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This overview of the molecular pathology of lung cancer includes a review of the most salient molecular alterations of the genome, transcriptome, and the epigenome. The insights provided by the growing use of next-generation sequencing (NGS) in lung cancer will be discussed, and interrelated concepts such as intertumor heterogeneity, intratumor heterogeneity, tumor mutational burden, and the advent of liquid biopsy will be explored. Moreover, this work describes how the evolving field of molecular pathology refines the understanding of different histologic phenotypes of non-small-cell lung cancer (NSCLC) and the underlying biology of small-cell lung cancer. This review will provide an appreciation for how ongoing scientific findings and technologic advances in molecular pathology are crucial for development of biomarkers, therapeutic agents, clinical trials, and ultimately improved patient care.

Background The standard of practice when a superficial lesion was identified during upper GI endoscopy is to take an endoscopic forceps biopsy (EFB) of the lesion. The histopathologic findings then will determine the management plan. Endoscopic submucosal dissection (ESD) enables en-bloc resection for early neoplasms of the gastrointestinal tract and provides an adequate specimen that permits a more reliable histopathologic assessment. The objective of this study was to determine the rate of histopathologic discrepancy between EFB and specimens resected by ESD, and to identify the predisposing risk factors for this discordance. Materials and methods This is a retrospective study, enrolling patients with superficial gastric neoplasms that underwent EFB followed by ESD. We divided cases to concordant or discordant group according to the histopathologic diagnosis of EFB and ESD specimens. We also analyzed the features that may have influenced the occurrence of histopathologic discordance and the association between discordant samples of adenocarcinoma and neoplastic invasion to deeper layers. Results A total of 115 gastric ESD procedures were performed with 84 patients meeting the inclusion criteria. Histopathologic discordance between EFB and ESD specimens were observed in 35.8% of cases (30/84 lesions). The univariant-bivariant analysis and multivariate logistic regression analysis showed that histologic discordance was closely related to the size of the lesions (P=0.028). Conclusion Histopathologic discrepancy between EFB and ESD specimens may occur in approximately one-third of cases, particularly for lesions over 20 mm, which may lead to crucial delays in gastric cancer precise diagnosis and treatment.

Background For patients with non-small cell lung cancer (NSCLC), targeted therapies are becoming part of the standard treatment. It is of question which information the clinicians provide on test requests and how the laboratories adapt test conclusions to this knowledge and regulations. Methods This study consisted of two components; 1) checking the presence of pre-defined elements (administrative and key for therapy-choice) on completed requests and corresponding reports in Belgian laboratories, both for tissue- and liquid biopsy (LB)-testing and b) opinion analysis from Belgian pathologists/molecular biologists and clinicians during national pathology/oncology meetings. Results Data from 4 out of 6 Belgian laboratories with ISO-accreditation for LB-testing were analyzed, of which 75% were university hospitals. On the scored requests (N = 4), 12 out of 19 ISO-required elements were present for tissue and 11 for LB-testing. Especially relevant patient history, such as line of therapy (for LB), tumor histology and the reason for testing were lacking. Similarly, 11 and 9 out of 18 elements were present in the reports (N = 4) for tissue and LB, respectively. Elements that pathologists/molecular biologists (N = 18) were missing on the request were the initial activating mutation, previous therapies, a clinical question and testing-related information. For reporting, an item considered important by both groups is the clinical interpretation of the test result. In addition, clinicians (N = 28) indicated that they also wish to read the percentage of neoplastic cells. Conclusions Communication flows between the laboratory and the clinician, together with possible pitfalls were identified. Based on the study results, templates for complete requesting and reporting were proposed.

The fifth edition of the World Health Organization (WHO) classification of digestive system tumours was published in 2019. The classification of invasive carcinoma is basically the same as in the fourth edition, but the description of each histological type has been updated, and some rare subtypes such as micropapillary carcinoma, gastric adenocarcinoma of the fundic gland type and undifferentiated carcinoma have been added and explained. Although this classification did not provide specific numerical criteria for the diagnosis of signet-ring cell carcinoma in poorly cohesive carcinoma, an additional study defined signet-ring cell carcinoma as having more than 90% signet-ring cells. The molecular classification of gastric cancer (Epstein-Barr virus-positive type, microsatellite instability type, genomically stable type, chromosomally unstable type) was additionally introduced. Many pages in the present classification have been devoted to precancerous lesions, and this article focuses on foveolar-type adenoma/dysplasia.Die fünfte Ausgabe der Klassifikation von Tumoren des Verdauungssystems durch die Weltgesundheitsorganisation (WHO) wurde 2019 veröffentlicht. Dabei ist die Klassifikation invasiver Karzinome prinzipiell gleich wie in der vierten Auflage, es erfolgte aber bei der Beschreibung der histologischen Typen eine Aktualisierung und die Ergänzung einiger seltener Subtypen wie das mikropapillärer Karzinom, das Magenadenokarzinom vom Fundusdrüsentyp und das undifferenzierte Karzinom. Obwohl diese Klassifikation keine spezifischen prozentualen Kriterien für die Diagnose des Siegelringzellkarzinoms bei nichtkohäsivem Karzinom lieferte, wurde in einer zusätzlichen Studie das Siegelringzellkarzinom als mehr als 90 % Siegelringzellen enthaltend definiert. Die molekulare Klassifikation von Magenkarzinomen (Epstein-Barr-Virus-positiver Typ – Mikrosatelliteninstabilitätstyp – genomisch stabiler Typ – chromosomal instabiler Typ) wurde zusätzlich eingeführt. In der WHO-Klassifikation wurde den Präkanzerosen viel Raum gewidmet. Der vorliegende Artikel konzentriert sich auf das Adenom/die Dysplasie vom foveolären Typ.

