Targeted RNA-sequencing analysis for fusion transcripts detection in tumor diagnostics: assessment of bioinformatic tools reliability in FFPE samples.

Aim: Diagnostic laboratories are progressively introducing next-generation sequencing (NGS) technologies in the routine workflow to meet the increasing clinical need for comprehensive molecular characterization in cancer patients for diagnosis and precision medicine, including fusion-transcripts detection. Nevertheless, the low quality of messenger RNA (mRNA) extracted from formalin-fixed paraffin-embedded (FFPE) samples may affect the transition from traditional single-gene testing approaches [like fluorescence hybridization (FISH), immunohistochemistry (IHC), or polymerase chain reaction (PCR)] to NGS. The present study is aimed at assessing the overall accuracy of RNA fusion transcripts detection by NGS analysis in FFPE samples in real-world diagnostics.

Diagnosis, treatment, and follow-up of a case of Wolman disease with hemophagocytic lymphohistiocytosis.

Wolman Disease (WD) is a severe multi-system metabolic disease due to lysosomal acid lipase (LAL) deficiency. We report on a WD infant who developed an unusual hemophagocytic lymphohistiocytosis (HLH) phenotype related to WD treated with sebelipase alfa. A male baby came to our attention at six months of life for respiratory insufficiency and sepsis, abdominal distension, severe hepatosplenomegaly, diarrhea, and severe growth retardation.

Use of DPB1 T-cell epitope algorithm among italian transplant centers: A survey on behalf of Associazione Italiana di Immunogenetica e Biologia dei Trapianti.

The HLA-DPB1 locus has been demonstrated to have a significant role on patients' outcome after allogeneic HSCT, and the so-called T-cell epitope (TCE) algorithm has been incorporated in international guidelines for the selection of unrelated donors. The purpose of the present study is to measure, through a national survey conducted on behalf of the Associazione Italiana di Immunogenetica e Biologia dei Trapianti (AIBT), the extent of awareness and use of HLA-DPB1 TCE-based algorithms during the donor search. 89% of the HLA laboratories answered to a short questionnaire and the results showed a progressive increase of the laboratories typing DPB1 in patients and their potential donors during the search (from 44% to 79% during the 2010-2019 period) as well as the application of a TCE-based algorithm for the donor choice whenever possible (from 24% to 65% during the same period).

A new case of myelodysplastic syndrome associated with t(3;3)(q21;q26) and inv(11)(p15q22).

Introduction: Myeloid malignancies are associated with a number of recurrent and sporadic rearrangements that may be oncogenic by ensuring growth advantage and/or increased survival. t(3;3)(q21;q26) has been recognized as a recurrent abnormality in myelodysplastic syndromes (MDS) with poor prognostic significance. Inversion of chr(11) engendering NUP98-DDX10 chimeric product is sporadic and usually associated with diseases with poor prognosis (therapy-related myeloid neoplasm).

Ucuùba () Fat-Based Nanostructured Lipid Carriers for Nail Drug Delivery of Ketoconazole: Development and Optimization Using Box-Behnken Design.

Ucuùba fat is fat obtained from a plant found in South America, mainly in Amazonian Brazil. Due to its biocompatibility and bioactivity, Ucuùba fat was used for the production of ketoconazole-loaded nanostructured lipid carriers (NLC) in view of an application for the treatment of onychomycosis and other persistent fungal infections. The development and optimization of Ucuùba fat-based NLC were performed using a Box-Behnken design of experiments.

Thymomatous myasthenia gravis: novel association with HLA DQB1*05:01 and strengthened evidence of high clinical and serological severity.

Background: The relative prevalence of myasthenia gravis (MG) subtypes is changing, and their differential features and association with HLA class II alleles are not completely understood.

Methods: Age at onset, presence/absence of autoantibodies (Ab) and thymoma were retrospectively considered in 230 adult Italian patients. Clinical severity, assessed by MGFA scale, and the highest Ab titer were recorded.

A new HLA-C*04 variant, HLA-C*04:01:106, discovered in an Italian hematopoietic stem cell donor.

HLA-C*04:01:106 differs from C*04:01:01:01 by a silent nucleotide substitution in exon 4.

Full-length extension of HLA allele sequences by HLA allele-specific hemizygous Sanger sequencing (SSBT).

