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Objective Combined immunohistochemical and molecular classification using the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) independently predicts prognosis in endometrial carcinoma (EC). As next-generation sequencing (NGS) is entering clinical practice, we evaluated whether more comprehensive immunomolecular profiling (CIMP), including NGS and extended immunohistochemical analysis, could further refine the current ProMisE classification. Methods A series of 120 consecutive ECs, classified according to ProMisE, was stained immunohistochemically for CD3, CD8, PD-L1, beta-catenin and L1CAM. An in-house 96 gene NGS panel was performed on a subset of 44 ECs, representing the 4 ProMisE subgroups (DNA polymerase epsilon catalytic subunit exonuclease domain mutated (POLEmut), mismatch repair deficient (MMRd), p53 abnormal (p53 abn) and no specific molecular profile (NSMP) ECs). Cases harboring non-hotspot POLE variants were analyzed with Illumina TruSight Oncology 500 NGS panel (TSO500) as a surrogate for whole-exome sequencing. Results Eight cases harbored POLE variants, half of which were hotspots. Using TSO500, non-hotspot POLE variants were classified as pathogenic (3) or variant of unknown significance (1). POLEmut and MMRd ECs typically showed higher numbers of CD3+/CD8+ tumor-infiltrating lymphocytes and higher PD-L1 expression in tumor-infiltrating immune cells. p53 abn ECs showed significantly higher L1CAM immunoreactivity and frequently harbored gene amplifications including HER2 (25%), but typically lacked ARID1A or PTEN variants. Beta-catenin-positivity and FGFR2 variants were predominantly found in NSMP ECs. Conclusions Our data show that CIMP adds significant value to EC characterization and may help to determine pathogenicity of non-hotspot POLE variants, encountered more frequently than expected in our series. In addition, CIMP may reveal ECs benefitting from immune checkpoint inhibition and allows upfront identification of targetable alterations, such as HER2 amplification in p53 abn ECs.

Post-transplantation lymphoproliferative disorder is an aggressive complication of transplantation, most frequently of diffuse large B-cell lymphoma morphology and associated with Epstein-Barr virus (EBV) infection/reactivation. In this study the microenvironment of EBV+ (n=23) and EBV- (n=9) post-transplant non-germinal center B-cell diffuse large B-cell lymphoma was characterized. Of EBV+ cases somatic hypermutation analysis, gene expression profiling, and extensive phenotyping were performed. Our results demonstrated variable cytotoxic T-cell infiltration and significantly increased CD163+ M2 macrophage infiltration in EBV+ compared with EBV- post-transplant diffuse large B-cell lymphoma. On the basis of IgM staining and hypermutation analysis, two EBV+ post-transplant diffuse large B-cell lymphoma subgroups were identified: IgM+ tumors lacking somatic hypermutations and IgM- tumors harboring somatic hypermutations. IgM- tumors arose late following transplantation (median interval: 16 months), mainly in kidney recipients. IgM+ tumors on the other hand arose early (median interval: 3 months, P-value=0.0032), almost exclusively following stem cell transplantation and were associated with worse outcome (median survival 1 month for IgM+ versus 41 months for IgM- tumors, log-rank/Wilcoxon P-value 0.07/0.04). Notably, IgM+ tumors were characterized by plasma cell features (monotypic kappa/lambda expression, high MUM1 expression, and partial CD138 expression) and a high proliferation index. Consistent with the plasma cell phenotype, unfolded protein response signaling was upregulated. In contrast, IgM- EBV+ post-transplant diffuse large B-cell lymphoma did not express kappa, lambda, IgD, or CD138 and expressed limited MUM1. In these tumors T-cell signaling was enhanced associated with increased T-cell infiltration compared with IgM+ cases. Overall, our results allow further molecular classification of EBV+ post-transplant diffuse large B-cell lymphoma and provide a rationale for the use of subtype-specific-targeted therapies (eg, bortezomib in IgM+ tumors). Our findings also provide a biological basis for the clinical differences between post-transplant lymphoproliferative disorder following solid organ and stem cell transplantation, which are regarded as different disorders.

Background & Aims: NEMO is the regulatory subunit of the IκB kinase (IKK) complex and is involved in controlling nuclear factor κB (NF-κB) activation. NEMO knockout mice die during embryogenesis due to massive hepatocyte apoptosis. Here we investigated the role of NEMO-dependent signaling in hepatocytes during acute liver injury. Methods: We generated conditional hepatocyte-specific NEMO knockout mice using the loxP system with the Cre recombinase under the control of the albumin promoter (NEMOΔLPC). In these mice, we studied mechanisms of tumor necrosis factor (TNF)- and ischemia/reperfusion-dependent liver cell damage. Results: In adult NEMOΔLPC animals, NEMO is specifically deleted in hepatocytes and no differences in survival, growth, and fertility were found when compared with wild-type (NEMOf/f) mice. TNF stimulation of NEMOΔLPC mice resulted in high serum transaminase levels and massive hepatocyte apoptosis, which were associated with lack of IκBα degradation, inhibition of NF-κB activation, and target gene transcription. Additionally, ischemia/reperfusion resulted in higher nonparenchymal cell–dependent induction of oxidative stress and stronger inflammation in NEMOΔLPC mice. This led to massive hepatocyte apoptosis and death of the animals, while NEMOf/f mice survived with significantly lesser liver damage, showing mainly necrotic cell death. Thus, complete inhibition of NF-κB activation in hepatocytes, in contrast to attenuation in hepatocyte-specific IKK2−/− mice, determines the type of liver cell damage during ischemia/reperfusion injury and is associated with a poor prognosis. Conclusions: Our results show that understanding of the fine tuning of NF-κB modulation during liver injury is essential to develop new therapeutic strategies.

Hepatitis virus replication in the liver is often accompanied by inflammation resulting in the formation of reactive oxygen species (ROS) and nitric oxide (NO) and these may induce cell death. We investigated whether the expression of HBx or HCV core protein in HepG2 cells has an influence on the sensitivity of these cells for oxidative radicals. Our previous study, using the inducible HBV model of HepAD38, revealed that oxidative-stress-related genes are upregulated by virus replication. In the present study, we examined the intracellular pro-oxidant status with dichlorofluorescein (DCF) in HepG2 cell lines transfected with HBx, HbsAg and HCV core. Baseline intracellular oxidative levels were not different in the cell lines expressing viral proteins as compared to control. However, when these cells were exposed to H(2)O(2), the viral protein expressing cells, especially those expressing HBx, showed a reduced level of ROS. This suggests that HBx and HCV core transfected cells can convert H(2)O(2) to less reactive compounds at a higher rate than the control cells. When HBx or HCV core expressing cells were exposed to peroxynitrite (a highly reactive product formed under physiological conditions through interaction of superoxide (O(2)(-)) with NO) these cells were less sensitive to induction of cell death. In addition, these cell lines were less prone to cell death when exposed to H(2)O(2) directly. In conclusion, HBx and HCV core expression in HepG2 cells leads to a survival benefit under oxidative stress which in vivo can be induced during inflammation.