AIMS Primary mediastinal germ cell tumours (PMGCTs) are rare mediastinal neoplasms, and their diagnosis can be challenging, owing to small biopsy samples. The aim of this study was to develop a diagnostic algorithm using immunohistochemical staining, with a focus on novel markers, and molecular analysis of isochromosome 12p [i(12p)]. METHODS AND RESULTS Paraffin-embedded tissues of 32 mediastinal tumours were analysed with immunohistochemical staining for sal-like transcription factor 4 (SALL4), Lin-28 homologue A (LIN28), octamer-binding transcription factor 3/4 (OCT3/4), D2-40, cluster of differentiation 117 (CD117), sex-determining region Y-box 17, sex-determining region Y-box 2 (SOX2), cluster of differentiation 30, the β-subunit of human chorionic gonadotropin (β-hCG), GATA-binding protein 3 (GATA3), forkhead box protein A2 (FOXA2), glypican-3 (GPC3), α-fetoprotein (AFP), terminal deoxynucleotidyl transferase (TdT), nuclear protein of the testis (NUT), and pan-cytokeratin. Quantitative real-time polymerase chain reaction was performed to investigate the i(12p) status. Fifteen seminomas, seven teratomas, one yolk sac tumour, one choriocarcinoma and seven mixed PMGCTs were diagnosed. Each entity had different immunohistochemical staining patterns, which helped to distinguish them: OCT3/4, D2-40, CD117 and TdT for seminoma; OCT3/4 and SOX2 for embryonal carcinoma; FOXA2, GPC3 and AFP for yolk sac tumour; and β-hCG and GATA3 for choriocarcinoma. Mature teratomas stained positively for pan-cytokeratin in epithelial components and focally for SALL4, SOX2, GATA3, D2-40, and FOXA2. Furthermore, a NUT carcinoma mimicking a PMGCT was diagnosed, showing strong nuclear SOX2 staining and speckled nuclear NUT staining. i(12p) was detected in 24 of 27 PMGCTs (89%). CONCLUSION A diagnostic algorithm is of great importance for a reliable diagnosis of PMGCT in, usually small, tissue biopsy samples. Therefore, a combination of three to four antibodies to identify the correct histological subtype is usually necessary, in addition to morphological features. The i(12p) status serves as an additional option to indicate a germ cell origin in selected cases.

Acute lymphoblastic leukaemia (ALL) remains a leading cause of non-traumatic death in children, and the majority of adults diagnosed will succumb to the disease. Recent advances in molecular biology and bioinformatics have enabled more detailed genomic analysis and a better understanding of the molecular biology of ALL. A number of recurrent genomic drivers have recently been described, which not only aid in diagnosis and prognostication, but also may offer opportunities for specific therapeutic targeting. The present review summarises B-ALL genomic pathology at diagnosis, including lesions detectable using traditional cytogenetic methods as well as those detected only through advanced molecular techniques.

Conventional tissue sampling can lead to misdiagnoses and repeated biopsies. Additionally, tissue processed for histopathology suffers from poor nucleic acid quality and/or quantity for downstream molecular profiling. Targeted micro-sampling of tissue can ensure accurate diagnosis and molecular profiling in the presence of spatial heterogeneity, especially in tumors, and facilitate acquisition of fresh tissue for molecular analysis. In this study, we explored the feasibility of performing 1–2 mm precision biopsies guided by high-resolution reflectance confocal microscopy (RCM) and optical coherence tomography (OCT), and reflective metallic grids for accurate spatial targeting. Accurate sampling was confirmed with either histopathology or molecular profiling through next generation sequencing (NGS) in 9 skin cancers in 7 patients. Imaging-guided 1–2 mm biopsies enabled spatial targeting for in vivo diagnosis, feature correlation and depth assessment, which were confirmed with histopathology. In vivo 1-mm targeted biopsies achieved adequate quantity and high quality of DNA for next-generation sequencing. Subsequent mutational profiling was confirmed on 1 melanoma in situ and 2 invasive melanomas, using a 505-gene mutational panel called Memorial Sloan Kettering-Integrated mutational profiling of actionable cancer targets (MSK-IMPACT). Differential mutational landscapes, in terms of number and types of mutations, were found between invasive and in situ melanomas in a single patient. Our findings demonstrate feasibility of accurate sampling of regions of interest for downstream histopathological diagnoses and molecular pathology in both in vivo and ex vivo settings with broad diagnostic, therapeutic and research potential in cutaneous diseases accessible by RCM-OCT imaging.

Epidermal growth factor receptor (EGFR) mutation testing in advanced non-small-cell lung cancer (NSCLC) has evolved rapidly over the past decade, largely triggered by the introduction of the targeted EGFR tyrosine kinase inhibitors (TKIs). Initially used to detect common EGFR mutations and determine the most appropriate first-line therapy at diagnosis, testing methodologies have expanded to test for multiple mutations at multiple time points throughout the disease course. Here we review the current mutation testing approaches, including types of biopsies, and the available assays commonly used in the clinic. Specific application of these approaches in advanced NSCLC, including current guideline recommendations, and potential future developments are discussed.

COVID-19 virus is a causative agent of viral pandemic in human beings which specifically targets respiratory system of humans and causes viral pneumonia. This unusual viral pneumonia is rapidly spreading to all parts of the world, currently affecting about 105 million people with 2.3 million deaths. Current review described history, genomic characteristics, replication, and pathogenesis of COVID-19 with special emphasis on Nigella sativum (N. sativum) as a treatment option. N. sativum seeds are historically and religiously used over the centuries, both for prevention and treatment of different diseases. This review summarizes the potential role of N. sativum seeds against COVID-19 infection at levels of in silico, cell lines and animal models. Electronic Supplementary Material Supplementary material (Appendixes 1) is available in the online version of this article at 10.1007/s11655-021-3311-z.

Sand flies are often collected in urban areas, which has several implications for the risk of transmission of Leishmania Ross, 1903, to humans and other mammals. Given this scenario, we describe the sand fly fauna of caves and their surroundings in Mangabeiras Municipal Park (MMP) and Paredão Serra do Curral Park (PSCP), both located in the urban area of Belo Horizonte, Minas Gerais, Brazil, an endemic focus of visceral and cutaneous leishmaniasis. Collections were conducted monthly from November 2011 to October 2012, using CDC light traps exposed for two consecutive nights in four caves and their surroundings. Nonsystematized collections using Shannon traps and active searches were also performed around the caves. The presence of Leishmania DNA in collected female sand flies was evaluated by ITS1-PCR. A total of 857 sand flies representing fourteen species were collected in MMP, of which Evandromyia edwardsi (Mangabeira, 1941) was the most abundant. Leishmania amazonensis was detected in Brumptomyia nitzulescui (Costa Lima, 1932) and Ev. edwardsi, with the latter also having Leishmania braziliensis, Leishmania infantum, and Leishmania sp. A total of 228 sand flies representing four species were collected in PSCP, of which Sciopemyia microps (Mangabeira, 1942) was the most abundant. No females from PSCP were positive for Leishmania-DNA. Studies aimed at describing sand fly faunas of cave environments and detecting Leishmania are essential to understanding the relationship between these insects and this ecotope and assessing and monitoring areas that may pose risks to the health of visitors and employees.

