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Obra ressenyada: Rudolf MUHR; Eugênia DUARTE; Amália MENDES; Carla AMORÓS; Juan A. THOMAS, Pluricentric Languages and Non-Dominant Varieties Worldwide. Part II : The Pluricentricity of Portuguese and Spanish. New Concepts and Descriptions. Frankfurt am Main: Peter Lang, 2016.

In May 2017, The University of Texas Hydrate Pressure Coring Expedition Gulf of Mexico 2-1 (UT-GOM2-1) drilled two adjacent holes in Green Canyon Block 955 in the deep-water Gulf of Mexico as part of The University of Texas at Austin and US Department of Energy Deepwater Methane Hydrate Characterization and Scientific Assessment. Expedition operations included testing two configurations of a rotary pressure-coring tool in a gas hydrate–bearing formation. In the first hole, an extended core barrel (cutting shoe) configuration of the Pressure Coring Tool with Ball Valve (PCTB-CS) was deployed, and in the second hole, the PCTB face bit configuration (PCTB-FB) was deployed. The PCTB-CS successfully recovered and maintained pressure for only one core out of eight deployments. A series of incremental modifications were made during and after the PCTB-CS deployment period that impacted the operations of the subsequent PCTB-FB deployments. Thus, in the second hole, the PCTB-FB successfully recovered and maintained pressure within the hydrate stability zone for 11 cores out of 13 deployments. The PCTB cored gas hydrate–bearing sandy silt interbedded with non–hydrate-bearing clayey silt within the main reservoir. The PCTB also recovered long intervals of unbroken, high-quality core with preserved sedimentary structures. We recovered one pressure core 130 m (437 ft) above the main hydrate reservoir in the silty clay. Pressure coring is the only available technology for recovering intact cores from sediment that is normally disturbed by gas expansion, dissolution, or dissociation; this allows a wide range of scientific measurements to be obtained with minimal disturbance to the core sediment fabric. Analysis of pressure cores has the potential to illuminate the in situ properties, gas saturation, and gas composition of a wide range of reservoirs including unconventional shale systems.

The University of Texas Hydrate Pressure Coring Expedition (UT-GOM2-1) recovered cores at near in situ formation pressures from a gas hydrate reservoir composed of sandy silt and clayey silt beds in Green Canyon Block 955 in the deep-water Gulf of Mexico. The expedition results are synthesized and linked to other detailed analyses presented in this volume. Millimeter- to meter-scale beds of sandy silt and clayey silt are interbedded on the levee of a turbidite channel. The hydrate saturation (the volume fraction of the pore space occupied by hydrate) in the sandy silts ranges from 79% to 93%, and there is little to no hydrate in the clayey silt. Gas from the hydrates is composed of nearly pure methane (99.99%) with less than 400 ppm of ethane or heavier hydrocarbons. The δ13C values from the methane are depleted (−60‰ to −65‰ Vienna Peedee belemnite), and it is interpreted that the gases were largely generated by primary microbial methanogenesis but that low concentrations of propane or heavier hydrocarbons record at least trace thermogenic components. The in situ pore-water salinity is very close to that of seawater. This suggests that the excess salinity generated during hydrate formation diffused away because the hydrate formed slowly or because it formed long ago. Because the sandy silt deposits have high hydrate concentration and high intrinsic permeability, they may represent a class of reservoir that can be economically developed. Results from this expedition will inform a new generation of reservoir simulation models that will illuminate how these reservoirs might be best produced.

