An in silico method to assess antibody fragment polyreactivity

Antibodies are essential biological research tools and important therapeutic agents, but some exhibit non-specific binding to off-target proteins and other biomolecules. Such polyreactive antibodies compromise screening pipelines, lead to incorrect and irreproducible experimental results, and are generally intractable for clinical development. We designed a set of experiments using a diverse naive synthetic camelid antibody fragment ('nanobody') library to enable machine learning models to accurately assess polyreactivity from protein sequence (AUC > 0.8). Moreover, our models provide quantitative scoring metrics that predict the effect of amino acid substitutions on polyreactivity. We experimentally tested our model's performance on three independent nanobody scaffolds, where over 90% of predicted substitutions successfully reduced polyreactivity. Importantly, the model allowed us to diminish the polyreactivity of an angiotensin II type I receptor antagonist nanobody, without compromising its pharmacological properties. We provide a companion web-server that provides a straightforward means of predicting polyreactivity and polyreactivity-reducing mutations for any given nanobody sequence.

Inflammatory breast cancer defined: proposed common diagnostic criteria to guide treatment and research

Purpose Inflammatory breast cancer is a deadly and aggressive type of breast cancer. A key challenge relates to the need for a more detailed, formal, objective definition of IBC, the lack of which compromises clinical care, hampers the conduct of clinical trials, and hinders the search for IBC-specific biomarkers and treatments because of the heterogeneity of patients considered to have IBC. Methods Susan G. Komen, the Inflammatory Breast Cancer Research Foundation, and the Milburn Foundation convened patient advocates, clinicians, and researchers to review the state of IBC and to propose initiatives to advance the field. After literature review of the defining clinical, pathologic, and imaging characteristics of IBC, the experts developed a novel quantitative scoring system for diagnosis. Results The experts identified through consensus several “defining characteristics” of IBC, including factors related to timing of onset and specific symptoms. These reflect common pathophysiologic changes, sometimes detectable on biopsy in the form of dermal lymphovascular tumor emboli and often reflected in imaging findings. Based on the importance and extent of these characteristics, the experts developed a scoring scale that yields a continuous score from 0 to 48 and proposed cut-points for categorization that can be tested in subsequent validation studies. Conclusion To move beyond subjective ‘clinical diagnosis’ of IBC, we propose a quantitative scoring system to define IBC, based on clinical, pathologic, and imaging features. This system is intended to predict outcome and biology, guide treatment decisions and inclusion in clinical trials, and increase diagnostic accuracy to aid basic research; future validation studies are necessary to evaluate its performance.

Comparison of Semi-Quantitative Scoring and Artificial Intelligence Aided Digital Image Analysis of Chromogenic Immunohistochemistry

Semi-quantitative scoring is a method that is widely used to estimate the quantity of proteins on chromogen-labelled immunohistochemical (IHC) tissue sections. However, it suffers from several disadvantages, including its lack of objectivity and the fact that it is a time-consuming process. Our aim was to test a recently established artificial intelligence (AI)-aided digital image analysis platform, Pathronus, and to compare it to conventional scoring by five observers on chromogenic IHC-stained slides belonging to three experimental groups. Because Pathronus operates on grayscale 0-255 values, we transformed the data to a seven-point scale for use by pathologists and scientists. The accuracy of these methods was evaluated by comparing statistical significance among groups with quantitative fluorescent IHC reference data on subsequent tissue sections. The pairwise inter-rater reliability of the scoring and converted Pathronus data varied from poor to moderate with Cohen’s kappa, and overall agreement was poor within every experimental group using Fleiss’ kappa. Only the original and converted that were obtained from Pathronus original were able to reproduce the statistical significance among the groups that were determined by the reference method. In this study, we present an AI-aided software that can identify cells of interest, differentiate among organelles, protein specific chromogenic labelling, and nuclear counterstaining after an initial training period, providing a feasible and more accurate alternative to semi-quantitative scoring.

High Prevalence of 5T4/Trophoblast Glycoprotein in Soft Tissue Sarcomas

The expression of 5T4/trophoblast glycoprotein was evaluated in several histological subtypes of soft tissue sarcoma (STS) to determine whether the prevalence and level of expression of this membrane-associated glycoprotein is sufficient for use in targeted therapies. Tumor tissue microarrays containing cores from different histological subtypes of STS were stained using a standardized immunohistochemical staining method to detect 5T4; the level of staining was assessed using a semi-quantitative scoring method. No 5T4 staining was seen in the angiosarcomas and liposarcomas investigated in this study. 5T4 staining in the other STS subtypes was seen in more than 50% of cases, warranting further investigation into whether this antigen could evoke an anti-tumor immune response or can be used as target for the delivery of more potent toxins through antibody drug conjugates.

