A protein complex network of Drosophila melanogaster.

Determining the composition of protein complexes is an essential step toward understanding the cell as an integrated system. Using coaffinity purification coupled to mass spectrometry analysis, we examined protein associations involving nearly 5,000 individual, FLAG-HA epitope-tagged Drosophila proteins. Stringent analysis of these data, based on a statistical framework designed to define individual protein-protein interactions, led to the generation of a Drosophila protein interaction map (DPiM) encompassing 556 protein complexes. The high quality of the DPiM and its usefulness as a paradigm for metazoan proteomes are apparent from the recovery of many known complexes, significant enrichment for shared functional attributes, and validation in human cells. The DPiM defines potential novel members for several important protein complexes and assigns functional links to 586 protein-coding genes lacking previous experimental annotation. The DPiM represents, to our knowledge, the largest metazoan protein complex map and provides a valuable resource for analysis of protein complex evolution.

JGI Plant Gene Atlas: an updateable transcriptome resource to improve functional gene descriptions across the plant kingdom.

Gene functional descriptions offer a crucial line of evidence for candidate genes underlying trait variation. Conversely, plant responses to environmental cues represent important resources to decipher gene function and subsequently provide molecular targets for plant improvement through gene editing. However, biological roles of large proportions of genes across the plant phylogeny are poorly annotated. Here we describe the Joint Genome Institute (JGI) Plant Gene Atlas, an updateable data resource consisting of transcript abundance assays spanning 18 diverse species. To integrate across these diverse genotypes, we analyzed expression profiles, built gene clusters that exhibited tissue/condition specific expression, and tested for transcriptional response to environmental queues. We discovered extensive phylogenetically constrained and condition-specific expression profiles for genes without any previously documented functional annotation. Such conserved expression patterns and tightly co-expressed gene clusters let us assign expression derived additional biological information to 64 495 genes with otherwise unknown functions. The ever-expanding Gene Atlas resource is available at JGI Plant Gene Atlas (https://plantgeneatlas.jgi.doe.gov) and Phytozome (https://phytozome.jgi.doe.gov/), providing bulk access to data and user-specified queries of gene sets. Combined, these web interfaces let users access differentially expressed genes, track orthologs across the Gene Atlas plants, graphically represent co-expressed genes, and visualize gene ontology and pathway enrichments.

A comprehensive population-based study of malignant ovarian tumors, including histologic and immunohistochemical review, in children and adolescents 0-19 years old in Sweden between 1970 and 2014.

OBJECTIVES: Ovarian tumors in the pediatric population are rare. The incidence and frequency of subtypes differ between children and adults. Although not all tumors are aggressive, they may still lead to morbidity. The goal of this study was a comprehensive review of malignant ovarian tumors in children and adolescents diagnosed and registered in Sweden. METHODS: Individuals were identified through a search in the National Cancer Register, limited for ages 0-19, years 1970-2014. Stored tumor diagnostic material from regional biobanks was retrieved and reviewed. RESULTS: The study includes 345 individuals with ovarian tumors and 70.7% of them were between 15 and 19 years at time of diagnosis. No differences in incidence over time or geographic location were identified. The average follow-up time was 21.2 years and 5-year survival was 88.4%. Survival was similar in the different time periods, except for 1970-1979. Review was possible for 260 cases, resulting in 85 epithelial tumors, 121 GCTs, 47 SCSTs and 7 others. For age 0-4 years SCSTs dominated (85.7%), for 5-9- and 10-14-years GCTs dominated (70,8% and 75.0% respectively), and for age 15-19 years epithelial tumors dominated (43.8%). There was a strong agreement between review diagnosis and original diagnosis (Cohen's κ 0.944). Differentiating between entities within the sex cord-stromal group posed the biggest diagnostic challenge. CONCLUSIONS: Ovarian tumors in children and adolescents are rare and distinct from their adult counterparts regarding incidence and frequency. There was a strong concurrence between original and review diagnoses. The greatest diagnostic difficulty was subtyping of epithelial tumors and differentiating between tumors within the SCST group.

Prognostic performance of the 2023 FIGO staging schema for endometrial cancer.

