Clinical Behavior and Molecular Landscape of Stage I p53-Abnormal Low-Grade Endometrioid Endometrial Carcinomas.

PURPOSE: The clinical significance of the p53-abnormal (p53abn) molecular subtype in stage I low-grade endometrioid endometrial carcinoma (EEC) is debated. We aimed to review pathologic and molecular characteristics, and outcomes of stage I low-grade p53abn EEC in a large international cohort. EXPERIMENTAL DESIGN: Previously diagnosed stage I p53abn EC (POLE-wild-type, mismatch repair-proficient) low-grade EEC from Canadian retrospective cohorts and PORTEC-1&2 trials were included. Pathology review was performed by six expert gynecologic pathologists blinded to p53 status. IHC profiling, next-generation sequencing, and shallow whole-genome sequencing was performed. Kaplan-Meier method was used for survival analysis. RESULTS: We identified 55 stage I p53abn low-grade EEC among 3,387 cases (2.5%). On pathology review, 17 cases (31%) were not diagnosed as low-grade EEC by any pathologists, whereas 26 cases (47%) were diagnosed as low-grade EEC by at least three pathologists. The IHC and molecular profile of the latter cases were consistent with low-grade EEC morphology (ER/PR positivity, patchy p16 expression, PIK3CA and PTEN mutations) but they also showed features of p53abn EC (TP53 mutations, many copy-number alterations). These cases had a clinically relevant risk of disease recurrence (5-year recurrence-free survival 77%), with pelvic and/or distant recurrences observed in 12% of the patients. CONCLUSIONS: A subset of p53abn EC is morphologically low-grade EEC and exhibit genomic instability. Even for stage I disease, p53abn low-grade EEC are at substantial risk of disease recurrence. These findings highlight the clinical relevance of universal p53-testing, even in low-grade EEC, to identify women at increased risk of recurrence.

Development and Implementation of an Asynchronous Online Interprofessional Course in LGBTQ+ Health.

BACKGROUND: The lesbian, gay, bisexual, transgender, and queer or questioning (LGBTQ+) community faces discrimination in health care. Nurses interact with LGBTQ+ individuals in a wide variety of health care settings. Baccalaureate nursing and health professions students do not receive adequate education on topics of LGBTQ+ health in academic or clinical settings, and additional nursing education is needed on LGBTQ+ health. METHOD: Existing courses were analyzed, and themes and structures were assessed. Course learning outcomes were developed, along with a timeline and a course outline. The course was structured as an asynchronous online course to be deployed in a learning management system. RESULTS: A senior-level asynchronous online nursing elective course was developed on LGBTQ+ health for an urban public university. The course was structured using a life-span approach with additional topics. CONCLUSION: Widespread development of courses on LGBTQ+ health should be implemented in all schools of nursing. [J Nurs Educ. 2023;62(10):584-588.].

Revising the Role of Integrin Subunit ß4 Expression in Colon Cancer Progression and Survival.

PURPOSE: Integrin subunit ß4 (ß4) has been proposed to play an important role in colon cancer progression through its involvement in hemidesmosome disassembly processes and tumor cell migration. However, the association between ß4 expression and clinicopathological outcomes in colon cancer remains unclear. METHODS: Expression of ß4 was assessed by immunohistochemistry in a large cohort of 651 colon cancer patients, the largest colon cancer cohort so far. Chi-squared tests were used to study the association between ß4 expression and clinicopathological features. Overall and disease-free survival were assessed by Cox proportional hazard models. RESULTS: Loss of ß4 expression was associated with local tumor invasion. Only 17.9% of the pT1 tumors displayed weak ß4 expression level versus 28.1% of pT4 tumors, and 25.0% of the pT1 tumors had a high expression level versus 8.6% of the pT4 tumors (p = 0.012). No association between ß4 expression and overall (p = 0.845) or disease-free survival (p = 0.767) was encountered, which disputes the role of ß4 as a biomarker of malignant behavior in colon cancer. CONCLUSION: Contradictory reports have suggested opposite roles for ß4 expression in (colon) cancer progression. In the present large cohort of colon cancer patients, we found that ß4 expression was not associated with worse clinical prognosis, but decreased with advanced pathological tumor stage. Future studies should establish whether loss of ß4 expression promotes invasive characteristics of colon cancer cells.

