Allele-specific CRISPR-Cas9 editing of dominant epidermolysis bullosa simplex in human epidermal stem cells.

Epidermolysis bullosa simplex (EBS) is a rare skin disease inherited mostly in an autosomal dominant manner. Patients display a skin fragility that leads to blisters and erosions caused by minor mechanical trauma. EBS phenotypic and genotypic variants are caused by genetic defects in intracellular proteins whose function is to provide the attachment of basal keratinocytes to the basement membrane zone and most EBS cases display mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes. Besides palliative treatments, there is still no long-lasting effective cure to correct the mutant gene and abolish the dominant negative effect of the pathogenic protein over its wild-type counterpart. Here, we propose a molecular strategy for EBS01 patient's keratinocytes carrying a monoallelic c.475/495del21 mutation in KRT14 exon 1. Through the CRISPR-Cas9 system, we perform a specific cleavage only on the mutant allele and restore a normal cellular phenotype and a correct intermediate filament network, without affecting the epidermal stem cell, referred to as holoclones, which play a crucial role in epidermal regeneration.

Canonical and uncanonical pathogenic germline variants in colorectal cancer patients by next-generation sequencing in a European referral center.

BACKGROUND: Despite increasing use of next-generation sequencing (NGS), data concerning the gain in germline pathogenic variants (PVs) remain scanty, especially with respect to uncanonical ones. We aimed to verify the impact of different cancer predisposition genes (CPGs) on colorectal cancer (CRC) in patients referred for genetic evaluation. MATERIALS AND METHODS: We enrolled for NGS, by Illumina TruSight Cancer panel comprising 94 CPGs, 190 consecutive subjects referred for microsatellite instability (MSI) CRC, polyposis, and/or family history. RESULTS: Overall, 51 (26.8%) subjects carried 64 PVs; PVs coexisted in 4 (7.8%) carriers. PVs in mismatch repair (MMR) genes accounted for one-third of variant burden (31.3%). Four Lynch syndrome patients (20%) harbored additional PVs (HOXB13, CHEK2, BRCA1, NF1 plus BRIP1); such multiple PVs occurred only in subjects with PVs in mismatch syndrome genes (4/20 versus 0/31; P = 0.02). Five of 22 (22.7%) patients with MSI cancers but wild-type MMR genes harbored PVs in unconventional genes (FANCL, FANCA, ATM, PTCH1, BAP1). In 10/63 patients (15.9%) with microsatellite stable CRC, 6 had MUTYH PVs (2 being homozygous) and 4 exhibited uncanonical PVs (BRCA2, BRIP1, MC1R, ATM). In polyposis, we detected PVs in 13 (25.5%) cases: 5 (9.8%) in APC, 6 (11.8%) with biallelic PVs in MUTYH, and 2 (3.9%) in uncanonical genes (FANCM, XPC). In subjects tested for family history only, we detected two carriers (18.2%) with PVs (ATM, MUTYH). CONCLUSION: Uncanonical variants may account for up to one-third of PVs, underlining the urgent need of consensus on clinical advice for incidental findings in cancer-predisposing genes not related to patient phenotype.

Patient-derived organoids (PDOs) as a novel in vitro model for neuroblastoma tumours.

BACKGROUND: Neuroblastoma (NB) is a paediatric tumour of the sympathetic nervous system. Half of all cases are defined high-risk with an overall survival less than 40% at 5 years from diagnosis. The lack of in vitro models able to recapitulate the intrinsic heterogeneity of primary NB tumours has hindered progress in understanding disease pathogenesis and therapy response. METHODS: Here we describe the establishment of 6 patient-derived organoids (PDOs) from cells of NB tumour biopsies capable of self-organising in a structure resembling the tissue of origin. RESULTS: PDOs recapitulate the histological architecture typical of the NB tumour. Moreover, PDOs expressed NB specific markers such as neural cell adhesion molecules, NB84 antigen, synaptophysin (SYP), chromogranin A (CHGA) and neural cell adhesion molecule NCAM (CD56). Analyses of whole genome genotyping array revealed that PDOs maintained patient-specific chromosomal aberrations such as MYCN amplification, deletion of 1p and gain of chromosome 17q. Furthermore, the PDOs showed stemness features and retained cellular heterogeneity reflecting the high heterogeneity of NB tumours. CONCLUSIONS: We were able to create a novel preclinical model for NB exhibiting self-renewal property and allowing to obtain a reservoir of NB patients' biological material useful for the study of NB molecular pathogenesis and to test drugs for personalised treatments.