OBJECTIVE: To assess the prognostic performance of the 2023 International Federation of Gynecology and Obstetrics (FIGO) endometrial cancer staging schema. METHODS: This retrospective cohort study queried the Commission-on-Cancer's National Cancer Database. Study population was 129,146 patients with stage I-IV endometrial cancer per the 2009 FIGO staging schema. Stage-shifting and overall survival (OS) were assessed according to the 2023 FIGO staging schema. RESULTS: Upstage (IA → II, 21.4 %; IB → II, 53.0 %) and downstage (IIIA→IA3, 22.2 %) occurred in both early and advanced diseases. Inter-stage prognostic performance improved in the 2023 schema with widened 5-year OS rate difference between the earliest and highest stages (68.2 % to 76.9 %). Stage IA1-IIB and IIC had distinct 5-year OS rate differences (85.8-96.1 % vs 75.4 %). The 5-year OS rate of the 2009 stage IIIA disease was 63.9 %; this was greater segregated in the 2023 schema: 88.0 %, 62.4 %, and 55.7 % for IIIA→IA3, IIIA1, and IIIA2, respectively (inter-substage rate-difference, 32.3 %). This 5-year OS rate of stage IA3 disease was comparable to the 2023 stage IB-IIB diseases (88.0 % vs 85.8-89.5 %). In the 2023 stage IIIC schema (micrometastasis rates: 29.6 % in IIIC1 and 15.6 % in IIIC2), micrometastasis and macrometastasis had the distinct 3-year OS rates in both pelvic (IIIC1-i vs IIIC1-ii, 84.9 % vs 71.1 %; rate-difference 13.8 %) and para-aortic (IIIC2-i vs IIIC2-ii, 82.9 % vs 65.2 %; rate-difference 17.7 %) nodal metastasis cases. The 5-year OS rate of the 2009 stage IVB disease was 23.4 %; this was segregated to 25.4 % for stage IVB and 19.2 % for stage IVC in the 2023 staging schema (rate-difference, 6.2 %). CONCLUSION: The 2023 FIGO endometrial cancer staging schema is a major revision from the 2009 FIGO schema. Almost doubled enriched sub-stages based on detailed anatomical metastatic site and incorporation of histological information enable more robust prognostication.

p53 Abnormal (Copy Number High) Endometrioid Endometrial Carcinoma Has a Prognosis Indistinguishable From Serous Carcinoma.

Among the 4 molecular subgroups of endometrial carcinoma, the p53 abnormal (copy number high) subgroup has the worst prognosis; however, the histologic characteristics of this subgroup are not well established. Also, it is not well established whether low-grade tumors can belong to the p53 abnormal molecular subgroup and if so, what is the prognostic significance of the p53-mutated molecular subgroup in low-grade tumors. In the current study, we included 146 p53-mutated endometrial carcinomas and performed molecular subgrouping either based on a combination of immunohistochemical studies for p53 and MMR protein expression and POLE mutation testing (81 cases) or based on array-based and sequencing-based technologies (65 cases). We excluded cases that belonged to the POLE mutant or MSI molecular subgroups and only studied p53 abnormal (molecular subgroup) endometrial carcinomas (125 cases). In 71 cases, the molecular subgroup was determined by a combination of immunohistochemical studies and POLE mutation testing, and in 54 cases by array-based and sequencing-based methods. We reviewed 1 to 2 representative digital slides from each case and recorded the morphologic characteristics as well as clinical, treatment, and survival follow-up data. Overall, 47 cases were classified as endometrioid carcinoma, 55 serous carcinoma, and 23 other histotypes. Eight cases were FIGO 1, 21 were FIGO 2, and 91 were FIGO 3. A significant proportion of the cases (24.2%) were histologically classified as low-grade (FIGO 1 or 2) endometrioid carcinoma. There was no morphologic characteristic that showed prognostic implication. There was no significant difference in survival among different histotypes (P=0.60). There was no significant difference in survival among low-grade endometrioid (FIGO 1 or 2) versus high-grade (FIGO 3) tumors (P=0.98). Early-stage (stage I), low-grade tumors showed no significant survival advantage over early-stage, high-grade tumors (P=0.16) and this was more evident in FIGO 2 tumors. Although not statistically significant, the FIGO 2 tumors showed a trend toward worse survival than FIGO 3 tumors. Among the cases with available treatment data, more patients with early-stage high-grade tumors received adjuvant treatment, compared to patients with early-stage low-grade tumors, possibly explaining this trend (P=0.03). In conclusion, the findings of our study suggest that low-grade p53 abnormal endometrioid endometrial carcinomas (especially FIGO 2 tumors) have an aggressive course, with a prognosis similar to high-grade tumors. Furthermore, our study suggests that patients who had early-stage low-grade p53 abnormal disease might have been undertreated because of the "low-grade" histotype.

