Integrated clinicopathologic and molecular analysis of endometrial carcinoma: Prognostic impact of the new ESGO-ESTRO-ESP endometrial cancer risk classification and proposal of histopathologic algorithm for its implementation in clinical practice

IntroductionThe European Society of Gynecologic Oncology/European Society of Radiation Therapy and Oncology/European Society of Pathology (ESGO/ESTRO/ESP) committee recently proposed a new risk stratification system for endometrial carcinoma (EC) patients that incorporates clinicopathologic and molecular features. The aim of the study is to compare the new ESGO/ESTRO/ESP risk classification system with the previous 2016 recommendations, evaluating the impact of molecular classification and defining a new algorithm for selecting cases for molecular analysis to assign the appropriate risk class.MethodsThe cohort included 211 consecutive EC patients. Immunohistochemistry and next-generation sequencing were used to assign molecular subgroups of EC: POLE mutant (POLE), mismatch repair deficient (MMRd), p53 mutant (p53abn), and no specific molecular profile (NSMP).ResultsImmuno-molecular analysis was successful in all cases, identifying the four molecular subgroups: 7.6% POLE, 32.2% MMRd, 20.9% p53abn, and 39.3% NSMP. The recent 2020 guidelines showed a 32.7% risk group change compared with the previous 2016 classification system: the reassignment is due to POLE mutations, abnormal p53 expression, and a better definition of lymphovascular space invasion. The 2020 system assigns more patients to lower-risk groups (42.2%) than the 2016 recommendation (25.6%). Considering the 2020 risk classification system that includes the difference between “unknown molecular classification” and “known,” the integration of molecular subgroups allowed 6.6% of patients to be recategorized into a different risk class. In addition, the use of the proposed algorithm based on histopathologic parameters would have resulted in a 62.6% reduction in molecular analysis, compared to applying molecular classification to all patients.ConclusionApplication of the new 2020 risk classification integrating clinicopathologic and molecular parameters provided more accurate identification of low-and high-risk patients, potentially allowing a more specific selection of patients for post-operative adjuvant therapy. The proposed histopathologic algorithm significantly decreases the number of tests needed and could be a promising tool for cost reduction without compromising prognostic stratification

Proteomics of black fungi : a sytems biology approach to investigate the adaptation to extreme environments

Schwarze Pilze gehören zu den stresstolerantesten Organismen auf der Erde. In dieser Studie wurde Proteomik von mikrokolonialen schwarzen Pilzen und pathogenen schwar-zen Hefen durchgeführt um die Auswirkung von Temperatur auf die Proteinexpression zu evaluieren. 2D-Proteinbandenmuster von Knufia perforans, Exophiala jeanselmei und von Friedmanniomyces endolithicus wurden mit dem Hyphomycet Penicillium chrysogenum verglichen. Während P. chrysogenum die höchste Zahl an Proteinen bei 40C und die niedrigste bei 1C exprimierte und somit einen sichtbaren Hinweis auf Temperaturab-hängigkeit gezeigt hat, verringerte sich die Anzahl der exprimierten Proteine bei schwar-zen Pilzen, als dieser Temperaturen von weit über dem optimalen Wachstum ausgesetzt wurden. Dieses Phänomen weist wahrscheinlich auf eine Herabregulierung von metabo-lischen Prozessen hin. Bei 1C stieg die Zahl der Proteine bei schwarzen Pilzstämmen an. Die Ergebnisse unserer Studie zeigen, dass schwarze Pilze eine andere Strategie verwenden als P. chrysogenum um die nicht optimalen Temperaturen zu bewältigen. 2D-DIGE wurde verwendet um die Charakterisierung der temperaturabhängigen Verän-derungen in der Gesamtproteinexpression der schwarzen Hefe Exophiala dermatitidis die Primär- und Sekundärinfektionen in gesunden menschlichen Wirten verursacht zu untersuchen. Drei Inkubationstemperaturen (37, 45, 1C) und zwei Zeitspannen (1 Std., 1 Woche) wurden ausgewählt um unterschiedliche Umweltbedingungen zu simulieren. 32 unterschiedliche Proteine wurden mittels Massenspektrometrie identifiziert. Interes-santerweise wurde bei 45C keine signifikante Stressreaktion festgestellt, wodurch bei 1C eine allgemeine Senkung des Metabolismus erreicht wurde. Unsere Ergebnisse lassen darauf schließen, dass unter erhöhten Temperaturen eine Feinregulation der Pro-teinexpression stattfindet, insbesondere bei Proteinsets, die in entscheidenden biologi-schen Prozessen involviert sind und das Zellüberleben sicherstellen.Black fungi are among the most stress tolerant organisms on Earth. In this study prote-omics of black rock microcolonial fungi and pathogenic black yeasts was performed in order to evaluate the impact of temperature on the protein expression profile. 2D-protein patterns of Knufia perforans, Exophiala jeanselmei and Friedmanniomyces endolithicus were compared with the hyphomycete Penicillium chrysogenum. Whereas P. chryso-genum showed the highest number of protein spots at 40C and the lowest at 1C, thus exhibiting real signs of temperature induced reaction, black fungi when exposed to temperatures far above their growth optimum decreased the spots number indicating a down-regulation of the metabolism. At 1C, an increase of protein spots occurred instead in all the black fungi strains. These results indicate a rather different strategy to cope with non-optimal temperature in rock inhabiting black fungi than in hyphomycetes as P. chrysogenum. 2D-DIGE was applied for characterization of temperature-related chang-es in total protein spot abundance in the black yeast Exophiala dermatitidis, an agent of primary and secondary diseases in both immunocompromised and healthy humans. Three incubation temperatures (37, 45, 1C) and two time spans (1h, 1 week) were se-lected to simulate different environmental conditions and evaluate the effects of short- and long-term exposure. A total of 32 variable proteins were identified by mass spec-trometry. Data about protein functions, localization and pathways were obtained. Inter-estingly, a typical stress response under non-optimal temperature was not observed at the proteome level, whereas a reduction of the metabolic activity was detected after ex-posure to cold. Our results suggests that under increased temperatures, a fine regulation of protein expression takes place, particularly concerning the protein sets which are in-volved in crucial biological processes, to guarantee the cell survival and maintenance.submitted by Donatella TeseiTitelzusatz sollte lauten: a systems biology approach to investigate the adaptation to extreme environmentsZusammenfassung in deutscher SpracheParalleltitel [Übersetzung des Autors]: Proteomik von schwarzen Pilzen: ein systembiologischer Ansatz um die Anpassung an extreme Umweltbedingungen zu untersuchenUniversität für Bodenkultur Wien, Dissertation, 2015OeBB(VLID)193029

