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This paper discusses the administrative aspects of molecular diagnostics in oncology, including US Food and Drug Administration (FDA) oversight, the regulatory approval process, clinical, and operational workflows, and payment models. Comprehensive molecular testing is important to deliver optimal oncology care and improve patient outcomes. Despite the potential benefits of testing, utilization remains low. The FDA regulatory approval process is reviewed for in vitro diagnostic products, which includes classification into three regulatory classes on the basis of risk. Companion diagnostic devices are used to guide treatment decisions. The clinical and operational challenges associated with molecular testing in oncology are also discussed, including the rapidly evolving landscape of precision oncology, the wide range of biomarker testing options, and complexities of test ordering, interpretation, and result delivery. There is a need for a multifaceted support approach involving education, technology enhancements, and workflow support to overcome these challenges. In terms of payment models, coverage policies between Medicare and commercial payers are compared with differences in coverage criteria, with Medicare focusing on FDA approval or clearance, whereas commercial payers consider additional factors such as National Comprehensive Cancer Network and ASCO guidelines. Commercial payers tend to cover smaller panels on the basis of guideline-recommended biomarkers, whereas coverage for broad tumor profiling is limited. Several strategies can increase the utilization of molecular testing, including integrating test results into electronic medical record platforms, standardizing billing practices, increasing clinical trials, and primary literature supporting the use of molecular testing, educating physicians, and using tumor boards for result interpretation and treatment discussions.

Background: While REIMS technology has successfully been demonstrated for the histological identification of ex-vivo breast tumor tissues, questions regarding the robustness of the approach and the possibility of tumor molecular diagnostics still remain unanswered. In the current study, we set out to determine whether it is possible to acquire cross-comparable REIMS datasets at multiple sites for the identification of breast tumors and subtypes., Methods: A consortium of four sites with three of them having access to fresh surgical tissue samples performed tissue analysis using identical REIMS setups and protocols. Overall, 21 breast cancer specimens containing pathology-validated tumor and adipose tissues were analyzed and results were compared using uni- and multivariate statistics on normal, WT and PIK3CA mutant ductal carcinomas., Results: Statistical analysis of data from standards showed significant differences between sites and individual users. However, the multivariate classification models created from breast cancer data elicited 97.1% and 98.6% correct classification for leave-one-site-out and leave-one-patient-out cross validation. Molecular subtypes represented by PIK3CA mutation gave consistent results across sites., Conclusions: The results clearly demonstrate the feasibility of creating and using global classification models for a REIMS-based margin assessment tool, supporting the clinical translatability of the approach., (© 2024. Waters Technologies Corporation and The Author(s).)

Cytopathology represents a well established diagnostic approach because of its limited cost, reliability, and minimal invasiveness with respect to other methodologies. The evolving complexity of the different classifications systems and the implementation of ancillary techniques to refine the diagnosis is progressively helping in the risk of malignancy stratification, and the adoption of next-generation sequencing techniques contributes to enrich this valuable tool with predictive information, which is always more essential in the tailored medicine era. The recent introduction of digital and computational pathology is further boosting the potentialities of cytopathology, aiding in the interpretation of samples to improve the cost effectiveness of large screening programs and the diagnostic efficiency within intermediate/atypical categories. Moreover, the adoption of artificial intelligence tools is promising to complement molecular investigations, representing a stimulating perspective in the cytopathology field. In this work, the authors tried to summarize the multifaceted nature of this complex and evolving field of pathology, synthesizing the most recent advances and providing the young pathologists' perspective on this fascinating world., (© 2024 American Cancer Society.)

Background: The advancements in artificial intelligence (AI) technology for image recognition were propelling molecular pathology research into a new era., Objective: To summarize the hot spots and research trends in the field of molecular pathology image recognition., Methods: Relevant articles from January 1st, 2010, to August 25th, 2023, were retrieved from the Web of Science Core Collection. Subsequently, CiteSpace was employed for bibliometric and visual analysis, generating diverse network diagrams illustrating keywords, highly cited references, hot topics, and research trends., Results: A total of 110 relevant articles were extracted from a pool of 10,205 articles. The overall publication count exhibited a rising trend each year. The leading contributors in terms of institutions, countries, and authors were Maastricht University (11 articles), the United States (38 articles), and Kather Jacob Nicholas (9 articles), respectively. Half of the top ten research institutions, based on publication volume, were affiliated with Germany. The most frequently cited article was authored by Nicolas Coudray et al. accumulating 703 citations. The keyword "Deep learning" had the highest frequency in 2019. Notably, the highlighted keywords from 2022 to 2023 included "microsatellite instability", and there were 21 articles focusing on utilizing algorithms to recognize microsatellite instability (MSI) in colorectal cancer (CRC) pathological images., Conclusion: The use of DL is expected to provide a new strategy to effectively solve the current problem of time-consuming and expensive molecular pathology detection. Therefore, further research is needed to address issues, such as data quality and standardization, model interpretability, and resource and infrastructure requirements., (© 2024. The Author(s).)

Cas12 and Cas13 are extensively utilized in molecular diagnostics for their trans-cleavage activities, yet their activation characteristics remain partially understood. Here, we conduct an in-depth investigation of Cas12a, Cas12f1, and Cas13a, uncovering the characteristics of their trans-DNase and trans-RNase activities with noncanonical activators. Our findings reveal that DNA can serve as a direct target for CRISPR-Cas13a, markedly increasing the detection sensitivity for single-base mismatches. Moreover, the trans-cleavage activities of Cas12a and Cas13a can be activated by diverse RNA:DNA and RNA:RNA duplexes, respectively, indicating that the presence of stem-loop structures in crRNAs is not essential for their activation. Notably, Cas12f1, unlike Cas12a, exhibits intrinsic RNase activity independently of activation. Leveraging these insights, we have improved the accuracy of a dual-gene target detection approach that employs the CRISPR-Cas12f1 and Cas13a systems. Our research advances the understanding of the noncanonical activation characteristics of Cas12 and Cas13a, contributing to the field of CRISPR-based diagnostics., (© 2024. The Author(s).)

Gliomas and Glioblastomas represent a significant portion of central nervous system (CNS) tumors associated with high mortality rates and variable prognosis. In 2021, the World Health Organization (WHO) updated its Glioma classification criteria, most notably incorporating molecular markers including CDKN2A/B homozygous deletion, TERT promoter mutation, EGFR amplification, + 7/-10 chromosome copy number changes, and others into the grading and classification of adult and pediatric Gliomas. The inclusion of these markers and the corresponding introduction of new Glioma subtypes has allowed for more specific tailoring of clinical interventions and has inspired a new wave of Radiogenomic studies seeking to leverage medical imaging information to explore the diagnostic and prognostic implications of these new biomarkers. Radiomics, deep learning, and combined approaches have enabled the development of powerful computational tools for MRI analysis correlating imaging characteristics with various molecular biomarkers integrated into the updated WHO CNS-5 guidelines. Recent studies have leveraged these methods to accurately classify Gliomas in accordance with these updated molecular-based criteria based solely on non-invasive MRI, demonstrating the great promise of Radiogenomic tools. In this review, we explore the relative benefits and drawbacks of these computational frameworks and highlight the technical and clinical innovations presented by recent studies in the landscape of fast evolving molecular-based Glioma subtyping. Furthermore, the potential benefits and challenges of incorporating these tools into routine radiological workflows, aiming to enhance patient care and optimize clinical outcomes in the evolving field of CNS tumor management, have been highlighted., (© 2024. The Author(s).)

