A deep learning model to triage and predict adenocarcinoma on pancreas cytology whole slide imaging.

Pancreatic fine-needle aspirations are the gold-standard diagnostic procedure for the evaluation of pancreatic ductal adenocarcinoma. A suspicion for malignancy can escalate towards chemotherapy followed by a major surgery and therefore is a high-stakes task for the pathologist. In this paper, we propose a deep learning framework, MIPCL, that can serve as a helpful screening tool, predicting the presence or absence of cancer. We also reproduce two deep learning models that have found success in surgical pathology for our cytopathology study. Our MIPCL significantly improves over both models across all evaluated metrics (F1-Score: 87.97% vs 88.70% vs 91.07%; AUROC: 0.9159 vs. 0.9051 vs 0.9435). Additionally, our model is able to recover the most contributing regions on the slide for the final prediction. We also present a dataset curation strategy that increases the number of training examples from an existing dataset, thereby reducing the resource burden tied to collecting and scanning additional cases.

Metastatic salivary gland neoplasms to pleural effusion: diagnostic challenges and prognostic significance in a series with 9 patients.

INTRODUCTION: Pleural effusions can present a diagnostic challenge as they are not always caused by malignancy in patients with a history of typical visceral primaries. MATERIAL AND METHODS: At 2 major academic medical centers, we have identified several cases in which salivary gland neoplasms metastasized to pleural effusions in patients who have been aggressively managed with various treatment modalities including chemotherapy, radiation, and/or surgical excision. RESULTS: Herein, we present a range of primary salivary gland tumors that metastasized to serous effusions and characterize their cytomorphology, immunoprofiles, and clinical courses. Our case series shows that many tumor types metastasize to pleural effusions and they present unique diagnostic challenges in each case. We found that metastasis of a salivary gland neoplasm to a pleural effusion is a late-stage event and is often associated with poor prognosis. CONCLUSIONS: This series serves as a resource to demonstrate the cytomorphologic and immunohistochemical features of malignant pleural effusions due to salivary gland neoplasms and draws attention to poor prognosis in cases of salivary duct carcinoma, mucoepidermoid carcinoma and adenoid cystic carcinoma.

Cystic Salivary Gland Neoplasms: Diagnostic Approach With a Focus on Ancillary Studies.

Cystic salivary gland cytology can be challenging due to the fact that a cystic mass can be the clinical presentation of both non-neoplastic and neoplastic conditions. Neoplastic lesions consist of both benign and malignant neoplasms. The cytomorphologic features of these entities can overlap and the cystic background may additionally contribute to the complexity of these lesions and their interpretation. Ancillary studies have been reported in several studies to be beneficial in further characterization of the cellular components and subsequent diagnosis of the cystic lesions of the salivary gland. Fluorescence in situ hybridization, real-time polymerase chain reaction, and next-generation sequencing are now being utilized to detect molecular alterations in salivary gland neoplasms. MALM2 rearrangement is the most common gene fusion in mucoepidermoid carcinoma. PLAG1 rearrangement is present in more than half of pleomorphic adenomas. AKT1:E17K mutation is the key diagnostic feature of the mucinous adenocarcinoma. NR4A3 overexpression is highly sensitive and specific for the diagnosis of acinic cell carcinoma. MYB fusion is noted in adenoid cystic carcinoma. ETV6:NTRK3 fusion is helpful in diagnosis of secretory carcinoma. p16 and human papillomavirus (HPV) studies differentiate HPV-related squamous cell carcinoma from non-HPV-related neoplasms with overlapping features. NCOA4:RET fusion protein is the main fusion in intraductal carcinoma.

Long read, isoform aware sequencing of mouse nucleus accumbens after chronic cocaine treatment.

To better understand the full-length transcriptome of the nucleus accumbens (NAc)-a key brain reward region-in chronic cocaine treatment, we perform the first single molecule, long-read sequencing analysis using the Iso-seq method to detect 42,114 unique transcripts from mouse NAc polyadenylated RNA. Using GENCODE annotation as a reference, we find that over half of the Iso-seq derived transcripts are annotated, while 46% of them harbor novel splicing events in known genes; around 1% of them correspond to other types of novel transcripts, such as fusion, antisense and intergenic. Approximately 34% of the novel transcripts are matched with a compiled transcriptome assembled from published short-read data from various tissues, with the remaining 69% being unique to NAc. These data provide a more complete picture of the NAc transcriptome than existing annotations and can serve as a comprehensive reference for future transcriptomic analyses of this important brain reward region.

p16 immunostaining in cytology specimens: its application, expression, interpretation, and challenges.