FOXL2 Mutation Status in Sex Cord-stromal Tumors Cannot be Predicted by Morphology.

Granulosa-cell tumors (GCTs) are the most common type of malignant ovarian sex cord-stromal tumor (SCST). The histopathologic diagnosis of these tumors can be challenging. A recurrent somatic mutation of the forkhead box L2 (FOXL2) gene has been identified in adult GCT. In this retrospective single-center study of 44 SCST, a morphologic review together with analysis of FOXL2 C134W was evaluated in relation to tumor morphology. In addition, TERT promoter mutation testing was performed. Twelve of 36 cases got an altered diagnosis based on morphology alone. The overarching architectural growth pattern in 32/44 (72.7%) tumors was diffuse/solid with several tumors showing markedly heterogeneous architecture. In correlation to FOXL2 C134W mutation status, cytoplasmic color, and nuclear shape, differed between the FOXL2 C134W positive and FOXL2 C134 W negative groups, but these differences were not significant when comparing them separately. Nineteen of 44 cases underwent TERT promoter sequencing with a positive result in 3 cases; 2 adult GCTs and 1 cellular fibroma. Three patients developed a recurrence of which 2 were FOXL2 C134W positive adult GCTs and the third was an unclassified SCST. In conclusion, the morphologic and immunohistochemical diagnosis of different SCSTs is challenging and one cannot reliably identify FOXL2 mutation-positive tumors solely by morphologic features. Therefore, broad use of molecular analysis of the FOXL2 C134W mutation is suggested for SCSTs, and further studies are needed to evaluate the clinical outcome of these tumors as well as the diagnostic and prognostic implications of TERT promoter mutations.

Exploring Molecular Drivers of PARPi Resistance in BRCA1-Deficient Ovarian Cancer: The Role of LY6E and Immunomodulation.

Approximately 50% of patients diagnosed with ovarian cancer harbor tumors with mutations in BRCA1, BRCA2, or other genes involved in homologous recombination repair (HR). The presence of homologous recombination deficiency (HRD) is an approved biomarker for poly-ADP-ribose polymerase inhibitors (PARPis) as a maintenance treatment following a positive response to initial platinum-based chemotherapy. Despite this treatment option, the development of resistance to PARPis is common among recurrent disease patients, leading to a poor prognosis. In this study, we conducted a comprehensive analysis using publicly available datasets to elucidate the molecular mechanisms driving PARPi resistance in BRCA1-deficient ovarian cancer. Our findings reveal a central role for the interferon (IFN) pathway in mediating resistance in the context of BRCA1 deficiency. Through integrative bioinformatics approaches, we identified LY6E, an interferon-stimulated gene, as a key mediator of PARPi resistance, with its expression linked to an immunosuppressive tumor microenvironment (TME) encouraging tumor progression and invasion. LY6E amplification correlates with poor prognosis and increased expression of immune-related gene signatures, which is predictive of immunotherapy response. Interestingly, LY6E expression upon PARPi treatment resistance was found to be dependent on BRCA1 status. Gene expression analysis in the Orien/cBioPortal database revealed an association between LY6E and genes involved in DNA repair, such as Rad21 and PUF60, emphasizing the interplay between DNA repair pathways and immune modulation. Moreover, PUF60, Rad21, and LY6E are located on chromosome 8q24, a locus often amplified and associated with the progression of ovarian cancer. Overall, our study provides novel insights into the molecular determinants of PARPi resistance and highlights LY6E as a promising prognostic biomarker in the management of HRD ovarian cancer. Future studies are needed to fully elucidate the molecular mechanisms underlying the role of LY6E in PARPi resistance.

Prognostic value of lymphovascular space invasion according to the molecular subgroups in endometrial cancer.