The gold standard for typing at the allele level of the highly polymorphic Human Leucocyte Antigen (HLA) gene system is sequence based typing. Since sequencing strategies have mainly focused on identification of the peptide binding groove, full-length sequence information is lacking for >90% of the HLA alleles. One of the goals of the 17th IHIWS workshop is to establish full-length sequences for as many HLA alleles as possible.

The HLA-B landscape of Africa: Signatures of pathogen-driven selection and molecular identification of candidate alleles to malaria protection.

Human leukocyte antigen (HLA) genes play a key role in the immune response to infectious diseases, some of which are highly prevalent in specific environments, like malaria in sub-Saharan Africa. Former case-control studies showed that one particular HLA-B allele, B*53, was associated with malaria protection in Gambia, but this hypothesis was not tested so far within a population genetics framework. In this study, our objective was to assess whether pathogen-driven selection associated with malaria contributed to shape the HLA-B genetic landscape of Africa.

Identification of the new HLA-DQA1*01:15N allele in an Italian patient.

The novel allele DQA1*01:15N differs from DQA1*01:03:01:01 by 1 nucleotide substitutions in exon 2.

Identification of the new C*07:555 allele in an unrelated donor for HSCT.

HLA-C*07:555 differs from C*07:01:01:01 by a C to G substitution in exon 2.

Characterization of the novel HLA-C*02:106 allele in a hematopoietic stem cell volunteer donor.

HLA-C*02:106 allele differs from C*02:02:02:01 by a C to T substitution in exon 4.

Deciphering intra-tumor heterogeneity of lung adenocarcinoma confirms that dominant, branching, and private gene mutations occur within individual tumor nodules.

While pulmonary adenocarcinoma (ADC) is morphologically heterogeneous, little is known about intra-tumor gene mutation heterogeneity (ITH). We therefore subjected 20 ADC nodules, 5 mutated for EGFR and 5 for KRAS, 5 with an ALK translocation, and 5 wild type (WT) for these alterations, to unsupervised next-generation sequencing of tumor regions from diverse architectural patterns. When 2 or more different gene mutations were found in a single tumor, this fulfilled the criteria for ITH.

HLA-C*18:01: A Rare Allele in the European Caucasian Population Coinciding with Difficult-to-Treat Plaque Psoriasis.

Background: Advances in knowledge about the metabolic pathways involved in the pathogenesis of psoriasis and related diseases have led to a search for new therapeutic targets and the development of new biological drugs. Several studies have focused on HLA-C*06 and investigated correlations between the genetic risk factors of psoriasis and clinical parameters such as the severity of the disease and the response to treatment.

Objective: Our objective is to share experience from our institution in the observation of two patients with severe chronic plaque psoriasis who were unresponsive to any anti-TNF-α treatment and only partially responsive to ustekinumab.

Doing more with less: fluorescence in situ hybridization and gene sequencing assays can be reliably performed on archival stained tumor tissue sections.

Little is known about molecular testing on tumor tissue retrieved from stained sections, for which there may be a clinical need. We retrospectively analyzed 112 sections from 56 tumor patients using either fluorescence in situ hybridization (FISH) with different probes (19 sections from 17 patients) or Sanger or targeted next generation sequencing for detection of BRAF, EGFR, KRAS, C-KIT, and TP53 mutations (93 sections from 39 patients). Tumor tissue sections had been stained by hematoxylin and eosin (H&E) (42 sections) or by immunohistochemistry for cytoplasmic or nuclear/nuclear-cytoplasmic markers (70 sections) with a peroxidase (P-IHC, with 3,3'-diaminobenzidine as chromogen) or alkaline phosphatase label (AP-IHC, with Warp Red™ as chromogen).

A nucleotide insertion in Exon 2 is responsible for a new HLA-DRB1 null allele, HLA-DRB1*14:166N.

DRB1*14 is identical to DRB1*14:54:01 except for a single nucleotide insertion of A in position 175 in Exon 2.

Mixed chimerism evolution is associated with T regulatory type 1 (Tr1) cells in a β-thalassemic patient after haploidentical haematopoietic stem cell transplantation.