INTRODUCTION: p16 immunostaining is considered as a surrogate marker for human papillomavirus (HPV)-related head and neck squamous cell carcinomas (HNSCC). Herein, the utility of p16 is evaluated in cytology specimens. MATERIAL AND METHODS: The electronic data of a large academic institution was searched for cytology cases accompanied by p16 (2014-2018). Cases were categorized based on body sites. P16 staining was quantified (negative [0%], focal/patchy, or diffusely positive [>70%]). HPV testing was correlated where available. RESULTS: A total of 372 cases were included (male:female, 239:133). The largest differences in application of p16 between men and women were in head/neck cases (209 versus 59) and the abdominal cases (1 versus 33), respectively. p16 diffuse staining is seen in most squamous cell carcinomas, small cell carcinomas, and gynecologic serous carcinomas. p16 expression was patchy or negative in most adenocarcinoma, neuroendocrine carcinoma, spindle cell neoplasms, and benign conditions. HPV testing was done on 217 cases including 138 cases with strong p16 (127 HPV+/11 HPV-), 20 cases with focal/patchy P16 staining (6 HPV+/14 HPV-) and 59 cases with negative p16 staining (3 HPV+/56 HPV-). CONCLUSIONS: Diffuse p16 staining aids in the diagnosis of HPV-related carcinomas, particularly HPV-related HNSCC, across the body and according to sex. In contrast, focal/patchy p16 staining does not correlate with HPV status across various body sites. In conclusion, intensity of p16 matters and should be correlated with cytomorphology, clinical history, and ancillary studies (eg, p40 immunostaining) for an accurate diagnosis and preventing diagnostic pitfalls.

Renal medullary carcinoma involving serous cavity fluids: a cytomorphologic study of 12 cases.

INTRODUCTION: Renal medullary carcinoma (RMC) is a highly lethal adenocarcinoma with a propensity for widespread metastatic disease in young patients. It is strongly associated with sickle cell trait and shows the loss of SMARCB1 (also known as INI1 or BAF47) protein expression. In the present study, we reviewed a series of 12 patients for whom the cytology specimens played a significant role in patient treatment. MATERIALS AND METHODS: We performed a retrospective case review of patients with a history of RMC from 3 large tertiary care pathology practices. RESULTS: A total of 12 patients were identified with histologically confirmed RMC who had had pleural, pericardial, or urine specimens involved by their disease or had undergone initial kidney fine needle aspiration. Patient age ranged from 13 to 37 years (median, 21.5 years). All 12 patients were black or of African descent, and 10 had a confirmed history of sickle cell trait. Of the 12 patients, 11 (92%) had fluid specimens involved by metastatic tumor at some point in their clinical course, and 4 (33%) had initially presented with pericardial and/or pleural effusions or urine specimens that were positive for malignancy. Cytologic examination predominantly showed fragments of 3-dimensional "tumor balls" with smooth borders, fine pale cytoplasm with vacuolization, and highly pleomorphic nuclei with irregular nuclear membranes and coarse to vesicular chromatin and single prominent nucleoli. CONCLUSIONS: The cytomorphology of RMC involving serous fluids is nonspecific and in keeping with metastatic high-grade adenocarcinoma. In a young patient presenting with no history of malignancy and a pleural or pericardial effusion, triaging the material for ancillary studies and a nuanced assessment of patient history and radiologic findings will be critical.

Dopaminergic Regulation of Nucleus Accumbens Cholinergic Interneurons Demarcates Susceptibility to Cocaine Addiction.