Optical Stability of High-translucency Resin-based Composites.

This study investigated the stability of the optical properties of high-translucent shades of dental resin-based composites. Four commercial materials (Filtek Z350 XT, Opallis, Amelogen Plus, and IPS Empress Direct) and 14 non-Vita shades were tested. Disc-shaped specimens for each resin-based composite-shade combination (n=6) were evaluated at T0 (baseline), T1 (after 30 days of storage in water), and T2 (after 30 days of storage each in water and a coffee solution). Color measurements were performed according to the L'C'h' color system. Translucency Parameter (TP) and CIEDE2000 color difference (ΔE00) were calculated. Data were statistically analyzed at α = 0.05. Baseline TP values varied from 43 ± 1 to 55 ± 1. Changes in TP at T1 varied from -3.0% (Opallis T-Neutral) to 4.2% (Amelogen Plus Trans Orange), with no major differences from T0. At T2, most resin-based composites showed significantly increased opacity, with changes varying between -15.0% (Empress Direct Trans 20) and -2.7% (Z350 XT Blue). However, the TP values were ≥40 throughout the study. Storage in water caused negligible color differences, with ΔE00 values at T1 ≤ 0.9 ± 0.6. At T2, all materials tested showed significant color difference, and ΔE00 ≥ 3.2 ± 0.2. The orange shades from Opallis and Amelogen Plus showed lower color variation than did the other shades. The most significant optical changes upon storage were detected in the hue and particularly the chroma color coordinate. In conclusion, the high-translucent resin-based composites showed large variability in the stability of their optical properties among the tested brands and different shades of the same material. Regardless of the storage condition, the tested resin-based composites retained their high-translucency character over time.

Primary mediastinal large B-cell lymphoma: Outcome of a series of pediatric patients treated with high-dose methotrexate and cytarabine plus anti-CD20.

Between 2007 and 2013, 13 children diagnosed with primary mediastinal large B-cell lymphoma (PMLBL) were treated according to a modified version of AIEOP (Associazione Italiana Ematologia Oncologia Pediatrica) LNH-97 protocol based on high-dose methotrexate, anthracyclines, and addition of anti-CD20. Ten patients achieved a continuous complete remission with front-line therapy. The overall 5-year survival was 91.7%, and event-free survival was 83.9%, with only one patient dying of progressive disease. Despite the few cases, these results demonstrate that this therapy, which includes anti-CD20, given in a multicenter setting, is feasible with acceptable toxicity in children with PMLBL.

Masking Colored Substrates Using Monolithic and Bilayer CAD-CAM Ceramic Structures.

OBJECTIVE: To evaluate the masking ability and translucency of monolithic and bilayer CAD-CAM ceramic structures. METHODS: Discs of high translucency (HT) and low translucency (LT) lithium disilicate-based ceramic (IPS e.max CAD) with different thicknesses (0.7, 1, 1.5, and 2 mm) were evaluated as a monolithic structure or combined (bilayer) with a 0.5-mm-thick zirconia framework (IPS e.max ZirCAD). The masking ability and translucency were calculated based on CIE L*a*b* color coordinates measured with a spectrophotometer (SP60, X-Rite). The translucency parameter (TP) was calculated using color coordinates measured over standard white-and-black backgrounds. The masking ability was calculated by CIEDE2000 color difference metric (ΔE00) for each specimen measured over a tooth-colored substrate (shade A2) compared to three darker backgrounds (shade C4 and two metal substrates). Confidence intervals (CI) for the means (95% CI) were calculated for TP and ΔE00. The Pearson correlation between ΔE00 and TP was investigated for monolithic and bilayer structures over all backgrounds. RESULTS: The thinner the lithium disilicate layer, the greater the translucency and the higher the ΔE00 values. The effect of ceramic thickness on both translucency and masking ability was more pronounced for the monolithic structures. In addition, monolayers always presented a greater color variation than their bilayer counterparts. The metallic background produced greater ΔE00 than the C4-shaded substrate. CONCLUSION: Monolithic veneers were able to mask C4-shaded background but did not mask metallic backgrounds. Bilayer structures showed greater shade masking ability than monolithic structures.