The use of CRISPR/Cas nucleases for the development of DNA diagnostic systems in out-of-laboratory conditions (point-of-need testing, PONT) has demonstrated rapid growth in the last few years, starting with the appearance in 2017-2018 of the first diagnostic platforms known as DETECTR and SHERLOCK. The platforms are based on a combination of methods of nucleic acid isothermal amplification with selective CRISPR/Cas detection of target amplicons. This significantly improves the sensitivity and specificity of PONT, making them comparable with or even superior to the sensitivity and specificity of polymerase chain reaction, considered as the "gold standard" of DNA diagnostics. The review considers modern approaches to the coupling of CRISPR/Cas detection using Cas9, Cas12a, Cas12b, Cas13a, Cas14, and Cas3 nucleases to various methods of nucleic acid isothermal amplification, with an emphasis on works in which sensitivity at the level of single molecules (attomolar and subattomolar concentrations of the target) is achieved. The properties of CRISPR/Cas nucleases used for targeted DNA diagnostics and the features of methods of nucleic acid isothermal amplification are briefly considered in the context of the development of diagnostic biosensing platforms. Special attention is paid to the most promising directions for the development of DNA diagnostics using CRISPR/Cas nuclease.

Diffuse mesotheliomas are characterized by recurrent genomic alterations involving tumor suppressors and epigenetic regulators such as BAP1, CDKN2A, MTAP, and NF2. Depending on the differential diagnosis as informed by histologic assessment, one can apply the appropriate immunohistochemical and/or molecular panels to reach the correct pathologic diagnosis, sometimes even in cases with limited tissues. Biomarkers aid in the diagnosis of mesothelioma in the following scenarios: 1) For a tumor that is overtly malignant, how can one distinguish mesothelioma from other tumors? 2) For a mesothelial proliferation, how can one distinguish mesothelioma from a reactive process? To distinguish mesotheliomas from carcinomas, at least two positive and two negative markers are currently recommended. To distinguish sarcomatoid mesothelioma from pleomorphic carcinoma, even more markers-and sometimes molecular testing-are needed. To distinguish mesothelioma from reactive mesothelial conditions, useful immunohistochemical biomarkers include BAP1, MTAP, and merlin, which serve as surrogates for the corresponding gene mutation status. In patients with unusual clinical history, for tumors with a peculiar microscopic appearance, and/or in cases with an equivocal immunophenotypic profile, molecular testing can help to exclude mimics and to confirm the pathologic diagnosis., (© 2024. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Mutations in the estrogen receptor alpha gene (ESR1) can lead to resistance to endocrine therapy (ET) in hormone receptor-positive (HR+)/ HER2- metastatic breast cancer (MBC). ESR1 mutations can be detected in up to 40 % of patients pretreated with ET in circulating tumor DNA (ctDNA). Data from prospective randomized trials highlight those patients with HR+/HER2- MBC with detectable ESR1 mutations experience better outcomes when receiving novel selective estrogen receptor degraders (SERDs). There is a high need for optimizing ESR1 testing strategies on liquid biopsy samples in HR+/HER2- MBC, including a hugh quality workflow implementation and molecular pathology reporting standardization. Our manuscript aims to elucidate the clinical and biological rationale for ESR1 testing in MBC, while critically examining the currently available guidelines and recommendations for this specific type of molecular testing on ctDNA. The objective will extend to the critical aspects of harmonization and standardization, specifically focusing on the pathology laboratory workflow. Finally, we propose a clear and comprehensive model for reporting ESR1 testing results on ctDNA in HR+/HER2- MBC., Competing Interests: Declaration of Competing Interest E.G-R. has relevant relationship (advisory fees, honoraria, travel accommodation and expenses, grants, and non-financial support) with AstraZeneca, Exact Sciences, GlaxoSmithKline (GSK), Novartis, Roche, Thermo Fisher Scientific unrelated to the current work. C.C. reported grants from Gilead, Seagen, personal fees from Pfizer, Lilly, Novartis, MSD, Daiichi Sankyo, and AstraZeneca outside the submitted work. G.C. reports funding from Astra Zeneca, Daichii Sankyo, Merck; consulting fees from BMS, Roche, Pfizer, Novartis, Lilly, Astra Zeneca, Daichii Sankyo, Merck, Seagen, Ellipsis; honoraria from Pfizer, Lilly; support for attending meetings from Roche, Pfizer. C.R. has received speaker honoraria from AstraZeneca, Roche, and MSD; advisory board honoraria from Inivata, Archer, Boston Pharmaceuticals, MD Serono, and Novartis; and institutional research funding for his work as Coordinating Principal Investigator from Pfizer and EMD Serono. U.M. has received personal fees (as consultant and/or speaker bureau) from Boehringer Ingelheim, Roche, MSD, Amgen, Thermo Fisher Scientific, Eli Lilly, Diaceutics, GSK, Merck and AstraZeneca, Janssen, Diatech, Novartis and Hedera. N.F. has received honoraria for consulting, advisory role, speaker bureau, travel, and/or research grants from Merck Sharp & Dohme (MSD), Merck, Novartis, AstraZeneca, Roche, Menarini, Daiichi Sankyo, GlaxoSmithKline (GSK), Gilead, Adicet Bio, Sysmex, Reply, Veracyte Inc., Sakura, Leica Biosystems, Lilly. These companies had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and/or in the decision to publish the results. All other authors declare no potential conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The increased accessibility and utilization of molecular testing including next-generation sequencing (NGS) has impacted the practice of pediatric pathology, with diagnostic, prognostic, and therapeutic implications for our patients. This survey is the first to describe the utilization of molecular testing in the routine practice of pediatric pathology for the care of children with known or suspected solid tumors., Procedure: The Society for Pediatric Pathology Practice Committee distributed a survey to our membership asking 25 questions about training, practice setting, molecular ordering practices, and barriers to testing., Results: Seventy-five pathologists responded to the survey. The survey provides valuable insight into the current use of molecular testing for the care of children with known or suspected solid tumors. Most respondents reported that they are increasingly using a variety of molecular techniques, with increased use over time, and that NGS is useful., Conclusions: These results highlight a variety of barriers to molecular testing, including cost, insurance coverage, turnaround time, limitations of available assays (including limited coverage of pediatric-specific alterations), and difficulty in determining the most appropriate test to order. These data may be useful in supporting pediatric pathologists in their practice., (© 2024 Wiley Periodicals LLC.)