OBJECTIVE: Lymphovascular space invasion (LVSI) is a known prognostic factor for oncological outcome in endometrial cancer patients. However, little is known about the prognostic value of LVSI among the different molecular subgroups. The aim of this study was to determine the prognostic dependence of LVSI from the molecular signature. METHODS: This study included endometrial cancer patients who underwent primary surgical treatment between February 2004 and February 2016 at the Karolinska University Hospital, Sweden and the Bern University Hospital, Switzerland (KImBer cohort). All cases had complete molecular analysis performed on the primary tumor according to the WHO Classification of Tumors, 5th edition. LVSI was reviewed by reference pathologists for all pathology slides. RESULTS: A total of 589 endometrial cancer patients were included in this study, consisting of 40 POLEmut (polymerase epsilon ultramutated), 198 MMRd (mismatch repair deficient), 83 p53abn (p53 abnormal), and 268 NSMP (non-specific molecular profile) cases. Altogether, 17% of tumors showed LVSI: 25% of the POLEmut, 19% of the MMRd, 30% of the p53abn, and 10% of the NSMP cases. There was a significant correlation of LVSI with lymph node metastasis in the entire study cohort (p<0.001), remaining significant in the MMRd (p=0.020), p53abn (p<0.001), and NSMP (p<0.001) subgroups. Mean follow-up was 89 months (95% CI 86 to 93). The presence of LVSI significantly decreased recurrence-free survival among patients with MMRd, p53abn, and NSMP endometrial cancer, and overall survival in patients with p53abn and NSMP tumors. In patients with NSMP endometrial cancer, evidence of substantial LVSI remained a significant independent predictor of recurrence in multivariable Cox regression analysis including tumor stage and grade (HR 7.5, 95% CI 2.2 to 25.5, p=o.001). CONCLUSION: The presence of LVSI was associated with recurrence in each subgroup of patients with MMRd, p53abn, and NSMP endometrial cancer, and LVSI remained an independent predictor of recurrence in NSMP endometrial cancer patients.

Genome evolution in the allotetraploid frog Xenopus laevis.

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

Determining independence and associations among various cardiovascular disease risk factors in 9-12 years old school-children: a cross sectional study.

BACKGROUND: Cardiovascular disease (CVD) risk assessment of children typically includes evaluating multiple CVD risk factors some of which tend to correlate each other. However, in older children and young adolescents, there are little data on the level of independence of CVD risk factors. The purpose of this study was to examine the relationships among various CVD risk factors to determine the level of independence of each risk factor in a sample of 5th-grade public school students. METHOD: A cross-sectional analysis of 1525 children (856 girls and 669 boys; age: 9-12 years) who participated in baseline CVD risk assessment for the (S)Partners for Heart Health program from 2010 - 2018. Thirteen CVD risk factor variables were used in the analysis and included blood lipids [low-density lipoprotein (LDL), high-density lipoprotein (HDL), total cholesterol (TC), and triglycerides], resting systolic and diastolic blood pressure (BP); anthropometrics [height, weight, body mass index (BMI), % body fat, waist circumference (WC)]. Additionally, acanthosis nigricans (a marker insulin resistance and diabetes), and cardiorespiratory fitness (VO2 ml/kg) was estimated using the PACER. Descriptive statistics, bivariate Pearson correlations, and principal component analysis were used to determine the relationships among these variables and the independence. RESULTS: Parallel analysis indicated two components should be extracted. Among the two components extracted, WC, % body fat, and BMI loaded highest on component 1, which explained 34% of the total variance. Systolic BP and diastolic BP loaded predominantly on component 2 and accounted for 17% of the variance. Cardiorespiratory fitness, acanthosis nigricans, HDL, and triglycerides loaded highest on the first component (loadings between 0.42 and 0.57) but still suggest some non-shared variance with this component. Low-density lipoprotein had low loadings on each component. Factor loadings were stable across sex. CONCLUSION: Among the various CVD risk indicators, measures of adiposity loaded highest on the component that explained the largest proportion of variability in the data reinforcing the importance of assessing adiposity in CVD risk assessment. In addition, blood pressure loaded highest on the second component, suggesting their relative independence when assessing CVD risk. The data also provide support and rationale for determining what CVD risk factors to include- based on resource needs. For example, researchers or public health programs may choose to assess WC instead of lipid profile for cardiovascular related problems if ease of assessment and cost are considerations.