The distinction between nevoid melanoma and a mitotically active nevus can be challenging at the microscopic level. In this study, we performed cytogenetic testing on a cohort of 25 mitotically active melanocytic proliferations resembling common melanocytic nevus from 25 patients. Based on cytogenetic findings, the lesions were classified as "nevoid melanoma" (n = 13) or "mitotically active nevus" (n = 12). Subsequently, we compared the clinicopathological features between these 2 groups. Nevoid melanomas occurred in older patients (P = 0.007); however, there were no significant differences in gender, size, or anatomical distribution between the 2 groups. Histologically, deep/marginal mitoses (P = 0.006), lack of maturation with depth (P = 0.036), and pseudo-maturation (P = 0.006) were significantly more common in nevoid melanomas. Immunohistochemically, complete loss of p16 was an important divisive feature (P = 0.0004), seen in 70% of nevoid melanomas, and highly correlated with loss of CDKN2A gene (chromosome 9p21). Our findings suggest that such reproducible immunomorphological differences can be of value in distinguishing nevoid melanoma from mitotically active nevus. Nevoid melanomas demonstrated a spectrum of chromosomal aberrations similar to those seen in common subtypes of melanoma, which can serve as a powerful adjunct diagnostic tool in morphologically challenging lesions.

Increasing evidence points to the presence of low-level de novo T790M mutations in patients with non-small cell lung carcinoma (NSCLC) harboring activating EGFR mutations. We utilized digital PCR (dPCR), a highly sensitive gene mutation detection method, to detect pre-treatment T790M mutations in NSCLC tumor samples and correlated the T790M status with clinical features and patient outcomes. DNA extracted from pre-treatment NSCLC tumor tissue with known activating EGFR mutations, diagnosed between October 2010 and May 2017 at PathWest laboratory, was used to perform targeted dPCR for quantitative detection of T790M mutations. T790M was detected in 42 of 109 pre-treatment samples (38.5%). Median variant allele frequency was 0.14% (range 0.02–28.5%). Overall response rate to first generation EGFR tyrosine kinase inhibitors (TKI) was 67% regardless of T790M status. The median progression free survival was 10.7 (IQR 5.6–19.9) versus 6.7 (IQR 3.5–20.8) months in T790M negative and positive patients respectively. T790M positivity correlated with increased rate of early disease progression. It also correlated with increased mortality (HR 3.1 95%CI 1.2–8.1, p = 0.022) in patients who did not respond to TKI treatment. We detected a significant rate of low-level pre-treatment T790M mutations in NSCLC using highly sensitive dPCR. Low-level pre-treatment T790M did not impact treatment response rate or overall survival, but was associated with increased rate of early progression on TKI therapy.

ContextPatient safety is a priority for healthcare organisations worldwide and is a key factor in providing high quality healthcare. Prescribing medications correctly is critical to ensuring safety, especially in the setting of a Paediatric Intensive Care Unit (PICU) where patients are vulnerable to being exposed to incidents due to highly complex care and illness severity. In our 21 bedded PICU, any prescribing errors detected by critical care pharmacists are recorded on a prescribing error database each day (Microsoft Access). Information inputted includes the drug involved in the error, the route of administration, prescriber identifier number, type of error and category of error based on the NCC MERP1classification system. Information is extracted monthly from this database to further populate a prescribing errors dashboard, highlighting the total number of prescribing errors each month and sub-categorising the number of errors according to drug cause and error type.Data collected in 2021 was analysed by our Trust’s Quality Improvement (QI) Team who generated pareto charts for the highest reported prescribing errors according to drug and error type. Although pharmacist data showed that many drugs were responsible for prescribing errors, pareto analysis by the QI team identified that Teicoplanin, Heparin, Fentanyl, Chloral Hydrate and Octenisan® were the drugs associated with the most frequent number of errors and causing the biggest cumulative impact on our prescribing error data. In terms of error type, pareto analysis identified that 80% of our cumulative errors were attributed to the wrong route, wrong dose or missing route of administration.ConclusionA pareto chart is a graph that indicates the frequency of defects as well as their cumulative impact. By applying this statistical control process to PICU prescribing error data for 2021, we were able to identify the drugs and error types responsible for the majority of our cumulative errors. Using the ‘Brilliant Basics’ methodology,2we followed a two-step approach in dissecting our data. For step one, we analysed the data that we had and then in step two, using this analysis, we were able to agree and introduce measures to our prescribing systems in order to mitigate the risk of the errors re-occurring. These measures have included redesigning our PICU prescription to add or adapt prescribing recommendations for Teicoplanin, Heparin and Fentanyl, updating prescribing advice in our PICU electronic drugs formulary for Chloral Hydrate and placing an additional daily task on our nurses’ electronic task list to ensure Octenisan® is used. In terms of error type, we have raised awareness of the prevalence of the errors causing the biggest impact on reported prescribing errors, through the medium of pharmacy newsletters, which are disseminated to all PICU staff and by educating new PICU prescribers as part of their induction to the unit. To assess whether the above changes have contributed to an improvement in our reported errors by drug and type, we will continue to perform statistical analysis on prescribing data collected throughout 2022.ReferencesNational Coordinating Council for Medication Error Reporting and Prevention. 2001. http://www.nccmerp.org/types-medication-errors Accessed 14 June 2022.A Catalyst for Better Health Strategy 2019/22. NHS Business Services Authority (V1) 03 2019. https://www.nhsbsa.nhs.uk/sites/default/files/2019-04/Strategy%202019-22%20%28V1%29%2003.2019.pdf