CRISPR-based (Clustered regularly interspaced short palindromic repeats-based) technologies have revolutionized molecular biology and diagnostics, offering unprecedented precision and versatility. However, challenges remain, such as high costs, demanding technical expertise, and limited quantification capabilities. To overcome these limitations, innovative microfluidic platforms are emerging as powerful tools for enhancing CRISPR diagnostics. This review explores the exciting intersection of CRISPR and microfluidics, highlighting their potential to revolutionize healthcare diagnostics. By integrating CRISPR's specificity with microfluidics' miniaturization and automation, researchers are developing more sensitive and portable diagnostic tools for a range of diseases. These microfluidic devices streamline sample processing, improve diagnostic performance, and enable point-of-care applications, allowing for rapid and accurate detection of pathogens, genetic disorders, and other health conditions. The review discusses various CRISPR/Cas systems, including Cas9, Cas12, and Cas13, and their integration with microfluidic platforms. It also examines the advantages and limitations of these systems, highlighting their potential for detecting DNA and RNA biomarkers. The review also explores the key challenges in developing and implementing CRISPR-driven microfluidic diagnostics, such as ensuring robustness, minimizing cross-contamination, and achieving robust quantification. Finally, it highlights potential future directions for this rapidly evolving field, emphasizing the transformative potential of these technologies for personalized medicine and global health., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Tumor mutational burden (TMB) has been recognized as a predictive biomarker for immunotherapy response in several tumor types. Several laboratories offer TMB testing, but there is significant variation in how TMB is calculated, reported, and interpreted among laboratories. TMB standardization efforts are underway, but no published guidance for TMB validation and reporting is currently available. Recognizing the current challenges of clinical TMB testing, the Association for Molecular Pathology convened a multidisciplinary collaborative working group with representation from the American Society of Clinical Oncology, the College of American Pathologists, and the Society for the Immunotherapy of Cancer to review the laboratory practices surrounding TMB and develop recommendations for the analytical validation and reporting of TMB testing based on survey data, literature review, and expert consensus. These recommendations encompass pre-analytical, analytical, and postanalytical factors of TMB analysis, and they emphasize the relevance of comprehensive methodological descriptions to allow comparability between assays., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Organ shortage is a major challenge in kidney transplantation but the use of older donors, often with co-morbidities, is hampered by inconsistent outcomes. Methods of accurately stratifying marginal donor organs by clinical and histological assessment are lacking. To better understand organ variability, we profiled the transcriptomes of 271 kidneys from deceased donors at retrieval. Following correction for biopsy composition, we assessed molecular pathways that associated with delayed, and sub-optimal one-year graft function. Analysis of cortical biopsies identified an adaptive immune gene-rich module that significantly associated with increasing age and worse outcomes. Cellular deconvolution using human kidney reference single cell transcriptomes confirmed an increase in kidney-specific B and T cell signatures, as well as kidney macrophage, myofibroblast and fibroblast gene sets in this module. Surprisingly, innate immune pathway and neutrophil gene signature enrichment was associated with better outcomes. Thus, our work uncovers cellular molecular features of pathological organ ageing, identifiable at kidney retrieval, with translational potential., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Objective: The molecular era of glioma diagnosis and treatment has arrived, and a single rapid histopathology is no longer sufficient for surgery. This study sought to present an automatic integrated gene detection system (AIGS), which enables rapid intraoperative detection of IDH/TERTp mutations., Methods: A total of 78 patients with gliomas were included in this study. IDH/TERTp mutations were detected intraoperatively using AIGS in 41 of these patients, and they were guided to surgical resection (AIGS detection group). The remaining 37 underwent histopathology-guided conventional surgical resection (non-AIGS detection group). The clinical utility of this technique was evaluated by comparing the accuracy of glioma subtype diagnosis before and after TERTp mutation results were obtained by pathologists and the extent of resection (EOR) and patient prognosis for molecular pathology-guided glioma surgery., Results: With NGS/Sanger sequencing and chromosome detection as the gold standard, the accuracy of AIGS results was 100%. And the timing was well matched to the intraoperative rapid pathology report. After obtaining the TERTp mutation detection results, the accuracy of the glioma subtype diagnosis made by the pathologists increased by 19.51%. Molecular pathology-guided surgical resection of gliomas significantly increased EOR (99.06% vs. 93.73%, p < 0.0001) and also improved median OS (26.77 vs. 13.47 months, p = 0.0289) and median PFS (15.90 vs. 10.57 months, p = 0.0181) in patients with glioblastoma., Interpretation: Using AIGS intraoperatively to detect IDH/TERTp mutations to accurately diagnose glioma subtypes can help achieve maximum safe resection of gliomas, which in turn improves the survival prognosis of patients., (© 2024 The Author(s). Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Robust, sensitive, and rapid molecular detection tools are essential prerequisites for disease diagnosis and epidemiological control. However, the current mainstream tests necessitate expensive equipment and specialized operators, impeding the advancement of molecular diagnostics. The CRISPR-Cas system is an integral component of the bacterial adaptive immune system, wherein Cas proteins recognize PAM sequences by binding to CRISPR RNA, subsequently triggering DNA or RNA cleavage. The discovery of the CRISPR-Cas system has invigorated molecular diagnostics. With further in-depth research on this system, its application in molecular diagnosis is flourishing. In this review, we provide a comprehensive overview of recent research progress on the CRISPR-Cas system, specifically focusing on its application in molecular diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Purpose: To investigate the discordance in sarcoma diagnoses between nonspecialized institutions following revision by dedicated sarcoma pathologists at a reference center in Brazil and the relevance of molecular pathology in this context., Methods: We conducted a retrospective analysis of sarcoma samples initially analyzed at outside laboratories and subsequently reviewed by two specialized pathologists between January 2014 and December 2020. After obtaining demographic and tumor characteristics, pathology results were matched and classified as complete discordance (CD; benign v malignant, sarcoma v other malignancies), partial concordance (similar diagnosis of connective tumor, but different grade/histological subtype/differentiation), and complete concordance (CC). The concordance for histology or grade, and the role of molecular assessments supporting the diagnosis were also independently determined. Statistical analyses were conducted through the kappa coefficient of agreement and adherence by χ 2 test, χ 2 test by Person, and Fisher exact test., Results: In total, 197 cases were included, with samples obtained predominately from male patients (57.9%) and localized/primary tumors (86.8%). Following revision, the most frequent final diagnoses were undifferentiated pleomorphic sarcoma (17.8%), well-differentiated/dedifferentiated liposarcoma (8.6%), and leiomyosarcoma (7.6%). CD was found in 13.2%, partial discordance in 45.2%, and CC in 41.6% of reviews ( P < .001). We found a concordance for histology or grade of 53.5% ( P < .001) and 51.8% ( P < .001), respectively. Molecular assessments, comprising next-generation sequencing panels (79.5%) and fluorescent in situ hybridization (20.5%), were performed in 44 (22.3%) cases, with findings classified as of diagnostic relevance in 31.8%., Conclusion: In nearly 60% of the cases, the initial sarcoma diagnosis was modified when revised by a reference center and dedicated pathologists, assisted by molecular pathology techniques. These results justify the assembly of referral networks in countries with limited health care resources.

The study presents a series of examples of magnetic nanoparticle systems designed for the diagnosis of viral diseases. In this interdisciplinary work, we describe one of the most comprehensive synthetic approaches for the preparation and functionalization of smart nanoparticle systems for rapid and effective RT-PCR diagnostics and isolation of viral RNA. Twelve different organic ligands and inorganic porous silica were used for surface functionalization of the Fe 3 O 4 magnetic core to increase the number of active centres for efficient RNA binding from human swab samples. Different nanoparticle systems with common beads were characterized by HRTEM, SEM, FT-IR, XRD, XPS and magnetic measurements. We demonstrate the application of the fundamental models modified to fit the experimental zero-field cooling magnetization data. We discuss the influence of the nanoparticle shell parameters (morphology, thickness, ligands) on the overall magnetic performance of the systems. The prepared nanoparticles were tested for the isolation of viral RNA from tissue samples infected with hepatitis E virus-HEV and from biofluid samples of SARS-CoV-2 positive patients. The efficiency of RNA isolation was quantified by RT-qPCR method., (© 2024. The Author(s).)