BACKGROUND: Cholinergic interneurons (ChINs) in the nucleus accumbens (NAc) play critical roles in processing information related to reward. However, the contribution of ChINs to the emergence of addiction-like behaviors and its underlying molecular mechanisms remain elusive. METHODS: We employed cocaine self-administration to identify two mouse subpopulations: susceptible and resilient to cocaine seeking. We compared the subpopulations for physiological responses with single-unit recording of NAc ChINs, and for gene expression levels with RNA sequencing of ChINs sorted using fluorescence-activated cell sorting. To provide evidence for a causal relationship, we manipulated the expression level of dopamine D2 receptor (DRD2) in ChINs in a cell type-specific manner. Using optogenetic activation combined with a double whole-cell recording, the effect of ChIN-specific DRD2 manipulation on each synaptic input was assessed in NAc medium spiny neurons in a pathway-specific manner. RESULTS: Susceptible mice showed higher levels of nosepoke responses under a progressive ratio schedule, and impairment in extinction and punishment procedures. DRD2 was highly abundant in the NAc ChINs of susceptible mice. Elevated abundance of DRD2 in NAc ChINs was sufficient and necessary to express high cocaine motivation, putatively through reduction of ChIN activity during cocaine exposure. DRD2 overexpression in ChINs mimicked cocaine-induced effects on the dendritic spine density and the ratios of excitatory inputs between two distinct medium spiny neuron cell types, while DRD2 depletion precluded cocaine-induced synaptic plasticity. CONCLUSIONS: These findings provide a molecular mechanism for dopaminergic control of NAc ChINs that can control the susceptibility to cocaine-seeking behavior.

Viral labeling of neurons synaptically connected to nucleus accumbens somatostatin interneurons.

The nucleus accumbens, a key brain reward region, receives synaptic inputs from a range of forebrain and brainstem regions. Many of these projections have been established using electrophysiology or fluorescent tract tracing. However, more recently developed viral tracing techniques have allowed for fluorescent labeling of synaptic afferents in a cell type-specific manner. Since the NAc is comprised of multiple cell types, these methods have enabled the delineation of the cell type-specific connectivity of principal medium spiny neurons in the region. The synaptic connectivity of somatostatin interneurons, which account for <5% of the neurons in the region, has been inferred from electrophysiological and immunohistochemical data, but has not yet been visualized using modern viral tracing techniques. Here, we use the pseudorabies virus (PRV)-Introvert-GFP virus, an alphaherpes virus previously shown to label synaptic afferents in a cell type-specific manner, to label first order afferents to NAc somatostatin interneurons. While we find GFP(+) labeling in several well established projections to the NAc, we also observe that several known projections to NAc did not contain GFP(+) cells, suggesting they do not innervate somatostatin interneurons in the region. A subset of the GFP(+) afferents are c-FOS(+) following acute administration of cocaine, showing that NAc somatostatin interneurons are innervated by some cells that respond to rewarding stimuli. These results provide a foundation for future studies aimed toward elucidating the cell type-specific connectivity of the NAc, and identify specific circuits that warrant future functional characterization.

Transcriptional and physiological adaptations in nucleus accumbens somatostatin interneurons that regulate behavioral responses to cocaine.

The role of somatostatin interneurons in nucleus accumbens (NAc), a key brain reward region, remains poorly understood due to the fact that these cells account for < 1% of NAc neurons. Here, we use optogenetics, electrophysiology, and RNA-sequencing to characterize the transcriptome and functioning of NAc somatostatin interneurons after repeated exposure to cocaine. We find that the activity of somatostatin interneurons regulates behavioral responses to cocaine, with repeated cocaine reducing the excitability of these neurons. Repeated cocaine also induces transcriptome-wide changes in gene expression within NAc somatostatin interneurons. We identify the JUND transcription factor as a key regulator of cocaine action and confirmed, by use of viral-mediated gene transfer, that JUND activity in somatostatin interneurons influences behavioral responses to cocaine. Our results identify alterations in NAc induced by cocaine in a sparse population of somatostatin interneurons, and illustrate the value of studying brain diseases using cell type-specific whole transcriptome RNA-sequencing.

Author Correction: Midbrain circuit regulation of individual alcohol drinking behaviors in mice.

The original version of this Article contained an error in the spelling of the author Scott Edwards, which was incorrectly given as Scott Edward. This has now been corrected in both the PDF and HTML versions of the Article.

Midbrain circuit regulation of individual alcohol drinking behaviors in mice.

Alcohol-use disorder (AUD) is the most prevalent substance-use disorder worldwide. There is substantial individual variability in alcohol drinking behaviors in the population, the neural circuit mechanisms of which remain elusive. Utilizing in vivo electrophysiological techniques, we find that low alcohol drinking (LAD) mice have dramatically higher ventral tegmental area (VTA) dopamine neuron firing and burst activity. Unexpectedly, VTA dopamine neuron activity in high alcohol drinking (HAD) mice does not differ from alcohol naive mice. Optogenetically enhancing VTA dopamine neuron burst activity in HAD mice decreases alcohol drinking behaviors. Circuit-specific recordings reveal that spontaneous activity of nucleus accumbens-projecting VTA (VTA-NAc) neurons is selectively higher in LAD mice. Specifically activating this projection is sufficient to reduce alcohol consumption in HAD mice. Furthermore, we uncover ionic and cellular mechanisms that suggest unique neuroadaptations between the alcohol drinking groups. Together, these data identify a neural circuit responsible for individual alcohol drinking behaviors.