Objective and Subjective Assessment of Bladder Function after Robot-assisted Laparoscopic Radical Hysterectomy for Early-stage Cervical Cancer.

STUDY OBJECTIVE: To examine whether objective bladder function after robot-assisted radical hysterectomy (RRH) for early-stage cervical cancer is correlated with subjective patient-reported outcomes and quality of life during the first year after RRH. DESIGN: Prospective observational study. SETTING: Karolinska University Hospital, Sweden. PATIENTS: Women with early-stage cervical cancer (International Federation of Gynecology and Obstetrics stage IA2-IB1) between July 2017 and May 2019 were assessed for eligibility. INTERVENTIONS: RRH. MEASUREMENTS AND MAIN RESULTS: Subjective bladder function was evaluated with the Female Lower Urinary Tract Symptoms and Urinary Incontinence Quality of Life modules of the International Consultation on Incontinence Questionnaire. Objective urinary function was characterized with urodynamic tests, and the nerves ablated at RRH were quantified by using immunohistochemical staining of biopsies from the resected paracervix, vesicouterine, and sacrouterine ligaments. Twenty-seven women were included for analysis at baseline, 2 weeks, 3 months, and 12 months after surgery. RRH caused hypotonia of the urinary bladder (p <.05). Patient-reported outcomes of voiding and filling dysfunction were most significant 2 weeks after surgery (p <.05) but for most of the women, bladder function recovered within 3 months. No correlations were found with either subjective or objective urinary function and the number of ablated nerves. CONCLUSION: For most women, objective and subjective urinary bladder dysfunction recovered within 3 months after RRH. The absence of correlation between functional outcomes and ablated autonomous nerves suggests that other underlying causes play a significant role. Early detection of bladder overextension after RRH is paramount, and the role of postoperative bladder catheterization needs further investigation.

Prognostic impact of tumor budding in endometrial carcinoma within distinct molecular subgroups.

Tumor budding is a robust prognostic parameter in several tumor entities but is rarely investigated in endometrial carcinoma. We applied the recently standardized counting method from the International Tumor Budding Consensus Conference for colorectal cancer (ITBCC) on a cohort of 255 endometrial carcinomas with known molecular profiles according to The Cancer Genome Atlas (TCGA) subgroups. Our investigation aims to clarify the potential prognostic role of tumor budding in endometrial carcinoma in contrast to other known prognostic factors, including molecular factors. In addition, the microcystic elongated and fragmented (MELF) pattern and tumor budding were compared with respect to their potential as markers for epithelial-mesenchymal transition (EMT). Tumor budding was found in n = 67 (26.3%) tumors, with a very low mean of 0.7 buds per ×20 HE field. Tumor budding was significantly associated with depth of invasion, nodal status, lymphatic invasion (each p < 0.001), grading (p = 0.004), and vascular invasion (p = 0.01). Tumor budding showed moderate inter-observer-variability with prognostic stratification irrespective of the observer (κ-value = 0.448). In multivariate analysis, tumor budding served as a significant independent prognosticator for worse outcomes in overall and recurrence-free survival (HR 2.376 and 2.736, p < 0.001), but not when the TCGA subgroups entered into the analysis. In consequence, dependency had to be clarified in the subgroup analysis for Polymerase E mutated (POLEmut), mismatch repair deficient (MMRdef), nonspecific mutation profile (NSMP), and P53 aberrant (P53abn) endometrial carcinomas. A particular impact was identified in the intermediate prognostic groups of NSMP and MMRdef carcinomas. Tumor budding outperformed the MELF pattern in single and combined prognostic information. In conclusion, the presence of tumor budding alone is a promising, robust, and easy-to-apply prognostic parameter in endometrial carcinoma. In a morpho-molecular approach, it exerts its prognostic potential in the most clinically relevant subgroups of endometrial carcinoma and serves as a good biomarker for EMT.

Performance of a HER2 testing algorithm specific for p53-abnormal endometrial cancer.