The article focuses on the Considerations related to the use of molecular diagnostic tests in veterinary clinical and regulatory practice . Topics discussed include technological advances are creating an ever widening array of options for detecting pathogens and diagnosing diseases of concern in clinical and regulatory veterinary medicine;and new technology can facilitate timelier responses and, in some instances, move assays from laboratory-based to patient-side.

Progeroid syndromes (PSs) are characterized by the premature onset of age-related pathologies. The genetic mutations underlying PSs are functionally linked to genome maintenance and repair, supporting the causative role of DNA damage accumulation in aging. Recent advances from studies in animal models of PSs have provided new insight into the role of DNA repair mechanisms in human disease and the physiological adaptations to accumulating DNA damage during aging. The molecular pathology of PSs is reminiscent of the natural aging process, highlighting the relevance for a wide range of age-related diseases. Recent progress has led to the development of novel therapeutic strategies against age-related diseases that are relevant to rare diseases as well as the general aging population.

Purpose To evaluate the diagnostic yield and clinical relevance of clinical genome sequencing (cGS) as a first genetic test for patients with suspected monogenic disorders. Methods We conducted a prospective randomized study with pediatric and adult patients recruited from genetics clinics at Massachusetts General Hospital who were undergoing planned genetic testing. Participants were randomized into two groups: standard-of-care genetic testing (SOC) only or SOC and cGS. Results Two hundred four participants were enrolled, 202 were randomized to one of the intervention arms, and 99 received cGS. In total, cGS returned 16 molecular diagnoses that fully or partially explained the indication for testing in 16 individuals (16.2% of the cohort, 95% confidence interval [CI] 8.9–23.4%), which was not significantly different from SOC (18.2%, 95% CI 10.6–25.8%, P = 0.71). An additional eight molecular diagnoses reported by cGS had uncertain relevance to the participant's phenotype. Nevertheless, referring providers considered 20/24 total cGS molecular diagnoses (83%) to be explanatory for clinical features or worthy of additional workup. Conclusion cGS is technically suitable as a first genetic test. In our cohort, diagnostic yield was not significantly different from SOC. Further studies addressing other variant types and implementation challenges are needed to support feasibility and utility of broad-scale cGS adoption.

Upper gastroesophageal carcinomas consist of cancers arising from the esophagus and stomach. Squamous cell carcinomas and adenocarcinomas are seen in the esophagus and despite arising from the same organ have different biology. Gastric adenocarcinomas are categorized into 4 molecular subtypes: high Epstein-Barr virus load, microsatellite unstable cancers, chromosomal unstable (CIN) cancers, and genomically stable cancers. Genomically stable gastric cancers correlate highly with histologically defined diffuse-type cancers. Esophageal carcinomas and CIN gastric cancers often are driven by high-level amplifications of oncogenes and contain a high degree of intratumoral heterogeneity. Targeted therapeutics is an active area of research for gastroesophageal cancers.

Patients with inborn errors of immunity (IEI) present with a heterogeneous clinical and immunological phenotype, therefore a correct molecular diagnosis is crucial for the classification and subsequent therapeutic management. On the other hand, IEI are a group of rare congenital diseases with highly diverse features and, in most cases, an as yet unknown genetic etiology. Next generation sequencing has facilitated genetic examinations of rare inherited disorders during the recent years, thus allowing a suitable molecular diagnosis in the IEI patients. This review aimed to investigate the current findings about these techniques in the field of IEI, suggesting an efficient stepwise approach to molecular diagnosis of inborn errors of immunity.

Purpose Several targeted agents demonstrated efficacy in early clinical trials for gastrointestinal (GI) cancers, but in many cases, phase-III trials and/or approval by the European Medicines Agency (EMA) are lacking. The primary focus of this study was to assess the regulatory processes associated with use and reimbursement of off-label treatment in precision oncology and to evaluate the benefit of targeted therapy in a real-world population in Germany. Methods Our cohort comprises 137 patients with GI cancers and is biased towards cancer entities with a high frequency of known targetable alterations, such as cholangiocarcinoma. Genetic testing was used to identify molecular targets, and therapy response was evaluated based on CT scans. Results A molecular target for precision oncology was identified in 53 patients and 43 requests for cost coverage were submitted to health insurance companies. 60% of the requests received approval after initial application and another 7% after appeal. Half of the rejected requests were denied despite ESCAT IA level evidence. The median time between initiation of molecular testing and start of therapy was 75 days. 35 patients received matched targeted therapies (n = 28) or, in the case of MSI, immunotherapy (IO) (n = 7). We observed a trend in favor of molecular therapy when compared to the immediate prior treatment. Conclusion Relevant treatment options were identified by molecular testing in a significant subset of patients. When targeted therapies that lack EMA approval are considered, treatment initiation may be delayed by the duration of the molecular analysis and the regulatory processes.

Aims There is a widespread perception among clinicians and pathologists that the histomorphological assessment of minor salivary gland (MinSG) tumours is more difficult and hampered by more misdiagnoses than that of major salivary gland tumours. This is based on a vague, subjective clinical impression, lacking scientific proof. The aim of the present study was to identify and statistically verify potential reasons that could explain this difference. Methods and results We identified 14 putative clinical, pathological and combined clinicopathological reasons that, altogether, could explain the phenomenon of the perceived greater diagnostic difficulty associated with MinSG tumours. We performed a comprehensive literature search and a statistical comparison of data from a large personal consultation series (biased for difficult cases) with cumulated data from straightforward, unselected (non-consultation) series from the literature. By performing this comparison, we identified, with statistical significance, a comprehensive series of reasons, as well as of consequences, of the greater difficulty in diagnosing MinSG tumours. Conclusions Among the 14 criteria, high frequencies of initial incisional biopsies and of a low-grade category in malignant tumours emerged as the two most important reasons for enhanced diagnostic difficulty. Very rare entities, unusual locations, shortcomings in clinicopathological communication, and pecularities of the special anatomical location of the hard palate, such as tumour necrosis, mucosal ulceration, pseudoinvasion, and the peculiar phenomenon of 'tumoral-mucosal fusion', contribute to further diagnostic difficulties. The awareness of these shortcomings and pitfalls enables us to provide a series of recommendations for clinicians and pathologists that might aid in assessment and reduce the rate of misdiagnosis of MinSG tumours.

