Detection of Human Papillomavirus Infection in Oral Cancers reported at Dental Facility: Assessing the Utility of FFPE tissues

Incidence of human papillomavirus (HPV)–associated oral cancers is on the rise. However, epidemiological data of this subset of cancers is limited. Dental hospital pose a unique advantage in detection of HPV-positive oral malignancies. We assessed the utility of formalin-fixed, paraffin-embedded (FFPE) tissues, which are readily available, for evaluation of high-risk HPV infection in oral cancer. For protocol standardization, we used 20 prospectively-collected, paired FFPE and fresh tissues of histopathologically-confirmed oral cancer cases reported in Oral Medicine department of a dental hospital for comparative study. Only short-PCRs (~200bp) of DNA isolated using a modified xylene-free method displayed a concordant HPV result. For HPV analysis, we used additional 30 retrospectively-collected FFPE tissues. DNA isolated from these specimens showed an overall 23.4% (11/47) HPV positivity with detection of HPV18. Comparison of HPV positivity from dental hospital FFPE-specimens with overall HPV positivity of freshly-collected oral cancer specimens (n-55) from three cancer care hospitals of the same region showed notable difference (12.7%; 7/55). Further, cancer hospital specimens showed HPV16 positivity and displayed a characteristic difference in reported sub-sites and patient spectrum. Overall, using a xylene-free FFPE-DNA isolation method clubbed with short amplicon PCR, we showed detection of HPV-positive oral cancer in dental hospitals.

P–123 How to develop accurate Computer Assisted Sperm Analysis (CASA) AI in the absence of protocol standardization and abundance of human error when performing semen analyses?

Study question How can an automation & artificial intelligent tools be developed to perform according to WHO recommendations? Summary answer Developing CASA performs at < 20% error margin requires AI trained with high quality datasets and a robotic system adheres to WHO guidelines. What is known already A survey of 40 andrology laboratories, in 22 countries, revealed that > 90% had nonconformities in correct use of equipment, standardisation of protocols and quality control, leading to a lack of compliance to WHO protocols. Conventional CASA systems can standardize analysis, but controversy has occurred due to differences between manual and automated analyses stemming from: 1) all cells in a semen sample are detected including debris; 2) protocol variation when compared to top-notch manual analysis. The first point can be addressed by AI. The second point can be addressed by robotics designed to adhere to WHO guidelines. Study design, size, duration A mojo AISA (AI-powered semen analysis) system was placed in four clinical laboratories mentioned above capturing images of over 300 samples, one million images were generated over a course of 2 years. Mojo AISA’s AI was trained on data collected from the four clinics using robotic system is developed according to WHO guidelines. Participants/materials, setting, methods For an AI to detect sperm accurately, sperm samples were captured using mojo AISA smart microscopy and then the extracted sperm images expertly annotated. To evaluate the system-ability for semen analysis, fresh sample were analysed for concentration and motility by a manual operator and compared to a mojo AISA test. Main results and the role of chance To train the sperm detection AI, representative sperm images were carefully captured using mojo AISA and processed according to the following criteria: the number of images and videos to train and to test the model: 50,000 spermatozoon head and tails with various variations the variety of images: data used to train the AI has to be representative of the population that will undergo the analysis: 1) wide concentration ranges from 0 to 300 M/ml, 2) high and low density of debris and cells, 3) Presence of slight aggregations careful and precise annotation: expert andrology scientists annotated sperm images and identify objects to exclude, such as debris in seminal plasma, Mojo AISA is an attempt strictly build CASA AI system to WHO-guidelines. The marriage of AI and robotics automation has shown a promising results to mimic humans when measuring a semen sample and attempt to obtain results comparable to the manual analysis. mojo AISA’s performance improved three-fold (from 0,85 to 0,95 Pearson sperm count correlation and from >100% means relative error to 25% mean relative error). Limitations, reasons for caution Lack of standardization for semen analysis laboratory process globally is a bottleneck towards building a robust multi-center study, on-site CASA testing and generating an actionable data pool for studying the causes behind male fertility declineWider implications of the findings: Key learnings for parties advancing developing AI based on images and videos for application in the fertility space. Trial registration number Not applicable