AIMS: Human epidermal growth factor receptor 2 (HER2) amplification in endometrial cancer (EC) is almost completely confined to the p53-abnormal (p53abn) molecular subtype and independent of histological subtype. HER2 testing should therefore be molecular subtype-directed. However, the most optimal approach for HER2 testing in EC has not been fully established. Therefore, we developed an EC-specific HER2 immunohistochemistry (IHC) scoring method and evaluated its reproducibility and performance to establish an optimal diagnostic HER2 testing algorithm for p53abn EC. METHODS AND RESULTS: HER2 IHC slides of 78 p53abn EC were scored by six gynaecopathologists according to predefined EC-specific IHC scoring criteria. Interobserver agreement was calculated using Fleiss' kappa and the first-order agreement coefficient (AC1). The consensus IHC score was compared with HER2 dual in-situ hybridisation (DISH) results. Sensitivity and specificity were calculated. A substantial interobserver agreement was found using three- or two-tiered scoring [κ = 0.675, 95% confidence interval (CI) = 0.633-0.717; AC1 = 0.723, 95% CI = 0.643-0.804 and κ = 0.771, 95% CI = 0.714-0.828; AC1 = 0.774, 95% CI = 0.684-0.865, respectively]. Sensitivity and specificity for the identification of HER2-positive EC was 100 and 97%, respectively, using a HER2 testing algorithm that recommends DISH in all cases with moderate membranous staining in >10% of the tumour (IHC+). Performing DISH on all IHC-2+ and -3+ cases yields a sensitivity and specificity of 100%. CONCLUSIONS: Our EC-specific HER2 IHC scoring method is reproducible. A screening strategy based on IHC scoring on all cases with subsequent DISH testing on IHC-2+/-3+ cases has perfect test accuracy for identifying HER2-positive EC.

Implementation of the 2021 molecular ESGO/ESTRO/ESP risk groups in endometrial cancer.

INTRODUCTION: In 2021, a joint ESGO/ESTRO/ESP committee updated their evidence-based guidelines for endometrial cancer, recommending a new risk grouping incorporating both clinicopathologic and molecular parameters. We applied the new risk grouping and compared the results to those of the prior 2016 clinicopathologic system. MATERIALS AND METHODS: We classified molecularly a cohort of 604 women diagnosed with endometrial cancer using immunohistochemistry for TP53 and MMR proteins on a tissue microarray, as well as Sanger sequencing for POLE mutations. These results, combined with clinicopathologic data, allowed the patients to be risk grouped using both the new 2021 molecular/clinicopathologic parameters and the prior 2016 clinicopathologic system. RESULTS: The application of the 2021 molecular markers shows Kaplan-Meier curves with a significant difference between the groups for all survival. Molecular classification under the 2021 guidelines revealed a total of 39 patients (39/594, 7%) with a change in risk group in relation to the 2016 classification system: the shift was alone due to either P53abn or POLEmut molecular marker. In order to ensure correct 2021 molecular risk classification, not all patients with endometrial cancer need a molecular diagnostic: 433 (72.9%) cases would need to be analyzed by TP53 IHC, only 46 (7.7%) by MMR IHC and 286 (48.1%) POLE sequencing reactions. CONCLUSION: Application of the 2021 molecular risk groups is feasible and shows significant differences in survival. IHC for TP53 and MMR and applying POLE sequencing is only needed in selected cases and leads to shifting risk groups both upward and downward for a sizeable number of patients. It is possible to significantly reduce the number of analyses required to implement the classification if resources are limited.

Interpretation of somatic POLE mutations in endometrial carcinoma.

