Safeguard function of PU.1 shapes the inflammatory epigenome of neutrophils.

Neutrophils are essential first-line defense cells against invading pathogens, yet when inappropriately activated, their strong immune response can cause collateral tissue damage and contributes to immunological diseases. However, whether neutrophils can intrinsically titrate their immune response remains unknown. Here we conditionally deleted the Spi1 gene, which encodes the myeloid transcription factor PU.1, from neutrophils of mice undergoing fungal infection and then performed comprehensive epigenomic profiling. We found that as well as providing the transcriptional prerequisite for eradicating pathogens, the predominant function of PU.1 was to restrain the neutrophil defense by broadly inhibiting the accessibility of enhancers via the recruitment of histone deacetylase 1. Such epigenetic modifications impeded the immunostimulatory AP-1 transcription factor JUNB from entering chromatin and activating its targets. Thus, neutrophils rely on a PU.1-installed inhibitor program to safeguard their epigenome from undergoing uncontrolled activation, protecting the host against an exorbitant innate immune response.

[Detection of BRAF V600E mutation in metastatic colorectal carcinoma : A QuIP round robin test]. / Nachweis der BRAF-V600E-Mutation beim metastasierten kolorektalen Karzinom : Ein QuIP-Ringversuch.

Round robin testing is an important instrument for quality assurance. Increasingly, this also applies to the results of molecular diagnostics in pathology, which directly influence therapy decisions in precision oncology. In metastatic colorectal carcinoma (mCRC), the focus has been on detecting KRAS and NRAS mutations, whose absence allows therapy with EGFR blocking antibodies. Recently, BRAF has been added as another predictive marker, since mCRC patients with BRAF V600E mutation benefit significantly from treatment with encorafenib (a BRAF inhibitor) in combination with cetuximab (anti-EGFR antibody) after systemic therapy. Due to the approval of this treatment in 2020, it is a pre-requisite that BRAF V600E mutation detection in diagnostic pathologies is reliably performed. Therefore, this round robin test with BRAF V600E testing either by immunohistochemistry or molecular methods was performed. The round robin test results demonstrate that molecular BRAF V600E detection is currently clearly superior to immunohistochemical detection.

CD163 expression defines specific, IRF8-dependent, immune-modulatory macrophages in the bone marrow.

BACKGROUND: Scavenger receptor CD163 is exclusively expressed on monocytes/macrophages and is widely used as a marker for alternatively activated macrophages. However, the role of CD163 is not yet clear. OBJECTIVES: We sought to examine the function of CD163 in steady-state as well as in sterile and infectious inflammation. METHODS: Expression of CD163 was analyzed under normal and inflammatory conditions in mice. Functional relevance of CD163 was investigated in models of inflammation in wild-type and CD163-/- mice. RESULTS: We describe a subpopulation of bone marrow-resident macrophages (BMRMs) characterized by a high expression of CD163 and functionally distinct from classical bone marrow-derived macrophages. Development of CD163+ BMRMs is strictly dependent on IFN regulatory factor-8. CD163+ BMRMs show a specific transcriptome and cytokine secretion pattern demonstrating a specific immunomodulatory profile of these cells. Accordingly, CD163-/- mice show a stronger inflammation in allergic contact dermatitis, indicating a regulatory role of CD163. However, CD163-/- mice are highly susceptible to S aureus infections, demonstrating the relevance of CD163 for antimicrobial defense as well. CONCLUSIONS: Our data indicate that anti-inflammatory and immunosuppressive mechanisms are not necessarily associated with a decreased antimicrobial activity. In contrast, our data define a novel macrophage population that controls overwhelming inflammation on one hand but is also necessary for an effective control of infections on the other hand.

[Quality assurance in dMMR and MSI diagnostics]. / Qualitätssicherung in der dMMR- und MSI-Diagnostik.

Deficient mismatch repair (dMMR) and microsatellite instability (MSI) have therapeutic relevance not only for colorectal carcinomas but also for carcinomas of other entities (endometrium, biliary tract, pancreas). In order to guarantee the knowledge and good technical quality necessary for adequate implementation of the corresponding analyses in pathology institutes, the Pathology Quality Assurance Initiative ("Die Qualitätssicherung-Initiative Pathologie") has been offering proficiency tests (PT) for years. It has been shown for the dMMR PT that various antibody clones from different manufacturers provide comparable results in immunohistological examinations, except for slight variations. The difficulty lies in the staining protocol (intensity of staining) and the interpretation of the staining results. The molecular pathological MSI PT has shown a positive trend at a high-quality level over the last three years. Success rates increased from 89 (2018) to 97% (2019/2020). The choice of assay, whether commercial or in-house tests with the designated cutoffs for this purpose, has not been shown to have a significant impact on the PTs in the selected EQA samples.

Myeloid leukemia with transdifferentiation plasticity developing from T-cell progenitors.