Isolation Method and Characterization of Outer Membranes Vesicles of Helicobacter pylori Grown in a Chemically Defined Medium

Outer membrane vesicles (OMVs) are small vesicles constitutively shed by all Gram-negative bacterium, which have been proposed to play a role in Helicobacter pylori persistence and pathogenesis. The methods currently available for the isolation of H. pylori OMVs are diverse and time-consuming, raising the need for a protocol standardization, which was the main aim of this study. Here, we showed that the chemically defined F12 medium, supplemented with cholesterol, nutritionally supports bacterial growth and maintains H. pylori viability for at least 72 h. Additionally, we developed an abridged protocol for isolation of OMVs from these bacterial cultures, which comprises a low-speed centrifugation, supernatant filtration through a 0.45 μm pore, and two ultracentrifugations for OMVs’ recovery and washing. Using this approach, a good yield of highly pure bona fide OMVs was recovered from cultures of different H. pylori strains and in different periods of bacterial growth, as assessed by nanoparticle tracking analysis, transmission electron microscopy (TEM), and proteomic analyses, confirming the reliability of the protocol. Analysis of the proteome of OMVs isolated from H. pylori F12-cholesterol cultures at different time points of bacterial growth revealed differentially expressed proteins, including the vacuolating cytotoxin VacA. In conclusion, this work proposes a time- and cost-efficient protocol for the isolation of H. pylori OMVs from a chemically defined culture medium that is suitable for implementation in research and in the biopharmaceutical field.

Plant-Derived Extracellular Vesicles: Current Findings,Challenges, and Future Applications

In recent years, plant-derived extracellular vesicles (PDEVs) have gained the interest of many experts in fields such as microbiology and immunology, and research in this field has exponentially increased. These nano-sized particles have provided researchers with a number of interesting findings, making their application in human health and disease very promising. Both in vitro and in vivo experiments have shown that PDEVs can exhibit a multitude of effects, suggesting that these vesicles may have many potential future applications, including therapeutics and nano-delivery of compounds. While the preliminary results are promising, there are still some challenges to face, such as a lack of protocol standardization, as well as knowledge gaps that need to be filled. This review aims to discuss various aspects of PDEV knowledge, including their preliminary findings, challenges, and future uses, giving insight into the complexity of conducting research in this field.

Diffusion weighted imaging of the breast: Performance of standardized breast tumor tissue selection methods in clinical decision making

Objectives In breast diffusion weighted imaging (DWI) protocol standardization, it is recently shown that no breast tumor tissue selection (BTTS) method outperformed the others. The purpose of this study is to analyze the feasibility of three fixed-size breast tumor tissue selection (BTTS) methods based on the reproducibility, accuracy and time-measurement in comparison to the largest oval and manual delineation in breast diffusion weighted imaging data. Methods This study is performed with a consecutive dataset of 116 breast lesions (98 malignant) of at least 1.0 cm, scanned in accordance with the EUSOBI breast DWI working group recommendations. Reproducibility of the maximum size manual (BTTS1) and of the maximal size round/oval (BTTS2) methods were compared with three smaller fixed-size circular BTTS methods in the middle of each lesion (BTTS3, 0.12 cm3 volume) and at lowest apparent diffusion coefficient (ADC) (BTTS4, 0.12 cm3; BTTS5, 0.24 cm3). Mean ADC values, intraclass-correlation-coefficients (ICCs), area under the curve (AUC) and measurement times (sec) of the 5 BTTS methods were assessed by two observers. Results Excellent inter- and intra-observer agreement was found for any BTTS (with ICC 0.88–0.92 and 0.92–0.94, respectively). Significant difference in ADCmean between any pair of BTTS methods was shown (p = <0.001–0.009), except for BTTS2 vs. BTTS3 for observer 1 (p = 0.10). AUCs were comparable between BTTS methods, with highest AUC for BTTS2 (0.89–0.91) and lowest for BTTS4 (0.76–0.85). However, as an indicator of clinical feasibility, BTTS2-3 showed shortest measurement times (10–15 sec) compared to BTTS1, 4–5 (19–39 sec). Conclusion The performance of fixed-size BTTS methods, as a potential tool for clinical decision making, shows equal AUC but shorter ADC measurement time compared to manual or oval whole lesion measurements. The advantage of a fixed size BTTS method is the excellent reproducibility. A central fixed breast tumor tissue volume of 0.12 cm3 is the most feasible method for use in clinical practice.