Cerebral amyloid angiopathy (CAA), a prevalent cerebral small vessel disease in the elderly and a common comorbidity of Alzheimer's disease, is characterized by cerebral vascular amyloid accumulation, cerebral infarction, microbleeds, and intracerebral hemorrhages and is a prominent contributor to vascular cognitive impairment and dementia. Here, we investigate proteome changes associated with specific pathological features in several brain regions of rTg-DI rats, a preclinical model of CAA. Whereas varying degrees of microvascular amyloid and associated neuroinflammation are found in several brain regions, the presence of microbleeds and occluded small vessels is largely restricted to the thalamic region of rTg-DI rats, indicating different levels of CAA and associated pathologies occur in distinct brain regions in this model. Here, using SWATHLC-MS/MS, we report specific proteomic analysis of isolated brain regions and employ pathway analysis to correlate regionally specific proteomic changes with uniquely implicated molecular pathways. Pathway analysis suggested common activation of tumor necrosis factor α (TNFα), abnormal nervous system morphology, and neutrophil degranulation in all three regions. Activation of transforming growth factor-β1 (TGF-β1) was common to the hippocampus and thalamus, which share high CAA loads, while the thalamus, which uniquely exhibits thrombotic events, additionally displayed activation of thrombin and aggregation of blood cells. Thus, we present significant and new insight into the cerebral proteome changes found in distinct brain regions with differential CAA-related pathologies of rTg-DI rats and provide new information on potential pathogenic mechanisms associated with these regional disease processes.

Dengue remains a major problem in the tropics. Several Asian countries have reported an increasing trend in the proportion of infants with dengue fever. However, most studies are limited to case reports or small case series from isolated outbreaks. We planned this study to look at clinico-laboratory profile, outcome, and predictors of severity in a large cohort of infants over a decade. Electronic medical records of infants admitted at a tertiary center of South India, with laboratory confirmed dengue infection between 2009 and 2019 were reviewed. Diagnosis was based on detection of NS-1 antigen and/or immunoglobulin M antibody against DENV(dengue virus) or positive DENV RNA polymerase chain reaction in infants presenting with acute febrile illness and clinical features consistent with dengue. Of 395 children with dengue admitted during study period, 99 (25%) were infants. A cyclical incidence pattern was noted, with higher cases in alternate years. Fever (99%) was most common, followed by gastrointestinal symptoms (vomiting, diarrhea—28%) and upper respiratory symptoms (cough, coryza—22%). Fifty-three infants had severe dengue, and 39 had shock. Fourteen children had multiorgan dysfunction syndrome, and 13 died. Infants with severe dengue were older than those with nonsevere disease, had lower serum albumin and greater frequency of severe thrombocytopenia, and had coagulopathy. On multivariable analysis, low serum albumin predicted development of severe dengue [P = 0.003, odds ratio 12.4 (95% confidence interval: 2.42–63.7)]. Dengue in infants may be challenging to recognize because of its undifferentiated presentation, with gastrointestinal and upper respiratory symptoms that are similar to other viral illness. Severe dengue is common in this sample, and lower serum albumin at presentation was predictive of severe disease.

INTRODUCTION: Molecular pathological epidemiology (MPE) is an integrative disciplinary area examining the relationships between various exposures and pathogenic signatures of diseases. In line with the accelerating advancements in MPE, social science and its health-related interdisciplinary areas have also developed rapidly. Accumulating evidence indicates the pathological role of social-demographic factors. We therefore initially proposed social MPE in 2015, which aims to elucidate etiological roles of social-demographic factors and address health inequalities globally. With the ubiquity of molecular diagnosis, there are ample opportunities for researchers to utilize and develop the social MPE framework. AREAS COVERED: Molecular subtypes of breast cancer have been investigated rigorously for understanding its etiologies rooted from social factors. Emerging evidence indicates pathogenic heterogeneity of neurological disorders such as Alzheimer’s disease. Presenting specific patterns of social-demographic factors across different molecular subtypes should be promising for advancing the screening, prevention, and treatment strategies of those heterogeneous disease. This article rigorously reviewed literatures investigating differences of race/ethnicity and socioeconomic status across molecular subtypes of breast cancer and Alzheimer’s disease to date. EXPERT OPINION: With advancements of the multi-omics technologies, we foresee a blooming of social MPE studies, which can address health disparities, advance personalized molecular medicine, and enhance public health.

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., Synthetic biology has enabled the development of engineered cells that can serve as ex vivo or in vivo diagnostic tools or therapeutic delivery systems. This Review discusses preclinical and clinical applications of bacterial and mammalian theranostic cells as well as their underlying biological designs and remaining hurdles to their successful clinical application.

Introduction Norovirus is highly contagious, and a few particles of this virus are sufficient to make people sick. It is desirable to develop quick and accurate laboratory methods to detect norovirus. Methods We evaluated two commercial molecular diagnostic assays, the Xpert Norovirus and the TRCReady NV, using clinical fecal samples. A reference method was performed using in-house real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR). Results The results of the real-time RT-PCR analysis of 60 suspected cases of norovirus infection showed 5 cases of Genogroup I (GI) positives and 21 cases of GII positives, among which was 1 GI and GII coinfection. The viral titers of the norovirus-positive samples ranged from 1.54 × 101 to 3.14 × 108 copies/μL. Norovirus GII.17 (12 cases, 48%) was the most frequently detected genotype in this study, followed by GII.4 (6 cases, 24%), GII.13 (2 cases, 8%), GI.2 (2 cases, 8%), GI.3 (2 cases, 8%), GI.1 (1 case, 4%), and GII.2 (1 case, 4%). The kappa coefficient was 1.000 (95% CI: 1.000–1.000) for Xpert Norovirus and 0.966 (95% CI: 0.896–1.000) for TRCReady NV, indicating a strong agreement. Conclusions Norovirus detection using Xpert Norovirus and TRCReady NV is highly useful for diagnosis and infection control because these assays are easy to operate, quick, and exhibit almost the same performance as that of real-time RT-PCR.

