The dynamics of circulating tumour DNA (ctDNA) during treatment reflects tumour response in advanced melanoma patients

Despite the introduction of targeted (BRAFi/MEKi) and immune checkpoint inhibitors (ICIs) has significantly reduced the recurrence rate and improved the overall survival (OS) of patients with Stage III and IV melanoma, only a percentage will benefit of durable disease control. The aim of this study was to examine whether the levels of circulating tumour DNA (ctDNA) in plasma of advanced melanoma patients undergoing BRAFi/MEKi or ICIs vary according to the patients' survival outcomes (i.e. progression-free survival (PFS) and OS) and disease progression. Plasma samples of Stage III-IV melanoma patients were collected at baseline (treatment initiation) and thereafter every 3 months. Circulating BRAFV600E/K and NRASQ61R/K mutations were analysed through droplet digital PCR (ddPCR, Bio-Rad) in a total of 177 plasma samples from 48 melanoma patients (19 Stage III, 29 Stage IV). Baseline ctDNA concentration was significantly associated with OS (HR = 1.003, 95% CI = 1.000–1.006, p = 0.043) and PFS (HR = 1.004, 95% CI = 1.000–1.007, p = 0.029) independent of clinical-prognostic confounders. For each unit increase in the ∆ctDNA (concentration difference between the last follow-up and baseline) there was a 24% increased risk of disease progression, irrespective of treatment type and stage at diagnosis (OR = 1.24, 95% CI = 1.03–1.49, p = 0.020, AUC = 0.93). Patients with reduction of ctDNA level from baseline to the last follow-up had longer OS (HR = 0.14; 95% CI = 0.05–0.44, p = 0.001) and PFS (HR = 0.08; 95% CI = 0.03–0.27, p < 0.0001) compared to patients with increased ctDNA, including adjustment for confounding factors. Our findings suggest that variation of ctDNA over time during melanoma treatment reflects the clinical outcome and tumour response to therapy and might be helpful in clinical monitoring

Peptidomics characterization of allergenic and non-allergenic tropomyosin orthologs

Background The cleavage and the digestion patterns of allergenic proteins play a key role in allergenicity. The transformation of food proteins starts with their denaturation by the acidic environment of the stomach. However, protein denaturation is not enough to completely remove allergenic properties of a protein, but it is necessary a complete enzymatic digestion. Whether the enzymatic digestion is not efficient, the persistence of bigger peptides can occur and put the basis for the development of the sensitization process.The hypothesis of the current work takes into consideration the probability that shrimp tropomyosin (TM) is not fully digested or presents a digestion pattern that generates some peptides that can be immunogenic.Therefore, the work plan designed, aims to study the cleavage pattern of: purified chicken TM, recombinant chicken TM, purified TM of Penaeus monodon (Pen m 1), recombinant TM of Penaeus monodon (rPen m 1) and recombinant TM of Crangon crangon (rCrac c 1). Methods One mg of each ortholog has been processed through simulated mouth, gastric and intestinal digestion. The sample was frozen to block the digestion and, after this step, concentrated and cleaned through protein precipitation. The protein pellet was processed for peptidomic analysis through 1D Tricine gel electrophoresis, 2D Tricine gel electrophoresis and mass spectrometry. Results Simulated gastric digestion pattern of shrimp TM highlighted the presence of a resistant band at an average MW of 25 kDa that could be involved in the immunogenic process. Conclusions This innovative approach (peptidomics study through 1D-2D Tricine/MS) could represent a milestone for the study of digestion patterns of allergenic proteins or for the study of allergenic potential of novel foods

Silver diagnosis in neuropathology: principles, practice and revised interpretation