Pharmacogenetics and induction/consolidation therapy toxicities in acute lymphoblastic leukemia patients treated with AIEOP-BFM ALL 2000 protocol.

Drug-related toxicities represent an important clinical concern in chemotherapy, genetic variants could help tailoring treatment to patient. A pharmacogenetic multicentric study was performed on 508 pediatric acute lymphoblastic leukemia patients treated with AIEOP-BFM 2000 protocol: 28 variants were genotyped by VeraCode and Taqman technologies, deletions of GST-M1 and GST-T1 by multiplex PCR. Toxicities were derived from a central database: 251 patients (49.4%) experienced at least one gastrointestinal (GI) or hepatic (HEP) or neurological (NEU) grade III/IV episode during the remission induction phase: GI occurred in 63 patients (12.4%); HEP in 204 (40.2%) and NEU in 44 (8.7%). Logistic regression model adjusted for sex, risk and treatment phase revealed that ITPA rs1127354 homozygous mutated patients showed an increased risk of severe GI and NEU. ABCC1 rs246240 and ADORA2A rs2236624 homozygous mutated genotypes were associated to NEU and HEP, respectively. These three variants could be putative predictive markers for chemotherapy-related toxicities in AIEOP-BFM protocols.

Characterization of children with FLT3-ITD acute myeloid leukemia: a report from the AIEOP AML-2002 study group.

Recurrent molecular markers have been routinely used in acute myeloid leukemia (AML) for risk assessment at diagnosis, whereas their post-induction monitoring still represents a debated issue. We evaluated the prognostic value and biological impact of minimal residual disease (MRD) and of the allelic ratio (AR) of FLT3-internal-tandem duplication (ITD) in childhood AML. We retrospectively screened 494 children with de novo AML for FLT3-ITD mutation, identifying 54 harboring the mutation; 51% of them presented high ITD-AR at diagnosis and had worse event-free survival (EFS, 19.2 versus 63.5% for low ITD-AR, <0.05). Forty-one percent of children with high levels of MRD after the 1st induction course, measured by a patient-specific real-time-PCR, had worse EFS (22.2 versus 59.4% in low-MRD patients, P<0.05). Next, we correlated these parameters with gene expression, showing that patients with high ITD-AR or persistent MRD had characteristic expression profiles with deregulated genes involved in methylation and acetylation. Moreover, patients with high CyclinA1 expression presented an unfavorable EFS (20.3 versus 51.2% in low CyclinA1 group, P<0.01). Our results suggest that ITD-AR levels and molecular MRD should be considered in planning clinical management of FLT3-ITD patients. Different transcriptional activation of epigenetic and oncogenic profiles may explain variability in outcome among these patients, for whom novel therapeutic approaches are desirable.