Extracellular vesicles are shed by every cell type and can be found in any biofluid. They contain different molecules that can be utilized as biomarkers, including several RNA species which they protect from degradation. Here, we present a pipeline for the development and analysis of extracellular vesicle-associated transcriptomic biomarkers that our group has successfully applied multiple times. We highlight the key steps of the pipeline and give particular emphasis to the necessary quality control checkpoints, which are linked to numerous available guidelines that should be considered along the workflow. Our pipeline starts with patient recruitment and continues with blood sampling and processing. The purification and characterization of extracellular vesicles is explained in detail, as well as the isolation and quality control of extracellular vesicle-associated RNA. We point out the possible pitfalls during library preparation and RNA sequencing and present multiple bioinformatic tools to pinpoint biomarker signature candidates from the sequencing data. Finally, considerations and pitfalls during the validation of the biomarker signature using RT-qPCR will be elaborated., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Histopathological assessment of tissue samples after prolonged formalin fixation has been described previously, but currently there is only limited knowledge regarding the feasibility of molecular pathology on such tissue. In this pilot study, we tested routine molecular pathology methods (DNA isolation, DNA pyrosequencing/next-generation sequencing, DNA methylation analysis, RT-PCR, clonality analysis and fluorescence in situ hybridization) on tissue samples from 11 tumor entities as well as non-neoplastic brain tissue from 43 body donors during the gross anatomy course at Ulm University (winter semester 2019/20 and 2020/21). The mean post mortem interval until fixation was 2.5 ± 1.6 days (range, 1-6 days). Fixation was performed with aqueous formaldehyde solution (formalin, 1.5-2%). The mean storage time of body donors was 12.8 ± 5.6 months (range, 7-25 months). While most diagnostic methods were successful, samples showed significant variability in DNA quality and evaluability. DNA pyrosequencing as well as next-generation sequencing was successful in all investigated samples. Methylation analyses were partially not successful in some extend due to limited intact DNA yield for these analyses. Taken together, the use of prolonged formalin-fixed tissue samples from body donors offers new avenues in research and education, as these samples could be used for morpho-molecular studies and the establishment of biobanks, especially for tissue types that cannot be preserved and studied in vivo. Pathological ward rounds, sample collection, and histopathological and molecular workup have been integrated in the gross anatomy course in Ulm as an integral part of the curriculum, linking anatomy and pathology and providing medical students early insight into the broad field of (molecular) pathology., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Digital PCR (dPCR) holds immense potential for precisely detecting nucleic acid markers essential for personalized medicine. However, its broader application is hindered by high consumable costs, complex procedures, and restricted multiplexing capabilities. To address these challenges, an all-in-one dPCR system is introduced that eliminates the need for microfabricated chips, offering fully automated operations and enhanced multiplexing capabilities. Using this innovative oscillation-induced droplet generation technique, OsciDrop, this system supports a comprehensive dPCR workflow, including precise liquid handling, pipette-based droplet printing, in situ thermocycling, multicolor fluorescence imaging, and machine learning-driven analysis. The system's reliability is demonstrated by quantifying reference materials and evaluating HER2 copy number variation in breast cancer. Its multiplexing capability is showcased with a quadruplex dPCR assay that detects key EGFR mutations, including 19Del, L858R, and T790M in lung cancer. Moreover, the digital stepwise melting analysis (dSMA) technique is introduced, enabling high-multiplex profiling of seven major EGFR variants spanning 35 subtypes. This innovative dPCR system presents a cost-effective and versatile alternative, overcoming existing limitations and paving the way for transformative advances in precision diagnostics., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Background/aim: Lung cancer, primarily non-small cell lung cancer (NSCLC), is the leading cause of cancer deaths globally. In Greece in 2020, 8,960 new cases were reported. NSCLC's 5-year survival rates range from 54% (stage I) to less than 2% (stage IV); however, innovative therapies like immune check points inhibitors (ICIs) and targeted treatments have notably enhanced outcomes. The aim of this study was to assess the 1 st and 2 nd line treatment patterns with the introduction of new treatment modalities. Additionally, we evaluated biomarker testing approaches in NSCLC., Patients and Methods: LACHESIS was a retrospective multinational study, collecting and analyzing data from adult patients from Russia, Bulgaria, and Greece with metastatic NSCLC either newly diagnosed or relapsed from earlier stages, who had the option to undergo biomarker testing (genetic alterations/programmed death-ligand 1 protein expression levels, PD-L1), and who received 1 st line treatment for squamous (SQ) or non-squamous (N-SQ) NSCLC. Subsequent lines of therapy were also reported., Results: The Greek site registered retrospective data from 250 NSCLC patients, of whom 206 were newly diagnosed (ND) metastatic NSCLC patients and 44 were patients relapsed from earlier stages. Seventy-two had SQ NSCLC and 169 had N-SQ NSCLC. For these patients, treatment patterns including immunotherapy±chemotherapy combinations were recorded. Biomarker testing patterns, including genetic alterations and PD-L1 expression levels were also documented., Conclusion: LACHESIS provides treatment patterns and biomarker testing data. Greek patients were treated according to international guidelines, with immunotherapy as a viable option, particularly for PD-L1 levels over 50%. Biomarker testing, crucial for non-squamous (N-SQ) cases, should yield timely results for driver mutations, prioritizing patient benefits., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Molecular pathological examinations of tumor samples encompass a wide range of diagnostic analyses. Especially in recent years, numerous new biomarkers have come to the forefront-the analysis of which is crucial for therapy decisions., Objectives: Within the field of molecular pathology, the demands of next generation sequencing (NGS)-based requirements have experienced massive growth in recent years. To meet this demand, methods are constantly being adapted and further developed. The following sections aim to illuminate how this trend arises and which analyses are gaining importance., Methods: The article provides an overview of the essential nucleic acid-based analysis techniques in the field of massive parallel sequencing. Terms such as DNA- and RNA-based techniques, as well as the associated analysis methods, are described, particularly with regard to their use in routine molecular pathological diagnostics., Results: The breadth of genomic sequencing has been steadily growing in recent years, particularly due to the increasing relevance of personalized medicine, along with the rising approvals of targeted therapeutics. This necessitates, among other things, the analysis of new biomarkers. The diagnostics as part of interdisciplinary molecular tumor boards (MTB) are now based on large gene panels (> 1 megabase). Furthermore, through the "Modellvorhaben Genomsequenzierung" § 64e, whole exome or whole genome sequencing has been made available for oncological patients. Given these developments, it is evident that future analyses will require the integration of additional omics fields, such as whole transcriptome analysis, epigenomics, and proteomics., Conclusion: The challenges of personalized medicine along with the necessity of simultaneously assessing numerous new biomarkers require the implementation and execution of new techniques in molecular pathology whose complexity is steadily increasing., (© 2024. The Author(s).)

The continuing evolution of treatment options in thoracic oncology requires the pathologist to regularly update diagnostic algorithms for management of tumor samples. It is essential to decide on the best way to use tissue biopsies, cytological samples, as well as liquid biopsies to identify the different mandatory predictive biomarkers of lung cancers in a short turnaround time. However, biological resources and laboratory member workforce are limited and may be not sufficient for the increased complexity of molecular pathological analyses and for complementary translational research development. In this context, the surgical pathologist is the only one who makes the decisions whether or not to send specimens to immunohistochemical and molecular pathology platforms. Moreover, the pathologist can rapidly contact the oncologist to obtain a new tissue biopsy and/or a liquid biopsy if he/she considers that the biological material is not sufficient in quantity or quality for assessment of predictive biomarkers. Inadequate control of algorithms and sampling workflow may lead to false negative, inconclusive, and incomplete findings, resulting in inappropriate choice of therapeutic strategy and potentially poor outcome for patients. International guidelines for lung cancer treatment are based on the results of the expression of different proteins and on genomic alterations. These guidelines have been established taking into consideration the best practices to be set up in clinical and molecular pathology laboratories. This review addresses the current predictive biomarkers and algorithms for use in thoracic oncology molecular pathology as well as the central role of the pathologist, notably in the molecular tumor board and her/his participation in the treatment decision-making. The perspectives in this setting will be discussed., (© 2023. The Author(s).)

Molecular pathological diagnostics plays a central role in personalized oncology and requires multidisciplinary teamwork. It is just as relevant for the individual patient who is being treated with an approved therapy method or an individual treatment attempt as it is for prospective clinical studies that require the identification of specific therapeutic target structures or complex biomarkers for study inclusion. It is also of crucial importance for the generation of real-world data, which is becoming increasingly important for drug development. Future developments will be significantly shaped by improvements in scalable molecular diagnostics, in which increasingly complex and multi-layered data sets must be quickly converted into clinically useful information. One focus will be on the development of adaptive diagnostic strategies in order to be able to depict the enormous plasticity of a cancer disease over time., Competing Interests: DK erklärt Vortragshonorare von AstraZeneca, Bristol-Myers Squibb, Pfizer, Lilly, Agilent, Takeda. MM erklärt keine Interessenskonflikte. JB erklärt Vortragshonorare von MSD. AS erklärt Beratergremien von Astra Zeneca, AGCT, Bayer, BMS, Eli Lilly, Illumina, Janssen, MSD, Novartis, Pfizer, Roche, Seattle Genetics, Takeda, Thermo Fisher; Forschungsunterstützung von Bayer, BMS, Chugai, Incyte., (Thieme. All rights reserved.)