Summary The current personalized oncology era has witnessed significant efforts to integrate clinical, pathological, and molecular classifications. The growing need for molecular biomarkers to feed personalized oncology, together with the unprecedented wealth of knowledge on the molecular basis of bladder cancer, has led to a novel approach to this disease, incorporating molecularly generated data in clinical practice for locally advanced or metastatic disease. Translational research allows a better understanding of the early events in the development of urothelial carcinoma in the urinary bladder. Thus, mutations in the KMT2D and KDM6A chromatin-modifying genes confer competitive advantages that drive cells to colonize larger regions of the urothelium. Additional mutations in TP53, PIK3CA, FGFR3, or RB1 genes then trigger the process of malignant transformation in the urothelium. In the current review, we provide an overview of what could be the expected transition from the morphology-based classification to a combined, molecularly enriched reporting of clinically meaningful parameters aiming to promote personalized oncology of urothelial carcinoma.

Background Plasmodium vivax controlled human malaria infection (PvCHMI) is an important tool for evaluation of drugs, vaccines, and pathologies associated with this parasite. However, there are few data on safety due to limited numbers of PvCHMIs performed. Methods We report clinical and laboratory data, including hematological and biochemical profiles and adverse events (AEs), following mosquito bite-induced PvCHMI in malaria-naive study participants. Malaria diagnosis and treatment initiation was based on microscopic analysis of Giemsa-stained slides. Exploratory molecular assays were used to detect parasites using real-time polymerase chain reaction (PCR). Results AEs were mild to moderate and no study-related severe AEs were observed in any study participants. The majority of symptoms were transient, resolving within 48 hours. Molecular diagnostic methods detected parasitemia in 100% of study participants before malaria diagnosis using microscopy. Of reported AEs, microscopy detected 67%–100%, quantitative PCR 79%–100%, and quantitative real-time reverse-transcription PCR 96%–100% of study participants prior to appearance of symptoms. Almost all symptoms appeared after initiation of treatment, likely as known consequence of drug treatment. Conclusions PvCHMI is safe with the majority of infections being detected prior to appearance of clinical symptoms, which can be further alleviated by using sensitive molecular methods for clinical diagnosis. Clinical Trials Registration. NCT01157897.

Progressive apraxia of speech is a neurodegenerative syndrome affecting spoken communication. Molecular pathology, biochemistry, genetics, and longitudinal imaging were investigated in 32 autopsy-confirmed patients with progressive apraxia of speech who were followed over 10 years. Corticobasal degeneration and progressive supranuclear palsy (4R-tauopathies) were the most common underlying pathologies. Perceptually distinct speech characteristics, combined with age-at-onset, predicted specific 4R-tauopathy; phonetic subtype and younger age predicted corticobasal degeneration, and prosodic subtype and older age predicted progressive supranuclear palsy. Phonetic and prosodic subtypes showed differing relationships within the cortico-striato-pallido-nigro-luysial network. Biochemical analysis revealed no distinct differences in aggregated 4R-tau while tau H1 haplotype frequency (69%) was lower compared to 1000+ autopsy-confirmed 4R-tauopathies. Corticobasal degeneration patients had faster rates of decline, greater cortical degeneration, and shorter illness duration than progressive supranuclear palsy. These findings help define the pathobiology of progressive apraxia of speech and may have consequences for development of 4R-tau targeting treatment., Progressive apraxia of speech (PAOS) is a neurodegenerative syndrome of multiple etiologies which affects spoken communication. Here, the authors characterized the molecular pathology, biochemistry, genetics and longitudinal neuroimaging of 32 autopsy-confirmed patients with PAOS who were followed over 10 years.

Context.— The presence of allogeneic contamination impacts clinical reporting in cancer next-generation sequencing specimens. Although consensus guidelines recommend the identification of contaminating DNA as a part of quality control, implementation of contamination assessment methods in clinical molecular diagnostic laboratories has not been reported in the literature. Objective.— To develop and implement a method to assess allogeneic contamination in clinical cancer next-generation sequencing specimens. Design.— We describe a method to detect contamination based on the evaluation of single-nucleotide polymorphic sites from tumor-only specimens. We validate this method and apply it to a large cohort of cancer sequencing specimens. Results.— Identification of specimen contamination was validated via in silico and in vitro mixtures, and reference range and reproducibility were established in a panel of normal specimens. The algorithm accurately detects an episode of systemic contamination due to reagent impurity. We prospectively applied this algorithm across 7571 clinical cancer specimens from a targeted next-generation sequencing panel, in which 262 specimens (3.5%) were predicted to be affected by greater than 5% contamination. Conclusions.— Allogeneic contamination can be inferred from intrinsic cancer next-generation sequencing data without paired normal sequencing. The adoption of this approach can be useful as a quality control measure for laboratories performing clinical next-generation sequencing.

The effectiveness of targeted therapies with tyrosine kinase inhibitors in non-small-cell lung cancer (NSCLC) depends on the accurate determination of the genomic status of the tumour. For this reason, molecular analyses to detect genetic rearrangements in some genes (ie, ALK, ROS1, RET and NTRK) have become standard in patients with advanced disease. Since immunohistochemistry is easier to implement and interpret, it is normally used as the screening procedure, while fluorescence in situ hybridisation (FISH) is used to confirm the rearrangement and decide on ambiguous immunostainings. Although FISH is considered the most sensitive method for the detection of ALK and ROS1 rearrangements, the interpretation of results requires detailed guidelines. In this review, we discuss the various technologies available to evaluate ALK and ROS1 genomic rearrangements using these techniques. Other techniques such as real-time PCR and next-generation sequencing have been developed recently to evaluate ALK and ROS1 gene rearrangements, but some limitations prevent their full implementation in the clinical setting. Similarly, liquid biopsies have the potential to change the treatment of patients with advanced lung cancer, but further research is required before this technology can be applied in routine clinical practice. We discuss the technical requirements of laboratories in the light of quality assurance programmes. Finally, we review the recent updates made to the guidelines for the determination of molecular biomarkers in patients with NSCLC.