CREB engages C/EBPδ to initiate leukemogenesis.

cAMP response element binding protein (CREB) is frequently overexpressed in acute myeloid leukemia (AML) and acts as a proto-oncogene; however, it is still debated whether such overactivation alone is able to induce leukemia as its pathogenetic downstream signaling is still unclear. We generated a zebrafish model overexpressing CREB in the myeloid lineage, which showed an aberrant regulation of primitive hematopoiesis, and in 79% of adult CREB-zebrafish a block of myeloid differentiation, triggering to a monocytic leukemia akin the human counterpart. Gene expression analysis of CREB-zebrafish revealed a signature of 20 differentially expressed human homologous CREB targets in common with pediatric AML. Among them, we demonstrated that CREB overexpression increased CCAAT-enhancer-binding protein-δ (C/EBPδ) levels to cause myeloid differentiation arrest, and the silencing of CREB-C/EBPδ axis restored myeloid terminal differentiation. Then, C/EBPδ overexpression was found to identify a subset of pediatric AML affected by a block of myeloid differentiation at monocytic stage who presented a significant higher relapse risk and the enrichment of aggressive signatures. Finally, this study unveils the aberrant activation of CREB-C/EBPδ axis concurring to AML onset by disrupting the myeloid cell differentiation process. We provide a novel in vivo model to perform high-throughput drug screening for AML cure improvement.

Reliability and failure behavior of CAD-on fixed partial dentures.

OBJECTIVES: To estimate the reliability and failure behavior of fixed partial dentures (FPDs) fabricated using the CAD-on technique. METHODS: FPDs (n=25) were fabricated using a CAD/CAM system: IPS e.max ZirCAD - Crystall./Connect and IPS e.max CAD (Ivoclar). The restoration type ("three-unit bridge") and design method ("multilayer") based on Biogenerics were used. Framework and porcelain structures were united using a fusion ceramic (Crystall./Connect, Ivoclar). Mechanical fatigue was tested in a servohydraulic load frame machine at a cyclic loading (frequency: 2Hz; load ratio: 0.1). Based on previous data from specimens tested in fast fracture, three different stress profiles were used. The lifetime data were analyzed using an inverse power law-Weibull cumulative damage model (ALTA PRO, Reliasoft). All failed specimens were examined under a field emission scanning electron microscope. RESULTS: Porcelain chipping was the predominant (60%) mode of failure for FPDs tested in fast fracture and connector failure was predominant (67%) under fatigue. For fast fracture data, the Weibull modulus (ß) of FPDs was 7.8 combining the two failure modes. When chipping and connector fracture data were analyzed separately, ß values were 7.9 and 2.9. For the step stress fatigue test, ß values were lower than estimated using fast fracture, being 1.6 for connector fracture and 1.3 for porcelain chipping. SIGNIFICANCE: The test method (fast fracture or fatigue) significantly influenced the reliability of FPDs fabricated using the CAD-on technique, but it did not influence their failure behavior.

An immediate transcriptional signature associated with response to the histone deacetylase inhibitor Givinostat in T acute lymphoblastic leukemia xenografts.

Despite some success with certain hematological malignancies and in contrast with the strong pro-apoptotic effects measured in vitro, the overall response rate of acute lymphoblastic leukemia (ALL) to histone deacetylase inhibitors (HDACis) is low. With the aim to improve the understanding of how HDACis work in vivo, we investigated the therapeutic efficacy of the clinically approved HDACi Givinostat in a collection of nine pediatric human T-ALL engrafted systemically in NOD/SCID mice. We observed highly heterogeneous antileukemia responses to Givinostat, associated with reduction of the percentage of infiltrating blasts in target organs, induction of apoptosis and differentiation. These effects were not associated with the T-ALL cytogenetic subgroup. Transcriptome analysis disclosed an immediate transcriptional signature enriched in genes involved in cell-cycle regulation and DNA repair, which was validated by quantitative RT-PCR and was associated with in vivo response to this HDACi. Increased phospho-H2AX levels, a marker of DNA damage, were measured in T-ALL cells from Givinostat responders. These results indicate that the induction of the DNA damage response could be an early biomarker of the therapeutic effects of Givinostat in T-ALL models. This information should be considered in the design of future clinical trials with HDACis in acute leukemia.

Calcineurin and GSK-3 inhibition sensitizes T-cell acute lymphoblastic leukemia cells to apoptosis through X-linked inhibitor of apoptosis protein degradation.