Unfavorable patient survival coincides with lineage plasticity observed in human acute leukemias. These cases are assumed to arise from hematopoietic stem cells, which have stable multipotent differentiation potential. However, here we report that plasticity in leukemia can result from instable lineage identity states inherited from differentiating progenitor cells. Using mice with enhanced c-Myc expression, we show, at the single-cell level, that T-lymphoid progenitors retain broad malignant lineage potential with a high capacity to differentiate into myeloid leukemia. These T-cell-derived myeloid blasts retain expression of a defined set of T-cell transcription factors, creating a lymphoid epigenetic memory that confers growth and propagates myeloid/T-lymphoid plasticity. Based on these characteristics, we identified a correlating human leukemia cohort and revealed targeting of Jak2/Stat3 signaling as a therapeutic possibility. Collectively, our study suggests the thymus as a source for myeloid leukemia and proposes leukemic plasticity as a driving mechanism. Moreover, our results reveal a pathway-directed therapy option against thymus-derived myeloid leukemogenesis and propose a model in which dynamic progenitor differentiation states shape unique neoplastic identities and therapy responses.

Quality assurance in neuropathology: Experiences from the round robin trials on IDH mutation and MGMT promoter methylation testing launched by the Quality Assurance Initiative Pathology (QuIP) in 2018 and 2019.

We here report on the first neuropathological round robin trials initiated by the Quality Assurance Initiative Pathology (QuIP) in Germany in the years 2018 and 2019. Testing services as external laboratory controls were offered for IDH1-R132H immunohistochemistry in 2018 followed by a molecular trial for IDH1 and IDH2 mutations in 2019 including the rare mutational variants. Also in 2019, a trial on MGMT promoter methylation testing was offered. On a national scale, trial offers were well received with around 40 participating institutions. The international announcement of the molecular IDH1/IDH2 mutational trial achieved only moderate European outspread. Success rates in all three trials were excellent (IDH1-R132H immunohistochemistry 2018: 94%, 18 out of 20 possible points required; IDH1/IDH2 mutational status 2019: 100%, 19 out of 20 possible points required; MGMT promoter methylation 2019: 94%, 19 out of 20 possible points required) indicating that quality standards are high in the broad majority of the institutions. Trial participation also involved filling in a questionnaire asking for background information on local testing procedures. We here present a first assessment of the information collected providing unique insights in the landscape of molecular testing in neuropathology. Derived from this information we identify future challenges and provide an outlook on the development of quality assurance in the field of neuropathology.

[Quality assurance in diagnostic in situ hybridization-experience of QuIP]. / Qualitätssicherung in der diagnostischen In-situ-Hybridisierung ­ Erfahrungen der QuIP.

The Quality Assurance Initiative Pathology (QuIP) gives pathologists the opportunity to check the methodological processes of immunohistological and molecular diagnostics in a result-oriented manner and obtain a certificate reflecting the quality. For in situ hybridization (ISH), 5 round robin tests were organized in 2019, two recurrent (HER2-ISH gastric carcinomas and HER2-ISH breast carcinomas) and three prototypical (ROS1-NSCLC, ALK1-NSCLC, NTRK). The different round robin tests, which were provided by QuIP, are based on the development in diagnostics and the importance of the therapeutic relevance of the molecules which are tested. The results of the round robin tests in 2019 showed a sensitivity of at least 94.4%, a specificity of at least 96.6%, and a success rate of 85-99%. This reflected the high standard of quality of the round robin test and the participating institutes.

Reproducibility of mRNA-Based Testing of ESR1, PGR, ERBB2, and MKI67 Expression in Invasive Breast Cancer-A Europe-Wide External Quality Assessment.

Estrogen receptor (ER), progesterone receptor (PgR), Ki-67, and HER2 immunohistochemistry (IHC) together with HER2 in situ hybridization (ISH) are utilized to classify invasive breast cancer (IBC) into predictive molecular subtypes. As IHC evaluation may be hampered by analytical errors, gene expression assays could offer a reliable alternative. In this first Europe-wide external quality assessment (EQA) study, we investigated performance of mRNA-based Xpert® Breast Cancer STRAT4 (CE-IVD) in five European laboratories. The cohort comprised ten pre-therapy IBC core biopsies diagnosed in the coordinating center (CC). STRAT4 binary (positive or negative) mRNA results of each marker (ESR1, PGR, ERBB2, MKI67) were compared with the gold standard IHC/ISH performed by the CC. Sensitivity, specificity, and accuracy of ESR1 and ERBB2 mRNA were 100% for all samples. In contrast, PGR expression was falsely negative for one case by two sites and MKI67 falsely negative for two cases (respectively by four and one sites). These cases had STRAT4 expression values close to assay cut-offs and immunohistochemically presented heterogeneous low positive PgR and heterogeneous Ki-67. Our EQA shows that STRAT4 mRNA assay may be a reproducible method to evaluate ER, PgR, HER2, and Ki-67 status. However, cases with expression values close to assay cut-offs should be carefully reviewed.