Nitro-Deficient Niclosamide Confers Reduced Genotoxicity and Retains Mitochondrial Uncoupling Activity for Cancer Therapy

Niclosamide is an oral anthelmintic drug, approved for use against tapeworm infections. Recent studies suggest however that niclosamide may have broader clinical applications in cancers, spurring increased interest in the functions and mechanisms of niclosamide. Previously, we reported that niclosamide targets a metabolic vulnerability in p53-deficient tumours, providing a basis for patient stratification and personalised treatment strategies. In the present study, we functionally characterised the contribution of the aniline 4′-NO2 group on niclosamide to its cellular activities. We demonstrated that niclosamide induces genome-wide DNA damage that is mechanistically uncoupled from its antitumour effects mediated through mitochondrial uncoupling. Elimination of the nitro group in ND-Nic analogue significantly reduced γH2AX signals and DNA breaks while preserving its antitumour mechanism mediated through a calcium signalling pathway and arachidonic acid metabolism. Lipidomics profiling further revealed that ND-Nic-treated cells retained a metabolite profile characteristic of niclosamide-treated cells. Notably, quantitative scoring of drug sensitivity suggests that elimination of its nitro group enhanced the target selectivity of niclosamide against p53 deficiency. Importantly, the results also raise concern that niclosamide may impose a pleiotropic genotoxic effect, which limits its clinical efficacy and warrants further investigation into alternative drug analogues that may ameliorate any potential unwanted side effects.

Bronchoalveolar lavage affects thorax computed tomography of healthy and SARS-CoV-2 infected rhesus macaques (Macaca mulatta)

Medical imaging as method to assess the longitudinal process of a SARS-CoV-2 infection in non-human primates is commonly used in research settings. Bronchoalveolar lavage (BAL) is regularly used to determine the local virus production and immune effects of SARS-CoV-2 in the lower respiratory tract. However, the potential interference of those two diagnostic modalities is unknown in non-human primates. The current study investigated the effect and duration of BAL on computed tomography (CT) in both healthy and experimentally SARS-CoV-2-infected female rhesus macaques (Macaca mulatta). In addition, the effect of subsequent BALs was reviewed. Thorax CTs and BALs were obtained from four healthy animals and 11 experimentally SARS-CoV-2-infected animals. From all animals, CTs were obtained just before BAL, and 24 hours post-BAL. Additionally, from the healthy animals, CTs immediately after, and four hours post-BAL were obtained. Thorax CTs were evaluated for alterations in lung density, measured in Hounsfield units, and a visual semi-quantitative scoring system. An increase in the lung density was observed on the immediately post-BAL CT but resolved within 24 hours in the healthy animals. In the infected animals, a significant difference in both the lung density and CT score was still found 24 hours after BAL. Furthermore, the differences between time points in CT score were increased for the second BAL. These results indicate that the effect of BAL on infected lungs is not resolved within the first 24 hours. Therefore, it is important to acknowledge the interference between BAL and CT in rhesus macaques.

Lung Ultrasound in COVID-19: Clinical Correlates and Comparison With Chest Computed Tomography

Background: lung ultrasound (LUS) and chest computed tomography (chest-CT) are largely employed to evaluate coronavirus-disease-19 (COVID-19) pneumonia. We investigated semi-quantitative LUS and CT scoring in hospitalized COVID-19 patients.Methods: LUS and chest-CT were performed within 24 hours upon admission. Both were analyzed according to semi-quantitative scoring systems. Subgroups were identified according to median LUS score.Results: patients within higher LUS-score group were older (79 vs 60 years, p<0.001), had higher C-reactive protein (CRP) (7.2 vs 1.3 mg/dl, p<0.001) and chest-CT score (10 vs 4, p=0.027) as well as lower PaO2/FiO2 (286 vs 356, p=0.029) as compared to patients within lower scores. We found a significant correlation between scores (r=0.390, p=0.023). Both LUS and CT scores correlated directly with patients age (r=0.586, p<0.001 and r=0.399, p=0.021 respectively) and CRP (r=0.472, p=0.002 and r=0.518, p=0.002 respectively), inversely with PaO2/FiO2 (r=-0.485, p=0.003 and r=-0.440, p=0.017 respectively). LUS-score only showed significant correlation with hs-Troponin T, NT-pro-BNP and creatinine (r=0.433, p=0.019; r=0.411, p=0.027 and r=0.497, p=0.001 respectively).Conclusions: semi-quantitative bedside LUS related to the severity of COVID-19 pneumonia similarly to chest-CT. Correlation of LUS-score with markers of cardiac and renal injury suggests that LUS might contribute to a more comprehensive evaluation of this heterogeneous population.

