The molecular analysis of BRCA1 and BRCA2: Next-generation sequencing supersedes conventional approaches

Abstract Background Accurate and sensitive detection of BRCA 1/2 germ-line mutations is crucial for the clinical management of women affected by breast cancer, for prevention and, notably, also for the identification of at-risk healthy relatives. The most widely used methods for BRCA1 / 2 molecular analysis are Sanger sequencing, and denaturing high performance liquid chromatography (dHPLC) followed by the Sanger method. However, recent findings suggest that next-generation sequencing (NGS)-based approaches may be an efficient tool for diagnostic purposes. In this context, we evaluated the effectiveness of NGS for BRCA gene analysis compared with dHPLC/Sanger sequencing. Methods Seventy women were screened for BRCA1/2 mutations by both dHPLC/Sanger sequencing and NGS, and the data were analyzed using a bioinformatic pipeline. Results Sequence data analysis showed that NGS is more sensitive in detecting BRCA 1/2 variants than the conventional procedure, namely, dHPLC/Sanger. Conclusion Next-generation sequencing is more sensitive, faster, easier to use and less expensive than the conventional Sanger method. Consequently, it is a reliable procedure for the routine molecular screening of the BRCA 1/2 genes

How to Improve the Quality of Life of Patients with Prostate Cancer Treated with Hormone Therapy?

Prostate cancer (PC) is a hormone-sensitive tumor. Androgen deprivation therapy (ADT) is the cornerstone of systemic therapy for patients with intermediate or high-risk localized, recurrent, and metastatic prostate cancer. Although generally well tolerated, ADT can lead to short- and long-term adverse events that can worsen the quality of life of patients with PC. In the last decade, the introduction of novel generation androgen receptor pathway inhibitors (ARPI) has resulted in an improvement in the prognosis of patients with metastatic PC when used in combination with ADT. The use of ARPI in increasingly early stages of the disease determines a longer exposure of patients to these treatments. Although ARPIs are normally well-tolerated drugs, they generally cause an increase in toxicity compared to ADT alone, being able to worsen some adverse events already induced by ADT or leading to the development of specific side effects. Although there are no specific treatments for all the adverse events induced by hormonal therapies, it is essential to know the possible toxicities induced by the different treatments and to start procedures to prevent and/or recognize and consequently treat them early in order to not compromise the quality of life of the patients with PC. The aim of this review is to describe the adverse events induced by hormonal therapies. We will first describe the side effects induced by both ADT and ARPI and then the specific adverse events of the different ARPIs. Furthermore, we will try to highlight the possible therapeutic options to prevent or mitigate the toxicity induced by hormone therapies in order to improve the quality of life of the patients with PC

Ultrasonography Tight Control and Monitoring in Crohn's Disease During Different Biological Therapies: A Multicenter Study

Background & Aims Bowel ultrasonography (BUS) is a noninvasive tool for evaluating bowel activity in Crohn's disease (CD) patients. Aim of our multicenter study was to assess whether BUS helps to monitor intestinal activity improvement/resolution following different biological therapies. Methods Adult CD patients were prospectively enrolled at 16 sites in Italy. Changes in BUS parameters [i.e. bowel wall thickening (BWT), lesion length, echo pattern, blood flow changes and transmural healing (TH: normalization of all BUS parameters)] were analyzed at baseline and after 3, 6 and 12 months of different biological therapies. Results One hundred eighty-eight out of 201 CD patients were enrolled and analyzed (116 males [62%]; median age 36 years). Fifty-five percent of patients were treated with adalimumab, 16% with infliximab, 13% with vedolizumab and 16% with ustekinumab. TH rates at 12 months were 27.5% with an NNT of 3.6. TH at 12 months after adalimumab was 26.8%, 37% after infliximab, 27.2% after vedolizumab and 20% after ustekinumab. Mean BWT improvement from baseline was statistically significant at 3 and 12 months (P Conclusions Data indicate that BUS is useful to monitor biologics-induced bowel activity improvement/resolution in CD

Disease-Modifying Therapies and Coronavirus Disease 2019 Severity in Multiple Sclerosis