Summary Ki-67 proliferative index (PI) has prognostic and predictive value in invasive breast carcinoma (IBC), but clinical uptake has been hampered by suboptimal accuracy, reproducibility and standardisation. Published guidelines have addressed pre-analytical and analytical factors to improve Ki-67 PI utility; however, practicalities of ongoing monitoring of Ki-67 PI quality in IBC reporting have not been established. We aimed to evaluate the internal and external quality of our established digital Ki-67 PI IBC reporting practice at a tertiary institution. In the 5 years since initial validation work, we've completed a series of internal and external quality assurance (QA) projects: (1) an interobserver agreement study, (2) a two site interlaboratory agreement study, (3) determination of the error of our Ki-67 results, (4) an audit of the year-to-year Ki-67 values, (5) an audit of Ki-67 in neoadjuvant chemotherapy (NAC) treated cases, and (6) comparison of our Ki-67 datasets with similar published datasets. There was excellent concordance (intra-class correlation = 0.98) and good agreement [kappa (κ) = 0.76–0.96] between pathologists, excellent concordance [Pearson correlation (R) = 0.94] and very good agreement (κ = 0.80) between laboratories and excellent concordance (R = 0.92–0.95) and good agreement (κ = 0.67–1.0) over time for our Ki-67 results. No significant difference was observed in Ki-67 data from year-to-year. Expected associations with clinico-pathological prognosticators, pathological complete response following NAC and mitotic index were evident. The median Ki-67 values from the overall and NAC treated datasets were within the range reported in other studies, and our data could be separated into similarly proportioned ‘high’ and ‘low’ Ki-67 PI groups when dichotomised as per protocols in other studies. Collectively, our work provides evidence of adequate internal and external quality control for our digital Ki-67 PI IBC reporting protocols. Given the paucity of formal Ki-67 QA programs, our approach could be emulated, and results compared between laboratories as a framework for internal and external Ki-67 QA.

Increasing evidence points to the presence of low-level de novo T790M mutations in patients with non-small cell lung carcinoma (NSCLC) harboring activating EGFR mutations. We utilized digital PCR (dPCR), a highly sensitive gene mutation detection method, to detect pre-treatment T790M mutations in NSCLC tumor samples and correlated the T790M status with clinical features and patient outcomes. DNA extracted from pre-treatment NSCLC tumor tissue with known activating EGFR mutations, diagnosed between October 2010 and May 2017 at PathWest laboratory, was used to perform targeted dPCR for quantitative detection of T790M mutations. T790M was detected in 42 of 109 pre-treatment samples (38.5%). Median variant allele frequency was 0.14% (range 0.02-28.5%). Overall response rate to first generation EGFR tyrosine kinase inhibitors (TKI) was 67% regardless of T790M status. The median progression free survival was 10.7 (IQR 5.6-19.9) versus 6.7 (IQR 3.5-20.8) months in T790M negative and positive patients respectively. T790M positivity correlated with increased rate of early disease progression. It also correlated with increased mortality (HR 3.1 95%CI 1.2-8.1, p = 0.022) in patients who did not respond to TKI treatment. We detected a significant rate of low-level pre-treatment T790M mutations in NSCLC using highly sensitive dPCR. Low-level pre-treatment T790M did not impact treatment response rate or overall survival, but was associated with increased rate of early progression on TKI therapy.

