Evaluating Enhancer Function and Transcription.

Cell-type- and condition-specific profiles of gene expression require coordination between protein-coding gene promoters and cis-regulatory sequences called enhancers. Enhancers can stimulate gene activity at great genomic distances from their targets, raising questions about how enhancers communicate with specific gene promoters and what molecular mechanisms underlie enhancer function. Characterization of enhancer loci has identified the molecular features of active enhancers that accompany the binding of transcription factors and local opening of chromatin. These characteristics include coactivator recruitment, histone modifications, and noncoding RNA transcription. However, it remains unclear which of these features functionally contribute to enhancer activity. Here, we discuss what is known about how enhancers regulate their target genes and how enhancers and promoters communicate. Further, we describe recent data demonstrating many similarities between enhancers and the gene promoters they control, and we highlight unanswered questions in the field, such as the potential roles of transcription at enhancers.

Human-Specific NOTCH2NL Genes Affect Notch Signaling and Cortical Neurogenesis.

Genetic changes causing brain size expansion in human evolution have remained elusive. Notch signaling is essential for radial glia stem cell proliferation and is a determinant of neuronal number in the mammalian cortex. We find that three paralogs of human-specific NOTCH2NL are highly expressed in radial glia. Functional analysis reveals that different alleles of NOTCH2NL have varying potencies to enhance Notch signaling by interacting directly with NOTCH receptors. Consistent with a role in Notch signaling, NOTCH2NL ectopic expression delays differentiation of neuronal progenitors, while deletion accelerates differentiation into cortical neurons. Furthermore, NOTCH2NL genes provide the breakpoints in 1q21.1 distal deletion/duplication syndrome, where duplications are associated with macrocephaly and autism and deletions with microcephaly and schizophrenia. Thus, the emergence of human-specific NOTCH2NL genes may have contributed to the rapid evolution of the larger human neocortex, accompanied by loss of genomic stability at the 1q21.1 locus and resulting recurrent neurodevelopmental disorders.

Integrator endonuclease drives promoter-proximal termination at all RNA polymerase II-transcribed loci.

RNA polymerase II (RNAPII) pausing in early elongation is critical for gene regulation. Paused RNAPII can be released into productive elongation by the kinase P-TEFb or targeted for premature termination by the Integrator complex. Integrator comprises endonuclease and phosphatase activities, driving termination by cleavage of nascent RNA and removal of stimulatory phosphorylation. We generated a degron system for rapid Integrator endonuclease (INTS11) depletion to probe the direct consequences of Integrator-mediated RNA cleavage. Degradation of INTS11 elicits nearly universal increases in active early elongation complexes. However, these RNAPII complexes fail to achieve optimal elongation rates and exhibit persistent Integrator phosphatase activity. Thus, only short transcripts are significantly upregulated following INTS11 loss, including transcription factors, signaling regulators, and non-coding RNAs. We propose a uniform molecular function for INTS11 across all RNAPII-transcribed loci, with differential effects on particular genes, pathways, or RNA biotypes reflective of transcript lengths rather than specificity of Integrator activity.

Co-depletion of NIPBL and WAPL balance cohesin activity to correct gene misexpression.

The relationship between cohesin-mediated chromatin looping and gene expression remains unclear. NIPBL and WAPL are two opposing regulators of cohesin activity; depletion of either is associated with changes in both chromatin folding and transcription across a wide range of cell types. However, a direct comparison of their individual and combined effects on gene expression in the same cell type is lacking. We find that NIPBL or WAPL depletion in human HCT116 cells each alter the expression of ~2,000 genes, with only ~30% of the genes shared between the conditions. We find that clusters of differentially expressed genes within the same topologically associated domain (TAD) show coordinated misexpression, suggesting some genomic domains are especially sensitive to both more or less cohesin. Finally, co-depletion of NIPBL and WAPL restores the majority of gene misexpression as compared to either knockdown alone. A similar set of NIPBL-sensitive genes are rescued following CTCF co-depletion. Together, this indicates that altered transcription due to reduced cohesin activity can be functionally offset by removal of either its negative regulator (WAPL) or the physical barriers (CTCF) that restrict loop-extrusion events.

