Ovarian endometrioid carcinoma with a sex cord-like pattern: a morphological, immunohistochemical, and molecular analysis.

Sex cord-like endometrioid carcinoma (SCLEC) is an uncommon entity which may constitute a diagnostic challenge. This study aimed to perform a clinicopathological, immunohistochemical, and molecular reappraisal of ovarian SCLEC. Consecutive ovarian SCLECs cases from a single institution were reviewed during a 13-year period. Twenty-three immunohistochemical markers were tested; 10 genes were analyzed by next-generation sequencing. Nine cases of ovarian SCLEC were identified. Mean patient age was 65.7 years; three cases showed extraovarian extension. Architectural pattern included sertoliform (n = 2), granulosa-like (n = 2), and mixed granulosa-like/sertoliform (n = 5). Eosinophilic changes accompanied by increased nuclear atypia were observed in four tumors. Endometrioid features (glands, squamous/morular differentiation) were observed in six cases. Most tumors were positive for cytokeratin-7 (8/9), EMA (9/9), estrogen and progesterone receptor (9/9), CD10 (7/9, including a luminal pattern reminiscent of mesonephric neoplasms), nuclear ß-catenin (8/9), and CDX2 (8/9). A minority of cases showed block-type p16 pattern (2/9), PAX8-positivity (3/9), and non-diffuse positivity for WT1 (1/9), inhibin (1/9), chromogranin (1/9), and synaptophysin (2/9). All cases were negative for GATA3, TTF1, calretinin, and SF1. Ki67 range was 15-90%. Six cases showed CTNNB1 exon 3 mutation. Eight cases were of "no specific molecular profile" (NSMP) and one was p53-abnormal. In conclusion, SCLECs frequently exhibit a mixed sertoliform/granulosa-like architecture and express epithelial markers, hormone receptors, nuclear ß-catenin, and CDX2, with luminal CD10 positivity and CTNNB1 mutations. PAX8 expression is often lost, while other mesonephric, sex cord, and neuroendocrine markers are negative.

Correlation between inflammatory biomarkers and disease control in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) may have a heterogeneous response to medical/surgical treatments based on endotypes. Data correlating biomarkers and severity of the disease are lacking. We aimed to determine if IL-5 and calprotectin may be useful in defining severity of disease and identifying uncontrolled patients. METHODS: This was a case-control study including 81 patients with diffuse CRSwNP who underwent at least one previous surgery and treated with intranasal steroids. We enrolled 39 uncontrolled patients (SNOT-22 ≥ 40 and two or more cycles of systemic corticosteroids in last year) (Group A) and 42 controlled one (SNOT-22 < 40 and less than two cycles of systemic corticosteroids in last year) (Group B). We analyzed IL-5 and calprotectin in both nasal secretions and nasal polyp tissue. RESULTS: Calprotectin and IL-5 were significantly higher in Group A in both secretions and tissue, and the higher the number of previous surgeries, the higher the levels detected in nasal secretions. At univariate analyses, smoking, asthma, non-steroidal anti-inflammatory drugs-exacerbated respiratory disease (NSAID-ERD), blood eosinophilia, neutrophils, and eosinophils at nasal cytology were significantly associated with uncontrolled disease. Multivariate analyses showed that asthma, NSAID-ERD, and IL-5 in nasal secretion/polyp tissue were significantly related to the risk of uncontrolled disease. CONCLUSIONS: Our data suggest that asthma, NSAID-ERD, and IL-5 in nasal secretions/tissue may be helpful to identify more severe patients, as they are related to the risk of uncontrolled disease. Nonetheless, high levels of calprotectin and neutrophilia were also observed in uncontrolled patients, especially after multiple surgeries.

Next-Generation Sequencing for Screening Analysis of Cystic Fibrosis: Spectrum and Novel Variants in a South-Central Italian Cohort.

The incidence of cystic fibrosis (CF) and the spectrum of cystic fibrosis transmembrane conductance regulator (CFTR) gene variants differ among geographic regions. Differences in CF carrier distribution are also reported among Italian regions. We described the spectrum of the CFTR variants observed in a large group of subjects belonging from central-southern Italy. We also provide a predictive evaluation of the novel variants identified. CFTR screening was performed in a south-central Italian cohort of 770 subjects. We adopted a next-generation sequencing (NGS) approach using the Devyser CFTR NGS kit on the Illumina MiSeq System coupled with Amplicon Suite data analysis. Bioinformatics evaluation of the impact of novel variants was described. Overall, the presence of at least one alternative allele in the CFTR gene was recorded for 23% of the subjects, with a carrier frequency of CF pathogenic variants of 1:12. The largest sub-group corresponded to the heterozygous carriers of a variant with a conflicting interpretation of pathogenicity. The common CFTR p.(Phe508del) pathogenic variants were identified in 37% of mutated subjects. Bioinformatics prediction supported a potential damaging effect for the three novel CFTR variants identified: p.(Leu1187Phe), p.(Pro22Thr), and c.744-3C > G. NGS applied to CF screening had the benefit of: effectively identifying asymptomatic carriers. It lies in a wide overview of CFTR variants and gives a comprehensive picture of the carrier prevalence. The identification of a high number of unclassified variants may represent a challenge whilst at the same time being of interest and relevance for clinicians.