Gene Network Dysregulation in Dorsolateral Prefrontal Cortex Neurons of Humans with Cocaine Use Disorder.

Metabolic and functional alterations of neurons in the dorsolateral prefrontal cortex (dlPFC) are thought to contribute to impulsivity, which is a hallmark of addictive behaviors that underlie compulsive drug seeking and taking in humans. To determine if there is a transcriptional signature in dlPFC neurons of humans with cocaine use disorder, we performed total RNA-sequencing on neuronal nuclei isolated from post-mortem dlPFC of cocaine addicts and healthy controls. Our results point toward a transcriptional mechanism whereby cocaine alters specific gene networks in dlPFC neurons. In particular, we identified an AP-1 regulated transcriptional network in dlPFC neurons associated with cocaine use disorder that contains several differentially expressed hub genes. Several of these hub genes are GWAS hits for traits that might involve dysfunction of brain reward circuitry (Body-Mass Index, Obesity) or dlPFC (Bipolar disorder, Schizophrenia). Further study is warranted to determine their potential pathophysiological role in cocaine addiction.

Dopaminergic dynamics underlying sex-specific cocaine reward.

Although both males and females become addicted to cocaine, females transition to addiction faster and experience greater difficulties remaining abstinent. We demonstrate an oestrous cycle-dependent mechanism controlling increased cocaine reward in females. During oestrus, ventral tegmental area (VTA) dopamine neuron activity is enhanced and drives post translational modifications at the dopamine transporter (DAT) to increase the ability of cocaine to inhibit its function, an effect mediated by estradiol. Female mice conditioned to associate cocaine with contextual cues during oestrus have enhanced mesolimbic responses to these cues in the absence of drug. Using chemogenetic approaches, we increase VTA activity to mechanistically link oestrous cycle-dependent enhancement of VTA firing to enhanced cocaine affinity at DAT and subsequent reward processing. These data have implications for sexual dimorphism in addiction vulnerability and define a mechanism by which cellular activity results in protein alterations that contribute to dysfunctional learning and reward processing.

Alterations of the Host Microbiome Affect Behavioral Responses to Cocaine.

Addiction to cocaine and other psychostimulants represents a major public health crisis. The development and persistence of addictive behaviors comes from a complex interaction of genes and environment - the precise mechanisms of which remain elusive. In recent years a surge of evidence has suggested that the gut microbiome can have tremendous impact on behavioral via the microbiota-gut-brain axis. In this study we characterized the influence of the gut microbiota on cocaine-mediated behaviors. Groups of mice were treated with a prolonged course of non-absorbable antibiotics via the drinking water, which resulted in a substantial reduction of gut bacteria. Animals with reduced gut bacteria showed an enhanced sensitivity to cocaine reward and enhanced sensitivity to the locomotor-sensitizing effects of repeated cocaine administration. These behavioral changes were correlated with adaptations in multiple transcripts encoding important synaptic proteins in the brain's reward circuitry. This study represents the first evidence that alterations in the gut microbiota affect behavioral response to drugs of abuse.

Role of Tet1 and 5-hydroxymethylcytosine in cocaine action.

Ten-eleven translocation (TET) enzymes mediate the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which is enriched in brain, and its ultimate DNA demethylation. However, the influence of TET and 5hmC on gene transcription in brain remains elusive. We found that ten-eleven translocation protein 1 (TET1) was downregulated in mouse nucleus accumbens (NAc), a key brain reward structure, by repeated cocaine administration, which enhanced behavioral responses to cocaine. We then identified 5hmC induction in putative enhancers and coding regions of genes that have pivotal roles in drug addiction. Such induction of 5hmC, which occurred similarly following TET1 knockdown alone, correlated with increased expression of these genes as well as with their alternative splicing in response to cocaine administration. In addition, 5hmC alterations at certain loci persisted for at least 1 month after cocaine exposure. Together, these reveal a previously unknown epigenetic mechanism of cocaine action and provide new insight into how 5hmC regulates transcription in brain in vivo.