A Paradoxical Role for Regulatory T Cells in the Tumor Microenvironment of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is considered to be a poorly immunogenic cancer type that combines a low mutation burden with a strong immunosuppressive tumor microenvironment. Regulatory T cells (Tregs) are major drivers of immune suppression but their prognostic role, particularly in gastrointestinal malignancies, remains controversial. Lymphocytic infiltration in 122 PDAC samples was assessed by multispectral immunofluorescence with anti-Keratin, -CD3, -CD8, -FOXP3 and -CD163 antibodies. Differential infiltration by Tregs was analyzed in the context of transcriptomic profiles that were available for 65 tumors. High infiltration of CD3+CD8- (mainly CD4+) T cells and, especially, of the subset expressing FOXP3 (Tregs) was associated with improved patient survival, whilst cytotoxic CD3+CD8+ T cell infiltration did not have an impact on overall survival. Transcriptomic analysis revealed three signatures in PDAC tumors comprising of epithelial-mesenchymal transition (EMT)/stromal, metabolic, and secretory/pancreatic signature. However, none of these signatures explained differences in Treg infiltration. We show that Tregs associate with improved overall survival in PDAC patients. This effect was independent of cytotoxic T cell infiltration and the transcriptomic profiles of their respective tumors. These findings provide a new layer of complexity in the study of PDAC tumor microenvironment that must be considered when developing immunotherapeutic interventions for this disease.

Increasing frequency of gene copy number aberrations is associated with immunosuppression and predicts poor prognosis in gastric adenocarcinoma.

BACKGROUND: Patients with Epstein-Barr virus-positive gastric cancers or those with microsatellite instability appear to have a favourable prognosis. However, the prognostic value of the chromosomal status (chromosome-stable (CS) versus chromosomal instable (CIN)) remains unclear in gastric cancer. METHODS: Gene copy number aberrations (CNAs) were determined in 16 CIN-associated genes in a retrospective study including test and validation cohorts of patients with gastric cancer. Patients were stratified into CS (no CNA), CINlow (1-2 CNAs) or CINhigh (3 or more CNAs). The relationship between chromosomal status, clinicopathological variables, and overall survival (OS) was analysed. The relationship between chromosomal status, p53 expression, and tumour infiltrating immune cells was also assessed and validated externally. RESULTS: The test and validation cohorts included 206 and 748 patients, respectively. CINlow and CINhigh were seen in 35.0 and 15.0 per cent of patients, respectively, in the test cohort, and 48.5 and 20.7 per cent in the validation cohort. Patients with CINhigh gastric cancer had the poorest OS in the test and validation cohorts. In multivariable analysis, CINlow, CINhigh and pTNM stage III-IV (P < 0.001) were independently associated with poor OS. CIN was associated with high p53 expression and low immune cell infiltration. CONCLUSION: CIN may be a potential new prognostic biomarker independent of pTNM stage in gastric cancer. Patients with gastric cancer demonstrating CIN appear to be immunosuppressed, which might represent one of the underlying mechanisms explaining the poor survival and may help guide future therapeutic decisions.

Targeting the NAD Salvage Synthesis Pathway as a Novel Therapeutic Strategy for Osteosarcomas with Low NAPRT Expression.

For osteosarcoma (OS), the most common primary malignant bone tumor, overall survival has hardly improved over the last four decades. Especially for metastatic OS, novel therapeutic targets are urgently needed. A hallmark of cancer is aberrant metabolism, which justifies targeting metabolic pathways as a promising therapeutic strategy. One of these metabolic pathways, the NAD+ synthesis pathway, can be considered as a potential target for OS treatment. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the classical salvage pathway for NAD+ synthesis, and NAMPT is overexpressed in OS. In this study, five OS cell lines were treated with the NAMPT inhibitor FK866, which was shown to decrease nuclei count in a 2D in vitro model without inducing caspase-driven apoptosis. The reduction in cell viability by FK866 was confirmed in a 3D model of OS cell lines (n = 3). Interestingly, only OS cells with low nicotinic acid phosphoribosyltransferase domain containing 1 (NAPRT1) RNA expression were sensitive to NAMPT inhibition. Using a publicly available (Therapeutically Applicable Research to Generate Effective Treatments (TARGET)) and a previously published dataset, it was shown that in OS cell lines and primary tumors, low NAPRT1 RNA expression correlated with NAPRT1 methylation around the transcription start site. These results suggest that targeting NAMPT in osteosarcoma could be considered as a novel therapeutic strategy, where low NAPRT expression can serve as a biomarker for the selection of eligible patients.

Targeting EML4-ALK gene fusion variant 3 in thyroid cancer.

