Extra-appendiceal mucinous neoplasms: A tumour with clinicopathologic similarities to low- and high-grade appendiceal counterpart.

AIMS: Appendiceal mucinous neoplasms feature neoplastic mucinous epithelium with pushing borders and densely fibrotic walls. We have identified five examples of analogous colorectal tumours. METHODS AND RESULTS: Slides, pathology reports, and clinical data were reviewed. Whole genome sequencing was performed in two cases. Three were women and the mean age was 70. Associated GI conditions included Crohn's disease [1], diverticulosis [2], and sarcoma of the terminal ileum [1]. Signs/symptoms included obstruction [2], nausea, vomiting, abdominal pain [1], and positive faecal immunohistochemical test [1]. Colonoscopic findings included narrowing [1], "fullness" [1], and caecal lesion concerning for GIST [1]. Tumours involved the rectosigmoid [2], sigmoid [1], transverse colon [1], and cecum [1] and ranged from 1.5 cm to 8.5 cm. All but one tumour arose in the setting of faecal stream abnormalities related to obstruction, diverticulosis, or bowel diversion. All cases showed columnar, variably mucinous epithelium associated with little-to-no lamina propria. All but one case showed fibrosis of the submucosa. Three cases had high-grade areas. Neoplastic glands and/or mucin dissected through the muscularis propria or subserosa in 3 examples. No extracolonic neoplastic cells/mucin, infiltrative invasion, or desmoplastic response were identified. Three patients with available follow-up [5.5-28 months] are alive. Whole genome sequencing identified pathogenic TP53 and ERBB2 variants, as well as ERBB2 copy number amplification in one high-grade example. CONCLUSIONS: Though these tumours share clinicopathologic characteristics with their appendiceal counterparts, our cohort is too small to draw solid conclusions. We propose the term "extra-appendiceal mucinous neoplasm [EAMN]" for these rare lesions.

Auditory Hair Cells and Spiral Ganglion Neurons Regenerate Synapses with Refined Release Properties In Vitro.

Ribbon synapses between inner hair cells (IHCs) and type I spiral ganglion neurons (SGNs) in the inner ear are damaged by noise trauma and with aging, causing 'synaptopathy 'and hearing loss. Co-cultures of neonatal denervated organs of Corti and newly introduced SGNs have been developed to find strategies for improving IHC synapse regeneration, but evidence of the physiological normality of regenerated synapses is missing. This study utilizes IHC optogenetic stimulation and SGN recordings, showing that newly formed IHC synapses are indeed functional, exhibiting glutamatergic excitatory postsynaptic currents. When older organs of Corti were plated, synaptic activity probed by deconvolution, showed more mature release properties, closer to the highly specialized mode of IHC synaptic transmission that is crucial for coding the sound signal. This newly developed functional assessment of regenerated IHC synapses provides a powerful tool for testing approaches to improve synapse regeneration.

Hepatitis B e Antigen-Negative Single Hepatocyte Analysis Shows Transcriptional Silencing and Slow Decay of Infected Cells With Treatment.

BACKGROUND: Nucleos(t)ide analogues (NUCs) rarely cure chronic hepatitis B (CHB) because they do not eliminate covalently closed circular deoxyribonucleic acid, the stable replication template. In hepatitis B e antigen (HBeAg)-positive CHB during NUCs, HBV-infected cells decline slowly and are transcriptionally silenced. Whether these occur in HBeAg-negative CHB is unknown. METHODS: Using paired liver biopsies separated by 2.7-3.7 years in 4 males with HIV and HBeAg-negative CHB at both biopsies and 1 male with HIV who underwent HBeAg seroconversion between biopsies, we quantified amounts of viral nucleic acids in hundreds of individual hepatocytes. RESULTS: In the 4 persistently HBeAg-negative participants, HBV-infected hepatocytes ranged from 6.2% to 17.7% (biopsy 1) and significantly declined in 3 of 4 by biopsy 2. In the HBeAg seroconverter, the proportion was 97.4% (biopsy 1) and declined to 81.9% at biopsy 2 (P < .05). We extrapolated that HBV eradication with NUCs would take >100 years. At biopsy 1 in the persistently HBeAg-negative participants, 23%-56.8% of infected hepatocytes were transcriptionally inactive-higher than we observed in HBeAg-positive CHB-and significantly declined in 1 of 4 at biopsy 2. CONCLUSIONS: In HBeAg-negative CHB on NUCs, the negligible decline in infected hepatocytes is similar to HBeAg-positive CHB, supporting the need for more potent therapeutics to achieve functional cure.