Pie chart of the summarized Gene Ontology (GO) terms for different conditions.

<p>(A) Over-represented BP-related GO terms for the genes upregulated at 1°C for 1 hour. (B) Over-represented GO terms related to biological processes (BP) for the genes up-regulated at 1°C for 1 week. (C) Over-represented BP-related GO terms for the genes down-regulated at 1°C for 1 week. (D) Over-represented CC-related GO terms for the genes up-regulated at 1°C for 1 week. (E) Over-represented BP-related GO terms for the genes down-regulated at 45°C for 1hour. (F) Over-represented BP-related GO terms for the genes up-regulated at 45°C for 1 week. (G) Over-represented BP-related GO terms for the genes down-regulated at 45°C for 1 week. (H) Over-represented BP-related GO terms for the genes up-regulated at both 45C1H and 45C1W.</p

From Glacier to Sauna: RNA-Seq of the Human Pathogen Black Fungus <i>Exophiala dermatitidis</i> under Varying Temperature Conditions Exhibits Common and Novel Fungal Response

<div><p><i>Exophiala dermatitidis (Wangiella dermatitidis</i>) belongs to the group of the so-called black yeasts. Thanks in part to its thick and strongly melanized cell walls, <i>E</i>. <i>dermatitidis</i> is extremely tolerant to various kinds of stress, including extreme pH, temperature and desiccation. <i>E</i>. <i>dermatitidis</i> is also the agent responsible for various severe illnesses in humans, such as pneumonia and keratitis, and might lead to fatal brain infections. Due to its association with the human environment, its poly-extremophilic lifestyle and its pathogenicity in humans, <i>E</i>. <i>dermatitidis</i> has become an important model organism. In this study we present the functional analysis of the transcriptional response of the fungus at 1°C and 45°C, in comparison with that at 37°C, for two different exposition times, i.e. 1 hour and 1 week. At 1°C, <i>E</i>. <i>dermatitidis</i> uses a large repertoire of tools to acclimatize, such as lipid membrane fluidization, trehalose production or cytoskeleton rearrangement, which allows the fungus to remain metabolically active. At 45°C, the fungus drifts into a replicative state and increases the activity of the Golgi apparatus. As a novel finding, our study provides evidence that, apart from the protein coding genes, non-coding RNAs, circular RNAs as well as fusion-transcripts are differentially regulated and that the function of the fusion-transcripts can be related to the corresponding temperature condition. This work establishes that <i>E</i>. <i>dermatitidis</i> adapts to its environment by modulating coding and non-coding gene transcription levels and through the regulation of chimeric and circular RNAs.</p></div

Protein functional analysis data in support of comparative proteomics of the pathogenic black yeast Exophiala dermatitidis under different temperature conditions

In the current study a comparative proteomic approach was used to investigate the response of the human pathogen black yeast Exophiala dermatitidis toward temperature treatment. Protein functional analysis – based on cellular process GO terms – was performed on the 32 temperature-responsive identified proteins. The bioinformatics analyses and data presented here provided novel insights into the cellular pathways at the base of the fungus temperature tolerance. A detailed analysis and interpretation of the data can be found in “Proteome of tolerance fine-tuning in the human pathogen black yeast Exophiala dermatitidis” by Tesei et al. (2015) [1]

Representation of up- and down-regulated genes for cold and hot conditions, compared to 37°C.

<p>Upwards and downwards arrows represent up- and down-regulated genes, respectively. Protein coding genes (A) show the highest number of differentially expressed genes following the treatment at 1°C for 1 week (1C1W), with a total of 609 and 288 down- and up-regulated genes, respectively. NcRNA genes (B) show the highest number of differentially expressed genes at the same experimental condition (1°C for 1 week, 1C1W), with a total of 30 and 6 up- and downregulated genes, respectively. For both coding and non-coding genes, the smallest number of differentially expressed genes is found when the fungus have been exposed at 45°C for 1 week (45C1W).</p

Graphical representation of the chimeric RNAs with the highest number of split reads for different temperatures.

<p>Fusion transcripts present at the following experimental conditions: 1°C for 1 week (A), 37°C (B) and 45°C for 1 week (C). The thick blue arrow represents the Supercontig with the corresponding scale. The start codon is represented by the green arrow. The stop codon is shown as a red vertical line. The splice connecting both genes is shown in black and the number of supporting split reads is located between both fused genes. The blue regions represent the Coding DNA Sequence (CDS), the dark grey regions represent the Untranslated Regions (UTRs), while the light gray regions represent introns. The splicing direction is given by the black arrow head.</p