Finding targetable gene fusions can expand the limited treatment options in radioactive iodine-refractory (RAI-r) thyroid cancer. To that end, we established a novel cell line 'JVE404' derived from an advanced RAI-r papillary thyroid cancer (PTC) patient, harboring an EML4-ALK gene fusion variant 3 (v3). Different EML4-ALK gene fusions can have different clinical repercussions. JVE404 cells were evaluated for cell viability and cell signaling in response to ALK inhibitors crizotinib, ceritinib and lorlatinib, in parallel to the patient's treatment. He received, after first-line lenvatinib, crizotinib (Drug Rediscovery Protocol (DRUP) trial), and lorlatinib (compassionate use). In vitro treatment with crizotinib or ceritinib decreased viability in JVE404, but most potently and significantly only with lorlatinib. Western blot analysis showed a near total decrease of 99% and 89%, respectively, in pALK and pERK expression levels in JVE404 cells with lorlatinib, in contrast to remaining signal intensities of a half and a third of control, respectively, with crizotinib. The patient had a 6-month lasting stable disease on crizotinib, but progressive disease occurred, including the finding of cerebral metastases, at 8 months. With lorlatinib, partial response, including clinical cerebral activity, was already achieved at 11 weeks' use and ongoing partial response at 7 months. To our best knowledge, this is the first reported case describing a patient-specific targeted treatment with lorlatinib based on an EML4-ALK gene fusion v3 in a thyroid cancer patient, and own cancer cell line. Tumor-agnostic targeted therapy may provide valuable treatment options in personalized medicine.

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.

Endometrial Cancer Risk in Women With Germline BRCA1 or BRCA2 Mutations: Multicenter Cohort Study.

BACKGROUND: Endometrial cancer (EC) risk in BReast CAncer gene 1/2 (BRCA1/2) mutation carriers is uncertain; therefore, we assessed this in a large Dutch nationwide cohort study. METHODS: We selected 5980 BRCA1/2 (3788 BRCA1, 2151 gBRCA2, 41 both BRCA1/BRCA2) and 8451 non-BRCA1/2 mutation carriers from the Hereditary Breast and Ovarian cancer study, the Netherlands cohort. Follow-up started at the date of the nationwide Dutch Pathology Registry coverage (January 1, 1989) or at the age of 25 years (whichever came last) and ended at date of EC diagnosis, last follow-up, or death (whichever came first). EC risk in BRCA1/2 mutation carriers was compared with 1) the general population, estimating standardized incidence ratios (SIRs) based on Dutch population-based incidence rates; and 2) non-BRCA1/2 mutation carriers, using Cox-regression analyses, expressed as hazard ratio (HR). Statistical tests were 2-sided. RESULTS: Fifty-eight BRCA1/2 and 33 non-BRCA1/2 mutation carriers developed EC over 119 296 and 160 841 person-years, respectively (SIR = 2.83, 95% confidence interval [CI] = 2.18 to 3.65; and HR = 2.37, 95% CI = 1.53 to 3.69, respectively). gBRCA1 mutation carriers showed increased risks for EC overall (SIR = 3.51, 95% CI = 2.61 to 4.72; HR = 2.91, 95% CI = 1.83 to 4.66), serous-like EC (SIR = 12.64, 95% CI = 7.62 to 20.96; HR = 10.48, 95% CI = 2.95 to 37.20), endometrioid EC (SIR = 2.63, 95% CI = 1.80 to 3.83; HR = 2.01, 95% CI = 1.18 to 3.45), and TP53-mutated EC (HR = 15.71, 95% CI = 4.62 to 53.40). For BRCA2 mutation carriers, overall (SIR = 1.70, 95% CI = 1.01 to 2.87) and serous-like EC risks (SIR = 5.11, 95% CI = 1.92 to 13.63) were increased compared with the general population. Absolute risks by 75 years remained low (overall EC = 3.0%; serous-like EC = 1.1%). CONCLUSIONS: BRCA1/2 mutation carriers have a two- to threefold increased risk for EC, with highest risk observed for the rare subgroups of serous-like and p53-abnormal EC in BRCA1 mutation carriers.

Non-IDH1-R132H IDH1/2 mutations are associated with increased DNA methylation and improved survival in astrocytomas, compared to IDH1-R132H mutations.