Context.—: The US Food and Drug Administration (FDA) approved immune checkpoint inhibitor therapy for patients with advanced solid tumors that have DNA mismatch repair defects or high levels of microsatellite instability; however, the FDA provided no guidance on which specific clinical assays should be used to determine mismatch repair status., Objective.—: To develop an evidence-based guideline to identify the optimal clinical laboratory test to identify defects in DNA mismatch repair in patients with solid tumor malignancies who are being considered for immune checkpoint inhibitor therapy., Design.—: The College of American Pathologists convened an expert panel to perform a systematic review of the literature and develop recommendations. Using the National Academy of Medicine-endorsed Grading of Recommendations Assessment, Development and Evaluation approach, the recommendations were derived from available evidence, strength of that evidence, open comment feedback, and expert panel consensus. Mismatch repair immunohistochemistry, microsatellite instability derived from both polymerase chain reaction and next-generation sequencing, and tumor mutation burden derived from large panel next-generation sequencing were within scope., Results.—: Six recommendations and 3 good practice statements were developed. More evidence and evidence of higher quality were identified for colorectal cancer and other cancers of the gastrointestinal (GI) tract than for cancers arising outside the GI tract., Conclusions.—: An optimal assay depends on cancer type. For most cancer types outside of the GI tract and the endometrium, there was insufficient published evidence to recommend a specific clinical assay. Absent published evidence, immunohistochemistry is an acceptable approach readily available in most clinical laboratories., Competing Interests: Authors' disclosures of potential conflicts of interest and author contributions are found in the Appendix at the end of this article.

Cancer heterogeneities hold the key to a deeper understanding of cancer etiology and progression and the discovery of more precise cancer therapy. Modern pathological and molecular technologies offer a powerful set of tools to profile tumor heterogeneities at multiple levels in large patient populations, from DNA to RNA, protein and epigenetics, and from tumor tissues to tumor microenvironment and liquid biopsy. When coupled with well-validated epidemiologic methodology and well-characterized epidemiologic resources, the rich tumor pathological and molecular tumor information provide new research opportunities at an unprecedented breadth and depth. This is the research space where Molecular Pathological Epidemiology (MPE) emerged over a decade ago and has been thriving since then. As a truly multidisciplinary field, MPE embraces collaborations from diverse fields including epidemiology, pathology, immunology, genetics, biostatistics, bioinformatics, and data science. Since first convened in 2013, the International MPE Meeting series has grown into a dynamic and dedicated platform for experts from these disciplines to communicate novel findings, discuss new research opportunities and challenges, build professional networks, and educate the next-generation scientists. Herein, we share the proceedings of the Fifth International MPE meeting, held virtually online, on May 24 and 25, 2021. The meeting consisted of 21 presentations organized into the three main themes, which were recent integrative MPE studies, novel cancer profiling technologies, and new statistical and data science approaches. Looking forward to the near future, the meeting attendees anticipated continuous expansion and fruition of MPE research in many research fronts, particularly immune-epidemiology, mutational signatures, liquid biopsy, and health disparities., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Odontogenic tumors (OTs) are rare, with an estimated incidence rate of less than 0.5 cases per 100,000 per year. The causes of OTs remain unclear. Nonetheless, the majority of OTs seem to arise de novo, without an apparent causative factor. Although the etiopathogenesis of most OTs remains unclear, there have been some recent advances in understanding the genetic basis relating to specific histologies and clinical features. Molecular analyses performed by different techniques, including Sanger sequencing, next-generation sequencing, and allele-specific PCR, have uncovered mutations in genes related to the oncogenic MAPK/ERK signaling pathway. Genetic mutations in these pathway genes have been reported in epithelial and mixed OTs, in addition to odontogenic carcinomas and sarcomas. Notably, B‑RAF proto-oncogene serine/threonine kinase (BRAF) and KRAS proto-oncogene GTPase (KRAS) pathogenic mutations have been reported in a high proportion of ameloblastoma and ameloblastoma-related tumors and adenomatoid odontogenic tumors, respectively., Objective: To discuss how molecular profiling aids in diagnostic classification of odontogenic tumors., Conclusion: Molecular profiling of odontogenic tumors helps to identify patients for neoadjuvant therapies and reduces postoperative morbidity., (© 2022. The Author(s).)

Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, an important disease in the pig industry. Accurate and sensitive diagnostics such as DNA-based diagnostics are essential for preventing or responding to an outbreak. The specificity of DNA-based diagnostics depends on species-specific markers. Previously, an insertion element was found within an A. pleuropneumoniae-specific gene commonly used for A. pleuropneumoniae detection, prompting the need for additional species-specific markers. Herein, 12 marker candidates highly conserved (99 - 100% identity) among 34 A. pleuropneumoniae genomes (covering 13 serovars) were identified to be A. pleuropneumoniae-specific in silico , as these sequences are distinct from 30 genomes of 13 other Actinobacillus and problematic [ Actinobacillus ] species and more than 1700 genomes of other bacteria in the Pasteurellaceae family. Five marker candidates are within the apxIVA gene, a known A. pleuropneumoniae-specific gene, validating our in silico marker discovery method. Seven other A. pleuropneumoniae-specific marker candidates within the eamA , nusG, sppA , xerD , ybbN , ycfL, and ychJ genes were validated by polymerase chain reaction (PCR) to be specific to 129 isolates of A. pleuropneumoniae (covering all 19 serovars), but not to four closely related Actinobacillus species, four [ Actinobacillus ] species, or seven other bacterial species. This is the first study to identify A. pleuropneumoniae-specific markers through genome mining. Seven novel A. pleuropneumoniae-specific DNA markers were identified by a combination of in silico and molecular methods and can serve as additional or alternative targets for A. pleuropneumoniae diagnostics, potentially leading to better control of the disease. IMPORTANCE Species-specific markers are crucial for infectious disease diagnostics. Mutations within a marker sequence can lead to false-negative results, inappropriate treatment, and economic loss. The availability of several species-specific markers is therefore desirable. In this study, 12 DNA markers specific to A. pleuropneumoniae, a pig pathogen, were simultaneously identified. Five marker candidates are within a known A. pleuropneumoniae-specific gene. Seven novel markers can be used as additional targets in DNA-based diagnostics, which in turn can expedite disease diagnosis, assist farm management, and lead to better animal health and food security. The marker discovery strategy outlined herein requires less time, effort, and cost, and results in more markers compared with conventional methods. Identification of species-specific markers of other pathogens and corresponding infectious disease diagnostics are possible, conceivably improving health care and the economy.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive bronchoscopic procedure, well established as a diagnostic modality of first choice for diagnosis and staging of non-small cell lung cancer (NSCLC). The therapeutic decisions for advanced NSCLC require comprehensive profiling of actionable mutations, which is currently considered to be an essential part of the diagnostic process. The purpose of this study was to evaluate the utility of EBUS-TBNA cytology specimen for histological subtyping, molecular profiling of NSCLC by massive parallel sequencing (MPS), as well as for PD-L1 analysis. A retrospective review of 806 EBUS bronchoscopies was performed, resulting in a cohort of 132 consecutive patients with EBUS-TBNA specimens showing NSCLC cells in lymph nodes. Data on patient demographics, radiology features of the suspected tumor and mediastinal engagement, lymph nodes sampled, the histopathological subtype of NSCLC, and performed molecular analysis were collected. The EBUS-TBNA specimen proved sufficient for subtyping NSCLC in 83% and analysis of treatment predictive biomarkers in 77% (MPS in 53%). The adequacy of the EBUS-TBNA specimen was 69% for EGFR gene mutation analysis, 49% for analysis of ALK rearrangement, 36% for ROS1 rearrangement, and 33% for analysis of PD-L1. The findings of our study confirm that EBUS-TBNA cytology aspirate is appropriate for diagnosis and subtyping of NSCLC and largely also for treatment predictive molecular testing, although more data is needed on the utility of EBUS cytology specimen for MPS and PD-L1 analysis., Competing Interests: The authors have declared that no competing interests exist.