DNAJA1 promotes cancer metastasis through interaction with mutant p53.

Accumulation of mutant p53 (mutp53) is crucial for its oncogenic gain of function activity. DNAJA1, a member of J-domain containing proteins or heat shock protein 40, is shown to prevent unfolded mutp53 from proteasomal degradation. However, the biological function of DNAJA1 remains largely unknown. Here we show that DNAJA1 promotes tumor metastasis by accumulating unfolded mutp53. Levels of DNAJA1 in head and neck squamous cell carcinoma (HNSCC) tissues were higher than those in normal tissues. Knockdown of DNAJA1 in HNSCC cell lines carrying unfolded mutp53 significantly decreased the levels of mutp53, filopodia/lamellipodia formation, migratory potential, and active forms of CDC42/RAC1, which were not observed in HNSCC cells with DNA contact mutp53, wild-type p53, or p53 null. Such mutp53-dependent functions of DNAJA1 were supported by the observation that DNAJA1 selectively bound to unfolded mutp53. Moreover, DNAJA1 knockdown in HNSCC cells carrying unfolded mutp53 inhibited primary tumor growth and metastases to the lymph nodes and lungs. Our study suggests that DNAJA1 promotes HNSCC metastasis mainly in a manner dependent on mutp53 status, suggesting DNAJA1 as a potential therapeutic target for HNSCC harboring unfolded mutp53.

Functional subgroups of cochlear inner hair cell ribbon synapses differently modulate their EPSC properties in response to stimulation.

Spiral ganglion neurons (SGNs) form single synapses on inner hair cells (IHCs), transforming sound-induced IHC receptor potentials into trains of action potentials. SGN neurons are classified by spontaneous firing rates as well as their threshold response to sound intensity levels. We investigated the hypothesis that synaptic specializations underlie mouse SGN response properties and vary with pillar versus modiloar synapse location around the hair cell. Depolarizing hair cells with 40 mM K+ increased the rate of postsynaptic responses. Pillar synapses matured later than modiolar synapses. Excitatory postsynaptic current (EPSC) amplitude, area, and number of underlying events per EPSC were similar between synapse locations at steady state. However, modiolar synapses produced larger monophasic EPSCs when EPSC rates were low and EPSCs became more multiphasic and smaller in amplitude when rates were higher, while pillar synapses produced more monophasic and larger EPSCs when the release rates were higher. We propose that pillar and modiolar synapses have different operating points. Our data provide insight into underlying mechanisms regulating EPSC generation.NEW & NOTEWORTHY Data presented here provide the first direct functional evidence of late synaptic maturation of the hair cell- spiral ganglion neuron synapse, where pillar synapses mature after postnatal day 20. Data identify a presynaptic difference in release during stimulation. This difference may in part drive afferent firing properties.

Resolution of subcomponents of synaptic release from postsynaptic currents in rat hair-cell/auditory-nerve fiber synapses.