Epigenetic basis of opiate suppression of Bdnf gene expression in the ventral tegmental area.

Brain-derived neurotrophic factor (BDNF) has a crucial role in modulating neural and behavioral plasticity to drugs of abuse. We found a persistent downregulation of exon-specific Bdnf expression in the ventral tegmental area (VTA) in response to chronic opiate exposure, which was mediated by specific epigenetic modifications at the corresponding Bdnf gene promoters. Exposure to chronic morphine increased stalling of RNA polymerase II at these Bdnf promoters in VTA and altered permissive and repressive histone modifications and occupancy of their regulatory proteins at the specific promoters. Furthermore, we found that morphine suppressed binding of phospho-CREB (cAMP response element binding protein) to Bdnf promoters in VTA, which resulted from enrichment of trimethylated H3K27 at the promoters, and that decreased NURR1 (nuclear receptor related-1) expression also contributed to Bdnf repression and associated behavioral plasticity to morphine. Our findings suggest previously unknown epigenetic mechanisms of morphine-induced molecular and behavioral neuroadaptations.

ß-catenin mediates stress resilience through Dicer1/microRNA regulation.

ß-catenin is a multi-functional protein that has an important role in the mature central nervous system; its dysfunction has been implicated in several neuropsychiatric disorders, including depression. Here we show that in mice ß-catenin mediates pro-resilient and anxiolytic effects in the nucleus accumbens, a key brain reward region, an effect mediated by D2-type medium spiny neurons. Using genome-wide ß-catenin enrichment mapping, we identify Dicer1-important in small RNA (for example, microRNA) biogenesis--as a ß-catenin target gene that mediates resilience. Small RNA profiling after excising ß-catenin from nucleus accumbens in the context of chronic stress reveals ß-catenin-dependent microRNA regulation associated with resilience. Together, these findings establish ß-catenin as a critical regulator in the development of behavioural resilience, activating a network that includes Dicer1 and downstream microRNAs. We thus present a foundation for the development of novel therapeutic targets to promote stress resilience.

Epigenetic regulation of RAC1 induces synaptic remodeling in stress disorders and depression.

Depression induces structural and functional synaptic plasticity in brain reward circuits, although the mechanisms promoting these changes and their relevance to behavioral outcomes are unknown. Transcriptional profiling of the nucleus accumbens (NAc) for Rho GTPase-related genes, which are known regulators of synaptic structure, revealed a sustained reduction in RAS-related C3 botulinum toxin substrate 1 (Rac1) expression after chronic social defeat stress. This was associated with a repressive chromatin state surrounding the proximal promoter of Rac1. Inhibition of class 1 histone deacetylases (HDACs) with MS-275 rescued both the decrease in Rac1 transcription after social defeat stress and depression-related behavior, such as social avoidance. We found a similar repressive chromatin state surrounding the RAC1 promoter in the NAc of subjects with depression, which corresponded with reduced RAC1 transcription. Viral-mediated reduction of Rac1 expression or inhibition of Rac1 activity in the NAc increases social defeat-induced social avoidance and anhedonia in mice. Chronic social defeat stress induces the formation of stubby excitatory spines through a Rac1-dependent mechanism involving the redistribution of synaptic cofilin, an actin-severing protein downstream of Rac1. Overexpression of constitutively active Rac1 in the NAc of mice after chronic social defeat stress reverses depression-related behaviors and prunes stubby spines. Taken together, our data identify epigenetic regulation of RAC1 in the NAc as a disease mechanism in depression and reveal a functional role for Rac1 in rodents in regulating stress-related behaviors.

Recent advancements in stem cell and gene therapies for neurological disorders and intractable epilepsy.

The potential applications of stem cell therapies for treating neurological disorders are enormous. Many laboratories are focusing on stem cell treatments for CNS diseases, including spinal cord injury, Amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, multiple sclerosis, stroke, traumatic brain injury, and epilepsy. Among the many stem cell types under testing for neurological treatments, the most common are fetal and adult brain stem cells, embryonic stem cells, induced pluripotent stem cells, and mesenchymal stem cells. An expanding toolbox of molecular probes is now available to allow analyses of neural stem cell fates prior to and after transplantation. Concomitantly, protocols are being developed to direct the fates of stem cell-derived neural progenitors, and also to screen stem cells for tumorigenicity and aneuploidy. The rapid progress in the field suggests that novel stem cell and gene therapies for neurological disorders are in the pipeline.