Besides promoting inflammation by mobilizing lipid mediators, group IIA secreted phospholipase A2 (sPLA2-IIA) prevents bacterial infection by degrading bacterial membranes. Here, we show that, despite the restricted intestinal expression of sPLA2-IIA in BALB/c mice, its genetic deletion leads to amelioration of cancer and exacerbation of psoriasis in distal skin. Intestinal expression of sPLA2-IIA is reduced after treatment with antibiotics or under germ-free conditions, suggesting its upregulation by gut microbiota. Metagenome, transcriptome, and metabolome analyses have revealed that sPLA2-IIA deficiency alters the gut microbiota, accompanied by notable changes in the intestinal expression of genes related to immunity and metabolism, as well as in the levels of various blood metabolites and fecal bacterial lipids, suggesting that sPLA2-IIA contributes to shaping of the gut microbiota. The skin phenotypes in Pla2g2a-/- mice are lost (a) when they are cohoused with littermate WT mice, resulting in the mixing of the microbiota between the genotypes, or (b) when they are housed in a more stringent pathogen-free facility, where Pla2g2a expression in WT mice is low and the gut microbial compositions in both genotypes are nearly identical. Thus, our results highlight a potentially new aspect of sPLA2-IIA as a modulator of gut microbiota, perturbation of which affects distal skin responses.

Secreted phospholipase A2-IIA (sPLA2-IIA) hydrolyzes phospholipids to liberate lysophospholipids and fatty acids. Given its poor activity toward eukaryotic cell membranes, its role in the generation of proinflammatory lipid mediators is unclear. Conversely, sPLA2-IIA efficiently hydrolyzes bacterial membranes. Here, we show that sPLA2-IIA affects the immune system by acting on the intestinal microbial flora. Using mice overexpressing transgene-driven human sPLA2-IIA, we found that the intestinal microbiota was critical for both induction of an immune phenotype and promotion of inflammatory arthritis. The expression of sPLA2-IIA led to alterations of the intestinal microbiota composition, but housing in a more stringent pathogen-free facility revealed that its expression could affect the immune system in the absence of changes to the composition of this flora. In contrast, untargeted lipidomic analysis focusing on bacteria-derived lipid mediators revealed that sPLA2-IIA could profoundly alter the fecal lipidome. The data suggest that a singular protein, sPLA2-IIA, produces systemic effects on the immune system through its activity on the microbiota and its lipidome.

Sensitive, reliable and fast diagnostic tools that are applicable in low-resource settings, at the point of care (PoC), are seen as crucial in the fight against visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Addressing the need for a PoC test, several diagnostic tests, including serological and molecular methods, have been developed and evaluated in the past. One promising molecular method, already implemented for diagnosis of a range of diseases, is the loop-mediated isothermal amplification (LAMP) protocol. In this systematic review and meta-analysis, using a comprehensive search strategy, we focus on studies evaluating the performance of LAMP for the diagnosis of leishmaniasis in humans and other mammals such as dogs, compared with microscopy and/or any other molecular diagnostic method. A meta-analysis, pooling sensitivity and specificity rates and calculating areas under the curve (AUCs) in summary receiver operating characteristic (SROC) plots, was conducted on datasets extracted from studies, grouped by clinical condition and sample type. We found high sensitivity and specificity for LAMP when compared with microscopy and PCR using blood samples, with pooled estimate values of > 90% for all subgroups, corresponding to calculated AUC values > 0.96, except for LAMP compared to microscopy for diagnosis of CL. However, only a limited number of studies were truly comparable. Most of the observed heterogeneity is likely based on true differences between the studies rather than sampling error only. Due to simple readout methods and low laboratory equipment requirements for sample preparation compared to other molecular methods, LAMP is a promising candidate for a molecular (near-)PoC diagnostic method for VL and CL., (© 2022. The Author(s).)

Background: The prevalence of Alzheimer's disease (AD) is greater in women compared to men, but the reasons for this remain unknown. This sex difference has been widely neglected in experimental studies using transgenic mouse models of AD., Objective: Here, we studied behavior and molecular pathology of 5-month-old 5XFAD mice, which express mutated human amyloid precursor protein and presenilin-1 on a C57BL/6J background, versus their wild-type littermate controls, to compare both sex- and genotype-dependent differences., Methods: A novel behavioral paradigm was utilized (OF-NO-SI), comprising activity measures (Open Field, OF) arena, followed by Novel Object exploration (NO) and Social Interaction (SI) of a sex-matched conspecific. Each segment consisted of two repeated trials to assess between-trial habituation. Subsequently, brain pathology (amyloid load, stress response and inflammation markers, synaptic integrity, trophic support) was assessed using qPCR and western blotting., Results: Female 5XFAD mice had higher levels of human APP and amyloid-β and heightened inflammation versus males. These markers correlated with hyperactivity observed in both sexes, yet only female 5XFAD mice presented with subtle deficits in object and social exploration. Male animals had higher expression of stress markers and neurotrophic factors irrespective of genotype, this correlated with cognitive performance., Conclusion: The impact of sex on AD-relevant phenotypes is in line with human data and emphasizes the necessity of appropriate study design and reporting. Differential molecular profiles observed in male versus female mice offer insights into possible protective mechanisms, and hence treatment strategies.

Objectives: To assess the concordance between lymphoma diagnoses made via tissue biopsy by local pathologists and also to assess the after review of these specimens by more specialized hematopathologists., Methods: A prospective, non-interventional and multicenter study was conducted at seven sites in Mexico from January 2017 to October 2017. Eligible biopsies were sampled from patients with a previous diagnosis of lymphoma on lymph node biopsy or a diagnosis of extranodal lymphoma, with adequate amount and tissue preservation for the review analysis. The biopsy tissues reviewed by local pathologists were also reviewed by hematopathologists participating in the study. The concordance in diagnosis results was classified into three categories: diagnostic agreement, minor discrepancy and major discrepancy., Results: Out of 111 samples received, 105 samples met the eligibility criteria and were included for full analysis. The median patient age (range) was 54 (16-94) years. A diagnostic agreement was observed in 23 (21.9%) biopsies, minor discrepancies were observed in 32 (30.5%) biopsies and major discrepancies were observed in 50 (47.6%) biopsies. Diagnostic concordance varied across the seven study sites; the rate of major discrepancies ranged from 0% to 100% and the rate of diagnostic agreement ranged from 0% to 81.8%. Out of the 105 reviewed biopsies, a total of 89 cases were diagnosed as lymphoma by hematopathologists., Conclusions: This study showed that major discrepancies were observed following the review by hematopathologists compared with that of the local pathologist's initial diagnosis in nearly one-half cases. In addition, there was a wide variation in the percentage of diagnostic agreements and discrepancies among different study sites.

