p16 Expression in Laryngeal Squamous Cell Carcinoma: A Surrogate or Independent Prognostic Marker?

Laryngeal squamous cell carcinoma (LSCC) is a common malignancy that, despite scientific advancements, has not seen an improvement in its prognosis in the last decades. Few promising predictive markers have been found and none are relevant in clinical practice. p16ink4a, an oncosuppressor protein involved in cell cycle arrest, with a prognostic impact on other cancers, has been widely used in the head and neck region as a surrogate marker of HPV infection. Published papers and recent meta-analyses seem to minimize the biological role of HPV in the context of LSCC's cancerogenesis, and to disprove the reliability of p16ink4a as a surrogate prognostic marker in this context, while still highlighting its potential role as an independent predictor of survival. Unfortunately, the available literature, in particular during the last two decades, is often not focused on its potential role as an independent biomarker and few relevant data are found in papers mainly focused on HPV. The available data suggest that future research should focus specifically on p16ink4a, taking into account both its potential inactivation and overexpression, different patterns of staining, and immunohistochemistry cutoffs, and should focus not on its potential role as a surrogate marker but on its independent role as a predictor of survival.

Discovery of a selective and biologically active low-molecular weight antagonist of human interleukin-1ß.

Human interleukin-1ß (hIL-1ß) is a pro-inflammatory cytokine involved in many diseases. While hIL-1ß directed antibodies have shown clinical benefit, an orally available low-molecular weight antagonist is still elusive, limiting the applications of hIL-1ß-directed therapies. Here we describe the discovery of a low-molecular weight hIL-1ß antagonist that blocks the interaction with the IL-1R1 receptor. Starting from a low affinity fragment-based screening hit 1, structure-based optimization resulted in a compound (S)-2 that binds and antagonizes hIL-1ß with single-digit micromolar activity in biophysical, biochemical, and cellular assays. X-ray analysis reveals an allosteric mode of action that involves a hitherto unknown binding site in hIL-1ß encompassing two loops involved in hIL-1R1/hIL-1ß interactions. We show that residues of this binding site are part of a conformationally excited state of the mature cytokine. The compound antagonizes hIL-1ß function in cells, including primary human fibroblasts, demonstrating the relevance of this discovery for future development of hIL-1ß directed therapeutics.

Preoperative Diagnosis of Warthin Tumors Combining Cytological, Clinical and Ultrasonographic Information within a Multidisciplinary Approach in a Lump Clinic.

(1) Background: Warthin tumors account for about 20% of all benign salivary tumors, approaching 50% if we consider only the parotid gland. Wait and see is considered a reasonable option, but the diagnosis should be certain. Diagnosis can be based on morphological and cytological data, but the sensitivity of the fine needle aspiration cytology (FNAC) is not absolute, with a high rate of non-diagnostic findings in the event of a Warthin tumor, hindering the counseling and therapeutic decisions. The aim of the study is to evaluate the reliability of FNAC and its combination with anamnestic, clinical, and ultrasonographic data in diagnosing Warthin tumors. (2) Methods: A total of 413 patients affected by masses within the major salivary gland and managed between 2017 and 2022 at our institution have been included in the present retrospective study. Each patient underwent fine needle aspiration biopsy (FNAB) with a subsequent cytological diagnosis; successively, for each patient, the clinician (otolaryngologist) and the histopathologist discussed the combination of cytological (even non-diagnostic), clinical, and ultrasonographic data in order to make a "multiparametric" diagnosis. A total of 214 cases were subsequently submitted to surgical resection and had a final histopathology report, to which the cytological and the multiparametric diagnoses can be compared. We extracted all the patients with a cytological, multiparametric, and/or histological diagnosis of Warthin tumors in order to assess the sensitivity and specificity of FNAC and of multiparametric analysis in diagnosing Warthin tumors in case of a major salivary gland mass. (3) Results: One hundred thirty-two cases had a cytological, multiparametric, and/or histological diagnosis of Warthin tumors. FNAC displays a sensitivity of 68.4% and a specificity of 98.7% in diagnosing Warthin tumors. The multiparametric evaluation allowed a considerable improvement in sensitivity (92.9% vs. 68.4%), minimizing the number of non-diagnostic results and preserving at the same time a similar value of specificity (95.5% vs. 98.7%). Notably, none of the patients with a cytological or multiparametric diagnosis of Warthin were affected by a malignant lesion in the final histopathological report. (4) Conclusions: In the case of Warthin tumors, a multiparametric evaluation encompassing anamnestic, clinical, and cytological data is effective in reducing the number of non-diagnostic reports and can safely guide the management of a tumor (e.g., antibiotic treatment of infectious complications, assign a low priority to surgery, even consider observation avoiding surgery) which is absolutely benign and can be associated with no clinically relevant issues.

Accuracy of p16 IHC in Classifying HPV-Driven OPSCC in Different Populations.

High-risk human papillomavirus (HPV) infection is a defined etiopathogenetic factor in oropharyngeal carcinogenesis with a clear prognostic value. The P16 IHC (immunohistochemistry) is a widely accepted marker for HPV-driven carcinogenesis in oropharyngeal squamous cell carcinoma (OPSCC); in the present paper, we discuss its reliability as a standalone marker in different populations. The literature suggests that rates of p16 IHC false positive results are inversely correlated with the prevalence of HPV-driven carcinogenesis in a population. We propose a formula that can calculate such a false positive rate while knowing the real prevalence of HPV-driven OPSCCs in a given population. As it has been demonstrated that p16 positive/HPV negative cases (i.e., false positives at p16 IHC) have the same prognosis as p16 negative OPSCC, we conclude that despite the valuable prognostic value of p16 IHC, relying only on a p16 IHC positive result to recommend treatment de-intensification could be risky. For this aim, confirmation with an HPV nucleic acid detection system, especially in areas with a low prevalence of HPV-related OPSCCs, should be pursued.

