, red cells present in the fine needle aspiration biopsy (FNAB) specimens caused ion suppression of other proteins during the MALDI-MSI analysis due to large amount of haemoglobin. Aim of this study was to set up a sample preparation workflow able to manage this haemoglobin interference. Three protocols were compared using ex vivo cytological samples collected from fresh thyroid nodules of 9 patients who underwent thyroidectomy: (A) conventional air-dried smears, (B) cytological smears immediately fixed in ethanol, and (C) ThinPrep liquid-based preparation. Protocols C and A were also evaluated using real FNABs. Results show that protocol C markedly decreased the amount of haemoglobin, with respect to protocols A and B. Protein profiles obtained with protocols A and B were characterised by high inter-patient variability, probably related to the abundance of the haemoglobin, whereas similar spectra were observed for protocol C, where haemoglobin contents were lower. Our findings suggest protocol C as the sample preparation method for MALDI-MSI analysis. Graphical abstract.

Use of Shear Wave Elastography in the Sonographic Triage of Thyroid Nodules: Feasibility Study in a Series of Lesions Already Selected for Fine Needle Aspiration.

OBJECTIVES: The aim of this study was to evaluate the application of shear wave elastography (SWE) in the routine management of thyroid nodules, as a possible additional tool to the standard sonographic triage. METHODS: A total of 248 consecutive patients scheduled for ultrasound-guided thyroid fine-needle aspiration were included in the study. The presence of a pure colloid lesion was an exclusion criterion. Absolute and relative SWE stiffness measurements on color-coded elastograms, expressed in kilopascals and meters per second, were correlated with radiologic and pathologic features. RESULTS: SWE values in thyroid nodules were significantly higher than normal thyroid tissue (P = .0001), proving the different elastic properties of the pathologic tissues. Regarding the radiologic characteristics of the nodules, SWE highest values were associated with the largest lesions (P = .0105) but independent from sonographic and Doppler findings. The SWE elasticity was not influenced by the characteristics of the biopsy smears. The final correlation between the SWE results and the pathologic diagnoses showed a trend in stiffness from tender tumors (follicular adenoma) to papillary thyroid carcinoma (P = .016). CONCLUSIONS: SWE allows the identification of nodules within normal parenchyma; however, the present study does not confirm the potential role in differentiating between benign and malignant thyroid nodules.

Update on: proteome analysis in thyroid pathology - part II: overview of technical and clinical enhancement of proteomic investigation of the thyroid lesions.

INTRODUCTION: An accurate diagnostic classification of thyroid lesions remains an important clinical aspect that needs to be addressed in order to avoid 'diagnostic' thyroidectomies. Among the several 'omics' techniques, proteomics is playing a pivotal role in the search for diagnostic markers. In recent years, different approaches have been used, taking advantage of the technical improvements related to mass spectrometry that have occurred. Areas covered: The review provides an update of the recent findings in diagnostic classification, in genetic definition and in the investigation of thyroid lesions based on different proteomics approaches and on different type of specimens: cytological, surgical and biofluid samples. A brief section will discuss how these findings can be integrated with those obtained by metabolomics investigations. Expert commentary: Among the several proteomics approaches able to deepen our knowledge of the molecular alterations of the different thyroid lesions, MALDI-MSI is strongly emerging above all. In fact, MS-imaging has also been demonstrated to be capable of distinguishing thyroid lesions, based on their different molecular signatures, using cytological specimens. The possibility to use the material obtained by the fine needle aspiration makes MALDI-MSI a highly promising technology that could be implemented into the clinical and pathological units.

GNPAT rs11558492 is not a Major Modifier of Iron Status: Study of Italian Hemochromatosis Patients and Blood Donors.

BACKGROUND AND AIM: HFE-related Hemochromatosis (HH) is characterized by marked phenotype heterogeneity, probably due to the combined action of acquired and genetic factors. Among them, GNPAT rs11558492 was proposed as genetic modifier of iron status, but results are still controversial. To shed light on these discrepancies, we genotyped 298 Italian p.C282Y homozygotes and 169 healthy controls. MATERIAL AND METHODS: Allele and genotype frequencies were analysed and compared with those reported in Exome Variant Server (EVS). To explore the role of rs11558492 as a potential modifier of iron status, serum ferritin (SF), liver iron concentration (LIC) and iron removed (IR) were studied according to allele and genotype frequencies. In addition, the effect of the SNP on liver fibrosis was examined comparing patients with absent/mild-moderate fibrosis to those with severe fibrosis-cirrhosis. RESULTS: GNPAT rs11558492 minor allele (G) frequency (MAF) was 20.3% in HFE-HH, 17.2% in controls and 20.6% in EVS database. Genotype frequencies were 64% and 69.2% (AA), 31.2% and 27.2% (AG), 4.8% and 3.6% (GG) in HFE-HH and controls, respectively. No significant differences were found comparing genotype and allele frequencies even selecting subgroups of only-males with extreme phenotypes and low alcohol intake. SF, IR and LIC levels did not significantly differ according to rs11558492 genotypes. Also, MAF did not differ between patients with absent/mild fibrosis and severe fibrosis/cirrhosis. CONCLUSIONS: Our findings indicate that GNPAT rs11558492 is not a major modifier of iron status and is not associated with liver fibrosis in HFE-HH patients.