Pitfalls in soft tissue cytopathology.

Fine needle aspiration biopsy (FNAB) is a diagnostic modality for the evaluation of suspicious soft tissue masses. Despite its reasonable sensitivity, specificity and positive predictive value in differentiating benign from malignant neoplasms, the exact subtyping of the primary soft tissue tumours can be challenging. Certain tumours constitute "pitfalls" and add to the diagnostic challenge. This review provides a detailed account of the diagnostic challenges in soft tissue cytopathology, including pitfalls and, more importantly, the ways to overcome these challenges by integrating clinical details, key cytomorphological features and judicious application of ancillary techniques.

Biopsy for molecular risk stratification in uveal melanoma: Yields and molecular characteristics in 119 patients.

BACKGROUND: Prognostic cytological and molecular features of uveal melanoma have been well researched and are essential in management. Samples can be obtained in vivo through fine needle aspirate biopsy, vitrector cutter, forceps or post-enucleation for off-site testing. This study aims to examine cytological and chromosome microarray yields of these samples. METHODS: A retrospective cohort analysis of 119 uveal melanoma biopsies submitted to our laboratory. Samples included those taken in vivo (n = 57) and post-enucleation (n = 62). Patient and tumour features were collected including age, sex, primary tumour location, basal diameter and tumour height. Prognostic outcomes measured include cell morphology, chromosomal status and immunohistochemistry. RESULTS: Post-enucleation biopsies accounted for just over half of our samples (52%). Post-enucleation samples had a more successful genetic yield than in vivo biopsies (77% vs. 50%, p = 0.04) though there was no difference for cytological yields. There was no difference in cytological or microarray yields between instruments. The vitrector biopsy group had the smallest tumour thickness (5 mm vs. 10 mm [fine-needle aspirate biopsy], p = 0.003). There was a strong correlation between monosomy 3, BAP1 aberrancy and epithelioid cell type in post-enucleation samples (Tb  = 0.742, p = 0.005). However, epithelioid morphology was not associated with either monosomy 3 (p = 0.07) or BAP1 aberrancy (p = 0.24) for in vivo biopsies. CONCLUSIONS: All three biopsy instruments provide similar cytological yields as post-enucleation sampling, although post-enucleation samples had a more successful chromosome microarray yield. Epithelioid cytomorphology alone is insufficient for prognostication in in vivo biopsies, immunohistochemistry would be a useful surrogate test.

Guidelines for the cytopathologic diagnosis of epithelioid and mixed-type malignant mesothelioma. Complementary statement from the International Mesothelioma Interest Group, also endorsed by the International Academy of Cytology and the Papanicolaou Society of Cytopathology.

OBJECTIVE: To provide practical guidelines for the cytopathologic diagnosis of malignant mesothelioma. DATA SOURCES: Cytopathologists with an interest in the field involved in the International Mesothelioma Interest Group (IMIG) and the International Academy of Cytology (IAC) contributed to this update. Reference material includes peer-reviewed publications and textbooks. RATIONALE: This article is the result of discussions during and after the IMIG 2012 conference in Boston, followed by thorough discussions during the 2013 IAC meeting in Paris. Additional contributions have been obtained from cytopathologists and scientists who could not attend these meetings, with final discussions and input during the IMIG 2014 conference in Cape Town.

Anncaliia algerae microsporidial myositis.

The insect microsporidian Anncaliia algerae was first described in 2004 as a cause of fatal myositis in an immunosuppressed person from Pennsylvania, USA. Two cases were subsequently reported, and we detail 2 additional cases, including the only nonfatal case. We reviewed all 5 case histories with respect to clinical characteristics, diagnosis, and management and summarized organism life cycle and epidemiology. Before infection, all case-patients were using immunosuppressive medications for rheumatoid arthritis or solid-organ transplantation. Four of the 5 case-patients were from Australia. All diagnoses were confirmed by skeletal muscle biopsy; however, peripheral nerves and other tissues may be infected. The surviving patient received albendazole and had a reduction of immunosuppressive medications and measures to prevent complications. Although insects are the natural hosts for A. algerae, human contact with water contaminated by spores may be a mode of transmission. A. algerae has emerged as a cause of myositis, particularly in coastal Australia.