Purpose: Due to relevant repercussions on reproductive medicine, we aimed to evaluate feasibility of RT-PCR as a detection method of SARS-CoV-2 RNA in seminal fluid., Methods: A qualitative determination of the RT-PCR assays in semen was performed through different approaches: (1) efficiency of RNA extraction from sperm and seminal plasma was determined using PRM1 and PRM2 mRNA and a heterologous system as control; (2) samples obtained by diluting viral preparation from a SARS-CoV-2 panel (virus cultured in Vero E6 cell lines) were tested; (3) viral presence in different fractions of seminal fluid (whole sample, seminal plasma and post-centrifugation pellet) was evaluated. Semen samples from mild and recovered COVID-19 subjects were collected by patients referring to the Infectious Disease Department of the Policlinico Umberto I Hospital - "Sapienza" University of Rome. Control subjects were recruited at the Laboratory of Seminology-Sperm Bank "Loredana Gandini'' of the same hospital., Results: The control panel using viral preparations diluted in saline and seminal fluid showed the capability to detect viral RNA presence with C t values depending on the initial viral concentration. All tested semen samples were negative for SARS-CoV-2, regardless of the nasopharyngeal swab result or seminal fluid fraction., Conclusion: These preliminary data show that RT-PCR for SARS-CoV-2 RNA testing appears to be a feasible method for the molecular diagnosis of SARS-CoV-2 in seminal fluid, supported by results of the control panel. The ability to detect SARS-CoV-2 in semen is extremely important for reproductive medicine, especially in assisted reproductive technology and sperm cryopreservation., (© 2021. The Author(s).)

Background: The difficulty in interpreting somatic alterations is correlated with the increase in sequencing panel size. To correctly guide the clinical management of patients with cancer, there needs to be accurate classification of pathogenicity followed by actionability assessment. Here, we describe a specific detailed workflow for the classification of the pathogenicity of somatic variants in cancer into five categories: benign, likely benign, unknown significance, likely pathogenic and pathogenic., Methods: Classification is obtained by combining a set of eight relevant criteria in favour of either a pathogenic or a benign effect (pathogenic stand-alone, pathogenic very strong, pathogenic strong, pathogenic moderate, pathogenic supporting, benign supporting, benign strong and benign stand-alone)., Results: Our guide is concordant with the ACMG/AMP 2015 guidelines for germline variants. Interpretation of somatic variants requires considering specific criteria, such as the disease and therapeutic context, co-occurring genomic events in the tumour when available and the use of cancer-specific variant databases. In addition, the gene role in tumorigenesis (oncogene or tumour suppressor gene) also needs to be taken into consideration., Conclusion: Our classification could contribute to homogenize best practices on somatic variant pathogenicity interpretation and improve interpretation consistency both within and between laboratories., Competing Interests: Conflict of interest statement The authors declare no conflict of interest., (Copyright © 2021. Published by Elsevier Ltd.)

CRISPR-Cas12a is a leading technology for development of model organisms, therapeutics, and diagnostics. These applications could benefit from chemical modifications that stabilize or tune enzyme properties. Here we chemically modify ribonucleotides of the AsCas12a CRISPR RNA 5' handle, a pseudoknot structure that mediates binding to Cas12a. Gene editing in human cells required retention of several native RNA residues corresponding to predicted 2'-hydroxyl contacts. Replacing these RNA residues with a variety of ribose-modified nucleotides revealed 2'-hydroxyl sensitivity. Modified 5' pseudoknots with as little as six out of nineteen RNA residues, with phosphorothioate linkages at remaining RNA positions, yielded heavily modified pseudoknots with robust cell-based editing. High trans activity was usually preserved with cis activity. We show that the 5' pseudoknot can tolerate near complete modification when design is guided by structural and chemical compatibility. Rules for modification of the 5' pseudoknot should accelerate therapeutic development and be valuable for CRISPR-Cas12a diagnostics., (© 2021. The Author(s).)

Blueberry stunt phytoplasma (BBSP; 'Candidatus Phytoplasma asteris') is an insect-vectored plant pathogen that causes severe yield losses in blueberry (Vaccinium corymbosum), which is the most valuable fruit crop in Canada. Rapid, field-based diagnostic assays are desirable tools for the control of BBSP, as part of an integrated, proactive approach to production management termed biovigilance. We designed and validated a chaperonin-60 (cpn60)-targeted LAMP assay for detection of BBSP, providing a rapid, low cost, field-deployable diagnostic option. Our validation demonstrates that the assay is reproducible, with high analytical specificity and improved sensitivity when compared with 16S rRNA nested PCR. We applied the validated LAMP assay to nearly 2000 blueberry samples from Québec and Nova Scotia over three growing seasons (2016-2018). Our surveys revealed that BBSP is present in most sites across both provinces, though detection of the pathogen in individual plants varied in different tissues across sampling dates and across years, and evidence of spread between plants was limited. To quantify pathogen load in select plants, we designed additional qPCR and ddPCR assays, also based on cpn60. We found that pathogen load fluctuates in individual plants, both within and between growing seasons. Finally, we designed an interactive map to visualize the results of our surveys. These results provide a validated diagnostic assay that can be used as part of a biovigilance strategy for detecting and controlling infections caused by BBSP., (© 2021. The Author(s).)

Warmblood Fragile Foal syndrome (WFFS) is an autosomal recessive condition that affects the maturation of collagen in affected foals. Foals affected with the disease typically die or are euthanised shortly after birth. WFFS is caused by a single nucleotide change at position 2032 of the equine PLOD1 gene, causing an impairment of the wild-type enzyme. A commercial test for the causative genetic mutation is currently available from companies operating under licence from Cornell University but it has limitations. This test requires amplification of a region of the PLOD1 gene encompassing the site of interest, followed by Sanger sequencing of that region and computational analysis. We describe here the development of an alternative, real-time PCR based assay that rapidly and reliably differentiates between the wild-type and WFFS associated nucleotides without the need for sequencing, thus increasing the potential for high throughput analysis of large numbers of samples in a cost-effective manner., Competing Interests: The authors have declared that no competing interests exist.

The last two decades have witnessed the emergence of three deadly coronaviruses (CoVs) in humans: severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). There are still no reliable and efficient therapeutics to manage the devastating consequences of these CoVs. Of these, SARS-CoV-2, the cause of the currently ongoing coronavirus disease 2019 (COVID-19) pandemic, has posed great global health concerns. The COVID-19 pandemic has resulted in an unprecedented crisis with devastating socio-economic and health impacts worldwide. This highlights the fact that CoVs continue to evolve and have the genetic flexibility to become highly pathogenic in humans and other mammals. SARS-CoV-2 carries a high genetic homology to the previously identified CoV (SARS-CoV), and the immunological and pathogenic characteristics of SARS-CoV-2, SARS-CoV, and MERS contain key similarities and differences that can guide therapy and management. This review presents salient and updated information on comparative pathology, molecular pathogenicity, immunological features, and genetic characterization of SARS-CoV, MERS-CoV, and SARS-CoV-2; this can help in the design of more effective vaccines and therapeutics for countering these pathogenic CoVs.