The synapse between inner hair cells and auditory nerve fiber dendrites shows large excitatory postsynaptic currents (EPSCs), which are either monophasic or multiphasic. Multiquantal or uniquantal (flickering) release of neurotransmitter has been proposed to underlie the unusual multiphasic waveforms. Here the nature of multiphasic waveforms is analyzed using EPSCs recorded in vitro in rat afferent dendrites. Spontaneous EPSCs were deconvolved into a sum of presumed release events having monophasic EPSC waveforms. Results include, first, the charge of EPSCs is about the same for multiphasic versus monophasic EPSCs. Second, EPSC amplitudes decline with the number of release events per EPSC. Third, there is no evidence of a mini-EPSC. Most results can be accounted for by versions of either uniquantal or multiquantal release. However, serial neurotransmitter release in multiphasic EPSCs shows properties that are not fully explained by either model, especially that the amplitudes of individual release events are established at the beginning of a multiphasic EPSC, constraining possible models of vesicle release.NEW & NOTEWORTHY How do monophasic and multiphasic waveshapes arise in auditory-nerve dendrites; mainly are they uniquantal, arising from release of a single vesicle, or multiquantal, requiring several vesicles? The charge injected by excitatory postsynaptic currents (EPSCs) is the same for monophasic or multiphasic EPSCs, supporting uniquantal release. Serial adaptation of responses to sequential EPSCs favors a multiquantal model. Finally, neurotransmitter partitioning into similar sized release boluses occurs at the first bolus in the EPSC, not easily explained with either model.

Suppression of pancreatic cancer liver metastasis by secretion-deficient ITIH5.

BACKGROUND: Previously, we identified ITIH5 as a suppressor of pancreatic ductal adenocarcinoma (PDAC) metastasis in experimental models. Expression of ITIH5 correlated with decreased cell motility, invasion and metastasis without significant inhibition of primary tumour growth. Here, we tested whether secretion of ITIH5 is required to suppress liver metastasis and sought to understand the role of ITIH5 in human PDAC. METHODS: We expressed mutant ITIH5 with deletion of the N-terminal secretion sequence (ITIH5Δs) in highly metastatic human PDAC cell lines. We used a human tissue microarray (TMA) to compare ITIH5 levels in uninvolved pancreas, primary and metastatic PDAC. RESULTS: Secretion-deficient ITIH5Δs was sufficient to suppress liver metastasis. Similar to secreted ITIH5, expression of ITIH5Δs was associated with rounded cell morphology, reduced cell motility and reduction of liver metastasis. Expression of ITIH5 is low in both human primary PDAC and matched metastases. CONCLUSIONS: Metastasis suppression by ITIH5 may be mediated by an intracellular mechanism. In human PDAC, loss of ITIH5 may be an early event and ITIH5-low PDAC cells in primary tumours may be selected for liver metastasis. Further defining the ITIH5-mediated pathway in PDAC could establish future therapeutic exploitation of this biology and reduce morbidity and mortality associated with PDAC metastasis.

Automated quantitative image analysis for ex vivo metastasis assays reveals differing lung composition requirements for metastasis suppression by KISS1.

Imaging is broadly used in biomedical research, but signal variation complicates automated analysis. Using the Pulmonary Metastasis Assay (PuMA) to study metastatic colonization by the metastasis suppressor KISS1, we cultured GFP-expressing melanoma cells in living mouse lung ex vivo for 3 weeks. Epifluorescence images of cells were used to measure growth, creating large datasets which were time consuming and challenging to quantify manually due to scattering of light from outside the focal plane. To address these challenges, we developed an automated workflow to standardize the measurement of disseminated cancer cell growth by applying statistical quality control to remove unanalyzable images followed and a filtering algorithm to quantify only in-focus cells. Using this tool, we demonstrate that expression of the metastasis suppressor KISS1 does not suppress growth of melanoma cells in the PuMA, in contrast to the robust suppression of lung metastasis observed in vivo. This result may suggest that a factor required for metastasis suppression is present in vivo but absent in the PuMA, or that KISS1 suppresses lung metastasis at a step in the metastatic cascade not tested by the PuMA. Together, these data provide a new tool for quantification of metastasis assays and further insight into the mechanism of KISS1 mediated metastasis suppression in the lung.

Clinicopathological variables of sporadic schwannomas of peripheral nerve in 291 patients and expression of biologically relevant markers.