The World Health Organization Reporting System for Pancreaticobiliary Cytopathology: Overview and Summary.

The recently published WHO Reporting System for Pancreaticobiliary Cytopathology (World Health Organization [WHO] System) is an international approach to the standardized reporting of pancreaticobiliary cytopathology, updating the Papanicolaou Society of Cytopathology System for Reporting Pancreaticobiliary Cytology (PSC System). Significant changes were made to the categorization of benign neoplasms, intraductal neoplasms, mucinous cystic neoplasms, and malignant neoplasms considered low grade. Benign neoplasms, such as serous cystadenoma, categorized as Neoplastic: benign in the PSC system, are categorized as Benign/negative for malignancy in the WHO system. Pancreatic neuroendocrine tumor, solid-pseudopapillary neoplasm, and gastrointestinal stromal tumor, categorized as Neoplastic: other in the PSC system, are categorized as Malignant in the WHO System in accord with their classification in the 5th edition WHO Classification of Digestive System Tumours (2019). The two new categories of Pancreaticobiliary Neoplasm Low-risk/grade and Pancreaticobiliary Neoplasm High-risk/grade are mostly limited to intraductal neoplasms and mucinous cystic neoplasms. Low-risk/grade lesions are mucinous cysts, with or without low-grade epithelial atypia. High-risk/grade lesions contain neoplastic epithelium with high-grade epithelial atypia. Correlation with clinical, imaging, and ancillary studies remains a key tenet. The sections for each entity are written to highlight key cytopathological features and cytopathological differential diagnoses with the pathologist working in low resource setting in mind. Each section also includes the most pertinent ancillary studies useful for the differential diagnosis. Sample reports are provided for each category. Finally, the book provides a separate section with risk of malignancy and management recommendations for each category to facilitate decision-making for clinicians.

Rapid cytological analysis of endobronchial ultrasound-guided aspirates in sarcoidosis.

Rapid on-site evaluation (ROSE) of endobronchial ultrasound-guided transbronchial needle aspirates (EBUS-TBNA) has not been compared to final detailed cytological analysis in patients with suspected sarcoidosis. To assess the diagnostic accuracy of EBUS-TBNA with ROSE in patients with suspected sarcoidosis, a prospective two-centre study performed EBUS-TBNA with ROSE of cellular material followed by transbronchial lung biopsy (TBLB) and endobronchial biopsy (EBB). The diagnostic accuracy of EBUS-TBNA with ROSE was compared to the final cytological assessment and to TBLB and EBB. Analysis confirmed 49 out of 60 cases of sarcoidosis. ROSE sensitivity was 87.8% (specificity 91%, positive predictive value 97.7%). ROSE slide interpretation in combination with the final fixed slide and cell block preparations had a sensitivity of 91.8% (specificity 100%, positive predictive value 100%). 67% of patients were confirmed as having sarcoidosis on TBLB and 29% on EBB. Interobserver agreement between cytotechnologists and pathologists was very good (κ=0.91, 95% CI 0.80-1.0 and κ=0.91, 95% CI 0.79-1.0, respectively). EBUS-TBNA with ROSE has high diagnostic accuracy and interobserver agreement and informs the bronchoscopist in theatre whether additional diagnostic procedures need to be undertaken. EBUS-TBNA with ROSE should therefore be considered as the first-line investigation of sarcoidosis.

The rationale for the development and publication of the World Health Organization reporting systems for cytopathology and a brief overview of the first editions of the lung and pancreaticobiliary systems.