Background: To reduce the high incidence and mortality of gastric cancer (GC), we aimed to develop deep learning-based models to assist in predicting the diagnosis and overall survival (OS) of GC patients using pathological images., Methods: 2333 hematoxylin and eosin-stained pathological pictures of 1037 GC patients were collected from two cohorts to develop our algorithms, Renmin Hospital of Wuhan University (RHWU) and the Cancer Genome Atlas (TCGA). Additionally, we gained 175 digital pictures of 91 GC patients from National Human Genetic Resources Sharing Service Platform (NHGRP), served as the independent external validation set. Two models were developed using artificial intelligence (AI), one named GastroMIL for diagnosing GC, and the other named MIL-GC for predicting outcome of GC., Findings: The discriminatory power of GastroMIL achieved accuracy 0.920 in the external validation set, superior to that of the junior pathologist and comparable to that of expert pathologists. In the prognostic model, C-indices for survival prediction of internal and external validation sets were 0.671 and 0.657, respectively. Moreover, the risk score output by MIL-GC in the external validation set was proved to be a strong predictor of OS both in the univariate (HR = 2.414, P < 0.0001) and multivariable (HR = 1.803, P = 0.043) analyses. The predicting process is available at an online website (https://baigao.github.io/Pathologic-Prognostic-Analysis/)., Interpretation: Our study developed AI models and contributed to predicting precise diagnosis and prognosis of GC patients, which will offer assistance to choose appropriate treatment to improve the survival status of GC patients., Funding: Not applicable., Competing Interests: Declaration of Competing Interest All authors have no conflicts of interest to disclose., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Synthetic biology seeks to redesign biological systems to perform novel functions in a predictable manner. Recent advances in bacterial and mammalian cell engineering include the development of cells that function in biological samples or within the body as minimally invasive diagnostics or theranostics for the real-time regulation of complex diseased states. Ex vivo and in vivo cell-based biosensors and therapeutics have been developed to target a wide range of diseases including cancer, microbiome dysbiosis and autoimmune and metabolic diseases. While probiotic therapies have advanced to clinical trials, chimeric antigen receptor (CAR) T cell therapies have received regulatory approval, exemplifying the clinical potential of cellular therapies. This Review discusses preclinical and clinical applications of bacterial and mammalian sensing and drug delivery platforms as well as the underlying biological designs that could enable new classes of cell diagnostics and therapeutics. Additionally, we describe challenges that must be overcome for more rapid and safer clinical use of engineered systems., (© 2021. Springer Nature Limited.)

Rapid diagnosis of microorganisms and antibiotic resistance is vital for the appropriate treatment of patients with lower respiratory infections, especially for patients in Intensive Care Unit. We conducted a multicenter prospective study to evaluate the ability of the Unyvero pneumonia system for rapid detection from bronchoalveolar lavage fluid (BALF) in China. Eighty-four patients with lower respiratory infections were enrolled, and their BALF samples were collected, and Unyvero, a rapid molecular diagnostic sample-to-answer solution based on multiple PCRs, was applied to detect 21 types of pathogens and 19 types of resistance markers, compared to a routine bacterial culture method. The overall concordance of Unyvero and routine culture was 69/84 (82.1%). Unyvero detected more microorganisms than routine culture (38.1% vs 27.4%, P<0.05) and reported multi-pathogens in more patients than routine culture (10.7% vs 2.4%, P=0.01). The overall sensitivity and specificity of Unyvero for bacteria detection were 84.0% and 98.0%. Besides, Unyvero showed a good performance for antibiotic-resistant bacteria, except Pseudomonas aeruginosa. The concordance was 87.5-100% for methicillin-resistant Staphylococcus aureus and carbapenem-resistant isolates but was only 20-33.3% for Pseudomonas aeruginosa. The high-level semi-quantitative signal intensity of microorganisms detected positive by Unyvero correlates well with positive bacterial cultures. For specimens that were exposed to antibiotic treatment, the Unyvero pneumonia system showed a high concordance with routine bacterial culture and performs well for the detection of antibiotic-resistant bacteria, especially, carbapenem-resistant Klebsiella pneumoniae. It shows promise in guiding the clinical use of antibiotics, such as ceftazidime/avibactam. However, the system needs improvement in detecting resistance markers of Pseudomonas aeruginosa., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Aneurysmal (ABC) and simple bone cysts (SBC) have been traditionally distinguished by radiological and histopathological features. However, there is some radiological and histopathological overlap between ABC and SBC. ABC is characterised by USP6 fusions while, recently, NFATC2 fusions have been found in a large proportion of SBC. Identifying these fusions may assist in confirming the diagnosis of either ABC or SBC. To elaborate the potential benefit of molecular testing, we report a prospective series of 19 consecutive bone cysts with comprehensive radiological, histopathological and molecular diagnostics. Integrating radiological, histopathological and molecular findings, 11 cysts were diagnosed as SBC and 8 as ABC. Radiologically, 6 of 11 SBC and 6 of 8 ABC were diagnosed as ABC. Fibrin-like collagen deposits were identified in 8 of 11 (73%) SBC and 3 of 8 (38%) ABC. Nodular fasciitis-like areas were identified in 6 of 8 (75%) ABC and in 7 of 11 (64%) SBC. A USP6 fusion was identified in all 8 ABC, including a novel RBM5-USP6 fusion. An NFATC2 fusion was found in 7 of 11 SBC (FUS-NFATC2 fusion in 5 and EWSR1-NFATC2 in 2 cases). There is radiological and histopathological overlap between SBC and ABC in a significant proportion of cases. A diagnosis of ABC is frequently suggested radiologically in SBC, and fibrin-like deposits, thought to be specific for SBC, may be found in some ABC. Molecular testing may significantly improve diagnostic accuracy in bone cysts., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Aims: Application of advanced molecular pathology in rare tumours is hindered by low sample numbers, access to specialised expertise/technologies and tissue/assay QC and rapid reporting requirements. We assessed the feasibility of co-ordinated real-time centralised pathology review (CPR), encompassing molecular diagnostics and contemporary genomics (RNA-seq/DNA methylation-array)., Methods: This nationwide trial in medulloblastoma (<80 UK diagnoses/year) introduced a national reference centre (NRC) and assessed its performance and reporting to World Health Organisation standards. Paired frozen/formalin-fixed, paraffin-embedded tumour material were co-submitted from 135 patients (16 referral centres)., Results: Complete CPR diagnostics were successful for 88% (120/135). Inadequate sampling was the most common cause of failure; biomaterials were typically suitable for methylation-array (129/135, 94%), but frozen tissues commonly fell below RNA-seq QC requirements (53/135, 39%). Late reporting was most often due to delayed submission. CPR assigned or altered histological variant (vs local diagnosis) for 40/135 tumours (30%). Benchmarking/QC of specific biomarker assays impacted test results; fluorescent in-situ hybridisation most accurately identified high-risk MYC/MYCN amplification (20/135, 15%), while combined methods (CTNNB1/chr6 status, methylation-array subgrouping) best defined favourable-risk WNT tumours (14/135; 10%). Engagement of a specialist pathologist panel was essential for consensus assessment of histological variants and immunohistochemistry. Overall, CPR altered clinical risk-status for 29% of patients., Conclusion: National real-time CPR is feasible, delivering robust diagnostics to WHO criteria and assignment of clinical risk-status, significantly altering clinical management. Recommendations and experience from our study are applicable to advanced molecular diagnostics systems, both local and centralised, across rare tumour types, enabling their application in biomarker-driven routine diagnostics and clinical/research studies., (© 2021 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)

Background: Adnexal skin tumors are diagnostically challenging with few known molecular signatures. Recently, however, YAP1-MAML2 and YAP1-NUTM1 fusions were identified in poroid adnexal skin tumors., Methods: Herein, we subjected eight poroid adnexal skin tumors (three poromas and five porocarcinomas) to fusion gene analysis by whole transcriptome sequencing and next-generation DNA sequencing analysis., Results: YAP1 fusions were identified in six cases. YAP1-NUTM1 fusions were identified in two poromas and three porocarcinomas. A single case of porocarcinoma harbored a YAP1-MAML2 fusion. Two cases were negative for gene fusion. All cases that harbored YAP1-NUTM1 fusions showed nuclear protein in testis (NUT) expression by immunohistochemistry, with NUT being negative in the YAP1-MAML2-positive case. In this case series, we provide a detailed histopathologic description of six YAP1-fused poroid skin tumors, which we show harbor reproducible histopathologic features, to include broad, bulbous tumor tongues with admixtures of basaloid, poroid cells punctuated by squamatized cuticles and ductules, with uniform tumor nuclei featuring frequent grooves and pseudonuclear inclusions., Conclusions: Awareness of the characteristic histopathologic features of YAP1-fused poroid adnexal skin tumor is a step toward a more reproducible classification of adnexal skin tumors as well as a step toward targeted therapy for metastatic and/or unresectable examples of this poroid group of neoplasms., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)