Somatic mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 occur at high frequency in several tumour types. Even though these mutations are confined to distinct hotspots, we show that gliomas are the only tumour type with an exceptionally high percentage of IDH1R132H mutations. Patients harbouring IDH1R132H mutated tumours have lower levels of genome-wide DNA-methylation, and an associated increased gene expression, compared to tumours with other IDH1/2 mutations ("non-R132H IDH1/2 mutations"). This reduced methylation is seen in multiple tumour types and thus appears independent of the site of origin. For 1p/19q non-codeleted glioma (astrocytoma) patients, we show that this difference is clinically relevant: in samples of the randomised phase III CATNON trial, patients harbouring tumours with IDH mutations other than IDH1R132H have a better outcome (hazard ratio 0.41, 95% CI [0.24, 0.71], p = 0.0013). Such non-R132H IDH1/2-mutated tumours also had a significantly lower proportion of tumours assigned to prognostically poor DNA-methylation classes (p < 0.001). IDH mutation-type was independent in a multivariable model containing known clinical and molecular prognostic factors. To confirm these observations, we validated the prognostic effect of IDH mutation type on a large independent dataset. The observation that non-R132H IDH1/2-mutated astrocytomas have a more favourable prognosis than their IDH1R132H mutated counterpart indicates that not all IDH-mutations are identical. This difference is clinically relevant and should be taken into account for patient prognostication.

Spatial profiling of gastric cancer patient-matched primary and locoregional metastases reveals principles of tumour dissemination.

OBJECTIVE: Endoscopic mucosal biopsies of primary gastric cancers (GCs) are used to guide diagnosis, biomarker testing and treatment. Spatial intratumoural heterogeneity (ITH) may influence biopsy-derived information. We aimed to study ITH of primary GCs and matched lymph node metastasis (LNmet). DESIGN: GC resection samples were annotated to identify primary tumour superficial (PTsup), primary tumour deep (PTdeep) and LNmet subregions. For each subregion, we determined (1) transcriptomic profiles (NanoString 'PanCancer Progression Panel', 770 genes); (2) next-generation sequencing (NGS, 225 gastrointestinal cancer-related genes); (3) DNA copy number profiles by multiplex ligation-dependent probe amplification (MLPA, 16 genes); and (4) histomorphological phenotypes. RESULTS: NanoString profiling of 64 GCs revealed no differences between PTsup1 and PTsup2, while 43% of genes were differentially expressed between PTsup versus PTdeep and 38% in PTsup versus LNmet. Only 16% of genes were differently expressed between PTdeep and LNmet. Several genes with therapeutic potential (eg IGF1, PIK3CD and TGFB1) were overexpressed in LNmet and PTdeep compared with PTsup. NGS data revealed orthogonal support of NanoString results with 40% mutations present in PTdeep and/or LNmet, but not in PTsup. Conversely, only 6% of mutations were present in PTsup and were absent in PTdeep and LNmet. MLPA demonstrated significant ITH between subregions and progressive genomic changes from PTsup to PTdeep/LNmet. CONCLUSION: In GC, regional lymph node metastases are likely to originate from deeper subregions of the primary tumour. Future clinical trials of novel targeted therapies must consider assessment of deeper subregions of the primary tumour and/or metastases as several therapeutically relevant genes are only mutated, overexpressed or amplified in these regions.

Beyond the Influence of IDH Mutations: Exploring Epigenetic Vulnerabilities in Chondrosarcoma.

Mutations in the isocitrate dehydrogenase (IDH1 or IDH2) genes are common in enchondromas and chondrosarcomas, and lead to elevated levels of the oncometabolite D-2-hydroxyglutarate causing widespread changes in the epigenetic landscape of these tumors. With the use of a DNA methylation array, we explored whether the methylome is altered upon progression from IDH mutant enchondroma towards high-grade chondrosarcoma. High-grade tumors show an overall increase in the number of highly methylated genes, indicating that remodeling of the methylome is associated with tumor progression. Therefore, an epigenetics compound screen was performed in five chondrosarcoma cell lines to therapeutically explore these underlying epigenetic vulnerabilities. Chondrosarcomas demonstrated high sensitivity to histone deacetylase (HDAC) inhibition in both 2D and 3D in vitro models, independent of the IDH mutation status or the chondrosarcoma subtype. siRNA knockdown and RNA expression data showed that chondrosarcomas rely on the expression of multiple HDACs, especially class I subtypes. Furthermore, class I HDAC inhibition sensitized chondrosarcoma to glutaminolysis and Bcl-2 family member inhibitors, suggesting that HDACs define the metabolic state and apoptotic threshold in chondrosarcoma. Taken together, HDAC inhibition may represent a promising targeted therapeutic strategy for chondrosarcoma patients, either as monotherapy or as part of combination treatment regimens.