The International Academy of Cytology has joined with the International Agency for Research on Cancer and the World Health Organization (WHO) to develop international systems for reporting the cytopathology of lung, pancreas and biliary tract, lymph nodes, soft tissue, liver, breast, and kidney and adrenal gland. The WHO recently published the reporting systems for lung and pancreaticobiliary cytopathology. The objectives of this collaboration are to standardize the reporting of cytopathology; improve the quality of reporting by establishing the key diagnostic cytopathological features of entities and neoplasms; provide detailed best-practice guidelines in sampling techniques, specimen handling and processing, and the use of ancillary techniques; and facilitate communication between cytopathologists and clinicians to improve patient care. Each WHO system has defined specific categories and terminology for reporting cytopathology, and each category has an estimated risk of malignancy as far as the current literature allows and a suggested diagnostic management algorithm to assist clinicians. The WHO systems recognize that local medical and pathology infrastructure will vary, particularly in low-income and middle-income countries, and the WHO systems and their diagnostic management recommendations have been developed to allow them to be applied worldwide in all resource settings. The process of the selection of editors and authors and the writing and editing responsibilities has used the same model as that used for the fifth edition WHO Classification of Tumours, to which the WHO cytopathology systems are directly linked. This review provides the rationale and history of this joint International Academy of Cytology, International Agency for Research on Cancer, and WHO cytopathology project and a brief overview of the WHO reporting systems for lung and pancreaticobiliary cytopathology.

Factors determining whether diffuse large B-cell lymphoma samples are detected by flow cytometry.

INTRODUCTION: Flow cytometry (FCM) is widely used in the diagnosis of mature B-cell neoplasms (MBN), and FCM data are usually consistent with morphological findings. However, diffuse large B-cell lymphoma (DLBCL), a common MBN, is sometimes not detected by FCM. This study aimed to explore factors that increase the likelihood of failure to detect DLBCL by FCM. METHODS: Cases with a final diagnosis of DLBCL that were analysed by eight-colour FCM were retrospectively collated. Clinical, FCM, histopathological and genetic data were compared between cases detected and cases not detected by FCM. RESULTS: DLBCL cases from 135 different patients were analysed, of which 22 (16%) were not detected by FCM. In samples not detected by flow cytometry, lymphocytes were a lower percentage of total events (p = 0.02), and T cells were a higher percentage of total lymphocytes (p = 0.01). Cases with high MYC protein expression on immunohistochemistry were less likely to be missed by FCM (p = 0.011). Detection of DLBCL was not different between germinal centre B-cell (GCB) and non-GCB subtypes, not significantly affected by the presence of necrosis or fibrosis, and not significantly different between biopsy specimens compared to fine-needle aspirates, or between samples from nodal compared to extranodal tissue. CONCLUSION: The study identifies several factors which affect the likelihood of DLBCL being missed by FCM. Even with eight-colour analysis, FCM fails to detect numerous cases of DLBCL.

The World Health Organization Reporting System for Lung Cytopathology.

The International Academy of Cytology has joined with the International Agency for Research on Cancer (IARC) to bring together a group of experts in lung cytopathology to develop a WHO Reporting System for Lung Cytopathology (WHO System). This WHO System defines five categories for reporting lung cytopathology, that is, "Insufficient"/"Inadequate"/"Non-diagnostic," "Benign," "Atypical," "Suspicious for malignancy," and "Malignant," each with a clear descriptive term for the category, a definition, a risk of malignancy and a suggested management algorithm. The key diagnostic cytopathology features of each of the lesions within each category have been established by consensus and will be presented more fully in a subsequent IARC e-book and published hard cover book.The WHO System provides the best practice application of ancillary testing, including immunocytochemistry and molecular pathology, and provides a review to guide sampling and processing techniques to optimize the handling and preparation of the cytopathology sample emphasizing the cytomorphological differential diagnosis to aid low-resourced settings. The authors recognize that local medical and pathology resources will vary, particularly in low- and middle-income countries, and have developed the WHO System to make it applicable worldwide based on cytomorphology with options for further diagnostic management of the patient.The online WHO System provides a direct link to the WHO Tumour Classification for Thoracic Tumours 5th Edition. It will raise the profile and use of cytopathology by increasing awareness of its current role and its potential role in the era of personalized medicine based on molecular pathology utilizing "small biopsies." Ultimately, the System will improve patient care and outcomes.This System aims to improve and standardize the reporting of cytopathology, facilitate communication between cytopathologists and clinicians and improve patient care. The System is based on the current role of lung cytopathology and synthesizes the existing evidence while highlighting areas requiring further research and the future potential role of lung cytopathology.