Electrocautery, Harmonic, and Thunderbeat Instruments in Parotid Surgery: A Retrospective Comparative Study.

The aim of this retrospective study has been to compare the surgical outcomes of patients undergoing superficial parotidectomy with three different instruments: bipolar electrocautery, ultrasound, and mixed energy instruments. The clinical records of 102 patients who had undergone superficial parotidectomy for benign tumors between January 2016 and April 2022 were considered. Based on the tool used during the surgery, the patients were divided into three study groups: classic electrocautery hemostasis group (CH group), ultrasonic instrument group (HA group), and combined energy instrument group (TB group). The duration of surgery, the total post-operative drainage volume, and the intra-operative blood loss were significantly higher in the CH group compared to the HA and the TB group, while the differences were not significant between the latter two groups. Facial nerve weakness was detected in 45.9% of the CH group, 12.5% of the HA group, and 21.2% of the TB group. The rate of facial nerve dysfunction in the CH group was significantly higher than in the HA group (0.011). In the patients who experienced post-operative facial nerve dysfunction, the recovery time was significantly shorter in the HA group compared to the CH and the TB group. The HA and TB groups have demonstrated comparable and significantly better surgical outcomes than bipolar electrocautery. Ultrasound instruments have been shown to cause, in comparison with the other techniques, a lower rate of temporary facial nerve dysfunction and, if this is present, lead to a faster spontaneous recovery time.

Fragment-Based Drug Discovery by NMR. Where Are the Successes and Where can It Be Improved?

Over the last century, the definitions of pharmaceutical drug and drug discovery have changed considerably. Evolving from an almost exclusively serendipitous approach, drug discovery nowadays involves several distinct, yet sometimes interconnected stages aimed at obtaining molecules able to interact with a defined biomolecular target, and triggering a suitable biological response. At each of the stages, a wide range of techniques are typically employed to obtain the results required to move the project into the next stage. High Throughput Screening (HTS) and Fragment Based Drug Design (FBDD) are the two main approaches used to identify drug-like candidates in the early stages of drug discovery. Nuclear Magnetic Resonance (NMR) spectroscopy has many applications in FBDD and is used extensively in industry as well as in academia. In this manuscript, we discuss the paths of both successful and unsuccessful molecules where NMR had a crucial part in their development. We specifically focus on the techniques used and describe strengths and weaknesses of each stage by examining several case studies. More precisely, we examine the development history from the primary screening to the final lead optimisation of AZD3839 interacting with BACE-1, ABT-199 interacting with BCL2/XL and S64315 interacting with MCL-1. Based on these studies, we derive observations and conclusions regarding the FBDD process by NMR and discuss its potential improvements.

CcpNmr AnalysisScreen, a new software programme with dedicated automated analysis tools for fragment-based drug discovery by NMR.

Fragment-based drug discovery or FBDD is one of the main methods used by industry and academia for identifying drug-like candidates in early stages of drug discovery. NMR has a significant impact at any stage of the drug discovery process, from primary identification of small molecules to the elucidation of binding modes for guiding optimisations. The essence of NMR as an analytical tool, however, requires the processing and analysis of relatively large amounts of single data items, e.g. spectra, which can be daunting when managed manually. One bottleneck in FBDD by NMR is a lack of adequate and well-integrated resources for NMR data analysis that are freely available to the community. Thus, scientists typically resort to manually inspecting large datasets and relying predominantly on subjective interpretations. In this manuscript, we present CcpNmr AnalysisScreen, a software package that provides computational tools for automated analysis of FBDD data by NMR. We outline how the quality of collected spectra can be evaluated quickly, and how robust workflows can be optimised for reliable and rapid hit identification. With an intuitive graphical user interface and powerful algorithms, AnalysisScreen enables easy analysis of the large datasets needed in the early process of drug discovery by NMR.

Simple high-resolution NMR spectroscopy as a tool in molecular biology.

NMR is one of the major techniques for investigating the structure, dynamics and interactions between biomolecules. However, non-experts often experience NMR experimentation and data analysis as intimidating. We discuss a simple yet powerful NMR technique, the so-called chemical shift perturbation (CSP) analysis, as a tool to elucidate macromolecular interactions in small- and medium-sized complexes, including protein-protein, protein-drug, and protein-DNA/RNA interactions. We discuss current software packages for NMR data analysis and present a new interactive graphical tool implemented in CcpNmr AnalysisAssign version-3, which can drastically reduce the time required for the CSP analysis. Lastly, we illustrate the usefulness of a protein three-dimensional structure for interpretation of the CSP data.

CcpNmr AnalysisAssign: a flexible platform for integrated NMR analysis.

NMR spectroscopy is an indispensably powerful technique for the analysis of biomolecules under ambient conditions, both for structural- and functional studies. However, in practice the complexity of the technique has often frustrated its application by non-specialists. In this paper, we present CcpNmr version-3, the latest software release from the Collaborative Computational Project for NMR, for all aspects of NMR data analysis, including liquid- and solid-state NMR data. This software has been designed to be simple, functional and flexible, and aims to ensure that routine tasks can be performed in a straightforward manner. We have designed the software according to modern software engineering principles and leveraged the capabilities of modern graphics libraries to simplify a variety of data analysis tasks. We describe the process of backbone assignment as an example of the flexibility and simplicity of implementing workflows, as well as the toolkit used to create the necessary graphics for this workflow. The package can be downloaded from www.ccpn.ac.uk/v3-software/downloads and is freely available to all non-profit organisations.