Objective: This study was undertaken to assess the impact of immunosuppressive and immunomodulatory therapies on the severity of coronavirus disease 2019 (COVID-19) in people with multiple sclerosis (PwMS). Methods: We retrospectively collected data of PwMS with suspected or confirmed COVID-19. All the patients had complete follow-up to death or recovery. Severe COVID-19 was defined by a 3-level variable: mild disease not requiring hospitalization versus pneumonia or hospitalization versus intensive care unit (ICU) admission or death. We evaluated baseline characteristics and MS therapies associated with severe COVID-19 by multivariate and propensity score (PS)-weighted ordinal logistic models. Sensitivity analyses were run to confirm the results. Results: Of 844 PwMS with suspected (n = 565) or confirmed (n = 279) COVID-19, 13 (1.54%) died; 11 of them were in a progressive MS phase, and 8 were without any therapy. Thirty-eight (4.5%) were admitted to an ICU; 99 (11.7%) had radiologically documented pneumonia; 96 (11.4%) were hospitalized. After adjusting for region, age, sex, progressive MS course, Expanded Disability Status Scale, disease duration, body mass index, comorbidities, and recent methylprednisolone use, therapy with an anti-CD20 agent (ocrelizumab or rituximab) was significantly associated (odds ratio [OR] = 2.37, 95% confidence interval [CI] = 1.18-4.74, p = 0.015) with increased risk of severe COVID-19. Recent use (<1 month) of methylprednisolone was also associated with a worse outcome (OR = 5.24, 95% CI = 2.20-12.53, p = 0.001). Results were confirmed by the PS-weighted analysis and by all the sensitivity analyses. Interpretation: This study showed an acceptable level of safety of therapies with a broad array of mechanisms of action. However, some specific elements of risk emerged. These will need to be considered while the COVID-19 pandemic persists

Extraction and annotation of human mitochondrial genomes from 1000 Genomes Whole Exome Sequencing data

Background Whole Exome Sequencing (WES) is one of the most used and cost-effective next generation technologies that allows sequencing of all nuclear exons. Off-target regions may be captured if they present high sequence similarity with baits. Bioinformatics tools have been optimized to retrieve a large amount of WES off-target mitochondrial DNA (mtDNA), by exploiting the aspecificity of probes, partially overlapping to Nuclear mitochondrial Sequences (NumtS). The 1000 Genomes project represents one of the widest resources to extract mtDNA sequences from WES data, considering the large effort the scientific community is undertaking to reconstruct human population history using mtDNA as marker, and the involvement of mtDNA in pathology. Results A previously published pipeline aimed at assembling mitochondrial genomes from off-target WES reads and further improved to detect insertions and deletions (indels) and heteroplasmy in a dataset of 1242 samples from the 1000 Genomes project, enabled to obtain a nearly complete mitochondrial genome from 943 samples (76% analyzed exomes). The robustness of our computational strategy was highlighted by the reduction of reads amount recognized as mitochondrial in the original annotation produced by the Consortium, due to NumtS filtering. An accurate survey was carried out on 1242 individuals. 215 indels, mostly heteroplasmic, and 3407 single base variants were mapped. A homogeneous mismatches distribution was observed along the whole mitochondrial genome, while a lower frequency of indels was found within protein-coding regions, where frameshift mutations may be deleterious. The majority of indels and mismatches found were not previously annotated in mitochondrial databases since conventional sequencing methods were limited to homoplasmy or quasi-homoplasmy detection. Intriguingly, upon filtering out non haplogroup-defining variants, we detected a widespread population occurrence of rare events predicted to be damaging. Eventually, samples were stratified into blood- and lymphoblastoid-derived to detect possibly different trends of mutability in the two datasets, an analysis which did not yield significant discordances. Conclusions To the best of our knowledge, this is likely the most extended population-scale mitochondrial genotyping in humans enriched with the estimation of heteroplasmies

A multi-parametric workflow for the prioritization of mitochondrial DNA variants of clinical interest