Identification and Molecular Characterization of a Novel Large-Scale Variant (Exons 4_18 Loss) in the LDLR Gene as a Cause of Familial Hypercholesterolaemia in an Italian Family.

Next-generation sequencing (NGS) is nowadays commonly used for clinical purposes, and represents an efficient approach for the molecular diagnosis of familial hypercholesterolemia (FH). Although the dominant form of the disease is mostly due to the low-density lipoprotein receptor (LDLR) small-scale pathogenic variants, the copy number variations (CNVs) represent the underlying molecular defects in approximately 10% of FH cases. Here, we reported a novel large deletion in the LDLR gene involving exons 4-18, identified by the bioinformatic analysis of NGS data in an Italian family. A long PCR strategy was employed for the breakpoint region analysis where an insertion of six nucleotides (TTCACT) was found. Two Alu sequences, identified within intron 3 and exon 18, could underlie the identified rearrangement by a nonallelic homologous recombination (NAHR) mechanism. NGS proved to be an effective tool suitable for the identification of CNVs, together with small-scale alterations in the FH-related genes. For this purpose, the use and implementation of this cost-effective, efficient molecular approach meets the clinical need for personalized diagnosis in FH cases.

High-grade serous ovarian carcinoma with a sertoliform pattern associated with BRCA mutation: a clinicopathological and molecular analysis.

Herein, we report a clinicopathological and molecular analysis of a case of tubo-ovarian high-grade serous carcinoma (HGSC) with a sertoliform pattern. A 45-year-old woman underwent surgery due to an advanced bilateral adnexal carcinoma with peritoneal and appendiceal metastases. Histological examination revealed an HGSC exhibiting a distinct sertoliform component. Such component showed diffuse PAX8, p53 (mutation-type), and p16 (block-type) expression, increased vimentin and decreased WT1 expression compared to the conventional HGSC component, membrane ß-catenin positivity, heterogeneous estrogen, and progesterone positivity, and retained PTEN and mismatch repair expression and negativity for GATA3, TTF1, inhibin, calretinin, CD10, CDX2, chromogranin, and synaptophysin. Molecular analysis showed a germline BRCA2 mutation; no mutations were detected in POLE, POLD1, MLH1, MSH2, MSH6, PMS2, APC, CTNNB1, MUTYH, and EPCAM. In conclusion, a sertoliform pattern can be part of the morphological spectrum of BRCA-related HGSC.

Corded and hyalinized endometrioid endometrial carcinoma with high-grade features: a clinicopathological and TCGA-based molecular analysis.

Corded and hyalinized endometrioid carcinoma (CHEC) typically shows low-grade features and "no specific molecular profile" (NSMP). This study aimed to perform a clinicopathological and molecular characterization of endometrial CHEC with high-grade features. Immunohistochemistry for cytokeratin AE1/AE3, e-cadherin, ß-catenin, estrogen receptor, progesterone receptor, p53, p16, and mismatch repair proteins was performed. A next-generation sequencing kit was used to assess POLE, POLD1, APC, MLH1, MSH2, MSH6, PMS2, MUTYH, EPCAM, and CTNNB1. Molecular groups, i.e., POLE-mutant, mismatch repair deficient (MMRd), p53-abnormal, and NSMP, were assigned according to the TCGA classifier. Six high-grade endometrial CHECs were identified. The mean age was 57.5 years; 5/6 cases were uterine-confined. Five cases showed a diffusely and markedly atypical corded component and a MMRd or p53-abnormal signature; additional features included single-cell keratinization, necrosis, osteoid or myxoid/chondro-myxoid matrix, foci of anaplasia, and nuclear ß-catenin expression. The remaining case showed a low mitotic count and a NSMP phenotype, with focal bizarre cells in an otherwise classical CH endometrioid carcinoma. All cases showed variably reduced expression of epithelial markers and hormone receptors in the corded component. No mutations were found in any of the analyzed genes. In conclusion, high-grade CHECs are a heterogeneous subset of biphasic endometrial carcinoma which show similarities and differences with classical CHEC and carcinosarcoma. These cases often show MMRd or p53-abnormal signatures.