Lack of myeloid cell infiltration as an acquired resistance strategy to immunotherapy.

BACKGROUND: Immunotherapy of cancer is successful but tumor regression often is incomplete and followed by escape. Understanding the mechanisms underlying this acquired resistance will aid the development of more effective treatments. METHODS: We exploited a mouse model where tumor-specific therapeutic vaccination results in tumor regression, followed by local recurrence and resistance. In depth studies on systemic, local and tumor intrinsic changes were performed with flow and mass cytometry, immunohistochemistry, transcriptomics and several perturbation studies with inhibitors or agonistic antibodies in mice. Main findings were recapitulated in vaccinated patients. RESULTS: Full tumor regression and cure of tumor-bearing mice is dependent on the magnitude of the vaccine-induced T-cell response. Recurrence of tumors did not involve classical immune escape mechanisms, such as antigen-presentation alterations, immune checkpoint expression, resistance to killing or local immune suppression. However, the recurrent tumors displayed a changed transcriptome with alterations in p53, tumor necrosis factor-α and transforming growth factor-ß signaling pathways and they became immunologically cold. Remarkably, ex vivo cell-sorted recurrent tumors, directly reinjected in naïve hosts retained their resistance to vaccination despite a strong infiltration with tumor-specific CD8+ T cells, similar to that of vaccine-responsive tumors. The influx of inflammatory mature myeloid effector cells in the resistant tumors, however, was impaired and this turned out to be the underlying mechanisms as restoration of inflammatory myeloid cell infiltration reinstated the sensitivity of these refractory tumors to vaccination. Notably, impaired myeloid cell infiltration after vaccination was also associated with vaccine resistance in patients. CONCLUSION: An immunotherapy-induced disability of tumor cells to attract innate myeloid effector cells formed a major mechanism underlying immune escape and acquired resistance. These data not only stresses the importance of myeloid effector cells during immunotherapy but also demands for new studies to harness their tumoricidal activities.

Developments in the Histopathological Classification of ANCA-Associated Glomerulonephritis.

BACKGROUND AND OBJECTIVES: The histopathologic classification for ANCA-associated GN distinguishes four classes on the basis of patterns of injury. In the original validation study, these classes were ordered by severity of kidney function loss as follows: focal, crescentic, mixed, and sclerotic. Subsequent validation studies disagreed on outcomes in the crescentic and mixed classes. This study, driven by the original investigators, provides several analyses in order to determine the current position of the histopathologic classification of ANCA-associated GN. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A validation study was performed with newly collected data from 145 patients from ten centers worldwide, including an analysis of interobserver agreement on the histopathologic evaluation of the kidney biopsies. This study also included a meta-analysis on previous validation studies and a validation of the recently proposed ANCA kidney risk score. RESULTS: The validation study showed that kidney failure at 10-year follow-up was significantly different between the histopathologic classes (P<0.001). Kidney failure at 10-year follow-up was 14% in the crescentic class versus 20% in the mixed class (P=0.98). In the meta-analysis, no significant difference in kidney failure was also observed when crescentic class was compared with mixed class (relative risk, 1.15; 95% confidence interval, 0.94 to 1.41). When we applied the ANCA kidney risk score to our cohort, kidney survival at 3 years was 100%, 96%, and 77% in the low-, medium-, and high-risk groups, respectively (P<0.001). These survival percentages are higher compared with the percentages in the original study. CONCLUSIONS: The crescentic and mixed classes seem to have a similar prognosis, also after adjusting for differences in patient populations, treatment, and interobserver agreement. However, at this stage, we are not inclined to merge the crescentic and mixed classes because the reported confidence intervals do not exclude important differences in prognosis and because an important histopathologic distinction would be lost.

Metabolic reprogramming related to whole-chromosome instability in models for Hürthle cell carcinoma.