Abstract. Objective: Fecal incontinence and constipation are common disorders in childhood. The enteric nervous system and the central nervous system are highly interactive along the brain-gut axis. The interaction is mainly afferent. These afferent pathways include centers that are involved in the central nervous processing of emotions as the mid/posterior insula and the anterior cingulate cortex. A previous study revealed altered processing of emotions in children with fecal incontinence. The present study replicates these results. Methods: In order to analyze the processing of emotions, we compared the event-related potentials of 25 children with fecal incontinence and constipation to those of 15 control children during the presentation of positive, negative, and neutral pictures. Results: Children with fecal incontinence and constipation showed altered processing of emotions, especially in the parietal and central cortical regions. Conclusions: The main study results of the previous study were replicated, increasing the certainty and validity of the findings.

Summary Distinction between melanocytic naevi and melanoma occasionally poses a diagnostic challenge in ambiguous cases showing overlapping histological features. Melanomas are characterised by the presence of multiple genomic copy number variants (CNVs), while this is not a feature of naevi. We assessed the feasibility and utility of array-based comparative genomic hybridisation (aCGH) to assess CNVs in melanocytic lesions. DNA was extracted from formalin fixed, paraffin embedded (FFPE) sections of unambiguous naevi ( n = 19) and melanomas ( n = 19). The test DNA and gender mismatched human reference DNA were differentially labelled with fluorophores. Equal quantities of the two DNA samples were mixed and co-hybridised to a SurePrint G3 Human CGH 8x60K array, and digitally scanned to capture and quantify the relative fluorescence intensities. The ratio of the fluorescence intensities was analysed by Cytogenomics software (Agilent). Frequent large CNVs were identified in 94.7% of melanoma samples, including losses of 9p (73.6%), 9q (52.6%), 10q (36.8%), 11q (36.8%), 3p (21%), and 10p (21%), and gains of 6p (42.1%), 7p (42.1%), 1q (36.8%), 8q (31.5%) and 20q (21%). Only one naevus showed two large copy number changes. Overall aCGH showed a specificity and sensitivity of 94.7% in separating naevi from melanomas. Based on our results, aCGH can be successfully used to analyse CNVs of melanocytic lesions utilising FFPE derived biopsy samples, providing a potentially useful adjunctive test for the classification of diagnostically challenging melanocytic proliferations.

Invasive lobular carcinoma (ILC) is almost always classified as Nottingham histological grade 2. Despite this, prognosis is markedly varied, with some ILCs behaving more akin to grade 3 invasive ductal carcinoma (IDC). Methods to separate these aggressive ILCs are needed. Digital image analysis (DIA) of the Ki-67 biomarker has potential in this regard; thus, we sought to determine the feasibility of its use for automated evaluation of ILC. An initial pilot study demonstrated no ILC specific changes were required to our Ki-67 DIA algorithm for reproducible results. Subsequently, 42 consecutive cases of ILC were evaluated by visual mitosis counting in HE p

Fecal incontinence and constipation are common disorders in childhood. The enteric nervous system and the central nervous system are highly interactive along the brain-gut axis. The interaction is mainly afferent. These afferent pathways include centers that are involved in the central nervous processing of emotions as the mid/posterior insula and the anterior cingulate cortex. A previous study revealed altered processing of emotions in children with fecal incontinence. The present study replicates these results.In order to analyze the processing of emotions, we compared the event-related potentials of 25 children with fecal incontinence and constipation to those of 15 control children during the presentation of positive, negative, and neutral pictures.Children with fecal incontinence and constipation showed altered processing of emotions, especially in the parietal and central cortical regions.The main study results of the previous study were replicated, increasing the certainty and validity of the findings.