Silver-staining methods are helpful for histological identification of pathological deposits. In spite of some ambiguities regarding their mechanism and interpretation, they are widely used for histopathological diagnosis. In this review, four major silver-staining methods, modified Bielschowsky, Bodian, Gallyas (GAL) and Campbell–Switzer (CS) methods, are outlined with respect to their principles, basic protocols and interpretations, thereby providing neuropathologists, technicians and neuroscientists with a common basis for comparing findings and identifying the issues that still need to be clarified. Some consider “argyrophilia” to be a homogeneous phenomenon irrespective of the lesion and the method. Thus, they seek to explain the differences among the methods by pointing to their different sensitivities in detecting lesions (quantitative difference). Comparative studies, however, have demonstrated that argyrophilia is heterogeneous and dependent not only on the method but also on the lesion (qualitative difference). Each staining method has its own lesion-dependent specificity and, within this specificity, its own sensitivity. This “method- and lesion-dependent” nature of argyrophilia enables operational sorting of disease-specific lesions based on their silver-staining profiles, which may potentially represent some disease-specific aspects. Furthermore, comparisons between immunohistochemical and biochemical data have revealed an empirical correlation between GAL+/CS-deposits and 4-repeat (4R) tau (corticobasal degeneration, progressive supranuclear palsy and argyrophilic grains) and its complementary reversal between GAL-/CS+deposits and 3-repeat (3R) tau (Pick bodies). Deposits containing both 3R and 4R tau (neurofibrillary tangles of Alzheimer type) are GAL+/CS+. Although no molecular explanations, other than these empiric correlations, are currently available, these distinctive features, especially when combined with immunohistochemistry, are useful because silver-staining methods and immunoreactions are complementary to each other

Let‐7a‐5p, mir‐100‐5p, mir‐101‐3p, and mir‐199a‐3p hyperexpression as potential predictive biomarkers in early breast cancer patients

Background: The aim of this study is to identify miRNAs able to predict the outcomes in breast cancer patients after neoadjuvant chemotherapy (NAC). Patients and methods: We retrospec-tively analyzed 24 patients receiving NAC and not reaching pathologic complete response (pCR). miRNAs were analyzed using an Illumina Next‐Generation‐Sequencing (NGS) system. Results: Event‐free survival (EFS) and overall survival (OS) were significantly higher in patients with up-regulation of let‐7a‐5p (EFS p = 0.006; OS p = 0.0001), mirR‐100‐5p (EFS s p = 0.01; OS p = 0.03), miR‐ 101‐3p (EFS p = 0.05; OS p = 0.01), and miR‐199a‐3p (EFS p = 0.02; OS p = 0.01) in post‐NAC samples, independently from breast cancer subtypes. At multivariate analysis, only let‐7a‐5p was significantly associated with EFS (p = 0.009) and OS (p = 0.0008). Conclusion: Up‐regulation of the above miRNAs could represent biomarkers in breast cancer

Stimulating TSH receptor autoantibodies immunoassay: analytical evaluation and clinical performance in Graves’ disease

Background Thyroid-stimulating hormone (TSH) receptor (TSHR) autoantibodies (TRAbs) are a heterogeneous group of antibodies (Abs) with different functionalities. Among all TRAbs, only the stimulating ones (S-TRAbs) are considered as the pathogenetic marker of Graves’ disease (GD). To date, the methods available for TRAbs testing are based on immunoassays (IMAs) which detect total serum TRAbs or bioassays which are not suitable in clinical practice, even though they discern Abs functionality. The aim of our work was to evaluate the analytical and clinical performance of a very recent IMA (Immulite TSI method), supposed to test only the serum concentration of S-TRAbs, in comparison with a current method for total TRAbs (Roche/Elecsys IMA). Methods We evaluated serum samples of 145 subjects: 46 with untreated (GD), 36 with chronic autoimmune thyroiditis, 3 with atrophic thyroiditis, 10 with multinodular non-toxic goiter and 50 healthy subjects. Results The method showed an optimal analytical sensitivity and high precision levels (LoB: 0.04 UI/L, LoD:0.07 UI/L, LoQ:0.14 UI/L, intra-assay CV: 4.2–5.9%, inter-assay: 4.5–7.2%). By receiver operating characteristics curve analysis, we obtained a value of 0.57 (sensitivity: 98.0%, specificity: 99.9%) as the best cut-off to distinguish GD, apart from four cases. Passing Bablok regression and Bland Altman analysis pointed out a good correlation and agreement with Roche method (R2 = 0.98, slope = 1.03, bias = −2.70). Conclusions The new method presents very promising analytical characteristics and could be adopted in clinical practice for GD diagnosis. Moreover, the test allows to accurately detect very low values of analyte with a further clinical utility in detecting earlier possible relapses. </jats:sec

Effect of Whole Body Hypothermia on Surfactant Function When Amniotic Fluid Is Meconium Stained

We provide the first complete biophysical study of surfactant by captive bubble surfactometry from a neonate with meconium-stained amniotic fluid under controlled whole body hypothermia. Surfactant function improves after 48 hours of hypothermia, as surfactant cholesterol decreases. These findings partially explain positive effect of hypothermia on respiratory outcomes during meconium aspiration syndrome. A larger study including several neonates with or without lung disease is being conducted to better define the effect of hypothermia on surfactant function