Pathogenic somatic missense mutations within the DNA polymerase epsilon (POLE) exonuclease domain define the important subtype of ultramutated tumours ('POLE-ultramutated') within the novel molecular classification of endometrial carcinoma (EC). However, clinical implementation of this classifier requires systematic evaluation of the pathogenicity of POLE mutations. To address this, we examined base changes, tumour mutational burden (TMB), DNA microsatellite instability (MSI) status, POLE variant frequency, and the results from six in silico tools on 82 ECs with whole-exome sequencing from The Cancer Genome Atlas (TCGA). Of these, 41 had one of five known pathogenic POLE exonuclease domain mutations (EDM) and showed characteristic genomic alterations: C>A substitution > 20%, T>G substitutions > 4%, C>G substitutions < 0.6%, indels < 5%, TMB > 100 mut/Mb. A scoring system to assess these alterations (POLE-score) was developed; based on their scores, 7/18 (39%) additional tumours with EDM were classified as POLE-ultramutated ECs, and the six POLE mutations present in these tumours were considered pathogenic. Only 1/23 (4%) tumours with non-EDM showed these genomic alterations, indicating that a large majority of mutations outside the exonuclease domain are not pathogenic. The infrequent combination of MSI-H with POLE EDM led us to investigate the clinical significance of this association. Tumours with pathogenic POLE EDM co-existent with MSI-H showed genomic alterations characteristic of POLE-ultramutated ECs. In a pooled analysis of 3361 ECs, 13 ECs with DNA mismatch repair deficiency (MMRd)/MSI-H and a pathogenic POLE EDM had a 5-year recurrence-free survival (RFS) of 92.3%, comparable to previously reported POLE-ultramutated ECs. Additionally, 14 cases with non-pathogenic POLE EDM and MMRd/MSI-H had a 5-year RFS of 76.2%, similar to MMRd/MSI-H, POLE wild-type ECs, suggesting that these should be categorised as MMRd, rather than POLE-ultramutated ECs for prognostication. This work provides guidance on classification of ECs with POLE mutations, facilitating implementation of POLE testing in routine clinical care. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Clinicopathological and molecular characterisation of 'multiple-classifier' endometrial carcinomas.

Endometrial carcinoma (EC) molecular classification based on four molecular subclasses identified in The Cancer Genome Atlas (TCGA) has gained relevance in recent years due to its prognostic utility and potential to predict benefit from adjuvant treatment. While most ECs can be classified based on a single classifier (POLE exonuclease domain mutations - POLEmut, MMR deficiency - MMRd, p53 abnormal - p53abn), a small but clinically relevant group of tumours harbour more than one molecular classifying feature and are referred to as 'multiple-classifier' ECs. We aimed to describe the clinicopathological and molecular features of multiple-classifier ECs with abnormal p53 (p53abn). Within a cohort of 3518 molecularly profiled ECs, 107 (3%) tumours displayed p53abn in addition to another classifier(s), including 64 with MMRd (MMRd-p53abn), 31 with POLEmut (POLEmut-p53abn), and 12 with all three aberrations (MMRd-POLEmut-p53abn). MMRd-p53abn ECs and POLEmut-p53abn ECs were mostly grade 3 endometrioid ECs, early stage, and frequently showed morphological features characteristic of MMRd or POLEmut ECs. 18/28 (60%) MMRd-p53abn ECs and 7/15 (46.7%) POLEmut-p53abn ECs showed subclonal p53 overexpression, suggesting that TP53 mutation was a secondary event acquired during tumour progression. Hierarchical clustering of TCGA ECs by single nucleotide variant (SNV) type and somatic copy number alterations (SCNAs) revealed that MMRd-p53abn tumours mostly clustered with single-classifier MMRd tumours (20/23) rather than single-classifier p53abn tumours (3/23), while POLEmut-p53abn tumours mostly clustered with single-classifier POLEmut tumours (12/13) and seldom with single-classifier p53abn tumours (1/13) (both p ≤ 0.001, chi-squared test). Finally, the clinical outcome of patients with MMRd-p53abn and POLEmut-p53abn ECs [stage I 5-year recurrence-free survival (RFS) of 92.2% and 94.1%, respectively] was significantly different from single-classifier p53abn EC (stage I RFS 70.8%, p = 0.024 and p = 0.050, respectively). Our results support the classification of MMRd-p53abn EC as MMRd and POLEmut-p53abn EC as POLEmut. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

High density of stroma-localized CD11c-positive macrophages is associated with longer overall survival in high-grade serous ovarian cancer.

OBJECTIVE: Pre-clinical studies have identified marker- and tumor compartment-defined functionally distinct macrophage subsets. Our study analyzes marker-defined macrophage subsets in different tumor compartments of high-grade serous ovarian cancer (HGSC). METHODS: A discovery cohort (N = 113) was subjected to immunohistochemistry (IHC) analyses. CD68-positivity was confirmed for CD11c-, CD80- and CD163-positive cells. Subset-marker-positive cells were scored in the total tumor and in four tumor compartments. Correlation analyses investigated co-expression of subsets, relationship to CD8+ cells and survival associations. A validation cohort (N = 121) was used to confirm selected findings from the discovery cohort. RESULTS: CD163-positve cells was the most abundant subtype in all compartments. CD11c and CD163 subsets were strongly correlated with each other in stroma and epithelial areas, whereas CD80 and CD163 were correlated in epithelial areas. CD80 and CD11c in perivascular areas showed low correlations. Strong associations were detected between CD8 and CD80 in the tumor epithelium-dominated areas, and between CD8 and CD11c in stroma areas. High stromal CD11c density was associated with a longer median overall survival in the discovery cohort (HR 0.39; CI 95%, 0.23-0.68; p = 0.001) and in the validation cohort (HR 0.46; CI 95%, 0.22-0.93; p = 0.03). CONCLUSIONS: Our study supports the existence of clinically relevant marker- and localization defined macrophage subsets in HGSC, which are independently regulated. Moreover, it suggests stromal CD11c as a novel prognostic marker in HGSC.