The use of rapid DNA sequencing technology in severely ill children in developed countries can accurately identify diagnoses and positively impact patient outcomes. This study sought to evaluate the outcome of Thai children and adults with unknown etiologies of critical illnesses with the deployment of rapid whole exome sequencing (rWES) in Thailand. We recruited 54 unrelated patients from 11 hospitals throughout Thailand. The median age was 3 months (range, 2 days-55 years) including 47 children and 7 adults with 52% males. The median time from obtaining blood samples to issuing the rWES report was 12 days (range, 5-27 days). A molecular diagnosis was established in 25 patients (46%), resulting in a change in clinical management for 24 patients (44%) resulting in improved clinical outcomes in 16 patients (30%). Four out of seven adult patients (57%) received the molecular diagnosis which led to a change in management. The 25 diagnoses comprised 23 different diseases. Of the 34 identified variants, 15 had never been previously reported. This study suggests that use of rWES as a first-tier investigation tool can provide tremendous benefits in critically ill patients with unknown etiology across age groups in Thailand.

Molecular testing has become part of the routine diagnostic workup of brain tumors after the implementation of integrated histomolecular diagnoses in the 2016 WHO classification update. It is important for every neuropathologist to be aware of practical preanalytical, analytical, and postanalytical factors that impact the performance and interpretation of molecular tests. Prior to testing, optimizing tumor purity and tumor amount increases the ability of the molecular test to detect the genetic alteration of interest. Recognizing basic molecular testing platform analytical characteristics allows selection of the optimal platform for each clinicopathological scenario. Finally, postanalytical considerations to properly interpret molecular test results include understanding the clinical significance of the detected genetic alteration, recognizing that detected clinically significant genetic alterations are occasionally germline constitutional rather than somatic tumor-specific, and being cognizant that recommended and commonly used genetic nomenclature may differ. Potential pitfalls in brain tumor molecular diagnosis are also discussed.

Context.— Molecular diagnostics play an increasing role in the diagnosis of Ewing sarcoma. The type of molecular testing used in clinical practice has been poorly described. Objective.— To describe patterns of translocation testing for newly diagnosed Ewing sarcoma. Design.— Children's Oncology Group (COG) trial AEWS1221 was a phase III randomized trial enrolling patients with newly diagnosed metastatic Ewing sarcoma from 2014 to 2019. Patients were required to have a histologic diagnosis of Ewing sarcoma, but translocation testing was not required. Sites provided types and results of any molecular diagnostics performed. Results.— Data from 305 enrolled patients were available. The most common type of molecular testing was fluorescence in situ hybridization (FISH) performed on the primary tumor (236 of 305 patients; 77.4%), with positive testing for an EWSR1 or FUS translocation in 211 (89.4%). Reverse transcription–polymerase chain reaction (RT-PCR) on the primary tumor was performed in 61 of 305 patients (20%), with positive results in 48 of 61 patients (78.7%). Next-generation sequencing was reported in 7 patients for the primary tumor and in 3 patients for metastatic sites. For all types of testing on either primary or metastatic tumor, 16 of 305 patients (5.2%) had no reported translocation testing. When evaluating all results from all testing, 44 of 305 patients (14.4%) lacked documentation of an abnormality consistent with a molecular diagnosis of Ewing sarcoma. Conclusions.— COG sites enrolling in a Ewing sarcoma trial have high rates of testing by FISH or PCR. A small proportion of patients have no translocation testing on either primary or metastatic sites. Next-generation sequencing techniques are not yet commonly used in this context.

Abdominal paragangliomas and pheochromocytomas (PPGLs) are rare neuroendocrine tumors of the infradiaphragmatic paraganglia and adrenal medulla, respectively. Although few pathologists outside of endocrine tertiary centers will ever diagnose such a lesion, the tumors are well known through the medical community—possible due to a combination of the sheer rarity, their often-spectacular presentation due to excess catecholamine secretion as well as their unrivaled coupling to constitutional susceptibility gene mutations and hereditary syndromes. All PPGLs are thought to harbor malignant potential, and therefore pose several challenges to the practicing pathologist. Specifically, a responsible diagnostician should recognize both the capacity and limitations of histological, immunohistochemical, and molecular algorithms to pinpoint high risk for future metastatic disease. This focused review aims to provide the surgical pathologist with a condensed update regarding the current strategies available in order to deliver an accurate prognostication of these enigmatic lesions.

Grade 3B follicular lymphoma (G3B FL) is rare, accounting for only 5-10% of FLs. Not only has it been routinely excluded from clinical trials, but data published on diagnosis, outcomes, choice of therapies and role of imaging are conflicting. With the advent of increasingly diverse treatment options for low-grade (G1-3A) FL, and the molecular subcategorisation of high-grade B-cell lymphomas, characterisation and treatment of G3B FL is ever more important as extrapolation of data becomes more difficult. New data have emerged exploring unique genetic characteristics, specific features on positron emission tomography imaging, choice of therapy, and outcomes of G3B FL in the current era. The present review will summarise and appraise these new data, and offer recommendations based on current evidence.

Environmental Mycobacterium ulcerans causes a disabling skin disease called Buruli ulcer. Recent studies completed the knowledge of the evolving geographic extension and epidemiology of Buruli ulcer in West Africa, where Côte d’Ivoire is reporting the highest number of cases. We report seven polymerase chain reaction-documented patients in Burkina Faso, a neighboring country of Côte d’Ivoire, where previously Buruli ulcer cases were confirmed primarily using clinical arguments.

The diagnosis of SARS-CoV-2 is based on the use of nucleic acid amplification tests (NAAT), especially rRT-PCR. The latter also allows us to quickly identify variants of concern. However, its use in follow-up of patients and the correlation between Ct value and the viability of the virus is controversial.