POS0842 LUNG ULTRASOUND TO ASSESS THE SEVERITY OF INTERSTITIAL LUNG DISEASE IN SYSTEMIC SCLEROSIS

Background:Interstitial lung disease (ILD) is one of the most common complications and one of the main causes of morbidity and mortality in Systemic Sclerosis (SSc). High-resolution computed tomography (HRCT) is the gold standard for the diagnosis of ILD and it allows its quantification. Among semi-quantitative methods, Goh et al proposed a semi-quantitative scoring system to visually quantify ILD extent, with categorical cut-off of 20% to distinguish limited and extensive parenchymal involvement with prognostic implications. More recently, the use of radiomics has allowed the objective quantification of ILD through the use of dedicated software, which calculate different parameters of lung density.Given the exposure to ionizing radiation that the procedure entails, other methods of ILD evaluation are being studied, among which lung ultrasound (LUS) identifies the B-lines as a main feature of ILD. So far, different evidences have proposed the use of LUS for the screening of ILD, even in the early phases of the disease and in subclinical lung involvement.Objectives:the aim of this study is to test the role of LUS in quantifying the severity of SSc-ILD, evaluated with both semi-quantitative visual radiological and quantitative radiomic scores.Methods:Adult SSc patients classified according to the ACR/EULAR 2013 criteria patients were assessed with pulmonary function test (PFTs), lung ultrasound and HRCT over 60 days. CT images were analysed qualitatively (by presence/absence of ILD), semi-quantitatively (categorical Goh score <20% vs> 20% of extent and the continuous extent Goh score made from 5 levels’ assessment– 0 to 100%) and quantitatively [with the densitometric radiomic data obtained through the Horos software - Mean lung attenuation (MLA), Standard Deviation (SD), Kurtosis, Skewness and Lung volume (LV)]. LUS was used to quantify the B-lines detected in each patient by scanning a total of 13 intercostal spaces, on both anterior and posterior chest wall.Results:Among 59 SSc patients (81% women, mean age 48±14 years, 45% anti-Scl70 positive), 23 (39%) presented ILD on HRCT, of which 14 limited and 9 extensive. The mean visual semi-quantitative score was 6%, ranging from 0 to 66%. Our data showed a significantly different number of B-Lines in ILD vs non-ILD patients (median 38 vs 9, p <.005), a result which was further confirmed among non-ILD vs ILD> 20% (median 47 vs 9, p=.001) and ILD <20% (median 36 vs 9, p=.001) patients. Conversely, the number of B-lines was not statistically different between patients with ILD <20% and >20% (median 47 vs 36, p=.78). We observed a significant negative correlation between the number of B-lines and FVC (r=-.472, p<.05) TLC (r=-.436, p=.003), DLco (r=-.515, p<.001), DLCO/VA (r=.-306, p=.03). Finally, the number of B-lines showed a statistically significant correlation with the Goh score on 5 levels (r=.437, p=.001), MLA (r=.571, p<.001), kurtosis (r=-.285, p=.028), skewness (r=-.370, p = .004) and LV (r=-.277, p=.033). All data were confirmed analysing anterior and posterior B-Lines separately.Conclusion:Our study confirms that LUS represents a useful tool for the identification of SSc-ILD. In addition, we showed that LUS may be useful also for the quantification of the severity of SSc-ILD, by correlating with PFT parameters, radiomics parameters and visual radiological evaluation. Together with the PFTs, LUS could be used to increase the accuracy of the screening and, potentially, of the follow-up of SSc-ILD patients.Disclosure of Interests:Cosimo Bruni: None declared, Lavinia Mattolini: None declared, Lorenzo Tofani: None declared, Luna Gargani Consultant of: GE Healthcare, Philips Healthcare and Caption Health, Nicholas Landini: None declared, Gemma Lepri: None declared, Martina Orlandi: None declared, Serena Guiducci: None declared, Silvia Bellando Randone: None declared, Marco Matucci-Cerinic: None declared