In a cohort of β-Thalassemia (β-Thal) transplanted with haploidentical-HSCT we identified one transplanted patient characterized by persistent mixed chimerism (PMC) for several months after HSCT. In this unique β-Thal patient we assessed the donor engraftment overtime after transplantation, the potential loss of the non-shared HLA haplotype, and the presence of CD49b(+)LAG-3(+) T regulatory type 1 (Tr1) cells, previously demonstrated to be associated with PMC after HLA-related HSCT for β-Thal. The majority of the patient's erythrocytes were of donor origin, whereas T cells were initially mostly derived from the recipient, no HLA loss, but an increased frequency of circulating Tr1 cells were observed.

Optimal Outcomes in Young Class 3 Patients With Thalassemia Undergoing HLA-Identical Sibling Bone Marrow Transplantation.

Background: Bone marrow transplantation (BMT) for class 3 patients with thalassemia is challenging due to high rates of graft rejection and transplant-related mortality. Since the first studies of BMT in the late 1980s, a number of conditioning regimens have been designed to improve outcomes, but with suboptimal results. Here we report the outcome of transplantation in class 3 patients using a modified protocol.

Dissecting Pulmonary Large-Cell Carcinoma by Targeted Next Generation Sequencing of Several Cancer Genes Pushes Genotypic-Phenotypic Correlations to Emerge.

Introduction: Little is known about genotypic and phenotypic correlations in undifferentiated large-cell carcinoma (LCC) of the lung.

Methods: Thirty LCC were dissected by unsupervised targeted next generation sequencing analysis for 50 cancer-associated oncogenes and tumor suppressor genes. Cell differentiation lineages were unveiled by using thyroid transcription factor-1 (TTF1) for adenocarcinoma (ADC) and p40 for squamous cell carcinoma (SQC), dichotomizing immunohistochemistry (IHC) results for TTF1 as negative or positive (whatever its extent) and for p40 as negative, positive, or focal (if <10% of reactive tumor cells).

HLA-A-B-C-DRB1-DQB1 phased haplotypes in 124 Nigerian families indicate extreme HLA diversity and low linkage disequilibrium in Central-West Africa.

The simultaneous typing of five-HLA loci at high resolution and the availability of pedigree data allowed us to characterize extended five-locus phased haplotypes in 124 Nigerian families and to compare the observed frequencies with those expected by an expectation-maximization algorithm for unphased data. Despite the occurrence of some frequent alleles at each locus (e.g.

Sequence-based typing characterization of the novel HLA-DRB3*01:16 allele, identified in an Italian family.

The novel DRB1*01:16 allele differs for G to T substitution at position 595 from DRB3*01:01P.

Haploidentical, G-CSF-primed, unmanipulated bone marrow transplantation for patients with high-risk hematological malignancies: an update.

Ninety-seven patients affected by high-risk hematological malignancies underwent G-CSF primed, unmanipulated bone marrow (BM) transplantation from a related, haploidentical donor. All patients were prepared with an identical conditioning regimen including Thiotepa, Busilvex, Fludarabine (TBF) and antithymocyte globulin given at myeloablative (MAC = 68) or reduced (reduced intensity conditioning (RIC) = 29) dose intensity and received the same GvHD prophylaxis consisting of the combination of methotrexate, cyclosporine, mycofenolate-mofetil and basiliximab. Patients were transplanted in 1st or 2nd CR (early phase: n = 60) or in > 2nd CR or active disease (advanced phase: n = 37).

Luminex-Based Methods in High-Resolution HLA Typing.

Luminex-based technology has been applied to discriminate between the different Human Leukocyte Antigens (HLA) alleles. The typing method consists in a reverse-SSO assay: Target DNA is PCR-amplified using biotinylated group-specific primers. A single PCR reaction is used for each HLA locus.

Efficacy of non-invasive mechanical ventilation in the general ward in patients with chronic obstructive pulmonary disease admitted for hypercapnic acute respiratory failure and pH < 7.35: a feasibility pilot study.

Aim: To date non-invasive (NIV) mechanical ventilation use is not recommended in chronic obstructive pulmonary disease (COPD) patients with acute respiratory failure (ARF) and pH < 7.30 outside a 'protected environment'. We assessed NIV efficacy and feasibility in improving arterial blood gases (ABG) and in-hospital outcome in patients with ARF and severe respiratory acidosis (RA) admitted to an experienced rural medical ward.