A Computational Framework for Comprehensive Genomic Profiling in Solid Cancers: The Analytical Performance of a High-Throughput Assay for Small and Copy Number Variants.

In January 2022, our institution launched a comprehensive cancer genome profiling program on 10 cancer types using a non-IVD solution named the TruSight Oncology 500 Assay provided by Illumina®. The assay analyzes both DNA and RNA, identifying Single-Nucleotide Variants (SNV)s and Insertion-Deletion (InDel) in 523 genes, as well as known and unknown fusions and splicing variants in 55 genes and Copy Number Alterations (CNVs), Mutational Tumor Burden (MTB) and Microsatellite Instability (MSI). According to the current European IVD Directive 98/79/EC, an internal validation was performed before running the test. A dedicated open-source bioinformatics pipeline was developed for data postprocessing, panel assessment and embedding in high-performance computing framework using the container technology to ensure scalability and reproducibility. Our protocols, applied to 71 DNA and 64 RNA samples, showed full agreement between the TruSight Oncology 500 assay and standard approaches, with only minor limitations, allowing to routinely perform our protocol in patient screening.

Sinonasal Biomarkers Defining Type 2-High and Type 2-Low Inflammation in Chronic Rhinosinusitis with Nasal Polyps.

The complex pathophysiology of chronic rhinosinusitis with nasal polyps (CRSwNP) generates a spectrum of phenotypes with a wide variety of inflammatory states. We enrolled 44 very-likely-to-be type 2 CRSwNP patients in order to evaluate the load of inflammation and to analyze human interleukins in nasal secretion. Clinical data were collected to evaluate the severity of the disease. High levels of IL-5, IL-4, IL-6, and IL-33 were detected in all type 2 CRSwNP patients. By analyzing type 2 cytokine profiles and local eosinophil count, we identified two coherent clusters: the first was characterized by high levels of IL-4, IL-5, IL-6, and a high-grade eosinophil count (type 2-high); the second had lower levels of cytokines and poor or absent eosinophilic inflammation (type-2 low). IL-5 levels were significantly higher within the type 2 cytokine and it was the most reliable biomarker for differentiating the two clusters. In type 2-high inflammatory profile clinical scores, the mean number of previous surgeries and need for systemic corticosteroids were significantly higher compared to type 2-low. Our research demonstrated the potential role of type 2 biomarkers, and in particular, of IL-5 in identifying patients with a more severe phenotype based on a high inflammatory load.

Calprotectin in nasal secretion: a new biomarker of non-type 2 inflammation in CRSwNP.

Objective: We analysed calprotectin in sinonasal secretions of different chronic rhinosinusitis with nasal polyps (CRSwNP) endotypes to assess its role as a biomarker of non-type 2 inflammation. Methods: We included primary diffuse CRSwNP patients (n = 41) and three different control groups [non-allergic rhinitis (NAR) (n = 13), non-allergic eosinophilic syndrome (NARES) (n = 10) and healthy subjects (n = 12)]. Calprotectin levels were detected in nasal secretions using a chemiluminescent immunoassay (CLIA). Results: Calprotectin levels in nasal secretions were significantly higher in all non-type 2 endotypes of CRSwNP compared to healthy controls (p < 0.05). In contrast, in type-2 CRSwNP calprotectin was significantly lower compared to controls (p < 0.05). A significant correlation between calprotectin levels and neutrophilic count/HPF was found in CRSwNP (p < 0.01). Clinically, mean levels of calprotectin and neutrophilia were significantly higher in patients who previously underwent 3 or more endoscopic sinus surgeries (p < 0.05). Conclusions: Calprotectin in nasal secretions may be a biomarker of non-type 2 inflammation. Low levels of calprotectin are indicative of a type-2 immune response in both CRSwNP and non-allergic rhinitis. We observed that calprotectin levels significantly increased based on the number of previous surgeries.

The Relationship between ACE, ACTN3 and MCT1 Genetic Polymorphisms and Athletic Performance in Elite Rugby Union Players: A Preliminary Study.