OBJECTIVE While sporadic peripheral schwannomas (SPSs) are generally well treated with surgery, their biology is not well understood. Consequently, treatment options are limited. The aim of this study was to provide a comprehensive description of SPS. The authors describe clinicopathological features and treatment outcomes of patients harboring these tumors, and they assess expression of biomarkers using a clinically annotated tissue microarray. Together, these data give new insight into the biology and management of SPS. METHODS Patients presenting with a primary SPS between 1993 and 2011 (n = 291) were selected from an institutional registry to construct a clinical database. All patients underwent follow-up, and short- and long-term outcomes were assessed. Expression of relevant biomarkers was assessed using a new tissue microarray (n = 121). RESULTS SPSs were generally large (mean 5.5 cm) and frequently painful at presentation (55%). Most patients were treated with surgery (80%), the majority of whom experienced complete resolution (52%) or improvement (18%) of their symptoms. Tumors that were completely resected (85%) did not recur. Some patients experienced short-term (16%) and long-term (4%) complications postoperatively. Schwannomas expressed higher levels of platelet-derived growth factor receptor-ß (2.1) than malignant peripheral nerve sheath tumors (MPNSTs) (1.5, p = 0.004) and neurofibromas (1.33, p = 0.007). Expression of human epidermal growth factor receptor-2 was greater in SPSs (0.91) than in MPNSTs (0.33, p = 0.002) and neurofibromas (0.33, p = 0.026). Epidermal growth factor receptor was expressed in far fewer SPS cells (10%) than in MPNSTs (58%, p < 0.0001) or neurofibromas (37%, p = 0.007). SPSs more frequently expressed cytoplasmic survivin (66% of tumor cells) than normal nerve (46% of cells), but SPS expressed nuclear survivin in fewer tumor cells than in MPNSTs (24% and 50%, respectively; p = 0.018). CONCLUSIONS Complete resection is curative for SPS. Left untreated, however, these tumors can cause significant morbidity, and not all patients are candidates for resection. SPSs express a pattern of biomarkers consistent with the dysregulation of the tumor suppressor merlin observed in neurofibromatosis Type 2-associated schwannomas, suggesting a shared etiology. This SPS pattern is distinct from that of other tumors of the peripheral nerve sheath.

Histopathologic features of melanoma in difficult-to-diagnose lesions: A case-control study.

BACKGROUND: Dermatopathology is considered the gold standard for melanoma diagnosis, but a subset of cases is difficult to diagnose by histopathology. OBJECTIVE: The goals of this study were to measure the accuracy of histopathologic features in difficult-to-diagnose melanocytic tumors and the interobserver agreement of those features. METHODS: This is a case-control study of histopathologic features of melanoma in 100 difficult-to-diagnose melanocytic neoplasms (40 melanomas and 60 nevi). Slides were blindly evaluated by 5 dermatopathologists. Frequencies, predictive values, and interobserver agreement were calculated. Univariate and multivariate logistic regression analyses were performed to identify the most influential features in arriving at a diagnosis of melanoma. RESULTS: Asymmetry, single-cell melanocytosis, solar elastosis, pagetoid melanocytosis, and broad surface diameter were most influential in arriving at a diagnosis of melanoma. Asymmetry and single-cell melanocytosis were most predictive of melanoma. Fleiss kappa was <0.6 for interobserver agreement in 9/10 histopathologic features of melanoma. LIMITATIONS: This study is limited by the small sample size, selection bias, and binary classification of melanocytic lesions. CONCLUSION: Our results indicate histopathologic features of melanoma in difficult-to-diagnose lesions vary in accuracy and reproducibility.

Genome-wide in vivo RNAi screen identifies ITIH5 as a metastasis suppressor in pancreatic cancer.

The overwhelming majority of pancreatic ductal adenocarcinoma (PDAC) is not diagnosed until the cancer has metastasized, leading to an abysmal average life expectancy (3-6 months post-diagnosis). Earlier detection and more effective treatments have been hampered by inadequate understanding of the underlying molecular mechanisms controlling metastasis. We hypothesized that metastasis suppressors are involved in controlling metastasis in pancreatic cancer. Using an unbiased genome-wide shRNA screen, an shRNA library was transduced into the non-metastatic PDAC line S2-028 followed by intrasplenic injection. Resulting liver metastases were individually isolated from these mice. One liver metastatic nodule contained shRNA for ITIH5 (Inter-alpha-trypsin inhibitor heavy chain 5), suggesting that ITIH5 may act as a metastasis suppressor. Consistent with this notion, metastatic PDAC cell lines had significantly lower protein expression of ITIH5 compared to immortalized pancreatic ductal epithelial cells and non-/poorly-metastatic PDAC cell lines. By manipulating expression of ITIH5 in different PDAC cell lines (over-expression in metastatic, knockdown in non-metastatic) functional and selective regulation of metastasis was observed for ITIH5. Orthotopic tumor growth of PDAC cells was not blocked following orthotopic injection. In vitro ITIH5 over-expression inhibited motility and invasion. Immunohistochemical analysis of a human PDAC tissue microarray revealed that ITIH5 expression inversely correlated with both survival and invasion/metastasis. ITIH5 is, therefore, functionally validated as a PDAC metastasis suppressor and shows promise as a prognostic biomarker.