The SPIRIT (Standard Protocol Items: Recommendations for Interventional Trials) 2013 Statement was developed to provide guidance for inclusion of key methodological components in clinical trial protocols. However, these standards do not include guidance specific to pathology input in clinical trials. This systematic review aims to synthesise existing recommendations specific to pathology practice in clinical trials for implementation in trial protocol design. Articles were identified from database searches and deemed eligible for inclusion if they contained: (1) guidance and/or a checklist, which was (2) pathology‐related, with (3) content relevant to clinical trial protocols or could influence a clinical trial protocol design from a pathology perspective and (4) were published in 1996 or later. The quality of individual papers was assessed using the AGREE‐GRS (Appraisal of Guidelines for REsearch & Evaluation – Global Rating Scale) tool, and the confidence in cumulative evidence was evaluated using the GRADE‐CERQual (Grading of Recommendations Assessment, Development and Evaluation–Confidence in Evidence from Reviews of Qualitative research) approach. Extracted recommendations were synthesised using the best fit framework method, which includes thematic analysis followed by a meta‐aggregative approach to synthesis within the framework. Of the 10 184 records screened and 199 full‐text articles reviewed, only 40 guidance resources met the eligibility criteria for inclusion. Recommendations extracted from 22 guidance documents were generalisable enough for data synthesis. Seven recommendation statements were synthesised as follows: (1) multidisciplinary collaboration in trial design with early involvement of pathologists, particularly with respect to the use of biospecimens and associated biomarker/analytical assays and in the evaluation of pathology‐related parameters; (2) funding and training for personnel undertaking trial work; (3) selection of an accredited laboratory with suitable facilities to undertake scheduled work; (4) quality assurance of pathology‐related parameters; (5) transparent reporting of pathology‐related parameters; (6) policies regarding informatics and tracking biospecimens across trial sites; and (7) informed consent for specimen collection and retention for future research.

Reparació per recombinació homòloga; Càncer de pròstata metastàtic; Inhibidors de la poli(ADP-ribosa) polimerasa Reparación por recombinación homóloga; Cáncer de próstata metastásico; Inhibidores de la poli(ADP-ribosa) polimerasa Homologous recombination repair; Metastatic prostate cancer; Poly(ADP-ribose) polymerase inhibitors Analysis of the genomic landscape of prostate cancer has identified different molecular subgroups with relevance for novel or existing targeted therapies. The recent approvals of the poly(ADP-ribose) polymerase (PARP) inhibitors olaparib and rucaparib in the metastatic castration-resistant prostate cancer (mCRPC) setting signal the need to embed molecular diagnostics in the clinical pathway of patients with mCRPC to identify those who can benefit from targeted therapies. Best practice guidelines in overall biospecimen collection and processing for molecular analysis are widely available for several tumour types. However, there is no standard protocol for molecular diagnostic testing in prostate cancer. Here, we provide a series of recommendations on specimen handling, sample pre-analytics, laboratory workflow, and testing pathways to maximise the success rates for clinical genomic analysis in prostate cancer. Early involvement of a multidisciplinary team of pathologists, urologists, oncologists, radiologists, nurses, molecular scientists, and laboratory staff is key to enable optimal workflow for specimen selection and preservation at the time of diagnosis so that samples are available for molecular analysis when required. Given the improved outcome of patients with mCRPC and homologous recombination repair gene alterations who have been treated with PARP inhibitors, there is an urgent need to incorporate high-quality genomic testing in the routine clinical pathway of these patients. JM has served on advisory boards for Amgen, AstraZeneca, Clovis Oncology, Janssen, Merck/MSD, and Roche; has participated in speaker bureaus for AstraZeneca, Pfizer, Janssen, Sanofi, and Astellas Pharma; and has received research funding from AstraZeneca and Pfizer Oncology through grants to his institution.

Craniofacial microsomia (CFM, OMIM%164 210) is one of the most common congenital facial abnormalities worldwide, but it’s genetic risk factors and environmental threats are poorly investigated, as well as their interaction, making the diagnosis and prenatal screening of CFM impossible. We perform a comprehensive association study on the largest CFM cohort of 6074 samples. We identify 15 significant (P

Objectives Tumor biomarker analyses accompanying immuno-oncology therapies are coupled with a tumor tissue journey aiming to guide tissue procurement and allow for accurate diagnosis and delivery of test results. The engagement of pathologists in the tumor tissue journey is essential because they are able to link the preanalytic requirements of this process with pathologic evaluation and clinical information, ultimately influencing treatment decisions for patients with cancer. The aim of this review is to provide suggestions on how cancer diagnosis and the delivery of molecular test results may be optimized, based on the needs and available resources of institutions, by placing the tumor tissue journey under the leadership of pathologists. Methods Literature searches on PubMed and personal experience provided the necessary material to satisfy the objectives of this review. Results Pathologists are usually involved across many steps of the tumor tissue journey and have the requisite knowledge to ensure its efficiency. Conclusions The expansion of oncology diagnostic testing emphasizes the need for pathologists to acquire a leadership role in the multidisciplinary effort to optimize the accuracy, completeness, and delivery of diagnoses guiding personalized treatments.

ObjectiveTo assess the benefits and limitations of whole genome sequencing (WGS) compared to exome sequencing (ES) or multigene panel (MGP) in the molecular diagnosis of developmental and epileptic encephalopathies (DEE).MethodsWe performed WGS of 30 comprehensively phenotyped DEE patient trios that were undiagnosed after first-tier testing, including chromosomal microarray and either research ES (n = 15) or diagnostic MGP (n = 15).ResultsEight diagnoses were made in the 15 individuals who received prior ES (53%): 3 individuals had complex structural variants; 5 had ES-detectable variants, which now had additional evidence for pathogenicity. Eleven diagnoses were made in the 15 MGP-negative individuals (68%); the majority (n = 10) involved genes not included in the panel, particularly in individuals with postneonatal onset of seizures and those with more complex presentations including movement disorders, dysmorphic features, or multiorgan involvement. A total of 42% of diagnoses were autosomal recessive or X-chromosome linked.ConclusionWGS was able to improve diagnostic yield over ES primarily through the detection of complex structural variants (n = 3). The higher diagnostic yield was otherwise better attributed to the power of re-analysis rather than inherent advantages of the WGS platform. Additional research is required to assist in the assessment of pathogenicity of novel noncoding and complex structural variants and further improve diagnostic yield for patients with DEE and other neurogenetic disorders.