Comet assay: a versatile but complex tool in genotoxicity testing

The comet assay is a versatile method for measuring DNA strand breaks in individual cells. It can also be applied to cells isolated from treated animals. In this review, we highlight advantages and limitations of this in vivo comet assay in a regulatory context. Modified versions of the standard protocol detect oxidized DNA bases and may be used to reveal sites of DNA base loss, DNA interstrand crosslinks, and the extent of DNA damage induced indirectly by reactive oxygen species elicited by chemical-induced oxidative stress. The assay is, however, at best semi-quantitative, and we discuss possible approaches to improving DNA damage quantitation and highlight the necessity of optimizing protocol standardization to enhance the comparability of results between laboratories. As a genotoxicity test in vivo, the in vivo comet assay has the advantage over the better established micronucleus erythrocyte test that it can be applied to any organ, including those that are specific targets of chemical carcinogens or those that are the first sites of contact of ingested or inhaled mutagens. We illustrate this by examples of its use in risk assessment for the food contaminants ochratoxin and furan. We suggest that improved quantitation is required to reveal the full potential of the comet assay and enhance its role in the battery of in vivo approaches to characterize the mechanisms of toxicity and carcinogenicity of chemicals and to aid the determination of safe human exposure limits.

Histopathological examination of surgical breast cancer specimens after neoadjuvant chemotherapy using digital radiography

The article provides a literature overview on significance, pathologic assessment of residual disease problems, and digital radiography (DR) potential in breast cancer (BC) after neoadjuvant therapy (NAT). Within the framework of the paper, the authors carry out an analysis of the Russian and English-language publications from PubMed, Google Scholar, ClinicalTrials.gov, eLibrary, and Cyberleninka. The comparison of the Russian clinical guidelines for BC diagnosis and the European and American guidelines revealed a lack of information on DR usage in the morphological assessment. The review showed the international experience in DR usage and demonstrated the relevance of the solution of morphological assessment problems in BC regression degree after NAT due to necessary clinical trial protocol standardization and increased predictive residual tumor class significance. The DR facilitated the morphological identification of metal markers implanted into the tumor bed, microcalcifications, altered foci, and improved tumor bed visibility, which is important for further objective status assessment of the resection margins and residual cancer burden class. The authors consider it necessary to conduct a study to optimize the residual tumor assessment using DR.

DICOM Format and Protocol Standardization—A Core Requirement for Digital Pathology Success

As the use of digital techniques in toxicologic pathology expands, challenges of scalability and interoperability come to the fore. Proprietary formats and closed single-vendor platforms prevail but depend on the availability and maintenance of multiformat conversion libraries. Expedient for small deployments, this is not sustainable at an industrial scale. Primarily known as a standard for radiology, the Digital Imaging and Communications in Medicine (DICOM) standard has been evolving to support other specialties since its inception, to become the single ubiquitous standard throughout medical imaging. The adoption of DICOM for whole slide imaging (WSI) has been sluggish. Prospects for widespread commercially viable clinical use of digital pathology change the incentives. Connectathons using DICOM have demonstrated its feasibility for WSI and virtual microscopy. Adoption of DICOM for digital and computational pathology will allow the reuse of enterprise-wide infrastructure for storage, security, and business continuity. The DICOM embedded metadata allows detached files to remain useful. Bright-field and multichannel fluorescence, Z-stacks, cytology, and sparse and fully tiled encoding are supported. External terminologies and standard compression schemes are supported. Color consistency is defined using International Color Consortium profiles. The DICOM files can be dual personality Tagged Image File Format (TIFF) for legacy support. Annotations for computational pathology results can be encoded.