Assigning a pathogenic role to mitochondrial DNA (mtDNA) variants and unveiling the potential involvement of the mitochondrial genome in diseases are challenging tasks in human medicine. Assuming that rare variants are more likely to be damaging, we designed a phylogeny-based prioritization workflow to obtain a reliable pool of candidate variants for further investigations. The prioritization workflow relies on an exhaustive functional annotation through the mtDNA extraction pipeline MToolBox and includes Macro Haplogroup Consensus Sequences to filter out fixed evolutionary variants and report rare or private variants, the nucleotide variability as reported in HmtDB and the disease score based on several predictors of pathogenicity for non-synonymous variants. Cutoffs for both the disease score as well as for the nucleotide variability index were established with the aim to discriminate sequence variants contributing to defective phenotypes. The workflow was validated on mitochondrial sequences from Leber's Hereditary Optic Neuropathy affected individuals, successfully identifying 23 variants including the majority of the known causative ones. The application of the prioritization workflow to cancer datasets allowed to trim down the number of candidate for subsequent functional analyses, unveiling among these a high percentage of somatic variants. Prioritization criteria were implemented in both standalone ( http://sourceforge.net/projects/mtoolbox/ ) and web version ( https://mseqdr.org/mtoolbox.php ) of MToolBox

MToolBox: a highly automated pipeline for heteroplasmy annotation and prioritization analysis of human mitochondrial variants in high-throughput sequencing

Motivation: The increasing availability of mitochondria-targeted and off-targeted sequencing data in Whole Exome and Genome Sequencing studies (WXS and WGS) has risen the demand of effective pipelines to accurately measure heteroplasmy and to easily recognize the most functionally important mitochondrial variants among a huge number of candidates. To this purpose we developed MToolBox, a highly automated pipeline to reconstruct and analyze human mitochondrial DNA from high-throughput sequencing data. Results: MToolBox implements an effective computational strategy for mitochondrial genomes assembling and haplogroup assignment also including a prioritization analysis of detected variants. MToolBox provides a Variant Call Format (VCF) file featuring, for the first time, allele-specific heteroplasmy and annotation files with prioritized variants. MToolBox was tested on simulated samples and applied on 1000 Genomes WXS datasets. Availability: MToolBox package is available at https://sourceforge.net/projects/mtoolbox/

HmtDB 2016: data update, a better performing query system and human mitochondrial DNA haplogroup predictor

The HmtDB resource hosts a database of human mitochondrial genome sequences from individuals with healthy and disease phenotypes. The database is intended to support both population geneticists as well as clinicians undertaking the task to assess the pathogenicity of specific mtDNA mutations. The wide application of next-generation sequencing (NGS) has provided an enormous volume of high-resolution data at a low price, increasing the availability of human mitochondrial sequencing data, which called for a cogent and significant expansion of HmtDB data content that has more than tripled in the current release. We here describe additional novel features, including: (i) a complete, user-friendly restyling of the web interface, (ii) links to the command-line stand-alone and web versions of the MToolBox package, an up-to-date tool to reconstruct and analyze human mitochondrial DNA from NGS data and (iii) the implementation of the Reconstructed Sapiens Reference Sequence (RSRS) as mitochondrial reference sequence. The overall update renders HmtDB an even more handy and useful resource as it enables a more rapid data access, processing and analysis. HmtDB is accessible at http://www.hmtdb.uniba.it/

MRI versus CT and PET/CT in the Preoperative Assessment of Hodgkin and Non-Hodgkin Lymphomas

(1) Background: The purpose of this study is to retrospectively compare CT, MRI, and PET/CT in detecting lymphadenopathies and extra-nodal lesions in lymphoma and in disease staging. (2) Methods: Inclusion criteria were the availability of TB (Total Body) CT and/or PET/CT performed before treatment; MRI performed no later than 2 weeks after TBCT; histological confirmation of lymphoma; clinical-diagnostic follow-up. Using these criteria, we included 64/353 patients with TBCT and MRI performed at our hospital; 20/64 had PET/CT performed in other hospitals. Histology and follow-up were gold standard. (3) Results: The sensitivity, specificity, and accuracy in lymph nodes detection was 84.5%, 94.4%, and 91% for CT and 95%, 98.9%, and 95.6% for MRI. High agreement was observed between CT and MRI regarding the number and size of positive lymph nodes and for disease staging. MRI identified eight more extra-nodal lesions than CT. In the subgroup of 20 patients, PET/CT did not show a significant superiority in sensitivity, specificity, accuracy, and staging ability than CT and MRI. (4) Conclusions: Our study demonstrates a mild superiority of MRI over CT in lymphoma staging. Although PET/CT remains the reference standard, MRI demonstrated a similar diagnostic accuracy, with the added value of being radiation-free