The World Health Organization Reporting System for Pancreaticobiliary Cytopathology.

The World Health Organization (WHO), the International Academy of Cytology, and the International Agency for Research on Cancer, with expert contributors from around the world, present an international approach to standardized reporting of pancreaticobiliary cytopathology. This reporting system is one of the first in a series from various body sites that mirror the WHO Classification of Tumours series and provides an evidence-based terminology system with associated risk of malignancy and diagnostic management recommendation per diagnostic category. The WHO Reporting System for Pancreaticobiliary Cytopathology (WHO system) revises the Papanicolaou Society of Cytopathology (PSC) system for Reporting Pancreaticobiliary Cytology published in 2015 and replaces the six-tiered system with a seven-tiered system: "insufficient/inadequate/nondiagnostic"; "benign (negative for malignancy)," "atypical," "pancreaticobiliary neoplasm of low risk/low grade," "pancreatic neoplasm of high risk/high grade," "suspicious for malignancy," and "malignant." The principal differences between the WHO and the PSC systems revolve around the classification of neoplasia. In the PSC system, there was a single category for "neoplastic" lesions that includes two groups, one for "benign neoplasms" [primarily serous cystadenoma] and one named "other," dominated by premalignant intraductal neoplasms (primarily intraductal papillary mucinous neoplasms) and low-grade malignant neoplasms [pancreatic neuroendocrine tumors (PanNETs) and solid pseudopapillary neoplasms (SPNs)]. In the WHO system, benign neoplasms with virtually no risk of malignancy are included in the "benign" category and low-grade malignancies (PanNET and SPN) are included in the "malignant" category, as per the WHO Classification of Digestive System Tumours, thus leaving in the "neoplasm" category primarily those noninvasive premalignant lesions of the ductal system. These neoplasms are divided by the cytomorphological grade of the epithelium into low risk/low-grade and high risk/high-grade, with distinctly different risks of malignancy. As with the PSC system, the WHO system advocates close correlation with imaging and encourages incorporation of ancillary testing into the final diagnosis, such as biochemical (CEA and amylase) and molecular testing of cyst fluid and bile duct brushings. Key diagnostic cytopathological features of specific lesions or neoplasms, ancillary studies for diagnostic and prognostic evaluation, and implications of diagnosis for patient care and management are discussed. In addition, the WHO system includes reporting and diagnostic management options that recognize the variations in the availability of diagnostic and prognostic ancillary testing modalities in low- and middle-income countries, where cytopathology is particularly useful and is increasingly available in the absence of histopathological services.

A brief review of the WHO reporting system for pancreaticobiliary cytopathology.

The World Health Organization (WHO), the International Academy of Cytology, and the International Agency for Research on Cancer have developed an approach to standardized reporting of pancreaticobiliary cytopathology. The WHO Reporting System for Pancreaticobiliary Cytopathology (WHO System) revises the Papanicolaou Society of Cytopathology (PSC) System for Reporting Pancreaticobiliary Cytology published in 2015 and replaces the 6 PSC categories with 7 categories: "Insufficient/Inadequate/Nondiagnostic"; "Benign/Negative for malignancy"; "Atypical"; "Pancreaticobiliary neoplasm, low risk/grade (PaN-low)"; "Pancreatic neoplasm, high risk/grade (PaN-High)"; "Suspicious for malignancy"; and "Malignant". In the PSC system, there is a single category for "Neoplastic" lesions that includes 2 groups, 1 for benign neoplasms and 1 named "Neoplastic-other", dominated by premalignant intraductal neoplasms primarily intraductal papillary mucinous neoplasms and low-grade malignant neoplasms (pancreatic neuroendocrine tumors (PanNET) and solid pseudopapillary neoplasms (SPN). In the WHO System, benign neoplasms with virtually no risk of malignancy are included in the "Benign" category and low-grade malignancies (PanNET and SPN) are included in the "Malignant" category, as per the 5th edition of the WHO Classification of Digestive System Tumors, while the non-invasive pre-malignant lesions of the ducts are divided by the cytomorphological grade of the epithelium into PaN-low and PaN-high with distinctly different risks of malignancy. Within each category, key diagnostic cytopathologic features and the ancillary studies for diagnostic and prognostic evaluation, as well as the implications of diagnosis for patient care and management, are outlined. Reporting and diagnostic management options recognize the variations in the availability of diagnostic and prognostic ancillary testing modalities in low- and middle-income countries.