Mitosis counting in HE stained sections is the most informative constituent of the Nottingham histological grade in breast carcinoma prognosis. Phosphohistone H3 (PHH3) immunohistochemistry (IHC) is a highly specific marker of mitoses, with practical application in identifying mitoses in poorly fixed or distorted tissue and is of prognostic significance in breast carcinoma. Our aim was to assess methods of PHH3 IHC mitosis counting in a tissue microarray (TMA) of 2 mm cores from 36 resected breast carcinomas. Mitoses in HE and PHH3 stained slides were manually scored by pathologist consensus and expressed as counts/2 mm. PHH3 stained cores were also evaluated by automated digital image analysis (DIA). Results were compared using Spearman correlation. A strong and significant correlation was observed between manual PHH3 and manual HE mitotic counts (correlation = 0.81; p 0.0001) and between automated PHH3 DIA and manual HE mitotic counts (correlation = 0.79; p 0.0001). More mitoses were identified with PHH3 IHC than with HE. Manual and DIA PHH3 counts were strongly and significantly correlated (correlation = 0.83; p 0.0001) and of similar absolute values. PHH3 DIA is a valid alternative to manual counting with potential application in breast cancer reporting and prognostication.

Hairy cell leukaemia (HCL) is a rare, indolent chronic B-cell leukaemia accounting for approximately 2% of all adult leukaemias. The recent association of the BRAF p.Val600Glu (V600E) mutation in HCL makes it a valuable molecular diagnostic marker. We compared the ability of Sanger sequencing, fluorescent single-strand conformational polymorphism (F-SSCP) and high resolution melting (HRM) analysis to detect BRAF mutations in 20 cases of HCL consisting of four archival Romanowsky stained air-dried peripheral blood and bone marrow aspirate smears, 12 mercury fixed decalcified bone marrow trephine biopsies, three formalin fixed, paraffin embedded (FFPE) splenectomy samples and one fresh peripheral blood sample. DNA was amplified and BRAF mutation status determined by the three methods above. V600E mutation was identified in 94%, 89% and 72% of HCL cases by F-SSCP, HRM and Sanger sequencing, respectively. In one case, in addition to the p.Val600Glu mutation, a p.Lys601Thr (K601T) mutation was identified. DNA from archival slide scrapings, mercury-fixed and FFPE tissue can be used to identify BRAF mutations with high sensitivity, especially using HRM/F-SSCP. The V600E mutation can be used as a supplementary molecular marker to aid in the diagnosis of HCL and the presence of the mutation may provide a target for therapy.

Serosal pathologies including malignant mesothelioma (MM) can show features of osseous and/or cartilaginous differentiation although the mechanism for its formation is unknown. Mesothelial cells have the capacity to differentiate into cells with myofibroblast, smooth muscle and endothelial cell characteristics. Whether they can differentiate into other cell types is unclear. This study tests the hypothesis that mesothelial cells can differentiate into cell lineages of the embryonic mesoderm including osteoblasts and adipocytes. To examine this, a functional assay of bone formation and an adipogenic assay were performed in vitro with primary rat and human mesothelial cells maintained in osteogenic or adipogenic medium (AM) for 0–26 days. Mesothelial cells expressed increasing levels of alkaline phosphatase, an early marker of the osteoblast phenotype, and formed mineralized bone-like nodules. Mesothelial cells also accumulated lipid indicative of a mature adipocyte phenotype when cultured in AM. All cells expressed several key osteoblast and adipocyte markers, including osteoblast-specific runt-related transcription factor 2, and demonstrated changes in mRNA expression consistent with epithelial-to-mesenchymal transition. In conclusion, these studies confirm that mesothelial cells have the capacity to differentiate into osteoblast- and adipocyte-like cells, providing definitive evidence of their multipotential nature. These data strongly support mesothelial cell differentiation as the potential source of different tissue types in MM tumours and other serosal pathologies, and add support for the use of mesothelial cells in regenerative therapies.