Athletic performance is influenced by many factors such as the environment, diet, training and endurance or speed in physical effort and by genetic predisposition. Just a few studies have analyzed the impact of genotypes on physical performance in rugby. The aim of this study was to verify the modulation of genetic influence on rugby-specific physical performance. Twenty-seven elite rugby union players were involved in the study during the in-season phase. Molecular genotyping was performed for: angiotensin-converting enzyme (ACE rs4646994), alfa-actinin-3 (ACTN3 rs1815739) and monocarboxylate transporter 1 (MCT1 rs1049434) and their variants. Lean mass index (from skinfolds), lower-limb explosive power (countermovement jump), agility (505), speed (20 m), maximal aerobic power (Yo-yo intermittent recovery test level 1) and repeated sprint ability (12 × 20 m) were evaluated. In our rugby union players ACE and ACTN3 variants did not show any influence on athletic performance. MCT1 analysis showed that TT-variant players had the highest peak vertical power (p = 0.037) while the ones with the AA genotype were the fastest in both agility and sprint tests (p = 0.006 and p = 0.012, respectively). Considering the T-dominant model, the AA genotype remains the fastest in both tests (agility: p = 0.013, speed: p = 0.017). Only the MCT1 rs1049434 A allele seems to be advantageous for elite rugby union players, particularly when power and speed are required.

Genetics and Sport Injuries: New Perspectives for Athletic Excellence in an Italian Court of Rugby Union Players.

Several genes are involved in sport performance, especially in injuries incidence. The aim of this study was to investigate the association of ACE, ACTN3, COL1A1, and MCT1 genotypes and injuries in rugby players in order to find a genotype/phenotype correlation and provide useful information improving athletic performance. One-hundred male professional and semiprofessional rugby players were selected. Analysis was performed genotyping the genes ACE, ACTN3, COL1A1, and MCT1 as candidate gene of interest involved in athletic performance. A control group of non-athletic Italian male participants was analyzed to compare the results. We found statistical significance of MCT1 rs1049434 AA for total injuries (χ2 = 0.115; p = 0.003) and bone injuries (χ2 = 0.603; p = 0.007) in the rugby athlete population. No statistical significance was found between injury incidence and ACE, ACTN3, COL1A1 genotypes. The MCT1 AA genotype is associated with the incidence of total and bone injuries in the rugby player population. Although environmental factors such as lifestyle, diet, training, and stress can influence athletic performance, our data demonstrated the importance of genetic study in sport aimed at developing personalized training and achieving the best possible athletic excellence.

A novel low-density lipoprotein receptor variant in a Ukrainian patient: a case report and overview of the disease-causing low-density lipoprotein receptor variants associated to familial hypercholesterolemia.

BACKGROUND: Familial hypercholesterolemia (FH) is characterized by high low-density lipoprotein-cholesterol levels and it is primarily caused by pathogenic/likely pathogenic variants (P/LPVs) in LDLR, APOB or PCSK9 genes. Next generation sequencing (NGS) technology allows the evaluation of more genes simultaneously, rising the diagnostic throughput of genomics laboratories. MATERIALS AND METHODS: We report a Ukrainian 37-year-old woman hypercholesterolemic since 2010. Despite a suggestive family history, FH was suspected only when the patient referred to the Endocrine and Metabolic Diseases Center of the Fondazione Policlinico Universitario A. Gemelli IRCCS in Rome. After specialist advice, genetic testing was offered to the patient at our Molecular and Genomic Diagnostics Unit. RESULTS: A targeted NGS-based pipeline highlighted a novel out-of-frame deletion in the LDLR gene. This variant has a clear deleterious effect on the LDLR protein and it can be classified as PV. CONCLUSIONS: The ideal model of care for FH is an evidence-based system aimed to provide the highest-quality health services to all FH patients. In fact, this study reports that the integrated care pathway adopted in our hospital for FH patients led successfully to the discovery of a novel LDLR PV in an Ukrainian patient. The finding of this LDLR variant allowed the clinical FH diagnosis in this patient and in her family, expanding the knowledge of FH-related genetic variants in the Ukrainian population.

Tumor BRCA testing in ovarian cancer and EQA scheme: our experience of a critical evaluation.

Next generation sequencing (NGS) is a widespread molecular biology method integrated into clinical practice to detect genetic variants, for diagnostic and prognostic purposes. The scheduled external quality assessments (EQA) is integral part of clinical molecular laboratory quality assurance. The EQA provides an efficient system to compare analytic test performances among different laboratories, which is essential to evaluate consistency of molecular test. EQA failures demands targeted corrective action plans. In this context, the complexity of the NGS techniques requires careful and continuous quality control procedures. We report a tumor BRCA1/2 (tBRCA) testing benchmark discrepancy provided by the European Molecular Genetics Quality Network in our laboratory during a round of EQA for somatic mutation testing of BRCA genes in relation to ovarian cancer. The critical analysis emerging from the tBRCA EQA is presented. We underline that harmonization processes are still required for the EQA in the molecular biology field, especially if applied to the evaluation of methods characterized by high complexity.