The calcineurin (Cn)-nuclear factor of activated T cells signaling pathway is critically involved in many aspects of normal T-cell physiology; however, its direct implication in leukemogenesis is still ill-defined. Glycogen synthase kinase-3ß (GSK-3ß) has recently been reported to interact with Cn in neuronal cells and is implicated in MLL leukemia. Our biochemical studies clearly demonstrated that Cn was able to interact with GSK-3ß in T-cell acute lymphoblastic leukemia (T-ALL) cells, and that this interaction was direct, leading to an increased catalytic activity of GSK-3ß, possibly through autophosphorylation of Y216. Sensitivity to GSK-3 inhibitor treatment correlated with altered GSK-3ß phosphorylation and was more prominent in T-ALL with Pre/Pro immunophenotype. In addition, dual Cn and GSK-3 inhibitor treatment in T-ALL cells promoted sensitization to apoptosis through proteasomal degradation of X-linked inhibitor of apoptosis protein (XIAP). Consistently, resistance to drug treatments in primary samples was strongly associated with higher XIAP protein levels. Finally, we showed that dual Cn and GSK-3 inhibitor treatment in vitro and in vivo is effective against available models of T-ALL, indicating an insofar untapped therapeutic opportunity.

Flexural strength and reliability of monolithic and trilayer ceramic structures obtained by the CAD-on technique.

OBJECTIVE: To evaluate the flexural strength, Weibull modulus, fracture toughness, and failure behavior of ceramic structures obtained by the CAD-on technique, testing the null hypothesis that trilayer structures show similar properties to monolithic structures. METHODS: Bar-shaped (1.8mm×4mm×16mm) monolithic specimens of zirconia (IPS e.max ZirCAD - Ivoclar Vivadent) and trilayer specimens of zirconia/fusion ceramic/lithium dissilicate (IPS e.max ZirCAD/IPS e.max CAD Crystall./Connect/IPS e.max CAD, Ivoclar Vivadent) were fabricated (n=30). Specimens were tested in flexure in 37°C deionized water using a universal testing machine at a crosshead speed of 0.5mm/min. Failure loads were recorded, and the flexural strength values were calculated. Fractography principles were used to examine the fracture surfaces under optical and scanning electron microscopy. Data were statistically analyzed using Student's t-test and Weibull statistics (α=0.05). RESULTS: Monolithic and trilayer specimens showed similar mean flexural strengths, characteristic strengths, and Weibull moduli. Trilayer structures showed greater mean critical flaw and fracture toughness values than monolithic specimens (p<0.001). Most critical flaws in the trilayer groups were located on the Y-TZP surface subjected to tension and propagated catastrophically. Trilayer structures showed no flaw deflection at the interface. SIGNIFICANCE: Considering the CAD-on technique, the trilayer structures showed greater fracture toughness than the monolithic zirconia specimens.

Focal nodular hyperplasia of the liver: an emerging complication of hematopoietic SCT in children.

Hepatic focal nodular hyperplasia (FNH) is a nonmalignant condition rarely affecting children previously treated for cancer, especially those who received hematopoietic SCT (HSCT). Some aspects of its pathogenesis still remain unclear and a strong association with specific risk factors has not yet been identified. We report here a single institution's case series of 17 patients who underwent HSCT and were diagnosed with FNH, analyzing retrospectively their clinical features and the radiological appearance of their hepatic lesions. We aimed to compare the diagnostic accuracy of ultrasound (US) and magnetic resonance imaging (MRI) and to explore the role of transient elastography (FibroScan) to evaluate the degree of hepatic fibrosis in FNH patients. Our analysis showed an association of FNH with age at transplant ⩽12 years (hazard ratio (HR) 9.10); chronic GVHD (HR 2.99); hormone-replacement therapy (HR 4.02) and abdominal radiotherapy (HR 4.37). MRI proved to be a more accurate diagnostic tool compared with US. Nine out of 12 patients who underwent FibroScan showed hepatic fibrosis. Our study points out that FNH is an emerging complication of HSCT, which requires a lifelong surveillance to follow its course in cancer patients.