Multiscale mapping of frequency sweep rate in mouse auditory cortex.

Functional organization is a key feature of the neocortex that often guides studies of sensory processing, development, and plasticity. Tonotopy, which arises from the transduction properties of the cochlea, is the most widely studied organizational feature in auditory cortex; however, in order to process complex sounds, cortical regions are likely specialized for higher order features. Here, motivated by the prevalence of frequency modulations in mouse ultrasonic vocalizations and aided by the use of a multiscale imaging approach, we uncover a functional organization across the extent of auditory cortex for the rate of frequency modulated (FM) sweeps. In particular, using two-photon Ca2+ imaging of layer 2/3 neurons, we identify a tone-insensitive region at the border of AI and AAF. This central sweep region behaves fundamentally differently from nearby neurons in AI and AII, responding preferentially to fast FM sweeps but not to tones or bandlimited noise. Together these findings define a second dimension of organization in the mouse auditory cortex for sweep rate complementary to that of tone frequency.

Maturation of Spontaneous Firing Properties after Hearing Onset in Rat Auditory Nerve Fibers: Spontaneous Rates, Refractoriness, and Interfiber Correlations.

Auditory nerve fibers (ANFs) exhibit a range of spontaneous firing rates (SRs) that are inversely correlated with threshold for sounds. To probe the underlying mechanisms and time course of SR differentiation during cochlear maturation, loose-patch extracellular recordings were made from ANF dendrites using acutely excised rat cochlear preparations of different ages after hearing onset. Diversification of SRs occurred mostly between the second and the third postnatal week. Statistical properties of ANF spike trains showed developmental changes that approach adult-like features in older preparations. Comparison with intracellularly recorded EPSCs revealed that most properties of ANF spike trains derive from the characteristics of presynaptic transmitter release. Pharmacological tests and waveform analysis showed that endogenous firing produces some fraction of ANF spikes, accounting for their unusual properties; the endogenous firing diminishes gradually during maturation. Paired recordings showed that ANFs contacting the same inner hair cell could have different SRs, with no correlation in their spike timing. SIGNIFICANCE STATEMENT: The inner hair cell (IHC)/auditory nerve fiber (ANF) synapse is the first synapse of the auditory pathway. Remarkably, each IHC is the sole partner of 10-30 ANFs with a range of spontaneous firing rates (SRs). Low and high SR ANFs respond to sound differently, and both are important for encoding sound information across varying acoustical environments. Here we demonstrate SR diversification after hearing onset by afferent recordings in acutely excised rat cochlear preparations. We describe developmental changes in spike train statistics and endogenous firing in immature ANFs. Dual afferent recordings provide the first direct evidence that fibers with different SRs contact the same IHCs and do not show correlated spike timing at rest. These results lay the groundwork for understanding the differential sensitivity of ANFs to acoustic trauma.

Histologic variability in solitary fibrous tumors reflects angiogenic and growth factor signaling pathway alterations.