Neuro-oncology is a rapidly developing field. A continuous evolution in the understanding of the molecular underpinnings of central nervous system tumors has helped reconfigure the classification of brain tumors. More importantly, it has laid the path forward for the development and investigation of new therapeutics. The authors discuss the classification of brain tumors and novel therapies in brain tumors as well as promising treatments underway.

Mixed germ cell-sex cord stromal tumours (MGC-SCSTs) of the testis and ovary differ significantly in their histological appearance, clinical behaviour, and molecular genetics. Until recently, the germ cells of testicular MGC-SCST were considered to be invariably histologically bland, whereas those from neoplasms that arise in the ovary have histological features characteristic of premalignancy. However, a recent histological and molecular genetic study demonstrated histological abnormalities and multiple chromosomal losses and gains in a small subset of testicular cases, thus providing the first evidence that testicular MGC-SCSTs can exceptionally show histological and molecular abnormalities. All cases of testicular MGC-SCST reported to date have been clinically benign, whereas ovarian examples are sometimes the precursor of a malignant germ cell neoplasm that can be clinically aggressive. Both genetic and epigenetic influences likely account for dissimilarities in these uncommon gonadal neoplasms.

The pandemic causing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has globally infected more than 50 million people and ∼1.2 million have succumbed to this deadly pathogen. With the vaccine trials still in clinical phases, mitigation of Coronavirus Disease 2019 (COVID-19) relies primarily on robust virus detection methods and subsequent quarantine measures. Hence, the importance of rapid, affordable and reproducible virus testing will serve the need to identify and treat infected subjects in a timely manner. Based on the type of diagnostic assay, the primary targets are viral genome (RNA) and encoded proteins. Currently, COVID-19 detection is performed using various molecular platforms as well as serodiagnostics that exhibit approximately 71% sensitivity. These methods encounter several limitations including sensitivity, specificity, availability of skilled expertise and instrument access. Saliva-based COVID-19 diagnostics are emerging as a superior alternative to nasal swabs because of the ease of sample collection, no interaction during sampling, and high viral titers during early stages of infection. In addition, SARS-CoV-2 is detected in the environment as aerosols associated with suspended particulate matter. Designing virus detection strategies in diverse samples will allow timely monitoring of virus spread in humans and its persistence in the environment. With the passage of time, advanced technologies are overcoming limitations associated with detection. Enhanced sensitivity and specificity of next-generation diagnostics are key features enabling improved prognostic care. In this comprehensive review, we analyze currently adopted advanced technologies and their concurrent use in the development of diagnostics for SARS-CoV-2 detection.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To counter COVID-19 spreading, an infrastructure to provide rapid and thorough molecular diagnostics and serology testing is the cornerstone of outbreak and pandemic management. We hereby review the clinical insights with regard to using molecular tests and immunoassays in the context of COVID-19 management life cycle: the preventive phase, the preparedness phase, the response phase and the recovery phase. The spatial and temporal distribution of viral RNA, antigens and antibodies during human infection is summarized to provide a biological foundation for accurate detection of the disease. We shared the lessons learned and the obstacles encountered during real world high-volume screening programs. Clinical needs are discussed to identify existing technology gaps in these tests. Leverage technologies, such as engineered polymerases, isothermal amplification, and direct amplification from complex matrices may improve the productivity of current infrastructure, while emerging technologies like CRISPR diagnostics, visual end point detection, and PCR free methods for nucleic acid sensing may lead to at-home tests. The lessons learned, and innovations spurred from the COVID-19 pandemic could upgrade our global public health infrastructure to better combat potential outbreaks in the future.

Background Astrovirus (AstV), Sapovirus (SaV) and Poliovirus (PV) are important pathogens that cause infections in children under five years of age. It is a very important task to systematically monitor and evaluate the diagnostic performance of these viruses in clinical laboratories. Methods In our study, we performed a multicenter evaluation study among 21 laboratories across China using simulated stool samples spiked with self-designed AstV, SaV and PV pseudoviral particles. Results The testing capability of 80.0% (16/20, AstV), 52.6% (10/19, SaV), and 25.0% (2/8, PV) of the participating laboratories were found to be “competent” in reporting correct results for all samples. The main type of errors were false negatives. None of the laboratories identified the subtypes of AstV and SaV, and six laboratories specifically identified the subtypes of PV. Lacking of well-trained personnel and adequate funding were the main challenges. From the questionnaire results, 55.6% laboratories (10/18) believe that training personnel could improve the laboratory testing performance. Conclusions The laboratories showed a competent diagnostic performance for AstV, but inferior diagnostic performances for SaV and PV. Sensitivity of detection and the ability for virus typing should be improved clinically. Professional and standardized personnel training is urgently needed to further improve laboratory performance.

Newer molecular diagnostics and improved understanding of cancer pathogenesis have identified multiple pathways that can be potentially targeted with the use of novel therapeutics in development. These developments have ushered cancer therapeutics in newer era of personalized medicine. Same is reflected on current management strategies for advanced gastrointestinal malignancies. Molecular profiling for BRAF and RAS is standard for colorectal cancer while Her2 and PDL1 status is needed for planning therapy of advanced gastroesophageal cancers. Tissue agnostic markers like MSI, TMB and NTRK are making headways in therapeutic armamentarium. While newer targeted therapies against FGFR, EGFR, PI3K-AKT, DDR pathways are showing promising results in initial studies. Here we review traditional as well as upcoming molecular markers in field of GI malignancies, methods of testing and evidence for rational use in clinical practice.

Thymomas exhibit a unique genomic landscape, comprising the lowest on average total mutational burden among adult human cancers; a unique point mutation in the GTF2I gene in WHO type A and AB thymomas (and rarely others); almost unique KMT2A-MAML2 translocations in rare WHO type B2 and B3 thymomas; a unique YAP1-MAML2 translocation in almost all metaplastic thymomas; and unique miRNA profiles in relation to GTF2I mutational status and WHO histotypes. While most thymomas can be diagnosed solely on the basis of morphological features, mutational analyses can solve challenging differential diagnostic problems. No molecular biomarkers have been identified that predict the response of unresectable thymomas to chemotherapy or agents with known molecular targets. Despite the common and strong expression of PDL1 in thymomas, immune checkpoint inhibitors are rarely applicable due to the poor predictability of common, life-threatening autoimmune side effects that are related to the unrivaled propensity of thymomas towards autoimmunity.