A brief review of the WHO reporting system for lung cytopathology.

The International Academy of Cytology has joined with the International Agency for Research on Cancer to bring together a group of experts in lung cytopathology to develop a WHO Reporting System for Lung Cytopathology (WHO System). This System aims to improve and standardize the reporting of cytopathology, facilitate communication between cytopathologists and clinicians, and improve patient care. The WHO System describes 5 categories for reporting lung cytopathology: 'Insufficient/Inadequate/Nondiagnostic', 'Benign', 'Atypical', 'Suspicious for malignancy', and 'Malignant', each one with a clear descriptive term, a definition, a risk of malignancy, and a suggested management algorithm. The key diagnostic cytopathologic features of each of the lesions within each category have been established by consensus through an Expert Editorial Board, who are also the authors of this review and selected for each reporting system and chosen based on their expertise in the field and/or diversity of geographical representation. Many other co-authors from around the world also contributed. The assignment of writing and editing responsibilities used the same model as that used for the WHO Classification of Tumours (https://whobluebooks.iarc.fr/about/faq/). The WHO System provides the best practice application of ancillary testing, including immunocytochemistry and molecular pathology, and guides in sampling and processing techniques to optimize the handling and preparation of specimens. The WHO System was created by the authors to be applicable globally and is based on cytomorphology with possibilities for additional diagnostic management of the patient. The authors are aware that local medical and pathology resources would differ, especially in low- and middle-income countries. The WHO Tumour Classification for Thoracic Tumors, Fifth Edition, is directly accessible through the online WHO System.

Digital Examination of LYmph node CYtopathology Using the Sydney system (DELYCYUS): An international, multi-institutional study.

BACKGROUND: After a series of standardized reporting systems in cytopathology, the Sydney system was recently introduced to address the need for reproducibility and standardization in lymph node cytopathology. Since then, the risk of malignancy for the categories of the Sydney system has been explored by several studies, but no studies have yet examined the interobserver reproducibility of the Sydney system. METHODS: The authors assessed interobserver reproducibility of the Sydney system on 85 lymph node fine-needle aspiration cytology cases reviewed by 15 cytopathologists from 12 institutions in eight different countries, resulting in 1275 diagnoses. In total, 186 slides stained with Diff-Quik, Papanicolaou, and immunocytochemistry were scanned. A subset of the cases included clinical data and results from ultrasound examinations, flow cytometry immunophenotyping, and fluorescence in situ hybridization analysis. The study participants assessed the cases digitally using whole-slide images. RESULTS: Overall, the authors observed an almost perfect agreement of cytopathologists with the ground truth (median weighted Cohen κ = 0.887; interquartile range, κ = 0.210) and moderate overall interobserver concordance (Fleiss κ = 0.476). There was substantial agreement for the inadequate and malignant categories (κ = 0.794 and κ = 0.729, respectively), moderate agreement for the benign category (κ = 0.490), and very slight agreement for the suspicious (κ = 0.104) and atypical (κ = 0.075) categories. CONCLUSIONS: The Sydney system for reporting lymph node cytopathology shows adequate interobserver concordance. Digital microscopy is an adequate means to assess lymph node cytopathology specimens.