This study aimed to evaluate expression of receptor tyrosine kinases, their ligands, and mutational status in solitary fibrous tumors, with correlation to histopathologic variants, tumor stage, and aggressive behavior. Immunohistochemical staining for PDGFα; PDGFß; PDGFR-α; PDGFR-ß; IGF1R; EGFR; VEGF; IGF2; c-Met; c-kit; c-erbB2; PTEN; and phosphorylated (p)AKT, pS6, and p4EBP1 was analyzed in 114 cases of solitary fibrous tumor using tissue microarray. Mutational analysis was performed using Sequenom MassARRAY-based platform. Multiple growth factors were overexpressed in most tumors, and increased numbers of overexpressed factors correlated with activation of the AKT pathway as measured by increased expression of p4EBP1(P = .0005). Compared to hypocellular tumors, localized hypercellular tumors were associated with high vascular endothelial growth factor (32% versus 8%; P = .008) and PDGFß (41% versus 13%; P = .008). Metastatic tumors more frequently overexpressed PDGFR-α compared to localized tumors (75% versus 31%; P < .001). None of the factors examined had prognostic significance in primary tumors. Single-nucleotide polymorphisms involving MET were identified in 4 patients; these do not appear to drive tumor behavior and were not reflected in c-Met expression levels. Simultaneous overexpression of multiple growth factors is common in solitary fibrous tumors; variability in expression may contribute to tumor phenotype and aggressive behavior.

Dorsal Cochlear Nucleus of the Rat: Representation of Complex Sounds in Ears Damaged by Acoustic Trauma.

Acoustic trauma damages the cochlea but secondarily modifies circuits of the central auditory system. Changes include decreases in inhibitory neurotransmitter systems, degeneration and rewiring of synaptic circuits, and changes in neural activity. Little is known about the consequences of these changes for the representation of complex sounds. Here, we show data from the dorsal cochlear nucleus (DCN) of rats with a moderate high-frequency hearing loss following acoustic trauma. Single-neuron recording was used to estimate the organization of neurons' receptive fields, the balance of inhibition and excitation, and the representation of the spectra of complex broadband stimuli. The complex stimuli had random spectral shapes (RSSs), and the responses were fit with a model that allows the quality of the representation and its degree of linearity to be estimated. Tone response maps of DCN neurons in rat are like those in other species investigated previously, suggesting the same general organization of this nucleus. Following acoustic trauma, abnormal response types appeared. These can be interpreted as reflecting degraded tuning in auditory nerve fibers plus loss of inhibitory inputs in DCN. Abnormal types are somewhat more prevalent at later times (103-376 days) following the exposure, but not significantly so. Inhibition became weaker in post-trauma neurons that retained inhibitory responses but also disappeared in many neurons. The quality of the representation of spectral shape, measured by sensitivity to the spectral shapes of RSS stimuli, was decreased following trauma; in fact, neurons with abnormal response types responded mainly to overall stimulus level, and not spectral shape.

Progressive loss of myogenic differentiation in leiomyosarcoma has prognostic value.

AIMS: Well-differentiated leiomyosarcomas show morphologically recognizable smooth muscle differentiation, whereas poorly differentiated tumours may form a spectrum with a subset of undifferentiated pleomorphic sarcomas. The expression of certain muscle markers has been reported to have prognostic impact. We investigated the correlation between the morphological spectrum and the muscle marker expression profile of leiomyosarcoma, and the impact of these factors on patient outcomes. METHODS AND RESULTS: Tissue microarrays including 202 non-uterine and 181 uterine leiomyosarcomas with a spectrum of tumour morphologies were evaluated for expression of immunohistochemical markers of muscle differentiation. Poorly differentiated tumours frequently lost one or more conventional smooth muscle markers [smooth muscle actin, desmin, h-caldesmon, and smooth muscle myosin (P < 0.0001)], as well as the more recently described markers SLMAP, MYLK, and ACTG2 (P < 0.0001). In primary tumours, both desmin and CFL2 expression predicted improved overall survival in multivariate analyses (P = 0.0111 and P = 0.043, respectively). Patients with muscle marker-enriched tumours (expressing all four conventional markers or any three of ACTG2, CFL2, CASQ2, MYLK, and SLMAP) had improved overall survival (P < 0.05) in univariate analyses. CONCLUSIONS: Morphologically and immunohistochemically, poorly differentiated leiomyosarcomas can masquerade as undifferentiated pleomorphic sarcomas with progressive loss of muscle markers. The expression of muscle markers has prognostic significance in primary leiomyosarcomas independently of tumour morphology.