Diversity and dynamics of the Drosophila transcriptome.

Animal transcriptomes are dynamic, with each cell type, tissue and organ system expressing an ensemble of transcript isoforms that give rise to substantial diversity. Here we have identified new genes, transcripts and proteins using poly(A)+ RNA sequencing from Drosophila melanogaster in cultured cell lines, dissected organ systems and under environmental perturbations. We found that a small set of mostly neural-specific genes has the potential to encode thousands of transcripts each through extensive alternative promoter usage and RNA splicing. The magnitudes of splicing changes are larger between tissues than between developmental stages, and most sex-specific splicing is gonad-specific. Gonads express hundreds of previously unknown coding and long non-coding RNAs (lncRNAs), some of which are antisense to protein-coding genes and produce short regulatory RNAs. Furthermore, previously identified pervasive intergenic transcription occurs primarily within newly identified introns. The fly transcriptome is substantially more complex than previously recognized, with this complexity arising from combinatorial usage of promoters, splice sites and polyadenylation sites.

Sarcoma Tumor Microenvironment.

Components of the tumor microenvironment (TME) are known to play an essential role during malignant progression, but often in a context-dependent manner. In bone and soft tissue sarcomas, disease-regulatory activities in the TME remain largely uncharacterized. This chapter introduces the cellular, structural, and chemical composition of the sarcoma TME from a pathobiological and therapeutic perspective.Sarcomas are malignant tumors with diverse features when it comes to primary tumor appearance, metastatic potential, and response to treatment. Many of the classic subtypes are mainly composed of malignant cells and are therefore assumed to be committed to autocrine signaling. Some of the tumors are infiltrated by immune cells and contain necrotic areas or excessive amounts of extracellular matrix (ECM) that regulates tissue stiffness and interstitial fluid pressure. Vascular invasion and blood vessel characteristics can in some instances be considered in the prognostic setting.Further insights into the disease-regulatory activities of the sarcoma TME will provide essential knowledge on how to develop successful combination treatments targeting not only malignant cells, but also their routes of nutrition and ability to shield themselves toward existing therapy.

Reclassifying endometrial carcinomas with a combined morphological and molecular approach.

PURPOSE OF REVIEW: Currently, endometrial carcinoma diagnosis is based on morphology, often supplemented by immunohistochemistry. However, especially with high-grade endometrial carcinomas, there is considerable interobserver variability in diagnosis calling into question the value of morphology in stratifying these tumours into different prognostic and therapeutic groups. The purpose of this review is to provide an update on the recently described molecular classification of endometrial carcinoma. RECENT FINDINGS: In 2013, the Cancer Genome Atlas (TCGA) published a seminal molecular study of endometrial carcinomas of endometrioid, serous and mixed types. This revealed that endometrial carcinoma consists of four intrinsic molecular subtypes: POLE (ultramutated), microsatellite instabilty (hypermutated), copy-number low (also referred to as microsatellite stable or no specific molecular profile) and copy-number high (serous-like). These four molecular subtypes are of prognostic significance with POLE tumours having the best and copy-number high, the worst prognosis. SUMMARY: It is likely that TCGA classification will become the mainstay of endometrial carcinoma diagnosis in the coming years and various strategies (Proactive Molecular Risk Classifier for Endometrial Cancer and the TransPORTEC classifiers) have been proposed for a combined morphological-molecular classification which can be undertaken in most pathology laboratories. This will necessitate routine undertaking of POLE mutation analysis in some endometrial carcinomas and require an appropriate infrastructure.