Duplex high resolution melting analysis (dHRMA) to detect two hot spot CYP24A1 pathogenic variants (PVs) associated to idiopathic infantile hypercalcemia (IIH).

Pathogenic variants (PVs) in CYP24A1 gene are associated with Idiopathic Infantile Hypercalcemia disease (IIH). The identification of CYP24A1 PVs can be a useful tool for the improvement of target therapeutic strategies. Aim of this study is to set up a rapid and inexpensive High Resolution Melting Analysis (HRMA)-based method for the simultaneous genotyping of two hot spot PVs in CYP24A1 gene, involved in IIH. A duplex-HRMA (dHRMA) was designed in order to detect simultaneously CYP24A1 c.428_430delAAG, p.(Glu143del) (rs777676129) and c.1186C > T, p.(Arg396Trp) (rs114368325), in peculiar cases addressed to our Laboratory. dHRMA was able to identify clearly and simultaneously both hot spot CYP24A1 PVs evaluating melting curve shape and melting temperature (Tm). This is the first dHRMA approach to rapidly screen the two most frequent CYP24A1 PVs in peculiar case, providing useful information for diagnosis and patient management in IIH disease.

Glucose-6-phosphate dehydrogenase deficiency and hydroxychloroquine in the COVID-19 era: a mini review.

The coronavirus disease 2019 (COVID-19) is until today a global health emergency. In an immense effort, effective drugs against COVID-19 are searched and intensive researches on possible repurposing of antiviral agents are performed. Since chloroquine (CQ) and hydroxychloroquine (HCQ) have shown in vitro anti- COVID-19 activities, the potential effect of CQ/HCQ to treat and/or prevent COVID-19 infection has caused global attention. However, concern regarding possible hemolysis in G6PD-deficient COVID-19 patients exists and for this reason, the association between HCQ and G6PD deficiency (G6PDD) is back in the limelight. This study aims to answer the question raised by Mastroianni et al. "Hydroxychloroquine: Culprit or Innocent Bystander in G6PD-Deficient Patients with COVID-19?", reporting all cases of HCQ in G6PD deficient COVID-19 patients published on PubMed (pubmed.ncbi.nlm.nih.gov), in addition to the Mastroianni's patient. In our opinion, after an accurate revision of these cases and responding the question raised by Mastroianni et al., we believe that it is difficult to reach a final verdict about the definitive role of HCQ in these patients. The COVID-19 pandemic has reopened attention on HCQ use and G6PDD. G6PD status is extremely important in modulating the level of reactive oxygen species and many cellular immune responses such as enhanced production of the pro-inflammatory cytokine and inflammasome activation. Since these processes are involved in COVID-19 infection, acute hemolytic anemia, a severe complication of the G6PDD, can occur in these patients. In this context, the role of HCQ, usually effective, safe, and well tolerated in G6PD deficient patients, must be redefined in these patients with COVID-19.As consequence, answering the question: "Hydroxychloroquine: Culprit or Innocent Bystander in G6PD-Deficient Patients with COVID-19?", we state that it is risky to believe that HCQ may be an "innocent bystander" in G6PD-deficient COVID-19 patients.

Identification of two novel LDLR variants by Next Generation Sequencing.

INTRODUCTION: Familial hypercholesterolemia (FH) is an autosomal dominant inherited disease characterized by elevated plasma low-density lipoprotein cholesterol (LDL-C). Targeted Next Generation Sequencing (NGS) is a new opportunity to expand the existing pathogenic variants (PVs) spectrum associated to FH. Our aim was to report a diagnostic NGS-based approach to detect variants associated to FH. METHODS: We report two patients: a 48-year-old Asian woman, without known history of hypercholesterolemia and a 46-year-old Caucasian man, with childhood hypercholesterolemia. RESULTS: An effective NGS-based pipeline, FH-Devyser kit/Amplicon Suite, beginning from sequencing to data analysis, did not identify known PVs in the LDLR, APOB, APOE, LDLRAP1, STAP1 and PCSK9 genes, but revealed two novel LDLR variants (c.1564A>T, p.Ile522Phe and c.1688C>T, p.Pro563Leu). DISCUSSION AND CONCLUSIONS: This study showed that an effective NGS-based pipeline led to a definitive diagnosis in two FH families, allowing to plan their therapeutic treatment. Although the functional consequence of the two LDLR variants needs to be assessed in vitro, the in silico analysis and high preservation of the two amino acid positions observed in the LDLR protein, across different animal species, suggest that both variants are deleterious.