Hürthle cell carcinoma (HCC) is a recurrent subtype of non-medullary thyroid cancer. HCC is characterized by profound whole-chromosome instability (w-CIN), resulting in a near-homozygous genome (NHG), a phenomenon recently attributed to reactive oxygen species (ROS) generated during mitosis by malfunctioning mitochondria. We studied shared metabolic traits during standard and glucose-depleted cell culture in thyroid cancer cell lines (TCCLs), with or without a NHG, using quantitative analysis of extra and intracellular metabolites and ROS production following inhibition of complex III with antimycin A. We found that the XTC.UC1 and FTC-236 cell lines (both NHG) are functionally impaired in complex I and produce significantly more superoxide radicals than SW579 and BHP 2-7 (non-NHG) after challenge with antimycin A. FTC-236 showed the lowest levels of glutathione and SOD2. XTC.UC1 and FTC-236 both exhibited reduced glycolytic activity and utilization of alternative sources to meet energy demands. Both cell lines also shared low levels of α-ketoglutarate and high levels of creatine, phosphocreatine, uridine diphosphate-N-acetylglucosamine, pyruvate and acetylcarnitine. Furthermore, the metabolism of XTC.UC1 was skewed towards the de novo synthesis of aspartate, an effect that persisted even in glucose-free media, pointing to reductive carboxylation. Our data suggests that metabolic reprogramming and a subtle balance between ROS generation and scavenging/conversion of intermediates may be involved in ROS-induced w-CIN in HCC and possibly also in rare cases of follicular thyroid cancer showing a NHG.

Genome-wide analysis of constitutional DNA methylation in familial melanoma.

BACKGROUND: Heritable epigenetic alterations have been proposed as an explanation for familial clustering of melanoma. Here we performed genome-wide DNA methylation analysis on affected family members not carrying pathogenic variants in established melanoma susceptibility genes, compared with healthy volunteers. RESULTS: All melanoma susceptibility genes showed the absence of epimutations in familial melanoma patients, and no loss of imprinting was detected. Unbiased genome-wide DNA methylation analysis revealed significantly different levels of methylation in single CpG sites. The methylation level differences were small and did not affect reported tumour predisposition genes. CONCLUSION: Our results provide no support for heritable epimutations as a cause of familial melanoma.

Genome-wide characterization of 5-hydoxymethylcytosine in melanoma reveals major differences with nevus.

Melanoma demonstrates altered patterns of DNA methylation that are associated with genetic instability and transcriptional repression of numerous genes. Active DNA demethylation is mediated by TET enzymes that catalyze conversion of 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC). Loss of hmC occurs in melanoma and correlates with disease progression. Here we analyzed the genomic distribution of hmC along with mC in nevus and melanoma using oxidative bisulfite chemistry combined with high-density arrays. HmC was enriched relative to mC at enhancers, 5'UTR regions and CpG shores in nevus and melanoma samples, pointing to specific TET enzyme activity. The proportion of interrogated CpG sites with high hmC levels was lower in melanoma (0.54%) than in nevus (2.0%). Depletion of hmC in melanoma was evident across all chromosomes and intragenic regions, being more pronounced in metastatic than in non-metastatic tumors. The patterns of hmC distribution in melanoma samples differed significantly from those in nevus samples, exceeding differences in mC patterns. We identified specific CpG sites and regions with significantly lower hmC levels in melanoma than in nevus that might serve as diagnostic markers. Differentially hydroxymethylated regions localized to cancer-related genes, including the PTEN gene promoter, suggesting that deregulated DNA hydroxymethylation may contribute to melanoma pathogenesis.

Reproducibility of lymphovascular space invasion (LVSI) assessment in endometrial cancer.

AIMS: Lymphovascular space invasion (LVSI) in endometrial cancer (EC) is an important prognostic variable impacting on a patient's individual recurrence risk and adjuvant treatment recommendations. Recent work has shown that grading the extent of LVSI further improves its prognostic strength in patients with stage I endometrioid EC. Despite this, there is little information on the reproducibility of LVSI assessment in EC. Therefore, we designed a study to evaluate interobserver agreement in discriminating true LVSI from LVSI mimics (Phase I) and reproducibility of grading extent of LVSI (Phase II). METHODS AND RESULTS: Scanned haematoxylin and eosin (H&E) slides of endometrioid EC (EEC) with a predefined possible LVSI focus were hosted on a website and assessed by a panel of six European gynaecological pathologists. In Phase I, 48 H&E slides were included for LVSI assessment and in Phase II, 42 H&E slides for LVSI grading. Each observer was instructed to apply the criteria for LVSI used in daily practice. The degree of agreement was measured using the two-way absolute agreement average-measures intraclass correlation coefficient (ICC). Reproducibility of LVSI assessment (ICC = 0.64, P < 0.001) and LVSI grading (ICC = 0.62, P < 0.001) in EEC was substantial among the observers. CONCLUSIONS: Given the good reproducibility of LVSI, this study further supports the important role of LVSI in decision algorithms for adjuvant treatment.

Correction to: KRAS status is related to histological phenotype in gastric cancer: results from a large multicentre study.

In the original publication of this article, Fig. 2 was published incorrectly. The correct Fig. 2 is given in this correction.